U.S. patent application number 12/292281 was filed with the patent office on 2009-08-27 for inducing cellular immune responses to human papillomavirus using peptide and nucleic acid compositions.
This patent application is currently assigned to Pharmexa Inc.. Invention is credited to Esteban Celis, Robert Chesnut, Howard M. Grey, Alessandro Sette, John Sidney, Scott Southwood.
Application Number | 20090214632 12/292281 |
Document ID | / |
Family ID | 26868370 |
Filed Date | 2009-08-27 |
United States Patent
Application |
20090214632 |
Kind Code |
A1 |
Sette; Alessandro ; et
al. |
August 27, 2009 |
Inducing cellular immune responses to human papillomavirus using
peptide and nucleic acid compositions
Abstract
This invention uses our knowledge of the mechanisms by which
antigen is recognized by T cells to identify and prepare human
papillomavirus (HPV) epitopes, and to develop epitope-based
vaccines directed towards HPV. More specifically, this application
communicates our discovery of pharmaceutical compositions and
methods of use in the prevention and treatment of HPV
infection.
Inventors: |
Sette; Alessandro; (La
Jolla, CA) ; Sidney; John; (San Diego, CA) ;
Southwood; Scott; (Santee, CA) ; Chesnut; Robert;
(Cardiff-by-the-Sea, CA) ; Celis; Esteban;
(Rochester, MN) ; Grey; Howard M.; (La Jolla,
CA) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
Pharmexa Inc.
|
Family ID: |
26868370 |
Appl. No.: |
12/292281 |
Filed: |
November 14, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10149136 |
Oct 29, 2002 |
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PCT/US00/33549 |
Dec 11, 2000 |
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12292281 |
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09641528 |
Aug 15, 2000 |
7026443 |
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10149136 |
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60172705 |
Dec 10, 1999 |
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Current U.S.
Class: |
424/450 ;
424/186.1; 435/372; 530/300; 536/23.72 |
Current CPC
Class: |
A61K 39/12 20130101;
A61K 2039/55555 20130101; A61P 31/12 20180101; C07K 14/005
20130101; A61P 35/00 20180101; C12N 2710/20022 20130101; A61K
2039/5158 20130101; A61K 2039/55516 20130101; A61P 31/20 20180101;
A61P 37/04 20180101; A61K 2039/57 20130101; C12N 2710/20034
20130101; A61K 2039/53 20130101 |
Class at
Publication: |
424/450 ;
530/300; 424/186.1; 435/372; 536/23.72 |
International
Class: |
A61K 39/12 20060101
A61K039/12; C07K 14/005 20060101 C07K014/005; A61K 9/127 20060101
A61K009/127; C12N 5/08 20060101 C12N005/08; C07H 21/00 20060101
C07H021/00 |
Claims
1. An isolated prepared human papilloma virus (HPV) epitope
consisting of a sequence selected from the group consisting of the
sequences set out in Tables VII-XX.
2. A composition of claim 1, wherein the epitope is admixed or
joined to a CTL epitope.
3. A composition of claim 2, wherein the CTL epitope is selected
from the group set out in claim 1.
4. A composition of claim 1, wherein the epitope is admixed or
joined to an HTL epitope.
5. A composition of claim 4, wherein the HTL epitope is selected
from the group set out in claim 1.
6. A composition of claim 4, wherein the HTL epitope is a pan-DR
binding molecule.
7. A composition of claim 1, comprising at least three epitopes
selected from the group set out in claim 1.
8. A composition of claim 1, further comprising a liposome, wherein
the epitope is on or within the liposome.
9. A composition of claim 1, wherein the epitope is joined to a
lipid.
10. A composition of claim 1, wherein the epitope is joined to a
linker.
11. A composition of claim 1, wherein the epitope is bound to an
HLA heavy chain, .beta.2-microglobulin, and strepavidin complex,
whereby a tetramer is formed.
12. A composition of claim 1, further comprising an antigen
presenting cell, wherein the epitope is on or within the antigen
presenting cell.
13. A composition of claim 12, wherein the epitope is bound to an
HLA molecule on the antigen presenting cell, whereby when a
cytotoxic lymphocyte (CTL) is present that is restricted to the HLA
molecule, a receptor of the CTL binds to a complex of the HLA
molecule and the epitope.
14. A clonal cytotoxic T lymphocyte (CTL), wherein the CTL is
cultured in vitro and binds to a complex of an epitope selected
from the group set out in Tables VII-XVIII, bound to an HLA
molecule.
15. A peptide comprising at least a first and a second epitope,
wherein the first epitope is selected from the group consisting of
the sequences set out in Tables VII-XX; wherein the peptide
comprise less than 50 contiguous amino acids that have 100%
identity with a native peptide sequence.
16. A composition of claim 15, wherein the first and the second
epitope are selected from the group of claim 14.
17. A composition of claim 16, further comprising a third epitope
selected from the group of claim 15.
18. A composition of claim 15, wherein the peptide is a
heteropolymer.
19. A composition of claim 15, wherein the peptide is a
homopolymer.
20. A composition of claim 15, wherein the second epitope is a CTL
epitope.
21. A composition of claim 15, wherein the second epitope is a
PanDR binding molecule.
22. A composition of claim 1, wherein the first epitope is linked
to an a linker sequence.
23. A vaccine composition comprising: a unit dose of a peptide that
comprises less than 50 contiguous amino acids that have 100%
identity with a native peptide sequence of HPV, the peptide
comprising at least a first epitope selected from the group
consisting of the sequences set out in Tables VII-XX; and; a
pharmaceutical excipient.
24. A vaccine composition in accordance with claim 23, further
comprising a second epitope.
25. A vaccine composition of claim 23, wherein the second epitope
is a PanDR binding molecule.
26. A vaccine composition of claim 23, wherein the pharmaceutical
excipient comprises an adjuvant.
27. An isolated nucleic acid encoding a peptide comprising an
epitope consisting of a sequence selected from the group consisting
of the sequences set out in Tables VII-XX.
28. An isolated nucleic acid encoding a peptide comprising at least
a first and a second epitope, wherein the first epitope is selected
from the group consisting of the sequences set out in Tables
VII-XX; and wherein the peptide comprises less than 50 contiguous
amino acids that have 100% identity with a native peptide
sequence.
29. An isolated nucleic acid of claim 28, wherein the peptide
comprises at least two epitopes selected from the sequences set out
in Tables VII-XX.
30. An isolated nucleic acid of claim 29, wherein the peptide
comprises at least three epitopes selected from the sequences set
out in Tables VII-XX.
31. An isolated nucleic acid of claim 28, wherein the second
peptide is a CTL epitope.
32. An isolated nucleic acid of claim 20, wherein the second
peptide is an HTL epitope.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/149,136, which is the national stage application of Intl.
Appl. No. PCT/US00/33549, filed on Dec. 11, 2000, which published
under PCT Article 21 (2) in English; said Intl. Appl. No.
PCT/US00/33549 claims the benefit of U.S. Prov. Appl. No.
60/172,705, filed on Dec. 10, 1999 and is a continuation-in-part of
U.S. application Ser. No. 09/641,528, filed on Aug. 15, 2000, now
U.S. Pat. No. 7,026,443; all of the above applications are
incorporated herein by reference in their entireties.
REFERENCE TO A SEQUENCE LISTING ON COMPACT DISC
[0002] The Sequence Listing written in file "sequence listing
ascii.txt," 184 kilobytes, created on Nov. 14, 2008, on two
identical copies of compact discs for the application filed
herewith, is herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] Human papillomavirus (HPV) is a member of the
papillomaviridae, a group of small DNA viruses that infect a
variety of higher vertebrates. More than 80 types of HPVs have been
identified. Of these, more than 30 can infect the genital tract.
Some types, generally types 6 and 11, may cause genital warts,
which are typically benign and rarely develop into cancer. Other
strains of HPV, "cancer-associated", or "high-risk" types, can more
frequently lead to the development of cancer. The primary mode of
transmission of these strains of HPV is through sexual contact.
[0004] The main manifestations of the genital warts are
cauliflower-like condylomata acuminata that usually involve moist
surfaces; keratotic and smooth papular warts, usually on dry
surfaces; and subclinical "flat" warts, which are found on any
mucosal or cutaneous surface (Handsfield, H., Am. J. Med. 102
(5A):16-20, 1997). These warts are typically benign but are a
source of inter-individual spread of the virus (Ponten, J. &
Guo, Z., Cancer Surv. 32:201-29, 1998). At least three HPV strains
associated with genital warts have been identified: type 6a (see,
e.g., Hofmann, K. J., et al., Virology 209(2):506-518, 1995), type
6b (see, e.g., Hofmann et al., supra) and type 11 (see, e.g.,
Dartmann, K. et al., Virology 151(1):124-130, 1986).
[0005] Cancer-associated HPVs have been linked with cancer in both
men and women; they include, but are not limited to, HPV-16,
HPV-18, HPV-31, HPV-45, HPV-33 and HPV-56. Other HPV strains,
including types 6 and 11 as well as others, e.g., HPV-5 and HPV-8,
are less frequently associated with cancer. The high risk types are
typically associated with the development of cervical carcinoma and
premalignant lesions of the cervix in women, but are also
associated with similar malignant and premalignant lesions at other
anatomic sites within the lower genital or anogenital tract. These
lesions include neoplasia of the vagina, vulva, perineum, the
penis, and the anus. HPV infection has also been associated with
respiratory tract papillomas, and rarely, cancer, as well as
abnormal growth or neoplasia in other epithelial tissues. See, e.g.
VIROLOGY, 2.sup.ND ED, Fields et al., Eds. Raven Press, New York,
1990, Chapters 58 and 59, for a review of HPV association with
cancer.
[0006] The HPV genome consists of three functional regions, the
early region, the late region, and the "long control region". The
early region gene products control viral replication, transcription
and cellular transformation They include the HPV E1 and E2
proteins, which play a role in HPV DNA replication, and the E6 and
E7 oncoproteins, which are involved in the control of cellular
proliferation. The late region include the genes that encode the
structural proteins L1 and L2, which are the major and minor capsid
proteins, respectively. The "long control region" contains such
sequences as enhancer and promoter regulatory regions.
[0007] HPV expresses different proteins at different stages of the
infection, for example early, as well as late, proteins. Even in
latent infections, however, early proteins are often expressed and
are therefore useful targets for vaccine-based therapies. For
example, high-grade dysplasia and cervical squamous cell carcinoma
continue to express E6 and E7, which therefore can be targeted to
treat disease at both early and late stages of infection.
[0008] Treatment for HPV infection is often unsatisfactory because
of persistence of virus after treatment and recurrence of
clinically apparent disease is common. The treatment may require
frequent visits to clinics and is not directed at elimination of
the virus but at clearing warts. Because of persistence of virus
after treatment, recurrence of clinically apparent disease is
common.
[0009] Thus, a need exists for an efficacious vaccine to both
prevent and treat HPV infection and to treat cancer that is
associated with HPV infection. Effective HPV vaccines would be a
significant advance in the control of sexually transmissable
infections and could also protect against clinical disease,
particularly cancers such as cervical cancer. (see, e.g., Rowen, P.
& Lacey, C., Dermatologic Clinics 16(4):835-838, 1998).
[0010] Virus-specific, human leukocyte antigen (HLA) class
I-restricted cytotoxic T lymphocytes (CTL) are known to play a
major role in the prevention and clearance of virus infections in
vivo (Oldstone et al., Nature 321:239, 1989; Jamieson et al., J.
Virol. 61:3930, 1987; Yap et al, Nature 273:238, 1978; Lukacher et
al., J. Exp. Med. 160:814, 1994; McMichael et al., N. Engl. J. Med.
309:13, 1983; Sethi et al., J. Gen. Virol. 64:443, 1983; Watari et
al., J. Exp. Med. 165:459, 1987; Yasukawa et al., J. Immunol.
143:2051, 1989; Tigges et al., J. Virol. 66:1622, 1993; Reddenhase
et al., J. Virol. 55:263, 1985; Quinnan et al., N. Engl. J. Med.
307:6, 1982). HLA class 1 molecules are expressed on the surface of
almost all nucleated cells. Following intracellular processing of
antigens, epitopes from the antigens are presented as a complex
with the HLA class 1 molecules on the surface of such cells. CTL
recognize the peptide-HLA class I complex, which then results in
the destruction of the cell bearing the HLA-peptide complex
directly by the CTL and/or via the activation of non-destructive
mechanisms e.g., the production of interferon, that inhibit viral
replication.
[0011] Virus-specific T helper lymphocytes are also known to be
critical for maintaining effective immunity in chronic viral
infections. Historically, HTL responses were viewed as primarily
supporting the expansion of specific CTL and B cell populations;
however, more recent data indicate that HTL may directly contribute
to the control of virus replication. For example, a decline in
CD4.sup.+ T cells and a corresponding loss in HTL function
characterize infection with HIV (Lane et al., New Engl. J. Med.
313:79, 1985). Furthermore, studies in HIV infected patients have
also shown that there is an inverse relationship between
virus-specific HTL responses and viral load, suggesting that HTL
plays a role in viremia (see, e.g., Rosenberg et al., Science
278:1447, 1997).
[0012] The development of vaccines with prophylactic and
therapeutic efficacy against HPV is ongoing. Early vaccine
development was hampered by the inability to culture HPV. With the
introduction of cloning techniques and protein expression, however,
some attempts have been made to stimulate humoral and CTL response
to HPV (See, e.g., Rowen, P. & Lacey, C., Dermatologic Clinics
16(4):835-838 (1998)). Studies to date, however, have been
inconclusive.
[0013] Activation of T helper cells and cytotoxic lymphocytes
(CTLs) in the development of vaccines has also been analyzed.
Lehtinen, M., et al. for instance, has shown that some peptides
from the E2 protein of HPV type 16 activate T helper cells and CTLs
(Biochem. Biophys. Res. Commun. 209(2):541-6 (1995). Similarly,
Tarpey et al, has shown that some peptides from HPV type 11 E7
protein can stimulate human HPV-specific CTLs in vitro (Immunology
81:222-227 (1994)) and Borysiewicz et al. have reported a
recombinant vaccinia virus expressing HPV 16 and HPV 17 E6 and E7
that stimulated CTL responses in at least one patient (Lancet
347:1347-1357, 1996).
[0014] The epitope approach, as we have described, allows the
incorporation of various antibody, CTL and HTL epitopes, from
various proteins, in a single vaccine composition. Such a
composition may simultaneously target multiple dominant and
subdominant epitopes and thereby be used to achieve effective
immunization in a diverse population.
[0015] The information provided in this section is intended to
disclose the presently understood state of the art as of the filing
date of the present application. Information is included in this
section which was generated subsequent to the priority date of this
application. Accordingly, information in this section is not
intended, in any way, to delineate the priority date for the
invention.
SUMMARY OF THE INVENTION
[0016] This invention applies our knowledge of the mechanisms by
which antigen is recognized by T cells, for example, to develop
epitope-based vaccines directed towards HPV. More specifically,
this application communicates our discovery of specific epitope
pharmaceutical compositions and methods of use in the prevention
and treatment of HPV infection.
[0017] Upon development of appropriate technology, the use of
epitope-based vaccines has several advantages over current
vaccines, particularly when compared to the use of whole antigens
in vaccine compositions. There is evidence that the immune response
to whole antigens is directed largely toward variable regions of
the antigen, allowing for immune escape due to mutations. The
epitopes for inclusion in an epitope-based vaccine may be selected
from conserved regions of viral or tumor-associated antigens, which
thereby reduces the likelihood of escape mutants. Furthermore,
immunosuppressive epitopes that may be present in whole antigens
can be avoided with the use of epitope-based vaccines.
[0018] An additional advantage of an epitope-based vaccine approach
is the ability to combine selected epitopes (CTL and HTL), and
further, to modify the composition of the epitopes, achieving, for
example, enhanced immunogenicity. Accordingly, the immune response
can be modulated, as appropriate, for the target disease. Similar
engineering of the response is not possible with traditional
approaches.
[0019] Another major benefit of epitope-based immune-stimulating
vaccines is their safety. The possible pathological side effects
caused by infectious agents or whole protein antigens, which might
have their own intrinsic biological activity, is eliminated.
[0020] An epitope-based vaccine also provides the ability to direct
and focus an immune response to multiple selected antigens from the
same pathogen. Thus, patient-by-patient variability in the immune
response to a particular pathogen may be alleviated by inclusion of
epitopes from multiple antigens from the pathogen in a vaccine
composition. In the case of HPV, epitopes derived from multiple
strains may also be included. A "pathogen" may be an infectious
agent or a tumor associated molecule.
[0021] One of the most formidable obstacles to the development of
broadly efficacious epitope-based immunotherapeutics, however, has
been the extreme polymorphism of HLA molecules. To date, effective
non-genetically biased coverage of a population has been a task of
considerable complexity; such coverage has required that epitopes
be used that are specific for HLA molecules corresponding to each
individual HLA allele. Impractically large numbers of epitopes
would therefore have to be used in order to cover ethnically
diverse populations. Thus, there has existed a need for peptide
epitopes that are bound by multiple HLA antigen molecules for use
in epitope-based vaccines. The greater the number of HLA antigen
molecules bound, the greater the breadth of population coverage by
the vaccine.
[0022] Furthermore, as described herein in greater detail, a need
has existed to modulate peptide binding properties, e.g., so that
peptides that are able to bind to multiple HLA antigens do so with
an affinity that will stimulate an immune response. Identification
of epitopes restricted by more than one HLA allele at an affinity
that correlates with immunogenicity is important to provide
thorough population coverage, and to allow the elicitation of
responses of sufficient vigor to prevent or clear an infection in a
diverse segment of the population. Such a response can also target
a broad array of epitopes. The technology disclosed herein provides
for such favored immune responses.
[0023] In a preferred embodiment, epitopes for inclusion in vaccine
compositions of the invention are selected by a process whereby
protein sequences of known antigens are evaluated for the presence
of motif or supermotif-bearing epitopes. Peptides corresponding to
a motif- or supermotif-bearing epitope are then synthesized and
tested for the ability to bind to the HLA molecule that recognizes
the selected motif. Those peptides that bind at an intermediate or
high affinity i.e., an IC.sub.50 (or a K.sub.D value) of 500 nM or
less for HLA class I molecules or an IC.sub.50 of 1000 nM or less
for HLA class II molecules, are further evaluated for their ability
to induce a CTL or HTL response. Immunogenic peptide epitopes are
selected for inclusion in vaccine compositions.
[0024] Supermotif-bearing peptides may additionally be tested for
the ability to bind to multiple alleles within the HLA supertype
family. Moreover, peptide epitopes may be analogued to modify
binding affinity and/or the ability to bind to multiple alleles
within an HLA supertype.
[0025] The invention also includes embodiments comprising methods
for monitoring or evaluating an immune response to HPV in a patient
having a known HLA-type. Such methods comprise incubating a T
lymphocyte sample from the patient with a peptide composition
comprising an HPV epitope that has an amino acid sequence described
in Tables VII to Table XX which binds the product of at least one
HLA allele present in the patient, and detecting for the presence
of a T lymphocyte that binds to the peptide. A CTL peptide epitope
may, for example, be used as a component of a tetrameric complex
for this type of analysis.
[0026] An alternative modality for defining the peptide epitopes in
accordance with the invention is to recite the physical properties,
such as length; primary structure; or charge, which are correlated
with binding to a particular allele-specific HLA molecule or group
of allele-specific HLA molecules. A further modality for defining
peptide epitopes is to recite the physical properties of an HLA
binding pocket, or properties shared by several allele-specific HLA
binding pockets (e.g. pocket configuration and charge distribution)
and reciting that the peptide epitope fits and binds to the pocket
or pockets.
[0027] As will be apparent from the discussion below, other methods
and embodiments are also contemplated. Further, novel synthetic
peptides produced by any of the methods described herein are also
part of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The peptides and corresponding nucleic acid compositions of
the present invention are useful for stimulating an immune response
to HPV by stimulating the production of CTL or HTL responses. The
peptide epitopes, which are derived directly or indirectly from
native HPV protein amino acid sequences, are able to bind to HLA
molecules and stimulate an immune response to HPV. The complete
sequence of the HPV proteins to be analyzed can be obtained from
Genbank. Epitopes and analogs thereof can also be readily
determined from sequence information that may subsequently be
discovered for heretofore unknown variants of HPV, as will be clear
from the disclosure provided below.
[0029] The epitopes of the invention have been identified in a
number of ways, as will be discussed below. Also discussed in
greater detail is that analog peptides have been derived and the
binding activity for HLA molecules modulated by modifying specific
amino acid residues to create peptide analogs exhibiting altered
immunogenicity. Further, the present invention provides
compositions and combinations of compositions that enable
epitope-based vaccines that are capable of interacting with HLA
molecules encoded by various genetic alleles to provide broader
population coverage than prior vaccines.
DEFINITIONS
[0030] The invention can be better understood with reference to the
following definitions, which are listed alphabetically:
[0031] A "computer" or "computer system" generally includes: a
processor; at least one information storage/retrieval apparatus
such as, for example, a hard drive, a disk drive or a tape drive;
at least one input apparatus such as, for example, a keyboard, a
mouse, a touch screen, or a microphone; and display structure.
Additionally, the computer may include a communication channel in
communication with a network. Such a computer may include more or
less than what is listed above.
[0032] A "construct" as used herein generally denotes a composition
that does not occur in nature. A construct can be produced by
synthetic technologies, e.g., recombinant DNA preparation and
expression or chemical synthetic techniques for nucleic or amino
acids. A construct can also be produced by the addition or
affiliation of one material with another such that the result is
not found in nature in that form.
[0033] "Cross-reactive binding" indicates that a peptide is bound
by more than one HLA molecule; a synonym is degenerate binding.
[0034] A "cryptic epitope" elicits a response by immunization with
an isolated peptide, but the response is not cross-reactive in
vitro when intact whole protein which comprises the epitope is used
as an antigen.
[0035] A "dominant epitope" is an epitope that induces an immune
response upon immunization with a whole native antigen (see, e.g.,
Sercarz, et al., Annu. Rev. Immunol. 11:729-766, 1993). Such a
response is cross-reactive in vitro with an isolated peptide
epitope.
[0036] With regard to a particular amino acid sequence, an
"epitope" is a set of amino acid residues which is involved in
recognition by a particular immunoglobulin, or in the context of T
cells, those residues necessary for recognition by T cell receptor
proteins and/or Major Histocompatibility Complex (MHC) receptors.
In an immune system setting, in vivo or in vitro, an epitope is the
collective features of a molecule, such as primary, secondary and
tertiary peptide structure, and charge, that together form a site
recognized by an immunoglobulin, T cell receptor or HLA molecule.
Throughout this disclosure epitope and peptide are often used
interchangeably. It is to be appreciated, however, that isolated or
purified protein or peptide molecules larger than and comprising an
epitope of the invention are still within the bounds of the
invention.
[0037] "Human Leukocyte Antigen" or "HLA" is a human class I or
class II Major Histocompatibility Complex (MHC) protein (see, e.g.,
Stites, et al., IMMUNOLOGY, 8.sup.TH ED., Lange Publishing, Los
Altos, Calif. (1994).
[0038] An "HLA supertype or family", as used herein, describes sets
of HLA molecules grouped on the basis of shared peptide-binding
specificities. HLA class I molecules that share somewhat similar
binding affinity for peptides bearing certain amino acid motifs are
grouped into HLA supertypes. The terms HLA superfamily, HLA
supertype family, HLA family, and HLA xx-like molecules (where xx
denotes a particular HLA type), are synonyms.
[0039] Throughout this disclosure, results are expressed in terms
of "IC.sub.50's." IC.sub.50 is the concentration of peptide in a
binding assay at which 50% inhibition of binding of a reference
peptide is observed. Given the conditions in which the assays are
run (i.e., limiting HLA proteins and labeled peptide
concentrations), these values approximate K.sub.D values. Assays
for determining binding are described in detail, e.g., in PCT
publications WO 94/20127 and WO 94/03205. It should be noted that
IC.sub.50 values can change, often dramatically, if the assay
conditions are varied, and depending on the particular reagents
used (e.g., HLA preparation, etc.). For example, excessive
concentrations of HLA molecules will increase the apparent measured
IC.sub.50 of a given ligand.
[0040] Alternatively, binding is expressed relative to a reference
peptide. Although as a particular assay becomes more, or less,
sensitive, the IC.sub.50's of the peptides tested may change
somewhat, the binding relative to the reference peptide will not
significantly change. For example, in an assay run under conditions
such that the IC.sub.50 of the reference peptide increases 10-fold,
the IC.sub.50 values of the test peptides will also shift
approximately 10-fold. Therefore, to avoid ambiguities, the
assessment of whether a peptide is a good, intermediate, weak, or
negative binder is generally based on its IC.sub.50, relative to
the IC.sub.50 of a standard peptide.
[0041] Binding may also be determined using other assay systems
including those using: live cells (e.g., Ceppellini et al., Nature
339:392, 1989; Christnick et al., Nature 352:67, 1991; Busch et
al., Int. Immunol. 2:443, 19990; Hill et al., J. Immunol. 147:189,
1991; del Guercio et al., J. Immunol. 154:685, 1995), cell free
systems using detergent lysates (e.g., Cerundolo et al., J.
Immunol. 21:2069, 1991), immobilized purified MHC (e.g., Hill et
al., J. Immunol. 152, 2890, 1994; Marshall et al., J. Immunol.
152:4946, 1994), ELISA systems (e.g., Reay et al., EMBO J. 11:2829,
1992), surface plasmon resonance (e.g., Khilko et al., J. Biol.
Chem. 268:15425, 1993); high flux soluble phase assays (Hammer et
al., J. Exp. Med. 180:2353, 1994), and measurement of class I MHC
stabilization or assembly (e.g., Ljunggren et al., Nature 346:476,
1990; Schumacher et al., Cell 62:563, 1990; Townsend et al., Cell
62:285, 1990; Parker et al., J. Immunol. 149:1896, 1992).
[0042] As used herein, "high affinity" with respect to HLA class 1
molecules is defined as binding with an IC.sub.50, or K.sub.D
value, of 50 nM or less; "intermediate affinity" is binding with an
IC.sub.50 or K.sub.D value of between about 50 and about 500 nM.
"High affinity" with respect to binding to HLA class II molecules
is defined as binding with an IC.sub.50 or K.sub.D value of 100 nM
or less; "intermediate affinity" is binding with an IC.sub.50 or
K.sub.D value of between about 100 and about 1000 nM.
[0043] The terms "identical" or percent "identity," in the context
of two or more peptide sequences, refer to two or more sequences or
subsequences that are the same or have a specified percentage of
amino acid residues that are the same, when compared and aligned
for maximum correspondence over a comparison window, as measured
using a sequence comparison algorithm or by manual alignment and
visual inspection.
[0044] An "immunogenic peptide" or "peptide epitope" is a peptide
that comprises an allele-specific motif or supermotif such that the
peptide will bind an HLA molecule and induce a CTL and/or HTL
response. Thus, immunogenic peptides of the invention are capable
of binding to an appropriate HLA molecule and thereafter inducing a
cytotoxic T cell response, or a helper T cell response, to the
antigen from which the immunogenic peptide is derived.
[0045] The phrases "isolated" or "biologically pure" refer to
material which is substantially or essentially free from components
which normally accompany the material as it is found in its native
state. Thus, isolated peptides in accordance with the invention
preferably do not contain materials normally associated with the
peptides in their in situ environment.
[0046] "Link" or "join" refers to any method known in the art for
functionally connecting peptides, including, without limitation,
recombinant fusion, covalent bonding, disulfide bonding, ionic
bonding, hydrogen bonding, and electrostatic bonding.
[0047] "Major Histocompatibility Complex" or "MHC" is a cluster of
genes that plays a role in control of the cellular interactions
responsible for physiologic immune responses. In humans, the MHC
complex is also known as the HLA complex. For a detailed
description of the MHC and HLA complexes, see, Paul, FUNDAMENTAL
IMMUNOLOGY, 3.sup.RD ED., Raven Press, New York, 1993.
[0048] The term "motif" refers to the pattern of residues in a
peptide of defined length, usually a peptide of from about 8 to
about 13 amino acids for a class I HLA motif and from about 6 to
about 25 amino acids for a class II HLA motif, which is recognized
by a particular HLA molecule. Peptide motifs are typically
different for each protein encoded by each human HLA allele and
differ in the pattern of the primary and secondary anchor
residues.
[0049] A "negative binding residue" or "deleterious residue" is an
amino acid which, if present at certain positions (typically not
primary anchor positions) in a peptide epitope, results in
decreased binding affinity of the peptide for the peptide's
corresponding HLA molecule.
[0050] A "non-native" sequence or "construct" refers to a sequence
that is not found in nature, i.e., is "non-naturally occurring".
Such sequences include, e.g., peptides that are lipidated or
otherwise modified, and polyepitopic compositions that contain
epitopes that are not contiguous in a native protein sequence.
[0051] The term "peptide" is used interchangeably with
"oligopeptide" in the present specification to designate a series
of residues, typically L-amino acids, connected one to the other,
typically by peptide bonds between the .alpha.-amino and carboxyl
groups of adjacent amino acids. The preferred CTL-inducing peptides
of the invention are 13 residues or less in length and usually
consist of between about 8 and about 11 residues, preferably 9 or
10 residues. The preferred HTL-inducing oligopeptides are less than
about 50 residues in length and usually consist of between about 6
and about 30 residues, more usually between about 12 and 25, and
often between about 15 and 20 residues.
[0052] It is to be appreciated that protein or peptide molecules
that comprise an epitope of the invention as well as additional
amino acid(s) are within the bounds of the invention. In certain
embodiments, there is a limitation on the length of a peptide of
the invention which is not otherwise a construct as defined herein.
An embodiment that is length-limited occurs when the
protein/peptide comprising an epitope of the invention comprises a
region (i.e., a contiguous series of amino acids) having 100%
identity with a native sequence. In order to avoid a recited
definition of epitope from reading, e.g., on whole natural
molecules, the length of any region that has 100% identity with a
native peptide sequence is limited. Thus, for a peptide comprising
an epitope of the invention and a region with 100% identity with a
native peptide sequence (and which is not otherwise a construct),
the region with 100% identity to a native sequence generally has a
length of: less than or equal to 600 amino acids, often less than
or equal to 500 amino acids, often less than or equal to 400 amino
acids, often less than or equal to 250 amino acids, often less than
or equal to 100 amino acids, often less than or equal to 85 amino
acids, often less than or equal to 75 amino acids, often less than
or equal to 65 amino acids, and often less than or equal to 50
amino acids. In certain embodiments, an "epitope" of the invention
which is not a construct is comprised by a peptide having a region
with less than 51 amino acids that has 100% identity to a native
peptide sequence, in any increment of (50, 49, 48, 47, 46, 45, 44,
43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27,
26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,
9, 8, 7, 6, 5) down to 5 amino acids.
[0053] Certain peptide or protein sequences longer than 600 amino
acids are within the scope of the invention. Such longer sequences
are within the scope of the invention so long as they do not
comprise any contiguous sequence of more than 600 amino acids that
have 100% identity with a native peptide sequence, or if longer
than 600 amino acids, they are a construct. For any peptide that
has five contiguous residues or less that correspond to a native
sequence, there is no limitation on the maximal length of that
peptide in order to fall within the scope of the invention. It is
presently preferred that a CTL epitope of the invention be less
than 600 residues long in any increment down to eight amino acid
residues.
[0054] "Pharmaceutically acceptable" refers to a non-toxic, inert,
and/or physiologically compatible composition.
[0055] A "pharmaceutical excipient" comprises a material such as an
adjuvant, a carrier, pH-adjusting and buffering agents, tonicity
adjusting agents, wetting agents, preservative, and the like.
[0056] A "primary anchor residue" is an amino acid at a specific
position along a peptide sequence which is understood to provide a
contact point between the immunogenic peptide and the HLA molecule.
One to three, usually two, primary anchor residues within a peptide
of defined length generally defines a "motif" for an immunogenic
peptide. These residues are understood to fit in close contact with
peptide binding grooves of an HLA molecule, with their side chains
buried in specific pockets of the binding grooves themselves. In
one embodiment, for example, the primary anchor residues are
located at position 2 (from the amino terminal position) and at the
carboxyl terminal position of a 9-residue peptide epitope in
accordance with the invention. The primary anchor positions for
each motif and supermotif are set forth in Table 1. For example,
analog peptides can be created by altering the presence or absence
of particular residues in these primary anchor positions. Such
analogs are used to modulate the binding affinity of a peptide
comprising a particular motif or supermotif.
[0057] "Promiscuous recognition" is where a distinct peptide is
recognized by the same T cell clone in the context of various HLA
molecules. Promiscuous recognition or binding is synonymous with
cross-reactive binding.
[0058] A "protective immune response" or "therapeutic immune
response" refers to a CTL and/or an HTL response to an antigen
derived from an infectious agent or a tumor antigen, which prevents
or at least partially arrests disease symptoms or progression. The
immune response may also include an antibody response which has
been facilitated by the stimulation of helper T cells.
[0059] The term "residue" refers to an amino acid or amino acid
mimetic incorporated into an oligopeptide by an amide bond or amide
bond mimetic.
[0060] A "secondary anchor residue" is an amino acid at a position
other than a primary anchor position in a peptide which may
influence peptide binding. A secondary anchor residue occurs at a
significantly higher frequency amongst bound peptides than would be
expected by random distribution of amino acids at one position. The
secondary anchor residues are said to occur at "secondary anchor
positions." A secondary anchor residue can be identified as a
residue which is present at a higher frequency among high or
intermediate affinity binding peptides, or a residue otherwise
associated with high or intermediate affinity binding. For example,
analog peptides can be created by altering the presence or absence
of particular residues in these secondary anchor positions. Such
analogs are used to finely modulate the binding affinity of a
peptide comprising a particular motif or supermotif.
[0061] A "subdominant epitope" is an epitope which evokes little or
no response upon immunization with whole antigens which comprise
the epitope, but for which a response can be obtained by
immunization with an isolated peptide, and this response (unlike
the case of cryptic epitopes) is detected when whole protein is
used to recall the response in vitro or in vivo.
[0062] A "supermotif" is a peptide binding specificity shared by
HLA molecules encoded by two or more HLA alleles. Preferably, a
supermotif-bearing peptide is recognized with high or intermediate
affinity (as defined herein) by two or more HLA antigens.
[0063] "Synthetic peptide" refers to a peptide that is man-made
using such methods as chemical synthesis or recombinant DNA
technology.
[0064] As used herein, a "vaccine" is a composition that contains
one or more peptides of the invention. There are numerous
embodiments of vaccines in accordance with the invention, such as
by a cocktail of one or more peptides; one or more epitopes of the
invention comprised by a polyepitopic peptide; or nucleic acids
that encode such peptides or polypeptides, e.g., a minigene that
encodes a polyepitopic peptide. The "one or more peptides" can
include any whole unit integer from 1-150, e.g., at least 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,
120, 125, 130, 135, 140, 145, or 150 or more peptides of the
invention. The peptides or polypeptides can optionally be modified,
such as by lipidation, addition of targeting or other sequences.
HLA class I-binding peptides of the invention can be admixed with,
or linked to, HLA class II-binding peptides, to facilitate
activation of both cytotoxic T lymphocytes and helper T
lymphocytes. Vaccines can also comprise peptide-pulsed antigen
presenting cells, e.g., dendritic cells.
[0065] The nomenclature used to describe peptide compounds follows
the conventional practice wherein the amino group is presented to
the left (the N-terminus) and the carboxyl group to the right (the
C-terminus) of each amino acid residue. When amino acid residue
positions are referred to in a peptide epitope they are numbered in
an amino to carboxyl direction with position one being the position
closest to the amino terminal end of the epitope, or the peptide or
protein of which it may be a part. In the formulae representing
selected specific embodiments of the present invention, the amino-
and carboxyl-terminal groups, although not specifically shown, are
in the form they would assume at physiologic pH values, unless
otherwise specified. In the amino acid structure formulae, each
residue is generally represented by standard three letter or single
letter designations. The L-form of an amino acid residue is
represented by a capital single letter or a capital first letter of
a three-letter symbol, and the D-form for those amino acids having
D-forms is represented by a lower case single letter or a lower
case three letter symbol. Glycine has no asymmetric carbon atom and
is simply referred to as "Gly" or G. The amino acid sequences of
peptides set forth herein are generally designated using the
standard single letter symbol. (A, Alanine; C, Cysteine; D,
Aspartic Acid; E, Glutamic Acid; F, Phenylalanine; G, Glycine; H,
Histidine; I, Isoleucine; K, Lysine; L, Leucine; M, Methionine; N,
Asparagine; P, Proline; Q, Glutamine; R, Arginine; S, Serine; T,
Threonine; V, Valine; W, Tryptophan; and Y, Tyrosine.) In addition
to these symbols, "B" in the single letter abbreviations used
herein designates .alpha.-amino butyric acid.
Stimulation of CTL and HTL Responses
[0066] The mechanism by which T cells recognize antigens has been
delineated during the past ten years. Based on our understanding of
the immune system we have developed efficacious peptide epitope
vaccine compositions that can induce a therapeutic or prophylactic
immune response to HPV in a broad population. For an understanding
of the value and efficacy of the claimed compositions, a brief
review of immunology-related technology is provided.
[0067] A complex of an HLA molecule and a peptidic antigen acts as
the ligand recognized by HLA-restricted T cells (Buus, S. et al.,
Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985;
Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989;
Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the
study of single amino acid substituted antigen analogs and the
sequencing of endogenously bound, naturally processed peptides,
critical residues that correspond to motifs required for specific
binding to HLA antigen molecules have been identified and are
described herein and are set forth in Tables I, II, and III (see
also, e.g., Southwood, et al., J. Immunol. 160:3363, 1998;
Rammensee, et al., Immunogenetics 41:178, 1995; Rammensee et al.,
SYFPEITHI, access via web at:
http://134.2.96.221/scripts.hlaserver.dll/home.htm; Sette, A. and
Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H.,
Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr.
Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr.
Biol. 6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et
al., J. Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol.
157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996;
Sette, A. and Sidney, J. Immunogenetics 1999 November; 50
(3-4):201-12, Review).
[0068] Furthermore, x-ray crystallographic analysis of HLA-peptide
complexes has revealed pockets within the peptide binding cleft of
HLA molecules which accommodate, in an allele-specific mode,
residues borne by peptide ligands; these residues in turn determine
the HLA binding capacity of the peptides in which they are present.
(See, e.g., Madden, D. R. Annu. Rev. Immunol. 13:587, 1995; Smith,
et al., Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998;
Stern et al., Structure 2:245, 1994; Jones, E. Y. Curr. Opin.
Immunol. 9:75, 1997; Brown, J. H. et al., Nature 364:33, 1993; Guo,
H. C. et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C.
et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367,
1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al.,
Cell 70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992;
Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol.
219:277, 1991.)
[0069] Accordingly, the definition of class I and class II
allele-specific HLA binding motifs, or class I or class II
supermotifs allows identification of regions within a protein that
have the potential of binding particular HLA antigen(s).
[0070] The present inventors have found that the correlation of
binding affinity with immunogenicity, which is disclosed herein, is
an important factor to be considered when evaluating candidate
peptides. Thus, by a combination of motif searches and HLA-peptide
binding assays, candidates for epitope-based vaccines have been
identified. After determining their binding affinity, additional
confirmatory work can be performed to select, amongst these vaccine
candidates, epitopes with preferred characteristics in terms of
population coverage, antigenicity, and immunogenicity.
[0071] Various strategies can be utilized to evaluate
immunogenicity, including:
[0072] 1) Evaluation of primary T cell cultures from normal
individuals (see, e.g., Wentworth, P. A. et al., Mol. Immunol.
32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105,
1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et
al., Human Immunol. 59:1, 1998); This procedure involves the
stimulation of peripheral blood lymphocytes (PBL) from normal
subjects with a test peptide in the presence of antigen presenting
cells in vitro over a period of several weeks. T cells specific for
the peptide become activated during this time and are detected
using, e.g., a lymphokine- or .sup.51 Cr-release assay involving
peptide sensitized target cells.
[0073] 2) Immunization of HLA transgenic mice (see, e.g.,
Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A.
et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J.
Immunol. 159:4753, 1997); In this method, peptides in incomplete
Freund's adjuvant are administered subcutaneously to HLA transgenic
mice. Several weeks following immunization, splenocytes are removed
and cultured in vitro in the presence of test peptide for
approximately one week. Peptide-specific T cells are detected
using, e.g., a .sup.51Cr-release assay involving peptide sensitized
target cells and target cells expressing endogenously generated
antigen.
[0074] 3) Demonstration of recall T cell responses from immune
individuals who have effectively been vaccinated, recovered from
infection, and/or from chronically infected patients (see, e.g.,
Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et
al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. Invest.
100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997;
Diepolder, H. M. et al., J. Virol. 71:6011, 1997); In applying this
strategy, recall responses are detected by culturing PBL from
subjects that have been naturally exposed to the antigen, for
instance through infection, and thus have generated an immune
response "naturally", or from patients who were vaccinated against
the infection. PBL from subjects are cultured in vitro for 1-2
weeks in the presence of test peptide plus antigen presenting cells
(APC) to allow activation of "memory" T cells, as compared to
"naive" T cells. At the end of the culture period, T cell activity
is detected using assays for T cell activity including .sup.51Cr
release involving peptide-sensitized targets, T cell proliferation,
or lymphokine release.
[0075] The following describes the peptide epitopes and
corresponding nucleic acids of the invention.
Binding Affinity of Peptide Epitopes for HLA Molecules
[0076] As indicated herein, the large degree of HLA polymorphism is
an important factor to be taken into account with the epitope-based
approach to vaccine development. To address this factor, epitope
selection encompassing identification of peptides capable of
binding at high or intermediate affinity to multiple HLA molecules
is preferably utilized, most preferably these epitopes bind at high
or intermediate affinity to two or more allele-specific HLA
molecules.
[0077] CTL-inducing peptides of interest for vaccine compositions
preferably include those that have an IC.sub.50 or binding affinity
value for class I HLA molecules of 500 nM or better (i.e., the
value is .ltoreq.500 nM). HTL-inducing peptides preferably include
those that have an IC.sub.50 or binding affinity value for class II
HLA molecules of 1000 nM or better, (i.e., the value is
.ltoreq.1,000 nM). For example, peptide binding is assessed by
testing the capacity of a candidate peptide to bind to a purified
HLA molecule in vitro. Peptides exhibiting high or intermediate
affinity are then considered for further analysis. Selected
peptides are tested on other members of the supertype family. In
preferred embodiments, peptides that exhibit cross-reactive binding
are then used in cellular screening analyses or vaccines.
[0078] As disclosed herein, higher HLA binding affinity is
correlated with greater immunogenicity. Greater immunogenicity can
be manifested in several different ways. Immunogenicity corresponds
to whether an immune response is elicited at all, and to the vigor
of any particular response, as well as to the extent of a
population in which a response is elicited. For example, a peptide
might elicit an immune response in a diverse array of the
population, yet in no instance produce a vigorous response. In
accordance with these principles, close to 90% of high binding
peptides have been found to be immunogenic, as contrasted with
about 50% of the peptides which bind with intermediate affinity.
Moreover, higher binding affinity peptides lead to more vigorous
immunogenic responses. As a result, less peptide is required to
elicit a similar biological effect if a high affinity binding
peptide is used. Thus, in preferred embodiments of the invention,
high affinity binding epitopes are particularly useful.
[0079] The relationship between binding affinity for HLA class I
molecules and immunogenicity of discrete peptide epitopes on bound
antigens has been determined for the first time in the art by the
present inventors. The correlation between binding affinity and
immunogenicity was analyzed in two different experimental
approaches (see, e.g., Sette, et al., J. Immunol. 153:5586-5592,
1994). In the first approach, the immunogenicity of potential
epitopes ranging in HLA binding affinity over a 10.000-fold range
was analyzed in HLA-A*0201 transgenic mice. In the second approach,
the antigenicity of approximately 100 different hepatitis B virus
(HBV)-derived potential epitopes, all carrying A*0201 binding
motifs, was assessed by using PBL from acute hepatitis patients.
Pursuant to these approaches, it was determined that an affinity
threshold value of approximately 500 nM (preferably 50 mM or less)
determines the capacity of a peptide epitope to elicit a CTL
response. These data are true for class I binding affinity
measurements for naturally processed peptides and for synthesized T
cell epitopes. These data also indicate the important role of
determinant selection in the shaping of T cell responses (see,
e.g., Schaeffer et al. Proc. Natl. Acad. Sci. USA 86:4649-4653,
1989).
[0080] An affinity threshold associated with immunogenicity in the
context of HLA class II DR molecules has also been delineated (see,
e.g., Southwood et al. J Immunology 160:3363-3373, 1998, and
co-pending U.S. Ser. No. 09/009,953 filed Jan. 21, 1998). In order
to define a biologically significant threshold of DR binding
affinity, a database of the binding affinities of 32 DR-restricted
epitopes for their restricting element (i.e., the HLA molecule that
binds the motif) was compiled. In approximately half of the cases
(15 of 32 epitopes), DR restriction was associated with high
binding affinities, i.e. binding affinity values of 100 nM or less.
In the other half of the cases (16 of 32), DR restriction was
associated with intermediate affinity (binding affinity values in
the 100-1000 nM range). In only one of 32 cases was DR restriction
associated with an IC.sub.50 of 1000 nM or greater. Thus, 1000 mM
can be defined as an affinity threshold associated with
immunogenicity in the context of DR molecules.
[0081] In the case of tumor-associated antigens (TAAs), many CTL
peptide epitopes that have been shown to induce CTL that lyse
peptide-pulsed target cells and tumor cell targets endogenously
expressing the epitope exhibit binding affinity or IC.sub.50 values
of 200 nM or less. In a study that evaluated the association of
binding affinity and immunogenicity of a small set of such TAA
epitopes, 100% ( 10/10) of the high binders, i.e., peptide epitopes
binding at an affinity of 50 nM or less, were immunogenic and 80% (
8/10) of them elicited CTLs that specifically recognized tumor
cells. In the 51 to 200 nM range, very similar figures were
obtained. With respect to analog peptides, CTL inductions positive
for wildtype peptide and tumor cells were noted for 86% ( 6/7) and
71% ( 5/7) of the peptides, respectively. In the 201-500 nM range,
most peptides (4/5 wildtype) were positive for induction of CTL
recognizing wildtype peptide, but tumor recognition was not
detected.
[0082] The binding affinity of peptides for HLA molecules can be
determined as described in Example 1, below.
Peptide Epitope Binding Motifs and Supermotifs
[0083] Through the study of single amino acid substituted antigen
analogs and the sequencing of endogenously bound, naturally
processed peptides, critical residues required for allele-specific
binding to HLA molecules have been identified. The presence of
these residues correlates with binding affinity for HLA molecules.
The identification of motifs and/or supermotifs that correlate with
high and intermediate affinity binding is an important issue with
respect to the identification of immunogenic peptide epitopes for
the inclusion in a vaccine. Kast et al. (J. Immunol. 152:3904-3912,
1994) have shown that motif-bearing peptides account for 90% of the
epitopes that bind to allele-specific HLA class I molecules. In
this study all possible peptides of 9 amino acids in length and
overlapping by eight amino acids (240 peptides), which cover the
entire sequence of the E6 and E7 proteins of human papillomavirus
type 16, were evaluated for binding to five allele-specific HLA
molecules that are expressed at high frequency among different
ethnic groups. This unbiased set of peptides allowed an evaluation
of the predictive value of HLA class I motifs. From the set of 240
peptides, 22 peptides were identified that bound to an
allele-specific HLA molecule with high or intermediate affinity. Of
these 22 peptides, 20 (i.e. 91%) were motif-bearing. Thus, this
study demonstrates the value of motifs for the identification of
peptide epitopes for inclusion in a vaccine: application of
motif-based identification techniques will identify about 90% of
the potential epitopes in a target antigen protein sequence.
[0084] Such peptide epitopes are identified in the Tables described
below.
[0085] Peptides of the present invention may also comprise epitopes
that bind to MHC class II DR molecules. A greater degree of
heterogeneity in both size and binding frame position of the motif,
relative to the N and C termini of the peptide, exists for class II
peptide ligands. This increased heterogeneity of HLA class II
peptide ligands is due to the structure of the binding groove of
the HLA class II molecule which, unlike its class I counterpart, is
open at both ends. Crystallographic analysis of HLA class II
DRB*0101-peptide complexes showed that the major energy of binding
is contributed by peptide residues complexed with complementary
pockets on the DRB*0101 molecules. An important anchor residue
engages the deepest hydrophobic pocket (see, e.g., Madden, D. R.
Ann. Rev. Immunol. 13:587, 1995) and is referred to as position 1
(P1). P1 may represent the N-terminal residue of a class II binding
peptide epitope, but more typically is flanked towards the
N-terminus by one or more residues. Other studies have also pointed
to an important role for the peptide residue in the 6.sup.th
position towards the C-terminus, relative to P1, for binding to
various DR molecules.
[0086] In the past few years evidence has accumulated to
demonstrate that a large fraction of HLA class I and class II
molecules can be classified into a relatively few supertypes, each
characterized by largely overlapping peptide binding repertoires,
and consensus structures of the main peptide binding pockets. Thus,
peptides of the present invention are identified by any one of
several HLA-specific amino acid motifs (see, e.g., Tables I-III),
or if the presence of the motif corresponds to the ability to bind
several allele-specific HLA antigens, a supermotif. The HLA
molecules that bind to peptides that possess a particular amino
acid supermotif are collectively referred to as an HLA
"supertype."
[0087] The peptide motifs and supermotifs described below, and
summarized in Tables I-III, provide guidance for the identification
and use of peptide epitopes in accordance with the invention.
[0088] Examples of peptide epitopes bearing a respective supermotif
or motif are included in Tables as designated in the description of
each motif or supermotif below. The Tables include a binding
affinity ratio listing for some of the peptide epitopes. The ratio
may be converted to IC.sub.50 by using the following formula:
IC.sub.50 of the standard peptide/ratio=IC.sub.50 of the test
peptide (i.e., the peptide epitope). The IC.sub.50 values of
standard peptides used to determine binding affinities for Class I
peptides are shown in Table IV. The IC.sub.50 values of standard
peptides used to determine binding affinities for Class II peptides
are shown in Table V. For example, where an HLA-A2.1 motif-bearing
peptide shows a relative binding ratio of 0.01 for HLA-A*0201, the
IC.sub.50 value is 500 nM, and where an HLA-A2.1 motif-bearing
peptide shows a relative binding ratio of 0.1 for HLA-A*0201, the
IC.sub.50 value is 50 nM.
[0089] The peptides used as standards for the binding assays
described herein are examples of standards; alternative standard
peptides can also be used when performing binding studies.
[0090] To obtain the peptide epitope sequences listed in Tables
VII-XX, protein sequence data for HPV types 6a, 6b, 11a, 16, 18,
31, 33, 45, and 56 were evaluated for the presence of the
designated supermotif or motif. Seven HPV structural and regulatory
proteins, E1, E2, E5, E6, E7, L1 and L2 were included in the
analysis. E4 was also included in the evaluation of some of the
strains. Peptide epitopes can additionally be evaluated on the
basis of their conservancy (i.e., the amount of variance) among the
available protein sequences for each HPV antigen.
[0091] In the Tables, motif- and/or supermotif-bearing amino acids
sequences identified in the indicated HPV strains are designated by
position number and length of the epitope with reference to the HPV
sequences and numbering provided below. For each sequence, the four
columns provide the following information: column 1 indicates the
HPV strain; column 2 indicates the HPV protein in which the
motif-bearing sequence is found, e.g., E1, E2, E4, E5, E6, E7, L1,
or L2; column 3 indicates the length of the epitope, or in the case
of HLA Class II epitopes, the length of the core sequence; and
column 4 designates the amino acid position in the HPV protein
sequence that corresponds to the first amino acid residue of the
epitope. For those sections of the Tables that include only three
columns, corresponding to columns 2, 3, and 4, the HPV strain is
indicated in the heading at the top of the page. For example, the
first peptide epitope listed in Table VII, i.e., the HLA-A1
supermotif, for HPV 16, protein E1 is a sequence of 10 residues in
length starting at position 206. Accordingly, the amino acid
sequence of the epitope is AMLAKFKELY.
[0092] For HPV strain 11, the number and position listed for
protein E5 refers to either the HPV 11 E5a or HPV 11 E5b sequence
set out below. Because the epitope must include the designated
motif or supermotif, e.g., HLA-A2, it can readily be determined
whether the sequence refers to HPV 11 E5a or E5b by checking the
amino acid sequences of both E5a and E5b and selecting the sequence
that conforms to the motif listed in Table I.
