U.S. patent application number 17/582682 was filed with the patent office on 2022-08-18 for arenavirus particles as cancer vaccines.
This patent application is currently assigned to Hookipa Biotech GmbH. The applicant listed for this patent is Hookipa Biotech GmbH. Invention is credited to Katherine Cohen, Klaus Orlinger, Sarah Schmidt.
Application Number | 20220257734 17/582682 |
Document ID | / |
Family ID | 1000006290969 |
Filed Date | 2022-08-18 |
United States Patent
Application |
20220257734 |
Kind Code |
A1 |
Schmidt; Sarah ; et
al. |
August 18, 2022 |
ARENAVIRUS PARTICLES AS CANCER VACCINES
Abstract
The present application relates generally to genetically
modified arenaviruses that are suitable vaccines against neoplastic
diseases, such as cancer. The arenaviruses described herein may be
suitable for vaccines and/or treatment of neoplastic diseases
and/or for the use in immunotherapies. In particular, provided
herein are methods and compositions for treating a neoplastic
disease by administering a genetically modified arenavirus in
combination with an immune checkpoint inhibitor, wherein the
arenavirus has been engineered to include a nucleotide sequence
encoding a tumor antigen, tumor associated antigen or antigenic
fragment thereof.
Inventors: |
Schmidt; Sarah; (Vienna,
AT) ; Orlinger; Klaus; (Wien, AT) ; Cohen;
Katherine; (Vienna, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hookipa Biotech GmbH |
Vienna |
|
AT |
|
|
Assignee: |
Hookipa Biotech GmbH
Vienna
AT
|
Family ID: |
1000006290969 |
Appl. No.: |
17/582682 |
Filed: |
January 24, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15775360 |
May 10, 2018 |
11266727 |
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PCT/EP2016/076668 |
Nov 4, 2016 |
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17582682 |
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62254654 |
Nov 12, 2015 |
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62254651 |
Nov 12, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/001195 20180801;
A61K 39/001149 20180801; A61K 39/001172 20180801; A61K 39/001151
20180801; A61K 39/001108 20180801; A61K 39/001139 20180801; A61K
39/001168 20180801; A61K 39/001184 20180801; A61K 39/001109
20180801; A61K 39/001197 20180801; A61K 39/001189 20180801; A61K
39/001104 20180801; A61K 39/001194 20180801; A61K 39/001157
20180801; A61K 39/001188 20180801; A61K 39/001159 20180801; A61P
35/00 20180101; A61K 39/00117 20180801; A61K 39/001174 20180801;
A61K 39/001186 20180801; A61K 39/001153 20180801; A61K 39/00118
20180801; A61K 39/001117 20180801; A61K 39/00119 20180801; A61K
2039/5256 20130101; A61K 39/001126 20180801; A61K 39/001164
20180801; A61K 39/001138 20180801; A61K 39/0011 20130101; A61K
39/001113 20180801; A61K 39/001193 20180801; A61K 39/001191
20180801; A61K 39/12 20130101; A61K 39/001103 20180801; C12N 15/62
20130101; A61K 39/001156 20180801; A61K 39/001135 20180801; A61K
39/001176 20180801; A61K 39/001152 20180801; A61K 39/001122
20180801; A61K 39/001106 20180801; A61K 39/001112 20180801; A61K
39/001182 20180801; A61K 39/001162 20180801; A61K 39/001171
20180801; A61K 39/001124 20180801; A61K 39/001166 20180801; A61K
39/001192 20180801; A61K 39/00116 20180801; A61K 39/00111
20180801 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61P 35/00 20060101 A61P035/00; A61K 39/12 20060101
A61K039/12; C12N 15/62 20060101 C12N015/62 |
Claims
1. A method for treating a neoplastic disease in a subject
comprising, administering to a subject in need thereof an
arenavirus particle and an immune checkpoint inhibitor, wherein
said arenavirus particle is engineered to contain an arenavirus
genomic segment comprising: (i) a nucleotide sequence encoding a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof; and (ii) at least one arenavirus open reading frame
("ORF") in a position other than the wild-type position of said
ORF, wherein said ORF encodes the glycoprotein ("GP"), the
nucleoprotein ("NP"), the matrix protein Z ("Z protein") or the RNA
dependent RNA polymerase L ("L protein") of said arenavirus
particle.
2. The method of claim 1, wherein said tumor antigen or tumor
associated antigen is selected from the group consisting of
oncogenic viral antigens, cancer-testis antigens, oncofetal
antigens, tissue differentiation antigens, mutant protein antigens,
neoantigens, Adipophilin, AIM-2, ALDH1AI, BCLX (L), BING-4, CALCA,
CD45, CPSF, cyclin D1, DKKI, ENAH (hMcna), Ga733 (EpCAM), EphA3,
EZH2, FGF5, glypican-3, G250/MN/CAIX, HER-2/neu, IDO1, IGF2B3,
IL13Ralpha2, Intestinal carboxyl esterase, alpha-foetoprotein,
Kallikrein 4, KIF20A, Lengsin, M-CSF, MCSP, mdm-2, Meloe, MMP-2,
MMP-7, MUC1, MUC5AC, p53 (non-mutant), PAX5, PBF, PRAME, PSMA,
RAGE, RAGE-1, RGS5, RhoC, RNF43, RU2AS, secernin 1, SOX1O, STEAP1
(six-transmembrane epithelial antigen of the prostate 1),
survivinn, Telomerase, VEGF, WT1, EGF-R, CEA, CD20, CD33, CD52,
glycoprotein 100 (GP100 or gp 100 protein), MELANA/MART1, MART2,
NY-ESO-1, p53, MAGE A1, MAGE A3, MAGE-4, MAGE-5, MAGE-6, CDK4,
alpha-actinin-4, ARTC1, BCR-ABL, BCR-ABL fusion protein (b3a2),
B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDKN2A, CLPP,
COA-1, dek-can fusion protein, EFTUD2, Elongation factor 2,
ETV6-AML, ETV6-AML1 fusion protein, FLT3-ITD, FN1, GPNMB,
LDLR-fucosyltransferaseAS fusion protein, NFYC, OGT, OS-9,
pml-RARalpha fusion protein, PRDX5, PTPRK, H-ras, K-ras (V-Ki-ras2
Kirsten rat sarcoma viral oncogene), N-ras, RBAF600, SIRT2, SNRPD1,
SSX, SSX2, SYT-SSX1 or -SSX2 fusion protein, TGF-betaRII,
Triosephosphate isomerase, ormdm-2, LMP2, HPV E6/E7, EGFRvIII
(epidermal growth factor variant III), Idiotype, GD2, ganglioside
G2), Ras-mutant, p53 (mutant), Proteinase3 (PR1), Tyrosinase, PSA,
hTERT, Sarcoma translocation breakpoints, EphA2, prostatic acid
phosphatase PAP, neo-PAP, ML-IAP, AFP, ERG (TMPRSS2 ETS Fusion
gene), NA17, PAX3, ALK, Androgen Receptor, Cyclin B1, Polysialic
acid, MYCN, TRP2, TRP2-Int2, GD3, Fucosyl GM1, Mesothelin, PSCA,
sLe(a), cyp1B1, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, SART3, STn,
Carbonic Anhydrase IX, OY-TES1, Sperm protein 17, LCK, high
molecular weight melanoma-associated antigen (HMWMAA), AKAP-4,
SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP,
PDGFR-beta, MAD-CT-2, For-related antigen 1, TRP1, CA-125, CA19-9,
Calretinin, Epithelial membrane antigen (EMA), Epithelial tumor
antigen (ETA), CD19, CD34, CD99, CD117, Chromogranin, Cytokeratin,
Desmin, Glial fibrillary acidic protein (GFAP), gross cystic
disease fluid protein (GCDFP-15), HMB-45 antigen, Myo-D1,
muscle-specific actin (MSA), neurofilament, neuron-specific enolase
(NSE), placental alkaline phosphatase, synaptophysis,
thyroglobulin, thyroid transcription factor-1, dimeric form of the
pyruvate kinase isoenzyme type M2 (tumor M2-PK), BAGE BAGE-1, CAGE,
CTAGE, FATE, GAGE, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,
GAGE-7, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-SAR-35,
SPANXB1, SPA17, SSX, SYCP1, TPTE, Carbohydrate/ganglioside GM2
(oncofetal antigen-immunogenic-1 OFA-I-1), GM3, CA 15-3 (CA
27.29\BCAA), CA 195, CA 242, CA 50, CAM 43, CEA, EBNA, EF2,
Epstein-Barr virus antigen, HLA-A2, HLA-A11, HSP70-2, KIAAO205,
MUM-1, MUM-2, MUM-3, Myosin class I, GnTV, Herv-K-mel, LAGE-1,
LAGE-2, (sperm protein) SP17, SCP-1, P15(58), Hom/Mel-40, E2A-PRL,
H4-RET, IGH-IGK, MYL-RAR, TSP-180, P185erbB2, p180erbB-3, c-met,
nm-23H1, TAG-72, TAG-72-4, CA-72-4, CAM 17.1, NuMa, 13-catenin,
P16, TAGE, CT7, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA,
CD68\KP1, CO-029, HTgp-175, M344, MG7-Ag, MOV18, NB\70K, NY-CO-1,
RCAS1, SDCCAG16, TA-90, TAAL6, TLP, TPS, CD22, CD27, CD30, CD70,
prostein, TARP (T cell receptor gamma alternate reading frame
protein), Trp-p8, integrin .alpha.v.beta.3 (CD61), galactin, or
Ral-B, CD123, CLL-1, CD38, CS-1, CD138, and ROR1.
3. The method of claim 1, wherein said nucleotide sequence encodes
two, three, four, five, six, seven, eight, nine, ten or more tumor
antigens or tumor associated antigens or antigenic fragments
thereof.
4. The method of claim 1, wherein said immune checkpoint inhibitor
inhibits, decreases or interferes with the activity of a negative
checkpoint regulator is selected from the group consisting of
Cytotoxic T-lymphocyte antigen-4 (CTLA-4), CD80, CD86, Programmed
cell death 1 (PD-1), Programmed cell death ligand 1 (PD-L1),
Programmed cell death ligand 2 (PD-L2), Lymphocyte activation
gene-3 (LAG-3; also known as CD223), Galectin-3, B and T lymphocyte
attenuator (BTLA), T-cell membrane protein 3 (TIM3), Galectin-9
(GAL9), B7-H1, B7-H3, B7-H4, T-Cell immunoreceptor with Ig and ITIM
domains (TIGIT/Vstm3/WUCAM/VSIG9), V-domain Ig suppressor of T-Cell
activation (VISTA), Glucocorticoid-induced tumor necrosis factor
receptor-related (GITR) protein, Herpes Virus Entry Mediator
(HVEM), OX40, CD27, CD28, CD137. CGEN-15001T, CGEN-15022,
CGEN-15027, CGEN-15049, CGEN-15052, and CGEN-15092.
5. The method of claim 1, to wherein said subject is suffering
from, is susceptible to, or is at risk for a neoplastic disease
selected from the group consisting of acute lymphoblastic leukemia;
acute lymphoblastic lymphoma; acute lymphocytic leukaemia; acute
myelogenous leukemia; acute myeloid leukemia (adult/childhood);
adrenocortical carcinoma; AIDS-related cancers; AIDS-related
lymphoma; anal cancer; appendix cancer; astrocytomas; atypical
teratoid/rhabdoid tumor; basal-cell carcinoma; bile duct cancer,
extrahepatic (cholangiocarcinoma); bladder cancer; bone
osteosarcoma/malignant fibrous histiocytoma; brain cancer
(adult/childhood); brain tumor, cerebellar astrocytoma
(adult/childhood); brain tumor, cerebral astrocytoma/malignant
glioma brain tumor; brain tumor, ependymoma; brain tumor,
medulloblastoma; brain tumor, supratentorial primitive
neuroectodermal tumors; brain tumor, visual pathway and
hypothalamic glioma; brainstem glioma; breast cancer; bronchial
adenomas/carcinoids; bronchial tumor; Burkitt lymphoma; cancer of
childhood; carcinoid gastrointestinal tumor; carcinoid tumor;
carcinoma of adult, unknown primary site; carcinoma of unknown
primary; central nervous system embryonal tumor; central nervous
system lymphoma, primary; cervical cancer; childhood adrenocortical
carcinoma; childhood cancers; childhood cerebral astrocytoma;
chordoma, childhood; chronic lymphocytic leukemia; chronic
myelogenous leukemia; chronic myeloid leukemia; chronic
myeloproliferative disorders; colon cancer; colorectal cancer;
craniopharyngioma; cutaneous T-cell lymphoma; desmoplastic small
round cell tumor; emphysema; endometrial cancer; ependymoblastoma;
ependymoma; esophageal cancer; ewing's sarcoma in the Ewing family
of tumors; extracranial germ cell tumor; extragonadal germ cell
tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric
(stomach) cancer; gastric carcinoid; gastrointestinal carcinoid
tumor; gastrointestinal stromal tumor; germ cell tumor:
extracranial, extragonadal, or ovarian gestational trophoblastic
tumor; gestational trophoblastic tumor, unknown primary site;
glioma; glioma of the brain stem; glioma, childhood visual pathway
and hypothalamic; hairy cell leukemia; head and neck cancer; heart
cancer; hepatocellular (liver) cancer; hodgkin lymphoma;
hypopharyngeal cancer; hypothalamic and visual pathway glioma;
intraocular melanoma; islet cell carcinoma (endocrine pancreas);
Kaposi Sarcoma; kidney cancer (renal cell cancer); langerhans cell
histiocytosis; laryngeal cancer; lip and oral cavity cancer;
liposarcoma; liver cancer (primary); lung cancer, non-small cell;
lung cancer, small cell; lymphoma, primary central nervous system;
macroglobulinemia, Waldenstrom; male breast cancer; malignant
fibrous histiocytoma of bone/osteosarcoma; medulloblastoma;
medulloepithelioma; melanoma; melanoma, intraocular (eye); merkel
cell cancer; merkel cell skin carcinoma; mesothelioma;
mesothelioma, adult malignant; metastatic squamous neck cancer with
occult primary; mouth cancer; multiple endocrine neoplasia
syndrome; multiple myeloma/plasma cell neoplasm; mycosis fungoides,
myelodysplastic syndromes; myelodysplastic/myeloproliferative
diseases; myelogenous leukemia, chronic; myeloid leukemia, adult
acute; myeloid leukemia, childhood acute; myeloma, multiple (cancer
of the bone-marrow); myeloproliferative disorders, chronic; nasal
cavity and paranasal sinus cancer; nasopharyngeal carcinoma;
neuroblastoma, non-small cell lung cancer; non-hodgkin lymphoma;
oligodendroglioma; oral cancer; oral cavity cancer; oropharyngeal
cancer; osteosarcoma/malignant fibrous histiocytoma of bone;
ovarian cancer; ovarian epithelial cancer (surface
epithelial-stromal tumor); ovarian germ cell tumor; ovarian low
malignant potential tumor; pancreatic cancer; pancreatic cancer,
islet cell; papillomatosis; paranasal sinus and nasal cavity
cancer; parathyroid cancer; penile cancer; pharyngeal cancer;
pheochromocytoma; pineal astrocytoma; pineal germinoma; pineal
parenchymal tumors of intermediate differentiation; pineoblastoma
and supratentorial primitive neuroectodermal tumors; pituitary
tumor; pituitary adenoma; plasma cell neoplasia/multiple myeloma;
pleuropulmonary blastoma; primary central nervous system lymphoma;
prostate cancer; rectal cancer; renal cell carcinoma (kidney
cancer); renal pelvis and ureter, transitional cell cancer;
respiratory tract carcinoma involving the NUT gene on chromosome
15; retinoblastoma; rhabdomyosarcoma, childhood; salivary gland
cancer; sarcoma, Ewing family of tumors; Sezary syndrome; skin
cancer (melanoma); skin cancer (non-melanoma); small cell lung
cancer; small intestine cancer soft tissue sarcoma; soft tissue
sarcoma; spinal cord tumor; squamous cell carcinoma; squamous neck
cancer with occult primary, metastatic; stomach (gastric) cancer;
supratentorial primitive neuroectodermal tumor; T-cell lymphoma,
cutaneous (Mycosis Fungoides and Sezary syndrome); testicular
cancer; throat cancer; thymoma; thymoma and thymic carcinoma;
thyroid cancer; thyroid cancer, childhood; transitional cell cancer
of the renal pelvis and ureter; urethral cancer; uterine cancer,
endometrial; uterine sarcoma; vaginal cancer; vulvar cancer; and
Wilms Tumor.
6. The method of claim 1, wherein said arenavirus particle and said
immune checkpoint inhibitor are co-administered simultaneously.
7. The method of claim 1, wherein said arenavirus particle is
administered: (i) prior to administration of said immune checkpoint
inhibitor; or (ii) after administration of said immune checkpoint
inhibitor.
8. (canceled)
9. The method of claim 7, wherein the interval between
administration of said arenavirus particle and said immune
checkpoint inhibitor is about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 10 hours, about 11 hours, about 12
hours, about 1 day, about 2 days, about 3 days, about 4 days, about
5 days, about 6 days, about 1 week, about 8 days, about 9 days,
about 10 days, about 11 days, about 12 days, about 13 days, about 2
weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks,
about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about
11 weeks, about 12 weeks, about 1 month, about 2 months, about 3
months, about 4 months, about 5 months, about 6 months, or
more.
10. The method of claim 1, wherein said arenavirus particle and
said immune checkpoint inhibitor are administered in a
therapeutically effective amount.
11. The method of claim 1, wherein said method comprises
administering to said subject a first arenavirus particle, and
administering to said subject, after a period of time, a second
arenavirus particle.
12. The method of claim 11, wherein said first arenavirus particle
and said second particle are derived from different arenavirus
species and/or comprise nucleotide sequences encoding different
tumor antigen, tumor associated antigens or antigenic fragments
thereof.
13. The method of claim 1, wherein said arenavirus genomic segment
is selected from the group consisting of: (i) an S segment, wherein
the ORF encoding the NP is under control of an arenavirus 5'
untranslated region ("UTR"); (ii) an S segment, wherein the ORF
encoding the Z protein is under control of an arenavirus 5' UTR;
(iii) an S segment, wherein the ORF encoding the L protein is under
control of an arenavirus 5' UTR; (iv) an S segment, wherein the ORF
encoding the GP is under control of an arenavirus 3' UTR; (v) an S
segment, wherein the ORF encoding the L protein is under control of
an arenavirus 3' UTR; (vi) an S segment, wherein the ORF encoding
the Z protein is under control of an arenavirus 3' UTR; (vii) an L
segment, wherein the ORF encoding the GP is under control of an
arenavirus 5' UTR; (viii) an L segment, wherein the ORF encoding
the NP is under control of an arenavirus 5' UTR; (ix) an L segment,
wherein the ORF encoding the L protein is under control of an
arenavirus 5' UTR; (x) an L segment, wherein the ORF encoding the
GP is under control of an arenavirus 3' UTR; (xi) an L segment,
wherein the ORF encoding the NP is under control of an arenavirus
3' UTR; and (xii) an L segment, wherein the ORF encoding the Z
protein is under control of an arenavirus 3' UTR, wherein said
arenavirus 3' UTR is the 3' UTR of the arenavirus S segment or the
arenavirus L segment, and wherein said arenavirus 5' UTR is the 5'
UTR of the arenavirus S segment or the arenavirus L segment.
14. (canceled)
15. The method of claim 1, wherein said arenavirus particle
comprises a second arenavirus genomic segment so that said
arenavirus particle comprises an S segment and an L segment.
16. The method of claim 15, wherein said arenavirus particle is
attenuated, infectious, and replication-competent.
17-25. (canceled)
26. The method of claim 1, wherein the arenavirus particle is a
tri-segmented arenavirus particle comprising either one L segment
and two S segments.
27-29. (canceled)
30. The method of claim 26, wherein propagation of said
tri-segmented arenavirus particle does not result in a
replication-competent bi-segmented viral particle after 70 days of
persistent infection in mice lacking type I interferon receptor,
type II interferon receptor and recombination activating gene 1
(RAG1) and having been infected with 10.sup.4 PFU of said
tri-segmented arenavirus particle.
31. The method of claim 26, wherein inter-segmental recombination
of two S segments, uniting two arenavirus ORFs on only one instead
of two separate segments, abrogates viral promoter activity.
32.-33. (canceled)
34. The method of claim 26, wherein the two S segments comprise:
(i) one or two nucleotide sequences each encoding a tumor antigen,
tumor associated antigen or an antigenic fragment thereof; or (ii)
one or two duplicated arenavirus ORFs; or (iii) one nucleotide
sequence encoding a tumor antigen, tumor associated antigen or an
antigenic fragment thereof and one duplicated arenavirus ORF.
35-37. (canceled)
38. The method of claim 1, wherein said arenavirus particle is
derived from lymphocytic choriomeningitis virus ("LCMV").
39. (canceled)
40. The method of claim 38, wherein said LCMV is MP strain, WE
strain, Armstrong strain, or Armstrong Clone 13 strain.
41.-42. (canceled)
43. The method of claim 1, wherein the growth or infectivity of
said arenavirus particle is not affected by said nucleotide
sequence encoding a tumor antigen, tumor associated antigen or an
antigenic fragment thereof.
44. A pharmaceutical composition comprising an arenavirus particle,
an immune checkpoint inhibitor and a pharmaceutically acceptable
carrier, wherein said arenavirus particle is engineered to contain
an arenavirus genomic segment comprising: (i) a nucleotide sequence
encoding a tumor antigen, tumor associated antigen or an antigenic
fragment thereof; and (ii) at least one arenavirus open reading
frame ("ORF") in a position other than the wild-type position of
said ORF, wherein said ORF encodes the glycoprotein ("GP"), the
nucleoprotein ("NP"), the matrix protein Z ("Z protein") or the RNA
dependent RNA polymerase L ("L protein") of said arenavirus
particle, and wherein said immune checkpoint inhibitor inhibits,
decreases or interferes with the activity of a negative checkpoint
regulator.
45-79. (canceled)
80. A kit comprising one or more containers and instructions for
use, wherein said one or more containers comprise said
pharmaceutical composition of claim 44.
81. A kit comprising two or more containers and instructions for
use, wherein one of said containers comprises an arenavirus
particle and another of said containers comprises an immune
checkpoint inhibitor, wherein said arenavirus particle is
engineered to contain an arenavirus genomic segment comprising: a.
a nucleotide sequence encoding a tumor antigen, tumor associated
antigen or an antigenic fragment thereof; and b. at least one
arenavirus open reading frame ("ORF") in a position other than the
wild-type position of said ORF, wherein said ORF encodes the
glycoprotein ("GP"), the nucleoprotein ("NP"), the matrix protein Z
("Z protein") or the RNA dependent RNA polymerase L ("L protein")
of said arenavirus particle, and wherein said immune checkpoint
inhibitor inhibits, decreases or interferes with the activity of a
negative checkpoint regulator.
82-116. (canceled)
117. The method of claim 1, wherein said immune checkpoint
inhibitor inhibits, decreases or interferes with the activity of a
negative checkpoint regulator.
118. The method of claim 1, wherein said immune checkpoint
inhibitor is an antibody that binds to or inhibits activity of
Programmed cell death 1 (PD1).
119. The method of claim 1, wherein said immune checkpoint
inhibitor is an antibody that binds to or inhibits activity of
Programmed cell death ligand 1 (PD-L1), Cytotoxic T-lymphocyte
antigen-4 (CTLA4), T-cell membrane protein 3 (TIM-3), Lymphocyte
activation gene-3 (LAG-3), or T-Cell immunoreceptor with Ig and
ITIM domains (TIGIT).
120. The method of claim 1, wherein said arenavirus particle
encodes a neoantigen.
121. (canceled)
122. The method of claim 1, wherein said arenavirus particle
encodes a melanoma antigen.
123-125. (canceled)
126. The method of claim 120, wherein said subject is suffering
from colon cancer.
127. The method of claim 122, wherein said subject is suffering
from melanoma.
128. The method of claim 120, wherein said immune checkpoint
inhibitor is an antibody that binds to or inhibits activity of
Programmed cell death 1 (PD 1).
129. (canceled)
Description
[0001] This application claims the benefit of priority of U.S.
Provisional Application No. 62/254,654, filed Nov. 12, 2015, and
U.S. Provisional Application No. 62/254,651, filed Nov. 12, 2015,
the entire contents of which are each incorporated herein by
reference.
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Nov. 2, 2016, is named 13194-017-228_Sequence_Listing.txt and is
112,465 bytes in size.
1. INTRODUCTION
[0003] The present application relates generally to genetically
modified arenaviruses that are suitable vaccines against neoplastic
diseases, such as cancer. The arenaviruses described herein may be
suitable for vaccines and/or treatment of neoplastic diseases
and/or for the use in immunotherapies. In particular, provided
herein are methods and compositions for treating a neoplastic
disease by administering a genetically modified arenavirus in
combination with an immune checkpoint inhibitor, wherein the
arenavirus has been engineered to include a nucleotide sequence
encoding a tumor antigen, tumor associated antigen or antigenic
fragment thereof.
2. BACKGROUND
2.1 Lymphocytic Choriomeningitis Virus Research and Human
Disease
[0004] Lymphocytic choriomeningitis virus (LCMV), a member of the
family arenaviridae, is a prototypic mouse model virus in research
on viral infections. Since its isolation in the 1930s (Rivers and
McNair Scott, 1935, Science, 81(2105): 439-440) studies using this
virus have uncovered many key concepts in viral immunology and
pathogenesis (summarized in Zinkernagel, 2002, Curr Top Microbiol
Immunol, 263:1-5; Oldstone, 2002, Curr Top Microbiol Immunol,
263:83-117). LCMV has been extensively used to investigate viral
molecular biology and immune responses particularly in the context
of persistent infection. The natural hosts of LCMV are mice,
however, several reports revealed that LCMV might also be a
neglected human pathogen (Barton, 1996, Clin. Infect. Dis,
22(1):197; Wright et al., 1997, Pediatrics 100(1): E9). Moreover,
numerous other members of the arenavirus family have been found in
rodent populations around the world. In addition to the Old World
arenavirus Lassa virus (LASV), which can be found in Africa,
several New World arenaviruses like Junin, Guanarito or Machupo are
prevalent in diverse rodent populations of South America (Johnson
et al., 1966, Am J Trop Med Hyg, 15(1): 103-106; Tesh et al., 1993,
Am J Trop Med Hyg 49(2):227-235; Mills et al., 1994, Trop Med Hyg
51(5): 554-562). Upon transmission to humans, many of those viruses
can cause viral hemorrhagic fever associated with high mortality
(Geisbert and Jahrling, 2004, Nat Med 10(12 Suppl): S110-121).
2.2 Genomic Organization of Lymphocytic Choriomeningitis Virus
[0005] Arenaviruses are enveloped viruses. Their genome consists of
two segments of single-stranded RNA of negative sense (L: 7.2 kb,
S: 3.4 kb). Each segment encodes for two viral genes in opposite
orientations. The short segment (S segment) encodes the viral
glycoprotein (GP) precursor (GP-C; 75 kDa) and the nucleoprotein
(NP; 63 kDa) (Salvato et al., 1988, Virology 164(2): 517-522). The
long segment (L segment) expresses the RNA-dependent RNA polymerase
(RdRp; L protein; approximately 200 kDa) and the matrix protein Z
(protein Z), a RING finger protein (11 kDa) (FIG. 1A) (Salvato et
al., 1988, Virology 164(2): 517-522). The GP precursor GP-C is
post-translationally cleaved into GP-1 and GP-2, which remain
non-covalently associated (Buchmeier and Oldstone 1979, Virology
99(1): 111-120). Trimers of GP-1 and GP-2 are assembled as spikes
on the surface of virions and are essential for mediating entry
into the host cells by interaction with the cellular surface
receptors. Binding and entry of the virus into host cells was long
claimed to be mediated by interaction of the LCMV GP with the
cellular receptor .alpha.-Dystroglycan as the only cellular
receptor for LCMV (Cao et al., 1998, Science, 282(5396):2079-2081).
Only very recently three additional human molecules (Ax1 and Tyro3
from the TAM family and dendritic cell-specific intracellular
adhesion molecule 3-grabbing nonintegrin) were postulated as
additional receptors for LCMV and LASV, a close relative of LCMV,
which enable entry of LCMV into cells independently of
.alpha.-Dystroglycan (Shimojima and Kawaoka 2012, J Vet Med,
74(10):1363-1366; Shimojima et al., 2012, J Virol 86(4):2067-2078).
NP binds to the viral RNA, forming the nucleocapsid, which serves
as a template for the viral L protein. The nucleocapsid associated
with the viral L protein forms the so-called ribonucleoprotein
complex, which is active both in replication and transcription and
represents the minimum unit of viral infectivity. It has been
shown, that NP and the L protein are the minimal trans-acting
factors necessary for viral RNA transcription and replication (Lee
et al., 2000, J Virol 74(8): 3470-3477). The two genes on each
segment are separated by a non-coding intergenic region (IGR) and
flanked by 5' and 3' untranslated regions (UTR). The IGR forms a
stable hairpin structure and has been shown to be involved in
structure-dependent termination of viral mRNA transcription
(Pinschewer et al., 2005, J Virol 79(7): 4519-4526). The terminal
nucleotides of the UTR show a high degree of complementarity,
resulting in the formation of secondary structures. These panhandle
structures are known to serve as the viral promoter for
transcription and replication, and their analysis by site-directed
mutagenesis has revealed sequence- and structure-dependence,
tolerating not even minor sequence changes (Perez and de la Torre,
2003, Virol 77(2): 1184-1194).
2.3 Reverse Genetic System
[0006] Isolated and purified RNAs of negative-strand viruses like
LCMV cannot directly serve as mRNA i.e., cannot be translated when
introduced into cells. Consequently transfection of cells with
viral RNA does not lead to production of infectious viral
particles. In order to generate infectious viral particles of
negative-stranded RNA viruses from cDNA in cultured permissive
cells, the viral RNA segment(s) must be trans-complemented with the
minimal factors required for transcription and replication. With
the help of a minigenome system which has been published several
years ago, viral cis-acting elements and transacting factors
involved in transcription, replication and formation of viral
particles could finally be analyzed (Lee et al., 2000, J Virol
74(8): 3470-3477; Lee et al., 2002, J Virol 76(12): 6393-6397;
Perez and de la Torre 2003, J Virol 77(2): 1184-1194; Pinschewer et
al., 2003, J Virol 77(6): 3882-3887; Pinschewer et al., 2005, J
Virol 79(7): 4519-4526.). Also for other arenaviruses like LASV and
Tacaribe virus reverse genetic systems have been established (Lopez
et al., 2001, J Virol 75(24): 12241-12251; Hass et al., 2004, J
Virol 78(24): 13793-13803). Two publications showed the recovery of
infectious LCMV entirely from cDNA using pol-I/-II or
T7/pol-II-driven plasmids, respectively (referred to as "viral
rescue") (Flatz et al., 2006, Proc Natl Acad Sci USA 103(12):
4663-4668; Sanchez and de la Torre, 2006, Virology 350(2):
370-380).
2.4 Recombinant LCMV Expressing Genes of Interest
[0007] The generation of recombinant negative-stranded RNA viruses
expressing foreign genes of interest has been pursued for a long
time. Different strategies have been published for other viruses
(Garcia-Sastre et al., 1994, J Virol 68(10): 6254-6261; Percy et
al., 1994, J Virol 68(7): 4486-4492; Flick and Hobom, 1999,
Virology 262(1): 93-103; Machado et al., 2003, Virology 313(1):
235-249). In the past it has been shown that it is possible to
introduce additional foreign genes into the genome of bi-segmented
LCMV particles (Emonet et al., 2009, PNAS, 106(9):3473-3478). Two
foreign genes of interest were inserted into the bi-segmented
genome of LCMV, resulting in tri-segmented LCMV particles (r3LCMV)
with two S segments and one L segment. In the tri-segmented virus,
published by Emonet et al., (2009), both NP and GP were kept in
their respective natural position in the S segment and thus were
expressed under their natural promoters in the flanking UTR.
2.5 Replication-Deficient Arenavirus
[0008] Recently, it has been shown that an infectious arenavirus
particle can be engineered to contain a genome with the ability to
amplify and express its genetic material in infected cells but
unable to produce further progeny in normal, not genetically
engineered cells (i.e., an infectious, replication-deficient
arenavirus particle) (International Publication No.: WO 2009/083210
A1 and International Publication No.: WO 2014/140301 A1).
2.6 Replication-Deficient Arenavirus Vectors Expressing Genes of
Interest
[0009] The use of infectious, replication-deficient arenaviruses as
vectors for the expression of antigens has been reported (see Flatz
et. al., 2010, Nat. Med., 16(3):339-345; Flatz et al., 2012, J.
Virol., 86(15), 7760-7770). These infectious, replication-deficient
arenaviruses can infect a host cell, i.e., attach to a host cell
and release their genetic material into the host cell. However,
they are replication-deficient, i.e., the arenavirus is unable to
produce further infectious progeny particles in a non-complementing
cell, due to a deletion or functional inactivation of an open
reading frame (ORF) encoding a viral protein, such as the GP
protein. Instead, the ORF is substituted with a nucleotide sequence
of an antigen of interest. In Flatz et al. 2010, the authors used
infectious, replication-deficient arenaviruses as vectors to
express OVA (SIINFEKL epitope). In Flatz et al. 2012, the authors
used replication deficient arenaviruses as vectors to express
HIV/SIV Env.
3. SUMMARY OF THE INVENTION
[0010] Provided herein are methods and compositions for treating a
neoplastic disease using an arenavirus particle comprising a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen or antigenic fragment thereof. Also provided herein are
methods and compositions for treating a neoplastic disease using an
immune checkpoint inhibitor. Such an immune check point inhibitor
can inhibit, decrease or interfere with the activity of a negative
checkpoint regulator. Thus, in certain embodiments, provided herein
are methods for treating a neoplastic disease using an arenavirus
particle comprising a nucleotide sequence encoding a tumor antigen,
tumor associated antigen or antigenic fragment thereof, and an
immune checkpoint inhibitor that inhibits, decreases or interferes
with the activity of a negative checkpoint regulator. Also, in
certain embodiments, provided herein are compositions comprising an
arenavirus particle comprising a nucleotide sequence encoding a
tumor antigen, tumor associated antigen or antigenic fragment
thereof, and an immune checkpoint inhibitor that inhibits,
decreases or interferes with the activity of a negative checkpoint
regulator.
[0011] In certain embodiments, the arenavirus particle provided
herein is engineered to contain an arenavirus genomic segment
having a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or antigenic fragment thereof and at least one
arenavirus open reading frame ("ORF") in a position other than the
wild-type position of the ORF. In certain embodiments, the
arenavirus particle provided herein is an infectious, replication
deficient arenavirus particle. In other embodiments, the arenavirus
particle provided herein is a tri-segmented arenavirus particle,
which can be replication-deficient or replication-competent. In
still other embodiments, the tri-segmented arenavirus particle
provided herein, when propagated, does not result in a
replication-competent bi-segmented viral particle.
3.1 Arenavirus Particles Having Non-Natural Open Reading Frame
[0012] In certain embodiments, provided herein are arenaviruses
with rearrangements of their ORFs in their genomes and a nucleotide
sequence encoding a tumor antigen, tumor associated antigen or an
antigenic fragment thereof provided herein. In a particular
embodiment, an arenavirus particle provided herein includes an
arenavirus genomic segment that has been engineered to carry an
arenavirus ORF in a position other than the wild-type position.
Thus, in certain particular embodiments, provided herein is an
arenavirus genomic segment comprising: a nucleotide sequence
encoding a tumor antigen, tumor associated antigen or an antigenic
fragment thereof; and at least one arenavirus ORF in a position
other than the wild-type position of said ORF, wherein the ORF
encodes the glycoprotein ("GP"), the nucleoprotein ("NP"), the
matrix protein Z ("Z protein") or the RNA dependent RNA polymerase
L ("L protein") of an arenavirus particle. Also provided herein is
an arenavirus particle that has been engineered to contain such an
arenavirus genomic segment.
[0013] In certain embodiments, an arenavirus particle provided
herein is infectious, i.e., it is capable of entering into or
injecting its genetic material into a host cell. In certain more
specific embodiments, an arenavirus particle as provided herein is
infectious, i.e., is capable of entering into or injecting its
genetic material into a host cell followed by amplification and
expression of its genetic information inside the host cell. In
certain embodiments, the arenavirus particle provided herein is
engineered to be an infectious, replication-deficient arenavirus
particle, i.e., it contains a genome with the ability to amplify
and express its genetic information in infected cells but unable to
produce further infectious progeny particles in non-complementing
cells.
[0014] The tumor antigen or tumor associated antigen encoded by the
nucleotide sequence included within an arenavirus genomic segment
or arenavirus particle provided herein can be one or more of the
tumor antigens or tumor associated antigens selected from the group
consisting of oncogenic viral antigens, cancer-testis antigens,
oncofetal antigens, tissue differentiation antigens, mutant protein
antigens, neoantigens, Adipophilin, AIM-2, ALDH1AI, BCLX (L),
BING-4, CALCA, CD45, CPSF, cyclin D1, DKKI, ENAH (hMcna), Ga733
(EpCAM), EphA3, EZH2, FGF5, glypican-3, G250/MN/CAIX, HER-2/neu,
IDOL IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase,
alpha-foetoprotein, Kallikrein 4, KIF20A, Lengsin, M-CSF, MCSP,
mdm-2, Meloe, MMP-2, MMP-7, MUC1, MUC5AC, p53 (non-mutant), PAX5,
PBF, PRAME, PSMA, RAGE, RAGE-1, RGS5, RhoC, RNF43, RU2AS, secernin
1, SOX1O, STEAP1 (six-transmembrane epithelial antigen of the
prostate 1), survivinn, Telomerase, VEGF, WT1, EGF-R, CEA, CD20,
CD33, CD52, glycoprotein 100 (GP100 or gp 100 protein),
MELANA/MART1, MART2, NY-ESO-1, p53, MAGE A1, MAGE A3, MAGE-4,
MAGE-5, MAGE-6, CDK4, alpha-actinin-4, ARTC1, BCR-ABL, BCR-ABL
fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27,
CDK4, CDKN2A, CLPP, COA-1, dek-can fusion protein, EFTUD2,
Elongation factor 2, ETV6-AML, ETV6-AML1 fusion protein, FLT3-ITD,
FN1, GPNMB, LDLR-fucosyltransferaseAS fusion protein, NFYC, OGT,
OS-9, pml-RARalpha fusion protein, PRDX5, PTPRK, H-ras, K-ras
(V-Ki-ras2 Kirsten rat sarcoma viral oncogene), N-ras, RBAF600,
SIRT2, SNRPD1, SSX, SSX2, SYT-SSX1 or -SSX2 fusion protein,
TGF-betaRII, Triosephosphate isomerase, ormdm-2, LMP2, HPV E6/E7,
EGFRvIII (epidermal growth factor variant III), Idiotype, GD2,
ganglioside G2), Ras-mutant, p53 (mutant), Proteinase3 (PR1),
Tyrosinase, PSA, hTERT, Sarcoma translocation breakpoints, EphA2,
prostatic acid phosphatase PAP, nco-PAP, ML-IAP, AFP, ERG (TMPRSS2
ETS Fusion gene), NA17, PAX3, ALK, Androgen Receptor, Cyclin B1,
Polysialic acid, MYCN, TRP2, TRP2-Int2, GD3, Fucosyl GM1,
Mesothelin, PSCA, sLe(a), cyp1B1, PLAC1, GM3, BORIS, Tn, GLoboH,
NY-BR-1, SART3, STn, Carbonic Anhydrase IX, OY-TES1, Sperm protein
17, LCK, high molecular weight melanoma-associated antigen
(HMWMAA), AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4,
VEGFR2, MAD-CT-1, FAP, PDGFR-beta, MAD-CT-2, For-related antigen 1,
TRP1, CA-125, CA19-9, Calretinin, Epithelial membrane antigen
(EMA), Epithelial tumor antigen (ETA), CD19, CD34, CD99, CD117,
Chromogranin, Cytokeratin, Desmin, Glial fibrillary acidic protein
(GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45
antigen, Myo-D1, muscle-specific actin (MSA), neurofilament,
neuron-specific enolase (NSE), placental alkaline phosphatase,
synaptophysis, thyroglobulin, thyroid transcription factor-1,
dimeric form of the pyruvate kinase isoenzyme type M2 (tumor
M2-PK), BAGE BAGE-1, CAGE, CTAGE, FATE, GAGE, GAGE-1, GAGE-2,
GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, HCA661, HOM-TES-85, MAGEA,
MAGEB, MAGEC, NA88, NY-SAR-35, SPANXB1, SPA17, SSX, SYCP1, TPTE,
Carbohydrate/ganglioside GM2 (oncofetal antigen-immunogenic-1
OFA-I-1), GM3, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA 50, CAM
43, CEA, EBNA, EF2, Epstein-Barr virus antigen, HLA-A2, HLA-A11,
HSP70-2, KIAAO205, MUM-1, MUM-2, MUM-3, Myosin class I, GnTV,
Herv-K-mel, LAGE-1, LAGE-2, (sperm protein) SP17, SCP-1, P15(58),
Hom/Mel-40, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, TSP-180, P185erbB2,
p180erbB-3, c-met, nm-23H1, TAG-72, TAG-72-4, CA-72-4, CAM 17.1,
NuMa, 13-catenin, P16, TAGE, CT7, 43-9F, 5T4, 791Tgp72, 13HCG,
BCA225, BTAA, CD68\KP1, CO-029, HTgp-175, M344, MG7-Ag, MOV18,
NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TLP, TPS, CD22,
CD27, CD30, CD70, prostein, TARP (T cell receptor gamma alternate
reading frame protein), Trp-p8, integrin .alpha.v.beta.3 (CD61),
galactin, or Ral-B, CD123, CLL-1, CD38, CS-1, CD138, and ROR1. In
certain embodiments, the nucleotide sequence encodes two, three,
four, five, six, seven, eight, nine, ten or more tumor antigens,
tumor associated antigens or antigenic fragments thereof. In
certain embodiments, an antigenic fragment of a tumor antigen or
tumor associated antigen provided herein is encoded by the
nucleotide sequence included within the arenavirus.
[0015] Accordingly, in certain embodiments, provided herein is an
arenavirus genomic segment comprising a nucleotide sequence
encoding a tumor antigen, tumor associated antigen or an antigenic
fragment thereof provided herein. In certain embodiments, the
genomic segment is engineered to carry an arenavirus ORF in a
position other than the wild-type position of the ORF. In some
embodiments, the arenavirus genomic segment is selected from the
group consisting of: [0016] (i) an S segment, wherein the ORF
encoding the NP is under control of an arenavirus 5' UTR; [0017]
(ii) an S segment, wherein the ORF encoding the Z protein is under
control of an arenavirus 5' UTR; [0018] (iii) an S segment, wherein
the ORF encoding the L protein is under control of an arenavirus 5'
UTR; [0019] (iv) an S segment, wherein the ORF encoding the GP is
under control of an arenavirus 3' UTR; [0020] (v) an S segment,
wherein the ORF encoding the L protein is under control of an
arenavirus 3' UTR; [0021] (vi) an S segment, wherein the ORF
encoding the Z protein is under control of an arenavirus 3' UTR;
[0022] (vii) an L segment, wherein the ORF encoding the GP is under
control of an arenavirus 5' UTR; [0023] (viii) an L segment,
wherein the ORF encoding the NP is under control of an arenavirus
5' UTR; [0024] (ix) an L segment, wherein the ORF encoding the L
protein is under control of an arenavirus 5' UTR; [0025] (x) an L
segment, wherein the ORF encoding the GP is under control of an
arenavirus 3' UTR; [0026] (xi) an L segment, wherein the ORF
encoding the NP is under control of an arenavirus 3' UTR; and
[0027] (xii) an L segment, wherein the ORF encoding the Z protein
is under control of an arenavirus 3' UTR.
[0028] In certain embodiments, the arenavirus 3' UTR is the 3' UTR
of the arenavirus S segment or the arenavirus L segment. In certain
embodiments, the arenavirus 5' UTR is the 5' UTR of the arenavirus
S segment or the arenavirus L segment.
[0029] In certain embodiments, the arenavirus particle provided
herein comprises a second arenavirus genomic segment so that the
arenavirus particle comprises an S segment and an L segment.
[0030] In certain embodiments, an arenavirus particle provided
herein is infectious, i.e., it is capable of entering into or
injecting its genetic material into a host cell. In certain more
specific embodiments, an arenavirus particle as provided herein is
infectious, i.e., is capable of entering into or injecting its
genetic material into a host cell followed by amplification and
expression of its genetic information inside the host cell. In
certain embodiments, the arenavirus particle is an infectious,
replication-deficient arenavirus particle engineered to contain a
genome with the ability to amplify and express its genetic
information in infected cells but unable to produce further
infectious progeny particles in non-complementing cells. In certain
embodiments, the arenavirus particle is replication-competent and
able to produce further infectious progeny particles in normal, not
genetically engineered cells. In certain more specific embodiments,
such a replication-competent particle is attenuated relative to the
wild type virus from which the replication-competent particle is
derived.
[0031] In certain embodiments, an arenavirus genomic segment
provided herein, including an arenavirus particle comprising the
arenavirus genomic segment, comprises at least one arenavirus ORF
that is at least partially removed or functionally inactivated. The
ORF can encode the GP, NP, Z protein, or L protein of an arenavirus
particle. Additionally, in certain embodiments, at least one ORF
encoding the GP, NP, Z protein, or L protein is removed and
replaced with a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof provided
herein. In certain embodiments, only one of the four ORFs encoding
GP, NP, Z protein, and L protein is removed. Thus, in certain
embodiments, the ORF encoding GP is removed. In certain
embodiments, the ORF encoding NP is removed. In certain
embodiments, the ORF encoding Z protein is removed. In certain
embodiments, the ORF encoding L protein is removed.
[0032] In certain embodiments, a bi-segmented infectious,
replication-deficient arenavirus particle provided herein comprises
at least one arenavirus ORF that is at least partially removed or
functionally inactivated. The ORF can encode GP, NP, Z protein, or
L protein. Additionally, in certain embodiments, at least one ORF
encoding the GP, NP, Z protein, or L protein is removed and
replaced with a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof provided
herein. In certain embodiments, only one of the four ORFs encoding
GP, NP, Z protein and L protein is removed. Thus, in certain
embodiments, the ORF encoding GP is removed. In certain
embodiments, the ORF encoding NP is removed. In certain
embodiments, the ORF encoding Z protein is removed. In certain
embodiments, the ORF encoding L protein is removed.
[0033] In certain embodiments, a bi-segmented infectious,
replication-deficient arenavirus particle comprising a nucleotide
sequence encoding a tumor antigen, tumor associated antigen or
antigenic fragment thereof as provided herein further comprises at
least one nucleotide sequence encoding at least one
immunomodulatory peptide, polypeptide or protein. In certain
embodiments, the immunomodulatory peptide, polypeptide or protein
is Calreticulin (CRT), or a fragment thereof; Ubiquitin or a
fragment thereof; Granulocyte-Macrophage Colony-Stimulating Factor
(GM-CSF), or a fragment thereof; Invariant chain (CD74) or an
antigenic fragment thereof; Mycobacterium tuberculosis Heat shock
protein 70 or an antigenic fragment thereof; Herpes simplex virus 1
protein VP22 or an antigenic fragment thereof; CD40 ligand or an
antigenic fragment thereof; or Fms-related tyrosine kinase 3 (Flt3)
ligand or an antigenic fragment thereof.
[0034] In certain embodiments, an arenavirus particle provided
herein is derived from a specific arenavirus species, such as
lymphocytic choriomeningitis virus ("LCMV") or Junin virus
("JUNV"). In other words, the genomic information encoding the
arenavirus particle is derived from a specific species of
arenavirus. Thus, in certain embodiments, the arenavirus particle
is derived from LCMV. In other embodiments, the arenavirus particle
is derived from JUNV. Additionally, is specific embodiments, the
LCMV is MP strain, WE strain, Armstrong strain, or Armstrong Clone
13 strain. In other specific embodiments, the JUNV is JUNV vaccine
Candid #1 strain, or JUNV vaccine XJ Clone 3 strain.
3.2 Tri-Segmented Arenaviruses
[0035] In certain embodiments, provided herein are tri-segmented
arenavirus particles comprising a nucleotide sequence encoding a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein. Thus, in certain embodiments, an
arenavirus particle provided herein can comprise one L segment and
two S segments or two L segments and one S segment. In certain
embodiments, the tri-segmented arenavirus particle provided herein
does not recombine into a replication-competent bi-segmented
arenavirus particle. Accordingly, in certain embodiments,
propagation of the tri-segmented arenavirus particle does not
result in a replication-competent bi-segmented particle after 70
days of persistent infection in mice lacking type I interferon
receptor, type II interferon receptor and recombination activating
gene 1 (RAG1) and having been infected with 10.sup.4 PFU of the
tri-segmented arenavirus particle. The tri-segmented arenavirus
particles provided herein, in certain embodiments, can be
engineered to improve genetic stability and ensure lasting
transgene expression. Moreover, in certain embodiments,
inter-segmental recombination of the two S segments or two L
segments, uniting two arenavirus ORFs on only one instead of two
separate segments, abrogates viral promoter activity.
[0036] In certain embodiments, a tri-segmented arenavirus particle,
as provided herein, is infectious, i.e., it is capable of entering
into or injecting its genetic material into a host cell. In certain
more specific embodiments, a tri-segmented arenavirus particle as
provided herein is infectious, i.e., is capable of entering into or
injecting its genetic material into a host cell followed by
amplification and expression of its genetic information inside the
host cell. In certain embodiments, the tri-segmented arenavirus
particle is an infectious, replication-deficient arenavirus
particle engineered to contain a genome with the ability to amplify
and express its genetic information in infected cells but unable to
produce further infectious progeny particles in non-complementing
cells. In certain embodiments, the tri-segmented arenavirus
particle is replication-competent and able to produce further
infectious progeny particles in normal, not genetically engineered
cells. In certain more specific embodiments, such a
replication-competent particle is attenuated relative to the wild
type virus from which the replication-competent particle is
derived.
[0037] The tumor antigen or tumor associated antigen encoded by the
nucleotide sequence included within a tri-segmented arenavirus
particle provided herein can be one or more of the tumor antigens
or tumor associated antigens selected from the group consisting of
oncogenic viral antigens, cancer-testis antigens, oncofetal
antigens, tissue differentiation antigens, mutant protein antigens,
neoantigens, Adipophilin, AIM-2, ALDH1AI, BCLX (L), BING-4, CALCA,
CD45, CPSF, cyclin D1, DKKI, ENAH (hMcna), Ga733 (EpCAM), EphA3,
EZH2, FGF5, glypican-3, G250/MN/CAIX, HER-2/neu, IDO1, IGF2B3,
IL13Ralpha2, Intestinal carboxyl esterase, alpha-foetoprotein,
Kallikrein 4, KIF20A, Lengsin, M-CSF, MCSP, mdm-2, Meloe, MMP-2,
MMP-7, MUC1, MUC5AC, p53 (non-mutant), PAX5, PBF, PRAME, PSMA,
RAGE, RAGE-1, RGS5, RhoC, RNF43, RU2AS, secernin 1, SOX1O, STEAP1
(six-transmembrane epithelial antigen of the prostate 1),
survivinn, Telomerase, VEGF, WT1, EGF-R, CEA, CD20, CD33, CD52,
glycoprotein 100 (GP100 or gp 100 protein), MELANA/MART1, MART2,
NY-ESO-1, p53, MAGE A1, MAGE A3, MAGE-4, MAGE-5, MAGE-6, CDK4,
alpha-actinin-4, ARTC1, BCR-ABL, BCR-ABL fusion protein (b3a2),
B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDKN2A, CLPP,
COA-1, dek-can fusion protein, EFTUD2, Elongation factor 2,
ETV6-AML, ETV6-AML1 fusion protein, FLT3-ITD, FN1, GPNMB,
LDLR-fucosyltransferaseAS fusion protein, NFYC, OGT, OS-9,
pml-RARalpha fusion protein, PRDX5, PTPRK, H-ras, K-ras (V-Ki-ras2
Kirsten rat sarcoma viral oncogene), N-ras, RBAF600, SIRT2, SNRPD1,
SSX, SSX2, SYT-SSX1 or -SSX2 fusion protein, TGF-betaRII,
Triosephosphate isomerase, ormdm-2, LMP2, HPV E6/E7, EGFRvIII
(epidermal growth factor variant III), Idiotype, GD2, ganglioside
G2), Ras-mutant, p53 (mutant), Proteinase3 (PR1), Tyrosinase, PSA,
hTERT, Sarcoma translocation breakpoints, EphA2, prostatic acid
phosphatase PAP, neo-PAP, ML-IAP, AFP, ERG (TMPRSS2 ETS Fusion
gene), NA17, PAX3, ALK, Androgen Receptor, Cyclin B1, Polysialic
acid, MYCN, TRP2, TRP2-Int2, GD3, Fucosyl GM1, Mesothelin, PSCA,
sLe(a), cyp1B1, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, SART3, STn,
Carbonic Anhydrase IX, OY-TES1, Sperm protein 17, LCK, high
molecular weight melanoma-associated antigen (HMWMAA), AKAP-4,
SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP,
PDGFR-beta, MAD-CT-2, For-related antigen 1, TRP1, CA-125, CA19-9,
Calretinin, Epithelial membrane antigen (EMA), Epithelial tumor
antigen (ETA), CD19, CD34, CD99, CD117, Chromogranin, Cytokeratin,
Desmin, Glial fibrillary acidic protein (GFAP), gross cystic
disease fluid protein (GCDFP-15), HMB-45 antigen, Myo-D1,
muscle-specific actin (MSA), neurofilament, neuron-specific enolase
(NSE), placental alkaline phosphatase, synaptophysis,
thyroglobulin, thyroid transcription factor-1, dimeric form of the
pyruvate kinase isoenzyme type M2 (tumor M2-PK), BAGE BAGE-1, CAGE,
CTAGE, FATE, GAGE, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,
GAGE-7, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-SAR-35,
SPANXB1, SPA17, SSX, SYCP1, TPTE, Carbohydrate/ganglioside GM2
(oncofetal antigen-immunogenic-1 OFA-I-1), GM3, CA 15-3 (CA
27.29\BCAA), CA 195, CA 242, CA 50, CAM 43, CEA, EBNA, EF2,
Epstein-Barr virus antigen, HLA-A2, HLA-A11, HSP70-2, KIAAO205,
MUM-1, MUM-2, MUM-3, Myosin class I, GnTV, Herv-K-mel, LAGE-1,
LAGE-2, (sperm protein) SP17, SCP-1, P15(58), Hom/Mel-40, E2A-PRL,
H4-RET, IGH-IGK, MYL-RAR, TSP-180, P185erbB2, p180erbB-3, c-met,
nm-23H1, TAG-72, TAG-72-4, CA-72-4, CAM 17.1, NuMa, 13-catenin,
P16, TAGE, CT7, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA,
CD68\KP1, CO-029, HTgp-175, M344, MG7-Ag, MOV18, NB\70K, NY-CO-1,
RCAS1, SDCCAG16, TA-90, TAAL6, TLP, TPS, CD22, CD27, CD30, CD70,
prostein, TARP (T cell receptor gamma alternate reading frame
protein), Trp-p8, integrin .alpha.v.beta.3 (CD61), galactin, or
Ral-B, CD123, CLL-1, CD38, CS-1, CD138, and ROR1. In certain
embodiments, the nucleotide sequence encodes two, three, four,
five, six, seven, eight, nine, ten or more tumor antigens, tumor
associated antigens or antigenic fragments thereof. In certain
embodiments, an antigenic fragment of a tumor antigen or tumor
associated antigen provided herein is encoded by the nucleotide
sequence included within the tri-segmented arenavirus.
[0038] In certain embodiments, provided herein are tri-segmented
arenaviruses with rearrangements of their ORFs in their genomes and
a nucleotide sequence encoding a tumor antigen, tumor associated
antigen or an antigenic fragment thereof provided herein. In a
particular embodiment, a tri-segmented arenavirus particle provided
herein has been engineered to carry an arenavirus ORF in a position
other than the wild-type position. Thus, in certain particular
embodiments, provided herein is a tri-segmented arenavirus
comprising: a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof; and at least
one arenavirus ORF in a position other than the wild-type position
of said ORF, wherein the ORF encodes the GP, NP, Z protein or L
protein of an arenavirus particle.
[0039] In certain embodiments, one of the two S segments included
in the tri-segmented arenavirus particle provided herein is
selected from the group consisting of: [0040] (i) an S segment,
wherein the ORF encoding the NP is under control of an arenavirus
5' UTR [0041] (ii) an S segment, wherein the ORF encoding the Z
protein is under control of an arenavirus 5' UTR; [0042] (iii) an S
segment, wherein the ORF encoding the L protein is under control of
an arenavirus 5' UTR; [0043] (iv) an S segment, wherein the ORF
encoding the GP is under control of an arenavirus 3' UTR; [0044]
(v) an S segment, wherein the ORF encoding the L protein is under
control of an arenavirus 3' UTR; and [0045] (vi) an S segment,
wherein the ORF encoding the Z protein is under control of an
arenavirus 3' UTR.
[0046] In certain embodiments, one of the two L segments included
in the tri-segmented arenavirus particle provided herein is
selected from the group consisting of: [0047] (i) an L segment,
wherein the ORF encoding the GP is under control of an arenavirus
5' UTR; [0048] (ii) an L segment, wherein the ORF encoding the NP
is under control of an arenavirus 5' UTR; [0049] (iii) an L
segment, wherein the ORF encoding the L protein is under control of
an arenavirus 5' UTR; [0050] (iv) an L segment, wherein the ORF
encoding the GP is under control of an arenavirus 3' UTR; [0051]
(v) an L segment, wherein the ORF encoding the NP is under control
of an arenavirus 3' UTR; and [0052] (vi) an L segment, wherein the
ORF encoding the Z protein is under control of an arenavirus 3'
UTR.
[0053] In certain embodiments, the tri-segmented arenavirus
particle 3' UTR is the 3' UTR of the arenavirus S segment or the
arenavirus L segment. In other embodiments, the tri-segmented
arenavirus particle 5' UTR is the 5' UTR of the arenavirus S
segment or the arenavirus L segment.
[0054] In certain embodiments, the two S segments comprise: (i) one
or two nucleotide sequences each encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof; or (ii) one or
two duplicated arenavirus ORFs; or (iii) one nucleotide sequence
encoding a tumor antigen, tumor associated antigen or an antigenic
fragment thereof and one duplicated arenavirus ORF.
[0055] In certain embodiments, the two L segments comprise (i) one
or two nucleotide sequences each encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof; or (ii) one or
two duplicated arenavirus ORFs; or (iii) one nucleotide sequence
encoding a tumor antigen, tumor associated antigen or an antigenic
fragment thereof and one duplicated arenavirus ORF.
[0056] In certain embodiments, a tri-segmented arenavirus particle
provided herein, comprises at least one arenavirus ORF that is at
least partially removed or functionally inactivated. The ORE can
encode the GP, NP, Z protein, or L protein of an arenavirus
particle. Additionally, in certain embodiments, at least one ORF
encoding the GP, NP, Z protein, or L protein is removed and
replaced with a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof provided
herein. In certain embodiments, only one of the four ORFs encoding
GP, NP, Z protein, and L protein is removed. Thus, in certain
embodiments, the ORF encoding GP is removed. In certain
embodiments, the ORF encoding NP is removed. In certain
embodiments, the ORF encoding Z protein is removed. In certain
embodiments, the ORF encoding L protein is removed.
[0057] In certain embodiments, a tri-segmented arenavirus particle
provided herein is derived from a specific arenavirus species, such
as lymphocytic choriomeningitis virus ("LCMV") or Junin virus
("JUNV"). In other words, the genomic information encoding the
tri-segmented arenavirus particle is derived from a specific
species of arenavirus. Thus, in certain embodiments, the
tri-segmented arenavirus particle is derived from LCMV. In other
embodiments, the tri-segmented arenavirus particle is derived from
JUNV. Additionally, is specific embodiments, the LCMV is MP strain,
WE strain, Armstrong strain, or Armstrong Clone 13 strain. In other
specific embodiments, the JUNV is JUNV vaccine Candid #1 strain, or
JUNV vaccine XJ Clone 3 strain
3.3 Methods for Treating a Neoplastic Disease
[0058] In certain embodiments, provided herein are methods of
treating a neoplastic disease in a subject. Such methods can
include administering to a subject in need thereof an arenavirus
particle, including a tri-segmented arenavirus particle, provided
herein and an immune checkpoint inhibitor provided herein.
[0059] In certain embodiments, the arenavirus particle used in the
methods is an infectious, replication-deficient arenavirus particle
provided herein. In certain embodiments, the arenavirus particle
used in the methods is a tri-segmented arenavirus particle provided
herein, including an infectious, replication-deficient
tri-segmented arenavirus particle or a replication-competent
tri-segmented arenavirus particle. Thus, in certain embodiments,
the arenavirus particle, including a tri-segmented arenavirus
particle, used in the methods is replication-deficient, wherein the
arenavirus particle is engineered to contain a genome comprising:
(1) a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof; and (2) the
ability to amplify and express its genetic information in infected
cells but unable to produce further infectious progeny particles in
non-complementing cells. Moreover, in certain embodiments, a
tri-segmented arenavirus particle used in the methods is
replication-competent, wherein the tri-segmented arenavirus
particle is engineered to contain a genome comprising: (1) a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen or an antigenic fragment thereof; (2) the ability to
amplify and express its genetic information in infected cells; and
(3) the ability to produce further infectious progeny particles in
normal, not genetically engineered cells. In certain embodiments,
the immune checkpoint inhibitor inhibits, decreases or interferes
with the activity of a negative checkpoint regulator. In certain
embodiments, the arenavirus particle used in the methods is a
bi-segmented infectious, replication-deficient arenavirus particle.
Thus, in certain embodiments, the infectious, replication-deficient
arenavirus particle used in the methods is engineered to contain a
genome comprising: (1) a nucleotide sequence encoding a tumor
antigen, tumor associated antigen or an antigenic fragment thereof;
and (2) the ability to amplify and express its genetic information
in infected cells but unable to produce further infectious progeny
particles in non-complementing cells. In certain embodiments, the
immune checkpoint inhibitor inhibits, decreases or interferes with
the activity of a negative checkpoint regulator.
[0060] In certain embodiments, the tumor antigen or tumor
associated antigen encoded by the nucleotide sequence included
within an arenavirus particle, including a tri-segmented arenavirus
particle, provided herein can be one or more of the tumor antigens
or tumor associated antigens selected from the group consisting of
oncogenic viral antigens, cancer-testis antigens, oncofetal
antigens, tissue differentiation antigens, mutant protein antigens,
neoantigens, Adipophilin, AIM-2, ALDH1AI, BCLX (L), BING-4, CALCA,
CD45, CPSF, cyclin D1, DKKI, ENAH (hMcna), Ga733 (EpCAM), EphA3,
EZH2, FGF5, glypican-3, G250/MN/CAIX, HER-2/neu, IDO1, IGF2B3,
IL13Ralpha2, Intestinal carboxyl esterase, alpha-foetoprotein,
Kallikrein 4, KIF20A, Lengsin, M-CSF, MCSP, mdm-2, Meloe, MMP-2,
MMP-7, MUC1, MUC5AC, p53 (non-mutant), PAX5, PBF, PRAME, PSMA,
RAGE, RAGE-1, RGS5, RhoC, RNF43, RU2AS, secernin 1, SOX1O, STEAP1
(six-transmembrane epithelial antigen of the prostate 1),
survivinn, Telomerase, VEGF, WT1, EGF-R, CEA, CD20, CD33, CD52,
glycoprotein 100 (GP100 or gp 100 protein), MELANA/MART1, MART2,
NY-ESO-1, p53, MAGE A1, MAGE A3, MAGE-4, MAGE-5, MAGE-6, CDK4,
alpha-actinin-4, ARTC1, BCR-ABL, BCR-ABL fusion protein (b3a2),
B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDKN2A, CLPP,
COA-1, dek-can fusion protein, EFTUD2, Elongation factor 2,
ETV6-AML, ETV6-AML1 fusion protein, FLT3-ITD, FN1, GPNMB,
LDLR-fucosyltransferaseAS fusion protein, NFYC, OGT, OS-9,
pml-RARalpha fusion protein, PRDX5, PTPRK, H-ras, K-ras (V-Ki-ras2
Kirsten rat sarcoma viral oncogene), N-ras, RBAF600, SIRT2, SNRPD1,
SSX, SSX2, SYT-SSX1 or -SSX2 fusion protein, TGF-betaRII,
Triosephosphate isomerase, ormdm-2, LMP2, HPV E6/E7, EGFRvIII
(epidermal growth factor variant III), Idiotype, GD2, ganglioside
G2), Ras-mutant, p53 (mutant), Proteinase3 (PR1), Tyrosinase, PSA,
hTERT, Sarcoma translocation breakpoints, EphA2, prostatic acid
phosphatase PAP, neo-PAP, ML-IAP, AFP, ERG (TMPRSS2 ETS Fusion
gene), NA17, PAX3, ALK, Androgen Receptor, Cyclin B1, Polysialic
acid, MYCN, TRP2, TRP2-Int2, GD3, Fucosyl GM1, Mesothelin, PSCA,
sLe(a), cyp1B1, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, SART3, STn,
Carbonic Anhydrase IX, OY-TES1, Sperm protein 17, LCK, high
molecular weight melanoma-associated antigen (HMWMAA), AKAP-4,
SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP,
PDGFR-beta, MAD-CT-2, For-related antigen 1, TRP1, CA-125, CA19-9,
Calretinin, Epithelial membrane antigen (EMA), Epithelial tumor
antigen (ETA), CD19, CD34, CD99, CD117, Chromogranin, Cytokeratin,
Desmin, Glial fibrillary acidic protein (GFAP), gross cystic
disease fluid protein (GCDFP-15), HMB-45 antigen, Myo-D1,
muscle-specific actin (MSA), neurofilament, neuron-specific enolase
(NSE), placental alkaline phosphatase, synaptophysis,
thyroglobulin, thyroid transcription factor-1, dimeric form of the
pyruvate kinase isoenzyme type M2 (tumor M2-PK), BAGE BAGE-1, CAGE,
CTAGE, FATE, GAGE, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,
GAGE-7, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-SAR-35,
SPANXB1, SPA17, SSX, SYCP1, TPTE, Carbohydrate/ganglioside GM2
(oncofetal antigen-immunogenic-1 OFA-I-1), GM3, CA 15-3 (CA
27.29\BCAA), CA 195, CA 242, CA 50, CAM 43, CEA, EBNA, EF2,
Epstein-Barr virus antigen, HLA-A2, HLA-A11, HSP70-2, KIAAO205,
MUM-1, MUM-2, MUM-3, Myosin class I, GnTV, Herv-K-mel, LAGE-1,
LAGE-2, (sperm protein) SP17, SCP-1, P15(58), Hom/Mel-40, E2A-PRL,
H4-RET, IGH-IGK, MYL-RAR, TSP-180, P185erbB2, p180crbB-3, c-met,
nm-23H1, TAG-72, TAG-72-4, CA-72-4, CAM 17.1, NuMa, 13-catenin,
P16, TAGE, CT7, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA,
CD68\KP1, CO-029, HTgp-175, M344, MG7-Ag, MOV18, NB\70K, NY-CO-1,
RCAS1, SDCCAG16, TA-90, TAAL6, TLP, TPS, CD22, CD27, CD30, CD70,
prostein, TARP (T cell receptor gamma alternate reading frame
protein), Trp-p8, integrin .alpha.v.beta.3 (CD61), galactin, or
Ral-B, CD123, CLL-1, CD38, CS-1, CD138, and ROR1. In certain
embodiments, the nucleotide sequence encodes two, three, four,
five, six, seven, eight, nine, ten or more tumor antigen, tumor
associated antigens or antigenic fragments thereof. In certain
embodiments, a tumor antigen, tumor associated antigen or an
antigenic fragment thereof, provided herein is encoded by the
nucleotide sequence included within the arenavirus, including a
tri-segmented arenavirus.
[0061] In certain embodiments, provided herein are methods for
treating a neoplastic disease in a subject by administering an
immune checkpoint inhibitor that inhibits, decreases or interferes
with the activity of a negative checkpoint regulator. In certain
embodiments, the negative checkpoint regulator is selected from the
group consisting of Cytotoxic T-lymphocyte antigen-4 (CTLA-4),
CD80, CD86, Programmed cell death 1 (PD-1), Programmed cell death
ligand 1 (PD-L1), Programmed cell death ligand 2 (PD-L2),
Lymphocyte activation gene-3 (LAG-3; also known as CD223),
Galectin-3, B and T lymphocyte attenuator (BTLA), T-cell membrane
protein 3 (TIM3), Galectin-9 (GAL9), B7-H1, B7-H3, B7-H4, T-Cell
immunoreceptor with Ig and ITIM domains (TIGIT/Vstm3/WUCAM/VSIG9),
V-domain Ig suppressor of T-Cell activation (VISTA),
Glucocorticoid-induced tumor necrosis factor receptor-related
(GITR) protein, Herpes Virus Entry Mediator (HVEM), OX40, CD27,
CD28, CD137. CGEN-15001T, CGEN-15022, CGEN-15027, CGEN-15049,
CGEN-15052, and CGEN-15092.
