U.S. patent application number 10/815340 was filed with the patent office on 2005-10-20 for mucosal cytotoxic t lymphocyte responses.
This patent application is currently assigned to Health and Human Services, The Government of the United States of America, as Represented by, Health and Human Services, The Government of the United States of America, as Represented by. Invention is credited to Belyakov, Igor M., Berzofsky, Jay A., Derby, Michael A., Kelsall, Brian L., Strober, Warren.
Application Number | 20050232897 10/815340 |
Document ID | / |
Family ID | 32397834 |
Filed Date | 2005-10-20 |
United States Patent
Application |
20050232897 |
Kind Code |
A1 |
Berzofsky, Jay A. ; et
al. |
October 20, 2005 |
Mucosal cytotoxic T lymphocyte responses
Abstract
The invention provides methods for induction of an
antigen-specific, mucosal cytotoxic T lymphocyte response useful in
preventing and treating infections with pathogens that gain entry
via a mucosal surface.
Inventors: |
Berzofsky, Jay A.;
(Bethesda, MD) ; Belyakov, Igor M.; (Gaithersburg,
MD) ; Derby, Michael A.; (Germantown, MD) ;
Kelsall, Brian L.; (Washington, DC) ; Strober,
Warren; (Bethesda, MD) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
8TH FLOOR
SAN FRANCISCO
CA
94111
US
|
Assignee: |
Health and Human Services, The
Government of the United States of America, as Represented
by
Rockville
MD
|
Family ID: |
32397834 |
Appl. No.: |
10/815340 |
Filed: |
March 30, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10815340 |
Mar 30, 2004 |
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09508552 |
Jun 12, 2000 |
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6749856 |
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09508552 |
Jun 12, 2000 |
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PCT/US98/19028 |
Sep 11, 1998 |
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60058523 |
Sep 11, 1997 |
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60074894 |
Feb 17, 1998 |
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Current U.S.
Class: |
424/85.1 ;
424/236.1; 424/241.1; 424/85.2; 424/85.6 |
Current CPC
Class: |
A61K 2039/55566
20130101; Y10S 530/826 20130101; A61K 39/21 20130101; A61K
2039/6037 20130101; A61K 2039/545 20130101; A61K 2039/55522
20130101; A61K 2039/55538 20130101; A61K 2039/5256 20130101; A61K
39/12 20130101; C07K 14/005 20130101; C12N 2740/16134 20130101;
C12N 2710/24143 20130101; A61K 2039/541 20130101; A61K 2039/55544
20130101; C12N 2740/16122 20130101 |
Class at
Publication: |
424/085.1 ;
424/085.2; 424/085.6; 424/241.1; 424/236.1 |
International
Class: |
A61K 039/108; A61K
039/02; A61K 038/21 |
Claims
1. A method for inducing a protective mucosal cytotoxic T
lymphocyte (CTL) response in a mammalian subject comprising
contacting a mucosal tissue of the subject with a composition
comprising a purified soluble antigen.
2. The method of claim 1, wherein the soluble antigen is an
antigenic peptide.
3. The method of claim 1, wherein said composition further
comprises an adjuvant.
4. The method of claim 3, wherein the adjuvant is selected from
cholera toxin (CT), mutant cholera toxin (MCT), or mutant-E. coli
heat labile enterotoxin (MLT).
5. The method of claim 1, further comprising administering a
purified cytokine to the subject.
6. The method of claim 1, wherein the cytokine is contacted with a
mucosal surface of the subject.
7. The method of claim 5, wherein the purified cytokine is selected
from granulocyte-macrophage colony-stimulating factor (GM-CSF),
interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12)
or tumor necrosis factor a (TNFa).
8. The method of claim 1, further comprising administering purified
interferon-.gamma. to the subject.
9. The method of claim 8, wherein the purified interferon-.gamma.
is contacted with a mucosal surface of the subject.
10. The method of claim 5, further comprising administering
purified interferon-.gamma. to the subject.
11. The method of claim 10, wherein the purified interferon-.gamma.
is contacted with a mucosal surface of the subject.
12. The method of claim 1, wherein said composition further
comprises a purified cytokine selected from granulocyte-macrophage
colony-stimulating factor (GM-CSF), interleukin-2 (IL-2),
interleukin-7 (IL-7), interleukin-12 (IL-12) or tumor necrosis
factor.
13. The method of claim 1, wherein said composition further
comprises purified interferon-.gamma..
14. The method of claim 12, wherein said composition further
comprises purified interferon-.gamma..
15. The method of claim 1, wherein the antigen is a peptide derived
from a pathogenic virus.
16. The method of claim 15, wherein the pathogenic virus is
HIV-1.
17. The method of claim 15, wherein the pathogenic virus is
influenza virus.
18. The method of claim 15, wherein the pathogenic virus is
rotavirus.
19. The method of claim 1, wherein the antigen is a peptide derived
from a pathogenic bacterium or protozoan.
20. The method of claim 1, wherein the antigen is a
tumor-associated peptide.
21. The method of claim 1, wherein the antigen is a peptide
comprising an HIV-1 cluster peptide vaccine construct (CLUVAC)
selected from the group consisting of:
3 (SEQ ID NO:1) EQMHEDIISLWDQSLKPCVKRIQRGPGRAFVTIGK, (SEQ ID NO:2)
KQIINMWQEVGKAMYAPPISGQIRRIQRGPGRAFVTIGK, (SEQ ID NO:3)
RDNWRSELYKYKVVKIEPLGVAPTRIQRGPGRAFV- TIGK, (SEQ ID NO:4)
AVAEGTDRVIEVVQGAYRAIRHIPRRIRQ- GLERRIQRGPGRAFVTIGK, (SEQ ID NO:5)
DRVIEVVQGAYRAIRHIPRRIRQGLERRIQRGPGRAFVTIGK, (SEQ ID NO:6)
DRVIEVVQGAYRAIRRIQRGPGRAFVTIGK, (SEQ ID NO:7)
AQGAYRAIRHIPRRIRRIQRGPGRAFVTIGK, (SEQ ID NO:8)
EQMHEDIISLWDQSLKPCVKRIHIGPGRAFYTTKN, (SEQ ID NO:9)
KQIINMWQEVGKAMYAPPISGQIRRIHIGPGRAFYTTKN, (SEQ ID NO:10)
RDNWRSELYKYKVVKIEPLGVAPTRIHIGPGRAFYTTKN, SEQ ID NO:11)
AVAEGTDRVIEVVQGAYRAIRHIPRRIRQGLERRIHIGPGRAF- YTTKN, (SEQ ID NO:12)
DRVIEVVQGAYRAIRHIPRRIRQGLER- RIHIGPGRAFYTTKN, (SEQ ID NO:13)
DRVIEVVQGAYRAIRRIHIGPGRAFYTTKN and (SEQ ID NO:14)
AQGAYRAIRHIPRRIRRIHIGPGRAFYTTKN.
22. (canceled)
23. The method of claim 21, wherein the HIV-1 CLUVAC is HIV-1
CLUVAC PCLUS3-18MN (SEQ ID NO:9).
24. The method of claim 21, wherein the HIV-1 CLUVAC is HIV-1
CLUVAC PCLUS 6.1-18MN (SEQ ID NO:12).
