U.S. patent application number 10/128711 was filed with the patent office on 2003-05-29 for compositions and methods for eliciting ctl immunity.
Invention is credited to Celis, Esteban, Chesnut, Robert W., Grey, Howard, Sette, Alessandro D., Vitiello, Maria A..
Application Number | 20030099634 10/128711 |
Document ID | / |
Family ID | 22729600 |
Filed Date | 2003-05-29 |
United States Patent
Application |
20030099634 |
Kind Code |
A1 |
Vitiello, Maria A. ; et
al. |
May 29, 2003 |
Compositions and methods for eliciting CTL immunity
Abstract
Cytotoxic T lymphocyte responses are effectively induced to an
antigen of interest, particularly viral, bacterial, parasitic and
tumor antigens. Compositions, including pharmaceutical
compositions, of CTL-inducing peptide and an adjuvant or a
lipidated peptide which induces a helper T cell (HTL) response
stimulate the antigen specific CTL response. Among the viral
antigens to which the CTL responses are effectively induced in
humans are those of hepatitis B. The CTL response may be optimized
by a regimen of two or more booster administrations. Cocktails of
two or more CTL inducing peptides are employed to optimize epitope
and/or MHC class I restricted coverage.
Inventors: |
Vitiello, Maria A.; (La
Jolla, CA) ; Chesnut, Robert W.; (Cardiff-by-the-Sea,
CA) ; Sette, Alessandro D.; (La Jolla, CA) ;
Celis, Esteban; (San Diego, CA) ; Grey, Howard;
(La Jolla, CA) |
Correspondence
Address: |
Bruce D. Grant
Morrison & Foerster LLP
Suite 500
3811 Valley Centre Drive
San Diego
CA
92130
US
|
Family ID: |
22729600 |
Appl. No.: |
10/128711 |
Filed: |
April 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10128711 |
Apr 22, 2002 |
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08197484 |
Feb 16, 1994 |
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6419931 |
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08197484 |
Feb 16, 1994 |
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07935811 |
Aug 26, 1992 |
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07935811 |
Aug 26, 1992 |
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07874491 |
Apr 27, 1992 |
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07874491 |
Apr 27, 1992 |
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07827682 |
Jan 29, 1992 |
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07827682 |
Jan 29, 1992 |
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07749568 |
Aug 26, 1991 |
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Current U.S.
Class: |
424/130.1 ;
424/133.1; 424/143.1; 424/147.1; 435/345; 530/300 |
Current CPC
Class: |
A61P 31/18 20180101;
A61P 37/04 20180101; C12N 2730/10122 20130101; C07K 14/005
20130101; A61P 31/12 20180101; C12N 2710/20022 20130101; C12N
2740/16022 20130101; C07K 14/705 20130101; C12N 2740/16322
20130101; C07K 14/33 20130101; C12N 2740/16122 20130101; C12N
2760/16122 20130101; A61K 39/00 20130101; C12N 2770/24222 20130101;
C12N 2740/16222 20130101 |
Class at
Publication: |
424/130.1 ;
424/143.1; 424/133.1; 424/147.1; 530/300; 435/345 |
International
Class: |
A61K 039/395; A61K
039/40; A61K 039/42; C07K 002/00; C07K 004/00; C07K 005/00; C07K
007/00; C07K 014/00; C07K 016/00; C07K 017/00; A61K 038/00; C12N
005/06; C12N 005/16 |
Goverment Interests
[0002] The invention may have been made with government support
under a contract with the National Institutes of Health and/or the
National Institute of Allergy and Infectious Disease. Therefore,
the government may have certain rights in the invention.
Foreign Application Data
Date |
Code |
Application Number |
Feb 16, 1995 |
US |
PCT/US95/02121 |
Claims
What is claimed is:
1. An immunogenically effective composition comprising: a first
peptide comprising an epitope, wherein the first peptide binds to
an HLA class I molecule to form an epitope-HLA complex recognized
by a human cytotoxic T cell; a second peptide comprising an
epitope, wherein the second peptide binds to an HLA class II
molecule to form an epitope-HLA complex recognized by a human
helper T cell; an adjuvant; and a physiologically acceptable
carrier.
2. The composition of claim 1, wherein the second peptide is
covalently linked to the first peptide.
3. The composition of claim 1, wherein the second peptide is not
linked to the first peptide.
4. The composition of claim 1, wherein the first peptide is linked
to the second peptide by a spacer molecule.
5. The composition of claim 1, wherein the epitope is a viral
epitope, a bacterial epitope, a parasitic epitope, or a tumor
epitope.
6. The composition of claim 1, wherein the first peptide, the
second peptide, or the first peptide and second peptide are each
from six to thirty amino acid residues in length.
7. The composition of claim 1, wherein the first peptide, the
second peptide, or the first peptide and second peptide comprises a
plurality of epitopic units.
8. The composition of claim 1, wherein the first peptide is elected
from the group consisting of LLAQFTSAI (SEQ ID NO:31), LLVPFVQWFV
(SEQ ID NO:32), WLSLLVPFV (SEQ ID NO:33), FLLAQFTSA (SEQ ID NO:34),
FLLSLGIHL (SEQ ID NO: 35), ALMPLYACI (SEQ ID NO:36), ILLLCLIFLL
(SEQ ID NO:37), KLHLYSHPI (SEQ ID NO:38), VLLDYQGML (SEQ ID NO:39),
LLPIFFCLWV (SEQ ID NO:40), VLQAGFFLL (SEQ ID NO:41), YLHTLWKAGI
(SEQ ID NO:42), YLHTLWKAGV (SEQ ID NO:43), PLLPIFFCL (SEQ ID
NO:44), ILSTLPETTV (SEQ ID NO:45), LLFNILGGWV (SEQ ID NO: 46),
LLALLSCLTV (SEQ ID NO:47), YLVAYQATV (SEQ ID NO:48), FLLLADARV (SEQ
ID NO:49), ILAGYGAGV (SEQ ID NO:50), DLMGYIPLV (SEQ ID NO:51),
YLLPRRGPRL (SEQ ID NO:52), ALSTGLIHL (SEQ ID NO:53), LLALLSCLTI
(SEQ ID NO:54), RLIVFPDLGV (SEQ ID NO:55), RLHGLSAFSL (SEQ ID
NO:56), ILGGWVAAQL (SEQ ID NO:57), SMVGNWAKV (SEQ ID NO:58),
YLVTRHADV (SEQ ID NO:59), VLAALAAYCL (SEQ ID NO:60), LLMGTLGIV (SEQ
ID NO:65), YMLDLQPET (SEQ ID NO:66), FAFRDLCIV (SEQ ID NO:67),
TLGIVCPIC (SEQ ID NO:68), TLHEYMLDL (SEQ ID NO:69), GTLGIVCPI (SEQ
ID NO:70), MLDLQPETT (SEQ ID NO:71), TIHDIILECV (SEQ ID NO:72),
VLAEAMSQV (SEQ ID NO:73), LLWKGEGAVV (SEQ ID NO:74), LLWKGEGAV (SEQ
ID NO:75), ILKEPVHGV (SEQ ID NO:76), IVGAETFYV (SEQ ID NO:77),
IIGAETFYV (SEQ ID NO:78), LWVTVYYGV (SEQ ID NO:79), LMVTVYYGV (SEQ
ID NO:80), KMVELVHFL (SEQ ID NO:81), KMVELVHFLL (SEQ ID NO:82),
LVFGIELMEV (SEQ ID NO:83), KVLEYVIKV (SEQ ID NO:84), KVADLVGFLL
(SEQ ID NO:85), KVAEFVHFL (SEQ ID NO:86), CILESLFRA (SEQ ID NO:87),
FLWGPRALA (SEQ ID NO:88), VMIAMEGGHA (SEQ ID NO:89), LVLGTLEEV (SEQ
ID NO:90), ALREEEEGV (SEQ ID NO:91), ALAETSYVKV (SEQ ID NO:92),
YVIKVSARV (SEQ ID NO:93), and RALAETSYV (SEQ ID NO:94).
9. The composition of claim 1, wherein the second peptide is
selected from the group consisting of QYIKANSKFIGITE (SEQ ID
NO:95), KIAKMEKASSVFNVVNS (SEQ ID NO:96), DIEKKIAKMEKASSVFNVVNS
(SEQ ID NO:97), ISQAVHAAHAEINE (SEQ ID NO:98), PKYVKQNTLKLAT (SEQ
ID NO:99), MDIDPYKEFGATVELLSFLP (SEQ ID NO:100),
PHHYALRQAILCWGELMYLA (SEQ ID NO:101), LLWFHISCLTFGRETVIEYL (SEQ ID
NO:102), EYLVSFGVWIRTPPA (SEQ ID NO:103), and VSFGVWIRTPPAYRPPNAPI
(SEQ ID NO:104).
10. The composition of claim 1, wherein the adjuvant is incomplete
Freund's adjuvant, complete Freund's adjuvant, alum, aluminum
hydroxide, or a lipid.
11. The composition of claim 1, wherein the adjuvant is a
lipid.
12. The composition of claim 11, wherein the lipid is linked to the
first peptide.
13. The composition of claim 11, wherein the lipid is linked to the
second peptide.
14. The composition of claim 11, wherein the lipid is linked to the
first and the second peptide.
15. A method for stimulating an immune response in a human against
an epitope, comprising the steps of: (a) providing a first peptide
comprising an epitope, wherein the first peptide binds to an HLA
class I molecule to form an epitope-HLA complex recognized by a
human cytotoxic T cell; (b) providing a second peptide comprising
an epitope, wherein the second peptide binds to an HLA class II
molecule to form an epitope-HLA complex recognized by a human
helper T cell; (c) providing an adjuvant; and (d) administering the
first and second peptides and the adjuvant to the human.
16. The method of claim 15, wherein the second peptide is
covalently linked to the first peptide.
17. The method of claim 15, wherein the second peptide is not
linked to the first peptide.
18. The method of claim 15, wherein the first peptide is linked to
the second peptide by a spacer molecule.
19. The method of claim 15, wherein the administration step
comprises administering the first peptide, second peptide and the
adjuvant concurrently.
20. The method of claim 15, which further comprises, following step
(d), a step (e) administering the first and second peptides to the
human, whereby the administration steps (d) and (e) are spaced a
sufficient interval apart to optimize development of said immune
response to the epitopes.
21. The method of claim 20, wherein the administration step (e)
comprises administering the second peptide and the first peptide
approximately four weeks after the administration step (d).
22. The method of claim 15, wherein the epitope is a viral epitope,
a bacterial epitope, a parasitic epitope, or a tumor epitope.
23. The method of claim 15, wherein the first and second peptides
are administered prophylactically.
24. The method of claim 15, wherein the first peptide and/or the
second peptide are each from six to thirty amino acid residues in
length.
25. The method of claim 15, wherein the first peptide, the second
peptide, or the first peptide and second peptide comprises a
plurality of epitopic units.
26. The composition of claim 15, wherein the first peptide is
selected from the group consisting of LLAQFTSAI (SEQ ID NO:31),
LLVPFVQWFV (SEQ ID NO:32), WLSLLVPFV (SEQ ID NO:33), FLLAQFTSA (SEQ
ID NO:34), FLLSLGIHL (SEQ ID NO: 35), ALMPLYACI (SEQ ID NO:36),
ILLLCLIFLL (SEQ ID NO:37), KLHLYSHPI (SEQ ID NO:38), VLLDYQGML (SEQ
ID NO:39), LLPIFFCLWV (SEQ ID NO:40), VLQAGFFLL (SEQ ID NO:41),
YLHTLWKAGI (SEQ ID NO:42), YLHTLWKAGV (SEQ ID NO:43), PLLPIFFCL
(SEQ ID NO:44), ILSTLPETTV (SEQ ID NO:45), LLFNILGGWV (SEQ ID NO:
46), LLALLSCLTV (SEQ ID NO:47), YLVAYQATV (SEQ ID NO:48), FLLLADARV
(SEQ ID NO:49), ILAGYGAGV (SEQ ID NO:50), DLMGYIPLV (SEQ ID NO:51),
YLLPRRGPRL (SEQ ID NO:52), ALSTGLIHL (SEQ ID NO:53), LLALLSCLTI
(SEQ ID NO:54), RLIVFPDLGV (SEQ ID NO:55), RLHGLSAFSL (SEQ ID
NO:56), ILGGWVAAQL (SEQ ID NO:57), SMVGNWAKV (SEQ ID NO:58),
YLVTRHADV (SEQ ID NO:59), VLAALAAYCL (SEQ ID NO:60), LLMGTLGIV (SEQ
ID NO:65), YMLDLQPET (SEQ ID NO:66), FAFRDLCIV (SEQ ID NO:67),
TLGIVCPIC (SEQ ID NO:68), TLHEYMLDL (SEQ ID NO:69), GTLGIVCPI (SEQ
ID NO:70), MLDLQPETT (SEQ ID NO:71), TIHDIILECV (SEQ ID NO:72),
VLAEAMSQV (SEQ ID NO:73), LLWKGEGAVV (SEQ ID NO:74), LLWKGEGAV (SEQ
ID NO:75), ILKEPVHGV (SEQ ID NO:76), IVGAETFYV (SEQ ID NO:77),
IIGAETFYV (SEQ ID NO:78), LWVTVYYGV (SEQ ID NO:79), LMVTVYYGV (SEQ
ID NO:80), KMVELVHFL (SEQ ID NO:81), KMVELVHFLL (SEQ ID NO:82),
LVFGIELMEV (SEQ ID NO:83), KVLEYVIKV (SEQ ID NO:84), KVADLVGFLL
(SEQ ID NO:85), KVAEFVHFL (SEQ ID NO:86), CILESLFRA (SEQ ID NO:87),
FLWGPRALA (SEQ ID NO:88), VMIAMEGGHA (SEQ ID NO:89), LVLGTLEEV (SEQ
ID NO:90), ALREEEEGV (SEQ ID NO:91), ALAETSYVKV (SEQ ID NO:92),
YVIKVSARV (SEQ ID NO:93), and RALAETSYV (SEQ ID NO:94).
27. The composition of claim 15, wherein the second peptide is
selected from the group consisting of QYIKANSKFIGITE (SEQ ID
NO:95), KIAKMEKASSVFNVVNS (SEQ ID NO:96), DIEKKIAKMEKASSVFNVVNS
(SEQ ID NO:97), ISQAVHAAHAEINE (SEQ ID NO:98), PKYVKQNTLKLAT (SEQ
ID NO:99), MDIDPYKEFGATVELLSFLP (SEQ ID NO:100),
PHHYALRQAILCWGELMYLA (SEQ ID NO:101), LLWFHISCLTFGRETVIEYL (SEQ ID
NO:102), EYLVSFGVWIRTPPA (SEQ ID NO:103), and VSFGVWIRTPPAYRPPNAPI
(SEQ ID NO:104).
28. The method of claim 15, wherein the first and the second
peptides are administered with a physiologically-acceptable
carrier.
29. The method of claim 15, wherein the adjuvant is alum, aluminum
hydroxide, or a lipid.
30. The method of claim 15, wherein the first peptide is
administered with the lipid.
31. The method of claim 30, wherein the adjuvant is a lipid and the
first peptide is linked to the lipid.
32. The method of claim 15, wherein the second peptide is
administered with the adjuvant.
33. The method of claim 32, wherein the second peptide is linked to
the adjuvant.
34. The method of claim 15, wherein the first peptide and the
second peptide are administered with an adjuvant.
35. The method of claim 34, wherein the first peptide and the
second peptide are both linked to the adjuvant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 08/197,484, filed Feb. 16, 1994 which is a
continuation-in-part of U.S. application Ser. No. 07/935,811, filed
Aug. 26, 1992, which is a continuation-in-part of U.S. application
Ser. No. 07/874,491, filed Apr. 27, 1992 and now abandoned, which
is a continuation-in-part of U.S. application Ser. No. 07/827,682,
filed Jan. 29, 1992 and now abandoned, which is a
continuation-in-part of U.S. application Ser. No. 07/749,568, filed
Aug. 26, 1991 and now abandoned, each of which is incorporated
herein by reference.
Background Art
[0003] Cytotoxic T lymphocytes ("CTL") represent an important
component of an animal's immune response against a variety of
pathogens and cancers. CTL which have been specifically activated
against a particular antigen are capable of killing the cell that
contains or expresses the antigen. CTL are particularly important
in providing an effective immune response against intracellular
pathogens, such as a wide variety of viruses, and some bacteria and
parasites. CTL responses are also believed to be capable of
contributing to anti-tumor responses in afflicted or susceptible
individuals.
[0004] The receptors on the surface of the CTL cannot recognize a
foreign antigen directly, however. The CTL express an
.alpha.-.beta.heterodimeric T cell receptor which is capable of
recognizing foreign antigen fragments bound to major
histocompatibility complex (MHC) class I molecules on the surface
of the effected (e.g., infected) cells. CTL also express the
non-polymorphic CD8 antigen. This cell surface protein interacts
with the third domain of the class I molecule on the antigen
presenting cells and plays a role in both stabilizing the
interaction between the CTL and the antigen presenting cell and in
CTL activation (Salter et al., Nature 345:41-46 (1990)).
[0005] There are a number of mechanisms by which CTL are thought to
disrupt the infectious or tumorigenic process. Among these, one
involves the production of lymphokines such as gamma interferon
(IFN.gamma.) and tumor necrosis factor alpha (TNF), which are known
to act directly on infected cells to inhibit viral replication
(Gilles et al., J. Virol. 66:3955-3960 (1992)). In addition,
IFN.gamma. causes increased expression of MHC class I molecules on
the surface of virus infected cells and enhances their ability to
be recognized by CTL and trigger immune intervention (Hayata et
al., Hepatology 13:1022-1028 (1991)).