[0062] In certain embodiments, the subject that is treated using
the methods provided herein is suffering from, is susceptible to,
or is at risk for a neoplastic disease. Thus, in some embodiments,
the subject is suffering from a neoplastic disease. In some
embodiments, the subject is susceptible to a neoplastic disease. In
some embodiments, the subject is at risk for a neoplastic disease.
In certain embodiments, the neoplastic disease that a subject
treatable by the methods provided herein is selected from the group
consisting of acute lymphoblastic leukemia; acute lymphoblastic
lymphoma; acute lymphocytic leukaemia; acute myelogenous leukemia;
acute myeloid leukemia (adult/childhood); adrenocortical carcinoma;
AIDS-related cancers; AIDS-related lymphoma; anal cancer; appendix
cancer; astrocytomas; atypical teratoid/rhabdoid tumor; basal-cell
carcinoma; bile duct cancer, extrahepatic (cholangiocarcinoma);
bladder cancer; bone osteosarcoma/malignant fibrous histiocytoma;
brain cancer (adult/childhood); brain tumor, cerebellar astrocytoma
(adult/childhood); brain tumor, cerebral astrocytoma/malignant
glioma brain tumor; brain tumor, ependymoma; brain tumor,
medulloblastoma; brain tumor, supratentorial primitive
neuroectodermal tumors; brain tumor, visual pathway and
hypothalamic glioma; brainstem glioma; breast cancer; bronchial
adenomas/carcinoids; bronchial tumor; Burkitt lymphoma; cancer of
childhood; carcinoid gastrointestinal tumor; carcinoid tumor;
carcinoma of adult, unknown primary site; carcinoma of unknown
primary; central nervous system embryonal tumor; central nervous
system lymphoma, primary; cervical cancer; childhood adrenocortical
carcinoma; childhood cancers; childhood cerebral astrocytoma;
chordoma, childhood; chronic lymphocytic leukemia; chronic
myelogenous leukemia; chronic myeloid leukemia; chronic
myeloproliferative disorders; colon cancer; colorectal cancer;
craniopharyngioma; cutaneous T-cell lymphoma; desmoplastic small
round cell tumor; emphysema; endometrial cancer; ependymoblastoma;
ependymoma; esophageal cancer; ewing's sarcoma in the Ewing family
of tumors; extracranial germ cell tumor; extragonadal germ cell
tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric
(stomach) cancer; gastric carcinoid; gastrointestinal carcinoid
tumor; gastrointestinal stromal tumor; germ cell tumor:
extracranial, extragonadal, or ovarian gestational trophoblastic
tumor; gestational trophoblastic tumor, unknown primary site;
glioma; glioma of the brain stem; glioma, childhood visual pathway
and hypothalamic; hairy cell leukemia; head and neck cancer; heart
cancer; hepatocellular (liver) cancer; hodgkin lymphoma;
hypopharyngeal cancer; hypothalamic and visual pathway glioma;
intraocular melanoma; islet cell carcinoma (endocrine pancreas);
Kaposi Sarcoma; kidney cancer (renal cell cancer); langerhans cell
histiocytosis; laryngeal cancer; lip and oral cavity cancer;
liposarcoma; liver cancer (primary); lung cancer, non-small cell;
lung cancer, small cell; lymphoma, primary central nervous system;
macroglobulinemia, Waldenstrom; male breast cancer; malignant
fibrous histiocytoma of bone/osteosarcoma; medulloblastoma;
medulloepithelioma; melanoma; melanoma, intraocular (eye); merkel
cell cancer; merkel cell skin carcinoma; mesothelioma;
mesothelioma, adult malignant; metastatic squamous neck cancer with
occult primary; mouth cancer; multiple endocrine neoplasia
syndrome; multiple myeloma/plasma cell neoplasm; mycosis fungoides,
myelodysplastic syndromes; myelodysplastic/myeloproliferative
diseases; myelogenous leukemia, chronic; myeloid leukemia, adult
acute; myeloid leukemia, childhood acute; myeloma, multiple (cancer
of the bone-marrow); myeloproliferative disorders, chronic; nasal
cavity and paranasal sinus cancer; nasopharyngeal carcinoma;
neuroblastoma, non-small cell lung cancer; non-hodgkin lymphoma;
oligodendroglioma; oral cancer; oral cavity cancer; oropharyngeal
cancer; osteosarcoma/malignant fibrous histiocytoma of bone;
ovarian cancer; ovarian epithelial cancer (surface
epithelial-stromal tumor); ovarian germ cell tumor; ovarian low
malignant potential tumor; pancreatic cancer; pancreatic cancer,
islet cell; papillomatosis; paranasal sinus and nasal cavity
cancer; parathyroid cancer; penile cancer; pharyngeal cancer;
pheochromocytoma; pineal astrocytoma; pineal germinoma; pineal
parenchymal tumors of intermediate differentiation; pineoblastoma
and supratentorial primitive neuroectodermal tumors; pituitary
tumor; pituitary adenoma; plasma cell neoplasia/multiple myeloma;
pleuropulmonary blastoma; primary central nervous system lymphoma;
prostate cancer; rectal cancer; renal cell carcinoma (kidney
cancer); renal pelvis and ureter, transitional cell cancer;
respiratory tract carcinoma involving the NUT gene on chromosome
15; retinoblastoma; rhabdomyosarcoma, childhood; salivary gland
cancer; sarcoma, Ewing family of tumors; Sezary syndrome; skin
cancer (melanoma); skin cancer (non-melanoma); small cell lung
cancer; small intestine cancer soft tissue sarcoma; soft tissue
sarcoma; spinal cord tumor; squamous cell carcinoma; squamous neck
cancer with occult primary, metastatic; stomach (gastric) cancer;
supratentorial primitive neuroectodermal tumor; T-cell lymphoma,
cutaneous (Mycosis Fungoides and Sezary syndrome); testicular
cancer; throat cancer; thymoma; thymoma and thymic carcinoma;
thyroid cancer; thyroid cancer, childhood; transitional cell cancer
of the renal pelvis and ureter; urethral cancer; uterine cancer,
endometrial; uterine sarcoma; vaginal cancer; vulvar cancer; and
Wilms Tumor.
[0063] In certain embodiments, the arenavirus particle, including a
tri-segmented arenavirus, provided herein and an immune checkpoint
inhibitor provided herein, which are used in the methods provided
herein, can be administered in a variety of different combinations.
Thus, in certain embodiments, the arenavirus particle and the
immune checkpoint inhibitor are co-administered simultaneously. In
other embodiments, the arenavirus particle is administered prior to
administration of the immune checkpoint inhibitor. In still other
embodiments, the arenavirus particle is administered after
administration of the immune checkpoint inhibitor. The interval
between administration of the arenavirus particle and the immune
checkpoint inhibitor can be hours, days, weeks or months. Thus, in
some embodiments, the interval is about 1 hour, about 2 hours,
about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 10 hours, about 11
hours, about 12 hours, about 1 day, about 2 days, about 3 days,
about 4 days, about 5 days, about 6 days, about 1 week, about 8
days, about 9 days, about 10 days, about 11 days, about 12 days,
about 13 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5
weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks,
about 10 weeks, about 11 weeks, about 12 weeks, about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months,
about 6 months, or more.
[0064] In certain embodiments, the method provided herein includes
administering an arenavirus particle, including a tri-segmented
arenavirus, provided herein and the immune checkpoint inhibitor
provided herein in a therapeutically effective amount. Thus, in
certain embodiments, provided herein is a method for treating a
neoplastic disease in a subject comprising, administering to a
subject in need thereof a therapeutically effective amount of an
arenavirus particle and a therapeutically effective amount of an
immune checkpoint inhibitor, wherein the arenavirus particle is
engineered to contain a genomic segment comprising: a nucleotide
sequence encoding a tumor antigen, tumor associated antigen or an
antigenic fragment thereof; and at least one arenavirus ORF in a
position other than the wild-type position of the ORF, wherein the
ORF encodes the GP, NP, Z protein or L protein of the arenavirus
particle, and wherein the immune checkpoint inhibitor inhibits,
decreases or interferes with the activity of a negative checkpoint
regulator.
[0065] In certain embodiments, provided herein is a method for
treating a neoplastic disease in a subject comprising,
administering to a subject in need thereof a therapeutically
effective amount of a bi-segmented infectious,
replication-deficient arenavirus particle and a therapeutically
effective amount of an immune checkpoint inhibitor, wherein the
arenavirus particle is engineered to contain a genome comprising: a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen or an antigenic fragment thereof; and the ability to
amplify and express its genetic information in infected cells but
unable to produce further infectious progeny particles in
non-complementing cells, and wherein the immune checkpoint
inhibitor inhibits, decreases or interferes with the activity of a
negative checkpoint regulator.
[0066] In certain embodiments, provided herein are methods of
treating a neoplastic disease in a subject comprising,
administering to the subject two or more arenaviruses, including a
tri-segmented arenavirus, provided herein expressing a tumor
antigen, tumor associated antigen or antigenic fragment thereof. In
a more specific embodiment, the method provided herein includes
administering to the subject a first arenavirus particle, and
administering to the subject, after a period of time, a second
arenavirus particle. In still another embodiment, the first
arenavirus particle and the second arenavirus particle are derived
from different arenavirus species and/or comprise nucleotide
sequences encoding different tumor antigen, tumor associated
antigens or antigenic fragments thereof.
[0067] In certain embodiments, a method described herein includes
administering to a subject in need thereof an arenavirus particle
encoding a neoantigen in combination with an immune checkpoint
inhibitor. In some embodiments, the neoantigen is ADP-dependent
glucokinase (Adpgk) having a R203M mutation. In some embodiments,
the subject has colon cancer. In some embodiments, the immune
checkpoint inhibitor is an antibody that binds to or inhibits
activity of Programmed cell death 1 ("PD1"). Accordingly, in one
embodiment, disclosed herein is a method for treating colon cancer
in a subject comprising administering to a subject in need thereof
an arenavirus particle and an immune checkpoint inhibitor, wherein
the arenavirus particle is engineered to contain an arenavirus
genomic segment comprising: (i) a nucleotide sequence encoding a
neoantigen or an antigenic fragment thereof; and (ii) at least one
arenavirus ORF in a position other than the wild-type position of
the ORF, wherein the ORF encodes the GP, the NP, the Z protein or
the L protein of the arenavirus particle, wherein the neoantigen is
Adpgk having a R203M mutation, the immune checkpoint inhibitor is
an antibody that binds to or inhibits PD1, the arenavirus particle
is derived from LCMV and is a tri-segmented arenavirus particle
comprising one L segment and two S segments, and wherein, in one of
the two S segments the ORF encoding the GP is under control of an
arenavirus 3' UTR, and each of the two S segments comprise a
nucleotide sequence encoding the neoantigen or antigenic fragment
thereof.
[0068] In certain embodiments, a method described herein includes
administering to a subject in need thereof an arenavirus particle
encoding a melanoma antigen in combination with an immune
checkpoint inhibitor. In some embodiments, the melanoma antigen is
glycoprotein 100 ("GP100"), tyrosinase-related protein 1 ("TRP1")
or tyrosinase-related protein 2 ("TRP2"). In some embodiments, the
subject has melanoma. In some embodiments, the immune checkpoint
inhibitor is an antibody that binds to or inhibits activity of PD1.
Accordingly, in one embodiment, disclosed herein is a method for
treating melanoma in a subject comprising administering to a
subject in need thereof an arenavirus particle and an immune
checkpoint inhibitor, wherein the arenavirus particle is engineered
to contain an arenavirus genomic segment comprising: (i) a
nucleotide sequence encoding a melanoma antigen or an antigenic
fragment thereof; and (ii) at least one arenavirus ORF in a
position other than the wild-type position of the ORF, wherein the
ORF encodes the GP, the NP, the Z protein or the L protein of the
arenavirus particle, wherein the melanoma antigen is selected from
the group consisting of GP100, TRP1 and TRP2, the immune checkpoint
inhibitor is an antibody that binds to or inhibits PD1, the
arenavirus particle is derived from LCMV and is a tri-segmented
arenavirus particle comprising one L segment and two S segments,
and wherein, in one of the two S segments the ORF encoding the GP
is under control of an arenavirus 3' UTR, and each of the two S
segments comprise a nucleotide sequence encoding the melanoma
antigen or antigenic fragment thereof.
[0069] In certain embodiments, provided herein is the use of an
arenavirus particle and an immune checkpoint inhibitor as described
herein in a method of treatment of a neoplastic disease in a
subject as described herein.
3.4 Pharmaceutical Compositions and Kits
[0070] In certain embodiments, provided herein are compositions,
e.g., pharmaceutical, immunogenic or vaccine compositions,
comprising an arenavirus particle, including a tri-segmented
arenavirus particle, provided herein, an immune checkpoint
inhibitor provided herein, and a pharmaceutically acceptable
carrier. Thus, in some embodiments, provided herein is a
pharmaceutical composition comprising an arenavirus particle as
provided herein, an immune checkpoint inhibitor as provided herein
and a pharmaceutically acceptable carrier.
[0071] In certain embodiments, the arenavirus particle contained
within the compositions is an infectious, replication-deficient
arenavirus particle provided herein. In certain embodiments, the
arenavirus particle contained within the compositions is a
tri-segmented arenavirus particle provided herein, including an
infectious, replication-deficient tri-segmented arenavirus particle
or a replication-competent tri-segmented arenavirus particle. Thus,
in certain embodiments, the compositions providing herein,
including a pharmaceutical, immunogenic or vaccine composition,
comprise an arenavirus particle, including a tri-segmented
arenavirus particle, that is replication-deficient, wherein the
arenavirus particle is engineered to contain a genome comprising:
(1) a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof; and (2) the
ability to amplify and express its genetic information in infected
cells but unable to produce further infectious progeny particles in
non-complementing cells. Moreover, in certain embodiments, the
compositions providing herein, including a pharmaceutical,
immunogenic or vaccine composition, comprise a tri-segmented
arenavirus particle that is replication-competent, wherein the
tri-segmented arenavirus particle is engineered to contain a genome
comprising: (1) a nucleotide sequence encoding a tumor antigen,
tumor associated antigen or an antigenic fragment thereof; (2) the
ability to amplify and express its genetic information in infected
cells; and (3) the ability to produce further infectious progeny
particles in normal, not genetically engineered cells. Tn certain
embodiments, the immune checkpoint inhibitor inhibits, decreases or
interferes with the activity of a negative checkpoint
regulator.
[0072] Thus, in some embodiments, provided herein is a
pharmaceutical composition comprising a bi-segmented infectious,
replication-deficient arenavirus particle as provided herein, an
immune checkpoint inhibitor as provided herein and a
pharmaceutically acceptable carrier. In specific certain
embodiments, the arenavirus particle is engineered to contain a
genome comprising: (1) a nucleotide sequence encoding a tumor
antigen, tumor associated antigen or an antigenic fragment thereof;
and (2) the ability to amplify and express its genetic information
in infected cells but unable to produce further infectious progeny
particles in non-complementing cells. In still further embodiments,
the immune checkpoint inhibitor inhibits, decreases or interferes
with the activity of a negative checkpoint regulator.
[0073] In certain embodiments, the tumor antigen or tumor
associated antigen encoded by the nucleotide sequence included
within an arenavirus particle provided herein can be one or more of
the tumor antigens or tumor associated antigens selected from the
group consisting of oncogenic viral antigens, cancer-testis
antigens, oncofetal antigens, tissue differentiation antigens,
mutant protein antigens, neoantigens, Adipophilin, AIM-2, ALDH1AI,
BCLX (L), BING-4, CALCA, CD45, CPSF, cyclin D1, DKKI, ENAH (hMcna),
Ga733 (EpCAM), EphA3, EZH2, FGF5, glypican-3, G250/MN/CAIX,
HER-2/neu, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase,
alpha-foetoprotein, Kallikrein 4, KIF20A, Lengsin, M-CSF, MCSP,
mdm-2, Meloe, MMP-2, MMP-7, MUC1, MUC5AC, p53 (non-mutant), PAX5,
PBF, PRAME, PSMA, RAGE, RAGE-1, RGS5, RhoC, RNF43, RU2AS, secernin
1, SOX1O, STEAP1 (six-transmembrane epithelial antigen of the
prostate 1), survivinn, Telomerase, VEGF, WT1, EGF-R, CEA, CD20,
CD33, CD52, glycoprotein 100 (GP100 or gp 100 protein),
MELANA/MART1, MART2, NY-ESO-1, p53, MAGE A1, MAGE A3, MAGE-4,
MAGE-5, MAGE-6, CDK4, alpha-actinin-4, ARTC1, BCR-ABL, BCR-ABL
fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27,
CDK4, CDKN2A, CLPP, COA-1, dek-can fusion protein, EFTUD2,
Elongation factor 2, ETV6-AML, ETV6-AML1 fusion protein, FLT3-ITD,
FN1, GPNMB, LDLR-fucosyltransferaseAS fusion protein, NFYC, OGT,
OS-9, pml-RARalpha fusion protein, PRDX5, PTPRK, H-ras, K-ras
(V-Ki-ras2 Kirsten rat sarcoma viral oncogene), N-ras, RBAF600,
SIRT2, SNRPD1, SSX, SSX2, SYT-SSX1 or -SSX2 fusion protein,
TGF-betaRII, Triosephosphate isomerase, ormdm-2, LMP2, HPV E6/E7,
EGFRvIII (epidermal growth factor variant III), Idiotype, GD2,
ganglioside G2), Ras-mutant, p53 (mutant), Proteinase3 (PRI),
Tyrosinase, PSA, hTERT, Sarcoma translocation breakpoints, EphA2,
prostatic acid phosphatase PAP, nco-PAP, ML-IAP, AFP, ERG (TMPRSS2
ETS Fusion gene), NA17, PAX3, ALK, Androgen Receptor, Cyclin B1,
Polysialic acid, MYCN, TRP2, TRP2-Int2, GD3, Fucosyl GM1,
Mesothelin, PSCA, sLe(a), cyp1B1, PLAC1, GM3, BORIS, Tn, GLoboH,
NY-BR-1, SART3, STn, Carbonic Anhydrase IX, OY-TES1, Sperm protein
17, LCK, high molecular weight melanoma-associated antigen
(HMWMAA), AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4,
VEGFR2, MAD-CT-1, FAP, PDGFR-beta, MAD-CT-2, For-related antigen 1,
TRP1, CA-125, CA19-9, Calretinin, Epithelial membrane antigen
(EMA), Epithelial tumor antigen (ETA), CD19, CD34, CD99, CD117,
Chromogranin, Cytokeratin, Desmin, Glial fibrillary acidic protein
(GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45
antigen, Myo-D1, muscle-specific actin (MSA), neurofilament,
neuron-specific enolase (NSE), placental alkaline phosphatase,
synaptophysis, thyroglobulin, thyroid transcription factor-1,
dimeric form of the pyruvate kinase isoenzyme type M2 (tumor
M2-PK), BAGE BAGE-1, CAGE, CTAGE, FATE, GAGE, GAGE-1, GAGE-2,
GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, HCA661, HOM-TES-85, MAGEA,
MAGEB, MAGEC, NA88, NY-SAR-35, SPANXB1, SPA17, SSX, SYCP1, TPTE,
Carbohydrate/ganglioside GM2 (oncofetal antigen-immunogenic-1
OFA-I-1), GM3, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA 50, CAM
43, CEA, EBNA, EF2, Epstein-Barr virus antigen, HLA-A2, HLA-A11,
HSP70-2, KIAAO205, MUM-1, MUM-2, MUM-3, Myosin class I, GnTV,
Herv-K-mel, LAGE-1, LAGE-2, (sperm protein) SP17, SCP-1, P15(58),
Hom/Mel-40, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, TSP-180, P185erbB2,
p180erbB-3, c-met, nm-23H1, TAG-72, TAG-72-4, CA-72-4, CAM 17.1,
NuMa, 13-catenin, P16, TAGE, CT7, 43-9F, 5T4, 791Tgp72, 13HCG,
BCA225, BTAA, CD68\KP1, CO-029, HTgp-175, M344, MG7-Ag, MOV18,
NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TLP, TPS, CD22,
CD27, CD30, CD70, prostein, TARP (T cell receptor gamma alternate
reading frame protein), Trp-p8, integrin .alpha.v.beta.3 (CD61),
galactin, or Ral-B, CD123, CLL-1, CD38, CS-1, CD138, and ROR1. In
certain embodiments, the nucleotide sequence encodes two, three,
four, five, six, seven, eight, nine, ten or more tumor antigen,
tumor associated antigens or antigenic fragments thereof. In
certain embodiments, an antigenic fragment of a tumor antigen or
tumor associated antigen provided herein is encoded by the
nucleotide sequence included within the arenavirus.
[0074] In certain embodiments, the composition provided herein,
including a pharmaceutical, immunogenic or vaccine composition,
includes an immune checkpoint inhibitor that inhibits, decreases or
interferes with the activity of a negative checkpoint regulator. In
certain embodiments, the native checkpoint regulator is selected
from the group consisting of Cytotoxic T-lymphocyte antigen-4
(CTLA-4), CD80, CD86, Programmed cell death 1 (PD-1), Programmed
cell death ligand 1 (PD-L1), Programmed cell death ligand 2
(PD-L2), Lymphocyte activation gene-3 (LAG-3; also known as CD223),
Galectin-3, B and T lymphocyte attenuator (BTLA), T-cell membrane
protein 3 (TIM3), Galectin-9 (GAL9), B7-H1, B7-H3, B7-H4, T-Cell
immunoreceptor with Ig and ITIM domains (TIGIT/Vstm3/WUCAM/VSIG9),
V-domain Ig suppressor of T-Cell activation (VISTA),
Glucocorticoid-induced tumor necrosis factor receptor-related
(GITR) protein, Herpes Virus Entry Mediator (HVEM), OX40, CD27,
CD28, CD137. CGEN-15001T, CGEN-15022, CGEN-15027, CGEN-15049,
CGEN-15052, and CGEN-15092.
[0075] In certain embodiments, the compositions provided herein, a
pharmaceutical, immunogenic or vaccine composition, can be used in
the methods described herein. Thus, in certain embodiments, the
compositions can be used for the treatment of a neoplastic disease.
In specific certain embodiments, the compositions provided herein
can be used for the treatment of a neoplastic disease selected from
the group consisting of acute lymphoblastic leukemia; acute
lymphoblastic lymphoma; acute lymphocytic leukaemia; acute
myelogenous leukemia; acute myeloid leukemia (adult/childhood);
adrenocortical carcinoma; AIDS-related cancers; AIDS-related
lymphoma; anal cancer; appendix cancer; astrocytomas; atypical
teratoid/rhabdoid tumor; basal-cell carcinoma; bile duct cancer,
extrahepatic (cholangiocarcinoma); bladder cancer; bone
osteosarcoma/malignant fibrous histiocytoma; brain cancer
(adult/childhood); brain tumor, cerebellar astrocytoma
(adult/childhood); brain tumor, cerebral astrocytoma/malignant
glioma brain tumor; brain tumor, ependymoma; brain tumor,
medulloblastoma; brain tumor, supratentorial primitive
neuroectodermal tumors; brain tumor, visual pathway and
hypothalamic glioma; brainstem glioma; breast cancer; bronchial
adenomas/carcinoids; bronchial tumor; Burkitt lymphoma; cancer of
childhood; carcinoid gastrointestinal tumor; carcinoid tumor;
carcinoma of adult, unknown primary site; carcinoma of unknown
primary; central nervous system embryonal tumor; central nervous
system lymphoma, primary; cervical cancer; childhood adrenocortical
carcinoma; childhood cancers; childhood cerebral astrocytoma;
chordoma, childhood; chronic lymphocytic leukemia; chronic
myelogenous leukemia; chronic myeloid leukemia; chronic
myeloproliferative disorders; colon cancer; colorectal cancer;
craniopharyngioma; cutaneous T-cell lymphoma; desmoplastic small
round cell tumor; emphysema; endometrial cancer; ependymoblastoma;
ependymoma; esophageal cancer; ewing's sarcoma in the Ewing family
of tumors; extracranial germ cell tumor; extragonadal germ cell
tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric
(stomach) cancer; gastric carcinoid; gastrointestinal carcinoid
tumor; gastrointestinal stromal tumor; germ cell tumor:
extracranial, extragonadal, or ovarian gestational trophoblastic
tumor; gestational trophoblastic tumor, unknown primary site;
glioma; glioma of the brain stem; glioma, childhood visual pathway
and hypothalamic; hairy cell leukemia; head and neck cancer; heart
cancer; hepatocellular (liver) cancer; hodgkin lymphoma;
hypopharyngeal cancer; hypothalamic and visual pathway glioma;
intraocular melanoma; islet cell carcinoma (endocrine pancreas);
Kaposi Sarcoma; kidney cancer (renal cell cancer); langerhans cell
histiocytosis; laryngeal cancer; lip and oral cavity cancer;
liposarcoma; liver cancer (primary); lung cancer, non-small cell;
lung cancer, small cell; lymphoma, primary central nervous system;
macroglobulinemia, Waldenstrom; male breast cancer; malignant
fibrous histiocytoma of bone/osteosarcoma; medulloblastoma;
medulloepithelioma; melanoma; melanoma, intraocular (eye); merkel
cell cancer; merkel cell skin carcinoma; mesothelioma;
mesothelioma, adult malignant; metastatic squamous neck cancer with
occult primary; mouth cancer; multiple endocrine neoplasia
syndrome; multiple myeloma/plasma cell neoplasm; mycosis fungoides,
myelodysplastic syndromes; myelodysplastic/myeloproliferative
diseases; myelogenous leukemia, chronic; myeloid leukemia, adult
acute; myeloid leukemia, childhood acute; myeloma, multiple (cancer
of the bone-marrow); myeloproliferative disorders, chronic; nasal
cavity and paranasal sinus cancer; nasopharyngeal carcinoma;
neuroblastoma, non-small cell lung cancer; non-hodgkin lymphoma;
oligodendroglioma; oral cancer; oral cavity cancer; oropharyngeal
cancer; osteosarcoma/malignant fibrous histiocytoma of bone;
ovarian cancer; ovarian epithelial cancer (surface
epithelial-stromal tumor); ovarian germ cell tumor; ovarian low
malignant potential tumor; pancreatic cancer; pancreatic cancer,
islet cell; papillomatosis; paranasal sinus and nasal cavity
cancer; parathyroid cancer; penile cancer; pharyngeal cancer;
pheochromocytoma; pineal astrocytoma; pineal germinoma; pineal
parenchymal tumors of intermediate differentiation; pineoblastoma
and supratentorial primitive neuroectodermal tumors; pituitary
tumor; pituitary adenoma; plasma cell neoplasia/multiple myeloma;
pleuropulmonary blastoma; primary central nervous system lymphoma;
prostate cancer; rectal cancer; renal cell carcinoma (kidney
cancer); renal pelvis and ureter, transitional cell cancer;
respiratory tract carcinoma involving the NUT gene on chromosome
15; retinoblastoma; rhabdomyosarcoma, childhood; salivary gland
cancer; sarcoma, Ewing family of tumors; Sezary syndrome; skin
cancer (melanoma); skin cancer (non-melanoma); small cell lung
cancer; small intestine cancer soft tissue sarcoma; soft tissue
sarcoma; spinal cord tumor; squamous cell carcinoma; squamous neck
cancer with occult primary, metastatic; stomach (gastric) cancer;
supratentorial primitive neuroectodermal tumor; T-cell lymphoma,
cutaneous (Mycosis Fungoides and Sezary syndrome); testicular
cancer; throat cancer; thymoma; thymoma and thymic carcinoma;
thyroid cancer; thyroid cancer, childhood; transitional cell cancer
of the renal pelvis and ureter; urethral cancer; uterine cancer,
endometrial; uterine sarcoma; vaginal cancer; vulvar cancer; and
Wilms Tumor.
[0076] Also provided herein are kits that can be used to perform
the methods described herein. Thus, in certain embodiments, the kit
provided herein includes one or more containers and instructions
for use, wherein the one or more containers comprise a composition
(e.g., pharmaceutical, immunogenic or vaccine composition) provided
herein. In other certain embodiments, a kit provided herein
includes containers that each contains the active ingredients for
performing the methods described herein. Thus, in certain
embodiments, the kit provided herein includes two or more
containers and instructions for use, wherein one of the containers
comprises an arenavirus particle, including a tri-segmented
arenavirus particle, provided herein and another container
comprises an immune checkpoint inhibitor provided herein. In a
specific embodiment, a kit provided herein includes two or more
containers and instructions for use, wherein one of the containers
comprises an arenavirus particle, including a tri-segmented
arenavirus particle, provided herein and another container
comprises an immune checkpoint inhibitor provided herein, wherein
the arenavirus particle is engineered to contain a genome
comprising: a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof; and the
ability to amplify and express its genetic information in infected
cells but unable to produce further infectious progeny particles in
non-complementing cells. Moreover, in certain embodiments, one of
the containers comprises a tri-segmented arenavirus particle that
is engineered to contain a genome comprising: a nucleotide sequence
encoding a tumor antigen, tumor associated antigen or an antigenic
fragment thereof; the ability to amplify and express its genetic
information in infected cells; and the ability to produce further
infectious progeny particles in normal, not genetically engineered
cells. In certain embodiments, the kit provided herein includes two
or more containers and instructions for use, wherein one of the
containers comprises a bi-segmented infectious,
replication-deficient arenavirus particle provided herein and
another container comprises an immune checkpoint inhibitor provided
herein. In a specific embodiment, a kit provided herein includes
two or more containers and instructions for use, wherein one of the
containers comprises a bi-segmented infectious,
replication-deficient arenavirus particle provided herein and
another container comprises an immune checkpoint inhibitor provided
herein, wherein the arenavirus particle is engineered to contain a
genome comprising: a nucleotide sequence encoding a tumor antigen,
tumor associated antigen or an antigenic fragment thereof; and the
ability to amplify and express its genetic information in infected
cells but unable to produce further infectious progeny particles in
non-complementing cells. In certain embodiments, the immune
checkpoint inhibitor inhibits, decreases or interferes with the
activity of a negative checkpoint regulator.
3.5 Conventions and Abbreviations
TABLE-US-00001 [0077] Abbreviation Convention APC Antigen
presenting cell C-cell Complementing cell line CD4 Cluster of
differentiation 4 CD8 Cluster of differentiation 8 CMI
cell-mediated immunity GP Glycoprotein GS-plasmid Plasmid
expressing genome segments IGR Intergenic region JUNV Junin virus L
protein RNA-dependent RNA polymerase L segment Long segment LCMV
Lymphocytic choriomeningitis virus MHC Major Histocompatibility
Complex NP Nucleoprotein ORF Open reading frame S segment Short
segment TF-plasmid Plasmid expressing transacting factors UTR
Untranslated region Z protein Matrix protein Z
4. BRIEF DESCRIPTION OF THE FIGURES
[0078] FIG. 1: Schematic representation of the genomic organization
of bi- and tri-segmented LCMV. The bi-segmented genome of wild-type
LCMV consists of one S segment encoding the GP and NP and one L
segment encoding the Z protein and the L protein (i). Both segments
are flanked by the respective 5' and 3' UTRs. The genome of
recombinant tri-segmented LCMVs (r3LCMV) consists of one L and two
S segments with one position where to insert a gene of interest
(here GFP, which can alternatively be a tumor antigen, tumor
associated antigen or antigenic fragment thereof as described
herein) into each one of the S segments. r3LCMV-GFP.sup.natural
(nat) has all viral genes in their natural position (ii), whereas
the GP ORF in r3LCMV-GFP.sup.artificial (art) is artificially
juxtaposed to and expressed under control of the 3' UTR (iii).
[0079] FIG. 2: The genome of wild type arenaviruses consists of a
short (1; .about.3.4 kb) and a large (2; .about.7.2 kb) RNA
segment. The short segment carries open reading frames encoding the
nucleoprotein (3) and glycoprotein (4). The large segment encodes
the RNA-dependent RNA polymerase L (5) and the matrix protein Z
(6). Wild type arenaviruses can be rendered replication-deficient
vaccine vectors by deleting the glycoprotein gene and inserting,
instead of the glycoprotein gene, a tumor antigen, tumor associated
antigen, or antigenic fragment thereof described herein (7) against
which immune responses are to be induced.
[0080] FIGS. 3A-3B depict exemplary results of a MC-38 OVA mouse
cancer model (C57BL/6 mice, implanted with MC-38 OVADim tumor
cells) treated with a tri-segmented replication competent
arenavirus vector encoding an OVA antigen (r3LCMV-OVA) and an
anti-PD1 antibody (aPD1) on tumor growth (FIG. 3A) and percent
survival (FIG. 3B). Symbols represent the mean.+-.SEM of five
(untreated) or six (other groups) mice per group.
[0081] FIGS. 4A-4C depict exemplary results of a MC-38 mouse cancer
model (C57BL/6 mice, implanted with MC-38 tumor cells) treated with
a tri-segmented replication competent (r3LCMV) or bi-segmented
replication-deficient (r2LCMV) arenavirus vector encoding an
Adpgk.sup.mut antigen (Adpgk.sup.mut), and an anti-PD1 antibody
(.alpha.-PD-1 or PD1) on the induction of AdpgK.sup.mut antigen
specific CD8+ cytotoxic T-cells (FIG. 4A), tumor growth (FIG. 4B)
and percent survival (FIG. 4C).
[0082] FIG. 5A graphically represents an exemplary treatment
regimen of a melanoma mouse model.
[0083] FIGS. 5B-5D depict exemplary results of a melanoma mouse
model (C57BL/6 mice injected with B16F10 melanoma cells) treated
with tri-segmented replication competent (r3LCMV) or bi-segmented
replication-deficient (rLCMV) arenavirus vectors encoding melanoma
antigens, and an anti-PD1 antibody (.alpha.-PD-1) using the
treatment regimen of FIG. 5A on tumor growth (FIG. 5B), percent of
animal survival (FIG. 5C) and days of survival (FIG. 5D). Gr.1
indicates group treated with r3LCMV and .alpha.-PD-1. Gr.2
indicates group treated with rLCMV and .alpha.-PD-1. Gr.3 indicates
group treated with buffer and .alpha.-PD-1. Gr.4 indicates group
treated with r3LCMV only. Gr.5 indicates group treated with rLCMV
only. Gr.6 indicates treated with buffer only. Symbols represent
the mean.+-.SEM of five mice per group.
5. DETAILED DESCRIPTION OF THE INVENTION
[0084] Provided herein are immunotherapies for treating a
neoplastic disease, such as cancer. The term "neoplastic" or
"neoplasm" refers to an abnormal new growth of cells or tissue.
This abnormal new growth can form a mass, also known as a tumor or
neoplasia. A neoplasm includes a benign neoplasm, an in situ
neoplasm, a malignant neoplasm, and a neoplasm of uncertain or
unknown behavior. In certain embodiments, the neoplastic disease
treated using the methods and compositions described herein is
cancer.
[0085] The immunotherapies provided herein include various methods
and compositions. More specifically, provided herein are arenavirus
particles or viral vectors that comprise a nucleotide sequence
encoding tumor antigen, tumor associated antigen or antigenic
fragment thereof. These genetically modified viruses can be
administered to a subject for the treatment of a neoplastic
disease, such as cancer. Detailed descriptions of the arenaviruses
provided herein, including the nucleotide sequences encoding a
tumor antigen, tumor associated antigen or antigenic fragment
thereof can be found in Sections 5.1, 5.2, 5.3, and 5.4.
Additionally, methods for generation of arenavirus particles or
viral vectors for use in the methods and compositions described
herein are described in more detail in Sections 5.5 and 5.6.
[0086] In addition to administering arenavirus particles or viral
vectors to a subject, the immunotherapies for treating a neoplastic
disease provided herein can include an immune checkpoint modulator.
The term "immune checkpoint modulator" (also referred to as
"checkpoint modulator" or as "checkpoint regulator") refers to a
molecule or to a compound that modulates (e.g., totally or
partially reduces, inhibits, interferes with, activates,
stimulates, increases, reinforces or supports) the function of one
or more checkpoint molecules. Thus, an immune checkpoint modulator
may be an immune checkpoint inhibitor or an immune checkpoint
activator.
[0087] An "immune checkpoint inhibitor" refers to a molecule that
inhibits, decreases or interferes with the activity of a negative
checkpoint regulator. In certain embodiments, immune checkpoint
inhibitors for use with the methods and compositions disclosed
herein can inhibit the activity of a negative checkpoint regulator
directly, or decrease the expression of a negative checkpoint
regulator, or interfere with the interaction of a negative
checkpoint regulator and a binding partner (e.g., a ligand). Immune
checkpoint inhibitors for use with the methods and compositions
disclosed herein include a protein, a polypeptide, a peptide, an
antisense oligonucleotide, an antibody, an antibody fragment, or an
inhibitory RNA molecule that targets the expression of a negative
checkpoint regulator.
[0088] A "negative checkpoint regulator" refers to a molecule that
down-regulates immune responses (e.g., T-cell activation) by
delivery of a negative signal to T-cells following their engagement
by ligands or counter-receptors. Exemplary functions of a
negative-checkpoint regulator are to prevent out-of-proportion
immune activation, minimize collateral damage, and/or maintain
peripheral self-tolerance. In certain embodiments, a negative
checkpoint regulator is a ligand or receptor expressed by an
antigen presenting cell. In certain embodiments, a negative
checkpoint regulator is a ligand or receptor expressed by a T-cell.
In certain embodiments, a negative checkpoint regulator is a ligand
or receptor expressed by both an antigen presenting cell and a
T-cell.
[0089] Thus, in certain embodiments, provided herein are methods
and compositions for treating a neoplastic disease using an
arenavirus particle or viral vector comprising a nucleotide
sequence encoding a tumor antigen, tumor associated antigen or
antigenic fragment thereof and an immune checkpoint inhibitor. Such
an immune check point inhibitor can inhibit, decrease or interfere
with the activity of a negative checkpoint regulator. Thus, in
certain embodiments, provided herein are methods for treating a
neoplastic disease using an arenavirus particle or viral vector
comprising a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or antigenic fragment thereof, and an immune
checkpoint inhibitor that inhibits, decreases or interferes with
the activity of a negative checkpoint regulator. Also, in certain
embodiments, provided herein are compositions comprising an
arenavirus particle or viral vector comprising a nucleotide
sequence encoding a tumor antigen, tumor associated antigen or
antigenic fragment thereof, and an immune checkpoint inhibitor that
inhibits, decreases or interferes with the activity of a negative
checkpoint regulator. In certain embodiments, the arenavirus
particle or viral vector provided herein is engineered to contain
an arenavirus genomic segment having a nucleotide sequence encoding
a tumor antigen, tumor associated antigen or antigenic fragment
thereof and at least one arenavirus open reading frame ("ORF") in a
position other than the wild-type position of the ORF. In certain
embodiments, the arenavirus particle or viral vector provided
herein is an infectious, replication deficient arenavirus particle
or viral vector. In other embodiments, the arenavirus particle
provided herein is a tri-segmented arenavirus particle or viral
vector, which can be replication-deficient or
replication-competent. In still other embodiments, the
tri-segmented arenavirus particle or viral vector provided herein,
when propagated, does not result in a replication-competent
bi-segmented viral particle. Methods and compositions for using an
arenavirus particle or viral vector and an immune checkpoint
inhibitor provided herein are described in more detail in Sections
5.8 and 5.9.
5.1 Arenaviruses with an Open Reading Frame in a Non-Natural
Position
[0090] Provided herein are arenaviruses with rearrangements of
their ORFs and a nucleotide sequence encoding a tumor antigen,
tumor associated antigen or an antigenic fragment thereof provided
herein. In certain embodiments, such arenaviruses are
replication-competent and infectious. Thus, in certain embodiments,
provided herein is an arenavirus genomic segment, wherein the
arenavirus genomic segment is engineered to carry an arenavirus ORF
in a position other than the position in which the respective gene
is found in viruses isolated from the wild, such as LCMV-MP
(referred to herein as "wild-type position") of the ORF (i.e., a
non-natural position) and a nucleotide sequence encoding a tumor
antigen, tumor associated antigen or an antigenic fragment thereof
provided herein.
[0091] The wild-type arenavirus genomic segments and ORFs are known
in the art. In particular, the arenavirus genome consists of an S
segment and an L segment. The S segment carries the ORFs encoding
the GP and the NP. The L segment encodes the L protein and the Z
protein. Both segments are flanked by the respective 5' and 3'
UTRs.
[0092] In certain embodiments, an arenavirus genomic segment can be
engineered to carry two or more arenavirus ORFs in a position other
than the wild-type position. In other embodiments, the arenavirus
genomic segment can be engineered to carry two arenavirus ORFs, or
three arenavirus ORFs, or four arenavirus ORFs in a position other
than the wild-type position.
[0093] In certain embodiments, an arenavirus genomic segment
provided herein can be: [0094] (i) an arenavirus S segment, wherein
the ORF encoding the NP is under control of an arenavirus 5' UTR;
[0095] (ii) an arenavirus S segment, wherein the ORF encoding the Z
protein is under control of an arenavirus 5' UTR; [0096] (iii) an
arenavirus S segment, wherein the ORF encoding the L protein is
under control of an arenavirus 5' UTR; [0097] (iv) an arenavirus S
segment, wherein the ORF encoding the GP is under control of an
arenavirus 3' UTR; [0098] (v) an arenavirus S segment, wherein the
ORF encoding the L protein is under control of an arenavirus 3'
UTR; [0099] (vi) an arenavirus S segment, wherein the ORF encoding
the Z protein is under control of an arenavirus 3' UTR; [0100]
(vii) an arenavirus L segment, wherein the ORF encoding the GP is
under control of an arenavirus 5' UTR; [0101] (viii) an arenavirus
L segment, wherein the ORF encoding the NP is under control of an
arenavirus 5' UTR; [0102] (ix) an arenavirus L segment, wherein the
ORF encoding the L protein is under control of an arenavirus 5'
UTR; [0103] (x) an arenavirus L segment, wherein the ORF encoding
the GP is under control of an arenavirus 3' UTR; [0104] (xi) an
arenavirus L segment, wherein the ORF encoding the NP is under
control of an arenavirus 3' UTR; and [0105] (xii) an arenavirus L
segment, wherein the ORF encoding the Z protein is under control of
an arenavirus 3' UTR.
[0106] In certain embodiments, the ORF that is in the non-natural
position of the arenavirus genomic segment described herein can be
under the control of an arenavirus 3' UTR or an arenavirus 5' UTR.
In more specific embodiments, the arenavirus 3' UTR is the 3' UTR
of the arenavirus S segment. In another specific embodiment, the
arenavirus 3' UTR is the 3'UTR of the arenavirus L segment. In more
specific embodiments, the arenavirus 5' UTR is the 5' UTR of the
arenavirus S segment. In other specific embodiments, the 5' UTR is
the 5' UTR of the L segment.
[0107] In other embodiments, the ORF that is in the non-natural
position of the arenavirus genomic segment described herein can be
under the control of the arenavirus conserved terminal sequence
element (the 5'- and 3'-terminal 19-20-nt regions) (see e.g., Perez
& de la Torre, 2003, J Virol. 77(2): 1184-1194).
[0108] In certain embodiments, the ORF that is in the non-natural
position of the arenavirus genomic segment can be under the control
of the promoter element of the 5' UTR (see e.g., Albarino et al.,
2011, J Virol., 85(8):4020-4). In another embodiment, the ORF that
is in the non-natural position of the arenavirus genomic segment
can be under the control of the promoter element of the 3' UTR (see
e.g., Albarino et al., 2011, J Virol., 85(8):4020-4). In more
specific embodiments, the promoter element of the 5' UTR is the 5'
UTR promoter element of the S segment or the L segment. In another
specific embodiment, the promoter element of the 3' UTR is the 3'
UTR the promoter element of the S segment or the L segment.
[0109] In certain embodiments, the ORF that is in the non-natural
position of the arenavirus genomic segment can be under the control
of a truncated arenavirus 3' UTR or a truncated arenavirus 5' UTR
(see e.g., Perez & de la Torre, 2003, J Virol. 77(2):
1184-1194; Albarino et al., 2011, J Virol., 85(8):4020-4). In more
specific embodiments, the truncated 3' UTR is the 3' UTR of the
arenavirus S segment or L segment. In more specific embodiments,
the truncated 5' UTR is the 5' UTR of the arenavirus S segment or L
segment.
[0110] Also provided herein, is an arenavirus particle comprising a
first genomic segment that has been engineered to carry an ORF in a
position other than the wild-type position of the ORF and a second
arenavirus genomic segment so that the arenavirus particle
comprises an S segment and an L segment. In specific embodiments,
the ORF in a position other than the wild-type position of the ORF
is one of the arenavirus ORFs.
[0111] In certain specific embodiments, the arenavirus particle can
comprise a full complement of all four arenavirus ORFs. In specific
embodiments, the second arenavirus genomic segment has been
engineered to carry an ORF in a position other than the wild-type
position of the ORF. In another specific embodiment, the second
arenavirus genomic segment can be the wild-type genomic segment
(i.e., comprises the ORFs on the segment in the wild-type
position).
[0112] In certain embodiments, the first arenavirus genomic segment
is an L segment and the second arenavirus genomic segment is an S
segment. In other embodiments, the first arenavirus genomic segment
is an S segment and the second arenavirus genomic segment is an L
segment.
[0113] Non-limiting examples of the arenavirus particle comprising
a genomic segment with an ORF in a position other than the
wild-type position of the ORF and a second genomic segment are
illustrated in Table 1.
TABLE-US-00002 TABLE 1 Arenavirus particle Position 1 Position 2
Position 3 Position 4 GP NP L Z GP Z L NP GP Z NP L GP L NP Z GP L
Z NP NP GP L Z NP GP Z L NP L GP Z NP L Z GP NP Z GP L NP Z L GP Z
GP L NP Z GP NP L Z NP GP L Z NP L GP Z L NP GP Z L GP NP L NP GP Z
L NP Z GP L GP Z NP L GP NP Z L Z NP GP L Z GP NP *Position 1 is
under the control of an arenavirus S segment 5' UTR; Position 2 is
under the control of an arenavirus S segment 3' UTR; Position 3 is
under the control of an arenavirus L segment 5' UTR; Position 4 is
under the control of an arenavirus L segment 3' UTR.
[0114] Also provided herein, is a cDNA of the arenavirus genomic
segment engineered to carry an ORF in a position other than the
wild-type position of the ORF and a nucleotide sequence encoding a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein. In more specific embodiments, provided
herein is a cDNA or a set of cDNAs of an arenavirus genome as set
forth in Table 1.
[0115] In certain embodiments, a cDNA of the arenavirus genomic
segment that is engineered to carry an ORF in a position other than
the wild-type position of the ORF is part of or incorporated into a
DNA expression vector. In a specific embodiment, a cDNA of the
arenavirus genomic segment that is engineered to carry an ORF in a
position other than the wild-type position of the ORF is part of or
incorporated into a DNA expression vector that facilitates
production of an arenavirus genomic segment as described herein. In
another embodiment, a cDNA described herein can be incorporated
into a plasmid. More detailed description of the cDNAs or nucleic
acids and expression systems are provided is Section 5.7.
Techniques for the production of a cDNA are routine and
conventional techniques of molecular biology and DNA manipulation
and production. Any cloning technique known to the skilled artesian
can be used. Such as techniques are well known and are available to
the skilled artesian in laboratory manuals such as, Sambrook and
Russell, Molecular Cloning: A laboratory Manual, 3.sup.rd edition,
Cold Spring Harbor Laboratory N.Y. (2001).
[0116] In certain embodiments, the cDNA of the arenavirus genomic
segment that is engineered to carry an ORF in a position other than
the wild-type position of the ORF and a nucleotide sequence
encoding a tumor antigen, tumor associated antigen or an antigenic
fragment thereof provided herein is introduced (e.g., transfected)
into a host cell. Thus, in some embodiments provided herein, is a
host cell comprising a cDNA of the arenavirus genomic segment that
is engineered to carry an ORF in a position other than the
wild-type position of the ORF (i.e., a cDNA of the genomic segment)
and a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof provided
herein. In other embodiments, the cDNA described herein is part of
or can be incorporated into a DNA expression vector and introduced
into a host cell. Thus, in some embodiments provided herein is a
host cell comprising a cDNA described herein that is incorporated
into a vector. In other embodiments, the arenavirus genomic segment
described herein is introduced into a host cell.
[0117] In certain embodiments, described herein is a method of
producing the arenavirus genomic segment comprising a nucleotide
sequence encoding a tumor antigen, tumor associated antigen or an
antigenic fragment thereof provided herein, wherein the method
comprises transcribing the cDNA of the arenavirus genomic segment.
In certain embodiments, a viral polymerase protein can be present
during transcription of the arenavirus genomic segment in vitro or
in vivo.
[0118] In certain embodiments transcription of the arenavirus
genomic segment is performed using a bi-directional promoter. In
other embodiments, transcription of the arenavirus genomic segment
is performed using a bi-directional expression cassette (see e.g.,
Ortiz-Riano et al., 2013, J Gen Virol., 94(Pt 6): 1175-1188). In
more specific embodiments the bi-directional expression cassette
comprises both a polymerase I and a polymerase II promoter reading
from opposite sides into the two termini of the inserted arenavirus
genomic segment, respectively. In yet more specific embodiments the
bi-directional expression cassette with pol-I and pol-II promoters
read from opposite sides into the L segment and S segment
[0119] In other embodiments, transcription of the cDNA of the
arenavirus genomic segment described herein comprises a promoter.
Specific examples of promoters include an RNA polymerase I
promoter, an RNA polymerase II promoter, an RNA polymerase III
promoter, a T7 promoter, an SP6 promote or a T3 promoter.
[0120] In certain embodiments, the method of producing the
arenavirus genomic segment can further comprise introducing into a
host cell the cDNA of the arenavirus genomic segment comprising a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen or an antigenic fragment thereof provided herein. In
certain embodiments, the method of producing the arenavirus genomic
segment can further comprise introducing into a host cell the cDNA
of the arenavirus genomic segment comprising a nucleotide sequence
encoding a tumor antigen, tumor associated antigen or an antigenic
fragment thereof provided herein, wherein the host cell expresses
all other components for production of the arenavirus genomic
segment; and purifying the arenavirus genomic segment from the
supernatant of the host cell. Such methods are well-known to those
skilled in the art.
[0121] Provided herein are cell lines, cultures and methods of
culturing cells infected with nucleic acids, vectors, and
compositions provided herein. More detailed description of nucleic
acids, vector systems and cell lines described herein is provided
in Section 5.7.
[0122] In certain embodiments, the arenavirus particle as described
herein results in an infectious and replication competent
arenavirus particle. In specific embodiments, the arenavirus
particle described herein is attenuated. In a particular
embodiment, the arenavirus particle is attenuated such that the
virus remains, at least partially, able to spread and can replicate
in vivo, but can only generate low viral loads resulting in
subclinical levels of infection that are non-pathogenic. Such
attenuated viruses can be used as an immunogenic composition.
Provided herein, are immunogenic compositions that comprise an
arenavirus with an ORF in a non-natural position as described in
Section 5.9.
5.1.1 Replication-Defective Arenavirus Particle with an Open
Reading Frame in a Non-Natural Position
[0123] In certain embodiments, provided herein is an arenavirus
particle in which (i) an ORF is in a position other than the
wild-type position of the ORF; and (ii) an ORF encoding GP, NP, Z
protein, and L protein has been removed or functionally inactivated
such that the resulting virus cannot produce further infectious
progeny virus particles. An arenavirus particle comprising a
genetically modified genome in which one or more ORFS has been
deleted or functionally inactivated can be produced in
complementing cells (i.e., cells that express the arenavirus ORF
that has been deleted or functionally inactivated). The genetic
material of the resulting arenavirus particle can be transferred
upon infection of a host cell into the host cell, wherein the
genetic material can be expressed and amplified. In addition, the
genome of the genetically modified arenavirus particle described
herein can encode a heterologous ORF from an organism other than an
arenavirus particle.
[0124] In certain embodiments, an ORF of the arenavirus is deleted
or functionally inactivated and replaced with a nucleotide sequence
encoding a tumor antigen or tumor associated antigen as described
herein. In a specific embodiment, the ORF that encodes the
glycoprotein GP of the arenavirus is deleted or functionally
inactivated. In certain embodiments, functional inactivation of a
gene eliminates any translation product. In certain embodiments,
functional inactivation refers to a genetic alteration that allows
some translation, the translation product, however, is not longer
functional and cannot replace the wild type protein.
[0125] In certain embodiments, at least one of the four ORFs
encoding GP, NP, Z protein, and L protein is removed and replaced
with a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof provided
herein. In another embodiment, at least one ORF, at least two ORFs,
at least three ORFs, or at least four ORFs encoding GP, NP, Z
protein and L protein can be removed and replaced with a nucleotide
sequence encoding a tumor antigen, tumor associated antigen or an
antigenic fragment thereof provided herein. In specific
embodiments, only one of the four ORFs encoding GP, NP, Z protein,
and L protein is removed and replaced with a nucleotide sequence
encoding a tumor antigen, tumor associated antigen or an antigenic
fragment thereof provided herein. In more specific embodiments, the
ORF that encodes GP of the arenavirus genomic segment is removed.
In another specific embodiment, the ORF that encodes the NP of the
arenavirus genomic segment is removed. In more specific
embodiments, the ORF that encodes the Z protein of the arenavirus
genomic segment is removed. In yet another specific embodiment, the
ORF encoding the L protein is removed.
[0126] Thus, in certain embodiments, the arenavirus particle
provided herein comprises a genomic segment that (i) is engineered
to carry an ORF in a non-natural position; (ii) an ORF encoding GP,
NP, Z protein, or L protein is removed; (iii) the ORF that is
removed is replaced with a nucleotide sequence encoding a tumor
antigen, tumor associated antigen or an antigenic fragment thereof
provided herein.
[0127] In certain embodiments, the fragment of the tumor antigen or
tumor associated antigen is antigenic when it is capable of (i)
eliciting an antibody immune response in a host (e.g., mouse,
rabbit, goat, donkey or human) wherein the resulting antibodies
bind specifically to an immunogenic protein expressed in or on a
neoplastic cell (e.g., a cancer cell); and/or (ii) eliciting a
specific T cell immune response.
[0128] In certain embodiments, the nucleotide sequence encoding an
antigenic fragment provided herein is 8 to 100 nucleotides in
length, 15 to 100 nucleotides in length, 25 to 100 nucleotides in
length, 50 to 200 nucleotide in length, 50 to 400 nucleotide in
length, 200 to 500 nucleotide in length, or 400 to 600 nucleotides
in length, 500 to 800 nucleotide in length. In other embodiments,
the nucleotide sequence encoding an antigenic fragment provided
herein is 750 to 900 nucleotides in length, 800 to 100 nucleotides
in length, 850 to 1000 nucleotides in length, 900 to 1200
nucleotides in length, 1000 to 1200 nucleotides in length, 1000 to
1500 nucleotides or 10 to 1500 nucleotides in length, 1500 to 2000
nucleotides in length, 1700 to 2000 nucleotides in length, 2000 to
2300 nucleotides in length, 2200 to 2500 nucleotides in length,
2500 to 3000 nucleotides in length, 3000 to 3200 nucleotides in
length, 3000 to 3500 nucleotides in length, 3200 to 3600
nucleotides in length, 3300 to 3800 nucleotides in length, 4000
nucleotides to 4400 nucleotides in length, 4200 to 4700 nucleotides
in length, 4800 to 5000 nucleotides in length, 5000 to 5200
nucleotides in length, 5200 to 5500 nucleotides in length, 5500 to
5800 nucleotides in length, 5800 to 6000 nucleotides in length,
6000 to 6400 nucleotides in length, 6200 to 6800 nucleotides in
length, 6600 to 7000 nucleotides in length, 7000 to 7200
nucleotides in lengths, 7200 to 7500 nucleotides in length, or 7500
nucleotides in length. In some embodiments, the nucleotide sequence
encodes a peptide or polypeptide that is 5 to 10 amino acids in
length, 10 to 25 amino acids in length, 25 to 50 amino acids in
length, 50 to 100 amino acids in length, 100 to 150 amino acids in
length, 150 to 200 amino acids in length, 200 to 250 amino acids in
length, 250 to 300 amino acids in length, 300 to 400 amino acids in
length, 400 to 500 amino acids in length, 500 to 750 amino acids in
length, 750 to 1000 amino acids in length, 1000 to 1250 amino acids
in length, 1250 to 1500 amino acids in length, 1500 to 1750 amino
acids in length, 1750 to 2000 amino acids in length, 2000 to 2500
amino acids in length, or more than 2500 or more amino acids in
length. In some embodiments, the nucleotide sequence encodes a
polypeptide that does not exceed 2500 amino acids in length. In
specific embodiments the nucleotide sequence does not contain a
stop codon. In certain embodiments, the nucleotide sequence is
codon-optimized. In certain embodiments the nucleotide composition,
nucleotide pair composition or both can be optimized. Techniques
for such optimizations are known in the art and can be applied to
optimize a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof provided
herein.
[0129] In certain embodiments, the growth and infectivity of the
arenavirus particle is not affected by the nucleotide sequence
encoding a tumor antigen, tumor associated antigen or an antigenic
fragment thereof provided herein.
[0130] Techniques known to one skilled in the art may be used to
produce an arenavirus particle comprising an arenavirus genomic
segment engineered to carry an arenavirus ORF in a position other
than the wild-type position and a nucleotide sequence encoding a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein. For example, reverse genetics techniques
may be used to generate such arenavirus particle. In other
embodiments, the replication-defective arenavirus particle (i.e.,
the arenavirus genomic segment engineered to carry an arenavirus
ORF in a position other than the wild-type position, wherein an ORF
encoding GP, NP, Z protein, L protein, has been deleted) can be
produced in a complementing cell.
[0131] In certain embodiments, an arenavirus particle or arenavirus
genomic segment provided herein comprising a nucleotide sequence
encoding a tumor antigen, tumor associated antigen or antigenic
fragment thereof as provided herein further comprises at least one
nucleotide sequence encoding at least one immunomodulatory peptide,
polypeptide or protein. In certain embodiments, the
immunomodulatory peptide, polypeptide or protein is Calreticulin
(CRT), or a fragment thereof; Ubiquitin or a fragment thereof;
Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), or a
fragment thereof; Invariant chain (CD74) or an antigenic fragment
thereof; Mycobacterium tuberculosis Heat shock protein 70 or an
antigenic fragment thereof; Herpes simplex virus 1 protein VP22 or
an antigenic fragment thereof; CD40 ligand or an antigenic fragment
thereof; or Fms-related tyrosine kinase 3 (Flt3) ligand or an
antigenic fragment thereof.
[0132] In certain embodiments, the arenavirus genomic segment or
the arenavirus particle used according to the present application
can be Old World viruses, for example Lassa virus, Lymphocytic
choriomeningitis virus (LCMV), Mobala virus, Mopeia virus, or Ippy
virus, or New World viruses, for example Amapari virus, Flexal
virus, Guanarito virus, Junin virus, Latino virus, Machupo virus,
Oliveros virus, Parana virus, Pichinde virus, Pirital virus, Sabia
virus, Tacaribe virus, Tamiami virus, Bear Canyon virus, or
Whitewater Arroyo virus.
[0133] In certain embodiments, the arenavirus particle as described
herein is suitable for use as a vaccine and methods of using such
arenavirus particle in a vaccination and treatment for a neoplastic
disease, for example, cancer, is provided. More detailed
description of the methods of using the arenavirus particle
described herein is provided in Section 5.8
[0134] In certain embodiments, the arenavirus particle as described
herein is suitable for use as a pharmaceutical composition and
methods of using such arenavirus particle in a vaccination and
treatment for a neoplastic disease, for example, cancer, is
provided. More detailed description of the methods of using the
arenavirus particle described herein is provided in Section
5.9.
5.2 Tri-Segmented Arenavirus Particle
[0135] Provided herein are tri-segmented arenavirus particles with
rearrangements of their ORFs and a nucleotide sequence encoding a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein. In one aspect, provided herein is a
tri-segmented arenavirus particle comprising one L segment and two
S segments or two L segments and one S segment. In certain
embodiments, the tri-segmented arenavirus particle does not
recombine into a replication competent bi-segmented arenavirus
particle. More specifically, in certain embodiments, two of the
genomic segments (e.g., the two S segments or the two L segments,
respectively) cannot recombine in a way to yield a single viral
segment that could replace the two parent segments. In specific
embodiments, the tri-segmented arenavirus particle comprises an ORF
in a position other than the wild-type position of the ORF and a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen or an antigenic fragment thereof provided herein. In yet
another specific embodiment, the tri-segmented arenavirus particle
comprises all four arenavirus ORFs. Thus, in certain embodiments,
the tri-segmented arenavirus particle is replication competent and
infectious. In other embodiments, the tri-segmented arenavirus
particle lacks one of the four arenavirus ORFs. Thus, in certain
embodiments, the tri-segmented arenavirus particle is infectious
but unable to produce further infectious progeny in
non-complementing cells.
[0136] In certain embodiments, the ORF encoding GP, NP, Z protein,
or the L protein of the tri-segmented arenavirus particle described
herein can be under the control of an arenavirus 3' UTR or an
arenavirus 5' UTR. In more specific embodiments, the tri-segmented
arenavirus 3' UTR is the 3' UTR of an arenavirus S segment(s). In
another specific embodiment, the tri-segmented arenavirus 3' UTR is
the 3' UTR of a tri-segmented arenavirus L segment(s). In more
specific embodiments, the tri-segmented arenavirus 5' UTR is the 5'
UTR of an arenavirus S segment(s). In other specific embodiments,
the 5' UTR is the 5' UTR of the L segment(s).
[0137] In other embodiments, the ORF encoding GP, NP, Z protein, or
the L protein of tri-segmented arenavirus particle described herein
can be under the control of the arenavirus conserved terminal
sequence element (the 5'- and 3'-terminal 19-20-nt regions) (see
e.g., Perez & de la Torre, 2003, J Virol. 77(2):
1184-1194).
[0138] In certain embodiments, the ORF encoding GP, NP, Z protein
or the L protein of the tri-segmented arenavirus particle can be
under the control of the promoter element of the 5' UTR (see e.g.,
Albarino et al., 2011, J Virol., 85(8):4020-4). In another
embodiment, the ORF encoding GP, NP Z protein, L protein of the
tri-segmented arenavirus particle can be under the control of the
promoter element of the 3' UTR (see e.g., Albarino et al., 2011, J
Virol., 85(8):4020-4). In more specific embodiments, the promoter
element of the 5' UTR is the 5' UTR promoter element of the S
segment(s) or the L segment(s). In another specific embodiment, the
promoter element of the 3' UTR is the 3' UTR the promoter element
of the S segment(s) or the L segment(s).
[0139] In certain embodiments, the ORF that encoding GP, NP, Z
protein or the L protein of the tri-segmented arenavirus particle
can be under the control of a truncated arenavirus 3' UTR or a
truncated arenavirus 5' UTR (see e.g., Perez & de la Torre,
2003, J Virol. 77(2): 1184-1194; Albarino et al., 2011, J Virol.,
85(8):4020-4). In more specific embodiments, the truncated 3' UTR
is the 3' UTR of the arenavirus S segment or L segment. In more
specific embodiments, the truncated 5' UTR is the 5' UTR of the
arenavirus S segment(s) or L segment(s).
[0140] Also provided herein, is a cDNA of the tri-segmented
arenavirus particle comprising a nucleotide sequence encoding a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein. In more specific embodiments, provided
herein is a DNA nucleotide sequence or a set of DNA nucleotide
sequences encoding a tri-segmented arenavirus particle as set forth
in Table 2 or Table 3.
[0141] In certain embodiments, the nucleic acids encoding the
tri-segmented arenavirus genome are part of or incorporated into
one or more DNA expression vectors. In a specific embodiment,
nucleic acids encoding the genome of the tri-segmented arenavirus
particle are part of or incorporated into one or more DNA
expression vectors that facilitate production of a tri-segmented
arenavirus particle as described herein. In another embodiment, a
cDNA described herein can be incorporated into a plasmid. More
detailed description of the cDNAs and expression systems are
provided is Section 5.7. Techniques for the production of a cDNA
routine and conventional techniques of molecular biology and DNA
manipulation and production. Any cloning technique known to the
skilled artesian can be used. Such techniques are well known and
are available to the skilled artesian in laboratory manuals such
as, Sambrook and Russell, Molecular Cloning: A laboratory Manual,
3.sup.rd edition, Cold Spring Harbor Laboratory N.Y. (2001).
[0142] In certain embodiments, the cDNA of the tri-segmented
arenavirus comprising a nucleotide sequence encoding a tumor
antigen, tumor associated antigen or an antigenic fragment thereof
provided herein is introduced (e.g., transfected) into a host cell.
Thus, in some embodiments provided herein, is a host cell
comprising a cDNA of the tri-segmented arenavirus particle (i.e., a
cDNA of the genomic segments of the tri-segmented arenavirus
particle) and a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof provided
herein. In other embodiments, the cDNA described herein that is
part of or can be incorporated into a DNA expression vector and
introduced into a host cell. Thus, in some embodiments provided
herein is a host cell comprising a cDNA described herein that is
incorporated into a vector. In other embodiments, the tri-segmented
arenavirus genomic segments (i.e., the L segment and/or S segment
or segments) described herein is introduced into a host cell.
[0143] In certain embodiments, described herein is a method of
producing the tri-segmented arenavirus particle, wherein the method
comprises transcribing the cDNA of the tri-segmented arenavirus
particle comprising a nucleotide sequence encoding a tumor antigen,
tumor associated antigen or an antigenic fragment thereof provided
herein. In certain embodiments, a viral polymerase protein can be
present during transcription of the tri-segmented arenavirus
particle in vitro or in vivo. In certain embodiments, transcription
of the arenavirus genomic segment is performed using a
bi-directional promoter.
[0144] In other embodiments, transcription of the arenavirus
genomic segment is performed using a bi-directional expression
cassette (see e.g., Ortiz-Riano et al., 2013, J Gen Virol., 94(Pt
6): 1175-1188). In more specific embodiments the bi-directional
expression cassette comprises both a polymerase I and a polymerase
II promoter reading from opposite sides into the two termini of the
inserted arenavirus genomic segment, respectively.
[0145] In other embodiments, transcription of the cDNA of the
arenavirus genomic segment described herein comprises a promoter.
Specific examples of promoters include an RNA polymerase I
promoter, an RNA polymerase II promoter, an RNA polymerase III
promoter, a T7 promoter, an SP6 promoter or a T3 promoter.
[0146] In certain embodiments, the method of producing the
tri-segmented arenavirus particle can further comprise introducing
into a host cell the cDNA of the tri-segmented arenavirus particle
comprising a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof provided
herein. In certain embodiments, the method of producing the
tri-segmented arenavirus particle can further comprise introducing
into a host cell the cDNA of the tri-segmented arenavirus particle
that comprises a nucleotide sequence encoding a tumor antigen,
tumor associated antigen or an antigenic fragment thereof provided
herein, wherein the host cell expresses all other components for
production of the tri-segmented arenavirus particle; and purifying
the tri-segmented arenavirus particle from the supernatant of the
host cell. Such methods are well-known to those skilled in the
art.
[0147] Provided herein are cell lines, cultures and methods of
culturing cells infected with nucleic acids, vectors, and
compositions provided herein. More detailed description of nucleic
acids, vector systems and cell lines described herein is provided
in Section 5.7.
[0148] In certain embodiments, the tri-segmented arenavirus
particle as described herein results in a infectious and
replication competent arenavirus particle. In specific embodiments,
the arenavirus particle described herein is attenuated. In a
particular embodiment, the tri-segmented arenavirus particle is
attenuated such that the virus remains, at least partially,
replication-competent and can replicate in vivo, but can only
generate low viral loads resulting in subclinical levels of
infection that are non-pathogenic. Such attenuated viruses can be
used as an immunogenic composition.
[0149] In certain embodiments, the tri-segmented arenavirus
particle has the same tropism as the bi-segmented arenavirus
particle.
[0150] Also provided herein, are compositions that comprise the
tri-segmented arenavirus particle as described in Section 5.8 and
5.9.