25. A method for inducing a protective mucosal CTL response in a
subject, comprising contacting a mucosal tissue of the subject with
a composition comprising a soluble antigen, wherein said
composition does not comprise an adjuvant.
26. The method of claim 25, further comprising administering a
purified cytokine to the subject.
27. The method of claim 25, wherein the cytokine is contacted with
a mucosal surface of the subject.
28. The method of claim 27, wherein the purified cytokine is
selected from granulocyte-macrophage colony-stimulating factor
(GM-CSF), interleukin-2 (IL-2), interleukin-7 (IL-7),
interleukin-12 (IL-12) or tumor necrosis factor a (TNFa).
29. The method of claim 25, further comprising administering
purified interferon-.gamma. to the subject.
30. The method of claim 29, wherein the purified interferon-.gamma.
is contacted with a mucosal surface of the subject.
31. The method of claim 26, further comprising administering
purified interferon-.gamma. to the subject.
32. The method of claim 31, wherein the purified interferon-.gamma.
is contacted with a mucosal surface of the subject.
33. The method of claim 25, wherein said composition further
comprises a purified cytokine selected from granulocyte-macrophage
colony-stimulating factor (GM-CSF), interleukin-2 (IL-2),
interleukin-7 (IL-7), interleukin-12 (IL-12) or tumor necrosis
factor.
34. The method of claim 25, wherein said composition further
comprises purified interferon-.gamma..
35. The method of claim 33, wherein said composition further
comprises purified interferon-.gamma..
36. The method of claim 25, wherein the antigen is a peptide
derived from a pathogenic virus.
37. The method of claim 36, wherein the pathogenic virus is
HIV-1.
38. The method of claim 36, wherein the pathogenic virus is
influenza virus.
39. The method of claim 36, wherein the pathogenic virus is
rotavirus.
40. The method of claim 25, wherein the antigen is a peptide
derived from a pathogenic bacterium or protozoan.
41. The method of claim 25, wherein the antigen is a
tumor-associated peptide.
42. The method of claim 25, wherein the antigen is a peptide
comprising an HIV-1 cluster peptide vaccine construct (CLUVAC)
selected from the group consisting of:
4 (SEQ ID NO:1) EQMHEDIISLWDQSLKPCVKRIQRGPGRAFVTIGK, (SEQ ID NO:2)
KQIINMWQEVGKAMYAPPISGQIRRIQRGPGRAFVTIGK, (SEQ ID NO:3)
RDNWRSELYKYKVVKIEPLGVAPTRIQRGPGRAFV- TIGK, (SEQ ID NO:4)
AVAEGTDRVIEVVQGAYRAIRHIPRRIRQ- GLERRIQRGPGRAFVTIGK, (SEQ ID NO:5)
DRVIEVVQGAYRAIRHIPRRIRQGLERRIQRGPGRAFVTIGK, (SEQ ID NO:6)
DRVIEVVQGAYRAIRRIQRGPGRAFVTIGK, (SEQ ID NO:7)
AQGAYRAIRHIPRRIRRIQRGPGRAFVTIGK, (SEQ ID NO:8)
EQMHEDIISLWDQSLKPCVKRIHIGPGRAFYTTKN, (SEQ ID NO:9)
KQIINMWQEVGKAMYAPPISGQIRRIHIGPGRAFYTTKN, (SEQ ID NO:10)
RDNWRSELYKYKVVKIEPLGVAPTRIHIGPGRAFYTTKN, (SEQ ID NO:11)
AVAEGTDRVIEVVQGAYRAIRHIPRRIRQGLERRIHIGPGRAFYTTKN, (SEQ ID NO:12)
DRVIEVVQGAYRAIRHIPRRIRQGLERRIHIGPG- RAFYTTKN, (SEQ ID NO:13)
DRVIEVVQGAYRAIRRIHIGPGRA- FYTTKN and (SEQ ID NO:14)
AQGAYRAIRHIPRRIRRIHIGPGRAFYTTKN.
43. (canceled)
44. The method of claim 42, wherein the HIV-1 CLUVAC is HIV-1
CLUVAC PCLUS3-18MN (SEQ ID NO:9).
45. The method of claim 42, wherein the HIV-1 CLUVAC is HIV-1
CLUVAC PCLUS 6.1-18MN (SEQ ID NO:12).
46. An immunogenic composition for inducing a protective mucosal
CTL response in a subject and adapted for intrarectal
administration comprising a purified soluble antigen formulated for
intrarectal delivery to the rectum, colon, sigmoid colon, or distal
colon.
47. The immunogenic composition of claim 46, which comprises a
rectal enema, foam, suppository, or topical gel.
48. The immunogenic composition of claim 46, further comprising a
base, carrier, or absorption-promoting agent adapted for
intrarectal delivery.
49. The immunogenic composition of claim 48, which includes a
rectal emulsion or gel preparation.
50. The immunogenic composition of claim 48, wherein the soluble
antigen is admixed with a homogenous gel carrier.
51. The immunogenic composition of claim 48, wherein the homogenous
gel carrier is a polyoxyethylene gel.
52. The immunogenic composition of claim 48, wherein the soluble
antigen is admixed with a rectally-compatible foam.
53. The immunogenic composition of claim 48, wherein the soluble
antigen is formulated in a suppository.
54. The immunogenic composition of claim 53, wherein the
suppository is comprised of a base selected from a
polyethyleneglycol, witepsol H15, witepsol W35, witepsol E85,
propyleneglycol dicaprylate (Sefsol 228), Miglyol810,
hydroxypropylcellulose-H (HPC), or carbopol-934P (CP).
55. The immunogenic composition of claim 53, comprising at least
two base materials.
56. The immunogenic composition of claim 46, including a
stabilizing agent to minimize intrarectal degradation of the
soluble antigen.
57. The immunogenic composition of claim 46, including an
absorption-promoting agent.
58. The immunogenic composition of claim 57, wherein the
absorption-promoting agent is selected from a surfactant, mixed
micelle, enamines, nitric oxide donor, sodium salicylate, glycerol
ester of acetoacetic acid, clyclodextrin or beta-cyclodextrin
derivative, or medium-chain fatty acid.
59. The immunogenic composition of claim 46, further comprising an
adjuvant.
60. The immunogenic composition of claim 59, wherein the adjuvant
is selected from cholera toxin (CT), mutant cholera toxin (MCT),
mutant-E. coli heat labile enterotoxin, o(Original) r pertussis
toxin.
61. The immunogenic composition of claim 59, wherein the adjuvant
is conjugated to a mucosal tissue or T cell binding agent.
62. The immunogenic composition of claim 61, wherein the mucosal
tissue or T cell binding agent is selected from protein A, an
antibody that binds a mucosal tissue- or T-cell-specific protein,
or a ligand or peptide that binds a mucosal tissue- or
T-cell-specific protein.
63. The immunogenic composition of claim 59, wherein the adjuvant
comprises a recombinant cholera toxin (CT) having a B chain of CT
substituted by protein A conjugated to a CT A chain to eliminate
toxicity and enhance mucosal tissue binding mediated by protein
A.
64. The immunogenic composition of claim 59, wherein the adjuvant
is conjugated to a protein or peptide that binds specifically to T
cells.
