U.S. patent application number 17/084841 was filed with the patent office on 2021-06-03 for chimeric vsv virus compositions and methods of use thereof for treatment of cancer.
The applicant listed for this patent is Yale University. Invention is credited to Anthony N. van den Pol, Guido Wollmann.
Application Number | 20210161979 17/084841 |
Document ID | / |
Family ID | 1000005399286 |
Filed Date | 2021-06-03 |
United States Patent
Application |
20210161979 |
Kind Code |
A1 |
van den Pol; Anthony N. ; et
al. |
June 3, 2021 |
CHIMERIC VSV VIRUS COMPOSITIONS AND METHODS OF USE THEREOF FOR
TREATMENT OF CANCER
Abstract
Methods of treating cancer including administering to a subject
with cancer a pharmaceutical composition including an effective
amount of a chimeric VSV virus are disclosed. The chimeric viruses
are based on a VSV background where the VSV G protein is replaced
with one or more heterologous viral glycoproteins. In the most
preferred embodiment, the VSV G protein is replaced with the
glycoprotein from Lassa virus or a functional fragment thereof. The
resulting chimeric virus is an oncolytic virus that is attenuated
and safe in the brain, yet still retains sufficient oncolytic
activity to infect and destroy cancer cells such glioblastoma, and
to generate an immune response against infected cancer cells.
Methods of using chimeric viruses as a platform for immunization
against other pathogenic microbes are also provided.
Inventors: |
van den Pol; Anthony N.;
(Branford, CT) ; Wollmann; Guido; (Innsbruck,
AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yale University |
New Haven |
CT |
US |
|
|
Family ID: |
1000005399286 |
Appl. No.: |
17/084841 |
Filed: |
October 30, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16235425 |
Dec 28, 2018 |
10821143 |
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17084841 |
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15037774 |
May 19, 2016 |
10179154 |
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PCT/US2014/067137 |
Nov 24, 2014 |
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16235425 |
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61907520 |
Nov 22, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2760/20232
20130101; C12N 7/04 20130101; C12N 7/00 20130101; A61K 2039/5152
20130101; A61K 2039/5256 20130101; A61K 9/0043 20130101; A61K 35/12
20130101; A61K 39/205 20130101; C12N 2760/20133 20130101; C12N
2760/20223 20130101; A61K 9/007 20130101; C12N 2760/20222 20130101;
A61K 35/766 20130101; A61K 9/0019 20130101; A61K 45/06 20130101;
C12N 2760/20234 20130101 |
International
Class: |
A61K 35/766 20060101
A61K035/766; C12N 7/04 20060101 C12N007/04; A61K 9/00 20060101
A61K009/00; A61K 35/12 20060101 A61K035/12; A61K 39/205 20060101
A61K039/205; A61K 45/06 20060101 A61K045/06; C12N 7/00 20060101
C12N007/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0001] This invention was made with government support under Grants
1R01CA161048 and RO1 CA175577 awarded by the National Institutes of
Health. The government has certain rights in the invention.
Claims
1. A method of treating cancer comprising administering to a
subject with cancer a pharmaceutical composition comprising an
effective amount of a chimeric Vesicular stomatitis Indiana virus
(VSV) virus to treat the cancer, wherein the chimeric VSV virus
comprises a VSV background with one or more heterologous viral
glycoproteins in place of the VSV G-protein, wherein the G protein
is not from Lymphocytic choriomeningitis (LCMV).
2.-4. (canceled)
5. The method of claim 1 wherein the VSV background is VSV Indiana,
VSV New Jersey, VSV Alagoas, (formerly Indiana 3), VSV Cocal
(formerly Indiana 2), VSV Chandipura, VSV Isfahan, VSV San Juan,
VSV Orsay, VSV Glasgow, or a recombinant VSV comprising at least 1
gene from two or more VSV strains or serotypes selected from the
group consisting of VSV Indiana, VSV New Jersey, VSV Alagoas,
(formerly Indiana 3), VSV Cocal (formerly Indiana 2), VSV
Chandipura, VSV Isfahan, VSV San Juan, VSV Orsay, and VSV
Glasgow.
6. (canceled)
7. The method of claim 1 wherein the chimeric VSV virus further
comprises one or more additional heterologous proteins.
8. The method of claim 7 wherein the chimeric VSV virus's genome
encodes the one or more heterologous genes.
9. The method of claim 7 wherein the one or more additional
heterologous proteins is a therapeutic protein, a reporter, a
vaccine antigen, a targeting moiety, or a combination thereof.
10.-11. (canceled)
12. The method of claim 1 wherein the cancer is selected from the
group consisting of multiple myeloma, bone, bladder, brain, breast,
cervical, colo-rectal, esophageal, kidney, liver, lung,
nasopharangeal, pancreatic, prostate, skin, stomach, and
uterine.
13. The method of claim 12 wherein the brain cancer is
oligodendroglioma, meningioma, supratentorial ependymona, pineal
region tumors, medulloblastoma, cerebellar astrocytoma,
infratentorial ependymona, brainstem glioma, schwannomas, pituitary
tumors, craniopharyngioma, optic glioma, and astrocytoma.
14. The method of claim 12 wherein the brain cancer is
glioblastoma.
15. The method of claim 1 wherein the virus is in a dosage of
between 10.sup.2 and 10.sup.12 PFU.
16. The method of claim 1 wherein the pharmaceutical composition is
administered locally to the site of the cancer.
17. (canceled)
18. The method of claim 1 wherein the pharmaceutical composition is
administered systemically to the subject.
19. (canceled)
20. (canceled)
21. The method of claim 1 further comprising administering the
subject a second therapeutic agent.
22. The method of claim 21 wherein the second therapeutic agent is
an anticancer agent, a therapeutic protein, or an
immunosuppressant.
23.-30. (canceled)
31. The method of claim 1 wherein the chimeric VSV show little or
no ability to infect normal or health neurons.
32. A method of treating a subject for cancer comprising (a)
infecting isolated cancer cells with an effective amount of a
chimeric VSV virus (b) administrating the infected cells to the
subject in an effective amount to induce an immune response against
the cancer cells in the subject; wherein the chimeric VSV virus
comprises a VSV background with one or more heterologous viral
glycoproteins in place of the VSV G-protein, wherein the G protein
is not from Lymphocytic choriomeningitis (LCMV).
33. The method of claim 32 further comprising irradiating the cells
to prevent their proliferation.
34. The method of claim 32 wherein the subject has cancer.
35.-43. (canceled)
44. A pharmaceutical dosage unit comprising an effective amount of
a chimeric VSV virus to treat cancer in subject in need thereof,
wherein the chimeric VSV virus comprises a VSV background with one
or more heterologous viral glycoproteins in place of the VSV
G-protein, wherein the G protein is not from Lymphocytic
choriomeningitis (LCMV).
45.-50. (canceled)
51. The method of claim 1, wherein the heterologous glycoprotein is
derived from an arenavirus.
52. The method of claim 51, wherein the arenavirus is Ippy virus.
Description
FIELD OF THE INVENTION
[0002] The invention is generally directed to recombinant vesicular
stomatitis virus (VSV) and methods of use thereof to treat cancer,
particularly glioblastoma.
BACKGROUND OF THE INVENTION
[0003] Patients diagnosed with glioblastoma (GBM) generally have
about a year to live. There is no cure for this malignant type of
brain cancer. The disease generally manifests with subtle changes
in brain function, and becomes worse with seizures, loss of
sensation or equilibrium, and progresses to loss of motor function
and mental ability, to finally, death. Surgery, focused radiation,
and medical treatment may slow the disease down and delay death by
several months, but unfortunately, this often comes at the expense
of normal function as neurons, glia, and axonal pathways are
damaged by the treatment) Wrensch, et al., Neuro-oncol., 4:278-299
(2002)).
[0004] Surgery is often done primarily to debulk the tumor, thereby
temporarily restoring function to surrounding brain regions that
were compressed by tumor expansion. A successful tumor resection
may remove billions of tumor cells. But inevitably, due to its
infiltrative nature, tens of millions of tumor cells remain within
the brain after surgery (Croteau, et al., Cancer Research, 67(6),
2840-2848 (2007)). A critical feature of GBMs is that tumor cells
are invasive, migrating away from the main tumor body, and
continuing to divide elsewhere in the brain. This is one reason why
approaches to treatment that focus on a tumor mass are ultimately
not successful at eliminating the disease.
[0005] A considerable number of viruses have been tested for
potential oncolytic activity against glioblastoma (Parker, et al.,
Neurotherapeutics, 6:558-569 (2009)). These include human
pathogenic viruses that were genetically attenuated to render them
safe enough for human application, for example HSV (Todo T, Front
Biosci., 13:2060-2064 (2008)) and adenovirus (Nandi, et al., Expert
Opin. Biol. Ther., 9:737-747 (2009); Chiocca, et al., Mol. Ther.,
16:618-626 (2008)), and viruses based on vaccination strains,
including polio (Dobrikova, et al., Mol. Ther., 16:1865-1872
(2008)), measles (Phuong, et al., Cancer Res, 63:2462-2469 (2003)),
and vaccinia (Lun, et al., Mol. Ther., 18:1927-1936 (2010)), or
adeno-associated viral vectors expressing various genes (Maguire,
et al., Mol. Ther., 16:1695-1702 (2008)). However, HSV and
retroviruses can either enter a latent mode and re-emerge later, or
can integrate into host chromosomes, enhancing an oncogenic
potential. Furthermore, many of these viruses share the potential
problem that most humans have been exposed to these viruses before
and their efficacy after systemic application may be challenged by
pre-existing immunity.
[0006] A promising alternative is the use of viruses that are
non-human pathogens but that display a tropism for tumors, as is
the case with myxoma (Lun, et al., Cancer Res, 65:9982-9990
(2005)), Newcastle disease virus (Freeman, et al., Mol. Ther.,
13:221-228 (2006), or VSV (Stojdl, et al., Cancer Cell., 4:263-275
(2003)). VSV infections do not integrate (and in fact do not even
enter the nucleus), and in animals, including humans, are
eliminated from the body within 1-2 weeks by the systemic immune
system. In regions of Central America where VSV is endemic, local
human populations are seropositive for VSV, with no obvious link to
substantive disease (Tesh, et al, 1969). VSV is rare in the US,
indicating a very low level of pre-existing immunity. VSV has been
approved for human clinical trials where VSV is used as a vaccine
vector to immunize people against dangerous viral or bacterial
pathogens (Roberts et al, 1999; Rose et al, 2000; Schwartz et al,
2010).
[0007] Another type of attenuated VSV, VSV-M51, shows a reduced
ability to block nuclear pores, thereby allowing normal cells to
up-regulate antiviral defenses, and has been described as showing
an enhanced safety profile (Stojdl, et al., Nat. Med., 6:821-825
(2000); Stojdl, et al., Cancer Cell, 4:263-275 (2003)). However,
this attenuated virus can still generate lethal outcomes after CNS
injection.
[0008] Some oncolytic viruses have been tested in early phase 1
clinical trials, but although the viruses were found to be safe,
little therapeutic effect was seen, and then only in a subset of
patients (Zemp, et al., Cytokine Growth Factor Rev., 21:103-117
(2010)), underlining the importance of continuing efforts to find
more effective oncolytic viruses and delivery strategies (Liu, et
al., Mol. Ther., 16:1006-1008 (2008)). Accordingly, despite the
advances in the development and use of oncolytic viruses for
treatment of cancer, there remains a need for improved virus and
methods of use therefore for safely and effective treating cancers
such as glioblastoma.
[0009] Therefore, it is an object of the invention to provide
recombinant oncolytic viruses with improved safety and efficacy
profiles.
[0010] It is a further object of the invention to provide
pharmaceutical compositions including an effective amount of
recombinant oncolytic viruses to treat cancer in a human
subject.
[0011] It is another object of the invention to provide methods of
using recombinant oncolytic virus to kill cancer cells.
[0012] It is a further object of the invention to increase the
body's immune response against cancer cells.
[0013] It is a further object to generate a safer virus-based
vaccine against other non-related microbial antigens.
SUMMARY OF THE INVENTION
[0014] Methods of treating cancer including administering to a
subject with cancer a pharmaceutical composition including an
effective amount of a chimeric VSV virus are disclosed. The
chimeric viruses are based on a VSV background where the VSV G
protein is replaced with one or more heterologous viral
glycoproteins. In the most preferred embodiment, the VSV G protein
is replaced with the glycoprotein from Lassa virus or a functional
fragment thereof. The Examples below show that replacement of the
VSV G protein with a heterologous glycoprotein, particularly the
glycoprotein from Lassa virus, results in an oncolytic virus that
is highly attenuated and safe in the brain, yet still retains
sufficient oncolytic activity to infect and destroy cancer cells
such as glioblastoma and intracranial melanoma metastases. The
chimeric virus can be further modified to express one or more
therapeutic proteins, reporters, vaccine antigens, or targeting
moieties. Exemplary therapeutic proteins and reporters include, but
are not limited to, IL-28, IL-2, FLT3L, GM-CSF, IL-4, IL-7, IL-12,
TRAIL, carcinoembryonic antigen, secreted alkaline phosphatase, the
beta subunit of chorionic gonadotropin, and green fluorescent
protein.
[0015] Methods can include administering to a subject an effective
amount of the virus to reduce one or more symptoms of cancer, for
example tumor burden. The cancer can be multiple myeloma, bone,
bladder, brain, breast, cervical, colo-rectal, esophageal, kidney,
liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach,
and uterine. In a preferred embodiment, the methods are used to
treat brain cancer and brain metastases. Brain cancers include, but
are not limited to, oligodendroglioma, meningioma, supratentorial
ependymona, pineal region tumors, medulloblastoma, cerebellar
astrocytoma, infratentorial ependymona, brainstem glioma,
schwannomas, pituitary tumors, craniopharyngioma, optic glioma, and
astrocytoma. In a particularly preferred embodiment, the cancer is
glioblastoma.
[0016] The virus is typically administered in a dosage of between
about 10.sup.2 and about 10.sup.12 PFU, more preferably between
about 10.sup.2 and about 10.sup.12 PFU. The pharmaceutical
composition can be administered locally to the site of the cancer.
For example, the composition can be injected into or adjacent to a
tumor in the subject, or via catheter into a tumor resection
cavity, for example, by convection-enhanced delivery (CED). The
pharmaceutical composition can be administered systemically to the
subject, for example by intravenous, intra-arterial, or intrathecal
injection or infusion.
[0017] The virus can be administered in combination with one or
more additional therapeutic agents. The one or more additional
therapeutic agents can be, for example, an anticancer agent such as
a chemotherapeutic agent, a therapeutic protein such as IL-2, or an
immunosuppressant. The immunosuppressant can be a histone
deacetylase (HDAC) inhibitor or an interferon blocker, for example,
valproate, the vacccinia protein B18R, Jak inhibitor 1, or
vorinostat, which can be used to reduce or delay the subject's
immune response to the virus.
[0018] The pharmaceutical composition can be administered in
combination with surgery. In some embodiments, the subject is
pre-treated with an immunizing composition including a virus
effective to immunize the subject to the chimeric VSV virus prior
to administration of the pharmaceutical composition. The virus in
the immunizing composition can be the chimeric VSV virus.
Immunizing the subject against the virus can increase the ability
of the subject's immune system to clear the virus following
therapeutic treatment if needed.
[0019] Other methods of treating cancer are also disclosed. For
example, a method of treating a subject for cancer can include (a)
infecting isolated cancer cells with an effective amount of a
chimeric VSV virus and (b) administrating the infected cells to the
subject in an effective amount to induce an immune response against
the cancer cells in the subject. In some embodiments, the method
includes irradiating the cells to prevent their proliferation in
the subject. The method can be used to therapeutically or
prophylactically treat cancer in the subject.
[0020] Methods of priming the immune system for attacking cancer
cells and adaptive T cell therapy are also disclosed. The priming
can occur in vitro or in vivo. A particular embodiment of preparing
cells for adaptive T cell therapy includes administering to a
subject with cancer a pharmaceutical composition including an
effective amount of a chimeric VSV virus to increase the number of
cytotoxic T cells (CTL) which can directly kill the cancer, or to
increase the number of CD4+ T and/or CD8+ T cells which can direct
an immune response against the cancer. The T cells can be isolated
from the subject and propagated in vitro. The T cells can be
administered back to the same subject, or another subject in need
thereof.
[0021] Pharmaceutical dosage units and kits including an effective
amount of the disclosed chimeric viruses are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1A is a histogram showing percent infected cells (%
GFP) for each of three gliomas tested (U87, U118 and CT2A, are
represented by different shading) 24 hours after infection with 0.1
multiplicity of infection (MOT) of a VSV ((control) also referred
to herein as VSV-wtG), or a chimeric VSV virus wherein the G
protein was replaced with the glycoprotein from Lassa (as referred
to herein as Lassa-VSV, VSV-LASV-G, and LASV), rabies (also
referred to herein as VSV-RABV-G and RABV), LCMV (also referred to
herein as VSV-LCMV-G), Ebola (also referred to herein as EBOV,
VSV-EBOV-G, and Ebola-VSV), or Marburg (also referred to herein as
MARV and VSV-MARV-G). FIG. 1B is a histogram showing the viral
replication (Titer, pfu/ml) VSV-wtG (control), or a chimeric VSV
virus wherein the G protein was replaced with the glycoprotein from
Lassa, rabies, LCMV, Ebola, or Marburg. FIG. 1C is an illustration
of the relative mean diameter and the small vertical line
surmounting each the SEM, of 60 randomly selected fluorescent
plaques measured and normalized to VSV-wtG plaque size on
monolayers of U87, U118 and normal human brain cells, 24 hpi with
VSV-wtG (control), or a chimeric VSV virus wherein the G protein
was replaced with the glycoprotein from Lassa, LCMV, or Ebola. FIG.
1D is a histogram showing the fraction of infected cells of mouse
brain cultures (% gila (top portion of the bar) versus % neurons
(bottom portion of the bar)) for cells infected with VSV-wtG
(control), or a chimeric VSV virus wherein the G protein was
replaced with the glycoprotein from Lassa, rabies, LCMV, Ebola, or
Marburg (MOI 5).
[0023] FIG. 2A is a Kaplan-Meier survival curve showing the %
survival of mice infected with chimeric Lassa-VSV, chimeric
Ebola-VSV, attenuated VSV-M.DELTA.51, or attenuated VSV-1'GFP over
time (in days post-inoculation) following intracranial inoculation
with virus. FIG. 2B is a Kaplan-Meier survival curve showing the %
survival of mice infected with chimeric Lassa-VSV or VSV-IFN over
time (in days post-inoculation) following intracranial inoculation
with virus.
[0024] FIG. 3A is a histogram showing infection of human neuronal
cultures with VSV-wtG or chimeric Lassa-VSV (GFP expression %) with
or without treatment with 100 IU/ml of interferon (IFN). FIG. 3B is
a Kaplan-Meier survival curve showing the % survival of normal mice
infected with chimeric Lassa-VSV, and IFN.alpha./.beta.-R knockout
(-/-) mice infected with VSV-wtG or Lassa-VSV (in days
post-inoculation) following intracranial inoculation with virus.
FIG. 3C is a histogram showing virus binding and internalization
(relative expression by qRT-PCR) of VSV-wtG and Lassa-VSV in
neurons and giloma cells at 4.degree. C. and 37.degree. C. FIG. 3D
is a dot plot showing the quantification of VSV-wtG viral
replication in neurons (.tangle-solidup.) and U87 glioma
(.diamond-solid.) cells assessed by plaque assay at 15 and 24
hpi.
[0025] FIG. 3E is a dot plot showing the quantification of
VSV-LASV-G viral replication in neurons (.tangle-solidup.) and U87
glioma (.diamond-solid.) cells assessed by plaque assay at 15 and
24 hpi.
[0026] FIG. 4 is a Kaplan-Meier survival curve showing the %
survival of mice infected with Lassa-VSV (solid line), and
Ebola-VSV (dotted line) and uninfected control (solid line),
respectively, over time (in days) following intracranial glioma
xenograph and subsequent inoculation with virus. The times of
i.c.U87 xenograft, and i.v. virus inoculation are indicated. The
increased longevity of tumor bearing mice receiving Lassa-VSV is
statistically significant (p<0.01; n=8 in each group).
[0027] FIG. 5 is a line graph showing the % body weight of mice
following intracranial glioma xenograph and subsequent systemic
infection with Lassa-VSV, Ebola-VSV and uninfected control,
respectively, over time (in days post-inoculation).
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0028] As used herein, the term "isolated" describes a compound of
interest (e.g., either a polynucleotide or a polypeptide) that is
in an environment different from that in which the compound
naturally occurs e.g., separated from its natural milieu such as by
concentrating a peptide to a concentration at which it is not found
in nature. "Isolated" includes compounds that are within samples
that are substantially enriched for the compound of interest and/or
in which the compound of interest is partially or substantially
purified. With respect to nucleic acids, the term "isolated"
includes any non-naturally-occurring nucleic acid sequence, since
such non-naturally-occurring sequences are not found in nature and
do not have immediately contiguous sequences in a
naturally-occurring genome.
[0029] As used herein, the term "nucleic acid(s)" refers to any
nucleic acid containing molecule, including, but not limited to,
DNA or RNA. The term encompasses sequences that include any of the
known base analogs of DNA and RNA including, but not limited to,
4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,
pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil,
5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxy-aminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine. In
accordance with standard nomenclature, nucleic acid sequences are
denominated by either a three letter, or single letter code as
indicated as follows: adenine (Ade, A), thymine (Thy, T), guanine
(Gua, G) cytosine (Cyt, C), uracil (Ura, U).
[0030] As used herein, the term "polynucleotide" refers to a chain
of nucleotides of any length, regardless of modification (e.g.,
methylation).
[0031] As used herein, the term "gene" refers to a nucleic acid
(e.g., DNA or RNA) sequence that comprises coding sequences
necessary for the production of a polypeptide, RNA (e.g., including
but not limited to, mRNA, tRNA and rRNA) or precursor. The
polypeptide, RNA, or precursor can be encoded by a full length
coding sequence or by any portion thereof. The term also
encompasses the coding region of a structural gene and the
sequences located adjacent to the coding region on both the 5' and
3' ends for a distance of about 1 kb on either end such that the
gene corresponds to the length of the full-length mRNA. The term
"gene" encompasses both cDNA and genomic forms of a gene, which may
be made of DNA, or RNA. A genomic form or clone of a gene may
contain the coding region interrupted with non-coding sequences
termed "introns" or "intervening regions" or "intervening
sequences." Introns are segments of a gene that are transcribed
into nuclear RNA (hnRNA); introns may contain regulatory elements
such as enhancers. Introns are removed or "spliced out" from the
nuclear or primary transcript; introns therefore are absent in the
messenger RNA (mRNA) transcript. The mRNA functions during
translation to specify the sequence or order of amino acids in a
nascent polypeptide.
[0032] As used herein, the term "nucleic acid molecule encoding,"
refers to the order or sequence of nucleotides along a strand of
nucleotides. The order of these nucleotides determines the order of
amino acids along the polypeptide (protein) chain. The nucleotide
sequence thus codes for the amino acid sequence.
[0033] As used herein, the term "polypeptide" refers to a chain of
amino acids of any length, regardless of modification (e.g.,
phosphorylation or glycosylation). In accordance with standard
nomenclature, amino acid residue sequences are denominated by
either a three letter or a single letter code as indicated as
follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N),
Aspartic Acid (Asp, D), Cysteine. (Cys, C), Glutamine (Gin, Q),
Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H),
Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine
(Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser,
S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and
Valine (Val, V).
[0034] As used herein, a "variant," "mutant," or "mutated"
polynucleotide contains at least one polynucleotide sequence
alteration as compared to the polynucleotide sequence of the
corresponding wild-type or parent polynucleotide. Mutations may be
natural, deliberate, or accidental. Mutations include
substitutions, deletions, and insertions.
[0035] As used herein, a "nucleic acid sequence alteration" can be,
for example, a substitution, a deletion, or an insertion of one or
more nucleotides. An "amino acid sequence alteration" can be, for
example, a substitution, a deletion, or an insertion of one or more
amino acids.
[0036] As used herein, the term "immunizing virus" includes
infectious virus, viral subunits, viral proteins and antigenic
fragments thereof, nucleic acids encoding viral subunits, antigenic
proteins or polypeptides, and expression vectors containing the
nucleic acids.
[0037] As used herein, a "vector" is a replicon, such as a plasmid,
phage, or cosmid, into which another DNA segment may be inserted so
as to bring about the replication of the inserted segment. The
vectors described herein can be expression vectors.
[0038] As used herein, the term "effective amount" or
"therapeutically effective amount" means a dosage sufficient to
treat, inhibit, or alleviate one or more symptoms of a disease
state being treated or to otherwise provide a desired pharmacologic
and/or physiologic effect. The precise dosage will vary according
to a variety of factors such as subject-dependent variables (e.g.,
age, immune system health, etc.), the disease, and the treatment
being effected.
[0039] As used herein, the, terms "neoplastic cells," "neoplasia,"
"tumor," "tumor cells," "cancer" and "cancer cells," (used
interchangeably) refer to cells which exhibit relatively autonomous
growth, so that they exhibit an aberrant growth phenotype
characterized by a significant loss of control of cell
proliferation (i.e., de-regulated cell division). Neoplastic cells
can be malignant or benign.
[0040] As used herein, an "immunogen" or "immunogenic amount"
refers to the ability of a substance (antigen) to induce an immune
response. An immune response is an alteration in the reactivity of
an organisms' immune system in response to an antigen. In
vertebrates this may involve antibody production, induction of
cell-mediated immunity, complement activation or development of
immunological tolerance.
[0041] As used herein, an "adjuvant" is a substance that increases
the ability of an antigen to stimulate the immune system.
[0042] As used herein, "attenuated" refers to refers to procedures
that weaken an agent of disease (a pathogen). An attenuated virus
is a weakened, less vigorous virus. A vaccine against a viral
disease can be made from an attenuated, less virulent strain of the
virus, a virus capable of stimulating an immune response and
creating immunity but not causing illness or less severe illness.
Attenuation can be achieved by chemical treatment of the pathogen,
through radiation, or by genetic modification, using methods known
to those skilled in the art. Attenuation may result in decreased
proliferation, attachment to host cells, or decreased production or
strength of toxins.
[0043] As used herein, "subject," "individual," and "patient" refer
to any individual who is the target of treatment using the
disclosed compositions. The subject can be a vertebrate, for
example, a mammal. Thus, the subject can be a human. The subjects
can be symptomatic or asymptomatic. The term does not denote a
particular age or sex. A subject can include a control subject or a
test subject.
[0044] As used herein, "identity," as known in the art, is a
relationship between two or more polypeptide sequences, as
determined by comparing the sequences. In the art, "identity" also
means the degree of sequence relatedness between polypeptide as
determined by the match between strings of such sequences.
"Identity" and "similarity" can be readily calculated by known
methods, including, but not limited to, those described in
(Computational Molecular Biology, Lesk, A. M., Ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part I, Griffin, A. M., and
Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
Eds., M Stockton Press, New York, 1991; and Carillo, H., and
Lipman, D., SIAM J Applied Math., 48: 1073 (1988).
[0045] Preferred methods to determine identity are designed to give
the largest match between the sequences tested. Methods to
determine identity and similarity are codified in publicly
available computer programs. The percent identity between two
sequences can be determined by using analysis software (i.e.,
Sequence Analysis Software Package of the Genetics Computer Group,
Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol.
Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST, and XBLAST). The
default parameters are used to determine the identity for the
polypeptides of the present disclosure.
[0046] By way of example, a polypeptide sequence may be identical
to the reference sequence, that is be 100% identical, or it may
include up, to a certain integer number of amino acid alterations
as compared to the reference sequence such that the % identity is
less than 100%. Such alterations are selected from: at least one
amino acid deletion, substitution, including conservative and
non-conservative substitution, or insertion, and wherein said
alterations may occur at the amino- or carboxy-terminal positions
of the reference polypeptide sequence or anywhere between those
terminal positions, interspersed either individually among the
amino acids in the reference sequence or in one or more contiguous
groups within the reference sequence. The number of amino acid
alterations for a given % identity is determined by multiplying the
total number of amino acids in the reference polypeptide by the
numerical percent of the respective percent identity (divided by
100) and then subtracting that product from said total number of
amino acids in the reference polypeptide.
[0047] As used herein "pharmaceutically acceptable carrier"
encompasses any of the standard pharmaceutical carriers, such as a
phosphate buffered saline solution, water and emulsions such as an
oil/water or water/oil emulsion, and various types of wetting
agents.
[0048] As used herein, "treatment" or "treating" means to
administer a composition to a subject or a system with an undesired
condition. The condition can include a disease. "Prevention" or
"preventing" means to administer a composition to a subject or a
system at risk for the condition. The condition can include a
predisposition to a disease. The effect of the administration of
the composition to the subject (either treating and/or preventing)
can be, but is not limited to, the cessation of one or more
symptoms of the condition, a reduction or prevention of one or more
symptoms of the condition, a reduction in the severity of the
condition, the complete ablation of the condition, a stabilization
or delay of the development or progression of a particular event or
characteristic, or minimization of the chances that a particular
event or characteristic will occur. It is understood that where
treat or prevent are used, unless specifically indicated otherwise,
the use of the other word is also expressly disclosed.
II. Compositions
[0049] VSV, a member of the Rhabdoviridae family, is enveloped and
has a negative-strand 11.2-kb RNA genome that comprises five
protein-encoding genes (N, P, M, G, and L) (Lyles, et al., Fields
virology, 5.sup.th ed., Lippincott Williams & Wilkins,
1363-1408 (2007)). It is a nonhuman pathogen which can cause mild
disease in livestock. Infection in humans is rare and usually
asymptomatic, with sporadic cases of mild flu-like symptoms. VSV
has a short replication cycle, which starts with attachment of the
viral glycoprotein spikes (G) to an unknown but ubiquitous cell
membrane receptor. Nonspecific electrostatic interactions have also
been proposed to facilitate viral binding (Lyles, et al., Fields
virology, 5.sup.th ed., Lippincott Williams & Wilkins,
1363-1408 (2007)). Upon internalization by clathrin-dependent
endocytosis, the virus-containing endosome acidifies, triggering
fusion of the viral membrane with the endosomal membrane. This
leads to release of the viral nucleocapsid (N) and viral RNA
polymerase complex (P and L) into the cytosol.
[0050] The viral polymerase initiates gene transcription at the 3'
end of the non-segmented genome, starting with expression of the
first VSV gene (N). This is followed by sequential gene
transcription, creating a gradient, with upstream genes expressed
more strongly than downstream genes. Newly produced VSV
glycoproteins are incorporated into the cellular membrane with a
large extracellular domain, a 20 amino acid trans-membrane domain,
and a cytoplasmic tail consisting of 29 amino acids. Trimers of G
protein accumulate in plasma membrane microdomains, several of
which congregate to form viral budding sites at the membrane
(Lyles, et al., Fields virology, 5.sup.th ed., Lippincott Williams
& Wilkins, 1363-1408 (2007)). Most cells activate antiviral
defense cascades upon viral entry, transcription, and replication,
which in turn are counteracted by VSV matrix protein (M). VSV M
protein's multitude of functions include virus assembly by linking
the nucleocapsid with the envelope membrane, induction of
cytopathic effects and apoptosis, inhibition of cellular gene
transcription, and blocking of host cell nucleocytoplasmic RNA
transfer, which includes blocking of antiviral cellular responses
(Ahmed, et al., Virology, 237:378-388 (1997)).
[0051] Certain native, engineered, and recombinant VSV strains have
been shown to target several tumor types, including gliomas, and
give a strong oncolytic action, both in vitro and in vivo (Paglino
and van den Pol, 2011) (Wollmann, et al, 2005; 2007; 2010; Ozduman
et al, 2008). However, there remains a need for improved
recombinant VSVs that are both efficacious for treating cancer and
exhibit low pathogenicity to healthy host cells. This is
particularly important in the brain where mature neurons do not
replicate, and once lost, are normally not replaced. Although some
evidence indicates that attenuated VSVs show reduced neurotoxicity,
CNS complications have been difficult to eliminate completely
(Obuchi et al, 2003; van den Pol et al, 2002; 2009).
[0052] It has been discovered that recombinant, chimeric VSV
viruses where the G gene is substituted with a gene encoding a
heterologous glycoprotein protein have oncolytic potential in
targeting and destroying cancer cells with little pathogenicity to
healthy host cells. Recombinant VSV viruses, pharmaceutical
compositions including recombinant VSV viruses, and methods of use
thereof for treating cancer are provided. As discussed in more
detail below, preferably, the virus targets and kills tumor cells,
and shows little or no infection of normal cells.
[0053] A. Chimeric G-Gene Substituted VSV Virus
[0054] The disclosed viruses are chimeric VSV viruses that are
typically based on a VSV background strain, also referred to herein
as a VSV backbone, wherein the G gene is substituted for a
heterologous glycoprotein. As discussed in more detail below, the
chimeric virus can also include additional genetic changes (e.g.,
additions, deletions, substitutions) relative to the background VSV
virus, and can have one or more additional transgenes.
[0055] 1. VSV Background Strain
[0056] Useful VSV virus background strains can be viruses that are
known in the art, or they can be mutants or variants of known
viruses. Any suitable VSV strain or serotype may be used,
including, but not limited to, VSV Indiana, VSV New Jersey, VSV
Alagoas, (formerly Indiana 3), VSV Cocal (formerly Indiana 2), VSV
Chandipura, VSV Isfahan, VSV San Juan, VSV Orsay, or VSV Glasgow.
The VSV virus background can be a naturally occurring virus, or a
virus modified, for example, to increase or decrease the virulence
of the virus, and/or increase the specificity or infectivity of the
virus compared to the parental strain or serotype. The virus can be
a recombinant virus that includes genes from two or more strains or
serotypes. For example, the VSV background strain can be a
recombinant VSV with all five genes of the Indiana serotype of VSV.
In another exemplary embodiments, the N, P, M, and L genes
originates from the San Juan strain, and the G gene from the Orsay
strain.
[0057] It may be desirable to further reduce the neurovirulence of
the viruses used in the disclosed methods, particularly the
virulence of the therapeutic virus, by using an attenuated virus. A
number of suitable VSV mutants have been described, see for example
(Clarke, et al., J. Virol., 81:2056-64 (2007), Flanagan, et al., J.
Virol., 77:5740-5748 (2003), Johnson, et al., Virology, 360:36-49
(2007), Simon, et al., J. Virol., 81:2078-82 (2007), Stojdl, et
al., Cancer Cell, 4:263-275 (2003)), Wollmann, et al., J. Virol,
84(3):1563-73 (2010) (epub 2010), WO 2010/080909, U.S. Published
Application No. 2007/0218078, and U.S. Published Application No
2009/0175906.
[0058] Recombinant VSVs derived from DNA plasmids also typically
show weakened virulence (Rose, et al., Cell, 106:539-549 (2001)).
Attenuation of VSV virulence can also be accomplished by one or
more nucleotide sequence alterations that result in substitution,
deletion, or insertion of one or more amino acids of the
polypeptide it encodes.
[0059] In some embodiments, the VSV background strain is a VSV
virus modified to attenuate virus growth or pathogenicity or to
reduce the ability to make infectious progeny viruses. VSV strains
and methods of making such VSV strains are known in the art, and
described in, for example, U.S. Published Application No.
2012/0171246.
[0060] For example, one strategy is to attenuate viral
pathogenicity by reducing the ability of the virus to suppress host
innate immune responses without compromising the yield of
infectious progeny. This can be accomplished by mutating the M
protein as described, for example, in Ahmed, J. Virol.,
82(18):9273-9277 (2008). The M protein is a multifunctional protein
that is involved in the shutoff of host transcription, nuclear
cytoplasmic transport, and translation during virus infection
(Lyles, Microbial. Mol. Biol. Rev. 64:709-724 (2000)). Mutation
and/or deletion of one or more amino acids from the M protein, for
example M.DELTA.51, or M51A mutants can result in viral protein
that is defective at inhibiting host gene expression. It may also
be desirable to switch or combine various substitutions, deletions,
and insertions to further modify the phenotype of the virus. For
example, the recombinant VSV background can have a deletion or
mutation in the M protein.
[0061] Altering the relative position of genes can also be used to
attenuate virus (Clarke, et al., J. Virol., 81:2056-2064, (2007),
Cooper, et al., J. Virol., 82:207-219 (2008), Flanagan, et al., J.
Virol., 75:6107-6114 (2001)). VSV is highly immunogenic, and a
substantial B and T cell response from the adaptive immune system
will ultimately limit VSV infection, which will halt long-lasting
viral infections. A virus that shows enhanced selectivity, and a
faster rate of infection, will have a greater likelihood of
eliminating cancer cells before the virus is eliminated by the
immune system. However, the use of VSV against cancer cells does
not have to be restricted to a single application. By molecular
substitution of the G-protein for enhancing immune responses
against foreign genes expressed by VSV, one could switch the
original G protein of the virus (e.g., Indiana VSV) with the G
protein from another strain or serotype (e.g., VSV New Jersey or
Chandipura), allowing a slightly different antigen presentation,
and reducing the initial response of the adaptive immune system to
second or third oncolytic inoculations with VSV.
[0062] Therefore, the disclosed chimeric viruses can have a VSV
genome that is rearranged compared to wildtype VSV. For example,
shifting the L-gene to the sixth position, by rearrangement or
insertion of an additional gene upstream, can result in attenuated
L-protein synthesis and a slight reduction in replication (Dalton
and Rose, Virology, 279(2):414-21 (2001)), an advantage when
considering treatment of the brain.
[0063] Repeat passaging of virulent strains under evolutionary
pressure can also be used to generate attenuated virus, increase
specificity of the virus for a particular target cell type, and/or
increase the oncolytic potential of the virus. For example,
VSV-rp30 ("30 times repeated passaging") is a wild-type-based VSV
with an enhanced oncolytic profile (Wollmann, et al., J. Virol.
79:6005-6022 (2005)). As described in WO 2010/080909, VSV-rp30 has
a preference for glioblastoma over control cells and an increased
cytolytic activity on brain tumor cells. Accordingly, in some
embodiments, the VSV background of the disclosed chimeric viruses
is one that has been modified to attenuate the virus, increase
specificity of the virus for a particular target cells, and/or
increase the oncolytic potential of the virus relative to a
wildtype or starting stain.
[0064] 2. Heterologous Glycoproteins
[0065] The disclosed chimeric VSV viruses have a heterologous
glycoprotein. Typically, the disclosed chimeric VSV viruses are
viruses that lack the G protein of VSV. Instead the chimeric VSV
viruses have a glycoprotein (e.g., G protein or GP protein) from a
distinct, non-VSV virus.
[0066] As demonstrated in the Examples below, glycoproteins for a
number of different viruses can be substituted into a VSV
background to create a chimeric VSV that can infect cancer cells.
Suitable glycoproteins can be from, for example, Lassa, rabies,
lymphocytic choriomeningitis virus (LCMV), Ebola, or Marburg virus.
The Examples below show that an Ebola-VSV, and even more so a
Lassa-VSV chimera, are particularly effective at killing brain
cancers with little or no toxicity to healthy or normal cells.
Other viral glycoprotein such as those from rabies, lymphocytic
choriomeningitis virus (LCMV), or Marburg virus may be more
suitable for targeting other cancer types, such as one or more of
the cancers discussed in more detail below. It is believed that VSV
chimeric viruses including an LCMV glycoprotein in place of the VSV
glycoprotein may show some advantages over the VSV glycoprotein in
infecting some cancer or sarcoma cells with enhanced innate
immunity, such as the virus-resistant sarcoma cells described in
Paglino and van den Pol, J. Virol., 85:9346-9358 (2011). In some
embodiments, the G protein in the VSV chimeric virus is a
heterologous G, wherein the G protein is not a G protein from LCMV.
In place of the Lassa glycoprotein which has a broad spectrum of
cells to which it binds, the VSV chimeric virus can have a
glycoprotein from another arena virus. Other arenaviruses may have
the same, similar, or different cellular binding receptors to
Lassa. In some embodiments, the glycoprotein is a viral
glycoprotein, preferably an arenavirus glycoprotein, that binds to
one or more of the same cell receptors as Lassa glycoprotein. In
some embodiments, the glycoprotein is a viral glycoprotein,
preferably an arenavirus glycoprotein that binds to one or more
similar cell receptors as Lassa glycoprotein. In some embodiments,
the glycoprotein is an areanvirus glycoprotein that binds to
different cell receptor(s) than Lassa glycoprotein. Such chimeric
viruses may also be safe viruses for use in oncolysis or as vaccine
vectors. Exemplary arenaviruses include, but are not limited to,
Old World complex arenaviruses such as Kodoko, Lujo, Mobala, Dank,
Gbagroube, Ippy, Merino Walk, Menekre, Mobala, and Mopeia, and New
World arenaviruses such as Guanarito, Junin, Machupo, Sabia,
Whitewater arroyo, Parana, Tamiami, Latino, plexal, and Chapare.
New World arenavirus glycoproteins may target receptors different
that those targeted by the Lassa glycoprotein.
[0067] a. Lassa G Proteins
[0068] In the most preferred embodiment, the G protein of VSV is
substituted with a glycoprotein from a Lassa virus. Lassa virus is
an Arenavirus. The genomic structure or Arenaviruses and the
genetic diversity of Lassa virus strains are discussed in Bowen, et
al., J. Virology, 6992-7004 (2000). Viruses of the genus
Arenavirus, family Arenaviridae, are enveloped viruses with a
genome consisting of two single stranded RNA species designated
small (S) and large (L). Each segment contains two non-overlapping
genes arranged in an ambisense orientation. The viral polymerase (L
protein) gene is encoded at the 3' end of the L RNA in the
genome-complementary sense, whereas the Z protein is encoded at the
5' end of the L RNA in the genomic sense. In a similar fashion, the
nucleoprotein (NP) gene is encoded at the 3' end of the S RNA,
whereas the glycoprotein precursor (GPC) is encoded at the 5' end
of the S RNA. The GPC is post-translationally cleaved into the
envelope glycoproteins GP1 and GP2. The arenaviruses have been
divided into two groups, the New World arenaviruses and the Old
World arenaviruses. Lassa virus is an Old World arenavirus.
[0069] The glycoprotein can come from any Lassa virus. The Lassa
virus glycoprotein can be from a naturally occurring virus, or a
virus modified, for example, to increase or decrease the virulence
of the virus, and/or increase the specificity or infectivity of the
virus compared to the parental strain or serotype. Suitable strains
and serotypes of Lassa virus from which the glycoprotein of the
chimeric VSV virus can be derived are known in the art and include,
for example, fifty-four strains identified and characterized in
Bowen, et al., J. Virology, 6992-7004 (2000). Common Lassa virus
stains include Lassa virus strain 803213, Lassa virus strain Acar
3080, Lassa virus strain AV, Lassa virus strain Josiah, Lassa virus
strain LP, Lassa virus strain Macenta, Lassa virus strain NL, Lassa
virus strain Pinneo, Lassa virus strain Weller, and Lassa virus
strain Z148.
[0070] Preferably, the chimeric virus's genome, or plasmid(s)
encoding the virus's genome encode the entire Lassa virus
glycoprotein precursor (GPC), such that both GP1 and GP2 are
expressed and contribute to formation of the chimeric virus's
envelope. In some embodiments, the chimeric virus's genome, or
plasmid(s) encoding the virus's genome encode less than the entire
Lassa virus glycoprotein precursor (GPC). For example, in some
embodiments, the viral genome or plasmid(s) encoding recombinant
viral genome encodes a glycoprotein that is a truncated GPC, or
only GP1 or only GP2.
[0071] The glycoprotein can be from Lassa strain Josiah. In a
particular embodiment, the chimeric viral genome includes the
nucleic acid sequence
TABLE-US-00001 1 cgcaccgggg atcctaggca tttttggttg cgcaattcaa
gtgtcctatt taaaatggga 61 caaatagtga cattcttcca ggaagtgcct
catgtaatag aagaggtgat gaacattgtt 121 ctcattgcac tgtctgtact
agcagtgctg aaaggtctgt acaattttgc aacgtgtggc 181 cttgttggtt
tggtcacttt cctcctgttg tgtggtaggt cttgcacaac cagtctttat 241
aaaggggttt atgagcttca gactctggaa ctaaacatgg agacactcaa tatgaccatg
301 cctctctcct gcacaaagaa caacagtcat cattatataa tggtgggcaa
tgagacagga 361 ctagaactga ccttgaccaa cacgagcatt attaatcaca
aattttgcaa tctgtctgat 421 gcccacaaaa agaacctcta tgaccacgct
cttatgagca taatctcaac tttccacttg 481 tccatcccca acttcaatca
gtatgaggca atgagctgcg attttaatgg gggaaagatt 541 agtgtgcagt
acaacctgag tcacagctat gctggggatg cagccaacca ttgtggtact 601
gttgcaaatg gtgtgttaca gacttttatg aggatggctt ggggtgggag ctacattgct
661 cttgactcag gccgtggcaa ctgggactgt attatgacta gttatcaata
tctgataatc 721 caaaatacaa cctgggaaga tcactgccaa ttctcgagac
catctcccat cggttatctc 781 gggctcctct cacaaaggac tagagatatt
tatattagta gaagattgct aggcacattc 841 acatggacac tgtcagattc
tgaaggtaaa gacacaccag ggggatattg tctgaccagg 901 tggatgctaa
ttgaggctga actaaaatgc ttcgggaaca cagctgtggc aaaatgtaat 961
gagaagcatg atgaggaatt ttgtgacatg ctgaggctgt ttgacttcaa caaacaagcc
1021 attcaaaggt tgaaagctga agcacaaatg agcattcagt tgatcaacaa
agcagtaaat 1081 gctttgataa atgaccaact tataatgaag aaccatctac
gggacatcat gggaattcca 1141 tactgtaatt acagcaagta ttggtacctc
aaccacacaa ctactgggag aacatcactg 1201 cccaaatgtt ggcttgtatc
aaatggttca tacttgaacg agacccactt ttctgatgat 1261 attgaacaac
aagctgacaa tatgatcact gagatgttac agaaggagta tatggagagg 1321
caggggaaga caccattggg tctagttgac ctctttgtgt tcagtacaag tttctatctt
1381 attagcatct tccttcacct agtcaaaata ccaactcata ggcatattgt
aggcaagtcg 1441 tgtcccaaac ctcacagatt gaatcatatg ggcatttgtt
cctgtggact ctacaaacag 1501 cctggtgtgc ctgtgaaatg gaagagatga
gacccttgtc agggcccccg tgacccaccg 1561 cctattggcg gtgggtcacg
ggggcgtcca tttacagaac gactctaggt gtcgatgttc 1621 tgaacaccat
atctctgggc agcactgctc tcaaaaccga tgtgttcagt cctcctgaca 1681
ctgctgcatc aaacatgatg cagtccatta gtgcacagtg aggggttatt tcctctttac
1741 cgcctctttt cttcttttca acaacgacac ctgtgtgcat gtggcataag
tctttatact 1801 ggtcccagac tgcattttca tacttcctgg aatcagtttt
gctgagggca atatcaatta 1861 gtttaatgtc ttttcttcct tgtgattcaa
ggagtttcct tatgtcatcg gacccctgac 1921 aggtaatgac catattccgg
gggagtgcat caatgacagc actggtcaag cccggttgtg 1981 tagcgaagag
gtctgtgaca tcaatcccat gtgagtactt agcatcctgc ttgaactgct 2041
ttaaatcagt aggttcacgg aagaagtgta tgtagcagcc tgaacttggt tgatagaggg
2101 caatttccac tggatcttca ggtcttcctt caatgtccat ccaggtctta
gcatttgggt 2161 caagttgcag cattgcatcc ttgagggtca tcagctgaga
ataggtaagc ccagcggtaa 2221 accctgccga ctgcagggat ttactggaat
tgttgctgtc agctttctgt ggcttcccat 2281 ctgattccag atcaacgaca
gtgttttccc aggcccttcc tgttattgag gttcttgatg 2341 caatatatgg
ccatccatct cctgacaaac aaatcttgta gagtatgttt tcataaggat 2401
tcctttcacc aggggtgtct gaaatgaaca ttccaagagc cttcttgacc tttaaaatgg
2461 atttgaggat accatccatt gtctgaggtg acaccttgat tgtctccaac
atattgccac 2521 catccagcat gcaagctcct gccttcacag ctgcacccaa
gctaaaatta taacctgaga 2581 tattcaaaga gcttttcttg gtgtcaatca
tatttaggat gggatgactt tgagtcagcc 2641 tgtctaagtc tgaagtgttg
ggatactttg ctgtgtagat caaacccaaa tctgtcaatg 2701 cttgtactgc
atcattcaag tcaacctgcc cctgttttgt cagacatgcc agtgtcagac 2761
ttggcatggt cccgaactga ttattgagca actctgcatt tttcacatcc caaactctca
2821 ccactccatc tctcccagcc cgagcccctt gattaccacc actcattcct
atcatattca 2881 ggagagctct tctttggtca agttgctgtg agcttaggtt
gcccatatag acacctgcac 2941 ttaatggcct ttctgttctg atcacctttg
actttaactt ctctagatca gcggctaaga 3001 ttaataagtc atctgaggtt
agagtcccaa ctctcagtat actcttttgt tgagttgatt 3061 ttaattcaac
aagattgttg accgcttgat ttaggtccct caaccgtttc aaatcattgt 3121
catcccttct ctccttgcgc atcaaccgtt gaacattact gacttcggag aagtcaagtc
3181 catgtaaaag agcctgggca tctttcacca cctgtagttt gatgttggag
cagtaaccag 3241 ataattccct cctcaaagat tgtgtccaca aaaaggattt
tatttccttt gaggcactca 3301 tcgccagatt gttgtgttgt atgcacgcaa
caaagaactg agactatctg ccaaaatgac 3361 aaaagcaaag cgcaatccaa
tagcctagga tccactgtgc g
(SEQ ID NO:1, Lassa virus strain recombinant Josiah segment S,
complete sequence GenBank: HQ688673.1), one or both of the open
reading frames thereof, or a fragment or fragments or variants
thereof encoding a functional glycoprotein. Variants can have at
least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence
identity to SEQ ID NO:1, or to the sequence encoding an open
reading frame thereof.
[0072] In some embodiments, the chimeric viral genome includes a
nucleic acid sequence encoding the polypeptide
TABLE-US-00002 MGQIVTFFQEVPHVIEEVMNIVLIALSVLAVLKGLYNFATCGLVGLVTFLL
LCGRSCTTSLYKGVYELQTLELNMETLNMTMPLSCTKNNSHHYIMVGNETG
LELTLTNTSIINHKFCNLSDAHKKNLYDHALMSIISTFHLSIPNFNQYEAM
SCDFNGGKISVQYNLSHSYAGDAANHCGTVANGVLQTFMRMAWGGSYIALD
SGRGNWDCIMTSYQYLIIQNTTWEDHCQFSRPSPIGYLGLLSQRTRDIYIS
RRLLGTFTWTLSDSEGKDTPGGYCLTRWMLIEAELKCFGNTAVAKCNEKHD
EEFCDMLRLFDFNKQAIQRLKAEAQMSIQLINKAVNALINDQLIMKNHLRD
IMGIPYCNYSKYWYLNHTTTGRTSLPKCWLVSNGSYLNETHFSDDIEQQAD
NMITEMLQKEYMERQGKTPLGLVDLFVFSTSFYLISIFLHLVKIPTHRHIV
GKSCPKPHRLNHMGICSCGLYKQPGVPVKWKR
(SEQ ID NO:2, GenBank: HQ688673.1), the polypeptide
TABLE-US-00003 MSASKEIKSFLWTQSLRRELSGYCSNIKLQVVKDAQALLHGLDFSEVSNVQ
RLMRKERRDDNDLKRLRDLNQAVNNLVELKSTQQKSILRVGTLTSDDLLIL
AADLEKLKSKVIRTERPLSAGVYMGNLSSQQLDQRRALLNMIGMSGGNQGA
RAGRDGVVRVWDVKNAELLNNQFGTMPSLTLACLTKQGQVDLNDAVQALTD
LGLIYTAKYPNTSDLDRLTQSHPILNMIDTKKSSLNISGYNFSLGAAVKAG
ACMLDGGNMLETIKVSPQTMDGILKSILKVKKALGMFISDTPGERNPYENI
LYKICLSGDGWPYIASRTSITGRAWENTVVDLESDGKPQKADSNNSSKSLQ
SAGFTAGLTYSQLMTLKDAMLQLDPNAKTWMDIEGRPEDPVEIALYQPSSG
CYIHFFREPTDLKQFKQDAKYSHGIDVTDLFATQPGLTSAVIDALPRNMVI
TCQGSDDIRKLLESQGRKDIKLIDIALSKTDSRKYENAVWDQYKDLCHMHT
GVVVEKKKRGGKEEITPHCALMDCIMFDAAVSGGLNTSVLRAVLPRDMVFR TSTPRVVL
(SEQ ID NO:3, GenBank: HQ688673.1), a combination thereof, or a one
or more functional fragments or variants thereof. Variants can have
at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more
sequence identity to SEQ ID NO:2 or 3.
[0073] b. Ebola G Proteins
[0074] In another preferred embodiment, the G protein of VSV is
substituted with a glycoprotein from an Ebola virus. Ebola virus,
along with Marburg virus, constitutes the family Filoviridae in the
order of Mononegavirales (reviewed in Feldmann and Geisbert,
Lancet, 377(9768): 849-862 (2011), and Sanchez, et al.,
Filoviridae: Marburg and Ebola viruses. In: Knipe, D M.; Howley, P
M., editors. Fields virology. Philadelphia: Lippincott Williams
& Wilkins; 2006. p. 1409-1448). Filoviruses are enveloped,
non-segmented, negative-stranded RNA viruses with filamentous
particles. Ebola virus particles have a uniform diameter of 80 nm
but can greatly vary in length, with lengths up to 14000 nm. The
genome includes seven genes in the order 3' leader, nucleoprotein,
virion protein (VP) 35, VP40, glycoprotein, VP30, VP24,
RNA-dependent RNA polymerase (L)-5' trailer. With the exception of
the glycoprotein gene, all genes are monocistronic, encoding for
one structural protein. The inner ribonucleoprotein complex of
virion particles consists of the RNA genome encapsulated by the
nucleoprotein, which associates with VP35, VP30, and RNA-dependent
RNA polymerase to form the functional transcriptase-replicase
complex. Additionally, the proteins of the ribonucleoprotein
complex have other functions, for example, VP35 is an antagonist of
interferon; VP40 is a matrix protein and modulates particle
formation; VP24, is structural, membrane-associated protein that
also interferes with interferon signaling.