5.2.1 Tri-Segmented Arenavirus Particle Comprising One L Segment
and Two S Segments
[0151] In one aspect, provided herein is a tri-segmented arenavirus
particle comprising one L segment and two S segments. In certain
embodiments, propagation of the tri-segmented arenavirus particle
comprising one L segment and two S segments does not result in a
replication-competent bi-segmented viral particle. In specific
embodiments, propagation of the tri-segmented arenavirus particle
comprising one L segment and two S segments does not result in a
replication-competent bi-segmented viral particle after at least 10
days, at least 20 days, at least 30 days, at least 40 days, at
least 50 days, at least 60 days, at least 70 days, at least 80
days, at least 90 days, or at least 100 days of persistent
infection in mice lacking type I interferon receptor, type II
interferon receptor and recombination activating gene (RAG1), and
having been infected with 10.sup.4 PFU of the tri-segmented
arenavirus particle (see Section 5.10.14). In other embodiments,
propagation of the tri-segmented arenavirus particle comprising one
L segment and two S segments does not result in a
replication-competent bi-segmented viral particle after at least 10
passages, at least 20 passages, at least 30 passages, at least 40
passages, or at least 50 passages.
[0152] The tri-segmented arenavirus particle with all viral genes
in their respective wild-type position is known in the art (e.g.,
Emonet et al., 2011 J. Virol., 85(4):1473; Popkin et al., 2011, J.
Virol, 85(15):7928). In particular, the tri-segmented arenavirus
genome consists of one L segment and two S segments, in which a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen or an antigenic fragment thereof provided herein is
inserted into one position on each S segment. More specifically,
one S segment encodes GP and a tumor antigen, tumor associated
antigen or an antigenic fragment thereof, respectively. The other S
segment encodes a tumor antigen, a tumor associated antigen or an
antigenic fragment thereof and NP, respectively. The L segment
encodes the L protein and Z protein. All segments are flanked by
the respective 5' and 3' UTRs.
[0153] In certain embodiments, inter-segmental recombination of the
two S segments of the tri-segmented arenavirus particle, provided
herein, that unities the two arenaviral ORFs on one instead of two
separate segments results in a non functional promoter (i.e., a
genomic segment of the structure: 5' UTR-5' UTR or a 3' UTR-3'
UTR), wherein each UTR forming one end of the genome is an inverted
repeat sequence of the other end of the same genome.
[0154] In certain embodiments, the tri-segmented arenavirus
particle comprising one L segment and two S segments has been
engineered to carry an arenavirus ORF in a position other than the
wild-type position of the ORF and a nucleotide sequence encoding a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein. In other embodiments, the tri-segmented
arenavirus particle comprising one L segment and two S segments has
been engineered to carry two arenavirus ORFs, or three arenavirus
ORFs, or four arenavirus ORFs, or five arenavirus ORFs, or six
arenavirus ORFs in a position other than the wild-type position. In
specific embodiments, the tri-segmented arenavirus particle
comprising one L segment and two S segments comprises a full
complement of all four arenavirus ORFs. Thus, in some embodiments,
the tri-segmented arenavirus particle is an infectious and
replication competent tri-segmented arenavirus particle. In
specific embodiments, the two S segments of the tri-segmented
arenavirus particle have been engineered to carry one of their ORFs
in a position other than the wild-type position. In more specific
embodiments, the two S segments comprise a full complement of the S
segment ORF's. In certain specific embodiments, the L segment has
been engineered to carry an ORF in a position other than the
wild-type position or the L segment can be the wild-type genomic
segment.
[0155] In certain embodiments, one of the two S segments can be:
[0156] (i) an arenavirus S segment, wherein the ORF encoding the Z
protein is under control of an arenavirus 5' UTR; [0157] (ii) an
arenavirus S segment, wherein the ORF encoding the L protein is
under control of an arenavirus 5' UTR; [0158] (iii) an arenavirus S
segment, wherein the ORF encoding the NP is under control of an
arenavirus 5' UTR; [0159] (iv) an arenavirus S segment, wherein the
ORF encoding the GP is under control of an arenavirus 3' UTR;
[0160] (v) an arenavirus S segment, wherein the ORF encoding the L
is under control of an arenavirus 3' UTR; and [0161] (vi) an
arenavirus S segment, wherein the ORF encoding the Z protein is
under control of an arenavirus 3' UTR.
[0162] In certain embodiments, the tri-segmented arenavirus
particle comprising one L segment and two S segments can comprise a
duplicate ORF (i.e., two wild-type S segment ORFs e.g., GP or NP).
In specific embodiments, the tri-segmented arenavirus particle
comprising one L segment and two S segments can comprise one
duplicate ORF (e.g., (GP, GP)) or two duplicate ORFs (e.g., (GP,
GP) and (NP, NP)).
[0163] Table 2A, below, is an illustration of the genome
organization of a tri-segmented arenavirus particle comprising one
L segment and two S segments, wherein intersegmental recombination
of the two S segments in the tri-segmented arenavirus genome does
not result in a replication-competent bi-segmented viral particle
and abrogates arenaviral promoter activity (i.e., the resulting
recombined S segment is made up of two 3 `UTRs instead of a 3` UTR
and a 5' UTR).
TABLE-US-00003 TABLE 2A Tri-segmented arenavirus particle
comprising one L segment and two S segments Position 1 Position 2
Position 3 Position 4 Position 5 Position 6 *ORF GP *ORF NP Z L
*ORF NP *ORF GP Z L *ORF NP *ORF GP L Z *ORF NP *ORF Z L GP *ORF NP
Z GP *ORF Z *ORF NP Z GP Z *ORF *ORF NP *ORF L Z GP *ORF L *ORF NP
Z GP *ORF L Z NP *ORF GP *ORF L *ORF GP Z NP *ORF L Z GP *ORF NP
*ORF Z L NP *ORF GP *ORF Z *ORF GP L NP *ORF Z L GP *ORF NP L GP
*ORF NP *ORF Z L GP *ORF *ORF Z NP L GP *ORF Z *ORF NP L *ORF Z GP
*ORF NP L GP *ORF NP *ORF Z L GP *ORF Z *ORF NP L GP Z NP *ORF *ORF
L GP Z NP *ORF *ORF L *ORF Z NP *ORF GP L NP *ORF Z *ORF GP L NP Z
*ORF GP *ORF L *ORF Z *ORF GP NP L NP Z GP *ORF *ORF L NP *ORF Z
*ORF GP L *ORF Z NP *ORF GP L Z *ORF GP *ORF NP L Z *ORF NP *ORF GP
Z GP *ORF NP *ORF L Z GP *ORF *ORF L NP Z GP *ORF L *ORF NP Z *ORF
L GP *ORF NP Z GP *ORF NP *ORF L Z GP *ORF L *ORF NP Z GP L NP *ORF
*ORF Z GP L NP *ORF *ORF Z *ORF L NP *ORF GP Z NP *ORF *ORF L GP Z
NP *ORF GP *ORF L Z NP *ORF *ORF L GP Z NP *ORF L *ORF GP Z NP L GP
*ORF *ORF Z *ORF L GP *ORF NP Z NP *ORF GP *ORF L Z NP *ORF L *ORF
GP Z *ORF L NP *ORF GP Z L *ORF GP *ORF NP Position 1 is under the
control of an arenavirus S segment 5' UTR; Position 2 is under the
control of an arenavirus S segment 3' UTR; Position 3 is under the
control of an arenavirus S segment 5' UTR; Position 4 under the
control of an arenavirus S segment 3' UTR; Position 5 is under the
control of an arenavirus L segment 5' UTR; Position 6 is under the
control of an arenavirus L segment 3' UTR. *ORF indicates that a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen or an antigenic fragment thereof provided herein has been
inserted.
[0164] In certain embodiments, the IGR between position one and
position two can be an arenavirus S segment or L segment IGR; the
IGR between position two and three can be an arenavirus S segment
or L segment IGR; and the IGR between the position five and six can
be an arenavirus L segment IGR. In a specific embodiment, the IGR
between position one and position two can be an arenavirus S
segment IGR; the IGR between position two and three can be an
arenavirus S segment IGR; and the IGR between the position five and
six can be an arenavirus L segment IGR. In certain embodiments,
other combinations are also possible. For example, a tri-segmented
arenavirus particle comprising one L segment and two S segments,
wherein intersegmental recombination of the two S segments in the
tri-segmented arenavirus genome does not result in a
replication-competent bi-segmented viral particle and abrogates
arenaviral promoter activity (i.e., the resulting recombined S
segment is made up of two 5'UTRs instead of a 3' UTR and a 5'
UTR).
[0165] In certain embodiments, intersegmental recombination of an S
segment and an L segment in the tri-segmented arenavirus particle
comprising one L segment and two S segments, restores a functional
segment with two viral genes on only one segment instead of two
separate segments. In other embodiments, intersegmental
recombination of an S segment and an L segment in the tri-segmented
arenavirus particle comprising one L segment and two S segments
does not result in a replication-competent bi-segmented viral
particle.
[0166] Table 2B, below, is an illustration of the genome
organization of a tri-segmented arenavirus particle comprising one
L segment and two S segments, wherein intersegmental recombination
of an S segment and an L segment in the tri-segmented arenavirus
genome does not result in a replication-competent bi-segmented
viral particle and abrogates arenaviral promoter activity (i.e.,
the resulting recombined S segment is made up of two 3'UTRs instead
of a 3' UTR and a 5' UTR).
TABLE-US-00004 TABLE 2B Tri-segmented arenavirus particle
comprising one L segment and two S segments Position 1 Position 2
Position 3 Position 4 Position 5 Position 6 L GP *ORF NP Z *ORF L
GP Z *ORF *ORF NP L GP *ORF NP Z *ORF L GP Z *ORF *ORF NP L NP *ORF
GP Z *ORF L NP Z *ORF *ORF GP L NP *ORF GP Z *ORF L NP Z *ORF *ORF
GP Z GP *ORF NP L *ORF Z GP L *ORF *ORF NP Z GP *ORF NP L *ORF Z NP
L *ORF *ORF GP Z NP *ORF GP L *ORF Z NP L *ORF *ORF GP Position 1
is under the control of an arenavirus S segment 5' UTR; Position 2
is under the control of an arenavirus S segment 3' UTR; Position 3
is under the control of an arenavirus S segment 5' UTR; Position 4
under the control of an arenavirus S segment 3' UTR; Position 5 is
under the control of an arenavirus L segment 5' UTR; Position 6 is
under the control of an arenavirus L segment 3' UTR. *ORF indicates
that a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof provided herein
has been inserted.
[0167] In certain embodiments, the IGR between position one and
position two can be an arenavirus S segment or L segment IGR; the
IGR between position two and three can be an arenavirus S segment
or L segment IGR; and the IGR between the position five and six can
be an arenavirus L segment IGR. In a specific embodiment, the IGR
between position one and position two can be an arenavirus S
segment IGR; the IGR between position two and three can be an
arenavirus S segment IGR; and the IGR between the position five and
six can be an arenavirus L segment IGR. In certain embodiments,
other combinations are also possible. For example, a tri-segmented
arenavirus particle comprising one L segment and two S segments,
wherein intersegmental recombination of the two S segments in the
tri-segmented arenavirus genome does not result in a
replication-competent bi-segmented viral particle and abrogates
arenaviral promoter activity (i.e., the resulting recombined S
segment is made up of two 5'UTRs instead of a 3' UTR and a 5'
UTR).
[0168] In certain embodiments, one of skill in the art could
construct an arenavirus genome with an organization as illustrated
in Table 2A or 2B and as described herein, and then use an assay as
described in Section 5.10 to determine whether the tri-segmented
arenavirus particle is genetically stable, i.e., does not result in
a replication-competent bi-segmented viral particle as discussed
herein.
5.2.2 Tri-Segmented Arenavirus Particle Comprising Two L Segments
and One S Segment
[0169] In one aspect, provided herein is a tri-segmented arenavirus
particle comprising two L segments and one S segment. In certain
embodiments, propagation of the tri-segmented arenavirus particle
comprising two L segments and one S segment does not result in a
replication-competent bi-segmented viral particle. In specific
embodiments, propagation of the tri-segmented arenavirus particle
comprising two L segments and one S segment does not result in a
replication-competent bi-segmented viral particle after at least 10
days, at least 20 days, at least 30 days, at least 40 days, or at
least 50 days, at least 60 days, at least 70 days, at least 80
days, at least 90 days, at least 100 days of persistent in mice
lacking type I interferon receptor, type II interferon receptor and
recombination activating gene (RAG1), and having been infected with
10.sup.4 PFU of the tri-segmented arenavirus particle (see Section
5.10.14). In other embodiments, propagation of the tri-segmented
arenavirus particle comprising two L segments and one S segment
does not result in a replication-competent bi-segmented viral
particle after at least 10 passages, 20 passages, 30 passages, 40
passages, or 50 passages.
[0170] In certain embodiments, inter-segmental recombination of the
two L segments of the tri-segmented arenavirus particle, provided
herein, that unities the two arenaviral ORFs on one instead of two
separate segments results in a non functional promoter (i.e., a
genomic segment of the structure: 5' UTR-5' UTR or a 3' UTR-3'
UTR), wherein each UTR forming one end of the genome is an inverted
repeat sequence of the other end of the same genome.
[0171] In certain embodiments, the tri-segmented arenavirus
particle comprising two L segments and one S segment has been
engineered to carry an arenavirus ORF in a position other than the
wild-type position of the ORF and a nucleotide sequence encoding a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein. In other embodiments, the tri-segmented
arenavirus particle comprising two L segments and one S segment has
been engineered to carry two arenavirus ORFs, or three arenavirus
ORFs, or four arenavirus ORFs, or five arenavirus ORFs, or six
arenavirus ORFs in a position other than the wild-type position. In
specific embodiments, the tri-segmented arenavirus particle
comprising two L segments and one S segment comprises a full
complement of all four arenavirus ORFs. Thus, in some embodiments,
the tri-segmented arenavirus particle is an infectious and
replication competent tri-segmented arenavirus particle. In
specific embodiments, the two L segments of the tri-segmented
arenavirus particle have been engineered to carry one of their ORFs
in a position other than the wild-type position. In more specific
embodiments, the two L segments comprise a full complement of the L
segment ORF's. In certain specific embodiments, the S segment has
been engineered to carry one of their ORFs in a position other than
the wild-type position or the S segment can be the wild-type
genomic segment.
[0172] In certain embodiments, one of the two L segments can be:
[0173] (i) an L segment, wherein the ORF encoding the GP is under
control of an arenavirus 5' UTR; [0174] (ii) an L segment, wherein
the ORF encoding NP is under control of an arenavirus 5' UTR;
[0175] (iii) an L segment, wherein the ORF encoding the L protein
is under control of an arenavirus 5' UTR; [0176] (iv) an L segment,
wherein the ORF encoding the GP is under control of an arenavirus
3' UTR; [0177] (v) an L segment, wherein the ORF encoding the NP is
under control of an arenavirus 3' UTR; and [0178] (vi) an L
segment, wherein the ORF encoding the Z protein is under control of
an arenavirus 3' UTR.
[0179] In certain embodiments, the tri-segmented arenavirus
particle comprising one L segment and two S segments can comprise a
duplicate ORF (i.e., two wild-type L segment ORFs e.g., Z protein
or L protein). In specific embodiments, the tri-segmented
arenavirus particle comprising two L segments and one S segment can
comprise one duplicate ORF (e.g., (Z protein, Z protein)) or two
duplicate ORFs (e.g., (Z protein, Z protein) and (L protein, L
protein)).
[0180] Table 3, below, is an illustration of the genome
organization of a tri-segmented arenavirus particle comprising two
L segments and one S segment, wherein intersegmental recombination
of the two L segments in the tri-segmented arenavirus genome does
not result in a replication-competent bi-segmented viral particle
and abrogates arenaviral promoter activity (i.e., the S segment is
made up of two 3'UTRs instead of a 3' UTR and a 5' UTR). Based on
Table 3 similar combinations could be predicted for generating an
arenavirus particle made up of two 5' UTRs instead of a 3' UTR and
a 5' UTR.
TABLE-US-00005 TABLE 3 Tri-segmented arenavirus particle comprising
two L segments and one S segment Position 1 Position 2 Position 3
Position 4 Position 5 Position 6 ORF* Z ORF* L NP GP ORF* Z ORF* L
GP NP ORF* Z GP L ORF* NP ORF* Z ORF* GP NP L ORF* Z GP ORF* NP L
ORF* Z NP ORF* GP L ORF* ORF* NP Z GP L ORF* Z GP NP ORF* L ORF* Z
NP GP ORF* L ORF* L ORF* Z NP GP ORF* L ORF* Z GP NP ORF* L ORF* GP
NP Z ORF* L GP Z ORF* NP ORF* L ORF* GP NP Z ORF* L NP Z ORF* GP
ORF* L GP NP ORF* Z ORF* L NP GP ORF* Z ORF* GP ORF* L NP Z ORF* GP
NP L ORF* Z ORF* GP ORF* Z NP L ORF* GP NP Z ORF* L ORF* NP ORF* L
GP Z ORF* NP GP L ORF* Z ORF* NP GP Z ORF* L ORF* NP ORF* Z GP L
ORF* L ORF* Z NP GP ORF* L ORF* Z GP NP ORF* L ORF* NP GP Z ORF* L
ORF* GP NP Z ORF* L NP Z ORF* GP ORF* Z ORF* GP NP L ORF* Z GP L
ORF* NP ORF* Z NP GP ORF* L ORF* Z GP NP ORF* L ORF* GP ORF* L NP Z
ORF* GP ORF* L Z NP ORF* GP ORF* Z GP L ORF* GP NP L ORF* Z GP L
ORF* Z ORF* NP GP L ORF* NP ORF* Z GP Z ORF* L ORF* NP GP Z ORF* L
ORF* NP GP Z ORF* NP ORF* L GP NP ORF* Z ORF* L NP L ORF* Z ORF* GP
NP L ORF* GP ORF* Z NP L ORF* Z ORF* GP *Position 1 is under the
control of an arenavirus L segment 5' UTR; position 2 is under the
control of an arenavirus L segment 3' UTR; position 3 is under the
control of an arenavirus L segment 5' UTR; position 4 is under the
control of an arenavirus L segment 3' UTR; position 5 is under the
control of an arenavirus S segment 5' UTR; position 6 is under the
control of an arenavirus S segment 3' UTR. *ORF indicates that a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen or an antigenic fragment thereof provided herein has been
inserted.
[0181] In certain embodiments, the IGR between position one and
position two cab be an arenavirus S segment or L segment IGR; the
IGR between position two and three can be an arenavirus S segment
or L segment IGR; and the IGR between the position five and six can
be an arenavirus L segment IGR. In a specific embodiment, the IGR
between position one and position two can be an arenavirus L
segment IGR; the IGR between position two and three can be an
arenavirus L segment IGR; and the IGR between the position five and
six can be an arenavirus S segment IGR. In certain embodiments,
other combinations are also possible.
[0182] In certain embodiments, intersegmental recombination of an L
segment and an S segment from the tri-segmented arenavirus particle
comprising two L segments and one S segment restores a functional
segment with two viral genes on only one segment instead of two
separate segments. In other embodiments, intersegmental
recombination of an L segment and an S segment in the tri-segmented
arenavirus particle comprising two L segments and one S segment
does not result in a replication-competent bi-segmented viral
particle.
[0183] Table 3B, below, is an illustration of the genome
organization of a tri-segmented arenavirus particle comprising two
L segments and one S segment, wherein intersegmental recombination
of an L segment and an S segment in the tri-segmented arenavirus
genome does not result in a replication-competent bi-segmented
viral particle and abrogates arenaviral promoter activity (i.e.,
the resulting recombined S segment is made up of two 3'UTRs instead
of a 3' UTR and a 5' UTR).
TABLE-US-00006 TABLE 3B Tri-segmented arenavirus particle
comprising two L segments and one S segment Position 1 Position 2
Position 3 Position 4 Position 5 Position 6 NP Z *ORF GP L *ORF NP
Z GP *ORF *ORF L NP Z *ORF GP L *ORF NP Z GP *ORF *ORF L NP L *ORF
GP Z *ORF NP L GP *ORF *ORF Z NP L *ORF GP Z *ORF NP L GP *ORF *ORF
Z GP Z *ORF NP L *ORF GP Z NP *ORF *ORF L GP Z *ORF NP L *ORF GP L
NP *ORF *ORF Z GP L *ORF NP Z *ORF GP L NP *ORF *ORF Z *Position 1
is under the control of an arenavirus L segment 5' UTR; position 2
is under the control of an arenavirus L segment 3' UTR; position 3
is under the control of an arenavirus L segment 5' UTR; position 4
is under the control of an arenavirus L segment 3' UTR; position 5
is under the control of an arenavirus S segment 5' UTR; position 6
is under the control of an arenavirus S segment 3' UTR. *ORF
indicates that a nucleotide sequence encoding a tumor antigen,
tumor associated antigen or an antigenic fragment thereof provided
herein has been inserted.
[0184] In certain embodiments, the IGR between position one and
position two cab be an arenavirus S segment or L segment IGR; the
IGR between position two and three can be an arenavirus S segment
or L segment IGR; and the IGR between the position five and six can
be an arenavirus L segment IGR. In a specific embodiment, the IGR
between position one and position two can be an arenavirus L
segment IGR; the IGR between position two and three can be an
arenavirus L segment IGR; and the IGR between the position five and
six can be an arenavirus S segment IGR. In certain embodiments,
other combinations are also possible.
[0185] In certain embodiments, one of skill in the art could
construct an arenavirus genome with an organization as illustrated
in Table 3A or 3B and as described herein, and then use an assay as
described in Section 5.10 to determine whether the tri-segmented
arenavirus particle is genetically stable, i.e., does not result in
a replication-competent bi-segmented viral particle as discussed
herein.
5.2.3 Replication-Defective Tri-Segmented Arenavirus Particle
[0186] In certain embodiments, provided herein is a tri-segmented
arenavirus particle in which (i) an ORF is in a position other than
the wild-type position of the ORF; and (ii) an ORF encoding GP, NP,
Z protein, or L protein has been removed or functionally
inactivated such that the resulting virus cannot produce further
infectious progeny virus particles (i.e., is replication
defective). In certain embodiments, the third arenavirus segment
can be an S segment. In other embodiments, the third arenavirus
segment can be an L segment. In more specific embodiments, the
third arenavirus segment can be engineered to carry an ORF in a
position other than the wild-type position of the ORF or the third
arenavirus segment can be the wild-type arenavirus genomic segment.
In yet more specific embodiments, the third arenavirus segment
lacks an arenavirus ORF encoding GP, NP, Z protein, or the L
protein.
[0187] In certain embodiments, a tri-segmented genomic segment
could be a S or a L segment hybrid (i.e., a genomic segment that
can be a combination of the S segment and the L segment). In other
embodiments, the hybrid segment is an S segment comprising an L
segment IGR. In another embodiment, the hybrid segment is an L
segment comprising an S segment IGR. In other embodiments, the
hybrid segment is an S segment UTR with and L segment IGR. In
another embodiment, the hybrid segment is an L segment UTR with an
S segment IGR. In specific embodiments, the hybrid segment is an S
segment 5' UTR with an L segment IGR or an S segment 3' UTR with an
L segment IGR. In other specific embodiments, the hybrid segment is
an L segment 5' UTR with an S segment IGR or an L segment 3' UTR
with an S segment IGR.
[0188] A tri-segmented arenavirus particle comprising a genetically
modified genome in which one or more ORFs has been deleted or
functionally inactivated can be produced in complementing cells
(i.e., cells that express the arenavirus ORF that has been deleted
or functionally inactivated). The genetic material of the resulting
arenavirus particle can be transferred upon infection of a host
cell into the host cell, wherein the genetic material can be
expressed and amplified. In addition, the genome of the genetically
modified arenavirus particle described herein can include a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen or an antigenic fragment thereof provided herein.
[0189] In certain embodiments, at least one of the four ORFs
encoding GP, NP, Z protein, and L protein is removed and replaced
with a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof provided
herein. In another embodiment, at least one ORF, at least two ORFs,
at least three ORFs, or at least four ORFs encoding GP, NP, Z
protein and L protein can be removed and replaced with a nucleotide
sequence encoding a tumor antigen, tumor associated antigen or an
antigenic fragment thereof provided herein. In specific
embodiments, only one of the four ORFs encoding GP, NP, Z protein,
and L protein is removed and replaced with a nucleotide sequence
encoding a tumor antigen, tumor associated antigen or an antigenic
fragment thereof provided herein. In more specific embodiments, the
ORF that encodes GP of the arenavirus genomic segment is removed.
In another specific embodiment, the ORF that encodes the NP of the
arenavirus genomic segment is removed. In more specific
embodiments, the ORF that encodes the Z protein of the arenavirus
genomic segment is removed. In yet another specific embodiment, the
ORF encoding the L protein is removed.
[0190] In certain embodiments, provided herein is a tri-segmented
arenavirus particle comprising one L segment and two S segments in
which (i) an ORF is in a position other than the wild-type position
of the ORF; and (ii) an ORF encoding GP or NP has been removed or
functionally inactivated, such that the resulting virus is
replication-defective and not infectious. In a specific embodiment,
one ORF is removed and replaced with a nucleotide sequence encoding
a tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein. In another specific embodiment, two ORFs
are removed and replaced with a nucleotide sequence encoding a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein. In other specific embodiments, three ORFs
are removed and replaced with a nucleotide sequence encoding a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein. In specific embodiments, the ORF encoding
GP is removed and replaced with a nucleotide sequence encoding a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein. In other specific embodiments, the ORF
encoding NP is removed and replaced with a nucleotide sequence
encoding a tumor antigen, tumor associated antigen or an antigenic
fragment thereof provided herein. In yet more specific embodiments,
the ORF encoding NP and the ORF encoding GP are removed and
replaced with one or two nucleotide sequences encoding tumor
antigens, tumor associated antigens or antigenic fragments thereof
provided herein. Thus, in certain embodiments the tri-segmented
arenavirus particle comprises (i) one L segment and two S segments;
(ii) an ORF in a position other than the wild-type position of the
ORF; (iii) one or more nucleotide sequences encoding tumor
antigens, tumor associated antigens or an antigenic fragments
thereof provided herein.
[0191] In certain embodiments, provided herein is a tri-segmented
arenavirus particle comprising two L segments and one S segment in
which (i) an ORF is in a position other than the wild-type position
of the ORF; and (ii) an ORF encoding the Z protein, and/or the L
protein has been removed or functionally inactivated, such that the
resulting virus replication-defective and not infectious. In a
specific embodiment, one ORF is removed and replaced with a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen or an antigenic fragment thereof provided herein. In
another specific embodiment, two ORFs are removed and replaced with
a nucleotide sequence encoding a tumor antigen, tumor associated
antigen or an antigenic fragment thereof provided herein. In
specific embodiments, the ORF encoding the Z protein is removed and
replaced with a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof provided
herein. In other specific embodiments, the ORF encoding the L
protein is removed and replaced with a nucleotide sequence encoding
a tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein. In yet more specific embodiments, the ORF
encoding the Z protein and the ORF encoding the L protein is
removed and replaced with a nucleotide sequence encoding a tumor
antigen, tumor associated antigen or an antigenic fragment thereof
provided herein. Thus, in certain embodiments the tri-segmented
arenavirus particle comprises (i) two L segments and one S segment;
(ii) an ORF in a position other than the wild-type position of the
ORF; (iii) a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof provided
herein.
[0192] Thus, in certain embodiments, the tri-segmented arenavirus
particle provided herein comprises a tri-segmented arenavirus
particle (i.e., one L segment and two S segments or two L segments
and one S segment) that i) is engineered to carry an ORF in a
non-natural position; ii) an ORF encoding GP, NP, Z protein, or L
protein is removed); iii) the ORF that is removed is replaced with
one or more nucleotide sequences encoding tumor antigens, tumor
associated antigens or antigenic fragments thereof provided
herein.
[0193] In certain embodiments, the nucleotide sequence encoding an
antigenic fragment provided herein is 8 to 100 nucleotides in
length, 15 to 100 nucleotides in length, 25 to 100 nucleotides in
length, 50 to 200 nucleotide in length, 50 to 400 nucleotide in
length, 200 to 500 nucleotide in length, or 400 to 600 nucleotides
in length, 500 to 800 nucleotide in length. In other embodiments,
the nucleotide sequence encoding an antigenic fragment provided
herein is 750 to 900 nucleotides in length, 800 to 100 nucleotides
in length, 850 to 1000 nucleotides in length, 900 to 1200
nucleotides in length, 1000 to 1200 nucleotides in length, 1000 to
1500 nucleotides or 10 to 1500 nucleotides in length, 1500 to 2000
nucleotides in length, 1700 to 2000 nucleotides in length, 2000 to
2300 nucleotides in length, 2200 to 2500 nucleotides in length,
2500 to 3000 nucleotides in length, 3000 to 3200 nucleotides in
length, 3000 to 3500 nucleotides in length, 3200 to 3600
nucleotides in length, 3300 to 3800 nucleotides in length, 4000
nucleotides to 4400 nucleotides in length, 4200 to 4700 nucleotides
in length, 4800 to 5000 nucleotides in length, 5000 to 5200
nucleotides in length, 5200 to 5500 nucleotides in length, 5500 to
5800 nucleotides in length, 5800 to 6000 nucleotides in length,
6000 to 6400 nucleotides in length, 6200 to 6800 nucleotides in
length, 6600 to 7000 nucleotides in length, 7000 to 7200
nucleotides in lengths, 7200 to 7500 nucleotides in length, or 7500
nucleotides in length. In some embodiments, the nucleotide sequence
encodes a peptide or polypeptide that is 5 to 10 amino acids in
length, 10 to 25 amino acids in length, 25 to 50 amino acids in
length, 50 to 100 amino acids in length, 100 to 150 amino acids in
length, 150 to 200 amino acids in length, 200 to 250 amino acids in
length, 250 to 300 amino acids in length, 300 to 400 amino acids in
length, 400 to 500 amino acids in length, 500 to 750 amino acids in
length, 750 to 1000 amino acids in length, 1000 to 1250 amino acids
in length, 1250 to 1500 amino acids in length, 1500 to 1750 amino
acids in length, 1750 to 2000 amino acids in length, 2000 to 2500
amino acids in length, or more than 2500 or more amino acids in
length. In some embodiments, the nucleotide sequence encodes a
polypeptide that does not exceed 2500 amino acids in length. In
specific embodiments the nucleotide sequence does not contain a
stop codon. In certain embodiments, the nucleotide sequence is
codon-optimized. In certain embodiments the nucleotide composition,
nucleotide pair composition or both can be optimized. Techniques
for such optimizations are known in the art and can be applied to
optimize a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof provided
herein.
[0194] Any nucleotide sequence encoding a tumor antigen, tumor
associated antigen or an antigenic fragment thereof provided herein
may be included in the tri-segmented arenavirus particle. In one
embodiment, the a nucleotide sequence encoding a tumor antigen,
tumor associated antigen or an antigenic fragment thereof provided
herein is capable of eliciting an immune response.
[0195] In certain embodiments, the growth and infectivity of the
arenavirus particle is not affected by the nucleotide sequence
encoding a tumor antigen, tumor associated antigen or an antigenic
fragment thereof provided herein.
[0196] Techniques known to one skilled in the art may be used to
produce an arenavirus particle comprising an arenavirus genomic
segment engineered to carry an arenavirus ORF in a position other
than the wild-type position and a nucleotide sequence encoding a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein. For example, reverse genetics techniques
may be used to generate such arenavirus particle. In other
embodiments, the replication-defective arenavirus particle (i.e.,
the arenavirus genomic segment engineered to carry an arenavirus
ORF in a position other than the wild-type position, wherein an ORF
encoding GP, NP, Z protein, L protein, has been deleted) can be
produced in a complementing cell.
[0197] In certain embodiments, a tri-segmented arenavirus particle
provided herein comprising a nucleotide sequence encoding a tumor
antigen, tumor associated antigen or antigenic fragment thereof as
provided herein further comprises at least one nucleotide sequence
encoding at least one immunomodulatory peptide, polypeptide or
protein. In certain embodiments, the immunomodulatory peptide,
polypeptide or protein is Calreticulin (CRT), or a fragment
thereof; Ubiquitin or a fragment thereof; Granulocyte-Macrophage
Colony-Stimulating Factor (GM-CSF), or a fragment thereof;
Invariant chain (CD74) or an antigenic fragment thereof;
Mycobacterium tuberculosis Heat shock protein 70 or an antigenic
fragment thereof; Herpes simplex virus 1 protein VP22 or an
antigenic fragment thereof; CD40 ligand or an antigenic fragment
thereof or Fms-related tyrosine kinase 3 (Flt3) ligand or an
antigenic fragment thereof.
[0198] Arenaviruses for use with the methods and compositions
provided herein can be Old World viruses, for example Lassa virus,
Lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia
virus, or Ippy virus, or New World viruses, for example Amapari
virus, Flexal virus, Guanarito virus, Junin virus, Latino virus,
Machupo virus, Oliveros virus, Parana virus, Pichinde virus,
Pirital virus, Sabia virus, Tacaribe virus, Tamiami virus, Bear
Canyon virus, or Whitewater Arroyo virus.
[0199] In certain embodiments, the tri-segmented arenavirus
particle as described herein is suitable for use as a vaccine and
methods of using such arenavirus particle in a vaccination and
treatment for a neoplastic disease, for example, cancer, is
provided. More detailed description of the methods of using the
arenavirus particle described herein is provided in Section 5.8
[0200] In certain embodiments, the tri-segmented arenavirus
particle as described herein is suitable for use as a
pharmaceutical composition and methods of using such arenavirus
particle in a vaccination and treatment for a neoplastic disease,
for example, cancer, is provided. More detailed description of the
methods of using the arenavirus particle described herein is
provided in Section 5.9.
5.3 Infectious, Replication-Deficient Arenavirus Particles
[0201] A genetically modified arenavirus provided herein includes
where the arenavirus: [0202] is infectious; [0203] cannot form
infectious progeny virus in a non-complementary cell (i.e., a cell
that does not express the functionality that is missing from the
replication-deficient arenavirus and causes it to be
replication-deficient); [0204] is capable of replicating its genome
and expressing its genetic information; and [0205] encodes a tumor
antigen, tumor associated antigen or an antigenic fragment
thereof.
[0206] A genetically modified arenavirus described herein is
infectious, i.e., it can attach to a host cell and release its
genetic material into the host cell. A genetically modified
arenavirus described herein is replication-deficient, i.e., the
arenavirus is unable to produce further infectious progeny
particles in a non-complementing cell. In particular, the genome of
the arenavirus is modified (e.g., by removal or functional
inactivation of an ORF) such that a virus carrying the modified
genome can no longer produce infectious progeny viruses. A
non-complementing cell is a cell that does not provide the
functionality that has been eliminated from the
replication-deficient arenavirus by modification of the virus
genome (e.g., if the ORF encoding the GP protein is removed or
functionally inactivated, a non-complementing cell does not provide
the GP protein). However, a genetically modified arenavirus
provided herein is capable of producing infectious progeny viruses
in complementing cells. Complementing cells are cells that provide
(in trans) the functionality that has been eliminated from the
replication-deficient arenavirus by modification of the virus
genome (e.g., if the ORF encoding the GP protein is removed or
functionally inactivated, a complementing cell does provide the GP
protein). Expression of the complementing functionality (e.g., the
GP protein) can be accomplished by any method known to the skilled
artisan (e.g., transient or stable expression). A genetically
modified arenavirus described herein can amplify and express its
genetic information in a cell that has been infected by the virus.
A genetically modified arenavirus provided herein can comprise a
nucleotide sequence that encodes a tumor antigen, tumor associated
antigen or an antigenic fragment thereof such as, but not limited
to, the tumor antigen, tumor associated antigen or an antigenic
fragment thereof described in Section 5.4.
[0207] In certain embodiments, provided herein is a genetically
modified arenavirus in which an ORF of the arenavirus genome is
removed or functionally inactivated such that the resulting virus
cannot produce further infectious progeny virus particles in
non-complementing cells. An arenavirus particle comprising a
genetically modified genome in which an ORF is removed or
functionally inactivated can be produced in complementing cells
(i.e., in cells that express the arenaviral ORF that has been
removed or functionally inactivated). The genetic material of the
resulting arenavirus particles can be transferred upon infection of
a host cell into the host cell, wherein the genetic material can be
expressed and amplified. In addition, the genome of the genetically
modified arenavirus particles provided herein encodes a tumor
antigen, tumor associated antigen or antigenic fragment thereof
that can be expressed in the host cell.
[0208] In certain embodiments, an ORF of the arenavirus is deleted
or functionally inactivated and replaced with a nucleotide encoding
a tumor antigen or tumor associated antigen as described herein. In
a specific embodiment, the ORF that encodes the glycoprotein GP of
the arenavirus is deleted or functionally inactivated. In certain
embodiments, functional inactivation of a gene eliminates any
translation product. In certain embodiments, functional
inactivation refers to a genetic alteration that allows some
translation, the translation product, however, is not longer
functional and cannot replace the wild type protein.
[0209] In certain embodiments, the ORF that encodes the
glycoprotein (GP) of the arenavirus is deleted to generate a
replication-deficient arenavirus for use in the methods and
compositions provided herein. In a specific embodiment, the
replication-deficient arenavirus comprises a genomic segment
comprising a nucleotide sequence encoding a tumor antigen, tumor
associated antigen or antigenic fragment thereof. Thus, in certain
embodiments, a genetically modified arenavirus particle provided
herein comprises a genomic segment that a) has a deletion or
functional inactivation of an ORF that is present in the wild type
form of the genomic segment; and b) encodes (either in sense or
antisense) a tumor antigen, tumor associated antigen or antigenic
fragment thereof.
[0210] In certain embodiments, the antigen encoded by the
nucleotide that is inserted into the genome of
replication-deficient arenavirus can encode, for example, a tumor
antigen, tumor associated antigen or antigenic fragment thereof or
combinations of tumor antigens, tumor associated antigens or
antigenic fragments thereof including, but not limited to,
oncogenic viral antigens, cancer-testis antigens, oncofetal
antigens, tissue differentiation antigens, mutant protein antigens,
neoantigens, Adipophilin, AIM-2, ALDH1AI, BCLX (L), BING-4, CALCA,
CD45, CPSF, cyclin D1, DKKI, ENAH (hMcna), Ga733 (EpCAM), EphA3,
EZH2, FGF5, glypican-3, G250/MN/CAIX, HER-2/neu, IDO1, IGF2B3,
IL13Ralpha2, Intestinal carboxyl esterase, alpha-foetoprotein,
Kallikrein 4, KIF20A, Lengsin, M-CSF, MCSP, mdm-2, Meloe, MMP-2,
MMP-7, MUC1, MUC5AC, p53 (non-mutant), PAX5, PBF, PRAME, PSMA,
RAGE, RAGE-1, RGS5, RhoC, RNF43, RU2AS, secernin 1, SOX1O, STEAP1
(six-transmembrane epithelial antigen of the prostate 1),
survivinn, Telomerase, VEGF, WT1, EGF-R, CEA, CD20, CD33, CD52,
glycoprotein 100 (GP100 or gp 100 protein), MELANA/MART1, MART2,
NY-ESO-1, p53, MAGE A1, MAGE A3, MAGE-4, MAGE-5, MAGE-6, CDK4,
alpha-actinin-4, ARTC1, BCR-ABL, BCR-ABL fusion protein (b3a2),
B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDKN2A, CLPP,
COA-1, dek-can fusion protein, EFTUD2, Elongation factor 2,
ETV6-AML, ETV6-AML1 fusion protein, FLT3-ITD, FN1, GPNMB,
LDLR-fucosyltransferaseAS fusion protein, NFYC, OGT, OS-9,
pml-RARalpha fusion protein, PRDX5, PTPRK, H-ras, K-ras (V-Ki-ras2
Kirsten rat sarcoma viral oncogene), N-ras, RBAF600, SIRT2, SNRPD1,
SSX, SSX2, SYT-SSX1 or -SSX2 fusion protein, TGF-betaRII,
Triosephosphate isomerase, ormdm-2, LMP2, HPV E6/E7, EGFRvIII
(epidermal growth factor variant III), Idiotype, GD2, ganglioside
G2), Ras-mutant, p53 (mutant), Proteinase3 (PR1), Tyrosinase, PSA,
hTERT, Sarcoma translocation breakpoints, EphA2, prostatic acid
phosphatase PAP, neo-PAP, ML-IAP, AFP, ERG (TMPRSS2 ETS Fusion
gene), NA17, PAX3, ALK, Androgen Receptor, Cyclin B1, Polysialic
acid, MYCN, TRP2, TRP2-Int2, GD3, Fucosyl GM1, Mesothelin, PSCA,
sLe(a), cyp1B1, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, SART3, STn,
Carbonic Anhydrase IX, OY-TES1, Sperm protein 17, LCK, high
molecular weight melanoma-associated antigen (HMWMAA), AKAP-4,
SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP,
PDGFR-beta, MAD-CT-2, For-related antigen 1, TRP1, CA-125, CA19-9,
Calretinin, Epithelial membrane antigen (EMA), Epithelial tumor
antigen (ETA), CD19, CD34, CD99, CD117, Chromogranin, Cytokeratin,
Desmin, Glial fibrillary acidic protein (GFAP), gross cystic
disease fluid protein (GCDFP-15), HMB-45 antigen, Myo-D1,
muscle-specific actin (MSA), neurofilament, neuron-specific enolase
(NSE), placental alkaline phosphatase, synaptophysis,
thyroglobulin, thyroid transcription factor-1, dimeric form of the
pyruvate kinase isoenzyme type M2 (tumor M2-PK), BAGE BAGE-1, CAGE,
CTAGE, FATE, GAGE, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,
GAGE-7, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-SAR-35,
SPANXB1, SPA17, SSX, SYCP1, TPTE, Carbohydrate/ganglioside GM2
(oncofetal antigen-immunogenic-1 OFA-I-1), GM3, CA 15-3 (CA
27.29\BCAA), CA 195, CA 242, CA 50, CAM 43, CEA, EBNA, EF2,
Epstein-Barr virus antigen, HLA-A2, HLA-A11, HSP70-2, KIAAO205,
MUM-1, MUM-2, MUM-3, Myosin class I, GnTV, Herv-K-mel, LAGE-1,
LAGE-2, (sperm protein) SP17, SCP-1, P15(58), Hom/Mel-40, E2A-PRL,
H4-RET, IGH-IGK, MYL-RAR, TSP-180, P185erbB2, p180erbB-3, c-met,
nm-23H1, TAG-72, TAG-72-4, CA-72-4, CAM 17.1, NuMa, 13-catenin,
P16, TAGE, CT7, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA,
CD68\KP1, CO-029, HTgp-175, M344, MG7-Ag, MOV18, NB\70K, NY-CO-1,
RCAS1, SDCCAG16, TA-90, TAAL6, TLP, TPS, CD22, CD27, CD30, CD70,
prostein, TARP (T cell receptor gamma alternate reading frame
protein), Trp-p8, integrin .alpha.v.beta.3 (CD61), galactin, or
Ral-B, CD123, CLL-1, CD38, CS-1, CD138, and ROR1. A detailed
description of the antigens described herein is provided in Section
5.4.
[0211] Arenaviruses for use with the methods and compositions
provided herein can be Old World viruses, for example Lassa virus,
Lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia
virus, or Ippy virus, or New World viruses, for example Amapari
virus, Flexal virus, Guanarito virus, Junin virus, Latino virus,
Machupo virus, Oliveros virus, Parana virus, Pichinde virus,
Pirital virus, Sabia virus, Tacaribe virus, Tamiami virus, Bear
Canyon virus, or Whitewater Arroyo virus.
[0212] The wild type arenavirus genome consists of a short
(.about.3.4 kb) and a large (.about.7.2 kb) RNA segment. The short
segment carries the ORFs encoding the nucleoprotein NP and
glycoprotein GP genes. The large segment comprises the
RNA-dependent RNA polymerase L and the matrix protein Z genes. Wild
type arenaviruses can be rendered replication-deficient to generate
vaccine vectors by substituting the glycoprotein gene for one or
more tumor antigens, tumor associated antigens or antigenic
fragments thereof, against which immune responses are to be
induced.
[0213] Infectious, replication-deficient arenavirus particles
expressing a tumor antigen, tumor associated antigen, or antigenic
fragment thereof, or a combination of tumor antigens, tumor
associated antigens or antigenic fragments thereof as described
herein, can be used to treat (in an immunotherapeutic manner)
subjects having a neoplastic disease described herein.
[0214] Arenavirus disease and immunosuppression in wild type
arenavirus infection are known to result from unchecked viral
replication. By abolishing replication, i.e., the ability to
produce infectious progeny virus particles, of arenavirus particles
by deleting from their genome, e.g., the Z gene which is required
for particle release, or the GP gene which is required for
infection of target cells, the total number of infected cells can
be limited by the inoculum administered, e.g., to a vaccine
recipient, or accidentally transmitted to personnel involved in
medical or biotechnological applications, or to animals. Therefore,
abolishing replication of arenavirus particles prevents
pathogenesis as a result of intentional or accidental transmission
of vector particles. In this invention, one important aspect
consists in exploiting the above necessity of abolishment of
replication in a beneficial way for the purpose of expressing tumor
antigens, tumor associated antigens or antigenic fragments thereof.
In certain embodiments, an arenavirus particle is rendered
replication deficient by genetic modification of its genome. Such
modifications to the genome can include: [0215] deletion of an ORF
(e.g., the ORF encoding the GP, NP, L, or Z protein); [0216]
functional inactivation of an ORF (e.g., the ORF encoding the GP,
NP, L, or Z protein). For example, this can be achieved by
introducing a missense or a nonsense mutation; [0217] change of the
sequence of the ORF (e.g., the exchange of an SIP cleavage site
with the cleavage site of another protease); [0218] mutagenesis of
one of the 5' or 3' termini of one of the genomic segments; [0219]
mutagenesis of an intergenic region (i.e., of the L or the S
genomic segment).
[0220] In certain embodiments, a bi-segmented infectious,
replication-deficient arenavirus expressing a tumor antigen, tumor
associated antigen or antigenic fragment thereof described herein
is a Lymphocytic choriomeningitis virus (LCMV) wherein the S
segment of the virus is modified by substituting the ORF encoding
the GP protein with an ORF encoding a tumor antigen, tumor
associated antigen or antigenic fragment thereof.
[0221] In certain embodiments, a wild type arenavirus vector genome
can be designed to retain at least the essential regulatory
elements on the 5' and 3' untranslated regions (UTRs) of both
segments, and/or also the intergenic regions (IGRs). Without being
bound by theory, the minimal transacting factors for gene
expression in infected cells remain in the vector genome as ORFs
that can be expressed, yet they can be placed differently in the
genome and can be placed under control of a different promoter than
naturally, or can be expressed from internal ribosome entry sites.
In certain embodiments, the nucleic acid encoding a tumor antigen,
tumor associated antigen or antigenic fragment thereof is
transcribed from one of the endogenous arenavirus promoters (i.e.,
5' UTR, 3' UTR of the S segment, 5' UTR, 3' UTR of the L segment).
In other embodiments, the nucleic acid encoding a tumor antigen,
tumor associated antigen or antigenic fragment thereof is expressed
from a heterologous introduced promoter sequences that can be read
by the viral RNA-dependent RNA polymerase, by cellular RNA
polymerase I, RNA polymerase II or RNA polymerase III, such as
duplications of viral promoter sequences that are naturally found
in the viral UTRs, the 28S ribosomal RNA promoter, the beta-actin
promoter or the 5S ribosomal RNA promoter, respectively. In certain
embodiments, ribonucleic acids coding for a tumor antigen, tumor
associated antigen or antigenic fragment thereof are transcribed
and translated either by themselves or as read-through by fusion to
arenavirus protein ORFs, and expression of proteins in the host
cell may be enhanced by introducing in the viral transcript
sequence at the appropriate place(s) one or more, e.g., two, three
or four, internal ribosome entry sites.
[0222] In certain embodiments, the vector generated to encode one
or more tumor antigens, tumor associated antigens or antigenic
fragments thereof may be based on a specific strain of LCMV.
Strains of LCMV include Clone 13, MP strain, Arm CA 1371, Arm
E-250, WE, UBC, Traub, Pasteur, 810885, CH-5692, Marseille #12,
HP65-2009, 200501927, 810362, 811316, 810316, 810366, 20112714,
Douglas, GR01, SN05, CABN and their derivatives. In certain
embodiments, the vector generated to encode one or more tumor
antigens, tumor associated antigens or antigenic fragments thereof
may be based on LCMV Clone 13. In other embodiments, the vector
generated to encode one or more tumor antigens, tumor associated
antigens or antigenic fragments thereof may be based on LCMV MP
strain.
[0223] In certain embodiments, the vector generated to encode one
or more tumor antigens, tumor associated antigens or antigenic
fragments thereof may be based on a specific strain of Junin virus.
Strains of Junin virus include vaccine strains XJ13, XJ#44, and
Candid#1 as well as IV4454, a human isolate. In certain
embodiments, the vector generated to encode one or more tumor
antigens, tumor associated antigens or antigenic fragments thereof
is based on Junin virus Candid #1 strain.
5.4 Tumor Antigens, Tumor Associated Antigens and Antigenic
Fragments
[0224] In certain embodiments, a tumor antigen or tumor associated
antigen for use with the methods and compositions described herein
is an immunogenic protein expressed in or on a neoplastic cell or
tumor, such as a cancer cell or malignant tumor. In certain
embodiments, a tumor antigen or tumor associated antigen for use
with the methods and compositions described herein is a
non-specific, mutant, overexpressed or abnormally expressed
protein, which can be present on both a neoplastic cell or tumor
and a normal cell or tissue. In certain embodiments, a tumor
antigen or tumor associated antigen for use with the methods and
compositions described herein is a tumor-specific antigen which is
restricted to tumor cells. In certain embodiments, a tumor antigen
for use with the methods and compositions described herein is a
cancer-specific antigen which is restricted to cancer cells.
[0225] In certain embodiments, a tumor antigen or tumor associated
antigen can exhibit one, two, three, or more, including all, of the
following characteristics: overexpressed/accumulated (i.e.,
expressed by both normal and neoplastic tissue, but highly
expressed in neoplasia), oncofetal (i.e., usually only expressed in
fetal tissues and in cancerous somatic cells), oncoviral or
oncogenic viral (i.e., encoded by tumorigenic transforming
viruses), cancer-testis (i.e., expressed only by cancer cells and
adult reproductive tissues, e.g., the testis), lineage-restricted
(i.e., expressed largely by a single cancer histotype), mutated
(i.e., only expressed in neoplastic tissue as a result of genetic
mutation or alteration in transcription), post-translationally
altered (e.g., tumor-associated alterations in glycosylation), or
idiotypic (i.e., developed from malignant clonal expansions of B or
T lymphocytes).
[0226] In certain embodiments, the tumor antigen or tumor
associated antigen for use with the methods and compositions
described herein includes antigens from neoplastic diseases
including acute lymphoblastic leukemia; acute lymphoblastic
lymphoma; acute lymphocytic leukaemia; acute myelogenous leukemia;
acute myeloid leukemia (adult/childhood); adrenocortical carcinoma;
AIDS-related cancers; AIDS-related lymphoma; anal cancer; appendix
cancer; astrocytomas; atypical teratoid/rhabdoid tumor; basal-cell
carcinoma; bile duct cancer, extrahepatic (cholangiocarcinoma);
bladder cancer; bone osteosarcoma/malignant fibrous histiocytoma;
brain cancer (adult/childhood); brain tumor, cerebellar astrocytoma
(adult/childhood); brain tumor, cerebral astrocytoma/malignant
glioma brain tumor; brain tumor, ependymoma; brain tumor,
medulloblastoma; brain tumor, supratentorial primitive
neuroectodermal tumors; brain tumor, visual pathway and
hypothalamic glioma; brainstem glioma; breast cancer; bronchial
adenomas/carcinoids; bronchial tumor; Burkitt lymphoma; cancer of
childhood; carcinoid gastrointestinal tumor; carcinoid tumor;
carcinoma of adult, unknown primary site; carcinoma of unknown
primary; central nervous system embryonal tumor; central nervous
system lymphoma, primary; cervical cancer; childhood adrenocortical
carcinoma; childhood cancers; childhood cerebral astrocytoma;
chordoma, childhood; chronic lymphocytic leukemia; chronic
myelogenous leukemia; chronic myeloid leukemia; chronic
myeloproliferative disorders; colon cancer; colorectal cancer;
craniopharyngioma; cutaneous T-cell lymphoma; desmoplastic small
round cell tumor; emphysema; endometrial cancer; ependymoblastoma;
ependymoma; esophageal cancer; ewing's sarcoma in the Ewing family
of tumors; extracranial germ cell tumor; extragonadal germ cell
tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric
(stomach) cancer; gastric carcinoid; gastrointestinal carcinoid
tumor; gastrointestinal stromal tumor; germ cell tumor:
extracranial, extragonadal, or ovarian gestational trophoblastic
tumor; gestational trophoblastic tumor, unknown primary site;
glioma; glioma of the brain stem; glioma, childhood visual pathway
and hypothalamic; hairy cell leukemia; head and neck cancer; heart
cancer; hepatocellular (liver) cancer; hodgkin lymphoma;
hypopharyngeal cancer; hypothalamic and visual pathway glioma;
intraocular melanoma; islet cell carcinoma (endocrine pancreas);
Kaposi Sarcoma; kidney cancer (renal cell cancer); langerhans cell
histiocytosis; laryngeal cancer; lip and oral cavity cancer;
liposarcoma; liver cancer (primary); lung cancer, non-small cell;
lung cancer, small cell; lymphoma, primary central nervous system;
macroglobulinemia, Waldenstrom; male breast cancer; malignant
fibrous histiocytoma of bone/osteosarcoma; medulloblastoma;
medulloepithelioma; melanoma; melanoma, intraocular (eye); merkel
cell cancer; merkel cell skin carcinoma; mesothelioma;
mesothelioma, adult malignant; metastatic squamous neck cancer with
occult primary; mouth cancer; multiple endocrine neoplasia
syndrome; multiple myeloma/plasma cell neoplasm; mycosis fungoides,
myelodysplastic syndromes; myelodysplastic/myeloproliferative
diseases; myelogenous leukemia, chronic; myeloid leukemia, adult
acute; myeloid leukemia, childhood acute; myeloma, multiple (cancer
of the bone-marrow); myeloproliferative disorders, chronic; nasal
cavity and paranasal sinus cancer; nasopharyngeal carcinoma;
neuroblastoma, non-small cell lung cancer; non-hodgkin lymphoma;
oligodendroglioma; oral cancer; oral cavity cancer; oropharyngeal
cancer; osteosarcoma/malignant fibrous histiocytoma of bone;
ovarian cancer; ovarian epithelial cancer (surface
epithelial-stromal tumor); ovarian germ cell tumor; ovarian low
malignant potential tumor; pancreatic cancer; pancreatic cancer,
islet cell; papillomatosis; paranasal sinus and nasal cavity
cancer; parathyroid cancer; penile cancer; pharyngeal cancer;
pheochromocytoma; pineal astrocytoma; pineal germinoma; pineal
parenchymal tumors of intermediate differentiation; pineoblastoma
and supratentorial primitive neuroectodermal tumors; pituitary
tumor; pituitary adenoma; plasma cell neoplasia/multiple myeloma;
pleuropulmonary blastoma; primary central nervous system lymphoma;
prostate cancer; rectal cancer; renal cell carcinoma (kidney
cancer); renal pelvis and ureter, transitional cell cancer;
respiratory tract carcinoma involving the NUT gene on chromosome
15; retinoblastoma; rhabdomyosarcoma, childhood; salivary gland
cancer; sarcoma, Ewing family of tumors; Sezary syndrome; skin
cancer (melanoma); skin cancer (non-melanoma); small cell lung
cancer; small intestine cancer soft tissue sarcoma; soft tissue
sarcoma; spinal cord tumor; squamous cell carcinoma; squamous neck
cancer with occult primary, metastatic; stomach (gastric) cancer;
supratentorial primitive neuroectodermal tumor; T-cell lymphoma,
cutaneous (Mycosis Fungoides and Sezary syndrome); testicular
cancer; throat cancer; thymoma; thymoma and thymic carcinoma;
thyroid cancer; thyroid cancer, childhood; transitional cell cancer
of the renal pelvis and ureter; urethral cancer; uterine cancer,
endometrial; uterine sarcoma; vaginal cancer; vulvar cancer; and
Wilms Tumor.
[0227] In certain embodiments, the tumor antigen or tumor
associated antigen for use with the methods and compositions
described herein includes oncogenic viral antigens, cancer-testis
antigens, oncofetal antigens, tissue differentiation antigens,
mutant protein antigens, neoantigens, Adipophilin, AIM-2, ALDH1AI,
BCLX (L), BING-4, CALCA, CD45, CPSF, cyclin D1, DKKI, ENAH (hMcna),
Ga733 (EpCAM), EphA3, EZH2, FGF5, glypican-3, G250/MN/CAIX,
HER-2/neu, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase,
alpha-foetoprotein, Kallikrein 4, KIF20A, Lengsin, M-CSF, MCSP,
mdm-2, Meloe, MMP-2, MMP-7, MUC1, MUC5AC, p53 (non-mutant), PAX5,
PBF, PRAME, PSMA, RAGE, RAGE-1, RGS5, RhoC, RNF43, RU2AS, secernin
1, SOX1O, STEAP1 (six-transmembrane epithelial antigen of the
prostate 1), survivinn, Telomerase, VEGF, WT1, EGF-R, CEA, CD20,
CD33, CD52, glycoprotein 100 (GP100 or gp 100 protein),
MELANA/MART1, MART2, NY-ESO-1, p53, MAGE A1, MAGE A3, MAGE-4,
MAGE-5, MAGE-6, CDK4, alpha-actinin-4, ARTC1, BCR-ABL, BCR-ABL
fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27,
CDK4, CDKN2A, CLPP, COA-1, dek-can fusion protein, EFTUD2,
Elongation factor 2, ETV6-AML, ETV6-AML1 fusion protein, FLT3-ITD,
FN1, GPNMB, LDLR-fucosyltransferaseAS fusion protein, NFYC, OGT,
OS-9, pml-RARalpha fusion protein, PRDX5, PTPRK, H-ras, K-ras
(V-Ki-ras2 Kirsten rat sarcoma viral oncogene), N-ras, RBAF600,
SIRT2, SNRPD1, SSX, SSX2, SYT-SSX1 or -SSX2 fusion protein,
TGF-betaRII, Triosephosphate isomerase, ormdm-2, LMP2, HPV E6/E7,
EGFRvIII (epidermal growth factor variant III), Idiotype, GD2,
ganglioside G2), Ras-mutant, p53 (mutant), Proteinase3 (PR1),
Tyrosinase, PSA, hTERT, Sarcoma translocation breakpoints, EphA2,
prostatic acid phosphatase PAP, neo-PAP, ML-IAP, AFP, ERG (TMPRSS2
ETS Fusion gene), NA17, PAX3, ALK, Androgen Receptor, Cyclin B1,
Polysialic acid, MYCN, TRP2, TRP2-Int2, GD3, Fucosyl GM1,
Mesothelin, PSCA, sLe(a), cyp1B1, PLAC1, GM3, BORIS, Tn, GLoboH,
NY-BR-1, SART3, STn, Carbonic Anhydrase IX, OY-TES1, Sperm protein
17, LCK, high molecular weight melanoma-associated antigen
(HMWMAA), AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4,
VEGFR2, MAD-CT-1, FAP, PDGFR-beta, MAD-CT-2, For-related antigen 1,
TRP1, CA-125, CA19-9, Calretinin, Epithelial membrane antigen
(EMA), Epithelial tumor antigen (ETA), CD19, CD34, CD99, CD117,
Chromogranin, Cytokeratin, Desmin, Glial fibrillary acidic protein
(GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45
antigen, Myo-D1, muscle-specific actin (MSA), neurofilament,
neuron-specific enolase (NSE), placental alkaline phosphatase,
synaptophysis, thyroglobulin, thyroid transcription factor-1,
dimeric form of the pyruvate kinase isoenzyme type M2 (tumor
M2-PK), BAGE BAGE-1, CAGE, CTAGE, FATE, GAGE, GAGE-1, GAGE-2,
GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, HCA661, HOM-TES-85, MAGEA,
MAGEB, MAGEC, NA88, NY-SAR-35, SPANXB1, SPA17, SSX, SYCP1, TPTE,
Carbohydrate/ganglioside GM2 (oncofetal antigen-immunogenic-1
OFA-I-1), GM3, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA 50, CAM
43, CEA, EBNA, EF2, Epstein-Barr virus antigen, HLA-A2, HLA-A11,
HSP70-2, KIAAO205, MUM-1, MUM-2, MUM-3, Myosin class I, GnTV,
Herv-K-mel, LAGE-1, LAGE-2, (sperm protein) SP17, SCP-1, P15(58),
Hom/Mel-40, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, TSP-180, P185erbB2,
p180erbB-3, c-met, nm-23H1, TAG-72, TAG-72-4, CA-72-4, CAM 17.1,
NuMa, 13-catenin, P16, TAGE, CT7, 43-9F, 5T4, 791Tgp72, 13HCG,
BCA225, BTAA, CD68\KP1, CO-029, HTgp-175, M344, MG7-Ag, MOV18,
NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TLP, TPS, CD22,
CD27, CD30, CD70, prostein, TARP (T cell receptor gamma alternate
reading frame protein), Trp-p8, integrin .alpha.v.beta.3 (CD61),
galactin, or Ral-B, CD123, CLL-1, CD38, CS-1, CD138, and ROR1.
[0228] In certain embodiments, the tumor antigen or tumor
associated antigen for use with the methods and compositions
described herein includes oncogenic viral antigens, wherein the
oncogenic virus antigens are antigens of human papillomavirus
(HPV), antigens of Kaposi's sarcoma-associated herpesvirus, such as
latency-associated nuclear antigen, antigens of Epstein-Barr virus,
such as EBV-EA, EBV-MA, or EBV-VCA, antigens of Merkel cell
polyomavirus, such as MCV T antigen, or antigens of human
T-lymphotropic virus, such as HTLV-1 Tax antigen.
[0229] In certain embodiments, the tumor antigen or tumor
associated antigen is a neoantigen. A "neoantigen," as used herein,
means an antigen that arises by mutation in a tumor cell and such
an antigen is not generally expressed in normal cells or tissue.
Without being bound by theory, because healthy tissues generally do
not posses these antigens, neoantigens represent a preferred
target. Additionally, without being bound by theory, in the context
of the present invention, since the T cells that recognize the
neoantigen may not have undergone negative thymic selection, such
cells can have high avidity to the antigen and mount a strong
immune response against tumors, while lacking the risk to induce
destruction of normal tissue and autoimmune damage. In certain
embodiments, the neoantigen is an MHC class I-restricted
neoantigen. In certain embodiments, the neoantigen is an MHC class
II-restricted neoantigen. In certain embodiments, a mutation in a
tumor cell of the patient results in a novel protein that produces
the neoantigen.
[0230] In certain embodiments, the tumor antigen or tumor
associated antigen can be an antigen ortholog, e.g., a mammalian
(i.e., non-human primate, pig, dog, cat, or horse) to a human tumor
antigen or tumor associated antigen.
[0231] In certain embodiments, an antigenic fragment of a tumor
antigen or tumor associated antigen described herein is encoded by
the nucleotide sequence included within the arenavirus. In certain
embodiments, a fragment is antigenic when it is capable of (i)
eliciting an antibody immune response in a host (e.g., mouse,
rabbit, goat, donkey or human) wherein the resulting antibodies
bind specifically to an immunogenic protein expressed in or on a
neoplastic cell (e.g., a cancer cell); and/or (ii) eliciting a
specific T cell immune response.
[0232] In certain embodiments, the nucleotide sequence encoding
antigenic fragment of a tumor antigen or tumor associated antigen
is 8 to 100 nucleotides in length, 15 to 100 nucleotides in length,
25 to 100 nucleotides in length, 50 to 200 nucleotide in length, 50
to 400 nucleotide in length, 200 to 500 nucleotide in length, or
400 to 600 nucleotides in length, 500 to 800 nucleotide in length.
In other embodiments, the nucleotide sequence is 750 to 900
nucleotides in length, 800 to 100 nucleotides in length, 850 to
1000 nucleotides in length, 900 to 1200 nucleotides in length, 1000
to 1200 nucleotides in length, 1000 to 1500 nucleotides or 10 to
1500 nucleotides in length, 1500 to 2000 nucleotides in length,
1700 to 2000 nucleotides in length, 2000 to 2300 nucleotides in
length, 2200 to 2500 nucleotides in length, 2500 to 3000
nucleotides in length, 3000 to 3200 nucleotides in length, 3000 to
3500 nucleotides in length, 3200 to 3600 nucleotides in length,
3300 to 3800 nucleotides in length, 4000 nucleotides to 4400
nucleotides in length, 4200 to 4700 nucleotides in length, 4800 to
5000 nucleotides in length, 5000 to 5200 nucleotides in length,
5200 to 5500 nucleotides in length, 5500 to 5800 nucleotides in
length, 5800 to 6000 nucleotides in length, 6000 to 6400
nucleotides in length, 6200 to 6800 nucleotides in length, 6600 to
7000 nucleotides in length, 7000 to 7200 nucleotides in lengths,
7200 to 7500 nucleotides in length, or 7500 nucleotides in length.
In some embodiments, the nucleotide sequence encodes a peptide or
polypeptide that is 5 to 10 amino acids in length, 10 to 25 amino
acids in length, 25 to 50 amino acids in length, 50 to 100 amino
acids in length, 100 to 150 amino acids in length, 150 to 200 amino
acids in length, 200 to 250 amino acids in length, 250 to 300 amino
acids in length, 300 to 400 amino acids in length, 400 to 500 amino
acids in length, 500 to 750 amino acids in length, 750 to 1000
amino acids in length, 1000 to 1250 amino acids in length, 1250 to
1500 amino acids in length, 1500 to 1750 amino acids in length,
1750 to 2000 amino acids in length, 2000 to 2500 amino acids in
length, or more than 2500 or more amino acids in length. In some
embodiments, the nucleotide sequence encodes a polypeptide that
does not exceed 2500 amino acids in length. In specific embodiments
the nucleotide sequence does not contain a stop codon. In certain
embodiments, the nucleotide sequence is codon-optimized. In certain
embodiments the nucleotide composition, nucleotide pair composition
or both can be optimized. Techniques for such optimizations are
known in the art and can be applied to optimize a nucleotide
sequence of a tumor antigen or tumor associated antigen.
[0233] In certain embodiments, the arenavirus genomic segment, the
arenavirus particle or the tri-segmented arenavirus particle can
comprise one or more nucleotide sequences encoding tumor antigens,
tumor associated antigens, or antigenic fragments thereof. In other
embodiments, the arenavirus genomic segment, the arenavirus
particle or the tri-segmented arenavirus particle can comprise at
least one nucleotide sequence encoding a tumor antigen, tumor
associated antigen, or antigenic fragment thereof, at least two
nucleotide sequences encoding tumor antigens, tumor associated
antigens, or antigenic fragments thereof, at least three nucleotide
sequences encoding tumor antigens, tumor associated antigens, or
antigenic fragments thereof, or more nucleotide sequences encoding
tumor antigens, tumor associated antigens, or antigenic fragments
thereof.
[0234] Nucleic acid sequences encoding a tumor antigen, tumor
associated antigen, or antigenic fragment thereof can be introduced
in the genome of a bi-segmented infectious, replication-deficient
arenavirus by substitution of the nucleic acid sequence of the ORF
of glycoprotein GP, the matrix protein Z, the nucleoprotein NP, or
the polymerase protein L. In other embodiments, the nucleic acid
sequence encoding the a tumor antigen, tumor associated antigen, or
antigenic fragment thereof is fused to the ORF of glycoprotein GP,
the matrix protein Z, the nucleoprotein NP, or the polymerase
protein L. The nucleotide sequence encoding the a tumor antigen,
tumor associated antigen, or antigenic fragment thereof, once
inserted into the genome of a bi-segmented infectious,
replication-deficient arenavirus, can be transcribed and/or
expressed under control of the four arenavirus promoters (5' UTR
and 3' UTR of the S segment, and 5' UTR and 3' UTR of the L
segment), as well as ribonucleic acids that can be inserted with
regulatory elements that can be read by the viral RNA-dependent RNA
polymerase, cellular RNA polymerase I, RNA polymerase II or RNA
polymerase III, such as duplications of viral promoter sequences
that are naturally found in the viral UTRs, the 28S ribosomal RNA
promoter, the beta-actin promoter or the 5S ribosomal RNA promoter,
respectively. The nucleic acids encoding the a tumor antigen, tumor
associated antigen, or antigenic fragment thereof can be
transcribed and/or expressed either by themselves or as
read-through by fusion to arenavirus ORFs and genes, respectively,
and/or in combination with one or more, e.g., two, three or four,
internal ribosome entry sites.