65. The immunogenic composition of claim 64, wherein the protein or
peptide binds to CD4 or CD8.
66. The immunogenic composition of claim 66, wherein the protein or
peptide is an HIV V3 loop or T cell-binding peptide fragment
thereof.
67. The immunogenic composition of claim 59, further comprising
purified IL-12.
68. The immunogenic composition of claim 59, further comprising
purified interferon-.gamma..
69. The immunogenic composition of claim 68, further comprising
purified IL-12.
Description
RELATED APPLICATIONS
[0001] The present application is a continuation-in-part
application of, and claims the benefit under Title 35 of U.S.
Provisional Application Nos. 60/058,523 filed on Sep. 11, 1997, and
60/074,894 filed on Feb. 17, 1998.
TECHNICAL FIELD
[0002] The present invention relates to methods and compositions
for stimulating immune responses in mammals. More particularly, the
invention relates to methods and compositions for stimulating
mucosal immunity.
BACKGROUND OF THE INVENTION
[0003] Many infectious pathogens, e.g., HIV-1, enter their
mammalian hosts via a mucosal tissue prior to establishing a
systemic infection. Veazey, et al., Science 280:427-431, 1998.
Accordingly, vaccines capable of protecting against HIV should be
capable of inducing long-term mucosal immune responses. A number of
recent studies have shown that such immune responses require direct
stimulation of mucosal tissues, and may be achieved with live
attenuated virus, Cranage, et al., Virology 229:143-154, 1997,
subunit SIV envelope Lehner, et al., Nature Medicine 2:767-775,
1996, HIV-recombinant viruses, including recombinant MVA 89.6 env
(Belyakov et al., unpublished), or HIV peptide constructs Belyakov,
et al., Proc. Nat. Acad. Sci. 95:1709-1714, 1998 (see also,
Gallichan, et al., J. Exp. Med. 184:1879-1890, 1996; Cranage, et
al., Virology 229:143-154, 1997; and Rosenthal, et al., Semin.
Immunol. 9:303-314, 1997).
[0004] Numerous questions remain, however, concerning which vaccine
candidates may afford the most effective protection against mucosal
challenge with virus, and what mechanisms may be involved in
mediating protective immunity. While a number of studies have shown
a role for CTL in protection against infections such as influenza
that have a mucosal component (Taylor and Askonas, Immunology
58:417-420, 1986; Epstein et al., J. Immunol. 160:322-327, 1998;
Kulkarni et al., J. Virol. 69:1261-1264, 1995), these reports have
not established whether the CTL need to be in a local mucosal site
to protect. Conversely, while other studies have shown the
induction of CTL in the mucosa, they have not established that
these cells have a role in protection (Gallichan and Rosenthal, J.
Exp. Med. 184:1879-1890, 1996; Bennink et al., Immunology
35:503-509, 1978; Lohman et al., J. Immunol. 155:5855-5860, 1995);
and Klavinskis, et al., J. Immunol. 157:2521-2527, 1996 J. Immunol.
155:5855-5860, 1995. Yet other studies have shown the induction by
vaccines of protective immunity in the mucosa, but in the face of
multiple immune responses, have not been able to sort out which
responses are involved in protection (Lehner et al., Nature
Medicine 2:767-775, 1996; Putkonen et al., J. Virol. 71:4981-4984,
1997; Miller et al., J. Virol. 71:1911-1921, 1997; Quesada-Rolander
et al., AIDS Res Hwn Retroviruses 12:993-999, 1996; Bender et al.,
J. Virol. 70:6418-6424, 1996; Wang et al., Vaccine 15:821-825,
1997).
[0005] Thus, although the role of CTL in protection against-mucosal
infections has been of interest for decades, especially in the case
of influenza virus, prior investigations have failed to identify
fundamental mechanisms linking immune responses to protection. In
this regard, because mucosal infection by virus induces a local IgA
response, it has been too readily assumed that this response, and
not a concomitant CTL response, was responsible for protection
against viral infection through the mucosal route. However, the
role of secretary IgA in neutralizing and protecting against
mucosal HIV challenge is also not clear.
[0006] CTL are crucial mediators of immunity to intracellular
microorganisms such as viruses as well as certain bacteria and
protozoan parasites. CTL specifically recognize "non-self"
antigenic peptides bound to major histocompatibility complex (MHC)
class I molecules on the surface of "target cells" and then kill
the target cells expressing the non-self antigenic peptides.
Non-self polypeptides from which the non-self peptides are derived
can be a) proteins-encoded by intracellular microbes, b)
host-encoded proteins whose expression is induced by a microbe, or
c) mutant host encoded proteins expressed by, for example, tumor
cells.
[0007] Thus, generation of CTL responses in the inductive and the
effector mucosal immune system may be important to establishing
effective protective immunity to intracellular microbial pathogens
that establish infection via the mucosal barriers. In some cases,
administration of antigens via parenteral routes (subcutaneous,
intramuscular, intravenous or intraperitoneal, for example) either
fails to induce mucosal immunity or does so extremely
inefficiently.
[0008] As noted above, previous reports of mucosal immune responses
elicited by mucosal challenge with viruses have disclosed that the
latter induces antiviral antibody responses, and in some cases CTL
responses, in the intraepithelial lymphoid populations. Chen et
al., J. Virol. 71:3431-3436, 1997; Sydora, et al., Cell Inununol.
167:161-169, 1996. However, it is not clear if either of such
responses is relevant to protection against viral infection in
general, or HIV infection in particular. Additional studies have
suggested a role for CTL in protection against infections that
involve the mucosa, such as influenza or respiratory syncytial
virus, Taylor et al., Immunology 58:417-420, 1986; Epstein et al.,
J. Immunol. 160:322-327, 1998; Kulkarni et al., J. Virol.
69:1261-1264, 1995. However, these studies have not addressed the
question of whether CTL must be present at the mucosal site of
infection, or if their principal activity occurs systemically.
[0009] Accordingly, a need exists in the art to better define the
roles and mechanisms of CTL in mediating immunity and to develop
new tools for mediating immune protection against HIV and other
pathogens, particularly by conferring immune protection at mucosal
sites where such pathogens initially proliferate.
SUMMARY OF THE INVENTION
[0010] The present invention is directed to methods and
compositions for inducing a protective mucosal CTL response in a
subject. The methods of the invention involve administering either
a soluble antigen itself, or a polynucleotide encoding the soluble
antigen, to a mucosal surface. The soluble antigens can be full
length, naturally occurring polypeptides or fragments (i.e.,
peptides) derived from them. Peptides to be administered can be any
length less than that of the naturally occurring polypeptide. They
can be, for example, five to one hundred amino acid residues long,
preferably twenty to seventy five amino acid residues long, more
preferably twenty five to sixty amino acid residues long and most
preferably thirty to fifty amino acid residues long.
[0011] The soluble antigen is administered with an adjuvant at the
mucosal site or without an adjuvant. Adjuvants can be, for example,
cholera toxin (CT), mutant CT (MCT), E. coli heat labile
enterotoxin (LT) or mutant LT (MLT). IL-12 and/or IFN.gamma. can be
administered with the soluble antigen either in the presence or
absence of an adjuvant. Alternatively, the two cytokines (IL-12
and/or IFN.gamma.) can be administered systemically and separately
from the soluble antigen which is administered mucosally,
optionally with adjuvant. Mucosal routes of administration include
IR, intranasal (IN), intragastric (IG), intravaginal (IVG) or
intratracheal (IT).
