U.S. patent application number 10/890526 was filed with the patent office on 2004-12-23 for cytotoxic t-cell epitopes of papillomavirus l1 protein and their use in diagnostics and therapy.
Invention is credited to Jochmus, Ingrid, Nieland, John.
Application Number | 20040258708 10/890526 |
Document ID | / |
Family ID | 7909963 |
Filed Date | 2004-12-23 |
United States Patent
Application |
20040258708 |
Kind Code |
A1 |
Jochmus, Ingrid ; et
al. |
December 23, 2004 |
Cytotoxic T-cell epitopes of papillomavirus L1 protein and their
use in diagnostics and therapy
Abstract
The present invention relates to a papillomavirus T-cell epitope
having an amino acid sequence ILVPKVSGL, RLVWACVGV, HLFNRAGTV,
YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT,
TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV, FYNPDTQRL, MHGDTPTLH,
ETTDLYCY, QAEPDRAHYN, SMVTSDAQI, and/or to a functionally active
variant thereof, and also to their use in diagnostics and
therapy.
Inventors: |
Jochmus, Ingrid;
(Grobenzell, DE) ; Nieland, John; (Munchen,
DE) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
7909963 |
Appl. No.: |
10/890526 |
Filed: |
July 13, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10890526 |
Jul 13, 2004 |
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09980177 |
May 2, 2002 |
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09980177 |
May 2, 2002 |
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PCT/EP00/05006 |
May 31, 2000 |
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Current U.S.
Class: |
424/186.1 ;
530/350 |
Current CPC
Class: |
A61K 39/00 20130101;
C07K 14/005 20130101; C12N 2710/20022 20130101; A61P 31/12
20180101; A61K 38/00 20130101 |
Class at
Publication: |
424/186.1 ;
530/350 |
International
Class: |
A61K 039/21; C07K
014/025; C07K 014/725 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 1, 1999 |
DE |
19925199.1 |
Claims
1-27. (Cancelled)
28. A T-cell epitope having an amino acid sequence ILVPKVSGL,
RLVWACVGV, HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL,
SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL,
FYNPDTQRL, MHGDTPTLH, ETTDLYCY, QAEPDRAHYN, SMVTSDAQI, and/or a
functionally active variant thereof.
29. The T-cell epitope as claimed in claim 28, wherein said variant
has a sequence homology to ILVPKVSGL, RLVWACVGV, HLFNRAGTV,
YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT,
TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL, FYNPDTQRL, MHGDTPTLH, ETTDLYCY,
QAEPDRAHYN or SMVTSDAQI of at least approx. 65%, preferably at
least approx. 75% and in particular at least approx. 85% at the
amino acid level.
30. The T-cell epitope as claimed in claim 28, wherein said variant
is structurally homologous to ILVPKVSGL, RLVWACVGV, HLFNRAGTV,
YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT,
TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL, FYNPDTQRL, MHGDTPTLH, ETTDLYCY,
QAEPDRAHYN or SMVTSDAQI.
31. The T-cell epitope as claimed in claim 28, wherein the T-cell
epitope is a cytotoxic T-cell epitope.
32. A compound comprising a T-cell epitope as claimed in claim 28,
wherein the compound is not a naturally occurring L1 protein of a
papillomavirus and not an exclusively N-terminal or an exclusively
C-terminal deletion mutant of a naturally occurring L1 protein of a
papillomavirus.
33. The compound as claimed in claim 32, wherein the compound is a
polypeptide, in particular a fusion protein.
34. The compound as claimed in claim 32, wherein the compound is a
polypeptide of at least 50 amino acids in length.
35. The compound as claimed in claim 32, wherein the compound is a
polypeptide of at least 35 amino acids in length.
36. The compound as claimed in claim 32, wherein the compound is a
polypeptide of at least approx. 20 amino acids in length.
37. The compound as claimed in claim 32, wherein the compound is a
polypeptide of at least 9-13 amino acids in length.
38. The compound as claimed in claim 32, wherein the compound
contains a label selected from the group consisting of a chemical,
radioactive, nonradioactive isotope and fluorescent label.
39. A nucleic acid, wherein the nucleic acid codes for a T-cell
epitope as claimed in claim 32.
40. A vector containing a nucleic acid as claimed in claim 39.
41. A cell containing at least one T-cell epitope as claimed in
claim 32.
42. The cell as claimed in claim 41, wherein the cell is
transfected, transformed, or infected with a nucleic acid as
claimed in claim 39.
43. The cell as claimed in claim 41, wherein the cell was incubated
with at least one compound as claimed in claim 32.
44. The cell as claimed in claim 41, wherein the cell is selected
from the group consisting of a B cell, a macrophage, a dendritic
cell, a fibroblast, in particular a JY, T2, CaSki cell and
EBV-transformed cell.
45. A complex comprising a T-cell epitope as claimed in claim 28
and at least one further compound.
46. The complex as claimed in claim 45, wherein the complex
contains at least one MHC class I molecule.
47. The complex as claimed in claim 46, wherein the complex
contains a human MHC class I molecule
48. The complex as claimed in claim 46, wherein the MHC class I
molecule is a HLA A2.01 molecule.
49. A method for in vitro detection of the activation of T cells by
at least one compound containing a T-cell epitope as claimed in
claim 28, which comprises the following steps: a) stimulating cells
using at least one said compound; b) adding at least one target
cell presenting a T-cell epitope as claimed in claims 28 or a
complex as claimed in claim 45, and c) determining T-cell
activation.
50. The method as claimed in claim 49, wherein it comprises, after
step a), the following additional step a'): a') coculturing of the
cells for at least 1 week with a substance selected from the group
consisting of: (i) at least one target cell loaded with a substance
selected from the group consisting of a compound as claimed in
claim 32, at least one complex as claimed in claim 45, at least one
capsomer, at least one stable capsomer, at least one VLP, at least
one CVLP, and at least one virus, (ii) at least one complex as
claimed in claim 45, and (iii) at least one target cell presenting
a T-cell epitope as claimed in claim 28, prior to step b).
51. A method for producing a target cell as claimed in claim 41,
wherein the target cell is incubated with at least one compound as
claimed in claim 32.
52. A method for producing a target cell as claimed in claim 41,
wherein the target cells is transfected, transformed or infected
with a nucleic acid as claimed in claim 39.
53. A method for producing a target cell as claimed in claim 51 or
52, wherein the target cell is selected from the group consisting
of a B cell, a macrophage, a dendritic cell, a fibroblast, in
particular a JY, T2, CaSki cell and EBV-transformed cell.
54. The method as claimed in claim 29, wherein instead of step a)
the following step a") is carried out: a") producing and preparing
samples containing T cells and subsequent culturing.
55. An assay system for in vitro detection of the activation of T
cells, comprising: a) a substance selected from the group
consisting of at least one T-cell epitope as claimed in claim 28,
at least one compound as claimed in claim 32, at least one vector
as claimed in claim 40, at least one cell as claimed in claim 41,
and at least one complex as claimed in claim 45, and b) effector
cells selected from the group consisting of the immune system, T
cells, cytotoxic T cells and T helper cells.
56. A method of causing or detecting an immune response using a
substance selected form the group consisting of at least one T-cell
epitope as claimed in claim 28, at least one compound as claimed in
claim 32, at least one vector as claimed in claim 40, at least one
cell as claimed in claim 41, and at least one complex as claimed in
claim 45.
57. A medicament or diagnostic agent, comprising a substance
selected from the group consisting of at least one compound as
claimed in claim 32, at least one vector as claimed in claim 40, at
least one cell as claimed in claim 41, and at least one complex as
claimed in claim 45.
58. The medicament or diagnostic agent as claimed in claim 57,
wherein a substance selected from the group consisting of at least
one compound as claimed in claim 32, at least one vector as claimed
in claim 40, at least one cell as claimed in claim 41, and at least
one complex as claimed in claim 45 is present in solution, bound to
a solid matrix or mixed with an adjuvant.
Description
[0001] The present invention relates to a papillomavirus T-cell
epitope having an amino acid sequence ILVPKVSGL, RLVWACVGV,
HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL,
NLASSNYFPT, TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV,
FYNPDTQRL, MHGDTPTLH, ETTDLYCY, QAEPDRAHYN, SMVTSDAQI, and/or to a
functionally active variant thereof, and also to its use in
diagnostics and therapy.
[0002] The papillomaviruses, also called wart viruses, are
double-stranded DNA viruses with a genome size of about 8000 base
pairs and an icosahedral capsid of approx. 55 nm in diameter. Up
until now, more than 100 different human-pathogenic papillomavirus
types (HPV) are known, some of which, for example HPV-16, HPV-18,
HPV-31, HPV-33, HPV-39, HPV-45, HPV-52 or HPV-58, may cause
malignant tumors and others, for example HPV-6, HPV-11 or HPV-42,
may cause benign tumors.
[0003] The papillomavirus genome can be divided into three parts:
the first part relates to a noncoding region containing regulatory
elements for virus transcription and replication. The second
region, the "E" (early) region, contains various protein-encoding
sections E1-E7 of which, for example, the E6 and E7 proteins are
responsible for transformation of epithelial cells and the E1
protein controls the DNA copy number. The E6 and E7 regions are
"oncogenes" which are also expressed in malignantly degenerate
cells. The third region, also called L (late) region, contains two
protein-encoding sections L1 and L2 which code for structural
components of the virus capsid. Over 90% of the L protein is
present in the viral capsid, the L1:L2 ratio generally being 30:1.
In accordance with the present invention, the term L1 protein means
the main capsid protein of papillomaviruses (Baker T. et al. (1991)
Biophys. J. 60, 1445).
[0004] In over 50% of cases, HPV-16 is connected with cervical
cancer (carcinoma of the cervix). HPV-16 is the main risk factor
for the formation of cervical neoplasms. The immune system plays an
important part in the progress of the disease. Thus, cellular
immune responses and in particular antigen-specific T lymphocytes
are presumably important for the defense mechanism. It has
furthermore been found that in high-grade malignant cervical
intraepithelial neoplasms (CIN II/III) and cervical tumors the E7
gene is expressed constitutively in all layers of the infected
epithelium. The E7 protein in particular is therefore considered as
a potential tumor antigen and as a target molecule for activated T
cells (see, for example, WO 93/20844). The E7-induced cellular
immune response in the patient, however, is apparently not strong
enough to influence the course of the disease. The immune response
may possibly be amplified by suitable vaccines.
