U.S. patent application number 12/004024 was filed with the patent office on 2008-05-08 for hybrid or chimeric polynucleotides, proteins, and compositions comprising hepatitis b virus sequences.
This patent application is currently assigned to INSTITUT PASTEUR. Invention is credited to Huseyin Firat, Pierre Langlade-demoyen, Francois Lemonnier, Marie-Louise Michel, Andreas A. Suhrbier.
Application Number | 20080107693 12/004024 |
Document ID | / |
Family ID | 22561753 |
Filed Date | 2008-05-08 |
United States Patent
Application |
20080107693 |
Kind Code |
A1 |
Firat; Huseyin ; et
al. |
May 8, 2008 |
Hybrid or chimeric polynucleotides, proteins, and compositions
comprising hepatitis B virus sequences
Abstract
H-2 class I negative, HLA-A2.1 transgenic HHD mice were used for
a comparative evaluation of the immunogenicity of HLA-A2.1
restricted human tumor-associated CTL epitopes. A hierarchy was
established among these epitopic peptides injected into mice in IFA
which correlates globally with their capacity to bind and stabilize
HLA-A2.1 molecules. Co-injection of a helper peptide enhanced most
CTL responses. In contrast, classical HLA class I transgenic mice
which still express their own class I molecules did not, in most
cases, develop H.A.-A2.1-restricted CTL responses under the same
experimental conditions. Different monoepitopic immunization
strategies of acceptable clinical usage were compared in HHD mice.
Recombinant Ty-virus-like particles, or DNA encoding epitopes fused
to the hepatitis B virus middle envelope protein gave the best
results. Using this latter approach and a melanoma-based
polyepitope construct, CTL responses against five distinct epitopes
could be elicited simultaneously in a single animal. Thus, HHD mice
provide a versatile animal model for preclinical evaluation of
peptide-based cancer immunotherapy.
Inventors: |
Firat; Huseyin; (Paris,
FR) ; Lemonnier; Francois; (Bourg-la-Reine, FR)
; Langlade-demoyen; Pierre; (Paris, FR) ; Michel;
Marie-Louise; (Paris, FR) ; Suhrbier; Andreas A.;
(Bunya, AU) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
INSTITUT PASTEUR
|
Family ID: |
22561753 |
Appl. No.: |
12/004024 |
Filed: |
December 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10388337 |
Mar 14, 2003 |
7345026 |
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12004024 |
Dec 20, 2007 |
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09671198 |
Sep 28, 2000 |
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10388337 |
Mar 14, 2003 |
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60156945 |
Sep 30, 1999 |
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Current U.S.
Class: |
424/277.1 ;
435/320.1; 514/44R; 536/23.72 |
Current CPC
Class: |
A61P 37/04 20180101;
A61K 2039/5258 20130101; A61K 39/001186 20180801; A61K 39/0011
20130101; A61K 48/00 20130101; C07K 2319/00 20130101; C12N
2730/10122 20130101; A61K 39/12 20130101; C12N 2730/10134 20130101;
A61K 39/001106 20180801; A61K 39/001191 20180801; A61K 39/292
20130101; C07K 14/005 20130101; A61K 39/001192 20180801; A61K
39/001151 20180801; C07K 2319/40 20130101; A61P 35/00 20180101;
A61K 39/001182 20180801; A61K 2039/57 20130101; A61K 39/001156
20180801 |
Class at
Publication: |
424/277.1 ;
536/023.72; 514/044; 435/320.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07H 21/04 20060101 C07H021/04; A61K 31/7088 20060101
A61K031/7088; A61P 37/04 20060101 A61P037/04; A61P 35/00 20060101
A61P035/00; C12N 15/63 20060101 C12N015/63 |
Claims
1. An isolated or purified polynucleotide containing at least: a
part of the coding sequence of the middle glycoprotein of the
hepatitis B virus (HBV), in which is inserted a DNA sequence coding
for at least one tumor epitope of a tumor antigen.
2. The polynucleotide according to claim 1, wherein the part of the
coding sequence of the middle glycoprotein of HBV is a part of the
preS2 sequence of HBV.
3. The polynucleotide according to claim 2, the DNA sequence coding
for at least one tumor epitope of a tumor antigen is a DNA sequence
coding for the surface antigen of HBV.
4. The polynucleotide according to claim 3, wherein the
polynucleotide comprises DNA sequences encoding from 1 to 30,
inclusive, epitopes, wherein none, some, or all of the epitopes are
the same.
5. The polynucleotide according to claim 1, wherein the
polynucleotide comprises DNA sequences encoding from 1 to 30,
inclusive, epitopes, wherein none, some, or all of the epitopes are
the same.
6. The polynucleotide according to claim 5, wherein one or more of
the epitopes are mutated due to a mutation in the DNA encoding the
epitopes.
7. A composition containing the polynucleotide according to claim
1, wherein expression of at least part of the polynucleotide in
vivo induces an immune response against at least one tumor specific
antigen or tissue specific antigen.
8. A vector comprising an early CMV promoter, preS2 and S
nucleotide sequences encoding preS2 and S antigens of HBV;
nucleotide sequences from the genome HBV containing
post-transcriptional regulatory elements (PREs) that direct nuclear
export of RNA corresponding to nucleotide 1,151 to nucleotide 1,684
of the HBV genome; signal sequences for polyadenylation of
messenger RNAs of HBV located at position nucleotide 1,921 to
nucleotide 1,955 of the HBV genome; and nucleotide sequences of
tumor epitopes surrounded up and down by alanine spacers.
9. The vector according to claim 8, wherein the vector, when
administered to an individual, induces an in vivo cellular
or/humoral immune response.
10. The vector according to claim 8, further comprising nucleotide
sequences encoding a B cell epitope fused to at least one tumor
sequence, wherein the B cell epitope sequences allow the detection
of hybrid proteins.
11. A process for treating an individual in vivo, said process
comprising: constructing a polynucleotide containing at least a
part of the coding sequence of the middle glycoprotein of the
hepatitis B virus (HBV), in which is inserted a DNA sequence coding
for at least one tumor epitope of a tumor antigen; injecting the
polynucleotide into an individual; and optionally, evaluating the
cytotoxic responses in the individual's lymphocyte population.
12. A composition comprising a hybrid protein, wherein the hybrid
protein comprises a preS2-S protein sequence and at least sequence
of at least one tumor antigen or epitope.
