U.S. patent application number 13/618996 was filed with the patent office on 2013-01-17 for inducing cellular immune responses to human papillomavirus using peptide and nucleic acid compositions.
The applicant listed for this patent is Esteban Celis, Robert Chestnut, Howard M. Grey, Alessandro SETTE, John Sidney, Scott Southwood. Invention is credited to Esteban Celis, Robert Chestnut, Howard M. Grey, Alessandro SETTE, John Sidney, Scott Southwood.
Application Number | 20130017253 13/618996 |
Document ID | / |
Family ID | 26868370 |
Filed Date | 2013-01-17 |
United States Patent
Application |
20130017253 |
Kind Code |
A1 |
SETTE; Alessandro ; et
al. |
January 17, 2013 |
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) ; Chestnut;
Robert; (Cardiff-by-the-Sea, CA) ; Celis;
Esteban; (Rochester, MN) ; Grey; Howard M.;
(La Jolla, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SETTE; Alessandro
Sidney; John
Southwood; Scott
Chestnut; Robert
Celis; Esteban
Grey; Howard M. |
La Jolla
San Diego
Santee
Cardiff-by-the-Sea
Rochester
La Jolla |
CA
CA
CA
CA
MN
CA |
US
US
US
US
US
US |
|
|
Family ID: |
26868370 |
Appl. No.: |
13/618996 |
Filed: |
September 14, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12292281 |
Nov 14, 2008 |
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13618996 |
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10149136 |
Oct 29, 2002 |
7572882 |
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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; 435/325; 530/324; 530/327; 530/328; 530/394;
536/23.72 |
Current CPC
Class: |
A61K 39/12 20130101;
A61K 2039/55555 20130101; C12N 2710/20022 20130101; C12N 2710/20034
20130101; A61K 2039/5158 20130101; A61P 35/00 20180101; A61P 31/12
20180101; A61K 2039/53 20130101; A61P 31/20 20180101; A61K 2039/57
20130101; A61P 37/04 20180101; C07K 14/005 20130101; A61K
2039/55516 20130101 |
Class at
Publication: |
424/450 ;
530/328; 530/327; 530/394; 530/324; 424/186.1; 536/23.72;
435/325 |
International
Class: |
C07K 7/06 20060101
C07K007/06; C07K 14/79 20060101 C07K014/79; C07K 14/025 20060101
C07K014/025; C12N 5/0783 20100101 C12N005/0783; C12N 15/37 20060101
C12N015/37; A61P 37/04 20060101 A61P037/04; C12N 5/078 20100101
C12N005/078; A61K 9/127 20060101 A61K009/127; A61K 39/12 20060101
A61K039/12 |
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 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 acuminate 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 I 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 I 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 hound, 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. 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.
[0034] 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.
[0035] 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.
[0036] "Human Leukocyte Antigen" or "HLA" is a human class I or
class II Major Histocompatibility Complex (MI-IC) protein (see,
e.g., Stites, et al., IMMUNOLOGY, 8.sup.TH ED., Lange Publishing,
Los Altos, Calif. (1994).
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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 plasmoin 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).
[0041] As used herein, "high affinity" with respect to HLA class I
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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] "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.
[0046] "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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] "Pharmaceutically acceptable" refers to a non-toxic, inert,
and/or physiologically compatible composition.
[0054] 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.
[0055] 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.
[0056] "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.
[0057] 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.
[0058] The term "residue" refers to an amino acid or amino acid
mimetic incorporated into an oligopeptide by an amide bond or amide
bond mimetic.
[0059] 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.
[0060] 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.
[0061] 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 HL A antigens.
[0062] "Synthetic peptide" refers to a peptide that is man-made
using such methods as chemical synthesis or recombinant DNA
technology.
[0063] 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.
[0064] 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
[0065] 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.
[0066] 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;
Ranmmensee, 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).
[0067] 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. R. et al., Science 257:919, 1992;
Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol.
219:277, 1991.)
[0068] 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).
[0069] 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.
[0070] Various strategies can be utilized to evaluate
immunogenicity, including:
[0071] 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.51Cr-release assay involving
peptide sensitized target cells.
[0072] 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.
[0073] 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.
[0074] The following describes the peptide epitopes and
corresponding nucleic acids of the invention.
Binding Affinity of Peptide Epitopes for HLA Molecules
[0075] 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.
[0076] 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.
[0077] 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. Is
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.
[0078] 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 nM 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).
[0079] 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 nM
can be defined as an affinity threshold associated with
immunogenicity in the context of DR molecules.
[0080] 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.
[0081] The binding affinity of peptides for HLA molecules can be
determined as described in Example 1, below.
Peptide Epitope Binding Motifs and Supermotifs
[0082] 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.
[0083] Such peptide epitopes are identified in the Tables described
below.
[0084] 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 position
towards the C-terminus, relative to P1, for binding to various DR
molecules.
[0085] 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."
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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, 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.
[0090] 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.