[0005] It has been possible to show that expression of the L1 gene
and/or coexpression of the L1 and L2 genes can lead to the
formation of capsomers, stable capsomers, capsids or virus-like
particles (VLPs) (see, for example, WO 93/02184, WO 94/20137 or WO
94/05792). Capsomers mean an oligomeric configuration which is
composed of five L1 proteins. The capsomer is the basic building
block of which viral capsids are composed. Stable capsomers mean
capsomers which are incapable of assembling to form capsids.
Capsids mean the papillomavirus coat which is, for example,
composed of 72 capsomers (Baker T. et al. (1991) Biophys. J. 60,
1445). VLP means a capsid which is morphologically and in its
antigenicity identical to an intact virus. It was possible to use
the VLPs in various animal systems for causing a humoral immune
response characterized by the formation of neutralizing antibodies.
The formation of virus-neutralizing antibodies against L1 and/or L2
protein, however, is of relatively low clinical importance if the
virus infection has already taken place, since for the elimination
of virus-infected cells a virus-specific cytotoxic T-cell (CTL)
response rather than antibodies seems to be necessary. And,
although VLPs are capable of causing a cytotoxic T-cell response,
an immune response exclusively directed against the capsid proteins
L1 and/or L2 appears unsuitable for controlling a tumor caused by
papillomaviruses.
[0006] Therefore, "chimeric papillomavirus-like particles" (CVLPs)
which comprise a fusion protein of the capsid protein L1 and the
potential tumor antigen E7 (WO 96/11272 and Muller, M. et al.
(1997) Virology, 234, 93) have been developed. The CVLPs caused
only to a small extent a humoral immune response directed against
the E7 protein (Muller, M. et al. (1997), supra). Some of the CVLPs
tested, however, do indeed induce the desired E7-specific cytotoxic
T-cell response in mice (see also Peng S. et al. (1998) Virology
240, 147-57). Therefore, CVLPs are of interest both for the
development of a vaccine and for the treatment of already
established infections and tumors resulting therefrom, since the E7
tumor cell peptides presented via MHC molecules of class I would
represent target molecules of cytotoxic T cells.
[0007] A vaccine comprising CVLPs is based on the principle of the
CVLPs pseudo-infecting cells. This means that CVLPs and viruses
alike get into the cell, are processed there to peptides, and the
peptides are then loaded onto MHC class I and II molecules and
finally presented to CD8- or CD4-positive T cells. As a consequence
of this stimulation, CD8 cells may differentiate into cytotoxic T
cells and then cause a cellular immune response, whereas CD4 cells
develop into T helper cells and stimulate B cells to give a humoral
or CD8-positive T cells to give a cytotoxic immune response and may
themselves induce lysis of infected cells.
[0008] Small peptides may bind to MHC class I molecules already on
the cell surface and then stimulate without further processing CD8-
or CD4-positive cells to give a cellular immune response. However,
a particular peptide can be bound only by particular MHC molecules.
Due to the large polymorphism of MHC molecules in natural
populations, a particular peptide can therefore be bound and
presented only by a small part of a population. In accordance with
the present invention, presentation means binding of a peptide or
protein fragment to an MHC molecule, it being possible for said
binding to take place, for example, in the endoplasmic reticulum,
the extracellular space, the endosomes, proendosomes, lysosomes or
protysosomes, and said MHC molecule-peptide complex then being
bound on the extracellular side of the cell membrane so that it can
be recognized specifically by immune cells.
[0009] Since CVLPs cause both a cellular and a humoral immune
response and are not MHC-restricted, this technology is generally
suitable for the development of vaccines, since an L1 portion
provides the ability to form particles and an additional antigen
portion is fused to said L1 portion.
[0010] For the development of CVLPs of this kind it is absolutely
necessary to have a functional assay system available which can be
used to study directly the immunogenicity of CVLPs. Such an assay
system should have the property that CVLPs with different antigen
proportions can be studied by using the same assay system. Since
the cellular immune response is of crucial importance for
immunological therapies of tumors or viral diseases, the object
arose to make it possible to measure the cellular immune response
caused by CVLPs.
[0011] This object was achieved by identifying T-cell epitopes
which in connection with MHC molecules, and in a particular
embodiment with HLA A2.01 MHC molecules, cause, for example, a
cytotoxic T-cell response in vivo and in vitro. Said peptides
preferably have the sequence ILVPKVSGL, RLVWACVGV, HLFNRAGTV,
YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT,
TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV, FYNPDTQRL, MHGDTPTLH,
ETTDLYCY, QAEPDRAHYN, SMVTSDAQI. These sequences are part of the L1
and E7 peptides of HPV16. They include the amino acid regions L1
86-94 (5104), L1 123-131 (5106), L1 285-293 (5107), L1 275-283
(5108), L1 238-246 (5109), L1 426-434 (5112), L1 28-39 (2016), L1
311-320 (2017), L1 408-417 (2018), L1 38-47 (2019), L1 396-404
(2020), L1 349-357 (2022), L1 298-306 (27/28), L1 90-98 (9), E7 1-9
(43), E7 18-25 (45) and E7 44-53 (47/48). The names of the relevant
epitopes is indicated in brackets.
[0012] The E7 peptides 43, 45 and 47/48 have already been published
as potential epitopes in Kast et al., (1994) Journal of Immunology
152, 3904-3912. However, this publication only shows that said
peptides can bind to HLA A1 molecules, but does not show that a
cytotoxic T-cell response can actually be caused. Furthermore, no
data are given which prove that T cells recognize the peptides as
part of a protein. For it has been shown many times that peptides
binding per se to HLA molecules are not necessarily also recognized
by T cells. Moreover, it is known that T cells, although
recognizing a peptide, which recognition can be measured by the
ability of a peptide to induce a T-cell response in said cells, do
not necessarily also recognize cells which have been loaded with
whole proteins containing the corresponding peptide. This can be
explained by the fact that peptides often contain protease
cleavage. sites within which the peptides, during processing of the
whole proteins in the cell, are cut and thus destroyed and thus
cannot be detected any longer by T cells. This problem is
confirmed, for example, in Feltkamp et al. (1993), Eur. J. Immunol.
23: 2242-2249.
[0013] The present invention therefore relates to a T-cell epitope
having an amino acid sequence ILVPKVSGL, RLVWACVGV, HLFNRAGTV,
YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT,
TLTADVMTYI; YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV; FYNPDTQRL, MHGDTPTLH,
ETTDLYCY, QAEPDRAHYN, SMVTSDAQI, and/or to a functionally active
variant thereof.
[0014] A functionally active variant of ILVPKVSGL, RLVWACVGV,
HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL,
NLASSNYFPT, TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV,
FYNPDTQRL, MHGDTPTLH, ETTDLYCY, QAEPDRAHYN or SMVTSDAQI means a
T-cell epitope which, in a T-cell cytotoxicity assay system (see,
for example, Examples 2-5 of the present invention), has a
cytotoxicity which, compared to the cytotoxicity of ILVPKVSGL,
RLVWACVGV, HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL,
SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL,
ICWGNQLFV, FYNPDTQRL, MHGDTPTLH, ETTDLYCY, QAEPDRAHYN or SMVTSDAQI,
corresponds to at least the sum of the average of the negative
controls and three times the standard deviation, preferably of at
least approx. 30%, in particular at least approx. 50% and
particularly preferably of at least approx. 80%.
[0015] An example of a preferred variant is a T-cell epitope having
a sequence homology to ILVPKVSGL, RLVWACVGV, HLFNRAGTV, YLRREQMFV,
TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYI,
YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV, FYNPDTQRL, MHGDTPTLH, ETTDLYCY,
QAEPDRAHYN or SMVTSDAQI of at least approx. 65%, preferably at
least approx. 75% and in particular at least approx. 85% at the
amino acid level. Other preferred variants are also T-cell epitopes
which are structurally homologous to ILVPKVSGL, RLVWACVGV,
HLFNRAGTV, YLRREQMFV, TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL,
NLASSNYFPT, TLTADVMTYI, YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV,
FYNPDTQRL, MHGDTPTLH, ETTDLYCY, QAEPDRAHYN or SMVTSDAQI. Such
epitopes may be found by generating specific T cells against the
T-cell epitopes ILVPKVSGL, RLVWACVGV, HLFNRAGTV, YLRREQMFV,
TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYI,
YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV, FYNPDTQRL, MHGDTPTLH, ETTDLYCY,
QAEPDRAHYN or SMVTSDAQI (DeBruijn M. L. et al. (1991) Eur. J.
Immunol. 21, 2963-70; and DeBruijn M. L. (1992) Eur. J. Immunol.
22, 3013-20) and assaying, for example, synthetically produced
peptides of choice for recognition by the peptide-specific T cells
(see examples). The T-cell epitopes in particular mean cytotoxic
T-cell epitopes. However, noncytotoxic T cells are also known which
can likewise recognize MHC I molecules so that the present
invention also includes noncytotoxic T-cell epitopes as
variant.
[0016] Another embodiment of the present invention is a T-cell
epitope which is part of a compound, the compound not being a
naturally occurring L1 protein of a papillomavirus and not being an
exclusively N-terminal or exclusively C-terminal deletion mutant of
a naturally occurring L1 protein of a papillomavirus.
[0017] In a particular embodiment, a T-cell epitope having an amino
acid sequence ILVPKVSGL, RLVWACVGV, HLFNRAGTV, YLRREQMFV,
TLQANKSEV, ILEDWNFGL, SLWLPSEATVYL, NLASSNYFPT, TLTADVMTYI,
YLPPVPVSKV, YDLQFIFQL, ICWGNQLFV, and/or a functionally active
variant may be contained in an L1 protein of a different
papillomavirus or in a chimeric L1 protein, for example an HPV18L1
E7 fusion protein. Such a compound of the invention may have the
ability to form CVLPs.
[0018] As part of a compound, said T-cell epitope may preferably be
a polypeptide which preferably contains further amino acid
sequences, and in particular a fusion protein. In particular, the
compound may be a polypeptide of at least approx. 50 amino acids,
preferably of at least approx. 35 amino acids, in particular of at
least approx. 20 amino acids and particularly preferably of at
least approx. 9-12 amino acids, in length.
[0019] In order to detect the compound or to modify its T-cell
binding activity, said compound may contain a chemical, radioactive
isotope, nonradioactive isotope and/or fluorescent label of the
T-cell epitope and/or of said fusion protein.
[0020] Examples of chemical substances known to the skilled worker,
which are suitable for chemical labeling according to the
invention, are: biotin, FITC (fluorescein isothiocyanate) or
streptavidin.