HPV Strains and Amino Acid Sequences of HPV Proteins
TABLE-US-00001 [0093] HPV6A E1 1
MADDSGTENEGSGCTGWFMVEAIVQHPTGTQISDDEDEEVEDSGYDMVDFIDDSNITHNS 60
LEAQALFNRQEADTHYATVQDLKRKYLGSPYVSPINTIAEAVESEISPRLDAIKLTRQPK 120
KVKRRLFQTRELTDSGYGYSEVEAGTGTQVEKHGVPENGGDGQEKDTGRDIEGEEHTEAE 180
APTNSVREHAGTAGILELLKCKDLRAALLGKFKECFGLSFIDLIRPFKSDKTTCADWVVA 240
GFGIHHSISEAFQKLIEPLSLYAHIQWLTNAWGMVLLVLVRFKVNKSRSTVARTLATLLN 300
IPDNQMLIEPPKIQSGVAALYWFRTGISNASTVIGEAPEWITRQTVIEHGLADSQFKLTE 360
MVQWAYDNDICEESEIAFEYAQRGDFDSNAPAFLNSNMQAKYVKDCATMCRHYKHAEMRK 420
MSIKQWIKHRGSKIEGTGNWKPIVQFLRHQNIEFIPFLSKFKLWLHGTPKKNCIAIVGPP 480
DTGKSYFCMSLISFLGGTVISHVNSSSHFWLQPLVDAKVALLDDATQPCWIYMDTYMRNL 540
LDGNPMSIDRKHKALTLIKCPPLLVTSNIDITKEEKYKYLHTRVTTFTFPNPFPFDRNGN 600
AVYELSNANWKCFFERLSSSLDIQDSEDEEDGSNSQAFRCVPGTVVRTL 649 HPV6A E2 1
MEAIAKRLDACQEQLLELYEENSTDLNKHVLHWKCMRHESVLLYKAKQMGLSHIGMQVVP 60
PLKVSEAKGHNAIEMQMHLESLLKTEYSMEPWTLQETSYEMWQTPPKRCFKKRGKTVEVK 120
FDGCANNTMDYVVWTDVYVQDTDSWVKVHSMVDAKGIYYTCGQFKTYYVNFVKEAEKYGS 180
TKQWEVCYGSTVICSPASVSSTTQEVSIPESTTYTPAQTSTPVSSSTQEDAVQTPPRKRA 240
RGVQQSPCNALCVAHIGPVDSGNHNLITNNHDQHQRRNNSNSSATPIVQFQGESNCLKCF 300
RYRLNDKHRHLFDLISSTWHWASPKAPHKHAIVTVTYHSEEQRQQFLNVVKIPPTIRHKL 360
GFMSLHLL 368 HPV6A E4 1
MAAQLYVLLHLYLALHKKYPFLNLLHTPPHRPPPLCPQAPRKTQCKRRLENEHEESNSHL 60
ATPCVWPTLDPWTVETTTSSLTITTSTKEGTTVTVQLRL 99 HPV6A E5 1
MEVVPVQIAAGTTSTLILPVIIAFVVCFVSIILIVWISDFIVYTSVLVLTLLLYLLLWLL 60
LTTPLQFFLLTLLVCYCPALYIHHYIVNTQQ 91 HPV6A E6 1
MESANASTSATTIDQLCKTFNLSMHTLQINCVFCKNALTTAEIYSYAYKQLKVLFRGGYP 60
YAACACCLEFHGKINQYRHFDYAGYATTVEEETKQDILDVLIRCYLCHKPLCEVEKVKHI 120
LTKARFIKLNCTWKGRCLHCWTTCMEDMLP 150 HPV6A E7 1
MHGRHVTLKDIVLDLQPPDPVGLHCYEQLVDSSEDEVDEVDGQDSQPLKQHFQIVTCCCG 60
CDSNVRLVVQCTETDIREVQQLLLGTLDIVCPICAPKT 98 HPV6A L1 1
MWRPSDSTVYVPPPNPVSKVVATDAYVTRTNIFYHASSSRLLAVGHPYFSIKRANKTVVP 60
KVSGYQYRVFKVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPFL 120
NKYDDVENSGSGGNPGQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGKQCTNTPVQAGD 180
CPPLELITSVIQDGDMVDTGFGAMNFADLQTNKSDVPIDICGTTCKYPDYLQMAADPYGD 240
RLFFFLRKEQMFARHFFNRAGEVGEPVPDTLIIKGSGNRTSVGSSIYVNTPSGSLVSSEA 300
QLFNKPYWLQKAQGHNNGICWGNQLFVTVVDTTRSTNMTLCASVTTSSTYTNSDYKEYMR 360
HVEEYDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGLSPPPNGTLEDTYRYVQSQA 420
ITCQKPTPEKEKPDPYKNLSFWEVNLKEKFSSELDQYPLGRKFLLQSGYRGRSSIRTGVK 480
RPAVSKASAAPKRKRAKTKR 500 HPV6A L2 1
MAHSRARRRKRASATQLYQTCKLTGTCPPDVIPKVEHNTIADQILKWGSLGVFFGGLGIG 60
TGSGTGGRTGYVPLGTSAKPSITSGPMARPPVVVEPVAPSDPSIVSLIEESAIINAGAPE 120
IVPPAHGGFTITSSETTTPAILDVSVTSHTTTSIFRNPVFTEPSVTQPQPPVEANGHILI 180
SAPTITSHPIEEIPLDTFVISSSDSGPTSSTPVPGTAPRPRVGLYSPALHQVQVTDPAFL 240
STPQRLITYDNPVYEGEDVSVQFSHDSIHNAPDEAFMDIIRLHRPAIASRRGLVRYSRIG 300
QRGSMHTRSGKHIGARIHYFYDISPIAQAAEEIEMHPLVAAQDDTFDIYAESFEPDINPT 360
QHPVTNISDTYLTSTPNTVTQPWGNTTVPLSSIPNDLFLQSGPDITFPTAPMGTPFSPVT 420
ALPTGPVFITGSGFYLHPAWYFARKRRKRIPLFFSDVAA 459 HPV6B E1 1
MADDSGTENEGSGCTGWFMVEAIVQHPTGTQISDDEDEEVEDSGYDMVDFIDDSNITHNS 60
LEAQALFNRQEADTHYATVQDLKRKYLGSPYVSPINTIAEAVESEISPRLDAIKLTRQPK 120
KVKRRLFQTRELTDSGYGYSEVEAGTGTQVEKHGVPENGGDGQEKDTGRDIEGEEHTEAE 180
APTNSVREHAGTAGILELLKCKDLRAALLGKFKECFGLSFIDLIRPFKSDKTTCLDWVVA 240
GFGIHHSISEAFQKLIEPLSLYAHIQWLTNAWGMVLLVLLRFKVNKSRSTVARTLATLLN 300
IPENQMLIEPPKIQSGVAALYWFRTGISNASTVIGEAPEWITRQTVIEHGLADSQFKLTE 360
MVQWAYDNDICEESEIAFEYAQRGDFDSNARAFLNSNMQAKYVKDCATMCRHYKHAEMRK 420
MSIKQWIKHRGSKIEGTGNWKPIVQFLRHQNTEFIPFLTKFKLWLHGTPKKNCIAIVGPP 480
DTGKSYFCMSLISFLGGTVISHVNSSSHFWLQPLVDAKVALLDDATQPCWIYMDTYMRNL 540
LDGNPMSIDRKHKALTLIKCPPLLVTSNIDITKEDKYKYLHTRVTTFTFPNPFPFDRNGN 600
AVYELSNTNWKCFFERLSSSLDIQDSEDEEDGSNSQAFRCVPGTVVTRTL 649 HPV6B E2 1
MEAIAKRLDACQEQLLELYEENSTDLHKHVLHWKCMRHESVLLYKAKQMGLSHIGMQVVP 60
PLKVSEAKGHNAIEMQMHLESLLRTEYSMEPWTLQETSYEMWQTPPKRCFKKRGKTVEVK 120
FDGCANNTMDYVVWTDVYVQDNDTWVKVHSMVDAKGIYYTCGQFKTYYVNFVKEAEKYGS 180
TKHWEVCYGSTVICSPASVSSTTQEVSIPESTTYTPAQTSTLVSSSTKEDAVQTPPRKRA 240
RGVQQSPCNALCVAHIGPVDSGNHNLITNNHDQHQRRNNSNSSATPIVQFQGESNCLKCF 300
RYRLNDRHRHLFDLISSTWHWASSKAPHKHAIVTVTYDSEEQRQQFLDVVKIPPTISHKL 360
GFMSLHLL 368 HPV6B E4 1
MGAPNIGKYVMAAQLYVLLHLYLALHKKYPFLNLLHTPPHRPPPLCPQAPRKTQCKRRLG 60
NEHEESNSPLATPCVWPTLDPWTVETTTSSLTITTSTKDGTTVTVQLRL 109 HPV6B E5A 1
MEVVPVQIAAGTTSTFILPVIIAFVVCFVSIILIVWISEFIVYTSVLVLTLLLYLLLWLL 60
LTTPLQFFLLTLLVCYCPALYIHYYIVTTQQ 91 HPV6B E5B 1
MMLTCQFNDGDTWLGLWLLCAFIVGMLGLLLMHYRAVQGDKHTKCKKCNKHNCNDDYVTM 60
HYTTDGDYIYMN 72 HPV6B E6 1
MESANASTSATTIDQLCKTFNLSMHTLQINCVFCKNALTTAEIYSYAYKHLKVLFRGGYP 60
YAACACCLEFHGKINQYRHFDYAGYATTVEEETKQDILDVLIRCYLCHKPLCEVEKVKHI 120
LTKARFIKLNCTWKGRCLHCWTTCMEDMLP 150 HPV6B E7 1
MHGRHVTLKDIVLDLQPPDPVGLHCYEQLVDSSEDEVDEVDGQDSQPLKQHFQIVTCCCG 60
CDSNVRLVVQCTETDIREVQQLLLGTLNIVCPICAPKT 98 HPV6A L1 1
MWRPSDSTVYVPPPNPVSKVVATDAYVTRTNIFYHASSSRLLAVGHPYFSIKRANKTVVP 60
KVSGYQYRVFKVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPFL 120
NKYDDVENSGSGGNPGQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGKQCTNTPVQAGD 180
CPPLELITSVIQDGDMVDTGFGAMNFADLQTNKSDVPIDICGTTCKYPDYLQMAADPYGD 240
RLFFFLRKEQMFARHFFNRAGEVGEPVPDTLIIKGSGNRTSVGSSIYVNTPSGSLVSSEA 300
QLFNKPYWLQKAQGHNNGICWGNQLFVTVVDTTRSTNMTLCASVTTSSTYTNSDYKEYMR 360
HVEEYDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGLSPPPNGTLEDTYRYVQSQA 420
ITCQKPTPEKEKPDPYKNLSFWEVNLKEKFSSELDQYPLGRKFLLQSGYRGRSSIRTGVK 480
RPAVSKASAAPKRKRAKTKR 500 HPV6B L2 1
MAHSRARRRKRASATQLYQTCKLTGTCPPDVIPKVEHNTIADQILKWGSLGVFFGGLGIG 60
TGSGTGGRTGYVPLQTSAKPSITSGPMARPPVVVEPVAPSDPSIVSLIEESAIINAGAPE 120
IVPPAHGGFTITSSETTTPAILDVSVTSHTTTSIFRNPVFTEPSVTQPQPPVEANGHILI 180
SAPTVTSHPIEEIPLDTFVVSSSDSGPTSSTPVPGTAPRPRVGLYSRALHQVQVTDPAFL 240
STPQRLITYDNPVYEGEDVSVQFSHDSIHNAPDEAFMDIIRLHRPAIASRRGLVRYSRIG 300
QRGSMHTRSGKHIGARIHYFYDISPIAQAAEEIEMHPLVAAQDDTFDIYAESFEPGINPT 360
QHPVTNISDTYLTSTPNTVTQPWGNTTVPLSLPNDLFLQSGPDITFPTAPMGTPFSPVTP 420
ALPTGPVFITGSGFYLHPAWYFARKRRKRIPLFFSDVAA 453 HPV11 E1 1
MADDSGTENEGSGCTGWFMVEAIVEHTTGTQISEDEEEEVEDSGYDMVDFIDDRHITQNS 60
VEAQALFNRQEADAHYATVQDLKRKYLGSPYVSPISNVANAVESEISPRLDAIKLTTQPK 120
KVKRRLFETRELTDSGYGYSEVEAATQVEKHGDPENGGDGQERDTGRDIEGEGVEHREAE 180
AVDDSTREHADTSGILELLKCKDIRSTLHGKFKDCFGLSFVDLIRPFKSDRTTCADWVVA 240
GFGIHHSIADAFQKLIEPLSLYAHIQWLTNAWGMVLLVLIRFKVNKSRCTVARTLGTLLN 300
IPENHMLIEPPKIQSGVRALYWFRTGISNASTVIGEAPEWITRQTVIEHSLADSQFKLTE 360
MVQWAYDNDICEESEIAFEYAQRGDFDSNAPAFLNSNMQAKYVKDCAIMCRHYKHAEMKK 420
MSIKQWIKYRGTKVDSVGNWKPIVQFLRHQNIEFIPFLSKLKLWLHGTPKKNCIAIVGPP 480
DTGKSCFCMSLIKFLGGTVISYVNSCSHFWLQPLTDAKVALLDDATQPCWTYMDTYMRNL 540
LDGNPMSIDRKHRALTLIKCPPLLVTSNIDISKEEKYKYLHSRVTTFTFPNPFPFDRNGN 600
AVYELSDANWKCFFERLSSSLDIEDSEDEEDGSNSQAFRCVPGSVVRTL 649 HPV11 E2 1
MEAIAKRLDACQDQLLELYEENSIDIHKHIMHWKCIRLESVLLHKAKQMGLSHIGLQVVP 60
PLTVSETKGHNAIEMQMHLESLAKTQYGVEPWTLQDTSYEMWLTPPKRCFKKQGNTVEVK 120
FDGCEDNVMEYVVWTHIYLQDNDSWVKVTSSVDAKGIYYTCGQFKTYYVNFNKEAQKYGS 180
TNHWEVCYGSTVICSPASVSSTVREVSIAEPTTYTPAQTTAPTVSACTTEDGVSAPPRKR 240
ARGPSTNNTLCVANIRSVDSTINNIVTDHYNKHQRRNNCHSAATPIVQLQGDSNCLKCFR 300
YRLNDKYKHLFELASSTWHWASPEAPHKNAIVTLTYSSEEQRQQFLNSVKIPPTIRHKVG 360
FMSLHLL 367 HPV11 E4 1
MVVPIIGKYVMAAQLYVLLHLYLALYEKYPLLNLLHTPPHRPPPLQCPPAPRKTACRRRL 60
GSEHVDRPLTTPCVWPTSDPWTVQSTTSSLTITTSTKEGTTVTVQLRL 108 HPV11 E5A 1
MEVVPVQIAAATTTTLILPVVIAFAVCILSIVLIILISDFVVYTSVLVLTLLLYLLLWLL 60
LTTPLQFFLLTLCVCYFPAFYIHIYIVQTQQ 91 HPV11 E5B 1
MVMLTCHLNDGDTWLFLWLFTAFVVAVLGLLLLHYRAVHGTEKTKCAKCKSNRNTTVDYV 60
YMSHGDNGDYVYMN 74
HPV11 E6 1
MESKDASTSATSIDQLCKTFNLSLHTLQIQCVFCRNALTTAEIYAYAYKNLKVVWRDNFP 60
FAACACCLELQGKINQYRHFNYAAYAPTVEEETNEDILKVLIRCYLCHKPLCEIEKLKHI 120
LGKARFIKLNNQWKGRCLHCWTTCMEDLLP 150 HPV11 E7 1
MHGRLVTLKDIVLDLQPPDPVGLHCYEQLEDSSEDEVDKVDKQDAQPLTQHYQILTCCCG 60
CDSNVRLVVECTDGDIRQLQDLLLGTLNIVCPICAPKP 98 HPV11 L1 1
MWRPSDSTVYVPPPNPVSKVVATDAYVKRTNIFYHASSSRLLAVGHPYYSIKKVNKTVVP 60
KVSGYQYRVFKVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPLL 120
NKYDDVENSGGYGGNPGQDNRVNVGMDYKQTQLCMVGCAPPLGEHWGKGTQCSNTSVQNG 180
DCPPLELITSVIQDGDMVDTGFGAMNFADLQTNKSDVPLDICGTVCKYPDYLQMAADPYG 240
DRLFFYLRKEQMFARHFFNRAGTVGEPVPDDLLVKGGNNRSSVASSIYVHTPSGSLVSSE 300
AQLFNKPYWLQKAQGHNNGICWGNHLFVTVVDTTRSTNMTLCASVSKSATYTNSDYKEYM 360
RHVEEFDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGLSPPPNGTLEDTYRYVQSQ 420
AITCQKPTPEKEKQDPYKDMSFWEVNLKEKFSSELDQFPLGRKFLLQSGYRGRTSARTGI 480
KRPAVSKPSTAPKRKRTKTKK 501 HPV11 L2 1
MKPRARRRKRASATQLYQTCKATGTCPPDVIPKVEHTTIADQILKWGSLGVFFGGLGIGT 60
GAGSGGRAGYIPLGSSPKPAITGGPAARPPVLVEPVAPSDPSIVSLIEESAIINAGAPEV 120
VPPTQGGFTITSSESTTPAILDVSVTNHTTTSVFQNPLFTEPSVIQPQPPVEASGHILIS 180
APTITSQHVEDIPLDTFVVSSSDSGPTSSTPLPRAFPRPRVGLYSRALQQVQVTDPAFLS 240
TPQRLVTYDNPVYEGEDVSLQFTHESIHNAPDEAFMDIIRLHRPAITSRRGLVRFSRIGQ 300
RGSMYTRSGQHIGARIHYFQDISPVTQAAEEIELHPLVAAENDTFDIYAEPFDPIPDPVQ 360
HSVTQSYLTSTPNTLSQSWGNTTVPLSIPSDWFVQSGPDITFPTASMGTPFSPVTPALPT 420
GPVFITGSDFYLHPTWYFARRRRKRIPLFFTDVAA 455 HPV16 E1 1
MADPAGTNGEEGTGCNGWFYVEAVVEKKTGDAISDDENENDSDTGEDLVDFIVNDNDYLT 60
QAETETAHALFTAQEAKQHRDAVQVLKRKYLVSPLSDISGCVDNNISPRLKAICIEKQSR 120
AAKRRLFESEDSGYGNTEVETQQMLQVEGRHETETPCSQYSGGSGGGCSQYSSGSGGEGV 180
SERHTICQTPLTNILNVLKTSNAKAAMLAKFKELYGVSFSELVRPFKSNKSTCCDWCIAA 240
FGLTPSIADSIKTLLQQYCLYLHIQSLACSWGMVVLLLVRYKCGKNRETIEKLLSKLLCV 300
SPMCMMIEPPKLRSTAAALYWYKTGISNISEVYGDTPEWIQRQTVLQHSFNDCTFELSQM 360
VQWAYDNDIVDDSEIAYKYAQLADTNSNASAFLKSNSQAKIVKDCATMCRHYKRAEKKQM 420
SMSQWIKYRCDRVDDGGDWKQIVMFLRYQGVEFMSFLTALKRFLQGIPKKNCILLYGAAN 480
TGKSLFGMSLMKFLQGSVICFVNSKSHFWLQPLADAKIGMLDDATVPCWNYIDDNLRNAL 540
DGNLVSMDVKHRPLVQLKCPPLLITSNINAGTDSRWPYLHNRLVVFTFPNEFPFDENGNP 600
VYELNDKNWKSFFSRTWSRLSLHEDEDKENDGDSLPTFKCVSGQNTNTL 649 HPV16 E2
Accession number W2WLHS 1
METLCQRLNVCQDKILTHYENDSTDLRDHIDYWKHMRLECAIYYKAREMGFKHINHQVVP 60
TLAVSKNKALQAIELQLTLETIYNSQYSNEKWTLQDVSLEVYLTAPTGCIKKHGYTVEVQ 120
FDGDICNTMHYTNWTHIYICEEASVTVVEGQVDYYGLYYVHEGIRTYFVQFKDDAEKYSK 180
NKVWEVHAGGQVILCPTSVFSSNEVSSPEIIRQHLANHPAATHTKAVALGTEETQTTIQR 240
PRSEPDTGNPCHTTKLLHRDSVDSAPILTAFNSSHKGRINCNSNTTPIVHLKGDANTLKC 300
LRYRFKKHCTLYTAVSSTWHWTGHNVKHKSAIVTLTYDSEWQRDQFLSQVKIPKTITVST 360
GFMSI 365 HPV16 E5 Accession number W5WLHS 1
MTNLDTASTTLLACFLLCFCVLLCVCLLIRPLLLSVSTYTSLIILVLLLWITAASAFRCF 60
IVYIIFVYIPLFLIHTHARFLIT 83 HPV16 E6 1
MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIV 60
YRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPE 120
EKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL 158 HPV16 E7 1
MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCK 60
CDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP 98 HPV16 L1 Accession number
AAD33259 1
MQVTFIYILVITCYENDVNVYHIFFQMSLWLPSEATVYLPPVPVSKVVSTDEYVARTNIY 60
YHAGTSRLLAVGHPYFPIKKPNNNKILVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPD 120
TQRLVWACVGVEVGRGQPLGVGISGHPLLNKLDDTENASAYAANAGVDNRECISMDYKQT 180
QLCLIGCKPPIGEHWGKGSPCTNVAVNPGDCPPLELINTVIQDGDMVDTGFGAMDFTTLQ 240
ANKSEVPLDICTSICKYPDYIKMVSEPYGDSLFFYLRREQMFVRHLFNRAGAVGENVPDD 300
LYIKGSGSTANLASSNYFPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTVV 360
DTTRSTNMSLCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKITLTADVMTYIHSM 420
NSTILEDWNFGLQPPPGGTLEDTYRFVTSQAIACQKHTPPAPKEDPLKKYTFWEVNLKEK 480
FSADLDQFPLGRKFLLQAGLKAKPKFTLGKRKATPTTSSTSTTAKRKKRKL 531 HPV16 L2
Accession number AAD33258 1
MRHKRSAKRTKRASATQLYKTCKQAGTCPPDIIPKVEGKTIADQILQYGSMGVFFGGLGI 60
GTGSGTGGRTGYIPLGTRPPTATDTLAPVRPPLTVDPVGPSDPSIVSLVEETSFIDAGAP 120
TSVPSIPPDVSGFSITTSTDTTPAILDINNTVTTVTTHNNPTFTDPSVLQPPTPAETGGH 180
FTLSSSTISTHNYEEIPMDTFIVSTNPNTVTSSTPIPGSRPVARLGLYSRTTQQVKVVDP 240
AFITTPTKLITYDNPAYEGIDVDNTLYFSSNDNSINIAPDPDFLDIVALHRPALTSRRTG 300
IRYSRIGNKQTLRTRSGKSIGAKVHYYYDFSTIDSAEEIELQTITPSTYTTTSHAALPTS 360
INNGLYDIYADDFITDTSTTPVPSVPSTSLSGYIPANTTIPFGGAYNIPLVSGPDIPINI 420
TDQAPSLIPIVPGSPQYTIIADAGDFYLHPSYYMLRKRRKRLPYFFSDVSLAA 473 HPV18 E1
1 MADPEGTDGEGTGCNGWFYVQAIVDKKTGDVISDDEDENATDTGSDMVDFIDTQGTFCEQ 60
AELETAQALFHAQEVHNDAQVLHVLKRKFAGGSTENSPLGERLEVDTELSPRLQEISLNS 120
GQKKAKRRLFTISDSGYGCSEVEATQIQVTTNGEHGGNVCSGGSTEAIDNGGTEGNNSSV 180
DGTSDNSNIENVNPQCTIAQLKDLLKVNNKQGAMLAVFKDTYGLSFTDLVRNFKSDKTTC 240
TDWVTAIFGVNPTIAEGFKTLIQPFILYAHIQCLDCKWGVLILALLRYKCGKSRLTVAKG 300
LSTLLHVPETCMLIQPPKLRSSVAALYWYRTGISNISEVMGDTPEWIQRLTIIQHGIDDS 360
NFDLSEMVQWAFDNELTDESDMAFEYALLADSNSNAAAFLKSNCQAKYLKDCATMCKHYR 420
RAQKRQMNMSQWIRFRCSKIDEGGDWRPIVQFLRYQQIEFITFLGALKSFLKGTPKKNCL 480
VFCGPANTGKSYFGMSFIHFIQGAVISFVNSTSHFWLEPLTDTKVAMLDDATTTCWTYFD 540
TYMRNALDGNPISIDRKHKPLIQLKCPPILLTTNIHPAKDNRWPYLESRITVFEFPNAFP 600
FDKNGNPVYEINDKNWKCFFERTWSRLDLHEEEEDADTEGNPFGTFKLRAGQNHRPL 657 HPV18
E2 Accession number W2WL18 1
MQTPKETLSERLSCVQDKIIDHYENDSKDIDSQIQYWQLIRWENAIFFAAREHGIQTLNH 60
QVVPAYNISKSKAHKAIELQMALQGLAQSRYKTEDWTLQDTCEELWNTEPTHCFKKGGQT 120
VQVYFDGNKDNCMTYVAWDSVYYMTDAGTWDKTATCVSHRGLYYVKEGYNTFYIEFKSEC 180
EKYGNTGTWEVHFGNNVIDCNDSMCSTSDDTVSATQLVKQLQHTPSPYSSTVSVGTAKTY 240
GQTSAATRPGHCGLAEKQHCGPVNPLLGAATPTGNNKRRKLCSGNTTPIIHLKGDRNSLK 300
CLRYRLRKHSDHYRDISSTWHWTGAGNEKTGILTVTYHSETQRTKFLNTVAIPDSVQILV 360
GYMTM 365 HPV18 E5 Accession number WSWL18 1
MLSLIFLFCFCVCMYVCCHVPLLPSVCMCAYAWVLVFVYIVVITSPATAFTVYVFCFLLP 60
MLLLHIHAILSLQ 73 HPV18 E6 1
MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSI 60
PHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLThTGLYNLLIRCLRCQKPLNPAEKLRH 120
LNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQV 158 HPV18 E7 1
MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHT 60
MLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPWCASQQ 105 HPV18 L1
Accession number CAA28671 1
MCLYTRVLILHYHLLPLYGPLYHPRPLPLHSILVYMVHIIICGHYIILFLRNVNVFPIFL 60
QMALWRPSDNTVYLPPPSVARVVNTDDYVTPTSIFYHAGSSRLLTVGNPYFRVPAGGGNK 120
QDIPKVSAYQYRVFRVQLPDPNKFGLPDTSIYNPETQRLVWACAGVEIGRGQPLGVGLSG 180
HPFYNKLDDTESSHAATSNVSEDVRDNVSVDYKQTQLCILGCAPAIGEHWAKGTACKSRP 240
LSQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLDICQSICKYPDYLQMSA 300
DPYGDSMFFCLRREQLFARHFWNRAGTMGDTVPQSLYIKGTGMPASPGSCVYSPSPSGSI 360
VTSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTPSTNLTICASTQSPVPGQYDAT 420
KFKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSILEDWNFGVPPPPTTSLVDTY 480
RFVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEKFSLDLDQYPLGRKFLVQAGLRRKP 540
TIGPRKRSAPSATTSSKPAKRVRVRARK 568 HPV18 L2 Accession number P2WL18 1
MVSHRAARRKRASVTDLYKTCKQSGTCPPDVVPKVEGTTLADKILQWSSLGIFLGGLGIG 60
TGSGTGGRTGYIPLGGRSNTVVDVGPTRPPVVIEPVGPTDPSIVTLIEDSSVVTSGAPRP 120
TFTGTSGFDITSAGTTTPAVLDITPSSTSVSISTTNFTNPAFSDPSIIEVPQTGEVAGNV 180
FVGTPTSGTHGYEEIPLQTFASSGTGEEPISSTPLPTVRRVAGPRLYSPAYQQVSVANPE 240
FLTRPSSLITYDNPAFEPVDTTLTFDPRSDVPDSDFMDIIRLHRPALTSRRGTVRFSRLG 300
QPATMFTRSGTQIGARVHFYHDISPIAPSPEYIELQPLVSATEDNDLFDIYADDMDPAVP 360
VPSRSTTSFAFFKYSPTISSASSYSNVTVPLTSSWDVPVYTGPDITLPSTTSVWPIVSPT 420
APASTQYIGIHGTHYYLWPLYYFIPKKRKRVPYFFADGFVAA 462 HPV31 E1 Accession
number W1WL31 1
MADPAGTDGEGTGCNGWFYVEAVIDRQTGDNISEDENEDSSDTGEDMVDFIDNCNVYNNQ 60
AEAETAQALFHAQEAEEHAEAVQVLKRKYVGSPLSDISSCVDYNISPRLKAICIENNSKT 120
AKRRLFELPDSGYGNTEVETQQMVQVEEQQTTLSCNGSDGTHSERENETPTRNILQVLKT 180
SNGKAAMLGKFKELYGVSFMELIRPFQSNKSTCTDWCVAAFGVTGTVAEGFKTLLQPYCL 240
YCHLQSLACSWGMVMLMLVRFKCAKNRITIEKLLEKLLCISTNCMLIQPPKLRSTAAALY 300
WYRTGMSNISDVYGETPEWIERQTVLQHSFNDTTFDLSQMVQWAYDNDVMDDSEIAYKYA 360
QLADSDSNACAFLKSNSQAKIVKDCGTMCRHYKRAEKRQMSMGQWIKSRCDKVSDEGDWR 420
DIVKFLRYQQIEFVSFLSALKLFLKGVPKKNCILIHGAPNTGKSYFGMSLISFLQGCIIS 480
YANSKSHFWLQPLADAKIGMLDDATTPCWHYIDNYLRNALDGNPVSIDVKHKALMQLKCP
540
PLLITSNINAGKDDRWPYLHSRLVVFTFPNPFPFDKNGNPVYELSDKNWKSFFSRTWCRL 600
NLHEEEDKENDGDSFSTFKCVSGQNIRTL 629 HPV31 E2 Accession number W2WL31
1 METLSQRLNVCQDKILEHYENDSKRLCDHIDYWKHIRLECVLMYKAREMGIHSINHQVVP 60
ALSVSKAKALQAIELQMMLETLNNTEYKNEDWTMQQTSLELYLTAPTGCLKKHGYTVEVQ 120
FDGDVHNTMHYTNWKFIYLCIDGQCTVVEGQVNCKGIYYVHEGHITYFVNFTEEAKKYGT 180
GKKWEVHAGGQVIVFPESVFSSDEISFAGIVTKLPTANNTTTSNSKTCALGTSEGVRRAT 240
TSTKRPRTEPEHRNTHHPNKLLRGDSVDSVNCGVISAAACTNQTRAVSCPATTPIIHLKG 300
DANILKCLRYRLSKYKQLYEQVSSTWHWTCTDGKHKNAIVTLTYISTSQRDDFLNTVKIP 360
NTVSVSTGYMTI 372 HPV31 E5 Accession number W5WL31 1
MIELNISTVSIVLCFLLCFCVLLFVCLVIRPLVLSVSVYATLLLLIVTLWVIATSPLRCF 60
CIYVVFIYTPLFVIHTHASFLSQQ 84 HPV31 E6 Accession number W6WL31 1
MFKNPAERPRKLHELSSALEIPYDELRLNCVYCKGQLTETEVLDFAFTDLTIVYRDDTPH 60
GVCTKCLRFYSKVSEFRWYRYSVYGTTLEKLTNKGICDLLIRCITCQRPLCPEEKQRHLD 120
KKKRFHNIGGRWTGRCIACWRRPRTETQV 149 HPV31 E7 Accession number W7WL31
1 MRGETPTLQDYVLDLQPEATDLHCYEQLPDSSDEEDVIDSPAGQAEPDTSNYNIVTFCCQ 60
CKSTLRLCVQSTQVDIRILQELLMGSFGIVCPNCSTRL 98 HPV31 L1 Accession number
P1WL31 1
MSLWRPSEATVYLPPVPVSKVVSTDEYVTRTNIYYHAGSARLLTVGHPYYSIPKSDNPKK 60
IVVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPETQRLVWACVGLEVGRGQPLGVGISG 120
HPLLNKFDDTENSNRYAGGPGTDNRECISMDYKQTQLCLLGCKPPIGEHWGKGSPCSNNA 180
ITPGDCPPLELKNSVIQDGDMVDTGFGAMDFTALQDTKSNVPLDICNSICKYPDYLKMVA 240
EPYGDTLFFYLRREQMFVRHFFNRSGTVGESVPTDLYIKGSGSTATLANSTYFPTPSGSM 300
VTSDAQIFNKPYWMQRAQGHNNGICWGNQLFVTVVDTTRSTNMSVCAAIANSDTTFKSSN 360
FKEYLRHGEEFDLQFIFQLCKITLSADIMTYIHSMNPAILEDWNFGLTTPPSGSLEDTYR 420
FVTSQAITCQKTAPQKPKEDPFKDYVFWEVNLKEKFSADLDQFPLGRKFLLQAGYRARPK 480
FKAGKRSAPSASTTTPAKRKKTKK 504 HPV31 L2 Accession number P2WL31 1
MRSKRSTKRTKRASATQLYQTCKAAGTCPSDVIPKIEHTTIADQILRYGSMGVFFGGLGI 60
GSGSGTGGRTGYVPLSTRPSTVSEASIPIRPPVSIDPVGPLDPSIVSLVEESGIVDVGAP 120
APIPHPPTTSGFDIATTADTTPAILDVTSVSTHENPTFTDPSVLQPPTPAETSGHLLLSS 180
SSISTHNYEEIPMDTFIVSTNNENITSSTPIPGVRRPARLGLYSKATQQVKVIDPTFLSA 240
PKQLITYENPAYETVNAEESLYFSNTSHNIAPDPDFLDIIALHRPALTSRRNTVRYSRLG 300
NKQTLRTRSGATIGARVHYYYDISSINPAGESIEMQPLGASATTTSTLNDGLYDIYADTD 360
FTVDTPATHNVSPSTAVQSTSAVSAYVPTNTTVPLSTGFDIPIFSGPDVPIEHAPTQVFP 420
FPLAPTTPQVSIFVDGGDFYLHPSYYMLKRRRKRVSYFFTDVSVAA 466 HPV45 E1
Accession number S36563 1
MADPEGTDGEGTGCNGWFFVETIVEKKTGDVISDDEDETATDTGSDMVDFIDTQLSICEQ 60
AEQETAQALFHAQEVQNDAQVLHLLKRKFAGGSKENSPLGEQLSVDTDLSPRLQEISLNS 120
GHKKAKRRLFTISDSGYGCSEVEAAETQVTVNTNAENGGSVHSTQSSGGDSSDNAENVDP 180
HCSITELKELLQASNKKAAMLAVFKDIYGLSFTDLVRNFKSDKTTCTDWVMAIFGVNPTV 240
AEGFKTLIKPATLYAHIQCLDCKWGVLILALLRYKCGKNRLTVAKGLSTLLHVPETCMLI 300
EPPKLRSSVAALYWYRTGISNISEVSGDTPEWIQRLTIIQHGIDDSNFDLSDMVQWAFDN 360
DLTDESDMAFQYAQLADCNSNAAAFLKSNCQAKYLKDCAVMCRHYKRAQKRQMNMSQWIK 420
YRCSKIDEGGDWRPIVQFLRYQGVEFISFLRALKEFLKGTPKKNCILLYGPANTGKSYFG 480
MSFIHFLQGAIISFVNSNSHFWLEPLADTKVANLDDATHTCWTYFDNYMRNALDGNPISI 540
DRKHKPLLQLKCPPILLTSNIDPAKDNKWPYLESRVTVFTFPHAFPFDKNGNPVYEINDK 600
NWKCFFERTWSRLDLHEDDEDADTEGIPFGTFKCVTGQNTRPL 643 HPV45 E2 Accession
number S36564
MKMQTPKESLSERLSALQDKILDHYENDSKDINSQISYWQLIRLENAILFTAREHGITKL 60
NHQVVPPINISKSKAHKAIELQMALKGLAQSKYNNEEWTLQDTCEELWNTEPSQCFKKGG 120
KTVHVYFDGNKDNCMNYVVWDSIYYITETGIWDKTAACVSYWGVYYIKDGDTTYYVQFKS 180
ECEKYGNSNTWEVQYGGNVIDCNDSMCSTSDDTVSATQIVRQLQHASTSTPKTASVGTPK 240
PHIQTPATKRPRQCGLTEQHHGRVNTHVHNPLLCSSTSNNKRRKVCSGNTTPIIHLKGDK 300
NSLKCLRYRLRKYADHYSEISSTWHWTGCNKNTGILTVTYNSEVQRNTFLDVVTIPNSVQ 360
ISVGYMTI 368 HPV45 E6 Accession number CAB44706 1
MARFDDPTQRPYKLPDLCTELNTSLQDVSIACVYCKATLERTEVYQFAFKDLFIVYRDCI 60
AYAACHKCIDFYSRIRELRYYSNSVYGETLEKITNTELYNLLIRCLRCQKPLNPAEKRRH 120
LKDKRRFHSIAGQYRGQCNTCCDQARQERLRRRRETQV 158 HPV45 E7 Accession
number CAB44707 1
MHGPRATLQEIVLHLEPQNELDPVDLLCYEQLSESEEENDEADGVSHAQLPARRAEPQRH 60
KILCVCCKCDGRIELTVESSADDLRTLQQLFLSTLSFVCPWCATNQ 106 HPV45 L1
Accession number CAB44705 1
MAHNIIYGHGIIIFLKNVNVFPIFLQMALWRPSDSTVYLPPPSVARVVNTDDYVSRTSIF 60
YHAGSSRLLTVGNPYFRVVPSGAGNKQAVPKVSAYQYRVFRVALPDPNKFGLPDSTIYNP 120
ETQRLVWACVGMEIGRGQPLGIGLSGHPFYNKLDDTESAHAATAVITQDVRDNVSVDYKQ 180
TQLCILGCVPAIGEHWAKGTLCKPAQLQPGDCPPLELKNTIIEDGDMVDTGYGAMDFSTL 240
QDTKCEVPLDICQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGVMGDTVPT 300
DLYIKGTSANMRETPGSCVYSPSPSGSITTSDSQLFNKPYWLHKAQGHNNGICWHNQLFV 360
TVVDTTRSTNLTLCASTQNPVPNTYDPTKFKHYSRHVEEYDLQFIFQLCTITLTAEVMSY 420
IHSMNSSILENWNFGVPPPPTTSLVDTYRFVQSVAVTCQKDTTPPEKQDPYDKLKFWTVD 480
LKEKFSSDLDQYPLGRKFLVQAGLRRRPTIGPRKRPAASTSTASRPAKRVRIRSKK 536 HPV45
L2 Accession number S36565 1
MVSHRAARRKRASATDLYRTCKQSGTCPPDVINKVEGTTLADKILQWSSLGIFLGGLGIG 60
TGSGSGGRTGYVPLGGRSNTVVDVGPTRPPVVIEPVGPTDPSIVTLVEDSSVVASGAPVP 120
TFTGTSGFEITSSGTTTPAVLDITPTVDSVSISSTSFTNPAFSDPSIIEVPQTGEVSGNI 180
FVGTPTSGSHGYEETPLQTFASSGSGTEPISSTPLPTVRRVRGPRLYSRANQQVRVSTSQ 240
FLTHPSSLVTFDNPAYEPLDTTLSFEPTSNVPDSDFMDIIRLHRPALSSRRGTVRFSRLG 300
QRATMFTRSGKQIGGRVHFYHDISPIAATEEIELQPLISATNDSDLFDVYADFPPPASTT 360
PSTIHKSFTYPKYSLTMPSTAASSYSNVTVPLTSAWDVPIYTGPDIILPSHTPMWPSTSP 420
TNASTTTYIGIHGTQYYLWPWYYYFPKKRKRIPYFFADGFVAA 463 HPV33 E1 Accession
number W1WL33 1
MADPEGTNGAGMGCTGWFEVEAVIERRTGDNISEDEDETADDSGTDLLEFIDDSMENSIQ 60
ADTEAARALFNIQEGEDDLNAVCALKRKFAACSQSAAEDVVDPAANPCRTSINKNKECTY 120
RKRKIDELEDSGYGNTEVETQQMVQQVESQNGDTNLNDLESSGVGDDSEVSCETNVDSCE 180
NVTLQEISNVLHSSNTKANILYKFKEAYGISFMELVRPFKSDKTSCTDWCITGYGISPSV 240
AESLKVLIKQHSLYTHLQCLTCDRGIIILLLIRFRCSKNRLTVAKLMSNLLSIPETCMVI 300
EPPKLRSQTCALYWFRTAMSNISDVQGTTPEWIDRLTVLQHSFNDNIFDLSEMVQWAYDN 360
ELTDDSDIAYYYAQLADSNSNAAAFLKSNSQAKIVKDCGIMCRHYKKAEKRKMSIGQWIQ 420
SRCEKTNDGGNWRPIVQLLRYQNIEFTAFLGAFKKFLKGIPKKSCMLICGPANTGKSYFG 480
MSLIQFLKGCVISCVNSKSHFWLQPLSDAKIGMIDDVTPISWTYIDDYMRNALDGNEISI 540
DVKHRALVQLKCPPLLLTSNTNAGTDSRWPYLHSRLTVFEFKNPFPFDENGNPVYAINDE 600
NWKSFFSRTWCKLDLIEEEDKENRGGNISTFKCSAGENTRSLRS 644 HPV33 E2 Accession
number W2WL33 1
MEEISARLNAVQEKILDLYEADKTDLPSQTEHWKLIRMECALLYTAKQMGFSHLCHQVVP 60
SLLASKTKAFQVIELQMALETLSKSQYSTSQWTLQQTSLEVWLCEPPKCFKKQGETVTVQ 120
YDNDKKNTMDYTNWGEIYIIEEDTCTMVTGKVDYIGMYYIHNCEKVYFKYFKEDAAKYSK 180
TQMWEVHVGGQVIVCPTSISSNQTSTTETADIQTDNDNRPPQAAAKRRRPADTTDTAQPL 240
TKLFCADPALDNRTARTATNCTNKQRTVCSSNVAPIVHLKGESNSLKCLRYRLKPYKELY 300
SSMSSTWHWTSDNKNSKNGIVTVTFVTEQQQQMFLGTVKIPPTVQISTGFMTL 353 HPV33 E5
Accession number W5WL33 1
MIFVFVLCFILFLCLSLLLRPLILSISTYAWLLVLVLLLWVFVGSPLKIFFCYLLFLYLP 60
MMCINFHAQHMTQQE 75 HPV33 E6 Accession number W6WL33 1
MFQDTEEKPRTLHDLCQALETTIHNIELQCVECKKPLQRSEVYDFAFADLTVVYREGNPF 60
GICKLCLRFLSKISEYRHYNYSVYGNTLEQTVKKPLNEILIRCIICQRPLCPQEKKRHVD 120
LNKRFHNISGRWAGRCAACWRSRRRETAL 149 HPV33 E7 Accession number W7WL33
1 MRGHKPTLKEYVLDLYPEPTDLYCYEQLSDSSDEDEGLDRPDGQAQPATADYYIVTCCHT 60
CNTTVRLCVNSTASDLRTIQQLLMGTVNIVCPTCAQQ 97 HPV33 L1 Accession number
P1WL33 1
MSVWRPSEATVYLPPVPVSKVVSTDEYVSRTSIYYYAGSSRLLAVGHPYFSIKNPTNAKK 60
LLVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPDTQRLVWACVGLEIGRGQPLGVGISG 120
HPLLNKFDDTETGNKYPGQPGADNPECLSMDYKQTQLCLLGCKPPTGEHWGKGVACTNAA 180
PANDCPPLELINTIIEDGDMVDTGFGCMDFKTLQANKSDVPIDICGSTCKYPDYLKMTSE 240
PYGDSLFFFLRREQMFVRHFFNRAGTLGEAVPDDLYIKGSGTTASIQSSAFFPTPSGSMV 300
TSESQLFNKPYWLQRAQGHNNGICWGNQVFVTVVDTTRSThMTLCTQVTSDSTYKNENFK 360
EYIRHVEEYDLQFVFQLCKVTLTAEVMTYIHAMNPDILEDWQFGLTPPPSASLQDTYRFV 420
TSQAITCQKTVPPKEKEDPLGKYTFWEVDLKEKFSADLDQFPLGRKFLLQAGLKAKPKLK 480
RAAPTSTRTSSAKRKKVKK 499 HPV33 L2 Accession number P2WL33 1
MRHKRSTRRKRASATQLYQTCKATGTCPPDVIPKVEGSTIADQILKYGSLGVFFGGLGIG 60
TGSGSGGRTGYVPIGTDPPTAAIPLQPIRPPVTVDTVGPLDSSIVSLIEETSFIEAGAPA 120
PSIPTPSGFDVTTSADTTPAIINVSSVGESSIQTISTHLNPTFTEPSVLHPPAPAEASGH 180
FIFSSPTVSTQSYENIPMDTFVVSTDSSNVTSSTPIPGSRPVARLGLYSRNTQQVKVVDP 240
AFLTSPHKLITYDNPAFESFDPEDTLQFQHSDISPAPDPDFLDIIALHRPAITSRRHTVR 300
FSRVGQKATLKTRSGKQIGARIHYYQDLSPIVPLDHTVPNEQYELQPLHDTSTSSYSIND 360
GLYDVYADDVDNVHTPMQHSYSTFATTRTSNVSIPLNTGFDTPVMSGPDIPSPLFPTSSP 420
FVPISPFFPFDTIVVDGADFVLHPSYFILRRRRKRFPYFFTDVRVAA 467 HPV56 E2
Accession number S36581 1
MVPCLQVCKAKACSAIEVQTALESLSTTIYNNEEWTLRDTCEELWLTEPKKCFKKEGQHI 60
EVWFDGSKNNCMQYVAWKYIYYNGDCGWQKVCSGVDYRGIYYVHDGHKTYYTDFEQEAKK 120
FGCKNIWEVHMENESIYCPDSVSSTCRYNVSPVETVNEYNTHKTTTTTSTSVGNQDAAVS 180
HRPGKRPRLRESEFDSSRESHAKCVTTHTHISDTDNTDSRSRSINNNNHPGDKTTPVVHL 240
KGEPNRLKCCRYRFQKYKTLFVDVTSTYHWTSTDNKNYSIITIIYKDETQRNSFLSHVKI 300
PVVYRLVWDK 310 HPV56 E6 Accession number W6WL56 1
MEPQFNNPQERPRSLHHLSEVLEIPLIDLRLSCVYCKKELTRAEVYNFACTELKLVYRDD 60
FPYAVCRVCLLFYSKVRKYRYYDYSVYGATLESITKKQLCDLLIRCYRCQSPLTPEEKQL 120
HCDRKRRFHLIAHGWTGSCLGCWRQTSREPRESTV 155 HPV56 E7 Accession number
S36580 1
MHGKVPTLQDVVLELTPQTEIDLQCNEQLDSSEDEDEDEVDHLQERPQQARQAKQHTCYL 60
IHVPCCECKFVVQLDIQSTKEDLRVVQQLLMGALTVTCPLCASSN 105 HPV56 L1
Accession number 538563 1
MMLPMMYIYRDPPLHYGLCIFLDVGAVNVFPIFLQMATWRPSENKVYLPPTPVSKVVATD 60
SYVKRTSIFYHAGSSRLLAVGHPYYSVTKDNTKTNIPKVSAYQYRVFRVRLPDPNKFGLP 120
DTNIYNPDQERLVWACVGLEVGRGQPLGAGLSGHPLFNRLDDTESSNLANNNVIEDSRDN 180
ISVDGKQTQLCIVGCTPAMGEHWTKGAVCKSTQVTTGDCPPLALINTPIEDGDMIDTGFG 240
AMDFKVLQESKAEVPLDIVQSTCKYPDYLKMSADAYGDSMWFYLRREQLFARHYFNRAGK 300
VGETIPAELYLKGSNGREPPPSSVYVATPSGSMITSEAQLFNKPYWLQRAQGHNNGICWG 360
NQLFVTVVDTTRSTNMTISTATEQLSKYDARKINQYLRHVEEYELQFVFQLCKITLSAEV 420
MAYLHNMNANLLEDWNIGLSPPVATSLEDKYRYVRSTAITCQREQPPTEKQDPLAKYKFW 480
DVNLQDSFSTDLDQFPLGRKFLMQLGTRSKPAVATSKKRSAPTSTSTPAKRKRR 534 HPV56 L2
Accession number 536582 1
MVAHRATRRKRASATQLYKTCKLSGTCPEDVVNKIEQKTWADKILQWGSLFTYFGGLGIG 60
TGTGSGGRAGYVPLGSRPSTIVDVTPARPPIVVESVGPTDPSIVTLVEESSVIESGAGIP 120
NFTGSGGFEITSSSTTTPAVLDITPTSSTVHVSSTHITNPLFIDPPVIEAPQTGEVSGNI 180
LISTPTSGIHSYEEIPMQTFAVHGSGTEPISSTPIPGFRRIAAPRLYRKAFQQVKVTDPA 240
FLDRPATLVSADNPLFEGTDTSLAFSPSGVAPDPDFMNIVALHRPAFTTRRGGVRFSRLG 300
RKATIQTRRGTQIGARVHYYYDISPIAQAEEIEMQPLLSANNSFDGLYDIYANIDDEAPG 360
LSSQSVATPSAHLPIKPSTLSFASNTTNVTAPLGNVWETPFYSGPDIVLPTGPSTWPFVP 420
QSPYDVTHDVYIQGSSFALWPVYFFRRRRRKRIPYFFADGDVAA 464
HLA Class I Motifs Indicative of CTL Inducing Peptide Epitopes
[0094] The primary anchor residues of the HLA class I peptide
epitope supermotifs and motifs delineated below are summarized in
Table I. The HLA class I motifs set out in Table I (a) are those
most particularly relevant to the invention claimed here. Primary
and secondary anchor positions are summarized in Table II.
Allele-specific HLA molecules that comprise HLA class I supertype
families are listed in Table VI. In some cases, peptide epitopes
may be listed in both a motif and a supermotif Table. The
relationship of a particular motif and respective supermotif is
indicated in the description of the individual motifs.
HLA-A1 Supermotif
[0095] The HLA-A1 supermotif is characterized by the presence in
peptide ligands of a small (T or S) or hydrophobic (L, I, V, or M)
primary anchor residue in position 2, and an aromatic (Y, F, or W)
primary anchor residue at the C-terminal position of the epitope.
The corresponding family of HLA molecules that bind to the A1
supermotif (i.e., the HLA-A1 supertype) is comprised of at least
A*0101, A*2601, A*2602, A*2501, and A*3201 (see, e.g., DiBrino, M.
et al., J. Immunol. 151:5930, 1993; DiBrino, M. et al., J. Immunol.
152:620, 1994; Kondo, A. et al., Immunogenetics 45:249, 1997).
Other allele-specific HLA molecules predicted to be members of the
A1 superfamily are shown in Table VI. Peptides binding to each of
the individual HLA proteins can be modulated by substitutions at
primary and/or secondary anchor positions, preferably choosing
respective residues specified for the supermotif.
[0096] Representative peptide epitopes that comprise the A1
supermotif are set forth in Table VII.
HLA-A2 Supermotif
[0097] Primary anchor specificities for allele-specific HLA-A2.1
molecules (see, e.g., Falk et al., Nature 351:290-296, 1991; Hunt
et al., Science 255:1261-1263, 1992; Parker et al., J. Immunol.
149:3580-3587, 1992; Ruppert et al., Cell 74:929-937, 1993) and
cross-reactive binding among HLA-A2 and -A28 molecules have been
described. (See, e.g., Fruci et al., Human Immunol. 38:187-192,
1993; Tanigaki et al., Human Immunol. 39:155-162, 1994; Del Guercio
et al., J. Immunol. 154:685-693, 1995; Kast et al., J. Immunol.
152:3904-3912, 1994 for reviews of relevant data.) These primary
anchor residues define the HLA-A2 supermotif; which presence in
peptide ligands corresponds to the ability to bind several
different HLA-A2 and -A28 molecules. The HLA-A2 supermotif
comprises peptide ligands with L, I, V, M, A, T, or Q as a primary
anchor residue at position 2 and L, I, V, M, A, or T as a primary
anchor residue at the C-terminal position of the epitope.
[0098] The corresponding family of HLA molecules (i.e., the HLA-A2
supertype that binds these peptides) is comprised of at least:
A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*0209,
A*0214, A*6802, and A*6901. Other allele-specific HLA molecules
predicted to be members of the A2 superfamily are shown in Table
VI. As explained in detail below, binding to each of the individual
allele-specific HLA molecules can be modulated by substitutions at
the primary anchor and/or secondary anchor positions, preferably
choosing respective residues specified for the supermotif.
[0099] Representative peptide epitopes that comprise an A2
supermotif are set forth in Table VIII. The motifs comprising the
primary anchor residues V, A, T, or Q at position 2 and L, I, V, A,
or T at the C-terminal position are those most particularly
relevant to the invention claimed herein.
HLA-A3 Supermotif
[0100] The HLA-A3 supermotif is characterized by the presence in
peptide ligands of A, L, I, V, M, S, or, T as a primary anchor at
position 2, and a positively charged residue, R or K, at the
C-terminal position of the epitope, e.g., in position 9 of 9-mers
(see, e.g., Sidney et al., Hum. Immunol. 45:79, 1996). Exemplary
members of the corresponding family of HLA molecules (the HLA-A3
supertype) that bind the A3 supermotif include at least A*0301,
A*1101, A*3101, A*3301, and A*6801. Other allele-specific HLA
molecules predicted to be members of the A3 supertype are shown in
Table VI. As explained in detail below, peptide binding to each of
the individual allele-specific HLA proteins can be modulated by
substitutions of amino acids at the primary and/or secondary anchor
positions of the peptide, preferably choosing respective residues
specified for the supermotif.
[0101] Representative peptide epitopes that comprise the A3
supermotif are set forth in Table IX.
HLA-A24 Supermotif
[0102] The HLA-A24 supermotif is characterized by the presence in
peptide ligands of an aromatic (F, W, or Y) or hydrophobic
aliphatic (L, I, V, M, or T) residue as a primary anchor in
position 2, and Y, F, W, L, I, or M as primary anchor at the
C-terminal position of the epitope (see, e.g., Sette and Sidney,
Immunogenetics 1999 November; 50 (3-4):201-12, Review). The
corresponding family of HLA molecules that bind to the A24
supermotif (i.e., the A24 supertype) includes at least A*2402,
A*3001, and A*2301. Other allele-specific HLA molecules predicted
to be members of the A24 supertype are shown in Table VI. Peptide
binding to each of the allele-specific HLA molecules can be
modulated by substitutions at primary and/or secondary anchor
positions, preferably choosing respective residues specified for
the supermotif.
[0103] Representative peptide epitopes that comprise the A24
supermotif are set forth in Table X.
HLA-B7 Supermotif
[0104] The HLA-B7 supermotif is characterized by peptides bearing
proline in position 2 as a primary anchor, and a hydrophobic or
aliphatic amino acid (L, I, V, M, A, F, W, or Y) as the primary
anchor at the C-terminal position of the epitope. The corresponding
family of HLA molecules that bind the B7 supermotif (i.e., the
HLA-B7 supertype) is comprised of at least twenty six HLA-B
proteins including: B*0702, B*0703, B*0704, B*0705, B*1508, B*3501,
B*3502, B*3503, B*3504, B*3505, B*3506, B*3507, B*3508, B*5101,
B*5102, B*5103, B*5104, B*5105, B*5301, B*5401, B*5501, B*5502,
B*5601, B*5602, B*6701, and B*7801 (see, e.g., Sidney, et al., J.
Immunol. 154:247, 1995; Barber, et al., Curr. Biol. 5:179, 1995;
Hill, et al., Nature 360:434, 1992; Rammensee, et al.,
Immunogenetics 41:178, 1995 for reviews of relevant data). Other
allele-specific HLA molecules predicted to be members of the B7
supertype are shown in Table VI. As explained in detail below,
peptide binding to each of the individual allele-specific HLA
proteins can be modulated by substitutions at the primary and/or
secondary anchor positions of the peptide, preferably choosing
respective residues specified for the supermotif.
[0105] Representative peptide epitopes that comprise the B7
supermotif are set forth in Table XI.
HLA-B27 Supermotif
[0106] The HLA-B27 supermotif is characterized by the presence in
peptide ligands of a positively charged (R, H, or K) residue as a
primary anchor at position 2, and a hydrophobic (F, Y, L, W, M, I,
A, or V) residue as a primary anchor at the C-terminal position of
the epitope (see, e.g., Sidney and Sette, Immunogenetics 1999
November; 50 (3-4):201-12, Review). Exemplary members of the
corresponding family of HLA molecules that bind to the B27
supermotif (i.e., the B27 supertype) include at least B*1401,
B*1402, B*1509, B*2702, B*2703, B*2704, B*2705, B*2706, B*3801,
B*3901, B*3902, and B*7301. Other allele-specific HLA molecules
predicted to be members of the B27 supertype are shown in Table VI.
Peptide binding to each of the allele-specific HLA molecules can be
modulated by substitutions at primary and/or secondary anchor
positions, preferably choosing respective residues specified for
the supermotif.
[0107] Representative peptide epitopes that comprise the B27
supermotif are set forth in Table XII.
HLA-B44 Supermotif
[0108] The HLA-B44 supermotif is characterized by the presence in
peptide ligands of negatively charged (D or E) residues as a
primary anchor in position 2, and hydrophobic residues (F, W, Y, L,
I, M, V, or A) as a primary anchor at the C-terminal position of
the epitope (see, e.g., Sidney et al., Immunol. Today 17:261,
1996). Exemplary members of the corresponding family of HLA
molecules that bind to the B44 supermotif (i.e., the B44 supertype)
include at least: B*1801, B*1802, B*3701, B*4001, B*4002, B*4006,
B*4402, B*4403, and B*4006. Peptide binding to each of the
allele-specific HLA molecules can be modulated by substitutions at
primary and/or secondary anchor positions; preferably choosing
respective residues specified for the supermotif.
HLA-B58 Supermotif
[0109] The HLA-B58 supermotif is characterized by the presence in
peptide ligands of a small aliphatic residue (A, S, or T) as a
primary anchor residue at position 2, and an aromatic or
hydrophobic residue (F, W, Y, L, I, V, M, or A) as a primary anchor
residue at the C-terminal position of the epitope (see, e.g.,
Sidney and Sette, Immunogenetics 1999 November; 50 (3-4):201-12,
Review). Exemplary members of the corresponding family of HLA
molecules that bind to the B58 supermotif (i.e., the B58 supertype)
include at least: B*1516, B*1517, B*5701, B*5702, and B*5801. Other
allele-specific HLA molecules predicted to be members of the B58
supertype are shown in Table VI. Peptide binding to each of the
allele-specific HLA molecules can be modulated by substitutions at
primary and/or secondary anchor positions, preferably choosing
respective residues specified for the supermotif.
[0110] Representative peptide epitopes that comprise the B58
supermotif are set forth in Table XIII.
HLA-B62 Supermotif
[0111] The HLA-B62 supermotif is characterized by the presence in
peptide ligands of the polar aliphatic residue Q or a hydrophobic
aliphatic residue (L, V, M, I, or P) as a primary anchor in
position 2, and a hydrophobic residue (F, W, Y, M, I, V, L, or A)
as a primary anchor at the C-terminal position of the epitope (see,
e.g., Sidney and Sette, Immunogenetics 1999 November; 50
(3-4):201-12, Review). Exemplary members of the corresponding
family of HLA molecules that bind to the B62 supermotif (i.e., the
B62 supertype) include at least: B*1501, B*1502, B*1513, and B5201.
Other allele-specific HLA molecules predicted to be members of the
B62 supertype are shown in Table VI. Peptide binding to each of the
allele-specific HLA molecules can be modulated by substitutions at
primary and/or secondary anchor positions, preferably choosing
respective residues specified for the supermotif.
[0112] Representative peptide epitopes that comprise the B62
supermotif are set forth in Table XIV.
HLA-A1 Motif
[0113] The HLA-A1 motif is characterized by the presence in peptide
ligands of T, S, or M as a primary anchor residue at position 2 and
the presence of Y as a primary anchor residue at the C-terminal
position of the epitope. An alternative allele-specific A1 motif is
characterized by a primary anchor residue at position 3 rather than
position 2. This motif is characterized by the presence of D, E, A,
or S as a primary anchor residue in position 3, and a Y as a
primary anchor residue at the C-terminal position of the epitope
(see, e.g., DiBrino et al., J. Immunol., 152:620, 1994; Kondo et
al., Immunogenetics 45:249, 1997; and Kubo et al., J. Immunol.