[0012] Soluble antigens can be derived from pathogenic viruses
(e.g., HIV-1, influenza virus or hepatitis A virus), bacteria (e.g,
Listeria monocytogenes), protozoans (e.g., Giardia lamblia).
Alternatively, the soluble antigen can be a tumor-associated
antigen, e.g., prostate specific antigen produced by prostate tumor
cells or tyrosinase produced by melanoma cells. Peptide antigens
can be cluster peptide vaccine constructs (CLUVAC). For example, an
HIV-1 CLUVAC can include one or more of the following
sequences:
1 (SEQ ID NO:1) EQMEDIISLWDQSLKPCVKIQRGPGRAFVTIGK, (SEQ ID NO:2)
KQIINMQEVGKAMYAPPISGQIRRIQRGPGRAFVTIGK, (SEQ ID NO:3)
RDNWRSELYKYKVVKIEPLGVAPTRIQRGPGRAFVTIG- K, SEQ ID NO:4)
AVAEGTDRVIEVVQGAYRAIRHIPRRIRQGLER- RIQRGPGRAFVTIGK, (SEQ ID NO:5)
DRVIEVVQGAYRAIRHIPRRIRQGLERRIQRGPGRAFVTIGK, (SEQ ID NO:6)
DRIVEVVQGAYRAIRRIQRGPGRAFVITGK, (SEQ ID NO:7)
AQGAYRAIRHIPRRIRRIQRGPGPRAFVTIGK, (SEQ ID NO:8)
EQMHEDIISLWDQSLKPCVKRIHIGPGRAFYTTKN, (SEQ ID NO:9)
KQIINMWQEVGKAMYAPPISGQIRRIHIGPGRAFYTTKN, (SEQ ID NO:10)
RDNWRSELYKYKVVKIEPLGVAPTRIHIGPGRAFYTTKN, (SEQ ID NO:11)
AVAEGTDRVIEVVQGAYRAIRHIPRRIRQGLERRIHIGPGRA- FYTTKN, (SEQ ID NO:12)
DRVIEVVQGAYRAIRHIPRRIRQGLE- RRIHIGPGRAFYTTKN, (SEQ ID NO:13)
DRVIEVVQGAYRAIRRIHIGPGRAFYTTKN or (SEQ ID NO:14)
AQGAYRAIRHIPRRIRRIHIGPGRAFYTTKN.
[0013] Preferably, the CLUVAC includes the amino acid sequence of
SEQ ID NO:2, SEQ ID NO:9 or SEQ ID NO:12. Antigenic peptides can be
longer than the length specified by the SEQ ID NOS. recited herein,
i.e., the peptide can be extended by adding one or more (e.g., 5,
10, 15, 20) amino acids at the amino and/or carboxy termini of the
peptide with any given SEQ ID NO.
[0014] The invention also encompasses methods for inducing a
protective mucosal CTL response in a subject in which the soluble
antigen is delivered IR. Preferably, the level of CTL activity
induced by IR immunization is at least 10% greater than that
induced by other routes of mucosal administration (e.g., IN). More
preferably, mucosal CTL activity induced by IR immunization is at
least 2-fold, more preferably, at least 5-fold, and most
preferably, at least 10-fold greater than that induced by other
routes of mucosal immunization (e.g., IN or IG).
[0015] Subjects to which the methods of the invention are applied
are mammals, e.g., humans, non-human primates, cats or mice.
[0016] Also provided within the invention are immunogenic
compositions for inducing a protective mucosal CTL response in a
subject which are adapted for intrarectal administration. The
compositions comprise a purified soluble antigen formulated for
intrarectal delivery to the rectum, colon, sigmoid colon, or distal
colon. They may be formulated as a rectal enema, foam, suppository,
or topical gel and generally comprise a base, carrier, or
aabsorption-promoting agent adapted for intrarectal delivery.
[0017] In more detailed aspects, the immunogenic compositions of
the invention may include a rectal emulsion or gel preparation,
preferably wherein the soluble antigen is admixed with a homogenous
emulsion or gel carrier, eg., a polyoxyethylene gel. Alternatively,
the soluble antigen may be admixed with a rectally-compatible
foam.
[0018] In other preferred aspects, the immunogenic compositions of
the invention are formulated in a suppository. The suppository is
comprised of a base or carrier specifically adapted for intrarectal
delivery of the antigen. Preferred bases may be selected from a
polyethyleneglycol, witepsol H15, witepsol W35, witepsol E85,
propyleneglycol dicaprylate (Sefsol 228), Miglyol810,
hydroxypropylcellulose-H (HPC), or carbopol-934P (CP). More
preferably, the suppository comprises at least two base materials
to optimize structural and delivery performance. In other aspects,
the suppository includes a stabilizing agent to minimize
intrarectal degradation of the soluble antigen.
[0019] To optimize intrarectal delivery, the immunogenic
compositions of the invention also preferably include an
absorption-promoting agent, for example a surfactant, mixed
micelle, enamine, nitric oxide donor, sodium salicylate, glycerol
ester of acetoacetic acid, clyclodextrin or beta-cyclodextrin
derivative, or medium-chain fatty acid.
[0020] In yet additional aspects of the invention, immunogenic
compositions are provided which include an adjuvant which enhances
the CTL response. Suitable adjuvants are detoxified bacterial
toxins, for example detoxified cholera toxin (CT), mutant cholera
toxin (MCT), mutant--E. coli heat labile enterotoxin, and pertussis
toxin. Preferably, the adjuvant is conjugated to a mucosal tissue
or T cell binding agent, such as protein A, an antibody that binds
a mucosal tissue- or T-cell-specific protein, or a ligand or
peptide that binds a mucosal tissue- or T-cell-specific protein. In
more preferred aspects, the adjuvant is a recombinant cholera toxin
(CT) having a B chain of CT substituted by protein A conjugated to
a CT A chain, which exhibits reduced toxicity and enhances mucosal
tissue binding mediated by protein A. Alternatively the adjuvant
may be conjugated to a protein or peptide that binds specifically
to T cells, for example by binding CD4 or CD8 (eg., the HIV V3 loop
or a T cell-binding peptide fragment of the HIV V3 loop).
[0021] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar to those described herein can be used
in the practice or testing of the present invention, suitable
methods and materials are described below. In case of conflict, the
present application, including definitions, will control. In
addition, the materials, methods, and examples described herein are
illustrative only and not intended to be limiting.
[0022] Other features and advantages of the invention, e.g.,
prevention of viral or other infectious diseases, will be apparent
from the following detailed description, from the drawings and from
the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a series of line graphs showing the results of a
CTL assay. IR HIV-1 peptide immunization induced long lasting
mucosal and systemic CTL activity.
[0024] FIG. 2 is a series of line graphs showing the results of a
CTL assay. CT enhanced (but was not essential) for induction of CTL
by IR administration of antigenic peptide.