[0021] In a possible embodiment a peptide is modified such that it
contains at least one lysine. In a manner known to the skilled
worker biotin or FITC (fluorescein isothiocyanate) is coupled to
said lysine. A peptide modified in this way is bound to an
appropriate MHC molecule or to a cell containing appropriate MHC
molecules. The peptide may then be detected via labeled avidin or
streptavidin or directly via FITC fluorescence.
[0022] Examples of isotopes known to the skilled worker, which are
suitable for radioactive isotope labeling according to the
invention are: .sup.3H, .sup.125I, .sup.131I .sup.32P, .sup.33P or
.sup.14C.
[0023] Examples of isotopes known to the skilled worker, which are
suitable for nonradioactive isotope labeling according to the
invention are: .sup.2H, or .sup.13C.
[0024] Examples of fluorescent substances known to the skilled
worker, which are suitable for fluorescence labeling according to
the invention are: .sup.152Eu, fluorescein isothiocyanate,
rhodamine, phycoerythrin, phycocyanin, allophycocyanin,
o-phtaldehyde or fluorescamine.
[0025] Further label not listed here, which may also be used for
labeling in accordance with this invention, are known to the
skilled worker.
[0026] Examples of inventive chemical modifications known to the
skilled worker are the transfer of acetyl, phosphate and/or
monosaccharide groups.
[0027] Inventive polypeptides of approx. 50 amino acids in length
may be prepared, for example, by chemical peptide synthesis. Longer
polypeptides are preferably generated by genetic engineering. The
present invention therefore further relates to a nucleic acid
construct for expressing said T-cell epitope or compounds
containing the following components: (a) at least one regulatory
element and (b) at least one nucleic acid coding for an amino acid
sequence of the compound of the invention. Said nucleic acid
construct is preferably made of DNA or RNA. Suitable regulatory
elements allow, for example, constitutive, regulatable,
tissue-specific, cell cycle-specific or metabolically specific
expression in eukaryotic cells or constitutive, metabolically
specific or regulatable expression in prokaryotic cells.
Regulatable elements according to the present invention are
promoters, activator sequences, enhancers, silencers, and/or
repressor sequences.
[0028] Examples of suitable regulatable elements which make
constitutive expression in eukaryotes possible are promoters
recognized by RNA polymerase III or viral promoters such as CMV
enhancer, CMV promoter, SV40 promoter and viral promoter and
activator sequences derived, for example, from HBV, HCV, HSV, HPV,
EBV, HTLV or HIV.
[0029] Examples of regulatable elements which make regulatable
expression in eukaryotes possible are the tetracyclin operator in
combination with a corresponding repressor (Gossen M. et al (1994)
Curr. Opin. Biotechnol. 5, 516-20).
[0030] Examples of regulatable elements which make tissue-specific
expression in eukaryotes possible are promoters or activator
sequences from promoters or enhancers of those genes coding for
proteins which are expressed only in particular cell types.
[0031] Examples of regulatable elements which make cell
cycle-specific expression in eukaryotes possible are the promoters
of the following genes: cdc25C, cyclin A, cyclin E, cdc2, E2F,
B-myb or DHFR (Zwicker J. and Muller R. (1997) Trends Genet. 13,
3-6).
[0032] Examples of regulatable elements which make metabolically
specific expression in eukaryotes possible are promoters regulated
by hypoxia, by glucose deficiency, by phosphate concentration or by
heat shock.
[0033] In order to make it possible to introduce said nucleic acid
and thus express the polypeptide in a eukaryotic or prokaryotic
cell by transfection, transformation or infection, the nucleic acid
may be present as plasmid, or as part of a viral or nonviral
vector. The present invention therefore further relates to a
vector, in particular an expression vector which contains a nucleic
acid coding for a polypeptide of the invention. Viral vectors
particularly suitable here are: baculo viruses, vaccinia viruses,
adenoviruses, adeno-associated viruses and herpes viruses. Nonviral
vectors particularly suitable here are: virosomes, liposomes,
cationic lipids or polylysine-conjugated DNA.
[0034] The present invention further relates to a cell containing,
preferably presenting, at least one T-cell epitope. In a particular
embodiment, the cell is transfected, transformed or infected by one
of the vectors mentioned. This cell expresses the polypeptide of
the invention under conditions known to a skilled worker which lead
to activation of the regulatable elements used in each case. The
polypeptide can then be isolated from said cell and purified, for
example by using one of the abovementioned labels. Cells which are
suitable for the preparation by genetic engineering and subsequent
purification of the expressed compounds of the invention are
prokaryotic and eukaryotic cells, in particular bacteria cells such
as, for example, E.coli, yeast cells such as, for example, S.
cerevisiae, insect cells such as, for example, Spodoptera
frugiperda cells (Sf-9) or Trichoplusia ni cells or mammalian cells
such as, for example, COS cells or HeLa cells.
[0035] A particular embodiment is using the cell itself which
expresses the polypeptide of the invention, and, in a particularly
preferred embodiment, the cell presents parts of the polypeptide of
the invention via MHC-1 molecules on the cell surface. Suitable
cells for preparing the cell of the invention are
antigen-presenting cells such as, for example, B cells,
macrophages, dendritic cells, fibroblasts or other HLA
A2.01-positive cells, in a preferred embodiment JY, T2, CaSki cells
or EBV-transformed B-cell lines. The cells of the invention which
present a polypeptide containing a T-cell epitope may be employed
as target cells for restimulating immune cells, in particular T
cells, and/or for measuring T-cell activation. A target cell means
in accordance with the present invention a cell which presents a
T-cell epitope via MHC molecules and thus specifically causes
T-cell activation, in particular a cytotoxic T-cell reaction
against the cell.
[0036] Furthermore, the T-cell epitope-containing compound may be
part of a complex which is characterized by the compound being
linked covalently or by hydrophobic interactions, ionic binding or
hydrogen bonds to at least one further species such as peptides,
proteins, peptoids, linear or branched oligo or polysaccharides and
nucleic acids.
[0037] The present invention therefore relates to a complex
containing a T-cell epitope or a compound and at least one further
compound. In a preferred embodiment, the polypeptide is linked to
MHC class I molecules, preferably as HLA A2.01 tetramer. Particular
preference is given to human MHC class I molecules. Using the
technique by Altman J. D. et al. (1996, Science 274, 94-6) it is
possible, for example, to prepare HLA A2.01 tetramers with the
appropriate bound peptides which are capable of binding to T-cell
receptors of peptide-specific cytotoxic T cells.
[0038] Another embodiment is immobilization of the compound of the
invention or of said complex to support materials. Examples of
suitable support materials are ceramic, metal, in particular noble
metal, glasses, plastics, crystalline materials or thin layers of
this support, in particular of said materials, or (bio)molecular
filaments such as cellulose or structural proteins.
[0039] In order to purify the complex of the invention, a component
of the complex may additionally also contain a protein tag. Protein
tags of the invention allow, for example, high-affinity absorption
to a matrix, stringent washing with suitable buffers with
negligible elution of the complex and subsequent specific elution
of the absorbed complex. Examples of protein tags known to the
skilled worker are an (HIS).sub.6 tag, a myc tag, a FLAG tag, a
hemagglutinin tag, glutathione transferase (GST) tag, intein with
chitin-binding affinity tag or maltose-binding protein (MBP) tag.
The protein tags of the invention may be located N-terminally,
C-terminally and/or internally.
[0040] The present invention also relates to a method for in vitro
detection of the activation of T cells by at least one compound
containing a T-cell epitope. A method of this kind preferably
comprises three steps:
[0041] a) In a first step, cells are stimulated by at least one
compounds containing a T-cell epitope. This compound may be at
least one inventive compound containing a T-cell epitope, at least
one inventive complex containing a T-cell epitope, at least one
capsomer, at least one stable capsomer, at least one VLP, at least
one CVLP, and/or at least one virus. In a preferred embodiment,
immune cells are stimulated by incubation with CVLPs. This
stimulation may be carried out, for example, in the form of a
vaccination or by incubating immune cells with CVLPs in vitro.
Immune cells stimulated in this way are obtained, for example,
after a vaccination or, in the case of a tumor patient, from the
blood, from tumors or from lymph nodes, and/or are cultured.
[0042] b) In a second step, the cells are incubated with at least
one T-cell epitope of the invention, at least one inventive
compound containing a T-cell epitope, at least one target cell
presenting a T-cell epitope and/or with at least one complex of the
invention.
[0043] c) In a third step, T-cell activation is determined.
Examples of methods suitable for this are detection of cytokine
production or secretion by the T cells, of the surface molecule
expression on T cells, of target cell lysis or of cell
proliferation. Examples of methods suitable for this are a
cytokinassay (Chapter 6.2 to 6.24 in Current Protocols in
Immunology (1999), edited by Coligan J. E., Kruisbeek A. M.,
Margulies D. H., Shevach E. M. and Strober W., John Wiley &
Sons), ELISPOT (Chapter 6.19 in Current Protocols in Immunology,
supra), a .sup.51Cr release assay (Chapter 3.11 in Current
Protocols in Immunology, supra) or detection of proliferation
(Chapter 3.12 in Current Protocols in Immunology, supra). Depending
on the method used, it is in this connection also possible to
distinguish between the immune cells such as cytotoxic T cells, T
helper cells, B cells, NK cells, and other cells. The use of
inventive compounds, complexes, and/or cells containing the labels
of the invention allows detection of T cells recognizing the T-cell
epitope via detection of the binding of labeled compounds,
complexes and/or cells to the T cells. In a preferred embodiment,
binding of inventive MHC-polypeptide complexes to the surface of T
cells is detected. This may be carried out such that the MHC
complexes are labeled themselves, for example fluorescently
labeled, or that, in a further step, an MHC-specific, labeled, for
example fluorescently labeled, antibody is used in order to detect
in turn the MHC complexes. The fluorescent label of the T cells can
then be measured and evaluated, for example, in a
fluorescence-activated cell sorter (FACS). Another possible way of
detecting binding of the complexes to the T cells is again
measuring T-cell activation (cytokine assay, Elispot, .sup.51Cr
release assay, proliferation, see above). However, this requires
simultaneous stimulation of coreceptors (e.g. CD28), for example by
coreceptor-specific antibodies (anti-CD28) and/or other unspecific
activators (IL-2).
[0044] The present invention also relates to a method containing an
additional step a') which is introduced after step a).