[0075] The glycoprotein is the only transmembrane surface protein
of the virus and forms trimeric spikes consisting of glycoprotein 1
and glycoprotein 2--two di-sulphide linked furin-cleavage fragments
(Sanchez, et al., Filoviridae: Marburg and Ebola viruses. In:
Knipe, D M.; Howley, P M., editors. Fields virology. Philadelphia:
Lippincott Williams & Wilkins; 2006. p. 1409-1448). The primary
product of the GP gene is a soluble glycoprotein (sGP) that is also
secreted from infected cells, a characteristic distinguishing it
from other Mononegavirales (Sanchez, et al., Proc Natl Acad Sci
USA, 93:3602-3607 (1996), Volchkov, et al., Virology, 214:421-430
(1995)). Nucleic acid sequences encoding Ebola glycoprotein, the
mechanism of transcription/translation yielding functional Ebola
glycoprotein, Ebola glycoprotein amino acid sequences, and the
structure and function of Ebola glycoprotein are well known in the
art and discussed in, for example, Lee and Saphire, Future
Virology, 4(6):621-635 (2009), Sanchez, Proc Natl Acad Sci USA.,
93(8):3602-3607 (1996), Volchkov, et al., Virology, 214(2):421-430
(1995), Gire et al, Science, 345: 1369-1372 (2014)).
[0076] The Ebola virus glycoprotein can be from a naturally
occurring virus, or a virus modified, for example, to increase or
decrease the virulence of the virus, and/or increase the
specificity or infectivity of the virus compared to the parental
strain or serotype. Suitable species of Ebola virus from which the
glycoprotein of the chimeric VSV virus can be derived are known in
the art and include, for example, Sudan ebolavirus (SEBOV), Zaire
ebolavirus (ZEBOV), Cote d'Ivoire ebolavirus (also known and here
referred to as Ivory Coast ebolavirus (ICEBOV)), Reston ebolavirus
(REBOV), and Bundigbugyo ebolavirus (BEBOV) (Geibert and Feldmann,
J. Infect. Dis., 204 (suppl 3): S1075-S1081 (2011)). Preferably,
the chimeric virus's genome, or plasmid(s) encoding the virus's
genome encode the entire Ebola virus glycoprotein (GP), such that
the glycoprotein is expressed and contributes to formation of the
chimeric virus's envelope. In some embodiments, the chimeric
virus's genome, or plasmid(s) encoding the virus's genome encode
less than the entire Ebola virus glycoprotein. For example, in some
embodiments, the viral genome or plasmid(s) encoding recombinant
viral genome encodes a glycoprotein that is a truncated or variant
GP. In some embodiments, the chimeric virus's genome, or plasmid(s)
encoding the virus's genome encode full length, truncated, or
variant GP1, GP2, or a combination thereof.
[0077] In some embodiment, the chimeric viral genome includes the
nucleic acid sequence
TABLE-US-00004 (SEQ ID NO: 6) 1 atgggcgtta caggaatatt gcagttacct
cgtgatcgat tcaagaggac atcattcttt 61 ctttgggtaa ttatcctttt
ccaaagaaca ttttccatcc cacttggagt catccacaat 121 agcacattac
aggttagtga tgtcgacaaa ctagtttgtc gtgacaaact gtcatccaca 181
aatcaattga gatcagttgg actgaatctc gaagggaatg gagtggcaac tgacgtgcca
241 tctgcaacta aaagatgggg cttcaggtcc ggtgtcccac caaaggtggt
caattatgaa 301 gctggtgaat gggctgaaaa ctgctacaat cttgaaatca
aaaaacctga cgggagtgag 361 tgtctaccag cagcgccaga cgggattcgg
ggcttccccc ggtgccggta tgtgcacaaa 421 gtatcaggaa cgggaccgtg
tgccggagac tttgccttcc ataaagaggg tgctttcttc 481 ctgtatgatc
gacttgcttc cacagttatc taccgaggaa cgactttcgc tgaaggtgtc 541
gttgcatttc tgatactgcc ccaagctaag aaggacttct tcagctcaca ccccttgaga
601 gagccggtca atgcaacgga ggacccgtct agtggctact attctaccac
aattagatat 661 caggctaccg gttttggaac caatgagaca gagtacttgt
tcgaggttga caatttgacc 721 tacgtccaac ttgaatcaag attcacacca
cagtttctgc tccagctgaa tgagacaata 781 tatacaagtg ggaaaagyag
caataccacg ggaaaactaa tttggaaggt caaccccgaa 841 attgatacaa
caatcgggga gtgggccttc tgggaaacta aaaaaaacct cactagaaaa 901
attcgcagtg aagagttgtc tttcacagtt gtatcaaacg gagccaaaaa catcagtggt
961 cagagtccgg cgcgaacttc ttccgaccca gggaccaaca caacaactga
agaccacaaa 1021 atcatggctt cagaaaattc ctctgcaatg gttcaagtgc
acagtcaagg aagggaagct 1081 gcagtgtcgc atctaacaac ccttgccaca
atctccacga gtccccaatc cctcacaacc 1141 aaaccaggtc cggacaacag
cacccataat acacccgtgt ataaacttga catctctgag 1201 gcaactcaag
ttgaacaaca tcaccgcaga acagacaacg acagcacagc ctccgacact 1261
ccctctgcca cgaccgcagc cggaccccca aaagcagaga acaccaacac gagcaagagc
1321 actgacttcc tggaccccgc caccacaaca agtccccaaa accacagcga
gaccgctggc 1381 aacaacaaca ctcatcacca agataccgga gaagagagtg
ccagcagcgg gaagctaggc 1441 ttaattacca atactattgc tggagtcgca
ggactgatca caggcgggag aagaactcga 1501 agagaagcaa ttgtcaatgc
tcaacccaaa tgcaacccta atttacatta ctggactact 1561 caggatgaag
gtgctgcaat cggactggcc tggataccat atttcgggcc agcagccgag 1621
ggaatttaca tagaggggct aatgcacaat caagatggtt taatctgtgg gttgagacag
1681 ctggccaacg agacgactca agctcttcaa ctgttcctga gagccacaac
tgagctacgc 1741 accttttcaa tcctcaaccg taaggcaatt gatttcttgc
tgcagcgatg gggcggcaca 1801 tgccacattc tgggaccgga ctgctgtatc
gaaccacatg attggaccaa gaacataaca 1861 gacaaaattg atcagattat
tcatgatttt gttgataaaa cccttccgga ccagggggac 1921 aatgacaatt
ggtggacagg atggagacaa tggataccgg caggtattgg agttacaggc 1981
gttataattg cagttatcgc tttattctgt atatgcaaat ttgtctttta g.
[0078] SEQ ID NO:6 a nucleic acid encoding a full-length
(non-secreted) glycoprotein gene found in GenBank accession
NC_002549 nt 6039-8068. Note that the GenBank sequence over this
region is 2030 nt long, whereas SEQ ID NO:6 is 2031 nt in length.
This difference derives from the fact that the GenBank sequence is
based on the genomic RNA sequence, whereas the sequence below is
based on the mRNA sequence that has been `edited` by the viral
polymerase to include an extra `A` nucleotide between 6918-6924 of
the GenBank sequence. Therefore, in some embodiments, the chimeric
viral genome includes a nucleic acid encoding SEQ ID NO:6, or the
variant thereof encoded by GenBank accession NC_002549 nt
6039-8068, or a fragment or variant thereof encoding a functional
glycoprotein. Variants can have at least 70%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:6, or to
the sequence encoding an open reading frame thereof.
[0079] In some embodiments, the chimeric viral genome includes a
nucleic acid sequence encoding the polypeptide
TABLE-US-00005 MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKL
VCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAG
EWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAF
HKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVN
ATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQ
LNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEEL
SFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQG
REAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHH
RRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETA
GNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPK
CNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQL
ANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEP
HDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVII
AVIALFCICKFVF
(SEQ ID NO:7), or a functional fragment or variant thereof.
Variants can have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99%, or more sequence identity to SEQ ID NO:7.
[0080] In some embodiments, the chimeric viral genome includes the
nucleic acid sequence
TABLE-US-00006 1 atgggcgtta caggaatatt gcagttacct cgtgatcgat
tcaagaggac atcattcttt 61 ctttgggtaa ttatcctttt ccaaagaaca
ttttccatcc cacttggagt catccacagt 121 agcacattac aggttagtga
tgtcgacaaa ctagtttgtc gtgacaaact gtcatccaca 181 aatcaattga
gatcagttgg actgaatctc gaagggaatg gagtggcaac tgacgtgcca 241
tctgcaacta aaagatgggg cttcaggtcc ggtgtcccac caaaggtggt caattatgaa
301 gctggtgaat gggctgaaaa ctgctacaat cttgaaatca aaaaacctga
cgggagtgag 361 tgtctaccag cagcgccaga cgggattcgg ggcttccccc
ggtgccggta tgtgcacaaa 421 gtatcaggaa cgggaccgtg tgccggagac
tttgccttcc ataaagaggg tgctttcttc 481 ctgtatgatc gacttgcttc
cacagttatc taccgaggaa cgactttcgc tgaaggtgtc 541 gttgcatttc
tgatactgcc ccaagctaag aaggacttct tcagctcaca ccccttgaga 601
gagccggtca atgcaacgga ggacccgtct agtggctact attctaccac aattagatat
661 caggctaccg gttttggaac caatgagaca gagtacttgt tcgaggttga
caatttgacc 721 tacgtccaac ttgaatcaag attcacacca cagtttctgc
tccagctgaa tgagacaata 781 tatacaagtg ggaaaaggag caataccacg
ggaaaactaa tttggaaggt caaccccgaa 841 attgatacaa caatcgggga
gtgggccttc tgggaaacta aaaaaaacct cactagaaaa 901 attcgcagtg
aagagttgtc tttctctaga gcaggactga tcacaggcgg gagaagaact 961
cgaagagaag caattgtcaa tgctcaaccc aaatgcaacc ctaatttaca ttactggact
1021 actcaggatg aaggtgctgc aatcggactg gcctggatac catatttcgg
gccagcagcc 1081 gagggaattt acatagaggg gctaatgcac aatcaagatg
gtttaatctg tgggttgaga 1141 cagctggcca acgagacgac tcaagctctt
caactgttcc tgagagccac aactgagcta 1201 cgcacctttt caatcctcaa
ccgtaaggca attgatttct tgctgcagcg atggggcggc 1261 acatgccaca
ttctgggacc ggactgctgt atcgaaccac atgattggac caagaacata 1321
acagacaaaa ttgatcagat tattcatgat tttgttgata aaacccttcc ggaccagggg
1381 gacaatgaca attggtggac aggatggaga caatggatac cggcaggtat
tggagttaca 1441 ggcgttgtaa ttgcagttat cgctttattc tgtatatgca
aatttgtctt ttag
(SEQ ID NO:8), or a fragment or variant thereof encoding a
functional glycoprotein. Variants can have at least 70%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID
NO:8.
[0081] In some embodiments, the chimeric viral genome includes a
nucleic acid sequence encoding the polypeptide
TABLE-US-00007 MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHSSTLQVSDVDKL
VCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAG
EWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAF
HKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVN
ATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQ
LNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEEL
SFSRAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFG
PAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNR
KAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQ
GDNDNWWTGWRQWIPAGIGVTGVVIAVIALFCICKFVF
(SEQ ID NO:9), or a functional fragment or variant thereof.
Variants can have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99%, or more sequence identity to SEQ ID NO:9.
[0082] In some embodiments, the Ebola G protein is lacking the
mucin domain, (e.g., about amino acids 309-489 of a full length G
protein), and/or has one or more substitutions (e.g. as described
in Wong, et al., J. Virol., 84(1):163-75 (2010), and in the
examples below).
[0083] 3. Additional Transgenes
[0084] Viruses can be modified to express one or more additional
transgenes, separately or as a part of other expressed proteins.
The viral genome of VSV has the capacity to accommodate additional
genetic material. At least two additional transcription units,
totaling 4.5 kb, can be added to the genome, and methods for doing
so are known in the art. The added genes are stably maintained in
the genome upon repeated passage (Schnell, et al., EMBO Journal,
17:1289-1296 (1998); Schnell, et al., PNAS, 93: 11359-11365 (1996);
Schnell, et al., Journal of Virology, 70:2318-2323 (1996); Kahn, et
al., Virology, 254, 81-91 (1999)).
[0085] In preferred embodiments the viruses are modified to include
a gene encoding a therapeutic protein, an antigen, a detectable
marker or reporter, a targeting moiety, or a combination thereof.
In some embodiments, the gene is placed in the first gene position
in the VSV background. Given the nature of VSV protein expression,
genes in the first position generate the highest expression of any
gene in the virus, with a 3' to 5' decrease in gene expression. The
chimeric VSV can also be constructed to contain two different and
independent genes placed in the first and second gene position of
VSV. For example, van den Pol and Davis, et al., J. Virol.,
87(2):1019-1034 (2013), describes the generation of a highly
attenuated VSV virus by adding two (reporter) genes to the 3' end
of the VSV genome, thereby shifting the NPMGL genes from positions
1 to 5 to positions 3 to 7. This strategy can be used to allow
strong expression of genes coding for any combination of two
heterologous proteins, for example two therapeutic proteins, a
therapeutic protein and reporter, or an immunogenic protein and a
reporter that could be useful to track the virus in a clinical
situation.
[0086] a. Therapeutic Proteins and Reporters
[0087] Chimeric VSV viruses can be engineered to include one or
more additional genes that encode a therapeutic protein or a
reporter. Suitable therapeutic proteins, such as cytokines or
chemokines, are known in the art, and can be selected depending on
the use or disease to be treated. Preferred cytokines include, but
are not limited to, granulocyte macrophage colony stimulating
factor (GM-CSF), tumor necrosis factor alpha (TNF.alpha.), tumor
necrosis factor beta (TNF.beta.), macrophage colony stimulating
factor (M-CSF), interleukin-1 (IL-1), interleukin-2 (IL-2),
interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6),
interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-15
(IL-15), interleukin-21 (IL-21), interferon alpha (IFN.alpha.),
interferon beta (IFN.beta.), interferon gamma (IFN.gamma.), and
IGIF, and variants and fragments thereof.
[0088] Suitable chemokines include, but are not limited to, an
alpha-chemokine or a beta-chemokine, including, but not limited to,
a C5a, interleukin-8 (IL-8), monocyte chemotactic protein I alpha
(MIP1.alpha.), monocyte chemotactic protein 1 beta (MIP1.beta.),
monocyte chemo-attractant protein 1 (MCP-1), monocyte
chemo-attractant protein 3 (MCP-3), platelet activating factor
(PAFR), N-formyl-methionyl-leucyl-[.sup.3H]phenylalanine (FMLPR),
leukotriene B.sub.4, gastrin releasing peptide (GRP), RANTES,
eotaxin, lymphotactin, IP10, I-309, ENA78, GCP-2, NAP-2 and
MGSA/gro, and variants and fragments thereof.
[0089] Particularly preferred genes include those that encode
proteins that up-regulate an immune attack on infected tumors such
as IL-28, IL-2, FLT3L, and GM-CSF (Ali, et al., Cancer Res,
65:7194-7204 (2005); Barzon, et al., Methods Mol. Biol.,
542:529-549 (2009); Wongthida, et al., Hum. Gene Ther., 22:1343-53
(2011). Other therapeutic proteins that have been successfully
engineered into VSV or other viruses include IL2, IL-4, IL-7,
IL-12, and TRAIL (Jinush, et al., Cancer Science, 100, 1389-1396.
(2009)). The virus can also be engineered to include one or more
genes encoding a reporter. The reporter can serve as a measure or
monitor of in vivo viral activity. Exemplary reporters are known in
the art and include, but are not limited to, carcinoembryonic
antigen, secreted alkaline phosphatase, and the beta subunit of
chorionic gonadotropin. These reporters are released by infected
cells into the blood, and can be measured peripherally to determine
viral activity, including viral activity in the brain (Phuong, et
al., Cancer Res., 63:2462-2469 (2003); Peng, et al., Nat. Med.,
8:527-531 (2002); Shashkova, et al., Cancer Gene Ther., 15:61-72
(2008); Hiramatsu, et al., Cancer Science, 100, 1389-1396
(2005)).
[0090] In some embodiments, the virus's genome is modified to
encode a detectable marker or reporter, preferably in the first
position. The detectable marker allows the user to detect and
monitor the location and efficacy of the virus in vivo and in
resected tissue ex vivo without the need for antibodies. Suitable
markers are known in the art and include, but are not limited to,
LacZ, GFP (or eGFP), and luciferase.
[0091] There have been reports of humoral immune response to eGFP
and rejection of eGFP transduced cells following subretinal
administration of AAV2 or lentivirus expressing eGFP in animals
(Bainbridge, et al., Gene Ther., 10(16):1336-44 (2003), and Doi,
K., J. Virol, 78(20): 11327-33 (2004)). Thus, the safety and in
vivo persistence of a virus including a detectable marker (e.g.,
one expressing eGFP) may be different than that of a virus without
the marker, however, these differences can be assessed by one of
skill in the art using methods known in the art and the methods
described in the Examples. As discussed in more detail above, in
the particular case of VSV, adding a gene added to the first
position typically attenuates the virulence of VSV (Wollmann, et
al., J. Virol., 84(3):1563-73 (2010)). Therefore, in some
embodiments, chimeric VSV that include a marker such as GFP in the
first position may have an improved safety profile compared viruses
without it.
[0092] b. Viruses Engineered to Deliver Vaccine Antigens
[0093] As discussed in more detail below, the virus can be a
vaccine vector that serves as an immunogen for eliciting an immune
response against a disease. This can be accomplished by cloning an
antigen of an unrelated disease into the chimeric VSV virus. VSV
viruses expressing foreign viral glycoproteins have shown promise
as a vaccine vectors (Roberts, et al., J. Virol. 73:3723-3732
(1999), Rose, et al., Cell, 106:539-549 (2001), Jones, et al., Nat.
Med. 11:786-790 (2005)). Additionally, recombinant VSVs are able to
accommodate large inserts and multiple genes in their genomes. This
ability to incorporate large gene inserts in replication-competent
viruses offers advantages over other RNA or DNA virus vectors, such
as those based on alphaviruses, REO virus, poliovirus, and
parvovirus.
[0094] VSV viruses can be engineered to incorporate one or more
nucleic acid sequences encoding one or more non-native or
heterologous immunogenic antigens. One or more native VSV genes may
be truncated or deleted to create additional space for the sequence
encoding the immunogenic antigen. When expressed by the VSV virus
administered to a patient in need thereof, the immunogenic antigen
produces prophylactic or therapeutic immunity against a disease or
disorder. Immunogenic antigens can be expressed as a fusion protein
with other polypeptides including, but not limited to, native VSV
polypeptides, or as a non-fusion protein. By way of non-limiting
examples, the antigen can be a protein or polypeptide derived from
a virus, bacterium, parasite, plant, protozoan, fungus, tissue or
transformed cell such as a cancer or leukemic cell. Antigens may be
expressed as single antigens or may be provided in combination.
[0095] Because the substitution of the Lassa glycoprotein for the
VSV glycoprotein generates a chimeric virus that appears far safer
than VSVs that contain the VSV glycoprotein, yet still retains the
broad spectrum of cells to which it can bind, the chimeric virus
can serve as a vaccination platform for a wide variety of microbial
pathogens, including but not limited to, HIV, influenza, polio,
measles, mumps, chicken pox, hendra, and others. Additionally, the
fact that the Lassa-VSV chimeric virus is safe even in the brains
of SCID mice lacking the normal T and B cell systemic immunity, as
demonstrated in the Examples below, indicates that a vaccine based
on chimeric Lassa-VSV would be safer than the corresponding vaccine
based on VSV that retained its VSV glycoprotein, and therefore the
chimeric Lassa-VSV might be useful in vaccinating people with
depressed immune systems, for instance those with AIDS or those
with genetically compromised immune systems, or patients with
attenuated immunity related to ongoing cancer. The target of the
vaccine could either be a type of cancer cell as a cancer
treatment. Alternately, the target could be any of a large number
of microbial pathogens.
[0096] c. Targeting Domains
[0097] Viruses can be engineered to include one or more additional
genes that target the virus to cells of interest, see for example
U.S. Pat. No. 7,429,481. In preferred embodiments, expression of
the gene results in expression of a ligand on the surface of the
virus containing one or more domains that bind to antigens, ligands
or receptors that are specific to tumor cells, or are up-regulated
in tumor cells compared to normal tissue. Appropriate targeting
ligands will depend on the target cell or cancer of interest and
will be known to those skilled in the art. For example, glioma stem
cells are reported to express CD133 and nestin. Accordingly, in
some embodiments, the viruses are engineered to express a targeting
moiety that bind to CD133 or nestin.
[0098] It is believed that the Lassa glycoprotein is important for
targeting chimeric Lassa-VSV virus to cells, and contributes to the
desirable oncolytic profile exhibited by the chimeric Lassa-VSV
virus. Accordingly, in preferred embodiments, any additional
targeting ligands or moieties engineered into the virus do not
reduce and preferably enhance the oncolytic activity or profile of
the virus.
[0099] 4. Exemplary Chimeric Viruses
[0100] Exemplary chimeric VSV viruses with Lassa virus glycoprotein
are known in the art. The viruses can be used in the disclosed
methods of use and treatment with or without one or more
modifications, such as those discussed above.
[0101] An exemplary virus is described in Jae, et al., Science,
340(6151):479-483 (2013). Briefly, recombinant VSV expressing eGFP
and the Lassa virus glycoprotein (rVSV-GP-LASV) was cloned and
recovered as follows: the open reading frame encoding LASV-GP
(strain Josiah, GenBank:HQ688673.1) was amplified using the
following primer sequences:
5'-GCGACGCGTACCATGGGACAAATAGTGACATTCT-3' (SEQ ID NO:4) and
5'-GGCGGCCGCTCATCTCTTCCATTTCACAGG-3' (SEQ ID NO:5). Subsequently,
the PCR product was sequenced and cloned into the MluI and NotI
sites of pVSVAG-eGFP-MN (Whelan, et al., Proc. Natl. Acad. Sci.
U.S.A., 92(18):8388-92 (1995), and Wong, et al., J. Virol.,
84(1):163-75 (2010)) thereby replacing the native VSV glycoprotein
G coding sequence. Recombinant virus was recovered and amplified as
described (Whelan, et al., supra).
[0102] The genome of this Lassa-VSV includes an open reading frame
encoding a GFP reporter in the first position. This allows easy
detection of which cells are infected, as they turn green. In
addition, having a gene added to the first position attenuates the
virulence of VSV (Wollmann, et al., J. Virol, 84(3):1563-73
(2010)). Accordingly, a chimeric VSV with a reporter or other
heterologous gene at the first position may be attenuated or less
virulent compared to the same virus without a reporter or other
heterologous gene at the first position. However, as discussed in
more detail in the Examples below, chimeric Lassa-VSV viruses are
both efficacious and safe with or without a reporter, or another
heterologous gene, in the first position. Therefore, the
heterologous gene in the first position is optional.
[0103] The construction of a recombinant VSV expressing eGFP and
the Ebola virus glycoprotein (rVSV-GP-EBOV) was also described in
Wong, et al., J. Virol., 84(1):163-75 (2010).
[0104] A second exemplary virus is discussed in Garbutt, et al., J.
Virol., 78(10): 5458-5465 (2004) and Geisbert, et al., PLOS,
2(6):537-545 (2005), which describe the construction of chimeric
VSV viruses having a VSV, Indiana serotype background and a
glycoprotein from Lassa virus, strain Josiah. A plasmid expressing
the positive-strand RNA complement of the VSV genome with a site
for foreign gene expression is described in Schnell, et al., J.
Virol., 70:2318-2323 (1996). This plasmid (VSVXN2) contains the
five VSV genes (nucleoprotein N, phosphoprotein P, matrixprotein M,
glycoprotein G, and polymerase L) in order, flanked by the
bacteriophage T7 promoter, the VSV leader, and the hepatitis delta
virus ribozyme, and the T7 terminator sequence. Between the G and
the L genes, a linker site (XhoI-NheI) is present, flanked by a
transcriptional start and stop signal for the additional gene to be
expressed. As discussed in Garbutt, et al., J. Virol., 78(10):
5458-5465 (2004), the plasmid can be modified to delete the G gene,
and the open reading frame encoding the transmembrane glycoprotein
of Lassa virus (GPC) can be prepared, for example by PCR, and
cloned into the XhoI and NheI sites of the modified vector where
the G gene has been deleted.
[0105] Following cloning, competent cells, for example, BSR-T7
cells, can be co-transfected with the vector and support plasmids
encoding the viral ribonucleoprotein constituents (e.g., pBS-VSV N,
pBS-VSV P, pBS-VSV L) to generate recombinant infectious virus that
can be recovered from the supernatant of the cultured cells.
Rescued rVSV can be passaged, on VeroE6 cells, for example, to
obtain a virus stock.
[0106] VSV-LCMV viruses are described in U.S. Patent Application
No. 2014/0301992, and 2011/0250188, and U.S. Pat. No. 6,440,730
[0107] Additional methods of making and recovering chimeric VSV
virus by expressing full-length cDNA from plasmid(s) are known in
the art and discussed in more detail below.
[0108] B. Pharmaceutical Compositions
[0109] Immunizing and therapeutic viruses are typically
administered to a patient in need thereof in a pharmaceutical
composition. Pharmaceutical compositions containing virus may be
for systemic or local administration, such as intratumoral. Dosage
forms for administration by parenteral (intramuscular (IM),
intraperitoneal (IP), intravenous (IV), intra-arterial, intrathecal
or subcutaneous injection (SC)), or transmucosal (nasal, vaginal,
pulmonary, or rectal) routes of administration can be formulated.
In some embodiments, a therapeutic virus is delivered by local
injection, for example intracranial injection preferably at or near
the tumor site. In a particular embodiment a therapeutic virus is
injected directly into the tumor. The compositions can be
formulated for and delivered via catheter into the tumor resection
cavity through convection-enhanced delivery (CED). In some
embodiments an immunizing virus is delivered peripherally,
intranasally or by intramuscular injection.
[0110] As discussed in more detail below, the virus can also be
used as an immunizing virus. The immunizing virus can be the same
as a therapeutic virus but administered prior to a therapeutic
administration so that the subject's immune system is primed to
eliminate the virus following the therapeutic administration.
Alternatively, the immunizing virus can be modified as discussed
above to carry a disease antigen and used as part of a vaccine
protocol. Immunizing viruses can be delivered peripherally, for
example, by the intranasal route or by intramuscular injection.
[0111] 1. Effective Amounts
[0112] As generally used herein, an "effective amount" is that
amount which is able to induce a desired result in a treated
subject. The desired results will depend on the disease or
condition to be treated. The precise dosage will vary according to
a variety of factors such as subject-dependent variables (e.g.,
age, immune system health, etc.), the disease, and the treatment
being effected. For example, an effective amount of immunizing
virus generally results in production of antibody and/or activated
T cells against an antigen, or that kill or limit proliferation of
or infection by a pathogen. An effective amount of the immunizing
virus can be an amount sufficient to reduce neurovirulence of the
therapeutic virus compared to administration of the therapeutic
virus without first administering the immunizing virus.