[0025] FIG. 3 is a series of line graphs showing the results of a
CTL assay. CTL induced by IR immunization with an HIV-1 gp160
peptide specifically lysed target cells transfected with and
expressing an HIV-1 gp160 gene.
[0026] FIG. 4 is a series of line graphs showing the results of a
CTL assay which indicated that IR induction of CTL was IL-12
dependent.
[0027] FIG. 5 is a bar graph showing that IR immunization protects
against IR challenge with HIV-1 gp160 expressing recombinant
vaccinia virus.
[0028] FIG. 6A is a line graph showing the results of a CTL assay
using SP cells obtained from animals six months after IR
immunization with an antigenic peptide.
[0029] FIG. 6B is a line graph showing the results of a CTL assay
using SP cells obtained from animals six months after intranasal
(IN) immunization with antigenic peptide.
[0030] FIG. 7A is a bar graph showing the results of a CTL assay
using PP as effector cells.
[0031] FIG. 7B is a bar graph showing the results of a CTL assay
using SP cells as effector cells. PP and SP cells were obtained
from animals thirty five days after mucosal (IR, IN, IG) or
systemic (subcutaneous (SC)) immunization with an antigenic
peptide.
[0032] FIG. 8A is a bar graph showing the results of a CTL assay
using effector cells from wild-type BALB/c mice.
[0033] FIG. 8B is a bar graph showing the results of a CTL assay
using effector cells from BALB/c mice which lack the ability to
produce functional IFN.gamma.. These experiments show the
dependence of IR induction of CTL on IFN.gamma..
[0034] FIG. 9A is a line graph showing the results of a CTL assay
using PP as effector cells.
[0035] FIG. 9B is a line graph showing the results of a CTL assay
using SP cells as effector cells. PP and SP cells were obtained
from BALB/c mice thirty five days after IR immunization with either
antigenic peptide, CT and IL-12 (composition A) or antigenic
peptide and CT (without IL-12; composition B).
[0036] FIGS. 10A and 10B demonstrate that IR immunization with
PCLUS3-18IIIB or PCLUS3-18MN induces both Peyer's patch (panel A)
and spleen (panel B) long-lasting immunity. However the levels of
the induction of CTL are different. Killing of P18IIIB-I10 (closed
squares) or P18MN-T10 peptide-pulsed targets (closed circles) is
compared with killing of unpulsed targets (open squares or
circles). In both panel A and panel B, SEM of triplicate were all
<5% of the mean.
[0037] FIG. 11 demonstrates that protection induced by mucosal
immunization with HIV-1 peptide vaccine is specific. On day 35,
mice were challenged intrarectally with 2.5.times.10.sup.7
plaque-forming units (pfu) of vaccinia virus expressing gp 160IIB
(vPE16) or with 2.5.times.10.sup.7 pfu of vaccinia virus expressing
.beta.-galactosidase (vSC8). Bars=SEM of five mice per group. The
difference is significant at P<0.01 by Student's test.
[0038] FIG. 12 demonstrates that protection induced by mucosal
immunization with HIV-1 peptide vaccine is long-lasting. On day 35
or 6 months after the start of the immunization, mice were
challenged intrarectally with 2.5.times.10.sup.7 pfu of vaccinia
virus expressing gp 160IIIB. Bars=SEM of five mice per group. The
difference is significant at P<0.01 by Student's test.
[0039] FIG. 13 demonstrates that protection induced by mucosal
immunization with HIV-peptide is dependent on CD8 positive T-cells.
BALB/c mice were treated IP with 0.5 mg monoclonal anti-CD8
antibody (clone 2.43, NIH Frederick, Md.) one day before and after
each immunization and also two days before and three days after the
challenge with vPE16. Mice were challenged intrarectally with
2.times.10.sup.7 pfu of vPE16 vaccinia-virus expressing gp
160IIIB.
[0040] FIGS. 14A and 14B demonstrate that mucosal immunization with
HIV-1 peptide induces mucosal CTL responses and stimulates
protective immunity against intrarectal recombinant HIV-1 vaccinia
challenge. FIG. 14A depicts induction of the mucosal and systemic
CTL responses by different routes of immunization with synthetic
peptide HIV vaccine. Killing of peptide-pulsed targets (closed
bars) is compared with killing of unpulsed targets (open bars) at
an effector-to-target ratio of 50:1. Panel A depicts results or
immunizing on day 35 (IR or SC-bar 1, no immunogen; bar 2, SC; bar
3, IR) BALB/c mice challenged intrarectally with 2.5.times.10.sup.7
plaque-forming units (pfu) of vaccinia virus expressing gp
160IIIB.
[0041] FIGS. 15A and 15B demonstrate enhancement of the mucosal
(FIG. A) and systemic (FIG. B) CTL responses to HIV-1 peptide by
the mucosal (not systemic) treatment with rmIL-12. BALB/c mice were
treated by the IP route (right panels) or IR route (left panels)
with 1 .mu.g of the rmIL-12 each day of the IR immunization with
PCLUS3-18IIIB (50 .mu.g/mice). On day 35 HIV-specific Peyer's patch
CTL (FIG. 15A) and spleen CTL (FIG. 15B) were studied.
[0042] FIG. 16 demonstrates that mucosal treatment with rmIL-12 in
DOTAP along with HIV peptide vaccine enhances protection against
mucosal challenge with vaccinia virus expressing gp160IIIB (vPE16).
Five mice per group were immunized IR on days 0, 7, 14 and 21 with
no immunogen (bar 1), with 50 .mu.g PCLUS3-18IIB alone (bar 2), or
with peptide plus 1 .mu.g rmIL-12 in DOTAP (bar 3), and challenged
on day 35 intrarectally with 5.times.10.sup.7 pfu of vaccinia virus
expressing gp 160IIIB. Viral pfu in the ovaries were determined six
days later.
[0043] FIGS. 17A and 17B demonstrate that IL-12 cannot act directly
in the induction of mucosal CD8+ CTL in the absence of IFN.gamma..
IFN.gamma..sup.-/- mice (BALB/c background) were immunized IR with
the rmIL-12 (1 .mu.g/mouse)+DOTAP together with peptide. On day 35
HIV-specific Peyer's patch CTL (FIG. 17A) and spleen CTL (FIG. 17B)
were studied. Killing of P18IIIB-I10 pulsed targets by effector
cells from immunized IFN.gamma..sup.-/- mice (closed squares) or
conventional BALB/c mice (closed circles) is compares with killing
of unpulsed targets (open squares or circles).
DETAILED DESCRIPTION
A. Induction of Mucosal Immunity
[0044] The present invention is based on the discovery that IR
administration of a peptide, e.g., a synthetic multideterminant
HIV-1 gp160 envelope glycoprotein peptide, to a mucosal surface can
induce an antigen-specific, protective CTL response in the mucosal
immune system, even in the absence of a mucosal adjuvant.
[0045] The exemplary synthetic multideterminant peptides (CLUVAC)
are composed of subregions containing epitopes that evoke some or
all of (a) a helper T cell response, (b) a CTL response and (c) a
high titer of neutralizing antibodies in multiple hosts of a given
species expressing a broad range of MHC haplotypes. IR immunization
with the HIV-1 CLTVAC PCLUS3-18IIIB (SEQ ID NO:2), and the mucosal
adjuvant CT induced peptide-specific CTL in both the inductive (PP)
and the effector (LP) sites of the mucosal immune system, as well
as in systemic lymphoid tissue, i.e., SP. In contrast, systemic
immunization with peptide vaccine produced HIV-1 peptide-specific
CTL only in the SP.