[0006] A second mechanism by which CTL combat infections or tumors
is through direct killing of the afflicted cell, e.g., those which
are infected by the targeted virus (Cohen et al., Ann. Rev.
Immunol. 10:267-293 (1992) and Henkart et al., Ann. Rev. Immunol.
3:31-58 (1985)). For example, since viruses must replicate within
the host cell the lysis of infected cells destroys virus production
prior to the liberation of infectious particles. The exact
mechanism(s) by which CTL kill infected target cells remains
unclear. Once CTL recognize an antigen presenting cell, close
contact between the cells is established over a large surface area.
A "direct hit" is then delivered by translocating enzymes present
in cytoplasmic vacuoles of CTL to the antigen presenting cell,
which enzymes kill the cell or perhaps induce programmed cell
death, "apoptosis". Once CTL have delivered their "lethal hit" to
the antigen presenting cell, they can detach and go on to kill
other antigen presenting cells through repetition of the
antigen-specific recognition, lymphokine release and target cell
killing mechanisms.
[0007] The means by which CTL distinguish infected from
non-infected cells is through the T cell receptor and its ability
to specifically recognize a peptide fragment of viral protein that
is bound to the peptide-binding cleft of the MHC class I molecule
(Monaco et al., Immunol. Today 13:173-179 (1992) and Townsend et
al., Ann. Rev. Immunol. 7:601-624 (1989)). Several viral fragments
that can serve as antigenic peptides have been identified.
[0008] The biochemical events that take place in the cytoplasm of
infected cells leading to CTL recognition are termed antigen
processing and presentation. While not completely defined, it seems
clear that during the synthesis and assembly of the infecting viral
or bacterial proteins, some proteolysis takes place in the
cytoplasm (Monaco et al., Immunol. Today 13:173-179 (1992)).
Structures called proteosomes cleave the foreign proteins into
peptide fragments. These fragments are then transported into the
endoplasmic reticulum (ER) by means of specific transporter
proteins where newly synthesized MHC class I molecules are present.
Those peptides that are capable of specifically binding to a given
MHC class I molecule do so in the ER. The non-polymorphic class I
.beta. chain, .beta..sub.2 microglobulin binds to the antigenic
peptide-class I complex, thus forming a stable trimolecular complex
that is transported to the cell surface and displayed as an
integral membrane component.
[0009] The selection of which peptides bind to a particular MHC
class I molecule is based on the ability of the peptide to bind
within the binding pocket or cleft which resides at the outermost
apex of the extra-cellular portion of the MHC molecule. For several
MHC molecules, this peptide binding pocket has been precisely
defined by X-ray crystallographic procedures allowing a
visualization of the types and location of the chemical bonds that
form to stabilize the interaction (Saper et al., J. Mol. Biol.
219:277-319 (1991)).
[0010] Because of the differences in the structure of the peptide
binding pocket between the diverse set of histocompatibility
alleles, e.g., the human HLA alleles, a distinct population of
antigenic peptides is bound by each allele, although in some cases
the population of antigenic peptides may overlap for closely
related alleles. Thus, the specificity of the CTL for a foreign
antigen resides at the level of the ability of MHC class I
molecules to bind to a specific peptide as well as for the T cell
receptor on the CTL to recognize the foreign protein fragment bound
to that specific MHC class I allele.
[0011] In animals, CT8+, MHC class I-restricted cytotoxic T cells
play an important role in the immune mediated clearance of viral
infections (e.g., Oldstone et al., Nature 321:239-243 (1986);
Mackenzie et al., Immunol. 67:375 (1989); and Robertson et al., J.
Virol. 66:3271-3277 (1992)). While similar studies have not been
possible in humans, and thus direct proof is still lacking, all of
the evidence points to a similar role for CTL.
[0012] The importance of CTL in viral clearance in animals is
evidenced by lymphocytic choriomeningitis virus (LCMV) infection in
mice (Oldstone et al., Nature 321:239-243 (1986); Mackenzie et al.,
Immunol. 67:375 (1989); Robertson et al., J. Virol. 66:3271-3277
(1992); and Ahmed et al., J. Virol. 61:3920-3929 (1987)). When LCMV
infects newborns or immune-suppressed adult animals, they become
chronically infected and virus is expressed in nearly all tissues
of the body. In contrast, adult mice infected with LCMV mount a
vigorous cellular and humoral response against the virus and clear
the infection within one to two weeks. When chronic carriers of
LCMV are adoptively treated by transfer of CD8+ LCMV-specific, MHC
class I-restricted CTL, the viral infection is cleared and the mice
become resistant to subsequent LCMV challenge. Additional studies
have shown that CTL are necessary and sufficient for LCMV clearance
and that other aspects of the immune system need not be functioning
(Oldstone et al., Nature 321:239-243 (1986) and Schulz et al. Proc.
Natl. Acad. Sci. USA 88:991-993 (1991)).
[0013] In addition to mediating the clearance of virus from
chronically infected animals, studies have demonstrated that CTL
generated in vivo against a synthetic peptide which presents an
antigenic epitope of LCMV are able to protect mice against acute
infection (Schulz et al., Proc. Natl. Acad. Sci. USA 88:991-993
(1991)). Mice injected with 100 .mu.g of a synthetic 15 amino acid
peptide in complete Freund's adjuvant were fully protected from a
lethal LCMV challenge.
[0014] With regard to the role of CTL in other viral infections,
studies with influenza virus and respiratory syncytial virus in
mice have similarly demonstrated the importance of CTL activation
in the rapid and effective recovery from these infections.
[0015] Strong evidence from animal studies indicates that an acute
infection can become chronic when there is an inadequate immune
response to clear the infection (Ahmed, Concepts in Viral
Pathogenesis III, Notkins and Oldstone eds., Springer-Verlag, New
York, 304-310 (1989)). Once the chronic infection has been
established, it appears to be more or less "tolerated" by the
host's immune system. Tolerance appears to be organism-specific
rather than a result of general immunosuppression (Fields et al.,
Fields Virology, Raven Press, New York, N.Y. 2:2137-2236 (1990)).
Studies examining which cells in the immune system are anergic or
tolerant to the infecting organism suggest that the CD4+, class
11-restricted T "helper" cells are dysfunctional (Schwartz, Cell
57:1073-1081 (1989)). Since class 11-restricted T helper cells play
a critical role in the initial priming of class I-restricted CTL
(Cassell et al., Ann. NY Acad. Sci. 532:51-60 (1988) and Fayolle et
al., J. Immunol. 174:4069 (1991)), diminished CD4 cell function may
impair the capacity of the immune system to respond adequately, and
may thus clear the way for chronic infection.
[0016] Decreased T helper cell activity has been shown in the case
of chronic hepatitis B infection in humans, although the fact that
some CD4+ T helper function is seen suggests that these cells are
not completely dysfunctional (see, e.g., Ahmed et al., J. Virol.
61:3920-3929 (1987); Alberti et al., Lancet 1:1421-1424 (1988);
Neurath et al., Nature 315:154-156 (1985); Celis et al., J.
Immunol. 132:1511-1516 (1984); and Ferrari et al., J. Immunol.
139:2050-2055 (1987)). Class I-restricted CTL can be detected in
patients with chronic HBV infection (Barnaba et al., J. Immunol.
143:2650-2654 (1989)).
[0017] The requirement for lymphokines such as IL-2 in the
generation of CD8+ CTL is well established, although the need for
activation of CD4+ T helper cells to provide these lymphokines
remains somewhat controversial. While the concept of linked T
helper-B cell recognition for antibody production has been firmly
defined, there is no compelling evidence for linked T helper-CTL
recognition for the in vivo induction of CD8+ CTL. See, e.g.,
Buller et al., Nature 328:77-79 (1987); Sarobe et al., Eur. J.
Immunol. 21:1555-1558 (1991); and Cassell and Forman, Annals N.Y.
Acad. Sci. :51-60 (1991).
[0018] Thus, the data available suggest that CD8+ class
I-restricted cytotoxic T cells specific for foreign antigens such
as viral proteins play a critical part in prevention of disease and
clearance of an established disease process. Therefore, the
challenge is to induce a sufficiently potent, antigen-specific,
cell-mediated immune response in humans and other mammals which, by
itself or in conjunction with chemotherapeutic agents or the like,
will either prevent a disease process such as an infection or tumor
from becoming established, or will eliminate or at least ameliorate
an infection or tumor which has already become established in the
host. Quite surprisingly, the present invention fulfills these and
other related needs.
DISCLOSURE OF THE INVENTION
[0019] The present invention provides compositions for inducing a
cytotoxic T lymphocyte response to an antigen of interest in a
mammal. The compositions comprise a peptide that induces a CTL
response to the antigen and a peptide that induces an HTL response.
The HTL-inducing peptide may be lipidated. The HTL-inducing peptide
is optionally linked to the CTL-inducing peptide or not linked.
When linked, the HTL-inducing peptide may be separated from the CTL
peptide by a spacer, such as Ala-Ala-Ala. The HTL-inducing peptide
will usually be linked at its C-terminal end to the CTL-inducing
peptide. Typically, the lipid is linked to the N-terminus of the
HTL-inducing peptide, where the linkage can optionally include a
spacer, such as Lys-Ser-Ser or the like.
[0020] The antigen to which the cytotoxic T lymphocyte response is
induced is selected from a viral, bacterial, parasitic or tumor
antigen. Among the viral antigens to which the CTL responses are
effectively induced are antigens of hepatitis B (such as envelope,
core or polymerase antigens), hepatitis C or human papilloma virus.
A particularly effective hepatitis B antigen is HBc18-27. Typically
the CTL inducing peptide will be from seven to fifteen residues,
and more usually from nine to eleven residues. The immunogenic
composition can further comprise a carrier, such as physiologic
saline, and an adjuvant, such as incomplete freunds adjuvant, alum
or MONTANIDE.RTM. (Seppic, Paris, France; oil-based adjuvant with
mannide oleate). When the peptide is lipidated, it may be modified
or unmodified. The lipid is preferably a linear alkyl chain of 6-22
carbons, and preferably is a linear alkyl chain of 16 carbons. In
some embodiments of the present invention the lipid is comprised of
palmitic acid attached to epsilon and alpha amino groups of a Lys
residue, wherein the Lys is linked to the amino terminus of the
HTL-inducing peptide by means of a linker.
[0021] In other embodiments the present invention comprises methods
for inducing a cytotoxic T lymphocyte response in a mammal against
an antigen such as a viral, bacterial, parasitic, or tumor antigen.
The method comprises administering to the mammal a peptide that
induces a CTL response to the antigen, and administering, either
separately or together, a lipidated peptide that induces an HTL
response. The HTL-inducing peptide is optionally linked to the
CTL-inducing peptide or unlinked. When unlinked, the HTL-inducing
peptide can be admixed with the CTL-inducing peptide. The
HTL-inducing peptide and the CTL-inducing peptide are typically
administered to the mammal in a regimen of two or more
administrations. These boosters are spaced a sufficient interval
apart to optimize development of a CTL response to the antigen of
interest, e.g., a second administration may be approximately four
weeks after the initial administration. In representative
embodiments described herein the antigen is hepatitis B antigen,
such as HBc18-27 and the mammal is a human, of the HLA-A2.1
histocompatibility type for the HBc18-27 CTL inducing peptide.
[0022] In yet other embodiments the invention provides methods for
treating or preventing a disease that is susceptible to treatment
by a CTL response by administering a CTL-inducing peptide to an
antigen associated with said disease, and an HTL-inducing peptide
conjugated to a lipid. The induction of a CTL response can be used
in the treatment or prevention of viral infection (e.g., hepatitis
B, hepatitis C or human papilloma virus), bacterial or parasitic
infection or tumors. When the disease is hepatitis B infection, for
example, the methods can be used to treat or prevent chronic or
acute infection.
[0023] In yet other embodiments the invention provides methods for
inducing a cytotoxic T lymphocyte response in a human against an
antigen of interest. The methods comprise administering a
composition which comprises a peptide that induces a CTL response
to said antigen in a human and an adjuvant. The method may further
comprise administering a peptide that induces an HTL response, and
in some embodiments the HTL inducing peptide is linked to the CTL
inducing peptide.
[0024] In other aspects the invention provides methods for inducing
a CTL response in a human against an antigen of interest by
administering a peptide that induces a CTL response to the antigen
and a peptide that induces an HTL response, where the CTL inducing
and/or the HTL inducing peptide is lipidated.
[0025] The CTL and HTL inducing peptides may be linked or
unlinked.
[0026] The HTL inducing peptide is preferably lipidated. The
lipidated HTL inducing peptide can be combined with a cocktail of
at least two CTL inducing peptides to optimize coverage of
individuals of different HLA types or, in some instances, different
antigen strains.
[0027] In a further aspect of the invention methods are described
for inducing an effective CTL response in a human against an
antigen of interest. According to these methods one or more
peptides that induce a CTL response to the antigen, such as a
viral, bacterial, parasitic or tumor antigen, is administered to a
human together or separately with a peptide that induces an HTL
response, where at least the CTL inducing and/or the HTL inducing
peptide is lipidated. In representative embodiments of such a
method described herein the CTL response is induced is to a viral
antigen, such as hepatitis B antigen.
[0028] Pharmaceutical compositions for the treatment of hepatitis B
infection are also provided. These compositions comprise a peptide
that induces a CTL response to hepatitis B and a peptide that
induces an HTL response, where the HTL-inducing peptide is
conjugated to a lipid, together with a pharmaceutically acceptable
carrier. The carrier can be a liposome, for example, and the
pharmaceutical composition may further comprise an adjuvant, such
as incomplete Freund's adjuvant, alum or MONTANIDE.RTM. (Seppic,
Paris, France; oil-based adjuvant with mannide oleate).
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1A-1H depict the results of induction of HBV
peptide-specific A2.1-restricted CTL by priming A2.1/K.sup.b
transgenic mice with syngeneic spleen cells "loaded" with HBV. FIG.
1A-1D: Splenocytes from HBV-primed transgenic mice were
restimulated in vitro with four mixtures of syngeneic LPS blasts
each coated with one of 13 different peptides. After 9 days
effector cells were assayed for lytic activity against .sup.5Cr
labeled Jurkat A2.1/K.sup.b target cells in the presence or absence
of the four different peptide mixtures used for induction. FIG.
1E-1H: Effector cells raised against the four different peptide
mixtures were restimulated in vitro against the same peptide
mixtures and assayed for lytic activity against .sup.51Cr labeled
Jurkat A2.1/Kb target cells in the presence or absence of the
individual peptides.
[0030] FIG. 2A-2H illustrates the HBV peptide specificity of A2.1
transgenic CTL. Transgenic CTL raised from HBV-primed transgenic
mice and restimulated in vitro twice with one of the four different
peptide mixtures were restimulated with individual HBV peptides and
assayed for lytic activity on .sup.51CR labelled Jurkat target
cells in the presence or absence of the HBV peptides used for the
restimulation. FIG. 2A-2H collectively illustrate the HBV peptide
specificity of A2.1 transgenic CTL. Transgenic CTL raised from
HBV-primed transgenic mice and restimulated in vitro twice with one
of the four different peptide mixtures were restimulated with
individual HBV peptides and assayed for lytic activity on .sup.51Cr
labeled Jurkat target cells in the presence or absence of the HBV
peptides used for the restimulation.
[0031] FIG. 3A-3L collectively illustrate the results of induction
of HBV peptide-specific A2.1-restricted CTL by priming A2.1/Kb
transgenic mice with HBV in IFA. Splenocytes from HBVprimed
transgenic mice were restimulated in vitro with syngeneic LPS
blasts coated with HBV peptides. After six days, effector cells
were assayed for lytic activity against .sup.51Cr labeled Jurkat
A2.1/Kb target cells in the presence or absence of the appropriate
HBV peptide. Each panel represents the CTL activity induced by the
indicated target peptide.
[0032] FIG. 4A-4L collectively show the results obtained when the
effector CTL of FIG. 3A-3L were restimulated with peptide coated
LPS blasts followed at one week interval by restimulation with
peptide coated Jurkat A2.1 /Kb cells. Six days after the last
restimulation effector cells were assayed for cytolytic activity
against .sup.51Cr labeled Jurkat A2.1/Kb target cells in the
absence or presence of the peptide used for the restimulation, plus
related peptides. Each panel represents the CTL activity induced by
the peptide indicated in the corresponding panel of FIG. 3A-3L. The
target peptides are indicated in each panel.
[0033] FIG. 5 illustrates that no HBc18-27-specific CTL response is
detected in mice primed with the HBc 875.23 T helper epitope alone.
Animals were primed subcutaneously with 100 .mu.g of 875.23 (T
helper epitope) in Complete Freund's Adjuvant (CFA) followed 9 days
later (subcutaneously) with IFA alone. Splenocytes were removed 3
weeks later, cultured for 6 days in the presence of LPS-blasts that
had been incubated with the CTL epitope (875.15), 100 .mu.g for 2
hrs before being washed and added to the culture as a source of
antigen presenting cells. The presence of HBc 18-27
(875.15)-specific CTL was determined using a standard 6 hr
.sup.15Cr release assay with Jurkat A2.1/K.sup.b cells as
targets.
[0034] FIG. 6 illustrates that no HBc 18-27-specific CTL response
was detected when mice were primed with HBc18-27 (875.15) in IFA.
Experimental protocol was similar to that described in FIG. 5,
except that mice received 100 .mu.g of peptide 875.15
subcutaneously in IFA rather than IFA alone for in vivo CTL
priming.
[0035] FIG. 7 illustrates that HBc18-27-specific CTL response was
detected in 50% of the mice primed with HBc T helper peptide
(875.23) mixed with HBc CTL inducing peptide (875.15) at a 1 to 1
ratio. The experimental protocol was similar to that described in
FIGS. 5 and 6.