13. The composition according to claim 12, wherein the composition
induces, in vivo, a CTL response against one or more of the
antigens or epitopes present on the hybrid protein.
14. The composition according to claim 12 wherein the hybrid
protein further comprises a tag B cell epitope.
15. Recombinant particles comprising the composition according to
claim 12 and the small envelope protein of HBV.
16. A process of treating cells of a host, said process
characterized by contacting the recombinant particles according to
claim 15 with the host's cells to create treated cells; and
optionally, injecting the treated cells into the host.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application depends on, and claims the benefit of, the
filing date of U.S. Provisional Application Ser. No. 60/156,945,
filed Sep. 30, 2000, the entire disclosure of which is hereby
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to polynucleotides and vectors, to
compositions containing the polynucleotides and vectors, to
polypeptides and polypeptide compositions, and to their use.
[0004] 2. Description of Related Art
[0005] CTL-mediated protection against tumors has been documented
in mouse experimental models (1). In view of our refined
comprehension of the molecular structures recognized by CTL, the
search for human tumor-derived CTL epitopes has been undertaken in
many laboratories, as best exemplified for melanomas (2).
Subsequently, clinical trials using peptide-based immunization
protocols provided encouraging results (3). However, the selection
of peptide(s) and vaccine strategy remain difficult in view of the
number of candidate peptides and variety of immunization
strategies. An animal model allowing a controlled evaluation of the
immunogenic potential of the epitopic peptides and of the
immunization strategies would be of interest before human
immunotherapeutic trials.
[0006] Classical H.A. class I transgenic mice (which still express
their own H-2 class I molecules) have been derived for such
purposes (4). However, unless the third domain of the human
molecules was substituted with the corresponding mouse domain, the
peripheral CTL repertoire of these mice was inefficiently mobilized
by the transgenic molecules due to poor interaction with mouse CD8
molecules (5). Accordingly, improved usage of H.A. class I
molecules has been documented in transgenic strains expressing
chimeric constructs (a1, a2 human, and a3 mouse) and exploited for
the study of CTL responses against certain viral and tumor epitopic
peptides (6, 7). Nevertheless, in such mice we observed a profound
bias in favor of H-2 restricted CTL responses (H. Firat,
unpublished observations). To circumvent that bias, we derived a
strain of mice in which the H-2 D.sup.b and mouse b2-microglobulin
(.beta.2m) genes have been disrupted and which expresses a chimeric
(a1, a2 human, and a3 mouse) HLA-A2.1 heavy chain covalently linked
to human b2m light chain. We named this chimeric molecule the HHD
molecule. In HHQ transgenic mice, the transgenic molecules are the
only class I molecules serologically detectable on cell surfaces
and are used efficiently by CTL in responses against viruses (8).
We report here the use of these mice to compare the immunogenic
potential of H.A.-A2.1-restricted human tumor-associated CTL
epitopes and different strategies of immunization.
SUMMARY OF THE INVENTION
[0007] Accordingly, this invention provides polynucleotides
containing at least part of the coding sequence of the middle
glycoprotein of the hepatitis B virus in which is inserted a DNA
sequence coding for an epitope comprising at least one tumoral
epitope of a tumor antigen.
[0008] This invention also provides a polynucleotide containing at
least a part of the preS2 sequence of the genome of HBV, in which
is inserted a DNA sequence coding for an epitope comprising at
least one tumor epitope of a tumor antigen; and a nucleotidic
sequence coding for the surface antigen of HBV. The epitope can
comprise 1 to 30 epitopes, identical or different, and in a wild
type or in a mutated configuration.
[0009] This invention also provides a composition containing the
polynucleotide sequence of the invention for inducing in vivo an
immune response against tumor specific antigens or tissue specific
antigens.
[0010] This invention also provides a vector for induction of an in
vivo cellular or humoral immune response using the polynucleotide
of the invention and an early CMV promoter, preS2 and S nucleotide
sequences encoding preS2 and S antigens of HBV; nucleotide
sequences derived from the genome of HBV containing
postranscriptional regulatory elements (PRE) and allowing nuclear
export of RNA corresponding to nt 1151 to nucleotide 1684 of the
HBV genome; and signal sequences for polyadenylation of messenger
RNAs of HBV located at position nt 1921 to nt 1955 of the HBV
genome; and nucleotide sequences of tumor epitopes surrounded up
and down by alanine spacers. The vector can also have nucleotide
sequences encoding a B cell epitope, which allows the detection of
the hybrid proteins, said B cell epitope sequence being fused to
the tumor sequences.
[0011] In addition, this invention provides a process of in vivo
treatment, characterized by construction of a recombinant or
synthetic polynucleotide sequence according to the invention;
injection of the composition according to the invention to a host;
and evaluation of cytotoxic response in host lymphocyte
population.
[0012] Further, this invention provides a composition comprising a
hybrid preS2-S protein containing tumor antigens (or epitope)
capable of inducing, in vivo, a CTL response against several
epitopes of one or more tumor antigens. The hybrid proteins in the
composition can also contain a tag B cell epitope.
[0013] Further, this invention provides a recombinant particle
comprising this composition and the small envelope protein of
HBV.
[0014] Further, this invention provides a process of treatment
cells of a host characterized by contacting the recombinant
particles of the invention with the host's cells, and reinjection
of treated cells into the host.
[0015] The PRE sequences are published in J. Virology 1996, pp.
4345-4351 (Donello et al.), the entire disclosure of which is
relied upon and incorporated by reference herein. The cell epitope
has a minimum of 5 amino acids. The CTL epitope has a minimum of 9
amino acids. The T helper epitope has a minimum of 12 amino
acids.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] This invention will be described in detail with reference to
the drawings in which:
[0017] FIG. 1: Representation of the pCMV-B10 constructs and CTL
responses of HHD mice injected with pCMV-B10 recombinant DNA coding
a melanoma-based polyepitope.
[0018] A. Monoepitopic Constructs. The pCMV-B10 vector is a pcDNA3
derivative (INVITROGEN, Costa Mesa, Calif.) in which the largely
overlapping coding and totally overlapping 3' untranslated
nucleotide sequences for hepatitis B virus middle (and, initiation
at ATG 900, termination at TAA 1744) and small (initiation at ATG
1066, termination at TAA 1744) envelope proteins have been inserted
downstream of a human CMV immediate early promotor (P.sub.CMV). The
central part of the coding sequence for the preS2 segment was
replaced by a polylinker and the coding sequence for a HIV
1-derived (MN isolate) V3 loop tag. The EcoRI and XhoI restriction
sites were used to insert oligonucleotides coding for the selected
epitopes.