[0046] IR immunization induced long-lasting protective immune
responses. For example, antigen-specific CTL were found in both
mucosal and systemic sites 6 months after immunization. IR
immunization with the antigenic peptide elicited significantly
stronger CTL responses than IN immunization with the same peptide.
While IR administration with PCLUS3-18IIIB (SEQ ID NO:2) induced a
significant response when administered alone, the response was
enhanced by the inclusion of CT. The CTL were CDB+ T lymphocytes
restricted by MHC class I molecules, recognizing MHC class I
positive target cells either endogenously expressing HIV-1 gp160 or
pulsed with an appropriate gp160 peptide. Induction of both mucosal
and systemic CTL response by IR immunization was IL-12-dependent,
as shown by inhibition of induction of CTL in mice treated i.p.
with anti-IL-12 antibody, Furthermore, inclusion of IL-12 in the
composition of antigenic peptide and CT used for IR immunization
resulted in enhanced mucosal and systemic CTL responses relative to
the responses elicited by antigenic peptide and CT without IL-12.
The dependence on IFN.gamma. of mucosal and systemic CTL generation
following IR immunization was demonstrated by the absence of such
responses in mice which lack the ability to produce functional
IFN.gamma., e.g., as the result of a premature stop codon in the
IFN.gamma.-encoding gene. The stop-codon mutation causes the gene
to encode a truncated protein lacking the activity of
IFN.gamma..
[0047] IR immunization with PCLUS3-18IIIB (SEQ ID NO:2) protected
mice against an IR challenge with a recombinant vaccinia virus
expressing HIV-1 IIIB gp160. Thus, an HIV-1 peptide induced CTL
responses in the mucosal and systemic immune systems after IR
immunization and protected against mucosal challenge with virus
expressing HIV-1 gp160.
[0048] The immunization method of the invention is useful to induce
a mucosal CTL response to any soluble antigen. Accordingly, the
invention provides a new method for vaccine administration to
elicit immunologic protection against viruses that enter through
mucosal barriers, including HIV-1. The method can also be applied
to achieving protection from infection by certain bacteria (e.g.,
L. monocytogenes) and protozoa (e.g., G. lamblia) that establish
infection via mucosae. In addition, since mucosal immunization
results in systemic generation of CTL activity, the protocol can
also be useful in prevention of infection by microorganisms that
enter is through non-mucosal routes. The method is also useful for
immunotherapy of infections that are mucosally or non-mucosally
established. Finally, the methods can be utilized for immunotherapy
of cancer, both in the region of and remote from a mucosal
surface.
B. Methods of Immunization
[0049] The invention features methods for protecting subjects from
infection by intracellular microorganisms such as viruses as well
as intracellular bacteria and protozoans. The methods involve
induction of CTL responses specific for antigenic peptides derived
from proteins encoded by genes of relevant microbes and expressed
in association with MHC class I molecules on the surface of
infected cells. This is achieved by delivery of an appropriate
soluble antigen to a mucosal surface, e.g., rectal, vaginal,
nasopharyngeal, gastric or tracheal mucosae. Relevant
microorganisms include but are not restricted to those that enter
their hosts via mucosal barriers, e.g. HIV-1, influenza virus,
enteric viruses such as rotaviruses, hepatitis A virus, papilloma
virus, feline immunodeficiency virus, feline leukemia virus, simian
immunodeficiency virus, intracellular bacterial pathogens, e.g., L.
monocytogenes and mycobacteria such as M. tuberculosis and M.
leprae. Since the responses elicited by mucosal immunization occur
in the systemic as well as the mucosal immune system, the methods
can also be applied to protection from infection by intracellular
pathogens that enter their hosts via non-mucosal (e.g., HIV-1 in
some scenarios such as a "stick" by a contaminated syringe needle,
rabies virus and malarial protozoans) as well as mucosal routes. In
light of the above considerations, immunization via mucosae can
also be used for immunotherapy of intracellular infections.
[0050] Finally, the mucosal immunotherapy of the invention can be
applied to subjects with cancer, particularly, but not limited to,
those with solid tumors in the region of the relevant mucosa, e.g.,
colonic, rectal, bladder, ovarian, uterine, vaginal, prostatic,
nasopharyngeal, lung or certain melanoma tumors. Tumor immunity is
substantially mediated by CTL with specificity for tumor associated
peptides (e.g., prostate specific antigen peptides in prostatic
cancer, carcinoembryonic antigen peptides in colon cancer, human
papilloma virus peptides in bladder cancer and MART1, gp100 and
tyrosinase peptides in melanoma) (Rosenberg et al. (1994) J.N.C.I.
76:1159; Kawakami et al. (1994) Proc. Natl. Acad. Sci. USA.
91:3515) bound to MHC class I molecules on the surface of tumor
cells.
[0051] B.1 Antigenic Polypeptides
[0052] A soluble antigen to be administered according to the
invention can be any soluble carbohydrate or peptide antigen, e.g.,
one containing all or part of the amino acid sequence of a peptide
which is naturally expressed in association with a MHC class I
molecule on the surface of a cell infected with relevant microbe or
expressed on a tumor cell. In the case of infected cells, the cell
surface expressed peptide is derived from a protein either encoded
by genes of the infectious agent or whose expression is induced by
the infectious agent. Thus, the soluble antigen can be a full
length, naturally occurring polypeptide, e.g., full length HIV-1
gp160 or gp120 or an antigenic fragment thereof.
[0053] Antigen-specific recognition by CTL involves interactions
between components of the antigen-specific T cell receptor on the
surface of the CTL and residues on both the antigenic peptide and
the MHC class I molecule to which the peptide is bound. Thus the
soluble antigen can also be a fragment (i.e., a peptide) of the
naturally occurring polypeptide that is either (a) itself capable
of binding to MHC class I molecules of multiple haplotypes on the
surface of antigen presenting cells (APC) and stimulating CD8+ T
cell responses in subjects expressing these MHC class I haplotypes
or (b) which can be proteolytically processed by APC into fragments
with these properties. Ways of establishing the ability of a
candidate peptide to stimulate a CTL response in the context of
multiple MHC class I haplotypes are well known to one of ordinary
skill in the art and are amply described in co-pending U.S. patent
application Ser. No. 08/060,988 incorporated herein by reference in
its entirety.
[0054] Antigenic peptides can be engineered to bind to MHC class II
molecules of multiple haplotypes and will be recognized by CD4+
helper T cell precursor cells of subjects expressing multiple MHC
class II haplotypes. Ways of establishing the ability of a
candidate peptide to stimulate a helper T cell response in the
context of multiple MHC class II haplotypes are well known to one
of ordinary skill in the art and are amply described in co-pending
U.S. patent application Ser. No. 08/060,988 incorporated herein by
reference in its entirety.