[0091] For HPV strain 11, the number and position listed for
protein E5 refers to either the HPV11 E5a or HPV11 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 HPV11 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 [0092] HPV6A E1 1
MADDSGTENEGSGCTGWFMVEAIVQHPTGTQISDDEDEEVEDSGYDMVDFIDDSNITHNS 60
LEAQALFNRQEADTHYATVQDLKRKYLGSPYVSPINTIAEAVESEISPRLDAIKLTRQPK 120
KVKRRLFQTRELTDSGYGYSEVEAGTGTQVEKHGVPENGGDGQEKDTGRDIEGEEHTEAE 180
APTNSVREHAGTAGILELLKCKDLRAALLGKFKECFGLSFIDLIRPFKSDKTTCADWVVA 240
GFGIHHSISEAFQKLIEPLSLYAHIQWLTNAWGMVLLVLVRFKVNKSRSTVARTLATLLN 300
IPDNQMLIEPPKIQSGVAALYWFRTGISNASTVIGEAPEWITRQTVIEHGLADSQFKLTE 360
MVQWAYDNDICEESEIAFEYAQRGDFDSNARAFLNSNMQAKYVKDCATMCRHYKHAEMRK 420
MSIKQWIKHRGSKIEGTGNWKPIVQFLRHQNIEFIPFLSKFKLWLHGTPKKNCIAIVGPP 480
DTGKSYFCMSLISFLGGTVISHVNSSSHFWLQPLVDAKVALLDDATQPCWIYMDTYMRNL 540
LDGNPMSIDRKHKALTLIKCPPLLVTSNIDITKEEKYKYLHTRVTTFTFPNPFPFDRNGN 600
AVYELSNANWKCFFERLSSSLDIQDSEDEEDGSNSQAFRCVPGTVVRTL 649 HPV6A E2 1
MEAIAKRLDACQEQLLELYEENSTDLNKHVLHWKCMRHESVLLYKAKQMGLSHIGMQVVP 60
PLKVSEAKGHNAIEMQMHLESLLKTEYSMEPWTLQETSYEMWQTPPKRCFKKRGKTVEVK 120
FDGCANNTMDYVVWTDVYVQDTDSWVKVHSMVDAKGIYYTCGQFKTYYVNFVKEAEKYGS 180
TKQWEVCYGSTVICSPASVSSTTQEVSIPESTTYTPAQTSTPVSSSTQEDAVQTPPRKRA 240
RGVQQSPCNALCVAHIGPVDSGNHNLITNNHDQHQRRNNSNSSATPIVQFQGESNCLKCF 300
RYRLNDKHRELFDLISSTWHWASPKAPHKHAIVTVTYHSEEQRQQFLNVVKIPPTIRHKL 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 HPV-6A L1 1
MWRPSDSTVYVPPPNPVSKVVATDAYVTRTNIFYHASSSRLLAVGHPYFSIKRANKTVVP 60
KVSGYQYRVFKVVLPDPNKFALPDSSLFDPTTQRLVWACTGLEVGRGQPLGVGVSGHPFL 120
NKYDDVENSGSGCNPOQDNRVNVGMDYKQTQLCMVSCAPPLGEHWGKGKQCTNTPVQAGD 180
CPPLELITSVIQDGDMVDTGFGAMNFADLQTNKSDVPIDICGTTCKYPDYLQMAADPYGD 240
RLFFFLRKEQMFARHFFNRAGEVGEPVPDTLIIKGSGNRTSVGSSIYVNTPSGSLVSSEA 380
QLFNKPYWLQKAQGHNNGICWGNQLFVTVVDTTRSTNMTLCASVTTSSTYTNSDYKEYMR 360
HVEEYDLQFIFQLCSITLSAEVMAYIHTMNPSVLEDWNFGLSPPPNGTLEDTYRYVQSQA 420
ITCQKPTPEKEKPDPYKNLSFWEVNLKEKFSSELDQYPLGRKFLLQSGYRGRSSIRTGVK 480
RPAVSKASAAPKRKRAKTKR 500 HPV6A L2 1
MAHSRARRRKRASATQLYQTCKLTGTCPPDVIPKVEHNTIADQILKWGSLGVFFGGLGTG 60
TGSGTGGRTGYVPLGTSAKPSITSGPMARPPVVVEPVAPSDPSIVSLIEESAIINAGAPE 120
IVPPAHGGFTITSSETTTPAILDVSVTSHTTTSIFRNPVFTEPSVTQPQPPVEANGHILI 180
SAPTITSHPIEEIPLDTFVISSSDSGPTSSTPVPGTAPRPRVGLYSRALHQVQVTDPAFL 240
STPQRLITYDNPVYEGEDVSVQFSHDSIHNAPDEAFMDIIRLHRPAIASRRGLVRYSRIG 300
QRGSMHTRSGKHIGARIHYFYDISPIAQAAEEIEMHPLVAAQDDTFDIYAESPEPDINPT 360
QHPVTNISDTYLTSTPNTVTQPWGNTTVPLSSIPNDLFLQSGPDITFPTAPMGTPFSPVT 420
ALPTGPVFITGSGFYLHPAWYFARKRRKRIPLFFSDVAA 459 HPV6B E1 1
MADDSGTENEGSGCTGWFMVEAIVQHPTGTQISDDEDEEVEDSGYDMVDFIDDSNITHNS 60
LEAQALFNRQEADTHYATVQDLKRKYLGSPYVSPINTIAEAVESEISPRLDAIKLTRQPK 120
KVKRRLFQTRELTDSGYGYSEVEAGTGTQVEKHGVPENGGDGQEKDTGRDIEGEEHTEAE 180
APTNSVREHAGTAGILELLKCKDLRAALLGKFKECFGLSFIDLIRPFKSDKTTCLDWVVA 240
GFGIHHSISEAFQKLIEPLSLYAHIQWLTNAWGMVLLVLLRFKVNKSRSTVARTLATLLN 300
IPENQMLIEPPKIQSGVAALYWFRTGISNASTVIGEAPEWITRQTVIEHGLADSQFKLTE 360