[0036] FIG. 8 illustrates that HBc-specific (875.15) CTL activity
was detected in mice primed with peptide 902.01 in which the HBc T
helper and CTL inducing peptide were linked via a peptide bond.
Experimental protocol was similar to that in FIGS. 5 and 6.
[0037] FIG. 9 illustrates that the greatest HBc18-27
(875.15)-specific CTL activity was detected in mice primed with
peptide 902.02 in which the HBc T helper and CTL epitopes were
linked via peptide bonds using an exemplary spacer such as
alanine-alanine-alanine. Protocol was similar to that in FIGS. 5
and 6.
[0038] FIG. 10A-10B illustrate that previous priming of helper T
cells was not required for in vivo priming of HBc 18-27-specific
CTL responses using peptide 902.01 and 902.02. CTL response is
shown from animals primed subcutaneously with peptide 902.01 (FIG.
10A) or 902.02 (FIG. 10B) alone without the previous priming with
peptide 875.23 in CFA.
[0039] FIG. 11 illustrates the induction of HBenv.sub.360-368
specific CTL response. A2.K.sup.b transgenic mice were injected
with 100 microliters of an emulsion (IFA) of 100 mg HBenv360-368
and 100 mg HBc128-140. Three weeks later, splenocytes were
restimulated with syngeneic LPS blasts coated with peptide
HBenv360-368. Effector cells were assayed for cytotoxicity against
.sup.51Cr labeled Jurkat A2/K.sup.1 target cells in the presence or
absence of HBenv 360-368.
[0040] FIG. 12 illustrates the induction of a CTL response specific
for HBc 18-27 by priming with a peptide containing HBc 18-27 linked
to tetanus toxoid 830-843 (human helper T cell epitope). Effector
cells were assayed against 51Cr labeled Jurkat A2-1/Kb target cells
in the presence or absence of HBc 18-27; Jy target cells in the
presence or absence of HBc 18-27 and Jy cells that had been
transfected with HBV core.
[0041] FIG. 13A-13B collectively illustrate the minimal sequence
for CTL recognition within HBV env 329-348 peptide (799.09). CTL
lines 110 and 113 were derived from splenocytes obtained from A2Kb
transgenic mice primed subcutaneously with HBV virus in IFA and in
vitro activated with 799.09 coated stimulator cells. 799.09
specific CTL lines 110 and 113 were assayed for lytic activity in a
6 hr .sup.51Cr release assay using JA2Kb cells as targets in the
presence of 799.09 peptide truncations (FIG. 13A=799.09 N-terminus
truncations; FIG. 13B=799.09 overlapping 9 mers and 10 mers).
[0042] FIG. 14A-14D show the 8Bc18-27 specific CTL response (d7
assay) from subjects immunized with placebo or CY-1899. The CTL
response against 8Bc18-27 was assessed by culturing
4.times.10.sup.6 PBMC/well in 24 well plates in the presence of
HBc18-27 peptide on day 3 and 6 after initiation cultures were fed
with 10 U/ml IL-2 (final concentration). On day 7, part of the
wells (2-3) were harvested and CTL activity was measured using
.sup.51Cr labeled .221 A2 target cells in the absence or presence
of HBc18-27 peptide and in the presence of a 20 fold excess of K562
cells (K562 cells were added in order to decrease background lysis
caused by NK cells). The data are expressed in lytic units/10.sup.6
cells where one lytic unit is defined as the number of lymphocytes
required to achieve 30% lysis of 10000 .221 A2 during a 6 hour
assay. Each bar represents the specific CTL activity (i.e. in the
presence of peptide and in the absence of peptide).
[0043] FIG. 15A-15D show the HBc18-27 specific CTL response (d14
assay) from subjects immunized with placebo or CY-1899. On day 7
after initiation of cultures (see FIG. 1A-1H), the remaining wells
(2-3) were harvested and cells were restimulated with HBc18-27
peptide-coated autologous adherent cells. Cultures were fed with 10
U/ml IL-2 on day 9 and thereafter as needed. CTL activity was
assayed on day 14 using the procedure described in FIG. 1A-1H.
[0044] FIG. 16 shows the mean and standard deviation of peak CTL
activity: the mean and standard deviation of peak CTL activity
after the first and second injections of different doses of
CY-1899.
[0045] FIG. 17A-17B show HBc18-27 specific CTL from subjects
injected with CY-1899 recognize endogenous processed antigen.
Effector CTL obtained from subjects 302 and 304 after 14 days of
culture were restimulated as described in FIG. 2I-2P. CTL activity
was assayed 7 days later as shown in FIG. 1A-1H using as targets
0.221 A.sub.2 cells in the absence (-.quadrature.-) or presence
(-.largecircle.-) of HBc18-27 peptide and 0.221 A.sub.2 cells
transfected with the HBV core protein (-.tangle-solidup.-).
[0046] FIG. 18A-18D show the proliferation response to T cells
specific for the TT 830-843 helper peptide from subjects immunized
with placebo or CY-1899. T cell proliferation response against the
helper peptide epitope was measured by culturing 1.5.times.10.sup.5
PBMC from each sample in flat-bottom 96/plate wells with or without
10 .mu.g/ml TT peptide. Seven days later cultures were fed with
medium containing recombinant IL-2 (20 U/ml final concentration) to
induce further proliferation of T cells which had been stimulated
against the peptide. On day 9 after initiation of culture, 1 RCi of
.sup.3H-thymidine was added to each well and 18 hr later cells in
each well were harvested onto glass fiber mats and counted for
.sup.3H-thymidine incorporation into DNA. Each bar represents the
difference in cpm .sup.3H-thymidine incorporation obtained from
wells which received peptide minus those which did not receive
peptide.
MODES OF CARRYING OUT THE INVENTION
[0047] The present invention provides compositions and methods for
inducing an effective CTL-mediated response to an antigen of
interest in humans and other mammals. The composition is comprised
of peptides that are capable of inducing MHC class I-restricted CTL
responses to the antigen of interest ("CTL peptide") and an
adjuvant. Another embodiment to the present invention is directed
to a composition comprised of said CTL peptide and a peptide
capable of eliciting a helper T lymphocyte (HTL) response. Another
embodiment of the invention is directed to either or both the CTL
and HTL peptide by lipidated, linked or unlinked and administered
with or without an adjuvant preparation. In particularly preferred
embodiments either the CTL peptide or the HTL peptide is lipidated,
linked or unlinked and administered without an adjuvant. In a
preferred embodiment the HTL epitope is lipidated and linked to the
CTL epitope and administered without an adjuvant. In another
preferred embodiment the lipidated HTL peptide is admixed with, but
not linked to, at least one CTL peptide.
[0048] By administering the compositions of the present invention
an effective CTL response is stimulated in the recipient mammal to
the antigen of interest. The cells which are targeted by the CTL
response can be involved in a wide variety of disease or potential
disease states, e.g., cells which are infected by viruses, bacteria
or parasites, cells which express certain tumor antigens, and cells
which express autoimmune antigens or other antigens that are
capable of being recognized as self by the mammal's CTL. The
specifically stimulated CTL attack the target cells by secreting
lymphokines (e.g., gamma interferon) and liberating products (e.g.,
proteolytic enzymes such as serine esterases) that inhibit
replication of the infecting organism in the cells and/or kill the
cells which express the antigen of interest, and thus are able to
interrupt or substantially prevent the disease of interest, e.g., a
viral infection, parasite or bacterial infection, a tumor or an
autoimmune disease process.
[0049] The CTL inducing peptides which are useful in the
compositions and methods of the present invention can be selected
from a variety of sources, depending of course on the targeted
antigen of interest. The CTL inducing peptides are typically small
peptides that are derived from selected epitopic regions of target
antigens associated with an effective CTL response to the disease
of interest. Thus, by "CTL inducing peptide" or "CTL peptide" of
the present invention is meant a chain of at least four amino acid
residues, preferably at least six, more preferably eight to ten,
sometimes eleven to fourteen residues, and usually fewer than about
thirty residues, more usually fewer than about twenty-five, and
preferably fewer than fifteen, e.g., eight to fourteen amino acid
residues derived from selected epitopic regions of the target
antigen(s).
[0050] Peptides that induce CTL responses are used in the methods
and compositions of the present invention irrespective of the
method or methods used to identify the epitope recognized by CTL.
The CTL epitope(s) contained in the CTL peptides can be identified
in one of several ways. In those cases where antigen-specific CTL
lines or clones are available, for example tumor-infiltrating
lymphocytes (TIL) or virus-specific CTL, these cells can be used to
screen for the presence of the relevant epitopes using target cells
prepared with specific antigens. Such targets can be prepared using
random, or selected synthetic peptide libraries, which would be
utilized to sensitize the target cells for lysis by the CTL.
Another approach to identify the relevant CTL epitope when CTL are
available is to use recombinant DNA methodologies. Gene, or
preferably cDNA, libraries from CTL-susceptible targets are first
prepared and transfected into non-susceptible target cells. This
allows the identification and cloning of the gene coding the
protein precursor to the peptide containing the CTL epitope. The
second step in this process is to prepare truncated genes from the
relevant cloned gene, in order to narrow down the region that
encodes for the CTL epitope. This step is optional if the gene is
not too large. The third step is to prepare synthetic peptides of
approximately 10-20 amino acids in length, overlapping by 5
residues, which are used to sensitize targets for the CTL. When a
peptide, or peptides, are shown to contain the relevant CTL
epitope, smaller peptides can be prepared to establish the peptide
of minimal size that contains the CTL epitope. These epitopes are
usually contained within 9-10 residues. Examples of peptides
containing known CTL epitopes identified in this way are listed
below.
1 SEQ HLA- ID RESTRIC- ANTIGEN SOURCE SEQUENCE NO: TION MAGE-1
EADPTGHSY 1 A1 HIV nef84-94 AVDLSHFLK 2 A11 EBNA4 416-424 IVTDFSVIK
3 A11 HBc18-27 FLPSDFFPSV 4 A2.1 HIV RT ILKIEPVHGV 5 A2.1 HTLV-1,
Tox 12-19 LFGYPVYV 6 A2.1 Influenza A, M1 58-66 GILGFVFTL 7 A2.1
HCMV, gB 619-628 IAGNSAYEYV 8 A2.1 p53 264-272 A8 LLGRNSFEV 9 A2.1
HBVadr-ENV (S Ag335- WLSLLVPFV 10 A2.1 343) c-ErbB2 (HER-2/neu)
RFRELVSEFSRMARDPQ 11 A2.1 HIV nef73-82 QVPLRPMTYK 12 A3,A11 HIV-1
NL43 env gp RLRDLLLIVTR 13 A3.1 41768-778 HCV 141-151 STLPETTVVRR
14 A31 Aw68 NP 383-391 SRYWAIRTR 15 B27 HIV gag p24 265-274
KRWIILGLNK 16 B27 P.falciparum circum KPKDELDY 17 B35 sp.368-375
P.falciparum circum KSKDLEDY 18 B35 sp.368-375 P.falciparum liverAg
KPNDKSLY 19 B35 1850-1857 HIV-2 TPYDINQML 20 B53 P.falciparum
liverAg KPIVQYDNF 21 B53 1786-1794 B53 self peptide YPAEITLTW 22
B53 HIV gp41 586-593 YLKDQQLL 23 B8 NP 380-388 ELRSRYWAI 24 B8 EBV
EBNA-3 FLRGRAYGI 25 B8 HIV gag261-269 GEIYKRWII 26 B8 HIV
gag331-339 DCKTILKAL 27 B8 HIV po1185-193 DPKVKQWPL 28 B8 HIV gp41
586-593 YLKDQQLYL 29 B8 HIV gap p17.3 GGKKKYKLK 30 B8
[0051] Another way of identifying a peptide containing a CTL
epitope, when CTLs are present, is to elute the peptide with an
acid or base. The peptides associated with MHC molecules are
present on the cells that are lysed by the CTL. The eluted peptides
are separated using a purification method such as HPLC, and
individual fractions are tested for their capacity to sensitize
targets for CTL lysis. When a fraction has been identified as
containing the CTL peptide, it is further purified and submitted to
sequence analysis. The peptide sequence can also be determined
using tandem mass spectrometry. A synthetic peptide is then
prepared and tested with the CTL to corroborate that the correct
sequence and peptide have been identified.
[0052] In some circumstances, where CTL are not available there are
other means to identify potential CTL epitopes. These methods rely
in the identification of MHC-binding peptides from known protein
sequences. These methods have been described in detail in pending
patent applications (U.S. patent application Ser. Nos. 08/159,339,
08/073,205 and EPO Patent Application No. 92201252.1, which are
herein incorporated by reference). Briefly, the protein sequences
for example from viral or tumor cell components are examined for
the presence of MHC-binding motifs. These binding motifs which
exist for each MHC allele, are conserved amino acid residues,
usually at positions 2 (or 3) and 9 (or 10) in peptides of 9-10
residues long. Synthetic peptides are then prepared of those
sequences bearing the MHC binding motifs, and subsequently are
tested for their ability to bind to MHC molecules. The MHC binding
assay can be done either using cells which express high number of
empty MHC molecules (cellular binding assay), or using purified MHC
molecules. The MHC binding peptides are then tested for their
capacity to induce a CTL response in naive individuals, either in
vitro using human lymphocytes, or in vivo using HLA-transgenic
animals. These CTL are tested using peptide-sensitized target
cells, and targets that naturally process the antigen, such as
viral infected cells or tumor cells. For example, an
HLA-A1-restricted CTL epitope for the tumor-associated antigen
MAGE-3 has been identified using this approach and is the subject
of a pending patent application, U.S. Patent Application No.
08/186,266, which is herein incorporated by reference.
[0053] Desirably, the CTL peptide will be as small as possible
while still maintaining substantially all of the biological
activity of a larger peptide. When possible, it may be desirable to
optimize peptides of the invention to a length of eight to twelve
amino acid residues, more usually nine or ten amino acid residues,
commensurate in size with endogenously processed antigen peptide
that is bound to MHC class I molecules on the cell surface. See
generally, Schumacher et al., Nature 350:703-706 (1991); Van Bleek
et al., Nature 348:213-216 (1990); Rotzschke et al., Nature
348:252-254 (1990); and Falk et al., Nature 351:290-296 (1991),
which are incorporated herein by reference. By biological activity
of a CTL inducing peptide is meant the ability to bind an
appropriate MHC molecule and, in the case of peptides useful for
stimulating CTL responses, induce a CTL response against the
selected antigen or antigen mimetic. By a CTL response is meant a
CD8.sup.+ T lymphocyte response specific for an antigen of
interest, wherein CD8.sup.+, MHC class I-restricted T lymphocytes
are activated. As noted above, the activated cytotoxic T
lymphocytes will secrete a variety of products which inhibit and
may or may not kill the targeted cell.
[0054] The compositions and methods of the present invention are
particularly preferred for targeting host cells infected by
viruses. CTL responses are an important component of the immune
responses of most mammals to a wide variety of viruses, and the
present invention provides a means to effectively stimulate a CTL
response to virus-infected cells and treat or prevent such an
infection in a host mammal. Thus the compositions and methods of
the present invention are applicable to any virus presenting
protein and/or peptide antigens. Such viruses include but are not
limited to the following, pathogenic viruses such as influenza A
and B viruses (FLU-A, FLU-B), human immunodeficiency type I and II
viruses (HIV-I, HIV-Il), Epstein-Barr virus (EBV), human T
lymphotropic (or T-cell leukemia) virus type I and type II (HTLV-I,
HTLV-II), human papillomaviruses types 1 to 18 (HPV-1 to HPV-18),
rubella (RV), varicella-zoster (VZV), hepatitis B (HBV), hepatitis
C (HCV), adenoviruses (AV), and herpes simplex viruses (HV). In
addition, cytomegalovirus (CMV), poliovirus, respiratory syncytial
(RSV), rhinovirus, rabies, mumps, rotavirus and measles
viruses.
[0055] In a like manner, the compositions and methods of the
present invention are applicable to tumor-associated proteins,
which could be sources for CTL epitopes. Such tumor proteins and/or
peptides include, but are not limited to, products of the MAGE-1,
-2 and -3 genes, products of the c-ErbB2 (HER-2/neu)
proto-oncogene, tumor suppressor and regulatory genes which could
be either mutated or overexpressed such as p53, ras, myc, and RB1.
Tissue specific proteins to target CTL responses to tumors such as
prostatic specific antigen (PSA) and prostatic acid phosphatase
(PAP) for prostate cancer, and tyrosinase for melanoma. In addition
viral related proteins associated with cell transformation into
tumor cells such as EBNA-1, HPV E6 and E7 are likewise applicable.
A large number of peptides from some of the above proteins have
been identified for the presence of MHC-binding motifs and for
their ability to bind with high efficiency to purified MHC
molecules and are the subject of pending patent applications (U.S.
patent application Ser. Nos. 08/159,339 and 08/073,205, previously
incorporated herein by reference).
[0056] The peptides can be prepared "synthetically," as described
hereinbelow, or by recombinant DNA technology. Although the peptide
will preferably be substantially free of other naturally occurring
viral, bacterial, parasitic, tumor or self proteins and fragments
thereof, in some embodiments the peptides can be synthetically
conjugated to native fragments or particles. The term peptide is
used interchangeably with polypeptide in the present specification
to designate a series of amino acids connected one to the other by
peptide bonds between the alpha-amino and alpha-carboxy groups of
adjacent amino acids. The polypeptides or peptides can be a variety
of lengths, either in their neutral (uncharged) forms or in forms
which are salts, and either free of modifications such as
glycosylation, side chain oxidation, or phosphorylation or
containing these modifications, subject to the condition that the
modification not destroy the biological activity of the
polypeptides as herein described.