[0055] In addition, antigenic peptides, can contain epitopes that
elicit neutralizing antibodies, i.e., those that bind to the
relevant microbe or a cell infected with the microbe and neutralize
or kill it ways of establishing these properties of a candidate
peptide are well known to one of ordinary skill in the art and are
amply described in copending U.S. patent application Ser. No.
08/060,988 incorporated herein by reference in its entirety.
[0056] Antigenic peptides can also elicit antibodies that induce
antibody-dependent cellular cytolysis (ADCC) of cells infected by
the appropriate microbe or tumor cells expressing at their surface
the protein from which the antigenic peptide was derived. ADCC is a
protective mechanism by which specialized cells of the immune
system (K cells) recognize the Fc portion of IgG antibody molecules
bound to the surface of a target cell and lyse the relevant target
cell. Sera, or other body fluids such as rectal lavages, from test
subjects mucosally immunized with a candidate peptide can be tested
for their ability to mediate ADCC by methods known to an ordinary
artisan. A standard cell mediated lympholysis (CML) assay is used.
Briefly, a source of lymphoid ADCC effector cells (e.g., peripheral
blood mononuclear cells (PBMC) or SP cells) is incubated in vitro
with target cells expressing the above described cell surface
protein in the presence of various dilutions of test sera. Lysis of
the target cells, which can be measured by the release of a
detectable label (.sup.51Cr, for example) from prelabeled target
cells, is an indication of the presence of ADCC inducing antibodies
in the test serum.
[0057] Peptides of the invention can be cluster peptide vaccine
constructs (CLUVAC). A CLUVAC is a chimeric peptide containing a) a
subregion with multiple overlapping helper T cell activating
epitopes that can be presented by multiple MHC class II molecules
(a cluster peptide), b) a subregion with a CTL activating epitope
and c) a subregion that elicits the production of a neutralizing
antibody. The peptide sequences containing these epitopes can be
derived from different parts of a microbial or tumor associated
polypeptide.
[0058] Alternatively, the CTL inducing and antibody neutralizing
epitopes can be located in one subregion of an antigen and the
helper epitope(s) can be in a second subregion. CLUVAC and their
design are extensively described in co-pending U.S. patent
application Ser. No. 08/060,988 incorporated herein by reference in
its entirety.
[0059] HIV-1 CLUVAC can include the following sequences:
2 (SEQ ID NO:1) EQMHEDIISLWDQSLKPCVKRIQRGPGRAFVTIGK (SEQ ID NO:2)
KQIINMWQEVGKAMYAPPISGQIRRIQRGPGRAFVTIGK (SEQ ID NO:3)
RDNWRSELYKYKVVKIEPLGVAPTRIQRGPGRAFVTI- GK (SEQ ID NO:4)
AVAEGTDRIEVVQGAYRAIRHIPRRIRQGLER- RIQRGPGRAFVTIGK (SEQ ID NO:5)
DRVIEVVQGAYRAIRHIPRRIRQGLERRIQRGPGRAFVTIGK (SEQ ID NO:6)
DRIEVVQGAYRAIRRIQRGPGRAFVTIGK (SEQ ID NO:7)
AQGAYRAIRHIPRRIRRIQRGPGRAFVTIGK (SEQ ID NO:8)
EQMHEDIISLWDQSLKPCVKRIHIGPGRAFYTTKN (SEQ ID NO:9)
KQIINMWQEVGKAMYAPPISGQIRRIHIGPGRAFYTTKN (SEQ ID NO:10)
RDNWRSELYKYKVVKIEPLGVAPTRIHIGPGRAFYTTKN (SEQ ID NO:11)
AVAEGTDRVIEVVQGAYRAIRHIPRRIRQGLERRIHIGPGRAFYTTKN (SEQ ID NO:12)
DRVIEVVQGAYRAIRHIPRRIRQGLERRIHIGPGRAFYTTKN (SEQ ID NO:13)
DRVIEVVQGAYRAIRRIHIGPGRAFYTTKN (SEQ ID NO:14)
AQGAYRAIRHIPRRIRRIHIGPGRAFYTTKN
[0060] It is possible, for example, to link a particular cluster
peptide to any peptide containing a CTL and/or neutralizing
antibody epitope. Examples of cluster peptides include: cluster
peptide 1 whose amino acid sequence (EQMHEDIISLWDQSLKPCVK) (SEQ ID
NO:17) is the first 20 amino acids of the HIV-1 CLUVAC with SEQ ID
NO:1; cluster peptide 3 whose amino acid sequence
(KQIINMWQEVGKAMYAPPISGQIR) (SEQ ID NO:18) is the first 24 amino
acids of the HIV-1 CLUVAC with SEQ ID NO: 2; cluster peptide 4
whose amino acid sequence (RDNWRSELYKYKVVKIEPLGVAPT) (SEQ ID NO:19)
is the first 24 amino acids of the HIV-1 CLUVAC with SEQ ID NO:3;
and cluster peptide 6 whose amino acid sequence
(AVAEGTDRVIEVVQGAYRAIRHIPRRIR- QGLER) (SEQ ID NO:20) is the first
33 amino acids of the HIV-1 CLUVAC with SEQ ID NO:4.
[0061] Of particular interest are peptides containing the amino
acid sequences of SEQ ID NO:2, SEQ ID NO:9 and SEQ ID NO:12.
Mucosal responses to the first (PCLUS3-18IIIB) are extensively
characterized in the Examples presented infra. PCLUS3-18MN (SEQ ID
NO:9) and PCLUS6.1-18MN (SEQ ID NO:12) have been tested in human
clinical trials. The CTL epitope within PCLUS3-18IIIB (SEQ ID NO:2)
is the amino acid sequence RIQRGPGRAFVTIGK (SEQ ID NO:15).
[0062] In some instances, mucosal adjuvants are co-administered at
the mucosal tissue site with the soluble antigens. Such adjuvants
include, but are not limited to, detoxified bacterial toxins, for
example detoxified cholera toxin (CT), E. coli heat labile toxin
(LT), mutant CT (MCT) (Yamamoto et al. J. Exp. Med. 185:1203
(1997); Yamamoto et al. Proc. Natl. Acad. Sci. USA 58:5267 (1997);
Douce et al. Infect. Immunity 65:2821 (1997)), mutant E. coli
heat-labile toxin (MLT) (Di Tommaso et al. Infect. Immunity 64:974
(1996); Partidos et al. Immunology 89:83 (1996), and pertussis
toxin. MCT and MLT contain point mutations that substantially
ablate toxicity without substantially compromising adjuvant
activity relative to that of the parent molecules. In other
preferred embodiments, the adjuvant is modified for increased
binding to mucosal tissues and/or T-cells. Thus, in one aspect of
the invention, CT or other bacterial toxins are conjugated to a
mucosal tissue binding agent, such as protein A, an antibody that
binds a mucosal tissue- or T-cell-specific protein (eg., a
receptor), or a ligand or peptide that binds a mucosal tissue- or
T-cell-specific protein (eg., CD4 or CD8). For example, the B chain
of CT may be substituted by protein A, conjugated to the CT A
chain, to eliminate toxicity and enhance mucosal tissue binding
mediated by protein A. Alternatively, the HIV V3 loop which binds
CD4 on T cells may be conjugated to the adjuvant to enhance
delivery to T cells.