MVQWAYDNDICEESEIAPEYAQRDGFDSNARAFLNSNMQAKYVKDCATMCRHYKHAEMRK 420
MSIKQWIKHRGSKIEGTGNWKPIVQFLRHQNIEPIPFLTKFKLWLHGTPKKNCIAIVGPP 480
DTGKSYFDMSLISFLGGTVISHVNSSSHFWLQPLVDAKVALLDDATQPCWIYMDTYMRNL 540
LDGNPMSIDRKHKALTLIKCPPLLVTSNIDITKEDKYKYLHTRVTTFTFPNPFPFDRNGN 600
AVYELSNTNWKCFFERLSSSLDIQDSEDEEDGSNSQAFRCVPGTVVRTL 649 HPV6B E2 1
MEAIAKRLDACQEQLLELYEENSTDLHKHVLHWKCMRHESVLLYKAKQMGLSHIGMQVVP 60
PLDVSEAKGHNAIEMQMHLESLLRTEYSMEPWTLQETSYEMWQTPPKRCFKKRGKTVEVK 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
MESANASTSATTIDQLCKTFNLSMHTLQINCVECKNALTTAEIYSYAYKHLKVLERGGYP 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
SAPTVTSRPIEEIPLDTFVVSSSDSGPTSSTPVPGTAPRPRVGLYSRALHQVQVTDPAFL 240
STPQRLITYDNPVYEGEDVSVQFSHDSIHNAPDEAFMDIIRLHRPAIASRRGLVRYSRIG 300
QRGSMHTRSGKHIGARIHYFYDISPIAQAAEEIEMHPLVAAQDDTFDIYAESFEPGINPT 360
QHFVTNISDTYLTSTPNTVTQPWGNTTVPLSLPNDLFLQSGPDITFPTAPMGTPFSPVTP 420
ALPTGPVFITGSGFYLHPAWYFARKRRKRIPLEPSDVAA 453 HPV11 E1 1
MADDSGTENEGSGCTGWFMVEAIVEHTTGTQISEDEEEEVEDSGYDMVDFIDDRHITQNS 60
VEAQALFNRQEADAHYATVQDLKRKYLGSPYVSPISNVANAVESEISPRLDAIKLTTQPK 120
KVKRRLPETRELTDSGYGYSEVEAATQVEKHGDPENGGDGQERDTGRDIEGEGVEHREAE 180
AVDDSTREHADTSGILELLKCKDIRSTLHGKFKDCFGLSFVDLIRPFKSDRTTCADWVVA 240
GPGIHHSIADAFQKLIEPLSLYAHIQWLTNANGMVLLVLIRFKVNKSRCTVARTLGTLLN 300
IPENHMLIEPPKIQSGVRALYWFRTGISNASTVIGEAPEWITRQTVISHSLADSQFKLTE 360
MVQWAYDNDICEESEIAPEYAQRGDFDSNARAFLNSNMQAKYVKDCAIMCRHYKHAEMKK 420
MSIKQWIKYRGTKVDSVGNWKPIVQFLRHQNIEFIPFLSKLKLWLHGTPKKNCTAIVGPP 480
DTGKSCFCMSLIKFLGGTVISYVNSCSHFWLQPLTDAKVALLDDATQPCWTYMDTYMRNL 540
LDGNPMSIDRKHRALTLIKCPPLLVTSNIDISKEEKYKYLHSRVTTFTFPNPFPFDRNGN 600
AVYELSDANWKCFFERLSSSLDIEDSEDEEDGSNSQAFRCVPGSVVRGL 649 HPV11 E2 1
MEAIAKRLDACQDQLLELYEENSIDIHKHIMHWKCIRLEWVLLHKAKQMGLSHIGLQVVP 60
PLTVSETKGHNAIEMQMHLESLAKTQYGVEPWTLQDTSYEMWLTPPKRCFKKQGNTVEVK 120
FDGCEDNVMEYVVWTHIYLQDNDSWYKVTSSVDAKGIYYTCGQFKTYYVNFNKEAQKYGS 180
TNHWEVCYGSTVICSPASVSSTVREVSIAEPTTYTPAQTTAPTVSACTTEDGVSAPPRKR 240
ARGPSTNNTLCVANIRSVDSTINNIVTDNYNKHQRRNNCHSAATPIVQLQGDSNCLKCFR 300
YRLNDKYKHLPELASSTWHWASPEAPHKNAIVTLTYSSEEQRQQFLNSVKIPPTIRHKVG 360
FMSLHLL 367 HPV11 E4 1
MVVPIIGKYVMAAQLYVLLHLYLALYEKYPLLNLLHTPPHRPPPLQCPPAPRKTACRRRL 60
GSEHVDRPLTTPCVWPTSDPWTVQSTTSSLTITTSTKEGTTVTVQLRL 108 HPV11 E5A 1
MEVVPVQIAAATTTTLILPVVIAFAVCILSIVLIILISDFVVYTSVLVLTLLLYLLLWLL 60
LTTPLQPFLLTLCVCYFPAFYIHIYIVQTQQ 91 HPV11 E5B 1
MVMLTCHLNDGDTWLFLWLFTAPVVAVLGLLLLHYRAVHGTEKTKCAKCKSNRNTTVDYV 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
MTNLDTASTTLLACFLLCFCVLLCVCLLIRPLLLSVSTYTSLIILVLLLWITAASAFRCP 60
IVYIIFVYIPLFLIHTBARFLIT 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
LYIKGSGSTANLASSNYPPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTVV 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
CLRYRLRKHSDHYRDISSTWHWTGAGNEKTGILTVTYHSETQRTKFLNTVATPDSVQILV 360
GYMTM 365 HPV18 E5 Accession number W5WL18 1