[0019] Amino acid sequence of the modified preS2 segment:
MQWNSAAA[epitope]AAA LEHIGPGRAFVVPLEEAWDPSRIGDPALNM (SEQ ID NO:1).
The residual preS2 residues are in bold characters and the V3 loop
tag residues in italics. The shaded area corresponds to the
introduced epitopic peptide with alanine spacers, other residues
originate from the residual polylinker nucleotides except the C
terminal methionine, which corresponds to position 1 of the
hepatitis B virus small envelope protein.
[0020] B. Amino-acid sequence of the melanoma polyepitope.
[0021] C. CTL responses against HHD-transfected RMAS cells. Spleen
cells of immunized mice were restimulated in vitro as indicated in
the legend of Table 4. On day 6, cells were assayed against targets
loaded with relevant (i.e., (NA17-A.nt38); MelanA/MART-1.27;
gp100.154; gp100.457; Tyrosinase.368-D) and control inf.m.58
peptides. No specific lysis was obtained for MAGE-3.271,
Tyrosinase. 1,gp100.209, gp100.280, Melan-A/MART-1.32 epitopic
peptides.
[0022] D. CTL responses against HHD-transfected HeLa target cells
producing endogeneously the melanoma polyepitope.
[0023] FIG. 2 is a graph, in two parts, depicting percents specific
lysis vs. E/T ratios.
[0024] FIG. 3 is a plasmid map of plasmid pCMV-B10, which is useful
in this invention.
DETAILED DESCRIPTION OF THE INVENTION
[0025] In one embodiment, this invention is related to the
construction of a recombinant DNA containing 10 melanoma epitopes
inserted in a plasmid (pCMV-B10), which expressed also the pre-S2
and HIBS Ag. More generally, the invention relates to new
recombinant HBS particles and nucleotidic sequences useful for the
in vivo antitumoral therapy, immunization against tumoral antigens
with a nucleotidic sequence or a vector encoding tumor epitopes and
preS or preS2-S or S antigen of HBV. The invention relates also to
the new particles comprising S antigen of HBV preS2-S and tumoral
peptides (or antigens tumoral).
[0026] The immunization of mice with this naked DNA elicited a very
good CTL response against the melanoma polyepitope. The plasmid
pCMV-B10 (FIG. 3) is derived from the plasmid pCMVHB-S2-S
(CNCMI-1410) described in the PCT/FR 9400483 (WO 95-11307)
corresponding to U.S. application Ser. No. 633,821 filed on Apr.
27, 1994, the entire disclosures of which are relied upon and
incorporated by reference herein.
[0027] The HIV1-V3 Loop inserted between nt 1011 and nt 985 is used
as a marker of expression. An insert of particular interest (in
case of melanoma) is located at the XhoI site (nt 979) and no 915
as shown in FIG. 1. The HIV1-V3 loop can be replaced by any B cell
epitope, which can be visualized. The size of the insert in this
plasmid is very flexible.
[0028] The polyepitope has been published by Firat et al. in Eur.
Journal of Immunology, 1999, 29:1-6, the entire disclosure of which
is relied upon and incorporated by reference herein, but the
expression is obtained in a recombinant poxvirus vaccine, and the
poxvaccine vectors are less safe than pre S2-S HBV particles.
(Described in U.S. Pat. Nos. 5,314,808 and 5,591,638). A
construction with the vector pCMV-S2-S and V3 loop of gp120 of
HIV-1 has been published in 1998 (Virology, 1998, 240:304-315), the
entire disclosure of which is relied upon and incorporated by
reference herein. The details for the construction of pCMV-B10 are
disclosed in the claims herein. Also relevant to the invention is
the hepatitis B virus post transcriptional regulatory element
composed of two sub-elements described in J. Virol. 70:4345-4351
(July 1996), the entire disclosure of which is relied upon and
incorporated by reference herein.
[0029] The possibility to introduce, without real size limitations,
DNA inserts in the preS2 segment of the middle hepatitis B
glycoprotein coding sequence will be exploited to further insert
epitopes presented by other HLA class I alleles.
[0030] The two first selected (HLA-A3.1, Embo. J. 1984, 3:887-894,
and H.A.-B7.2, PNAS, 1990, 87:2833-2837) should result in
polyepitopes of vaccinal interest for more than 80% of the
Caucasian human population.
[0031] More particularly, the inventors used H-2 class I negative
HLA-A2.1 transgenic HHD mice to evaluate the immunogenic potential
of 19 human tumor-associated CTL epitopes and to compare different
immunization strategies. A parallel study of the CTL responses of
H-2 positive A2A2K.sup.b classical transgenic mice illustrated the
improved capacity of HHD transgenic mice to develop
H.A.-A2.1-restricted CTL responses. This advantage was previously
documented by analyzing antiviral responses (8) but could have
resulted from the preferential development in classical transgenic
mice of H-2-restricted CTL responses against other viral epitopes.
This explanation cannot apply to the responses induced by
H.A.-A2.1-restricted synthetic peptides. The present results
suggest that the size of the H.A.-A2.1-educated CD8.sup.+
peripheral T cell repertoire is larger in HHD than in A2A2 K.sup.b
transgenic mice, in spite of a 10-fold lower cell surface
expression of the transgenic HHD molecules and 5-fold lower
CD8.sup.+ T cell number in the periphery (data not shown). The HHD
and A2A2K.sup.b molecules being functionally equivalent in terms of
antigen presentation (8), we have to postulate that in classical
transgenic mice, the co-expression of the A2A2 K.sup.b and H-2
class I molecules at the thymic level results in preferential
H-2-education. This could be due to the fact, documented for
HLA-A2.1 molecules, that residues in the second domain of the heavy
chain and some of .beta.2m, participate in the CD8 accessory
interaction (23). Assuming the affinity of the mouse CD8 molecules
to remain higher for H-2 than for chimeric A2A2K.sup.b class I
molecules, and the thymic education to be a saturable process, one
can conceive the quantitative advantage, in terms of HLA education,
that would result from the absence of H-2 class I molecules.
[0032] Immunizing HHD mice with human tumor-derived CD8 epitopic
pepticles alone in IFA, lead us to devise a hierarchy which
correlates, although imperfectly, with their binding and
stabilizing capacities of HLA-A2.1 molecules. One might wonder
whether this hierarchy also applies to humans. It appears unlikely
that TcR differences between human and mouse could be of any
significant influence considering the huge diversity of the T cell
repertoire in both species and the absence of species-specific
structural features in the variable TcR segments (24).