[0113] Therapeutically effective amounts of the therapeutic viruses
disclosed herein used in the treatment of cancer will generally
kill tumor cells or inhibit proliferation or metastasis of the
tumor cells. Symptoms of cancer may be physical, such as tumor
burden, or biological such as proliferation of cancer cells. The
actual effective amounts of virus can vary according to factors
including the specific virus administered, the particular
composition formulated, the mode of administration, and the age,
weight, condition of the subject being treated, as well as the
route of administration and the disease or disorder.
[0114] An effective amount of the virus can be compared to a
control. Suitable controls are known in the art. A typical control
is a comparison of a condition or symptom of a subject prior to and
after administration of the virus. The condition or symptom can be
a biochemical, molecular, physiological, or pathological readout.
In another embodiment, the control is a matched subject that is
administered a different therapeutic agent. Accordingly, the
compositions disclosed here can be compared to other art recognized
treatments for the disease or condition to be treated.
[0115] For example, the virus can be administered in an amount
effective to infect and kill cancer cells, improve survival of a
subject with cancer, or a combination thereof. In a particular
embodiment, the cancer is glioblastoma.
[0116] One of the advantages of the disclosed viruses is that they
show little or no toxicity to normal or healthy cells (e.g.,
non-cancerous cells). Therefore, in some embodiments the effective
amount of virus causes little or no destruction of non-cancerous
cells. The level of pathogenicity to normal cells can be compared
to the level of pathogenicity of other VSV oncolytic viruses that
do not have G gene replaced with a heterologous G gene. Such
viruses are known in the art and include, for example, VSV-1'GFP,
VSV-rp30, or VSV-.DELTA.M51.
[0117] One important index of oncolytic potential is the ratio of
viral replication in normal/control cells versus tumor or cancer
cells. These ratios serve as an important index of the relative
levels of viral replication in normal and tumor cells. A large
ratio indicates greater replication in cancer cells than in control
cells. In preferred embodiments, the ratio of replication of normal
cells:target cells is greater than about 1:100, preferably greater
than about 1:250, more preferable greater than about 1:500, most
preferably greater than about 1:1000. In some embodiments, the
oncolytic potential of the disclosed viruses is larger than the
oncolytic potential of other VSV oncolytic viruses that do not have
G gene replaced with a heterologous G gene, for example, VSV-1'GFP,
VSV-rp30, or VSV-.DELTA.M51.
[0118] 2. Dosages
[0119] Appropriate dosages can be determined by a person skilled in
the art, considering the therapeutic context, age, and general
health of the recipient. The selected dosage depends upon the
desired therapeutic effect, on the route of administration, and on
the duration of the treatment desired. Active virus can also be
measured in terms of plaque-forming units (PFU). A plaque-forming
unit can be defined as areas of cell lysis (CPE) in monolayer cell
culture, under overlay conditions, initiated by infection with a
single virus particle. Generally dosage levels of virus between
10.sup.2 and 10.sup.12 PFU are administered to humans. Virus is
typically administered in a liquid suspension, in a volume ranging
between 10 .mu.l and 100 ml depending on the route of
administration. Generally, dosage and volume will be lower for
intratumoral injection as compared to systemic administration or
infusion. The dose may be administered once or multiple times. When
administered locally, virus can be administered to humans at dosage
levels between 10.sup.2 and 10.sup.8 PFU. Virus can be administered
in a liquid suspension, in a low volume. For example, the volume
for local administration can range from about 20 .mu.l to about
2000. Multiple doses can be administered. In some embodiment,
multiple injections are used to achieve a single dose. Systemic or
regional administration via subcutaneous, intramuscular,
intra-organ, or intravenous administration can have higher volumes,
for example, 10 to 100 ml.
[0120] 3. Formulations
[0121] The term "pharmaceutically acceptable" means a non-toxic
material that does not interfere with the effectiveness of the
biological activity of the active ingredients. The term
"pharmaceutically-acceptable carrier" means one or more compatible
solid or liquid fillers, diluents or encapsulating substances which
are suitable for administration to a human or other vertebrate
animal. The term "carrier" refers to an organic or inorganic
ingredient, natural or synthetic, with which the active ingredient
is combined to facilitate the application.
[0122] Pharmaceutical compositions may be formulated in a
conventional manner using one or more physiologically acceptable
carriers including excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. The compositions may be administered in
combination with one or more physiologically or pharmaceutically
acceptable carriers, thickening agents, co-solvents, adhesives,
antioxidants, buffers, viscosity and absorption enhancing agents
and agents capable of adjusting osmolarity of the formulation.
Proper formulation is dependent upon the route of administration
chosen. If desired, the compositions may also contain minor amounts
of nontoxic auxiliary substances such as wetting or emulsifying
agents, dyes, pH buffering agents, or preservatives. The
formulations should not include membrane disrupting agents which
could kill or inactivate the virus.
[0123] a. Formulations for Local or Parenteral Administration
[0124] In a preferred embodiment, compositions including oncolytic
viruses disclosed herein, are administered in an aqueous solution,
by parenteral injection. Injection includes, but it not limited to,
local, intratumoral, intravenous, intraperitoneal, intramuscular,
or subcutaneous injection. The formulation may also be in the form
of a suspension or emulsion. In general, pharmaceutical
compositions are provided including effective amounts of virus, and
optionally include pharmaceutically acceptable diluents,
preservatives, solubilizers, emulsifiers, adjuvants and/or
carriers. Such compositions include diluents such as sterile water,
buffered saline of various buffer content (e.g., Tris-HCl, acetate,
phosphate), pH and ionic strength; and optionally, additives such
as anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and
preservatives and bulking substances (e.g., lactose, mannitol).
Examples of non-aqueous solvents or vehicles are propylene glycol,
polyethylene glycol, vegetable oils, such as olive oil and corn
oil, gelatin, and injectable organic esters such as ethyl oleate. A
preferred solution is phosphate buffered saline or sterile
saline.
[0125] b. Formulations for Mucosal Administration
[0126] In some embodiments, the compositions are formulated for
mucosal administration, such as through nasal, pulmonary, or buccal
delivery.
[0127] Mucosal formulations may include one or more agents for
enhancing delivery through the nasal mucosa. Agents for enhancing
mucosal delivery are known in the art, see, for example, U.S.
Patent Application No. 200910252672 to Eddington, and U.S. Patent
Application No. 2009/0047234 to Touitou. Acceptable agents include,
but are not limited to, chelators of calcium (EDTA), inhibitors of
nasal enzymes (boro-leucin, aprotinin), inhibitors of muco-ciliar
clearance (preservatives), solubilizers of nasal membrane
(cyclodextrin, fatty acids, surfactants) and formation of micelles
(surfactants such as bile acids, Laureth 9 and taurodehydrofusidate
(STDHF)). Compositions may include one or more absorption
enhancers, including surfactants, fatty acids, and chitosan
derivatives, which can enhance delivery by modulation of the tight
junctions (TJ) (B. J. Aungst, et al., J. Pharm. Sci. 89(4):429-442
(2000)). In general, the optimal absorption enhancer should possess
the following qualities: its effect should be reversible, it should
provide a rapid permeation enhancing effect on the cellular
membrane of the mucosa, and it should be non-cytotoxic at the
effective concentration level and without deleterious and/or
irreversible effects on the cellular or virus membrane, Intranasal
compositions maybe administered using devices known in the art, for
example a nebulizer.
III. Methods of Use
[0128] A. Methods of Treatment
[0129] 1. Administration of Therapeutic Virus
[0130] The disclosed chimeric VSV viruses can be administered to a
subject in need thereof in an amount effective to treat a disease
or disorder, for example, cancer. Pharmaceutical compositions
including a chimeric virus may be administered once or more than
once, for example 2, 3, 4, 5, or more times. Serial administration
of chimeric virus may occur days, weeks, or months apart. As
discussed in more detail below, boosters of immunizing virus may be
administered between therapeutic treatments. It may be particularly
preferable to administer a booster of immunizing virus if there are
lengthy delays between treatments with therapeutic virus, for
example, one or more years.
[0131] Virus can be administered peripherally, or can be injected
directly into a tumor, for example a tumor within the brain. In
addition, virus can be used after resection of the main body of the
tumor, for example by administering directly to the remaining
adjacent tissue after surgery, or after a period of one to two
weeks to allow recovery of local damage. Adding virus after
surgical resection would eliminate any remaining tumor cells that
the neurosurgeon did not remove. The injections can be given at
one, or multiple locations. It is also believed that virus
administered systemically can target and kill brain cancers.
[0132] In some embodiments, it may be desirable to administer the
chimeric virus after or in combination with an immunosuppressant.
Treatment with an immunosuppressant during administration with a
therapeutic virus allows controlled suppression of the subject's
immune system during administration of the therapeutic virus. This
may be desirable, for example, if the capacity of the oncolytic
virus to kill cancer is reduced due to an earlier administration of
the immunizing virus. Treatment with the immunosuppressant is
typically transient, and occurs during administration of the virus,
particularly when the virus is being used to treat tumors and/or
cancer. Following treatment with the chimeric virus, treatment with
the immunosuppressant is discontinued and the patient's immunity
returns. The duration of immunosuppressive treatment will depend on
the condition to be treated. Typically the immunosuppressive
treatment will be long enough for the oncolytic virus to kill
cancer cells, reduce tumor size, or inhibit tumor progression.
[0133] 2. Peripheral Administration of Immunizing Virus
[0134] One or more peripheral administrations with an immunizing
virus can elicit an adaptive immune response that protects the
brain from potential side-effects of oncolytic virus therapy. The
term immunizing virus includes live virus as well as viral
subunits, proteins and fragments thereof, antigenic polypeptides,
nucleic acids, and expression vectors containing nucleic acids
encoding viral subunits, proteins, or fragments thereof, or
antigenic polypeptides which can be useful in eliciting an immune
response. For example, if the immunizing virus is a VSV virus, the
immunizing virus includes, but is not limited to, live VSV virus,
the N, P, M, G, or L proteins, or combinations thereof.
[0135] The immunizing virus may be the same virus, or a different
virus than the therapeutic virus. The immunizing virus should
initiate an adaptive immune response that is sufficient to
attenuate, reduce, or prevent the neurovirulence of the therapeutic
virus. The therapeutic virus administered after a first
administration of immunizing virus should have reduced
neurovirulence compared to therapeutic virus administered without a
first administration of immunizing virus. In preferred embodiments,
the immunizing virus is similar to the therapeutic virus. For
example if the therapeutic virus is a VSV, the immunizing virus is
preferably a VSV, or an antigenic protein or nucleic acid component
thereof. In some embodiments the immunizing virus has an attenuated
phenotype compared to the therapeutic virus. As described above,
suitable immunizing viruses include wildtype viruses, as well as
mutant and variants thereof. In one preferred embodiment, the
immunizing virus is a wildtype virus or an antigenic protein or
nucleic acid component thereof, while the therapeutic virus is a
mutant, variant, chimeric virus having the same virus background
but reduced neurovirulence compared to wildtype. In some
embodiments, therapeutic viruses may be engineered to express
therapeutic proteins or targeting molecules. Immunizing viruses may
also be engineered to express additional proteins, but preferably
are not. VSV-G/GFP is a suitable immunizing virus. The nucleotide
sequence for VSV-G/GFP is GenBank Accession FJ478454.
[0136] Immunizing viruses are administered sufficiently prior to
therapeutic viruses to elicit an adaptive immune response.
Immunizing viruses are typically administered one or more times at
least about 5 days, preferably 1 week, more preferably greater than
one week before administration of the therapeutic virus. Immunizing
viruses can be administered up to 1, 2, 3, 4, 5, or more weeks
before the therapeutic virus. Immunizing viruses can be
administered up to 1, 2, 3, 4, 5, or more months before the
therapeutic virus. Most preferably the immunizing virus is
administered between about ten days and 12 weeks before the
therapeutic virus.
[0137] After an initial administration of the immunizing virus,
subsequent booster immunizations can be administered. For example,
it may be desirable to administer the immunizing virus two or more
times. A first administration of the immunizing virus is typically
provided to a patient in need therefore prior to a first
administration of the therapeutic virus. Subsequent administrations
of the immunizing virus may occur before and/or after a first
administration of the therapeutic virus. In preferred embodiments
the immunizing virus is administered two or more times before the
first administration of the therapeutic virus. In a non-limiting
example, the immunizing virus is first administered on day 1, a
booster of immunizing virus is administered six weeks later on
about day 43, and the therapeutic virus is first administered two
weeks later on about day 57.
[0138] Various factors may be considered when determining the
frequency, dosage, duration, and number of administrations of
immunizing virus, as well as the duration between administration of
the immunizing virus and first administration of therapeutic virus.
For example, the subject's adaptive immune response can be
monitored to assess the effectiveness of the immunization. Methods
of measuring adaptive immune activation are known in the art and
include antibody profiling, serum analysis for changes in levels of
antibodies, cytokines, chemokines, or other inflammatory molecules,
and cell counts and/or cell profiling using extracellular markers
to assess the numbers and types of immune cells such as B cells and
T cells.
[0139] Immunizing virus is most typically delivered to a subject in
need thereof by peripheral administration, and not directly or
locally to the site in need of treatment by therapeutic virus.
Peripheral administration includes intravenous, by injection or
infusion, intraperitoneal, intramuscular, subcutaneous, and mucosal
such as intranasal delivery. In some embodiments, the composition
is delivered systemically, by injection or infusion into the
circulatory system (i.e. intravenous) or an appropriate lymphoid
tissue, such as the spleen, lymph nodes or mucosal-associated
lymphoid tissue. The injections can be given at one, or multiple
locations. Preferably the immunizing virus is administered
intranasally or by intramuscular injection, most preferably by
intranasal delivery.
[0140] Generally immunizing virus is administered to humans at
dosage levels between 10.sup.2 and 10.sup.12 PFU. Virus is
typically administered in a liquid suspension, in a volume ranging
between 10 .mu.l and 100 ml depending on the route of
administration.
[0141] It may also be desirable to administer the immunizing virus
in combination with one or more adjuvants. These can be
incorporated into, administered with, or administered separately
from, the immunogenizing virus. Depending on whether or not the
individual is a human or an animal, the adjuvant can be, but is not
limited to, one or more of the following: oil emulsions (e.g.,
Freund's adjuvant); saponin formulations; virosomes and viral-like
particles; bacterial and microbial derivatives; immunostimulatory
oligonucleotides; ADP-ribosylating toxins and detoxified
derivatives; alum; BCG; mineral-containing compositions (e.g.,
mineral salts, such as aluminium salts and calcium salts,
hydroxides, phosphates, sulfates, etc.); bioadhesives and/or
mucoadhesives; microparticles; liposomes; polyoxyethylene ether and
polyoxyethylene ester formulations; polyphosphazene; muramyl
peptides; imidazoquinolone compounds; and surface active substances
(e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanin, and dinitrophenol).
[0142] 3. Vaccination
[0143] The chimeric viruses can also serve as an immunogen for
generating an immune response against other antigens administered
with or cloned into virus. The safety profile of the disclosed
Lassa-VSVs make them particularly attractive for use as part of a
vaccine. Other VSVs can lead to adverse consequence in brain,
whereas a Lassa-VSV with another antigen, for example, an influenza
antigen, would be safer, yet effective.
[0144] For example, in some embodiments, the chimeric virus is a
vaccine vector. Experiments conducted with the Lassa-VSV including
a GFP reporter discussed below, show that the chimeric virus
generates a strong immune response against the virus, and also
against the GFP reporter. Accordingly, other proteins could be
substituted for GFP. These could include proteins from pathogenic
microbes unrelated to Lassa virus or VSV; the Lassa-VSV could serve
as a safe vaccine platform against many different pathogenic
microbes. As described above, VSV can be engineered to express one
or more immunogenic antigens. Expression of these antigens in a
patient in need thereof presents the antigen to the immune system
and provokes an immune response. Vaccines can be administered
prophylactically or therapeutically. Vaccines can also be
administered according to a vaccine schedule. A vaccine schedule is
a series of vaccinations, including the timing of all doses. Many
vaccines require multiple doses for maximum effectiveness, either
to produce sufficient initial immune response or to boost response
that fades over time. Vaccine schedules are known in the art, and
are designed to achieve maximum effectiveness. The adaptive immune
response can be monitored using methods known in the art to measure
the effectiveness of the vaccination protocol.
[0145] 4. Immunotherapy
[0146] Chimeric VSV viruses wherein the G protein is replaced with
a heterologous glycoprotein, for example the glycoprotein from
Lassa virus, have been shown to be immunogenic and initiate an
up-regulation of both humoral and cellular immunity toward the
virus (Geisbert, et al., PLoS Med., 2:e183 (2005) and the Examples
below. Therefore, methods of initiating an immune response against
the infected tumor are disclosed. It is believed that the disclosed
chimeric viruses will not only infect and kill cancer cells, but
will enhance an attack by the systemic immune system on the
infected cell-type both during and after the virus is eliminated.
In this way, the virus can be used to induce an immune response
against non-infected target cells. In this way, treatment with the
disclosed VSV virus may delay, reduce, or prevent reoccurrence of
the cancer being treated.
[0147] In some methods, the chimeric virus is used to infect
targets cells, and the infected target cells or antigens isolated
therefrom are used for peripheral immunization of the subject
against the target cells, or antigens thereof. For example, target
cells against which an immune response is desired are implanted
into a subject. The cells are injected with virus which kills the
cells and leads to an immune response against antigens of the
cells. The cells can be infected with virus before or after
implantation. For example, the cells are infected with virus in
vitro prior to injection into the subject. In another embodiment,
the subject is immunized with antigen(s) isolated from tumor cells
infected with virus in vitro.
[0148] The target cell can be any cell to which an immune response
is desired. For example, the target cells can be cancer cells
against which an immune response is desired. The cancer cells can
be from an established cell line or primary cancer cells isolated
from a subject. For example, the target cells can be cancer cells
isolated from a subject in a biopsy or during surgery to remove a
tumor. As discussed above, the target cells can be infected in
vitro prior to administration to the subject, or the target cells
can be inject by local injection of the virus into the subject at
the site of implantation of the target cells. The cells can be
harvested from and administered back to the same subject.
Alternatively, the cells can be harvested from one subject and
administered to a different subject. In this way, the virus can be
used to induce an immune response against a cancer or tumor in a
subject that has the cancer or tumor, or prophylactically prime the
immune system to attack a future cancer or tumor that the subject
does not yet have. Accordingly, the treatment can be therapeutic,
prophylactic, or a combination thereof.
[0149] In a particular embodiment, this strategy is employed in
combination with surgery in which a tumor is removed from a
subject. Cells are isolated from the tumor, infected with virus,
and implanted in the subject. In this way, an immune response is
induced against any cancer cells that remain in the subject, for
example in the margins and other tissue at the site from which the
tumor removed, as well as circulating cancer cells and metastases
throughout the body including those sites distant from the tumor
that was removed. The method can also reduce, delay, or prevent
recurrence of the cancer.
[0150] In some embodiments the isolated target cells are irradiated
in amount effective to prevent cell division, but not to kill the
cells, to avoid concerns about in vivo replication of the target
cells following implantation. Typically, the cells are implanted
into the subject peripherally. For example, the cells can be
injected into the subject subcutaneously, intramuscularly,
intranasally, intravenously, intraperitoneally, or using another
suitable method of peripheral administration, such as those
discussed above. In some embodiments, the tumor cells are expanded
in culture for one or generations or passages between isolation and
implantation in the subject.
[0151] It is believed that VSV infection will increase
tumor-specific cytotoxic effector CD8+ T cells, increase CD4+ T
cells, increase production of tumor specific antibodies, or a
combination thereof. Therefore, in some embodiments, tumor-specific
cytotoxic effector CD8+ T cells primed by chimeric VSV infected
tumor cells are administered to a subject in need thereof. The T
cells can be harvested from a treated subject, and optionally
expanded in culture, or primed and expanded in vitro.
[0152] For example, in a particular embodiment, the method is one
of adaptive T cell therapy. Methods of adoptive T cell therapy are
known in the art and used in clinical practice. Generally adoptive
T cell therapy involves the isolation and ex vivo expansion of
tumor specific T cells to achieve greater number of T cells than
what could be obtained by vaccination alone. The tumor specific T
cells are then infused into patients with cancer in an attempt to
give their immune system the ability to overwhelm remaining tumor
via T cells which can attack and kill cancer. Several forms of
adoptive T cell therapy can be used for cancer treatment including,
but not limited to, culturing tumor infiltrating lymphocytes or
TIL; isolating and expanding one particular T cell or clone; and
using T cells that have been engineered to recognize and attack
tumors. In the disclosed methods, the tumors infected with the
chimeric VSV, or isolated components thereof, are used to prime the
T cells. In some embodiments, the T cells are taken directly from
the patient's blood after they have received treatment or
immunization with the virus. Methods of priming and activating T
cells in vitro for adaptive T cell cancer therapy are known in the
art. See, for example, Wang, et al., Blood, 109(11):4865-4872
(2007) and Hervas-Stubbs, et al., J. Immunol., 189(7):3299-310
(2012). The methods can be used in conjunction with virus infected
cancer cells, or antigens isolated therefrom, to prime and activate
T cells against the cancer.
[0153] Historically, adoptive T cell therapy strategies have
largely focused on the infusion of tumor antigen specific cytotoxic
T cells (CTL) which can directly kill tumor cells. However, CD4+ T
helper (Th) cells can also be used. Th can activate
antigen-specific effector cells and recruit cells of the innate
immune system such as macrophages and dendritic cells to assist in
antigen presentation (APC), and antigen primed Th cells can
directly activate tumor antigen-specific CTL. As a result of
activating APC, antigen specific Th1 have been implicated as the
initiators of epitope or determinant spreading which is a
broadening of immunity to other antigens in the tumor. The ability
to elicit epitope spreading broadens the immune response to many
potential antigens in the tumor and can lead to more efficient
tumor cell kill due to the ability to mount a heterogeneic
response. In this way, adoptive T cell therapy can used to
stimulate endogenous immunity.
[0154] B. Subjects to be Treated
[0155] In general, the disclosed chimeric viruses and methods of
treatment thereof are useful in the context of cancer, including
tumor therapy, particular brain tumor therapy.
[0156] In a mature animal, a balance usually is maintained between
cell renewal and cell death in most organs and tissues. The various
types of mature cells in the body have a given life span; as these
cells die, new cells are generated by the proliferation and
differentiation of various types of stem cells. Under normal
circumstances, the production of new cells is so regulated that the
numbers of any particular type of cell remain constant.
Occasionally, though, cells arise that are no longer responsive to
normal growth-control mechanisms. These cells give rise to clones
of cells that can expand to a considerable size, producing a tumor
or neoplasm. A tumor that is not capable of indefinite growth and
does not invade the healthy surrounding tissue extensively is
benign. A tumor that continues to grow and becomes progressively
invasive is malignant. The term cancer refers specifically to a
malignant tumor. In addition to uncontrolled growth, malignant
tumors exhibit metastasis. In this process, small clusters of
cancerous cells dislodge from a tumor, invade the blood or
lymphatic vessels, and are carried to other tissues, where they
continue to proliferate. In this way a primary tumor at one site
can give rise to a secondary tumor at another site.
[0157] The compositions and methods described herein are useful for
treating subjects having benign or malignant tumors by delaying or
inhibiting the growth of a tumor in a subject, reducing the growth
or size of the tumor, inhibiting or reducing metastasis of the
tumor, and/or inhibiting or reducing symptoms associated with tumor
development or growth. The examples below indicate that the viruses
and methods disclosed herein are useful for treating cancer,
particular brain tumors, in vivo.
[0158] Malignant tumors which may be treated are classified herein
according to the embryonic origin of the tissue from which the
tumor is derived. Carcinomas are tumors arising from endodermal or
ectodermal tissues such as skin or the epithelial lining of
internal organs and glands. The disclosed compositions are
particularly effective in treating carcinomas. Sarcomas, which
arise less frequently, are derived from mesodermal connective
tissues such as bone, fat, and cartilage. The leukemias and
lymphomas are malignant tumors of hematopoietic cells of the bone
marrow. Leukemias proliferate as single cells, whereas lymphomas
tend to grow as tumor masses. Malignant tumors may show up at
numerous organs or tissues of the body to establish a cancer.
[0159] The types of cancer that can be treated with the provided
compositions and methods include, but are not limited to, cancers
such as vascular cancer such as multiple myeloma, adenocarcinomas
and sarcomas, of bone, bladder, brain, breast, cervical,
colo-rectal, esophageal, kidney, liver, lung, nasopharangeal,
pancreatic, prostate, skin, stomach, and uterine. In some
embodiments, the disclosed compositions are used to treat multiple
cancer types concurrently. The compositions can also be used to
treat metastases or tumors at multiple locations.
[0160] The disclosed methods are particularly useful in treating
brain tumors. Brain tumors include all tumors inside the cranium or
in the central spinal canal. They are created by an abnormal and
uncontrolled cell division, normally either in the brain itself
(neurons, glial cells (astrocytes, oligodendrocytes, ependymal
cells, myelin-producing Schwann cells, lymphatic tissue, blood
vessels), in the cranial nerves, in the brain envelopes (meninges),
skull, pituitary and pineal gland, or spread from cancers primarily
located in other organs (metastatic tumors). Examples of brain
tumors include, but are not limited to, oligodendroglioma,
meningioma, supratentorial ependymona, pineal region tumors,
medulloblastoma, cerebellar astrocytoma, infratentorial ependymona,
brainstem glioma, schwannomas, pituitary tumors, craniopharyngioma,
optic glioma, and astrocytoma.
[0161] "Primary" brain tumors originate in the brain and
"secondary" (metastatic) brain tumors originate from cancer cells
that have migrated from other parts of the body. Primary brain
cancer rarely spreads beyond the central nervous system, and death
results from uncontrolled tumor growth within the limited space of
the skull. Metastatic brain cancer indicates advanced disease and
has a poor prognosis. Primary brain tumors can be cancerous or
noncancerous. Both types take up space in the brain and may cause
serious symptoms (e.g., vision or hearing loss) and complications
(e.g., stroke). All cancerous brain tumors are life threatening
(malignant) because they have an aggressive and invasive nature. A
noncancerous primary brain tumor is life threatening when it
compromises vital structures (e.g., an artery). In a particular
embodiment, the disclosed compositions and methods are used to
treat cancer cells or tumors that have metastasized from outside
the brain (e.g., lung, breast, melanoma) and migrated into the
brain.
[0162] The Examples below illustrate that Lassa-VSV is oncolytic,
but also non-toxic to health or normal cells, even when
administered directly to the brain. Therefore, the disclosed
viruses are particularly useful for treating brain cancer, cancer
that can metastasize to the brains, for example lung cancer, breast
cancer, and skin cancer such as melanoma.
[0163] Although the viruses are particularly safe and useful for
treating cancer in the brain, the cancer does not have to be in the
brain. It is believed that the chimeric virus are also effective
for treating other cancer outside the brain, and can thereof be
administered systemically in or locally outside the brain. In a
particular embodiment, a chimeric virus is used to treat a cancer
that could, but has not yet metastasized to the brain. See, for
example, Yarde, et al., Cancer Gene Ther., 2013 Nov. 1. doi:
10.1038/cgt.2013.63, which describes that intravenously
administered VSVs encoding IFN-.beta. have potent activity against
subcutaneous tumors in the 5TGM1 mouse myeloma model, without
attendant neurotoxicity. However, when 5TGM1 tumor cells were
seeded intravenously, virus-treated mice with advanced myeloma
developed clinical signs suggestive of meningoencephalitis, and
leading to deaths that are believed to be associated with viral
toxicity. Histological analysis revealed that systemically
administered 5TGM1 cells seed to the CNS, forming meningeal tumor
deposits, and that VSV infects and destroys these tumors. Death is
presumably a consequence of meningeal damage and/or direct
transmission of virus to adjacent neural tissue.