MLSLIFLFCFCVCMYVCCHVPLLPSVCMCAYAWVLVFVYIVVITSPATAFTVYVFCFLLP 60
MLLLHIHAILSLQ 73 HPV18 E6 1
MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSI 60
PHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRH 120
LNEKRRPHNIAGHYRGQCHSCCNRARQERLQRRRETQV 158 HPV18 E7 1
MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHT 60
MLCMCCKCEARIKLVVESSADDLRAFQQLFLNTLSFVCPWCASQQ 105 HPV18 L1
Accession number CAA28671 1
MCLYTRVLILHYHLLPLYGPLYHPRPLPLHSILVYMVHIIICGHYIILFLRNVNVFPIFL 60
QMALWRPSDNTVYLPPPSVARVVNTDDYVTPTSIPYHAGSSRLLTVGNPYFRVPAGGGNK 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
FVGTPTSGTHGYEEIPLQTFASSGTGEEPISSTPLPTVRRVAGPRLYSRAYQQVSVANPE 240
FLTRPSSLITYDNPAFEPVDTTLTFDPRSDVPDSDFMDIIRLHRPALTSRRGTVRFSRLG 300
QRATMFTRSGTQIGARVHFYHDISPIAPSPEYIELQPLVSATEDNDLFDIYADDMDPAVP 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
NLHEEEDKENDGDSFTFKCVSGQNIRTL 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
MIELNISTVSIVLCFLLCFCVLLFVCLVIRPLVLSVSVYATLLLLIVILWVIATSPLRCF 60
CIYVVFIYIPLFVIHTHASFLSQQ 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
AEQETAQALFHAQEVQNDAQVLHLLKRKPAGGSKENSPLGEQLSVDTDLSPRLQEISLNS 120
GHKKAKRRLFTISDSGYGCSEVEAAETQVTVNTNAENGGSVHSTQSSGGDSSDNAENVDP 180
HCSITELKELLQASNKKAAMLAVFKDIYGLSFTDLVRNFKSDKTTCTDWVMAIPGVNPTV 240
AEGFKTLIKPATLYAHIQCLDCKWGVLILALLRYKCGKNRLTVAKGLSTLLHVPETCMLI 300
EPPKLRSSVAALYWYRTGISNISEVSGDTPEWIQRLTIIQHGIDDSNFDLSDMVQWAPDN 360
DLTDESDMAFQYAQLADCNSNAAAFLKSNCQAKYLKDCAVMCRHYKRAQKRQMNMSQWIK 420
YRCSKIDEGGDWRPIVQFLRYQGVEFISFLRALKEFLKGTPKKNCILLYGPANTGKSYFG 480
MSFIHFLQGAIISPVNSNSHPWLEPLADTKVAMLDDATHTCWTYPDNYWRNALDGNPISI 540
DRKHKPLLQLKCPPILLTSNIDPAKDNKWPYLESRVTVFTFPHAFPFDKNGNPVYEINDK 600
NWKCFFERTWSRLDLHEDDEDADTEGIPFGTFKCVTGQNTRPL 643 HPV45 E2 Accession
number S36564
MKMQTPKESLSERLSALQDKILDHYENDSKDINSQISYWQLIRLENAILFTAREHGITKL 60
NHQVVPPINISKSKAHKAIELQMALKGLAQSKYNNEEWTLQDTCEELWNTEPSQCFKKGG 120
KTVHVYFDGNYDNCMNYVVWDSIYYITETGIWDKTAACVSYWGVYYIKDGDTTYYVQFKS 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
MGGPRATLQEIVLHLEPQNELDPVDLLCYEQLSESEEENDEADGVSHAQLPARRAEPQRH 60
KILCVCCKCDGRIELTVESSADDLRTLQQLFLSTLSFVCPWCATNQ 106 HP45 L1
Accession number CAB44705 1
MAHNIIYGHGIIIFLKNVNVFPIFLQMALWRPSDSTVYLPPPSVARVVNTDDYVSRTSIF 60
YHAGSSRLLTVGNPYFRVVPSGAGNKQAVPKVSAYQYRVFRVALPDPNKFGLPDSTIYNP 120
ETQRLVWACVGMEIGRGQPLGIGLSGHPFYNKLDDTESAHAATAVITQDVRDNVSVDYKQ 180
TQLCILGCVPAIGEHWAKGTLCKPAQLQPGDCPPLELKNTIIEDGDMVDTGYGAMDFSTL 240
QDTKCEVPLDICQSICKYPDYLQMSADPYGDSMFFCLRREQLFARHFWNRAGVMGDTVPT 300
DLYIKGTSANMRETPGSCVYSPSPSGSITTSDSQLFNKPYWLHKAQGHNNGICWHNQLFV 360
TVVDTTRSTNLTLCASTQNPVPNTYDPTKFKHYSRHVEEYDLQFIFQLCTITLTAEVMSY 420
IHSMNSSILEINWFGVPPPPTTSLVDTYRFVQSVAVTCQKDTTPPEKQDPYDKLKFWTVD 480
LKEKFSSDLDQYPLGRKFLVQAGLRRRPTIGPRKRPAASTSTASRPAKRVRIRSKK 536 HPV45
L2 Accession number S36565 1
MVSHRAARRKRASATDLYRTCKQSGTCPPDVINKVEGTTLADKILQWSSLGIFLGGLGIG 60
TGSGSGGRTGYVPLGGRSNTVVDVGPTRPPVVIEPVGPTDPSIVTLVEDSSVVASGAPVP 120