[0045] a') In this additional step a') which follows step a), the
isolated or cultured cells are cocultured with at least one target
cell loaded with an inventive compound containing a T-cell epitope,
at least one inventive complex containing a T-cell epitope, at
least one capsomer, at least one stable capsomer, at least one VLP,
at least one CVLP and/or at least one virus, with at least one
inventive complex containing a T-cell epitope, and/or at least one
target cell presenting a T-cell epitope for at least approx. 8
weeks, in particular for at least approx. 1 week, prior to step
b)
[0046] Coculturing means growing cells:
[0047] (i) in the presence of at least one target cell loaded with
an inventive compound containing a T-cell epitope, at least one
inventive complex containing a T-cell epitope, at least one
capsomer, at least one stable capsomer, at least one VLP, at least
one CVLP, and/or at least one virus,
[0048] (ii) in the presence of at least one inventive complex
containing a T-cell epitope,
[0049] (iii) in the presence of at least one target cell presenting
a T-cell epitope,
[0050] in the same growth medium and the same tissue culture
container.
[0051] The present invention further relates to a method for
preparing a target cell presenting a T-cell epitope. It is possible
here to load the target cell with combinations of different T-cell
epitopes. In a preferred embodiment, the target cell is incubated
with at least one compound containing a T-cell epitope and/or at
least one complex containing a T-cell epitope. In a particularly
preferred embodiment, the target cell is incubated in growth medium
containing polypeptides of the invention or with MHC class I
complexes with bound polypeptides of the invention. The MHC class I
complexes may be present for example as HLA A2.01 tetramers. In
this connection, a tetramer normally binds four peptides. These can
be identical or else represent different peptide species. In a
further preferred embodiment, the target cell is transfected,
transformed and/or infected with a nucleic acid and/or a vector. In
a particularly preferred embodiment, the target cell is infected
with a vaccinia virus vector. The method of the invention is
carried out using antigen-presenting cells, for example B cells,
macrophages, dendritic cells, embryonal cells or fibroblasts or
other HLA A2.01-positive cells, and, in a preferred embodiment,
using JY, T2, CaSki cells or EBV-transformed B-cell lines.
[0052] The CVLPs used contain a papillomavirus L1 protein or
variants thereof, in particular HPV16 L1 protein and, but not
necessarily, a protein heterologous to an L1 or variants thereof.
The two proteins may be bound directly or indirectly. In accordance
with the invention, directly bound means that the two proteins are
covalently bound to one another, for example via a peptide bond or
a disulfide bond. Indirectly bound means that the proteins are
bound via noncovalent bonds, for example hydrophobic interactions,
ionic bonds or hydrogen bonds. In a further embodiment, the CVLPs
contain, in addition to L1 protein or variants thereof, a
papillomavirus L2 protein.
[0053] Examples of a preferred embodiment of the L1 protein of the
present invention are L1 proteins having one or more deletions, in
particular a C-terminal deletion. A C-terminal deletion has the
advantage that it is possible to increase the efficiency of
virus-like particle formation, since the nuclear localization
signal located at the C terminus is deleted. The C-terminal
deletion is therefore preferably up to approx. 35 amino acids, in
particular approx. 25 to approx. 35 amino acids, especially approx.
32 to approx. 34 amino acids. For example, a 32 amino acid long
C-terminal deletion of the HPV16 L1 protein is sufficient in order
to be able to increase the formation of virus-like particles at
least approx. ten times. Furthermore, the L1 protein may carry one
or more mutations or the L1 portion may be composed of L1 proteins
of various papillomaviruses. A common characteristic of the L1
proteins of the invention is the fact that they permit the
formation of VLPs or CVLPs and that they contain at least one
T-cell epitope of the invention.
[0054] In a preferred embodiment, the L1 protein or variants
thereof and the protein heterologous to L1 are a fusion protein.
Heterologous proteins which are composed of a plurality of various
proteins or parts thereof are also included. These may also be, for
example, epitopes, in particular cytotoxic T-cell epitopes, of
proteins. In this connection, epitopes in accordance with the
invention may also be part of a synthetic polypeptide of approx. 50
amino acids, preferably of at least approx. 35 amino acids, in
particular of at least approx. 20 amino acids and particularly
preferably of at least approx. 9 amino acids, in length.
[0055] Preference is given to proteins heterologous to L1, which
are derived from a viral protein, for example derived from HIV, HBV
or HCV, preferably from papillomaviruses, in particular from human
papillomaviruses.
[0056] In a preferred embodiment, said viral protein is a
papillomavirus E protein, preferably an E6 and/or E7 protein. It is
particularly preferred if the E protein is a deleted E protein,
preferably a C-terminally deleted, in particular a C-terminally
deleted E7 protein, since these constructs in connection with
deleted L1 protein can form preferably virus-like particles.
Particular preference is given to deletions of up to 55 amino
acids, preferably approx. 5 to approx. 55 amino acids, in
particular approx. 38 to approx. 55 amino acids.
[0057] In a further embodiment, the protein heterologous to L1 may
originate from antigens of nonviral pathogens. Likewise, they may
be derived from autoimmune antigens such as, for example,
thyroglobulin, myelin basic protein or zona pellucida glycoprotein
3 (ZP.sub.3), which are associated with particular autoimmune
diseases such as, for example, thyroiditis, multiple sclerosis,
oophoritis or rheumatoid arthritis. In a preferred embodiment, the
protein heterologous to L1 originates from tumor antigens,
preferably melanoma antigens such as MART, ovarian carcinoma
antigens such as Her2 neu (c-erbB2), BCRA-1 or CA125, colon
carcinoma antigens such as CA125 or breast carcinoma antigens such
as Her2 neu (c-erbB2), BCRA-1, BCRA-2.
[0058] The present invention further relates to a method for in
vitro detection of the activation of T cells which are obtained by
preparation from samples. This method makes it possible to
determine if a sample, for example a blood sample of a patient, or
tumors or lymph nodes of a tumor patient contain papillomavirus
L1-protein-specific cytotoxic T cells. A detection method of this
kind comprises the following steps:
[0059] a") In a first step, cells are obtained, for example by
taking blood from a patient or by preparation, for example, of
tumors or lymph nodes. Subsequently, the cells are taken up in
growth medium and cultured.
[0060] b) In a second step, cells are incubated with at least one
target cell presenting a T-cell epitope or with at least one
complex which comprises as a component a compound containing a
T-cell epitope.
[0061] c) In a third step, T-cell activation is determined.
Examples of methods suitable for this are detection of cytokine
production or secretion by the T cells, of the surface molecule
expression on T cells, of target cell lysis or of cell
proliferation. Examples of methods suitable for this are a
cytokinassay (Chapter 6.2 to 6.24 in Current Protocols in
Immunology (1999), edited by Coligan J. E., Kruisbeek A. M.,
Margulies D. H., Shevach E. M. and Strober W., John Wiley &
Sons), ELISPOT (Chapter 6.19 in Current Protocols in Immunology,
supra), a .sup.51Cr release assay (Chapter 3.11 in Current
Protocols in Immunology, supra) or detection of proliferation
(Chapter 3.12 in Current Protocols in Immunology, supra). Depending
on the method used, it is in this connection also possible to
distinguish between the immune cells such as cytotoxic T cells, T
helper cells, B cells, NK cells, and other cells. The use of
inventive compounds, complexes, and/or cells containing the labels
of the invention allows detection of T cells recognizing the T-cell
epitope via detection of the binding of labeled compounds,
complexes and/or cells to the T cells. In a preferred embodiment,
binding of inventive MHC-polypeptide complexes to the surface of T
cells is detected. This may be carried out such that the MHC
complexes are labeled themselves, for example fluorescently
labeled, or that, in a further step, an MHC-specific, labeled, for
example fluorescently labeled, antibody is used in order to detect
in turn the MHC complexes. The fluorescent label of the T cells can
then be measured and evaluated, for example, in a
fluorescence-activated cell sorter (FACS). Another possible way of
detecting binding of the complexes to the T cells is again
measuring T-cell activation (cytokine assay, Elispot, .sup.51Cr
release assay, proliferation, see above). However, this requires
simultaneous stimulation of coreceptors (e.g. CD28), for example by
coreceptor-specific antibodies (anti-CD28) and/or other unspecific
activators (IL-2).
[0062] The present invention also relates to a method containing an
additional step a') which is introduced after step a").
[0063] a') In this additional step a') which follows step a"), the
isolated or cultured cells are cocultured with at least one target
cell loaded with an inventive compound containing a T-cell epitope,
at least one inventive complex containing a T-cell epitope, at
least one capsomer, at least one stable capsomer, at least one VLP,
at least one CVLP and/or at least one virus, with at least one
inventive complex containing a T-cell epitope, and/or at least one
target cell presenting a T-cell epitope for at least approx. 8
weeks, in particular for at least approx. 1 week, prior to step
b).
[0064] Coculturing means growing cells:
[0065] (i) in the presence of at least one target cell loaded with
an inventive compound containing a T-cell epitope, at least one
inventive complex containing a T-cell epitope, at least one
capsomer, at least one stable capsomer, at least one VLP, at least
one CVLP, and/or at least one virus,
[0066] (ii) in the presence of at least one inventive complex
containing a T-cell epitope,
[0067] (iii) in the presence of at least one target cell presenting
a T-cell epitope,
[0068] in the same growth medium and the same tissue culture
container.
[0069] The invention further relates to an assay system (kit) for
in vitro detection of the activation of T cells, comprising:
[0070] a) at least one T-cell epitope of the invention, at least
one compound of the invention, at least one vector of the
invention, at least one cell of the invention, and/or at least one
complex of the invention, and
[0071] b) effector cells of the immune system, preferably T cells,
in particular cytotoxic T cells or T helper cells.
[0072] In a particular embodiment, the assay system is used for
determining the L1 protein-specific cytotoxic T cells which are
present, for example, in a patient's blood sample or in tumors or
lymph nodes of a tumor patient. In this case, the cells described
in b) are control cells contained in the assay system, whose
activation by the first kit component, the substances mentioned
under a), serves as a standard. The activation observed in this
reaction is compared with the T-cell activation of cells, which
have been isolated from patients, by kit component a).
[0073] In a further particular embodiment, the assay system is
used, for example, for determining the L1 protein-specific
antigenicity of a compound containing a T-cell epitope, a complex
containing a T-cell epitope, a capsomer, a stable capsomer, a VLP,
a CVLP and/or a virus. In this case, the substances described in a)
are control substances whose activating effect on the second kit
component, the cells mentioned under b), serves as a standard. The
activation observed in this reaction is compared with the
activating effect of a compound containing a T-cell epitope, a
complex comprising a T-cell epitope, a capsomer, a stable capsomer,
a VLP, a CVLP, and/or a virus on kit component b).