152:3913, 1994 for reviews of relevant data). Peptide binding to
HLA A1 can be modulated by substitutions at primary and/or
secondary anchor positions, preferably choosing respective residues
specified for the motif.
[0114] Representative peptide epitopes that comprise either A1
motif are set forth in Table XV. Those epitopes comprising T, S, or
M at position 2 and Y at the C-terminal position are also included
in the listing of HLA-A1 supermotif-bearing peptides listed in
Table VII, as these residues are a subset of the A1 supermotif
primary anchors.
HLA-A*0201 Motif
[0115] An HLA-A2*0201 motif was determined to be characterized by
the presence in peptide ligands of L or M as a primary anchor
residue in position 2, and L or V as a primary anchor residue at
the C-terminal position of a 9-residue peptide (see, e.g., Falk et
al., Nature 351:290-296, 1991) and was further found to comprise an
I at position 2 and I or A at the C-terminal position of a nine
amino acid peptide (see, e.g., Hunt et al., Science 255:1261-1263,
Mar. 6, 1992; Parker et al., J. Immunol. 149:3580-3587, 1992). The
A*0201 allele-specific motif has also been defined by the present
inventors to additionally comprise V, A, T, or Q as a primary
anchor residue at position 2, and M or T as a primary anchor
residue at the C-terminal position of the epitope (see, e.g., Kast
et al., J. Immunol. 152:3904-3912, 1994). Thus, the HLA-A*0201
motif comprises peptide ligands with L, I, V, M, A, T, or Q as
primary anchor residues at position 2 and L, I, V, M, A, or T as a
primary anchor residue at the C-terminal position of the epitope.
The preferred and tolerated residues that characterize the primary
anchor positions of the HLA-A*0201 motif are identical to the
residues describing the A2 supermotif. (For reviews of relevant
data, see, e.g., Del Guercio et al., J. Immunol. 154:685-693, 1995;
Ruppert et al., Cell 74:929-937, 1993; Sidney et al., Immunol.
Today 17:261-266, 1996; Sette and Sidney, Curr. Opin. in Immunol.
10:478-482, 1998). Secondary anchor residues that characterize the
A*0201 motif have additionally been defined (see, e.g., Ruppert et
al., Cell 74:929-937, 1993). These are shown in Table II. Peptide
binding to HLA-A*0201 molecules can be modulated by substitutions
at primary and/or secondary anchor positions, preferably choosing
respective residues specified for the motif.
[0116] Representative peptide epitopes that comprise an A*0201
motif are set forth in Table VIII. The A*0201 motifs comprising the
primary anchor residues V, A, T, or Q at position 2 and L, I, V, A,
or T at the C-terminal position are those most particularly
relevant to the invention claimed herein.
HLA-A3 Motif
[0117] The HLA-A3 motif is characterized by the presence in peptide
ligands of L, M, V, I, S, A, T, F, C, G, or D as a primary anchor
residue at position 2, and the presence of K, Y, R, H, F, or A as a
primary anchor residue at the C-terminal position of the epitope
(see, e.g., DiBrino et al., Proc. Natl. Acad. Sci. USA 90:1508,
1993; and Kubo et al., J. Immunol. 152:3913-3924, 1994). Peptide
binding to HLA-A3 can be modulated by substitutions at primary
and/or secondary anchor positions, preferably choosing respective
residues specified for the motif.
[0118] Representative peptide epitopes that comprise the A3 motif
are set forth in Table XVI. Those epitopes that also comprise the
A3 supermotif are also listed in Table IX. The A3 supermotif
primary anchor residues comprise a subset of the A3- and A
11-allele specific motif primary anchor residues.
HLA-A 11 Motif
[0119] The HLA-A11 motif is characterized by the presence in
peptide ligands of V, T, M, L, I, S, A, G, N, C, D, or F as a
primary anchor residue in position 2, and K, R, Y, or H as a
primary anchor residue at the C-terminal position of the epitope
(see, e.g., Zhang et al., Proc. Natl. Acad. Sci. USA 90:2217-2221,
1993; and Kubo et al., J. Immunol. 152:3913-3924, 1994). Peptide
binding to HLA-A11 can be modulated by substitutions at primary
and/or secondary anchor positions, preferably choosing respective
residues specified for the motif.
[0120] Representative peptide epitopes that comprise the A11 motif
are set forth in Table XVII; peptide epitopes comprising the A3
allele-specific motif are also present in this Table because of the
extensive overlap between the A3 and A11 motif primary anchor
specificities. Further, those peptide epitopes that comprise the A3
supermotif are also listed in Table IX.
HLA-A24 Motif
[0121] The HLA-A24 motif is characterized by the presence in
peptide ligands of Y, F, W, or M as a primary anchor residue in
position 2, and F, L, I, or W as a primary anchor residue at the
C-terminal position of the epitope (see, e.g., Kondo et al., J.
Immunol. 155:4307-4312, 1995; and Kubo et al., J. Immunol.
152:3913-3924, 1994). Peptide binding to HLA-A24 molecules can be
modulated by substitutions at primary and/or secondary anchor
positions; preferably choosing respective residues specified for
the motif.
[0122] Representative peptide epitopes that comprise the A24 motif
are set forth in Table XVIII. These epitopes are also listed in
Table X, which sets forth HLA-A24-supermotif-bearing peptides, as
the primary anchor residues characterizing the A24 allele-specific
motif comprise a subset of the A24 supermotif primary anchor
residues.
Motifs Indicative of Class II HTL Inducing Peptide Epitopes
[0123] The primary and secondary anchor residues of the HLA class
II peptide epitope supermotifs and motifs delineated below are
summarized in Table III.
HLA DR-1-4-7 Supermotif
[0124] Motifs have also been identified for peptides that bind to
three common HLA class II allele-specific HLA molecules: HLA
DRB1*0401, DRB1*0101, and DRB1*0701 (see, e.g., the review by
Southwood et al. J Immunology 160:3363-3373, 1998). Collectively,
the common residues from these motifs delineate the HLA DR-1-4-7
supermotif. Peptides that bind to these DR molecules carry a
supermotif characterized by a large aromatic or hydrophobic residue
(Y, F, W, L, I, V, or M) as a primary anchor residue in position 1,
and a small, non-charged residue (S, T, C, A, P, V, I, L, or M) as
a primary anchor residue in position 6 of a 9-mer core region.
Allele-specific secondary effects and secondary anchors for each of
these HLA types have also been identified (Southwood et al.,
supra). These are set forth in Table III. Peptide binding to
HLA-DRB1*0401, DRB1*0101, and/or DRB1*0701 can be modulated by
substitutions at primary and/or secondary anchor positions,
preferably choosing respective residues specified for the
supermotif.
[0125] Representative 9-mer epitopes comprising the DR-1-4-7
supermotif, wherein position 1 of the supermotif is at position 1
of the nine-residue core, are set forth in Table XIX. Exemplary
epitopes of 15 amino acids in length that comprises the nine
residue core include the three residues on either side that flank
the nine residue core. HTL epitopes that comprise the core
sequences can also be of lengths other than 15 amino acids, supra.
Accordingly, epitopes of the invention include sequences that
typically comprise the nine residue core plus 1, 2, 3 (as in the
exemplary 15-mer), 4, or 5 flanking residues on either side of the
nine residue core.
HLA DR3 Motifs
[0126] Two alternative motifs (i.e., submotifs) characterize
peptide epitopes that bind to HLA-DR3 molecules (see, e.g., Geluk
et al., J. Immunol. 152:5742, 1994). In the first motif (submotif
DR3A) a large, hydrophobic residue (L, I, V, M, F, or Y) is present
in anchor position 1 of a 9-mer core, and D is present as an anchor
at position 4, towards the carboxyl terminus of the epitope. As in
other class II motifs, core position 1 may or may not occupy the
peptide N-terminal position.
[0127] The alternative DR3 submotif provides for lack of the large,
hydrophobic residue at anchor position 1, and/or lack of the
negatively charged or amide-like anchor residue at position 4, by
the presence of a positive charge at position 6 towards the
carboxyl terminus of the epitope. Thus, for the alternative
allele-specific DR3 motif (submotif DR3B): L, I, V, M, F, Y, A, or
Y is present at anchor position 1; D, N, Q, E, S, or T is present
at anchor position 4; and K, R, or H is present at anchor position
6. Peptide binding to HLA-DR3 can be modulated by substitutions at
primary and/or secondary anchor positions, preferably choosing
respective residues specified for the motif.
[0128] Representative 9-mer epitopes corresponding to a nine
residue sequence comprising the DR3a and DR3b submotifs (wherein
position 1 of the motif is at position 1 of the nine residue core)
are set forth in Table XXa and b. Exemplary epitopes of 15 amino
acids in length that comprises the nine residue core include the
three residues on either side that flank the nine residue core. HTL
epitopes that comprises the cores sequences can also be of lengths
other than 15 amino acids, supra. Accordingly, epitopes of the
invention include sequences that typically comprise the nine
residue core plus 1, 2, 3 (as in the exemplary 15-mer), 4, or 5
flanking residues on either side of the nine residue core.
[0129] Each of the HLA class I or class II epitopes set out in the
Tables herein are deemed singly to be an inventive aspect of this
application. Further, it is also an inventive aspect of this
application that each epitope may be used in combination with any
other epitope.
Enhancing Population Coverage of the Vaccine
[0130] Vaccines that have broad population coverage are preferred
because they are more commercially viable and generally applicable
to the most people. Broad population coverage can be obtained using
the peptides of the invention (and nucleic acid compositions that
encode such peptides) through selecting peptide epitopes that bind
to HLA alleles which, when considered in total, are present in most
of the population. The Table below lists the overall frequencies of
the HLA class I supertypes in various ethnicities (section a) and
the combined population coverage achieved by the A2-, A3-, and
B7-supertypes (section b). The A2-, A3-, and B7 supertypes are each
present on the average of over 40% in each of these five major
ethnic groups. Coverage in excess of 80% is achieved with a
combination of these supermotifs. These results suggest that
effective and non-ethnically biased population coverage is achieved
upon use of a limited number of cross-reactive peptides. Although
the population coverage reached with these three main peptide
specificities is high, coverage can be expanded to reach 95%
population coverage and above, and more easily achieve truly
multispecific responses upon use of additional supermotif or
allele-specific motif bearing peptides.
[0131] The B44-, A1-, and A24-supertypes are each present, on
average, in a range from 25% to 40% in these major ethnic
populations (section a). While less prevalent overall, the B27-,
B58-, and B62 supertypes are each present with a frequency >25%
in at least one major ethnic group (section a). In section b, the
Table summarizes the estimated prevalence of combinations of HLA
supertypes that have been identified in five major ethnic groups.
The incremental coverage obtained by the inclusion of A1-, A24-,
and B44-supertypes to the A2, A3, and B7 coverage and coverage
obtained with all of the supertypes described herein, is shown.
[0132] The data presented herein, together with the previous
definition of the A2-, A3-, and B7-supertypes, indicates that all
antigens, with the possible exception of A29, B8, and B46, can be
classified into a total of nine HLA supertypes. By including
epitopes from the six most frequent supertypes, an average
population coverage of 99% is obtained for five major ethnic
groups.
Population Coverage with Combined HLA Supertypes
TABLE-US-00002 PHENOTYPIC FREQUENCY North HLA- American SUPERTYPES
Caucasian Black Japanese Chinese Hispanic Average a. Individual
Supertypes A2 45.8 39.0 42.4 45.9 43.0 43.2 A3 37.5 42.1 45.8 52.7
43.1 44.2 B7 43.2 55.1 57.1 43.0 49.3 49.5 A1 47.1 16.1 21.8 14.7
26.3 25.2 A24 23.9 38.9 58.6 40.1 38.3 40.0 B44 43.0 21.2 42.9 39.1
39.0 37.0 B27 28.4 26.1 13.3 13.9 35.3 23.4 B62 12.6 4.8 36.5 25.4
11.1 18.1 B58 10.0 25.1 1.6 9.0 5.9 10.3 b. Combined Supertypes A2,
A3, B7 84.3 86.8 89.5 89.8 86.8 87.4 A2, A3, B7, A24, 99.5 98.1
100.0 99.5 99.4 99.3 B44, A1 A2, A3, B7, A24, 99.9 99.6 100.0 99.8
99.9 99.8 B44, A1, B27, B62, B58
Immune Response-Stimulating Peptide Analogs
[0133] In general, CTL and HTL responses to whole antigens are not
directed against all possible epitopes. Rather, they are restricted
to a few "immunodominant" determinants (Zinkernagel, et al., Adv.
Immunol. 27:5159, 1979; Bennink, et al., J. Exp. Med. 168:19351939,
1988; Rawle, et al., J. Immunol. 146:3977-3984, 1991). It has been
recognized that immunodominance (Benacerraf, et al., Science
175:273-279, 1972) could be explained by either the ability of a
given epitope to selectively bind a particular HLA protein
(determinant selection theory) (Vitiello, et al., J. Immunol.
131:1635, 1983); Rosenthal, et al., Nature 267:156-158, 1977), or
to be selectively recognized by the existing TCR (T cell receptor)
specificities (repertoire theory) (Klein, J., IMMUNOLOGY, THE
SCIENCE OF SELFNONSELF DISCRIMINATION, John Wiley & Sons, New
York, pp. 270-310, 1982). It has been demonstrated that additional
factors, mostly linked to processing events, can also play a key
role in dictating, beyond strict immunogenicity, which of the many
potential determinants will be presented as immunodominant
(Sercarz, et al., Annu. Rev. Immunol. 11:729-766, 1993).
[0134] The concept of dominance and subdominance is relevant to
immunotherapy of both infectious diseases and cancer. For example,
in the course of chronic viral disease, recruitment of subdominant
epitopes can be important for successful clearance of the
infection, especially if dominant CTL or HTL specificities have
been inactivated by functional tolerance, suppression, mutation of
viruses and other mechanisms (Franco, et al., Curr. Opin. Immunol.
7:524-531, 1995). In the case of cancer and tumor antigens, CTLs
recognizing at least some of the highest binding affinity peptides
might be functionally inactivated. Lower binding affinity peptides
are preferentially recognized at these times, and may therefore be
preferred in therapeutic or prophylactic anti-cancer vaccines.
[0135] In particular, it has been noted that a significant number
of epitopes derived from known non-viral tumor associated antigens
(TAA) bind HLA class I with intermediate affinity (IC.sub.50 in the
50-500 nM range). For example, it has been found that 8 of 15 known
TAA peptides recognized by tumor infiltrating lymphocytes (TIL) or
CTL bound in the 50-500 nM range. (These data are in contrast with
estimates that 90% of known viral antigens were bound by HLA class
I molecules with IC.sub.50 of 50 nM or less, while only
approximately 10% bound in the 50-500 nM range (Sette, et al., J.
Immunol., 153:558-5592, 1994). In the cancer setting this
phenomenon is probably due to elimination or functional inhibition
of the CTL recognizing several of the highest binding peptides,
presumably because of T cell tolerization events.
[0136] Without intending to be bound by theory, it is believed that
because T cells to dominant epitopes may have been clonally
deleted, selecting subdominant epitopes may allow existing T cells
to be recruited, which will then lead to a therapeutic or
prophylactic response. However, the binding of HLA molecules to
subdominant epitopes is often less vigorous than to dominant ones.
Accordingly, there is a need to be able to modulate the binding
affinity of particular immunogenic epitopes for one or more HLA
molecules, and thereby to modulate the immune response elicited by
the peptide, for example to prepare analog peptides which elicit a
more vigorous response. This ability would greatly enhance the
usefulness of peptide epitope-based vaccines and therapeutic
agents.
[0137] Although peptides with suitable cross-reactivity among all
alleles of a superfamily are identified by the screening procedures
described above, cross-reactivity is not always as complete as
possible, and in certain cases procedures to increase
cross-reactivity of peptides can be useful; moreover, such
procedures can also be used to modify other properties of the
peptides such as binding affinity or peptide stability. Having
established the general rules that govern cross-reactivity of
peptides for HLA alleles within a given motif or supermotif,
modification (i.e., analoging) of the structure of peptides of
particular interest in order to achieve broader (or otherwise
modified) HLA binding capacity can be performed. More specifically,
peptides which exhibit the broadest cross-reactivity patterns, can
be produced in accordance with the teachings herein. The present
concepts related to analog generation are set forth in greater
detail in co-pending U.S. Ser. No. 09/226,775 filed Jan. 6,
1999.
[0138] In brief, the strategy employed utilizes the motifs or
supermotifs which correlate with binding to certain HLA molecules.
The motifs or supermotifs are defined by having primary anchors,
and in many cases secondary anchors. Analog peptides can be created
by substituting amino acid residues at primary anchor, secondary
anchor, or at primary and secondary anchor positions. Generally,
analogs are made for peptides that already bear a motif or
supermotif. Preferred secondary anchor residues of supermotifs and
motifs that have been defined for HLA class I and class II binding
peptides are shown in Tables II and III, respectively.
[0139] For a number of the motifs or supermotifs in accordance with
the invention, residues are defined which are deleterious to
binding to allele-specific HLA molecules or members of HLA
supertypes that bind the respective motif or supermotif (Tables II
and III). Accordingly, removal of such residues that are
detrimental to binding can be performed in accordance with the
present invention. For example, in the case of the A3 supertype,
when all peptides that have such deleterious residues are removed
from the population of peptides used in the analysis, the incidence
of cross-reactivity increased from 22% to 37% (see, e.g., Sidney,
J. et al., Hu. Immunol. 45:79, 1996). Thus, one strategy to improve
the cross-reactivity of peptides within a given supermotif is
simply to delete one or more of the deleterious residues present
within a peptide and substitute a small "neutral" residue such as
Ala (that may not influence T cell recognition of the peptide). An
enhanced likelihood of cross-reactivity is expected if, together
with elimination of detrimental residues within a peptide,
"preferred" residues associated with high affinity binding to an
allele-specific HLA molecule or to multiple HLA molecules within a
superfamily are inserted.
[0140] To ensure that an analog peptide, when used as a vaccine,
actually elicits a CTL response to the native epitope in vivo (or,
in the case of class II epitopes, elicits helper T cells that
cross-react with the wild type peptides), the analog peptide may be
used to immunize T cells in vitro from individuals of the
appropriate HLA allele. Thereafter, the immunized cells' capacity
to induce lysis of wild type peptide sensitized target cells is
evaluated. It will be desirable to use as antigen presenting cells,
cells that have been either infected, or transfected with the
appropriate genes, or, in the case of class II epitopes only, cells
that have been pulsed with whole protein antigens, to establish
whether endogenously produced antigen is also recognized by the
relevant T cells.
[0141] Another embodiment of the invention is to create analogs of
weak binding peptides, to thereby ensure adequate numbers of
cross-reactive cellular binders. Class I binding peptides
exhibiting binding affinities of 500-5000 nM, and carrying an
acceptable but suboptimal primary anchor residue at one or both
positions can be "fixed" by substituting preferred anchor residues
in accordance with the respective supertype. The analog peptides
can then be tested for crossbinding activity.
[0142] Another embodiment for generating effective peptide analogs
involves the substitution of residues that have an adverse impact
on peptide stability or solubility in, e.g., a liquid environment.
This substitution may occur at any position of the peptide epitope.
For example, a cysteine (C) can be substituted out in favor of
.alpha.-amino butyric acid. Due to its chemical nature, cysteine
has the propensity to form disulfide bridges and sufficiently alter
the peptide structurally so as to reduce binding capacity.
Substituting .alpha.-amino butyric acid for C not only alleviates
this problem, but actually improves binding and crossbinding
capability in certain instances (see, e.g., the review by Sette et
al., In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen,
John Wiley & Sons, England, 1999). Substitution of cysteine
with .alpha.-amino butyric acid may occur at any residue of a
peptide epitope, i.e. at either anchor or non-anchor positions.
Computer Screening of Protein Sequences from Disease-Related
Antigens for Supermotif- or Motif-Bearing Peptides
[0143] In order to identify supermotif- or motif-bearing epitopes
in a target antigen, a native protein sequence, e.g., a
tumor-associated antigen, or sequences from an infectious organism,
or a donor tissue for transplantation, is screened using a means
for computing, such as an intellectual calculation or a computer,
to determine the presence of a supermotif or motif within the
sequence. The information obtained from the analysis of native
peptide can be used directly to evaluate the status of the native
peptide or may be utilized subsequently to generate the peptide
epitope.
[0144] Computer programs that allow the rapid screening of protein
sequences for the occurrence of the subject supermotifs or motifs
are encompassed by the present invention; as are programs that
permit the generation of analog peptides. These programs are
implemented to analyze any identified amino acid sequence or
operate on an unknown sequence and simultaneously determine the
sequence and identify motif-bearing epitopes thereof; analogs can
be simultaneously determined as well. Generally, the identified
sequences will be from a pathogenic organism or a tumor-associated
peptide. For example, the target molecules considered herein
include, without limitation, the E1, E2, E4, E5a, E5b, E6, E7, L1
and L2 proteins of HPV.
[0145] In cases where the sequences of multiple variants of the
same target protein are available, potential peptide epitopes can
also be selected on the basis of their conservancy. For example, a
criterion for conservancy may define that the entire sequence of an
HLA class I binding peptide or the entire 9-mer core of a class II
binding peptide, be conserved in a designated percentage, of the
sequences evaluated for a specific protein antigen.
[0146] To target a broad population that may be infected with a
number of different strains, it is preferable to include in vaccine
compositions epitopes that are representative of HPV antigen
sequences from different HPV strains. As appreciated by those in
the art, regions with greater or lessor degrees of conservancy
among HPv strains can be employed as appropriate for a given
antigenic target.
[0147] It is important that the selection criteria utilized for
prediction of peptide binding are as accurate as possible, to
correlate most efficiently with actual binding. Prediction of
peptides that bind, for example, to HLA-A*0201, on the basis of the
presence of the appropriate primary anchors, is positive at about a
30% rate (see, e.g., Ruppert, J. et al. Cell 74:929, 1993).
However, by extensively analyzing peptide-HLA binding data
disclosed herein, data in related patent applications, and data in
the art, the present inventors have developed a number of
allele-specific polynomial algorithms that dramatically increase
the predictive value over identification on the basis of the
presence of primary anchor residues alone. These algorithms take
into account not only the presence or absence of primary anchors,
but also consider the positive or deleterious presence of secondary
anchor residues (to account for the impact of different amino acids
at different positions). The algorithms are essentially based on
the premise that the overall affinity (or .DELTA.G) of peptide-HLA
interactions can be approximated as a linear polynomial function of
the type:
.DELTA.G=a.sub.1i.times.a.sub.2i.times.a.sub.3i . . .
.times.a.sub.ni
[0148] where a.sub.ji is a coefficient that represents the effect
of the presence of a given amino acid (j) at a given position (i)
along the sequence of a peptide of n amino acids. An important
assumption of this method is that the effects at each position are
essentially independent of each other. This assumption is justified
by studies that demonstrated that peptides are bound to HLA
molecules and recognized by T cells in essentially an extended
conformation. Derivation of specific algorithm coefficients has
been described, for example, in Gulukota, K. et al., J. Mol. Biol.
267:1258, 1997.
[0149] Additional methods to identify preferred peptide sequences,
which also make use of specific motifs, include the use of neural
networks and molecular modeling programs (see, e.g., Milik et al.,
Nature Biotechnology 16:753, 1998; Altuvia et al., Hum. Immunol.
58:1, 1997; Altuvia et al, J. Mol. Biol. 249:244, 1995; Buus, S.
Curr. Opin. Immunol. 11:209-213, 1999; Brusic, V. et al.,
Bioinformatics 14:121-130, 1998; Parker et al., J. Immunol.
152:163, 1993; Meister et al., Vaccine 13:581, 1995; Hammer et al.,
J. Exp. Med. 180:2353, 1994; Sturniolo et al., Nature Biotechnol.
17:555 1999).
[0150] For example, it has been shown that in sets of A*0201
motif-bearing peptides containing at least one preferred secondary
anchor residue while avoiding the presence of any deleterious
secondary anchor residues, 69% of the peptides will bind A*0201
with an IC.sub.50 less than 500 nM (Ruppert, J. et al. Cell 74:929,
1993). These algorithms are also flexible in that cut-off scores
may be adjusted to select sets of peptides with greater or lower
predicted binding properties, as desired.
[0151] In utilizing computer screening to identify peptide
epitopes, a protein sequence or translated sequence may be analyzed
using software developed to search for motifs, for example the
"FINDPATTERNS` program (Devereux, et al. Nucl. Acids Res.
12:387-395, 1984) or MotifSearch 1.4 software program (D. Brown,
San Diego, Calif.) to identify potential peptide sequences
containing appropriate HLA binding motifs. The identified peptides
can be scored using customized polynomial algorithms to predict
their capacity to bind specific HLA class I or class II alleles. As
appreciated by one of ordinary skill in the art, a large array of
computer programming software and hardware options are available in
the relevant art which can be employed to implement the motifs of
the invention in order to evaluate (e.g., without limitation, to
identify epitopes, identify epitope concentration per peptide
length, or to generate analogs) known or unknown peptide
sequences.
[0152] In accordance with the procedures described above, HPV
peptide epitopes that are able to bind HLA supertype groups or
allele-specific HLA molecules have been identified (Tables
VII-XX).
Preparation of Peptide Epitopes
[0153] Peptides in accordance with the invention can be prepared
synthetically, by recombinant DNA technology or chemical synthesis,
or from natural sources such as native tumors or pathogenic
organisms. Peptide epitopes may be synthesized individually or as
polyepitopic peptides. Although the peptide will preferably be
substantially free of other naturally occurring host cell proteins
and fragments thereof, in some embodiments the peptides may be
synthetically conjugated to native fragments or particles.
[0154] The peptides in accordance with the invention can be a
variety of lengths, and either in their neutral (uncharged) forms
or in forms which are salts. The peptides in accordance with the
invention are either free of modifications such as glycosylation,
side chain oxidation, or phosphorylation; or they contain these
modifications, subject to the condition that modifications do not
destroy the biological activity of the peptides as described
herein.
[0155] When possible, it may be desirable to optimize HLA class I
binding epitopes of the invention, such as can be used in a
polyepitopic construct, to a length of about 8 to about 13 amino
acid residues, often 8 to 11, preferably 9 to 10. HLA class II
binding peptide epitopes of the invention may be optimized to a
length of about 6 to about 30 amino acids in length, preferably to
between about 13 and about 20 residues. Preferably, the peptide
epitopes are commensurate in size with endogenously processed
pathogen-derived peptides or tumor cell peptides that are bound to
the relevant HLA molecules, however, the identification and
preparation of peptides that comprise epitopes of the invention can
also be carried out using the techniques described herein.
[0156] In alternative embodiments, epitopes of the invention can be
linked as a polyepitopic peptide, or as a minigene that encodes a
polyepitopic peptide.
[0157] In another embodiment, it is preferred to identify native
peptide regions that contain a high concentration of class I and/or
class II epitopes. Such a sequence is generally selected on the
basis that it contains the greatest number of epitopes per amino
acid length. It is to be appreciated that epitopes can be present
in a nested or overlapping manner, e.g. a 10 amino acid long
peptide could contain two 9 amino acid long epitopes and one 10
amino acid long epitope; upon intracellular processing, each
epitope can be exposed and bound by an HLA molecule upon
administration of such a peptide. This larger, preferably
multi-epitopic, peptide can be generated synthetically,
recombinantly, or via cleavage from the native source.
[0158] The peptides of the invention can be prepared in a wide
variety of ways. For the preferred relatively short size, the
peptides can be synthesized in solution or on a solid support in
accordance with conventional techniques. Various automatic
synthesizers are commercially available and can be used in
accordance with known protocols. (See, for example, Stewart &
Young, SOLID PHASE PEPTIDE SYNTHESIS, 2D. ED., Pierce Chemical Co.,
1984). Further, individual peptide epitopes can be joined using
chemical ligation to produce larger peptides that are still within
the bounds of the invention.
[0159] Alternatively, recombinant DNA technology can be employed
wherein a nucleotide sequence which encodes an immunogenic peptide
of interest is inserted into an expression vector, transformed or
transfected into an appropriate host cell and cultivated under
conditions suitable for expression. These procedures are generally
known in the art, as described generally in Sambrook et al.,
MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Press,
Cold Spring Harbor, N.Y. (1989). Thus, recombinant polypeptides
which comprise one or more peptide sequences of the invention can
be used to present the appropriate T cell epitope.
[0160] The nucleotide coding sequence for peptide epitopes of the
preferred lengths contemplated herein can be synthesized by
chemical techniques, for example, the phosphotriester method of
Matteucci, et al., J. Am. Chem. Soc. 103:3185 (1981). Peptide
analogs can be made simply by substituting the appropriate and
desired nucleic acid base(s) for those that encode the native
peptide sequence; exemplary nucleic acid substitutions are those
that encode an amino acid defined by the motifs/supermotifs herein.
The coding sequence can then be provided with appropriate linkers
and ligated into expression vectors commonly available in the art,
and the vectors used to transform suitable hosts to produce the
desired fusion protein. A number of such vectors and suitable host
systems are now available. For expression of the fusion proteins,
the coding sequence will be provided with operably linked start and
stop codons, promoter and terminator regions and usually a
replication system to provide an expression vector for expression
in the desired cellular host. For example, promoter sequences
compatible with bacterial hosts are provided in plasmids containing
convenient restriction sites for insertion of the desired coding
sequence. The resulting expression vectors are transformed into
suitable bacterial hosts. Of course, yeast, insect or mammalian
cell hosts may also be used, employing suitable vectors and control
sequences.
Assays to Detect T-Cell Responses
[0161] Once HLA binding peptides are identified, they can be tested
for the ability to elicit a T-cell response. The preparation and
evaluation of motif-bearing peptides are described in PCT
publications WO 94/20127 and WO 94/03205. Briefly, peptides
comprising epitopes from a particular antigen are synthesized and
tested for their ability to bind to the appropriate HLA proteins.
These assays may involve evaluating the binding of a peptide of the
invention to purified HLA class I molecules in relation to the
binding of a radioiodinated reference peptide. Alternatively, cells
expressing empty class I molecules (i.e. lacking peptide therein)
may be evaluated for peptide binding by immunofluorescent staining
and flow microfluorimetry. Other assays that may be used to
evaluate peptide binding include peptide-dependent class I assembly
assays and/or the inhibition of CTL recognition by peptide
competition. Those peptides that bind to the class I molecule,
typically with an affinity of 500 nM or less, are further evaluated
for their ability to serve as targets for CTLs derived from
infected or immunized individuals, as well as for their capacity to
induce primary in vitro or in vivo CTL responses that can give rise
to CTL populations capable of reacting with selected target cells
associated with a disease.
[0162] Analogous assays are used for evaluation of HLA class II
binding peptides. HLA class II motif-bearing peptides that are
shown to bind, typically at an affinity of 1000 nM or less, are
further evaluated for the ability to stimulate HTL responses.
[0163] Conventional assays utilized to detect T cell responses
include proliferation assays, lymphokine secretion assays, direct
cytotoxicity assays, and limiting dilution assays. For example,
antigen-presenting cells that have been incubated with a peptide
can be assayed for the ability to induce CTL responses in responder
cell populations. Antigen-presenting cells can be normal cells such
as peripheral blood mononuclear cells or dendritic cells.
Alternatively, mutant non-human mammalian cell lines that are
deficient in their ability to load class 1 molecules with
internally processed peptides and that have been transfected with
the appropriate human class I gene, may be used to test for the
capacity of the peptide to induce in vitro primary CTL
responses.
[0164] Peripheral blood mononuclear cells (PBMCs) may be used as
the responder cell source of CTL precursors. The appropriate
antigen-presenting cells are incubated with peptide, after which
the peptide-loaded antigen-presenting cells are then incubated with
the responder cell population under optimized culture conditions.
Positive CTL activation can be determined by assaying the culture
for the presence of CTLs that kill radio-labeled target cells, both
specific peptide-pulsed targets as well as target cells expressing
endogenously processed forms of the antigen from which the peptide
sequence was derived.
[0165] Additionally, a method has been devised which allows direct
quantification of antigen-specific T cells by staining with
Fluorescein-labelled HLA tetrameric complexes (Altman, J. D. et
al., Proc. Natl. Acad. Sci. USA 90:10330, 1993; Altman, J. D. et
al., Science 274:94, 1996). Other relatively recent technical
developments include staining for intracellular lymphokines, and
interferon release assays or ELISPOT assays. Tetramer staining,
intracellular lymphokine staining and ELISPOT assays all appear to
be at least 10-fold more sensitive than more conventional assays
(Lalvani, A. et al., J. Exp. Med. 186:859, 1997; Dunbar, P. R. et
al., Curr. Biol. 8:413, 1998; Murali-Krishna, K. et al., Immunity
8:177, 1998).
[0166] HTL activation may also be assessed using such techniques
known to those in the art such as T cell proliferation and
secretion of lymphokines, e.g. IL-2 (see, e.g. Alexander et al.,
Immunity 1:751-761, 1994).
[0167] Alternatively, immunization of HLA transgenic mice can be
used to determine immunogenicity of peptide epitopes. Several
transgenic mouse models including mice with human A2.1, A11 (which
can additionally be used to analyze HLA-A3 epitopes), and B7
alleles have been characterized and others (e.g., transgenic mice
for HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse
models have also been developed. Additional transgenic mouse models
with other HLA alleles may be generated as necessary. Mice may be
immunized with peptides emulsified in Incomplete Freund's Adjuvant
and the resulting T cells tested for their capacity to recognize
peptide-pulsed target cells and target cells transfected with
appropriate genes. CTL responses may be analyzed using cytotoxicity
assays described above. Similarly, HTL responses may be analyzed
using such assays as T cell proliferation or secretion of
lymphokines.
Use of Peptide Epitopes as Diagnostic Agents and for Evaluating
Immune Responses
[0168] In one aspect of the invention, HLA class I and class II
binding peptides as described herein can be used as reagents to
evaluate an immune response. The immune response to be evaluated is
induced by using as an immunogen any agent that may result in the
production of antigen-specific CTLs or HTLs that recognize and bind
to the peptide epitope(s) to be employed as the reagent. The
peptide reagent need not be used as the immunogen. Assay systems
that are used for such an analysis include relatively recent
technical developments such as tetramers, staining for
intracellular lymphokines and interferon release assays, or ELISPOT
assays.
[0169] For example, a peptide of the invention is used in a
tetramer staining assay to assess peripheral blood mononuclear
cells for the presence of antigen-specific CTLs following exposure
to a pathogen or immunogen. The HLA-tetrameric complex is used to
directly visualize antigen-specific CTLs (see, e.g., Ogg et al.,
Science 279:2103-2106, 1998; and Altman et al., Science 174:94-96,
1996) and determine the frequency of the antigen-specific CTL
population in a sample of peripheral blood mononuclear cells.
[0170] A tetramer reagent using a peptide of the invention is
generated as follows: A peptide that binds to an HLA molecule is
refolded in the presence of the corresponding HLA heavy chain and
.beta..sub.2-microglobulin to generate a trimolecular complex. The
complex is biotinylated at the carboxyl terminal end of the heavy
chain at a site that was previously engineered into the protein.
Tetramer formation is then induced by the addition of streptavidin.
By means of fluorescently labeled streptavidin, the tetramer can be
used to stain antigen-specific cells. The cells can then be readily
identified, for example, by flow cytometry. Such procedures are
used for diagnostic or prognostic purposes. Cells identified by the
procedure can also be used for therapeutic purposes.
[0171] Peptides of the invention are also used as reagents to
evaluate immune recall responses. (see, e.g., Bertoni et al., J.
Clin. Invest. 100:503-513, 1997 and Penna et al., J. Exp. Med.
174:1565-1570, 1991.) For example, patient PBMC samples from
individuals infected with HPV are analyzed for the presence of
antigen-specific CTLs or HTLs using specific peptides. A blood
sample containing mononuclear cells may be evaluated by cultivating
the PBMCs and stimulating the cells with a peptide of the
invention. After an appropriate cultivation period, the expanded
cell population may be analyzed, for example, for CTL or for HTL
activity.
[0172] The peptides are also used as reagents to evaluate the
efficacy of a vaccine. PBMCs obtained from a patient vaccinated
with an immunogen are analyzed using, for example, either of the
methods described above. The patient is HLA typed, and peptide
epitope reagents that recognize the allele-specific molecules
present in that patient are selected for the analysis. The
immunogenicity of the vaccine is indicated by the presence of HPV
epitope-specific CTLs and/or HTLs in the PBMC sample.
[0173] The peptides of the invention are also be used to make
antibodies, using techniques well known in the art (see, e.g.
CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, N.Y.; and Antibodies
A Laboratory Manual Harlow, Harlow and Lane, Cold Spring Harbor
Laboratory Press, 1989), which may be useful as reagents to
diagnose HPV infection. Such antibodies include those that
recognize a peptide in the context of an HLA molecule, i.e.,
antibodies that bind to a peptide-MHC complex.
Vaccine Compositions
[0174] Vaccines and methods of preparing vaccines that contain an
immunogenically effective amount of one or more peptides as
described herein are further embodiments of the invention. Once
appropriately immunogenic epitopes have been defined, they can be
sorted and delivered by various means, herein referred to as
"vaccine" compositions. Such vaccine compositions can include, for
example, lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest.
95:341, 1995), peptide compositions encapsulated in
poly(DL-lactide-co-glycolide) ("PLG") microspheres (see, e.g.,
Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al.,
Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995),
peptide compositions contained in immune stimulating complexes
(ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu
et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigen
peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad.
Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P., J. Immunol. Methods
196:17-32, 1996), peptides formulated as multivalent peptides;
peptides for use in ballistic delivery systems, typically
crystallized peptides, viral delivery vectors (Perkus, M. E. et
al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed.,
p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S.
L. et al., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS
Bio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis.
124:148, 1971; Chanda, P. K. et al., Virology 175:535, 1990),
particles of viral or synthetic origin (e.g., Kofler, N. et al., J.
Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem.
Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649,
1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A.
Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine
11:293, 1993), liposomes (Reddy, R. et al., J. Immunol. 148:1585,
1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked or
particle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745,
1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine
11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine
development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B.,
and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and
Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted
delivery technologies, also known as receptor mediated targeting,
such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.)
may also be used.
[0175] Vaccine compositions of the invention include nucleic
acid-mediated modalities. DNA or RNA encoding one or more of the
peptides of the invention can also be administered to a patient.
This approach is described, for instance, in Wolff et. al., Science
247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466;
5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720; and in
more detail below. Examples of DNA-based delivery technologies
include "naked DNA", facilitated (bupivicaine, polymers,
peptide-mediated) delivery, cationic lipid complexes, and
particle-mediated ("gene gun") or pressure-mediated delivery (see,
e.g., U.S. Pat. No. 5,922,687).
[0176] For therapeutic or prophylactic immunization purposes, the
peptides of the invention can be expressed by viral or bacterial
vectors. Examples of expression vectors include attenuated viral
hosts, such as vaccinia or fowlpox. This approach involves the use
of vaccinia virus, for example, as a vector to express nucleotide
sequences that encode the peptides of the invention. Upon
introduction into an acutely or chronically infected host or into a
non-infected host, the recombinant vaccinia virus expresses the
immunogenic peptide, and thereby elicits a host CTL and/or HTL
response. Vaccinia vectors and methods useful in immunization
protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another
vector is BCG (Bacille Calmette Guerin). BCG vectors are described
in Stover et al., Nature 351:456-460 (1991). A wide variety of
other vectors useful for therapeutic administration or immunization
of the peptides of the invention, e.g. adeno and adeno-associated
virus vectors, retroviral vectors, Salmonella typhi vectors,
detoxified anthrax toxin vectors, and the like, will be apparent to
those skilled in the art from the description herein.
[0177] Furthermore, vaccines in accordance with the invention
encompass compositions of one or more of the claimed peptides. A
peptide can be present in a vaccine individually. Alternatively,
the peptide can exist as a homopolymer comprising multiple copies
of the same peptide, or as a heteropolymer of various peptides.
Polymers have the advantage of increased immunological reaction
and, where different peptide epitopes are used to make up the
polymer, the additional ability to induce antibodies and/or CTLs
that react with different antigenic determinants of the pathogenic
organism or tumor-related peptide targeted for an immune response.
The composition can be a naturally occurring region of an antigen
or can be prepared, e.g., recombinantly or by chemical
synthesis.
[0178] Carriers that can be used with vaccines of the invention are
well known in the art, and include, e.g., thyroglobulin, albumins
such as human serum albumin, tetanus toxoid, polyamino acids such
as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B
virus core protein, and the like. The vaccines can contain a
physiologically tolerable (i.e., acceptable) diluent such as water,
or saline, preferably phosphate buffered saline. The vaccines also
typically include an adjuvant. Adjuvants such as incomplete
Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum
are examples of materials well known in the art. Additionally, as
disclosed herein, CTL responses can be primed by conjugating
peptides of the invention to lipids, such as
tripalmitoyl-S-glycerylcysteinlyseryl-serine (P.sub.3CSS).
[0179] Upon immunization with a peptide composition in accordance
with the invention, via injection, aerosol, oral, transdermal,
transmucosal, intrapleural, intrathecal, or other suitable routes,
the immune system of the host responds to the vaccine by producing
large amounts of CTLs and/or HTLs specific for the desired antigen.
Consequently, the host becomes at least partially immune to later
infection, or at least partially resistant to developing an ongoing
chronic infection, or derives at least some therapeutic benefit
when the antigen was tumor-associated.
[0180] In some embodiments, it may be desirable to combine the
class I peptide components with components that induce or
facilitate neutralizing antibody and or helper T cell responses to
the target antigen of interest. A preferred embodiment of such a
composition comprises class I and class II epitopes in accordance
with the invention. An alternative embodiment of such a composition
comprises a class I and/or class II epitope in accordance with the
invention, along with a cross reactive HTL epitope such as
PADRE.TM. (Epimmune, San Diego, Calif.) molecule (described e.g.,
in U.S. Pat. No. 5,736,142).
[0181] A vaccine of the invention can also include
antigen-presenting cells (APC), such as dendritic cells (DC), as a
vehicle to present peptides of the invention. Vaccine compositions
can be created in vitro, following dendritic cell mobilization and
harvesting, whereby loading of dendritic cells occurs in vitro. For
example, dendritic cells are transfected, e.g., with a minigene in
accordance with the invention, or are pulsed with peptides. The
dendritic cell can then be administered to a patient to elicit
immune responses in vivo.
[0182] Vaccine compositions, either DNA- or peptide-based, can also
be administered in vivo in combination with dendritic cell
mobilization whereby loading of dendritic cells occurs in vivo.
[0183] Antigenic peptides are used to elicit a CTL and/or HTL
response ex vivo, as well. The resulting CTL or HTL cells, can be
used to treat chronic infections, or tumors in patients that do not
respond to other conventional forms of therapy, or will not respond
to a therapeutic vaccine peptide or nucleic acid in accordance with
the invention. Ex vivo CTL or HTL responses to a particular antigen
(infectious or tumor-associated antigen) are induced by incubating
in tissue culture the patient's, or genetically compatible, CTL or
HTL precursor cells together with a source of antigen-presenting
cells (APC), such as dendritic cells, and the appropriate
immunogenic peptide. After an appropriate incubation time
(typically about 7-28 days), in which the precursor cells are
activated and expanded into effector cells, the cells are infused
back into the patient, where they will destroy (CTL) or facilitate
destruction (HTL) of their specific target cell (an infected cell
or a tumor cell). Transfected dendritic cells may also be used as
antigen presenting cells.
[0184] The vaccine compositions of the invention may also be used
in combination with other procedures to remove warts or treat HPV
infections. Such procedures include cryosurgery, application of
caustic agents, electrodessication, surgical excision and laser
ablation (Fauci et al. HARRISON'S PRINCIPLES OF INTERNAL MEDICINE,
14th ED., McGraw-Hill Co., Inc, 1998), as well as treatment with
antiviral drugs such as interferon-.alpha. (see, e.g., Stellato,
G., et al., Clin. Diagn. Virol. 7(3):167-72 (1997)) or
interferon-inducing drugs such as imiquimod. Topical
antimetabolites such a 5-fluorouracil may also be applied.
[0185] In patients with HPV-associated cancer, the vaccine
compositions of the invention can also be used in conjunction with
other treatments used for cancer, e.g., surgery, chemotherapy, drug
therapies, radiation therapies, etc. including use in combination
with immune adjuvants such as IL-2, IL-12, GM-CSF, and the
like.
[0186] Preferably, the following principles are utilized when
selecting an array of epitopes for inclusion in a polyepitopic
composition for use in a vaccine, or for selecting discrete
epitopes to be included in a vaccine and/or to be encoded by
nucleic acids such as a minigene. It is preferred that each of the
following principles are balanced in order to make the selection.
The multiple epitopes to be incorporated in a given vaccine
composition may be, but need not be, contiguous in sequence in the
native antigen from which the epitopes are derived.
[0187] 1.) Epitopes are selected which, upon administration, mimic
immune responses that have been observed to be correlated with
clearance of HPV infection or tumor clearance. For HLA Class I this
includes 3-4 epitopes that come from at least one TAA. For HLA
Class II a similar rationale is employed; again 3-4 epitopes are
selected from at least one TAA (see, e.g., Rosenberg et al.,
Science 278:1447-1450). Epitopes from one TAA may be used in
combination with epitopes from one or more additional TAAs to
produce a vaccine that targets tumors with varying expression
patterns of frequently-expressed TAAs as described, e.g., in
Example 15.
[0188] 2.) Epitopes are selected that have the requisite binding
affinity established to be correlated with immunogenicity: for HLA
Class I an IC.sub.50 of 500 nM or less, often 200 nM or less; and
for Class II an IC.sub.50 of 1000 nM or less.
[0189] 3.) Sufficient supermotif bearing-peptides, or a sufficient
array of allele-specific motif-bearing peptides, are selected to
give broad population coverage. For example, it is preferable to
have at least 80% population coverage. A Monte Carlo analysis, a
statistical evaluation known in the art, can be employed to assess
the breadth, or redundancy of, population coverage.
[0190] 4.) When selecting epitopes from cancer-related antigens it
is often useful to select analogs because the patient may have
developed tolerance to the native epitope. When selecting epitopes
for infectious disease-related antigens it is preferable to select
either native or analoged epitopes.
[0191] 5.) Of particular relevance are epitopes referred to as
"nested epitopes." Nested epitopes occur where at least two
epitopes overlap in a given peptide sequence. A nested peptide
sequence can comprise both HLA class I and HLA class II epitopes.
When providing nested epitopes, a general objective is to provide
the greatest number of epitopes per sequence. Thus, an aspect is to
avoid providing a peptide that is any longer than the amino
terminus of the amino terminal epitope and the carboxyl terminus of
the carboxyl terminal epitope in the peptide. When providing a
multi-epitopic sequence, such as a sequence comprising nested
epitopes, it is generally important to screen the sequence in order
to insure that it does not have pathological or other deleterious
biological properties.
[0192] 6.) If a polyepitopic protein is created, or when creating a
minigene, an objective is to generate the smallest peptide that
encompasses the epitopes of interest. This principle is similar, if
not the same as that employed when selecting a peptide comprising
nested epitopes. However, with an artificial polyepitopic peptide,
the size minimization objective is balanced against the need to
integrate any spacer sequences between epitopes in the polyepitopic
protein. Spacer amino acid residues can, for example, be introduced
to avoid junctional epitopes (an epitope recognized by the immune
system, not present in the target antigen, and only created by the
man-made juxtaposition of epitopes), or to facilitate cleavage
between epitopes and thereby enhance epitope presentation.
Junctional epitopes are generally to be avoided because the
recipient may generate an immune response to that non-native
epitope. Of particular concern is a junctional epitope that is a
"dominant epitope." A dominant epitope may lead to such a zealous
response that immune responses to other epitopes are diminished or
suppressed.
[0193] 7.) In cases where the sequences of multiple variants of the
same target protein are available, potential peptide epitopes can
also be selected on the basis of their conservancy. For example, a
criterion for conservancy may define that the entire sequence of an
HLA class I binding peptide or the entire 9-mer core of a class II
binding peptide be conserved in a designated percentage of the
sequences evaluated for a specific protein antigen.
[0194] 8.) When selecting an array of epitopes of an infectious
agent, it is preferred that at least some of the epitopes are
derived from early and late proteins. The early proteins of HPV are
expressed when the virus is replicating, either following acute or
dormant infection. Therefore, it is particularly preferred to use
epitopes from early stage proteins to alleviate disease
manifestations at the earliest stage possible.
Minigene Vaccines
[0195] A number of different approaches are available which allow
simultaneous delivery of multiple epitopes. Nucleic acids encoding
the peptides of the invention are a particularly useful embodiment
of the invention. Epitopes for inclusion in a minigene are
preferably selected according to the guidelines set forth in the
previous section. A preferred means of administering nucleic acids
encoding the peptides of the invention uses minigene constructs
encoding a peptide comprising one or multiple epitopes of the
invention.
[0196] The use of multi-epitope minigenes is described below and
in, e.g., co-pending application U.S. Ser. No. 09/311,784; Ishioka
et al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L.,
J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol.
157:822, 1996; Whitton, J. L. et al., J. Virol. 67:348, 1993;
Hanke, R. et al., Vaccine 16:426, 1998. For example, a
multi-epitope DNA plasmid encoding supermotif- and/or motif-bearing
epitopes derived from multiple regions of one or more HPV antigens,
the PADRE.quadrature. universal helper T cell epitope (or multiple
HTL epitopes from HPV antigens), and an endoplasmic
reticulum-translocating signal sequence can be engineered. A
vaccine may also comprise epitopes that are derived from other
TAAs.
[0197] The immunogenicity of a multi-epitopic minigene can be
tested in transgenic mice to evaluate the magnitude of CTL
induction responses against the epitopes tested. Further, the
immunogenicity of DNA-encoded epitopes in vivo can be correlated
with the in vitro responses of specific CTL lines against target
cells transfected with the DNA plasmid. Thus, these experiments can
show that the minigene serves to both: 1.) generate a CTL response
and 2.) that the induced CTLs recognized cells expressing the
encoded epitopes.
[0198] For example, to create a DNA sequence encoding the selected
epitopes (minigene) for expression in human cells, the amino acid
sequences of the epitopes may be reverse translated. A human codon
usage table can be used to guide the codon choice for each amino
acid. These epitope-encoding DNA sequences may be directly
adjoined, so that when translated, a continuous polypeptide
sequence is created. To optimize expression and/or immunogenicity,
additional elements can be incorporated into the minigene design.
Examples of amino acid sequences that can be reverse translated and
included in the minigene sequence include: HLA class I epitopes,
HLA class II epitopes, a ubiquitination signal sequence, and/or an
endoplasmic reticulum targeting signal. In addition, HLA
presentation of CTL and HTL epitopes may be improved by including
synthetic (e.g. poly-alanine) or naturally-occurring flanking
sequences adjacent to the CTL or HTL epitopes; these larger
peptides comprising the epitope(s) are within the scope of the
invention.
[0199] The minigene sequence may be converted to DNA by assembling
oligonucleotides that encode the plus and minus strands of the
minigene. Overlapping oligonucleotides (30-100 bases long) may be
synthesized, phosphorylated, purified and annealed under
appropriate conditions using well known techniques. The ends of the
oligonucleotides can be joined, for example, using T4 DNA ligase.
This synthetic minigene, encoding the epitope polypeptide, can then
be cloned into a desired expression vector.
[0200] Standard regulatory sequences well known to those of skill
in the art are preferably included in the vector to ensure
expression in the target cells. Several vector elements are
desirable: a promoter with a down-stream cloning site for minigene
insertion; a polyadenylation signal for efficient transcription
termination; an E. coli origin of replication; and an E. coli
selectable marker (e.g. ampicillin or kanamycin resistance).
Numerous promoters can be used for this purpose, e.g., the human
cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos.
5,580,859 and 5,589,466 for other suitable promoter sequences.
[0201] Additional vector modifications may be desired to optimize
minigene expression and immunogenicity. In some cases, introns are
required for efficient gene expression, and one or more synthetic
or naturally-occurring introns could be incorporated into the
transcribed region of the minigene. The inclusion of mRNA
stabilization sequences and sequences for replication in mammalian
cells may also be considered for increasing minigene
expression.
[0202] Once an expression vector is selected, the minigene is
cloned into the polylinker region downstream of the promoter. This
plasmid is transformed into an appropriate E. coli strain, and DNA
is prepared using standard techniques. The orientation and DNA
sequence of the minigene, as well as all other elements included in
the vector, are confirmed using restriction mapping and DNA
sequence analysis. Bacterial cells harboring the correct plasmid
can be stored as a master cell bank and a working cell bank.
[0203] In addition, immunostimulatory sequences (ISSs or CpGs)
appear to play a role in the immunogenicity of DNA vaccines. These
sequences may be included in the vector, outside the minigene
coding sequence, if desired to enhance immunogenicity.
[0204] In some embodiments, a bi-cistronic expression vector which
allows production of both the minigene-encoded epitopes and a
second protein (included to enhance or decrease immunogenicity) can
be used. Examples of proteins or polypeptides that could
beneficially enhance the immune response if co-expressed include
cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules
(e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR
binding proteins (PADRE.TM., Epimmune, San Diego, Calif.). Helper
(HTL) epitopes can be joined to intracellular targeting signals and
expressed separately from expressed CTL epitopes; this allows
direction of the HTL epitopes to a cell compartment different than
that of the CTL epitopes. If required, this could facilitate more
efficient entry of HTL epitopes into the HLA class II pathway,
thereby improving HTL induction. In contrast to HTL or CTL
induction, specifically decreasing the immune response by
co-expression of immunosuppressive molecules (e.g. TGF-.beta.) may
be beneficial in certain diseases.
[0205] Therapeutic quantities of plasmid DNA can be produced for
example, by fermentation in E. coli, followed by purification.
Aliquots from the working cell bank are used to inoculate growth
medium, and grown to saturation in shaker flasks or a bioreactor
according to well known techniques. Plasmid DNA can be purified
using standard bioseparation technologies such as solid phase
anion-exchange resins supplied by QIAGEN, Inc. (Valencia, Calif.).
If required, supercoiled DNA can be isolated from the open circular
and linear forms using gel electrophoresis or other methods.
[0206] Purified plasmid DNA can be prepared for injection using a
variety of formulations. The simplest of these is reconstitution of
lyophilized DNA in sterile phosphate-buffer saline (PBS). This
approach, known as "naked DNA," is currently being used for
intramuscular (IM) administration in clinical trials. To maximize
the immunotherapeutic effects of minigene DNA vaccines, an
alternative method for formulating purified plasmid DNA may be
desirable. A variety of methods have been described, and new
techniques may become available. Cationic lipids, glycolipids, and
fusogenic liposomes can also be used in the formulation (see, e.g.,
as described by WO 93/24640; Mannino & Gould-Fogerite,
BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO
91/06309; and Felgner, et al, Proc. Nat'l Acad. Sci. USA 84:7413
(1987). In addition, peptides and compounds referred to
collectively as protective, interactive, non-condensing compounds
(PINC) could also be complexed to purified plasmid DNA to influence
variables such as stability, intramuscular dispersion, or
trafficking to specific organs or cell types.
[0207] Target cell sensitization can be used as a functional assay
for expression and HLA class I presentation of minigene-encoded CTL
epitopes. For example, the plasmid DNA is introduced into a
mammalian cell line that is suitable as a target for standard CTL
chromium release assays. The transfection method used will be
dependent on the final formulation. Electroporation can be used for
"naked" DNA, whereas cationic lipids allow direct in vitro
transfection. A plasmid expressing green fluorescent protein (GFP)
can be co-transfected to allow enrichment of transfected cells
using fluorescence activated cell sorting (FACS). These cells are
then chromium-51 (.sup.51Cr) labeled and used as target cells for
epitope-specific CTL lines; cytolysis, detected by .sup.51Cr
release, indicates both production of, and HLA presentation of,
minigene-encoded CTL epitopes. Expression of HTL epitopes may be
evaluated in an analogous manner using assays to assess HTL
activity.
[0208] In vivo immunogenicity is a second approach for functional
testing of minigene DNA formulations. Transgenic mice expressing
appropriate human HLA proteins are immunized with the DNA product.
The dose and route of administration are formulation dependent
(e.g., IM for DNA in PBS, intraperitoneal (i.p.) for
lipid-complexed DNA). Twenty-one days after immunization,
splenocytes are harvested and restimulated for one week in the
presence of peptides encoding each epitope being tested.
Thereafter, for CTL effector cells, assays are conducted for
cytolysis of peptide-loaded, .sup.51Cr-labeled target cells using
standard techniques. Lysis of target cells that were sensitized by
HLA loaded with peptide epitopes, corresponding to minigene-encoded
epitopes, demonstrates DNA vaccine function for in vivo induction
of CTLs. Immunogenicity of HTL epitopes is evaluated in transgenic
mice in an analogous manner.