TFTGTSGFEITSSGTTTPAVLDITPTVDSVSISSTSFTNPAFSDPSIIEVPQTGEVSGNI 180
FVGTPTSGSHGYEEIPLQTFASSGSGTEPISSTPLPTVRRVRGPRLYSRANQQVRVSTSQ 240
FLTHPSSLVTFDNPAYEPLDTTLSFEPTSNVPDSDFMDIIRLHRPALSSRRGTVRFSRLG 300
QRATMFTRSGKQIGGRVHFYHDISPIAATEEIELQPLISATNDSDLFDVYADFPPPASTT 360
PSTIHKSFTYPKYSLTMPSTAASSYSNVTVPLTSAWDVPIYTGPDIILPSHTPMWPSTSP 420
TNASTTTYIGIHGTQYYLWPWYYYFPKKRKRIPYFFADGFVAA 463 HPV33 EQ Accession
number W1WL33 1
MADPEGTNGAGMGCTGWFEVEAVIERRTGDNISEDEDETADDSGTDLLEFIDDSMENSIQ 60
ADTEAARALFNIQEGEDDLNAVCALKRKFAACSQSAAEDVVDRAANPCRTSINKNKECTY 120
RKRKIDELED$GYGNTEVETQQMVQQVESQNGDTNLNDLESSGVGDDSEVSCETNVDSCE 180
NVTLQEISNVLHSSNTKANILYKFKEAYGISFMELVRPFKSDKTSCTDWCITGYGISPSV 240
AESLKVLIKQHSLYTHLQCLTCDRGIIILLLIRFRCSKNRLTVAKLMSNLLSIPETCMVI 300
EPPKLRSQTCALYWFRTAMSNISDVQGTTPEWIDRLTVLQHSFNDNIFDLSEMVQWAYDN 360
ELTDDSDIAYYYAQLADSNSNAAAFLKSNSQAKIVKDCGIMCRHYKKAEKRKMSIGQWIQ 420
SRCEKTNDGGNWRPIVQLLRYQNIEFTAFLGAFKKFLKGIPKKSCMLICGPANTGKSYFG 480
MSLIQFLKGCVISCVNSKSHFWLQPLSDAKIGMIDDVTPISWTYIDDYMRNALDGNEISI 540
DVKHRALVQLKCPPLLLTSNTNAGTDSRWPYLHSRLTVFEFKNPFPFDENGNPVYAINDE 600
NWKSFFSRTWCKLDLIEEEDKENHGGNISTFKCSAGENTRSLRS 644 HPV33 E2 Accession
number W2WL33 1
MEEISARLNAVQEKILDLYEADKTDLPSQIEHWKLIRMECALLYTAKQMGFSHLCHQVVP 60
SLLASKTKAFQVIELQMALETLSKSQYSTSQWTLQQTSLEVWLCEPPKCFKKQGETVTVQ 120
YDNDKKNTMDYTNWGEIYIIEEDTCTMVTGKVDYIGMYYIHNCEKVYFKYFKEDAAKYSK 180
TQMWEVHVGGQVIVCPTSISSNQISTTETADIQTDNDNRPPQAAAKRRRPADTTDTAQPL 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 MRGKKPTLKEYVLDLYPEPTDLYCYEQLSDSSDEDEGLDRPDGQAQPATADYYIVTCCHT 60
CNTTVRLCVNSTASDLRTIQQLLMGTVNIVCPTCAQQ 97 HPV33 L1 Acession number
P1WL33 1
MSVWRPSEATVYLPPVPVSKVVSTDEYVSRTSIYYYAGSSRLLAVGHPYFSIKNPTNAKK 60
LLVPKVSGLQYRVFRVRLPDPNKFGFPDTSFYNPDTQRLVWACVGLEIGRGQPLGVGISG 120
HPLLNKFDDTETGNKYPGQPGADNRECLSMDYKQTQLCLLGCKPPTGEHWGKGVACTNAA 180
PANDCPPLELINTIIEDGDMVDTGFGCMDFKTLQANKSDVPIDICGSTCKYPDVLKMTSE 240
PYGDSLFFFLRREQMFVRHFFNRAGTLGEAVPDDLYIKGSGTTASIQSSAFFPTPSGSMV 300
TSESQLFNKPYWLQRAQGHNNGICWGNQVFVTVVDTTRSTNMTLCTQVTSDSTYKNENFK 360
EYIRHVEEYDLQFVFQLCKVTLTAEVMTYIHAMNPDILEDWQFGLTPPPSASLQDTYRFV 420
TSQAITCQKTVPPKEKEDPLGKYTFWEVDLKEKFSADLDQFPLGRKFLLQAGLKAKPKLK 480
RAAPTSTRTSSAKRKKVKK 499 HPV33 L2 Accession number P2WL33 1
MRHKRSTRRKRASATQLYQTCKATGTCPPDVIPKVEGSTIADQILKYGSLGVFFGGLGIG 60
TGSGSGGRTGYVPIGTDPPTAAIPLQPIRPPVTVDTVGPLDSSIVSLIEETSFIEAGAPA 120
PSIPTPSGFDVTTSADTTPAIINVSSVGESSIQTISTHLNPTFTEPSVLHPPAPAEASGH 180
FIPSSPTVSTQSYENIPMDTFVVSTDSSNVTSSTPIPGSRPVARLGLYSRNTQQVKVVDP 240
AFLTSPHKLITYDNPAFESFDPEDTLQFQHSDISPAPDPDFLDIIALHRPAITSRRHTVR 300
FSRVGQKATLKTRSGKQIGARIGYYQDLSPIVPLDHTVPNEQYELQPLHDTSTSSYSIND 360
GLYDVYADDVDNVHTPMQHSYSTFATTRTSNVSIPLNTGFDTPVMSGPDIPSPLFPTSSP 420
FVPISPFFPFDTIVVDGADFVLHPSYFILRRRRKRFPYFFTDVRVAA 467 HPV56 E2
Accession number S36581 1
MVPCLQVCKAKACSAIEVQIALESLSTTIYNNEEWTLRDTCEELWLTEPKKCFKKEGQHI 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 S38563 1