[0057] The terms "homologous", "substantially homologous", and
"substantial homology" as used herein denote a sequence of amino
acids having at least 50% identity wherein one sequence is compared
to a reference sequence of amino acids. The percentage of sequence
identity or homology is calculated by comparing one to another when
aligned to corresponding portions of the reference sequence.
[0058] The peptides useful in the present invention can be
optionally flanked and/or modified at one or both of the N- and
C-termini, as desired, by amino acids from the naturally occurring
(e.g., HBV) sequences, amino acids added to facilitate linking to
another peptide or to a lipid, other N- and C-terminal
modifications, linked to carriers, etc., as further described
herein. Additional amino acids can be added to the termini of a
peptide to provide for modifying the physical or chemical
properties of the peptide 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. In addition, the peptide sequences can differ from
the natural sequence by being modified by terminal-NH.sub.2
acylation, e.g., by alkanoyl (C.sub.1-C.sub.20) or thioglycolyl
acetylation, terminal-carboxy amidation, e.g., ammonia,
methylamine, etc. In some instances these modifications may provide
sites for linking to a support or other molecule.
[0059] It will be understood that the peptides of the present
invention or analogs thereof which have CTL stimulating activity
may be modified to provide other desired attributes, e.g., improved
pharmacological characteristics, while increasing or at least
retaining substantially all of the biological activity of the
unmodified peptide. For instance, the peptides can be modified by
extending, decreasing or substituting amino acid sequences by,
e.g., the addition or deletion of amino acids on either the amino
terminal or carboxy terminal end, or both, of peptides derived from
the sequences disclosed herein.
[0060] With respect to treatment or prevention of hepatitis B
infection in humans, selection of a CTL inducing peptide(s) useful
in the present invention can be as set forth in more detail in
copending applications U.S. patent application Ser. Nos.
07/935,811, 07/935,898 and 08/024,120 which are incorporated herein
by reference. These applications provide the ability to select one
or more peptides that induce CTL responses to a hepatitis B
antigen, which responses are capable of killing (or inhibiting)
cells which are infected by or otherwise express (in the case of
transfected cells) the native HBV antigens. The HBV CTL inducing
peptide will usually have at least four, sometimes six, often seven
or more residues, or a majority of amino acids of that peptide,
that are identical or homologous when compared to the corresponding
portion of the naturally occurring HBV sequence. For example,
peptides preferred for stimulating HBV CTL responses include the
following:
2 +HC,10 SEQ ID BINDING SOURCE POSITION SEQUENCE SIZE NO: A2 HBV
POL 1117 LLAQFTSAI 9 31 9.6000 HBV ENV 338 LLVPFVQWFV 10 32 1.6000
HBV ENV 335 WLSLLVPFV 9 33 0.9600 HBV ENV 1116 FLLAQFTSA 9 34
0.6600 HBV POL 1147 FLLSLGIHL 9 35 0.5200 HBV POL 1245 ALMPLYACI 9
36 0.5000 HBV ENV 249 ILLLCLIFLL 10 37 0.3000 HBV POL 1092
KLHLYSHPI 9 38 0.2900 HBV ENV 259 VLLDYQGML 9 39 0.1100 HBV ENV 378
LLPIFFCLWV 10 40 0.1000 HBV ENV 177 VLQAGFFLL 9 41 0.0660 HBV POL
721 YLHTLWKAGI 10 42 0.0560 HBV POL 721 YLHTLWKAGV 10 43 0.1300 HBV
ENV 377 PLLPIFFCL 9 44 0.0310 HBV NUC (CORE) 529 ILSTLPETTV 10 45
0.0220
[0061] Other HBV CTL stimulating peptides include HBenv.sub.309-328
(peptide 799.08), HBenv.sub.329-349 (peptide 799.09) or
HBenv.sub.349-368 (peptide 799.10), and the HBc region HBc91-110
(peptide 802.03).
[0062] For example, a CTL inducing HBc peptide comprises from six
to thirty amino acids and is derived from the region HBc18-27,
contains at least one CTL epitopic site, and has at least seven
amino acids wherein a majority of amino acids of the peptide will
be identical or substantially homologous, when compared to the
amino acids comprising the corresponding portion of the naturally
occurring HBc18-27 sequence. A representative peptide of this
region is peptide HBc18-27, which has the following sequence (for
HBV subtype ayw): FLPSDFFPSV (SEQ ID NO:4).
[0063] With respect to treatment or prevention of hepatitis C
infection in mammals, one or more peptides that induce a CTL
response to a hepatitis C antigen may be selected. The HCV
CTL-inducing peptide will usually have at least four, sometimes
six, often seven or more residues, or a majority of amino acids of
that peptide that are identical or homologous when compared to the
corresponding portion of the naturally occurring HCV sequence. For
example, those peptides which are preferred for stimulating HCV CTL
responses include sequences contained within copending U.S patent
application Ser. Nos. 08/159,339 and 08/073,205, previously
incorporated herein by reference, in particular peptides 1073.05
(LLFNILGGWV, SEQ ID NO: 46), 1090.18 (FLLLADARV, SEQ ID NO:49),
939.20 (LLALLSCLTV, SEQ ID NO:47), 1073.07 (YLLPRRGPRL, SEQ ID
NO:52), 1013.10 (DLMGYIPLV, SEQ ID NO:51), 1073.10 (GVAGALVAFK, SEQ
ID NO:61). Also suitable are other peptides identified by other
methods, such as STNPKPQK (SEQ ID NO:62) and GPRLGVRAT (SEQ ID
NO:63) (Koziel et al., J. Virol. 67:7522-7535, 1993), and
YPWPLYGNEGLGWAGWLLSP (SEQ ID NO:64) (Kita et al., Abstract #631,
1993 Am. Assoc. For Study of Liver Diseases Meeting). Other HCV
derived peptides for stimulating HCV CTL responses include the
following:
3 SEQ ID BINDING SOURCE POSITION SEQUENCE SIZE NO: A2 HCV NS4 1807
LLFNILGGWV 10 46 3.5000 HCV CORE 178 LLALLSCLTV 10 47 0.6050 HCV
NS4 1585 YLVAYQATV 9 48 0.2450 HCV NS1/ENV 725 FLLLADARV 9 49
0.2250 HCV NS4 1851 ILAGYGAGV 9 50 0.2150 HCV CORE 132 DLMGYIPLV 9
51 0.0835 HCV CORE 35 YLLPRRGPRL 10 52 0.0725 NS1/ENV2 686
ALSTGLIHL 9 53 0.0415 HCV CORE 178 LLALLSCLTI 10 54 0.0340 HCV NS5
2578 RLIVFPDLGV 10 55 0.0320 HCV NS5 2885 RLHGLSAFSL 10 56 0.0200
HCV NS4 1811 ILGGWVAAQL 10 57 0.0180 HCV ENV1 364S SMVGNWAKV 9 58
0.0155 HCV NS3 1131 YLVTRHADV 9 59 0.0109 HCV NS4 1666 VLAALAAYCL
10 60 0.0106
[0064] With respect to treatment or prevention of HPV infections in
mammals, one or more peptides that induce a CTL response to a HPV
may be selected. The HPV CTL-inducing peptide will usually have at
least four, sometimes six, often seven or more residues, or a
majority of amino acids of that peptide that are identical or
homologous when compared to the corresponding portion of the
naturally occurring HPV sequence. For example, those peptides which
are preferred for stimulating HPV CTL responses include sequences
contained within copending U.S. patent application Ser. Nos.
08/159,339 and 08/073,205 and EPO Patent Application 92201252.1,
previously incorporated by reference, in particular the following
peptides:
4 SEQ ID BINDING SOURCE POSITION SEQUENCE SIZE NO: A2 HPV16 E7 82
LLMGTLGIV 9 65 0.0240 HPV16 E7 11 YMLDLQPET 9 66 0.1400 HPV16 E6 52
FAFRDLCIV 9 67 0.0570 HPV16 E7 86 TLGIVCPIC 9 68 0.0750 HPV16 E7 7
TLHEYMLDL 9 69 0.0070 HPV16 E7 85 GTLGIVCPI 9 70 0.0820 HPV16 E7 12
MLDLQPETT 9 71 0.0028 HPV16 E6 29 TIHDIILECV 10 72 0.0210
[0065] With respect to treatment or prevention of human
immunodeficiency virus 1 and 2 in humans, one or more peptides that
induce a CTL response to a HIV 1 or 2 antigen may be selected. The
HIV CTL-inducing peptide will usually have at least four, sometimes
six, often seven or more residues, or a majority of amino acids of
that peptide that are identical or homologous when compared to the
corresponding portion of the naturally occurring HIV sequence. For
example, those peptides which are preferred for stimulating HIV CTL
responses include the following peptides:
5 SEQ ID BINDING SOURCE POSITION SEQUENCE SIZE NO: A2 HIV 367
VLAEAMSQV 9 73 0.1100 HIV 1496 LLWKGEGAVV 10 74 0.0360 HIV 1496
LLWKGEGAV 9 75 0.0230 HIV 1004 ILKEPVHGV 9 76 0.0190 HIV 1129
IVGAETFYV 9 77 0.0099 HIV 1129 IIGAETFYV 9 78 0.0260 HIV 2182
LWVTVYYGV 9 79 0.0014 HIV 2182 LMVTVYYGV 9 80 0.4400
[0066] Several tumor associated antigens have also been correlated
with CTL responses, including, but not limited to, renal cell
carcinoma antigens, breast cancer antigens, carcinoembryonic
antigen (CEA), melanoma (MAGE-1 and MAGE-3) antigens, prostate
cancer specific antigen and others. For example, an
HLA-A1-restricted CTL epitope for the tumor-associated antigen
MAGE-3 has been identified using this approach and is the subject
of a pending patent application, U.S. patent application Ser. No.
08/186,266, previously incorporated herein by reference.
[0067] Peptides which stimulate CTL responses to tumor antigens and
which can be used in the methods and compositions of the present
invention can be selected as described in, for example, U.S. patent
applications Ser. Nos. 08/159,339 and 08/073,205, previously
incorporated by reference. For example, representative peptides
which are preferred for inducing MAGE-3 and -1 CTL responses
include the following:
6 SEQ ID BINDING SOURCE POSITION SEQUENCE SIZE NO: A2 MAGE2 105
KMVELVHFL 9 81 0.5100 MAGE2 105 KMVELVHFLL 10 82 0.2200 MAGE3 153
LVFGIELMEV 10 83 0.1100 MAGE1 278 KVLEYVIKV 9 84 0.0900 MAGE1 105
KVADLVGFLL 10 85 0.0560 MAGE3 105 KVAEFVHFL 9 86 0.0550 MAGE1 92
CILESLFRA 9 87 0.0460 MAGE1 264 FLWGPRALA 9 88 0.0420 MAGE1 200
VMIAMEGGHA 10 89 0.0360 MAGE1 38 LVLGTLEEV 9 90 0.0320 MAGE1 301
ALREEEEGV 9 91 0.0210 MAGE1 270 ALAETSYVKV 10 92 0.0150 MAGE1 282
YVIKVSARV 9 93 0.0140 MAGE1 269 RALAETSYV 9 94 0.0100
[0068] The present invention enhances the effectiveness of a
CTL-inducing peptide by co-delivery with a sequence which contains
at least one epitope that is capable of inducing an HTL response.
By an HTL response is meant a CD4.sup.+ T lymphocyte response
wherein CD4.sup.+ T lymphocytes are activated. The HTLs stimulated
by the HTL-inducing peptide can be the T-helper 1 and/or T-helper 2
phenotype, for example. The activated T helper lymphocytes will
secrete a variety of products, including, for example,
interleukin-2, which may facilitate expression of the T cell
receptor and promote recognition by activated CTLs.
[0069] HTL-inducing epitopes can be provided by peptides which
correspond substantially to the antigen targeted by the
CTL-inducing peptide, or more preferably peptides to a more widely
recognized antigen, and preferably not specific for a particular
histocompatibility antigen restriction. Peptides which are
recognized by most individuals regardless of their MHC class II
phenotype ("promiscuous") may be particularly advantageous. The HTL
peptide will typically comprise from six to thirty amino acids and
contain an HTL-inducing epitope. For example, illustrative peptides
useful in the present invention are those which contain HTL
inducing epitopes within an HTL peptide from tetanus toxoid 830-843
having the sequence Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-I-
le-Thr-Glu (QYIKANSKFIGITE) [SEQ ID NO:95], malaria
circumsporozoite 382-398
Lys-Ile-Ala-Lys-Met-Lys-Ala-Ser-Ser-Val-Phe-Asn-Val-Val-Asn-Ser
(KIAKMEKASSVFNVVNS) [SEQ ID NO:96]; malaria circumsporozoite
.sub.378-398
Asp-Ile-Glu-Lys-Lys-Ile-Ala-Lys-Met-Lys-Ala-Ser-Ser-Val-Phe-Asn-Val-Val-A-
sn-Ser (DIEKKIAKMEKASSVFNVVNS) [SEQ ID NO:97], and ovalbumin
323-336 ile-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu
[SEQ ID NO:98] and the influenza epitope 307-319
Pro-Lys-Tyr-Val-Lys-Gln-Asn-Thr-Leu-Lys- -Leu-Ala-Thr [SEQ ID
NO:99]. In addition suitable T helper peptides have been identified
as described in pending U.S. patent application Ser. No.
08/121,101, incorporated herein by reference.
[0070] Other examples of HTL-inducing peptides are those which are
specific for the antigen (virus or other organism, tumor, etc.)
being targeted by the CTL. For example, several HTL-inducing
peptides specific for HBV have been described, such as
HBc.sub.1-20, having the sequence:
Met-Asp-Ile-Asp-Pro-Tyr-Lys-Glu-Phe-Gly-Ala-Thr-Val-Glu-Leu-Leu-Ser-Phe-L-
eu-Pro [SEQ ID NO:100]; peptides from the region HBc.sub.50-69,
which has the sequence
Pro-His-His-Tyr-Ala-Leu-Arg-Gln-Ala-Ile-Leu-Cys-Trp-Gly-Glu--
Leu-Met-Tyr-Leu-Ala [SEQ ID NO:101], and from the region of
HBc,.sub.100-139, including HBc.sub.100-119 having the sequence
Leu-Leu-Trp-Phe-His-Ile-Ser-Cys-Leu-Thr-Phe-Gly-Arg-Glu-Thr-Val-Ile-Glu-T-
yr-Leu [SEQ ID NO:102] (where Ile.sub.116 is Leu in the HBV adw
subtype), HBc.sub.117-.sub.131, having the sequence
Glu-Tyr-Leu-Val-Ser-Phe-Gly-Val- -Trp-Ile-Arg-Thr-Pro-Pro-Ala [SEQ
ID NO:103], and peptide HBc.sub.120-139 having the sequence
Val-Ser-Phe-Gly-Val-Trp-Ile-Arg-Thr-Pro-Pro-Ala-Tyr-A-
rg-Pro-Pro-Asn-Ala-Pro-IIe [SEQ ID NO:104]. See, Ferrari et al., J.
Clin. Invest. 88:214-222 (1991), and U.S. Pat. Nos. 4,882,145, and
5,143,726, each of which is incorporated herein by reference.
[0071] The CTL or HTL inducing peptides employed in the
compositions and methods of the present invention need not be
identical to specific peptides disclosed in aforementioned
disclosures, and can be selected by a variety of techniques, for
example, according to certain motifs as described above. Therefore,
the peptides may be subject to various changes, such as insertions,
deletions, and substitutions, either conservative or
non-conservative, where such changes might provide for certain
advantages in their use. By conservative substitutions is meant
replacing an amino acid residue with another which is biologically
and/or chemically similar, e.g., one hydrophobic residue for
another, or one polar residue for another. The substitutions
include combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu;
Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Usually, the portion of
the sequence which is intended to substantially mimic a CTL or HTL
stimulating epitope will not differ by more than about 20% from the
corresponding sequence of a native antigen, when known, except
where additional amino acids may be added at either terminus for
the purpose of modifying the physical or chemical properties of the
peptide for, e.g., ease of linking or coupling, and the like. In
those situations where regions of the peptide sequences are found
to be polymorphic among antigen subtypes, it may be desirable to
vary one or more particular amino acids to more effectively mimic
differing CTL or HTL epitopes of different antigen strains.
[0072] In some instances it may be desirable to combine two or more
peptides which contribute to stimulating specific CTL responses in
one or more patients or histocompatibility types. The peptides in
the composition can be identical or different, and together they
should provide equivalent or greater biological activity than the
parent peptide(s). For example, using the methods described herein,
two or more peptides may define different or overlapping CTL
epitopes from a particular region, e.g., the peptide region 799.08
(HBenv.sub.309-328), peptide region, 799.09 (HBenv.sub.329-349),
799.10 (HBenv.sub.349-368), or peptide region 802.03
(HBc.sub.91-110), peptides can be combined in a "cocktail" to
provide enhanced immunogenicity of CTL responses, and peptides can
be combined with peptides having different MHC restriction
elements. This composition can be used to effectively broaden the
immunological coverage provided by therapeutic, vaccine or
diagnostic methods and compositions of the invention among a
diverse population.
[0073] In some embodiments the CTL inducing peptides of the
invention are linked to the HTL inducing peptides. CTL inducing
peptides/T helper conjugates can be linked by a spacer molecule, or
the CTL peptide may be linked to the HTL peptide without a spacer.
When present, 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 and may have linear or branched side chains. The spacers
are typically selected from, e.g., Ala, Gly, or other neutral
spacers of nonpolar amino acids or neutral polar amino acids. In
certain preferred embodiments herein the neutral spacer is Ala. 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. Preferred exemplary spacers are homo-oligomers of
Ala. When present, the spacer will usually be at least one or two
residues, more usually three to six residues. When the HTL-inducing
peptide is conjugated to the CTL-inducing peptide, in the presence
or absence of a spacer, preferably with the HTL peptide is
positioned at the amino end of the conjugate.