[0209] Alternatively, the nucleic acids can be administered using
ballistic delivery as described, for instance, in U.S. Pat. No.
5,204,253. Using this technique, particles comprised solely of DNA
are administered. In a further alternative embodiment, DNA can be
adhered to particles, such as gold particles.
[0210] Minigenes can also be delivered using other bacterial or
viral delivery systems well known in the art, e.g., an expression
construct encoding epitopes of the invention can be incorporated
into a viral vector such as vaccinia.
Combinations of CTL Peptides with Helper Peptides
[0211] Vaccine compositions comprising CTL peptides of the
invention can be modified to provide desired attributes, such as
improved serum half life, broadened population coverage or enhanced
immunogenicity.
[0212] For instance, the ability of a peptide to induce CTL
activity can be enhanced by linking the peptide to a sequence which
contains at least one epitope that is capable of inducing a T
helper cell response. The use of T helper epitopes in conjunction
with CTL epitopes to enhance immunogenicity is illustrated, for
example, in the co-pending applications U.S. Ser. No. 08/820,360,
U.S. Ser. No. 08/197,484, and U.S. Ser. No. 08/464,234.
[0213] Although a CTL peptide can be directly linked to a T helper
peptide, often CTL epitope/HTL epitope conjugates are linked by a
spacer molecule. The spacer is typically comprised of relatively
small, neutral molecules, such as amino acids or amino acid
mimetics, which are substantially uncharged under physiological
conditions. The spacers are typically selected from, e.g., Ala,
Gly, or other neutral spacers of nonpolar amino acids or neutral
polar amino acids. It will be understood that the optionally
present spacer need not be comprised of the same residues and thus
may be a hetero- or homo-oligomer. When present, the spacer will
usually be at least one or two residues, more usually three to six
residues and sometimes 10 or more residues. The CTL peptide epitope
can be linked to the T helper peptide epitope either directly or
via a spacer either at the amino or carboxy terminus of the CTL
peptide. The amino terminus of either the immunogenic peptide or
the T helper peptide may be acylated.
[0214] In certain embodiments, the T helper peptide is one that is
recognized by T helper cells present in the majority of the
population. This can be accomplished by selecting peptides that
bind to many, most, or all of the HLA class II molecules. These are
known as "loosely HLA-restricted" or "promiscuous" T helper
sequences. Examples of amino acid sequences that are promiscuous
include sequences from antigens such as tetanus toxoid at positions
830-843 (QYIKANSKFIGITE; SEQ ID NO: 51484), Plasmodium falciparum
circumsporozoite (CS) protein at positions 378-398
(DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 51485), and Streptococcus 18 kD
protein at positions 116 (GAVDSILGGVATYGAA; SEQ ID NO: 51486).
Other examples include peptides bearing a DR 1-4-7 supermotif, or
either of the DR3 motifs.
[0215] Alternatively, it is possible to prepare synthetic peptides
capable of stimulating T helper lymphocytes, in a loosely
HLA-restricted fashion, using amino acid sequences not found in
nature (see, e.g., PCT publication WO 95/07707). These synthetic
compounds called Pan-DR-binding epitopes (e.g., PADRE.TM.,
Epimmune, Inc., San Diego, Calif.) are designed to most preferrably
bind most HLA-DR (human HLA class II) molecules. For instance, a
pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAAa,
where "X" is either cyclohexylalanine, phenylalanine, or tyrosine,
and a is either D-alanine or L-alanine, has been found to bind to
most HLA-DR alleles, and to stimulate the response of T helper
lymphocytes from most individuals, regardless of their HLA type. An
alternative of a pan-DR binding epitope comprises all "L" natural
amino acids and can be provided in the form of nucleic acids that
encode the epitope.
[0216] HTL peptide epitopes can also be modified to alter their
biological properties. For example, they can be modified to include
D-amino acids to increase their resistance to proteases and thus
extend their serum half life, or they can be conjugated to other
molecules such as lipids, proteins, carbohydrates, and the like to
increase their biological activity. For example, a T helper peptide
can be conjugated to one or more palmitic acid chains at either the
amino or carboxyl termini.
Combinations of CTL Peptides with T Cell Priming Agents
[0217] In some embodiments it may be desirable to include in the
pharmaceutical compositions of the invention at least one component
which primes cytotoxic T lymphocytes. Lipids have been identified
as agents capable of priming CTL in vivo against viral antigens.
For example, palmitic acid residues can be attached to the
.epsilon.- and .alpha.-amino groups of a lysine residue and then
linked, e.g., via one or more linking residues such as Gly,
Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The
lipidated peptide can then be administered either directly in a
micelle or particle, incorporated into a liposome, or emulsified in
an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred
embodiment, a particularly effective immunogenic composition
comprises palmitic acid attached to .epsilon.- and .alpha.-amino
groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the
amino terminus of the immunogenic peptide.
[0218] As another example of lipid priming of CTL responses, E.
coli lipoproteins, such as
tripalmitoyl-S-glycerylcysteinlyseryl-serine (P.sub.3CSS) can be
used to prime virus specific CTL when covalently attached to an
appropriate peptide (see, e.g., Deres, et al., Nature 342:561,
1989). Peptides of the invention can be coupled to P.sub.3CSS, for
example, and the lipopeptide administered to an individual to
specifically prime a CTL response to the target antigen. Moreover,
because the induction of neutralizing antibodies can also be primed
with P.sub.3CSS-conjugated epitopes, two such compositions can be
combined to more effectively elicit both humoral and cell-mediated
responses.
[0219] CTL and/or HTL peptides can also be modified by the addition
of amino acids to the termini of a peptide to provide for ease of
linking peptides one to another, for coupling to a carrier support
or larger peptide, for modifying the physical or chemical
properties of the peptide or oligopeptide, or the like. Amino acids
such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or
the like, can be introduced at the C- or N-terminus of the peptide
or oligopeptide, particularly class I peptides. However, it is to
be noted that modification at the carboxyl terminus of a CTL
epitope may, in some cases, alter binding characteristics of the
peptide. In addition, the peptide or oligopeptide sequences can
differ from the natural sequence by being modified by
terminal-NH.sub.2 acylation, e.g., by alkanoyl (C1-C20) or
thioglycolyl acetylation, terminal-carboxyl amidation, e.g.,
ammonia, methylamine, etc. In some instances these modifications
may provide sites for linking to a support or other molecule.
Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL
Peptides
[0220] An embodiment of a vaccine composition in accordance with
the invention comprises ex vivo administration of a cocktail of
epitope-bearing peptides to PBMC, or isolated DC therefrom, from
the patient's blood. A pharmaceutical to facilitate harvesting of
DC can be used, such as Progenipoietin.quadrature. (Monsanto, St.
Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC with peptides and
prior to reinfusion into patients, the DC are washed to remove
unbound peptides. In this embodiment, a vaccine comprises
peptide-pulsed DCs which present the pulsed peptide epitopes
complexed with HLA molecules on their surfaces.
[0221] The DC can be pulsed ex vivo with a cocktail of peptides,
some of which stimulate CTL responses to one or more HPV antigens
of interest. Optionally, a helper T cell (HTL) peptide such as a
PADRE family molecule, can be included to facilitate the CTL
response. Thus, a vaccine in accordance with the invention,
preferably comprising epitopes from multiple HPV antigens, is used
to treat HPV infection or cancer resulting from HPV infection.
Administration of Vaccines for Therapeutic or Prophylactic
Purposes
[0222] The peptides of the present invention and pharmaceutical and
vaccine compositions of the invention are typically used to treat
and/or prevent cancer associated with HPV infection. Vaccine
compositions containing the peptides of the invention are
administered to a patient infected with HPV or to an individual
susceptible to, or otherwise at risk for, HPV infection to elicit
an immune response against HPV antigens and thus enhance the
patient's own immune response capabilities.
[0223] As noted above, peptides comprising CTL and/or HTL epitopes
of the invention induce immune responses when presented by HLA
molecules and contacted with a CTL or HTL specific for an epitope
comprised by the peptide. The peptides (or DNA encoding them) can
be administered individually or as fusions of one or more peptide
sequences. The manner in which the peptide is contacted with the
CTL or HTL is not critical to the invention. For instance, the
peptide can be contacted with the CTL or HTL either in vivo or in
vitro. If the contacting occurs in vivo, the peptide itself can be
administered to the patient, or other vehicles, e.g., DNA vectors
encoding one or more peptides, viral vectors encoding the
peptide(s), liposomes and the like, can be used, as described
herein.
[0224] When the peptide is contacted in vitro, the vaccinating
agent can comprise a population of cells, e.g., peptide-pulsed
dendritic cells, or HPV-specific CTLs, which have been induced by
pulsing antigen-presenting cells in vitro with the peptide or by
transfecting antigen-presenting cells with a minigene of the
invention. Such a cell population is subsequently administered to a
patient in a therapeutically effective dose.
[0225] In therapeutic applications, peptide and/or nucleic acid
compositions are administered to a patient in an amount sufficient
to elicit an effective CTL and/or HTL response to the virus antigen
and to cure or at least partially arrest or slow symptoms and/or
complications. An amount adequate to accomplish this is defined as
"therapeutically effective dose." Amounts effective for this use
will depend on, e.g., the particular composition administered, the
manner of administration, the stage and severity of the disease
being treated, the weight and general state of health of the
patient, and the judgment of the prescribing physician.
[0226] For pharmaceutical compositions, the immunogenic peptides of
the invention, or DNA encoding them, are generally administered to
an individual already infected with HPV. The peptides or DNA
encoding them can be administered individually or as fusions of one
or more peptide sequences. HPV-infected patients, with or without
neoplasia, can be treated with the immunogenic peptides separately
or in conjunction with other treatments, such as surgery, as
appropriate.
[0227] For therapeutic use, administration should generally begin
at the first diagnosis of HPV infection or HPV-associated cancer.
This is followed by boosting doses until at least symptoms are
substantially abated and for a period thereafter. The embodiment of
the vaccine composition (i.e., including, but not limited to
embodiments such as peptide cocktails, polyepitopic polypeptides,
minigenes, or TAA-specific CTLs or pulsed dendritic cells)
delivered to the patient may vary according to the stage of the
disease or the patient's health status. For example, in a patient
with a tumor that expresses HPV antigens, a vaccine comprising
HPV-specific CTL may be more efficacious in killing tumor cells in
patient with advanced disease than alternative embodiments.
[0228] Where susceptible individuals are identified prior to or
during infection, the composition can be targeted to them, thus
minimizing the need for administration to a larger population.
Susceptible populations include those individuals who are sexually
active.
[0229] The peptide or other compositions used for the treatment or
prophylaxis of HPV infection can be used, e.g., in persons who have
not manifested symptoms, e.g., genital warts or neoplastic growth.
In this context, it is generally important to provide an amount of
the peptide epitope delivered by a mode of administration
sufficient to effectively stimulate a cytotoxic T cell response;
compositions which stimulate helper T cell responses can also be
given in accordance with this embodiment of the invention.
[0230] The dosage for an initial therapeutic immunization generally
occurs in a unit dosage range where the lower value is about 1, 5,
50, 500, or 1,000 .mu.g and the higher value is about 10,000;
20,000; 30,000; or 50,000 .mu.g. Dosage values for a human
typically range from about 500 .mu.g to about 50,000 .mu.g per 70
kilogram patient. Boosting dosages of between about 1.0 .mu.g to
about 50,000 .mu.g of peptide pursuant to a boosting regimen over
weeks to months may be administered depending upon the patient's
response and condition as determined by measuring the specific
activity of CTL and HTL obtained from the patient's blood.
Administration should continue until at least clinical symptoms or
laboratory tests indicate that the viral infection, or neoplasia,
has been eliminated or reduced and for a period thereafter. The
dosages, routes of administration, and dose schedules are adjusted
in accordance with methodologies known in the art.
[0231] In certain embodiments, the peptides and compositions of the
present invention are employed in serious disease states, that is,
life-threatening or potentially life threatening situations. In
such cases, as a result of the minimal amounts of extraneous
substances and the relative nontoxic nature of the peptides in
preferred compositions of the invention, it is possible and may be
felt desirable by the treating physician to administer substantial
excesses of these peptide compositions relative to these stated
dosage amounts.
[0232] The vaccine compositions of the invention can also be used
purely as prophylactic agents. Generally the dosage for an initial
prophylactic immunization generally occurs in a unit dosage range
where the lower value is about 1, 5, 50, 500, or 1000 .mu.g and the
higher value is about 10,000; 20,000; 30,000; or 50,000 .mu.g.
Dosage values for a human typically range from about 500 .mu.g to
about 50,000 .mu.g per 70 kilogram patient. This is followed by
boosting dosages of between about 1.0 .mu.g to about 50,000 .mu.g
of peptide administered at defined intervals from about four weeks
to six months after the initial administration of vaccine. The
immunogenicity of the vaccine can be assessed by measuring the
specific activity of CTL and HTL obtained from a sample of the
patient's blood.
[0233] The pharmaceutical compositions for therapeutic treatment
are intended for parenteral, topical, oral, intrathecal, or local
(e.g. as a cream or topical ointment) administration. Preferably,
the pharmaceutical compositions are administered parentally, e.g.,
intravenously, subcutaneously, intradermally, or intramuscularly.
Thus, the invention provides compositions for parenteral
administration which comprise a solution of the immunogenic
peptides dissolved or suspended in an acceptable carrier,
preferably an aqueous carrier. A variety of aqueous carriers may be
used, e.g., water, buffered water, 0.8% saline, 0.3% glycine,
hyaluronic acid and the like. These compositions may be sterilized
by conventional, well known sterilization techniques, or may be
sterile filtered. The resulting aqueous solutions may be packaged
for use as is, or lyophilized, the lyophilized preparation being
combined with a sterile solution prior to administration. The
compositions may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions,
such as pH-adjusting and buffering agents, tonicity adjusting
agents, wetting agents, preservatives, and the like, for example,
sodium acetate, sodium lactate, sodium chloride, potassium
chloride, calcium chloride, sorbitan monolaurate, triethanolamine
oleate, etc.
[0234] The concentration of peptides of the invention in the
pharmaceutical formulations can vary widely, i.e., from less than
about 0.1%, usually at or at least about 2% to as much as 20% to
50% or more by weight, and will be selected primarily by fluid
volumes, viscosities, etc., in accordance with the particular mode
of administration selected.
[0235] A human unit dose form of the peptide composition is
typically included in a pharmaceutical composition that comprises a
human unit dose of an acceptable carrier, preferably an aqueous
carrier, and is administered in a volume of fluid that is known by
those of skill in the art to be used for administration of such
compositions to humans (see, e.g., Remington's Pharmaceutical
Sciences, 17th Edition, A. Gennaro, Editor, Mack Publishing Co.,
Easton, Pa., 1985).
[0236] The peptides of the invention, and/or nucleic acids encoding
the peptides, can also be administered via liposomes, which may
also serve to target the peptides to a particular tissue, such as
lymphoid tissue, or to target selectively to infected cells, as
well as to increase the half-life of the peptide composition.
Liposomes include emulsions, foams, micelles, insoluble monolayers,
liquid crystals, phospholipid dispersions, lamellar layers and the
like. In these preparations, the peptide to be delivered is
incorporated as part of a liposome, alone or in conjunction with a
molecule which binds to a receptor prevalent among lymphoid cells,
such as monoclonal antibodies which bind to the CD45 antigen, or
with other therapeutic or immunogenic compositions. Thus, liposomes
either filled or decorated with a desired peptide of the invention
can be directed to the site of lymphoid cells, where the liposomes
then deliver the peptide compositions. Liposomes for use in
accordance with the invention are formed from standard
vesicle-forming lipids, which generally include neutral and
negatively charged phospholipids and a sterol, such as cholesterol.
The selection of lipids is generally guided by consideration of,
e.g., liposome size, acid lability and stability of the liposomes
in the blood stream. A variety of methods are available for
preparing liposomes, as described in, e.g., Szoka, et al., Ann.
Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat. Nos. 4,235,871,
4,501,728, 4,837,028, and 5,019,369.
[0237] For targeting cells of the immune system, a ligand to be
incorporated into the liposome can include, e.g., antibodies or
fragments thereof specific for cell surface determinants of the
desired immune system cells. A liposome suspension containing a
peptide may be administered intravenously, locally, topically, etc.
in a dose which varies according to, inter alia, the manner of
administration, the peptide being delivered, and the stage of the
disease being treated.
[0238] For solid compositions, conventional nontoxic solid carriers
may be used which include, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the
like. For oral administration, a pharmaceutically acceptable
nontoxic composition is formed by incorporating any of the normally
employed excipients, such as those carriers previously listed, and
generally 10-95% of active ingredient, that is, one or more
peptides of the invention, and more preferably at a concentration
of 25%-75%.
[0239] For aerosol administration, the immunogenic peptides are
preferably supplied in finely divided form along with a surfactant
and propellant. Typical percentages of peptides are 0.01%-20% by
weight, preferably 1%-10%. The surfactant must, of course, be
nontoxic, and preferably soluble in the propellant. Representative
of such agents are the esters or partial esters of fatty acids
containing from 6 to 22 carbon atoms, such as caproic, octanoic,
lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic
acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
Mixed esters, such as mixed or natural glycerides may be employed.
The surfactant may constitute 0.1%-20% by weight of the
composition, preferably 0.25-5%. The balance of the composition is
ordinarily propellant. A carrier can also be included, as desired,
as with, e.g., lecithin for intranasal delivery.
HLA Expression: Implications for T Cell-Based Immunotherapy
Disease Progression in Cancer and Infectious Disease
[0240] It is well recognized that a dynamic interaction between
exists between host and disease, both in the cancer and infectious
disease settings. In the infectious disease setting, it is well
established that pathogens evolve during disease. The strains that
predominate early in HIV infection are different from the ones that
are associated with AIDS and later disease stages (NS versus S
strains). It has long been hypothesized that pathogen forms that
are effective in establishing infection may differ from the ones
most effective in terms of replication and chronicity.
[0241] Similarly, it is widely recognized that the pathological
process by which an individual succumbs to a neoplastic disease is
complex. During the course of disease, many changes occur in cancer
cells. The tumor accumulates alterations which are in part related
to dysfunctional regulation of growth and differentiation, but also
related to maximizing its growth potential, escape from drug
treatment and/or the body's immunosurveillance. Neoplastic disease
results in the accumulation of several different biochemical
alterations of cancer cells, as a function of disease progression.
It also results in significant levels of intra- and inter-cancer
heterogeneity, particularly in the late, metastatic stage.
[0242] Familiar examples of cellular alterations affecting
treatment outcomes include the outgrowth of radiation or
chemotherapy resistant tumors during the course of therapy.
[0243] These examples parallel the emergence of drug resistant
viral strains as a result of aggressive chemotherapy, e.g., of
chronic HBV and HIV infection, and the current resurgence of drug
resistant organisms that cause Tuberculosis and Malaria. It appears
that significant heterogeneity of responses is also associated with
other approaches to cancer therapy, including anti-angiogenesis
drugs, passive antibody immunotherapy, and active T cell-based
immunotherapy. Thus, in view of such phenomena, epitopes from
multiple disease-related antigens can be used in vaccines and
therapeutics thereby counteracting the ability of diseased cells to
mutate and escape treatment.
The Interplay Between Disease and the Immune System
[0244] One of the main factors contributing to the dynamic
interplay between host and disease is the immune response mounted
against the pathogen, infected cell, or malignant cell. In many
conditions such immune responses control the disease. Several
animal model systems and prospective studies of natural infection
in humans suggest that immune responses against a pathogen can
control the pathogen, prevent progression to severe disease and/or
eliminate the pathogen. A common theme is the requirement for a
multispecific T cell response, and that narrowly focused responses
appear to be less effective. These observations guide skilled
artisan as to embodiments of methods and compositions of the
present invention that provide for a broad immune response.
[0245] In the cancer setting there are several findings that
indicate that immune responses can impact neoplastic growth:
[0246] First, the demonstration in many different animal models,
that anti-tumor T cells, restricted by MHC class I, can prevent or
treat tumors.
[0247] Second, encouraging results have come from immunotherapy
trials.
[0248] Third, observations made in the course of natural disease
correlated the type and composition of T cell infiltrate within
tumors with positive clinical outcomes (Coulie P G, et al.
Antitumor immunity at work in a melanoma patient In Advances in
Cancer Research, 213-242, 1999).
[0249] Finally, tumors commonly have the ability to mutate, thereby
changing their immunological recognition. For example, the presence
of monospecific CTL was also correlated with control of tumor
growth, until antigen loss emerged (Riker A, et al., Immune
selection after antigen-specific immunotherapy of melanoma Surgery,
August: 126(2):112-20, 1999; Marchand M, et al., Tumor regressions
observed in patients with metastatic melanoma treated with an
antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1
Int. J. Cancer 80(2):219-30, Jan. 18, 1999). Similarly, loss of
beta 2 microglobulin was detected in 5/13 lines established from
melanoma patients after receiving immunotherapy at the NCI (Restifo
N P, et al., Loss of functional Beta2-microglobulin in metastatic
melanomas from five patients receiving immunotherapy Journal of the
National Cancer Institute, Vol. 88 (2), 100-108, January 1996). It
has long been recognized that HLA class I is frequently altered in
various tumor types. This has led to a hypothesis that this
phenomenon might reflect immune pressure exerted on the tumor by
means of class I restricted CTL. The extent and degree of
alteration in HLA class I expression appears to be reflective of
past immune pressures, and may also have prognostic value (van
Duinen S G, et al., Level of HLA antigens in locoregional
metastases and clinical course of the disease in patients with
melanoma Cancer Research 48, 1019-1025, February 1988; Moller P, et
al., Influence of major histocompatibility complex class I and II
antigens on survival in colorectal carcinoma Cancer Research 51,
729-736, January 1991). Taken together, these observations provide
a rationale for immunotherapy of cancer and infectious disease, and
suggest that effective strategies need to account for the complex
series of pathological changes associated with disease.
The three main types of alterations in HLA expression in tumors and
their functional significance
[0250] The level and pattern of expression of HLA class I antigens
in tumors has been studied in many different tumor types and
alterations have been reported in all types of tumors studied. The
molecular mechanisms underlining HLA class I alterations have been
demonstrated to be quite heterogeneous. They include alterations in
the TAP/processing pathways, mutations of .beta.2-microglobulin and
specific HLA heavy chains, alterations in the regulatory elements
controlling over class I expression and loss of entire chromosome
sections. There are several reviews on this topic, see, e.g.,:
Gamido F, et al., Natural history of HLA expression during tumour
development Immunol Today 14(10):491-499, 1993; Kaklamanis L, et
al., Loss of HLA class-I alleles, heavy chains and
.beta.2-microglobulin in colorectal cancer Int. J. Cancer,
51(3):379-85, May 28, 1992. There are three main types of HLA Class
I alteration (complete loss, allele-specific loss and decreased
expression). The functional significance of each alteration is
discussed separately:
Complete Loss of HLA Expression
[0251] Complete loss of HLA expression can result from a variety of
different molecular mechanisms, reviewed in (Algarra I, et al., The
HLA crossroad in tumor immunology Human Immunology 61, 65-73, 2000;
Browning M, et al., Mechanisms of loss of HLA class I expression on
colorectal tumor cells Tissue Antigens 47:364-371, 1996; Ferrone S,
et al., Loss of HLA class I antigens by melanoma cells: molecular
mechanisms, functional significance and clinical relevance
Immunology Today, 16(10): 487-494, 1995; Gamido F, et al., Natural
history of HLA expression during tumour development Immunology
Today 14(10):491-499, 1993; Tait, B D, HLA Class I expression on
human cancer cells: Implications for effective immunotherapy Hum
Immunol 61, 158-165, 2000). In functional terms, this type of
alteration has several important implications.
[0252] While the complete absence of class I expression will
eliminate CTL recognition of those tumor cells, the loss of HLA
class I will also render the tumor cells extraordinary sensitive to
lysis from NK cells (Ohnmacht, Ga., et al., Heterogeneity in
expression of human leukocyte antigens and melanoma-associated
antigens in advanced melanoma J Cellular Phys 182:332-338, 2000;
Liunggren H G, et al., Host resistance directed selectively against
H-2 deficient lymphoma variants: Analysis of the mechanism J. Exp.
Med., December 1; 162(6):1745-59, 1985; Maio M, et al., Reduction
in susceptibility to natural killer cell-mediated lysis of human
FO-1 melanoma cells after induction of HLA class I antigen
expression by transfection with B2m gene J. Clin. Invest.
88(1):282-9, July 1991; Schrier P I, et al., Relationship between
myc oncogene activation and MHC class I expression Adv. Cancer
Res., 60:181-246, 1993).
[0253] The complementary interplay between loss of HLA expression
and gain in NK sensitivity is exemplified by the classic studies of
Coulie and coworkers (Coulie, P G, et al., Antitumor immunity at
work in a melanoma patient. In Advances in Cancer Research,
213-242, 1999) which described the evolution of a patient's immune
response over the course of several years. Because of increased
sensitivity to NK lysis, it is predicted that approaches leading to
stimulation of innate immunity in general and NK activity in
particular would be of special significance. An example of such
approach is the induction of large amounts of dendritic cells (DC)
by various hematopoietic growth factors, such as Flt3 ligand or
ProGP. The rationale for this approach resides in the well known
fact that dendritic cells produce large amounts of IL-12, one of
the most potent stimulators for innate immunity and NK activity in
particular. Alternatively, IL-12 is administered directly, or as
nucleic acids that encode it. In this light, it is interesting to
note that Flt3 ligand treatment results in transient tumor
regression of a class I negative prostate murine cancer model
(Ciavarra R P, et al., Flt3-Ligand induces transient tumor
regression in an ectopic treatment model of major
histocompatibility complex-negative prostate cancer Cancer Res
60:2081-84, 2000). In this context, specific anti-tumor vaccines in
accordance with the invention synergize with these types of
hematopoietic growth factors to facilitate both CTL and NK cell
responses, thereby appreciably impairing a cell's ability to mutate
and thereby escape efficacious treatment. Thus, an embodiment of
the present invention comprises a composition of the invention
together with a method or composition that augments functional
activity or numbers of NK cells. Such an embodiment can comprise a
protocol that provides a composition of the invention sequentially
with an NK-inducing modality, or contemporaneous with an
NK-inducing modality.
[0254] Secondly, complete loss of HLA frequently occurs only in a
fraction of the tumor cells, while the remainder of tumor cells
continue to exhibit normal expression. In functional terms, the
tumor would still be subject, in part, to direct attack from a CTL
response; the portion of cells lacking HLA subject to an NK
response. Even if only a CTL response were used, destruction of the
HLA expressing fraction of the tumor has dramatic effects on
survival times and quality of life.
[0255] It should also be noted that in the case of heterogeneous
HLA expression, both normal HLA-expressing as well as defective
cells are predicted to be susceptible to immune destruction based
on "bystander effects." Such effects were demonstrated, e.g., in
the studies of Rosendahl and colleagues that investigated in vivo
mechanisms of action of antibody targeted superantigens (Rosendahl
A, et al., Perforin and IFN-gamma are involved in the antitumor
effects of antibody-targeted superantigens J. Immunol.
160(11):5309-13, Jun. 1, 1998). The bystander effect is understood
to be mediated by cytokines elicited from, e.g., CTLs acting on an
HLA-bearing target cell, whereby the cytokines are in the
environment of other diseased cells that are concomitantly
killed.
Allele-Specific Loss
[0256] One of the most common types of alterations in class I
molecules is the selective loss of certain alleles in individuals
heterozygous for HLA. Allele-specific alterations might reflect the
tumor adaptation to immune pressure, exerted by an immunodominant
response restricted by a single HLA restriction element. This type
of alteration allows the tumor to retain class I expression and
thus escape NK cell recognition, yet still be susceptible to a
CTL-based vaccine in accordance with the invention which comprises
epitopes corresponding to the remaining HLA type. Thus, a practical
solution to overcome the potential hurdle of allele-specific loss
relies on the induction of multispecific responses. Just as the
inclusion of multiple disease-associated antigens in a vaccine of
the invention guards against mutations that yield loss of a
specific disease antigens, simultaneously targeting multiple HLA
specificities and multiple disease-related antigens prevents
disease escape by allele-specific losses.
Decrease in Expression (Allele-Specific or not)
[0257] The sensitivity of effector CTL has long been demonstrated
(Brower, R C, et al., Minimal requirements for peptide mediated
activation of CD8+ CTL Mol. Immunol., 31; 1285-93, 1994;
Chriustnick, E T, et al. Low numbers of MHC class I-peptide
complexes required to trigger a T cell response Nature 352:67-70,
1991; Sykulev, Y, et al., Evidence that a single peptide-MHC
complex on a target cell can elicit a cytolytic T cell response
Immunity, 4(6):565-71, June 1996). Even a single peptide/MHC
complex can result in tumor cells lysis and release of anti-tumor
lymphokines. The biological significance of decreased HLA
expression and possible tumor escape from immune recognition is not
fully known. Nevertheless, it has been demonstrated that CTL
recognition of as few as one MHC/peptide complex is sufficient to
lead to tumor cell lysis.
[0258] Further, it is commonly observed that expression of HLA can
be upregulated by gamma IFN, commonly secreted by effector CTL.
Additionally, HLA class I expression can be induced in vivo by both
alpha and beta IFN (Halloran, et al. Local T cell responses induce
widespread MHC expression. J Immunol 148:3837, 1992; Pestka, S, et
al., Interferons and their actions Annu. Rev. Biochem. 56:727-77,
1987). Conversely, decreased levels of HLA class I expression also
render cells more susceptible to NK lysis.
[0259] With regard to gamma IFN, Torres et al (Torres, M J, et al.,
Loss of an HLA haplotype in pancreas cancer tissue and its
corresponding tumor derived cell line. Tissue Antigens 47:372-81,
1996) note that HLA expression is upregulated by gamma IFN in
pancreatic cancer, unless a total loss of haplotype has occurred.
Similarly, Rees and Mian note that allelic deletion and loss can be
restored, at least partially, by cytokines such as IFN-gamma (Rees,
R., et al. Selective MHC expression in tumours modulates adaptive
and innate antitumour responses Cancer Immunol Immunother
48:374-81, 1999). It has also been noted that IFN-gamma treatment
results in upregulation of class 1 molecules in the majority of the
cases studied (Browning M, et al., Mechanisms of loss of HLA class
I expression on colorectal tumor cells. Tissue Antigens 47:364-71,
1996). Kaklamakis, et al. also suggested that adjuvant
immunotherapy with IFN-gamma may be beneficial in the case of HLA
class I negative tumors (Kaklamanis L, Loss of transporter in
antigen processing 1 transport protein and major histocompatibility
complex class I molecules in metastatic versus primary breast
cancer. Cancer Research 55:5191-94, November 1995). It is important
to underline that IFN-gamma production is induced and
self-amplified by local inflammation/immunization (Halloran, et al.
Local T cell responses induce widespread MHC expression J Immunol
148:3837, 1992), resulting in large increases in MHC expressions
even in sites distant from the inflammatory site.
[0260] Finally, studies have demonstrated that decreased HLA
expression can render tumor cells more susceptible to NK lysis
(Ohnmacht, Ga., et al., Heterogeneity in expression of human
leukocyte antigens and melanoma-associated antigens in advanced
melanoma J Cellular Phys 182:332-38, 2000; Liunggren H G, et al.,
Host resistance directed selectively against H-2 deficient lymphoma
variants: Analysis of the mechanism J. Exp. Med., 162(6):1745-59,
Dec. 1, 1985; Maio M, et al., Reduction in susceptibility to
natural killer cell-mediated lysis of human FO-1 melanoma cells
after induction of HLA class I antigen expression by transfection
with .beta.2m gene J. Clin. Invest. 88(1):282-9, July 1991; Schrier
P I, et al., Relationship between myc oncogene activation and MHC
class I expression Adv. Cancer Res., 60:181-246, 1993). If
decreases in HLA expression benefit a tumor because it facilitates
CTL escape, but render the tumor susceptible to NK lysis, then a
minimal level of HLA expression that allows for resistance to NK
activity would be selected for (Gamido F, et al., Implications for
immunosurveillance of altered HLA class I phenotypes in human
tumours Immunol Today 18(2):89-96, February 1997). Therefore, a
therapeutic compositions or methods in accordance with the
invention together with a treatment to upregulate HLA expression
and/or treatment with high affinity T-cells renders the tumor
sensitive to CTL destruction.
Frequency of Alterations in HLA Expression
[0261] The frequency of alterations in class I expression is the
subject of numerous studies (Algarra I, et al., The HLA crossroad
in tumor immunology Human Immunology 61, 65-73, 2000). Rees and
Mian estimate allelic loss to occur overall in 3-20% of tumors, and
allelic deletion to occur in 15-50% of tumors. It should be noted
that each cell carries two separate sets of class I genes, each
gene carrying one HLA-A and one HLA-B locus. Thus, fully
heterozygous individuals carry two different HLA-A molecules and
two different HLA-B molecules. Accordingly, the actual frequency of
losses for any specific allele could be as little as one quarter of
the overall frequency. They also note that, in general, a gradient
of expression exists between normal cells, primary tumors and tumor
metastasis. In a study from Natali and coworkers (Natali P G, et
al., Selective changes in expression of HLA class I polymorphic
determinants in human solid tumors PNAS USA 86:6719-6723, September
1989), solid tumors were investigated for total HLA expression,
using W6/32 antibody, and for allele-specific expression of the A2
antigen, as evaluated by use of the BB7.2 antibody. Tumor samples
were derived from primary cancers or metastasis, for 13 different
tumor types, and scored as negative if less than 20%, reduced if in
the 30-80% range, and normal above 80%. All tumors, both primary
and metastatic, were HLA positive with W6/32. In terms of A2
expression, a reduction was noted in 16.1% of the cases, and A2 was
scored as undetectable in 39.4% of the cases. Gamido and coworkers
(Gamido F, et al., Natural history of HLA expression during tumour
development Immunol Today 14(10):491-99, 1993) emphasize that HLA
changes appear to occur at a particular step in the progression
from benign to most aggressive. Jiminez et al (Jiminez P, et al.,
Microsatellite instability analysis in tumors with different
mechanisms for total loss of HLA expression. Cancer Immunol
Immunother 48:684-90, 2000) have analyzed 118 different tumors (68
colorectal, 34 laryngeal and 16 melanomas). The frequencies
reported for total loss of HLA expression were 11% for colon, 18%
for melanoma and 13% for larynx. Thus, HLA class I expression is
altered in a significant fraction of the tumor types, possibly as a
reflection of immune pressure, or simply a reflection of the
accumulation of pathological changes and alterations in diseased
cells.
Immunotherapy in the Context of HLA Loss
[0262] A majority of the tumors express HLA class I, with a general
tendency for the more severe alterations to be found in later stage
and less differentiated tumors. This pattern is encouraging in the
context of immunotherapy, especially considering that: 1) the
relatively low sensitivity of immunohistochemical techniques might
underestimate HLA expression in tumors; 2) class I expression can
be induced in tumor cells as a result of local inflammation and
lymphokine release; and, 3) class I negative cells are sensitive to
lysis by NK cells.
[0263] Accordingly, various embodiments of the present invention
can be selected in view of the fact that there can be a degree of
loss of HLA molecules, particularly in the context of neoplastic
disease. For example, the treating physician can assay a patient's
tumor to ascertain whether HLA is being expressed. If a percentage
of tumor cells express no class I HLA, then embodiments of the
present invention that comprise methods or compositions that elicit
NK cell responses can be employed. As noted herein, such
NK-inducing methods or composition can comprise a Flt3 ligand or
ProGP which facilitate mobilization of dendritic cells, the
rationale being that dendritic cells produce large amounts of
IL-12. IL-12 can also be administered directly in either amino acid
or nucleic acid form. It should be noted that compositions in
accordance with the invention can be administered concurrently with
NK cell-inducing compositions, or these compositions can be
administered sequentially.
[0264] In the context of allele-specific HLA loss, a tumor retains
class I expression and may thus escape NK cell recognition, yet
still be susceptible to a CTL-based vaccine in accordance with the
invention which comprises epitopes corresponding to the remaining
HLA type. The concept here is analogous to embodiments of the
invention that include multiple disease antigens to guard against
mutations that yield loss of a specific antigen. Thus, one can
simultaneously target multiple HLA specificities and epitopes from
multiple disease-related antigens to prevent tumor escape by
allele-specific loss as well as disease-related antigen loss. In
addition, embodiments of the present invention can be combined with
alternative therapeutic compositions and methods. Such alternative
compositions and methods comprise, without limitation, radiation,
cytotoxic pharmaceuticals, and/or compositions/methods that induce
humoral antibody responses.
[0265] Moreover, it has been observed that expression of HLA can be
upregulated by gamma IFN, which is commonly secreted by effector
CTL, and that HLA class I expression can be induced in vivo by both
alpha and beta IFN. Thus, embodiments of the invention can also
comprise alpha, beta and/or gamma IFN to facilitate upregulation of
HLA.
Reprieve Periods from Therapies that Induce Side Effects:
"Scheduled Treatment Interruptions or Drug Holidays"
[0266] Recent evidence has shown that certain patients infected
with a pathogen, whom are initially treated with a therapeutic
regimen to reduce pathogen load, have been able to maintain
decreased pathogen load when removed from the therapeutic regimen,
i.e., during a "drug holiday" (Rosenberg, E., et al., Immune
control of HIV-1 after early treatment of acute infection Nature
407:523-26, Sep. 28, 2000) As appreciated by those skilled in the
art, many therapeutic regimens for both pathogens and cancer have
numerous, often severe, side effects. During the drug holiday, the
patient's immune system is keeping the disease in check. Methods
for using compositions of the invention are used in the context of
drug holidays for cancer and pathogenic infection.
[0267] For treatment of an infection, where therapies are not
particularly immunosuppressive, compositions of the invention are
administered concurrently with the standard therapy. During this
period, the patient's immune system is directed to induce responses
against the epitopes comprised by the present inventive
compositions. Upon removal from the treatment having side effects,
the patient is primed to respond to the infectious pathogen should
the pathogen load begin to increase. Composition of the invention
can be provided during the drug holiday as well.
[0268] For patients with cancer, many therapies are
immunosuppressive. Thus, upon achievement of a remission or
identification that the patient is refractory to standard
treatment, then upon removal from the immunosuppressive therapy, a
composition in accordance with the invention is administered.
Accordingly, as the patient's immune system reconstitutes, precious
immune resources are simultaneously directed against the cancer.
Composition of the invention can also be administered concurrently
with an immunosuppressive regimen if desired.
Kits
[0269] The peptide and nucleic acid compositions of this invention
can be provided in kit form together with instructions for vaccine
administration. Typically the kit would include desired peptide
compositions in a container, preferably in unit dosage form and
instructions for administration. An alternative kit would include a
minigene construct with desired nucleic acids of the invention in a
container, preferably in unit dosage form together with
instructions for administration. Lymphokines such as IL-2 or IL-12
may also be included in the kit. Other kit components that may also
be desirable include, for example, a sterile syringe, booster
dosages, and other desired excipients.
Overview
[0270] Epitopes in accordance with the present invention were
successfully used to induce an immune response. Immune responses
with these epitopes have been induced by administering the epitopes
in various forms. The epitopes have been administered as peptides,
as nucleic acids, and as viral vectors comprising nucleic acids
that encode the epitope(s) of the invention. Upon administration of
peptide-based epitope forms, immune responses have been induced by
direct loading of an epitope onto an empty HLA molecule that is
expressed on a cell, and via internalization of the epitope and
processing via the HLA class I pathway; in either event, the HLA
molecule expressing the epitope was then able to interact with and
induce a CTL response. Peptides can be delivered directly or using
such agents as liposomes. They can additionally be delivered using
ballistic delivery, in which the peptides are typically in a
crystalline form. When DNA is used to induce an immune response, it
is administered either as naked DNA, generally in a dose range of
approximately 1-5 mg, or via the ballistic "gene gun" delivery,
typically in a dose range of approximately 10-100 .mu.g. The DNA
can be delivered in a variety of conformations, e.g., linear,
circular etc. Various viral vectors have also successfully been
used that comprise nucleic acids which encode epitopes in
accordance with the invention.
[0271] Accordingly compositions in accordance with the invention
exist in several forms. Embodiments of each of these composition
forms in accordance with the invention have been successfully used
to induce an immune response.
[0272] One composition in accordance with the invention comprises a
plurality of peptides. This plurality or cocktail of peptides is
generally admixed with one or more pharmaceutically acceptable
excipients. The peptide cocktail can comprise multiple copies of
the same peptide or can comprise a mixture of peptides. The
peptides can be analogs of naturally occurring epitopes. The
peptides can comprise artificial amino acids and/or chemical
modifications such as addition of a surface active molecule, e.g.,
lipidation; acetylation, glycosylation, biotinylation,
phosphorylation etc. The peptides can be CTL or HTL epitopes. In a
preferred embodiment the peptide cocktail comprises a plurality of
different CTL epitopes and at least one HTL epitope. The HTL
epitope can be naturally or non-naturally (e.g., PADRE.RTM.,
Epimmune Inc., San Diego, Calif.). The number of distinct epitopes
in an embodiment of the invention is generally a whole unit integer
from one through one hundred fifty (e.g., 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 100, or 150).
[0273] An additional embodiment of a composition in accordance with
the invention comprises a polypeptide multi-epitope construct,
i.e., a polyepitopic peptide. Polyepitopic peptides in accordance
with the invention are prepared by use of technologies well-known
in the art. By use of these known technologies, epitopes in
accordance with the invention are connected one to another. The
polyepitopic peptides can be linear or non-linear, e.g.,
multivalent. These polyepitopic constructs can comprise artificial
amino acids, spacing or spacer amino acids, flanking amino acids,
or chemical modifications between adjacent epitope units. The
polyepitopic construct can be a heteropolymer or a homopolymer. The
polyepitopic constructs generally comprise epitopes in a quantity
of any whole unit integer between 2-150 (e.g., 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 100, or 150). The polyepitopic construct
can comprise CTL and/or HTL epitopes. One or more of the epitopes
in the construct can be modified, e.g., by addition of a surface
active material, e.g. a lipid, or chemically modified, e.g.,
acetylation, etc. Moreover, bonds in the multiepitopic construct
can be other than peptide bonds, e.g., covalent bonds, ester or
ether bonds, disulfide bonds, hydrogen bonds, ionic bonds etc.
[0274] Alternatively, a composition in accordance with the
invention comprises construct which comprises a series, sequence,
stretch, etc., of amino acids that have homology to (i.e.,
corresponds to or is contiguous with) to a native sequence. This
stretch of amino acids comprises at least one subsequence of amino
acids that, if cleaved or isolated from the longer series of amino
acids, functions as an HLA class I or HLA class II epitope in
accordance with the invention. In this embodiment, the peptide
sequence is modified, so as to become a construct as defined
herein, by use of any number of techniques known or to be provided
in the art. The polyepitopic constructs can contain homology to a
native sequence in any whole unit integer increment from 70-100%,
e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or, 100
percent.
[0275] A further embodiment of a composition in accordance with the
invention is an antigen presenting cell that comprises one or more
epitopes in accordance with the invention. The antigen presenting
cell can be a "professional" antigen presenting cell, such as a
dendritic cell. The antigen presenting cell can comprise the
epitope of the invention by any means known or to be determined in
the art. Such means include pulsing of dendritic cells with one or
more individual epitopes or with one or more peptides that comprise
multiple epitopes, by nucleic acid administration such as ballistic
nucleic acid delivery or by other techniques in the art for
administration of nucleic acids, including vector-based, e.g. viral
vector, delivery of nucleic acids.
[0276] Further embodiments of compositions in accordance with the
invention comprise nucleic acids that encode one or more peptides
of the invention, or nucleic acids which encode a polyepitopic
peptide in accordance with the invention. As appreciated by one of
ordinary skill in the art, various nucleic acids compositions will
encode the same peptide due to the redundancy of the genetic code.
Each of these nucleic acid compositions falls within the scope of
the present invention. This embodiment of the invention comprises
DNA or RNA, and in certain embodiments a combination of DNA and
RNA. It is to be appreciated that any composition comprising
nucleic acids that will encode a peptide in accordance with the
invention or any other peptide based composition in accordance with
the invention, falls within the scope of this invention.
[0277] It is to be appreciated that peptide-based forms of the
invention (as well as the nucleic acids that encode them) can
comprise analogs of epitopes of the invention generated using
principles already known, or to be known, in the art. Principles
related to analoging are now known in the art, and are disclosed
herein; moreover, analoging principles (heteroclitic analoging) are
disclosed in co-pending application serial number U.S. Ser. No.
09/226,775 filed 6 Jan. 1999. Generally the compositions of the
invention are isolated or purified.
[0278] The invention will be described in greater detail by way of
specific examples. The following examples are offered for
illustrative purposes, and are not intended to limit the invention
in any manner. Those of skill in the art will readily recognize a
variety of non-critical parameters that can be changed or modified
to yield alternative embodiments in accordance with the
invention.
Examples
[0279] The following example of peptide binding to HLA molecules
demonstrates quantification of binding affinities of HLA class I
and class II peptides. Binding assays can be performed with
peptides that are either motif-bearing or not motif-bearing.
Example 1
HLA Class I and Class II Binding Assays
[0280] The following example of peptide binding to HLA molecules
demonstrates quantification of binding affinities of HLA class I
and class II peptides. Binding assays can be performed with
peptides that are either motif-bearing or not motif-bearing.
[0281] HLA class I and class II binding assays using purified HLA
molecules were performed in accordance with disclosed protocols
(e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney et al.,
Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J.
Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813
(1994)). Briefly, purified MHC molecules (5 to 500 nM) were
incubated with various unlabeled peptide inhibitors and 1-10 nM
.sup.125I-radiolabeled probe peptides as described. Following
incubation, MHC-peptide complexes were separated from free peptide
by gel filtration and the fraction of peptide bound was determined.
Typically, in preliminary experiments, each MHC preparation was
titered in the presence of fixed amounts of radiolabeled peptides
to determine the concentration of HLA molecules necessary to bind
10-20% of the total radioactivity. All subsequent inhibition and
direct binding assays were performed using these HLA
concentrations.
[0282] Since under these conditions [label]<[HLA] and
IC.sub.50.gtoreq.[HLA], the measured IC.sub.50 values are
reasonable approximations of the true K.sub.D values. Peptide
inhibitors are typically tested at concentrations ranging from 120
.mu.g/ml to 1.2 ng/ml, and are tested in two to four completely
independent experiments. To allow comparison of the data obtained
in different experiments, a relative binding figure is calculated
for each peptide by dividing the IC.sub.50 of a positive control
for inhibition by the IC.sub.50 for each tested peptide (typically
unlabeled versions of the radiolabeled probe peptide). For database
purposes, and inter-experiment comparisons, relative binding values
are compiled. These values can subsequently be converted back into
IC.sub.50 nM values by dividing the IC.sub.50 nM of the positive
controls for inhibition by the relative binding of the peptide of
interest. This method of data compilation has proven to be the most
accurate and consistent for comparing peptides that have been
tested on different days, or with different lots of purified
MHC.
[0283] Binding assays as outlined above may be used to analyze
supermotif and/or motif-bearing epitopes as, for example, described
in Example 2.
Example 2
Identification of HLA Supermotif- and Motif-Bearing CTL Candidate
Epitopes
[0284] Vaccine compositions of the invention can include multiple
epitopes that comprise multiple HLA supermotifs or motifs to
achieve broad population coverage. This example illustrates the
identification of supermotif- and motif-bearing epitopes for the
inclusion in such a vaccine composition. Calculation of population
coverage was performed using the strategy described below.
Computer Searches and Algorithms for Identification of Supermotif
and/or Motif-Bearing Epitopes
[0285] The searches performed to identify the motif-bearing peptide
sequences in Examples 2 and 5 employed the protein sequence data
from seven proteins (E1, E2, E5, E6, E7, L1 and L2) from HPV types
16, 18, 31, 33, 45, and 56.
TABLE-US-00003 Accession numbers for HPV types Protein 6a 6b 11 E1
Q84293 P03113 W1WL11 AAA74213 CAA25020 P04014 W1WL6 AAA46929 E2
Q84294 P03119 AAA46930 AAA74214 CAA25021 W2WLI1 W2WL6 P04015 E4
Q84295 CAA25022 P04016 AAA74215 W4WL6 W4WL11 AAA46931 E5a Q84296
P06460 W5WL11 AAA74216 CAA25023 P04017 W5WL6A AAA46932 E5b N.A.
P06461 W5WL1B CAA25024 P04018 W5WLB AAA46933 E6 Q84291 P06462
W6WL11 AAA74211 CAA250I8 P04019 W6WL6 AAA21703 AAA46927 E7 Q84929
P06464 AAA46928 AAA74212 CAA25019 AAA21704 W7WL6 W7WL11 P04020 L1
P03100 P03100 P04012 AAA74218 CAA25026 P1WL11 P1WL6 AAA4635 L2
Q84297 P03106 P2WL11 CAA25025 AAA46934 P2WL6 P040I3 Strain Protein
Antigen Accession number HPV16 E1 W1SLHS HPV16 E2 W2WLHS HPV16 E5
W5WLHS HPV16 E6 W6WLHS HPV16 E7 W7WLHS HPV16 L1 AAD33259 HPV16 L2
AAD33258 HPV18 E1 W1WL18 HPV18 E2 WL18 HPV18 E5 W5WL18 HPV18 E6
W6WL18 HPV18 E7 PO6788 HPV18 L1 CAA28671 HPV18 L2 P2WL18 HPV31 E1
W1WL31 HPV31 E2 W2WL3 HPV31 E5 W5WL31 HPV31 E6 W6WL31 HPV31 E7
W7WL31 HPV31 L1 P1WL31 HPV31 L2 P2WL31 HPV45 E1 S36563 HPV45 E2
S36564 HPV45 E6 CAB44706 HPV45 E7 CAB44707 HPV45 L1 CAB44705 HPV45
L2 S36565 HPV33 E1 W1WL33 HPV33 E2 W2WL33 HPV33 E5 W5WL33 HPV33 E6
W6WL33 HPV33 E7 W7WL33 HPV33 L1 P1WL33 HPV33 L2 P2WL33 HPV56 E2
S36581 HPV56 E6 W6WL56 HPV56 E7 S36580 HPV56 L1 S38563 HPV56 L2
S36582
[0286] Computer searches for epitopes bearing HLA Class I or Class
II supermotifs or motifs were performed as follows. All translated
HPV protein sequences were analyzed using a text string search
software program, e.g., MotifSearch 1.4 (D. Brown, San Diego) to
identify potential peptide sequences containing appropriate HLA
binding motifs; alternative programs are readily produced in
accordance with information in the art in view of the
motif/supermotif disclosure herein. Furthermore, such calculations
can be made mentally.
[0287] Identified A2-, A3-, and DR-supermotif sequences were scored
using polynomial algorithms to predict their capacity to bind to
specific HLA-Class I or Class II molecules. These polynomial
algorithms take into account both extended and refined motifs (that
is, to account for the impact of different amino acids at different
positions), and are essentially based on the premise that the
overall affinity (or .DELTA.G) of peptide-HLA molecule interactions
can be approximated as a linear polynomial function of the
type:
".DELTA.G"=a.sub.1i.times.a.sub.2i.times.a.sub.3i . . .
.times.a.sub.ni
where a.sub.ji is a coefficient which represents the effect of the
presence of a given amino acid (j) at a given position (i) along
the sequence of a peptide of n amino acids. The crucial assumption
of this method is that the effects at each position are essentially
independent of each other (i.e., independent binding of individual
side-chains). When residue j occurs at position i in the peptide,
it is assumed to contribute a constant amount j.sub.i to the free
energy of binding of the peptide irrespective of the sequence of
the rest of the peptide. This assumption is justified by studies
from our laboratories that demonstrated that peptides are bound to
MHC and recognized by T cells in essentially an extended
conformation (data omitted herein).
[0288] The method of derivation of specific algorithm coefficients
has been described in Gulukota et al., J. Mol. Biol. 267:1258-126,
1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and
Southwood et al., J. Immunol. 160:3363-3373, 1998). Briefly, for
all i positions, anchor and non-anchor alike, the geometric mean of
the average relative binding (ARB) of all peptides carrying j is
calculated relative to the remainder of the group, and used as the
estimate of j.sub.i. For Class II peptides, if multiple alignments
are possible, only the highest scoring alignment is utilized,
following an iterative procedure. To calculate an algorithm score
of a given peptide in a test set, the ARB values corresponding to
the sequence of the peptide are multiplied. If this product exceeds
a chosen threshold, the peptide is predicted to bind. Appropriate
thresholds are chosen as a function of the degree of stringency of
prediction desired.
Selection of HLA-A2 Supertype Cross-Reactive Peptides
[0289] Complete protein sequences from the seven HPV structural and
regulatory proteins of the HPV strains listed above were aligned,
then scanned, utilizing motif identification software, to identify
9- and 10-mer sequences containing the HLA-A2-supermotif main
anchor specificity.
[0290] HLA-A2 supermotif-bearing sequences are shown in Table VIII.
Typically, these sequences are then scored using the A2 algorithm
and the peptides corresponding to the positive-scoring sequences
are synthesized and tested for their capacity to bind purified
HLA-A*0201 molecules in vitro (HLA-A*0201 is considered a prototype
A2 supertype molecule).
[0291] Examples of peptides that bind to HLA-A*0201 with IC.sub.50
values .ltoreq.500 nM are shown in Table VIII. These peptides are
then tested for the capacity to bind to additional A2-supertype
molecules (A*0202, A*0203, A*0206, and A*6802). Peptides that bind
to at least three of the five A2-supertype alleles tested are
typically deemed A2-supertype cross-reactive binders. Preferred
peptides bind at an affinity equal to or less than 500 nM to three
or more HLA-A2 supertype molecules.
Selection of HLA-A3 Supermotif-Bearing Epitopes
[0292] The HPV protein sequences scanned above were also examined
for the presence of peptides with the HLA-A3-supermotif primary
anchors (Table IX).
[0293] Peptides corresponding to the supermotif-bearing sequences
are then synthesized and tested for binding to HLA-A*0301 and
HLA-A*1101 molecules, the two most prevalent A3-supertype alleles.
The peptides that are found to bind one of the two alleles with
binding affinities of .ltoreq.500 nM, often .ltoreq.200 nM, are
then tested for binding cross-reactivity to the other common
A3-supertype alleles (A*3101, A*3301, and A*6801) to identify those
that can bind at least three of the five HLA-A3-supertype molecules
tested.
Selection of HLA-B7 Supermotif Bearing Epitopes
[0294] The same HPV target antigen protein sequences were also
analyzed for the presence of 9- or 10-mer peptides with the
HLA-B7-supermotif (Table XI).
[0295] Corresponding peptides are synthesized and tested for
binding to HLA-B*0702, the most common B7-supertype allele (i.e.,
the prototype B7 supertype allele). Peptides binding B*0702 with
IC.sub.50 of .ltoreq.500 nM are identified using standard methods.
These peptides are then tested for binding to other common
B7-supertype molecules (B*3501, B*5101, B*5301, and B*5401).
Peptides capable of binding to three or more of the five
B7-supertype alleles tested are thereby identified.
Selection of A1 and A24 Motif-Bearing Epitopes
[0296] To further increase population coverage, HLA-A1 and -A24
epitopes can, for example, also be incorporated into potential
vaccine constructs. An analysis of the protein sequence data from
the HPV target antigens utilized above can also be performed to
identify HLA-A1- and A24-motif-containing sequences.
[0297] High affinity and/or cross-reactive binding epitopes that
bear other motif and/or supermotifs are identified using analogous
methodology.
Example 3
Confirmation of Immunogenicity
[0298] Cross-reactive candidate CTL A2-supermotif-bearing peptides
that are identified as described in Example 2 were selected for in
vitro immunogenicity testing. Testing was performed using the
following methodology:
Target Cell Lines for Cellular Screening:
[0299] The 0.221A2.1 cell line, produced by transferring the
HLA-A2.1 gene into the HLA-A, -B, -C null mutant human
B-lymphoblastoid cell line 721.221, is used as the peptide-loaded
target to measure activity of HLA-A2.1-restricted CTL. This cell
line is grown in RPMI-1640 medium supplemented with antibiotics,
sodium pyruvate, nonessential amino acids and 10% (v/v) heat
inactivated FCS. Cells that express an antigen of interest, or
transfectants comprising the gene encoding the antigen of interest,
can be used as target cells to test the ability of peptide-specific
CTLs to recognize endogenous antigen.
Primary CTL Induction Cultures:
[0300] Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI
with 30 .mu.g/ml DNAse, washed twice and resuspended in complete
medium (RPMI-1640 plus 5% AB human serum, non-essential amino
acids, sodium pyruvate, L-glutamine and penicillin/strpetomycin).