MMLPMMYIYRDPPLHYGLCIFLDVGAVNVFPIFLQMATWRPSENKVYLPPTPVSKVVATD 60
SYVKRTSIFYHAGSSRLLAVGHPYYSVTKDNTKTNIPKVSAYQYRVFRVRLPDPNKFGLP 120
DTNIYNPDQERLVWACVGLEVGRGQPLGAGLSGHPLFNRLDDTESSNLANNNVIEDSRDN 180
ISVDGKQTQLCIVGCTPAMGEHWTKGAVCKSTQVTTGDCPPLALINTPIEDGDMIDTGFG 240
AMDFKVLQESKAEVPLDIVQSTCKYPDYLKMSADAYGDSMWFYLRREQLFARHYFNRAGK 300
VGETIPAELYLKGSNGREPPPSSVYVATPSGSMITSEAQLFNKPYWLQRAQGHNNGICWG 360
NQLFVTVVDTTRSTNMTISTATEQLSKYDARKINQYLRHVEEYELQFVFQLCKITLSAEV 420
MAYLHNMNANLLEDWNIGLSPPVATSLEDKYRYVRSTAITCQREQPPTEKQDPLAKYKFW 480
DVNLQDSFSTDLDQFPLGRKFLMQLGTRSKPAVATSKKRSAPTSTSTPAKRKRR 534 HPV56 L2
Accession number S36582 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
[0093] 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
[0094] 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*1101, 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.
[0095] Representative peptide epitopes that comprise the A1
supermotif are set forth in Table VII.
HLA-A2 Supermotif
[0096] 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.
[0097] 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.
[0098] 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
[0099] 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.
[0100] Representative peptide epitopes that comprise the A3
supermotif are set forth in Table IX.
HLA-A24 Supermotif
[0101] 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.
[0102] Representative peptide epitopes that comprise the A24
supermotif are set forth in Table X.
HLA-B37 Supermotif
[0103] 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*1058, 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.
[0104] Representative peptide epitopes that comprise the B7
supermotif are set forth in Table XI.
HLA-B27 Supermotif
[0105] 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.
[0106] Representative peptide epitopes that comprise the B27
supermotif are set forth in Table XII.
HLA-B44 Supermotif
[0107] 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
[0108] 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.
[0109] Representative peptide epitopes that comprise the B58
supermotif are set forth in Table XIII.
HLA-B62 Supermotif
[0110] 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.
[0111] Representative peptide epitopes that comprise hie B62
supermotif are set forth in Table XIV.
HLA-A1 Motif
[0112] 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.
[0113] 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
[0114] An HLA-A*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.
[0115] 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
[0116] 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.
[0117] 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 DC. The A3 supermotif
primary anchor residues comprise a subset of the A3- and A11-allele
specific motif primary anchor residues.