[0164] The disclosed chimeric Lassa-VSV viruses have reduced
toxicity for normal and healthy cells including neurons. Therefore,
these viruses are a safer, less toxic alternative for treating
systemic cancers that can potential traffic virus into the brain
and cause neurotoxicity and even death.
[0165] As shown the examples below, the chimeric Lassa-VSV was safe
in the brains of SCID mice that have a depressed systemic immunity
due to substantially reduced T and B cells. Thus, Lassa-VSV should
be far safer than VSV with its normal VSV glycoprotein. This may
enable Lassa-VSV to be used in patients showing depressed immunity,
typical of many cancer patients, and also of patients with AIDS, or
with genetic immune depression. The enhanced safety in the brain
may also be of benefit in patients with compromised blood brain
barriers where Lassa-VSV would be safer than VSV in both cancer
treatment, and for vaccination against either a cancer cell type,
or against unrelated (e.g., non-Lassa, non-VSV) pathogenic
microbes.
[0166] The Examples below also show that the chimeric VSV are
effective at infecting and replicating in colon, prostate, breast,
bone, and bladder cancer cells, indicating its oncolytic potential
was not restricted to glioma and melanoma brain tumors. It is
believed that the disclosed viruses are effective for treating both
primary and secondary brain tumors, but as peripheral (non-brain)
cancers and tumors.
[0167] C. Combination Therapies
[0168] Administration of the disclosed compositions containing
oncolytic viruses may be coupled with surgical, radiologic, other
therapeutic approaches to treatment of tumors and cancers.
[0169] 1. Surgery
[0170] The disclosed compositions and methods can be used as an
adjunct to surgery. Surgery is a common treatment for many types of
benign and malignant tumors. As it is often not possible to remove
all the tumor cells from during surgery, the disclosed compositions
containing oncolytic virus are particularly useful subsequent to
resection of the primary tumor mass, and would be able to infect
and destroy even dispersed tumor cells.
[0171] In a preferred embodiment, the disclosed compositions and
methods are used as an adjunct or alternative to neurosurgery. The
compositions are particularly well suited to treat areas of the
brain that is difficult to treat surgically, for instance high
grade tumors of the brain stem, motor cortex, basal ganglia, or
internal capsule. High grade gliomas in these locations are
generally considered inoperable. An additional situation where an
oncolytic virus may be helpful is in regions where the tumor is
either wrapped around critical vasculature, or in an area that is
difficult to treat surgically.
[0172] 2. Therapeutic Agents
[0173] The viral compositions can be administered to a subject in
need thereof alone or in combination with one or more additional
therapeutic agents selected based on the condition, disorder or
disease to be treated. A description of the various classes of
suitable pharmacological agents and drugs may be found in Goodman
and Gilman, The Pharmacological Basis of Therapeutics, (11th Ed.,
McGraw-Hill Publishing Co.) (2005).
[0174] Additional therapeutic agents include conventional cancer
therapeutics such as chemotherapeutic agents, cytokines,
chemokines, and radiation therapy. The majority of chemotherapeutic
drugs can be divided into: alkylating agents, antimetabolites,
anthracyclines, plant alkaloids, topoisomerase inhibitors, and
other antitumour agents. All of these drugs affect cell division or
DNA synthesis and function in some way. Additional therapeutics
include monoclonal antibodies and the tyrosine kinase inhibitors
e.g., imatinib mesylate (GLEEVEC.RTM. or GLIVEC.RTM.), which
directly targets a molecular abnormality in certain types of cancer
(chronic myelogenous leukemia, gastrointestinal stromal
tumors).
[0175] Representative chemotherapeutic agents include, but are not
limited to, amsacrine, bleomycin, busulfan, capecitabine,
carboplatin, carmustine, chlorambucil, cisplatin, cladribine,
clofarabine, crisantaspase, cyclophosphamide, cytarabine,
dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin,
epipodophyllotoxins, epirubicin, etoposide, etoposide phosphate,
fludarabine, fluorouracil, gemcitabine, hydroxycarbamide,
idarubicin, ifosfamide, irinotecan, leucovorin, liposomal
doxorubicin, liposomal daunorubicin, lomustine, mechlorethamine,
melphalan, mercaptopurine, mesna, methotrexate, mitomycin,
mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin,
procarbazine, raltitrexed, satraplatin, streptozocin, teniposide,
tegafur-uracil, temozolomide, teniposide, thiotepa, tioguanine,
topotecan, treosulfan, vinblastine, vincristine, vindesine,
vinorelbine, taxol and derivatives thereof, trastuzumab
(HERCEPTIN.RTM.), cetuximab, and rituximab (RITUXAN.RTM. or
MABTHERA.RTM.), bevacizumab (AVASTIN.RTM.), and combinations
thereof. Representative pro-apoptotic agents include, but are not
limited to, fludarabinetaurosporine, cycloheximide, actinomycin D,
lactosylceramide, 15d-PGJ(2), and combinations thereof.
[0176] Preferred chemotherapeutics will affect tumors or cancer
cells, without diminishing the activity of the virus. For example,
in a preferred embodiment, the additional therapeutic agent
inhibits proliferation of cancer cells without affecting targeting,
infectivity, or replication of the virus.
[0177] a. Anticancer Agents
[0178] The compositions can be administered with an antibody or
antigen binding fragment thereof specific for growth factor
receptors or tumor specific antigens. Representative growth factors
receptors include, but are not limited to, epidermal growth factor
receptor (EGFR; HER1); c-erbB2 (HER2); c-erbB3 (HER3); c-erbB4
(HER4); insulin receptor; insulin-like growth factor receptor 1
(IGF-1R); insulin-like growth factor receptor 2/Mannose-6-phosphate
receptor (IGF-II RIM-6-P receptor); insulin receptor related kinase
(IRRK); platelet-derived growth factor receptor (PDGFR);
colony-stimulating factor-1receptor (CSF-1R) (c-Fms); steel
receptor (c-Kit); Flk2/Flt3; fibroblast growth factor receptor 1
(Flg/Cek1); fibroblast growth factor receptor 2 (Bek/Cek3/K-Sam);
Fibroblast growth factor receptor 3; Fibroblast growth factor
receptor 4; nerve growth factor receptor (NGFR) (TrkA); BDNF
receptor (TrkB); NT-3-receptor (TrkC); vascular endothelial growth
factor receptor 1 (Flt1); vascular endothelial growth factor
receptor 2/Flk1/KDR; hepatocyte growth factor receptor (HGF-R/Met);
Eph; Eck; Eek; Cek4/Mek4/HEK; Cek5; Elk/Cek6; Cek7; Sek/Cek8; Cek9;
Cek10; HEK11; 9 Ror1; Ror2; Ret; Axl; RYK; DDR; and Tie.
[0179] b. Therapeutic Proteins
[0180] It may be desirable to administer the disclosed compositions
in combination with therapeutic proteins. VSV is an effective
oncolytic virus, in-part, by taking advantage of defects in the
interferon system. Administration of therapeutic proteins such as
IFN-.alpha., or IFN-.alpha./3 pathway inducer polyriboinosinic
polyribocytidylic acid [poly(I:C)] are effective in protecting
normal cells from the oncolytic activity, while leaving the tumor
cells susceptible to infection and death (Wollmann, et al. J.
Virol., 81(3): 1479-1491 (2007). Therefore, in some embodiments,
the disclosed compositions are administered in combination with a
therapeutic protein to, reduce infectivity and death of normal
cells.
[0181] Other therapeutic proteins that can be administered in
combination with the disclosed viruses include those provided above
as therapeutic proteins that can be engineered into the virus.
Accordingly, the therapeutic virus can be part of the virus itself,
or administered separately. In some embodiments, the virus includes
one or more therapeutic proteins and one more therapeutic proteins
are administered separately.
[0182] c. Immuno-Suppressants
[0183] As discussed throughout and demonstrated in the Examples
below, the disclosed chimeric VSV viruses generally, show a reduced
probability of infecting normal brain cells, but still have a good
oncolytic capacity. One limitation of oncolytic viruses in general
is that the adaptive immune system can up-regulate its antiviral
response and eliminate the virus before the virus has had a chance
to maximally infect tumor cells. Although it is important for the
adaptive immune system to eliminate the chimeric VSV from the
subject, the virus should remain in the subject long enough to
infect and kill as many tumor cells as possible balanced against
the pathogenicity of the virus to normal cells of the subject.
Temporary concomitant immune-suppression has been identified as a
strategy to enhance the efficacy of other oncolytic viruses (HSV,
adenovirus, vaccinia) that are human pathogens and face
pre-existing immunity (Fukuhara, et al., Curr. Cancer Drug Targets,
7:149-155 (2007); Lun, et al., Clin. Cancer Res., 15:2777-2788
(2009)). Therefore, the virus can be administered to the subject in
combination with temporary concomitant immune suppression.
[0184] In some embodiments, the virus is administered in
combination with an agent that reduces or attenuates the intrinsic
IFN-mediated immune responses that can eliminate the virus before
it has achieved maximal tumor destruction. In preferred
embodiments, the attenuation of the intrinsic IFN-mediated immune
responses enhances the rate of recombinant VSV-mediated tumor
destruction without increasing infection of normal cells. This
strategy should also reduce the initiation of the adaptive immune
response which is enhanced by the innate immune response, giving
the virus more time to complete its oncolytic actions.
[0185] Paglino, et al., J. Virol., 85:9346-58 (2011) showed that a
cancer cell highly resistant to VSV could be infected by blocking
the IFN response to VSV with one of three IFN blockers, valproate,
the vacccinia protein B18R, or Jak inhibitor 1. Valproate crosses
the blood brain barrier as evident in its use to treat epilepsy. It
is already approved for clinical use in humans (for attenuating
epilepsy), and like many other histone deacetylase (HDAC)
inhibitors, it has an intrinsic anti-tumor property, independent of
oncolytic virus infection, that reduces glioma and other tumor
growth in the brain (Chateauvieux, et al., J. Biomed. Biotechnol.,
479364. Epub 2010 Jul. 29 (2010); Fu, et al., Neuro. Oncol.,
12:328-340 (2010); Su, et al., Clin. Cancer Res., 17:589-597
(2011). Similarly, the HDAC inhibitor vorinostat (ZOLLINZA.RTM.) is
approved by the FDA for the treatment of cutaneous T-cell lymphoma
(Glaser K B, Biochem. Pharmacal., 74:659-671 (2007)). Vorinostat on
its own appears to penetrate brain tumors and to increase survival
of patients with glioblastoma, and animal studies have shown that
valproate can increase infection by viruses in tumors with minimal
increased collateral damage. Valproate increased survival
substantially in tumor bearing animals treated with HSV (Otsuki, et
al., Mol. Ther., 16:1546-1555 (2008)). In one particular ease study
a pediatric anaplastic astrocytoma that was resistant to
chemotherapy and irradiation, underwent a substantial regression
after combined treatment with oral valproate and oncolytic
attenuated Newcastle disease virus Wagner, et al., APMIS,
114:731-743 (2006)).
[0186] Other HDAC inhibitors have been shown to enhance viral
cancer cell targeting and viral replication by vaccinia (MacTavish,
et al., PLoS One, 5:e14462 (2010) and VSV (Nguyen, et al., Proc.
Natl. Acad. Sci., USA 105:14981-14986 (2008)) without substantially
altering infection in normal non-cancer cells. Valproate inhibited
the induction of several antiviral genes after oncolytic HSV
infection, and resulted in enhanced viral propagation in glioma
cells, even in the presence of IFN (Otsuki, et at, Mol. Ther.,
16:1546-1555 (2008)). Importantly, valproate treatment had no
augmenting effect on viral yield in normal human astrocytes.
Valproate pretreatment was also shown to enhance HSV propagation in
tumors 10-fold in vivo and improved the survival of nude mice
bearing U87delta-EGFR brain tumors.
[0187] Therefore, in some embodiments, the virus is administered in
combination with an HDAC inhibitor. In some embodiments, the virus
is administered in combination with valproate, the vacccinia
protein B18R, Jak inhibitor 1, or vorinostat.
[0188] Other immunosuppressants such as cyclosporin, prednisone,
dexamethasone, or other steroidal anti-inflammatory, can also be
used to reduce the immune response immediately before, during, or
shortly after administration of the therapeutic virus. The
immunosuppressant is then discontinued or decreased to allow the
patient's immune system to prevent inflammation and/or killing of
the virus after it has competed the desired killing of tumor or
diseased tissue.
[0189] Suitable immunosuppressants are known in the art and include
glucocorticoids, cytostatics (such as alkylating agents,
antimetabolites, and cytotoxic antibodies), antibodies (such as
those directed against T-cell recepotors or 11-2 receptors), drugs
acting on immunophilins (such as cyclosporine, tacrolimus, and
sirolimus) and other drugs (such as interferons, opioids, TNF
binding proteins, mycophenolate, and other small molecules such as
fingolimod). The dosage ranges for immunosuppressant agents are
known in the art. The specific dosage will depend upon the desired
therapeutic effect, the route of administration, and on the
duration of the treatment desired. For example, when used as an
immunosuppressant, a cytostatic maybe administered at a lower
dosage than when used in chemotherapy. Immunosuppressants include,
but are not limited to, FK506, prednisone, methylprednisolone,
cyclophosphamide, thalidomide, azathioprine, and daclizumab,
physalin B, physalin F, physalin G, seco-steroids purified from
Physalis angulata L., 15-deoxyspergualin, MMF, rapamycin and its
derivatives, CCI-779, FR 900520, FR 900523, NK86-1086,
depsidomycin, kanglemycin-C, spergualin, prodigiosin25-c,
cammunomicin, demethomycin, tetranactin, tranilast, stevastelins,
myriocin, gliotoxin, FR 651814, SDZ214-104, bredinin, WS9482,
mycophenolic acid, mimoribine, misoprostol, OKT3, anti-IL-2
receptor antibodies, azasporine, leflunomide, mizoribine,
azaspirane, paclitaxel, altretamine, busulfan, chlorambucil,
ifosfamide, mechlorethamine, melphalan, thiotepa, cladribine,
fluorouracil, floxuridine, gemcitabine, thioguanine, pentostatin,
methotrexate, 6-mercaptopurine, cytarabine, carmustine, lomustine,
streptozotocin, carboplatin, cisplatin, oxaliplatin, iproplatin,
tetraplatin, lobaplatin, JM216, JM335, fludarabine,
aminoglutethimide, flutamide, goserelin, leuprolide, megestrol
acetate, cyproterone acetate, tamoxifen, anastrozole, bicalutamide,
dexamethasone, diethylstilbestrol, bleomycin, dactinomycin,
daunorubicin, doxirubicin, idarubicin, mitoxantrone, losoxantrone,
mitomycin-c, plicamycin, paclitaxel, docetaxel, topotecan,
irinotecan, 9-amino camptothecan, 9-nitro camptothecan, GS-211,
etoposide, teniposide, vinblastine, vincristine, vinorelbine,
procarbazine, asparaginase, pegaspargase, octreotide, estramustine,
and hydroxyurea, and combinations thereof. Preferred
immunosuppressants will preferentially reduce or inhibit the
subject's immune response, without reducing or inhibiting the
activity of the virus.
IV. Kits
[0190] Dosage units including virus in a pharmaceutically
acceptable carrier for shipping and storage and/or administration
are also disclosed. Active virus should be shipped and stored using
a method consistent with viability such as in cooler containing dry
ice so that viruses are maintained below 4.degree. C., and
preferably below -20.degree. C. VSV virus should not be
lyophilized. Components of the kit may be packaged individually and
can be sterile. In one embodiment, a pharmaceutically acceptable
carrier containing an effective amount of virus is shipped and
stored in a sterile vial. The sterile vial may contain enough virus
for one or more doses. Virus may be shipped and stored in a volume
suitable for administration, or may be provided in a concentrated
titer that is diluted prior to administration. In another
embodiment, a pharmaceutically acceptable carrier containing an
effective amount of virus can be shipped and stored in a
syringe.
[0191] Typical concentrations of concentrated viral particles in
the sterile saline, phosphate buffered saline or other suitable
media for the virus is in the range of 10.sup.8 to 10.sup.9 with a
maximum of 10.sup.12. Dosage units should not contain membrane
disruptive agents nor should the viral solution be frozen and dried
(i.e., lyophilized), which could kill the virus.
[0192] Kits containing syringes of various capacities or vessels
with deformable sides (e.g., plastic vessels or plastic-sided
vessels) that can be squeezed to force a liquid composition out of
an orifice are provided. The size and design of the syringe will
depend on the route of administration. For example, in one
embodiment, a syringe for administering virus intratumorally, is
capable of accurately delivering a smaller volume (such as 1 to 100
.mu.l). Typically, a larger syringe, pump or catheter will be used
to administer virus systemically. Any of the kits can include
instructions for use.
V. Methods of Manufacture
[0193] A. Engineering Recombinant VSV Viruses
[0194] The native VSV genome is a single negative-sense,
non-segmented stand of RNA that contains five genes (N, L, P, M,
and G) and has a total size of 11.161 kb. Methods of engineering
recombinant viruses by reconstituting VSV from DNA encoding a
positive-sense stand of RNA are known in the art (Lawson, et al.,
PNAS, 92:4477-4481 (1995), Dalton and Rose, Virology., 279:414421
(2001)). For example, recombinant DNA can be transcribed by T7 RNA
polymerase to generate a full-length positive-strand RNA
complimentary to the viral genome. Expression of this RNA in cells
also expressing the VSV nucleocapsid protein and the two VSV
polymerase subunits results in production of VSV virus (Lawson, et
al., PNAS, 92:4477-4481 (1995)). In this way, VSV viruses can be
engineered to express variant proteins, additional proteins,
foreign antigens, targeting proteins, or therapeutic proteins using
known cloning methods. Methods of preparing exemplary suitable VSV
viruses where the gene encoding the VSV G protein is deleted and
replaced with a gene encoding the Lassa virus glycoprotein are
described in more detail above.
[0195] In some embodiments, the chimeric VSV is prepared by
substituting the sequence encoding the G protein on the plasmid
referred as VSVXN2 (Schnell, et al., J. Virol., 70:2318-2323
(1996)) with a heterologous glycoprotein, such as the glycoprotein
from Lassa virus.
[0196] In other embodiments the chimeric VSV is prepared by
substituting the sequence encoding the G protein on plasmid pVSV(+)
described in Whelan, et al., Proc. Natl. Acad. Sci. USA.,
92(18):8388-92 (1995). Whelan describes the constructions of a
full-length cDNA clone of VSV assembled from clones of each of the
VSV genes and intergenic junctions. These clones were assembled
into a full-length cDNA and inserted in both orientations between
the bacteriophage T7 promoter and a cDNA copy of the self-cleaving
ribozyme from the antigenomic strand of HDV. The resulting plasmids
were named pVSV1(+) and pVSV1(-) to reflect the polarity of the T7
transcript they generated: VSV antigenomic or genomic RNA,
respectively.
[0197] The T7 transcripts contained two non-VSV nucleotides (GG) at
their 5' ends but were cleaved by the HDV ribozyme to generate a 3'
terminus which corresponded precisely to the 3' end of the VSV
antigenomic or genomic sequence, an important requirement for VSV
RNA replication. Transfection of plasmids into BHK21 cells infected
with vTF7-3 was performed under the conditions and with quantities
of support plasmids as described (Pattnaik, et al., Cell,
69:1011-1020 (1992)), and up to 5 ug of pVSV1(+) or pVSV1(-).
Transfected cells were incubated at 31.degree. C. or 37.degree. C.
For some experiments, pVSV1(+) and pVSV1(-) were linearized by
digestion at a unique Nhe I site located downstream of the T7
terminator in the pGEM-3-based plasmids.
[0198] To identify cDNA-derived virus unambiguously, several
genetic markers were incorporated into the full-length cDNA clones.
All five genes were of the Indiana serotype of VSV, but whereas the
N, P, M, and L genes originated from the San Juan strain, the G
gene was from the Orsay strain. In addition, the functional P clone
has 28 nucleotide sequence differences from the published San Juan
sequence and in the case of pVSV1(+) the 516 nt at the 5' end of
the VSV genome originated from pDI, the clone of DI-T RNA
(Pattnaik, et al., Cell, 69:1011-1020 (1992)).
[0199] B. Creating Mutant VSV Virus
[0200] RNA viruses are prone to spontaneous genetic variation. The
mutation rate of VSV is about 10.sup.-4 per nucleotide replicated,
which is approximately one nucleotide change per genome (Drake, et
al., Proc. Natl. Acad. Sci. USA, 96:13910-13913). Therefore, mutant
VSV viruses exhibiting desired properties can be developed by
applying selective pressure. Methods for adaption of VSV viruses
through repeated passaging is described in the art. See, for
example, Wollmann, et al., J. Virol., 79(10): 6005-6022 (2005).
Selective pressure can be applied by repeated passaging and
enhanced selection to create mutant virus with desirable traits
such as increased infectivity and oncolytic potential for a cell
type of interest. The cell type of interest could be general, such
as cancer cells, or specific such as glioblastoma cells. Mutant
virus can also be selected based on reduced toxicity to normal
cells. Methods of enhanced selection include, but are not limited
to, short time for viral attachment to cells, collection of early
viral progeny, and preabsorption of viral particles with high
affinity of undesirable cells (such as normal cells). Mutations can
be identified by sequencing the viral genome and comparing the
sequence to the sequence of the parental strain.
[0201] DNA encoding the VSV genome can also be used as a substrate
for random or site directed mutagenesis to develop VSV mutant
viruses. Mutagenesis can be accomplished by a variety of standard,
mutagenic procedures. Changes in single genes may be the
consequence of point mutations that involve the removal, addition
or substitution of a single nucleotide base within a DNA sequence,
or they may be the consequence of changes involving the insertion
or deletion of large numbers of nucleotides.
[0202] Mutations can arise spontaneously as a result of events such
as errors in the fidelity of nucleic acid replication or the
movement of transposable genetic elements (transposons) within the
genome. They also are induced following exposure to chemical or
physical mutagens. Such mutation-inducing agents include ionizing
radiations, ultraviolet light and a diverse array of chemicals such
as alkylating agents and polycyclic aromatic hydrocarbons all of
which are capable of interacting either directly or indirectly
(generally following some metabolic biotransformations) with
nucleic acids. The nucleic acid lesions induced by such
environmental agents may lead to modifications of base sequence
when the affected DNA is replicated or repaired and thus to a
mutation. Mutation also can be site-directed through the use of
particular targeting methods. Various types of mutagenesis such as
random mutagenesis, e.g., insertional mutagenesis, chemical
mutagenesis, radiation mutagenesis, in vitro scanning mutagenesis,
random mutagenesis by fragmentation and reassembly, and site
specific mutagenesis, e.g., directed evolution, are described in
U.S. Patent Application No. 2007/0026012.
[0203] Mutant viruses can be prepared by site specific mutagenesis
of nucleotides in the DNA encoding the protein, thereby producing
DNA encoding the mutant. Techniques for making substitution
mutations at predetermined sites in DNA having a known sequence are
well known, for example M13 primer mutagenesis and PCR mutagenesis.
Amino acid substitutions are typically of single residues, but can
occur at a number of different locations at once. Insertions
usually will be on the order of about from 1 to 10 amino acid
residues; and deletions will range about from 1 to 30 residues.
Substitutions, deletions, insertions or any combination thereof can
be combined to arrive at a final construct. The mutations must not
place the sequence out of reading frame and preferably will not
create complementary regions that could produce secondary mRNA
structure. Substitution variants are those in which at least one
residue has been removed and a different residue inserted in its
place.
EXAMPLES
Example 1: Chimeric Lassa-VSV Virus Infected and Killed Gliomas
without Causing Neurological Dysfunction
Materials and Methods
[0204] Viruses and Cells
[0205] VSV-wtG and chimeric VSV's expressing the G protein from
either Lassa fever virus (VSV-LASV-G), rabies virus (VSV-RABV),
lymphocytic choriomeningitis virus (VSV-LCMV-G), Ebola virus
(VSV-EBOV-G), or Marburg virus (VSV-MARV-G) were generated as
described previously (Beier, et al., Proc. Natl. Acad. Sci. USA,
108:15414-15419 (2011); Jae, et al., Science, 344:1506-1510 (2014);
Krishnan, et al., Viruses, 4:2471-2484. A GFP reporter gene was
engineered into the first genome position of these VSVs. VSV-EBOV-G
and VSV-LASV-G that did not express any reporter genes were also
tested in some assays (Garbutt, et al., J. Virol., 78(10):5458-65
(2004)).
[0206] VSV-EBOV
[0207] The EBOV glycoprotein sequence expressed in VSV-EBOV is
derived from the Mayinga strain of Zaire Ebola virus as described
in the methods of Garbutt, et al. (2004) J. Virol 78: 5458-5465.
The nucleotide sequence shown below was determined by direct
sequencing of VSV-EBOV samples and matches the sequence for the
full-length (non-secreted) glycoprotein gene found in GenBank
accession NC_002549 nt 6039-8068. Note that the GenBank sequence
over this region is 2030 nt long, whereas the sequence below is
2031 nt in length. This difference derives from the fact that the
GenBank sequence is based on the genomic RNA sequence, whereas the
sequence below is based on the mRNA sequence that has been `edited`
by the viral polymerase to include an extra `A` nucleotide between
6918-6924 of the GenBank sequence.