The monocytes are purified by plating 10.times.10.sup.6 PBMC/well
in a 6-well plate. After 2 hours at 37.degree. C., the non-adherent
cells are removed by gently shaking the plates and aspirating the
supernatants. The wells are washed a total of three times with 3 ml
RPMI to remove most of the non-adherent and loosely adherent cells.
Three ml of complete medium containing 50 ng/ml of GM-CSF and 1,000
U/ml of IL-4 are then added to each well. TNF.quadrature. is added
to the DCs on day 6 at 75 ng/ml and the cells are used for CTL
induction cultures on day 7.
[0301] Induction of CTL with DC and Peptide: CD8+ T-cells are
isolated by positive selection with Dynal immunomagnetic beads
(Dynabeads.RTM. M-450) and the Detacha-Bead.RTM. reagent. Typically
about 200-250.times.10.sup.6 PBMC are processed to obtain
24.times.10.sup.6 CD8.sup.+ T-cells (enough for a 48-well plate
culture). Briefly, the PBMCs are thawed in RPMI with 30 .mu.g/ml
DNAse, washed once with PBS containing 1% human AB serum and
resuspended in PBS/1% AB serum at a concentration of
20.times.10.sup.6 cells/ml. The magnetic beads are washed 3 times
with PBS/AB serum, added to the cells (140 .mu.l
beads/20.times.10.sup.6 cells) and incubated for 1 hour at
4.degree. C. with continuous mixing. The beads and cells are washed
4.times. with PBS/AB serum to remove the nonadherent cells and
resuspended at 100.times.10.sup.6 cells/ml (based on the original
cell number) in PBS/AB serum containing 100 .mu.l/ml
Detacha-Bead.RTM. reagent and 30 .mu.g/ml DNAse. The mixture is
incubated for 1 hour at room temperature with continuous mixing.
The beads are washed again with PBS/AB/DNAse to collect the CD8+
T-cells. The DC are collected and centrifuged at 1300 rpm for 5-7
minutes, washed once with PBS with 1% BSA, counted and pulsed with
40 .mu.g/ml of peptide at a cell concentration of
1-2.times.10.sup.6/ml in the presence of 3 .mu.g/ml
.beta..sub.2-microglobulin for 4 hours at 20.degree. C. The DC are
then irradiated (4,200 rads), washed 1 time with medium and counted
again.
[0302] Setting up induction cultures: 0.25 ml cytokine-generated DC
(@1.times.10.sup.5 cells/ml) are co-cultured with 0.25 ml of CD8+
T-cells (@2.times.10.sup.6 cell/ml) in each well of a 48-well plate
in the presence of 10 ng/ml of IL-7. Recombinant human IL10 is
added the next day at a final concentration of 10 ng/ml and rhuman
IL2 is added 48 hours later at 10 IU/ml.
[0303] Restimulation of the induction cultures with peptide-pulsed
adherent cells: Seven and fourteen days after the primary induction
the cells are restimulated with peptide-pulsed adherent cells. The
PBMCS are thawed and washed twice with RPMI and DNAse. The cells
are resuspended at 5.times.10.sup.6 cells/ml and irradiated at
.about.4200 rads. The PBMCs are plated at 2.times.10.sup.6 in 0.5
ml complete medium per well and incubated for 2 hours at 37.degree.
C. The plates are washed twice with RPMI by tapping the plate
gently to remove the nonadherent cells and the adherent cells
pulsed with 10 .mu.g/ml of peptide in the presence of 3 .mu.g/ml
.beta..sub.2 microglobulin in 0.25 ml RPMI/5% AB per well for 2
hours at 37.degree. C. Peptide solution from each well is aspirated
and the wells are washed once with RPMI. Most of the media is
aspirated from the induction cultures (CD8+ cells) and brought to
0.5 ml with fresh media. The cells are then transferred to the
wells containing the peptide-pulsed adherent cells. Twenty four
hours later rhuman IL10 is added at a final concentration of 10
ng/ml and rhuman IL2 is added the next day and again 2-3 days later
at 50 IU/ml (Tsai et al., Critical Reviews in Immunology 18
(1-2):65-75, 1998). Seven days later the cultures are assayed for
CTL activity in a .sup.51Cr release assay. In some experiments the
cultures are assayed for peptide-specific recognition in the in
situ IFN.gamma. ELISA at the time of the second restimulation
followed by assay of endogenous recognition 7 days later. After
expansion, activity is measured in both assays for a side by side
comparison.
Measurement of CTL Lytic Activity by .sup.51Cr Release.
[0304] Seven days after the second restimulation, cytotoxicity is
determined in a standard (5 hr) .sup.51Cr release assay by assaying
individual wells at a single E:T. Peptide-pulsed targets are
prepared by incubating the cells with 10 .mu.g/ml peptide overnight
at 37.degree. C.
[0305] Adherent target cells are removed from culture flasks with
trypsin-EDTA. Target cells are labelled with 200 .mu.Ci of
.sup.51Cr sodium chromate (Dupont, Wilmington, Del.) for 1 hour at
37.degree. C. Labelled target cells are resuspended at 10.sup.6 per
ml and diluted 1:10 with K562 cells at a concentration of
3.3.times.10.sup.6/ml (an NK-sensitive erythroblastoma cell line
used to reduce non-specific lysis). Target cells (100 .mu.l) and
100 .mu.l of effectors are plated in 96 well round-bottom plates
and incubated for 5 hours at 37.degree. C. At that time, 100 .mu.l
of supernatant are collected from each well and percent lysis is
determined according to the formula: [(cpm of the test sample-cpm
of the spontaneous .sup.51Cr release sample)/(cpm of the maximal
.sup.51Cr release sample-cpm of the spontaneous .sup.51Cr release
sample)].times.100. Maximum and spontaneous release are determined
by incubating the labelled targets with 1% Trition X-100 and media
alone, respectively. A positive culture is defined as one in which
the specific lysis (sample-background) is 10% or higher in the case
of individual wells and is 15% or more at the 2 highest E:T ratios
when expanded cultures are assayed.
In situ Measurement of Human .gamma.IFN Production as an Indicator
of Peptide-Specific and Endogenous Recognition
[0306] Immulon 2 plates are coated with mouse anti-human IFN.gamma.
monoclonal antibody (4 .mu.g/ml 0.1M NaHCO.sub.3, pH8.2) overnight
at 4.degree. C. The plates are washed with Ca.sup.2+,
Mg.sup.2+-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for
2 hours, after which the CTLs (100 .mu.l/well) and targets (100
.mu.l/well) are added to each well, leaving empty wells for the
standards and blanks (which received media only). The target cells,
either peptide-pulsed or endogenous targets, are used at a
concentration of 1.times.10.sup.6 cells/ml. The plates are
incubated for 48 hours at 37.degree. C. with 5% CO.sub.2.
[0307] Recombinant human IFN.gamma. is added to the standard wells
starting at 400 pg or 1200 pg/100 .mu.l/well and the plate
incubated for 2 hours at 37.degree. C. The plates are washed and
100 .mu.l of biotinylated mouse anti-human IFN.gamma. monoclonal
antibody (2 .mu.g/ml in PBS/3% FCS/0.05% Tween 20) are added and
incubated for 2 hours at room temperature. After washing again, 100
.mu.l HRP-streptavidin (1:4000) are added and the plates incubated
for 1 hour at room temperature. The plates are then washed 6.times.
with wash buffer, 100 .mu.l/well developing solution (TMB 1:1) are
added, and the plates allowed to develop for 5-15 minutes. The
reaction is stopped with 50 .mu.l/well 1M H.sub.3PO.sub.4 and read
at OD450. A culture is considered positive if it measured at least
50 pg of IFN.gamma./well above background and is twice the
background level of expression.
[0308] CTL Expansion. Those cultures that demonstrate specific
lytic activity against peptide-pulsed targets and/or tumor targets
are expanded over a two week period with anti-CD3. Briefly,
5.times.10.sup.4 CD8+ cells are added to a T25 flask containing the
following: 1.times.10.sup.6 irradiated (4,200 rad) PBMC (autologous
or allogeneic) per ml, 2.times.10.sup.5 irradiated (8,000 rad)
EBV-transformed cells per ml, and OKT3 (anti-CD3) at 30 ng per ml
in RPMI-1640 containing 10% (v/v) human AB serum, non-essential
amino acids, sodium pyruvate, 25 .mu.M 2-mercaptoethanol,
L-glutamine and penicillin/streptomycin. Rhuman IL2 is added 24
hours later at a final concentration of 2001 U/ml and every 3 days
thereafter with fresh media at 50 IU/ml. The cells are split if the
cell concentration exceeded 1.times.10.sup.6/ml and the cultures
are assayed between days 13 and 15 at E:T ratios of 30, 10, 3 and
1:1 in the .sup.51Cr release assay or at 1.times.10.sup.6/ml in the
in situ IFN.quadrature. assay using the same targets as before the
expansion.
[0309] Cultures are expanded in the absence of anti-CD3.sup.+ as
follows. Those cultures that demonstrate specific lytic activity
against peptide and endogenous targets are selected and
5.times.10.sup.4 CD8.sup.+ cells are added to a T25 flask
containing the following: 1.times.10.sup.6 autologous PBMC per ml
which have been peptide-pulsed with 10 .mu.g/ml peptide for 2 hours
at 37.degree. C. and irradiated (4,200 rad); 2.times.10.sup.5
irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640
containing 10% (v/v) human AB serum, non-essential AA, sodium
pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.
Immunogenicity of A2 Supermotif-Bearing Peptides
[0310] A2-supermotif cross-reactive binding peptides are tested in
the cellular assay for the ability to induce peptide-specific CTL
in normal individuals. In this analysis, a peptide is typically
considered to be an epitope if it induces peptide-specific CTLs in
at least 2 donors (unless otherwise noted) and preferably, also
recognizes the endogenously expressed peptide.
[0311] Immunogenicity is additionally confirmed using PBMCs
isolated from HPV-infected patients. Briefly, PBMCs are isolated
from patients, re-stimulated with peptide-pulsed monocytes and
assayed for the ability to recognize peptide-pulsed target cells as
well as transfected cells endogenously expressing the antigen.
Evaluation of A*03/A11 Immunogenicity
[0312] HLA-A3 supermotif-bearing cross-reactive binding peptides
are also evaluated for immunogenicity using methodology analogous
for that used to evaluate the immunogenicity of the HLA-A2
supermotif peptides.
Evaluation of B7 Immunogenicity
[0313] Immunogenicity screening of the B7-supertype cross-reactive
binding peptides identified in Example 2 are evaluated in a manner
analogous to the evaluation of A2- and A3-supermotif-bearing
peptides.
[0314] Peptides bearing other supermotifs/motifs, e.g., HLA-A1,
HLA-A24 etc. are also evaluated using similar methodology
Example 4
Implementation of the Extended Supermotif to Improve the Binding
Capacity of Native Epitopes by Creating Analogs
[0315] HLA motifs and supermotifs (comprising primary and/or
secondary residues) are useful in the identification and
preparation of highly cross-reactive native peptides, as
demonstrated herein. Moreover, the definition of HLA motifs and
supermotifs also allows one to engineer highly cross-reactive
epitopes by identifying residues within a native peptide sequence
which can be analoged, or "fixed" to confer upon the peptide
certain characteristics, e.g. greater cross-reactivity within the
group of HLA molecules that comprise a supertype, and/or greater
binding affinity for some or all of those HLA molecules. Examples
of analoging peptides to exhibit modulated binding affinity are set
forth in this example.
Analoging at Primary Anchor Residues
[0316] Peptide engineering strategies are implemented to further
increase the cross-reactivity of the epitopes. For example, on the
basis of the data disclosed, e.g., in related and co-pending
U.S.S.N 09/226,775, the main anchors of A2-supermotif-bearing
peptides are altered, for example, to introduce a preferred L, I,
V, or M at position 2, and I or V at the C-terminus.
[0317] To analyze the cross-reactivity of the analog peptides, each
engineered analog is initially tested for binding to the prototype
A2 supertype allele A*0201, then, if A*0201 binding capacity is
maintained, for A2-supertype cross-reactivity.
[0318] Alternatively, a peptide is tested for binding to one or all
supertype members and then analogued to modulate binding affinity
to any one (or more) of the supertype members to add population
coverage.
[0319] The selection of analogs for immunogenicity in a cellular
screening analysis is typically further restricted by the capacity
of the parent peptide to bind at least weakly, i.e., bind at an
IC.sub.50 of 500 nM or less, to three of more A2 supertype alleles.
The rationale for this requirement is that the WT peptides must be
present endogenously in sufficient quantity to be biologically
relevant. Analoged peptides have been shown to have increased
immunogenicity and cross-reactivity by T cells specific for the
parent epitope (see, e.g., Parkhurst et al., J. Immunol. 157:2539,
1996; and Pogue et al., Proc. Natl. Acad. Sci. USA 92:8166,
1995).
[0320] In the cellular screening of these peptide analogs, it is
important to demonstrate that analog-specific CTLs are also able to
recognize the wild-type peptide and, when possible, target cells
that endogenously express the epitope.
Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides
[0321] Analogs of HLA-A3 supermotif-bearing epitopes are generated
using strategies similar to those employed in analoging HLA-A2
supermotif-bearing peptides. For example, peptides binding to 3/5
of the A3-supertype molecules are engineered at primary anchor
residues to possess a preferred residue (V, S, M, or A) at position
2.
[0322] The analog peptides are then tested for the ability to bind
A*03 and A*11 (prototype A3 supertype alleles). Those peptides that
demonstrate .ltoreq.500 nM binding capacity are then tested for
A3-supertype cross-reactivity.
[0323] Similarly to the A2- and A3-motif bearing peptides, peptides
binding 3 or more B7-supertype alleles can be improved, where
possible, to achieve increased cross-reactive binding. B7
supermotif-bearing peptides are, for example, engineered to possess
a preferred residue (V, I, L, or F) at the C-terminal primary
anchor position, as demonstrated by Sidney et al. (J. Immunol.
157:3480-3490, 1996).
[0324] Analoguing at primary anchor residues of other motif and/or
supermotif-bearing epitopes is performed in a like manner.
[0325] The analog peptides are then be tested for immunogenicity,
typically in a cellular screening assay. Again, it is generally
important to demonstrate that analog-specific CTLs are also able to
recognize the wild-type peptide and, when possible, targets that
endogenously express the epitope.
Analoging at Secondary Anchor Residues
[0326] Moreover, HLA supermotifs are of value in engineering highly
cross-reactive peptides and/or peptides that bind HLA molecules
with increased affinity by identifying particular residues at
secondary anchor positions that are associated with such
properties. For example, the binding capacity of a B7
supermotif-bearing peptide with an F residue at position 1 is
analyzed. The peptide is then analoged to, for example, substitute
L for F at position 1. The analoged peptide is evaluated for
increased binding affinity/and or increased cross-reactivity. Such
a procedure identifies analoged peptides with modulated binding
affinity.
[0327] Engineered analogs with sufficiently improved binding
capacity or cross-reactivity can also be tested for immunogenicity
in HLA-B7-transgenic mice, following for example, IFA immunization
or lipopeptide immunization. Analogued peptides are additionally
tested for the ability to stimulate a recall response using PBMC
from HPV-infected patients.
Other Analoguing strategies
[0328] Another form of peptide analoguing, unrelated to the anchor
positions, involves the substitution of a cysteine with
.alpha.-amino butyric acid. Due to its chemical nature, cysteine
has the propensity to form disulfide bridges and sufficiently alter
the peptide structurally so as to reduce binding capacity.
Substitution of .alpha.-amino butyric acid for cysteine not only
alleviates this problem, but has been shown to improve binding and
crossbinding capabilities in some instances (see, e.g., the review
by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and
I. Chen, John Wiley & Sons, England, 1999).
[0329] Thus, by the use of even single amino acid substitutions,
the binding affinity and/or cross-reactivity of peptide ligands for
HLA supertype molecules can be modulated.
Example 5
Identification of HPV-Derived Sequences with HLA-DR Binding
Motifs
[0330] Peptide epitopes bearing an HLA class II supermotif or motif
are identified as outlined below using methodology similar to that
described in Examples 1-3.
Selection of HLA-DR-Supermotif-Bearing Epitopes.
[0331] To identify HPV-derived, HLA class II HTL epitopes, the
protein sequences from the same HPV antigens used for the
identification of HLA Class I supermotif/motif sequences were
analyzed for the presence of sequences bearing an HLA-DR-motif or
supermotif. Specifically, 15-mer sequences were selected comprising
a DR-supermotif, further comprising a 9-mer core, and three-residue
N- and C-terminal flanking regions (15 amino acids total).
[0332] Protocols for predicting peptide binding to DR molecules
have been developed (Southwood et al., J. Immunol. 160:3363-3373,
1998). These protocols, specific for individual DR molecules, allow
the scoring, and ranking, of 9-mer core regions. Each protocol not
only scores peptide sequences for the presence of DR-supermotif
primary anchors (i.e., at position 1 and position 6) within a 9-mer
core, but additionally evaluates sequences for the presence of
secondary anchors. Using allele specific selection tables (see,
e.g., Southwood et al., ibid.), it has been found that these
protocols efficiently select peptide sequences with a high
probability of binding a particular DR molecule. Additionally, it
has been found that performing these protocols in tandem,
specifically those for DR1, DR4w4, and DR7, can efficiently select
DR cross-reactive peptides.
[0333] The HPV-derived peptides identified above are tested for
their binding capacity for various common HLA-DR molecules. All
peptides are initially tested for binding to the DR molecules in
the primary panel: DR1, DR4w4, and DR7. Peptides binding at least 2
of these 3 DR molecules are then tested for binding to DR2w2
.beta.1, DR2w2 .beta.2, DR6w19, and DR9 molecules in secondary
assays. Finally, peptides binding at least 2 of the 4 secondary
panel DR molecules, and thus cumulatively at least 4 of 7 different
DR molecules, are screened for binding to DR4w15, DR5w11, and DR8w2
molecules in tertiary assays. Peptides binding at least 7 of the 10
DR molecules comprising the primary, secondary, and tertiary
screening assays are considered cross-reactive DR binders.
HPV-derived peptides found to bind common HLA-DR alleles are of
particular interest.
Selection of DR3 Motif Peptides
[0334] Because HLA-DR3 is an allele that is prevalent in Caucasian,
Black, and Hispanic populations, DR3 binding capacity is an
important criterion in the selection of HTL epitopes. However, data
generated previously indicated that DR3 only rarely cross-reacts
with other DR alleles (Sidney et al., J. Immunol. 149:2634-2640,
1992; Geluk et al., J. Immunol. 152:5742-5748, 1994; Southwood et
al., J. Immunol. 160:3363-3373, 1998). This is not entirely
surprising in that the DR3 peptide-binding motif appears to be
distinct from the specificity of most other DR alleles. For maximum
efficiency in developing vaccine candidates it would be desirable
for DR3 motifs to be clustered in proximity with DR supermotif
regions. Thus, peptides shown to be candidates may also be assayed
for their DR3 binding capacity. However, in view of the distinct
binding specificity of the DR3 motif, peptides binding only to DR3
can also be considered as candidates for inclusion in a vaccine
formulation.
[0335] To efficiently identify peptides that bind DR3, target HPV
antigens are analyzed for sequences carrying one of the two DR3
specific binding motifs reported by Geluk et al. (J. Immunol.
152:5742-5748, 1994). The corresponding peptides are then
synthesized and tested for the ability to bind DR3 with an affinity
of 1 .mu.M or better, i.e., less than 1 .mu.M. Peptides are found
that meet this binding criterion and qualify as HLA class II high
affinity binders.
[0336] DR3 binding epitopes identified in this manner are included
in vaccine compositions with DR supermotif-bearing peptide
epitopes.
[0337] Similarly to the case of HLA class I motif-bearing peptides,
the class II motif-bearing peptides are analoged to improve
affinity or cross-reactivity. For example, aspartic acid at
position 4 of the 9-mer core sequence is an optimal residue for DR3
binding, and substitution for that residue often improves DR 3
binding.
Example 6
Immunogenicity of HPV-Derived HTL Epitopes
[0338] This example determines immunogenic DR supermotif- and DR3
motif-bearing epitopes among those identified using the methodology
in Example 5.
[0339] Immunogenicity of HTL epitopes are evaluated in a manner
analogous to the determination of immunogenicity of CTL epitopes by
assessing the ability to stimulate HTL responses and/or by using
appropriate transgenic mouse models. Immunogenicity is determined
by screening for: 1.) in vitro primary induction using normal PBMC
or 2.) recall responses from cancer patient PBMCs.
Example 7
Calculation of Phenotypic Frequencies of HLA-Supertypes in Various
Ethnic Backgrounds to Determine Breadth of Population Coverage
[0340] This example illustrates the assessment of the breadth of
population coverage of a vaccine composition comprised of multiple
epitopes comprising multiple supermotifs and/or motifs.
[0341] In order to analyze population coverage, gene frequencies of
HLA alleles were determined. Gene frequencies for each HLA allele
were calculated from antigen or allele frequencies utilizing the
binomial distribution formulae gf=1-(SQRT(1-af)) (see, e.g., Sidney
et al., Human Immunol. 45:79-93, 1996). To obtain overall
phenotypic frequencies, cumulative gene frequencies were
calculated, and the cumulative antigen frequencies derived by the
use of the inverse formula [af=1-(1-Cgf).sup.2].
[0342] Where frequency data was not available at the level of DNA
typing, correspondence to the serologically defined antigen
frequencies was assumed. To obtain total potential supertype
population coverage no linkage disequilibrium was assumed, and only
alleles confirmed to belong to each of the supertypes were included
(minimal estimates). Estimates of total potential coverage achieved
by inter-loci combinations were made by adding to the A coverage
the proportion of the non-A covered population that could be
expected to be covered by the B alleles considered (e.g.,
total=A+B*(1-A)). Confirmed members of the A3-like supertype are
A3, A11l, A31, A*3301, and A*6801. Although the A3-like supertype
may also include A34, A66, and A*7401, these alleles were not
included in overall frequency calculations. Likewise, confirmed
members of the A2-like supertype family are A*0201, A*0202, A*0203,
A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. Finally, the
B7-like supertype-confirmed alleles are: B7, B*3501-03, B51,
B*5301, B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially
also B*1401, B*3504-06, B*4201, and B*5602).
[0343] Population coverage achieved by combining the A2-, A3- and
B7-supertypes is approximately 86% in five major ethnic groups,
supra. Coverage may be extended by including peptides bearing the
A1 and A24 motifs. On average, A1 is present in 12% and A24 in 29%
of the population across five different major ethnic groups
(Caucasian, North American Black, Chinese, Japanese, and Hispanic).
Together, these alleles are represented with an average frequency
of 39% in these same ethnic populations. The total coverage across
the major ethnicities when A1 and A24 are combined with the
coverage of the A2-, A3- and B7-supertype alleles is >95%. An
analagous approach can be used to estimate population coverage
achieved with combinations of class II motif-bearing epitopes.
[0344] Immunogenicity studies in humans (e.g., Bertoni et al., J.
Clin. Invest. 100:503, 1997; Doolan et al., Immunity 7:97, 1997;
and Threlkeld et al., J. Immunol. 159:1648, 1997) have shown that
highly cross-reactive binding peptides are almost always recognized
as epitopes. The use of highly cross-reactive binding peptides is
an important selection criterion in identifying candidate epitopes
for inclusion in a vaccine that is immunogenic in a diverse
population.
[0345] With a sufficient number of epitopes (as disclosed herein
and from the art), an average population coverage is predicted to
be greater than 95% in each of five major ethnic populations. The
game theory Monte Carlo simulation analysis, which is known in the
art (see e.g., Osborne, M. J. and Rubinstein, A. "A course in game
theory" MIT Press, 1994), can be used to estimate what percentage
of the individuals in a population comprised of the Caucasian,
North American Black, Japanese, Chinese, and Hispanic ethnic groups
would recognize the vaccine epitopes described herein. A preferred
percentage is 90%. A more preferred percentage is 95%.
Example 8
CTL Recognition Of Endogenous Processed Antigens After Priming
[0346] This example determines that CTL induced by native or
analogued peptide epitopes identified and selected as described in
Examples 1-6 recognize endogenously synthesized, i.e., native
antigens.
[0347] Effector cells isolated from transgenic mice that are
immunized with peptide epitopes as in Example 3, for example HLA-A2
supermotif-bearing epitopes, are re-stimulated in vitro using
peptide-coated stimulator cells. Six days later, effector cells are
assayed for cytotoxicity and the cell lines that contain
peptide-specific cytotoxic activity are further re-stimulated. An
additional six days later, these cell lines are tested for
cytotoxic activity on .sup.51Cr labeled Jurkat-A2.1/K.sup.b target
cells in the absence or presence of peptide, and also tested on
.sup.51Cr labeled target cells bearing the endogenously synthesized
antigen, i.e. cells that are stably transfected with HPV expression
vectors.
[0348] The result will demonstrate that CTL lines obtained from
animals primed with peptide epitope recognize endogenously
synthesized HPV antigen. The choice of transgenic mouse model to be
used for such an analysis depends upon the epitope(s) that is being
evaluated. In addition to HLA-A*0201/K.sup.b transgenic mice,
several other transgenic mouse models including mice with human
A11, which may also be used to evaluate A3 epitopes, and B7 alleles
have been characterized and others (e.g., transgenic mice for
HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse
models have also been developed, which may be used to evaluate HTL
epitopes.
Example 9
Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice
[0349] This example illustrates the induction of CTLs and HTLs in
transgenic mice by use of a tumor associated antigen CTL/HTL
peptide conjugate whereby the vaccine composition comprises
peptides to be administered to an HPV-infected patient. The peptide
composition can comprise multiple CTL and/or HTL epitopes and
further, can comprise epitopes selected from multiple HPV target
antigens. The epitopes are identified using methodology as
described in Examples 1-6 This analysis demonstrates the enhanced
immunogenicity that can be achieved by inclusion of one or more HTL
epitopes in a vaccine composition. Such a peptide composition can
comprise an HTL epitope conjugated to a preferred CTL epitope
containing, for example, at least one CTL epitope that binds to
multiple HLA family members at an affinity of 500 nM or less, or
analogs of that epitope. The peptides may be lipidated, if
desired.
[0350] Immunization procedures: Immunization of transgenic mice is
performed as described (Alexander et al., J. Immunol.
159:4753-4761, 1997). For example, A2/K.sup.b mice, which are
transgenic for the human HLA A2.1 allele and are useful for the
assessment of the immunogenicity of HLA-A*0201 motif- or HLA-A2
supermotif-bearing epitopes, are primed subcutaneously (base of the
tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or
if the peptide composition is a lipidated CTL/HTL conjugate, in
DMSO/saline or if the peptide composition is a polypeptide, in PBS
or Incomplete Freund's Adjuvant. Seven days after priming,
splenocytes obtained from these animals are restimulated with
syngenic irradiated LPS-activated lymphoblasts coated with
peptide.
[0351] Cell lines: Target cells for peptide-specific cytotoxicity
assays are Jurkat cells transfected with the HLA-A2.1/K.sup.b
chimeric gene (e.g., Vitiello et al., J. Exp. Med. 173:1007,
1991)
[0352] In vitro CTL activation: One week after priming, spleen
cells (30.times.10.sup.6 cells/flask) are co-cultured at 37.degree.
C. with syngeneic, irradiated (3000 rads), peptide coated
lymphoblasts (10.times.10.sup.6 cells/flask) in 10 ml of culture
medium/T25 flask. After six days, effector cells are harvested and
assayed for cytotoxic activity.
[0353] Assay for cytotoxic activity: Target cells (1.0 to
1.5.times.10.sup.6) are incubated at 37.degree. C. in the presence
of 200 .mu.l of .sup.51Cr. After 60 minutes, cells are washed three
times and resuspended in R10 medium. Peptide is added where
required at a concentration of 1 .mu.g/ml. For the assay, 10.sup.4
51 Cr-labeled target cells are added to different concentrations of
effector cells (final volume of 200 .mu.l) in U-bottom 96-well
plates. After a 6 hour incubation period at 37.degree. C., a 0.1 ml
aliquot of supernatant is removed from each well and radioactivity
is determined in a Micromedic automatic gamma counter. The percent
specific lysis is determined by the formula: percent specific
release=100.times.(experimental release-spontaneous
release)/(maximum release-spontaneous release). To facilitate
comparison between separate CTL assays run under the same
conditions, % .sup.51Cr release data is expressed as lytic
units/10.sup.6 cells. One lytic unit is arbitrarily defined as the
number of effector cells required to achieve 30% lysis of 10,000
target cells in a 6 hour .sup.51Cr release assay. To obtain
specific lytic units/10.sup.6, the lytic units/10.sup.6 obtained in
the absence of peptide is subtracted from the lytic units/10.sup.6
obtained in the presence of peptide. For example, if 30% .sup.51Cr
release is obtained at the effector (E): target (T) ratio of 50:1
(i.e., 5.times.10.sup.5 effector cells for 10,000 targets) in the
absence of peptide and 5:1 (i.e., 5.times.10.sup.4 effector cells
for 10,000 targets) in the presence of peptide, the specific lytic
units would be: [( 1/50,000)-( 1/500,000)].times.10.sup.6=18
LU.
[0354] The results are analyzed to assess the magnitude of the CTL
responses of animals injected with the immunogenic CTL/HTL
conjugate vaccine preparation and are compared to the magnitude of
the CTL response achieved using the CTL epitope as outlined in
Example 3. Analyses similar to this may be performed to evaluate
the immunogenicity of peptide conjugates containing multiple CTL
epitopes and/or multiple HTL epitopes. In accordance with these
procedures it is found that a CTL response is induced, and
concomitantly that an HTL response is induced upon administration
of such compositions.
Example 10
Selection of CTL and HTL Epitopes for Inclusion in an HPV-Specific
Vaccine
[0355] This example illustrates the procedure for the selection of
peptide epitopes for vaccine compositions of the invention. The
peptides in the composition can be in the form of a nucleic acid
sequence, either single or one or more sequences (i.e., minigene)
that encodes peptide(s), or can be single and/or polyepitopic
peptides.
[0356] The following principles are utilized when selecting an
array of epitopes for inclusion in a vaccine composition. Each of
the following principles is balanced in order to make the
selection.
[0357] Epitopes are selected which, upon administration, mimic
immune responses that have been observed to be correlated with HPV
clearance. The number of epitopes used depends on observations of
patients who spontaneously clear HPV. For example, if it has been
observed that patients who spontaneously clear HPV generate an
immune response to at least 3 epitopes on at least one HPV antigen,
then 3-4 epitopes should be included for HLA class I. A similar
rationale is used to determine HLA class II epitopes.
[0358] When selecting an array of HPV epitopes, it is preferred
that at least some of the epitopes are derived from early and late
proteins. The early proteins of HPV are expressed when the virus is
replicating, either following acute or dormant infection.
Therefore, it is particularly preferred to use epitopes from early
stage proteins to alleviate disease manifestations at the earliest
stage possible.
[0359] Epitopes are often selected that have a binding affinity of
an IC.sub.50 of 500 nM or less for an HLA class 1 molecule, or for
class II, an IC.sub.50 of 1000 nM or less.
[0360] Sufficient supermotif bearing peptides, or a sufficient
array of allele-specific motif bearing peptides, are selected to
give broad population coverage. For example, epitopes are selected
to provide at least 80% population coverage. A Monte Carlo
analysis, a statistical evaluation known in the art, can be
employed to assess breadth, or redundancy, of population
coverage.
[0361] When creating a polyepitopic compositions, e.g. a minigene,
it is typically desirable to generate the smallest peptide possible
that encompasses the epitopes of interest. The principles employed
are similar, if not the same, as those employed when selecting a
peptide comprising nested epitopes.
[0362] In cases where the sequences of multiple variants of the
same target protein are available, potential peptide epitopes can
also be selected on the basis of their conservancy. For example, a
criterion for conservancy may define that the entire sequence of an
HLA class I binding peptide or the entire 9-mer core of a class II
binding peptide be conserved in a designated percentage of the
sequences evaluated for a specific protein antigen.
[0363] A vaccine composition comprised of selected peptides, when
administered, is safe, efficacious, and elicits an immune response
similar in magnitude to an immune response that controls or clears
an acute HPV infection.
Example 11
Construction of Minigene Multi-Epitope DNA Plasmids
[0364] This example provides general guidance for the construction
of a minigene expression plasmid. Minigene plasmids may, of course,
contain various configurations of CTL and/or HTL epitopes or
epitope analogs as described herein. Examples of the construction
and evaluation of expression plasmids are described, for example,
in co-pending U.S. Ser. No. 09/311,784 filed May 13, 1999.
[0365] A minigene expression plasmid typically includes multiple
CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3,
-B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24
motif-bearing peptide epitopes are used in conjunction with DR
supermotif-bearing epitopes and/or DR3 epitopes. HLA class I
supermotif or motif-bearing peptide epitopes derived from multiple
HPV antigens, preferably including both early and late phase
antigens, are selected such that multiple supermotifs/motifs are
represented to ensure broad population coverage. Similarly, HLA
class II epitopes are selected from multiple HPV antigens to
provide broad population coverage, i.e. both HLA DR-1-4-7
supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are
selected for inclusion in the minigene construct. The selected CTL
and HTL epitopes are then incorporated into a minigene for
expression in an expression vector.
[0366] Such a construct may additionally include sequences that
direct the HTL epitopes to the endoplasmic reticulum. For example,
the Ii protein may be fused to one or more HTL epitopes as
described in co-pending application U.S. Ser. No. 09/311,784 filed
May 13, 1999, wherein the CLIP sequence of the Ii protein is
removed and replaced with an HLA class II epitope sequence so that
HLA class II epitope is directed to the endoplasmic reticulum,
where the epitope binds to an HLA class II molecules.
[0367] This example illustrates the methods to be used for
construction of a minigene-bearing expression plasmid. Other
expression vectors that may be used for minigene compositions are
available and known to those of skill in the art.
[0368] The minigene DNA plasmid of this example contains a
consensus Kozak sequence and a consensus murine kappa Ig-light
chain signal sequence followed by CTL and/or HTL epitopes selected
in accordance with principles disclosed herein. The sequence
encodes an open reading frame fused to the Myc and H is antibody
epitope tag coded for by the pcDNA 3.1 Myc-His vector.
[0369] Overlapping oligonucleotides that can, for example, average
about 70 nucleotides in length with 15 nucleotide overlaps, are
synthesized and HPLC-purified. The oligonucleotides encode the
selected peptide epitopes as well as appropriate linker
nucleotides, Kozak sequence, and signal sequence. The final
multiepitope minigene is assembled by extending the overlapping
oligonucleotides in three sets of reactions using PCR. A
Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are
performed using the following conditions: 95.degree. C. for 15 sec,
annealing temperature (5.degree. below the lowest calculated Tm of
each primer pair) for 30 sec, and 72.degree. C. for 1 min.
[0370] For example, a minigene can be prepared as follows. For a
first PCR reaction, 5 .mu.g of each of two oligonucleotides are
annealed and extended: In an example using eight oligonucleotides,
i.e., four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and
7+8 are combined in 100 .mu.l reactions containing Pfu polymerase
buffer (1.times.=10 mM KCL, 10 mM (NH.sub.4).sub.2SO.sub.4, 20 mM
Tris-chloride, pH 8.75, 2 mM MgSO.sub.4, 0.1% Triton X-100, 100
.mu.g/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The
full-length dimer products are gel-purified, and two reactions
containing the product of 1+2 and 3+4, and the product of 5+6 and
7+8 are mixed, annealed, and extended for 10 cycles. Half of the
two reactions are then mixed, and 5 cycles of annealing and
extension carried out before flanking primers are added to amplify
the full length product. The full-length product is gel-purified
and cloned into pCR-blunt (Invitrogen) and individual clones are
screened by sequencing.
Example 12
The Plasmid Construct and the Degree to which it Induces
Immunogenicity
[0371] The degree to which a plasmid construct, for example a
plasmid constructed in accordance with Example 11, is able to
induce immunogenicity can be evaluated in vitro by testing for
epitope presentation by APC following transduction or transfection
of the APC with an epitope-expressing nucleic acid construct. Such
a study determines "antigenicity" and allows the use of human APC.
The assay determines the ability of the epitope to be presented by
the APC in a context that is recognized by a T cell by quantifying
the density of epitope-HLA class I complexes on the cell surface.
Quantitation can be performed by directly measuring the amount of
peptide eluted from the APC (see, e.g., Sijts et al., J. Immunol.
156:683-692, 1996; Demotz et al., Nature 342:682-684, 1989); or the
number of peptide-HLA class I complexes can be estimated by
measuring the amount of lysis or lymphokine release induced by
infected or transfected target cells, and then determining the
concentration of peptide necessary to obtained equivalent levels of
lysis or lymphokine release (see, e.g., Kageyama et al., J.
Immunol. 154:567-576, 1995).
[0372] Alternatively, immunogenicity can be evaluated through in
vivo injections into mice and subsequent in vitro assessment of CTL
and HTL activity, which are analysed using cytotoxicity and
proliferation assays, respectively, as detailed e.g., in copending
U.S. Ser. No. 09/311,784 filed May 13, 1999 and Alexander et al.,
Immunity 1:751-761, 1994.
[0373] For example, to assess the capacity of a DNA minigene
construct (e.g., a pMin minigene construct generated as described
in U.S. Ser. No. 09/311,784) containing at least one HLA-A2
supermotif peptide to induce CTLs in vivo, HLA-A2.1/K.sup.b
transgenic mice, for example, are immunized intramuscularly with
100 .mu.g of naked cDNA. As a means of comparing the level of CTLs
induced by cDNA immunization, a control group of animals is also
immunized with an actual peptide composition that comprises
multiple epitopes synthesized as a single polypeptide as they would
be encoded by the minigene.
[0374] Splenocytes from immunized animals are stimulated twice with
each of the respective compositions (peptide epitopes encoded in
the minigene or the polyepitopic peptide), then assayed for
peptide-specific cytotoxic activity in a .sup.51Cr release assay.
The results indicate the magnitude of the CTL response directed
against the A2-restricted epitope, thus indicating the in vivo
immunogenicity of the minigene vaccine and polyepitopic vaccine. It
is, therefore, found that the minigene elicits immune responses
directed toward the HLA-A2 supermotif peptide epitopes as does the
polyepitopic peptide vaccine. A similar analysis is also performed
using other HLA-A3 and HLA-B7 transgenic mouse models to assess CTL
induction by HLA-A3 and HLA-B7 motif or supermotif epitopes.
[0375] To assess the capacity of a class II epitope encoding
minigene to induce HTLs in vivo, DR transgenic mice, or for those
epitope that cross react with the appropriate mouse MHC molecule,
I-A.sup.b-restricted mice, for example, are immunized
intramuscularly with 100 .mu.g of plasmid DNA. As a means of
comparing the level of HTLs induced by DNA immunization, a group of
control animals is also immunized with an actual peptide
composition emulsified in complete Freund's adjuvant. CD4+ T cells,
i.e. HTLs, are purified from splenocytes of immunized animals and
stimulated with each of the respective compositions (peptides
encoded in the minigene). The HTL response is measured using a
.sup.3H-thymidine incorporation proliferation assay, (see, e.g.,
Alexander et al. Immunity 1:751-761, 1994). The results indicate
the magnitude of the HTL response, thus demonstrating the in vivo
immunogenicity of the minigene.
[0376] DNA minigenes, constructed as described in Example 11, may
also be evaluated as a vaccine in combination with a boosting agent
using a prime boost protocol. The boosting agent can consist of
recombinant protein (e.g., Barnett et al., Aids Res. and Human
Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant
vaccinia, for example, expressing a minigene or DNA encoding the
complete protein of interest (see, e.g., Hanke et al., Vaccine
16:439-445, 1998; Sedegah et al., Proc. Natl. Acad. Sci. USA
95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181,
1999; and Robinson et al., Nature Med. 5:526-34, 1999).
[0377] For example, the efficacy of the DNA minigene used in a
prime boost protocol is initially evaluated in transgenic mice. In
this example, A2.1/K.sup.b transgenic mice are immunized IM with
100 .mu.g of a DNA minigene encoding the immunogenic peptides
including at least one HLA-A2 supermotif-bearing peptide. After an
incubation period (ranging from 3-9 weeks), the mice are boosted IP
with 10.sup.7 pfu/mouse of a recombinant vaccinia virus expressing
the same sequence encoded by the DNA minigene. Control mice are
immunized with 100 .mu.g of DNA or recombinant vaccinia without the
minigene sequence, or with DNA encoding the minigene, but without
the vaccinia boost. After an additional incubation period of two
weeks, splenocytes from the mice are immediately assayed for
peptide-specific activity in an ELISPOT assay. Additionally,
splenocytes are stimulated in vitro with the A2-restricted peptide
epitopes encoded in the minigene and recombinant vaccinia, then
assayed for peptide-specific activity in an IFN-.quadrature.
ELISA.
[0378] It is found that the minigene utilized in a prime-boost
protocol elicits greater immune responses toward the HLA-A2
supermotif peptides than with DNA alone. Such an analysis can also
be performed using HLA-A11 or HLA-B7 transgenic mouse models to
assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif
epitopes.
[0379] The use of prime boost protocols in humans is described in
Example 20.
Example 13
Peptide Composition for Prophylactic Uses
[0380] Vaccine compositions of the present invention can be used to
prevent HPV infection in persons who are at risk for such
infection. For example, a polyepitopic peptide epitope composition
(or a nucleic acid comprising the same) containing multiple CTL and
HTL epitopes such as those selected in Examples 9 and/or 10, which
are also selected to target greater than 80% of the population, is
administered to individuals at risk for HPV infection.
[0381] For example, a peptide-based composition can be provided as
a single polypeptide that encompasses multiple epitopes. The
vaccine is typically administered in a physiological solution that
comprises an adjuvant, such as Incomplete Freunds Adjuvant. The
dose of peptide for the initial immunization is from about 1 to
about 50,000 .mu.g, generally 100-5,000 .mu.g, for a 70 kg patient.
The initial administration of vaccine is followed by booster
dosages at 4 weeks followed by evaluation of the magnitude of the
immune response in the patient, by techniques that determine the
presence of epitope-specific CTL populations in a PBMC sample.
Additional booster doses are administered as required. The
composition is found to be both safe and efficacious as a
prophylaxis against HPV infection.
[0382] Alternatively, a composition typically comprising
transfecting agents can be used for the administration of a nucleic
acid-based vaccine in accordance with methodologies known in the
art and disclosed herein.
Example 14
Polyepitopic Vaccine Compositions Derived from Native HPV
Sequences
[0383] A native HPV polyprotein sequence is screened, preferably
using computer algorithms defined for each class I and/or class II
supermotif or motif, to identify "relatively short" regions of the
polyprotein that comprise multiple epitopes and is preferably less
in length than an entire native antigen. This relatively short
sequence that contains multiple distinct, even overlapping,
epitopes is selected and used to generate a minigene construct. The
construct is engineered to express the peptide, which corresponds
to the native protein sequence. The "relatively short" peptide is
generally less than 250 amino acids in length, often less than 100
amino acids in length, preferably less than 75 amino acids in
length, and more preferably less than 50 amino acids in length. The
protein sequence of the vaccine composition is selected because it
has maximal number of epitopes contained within the sequence, i.e.,
it has a high concentration of epitopes. As noted herein, epitope
motifs may be nested or overlapping (i.e., frame shifted relative
to one another). For example, with f overlapping epitopes, two
9-mer epitopes and one 10-mer epitope can be present in a 10 amino
acid peptide. Such a vaccine composition is administered for
therapeutic or prophylactic purposes.
[0384] The vaccine composition will include, for example, three CTL
epitopes from at least one HPV target antigen and at least one HTL
epitope. This polyepitopic native sequence is administered either
as a peptide or as a nucleic acid sequence which encodes the
peptide. Alternatively, an analog can be made of this native
sequence, whereby one or more of the epitopes comprise
substitutions that alter the cross-reactivity and/or binding
affinity properties of the polyepitopic peptide.
[0385] The embodiment of this example provides for the possibility
that an as yet undiscovered aspect of immune system processing will
apply to the native nested sequence and thereby facilitate the
production of therapeutic or prophylactic immune response-inducing
vaccine compositions. Additionally such an embodiment provides for
the possibility of motif-bearing epitopes for an HLA makeup that is
presently unknown. Furthermore, this embodiment (absent analogs)
directs the immune response to multiple peptide sequences that are
actually present in native HPV antigens thus avoiding the need to
evaluate any junctional epitopes. Lastly, the embodiment provides
an economy of scale when producing nucleic acid vaccine
compositions.
[0386] Related to this embodiment, computer programs can be derived
in accordance with principles in the art, which identify in a
target sequence, the greatest number of epitopes per sequence
length.
Example 15
Polyepitopic Vaccine Compositions from Multiple Antigens
[0387] The HPV peptide epitopes of the present invention are used
in conjunction with peptide epitopes from other target
tumor-associated antigens to create a vaccine composition that is
useful for the prevention or treatment of cancer resulting from HPV
infection in multiple patients.
[0388] For example, a vaccine composition can be provided as a
single polypeptide that incorporates multiple epitopes from HPV
antigens as well as tumor-associated antigens that are often
expressed with a target cancer, e.g., cervical cancer, associated
with HPV infection, or can be administered as a composition
comprising one or more discrete epitopes. Alternatively, the
vaccine can be administered as a minigene construct or as dendritic
cells which have been loaded with the peptide epitopes in
vitro.
Example 16
Use of Peptides to Evaluate an Immune Response
[0389] Peptides of the invention may be used to analyze an immune
response for the presence of specific CTL or HTL populations
directed to HPV. Such an analysis may be performed in a manner as
that described by Ogg et al., Science 279:2103-2106, 1998. In the
following example, peptides in accordance with the invention are
used as a reagent for diagnostic or prognostic purposes, not as an
immunogen.
[0390] In this example highly sensitive human leukocyte antigen
tetrameric complexes ("tetramers") are used for a cross-sectional
analysis of, for example, HPV HLA-A*0201-specific CTL frequencies
from HLA A*0201-positive individuals at different stages of
infection or following immunization using an HPV peptide containing
an A*0201 motif. Tetrameric complexes are synthesized as described
(Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified
HLA heavy chain (A*0201 in this example) and .beta.2-microglobulin
are synthesized by means of a prokaryotic expression system. The
heavy chain is modified by deletion of the transmembrane-cytosolic
tail and COOH-terminal addition of a sequence containing a BirA
enzymatic biotinylation site. The heavy chain,
.beta.2-microglobulin, and peptide are refolded by dilution. The
45-kD refolded product is isolated by fast protein liquid
chromatography and then biotinylated by BirA in the presence of
biotin (Sigma, St. Louis, Mo.), adenosine 5' triphosphate and
magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4
molar ratio, and the tetrameric product is concentrated to 1 mg/ml.
The resulting product is referred to as tetramer-phycoerythrin.
[0391] For the analysis of patient blood samples, approximately one
million PBMCs are centrifuged at 300 g for 5 minutes and
resuspended in 50 .mu.l of cold phosphate-buffered saline.
Tri-color analysis is performed with the tetramer-phycoerythrin,
along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are
incubated with tetramer and antibodies on ice for 30 to 60 min and
then washed twice before formaldehyde fixation. Gates are applied
to contain >99.98% of control samples. Controls for the
tetramers include both A*0201-negative individuals and
A*0201-positive uninfected donors. The percentage of cells stained
with the tetramer is then determined by flow cytometry. The results
indicate the number of cells in the PBMC sample that contain
epitope-restricted CTLs, thereby readily indicating the extent of
immune response to the HPV epitope, and thus the stage of infection
with HPV, the status of exposure to HPV, or exposure to a vaccine
that elicits a protective or therapeutic response.
Example 17
Use of Peptide Epitopes to Evaluate Recall Responses
[0392] The peptide epitopes of the invention are used as reagents
to evaluate T cell responses, such as acute or recall responses, in
patients. Such an analysis may be performed on patients who have
recovered from infection, who are chronically infected with HPV, or
who have been vaccinated with an HPV vaccine.
[0393] For example, the class I restricted CTL response of persons
who have been vaccinated may be analyzed. The vaccine may be any
HPV vaccine. PBMC are collected from vaccinated individuals and HLA
typed. Appropriate peptide epitopes of the invention that,
optimally, bear supermotifs to provide cross-reactivity with
multiple HLA supertype family members, are then used for analysis
of samples derived from individuals who bear that HLA type.
[0394] PBMC from vaccinated individuals are separated on
Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis,
Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended
in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2
mM), penicillin (50 U/ml), streptomycin (50 .mu.g/ml), and Hepes
(10 mM) containing 10% heat-inactivated human AB serum (complete
RPMI) and plated using microculture formats. A synthetic peptide
comprising an epitope of the invention is added at 10 .mu.g/ml to
each well and HBV core 128-140 epitope is added at 1 .mu.g/ml to
each well as a source of T cell help during the first week of
stimulation.
[0395] In the microculture format, 4.times.10.sup.5 PBMC are
stimulated with peptide in 8 replicate cultures in 96-well round
bottom plate in 100 .mu.l/well of complete RPMI. On days 3 and 10,
100 ul of complete RPMI and 20 U/ml final concentration of rIL-2
are added to each well. On day 7 the cultures are transferred into
a 96-well flat-bottom plate and restimulated with peptide, rIL-2
and 10.sup.5 irradiated (3,000 rad) autologous feeder cells. The
cultures are tested for cytotoxic activity on day 14. A positive
CTL response requires two or more of the eight replicate cultures
to display greater than 10% specific .sup.51Cr release, based on
comparison with uninfected control subjects as previously described
(Rehermann, et al., Nature Med. 2:1104, 1108, 1996; Rehermann et
al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J.
Clin. Invest. 98:1432-1440, 1996).
[0396] Target cell lines are autologous and allogeneic
EBV-transformed B-LCL that are either purchased from the American
Society for Histocompatibility and Immunogenetics (ASHI, Boston,
Mass.) or established from the pool of patients as described
(Guilhot, et al. J. Virol. 66:2670-2678, 1992).
[0397] Cytotoxicity assays are performed in the following manner.
Target cells consist of either allogeneic HLA-matched or autologous
EBV-transformed B lymphoblastoid cell line that are incubated
overnight with the synthetic peptide epitope of the invention at 10
.mu.M, and labeled with 100 .mu.Ci of .sup.51Cr (Amersham Corp.,
Arlington Heights, Ill.) for 1 hour after which they are washed
four times with HBSS.
[0398] Cytolytic activity is determined in a standard 4-h, split
well .sup.51Cr release assay using U-bottomed 96 well plates
containing 3,000 targets/well. Stimulated PBMC are tested at
effector/target (E/T) ratios of 20-50:1 on day 14. Percent
cytotoxicity is determined from the formula:
100.times.[(experimental release-spontaneous release)/maximum
release-spontaneous release)]. Maximum release is determined by
lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co.,
St. Louis, Mo.). Spontaneous release is <25% of maximum release
for all experiments.
[0399] The results of such an analysis indicate the extent to which
HLA-restricted CTL populations have been stimulated by previous
exposure to HPV or an HPV vaccine.
[0400] The class II restricted HTL responses may also be analyzed.
Purified PBMC are cultured in a 96-well flat bottom plate at a
density of 1.5.times.10.sup.5 cells/well and are stimulated with 10
.mu.g/ml synthetic peptide, whole antigen, or PHA. Cells are
routinely plated in replicates of 4-6 wells for each condition.
After seven days of culture, the medium is removed and replaced
with fresh medium containing 10 U/ml IL-2. Two days later, 1 .mu.Ci
.sup.3H-thymidine is added to each well and incubation is continued
for an additional 18 hours. Cellular DNA is then harvested on glass
fiber mats and analyzed for .sup.3H-thymidine incorporation.
Antigen-specific T cell proliferation is calculated as the ratio of
.sup.3H-thymidine incorporation in the presence of antigen divided
by the .sup.3H-thymidine incorporation in the absence of
antigen.
Example 18
Induction of Specific CTL Response in Humans
[0401] A human clinical trial for an immunogenic composition
comprising CTL and HTL epitopes of the invention is set up as an
IND Phase I, dose escalation study and carried out as a randomized,
double-blind, placebo-controlled trial. Such a trial is designed,
for example, as follows:
[0402] A total of about 27 individuals are enrolled and divided
into 3 groups:
[0403] Group I: 3 subjects are injected with placebo and 6 subjects
are injected with 5 .mu.g of peptide composition;
[0404] Group II: 3 subjects are injected with placebo and 6
subjects are injected with 50 .mu.g peptide composition;
[0405] Group III: 3 subjects are injected with placebo and 6
subjects are injected with 500 .mu.g of peptide composition.
[0406] After 4 weeks following the first injection, all subjects
receive a booster inoculation at the same dosage.
[0407] The endpoints measured in this study relate to the safety
and tolerability of the peptide composition as well as its
immunogenicity. Cellular immune responses to the peptide
composition are an index of the intrinsic activity of this the
peptide composition, and can therefore be viewed as a measure of
biological efficacy. The following summarize the clinical and
laboratory data that relate to safety and efficacy endpoints.
[0408] Safety: The incidence of adverse events is monitored in the
placebo and drug treatment group and assessed in terms of degree
and reversibility.
[0409] Evaluation of Vaccine Efficacy: For evaluation of vaccine
efficacy, subjects are bled before and after injection. Peripheral
blood mononuclear cells are isolated from fresh heparinized blood
by Ficoll-Hypaque density gradient centrifugation, aliquoted in
freezing media and stored frozen. Samples are assayed for CTL and
HTL activity.
[0410] The vaccine is found to be both safe and efficacious.
Example 19
Phase II Trials In Patients Infected With HPV
[0411] Phase II trials are performed to study the effect of
administering the CTL-HTL peptide compositions to patients having
cancer associated with HPV infection. The main objectives of the
trials are to determine an effective dose and regimen for inducing
CTLs in HPV-infected patients with cancer, to establish the safety
of inducing a CTL and HTL response in these patients, and to see to
what extent activation of CTLs improves the clinical picture of
chronically infected HPV patients, as manifested by a reduction in
viral load, e.g., the reduction and/or shrinking of lesions. Such a
study is designed, for example, as follows:
[0412] The studies are performed in multiple centers. The trial
design is an open-label, uncontrolled, dose escalation protocol
wherein the peptide composition is administered as a single dose
followed six weeks later by a single booster shot of the same dose.
The dosages are 50, 500 and 5,000 micrograms per injection.
Drug-associated adverse effects (severity and reversibility) are
recorded.
[0413] There are three patient groupings. The first group is
injected with 50 micrograms of the peptide composition and the
second and third groups with 500 and 5,000 micrograms of peptide
composition, respectively. The patients within each group range in
age from 21-65 and represent diverse ethnic backgrounds. All of
them are infected with HPV and are HIV, HCV, HBV and delta
hepatitis virus (HDV) negative, but are positive for HPV DNA as
monitored by PCR.
[0414] Clinical manifestations or antigen-specific T-cell responses
are monitored to assess the effects of administering the peptide
compositions. The vaccine composition is found to be both safe and
efficacious in the treatment of HPV infection.
Example 20
Induction of CTL Responses Using a Prime Boost Protocol
[0415] A prime boost protocol similar in its underlying principle
to that used to evaluate the efficacy of a DNA vaccine in
transgenic mice, such as described in Example 12, can also be used
for the administration of the vaccine to humans. Such a vaccine
regimen can include an initial administration of, for example,
naked DNA followed by a boost using recombinant virus encoding the
vaccine, or recombinant protein/polypeptide or a peptide mixture
administered in an adjuvant.
[0416] For example, the initial immunization may be performed using
an expression vector, such as that constructed in Example 11, in
the form of naked nucleic acid administered IM (or SC or ID) in the
amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to
1000 .mu.g) can also be administered using a gene gun. Following an
incubation period of 3-4 weeks, a booster dose is then
administered. The booster can be recombinant fowlpox virus
administered at a dose of 5-10.sup.7 to 5.times.10.sup.9 pfu. An
alternative recombinant virus, such as an MVA, canarypox,
adenovirus, or adeno-associated virus, can also be used for the
booster, or the polyepitopic protein or a mixture of the peptides
can be administered. For evaluation of vaccine efficacy, patient
blood samples will be obtained before immunization as well as at
intervals following administration of the initial vaccine and
booster doses of the vaccine. Peripheral blood mononuclear cells
are isolated from fresh heparinized blood by Ficoll-Hypaque density
gradient centrifugation, aliquoted in freezing media and stored
frozen. Samples are assayed for CTL and HTL activity.
[0417] Analysis of the results indicates that a magnitude of
response sufficient to achieve protective immunity against HPV is
generated.