HLA-A11 Motif
[0118] 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.
[0119] Representative peptide epitopes that comprise the A1 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
[0120] 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., 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.
[0121] 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
[0122] 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
[0123] 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.
[0124] 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
[0125] 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 I 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 I may or may not occupy the
peptide N-terminal position.
[0126] 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.
[0127] 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.
[0128] 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
[0129] 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.
[0130] 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.
[0131] 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 HLA- North SUPER- Cau- American
Japa- TYPES casian Black nese 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,
99.5 98.1 100.0 99.5 99.4 99.3 A24, B44, A1 A2, A3, B7, 99.9 99.6
100.0 99.8 99.9 99.8 A24, B44, A1, B27, B62, B58
Immune Response-Stimulating Peptide Analogs
[0132] In general, CTL and HTL responses to whole antigens are not
directed against all possible epitopes. Rather, they axe restricted
to a few "immunodominant" determinants (Zinkernagel, et al., Adv.
Immunol. 27:5159, 1979; Beanink, et al., J. Exp. Med. 168:19351939,
1988; Rawle, et al., J. Immuno. 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).
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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.
[0138] 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.
[0139] 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.
[0140] 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.
[0141] 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
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 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.
[0146] 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
[0147] 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.
[0148] 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).
[0149] 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.
[0150] 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.
[0151] 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
[0152] 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.
[0153] 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.
[0154] 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.
[0155] In alternative embodiments, epitopes of the invention can be
linked as a polyepitopic peptide, or as a minigene that encodes a
polyepitopic peptide.
[0156] 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.
[0157] 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.
[0158] 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.
[0159] 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
[0160] 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.
[0161] 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.
[0162] 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 I 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.
[0163] 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.
[0164] 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).
[0165] 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).
[0166] 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
[0167] 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.
[0168] 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.
[0169] 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.
[0170] 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.
[0171] 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.
[0172] 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
[0173] 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. 3016, 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, K. 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.
[0174] 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).
[0175] 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.
[0176] 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.
[0177] 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.3 CSS).
[0178] 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.
[0179] 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).
[0180] 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.
[0181] 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.
[0182] 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 patients, 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.
[0183] 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.
[0184] 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.
[0185] 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.
[0186] 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.
[0187] 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.
[0188] 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.
[0189] 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.
[0190] 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.
[0191] 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.
[0192] 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.
[0193] 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
[0194] 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.
[0195] 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.
[0196] 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.
[0197] 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.
[0198] The minigene sequence may be converted to DNA by assembling
oligonucleotides that encode the plus and minus stands 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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.
[0206] 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-ee-encoded CTL epitopes. Expression of HTL epitopes may be
evaluated in an analogous manner using assays to assess HTL
activity.
[0207] In viva 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.
[0208] 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.
[0209] 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
[0210] 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.
[0211] 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.
[0212] 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.
[0213] 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.
[0214] 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-alamine, 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.
[0215] 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
[0216] 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 .delta.-
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.
[0217] As another example of lipid priming of CTL responses, E.
coli lipoproteins, such as
tripalmitoyl-5-glycerylcysteinlyseryl-serine (P.sub.3 CSS) 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.
[0218] 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
[0219] 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.
[0220] 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
[0221] 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.
[0222] 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.
[0223] 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.
[0224] 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.
[0225] 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.
[0226] 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.
[0227] 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.
[0228] 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.
[0229] 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.
[0230] 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.
[0231] 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.
[0232] 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.
[0233] 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.
[0234] 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).
[0235] 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.
[0236] 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.
[0237] 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%.
[0238] 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
[0239] 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. 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
[0243] 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.
[0244] In the cancer setting there are several findings that
indicate that immune responses can impact neoplastic growth:
[0245] First, the demonstration in many different animal models,
that anti-tumor T cells, restricted by MHC class I, can prevent or
treat tumors.
[0246] Second, encouraging results have come from immunotherapy
trials.
[0247] 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).
[0248] 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: 26(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 (Reslifo
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
[0249] 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
[0250] 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.
[0251] 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 (Ohmnachi, G A, 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).
[0252] 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 inmate 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.
[0253] 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.
[0254] 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
[0255] 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)
[0256] 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.
[0257] 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.
[0258] 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 tumors 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 I 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). Kaklamnakis, et al. also suggested that adjuvant
immunotherapy with IFN-gamma may b 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.
[0259] Finally, studies have demonstrated that decreased HLA
expression can render tumor cells more susceptible to NK lysis
(Ohnmacht, G A, 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
[0260] 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
[0261] 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.
[0262] 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.
[0263] 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.
[0264] 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"
[0265] 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.
[0266] 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.
[0267] 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
[0268] 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
[0269] 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.
[0270] 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.
[0271] 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).
[0272] 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 constructs can be a heteropolymer or a homopolymer.
The polyepitopic constructs generally comprise epitopes in a
quantity of any whole unit integer between 2-50 (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,
este or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds
etc.
[0273] 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.
[0274] 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.
[0275] 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.
[0276] 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.
[0277] 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
[0278] 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
[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.
[0280] 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 183.1 (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.
[0281] 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.
[0282] 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
[0283] 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
[0284] 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
[0285] 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.