[0074] The peptides of the invention can be combined via linkage to
form polymers (multimers), or can be formulated in a composition
without linkage, as an admixture. Where the same peptide is linked
to itself, thereby forming a homopolymer, a plurality of repeating
epitopic units are presented. When the peptides differ, e.g., a
cocktail representing different antigen strains or subtypes,
different epitopes within a subtype, different histocompatibility
restriction specificities, or peptides which contain HTL epitopes,
heteropolymers with repeating units are provided. In addition to
covalent linkages, noncovalent linkages capable of forming
intermolecular and intrastructural bonds are also contemplated.
[0075] Linkages for homo- or hetero-polymers or for coupling to
carriers can be provided in a variety of ways. For example,
cysteine residues can be added at both the amino- and
carboxy-termini, where the peptides are covalently bonded via
controlled oxidation of the cysteine residues. Also useful are a
large number of heterobifunctional agents which generate a
disulfide link at one functional group end and a peptide link at
the other, including N-succidimidyl-3-(2-pyridyldithio) proprionate
(SPDP). This reagent creates a disulfide linkage between itself and
a cysteine residue in one protein and an amide linkage through the
amino on a lysine or other free amino group in the other. A variety
of such disulfide/amide forming agents are known. See, for example,
Immun. Rev. 62:185 (1982). Other bifunctional coupling agents form
a thioether rather than a disulfide linkage. Many of these
thioether forming agents are commercially available and include
reactive esters of 6-maleimidocaproic acid, 2 bromoacetic acid,
2-iodoacetic acid, 4-(N-maleimido-methyl) cyclohexane-1-carboxylic
acid and the like. The carboxyl groups can be activated by
combining them with succinimide or 1-hydroxy-2-nitro-4-sulfo- nic
acid, sodium salt. A particularly preferred coupling agent is
succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate
(SMCC). Of course, it will be understood that linkage should not
substantially interfere with either of the linked groups to
function as described, e.g., to function as a CTL determinant or
HTL determinant.
[0076] As a further aspect of the present invention the
HTL-inducing peptide(s) and CTL-inducing peptide(s) can be
delivered to the patient in the presence of a lipid. The lipid
residue, such as palmitic acid or the like (as described further
below, which is attached to alpha and epsilon amino groups of a Lys
residue ((PAM).sub.2Lys), is attached to the amino terminus of the
HTL-inducing peptide. The lipid can be attached directly to the HTL
peptide, or, more typically, indirectly via a linkage, such as a
Ser-Ser, Gly, Gly-Gly, Ser linkage or the like.
[0077] As another example of lipid-HTL priming of CTL responses, E.
coli lipoprotein, such as
tripahnitoyl-S-glycerylcysteinly-seryl-serine (P.sub.3CSS), can be
used to prime specific CTL when covalently attached to an
appropriate HTL peptide. See, Deres et al., Nature 342:561-564
(1989), incorporated herein by reference. The HTL peptides can be
coupled to P.sub.3CSS, for example, and the lipopeptide
administered in conjunction with the CTL inducing peptide to an
mammal to specifically prime a CTL response to the antigen of
interest.
[0078] Yet another example of lipid priming of CTL response is
achieved by conjugating the CTL/T helper-peptide-conjugate with
uncharged fatty acid residues of different chain lengths and
degrees of unsaturation, ranging from acetic to stearic acid as
well as to negatively charged succinyl residues via the appropriate
carboxylic acid anhydrides.
[0079] The lipid may be linked to other peptides which present HTL
epitopes which are then combined with the lipid. When the HTL and
CTL are linked in a conjugate, the arrangement of the components of
the conjugate comprising the CTL inducing peptide/T helper
peptide/lipid can be varied. In one case, the lipid moiety can be
linked to the amino terminal end of the CTL inducing peptide, which
in turn is linked at its carboxy terminal to the T helper peptide.
In another case, the lipid is linked at the amino terminal end of
the T helper peptide, which is linked at its carboxy terminal to
the CTL inducing peptide. In each case, a spacer molecule can be
selectively inserted between the lipid moiety and the CTL or T
helper peptide, as well as between the T helper and the CTL
inducing peptides. In the case of the spacer between the lipid and
the T helper or CTL inducing peptide, a preferred example comprises
Lys-Ser-Ser, although other spacers are described herein. An
example of a spacer between the T helper and CTL inducing peptides
will be Ala-Ala-Ala, as also described in further detail
herein.
[0080] As further described herein, the lipidated HTL peptide and
CTL peptide can then be emulsified in an adjuvant, e.g., incomplete
Freund's adjuvant, alum or montanide.
[0081] In an exemplary embodiment described below, a T helper
peptide from substantially within TT830-843 was lipidated at its
N-terminus (with (PAM)2) via a linker (KSS) and then linked at its
C-terminus (via a linker AAA) with a HBV CTL inducing peptide,
HBc18-27. Thus, the structure of the peptide was (PAM).sub.2KSS-T
helper-AAA-CTL and had the sequence of
(PAM).sub.2KSS-ISQAVHAAHAEINE-AAA-TYQRTRALV. This conjugate, when
administered to transgenic animals expressing the HLA2.1 antigen,
was shown to induce specific CTL priming of animals. It was also
established that the CTL induced by the peptide recognized
endogenously synthesized HBcore antigens. When the same
lipid-THL-CTL peptide conjugate was administered to humans an
induction of specific CTL response was observed, where the response
dose dependent both in proportion of subjects exhibiting a positive
response as well as in the magnitude of the response obtained.
[0082] The peptides of the invention can be prepared in a wide
variety of ways. Because of their 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 and
Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co.
(1984); Tam et al., J. Am. Chem. Soc. 105:6442 (1983); Merrifield,
Science 232:341-347 (1986); and Barany and Merrifield, The
Peptides, Gross and Meienhofer, eds., Academic Press, New York, pp.
1-284 (1979), each of which is incorporated herein by
reference.
[0083] Alternatively, recombinant DNA technology may be employed
wherein a nucleotide sequence which encodes a CTL peptide and/or T
helper 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. (1982), and Ausubel
et al., (ed.) Current Protocols in Molecular Biology, John Wiley
and Sons, Inc., New York (1987), and U.S. Pat. Nos. 4,237,224,
4,273,875, 4,431,739, 4,363,877 and 4,428,941, for example, which
disclosures are incorporated herein by reference. Thus, fusion
proteins which comprise one or more peptide sequences can be used
to present the CTL and HTL determinants. For example, a recombinant
HBV surface antigen protein is prepared in which the HBenv amino
acid sequence is altered so as to more effectively present epitopes
of peptide regions described herein to stimulate a CTL response. By
this means a polypeptide is used which incorporates several CTL and
HTL epitopes.
[0084] As the coding sequence for peptides of the length
contemplated herein can be synthesized by chemical techniques, for
example, the phosphotriester method of Matteucci et al., J. Am.
Chem. Soc. 103:3185 (1981), modification can be made simply by
substituting the appropriate base(s) for those encoding the native
peptide sequence. 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 or
mammalian cell hosts may also be used, employing suitable vectors
and control sequences.
[0085] The peptides of the present invention and pharmaceutical and
vaccine compositions thereof are useful for administration to
mammals, particularly humans, to treat and/or prevent viral,
bacterial, and parasitic infections. As the peptides are used to
stimulate cytotoxic T-lymphocyte responses to HBV infected cells,
the compositions can be used to treat or prevent acute and/or
chronic HBV infection.
[0086] For pharmaceutical compositions, the peptides, i.e., the
compositions of lipidated HTL/CTL peptides of the invention as
described above will be administered to a mammal already suffering
from or susceptible to the disease being treated. Those in the
incubation phase or the acute phase of disease such as a viral
infection, e.g., HBV, can be treated with the immunogenic peptides
separately or in conjunction with other treatments, as appropriate.
In therapeutic applications, compositions are administered to a
patient in an amount sufficient to elicit an effective CTL response
to the disease and to cure or at least partially arrest its
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 peptide
composition, 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, but generally range for the initial immunization (that
is for therapeutic or prophylactic administration) from about 1.0
.mu.g to about 50 mg, preferably 1 .mu.g to 500 .mu.g, most
preferably 1 .mu.g to 250 .mu.g followed by boosting dosages of
from about 1.0 .mu.g to 50 mg, preferably 1 .mu.g to 500 .mu.g, and
more preferably 1 .mu.g to about 250 .mu.g of peptide pursuant to a
boosting regimen over weeks to months depending upon the patient's
response and condition by measuring specific CTL activity in the
patient's blood. It must be kept in mind that the peptides and
compositions of the present invention may generally be employed in
serious disease states, that is, life-threatening or potentially
life threatening situations. In such cases, in view of the
minimization of extraneous substances and the relative nontoxic
nature of the peptides, it is possible and may be felt desirable by
the treating physician to administer substantial excesses of these
peptide compositions.
[0087] Single or multiple administrations of the compositions can
be carried out with dose levels and pattern being selected by the
treating physician. In any event, the pharmaceutical formulations
should provide a quantity of cytotoxic T-lymphocyte stimulatory
peptides of the invention sufficient to effectively treat the
patient.
[0088] For therapeutic use, administration should begin at the
first sign of disease (e.g., HBV infection), to be followed by
boosting doses until at least symptoms are substantially abated and
for a period thereafter. In cases of established or chronic
disease, such as chronic HBV infection, loading doses followed by
boosting doses may be required. The elicitation of an effective CTL
response during early treatment of an acute disease stage will
minimize the possibility of subsequent development of chronic
disease such as hepatitis, HBV carrier stage, and ensuing
hepatocellular carcinoma.
[0089] Treatment of an infected mammal with the compositions of the
invention may hasten resolution of the disease in acutely afflicted
mammals. For those mammals susceptible (or predisposed) to
developing chronic disease the compositions of the present
invention are particularly useful in methods for preventing the
evolution from acute to chronic disease. Where the susceptible
individuals are identified prior to or during infection, for
instance, as described herein, the composition can be targeted to
them, minimizing need for administration to a larger
population.
[0090] The peptide compositions can also be used for the treatment
of chronic or established disease such as viral hepatitis and to
stimulate the immune system to eliminate virus-infected cells.
Those with chronic hepatitis can be identified as testing positive
for virus from about 3-6 months after infection. As individuals may
develop chronic HBV infection because of an inadequate (or absent)
CTL response during the acute phase of their infection, it is
important to provide an amount of immuno-potentiating peptide
compositions of the invention in a formulation and mode of
administration sufficient to effectively stimulate a CTL response.
Thus, for treatment of chronic hepatitis, a representative dose is
in the range of about 1.0 .mu.g to about 50 mg, preferably 1 .mu.g
to 500 .mu.g, most preferably 1 .mu.g to 250 .mu.g followed by
boosting dosages of from about 1.0 .mu.g to 50 mg, preferably 1
.mu.g to 500 .mu.g, and more preferably 1 .mu.g to about 250 .mu.g
per dose. Administration should continue until at least clinical
symptoms or laboratory indicators indicate that the HBV infection
has been eliminated or substantially abated and for a period
thereafter. Immunizing doses followed by boosting doses at
established intervals, e.g., from one to four weeks, may be
required, possibly for a prolonged period of time, as necessary to
resolve the infection. For the treatment of chronic and carrier HBV
infection it may also be desirable to combine the CTL and HTL
peptides with other peptides or proteins that induce immune
response to other HBV antigens, such as HBsAg.
[0091] The pharmaceutical compositions for therapeutic treatment
are intended for parenteral, topical, oral or local administration.
Preferably, the pharmaceutical compositions are administered
parenterally, e.g., intravenously, subcutaneously, intradermally,
or intramuscularly. Thus, the invention provides compositions for
parenteral administration which comprise a solution of the HTL and
CTL stimulatory 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, 0.4% 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 and the like, for example, sodium acetate,
sodium lactate, sodium chloride, potassium chloride, calcium
chloride, sorbitan monolaurate, triethanolamine oleate, methanol,
and dissolving agents such as DMSO and other like agents.
[0092] The concentration of HTL and CTL stimulatory peptides of the
invention in the pharmaceutical formulations can vary widely, i.e.,
from less than about 1%, usually at or at least about 10% 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.
[0093] Thus, a typical pharmaceutical composition for intravenous
infusion could be made up to contain 250 ml of sterile Ringer's
solution, and 50 mg of peptide. Actual methods for preparing
parenterally administrable compounds will be known or apparent to
those skilled in the art and are described in more detail in for
example, Remington's Pharmaceutical Sciences, 17th ed., Mack
Publishing Company, Easton, Pa. (1985), which is incorporated
herein by reference.
[0094] The peptides of the invention may also be administered via
liposomes, which serve to target the peptides to a particular
tissue, such as lymphoid tissue, or targeted selectively to
infected cells, as well as 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, e.g., 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 filled with a desired peptide of the
invention can be directed to the site of lymphoid cells, where the
liposomes then deliver the selected therapeutic/immunogenic peptide
compositions. Liposomes for use in 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 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), U.S. Pat. Nos. 4,235,871,
4,501,728, 4,837,028, and 5,019,369, incorporated herein by
reference. For targeting to the immune cells, 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, and
in other manners 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.
[0095] 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 peptide
compositions of the invention, and more preferably at a
concentration of 25%-75%.
[0096] For aerosol administration, the HTL and CTL stimulatory
peptide compositions 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.
[0097] In another aspect the present invention is directed to
vaccines which contain as an active ingredient an immunogenically
effective amount of a composition of HTL and CTL stimulating
peptides as described herein. The peptide(s) may be introduced into
a mammalian host, including humans, linked to its own carrier or as
a homopolymer or heteropolymer of active peptide units. Such a
polymer has the advantage of increased immunological reaction and,
where different peptides are used to make up the polymer, the
additional ability to induce antibodies and/or cytotoxic T cells
that react with different antigenic determinants of the virus.
Useful carriers are well known in the art, and include, e.g.,
thyroglobulin, albumins such as human serum albumin, tetanus
toxoid, polyamino acids such as poly(D-lysine:D-glutamic acid),
influenza protein and the like. The vaccines can also contain a
physiologically tolerable (acceptable) diluent such as water,
phosphate buffered saline, or saline, and further typically include
an adjuvant. Adjuvants such as incomplete Freund's adjuvant,
aluminum phosphate, aluminum hydroxide, alum, or
MONTANIDE.RTM.(Seppic, Paris, France; oil-based adjuvant with
mannide oleate) are materials well known in the art. Upon
immunization with a peptide composition as described herein, via
injection, aerosol, oral, transdermal or other route, the immune
system of the host responds to the vaccine by producing large
amounts of CTLs specific for the disease associated antigen, and
the host becomes at least partially immune to the disease, e.g.,
HBV infection, or resistant to developing chronic disease.
[0098] Vaccine compositions containing the peptides of the
invention are administered to a patient susceptible to or otherwise
at risk of disease, e.g., viral infection, to enhance the patient's
own immune response capabilities. Such an amount is defined to be a
"immunogenically effective dose." In this use, the precise amounts
depend on the patient's state of health, age, the mode of
administration, the nature of the formulation, and other
parameters. The peptides are administered to individuals of an
appropriate HLA type, e.g., for vaccine compositions of peptide HBc
18-27, these will be administered to HLA-A2 individuals.
[0099] In some instances it may be desirable to combine the peptide
vaccines of the invention with vaccines which induce neutralizing
antibody responses to the disease, e.g., HBV, particularly to HBV
envelope antigens, such as recombinant HBV env-encoded antigens or
vaccines prepared from purified plasma preparations obtained from
HBV-infected individuals. A variety of HBV vaccine preparations
have been described, and are based primarily on HBsAg and
polypeptide fragments thereof. For examples of vaccines which can
be formulated with the peptides of the present invention, see
generally, European Patent publications EP 154,902 and EP 291,586,
and U.S. Pat. Nos. 4,565,697, 4,624,918, 4,599,230, 4,599,231,
4,803,164, 4,882,145, 4,977,092, 5,017,558 and 5,019,386, each of
which is incorporated herein by reference. The vaccines can be
combined and administered concurrently, or as separate
preparations.
[0100] The peptides may also find use as diagnostic reagents. For
example, a peptide of the invention may be used to determine the
susceptibility of a particular individual to a treatment regimen
which employs the peptide or related peptides, and thus may be
helpful in modifying an existing treatment protocol or in
determining a prognosis for an affected individual. In addition,
the peptides may also be used to predict which individuals will be
at substantial risk for developing chronic HBV infection.
[0101] The following examples are offered by way of illustration,
not by way of limitation.
EXAMPLE 1
Identification of CTL-specific HBV Epitopes
[0102] A line of transgenic mice which express a mouse-human
chimeric class I molecule was used to define HBV core and surface
antigen sequences that represent CTL-specific epitopes.
[0103] The transgenic mouse line 66 obtained from Scripps Clinic
and Research Foundation expresses a chimeric class I molecule
composed of the .alpha.1 and .alpha.2 domains of human HLA-A2.1
antigen and the 3 transmembrane and cytoplasmic domains of
H-2K.sup.b. The transgenic mice were prepared as generally
described in Vitiello et al., J. Exp. Med. 173:1007-1015 (1991),
which is incorporated herein by reference. When these mice are
primed in vivo with the influenza virus, they generate a CTL
response that is specific for virtually the same epitopes as those
recognized by human influenza-specific CTL. Thus, these transgenic
animals can be used to determine HBV epitopes recognized by human T
cells.
[0104] To define which sequence regions within HBV surface and core
proteins represented CTL epitopes, synthetic peptides derived from
the two proteins were prepared and tested for their ability to bind
to human HLA-A2.1. Binding was determined by the relative capacity
of different peptide concentrations to inhibit recognition of A2.1
target cells in the presence of the influenza matrix peptide 57-68
by the CTL line 219, as determined by the inhibition of release of
serine esterase from the cells. The 219 line was derived from A2.1
transgenic mice and is specific for the matrix peptide 57-68 in the
context of HLA-A2.1.