[0074] The invention further relates to the use of at least one
T-cell epitope, at least one inventive compound containing a T-cell
epitope, at least one inventive vector containing a nucleic acid
coding for a T-cell epitope-containing compound, at least one
inventive cell containing a T-cell epitope for, and/or at least one
inventive complex containing a T-cell epitope for causing or
detecting an immune response.
[0075] Suitable cells for immune cell stimulation in vitro as well
as in vivo are in particular cells which present at least one of
the molecules of the invention via their MHC class I molecules.
Examples of cells suitable for antigen presentation are B cells,
dendritic cells, macrophages, fibroblasts or other HLA
A2.01-positive cells which, by being cultured together with immune
cells, can stimulate specific T cells.
[0076] In a particular embodiment, it is possible to use a compound
of the invention, for example an HPV18 L1E7 fusion protein which
additionally contains a T-cell epitope of the invention, for
detecting an immune response. Such a compound of the invention may
have the ability to form CVLPs.
[0077] The invention further relates to a medicament or diagnostic
agent which contains at least one inventive compound containing a
T-cell epitope, at least one vector containing a nucleic acid
coding for a T-cell epitope-containing compound, at least one
inventive cell containing a T-cell epitope, and/or at least one
inventive complex containing a T-cell epitope and, where
appropriate, a pharmaceutically acceptable carrier.
[0078] Examples of carriers known to the skilled worker are glass,
polystyrene, polypropylene, polyethylene, dextran, nylon, amylase,
natural or modified cellulose, polyacrylamides, agarose, aluminum
hydroxide or magnetite.
[0079] A medicament or diagnostic agent of the invention may be
present in solution, bound to a solid matrix, and/or mixed with an
adjuvant.
[0080] The medicament or diagnostic agent .-ay be administered in
different ways. Examples of a-ministration forms known to the
skilled worker are parenteral, local and/or systemic administration
by, for example, oral, intranasal, intravenous, intramuscular,
and/or topical administration. The preferred administration form is
influenced, for example, by the natural path of infection of the
particular papillomavirus infection. The amount administered
depends on the age, weight and general state of health of the
patient and the type of papillomavirus infection. The medicament or
diagnostic agent may be administered in the form of capsules, a
solution, suspension, elixir (for oral administration) or sterile
solutions or suspensions (for parenteral or intranasal
administration). An inert and immunologically acceptable carrier
which may be used is, for example, a saline or phosphate-buffered
saline. The medicament is administered in therapeutically effective
amounts. These are amounts which are sufficient for causing a
protective immunological response.
[0081] In a particular embodiment, it is possible to use a compound
of the invention, for example an HPV18 L1E7 fusion protein which
additionally contains a T-cell epitope of the invention, as
medicament or diagnostic agent. Such a compound of the invention
may have the ability to form CVLPs.
[0082] The figures and the following examples are intended to
illustrate the invention in more detail, without restricting
it.
[0083] FIG. 1 shows the graphical analyses of MTT staining,
measured as absorption at 595 nm, of WEHI-cell lysates which had
been incubated with supernatants of T cells which in turn had been
stimulated by different, antigen-presenting peripheral blood
lymphocytes (PBLs). T cells stimulated by specific,
antigen-presenting PBLs release TNF.alpha.. This induces apoptosis
in WEHI cells so that these cells are no longer able co process MTT
into a brownish dye. Low absorption means low dye production and
thus many apoptotic cells which had thus been exposed to a lot of
TNF.alpha. so that the corresponding T cells had been stimulated.
Thus, stimulation of T cells improves with decreasing absorption at
595 nm for a particular antigen.
[0084] FIG. 2 shows the graphical analysis of the fluorescence,
measured in an FACS analysis, of T2 cells whose MHC-1 molecules
located on the cell surface had been labeled with an FITC-labeled
antibody. Cells whose MHC-1 molecules can specifically bind those
peptides listed on the X axis, have an increased number of MHC-1
molecules, since the specific binding stabilizes the MHC complexes
so that they can accumulate on the cell surface.
[0085] FIG. 3 shows the analysis of three FACScan experiments after
restimulating CVLP-specific human T cells with peripheral blood
mononuclear cells (PBMCs) which present different antigens. The
content of T cell-specific CD3 for each experiment is listed from
left to right and the content of human y interferon which is
specific for activated cells is listed from bottom to top.
[0086] FIG. 4 shows the analysis of FACScan experiments after
restimulating specific human T cells of an HLA A1-positive donor
with peripheral blood mononuclear cells (PBMCs) which present
different antigens. The name of the particular peptide with which
the PBMCs were loaded is listed from left to right. "None"
represents PBMCs which were incubated only with buffer. The Y axis
shows the proportion of CD8-positive T cells which were classified
as reactive in the FACScan experiment on the basis of
.gamma.-interferon expression.
[0087] FIG. 5 shows the analysis of FACScan experiments after
restimulating specific human T cells of a non-HLA-classified donor
with peripheral blood mononuclear cells (PBMCs) which present
different antigens. The name of the particular peptide with which
the PBMCs were loaded is listed from left to right. "None"
represents PBMCs which were incubated only with buffer. The Y axis
shows the proportion of CD4-positive T cells which were classified
as reactive in the FACScan experiment on the basis of
.gamma.-interferon expression.
[0088] FIG. 6 shows the analysis of FACScan experiments after
restimulating specific human T cells of an HLA A1-positive donor
with peripheral blood mononuclear cells (PBMCs) which present
different antigens. The name of the particular peptide pool with
which the PBMCs were loaded is listed from left to right. "None"
represents PBMCs which were incubated only with buffer. The Y axis
shows the proportion of CD8-positive T cells which were classified
as reactive in the FACScan experiment on the basis of
.gamma.-interferon expression.
[0089] FIG. 7 shows the analysis of FACScan experiments after
restimulating specific human T cells of an HLA A24-positive donor
with peripheral blood mononuclear cells (BLCLs) which present
different antigens. The name of the particular peptide pool with
which the BLCLs were loaded is listed from left to right. "None"
represents BLCLs which were incubated only with buffer. The Y axis
shows the proportion of CD8-positive T cells which were classified
as reactive in the FACScan experiment on the basis of
.gamma.-interferon expression.
[0090] FIG. 8 shows the analysis of a .sup.51Cr release experiment
after loading BLCL cells of an HLA A24-positive donor (=target
cells) with peptide 9. The target cells were lysed by T cells
stimulated with peptides 1-43 (=effector cells). The X axis shows
the ratio of the effector cells used to the target cells used, and
the Y axis shows the % of specifically lysed target cells,
determined by .sup.51Cr release from the target cells. The % values
were calculated according to the formula given in Example 7.
EXAMPLES
1. Description of Starting Materials
[0091] The preparation of HPV16
L1.sub..DELTA.c.multidot.E7.sub.1-55 CVLPs was carried out
according to the German patent application DE 198 12 941, see also
Muller M. et al. (1997) Virology 234, 93-111.
[0092] The preparation of L1 VLPs was carried out according to
Muller M. et al. (1997) Virology 234, 93-111.
[0093] T2 cells which can be obtained under ATCC number: CRL-1992
have a defect in the antigen processing-associated transport, which
stops the loading of MHC-1 molecules in the endoplasmic reticulum.
The unloaded MHC-1 molecules which are nevertheless present on the
cell surface may be loaded, for example, by incubating the cells in
peptide-containing media so that these cells are very suitable for
presenting an antigen.
[0094] WEHI cells can be obtained under ATCC number CRL-2148.
[0095] PMBC means peripheral blood mononuclear cells whose
isolation is described, for example, in Rudolf M. P. et al. (1999),
Biol. Chem. 380, 335-40.
[0096] BLCL means a B-cell line transformed with the aid of
Epstein-Barr virus (obtained from Dr. Andreas Kaufmann,
Friedrich-Schiller University, Jena, Germany).
[0097] BB7.2 means an .alpha.-HLA A2.01-specific monoclonal mouse
antibody (ATCC HB-82).
[0098] .alpha.-hum CD28 means a monoclonal mouse antibody which is
directed against the extracellular part of human CD28.
[0099] .alpha.-hum CD3/PE means a monoclonal mouse antibody which
is directed against the extracellular part of human CD3 (.epsilon.)
and contains the fluorescent marker phycoerythrin (Medac, Hamburg,
Germany).
[0100] .alpha.-hum CD4/Cychrome means a monoclonal mouse antibody
which is directed against the extracellular part of human CD4 and
contains the fluorescent marker Cychrome (DAKO; Glostrup,
Denmark).
[0101] .alpha.-hum .gamma. Interferon/FITC means a monoclonal rat
antibody which is directed against human .gamma. interferon and
contains the fluorescent marker FITC (Medac, Hamburg, Germany).
[0102] .alpha.-hum CD8/PE means a monoclonal mouse antibody which
is directed against the extracellular part of human CD8 and
contains the fluorescent marker phycoerythrin (Pharmingen,
Heidelberg, Germany).
[0103] InfluenzaMP means amino acids 58-66 GILGFVFTL of the
influenza matrix protein (see Dunbar P. R. et al. (1998) Curr.
Biol. 26, 413-6).
[0104] HPV16E7 peptide means amino acids 11-20 YMLDLQPETT of human
papillomavirus E7 protein.
[0105] On the basis of the algorithm for potential HLA
A2.01-binding peptides (Parker, K C et al. (1994) J. Immunol.
152:163), carried out in the peptide prediction program by Parker
under http://www-bimas.dcrt.nih.- gov/molbio/hla_bind/index.html,
the peptides below were identified as candidates for HPV16 L1 and
synthesized. The stated amino acid positions of the particular
peptide are shown in relation to Met(+1) of the L1 sequence
deposited under GenBank accession number k02718 (see Table 1
below).
1TABLE 1 Potential HLA A2.01-binding peptides of HPV16 L1 Peptide
name Sequence Relative L1 position 5104 ILVPKVSGL (86-94) 5105
SMDYKQTQL (174-182) 5106 RLVWACVGV (123-131) 5107 HLFNRAGTV
(285-293) 5108 YLRREQMFV (275-283) 5109 TLQANKSEV (238-246) 5112
ILEDWNFGL (426-434) 5113 TLEDTYRFV (441-449) 2016 SLWLPSEATVYL
(28-39) 2017 NLASSNYFPT (311-320) 2018 TLTADVMTYI (408-417) 2019
YLPPVPVSKV (38-47) 2020 YDLQFIFQL (396-404) 2021 FQLCKITLT
(402-410) 2022 ICWGNQLFV (349-357) 2023 KVVSTDEYV (46-54) 2024
QLFVTVVDT (354-362) 2025 GLQYRVFRI (93-101)
[0106] Furthermore, 20 mer peptides which overlap by in each case 9
amino acids and which include the sequence of HPV16 L1 and E7
proteins were synthesized. The peptides were numbered consecutively
from 1 to 52. Their name and their sequence are summarized in the
following table in which "restr." means restricted.