[0235] In certain embodiments, an arenavirus particle comprising a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen or antigenic fragment thereof as provided herein further
comprises at least one nucleotide sequence encoding at least one
immunomodulatory peptide, polypeptide or protein. In certain
embodiments, the immunomodulatory peptide, polypeptide or protein
is Calreticulin (CRT), or a fragment thereof; Ubiquitin or a
fragment thereof; Granulocyte-Macrophage Colony-Stimulating Factor
(GM-CSF), or a fragment thereof; Invariant chain (CD74) or an
antigenic fragment thereof; Mycobacterium tuberculosis Heat shock
protein 70 or an antigenic fragment thereof; Herpes simplex virus 1
protein VP22 or an antigenic fragment thereof; CD40 ligand or an
antigenic fragment thereof; or Fms-related tyrosine kinase 3 (Flt3)
ligand or an antigenic fragment thereof.
[0236] In certain embodiments, an arenavirus particle provided
herein comprises a genomic segment that a) has a removal or
functional inactivation of an ORF that is present in the wild type
form of the genomic segment; and b) encodes (either in sense or
antisense): (i) one or more tumor antigen, tumor associated antigen
or an antigenic fragment thereof provided herein, and (ii) one or
more immunomodulatory peptide, polypeptide or protein provided
herein.
[0237] In certain embodiments, the nucleotide sequence encoding the
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein, and the nucleotide sequence encoding the
immunomodulatory peptide, polypeptide or protein provided herein,
are on the same position of the viral genome. In certain
embodiments, the nucleotide sequence encoding the tumor antigen,
tumor associated antigen or an antigenic fragment thereof provided
herein, and the nucleotide sequence encoding the immunomodulatory
peptide, polypeptide or protein provided herein, are on different
positions of the viral genome.
[0238] In certain embodiments, the nucleotide sequence encoding the
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein, and the nucleotide sequence encoding the
immunomodulatory peptide, polypeptide or protein provided herein,
are separated via a spacer sequence. In certain embodiments, the
sequence encoding the tumor antigen, tumor associated antigen or an
antigenic fragment thereof provided herein, and the nucleotide
sequence encoding the immunomodulatory peptide, polypeptide or
protein provided herein, are separated by an internal ribosome
entry site, or a sequence encoding a protease cleavage site. In
certain embodiments, the nucleotide sequence encoding the tumor
antigen, tumor associated antigen or an antigenic fragment thereof
provided herein, and the nucleotide sequence encoding the
immunomodulatory peptide, polypeptide or protein provided herein,
are separated by a nucleotide sequence encoding a linker or a
self-cleaving peptide. Any linker peptide or self-cleaving peptide
known to the skilled artisan can be used with the compositions and
methods provided herein. A non-limiting example of a peptide linker
is GSG. Non-limiting examples of a self-cleaving peptide are
Porcine teschovirus-1 2A peptide, Thoseaasignavirus 2A peptide, or
Foot-and-mouth disease virus 2A peptide.
[0239] In certain embodiments, the tumor antigen, tumor associated
antigen or an antigenic fragment thereof provided herein, and the
immunomodulatory peptide, polypeptide or protein provided herein,
are directly fused together. In certain embodiments, the tumor
antigen, tumor associated antigen or an antigenic fragment thereof
provided herein, and the immunomodulatory peptide, polypeptide or
protein provided herein, are fused together via a peptide linker.
In certain embodiments, the tumor antigen, tumor associated antigen
or an antigenic fragment thereof provided herein, and the
immunomodulatory peptide, polypeptide or protein provided herein
are separated from each other via a self-cleaving peptide. A
non-limiting example of a peptide linker is GSG. Non-limiting
examples of a self-cleaving peptide are Porcine teschovirus-1 2A
peptide, Thoseaasignavirus 2A peptide, or Foot-and-mouth disease
virus 2A peptide.
[0240] In certain embodiments, the tumor antigen, tumor associated
antigen or an antigenic fragment thereof provided herein, and the
immunomodulatory peptide, polypeptide or protein provided herein
are expressed on the same arenavirus particle. In certain
embodiments, the tumor antigen, tumor associated antigen or an
antigenic fragment thereof provided herein, and the
immunomodulatory peptide, polypeptide or protein provided herein
are expressed on different arenavirus particles. In certain
embodiments, the tumor antigen, tumor associated antigen or an
antigenic fragment thereof provided herein, and the
immunomodulatory peptide, polypeptide or protein provided herein
are expressed on different viruses of the same strain. In certain
embodiments, the tumor antigen, tumor associated antigen or an
antigenic fragment thereof provided herein, and the
immunomodulatory peptide, polypeptide or protein provided herein
are expressed on different viruses of different strains.
[0241] In certain embodiments, an arenavirus particle generated to
encode one or more tumor antigens, tumor associated antigens or
antigenic fragments thereof comprises one or more nucleotide
sequences encoding tumor antigens, tumor associated antigens or
antigenic fragments thereof provided herein. In specific
embodiments the tumor antigens, tumor associated antigens or
antigenic fragments thereof provided herein are separated by
various one or more linkers, spacers, or cleavage sites as
described herein.
5.5 Generation of an Arenavirus Particle and a Tri-Segmented
Arenavirus Particle
[0242] Generally, arenavirus particles can be recombinantly
produced by standard reverse genetic techniques as described for
LCMV (see Flatz et al., 2006, Proc Natl Acad Sci USA 103:4663-4668;
Sanchez et al., 2006, Virology 350:370; Ortiz-Riano et al., 2013, J
Gen Virol. 94:1175-88, which are incorporated by reference herein).
To generate the arenavirus particles provided herein, these
techniques can be applied as described below. The genome of the
viruses can be modified as described herein.
5.5.1 Non-Natural Position Open Reading Frame
[0243] The generation of an arenavirus particle comprising a
genomic segment that has been engineered to carry a viral ORF in a
position other than the wild-type position of the ORF and a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen or antigenic fragment thereof can be recombinantly produced
by any reverse genetic techniques known to one skilled in the
art.
[0244] (i) Infectious and Replication Competent Arenavirus
Particle
[0245] In certain embodiments, the method of generating the
arenavirus particle comprises (i) transfecting into a host cell the
cDNA of the first arenavirus genomic segment; (ii) transfecting
into a host cell the cDNA of the second arenavirus genomic segment;
(iii) transfecting into a host cell plasmids expressing the
arenavirus' minimal trans-acting factors NP and L; (iv) maintaining
the host cell under conditions suitable for virus formation; and
(v) harvesting the arenavirus particle. In certain more specific
embodiments, the cDNA is comprised in a plasmid.
[0246] Once generated from cDNA, arenavirus particles (e.g.,
infectious and replication competent) can be propagated. In certain
embodiments, the arenavirus particle can be propagated in any host
cell that allows the virus to grow to titers that permit the uses
of the virus as described herein. In one embodiment, the host cell
allows the arenavirus particle to grow to titers comparable to
those determined for the corresponding wild-type.
[0247] In certain embodiments, the arenavirus particle may be
propagated in host cells. Specific examples of host cells that can
be used include BHK-21, HEK 293, VERO or other. In a specific
embodiment, the arenavirus particle may be propagated in a cell
line.
[0248] In certain embodiments, the host cells are kept in culture
and are transfected with one or more plasmid(s). The plasmid(s)
express the arenavirus genomic segment(s) to be generated under
control of one or more expression cassettes suitable for expression
in mammalian cells, e.g., consisting of a polymerase I promoter and
terminator.
[0249] Plasmids that can be used for the generation of the
arenavirus particle can include: i) a plasmid encoding the S
genomic segment e.g., pol-I S, ii) a plasmid encoding the L genomic
segment e.g., pol-I L. In certain embodiments, the plasmid encoding
an arenavirus polymerase that direct intracellular synthesis of the
viral L and S segments can be incorporated into the transfection
mixture. For example, a plasmid encoding the L protein and/or a
plasmid encoding NP (pC-L and pC-NP, respectively) can be present.
The L protein and NP are the minimal trans-acting factors necessary
for viral RNA transcription and replication. Alternatively,
intracellular synthesis of viral L and S segments, together with NP
and L protein can be performed using an expression cassette with
pol-I and pol-II promoters reading from opposite sides into the L
and S segment cDNAs of two separate plasmids, respectively.
[0250] In certain embodiments, the arenavirus genomic segments are
under the control of a promoter. Typically, RNA polymerase I-driven
expression cassettes, RNA polymerase II-driven cassettes or T7
bacteriophage RNA polymerase driven cassettes can be used. In
certain embodiments, the plasmid(s) encoding the arenavirus genomic
segments can be the same, i.e., the genome sequence and transacting
factors can be transcribed by a promoter from one plasmid. Specific
examples of promoters include an RNA polymerase I promoter, an RNA
polymerase II promoter, an RNA polymerase III promoter, a T7
promoter, an SP6 promoter or a T3 promoter.
[0251] In addition, the plasmid(s) can feature a mammalian
selection marker, e.g., puromycin resistance, under control of an
expression cassette suitable for gene expression in mammalian
cells, e.g., polymerase II expression cassette as above, or the
viral gene transcript(s) are followed by an internal ribosome entry
site, such as the one of encephalomyocarditis virus, followed by
the mammalian resistance marker. For production in E. coli, the
plasmid additionally features a bacterial selection marker, such as
an ampicillin resistance cassette.
[0252] Transfection of a host cell with a plasmid(s) can be
performed using any of the commonly used strategies such as
calcium-phosphate, liposome-based protocols or electroporation. A
few days later the suitable selection agent, e.g., puromycin, is
added in titrated concentrations. Surviving clones are isolated and
subcloned following standard procedures, and high-expressing clones
are identified using Western blot or flow cytometry procedures with
antibodies directed against the viral protein(s) of interest.
[0253] For recovering the arenavirus particle described herein, the
following procedures are envisaged. First day: cells, typically 80%
confluent in M6-well plates, are transfected with a mixture of the
plasmids, as described above. For this one can exploit any commonly
used strategies such as calcium-phosphate, liposome-based protocols
or electroporation.
[0254] 3-5 days later: The cultured supernatant (arenavirus vector
preparation) is harvested, aliquoted and stored at 4.degree. C.,
-20.degree. C., or -80.degree. C., depending on how long the
arenavirus vector should be stored prior use. The arenavirus vector
preparation's infectious titer is assessed by an immunofocus assay.
Alternatively, the transfected cells and supernatant may be
passaged to a larger vessel (e.g., a T75 tissue culture flask) on
day 3-5 after transfection, and culture supernatant is harvested up
to five days after passage.
[0255] The present application furthermore relates to expression of
a heterologous ORF, wherein a plasmid encoding the genomic segment
is modified to incorporated a heterologous ORF. The heterologous
ORF can be incorporated into the plasmid using restriction enzymes.
[0256] (ii) Infectious, Replication-Defective Arenavirus
Particle
[0257] Infectious, replication-defective arenavirus particles can
be rescued as described above. However, once generated from cDNA,
the infectious, replication-deficient arenaviruses provided herein
can be propagated in complementing cells. Complementing cells are
cells that provide the functionality that has been eliminated from
the replication-deficient arenavirus by modification of its genome
(e.g., if the ORF encoding the GP protein is deleted or
functionally inactivated, a complementing cell does provide the GP
protein).
[0258] Owing to the removal or functional inactivation of one or
more of the ORFs in arenavirus vectors (here deletion of the
glycoprotein, GP, will be taken as an example), arenavirus vectors
can be generated and expanded in cells providing in trans the
deleted viral gene(s), e.g., the GP in the present example. Such a
complementing cell line, henceforth referred to as C-cells, is
generated by transfecting a cell line such as BHK-21, HEK 293, VERO
or other with one or more plasmid(s) for expression of the viral
gene(s) of interest (complementation plasmid, referred to as
C-plasmid). The C-plasmid(s) express the viral gene(s) deleted in
the arenavirus vector to be generated under control of one or more
expression cassettes suitable for expression in mammalian cells,
e.g., a mammalian polymerase II promoter such as the EF1alpha
promoter with a polyadenylation signal. In addition, the
complementation plasmid features a mammalian selection marker,
e.g., puromycin resistance, under control of an expression cassette
suitable for gene expression in mammalian cells, e.g., polymerase
II expression cassette as above, or the viral gene transcript(s)
are followed by an internal ribosome entry site, such as the one of
encephalomyocarditis virus, followed by the mammalian resistance
marker. For production in E. coli, the plasmid additionally
features a bacterial selection marker, such as an ampicillin
resistance cassette.
[0259] Cells that can be used, e.g., BHK-21, HEK 293, MC57G or
other, are kept in culture and are transfected with the
complementation plasmid(s) using any of the commonly used
strategies such as calcium-phosphate, liposome-based protocols or
electroporation. A few days later the suitable selection agent,
e.g., puromycin, is added in titrated concentrations. Surviving
clones are isolated and subcloned following standard procedures,
and high-expressing C-cell clones are identified using Western blot
or flow cytometry procedures with antibodies directed against the
viral protein(s) of interest. As an alternative to the use of
stably transfected C-cells transient transfection of normal cells
can complement the missing viral gene(s) in each of the steps where
C-cells will be used below. In addition, a helper virus can be used
to provide the missing functionality in trans.
[0260] Plasmids can be of two types: i) two plasmids, referred to
as TF-plasmids for expressing intracellularly in C-cells the
minimal transacting factors of the arenavirus, is derived from
e.g., NP and L proteins of LCMV in the present example; and ii)
plasmids, referred to as GS-plasmids, for expressing
intracellularly in C-cells the arenavirus vector genome segments,
e.g., the segments with designed modifications. TF-plasmids express
the NP and L proteins of the respective arenavirus vector under
control of an expression cassette suitable for protein expression
in mammalian cells, typically e.g., a mammalian polymerase II
promoter such as the CMV or EF 1 alpha promoter, either one of them
preferentially in combination with a polyadenylation signal.
GS-plasmids express the small (S) and the large (L) genome segments
of the vector. Typically, polymerase I-driven expression cassettes
or T7 bacteriophage RNA polymerase (T7-) driven expression
cassettes can be used, the latter preferentially with a 3'-terminal
ribozyme for processing of the primary transcript to yield the
correct end. In the case of using a T7-based system, expression of
T7 in C-cells must be provided by either including in the recovery
process an additional expression plasmid, constructed analogously
to TF-plasmids, providing T7, or C-cells are constructed to
additionally express T7 in a stable manner. In certain embodiments,
TF and GS plasmids can be the same, i.e., the genome sequence and
transacting factors can be transcribed by T7, polI and polII
promoters from one plasmid.
[0261] For recovering of the arenavirus vector, the following
procedures can be used. First day: C-cells, typically 80% confluent
in M6-well plates, are transfected with a mixture of the two
TF-plasmids plus the two GS-plasmids. In certain embodiments, the
TF and GS plasmids can be the same, i.e., the genome sequence and
transacting factors can be transcribed by T7, polI and polII
promoters from one plasmid. For this one can exploit any of the
commonly used strategies such as calcium-phosphate, liposome-based
protocols or electroporation.
[0262] 3-5 days later: The culture supernatant (arenavirus vector
preparation) is harvested, aliquoted and stored at 4.degree. C.,
-20.degree. C. or -80.degree. C. depending on how long the
arenavirus vector should be stored prior to use. Then the
arenavirus vector preparation's infectious titer is assessed by an
immunofocus assay on C-cells. Alternatively, the transfected cells
and supernatant may be passaged to a larger vessel (e.g., a T75
tissue culture flask) on day 3-5 after transfection, and culture
supernatant is harvested up to five days after passage.
[0263] The invention furthermore relates to expression of a antigen
in a cell culture wherein the cell culture is infected with an
infectious, replication-deficient arenavirus expressing an antigen.
When used for expression of a antigen in cultured cells, the
following two procedures can be used:
[0264] i) The cell type of interest is infected with the arenavirus
vector preparation described herein at a multiplicity of infection
(MOI) of one or more, e.g., two, three or four, resulting in
production of the antigen in all cells already shortly after
infection.
[0265] ii) Alternatively, a lower MOI can be used and individual
cell clones can be selected for their level of virally driven
antigen expression. Subsequently individual clones can be expanded
infinitely owing to the non-cytolytic nature of arenavirus vectors.
Irrespective of the approach, the antigen can subsequently be
collected (and purified) either from the culture supernatant or
from the cells themselves, depending on the properties of the
antigen produced. However, the invention is not limited to these
two strategies, and other ways of driving expression of antigen
using infectious, replication-deficient arenaviruses as vectors may
be considered.
5.5.2 Generation of a Tri-Segmented Arenavirus Particle
[0266] A tri-segmented arenavirus particle comprising a nucleotide
sequence encoding a tumor antigen, tumor associated antigen or
antigenic fragment thereof can be recombinantly produced by reverse
genetic techniques known in the art, for example as described by
Emonet et al., 2008, PNAS, 106(9):3473-3478; Popkin et al., 2011,
J. Virol., 85 (15):7928-7932, which are incorporated by reference
herein. The generation of the tri-segmented arenavirus particle
provided herein can be modified as described in Section 5.2.
[0267] (i) Infectious and Replication Competent Tri-Segmented
Arenavirus Particle
[0268] In certain embodiments, the method of generating the
tri-segmented arenavirus particle comprises (i) transfecting into a
host cell the cDNAs of the one L segment and two S segments or two
L segments and one S segment; (ii) transfecting into a host cell
plasmids expressing the arenavirus' minimal trans-acting factors NP
and L; (iii) maintaining the host cell under conditions suitable
for virus formation; and (iv) harvesting the arenavirus
particle.
[0269] Once generated from cDNA, the tri-segmented arenavirus
particle (i.e., infectious and replication competent) can be
propagated. In certain embodiments tri-segmented arenavirus
particle can be propagated in any host cell that allows the virus
to grow to titers that permit the uses of the virus as described
herein. In one embodiment, the host cell allows the tri-segmented
arenavirus particle to grow to titers comparable to those
determined for the corresponding wild-type.
[0270] In certain embodiments, the tri-segmented arenavirus
particle may be propagated in host cells. Specific examples of host
cells that can be used include BHK-21, HEK 293, VERO or other. In a
specific embodiment, the tri-segmented arenavirus particle may be
propagated in a cell line.
[0271] In certain embodiments, the host cells are kept in culture
and are transfected with one or more plasmid(s). The plasmid(s)
express the arenavirus genomic segment(s) to be generated under
control of one or more expression cassettes suitable for expression
in mammalian cells, e.g., consisting of a polymerase I promoter and
terminator.
[0272] In specific embodiments, the host cells are kept in culture
and are transfected with one or more plasmid(s). The plasmid(s)
express the viral gene(s) to be generated under control of one or
more expression cassettes suitable for expression in mammalian
cells, e.g., consisting of a polymerase I promoter and
terminator.
[0273] Plasmids that can be used for generating the tri-segmented
arenavirus comprising one L segment and two S segments can include:
i) two plasmids each encoding the S genome segment e.g., pol-I S,
ii) a plasmid encoding the L genome segment e.g., pol-I L. Plasmids
needed for the tri-segmented arenavirus comprising two L segments
and one S segments are: i) two plasmids each encoding the L genome
segment e.g., pol-L, ii) a plasmid encoding the S genome segment
e.g., pol-I S.
[0274] In certain embodiments, plasmids encoding an arenavirus
polymerase that direct intracellular synthesis of the viral L and S
segments can be incorporated into the transfection mixture. For
example, a plasmid encoding the L protein and a plasmid encoding NP
(pC-L and pC-NP, respectively). The L protein and NP are the
minimal trans-acting factors necessary for viral RNA transcription
and replication. Alternatively, intracellular synthesis of viral L
and S segments, together with NP and L protein can be performed
using an expression cassette with pol-I and pol-II promoters
reading from opposite sides into the L and S segment cDNAs of two
separate plasmids, respectively.
[0275] In addition, the plasmid(s) features a mammalian selection
marker, e.g., puromycin resistance, under control of an expression
cassette suitable for gene expression in mammalian cells, e.g.,
polymerase II expression cassette as above, or the viral gene
transcript(s) are followed by an internal ribosome entry site, such
as the one of encephalomyocarditis virus, followed by the mammalian
resistance marker. For production in E. coli, the plasmid
additionally features a bacterial selection marker, such as an
ampicillin resistance cassette.
[0276] Transfection of BHK-21 cells with a plasmid(s) can be
performed using any of the commonly used strategies such as
calcium-phosphate, liposome-based protocols or electroporation. A
few days later the suitable selection agent, e.g., puromycin, is
added in titrated concentrations. Surviving clones are isolated and
subcloned following standard procedures, and high-expressing clones
are identified using Western blot or flow cytometry procedures with
antibodies directed against the viral protein(s) of interest.
[0277] Typically, RNA polymerase I-driven expression cassettes, RNA
polymerase II-driven cassettes or T7 bacteriophage RNA polymerase
driven cassettes can be used, the latter preferentially with a
3'-terminal ribozyme for processing of the primary transcript to
yield the correct end. In certain embodiments, the plasmids
encoding the arenavirus genomic segments can be the same, i.e., the
genome sequence and transacting factors can be transcribed by T7,
polI and polII promoters from one plasmid.
[0278] For recovering the arenavirus the tri-segmented arenavirus
vector, the following procedures are envisaged. First day: cells,
typically 80% confluent in M6-well plates, are transfected with a
mixture of the plasmids, as described above. For this one can
exploit any commonly used strategies such as calcium-phosphate,
liposome-based protocols or electroporation.
[0279] 3-5 days later: The cultured supernatant (arenavirus vector
preparation) is harvested, aliquoted and stored at 4.degree. C.,
-20.degree. C., or -80.degree. C., depending on how long the
arenavirus vector should be stored prior use. The arenavirus vector
preparation's infectious titer is assessed by an immunofocus assay.
Alternatively, the transfected cells and supernatant may be
passaged to a larger vessel (e.g., a T75 tissue culture flask) on
day 3-5 after transfection, and culture supernatant is harvested up
to five days after passage.
[0280] In certain embodiments, expression of a nucleotide sequence
encoding a tumor antigen, tumor associated antigen or antigenic
fragment thereof is provided, wherein a plasmid encoding the
genomic segment is modified to incorporated a nucleotide sequence
encoding a tumor antigen, tumor associated antigen or antigenic
fragment thereof. The nucleotide sequence encoding a tumor antigen,
tumor associated antigen or antigenic fragment thereof can be
incorporated into the plasmid using restriction enzymes.
[0281] (ii) Infectious, Replication-Defective Tri-Segmented
Arenavirus Particle
[0282] Infectious, replication-defective tri-segmented arenavirus
particles can be rescued as described above. However, once
generated from cDNA, the infectious, replication-deficient
arenaviruses provided herein can be propagated in complementing
cells. Complementing cells are cells that provide the functionality
that has been eliminated from the replication-deficient arenavirus
by modification of its genome (e.g., if the ORF encoding the GP
protein is deleted or functionally inactivated, a complementing
cell does provide the GP protein).
[0283] Owing to the removal or functional inactivation of one or
more of the ORFs in arenavirus vectors (here deletion of the
glycoprotein, GP, will be taken as an example), arenavirus vectors
can be generated and expanded in cells providing in trans the
deleted viral gene(s), e.g., the GP in the present example. Such a
complementing cell line, henceforth referred to as C-cells, is
generated by transfecting a mammalian cell line such as BHK-21, HEK
293, VERO or other (here BHK-21 will be taken as an example) with
one or more plasmid(s) for expression of the viral gene(s) of
interest (complementation plasmid, referred to as C-plasmid). The
C-plasmid(s) express the viral gene(s) deleted in the arenavirus
vector to be generated under control of one or more expression
cassettes suitable for expression in mammalian cells, e.g., a
mammalian polymerase II promoter such as the CMV or EF1alpha
promoter with a polyadenylation signal. In addition, the
complementation plasmid features a mammalian selection marker,
e.g., puromycin resistance, under control of an expression cassette
suitable for gene expression in mammalian cells, e.g., polymerase
II expression cassette as above, or the viral gene transcript(s)
are followed by an internal ribosome entry site, such as the one of
encephalomyocarditis virus, followed by the mammalian resistance
marker. For production in E. coli, the plasmid additionally
features a bacterial selection marker, such as an ampicillin
resistance cassette.
[0284] Cells that can be used, e.g., BHK-21, HEK 293, MC57G or
other, are kept in culture and are transfected with the
complementation plasmid(s) using any of the commonly used
strategies such as calcium-phosphate, liposome-based protocols or
electroporation. A few days later the suitable selection agent,
e.g., puromycin, is added in titrated concentrations. Surviving
clones are isolated and subcloned following standard procedures,
and high-expressing C-cell clones are identified using Western blot
or flow cytometry procedures with antibodies directed against the
viral protein(s) of interest. As an alternative to the use of
stably transfected C-cells transient transfection of normal cells
can complement the missing viral gene(s) in each of the steps where
C-cells will be used below. In addition, a helper virus can be used
to provide the missing functionality in trans.
[0285] Plasmids of two types can be used: i) two plasmids, referred
to as TF-plasmids for expressing intracellularly in C-cells the
minimal transacting factors of the arenavirus, is derived from
e.g., NP and L proteins of LCMV in the present example; and ii)
plasmids, referred to as GS-plasmids, for expressing
intracellularly in C-cells the arenavirus vector genome segments,
e.g., the segments with designed modifications. TF-plasmids express
the NP and L proteins of the respective arenavirus vector under
control of an expression cassette suitable for protein expression
in mammalian cells, typically e.g., a mammalian polymerase II
promoter such as the CMV or EF1alpha promoter, either one of them
preferentially in combination with a polyadenylation signal.
GS-plasmids express the small (S) and the large (L) genome segments
of the vector. Typically, polymerase I-driven expression cassettes
or T7 bacteriophage RNA polymerase (T7-) driven expression
cassettes can be used, the latter preferentially with a 3'-terminal
ribozyme for processing of the primary transcript to yield the
correct end. In the case of using a T7-based system, expression of
T7 in C-cells must be provided by either including in the recovery
process an additional expression plasmid, constructed analogously
to TF-plasmids, providing T7, or C-cells are constructed to
additionally express T7 in a stable manner. In certain embodiments,
TF and GS plasmids can be the same, i.e., the genome sequence and
transacting factors can be transcribed by T7, polI and polI
promoters from one plasmid.
[0286] For recovering of the arenavirus vector, the following
procedures can be used. First day: C-cells, typically 80% confluent
in M6-well plates, are transfected with a mixture of the two
TF-plasmids plus the two GS-plasmids. In certain embodiments, the
TF and GS plasmids can be the same, i.e., the genome sequence and
transacting factors can be transcribed by T7, polI and polII
promoters from one plasmid. For this one can exploit any of the
commonly used strategies such as calcium-phosphate, liposome-based
protocols or electroporation.
[0287] 3-5 days later: The culture supernatant (arenavirus vector
preparation) is harvested, aliquoted and stored at 4.degree. C.,
-20.degree. C. or -80.degree. C. depending on how long the
arenavirus vector should be stored prior to use. Then the
arenavirus vector preparation's infectious titer is assessed by an
immunofocus assay on C-cells. Alternatively, the transfected cells
and supernatant may be passaged to a larger vessel (e.g., a T75
tissue culture flask) on day 3-5 after transfection, and culture
supernatant is harvested up to five days after passage.
[0288] The invention furthermore relates to expression of an
antigen in a cell culture wherein the cell culture is infected with
an infectious, replication-deficient tri-segmented arenavirus
expressing a antigen. When used for expression of a CMV antigen in
cultured cells, the following two procedures can be used:
[0289] i) The cell type of interest is infected with the arenavirus
vector preparation described herein at a multiplicity of infection
(MOI) of one or more, e.g., two, three or four, resulting in
production of the tumor antigen, tumor associated antigen, or
antigenic fragment thereof in all cells already shortly after
infection.
[0290] ii) Alternatively, a lower MOI can be used and individual
cell clones can be selected for their level of virally driven
expression of a tumor antigen, tumor associated antigen or
antigenic fragment thereof. Subsequently individual clones can be
expanded infinitely owing to the non-cytolytic nature of arenavirus
vectors. Irrespective of the approach, the tumor antigen, tumor
associated antigen or antigenic fragment thereof can subsequently
be collected (and purified) either from the culture supernatant or
from the cells themselves, depending on the properties of the tumor
antigen, tumor associated antigen or antigenic fragment produced.
However, the invention is not limited to these two strategies, and
other ways of driving expression of tumor antigen, tumor associated
antigen or antigenic fragment thereof using infectious,
replication-deficient arenaviruses as vectors may be
considered.
5.6 Generation of Infectious, Replication-Deficient Arenavirus
Expressing a Tumor Antigen, Tumor Associated Antigen or Antigenic
Fragment Thereof
[0291] Generally, arenavirus particles can be recombinantly
produced by standard reverse genetic techniques as described for
LCMV (L. Flatz, A. Bergthaler, J. C. de la Torre, and D. D.
Pinschewer, Proc Natl Acad Sci USA 103:4663-4668, 2006; A. B.
Sanchez and J. C. de la Torre, Virology 350:370, 2006; E.
Ortiz-Riano, B. Y. Cheng, J. C. de la Torre, L. Martinez-Sobrido. J
Gen Virol. 94:1175-88, 2013). To generate infectious,
replication-deficient arenaviruses for use with the present
invention these techniques can be used, however, the genome of the
rescued virus is modified as described herein. These modifications
can be: i) one or more, e.g., two, three or four, of the four
arenavirus ORFs (glycoprotein (GP); nucleoprotein (NP); the matrix
protein Z; the RNA-dependent RNA polymerase L) are removed or
functionally inactivated to prevent formation of infectious
particles in normal cells albeit still allowing gene expression in
arenavirus vector-infected host cells; and ii) nucleotides encoding
for a tumor antigen, tumor associated antigen, or antigenic
fragment thereof can be introduced. Infectious,
replication-deficient viruses as described herein can be produced
as described in International Patent Application Publication No. WO
2009/083210 (application number PCT/EP2008/010994) and
International Patent Application Publication No. WO 2014/140301
(application number PCT/EP2014/055144), each of which is
incorporated by reference herein in its entirety.
[0292] Once generated from cDNA, the infectious,
replication-deficient arenaviruses provided herein can be
propagated in complementing cells. Complementing cells are cells
that provide the functionality that has been eliminated from the
replication-deficient arenavirus by modification of its genome
(e.g., if the ORF encoding the GP protein is deleted or
functionally inactivated, a complementing cell does provide the GP
protein).
[0293] Owing to the removal or functional inactivation of one or
more of the viral genes in arenavirus vectors (here deletion of the
glycoprotein, GP, will be taken as an example), arenavirus vectors
can be generated and expanded in cells providing in trans the
deleted viral gene(s), e.g., the GP in the present example. Such a
complementing cell line, henceforth referred to as C-cells, is
generated by transfecting a mammalian cell line such as BHK-21, HEK
293, VERO or other (here BHK-21 will be taken as an example) with
one or more plasmid(s) for expression of the viral gene(s) of
interest (complementation plasmid, referred to as C-plasmid). The
C-plasmid(s) express the viral gene(s) deleted in the arenavirus
vector to be generated under control of one or more expression
cassettes suitable for expression in mammalian cells, e.g., a
mammalian polymerase II promoter such as the CMV or EF1alpha
promoter with a polyadenylation signal. In addition, the
complementation plasmid features a mammalian selection marker,
e.g., puromycin resistance, under control of an expression cassette
suitable for gene expression in mammalian cells, e.g., polymerase
II expression cassette as above, or the viral gene transcript(s)
are followed by an internal ribosome entry site, such as the one of
encephalomyocarditis virus, followed by the mammalian resistance
marker. For production in E. coli, the plasmid additionally
features a bacterial selection marker, such as an ampicillin
resistance cassette.
[0294] Cells that can be used, e.g., BHK-21, HEK 293, MC57G or
other, are kept in culture and are transfected with the
complementation plasmid(s) using any of the commonly used
strategies such as calcium-phosphate, liposome-based protocols or
electroporation. A few days later the suitable selection agent,
e.g., puromycin, is added in titrated concentrations. Surviving
clones are isolated and subcloned following standard procedures,
and high-expressing C-cell clones are identified using Western blot
or flow cytometry procedures with antibodies directed against the
viral protein(s) of interest. As an alternative to the use of
stably transfected C-cells transient transfection of normal cells
can complement the missing viral gene(s) in each of the steps where
C-cells will be used below. In addition, a helper virus can be used
to provide the missing functionality in trans.
[0295] Plasmids that can be used can be of two types: i) Two
plasmids, referred to as TF-plasmids for expressing intracellularly
in C-cells the minimal transacting factors of the arenavirus, is
derived from e.g., NP and L proteins of LCMV in the present
example; and ii) Plasmids, referred to as GS-plasmids, for
expressing intracellularly in C-cells the arenavirus vector genome
segments, e.g., the segments with designed modifications.
TF-plasmids express the NP and L proteins of the respective
arenavirus vector under control of an expression cassette suitable
for protein expression in mammalian cells, typically e.g., a
mammalian polymerase II promoter such as the CMV or EF1alpha
promoter, either one of them preferentially in combination with a
polyadenylation signal. GS-plasmids express the small (S) and the
large (L) genome segments of the vector. Typically, polymerase
I-driven expression cassettes or T7 bacteriophage RNA polymerase
(T7-) driven expression cassettes can be used, the latter
preferentially with a 3'-terminal ribozyme for processing of the
primary transcript to yield the correct end. In the case of using a
T7-based system, expression of T7 in C-cells must be provided by
either including in the recovery process an additional expression
plasmid, constructed analogously to TF-plasmids, providing T7, or
C-cells are constructed to additionally express T7 in a stable
manner. In certain embodiments, TF and GS plasmids can be the same,
i.e. the genome sequence and transacting factors can be transcribed
by T7, polI and polII promoters from one plasmid.
[0296] For recovering of the arenavirus vector, the following
procedures can be used. First day: C-cells, typically 80% confluent
in M6-well plates, are transfected with a mixture of the two
TF-plasmids plus the two GS-plasmids. In certain embodiments, the
TF and GS plasmids can be the same, i.e. the genome sequence and
transacting factors can be transcribed by T7, polI and polII
promoters from one plasmid. For this one can exploit any of the
commonly used strategies such as calcium-phosphate, liposome-based
protocols or electroporation.
[0297] 3-5 days later: The culture supernatant (arenavirus vector
preparation) is harvested, aliquoted and stored at 4.degree. C.,
-20.degree. C. or -80.degree. C. depending on how long the
arenavirus vector should be stored prior to use. Then the
arenavirus vector preparation's infectious titer is assessed by an
immunofocus assay on C-cells.
[0298] The invention furthermore relates to expression of a tumor
antigen, tumor associated antigen, or antigenic fragment thereof in
a cell culture wherein the cell culture is infected with a
bi-segmented infectious, replication-deficient arenavirus
expressing a tumor antigen, tumor associated antigen, or antigenic
fragment thereof. When used for expression of a tumor antigen,
tumor associated antigen, or antigenic fragment thereof in cultured
cells, the following two procedures can be used:
[0299] i) The cell type of interest is infected with the arenavirus
vector preparation described herein at a multiplicity of infection
(MOI) of one or more, e.g., two, three or four, resulting in
production of the tumor antigen, tumor associated antigen, or
antigenic fragment thereof in all cells already shortly after
infection.
[0300] ii) Alternatively, a lower MOI can be used and individual
cell clones can be selected for their level of virally driven
expression of a tumor antigen, tumor associated antigen, or
antigenic fragment thereof. Subsequently individual clones can be
expanded infinitely owing to the non-cytolytic nature of arenavirus
vectors. Irrespective of the approach, the tumor antigen, tumor
associated antigen, or antigenic fragment thereof can subsequently
be collected (and purified) either from the culture supernatant or
from the cells themselves, depending on the properties of the tumor
antigen, tumor associated antigen, or antigenic fragment thereof
produced. However, the invention is not limited to these two
strategies, and other ways of driving expression of a tumor
antigen, tumor associated antigen, or antigenic fragment thereof
using infectious, replication-deficient arenaviruses as vectors may
be considered.
[0301] Alternatively, a rescue system consisting of three plasmids
can be used: (1) the first plasmid expresses the protein NP by
transcription via Polymerase II and subsequent translation in
transfected cells; (2) the second plasmid gives rise to the
(negative-stranded) L-Segment of the LCMV genome by transcription
via Polymerase I as well as the L protein by transcription via
Polymerase II from the same template in the opposite direction of
the Polymerase I promoter; (3) the third plasmid gives rise to the
S-segment of the LCMV genome (encoding the antigen coding sequence
instead of the LCMV glycoprotein) via transcription by Polymerase
I. 3 .mu.g of each plasmid is used for electroporation of C-cells,
followed by seeding of cells in 6-well plates and incubation at
37.degree. C. After incubation, cells and supernatant from
transfections are combined with freshly seeded C-cells, and vectors
are harvested and cleared from cells & debris at a defined
timepoint post infection. Once the vector has been generated, a
nucleic acid encoding a tumor antigen, tumor associated antigen, or
antigenic fragment thereof can be inserted into a plasmid from
which a genomic segment of an infectious replication-deficient
vector is transcribed by any technique known to the skilled
artisan.
[0302] Owing to the removal or functional inactivation of one or
more of the viral genes in arenavirus vectors (here deletion of the
glycoprotein, GP, will be taken as an example) arenavirus vectors
can be generated and expanded in cells that provide the deleted or
functionally inactivated viral gene(s) (e.g., the GP) in trans. The
resulting virus itself is infectious but is unable to produce
further infectious progeny particles in non-complementing cells due
to the lack of the deleted or functionally inactivated viral
gene(s) (e.g., the GP). The complementing cell can provide the
missing functionality either by stable transfection, transient
transfection, or by infection with a helper virus that expresses
the missing functionality.
[0303] In certain embodiments, the complementing cell provides the
viral gene that has been deleted or functionally inactivated from
the arenavirus vector genome. In a specific embodiment, the
complementing cell provides the viral gene from a viral strain that
is the same as the viral strain that was used to generate the
genome of the arenavirus vector. In another embodiment, the
complementing cell provides the viral gene from a viral strain that
is different from the viral strain that was used to generate the
genome of the arenavirus vector. For example, the viral gene
provided in the complementing cell is obtained from the MP strain
of LCMV. In another example, the viral gene provided in the
complementing cell is obtained from the Clone 13 strain of LCMV. In
another example, the viral gene provided in the complementing cell
is obtained from the WE strain of LCMV.
[0304] In a specific embodiment, the complementing cell provides
the GP of the MP strain of LCMV and the arenavirus vector comprises
an ORF of a tumor antigen, tumor associated antigen, or antigenic
fragment thereof as described herein in place of the ORF encoding
the GP protein. In an even more specific embodiment, the
complementing cell provides the GP of the MP strain of LCMV and the
arenavirus vector is obtained from LCMV Clone 13 and comprises an
ORF of a tumor antigen, tumor associated antigen, or antigenic
fragment thereof as described herein in place of the ORF encoding
the GP protein.
[0305] In a specific embodiment, the complementing cell provides
the GP of the Clone 13 strain of LCMV and the arenavirus vector
comprises an ORF of a tumor antigen, tumor associated antigen, or
antigenic fragment thereof as described herein in place of the ORF
encoding the GP protein. In an even more specific embodiment, the
complementing cell provides the GP of the Clone 13 strain of LCMV
and the arenavirus vector is obtained from LCMV MP strain and
comprises an ORF of a tumor antigen, tumor associated antigen, or
antigenic fragment thereof as described herein in place of the ORF
encoding the GP protein.
[0306] In a specific embodiment, the complementing cell provides
the GP of the WE strain of LCMV and the arenavirus vector comprises
an ORF of a tumor antigen, tumor associated antigen, or antigenic
fragment thereof as described herein in place of the ORF encoding
the GP protein. In an even more specific embodiment, the
complementing cell provides the GP of the WE strain of LCMV and the
arenavirus vector is obtained from LCMV Clone 13 and comprises an
ORF of a tumor antigen, tumor associated antigen, or antigenic
fragment thereof as described herein in place of the ORF encoding
the GP protein.
[0307] In a specific embodiment, the complementing cell provides
the GP of the WE strain of LCMV and the arenavirus vector comprises
an ORF of a tumor antigen, tumor associated antigen, or antigenic
fragment thereof as described herein in place of the ORF encoding
the GP protein. In an even more specific embodiment, the
complementing cell provides the GP of the WE strain of LCMV and the
arenavirus vector is obtained from LCMV MP strain and comprises an
ORF of a tumor antigen, tumor associated antigen, or antigenic
fragment thereof as described herein in place of the ORF encoding
the GP protein.
5.7 Nucleic Acids, Vector Systems and Cell Lines
[0308] In certain embodiments, provided herein are cDNAs comprising
or consisting of the arenavirus genomic segment or the
tri-segmented arenavirus particle as described herein.
5.7.1 Non-Natural Position Open Reading Frame
[0309] In one embodiment, provided herein are nucleic acids that
encode an arenavirus genomic segment as described in Section 5.1.
In more specific embodiments, provided herein is a DNA nucleotide
sequence or a set of DNA nucleotide sequences as set forth in Table
1. Host cells that comprise such nucleic acids are also provided
Section 5.1.
[0310] In specific embodiments, provided herein is a cDNA of the
arenavirus genomic segment engineered to carry an ORF in a position
other than the wild-type position of the ORF and a nucleotide
sequence encoding a tumor antigen, tumor associated antigen or
antigenic fragment thereof, wherein the arenavirus genomic segment
encodes a heterologous ORF as described in Section 5.1
[0311] In one embodiment, provided herein is a DNA expression
vector system that encodes the arenavirus genomic segment
engineered to carry an ORF in a position other than the wild-type
position of the ORF and a nucleotide sequence encoding a tumor
antigen, tumor associated antigen or antigenic fragment thereof.
Specifically, provided herein is a DNA expression vector system
wherein one or more vectors encodes two arenavirus genomic
segments, namely, an L segment and an S segment, of an arenavirus
particle described herein. Such a vector system can encode a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen or antigenic fragment thereof.
[0312] In another embodiment, provided herein is a cDNA of the
arenavirus S segment that has been engineered to carry an ORF in a
position other than the wild-type position and a nucleotide
sequence encoding a tumor antigen, tumor associated antigen or
antigenic fragment thereof that is part of or incorporated into a
DNA expression system. In other embodiments, provided herein is a
cDNA of the arenavirus L segment that has been engineered to carry
an ORF in a position other than the wild-type position and a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen or antigenic fragment thereof that is part of or
incorporated into a DNA expression system. In certain embodiments,
is a cDNA of the arenavirus genomic segment that has been
engineered to carry (i) an ORF in a position other than the
wild-type position of the ORF; and (ii) and ORF encoding GP, NP, Z
protein, or L protein has been removed and replaced with a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen or antigenic fragment thereof.
[0313] In certain embodiments, the cDNA provided herein can be
derived from a particular strain of LCMV. Strains of LCMV include
Clone 13, MP strain, Arm CA 1371, Arm E-250, WE, UBC, Traub,
Pasteur, 810885, CH-5692, Marseille #12, HP65-2009, 200501927,
810362, 811316, 810316, 810366, 20112714, Douglas, GR01, SN05, CABN
and their derivatives. In specific embodiments, the cDNA is derived
from LCMV Clone 13. In other specific embodiments, the cDNA is
derived from LCMV MP strain.
[0314] In certain embodiments, the vector generated to encode an
arenavirus particle or a tri-segmented arenavirus particle as
described herein may be based on a specific strain of LCMV. Strains
of LCMV include Clone 13, MP strain, Arm CA 1371, Arm E-250, WE,
UBC, Traub, Pasteur, 810885, CH-5692, Marseille #12, HP65-2009,
200501927, 810362, 811316, 810316, 810366, 20112714, Douglas, GR01,
SN05, CABN and their derivatives. In certain embodiments, an
arenavirus particle or a tri-segmented arenavirus particle as
described herein may be based on LCMV Clone 13. In other
embodiments, the vector generated to encode an arenavirus particle
or a tri-segmented arenavirus particle as described herein LCMV MP
strain.
[0315] In another embodiment, provided herein is a cell, wherein
the cell comprises a cDNA or a vector system described above in
this section. Cell lines derived from such cells, cultures
comprising such cells, methods of culturing such cells infected are
also provided herein. In certain embodiments, provided herein is a
cell, wherein the cell comprises a cDNA of the arenavirus genomic
segment that has been engineered to carry an ORF in a position
other than the wild-type position of the ORF and a nucleotide
sequence encoding a tumor antigen, tumor associated antigen or
antigenic fragment thereof. In some embodiments, the cell comprises
the S segment and/or the L segment.
5.7.2 Tri-Segmented Arenavirus Particle
[0316] In one embodiment, provided herein are nucleic acids that
encode a tri-segmented arenavirus particle as described in Section
5.2. In more specific embodiments, provided herein is a DNA
nucleotide sequence or a set of DNA nucleotide sequences, for
example, as set forth in Table 2 or Table 3. Host cells that
comprise such nucleic acids are also provided Section 5.2.
[0317] In specific embodiments, provided herein is a cDNA
consisting of a cDNA of the tri-segmented arenavirus particle that
has been engineered to carry an ORF in a position other than the
wild-type position of the ORF. In other embodiments, is a cDNA of
the tri-segmented arenavirus particle that has been engineered to
(i) carry an arenavirus ORF in a position other than the wild-type
position of the ORF; and (ii) wherein the tri-segmented arenavirus
particle encodes a heterologous ORF as described in Section
5.2.
[0318] In one embodiment, provided herein is a DNA expression
vector system that together encode the tri-segmented arenavirus
particle comprising a nucleotide sequence encoding a tumor antigen,
tumor associated antigen or antigenic fragment thereof as described
herein. Specifically, provided herein is a DNA expression vector
system wherein one or more vectors encode three arenavirus genomic
segments, namely, one L segment and two S segments or two L
segments and one S segment of a tri-segmented arenavirus particle
described herein. Such a vector system can encode a tumor antigen,
tumor associated antigen or antigenic fragment thereof.
[0319] In another embodiment, provided herein is a cDNA of the
arenavirus S segment(s) that has been engineered to carry an ORF in
a position other than the wild-type position and a nucleotide
sequence encoding a tumor antigen, tumor associated antigen or
antigenic fragment thereof that is part of or incorporated into a
DNA expression system. In other embodiments, a cDNA of the
arenavirus L segment(s) that has been engineered to carry an ORF in
a position other than the wild-type position and a nucleotide
sequence encoding a tumor antigen, tumor associated antigen or
antigenic fragment thereof that is part of or incorporated into a
DNA expression system. In certain embodiments, is a cDNA of the
tri-segmented arenavirus particle that has been engineered to carry
(i) an ORF in a position other than the wild-type position of the
ORF; and (ii) an ORF encoding GP, NP, Z protein, or L protein has
been removed and replaced with a nucleotide sequence encoding a
tumor antigen, tumor associated antigen or antigenic fragment
thereof.
[0320] In certain embodiments, the cDNA provided herein can be
derived from a particular strain of LCMV. Strains of LCMV include
Clone 13, MP strain, Arm CA 1371, Arm E-250, WE, UBC, Traub,
Pasteur, 810885, CH-5692, Marseille #12, HP65-2009, 200501927,
810362, 811316, 810316, 810366, 20112714, Douglas, GR01, SN05, CABN
and their derivatives. In specific embodiments, the cDNA is derived
from LCMV Clone 13. In other specific embodiments, the cDNA is
derived from LCMV MP strain.
[0321] In certain embodiments, the vector generated to encode an
arenavirus particle or a tri-segmented arenavirus particle as
described herein may be based on a specific strain of LCMV. Strains
of LCMV include Clone 13, MP strain, Arm CA 1371, Arm E-250, WE,
UBC, Traub, Pasteur, 810885, CH-5692, Marseille #12, HP65-2009,
200501927, 810362, 811316, 810316, 810366, 20112714, Douglas, GR01,
SN05, CABN and their derivatives. In certain embodiments, an
arenavirus particle or a tri-segmented arenavirus particle as
described herein may be based on LCMV Clone 13. In other
embodiments, the vector generated to encode an arenavirus particle
or a tri-segmented arenavirus particle as described herein LCMV MP
strain.
[0322] In another embodiment, provided herein is a cell, wherein
the cell comprises a cDNA or a vector system described above in
this section. Cell lines derived from such cells, cultures
comprising such cells, methods of culturing such cells infected are
also provided herein. In certain embodiments, provided herein is a
cell, wherein the cell comprises a cDNA of the tri-segmented
arenavirus particle. In some embodiments, the cell comprises the S
segment and/or the L segment.
5.7.3 Replication-Deficient Arenavirus
[0323] In one embodiment, described herein is a nucleic acid
sequence which is the cDNA of the large genomic segment (L segment)
of a bi-segmented infectious, replication-deficient arenavirus
described herein, in which one ORF of the genomic segment is
deleted or functionally inactivated, and the genomic segment
comprises a nucleotide sequence encoding a tumor antigen, tumor
associated antigen, or antigenic fragment thereof.
[0324] In one embodiment, described herein is a nucleic acid
sequence that encodes the short genomic segment (S segment) of a
bi-segmented infectious, replication-deficient arenavirus described
herein, in which one ORF of the genomic segment is deleted or
functionally inactivated and wherein the short genomic segment
comprises a nucleotide sequence encoding a tumor antigen, tumor
associated antigen, or antigenic fragment thereof. In another
embodiment, described herein is a nucleic acid sequence that
encodes the short genomic segment (S segment) of a bi-segmented
infectious, replication-deficient arenavirus described herein, in
which the ORF of the glycoprotein gene is deleted or functionally
inactivated and wherein the short genomic segment comprises a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen, or antigenic fragment thereof. In certain, more specific
embodiments, the tumor antigen, tumor associated antigen, or
antigenic fragment thereof is an antigen described in Section
5.4.
[0325] In certain embodiments, the nucleic acid sequences provided
herein can be derived from a particular strain of LCMV. Strains of
LCMV include Clone 13, MP strain, Arm CA 1371, Arm E-250, WE, UBC,
Traub, Pasteur, 810885, CH-5692, Marseille #12, HP65-2009,
200501927, 810362, 811316, 810316, 810366, 20112714, Douglas, GR01,
SN05, CABN and their derivatives. In specific embodiments, the
nucleic acid is derived from LCMV Clone 13. In other specific
embodiments, the nucleic acid is derived from LCMV MP strain.
[0326] In a more specific embodiment, provided herein is a nucleic
acid that comprises an arenavirus genomic segment; and (ii) a
nucleotide sequence encoding a tumor antigen, tumor associated
antigen, or antigenic fragment thereof.
[0327] In one embodiment, described herein is a vector system
comprising one or more vectors that together comprise the genome of
a bi-segmented infectious, replication-deficient arenavirus
particle described herein. Specifically, provided herein is a
vector system wherein the one or more vectors comprise two
arenavirus genomic segments, namely an L segment and an S segment,
of a bi-segmented infectious, replication-deficient arenavirus
described herein. Such a vector system can comprise (on one or more
separate DNA molecules): [0328] An arenavirus S genomic segment
that is modified such that an arenavirus particle carrying this
modified S genomic segment cannot produce infectious progeny virus
particles and an arenavirus L genomic segment that comprises a
nucleotide sequence encoding (in sense or antisense) a tumor
antigen, tumor associated antigen, or antigenic fragment thereof;
[0329] An arenavirus L genomic segment that is modified such that
an arenavirus particle carrying this modified L genomic segment
cannot produce infectious progeny virus particles and an arenavirus
S genomic segment that comprises a nucleotide sequence encoding (in
sense or antisense) a tumor antigen, tumor associated antigen, or
antigenic fragment thereof; [0330] An arenavirus S genomic segment
that is modified such that an arenavirus particle carrying this
modified S genomic segment cannot produce infectious progeny virus
particles and wherein the arenavirus S genomic segment comprises a
nucleotide sequence encoding (in sense or antisense) a tumor
antigen, tumor associated antigen, or antigenic fragment thereof
and comprising a wild type arenavirus L genomic segment; or [0331]
An arenavirus L genomic segment that is modified such that an
arenavirus particle carrying this modified L genomic segment cannot
produce infectious progeny virus particles and wherein the
arenavirus L genomic segment comprises a nucleotide sequence
encoding (in sense or antisense) a tumor antigen, tumor associated
antigen, or antigenic fragment thereof and comprising a wild type
arenavirus S genomic segment.
[0332] In certain embodiments, described herein is a nucleic acid
sequence comprising an arenavirus (e.g., LCMV) genomic segment in
which the ORF encoding the GP of the S genomic segment is
substituted with a nucleotide sequence encoding a tumor antigen,
tumor associated antigen, or antigenic fragment thereof, which is
selected from the group consisting of oncogenic viral antigens,
cancer-testis antigens, oncofetal antigens, tissue differentiation
antigens, mutant protein antigens, neoantigens, Adipophilin, AIM-2,
ALDH1AI, BCLX (L), BING-4, CALCA, CD45, CPSF, cyclin D1, DKKI, ENAH
(hMcna), Ga733 (EpCAM), EphA3, EZH2, FGF5, glypican-3,
G250/MN/CAIX, HER-2/neu, IDO1, IGF2B3, IL13Ralpha2, Intestinal
carboxyl esterase, alpha-foetoprotein, Kallikrein 4, KIF20A,
Lengsin, M-CSF, MCSP, mdm-2, Meloe, MMP-2, MMP-7, MUC1, MUC5AC, p53
(non-mutant), PAX5, PBF, PRAME, PSMA, RAGE, RAGE-1, RGS5, RhoC,
RNF43, RU2AS, secernin 1, SOX1O, STEAP1 (six-transmembrane
epithelial antigen of the prostate 1), survivinn, Telomerase, VEGF,
WT1, EGF-R, CEA, CD20, CD33, CD52, glycoprotein 100 (GP100 or gp
100 protein), MELANA/MART1, MART2, NY-ESO-1, p53, MAGE A1, MAGE A3,
MAGE-4, MAGE-5, MAGE-6, CDK4, alpha-actinin-4, ARTC1, BCR-ABL,
BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin,
Cdc27, CDK4, CDKN2A, CLPP, COA-1, dek-can fusion protein, EFTUD2,
Elongation factor 2, ETV6-AML, ETV6-AML1 fusion protein, FLT3-ITD,
FN1, GPNMB, LDLR-fucosyltransferaseAS fusion protein, NFYC, OGT,
OS-9, pml-RARalpha fusion protein, PRDX5, PTPRK, H-ras, K-ras
(V-Ki-ras2 Kirsten rat sarcoma viral oncogene), N-ras, RBAF600,
SIRT2, SNRPD1, SSX, SSX2, SYT-SSX1 or -SSX2 fusion protein,
TGF-betaRII, Triosephosphate isomerase, ormdm-2, LMP2, HPV E6/E7,
EGFRvIII (epidermal growth factor variant III), Idiotype, GD2,
ganglioside G2), Ras-mutant, p53 (mutant), Proteinase3 (PR1),
Tyrosinase, PSA, hTERT, Sarcoma translocation breakpoints, EphA2,
prostatic acid phosphatase PAP, nco-PAP, ML-IAP, AFP, ERG (TMPRSS2
ETS Fusion gene), NA17, PAX3, ALK, Androgen Receptor, Cyclin B1,
Polysialic acid, MYCN, TRP2, TRP2-Int2, GD3, Fucosyl GM1,
Mesothelin, PSCA, sLe(a), cyp1B1, PLAC1, GM3, BORIS, Tn, GLoboH,
NY-BR-1, SART3, STn, Carbonic Anhydrase IX, OY-TES1, Sperm protein
17, LCK, high molecular weight melanoma-associated antigen
(HMWMAA), AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4,
VEGFR2, MAD-CT-1, FAP, PDGFR-beta, MAD-CT-2, For-related antigen 1,
TRP1, CA-125, CA19-9, Calretinin, Epithelial membrane antigen
(EMA), Epithelial tumor antigen (ETA), CD19, CD34, CD99, CD117,
Chromogranin, Cytokeratin, Desmin, Glial fibrillary acidic protein
(GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45
antigen, Myo-D1, muscle-specific actin (MSA), neurofilament,
neuron-specific enolase (NSE), placental alkaline phosphatase,
synaptophysis, thyroglobulin, thyroid transcription factor-1,
dimeric form of the pyruvate kinase isoenzyme type M2 (tumor
M2-PK), BAGE BAGE-1, CAGE, CTAGE, FATE, GAGE, GAGE-1, GAGE-2,
GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, HCA661, HOM-TES-85, MAGEA,
MAGEB, MAGEC, NA88, NY-SAR-35, SPANXB1, SPA17, SSX, SYCP1, TPTE,
Carbohydrate/ganglioside GM2 (oncofetal antigen-immunogenic-1
OFA-I-1), GM3, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA 50, CAM
43, CEA, EBNA, EF2, Epstein-Barr virus antigen, HLA-A2, HLA-A11,
HSP70-2, KIAAO205, MUM-1, MUM-2, MUM-3, Myosin class I, GnTV,
Herv-K-mel, LAGE-1, LAGE-2, (sperm protein) SP17, SCP-1, P15(58),
Hom/Mel-40, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, TSP-180, P185erbB2,
p180erbB-3, c-met, nm-23H1, TAG-72, TAG-72-4, CA-72-4, CAM 17.1,
NuMa, 13-catenin, P16, TAGE, CT7, 43-9F, 5T4, 791Tgp72, 13HCG,
BCA225, BTAA, CD68\KP1, CO-029, HTgp-175, M344, MG7-Ag, MOV18,
NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TLP, TPS, CD22,
CD27, CD30, CD70, prostein, TARP (T cell receptor gamma alternate
reading frame protein), Trp-p8, integrin .alpha.v.beta.3 (CD61),
galactin, or Ral-B, CD123, CLL-1, CD38, CS-1, CD138, and ROR1.
[0333] In certain embodiments, described herein is a nucleic acid
sequence comprising an arenavirus (e.g., LCMV) genomic segment in
which the ORF encoding the GP of the S genomic segment is
substituted with a nucleotide sequence encoding one or more a tumor
antigen, tumor associated antigen, or antigenic fragment thereof
(e.g., one or more of those listed in the above paragraph).
[0334] In another embodiment, provided herein is a cell wherein the
cell comprises a nucleic acid or a vector system described above in
this section. Cell lines derived from such cells, cultures
comprising such cells, and methods of culturing such cells infected
with nucleic acids or vector systems are also provided herein. In
certain embodiments, provided herein is a cell wherein the cell
comprises a nucleic acid comprising the large genomic segment (L
segment) of a bi-segmented infectious, replication-deficient
arenavirus described herein, in which one ORF of the genomic
segment is deleted or functionally inactivated, and the genomic
segment comprises a nucleotide sequence encoding a tumor antigen,
tumor associated antigen, or antigenic fragment thereof.
[0335] In other embodiments, provided herein is a cell wherein the
cell comprises a nucleic acid sequence that comprises the short
genomic segment (S segment) of a bi-segmented infectious,
replication-deficient arenavirus described herein, in which one ORF
of the genomic segment is deleted or functionally inactivated and
wherein the short genomic segment comprises a nucleotide sequence
encoding a tumor antigen, tumor associated antigen, or antigenic
fragment thereof.
[0336] In another embodiment, provided herein is a cell wherein the
cell comprises two nucleic acids or vector systems described
herein. Cell lines derived from such cells, cultures comprising
such cells, and methods of culturing such cells infected with
nucleic acids or vector systems are also provided herein.
5.8 Methods of Use
[0337] Vaccines have been successful for preventing and/or treating
infectious diseases, such as those for polio virus and measles.
However, therapeutic immunization in the setting of established,
chronic disease, including cancer has been less successful. The
ability to generate an arenavirus particle that is used in
combination with an immune checkpoint inhibitor represents a new
novel vaccine strategy.
[0338] In certain embodiments, provided herein are methods of
treating a neoplastic disease in a subject. Such methods can
include administering to a subject in need thereof an arenavirus
particle provided herein and an immune checkpoint inhibitor
provided herein. In certain embodiments, the arenavirus particle
used in the methods is an infectious, replication-deficient
arenavirus particle provided herein. In certain embodiments, the
arenavirus particle used in the methods is a tri-segmented
arenavirus particle provided herein, including an infectious,
replication-deficient tri-segmented arenavirus particle or a
replication-competent tri-segmented arenavirus particle. Thus, in
certain embodiments, the arenavirus particle, including a
tri-segmented arenavirus particle, used in the methods is
replication-deficient, wherein the arenavirus particle is
engineered to contain a genome comprising: (1) a nucleotide
sequence encoding a tumor antigen, tumor associated antigen or an
antigenic fragment thereof; and (2) the ability to amplify and
express its genetic information in infected cells but unable to
produce further infectious progeny particles in non-complementing
cells. Moreover, in certain embodiments, a tri-segmented arenavirus
particle used in the methods is replication-competent, wherein the
tri-segmented arenavirus particle is engineered to contain a genome
comprising: (1) a nucleotide sequence encoding a tumor antigen,
tumor associated antigen or an antigenic fragment thereof; (2) the
ability to amplify and express its genetic information in infected
cells; and (3) the ability to produce further infectious progeny
particles in normal, not genetically engineered cells. In certain
embodiments, the arenavirus particle used in the methods is a
bi-segmented infectious, replication-deficient arenavirus particle.
Thus, in certain embodiments, the infectious, replication-deficient
arenavirus particle used in the methods is engineered to contain a
genome comprising: (1) a nucleotide sequence encoding a tumor
antigen, tumor associated antigen or an antigenic fragment thereof;
and (2) the ability to amplify and express its genetic information
in infected cells but unable to produce further infectious progeny
particles in non-complementing cells. In certain embodiments, the
immune checkpoint inhibitor inhibits, decreases or interferes with
the activity of a negative checkpoint regulator.
[0339] In one embodiment, provided herein are methods of treating a
neoplastic disease in a subject comprising administering to the
subject one or more arenavirus particles expressing a tumor
antigen, tumor associated antigen or an antigenic fragment thereof
as provided herein or a composition thereof, and an immune
checkpoint inhibitor provided herein. In a specific embodiment, a
method for treating a neoplastic disease described herein comprises
administering to a subject in need thereof a therapeutically
effective amount of one or more arenavirus particles expressing a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein or a composition thereof, and an immune
checkpoint inhibitor provided herein. The subject can be a mammal,
such as but not limited to a human, a mouse, a rat, a guinea pig, a
domesticated animal, such as, but not limited to, a cow, a horse, a
sheep, a pig, a goat, a cat, a dog, a hamster, a donkey. In a
specific embodiment, the subject is a human.
[0340] In another embodiment, provided herein are methods for
inducing an immune response against a neoplastic cell or tissue,
such as a cancer cell or tumor, in a subject comprising
administering to the subject an arenavirus particle expressing a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein, or a composition thereof, and an immune
checkpoint inhibitor provided herein.
[0341] In another embodiment, the subjects to whom an arenavirus
particle expressing a tumor antigen, tumor associated antigen or an
antigenic fragment thereof provided herein, or a composition
thereof, and an immune checkpoint inhibitor provided herein is
administered have, are susceptible to, or are at risk for a
neoplastic disease.
[0342] In another embodiment, the subjects to whom an arenavirus
particle expressing a tumor antigen, tumor associated antigen or an
antigenic fragment thereof provided herein, or a composition
thereof, and an immune checkpoint inhibitor provided herein is
administered have, are susceptible to, or are at risk for
development of a neoplastic disease, such as cancer, or exhibit a
pre-cancerous tissue lesion. In another specific embodiment, the
subjects to whom arenavirus particle expressing a tumor antigen,
tumor associated antigen or an antigenic fragment thereof provided
herein, or a composition thereof, and an immune checkpoint
inhibitor provided herein is administered are diagnosed with a
neoplastic disease, such as cancer, or exhibit a pre-cancerous
tissue lesion.
[0343] In another embodiment, the subjects to whom an arenavirus
particle expressing a tumor antigen, tumor associated antigen or an
antigenic fragment thereof provided herein, or a composition
thereof, and an immune checkpoint inhibitor provided herein is
administered are suffering from, are susceptible to, or are at risk
for, a neoplastic disease selected from, but not limited to, acute
lymphoblastic leukemia; acute lymphoblastic lymphoma; acute
lymphocytic leukaemia; acute myelogenous leukemia; acute myeloid
leukemia (adult/childhood); adrenocortical carcinoma; AIDS-related
cancers; AIDS-related lymphoma; anal cancer; appendix cancer;
astrocytomas; atypical teratoid/rhabdoid tumor; basal-cell
carcinoma; bile duct cancer, extrahepatic (cholangiocarcinoma);
bladder cancer; bone osteosarcoma/malignant fibrous histiocytoma;
brain cancer (adult/childhood); brain tumor, cerebellar astrocytoma
(adult/childhood); brain tumor, cerebral astrocytoma/malignant
glioma brain tumor; brain tumor, ependymoma; brain tumor,
medulloblastoma; brain tumor, supratentorial primitive
neuroectodermal tumors; brain tumor, visual pathway and
hypothalamic glioma; brainstem glioma; breast cancer; bronchial
adenomas/carcinoids; bronchial tumor; Burkitt lymphoma; cancer of
childhood; carcinoid gastrointestinal tumor; carcinoid tumor;
carcinoma of adult, unknown primary site; carcinoma of unknown
primary; central nervous system embryonal tumor; central nervous
system lymphoma, primary; cervical cancer; childhood adrenocortical
carcinoma; childhood cancers; childhood cerebral astrocytoma;
chordoma, childhood; chronic lymphocytic leukemia; chronic
myelogenous leukemia; chronic myeloid leukemia; chronic
myeloproliferative disorders; colon cancer; colorectal cancer;
craniopharyngioma; cutaneous T-cell lymphoma; desmoplastic small
round cell tumor; emphysema; endometrial cancer; ependymoblastoma;
ependymoma; esophageal cancer; ewing's sarcoma in the Ewing family
of tumors; extracranial germ cell tumor; extragonadal germ cell
tumor; extrahepatic bile duct cancer; gallbladder cancer; gastric
(stomach) cancer; gastric carcinoid; gastrointestinal carcinoid
tumor; gastrointestinal stromal tumor; germ cell tumor:
extracranial, extragonadal, or ovarian gestational trophoblastic
tumor; gestational trophoblastic tumor, unknown primary site;
glioma; glioma of the brain stem; glioma, childhood visual pathway
and hypothalamic; hairy cell leukemia; head and neck cancer; heart
cancer; hepatocellular (liver) cancer; hodgkin lymphoma;
hypopharyngeal cancer; hypothalamic and visual pathway glioma;
intraocular melanoma; islet cell carcinoma (endocrine pancreas);
Kaposi Sarcoma; kidney cancer (renal cell cancer); langerhans cell
histiocytosis; laryngeal cancer; lip and oral cavity cancer;
liposarcoma; liver cancer (primary); lung cancer, non-small cell;
lung cancer, small cell; lymphoma, primary central nervous system;
macroglobulinemia, Waldenstrom; male breast cancer; malignant
fibrous histiocytoma of bone/osteosarcoma; medulloblastoma;
medulloepithelioma; melanoma; melanoma, intraocular (eye); merkel
cell cancer; merkel cell skin carcinoma; mesothelioma;
mesothelioma, adult malignant; metastatic squamous neck cancer with
occult primary; mouth cancer; multiple endocrine neoplasia
syndrome; multiple myeloma/plasma cell neoplasm; mycosis fungoides,
myelodysplastic syndromes; myelodysplastic/myeloproliferative
diseases; myelogenous leukemia, chronic; myeloid leukemia, adult
acute; myeloid leukemia, childhood acute; myeloma, multiple (cancer
of the bone-marrow); myeloproliferative disorders, chronic; nasal
cavity and paranasal sinus cancer; nasopharyngeal carcinoma;
neuroblastoma, non-small cell lung cancer; non-hodgkin lymphoma;
oligodendroglioma; oral cancer; oral cavity cancer; oropharyngeal
cancer; osteosarcoma/malignant fibrous histiocytoma of bone;
ovarian cancer; ovarian epithelial cancer (surface
epithelial-stromal tumor); ovarian germ cell tumor; ovarian low
malignant potential tumor; pancreatic cancer; pancreatic cancer,
islet cell; papillomatosis; paranasal sinus and nasal cavity
cancer; parathyroid cancer; penile cancer; pharyngeal cancer;
pheochromocytoma; pineal astrocytoma; pineal germinoma; pineal
parenchymal tumors of intermediate differentiation; pineoblastoma
and supratentorial primitive neuroectodermal tumors; pituitary
tumor; pituitary adenoma; plasma cell neoplasia/multiple myeloma;
pleuropulmonary blastoma; primary central nervous system lymphoma;
prostate cancer; rectal cancer; renal cell carcinoma (kidney
cancer); renal pelvis and ureter, transitional cell cancer;
respiratory tract carcinoma involving the NUT gene on chromosome
15; retinoblastoma; rhabdomyosarcoma, childhood; salivary gland
cancer; sarcoma, Ewing family of tumors; Sezary syndrome; skin
cancer (melanoma); skin cancer (non-melanoma); small cell lung
cancer; small intestine cancer soft tissue sarcoma; soft tissue
sarcoma; spinal cord tumor; squamous cell carcinoma; squamous neck
cancer with occult primary, metastatic; stomach (gastric) cancer;
supratentorial primitive neuroectodermal tumor; T-cell lymphoma,
cutaneous (Mycosis Fungoides and Sezary syndrome); testicular
cancer; throat cancer; thymoma; thymoma and thymic carcinoma;
thyroid cancer; thyroid cancer, childhood; transitional cell cancer
of the renal pelvis and ureter; urethral cancer; uterine cancer,
endometrial; uterine sarcoma; vaginal cancer; vulvar cancer; and
Wilms Tumor.