[0105] Briefly, peptides to be assayed for CTL epitopes were
dissolved in DMSO at a concentration of 20 mg/ml. Just before the
assay, peptides were diluted in RPMI 1640 buffered with 25 .mu.M
Hepes and containing 0.05% BSA (assay media). Fifty microliters of
a 200 .mu.g/ml, 66 .mu.g/ml, or 22 .mu.g/ml of peptide solution
were added to wells of 96 round-bottomed plates containing
4.times.10.sup.5 Jurkat A2.1/K.sup.b cells in a volume of 50 .mu.l
of assay media. Plates were incubated for 30 min. at 37.degree. C.
Fifty .mu.l of 2.5 .mu.g/ml solution of the index peptide (matrix
peptide 57-68 from PR8 influenza virus) were then added to the
cells, followed by 50 .mu.l containing 5.times.10.sup.4 line 219
CTL, where the concentration of index peptide used was chosen as
that which induced 75% serine esterase release from CTL 219, as
determined by titration of the peptide. After 4 hours incubation at
37.degree. C., plates were centrifuged for 5 min. at 1000 RPM, and
20 .mu.l supernatant transferred to flat-bottomed 96-well plates.
Esterase activity in the supernatant was measured by adding 180
.mu.l of a reaction mixture consisting of 0.2M TrisHCl pH 8.1,
2.0.times.10.sup.-4 N-benzyloxycarbonyl-L-Lysine thiobenzyl ester
(BLT) and 2.2.times.10.sup.-4 M dithiobis (nitrobenzoic acid).
Plates were incubated for 1 hour at 37.degree. C. and absorbance
read at 412 nm. Percent inhibition was calculated by the following
formula:
% inhibition=100-A.sub.412(test+index) peptide-A.sub.412test
peptide alone.times.100A.sub.412index peptide-A.sub.412no
peptide
[0106] Those peptides which bound to A2.1 and caused more than 24%
inhibition of serine esterase release by the cells were assayed in
vitro for the ability to restimulate a CTL response from
splenocytes derived from HBV primed A2.1 transgenic mice. (Sette,
A. et al., J. Immunol. 147:3893 (1991)). HBV priming was performed
by injecting A2.1 spleen cells "loaded" with HBV virus as described
by Carbone and Bevan, J. Exp. Med. 171:377-387 (1990).
[0107] Briefly, red blood cell depleted splenocytes were suspended
in 0.4 ml of a solution composed of 200 .mu.l of HBV purified virus
and 200 .mu.l of a 2.times.hypertonic solution (0.5 M sucrose, 10%
w/v polyethylene glycol 1000, 10 mM Hepes, pH 7.2, in RPMI 1640
medium), for 10.degree. C. min. at 37.degree. C. The cell
suspension was then rapidly diluted in prewarmed hypotonic media
(60% HBSS and 40% water), incubated for 2 min. at 37.degree. C.,
pelleted, washed twice in HBSS and irradiated (1,000 rad.). Mice
were then injected with 5.0.times.10.sup.6 loaded cells in a volume
of 200 .mu.l. Mice were boosted with HBV-loaded spleen cells 10
days later.
[0108] After about 2 weeks, spleen cells from primed mice
(5.times.10.sup.6 cells/well in 24 well plates) were cultured with
4 different mixtures of syngeneic irradiated (3000 rads) LPS blasts
(2.times.10.sup.6cells/well) that had been independently coated
with 13 different peptides. Coating was achieved by incubating
aliquots of 25.times.10.sup.6 LPS blasts in tubes each with 100
.mu.g of one of the 13 HBV synthetic peptides in one ml for 1-2 hrs
at 37.degree. C.; the contents of the different tubes were then
pooled to give 4 mixtures.
7 Mixture No. Peptide No. Peptide Location 1 800.04 HBenv47-63
802.01 HBc11-27 802.06 HBc162-176 2 801.02 HBenv141-157 799.02
HBenv194-213 802.03 HBc91-110 3 799.09 HBenv329-348 799.10
HBenv349-368 802.04 HBc111-125 4 799.04 HBenv234-253 799.05
HBenv246-265 799.08 HBenv309-328 800.05 HBenv63-77
[0109] The mixture of cells was washed once, diluted at the
required concentration and plated. The medium used for the cultures
was RPMI 1640 supplemented with 10% FCS, 50.mu.g/ml gentamicin, 2mM
glutamine and 5.times.10.sup.31 5 M 2-mercaptoethanol (R10). After
nine days, effector cells were assayed for cytotoxicity against
Jurkat A.sub.2/k.sup.b target cells in the presence of different
peptide mixtures corresponding to those used in the cultures. The
results obtained are shown in FIG. 1 panels, A through D. The
effector cells (0.2.times.10.sup.6 cells/well) obtained from these
cultures were restimulated with irradiated (20,000 rads),
peptide-coated Jurkat A.sub.2/K.sup.b cells (0.2.times.10.sup.6
cells/well) in the presence of 3.times.10.sup.6 feeder cells/well
(C57BL/6 irradiated spleen cells) in R10 supplemented with 5%-rat
ConA supernatant. After 6 days, these effector cells were assayed
for cytotoxicity against .sup.51Cr labeled Jurkat A.sub.2/K.sup.b
target cells in the presence of the 13 individual peptides.
Peptides that induced CTL lysis of Jurkat A.sub.2/K.sup.b target
cells above background (FIG. 1, panels E through H) i.e., HBenv
47-63, HBc 11-27 (panel E) HBenv 141-157, HBenv 194-213, HBc 91-110
panel F), HBenv 329-348 and 349-368 panel G) and HBenv 309-328
(panel H) were independently used to restimulate the effector cells
generated with the peptide mixtures. After six days in culture, the
effector cells were tested for cytotoxicity against .sup.51Cr
Jurkat A.sub.2/K.sup.b cells in the presence of the peptide used
for the restimulation (FIG. 1). The set of experiments, outlined in
this example allow us to determine that HBV peptides HBc 11-27
(FIG. 1 panels A, E; FIG. 2 panel A) HBc 91-110 (FIG. 1 panels B,
F; FIG. 2 panel E), HBenv 329-348 (FIG. 1 panels C, G; FIG. 2 panel
F) HBenv 349-368 (FIG. 1 panels C, G; FIG. 2 panel G) and HBenv
309-328 (FIG. 1 panels D, H; FIG. 2 panel H) clearly represent CTL
epitopes.
EXAMPLE 1A
Induction of Ovalbumin-specific CTL Response in Mice
[0110] B6 mice were injected with 10, 50, or 200 .mu.g of ovalbumin
in HBSS intravenously, intraperitoneally and subcutaneously with
10, 50, 200 .mu.g ovalbumin subcutaneously in IFA. Ten days later,
splenocytes from primed animals were stimulated in vitro with
irradiated EG7 cells (EL-4 cells transfected with OVA). Six days
later, the effector cells were tested for cytolytic activity
against .sup.51Cr labelled EL-4 and EG7 cells. No CTL activity was
induced by injection of ovalbumin in HBSS either intravenously or
interperitoneally. Some CTL induction was seen at the 200 .mu.g
dose for subcutaneous injection of ovalbumin in HBSS. Strong CTL in
vivo induction was seen when ovalbumin was administered with IFA,
optimal induction occurred with the 10 .mu.g dose given
subcutaneously.
EXAMPLE 2
Induction of A2.1-Restricted CTL by Subcutaneous Priming With
Purified HBV in Incomplete Freund's Adjuvant (IFA)
[0111] Injection of ovalbumin (OVA) in IFA subcutaneously induces
an ovalbumin-specific CTL response in mice, while injection of OVA
either i.v. or i.p. generally does not lead to the generation of
CTL. This technique was used to induce HBV-specific CTL in A2.1
transgenic mice.
[0112] Priming and In Vitro Restimulation: A2.1/K.sup.b transgenic
mice were injected with 100 microliters of an emulsion of purified
HBV virus in incomplete Freund's adjuvant (IFA). This emulsion was
prepared by mixing purified HBV (1 mg protein/ml) diluted 1:5 in
HBSS with an equal volume of IFA. Seven days after priming,
splenocytes (5.times.10.sup.6 cells/well in a 24 well plate)
obtained from these animals were restimulated with syngeneic
irradiated LPS blasts (2.times.10.sup.6/well) coated with each of
the following peptides:
8 799.09 HBenv 329-348 802.03 HBc91-110 875.20 HBenv 335-343 883.02
HBc92-101 875.21 HBenv 338-347 883.03 HBc93-102 799.10 HBenv
349-368 875.15 HBc18-27 884.01 HBenv 348-357 875.18 HBc107-115
884.02 HBenv 349-358 875.19 HBc139-148
[0113] These peptides were chosen because: 1) They had been defined
as containing CTL epitopes in Example I (peptides 799.10, 799.09,
802.03); 2) they represent truncations of peptides defined in
Example I that are recognized by the CTL raised against the larger
epitopes (i.e., peptides 875.15, 884.02, 883.02, 883.03); or 3)
they contain the A2.1 binding motif as described by Falk et al.
(Nature 351:290-296 (1991)), i.e., leucine or methionine in
position 2, and either leucine or valine in position 9 or valine in
position 10, (i.e., peptides 884.01, 875.20, 875.21, 875.18 and
875.19). Coating was achieved by incubating 50 .mu.g of each
individual peptide with 12.times.10.sup.6 LPS blasts in a volume of
0.4 ml of RPMI medium supplemented with 10% FCS for 1 h at
37.degree. C. The cells were washed once. After 6 days, effector
cells were assayed for cytotoxicity against .sup.51Cr labelled
Jurkat A.sub.2/K.sup.b cells in the presence of the appropriate
peptides. The results are shown in FIG. 3.
[0114] These effector cells (0.2.times.10.sup.6 cells/well) were
restimulated at weekly intervals. For the first restimulation,
peptide-coated LPS blasts were used, followed by peptide-coated
Jurkat A2.1/K.sup.b cells. Six days after restimulation, effector
cells were assayed for cytotoxicity against .sup.51Cr labelled
Jurkat A2/K.sup.b target cells in the presence of the appropriate
peptides. The results obtained are shown in FIG. 4.
[0115] Peptides clearly able to induce in vitro CTL from
splenocytes of HBV-primed mice are FIG. 3 and 4, panel A: HBc
18-27; FIG. 3 and 4, panel B: HBenv 349-368; FIG. 3 and 4, panel D:
HBenv 349-358; FIG. 3 and 4, panel F: HBenv 329-348; FIG. 3 and 4,
panel I: HBc 91-110; FIG. 3 and 4, panel J: HBc 92-102; and FIG. 3
and 4, panel K: HBc 93-102. Truncation peptides recognized by CTL
raised against the larger peptide and as such should contain at
least part of a CTL epitope are: FIG. 3F, 4F: HBenv 335-343 and
HBenv 338-347.
EXAMPLE 2A
Other HBV CTL Epitopes
[0116] The following peptides were identified following procedures
disclosed in pending U.S. patent application Ser. Nos. 08/159,339
and 08/073,205, incorporated herein by reference.
9 CTL Epitopes Position Sequence SEQ ID NO: HBV POL 561
FLLSLGIHL-COOH 35 HBV POL 61 GLYSSTVPV-COOH 106 HBV POL 411
NLSWLSLDV-COOH 107 HBV POL 491 HLYSHPIIL-COOH 108
EXAMPLE 3
Synthesis of Peptides
[0117] Peptides were synthesized on an Applied Biosystems (Foster
City, Calif.) 430A peptides synthesizer using Fmoc protected amino
acids and 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) esters for amino acid activation. Each
amino acid was routinely triple coupled. Fmoc protected amino acids
and Hydroxybenzotriazole were purchased from Burdick and Jackson.
HBTU was purchased from Richelieu Biotechnologies (St-Hyacinthe,
Canada). Piperidine and trifluoroacetic acid, acetic anhydride, and
ethanedithiol were purchased from Sigma Chemical Corporation.
[0118] a. Peptide Phe-Leu-Pro-Ser-Asp-Phe-Phe-Pro-Ser-Val-OH [SEQ
ID NO:4]
[0119] L-Valine coupled to SASRIN.RTM. resin (Bachem Biosciences)
was loaded into the peptide synthesis reaction vessel and washed
one time with N-methylpyrolidone (NMP). The following operations
were then sequentially performed:
[0120] 1. The Fmoc protecting group was removed by treatment of the
resin bound amino acid with 25% piperidine in NMP.
[0121] 2. The resin was washed 5 times with NMP.
[0122] 3. A mixture containing Fmoc-serine, diisopropylethylamine,
HBTU and NMP was added to the reaction vessel and allowed to react
for 30 minutes, under vortex agitation.
[0123] 4. The solvent was drained, and the resin was washed three
times with NMP.
[0124] 5. Steps (3) and (4) were repeated two more times.
[0125] 6. The resin was washed four more times with NMP.
[0126] Steps 1-6 were repeated for each amino acid of the peptide.
Following the final coupling cycle, the resin-bound peptide was
deproteced by reaction with 25% piperidine in NMP, washed 7 times
with NMP, and washed 2 times with dichloromethane. The resin was
dried in vacuo for 24 hours. The peptide was cleaved from the
SASRIN.RTM. resin by treatment with trifluoroacetic acid containing
2.5% ethanedithiol and 5% water. The polystyrene resin was
separated from the trifluoroacetic acid solution by filtration.
Trifluoroacetic acid was removed by evaporation in vacuo. The crude
peptide was triturated with diethylether and dissolved in water.
The water was removed by lyophilization. The peptide was then
purified by reverse phase HPLC on a C.sub.8 column (VYDAC) using a
gradient of acetonitrile, water, each containing 0.1% TFA as
modifier.
[0127] b. Peptide
(Pal).sub.2-Lys-Ser-Ser-Phe-Leu-Pro-Ser-Asp-Phe-Phe-Pro--
Ser-Val-OH [SEQ ID NO:109]
[0128] The resin bound peptide described in section (a) was
extended by the addition of two serine residues according to the
above described procedure. The following operations were then
performed:
[0129] 1. The Fmoc protecting group was removed by treatment of the
resin bound amino acid with 25% piperidine in NMP.
[0130] 2. The resin was washed 5 times with NMP.
[0131] 3. Bis-Fmoc-Lysine was converted to the corresponding
symmetrical anhydride by treatment with diisopropylcarbodiimide in
NMP. The resin bound peptide was allowed to react with the
resulting anhydride.
[0132] 4. The resin was washed 5 times with NMP.
[0133] 5. The Fmoc protecting group was removed by treatment of the
resin bound amino acid with 25% piperidine in NMP.
[0134] 6. Palmitic acid was reacted with hydroxybenzotriazole and
diisopropylcarbodiimide in NMP. The resin bound peptide was allowed
to react with the resulting solution.
[0135] 7. The resin was washed 5 times with NMP. Finally, the
peptide was cleaved from the resin as described above.
[0136] c. Peptide
Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile-Thr-Glu--
Phe-Leu-Pro-Ser-Asp-Phe-Phe-Pro-Ser-Val-OH [SEQ ID NO:110]. The
resin bound peptide described in section (a) was chain extended by
the addition of Glu, Thr, Ile, Gly, Ile, Phe, Lys, Ser, Asn, Ala,
Lys, Ile, Tyr, and Gln residues, according to the procedure
described in section (a). Cleavage and purification were performed
as described above.
[0137] d. Peptide
Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile-Thr-Glu--
Ala-Ala-Ala-Phe-Leu-Pro-Ser-Asp-Phe-Phe-Pro-Ser-Val-OH [SEQ ID
NO:111]. The resin bound peptide described in section (a) was chain
extended by the sequential addition of Ala, Ala, Ala, Glu, Thr,
Ile, Gly, Ile, Phe, Lys, Ser, Asn, Ala, Lys, Ile, Tyr, and Gln
residues, according to the procedure described in section (a).
Cleavage and purification were performed as described above.
[0138] e. Peptide
Ac-Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile-Thr-G-
lu-Ala-Ala-Ala-Phe-Leu-Pro-Ser-Asp-Phe-Phe-Pro-Ser-Val-OH [SEQ ID
NO:112]. The resin bound peptide described in section (d) was
acetylated by reaction with acetic anhydride in NMP. Cleavage and
purification were performed as described above.
EXAMPLE 4
Induction of CTL by Combining CTL and T-helper Epitopes
[0139] This example describes experiments which define the relative
in vivo HBV-specific CTL priming efficiency of peptides expressing
HBV CTL epitopes alone, CTL epitopes mixed with peptides containing
T helper epitopes or CTL epitopes physically linked to T helper
epitopes.
[0140] Transgenic mice (HLA-A2.1/K.sup.b) were primed
subcutaneously (base of tail) with 100 .mu.g of peptide 875.23
(Ia.sup.b-restricted helper epitope HBc 128-140 TPPAYRPPNAPIL) in
complete Freund's adjuvant (CFA). Nine days later each of the
following peptides were injected subcutaneously into two unprimed
and two helper-primed mice, 100 .mu.g/mouse in incomplete Freund's
adjuvant (IFA).
10 Peptide T Helper (HBc128-140) CTL (HBc18-27) 1. 875.23
TPPAYRPPNAPIL 2. 875.15 FLPSDFFPSV 3. 875.23 + 875.15 TPPAYRPPNAPIL
+ FLPSDFFPSV 4. 902.01 TPPAYRPPNAPILFLPSDFFPSV-NH.sub.2 5. 902.02
TPPAYRPPNAPILAAAFLPSDFFPSV-NH.sub.2 6. No peptide
[0141] Three weeks after priming with the CTL epitope, splenocytes
were in vitro restimulated with LPS blasts coated with HBc 18-27
(coating was achieved by incubating 30.times.10.sup.6 LPS blasts
with 100 .mu.g of HBc18-27 in one ml of medium; after 1-2 hr at
37.degree. C., the cells were washed). After 6 days, effector cells
were assayed for lytic activity against .sup.51Cr labelled Jurkat
A.sub.2/K.sup.b target cells in the presence or absence of
HBc18-27.