Self-tolerance and accumulated phylogenic protein differences are
more likely to modulate in a species specific manner this epitopic
hierarchy. However, the human genetic polymorphism should also, to
some extent, have the same consequences at the individual level.
Using antigenic formulations which need to be processed, such as
Ty-VLP and recombinant HBs particles, one should finally consider
the possibility that the cell processing machinery of mouse and man
could be functionally different. One such difference concerns the
TAP pumps, the human one transporting more efficiently than its
mouse counterpart peptides with positively charged C-terminal
residues (25). This is of no relevance for H.A.-A2.1 transgenic
mice, since this molecule binds peptides with leucine or methionine
C-termini which are efficiently transported by the mouse TAP pump
(26). In fact, so far, all reported observations, except those
concerning TAP, suggest a large functional redundancy between the
mouse and human processing machineries (6, 27).
[0033] The potent CTL responses induced either by Ty-VLP or
recombinant pCMV-B10 (HBs) DNA were anticipated. Particulate
antigens, which should also be released in the organism after
intramuscular injection of the recombinant HBs DNA, are good
immunogens (41). Endosomal processing of the p1 and HBs proteins
included in these particulate antigens might also result in the
production of helper peptides which facilitate the development of
cytotoxic responses. Of special interest, was the possibility of
simultaneously inducing, in a single mouse, CTL responses against 5
different peptides included in the melanoma-based polyepitopic
construct. This should compensate for the differences in expression
of the molecules of immunological interest among melanomas (9) and
reduce the risk of tumor escape. Among the 5 epitopic peptides for
which CTL responses were documented, three (gp100.154, NA17-A.nt38,
and MelanA/MART-1.27) are of special interest since they are
expressed in 40, 50, and 40% respectively, of melanomas, with the
expression of the NA17-A.nt38 epitope restricted to malignant
cells. The 5 epitopes which did not induce CTL responses after
polyepitopic immunization are poor binders and poor stabilizers.
Modifications are currently tested to enhance their binding and
stabilizing capacities and HHD mice are used to verify that the CTL
responses they induce cross-recognize the wild-type epitopes.
[0034] The weakness of the CTL responses induced by peptide-loaded
dendritic cells generated in vitro, was unexpected. This strategy
has been documented as very efficient in many situations (28, 29,
30). One explanation could be that HHD mice are not congenic. Minor
histocompatibility antigen differences could result in rapid
destruction of the injected cells by the recipient mice.
Backcrosses are underway to reach a B6 homogeneous genetic
background and evaluate such a possibility. Such B6 congenic HHD
mice would also provide us with the possibility to evaluate, using
EL4 .beta.2m negative HHD.sup.+ S3.sup.-Rob transfectants (8), the
protection conferred by the elicited CTL responses.
EXAMPLES
[0035] This invention will be described in greater detail in the
following examples, which are exemplary only, and do not in any way
limit the scope of the invention.
Materials and Methods
Mice
[0036] HHD mice express a transgenic monochain histocompatibility
class I molecule in which the C terminus of the human .beta.2m is
covalently linked to the N terminus of a chimeric heavy chain
(HLA-A2.1 .alpha.1-.alpha.2, H-2 D.sup.b .alpha.3-transmembrane,
and intracytoplasmic domains). The H-2D.sup.b and mouse .beta.2m
genes of these mice have been disrupted by homologous recombination
resulting in complete lack of serologically detectable cell surface
expression of mouse histocompatibility class I molecules.
A2A2K.sup.b mice were obtained from HARLAN SPRAGUE DAWLEY
(Indianapolis, Ind.). These mice express chimeric heavy chain
(HLA-A2.1 .alpha.1.alpha.2, H-2k.sup.b .alpha.3 transmembrane, and
cytoplasmic domains) in non-covalent association with mouse
.beta.2m. They additionally express a full set of C57BI/6 derived
(H-2b) class la and lb mouse histocompatibility molecules. All mice
used were bred in our animal facility.
Peptides, Lipopeptides and Immunization Procedures
[0037] Peptides, purchased from either NEOSYSTEM (Strasbourg,
France) or SYNT:EM (Nimes, France), were dissolved in
dimethylsulfoxide (DMSO, 20 .mu.l/mg of peptide) and subsequently
diluted in PBS 1.times. (2 mg/ml). Mice were injected
subcutaneously at the base of the tail with 100 .mu.g of a
H.A.-A2.1-restricted peptide, with or without 140 .mu.g of the
helper peptide, emulsified (v/v) in IFA (DIFCO, Detroit, Mich.) 7
days before in vitro re-stimulation. Lipopeptides were synthesized
as already described (21) resulting in covalent linkage of the
peptide N terminus to a S-[2.3
palmitoyloxy-(2-R)-propyl]-N-palmitoyl-(R)-Cysteine moiety (P3C)
via a two serine spacer. Lipopeptides were dissolved in DMSO (20
.mu.l/mg), then diluted in PBS 1.times. (2 mg/ml). One hundred
.mu.g were injected i.p. 2 weeks before in vitro restimulation.
Recombinant HBs DNA Constructs and Immunization Procedure
[0038] Synthetic complementary oligonucleotides corresponding to
the selected T cell epitopes were individually inserted into the
pre-S2 segment of the hepatitis B surface (HBs) middle protein
using a pCMV-B10 mammalian cell-expression vector (FIG. 1A) (20).
Each epitope was flanked on both sides by a 3 alanine spacer. A
HIV-1 derived V3 loop tag was inserted in the pre-S2 sequence just
after the C-terminal alanine spacer. Recombinant plasmids were
purified on LPS-free QIAGEN columns (QIAGEN, Hilden, Germany). Mice
were injected i.m. with 10 .mu.M cardiotoxin (LATOXAN, Rosans,
France) in 50 .mu.l PBS 1.times. and, 5 days later, with 50 .mu.g
of pCMV-B10 DNA for a 21 day priming.
Recombinant Ty-VLP and Immunization Procedure
[0039] Construction and purification of recombinant Ty-VLP were
performed as previously described using a pOGS40 yeast-expression
vector (31). PCR-amplified oligonucleotides corresponding to the
selected epitopes were produced from the recombinant pCMV-B10
constructs, in order to include the two 3 alanine spacers and the
HIV 1-derived V3 loop tag. They were introduced in frame in a BamHI
site at the 3' end of the coding sequence of the Ty p1 protein.