[0344] In another embodiment, an arenavirus particle expressing a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein, or a composition thereof, and an immune
checkpoint inhibitor provided herein is administered to a subject
of any age group suffering from, are susceptible to, or are at risk
for a neoplastic disease. In a specific embodiment, an arenavirus
particle expressing a tumor antigen, tumor associated antigen or an
antigenic fragment thereof provided herein, or a composition
thereof, and an immune checkpoint inhibitor provided herein is
administered to a subject with a compromised immune system, a
pregnant subject, a subject undergoing an organ or bone marrow
transplant, a subject taking immunosuppressive drugs, a subject
undergoing hemodialysis, a subject who has cancer, or a subject who
is suffering from, are susceptible to, or are at risk for a
neoplastic disease. In a more specific embodiment, an arenavirus
particle expressing a tumor antigen, tumor associated antigen or an
antigenic fragment thereof provided herein, or a composition
thereof, and an immune checkpoint inhibitor provided herein is
administered to a subject who is a child of 0, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 years of age suffering
from, are susceptible to, or are at risk for a neoplastic disease.
In yet another specific embodiment, an arenavirus particle
expressing a tumor antigen, tumor associated antigen or an
antigenic fragment thereof provided herein, or a composition
thereof, and an immune checkpoint inhibitor provided herein is
administered to a subject who is an infant suffering from, is
susceptible to, or is at risk for a neoplastic disease. In yet
another specific embodiment, an arenavirus particle expressing a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein, or a composition thereof, and an immune
checkpoint inhibitor provided herein is administered to a subject
who is an infant of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12
months of age suffering from, is susceptible to, or is at risk for
a neoplastic disease. In yet another specific embodiment, an
arenavirus particle expressing a tumor antigen, tumor associated
antigen or an antigenic fragment thereof provided herein, or a
composition thereof, and an immune checkpoint inhibitor provided
herein is administered to an elderly subject who is suffering from,
is susceptible to, or is at risk for a neoplastic disease. In a
more specific embodiment, an arenavirus particle expressing a tumor
antigen, tumor associated antigen or an antigenic fragment thereof
provided herein, or a composition thereof, and an immune checkpoint
inhibitor provided herein is administered to a subject who is a
senior subject of 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 years of
age.
[0345] In another embodiment, an arenavirus particle expressing a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein, or a composition thereof, and an immune
checkpoint inhibitor provided herein is administered to subjects
with a heightened risk of cancer metastasis. In a specific
embodiment, an arenavirus particle expressing a tumor antigen,
tumor associated antigen or an antigenic fragment thereof provided
herein, or a composition thereof, and an immune checkpoint
inhibitor provided herein is administered to subjects in the
neonatal period with a neonatal and therefore immature immune
system.
[0346] In another embodiment, an arenavirus particle expressing a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein, or a composition thereof, and an immune
checkpoint inhibitor provided herein is administered to a subject
having grade 0 (i.e., in situ neoplasm), grade 1, grade 2, grade 3
or grade 4 cancer or a subcategory thereof, such as grade 3A, 3B,
or 3C, or an equivalent thereof.
[0347] In another embodiment, an arenavirus particle expressing a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein, or a composition thereof, and an immune
checkpoint inhibitor provided herein is administered to a subject
having cancer at a Tumor, Node, Metastasis (TNM) stage of any
combination selected from Tumor T1, T2, T3, and T4, and Node N0,
N1, N2, or N3, and Metastasis M0 and M1.
[0348] Successful treatment of a cancer patient can be assessed as
prolongation of expected survival, induction of an anti-tumor
immune response, or improvement of a particular characteristic of a
cancer. Examples of characteristics of a cancer that might be
improved include tumor size (e.g., T0, T is, or T1-4), state of
metastasis (e.g., M0, M1), number of observable tumors, node
involvement (e.g., N0, N1-4, Nx), grade (i.e., grades 1, 2, 3, or
4), stage (e.g., 0, I, II, III, or IV), presence or concentration
of certain markers on the cells or in bodily fluids (e.g., AFP,
B2M, beta-HCG, BTA, CA 15-3, CA 27.29, CA 125, CA 72.4, CA 19-9,
calcitonin, CEA, chromgrainin A, EGFR, hormone receptors, HER2,
HCG, immunoglobulins, NSE, NMP22, PSA, PAP, PSMA, S-100, TA-90, and
thyroglobulin), and/or associated pathologies (e.g., ascites or
edema) or symptoms (e.g., cachexia, fever, anorexia, or pain). The
improvement, if measureable by percent, can be at least 5, 10, 15,
20, 25, 30, 40, 50, 60, 70, 80, or 90% (e.g., survival, or volume
or linear dimensions of a tumor).
[0349] In another embodiment, an arenavirus particle expressing a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein, or a composition thereof, and an immune
checkpoint inhibitor provided herein is administered to a subject
having a dormant cancer (e.g., the subject is in remission). Thus,
provided herein is a method for preventing reactivation of a
cancer.
[0350] In another embodiment, an arenavirus particle expressing a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein, or a composition thereof, and an immune
checkpoint inhibitor provided herein is administered to a subject
having a recurrent a cancer.
[0351] In another embodiment, an arenavirus particle expressing a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein, or a composition thereof, and an immune
checkpoint inhibitor provided herein is administered to a subject
with a genetic predisposition for a cancer. In another embodiment,
an arenavirus particle expressing a tumor antigen, tumor associated
antigen or an antigenic fragment thereof provided herein, or a
composition thereof, and an immune checkpoint inhibitor provided
herein is administered to a subject with risk factors. Exemplary
risk factors include, aging, tobacco, sun exposure, radiation
exposure, chemical exposure, family history, alcohol, poor diet,
lack of physical activity, or being overweight.
[0352] In another embodiment, an arenavirus particle expressing a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein, or a composition thereof, and an immune
checkpoint inhibitor provided herein is administered to subjects
who suffer from one or more types of cancers. In other embodiments,
any type of neoplastic disease, such as cancer, that is susceptible
to treatment with the compositions described herein might be
targeted.
[0353] In another embodiment, administering an arenavirus particle
expressing a tumor antigen, tumor associated antigen or an
antigenic fragment thereof provided or a composition thereof to
subjects confer cell-mediated immunity (CMI) against a neoplastic
cell or tumor, such as a cancer cell or tumor. Without being bound
by theory, in another embodiment, an arenavirus particle expressing
a tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided or a composition thereof infects and expresses
antigens of interest in antigen presenting cells (APC) of the host
(e.g., macrophages) for direct presentation of antigens on Major
Histocompatibility Complex (MHC) class I and II. In another
embodiment, administering an arenavirus particle expressing a tumor
antigen, tumor associated antigen or an antigenic fragment thereof
provided herein, or a composition thereof, to subjects induces
plurifunctional IFN-.gamma. and TNF-.alpha. co-producing
cancer-specific CD4+ and CD8+ T cell responses (IFN-.gamma. is
produced by CD4+ and CD8+ T cells and TNF-.alpha. is produced by
CD4+ T cells) of high magnitude to treat a neoplastic disease.
[0354] In another embodiment, administering an arenavirus particle
expressing a tumor antigen, tumor associated antigen or an
antigenic fragment thereof provided herein, or a composition
thereof, and an immune checkpoint inhibitor provided herein
increases or improves one or more clinical outcome for cancer
treatment. Non-limiting examples of such outcomes are overall
survival, progression-free survival, time to progression, time to
treatment failure, event-free survival, time to next treatment,
overall response rate and duration of response. The increase or
improvement in one or more of the clinical outcomes can be by at
least about 10%, at least about 20%, at least about 25%, at least
about 30%, at least about 35%, at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, or more, compared to a patient or group of
patients having the same neoplastic disease in the absence of such
treatment.
[0355] Changes in cell-mediated immunity (CMI) response function
against a neoplastic cell or tumor, including a cancer cell or
tumor, induced by administering an arenavirus particle expressing a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided, or a composition thereof, in subjects can be
measured by any assay known to the skilled artisan including, but
not limited to flow cytometry (see, e.g., Perfetto S. P. et al.,
Nat Rev Immun. 2004; 4(8):648-55), lymphocyte proliferation assays
(see, e.g., Bonilla F. A. et al., Ann Allergy Asthma Immunol. 2008;
101:101-4; and Hicks M. J. et al., Am J Clin Pathol. 1983;
80:159-63), assays to measure lymphocyte activation including
determining changes in surface marker expression following
activation of measurement of cytokines of T lymphocytes (see, e.g.,
Caruso A. et al., Cytometry. 1997; 27:71-6), ELISPOT assays (see,
e.g., Czerkinsky C. C. et al., J Immunol Methods. 1983; 65:109-121;
and Hutchings P. R. Et al., J Immunol Methods. 1989; 120:1-8), or
Natural killer cell cytotoxicity assays (see, e.g., Bonilla F. A.
et al., Ann Allergy Asthma Immunol. 2005 May; 94(5 Suppl
1):S1-63).
[0356] Immune checkpoint inhibitors that can be used with the
methods and compositions described herein can target any negative
checkpoint regulator. In certain embodiments, such a negative
checkpoint regulator is a protein involved in T-Cell activation. In
certain, more specific embodiments, such a negative checkpoint
regulator is Cytotoxic T-lymphocyte antigen-4 (CTLA-4), CD80, CD86,
Programmed cell death 1 (PD-1), Programmed cell death ligand 1
(PD-L1), Programmed cell death ligand 2 (PD-L2), Lymphocyte
activation gene-3 (LAG-3; also known as CD223), Galectin-3, B and T
lymphocyte attenuator (BTLA), T-cell membrane protein 3 (TIM3),
Galectin-9 (GAL9), B7-H1, B7-H3, B7-H4, T-Cell immunoreceptor with
Ig and ITIM domains (TIGIT/Vstm3/WUCAM/VSIG9), V-domain Ig
suppressor of T-Cell activation (VISTA), Glucocorticoid-induced
tumor necrosis factor receptor-related (GITR) protein, Herpes Virus
Entry Mediator (HVEM), OX40, CD27, CD28, CD137. CGEN-15001T,
CGEN-15022, CGEN-15027, CGEN-15049, CGEN-15052, or CGEN-15092. An
overview such checkpoint regulators and drugs that target them is
set forth in Table 4.
TABLE-US-00007 TABLE 4 Target for Commercial FDA Indication
Immunotherapy Mode of Action Name Approval (human) Preclinical
Evaluation PD1/ Antibodies that bind Nivolumab Yes Metastatic Yes.
PD1-L(1,2) to PD1 (Block (Opdivo-Bristol Melanoma, Non- PD1 Ab
alone and in (programmed receptor) Myers Squibb) Small cell lung
combination with CTLA4 have cell death cancer been systematically
evaluated protein-1) Pembrolizumab Yes Metastatic in 7 different
mouse models. (Keytruda, MK- Melanoma (Barnes et al, AACR Annual
3475 Merck) (Clinical trials for Meeting Poster # 3362 (2015)) lung
cancer, P815-B7-H1-modified lymphoma, (Mastocytoma) have also been
mesothelioma) evaluated. (See Hirano et al., Pidilizumab (CT- No
Clinical Trials - Cancer Res, 65(3), 1089-96 011, Cure Tech)
multiple cancers (2005)) Antibodies bind PD- BMS936559 No Melanoma,
Non- L1 (inhibit receptor (Bristol Myers- Small cell lung binding)
Squibb) cancer, Ovarian Cancer Phase I: HIV MPDL328OA No NSCLC and
(Roche) melanoma (locally advanced or metatstatic tumor) CTLA4 By
binding to Ipilimumab Yes Melanoma Yes. (cytotoxic T CTLA4, the
(MDX010, See Simpson et al. J. Exp. Med. lymphocyte- compounds
enhance Yervoy; Bristol 2013: 210(9): 1695-710 associated T-cell
activation and Myers-Squibb) variable abilities were allocated
antigen 4) block B7-1 and B7-2 Tremelimumab No Clinical trials - to
different Ab clones. T-cell co-stimulatory (CP-675, 206, Multiple
Cancers pathways Pfizer) TIM-3 Anti-TIM-3 IgGs n. a. No n. a. Yes.
(T-Cell inhibit binding of (See Sakuishi et al., J Exp.
immunoglobulin TIM-3 to its receptor Med., 207(10), 2187-94 and
mucin- and/or Ligand (2010)) containing (perhaps galectin-9) CT26,
4T1 and B16 were protein 3) tested in their respective background.
LAG-3/ Anti-LAG-3 n. a. No n. a. Yes. Galectin-3
antibodies/depletion NT2.5 (neu-expressing tumor (lymphocyte- of
galectin-3 cell line) reported in Kouo et activated gene-3) al.
Cancer Immunol Res. 3(4): 412-23 (2015). B16 and MC38 reported in
Woo et al. Cancer Res 72(4)_917-27 (.2012). TIGIT Antibodies
against Under No n. a. Yes. (T-Cell TIGIT inhibit binding
development (See Johnston et al., Cancer immunoreceptor to PVR
(receptor) (Genentech) Cell. 26(6): 923-37 (2014)). with Ig and
ITIM disturbing the domains) inhibitory signaling. Further CD226
can bind to PVR instead and deploy its T-Cell activating function/
Might be also involved in NK cell inhibition BTLA (B and T
lymphocyte attenuator) VISTA (V-domain Ig suppressor of T- Cell
activation)
[0357] In certain embodiments, an arenavirus particle expressing a
tumor antigen, tumor associated antigen or an antigenic fragment
thereof provided herein, or a composition thereof, and an immune
checkpoint inhibitor provided herein is preferably administered in
multiple injections (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,
14, 16, 18, 20, 25, 30, 40, 45, or 50 injections) or by continuous
infusion (e.g., using a pump) at multiple sites (e.g., at least 2,
3, 4, 5, 6, 7, 8, 9, 10, 12, or 14 sites). In certain embodiments,
the arenavirus particle expressing a tumor antigen, tumor
associated antigen or an antigenic fragment thereof provided
herein, or a composition thereof, is administered in two or more
separate injections over a 6-month period, a 12-month period, a
24-month period, or a 48-month period. In certain embodiments, the
arenavirus particle expressing a tumor antigen, tumor associated
antigen or an antigenic fragment thereof provided herein, or a
composition thereof, is administered with a first dose at an
elected date, a second dose at least 2 months after the first dose,
and a third does 6 months after the first dose.
[0358] In one example, cutaneous injections are performed at
multiple body sites to reduce extent of local skin reactions. On a
given vaccination day, the patient receives the assigned total dose
administered from one syringe in 3 to 5 separate intradermal
injections of the dose (e.g., at least 0.4 ml, 0.2 ml, or 0.1 ml)
each in an extremity spaced at least about 5 cm (e.g., at least
4.5, 5, 6, 7, 8, 9, or cm) at needle entry from the nearest
neighboring injection. On subsequent vaccination days, the
injection sites are rotated to different limbs in a clockwise or
counter-clockwise manner.
[0359] In certain embodiments, the methods further comprise
co-administration of the arenavirus particle provided herein and an
immune checkpoint inhibitor. In certain embodiments, the
co-administration is simultaneous. In another embodiment, the
arenavirus particle is administered prior to administration of the
immune checkpoint inhibitor. In other embodiments, the arenavirus
particle is administered after administration of the immune
checkpoint inhibitor. In certain embodiments, the interval between
administration of the arenavirus particle and the immune checkpoint
inhibitor is about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 10 hours, about 11 hours, or about 12 hours.
In certain embodiments, the interval between administration of the
arenavirus particle and the immune checkpoint inhibitor is about 1
day, about 2 days, about 2 days, about 3 days, about 4 days, about
5 days, about 6 days, about 1 week, about 8 days, about 9 days,
about 10 days, about 11 days, about 12 days, about 13 days, about 2
weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks,
about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about
11 weeks, about 12 weeks. In certain embodiments, the interval
between administration of the arenavirus particle and the immune
checkpoint inhibitor is about 1 month, about 2 months, about 3
months, about 4 months, about 5 months, or about 6 months. In some
embodiments, the method further includes administering at least one
additional therapy.
[0360] In certain embodiments, provided herein are methods of
treating a neoplastic disease in a subject comprising administering
to the subject one or more arenavirus particles provided herein and
an indoleamine-2,3 dioxygenase ("IDO") inhibitor. The method of
treating a neoplastic disease in a subject can include
administering to the subject one or more arenavirus particles
expressing a tumour antigen, tumor associated antigen or an
antigenic fragment thereof as provided herein, or a composition
thereof, and an IDO inhibitor. In certain embodiments, provided
herein are methods for inducing an immune response against a
neoplastic cell or tissue, such as a cancer cell or tumor, in a
subject comprising administration to the subject an arenavirus
particle expressing a tumour antigen, tumor associated antigen or
an antigenic fragment thereof as provided herein, or a composition
thereof, and an IDO inhibitor. In certain embodiments, the methods
comprise co-administration of an arenavirus particle provided
herein and an IDO inhibitor. In certain embodiments, the methods
comprise co-administration of the arenavirus particle provided
herein, an immune checkpoint inhibitor provided herein and an IDO
inhibitor.
[0361] In another embodiment, two arenavirus particles are
administered in a treatment regime at molar ratios ranging from
about 1:1 to 1:1000, in particular including: 1:1 ratio, 1:2 ratio,
1:5 ratio, 1:10 ratio, 1:20 ratio, 1:50 ratio, 1:100 ratio, 1:200
ratio, 1:300 ratio, 1:400 ratio, 1:500 ratio, 1:600 ratio, 1:700
ratio, 1:800 ratio, 1:900 ratio, 1:1000 ratio.
[0362] In certain embodiments, provided herein is a method of
treating neoplastic disease wherein a first arenavirus particle is
administered first as a "prime," and a second arenavirus particle
is administered as a "boost." The first and the second arenavirus
particles can express the same or different tumor antigens, tumor
associated antigens or antigenic fragments thereof. Alternatively,
or additionally, some certain embodiments, the "prime" and "boost"
administration are performed with an arenavirus particle derived
from different species. In certain specific embodiments, the
"prime" administration is performed with an arenavirus particle
derived from LCMV, and the "boost" is performed with an arenavirus
particle derived from Junin virus. In certain specific embodiments,
the "prime" administration is performed with an arenavirus particle
derived from Junin virus, and the "boost" is performed with an
arenavirus particle derived from LCMV.
[0363] In certain embodiments, administering a first arenavirus
particle expressing a tumor antigen, tumor associated antigen or
antigenic fragment thereof, followed by administering a second
arenavirus particle expressing a tumor antigen, tumor associated
antigen or antigenic fragment thereof results in a greater antigen
specific CD8+ T cell response than administering a single
arenavirus particle expressing a tumor antigen, tumor associated
antigen or antigenic fragment thereof. In certain embodiments, the
antigen specific CD8+ T cell count increases by 50%, 100%, 150% or
200% after the second administration compared to the first
administration. In certain embodiments, administering a third
arenavirus particle expressing a tumor antigen, tumor associated
antigen or antigenic fragment thereof results in a greater antigen
specific CD8+ T cell response than administering two consecutive
arenavirus particles expressing a tumor antigen, tumor associated
antigen or antigenic fragment thereof. In certain embodiments, the
antigen specific CD8+ T cell count increases by about 50%, about
100%, about 150%, about 200% or about 250% after the third
administration compared to the first administration.
[0364] In certain embodiments, provided herein are methods for
treating a neoplastic disease comprising administering two or more
arenavirus particles, wherein the two or more arenavirus particles
are homologous, and wherein the time interval between each
administration is about 1 week, about 2 weeks, about 3 week, about
4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8
weeks, about 3 months, about 4 months, about 5 months, about 6
months, about 7 months, about 8 months, about 9 months, about 10
months, about 11 months, about 12 months, about 18 months, or about
24 months.
[0365] In certain embodiments, administering a first arenavirus
particle expressing a tumor antigen, tumor associated antigen or
antigenic fragment thereof and a second, heterologous, arenavirus
particle expressing a tumor antigen, tumor associated antigen or
antigenic fragment thereof elicits a greater CD8+ T cell response
than administering a first arenavirus particle expressing a tumor
antigen, tumor associated antigen or antigenic fragment thereof and
a second, homologous, arenavirus particle expressing a tumor
antigen, tumor associated antigen or antigenic fragment
thereof.
5.9 Compositions, Administration, and Dosage
[0366] Also provided herein are vaccines, immunogenic compositions
(e.g., vaccine formulations), and pharmaceutical compositions
comprising an arenavirus particle provided herein, and, in certain
embodiments, an immune checkpoint inhibitor provided herein. Such
vaccines, immunogenic compositions and pharmaceutical compositions
can be formulated according to standard procedures in the art.
[0367] In another embodiment, provided herein are compositions
comprising an infectious, replication-deficient arenavirus particle
described herein, and, in certain embodiments, an immune checkpoint
inhibitor provided herein. Such compositions can be used in methods
of treating a neoplastic disease. In another specific embodiment,
the immunogenic compositions provided herein can be used to induce
an immune response in a host to whom the composition is
administered. The immunogenic compositions described herein can be
used as vaccines and can accordingly be formulated as
pharmaceutical compositions. In a specific embodiment, the
immunogenic compositions described herein are used in the treatment
of a neoplastic disease a subject (e.g., human subject). In other
embodiments, the vaccine, immunogenic composition or pharmaceutical
composition are suitable for veterinary and/or human
administration.
[0368] In certain embodiments, provided herein are immunogenic
compositions comprising an arenavirus particle (or a combination of
different arenavirus particles) as described herein. In certain
embodiments, such an immunogenic composition further comprises a
pharmaceutically acceptable excipient. In certain embodiments, such
an immunogenic composition further comprises an adjuvant. The
adjuvant for administration in combination with a composition
described herein may be administered before, concomitantly with, or
after administration of said composition. In some embodiments, the
term "adjuvant" refers to a compound that when administered in
conjunction with or as part of a composition described herein
augments, enhances and/or boosts the immune response to an
infectious, replication-deficient arenavirus particle, but when the
compound is administered alone does not generate an immune response
to the infectious, replication-deficient arenavirus particle. In
some embodiments, the adjuvant generates an immune response to the
infectious, replication-deficient arenavirus particle and does not
produce an allergy or other adverse reaction. Adjuvants can enhance
an immune response by several mechanisms including, e.g.,
lymphocyte recruitment, stimulation of B and/or T cells, and
stimulation of macrophages. When a vaccine or immunogenic
composition of the invention comprises adjuvants or is administered
together with one or more adjuvants, the adjuvants that can be used
include, but are not limited to, mineral salt adjuvants or mineral
salt gel adjuvants, particulate adjuvants, microparticulate
adjuvants, mucosal adjuvants, and immunostimulatory adjuvants.
Examples of adjuvants include, but are not limited to, aluminum
salts (alum) (such as aluminum hydroxide, aluminum phosphate, and
aluminum sulfate), 3 De-O-acylated monophosphoryl lipid A (MPL)
(see GB 2220211), MF59 (Novartis), AS03 (GlaxoSmithKline), AS04
(GlaxoSmithKline), polysorbate 80 (Tween 80; ICL Americas, Inc.),
imidazopyridine compounds (see International Application No.
PCT/US2007/064857, published as International Publication No.
WO2007/109812), imidazoquinoxaline compounds (see International
Application No. PCT/US2007/064858, published as International
Publication No. WO2007/109813) and saponins, such as QS21 (see
Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach
(eds. Powell & Newman, Plenum Press, N Y, 1995); U.S. Pat. No.
5,057,540). In some embodiments, the adjuvant is Freund's adjuvant
(complete or incomplete). Other adjuvants are oil in water
emulsions (such as squalene or peanut oil), optionally in
combination with immune stimulants, such as monophosphoryl lipid A
(see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)).
[0369] The compositions comprise the infectious,
replication-deficient arenavirus particles described herein alone
or together with a pharmaceutically acceptable carrier and/or an
immune checkpoint inhibitor. Suspensions or dispersions of
genetically engineered arenavirus particles, especially isotonic
aqueous suspensions or dispersions, can be used. The pharmaceutical
compositions may be sterilized and/or may comprise excipients,
e.g., preservatives, stabilizers, wetting agents and/or
emulsifiers, solubilizers, salts for regulating osmotic pressure
and/or buffers and are prepared in a manner known per se, for
example by means of conventional dispersing and suspending
processes. In certain embodiments, such dispersions or suspensions
may comprise viscosity-regulating agents. The suspensions or
dispersions are kept at temperatures around 2-8.degree. C., or
preferentially for longer storage may be frozen and then thawed
shortly before use. For injection, the vaccine or immunogenic
preparations may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks's solution,
Ringer's solution, or physiological saline buffer. The solution may
contain formulatory agents such as suspending, stabilizing and/or
dispersing agents.
[0370] In certain embodiments, the compositions described herein
additionally comprise a preservative, e.g., the mercury derivative
thimerosal. In a specific embodiment, the pharmaceutical
compositions described herein comprise 0.001% to 0.01% thimerosal.
In other embodiments, the pharmaceutical compositions described
herein do not comprise a preservative.
[0371] The pharmaceutical compositions comprise from about 10.sup.3
to about 10.sup.11 focus forming units of the genetically
engineered arenavirus particles. Unit dose forms for parenteral
administration are, for example, ampoules or vials, e.g., vials
containing from about 10.sup.3 to 10.sup.10 focus forming units or
10.sup.5 to 10.sup.15 physical particles of genetically engineered
arenavirus particles.
[0372] In another embodiment, a vaccine or immunogenic composition
provided herein is administered to a subject by, including but not
limited to, oral, intradermal, intramuscular, intraperitoneal,
intravenous, topical, subcutaneous, percutaneous, intranasal and
inhalation routes, and via scarification (scratching through the
top layers of skin, e.g., using a bifurcated needle). Specifically,
subcutaneous, intramuscular or intravenous routes can be used.
[0373] For administration intranasally or by inhalation, the
preparation for use according to the present invention can be
conveniently delivered in the form of an aerosol spray presentation
from pressurized packs or a nebulizer, with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of, e.g., gelatin for use in an inhaler or insufflators
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0374] The dosage of the active ingredient depends upon the type of
vaccination and upon the subject, and their age, weight, individual
condition, the individual pharmacokinetic data, and the mode of
administration.
[0375] In certain embodiments, the compositions can be administered
to the patient in a single dosage comprising a therapeutically
effective amount of the arenavirus particle and/or a
therapeutically effective amount of an immune checkpoint inhibitor.
In some embodiments, the arenavirus particle can be administered to
the patient in a single dose comprising an arenavirus particle and
an immune checkpoint inhibitor, each in a therapeutically effective
amount.
[0376] In certain embodiments, the composition is administered to
the patient as a single dose followed by a second dose three to six
weeks later. In accordance with these embodiments, the booster
inoculations may be administered to the subjects at six to twelve
month intervals following the second inoculation. In certain
embodiments, the booster inoculations may utilize a different
arenavirus particle or composition thereof. In some embodiments,
the administration of the same composition as described herein may
be repeated and separated by at least 1 day, 2 days, 3 days, 4
days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75
days, 3 months, or at least 6 months.
[0377] In certain embodiments, the vaccine, immunogenic
composition, or pharmaceutical composition comprising an arenavirus
particle can be used as a live vaccination. Exemplary doses for a
live arenavirus particle may vary from 10-100, or more, PFU of live
virus per dose. In some embodiments, suitable dosages of an
arenavirus particle or the tri-segmented arenavirus particle are
10.sup.2, 5.times.10.sup.2, 10.sup.3, 5.times.10.sup.3, 10.sup.4,
5.times.10.sup.4, 10.sup.5, 5.times.10.sup.5, 10.sup.6,
5.times.10.sup.6, 10.sup.7, 5.times.10.sup.7, 10.sup.8,
5.times.10.sup.8, 1.times.10.sup.9, 5.times.10.sup.9,
1.times.10.sup.10, 5.times.10.sup.10, 1.times.10.sup.11,
5.times.10.sup.11 or 10.sup.12 pfu, and can be administered to a
subject once, twice, three or more times with intervals as often as
needed. In another embodiment, a live arenavirus is formulated such
that a 0.2-mL dose contains 10.sup.6.5-10.sup.7.5 fluorescent focal
units of live arenavirus particle. In another embodiment, an
inactivated vaccine is formulated such that it contains about 15
.mu.g to about 100 .mu.g, about 15 .mu.g to about 75 .mu.g, about
15 .mu.g to about 50 .mu.g, or about 15 .mu.g to about 30 .mu.g of
an arenavirus
[0378] Also provided are processes and uses of an arenavirus
particle and an immune checkpoint inhibitor for the manufacture of
vaccines in the form of pharmaceutical preparations, which comprise
the arenavirus particle and the immune checkpoint inhibitor as an
active ingredient. Still further provided is a combination of an
arenavirus particle provided herein and an immune checkpoint
inhibitor provided herein for use in the treatment of a neoplastic
disease described herein. In certain embodiments, the combination
is in the same pharmaceutical compostions. In certain embodiments,
the combination is not in the same pharmaceutical composition, such
as when the arenavirus particle and the immune checkpoint inhibitor
are to be separately administered. The pharmaceutical compositions
of the present application are prepared in a manner known per se,
for example by means of conventional mixing and/or dispersing
processes.
[0379] In certain embodiments, the methods and compositions
provided herein are used in combination with personalized medicine.
Personalized medicine seeks to benefit patients by using
information from a patient's unique genetic and/or epigenetic
profile to predict a patient's response to different therapies and
identify which therapies are more likely to be effective.
Techniques that can be used in combination with the methods and
compositions provided herein to obtain a patient's unique genetic
and/or epigenetic profile include, but are not limited to, genome
sequencing, RNA sequencing, gene expression analysis and
identification of a tumor antigen (e.g., neoantigen), tumor
associated antigen or an antigenic fragment thereof. In certain
embodiments, the selection of an arenavirus tumor antigen or tumor
associated antigen for use in the methods and compositions provided
herein is performed based on the genetic profile of the patient. In
certain embodiments, the selection of an arenavirus tumor antigen
or tumor associated antigen for use in the methods and compositions
provided herein is performed based on the genetic profile of a
tumor or tumor cell.
[0380] Also provided herein are kits that can be used to perform
the methods described herein. In certain embodiments, the kit
provided herein can include one or more containers. These
containers can hold for storage the compositions (e.g.,
pharmaceutical, immunogenic or vaccine composition) provided
herein. Also included in the kit are instructions for use. These
instructions describe, in sufficient detail, a treatment protocol
for using the compositions contained therein. For example, the
instructions can include dosing and administration instructions as
provided herein for the methods of treating a neoplastic
disease.
[0381] In certain embodiments, a kit provided herein includes
containers that each contains the active ingredients for performing
the methods described herein. Thus, in certain embodiments, the kit
provided herein includes two or more containers and instructions
for use, wherein one of the containers comprises an infectious,
replication-deficient arenavirus particle provided herein and
another container that comprises an immune checkpoint inhibitor
provided herein.
5.10 Assays
5.10.1 Arenavirus Detection Assays
[0382] The skilled artesian could detect an arenavirus genomic
segment or tri-segmented arenavirus particle, as described herein
using techniques known in the art. For example, RT-PCR can be used
with primers that are specific to an arenavirus to detect and
quantify an arenavirus genomic segment that has been engineered to
carry an ORF in a position other than the wild-type position of the
ORF or a tri-segmented arenavirus particle. Western blot, ELISA,
radioimmunoassay, immunoprecipitation, immunocytochemistry, or
immunocytochemistry in conjunction with FACS can be used to
quantify the gene products of the arenavirus genomic segment or
tri-segmented arenavirus particle.
5.10.2 Assay to Measure Infectivity
[0383] Any assay known to the skilled artisan can be used for
measuring the infectivity of an arenavirus vector preparation. For
example, determination of the virus/vector titer can be done by a
"focus forming unit assay" (FFU assay). In brief, complementing
cells, e.g., MC57 or HEK293 cells optionally expressing LCMV GP
protein, are plated and inoculated with different dilutions of a
virus/vector sample. After an incubation period, to allow cells to
form a monolayer and virus to attach to cells, the monolayer is
covered with Methylcellulose. When the plates are further
incubated, the original infected cells release viral progeny. Due
to the Methylcellulose overlay the spread of the new viruses is
restricted to neighboring cells. Consequently, each infectious
particle produces a circular zone of infected cells called a Focus.
Such Foci can be made visible and by that countable using
antibodies against LCMV-NP or another protein expressed by the
arenavirus particle or the tri-segmented arenavirus particle and a
HRP-based color reaction. The titer of a virus/vector can be
calculated in focus-forming units per milliliter (FFU/mL).
5.10.3 Growth of an Arenavirus Particle
[0384] Growth of an arenavirus particle described herein can be
assessed by any method known in the art or described herein (e.g.,
cell culture). Viral growth may be determined by inoculating serial
dilutions of an arenavirus particle described herein into cell
cultures (e.g., Vero cells or BHK-21 cells). After incubation of
the virus for a specified time, the virus is isolated using
standard methods.
5.10.4 Serum ELISA
[0385] Determination of the humoral immune response upon
vaccination of animals (e.g., mice, guinea pigs) can be done by
antigen-specific serum ELISA's (enzyme-linked immunosorbent
assays). In brief, plates are coated with antigen (e.g.,
recombinant protein), blocked to avoid unspecific binding of
antibodies and incubated with serial dilutions of sera. After
incubation, bound serum-antibodies can be detected, e.g., using an
enzyme-coupled anti-species (e.g., mouse, guinea pig)-specific
antibody (detecting total IgG or IgG subclasses) and subsequent
color reaction. Antibody titers can be determined as, e.g.,
endpoint geometric mean titer.
[0386] Immunocapture ELISA (IC-ELISA) may also be performed (see
Shanmugham et al., 2010, Clin. Vaccine Immunol. 17(8):1252-1260),
wherein the capture agents are cross-linked to beads.
5.10.5 Assay to Measure the Neutralizing Activity of Induced
Antibodies
[0387] Determination of the neutralizing antibodies in sera is
performed with the following cell assay using ARPE-19 cells from
ATCC and a GFP-tagged virus. In addition supplemental serum (e.g.,
guinea pig serum) as a source of exogenous complement is used. The
assay is started with seeding of 6.5.times.10.sup.3 cells/well (50
.mu.l/well) in a 384 well plate one or two days before using for
neutralization. The neutralization is done in 96-well sterile
tissue culture plates without cells for 1 h at 37.degree. C. After
the neutralization incubation step the mixture is added to the
cells and incubated for additional 4 days for GFP-detection with a
plate reader. A positive neutralizing human sera is used as assay
positive control on each plate to check the reliability of all
results. Titers (EC50) are determined using a 4 parameter logistic
curve fitting. As additional testing the wells are checked with a
fluorescence microscope.
5.10.6 Plaque Reduction Assay
[0388] In brief, plaque reduction (neutralization) assays for LCMV
can be performed by use of a replication-competent or -deficient
LCMV that is tagged with green fluorescent protein, 5% rabbit serum
may be used as a source of exogenous complement, and plaques can be
enumerated by fluorescence microscopy. Neutralization titers may be
defined as the highest dilution of serum that results in a 50%,
75%, 90% or 95% reduction in plaques, compared with that in control
(pre-immune) serum samples.
[0389] qPCR LCMV RNA genomes are isolated using QIAamp Viral RNA
mini Kit (QIAGEN), according to the protocol provided by the
manufacturer. LCMV RNA genome equivalents are detected by
quantitative PCR carried out on an StepOnePlus Real Time PCR System
(Applied Biosystems) with SuperScript.RTM. III Platinum.RTM.
One-Step qRT-PCR Kit (Invitrogen) and primers and probes (FAM
reporter and NFQ-MGB Quencher) specific for part of the LCMV NP
coding region or another genomic stretch of the arenavirus particle
or the tri-segmented arenavirus particle. The temperature profile
of the reaction may be: 30 min at 60.degree. C., 2 min at
95.degree. C., followed by 45 cycles of 15 s at 95.degree. C., 30 s
at 56.degree. C. RNA can be quantified by comparison of the sample
results to a standard curve prepared from a log 10 dilution series
of a spectrophotometrically quantified, in vitro-transcribed RNA
fragment, corresponding to a fragment of the LCMV NP coding
sequence or another genomic stretch of the arenavirus particle or
the tri-segmented arenavirus particle containing the primer and
probe binding sites.
5.10.7 Neutralization Assay in Guinea Pig Lung Fibroblast (GPL)
Cells
[0390] In brief, serial dilutions of test and control
(pre-vaccination) sera were prepared in GPL complete media with
supplemental rabbit serum (1%) as a source of exogenous complement.
The dilution series spanned 1:40 through 1:5120. Serum dilutions
were incubated with eGFP tagged virus (100-200 pfu per well) for 30
min at 37.degree. C., and then transferred to 12-well plates
containing confluent GPL cells. Samples were processed in
triplicate. After 2 hours incubation at 37.degree. C. the cells
were washed with PBS, re-fed with GPL complete media and incubated
at 37.degree. C./5% CO.sub.2 for 5 days. Plaques were visualized by
fluorescence microscopy, counted, and compared to control wells.
That serum dilution resulting in a 50% reduction in plaque number
compared to controls was designated as the neutralizing titer.
5.10.8 Western Blotting
[0391] Infected cells grown in tissue culture flasks or in
suspension are lysed at indicated timepoints post infection using
RIPA buffer (Thermo Scientific) or used directly without
cell-lysis. Samples are heated to 99.degree. C. for 10 minutes with
reducing agent and NuPage LDS Sample buffer (NOVEX) and chilled to
room temperature before loading on 4-12% SDS-gels for
electrophoresis. Proteins are blotted onto membranes using
Invitrogens iBlot Gel transfer Device and visualized by Ponceau
staining Finally, the preparations are probed with a primary
antibodies directed against proteins of interest and alkaline
phosphatase conjugated secondary antibodies followed by staining
with 1-Step NBT/BCIP solution (INVITROGEN).
5.10.9 MHC-Peptide Multimer Staining Assay for Detection of
Antigen-Specific CD8+ T-Cell Proliferation
[0392] Any assay known to the skilled artisan can be used to test
antigen-specific CD8+ T-cell responses. For example, the
MHC-peptide tetramer staining assay can be used (see, e.g., Altman
J. D. et al., Science. 1996; 274:94-96; and Murali-Krishna K. et
al., Immunity. 1998; 8:177-187). Briefly, the assay comprises the
following steps, a tetramer assay is used to detect the presence of
antigen specific T-cells. In order for a T-cell to detect the
peptide to which it is specific, it must both recognize the peptide
and the tetramer of MHC molecules custom made for a defined antigen
specificity and MHC haplotype of T-cells (typically fluorescently
labeled). The tetramer is then detected by flow cytometry via the
fluorescent label.
5.10.10 ELISPOT Assay for Detection of Antigen-Specific CD4+ T-Cell
Proliferation
[0393] Any assay known to the skilled artisan can be used to test
antigen-specific CD4+ T-cell responses. For example, the ELISPOT
assay can be used (see, e.g., Czerkinsky C. C. et al., J Immunol
Methods. 1983; 65:109-121; and Hutchings P. R. et al., J Immunol
Methods. 1989; 120:1-8). Briefly, the assay comprises the following
steps: An immunospot plate is coated with an anti-cytokine
antibody. Cells are incubated in the immunospot plate. Cells
secrete cytokines and are then washed off. Plates are then coated
with a second biotinylated-anticytokine antibody and visualized
with an avidin-HRP system.
5.10.11 Intracellular Cytokine Assay for Detection of Functionality
of CD8+ and CD4+ T-Cell Responses
[0394] Any assay known to the skilled artisan can be used to test
the functionality of CD8+ and CD4+ T cell responses. For example,
the intracellular cytokine assay combined with flow cytometry can
be used (see, e.g., Suni M. A. et al., J Immunol Methods. 1998;
212:89-98; Nomura L. E. et al., Cytometry. 2000; 40:60-68; and
Ghanekar S. A. et al., Clinical and Diagnostic Laboratory
Immunology. 2001; 8:628-63). Briefly, the assay comprises the
following steps: activation of cells via specific peptides or
protein, an inhibition of protein transport (e.g., brefeldin A) is
added to retain the cytokines within the cell. After a defined
period of incubation, typically 5 hours, a washing steps follows,
and antibodies to other cellular markers can be added to the cells.
Cells are then fixed and permeabilized. The fluorochrome-conjugated
anti-cytokine antibodies are added and the cells can be analyzed by
flow cytometry.
5.10.12 Assay for Confirming Replication-Deficiency of Viral
Vectors
[0395] Any assay known to the skilled artisan that determines
concentration of infectious and replication-competent virus
particles can also be used as a to measure replication-deficient
viral particles in a sample. For example, FFU assays with
non-complementing cells can be used for this purpose.
[0396] Furthermore, plaque-based assays are the standard method
used to determine virus concentration in terms of plaque forming
units (PFU) in a virus sample. Specifically, a confluent monolayer
of non-complementing host cells is infected with the virus at
varying dilutions and covered with a semi-solid medium, such as
agar to prevent the virus infection from spreading
indiscriminately. A viral plaque is formed when a virus
successfully infects and replicates itself in a cell within the
fixed cell monolayer, and spreads to surrounding cells (see, e.g.,
Kaufmann, S. H.; Kabelitz, D. (2002). Methods in Microbiology Vol.
32: Immunology of Infection. Academic Press. ISBN 0-12-521532-0).
Plaque formation can take 2-14 days, depending on the virus being
analyzed. Plaques are generally counted manually and the results,
in combination with the dilution factor used to prepare the plate,
are used to calculate the number of plaque forming units per sample
unit volume (PFU/mL). The PFU/mL result represents the number of
infective replication-competent particles within the sample. When
C-cells are used, the same assay can be used to titrate
replication-deficient arenavirus particles or tri-segmented
arenavirus particles.
5.10.13 Assay for Expression of Viral Antigen
[0397] Any assay known to the skilled artisan can be used for
measuring expression of viral antigens. For example, FFU assays can
be performed. For detection, mono- or polyclonal antibody
preparation(s) against the respective viral antigens are used
(transgene-specific FFU).
5.10.14 Animal Models
[0398] To investigate recombination and infectivity of an
arenavirus particle described herein in vivo animal models can be
used. In certain embodiments, the animal models that can be used to
investigate recombination and infectivity of a tri-segmented
arenavirus particle include mouse, guinea pig, rabbit, and monkeys.
In a preferred embodiment, the animal models that can be used to
investigate recombination and infectivity of an arenavirus include
mouse. In a more specific embodiment, the mice can be used to
investigate recombination and infectivity of an arenavirus particle
are triple-deficient for type I interferon receptor, type II
interferon receptor and recombination activating gene 1 (RAG1).
[0399] In certain embodiments, the animal models can be used to
determine arenavirus infectivity and transgene stability. In some
embodiments, viral RNA can be isolated from the serum of the animal
model. Techniques are readily known by those skilled in the art.
The viral RNA can be reverse transcribed and the cDNA carrying the
arenavirus ORFs can be PCR-amplified with gene-specific primers.
Flow cytometry can also be used to investigate arenavirus
infectivity and transgene stability.
[0400] The safety, tolerance and immunogenic effectiveness of
vaccines comprising of a bi-segmented infectious,
replication-deficient arenavirus expressing a tumor antigen, tumor
associate antigen or antigenic fragment thereof described herein or
a composition thereof can be tested in animals models. In certain
embodiments, the animal models that can be used to test the safety,
tolerance and immunogenic effectiveness of the vaccines and
compositions thereof used herein include mouse, guinea pig, rat,
monkey, and chimpanzee. In a preferred embodiment, the animal
models that can be used to test the safety, tolerance and
immunogenic effectiveness of the vaccines and compositions thereof
used herein include mouse.
5.10.15 Immune Checkpoint Inhibitor Assays
[0401] A number of assays have been devised to assess properties of
proposed checkpoint inhibitors, for example as described in Lechner
et al., J. Immunother. 36(9), 477-89 (2013). Tumor models that can
be used to test the methods and compositions described herein
include Colon26 (CT26), MC38 (mouse colon adenocarcinoma), B16F10
(B16), Lewis Lung (LLC), Madison109 (Mad 109), EMT-6 (murine breast
cancer), 4T1 (4T1) (murine breast cancer), and (RENCA) (murine
renal cancer).
[0402] In certain embodiments, in these model systems,
"transplantable tumors" can be generated by subcutaneous (e.g.,
CT26, 4T1, MAD109, RENCA, LLC, or B16) or intracerebral (e.g.,
GL261, ONC26M4) inoculation of tumor cell lines into rodents, for
example in adult female mice. Tumors can be developed over
pre-determined time intervals, for example several days. These
tumors are grown in syngeneic, immunocompetent rodent, e.g., mouse,
strains. For example CT26, 4T1, MAD109, and RENCA can be grown in
BALB/c mice, LLC, B16, and GL261 can be grown in C57BL/6 mice, and
ONC26M4 can be grown in FVBN mice. "Spontaneous tumors" can be
generated by intracerebral injection of DNA plasmids encoding a
number (e.g., one, two, three or more) of oncogenes and encoding
one or more reporter, e.g., firefly luciferase reporter, into
neonatal C57BL/6 or FVBN mice to transform endogenous brain cells.
Growth of gliomas can be monitored by techniques known in the art,
e.g., bioluminescence imaging. Growth of subcutaneous tumors can be
monitored by techniques known in the art, e.g., caliper
measurements in three dimensions at specified time intervals.
6. EQUIVALENTS
[0403] The viruses, nucleic acids, methods, host cells, and
compositions disclosed herein are not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the viruses, nucleic acids, methods, host cells,
and compositions in addition to those described will become
apparent to those skilled in the art from the foregoing description
and accompanying figures. Such modifications are intended to fall
within the scope of the appended claims.
[0404] Various publications, patents and patent applications are
cited herein, the disclosures of which are incorporated by
reference in their entireties.
[0405] Various publications, patents and patent applications are
cited herein, the disclosures of which are incorporated by
reference in their entireties.
7. EXAMPLES
[0406] The examples in this section are offered by way of
illustration, and not by way of limitation.
7.1 Treatment with Tri-Segmented Arenavirus Vector and Immune
Checkpoint Inhibitor in a Colon Adenocarcinoma Tumor Model
[0407] This example demonstrates that treatment with an arenavirus
vector encoding an exemplary tumor specific antigen (ovalbumin
(OVA)) and an immune checkpoint inhibitor (anti-PD1 antibody)
resulted in a synergistic effect on tumor growth and percent
survival.
[0408] To determine the effects of treatment with a tri-segmented
replication-competent LCMV vector encoding ovalbumin (OVA)
(r3LCMV-OVA) and anti-PD1 checkpoint inhibitor (anti-PD1 antibody),
a subcutaneous MC38-OVA mouse model was used. On day 0,
MC38-OVA.sup.dim colon carcinomas tumor cells (5.times.10.sup.5)
were implanted subcutaneously into the flank of C57BL/6 mice. When
the tumors became palpable (at day 7), mice were either left
untreated (group 1), treated with 12.5 mg/kg anti-PD1 antibody
(clone RMP1-14, BioXCell) (administered intraperitoneally) on days
13, 17, 20 and 24 (group 2), injected intravenously with 10.sup.5
PFU r3LCMV-OVA on day 7 (group 3), or treated with a combination of
r3LCMV-OVA and anti-PD1 antibody using the same treatment regimen
(group 4). Five mice were left untreated, whereas all other groups
included six mice per group. Tumor growth over time as well as
animal survival was monitored.
[0409] The above experiment showed that established tumors from
OVA-expressing MC38 colon adenocarcinoma cells showed limited
response to either anti-PD1 antibody or r3LCMV-OVA treatment alone,
whereas a combination of anti-PD1 antibody and r3LCMV-OVA
immunotherapy resulted in a synergy effect that lead to a
significant reduction in tumor growth (FIG. 3A) as well as
increased percent survival (FIG. 3B).
7.2. Treatment with Tri-Segmented or Bi-Segmented Arenavirus
Vectors Encoding a Tumor Neoantigen and Immune Checkpoint Inhibitor
in a Colon Adenocarcinoma Tumor Model
[0410] This example demonstrates that treatment with an arenavirus
vector encoding a neoantigen (MC-38 Adpgk.sup.mut) and an immune
checkpoint inhibitor (anti-PD1 antibody) resulted in a synergistic
effect on induction of antigen specific CD8+ T cells, tumor growth
and percent survival.
[0411] To determine the immunogenicity and effects of treatment
with replication competent and replication-deficient
arenavirus-based vectors encoding a tumor neoantigen (e.g., MC-38
Adpgk.sup.mut (R204M mutation in ADP-dependent glucokinase
(Adpgk)); Yadav et al., 2014, Nature 515:572-576), mice implanted
with MC-38 tumor cells were evaluated after treatment with
Adpgk.sup.mut-encoding tri-segmented replication competent
(r3LCMV-Adpgk.sup.mut) or replication-deficient bi-segmented
(r2LCMV-Adpgk.sup.mut) arenavirus vectors and PD1 checkpoint
inhibition (anti-PD1 antibody).
[0412] On day -7 of the experiment MC-38 tumor cells (1.times.106)
were implanted subcutaneously into the flank of C57BL/6 mice. Seven
days later, on day 0 of the experiment, mice were immunized once by
intravenous injection of 5.5.times.10.sup.5 PFU of
r2LCMV-Adpgk.sup.mut (groups 1 and 2) or 5.5.times.10.sup.4 PFU of
r3LCMV-Adpgk.sup.mut corresponding to a total of 5.5.times.10.sup.5
viral particles (groups 3 and 4) or buffer (groups 5 and 6).
Animals in groups 1, 3 and 5 were additionally treated by
intraperitoneal injection of 6.8 mg/kg anti-PD1 antibody clone
RMP1-14 on days 0, 3, 6 and 9. The induction of
Adpgk.sup.mut-specific CD8.sup.+ T-cells was subsequently analyzed
with MC-38 Adpgk.sup.mut specific pentamers (F4B-E-H-2db-ASMTNMELM,
ProImmune) in peripheral blood samples on days 7 and 14 after
vaccination.
[0413] The above experiments showed that neoantigen-specific CD8
T-cell responses of considerable magnitude were induced in some
animals treated with Adpgk.sup.mut-expressing LCMV vectors (groups
1-4) (FIG. 4A). Significantly higher CD8.sup.+ T-cell frequencies
were, however, observed in animals treated with the tri-segmented
replicating vector (groups 3 and 4) compared to the bi-segmented
replication-deficient vector (groups 1 and 2) (FIG. 4A). Highest
neoantigen-specific CD8.sup.+ T-cell responses were induced in mice
treated with a Adpgk.sup.mut-expressing replication competent LCMV
vector and anti-PD1 antibody (group 3), pointing to a synergistic
effect of the combination treatment on the immunogenicity of
neoantigens (FIG. 4A). The highest level of tumor control and best
survival rates were observed in animals treated with a combination
of r3LCMV-Adpgk.sup.mut and anti-PD1 antibody (FIGS. 4B and
4C).
7.3. Treatment with Tri-Segmented or Bi-Segmented Arenavirus
Vectors and Immune Checkpoint Inhibitor in a Melanoma Model
[0414] This example demonstrates that treatment with a mixture of
tri-segmented arenavirus vectors encoding melanoma antigens
(glycoprotein 100 ("GP100"), tyrosinase-related protein 1 ("TRP1")
and tyrosinase-related protein 2 ("TRP2")) with and without an
immune checkpoint inhibitor (anti-PD1 antibody) resulted in reduced
tumor size and increased animal survival (percent survival and
overall days survived).
[0415] To determine the effects of treatment with replication
competent and replication-deficient arenavirus-based vectors
encoding melanoma antigens and PD1 checkpoint inhibition, a mouse
B16F10 melanoma model was used.
[0416] On day 0 of the experiment B16F10 melanoma cells
(1.times.10.sup.5) were implanted subcutaneously into the flank of
C57BL/6 mice (FIG. 5A). On day 7 of the experiment mice were
injected intravenously with 1.2.times.10.sup.5 RCV PFU (in total)
of a mix of replication-competent tri-segmented arenavirus vectors
(4.times.10.sup.4 PFU of each r3LCMV-GP100, r3LCMV-Trp1 and
r3LCMV-Trp2) on day 7 (groups 1 and 4), with 1.2.times.10.sup.6 PFU
(in total) of a mix of replication-deficient bi-segmented
arenavirus vectors (4.times.10.sup.5 PFU of each rLCMV-GP100,
rLCMV-Trp1 and rLCMV-Trp2) on day 7 (groups 2 and 5), or with
buffer only (groups 3 and 6). On days 10, 13, 16, 19 and 22 of the
experiment mice in groups 1, 2 and 3 were additionally treated with
200 .mu.g anti-PD1 antibody (clone 29F.1A12, BioXCell),
administered intraperitoneally. Tumor growth over time as well as
percent of animal survival and days of survival were monitored.
There were five mice per group.
[0417] The above experiments showed that established melanoma
tumors responded to a single treatment with a mix of tri-segmented
replication-competent LCMV vectors expressing melanoma antigens
GP100, Trp1 and Trp2 (group 4), as shown by reduced tumor volumes
(FIG. 5B) and increased survival rates compared to control animals
(group 6) (FIGS. 5C and 5D). In contrast, there was no response to
treatment with a mix of bi-segmented replication-deficient LCMV
vectors expressing the same melanoma antigens, independent of the
presence or absence of checkpoint inhibition (groups 2 and 5)
(FIGS. 5B-5D). Anti-PD1 blockade alone also had no effect on tumor
growth or survival of the animals (group 3) (FIGS. 5B-5D). However,
in mice treated with tri-segmented replication-competent melanoma
antigen-expressing LCMV vectors in combination with anti-PD1
antibodies (group 1) an increase in survival rates that correlated
with a decrease in tumor volumes was observed, compared to animals
treated with a mix of tri-segmented replication-competent LCMV
vectors alone, pointing to a synergistic effect of these treatment
modalities (FIGS. 5B-5D).
8. SEQUENCES
[0418] The sequences in Table 5 are illustrative amino acid
sequences and nucleotide sequences that can be used with the
methods and compositions described herein. In some instances a DNA
sequence is used to describe the RNA sequence of a viral genomic
segment. The RNA sequence can be readily deduced from the DNA
sequence.
TABLE-US-00008 TABLE 5 SEQ ID NO. Description Sequence 1
Lymphocytic GCGCACCGGGGATCCTAGGCGTTTAGTTGCGCTGTTTGGT
choriomeningitis TGCACAACTTTCTTCGTGAGGCTGTCAGAAGTGGACCTGG virus
clone 13 CTGATAGCGATGGGTCAAGGCAAGTCCAGAGAGGAGAAAG segment L,
complete GCACCAATAGTACAAACAGGGCCGAAATCCTACCAGATAC sequence
(GenBank: CACCTATCTTGGCCCTTTAAGCTGCAAATCTTGCTGGCAG DQ361066.1)
AAATTTGACAGCTTGGTAAGATGCCATGACCACTACCTTT (The genomic
GCAGGCACTGTTTAAACCTTCTGCTGTCAGTATCCGACAG segment is RNA, the
GTGTCCTCTTTGTAAATATCCATTACCAACCAGATTGAAG sequence in SEQ ID
ATATCAACAGCCCCAAGCTCTCCACCTCCCTACGAAGAGT NO: 1 is shown for
AACACCGTCCGGCCCCGGCCCCGACAAACAGCCCAGCACA DNA; however,
AGGGAACCGCACGTCaCCCAACGCACACAGACACAGCACC exchanging all
CAACACAGAACACGCACACACACACACACACACACCCACA thymidines ("T") in
CGCACGCGCCCCCACCACCGGGGGGCGCCCCCCCCCGGGG SEQ ID NO: 1 for
GGCGGCCCCCCGGGAGCCCGGGCGGAGCCCCACGGAGATG uridines ("U")
CCCATCAGTCGATGTCCTCGGCCACCGACCCGCCcAGCCA provides the RNA
ATCGTCGCAGGACCTCCCCTTGAGTCTAAACCTGCCCCCC sequence.)
ACTgTTTCATACATCAAAGTGCTCCTAGATTTGCTAAAAC
AAAGTCTGCAATCCTTAAAGGCGAACCAGTCTGGCAAAAG
CGACAGTGGAATCAGCAGAATAGATCTGTCTATACATAGT
TCCTGGAGGATTACACTTATCTCTGAACCCAACAAATGTT
CACCAGTTCTGAATCGATGCAGGAAGAGGTTCCCAAGGAC
ATCACTAATCTTTTCATAGCCCTCAAGTCCTGCTAGAAAG
ACTTTCATGTCCTTGGTCTCCAGCTTCACAATGATATTTT
GGACAAGGTTTCTTCCTTCAAAAAGGGCACCCATCTTTAC
AGTCAGTGGCACAGGCTCCCACTCAGGTCCAACTCTCTCA
AAGTCAATAGATCTAATCCCATCCAGTATTCTTTTGGAGC
CCAACAACTCAAGCTCAAGAGAATCACCAAGTATCAAGGG
ATCTTCCATGTAATCCTCAAACTCTTCAGATCTGATATCA
AAGACACCATCGTTCACCTTGAAGACAGAGTCTGTCCTCA
GTAAGTGGAGGCATTCATCCAACATTCTTCTATCTATCTC
ACCCTTAAAGAGGTGAGAGCATGATAAAAGTTCAGCCACA
CCTGGATTCTGTAATTGGCACCTAACCAAGAATATCAATG
AAAATTTCCTTAAACAGTCAGTATTATTCTGATTGTGCGT
AAAGTCCACTGAAATTGAAAACTCCAATACCCCTTTTGTG
TAGTTGAGCATGTAGTCCCACAGATCCTTTAAGGATTTAA
ATGCCTTTGGGTTTGTCAGGCCCTGCCTAATCAACATGGC
AGCATTACACACAACATCTCCCATTCGGTAAGAGAACCAC
CCAAAACCAAACTGCAAATCATTCCTAAACATAGGCCTCT
CCACATTTTTGTTCACCACCTTTGAGACAAATGATTGAAA
GGGGCCCAGTGCCTCAGCACCATCTTCAGATGGCATCATT
TCTTTATGAGGGAACCATGAAAAATTGCCTAATGTCCTGG
TTGTTGCAACAAATTCTCGAACAAATGATTCAAAATACAC
CTGTTTTAAGAAGTTCTTGCAGACATCCCTCGTGCTAACA
ACAAATTCATCAACCAGACTGGAGTCAGATCGCTGATGAG
AATTGGCAAGGTCAGAAAACAGAACAGTGTAATGTTCATC
CCTTTTCCACTTAACAACATGAGAAATGAGTGACAAGGAT
TCTGAGTTAATATCAATTAAAACACAGAGGTCAAGGAATT
TAATTCTGGGACTCCACCTCATGTTTTTTGAGCTCATGTC
AGACATAAATGGAAGAAGCTGATCCTCAAAGATCTTGGGA
TATAGCCGCCTCACAGATTGAATCACTTGGTTCAAATTCA
CTTTGTCCTCCAGTAGCCTTGAGCTCTCAGGCTTTCTTGC
TACATAATCACATGGGTTTAAGTGCTTAAGAGTTAGGTTC
TCACTGTTATTCTTCCCTTTGGTCGGTTCTGCTAGGACCC
AAACACCCAACTCAAAAGAGTTGCTCAATGAAATACAAAT
GTAGTCCCAAAGAAGAGGCCTTAAAAGGCATATATGATCA
CGGTGGGCTTCTGGATGAGACTGTTTGTCACAAATGTACA
GCGTTATACCATCCCGATTGCAAACTCTTGTCACATGATC
ATCTGTGGTTAGATCCTCAAGCAGCTTTTTGATATACAGA
TTTTCCCTATTTTTGTTTCTCACACACCTGCTTCCTAGAG
TTTTGCAAAGGCCTATAAAGCCAGATGAGATACAACTCTG
GAAAGCTGACTTGTTGATTGCTTCTGACAGCAGCTTCTGT
GCACCCCTTGTGAATTTACTACAAAGTTTGTTCTGGAGTG
TCTTGATCAATGATGGGATTCTTTCCTCTTGGAAAGTCAT
CACTGATGGATAAACCACCTTTTGTCTTAAAACCATCCTT
AATGGGAACATTTCATTCAAATTCAACCAGTTAACATCTG
CTAACTGATTCAGATCTTCTTCAAGACCGAGGAGGTCTCC
CAATTGAAGAATGGCCTCCtTTTTATCTCTGTTAAATAGG
TCTAAGAAAAATTCTTCATTAAATTCACCATTTTTGAGCT
TATGATGCAGTTTCCTTACAAGCTTTCTTACAACCTTTGT
TTCATTAGGACACAGTTCCTCAATGAGTCTTTGTATTCTG
TAACCTCTAGAACCATCCAGCCAATCTTTCACATCAGTGT
TGGTATTCAGTAGAAATGGATCCAAAGGGAAATTGGCATA
CTTTAGGAGGTCCAGTGTTCTCCTTTGGATACTATTAACT
AGGGAGACTGGGACGCCATTTGCGATGGCTTGATCTGCAA
TTGTATCTATTGTTTCACAAAGTTGATGTGGCTCTTTACA
CTTGACATTGTGTAGCGCTGCAGATACAAACTTTGTGAGA
AGAGGGACTTCCTCCCCCCATACATAGAATCTAGATTTAA
ATTCTGCAGCGAACCTCCCAGCCACACTTTTTGGGCTGAT
AAATTTGTTTAACAAGCCGCTCAGATGAGATTGGAATTCC
AACAGGACAAGGACTTCCTCCGGATCACTTACAACCAGGT
CACTCAGCCTCCTATCAAATAAAGTGATCTGATCATCACT
TGATGTGTAAGCCTCTGGTCTTTCGCCAAAGATAACACCA
ATGCAGTAGTTGATGAACCTCTCGCTAAGCAAACCATAGA
AGTCAGAAGCATTATGCAAGATTCCCTGCCCCATATCAAT
AAGGCTGGATATATGGGATGGCACTATCCCCATTTCAAAA
TATTGTCTGAAAATTCTCTCAGTAACAGTTGTTTCTGAAC
CCCTGAGAAGTTTTAGCTTCGACTTGACATATGATTTCAT
CATTGCATTCACAACAGGAAAGGGGACCTCGACAAGCTTA
TGCATGTGCCAAGTTAACAAAGTGCTAACATGATCTTTCC
CGGAACGCACATACTGGTCATCACCTAGTTTGAGATTTTG
TAGAAACATTAAGAACAAAAATGGGCACATCATTGGTCCC
CATTTGCTGTGATCCATACTATAGTTTAAGAACCCTTCCC
GCACATTGATAGTCATTGACAAGATTGCATTTTCAAATTC
CTTATCATTGTTTAAACAGGAGCCTGAAAAGAAACTTGAA
AAAGACTCAAAATAATCTTCTATTAACCTTGTGAACATTT
TTGTCCTCAAATCTCCAATATAGAGTTCTCTATTTCCCCC
AACCTGCTCTTTATAAGATAGTGCAAATTTCAGCCTTCCA
GAGTCAGGACCTACTGAGGTGTATGATGTTGGTGATTCTT
CTGAGTAGAAGCACAGATTTTTCAAAGCAGCACTCATACA
TTgTGTCAACGACAGAGCTTTACTAAGGGACTCAGAATTA
CTTTCCCTCTCACTGATTCTCACGTCTTCTTCCAGTTTGT
CCCAGTCAAATTTGAAATTCAAGCCTTGCCTTTGCATATG
CCTGTATTTCCCTGAGTACGCATTTGCATTCATTTGCAAC
AGAATCATCTTCATGCAAGAAAACCAATCATTCTCAGAAA
AGAACTTTCTACAAAGGTTTTTTGCCATCTCATCGAGGCC
ACACTGATCTTTAATGACTGAGGTGAAATACAAAGGTGAC
AGCTCTGTGGAACCCTCAACAGCCTCACAGATAAATTTCA
TGTCATCATTGGTTAGACATGATGGGTCAAAGTCTTCTAC
TAAATGGAAAGATATTTCTGACAAGATAACTTTTCTTAAG
TGAGCCATCTTCCCTGTTAGAATAAGCTGTAAATGATGTA
GTCCTTTTGTATTTGTAAGTTTTTCTCCATCTCCTTTGTC
ATTGGCCCTCCTACCTCTTCTGTACCGTGCTATTGTGGTG
TTGACCTTTTCTTCGAGACTTTTGAAGAAGCTTGTCTCTT
CTTCTCCATCAAAACATATTTCTGCCAGGTTGTCTTCCGA
TCTCCCTGTCTCTTCTCCCTTGGAACCGATGACCAATCTA
GAGACTAACTTGGAAACTTTATATTCATAGTCTGAGTGGC
TCAACTTATACTTTTGTTTTCTTACGAAACTCTCCGTAAT
TTGACTCACAGCACTAACAAGCAATTTGTTAAAGTCATAT
TCCAGAAGTCGTTCTCCATTTAGATGCTTATTAACCACCA
CACTTTTGTTACTAGCAAGATCTAATGCTGTCGCACATCC
AGAGTTAGTCATGGGATCTAGGCTGTTTAGCTTCTTCTCT
CCTTTGAAAATTAAAGTGCCGTTGTTAAATGAAGACACCA
TTAGGCTAAAGGCTTCCAGATTAACACCTGGAGTTGTATG
CTGACAGTCAATTTCTTTACTAGTGAATCTCTTCATTTGC
TCATAGAACACACATTCTTCCTCAGGAGTGATTGCTTCCT
TGGGGTTGACAAAAAAACCAAATTGACTTTTGGGCTCAAA
GAACTTTTCAAAACATTTTATCTGATCTGTTAGCCTGTCA
GGGGTCTCCTTTGTGATCAAATGACACAGGTATGACACAT
TCAACATAAATTTAAATTTTGCACTCAACAACACCTTCTC
ACCAGTACCAAAAATAGTTTTTATTAGGAATCTAAGCAGC
TTATACACCACCTTCTCAGCAGGTGTGATCAGATCCTCCC
TCAACTTATCCATTAATGATGTAGATGAAAAATCTGACAC
TATTGCCATCACCAAATATCTGACACTCTGTACCTGCTTT
TGATTTCTCTTTGTTGGGTTGGTGAGCATTAGCAACAATA
GGGTCCTCAGTGCAACCTCAATGTCGGTGAGACAGTCTTT
CAAATCAGGACATGATCTAATCCATGAAATCATGATGTCT
ATCATATTGTATAAGACCTCATCTGAAAAAATTGGTAAAA
AGAACCTTTTAGGATCTGCATAGAAGGAAATTAAATGACC
ATCCGGGCCTTGTATGGAGTAGCACCTTGAAGATTCTCCA
GTCTTCTGGTATAATAGGTGGTATTCTTCAGAGTCCAGTT
TTATTACTTGGCAAAACACTTCTTTGCATTCTACCACTTG
ATATCTCACAGACCCTATTTGATTTTGCCTTAGTCTAGCA
ACTGAGCTAGTTTTCATACTGTTTGTTAAGGCCAGACAAA
CAGATGATAATCTTCTCAGGCTCTGTATGTTCTTCAGCTG
CTCTGTGCTGGGTTGGAAATTGTAATCTTCAAACTTCGTA
TAATACATTATCGGGTGAGCTCCAATTTTCATAAAGTTCT
CAAATTCAGTGAATGGTATGTGGCATTCTTGCTCAAGGTG
TTCAGACAGTCCGTAATGCTCGAAACTCAGTCCCACCACT
AACAGGCATTTTTGAATTTTTGCAATGAACTCACTAATAG
AtGCCCTAAACAATTCCTCAAAAGACACCTTTCTAAACAC
CTTTGACTTTTTTCTATTCCTCAAAAGTCTAATGAACTCC
TCTTTAGTGCTGTGAAAGCTTACCAGCCTATCATTCACAC
TACTATAGCAACAACCCACCCAGTGTTTATCATTTTTTAA
CCCTTTGAATTTCGACTGTTTTATCAATGAGGAAAGACAC
AAAACATCCAGATTTAACAACTGTCTCCTTCTAGTATTCA
ACAGTTTCAAACTCTTGACTTTGTTTAACATAGAGAGGAG
CCTCTCATATTCAGTGCTAGTCTCACTTCCCCTTTCGTGC
CCATGGGTCTCTGCAGTTATGAATCTCATCAAAGGACAGG
ATTCGACTGCCTCCCTGCTTAATGTTAAGATATCATCACT
ATCAGCAAGGTTTTCATAGAGCTCAGAGAATTCCTTGATC
AAGCCTTCAGGGTTTACTTTCTGAAAGTTTCTCTTTAATT
TCCCACTTTCTAAATCTCTTCTAAACCTGCTGAAAAGAGA
GTTTATTCCAAAAACCACATCATCACAGCTCATGTTGGGG
TTGATGCCTTCGTGGCACATCCTCATAATTTCATCATTGT
GAGTTGACCTCGCATCTTTCAGAATTTTCATAGAGTCCAT
ACCGGAGCGCTTGTCGATAGTAGTCTTCAGGGACTCACAG
AGTCTAAAATATTCAGACTCTTCAAAGACTTTCTCATTTT
GGTTAGAATACTCCAAAAGTTTGAATAAAAGGTCTCTAAA
TTTGAAGTTTGCCCACTCTGGCATAAAACTATTATCATAA
TCACAACGACCATCTACTATTGGAACTAATGTGACACCCG
CAACAGCAAGGTCTTCCCTGATGCATGCCAATTTGTTAGT
GTCCTCTATAAATTTCTTCTCAAAACTGGCTGGaGtGCTC
CTAACAAAACACTCAAGAAGAATGAGAGAATTGTCTATCA
GCTTGTAACCATCAGGAATGATAAGTGGTAGTCCTGGGCA
TACAATTCCAGACTCCACCAAAATTGTTTCCACAGACTTA
TCGTCGTGGTTGTGTGTGCAGCCACTCTTGTCTGCACTGT
CTATTTCAATGCAGCGTGACAGCAACTTGAGTCCCTCAAT
CAGAACCATTCTGGGTTCCCTTTGTCCCAGAAAGTTGAGT
TTCTGCCTTGACAACCTCTCATCCTGTTCTATATAGTTTA
AACATAACTCTCTCAATTCTGAGATGATTTCATCCATTGC
GCATCAAAAAGCCTAGGATCCTCGGTGCG 2 Lymphocytic
CGCACCGGGGATCCTAGGCTTTTTGGATTGCGCTTTCCTC choriomeningitis
TAGATCAACTGGGTGTCAGGCCCTATCCTACAGAAGGATG virus segment S,
GGTCAGATTGTGACAATGTTTGAGGCTCTGCCTCACATCA complete sequence
TCGATGAGGTGATCAACATTGTCATTATTGTGCTTATCGT (The genomic
GATCACGGGTATCAAGGCTGTCTACAATTTTGCCACCTGT segment is RNA, the
GGGATATTCGCATTGATCAGTTTCCTACTTCTGGCTGGCA sequence in SEQ ID
GGTCCTGTGGCATGTACGGTCTTAAGGGACCCGACATTTA NO: 2 is shown for
CAAAGGAGTTTACCAATTTAAGTCAGTGGAGTTTGATATG DNA; however,
TCACATCTGAACCTGACCATGCCCAACGCATGTTCAGCCA exchanging all
ACAACTCCCACCATTACATCAGTATGGGGACTTCTGGACT thymidines ("T") in
AGAATTGACCTTCACCAATGATTCCATCATCAGTCACAAC SEQ ID NO: 2 for
TTTTGCAATCTGACCTCTGCCTTCAACAAAAAGACCTTTG uridines ("U")
ACCACACACTCATGAGTATAGTTTCGAGCCTACACCTCAG provides the RNA
TATCAGAGGGAACTCCAACTATAAGGCAGTATCCTGCGAC sequence.)