2TABLE 2 Synthetic overlapping 20mer peptides of HPV16 L1 and E7
Peptide Relative Epitope name Sequence position information
L1-Peptide 1 MSLWLPSEATVYLPPVPVSK (1-20) 2 YLPPVPVSKVVSTDEYVART
(12-31) 3 STDEYVARTNIYYHAGTSRL (23-42) 4 YYHAGTSRLLAVGHPYFPIK
(34-53) 5 VGHPYFPIKKPNNNKILVPK (45-64) 6 NNNKILVPKVSGLQYRVFRI
(56-75) 7 GLQYRVFRIHLPDPNKFGFP (67-86) 8 PDPNKFGFPDTSFYNPDTQR
(78-97) 9 SFYNPDTQRLVWACVGVEVG (89-108) cytotoxic epitope HLA A24
restr. 10 WACVGVEVGRGQPLGVGISG (100-119) 11 QPLGVGISGHPLLNKLDDTE
(111-130) 12 LLNKLDDTENASAYAANAGV (122-141) 13 SAYAANAGVDNRECISMDYK
(133-152) 14 RECISMDYKQTQLCLIGCKP (144-163) 15 QLCLIGCKPPIGEHWGKGSP
(155-174) 16 GEHWGKGSPCTNVAVNPGDC (166-185) 17 NVAVNPGDCPPLELINTVIQ
(177-196) 18 LELINTVIQDGDMVDTGFGA (188-207) 19 DMVDTGFGAMDFTTLQANKS
(199-218) 20 FTTLQANKSEVPLDICTSIC (210-229) 21 PLDICTSICKYPDYIKMVSE
(221-240) 22 PDYIKMVSEPYGDSLFFYLR (232-251) 23 GDSLFFYLRREQMFVRHLFN
(243-262) 24 QMFVRHLFNRAGAVGENVPD (254-273) 25 GAVGENVPDDLYIKGSGSTA
(265-284) 26 YIKGSGSTANLASSNYFPTP (276-295) 27 ASSNYFPTPSGSMVTSDAQI
(287-306) T-helper epitope 28 SMVTSDAQIFNKPYWLQRAQ (298-317)
T-helper epitope 29 KPYWLQRAQGHNNGICWGNQ (309-328) 30
NNGICWGNQLFVTVVDTTRS (320-339) 31 VTVVDTTRSTNMSLCAAIST (331-350) 32
MSLCAAISTSETTYKNTNFK (342-361) 33 TTYKNTNFKEYLRHGEEYDL (353-372) 34
LRHGEEYDLQFIFQLCKITL (364-383) 35 IFQLCKITLTADVMTYIHSM (375-394) 36
DVMTYIHSMNSTILEDWNFG (386-405) 37 TILEDWNFGLQPPPGGTLED (397-416) 38
PPPGGTLEDTYRFVTSQAIA (408-427) 39 RFVTSQAIACQKHTPPAPKE (419-438) 40
KHTPPAPKEDPLKKYTFWEV (430-449) 41 LKKYTFWEVNLKEKFSADLD (441-460) 42
KEKFSADLDQFPLGRKFLLQ (452-471) 43 PLGRKFLLQAGMHGDTPTLH (463-482)
cytotoxic epitope HLA A1 restr. E7 Peptides 44 MHGDTPTLHEYMLDLQPETT
(1-20) 45 MLDLQPETTDLYCYEQLNDS (12-31) cytotoxic epitope HLA A1
restr. 46 YCYEQLNDSSEEEDEIDGPA (23-42) 47 EEDEIDGPAGQAEPDRAHYN
(34-53) cytotoxic epitope HLA A1 restr. 48 AEPDRAHYNIVTFCCKCDST
(45-64) cytotoxic epitope HLA A1 restr. 49 TFCCKCDSTLRLCVQSTHVD
(56-75) 50 LCVQSTHVDIRTLEDLLMGT (67-86) 51 TLEDLLMGTLGIVCPICSQKP
(78-97) Influenza control peptide 52 KEYLRHGEEGILGFVFTLCK
[0107] Golgi Plug is obtainable through Pharmingen (Hamburg,
Germany).
[0108] Monensin is obtainable through Sigma (Deisenhofen,
Germany).
[0109] IL-2 was obtained from Becton Dickinson (Hamburg,
Germany).
[0110] MTT solution in PBS means 2.5 mg/ml
3-[4,5-dimethylthiazol-2-yl]-2,- 5-diphenyltetrazolium bromide in
PBS (Sigma, Deisenhofen, Germany).
[0111] PBS means phosphate-buffered saline and consists of 16.6 mM
Na.sub.2HPO.sub.4 8.4 mM NaH.sub.2PO.sub.4, 150 mM NaCl pH 7.4.
[0112] Cells were cultured in each case at 37.degree. C. and 5%
CO.sub.2 in RPMI medium (Gibco BRL, Eggenstein; Germany) with 10%
fetal calf serum, kanamycin and ampicillin.
[0113] Luma plates and the Canberra-Packerd B-plate counter were
obtained from Canberra-Packerd, Dreieich, Germany.
[0114] FACScan calibur means fluorescence-activated cell sorter;
the apparatus was obtained from Becton Dickenson (Hamburg,
Germany).
[0115] Cellquest software was obtained from Becton Dickinson
(Hamburg, Germany).
2. Peptide-Specific TNFA Secretion by CVLP-Stimulated T Cells
[0116] a) Preparation of CVLP-Specific T Cells Human T cells
(4.times.10.sup.5) of an HLA A2.01-positive donor were stimulated
with HPV16 L1.sup..DELTA.C.multidot.E7.sup.1-55 CVLPs at 37.degree.
C. for 8 weeks with weekly addition of 1 .mu.g/ml CVLPs, 10.sup.5
irradiated peripheral blood mononuclear cells (PMBCs) and 10 IU/ml
IL-2, and harvested.
[0117] b) Stimulation with Antigens
[0118] The cells were stimulated in 100 .mu.l of medium at
37.degree. C. overnight with different antigens (PMBC+E7 peptide;
PMBC+HPV16 L1 .sub..DELTA.C.multidot.E7.sub.1-55 (CVLP); PMBC+5104,
5105, 5106, 5107, 5108, 5109, 5112, 5113, 10 .mu.g/ml each) in the
presence of 10 .mu.U/ml IL-2. During this time, stimulated cells
produce TNF.alpha..
[0119] c) Detection of TNF.alpha.
[0120] The following day, 50 .mu.l of supernatant were removed,
frozen, thawed again (in order to destroy possibly coremoved cells)
and added to 50 .mu.l of a cell suspension containing
0.9.times.10.sup.6 WEHI cells, 2 .mu.g/ml actinomycin D and 400 mM
LiCl. The cells were incubated at 37.degree. C. for 24 h. During
this time, TNF .DELTA. (if present in the supernatant) induces
apoptosis of WEHI cells. Addition of 50 .mu.l of a 2.5 mg/ml MTT
solution in PBS stained non-apoptotic cells brown within three
hours, whereas apoptotic cells remained yellow. All cells were
lysed by adding 100 .mu.l of lysis buffer (34%
N,N-dimethylformamide, 20% sodium dodecyl sulfate) and incubating
at 37.degree. for at least 6 hours so that the dyes were released.
Finally, absorption of the solution was measured at 595 nm.
[0121] FIG. 1 shows the absorption measured at 595 nm as a function
of the different antigens. Low absorption means low dye production
and thus many apoptotic cells which had thus been exposed to a lot
of TNF.alpha. so that the corresponding T cells had been
stimulated. Thus, T-cell stimulation improved with decreasing
absorption at 595 nm.
[0122] Result: peptides 5104, 5106, 5107, 5108, 5109 and 5112 were
capable of stimulating CVLP-specific T cells.
3. Binding of Peptides to T2 Cells
[0123] a) Loading of T2 cells 2.5.times.10.sup.6 T2 cells of an HLA
A2.01-positive donor were incubated in medium containing 2% human
serum at 37.degree. C. overnight in the presence of 0, 10 or 100
.mu.g/ml 5105, 5106, 5107, 5109, 5112, 5113, InfluenzaMP, 2016,
2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025 peptide.
During this time, fitting peptides can bind to the
unphysiologically empty MHC-1 molecules and thus stabilize said
molecules, whereas MHC-1 molecules without fitting peptides are
relatively quickly reabsorbed into the cell. Thus, specifically
binding peptides increase the number of MHC-1 molecules on the cell
surface.
[0124] b) Detection of Peptide Binding to MHC-1 Complexes of T2
Cells
[0125] The following morning, the cells were harvested, washed with
PBS containing 0.5% bovine serum albumin (BSA) and the MHC-1
molecules were detected. This is carried out by incubating with
antibody BB7.2 on ice for 30 min, washing and staining with an
.alpha.-mouse FITC antibody on ice for a further 30 min. The cells
were washed again, measured in a FACScan calibur and analyzed using
Cellquest software.
[0126] FIG. 2 shows the measured fluorescence as a function of the
various peptides.
[0127] Result: T2 cells, after incubation with L1 peptides 5106,
5107, 5109, 5112, 2016, 2017, 2018, 2019, 2020 and 2022 show, as
after incubation with the known influenza peptide MP, significantly
more MHC molecules on the cell surface, indicating binding of the
corresponding peptides to the MHC molecules.
4. Restimulation of CVLP-Stimulated T Cells with Different
Antigen-Presenting Cells
[0128] Human T cells (4.times.10.sup.5) of an HLA A2.01-positive
donor were stimulated with HPV16
L1.sub..DELTA.C.multidot.E7.sub.1-55 CVLPs at 37.degree. C. for 8
weeks with weekly addition of 1 .mu.g/ml CVLPs and 10.sup.5
antigen-presenting cells (irradiated PMBCs), and harvested. The
cells were then restimulated in 100 82 l of medium at 37.degree. C.
with 10 .mu.g/ml various antigens in the presence of 10 IU/ml IL2
and 0.5 .mu.g/ml .alpha.-human CD28:
[0129] a) with CVLP-incubated PBMCs overnight
[0130] b) with L1 2022 peptide-incubated PBMCs overnight
[0131] c) with L1 2025 peptide (control peptide)-incubated PBMCs
overnight
[0132] After one hour, 1 .mu.l of Golgi Plug was added. The cells
were incubated at 37.degree. C. for a further 5 hours. The cells
were then fixed and permeabilized, and stained with .alpha.-hum
CD3/PE, with .alpha.-hum CD4/Cychrome and with .alpha.-hum
.gamma.-interferon/FITC. The cells were examined in a FACScan
calibur with respect to their label and the data were analyzed with
the aid of Cellquest software.