Example 21
Administration of Vaccine Compositions Using Dendritic Cells
(DC)
[0418] Vaccines comprising peptide epitopes of the invention can be
administered using APCs, or "professional" APCs such as DC. In this
example, the peptide-pulsed DC are administered to a patient to
stimulate a CTL response in vivo. In this method, dendritic cells
are isolated, expanded, and pulsed with a vaccine comprising
peptide CTL and HTL epitopes of the invention. The dendritic cells
are infused back into the patient to elicit CTL and HTL responses
in vivo. The induced CTL and HTL then destroy or facilitate
destruction of the specific target cells that bear the proteins
from which the epitopes in the vaccine are derived.
[0419] For example, a cocktail of epitope-bearing peptides is
administered ex vivo to PBMC, or isolated DC therefrom. A
pharmaceutical to facilitate harvesting of DC can be used, such as
Progenipoietin.quadrature. (Monsanto, St. Louis, Mo.) or
GM-CSF/IL-4. After pulsing the DC with peptides and prior to
reinfusion into patients, the DC are washed to remove unbound
peptides.
[0420] As appreciated clinically, and readily determined by one of
skill based on clinical outcomes, the number of DC reinfused into
the patient can vary (see, e.g., Nature Med. 4:328, 1998; Nature
Med. 2:52, 1996 and Prostate 32:272, 1997). Although
2-50.times.10.sup.6 DC per patient are typically administered,
larger number of DC, such as 10.sup.7 or 10.sup.8 can also be
provided. Such cell populations typically contain between 50-90%
DC.
[0421] In some embodiments, peptide-loaded PBMC are injected into
patients without purification of the DC. For example, PBMC
containing DC generated after treatment with an agent such as
Progenipoietin.quadrature. are injected into patients without
purification of the DC. The total number of PBMC that are
administered often ranges from 10.sup.8 to 10.sup.10. Generally,
the cell doses injected into patients is based on the percentage of
DC in the blood of each patient, as determined, for example, by
immunofluorescence analysis with specific anti-DC antibodies. Thus,
for example, if Progenipoietin.TM. mobilizes 2% DC in the
peripheral blood of a given patient, and that patient is to receive
5.times.10.sup.6 DC, then the patient will be injected with a total
of 2.5.times.10.sup.8 peptide-loaded PBMC. The percent DC mobilized
by an agent such as Progenipoietin.TM. is typically estimated to be
between 2-10%, but can vary as appreciated by one of skill in the
art.
Ex Vivo Activation of CTL/HTL Responses
[0422] Alternatively, ex vivo CTL or HTL responses to HPV antigens
can be induced by incubating in tissue culture the patient's, or
genetically compatible, CTL or HTL precursor cells together with a
source of APC, such as DC, and the appropriate immunogenic
peptides. After an appropriate incubation time (typically about
7-28 days), in which the precursor cells are activated and expanded
into effector cells, the cells are infused back into the patient,
where they will destroy (CTL) or facilitate destruction (HTL) of
their specific target cells, i.e., tumor cells.
Example 22
Alternative Method of Identifying Motif-Bearing Peptides
[0423] Another method of identifying motif-bearing peptides is to
elute them from cells bearing defined MHC molecules. For example,
EBV transformed B cell lines used for tissue typing have been
extensively characterized to determine which HLA molecules they
express. In certain cases these cells express only a single type of
HLA molecule. These cells can be infected with a pathogenic
organism or transfected with nucleic acids that express the antigen
of interest, e.g. HPV regulatory or structural proteins. Peptides
produced by endogenous antigen processing of peptides produced
consequent to infection (or as a result of transfection) will then
bind to HLA molecules within the cell and be transported and
displayed on the cell surface. Peptides are then eluted from the
HLA molecules by exposure to mild acid conditions and their amino
acid sequence determined, e.g., by mass spectral analysis (e.g.,
Kubo et al., J. Immunol. 152:3913, 1994). Because the majority of
peptides that bind a particular HLA molecule are motif-bearing,
this is an alternative modality for obtaining the motif-bearing
peptides correlated with the particular HLA molecule expressed on
the cell.
[0424] Alternatively, cell lines that do not express endogenous HLA
molecules can be transfected with an expression construct encoding
a single HLA allele. These cells can then be used as described,
i.e., they can be infected with a pathogen or transfected with
nucleic acid encoding an antigen of interest to isolate peptides
corresponding to the pathogen or antigen of interest that have been
presented on the cell surface. Peptides obtained from such an
analysis will bear motif(s) that correspond to binding to the
single HLA allele that is expressed in the cell.
[0425] As appreciated by one in the art, one can perform a similar
analysis on a cell bearing more than one HLA allele and
subsequently determine peptides specific for each HLA allele
expressed. Moreover, one of skill would also recognize that means
other than infection or transfection, such as loading with a
protein antigen, can be used to provide a source of antigen to the
cell.
[0426] The above examples are provided to illustrate the invention
but not to limit its scope. For example, the human terminology for
the Major Histocompatibility Complex, namely HLA, is used
throughout this document. It is to be appreciated that these
principles can be extended to other species as well. Thus, other
variants of the invention will be readily apparent to one of
ordinary skill in the art and are encompassed by the appended
claims. All publications, patents, and patent application cited
herein are hereby incorporated by reference for all purposes.
Sequence CWU 1
1
88110PRTArtificial SequenceDescription of Artificial
Sequencepeptide epitope HPV 16 protein E1 starting at position 206
1Ala Met Leu Ala Lys Phe Lys Glu Leu Tyr1 5 102649PRTHuman
papillomavirus type 6a 2Met Ala Asp Asp Ser Gly Thr Glu Asn Glu Gly
Ser Gly Cys Thr Gly1 5 10 15Trp Phe Met Val Glu Ala Ile Val Gln His
Pro Thr Gly Thr Gln Ile20 25 30Ser Asp Asp Glu Asp Glu Glu Val Glu
Asp Ser Gly Tyr Asp Met Val35 40 45Asp Phe Ile Asp Asp Ser Asn Ile
Thr His Asn Ser Leu Glu Ala Gln50 55 60Ala Leu Phe Asn Arg Gln Glu
Ala Asp Thr His Tyr Ala Thr Val Gln65 70 75 80Asp Leu Lys Arg Lys
Tyr Leu Gly Ser Pro Tyr Val Ser Pro Ile Asn85 90 95Thr Ile Ala Glu
Ala Val Glu Ser Glu Ile Ser Pro Arg Leu Asp Ala100 105 110Ile Lys
Leu Thr Arg Gln Pro Lys Lys Val Lys Arg Arg Leu Phe Gln115 120
125Thr Arg Glu Leu Thr Asp Ser Gly Tyr Gly Tyr Ser Glu Val Glu
Ala130 135 140Gly Thr Gly Thr Gln Val Glu Lys His Gly Val Pro Glu
Asn Gly Gly145 150 155 160Asp Gly Gln Glu Lys Asp Thr Gly Arg Asp
Ile Glu Gly Glu Glu His165 170 175Thr Glu Ala Glu Ala Pro Thr Asn
Ser Val Arg Glu His Ala Gly Thr180 185 190Ala Gly Ile Leu Glu Leu
Leu Lys Cys Lys Asp Leu Arg Ala Ala Leu195 200 205Leu Gly Lys Phe
Lys Glu Cys Phe Gly Leu Ser Phe Ile Asp Leu Ile210 215 220Arg Pro
Phe Lys Ser Asp Lys Thr Thr Cys Ala Asp Trp Val Val Ala225 230 235
240Gly Phe Gly Ile His His Ser Ile Ser Glu Ala Phe Gln Lys Leu
Ile245 250 255Glu Pro Leu Ser Leu Tyr Ala His Ile Gln Trp Leu Thr
Asn Ala Trp260 265 270Gly Met Val Leu Leu Val Leu Val Arg Phe Lys
Val Asn Lys Ser Arg275 280 285Ser Thr Val Ala Arg Thr Leu Ala Thr
Leu Leu Asn Ile Pro Asp Asn290 295 300Gln Met Leu Ile Glu Pro Pro
Lys Ile Gln Ser Gly Val Ala Ala Leu305 310 315 320Tyr Trp Phe Arg
Thr Gly Ile Ser Asn Ala Ser Thr Val Ile Gly Glu325 330 335Ala Pro
Glu Trp Ile Thr Arg Gln Thr Val Ile Glu His Gly Leu Ala340 345
350Asp Ser Gln Phe Lys Leu Thr Glu Met Val Gln Trp Ala Tyr Asp
Asn355 360 365Asp Ile Cys Glu Glu Ser Glu Ile Ala Phe Glu Tyr Ala
Gln Arg Gly370 375 380Asp Phe Asp Ser Asn Ala Arg Ala Phe Leu Asn
Ser Asn Met Gln Ala385 390 395 400Lys Tyr Val Lys Asp Cys Ala Thr
Met Cys Arg His Tyr Lys His Ala405 410 415Glu Met Arg Lys Met Ser
Ile Lys Gln Trp Ile Lys His Arg Gly Ser420 425 430Lys Ile Glu Gly
Thr Gly Asn Trp Lys Pro Ile Val Gln Phe Leu Arg435 440 445His Gln
Asn Ile Glu Phe Ile Pro Phe Leu Ser Lys Phe Lys Leu Trp450 455
460Leu His Gly Thr Pro Lys Lys Asn Cys Ile Ala Ile Val Gly Pro
Pro465 470 475 480Asp Thr Gly Lys Ser Tyr Phe Cys Met Ser Leu Ile
Ser Phe Leu Gly485 490 495Gly Thr Val Ile Ser His Val Asn Ser Ser
Ser His Phe Trp Leu Gln500 505 510Pro Leu Val Asp Ala Lys Val Ala
Leu Leu Asp Asp Ala Thr Gln Pro515 520 525Cys Trp Ile Tyr Met Asp
Thr Tyr Met Arg Asn Leu Leu Asp Gly Asn530 535 540Pro Met Ser Ile
Asp Arg Lys His Lys Ala Leu Thr Leu Ile Lys Cys545 550 555 560Pro
Pro Leu Leu Val Thr Ser Asn Ile Asp Ile Thr Lys Glu Glu Lys565 570
575Tyr Lys Tyr Leu His Thr Arg Val Thr Thr Phe Thr Phe Pro Asn
Pro580 585 590Phe Pro Phe Asp Arg Asn Gly Asn Ala Val Tyr Glu Leu
Ser Asn Ala595 600 605Asn Trp Lys Cys Phe Phe Glu Arg Leu Ser Ser
Ser Leu Asp Ile Gln610 615 620Asp Ser Glu Asp Glu Glu Asp Gly Ser
Asn Ser Gln Ala Phe Arg Cys625 630 635 640Val Pro Gly Thr Val Val
Arg Thr Leu6453368PRTHuman papillomavirus type 6a 3Met Glu Ala Ile
Ala Lys Arg Leu Asp Ala Cys Gln Glu Gln Leu Leu1 5 10 15Glu Leu Tyr
Glu Glu Asn Ser Thr Asp Leu Asn Lys His Val Leu His20 25 30Trp Lys
Cys Met Arg His Glu Ser Val Leu Leu Tyr Lys Ala Lys Gln35 40 45Met
Gly Leu Ser His Ile Gly Met Gln Val Val Pro Pro Leu Lys Val50 55
60Ser Glu Ala Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu65
70 75 80Ser Leu Leu Lys Thr Glu Tyr Ser Met Glu Pro Trp Thr Leu Gln
Glu85 90 95Thr Ser Tyr Glu Met Trp Gln Thr Pro Pro Lys Arg Cys Phe
Lys Lys100 105 110Arg Gly Lys Thr Val Glu Val Lys Phe Asp Gly Cys
Ala Asn Asn Thr115 120 125Met Asp Tyr Val Val Trp Thr Asp Val Tyr
Val Gln Asp Thr Asp Ser130 135 140Trp Val Lys Val His Ser Met Val
Asp Ala Lys Gly Ile Tyr Tyr Thr145 150 155 160Cys Gly Gln Phe Lys
Thr Tyr Tyr Val Asn Phe Val Lys Glu Ala Glu165 170 175Lys Tyr Gly
Ser Thr Lys Gln Trp Glu Val Cys Tyr Gly Ser Thr Val180 185 190Ile
Cys Ser Pro Ala Ser Val Ser Ser Thr Thr Gln Glu Val Ser Ile195 200
205Pro Glu Ser Thr Thr Tyr Thr Pro Ala Gln Thr Ser Thr Pro Val
Ser210 215 220Ser Ser Thr Gln Glu Asp Ala Val Gln Thr Pro Pro Arg
Lys Arg Ala225 230 235 240Arg Gly Val Gln Gln Ser Pro Cys Asn Ala
Leu Cys Val Ala His Ile245 250 255Gly Pro Val Asp Ser Gly Asn His
Asn Leu Ile Thr Asn Asn His Asp260 265 270Gln His Gln Arg Arg Asn
Asn Ser Asn Ser Ser Ala Thr Pro Ile Val275 280 285Gln Phe Gln Gly
Glu Ser Asn Cys Leu Lys Cys Phe Arg Tyr Arg Leu290 295 300Asn Asp
Lys His Arg His Leu Phe Asp Leu Ile Ser Ser Thr Trp His305 310 315
320Trp Ala Ser Pro Lys Ala Pro His Lys His Ala Ile Val Thr Val
Thr325 330 335Tyr His Ser Glu Glu Gln Arg Gln Gln Phe Leu Asn Val
Val Lys Ile340 345 350Pro Pro Thr Ile Arg His Lys Leu Gly Phe Met
Ser Leu His Leu Leu355 360 365499PRTHuman papillomavirus type 6a
4Met Ala Ala Gln Leu Tyr Val Leu Leu His Leu Tyr Leu Ala Leu His1 5
10 15Lys Lys Tyr Pro Phe Leu Asn Leu Leu His Thr Pro Pro His Arg
Pro20 25 30Pro Pro Leu Cys Pro Gln Ala Pro Arg Lys Thr Gln Cys Lys
Arg Arg35 40 45Leu Glu Asn Glu His Glu Glu Ser Asn Ser His Leu Ala
Thr Pro Cys50 55 60Val Trp Pro Thr Leu Asp Pro Trp Thr Val Glu Thr
Thr Thr Ser Ser65 70 75 80Leu Thr Ile Thr Thr Ser Thr Lys Glu Gly
Thr Thr Val Thr Val Gln85 90 95Leu Arg Leu591PRTHuman
papillomavirus type 6a 5Met Glu Val Val Pro Val Gln Ile Ala Ala Gly
Thr Thr Ser Thr Leu1 5 10 15Ile Leu Pro Val Ile Ile Ala Phe Val Val
Cys Phe Val Ser Ile Ile20 25 30Leu Ile Val Trp Ile Ser Asp Phe Ile
Val Tyr Thr Ser Val Leu Val35 40 45Leu Thr Leu Leu Leu Tyr Leu Leu
Leu Trp Leu Leu Leu Thr Thr Pro50 55 60Leu Gln Phe Phe Leu Leu Thr
Leu Leu Val Cys Tyr Cys Pro Ala Leu65 70 75 80Tyr Ile His His Tyr
Ile Val Asn Thr Gln Gln85 906150PRTHuman papillomavirus type 6a
6Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln Leu1 5
10 15Cys Lys Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys
Val20 25 30Phe Cys Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr
Ala Tyr35 40 45Lys Gln Leu Lys Val Leu Phe Arg Gly Gly Tyr Pro Tyr
Ala Ala Cys50 55 60Ala Cys Cys Leu Glu Phe His Gly Lys Ile Asn Gln
Tyr Arg His Phe65 70 75 80Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu
Glu Glu Thr Lys Gln Asp85 90 95Ile Leu Asp Val Leu Ile Arg Cys Tyr
Leu Cys His Lys Pro Leu Cys100 105 110Glu Val Glu Lys Val Lys His
Ile Leu Thr Lys Ala Arg Phe Ile Lys115 120 125Leu Asn Cys Thr Trp
Lys Gly Arg Cys Leu His Cys Trp Thr Thr Cys130 135 140Met Glu Asp
Met Leu Pro145 150798PRTHuman papillomavirus type 6a 7Met His Gly
Arg His Val Thr Leu Lys Asp Ile Val Leu Asp Leu Gln1 5 10 15Pro Pro
Asp Pro Val Gly Leu His Cys Tyr Glu Gln Leu Val Asp Ser20 25 30Ser
Glu Asp Glu Val Asp Glu Val Asp Gly Gln Asp Ser Gln Pro Leu35 40
45Lys Gln His Phe Gln Ile Val Thr Cys Cys Cys Gly Cys Asp Ser Asn50
55 60Val Arg Leu Val Val Gln Cys Thr Glu Thr Asp Ile Arg Glu Val
Gln65 70 75 80Gln Leu Leu Leu Gly Thr Leu Asp Ile Val Cys Pro Ile
Cys Ala Pro85 90 95Lys Thr8500PRTHuman papillomavirus type 6a 8Met
Trp Arg Pro Ser Asp Ser Thr Val Tyr Val Pro Pro Pro Asn Pro1 5 10
15Val Ser Lys Val Val Ala Thr Asp Ala Tyr Val Thr Arg Thr Asn Ile20
25 30Phe Tyr His Ala Ser Ser Ser Arg Leu Leu Ala Val Gly His Pro
Tyr35 40 45Phe Ser Ile Lys Arg Ala Asn Lys Thr Val Val Pro Lys Val
Ser Gly50 55 60Tyr Gln Tyr Arg Val Phe Lys Val Val Leu Pro Asp Pro
Asn Lys Phe65 70 75 80Ala Leu Pro Asp Ser Ser Leu Phe Asp Pro Thr
Thr Gln Arg Leu Val85 90 95Trp Ala Cys Thr Gly Leu Glu Val Gly Arg
Gly Gln Pro Leu Gly Val100 105 110Gly Val Ser Gly His Pro Phe Leu
Asn Lys Tyr Asp Asp Val Glu Asn115 120 125Ser Gly Ser Gly Gly Asn
Pro Gly Gln Asp Asn Arg Val Asn Val Gly130 135 140Met Asp Tyr Lys
Gln Thr Gln Leu Cys Met Val Gly Cys Ala Pro Pro145 150 155 160Leu
Gly Glu His Trp Gly Lys Gly Lys Gln Cys Thr Asn Thr Pro Val165 170
175Gln Ala Gly Asp Cys Pro Pro Leu Glu Leu Ile Thr Ser Val Ile
Gln180 185 190Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met Asn
Phe Ala Asp195 200 205Leu Gln Thr Asn Lys Ser Asp Val Pro Ile Asp
Ile Cys Gly Thr Thr210 215 220Cys Lys Tyr Pro Asp Tyr Leu Gln Met
Ala Ala Asp Pro Tyr Gly Asp225 230 235 240Arg Leu Phe Phe Phe Leu
Arg Lys Glu Gln Met Phe Ala Arg His Phe245 250 255Phe Asn Arg Ala
Gly Glu Val Gly Glu Pro Val Pro Asp Thr Leu Ile260 265 270Ile Lys
Gly Ser Gly Asn Arg Thr Ser Val Gly Ser Ser Ile Tyr Val275 280
285Asn Thr Pro Ser Gly Ser Leu Val Ser Ser Glu Ala Gln Leu Phe
Asn290 295 300Lys Pro Tyr Trp Leu Gln Lys Ala Gln Gly His Asn Asn
Gly Ile Cys305 310 315 320Trp Gly Asn Gln Leu Phe Val Thr Val Val
Asp Thr Thr Arg Ser Thr325 330 335Asn Met Thr Leu Cys Ala Ser Val
Thr Thr Ser Ser Thr Tyr Thr Asn340 345 350Ser Asp Tyr Lys Glu Tyr
Met Arg His Val Glu Glu Tyr Asp Leu Gln355 360 365Phe Ile Phe Gln
Leu Cys Ser Ile Thr Leu Ser Ala Glu Val Met Ala370 375 380Tyr Ile
His Thr Met Asn Pro Ser Val Leu Glu Asp Trp Asn Phe Gly385 390 395
400Leu Ser Pro Pro Pro Asn Gly Thr Leu Glu Asp Thr Tyr Arg Tyr
Val405 410 415Gln Ser Gln Ala Ile Thr Cys Gln Lys Pro Thr Pro Glu
Lys Glu Lys420 425 430Pro Asp Pro Tyr Lys Asn Leu Ser Phe Trp Glu
Val Asn Leu Lys Glu435 440 445Lys Phe Ser Ser Glu Leu Asp Gln Tyr
Pro Leu Gly Arg Lys Phe Leu450 455 460Leu Gln Ser Gly Tyr Arg Gly
Arg Ser Ser Ile Arg Thr Gly Val Lys465 470 475 480Arg Pro Ala Val
Ser Lys Ala Ser Ala Ala Pro Lys Arg Lys Arg Ala485 490 495Lys Thr
Lys Arg5009459PRTHuman papillomavirus type 6a 9Met Ala His Ser Arg
Ala Arg Arg Arg Lys Arg Ala Ser Ala Thr Gln1 5 10 15Leu Tyr Gln Thr
Cys Lys Leu Thr Gly Thr Cys Pro Pro Asp Val Ile20 25 30Pro Lys Val
Glu His Asn Thr Ile Ala Asp Gln Ile Leu Lys Trp Gly35 40 45Ser Leu
Gly Val Phe Phe Gly Gly Leu Gly Ile Gly Thr Gly Ser Gly50 55 60Thr
Gly Gly Arg Thr Gly Tyr Val Pro Leu Gly Thr Ser Ala Lys Pro65 70 75
80Ser Ile Thr Ser Gly Pro Met Ala Arg Pro Pro Val Val Val Glu Pro85
90 95Val Ala Pro Ser Asp Pro Ser Ile Val Ser Leu Ile Glu Glu Ser
Ala100 105 110Ile Ile Asn Ala Gly Ala Pro Glu Ile Val Pro Pro Ala
His Gly Gly115 120 125Phe Thr Ile Thr Ser Ser Glu Thr Thr Thr Pro
Ala Ile Leu Asp Val130 135 140Ser Val Thr Ser His Thr Thr Thr Ser
Ile Phe Arg Asn Pro Val Phe145 150 155 160Thr Glu Pro Ser Val Thr
Gln Pro Gln Pro Pro Val Glu Ala Asn Gly165 170 175His Ile Leu Ile
Ser Ala Pro Thr Ile Thr Ser His Pro Ile Glu Glu180 185 190Ile Pro
Leu Asp Thr Phe Val Ile Ser Ser Ser Asp Ser Gly Pro Thr195 200
205Ser Ser Thr Pro Val Pro Gly Thr Ala Pro Arg Pro Arg Val Gly
Leu210 215 220Tyr Ser Arg Ala Leu His Gln Val Gln Val Thr Asp Pro
Ala Phe Leu225 230 235 240Ser Thr Pro Gln Arg Leu Ile Thr Tyr Asp
Asn Pro Val Tyr Glu Gly245 250 255Glu Asp Val Ser Val Gln Phe Ser
His Asp Ser Ile His Asn Ala Pro260 265 270Asp Glu Ala Phe Met Asp
Ile Ile Arg Leu His Arg Pro Ala Ile Ala275 280 285Ser Arg Arg Gly
Leu Val Arg Tyr Ser Arg Ile Gly Gln Arg Gly Ser290 295 300Met His
Thr Arg Ser Gly Lys His Ile Gly Ala Arg Ile His Tyr Phe305 310 315
320Tyr Asp Ile Ser Pro Ile Ala Gln Ala Ala Glu Glu Ile Glu Met
His325 330 335Pro Leu Val Ala Ala Gln Asp Asp Thr Phe Asp Ile Tyr
Ala Glu Ser340 345 350Phe Glu Pro Asp Ile Asn Pro Thr Gln His Pro
Val Thr Asn Ile Ser355 360 365Asp Thr Tyr Leu Thr Ser Thr Pro Asn
Thr Val Thr Gln Pro Trp Gly370 375 380Asn Thr Thr Val Pro Leu Ser
Ser Ile Pro Asn Asp Leu Phe Leu Gln385 390 395 400Ser Gly Pro Asp
Ile Thr Phe Pro Thr Ala Pro Met Gly Thr Pro Phe405 410 415Ser Pro
Val Thr Ala Leu Pro Thr Gly Pro Val Phe Ile Thr Gly Ser420 425
430Gly Phe Tyr Leu His Pro Ala Trp Tyr Phe Ala Arg Lys Arg Arg
Lys435 440 445Arg Ile Pro Leu Phe Phe Ser Asp Val Ala Ala450
45510649PRTHuman papillomavirus type 6b 10Met Ala Asp Asp Ser Gly
Thr Glu Asn Glu Gly Ser Gly Cys Thr Gly1 5 10 15Trp Phe Met Val Glu
Ala Ile Val Gln His Pro Thr Gly Thr Gln Ile20 25 30Ser Asp Asp Glu
Asp Glu Glu Val Glu Asp Ser Gly Tyr Asp Met Val35 40 45Asp Phe Ile
Asp Asp Ser Asn Ile Thr His Asn Ser Leu Glu Ala Gln50 55 60Ala Leu
Phe Asn Arg Gln Glu Ala Asp Thr His Tyr Ala Thr Val Gln65 70 75
80Asp Leu Lys Arg Lys Tyr Leu Gly Ser Pro Tyr Val Ser Pro Ile Asn85
90 95Thr Ile Ala Glu Ala Val Glu Ser Glu Ile Ser Pro Arg Leu Asp
Ala100 105 110Ile Lys Leu Thr Arg Gln Pro Lys Lys Val Lys Arg Arg
Leu Phe Gln115 120 125Thr Arg Glu Leu Thr Asp Ser Gly Tyr Gly Tyr
Ser Glu Val Glu Ala130 135 140Gly Thr Gly Thr Gln Val Glu Lys His
Gly Val Pro Glu Asn Gly Gly145 150 155 160Asp Gly Gln Glu Lys Asp
Thr Gly Arg Asp Ile Glu Gly Glu Glu His165 170 175Thr
Glu Ala Glu Ala Pro Thr Asn Ser Val Arg Glu His Ala Gly Thr180 185
190Ala Gly Ile Leu Glu Leu Leu Lys Cys Lys Asp Leu Arg Ala Ala
Leu195 200 205Leu Gly Lys Phe Lys Glu Cys Phe Gly Leu Ser Phe Ile
Asp Leu Ile210 215 220Arg Pro Phe Lys Ser Asp Lys Thr Thr Cys Leu
Asp Trp Val Val Ala225 230 235 240Gly Phe Gly Ile His His Ser Ile
Ser Glu Ala Phe Gln Lys Leu Ile245 250 255Glu Pro Leu Ser Leu Tyr
Ala His Ile Gln Trp Leu Thr Asn Ala Trp260 265 270Gly Met Val Leu
Leu Val Leu Leu Arg Phe Lys Val Asn Lys Ser Arg275 280 285Ser Thr
Val Ala Arg Thr Leu Ala Thr Leu Leu Asn Ile Pro Glu Asn290 295
300Gln Met Leu Ile Glu Pro Pro Lys Ile Gln Ser Gly Val Ala Ala
Leu305 310 315 320Tyr Trp Phe Arg Thr Gly Ile Ser Asn Ala Ser Thr
Val Ile Gly Glu325 330 335Ala Pro Glu Trp Ile Thr Arg Gln Thr Val
Ile Glu His Gly Leu Ala340 345 350Asp Ser Gln Phe Lys Leu Thr Glu
Met Val Gln Trp Ala Tyr Asp Asn355 360 365Asp Ile Cys Glu Glu Ser
Glu Ile Ala Phe Glu Tyr Ala Gln Arg Gly370 375 380Asp Phe Asp Ser
Asn Ala Arg Ala Phe Leu Asn Ser Asn Met Gln Ala385 390 395 400Lys
Tyr Val Lys Asp Cys Ala Thr Met Cys Arg His Tyr Lys His Ala405 410
415Glu Met Arg Lys Met Ser Ile Lys Gln Trp Ile Lys His Arg Gly
Ser420 425 430Lys Ile Glu Gly Thr Gly Asn Trp Lys Pro Ile Val Gln
Phe Leu Arg435 440 445His Gln Asn Ile Glu Phe Ile Pro Phe Leu Thr
Lys Phe Lys Leu Trp450 455 460Leu His Gly Thr Pro Lys Lys Asn Cys
Ile Ala Ile Val Gly Pro Pro465 470 475 480Asp Thr Gly Lys Ser Tyr
Phe Cys Met Ser Leu Ile Ser Phe Leu Gly485 490 495Gly Thr Val Ile
Ser His Val Asn Ser Ser Ser His Phe Trp Leu Gln500 505 510Pro Leu
Val Asp Ala Lys Val Ala Leu Leu Asp Asp Ala Thr Gln Pro515 520
525Cys Trp Ile Tyr Met Asp Thr Tyr Met Arg Asn Leu Leu Asp Gly
Asn530 535 540Pro Met Ser Ile Asp Arg Lys His Lys Ala Leu Thr Leu
Ile Lys Cys545 550 555 560Pro Pro Leu Leu Val Thr Ser Asn Ile Asp
Ile Thr Lys Glu Asp Lys565 570 575Tyr Lys Tyr Leu His Thr Arg Val
Thr Thr Phe Thr Phe Pro Asn Pro580 585 590Phe Pro Phe Asp Arg Asn
Gly Asn Ala Val Tyr Glu Leu Ser Asn Thr595 600 605Asn Trp Lys Cys
Phe Phe Glu Arg Leu Ser Ser Ser Leu Asp Ile Gln610 615 620Asp Ser
Glu Asp Glu Glu Asp Gly Ser Asn Ser Gln Ala Phe Arg Cys625 630 635
640Val Pro Gly Thr Val Val Arg Thr Leu64511368PRTHuman
papillomavirus type 6b 11Met Glu Ala Ile Ala Lys Arg Leu Asp Ala
Cys Gln Glu Gln Leu Leu1 5 10 15Glu Leu Tyr Glu Glu Asn Ser Thr Asp
Leu His Lys His Val Leu His20 25 30Trp Lys Cys Met Arg His Glu Ser
Val Leu Leu Tyr Lys Ala Lys Gln35 40 45Met Gly Leu Ser His Ile Gly
Met Gln Val Val Pro Pro Leu Lys Val50 55 60Ser Glu Ala Lys Gly His
Asn Ala Ile Glu Met Gln Met His Leu Glu65 70 75 80Ser Leu Leu Arg
Thr Glu Tyr Ser Met Glu Pro Trp Thr Leu Gln Glu85 90 95Thr Ser Tyr
Glu Met Trp Gln Thr Pro Pro Lys Arg Cys Phe Lys Lys100 105 110Arg
Gly Lys Thr Val Glu Val Lys Phe Asp Gly Cys Ala Asn Asn Thr115 120
125Met Asp Tyr Val Val Trp Thr Asp Val Tyr Val Gln Asp Asn Asp
Thr130 135 140Trp Val Lys Val His Ser Met Val Asp Ala Lys Gly Ile
Tyr Tyr Thr145 150 155 160Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn
Phe Val Lys Glu Ala Glu165 170 175Lys Tyr Gly Ser Thr Lys His Trp
Glu Val Cys Tyr Gly Ser Thr Val180 185 190Ile Cys Ser Pro Ala Ser
Val Ser Ser Thr Thr Gln Glu Val Ser Ile195 200 205Pro Glu Ser Thr
Thr Tyr Thr Pro Ala Gln Thr Ser Thr Leu Val Ser210 215 220Ser Ser
Thr Lys Glu Asp Ala Val Gln Thr Pro Pro Arg Lys Arg Ala225 230 235
240Arg Gly Val Gln Gln Ser Pro Cys Asn Ala Leu Cys Val Ala His
Ile245 250 255Gly Pro Val Asp Ser Gly Asn His Asn Leu Ile Thr Asn
Asn His Asp260 265 270Gln His Gln Arg Arg Asn Asn Ser Asn Ser Ser
Ala Thr Pro Ile Val275 280 285Gln Phe Gln Gly Glu Ser Asn Cys Leu
Lys Cys Phe Arg Tyr Arg Leu290 295 300Asn Asp Arg His Arg His Leu
Phe Asp Leu Ile Ser Ser Thr Trp His305 310 315 320Trp Ala Ser Ser
Lys Ala Pro His Lys His Ala Ile Val Thr Val Thr325 330 335Tyr Asp
Ser Glu Glu Gln Arg Gln Gln Phe Leu Asp Val Val Lys Ile340 345
350Pro Pro Thr Ile Ser His Lys Leu Gly Phe Met Ser Leu His Leu
Leu355 360 36512109PRTHuman papillomavirus type 6b 12Met Gly Ala
Pro Asn Ile Gly Lys Tyr Val Met Ala Ala Gln Leu Tyr1 5 10 15Val Leu
Leu His Leu Tyr Leu Ala Leu His Lys Lys Tyr Pro Phe Leu20 25 30Asn
Leu Leu His Thr Pro Pro His Arg Pro Pro Pro Leu Cys Pro Gln35 40
45Ala Pro Arg Lys Thr Gln Cys Lys Arg Arg Leu Gly Asn Glu His Glu50
55 60Glu Ser Asn Ser Pro Leu Ala Thr Pro Cys Val Trp Pro Thr Leu
Asp65 70 75 80Pro Trp Thr Val Glu Thr Thr Thr Ser Ser Leu Thr Ile
Thr Thr Ser85 90 95Thr Lys Asp Gly Thr Thr Val Thr Val Gln Leu Arg
Leu100 1051391PRTHuman papillomavirus type 6b 13Met Glu Val Val Pro
Val Gln Ile Ala Ala Gly Thr Thr Ser Thr Phe1 5 10 15Ile Leu Pro Val
Ile Ile Ala Phe Val Val Cys Phe Val Ser Ile Ile20 25 30Leu Ile Val
Trp Ile Ser Glu Phe Ile Val Tyr Thr Ser Val Leu Val35 40 45Leu Thr
Leu Leu Leu Tyr Leu Leu Leu Trp Leu Leu Leu Thr Thr Pro50 55 60Leu
Gln Phe Phe Leu Leu Thr Leu Leu Val Cys Tyr Cys Pro Ala Leu65 70 75
80Tyr Ile His Tyr Tyr Ile Val Thr Thr Gln Gln85 901472PRTHuman
papillomavirus type 6b 14Met Met Leu Thr Cys Gln Phe Asn Asp Gly
Asp Thr Trp Leu Gly Leu1 5 10 15Trp Leu Leu Cys Ala Phe Ile Val Gly
Met Leu Gly Leu Leu Leu Met20 25 30His Tyr Arg Ala Val Gln Gly Asp
Lys His Thr Lys Cys Lys Lys Cys35 40 45Asn Lys His Asn Cys Asn Asp
Asp Tyr Val Thr Met His Tyr Thr Thr50 55 60Asp Gly Asp Tyr Ile Tyr
Met Asn65 7015150PRTHuman papillomavirus type 6b 15Met Glu Ser Ala
Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln Leu1 5 10 15Cys Lys Thr
Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys Val20 25 30Phe Cys
Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr35 40 45Lys
His Leu Lys Val Leu Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys50 55
60Ala Cys Cys Leu Glu Phe His Gly Lys Ile Asn Gln Tyr Arg His Phe65
70 75 80Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu Glu Glu Thr Lys Gln
Asp85 90 95Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His Lys Pro
Leu Cys100 105 110Glu Val Glu Lys Val Lys His Ile Leu Thr Lys Ala
Arg Phe Ile Lys115 120 125Leu Asn Cys Thr Trp Lys Gly Arg Cys Leu
His Cys Trp Thr Thr Cys130 135 140Met Glu Asp Met Leu Pro145
1501698PRTHuman papillomavirus type 6b 16Met His Gly Arg His Val
Thr Leu Lys Asp Ile Val Leu Asp Leu Gln1 5 10 15Pro Pro Asp Pro Val
Gly Leu His Cys Tyr Glu Gln Leu Val Asp Ser20 25 30Ser Glu Asp Glu
Val Asp Glu Val Asp Gly Gln Asp Ser Gln Pro Leu35 40 45Lys Gln His
Phe Gln Ile Val Thr Cys Cys Cys Gly Cys Asp Ser Asn50 55 60Val Arg
Leu Val Val Gln Cys Thr Glu Thr Asp Ile Arg Glu Val Gln65 70 75
80Gln Leu Leu Leu Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro85
90 95Lys Thr17500PRTHuman papillomavirus type 6a 17Met Trp Arg Pro
Ser Asp Ser Thr Val Tyr Val Pro Pro Pro Asn Pro1 5 10 15Val Ser Lys
Val Val Ala Thr Asp Ala Tyr Val Thr Arg Thr Asn Ile20 25 30Phe Tyr
His Ala Ser Ser Ser Arg Leu Leu Ala Val Gly His Pro Tyr35 40 45Phe
Ser Ile Lys Arg Ala Asn Lys Thr Val Val Pro Lys Val Ser Gly50 55
60Tyr Gln Tyr Arg Val Phe Lys Val Val Leu Pro Asp Pro Asn Lys Phe65
70 75 80Ala Leu Pro Asp Ser Ser Leu Phe Asp Pro Thr Thr Gln Arg Leu
Val85 90 95Trp Ala Cys Thr Gly Leu Glu Val Gly Arg Gly Gln Pro Leu
Gly Val100 105 110Gly Val Ser Gly His Pro Phe Leu Asn Lys Tyr Asp
Asp Val Glu Asn115 120 125Ser Gly Ser Gly Gly Asn Pro Gly Gln Asp
Asn Arg Val Asn Val Gly130 135 140Met Asp Tyr Lys Gln Thr Gln Leu
Cys Met Val Gly Cys Ala Pro Pro145 150 155 160Leu Gly Glu His Trp
Gly Lys Gly Lys Gln Cys Thr Asn Thr Pro Val165 170 175Gln Ala Gly
Asp Cys Pro Pro Leu Glu Leu Ile Thr Ser Val Ile Gln180 185 190Asp
Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met Asn Phe Ala Asp195 200
205Leu Gln Thr Asn Lys Ser Asp Val Pro Ile Asp Ile Cys Gly Thr
Thr210 215 220Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ala Ala Asp Pro
Tyr Gly Asp225 230 235 240Arg Leu Phe Phe Phe Leu Arg Lys Glu Gln
Met Phe Ala Arg His Phe245 250 255Phe Asn Arg Ala Gly Glu Val Gly
Glu Pro Val Pro Asp Thr Leu Ile260 265 270Ile Lys Gly Ser Gly Asn
Arg Thr Ser Val Gly Ser Ser Ile Tyr Val275 280 285Asn Thr Pro Ser
Gly Ser Leu Val Ser Ser Glu Ala Gln Leu Phe Asn290 295 300Lys Pro
Tyr Trp Leu Gln Lys Ala Gln Gly His Asn Asn Gly Ile Cys305 310 315
320Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser
Thr325 330 335Asn Met Thr Leu Cys Ala Ser Val Thr Thr Ser Ser Thr
Tyr Thr Asn340 345 350Ser Asp Tyr Lys Glu Tyr Met Arg His Val Glu
Glu Tyr Asp Leu Gln355 360 365Phe Ile Phe Gln Leu Cys Ser Ile Thr
Leu Ser Ala Glu Val Met Ala370 375 380Tyr Ile His Thr Met Asn Pro
Ser Val Leu Glu Asp Trp Asn Phe Gly385 390 395 400Leu Ser Pro Pro
Pro Asn Gly Thr Leu Glu Asp Thr Tyr Arg Tyr Val405 410 415Gln Ser
Gln Ala Ile Thr Cys Gln Lys Pro Thr Pro Glu Lys Glu Lys420 425
430Pro Asp Pro Tyr Lys Asn Leu Ser Phe Trp Glu Val Asn Leu Lys
Glu435 440 445Lys Phe Ser Ser Glu Leu Asp Gln Tyr Pro Leu Gly Arg
Lys Phe Leu450 455 460Leu Gln Ser Gly Tyr Arg Gly Arg Ser Ser Ile
Arg Thr Gly Val Lys465 470 475 480Arg Pro Ala Val Ser Lys Ala Ser
Ala Ala Pro Lys Arg Lys Arg Ala485 490 495Lys Thr Lys
Arg50018459PRTHuman papillomavirus type 6b 18Met Ala His Ser Arg
Ala Arg Arg Arg Lys Arg Ala Ser Ala Thr Gln1 5 10 15Leu Tyr Gln Thr
Cys Lys Leu Thr Gly Thr Cys Pro Pro Asp Val Ile20 25 30Pro Lys Val
Glu His Asn Thr Ile Ala Asp Gln Ile Leu Lys Trp Gly35 40 45Ser Leu
Gly Val Phe Phe Gly Gly Leu Gly Ile Gly Thr Gly Ser Gly50 55 60Thr
Gly Gly Arg Thr Gly Tyr Val Pro Leu Gln Thr Ser Ala Lys Pro65 70 75
80Ser Ile Thr Ser Gly Pro Met Ala Arg Pro Pro Val Val Val Glu Pro85
90 95Val Ala Pro Ser Asp Pro Ser Ile Val Ser Leu Ile Glu Glu Ser
Ala100 105 110Ile Ile Asn Ala Gly Ala Pro Glu Ile Val Pro Pro Ala
His Gly Gly115 120 125Phe Thr Ile Thr Ser Ser Glu Thr Thr Thr Pro
Ala Ile Leu Asp Val130 135 140Ser Val Thr Ser His Thr Thr Thr Ser
Ile Phe Arg Asn Pro Val Phe145 150 155 160Thr Glu Pro Ser Val Thr
Gln Pro Gln Pro Pro Val Glu Ala Asn Gly165 170 175His Ile Leu Ile
Ser Ala Pro Thr Val Thr Ser His Pro Ile Glu Glu180 185 190Ile Pro
Leu Asp Thr Phe Val Val Ser Ser Ser Asp Ser Gly Pro Thr195 200
205Ser Ser Thr Pro Val Pro Gly Thr Ala Pro Arg Pro Arg Val Gly
Leu210 215 220Tyr Ser Arg Ala Leu His Gln Val Gln Val Thr Asp Pro
Ala Phe Leu225 230 235 240Ser Thr Pro Gln Arg Leu Ile Thr Tyr Asp
Asn Pro Val Tyr Glu Gly245 250 255Glu Asp Val Ser Val Gln Phe Ser
His Asp Ser Ile His Asn Ala Pro260 265 270Asp Glu Ala Phe Met Asp
Ile Ile Arg Leu His Arg Pro Ala Ile Ala275 280 285Ser Arg Arg Gly
Leu Val Arg Tyr Ser Arg Ile Gly Gln Arg Gly Ser290 295 300Met His
Thr Arg Ser Gly Lys His Ile Gly Ala Arg Ile His Tyr Phe305 310 315
320Tyr Asp Ile Ser Pro Ile Ala Gln Ala Ala Glu Glu Ile Glu Met
His325 330 335Pro Leu Val Ala Ala Gln Asp Asp Thr Phe Asp Ile Tyr
Ala Glu Ser340 345 350Phe Glu Pro Gly Ile Asn Pro Thr Gln His Pro
Val Thr Asn Ile Ser355 360 365Asp Thr Tyr Leu Thr Ser Thr Pro Asn
Thr Val Thr Gln Pro Trp Gly370 375 380Asn Thr Thr Val Pro Leu Ser
Leu Pro Asn Asp Leu Phe Leu Gln Ser385 390 395 400Gly Pro Asp Ile
Thr Phe Pro Thr Ala Pro Met Gly Thr Pro Phe Ser405 410 415Pro Val
Thr Pro Ala Leu Pro Thr Gly Pro Val Phe Ile Thr Gly Ser420 425
430Gly Phe Tyr Leu His Pro Ala Trp Tyr Phe Ala Arg Lys Arg Arg
Lys435 440 445Arg Ile Pro Leu Phe Phe Ser Asp Val Ala Ala450
45519649PRTHuman papillomavirus type 11 19Met Ala Asp Asp Ser Gly
Thr Glu Asn Glu Gly Ser Gly Cys Thr Gly1 5 10 15Trp Phe Met Val Glu
Ala Ile Val Glu His Thr Thr Gly Thr Gln Ile20 25 30Ser Glu Asp Glu
Glu Glu Glu Val Glu Asp Ser Gly Tyr Asp Met Val35 40 45Asp Phe Ile
Asp Asp Arg His Ile Thr Gln Asn Ser Val Glu Ala Gln50 55 60Ala Leu
Phe Asn Arg Gln Glu Ala Asp Ala His Tyr Ala Thr Val Gln65 70 75
80Asp Leu Lys Arg Lys Tyr Leu Gly Ser Pro Tyr Val Ser Pro Ile Ser85
90 95Asn Val Ala Asn Ala Val Glu Ser Glu Ile Ser Pro Arg Leu Asp
Ala100 105 110Ile Lys Leu Thr Thr Gln Pro Lys Lys Val Lys Arg Arg
Leu Phe Glu115 120 125Thr Arg Glu Leu Thr Asp Ser Gly Tyr Gly Tyr
Ser Glu Val Glu Ala130 135 140Ala Thr Gln Val Glu Lys His Gly Asp
Pro Glu Asn Gly Gly Asp Gly145 150 155 160Gln Glu Arg Asp Thr Gly
Arg Asp Ile Glu Gly Glu Gly Val Glu His165 170 175Arg Glu Ala Glu
Ala Val Asp Asp Ser Thr Arg Glu His Ala Asp Thr180 185 190Ser Gly
Ile Leu Glu Leu Leu Lys Cys Lys Asp Ile Arg Ser Thr Leu195 200
205His Gly Lys Phe Lys Asp Cys Phe Gly Leu Ser Phe Val Asp Leu
Ile210 215 220Arg Pro Phe Lys Ser Asp Arg Thr Thr Cys Ala Asp Trp
Val Val Ala225 230 235 240Gly Phe Gly Ile His His Ser Ile Ala Asp
Ala Phe Gln Lys Leu Ile245 250 255Glu Pro Leu Ser Leu Tyr Ala His
Ile Gln Trp Leu Thr Asn Ala Trp260 265 270Gly Met Val Leu Leu Val
Leu Ile Arg Phe Lys Val Asn Lys Ser Arg275 280 285Cys Thr Val Ala
Arg Thr Leu Gly Thr Leu Leu Asn Ile Pro Glu Asn290 295
300His Met Leu Ile Glu Pro Pro Lys Ile Gln Ser Gly Val Arg Ala
Leu305 310 315 320Tyr Trp Phe Arg Thr Gly Ile Ser Asn Ala Ser Thr
Val Ile Gly Glu325 330 335Ala Pro Glu Trp Ile Thr Arg Gln Thr Val
Ile Glu His Ser Leu Ala340 345 350Asp Ser Gln Phe Lys Leu Thr Glu
Met Val Gln Trp Ala Tyr Asp Asn355 360 365Asp Ile Cys Glu Glu Ser
Glu Ile Ala Phe Glu Tyr Ala Gln Arg Gly370 375 380Asp Phe Asp Ser
Asn Ala Arg Ala Phe Leu Asn Ser Asn Met Gln Ala385 390 395 400Lys
Tyr Val Lys Asp Cys Ala Ile Met Cys Arg His Tyr Lys His Ala405 410
415Glu Met Lys Lys Met Ser Ile Lys Gln Trp Ile Lys Tyr Arg Gly
Thr420 425 430Lys Val Asp Ser Val Gly Asn Trp Lys Pro Ile Val Gln
Phe Leu Arg435 440 445His Gln Asn Ile Glu Phe Ile Pro Phe Leu Ser
Lys Leu Lys Leu Trp450 455 460Leu His Gly Thr Pro Lys Lys Asn Cys
Ile Ala Ile Val Gly Pro Pro465 470 475 480Asp Thr Gly Lys Ser Cys
Phe Cys Met Ser Leu Ile Lys Phe Leu Gly485 490 495Gly Thr Val Ile
Ser Tyr Val Asn Ser Cys Ser His Phe Trp Leu Gln500 505 510Pro Leu
Thr Asp Ala Lys Val Ala Leu Leu Asp Asp Ala Thr Gln Pro515 520
525Cys Trp Thr Tyr Met Asp Thr Tyr Met Arg Asn Leu Leu Asp Gly
Asn530 535 540Pro Met Ser Ile Asp Arg Lys His Arg Ala Leu Thr Leu
Ile Lys Cys545 550 555 560Pro Pro Leu Leu Val Thr Ser Asn Ile Asp
Ile Ser Lys Glu Glu Lys565 570 575Tyr Lys Tyr Leu His Ser Arg Val
Thr Thr Phe Thr Phe Pro Asn Pro580 585 590Phe Pro Phe Asp Arg Asn
Gly Asn Ala Val Tyr Glu Leu Ser Asp Ala595 600 605Asn Trp Lys Cys
Phe Phe Glu Arg Leu Ser Ser Ser Leu Asp Ile Glu610 615 620Asp Ser
Glu Asp Glu Glu Asp Gly Ser Asn Ser Gln Ala Phe Arg Cys625 630 635
640Val Pro Gly Ser Val Val Arg Thr Leu64520367PRTHuman
papillomavirus type 11 20Met Glu Ala Ile Ala Lys Arg Leu Asp Ala
Cys Gln Asp Gln Leu Leu1 5 10 15Glu Leu Tyr Glu Glu Asn Ser Ile Asp
Ile His Lys His Ile Met His20 25 30Trp Lys Cys Ile Arg Leu Glu Ser
Val Leu Leu His Lys Ala Lys Gln35 40 45Met Gly Leu Ser His Ile Gly
Leu Gln Val Val Pro Pro Leu Thr Val50 55 60Ser Glu Thr Lys Gly His
Asn Ala Ile Glu Met Gln Met His Leu Glu65 70 75 80Ser Leu Ala Lys
Thr Gln Tyr Gly Val Glu Pro Trp Thr Leu Gln Asp85 90 95Thr Ser Tyr
Glu Met Trp Leu Thr Pro Pro Lys Arg Cys Phe Lys Lys100 105 110Gln
Gly Asn Thr Val Glu Val Lys Phe Asp Gly Cys Glu Asp Asn Val115 120
125Met Glu Tyr Val Val Trp Thr His Ile Tyr Leu Gln Asp Asn Asp
Ser130 135 140Trp Val Lys Val Thr Ser Ser Val Asp Ala Lys Gly Ile
Tyr Tyr Thr145 150 155 160Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn
Phe Asn Lys Glu Ala Gln165 170 175Lys Tyr Gly Ser Thr Asn His Trp
Glu Val Cys Tyr Gly Ser Thr Val180 185 190Ile Cys Ser Pro Ala Ser
Val Ser Ser Thr Val Arg Glu Val Ser Ile195 200 205Ala Glu Pro Thr
Thr Tyr Thr Pro Ala Gln Thr Thr Ala Pro Thr Val210 215 220Ser Ala
Cys Thr Thr Glu Asp Gly Val Ser Ala Pro Pro Arg Lys Arg225 230 235
240Ala Arg Gly Pro Ser Thr Asn Asn Thr Leu Cys Val Ala Asn Ile
Arg245 250 255Ser Val Asp Ser Thr Ile Asn Asn Ile Val Thr Asp Asn
Tyr Asn Lys260 265 270His Gln Arg Arg Asn Asn Cys His Ser Ala Ala
Thr Pro Ile Val Gln275 280 285Leu Gln Gly Asp Ser Asn Cys Leu Lys
Cys Phe Arg Tyr Arg Leu Asn290 295 300Asp Lys Tyr Lys His Leu Phe
Glu Leu Ala Ser Ser Thr Trp His Trp305 310 315 320Ala Ser Pro Glu
Ala Pro His Lys Asn Ala Ile Val Thr Leu Thr Tyr325 330 335Ser Ser
Glu Glu Gln Arg Gln Gln Phe Leu Asn Ser Val Lys Ile Pro340 345
350Pro Thr Ile Arg His Lys Val Gly Phe Met Ser Leu His Leu Leu355
360 36521108PRTHuman papillomavirus type 11 21Met Val Val Pro Ile
Ile Gly Lys Tyr Val Met Ala Ala Gln Leu Tyr1 5 10 15Val Leu Leu His
Leu Tyr Leu Ala Leu Tyr Glu Lys Tyr Pro Leu Leu20 25 30Asn Leu Leu
His Thr Pro Pro His Arg Pro Pro Pro Leu Gln Cys Pro35 40 45Pro Ala
Pro Arg Lys Thr Ala Cys Arg Arg Arg Leu Gly Ser Glu His50 55 60Val
Asp Arg Pro Leu Thr Thr Pro Cys Val Trp Pro Thr Ser Asp Pro65 70 75
80Trp Thr Val Gln Ser Thr Thr Ser Ser Leu Thr Ile Thr Thr Ser Thr85
90 95Lys Glu Gly Thr Thr Val Thr Val Gln Leu Arg Leu100
1052291PRTHuman papillomavirus type 11 22Met Glu Val Val Pro Val
Gln Ile Ala Ala Ala Thr Thr Thr Thr Leu1 5 10 15Ile Leu Pro Val Val
Ile Ala Phe Ala Val Cys Ile Leu Ser Ile Val20 25 30Leu Ile Ile Leu
Ile Ser Asp Phe Val Val Tyr Thr Ser Val Leu Val35 40 45Leu Thr Leu
Leu Leu Tyr Leu Leu Leu Trp Leu Leu Leu Thr Thr Pro50 55 60Leu Gln
Phe Phe Leu Leu Thr Leu Cys Val Cys Tyr Phe Pro Ala Phe65 70 75
80Tyr Ile His Ile Tyr Ile Val Gln Thr Gln Gln85 902374PRTHuman
papillomavirus type 11 23Met Val Met Leu Thr Cys His Leu Asn Asp
Gly Asp Thr Trp Leu Phe1 5 10 15Leu Trp Leu Phe Thr Ala Phe Val Val
Ala Val Leu Gly Leu Leu Leu20 25 30Leu His Tyr Arg Ala Val His Gly
Thr Glu Lys Thr Lys Cys Ala Lys35 40 45Cys Lys Ser Asn Arg Asn Thr
Thr Val Asp Tyr Val Tyr Met Ser His50 55 60Gly Asp Asn Gly Asp Tyr
Val Tyr Met Asn65 7024150PRTHuman papillomavirus type 11 24Met Glu
Ser Lys Asp Ala Ser Thr Ser Ala Thr Ser Ile Asp Gln Leu1 5 10 15Cys