TTCAACAATGGCATAACCATCCAATACAACTTGACATTCT
CAGATCGACAAAGTGCTCAGAGCCAGTGTAGAACCTTCAG
AGGTAGAGTCCTAGATATGTTTAGAACTGCCTTCGGGGGG
AAATACATGAGGAGTGGCTGGGGCTGGACAGGCTCAGATG
GCAAGACCACCTGGTGTAGCCAGACGAGTTACCAATACCT
GATTATACAAAATAGAACCTGGGAAAACCACTGCACATAT
GCAGGTCCTTTTGGGATGTCCAGGATTCTCCTTTCCCAAG
AGAAGACTAAGTTCTTCACTAGGAGACTAGCGGGCACATT
CACCTGGACTTTGTCAGACTCTTCAGGGGTGGAGAATCCA
GGTGGTTATTGCCTGACCAAATGGATGATTCTTGCTGCAG
AGCTTAAGTGTTTCGGGAACACAGCAGTTGCGAAATGCAA
TGTAAATCATCATGCCGAATTCTCTGACATGCTGCGACTA
ATTGACTACAACAAGGCTGCTTTGAGTAAGTTCAAAGAGG
ACGTAGAATCTGCCTTGCACTTATTCAAAACAACAGTGAA
TTCTTTGATTTCAGATCAACTACTGATGAGGAACCACTTG
AGAGATCTGATGGGGGTGCCATATTGCAATTACTCAAAGT
TTTGGTACCTAGAACATGCAAAGACCGGCGAAACTAGTGT
CCCCAAGTGCTGGCTTGTCACCAATGGTTCTTACTTAAAT
GAGACCCACTTCAGTGATCAAATCGAACAGGAAGCCGATA
ACATGATTACAGAGATGTTGAGGAAGGATTACATAAAGAG
GCAGGGGAGTACCCCCCTAGCATTGATGGACCTTCTGATG
TTTTCCACATCTGCATATCTAGTCAGCATCTTCCTGCACC
TTGTCAAAATACCAACACACAGGCACATAAAAGGTGGCTC
ATGTCCAAAGCCACACCGATTAACCAACAAAGGAATTTGT
AGTTGTGGTGCATTTAAGGTGCCTGGTGTAAAAACCGTCT
GGAAAAGACGCTGAAGAACAGCGCCTCCCTGACTCTCCAC
CTCGAAAGAGGTGGAGAGTCAGGGAGGCCCAGAGGGTCTT
AGAGTGTCACAACATTTGGGCCTCTAAAAATTAGGTCATG
TGGCAGAATGTTGTGAACAGTTTTCAGATCTGGGAGCCTT
GCTTTGGAGGCGCTTTCAAAAATGATGCAGTCCATGAGTG
CACAGTGCGGGGTGATCTCTTTCTTCTTTTTGTCCCTTAC
TATTCCAGTATGCATCTTACACAACCAGCCATATTTGTCC
CACACTTTGTCTTCATACTCCCTCGAAGCTTCCCTGGTCA
TTTCAACATCGATAAGCTTAATGTCCTTCCTATTCTGTGA
GTCCAGAAGCTTTCTGATGTCATCGGAGCCTTGACAGCTT
AGAACCATCCCCTGCGGAAGAGCACCTATAACTGACGAGG
TCAACCCGGGTTGCGCATTGAAGAGGTCGGCAAGATCCAT
GCCGTGTGAGTACTTGGAATCTTGCTTGAATTGTTTTTGA
TCAACGGGTTCCCTGTAAAAGTGTATGAACTGCCCGTTCT
GTGGTTGGAAAATTGCTATTTCCACTGGATCATTAAATCT
ACCCTCAATGTCAATCCATGTAGGAGCGTTGGGGTCAATT
CCTCCCATGAGGTCTTTTAAAAGCATTGTCTGGCTGTAGC
TTAAGCCCACCTGAGGTGGACCTGCTGCTCCAGGCGCTGG
CCTGGGTGAATTGACTGCAGGTTTCTCGCTTGTGAGATCA
ATTGTTGTGTTTTCCCATGCTCTCCCCACAATCGATGTTC
TACAAGCTATGTATGGCCATCCTTCACCTGAAAGGCAAAC
TTTATAGAGGATGTTTTCATAAGGGTTCCTGTCCCCAACT
TGGTCTGAAACAAACATGTTGAGTTTTCTCTTGGCCCCGA
GAACTGCCTTCAAGAGGTCCTCGCTGTTGCTTGGCTTGAT
CAAAATTGACTCTAACATGTTACCCCCATCCAACAGGGCT
GCCCCTGCCTTCACGGCAGCACCAAGACTAAAGTTATAGC
CAGAAATGTTGATGCTGGACTGCTGTTCAGTGATGACCCC
CAGAACTGGGTGCTTGTCTTTCAGCCTTTCAAGATCATTA
AGATTTGGATACTTGACTGTGTAAAGCAAGCCAAGGTCTG
TGAGCGCTTGTACAACGTCATTGAGCGGAGTCTGTGACTG
TTTGGCCATACAAGCCATAGTTAGACTTGGCATTGTGCCA
AATTGATTGTTCAAAAGTGATGAGTCTTTCACATCCCAAA
CTCTTACCACACCACTTGCACCCTGCTGAGGCTTTCTCAT
CCCAACTATCTGTAGGATCTGAGATCTTTGGTCTAGTTGC
TGTGTTGTTAAGTTCCCCATATATACCCCTGAAGCCTGGG
GCCTTTCAGACCTCATGATCTTGGCCTTCAGCTTCTCAAG
GTCAGCCGCAAGAGACATCAGTTCTTCTGCACTGAGCCTC
CCCACTTTCAAAACATTCTTCTTTGATGTTGACTTTAAAT
CCACAAGAGAATGTACAGTCTGGTTGAGACTTCTGAGTCT
CTGTAGGTCTTTGTCATCTCTCTTTTCCTTCCTCATGATC
CTCTGAACATTGCTGACCTCAGAGAAGTCCAACCCATTCA
GAAGGTTGGTTGCATCCTTAATGACAGCAGCCTTCACATC
TGATGTGAAGCTCTGCAATTCTCTTCTCAATGCTTGCGTC
CATTGGAAGCTCTTAACTTCCTTAGACAAGGACATCTTGT
TGCTCAATGGTTTCTCAAGACAAATGCGCAATCAAATGCC TAGGATCCACTGTGCG 3
Lymphocytic GCGCACCGGGGATCCTAGGCTTTTTGGATTGCGCTTTCCT
choriomeningitis CTAGATCAACTGGGTGTCAGGCCCTATCCTACAGAAGGAT virus
clone 13 GGGTCAGATTGTGACAATGTTTGAGGCTCTGCCTCACATC segment S,
complete ATCGATGAGGTGATCAACATTGTCATTATTGTGCTTATCG sequence
(GenBank: TGATCACGGGTATCAAGGCTGTCTACAATTTTGCCACCTG DQ361065.2)
TGGGATATTCGCATTGATCAGTTTCCTACTTCTGGCTGGC (The genomic
AGGTCCTGTGGCATGTACGGTCTTAAGGGACCCGACATTT segment is RNA, the
ACAAAGGAGTTTACCAATTTAAGTCAGTGGAGTTTGATAT sequence in SEQ ID
GTCACATCTGAACCTGACCATGCCCAACGCATGTTCAGCC NO: 3 is shown for
AACAACTCCCACCATTACATCAGTATGGGGACTTCTGGAC DNA; however,
TAGAATTGACCTTCACCAATGATTCCATCATCAGTCACAA exchanging all
CTTTTGCAATCTGACCTCTGCCTTCAACAAAAAGACCTTT thymidines ("T") in
GACCACACACTCATGAGTATAGTTTCGAGCCTACACCTCA SEQ ID NO: 3 for
GTATCAGAGGGAACTCCAACTATAAGGCAGTATCCTGCGA uridines ("U")
CTTCAACAATGGCATAACCATCCAATACAACTTGACATTC provides the RNA
TCAGATGCACAAAGTGCTCAGAGCCAGTGTAGAACCTTCA sequence.)
GAGGTAGAGTCCTAGATATGTTTAGAACTGCCTTCGGGGG
GAAATACATGAGGAGTGGCTGGGGCTGGACAGGCTCAGAT
GGCAAGACCACCTGGTGTAGCCAGACGAGTTACCAATACC
TGATTATACAAAATAGAACCTGGGAAAACCACTGCACATA
TGCAGGTCCTTTTGGGATGTCCAGGATTCTCCTTTCCCAA
GAGAAGACTAAGTTCCTCACTAGGAGACTAGCGGGCACAT
TCACCTGGACTTTGTCAGACTCTTCAGGGGTGGAGAATCC
AGGTGGTTATTGCCTGACCAAATGGATGATTCTTGCTGCA
GAGCTTAAGTGTTTCGGGAACACAGCAGTTGCGAAATGCA
ATGTAAATCATGATGAAGAATTCTGTGACATGCTGCGACT
AATTGACTACAACAAGGCTGCTTTGAGTAAGTTCAAAGAG
GACGTAGAATCTGCCTTGCACTTATTCAAAACAACAGTGA
ATTCTTTGATTTCAGATCAACTACTGATGAGGAACCACTT
GAGAGATCTGATGGGGGTGCCATATTGCAATTACTCAAAG
TTTTGGTACCTAGAACATGCAAAGACCGGCGAAACTAGTG
TCCCCAAGTGCTGGCTTGTCACCAATGGTTCTTACTTAAA
TGAGACCCACTTCAGTGACCAAATCGAACAGGAAGCCGAT
AACATGATTACAGAGATGTTGAGGAAGGATTACATAAAGA
GGCAGGGGAGTACCCCCCTAGCATTGATGGACCTTCTGAT
GTTTTCCACATCTGCATATCTAGTCAGCATCTTCCTGCAC
CTTGTCAAAATACCAACACACAGGCACATAAAAGGTGGCT
CATGTCCAAAGCCACACCGATTAACCAACAAAGGAATTTG
TAGTTGTGGTGCATTTAAGGTGCCTGGTGTAAAAACCGTC
TGGAAAAGACGCTGAAGAACAGCGCCTCCCTGACTCTCCA
CCTCGAAAGAGGTGGAGAGTCAGGGAGGCCCAGAGGGTCT
TAGAGTGTCACAACATTTGGGCCTCTAAAAATTAGGTCAT
GTGGCAGAATGTTGTGAACAGTTTTCAGATCTGGGAGCCT
TGCTTTGGAGGCGCTTTCAAAAATGATGCAGTCCATGAGT
GCACAGTGCGGGGTGATCTCTTTCTTCTTTTTGTCCCTTA
CTATTCCAGTATGCATCTTACACAACCAGCCATATTTGTC
CCACACTTTGTCTTCATACTCCCTCGAAGCTTCCCTGGTC
ATTTCAACATCGATAAGCTTAATGTCCTTCCTATTCTGTG
AGTCCAGAAGCTTTCTGATGTCATCGGAGCCTTGACAGCT
TAGAACCATCCCCTGCGGAAGAGCACCTATAACTGACGAG
GTCAACCCGGGTTGCGCATTGAAGAGGTCGGCAAGATCCA
TGCCGTGTGAGTACTTGGAATCTTGCTTGAATTGTTTTTG
ATCAACGGGTTCCCTGTAAAAGTGTATGAACTGCCCGTTC
TGTGGTTGGAAAATTGCTATTTCCACTGGATCATTAAATC
TACCCTCAATGTCAATCCATGTAGGAGCGTTGGGGTCAAT
TCCTCCCATGAGGTCTTTTAAAAGCATTGTCTGGCTGTAG
CTTAAGCCCACCTGAGGTGGACCTGCTGCTCCAGGCGCTG
GCCTGGGTGAATTGACTGCAGGTTTCTCGCTTGTGAGATC
AATTGTTGTGTTTTCCCATGCTCTCCCCACAATCGATGTT
CTACAAGCTATGTATGGCCATCCTTCACCTGAAAGGCAAA
CTTTATAGAGGATGTTTTCATAAGGGTTCCTGTCCCCAAC
TTGGTCTGAAACAAACATGTTGAGTTTTCTCTTGGCCCCG
AGAACTGCCTTCAAGAGGTCCTCGCTGTTGCTTGGCTTGA
TCAAAATTGACTCTAACATGTTACCCCCATCCAACAGGGC
TGCCCCTGCCTTCACGGCAGCACCAAGACTAAAGTTATAG
CCAGAAATGTTGATGCTGGACTGCTGTTCAGTGATGACCC
CCAGAACTGGGTGCTTGTCTTTCAGCCTTTCAAGATCATT
AAGATTTGGATACTTGACTGTGTAAAGCAAGCCAAGGTCT
GTGAGCGCTTGTACAACGTCATTGAGCGGAGTCTGTGACT
GTTTGGCCATACAAGCCATAGTTAGACTTGGCATTGTGCC
AAATTGATTGTTCAAAAGTGATGAGTCTTTCACATCCCAA
ACTCTTACCACACCACTTGCACCCTGCTGAGGCTTTCTCA
TCCCAACTATCTGTAGGATCTGAGATCTTTGGTCTAGTTG
CTGTGTTGTTAAGTTCCCCATATATACCCCTGAAGCCTGG
GGCCTTTCAGACCTCATGATCTTGGCCTTCAGCTTCTCAA
GGTCAGCCGCAAGAGACATCAGTTCTTCTGCACTGAGCCT
CCCCACTTTCAAAACATTCTTCTTTGATGTTGACTTTAAA
TCCACAAGAGAATGTACAGTCTGGTTGAGACTTCTGAGTC
TCTGTAGGTCTTTGTCATCTCTCTTTTCCTTCCTCATGAT
CCTCTGAACATTGCTGACCTCAGAGAAGTCCAACCCATTC
AGAAGGTTGGTTGCATCCTTAATGACAGCAGCCTTCACAT
CTGATGTGAAGCTCTGCAATTCTCTTCTCAATGCTTGCGT
CCATTGGAAGCTCTTAACTTCCTTAGACAAGGACATCTTG
TTGCTCAATGGTTTCTCAAGACAAATGCGCAATCAAATGC CTAGGATCCACTGTGCG 4
Lymphocytic GCGCACCGGGGATCCTAGGCATTTTTGTTGCGCATTTTGT
choriomeningitis TGTGTTATTTGTTGCACAGCCCTTCATCGTGGGACCTTCA strain MP
segment CAAACAAACCAAACCACCAGCCATGGGCCAAGGCAAGTCC L, complete
AAAGAGGGAAGGGATGCCAGCAATACGAGCAGAGCTGAAA sequence
TTCTGCCAGACACCACCTATCTCGGACCTCTGAACTGCAA (The genomic
GTCATGCTGGCAGAGATTTGACAGTTTAGTCAGATGCCAT segment is RNA, the
GACCACTATCTCTGCAGACACTGCCTGAACCTCCTGCTGT sequence in SEQ ID
CAGTCTCCGACAGGTGCCCTCTCTGCAAACATCCATTGCC NO: 4 is shown for
AACCAAACTGAAAATATCCACGGCCCCAAGCTCTCCACCC DNA; however,
CCTTACGAGGAGTGACGCCCCGAGCCCCAACACCGACACA exchanging all
AGGAGGCCACCAACACAACGCCCAACACGGAACACACACA thymidines ("T") in
CACACACCCACACACACATCCACACACACGCGCCCCCACA SEQ ID NO: 4 for
ACGGGGGCGCCCCCCCGGGGGTGGCCCCCCGGGTGCTCGG uridines ("U")
GCGGAGCCCCACGGAGAGGCCAATTAGTCGATCTCCTCGA provides the RNA
CCACCGACTTGGTCAGCCAGTCATCACAGGACTTGCCCTT sequence.)
AAGTCTGTACTTGCCCACAACTGTTTCATACATCACCGTG
TTCTTTGACTTACTGAAACATAGCCTACAGTCTTTGAAAG
TGAACCAGTCAGGCACAAGTGACAGCGGTACCAGTAGAAT
GGATCTATCTATACACAACTCTTGGAGAATTGTGCTAATT
TCCGACCCCTGTAGATGCTCACCAGTTCTGAATCGATGTA
GAAGAAGGCTCCCAAGGACGTCATCAAAATTTCCATAACC
CTCGAGCTCTGCCAAGAAAACTCTCATATCCTTGGTCTCC
AGTTTCACAACGATGTTCTGAACAAGGCTTCTTCCCTCAA
AAAGAGCACCCATTCTCACAGTCAAGGGCACAGGCTCCCA
TTCAGGCCCAATCCTCTCAAAATCAAGGGATCTGATCCCG
TCCAGTATTTTCCTTGAGCCTATCAGCTCAAGCTCAAGAG
AGTCACCGAGTATCAGGGGGTCCTCCATATAGTCCTCAAA
CTCTTCAGACCTAATGTCAAAAACACCATCGTTCACCTTG
AAGATAGAGTCTGATCTCAACAGGTGGAGGCATTCGTCCA
AGAACCTTCTGTCCACCTCACCTTTAAAGAGGTGAGAGCA
TGATAGGAACTCAGCTACACCTGGACCTTGTAACTGGCAC
TTCACTAAAAAGATCAATGAAAACTTCCTCAAACAATCAG
TGTTATTCTGGTTGTGAGTGAAATCTACTGTAATTGAGAA
CTCTAGCACTCCCTCTGTATTATTTATCATGTAATCCCAC
AAGTTTCTCAAAGACTTGAATGCCTTTGGATTTGTCAAGC
CTTGTTTGATTAGCATGGCAGCATTGCACACAATATCTCC
CAATCGGTAAGAGAACCATCCAAATCCAAATTGCAAGTCA
TTCCTAAACATGGGCCTCTCCATATTTTTGTTCACTACTT
TTAAGATGAATGATTGGAAAGGCCCCAATGCTTCAGCGCC
ATCTTCAGATGGCATCATGTCTTTATGAGGGAACCATGAA
AAACTTCCTAGAGTTCTGCTTGTTGCTACAAATTCTCGTA
CAAATGACTCAAAATACACTTGTTTTAAAAAGTTTTTGCA
GACATCCCTTGTACTAACGACAAATTCATCAACAAGGCTT
GAGTCAGAGCGCTGATGGGAATTTACAAGATCAGAAAATA
GAACAGTGTAGTGTTCGTCCCTCTTCCACTTAACTACATG
AGAAATGAGCGATAAAGATTCTGAATTGATATCGATCAAT
ACGCAAAGGTCAAGGAATTTGATTCTGGGACTCCATCTCA
TGTTTTTTGAGCTCATATCAGACATGAAGGGAAGCAGCTG
ATCTTCATAGATTTTAGGGTACAATCGCCTCACAGATTGG
ATTACATGGTTTAAACTTATCTTGTCCTCCAGTAGCCTTG
AACTCTCAGGCTTCCTTGCTACATAATCACATGGGTTCAA
GTGCTTGAGGCTTGAGCTTCCCTCATTCTTCCCTTTCACA
GGTTCAGCTAAGACCCAAACACCCAACTCAAAGGAATTAC
TCAGTGAGATGCAAATATAGTCCCAAAGGAGGGGCCTCAA
GAGACTGATGTGGTCGCAGTGAGCTTCTGGATGACTTTGC
CTGTCACAAATGTACAACATTATGCCATCATGTCTGTGGA
TTGCTGTCACATGCGCATCCATAGCTAGATCCTCAAGCAC
TTTTCTAATGTATAGATTGTCCCTATTTTTATTTCTCACA
CATCTACTTCCCAAAGTTTTGCAAAGACCTATAAAGCCTG
ATGAGATGCAACTTTGAAAGGCTGACTTATTGATTGCTTC
TGACAGCAACTTCTGTGCACCTCTTGTGAACTTACTGCAG
AGCTTGTTCTGGAGTGTCTTGATTAATGATGGGATTCTTT
CCTCTTGGAAAGTCATTACTGATGGATAAACCACTTTCTG
CCTCAAGACCATTCTTAATGGGAACAACTCATTCAAATTC
AGCCAATTTATGTTTGCCAATTGACTTAGATCCTCTTCGA
GGCCAAGGATGTTTCCCAACTGAAGAATGGCTTCCTTTTT
ATCCCTATTGAAGAGGTCTAAGAAGAATTCTTCATTGAAC
TCACCATTCTTGAGCTTATGATGTAGTCTCCTTACAAGCC
TTCTCATGACCTTCGTTTCACTAGGACACAATTCTTCAAT
AAGCCTTTGGATTCTGTAACCTCTAGAGCCATCCAACCAA
TCCTTGACATCAGTATTAGTGTTAAGCAAAAATGGGTCCA
AGGGAAAGTTGGCATATTTTAAGAGGTCTAATGTTCTCTT
CTGGATGCAGTTTACCAATGAAACTGGAACACCATTTGCA
ACAGCTTGATCGGCAATTGTATCTATTGTTTCACAGAGTT
GGTGTGGCTCTTTACACTTAACGTTGTGTAATGCTGCTGA
CACAAATTTTGTTAAAAGTGGGACCTCTTCCCCCCACACA
TAAAATCTGGATTTAAATTCTGCAGCAAATCGCCCCACCA
CACTTTTCGGACTGATGAACTTGTTAAGCAAGCCACTCAA
ATGAGAATGAAATTCCAGCAATACAAGGACTTCCTCAGGG
TCACTATCAACCAGTTCACTCAATCTCCTATCAAATAAGG
TGATCTGATCATCACTTGATGTGTAAGATTCTGGTCTCTC
ACCAAAAATGACACCGATACAATAATTAATGAATCTCTCA
CTGATTAAGCCGTAAAAGTCAGAGGCATTATGTAAGATTC
CCTGTCCCATGTCAATGAGACTGCTTATATGGGAAGGCAC
TATTCCTAATTCAAAATATTCTCGAAAGATTCTTTCAGTC
ACAGTTGTCTCTGAACCCCTAAGAAGTTTCAGCTTTGATT
TGATATATGATTTCATCATTGCATTCACAACAGGAAAAGG
GACCTCAACAAGTTTGTGCATGTGCCAAGTTAATAAGGTG
CTGATATGATCCTTTCCGGAACGCACATACTGGTCATCAC
CCAGTTTGAGATTTTGAAGGAGCATTAAAAACAAAAATGG
GCACATCATTGGCCCCCATTTGCTATGATCCATACTGTAG
TTCAACAACCCCTCTCGCACATTGATGGTCATTGATAGAA
TTGCATTTTCAAATTCTTTGTCATTGTTTAAGCATGAACC
TGAGAAGAAGCTAGAAAAAGACTCAAAATAATCCTCTATC
AATCTTGTAAACATTTTTGTTCTCAAATCCCCAATATAAA
GTTCTCTGTTTCCTCCAACCTGCTCTTTGTATGATAACGC
AAACTTCAACCTTCCGGAATCAGGACCAACTGAAGTGTAT
GACGTTGGTGACTCCTCTGAGTAAAAACATAAATTCTTTA
AAGCAGCACTCATGCATTTTGTCAATGATAGAGCCTTACT
TAGAGACTCAGAATTACTTTCCCTTTCACTAATTCTAACA
TCTTCTTCTAGTTTGTCCCAGTCAAACTTGAAATTCAGAC
CTTGTCTTTGCATGTGCCTGTATTTCCCTGAGTATGCATT
TGCATTCATTTGCAGTAGAATCATTTTCATACACGAAAAC
CAATCACCCTCTGAAAAAAACTTCCTGCAGAGGTTTTTTG
CCATTTCATCCAGACCACATTGTTCTTTGACAGCTGAAGT
GAAATACAATGGTGACAGTTCTGTAGAAGTTTCAATAGCC
TCACAGATAAATTTCATGTCATCATTGGTGAGACAAGATG
GGTCAAAATCTTCCACAAGATGAAAAGAAATTTCTGATAA
GATGACCTTCCTTAAATATGCCATTTTACCTGACAATATA
GTCTGAAGGTGATGCAATCCTTTTGTATTTTCAAACCCCA
CCTCATTTTCCCCTTCATTGGTCTTCTTGCTTCTTTCATA
CCGCTTTATTGTGGAGTTGACCTTATCTTCTAAATTCTTG
AAGAAACTTGTCTCTTCTTCCCCATCAAAGCATATGTCTG
CTGAGTCACCTTCTAGTTTCCCAGCTTCTGTTTCTTTAGA
GCCGATAACCAATCTAGAGACCAACTTTGAAACCTTGTAC
TCGTAATCTGAGTGGTTCAATTTGTACTTCTGCTTTCTCA
TGAAGCTCTCTGTGATCTGACTCACAGCACTAACAAGCAA
TTTGTTAAAATCATACTCTAGGAGCCGTTCCCCATTTAAA
TGTTTGTTAACAACCACACTTTTGTTGCTGGCAAGGTCTA
ATGCTGTTGCACACCCAGAGTTAGTCATGGGATCCAAGCT
ATTGAGCCTCTTCTCCCCTTTGAAAATCAAAGTGCCATTG
TTGAATGAGGACACCATCATGCTAAAGGCCTCCAGATTGA
CACCTGGGGTTGTGCGCTGACAGTCAACTTCTTTCCCAGT
GAACTTCTTCATTTGGTCATAAAAAACACACTCTTCCTCA
GGGGTGATTGACTCTTTAGGGTTAACAAAGAAGCCAAACT
CACTTTTAGGCTCAAAGAATTTCTCAAAGCATTTAATTTG
ATCTGTCAGCCTATCAGGGGTTTCCTTTGTGATTAAATGA
CACAGGTATGACACATTCAACATGAACTTGAACTTTGCGC
TCAACAGTACCTTTTCACCAGTCCCAAAAACAGTTTTGAT
CAAAAATCTGAGCAATTTGTACACTACTTTCTCAGCAGGT
GTGATCAAATCCTCCTTCAACTTGTCCATCAATGATGTGG
ATGAGAAGTCTGAGACAATGGCCATCACTAAATACCTAAT
GTTTTGAACCTGTTTTTGATTCCTCTTTGTTGGGTTGGTG
AGCATGAGTAATAATAGGGTTCTCAATGCAATCTCAACAT
CATCAATGCTGTCCTTCAAGTCAGGACATGATCTGATCCA
TGAGATCATGGTGTCAATCATGTTGTGCAACACTTCATCT
GAGAAGATTGGTAAAAAGAACCTTTTTGGGTCTGCATAAA
AAGAGATTAGATGGCCATTGGGACCTTGTATAGAATAACA
CCTTGAGGATTCTCCAGTCTTTTGATACAGCAGGTGATAT
TCCTCAGAGTCCAATTTTATCACTTGGCAAAATACCTCTT
TACATTCCACCACTTGATACCTTACAGAGCCCAATTGGTT
TTGTCTTAATCTAGCAACTGAACTTGTTTTCATACTGTTT
GTCAAAGCTAGACAGACAGATGACAATCTTTTCAAACTAT
GCATGTTCCTTAATTGTTCCGTATTAGGCTGGAAATCATA
ATCTTCAAACTTTGTATAATACATTATAGGATGAGTTCCG
GACCTCATGAAATTCTCAAACTCAATAAATGGTATGTGGC
ACTCATGCTCAAGATGTTCAGACAGACCATAGTGCCCAAA
ACTAAGTCCCACCACTGACAAGCACCTTTGAACTTTTAAA
ATGAACTCATTTATGGATGTTCTAAACAAATCCTCAAGAG
ATACCTTTCTATACGCCTTTGACTTTCTCCTGTTCCTTAG
AAGTCTGATGAACTCTTCCTTGGTGCTATGAAAGCTCACC
AACCTATCATTCACACTCCCATAGCAACAACCAACCCAGT
GCTTATCATTTTTTGACCCTTTGAGTTTAGACTGTTTGAT
CAACGAAGAGAGACACAAGACATCCAAATTCAGTAACTGT
CTCCTTCTGGTGTTCAATAATTTTAAACTTTTAACTTTGT
TCAACATAGAGAGGAGCCTCTCATACTCAGTGCTAGTCTC
ACTTCCTCTCTCATAACCATGGGTATCTGCTGTGATAAAT
CTCATCAAAGGACAGGATTCAACTGCCTCCTTGCTTAGTG
CTGAAATGTCATCACTGTCAGCAAGAGTCTCATAAAGCTC
AGAGAATTCCTTAATTAAATTTCCGGGGTTGATTTTCTGA
AAACTCCTCTTGAGCTTCCCAGTTTCCAAGTCTCTTCTAA
ACCTGCTGTAAAGGGAGTTTATGCCAAGAACCACATCATC
GCAGTTCATGTTTGGGTTGACACCATCATGGCACATTTTC
ATAATTTCATCATTGTGAAATGATCTTGCATCTTTCAAGA
TTTTCATAGAGTCTATACCGGAACGCTTATCAACAGTGGT
CTTGAGAGATTCGCAAAGTCTGAAGTACTCAGATTCCTCA
AAGACTTTCTCATCTTGGCTAGAATACTCTAAAAGTTTAA
ACAGAAGGTCTCTGAACTTGAAATTCACCCACTCTGGCAT
AAAGCTGTTATCATAATCACACCGACCATCCACTATTGGG
ACCAATGTGATACCCGCAATGGCAAGGTCTTCTTTGATAC
AGGCTAGTTTATTGGTGTCCTCTATAAATTTCTTCTCAAA
ACTAGCTGGTGTGCTTCTAACGAAGCACTCAAGAAGAATG
AGGGAATTGTCAATCAGTTTATAACCATCAGGAATGATCA
AAGGCAGTCCCGGGCACACAATCCCAGACTCTATTAGAAT
TGCCTCAACAGATTTATCATCATGGTTGTGTATGCAGCCG
CTCTTGTCAGCACTGTCTATCTCTATACAACGCGACAAAA
GTTTGAGTCCCTCTATCAATACCATTCTGGGTTCTCTTTG
CCCTAAAAAGTTGAGCTTCTGCCTTGACAACCTCTCATCT
TGTTCTATGTGGTTTAAGCACAACTCTCTCAACTCCGAAA
TAGCCTCATCCATTGCGCATCAAAAAGCCTAGGATCCTCG GTGCG 5 Lymphocytic
CGCACCGGGGATCCTAGGCTTTTTGGATTGCGCTTTCCTC choriomeningitis
AGCTCCGTCTTGTGGGAGAATGGGTCAAATTGTGACGATG strain MP segment
TTTGAGGCTCTGCCTCACATCATTGATGAGGTCATTAACA S, complete
TTGTCATTATCGTGCTTATTATCATCACGAGCATCAAAGC sequence
TGTGTACAATTTCGCCACCTGCGGGATACTTGCATTGATC (The genomic
AGCTTTCTTTTTCTGGCTGGCAGGTCCTGTGGAATGTATG segment is RNA, the
GTCTTGATGGGCCTGACATTTACAAAGGGGTTTACCGATT sequence in SEQ ID
CAAGTCAGTGGAGTTTGACATGTCTTACCTTAACCTGACG NO: 5 is shown for
ATGCCCAATGCATGTTCGGCAAACAACTCCCATCATTATA DNA; however,
TAAGTATGGGGACTTCTGGATTGGAGTTAACCTTCACAAA exchanging all
TGACTCCATCATCACCCACAACTTTTGTAATCTGACTTCC thymidines ("T") in
GCCCTCAACAAGAGGACTTTTGACCACACACTTATGAGTA SEQ ID NO: 5 for
TAGTCTCAAGTCTGCACCTCAGCATTAGAGGGGTCCCCAG uridines ("U")
CTACAAAGCAGTGTCCTGTGATTTTAACAATGGCATCACT provides the RNA
ATTCAATACAACCTGTCATTTTCTAATGCACAGAGCGCTC sequence.)
TGAGTCAATGTAAGACCTTCAGGGGGAGAGTCCTGGATAT
GTTCAGAACTGCTTTTGGAGGAAAGTACATGAGGAGTGGC
TGGGGCTGGACAGGTTCAGATGGCAAGACTACTTGGTGCA
GCCAGACAAACTACCAATATCTGATTATACAAAACAGGAC
TTGGGAAAACCACTGCAGGTACGCAGGCCCTTTCGGAATG
TCTAGAATTCTCTTCGCTCAAGAAAAGACAAGGTTTCTAA
CTAGAAGGCTTGCAGGCACATTCACTTGGACTTTATCAGA
CTCATCAGGAGTGGAGAATCCAGGTGGTTACTGCTTGACC
AAGTGGATGATCCTCGCTGCAGAGCTCAAGTGTTTTGGGA
ACACAGCTGTTGCAAAGTGCAATGTAAATCATGATGAAGA
GTTCTGTGATATGCTACGACTGATTGATTACAACAAGGCT
GCTTTGAGTAAATTCAAAGAAGATGTAGAATCCGCTCTAC
ATCTGTTCAAGACAACAGTGAATTCTTTGATTTCTGATCA
GCTTTTGATGAGAAATCACCTAAGAGACTTGATGGGAGTG
CCATACTGCAATTACTCGAAATTCTGGTATCTAGAGCATG
CAAAGACTGGTGAGACTAGTGTCCCCAAGTGCTGGCTTGT
CAGCAATGGTTCTTATTTGAATGAAACCCATTTCAGCGAC
CAAATTGAGCAGGAAGCAGATAATATGATCACAGAAATGC
TGAGAAAGGACTACATAAAAAGGCAAGGGAGTACCCCTCT
AGCCTTGATGGATCTATTGATGTTTTCTACATCAGCATAT
TTGATCAGCATCTTTCTGCATCTTGTGAGGATACCAACAC
ACAGACACATAAAGGGCGGCTCATGCCCAAAACCACATCG
GTTAACCAGCAAGGGAATCTGTAGTTGTGGTGCATTTAAA
GTACCAGGTGTGGAAACCACCTGGAAAAGACGCTGAACAG
CAGCGCCTCCCTGACTCACCACCTCGAAAGAGGTGGTGAG
TCAGGGAGGCCCAGAGGGTCTTAGAGTGTTACGACATTTG
GACCTCTGAAGATTAGGTCATGTGGTAGGATATTGTGGAC
AGTTTTCAGGTCGGGGAGCCTTGCCTTGGAGGCGCTTTCA
AAGATGATACAGTCCATGAGTGCACAGTGTGGGGTGACCT
CTTTCTTTTTCTTGTCCCTCACTATTCCAGTGTGCATCTT
GCATAGCCAGCCATATTTGTCCCAGACTTTGTCCTCATAT
TCTCTTGAAGCTTCTTTAGTCATCTCAACATCGATGAGCT
TAATGTCTCTTCTGTTTTGTGAATCTAGGAGTTTCCTGAT
GTCATCAGATCCCTGACAACTTAGGACCATTCCCTGTGGA
AGAGCACCTATTACTGAAGATGTCAGCCCAGGTTGTGCAT
TGAAGAGGTCAGCAAGGTCCATGCCATGTGAGTATTTGGA
GTCCTGCTTGAATTGTTTTTGATCAGTGGGTTCTCTATAG
AAATGTATGTACTGCCCATTCTGTGGCTGAAATATTGCTA
TTTCTACCGGGTCATTAAATCTGCCCTCAATGTCAATCCA
TGTAGGAGCGTTAGGGTCAATACCTCCCATGAGGTCCTTC
AGCAACATTGTTTGGCTGTAGCTTAAGCCCACCTGAGGTG
GGCCCGCTGCCCCAGGCGCTGGTTTGGGTGAGTTGGCCAT
AGGCCTCTCATTTGTCAGATCAATTGTTGTGTTCTCCCAT
GCTCTCCCTACAACTGATGTTCTACAAGCTATGTATGGCC
ACCCCTCCCCTGAAAGACAGACTTTGTAGAGGATGTTCTC
GTAAGGATTCCTGTCTCCAACCTGATCAGAAACAAACATG
TTGAGTTTCTTCTTGGCCCCAAGAACTGCTTTCAGGAGAT
CCTCACTGTTGCTTGGCTTAATTAAGATGGATTCCAACAT
GTTACCCCCATCTAACAAGGCTGCCCCTGCTTTCACAGCA
GCACCGAGACTGAAATTGTAGCCAGATATGTTGATGCTAG
ACTGCTGCTCAGTGATGACTCCCAAGACTGGGTGCTTGTC
TTTCAGCCTTTCAAGGTCACTTAGGTTCGGGTACTTGACT
GTGTAAAGCAGCCCAAGGTCTGTGAGTGCTTGCACAACGT
CATTGAGTGAGGTTTGTGATTGTTTGGCCATACAAGCCAT
TGTTAAGCTTGGCATTGTGCCGAATTGATTGTTCAGAAGT
GATGAGTCCTTCACATCCCAGACCCTCACCACACCATTTG
CACTCTGCTGAGGTCTCCTCATTCCAACCATTTGCAGAAT
CTGAGATCTTTGGTCAAGCTGTTGTGCTGTTAAGTTCCCC
ATGTAGACTCCAGAAGTTAGAGGCCTTTCAGACCTCATGA
TTTTAGCCTTCAGTTTTTCAAGGTCAGCTGCAAGGGACAT
CAGTTCTTCTGCACTAAGCCTCCCTACTTTTAGAACATTC
TTTTTTGATGTTGACTTTAGGTCCACAAGGGAATACACAG
TTTGGTTGAGGCTTCTGAGTCTCTGTAAATCTTTGTCATC
CCTCTTCTCTTTCCTCATGATCCTCTGAACATTGCTCACC
TCAGAGAAGTCTAATCCATTCAGAAGGCTGGTGGCATCCT
TGATCACAGCAGCTTTCACATCTGATGTGAAGCCTTGAAG
CTCTCTCCTCAATGCCTGGGTCCATTGAAAGCTTTTAACT
TCTTTGGACAGAGACATTTTGTCACTCAGTGGATTTCCAA
GTCAAATGCGCAATCAAAATGCCTAGGATCCACTGTGCG 6 Amino acid sequence
MSLSKEVKSFQWTQALRRELQGFTSDVKAAVIKDATSLLN of the NP protein
GLDFSEVSNVQRIMRKEKRDDKDLQRLRSLNQTVYSLVDL of the MP strain of
KSTSKKNVLKVGRLSAEELMSLAADLEKLKAKIMRSERPL LCMV
TSGVYMGNLTAQQLDQRSQILQMVGMRRPQQSANGVVRVW
DVKDSSLLNNQFGTMPSLTMACMAKQSQTSLNDVVQALTD
LGLLYTVKYPNLSDLERLKDKHPVLGVITEQQSSINISGY
NFSLGAAVKAGAALLDGGNMLESILIKPSNSEDLLKAVLG
AKKKLNMFVSDQVGDRNPYENILYKVCLSGEGWPYIACRT
SVVGRAWENTTIDLTNERPMANSPKPAPGAAGPPQVGLSY
SQTMLLKDLMGGIDPNAPTWIDIEGRFNDPVEIAIFQPQN
GQYIHEYREPTDQKQFKQDSKYSHGMDLADLENAQPGLTS
SVIGALPQGMVLSCQGSDDIRKLLDSQNRRDIKLIDVEMT
KEASREYEDKVWDKYGWLCKMHTGIVRDKKKKEVTPHCAL
MDCIIFESASKARLPDLKTVHNILPHDLIFRGPNVVTL 7 Amino acid sequence
MGQIVTMFEALPHIIDEVINIVIIVLIIITSIKAVYNFAT of the GP protein
CGILALISFLFLAGRSCGMYGLDGPDIYKGVYRFKSVEFD of the MP strain of
MSYLNLTMPNACSANNSHHYISMGTSGLELTFTNDSIITH LCMV
NFCNLTSALNKRTFDHTLMSIVSSLHLSIRGVPSYKAVSC
DFNNGITIQYNLSFSNAQSALSQCKTFRGRVLDMFRTAFG
GKYMRSGWGWTGSDGKTTWCSQTNYQYLIIQNRTWENHCR
YAGPFGMSRILFAQEKTRFLTRRLAGTFTWTLSDSSGVEN
PGGYCLTKWMILAAELKCFGNTAVAKCNVNHDEEFCDMLR
LIDYNKAALSKFKEDVESALHLFKTTVNSLISDQLLMRNH
LRDLMGVPYCNYSKFWYLEHAKTGETSVPKCWLVSNGSYL
NETHFSDQIEQEADNMITEMLRKDYIKRQGSTPLALMDLL
MFSTSAYLISIFLHLVRIPTHRHIKGGSCPKPHRLTSKGI CSCGAFKVPGVETTWKRR 8 amino
acid sequence MDEAISELRELCLNHIEQDERLSRQKLNFLGQREPRMVLI of the L
protein of EGLKLLSRCIEIDSADKSGCIHNHDDKSVEAILIESGIVC the MP strain
of PGLPLIIPDGYKLIDNSLILLECFVRSTPASFEKKFIEDT LCMV
NKLACIKEDLAIAGITLVPIVDGRCDYDNSFMPEWVNFKF
RDLLFKLLEYSSQDEKVFEESEYFRLCESLKTTVDKRSGI
DSMKILKDARSFHNDEIMKMCHDGVNPNMNCDDVVLGINS
LYSRFRRDLETGKLKRSFQKINPGNLIKEFSELYETLADS
DDISALSKEAVESCPLMRFITADTHGYERGSETSTEYERL
LSMLNKVKSLKLLNTRRRQLLNLDVLCLSSLIKQSKLKGS
KNDKHWVGCCYGSVNDRLVSFHSTKEEFIRLLRNRRKSKA
YRKVSLEDLFRTSINEFILKVQRCLSVVGLSFGHYGLSEH
LEHECHIPFIEFENFMRSGTHPIMYYTKFEDYDFQPNTEQ
LRNMHSLKRLSSVCLALTNSMKTSSVARLRQNQLGSVRYQ
VVECKEVFCQVIKLDSEEYHLLYQKTGESSRCYSIQGPNG
HLISFYADPKRFFLPIFSDEVLHNMIDTMISWIRSCPDLK
DSIDDVEIALRTLLLLMLTNPTKRNQKQVQNIRYLVMAIV
SDFSSTSLMDKLKEDLITPAEKVVYKLLRFLIKTVFGTGE
KVLLSAKFKFMLNVSYLCHLITKETPDRLTDQIKCFEKFF
EPKSEFGFFVNPKESITPEEECVFYDQMKKFTGKEVDCQR
TTPGVNLEAFSMMVSSFNNGTLIFKGEKRLNSLDPMTNSG
CATALDLASNKSVVVNKHLNGERLLEYDFNKLLVSAVSQI
TESFMRKQKYKLNHSDYEYKVSKLVSRLVIGSKETEAGKL
EGDSADICFDGEEETSFFKNLEDKVNSTIKRYERSKKTNE
GENEVGFENTKGLHHLQTILSGKMAYLRKVILSEISFHLV
EDFDPSCLTNDDMKFICEAIETSTELSPLYFTSAVKEQCG
LDEMAKNLCRKFFSEGDWFSCMKMILLQMNANAYSGKYRH
MQRQGLNFKFDWDKLEEDVRISERESNSESLSKALSLTKC
MSAALKNLCFYSEESPTSYTSVGPDSGRLKFALSYKEQVG
GNRELYIGDLRTKMFTRLIEDYFESFSSFFSGSCLNNDKE
FENAILSMTINVREGLLNYSMDHSKWGPMMCPFLFLMLLQ
NLKLGDDQYVRSGKDHISTLLTWHMHKLVEVPFPVVNAMM
KSYIKSKLKLLRGSETTVTERIFREYFELGIVPSHISSLI
DMGQGILHNASDFYGLISERFINYCIGVIFGERPESYTSS
DDQITLFDRRLSELVDSDPEEVLVLLEFHSHLSGLLNKFI
SPKSVVGRFAAEFKSRFYVWGEEVPLLTKFVSAALHNVKC
KEPHQLCETIDTIADQAVANGVPVSLVNCIQKRTLDLLKY
ANFPLDPFLLNTNTDVKDWLDGSRGYRIQRLIEELCPSET
KVMRRLVRRLHHKLKNGEFNEEFFLDLFNRDKKEAILQLG
NILGLEEDLSQLANINWLNLNELFPLRMVLRQKVVYPSVM
TFQEERIPSLIKTLQNKLCSKFTRGAQKLLSEAINKSAFQ
SCISSGFIGLCKTLGSRCVRNKNRDNLYIRKVLEDLAMDA
HVTAIHRHDGIMLYICDRQSHPEAHCDHISLLRPLLWDYI
CISLSNSFELGVWVLAEPVKGKNEGSSSLKHLNPCDYVAR
KPESSRLLEDKISLNHVIQSVRRLYPKIYEDQLLPFMSDM
SSKNMRWSPRIKFLDLCVLIDINSESLSLISHVVKWKRDE
HYTVLFSDLVNSHQRSDSSLVDEFVVSTRDVCKNFLKQVY
FESFVREFVATSRTLGSFSWFPHKDMMPSEDGAEALGPFQ
SFILKVVNKNMERPMFRNDLQFGFGWFSYRLGDIVCNAAM
LIKQGLTNPKAFKSLRNLWDYMINNTEGVLEFSITVDFTH
NQNNTDCLRKFSLIFLVKCQLQGPGVAEFLSCSHLFKGEV
DRRFLDECLHLLRSDSIFKVNDGVFDIRSEEFEDYMEDPL
ILGDSLELELIGSRKILDGIRSLDFERIGPEWEPVPLTVR
MGALFEGRSLVQNIVVKLETKDMRVFLAELEGYGNFDDVL
GSLLLHRFRTGEHLQGSEISTILQELCIDRSILLVPLSLV
PDWFTFKDCRLCFSKSKNTVMYETVVGKYRLKGKSCDDWL TKSVVEEID 9 Amino acid
sequence MGQGKSKEGRDASNTSRAEILPDTTYLGPLNCKSCWQRFD of the Z protein
of SLVRCHDHYLCRHCLNLLLSVSDRCPLCKHPLPTKLKIST the MP strain of
APSSPPPYEE LCMV 10 Junin virus
GCGCACCGGGGATCCTAGGCGTAACTTCATCATTAAAATC Candid#1 L segment
TCAGATTCTGCTCTGAGTGTGACTTACTGCGAAGAGGCAG
ACAAATGGGCAACTGCAACGGGGCATCCAAGTCTAACCAG
CCAGACTCCTCAAGAGCCACACAGCCAGCCGCAGAATTTA
GGAGGGTAGCTCACAGCAGTCTATATGGTAGATATAACTG
TAAGTGCTGCTGGTTTGCTGATACCAATTTGATAACCTGT
AATGATCACTACCTTTGTTTAAGGTGCCATCAGGGTATGT
TAAGGAATTCAGATCTCTGCAATATCTGCTGGAAGCCCCT
GCCCACCACAATCACAGTACCGGTGGAGCCAACAGCACCA
CCACCATAGGCAGACTGCACAGGGTCAGACCCGACCCCCC
GGGGGGCCCCCATGGGGACCCCCCGTGGGGGAACCCCGGG
GGTGATGCGCCATTAGTCAATGTCTTTGATCTCGACTTTG
TGCTTCAGTGGCCTGCATGTCACCCCTTTCAATCTGAACT
GCCCTTGGGGATCTGATATCAGCAGGTCATTTAAAGATCT
GCTGAATGCCACCTTGAAATTTGAGAATTCCAACCAGTCA
CCAAATTTATCAAGTGAACGGATCAACTGCTCTTTGTGTA
GATCATAAACGAGGACAAAGTCCTCTTGCTGAAATAATAT
TGTTTGTGATGTTGTTTTTAGATAAGGCCATAGTTGGCTT
AATAAGGTTTCCACACTATCAATGTCCTCTAGTGCTCCAA
TTGCCTTGACTATGACATCCCCAGACAACTCAACTCTATA
TGTTGACAACCTTTCATTACCTCTGTAAAAGATACCCTCT
TTCAAGACAAGAGGTTCTCCTGGGTTATCTGGCCCAATGA
GGTCATATGCATACTTGTTACTTAGTTCAGAATAAAAGTC
ACCAAAGTTGAACTTAACATGGCTCAGAATATTGTCATCA
TTTGTCGCAGCGTAGCCTGCATCAATAAACAAGCCAGCTA
GGTCAAAGCTCTCATGGCCTGTGAACAATGGTAGGCTAGC
GATAACCAGTGCACCATCCAACAATGAGTGGCTTCCCTCA
GACCCAGAAACACATTGACTCATTGCATCCACATTCAGCT
CTAATTCAGGGGTACCGACATCATCCACTCCTAGTGAACT
GACAATGGTGTAACTGTACACCATCTTTCTTCTAAGTTTA
AATTTTGTCGAAACTCGTGTGTGTTCTACTTGAATGATCA
ATTTTAGTTTCACAGCTTCTTGGCAAGCAACATTGCGCAA
CACAGTGTGCAGGTCCATCATGTCTTCCTGAGGCAACAAG
GAGATGTTGTCAACAGAGACACCCTCAAGGAAAACCTTGA
TATTATCAAAGCTAGAAACTACATAACCCATTGCAATGTC
TTCAACAAACATTGCTCTTGATACTTTATTATTCCTAACT
GACAAGGTAAAATCTGTGAGTTCAGCTAGATCTACTTGAC
TGTCATCTTCTAGATCTAGAACTTCATTGAACCAAAAGAA
GGATTTGAGACACGATGTTGACATGACTAGTGGGTTTATC
ATCGAAGATAAGACAACTTGCACCATGAAGTTCCTGCAAA
CTTGCTGTGGGCTGATGCCAACTTCCCAATTTGTATACTC
TGACTGTCTAACATGGGCTGAAGCGCAATCACTCTGTTTC
ACAATATAAACATTATTATCTCTTACTTTCAATAAGTGAC
TTATAATCCCTAAGTTTTCATTCATCATGTCTAGAGCCAC
ACAGACATCTAGAAACTTGAGTCTTCCACTATCCAAAGAT
CTGTTCACTTGAAGATCATTCATAAAGGGTGCCAAATGTT
CTTCAAATAGTTTGGGGTAATTTCTTCGTATAGAATGCAA
TACATGGTTCATGCCTAATTGGTCTTCTATCTGTCGTACT
GCTTTGGGTTTAACAGCCCAGAAGAAATTCTTATTACATA
AGACCAGAGGGGCCTGTGGACTCTTAATAGCAGAAAACAC
CCACTCCCCTAACTCACAGGCATTTGTCAGCACCAAAGAG
AAGTAATCCCACAAAATTGGTTTAGAAAATTGGTTAACTT
CTTTAAGTGATTTTTGACAGTAAATAACTTTAGGCTTTCT
CTCACAAATTCCACAAAGACATGGCATTATTCGAGTAAAT
ATGTCCTTTATATACAGAAATCCGCCTTTACCATCCCTAA
CACACTTACTCCCCATACTCTTACAAAACCCAATGAAGCC
TGAGGCAACAGAAGACTGAAATGCAGATTTGTTGATTGAC
TCTGCCAAGATCTTCTTCACGCCTTTTGTGAAATTTCTTG
ACAGCCTGGACTGTATTGTCCTTATCAATGTTGGCATCTC
TTCTTTCTCTAACACTCTTCGACTTGTCATGAGTTTGGTC
CTCAAGACCAACCTCAAGTCCCCAAAGCTCGCTAAATTGA
CCCATCTGTAGTCTAGAGTTTGTCTGATTTCATCTTCACT
ACACCCGGCATATTGCAGGAATCCGGATAAAGCCTCATCC
CCTCCCCTGCTTATCAAGTTGATAAGGTTTTCCTCAAAGA
TTTTGCCTCTCTTAATGTCATTGAACACTTTCCTCGCGCA
GTTCCTTATAAACATTGTCTCCTTATCATCAGAAAAAATA
GCTTCAATTTTCCTCTGTAGACGGTACCCTCTAGACCCAT
CAACCCAGTCTTTGACATCTTGTTCTTCAATAGCTCCAAA
CGGAGTCTCTCTGTATCCAGAGTATCTAATCAATTGGTTG
ACTCTAATGGAAATCTTTGACACTATATGAGTGCTAACCC
CATTAGCAATACATTGATCACAAATTGTGTCTATGGTCTC
TGACAGTTGTGTTGGAGTTTTACACTTAACGTTGTGTAGA
GCAGCAGACACAAACTTGGTGAGTAAAGGAGTCTCTTCAC
CCATGACAAAAAATCTTGACTTAAACTCAGCAACAAAAGT
TCCTATCACACTCTTTGGGCTGATAAACTTGTTTAATTTA
GAAGATAAGAATTCATGGAAGCACACCATTTCCAGCAGTT
CTGTCCTGTCTTGAAACTTTTCATCACTAAGGCAAGGAAT
TTTTATAAGGCTAACCTGGTCATCGCTGGAGGTATAAGTG
ACAGGTATCACATCATACAATAAGTCAAGTGCATAACACA
GAAATTGTTCAGTAATTAGCCCATATAAATCTGATGTGTT
GTGCAAGATTCCCTGGCCCATGTCCAAGACAGACATTATA
TGGCTGGGGACCTGGTCCCTTGACTGCAGATACTGGTGAA
AAAACTCTTCACCAACACTAGTACAGTCACAACCCATTAA
ACCTAAAGATCTCTTCAATTTCCCTACACAGTAGGCTTCT
GCAACATTAATTGGAACTTCAACGACCTTATGAAGATGCC
ATTTGAGAATGTTCATTACTGGTTCAAGATTCACCTTTGT
TCTATCTCTGGGATTCTTCAATTCTAATGTGTACAAAAAA
GAAAGGAAAAGTGCTGGGCTCATAGTTGGTCCCCATTTGG
AGTGGTCATATGAACAGGACAAGTCACCATTGTTAACAGC
CATTTTCATATCACAGATTGCACGTTCGAATTCCTTTTCT
GAATTCAAGCATGTGTATTTCATTGAACTACCCACAGCTT
CTGAGAAGTCTTCAACTAACCTGGTCATCAGCTTAGTGTT
GAGGTCTCCCACATACAGTTCTCTATTTGAGCCAACCTGC
TCCTTATAACTTAGTCCAAATTTCAAGTTCCCTGTATTTG
AGCTGATGCTTGTGAACTCTGTAGGAGAGTCGTCTGAATA
GAAACATAAATTCCGTAGGGCTGCATTTGTAAAATAACTT
TTGTCTAGCTTATCAGCAATGGCTTCAGAATTGCTTTCCC
TGGTACTAAGCCGAACCTCATCCTTTAGTCTCAGAACTTC
ACTGGAAAAGCCCAATCTAGATCTACTTCTATGCTCATAA
CTACCCAATTTCTGATCATAATGTCCTTGAATTAAAAGAT
ACTTGAAGCATTCAAAGAATTCATCTTCTTGGTAGGCTAT
TGTTGTCAAATTTTTTAATAACAAACCCAAAGGGCAGATG
TCCTGCGGTGCTTCAAGAAAATAAGTCAATTTAAATGGAG
ATAGATAAACAGCATCACATAACTCTTTATACACATCAGA
CCTGAGCACATCTGGATCAAAATCCTTCACCTCATGCATT
GACACCTCTGCTTTAATCTCTCTCAACACTCCAAAAGGGG
CCCACAATGACTCAAGAGACTCTCGCTCATCAACAGATGG
ATTTTTTGATTTCAACTTGGTGATCTCAACTTTTGTCCCC
TCACTATTAGCCATCTTGGCTAGTGTCATTTGTACGTCAT
TTCTAATACCCTCAAAGGCCCTTACTTGATCCTCTGTTAA
ACTCTCATACATCACTGATAATTCTTCTTGATTGGTTCTG
GTTCTTGAACCGGTGCTCACAAGACCTGTTAGATTTTTTA
ATATTAAGTAGTCCATGGAATCAGGATCAAGATTATACCT
GCCTTTTGTTTTAAACCTCTCAGCCATAGTAGAAACGCAT
GTTGAAACAAGTTTCTCCTTATCATAAACAGAAAGAATAT
TTCCAAGTTCGTCGAGCTTGGGGATTACCACACTTTTATT
GCTTGACAGATCCAGAGCTGTGCTAGTGATGTTAGGCCTG
TAGGGATTGCTTTTCAGTTCACCTGTAACTTTAAGTCTTC
CTCTATTGAAGAGAGAAATGCAGAAGGACAAAATCTCTTT
ACACACTCCTGGAATTTGAGTATCTGAGGAAGTCTTAGCC
TCTTTGGAAAAGAATCTGTCCAATCCTCTTATCATGGTGT
CCTCTTGTTCCAGTGTTAGACTCCCACTTAGAGGGGGGTT
TACAACAACACAATCAAACTTGACTTTGGGCTCAATAAAC
TTCTCAAAACACTTTATTTCATCTGTCACGCCATCAGGTG
TCTCTTTCGTTACCAAGTGACACAGATAACTAACATTTAA
TAGATATTTAAACCTTCTTGCAAAGTAAAGATCTGCATCT
TCCCCTTCACCCAAAATTGTCTGGAAAAGTTCCACAGCCA
TCCTCTGAATCAGCACCTCTGATCCAGACATGCAGTCGAC
CCTTAACTTTGACATCAAATCCACATGATGGATTTGATTT
GCATATGCCATCAAGAAATATCTTAGACCTTGTAAAAATG
TCTGGTTCCTTTTGGAAGGGGAACAGAGTACAGCTAACAC
TAACAATCTTAATATTGGCCTTGTCATTGTCATGAGTTCG
TGGCTAAAATCCAACCAGCTGGTCATTTCCTCACACATTT
CAATTAACACATCCTCCGAAAATATAGGCAGGAAAAATCT
CTTTGGATCACAGTAAAAAGAGCCTTGTTCTTCCAATACC
CCATTGATGGATAGATAGATAGAATAGCACCTTGACTTCT
CACCTGTTTTTTGGTAAAACAAGAGACCAAATGTATTCTT
TGTCAGATGAAATCTTTGTACATAACACTCTCTTAGTCTA
ACATTCCCAAAATATCTAGAATACTCTCTTTCATTGATTA
ACAATCGGGAGGAAAATGATGTCTTCATCGAGTTGACCAA
TGCAAGGGAAATGGAGGACAAAATCCTAAATAATTTCTTC
TGCTCACCTTCCACTAAGCTGCTGAATGGCTGATGTCTAC
AGATTTTCTCAAATTCCTTGTTAATAGTATATCTCATCAC
TGGTCTGTCAGAAACAAGTGCCTGAGCTAAAATCATCAAG
CTATCCATATCAGGGTGTTTTATTAGTTTTTCCAGCTGTG
ACCAGAGATCTTGATGAGAGTTCTTCAATGTTCTGGAACA
CGCTTGAACCCACTTGGGGCTGGTCATCAATTTCTTCCTT
ATTAGTTTAATCGCCTCCAGAATATCTAGAAGTCTGTCAT
TGACTAACATTAACATTTGTCCAACAACTATTCCCGCATT
TCTTAACCTTACAATTGCATCATCATGCGTTTTGAAAAGA
TCACAAAGTAAATTGAGTAAAACTAAGTCCAGAAACAGTA
AAGTGTTTCTCCTGGTGTTGAAAACTTTTAGACCTTTCAC
TTTGTTACACACGGAAAGGGCTTGAAGATAACACCTCTCT
ACAGCATCAATAGATATAGAATTCTCATCTGACTGGCTTT
CCATGTTGACTTCATCTATTGGATGCAATGCGATAGAGTA
GACTACATCCATCAACTTGTTTGCACAAAAAGGGCAGCTG
GGCACATCACTGTCTTTGTGGCTTCCTAATAAGATCAAGT
CATTTATAAGCTTAGACTTTTGTGAAAATTTGAATTTCCC
CAACTGCTTGTCAAAAATCTCCTTCTTAAACCAAAACCTT
AACTTTATGAGTTCTTCTCTTATGACAGATTCTCTAATGT
CTCCTCTAACCCCAACAAAGAGGGATTCATTTAACCTCTC
ATCATAACCCAAAGAATTCTTTTTCAAGCATTCGATGTTT
TCTAATCCCAAGCTCTGGTTTTTTGTGTTGGACAAACTAT
GGATCAATCGCTGGTATTCTTGTTCTTCAATATTAATCTC
TTGCATAAATTTTGATTTCTTTAGGATGTCGATCAGCAAC
CACCGAACTCTTTCAACAACCCAATCAGCAAGGAATCTAT
TGCTGTAGCTAGATCTGCCATCAACCACAGGAACCAACGT
AATCCCTGCCCTTAGTAGGTCGGACTTTAGGTTTAAGAGC
TTTGACATGTCACTCTTCCATTTTCTCTCAAACTCATCAG
GATTGACCCTAACAAAGGTTTCCAATAGGATGAGTGTTTT
CCCTGTGAGTTTGAAGCCATCCGGAATGACTTTTGGAAGG
GTGGGACATAGTATGCCATAGTCAGACAGGATCACATCAA
CAAACTTCTGATCTGAATTGATCTGACAGGCGTGTGCCTC
ACAGGACTCAAGCTCTACTAAACTTGACAGAAGTTTGAAC
CCTTCCAACAACAGAGAGCTGGGGTGATGTTGAGATAAAA
AGATGTCCCTTTGGTATGCTAGCTCCTGTCTTTCTGGAAA
ATGCTTTCTAATAAGGCTTTTTATTTCATTTACTGATTCC
TCCATGCTCAAGTGCCGCCTAGGATCCTCGGTGCG 11 Junin virus
GCGCACCGGGGATCCTAGGCGATTTTGGTTACGCTATAAT Candid#1 S segment
TGTAACTGTTTTCTGTTTGGACAACATCAAAAACATCCAT
TGCACAATGGGGCAGTTCATTAGCTTCATGCAAGAAATAC
CAACCTTTTTGCAGGAGGCTCTGAACATTGCTCTTGTTGC
AGTCAGTCTCATTGCCATCATTAAGGGTATAGTGAACTTG
TACAAAAGTGGTTTATTCCAATTCTTTGTATTCCTAGCGC
TTGCAGGAAGATCCTGCACAGAAGAAGCTTTCAAAATCGG
ACTGCACACTGAGTTCCAGACTGTGTCCTTCTCAATGGTG
GGTCTCTTTTCCAACAATCCACATGACCTACCTTTGTTGT
GTACCTTAAACAAGAGCCATCTTTACATTAAGGGGGGCAA
TGCTTCATTTCAGATCAGCTTTGATGATATTGCAGTATTG
TTGCCACAGTATGATGTTATAATACAACATCCAGCAGATA
TGAGCTGGTGTTCCAAAAGTGATGATCAAATTTGGTTGTC
TCAGTGGTTCATGAATGCTGTGGGACATGATTGGCATCTA
GACCCACCATTTCTGTGTAGGAACCGTGCAAAGACAGAAG
GCTTCATCTTTCAAGTCAACACCTCCAAGACTGGTGTCAA
TGGAAATTATGCTAAGAAGTTTAAGACTGGCATGCATCAT
TTATATAGAGAATATCCTGACCCTTGCTTGAATGGCAAAC
TGTGCTTAATGAAGGCACAACCTACCAGTTGGCCTCTCCA
ATGTCCACTCGACCACGTTAACACATTACACTTCCTTACA
AGAGGTAAAAACATTCAACTTCCAAGGAGGTCCTTGAAAG
CATTCTTCTCCTGGTCTTTGACAGACTCATCCGGCAAGGA
TACCCCTGGAGGCTATTGTCTAGAAGAGTGGATGCTCGTA
GCAGCCAAAATGAAGTGTTTTGGCAATACTGCTGTAGCAA
AATGCAATTTGAATCATGACTCTGAATTCTGTGACATGTT
GAGGCTCTTTGATTACAACAAAAATGCTATCAAAACCCTA
AATGATGAAACTAAGAAACAAGTAAATCTGATGGGGCAGA
CAATCAATGCCCTGATATCTGACAATTTATTGATGAAAAA
CAAAATTAGGGAACTGATGAGTGTCCCTTACTGCAATTAC
ACAAAATTTTGGTATGTCAACCACACACTTTCAGGACAAC
ACTCATTACCAAGGTGCTGGTTAATAAAAAACAACAGCTA
TTTGAACATCTCTGACTTCCGTAATGACTGGATATTAGAA
AGTGACTTCTTAATTTCTGAAATGCTAAGCAAAGAGTATT
CGGACAGGCAGGGTAAAACTCCTTTGACTTTAGTTGACAT
CTGTATTTGGAGCACAGTATTCTTCACAGCGTCACTCTTC
CTTCACTTGGTGGGTATACCCTCCCACAGACACATCAGGG
GCGAAGCATGCCCTTTGCCACACAGGTTGAACAGCTTGGG
TGGTTGCAGATGTGGTAAGTACCCCAATCTAAAGAAACCA
ACAGTTTGGCGTAGAGGACACTAAGACCTCCTGAGGGTCC
CCACCAGCCCGGGCACTGCCCGGGCTGGTGTGGCCCCCCA
GTCCGCGGCCTGGCCGCGGACTGGGGAGGCACTGCTTACA
GTGCATAGGCTGCCTTCGGGAGGAACAGCAAGCTCGGTGG
TAATAGAGGTGTAGGTTCCTCCTCATAGAGCTTCCCATCT
AGCACTGACTGAAACATTATGCAGTCTAGCAGAGCACAGT
GTGGTTCACTGGAGGCCAACTTGAAGGGAGTATCCTTTTC
CCTCTTTTTCTTATTGACAACCACTCCATTGTGATATTTG
CATAAGTGACCATATTTCTCCCAGACCTGTTGATCAAACT
GCCTGGCTTGTTCAGATGTGAGCTTAACATCAACCAGTTT
AAGATCTCTTCTTCCATGGAGGTCAAACAACTTCCTGATG
TCATCGGATCCTTGAGTAGTCACAACCATGTCTGGAGGCA
GCAAGCCGATCACGTAACTAAGAACTCCTGGCATTGCATC
TTCTATGTCCTTCATTAAGATGCCGTGAGAGTGTCTGCTA
CCATTTTTAAACCCTTTCTCATCATGTGGTTTTCTGAAGC
AGTGAATGTACTGCTTACCTGCAGGTTGGAATAATGCCAT
CTCAACAGGGTCAGTGGCTGGTCCTTCAATGTCGAGCCAA
AGGGTGTTGGTGGGGTCGAGTTTCCCCACTGCCTCTCTGA
TGACAGCTTCTTGTATCTCTGTCAAGTTAGCCAATCTCAA
ATTCTGACCGTTTTTTTCCGGCTGTCTAGGACCAGCAACT
GGTTTCCTTGTCAGATCAATACTTGTGTTGTCCCATGACC
TGCCTGTGATTTGTGATCTAGAACCAATATAAGGCCAACC
ATCGCCAGAAAGACAAAGTTTGTACAAAAGGTTTTCATAA
GGATTTCTATTGCCTGGTTTCTCATCAATAAACATGCCTT
CTCTTCGTTTAACCTGAATGGTTGATTTTATGAGGGAAGA
GAAGTTTTCTGGGGTGACTCTGATTGTTTCCAACATGTTT
CCACCATCAAGAATAGATGCTCCAGCCTTTACTGCAGCTG
AAAGACTGAAGTTGTAACCAGAAATATTGATGGAGCTTTC
ATCTTTAGTCACAATCTGAAGGCAGTCATGTTCCTGAGTC
AGTCTGTCAAGGTCACTTAAGTTTGGATACTTCACAGTGT
ATAGAAGCCCAAGTGAGGTTAAAGCTTGTATGACACTGTT
CATTGTCTCACCTCCTTGAACAGTCATGCATGCAATTGTC
AATGCAGGAACAGAGCCAAACTGATTGTTTAGCTTTGAAG
GGTCTTTAACATCCCATATCCTCACCACACCATTTCCCCC
AGTCCCTTGCTGTTGAAATCCCAGTGTTCTCAATATCTCT
GATCTTTTAGCAAGTTGTGACTGGGACAAGTTACCCATGT
AAACCCCCTGAGAGCCTGTCTCTGCTCTTCTTATCTTGTT
TTTTAATTTCTCAAGGTCAGACGCCAACTCCATCAGTTCA
TCCCTCCCCAGATCTCCCACCTTGAAAACTGTGTTTCGTT
GAACACTCCTCATGGACATGAGTCTGTCAACCTCTTTATT
CAGGTCCCTCAACTTGTTGAGGTCTTCTTCCCCCTTTTTA
GTCTTTCTGAGTGCCCGCTGCACCTGTGCCACTTGGTTGA
AGTCGATGCTGTCAGCAATTAGCTTGGCGTCCTTCAAAAC
ATCTGACTTGACAGTCTGAGTGAATTGGCTCAAACCTCTC
CTTAAGGACTGAGTCCATCTAAAGCTTGGAACCTCCTTGG
AGTGTGCCATGCCAGAAGTTCTGGTGATTTTGATCTAGAA
TAGAGTTGCTCAGTGAAAGTGTTAGACACTATGCCTAGGA TCCACTGTGCG 12 Amino acid
sequence MSLSKEVKSFQWTQALRRELQSFTSDVKAAVIKDATNLLN of the NP protein
GLDFSEVSNVQRIMRKEKRDDKDLQRLRSLNQTVHSLVDL of the Clone 13
KSTSKKNVLKVGRLSAEELMSLAADLEKLKAKIMRSERPQ strain of LCMV
ASGVYMGNLTTQQLDQRSQILQIVGMRKPQQGASGVVRVW (GenBank Accession
DVKDSSLLNNQFGTMPSLTMACMAKQSQTPLNDVVQALTD No. ABC96002.1;
LGLLYTVKYPNLNDLERLKDKHPVLGVITEQQSSINISGY GI:86440166)
NFSLGAAVKAGAALLDGGNMLESILIKPSNSEDLLKAVLG
AKRKLNMFVSDQVGDRNPYENILYKVCLSGEGWPYIACRT
SIVGRAWENTTIDLTSEKPAVNSPRPAPGAAGPPQVGLSY
SQTMLLKDLMGGIDPNAPTWIDIEGRFNDPVEIAIFQPQN
GQFIHFYREPVDQKQFKQDSKYSHGMDLADLFNAQPGLTS
SVIGALPQGMVLSCQGSDDIRKLLDSQNRKDIKLIDVEMT
REASREYEDKVWDKYGWLCKMHTGIVRDKKKKEITPHCAL
MDCIIFESASKARLPDLKTVHNILPHDLIFRGPNVVTL 13 Amino acid sequence
MGQIVTMFEALPHIIDEVINIVIIVLIVITGIKAVYNFAT of the GP protein
CGIFALISFLLLAGRSCGMYGLKGPDIYKGVYQFKSVEFD of the Clone 13
MSHLNLTMPNACSANNSHHYISMGTSGLELTFTNDSIISH
strain of LCMV NFCNLTSAFNKKTFDHTLMSIVSSLHLSIRGNSNYKAVSC (GenBank
Accession DFNNGITIQYNLTFSDAQSAQSQCRTFRGRVLDMFRTAFG No. ABC96001.2;
GKYMRSGWGWTGSDGKTTWCSQTSYQYLIIQNRTWENHCT GI:116563462)
YAGPFGMSRILLSQEKTKFLTRRLAGTFTWTLSDSSGVEN
PGGYCLIKWMILAAELKCFGNTAVAKCNVNHDEEFCDMLR
LIDYNKAALSKFKEDVESALHLFKTTVNSLISDQLLMRNH
LRDLMGVPYCNYSKFWYLEHAKTGETSVPKCWLVTNGSYL
NETHFSDQIEQEADNMITEMLRKDYIKRQGSTPLALMDLL
MFSTSAYLVSIFLHLVKIPTHRHIKGGSCPKPHRLTNKGI CSCGAFKVPGVKTVWKRR 14
amino acid sequence MDEIISELRELCLNYIEQDERLSRQKLNFLGQREPRMVLI of the
L protein of EGLKLLSRCIEIDSADKSGCTHNHDDKSVETILVESGIVC the Clone 13
strain PGLPLIIPDGYKLIDNSLILLECFVRSTPASFEKKFIEDT of LCMV
NKLACIREDLAVAGVTLVPIVDGRCDYDNSFMPEWANFKF (GenBank Accession
RDLLFKLLEYSNQNEKVFEESEYFRLCESLKTTIDKRSGM No. ABC96004.1;
DSMKILKDARSTHNDEIMRMCHEGINPNMSCDDVVFGINS GI:86440169)
LFSRFRRDLESGKLKRNFQKVNPEGLIKEFSELYENLADS
DDILTLSREAVESCPLMRFITAETHGHERGSETSTEYERL
LSMLNKVKSLKLLNTRRRQLLNLDVLCLSSLIKQSKFKGL
KNDKHWVGCCYSSVNDRLVSFHSTKEEFIRLLRNRKKSKV
FRKVSFEELFRASISEFIAKIQKCLLVVGLSFEHYGLSEH
LEQECHIPFTEFENFMKIGAHPIMYYTKFEDYNFQPSTEQ
LKNIQSLRRLSSVCLALTNSMKTSSVARLRQNQIGSVRYQ
VVECKEVFCQVIKLDSEEYHLLYQKTGESSRCYSIQGPDG
HLISFYADPKRFFLPIFSDEVLYNMIDIMISWIRSCPDLK
DCLTDIEVALRTLLLLMLTNPTKRNQKQVQSVRYLVMAIV
SDFSSTSLMDKLREDLITPAEKVVYKLLRFLIKTIFGTGE
KVLLSAKFKFMLNVSYLCHLITKETPDRLTDQIKCFEKFF
EPKSQFGFFVNPKEAITPEEECVFYEQMKRFTSKEIDCQH
TTPGVNLEAFSLMVSSFNNGTLIFKGEKKLNSLDPMTNSG
CATALDLASNKSVVVNKHLNGERLLEYDFNKLLVSAVSQI
TESFVRKQKYKLSHSDYEYKVSKLVSRLVIGSKGEETGRS
EDNLAEICFDGEEETSFFKSLEEKVNTTIARYRRGRRAND
KGDGEKLTNTKGLHHLQLILTGKMAHLRKVILSEISFHLV
EDFDPSCLTNDDMKFICEAVEGSTELSPLYFTSVIKDQCG
LDEMAKNLCRKFFSENDWFSCMKMILLQMNANAYSGKYRH
MQRQGLNFKFDWDKLEEDVRISERESNSESLSKALSLTQC
MSAALKNLCFYSEESPTSYTSVGPDSGRLKFALSYKEQVG
GNRELYIGDLRTKMFTRLIEDYFESFSSFFSGSCLNNDKE
FENAILSMTINVREGFLNYSMDHSKWGPMMCPFLFLMFLQ
NLKLGDDQYVRSGKDHVSTLLTWHMHKLVEVPFPVVNAMM
KSYVKSKLKLLRGSETTVTERIFRQYFEMGIVPSHISSLI
DMGQGILHNASDFYGLLSERFINYCIGVIFGERPEAYTSS
DDQITLFDRRLSDLVVSDPEEVLVLLEFQSHLSGLLNKFI
SPKSVAGRFAAEFKSRFYVWGEEVPLLTKFVSAALHNVKC
KEPHQLCETIDTIADQAIANGVPVSLVNSIQRRTLDLLKY
ANFPLDPFLLNTNTDVKDWLDGSRGYRIQRLIEELCPNET
KVVRKLVRKLHHKLKNGEFNEEFFLDLFNRDKKEAILQLG
DLLGLEEDLNQLADVNWLNLNEMFPLRMVLRQKVVYPSVM
TFQEERIPSLIKTLQNKLCSKFTRGAQKLLSEAINKSAFQ
SCISSGFIGLCKTLGSRCVRNKNRENLYIKKLLEDLTTDD
HVTRVCNRDGITLYICDKQSHPEAHRDHICLLRPLLWDYI
CISLSNSFELGVWVLAEPTKGKNNSENLTLKHLNPCDYVA
RKPESSRLLEDKVNLNQVIQSVRRLYPKIFEDQLLPFMSD
MSSKNMRWSPRIKFLDLCVLIDINSESLSLISHVVKWKRD
EHYTVLFSDLANSHQRSDSSLVDEFVVSTRDVCKNFLKQV
YFESFVREFVATTRTLGNFSWFPHKEMMPSEDGAEALGPF
QSFVSKVVNKNVERPMFRNDLQFGFGWFSYRMGDVVCNAA
MLIRQGLTNPKAFKSLKDLWDYMLNYTKGVLEFSISVDFT
HNQNNTDCLRKFSLIFLVRCQLQNPGVAELLSCSHLFKGE
IDRRMLDECLHLLRTDSVFKVNDGVFDIRSEEFEDYMEDP
LILGDSLELELLGSKRILDGIRSIDFERVGPEWEPVPLTV
KMGALFEGRNLVQNIIVKLETKDMKVFLAGLEGYEKISDV
LGNLFLHRFRTGEHLLGSEISVILQELCIDRSILLIPLSL
LPDWFAFKDCRLCFSKSRSTLMYETVGGRFRLKGRSCDDW LGGSVAEDID 15 Amino acid
MGQGKSREEKGTNSTNRAEILPDTTYLGPLSCKSCWQKFD sequence of the Z
SLVRCHDHYLCRHCLNLLLSVSDRCPLCKYPLPTRLKIST protein of the APSSPPPYEE
Clone 13 strain of LCMV (GenBank Accession No. ABC96003.1;
GI:86440168) 16 Amino acid sequence
MGQIVTMFEALPHIIDEVINIVIIVLIIITSIKAVYNFAT of the GP protein
CGILALVSFLFLAGRSCGMYGLNGPDIYKGVYQFKSVEFD of the WE strain of
MSHLNLTMPNACSANNSHHYISMGSSGLELTFTNDSILNH LCMV
NFCNLTSAFNKKTFDHTLMSIVSSLHLSIRGNSNHKAVSC
DFNNGITIQYNLSFSDPQSAISQCRTFRGRVLDMFRTAFG
GKYMRSGWGWAGSDGKTTWCSQTSYQYLIIQNRTWENHCR
YAGPFGMSRILFAQEKTKFLTRRLAGTFTWTLSDSSGVEN
PGGYCLTKWMILAAELKCFGNTAVAKCNVNHDEEFCDMLR
LIDYNKAALSKFKQDVESALHVFKTTVNSLISDQLLMRNH
LRDLMGVPYCNYSKFWYLEHAKTGETSVPKCWLVTNGSYL
NETHFSDQIEQEADNMITEMLRKDYIKRQGSTPLALMDLL
MFSTSAYLISIFLHLVKIPTHRHIKGGSCPKPHRLTNKGI CSCGAFKVPGVKTIWKRR 17 WE
specific primer 5'AATCGTCTCTAAGGATGGGTCAGATTGTGACAATG-3' 18 WE
specific fusion- 5'AATCGTCTCTAAGGATGGGTCAGATTGTGACAATG-3' primer
carrying an overhang complementary to the WET-specific primer 19 WE
specific primer 5'CTCGGTGATCATGTTATCTGCTTCTTGTTCGATTTG A-3' 20 WE
specific fusion- 5'AATCGTCTCTTTCTTTATCTCCTCTTCCAGATGG-3' primer
complementary to the WE-sequence 21 Primer specific for
5'-GGCTCCCAGATCTGAAAACTGTT-3' LCMV NP 22 NP- and GP-specific
5'-GCTGGCTTGTCACTAATGGCTC-3' primers; NP- specific: same as in RT
reaction, GP- specific: 5'
Sequence CWU 1
1
2217229DNAArtificial SequenceLymphocytic choriomeningitis virus
(LCMV) clone 13 segment L (GenBank DQ361066.1) 1gcgcaccggg
gatcctaggc gtttagttgc gctgtttggt tgcacaactt tcttcgtgag 60gctgtcagaa
gtggacctgg ctgatagcga tgggtcaagg caagtccaga gaggagaaag
120gcaccaatag tacaaacagg gccgaaatcc taccagatac cacctatctt
ggccctttaa 180gctgcaaatc ttgctggcag aaatttgaca gcttggtaag
atgccatgac cactaccttt 240gcaggcactg tttaaacctt ctgctgtcag
tatccgacag gtgtcctctt tgtaaatatc 300cattaccaac cagattgaag
atatcaacag ccccaagctc tccacctccc tacgaagagt 360aacaccgtcc
ggccccggcc ccgacaaaca gcccagcaca agggaaccgc acgtcaccca
420acgcacacag acacagcacc caacacagaa cacgcacaca cacacacaca
cacacccaca 480cgcacgcgcc cccaccaccg gggggcgccc ccccccgggg
ggcggccccc cgggagcccg 540ggcggagccc cacggagatg cccatcagtc