[0133] Result: FIG. 3 shows that CVLP-incubated PBMCs as well as L1
peptide 2022-incubated PBMCs, but not control peptide-incubated
PBMCs, effected restimulation of CVLP-stimulated T cells.
5. Restimulation of CVLP-Stimulated T Cells with Different
Antigen-Presenting Cells
[0134] Human T cells (4.times.10.sup.5) of an HLA A1-positive donor
were stimulated with HPV16 L1.sub..DELTA.C.multidot.E7.sub.1-55
CVLPs at 37.degree. C. for 5 weeks with weekly addition of 1
.mu.g/ml CVLPs and 10.sup.5 antigen-presenting cells (irradiated
PMBCs), and harvested. The cells were then restimulated in 100
.mu.l of medium at 37.degree. C. with 10 .mu.g/ml of the peptides
listed along the X axis of FIG. 4 in the presence of 10 IU/ml IL2.
Cells incubated only with buffer served as a negative control.
[0135] After one hour, 1 .mu.l of Monensin (300 .mu.M) was added.
The cells were incubated at 37.degree. C. for a further 5 hours.
The cells were then fixed and permeabilized, and stained with
.alpha.-hum CD8/PE, with .alpha.-hum CD4/Cychrome and with a
c.alpha.-hum .gamma.-interferon/FITC. The cells were examined in a
FACScan calibur with respect to their label and the data were
analyzed with the aid of Cellquest software.
[0136] Result: FIG. 4 shows that PBMCs incubated with peptides 43,
47 and 48, but not PBMCs incubated with the remaining peptides,
effected restimulation of CVLP-stimulated T cells. Peptide 43
contains the 9 mer peptide of the sequence MHGDTPTLH, and the two
overlapping peptides 47 and 48 contain the 10 mer peptide of the
sequence QAEPDRAHYN, which in each case have been described as HLA
A1-binding peptides in Kast et al. (supra), but for which it has
been impossible so far to carry out a functional detection.
6. Restimulation of CVLP-Stimulated T Cells with Different
Antigen-Presenting Cells
[0137] Human T cells (4.times.10.sup.5) of a non-HLA-classified
donor were stimulated with HPV16
L1.sub..DELTA.C.multidot.E7.sub.1-55 CVLPs at 37.degree. C. for 6
weeks with weekly addition of 1 .mu.g/ml CVLPs and 10.sup.5
antigen-presenting cells (irradiated PMBCs), and harvested. The
cells were then restimulated in 100 .mu.l of medium at 37.degree.
C. with 10 .mu.g/ml of the peptides listed along the X axis of FIG.
5 in the presence of 10 IU/ml IL2. Cells incubated only with buffer
served as a negative control.
[0138] After one hour, 1 .mu.l of Monensin (300 .mu.M) was added.
The cells were incubated at 37.degree. C. for a further 5 hours.
The cells were then fixed and permeabilized, and stained with
.alpha.-hum CD8/PE, with .alpha.-hum CD4/Cychrome and with
.alpha.-hum .gamma.-interferon/FITC. The cells were examined in a
FACScan calibur with respect to their label and the data were
analyzed with the aid of Cellquest software.
[0139] Result: FIG. 5 shows that PBMCs incubated with peptides 27
and 28, but not PBMCs incubated with the remaining peptides,
effected restimulation of CVLP-stimulated T cells. The two
overlapping peptides 27 and 28 contain the peptide of the sequence
SMVTSDAQI so that the actually recognized peptide essentially must
include this sequence.
7. Restimulation of CVLP-Stimulated T Cells with Different
Antigen-Presenting Cells
[0140] Human T cells (4.times.10.sup.5) of an HLA A1-positive donor
were stimulated with HPV16 L1.sub..DELTA.C.multidot.E7.sub.1-55
CVLPs at 37.degree. C. for 6 weeks with weekly addition of 1
.mu.g/ml CVLPs and 10.sup.5 antigen-presenting cells (irradiated
PMBCs), and harvested.
[0141] The 20 mer peptides 1 to 51 were combined in peptide pools A
to H and 1 to 7 according to the matrix
3 Pools A B C D E F G H 1 1 2 3 4 5 6 7 8 2 9 10 11 12 13 14 15 16
3 17 18 19 20 21 22 23 24 4 25 26 27 28 29 30 31 32 5 33 34 35 36
37 38 39 40 6 41 42 43 44 45 46 47 48 7 49 50 51
[0142] The T cells of an HLA A1-positive donor were then
restimulated in 100 .mu.l of medium at 37.degree. C. with the
peptide pools in the presence of 10 IU/ml IL2. In this connection,
such amounts of peptide pools were used that for each individual
peptide 1 .mu.g/ml was added. Cells incubated only with buffer
served as a negative control.
[0143] After one hour, 1 .mu.l of Golgi Plug was added. The cells
were incubated at 37.degree. C. for a further 5 hours. The cells
were then fixed and permeabilized, and stained with .alpha.-hum
CD8/PE, with .alpha.-hum CD4/Cychrome and with .alpha.-hum
.gamma.-interferon/FITC. The cells were examined in a FACScan
calibur with respect to their label and the data were analyzed with
the aid of Cellquest software.
[0144] Result: FIG. 6 shows that PBMCs incubated with peptide pools
E and 6, but not PBMCs incubated with the remaining peptide pools,
effected restimulation of CVLP-stimulated T cells. Peptide pools E
and 6 both contain peptide 45 which thus is in all probability
responsible for restimulation of CVLP-stimulated T cells. Peptide
45 in turn contains peptide ETTDLYCY which has been described as
HLA A1-binding peptide by Kast et al. (supra), but for which it has
been impossible so far to carry out a functional detection.
[0145] Furthermore, the T cells of an HLA A24-positive donor were
restimulated with the peptide pools as above and analyzed.
[0146] Result: FIG. 7 shows that PBMCs incubated with peptide pools
A and 2, but not PBMCs incubated with the remaining peptide pools,
effected restimulation of CVLP-stimulated T cells. Peptide pools A
and 2 both contain peptide 9 which thus is in all probability
responsible for restimulation of CVLP-stimulated T cells. The
prediction according to Parker et al. (supra) results in a
potential peptide for HLA A24 which has the sequence FYNPDTQRL and
is thus probably responsible for the activity of peptide 9.
8. Lysis of BLCL Cells Loaded with Peptide 9
[0147] BLCL cells of an HLA A24-positive donor were incubated with
.sup.15Cr at 37.degree. C. for one hour, washed three times with
medium and divided into 2 aliquots. 10 .mu.g/ml of peptide 9 were
added to one aliquot of the cells, and the other aliquot served as
a negative control in the absence of a peptide. Subsequently, in
each case 2000 cells (=target cells) were added to increasing
amounts of T cells (=effector cells) in a total volume of 150
.mu.l. The T cells had been stimulated previously over 5 weeks with
a mixture of 43 peptides (peptides 1-43, 1 .mu.g/ml each). Reaction
mixtures for spontaneous and maximum cell lysis were set up in
parallel. For spontaneous lysis, target cells which were incubated
in medium were used, and for maximum lysis target cells which were
incubated with 0.5% Triton were used. The mixtures were incubated
at 37.degree. C. for 5 h. 50 .mu.l of mixture supernatant were
applied to Luma plates and dried at room temperature overnight. On
the following morning, the amount of radioactive .sup.51Cr was
determined with the aid of a Canberra-Packerd B-plate counter
(counts) and compared to the maximal lysed cells of the Triton
mixture. The percentage of specific lysis was determined according
to the formula:
x=100.multidot.(counts-spontaneous counts)/(maximal
counts-spontaneous counts).
[0148] FIG. 8 shows that it was possible for the T cells to lyse
BLCL cells loaded with peptide 9 effectively, but not unloaded BLCL
cells. Peptide 9 is thus an HLA A24-restricted cytotoxic T-cell
epitope.