TABLE-US-00008 VSV-EBOV glycoprotein gene nucleotide sequence (2031
nt) (SEQ ID NO: 6)
ATGGGCGTTACAGGAATATTGCAGTTACCTCGTGATCGATTCAAGAGGACA
TCATTCTTTCTTTGGGTAATTATCCTTTTCCAAAGAACATTTTCCATCCCA
CTTGGAGTCATCCACAATAGCACATTACAGGTTAGTGATGTCGACAAACTA
GTTTGTCGTGACAAACTGTCATCCACAAATCAATTGAGATCAGTTGGACTG
AATCTCGAAGGGAATGGAGTGGCAACTGACGTCCCATCTGCAACTAAAAGA
TGGGGCTTCAGGTCCGGTGTCCCACCAAAGGTGGTCAATTATGAAGCTGGT
GAATGGGCTGAAAACTGCTACAATCTTGAAATCAAAAAACCTGACGGGAGT
GAGTGTCTACCAGCAGCGCCAGACGGGATTCGGGGCTTCCCCCGGTGCCGG
TATGTGCACAAAGTATCAGGAACGGGACCGTGTGCCGGAGACTTTGCCTTC
CATAAAGAGGGTGCTTTCTTCCTGTATGATCGACTTGCTTCCACAGTTATC
TACCGAGGAACGACTTTCGCTGAAGGTGTCGTTGCATTTCTGATACTGCCC
CAAGCTAAGAAGGACTTCTTCAGCTCACACCCCTTGAGAGAGCCGGTCAAT
GCAACGGAGGACCCGTCTAGTGGCTACTATTCTACCACAATTAGATATCAG
GCTACCGGTTTTGGAACCAATGAGACAGAGTACTTGTTCGAGGTTGACAAT
TTGACCTACGTCCAACTTGAATCAAGATTCACACCACAGTTTCTGCTCCAG
CTGAATGAGACAATATATACAAGTGGGAAAAGGAGCAATACCACGGGAAAA
CTAATTTGGAAGGTCAACCCCGAAATTGATACAACAATCGGGGAGTGGGCC
TTCTGGGAAACTAAAAAAAACCTCACTAGAAAAATTCGCAGTGAAGAGTTG
TCTTTCACAGTTGTATCAAACGGAGCCAAAAACATCAGTGGTCAGAGTCCG
GCGCGAACTTCTTCCGACCCAGGGACCAACACAACAACTGAAGACCACAAA
ATCATGGCTTCAGAAAATTCCTCTGCAATGGTTCAAGTGCACAGTCAAGGA
AGGGAAGCTGCAGTGTCGCATCTAACAACCCTTGCCACAATCTCCACGAGT
CCCCAATCCCTCACAACCAAACCAGGTCCGGACAACAGCACCCATAATACA
CCCGTGTATAAACTTGACATCTCTGAGGCAACTCAAGTTGAACAACATCAC
CGCAGAACAGACAACGACAGCACAGCCTCCGACACTCCCTCTGCCACGACC
GCAGCCGGACCCCCAAAAGCAGAGAACACCAACACGAGCAAGAGCACTGAC
TTCCTGGACCCCGCCACCACAACAAGTCCCCAAAACCACAGCGAGACCGCT
GGCAACAACAACACTCATCACCAAGATACCGGAGAAGAGAGTGCCAGCAGC
GGGAAGCTAGGCTTAATTACCAATACTATTGCTGGAGTCGCAGGACTGATC
ACAGGCGGGAGAAGAACTCGAAGAGAAGCAATTGTCAATGCTCAACCCAAA
TGCAACCCTAATTTACATTACTGGACTACTCAGGATGAAGGTGCTGCAATC
GGACTGGCCTGGATACCATATTTCGGGCCAGCAGCCGAGGGAATTTACATA
GAGGGGCTAATGCACAATCAAGATGGTTTAATCTGTGGGTTGAGACAGCTG
GCCAACGAGACGACTCAAGCTCTTCAACTGTTCCTGAGAGCCACAACTGAG
CTACGCACCTTTTCAATCCTCAACCGTAAGGCAATTGATTTCTTGCTGCAG
CGATGGGGCGGCACATGCCACATTCTGGGACCGGACTGCTGTATCGAACCA
CATGATTGGACCAAGAACATAACAGACAAAATTGATCAGATTATTCATGAT
TTTGTTGATAAAACCCTTCCGGACCAGGGGGACAATGACAATTGGTGGACA
GGATGGAGACAATGGATACCGGCAGGTATTGGAGTTACAGGCGTTATAATT
GCAGTTATCGCTTTATTCTGTATATGCAAATTTGTCTTTTAG VSV-EBOV glycoprotein
amino acid sequence (676 a.a.) (SEQ ID NO: 7)
MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKL
VCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAG
EWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAF
HKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVN
ATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQ
LNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEEL
SFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQG
REAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHH
RRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETA
GNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPK
CNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYTEGLMHNQDGLICGLRQL
ANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEP
HDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVII
AVIALFCICKFVF
[0208] VSV-1'GFP-EBOV
[0209] The EBOV glycoprotein sequence expressed in VSV-1'GFP-EBOV
is derived from the Mayinga strain of Zaire Ebola virus as
described in the methods of Wong, et al. (2010) J. Virol 84:
163-175. The nucleotide sequence shown below was determined by
direct sequencing of VSV-1'GFP-EBOV samples and is similar (but not
identical) to the sequence for the full-length (non-secreted)
glycoprotein gene found in GenBank accession AF086833 nt 6039-8068.
The mucin domain (a.a. 309-489) has been deleted from the protein
and two amino acids are different (N40S and I662V) relative to the
GenBank sequence. Whereas the mucin deletion and I662V mutations
were intentionally introduced and are described in the methods of
Wong, et al. (2010), the N40S mutation was not described and it is
unknown whether this mutation existed within the original
recombinant plasmid used to generate the virus or emerged after
recovery through repeated passages.
TABLE-US-00009 VSV-1'GFP-EBOV glycoprotein gene nucleotide sequence
(1494 nt) (SEQ ID NO: 8)
ATGGGCGTTACAGGAATATTGCAGTTACCTCGTGATCGATTCAAGAGGACA
TCATTCTTTCTTTGGGTAATTATCCTTTTCCAAAGAACATTTTCCATCCCA
CTTGGAGTCATCCACAGTAGCACATTACAGGTTAGTGATGTCGACAAACTA
GTTTGTCGTGACAAACTGTCATCCACAAATCAATTGAGATCAGTTGGACTG
AATCTCGAAGGGAATGGAGTGGCAACTGACGTGCCATCTGCAACTAAAAGA
TGGGGCTTCAGGTCCGGTGTCCCACCAAAGGTGGTCAATTATGAAGCTGGT
GAATGGGCTGAAAACTGCTACAATCTTGAAATCAAAAAACCTGACGGGAGT
GAGTGTCTACCAGCAGCGCCAGACGGGATTCGGGGCTTCCCCCGGTGCCGG
TATGTGCACAAAGTATCAGGAACGGGACCGTGTGCCGGAGACTTTGCCTTC
CATAAAGAGGGTGCTTTCTTCCTGTATGATCGACTTGCTTCCACAGTTATC
TACCGAGGAACGACTTTCGCTGAAGGTGTCGTTGCATTTCTGATACTGCCC
CAAGCTAAGAAGGACTTCTTCAGCTCACACCCCTTGAGAGAGCCGGTCAAT
GCAACGGAGGACCCGTCTAGTGGCTACTATTCTACCACAATTAGATATCAG
GCTACCGGTTTTGGAACCAATGAGACAGAGTACTTGTTCGAGGTTGACAAT
TTGACCTACGTCCAACTTGAATCAAGATTCACACCACAGTTTCTGCTCCAG
CTGAATGAGACAATATATACAAGTGGGAAAAGGAGCAATACCACGGGAAAA
CTAATTTGGAAGGTCAACCCCGAAATTGATACAACAATCGGGGAGTGGGCC
TTCTGGGAAACTAAAAAAAACCTCACTAGAAAAATTCGCAGTGAAGAGTTG
TCTTTCTCTAGAGCAGGACTGATCACAGGCGGGAGAAGAACTCGAAGAGAA
GCAATTGTCAATGCTCAACCCAAATGCAACCCTAATTTACATTACTGGACT
ACTCAGGATGAAGOTGCTGCAATCGGACTGGCCTGGATACCATATTTCGGG
CCAGCAGCCGAGGGAATTTACATAGAGGGGCTAATGCACAATCAAGATGGT
TTAATCTGTGGGTTGAGACACCTGGCCAACGAGACGACTCAAGCTCTTGAA
CTGTTCCTGAGAGCCACAACTGAGCTACGCACCTTTTCAATCCTCAACCGT
AAGGCAATTGATTTCTTGCTGCAGCGATGGGGCGGCACATGCCACATTCTG
GGACCGGACTGCTGTATCGAACCACATGATTGGACCAAGAACATAACAGAC
AAAATTGATCAGATTATTCATGATTTTGTTGATAAAACGCTTCCGGACCAG
GGGGACAATGACAATTGGTGGACAGGATGGAGACAATGGATACCGGCAGGT
ATTGGAGTTACAGGCGTTGTAATTGCAGTTATCGCTTTATTCTGTATATGC AAATTTGTCTTTTAG
VSV-1'GFP-EBOV glycoprotein amino acid sequence (497 aa.) (SEQ ID
NO: 9) MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHSSTLQVSDVDKL
VCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAG
EWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAF
HKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVN
ATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQ
LNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEEL
SFSRAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFG
PAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNR
KAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQ
GDNDNWWTGWRQWIPAGIGVTGVVIAVIALFCICKFVF
[0210] VSV-LASV
[0211] The viral genome is a VSV background where the sequence
encoding the VSV G protein is substituted for a sequence encoding
the glycoprotein from Lassa virus, and a sequence encoding GFP is
inserted into the first position of the viral genome. The LASV
glycoprotein sequence expressed in VSV-LASV is derived from the
Josiah strain of Lassa virus as described in the methods of
Garbutt, et al. (2004) 1 Virol 78: 5458-5465.
[0212] VSV-1'GFP-LASV
[0213] The LASV glycoprotein sequence expressed in VSV-1'GFP-LASV
is derived from the Josiah strain of Lassa virus as described in
the methods of Jae, et al. (2013) Science 340: 479-483.
[0214] Viral infection was detected by the GFP reporter, using
fluorescent and phase contrast imaging under a microscope. All
viruses were used at an MOI of 0.1; thus Lassa-VSV replicated in
gliomas in order to infect all cells tested.
[0215] For in vitro experiments, viruses were propagated on Vero
cells, purified, and concentrated using sucrose gradient
centrifugation (Cureton, et al., PLoS Pathog, 6, e1001127 (2010).
For in vivo experiments, viruses were propagated on Vero cells and
filter-purified according to a protocol described previously
(Lawson, et al., Proc. Natl. Acad. Sci. USA, 92:4477-4481 (1995).
All viruses were plaque-titered on Vero cells prior to experiments.
Human glioma U87 and U118 were obtained from ATCC (Manassas, Va.),
mouse glioma CT2A was a gift from Dr T Seyfried (Boston College,
Chestnut Hill, Mass.), human melanoma YUMAC and normal human
melanocytes were provided by Yale SPORE in Skin Cancer. Normal
human glial cells were derived from human temporal lobectomies
(Ozduman, et al., J. Neurosci, 28:1882-1893 (2008)). Normal human
dermal fibroblasts were purchased from Cambrex (Walkersville, Md.).
Normal human embryonic neurons were purchased from Sciencell
(Carlsbad, Calif.). The human cancer cell lines SJSA-1, BT-549,
T-47D, HCT116, SW480, T24, and RT4, and DU-145 lines were kindly
provided. Stably transfected tumor cells expressing red fluorescent
protein RFP (rU87 and rYUMAC) were generated as described earlier
(Wollmann, et al., J. Virol., 87:6644-6659 (2013)).
[0216] Primary cultures of mouse brain were generated by
dissociating cortex of E17 mice for predominantly neuronal cultures
and whole brain tissue of P1 mice for mixed neuronal/glia cultures.
Cells were plated in MEM (Invitrogen, Carlsbad, Calif.)
supplemented with 10% FBS overnight before medium was replaced with
Neurobasal/B27 medium (Invitrogen). Melanoma cells were maintained
in Opti-MEM/5% FBS, melanocyte medium containing additional
supplements listed elsewhere (Wollmann, et al., J. Virol.,
87:6644-6659 (2013)). Glioma cells, human glia and fibroblasts were
maintained in MEM/10% FBS (Invitrogen). All cultures were kept in
humidified atmosphere containing 5% CO.sub.2 at 37.degree. C.
[0217] In Vitro Experiments
[0218] Viral infection of mixed neuronal/glial cultures, and U87,
U118, CT2A, and YUMAC tumor cultures was monitored by quantifying
GFP expression of infected cells compared to total number of cells
on at least 10 microscopic fields using fluorescence microscopy.
Cultures were assessed for presence of cytopathic effects before
and after virus application. Cytopathic effects were noted as the
appearance of rounding, blebbing and syncitia formation. For
analysis of infection characteristic of chimeric VSVs on mixed
neuronal cultures, morphology of infected cells was used as a guide
for identifying neuronal or glia infection by the virus.
Identification of cell type was later corroborated by
immunohistochemistry for NeuN and GFAP. Mixed human brain cultures
were established by first plating a glia monolayer. Two days later,
human neurons were seeded onto the glia monolayer. After 7 days in
culture and morphological confirmation of neuron process outgrowth,
cultures were inoculated with VSV-wtG or VSVLASV-G (MOI 1). GFP
expression was quantified 24 hours later.
[0219] Plaque assay was used to assess viral replication. In short,
cells were infected at an MOI of 0.1, residual virus was removed by
replacing supernatant, and progeny virus was collected from the
supernatant at the indicated time points. Serial dilutions were
plated on monolayers of Vero or BHK cells, respectively. 2.5% Agar
solution in MEM was used as semi-solid overlay. For IFN
pretreatment, cultures were incubated for 8 hours with recombinant
hybrid interferon type 1 IFN.alpha.A/D (Sigma-Aldrich; catalog no.
I4401) at a concentration of 100 IU/ml. To test generalization of
the oncolytic effect of VSV-LASV-G to other human cancer types, 8
human tumor cell types were infected at an MOI 3 (primary
inoculation). 2 hours later inoculum was removed and cultures were
washed 3 times with PBS before addition of fresh medium. 24 hours
later infection rates were determined by GFP expression. To assess
the capability of VSV-LASV-G to propagate in these tumor cultures
the 24 hpi supernatant was filtered to remove cellular debris and
transferred onto an uninfected monolayer of the same tumor
designation (secondary inoculation).
[0220] Image Analysis Virus infected cultures and histological
sections of mouse brain were analyzed using a fluorescence
microscope (IX 71, Olympus Optical, Tokyo, Japan). A fluorescence
stereomicroscope (SZX12, Olympus Optical) was used for whole brain
scanning before sectioning.
Results
[0221] To test the efficacy of different binding glycoproteins, 6
recombinant VSVs were compared, each expressing a different binding
glycoprotein (FIGS. 1A-1C). Five chimeric VSVs were tested in which
the VSV glycoprotein was replaced (Beier, et al., Proc. Natl. Acad.
Sci. USA, 108:15414-15419 (2011); Jae, et al., Science, 340:479-483
(2013); Krishnan, et al., Viruses, 4:2471-2484 (2012)) by the G
genes from other viruses, including Lassa fever, rabies,
lymphocytic choriomeningitis, Ebola, or Marburg virus. These
chimeric viruses were compared with control VSVs that retained the
normal VSV glycoprotein. Chimeric viruses, which like VSV-wtG, also
encoded GFP in the first genomic position, were tested first in
human and mouse glioblastoma (GBM). All six viruses infected GBM
cells in vitro FIG. 1A), with a greater level of infection of human
GBM (U87 and U118) than mouse GBM (CT2A) 24hpi (FIG. 1A). Generally
speaking, Lassa-VSV showed the most robust infection of the 3
gliomas tested (U87, U118, CT2A) at 24 hrs, and earlier, while
other viruses also infected some of the gliomas, but not all of
them. Specifically, in U87 glioma, all viruses showed good
infection except VSV-MARV (FIG. 1A). In U118 glioma, VSV-LASV,
VSV-EBOV, and VSV-RABV showed high levels of infection, whereas
VSV-MARV and VSV-LCMV significantly less. In mouse CT-2A glioma,
VSV-LASV performed the best; VSV-EBOV infected almost as well as
VSV-LASV (FIG. 1A). Lassa-VSV (VSV-La-G) infected and killed 100%
of two human gliomas, U87 and U118.
[0222] All six viruses replicated on human GBMs, as seen at 24 hrs
post inoculation (hpi) (FIG. 1B). Typical of VSV-infected cells
(Ozduman, et al., J. Neurosci, 28:1882-1893 (2008); Wollmann, et
al., J. Virol, 81:1479-1491 (2007)), infected cells died, as
confirmed by staining with membrane impermeant dyes.
[0223] The relative infection of all six viruses on mouse and human
brain cultures that included neurons and glia was also tested. The
relative infection of mouse neurons vs glia is shown in FIG. 1D.
VSV-wtG showed the greatest level of neuronal infection (around
90%). VSV-LASV-G and VSV-LCMV-G showed the least neuronal
infection. When plaque size was examined as a measure of
infectivity and replication, VSV-LASV plaques were bigger than
VSV-LCMV and VSV-EBOV on human gliomas U87 and U118 (FIG. 1C),
indicating enhanced infection and replication. VSV generated the
largest plaques on tumor cells, but also showed large plaques on
normal brain cells, an unwanted phenotype. Among the chimeric
viruses, VSV-LASV and VSV-EBOV generated smaller plaques on human
brain than VSV-wtG and VSV-LCMV (FIG. 1C), and showed much weaker
infection of pure human neuronal cultures than VSV-wtG (FIG.
3A).
[0224] Lassa-VSV, which was the superior oncolytic candidate from
an analysis of glycoproteins from 5 unrelated viruses that were
incorporated into the VSV genome, was also compared with other VSVs
that expressed the normal VSV glycoprotein. Of 39 potential
oncolytic viruses, including 17 variants of VSV, (Wollmann, et al.,
J Virol., 79: 6005-6022 (2005); Wollmann, et al., J. Virol.,
81:1479-1491 (2007); Wollmann, et al., J 84:1563-73 (2010);
Wollmann, et al., Cancer J., 18:69-81 (2012); Paglino, et al., J.
Virol., 85:9346-58 (2011); Ozduman, et al., J. Virology,
83:11540-11549 (2009); Ozduman, et al., J. Neurosci., 28:1882-1893
(2008)), Lassa-VSV was identified as a superior candidate in terms
of a strong safety profile, as well as an effective potential to
selectively target and kill GBMs and to precipitate an immune
attack on the glioma.
[0225] Together, the in vitro data point to VSV-LASV as having the
best combination of glioma infectivity (FIGS. 1A and 1C) and low
neuronal tropism (FIG. 1D). VSV-EBOV was selected as a second
candidate to include for in vivo testing being another virus with
broad glioma tropism and evidence of reduced neurotropsim compared
to VSVwtG in both mouse and human brain, although not as reduced as
VSV-LASV. VSV-Lassa showed the least infection of neurons, coupled
with a strong infection of human glioma. In contrast, a control
rabies-VSV chimeric virus showed strong infection of neurons and
would be a relatively very poor candidate for glioma treatment.
Recombinant VSVs with minor changes to the genome, for instance
VSV-M51, can potentially revert to the more aggressive wild type
virus by gene mutation. In contrast, since the entire binding of
VSV to cells has been switched to Lassa glycoprotein, and the VSV
glycoprotein gene is deleted, this virus cannot revert to the wild
type VSV, a substantial benefit. A primary mechanism of targeting
is the absence or attenuation of the intrinsic anti-viral response
in the majority of cancer cells, including gliomas, compared to
normal cells that have an intrinsic interferon mediated antiviral
response to VSV and other viruses (Stojdl, et al., Nat. Med.,
6:821-825 (2000); Stojdl, et al., Cancer Cell., 4:263-275 (2003);
Wollmann, et al., J. Virol., 81:1479-1491 (2007)).
[0226] Furthermore, although there is not full agreement as to the
origin of cancer stem cells, with some indications that the cells
initiate cancer, or that they arise from multiple mutations in
dividing tumor cells, there is more agreement that cells that
express antigens typical of stem cells do show resistance to
small-molecule cancer therapeutics, and to radiation treatment.
Glioma stem cells are reported to express CD133 and nestin. In
preliminary experiments, using cells expressing CD133, Lassa-VSV
was found to infect and kill these stem-like cells in culture,
indicating another potential attribute of the use of this virus in
the treatment of cancer.
Example 2: Lassa-VSV is Safe in the Brain
Materials and Methods
[0227] Mouse Procedures
[0228] 6-8 week old male Swiss Webster mice received the following
virus doses: I.C.: 3.6.times.10.sup.4 pfu in 1 ml into the right
striatum (2 mm lateral, 0.5 mm rostral to Bregma at 3 mm depth),
I.V.: 10.sup.6 pfu in 100 .mu.l via tail vein injection, I.N.:
2.5.times.10.sup.5 pfu in 25 .mu.l in each nostril. Stereotactic
application of virus or tumor cells was performed under full
anesthesia using ketamine/xylazine (100 and 10 mg/kg, respectively)
applied via i.p. route. Uni- and bilateral intracranial glioma and
melanoma xenografts were established in 4-6 week old male CB17 SCID
mice by injection of 5.times.10.sup.4 cells each into the left and
right striatum (Ozduman, et al., J. Neurosci, 28:1882-1893 (2008);
Wollmann, et al., J. Virol, 87:6644-6659 (2013)). 15 days post
tumor placement mice received virus either via a unilateral
intratumoral injection (3.6.times.10.sup.4 pfu in 1 .mu.l) or via
tail vein injection (10.sup.6 pfu in 100 .mu.l). Mice were
monitored daily and sacrificed if one of the following conditions
were observed: A) weight loss of 25% or more, B) immobility, C)
occurrence of adverse neurological symptoms, or D) reaching the end
of the observation period of the survival study.
[0229] For histologic analysis of early states of viral infection
mice were sacrificed at 2 or 8 days post viral inoculation. After
overdose with anesthetic, mice were transcardially perfused with 4%
paraformaldehyde. Brains were harvested and stored in 4%
paraformaldehyde, dehydrated in 30% sucrose solution and cut in 20
to 30 .mu.m coronal sections with a cryostat. For detection of live
virus in designated mice after short (2 days) and long (>60
days) exposure to VSV-LASV-G, tissue samples were collected under
sterile conditions from brain, lung, blood, and liver after
euthanasia. Tissues were mechanically homogenized in PBS using a
microcentrifuge tube tissue grinder. Part of the resultant tissue
suspension was plated onto BHK monolayers and assessed for presence
of GFP positive cells 24 hours later. To test the capability of
VSV-LASV-G to induce antibody production, adult Swiss Webster mice
received an intranasal and intramuscular primary VSV-LASV-G
inoculation (at concentrations listed above) followed by a boost 4
weeks later. 2 weeks later, mice were euthanized, bled, and serum
was collected. Antibody containing serum was diluted 1:50 to
1:10,000.
[0230] Brain sections from transgenic mice expressing GFP in
hypocretin neurons were used to target GFP. Brain sections of
IFN.alpha./.beta.-R knock-out mice infected with VSV-LASV-G were
used to target VSV-LASV-G expression. rU87 and rYUMAC cells were
tested for pathogens before tumor grafting and found to be pathogen
free. All animal experiments adhered to institutional guidelines
and were approved by the Yale University Animal Care and Use
Committee.
[0231] Rat Procedures
[0232] Safety of intracranial VSV-LASV-G in rats was tested.
Stereotactic coordinates and applied virus dose equaled those in
rat tumor models described in the following. Syngeneic brain tumor
models were established via stereotactic injection of 50,000 cells
in 3 .mu.l suspension into the right striatum (0.7 mm rostral of
Bregma, 3.5 mm lateral, 5 mm deep) of 7-8 week old Lewis rats (rat
CNS-1 glioma). 7 days post tumor placement, rats received a single
intracranial injection of 3 .mu.l suspension containing
1.2.times.105 PFU of VSV-LASV-G into the area of the tumor. Rats
were euthanized at 3 dpi and brains harvested.
[0233] Intracranial Injection Safety Assays
[0234] The Lassa-VSV and Ebola-VSV viruses were injected directly
into the brains of 6 week old Swiss-Webster mice (n=10 mice for
each virus) using stereotactic injection procedures. A Hamilton
syringe controlled by a Stoelting stereotactic injector (Stoelting,
Wood Dale, Ill.) was used to inject 1 .mu.l of virus solution into
the striatum (2 mm lateral, 0.4 mm rostral to bregma, at 3 mm
depth). As a control, attenuated VSV-1'GFP and attenuated VSV-M51
viruses were injected directly into the brains of mice (n=6 mice
for each virus) using the same procedure. All injections were
3.6.times.10.sup.4 pfu in 1 Animals were monitored daily for body
weight, grooming, and overall health. Mice were euthanized with a
pentobarbital overdose when neurological symptoms were detected or
when body weight dropped below 75%.
[0235] Statistical Analysis
[0236] Statistical significance was determined by Student's t-test,
ANOVA, and Chi-Square test. P-values<0.05 were considered
statistically significant. Survival studies were non-blinded. Mice
were allocated to experimental or control group based on cage
number. Power analysis was employed to determine group size for
survival experiments of tumor-bearing mice and of virus injected
mice.
Results
[0237] To corroborate further the relative lack of infection of
neurons of the chimeric viruses, VSV-LASV and VSV-EBOV were
compared to two VSVs that expressed the VSV glycoprotein in vivo;
one of the VSVs was further attenuated by including an M51 mutation
which enhances the antiviral innate immune response against the
virus (Waibler et al, 2007). Injection of either VSV with normal
wild-type G (VSV-wtG) into the normal brain generated lethal
consequences, with a median survival of 3.5 days for VSV-wtG (n=6)
and 8 days for the attenuated strain VSV-M51 (n=6) (FIG. 2A)
consistent with previous observations (Ozduman et al., 2009). This
underlines the neurotoxicity of VSV with normal VSV-G protein, even
when attenuated. In striking contrast, direct injections of
VSV-LASV-G or VSV-EBOV-G, (3.6.times.10.sup.4 pfu in 1 .mu.l) into
normal mouse brains exerted no adverse effect in >112 days (n=8
each virus; p<0.001; Chi square test) (FIG. 2A).
[0238] In another experiment VSV-LASV-G was compared with VSV-IFN,
an IFN-expressing virus modeled after one (Obuchi, et al., J.
Virol., 77:8843-8856 (2003) currently in clinical trials for the
treatment of liver cancer (NCT01628640). All mice (n=9) receiving
intracranial VSV-IFN died within 12 days consistent with reports of
this virus infecting brain meninges (Yarde, et al., Cancer Gene
Ther., 20:616-621 (2013); all mice that received VSV-LASV-G (n=4
here) survived with no adverse side effects (FIG. 2B). Similarly,
VSV-LASV-G injected into the rat brain also showed no sign of
neurotoxicity (>80 days, n=3). No virus or infected cells were
detected in the brain or elsewhere (liver, spleen, blood) by
histological analysis or culture of inoculated mouse tissue at the
conclusion of the experiment, indicating the total elimination of
the virus. The injections of VSV-LASV-G and VSV-EBOV-G were viable,
as euthanasia at 2 dpi revealed limited infection of glia within
the brain. However, by 8 dpi, few or no infected cells could be
found. VSV-LASV-G and VSV-EBOV-G were also injected intravenously
(n=5 each), and an additional set of mice were inoculated at
combined intranasal/intramuscular/subcutaneous (n=5, total
8.times.10.sup.6 pfu VSV-LASV-G) sites; none of these mice showed
signs of viral pathogenicity and no virus could be harvested from
these mice 2 months postinoculation.
[0239] A different VSV-LASV-G that contained no GFP (Garbutt, et
al., J. Virol., 78:5458-5465 (2004) was also tested and found to
have no adverse effect after injection into the mouse brain (n=5)
and followed for 6 weeks, showing that the attenuation mediated by
inclusion of the GFP gene in the first genomic position was not
critical for CNS safety. The 100% survival of mice injected with
VSV-LASV-G and VSV-EBOV-G correlated with a very low infectivity of
these viruses in pure neuronal cultures, particularly when compared
to VSV with the normal VSV G protein (FIG. 2A-2B).