Ty-VLP and purification were monitored by western blotting using
mAb F5.5 against the V3 loop tag (HYBRIDOLAB, Institut Pasteur,
Paris, France). The hybrid Ty-VLPs were injected (100 .mu.g/mouse)
s.c. into mice for a 14 day in vivo immunization.
Polyepitopic Recombinant HBs DNA Construct
[0040] A DNA sequence encoding 10 melanoma-derived
H.A.-A2.1-restricted CTL epitopes (FIG. 1B) was amplified by PCR
(Mateo L, in preparation). The polyepitope sequence was inserted in
frame between the EcoRI-XhoI sites of the pCMV-B10 expression
vector. Immunizations with naked DNA and in vitro restimulations
were performed as described above, except that 10% TCGF was added
to the culture medium for the last two days of culture.
Generation of Dendritic Cells and Immunization Procedure
[0041] Bone marrow-derived dendritic cells were obtained as
previously described (32) with some modifications. Bone marrow
mononuclear cells were cultured in RPMI supplemented with 10% FCS,
2 mM L glutamine, 50 U/ml penicillin, 50 .mu.g/ml streptomycin, and
2-mercaptoethanol (complete RPMI medium), further supplemented with
20 ng/ml of recombinant mouse GM-CSF and 100 ng/ml recombinant
mouse IL4 (both from GENZYME, Cambridge, Mass.). On days 2 and 6,
non-adherent cells were removed, and fresh complete RPMI medium,
supplemented with 10 ng/ml mouse GM-CSF and 50 ng Mouse IL4, was
added. On day 7, the culture medium was replaced by complete RPMI
medium supplemented with 100 U/ml of mouse TNF.alpha.. Dendritic
cells, collected on day 9, were more than 95% pure (IA.sup.b+,
HHD.sup.+, CD3.sup.-, 33D1.sup.+, NDL145.sup.+, and CD 11c.sup.+)
as assessed with appropriate mAb. Those dendritic cells were loaded
with peptides (2.times.10.sup.6 cells/ml, 10 .mu.g/ml of peptides,
2 h at RT in FCS-free RPMI medium), then washed (.times.3) and
injected (1.times.10.sup.6 cells/mouse) i.v. into recipient mice
for in vivo priming 14 days before in vitro restimulation.
In Vitro Restimulation and Cytolytic Assays
[0042] Spleen cells from primed mice were restimulated using
irradiated (5000 rads) peptide-loaded (5.10.sup.6 cells/ml, 10
.mu.g/ml peptide, 2 h at RT in FCS-free RPMI medium), LPS-induced
(25 .mu.g/ml LPS, 7 .mu.g/ml dextran sulfate, in complete RPMI
medium, 48 h of culture) HHD lymphoblasts. On day 6, cultured cells
were tested in a 4 h .sup.51Cr-release assay, using as targets
HHD-transfected TAP.sup.- RMA-S cells loaded with relevant or
negative control influenza matrix 58-66 (Inf.m.58) peptides (10
.mu.g/ml, 5.10.sup.6 cells/ml, in FCS-free RPMI medium, 2 h at RT).
Specific lysis was calculated as follows: (experimental
release-spontaneous release)/total release-spontaneous
release).times.100.
Peptide Binding and Stabilization of H.A.-A2.1 Molecules
[0043] T2 (TAP.sup.-, H.A.-A2.1.sup.+) cells were incubated
overnight at 37.degree. C. (1.times.10.sup.6 cells/ml) in FCS-free
RPMI medium supplemented with 100 ng/ml of human .beta.2m (SIGMA,
St Louis, Mo.) in the absence (negative control) or presence of
either reference HIV 1 reverse transcriptase 476-484 (HIV 1 rt.476)
or tested peptides at various final concentrations (100, 10, 1, and
0.1 .mu.M). Following a 1 h incubation with brefeldine A (0.5
.mu.g/ml, SIGMA), T2 Cells were labeled (30 min, 4.degree. C.) with
a saturating concentration of anti-H.A.-A2.1 (BB7.2) mAb, then
washed twice. The cells were then incubated (30 min, 4.degree. C.)
with saturating concentration of FITC-conjugated goat IgG F(ab')2
anti-mouse Ig (CALTAG, South San Francisco, Calif.), washed
(.times.2), fixed in PBS 1.times., 1% paraformaldehyde and analyzed
using a FACs Calibur cytofluorometer (BECTON DICKINSON,
IMMUNOCYTOMETRY SYSTEMS, San Jose, Calif.). The mean intensity of
fluorescence (MIF), observed for each peptide concentration (after
subtraction of the MIF observed without peptide), was used as an
estimate of peptide binding. For each peptide, the concentration
needed to reach 20% of the maximal binding (as defined with HIV 1
rt.476 peptide) was calculated. Relative affinity (RA) is the ratio
of the concentrations of tested and HIV 1 rt.476 reference peptides
needed to reach this value. The lower the RA, the stronger the
binding. Stabilization assays were performed similarly. Following
initial evaluation of peptide binding (t0), cells were washed in
RPMI complete medium to remove free peptides and incubated, in the
continuous presence of brefeldine A (0.5 .mu.g/ml) for 2, 4, 6, and
8 h. The amount of stable peptide-HLA-A2.1 complexes was estimated,
as described above, by indirect immunofluorescence analysis. The
half-life of complexes (DC50) is the time required for a 50%
reduction of the t0 MIF value.
Example 1
Comparative Evaluation of the Immunogenic Potential of Human
Tumor-Derived CD8 Epitopic Peptides with HHD and A2A2 K.sup.b
Mice
[0044] Nineteen HLA-A2.1 restricted synthetic epitopic peptides (9,
10, 11, 12) listed in Table 1 were injected s.c. in IFA in at least
six H-2 negative HHD mice and six H-2 positive, A2A2K.sup.b
transgenic mice. Seven days later, spleen cells from each animal
were separately restimulated in vitro and then tested against
Transporter associated with Antigen Presentation (TAP)-deficient
HHD-transfected RMA-S peptide-loaded target cells.
[0045] Only 3 peptides elicited H.A.-A2.1-restricted CTL responses
in A2A2 K.sup.b mice whereas 12 did so in HHD mice (Table 2).