Sequence CWU 1
1
77 1 9 PRT Human papillomavirus type 16 1 Ile Leu Val Pro Lys Val
Ser Gly Leu 1 5 2 9 PRT Human papillomavirus type 16 2 Arg Leu Val
Trp Ala Cys Val Gly Val 1 5 3 9 PRT Human papillomavirus type 16 3
His Leu Phe Asn Arg Ala Gly Thr Val 1 5 4 9 PRT Human
papillomavirus type 16 4 Tyr Leu Arg Arg Glu Gln Met Phe Val 1 5 5
9 PRT Human papillomavirus type 16 5 Thr Leu Gln Ala Asn Lys Ser
Glu Val 1 5 6 9 PRT Human papillomavirus type 16 6 Ile Leu Glu Asp
Trp Asn Phe Gly Leu 1 5 7 12 PRT Human papillomavirus type 16 7 Ser
Leu Trp Leu Pro Ser Glu Ala Thr Val Tyr Leu 1 5 10 8 10 PRT Human
papillomavirus type 16 8 Asn Leu Ala Ser Ser Asn Tyr Phe Pro Thr 1
5 10 9 10 PRT Human papillomavirus type 16 9 Thr Leu Thr Ala Asp
Val Met Thr Tyr Ile 1 5 10 10 10 PRT Human papillomavirus type 16
10 Tyr Leu Pro Pro Val Pro Val Ser Lys Val 1 5 10 11 9 PRT Human
papillomavirus type 16 11 Tyr Asp Leu Gln Phe Ile Phe Gln Leu 1 5
12 9 PRT Human papillomavirus type 16 12 Ile Cys Trp Gly Asn Gln
Leu Phe Val 1 5 13 9 PRT Human papillomavirus type 16 13 Phe Tyr
Asn Pro Asp Thr Gln Arg Leu 1 5 14 9 PRT Human papillomavirus type
16 14 Met His Gly Asp Thr Pro Thr Leu His 1 5 15 8 PRT Human
papillomavirus type 16 15 Glu Thr Thr Asp Leu Tyr Cys Tyr 1 5 16 10
PRT Human papillomavirus type 16 16 Gln Ala Glu Pro Asp Arg Ala His
Tyr Asn 1 5 10 17 9 PRT Human papillomavirus type 16 17 Ser Met Val
Thr Ser Asp Ala Gln Ile 1 5 18 9 PRT Human papillomavirus type 16
18 Gly Ile Leu Gly Phe Val Phe Thr Leu 1 5 19 10 PRT Human
papillomavirus type 16 19 Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr 1
5 10 20 9 PRT Human papillomavirus type 16 20 Ser Met Asp Tyr Lys
Gln Thr Gln Leu 1 5 21 9 PRT Human papillomavirus type 16 21 Thr
Leu Glu Asp Thr Tyr Arg Phe Val 1 5 22 9 PRT Human papillomavirus
type 16 22 Phe Gln Leu Cys Lys Ile Thr Leu Thr 1 5 23 9 PRT Human
papillomavirus type 16 23 Lys Val Val Ser Thr Asp Glu Tyr Val 1 5
24 9 PRT Human papillomavirus type 16 24 Gln Leu Phe Val Thr Val
Val Asp Thr 1 5 25 9 PRT Human papillomavirus type 16 25 Gly Leu
Gln Tyr Arg Val Phe Arg Ile 1 5 26 20 PRT Human papillomavirus type
16 26 Met Ser Leu Tyr Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro
Val 1 5 10 15 Pro Val Ser Lys 20 27 20 PRT Human papillomavirus
type 16 27 Tyr Leu Pro Pro Val Pro Val Ser Lys Val Val Ser Thr Asp
Glu Tyr 1 5 10 15 Val Ala Arg Thr 20 28 20 PRT Human papillomavirus
type 16 28 Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn Ile Tyr Tyr His
Ala Gly 1 5 10 15 Thr Ser Arg Leu 20 29 20 PRT Human papillomavirus
type 16 29 Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His
Pro Tyr 1 5 10 15 Phe Pro Ile Lys 20 30 20 PRT Human papillomavirus
type 16 30 Val Gly His Pro Tyr Phe Pro Ile Lys Lys Pro Asn Asn Asn
Lys Ile 1 5 10 15 Leu Val Pro Lys 20 31 20 PRT Human papillomavirus
type 16 31 Asn Asn Asn Lys Ile Leu Val Pro Lys Val Ser Gly Leu Gln
Tyr Arg 1 5 10 15 Val Phe Arg Ile 20 32 20 PRT Human papillomavirus
type 16 32 Gly Leu Gln Tyr Arg Val Phe Arg Ile His Leu Pro Asp Pro
Asn Lys 1 5 10 15 Phe Gly Phe Pro 20 33 20 PRT Human papillomavirus
type 16 33 Pro Asp Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr
Asn Pro 1 5 10 15 Asp Thr Gln Arg 20 34 20 PRT Human papillomavirus
type 16 34 Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp Ala Cys
Val Gly 1 5 10 15 Val Glu Val Gly 20 35 20 PRT Human papillomavirus
type 16 35 Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro Leu
Gly Val 1 5 10 15 Gly Ile Ser Gly 20 36 20 PRT Human papillomavirus
type 16 36 Gln Pro Leu Gly Val Gly Ile Ser Gly His Pro Leu Leu Asn
Lys Leu 1 5 10 15 Asp Asp Thr Glu 20 37 20 PRT Human papillomavirus
type 16 37 Leu Leu Asn Lys Leu Asp Asp Thr Glu Asn Ala Ser Ala Tyr
Ala Ala 1 5 10 15 Asn Ala Gly Val 20 38 20 PRT Human papillomavirus
type 16 38 Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg Glu Cys
Ile Ser 1 5 10 15 Met Asp Tyr Lys 20 39 20 PRT Human papillomavirus
type 16 39 Arg Glu Cys Ile Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys
Leu Ile 1 5 10 15 Gly Cys Lys Pro 20 40 20 PRT Human papillomavirus
type 16 40 Gln Leu Cys Leu Ile Gly Cys Lys Pro Pro Ile Gly Glu His
Trp Gly 1 5 10 15 Lys Gly Ser Pro 20 41 20 PRT Human papillomavirus
type 16 41 Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr Asn Val Ala
Val Asn 1 5 10 15 Pro Gly Asp Cys 20 42 20 PRT Human papillomavirus
type 16 42 Asn Val Ala Val Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu
Ile Asn 1 5 10 15 Thr Val Ile Gln 20 43 20 PRT Human papillomavirus
type 16 43 Leu Glu Leu Ile Asn Thr Val Ile Gln Asp Gly Asp Met Val
Asp Thr 1 5 10 15 Gly Phe Gly Ala 20 44 20 PRT Human papillomavirus
type 16 44 Asp Met Val Asp Thr Gly Phe Gly Ala Met Asp Phe Thr Thr
Leu Gln 1 5 10 15 Ala Asn Lys Ser 20 45 20 PRT Human papillomavirus
type 16 45 Phe Thr Thr Leu Gln Ala Asn Lys Ser Glu Val Pro Leu Asp
Ile Cys 1 5 10 15 Thr Ser Ile Cys 20 46 20 PRT Human papillomavirus
type 16 46 Pro Leu Asp Ile Cys Thr Ser Ile Cys Lys Tyr Pro Asp Tyr
Ile Lys 1 5 10 15 Met Val Ser Glu 20 47 20 PRT Human papillomavirus
type 16 47 Pro Asp Tyr Ile Lys Met Val Ser Glu Pro Tyr Gly Asp Ser
Leu Phe 1 5 10 15 Phe Tyr Leu Arg 20 48 20 PRT Human papillomavirus
type 16 48 Gly Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe
Val Arg 1 5 10 15 His Leu Phe Asn 20 49 20 PRT Human papillomavirus
type 16 49 Gln Met Phe Val Arg His Leu Phe Asn Arg Ala Gly Ala Val
Gly Glu 1 5 10 15 Asn Val Pro Asp 20 50 20 PRT Human papillomavirus
type 16 50 Gly Ala Val Gly Glu Asn Val Pro Asp Asp Leu Tyr Ile Lys
Gly Ser 1 5 10 15 Gly Ser Thr Ala 20 51 20 PRT Human papillomavirus
type 16 51 Tyr Ile Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser Ser
Asn Tyr 1 5 10 15 Phe Pro Thr Pro 20 52 20 PRT Human papillomavirus
type 16 52 Ala Ser Ser Asn Tyr Phe Pro Thr Pro Ser Gly Ser Met Val
Thr Ser 1 5 10 15 Asp Ala Gln Ile 20 53 20 PRT Human papillomavirus
type 16 53 Ser Met Val Thr Ser Asp Ala Gln Ile Phe Asn Lys Pro Tyr
Trp Leu 1 5 10 15 Gln Arg Ala Gln 20 54 20 PRT Human papillomavirus
type 16 54 Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly
Ile Cys 1 5 10 15 Trp Gly Asn Gln 20 55 20 PRT Human papillomavirus
type 16 55 Asn Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val
Val Asp 1 5 10 15 Thr Thr Arg Ser 20 56 20 PRT Human papillomavirus
type 16 56 Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met Ser Leu
Cys Ala 1 5 10 15 Ala Ile Ser Thr 20 57 20 PRT Human papillomavirus
type 16 57 Met Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu Thr Thr Tyr
Lys Asn 1 5 10 15 Thr Asn Phe Lys 20 58 20 PRT Human papillomavirus
type 16 58 Thr Thr Tyr Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His
Gly Glu 1 5 10 15 Glu Tyr Asp Leu 20 59 20 PRT Human papillomavirus
type 16 59 Leu Arg His Gly Glu Glu Tyr Asp Leu Gln Phe Ile Phe Gln
Leu Cys 1 5 10 15 Lys Ile Thr Leu 20 60 20 PRT Human papillomavirus
type 16 60 Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Asp Val Met
Thr Tyr 1 5 10 15 Ile His Ser Met 20 61 20 PRT Human papillomavirus
type 16 61 Asp Val Met Thr Tyr Ile His Ser Met Asn Ser Thr Ile Leu
Glu Asp 1 5 10 15 Trp Asn Phe Gly 20 62 20 PRT Human papillomavirus
type 16 62 Thr Ile Leu Glu Asp Trp Asn Phe Gly Leu Gln Pro Pro Pro
Gly Gly 1 5 10 15 Thr Leu Glu Asp 20 63 20 PRT Human papillomavirus
type 16 63 Pro Pro Pro Gly Gly Thr Leu Glu Asp Thr Tyr Arg Phe Val
Thr Ser 1 5 10 15 Gln Ala Ile Ala 20 64 20 PRT Human papillomavirus
type 16 64 Arg Phe Val Thr Ser Gln Ala Ile Ala Cys Gln Lys His Thr
Pro Pro 1 5 10 15 Ala Pro Lys Glu 20 65 20 PRT Human papillomavirus
type 16 65 Lys His Thr Pro Pro Ala Pro Lys Glu Asp Pro Leu Lys Lys
Tyr Thr 1 5 10 15 Phe Trp Glu Val 20 66 20 PRT Human papillomavirus
type 16 66 Leu Lys Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys
Phe Ser 1 5 10 15 Ala Asp Leu Asp 20 67 20 PRT Human papillomavirus
type 16 67 Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly
Arg Lys 1 5 10 15 Phe Leu Leu Gln 20 68 20 PRT Human papillomavirus
type 16 68 Pro Leu Gly Arg Lys Phe Leu Leu Gln Ala Gly Met His Gly
Asp Thr 1 5 10 15 Pro Thr Leu His 20 69 20 PRT Human papillomavirus
type 16 69 Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp
Leu Gln 1 5 10 15 Pro Glu Thr Thr 20 70 20 PRT Human papillomavirus
type 16 70 Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys Tyr
Glu Gln 1 5 10 15 Leu Asn Asp Ser 20 71 20 PRT Human papillomavirus
type 16 71 Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp
Glu Ile 1 5 10 15 Asp Gly Pro Ala 20 72 20 PRT Human papillomavirus
type 16 72 Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro
Asp Arg 1 5 10 15 Ala His Tyr Asn 20 73 20 PRT Human papillomavirus
type 16 73 Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys
Cys Lys 1 5 10 15 Cys Asp Ser Thr 20 74 20 PRT Human papillomavirus
type 16 74 Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val
Gln Ser 1 5 10 15 Thr His Val Asp 20 75 20 PRT Human papillomavirus
type 16 75 Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu
Asp Leu 1 5 10 15 Leu Met Gly Thr 20 76 21 PRT Human papillomavirus
type 16 76 Thr Leu Glu Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys
Pro Ile 1 5 10 15 Cys Ser Gln Lys Pro 20 77 20 PRT Influenza virus
type A 77 Lys Glu Tyr Leu Arg His Gly Glu Glu Gly Ile Leu Gly Phe
Val Phe 1 5 10 15 Thr Leu Cys Lys 20
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References