Lys Thr Phe Asn Leu Ser Leu His Thr Leu Gln Ile Gln Cys Val20 25
30Phe Cys Arg Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ala Tyr Ala Tyr35
40 45Lys Asn Leu Lys Val Val Trp Arg Asp Asn Phe Pro Phe Ala Ala
Cys50 55 60Ala Cys Cys Leu Glu Leu Gln Gly Lys Ile Asn Gln Tyr Arg
His Phe65 70 75 80Asn Tyr Ala Ala Tyr Ala Pro Thr Val Glu Glu Glu
Thr Asn Glu Asp85 90 95Ile Leu Lys Val Leu Ile Arg Cys Tyr Leu Cys
His Lys Pro Leu Cys100 105 110Glu Ile Glu Lys Leu Lys His Ile Leu
Gly Lys Ala Arg Phe Ile Lys115 120 125Leu Asn Asn Gln Trp Lys Gly
Arg Cys Leu His Cys Trp Thr Thr Cys130 135 140Met Glu Asp Leu Leu
Pro145 1502598PRTHuman papillomavirus type 11 25Met His Gly Arg Leu
Val Thr Leu Lys Asp Ile Val Leu Asp Leu Gln1 5 10 15Pro Pro Asp Pro
Val Gly Leu His Cys Tyr Glu Gln Leu Glu Asp Ser20 25 30Ser Glu Asp
Glu Val Asp Lys Val Asp Lys Gln Asp Ala Gln Pro Leu35 40 45Thr Gln
His Tyr Gln Ile Leu Thr Cys Cys Cys Gly Cys Asp Ser Asn50 55 60Val
Arg Leu Val Val Glu Cys Thr Asp Gly Asp Ile Arg Gln Leu Gln65 70 75
80Asp Leu Leu Leu Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro85
90 95Lys Pro26501PRTHuman papillomavirus type 11 26Met Trp Arg Pro
Ser Asp Ser Thr Val Tyr Val Pro Pro Pro Asn Pro1 5 10 15Val Ser Lys
Val Val Ala Thr Asp Ala Tyr Val Lys Arg Thr Asn Ile20 25 30Phe Tyr
His Ala Ser Ser Ser Arg Leu Leu Ala Val Gly His Pro Tyr35 40 45Tyr
Ser Ile Lys Lys Val Asn Lys Thr Val Val Pro Lys Val Ser Gly50 55
60Tyr Gln Tyr Arg Val Phe Lys Val Val Leu Pro Asp Pro Asn Lys Phe65
70 75 80Ala Leu Pro Asp Ser Ser Leu Phe Asp Pro Thr Thr Gln Arg Leu
Val85 90 95Trp Ala Cys Thr Gly Leu Glu Val Gly Arg Gly Gln Pro Leu
Gly Val100 105 110Gly Val Ser Gly His Pro Leu Leu Asn Lys Tyr Asp
Asp Val Glu Asn115 120 125Ser Gly Gly Tyr Gly Gly Asn Pro Gly Gln
Asp Asn Arg Val Asn Val130 135 140Gly Met Asp Tyr Lys Gln Thr Gln
Leu Cys Met Val Gly Cys Ala Pro145 150 155 160Pro Leu Gly Glu His
Trp Gly Lys Gly Thr Gln Cys Ser Asn Thr Ser165 170 175Val Gln Asn
Gly Asp Cys Pro Pro Leu Glu Leu Ile Thr Ser Val Ile180 185 190Gln
Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met Asn Phe Ala195 200
205Asp Leu Gln Thr Asn Lys Ser Asp Val Pro Leu Asp Ile Cys Gly
Thr210 215 220Val Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ala Ala Asp
Pro Tyr Gly225 230 235 240Asp Arg Leu Phe Phe Tyr Leu Arg Lys Glu
Gln Met Phe Ala Arg His245 250 255Phe Phe Asn Arg Ala Gly Thr Val
Gly Glu Pro Val Pro Asp Asp Leu260 265 270Leu Val Lys Gly Gly Asn
Asn Arg Ser Ser Val Ala Ser Ser Ile Tyr275 280 285Val His Thr Pro
Ser Gly Ser Leu Val Ser Ser Glu Ala Gln Leu Phe290 295 300Asn Lys
Pro Tyr Trp Leu Gln Lys Ala Gln Gly His Asn Asn Gly Ile305 310 315
320Cys Trp Gly Asn His Leu Phe Val Thr Val Val Asp Thr Thr Arg
Ser325 330 335Thr Asn Met Thr Leu Cys Ala Ser Val Ser Lys Ser Ala
Thr Tyr Thr340 345 350Asn Ser Asp Tyr Lys Glu Tyr Met Arg His Val
Glu Glu Phe Asp Leu355 360 365Gln Phe Ile Phe Gln Leu Cys Ser Ile
Thr Leu Ser Ala Glu Val Met370 375 380Ala Tyr Ile His Thr Met Asn
Pro Ser Val Leu Glu Asp Trp Asn Phe385 390 395 400Gly Leu Ser Pro
Pro Pro Asn Gly Thr Leu Glu Asp Thr Tyr Arg Tyr405 410 415Val Gln
Ser Gln Ala Ile Thr Cys Gln Lys Pro Thr Pro Glu Lys Glu420 425
430Lys Gln Asp Pro Tyr Lys Asp Met Ser Phe Trp Glu Val Asn Leu
Lys435 440 445Glu Lys Phe Ser Ser Glu Leu Asp Gln Phe Pro Leu Gly
Arg Lys Phe450 455 460Leu Leu Gln Ser Gly Tyr Arg Gly Arg Thr Ser
Ala Arg Thr Gly Ile465 470 475 480Lys Arg Pro Ala Val Ser Lys Pro
Ser Thr Ala Pro Lys Arg Lys Arg485 490 495Thr Lys Thr Lys
Lys50027455PRTHuman papillomavirus type 11 27Met Lys Pro Arg Ala
Arg Arg Arg Lys Arg Ala Ser Ala Thr Gln Leu1 5 10 15Tyr Gln Thr Cys
Lys Ala Thr Gly Thr Cys Pro Pro Asp Val Ile Pro20 25 30Lys Val Glu
His Thr Thr Ile Ala Asp Gln Ile Leu Lys Trp Gly Ser35 40 45Leu Gly
Val Phe Phe Gly Gly Leu Gly Ile Gly Thr Gly Ala Gly Ser50 55 60Gly
Gly Arg Ala Gly Tyr Ile Pro Leu Gly Ser Ser Pro Lys Pro Ala65 70 75
80Ile Thr Gly Gly Pro Ala Ala Arg Pro Pro Val Leu Val Glu Pro Val85
90 95Ala Pro Ser Asp Pro Ser Ile Val Ser Leu Ile Glu Glu Ser Ala
Ile100 105 110Ile Asn Ala Gly Ala Pro Glu Val Val Pro Pro Thr Gln
Gly Gly Phe115 120 125Thr Ile Thr Ser Ser Glu Ser Thr Thr Pro Ala
Ile Leu Asp Val Ser130 135 140Val Thr Asn His Thr Thr Thr Ser Val
Phe Gln Asn Pro Leu Phe Thr145 150 155 160Glu Pro Ser Val Ile Gln
Pro Gln Pro Pro Val Glu Ala Ser Gly His165 170 175Ile Leu Ile Ser
Ala Pro Thr Ile Thr Ser Gln His Val Glu Asp Ile180 185 190Pro Leu
Asp Thr Phe Val Val Ser Ser Ser Asp Ser Gly Pro Thr Ser195 200
205Ser Thr Pro Leu Pro Arg Ala Phe Pro Arg Pro Arg Val Gly Leu
Tyr210 215 220Ser Arg Ala Leu Gln Gln Val Gln Val Thr Asp Pro Ala
Phe Leu Ser225 230 235 240Thr Pro Gln Arg Leu Val Thr Tyr Asp Asn
Pro Val Tyr Glu Gly Glu245 250 255Asp Val Ser Leu Gln Phe Thr His
Glu Ser Ile His Asn Ala Pro Asp260 265 270Glu Ala Phe Met Asp Ile
Ile Arg Leu His Arg Pro Ala Ile Thr Ser275 280 285Arg Arg Gly Leu
Val Arg Phe Ser Arg Ile Gly Gln Arg Gly Ser Met290 295 300Tyr Thr
Arg Ser Gly Gln His Ile Gly Ala Arg Ile His Tyr Phe Gln305 310 315
320Asp Ile Ser Pro Val Thr Gln Ala Ala Glu Glu Ile Glu Leu His
Pro325 330 335Leu Val Ala Ala Glu Asn Asp Thr Phe Asp Ile Tyr Ala
Glu Pro Phe340 345 350Asp Pro Ile Pro Asp Pro Val Gln His Ser Val
Thr Gln Ser Tyr Leu355 360 365Thr Ser Thr Pro Asn Thr Leu Ser Gln
Ser Trp Gly Asn Thr Thr Val370 375 380Pro Leu Ser Ile Pro Ser Asp
Trp Phe Val Gln Ser Gly Pro Asp Ile385 390 395 400Thr Phe Pro Thr
Ala Ser Met Gly Thr Pro Phe Ser Pro Val Thr Pro405 410 415Ala Leu
Pro Thr Gly Pro Val Phe Ile Thr Gly Ser Asp Phe Tyr Leu420 425
430His Pro Thr Trp Tyr Phe Ala Arg Arg Arg Arg Lys Arg Ile Pro
Leu435 440 445Phe Phe Thr Asp Val Ala Ala450 45528649PRTHuman
papillomavirus type 16 28Met Ala Asp Pro Ala Gly Thr Asn Gly Glu
Glu Gly Thr Gly Cys Asn1 5 10 15Gly Trp Phe Tyr Val Glu Ala Val Val
Glu Lys Lys Thr Gly Asp Ala20 25 30Ile Ser Asp Asp Glu Asn Glu Asn
Asp Ser Asp Thr Gly Glu Asp Leu35 40 45Val Asp Phe Ile Val Asn Asp
Asn Asp Tyr Leu Thr Gln Ala Glu Thr50 55 60Glu Thr Ala His Ala Leu
Phe Thr Ala Gln Glu Ala Lys Gln His Arg65 70 75 80Asp Ala Val Gln
Val Leu Lys Arg Lys Tyr Leu Val Ser Pro Leu Ser85 90 95Asp Ile Ser
Gly Cys Val Asp Asn Asn Ile Ser Pro Arg Leu Lys Ala100 105 110Ile
Cys Ile Glu Lys Gln Ser Arg Ala Ala Lys Arg Arg Leu Phe Glu115 120
125Ser Glu Asp Ser Gly Tyr Gly Asn Thr Glu Val Glu Thr Gln Gln
Met130 135 140Leu Gln Val Glu Gly Arg His Glu Thr Glu Thr Pro Cys
Ser Gln Tyr145 150 155 160Ser Gly Gly Ser Gly Gly Gly Cys Ser Gln
Tyr Ser Ser Gly Ser Gly165 170 175Gly Glu Gly Val Ser Glu Arg His
Thr Ile Cys Gln Thr Pro Leu Thr180 185 190Asn Ile Leu Asn Val Leu
Lys Thr Ser Asn Ala Lys Ala Ala Met Leu195 200 205Ala Lys Phe Lys
Glu Leu Tyr Gly Val Ser Phe Ser Glu Leu Val Arg210 215 220Pro Phe
Lys Ser Asn Lys Ser Thr Cys Cys Asp Trp Cys Ile Ala Ala225 230 235
240Phe Gly Leu Thr Pro Ser Ile Ala Asp Ser Ile Lys Thr Leu Leu
Gln245 250 255Gln Tyr Cys Leu Tyr Leu His Ile Gln Ser Leu Ala Cys
Ser Trp Gly260 265 270Met Val Val Leu Leu Leu Val Arg Tyr Lys Cys
Gly Lys Asn Arg Glu275 280 285Thr Ile Glu Lys Leu Leu Ser Lys Leu
Leu Cys Val Ser Pro Met Cys290 295 300Met Met Ile Glu Pro Pro Lys
Leu Arg Ser Thr Ala Ala Ala Leu Tyr305 310 315 320Trp Tyr Lys Thr
Gly Ile Ser Asn Ile Ser Glu Val Tyr Gly Asp Thr325 330 335Pro Glu
Trp Ile Gln Arg Gln Thr Val Leu Gln His Ser Phe Asn Asp340 345
350Cys Thr Phe Glu Leu Ser Gln Met Val Gln Trp Ala Tyr Asp Asn
Asp355 360 365Ile Val Asp Asp Ser Glu Ile Ala Tyr Lys Tyr Ala Gln
Leu Ala Asp370 375 380Thr Asn Ser Asn Ala Ser Ala Phe Leu Lys Ser
Asn Ser Gln Ala Lys385 390 395 400Ile Val Lys Asp Cys Ala Thr Met
Cys Arg His Tyr Lys Arg Ala Glu405 410 415Lys Lys Gln Met Ser Met
Ser Gln Trp Ile Lys Tyr Arg Cys Asp Arg420
425 430Val Asp Asp Gly Gly Asp Trp Lys Gln Ile Val Met Phe Leu Arg
Tyr435 440 445Gln Gly Val Glu Phe Met Ser Phe Leu Thr Ala Leu Lys
Arg Phe Leu450 455 460Gln Gly Ile Pro Lys Lys Asn Cys Ile Leu Leu
Tyr Gly Ala Ala Asn465 470 475 480Thr Gly Lys Ser Leu Phe Gly Met
Ser Leu Met Lys Phe Leu Gln Gly485 490 495Ser Val Ile Cys Phe Val
Asn Ser Lys Ser His Phe Trp Leu Gln Pro500 505 510Leu Ala Asp Ala
Lys Ile Gly Met Leu Asp Asp Ala Thr Val Pro Cys515 520 525Trp Asn
Tyr Ile Asp Asp Asn Leu Arg Asn Ala Leu Asp Gly Asn Leu530 535
540Val Ser Met Asp Val Lys His Arg Pro Leu Val Gln Leu Lys Cys
Pro545 550 555 560Pro Leu Leu Ile Thr Ser Asn Ile Asn Ala Gly Thr
Asp Ser Arg Trp565 570 575Pro Tyr Leu His Asn Arg Leu Val Val Phe
Thr Phe Pro Asn Glu Phe580 585 590Pro Phe Asp Glu Asn Gly Asn Pro
Val Tyr Glu Leu Asn Asp Lys Asn595 600 605Trp Lys Ser Phe Phe Ser
Arg Thr Trp Ser Arg Leu Ser Leu His Glu610 615 620Asp Glu Asp Lys
Glu Asn Asp Gly Asp Ser Leu Pro Thr Phe Lys Cys625 630 635 640Val
Ser Gly Gln Asn Thr Asn Thr Leu64529365PRTHuman papillomavirus type
16 29Met Glu Thr Leu Cys Gln Arg Leu Asn Val Cys Gln Asp Lys Ile
Leu1 5 10 15Thr His Tyr Glu Asn Asp Ser Thr Asp Leu Arg Asp His Ile
Asp Tyr20 25 30Trp Lys His Met Arg Leu Glu Cys Ala Ile Tyr Tyr Lys
Ala Arg Glu35 40 45Met Gly Phe Lys His Ile Asn His Gln Val Val Pro
Thr Leu Ala Val50 55 60Ser Lys Asn Lys Ala Leu Gln Ala Ile Glu Leu
Gln Leu Thr Leu Glu65 70 75 80Thr Ile Tyr Asn Ser Gln Tyr Ser Asn
Glu Lys Trp Thr Leu Gln Asp85 90 95Val Ser Leu Glu Val Tyr Leu Thr
Ala Pro Thr Gly Cys Ile Lys Lys100 105 110His Gly Tyr Thr Val Glu
Val Gln Phe Asp Gly Asp Ile Cys Asn Thr115 120 125Met His Tyr Thr
Asn Trp Thr His Ile Tyr Ile Cys Glu Glu Ala Ser130 135 140Val Thr
Val Val Glu Gly Gln Val Asp Tyr Tyr Gly Leu Tyr Tyr Val145 150 155
160His Glu Gly Ile Arg Thr Tyr Phe Val Gln Phe Lys Asp Asp Ala
Glu165 170 175Lys Tyr Ser Lys Asn Lys Val Trp Glu Val His Ala Gly
Gly Gln Val180 185 190Ile Leu Cys Pro Thr Ser Val Phe Ser Ser Asn
Glu Val Ser Ser Pro195 200 205Glu Ile Ile Arg Gln His Leu Ala Asn
His Pro Ala Ala Thr His Thr210 215 220Lys Ala Val Ala Leu Gly Thr
Glu Glu Thr Gln Thr Thr Ile Gln Arg225 230 235 240Pro Arg Ser Glu
Pro Asp Thr Gly Asn Pro Cys His Thr Thr Lys Leu245 250 255Leu His
Arg Asp Ser Val Asp Ser Ala Pro Ile Leu Thr Ala Phe Asn260 265
270Ser Ser His Lys Gly Arg Ile Asn Cys Asn Ser Asn Thr Thr Pro
Ile275 280 285Val His Leu Lys Gly Asp Ala Asn Thr Leu Lys Cys Leu
Arg Tyr Arg290 295 300Phe Lys Lys His Cys Thr Leu Tyr Thr Ala Val
Ser Ser Thr Trp His305 310 315 320Trp Thr Gly His Asn Val Lys His
Lys Ser Ala Ile Val Thr Leu Thr325 330 335Tyr Asp Ser Glu Trp Gln
Arg Asp Gln Phe Leu Ser Gln Val Lys Ile340 345 350Pro Lys Thr Ile
Thr Val Ser Thr Gly Phe Met Ser Ile355 360 3653083PRTHuman
papillomavirus type 16 30Met Thr Asn Leu Asp Thr Ala Ser Thr Thr
Leu Leu Ala Cys Phe Leu1 5 10 15Leu Cys Phe Cys Val Leu Leu Cys Val
Cys Leu Leu Ile Arg Pro Leu20 25 30Leu Leu Ser Val Ser Thr Tyr Thr
Ser Leu Ile Ile Leu Val Leu Leu35 40 45Leu Trp Ile Thr Ala Ala Ser
Ala Phe Arg Cys Phe Ile Val Tyr Ile50 55 60Ile Phe Val Tyr Ile Pro
Leu Phe Leu Ile His Thr His Ala Arg Phe65 70 75 80Leu Ile
Thr31158PRTHuman papillomavirus type 16 31Met His Gln Lys Arg Thr
Ala Met Phe Gln Asp Pro Gln Glu Arg Pro1 5 10 15Arg Lys Leu Pro Gln
Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp20 25 30Ile Ile Leu Glu
Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu35 40 45Val Tyr Asp
Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly50 55 60Asn Pro
Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile65 70 75
80Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu85
90 95Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile
Asn100 105 110Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His
Leu Asp Lys115 120 125Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp
Thr Gly Arg Cys Met130 135 140Ser Cys Cys Arg Ser Ser Arg Thr Arg
Arg Glu Thr Gln Leu145 150 1553298PRTHuman papillomavirus type 16
32Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln1
5 10 15Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser
Ser20 25 30Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu
Pro Asp35 40 45Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys
Asp Ser Thr50 55 60Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile
Arg Thr Leu Glu65 70 75 80Asp Leu Leu Met Gly Thr Leu Gly Ile Val
Cys Pro Ile Cys Ser Gln85 90 95Lys Pro33531PRTHuman papillomavirus
type 16 33Met Gln Val Thr Phe Ile Tyr Ile Leu Val Ile Thr Cys Tyr
Glu Asn1 5 10 15Asp Val Asn Val Tyr His Ile Phe Phe Gln Met Ser Leu
Trp Leu Pro20 25 30Ser Glu Ala Thr Val Tyr Leu Pro Pro Val Pro Val
Ser Lys Val Val35 40 45Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn Ile
Tyr Tyr His Ala Gly50 55 60Thr Ser Arg Leu Leu Ala Val Gly His Pro
Tyr Phe Pro Ile Lys Lys65 70 75 80Pro Asn Asn Asn Lys Ile Leu Val
Pro Lys Val Ser Gly Leu Gln Tyr85 90 95Arg Val Phe Arg Ile His Leu
Pro Asp Pro Asn Lys Phe Gly Phe Pro100 105 110Asp Thr Ser Phe Tyr
Asn Pro Asp Thr Gln Arg Leu Val Trp Ala Cys115 120 125Val Gly Val
Glu Val Gly Arg Gly Gln Pro Leu Gly Val Gly Ile Ser130 135 140Gly
His Pro Leu Leu Asn Lys Leu Asp Asp Thr Glu Asn Ala Ser Ala145 150
155 160Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg Glu Cys Ile Ser Met
Asp165 170 175Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys Pro
Pro Ile Gly180 185 190Glu His Trp Gly Lys Gly Ser Pro Cys Thr Asn
Val Ala Val Asn Pro195 200 205Gly Asp Cys Pro Pro Leu Glu Leu Ile
Asn Thr Val Ile Gln Asp Gly210 215 220Asp Met Val Asp Thr Gly Phe
Gly Ala Met Asp Phe Thr Thr Leu Gln225 230 235 240Ala Asn Lys Ser
Glu Val Pro Leu Asp Ile Cys Thr Ser Ile Cys Lys245 250 255Tyr Pro
Asp Tyr Ile Lys Met Val Ser Glu Pro Tyr Gly Asp Ser Leu260 265
270Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe Val Arg His Leu Phe
Asn275 280 285Arg Ala Gly Ala Val Gly Glu Asn Val Pro Asp Asp Leu
Tyr Ile Lys290 295 300Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser Ser
Asn Tyr Phe Pro Thr305 310 315 320Pro Ser Gly Ser Met Val Thr Ser
Asp Ala Gln Ile Phe Asn Lys Pro325 330 335Tyr Trp Leu Gln Arg Ala
Gln Gly His Asn Asn Gly Ile Cys Trp Gly340 345 350Asn Gln Leu Phe
Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met355 360 365Ser Leu
Cys Ala Ala Ile Ser Thr Ser Glu Thr Thr Tyr Lys Asn Thr370 375
380Asn Phe Lys Glu Tyr Leu Arg His Gly Glu Glu Tyr Asp Leu Gln
Phe385 390 395 400Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Asp
Val Met Thr Tyr405 410 415Ile His Ser Met Asn Ser Thr Ile Leu Glu
Asp Trp Asn Phe Gly Leu420 425 430Gln Pro Pro Pro Gly Gly Thr Leu
Glu Asp Thr Tyr Arg Phe Val Thr435 440 445Ser Gln Ala Ile Ala Cys
Gln Lys His Thr Pro Pro Ala Pro Lys Glu450 455 460Asp Pro Leu Lys
Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys465 470 475 480Phe
Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu485 490
495Gln Ala Gly Leu Lys Ala Lys Pro Lys Phe Thr Leu Gly Lys Arg
Lys500 505 510Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr Thr Ala Lys
Arg Lys Lys515 520 525Arg Lys Leu53034473PRTHuman papillomavirus
type 16 34Met Arg His Lys Arg Ser Ala Lys Arg Thr Lys Arg Ala Ser
Ala Thr1 5 10 15Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro
Pro Asp Ile20 25 30Ile Pro Lys Val Glu Gly Lys Thr Ile Ala Asp Gln
Ile Leu Gln Tyr35 40 45Gly Ser Met Gly Val Phe Phe Gly Gly Leu Gly
Ile Gly Thr Gly Ser50 55 60Gly Thr Gly Gly Arg Thr Gly Tyr Ile Pro
Leu Gly Thr Arg Pro Pro65 70 75 80Thr Ala Thr Asp Thr Leu Ala Pro
Val Arg Pro Pro Leu Thr Val Asp85 90 95Pro Val Gly Pro Ser Asp Pro
Ser Ile Val Ser Leu Val Glu Glu Thr100 105 110Ser Phe Ile Asp Ala
Gly Ala Pro Thr Ser Val Pro Ser Ile Pro Pro115 120 125Asp Val Ser
Gly Phe Ser Ile Thr Thr Ser Thr Asp Thr Thr Pro Ala130 135 140Ile
Leu Asp Ile Asn Asn Thr Val Thr Thr Val Thr Thr His Asn Asn145 150
155 160Pro Thr Phe Thr Asp Pro Ser Val Leu Gln Pro Pro Thr Pro Ala
Glu165 170 175Thr Gly Gly His Phe Thr Leu Ser Ser Ser Thr Ile Ser
Thr His Asn180 185 190Tyr Glu Glu Ile Pro Met Asp Thr Phe Ile Val
Ser Thr Asn Pro Asn195 200 205Thr Val Thr Ser Ser Thr Pro Ile Pro
Gly Ser Arg Pro Val Ala Arg210 215 220Leu Gly Leu Tyr Ser Arg Thr
Thr Gln Gln Val Lys Val Val Asp Pro225 230 235 240Ala Phe Ile Thr
Thr Pro Thr Lys Leu Ile Thr Tyr Asp Asn Pro Ala245 250 255Tyr Glu
Gly Ile Asp Val Asp Asn Thr Leu Tyr Phe Ser Ser Asn Asp260 265
270Asn Ser Ile Asn Ile Ala Pro Asp Pro Asp Phe Leu Asp Ile Val
Ala275 280 285Leu His Arg Pro Ala Leu Thr Ser Arg Arg Thr Gly Ile
Arg Tyr Ser290 295 300Arg Ile Gly Asn Lys Gln Thr Leu Arg Thr Arg
Ser Gly Lys Ser Ile305 310 315 320Gly Ala Lys Val His Tyr Tyr Tyr
Asp Phe Ser Thr Ile Asp Ser Ala325 330 335Glu Glu Ile Glu Leu Gln
Thr Ile Thr Pro Ser Thr Tyr Thr Thr Thr340 345 350Ser His Ala Ala
Leu Pro Thr Ser Ile Asn Asn Gly Leu Tyr Asp Ile355 360 365Tyr Ala
Asp Asp Phe Ile Thr Asp Thr Ser Thr Thr Pro Val Pro Ser370 375
380Val Pro Ser Thr Ser Leu Ser Gly Tyr Ile Pro Ala Asn Thr Thr
Ile385 390 395 400Pro Phe Gly Gly Ala Tyr Asn Ile Pro Leu Val Ser
Gly Pro Asp Ile405 410 415Pro Ile Asn Ile Thr Asp Gln Ala Pro Ser
Leu Ile Pro Ile Val Pro420 425 430Gly Ser Pro Gln Tyr Thr Ile Ile
Ala Asp Ala Gly Asp Phe Tyr Leu435 440 445His Pro Ser Tyr Tyr Met
Leu Arg Lys Arg Arg Lys Arg Leu Pro Tyr450 455 460Phe Phe Ser Asp
Val Ser Leu Ala Ala465 47035657PRTHuman papillomavirus type 18
35Met Ala Asp Pro Glu Gly Thr Asp Gly Glu Gly Thr Gly Cys Asn Gly1
5 10 15Trp Phe Tyr Val Gln Ala Ile Val Asp Lys Lys Thr Gly Asp Val
Ile20 25 30Ser Asp Asp Glu Asp Glu Asn Ala Thr Asp Thr Gly Ser Asp
Met Val35 40 45Asp Phe Ile Asp Thr Gln Gly Thr Phe Cys Glu Gln Ala
Glu Leu Glu50 55 60Thr Ala Gln Ala Leu Phe His Ala Gln Glu Val His
Asn Asp Ala Gln65 70 75 80Val Leu His Val Leu Lys Arg Lys Phe Ala
Gly Gly Ser Thr Glu Asn85 90 95Ser Pro Leu Gly Glu Arg Leu Glu Val
Asp Thr Glu Leu Ser Pro Arg100 105 110Leu Gln Glu Ile Ser Leu Asn
Ser Gly Gln Lys Lys Ala Lys Arg Arg115 120 125Leu Phe Thr Ile Ser
Asp Ser Gly Tyr Gly Cys Ser Glu Val Glu Ala130 135 140Thr Gln Ile
Gln Val Thr Thr Asn Gly Glu His Gly Gly Asn Val Cys145 150 155
160Ser Gly Gly Ser Thr Glu Ala Ile Asp Asn Gly Gly Thr Glu Gly
Asn165 170 175Asn Ser Ser Val Asp Gly Thr Ser Asp Asn Ser Asn Ile
Glu Asn Val180 185 190Asn Pro Gln Cys Thr Ile Ala Gln Leu Lys Asp
Leu Leu Lys Val Asn195 200 205Asn Lys Gln Gly Ala Met Leu Ala Val
Phe Lys Asp Thr Tyr Gly Leu210 215 220Ser Phe Thr Asp Leu Val Arg
Asn Phe Lys Ser Asp Lys Thr Thr Cys225 230 235 240Thr Asp Trp Val
Thr Ala Ile Phe Gly Val Asn Pro Thr Ile Ala Glu245 250 255Gly Phe
Lys Thr Leu Ile Gln Pro Phe Ile Leu Tyr Ala His Ile Gln260 265
270Cys Leu Asp Cys Lys Trp Gly Val Leu Ile Leu Ala Leu Leu Arg
Tyr275 280 285Lys Cys Gly Lys Ser Arg Leu Thr Val Ala Lys Gly Leu
Ser Thr Leu290 295 300Leu His Val Pro Glu Thr Cys Met Leu Ile Gln
Pro Pro Lys Leu Arg305 310 315 320Ser Ser Val Ala Ala Leu Tyr Trp
Tyr Arg Thr Gly Ile Ser Asn Ile325 330 335Ser Glu Val Met Gly Asp
Thr Pro Glu Trp Ile Gln Arg Leu Thr Ile340 345 350Ile Gln His Gly
Ile Asp Asp Ser Asn Phe Asp Leu Ser Glu Met Val355 360 365Gln Trp
Ala Phe Asp Asn Glu Leu Thr Asp Glu Ser Asp Met Ala Phe370 375
380Glu Tyr Ala Leu Leu Ala Asp Ser Asn Ser Asn Ala Ala Ala Phe
Leu385 390 395 400Lys Ser Asn Cys Gln Ala Lys Tyr Leu Lys Asp Cys
Ala Thr Met Cys405 410 415Lys His Tyr Arg Arg Ala Gln Lys Arg Gln
Met Asn Met Ser Gln Trp420 425 430Ile Arg Phe Arg Cys Ser Lys Ile
Asp Glu Gly Gly Asp Trp Arg Pro435 440 445Ile Val Gln Phe Leu Arg
Tyr Gln Gln Ile Glu Phe Ile Thr Phe Leu450 455 460Gly Ala Leu Lys
Ser Phe Leu Lys Gly Thr Pro Lys Lys Asn Cys Leu465 470 475 480Val
Phe Cys Gly Pro Ala Asn Thr Gly Lys Ser Tyr Phe Gly Met Ser485 490
495Phe Ile His Phe Ile Gln Gly Ala Val Ile Ser Phe Val Asn Ser
Thr500 505 510Ser His Phe Trp Leu Glu Pro Leu Thr Asp Thr Lys Val
Ala Met Leu515 520 525Asp Asp Ala Thr Thr Thr Cys Trp Thr Tyr Phe
Asp Thr Tyr Met Arg530 535 540Asn Ala Leu Asp Gly Asn Pro Ile Ser
Ile Asp Arg Lys His Lys Pro545 550 555 560Leu Ile Gln Leu Lys Cys
Pro Pro Ile Leu Leu Thr Thr Asn Ile His565 570 575Pro Ala Lys Asp
Asn Arg Trp Pro Tyr Leu Glu Ser Arg Ile Thr Val580 585 590Phe Glu
Phe Pro Asn Ala Phe Pro Phe Asp Lys Asn Gly Asn Pro Val595 600
605Tyr Glu Ile Asn Asp Lys Asn Trp Lys Cys Phe Phe Glu Arg Thr
Trp610 615 620Ser Arg Leu Asp Leu His Glu Glu Glu Glu Asp Ala Asp
Thr Glu Gly625 630 635 640Asn Pro Phe Gly Thr Phe Lys Leu Arg Ala
Gly Gln Asn His Arg Pro645 650 655Leu36365PRTHuman papillomavirus
type 18 36Met Gln Thr Pro Lys Glu Thr Leu Ser Glu Arg Leu Ser Cys
Val Gln1 5 10 15Asp Lys Ile Ile Asp His Tyr Glu Asn Asp Ser Lys Asp
Ile Asp Ser20 25
30Gln Ile Gln Tyr Trp Gln Leu Ile Arg Trp Glu Asn Ala Ile Phe Phe35
40 45Ala Ala Arg Glu His Gly Ile Gln Thr Leu Asn His Gln Val Val
Pro50 55 60Ala Tyr Asn Ile Ser Lys Ser Lys Ala His Lys Ala Ile Glu
Leu Gln65 70 75 80Met Ala Leu Gln Gly Leu Ala Gln Ser Arg Tyr Lys
Thr Glu Asp Trp85 90 95Thr Leu Gln Asp Thr Cys Glu Glu Leu Trp Asn
Thr Glu Pro Thr His100 105 110Cys Phe Lys Lys Gly Gly Gln Thr Val
Gln Val Tyr Phe Asp Gly Asn115 120 125Lys Asp Asn Cys Met Thr Tyr
Val Ala Trp Asp Ser Val Tyr Tyr Met130 135 140Thr Asp Ala Gly Thr
Trp Asp Lys Thr Ala Thr Cys Val Ser His Arg145 150 155 160Gly Leu
Tyr Tyr Val Lys Glu Gly Tyr Asn Thr Phe Tyr Ile Glu Phe165 170
175Lys Ser Glu Cys Glu Lys Tyr Gly Asn Thr Gly Thr Trp Glu Val
His180 185 190Phe Gly Asn Asn Val Ile Asp Cys Asn Asp Ser Met Cys
Ser Thr Ser195 200 205Asp Asp Thr Val Ser Ala Thr Gln Leu Val Lys
Gln Leu Gln His Thr210 215 220Pro Ser Pro Tyr Ser Ser Thr Val Ser
Val Gly Thr Ala Lys Thr Tyr225 230 235 240Gly Gln Thr Ser Ala Ala
Thr Arg Pro Gly His Cys Gly Leu Ala Glu245 250 255Lys Gln His Cys
Gly Pro Val Asn Pro Leu Leu Gly Ala Ala Thr Pro260 265 270Thr Gly
Asn Asn Lys Arg Arg Lys Leu Cys Ser Gly Asn Thr Thr Pro275 280
285Ile Ile His Leu Lys Gly Asp Arg Asn Ser Leu Lys Cys Leu Arg
Tyr290 295 300Arg Leu Arg Lys His Ser Asp His Tyr Arg Asp Ile Ser
Ser Thr Trp305 310 315 320His Trp Thr Gly Ala Gly Asn Glu Lys Thr
Gly Ile Leu Thr Val Thr325 330 335Tyr His Ser Glu Thr Gln Arg Thr
Lys Phe Leu Asn Thr Val Ala Ile340 345 350Pro Asp Ser Val Gln Ile
Leu Val Gly Tyr Met Thr Met355 360 3653773PRTHuman papillomavirus
type 18 37Met Leu Ser Leu Ile Phe Leu Phe Cys Phe Cys Val Cys Met
Tyr Val1 5 10 15Cys Cys His Val Pro Leu Leu Pro Ser Val Cys Met Cys
Ala Tyr Ala20 25 30Trp Val Leu Val Phe Val Tyr Ile Val Val Ile Thr
Ser Pro Ala Thr35 40 45Ala Phe Thr Val Tyr Val Phe Cys Phe Leu Leu
Pro Met Leu Leu Leu50 55 60His Ile His Ala Ile Leu Ser Leu Gln65
7038158PRTHuman papillomavirus type 18 38Met Ala Arg Phe Glu Asp
Pro Thr Arg Arg Pro Tyr Lys Leu Pro Asp1 5 10 15Leu Cys Thr Glu Leu
Asn Thr Ser Leu Gln Asp Ile Glu Ile Thr Cys20 25 30Val Tyr Cys Lys
Thr Val Leu Glu Leu Thr Glu Val Phe Glu Phe Ala35 40 45Phe Lys Asp
Leu Phe Val Val Tyr Arg Asp Ser Ile Pro His Ala Ala50 55 60Cys His
Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu Arg His65 70 75
80Tyr Ser Asp Ser Val Tyr Gly Asp Thr Leu Glu Lys Leu Thr Asn Thr85
90 95Gly Leu Tyr Asn Leu Leu Ile Arg Cys Leu Arg Cys Gln Lys Pro
Leu100 105 110Asn Pro Ala Glu Lys Leu Arg His Leu Asn Glu Lys Arg
Arg Phe His115 120 125Asn Ile Ala Gly His Tyr Arg Gly Gln Cys His
Ser Cys Cys Asn Arg130 135 140Ala Arg Gln Glu Arg Leu Gln Arg Arg
Arg Glu Thr Gln Val145 150 15539105PRTHuman papillomavirus type 18
39Met His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu1
5 10 15Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Cys His Glu Gln Leu
Ser20 25 30Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn His
Gln His35 40 45Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met
Leu Cys Met50 55 60Cys Cys Lys Cys Glu Ala Arg Ile Lys Leu Val Val
Glu Ser Ser Ala65 70 75 80Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe
Leu Asn Thr Leu Ser Phe85 90 95Val Cys Pro Trp Cys Ala Ser Gln
Gln100 10540568PRTHuman papillomavirus type 18 40Met Cys Leu Tyr
Thr Arg Val Leu Ile Leu His Tyr His Leu Leu Pro1 5 10 15Leu Tyr Gly
Pro Leu Tyr His Pro Arg Pro Leu Pro Leu His Ser Ile20 25 30Leu Val
Tyr Met Val His Ile Ile Ile Cys Gly His Tyr Ile Ile Leu35 40 45Phe
Leu Arg Asn Val Asn Val Phe Pro Ile Phe Leu Gln Met Ala Leu50 55
60Trp Arg Pro Ser Asp Asn Thr Val Tyr Leu Pro Pro Pro Ser Val Ala65
70 75 80Arg Val Val Asn Thr Asp Asp Tyr Val Thr Pro Thr Ser Ile Phe
Tyr85 90 95His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly Asn Pro Tyr
Phe Arg100 105 110Val Pro Ala Gly Gly Gly Asn Lys Gln Asp Ile Pro
Lys Val Ser Ala115 120 125Tyr Gln Tyr Arg Val Phe Arg Val Gln Leu
Pro Asp Pro Asn Lys Phe130 135 140Gly Leu Pro Asp Thr Ser Ile Tyr
Asn Pro Glu Thr Gln Arg Leu Val145 150 155 160Trp Ala Cys Ala Gly
Val Glu Ile Gly Arg Gly Gln Pro Leu Gly Val165 170 175Gly Leu Ser
Gly His Pro Phe Tyr Asn Lys Leu Asp Asp Thr Glu Ser180 185 190Ser
His Ala Ala Thr Ser Asn Val Ser Glu Asp Val Arg Asp Asn Val195 200
205Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Leu Gly Cys Ala
Pro210 215 220Ala Ile Gly Glu His Trp Ala Lys Gly Thr Ala Cys Lys
Ser Arg Pro225 230 235 240Leu Ser Gln Gly Asp Cys Pro Pro Leu Glu
Leu Lys Asn Thr Val Leu245 250 255Glu Asp Gly Asp Met Val Asp Thr
Gly Tyr Gly Ala Met Asp Phe Ser260 265 270Thr Leu Gln Asp Thr Lys
Cys Glu Val Pro Leu Asp Ile Cys Gln Ser275 280 285Ile Cys Lys Tyr
Pro Asp Tyr Leu Gln Met Ser Ala Asp Pro Tyr Gly290 295 300Asp Ser
Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe Ala Arg His305 310 315
320Phe Trp Asn Arg Ala Gly Thr Met Gly Asp Thr Val Pro Gln Ser
Leu325 330 335Tyr Ile Lys Gly Thr Gly Met Pro Ala Ser Pro Gly Ser
Cys Val Tyr340 345 350Ser Pro Ser Pro Ser Gly Ser Ile Val Thr Ser
Asp Ser Gln Leu Phe355 360 365Asn Lys Pro Tyr Trp Leu His Lys Ala
Gln Gly His Asn Asn Gly Val370 375 380Cys Trp His Asn Gln Leu Phe
Val Thr Val Val Asp Thr Thr Pro Ser385 390 395 400Thr Asn Leu Thr
Ile Cys Ala Ser Thr Gln Ser Pro Val Pro Gly Gln405 410 415Tyr Asp
Ala Thr Lys Phe Lys Gln Tyr Ser Arg His Val Glu Glu Tyr420 425
430Asp Leu Gln Phe Ile Phe Gln Leu Cys Thr Ile Thr Leu Thr Ala
Asp435 440 445Val Met Ser Tyr Ile His Ser Met Asn Ser Ser Ile Leu
Glu Asp Trp450 455 460Asn Phe Gly Val Pro Pro Pro Pro Thr Thr Ser
Leu Val Asp Thr Tyr465 470 475 480Arg Phe Val Gln Ser Val Ala Ile
Thr Cys Gln Lys Asp Ala Ala Pro485 490 495Ala Glu Asn Lys Asp Pro
Tyr Asp Lys Leu Lys Phe Trp Asn Val Asp500 505 510Leu Lys Glu Lys
Phe Ser Leu Asp Leu Asp Gln Tyr Pro Leu Gly Arg515 520 525Lys Phe
Leu Val Gln Ala Gly Leu Arg Arg Lys Pro Thr Ile Gly Pro530 535
540Arg Lys Arg Ser Ala Pro Ser Ala Thr Thr Ser Ser Lys Pro Ala
Lys545 550 555 560Arg Val Arg Val Arg Ala Arg Lys56541462PRTHuman
papillomavirus type 18 41Met Val Ser His Arg Ala Ala Arg Arg Lys
Arg Ala Ser Val Thr Asp1 5 10 15Leu Tyr Lys Thr Cys Lys Gln Ser Gly
Thr Cys Pro Pro Asp Val Val20 25 30Pro Lys Val Glu Gly Thr Thr Leu
Ala Asp Lys Ile Leu Gln Trp Ser35 40 45Ser Leu Gly Ile Phe Leu Gly
Gly Leu Gly Ile Gly Thr Gly Ser Gly50 55 60Thr Gly Gly Arg Thr Gly
Tyr Ile Pro Leu Gly Gly Arg Ser Asn Thr65 70 75 80Val Val Asp Val
Gly Pro Thr Arg Pro Pro Val Val Ile Glu Pro Val85 90 95Gly Pro Thr
Asp Pro Ser Ile Val Thr Leu Ile Glu Asp Ser Ser Val100 105 110Val
Thr Ser Gly Ala Pro Arg Pro Thr Phe Thr Gly Thr Ser Gly Phe115 120
125Asp Ile Thr Ser Ala Gly Thr Thr Thr Pro Ala Val Leu Asp Ile
Thr130 135 140Pro Ser Ser Thr Ser Val Ser Ile Ser Thr Thr Asn Phe
Thr Asn Pro145 150 155 160Ala Phe Ser Asp Pro Ser Ile Ile Glu Val
Pro Gln Thr Gly Glu Val165 170 175Ala Gly Asn Val Phe Val Gly Thr
Pro Thr Ser Gly Thr His Gly Tyr180 185 190Glu Glu Ile Pro Leu Gln
Thr Phe Ala Ser Ser Gly Thr Gly Glu Glu195 200 205Pro Ile Ser Ser
Thr Pro Leu Pro Thr Val Arg Arg Val Ala Gly Pro210 215 220Arg Leu
Tyr Ser Arg Ala Tyr Gln Gln Val Ser Val Ala Asn Pro Glu225 230 235
240Phe Leu Thr Arg Pro Ser Ser Leu Ile Thr Tyr Asp Asn Pro Ala
Phe245 250 255Glu Pro Val Asp Thr Thr Leu Thr Phe Asp Pro Arg Ser
Asp Val Pro260 265 270Asp Ser Asp Phe Met Asp Ile Ile Arg Leu His
Arg Pro Ala Leu Thr275 280 285Ser Arg Arg Gly Thr Val Arg Phe Ser
Arg Leu Gly Gln Arg Ala Thr290 295 300Met Phe Thr Arg Ser Gly Thr
Gln Ile Gly Ala Arg Val His Phe Tyr305 310 315 320His Asp Ile Ser
Pro Ile Ala Pro Ser Pro Glu Tyr Ile Glu Leu Gln325 330 335Pro Leu
Val Ser Ala Thr Glu Asp Asn Asp Leu Phe Asp Ile Tyr Ala340 345
350Asp Asp Met Asp Pro Ala Val Pro Val Pro Ser Arg Ser Thr Thr
Ser355 360 365Phe Ala Phe Phe Lys Tyr Ser Pro Thr Ile Ser Ser Ala
Ser Ser Tyr370 375 380Ser Asn Val Thr Val Pro Leu Thr Ser Ser Trp
Asp Val Pro Val Tyr385 390 395 400Thr Gly Pro Asp Ile Thr Leu Pro
Ser Thr Thr Ser Val Trp Pro Ile405 410 415Val Ser Pro Thr Ala Pro
Ala Ser Thr Gln Tyr Ile Gly Ile His Gly420 425 430Thr His Tyr Tyr
Leu Trp Pro Leu Tyr Tyr Phe Ile Pro Lys Lys Arg435 440 445Lys Arg
Val Pro Tyr Phe Phe Ala Asp Gly Phe Val Ala Ala450 455
46042629PRTHuman papillomavirus type 31 42Met Ala Asp Pro Ala Gly
Thr Asp Gly Glu Gly Thr Gly Cys Asn Gly1 5 10 15Trp Phe Tyr Val Glu
Ala Val Ile Asp Arg Gln Thr Gly Asp Asn Ile20 25 30Ser Glu Asp Glu
Asn Glu Asp Ser Ser Asp Thr Gly Glu Asp Met Val35 40 45Asp Phe Ile
Asp Asn Cys Asn Val Tyr Asn Asn Gln Ala Glu Ala Glu50 55 60Thr Ala
Gln Ala Leu Phe His Ala Gln Glu Ala Glu Glu His Ala Glu65 70 75
80Ala Val Gln Val Leu Lys Arg Lys Tyr Val Gly Ser Pro Leu Ser Asp85
90 95Ile Ser Ser Cys Val Asp Tyr Asn Ile Ser Pro Arg Leu Lys Ala
Ile100 105 110Cys Ile Glu Asn Asn Ser Lys Thr Ala Lys Arg Arg Leu
Phe Glu Leu115 120 125Pro Asp Ser Gly Tyr Gly Asn Thr Glu Val Glu
Thr Gln Gln Met Val130 135 140Gln Val Glu Glu Gln Gln Thr Thr Leu
Ser Cys Asn Gly Ser Asp Gly145 150 155 160Thr His Ser Glu Arg Glu
Asn Glu Thr Pro Thr Arg Asn Ile Leu Gln165 170 175Val Leu Lys Thr
Ser Asn Gly Lys Ala Ala Met Leu Gly Lys Phe Lys180 185 190Glu Leu
Tyr Gly Val Ser Phe Met Glu Leu Ile Arg Pro Phe Gln Ser195 200
205Asn Lys Ser Thr Cys Thr Asp Trp Cys Val Ala Ala Phe Gly Val
Thr210 215 220Gly Thr Val Ala Glu Gly Phe Lys Thr Leu Leu Gln Pro
Tyr Cys Leu225 230 235 240Tyr Cys His Leu Gln Ser Leu Ala Cys Ser
Trp Gly Met Val Met Leu245 250 255Met Leu Val Arg Phe Lys Cys Ala
Lys Asn Arg Ile Thr Ile Glu Lys260 265 270Leu Leu Glu Lys Leu Leu
Cys Ile Ser Thr Asn Cys Met Leu Ile Gln275 280 285Pro Pro Lys Leu
Arg Ser Thr Ala Ala Ala Leu Tyr Trp Tyr Arg Thr290 295 300Gly Met
Ser Asn Ile Ser Asp Val Tyr Gly Glu Thr Pro Glu Trp Ile305 310 315
320Glu Arg Gln Thr Val Leu Gln His Ser Phe Asn Asp Thr Thr Phe
Asp325 330 335Leu Ser Gln Met Val Gln Trp Ala Tyr Asp Asn Asp Val
Met Asp Asp340 345 350Ser Glu Ile Ala Tyr Lys Tyr Ala Gln Leu Ala
Asp Ser Asp Ser Asn355 360 365Ala Cys Ala Phe Leu Lys Ser Asn Ser
Gln Ala Lys Ile Val Lys Asp370 375 380Cys Gly Thr Met Cys Arg His
Tyr Lys Arg Ala Glu Lys Arg Gln Met385 390 395 400Ser Met Gly Gln
Trp Ile Lys Ser Arg Cys Asp Lys Val Ser Asp Glu405 410 415Gly Asp
Trp Arg Asp Ile Val Lys Phe Leu Arg Tyr Gln Gln Ile Glu420 425
430Phe Val Ser Phe Leu Ser Ala Leu Lys Leu Phe Leu Lys Gly Val
Pro435 440 445Lys Lys Asn Cys Ile Leu Ile His Gly Ala Pro Asn Thr
Gly Lys Ser450 455 460Tyr Phe Gly Met Ser Leu Ile Ser Phe Leu Gln
Gly Cys Ile Ile Ser465 470 475 480Tyr Ala Asn Ser Lys Ser His Phe
Trp Leu Gln Pro Leu Ala Asp Ala485 490 495Lys Ile Gly Met Leu Asp
Asp Ala Thr Thr Pro Cys Trp His Tyr Ile500 505 510Asp Asn Tyr Leu
Arg Asn Ala Leu Asp Gly Asn Pro Val Ser Ile Asp515 520 525Val Lys
His Lys Ala Leu Met Gln Leu Lys Cys Pro Pro Leu Leu Ile530 535
540Thr Ser Asn Ile Asn Ala Gly Lys Asp Asp Arg Trp Pro Tyr Leu
His545 550 555 560Ser Arg Leu Val Val Phe Thr Phe Pro Asn Pro Phe
Pro Phe Asp Lys565 570 575Asn Gly Asn Pro Val Tyr Glu Leu Ser Asp
Lys Asn Trp Lys Ser Phe580 585 590Phe Ser Arg Thr Trp Cys Arg Leu
Asn Leu His Glu Glu Glu Asp Lys595 600 605Glu Asn Asp Gly Asp Ser
Phe Ser Thr Phe Lys Cys Val Ser Gly Gln610 615 620Asn Ile Arg Thr
Leu62543372PRTHuman papillomavirus type 31 43Met Glu Thr Leu Ser
Gln Arg Leu Asn Val Cys Gln Asp Lys Ile Leu1 5 10 15Glu His Tyr Glu
Asn Asp Ser Lys Arg Leu Cys Asp His Ile Asp Tyr20 25 30Trp Lys His
Ile Arg Leu Glu Cys Val Leu Met Tyr Lys Ala Arg Glu35 40 45Met Gly
Ile His Ser Ile Asn His Gln Val Val Pro Ala Leu Ser Val50 55 60Ser
Lys Ala Lys Ala Leu Gln Ala Ile Glu Leu Gln Met Met Leu Glu65 70 75
80Thr Leu Asn Asn Thr Glu Tyr Lys Asn Glu Asp Trp Thr Met Gln Gln85
90 95Thr Ser Leu Glu Leu Tyr Leu Thr Ala Pro Thr Gly Cys Leu Lys
Lys100 105 110His Gly Tyr Thr Val Glu Val Gln Phe Asp Gly Asp Val
His Asn Thr115 120 125Met His Tyr Thr Asn Trp Lys Phe Ile Tyr Leu
Cys Ile Asp Gly Gln130 135 140Cys Thr Val Val Glu Gly Gln Val Asn
Cys Lys Gly Ile Tyr Tyr Val145 150 155 160His Glu Gly His Ile Thr
Tyr Phe Val Asn Phe Thr Glu Glu Ala Lys165 170 175Lys Tyr Gly Thr
Gly Lys Lys Trp Glu Val His Ala Gly Gly Gln Val180 185 190Ile Val
Phe Pro Glu Ser Val Phe Ser Ser Asp Glu Ile Ser Phe Ala195 200
205Gly Ile Val Thr Lys Leu Pro Thr Ala Asn Asn Thr Thr Thr Ser
Asn210 215 220Ser Lys Thr Cys Ala Leu Gly Thr Ser Glu Gly Val Arg
Arg Ala Thr225 230 235 240Thr Ser Thr Lys Arg Pro Arg Thr Glu Pro
Glu His Arg Asn Thr His245 250 255His Pro Asn Lys Leu Leu Arg Gly
Asp Ser Val Asp Ser Val Asn Cys260 265 270Gly Val Ile Ser Ala Ala
Ala Cys Thr Asn Gln Thr Arg Ala Val Ser275 280 285Cys Pro Ala Thr
Thr Pro Ile Ile His Leu Lys Gly Asp Ala Asn Ile290 295
300Leu Lys Cys Leu Arg Tyr Arg Leu Ser Lys Tyr Lys Gln Leu Tyr
Glu305 310 315 320Gln Val Ser Ser Thr Trp His Trp Thr Cys Thr Asp
Gly Lys His Lys325 330 335Asn Ala Ile Val Thr Leu Thr Tyr Ile Ser
Thr Ser Gln Arg Asp Asp340 345 350Phe Leu Asn Thr Val Lys Ile Pro
Asn Thr Val Ser Val Ser Thr Gly355 360 365Tyr Met Thr
Ile3704484PRTHuman papillomavirus type 31 44Met Ile Glu Leu Asn Ile
Ser Thr Val Ser Ile Val Leu Cys Phe Leu1 5 10 15Leu Cys Phe Cys Val
Leu Leu Phe Val Cys Leu Val Ile Arg Pro Leu20 25 30Val Leu Ser Val
Ser Val Tyr Ala Thr Leu Leu Leu Leu Ile Val Ile35 40 45Leu Trp Val
Ile Ala Thr Ser Pro Leu Arg Cys Phe Cys Ile Tyr Val50 55 60Val Phe
Ile Tyr Ile Pro Leu Phe Val Ile His Thr His Ala Ser Phe65 70 75
80Leu Ser Gln Gln45149PRTHuman papillomavirus type 31 45Met Phe Lys
Asn Pro Ala Glu Arg Pro Arg Lys Leu His Glu Leu Ser1 5 10 15Ser Ala
Leu Glu Ile Pro Tyr Asp Glu Leu Arg Leu Asn Cys Val Tyr20 25 30Cys
Lys Gly Gln Leu Thr Glu Thr Glu Val Leu Asp Phe Ala Phe Thr35 40
45Asp Leu Thr Ile Val Tyr Arg Asp Asp Thr Pro His Gly Val Cys Thr50
55 60Lys Cys Leu Arg Phe Tyr Ser Lys Val Ser Glu Phe Arg Trp Tyr
Arg65 70 75 80Tyr Ser Val Tyr Gly Thr Thr Leu Glu Lys Leu Thr Asn
Lys Gly Ile85 90 95Cys Asp Leu Leu Ile Arg Cys Ile Thr Cys Gln Arg
Pro Leu Cys Pro100 105 110Glu Glu Lys Gln Arg His Leu Asp Lys Lys
Lys Arg Phe His Asn Ile115 120 125Gly Gly Arg Trp Thr Gly Arg Cys
Ile Ala Cys Trp Arg Arg Pro Arg130 135 140Thr Glu Thr Gln
Val1454698PRTHuman papillomavirus type 31 46Met Arg Gly Glu Thr Pro
Thr Leu Gln Asp Tyr Val Leu Asp Leu Gln1 5 10 15Pro Glu Ala Thr Asp
Leu His Cys Tyr Glu Gln Leu Pro Asp Ser Ser20 25 30Asp Glu Glu Asp
Val Ile Asp Ser Pro Ala Gly Gln Ala Glu Pro Asp35 40 45Thr Ser Asn
Tyr Asn Ile Val Thr Phe Cys Cys Gln Cys Lys Ser Thr50 55 60Leu Arg
Leu Cys Val Gln Ser Thr Gln Val Asp Ile Arg Ile Leu Gln65 70 75
80Glu Leu Leu Met Gly Ser Phe Gly Ile Val Cys Pro Asn Cys Ser Thr85
90 95Arg Leu47504PRTHuman papillomavirus type 31 47Met Ser Leu Trp
Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val1 5 10 15Pro Val Ser
Lys Val Val Ser Thr Asp Glu Tyr Val Thr Arg Thr Asn20 25 30Ile Tyr
Tyr His Ala Gly Ser Ala Arg Leu Leu Thr Val Gly His Pro35 40 45Tyr
Tyr Ser Ile Pro Lys Ser Asp Asn Pro Lys Lys Ile Val Val Pro50 55
60Lys Val Ser Gly Leu Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp65
70 75 80Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Glu
Thr85 90 95Gln Arg Leu Val Trp Ala Cys Val Gly Leu Glu Val Gly Arg
Gly Gln100 105 110Pro Leu Gly Val Gly Ile Ser Gly His Pro Leu Leu
Asn Lys Phe Asp115 120 125Asp Thr Glu Asn Ser Asn Arg Tyr Ala Gly
Gly Pro Gly Thr Asp Asn130 135 140Arg Glu Cys Ile Ser Met Asp Tyr
Lys Gln Thr Gln Leu Cys Leu Leu145 150 155 160Gly Cys Lys Pro Pro
Ile Gly Glu His Trp Gly Lys Gly Ser Pro Cys165 170 175Ser Asn Asn
Ala Ile Thr Pro Gly Asp Cys Pro Pro Leu Glu Leu Lys180 185 190Asn
Ser Val Ile Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala195 200
205Met Asp Phe Thr Ala Leu Gln Asp Thr Lys Ser Asn Val Pro Leu
Asp210 215 220Ile Cys Asn Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Lys