Considering the number of responding mice and the level of specific
lysis, a hierarchy could be devised with strong (gp100.154 and
CEA.571), intermediate (Tyrosinase.368-N, NA17-A.nt38, p53.65, and
Her2/neu.369), weak (gp100.209, gp100.280, gp100.476,
Melan-A/MART-1.27, Tyrosinase.368-D, MAGE-3.271, and Her2/neu.654),
and inefficient (gp100.457, Melan-A/MART-1.32, Tyrosinase.1,
p53.149, p53.264, and HPV E7.86) CTL inducers. TABLE-US-00001 TABLE
1 List of the epitopic peptides tested.sup.a Epitopic Protein
peptide Sequence Human melanoma gp100 154-162 KTWGQYWQV (SEQ ID NO:
2) 209-217 ITDQVPFSV (SEQ ID NO: 3) 280-288 YLEPGPVTA (SEQ ID NO:
4) 457-466 LLDGTATLRL (SEQ ID NO: 5) 476-485 VLYRYGSFSV (SEQ ID NO:
6) Melan-A/MART-1 27-35 AAGIGILTV (SEQ ID NO: 7) 32-40 ILTVILGVL
(SEQ ID NO: 8) Tyrosinase 1-9 MLLAVLYCL (SEQ ID NO: 9)
368-376-D.sup.b YMDGTMSQV (SEQ ID NO: 10) 368-376-N.sup.b YMNGTMSOV
(SEQ ID NO: 11) NA17-A nt38-64.sup.c VLPDVFIRC (SEQ ID NO: 12)
MAGE-3 271-279 FLWGPRALV (SEQ ID NO: 13) other human tumors CEA
571-579 YLSGANLNL (SEQ ID NO: 14) p53 65-73 RMPEAAPPV (SEQ ID NO:
15) 149-157 STPPPGTRV (SEQ ID NO: 16) 264-272 LLGRNSFEV (SEQ ID NO:
17) Her2/neu 369-377 XIFGSLAFL (SEQ ID NO: 18) 654-662 IISAVVGIL
(SEQ ID NO: 19) HPV16 E7 86-93 TLGIVCPI (SEQ ID NO: 20) Viruses
Inf. m 58-66 GILGFVFTL (SEQ ID NO: 21) HBVc 128-140 TPPAYRPPNAPIL
(SEQ ID NO: 22) HIV 1 rt 476-484 ILKEPVHGV (SEQ ID NO: 23)
.sup.aHuman melanoma and other tumor epitopic peptides have been
reviewed recently (9). Influenza matrix (Inf.m.58), hepatitis B
virus core (HBVc) and HIV 1 reverse transcriptase (rt) epitopic
peptides are from references 10-12 respectively. .sup.bAsparagine
370 being a glycosylation site, cytosolic deglycosylation results
in presentation to CTL of the 368-376 epitopic pepticle with a
Aspartate 370 residue. .sup.cEpitopic peptide corresponding to a
tumor-specific transcript initiated by a cryptic promotor and
resulting in the translation of intronic nucleotides (38 to 64) of
the N-Acetyl glucosaminyl-Transferase-V gene.
Example 2
HLA-A2.1 Binding and Stabilizing Capacities of the Epitopic
Peptides
[0046] The immunogenicity of CD8 epitopic peptides largely reflects
their binding and stabilizing capacities, with most of the strong
CTL-inducers being both good binders and stabilizers (13, 14).
Using TAP-deficient, H.A.-A2.1.sup.+ T2 cells, we evaluated these
parameters in an immunofluorescence assay as indicated in Materials
and Methods section.
[0047] The results shown in Table 2 demonstrate that as a rule,
strong and intermediate CTL-inducers fell into the strong binder,
strong stabilizer group (RA<3, DC50>4 h). There were,
however, exceptions, most of which concern peptides such as
MAGE-3.271, and p53.264 with high binding and stabilizing
capacities, but poor CTL-induction capacity. Thus, H.A.-A2.1
binding and stabilizing capacities of epitopic peptides correlate
well, but not completely, with peptide immunogenicity.
TABLE-US-00002 TABLE 2 CTL responses against tumor epitopic
peptides in IFA and H.A.-A2.1 peptide binding and stabilizing
capacities. HHD mice.sup.a A2A2Kb mice.sup.a Peptide R/T (lysis in
%).sup.b R/T (lysis in %).sup.b RA.sup.c D50(h).sup.d gp100.154 4/6
(39, 51, 57, 60) 5/6 (22, 34, 39, 44, 2.28 6-8 50) gp100.209 1/6
(23) 0/6 1.32 4 gp100.280 3/16 (13, 23, 47) 0/6 1.35 4 gp100.457
0/6 0/6 1.65 2-4 gp100.476 2/6 (54, 70) 1/6 (29) 10 4-6
Melan-A/MART-1.27 2/8 (15, 19) 0/6 2.16 4 Melan-A/MART-1.32 0/6 0/6
21.1 2-4 Tyrosinase.1 0/10 0/6 >60 2-4 Tyrosinase.368-D 1/6 (10)
0/6 2.27 >6 Tyrosinase.368-N 4/15 (12, 12, 20, 29) 0/6 2.2 >8
NA17-A.nt38 4/7 (15, 23, 25, 30) 0/6 1.52 >8 MAGE-3.271 1/6 (33)
0/6 0.91 6 CEA.57-1 6/6 (64, 67, 70, 71, 73, 0/6 2.8 >8 75)
p.53.65 4/6 (10, 12, 26, 60) 0/6 0.91 6-8 p53.149 0/6 0/6 36.6
<2 p53.264 0/7 0/6 2.09 6-8 Her2/neu.369 5/6 (12, 18, 22, 33,
39) 0/6 2.24 6-8 Her2/neu.654 1/6 (42) 0/6 11 4 HPV E7.88 0/6 2/6
(10, 13) 0.9 >8 .sup.aSpleen cells from mice injected s.c. with
peptide In IFA 7 days before were in vitro restimulated and assayed
6 days later against HHD-transfected RMA-S cells loaded with
relevant or control (inf.m.58) peptides. .sup.bR/T: responder
versus tested mice. Mice were considered as responders when at
least 10% specific lysis was observed. The values in parenthesis
correspond to the maximal lysis observed for each responder mouse,
usually at a 60:1 E/T ratio. .sup.cRelative affinity (RA) is the
ratio of the concentrations of tested versus reference peptides
needed to reach 20% of the maximal amount of stabilized molecules
as defined with high concentrations of reference peptide.