gatgtcctcg gccaccgacc cgcccagcca 600atcgtcgcag gacctcccct
tgagtctaaa cctgcccccc actgtttcat acatcaaagt 660gctcctagat
ttgctaaaac aaagtctgca atccttaaag gcgaaccagt ctggcaaaag
720cgacagtgga atcagcagaa tagatctgtc tatacatagt tcctggagga
ttacacttat 780ctctgaaccc aacaaatgtt caccagttct gaatcgatgc
aggaagaggt tcccaaggac 840atcactaatc ttttcatagc cctcaagtcc
tgctagaaag actttcatgt ccttggtctc 900cagcttcaca atgatatttt
ggacaaggtt tcttccttca aaaagggcac ccatctttac 960agtcagtggc
acaggctccc actcaggtcc aactctctca aagtcaatag atctaatccc
1020atccagtatt cttttggagc ccaacaactc aagctcaaga gaatcaccaa
gtatcaaggg 1080atcttccatg taatcctcaa actcttcaga tctgatatca
aagacaccat cgttcacctt 1140gaagacagag tctgtcctca gtaagtggag
gcattcatcc aacattcttc tatctatctc 1200acccttaaag aggtgagagc
atgataaaag ttcagccaca cctggattct gtaattggca 1260cctaaccaag
aatatcaatg aaaatttcct taaacagtca gtattattct gattgtgcgt
1320aaagtccact gaaattgaaa actccaatac cccttttgtg tagttgagca
tgtagtccca 1380cagatccttt aaggatttaa atgcctttgg gtttgtcagg
ccctgcctaa tcaacatggc 1440agcattacac acaacatctc ccattcggta
agagaaccac ccaaaaccaa actgcaaatc 1500attcctaaac ataggcctct
ccacattttt gttcaccacc tttgagacaa atgattgaaa 1560ggggcccagt
gcctcagcac catcttcaga tggcatcatt tctttatgag ggaaccatga
1620aaaattgcct aatgtcctgg ttgttgcaac aaattctcga acaaatgatt
caaaatacac 1680ctgttttaag aagttcttgc agacatccct cgtgctaaca
acaaattcat caaccagact 1740ggagtcagat cgctgatgag aattggcaag
gtcagaaaac agaacagtgt aatgttcatc 1800ccttttccac ttaacaacat
gagaaatgag tgacaaggat tctgagttaa tatcaattaa 1860aacacagagg
tcaaggaatt taattctggg actccacctc atgttttttg agctcatgtc
1920agacataaat ggaagaagct gatcctcaaa gatcttggga tatagccgcc
tcacagattg 1980aatcacttgg ttcaaattca ctttgtcctc cagtagcctt
gagctctcag gctttcttgc 2040tacataatca catgggttta agtgcttaag
agttaggttc tcactgttat tcttcccttt 2100ggtcggttct gctaggaccc
aaacacccaa ctcaaaagag ttgctcaatg aaatacaaat 2160gtagtcccaa
agaagaggcc ttaaaaggca tatatgatca cggtgggctt ctggatgaga
2220ctgtttgtca caaatgtaca gcgttatacc atcccgattg caaactcttg
tcacatgatc 2280atctgtggtt agatcctcaa gcagcttttt gatatacaga
ttttccctat ttttgtttct 2340cacacacctg cttcctagag ttttgcaaag
gcctataaag ccagatgaga tacaactctg 2400gaaagctgac ttgttgattg
cttctgacag cagcttctgt gcaccccttg tgaatttact 2460acaaagtttg
ttctggagtg tcttgatcaa tgatgggatt ctttcctctt ggaaagtcat
2520cactgatgga taaaccacct tttgtcttaa aaccatcctt aatgggaaca
tttcattcaa 2580attcaaccag ttaacatctg ctaactgatt cagatcttct
tcaagaccga ggaggtctcc 2640caattgaaga atggcctcct ttttatctct
gttaaatagg tctaagaaaa attcttcatt 2700aaattcacca tttttgagct
tatgatgcag tttccttaca agctttctta caacctttgt 2760ttcattagga
cacagttcct caatgagtct ttgtattctg taacctctag aaccatccag
2820ccaatctttc acatcagtgt tggtattcag tagaaatgga tccaaaggga
aattggcata 2880ctttaggagg tccagtgttc tcctttggat actattaact
agggagactg ggacgccatt 2940tgcgatggct tgatctgcaa ttgtatctat
tgtttcacaa agttgatgtg gctctttaca 3000cttgacattg tgtagcgctg
cagatacaaa ctttgtgaga agagggactt cctcccccca 3060tacatagaat
ctagatttaa attctgcagc gaacctccca gccacacttt ttgggctgat
3120aaatttgttt aacaagccgc tcagatgaga ttggaattcc aacaggacaa
ggacttcctc 3180cggatcactt acaaccaggt cactcagcct cctatcaaat
aaagtgatct gatcatcact 3240tgatgtgtaa gcctctggtc tttcgccaaa
gataacacca atgcagtagt tgatgaacct 3300ctcgctaagc aaaccataga
agtcagaagc attatgcaag attccctgcc ccatatcaat 3360aaggctggat
atatgggatg gcactatccc catttcaaaa tattgtctga aaattctctc
3420agtaacagtt gtttctgaac ccctgagaag ttttagcttc gacttgacat
atgatttcat 3480cattgcattc acaacaggaa aggggacctc gacaagctta
tgcatgtgcc aagttaacaa 3540agtgctaaca tgatctttcc cggaacgcac
atactggtca tcacctagtt tgagattttg 3600tagaaacatt aagaacaaaa
atgggcacat cattggtccc catttgctgt gatccatact 3660atagtttaag
aacccttccc gcacattgat agtcattgac aagattgcat tttcaaattc
3720cttatcattg tttaaacagg agcctgaaaa gaaacttgaa aaagactcaa
aataatcttc 3780tattaacctt gtgaacattt ttgtcctcaa atctccaata
tagagttctc tatttccccc 3840aacctgctct ttataagata gtgcaaattt
cagccttcca gagtcaggac ctactgaggt 3900gtatgatgtt ggtgattctt
ctgagtagaa gcacagattt ttcaaagcag cactcataca 3960ttgtgtcaac
gacagagctt tactaaggga ctcagaatta ctttccctct cactgattct
4020cacgtcttct tccagtttgt cccagtcaaa tttgaaattc aagccttgcc
tttgcatatg 4080cctgtatttc cctgagtacg catttgcatt catttgcaac
agaatcatct tcatgcaaga 4140aaaccaatca ttctcagaaa agaactttct
acaaaggttt tttgccatct catcgaggcc 4200acactgatct ttaatgactg
aggtgaaata caaaggtgac agctctgtgg aaccctcaac 4260agcctcacag
ataaatttca tgtcatcatt ggttagacat gatgggtcaa agtcttctac
4320taaatggaaa gatatttctg acaagataac ttttcttaag tgagccatct
tccctgttag 4380aataagctgt aaatgatgta gtccttttgt atttgtaagt
ttttctccat ctcctttgtc 4440attggccctc ctacctcttc tgtaccgtgc
tattgtggtg ttgacctttt cttcgagact 4500tttgaagaag cttgtctctt
cttctccatc aaaacatatt tctgccaggt tgtcttccga 4560tctccctgtc
tcttctccct tggaaccgat gaccaatcta gagactaact tggaaacttt
4620atattcatag tctgagtggc tcaacttata cttttgtttt cttacgaaac
tctccgtaat 4680ttgactcaca gcactaacaa gcaatttgtt aaagtcatat
tccagaagtc gttctccatt 4740tagatgctta ttaaccacca cacttttgtt
actagcaaga tctaatgctg tcgcacatcc 4800agagttagtc atgggatcta
ggctgtttag cttcttctct cctttgaaaa ttaaagtgcc 4860gttgttaaat
gaagacacca ttaggctaaa ggcttccaga ttaacacctg gagttgtatg
4920ctgacagtca atttctttac tagtgaatct cttcatttgc tcatagaaca
cacattcttc 4980ctcaggagtg attgcttcct tggggttgac aaaaaaacca
aattgacttt tgggctcaaa 5040gaacttttca aaacatttta tctgatctgt
tagcctgtca ggggtctcct ttgtgatcaa 5100atgacacagg tatgacacat
tcaacataaa tttaaatttt gcactcaaca acaccttctc 5160accagtacca
aaaatagttt ttattaggaa tctaagcagc ttatacacca ccttctcagc
5220aggtgtgatc agatcctccc tcaacttatc cattaatgat gtagatgaaa
aatctgacac 5280tattgccatc accaaatatc tgacactctg tacctgcttt
tgatttctct ttgttgggtt 5340ggtgagcatt agcaacaata gggtcctcag
tgcaacctca atgtcggtga gacagtcttt 5400caaatcagga catgatctaa
tccatgaaat catgatgtct atcatattgt ataagacctc 5460atctgaaaaa
attggtaaaa agaacctttt aggatctgca tagaaggaaa ttaaatgacc
5520atccgggcct tgtatggagt agcaccttga agattctcca gtcttctggt
ataataggtg 5580gtattcttca gagtccagtt ttattacttg gcaaaacact
tctttgcatt ctaccacttg 5640atatctcaca gaccctattt gattttgcct
tagtctagca actgagctag ttttcatact 5700gtttgttaag gccagacaaa
cagatgataa tcttctcagg ctctgtatgt tcttcagctg 5760ctctgtgctg
ggttggaaat tgtaatcttc aaacttcgta taatacatta tcgggtgagc
5820tccaattttc ataaagttct caaattcagt gaatggtatg tggcattctt
gctcaaggtg 5880ttcagacagt ccgtaatgct cgaaactcag tcccaccact
aacaggcatt tttgaatttt 5940tgcaatgaac tcactaatag atgccctaaa
caattcctca aaagacacct ttctaaacac 6000ctttgacttt tttctattcc
tcaaaagtct aatgaactcc tctttagtgc tgtgaaagct 6060taccagccta
tcattcacac tactatagca acaacccacc cagtgtttat cattttttaa
6120ccctttgaat ttcgactgtt ttatcaatga ggaaagacac aaaacatcca
gatttaacaa 6180ctgtctcctt ctagtattca acagtttcaa actcttgact
ttgtttaaca tagagaggag 6240cctctcatat tcagtgctag tctcacttcc
cctttcgtgc ccatgggtct ctgcagttat 6300gaatctcatc aaaggacagg
attcgactgc ctccctgctt aatgttaaga tatcatcact 6360atcagcaagg
ttttcataga gctcagagaa ttccttgatc aagccttcag ggtttacttt
6420ctgaaagttt ctctttaatt tcccactttc taaatctctt ctaaacctgc
tgaaaagaga 6480gtttattcca aaaaccacat catcacagct catgttgggg
ttgatgcctt cgtggcacat 6540cctcataatt tcatcattgt gagttgacct
cgcatctttc agaattttca tagagtccat 6600accggagcgc ttgtcgatag
tagtcttcag ggactcacag agtctaaaat attcagactc 6660ttcaaagact
ttctcatttt ggttagaata ctccaaaagt ttgaataaaa ggtctctaaa
6720tttgaagttt gcccactctg gcataaaact attatcataa tcacaacgac
catctactat 6780tggaactaat gtgacacccg caacagcaag gtcttccctg
atgcatgcca atttgttagt 6840gtcctctata aatttcttct caaaactggc
tggagtgctc ctaacaaaac actcaagaag 6900aatgagagaa ttgtctatca
gcttgtaacc atcaggaatg ataagtggta gtcctgggca 6960tacaattcca
gactccacca aaattgtttc cacagactta tcgtcgtggt tgtgtgtgca
7020gccactcttg tctgcactgt ctatttcaat gcagcgtgac agcaacttga
gtccctcaat 7080cagaaccatt ctgggttccc tttgtcccag aaagttgagt
ttctgccttg acaacctctc 7140atcctgttct atatagttta aacataactc
tctcaattct gagatgattt catccattgc 7200gcatcaaaaa gcctaggatc
ctcggtgcg 722923376DNAArtificial SequenceLymphocytic
choriomeningitis virus (LCMV) segment S 2cgcaccgggg atcctaggct
ttttggattg cgctttcctc tagatcaact gggtgtcagg 60ccctatccta cagaaggatg
ggtcagattg tgacaatgtt tgaggctctg cctcacatca 120tcgatgaggt
gatcaacatt gtcattattg tgcttatcgt gatcacgggt atcaaggctg
180tctacaattt tgccacctgt gggatattcg cattgatcag tttcctactt
ctggctggca 240ggtcctgtgg catgtacggt cttaagggac ccgacattta
caaaggagtt taccaattta 300agtcagtgga gtttgatatg tcacatctga
acctgaccat gcccaacgca tgttcagcca 360acaactccca ccattacatc
agtatgggga cttctggact agaattgacc ttcaccaatg 420attccatcat
cagtcacaac ttttgcaatc tgacctctgc cttcaacaaa aagacctttg
480accacacact catgagtata gtttcgagcc tacacctcag tatcagaggg
aactccaact 540ataaggcagt atcctgcgac ttcaacaatg gcataaccat
ccaatacaac ttgacattct 600cagatcgaca aagtgctcag agccagtgta
gaaccttcag aggtagagtc ctagatatgt 660ttagaactgc cttcgggggg
aaatacatga ggagtggctg gggctggaca ggctcagatg 720gcaagaccac
ctggtgtagc cagacgagtt accaatacct gattatacaa aatagaacct
780gggaaaacca ctgcacatat gcaggtcctt ttgggatgtc caggattctc
ctttcccaag 840agaagactaa gttcttcact aggagactag cgggcacatt
cacctggact ttgtcagact 900cttcaggggt ggagaatcca ggtggttatt
gcctgaccaa atggatgatt cttgctgcag 960agcttaagtg tttcgggaac
acagcagttg cgaaatgcaa tgtaaatcat gatgccgaat 1020tctgtgacat
gctgcgacta attgactaca acaaggctgc tttgagtaag ttcaaagagg
1080acgtagaatc tgccttgcac ttattcaaaa caacagtgaa ttctttgatt
tcagatcaac 1140tactgatgag gaaccacttg agagatctga tgggggtgcc
atattgcaat tactcaaagt 1200tttggtacct agaacatgca aagaccggcg
aaactagtgt ccccaagtgc tggcttgtca 1260ccaatggttc ttacttaaat
gagacccact tcagtgatca aatcgaacag gaagccgata 1320acatgattac
agagatgttg aggaaggatt acataaagag gcaggggagt acccccctag
1380cattgatgga ccttctgatg ttttccacat ctgcatatct agtcagcatc
ttcctgcacc 1440ttgtcaaaat accaacacac aggcacataa aaggtggctc
atgtccaaag ccacaccgat 1500taaccaacaa aggaatttgt agttgtggtg
catttaaggt gcctggtgta aaaaccgtct 1560ggaaaagacg ctgaagaaca
gcgcctccct gactctccac ctcgaaagag gtggagagtc 1620agggaggccc
agagggtctt agagtgtcac aacatttggg cctctaaaaa ttaggtcatg
1680tggcagaatg ttgtgaacag ttttcagatc tgggagcctt gctttggagg
cgctttcaaa 1740aatgatgcag tccatgagtg cacagtgcgg ggtgatctct
ttcttctttt tgtcccttac 1800tattccagta tgcatcttac acaaccagcc
atatttgtcc cacactttgt cttcatactc 1860cctcgaagct tccctggtca
tttcaacatc gataagctta atgtccttcc tattctgtga 1920gtccagaagc
tttctgatgt catcggagcc ttgacagctt agaaccatcc cctgcggaag
1980agcacctata actgacgagg tcaacccggg ttgcgcattg aagaggtcgg
caagatccat 2040gccgtgtgag tacttggaat cttgcttgaa ttgtttttga
tcaacgggtt ccctgtaaaa 2100gtgtatgaac tgcccgttct gtggttggaa
aattgctatt tccactggat cattaaatct 2160accctcaatg tcaatccatg
taggagcgtt ggggtcaatt cctcccatga ggtcttttaa 2220aagcattgtc
tggctgtagc ttaagcccac ctgaggtgga cctgctgctc caggcgctgg
2280cctgggtgaa ttgactgcag gtttctcgct tgtgagatca attgttgtgt
tttcccatgc 2340tctccccaca atcgatgttc tacaagctat gtatggccat
ccttcacctg aaaggcaaac 2400tttatagagg atgttttcat aagggttcct
gtccccaact tggtctgaaa caaacatgtt 2460gagttttctc ttggccccga
gaactgcctt caagaggtcc tcgctgttgc ttggcttgat 2520caaaattgac
tctaacatgt tacccccatc caacagggct gcccctgcct tcacggcagc
2580accaagacta aagttatagc cagaaatgtt gatgctggac tgctgttcag
tgatgacccc 2640cagaactggg tgcttgtctt tcagcctttc aagatcatta
agatttggat acttgactgt 2700gtaaagcaag ccaaggtctg tgagcgcttg
tacaacgtca ttgagcggag tctgtgactg 2760tttggccata caagccatag
ttagacttgg cattgtgcca aattgattgt tcaaaagtga 2820tgagtctttc
acatcccaaa ctcttaccac accacttgca ccctgctgag gctttctcat
2880cccaactatc tgtaggatct gagatctttg gtctagttgc tgtgttgtta
agttccccat 2940atatacccct gaagcctggg gcctttcaga cctcatgatc
ttggccttca gcttctcaag 3000gtcagccgca agagacatca gttcttctgc
actgagcctc cccactttca aaacattctt 3060ctttgatgtt gactttaaat
ccacaagaga atgtacagtc tggttgagac ttctgagtct 3120ctgtaggtct
ttgtcatctc tcttttcctt cctcatgatc ctctgaacat tgctgacctc
3180agagaagtcc aacccattca gaaggttggt tgcatcctta atgacagcag
ccttcacatc 3240tgatgtgaag ctctgcaatt ctcttctcaa tgcttgcgtc
cattggaagc tcttaacttc 3300cttagacaag gacatcttgt tgctcaatgg
tttctcaaga caaatgcgca atcaaatgcc 3360taggatccac tgtgcg
337633377DNAArtificial SequenceLymphocytic choriomeningitis virus
(LCMV) clone 13 segment S (GenBank DQ361065.2) 3gcgcaccggg
gatcctaggc tttttggatt gcgctttcct ctagatcaac tgggtgtcag 60gccctatcct
acagaaggat gggtcagatt gtgacaatgt ttgaggctct gcctcacatc
120atcgatgagg tgatcaacat tgtcattatt gtgcttatcg tgatcacggg
tatcaaggct 180gtctacaatt ttgccacctg tgggatattc gcattgatca
gtttcctact tctggctggc 240aggtcctgtg gcatgtacgg tcttaaggga
cccgacattt acaaaggagt ttaccaattt 300aagtcagtgg agtttgatat
gtcacatctg aacctgacca tgcccaacgc atgttcagcc 360aacaactccc
accattacat cagtatgggg acttctggac tagaattgac cttcaccaat
420gattccatca tcagtcacaa cttttgcaat ctgacctctg ccttcaacaa
aaagaccttt 480gaccacacac tcatgagtat agtttcgagc ctacacctca
gtatcagagg gaactccaac 540tataaggcag tatcctgcga cttcaacaat
ggcataacca tccaatacaa cttgacattc 600tcagatgcac aaagtgctca
gagccagtgt agaaccttca gaggtagagt cctagatatg 660tttagaactg
ccttcggggg gaaatacatg aggagtggct ggggctggac aggctcagat
720ggcaagacca cctggtgtag ccagacgagt taccaatacc tgattataca
aaatagaacc 780tgggaaaacc actgcacata tgcaggtcct tttgggatgt
ccaggattct cctttcccaa 840gagaagacta agttcctcac taggagacta
gcgggcacat tcacctggac tttgtcagac 900tcttcagggg tggagaatcc
aggtggttat tgcctgacca aatggatgat tcttgctgca 960gagcttaagt
gtttcgggaa cacagcagtt gcgaaatgca atgtaaatca tgatgaagaa
1020ttctgtgaca tgctgcgact aattgactac aacaaggctg ctttgagtaa
gttcaaagag 1080gacgtagaat ctgccttgca cttattcaaa acaacagtga
attctttgat ttcagatcaa 1140ctactgatga ggaaccactt gagagatctg
atgggggtgc catattgcaa ttactcaaag 1200ttttggtacc tagaacatgc
aaagaccggc gaaactagtg tccccaagtg ctggcttgtc 1260accaatggtt
cttacttaaa tgagacccac ttcagtgacc aaatcgaaca ggaagccgat
1320aacatgatta cagagatgtt gaggaaggat tacataaaga ggcaggggag
taccccccta 1380gcattgatgg accttctgat gttttccaca tctgcatatc
tagtcagcat cttcctgcac 1440cttgtcaaaa taccaacaca caggcacata
aaaggtggct catgtccaaa gccacaccga 1500ttaaccaaca aaggaatttg
tagttgtggt gcatttaagg tgcctggtgt aaaaaccgtc 1560tggaaaagac
gctgaagaac agcgcctccc tgactctcca cctcgaaaga ggtggagagt
1620cagggaggcc cagagggtct tagagtgtca caacatttgg gcctctaaaa
attaggtcat 1680gtggcagaat gttgtgaaca gttttcagat ctgggagcct
tgctttggag gcgctttcaa 1740aaatgatgca gtccatgagt gcacagtgcg
gggtgatctc tttcttcttt ttgtccctta 1800ctattccagt atgcatctta
cacaaccagc catatttgtc ccacactttg tcttcatact 1860ccctcgaagc
ttccctggtc atttcaacat cgataagctt aatgtccttc ctattctgtg
1920agtccagaag ctttctgatg tcatcggagc cttgacagct tagaaccatc
ccctgcggaa 1980gagcacctat aactgacgag gtcaacccgg gttgcgcatt
gaagaggtcg gcaagatcca 2040tgccgtgtga gtacttggaa tcttgcttga
attgtttttg atcaacgggt tccctgtaaa 2100agtgtatgaa ctgcccgttc
tgtggttgga aaattgctat ttccactgga tcattaaatc 2160taccctcaat
gtcaatccat gtaggagcgt tggggtcaat tcctcccatg aggtctttta
2220aaagcattgt ctggctgtag cttaagccca cctgaggtgg acctgctgct
ccaggcgctg 2280gcctgggtga attgactgca ggtttctcgc ttgtgagatc
aattgttgtg ttttcccatg 2340ctctccccac aatcgatgtt ctacaagcta
tgtatggcca tccttcacct gaaaggcaaa 2400ctttatagag gatgttttca
taagggttcc tgtccccaac ttggtctgaa acaaacatgt 2460tgagttttct
cttggccccg agaactgcct tcaagaggtc ctcgctgttg cttggcttga
2520tcaaaattga ctctaacatg ttacccccat ccaacagggc tgcccctgcc
ttcacggcag 2580caccaagact aaagttatag ccagaaatgt tgatgctgga
ctgctgttca gtgatgaccc 2640ccagaactgg gtgcttgtct ttcagccttt
caagatcatt aagatttgga tacttgactg 2700tgtaaagcaa gccaaggtct
gtgagcgctt gtacaacgtc attgagcgga gtctgtgact 2760gtttggccat
acaagccata gttagacttg gcattgtgcc aaattgattg ttcaaaagtg
2820atgagtcttt cacatcccaa actcttacca caccacttgc accctgctga
ggctttctca 2880tcccaactat ctgtaggatc tgagatcttt ggtctagttg
ctgtgttgtt aagttcccca 2940tatatacccc tgaagcctgg ggcctttcag
acctcatgat cttggccttc agcttctcaa 3000ggtcagccgc aagagacatc
agttcttctg cactgagcct ccccactttc aaaacattct 3060tctttgatgt
tgactttaaa tccacaagag aatgtacagt ctggttgaga cttctgagtc
3120tctgtaggtc tttgtcatct ctcttttcct tcctcatgat cctctgaaca
ttgctgacct 3180cagagaagtc caacccattc agaaggttgg ttgcatcctt
aatgacagca gccttcacat 3240ctgatgtgaa gctctgcaat tctcttctca
atgcttgcgt ccattggaag ctcttaactt 3300ccttagacaa ggacatcttg
ttgctcaatg gtttctcaag acaaatgcgc aatcaaatgc 3360ctaggatcca ctgtgcg
337747205DNAArtificial SequenceLymphocytic choriomeningitis strain
MP segment L 4gcgcaccggg gatcctaggc atttttgttg cgcattttgt
tgtgttattt gttgcacagc 60ccttcatcgt gggaccttca caaacaaacc aaaccaccag
ccatgggcca aggcaagtcc 120aaagagggaa gggatgccag caatacgagc
agagctgaaa ttctgccaga caccacctat 180ctcggacctc tgaactgcaa
gtcatgctgg cagagatttg acagtttagt cagatgccat 240gaccactatc
tctgcagaca ctgcctgaac ctcctgctgt cagtctccga caggtgccct
300ctctgcaaac atccattgcc aaccaaactg aaaatatcca cggccccaag
ctctccaccc 360ccttacgagg agtgacgccc cgagccccaa caccgacaca
aggaggccac caacacaacg 420cccaacacgg aacacacaca cacacaccca
cacacacatc cacacacacg cgcccccaca 480acgggggcgc ccccccgggg
gtggcccccc gggtgctcgg gcggagcccc acggagaggc 540caattagtcg
atctcctcga ccaccgactt ggtcagccag tcatcacagg acttgccctt
600aagtctgtac
ttgcccacaa ctgtttcata catcaccgtg ttctttgact tactgaaaca
660tagcctacag tctttgaaag tgaaccagtc aggcacaagt gacagcggta
ccagtagaat 720ggatctatct atacacaact cttggagaat tgtgctaatt
tccgacccct gtagatgctc 780accagttctg aatcgatgta gaagaaggct
cccaaggacg tcatcaaaat ttccataacc 840ctcgagctct gccaagaaaa
ctctcatatc cttggtctcc agtttcacaa cgatgttctg 900aacaaggctt
cttccctcaa aaagagcacc cattctcaca gtcaagggca caggctccca
960ttcaggccca atcctctcaa aatcaaggga tctgatcccg tccagtattt
tccttgagcc 1020tatcagctca agctcaagag agtcaccgag tatcaggggg
tcctccatat agtcctcaaa 1080ctcttcagac ctaatgtcaa aaacaccatc
gttcaccttg aagatagagt ctgatctcaa 1140caggtggagg cattcgtcca
agaaccttct gtccacctca cctttaaaga ggtgagagca 1200tgataggaac
tcagctacac ctggaccttg taactggcac ttcactaaaa agatcaatga
1260aaacttcctc aaacaatcag tgttattctg gttgtgagtg aaatctactg
taattgagaa 1320ctctagcact ccctctgtat tatttatcat gtaatcccac
aagtttctca aagacttgaa 1380tgcctttgga tttgtcaagc cttgtttgat
tagcatggca gcattgcaca caatatctcc 1440caatcggtaa gagaaccatc
caaatccaaa ttgcaagtca ttcctaaaca tgggcctctc 1500catatttttg
ttcactactt ttaagatgaa tgattggaaa ggccccaatg cttcagcgcc
1560atcttcagat ggcatcatgt ctttatgagg gaaccatgaa aaacttccta
gagttctgct 1620tgttgctaca aattctcgta caaatgactc aaaatacact
tgttttaaaa agtttttgca 1680gacatccctt gtactaacga caaattcatc
aacaaggctt gagtcagagc gctgatggga 1740atttacaaga tcagaaaata
gaacagtgta gtgttcgtcc ctcttccact taactacatg 1800agaaatgagc
gataaagatt ctgaattgat atcgatcaat acgcaaaggt caaggaattt
1860gattctggga ctccatctca tgttttttga gctcatatca gacatgaagg
gaagcagctg 1920atcttcatag attttagggt acaatcgcct cacagattgg
attacatggt ttaaacttat 1980cttgtcctcc agtagccttg aactctcagg
cttccttgct acataatcac atgggttcaa 2040gtgcttgagg cttgagcttc
cctcattctt ccctttcaca ggttcagcta agacccaaac 2100acccaactca
aaggaattac tcagtgagat gcaaatatag tcccaaagga ggggcctcaa
2160gagactgatg tggtcgcagt gagcttctgg atgactttgc ctgtcacaaa
tgtacaacat 2220tatgccatca tgtctgtgga ttgctgtcac atgcgcatcc
atagctagat cctcaagcac 2280ttttctaatg tatagattgt ccctattttt
atttctcaca catctacttc ccaaagtttt 2340gcaaagacct ataaagcctg
atgagatgca actttgaaag gctgacttat tgattgcttc 2400tgacagcaac
ttctgtgcac ctcttgtgaa cttactgcag agcttgttct ggagtgtctt
2460gattaatgat gggattcttt cctcttggaa agtcattact gatggataaa
ccactttctg 2520cctcaagacc attcttaatg ggaacaactc attcaaattc
agccaattta tgtttgccaa 2580ttgacttaga tcctcttcga ggccaaggat
gtttcccaac tgaagaatgg cttccttttt 2640atccctattg aagaggtcta
agaagaattc ttcattgaac tcaccattct tgagcttatg 2700atgtagtctc
cttacaagcc ttctcatgac cttcgtttca ctaggacaca attcttcaat
2760aagcctttgg attctgtaac ctctagagcc atccaaccaa tccttgacat
cagtattagt 2820gttaagcaaa aatgggtcca agggaaagtt ggcatatttt
aagaggtcta atgttctctt 2880ctggatgcag tttaccaatg aaactggaac
accatttgca acagcttgat cggcaattgt 2940atctattgtt tcacagagtt
ggtgtggctc tttacactta acgttgtgta atgctgctga 3000cacaaatttt
gttaaaagtg ggacctcttc cccccacaca taaaatctgg atttaaattc
3060tgcagcaaat cgccccacca cacttttcgg actgatgaac ttgttaagca
agccactcaa 3120atgagaatga aattccagca atacaaggac ttcctcaggg
tcactatcaa ccagttcact 3180caatctccta tcaaataagg tgatctgatc
atcacttgat gtgtaagatt ctggtctctc 3240accaaaaatg acaccgatac
aataattaat gaatctctca ctgattaagc cgtaaaagtc 3300agaggcatta
tgtaagattc cctgtcccat gtcaatgaga ctgcttatat gggaaggcac
3360tattcctaat tcaaaatatt ctcgaaagat tctttcagtc acagttgtct
ctgaacccct 3420aagaagtttc agctttgatt tgatatatga tttcatcatt
gcattcacaa caggaaaagg 3480gacctcaaca agtttgtgca tgtgccaagt
taataaggtg ctgatatgat cctttccgga 3540acgcacatac tggtcatcac
ccagtttgag attttgaagg agcattaaaa acaaaaatgg 3600gcacatcatt
ggcccccatt tgctatgatc catactgtag ttcaacaacc cctctcgcac
3660attgatggtc attgatagaa ttgcattttc aaattctttg tcattgttta
agcatgaacc 3720tgagaagaag ctagaaaaag actcaaaata atcctctatc
aatcttgtaa acatttttgt 3780tctcaaatcc ccaatataaa gttctctgtt
tcctccaacc tgctctttgt atgataacgc 3840aaacttcaac cttccggaat
caggaccaac tgaagtgtat gacgttggtg actcctctga 3900gtaaaaacat
aaattcttta aagcagcact catgcatttt gtcaatgata gagccttact
3960tagagactca gaattacttt ccctttcact aattctaaca tcttcttcta
gtttgtccca 4020gtcaaacttg aaattcagac cttgtctttg catgtgcctg
tatttccctg agtatgcatt 4080tgcattcatt tgcagtagaa tcattttcat
acacgaaaac caatcaccct ctgaaaaaaa 4140cttcctgcag aggttttttg
ccatttcatc cagaccacat tgttctttga cagctgaagt 4200gaaatacaat
ggtgacagtt ctgtagaagt ttcaatagcc tcacagataa atttcatgtc
4260atcattggtg agacaagatg ggtcaaaatc ttccacaaga tgaaaagaaa
tttctgataa 4320gatgaccttc cttaaatatg ccattttacc tgacaatata
gtctgaaggt gatgcaatcc 4380ttttgtattt tcaaacccca cctcattttc
cccttcattg gtcttcttgc ttctttcata 4440ccgctttatt gtggagttga
ccttatcttc taaattcttg aagaaacttg tctcttcttc 4500cccatcaaag
catatgtctg ctgagtcacc ttctagtttc ccagcttctg tttctttaga
4560gccgataacc aatctagaga ccaactttga aaccttgtac tcgtaatctg
agtggttcaa 4620tttgtacttc tgctttctca tgaagctctc tgtgatctga
ctcacagcac taacaagcaa 4680tttgttaaaa tcatactcta ggagccgttc
cccatttaaa tgtttgttaa caaccacact 4740tttgttgctg gcaaggtcta
atgctgttgc acacccagag ttagtcatgg gatccaagct 4800attgagcctc
ttctcccctt tgaaaatcaa agtgccattg ttgaatgagg acaccatcat
4860gctaaaggcc tccagattga cacctggggt tgtgcgctga cagtcaactt
ctttcccagt 4920gaacttcttc atttggtcat aaaaaacaca ctcttcctca
ggggtgattg actctttagg 4980gttaacaaag aagccaaact cacttttagg
ctcaaagaat ttctcaaagc atttaatttg 5040atctgtcagc ctatcagggg
tttcctttgt gattaaatga cacaggtatg acacattcaa 5100catgaacttg
aactttgcgc tcaacagtac cttttcacca gtcccaaaaa cagttttgat
5160caaaaatctg agcaatttgt acactacttt ctcagcaggt gtgatcaaat
cctccttcaa 5220cttgtccatc aatgatgtgg atgagaagtc tgagacaatg
gccatcacta aatacctaat 5280gttttgaacc tgtttttgat tcctctttgt
tgggttggtg agcatgagta ataatagggt 5340tctcaatgca atctcaacat
catcaatgct gtccttcaag tcaggacatg atctgatcca 5400tgagatcatg
gtgtcaatca tgttgtgcaa cacttcatct gagaagattg gtaaaaagaa
5460cctttttggg tctgcataaa aagagattag atggccattg ggaccttgta
tagaataaca 5520ccttgaggat tctccagtct tttgatacag caggtgatat
tcctcagagt ccaattttat 5580cacttggcaa aatacctctt tacattccac
cacttgatac cttacagagc ccaattggtt 5640ttgtcttaat ctagcaactg
aacttgtttt catactgttt gtcaaagcta gacagacaga 5700tgacaatctt
ttcaaactat gcatgttcct taattgttcc gtattaggct ggaaatcata
5760atcttcaaac tttgtataat acattatagg atgagttccg gacctcatga
aattctcaaa 5820ctcaataaat ggtatgtggc actcatgctc aagatgttca
gacagaccat agtgcccaaa 5880actaagtccc accactgaca agcacctttg
aacttttaaa atgaactcat ttatggatgt 5940tctaaacaaa tcctcaagag
atacctttct atacgccttt gactttctcc tgttccttag 6000aagtctgatg
aactcttcct tggtgctatg aaagctcacc aacctatcat tcacactccc
6060atagcaacaa ccaacccagt gcttatcatt ttttgaccct ttgagtttag
actgtttgat 6120caacgaagag agacacaaga catccaaatt cagtaactgt
ctccttctgg tgttcaataa 6180ttttaaactt ttaactttgt tcaacataga
gaggagcctc tcatactcag tgctagtctc 6240acttcctctc tcataaccat
gggtatctgc tgtgataaat ctcatcaaag gacaggattc 6300aactgcctcc
ttgcttagtg ctgaaatgtc atcactgtca gcaagagtct cataaagctc
6360agagaattcc ttaattaaat ttccggggtt gattttctga aaactcctct
tgagcttccc 6420agtttccaag tctcttctaa acctgctgta aagggagttt
atgccaagaa ccacatcatc 6480gcagttcatg tttgggttga caccatcatg
gcacattttc ataatttcat cattgtgaaa 6540tgatcttgca tctttcaaga
ttttcataga gtctataccg gaacgcttat caacagtggt 6600cttgagagat
tcgcaaagtc tgaagtactc agattcctca aagactttct catcttggct
6660agaatactct aaaagtttaa acagaaggtc tctgaacttg aaattcaccc
actctggcat 6720aaagctgtta tcataatcac accgaccatc cactattggg
accaatgtga tacccgcaat 6780ggcaaggtct tctttgatac aggctagttt
attggtgtcc tctataaatt tcttctcaaa 6840actagctggt gtgcttctaa
cgaagcactc aagaagaatg agggaattgt caatcagttt 6900ataaccatca
ggaatgatca aaggcagtcc cgggcacaca atcccagact ctattagaat
6960tgcctcaaca gatttatcat catggttgtg tatgcagccg ctcttgtcag
cactgtctat 7020ctctatacaa cgcgacaaaa gtttgagtcc ctctatcaat
accattctgg gttctctttg 7080ccctaaaaag ttgagcttct gccttgacaa
cctctcatct tgttctatgt ggtttaagca 7140caactctctc aactccgaaa
tagcctcatc cattgcgcat caaaaagcct aggatcctcg 7200gtgcg
720553359DNAArtificial SequenceLymphocytic choriomeningitis strain
MP segment S 5cgcaccgggg atcctaggct ttttggattg cgctttcctc
agctccgtct tgtgggagaa 60tgggtcaaat tgtgacgatg tttgaggctc tgcctcacat
cattgatgag gtcattaaca 120ttgtcattat cgtgcttatt atcatcacga
gcatcaaagc tgtgtacaat ttcgccacct 180gcgggatact tgcattgatc
agctttcttt ttctggctgg caggtcctgt ggaatgtatg 240gtcttgatgg
gcctgacatt tacaaagggg tttaccgatt caagtcagtg gagtttgaca
300tgtcttacct taacctgacg atgcccaatg catgttcggc aaacaactcc
catcattata 360taagtatggg gacttctgga ttggagttaa ccttcacaaa
tgactccatc atcacccaca 420acttttgtaa tctgacttcc gccctcaaca
agaggacttt tgaccacaca cttatgagta 480tagtctcaag tctgcacctc
agcattagag gggtccccag ctacaaagca gtgtcctgtg 540attttaacaa
tggcatcact attcaataca acctgtcatt ttctaatgca cagagcgctc
600tgagtcaatg taagaccttc agggggagag tcctggatat gttcagaact
gcttttggag 660gaaagtacat gaggagtggc tggggctgga caggttcaga
tggcaagact acttggtgca 720gccagacaaa ctaccaatat ctgattatac
aaaacaggac ttgggaaaac cactgcaggt 780acgcaggccc tttcggaatg
tctagaattc tcttcgctca agaaaagaca aggtttctaa 840ctagaaggct
tgcaggcaca ttcacttgga ctttatcaga ctcatcagga gtggagaatc
900caggtggtta ctgcttgacc aagtggatga tcctcgctgc agagctcaag
tgttttggga 960acacagctgt tgcaaagtgc aatgtaaatc atgatgaaga
gttctgtgat atgctacgac 1020tgattgatta caacaaggct gctttgagta
aattcaaaga agatgtagaa tccgctctac 1080atctgttcaa gacaacagtg
aattctttga tttctgatca gcttttgatg agaaatcacc 1140taagagactt
gatgggagtg ccatactgca attactcgaa attctggtat ctagagcatg
1200caaagactgg tgagactagt gtccccaagt gctggcttgt cagcaatggt
tcttatttga 1260atgaaaccca tttcagcgac caaattgagc aggaagcaga
taatatgatc acagaaatgc 1320tgagaaagga ctacataaaa aggcaaggga
gtacccctct agccttgatg gatctattga 1380tgttttctac atcagcatat
ttgatcagca tctttctgca tcttgtgagg ataccaacac 1440acagacacat
aaagggcggc tcatgcccaa aaccacatcg gttaaccagc aagggaatct
1500gtagttgtgg tgcatttaaa gtaccaggtg tggaaaccac ctggaaaaga
cgctgaacag 1560cagcgcctcc ctgactcacc acctcgaaag aggtggtgag
tcagggaggc ccagagggtc 1620ttagagtgtt acgacatttg gacctctgaa
gattaggtca tgtggtagga tattgtggac 1680agttttcagg tcggggagcc
ttgccttgga ggcgctttca aagatgatac agtccatgag 1740tgcacagtgt
ggggtgacct ctttcttttt cttgtccctc actattccag tgtgcatctt
1800gcatagccag ccatatttgt cccagacttt gtcctcatat tctcttgaag
cttctttagt 1860catctcaaca tcgatgagct taatgtctct tctgttttgt
gaatctagga gtttcctgat 1920gtcatcagat ccctgacaac ttaggaccat
tccctgtgga agagcaccta ttactgaaga 1980tgtcagccca ggttgtgcat
tgaagaggtc agcaaggtcc atgccatgtg agtatttgga 2040gtcctgcttg
aattgttttt gatcagtggg ttctctatag aaatgtatgt actgcccatt
2100ctgtggctga aatattgcta tttctaccgg gtcattaaat ctgccctcaa
tgtcaatcca 2160tgtaggagcg ttagggtcaa tacctcccat gaggtccttc
agcaacattg tttggctgta 2220gcttaagccc acctgaggtg ggcccgctgc
cccaggcgct ggtttgggtg agttggccat 2280aggcctctca tttgtcagat
caattgttgt gttctcccat gctctcccta caactgatgt 2340tctacaagct
atgtatggcc acccctcccc tgaaagacag actttgtaga ggatgttctc
2400gtaaggattc ctgtctccaa cctgatcaga aacaaacatg ttgagtttct
tcttggcccc 2460aagaactgct ttcaggagat cctcactgtt gcttggctta
attaagatgg attccaacat 2520gttaccccca tctaacaagg ctgcccctgc
tttcacagca gcaccgagac tgaaattgta 2580gccagatatg ttgatgctag
actgctgctc agtgatgact cccaagactg ggtgcttgtc 2640tttcagcctt
tcaaggtcac ttaggttcgg gtacttgact gtgtaaagca gcccaaggtc
2700tgtgagtgct tgcacaacgt cattgagtga ggtttgtgat tgtttggcca
tacaagccat 2760tgttaagctt ggcattgtgc cgaattgatt gttcagaagt
gatgagtcct tcacatccca 2820gaccctcacc acaccatttg cactctgctg
aggtctcctc attccaacca tttgcagaat 2880ctgagatctt tggtcaagct
gttgtgctgt taagttcccc atgtagactc cagaagttag 2940aggcctttca
gacctcatga ttttagcctt cagtttttca aggtcagctg caagggacat
3000cagttcttct gcactaagcc tccctacttt tagaacattc ttttttgatg
ttgactttag 3060gtccacaagg gaatacacag tttggttgag gcttctgagt
ctctgtaaat ctttgtcatc 3120cctcttctct ttcctcatga tcctctgaac
attgctcacc tcagagaagt ctaatccatt 3180cagaaggctg gtggcatcct
tgatcacagc agctttcaca tctgatgtga agccttgaag 3240ctctctcctc
aatgcctggg tccattgaaa gcttttaact tctttggaca gagacatttt
3300gtcactcagt ggatttccaa gtcaaatgcg caatcaaaat gcctaggatc
cactgtgcg 33596558PRTLymphocytic choriomeningitis virusNP protein
of the MP strain of LCMV 6Met Ser Leu Ser Lys Glu Val Lys Ser Phe
Gln Trp Thr Gln Ala Leu1 5 10 15Arg Arg Glu Leu Gln Gly Phe Thr Ser
Asp Val Lys Ala Ala Val Ile 20 25 30Lys Asp Ala Thr Ser Leu Leu Asn
Gly Leu Asp Phe Ser Glu Val Ser 35 40 45Asn Val Gln Arg Ile Met Arg
Lys Glu Lys Arg Asp Asp Lys Asp Leu 50 55 60Gln Arg Leu Arg Ser Leu
Asn Gln Thr Val Tyr Ser Leu Val Asp Leu65 70 75 80Lys Ser Thr Ser
Lys Lys Asn Val Leu Lys Val Gly Arg Leu Ser Ala 85 90 95Glu Glu Leu
Met Ser Leu Ala Ala Asp Leu Glu Lys Leu Lys Ala Lys 100 105 110Ile
Met Arg Ser Glu Arg Pro Leu Thr Ser Gly Val Tyr Met Gly Asn 115 120
125Leu Thr Ala Gln Gln Leu Asp Gln Arg Ser Gln Ile Leu Gln Met Val
130 135 140Gly Met Arg Arg Pro Gln Gln Ser Ala Asn Gly Val Val Arg
Val Trp145 150 155 160Asp Val Lys Asp Ser Ser Leu Leu Asn Asn Gln
Phe Gly Thr Met Pro 165 170 175Ser Leu Thr Met Ala Cys Met Ala Lys
Gln Ser Gln Thr Ser Leu Asn 180 185 190Asp Val Val Gln Ala Leu Thr
Asp Leu Gly Leu Leu Tyr Thr Val Lys 195 200 205Tyr Pro Asn Leu Ser
Asp Leu Glu Arg Leu Lys Asp Lys His Pro Val 210 215 220Leu Gly Val
Ile Thr Glu Gln Gln Ser Ser Ile Asn Ile Ser Gly Tyr225 230 235
240Asn Phe Ser Leu Gly Ala Ala Val Lys Ala Gly Ala Ala Leu Leu Asp
245 250 255Gly Gly Asn Met Leu Glu Ser Ile Leu Ile Lys Pro Ser Asn
Ser Glu 260 265 270Asp Leu Leu Lys Ala Val Leu Gly Ala Lys Lys Lys
Leu Asn Met Phe 275 280 285Val Ser Asp Gln Val Gly Asp Arg Asn Pro
Tyr Glu Asn Ile Leu Tyr 290 295 300Lys Val Cys Leu Ser Gly Glu Gly
Trp Pro Tyr Ile Ala Cys Arg Thr305 310 315 320Ser Val Val Gly Arg
Ala Trp Glu Asn Thr Thr Ile Asp Leu Thr Asn 325 330 335Glu Arg Pro
Met Ala Asn Ser Pro Lys Pro Ala Pro Gly Ala Ala Gly 340 345 350Pro
Pro Gln Val Gly Leu Ser Tyr Ser Gln Thr Met Leu Leu Lys Asp 355 360
365Leu Met Gly Gly Ile Asp Pro Asn Ala Pro Thr Trp Ile Asp Ile Glu
370 375 380Gly Arg Phe Asn Asp Pro Val Glu Ile Ala Ile Phe Gln Pro
Gln Asn385 390 395 400Gly Gln Tyr Ile His Phe Tyr Arg Glu Pro Thr
Asp Gln Lys Gln Phe 405 410 415Lys Gln Asp Ser Lys Tyr Ser His Gly
Met Asp Leu Ala Asp Leu Phe 420 425 430Asn Ala Gln Pro Gly Leu Thr
Ser Ser Val Ile Gly Ala Leu Pro Gln 435 440 445Gly Met Val Leu Ser
Cys Gln Gly Ser Asp Asp Ile Arg Lys Leu Leu 450 455 460Asp Ser Gln
Asn Arg Arg Asp Ile Lys Leu Ile Asp Val Glu Met Thr465 470 475
480Lys Glu Ala Ser Arg Glu Tyr Glu Asp Lys Val Trp Asp Lys Tyr Gly
485 490 495Trp Leu Cys Lys Met His Thr Gly Ile Val Arg Asp Lys Lys
Lys Lys 500 505 510Glu Val Thr Pro His Cys Ala Leu Met Asp Cys Ile
Ile Phe Glu Ser 515 520 525Ala Ser Lys Ala Arg Leu Pro Asp Leu Lys
Thr Val His Asn Ile Leu 530 535 540Pro His Asp Leu Ile Phe Arg Gly
Pro Asn Val Val Thr Leu545 550 5557498PRTLymphocytic
choriomeningitis virusGP protein of the MP strain of LCMV 7Met Gly
Gln Ile Val Thr Met Phe Glu Ala Leu Pro His Ile Ile Asp1 5 10 15Glu
Val Ile Asn Ile Val Ile Ile Val Leu Ile Ile Ile Thr Ser Ile 20 25
30Lys Ala Val Tyr Asn Phe Ala Thr Cys Gly Ile Leu Ala Leu Ile Ser
35 40 45Phe Leu Phe Leu Ala Gly Arg Ser Cys Gly Met Tyr Gly Leu Asp
Gly 50 55 60Pro Asp Ile Tyr Lys Gly Val Tyr Arg Phe Lys Ser Val Glu
Phe Asp65 70 75 80Met Ser Tyr Leu Asn Leu Thr Met Pro Asn Ala Cys
Ser Ala Asn Asn 85 90 95Ser His His Tyr Ile Ser Met Gly Thr Ser Gly
Leu Glu Leu Thr Phe 100 105 110Thr Asn Asp Ser Ile Ile Thr His Asn
Phe Cys Asn Leu Thr Ser Ala 115 120 125Leu Asn Lys Arg Thr Phe Asp
His Thr Leu Met Ser Ile Val Ser Ser 130 135 140Leu His Leu Ser Ile
Arg Gly Val Pro Ser Tyr Lys Ala Val Ser Cys145 150 155 160Asp Phe
Asn Asn Gly Ile Thr Ile Gln Tyr Asn Leu Ser Phe Ser Asn 165 170
175Ala Gln Ser Ala Leu Ser Gln Cys Lys Thr Phe Arg Gly Arg Val Leu
180 185 190Asp Met Phe Arg Thr Ala Phe Gly Gly Lys Tyr Met Arg Ser
Gly Trp 195 200 205Gly Trp Thr Gly Ser Asp Gly Lys Thr Thr Trp Cys
Ser Gln Thr Asn 210 215 220Tyr Gln Tyr Leu Ile Ile Gln Asn Arg Thr
Trp Glu Asn His Cys Arg225 230 235
240Tyr Ala Gly Pro Phe Gly Met Ser Arg Ile Leu Phe Ala Gln Glu Lys
245 250 255Thr Arg Phe Leu Thr Arg Arg Leu Ala Gly Thr Phe Thr Trp
Thr Leu 260 265 270Ser Asp Ser Ser Gly Val Glu Asn Pro Gly Gly Tyr
Cys Leu Thr Lys 275 280 285Trp Met Ile Leu Ala Ala Glu Leu Lys Cys
Phe Gly Asn Thr Ala Val 290 295 300Ala Lys Cys Asn Val Asn His Asp
Glu Glu Phe Cys Asp Met Leu Arg305 310 315 320Leu Ile Asp Tyr Asn
Lys Ala Ala Leu Ser Lys Phe Lys Glu Asp Val 325 330 335Glu Ser Ala
Leu His Leu Phe Lys Thr Thr Val Asn Ser Leu Ile Ser 340 345 350Asp
Gln Leu Leu Met Arg Asn His Leu Arg Asp Leu Met Gly Val Pro 355 360
365Tyr Cys Asn Tyr Ser Lys Phe Trp Tyr Leu Glu His Ala Lys Thr Gly
370 375 380Glu Thr Ser Val Pro Lys Cys Trp Leu Val Ser Asn Gly Ser
Tyr Leu385 390 395 400Asn Glu Thr His Phe Ser Asp Gln Ile Glu Gln
Glu Ala Asp Asn Met 405 410 415Ile Thr Glu Met Leu Arg Lys Asp Tyr
Ile Lys Arg Gln Gly Ser Thr 420 425 430Pro Leu Ala Leu Met Asp Leu
Leu Met Phe Ser Thr Ser Ala Tyr Leu 435 440 445Ile Ser Ile Phe Leu
His Leu Val Arg Ile Pro Thr His Arg His Ile 450 455 460Lys Gly Gly
Ser Cys Pro Lys Pro His Arg Leu Thr Ser Lys Gly Ile465 470 475
480Cys Ser Cys Gly Ala Phe Lys Val Pro Gly Val Glu Thr Thr Trp Lys
485 490 495Arg Arg82209PRTLymphocytic choriomeningitis virusL
protein of the MP strain of LCMV 8Met Asp Glu Ala Ile Ser Glu Leu
Arg Glu Leu Cys Leu Asn His Ile1 5 10 15Glu Gln Asp Glu Arg Leu Ser
Arg Gln Lys Leu Asn Phe Leu Gly Gln 20 25 30Arg Glu Pro Arg Met Val
Leu Ile Glu Gly Leu Lys Leu Leu Ser Arg 35 40 45Cys Ile Glu Ile Asp
Ser Ala Asp Lys Ser Gly Cys Ile His Asn His 50 55 60Asp Asp Lys Ser
Val Glu Ala Ile Leu Ile Glu Ser Gly Ile Val Cys65 70 75 80Pro Gly
Leu Pro Leu Ile Ile Pro Asp Gly Tyr Lys Leu Ile Asp Asn 85 90 95Ser
Leu Ile Leu Leu Glu Cys Phe Val Arg Ser Thr Pro Ala Ser Phe 100 105
110Glu Lys Lys Phe Ile Glu Asp Thr Asn Lys Leu Ala Cys Ile Lys Glu
115 120 125Asp Leu Ala Ile Ala Gly Ile Thr Leu Val Pro Ile Val Asp
Gly Arg 130 135 140Cys Asp Tyr Asp Asn Ser Phe Met Pro Glu Trp Val
Asn Phe Lys Phe145 150 155 160Arg Asp Leu Leu Phe Lys Leu Leu Glu
Tyr Ser Ser Gln Asp Glu Lys 165 170 175Val Phe Glu Glu Ser Glu Tyr
Phe Arg Leu Cys Glu Ser Leu Lys Thr 180 185 190Thr Val Asp Lys Arg
Ser Gly Ile Asp Ser Met Lys Ile Leu Lys Asp 195 200 205Ala Arg Ser
Phe His Asn Asp Glu Ile Met Lys Met Cys His Asp Gly 210 215 220Val
Asn Pro Asn Met Asn Cys Asp Asp Val Val Leu Gly Ile Asn Ser225 230
235 240Leu Tyr Ser Arg Phe Arg Arg Asp Leu Glu Thr Gly Lys Leu Lys
Arg 245 250 255Ser Phe Gln Lys Ile Asn Pro Gly Asn Leu Ile Lys Glu
Phe Ser Glu 260 265 270Leu Tyr Glu Thr Leu Ala Asp Ser Asp Asp Ile
Ser Ala Leu Ser Lys 275 280 285Glu Ala Val Glu Ser Cys Pro Leu Met
Arg Phe Ile Thr Ala Asp Thr 290 295 300His Gly Tyr Glu Arg Gly Ser
Glu Thr Ser Thr Glu Tyr Glu Arg Leu305 310 315 320Leu Ser Met Leu
Asn Lys Val Lys Ser Leu Lys Leu Leu Asn Thr Arg 325 330 335Arg Arg
Gln Leu Leu Asn Leu Asp Val Leu Cys Leu Ser Ser Leu Ile 340 345
350Lys Gln Ser Lys Leu Lys Gly Ser Lys Asn Asp Lys His Trp Val Gly
355 360 365Cys Cys Tyr Gly Ser Val Asn Asp Arg Leu Val Ser Phe His
Ser Thr 370 375 380Lys Glu Glu Phe Ile Arg Leu Leu Arg Asn Arg Arg
Lys Ser Lys Ala385 390 395 400Tyr Arg Lys Val Ser Leu Glu Asp Leu
Phe Arg Thr Ser Ile Asn Glu 405 410 415Phe Ile Leu Lys Val Gln Arg
Cys Leu Ser Val Val Gly Leu Ser Phe 420 425 430Gly His Tyr Gly Leu
Ser Glu His Leu Glu His Glu Cys His Ile Pro 435 440 445Phe Ile Glu
Phe Glu Asn Phe Met Arg Ser Gly Thr His Pro Ile Met 450 455 460Tyr
Tyr Thr Lys Phe Glu Asp Tyr Asp Phe Gln Pro Asn Thr Glu Gln465 470
475 480Leu Arg Asn Met His Ser Leu Lys Arg Leu Ser Ser Val Cys Leu
Ala 485 490 495Leu Thr Asn Ser Met Lys Thr Ser Ser Val Ala Arg Leu
Arg Gln Asn 500 505 510Gln Leu Gly Ser Val Arg Tyr Gln Val Val Glu
Cys Lys Glu Val Phe 515 520 525Cys Gln Val Ile Lys Leu Asp Ser Glu
Glu Tyr His Leu Leu Tyr Gln 530 535 540Lys Thr Gly Glu Ser Ser Arg
Cys Tyr Ser Ile Gln Gly Pro Asn Gly545 550 555 560His Leu Ile Ser
Phe Tyr Ala Asp Pro Lys Arg Phe Phe Leu Pro Ile 565 570 575Phe Ser
Asp Glu Val Leu His Asn Met Ile Asp Thr Met Ile Ser Trp 580 585
590Ile Arg Ser Cys Pro Asp Leu Lys Asp Ser Ile Asp Asp Val Glu Ile
595 600 605Ala Leu Arg Thr Leu Leu Leu Leu Met Leu Thr Asn Pro Thr
Lys Arg 610 615 620Asn Gln Lys Gln Val Gln Asn Ile Arg Tyr Leu Val
Met Ala Ile Val625 630 635 640Ser Asp Phe Ser Ser Thr Ser Leu Met
Asp Lys Leu Lys Glu Asp Leu 645 650 655Ile Thr Pro Ala Glu Lys Val
Val Tyr Lys Leu Leu Arg Phe Leu Ile 660 665 670Lys Thr Val Phe Gly
Thr Gly Glu Lys Val Leu Leu Ser Ala Lys Phe 675 680 685Lys Phe Met
Leu Asn Val Ser Tyr Leu Cys His Leu Ile Thr Lys Glu 690 695 700Thr
Pro Asp Arg Leu Thr Asp Gln Ile Lys Cys Phe Glu Lys Phe Phe705 710
715 720Glu Pro Lys Ser Glu Phe Gly Phe Phe Val Asn Pro Lys Glu Ser
Ile 725 730 735Thr Pro Glu Glu Glu Cys Val Phe Tyr Asp Gln Met Lys
Lys Phe Thr 740 745 750Gly Lys Glu Val Asp Cys Gln Arg Thr Thr Pro
Gly Val Asn Leu Glu 755 760 765Ala Phe Ser Met Met Val Ser Ser Phe
Asn Asn Gly Thr Leu Ile Phe 770 775 780Lys Gly Glu Lys Arg Leu Asn
Ser Leu Asp Pro Met Thr Asn Ser Gly785 790 795 800Cys Ala Thr Ala
Leu Asp Leu Ala Ser Asn Lys Ser Val Val Val Asn 805 810 815Lys His
Leu Asn Gly Glu Arg Leu Leu Glu Tyr Asp Phe Asn Lys Leu 820 825
830Leu Val Ser Ala Val Ser Gln Ile Thr Glu Ser Phe Met Arg Lys Gln
835 840 845Lys Tyr Lys Leu Asn His Ser Asp Tyr Glu Tyr Lys Val Ser
Lys Leu 850 855 860Val Ser Arg Leu Val Ile Gly Ser Lys Glu Thr Glu
Ala Gly Lys Leu865 870 875 880Glu Gly Asp Ser Ala Asp Ile Cys Phe
Asp Gly Glu Glu Glu Thr Ser 885 890 895Phe Phe Lys Asn Leu Glu Asp
Lys Val Asn Ser Thr Ile Lys Arg Tyr 900 905 910Glu Arg Ser Lys Lys
Thr Asn Glu Gly Glu Asn Glu Val Gly Phe Glu 915 920 925Asn Thr Lys
Gly Leu His His Leu Gln Thr Ile Leu Ser Gly Lys Met 930 935 940Ala
Tyr Leu Arg Lys Val Ile Leu Ser Glu Ile Ser Phe His Leu Val945 950
955 960Glu Asp Phe Asp Pro Ser Cys Leu Thr Asn Asp Asp Met Lys Phe
Ile 965 970 975Cys Glu Ala Ile Glu Thr Ser Thr Glu Leu Ser Pro Leu
Tyr Phe Thr 980 985 990Ser Ala Val Lys Glu Gln Cys Gly Leu Asp Glu
Met Ala Lys Asn Leu 995 1000 1005Cys Arg Lys Phe Phe Ser Glu Gly
Asp Trp Phe Ser Cys Met Lys 1010 1015 1020Met Ile Leu Leu Gln Met