[0240] The most problematic aspect of using VSV either as an
oncolytic virus or as a vaccine vector against more dangerous
viruses including Ebola (Garbutt, et al., J. Virol., 78:5458-5465
(2004), HIV, and other pathogenic viruses, has been the concern
about adverse effects of the virus in the brain (Ozduman, et al.,
J. Virol., 83:11540-11549 (2009); Huneycutt, et al., Brain Res.,
635:81-95 (1994)). A number of previous studies have described the
potential for VSV to selectively target and destroy many different
types of tumors. However, a substantial problem with the use of VSV
as an oncolytic virus, both outside and within the brain, has been
the possibility of adverse effects within the brain potentially
leading to motor dysfunction (Huneycutt, et al., Brain Res.,
635:81-95 (1994), behavioral disturbances (Lundh, et al., J
Neuropathol. Exp. Neurol., 47:497-506 (1988) or death (Huneycutt,
et al., Brain Res., 635:81-95 (1994); van den Pol, et al., J.
Virol., 76:1309-1327 (2002). The data show that eliminating the
native glycoprotein from the VSV genome and substituting binding
glycoproteins from other viruses greatly reduces infection and
cytolysis of neurons. All five chimeric VSVs tested showed
considerably reduced neuron tropism and replication compared with
the natural VSV glycoprotein. Not only were the chimeric viruses
tested safe within the brain, but direct injection of VSV-LASV-G
into the brains of SCID mice lacking the normal complement of B and
T immune cells generated no adverse effect, and the innate immune
system within the brain eliminated the virus.
[0241] Importantly, after intravenous inoculation, the chimeric
viruses VSV-LASV-G and VSV-EBOV-G were able to cross the blood
brain barrier and selectively infect brain tumors with little or no
infection of normal neurons or glia, and no adverse effects.
VSV-LASV-G completely eliminated brain tumors and prolonged the
lives of tumor-bearing mice indefinitely. VSV containing the Ebola
glycoprotein also crossed the blood brain barrier and targeted the
brain tumor, but showed only partial infection of the glioma.
[0242] VSV neurotoxicity can be reduced by generating peripheral
immunity in advance of intracerebral inoculation (Ozduman, et al.,
J. Virol., 83:11540-11549 (2009) or by administering exogenous type
1 interferon, or via intra cerebral viral vectors that generate
interferon (Wollmann, et al., Virology, in press. (2014).
Attenuated VSVs have been constructed by a number of molecular
alterations, including reduction of cytoplasmic amino acids in the
G protein, mutations in the M gene particularly at M51, adding
genes upstream of viral genes (Roberts, et al., J. Virol.,
73:3723-3732 (1999); Ahmed, et al., Virology, 33:34-39 (2004);
Ramsburg, et al., J. Virol., 79:15043-15053 (2005)), but most of
these resultant VSVs still retain negative side effects in the
brain due to neuronal infection. Even a VSV currently in clinical
tests that expresses interferon (VSV-IFN), although attenuated, is
problematic and can be lethal if it gains access to the brain. None
of these recombinant strategies directly eliminate the lethal
neurotropism of the virus within the brain conferred by the VSV-G
protein. In contrast, all five G protein chimeric viruses used in
the disclosed studies showed reduced neuron infection, and the two
tested in vivo showed complete safety within the brain. Even
substitution of the glycoprotein from rabies, a virus with
well-known neuronal targeting, although it still showed an
unacceptably high neuron targeting, still showed reduced infection
of neurons compared with the VSV glycoprotein, and has been shown
relatively safe in the brain (Beier, et al., Proc. Natl. Acad. Sci.
USA, 108:15414-15419 (2011), underlining the importance of
eliminating the VSV glycoprotein.
[0243] Neither VSV-EBOV-G nor VSV-LASV-G injected intracerebrally
into normal mice, immunocompromised SCID mice, or rats evoked any
adverse action, whereas similar concentrations of other VSVs with
the native G, including attenuated VSV-CT9-M51 (Wollmann, et al.,
Virology, in press. (2014), VSV-1' GFP, VSVmIFN.alpha., and VSV-M51
(FIG. 2A-2B) were lethal.
[0244] This safety is also corroborated with previous studies that
have used similar virus recombinants with Lassa (or Ebola)
glycoproteins in place of the VSV G-protein as immunization
vehicles to protect against wild-type Lassa or Ebola viruses; and
deletion of genes other that the glycoprotein gene eliminates the
toxicity and disease potential associated with wild type Ebola and
Lassa. Lassa and Ebola virus may have arisen from a common ancestor
virus (Gallaher, et al., BMC Microbial., 1:1, 6 pages, (2001)).
VSV-LASV-G was previously shown safe in rodents after
intraperitoneal injection, but intracranial safety was not
investigated (Garbutt, et al., J. Virol., 78:5458-5465 (2004). One
human has been injected with Ebola-VSV as a vaccine after a lab
accident, with no adverse consequence (Gunther, et al., J. Infect.
Dis., 204 Suppl 3:S785-S790 (2011)). The results disclosed herein
show that both VSV-LASV-G and VSV-EBOV-G are safe in the rodent
brain. The GFP reporter gene in both VSV-LASV-G and VSV-wtG
provides some attenuation to both viruses by reducing expression of
other VSV genes; in spite of this, VSV with the wild type
glycoprotein was still lethal within the brain even when carrying
the attenuating GFP gene, whereas VSV-LASV-G and -EBOV-G were safe
in the brain indicating that safety in the brain is due to the
absence of the neurotoxic VSV glycoprotein. This view is supported
by the data showing safety of VSV-LASV in chimeric viruses not
containing the GFP gene. The lack of VSV-LASV-G infection of
neurons in the rodent brain parallels the in vitro studies with
human neurons showing a lack of infection, and indicating that the
virus may also be safe in the human brain. This is consistent with
the findings which did not consider the oncolytic potential, but
did show that these chimeric viruses evoked a strong humoral and
cellular immune response, and importantly, evoked no adverse health
consequences, even when injected directly into the brains of
rodents or non-human primates (Geisbert, et al., PLoS Med, 2:e183
(2005); Geisbert, et al., PLoS Pathog., 4:e1000225 (2008);
Geisbert, et al., J. Virol., 83(14):7296-7304 (2009); Mire, et al.,
PLoS Negl. Trop Dis, 6:e1567 (2012)). Initiation of a strong immune
response is another benefit, and can generate a secondary immune
attack on the brain tumor.
[0245] Such strong attenuation of virus infection on normal cells
might also lead to lack of efficiency in tumor destruction.
Lassa-VSV was nonetheless capable of selectively infecting and
killing GBM cells in vitro and in the mouse brain after intravenous
or intracerebral virus administration, and substantially prolonged
cancer survival far beyond that of control tumor-bearing mice that
received no virus. In fact, at the time when all tumor bearing
control mice had died from the expanding brain tumor, none of the
mice in which tumors were treated with VSV-Lassa showed any obvious
symptoms from the tumor, or from the virus.
[0246] In conclusion, other VSVs, even those attenuated, can lead
to adverse neurological consequences or death. In contrast,
Lassa-VSV shows no detectable adverse effects when injected
directly into the brain.
Example 3: IFN has Little Effect in Attenuating Infection of
Gliomas by VSV-LASV-G
Materials and Methods
[0247] Quantitative RT-PCR
[0248] Mouse neuronal cultures and human U87 glioma cells were
cultured in 6-well plates. VSV-LASV-G or VSV-wtG was added at an
MOI of 1 and cultures were incubated for 20 minutes at 4.degree. C.
to test virus binding or for 30 minutes at 37.degree. C. to test
virus binding+internalization, respectively, as described elsewhere
(Ozduman et al, 2008). Experiments were performed in duplicate.
Cells were washed five times with PBS prior to RNA isolation using
TRIzol Reagent (Invitrogen, Carlsbad, Calif.). Stratascript reverse
transcriptase kit (Stratagene) was used for cDNA generation. TaqMan
gene expression assays (Applied Biosystems, Foster City, Calif.)
were used to quantify the expression of .beta.-actin and VSV
genomes using an ICycler iQ Real time PCR system (Bio-Rad,
Hercules, Calif.). For specific VSV genome detection (excluding
viral mRNA) primers were designed to yield a product that spanned
the junction between N and P genes. PCR samples were measured in
triplicates, normalized to 3-actin expression and compared to
expression of VSVLASV-G binding to neurons as reference
(.DELTA..DELTA.Ct method).
Results
[0249] The results described herein show that the identity of the G
protein has a profound effect on neuronal toxicity in the context
of chimeric VSV, with viruses bearing LASV-G or LCMV-G being the
least neurotropic. To elucidate mechanisms whereby the chimeric
viruses appeared to show strong infection of glioma, but less
infection of normal neurons and glia, the innate immune response
was examined. In contrast to the protective effect of IFN on
cultures of normal human neurons, glia, or fibroblasts, microscopy
reveled that IFN had little effect in attenuating infection by
VSV-LASV-G of U118 and U87 human gliomas, as virtually all glioma
cells were infected and GFP-positive by 24 hours post-inoculation
(FIG. 3A).
[0250] These data are consistent with the view that a primary
mechanism underlying the selective VSV-LASV-G infection of cancer
cells over normal glia is related to deficiencies in innate
immunity. The lack of protection by added IFN points to a deficient
IFN response among VSV-LASV-G susceptible tumors, similar to the
mechanism described for the enhanced relative susceptibility of a
number of tumor types to native VSV and to several other viruses
including Newcastle disease virus, reovirus and myxoma virus
(Phuangsab, et al., Cancer Lett., 172:27-36 (2001); Strong, et al.,
EMBO J., 17:3351-3362 (1998); Wang, et al., Nature Immunol.,
5:1266-1274 (2004)). The observation that normal brain cells in
SCID mice which are deficient in T- and B-cell antiviral defenses
showed little infection and no adverse effects from VSV-LASV-G in
the brain further supports the view that an innate immune mechanism
is protective of normal brain cells.
[0251] To test whether the intrinsic IFN system is important for
the resistance of the brain to VSV-LASV-G, VSV-LASV-G was injected
into the brains of mice (n=5) lacking the type I IFN receptor.
Although IFN.alpha./.beta.-R-/- mice with intracerebral infection
from VSV-LASV-G survived longer than those with VSV-wtG (n=3) (FIG.
3B), all these mice ultimately died within a week of CNS
inoculation. These data support the view that an innate
IFN-mediated immune response is important for both short and long
term survival after VSV-LASV-G infection of the brain.
[0252] This motivated a comparison of binding of VSV-wtG and
VSV-LASV-G to glioma and neurons. As judged by quantitative RT-PCR,
both viruses bound similarly well to glioma cells and to neurons at
4.degree. C. (FIG. 3C), a temperature at which endocytosis is
inhibited. At 37.degree. C., approximately 2-logs more virus
becomes cell-associated than at 4.degree. C., indicating effective
and similar rates of internalization in both cell types and for
both viruses.
[0253] Next, the ability of infected glioma cells and neurons to
generate progeny virions was examined. On gliomas, both VSV-wtG
(FIG. 3D) and VSV-LASV-G (FIG. 3E) show substantial virus
replication. In contrast, replication of VSV-LASV-G was greatly
attenuated in neurons, by 4.5 logs relative to VSV-wtG which showed
robust replication in neuron cultures (FIG. 3D-3E). Together, these
data indicate that although both VSV-wtG and VSV-LASV-G bind to
glioma cells and neurons, VSV-LASV-G replicates poorly in neurons.
In neurons, a block to replication appears most likely to occur at
the point of endosomal escape and uncoating, which is G-protein
mediated, or possibly a subsequent step.
[0254] These data support a conclusion that the advantage of the
Lassa virus glycoprotein is that it produces a selective block to
replication in neurons but not in a wide variety of human tumors.
This block to neuron infection appears not to be at the binding or
internalization steps but at a step further downstream in the life
cycle, possibly at the uncoating/endosomal escape step in neurons,
given the importance of the glycoprotein to this process (Rigaut,
et al., J. Virol., 65:2622-2628 (1991).
[0255] Type 1 IFN is important to the selectivity and safety of
VSV-LASV; mice lacking IFN receptor succumbed to virus inoculation.
Previous reports indicate that whereas type 2 and 3 IFN may also
contribute to immunity in the brain (van den Pol, et al., J.
Virol., 88:3695-3704 (2014), type 1 IFN is necessary for survival.
The impairment of innate immunity characteristic of ontogenically
transformed cells allows VSV-LASV to replicate rapidly, with
cytolytic consequences for tumor cells. Addition of IFN to glioma
had little to no effect on the infectivity of VSV-LASV, but
provided additional protection to normal neurons and glia. The
exquisite tumor-selectivity of VSV-LASV is thus a consequence of
both reduced neurotropsism (via substitution of the Lassa G for the
VSV G-protein) and virus susceptibility to IFN in normal cells;
thus the virus is able to very selectively infect and destroy
tumors whether entering from the blood stream into the brain, or
crossing from an infected tumor to infect a distant locus of tumor
cell growth in the contralateral brain, all without infecting the
intervening healthy brain tissue. The fact that the innate immune
system appears able to contain VSV-LASV-G indicates that the virus
may prove safe to treat the increased incidence of cancers found in
patients with compromised adaptive immune systems, for instance
with AIDS.
Example 4: The Chimeric Virus Lassa-VSV Selectively Infects and
Kills Glioma
Materials and Methods
[0256] Migration Assays
[0257] Red fluorescence marker RFP expressing tumor cells (U87
human glioblastoma cells and YUMAC human melanoma cells,
respectively) were implanted into mice: two tumors per mouse--one
in the left striatum, and a second in the right striatum.
[0258] 14 days after tumor placement, Lassa-VSV expressing a green
reporter gene GFP was then injected into the right tumor only.
[0259] Eight days later, mice were euthanized and perfused
transcardially with 4% paraformaldehyde. Brains were harvested and
analyzed for tumor expansion and virus infection using red and
green fluorescence on a stereomicroscope for whole brain analysis
and on an Olympus microscope for analysis of brain sections.
Results
[0260] The ability of chimeric virus to target and destroy brain
tumors was also tested. Fifteen days after implant of red
fluorescent human glioma into the SCID mouse brain, VSV-LASV-G
(10.sup.6 pfu in 100 ul) was injected intravenously (tail vein).
This resulted in highly selective tumor infection and complete
destruction of tumor cells within the brain. Mice with brain tumors
survived only if treated with VSVLASV-G, with no adverse effect as
of 80 days (FIG. 4). Mice with tumors that did not receive virus
all died from the tumor (median 29 day survival; n=8; FIG. 4). The
chimeric virus VSV-EBOV-G also targeted brain tumors after
intravenous inoculation (FIG. 4); VSV-EBOV-G extended life
minimally (median survival 34 days; n=8) (FIG. 4).
[0261] Histological analysis showed large tumors in mice not
treated with virus, and incomplete but selective infection of the
tumor treated with VSV-EBOV-G. In contrast, few tumor cells if any
were found following treatment with VSV-LASV-G, indicating highly
selective infection and destruction of glioma.
[0262] In another short term experiment, mice showed near-complete
infection of bilateral tumor masses 8 days post-inoculation, with
little infection outside the tumor area indicating a rapid and
selective VSV-LASV-G anti-tumor action (n=4).
[0263] Using a syngeneic rodent tumor model, the ability of
VSV-LASV-G to infect rat glioma in immunocompetent rats was tested.
VSV-LASV-G showed selective strong cytolytic infection of CNS-1
glioma tumors 3 days after intracerebral inoculation, with little
detectable infection of normal brain, demonstrating the VSV-LASV-G
selectively infects glioma not only in mice, but also in
immunocompetent rats.
[0264] A major problem with gliomas is tumor cell migration within
the brain. Thus skilled neurosurgeons can debulk the tumor, but
cannot eliminate migrating cells or tumors that arise from
migrating cells. To model this, human glioma were implanted in the
left and right side of the SCID mouse brain with (in striatum).
Fifteen days later VSV-LASV-G was stereotactically injected
unilaterally, only into the tumor on the right side.
[0265] Eight days later, the brains were examined. VSV-LASV-G had
completely destroyed the inoculated tumor on the right side of the
brain, and the virus had migrated to the contralateral left tumor
and begun the process of infection and destruction without
infecting the intervening normal brain. Accordingly, in addition to
completely destroying the right tumor, Lassa-VSV migrated within
the brain, and had begun to infect and kill the left tumor without
detectable infection of normal brain cells surrounding the tumors.
Remarkably, VSV-LASV-G selectively destroyed the brain tumor with
no adverse effects to the SCID mouse brain. These observations
indicate that the virus was suppressed in the brain by a T- and
B-cell-independent mechanism.
[0266] This is exactly the type of phenotype needed, that is, a
virus that can infect multiple tumors in different CNS sites with
no collateral damage to normal brain. Peripheral immunization has
been shown to protect the brain from intracranial VSV (Ozduman, et
al., J. Virology, 83:11540-11549 (2009)). Even without
immunization, Lassa-VSV showed no neurotoxicity, whereas other VSVs
did show intracranial neurotoxicity. Lassa-VSV was substantially
more attenuated than the most attenuated VSV used (Ozduman, et al.,
J. Virology, 83:11540-11549 (2009); Wollmann, et al., J. Virol,
84:1563-73 (2010)), underlining its safety in the brain.
[0267] One potential limitation of VSVs with point and some other
types of mutations (VSV-M51, VSV1'2'-GFP, and others) is that
additional spontaneous mutations can cause the virus to revert back
to a more aggressive wild type virus. That is unlikely in
Lassa-VSV, as there is no mechanism whereby the Lassa-glycoprotein
can mutate into the VSV glycoprotein, or where the VSV G-protein
can be reconstituted, and also no way in which the non-glycoprotein
genes of Lassa could appear in the recombinant virus.
Example 5: The Chimeric Virus Lassa-VSV Infects and Kills
Melanoma
[0268] Next experiments were designed to determine if VSV-LASV-G is
selective for gliomas, or is able to target other types of
metastatic brain cancer. Melanomas are the deadliest form of skin
cancer, and one of the chief problems is metastasis into the brain
(Carlino, et al., Cancer J, 18:208-212 (2012). Red fluorescent
human melanoma was injected into the left and right side of the
brain, similar to the glioma experiments described above.
VSV-LASV-G was subsequently injected unilaterally into the right
side melanoma. VSV-LASV-G not only caused complete destruction of
the injected tumor mass, it also diffused across the brain midline,
and showed strong infection of the non-injected contralateral
melanoma, with no neuron infection in the intervening (SCID)
brain.
[0269] Because VSV-LASV-G infected two unrelated brain tumor types,
glioma and melanoma, it was also tested on other types of human
cancer cells. VSV-LASV-G infected and replicated in colon,
prostate, breast, bone, and bladder cancer cells, indicating its
oncolytic potential was not restricted to glioma and melanoma brain
tumors.
[0270] These results indicate that VSV-LASV-G is effective not only
against glioma (which arise within the brain) but also against
melanoma, a cancer that arises in the skin but metastasizes into
the brain resulting in death within a few months of entering the
brain. There is also a strong viral tropism for other types of
human cancer cells including prostate, breast, colon, and bladder
which can sometimes metastasize into the brain. The broad
infectivity and cytolysis of multiple types of cancer cells
indicate that VSV-LASV-G may also be effective in targeting other
types of brain tumors not tested here, including meningioma,
astrocytoma, ependymoma, and oligodendroglioma. The elimination of
neurotropism by substitution of the Lassa glycoprotein for the VSV
glycoprotein would provide an increased level of brain safety even
if the virus were used to attack peripheral cancers.
[0271] In vitro testing indicates VSV-LASV had the best combination
of reduced neurotropism and broad infectivity across different
gliomas. VSV-EBOV also demonstrated broad glioma infectivity in
vitro; although VSV-EBOV targeted tumors in the brain after
intravenous application, the virus was not very effective in
destroying the tumor. VSV-LCMV had a reduced neurotropism similar
to VSV-LASV, but did not show as great or broad a potential for
infectivity of gliomas, and formed smaller plaques than VSV-LASV in
the human gliomas tested.
Example 6: VSV-LASV-G Evokes a Humoral Immune Response
[0272] To test whether VSV-LASV-G would evoke a humoral immune
response, mice were inoculated intranasal and intramuscularly with
this virus. VSV-LASV-G generated high titer antisera to VSV-LASV-G
infected cells and also to the GFP transgene. Antisera with
dilutions out to 1:10,000 generated positive immunostaining on
GFP-expressing transgenic mice, indicating the potential for a
secondary systemic immune response against VSV-LASV-G infected
gliomas as reported for wildtype VSVs (Publicover, et al., J.
Virol., 78:9317-9324 (2004). It is interesting to note that
chimeric VSV-LCMV/G was shown to be only weakly immunogenic with
regard to generation of neutralizing antibodies against the virus
(Muik, et al., Cancer Res., 74:3567-3578 (2014). VSV-LASV-G
appeared to completely eliminate human brain tumors in these
experiments; VSVs also generated a strong immune response against
tumors which could serve to augment tumor eradication in the event
of incomplete direct tumor destruction (Wongthida, et al., Hum.
Gene Ther., 22:1343-1353 (2011). Chimeric VSVs that generate a
strong immune response may also be beneficial in terms of evoking a
strong secondary immune response against tumor related
antigens.
[0273] VSV is a very promising vaccine platform, and even single
inoculations generate strong cellular and humoral immunity
(Publicover, et al, J. Virol., 79:13231-13238 (2005); Buonocore, et
al., J. Virol., 76:6865-6872 (2002); Schell, et al., J. Virol.,
85:5764-5772 (2011)), but would advance further with the risk of
neurotoxicity effectively eliminated, for example by substitution
of the Lassa glycoprotein for the VSV glycoprotein. VSV-LASV-G may
prove beneficial as a vaccine vector, given its potent
immunogenicity. Immunogenic viral vectors could be generated
against other pathogenic organisms by substituting a gene coding
for a protein from the pathogenic microbe in place of the GFP gene
within VSV-LASV-G; use of the Lassa glycoprotein in place of the
VSV glycoprotein could provide a safer vector for immunization than
wildtype VSV.
Example 7: The Chimeric Virus Lassa-VSV Protects from Glioma
Materials and Methods
[0274] Three groups of SCID mice received human glioma implants of
similar size. 15 days later, one group received Lassa-VSV
intravenously (tail vein), the second group received Ebola-VSV
intravenously, and the third group served as a tumor-only control.
Mice were monitored for changes in body weight. Mice showing body
weight <75% of pre-tumor body weight were euthanized, as per
animal use regulations.
Results
[0275] A mouse weight graph (FIG. 5) showed that mice receiving
Lassa-VSV showed no strong reduction in body weight, whereas
control mice with tumors show body weight reduction from the
expanding tumors. Mice with tumors inoculated with Ebola-VSV
(VSV-Eb-G) showed a reduced body weight from the expanding tumor.
Lassa-VSV is safe in the brain, as mice maintained a normal body
weight.
[0276] A mouse survival graph (FIG. 4) showed that Lassa-VSV
protected mice from an implanted glioma. In contrast, control mice
with the same tumor showed a median survival of 29 days post-glioma
injection. All controls were dead by day 33 after tumor implant.
The Ebola-VSV gave some protection against the tumor with a median
survival of 34 days post-glioma implant, but ultimately, all were
dead by 57 days after tumor implant. All mice treated with
Lassa-VSV survived >43 days after Lassa-VSV inoculation, and
>80 days after glioma implant. The increased longevity of tumor
bearing mice receiving Lassa-VSV was statistically significant
(p<0.01; n=8 in each group). Ultimately all controls died from
the glioma. In striking contrast, Lassa-VSV crossed the blood brain
barrier, infected and destroyed the tumor, and these mice all
survived. Upon histological examination after euthanasia, large red
gliomas were found in control mice, but were absent from
Lassa-VSV-treated mice. Furthermore, since these SCID mice had
severely attenuated systemic immune system (necessary for
implantation of human glioma), but still survived peripheral and
CNS infection with the virus, the chimeric Lassa-VSV is remarkably
safe within the body, and particularly within the brain.
[0277] When injected into a brain tumor, Lassa-VSV kills tumor
cells, and prolongs life with no detectable neurological
consequences. When injected intravenously, the virus crosses the
blood brain barrier and selectively infects gliomas.