.sup.dHalf-life of stabilized peptide-HLA-A2.1 complexes (DC 50)
was evaluated following T2 cells and pepticle overnight incubation
by measuring the amount of residual cell surface peptide-HLA-A2.1
complexes at time intervals (0, 2, 4, 6, 8 h) using indirect
immunofluorescence and FACS analysis.
Example 3
Co-Immunization with Helper Hepatitis B Virus Core (HBVc 128-140)
Peptide
[0048] Whereas the sole immunization with class I-restricted
synthetic peptides of optimal size is sufficient for the induction
of CTL responses in some cases (15), the need for help has been
documented in other circumstances (16). Therefore, the CTL
responses of HHD mice which express H-2.sup.b class II molecules
were tested by co-injecting tumor-associated peptides and the
1A.sup.b- restricted HBVc.128 peptide (11).
[0049] Globally, all cytolytic responses were either induced or
improved, except in the case of the Her2/neu.654 peptide (Table 3).
Peptides of the weak and inefficient CTL-inducer groups (gp100.457,
Tyrosinase.1, and MAGE-3.271) elicited good CTL responses in a
large proportion of mice when co-injected with the helper peptide
and peptides of the strong or intermediate CTL-inducer group
(gp100.154, NA17-A.nt38, p53.65 and Her2/neu.368) elicited stronger
responses in a larger proportion of mice. However, one noticeable
exception, the CEA.571 peptide, turned out to be less immunogenic
when co-injected with the helper peptide. TABLE-US-00003 TABLE 3
CTL responses of HHD mice co-immunized in IFA with HBVC.128 helper
peptide.sup.a Peptide R/T.sup.b (lysis in %) gp100.154 9/11 (43,
59, 60, 64, 77. 77, 80, 82, 85) gp100.209 3/6 (31, 36, 56)
gp100.280 2/8 (12, 16) gp100.457 5/6 (14, 35, 43, 77, 79) gp100.476
6/7 (20, 22, 59, 63, 75, 79) Melan-A/MART-1.27 4/5 (10, 19, 20, 30)
Melan-A/MART-1.32 1/6 (10) Tyrosinase.1 5/6 (27, 33, 40, 42, 51)
Tyrosinase.368-N 5/12 (21, 36, 70, 72, 78) Tyrosinase.368-D 2/6
(13, 15) NA7-A.nt38 5/6 (36, 39, 61, 64, 71) MAGE-3.271 6/6 (34,
38, 59, 63, 64, 79) CEA.571 3/6 (20, 32, 46) p53.65 5/6 (29, 31,
40, 41, 55) p53.149 3/6 (20, 51, 71) p53.264 2/3 (37, 64)
Her2/neu.369 7/8 (21, 23, 25, 39, 40, 72, 75) Her2/neu.654 0/6 HPV
16 E7.86 2/6 (34, 48) .sup.aSpleen cells from mice, co-injected
s.c. with CD8 epitopic (50 .mu.g) and helper (140 .mu.g) peptides
in IFA 7 days before, were in vitro restimulated with
peptide-loaded LPS-lymphoblasts and assayed 6 days later at
different E/T ratios against HHD-transfected RMA-S target cells
loaded with relevant or control (Inf.m.58) peptides. .sup.bR/T,
responder versus tested mice (see table 2.sup.b)
Example 4
Comparison of Monoepitopic Immunization Strategies
[0050] Five peptides, one strong (CEA.571), two intermediate
(NA17-A.nt38, Tyrosinase.368-N), and two weak (gp100.280,
Tyrosinase.368-D) CTL-inducers were selected for this study. Four
immunization strategies were compared: peptide-loaded, in vitro
generated dendritic cells (17, 18), yeast-derived hybrid Ty-virus
like particles (VLP) (19), recombinant Hepatitis B surface (HBs)
middle protein encoding DNA (pCMV-B10-DNA) (20) and lipopeptides
(21). Following in vitro restimulation, spleen cells of HHD mice
were individually tested in a CTL assay.
[0051] Immunization with recombinant Ty-VLP and recombinant
pCMV-B10-DNA gave the best results with strong CTL responses
against CEA.571, and NA17-A.nt38 T cell epitopes in all animals
tested (Table 4). Some responses of weak to strong magnitude could
even be elicited against the weak CTL-inducers gp100.280 and
Tyrosinase.368-D. Immunization with lipopeptides resulted in CTL
responses but with large inter-individual differences.
Surprisingly, peptide-loaded dendritic cells (95% pure), gave poor
results. It is noteworthy that the weak CTL responses observed in
this latter case were clearly evidenced following a second in vitro
restimulation in the presence of IL2. TABLE-US-00004 TABLE 4 CTL
responses against tumor epitopic peptides in IFA and H.A.-A2.1
peptide binding and stabilizing capacities. Tyrosinase Tyrosinase
NA gp100.280 368-D lysis 368-N 17-A.nt38 CEA.571 Micc lysis in % in
% lysis in % lysis in % lysis in % Dendritic cells 1 0 (0) 7 (68)
15 (61) 6 (7) 2 (0) 2 0 (0) 23 (56) 0 (1) 0 (2) 4 (22) 3 0 (1) 12
(64) 0 (3) 0 (6) 4 (1) 4 0 (0) 20 (64) 0 (17) 3 (5) 1 (5) 5 0 (1) 8
(92) 0 (30) 5 (24) 1 (2) 6 0 (1) 3 (71) 8 (80) 3 (9) 0 (0) Ty-VLP 1
8 nd 0 55 56 2 3 nd 61 39 59 3 1 nd 58 50 51 4 8 nd 39 60 58 5 4 nd
29 59 46 6 18 nd 17 61 62 pCMV-B10 DNA 1 0 57 6 35 19 2 0 55 12 43
66 3 8 28 16 47 40 4 36 58 13 44 65 5 21 56 11 40 62 6 9 28 38 54
55 Lipopeptides 1 0 0 51 0 0 2 8 10 20 12 8 3 40 1 0 0 8 4 8 4 5 68
34 5 0 0 11 65 3 6 21 0 12 47 0 Spleen cells from mice previously
injected with either peptide-loaded dendritic cells differentiated
in vitro, on purified TY-VLP, on naked pCMV-B10 DNA encoding
recombinant HBs particles, or lipopeptides were restimulated in
vitro using peptide-loaded, irradiated LPS-lymphoblasts. Six days
later, they were assayed at different E/T ratios against
HHD-transfected RMAS cells loaded with relevant or control
(Inf.m.58) peptides. # The values correspond to the highest
specific lysis observed, usually at a 60/1 E/T ratio. ND: not done.