Asn Ala Asn Ala Tyr Ser Gly Lys Tyr 1025 1030 1035Arg His Met Gln
Arg Gln Gly Leu Asn Phe Lys Phe Asp Trp Asp 1040 1045 1050Lys Leu
Glu Glu Asp Val Arg Ile Ser Glu Arg Glu Ser Asn Ser 1055 1060
1065Glu Ser Leu Ser Lys Ala Leu Ser Leu Thr Lys Cys Met Ser Ala
1070 1075 1080Ala Leu Lys Asn Leu Cys Phe Tyr Ser Glu Glu Ser Pro
Thr Ser 1085 1090 1095Tyr Thr Ser Val Gly Pro Asp Ser Gly Arg Leu
Lys Phe Ala Leu 1100 1105 1110Ser Tyr Lys Glu Gln Val Gly Gly Asn
Arg Glu Leu Tyr Ile Gly 1115 1120 1125Asp Leu Arg Thr Lys Met Phe
Thr Arg Leu Ile Glu Asp Tyr Phe 1130 1135 1140Glu Ser Phe Ser Ser
Phe Phe Ser Gly Ser Cys Leu Asn Asn Asp 1145 1150 1155Lys Glu Phe
Glu Asn Ala Ile Leu Ser Met Thr Ile Asn Val Arg 1160 1165 1170Glu
Gly Leu Leu Asn Tyr Ser Met Asp His Ser Lys Trp Gly Pro 1175 1180
1185Met Met Cys Pro Phe Leu Phe Leu Met Leu Leu Gln Asn Leu Lys
1190 1195 1200Leu Gly Asp Asp Gln Tyr Val Arg Ser Gly Lys Asp His
Ile Ser 1205 1210 1215Thr Leu Leu Thr Trp His Met His Lys Leu Val
Glu Val Pro Phe 1220 1225 1230Pro Val Val Asn Ala Met Met Lys Ser
Tyr Ile Lys Ser Lys Leu 1235 1240 1245Lys Leu Leu Arg Gly Ser Glu
Thr Thr Val Thr Glu Arg Ile Phe 1250 1255 1260Arg Glu Tyr Phe Glu
Leu Gly Ile Val Pro Ser His Ile Ser Ser 1265 1270 1275Leu Ile Asp
Met Gly Gln Gly Ile Leu His Asn Ala Ser Asp Phe 1280 1285 1290Tyr
Gly Leu Ile Ser Glu Arg Phe Ile Asn Tyr Cys Ile Gly Val 1295 1300
1305Ile Phe Gly Glu Arg Pro Glu Ser Tyr Thr Ser Ser Asp Asp Gln
1310 1315 1320Ile Thr Leu Phe Asp Arg Arg Leu Ser Glu Leu Val Asp
Ser Asp 1325 1330 1335Pro Glu Glu Val Leu Val Leu Leu Glu Phe His
Ser His Leu Ser 1340 1345 1350Gly Leu Leu Asn Lys Phe Ile Ser Pro
Lys Ser Val Val Gly Arg 1355 1360 1365Phe Ala Ala Glu Phe Lys Ser
Arg Phe Tyr Val Trp Gly Glu Glu 1370 1375 1380Val Pro Leu Leu Thr
Lys Phe Val Ser Ala Ala Leu His Asn Val 1385 1390 1395Lys Cys Lys
Glu Pro His Gln Leu Cys Glu Thr Ile Asp Thr Ile 1400 1405 1410Ala
Asp Gln Ala Val Ala Asn Gly Val Pro Val Ser Leu Val Asn 1415 1420
1425Cys Ile Gln Lys Arg Thr Leu Asp Leu Leu Lys Tyr Ala Asn Phe
1430 1435 1440Pro Leu Asp Pro Phe Leu Leu Asn Thr Asn Thr Asp Val
Lys Asp 1445 1450 1455Trp Leu Asp Gly Ser Arg Gly Tyr Arg Ile Gln
Arg Leu Ile Glu 1460 1465 1470Glu Leu Cys Pro Ser Glu Thr Lys Val
Met Arg Arg Leu Val Arg 1475 1480 1485Arg Leu His His Lys Leu Lys
Asn Gly Glu Phe Asn Glu Glu Phe 1490 1495 1500Phe Leu Asp Leu Phe
Asn Arg Asp Lys Lys Glu Ala Ile Leu Gln 1505 1510 1515Leu Gly Asn
Ile Leu Gly Leu Glu Glu Asp Leu Ser Gln Leu Ala 1520 1525 1530Asn
Ile Asn Trp Leu Asn Leu Asn Glu Leu Phe Pro Leu Arg Met 1535 1540
1545Val Leu Arg Gln Lys Val Val Tyr Pro Ser Val Met Thr Phe Gln
1550 1555 1560Glu Glu Arg Ile Pro Ser Leu Ile Lys Thr Leu Gln Asn
Lys Leu 1565 1570 1575Cys Ser Lys Phe Thr Arg Gly Ala Gln Lys Leu
Leu Ser Glu Ala 1580 1585 1590Ile Asn Lys Ser Ala Phe Gln Ser Cys
Ile Ser Ser Gly Phe Ile 1595 1600 1605Gly Leu Cys Lys Thr Leu Gly
Ser Arg Cys Val Arg Asn Lys Asn 1610 1615 1620Arg Asp Asn Leu Tyr
Ile Arg Lys Val Leu Glu Asp Leu Ala Met 1625 1630 1635Asp Ala His
Val Thr Ala Ile His Arg His Asp Gly Ile Met Leu 1640 1645 1650Tyr
Ile Cys Asp Arg Gln Ser His Pro Glu Ala His Cys Asp His 1655 1660
1665Ile Ser Leu Leu Arg Pro Leu Leu Trp Asp Tyr Ile Cys Ile Ser
1670 1675 1680Leu Ser Asn Ser Phe Glu Leu Gly Val Trp Val Leu Ala
Glu Pro 1685 1690 1695Val Lys Gly Lys Asn Glu Gly Ser Ser Ser Leu
Lys His Leu Asn 1700 1705 1710Pro Cys Asp Tyr Val Ala Arg Lys Pro
Glu Ser Ser Arg Leu Leu 1715 1720 1725Glu Asp Lys Ile Ser Leu Asn
His Val Ile Gln Ser Val Arg Arg 1730 1735 1740Leu Tyr Pro Lys Ile
Tyr Glu Asp Gln Leu Leu Pro Phe Met Ser 1745 1750 1755Asp Met Ser
Ser Lys Asn Met Arg Trp Ser Pro Arg Ile Lys Phe 1760 1765 1770Leu
Asp Leu Cys Val Leu Ile Asp Ile Asn Ser Glu Ser Leu Ser 1775 1780
1785Leu Ile Ser His Val Val Lys Trp Lys Arg Asp Glu His Tyr Thr
1790 1795 1800Val Leu Phe Ser Asp Leu Val Asn Ser His Gln Arg Ser
Asp Ser 1805 1810 1815Ser Leu Val Asp Glu Phe Val Val Ser Thr Arg
Asp Val Cys Lys 1820 1825 1830Asn Phe Leu Lys Gln Val Tyr Phe Glu
Ser Phe Val Arg Glu Phe 1835 1840 1845Val Ala Thr Ser Arg Thr Leu
Gly Ser Phe Ser Trp Phe Pro His 1850 1855 1860Lys Asp Met Met Pro
Ser Glu Asp Gly Ala Glu Ala Leu Gly Pro 1865 1870 1875Phe Gln Ser
Phe Ile Leu Lys Val Val Asn Lys Asn Met Glu Arg 1880 1885 1890Pro
Met Phe Arg Asn Asp Leu Gln Phe Gly Phe Gly Trp Phe Ser 1895 1900
1905Tyr Arg Leu Gly Asp Ile Val Cys Asn Ala Ala Met Leu Ile Lys
1910 1915 1920Gln Gly Leu Thr Asn Pro Lys Ala Phe Lys Ser Leu Arg
Asn Leu 1925 1930 1935Trp Asp Tyr Met Ile Asn Asn Thr Glu Gly Val
Leu Glu Phe Ser 1940 1945 1950Ile Thr Val Asp Phe Thr His Asn Gln
Asn Asn Thr Asp Cys Leu 1955 1960 1965Arg Lys Phe Ser Leu Ile Phe
Leu Val Lys Cys Gln Leu Gln Gly 1970 1975 1980Pro Gly Val Ala Glu
Phe Leu Ser Cys Ser His Leu Phe Lys Gly 1985 1990 1995Glu Val Asp
Arg Arg Phe Leu Asp Glu Cys Leu His Leu Leu Arg 2000 2005 2010Ser
Asp Ser Ile Phe Lys Val Asn Asp Gly Val Phe Asp Ile Arg 2015 2020
2025Ser Glu Glu Phe Glu Asp Tyr Met Glu Asp Pro Leu Ile Leu Gly
2030 2035 2040Asp Ser Leu Glu Leu Glu Leu Ile Gly Ser Arg Lys Ile
Leu Asp 2045 2050 2055Gly Ile Arg Ser Leu Asp Phe Glu Arg Ile Gly
Pro Glu Trp Glu 2060 2065 2070Pro Val Pro Leu Thr Val Arg Met Gly
Ala Leu Phe Glu Gly Arg 2075 2080 2085Ser Leu Val Gln Asn Ile Val
Val Lys Leu Glu Thr Lys Asp Met 2090 2095 2100Arg Val Phe Leu Ala
Glu Leu Glu Gly Tyr Gly Asn Phe Asp Asp 2105 2110 2115Val Leu Gly
Ser Leu Leu Leu His Arg Phe Arg Thr Gly Glu His 2120 2125 2130Leu
Gln Gly Ser Glu Ile Ser Thr Ile Leu Gln Glu Leu Cys Ile 2135 2140
2145Asp Arg Ser Ile Leu Leu Val Pro Leu Ser Leu Val Pro Asp Trp
2150 2155 2160Phe Thr Phe Lys Asp Cys Arg Leu Cys Phe Ser Lys Ser
Lys Asn 2165 2170 2175Thr Val Met Tyr Glu Thr Val Val Gly Lys Tyr
Arg Leu Lys Gly
2180 2185 2190Lys Ser Cys Asp Asp Trp Leu Thr Lys Ser Val Val Glu
Glu Ile 2195 2200 2205Asp990PRTLymphocytic choriomeningitis virusZ
protein of the MP strain of LCMV 9Met Gly Gln Gly Lys Ser Lys Glu
Gly Arg Asp Ala Ser Asn Thr Ser1 5 10 15Arg Ala Glu Ile Leu Pro Asp
Thr Thr Tyr Leu Gly Pro Leu Asn Cys 20 25 30Lys Ser Cys Trp Gln Arg
Phe Asp Ser Leu Val Arg Cys His Asp His 35 40 45Tyr Leu Cys Arg His
Cys Leu Asn Leu Leu Leu Ser Val Ser Asp Arg 50 55 60Cys Pro Leu Cys
Lys His Pro Leu Pro Thr Lys Leu Lys Ile Ser Thr65 70 75 80Ala Pro
Ser Ser Pro Pro Pro Tyr Glu Glu 85 90107115DNAArtificial
SequenceJunin virus Candid#1 L segment 10gcgcaccggg gatcctaggc
gtaacttcat cattaaaatc tcagattctg ctctgagtgt 60gacttactgc gaagaggcag
acaaatgggc aactgcaacg gggcatccaa gtctaaccag 120ccagactcct
caagagccac acagccagcc gcagaattta ggagggtagc tcacagcagt
180ctatatggta gatataactg taagtgctgc tggtttgctg ataccaattt
gataacctgt 240aatgatcact acctttgttt aaggtgccat cagggtatgt
taaggaattc agatctctgc 300aatatctgct ggaagcccct gcccaccaca
atcacagtac cggtggagcc aacagcacca 360ccaccatagg cagactgcac
agggtcagac ccgacccccc ggggggcccc catggggacc 420ccccgtgggg
gaaccccggg ggtgatgcgc cattagtcaa tgtctttgat ctcgactttg
480tgcttcagtg gcctgcatgt cacccctttc aatctgaact gcccttgggg
atctgatatc 540agcaggtcat ttaaagatct gctgaatgcc accttgaaat
ttgagaattc caaccagtca 600ccaaatttat caagtgaacg gatcaactgc
tctttgtgta gatcataaac gaggacaaag 660tcctcttgct gaaataatat
tgtttgtgat gttgttttta gataaggcca tagttggctt 720aataaggttt
ccacactatc aatgtcctct agtgctccaa ttgccttgac tatgacatcc
780ccagacaact caactctata tgttgacaac ctttcattac ctctgtaaaa
gataccctct 840ttcaagacaa gaggttctcc tgggttatct ggcccaatga
ggtcatatgc atacttgtta 900cttagttcag aataaaagtc accaaagttg
aacttaacat ggctcagaat attgtcatca 960tttgtcgcag cgtagcctgc
atcaataaac aagccagcta ggtcaaagct ctcatggcct 1020gtgaacaatg
gtaggctagc gataaccagt gcaccatcca acaatgagtg gcttccctca
1080gacccagaaa cacattgact cattgcatcc acattcagct ctaattcagg
ggtaccgaca 1140tcatccactc ctagtgaact gacaatggtg taactgtaca
ccatctttct tctaagttta 1200aattttgtcg aaactcgtgt gtgttctact
tgaatgatca attttagttt cacagcttct 1260tggcaagcaa cattgcgcaa
cacagtgtgc aggtccatca tgtcttcctg aggcaacaag 1320gagatgttgt
caacagagac accctcaagg aaaaccttga tattatcaaa gctagaaact
1380acataaccca ttgcaatgtc ttcaacaaac attgctcttg atactttatt
attcctaact 1440gacaaggtaa aatctgtgag ttcagctaga tctacttgac
tgtcatcttc tagatctaga 1500acttcattga accaaaagaa ggatttgaga
cacgatgttg acatgactag tgggtttatc 1560atcgaagata agacaacttg
caccatgaag ttcctgcaaa cttgctgtgg gctgatgcca 1620acttcccaat
ttgtatactc tgactgtcta acatgggctg aagcgcaatc actctgtttc
1680acaatataaa cattattatc tcttactttc aataagtgac ttataatccc
taagttttca 1740ttcatcatgt ctagagccac acagacatct agaaacttga
gtcttccact atccaaagat 1800ctgttcactt gaagatcatt cataaagggt
gccaaatgtt cttcaaatag tttggggtaa 1860tttcttcgta tagaatgcaa
tacatggttc atgcctaatt ggtcttctat ctgtcgtact 1920gctttgggtt
taacagccca gaagaaattc ttattacata agaccagagg ggcctgtgga
1980ctcttaatag cagaaaacac ccactcccct aactcacagg catttgtcag
caccaaagag 2040aagtaatccc acaaaattgg tttagaaaat tggttaactt
ctttaagtga tttttgacag 2100taaataactt taggctttct ctcacaaatt
ccacaaagac atggcattat tcgagtaaat 2160atgtccttta tatacagaaa
tccgccttta ccatccctaa cacacttact ccccatactc 2220ttacaaaacc
caatgaagcc tgaggcaaca gaagactgaa atgcagattt gttgattgac
2280tctgccaaga tcttcttcac gccttttgtg aaatttcttg acagcctgga
ctgtattgtc 2340cttatcaatg ttggcatctc ttctttctct aacactcttc
gacttgtcat gagtttggtc 2400ctcaagacca acctcaagtc cccaaagctc
gctaaattga cccatctgta gtctagagtt 2460tgtctgattt catcttcact
acacccggca tattgcagga atccggataa agcctcatcc 2520cctcccctgc
ttatcaagtt gataaggttt tcctcaaaga ttttgcctct cttaatgtca
2580ttgaacactt tcctcgcgca gttccttata aacattgtct ccttatcatc
agaaaaaata 2640gcttcaattt tcctctgtag acggtaccct ctagacccat
caacccagtc tttgacatct 2700tgttcttcaa tagctccaaa cggagtctct
ctgtatccag agtatctaat caattggttg 2760actctaatgg aaatctttga
cactatatga gtgctaaccc cattagcaat acattgatca 2820caaattgtgt
ctatggtctc tgacagttgt gttggagttt tacacttaac gttgtgtaga
2880gcagcagaca caaacttggt gagtaaagga gtctcttcac ccatgacaaa
aaatcttgac 2940ttaaactcag caacaaaagt tcctatcaca ctctttgggc
tgataaactt gtttaattta 3000gaagataaga attcatggaa gcacaccatt
tccagcagtt ctgtcctgtc ttgaaacttt 3060tcatcactaa ggcaaggaat
ttttataagg ctaacctggt catcgctgga ggtataagtg 3120acaggtatca
catcatacaa taagtcaagt gcataacaca gaaattgttc agtaattagc
3180ccatataaat ctgatgtgtt gtgcaagatt ccctggccca tgtccaagac
agacattata 3240tggctgggga cctggtccct tgactgcaga tactggtgaa
aaaactcttc accaacacta 3300gtacagtcac aacccattaa acctaaagat
ctcttcaatt tccctacaca gtaggcttct 3360gcaacattaa ttggaacttc
aacgacctta tgaagatgcc atttgagaat gttcattact 3420ggttcaagat
tcacctttgt tctatctctg ggattcttca attctaatgt gtacaaaaaa
3480gaaaggaaaa gtgctgggct catagttggt ccccatttgg agtggtcata
tgaacaggac 3540aagtcaccat tgttaacagc cattttcata tcacagattg
cacgttcgaa ttccttttct 3600gaattcaagc atgtgtattt cattgaacta
cccacagctt ctgagaagtc ttcaactaac 3660ctggtcatca gcttagtgtt
gaggtctccc acatacagtt ctctatttga gccaacctgc 3720tccttataac
ttagtccaaa tttcaagttc cctgtatttg agctgatgct tgtgaactct
3780gtaggagagt cgtctgaata gaaacataaa ttccgtaggg ctgcatttgt
aaaataactt 3840ttgtctagct tatcagcaat ggcttcagaa ttgctttccc
tggtactaag ccgaacctca 3900tcctttagtc tcagaacttc actggaaaag
cccaatctag atctacttct atgctcataa 3960ctacccaatt tctgatcata
atgtccttga attaaaagat acttgaagca ttcaaagaat 4020tcatcttctt
ggtaggctat tgttgtcaaa ttttttaata acaaacccaa agggcagatg
4080tcctgcggtg cttcaagaaa ataagtcaat ttaaatggag atagataaac
agcatcacat 4140aactctttat acacatcaga cctgagcaca tctggatcaa
aatccttcac ctcatgcatt 4200gacacctctg ctttaatctc tctcaacact
ccaaaagggg cccacaatga ctcaagagac 4260tctcgctcat caacagatgg
attttttgat ttcaacttgg tgatctcaac ttttgtcccc 4320tcactattag
ccatcttggc tagtgtcatt tgtacgtcat ttctaatacc ctcaaaggcc
4380cttacttgat cctctgttaa actctcatac atcactgata attcttcttg
attggttctg 4440gttcttgaac cggtgctcac aagacctgtt agatttttta
atattaagta gtccatggaa 4500tcaggatcaa gattatacct gccttttgtt
ttaaacctct cagccatagt agaaacgcat 4560gttgaaacaa gtttctcctt
atcataaaca gaaagaatat ttccaagttc gtcgagcttg 4620gggattacca
cacttttatt gcttgacaga tccagagctg tgctagtgat gttaggcctg
4680tagggattgc ttttcagttc acctgtaact ttaagtcttc ctctattgaa
gagagaaatg 4740cagaaggaca aaatctcttt acacactcct ggaatttgag
tatctgagga agtcttagcc 4800tctttggaaa agaatctgtc caatcctctt
atcatggtgt cctcttgttc cagtgttaga 4860ctcccactta gaggggggtt
tacaacaaca caatcaaact tgactttggg ctcaataaac 4920ttctcaaaac
actttatttg atctgtcagg cgatcaggtg tctctttggt taccaagtga
4980cacagataac taacatttaa tagatattta aaccttcttg caaagtaaag
atctgcatct 5040tccccttcac ccaaaattgt ctggaaaagt tccacagcca
tcctctgaat cagcacctct 5100gatccagaca tgcagtcgac ccttaacttt
gacatcaaat ccacatgatg gatttgattt 5160gcatatgcca tcaagaaata
tcttagacct tgtaaaaatg tctggttcct tttggaaggg 5220gaacagagta
cagctaacac taacaatctt aatattggcc ttgtcattgt catgagttcg
5280tggctaaaat ccaaccagct ggtcatttcc tcacacattt caattaacac
atcctccgaa 5340aatataggca ggaaaaatct ctttggatca cagtaaaaag
agccttgttc ttccaatacc 5400ccattgatgg atagatagat agaatagcac
cttgacttct cacctgtttt ttggtaaaac 5460aagagaccaa atgtattctt
tgtcagatga aatctttgta cataacactc tcttagtcta 5520acattcccaa
aatatctaga atactctctt tcattgatta acaatcggga ggaaaatgat
5580gtcttcatcg agttgaccaa tgcaagggaa atggaggaca aaatcctaaa
taatttcttc 5640tgctcacctt ccactaagct gctgaatggc tgatgtctac
agattttctc aaattccttg 5700ttaatagtat atctcatcac tggtctgtca
gaaacaagtg cctgagctaa aatcatcaag 5760ctatccatat cagggtgttt
tattagtttt tccagctgtg accagagatc ttgatgagag 5820ttcttcaatg
ttctggaaca cgcttgaacc cacttggggc tggtcatcaa tttcttcctt
5880attagtttaa tcgcctccag aatatctaga agtctgtcat tgactaacat
taacatttgt 5940ccaacaacta ttcccgcatt tcttaacctt acaattgcat
catcatgcgt tttgaaaaga 6000tcacaaagta aattgagtaa aactaagtcc
agaaacagta aagtgtttct cctggtgttg 6060aaaactttta gacctttcac
tttgttacac acggaaaggg cttgaagata acacctctct 6120acagcatcaa
tagatataga attctcatct gactggcttt ccatgttgac ttcatctatt
6180ggatgcaatg cgatagagta gactacatcc atcaacttgt ttgcacaaaa
agggcagctg 6240ggcacatcac tgtctttgtg gcttcctaat aagatcaagt
catttataag cttagacttt 6300tgtgaaaatt tgaatttccc caactgcttg
tcaaaaatct ccttcttaaa ccaaaacctt 6360aactttatga gttcttctct
tatgacagat tctctaatgt ctcctctaac cccaacaaag 6420agggattcat
ttaacctctc atcataaccc aaagaattct ttttcaagca ttcgatgttt
6480tctaatccca agctctggtt ttttgtgttg gacaaactat ggatcaatcg
ctggtattct 6540tgttcttcaa tattaatctc ttgcataaat tttgatttct
ttaggatgtc gatcagcaac 6600caccgaactc tttcaacaac ccaatcagca
aggaatctat tgctgtagct agatctgcca 6660tcaaccacag gaaccaacgt
aatccctgcc cttagtaggt cggactttag gtttaagagc 6720tttgacatgt
cactcttcca ttttctctca aactcatcag gattgaccct aacaaaggtt
6780tccaatagga tgagtgtttt ccctgtgagt ttgaagccat ccggaatgac
ttttggaagg 6840gtgggacata gtatgccata gtcagacagg atcacatcaa
caaacttctg atctgaattg 6900atctgacagg cgtgtgcctc acaggactca
agctctacta aacttgacag aagtttgaac 6960ccttccaaca acagagagct
ggggtgatgt tgagataaaa agatgtccct ttggtatgct 7020agctcctgtc
tttctggaaa atgctttcta ataaggcttt ttatttcatt tactgattcc
7080tccatgctca agtgccgcct aggatcctcg gtgcg 7115113411DNAArtificial
SequenceJunin virus Candid#1 S segment 11gcgcaccggg gatcctaggc
gattttggtt acgctataat tgtaactgtt ttctgtttgg 60acaacatcaa aaacatccat
tgcacaatgg ggcagttcat tagcttcatg caagaaatac 120caaccttttt
gcaggaggct ctgaacattg ctcttgttgc agtcagtctc attgccatca
180ttaagggtat agtgaacttg tacaaaagtg gtttattcca attctttgta
ttcctagcgc 240ttgcaggaag atcctgcaca gaagaagctt tcaaaatcgg
actgcacact gagttccaga 300ctgtgtcctt ctcaatggtg ggtctctttt
ccaacaatcc acatgaccta cctttgttgt 360gtaccttaaa caagagccat
ctttacatta aggggggcaa tgcttcattt cagatcagct 420ttgatgatat
tgcagtattg ttgccacagt atgatgttat aatacaacat ccagcagata
480tgagctggtg ttccaaaagt gatgatcaaa tttggttgtc tcagtggttc
atgaatgctg 540tgggacatga ttggcatcta gacccaccat ttctgtgtag
gaaccgtgca aagacagaag 600gcttcatctt tcaagtcaac acctccaaga
ctggtgtcaa tggaaattat gctaagaagt 660ttaagactgg catgcatcat
ttatatagag aatatcctga cccttgcttg aatggcaaac 720tgtgcttaat
gaaggcacaa cctaccagtt ggcctctcca atgtccactc gaccacgtta
780acacattaca cttccttaca agaggtaaaa acattcaact tccaaggagg
tccttgaaag 840cattcttctc ctggtctttg acagactcat ccggcaagga
tacccctgga ggctattgtc 900tagaagagtg gatgctcgta gcagccaaaa
tgaagtgttt tggcaatact gctgtagcaa 960aatgcaattt gaatcatgac
tctgaattct gtgacatgtt gaggctcttt gattacaaca 1020aaaatgctat
caaaacccta aatgatgaaa ctaagaaaca agtaaatctg atggggcaga
1080caatcaatgc cctgatatct gacaatttat tgatgaaaaa caaaattagg
gaactgatga 1140gtgtccctta ctgcaattac acaaaatttt ggtatgtcaa
ccacacactt tcaggacaac 1200actcattacc aaggtgctgg ttaataaaaa
acaacagcta tttgaacatc tctgacttcc 1260gtaatgactg gatattagaa
agtgacttct taatttctga aatgctaagc aaagagtatt 1320cggacaggca
gggtaaaact cctttgactt tagttgacat ctgtatttgg agcacagtat
1380tcttcacagc gtcactcttc cttcacttgg tgggtatacc ctcccacaga
cacatcaggg 1440gcgaagcatg ccctttgcca cacaggttga acagcttggg
tggttgcaga tgtggtaagt 1500accccaatct aaagaaacca acagtttggc
gtagaggaca ctaagacctc ctgagggtcc 1560ccaccagccc gggcactgcc
cgggctggtg tggcccccca gtccgcggcc tggccgcgga 1620ctggggaggc
actgcttaca gtgcataggc tgccttcggg aggaacagca agctcggtgg
1680taatagaggt gtaggttcct cctcatagag cttcccatct agcactgact
gaaacattat 1740gcagtctagc agagcacagt gtggttcact ggaggccaac
ttgaagggag tatccttttc 1800cctctttttc ttattgacaa ccactccatt
gtgatatttg cataagtgac catatttctc 1860ccagacctgt tgatcaaact
gcctggcttg ttcagatgtg agcttaacat caaccagttt 1920aagatctctt
cttccatgga ggtcaaacaa cttcctgatg tcatcggatc cttgagtagt
1980cacaaccatg tctggaggca gcaagccgat cacgtaacta agaactcctg
gcattgcatc 2040ttctatgtcc ttcattaaga tgccgtgaga gtgtctgcta
ccatttttaa accctttctc 2100atcatgtggt tttctgaagc agtgaatgta
ctgcttacct gcaggttgga ataatgccat 2160ctcaacaggg tcagtggctg
gtccttcaat gtcgagccaa agggtgttgg tggggtcgag 2220tttccccact
gcctctctga tgacagcttc ttgtatctct gtcaagttag ccaatctcaa
2280attctgaccg tttttttccg gctgtctagg accagcaact ggtttccttg
tcagatcaat 2340acttgtgttg tcccatgacc tgcctgtgat ttgtgatcta
gaaccaatat aaggccaacc 2400atcgccagaa agacaaagtt tgtacaaaag
gttttcataa ggatttctat tgcctggttt 2460ctcatcaata aacatgcctt
ctcttcgttt aacctgaatg gttgatttta tgagggaaga 2520gaagttttct
ggggtgactc tgattgtttc caacatgttt ccaccatcaa gaatagatgc
2580tccagccttt actgcagctg aaagactgaa gttgtaacca gaaatattga
tggagctttc 2640atctttagtc acaatctgaa ggcagtcatg ttcctgagtc
agtctgtcaa ggtcacttaa 2700gtttggatac ttcacagtgt atagaagccc
aagtgaggtt aaagcttgta tgacactgtt 2760cattgtctca cctccttgaa
cagtcatgca tgcaattgtc aatgcaggaa cagagccaaa 2820ctgattgttt
agctttgaag ggtctttaac atcccatatc ctcaccacac catttccccc
2880agtcccttgc tgttgaaatc ccagtgttct caatatctct gatcttttag
caagttgtga 2940ctgggacaag ttacccatgt aaaccccctg agagcctgtc
tctgctcttc ttatcttgtt 3000ttttaatttc tcaaggtcag acgccaactc
catcagttca tccctcccca gatctcccac 3060cttgaaaact gtgtttcgtt
gaacactcct catggacatg agtctgtcaa cctctttatt 3120caggtccctc
aacttgttga ggtcttcttc ccccttttta gtctttctga gtgcccgctg
3180cacctgtgcc acttggttga agtcgatgct gtcagcaatt agcttggcgt
ccttcaaaac 3240atctgacttg acagtctgag tgaattggct caaacctctc
cttaaggact gagtccatct 3300aaagcttgga acctccttgg agtgtgccat
gccagaagtt ctggtgattt tgatctagaa 3360tagagttgct cagtgaaagt
gttagacact atgcctagga tccactgtgc g 341112558PRTLymphocytic
choriomeningitis virusNP protein of the Clone 13 strain of LCMV
(GenBank Accession No. ABC96002.1; GI86440166) 12Met Ser Leu Ser
Lys Glu Val Lys Ser Phe Gln Trp Thr Gln Ala Leu1 5 10 15Arg Arg Glu
Leu Gln Ser Phe Thr Ser Asp Val Lys Ala Ala Val Ile 20 25 30Lys Asp
Ala Thr Asn Leu Leu Asn Gly Leu Asp Phe Ser Glu Val Ser 35 40 45Asn
Val Gln Arg Ile Met Arg Lys Glu Lys Arg Asp Asp Lys Asp Leu 50 55
60Gln Arg Leu Arg Ser Leu Asn Gln Thr Val His Ser Leu Val Asp Leu65
70 75 80Lys Ser Thr Ser Lys Lys Asn Val Leu Lys Val Gly Arg Leu Ser
Ala 85 90 95Glu Glu Leu Met Ser Leu Ala Ala Asp Leu Glu Lys Leu Lys
Ala Lys 100 105 110Ile Met Arg Ser Glu Arg Pro Gln Ala Ser Gly Val
Tyr Met Gly Asn 115 120 125Leu Thr Thr Gln Gln Leu Asp Gln Arg Ser
Gln Ile Leu Gln Ile Val 130 135 140Gly Met Arg Lys Pro Gln Gln Gly
Ala Ser Gly Val Val Arg Val Trp145 150 155 160Asp Val Lys Asp Ser
Ser Leu Leu Asn Asn Gln Phe Gly Thr Met Pro 165 170 175Ser Leu Thr
Met Ala Cys Met Ala Lys Gln Ser Gln Thr Pro Leu Asn 180 185 190Asp
Val Val Gln Ala Leu Thr Asp Leu Gly Leu Leu Tyr Thr Val Lys 195 200
205Tyr Pro Asn Leu Asn Asp Leu Glu Arg Leu Lys Asp Lys His Pro Val
210 215 220Leu Gly Val Ile Thr Glu Gln Gln Ser Ser Ile Asn Ile Ser
Gly Tyr225 230 235 240Asn Phe Ser Leu Gly Ala Ala Val Lys Ala Gly
Ala Ala Leu Leu Asp 245 250 255Gly Gly Asn Met Leu Glu Ser Ile Leu
Ile Lys Pro Ser Asn Ser Glu 260 265 270Asp Leu Leu Lys Ala Val Leu
Gly Ala Lys Arg Lys Leu Asn Met Phe 275 280 285Val Ser Asp Gln Val
Gly Asp Arg Asn Pro Tyr Glu Asn Ile Leu Tyr 290 295 300Lys Val Cys
Leu Ser Gly Glu Gly Trp Pro Tyr Ile Ala Cys Arg Thr305 310 315
320Ser Ile Val Gly Arg Ala Trp Glu Asn Thr Thr Ile Asp Leu Thr Ser
325 330 335Glu Lys Pro Ala Val Asn Ser Pro Arg Pro Ala Pro Gly Ala
Ala Gly 340 345 350Pro Pro Gln Val Gly Leu Ser Tyr Ser Gln Thr Met
Leu Leu Lys Asp 355 360 365Leu Met Gly Gly Ile Asp Pro Asn Ala Pro
Thr Trp Ile Asp Ile Glu 370 375 380Gly Arg Phe Asn Asp Pro Val Glu
Ile Ala Ile Phe Gln Pro Gln Asn385 390 395 400Gly Gln Phe Ile His
Phe Tyr Arg Glu Pro Val Asp Gln Lys Gln Phe 405 410 415Lys Gln Asp
Ser Lys Tyr Ser His Gly Met Asp Leu Ala Asp Leu Phe 420 425 430Asn
Ala Gln Pro Gly Leu Thr Ser Ser Val Ile Gly Ala Leu Pro Gln 435 440
445Gly Met Val Leu Ser Cys Gln Gly Ser Asp Asp Ile Arg Lys Leu Leu
450 455 460Asp Ser Gln Asn Arg Lys Asp Ile Lys Leu Ile Asp Val Glu
Met Thr465 470 475 480Arg Glu Ala Ser Arg Glu Tyr Glu Asp Lys Val
Trp Asp Lys Tyr Gly 485 490 495Trp Leu Cys Lys Met His Thr Gly Ile
Val Arg Asp Lys Lys Lys Lys 500 505 510Glu Ile Thr Pro His Cys Ala
Leu Met Asp Cys Ile Ile Phe Glu Ser 515 520 525Ala Ser Lys Ala Arg
Leu Pro Asp Leu Lys Thr Val His Asn Ile Leu 530 535 540Pro His Asp
Leu Ile Phe Arg Gly Pro Asn Val Val Thr Leu545 550
55513498PRTLymphocytic choriomeningitis virusGP protein of the
Clone 13 strain of LCMV (GenBank Accession No. ABC96001.2;
GI116563462) 13Met Gly Gln Ile Val Thr Met Phe Glu Ala Leu
Pro His Ile Ile Asp1 5 10 15Glu Val Ile Asn Ile Val Ile Ile Val Leu
Ile Val Ile Thr Gly Ile 20 25 30Lys Ala Val Tyr Asn Phe Ala Thr Cys
Gly Ile Phe Ala Leu Ile Ser 35 40 45Phe Leu Leu Leu Ala Gly Arg Ser
Cys Gly Met Tyr Gly Leu Lys Gly 50 55 60Pro Asp Ile Tyr Lys Gly Val
Tyr Gln Phe Lys Ser Val Glu Phe Asp65 70 75 80Met Ser His Leu Asn
Leu Thr Met Pro Asn Ala Cys Ser Ala Asn Asn 85 90 95Ser His His Tyr
Ile Ser Met Gly Thr Ser Gly Leu Glu Leu Thr Phe 100 105 110Thr Asn
Asp Ser Ile Ile Ser His Asn Phe Cys Asn Leu Thr Ser Ala 115 120
125Phe Asn Lys Lys Thr Phe Asp His Thr Leu Met Ser Ile Val Ser Ser
130 135 140Leu His Leu Ser Ile Arg Gly Asn Ser Asn Tyr Lys Ala Val
Ser Cys145 150 155 160Asp Phe Asn Asn Gly Ile Thr Ile Gln Tyr Asn
Leu Thr Phe Ser Asp 165 170 175Ala Gln Ser Ala Gln Ser Gln Cys Arg
Thr Phe Arg Gly Arg Val Leu 180 185 190Asp Met Phe Arg Thr Ala Phe
Gly Gly Lys Tyr Met Arg Ser Gly Trp 195 200 205Gly Trp Thr Gly Ser
Asp Gly Lys Thr Thr Trp Cys Ser Gln Thr Ser 210 215 220Tyr Gln Tyr
Leu Ile Ile Gln Asn Arg Thr Trp Glu Asn His Cys Thr225 230 235
240Tyr Ala Gly Pro Phe Gly Met Ser Arg Ile Leu Leu Ser Gln Glu Lys
245 250 255Thr Lys Phe Leu Thr Arg Arg Leu Ala Gly Thr Phe Thr Trp
Thr Leu 260 265 270Ser Asp Ser Ser Gly Val Glu Asn Pro Gly Gly Tyr
Cys Leu Thr Lys 275 280 285Trp Met Ile Leu Ala Ala Glu Leu Lys Cys
Phe Gly Asn Thr Ala Val 290 295 300Ala Lys Cys Asn Val Asn His Asp
Glu Glu Phe Cys Asp Met Leu Arg305 310 315 320Leu Ile Asp Tyr Asn
Lys Ala Ala Leu Ser Lys Phe Lys Glu Asp Val 325 330 335Glu Ser Ala
Leu His Leu Phe Lys Thr Thr Val Asn Ser Leu Ile Ser 340 345 350Asp
Gln Leu Leu Met Arg Asn His Leu Arg Asp Leu Met Gly Val Pro 355 360
365Tyr Cys Asn Tyr Ser Lys Phe Trp Tyr Leu Glu His Ala Lys Thr Gly
370 375 380Glu Thr Ser Val Pro Lys Cys Trp Leu Val Thr Asn Gly Ser
Tyr Leu385 390 395 400Asn Glu Thr His Phe Ser Asp Gln Ile Glu Gln
Glu Ala Asp Asn Met 405 410 415Ile Thr Glu Met Leu Arg Lys Asp Tyr
Ile Lys Arg Gln Gly Ser Thr 420 425 430Pro Leu Ala Leu Met Asp Leu
Leu Met Phe Ser Thr Ser Ala Tyr Leu 435 440 445Val Ser Ile Phe Leu
His Leu Val Lys Ile Pro Thr His Arg His Ile 450 455 460Lys Gly Gly
Ser Cys Pro Lys Pro His Arg Leu Thr Asn Lys Gly Ile465 470 475
480Cys Ser Cys Gly Ala Phe Lys Val Pro Gly Val Lys Thr Val Trp Lys
485 490 495Arg Arg142210PRTLymphocytic choriomeningitis virusL
protein of the Clone 13 strain of LCMV (GenBank Accession No.
ABC96004.1; GI86440169) 14Met Asp Glu Ile Ile Ser Glu Leu Arg Glu
Leu Cys Leu Asn Tyr Ile1 5 10 15Glu Gln Asp Glu Arg Leu Ser Arg Gln
Lys Leu Asn Phe Leu Gly Gln 20 25 30Arg Glu Pro Arg Met Val Leu Ile
Glu Gly Leu Lys Leu Leu Ser Arg 35 40 45Cys Ile Glu Ile Asp Ser Ala
Asp Lys Ser Gly Cys Thr His Asn His 50 55 60Asp Asp Lys Ser Val Glu
Thr Ile Leu Val Glu Ser Gly Ile Val Cys65 70 75 80Pro Gly Leu Pro
Leu Ile Ile Pro Asp Gly Tyr Lys Leu Ile Asp Asn 85 90 95Ser Leu Ile
Leu Leu Glu Cys Phe Val Arg Ser Thr Pro Ala Ser Phe 100 105 110Glu
Lys Lys Phe Ile Glu Asp Thr Asn Lys Leu Ala Cys Ile Arg Glu 115 120
125Asp Leu Ala Val Ala Gly Val Thr Leu Val Pro Ile Val Asp Gly Arg
130 135 140Cys Asp Tyr Asp Asn Ser Phe Met Pro Glu Trp Ala Asn Phe
Lys Phe145 150 155 160Arg Asp Leu Leu Phe Lys Leu Leu Glu Tyr Ser
Asn Gln Asn Glu Lys 165 170 175Val Phe Glu Glu Ser Glu Tyr Phe Arg
Leu Cys Glu Ser Leu Lys Thr 180 185 190Thr Ile Asp Lys Arg Ser Gly
Met Asp Ser Met Lys Ile Leu Lys Asp 195 200 205Ala Arg Ser Thr His
Asn Asp Glu Ile Met Arg Met Cys His Glu Gly 210 215 220Ile Asn Pro
Asn Met Ser Cys Asp Asp Val Val Phe Gly Ile Asn Ser225 230 235
240Leu Phe Ser Arg Phe Arg Arg Asp Leu Glu Ser Gly Lys Leu Lys Arg
245 250 255Asn Phe Gln Lys Val Asn Pro Glu Gly Leu Ile Lys Glu Phe
Ser Glu 260 265 270Leu Tyr Glu Asn Leu Ala Asp Ser Asp Asp Ile Leu
Thr Leu Ser Arg 275 280 285Glu Ala Val Glu Ser Cys Pro Leu Met Arg
Phe Ile Thr Ala Glu Thr 290 295 300His Gly His Glu Arg Gly Ser Glu
Thr Ser Thr Glu Tyr Glu Arg Leu305 310 315 320Leu Ser Met Leu Asn
Lys Val Lys Ser Leu Lys Leu Leu Asn Thr Arg 325 330 335Arg Arg Gln
Leu Leu Asn Leu Asp Val Leu Cys Leu Ser Ser Leu Ile 340 345 350Lys
Gln Ser Lys Phe Lys Gly Leu Lys Asn Asp Lys His Trp Val Gly 355 360
365Cys Cys Tyr Ser Ser Val Asn Asp Arg Leu Val Ser Phe His Ser Thr
370 375 380Lys Glu Glu Phe Ile Arg Leu Leu Arg Asn Arg Lys Lys Ser
Lys Val385 390 395 400Phe Arg Lys Val Ser Phe Glu Glu Leu Phe Arg
Ala Ser Ile Ser Glu 405 410 415Phe Ile Ala Lys Ile Gln Lys Cys Leu
Leu Val Val Gly Leu Ser Phe 420 425 430Glu His Tyr Gly Leu Ser Glu
His Leu Glu Gln Glu Cys His Ile Pro 435 440 445Phe Thr Glu Phe Glu
Asn Phe Met Lys Ile Gly Ala His Pro Ile Met 450 455 460Tyr Tyr Thr
Lys Phe Glu Asp Tyr Asn Phe Gln Pro Ser Thr Glu Gln465 470 475
480Leu Lys Asn Ile Gln Ser Leu Arg Arg Leu Ser Ser Val Cys Leu Ala
485 490 495Leu Thr Asn Ser Met Lys Thr Ser Ser Val Ala Arg Leu Arg
Gln Asn 500 505 510Gln Ile Gly Ser Val Arg Tyr Gln Val Val Glu Cys
Lys Glu Val Phe 515 520 525Cys Gln Val Ile Lys Leu Asp Ser Glu Glu
Tyr His Leu Leu Tyr Gln 530 535 540Lys Thr Gly Glu Ser Ser Arg Cys
Tyr Ser Ile Gln Gly Pro Asp Gly545 550 555 560His Leu Ile Ser Phe
Tyr Ala Asp Pro Lys Arg Phe Phe Leu Pro Ile 565 570 575Phe Ser Asp
Glu Val Leu Tyr Asn Met Ile Asp Ile Met Ile Ser Trp 580 585 590Ile
Arg Ser Cys Pro Asp Leu Lys Asp Cys Leu Thr Asp Ile Glu Val 595 600
605Ala Leu Arg Thr Leu Leu Leu Leu Met Leu Thr Asn Pro Thr Lys Arg
610 615 620Asn Gln Lys Gln Val Gln Ser Val Arg Tyr Leu Val Met Ala
Ile Val625 630 635 640Ser Asp Phe Ser Ser Thr Ser Leu Met Asp Lys
Leu Arg Glu Asp Leu 645 650 655Ile Thr Pro Ala Glu Lys Val Val Tyr
Lys Leu Leu Arg Phe Leu Ile 660 665 670Lys Thr Ile Phe Gly Thr Gly
Glu Lys Val Leu Leu Ser Ala Lys Phe 675 680 685Lys Phe Met Leu Asn
Val Ser Tyr Leu Cys His Leu Ile Thr Lys Glu 690 695 700Thr Pro Asp
Arg Leu Thr Asp Gln Ile Lys Cys Phe Glu Lys Phe Phe705 710 715
720Glu Pro Lys Ser Gln Phe Gly Phe Phe Val Asn Pro Lys Glu Ala Ile
725 730 735Thr Pro Glu Glu Glu Cys Val Phe Tyr Glu Gln Met Lys Arg
Phe Thr 740 745 750Ser Lys Glu Ile Asp Cys Gln His Thr Thr Pro Gly
Val Asn Leu Glu 755 760 765Ala Phe Ser Leu Met Val Ser Ser Phe Asn
Asn Gly Thr Leu Ile Phe 770 775 780Lys Gly Glu Lys Lys Leu Asn Ser
Leu Asp Pro Met Thr Asn Ser Gly785 790 795 800Cys Ala Thr Ala Leu
Asp Leu Ala Ser Asn Lys Ser Val Val Val Asn 805 810 815Lys His Leu
Asn Gly Glu Arg Leu Leu Glu Tyr Asp Phe Asn Lys Leu 820 825 830Leu
Val Ser Ala Val Ser Gln Ile Thr Glu Ser Phe Val Arg Lys Gln 835 840
845Lys Tyr Lys Leu Ser His Ser Asp Tyr Glu Tyr Lys Val Ser Lys Leu
850 855 860Val Ser Arg Leu Val Ile Gly Ser Lys Gly Glu Glu Thr Gly
Arg Ser865 870 875 880Glu Asp Asn Leu Ala Glu Ile Cys Phe Asp Gly
Glu Glu Glu Thr Ser 885 890 895Phe Phe Lys Ser Leu Glu Glu Lys Val
Asn Thr Thr Ile Ala Arg Tyr 900 905 910Arg Arg Gly Arg Arg Ala Asn
Asp Lys Gly Asp Gly Glu Lys Leu Thr 915 920 925Asn Thr Lys Gly Leu
His His Leu Gln Leu Ile Leu Thr Gly Lys Met 930 935 940Ala His Leu
Arg Lys Val Ile Leu Ser Glu Ile Ser Phe His Leu Val945 950 955
960Glu Asp Phe Asp Pro Ser Cys Leu Thr Asn Asp Asp Met Lys Phe Ile
965 970 975Cys Glu Ala Val Glu Gly Ser Thr Glu Leu Ser Pro Leu Tyr
Phe Thr 980 985 990Ser Val Ile Lys Asp Gln Cys Gly Leu Asp Glu Met
Ala Lys Asn Leu 995 1000 1005Cys Arg Lys Phe Phe Ser Glu Asn Asp
Trp Phe Ser Cys Met Lys 1010 1015 1020Met Ile Leu Leu Gln Met Asn
Ala Asn Ala Tyr Ser Gly Lys Tyr 1025 1030 1035Arg His Met Gln Arg
Gln Gly Leu Asn Phe Lys Phe Asp Trp Asp 1040 1045 1050Lys Leu Glu
Glu Asp Val Arg Ile Ser Glu Arg Glu Ser Asn Ser 1055 1060 1065Glu
Ser Leu Ser Lys Ala Leu Ser Leu Thr Gln Cys Met Ser Ala 1070 1075
1080Ala Leu Lys Asn Leu Cys Phe Tyr Ser Glu Glu Ser Pro Thr Ser
1085 1090 1095Tyr Thr Ser Val Gly Pro Asp Ser Gly Arg Leu Lys Phe
Ala Leu 1100 1105 1110Ser Tyr Lys Glu Gln Val Gly Gly Asn Arg Glu
Leu Tyr Ile Gly 1115 1120 1125Asp Leu Arg Thr Lys Met Phe Thr Arg
Leu Ile Glu Asp Tyr Phe 1130 1135 1140Glu Ser Phe Ser Ser Phe Phe
Ser Gly Ser Cys Leu Asn Asn Asp 1145 1150 1155Lys Glu Phe Glu Asn
Ala Ile Leu Ser Met Thr Ile Asn Val Arg 1160 1165 1170Glu Gly Phe
Leu Asn Tyr Ser Met Asp His Ser Lys Trp Gly Pro 1175 1180 1185Met
Met Cys Pro Phe Leu Phe Leu Met Phe Leu Gln Asn Leu Lys 1190 1195
1200Leu Gly Asp Asp Gln Tyr Val Arg Ser Gly Lys Asp His Val Ser
1205 1210 1215Thr Leu Leu Thr Trp His Met His Lys Leu Val Glu Val
Pro Phe 1220 1225 1230Pro Val Val Asn Ala Met Met Lys Ser Tyr Val
Lys Ser Lys Leu 1235 1240 1245Lys Leu Leu Arg Gly Ser Glu Thr Thr
Val Thr Glu Arg Ile Phe 1250 1255 1260Arg Gln Tyr Phe Glu Met Gly
Ile Val Pro Ser His Ile Ser Ser 1265 1270 1275Leu Ile Asp Met Gly
Gln Gly Ile Leu His Asn Ala Ser Asp Phe 1280 1285 1290Tyr Gly Leu
Leu Ser Glu Arg Phe Ile Asn Tyr Cys Ile Gly Val 1295 1300 1305Ile
Phe Gly Glu Arg Pro Glu Ala Tyr Thr Ser Ser Asp Asp Gln 1310 1315
1320Ile Thr Leu Phe Asp Arg Arg Leu Ser Asp Leu Val Val Ser Asp
1325 1330 1335Pro Glu Glu Val Leu Val Leu Leu Glu Phe Gln Ser His
Leu Ser 1340 1345 1350Gly Leu Leu Asn Lys Phe Ile Ser Pro Lys Ser
Val Ala Gly Arg 1355 1360 1365Phe Ala Ala Glu Phe Lys Ser Arg Phe
Tyr Val Trp Gly Glu Glu 1370 1375 1380Val Pro Leu Leu Thr Lys Phe
Val Ser Ala Ala Leu His Asn Val 1385 1390 1395Lys Cys Lys Glu Pro
His Gln Leu Cys Glu Thr Ile Asp Thr Ile 1400 1405 1410Ala Asp Gln
Ala Ile Ala Asn Gly Val Pro Val Ser Leu Val Asn 1415 1420 1425Ser
Ile Gln Arg Arg Thr Leu Asp Leu Leu Lys Tyr Ala Asn Phe 1430 1435
1440Pro Leu Asp Pro Phe Leu Leu Asn Thr Asn Thr Asp Val Lys Asp
1445 1450 1455Trp Leu Asp Gly Ser Arg Gly Tyr Arg Ile Gln Arg Leu
Ile Glu 1460 1465 1470Glu Leu Cys Pro Asn Glu Thr Lys Val Val Arg
Lys Leu Val Arg 1475 1480 1485Lys Leu His His Lys Leu Lys Asn Gly
Glu Phe Asn Glu Glu Phe 1490 1495 1500Phe Leu Asp Leu Phe Asn Arg
Asp Lys Lys Glu Ala Ile Leu Gln 1505 1510 1515Leu Gly Asp Leu Leu
Gly Leu Glu Glu Asp Leu Asn Gln Leu Ala 1520 1525 1530Asp Val Asn
Trp Leu Asn Leu Asn Glu Met Phe Pro Leu Arg Met 1535 1540 1545Val
Leu Arg Gln Lys Val Val Tyr Pro Ser Val Met Thr Phe Gln 1550 1555
1560Glu Glu Arg Ile Pro Ser Leu Ile Lys Thr Leu Gln Asn Lys Leu
1565 1570 1575Cys Ser Lys Phe Thr Arg Gly Ala Gln Lys Leu Leu Ser
Glu Ala 1580 1585 1590Ile Asn Lys Ser Ala Phe Gln Ser Cys Ile Ser
Ser Gly Phe Ile 1595 1600 1605Gly Leu Cys Lys Thr Leu Gly Ser Arg
Cys Val Arg Asn Lys Asn 1610 1615 1620Arg Glu Asn Leu Tyr Ile Lys
Lys Leu Leu Glu Asp Leu Thr Thr 1625 1630 1635Asp Asp His Val Thr
Arg Val Cys Asn Arg Asp Gly Ile Thr Leu 1640 1645 1650Tyr Ile Cys
Asp Lys Gln Ser His Pro Glu Ala His Arg Asp His 1655 1660 1665Ile
Cys Leu Leu Arg Pro Leu Leu Trp Asp Tyr Ile Cys Ile Ser 1670 1675
1680Leu Ser Asn Ser Phe Glu Leu Gly Val Trp Val Leu Ala Glu Pro
1685 1690 1695Thr Lys Gly Lys Asn Asn Ser Glu Asn Leu Thr Leu Lys
His Leu 1700 1705 1710Asn Pro Cys Asp Tyr Val Ala Arg Lys Pro Glu
Ser Ser Arg Leu 1715 1720 1725Leu Glu Asp Lys Val Asn Leu Asn Gln
Val Ile Gln Ser Val Arg 1730 1735 1740Arg Leu Tyr Pro Lys Ile Phe
Glu Asp Gln Leu Leu Pro Phe Met 1745 1750 1755Ser Asp Met Ser Ser
Lys Asn Met Arg Trp Ser Pro Arg Ile Lys 1760 1765 1770Phe Leu Asp
Leu Cys Val Leu Ile Asp Ile Asn Ser Glu Ser Leu 1775 1780 1785Ser
Leu Ile Ser His Val Val Lys Trp Lys Arg Asp Glu His Tyr 1790 1795
1800Thr Val Leu Phe Ser Asp Leu Ala Asn Ser His Gln Arg Ser Asp
1805 1810 1815Ser Ser Leu Val Asp Glu Phe Val Val Ser Thr Arg Asp
Val Cys 1820 1825 1830Lys Asn Phe Leu Lys Gln Val Tyr Phe Glu Ser
Phe Val Arg Glu 1835 1840 1845Phe Val Ala Thr Thr Arg Thr Leu Gly
Asn Phe Ser Trp Phe Pro 1850 1855 1860His Lys Glu Met Met Pro Ser
Glu Asp Gly Ala Glu Ala Leu Gly 1865 1870 1875Pro Phe Gln Ser Phe
Val Ser Lys Val Val Asn Lys Asn Val Glu 1880 1885 1890Arg Pro Met
Phe Arg Asn Asp Leu Gln Phe Gly Phe Gly Trp Phe 1895 1900 1905Ser
Tyr Arg Met Gly Asp Val Val Cys Asn Ala Ala Met Leu Ile 1910 1915
1920Arg Gln Gly Leu Thr Asn Pro Lys Ala Phe Lys Ser Leu Lys Asp
1925 1930 1935Leu Trp Asp Tyr Met Leu Asn Tyr Thr Lys Gly Val Leu
Glu Phe
1940 1945 1950Ser Ile Ser Val Asp Phe Thr His Asn Gln Asn Asn Thr
Asp Cys 1955 1960 1965Leu Arg Lys Phe Ser Leu Ile Phe Leu Val Arg
Cys Gln Leu Gln 1970 1975 1980Asn Pro Gly Val Ala Glu Leu Leu Ser
Cys Ser His Leu Phe Lys 1985 1990 1995Gly Glu Ile Asp Arg Arg Met
Leu Asp Glu Cys Leu His Leu Leu 2000 2005 2010Arg Thr Asp Ser Val
Phe Lys Val Asn Asp Gly Val Phe Asp Ile 2015 2020 2025Arg Ser Glu
Glu Phe Glu Asp Tyr Met Glu Asp Pro Leu Ile Leu 2030 2035 2040Gly
Asp Ser Leu Glu Leu Glu Leu Leu Gly Ser Lys Arg Ile Leu 2045 2050
2055Asp Gly Ile Arg Ser Ile Asp Phe Glu Arg Val Gly Pro Glu Trp
2060 2065 2070Glu Pro Val Pro Leu Thr Val Lys Met Gly Ala Leu Phe
Glu Gly 2075 2080 2085Arg Asn Leu Val Gln Asn Ile Ile Val Lys Leu
Glu Thr Lys Asp 2090 2095 2100Met Lys Val Phe Leu Ala Gly Leu Glu
Gly Tyr Glu Lys Ile Ser 2105 2110 2115Asp Val Leu Gly Asn Leu Phe
Leu His Arg Phe Arg Thr Gly Glu 2120 2125 2130His Leu Leu Gly Ser
Glu Ile Ser Val Ile Leu Gln Glu Leu Cys 2135 2140 2145Ile Asp Arg
Ser Ile Leu Leu Ile Pro Leu Ser Leu Leu Pro Asp 2150 2155 2160Trp
Phe Ala Phe Lys Asp Cys Arg Leu Cys Phe Ser Lys Ser Arg 2165 2170
2175Ser Thr Leu Met Tyr Glu Thr Val Gly Gly Arg Phe Arg Leu Lys
2180 2185 2190Gly Arg Ser Cys Asp Asp Trp Leu Gly Gly Ser Val Ala
Glu Asp 2195 2200 2205Ile Asp 22101590PRTLymphocytic
choriomeningitis virusZ protein of the Clone 13 strain of LCMV
(GenBank Accession No. ABC96003.1; GI86440168) 15Met Gly Gln Gly
Lys Ser Arg Glu Glu Lys Gly Thr Asn Ser Thr Asn1 5 10 15Arg Ala Glu
Ile Leu Pro Asp Thr Thr Tyr Leu Gly Pro Leu Ser Cys 20 25 30Lys Ser
Cys Trp Gln Lys Phe Asp Ser Leu Val Arg Cys His Asp His 35 40 45Tyr
Leu Cys Arg His Cys Leu Asn Leu Leu Leu Ser Val Ser Asp Arg 50 55
60Cys Pro Leu Cys Lys Tyr Pro Leu Pro Thr Arg Leu Lys Ile Ser Thr65
70 75 80Ala Pro Ser Ser Pro Pro Pro Tyr Glu Glu 85
9016498PRTLymphocytic choriomeningitis virusGP protein of the WE
strain of LCMV 16Met Gly Gln Ile Val Thr Met Phe Glu Ala Leu Pro
His Ile Ile Asp1 5 10 15Glu Val Ile Asn Ile Val Ile Ile Val Leu Ile
Ile Ile Thr Ser Ile 20 25 30Lys Ala Val Tyr Asn Phe Ala Thr Cys Gly
Ile Leu Ala Leu Val Ser 35 40 45Phe Leu Phe Leu Ala Gly Arg Ser Cys
Gly Met Tyr Gly Leu Asn Gly 50 55 60Pro Asp Ile Tyr Lys Gly Val Tyr
Gln Phe Lys Ser Val Glu Phe Asp65 70 75 80Met Ser His Leu Asn Leu
Thr Met Pro Asn Ala Cys Ser Ala Asn Asn 85 90 95Ser His His Tyr Ile
Ser Met Gly Ser Ser Gly Leu Glu Leu Thr Phe 100 105 110Thr Asn Asp
Ser Ile Leu Asn His Asn Phe Cys Asn Leu Thr Ser Ala 115 120 125Phe
Asn Lys Lys Thr Phe Asp His Thr Leu Met Ser Ile Val Ser Ser 130 135
140Leu His Leu Ser Ile Arg Gly Asn Ser Asn His Lys Ala Val Ser
Cys145 150 155 160Asp Phe Asn Asn Gly Ile Thr Ile Gln Tyr Asn Leu
Ser Phe Ser Asp 165 170 175Pro Gln Ser Ala Ile Ser Gln Cys Arg Thr
Phe Arg Gly Arg Val Leu 180 185 190Asp Met Phe Arg Thr Ala Phe Gly
Gly Lys Tyr Met Arg Ser Gly Trp 195 200 205Gly Trp Ala Gly Ser Asp
Gly Lys Thr Thr Trp Cys Ser Gln Thr Ser 210 215 220Tyr Gln Tyr Leu
Ile Ile Gln Asn Arg Thr Trp Glu Asn His Cys Arg225 230 235 240Tyr
Ala Gly Pro Phe Gly Met Ser Arg Ile Leu Phe Ala Gln Glu Lys 245 250
255Thr Lys Phe Leu Thr Arg Arg Leu Ala Gly Thr Phe Thr Trp Thr Leu
260 265 270Ser Asp Ser Ser Gly Val Glu Asn Pro Gly Gly Tyr Cys Leu
Thr Lys 275 280 285Trp Met Ile Leu Ala Ala Glu Leu Lys Cys Phe Gly
Asn Thr Ala Val 290 295 300Ala Lys Cys Asn Val Asn His Asp Glu Glu
Phe Cys Asp Met Leu Arg305 310 315 320Leu Ile Asp Tyr Asn Lys Ala
Ala Leu Ser Lys Phe Lys Gln Asp Val 325 330 335Glu Ser Ala Leu His
Val Phe Lys Thr Thr Val Asn Ser Leu Ile Ser 340 345 350Asp Gln Leu
Leu Met Arg Asn His Leu Arg Asp Leu Met Gly Val Pro 355 360 365Tyr
Cys Asn Tyr Ser Lys Phe Trp Tyr Leu Glu His Ala Lys Thr Gly 370 375
380Glu Thr Ser Val Pro Lys Cys Trp Leu Val Thr Asn Gly Ser Tyr
Leu385 390 395 400Asn Glu Thr His Phe Ser Asp Gln Ile Glu Gln Glu
Ala Asp Asn Met 405 410 415Ile Thr Glu Met Leu Arg Lys Asp Tyr Ile
Lys Arg Gln Gly Ser Thr 420 425 430Pro Leu Ala Leu Met Asp Leu Leu
Met Phe Ser Thr Ser Ala Tyr Leu 435 440 445Ile Ser Ile Phe Leu His
Leu Val Lys Ile Pro Thr His Arg His Ile 450 455 460Lys Gly Gly Ser
Cys Pro Lys Pro His Arg Leu Thr Asn Lys Gly Ile465 470 475 480Cys
Ser Cys Gly Ala Phe Lys Val Pro Gly Val Lys Thr Ile Trp Lys 485 490
495Arg Arg1735DNAArtificial SequenceWE specific primer 17aatcgtctct
aaggatgggt cagattgtga caatg 351835DNAArtificial SequenceWE specific
fusion-primer carrying an overhang complementary to the
WET-specific primer 18aatcgtctct aaggatgggt cagattgtga caatg
351937DNAArtificial SequenceWE specific primer 19ctcggtgatc
atgttatctg cttcttgttc gatttga 372034DNAArtificial SequenceWE
specific fusion-primer complementary to the WE-sequence
20aatcgtctct ttctttatct cctcttccag atgg 342123DNAArtificial
SequencePrimer specific for LCMV NP 21ggctcccaga tctgaaaact gtt
232222DNAArtificial SequenceNP- and GP-specific primers;
NP-specific same as in RT reaction, GP- specific 5 prime end
22gctggcttgt cactaatggc tc 22
* * * * *