[0286] 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
[0287] where a.sub.ji is a coefficient which represents the effect
of the presence of a given amino acid (i) 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 <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 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):
[0301] 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/streptomycin). 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.
[0302] Induction of CTL with DC and Peptide:
[0303] 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 (1401
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 3 .mu.g/1 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.l/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.
[0304] Setting Up Induction Cultures:
[0305] 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.
[0306] Restimulation of the Induction Cultures with Peptide-Pulsed
Adherent Cells:
[0307] 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.
[0308] 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.
[0309] 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
[0310] 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.
[0311] 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 fbr 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.
[0312] CTL Expansion.
[0313] 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 501 U/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.
[0314] 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
[0315] 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.
[0316] 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
[0317] 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
[0318] 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.
[0319] 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
[0320] 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
[0321] 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.
Ser. No. 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.
[0322] 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.
[0323] 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.
[0324] 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 5000 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).
[0325] 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
[0326] 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.
[0327] 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.
[0328] 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).
[0329] Analoguing at primary anchor residues of other motif and/or
supermotif-bearing epitopes is performed in a like manner.
[0330] 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
[0331] 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.
[0332] 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
[0333] 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
L Chen, John Wiley & Sons, England, 1999).
[0334] 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
[0335] 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.
[0336] 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).
[0337] 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.
[0338] 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
[0339] 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.
[0340] 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.
[0341] DR3 binding epitopes identified in this manner are included
in vaccine compositions with DR supermotif-bearing peptide
epitopes.
[0342] 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
[0343] This example determines immunogenic DR supermotif- and DR3
motif-bearing epitopes among those identified using the methodology
in Example 5.
[0344] 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
[0345] 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.
[0346] 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].
[0347] 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, A11, 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
17-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*350406, B*4201, and B*5602).
[0348] 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.
[0349] 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.
[0350] 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
[0351] 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.
[0352] 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.
[0353] 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
[0354] 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 .mu.M or less, or
analogs of that epitope. The peptides may be lipidated, if
desired.
[0355] Immunization procedures: Immunization of transgenic mice is
performed as described (Alexander et al., J. Immunol.
159:4753-4761, 1997). For example, A2/Kb 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.
[0356] 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)
[0357] 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.
[0358] 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
51Cr-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.
[0359] 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
[0360] 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.
[0361] 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.
[0362] 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.
[0363] 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.
[0364] Epitopes are often selected that have a binding affinity of
an IC.sub.50 of 500 M or less for an HLA class I molecule, or for
class II, an IC.sub.50 of 1000 nM or less.
[0365] 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.
[0366] 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.
[0367] 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.
[0368] 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
[0369] 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.
[0370] 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.
[0371] 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.
[0372] 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.
[0373] 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 His antibody
epitope tag coded for by the pcDNA 3.1 Myc-His vector.
[0374] 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 see,
annealing temperature (5.degree. below the lowest calculated Tm of
each primer pair) for 30 sec, and 72.degree. C. for min.
[0375] 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
[0376] 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).
[0377] 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.
[0378] 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.
[0379] 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.
[0380] 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.
[0381] 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).
[0382] 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.
[0383] 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.
[0384] The use of prime boost protocols in humans is described in
Example 20.
Example 13
Peptide Composition for Prophylactic Uses
[0385] 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.
[0386] 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.
[0387] 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
[0388] 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.
[0389] 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.
[0390] 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.
[0391] 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
[0392] 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.
[0393] 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
[0394] 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.
[0395] 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.
[0396] 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
[0397] 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.
[0398] 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.
[0399] 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.
[0400] 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).
[0401] 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).
[0402] 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.
[0403] 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.
[0404] 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.
[0405] 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 IL22. 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
H.sup.3-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
[0406] A human clinical trial for an immunogenic composition
comprising CTL and HT epitopes of the invention is set up as an IND
Phase I, dose escalation study and carried cut as a randomized,
double-blind, placebo-controlled trial. Such a trial is designed,
for example, as follows:
[0407] A total of about 27 individuals are c rolled and divided
into 3 groups:
[0408] Group I: 3 subjects are injected with placebo and 6 subjects
are injected with 5 .mu.g of peptide composition;
[0409] Group II: 3 subjects are injected with placebo and 6
subjects are injected with 50 .mu.g peptide composition;
[0410] Group III: 3 subjects are injected with placebo and 6
subjects are injected with 500 .mu.g of peptide composition,
[0411] After 4 weeks following the first injection, all subjects
receive a booster inoculation at the same dosage.
[0412] 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.
[0413] Safety: The incidence of adverse events is monitored in the
placebo and drug treatment group and assessed in terms of degree
and reversibility.
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.
[0414] The vaccine is found to be both safe and efficacious.
Example 19
Phase II Trials in Patients Infected with HPV
[0415] 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:
[0416] 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.
[0417] 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.
[0418] 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
[0419] 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.
[0420] 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 recombinat 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.
[0421] 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)
[0422] 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 viva. 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.
[0423] 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.
[0424] 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 or 10 can also be provided. Such
cell populations typically contain between 50-90% DC.
[0425] 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
Progenipcietin.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
[0426] 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
[0427] 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.
[0428] 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.
[0429] 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.
[0430] 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.
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References