Numbers in parentheses: specific lysis following a second in vitro
restimulation of effector cells in the presence of 10% TCGF.
Example 5
Melanoma Polyepitopic Immunization
[0052] Using a polyepitope construct (22), we evaluated the
possibility of simultaneously inducing in a single mouse CTL
responses against several melanoma epitopes. Six HHD mice were
injected with pCMV-B10 DNA encoding recombinant preS2/S
glycoproteins containing a polyepitopic melanoma-derived motif
(FIGS. 1A and B). Following separate in vitro splenocyte
restimulation by each epitopic peptide, they were individually
assayed against peptide-loaded HHD-transfected RMA-S cells and
HHD-transfected human HeLa cells further transfected with a HIV-1
derived polyepitope expression vector (H. Firat, in
preparation).
[0053] Whether peptide-loaded cells or cells endogenously
expressing the polyepitopic construct were used as target, specific
CTL responses were regularly induced against 4 to 5 out of the 10
melanoma epitopes included in the polyepitopic motif (FIGS. 1C and
D). Strong responses were elicited against gp100.154 and
NA17-A.nt38 epitopic peptides classified as strong and intermediate
CTIL-inducers, respectively. Significant responses were observed
against gp100.45.sup.7 and Melan-A/MART.1.27 (strong CTL-inducers
when associated with a helper peptide). Tyrosinase.368-D or
gp100.209 also elicited CTL responses depending on mice. The 4
epitopes (gp100.280, gp100.476, Melan-A/MART.1.27,
Melan-A/MART-1.32, and Tyrosinase.1) which did not elicit CTL
responses all fall into the weak and non CTL-inducer groups when
administered as synthetic peptide in IFA with or without helper
peptide. In mice assayed 17 weeks after injection of the
polyepitope, 4 out of the 5 CTL responses could still be documented
(data not shown). This suggests that memory CTL can be elicited
using pCMV-B10 DNA polyepitope immunization.
[0054] Abbreviations used: .beta.2m, .beta.2-microglobulin; DC50,
Decay complexes 50 (half-life of peptide-HLA-A2.1 complexes); HBs
middle protein, Hepatitis B surface middle protein; RA, relative
affinity; TAP, Transporter associated with Antigen Presentation;
VLP, virus-like particles.
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Sequence CWU 1
1
25 1 8 PRT Hepatitis B virus 1 Met Gln Trp Asn Ser Ala Ala Ala 1 5
2 9 PRT Homo sapiens 2 Lys Thr Trp Gly Gln Tyr Trp Gln Val 1 5 3 9
PRT Homo sapiens 3 Ile Thr Asp Gln Val Pro Phe Ser Val 1 5 4 9 PRT
Homo sapiens 4 Tyr Leu Glu Pro Gly Pro Val Thr Ala 1 5 5 10 PRT
Homo sapiens 5 Leu Leu Asp Gly Thr Ala Thr Leu Arg Leu 1 5 10 6 10
PRT Homo sapiens 6 Val Leu Tyr Arg Tyr Gly Ser Phe Ser Val 1 5 10 7
9 PRT Homo sapiens 7 Ala Ala Gly Ile Gly Ile Leu Thr Val 1 5 8 9
PRT Homo sapiens 8 Ile Leu Thr Val Ile Leu Gly Val Leu 1 5 9 9 PRT
Homo sapiens 9 Met Leu Leu Ala Val Leu Tyr Cys Leu 1 5 10 9 PRT
Homo sapiens 10 Tyr Met Asp Gly Thr Met Ser Gln Val 1 5 11 8 PRT
Homo sapiens 11 Tyr Met Asn Gly Thr Met Ser Val 1 5 12 9 PRT Homo
sapiens 12 Val Leu Pro Asp Val Phe Ile Arg Cys 1 5 13 9 PRT Homo
sapiens 13 Phe Leu Trp Gly Pro Arg Ala Leu Val 1 5 14 9 PRT Homo
sapiens 14 Tyr Leu Ser Gly Ala Asn Leu Asn Leu 1 5 15 9 PRT Homo
sapiens 15 Arg Met Pro Glu Ala Ala Pro Pro Val 1 5 16 9 PRT Homo
sapiens 16 Ser Thr Pro Pro Pro Gly Thr Arg Val 1 5 17 9 PRT Homo
sapiens 17 Leu Leu Gly Arg Asn Ser Phe Glu Val 1 5 18 9 PRT Homo
sapiens 18 Lys Ile Phe Gly Ser Leu Ala Phe Leu 1 5 19 9 PRT Homo
sapiens 19 Ile Ile Ser Ala Val Val Gly Ile Leu 1 5 20 8 PRT Homo
sapiens 20 Thr Leu Gly Ile Val Cys Pro Ile 1 5 21 9 PRT Influenza
virus 21 Gly Ile Leu Gly Phe Val Phe Thr Leu 1 5 22 13 PRT
Hepatitis B virus 22 Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro
Ile Leu 1 5 10 23 9 PRT Human immunodeficiency virus type 1 23 Ile
Leu Lys Glu Pro Val His Gly Val 1 5 24 92 PRT Artificial Sequence
Description of Artificial Sequence AMINO ACID SEQUENCE OF A
MODIFIED PREX2 SEGMENT FROM HEPATITIS B VIRUS 24 Ala Ala Gly Ile
Gly Ile Leu Thr Val Phe Leu Trp Gly Pro Arg Ala 1 5 10 15 Leu Val
Met Glu Leu Ala Val Leu Tyr Cys Leu Leu Leu Asp Gly Thr 20 25 30
Ala Thr Leu Arg Leu Lys Thr Trp Gly Gln Tyr Trp Gln Val Tyr Met 35
40 45 Asp Gly Thr Met Ser Asp Val Ile Thr Asp Gln Val Pro Phe Ser
Val 50 55 60 Tyr Leu Glu Phe Gly Pro Val Thr Ala Ile Leu Thr Val
Ile Leu Gly 65 70 75 80 Val Leu Val Leu Pro Asp Val Phe Ile Arg Cys
Val 85 90 25 33 PRT Hepatitis B virus 25 Ala Ala Ala Leu Glu His
Ile Gly Pro Gly Arg Ala Phe Val Val Pro 1 5 10 15 Leu Glu Glu Ala
Trp Asp Pro Ser Arg Ile Gly Asp Pro Ala Leu Asn 20 25 30 Met
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