[0142] The results showed that in 50% of the animals studied in
which the T helper and CTL epitope peptides were simply mixed
(i.e., not linked) and administered in an immunizing dose,
induction of some detectible antigen-specific CTL activity above
the level of background killing was seen. An example of the
response detected is shown in FIG. 7. Surprisingly, when animals
were primed with the T helper epitope linked to the CTL epitope,
100% showed evidence of specific CTL priming (FIG. 8), the
magnitude of which was greater than that detected when the epitopes
were administered non-linked (FIG. 7). Quite unexpectedly, as shown
in FIG. 9, it was found that linking the T helper and CTL epitopes
via an alanine-alanine-alanine spacer (i.e., T helper-AAA-CTL)
resulted in the induction of specific CTL activity greater than
that detected by linking the T helper and CTL determinants alone.
Priming with the T helper peptide or CTL peptide alone did not
induce HBc-specific CTL (FIGS. 5 and 6). Also, prior immunization
of animals to induce T helper-specific immunity did not appear to
be essential for priming for CTL using either the T helper and CTL
mixture or the T helper-CTL conjugate, since immunization was
detected when naive animals were primed with the appropriate
conjugate (FIGS. 10A and B).
EXAMPLE 4A
Recognition of Generation of Endogenously Processed Antigens After
Priming
[0143] This example discloses that CTL induced by in vivo priming
with peptide (as disclosed in Example 4) recognize endogenously
synthesized antigens.
[0144] A fraction of the effector cells from the procedure
disclosed in Example 4 whose CTL function is represented in FIGS.
7, 8 and 9, were restimulated in vitro using HBcAg 18-27-coated
JA2.1 /K.sup.b stimulator cells. Six days later, effector cells
were assayed for cytotoxicity and the cell lines that contained
HBcAg 18-27-specific cytotoxic activity were further restimulated.
Six days later, these cell lines were tested for cytotoxic activity
on .sup.51Cr labeled Jy S1 target cells in the absence or presence
of HBcAg 18-27 and on .sup.51Cr labeled Jy core target cells, as
demonstrated in the following table. CTL lines obtained from
animals primed with HBcAG 18-27 peptide derived recognize
endogenously synthesized HBc antigen.
11 In vivo Primed Jv S.sub.1 Animal No. Day 0 Day 9 E:T NoAg 875.15
Jy Core H21 -- 902.01 0.1:1 1 43 3 0.5:1 3 66 2 2:1 6 73 9 8:1 11
74 1.5 H5-2 -- 902.02 0.3:1 2 55 21 1:1 1 78 35 4.5:1 5 77 44 18:1
12 82 54 H6-3 -- 875.15 0.4:1 4 67 9 +875.23 1.5:1 6 78 17 6:1 11
75 23 24:1 18 79 29 H4-6 875.23 902.01 0.15:1 2 29 9 0.6:1 5 51 21
2.5:1 12 63 32 10:1 21 62 42 H4-2 875.23 902.02 0.6:1 1 74 37 2.5:1
3 80 55 10:1 6 80 59 40:1 14 77 63
[0145] All CTL lines were specific for HBcAg 18-27 and recognized
Jy core albeit to a different extent. The much higher level of
lysis obtained in the presence of exogenously added peptide as
compared to endogenously synthesized, is explained by the
relatively greater concentration of peptide that can be added
exogenously. Because CTL lines derived from both animals primed
with 902.02 had the highest affinity for endogenous antigen
(producing a 35% and 37% specific lysis of Jy core at the E:T of
1:1 and 0.6: 1, respectively), it is demonstrated that this
construct, i.e., T helper epitope AAA HBcAg 18-27 possesses
immunogenic characteristics suitable for induction of cells that
recognize HBV infected cells.
EXAMPLE 5
Induction of A2.1-Restricted Ctl-Specific for HBenv.sub.360-368
[0146] A2/K.sup.b transgenic mice were injected with 100
microliters of an emulsion of 100 .mu.g HBenv.sub.360-368 and 100
.mu.g HBc.sub.128-140 helper epitope in incomplete Freund's
adjuvant (IFA). (This emulsion was prepared by mixing 500 .mu.g of
both peptides in PBS with an equal volume of IFA.) Twenty-one days
after priming, splenocytes (5.times.10.sup.6 cells/well in a
24-well plate) obtained from these animals were restimulated with
syngeneic LPS blasts (2.times.10.sup.6/well) coated with the
peptide HBenv.sub.360-368. These effector cells
(0.2.times.10.sup.6/well) were restimulated at weekly intervals.
For the first and second restimulations, HBenv.sub.360-368 coated
LPS blasts were used, followed by HBenv.sub.360-368 coated Jurkat
A2.1/K.sup.b cells. Six days after restimulation, effector cells
were assayed for cytotoxicity against .sup.51Cr labelled Jurkat
A.sub.2/K.sup.b target cells in the presence and absence of
HBenv.sub.360-368 (see FIG. 11). Therefore, HBenv.sub.360-368 is a
CTL specific epitope.
EXAMPLE 6
Testing of Linked Tetanus Toxoid T Helper and HBc Cytotoxic T Cell
Epitopes for in vivo Priming
[0147] Transgenic mice (HLA-A2-1/kb) were primed subcutaneously
(base of the tail) with 200 mg (0.07 mM) of peptide 934.02.
12 Tetanus Toxoid 830-843 HBV core 18-27 Peptide T Helper Epitope
Linker CTL Epitope 934.02 AC-QYIKANSKFIGITE AAA FLPSDFFPSV (SEQ ID
NO:95) (SEQ ID NO:4)
[0148] Three weeks after priming, splenocytes were restimulated in
vitro with LPS blasts coated with HBc 18-27 (as described in
example I). After 7 days cells were restimulated with jurkat A2/Kb
cells coated with HBc 18-27 (as described in Example 1)-After 6
days these effector cells were assayed for cytotoxicity against
.sup.51Cr labeled jurkat A2/Kb target cells in the presence or
absence of HBc 18-27, Jy target cells in the presence or absence of
HBc 18-27 and Jy cells transfected with HBV core. The results,
shown in FIG. 12, indicate that peptide 934.02 effectively induces
CTL specific for HBc 18-27. Moreover, these CTL recognize and kill
endogenously presented antigen (Jy core).
EXAMPLE 7
Comparison of CTL Immunity Induced by Peptide Immunization
[0149] Various modifications and formulations of an antigenic CTL
peptide were tested in an effort to enhance its immunogenicity.
BALB/c mice were primed subcutaneously in the base of the tail with
one of the following peptides or peptide mixtures:
13 Peptide or Dose Number Peptides .mu.M/mouse Formulation 932.01
CTL (Flu NP 147-155) 0.1, 0.01 Saline, Alum, IFA TYQRTRALV 932.07
(PAM).sub.2KSS-CTL 0.1, 0.01 Saline, Alum, IFA
(PAM).sub.2KSSTYQRTRNLV 932.01 CTL + T helper(OVA 323-336) 0.1,
0.01 ea. Saline, Alum, IFA +577.01 TYQRTRALV-ISQAVHAAHAEINE 932.07
(PAM).sub.2KSS-CTL + T helper 0.1, 0.01 Saline, Alum, IFA +577.01
(PAM).sub.2KSSTYQRTRALV- -ISQAVHAAHAETNIE 932.02 T helper-CTL 0.1,
0.01 Saline, Alum, IFA ISQAVHAAHAEINE-TYQRTRALV 932.04
(PAM).sub.2KSS-T helper-CTL 0.1, 0.01 Saline, Alum, IFA
(PAM).sub.2KSSISQAVHAAHAEI- NE-TYQRTRALV 932.03 T helper-AAA-CTL
0.1, 0.01 Saline, Alum, IFA ISQAVHAAHAEINE-AAA-TYQRTRALV 932.05
(PAM).sub.2KSS-T helper-AAA-CTL 0.1, 0.01 Saline, Alum, IFA
(PAM).sub.2KSS-ISQAVHAAHAEINE-AAA-TYQRTRALV
[0150] Three weeks after immunization splenocytes were removed and
stimulated in vitro with the flu 147-155 peptide. CTL activity was
assayed one week later using .sup.51Cr-labeled B10.D2 fibroblasts
as targets. Target cells were tested in the absence of antigen, in
the presence of Flu 147-155 peptide or following infection with
influenza PR8 virus. Representative results obtained from one of
four independently run experiments are summarized in the following
table.
[0151] CTL Immunogenicity of Various Modifications and Formulations
of PR8-NP 148-155
14 Formulation Saline Alum IFA Peptide 0.01.sup.a 0.1 0.01 0.1 0.01
0.1 932.01 -b - - - + +++ 932.07 ++ + - - - - 932.01 + 577.01 - ++
- - +++ ++ 932.07 + 577.01 + - - - - .+-. 932.02 - - - +++ +++ +++
932.04 +++ +++ +++ +++ +++ +++ 932.03 - - - + +++ ++ 932.05 +++ +++
+++ +++ +++ +++ .sup.a = Dose (.mu.M/mouse) b = CTL immunogenicity
of various modifications and formulations of NP 148-155
(nucleoprotein of PR8 influenza virus). Each symbol represents the
result obtained from spleen cells derived from a single Balb/c
mouse and reflects the effector to target ratio (E:T) required to
induce 40% antigen specific lysis of .sup.51Cr labeled B10D2 target
cells in the presence of ND.sub.148-155 peptide; +++ E:T below
10:1; ++ E + T between 10 + 1 # and 30:1; + E:T greater than 30:1;
- not achieved at any E:T tested.
[0152] A discussion of the results follows:
[0153] IFA formulation: The HTL-CTL linked peptides either
lipidated (i.e., 932.04 and 932.05) or unlipidated (932.02 and
932.03) were all very active, inducing good CTL activity in all
animals at both injection doses. The mixture of HTL (577.01) and
CTL (932.01) peptides also induced CTL activity in all animals
injected. The CTL peptide (932.01) demonstrated good activity at
the 100 nmoles dose, however, only one out of four animals
responded at the 10 nmoles dose. The lipidated CTL peptide (932.07)
was completely inactive but some activity, albeit low, was observed
when peptide 932.07 was mixed with the HTL peptide (577.01).
[0154] Saline formulation: The PAM.sub.2-HTL-CTL linked peptides
(932.04 and 932.05) induced good CTL activity in all animals
injected and were far superior to all other peptide or peptide
combinations tested. The unlipidated versions of those peptides
(932.02 and 932.03) were totally ineffective at the 10 nmoles dose
and only marginally active at the 100 nmoles dose underlying the
importance of peptide lipidation for priming activity of saline
formulations. For the other peptide combinations tested lipidation
appeared to affect activity in the opposite direction as it did in
IFA in that the CTL peptide (932.01) was completely inactive but
its lipidated version (932.07) induced CTL activity in some of the
animals injected. The activity of both peptides improved when mixed
with the HTL peptide.
[0155] Alum formulation: The results obtained were similar to the
ones obtained with peptides formulated in saline with the exception
of peptide 932.07 that was inactive when injected in alum.
[0156] Accordingly, the constructs (PAM.sub.2KSS-T helper-CTL and
(PAM).sub.2KSS-T helper-AAA-CTL were superior when injected in
saline or Alum compared to all of the other combinations. The
peptides T helper-CTL and T helper-AAA-CTL were superior to mixing
the T helper+CTL (i.e., non-linked) and worked well in IFA, but not
well in saline or Alum. Thus, for vaccine development, linking the
(PAM).sub.2KSS to a T helper peptide which is linked to the CTL
peptide appears to be advantageous for inducing CTL immunity.
EXAMPLE 8
Definition of the Minimal Optimal Sequence with the CTL Peptide
Epitope 799.09
[0157] Transgenic mice (A2-1/Kb) were primed with HBV virus in IFA
substantially as described in Example 2. Seven days after priming,
splenocytes obtained from these animals were restimulated with
syngeneic irradiated LPS blasts coated with peptide 799.09 (as
described in Example 1). After 6 cycles of restimulation with
799.09 coated cells (as described in Example 2) the effector cells
were cloned by limiting dilution using syngeneic irradiated LPS
blasts coated with peptide 799.09 (0.2.times.10.sup.6 cells/well of
a 96 well plate) and media supplemented with 10% rat Con A
supernatant. Two CTL lines were obtained, line 110 and 113 that
killed JA2Kb target cells coated with peptide 799.09. These lines
were tested on a panel of 799.09 N-terminus truncated peptides and
overlapping 9mers and 10mers covering the entire 799.09 sequence.
As shown in FIG. 13 panel A the minimal N-terminus truncated
peptide recognized by line 113 and 110 were respectively peptides
HBV env. 333-348 (923-09) and 335-348 (923.07). FIG. 13 panel B
shows that none of the 9mers or 10mers were recognized by line 113
implicating a longer peptide as the minimal sequence required for
recognition by this CTL line. The minimal sequence recognized by
line 110 is represented by peptides HBV env. 333-341 (923.26) and
HBV env 335-343 (923.22) indicating that possibly two distinct but
overlapping peptides can serve as antigenic determinants for 799.09
specific CTL.
EXAMPLE 9
Priming of Peptide 934.05 ("Cy-1899") in Transgenic Mice
[0158] This example discloses that peptide 934.02 (i.e., tetanus
toxoid 830-843-AAA-HBcAg 18-27) when modified by linking
(PAM).sub.2KSS to the tetanus toxoid end (resulting in "CY-1899")
and formulated in DMSO/saline induced CTLs in transgenic mice.
[0159] Lipopeptides were prepared by coupling the appropriate fatty
acid to the amino terminus of the resin bound peptide. A typical
procedure was as follows: A dichloromethane solution of a 4 fold
excess of a pre-formed symmetrical anhydride of the appropriate
fatty acid was added to the resin an the mixture was allowed to
react for 2 hrs. The resin was washed with dichloromethane and
dried. The resin was then treated with trifluoroacetic acid in the
presence of appropriate scavengers [e.g. 5% (v/v) water] for 60
minutes at 20.degree. C. After evaporation of excess
trifluoroacetic acid, the crude peptide was washed with diethyl
ether, dissolved in methanol and precipitated by the addition of
water. The peptide was collected by filtration and dried.
[0160] Preparation of peptides for immunization: Peptides were
routinely resuspended in DMSO at a concentration of 20 mg/ml.
Before use, peptides were prepared at the required concentration by
dilution in saline or the appropriate medium. For selected
experiments, CY-1 899 and 934.02 were also prepared as saline
suspensions (in the complete absence of DMSO).
[0161] Immunization procedures: Transgenic mice were primed
subcutaneously (base of the tail) with 0.1 ml of the appropriate
peptide formulated in saline, or DMSO/saline.
[0162] Media:
[0163] RPMI-1640 supplemented with 10% fetal calf serum (FCS) 2 mM
Glutamine, 50 .mu.g/ml Gentamicin and 5.times.10.sup.-5
2-mercaptoethanol served as culture medium and will be referred to
as R10 medium.
[0164] b. RPMI-1640 containing 25 mM HEPES buffer and supplemented
with 2% (FCS) was used as cell washing medium.
[0165] Cell lines:
[0166] Jurkat A2.1/K.sup.b (5) is a stable transfectant of the
human T cell leukemia line, Jurkat. Jurkat A2.1/K.sup.b cells were
routinely grown in R10 medium supplemented with 400 .mu.g/ml of
G418.
[0167] LPS-activated lymphoblasts: Splenocytes obtained from
A2.1/K.sup.b transgenic mice were resuspended at a concentration of
1-1.5.times.10.sup.6/ml in R10 medium supplemented with 25 .mu.g/ml
LPS and 7 .mu.g/ml dextran sulfate in 75 cm.sup.2 tissue culture
flasks. After 72 hr at 37.degree. C., the lymphoblasts were
collected for use by centrifugation.
[0168] Peptide coating of lymphoblasts: Peptide coating of the LPS
activated lymphoblasts was achieved by incubating 30.times.10.sup.6
irradiated (3000 rads) lymphoblasts with 100 .mu.g of peptide in 1
ml of R10 medium for 1 hr at 37.degree. C. Cells were then washed
once and resuspended in R10 medium at the desired
concentration.
[0169] In vitro CTL activation: One to four weeks after priming
spleen cells (30.times.10.sup.6 cells/flask) were 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 R10
medium/T25 flask. After 6 days, the effector cells were harvested
and assayed for cytotoxic activity.
[0170] Assay for cytotoxic activity: Target cells
(1.0-1.5.times.10.sup.6) were incubated at 37.degree. C. in the
presence of 200 .mu.l of sodium .sup.51Cr chromate. After 60
minutes, cells were washed three times and resuspended in R10
medium. Peptide 875.15 was added where required at a concentration
of 1 .mu.tg/ml. For the assay, 10.sup.4 51Cr-labeled target cells
were 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
was removed from each well and radioactivity was determined in a
Micromedic automatic gamma counter. The percent specific lysis was
determined by the formula: percent specific
release=100.times.(experimental release-spontaneous
release)/(maximum release-spontaneous release). For the purpose of
easy comparison between separate CTL assays run under the same
conditions, % .sup.51Cr release data was 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
Jurkat A.sub.2.1/K.sup.b 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 E:T 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.times.10.sup.6.div.5.times-
.10.sup.4)-(1.times.10.sup.6.div.5.times.10.sup.5)=18LU/10.sup.6.