Sequence CWU 1
1
913401DNALassa virus 1cgcaccgggg atcctaggca tttttggttg cgcaattcaa
gtgtcctatt taaaatggga 60caaatagtga cattcttcca ggaagtgcct catgtaatag
aagaggtgat gaacattgtt 120ctcattgcac tgtctgtact agcagtgctg
aaaggtctgt acaattttgc aacgtgtggc 180cttgttggtt tggtcacttt
cctcctgttg tgtggtaggt cttgcacaac cagtctttat 240aaaggggttt
atgagcttca gactctggaa ctaaacatgg agacactcaa tatgaccatg
300cctctctcct gcacaaagaa caacagtcat cattatataa tggtgggcaa
tgagacagga 360ctagaactga ccttgaccaa cacgagcatt attaatcaca
aattttgcaa tctgtctgat 420gcccacaaaa agaacctcta tgaccacgct
cttatgagca taatctcaac tttccacttg 480tccatcccca acttcaatca
gtatgaggca atgagctgcg attttaatgg gggaaagatt 540agtgtgcagt
acaacctgag tcacagctat gctggggatg cagccaacca ttgtggtact
600gttgcaaatg gtgtgttaca gacttttatg aggatggctt ggggtgggag
ctacattgct 660cttgactcag gccgtggcaa ctgggactgt attatgacta
gttatcaata tctgataatc 720caaaatacaa cctgggaaga tcactgccaa
ttctcgagac catctcccat cggttatctc 780gggctcctct cacaaaggac
tagagatatt tatattagta gaagattgct aggcacattc 840acatggacac
tgtcagattc tgaaggtaaa gacacaccag ggggatattg tctgaccagg
900tggatgctaa ttgaggctga actaaaatgc ttcgggaaca cagctgtggc
aaaatgtaat 960gagaagcatg atgaggaatt ttgtgacatg ctgaggctgt
ttgacttcaa caaacaagcc 1020attcaaaggt tgaaagctga agcacaaatg
agcattcagt tgatcaacaa agcagtaaat 1080gctttgataa atgaccaact
tataatgaag aaccatctac gggacatcat gggaattcca 1140tactgtaatt
acagcaagta ttggtacctc aaccacacaa ctactgggag aacatcactg
1200cccaaatgtt ggcttgtatc aaatggttca tacttgaacg agacccactt
ttctgatgat 1260attgaacaac aagctgacaa tatgatcact gagatgttac
agaaggagta tatggagagg 1320caggggaaga caccattggg tctagttgac
ctctttgtgt tcagtacaag tttctatctt 1380attagcatct tccttcacct
agtcaaaata ccaactcata ggcatattgt aggcaagtcg 1440tgtcccaaac
ctcacagatt gaatcatatg ggcatttgtt cctgtggact ctacaaacag
1500cctggtgtgc ctgtgaaatg gaagagatga gacccttgtc agggcccccg
tgacccaccg 1560cctattggcg gtgggtcacg ggggcgtcca tttacagaac
gactctaggt gtcgatgttc 1620tgaacaccat atctctgggc agcactgctc
tcaaaaccga tgtgttcagt cctcctgaca 1680ctgctgcatc aaacatgatg
cagtccatta gtgcacagtg aggggttatt tcctctttac 1740cgcctctttt
cttcttttca acaacgacac ctgtgtgcat gtggcataag tctttatact
1800ggtcccagac tgcattttca tacttcctgg aatcagtttt gctgagggca
atatcaatta 1860gtttaatgtc ttttcttcct tgtgattcaa ggagtttcct
tatgtcatcg gacccctgac 1920aggtaatgac catattccgg gggagtgcat
caatgacagc actggtcaag cccggttgtg 1980tagcgaagag gtctgtgaca
tcaatcccat gtgagtactt agcatcctgc ttgaactgct 2040ttaaatcagt
aggttcacgg aagaagtgta tgtagcagcc tgaacttggt tgatagaggg
2100caatttccac tggatcttca ggtcttcctt caatgtccat ccaggtctta
gcatttgggt 2160caagttgcag cattgcatcc ttgagggtca tcagctgaga
ataggtaagc ccagcggtaa 2220accctgccga ctgcagggat ttactggaat
tgttgctgtc agctttctgt ggcttcccat 2280ctgattccag atcaacgaca
gtgttttccc aggcccttcc tgttattgag gttcttgatg 2340caatatatgg
ccatccatct cctgacaaac aaatcttgta gagtatgttt tcataaggat
2400tcctttcacc aggggtgtct gaaatgaaca ttccaagagc cttcttgacc
tttaaaatgg 2460atttgaggat accatccatt gtctgaggtg acaccttgat
tgtctccaac atattgccac 2520catccagcat gcaagctcct gccttcacag
ctgcacccaa gctaaaatta taacctgaga 2580tattcaaaga gcttttcttg
gtgtcaatca tatttaggat gggatgactt tgagtcagcc 2640tgtctaagtc
tgaagtgttg ggatactttg ctgtgtagat caaacccaaa tctgtcaatg
2700cttgtactgc atcattcaag tcaacctgcc cctgttttgt cagacatgcc
agtgtcagac 2760ttggcatggt cccgaactga ttattgagca actctgcatt
tttcacatcc caaactctca 2820ccactccatc tctcccagcc cgagcccctt
gattaccacc actcattcct atcatattca 2880ggagagctct tctttggtca
agttgctgtg agcttaggtt gcccatatag acacctgcac 2940ttaatggcct
ttctgttctg atcacctttg actttaactt ctctagatca gcggctaaga
3000ttaataagtc atctgaggtt agagtcccaa ctctcagtat actcttttgt
tgagttgatt 3060ttaattcaac aagattgttg accgcttgat ttaggtccct
caaccgtttc aaatcattgt 3120catcccttct ctccttgcgc atcaaccgtt
gaacattact gacttcggag aagtcaagtc 3180catgtaaaag agcctgggca
tctttcacca cctgtagttt gatgttggag cagtaaccag 3240ataattccct
cctcaaagat tgtgtccaca aaaaggattt tatttccttt gaggcactca
3300tcgccagatt gttgtgttgt atgcacgcaa caaagaactg agactatctg
ccaaaatgac 3360aaaagcaaag cgcaatccaa tagcctagga tccactgtgc g
34012491PRTLassa virus 2Met Gly Gln Ile Val Thr Phe Phe Gln Glu Val
Pro His Val Ile Glu1 5 10 15Glu Val Met Asn Ile Val Leu Ile Ala Leu
Ser Val Leu Ala Val Leu 20 25 30Lys Gly Leu Tyr Asn Phe Ala Thr Cys
Gly Leu Val Gly Leu Val Thr 35 40 45Phe Leu Leu Leu Cys Gly Arg Ser
Cys Thr Thr Ser Leu Tyr Lys Gly 50 55 60Val Tyr Glu Leu Gln Thr Leu
Glu Leu Asn Met Glu Thr Leu Asn Met65 70 75 80Thr Met Pro Leu Ser
Cys Thr Lys Asn Asn Ser His His Tyr Ile Met 85 90 95Val Gly Asn Glu
Thr Gly Leu Glu Leu Thr Leu Thr Asn Thr Ser Ile 100 105 110Ile Asn
His Lys Phe Cys Asn Leu Ser Asp Ala His Lys Lys Asn Leu 115 120
125Tyr Asp His Ala Leu Met Ser Ile Ile Ser Thr Phe His Leu Ser Ile
130 135 140Pro Asn Phe Asn Gln Tyr Glu Ala Met Ser Cys Asp Phe Asn
Gly Gly145 150 155 160Lys Ile Ser Val Gln Tyr Asn Leu Ser His Ser
Tyr Ala Gly Asp Ala 165 170 175Ala Asn His Cys Gly Thr Val Ala Asn
Gly Val Leu Gln Thr Phe Met 180 185 190Arg Met Ala Trp Gly Gly Ser
Tyr Ile Ala Leu Asp Ser Gly Arg Gly 195 200 205Asn Trp Asp Cys Ile
Met Thr Ser Tyr Gln Tyr Leu Ile Ile Gln Asn 210 215 220Thr Thr Trp
Glu Asp His Cys Gln Phe Ser Arg Pro Ser Pro Ile Gly225 230 235
240Tyr Leu Gly Leu Leu Ser Gln Arg Thr Arg Asp Ile Tyr Ile Ser Arg
245 250 255Arg Leu Leu Gly Thr Phe Thr Trp Thr Leu Ser Asp Ser Glu
Gly Lys 260 265 270Asp Thr Pro Gly Gly Tyr Cys Leu Thr Arg Trp Met
Leu Ile Glu Ala 275 280 285Glu Leu Lys Cys Phe Gly Asn Thr Ala Val
Ala Lys Cys Asn Glu Lys 290 295 300His Asp Glu Glu Phe Cys Asp Met
Leu Arg Leu Phe Asp Phe Asn Lys305 310 315 320Gln Ala Ile Gln Arg
Leu Lys Ala Glu Ala Gln Met Ser Ile Gln Leu 325 330 335Ile Asn Lys
Ala Val Asn Ala Leu Ile Asn Asp Gln Leu Ile Met Lys 340 345 350Asn
His Leu Arg Asp Ile Met Gly Ile Pro Tyr Cys Asn Tyr Ser Lys 355 360
365Tyr Trp Tyr Leu Asn His Thr Thr Thr Gly Arg Thr Ser Leu Pro Lys
370 375 380Cys Trp Leu Val Ser Asn Gly Ser Tyr Leu Asn Glu Thr His
Phe Ser385 390 395 400Asp Asp Ile Glu Gln Gln Ala Asp Asn Met Ile
Thr Glu Met Leu Gln 405 410 415Lys Glu Tyr Met Glu Arg Gln Gly Lys
Thr Pro Leu Gly Leu Val Asp 420 425 430Leu Phe Val Phe Ser Thr Ser
Phe Tyr Leu Ile Ser Ile Phe Leu His 435 440 445Leu Val Lys Ile Pro
Thr His Arg His Ile Val Gly Lys Ser Cys Pro 450 455 460Lys Pro His
Arg Leu Asn His Met Gly Ile Cys Ser Cys Gly Leu Tyr465 470 475
480Lys Gln Pro Gly Val Pro Val Lys Trp Lys Arg 485 4903569PRTLassa
virus 3Met Ser Ala Ser Lys Glu Ile Lys Ser Phe Leu Trp Thr Gln Ser
Leu1 5 10 15Arg Arg Glu Leu Ser Gly Tyr Cys Ser Asn Ile Lys Leu Gln
Val Val 20 25 30Lys Asp Ala Gln Ala Leu Leu His Gly Leu Asp Phe Ser
Glu Val Ser 35 40 45Asn Val Gln Arg Leu Met Arg Lys Glu Arg Arg Asp
Asp Asn Asp Leu 50 55 60Lys Arg Leu Arg Asp Leu Asn Gln Ala Val Asn
Asn Leu Val Glu Leu65 70 75 80Lys Ser Thr Gln Gln Lys Ser Ile Leu
Arg Val Gly Thr Leu Thr Ser 85 90 95Asp Asp Leu Leu Ile Leu Ala Ala
Asp Leu Glu Lys Leu Lys Ser Lys 100 105 110Val Ile Arg Thr Glu Arg
Pro Leu Ser Ala Gly Val Tyr Met Gly Asn 115 120 125Leu Ser Ser Gln
Gln Leu Asp Gln Arg Arg Ala Leu Leu Asn Met Ile 130 135 140Gly Met
Ser Gly Gly Asn Gln Gly Ala Arg Ala Gly Arg Asp Gly Val145 150 155
160Val Arg Val Trp Asp Val Lys Asn Ala Glu Leu Leu Asn Asn Gln Phe
165 170 175Gly Thr Met Pro Ser Leu Thr Leu Ala Cys Leu Thr Lys Gln
Gly Gln 180 185 190Val Asp Leu Asn Asp Ala Val Gln Ala Leu Thr Asp
Leu Gly Leu Ile 195 200 205Tyr Thr Ala Lys Tyr Pro Asn Thr Ser Asp
Leu Asp Arg Leu Thr Gln 210 215 220Ser His Pro Ile Leu Asn Met Ile
Asp Thr Lys Lys Ser Ser Leu Asn225 230 235 240Ile Ser Gly Tyr Asn
Phe Ser Leu Gly Ala Ala Val Lys Ala Gly Ala 245 250 255Cys Met Leu
Asp Gly Gly Asn Met Leu Glu Thr Ile Lys Val Ser Pro 260 265 270Gln
Thr Met Asp Gly Ile Leu Lys Ser Ile Leu Lys Val Lys Lys Ala 275 280
285Leu Gly Met Phe Ile Ser Asp Thr Pro Gly Glu Arg Asn Pro Tyr Glu
290 295 300Asn Ile Leu Tyr Lys Ile Cys Leu Ser Gly Asp Gly Trp Pro
Tyr Ile305 310 315 320Ala Ser Arg Thr Ser Ile Thr Gly Arg Ala Trp
Glu Asn Thr Val Val 325 330 335Asp Leu Glu Ser Asp Gly Lys Pro Gln
Lys Ala Asp Ser Asn Asn Ser 340 345 350Ser Lys Ser Leu Gln Ser Ala
Gly Phe Thr Ala Gly Leu Thr Tyr Ser 355 360 365Gln Leu Met Thr Leu
Lys Asp Ala Met Leu Gln Leu Asp Pro Asn Ala 370 375 380Lys Thr Trp
Met Asp Ile Glu Gly Arg Pro Glu Asp Pro Val Glu Ile385 390 395
400Ala Leu Tyr Gln Pro Ser Ser Gly Cys Tyr Ile His Phe Phe Arg Glu
405 410 415Pro Thr Asp Leu Lys Gln Phe Lys Gln Asp Ala Lys Tyr Ser
His Gly 420 425 430Ile Asp Val Thr Asp Leu Phe Ala Thr Gln Pro Gly
Leu Thr Ser Ala 435 440 445Val Ile Asp Ala Leu Pro Arg Asn Met Val
Ile Thr Cys Gln Gly Ser 450 455 460Asp Asp Ile Arg Lys Leu Leu Glu
Ser Gln Gly Arg Lys Asp Ile Lys465 470 475 480Leu Ile Asp Ile Ala
Leu Ser Lys Thr Asp Ser Arg Lys Tyr Glu Asn 485 490 495Ala Val Trp
Asp Gln Tyr Lys Asp Leu Cys His Met His Thr Gly Val 500 505 510Val
Val Glu Lys Lys Lys Arg Gly Gly Lys Glu Glu Ile Thr Pro His 515 520
525Cys Ala Leu Met Asp Cys Ile Met Phe Asp Ala Ala Val Ser Gly Gly
530 535 540Leu Asn Thr Ser Val Leu Arg Ala Val Leu Pro Arg Asp Met
Val Phe545 550 555 560Arg Thr Ser Thr Pro Arg Val Val Leu
565434DNAArtificial SequenceDNA Primer 4gcgacgcgta ccatgggaca
aatagtgaca ttct 34530DNAArtificial SequenceDNA Primer 5ggcggccgct
catctcttcc atttcacagg 3062031DNAEbola virus Zaire strain
6atgggcgtta caggaatatt gcagttacct cgtgatcgat tcaagaggac atcattcttt
60ctttgggtaa ttatcctttt ccaaagaaca ttttccatcc cacttggagt catccacaat
120agcacattac aggttagtga tgtcgacaaa ctagtttgtc gtgacaaact
gtcatccaca 180aatcaattga gatcagttgg actgaatctc gaagggaatg
gagtggcaac tgacgtgcca 240tctgcaacta aaagatgggg cttcaggtcc
ggtgtcccac caaaggtggt caattatgaa 300gctggtgaat gggctgaaaa
ctgctacaat cttgaaatca aaaaacctga cgggagtgag 360tgtctaccag
cagcgccaga cgggattcgg ggcttccccc ggtgccggta tgtgcacaaa
420gtatcaggaa cgggaccgtg tgccggagac tttgccttcc ataaagaggg
tgctttcttc 480ctgtatgatc gacttgcttc cacagttatc taccgaggaa
cgactttcgc tgaaggtgtc 540gttgcatttc tgatactgcc ccaagctaag
aaggacttct tcagctcaca ccccttgaga 600gagccggtca atgcaacgga
ggacccgtct agtggctact attctaccac aattagatat 660caggctaccg
gttttggaac caatgagaca gagtacttgt tcgaggttga caatttgacc
720tacgtccaac ttgaatcaag attcacacca cagtttctgc tccagctgaa
tgagacaata 780tatacaagtg ggaaaaggag caataccacg ggaaaactaa
tttggaaggt caaccccgaa 840attgatacaa caatcgggga gtgggccttc
tgggaaacta aaaaaaacct cactagaaaa 900attcgcagtg aagagttgtc
tttcacagtt gtatcaaacg gagccaaaaa catcagtggt 960cagagtccgg
cgcgaacttc ttccgaccca gggaccaaca caacaactga agaccacaaa
1020atcatggctt cagaaaattc ctctgcaatg gttcaagtgc acagtcaagg
aagggaagct 1080gcagtgtcgc atctaacaac ccttgccaca atctccacga
gtccccaatc cctcacaacc 1140aaaccaggtc cggacaacag cacccataat
acacccgtgt ataaacttga catctctgag 1200gcaactcaag ttgaacaaca
tcaccgcaga acagacaacg acagcacagc ctccgacact 1260ccctctgcca
cgaccgcagc cggaccccca aaagcagaga acaccaacac gagcaagagc
1320actgacttcc tggaccccgc caccacaaca agtccccaaa accacagcga
gaccgctggc 1380aacaacaaca ctcatcacca agataccgga gaagagagtg
ccagcagcgg gaagctaggc 1440ttaattacca atactattgc tggagtcgca
ggactgatca caggcgggag aagaactcga 1500agagaagcaa ttgtcaatgc
tcaacccaaa tgcaacccta atttacatta ctggactact 1560caggatgaag
gtgctgcaat cggactggcc tggataccat atttcgggcc agcagccgag
1620ggaatttaca tagaggggct aatgcacaat caagatggtt taatctgtgg
gttgagacag 1680ctggccaacg agacgactca agctcttcaa ctgttcctga
gagccacaac tgagctacgc 1740accttttcaa tcctcaaccg taaggcaatt
gatttcttgc tgcagcgatg gggcggcaca 1800tgccacattc tgggaccgga
ctgctgtatc gaaccacatg attggaccaa gaacataaca 1860gacaaaattg
atcagattat tcatgatttt gttgataaaa cccttccgga ccagggggac
1920aatgacaatt ggtggacagg atggagacaa tggataccgg caggtattgg
agttacaggc 1980gttataattg cagttatcgc tttattctgt atatgcaaat
ttgtctttta g 20317676PRTEbola virus Zaire strain 7Met Gly Val Thr
Gly Ile Leu Gln Leu Pro Arg Asp Arg Phe Lys Arg1 5 10 15Thr Ser Phe
Phe Leu Trp Val Ile Ile Leu Phe Gln Arg Thr Phe Ser 20 25 30Ile Pro
Leu Gly Val Ile His Asn Ser Thr Leu Gln Val Ser Asp Val 35 40 45Asp
Lys Leu Val Cys Arg Asp Lys Leu Ser Ser Thr Asn Gln Leu Arg 50 55
60Ser Val Gly Leu Asn Leu Glu Gly Asn Gly Val Ala Thr Asp Val Pro65
70 75 80Ser Ala Thr Lys Arg Trp Gly Phe Arg Ser Gly Val Pro Pro Lys
Val 85 90 95Val Asn Tyr Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn
Leu Glu 100 105 110Ile Lys Lys Pro Asp Gly Ser Glu Cys Leu Pro Ala
Ala Pro Asp Gly 115 120 125Ile Arg Gly Phe Pro Arg Cys Arg Tyr Val
His Lys Val Ser Gly Thr 130 135 140Gly Pro Cys Ala Gly Asp Phe Ala
Phe His Lys Glu Gly Ala Phe Phe145 150 155 160Leu Tyr Asp Arg Leu
Ala Ser Thr Val Ile Tyr Arg Gly Thr Thr Phe 165 170 175Ala Glu Gly
Val Val Ala Phe Leu Ile Leu Pro Gln Ala Lys Lys Asp 180 185 190Phe
Phe Ser Ser His Pro Leu Arg Glu Pro Val Asn Ala Thr Glu Asp 195 200
205Pro Ser Ser Gly Tyr Tyr Ser Thr Thr Ile Arg Tyr Gln Ala Thr Gly
210 215 220Phe Gly Thr Asn Glu Thr Glu Tyr Leu Phe Glu Val Asp Asn
Leu Thr225 230 235 240Tyr Val Gln Leu Glu Ser Arg Phe Thr Pro Gln
Phe Leu Leu Gln Leu 245 250 255Asn Glu Thr Ile Tyr Thr Ser Gly Lys
Arg Ser Asn Thr Thr Gly Lys 260 265 270Leu Ile Trp Lys Val Asn Pro
Glu Ile Asp Thr Thr Ile Gly Glu Trp 275 280 285Ala Phe Trp Glu Thr
Lys Lys Asn Leu Thr Arg Lys Ile Arg Ser Glu 290 295 300Glu Leu Ser
Phe Thr Val Val Ser Asn Gly Ala Lys Asn Ile Ser Gly305 310 315
320Gln Ser Pro Ala Arg Thr Ser Ser Asp Pro Gly Thr Asn Thr Thr Thr
325 330 335Glu Asp His Lys Ile Met Ala Ser Glu Asn Ser Ser Ala Met
Val Gln 340 345 350Val His Ser Gln Gly Arg Glu Ala Ala Val Ser His
Leu Thr Thr Leu 355 360 365Ala Thr Ile Ser Thr Ser Pro Gln Ser Leu
Thr Thr Lys Pro Gly Pro 370 375 380Asp Asn Ser Thr His Asn Thr Pro
Val Tyr Lys Leu Asp Ile Ser Glu385 390 395 400Ala Thr Gln Val Glu
Gln His His Arg Arg Thr Asp Asn Asp Ser Thr 405 410 415Ala Ser Asp
Thr Pro Ser Ala Thr Thr Ala Ala Gly Pro Pro Lys Ala 420 425 430Glu
Asn Thr Asn Thr Ser Lys Ser Thr Asp Phe Leu Asp Pro Ala Thr 435 440
445Thr Thr Ser Pro Gln Asn His Ser Glu Thr Ala Gly Asn Asn Asn Thr
450 455 460His His Gln Asp Thr Gly Glu Glu Ser Ala Ser Ser Gly Lys
Leu Gly465
470 475 480Leu Ile Thr Asn Thr Ile Ala Gly Val Ala Gly Leu Ile Thr
Gly Gly 485 490 495Arg Arg Thr Arg Arg Glu Ala Ile Val Asn Ala Gln
Pro Lys Cys Asn 500 505 510Pro Asn Leu His Tyr Trp Thr Thr Gln Asp
Glu Gly Ala Ala Ile Gly 515 520 525Leu Ala Trp Ile Pro Tyr Phe Gly
Pro Ala Ala Glu Gly Ile Tyr Ile 530 535 540Glu Gly Leu Met His Asn
Gln Asp Gly Leu Ile Cys Gly Leu Arg Gln545 550 555 560Leu Ala Asn
Glu Thr Thr Gln Ala Leu Gln Leu Phe Leu Arg Ala Thr 565 570 575Thr
Glu Leu Arg Thr Phe Ser Ile Leu Asn Arg Lys Ala Ile Asp Phe 580 585
590Leu Leu Gln Arg Trp Gly Gly Thr Cys His Ile Leu Gly Pro Asp Cys
595 600 605Cys Ile Glu Pro His Asp Trp Thr Lys Asn Ile Thr Asp Lys
Ile Asp 610 615 620Gln Ile Ile His Asp Phe Val Asp Lys Thr Leu Pro
Asp Gln Gly Asp625 630 635 640Asn Asp Asn Trp Trp Thr Gly Trp Arg
Gln Trp Ile Pro Ala Gly Ile 645 650 655Gly Val Thr Gly Val Ile Ile
Ala Val Ile Ala Leu Phe Cys Ile Cys 660 665 670Lys Phe Val Phe
67581494DNAEbola virus Zaire strain 8atgggcgtta caggaatatt
gcagttacct cgtgatcgat tcaagaggac atcattcttt 60ctttgggtaa ttatcctttt
ccaaagaaca ttttccatcc cacttggagt catccacagt 120agcacattac
aggttagtga tgtcgacaaa ctagtttgtc gtgacaaact gtcatccaca
180aatcaattga gatcagttgg actgaatctc gaagggaatg gagtggcaac
tgacgtgcca 240tctgcaacta aaagatgggg cttcaggtcc ggtgtcccac
caaaggtggt caattatgaa 300gctggtgaat gggctgaaaa ctgctacaat
cttgaaatca aaaaacctga cgggagtgag 360tgtctaccag cagcgccaga
cgggattcgg ggcttccccc ggtgccggta tgtgcacaaa 420gtatcaggaa
cgggaccgtg tgccggagac tttgccttcc ataaagaggg tgctttcttc
480ctgtatgatc gacttgcttc cacagttatc taccgaggaa cgactttcgc
tgaaggtgtc 540gttgcatttc tgatactgcc ccaagctaag aaggacttct
tcagctcaca ccccttgaga 600gagccggtca atgcaacgga ggacccgtct
agtggctact attctaccac aattagatat 660caggctaccg gttttggaac
caatgagaca gagtacttgt tcgaggttga caatttgacc 720tacgtccaac
ttgaatcaag attcacacca cagtttctgc tccagctgaa tgagacaata
780tatacaagtg ggaaaaggag caataccacg ggaaaactaa tttggaaggt
caaccccgaa 840attgatacaa caatcgggga gtgggccttc tgggaaacta
aaaaaaacct cactagaaaa 900attcgcagtg aagagttgtc tttctctaga
gcaggactga tcacaggcgg gagaagaact 960cgaagagaag caattgtcaa
tgctcaaccc aaatgcaacc ctaatttaca ttactggact 1020actcaggatg
aaggtgctgc aatcggactg gcctggatac catatttcgg gccagcagcc
1080gagggaattt acatagaggg gctaatgcac aatcaagatg gtttaatctg
tgggttgaga 1140cagctggcca acgagacgac tcaagctctt caactgttcc
tgagagccac aactgagcta 1200cgcacctttt caatcctcaa ccgtaaggca
attgatttct tgctgcagcg atggggcggc 1260acatgccaca ttctgggacc
ggactgctgt atcgaaccac atgattggac caagaacata 1320acagacaaaa
ttgatcagat tattcatgat tttgttgata aaacccttcc ggaccagggg
1380gacaatgaca attggtggac aggatggaga caatggatac cggcaggtat
tggagttaca 1440ggcgttgtaa ttgcagttat cgctttattc tgtatatgca
aatttgtctt ttag 14949497PRTEbola virus Zaire strain 9Met Gly Val
Thr Gly Ile Leu Gln Leu Pro Arg Asp Arg Phe Lys Arg1 5 10 15Thr Ser
Phe Phe Leu Trp Val Ile Ile Leu Phe Gln Arg Thr Phe Ser 20 25 30Ile
Pro Leu Gly Val Ile His Ser Ser Thr Leu Gln Val Ser Asp Val 35 40
45Asp Lys Leu Val Cys Arg Asp Lys Leu Ser Ser Thr Asn Gln Leu Arg
50 55 60Ser Val Gly Leu Asn Leu Glu Gly Asn Gly Val Ala Thr Asp Val
Pro65 70 75 80Ser Ala Thr Lys Arg Trp Gly Phe Arg Ser Gly Val Pro
Pro Lys Val 85 90 95Val Asn Tyr Glu Ala Gly Glu Trp Ala Glu Asn Cys
Tyr Asn Leu Glu 100 105 110Ile Lys Lys Pro Asp Gly Ser Glu Cys Leu
Pro Ala Ala Pro Asp Gly 115 120 125Ile Arg Gly Phe Pro Arg Cys Arg
Tyr Val His Lys Val Ser Gly Thr 130 135 140Gly Pro Cys Ala Gly Asp
Phe Ala Phe His Lys Glu Gly Ala Phe Phe145 150 155 160Leu Tyr Asp
Arg Leu Ala Ser Thr Val Ile Tyr Arg Gly Thr Thr Phe 165 170 175Ala
Glu Gly Val Val Ala Phe Leu Ile Leu Pro Gln Ala Lys Lys Asp 180 185
190Phe Phe Ser Ser His Pro Leu Arg Glu Pro Val Asn Ala Thr Glu Asp
195 200 205Pro Ser Ser Gly Tyr Tyr Ser Thr Thr Ile Arg Tyr Gln Ala
Thr Gly 210 215 220Phe Gly Thr Asn Glu Thr Glu Tyr Leu Phe Glu Val
Asp Asn Leu Thr225 230 235 240Tyr Val Gln Leu Glu Ser Arg Phe Thr
Pro Gln Phe Leu Leu Gln Leu 245 250 255Asn Glu Thr Ile Tyr Thr Ser
Gly Lys Arg Ser Asn Thr Thr Gly Lys 260 265 270Leu Ile Trp Lys Val
Asn Pro Glu Ile Asp Thr Thr Ile Gly Glu Trp 275 280 285Ala Phe Trp
Glu Thr Lys Lys Asn Leu Thr Arg Lys Ile Arg Ser Glu 290 295 300Glu
Leu Ser Phe Ser Arg Ala Gly Leu Ile Thr Gly Gly Arg Arg Thr305 310
315 320Arg Arg Glu Ala Ile Val Asn Ala Gln Pro Lys Cys Asn Pro Asn
Leu 325 330 335His Tyr Trp Thr Thr Gln Asp Glu Gly Ala Ala Ile Gly
Leu Ala Trp 340 345 350Ile Pro Tyr Phe Gly Pro Ala Ala Glu Gly Ile
Tyr Ile Glu Gly Leu 355 360 365Met His Asn Gln Asp Gly Leu Ile Cys
Gly Leu Arg Gln Leu Ala Asn 370 375 380Glu Thr Thr Gln Ala Leu Gln
Leu Phe Leu Arg Ala Thr Thr Glu Leu385 390 395 400Arg Thr Phe Ser
Ile Leu Asn Arg Lys Ala Ile Asp Phe Leu Leu Gln 405 410 415Arg Trp
Gly Gly Thr Cys His Ile Leu Gly Pro Asp Cys Cys Ile Glu 420 425
430Pro His Asp Trp Thr Lys Asn Ile Thr Asp Lys Ile Asp Gln Ile Ile
435 440 445His Asp Phe Val Asp Lys Thr Leu Pro Asp Gln Gly Asp Asn
Asp Asn 450 455 460Trp Trp Thr Gly Trp Arg Gln Trp Ile Pro Ala Gly
Ile Gly Val Thr465 470 475 480Gly Val Val Ile Ala Val Ile Ala Leu
Phe Cys Ile Cys Lys Phe Val 485 490 495Phe
* * * * *