Met Val Ala225 230 235 240Glu Pro Tyr Gly Asp Thr Leu Phe Phe Tyr
Leu Arg Arg Glu Gln Met245 250 255Phe Val Arg His Phe Phe Asn Arg
Ser Gly Thr Val Gly Glu Ser Val260 265 270Pro Thr Asp Leu Tyr Ile
Lys Gly Ser Gly Ser Thr Ala Thr Leu Ala275 280 285Asn Ser Thr Tyr
Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp290 295 300Ala Gln
Ile Phe Asn Lys Pro Tyr Trp Met Gln Arg Ala Gln Gly His305 310 315
320Asn Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val
Asp325 330 335Thr Thr Arg Ser Thr Asn Met Ser Val Cys Ala Ala Ile
Ala Asn Ser340 345 350Asp Thr Thr Phe Lys Ser Ser Asn Phe Lys Glu
Tyr Leu Arg His Gly355 360 365Glu Glu Phe Asp Leu Gln Phe Ile Phe
Gln Leu Cys Lys Ile Thr Leu370 375 380Ser Ala Asp Ile Met Thr Tyr
Ile His Ser Met Asn Pro Ala Ile Leu385 390 395 400Glu Asp Trp Asn
Phe Gly Leu Thr Thr Pro Pro Ser Gly Ser Leu Glu405 410 415Asp Thr
Tyr Arg Phe Val Thr Ser Gln Ala Ile Thr Cys Gln Lys Thr420 425
430Ala Pro Gln Lys Pro Lys Glu Asp Pro Phe Lys Asp Tyr Val Phe
Trp435 440 445Glu Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp
Gln Phe Pro450 455 460Leu Gly Arg Lys Phe Leu Leu Gln Ala Gly Tyr
Arg Ala Arg Pro Lys465 470 475 480Phe Lys Ala Gly Lys Arg Ser Ala
Pro Ser Ala Ser Thr Thr Thr Pro485 490 495Ala Lys Arg Lys Lys Thr
Lys Lys50048466PRTHuman papillomavirus type 31 48Met Arg Ser Lys
Arg Ser Thr Lys Arg Thr Lys Arg Ala Ser Ala Thr1 5 10 15Gln Leu Tyr
Gln Thr Cys Lys Ala Ala Gly Thr Cys Pro Ser Asp Val20 25 30Ile Pro
Lys Ile Glu His Thr Thr Ile Ala Asp Gln Ile Leu Arg Tyr35 40 45Gly
Ser Met Gly Val Phe Phe Gly Gly Leu Gly Ile Gly Ser Gly Ser50 55
60Gly Thr Gly Gly Arg Thr Gly Tyr Val Pro Leu Ser Thr Arg Pro Ser65
70 75 80Thr Val Ser Glu Ala Ser Ile Pro Ile Arg Pro Pro Val Ser Ile
Asp85 90 95Pro Val Gly Pro Leu Asp Pro Ser Ile Val Ser Leu Val Glu
Glu Ser100 105 110Gly Ile Val Asp Val Gly Ala Pro Ala Pro Ile Pro
His Pro Pro Thr115 120 125Thr Ser Gly Phe Asp Ile Ala Thr Thr Ala
Asp Thr Thr Pro Ala Ile130 135 140Leu Asp Val Thr Ser Val Ser Thr
His Glu Asn Pro Thr Phe Thr Asp145 150 155 160Pro Ser Val Leu Gln
Pro Pro Thr Pro Ala Glu Thr Ser Gly His Leu165 170 175Leu Leu Ser
Ser Ser Ser Ile Ser Thr His Asn Tyr Glu Glu Ile Pro180 185 190Met
Asp Thr Phe Ile Val Ser Thr Asn Asn Glu Asn Ile Thr Ser Ser195 200
205Thr Pro Ile Pro Gly Val Arg Arg Pro Ala Arg Leu Gly Leu Tyr
Ser210 215 220Lys Ala Thr Gln Gln Val Lys Val Ile Asp Pro Thr Phe
Leu Ser Ala225 230 235 240Pro Lys Gln Leu Ile Thr Tyr Glu Asn Pro
Ala Tyr Glu Thr Val Asn245 250 255Ala Glu Glu Ser Leu Tyr Phe Ser
Asn Thr Ser His Asn Ile Ala Pro260 265 270Asp Pro Asp Phe Leu Asp
Ile Ile Ala Leu His Arg Pro Ala Leu Thr275 280 285Ser Arg Arg Asn
Thr Val Arg Tyr Ser Arg Leu Gly Asn Lys Gln Thr290 295 300Leu Arg
Thr Arg Ser Gly Ala Thr Ile Gly Ala Arg Val His Tyr Tyr305 310 315
320Tyr Asp Ile Ser Ser Ile Asn Pro Ala Gly Glu Ser Ile Glu Met
Gln325 330 335Pro Leu Gly Ala Ser Ala Thr Thr Thr Ser Thr Leu Asn
Asp Gly Leu340 345 350Tyr Asp Ile Tyr Ala Asp Thr Asp Phe Thr Val
Asp Thr Pro Ala Thr355 360 365His Asn Val Ser Pro Ser Thr Ala Val
Gln Ser Thr Ser Ala Val Ser370 375 380Ala Tyr Val Pro Thr Asn Thr
Thr Val Pro Leu Ser Thr Gly Phe Asp385 390 395 400Ile Pro Ile Phe
Ser Gly Pro Asp Val Pro Ile Glu His Ala Pro Thr405 410 415Gln Val
Phe Pro Phe Pro Leu Ala Pro Thr Thr Pro Gln Val Ser Ile420 425
430Phe Val Asp Gly Gly Asp Phe Tyr Leu His Pro Ser Tyr Tyr Met
Leu435 440 445Lys Arg Arg Arg Lys Arg Val Ser Tyr Phe Phe Thr Asp
Val Ser Val450 455 460Ala Ala46549643PRTHuman papillomavirus type
45 49Met Ala Asp Pro Glu Gly Thr Asp Gly Glu Gly Thr Gly Cys Asn
Gly1 5 10 15Trp Phe Phe Val Glu Thr Ile Val Glu Lys Lys Thr Gly Asp
Val Ile20 25 30Ser Asp Asp Glu Asp Glu Thr Ala Thr Asp Thr Gly Ser
Asp Met Val35 40 45Asp Phe Ile Asp Thr Gln Leu Ser Ile Cys Glu Gln
Ala Glu Gln Glu50 55 60Thr Ala Gln Ala Leu Phe His Ala Gln Glu Val
Gln Asn Asp Ala Gln65 70 75 80Val Leu His Leu Leu Lys Arg Lys Phe
Ala Gly Gly Ser Lys Glu Asn85 90 95Ser Pro Leu Gly Glu Gln Leu Ser
Val Asp Thr Asp Leu Ser Pro Arg100 105 110Leu Gln Glu Ile Ser Leu
Asn Ser Gly His Lys Lys Ala Lys Arg Arg115 120 125Leu Phe Thr Ile
Ser Asp Ser Gly Tyr Gly Cys Ser Glu Val Glu Ala130 135 140Ala Glu
Thr Gln Val Thr Val Asn Thr Asn Ala Glu Asn Gly Gly Ser145 150 155
160Val His Ser Thr Gln Ser Ser Gly Gly Asp Ser Ser Asp Asn Ala
Glu165 170 175Asn Val Asp Pro His Cys Ser Ile Thr Glu Leu Lys Glu
Leu Leu Gln180 185 190Ala Ser Asn Lys Lys Ala Ala Met Leu Ala Val
Phe Lys Asp Ile Tyr195 200 205Gly Leu Ser Phe Thr Asp Leu Val Arg
Asn Phe Lys Ser Asp Lys Thr210 215 220Thr Cys Thr Asp Trp Val Met
Ala Ile Phe Gly Val Asn Pro Thr Val225 230 235 240Ala Glu Gly Phe
Lys Thr Leu Ile Lys Pro Ala Thr Leu Tyr Ala His245 250 255Ile Gln
Cys Leu Asp Cys Lys Trp Gly Val Leu Ile Leu Ala Leu Leu260 265
270Arg Tyr Lys Cys Gly Lys Asn Arg Leu Thr Val Ala Lys Gly Leu
Ser275 280 285Thr Leu Leu His Val Pro Glu Thr Cys Met Leu Ile Glu
Pro Pro Lys290 295 300Leu Arg Ser Ser Val Ala Ala Leu Tyr Trp Tyr
Arg Thr Gly Ile Ser305 310 315 320Asn Ile Ser Glu Val Ser Gly Asp
Thr Pro Glu Trp Ile Gln Arg Leu325 330 335Thr Ile Ile Gln His Gly
Ile Asp Asp Ser Asn Phe Asp Leu Ser Asp340 345 350Met Val Gln Trp
Ala Phe Asp Asn Asp Leu Thr Asp Glu Ser Asp Met355 360 365Ala Phe
Gln Tyr Ala Gln Leu Ala Asp Cys Asn Ser Asn Ala Ala Ala370 375
380Phe Leu Lys Ser Asn Cys Gln Ala Lys Tyr Leu Lys Asp Cys Ala
Val385 390 395 400Met Cys Arg His Tyr Lys Arg Ala Gln Lys Arg Gln
Met Asn Met Ser405 410 415Gln Trp Ile Lys Tyr Arg Cys Ser Lys Ile
Asp Glu Gly Gly Asp Trp420 425 430Arg Pro Ile Val Gln Phe Leu Arg
Tyr Gln Gly Val Glu Phe Ile Ser435 440 445Phe Leu Arg Ala Leu Lys
Glu Phe Leu Lys Gly Thr Pro Lys Lys Asn450 455 460Cys Ile Leu Leu
Tyr Gly Pro Ala Asn Thr Gly Lys Ser Tyr Phe Gly465 470 475 480Met
Ser Phe Ile His Phe Leu Gln Gly Ala Ile Ile Ser Phe Val Asn485 490
495Ser Asn Ser His Phe Trp Leu Glu Pro Leu Ala Asp Thr Lys Val
Ala500 505 510Met Leu Asp Asp Ala Thr His Thr Cys Trp Thr Tyr Phe
Asp Asn Tyr515 520 525Met Arg Asn Ala Leu Asp Gly Asn Pro Ile Ser
Ile Asp Arg Lys His530 535 540Lys Pro Leu Leu Gln Leu Lys Cys Pro
Pro Ile Leu Leu Thr Ser Asn545 550 555 560Ile Asp Pro Ala Lys Asp
Asn Lys Trp Pro Tyr Leu Glu Ser Arg Val565 570 575Thr Val Phe Thr
Phe Pro His Ala Phe Pro Phe Asp Lys Asn Gly Asn580 585 590Pro Val
Tyr Glu Ile Asn Asp Lys Asn Trp Lys Cys Phe Phe Glu Arg595 600
605Thr Trp Ser Arg Leu Asp Leu His Glu Asp Asp Glu Asp Ala Asp
Thr610 615 620Glu Gly Ile Pro Phe Gly Thr Phe Lys Cys Val Thr Gly
Gln Asn Thr625 630 635 640Arg Pro Leu50368PRTHuman papillomavirus
type 45 50Met Lys Met Gln Thr Pro Lys Glu Ser Leu Ser Glu Arg Leu
Ser Ala1 5 10 15Leu Gln Asp Lys Ile Leu Asp His Tyr Glu Asn Asp Ser
Lys Asp Ile20 25 30Asn Ser Gln Ile Ser Tyr Trp Gln Leu Ile Arg Leu
Glu Asn Ala Ile35 40 45Leu Phe Thr Ala Arg Glu His Gly Ile Thr Lys
Leu Asn His Gln Val50 55 60Val Pro Pro Ile Asn Ile Ser Lys Ser Lys
Ala His Lys Ala Ile Glu65 70 75 80Leu Gln Met Ala Leu Lys Gly Leu
Ala Gln Ser Lys Tyr Asn Asn Glu85 90 95Glu Trp Thr Leu Gln Asp Thr
Cys Glu Glu Leu Trp Asn Thr Glu Pro100 105 110Ser Gln Cys Phe Lys
Lys Gly Gly Lys Thr Val His Val Tyr Phe Asp115 120 125Gly Asn Lys
Asp Asn Cys Met Asn Tyr Val Val Trp Asp Ser Ile Tyr130 135 140Tyr
Ile Thr Glu Thr Gly Ile Trp Asp Lys Thr Ala Ala Cys Val Ser145 150
155 160Tyr Trp Gly Val Tyr Tyr Ile Lys Asp Gly Asp Thr Thr Tyr Tyr
Val165 170 175Gln Phe Lys Ser Glu Cys Glu Lys Tyr Gly Asn Ser Asn
Thr Trp Glu180 185 190Val Gln Tyr Gly Gly Asn Val Ile Asp Cys Asn
Asp Ser Met Cys Ser195 200 205Thr Ser Asp Asp Thr Val Ser Ala Thr
Gln Ile Val Arg Gln Leu Gln210 215 220His Ala Ser Thr Ser Thr Pro
Lys Thr Ala Ser Val Gly Thr Pro Lys225 230 235 240Pro His Ile Gln
Thr Pro Ala Thr Lys Arg Pro Arg Gln Cys Gly Leu245 250 255Thr Glu
Gln His His Gly Arg Val Asn Thr His Val His Asn Pro Leu260 265
270Leu Cys Ser Ser Thr Ser Asn Asn Lys Arg Arg Lys Val Cys Ser
Gly275 280 285Asn Thr Thr Pro Ile Ile His Leu Lys Gly Asp Lys Asn
Ser Leu Lys290 295 300Cys Leu Arg Tyr Arg Leu Arg Lys Tyr Ala Asp
His Tyr Ser Glu Ile305 310 315 320Ser Ser Thr Trp His Trp Thr Gly
Cys Asn Lys Asn Thr Gly Ile Leu325 330 335Thr Val Thr Tyr Asn Ser
Glu Val Gln Arg Asn Thr Phe Leu Asp Val340 345 350Val Thr Ile Pro
Asn Ser Val Gln Ile Ser Val Gly Tyr Met Thr Ile355 360
36551158PRTHuman papillomavirus type 45 51Met Ala Arg Phe Asp Asp
Pro Thr Gln Arg Pro Tyr Lys Leu Pro Asp1 5 10 15Leu Cys Thr Glu Leu
Asn Thr Ser Leu Gln Asp Val Ser Ile Ala Cys20 25 30Val Tyr Cys Lys
Ala Thr Leu Glu Arg Thr Glu Val Tyr Gln Phe Ala35 40 45Phe Lys Asp
Leu Phe Ile Val Tyr Arg Asp Cys Ile Ala Tyr Ala Ala50 55 60Cys His
Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu Arg Tyr65 70 75
80Tyr Ser Asn Ser Val Tyr Gly Glu Thr Leu Glu Lys Ile Thr Asn Thr85
90 95Glu Leu Tyr Asn Leu Leu Ile Arg Cys Leu Arg Cys Gln Lys Pro
Leu100 105 110Asn Pro Ala Glu Lys Arg Arg His Leu Lys Asp Lys Arg
Arg Phe His115 120 125Ser Ile Ala Gly Gln Tyr Arg Gly Gln Cys Asn
Thr Cys Cys Asp Gln130 135 140Ala Arg Gln Glu Arg Leu Arg Arg Arg
Arg Glu Thr Gln Val145 150 15552106PRTHuman papillomavirus type 45
52Met His Gly Pro Arg Ala Thr Leu Gln Glu Ile Val Leu His Leu Glu1
5 10 15Pro Gln Asn Glu Leu Asp Pro Val Asp Leu Leu Cys Tyr Glu Gln
Leu20 25 30Ser Glu Ser Glu Glu Glu Asn Asp Glu Ala Asp Gly Val Ser
His Ala35 40 45Gln Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Lys
Ile Leu Cys50 55 60Val Cys Cys Lys Cys Asp Gly Arg Ile Glu Leu Thr
Val Glu Ser Ser65 70 75
80Ala Asp Asp Leu Arg Thr Leu Gln Gln Leu Phe Leu Ser Thr Leu Ser85
90 95Phe Val Cys Pro Trp Cys Ala Thr Asn Gln100 10553536PRTHuman
papillomavirus type 45 53Met Ala His Asn Ile Ile Tyr Gly His Gly
Ile Ile Ile Phe Leu Lys1 5 10 15Asn Val Asn Val Phe Pro Ile Phe Leu
Gln Met Ala Leu Trp Arg Pro20 25 30Ser Asp Ser Thr Val Tyr Leu Pro
Pro Pro Ser Val Ala Arg Val Val35 40 45Asn Thr Asp Asp Tyr Val Ser
Arg Thr Ser Ile Phe Tyr His Ala Gly50 55 60Ser Ser Arg Leu Leu Thr
Val Gly Asn Pro Tyr Phe Arg Val Val Pro65 70 75 80Ser Gly Ala Gly
Asn Lys Gln Ala Val Pro Lys Val Ser Ala Tyr Gln85 90 95Tyr Arg Val
Phe Arg Val Ala Leu Pro Asp Pro Asn Lys Phe Gly Leu100 105 110Pro
Asp Ser Thr Ile Tyr Asn Pro Glu Thr Gln Arg Leu Val Trp Ala115 120
125Cys Val Gly Met Glu Ile Gly Arg Gly Gln Pro Leu Gly Ile Gly
Leu130 135 140Ser Gly His Pro Phe Tyr Asn Lys Leu Asp Asp Thr Glu
Ser Ala His145 150 155 160Ala Ala Thr Ala Val Ile Thr Gln Asp Val
Arg Asp Asn Val Ser Val165 170 175Asp Tyr Lys Gln Thr Gln Leu Cys
Ile Leu Gly Cys Val Pro Ala Ile180 185 190Gly Glu His Trp Ala Lys
Gly Thr Leu Cys Lys Pro Ala Gln Leu Gln195 200 205Pro Gly Asp Cys
Pro Pro Leu Glu Leu Lys Asn Thr Ile Ile Glu Asp210 215 220Gly Asp
Met Val Asp Thr Gly Tyr Gly Ala Met Asp Phe Ser Thr Leu225 230 235
240Gln Asp Thr Lys Cys Glu Val Pro Leu Asp Ile Cys Gln Ser Ile
Cys245 250 255Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp Pro Tyr
Gly Asp Ser260 265 270Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe
Ala Arg His Phe Trp275 280 285Asn Arg Ala Gly Val Met Gly Asp Thr
Val Pro Thr Asp Leu Tyr Ile290 295 300Lys Gly Thr Ser Ala Asn Met
Arg Glu Thr Pro Gly Ser Cys Val Tyr305 310 315 320Ser Pro Ser Pro
Ser Gly Ser Ile Thr Thr Ser Asp Ser Gln Leu Phe325 330 335Asn Lys
Pro Tyr Trp Leu His Lys Ala Gln Gly His Asn Asn Gly Ile340 345
350Cys Trp His Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg
Ser355 360 365Thr Asn Leu Thr Leu Cys Ala Ser Thr Gln Asn Pro Val
Pro Asn Thr370 375 380Tyr Asp Pro Thr Lys Phe Lys His Tyr Ser Arg
His Val Glu Glu Tyr385 390 395 400Asp Leu Gln Phe Ile Phe Gln Leu
Cys Thr Ile Thr Leu Thr Ala Glu405 410 415Val Met Ser Tyr Ile His
Ser Met Asn Ser Ser Ile Leu Glu Asn Trp420 425 430Asn Phe Gly Val
Pro Pro Pro Pro Thr Thr Ser Leu Val Asp Thr Tyr435 440 445Arg Phe
Val Gln Ser Val Ala Val Thr Cys Gln Lys Asp Thr Thr Pro450 455
460Pro Glu Lys Gln Asp Pro Tyr Asp Lys Leu Lys Phe Trp Thr Val
Asp465 470 475 480Leu Lys Glu Lys Phe Ser Ser Asp Leu Asp Gln Tyr
Pro Leu Gly Arg485 490 495Lys Phe Leu Val Gln Ala Gly Leu Arg Arg
Arg Pro Thr Ile Gly Pro500 505 510Arg Lys Arg Pro Ala Ala Ser Thr
Ser Thr Ala Ser Arg Pro Ala Lys515 520 525Arg Val Arg Ile Arg Ser
Lys Lys530 53554463PRTHuman papillomavirus type 45 54Met Val Ser
His Arg Ala Ala Arg Arg Lys Arg Ala Ser Ala Thr Asp1 5 10 15Leu Tyr
Arg Thr Cys Lys Gln Ser Gly Thr Cys Pro Pro Asp Val Ile20 25 30Asn
Lys Val Glu Gly Thr Thr Leu Ala Asp Lys Ile Leu Gln Trp Ser35 40
45Ser Leu Gly Ile Phe Leu Gly Gly Leu Gly Ile Gly Thr Gly Ser Gly50
55 60Ser Gly Gly Arg Thr Gly Tyr Val Pro Leu Gly Gly Arg Ser Asn
Thr65 70 75 80Val Val Asp Val Gly Pro Thr Arg Pro Pro Val Val Ile
Glu Pro Val85 90 95Gly Pro Thr Asp Pro Ser Ile Val Thr Leu Val Glu
Asp Ser Ser Val100 105 110Val Ala Ser Gly Ala Pro Val Pro Thr Phe
Thr Gly Thr Ser Gly Phe115 120 125Glu Ile Thr Ser Ser Gly Thr Thr
Thr Pro Ala Val Leu Asp Ile Thr130 135 140Pro Thr Val Asp Ser Val
Ser Ile Ser Ser Thr Ser Phe Thr Asn Pro145 150 155 160Ala Phe Ser
Asp Pro Ser Ile Ile Glu Val Pro Gln Thr Gly Glu Val165 170 175Ser
Gly Asn Ile Phe Val Gly Thr Pro Thr Ser Gly Ser His Gly Tyr180 185
190Glu Glu Ile Pro Leu Gln Thr Phe Ala Ser Ser Gly Ser Gly Thr
Glu195 200 205Pro Ile Ser Ser Thr Pro Leu Pro Thr Val Arg Arg Val
Arg Gly Pro210 215 220Arg Leu Tyr Ser Arg Ala Asn Gln Gln Val Arg
Val Ser Thr Ser Gln225 230 235 240Phe Leu Thr His Pro Ser Ser Leu
Val Thr Phe Asp Asn Pro Ala Tyr245 250 255Glu Pro Leu Asp Thr Thr
Leu Ser Phe Glu Pro Thr Ser Asn Val Pro260 265 270Asp Ser Asp Phe
Met Asp Ile Ile Arg Leu His Arg Pro Ala Leu Ser275 280 285Ser Arg
Arg Gly Thr Val Arg Phe Ser Arg Leu Gly Gln Arg Ala Thr290 295
300Met Phe Thr Arg Ser Gly Lys Gln Ile Gly Gly Arg Val His Phe
Tyr305 310 315 320His Asp Ile Ser Pro Ile Ala Ala Thr Glu Glu Ile
Glu Leu Gln Pro325 330 335Leu Ile Ser Ala Thr Asn Asp Ser Asp Leu
Phe Asp Val Tyr Ala Asp340 345 350Phe Pro Pro Pro Ala Ser Thr Thr
Pro Ser Thr Ile His Lys Ser Phe355 360 365Thr Tyr Pro Lys Tyr Ser
Leu Thr Met Pro Ser Thr Ala Ala Ser Ser370 375 380Tyr Ser Asn Val
Thr Val Pro Leu Thr Ser Ala Trp Asp Val Pro Ile385 390 395 400Tyr
Thr Gly Pro Asp Ile Ile Leu Pro Ser His Thr Pro Met Trp Pro405 410
415Ser Thr Ser Pro Thr Asn Ala Ser Thr Thr Thr Tyr Ile Gly Ile
His420 425 430Gly Thr Gln Tyr Tyr Leu Trp Pro Trp Tyr Tyr Tyr Phe
Pro Lys Lys435 440 445Arg Lys Arg Ile Pro Tyr Phe Phe Ala Asp Gly
Phe Val Ala Ala450 455 46055644PRTHuman papillomavirus type 33
55Met Ala Asp Pro Glu Gly Thr Asn Gly Ala Gly Met Gly Cys Thr Gly1
5 10 15Trp Phe Glu Val Glu Ala Val Ile Glu Arg Arg Thr Gly Asp Asn
Ile20 25 30Ser Glu Asp Glu Asp Glu Thr Ala Asp Asp Ser Gly Thr Asp
Leu Leu35 40 45Glu Phe Ile Asp Asp Ser Met Glu Asn Ser Ile Gln Ala
Asp Thr Glu50 55 60Ala Ala Arg Ala Leu Phe Asn Ile Gln Glu Gly Glu
Asp Asp Leu Asn65 70 75 80Ala Val Cys Ala Leu Lys Arg Lys Phe Ala
Ala Cys Ser Gln Ser Ala85 90 95Ala Glu Asp Val Val Asp Arg Ala Ala
Asn Pro Cys Arg Thr Ser Ile100 105 110Asn Lys Asn Lys Glu Cys Thr
Tyr Arg Lys Arg Lys Ile Asp Glu Leu115 120 125Glu Asp Ser Gly Tyr
Gly Asn Thr Glu Val Glu Thr Gln Gln Met Val130 135 140Gln Gln Val
Glu Ser Gln Asn Gly Asp Thr Asn Leu Asn Asp Leu Glu145 150 155
160Ser Ser Gly Val Gly Asp Asp Ser Glu Val Ser Cys Glu Thr Asn
Val165 170 175Asp Ser Cys Glu Asn Val Thr Leu Gln Glu Ile Ser Asn
Val Leu His180 185 190Ser Ser Asn Thr Lys Ala Asn Ile Leu Tyr Lys
Phe Lys Glu Ala Tyr195 200 205Gly Ile Ser Phe Met Glu Leu Val Arg
Pro Phe Lys Ser Asp Lys Thr210 215 220Ser Cys Thr Asp Trp Cys Ile
Thr Gly Tyr Gly Ile Ser Pro Ser Val225 230 235 240Ala Glu Ser Leu
Lys Val Leu Ile Lys Gln His Ser Leu Tyr Thr His245 250 255Leu Gln
Cys Leu Thr Cys Asp Arg Gly Ile Ile Ile Leu Leu Leu Ile260 265
270Arg Phe Arg Cys Ser Lys Asn Arg Leu Thr Val Ala Lys Leu Met
Ser275 280 285Asn Leu Leu Ser Ile Pro Glu Thr Cys Met Val Ile Glu
Pro Pro Lys290 295 300Leu Arg Ser Gln Thr Cys Ala Leu Tyr Trp Phe
Arg Thr Ala Met Ser305 310 315 320Asn Ile Ser Asp Val Gln Gly Thr
Thr Pro Glu Trp Ile Asp Arg Leu325 330 335Thr Val Leu Gln His Ser
Phe Asn Asp Asn Ile Phe Asp Leu Ser Glu340 345 350Met Val Gln Trp
Ala Tyr Asp Asn Glu Leu Thr Asp Asp Ser Asp Ile355 360 365Ala Tyr
Tyr Tyr Ala Gln Leu Ala Asp Ser Asn Ser Asn Ala Ala Ala370 375
380Phe Leu Lys Ser Asn Ser Gln Ala Lys Ile Val Lys Asp Cys Gly
Ile385 390 395 400Met Cys Arg His Tyr Lys Lys Ala Glu Lys Arg Lys
Met Ser Ile Gly405 410 415Gln Trp Ile Gln Ser Arg Cys Glu Lys Thr
Asn Asp Gly Gly Asn Trp420 425 430Arg Pro Ile Val Gln Leu Leu Arg
Tyr Gln Asn Ile Glu Phe Thr Ala435 440 445Phe Leu Gly Ala Phe Lys
Lys Phe Leu Lys Gly Ile Pro Lys Lys Ser450 455 460Cys Met Leu Ile
Cys Gly Pro Ala Asn Thr Gly Lys Ser Tyr Phe Gly465 470 475 480Met
Ser Leu Ile Gln Phe Leu Lys Gly Cys Val Ile Ser Cys Val Asn485 490
495Ser Lys Ser His Phe Trp Leu Gln Pro Leu Ser Asp Ala Lys Ile
Gly500 505 510Met Ile Asp Asp Val Thr Pro Ile Ser Trp Thr Tyr Ile
Asp Asp Tyr515 520 525Met Arg Asn Ala Leu Asp Gly Asn Glu Ile Ser
Ile Asp Val Lys His530 535 540Arg Ala Leu Val Gln Leu Lys Cys Pro
Pro Leu Leu Leu Thr Ser Asn545 550 555 560Thr Asn Ala Gly Thr Asp
Ser Arg Trp Pro Tyr Leu His Ser Arg Leu565 570 575Thr Val Phe Glu
Phe Lys Asn Pro Phe Pro Phe Asp Glu Asn Gly Asn580 585 590Pro Val
Tyr Ala Ile Asn Asp Glu Asn Trp Lys Ser Phe Phe Ser Arg595 600
605Thr Trp Cys Lys Leu Asp Leu Ile Glu Glu Glu Asp Lys Glu Asn
His610 615 620Gly Gly Asn Ile Ser Thr Phe Lys Cys Ser Ala Gly Glu
Asn Thr Arg625 630 635 640Ser Leu Arg Ser56353PRTHuman
papillomavirus type 33 56Met Glu Glu Ile Ser Ala Arg Leu Asn Ala
Val Gln Glu Lys Ile Leu1 5 10 15Asp Leu Tyr Glu Ala Asp Lys Thr Asp
Leu Pro Ser Gln Ile Glu His20 25 30Trp Lys Leu Ile Arg Met Glu Cys
Ala Leu Leu Tyr Thr Ala Lys Gln35 40 45Met Gly Phe Ser His Leu Cys
His Gln Val Val Pro Ser Leu Leu Ala50 55 60Ser Lys Thr Lys Ala Phe
Gln Val Ile Glu Leu Gln Met Ala Leu Glu65 70 75 80Thr Leu Ser Lys
Ser Gln Tyr Ser Thr Ser Gln Trp Thr Leu Gln Gln85 90 95Thr Ser Leu
Glu Val Trp Leu Cys Glu Pro Pro Lys Cys Phe Lys Lys100 105 110Gln
Gly Glu Thr Val Thr Val Gln Tyr Asp Asn Asp Lys Lys Asn Thr115 120
125Met Asp Tyr Thr Asn Trp Gly Glu Ile Tyr Ile Ile Glu Glu Asp
Thr130 135 140Cys Thr Met Val Thr Gly Lys Val Asp Tyr Ile Gly Met
Tyr Tyr Ile145 150 155 160His Asn Cys Glu Lys Val Tyr Phe Lys Tyr
Phe Lys Glu Asp Ala Ala165 170 175Lys Tyr Ser Lys Thr Gln Met Trp
Glu Val His Val Gly Gly Gln Val180 185 190Ile Val Cys Pro Thr Ser
Ile Ser Ser Asn Gln Ile Ser Thr Thr Glu195 200 205Thr Ala Asp Ile
Gln Thr Asp Asn Asp Asn Arg Pro Pro Gln Ala Ala210 215 220Ala Lys
Arg Arg Arg Pro Ala Asp Thr Thr Asp Thr Ala Gln Pro Leu225 230 235
240Thr Lys Leu Phe Cys Ala Asp Pro Ala Leu Asp Asn Arg Thr Ala
Arg245 250 255Thr Ala Thr Asn Cys Thr Asn Lys Gln Arg Thr Val Cys
Ser Ser Asn260 265 270Val Ala Pro Ile Val His Leu Lys Gly Glu Ser
Asn Ser Leu Lys Cys275 280 285Leu Arg Tyr Arg Leu Lys Pro Tyr Lys
Glu Leu Tyr Ser Ser Met Ser290 295 300Ser Thr Trp His Trp Thr Ser
Asp Asn Lys Asn Ser Lys Asn Gly Ile305 310 315 320Val Thr Val Thr
Phe Val Thr Glu Gln Gln Gln Gln Met Phe Leu Gly325 330 335Thr Val
Lys Ile Pro Pro Thr Val Gln Ile Ser Thr Gly Phe Met Thr340 345
350Leu5775PRTHuman papillomavirus type 33 57Met Ile Phe Val Phe Val
Leu Cys Phe Ile Leu Phe Leu Cys Leu Ser1 5 10 15Leu Leu Leu Arg Pro
Leu Ile Leu Ser Ile Ser Thr Tyr Ala Trp Leu20 25 30Leu Val Leu Val
Leu Leu Leu Trp Val Phe Val Gly Ser Pro Leu Lys35 40 45Ile Phe Phe
Cys Tyr Leu Leu Phe Leu Tyr Leu Pro Met Met Cys Ile50 55 60Asn Phe
His Ala Gln His Met Thr Gln Gln Glu65 70 7558149PRTHuman
papillomavirus type 33 58Met Phe Gln Asp Thr Glu Glu Lys Pro Arg
Thr Leu His Asp Leu Cys1 5 10 15Gln Ala Leu Glu Thr Thr Ile His Asn
Ile Glu Leu Gln Cys Val Glu20 25 30Cys Lys Lys Pro Leu Gln Arg Ser
Glu Val Tyr Asp Phe Ala Phe Ala35 40 45Asp Leu Thr Val Val Tyr Arg
Glu Gly Asn Pro Phe Gly Ile Cys Lys50 55 60Leu Cys Leu Arg Phe Leu
Ser Lys Ile Ser Glu Tyr Arg His Tyr Asn65 70 75 80Tyr Ser Val Tyr
Gly Asn Thr Leu Glu Gln Thr Val Lys Lys Pro Leu85 90 95Asn Glu Ile
Leu Ile Arg Cys Ile Ile Cys Gln Arg Pro Leu Cys Pro100 105 110Gln
Glu Lys Lys Arg His Val Asp Leu Asn Lys Arg Phe His Asn Ile115 120
125Ser Gly Arg Trp Ala Gly Arg Cys Ala Ala Cys Trp Arg Ser Arg
Arg130 135 140Arg Glu Thr Ala Leu1455997PRTHuman papillomavirus
type 33 59Met Arg Gly His Lys Pro Thr Leu Lys Glu Tyr Val Leu Asp
Leu Tyr1 5 10 15Pro Glu Pro Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Ser
Asp Ser Ser20 25 30Asp Glu Asp Glu Gly Leu Asp Arg Pro Asp Gly Gln
Ala Gln Pro Ala35 40 45Thr Ala Asp Tyr Tyr Ile Val Thr Cys Cys His
Thr Cys Asn Thr Thr50 55 60Val Arg Leu Cys Val Asn Ser Thr Ala Ser
Asp Leu Arg Thr Ile Gln65 70 75 80Gln Leu Leu Met Gly Thr Val Asn
Ile Val Cys Pro Thr Cys Ala Gln85 90 95Gln60499PRTHuman
papillomavirus type 33 60Met Ser Val Trp Arg Pro Ser Glu Ala Thr
Val Tyr Leu Pro Pro Val1 5 10 15Pro Val Ser Lys Val Val Ser Thr Asp
Glu Tyr Val Ser Arg Thr Ser20 25 30Ile Tyr Tyr Tyr Ala Gly Ser Ser
Arg Leu Leu Ala Val Gly His Pro35 40 45Tyr Phe Ser Ile Lys Asn Pro
Thr Asn Ala Lys Lys Leu Leu Val Pro50 55 60Lys Val Ser Gly Leu Gln
Tyr Arg Val Phe Arg Val Arg Leu Pro Asp65 70 75 80Pro Asn Lys Phe
Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr85 90 95Gln Arg Leu
Val Trp Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln100 105 110Pro
Leu Gly Val Gly Ile Ser Gly His Pro Leu Leu Asn Lys Phe Asp115 120
125Asp Thr Glu Thr Gly Asn Lys Tyr Pro Gly Gln Pro Gly Ala Asp
Asn130 135 140Arg Glu Cys Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu
Cys Leu Leu145 150 155 160Gly Cys Lys Pro Pro Thr Gly Glu His Trp
Gly Lys Gly Val Ala Cys165 170 175Thr Asn Ala Ala Pro Ala Asn Asp
Cys Pro Pro Leu Glu Leu Ile Asn180 185 190Thr Ile Ile Glu Asp Gly
Asp Met Val Asp Thr Gly Phe Gly Cys Met195 200 205Asp Phe Lys Thr
Leu Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile210 215 220Cys Gly
Ser Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Thr Ser Glu225 230 235
240Pro Tyr Gly Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met
Phe245 250 255Val Arg His Phe Phe Asn Arg Ala Gly Thr Leu Gly Glu
Ala Val Pro260 265 270Asp Asp Leu Tyr Ile Lys Gly Ser Gly Thr Thr
Ala
Ser Ile Gln Ser275 280 285Ser Ala Phe Phe Pro Thr Pro Ser Gly Ser
Met Val Thr Ser Glu Ser290 295 300Gln Leu Phe Asn Lys Pro Tyr Trp
Leu Gln Arg Ala Gln Gly His Asn305 310 315 320Asn Gly Ile Cys Trp
Gly Asn Gln Val Phe Val Thr Val Val Asp Thr325 330 335Thr Arg Ser
Thr Asn Met Thr Leu Cys Thr Gln Val Thr Ser Asp Ser340 345 350Thr
Tyr Lys Asn Glu Asn Phe Lys Glu Tyr Ile Arg His Val Glu Glu355 360
365Tyr Asp Leu Gln Phe Val Phe Gln Leu Cys Lys Val Thr Leu Thr
Ala370 375 380Glu Val Met Thr Tyr Ile His Ala Met Asn Pro Asp Ile
Leu Glu Asp385 390 395 400Trp Gln Phe Gly Leu Thr Pro Pro Pro Ser
Ala Ser Leu Gln Asp Thr405 410 415Tyr Arg Phe Val Thr Ser Gln Ala
Ile Thr Cys Gln Lys Thr Val Pro420 425 430Pro Lys Glu Lys Glu Asp
Pro Leu Gly Lys Tyr Thr Phe Trp Glu Val435 440 445Asp Leu Lys Glu
Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly450 455 460Arg Lys
Phe Leu Leu Gln Ala Gly Leu Lys Ala Lys Pro Lys Leu Lys465 470 475
480Arg Ala Ala Pro Thr Ser Thr Arg Thr Ser Ser Ala Lys Arg Lys
Lys485 490 495Val Lys Lys61467PRTHuman papillomavirus type 33 61Met
Arg His Lys Arg Ser Thr Arg Arg Lys Arg Ala Ser Ala Thr Gln1 5 10
15Leu Tyr Gln Thr Cys Lys Ala Thr Gly Thr Cys Pro Pro Asp Val Ile20
25 30Pro Lys Val Glu Gly Ser Thr Ile Ala Asp Gln Ile Leu Lys Tyr
Gly35 40 45Ser Leu Gly Val Phe Phe Gly Gly Leu Gly Ile Gly Thr Gly
Ser Gly50 55 60Ser Gly Gly Arg Thr Gly Tyr Val Pro Ile Gly Thr Asp
Pro Pro Thr65 70 75 80Ala Ala Ile Pro Leu Gln Pro Ile Arg Pro Pro
Val Thr Val Asp Thr85 90 95Val Gly Pro Leu Asp Ser Ser Ile Val Ser
Leu Ile Glu Glu Thr Ser100 105 110Phe Ile Glu Ala Gly Ala Pro Ala
Pro Ser Ile Pro Thr Pro Ser Gly115 120 125Phe Asp Val Thr Thr Ser
Ala Asp Thr Thr Pro Ala Ile Ile Asn Val130 135 140Ser Ser Val Gly
Glu Ser Ser Ile Gln Thr Ile Ser Thr His Leu Asn145 150 155 160Pro
Thr Phe Thr Glu Pro Ser Val Leu His Pro Pro Ala Pro Ala Glu165 170
175Ala Ser Gly His Phe Ile Phe Ser Ser Pro Thr Val Ser Thr Gln
Ser180 185 190Tyr Glu Asn Ile Pro Met Asp Thr Phe Val Val Ser Thr
Asp Ser Ser195 200 205Asn Val Thr Ser Ser Thr Pro Ile Pro Gly Ser
Arg Pro Val Ala Arg210 215 220Leu Gly Leu Tyr Ser Arg Asn Thr Gln
Gln Val Lys Val Val Asp Pro225 230 235 240Ala Phe Leu Thr Ser Pro
His Lys Leu Ile Thr Tyr Asp Asn Pro Ala245 250 255Phe Glu Ser Phe
Asp Pro Glu Asp Thr Leu Gln Phe Gln His Ser Asp260 265 270Ile Ser
Pro Ala Pro Asp Pro Asp Phe Leu Asp Ile Ile Ala Leu His275 280
285Arg Pro Ala Ile Thr Ser Arg Arg His Thr Val Arg Phe Ser Arg
Val290 295 300Gly Gln Lys Ala Thr Leu Lys Thr Arg Ser Gly Lys Gln
Ile Gly Ala305 310 315 320Arg Ile His Tyr Tyr Gln Asp Leu Ser Pro
Ile Val Pro Leu Asp His325 330 335Thr Val Pro Asn Glu Gln Tyr Glu
Leu Gln Pro Leu His Asp Thr Ser340 345 350Thr Ser Ser Tyr Ser Ile
Asn Asp Gly Leu Tyr Asp Val Tyr Ala Asp355 360 365Asp Val Asp Asn
Val His Thr Pro Met Gln His Ser Tyr Ser Thr Phe370 375 380Ala Thr
Thr Arg Thr Ser Asn Val Ser Ile Pro Leu Asn Thr Gly Phe385 390 395
400Asp Thr Pro Val Met Ser Gly Pro Asp Ile Pro Ser Pro Leu Phe
Pro405 410 415Thr Ser Ser Pro Phe Val Pro Ile Ser Pro Phe Phe Pro
Phe Asp Thr420 425 430Ile Val Val Asp Gly Ala Asp Phe Val Leu His
Pro Ser Tyr Phe Ile435 440 445Leu Arg Arg Arg Arg Lys Arg Phe Pro
Tyr Phe Phe Thr Asp Val Arg450 455 460Val Ala Ala46562310PRTHuman
papillomavirus type 56 62Met Val Pro Cys Leu Gln Val Cys Lys Ala
Lys Ala Cys Ser Ala Ile1 5 10 15Glu Val Gln Ile Ala Leu Glu Ser Leu
Ser Thr Thr Ile Tyr Asn Asn20 25 30Glu Glu Trp Thr Leu Arg Asp Thr
Cys Glu Glu Leu Trp Leu Thr Glu35 40 45Pro Lys Lys Cys Phe Lys Lys
Glu Gly Gln His Ile Glu Val Trp Phe50 55 60Asp Gly Ser Lys Asn Asn
Cys Met Gln Tyr Val Ala Trp Lys Tyr Ile65 70 75 80Tyr Tyr Asn Gly
Asp Cys Gly Trp Gln Lys Val Cys Ser Gly Val Asp85 90 95Tyr Arg Gly
Ile Tyr Tyr Val His Asp Gly His Lys Thr Tyr Tyr Thr100 105 110Asp
Phe Glu Gln Glu Ala Lys Lys Phe Gly Cys Lys Asn Ile Trp Glu115 120
125Val His Met Glu Asn Glu Ser Ile Tyr Cys Pro Asp Ser Val Ser
Ser130 135 140Thr Cys Arg Tyr Asn Val Ser Pro Val Glu Thr Val Asn
Glu Tyr Asn145 150 155 160Thr His Lys Thr Thr Thr Thr Thr Ser Thr
Ser Val Gly Asn Gln Asp165 170 175Ala Ala Val Ser His Arg Pro Gly
Lys Arg Pro Arg Leu Arg Glu Ser180 185 190Glu Phe Asp Ser Ser Arg
Glu Ser His Ala Lys Cys Val Thr Thr His195 200 205Thr His Ile Ser
Asp Thr Asp Asn Thr Asp Ser Arg Ser Arg Ser Ile210 215 220Asn Asn
Asn Asn His Pro Gly Asp Lys Thr Thr Pro Val Val His Leu225 230 235
240Lys Gly Glu Pro Asn Arg Leu Lys Cys Cys Arg Tyr Arg Phe Gln
Lys245 250 255Tyr Lys Thr Leu Phe Val Asp Val Thr Ser Thr Tyr His
Trp Thr Ser260 265 270Thr Asp Asn Lys Asn Tyr Ser Ile Ile Thr Ile
Ile Tyr Lys Asp Glu275 280 285Thr Gln Arg Asn Ser Phe Leu Ser His
Val Lys Ile Pro Val Val Tyr290 295 300Arg Leu Val Trp Asp Lys305
31063155PRTHuman papillomavirus type 56 63Met Glu Pro Gln Phe Asn
Asn Pro Gln Glu Arg Pro Arg Ser Leu His1 5 10 15His Leu Ser Glu Val
Leu Glu Ile Pro Leu Ile Asp Leu Arg Leu Ser20 25 30Cys Val Tyr Cys
Lys Lys Glu Leu Thr Arg Ala Glu Val Tyr Asn Phe35 40 45Ala Cys Thr
Glu Leu Lys Leu Val Tyr Arg Asp Asp Phe Pro Tyr Ala50 55 60Val Cys
Arg Val Cys Leu Leu Phe Tyr Ser Lys Val Arg Lys Tyr Arg65 70 75
80Tyr Tyr Asp Tyr Ser Val Tyr Gly Ala Thr Leu Glu Ser Ile Thr Lys85
90 95Lys Gln Leu Cys Asp Leu Leu Ile Arg Cys Tyr Arg Cys Gln Ser
Pro100 105 110Leu Thr Pro Glu Glu Lys Gln Leu His Cys Asp Arg Lys
Arg Arg Phe115 120 125His Leu Ile Ala His Gly Trp Thr Gly Ser Cys
Leu Gly Cys Trp Arg130 135 140Gln Thr Ser Arg Glu Pro Arg Glu Ser
Thr Val145 150 15564105PRTHuman papillomavirus type 56 64Met His
Gly Lys Val Pro Thr Leu Gln Asp Val Val Leu Glu Leu Thr1 5 10 15Pro
Gln Thr Glu Ile Asp Leu Gln Cys Asn Glu Gln Leu Asp Ser Ser20 25
30Glu Asp Glu Asp Glu Asp Glu Val Asp His Leu Gln Glu Arg Pro Gln35
40 45Gln Ala Arg Gln Ala Lys Gln His Thr Cys Tyr Leu Ile His Val
Pro50 55 60Cys Cys Glu Cys Lys Phe Val Val Gln Leu Asp Ile Gln Ser
Thr Lys65 70 75 80Glu Asp Leu Arg Val Val Gln Gln Leu Leu Met Gly
Ala Leu Thr Val85 90 95Thr Cys Pro Leu Cys Ala Ser Ser Asn100
10565534PRTHuman papillomavirus type 56 65Met Met Leu Pro Met Met
Tyr Ile Tyr Arg Asp Pro Pro Leu His Tyr1 5 10 15Gly Leu Cys Ile Phe
Leu Asp Val Gly Ala Val Asn Val Phe Pro Ile20 25 30Phe Leu Gln Met
Ala Thr Trp Arg Pro Ser Glu Asn Lys Val Tyr Leu35 40 45Pro Pro Thr
Pro Val Ser Lys Val Val Ala Thr Asp Ser Tyr Val Lys50 55 60Arg Thr
Ser Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Ala Val65 70 75
80Gly His Pro Tyr Tyr Ser Val Thr Lys Asp Asn Thr Lys Thr Asn Ile85
90 95Pro Lys Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Arg Leu
Pro100 105 110Asp Pro Asn Lys Phe Gly Leu Pro Asp Thr Asn Ile Tyr
Asn Pro Asp115 120 125Gln Glu Arg Leu Val Trp Ala Cys Val Gly Leu
Glu Val Gly Arg Gly130 135 140Gln Pro Leu Gly Ala Gly Leu Ser Gly
His Pro Leu Phe Asn Arg Leu145 150 155 160Asp Asp Thr Glu Ser Ser
Asn Leu Ala Asn Asn Asn Val Ile Glu Asp165 170 175Ser Arg Asp Asn
Ile Ser Val Asp Gly Lys Gln Thr Gln Leu Cys Ile180 185 190Val Gly
Cys Thr Pro Ala Met Gly Glu His Trp Thr Lys Gly Ala Val195 200
205Cys Lys Ser Thr Gln Val Thr Thr Gly Asp Cys Pro Pro Leu Ala
Leu210 215 220Ile Asn Thr Pro Ile Glu Asp Gly Asp Met Ile Asp Thr
Gly Phe Gly225 230 235 240Ala Met Asp Phe Lys Val Leu Gln Glu Ser
Lys Ala Glu Val Pro Leu245 250 255Asp Ile Val Gln Ser Thr Cys Lys
Tyr Pro Asp Tyr Leu Lys Met Ser260 265 270Ala Asp Ala Tyr Gly Asp
Ser Met Trp Phe Tyr Leu Arg Arg Glu Gln275 280 285Leu Phe Ala Arg
His Tyr Phe Asn Arg Ala Gly Lys Val Gly Glu Thr290 295 300Ile Pro
Ala Glu Leu Tyr Leu Lys Gly Ser Asn Gly Arg Glu Pro Pro305 310 315
320Pro Ser Ser Val Tyr Val Ala Thr Pro Ser Gly Ser Met Ile Thr
Ser325 330 335Glu Ala Gln Leu Phe Asn Lys Pro Tyr Trp Leu Gln Arg
Ala Gln Gly340 345 350His Asn Asn Gly Ile Cys Trp Gly Asn Gln Leu
Phe Val Thr Val Val355 360 365Asp Thr Thr Arg Ser Thr Asn Met Thr
Ile Ser Thr Ala Thr Glu Gln370 375 380Leu Ser Lys Tyr Asp Ala Arg
Lys Ile Asn Gln Tyr Leu Arg His Val385 390 395 400Glu Glu Tyr Glu
Leu Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu405 410 415Ser Ala
Glu Val Met Ala Tyr Leu His Asn Met Asn Ala Asn Leu Leu420 425
430Glu Asp Trp Asn Ile Gly Leu Ser Pro Pro Val Ala Thr Ser Leu
Glu435 440 445Asp Lys Tyr Arg Tyr Val Arg Ser Thr Ala Ile Thr Cys
Gln Arg Glu450 455 460Gln Pro Pro Thr Glu Lys Gln Asp Pro Leu Ala
Lys Tyr Lys Phe Trp465 470 475 480Asp Val Asn Leu Gln Asp Ser Phe
Ser Thr Asp Leu Asp Gln Phe Pro485 490 495Leu Gly Arg Lys Phe Leu
Met Gln Leu Gly Thr Arg Ser Lys Pro Ala500 505 510Val Ala Thr Ser
Lys Lys Arg Ser Ala Pro Thr Ser Thr Ser Thr Pro515 520 525Ala Lys
Arg Lys Arg Arg53066464PRTHuman papillomavirus type 56 66Met Val
Ala His Arg Ala Thr Arg Arg Lys Arg Ala Ser Ala Thr Gln1 5 10 15Leu
Tyr Lys Thr Cys Lys Leu Ser Gly Thr Cys Pro Glu Asp Val Val20 25
30Asn Lys Ile Glu Gln Lys Thr Trp Ala Asp Lys Ile Leu Gln Trp Gly35
40 45Ser Leu Phe Thr Tyr Phe Gly Gly Leu Gly Ile Gly Thr Gly Thr
Gly50 55 60Ser Gly Gly Arg Ala Gly Tyr Val Pro Leu Gly Ser Arg Pro
Ser Thr65 70 75 80Ile Val Asp Val Thr Pro Ala Arg Pro Pro Ile Val
Val Glu Ser Val85 90 95Gly Pro Thr Asp Pro Ser Ile Val Thr Leu Val
Glu Glu Ser Ser Val100 105 110Ile Glu Ser Gly Ala Gly Ile Pro Asn
Phe Thr Gly Ser Gly Gly Phe115 120 125Glu Ile Thr Ser Ser Ser Thr
Thr Thr Pro Ala Val Leu Asp Ile Thr130 135 140Pro Thr Ser Ser Thr
Val His Val Ser Ser Thr His Ile Thr Asn Pro145 150 155 160Leu Phe
Ile Asp Pro Pro Val Ile Glu Ala Pro Gln Thr Gly Glu Val165 170
175Ser Gly Asn Ile Leu Ile Ser Thr Pro Thr Ser Gly Ile His Ser
Tyr180 185 190Glu Glu Ile Pro Met Gln Thr Phe Ala Val His Gly Ser
Gly Thr Glu195 200 205Pro Ile Ser Ser Thr Pro Ile Pro Gly Phe Arg
Arg Ile Ala Ala Pro210 215 220Arg Leu Tyr Arg Lys Ala Phe Gln Gln
Val Lys Val Thr Asp Pro Ala225 230 235 240Phe Leu Asp Arg Pro Ala
Thr Leu Val Ser Ala Asp Asn Pro Leu Phe245 250 255Glu Gly Thr Asp
Thr Ser Leu Ala Phe Ser Pro Ser Gly Val Ala Pro260 265 270Asp Pro
Asp Phe Met Asn Ile Val Ala Leu His Arg Pro Ala Phe Thr275 280
285Thr Arg Arg Gly Gly Val Arg Phe Ser Arg Leu Gly Arg Lys Ala
Thr290 295 300Ile Gln Thr Arg Arg Gly Thr Gln Ile Gly Ala Arg Val
His Tyr Tyr305 310 315 320Tyr Asp Ile Ser Pro Ile Ala Gln Ala Glu
Glu Ile Glu Met Gln Pro325 330 335Leu Leu Ser Ala Asn Asn Ser Phe
Asp Gly Leu Tyr Asp Ile Tyr Ala340 345 350Asn Ile Asp Asp Glu Ala
Pro Gly Leu Ser Ser Gln Ser Val Ala Thr355 360 365Pro Ser Ala His
Leu Pro Ile Lys Pro Ser Thr Leu Ser Phe Ala Ser370 375 380Asn Thr
Thr Asn Val Thr Ala Pro Leu Gly Asn Val Trp Glu Thr Pro385 390 395
400Phe Tyr Ser Gly Pro Asp Ile Val Leu Pro Thr Gly Pro Ser Thr
Trp405 410 415Pro Phe Val Pro Gln Ser Pro Tyr Asp Val Thr His Asp
Val Tyr Ile420 425 430Gln Gly Ser Ser Phe Ala Leu Trp Pro Val Tyr
Phe Phe Arg Arg Arg435 440 445Arg Arg Lys Arg Ile Pro Tyr Phe Phe
Ala Asp Gly Asp Val Ala Ala450 455 4606714PRTArtificial
SequenceDescription of Artificial Sequencetetanus toxoid positions
830-843 67Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu1
5 106821PRTArtificial SequenceDescription of Artificial
SequencePlasmodium falciparum circumsporozoite (CS) protein
positions 378-398 68Asp Ile Glu Lys Lys Ile Ala Lys Met Glu Lys Ala
Ser Ser Val Phe1 5 10 15Asn Val Val Asn Ser206916PRTArtificial
SequenceDescription of Artificial Sequence Streptococcus18kD
protein position 116 69Gly Ala Val Asp Ser Ile Leu Gly Gly Val Ala
Thr Tyr Gly Ala Ala1 5 10 157013PRTArtificial SequenceDescription
of Artificial Sequencepan-DR binding epitope peptide 70Ala Lys Xaa
Val Ala Ala Trp Thr Leu Lys Ala Ala Ala1 5 10719PRTArtificial
SequenceDescription of Artificial Sequencestandard peptide 944.02
71Tyr Leu Glu Pro Ala Ile Ala Lys Tyr1 57210PRTArtificial
SequenceDescription of Artificial Sequencestandard peptide 941.01
72Phe Leu Pro Ser Asp Tyr Phe Pro Ser Val1 5 10739PRTArtificial
SequenceDescription of Artificial Sequencestandard peptide 1072.34
73Tyr Val Ile Lys Val Ser Ala Arg Val1 57410PRTArtificial
SequenceDescription of Artificial Sequencestandard peptide 941.12
74Lys Val Phe Pro Tyr Ala Leu Ile Asn Lys1 5 10759PRTArtificial
SequenceDescription of Artificial Sequencestandard peptide 940.06
75Ala Val Asp Leu Tyr His Phe Leu Lys1 57611PRTArtificial
SequenceDescription of Artificial Sequencestandard peptide 1083.02
76Ser Thr Leu Pro Glu Thr Tyr Val Val Arg Arg1 5 10779PRTArtificial
SequenceDescription of Artificial Sequencestandard peptide 979.02
77Ala Tyr Ile Asp Asn Tyr Asn Lys Phe1 5789PRTArtificial
SequenceDescription of Artificial Sequencestandard peptide 1075.23
78Ala Pro Arg Thr Leu Val Tyr Leu Leu1 5799PRTArtificial
SequenceDescription of Artificial Sequencestandard peptide 1021.05
79Phe Pro Phe Lys Tyr Ala Ala Ala Phe1 58013PRTArtificial
SequenceDescription of Artificial Sequencestandard peptide 515.01
80Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr1 5
108112PRTArtificial SequenceDescription of Artificial
Sequencestandard
peptide 829.02 81Tyr Lys Thr Ile Ala Phe Asp Glu Glu Ala Arg Arg1 5
108214PRTArtificial SequenceDescription of Artificial
Sequencestandard peptide 717.01 82Tyr Ala Arg Phe Gln Ser Gln Thr
Thr Leu Lys Gln Lys Thr1 5 108315PRTArtificial SequenceDescription
of Artificial Sequencestandard peptide 1200.05 83Glu Ala Leu Ile
His Gln Leu Lys Ile Asn Pro Tyr Val Leu Ser1 5 10
158414PRTArtificial SequenceDescription of Artificial
Sequencestandard peptide 650.22 84Gln Tyr Ile Lys Ala Asn Ala Lys
Phe Ile Gly Ile Thr Glu1 5 108524PRTArtificial SequenceDescription
of Artificial Sequencestandard peptide 507.02 85Gly Arg Thr Gln Asp
Glu Asn Pro Val Val His Phe Phe Lys Asn Ile1 5 10 15Val Thr Pro Arg
Thr Pro Pro Pro208613PRTArtificial SequenceDescription of
Artificial Sequencestandard peptide 511 86Asn Gly Gln Ile Gly Asn
Asp Pro Asn Arg Asp Ile Leu1 5 10879PRTArtificial SequenceDR7
preferred motif 87Xaa Met Trp Ala Xaa Xaa Met Xaa Xaa1
5889PRTArtificial SequenceDR7 deleterious motif 88Xaa Cys Xaa Gly
Xaa Xaa Xaa Asn Gly1 5
* * * * *
References