[0171] Results
[0172] The results indicated that CY 1899 (i.e., tetanus toxoid
830-843-AAA-HBcAg 18-27) formulated in DMSO/saline is a strong
immunogenic preparation inducing a good CTL response in all animals
injected (six out of six).
[0173] CY 1899 in saline (without prior solubilization induced a
poor CTL response in that only one of six animals injected had
detectible CTL activity.
[0174] Peptide 934.02 in saline induced a marginal CTL response in
that only one of the four animals tested had detectable CTL
activity.
[0175] No CTL activity was detected in any of the animals injected
with peptide 934.02 in DMSO/saline.
[0176] CTL induced by CY-1899 were shown to be able to kill cells
expressing endogenous HBc antigen.
EXAMPLE 10
Priming of Peptide 934.05 ("Cy-1899") in Transgenic Mice Epitope on
CTL Induction
[0177] This example discloses that the desired CTL activity is
induced in the absence of adjuvant (IFA or Alum) by lipidation of
the T-Helper epitope where the T-Helper epitope and the CTL epitope
are not linked.
[0178] The effect of lipidation of the HTL peptide epitope on the
immunogenicity of PR8-NP 147-155 CTL peptide epitope was
determined. Peptides used in this Example were as follows:
15 ID Description Sequence 932-01 CTL TYQRTRALV (PR8-NIP 147-155)
932.07 (PAM).sub.2-CTL (PAM)2 577.01 HTL ISQAVHAAHAE1NE 932.06
(PAM)2-HTL (PAM).sub.2KSSISQAVHAAHAEINE 932.04 or (PAM)2-HTL-CTL
(PAM).sub.2KSSISQAVHAAHAEINETYQRTRALV
[0179] Balb/c mice were injected with the different combinations
indicated above of HTL (100 nmoles/mouse) and CTL (10 nmoles/mouse)
peptide epitope in saline. Four animals were injected with each
different preparation. Ten days after immunization, splenocytes
were stimulated in vitro with NP 147-155 peptide. CTL activity was
assayed 6 days later using .sup.51Cr-labeled B10.D2 fibroblasts as
targets in the absence or presence of NP 147-155 peptide. The data
are expressed in lytic units/10.sup.6 cells. One lytic unit is
arbitrarily defined as the number of lymphocytes required to
achieve 30% lysis of 5000 B10.D2 .sup.51Cr-labeled target cells
within 6 hours, in the absence or presence of NP 147-155 peptide.
(a) Each number represents the specific CTL activity (LU
30/10.sup.6 cells obtained in the presence of NP 147-155-LU
30/10.sup.6 cells obtained in the absence of NP 147-0155) from an
individual mouse.
Effect of HTL Peptide Epitope and Peptide Liquid on the
Immunogenicity of PR8-NP 147-155 CTL Peptide Epitope
[0180]
16 Priming Peptide LU30 Cytel ID Number Description (Geometric
Mean) 932.01 CTL 0 (a) 0.2 0 0 (0.1x/+1.4) 932.01 + CTL + HTL 0 0.3
577.01 0 0 (0.1x/+1.7) 932.07 (PAM).sub.2-CTL 0 0 0.5 0
(0.15x/+2.2) 932.07 + (PAM).sub.2-CTL + HTL 1 1.1 577.01 12.4 0
(1.1x/+7.2) 932.01 + CTL + (PAM).sub.2-HTL 9.3 74.1 932.06 1.7 69.5
(16.9x/+6.1) 932.07 +(PAM).sub.2-CTL + (PAM).sub.2-HTL 2.5 932.06 1
1 11.7 (2.3x/+6.1) 932.04 (PAM).sub.2-HTL-CTL 4.7 2 21 1.2
(3.9x/+3.5)
[0181] The results obtained indicate that, 1) unlipidated peptides
(932.01 and 932.01+577.01) are ineffective at the concentrations
tested when injected in saline; 2) the presence of both the HTL and
the CTL peptide epitope and the lipidation of at least one peptide
is necessary for CTL induction when the peptides are formulated in
saline.
[0182] The preferred combination appeared to be the lipidated HTL
peptides unlinked to the unlipidated CTL peptide. Thus, a titration
of the CTL epitope and the lipidated HTL epitope in mixtures was
performed (i.e., the titration of (PAM).sub.2-HTL peptide epitope
and CTL peptide epitope in mixtures).
[0183] Balb/c mice were injected with 10 nmoles of the linked
[(PAM).sub.2-HTL-CTL] peptide immunogen and with mixtures
containing 0, 1, 10, or 100 umoles CTL epitope (932.01) and 0, 1,
10, or 100 nmoles PAM-helper epitope (932.06) per mouse as
indicated in the table. Four animals were injected with each
different preparation. Fourteen days after immunization,
splenocytes were removed and stimulated in vitro with the NP
147-155 peptide. CTL activity was assayed 6 days later using
.sup.51Cr-labeled B 10.D2 fibroblasts as targets. Target cells were
tested in the absence or presence of antigen (NP 147-155 peptide).
The data are expressed in lytic units/10.sup.6 cells. One lytic
unit is arbitrarily defined as the number of lymphocytes required
to achieve 30% lysis of 5000 B10.D2 .sup.51Cr-labeled target cells
within 6 hours, in the absence or presence of antigen (NP 147-155
peptide). (a) Each number represents the specific CTL activity (LU
30/10.sup.6 cells; obtained in the presence of NP 147-155-LU
30/10.sup.6 cells obtained in the absence of NP 147 155) from an
individual mouse.
Titration of (PAM).sub.2-HTL Peptide Epitope and CTL Peptide
Epitope Contained in Mixtures
[0184]
17 nmoles nmoles CTL epitope, 932.01 (PAM).sup.2-HTL 0 1 10 100
epitope LU30 LU30 LU30 LU30 932.06 Geo Mean Geo Mean Geo Mean Geo
Mean 0 3 .8 (a) 0. .8 0 .3 1 .3 1.0x/+2.9 1 3.8 2.5 3 23.9 4.4 7.4
2.6 0.2 2.6 0.2 4.4 2.6 2.6x/+7.2 1.8x/+4.4 3.5x/+1.7 10 >58 50
68 29.5 20.3 53 2.2 7.3 >64 53.5 27.5 >68 21.2x/+4.7
21.2x/+2.2 62.9x/+1.1 1001.5 26 69 64 0.8 61 >58 >64 0.7 63
>41 73 0 >54 7.9 62 0.5x/+3.2 48.2x/+1.5 33.7x/+2.7
65.6x/+1.1 (PAM).sub.2-HTL- 90 CTL 65 17.8 10 nmoles 25.4 1053.05
40.3x/+2.1
[0185] The results obtained indicate that optimal CTL induction
(i.e., comparable to that achieved by immunization with 10 nmoles
of the linked immunogen 1053.05) is achieved with mixtures
containing 100 nmoles of the 932.06 [(PAM).sub.2 HTL] peptide and
1, 10 or 100 nmoles of the 932.01 (CTL) peptide. In the presence of
10 nmoles of 932.06, optimal CTL induction is achieved by the
mixture containing 100 nmoles of 932.01 while a slight decrease was
observed in the mixtures containing either 10 or 1 nmoles of the
CTL peptide. Mixtures containing 1 nmole of the HTL peptide
resulted rather inefficiently.
[0186] Lipidated T-Helper Epitopes With Multiple CTL Epitopes
[0187] By following the procedures described in this Example 10, a
composition of matter comprising of a lipidated HTL admixed with
multiple CTL epitopes can be prepared. Optionally, the CTL epitopes
can be packaged in different vials from the HTL epitope(s).
EXAMPLE 11
Induction of Specific CTL Response in Humans
[0188] The human clinical trial for CY-1899 was set up as an IDN
Phase I dose escalation study (5, 50 and 500 .mu.g) and was carried
out in a single center as a randomized double-blind placebo
controlled trial.
[0189] A total of 27 subjects were enrolled and divided into 3
groups:
[0190] Group I: 3 subjects were injected with placebo and 6
subjects were injected with 5 .mu.g of CY-1899;
[0191] Group II: 3 subjects were injected with placebo and 6
subjects were injected with 50 .mu.g of CY-1899;
[0192] Group III: 3 subjects were injected with placebo and 6
subjects were injected with 500 .mu.g of CY-1899.
[0193] After 5 weeks (GI and GII) or 4 weeks (GIII) following the
first injection, all subjects received a booster inoculation at the
same dosage.
[0194] The endpoints measured in this study relate to the safety
and tolerability of CY-1899 (also known as Theradigm-HBV) as well
as its immunogenicity. Cellular immune responses to CY-1899 are an
index of the intrinsic activity of this drug, and can therefore be
viewed as a measure of biological efficacy. The following
summarized the clinical and laboratory data relating to safety and
efficacy endpoints.
[0195] Safety: Adverse events were observed with similar frequency
in the placebo and drug treatment groups. All adverse events were
judged to be mild in degree and completely reversible. No
relationship between adverse events and dose level were found. The
most common adverse events were headache (4 subjects) and skin
reaction at the site of inoculation (3 subjects).
[0196] Evaluation of Vaccine Efficacy: For evaluation of vaccine
efficacy subjects were bled before and after injection. Peripheral
blood mononuclear cells were isolated from fresh heparinized blood
by Ficoll-Hypaque density gradient centrifugation, aliquoted in
freezing media and stored frozen. Samples were assayed for CTL and
HTL activity.
[0197] Data:Injection of CY-1899 in normal volunteers resulted in
induction of HBc18-27 specific CTL as disclosed in FIGS. 14 and 15.
The CTL response observed was dose dependent both in the proportion
of subjections exhibiting a positive response as well as in the
magnitude of the response obtained. The dose response effect is
demonstrated in FIG. 16 where the mean peak CTL activity per each
group of subjects after the first and second injections are
shown.
[0198] The peptide-induced CTL responses were capable of killing
cells expressing endogenous antigen (as disclosed in FIG. 17)
validating the concept of peptide priming for induction of virus
specific CTL.
[0199] The proliferative T cell response against the helper TT
peptide among the subjects receiving the three doses of CY-1899
correlates with their CTL responses (as disclosed in. FIG. 18). The
most striking T cell responses against the TT helper peptide were
observed in individuals receiving the highest vaccine dose. In
three out of four subjects in this dose group a strong T cell
proliferative response against TT peptide was correlated with high
levels of CTL induction following vaccination with CY-1899. The
only instance where a strong CTL response was observed in the
absence of helper T cell induction was in the case of an individual
who demonstrated a late CTL response 14 days after boosting.
EXAMPLE 12
Specific CTL Inducing Vaccine for Preventing and/or Treating
HCV
[0200] By following the procedures disclosed in Examples 1-11 a
therapeutic composition of matter useful for treating mammals with
HCV infections by inducing specific CTLs can be made.
[0201] For example, following the procedures in Examples 1, and 2,
or alternatively Example 2A, peptides suitable for inducing CTLs
can be identified, for example, the following peptides:
18 SEQ ID SOURCE POSITION SEQUENCE SIZE NO: BINDING A2 HCV NS4 1807
LLFNILGGWV 10 125 3.5000 HCV CORE 178 LLFNILGGWV 10 126 0.6050 HCV
N54 1585 YLVAYQATV 9 127 0.2450 HCV NS1/ENV 725 FLLLADARV 9 128
0.2250 HCV NS4 1851 ILAGYGAGV 9 129 0.2150 HCV CORE 132 DLMGYIPLV 9
130 0.0835 HCV CORE 35 YLLPRRGPRL 10 131 0.0725 NS1/ENV2 686
ALSTGLIHL 9 132 0.0415 HCV CORE 178 LLALLSCLTI 10 133 0.0340 HCV
N55 2578 RLIVFPDLGV 10 134 0.0320 HCV N55 2885 RLHGLSAFSL 10 135
0.0200 HCV NS4 1811 ILGGWVAAQL 10 136 0.0180 HCV ENV1 364 SMVGNWAKV
9 137 0.0155 HCV NS3 1131 YLVTRHADV 9 138 0.0109 HCV NS4 1666
VLAALAAYCL 10 139 0.0106
[0202] Once identified, such desired peptides can be obtained
either commercially or prepared following the procedures disclosed
in Example 3.
[0203] The preferability of the identified peptides is elaborated
by optimization of the configuration of the composition of matter
(i.e., whether the T-Helper and CTL peptides are linked or
unlinked, and whether the peptides are delivered in a saline, alum
or IFA medium, lipidated T-Helper linked or unlinked to the CTL in
saline, or linked and unlinked T-Helper with CTL in IFA).
[0204] By following the procedures disclosed above and in the
preceding examples a vaccine capable of treating HCV can be
obtained.
EXAMPLE 13
Specific CTL Inducing Vaccine for Preventing and/or Treating
Melanoma
[0205] By following the procedures disclosed in Examples 1-10 a
therapeutic composition of matter useful for treating mammals with
melanomas by inducing specific CTLs can be made.
[0206] For example, following the procedures in Examples 1, and 2,
or alternatively Example 2A, peptides suitable for inducing CTLs
can be identified, for example, the following peptides:
19 SEQ ID BINDING SOURCE POSITION SEQUENCE SIZE NO: A2 MAGE2 105
KMVELVHFL 9 140 0.5100 MAGE2 105 KMVELVHFLL 10 141 0.2200 MAGE3 153
LVFGIELMEV 10 142 0.1100 MAGE1 278 KVLEYVIKV 9 143 0.0900 MAGE1 105
KVADLVGFLL 10 144 0.0560 MAGE3 105 KVAEFVHFL 9 145 0.0550 MAGE1 92
CILESLFRA 9 146 0.0460 MAGE1 264 FLWGPRALA 9 147 0.0420 MAGE1 200
VMIAMEGGHA 10 148 0.0360 MAGE1 38 LVLGTLEEV 9 149 0.0320 MAGE1 301
ALREEEEGV 9 150 0.0210 MAGE1 270 ALAETSYVKV 10 151 0.0150 MAGE1 282
YVIKVSARV 9 152 0.0140 MAGE1 269 RALAETSYV 9 153 0.0100
[0207] Once identified, such desired peptides can be obtained
either commercially or prepared following the procedures disclosed
in Example 3.
[0208] The preferability of the identified peptides is elaborated
by optimization of the configuration of the composition of matter
(i.e., whether the T-Helper and CTL peptides are linked or
unlinked, and whether the peptides are delivered in a saline, alum
or IFA medium, lipidated T-Helper linked or unlinked to the CTL in
saline, or linked and unlinked T-Helper with CTL in IFA).
[0209] By following the procedures disclosed above and in the
preceding examples a vaccine capable of treating melanomas can be
obtained.
EXAMPLE 14
Specific CTL Inducing Vaccine for Preventing and/or Treating
HPV
[0210] By following the procedures disclosed in Examples 1-10 a
therapeutic composition of matter useful for treating mammals with
HPV infections by inducing specific CTLs can be made.
[0211] For example, following the procedures in Examples 1, and 2,
or alternatively Example 2A, peptides suitable for inducing CTLs
can be identified, for example, the following peptides:
20 SEQ ID BINDING SOURCE POSITION SEQUENCE SIZE NO: A2 HPV16 E7 82
LLMGTLGIV 9 65 0.0240 HPV16 E7 11 YMLDLQPET 9 66 0.1400 HPV16 E6 52
FAFRDLCIV 9 67 0.0570 HPV16 E7 86 TLGIVCPIC 9 68 0.0750 HPV16 E7 7
TLHEYMLDL 9 69 0.0070 HPV16 E7 85 GTLGIVCPI 9 70 0.0820 HPV16 E7 12
MLDLQPETT 9 71 0.0028 HPV16 E6 29 TIHDIILECV 10 72 0.0210
[0212] Once identified, such desired peptides can be obtained
either commercially or prepared following the procedures disclosed
in Example 3.
[0213] The preferability of the identified peptides is elaborated
by optimization of the configuration of the composition of matter
(i.e., whether the T-Helper and CTL peptides are linked or
unlinked, and whether the peptides are delivered in a saline, alum
or IFA medium, lipidated T-Helper linked or unlinked to the CTL in
saline, or linked and unlinked T-Helper with CTL in IFA).
[0214] By following the procedures disclosed above and in the
preceding examples a vaccine capable of treating HPV can be
obtained.
EXAMPLE 15
Specific CTL Inducing Vaccine for Preventing and/or Treating
HIV
[0215] By following the procedures disclosed in Examples 1-10 a
therapeutic composition of matter useful for treating humans with
HIV infections by inducing specific CTLs can be made.
[0216] For example, following the procedures in Examples 1, and 2,
or alternatively Example 2A, peptides suitable for inducing CTLs
can be identified, for example, the following peptides:
21 SEQ ID BINDING SOURCE POSITION SEQUENCE SIZE NO: A2 HIV 367
VLAEAMSQV 9 73 0.1100 HIV 1496 LLWKGEGAVV 10 74 0.0360 HIV 1496
LLWKGEGAV 9 75 0.0230 HIV 1004 ILKLEPVHGV 9 76 0.0190 HIV 1129
IVGAETFYV 9 77 0.0099 HIV 1129 IIGAETFYV 9 78 0.0260 HIV 2182
LWVTVYYGV 9 79 0.0014 HIV 2182 LMVTVYYGV 9 80 0.4400
[0217] Once identified, such desired peptides can be obtained
either commercially or prepared following the procedures disclosed
in Example 3.
[0218] The preferability of the identified peptides is elaborated
by optimization of the configuration of the composition of matter
(i.e., whether the T-Helper and CTL peptides are linked or
unlinked, and whether the peptides are delivered in a saline, alum
or IFA medium, lipidated T-Helper linked or unlinked to the CTL in
saline, or linked and unlinked T-Helper with CTL in IFA).
[0219] By following the procedures disclosed above and in the
preceding examples a vaccine capable of treating HIV can be
obtained.
[0220] From the foregoing it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
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