[0063] The CTL augmenting cytokines, eg., IL-12 and IFN.gamma.,
are, in some instances, either administered systemically or,
preferably, coadministered at the mucosal tissue site with the
soluble antigen.
[0064] Variants of disclosed antigenic peptides can contain
different amino acids (preferably conservative changes) from the
parental molecules but retain the biological activity of the
parental molecules, e.g., the ability to induce specific CTL
responses subsequent to mucosal immunization. Such variants can be
synthesized by standard means, and are readily tested in assays
known to those in the art. Antigenic polypeptides are typically
longer than the length of the nominal SEQ ID NOS. recited herein,
i.e., the polypeptide can be extended by adding amino-acids to the
amino or carboxy termini of the peptide defined by any given SEQ ID
NO.
[0065] Peptides and polypeptides of the invention will also include
those described above but modified for in vivo use by:
[0066] (a) chemical or recombinant DNA methods to include mammalian
signal peptides (Lin et al., J. Biol. Chem. 270:14255 (1995)) or
the bacterial peptide, penetratin (Joliot et al., Proc. Natl. Acad.
Sci. USA 88:1864 (1991)), that will serve to direct the peptide
across cell and cytoplasmic membranes and/or traffic it to the
endoplasmic reticulum (ER) of antigen presenting cells (APC), e.g.,
dendritic cells which are potent CTL inducers;
[0067] (b) addition of a biotin residue which serves to direct the
polypeptides or peptide across cell membranes by virtue of its
ability to bind specifically to a translocator present on the
surface of cells (Chen et al., Analytical Biochem. 227:168
(1995));
[0068] (c) addition at either or both the amino- and
carboxy-terminal ends, of a blocking agent in order to facilitate
survival of the relevant polypeptide or peptide in vivo. This can
be useful in those situations in which the termini tend to be
degraded ("nibbled") by proteases prior to cellular or ER uptake.
Such blocking agents can include, without limitation, additional
related or unrelated peptide sequences that can be attached to the
amino and/or carboxy terminal residues of the polypeptide or
peptide to be administered. This can be done either chemically
during the synthesis of the peptide or by recombinant DNA
technology (see Section 3.3 infra). Alternatively, blocking agents
such as pyroglutamic acid or other molecules known to those of
average skill in the art can be attached to the amino and/or
carboxy terminal residues, or the amino group at the amino terminus
or carboxyl group at the carboxy terminus replaced with a different
moiety. Likewise, the polypeptides or peptides can be covalently or
noncovalently coupled to pharmaceutically acceptable "carrier"
proteins prior to administration.
[0069] Also of interest are peptidomimetic compounds based upon the
amino acid sequence of the peptides of the invention.
Peptidomimetic compounds are synthetic compounds having a
three-dimensional structure (i.e. a "peptide motif") based upon the
three-dimensional structure of a selected peptide. The peptide
motif provides the peptidomimetic compound with the activity of
binding to MHC molecules of multiple haplotypes and activating
CD8.sup.+ and CD4.sup.+ T cells from subjects expressing such MHC
molecules that is the same or greater than the activity of the
peptide from which the peptidomimetic was derived. Peptidomimetic
compounds can have additional characteristics that enhance their
therapeutic application such as increased cell permeability,
greater affinity and/or avidity and prolonged biological half-life.
The peptidomimetics of the invention typically have a backbone that
is partially or completely non-peptide, but with side groups
identical to the side groups of the amino acid residues that occur
in the peptide on which the peptidomimetic is based. Several types
of chemical bonds,.e.g. ester, thioester, thioamide, retroamide,
reduced carbonyl, dimethylene and ketomethylene bonds, are known in
the art to be generally useful substitutes for peptide bonds in the
construction of protease-resistant peptidomimetics.
[0070] B.2 In vivo Methods to Deliver Soluble Antigens to Mucosae
of a Subject
[0071] In methods of the invention that induce mucosal CTL
responses, a soluble antigen is delivered to antigen presenting
cells of the inductive mucosal immune system (e.g., PP of the
intestine). Delivery involves administering to a subject either the
soluble antigen itself, e.g., an antigenic peptide, an expression
vector encoding the soluble antigen, or cells transfected or
transduced with the vector.
[0072] B.2.1 Administration of Soluble Antigen
[0073] Soluble antigens can be delivered to the mucosal immune
system of a mammal using techniques substantially the same as those
described infra for delivery to human subjects. Examples of
appropriate mammals include but are not restricted to humans,
non-human primates, horses, cattle, sheep, dogs, cats, mice, rats,
guinea pigs, hamsters, rabbits and goats.
[0074] A soluble antigen of the invention can be delivered to the
mucosal immune system of a human in its unmodified state, dissolved
in an appropriate physiological solution, e.g., physiological
saline. Alternatively, it can be modified as detailed in Section
B.1 in order to facilitate transport across cell and/or
intracellular membranes and to prevent extracellular or
intracellular degradation. Its transport across biological
membranes can also be enhanced by delivering it encapsulated in
liposomes using known methods (Gabizon et al., Cancer Res. 50:6371
(1990); Ranade, J. Clin. Pharmacol. 29:685 (1989)) or an
appropriate biodegradable polymeric microparticle (also referred to
as a "microsphere", "nanosphere", "nanoparticle, or
"microcapsule"). Naturally, it is desirable that the soluble
antigens be selectively targeted to the mucosae. This can be
achieved by contacting the soluble antigens directly with the
relevant mucosal surface, e.g., by IR, IVG, IN, intrapharyngeal
(IPG) or IT infusion or implantation. CTL activity (systemic or
mucosal) induced by IR immunization can be two-fold, preferably
five-fold, more preferably twenty-fold, even more preferably
fifty-fold and most preferably two hundred-fold greater than that
induced by IN immunization.
[0075] Soluble antigens of the invention can be delivered in
liposomes into which have been incorporated ligands for receptors
on relevant cells (e.g., dendritic cell or, macrophage APC) or
antibodies to cell-surface markers expressed by these cells. Thus,
for example, an antibody specific for a dendritic cell surface
marker can direct liposomes containing both the anti-dendritic cell
antibody and the relevant soluble antigen to dendritic cells.
[0076] The soluble antigens can be administered mucosally either
alone or together with a mucosal adjuvant. Suitable adjuvants
include, but are not restricted to, CT, MCT and MLT. IL-12 and/or
IFN.gamma. can also be administered to the subject. Thus, a soluble
antigen can be administered with an adjuvant, with IL-12 (in the
absence of an adjuvant), with IFN.gamma. (in the absence of an
adjuvant), with both IL-12 and IFN.gamma. (in the absence of an
adjuvant), with an adjuvant and IL-12, with an adjuvant and
IFN.gamma., or with an adjuvant and both IL-12 and IFN.gamma.. The
IL-12 and IFN.gamma. can be administered systemically or
co-administered mucosally with the peptide. When the soluble
antigen is administered encapsulated in liposomes or
microparticles, the IL-12 and/or IFN.gamma. can be co-incorporated
into the same liposomes/microparticles or incorporated into
separate liposomes/microparticles. Alternatively, the cytokines can
be administered in a free, soluble form. Examples of other
cytokines that can be used, singly or in combination, are
granulocyte-macrophage colony-stimulating factor (GM-CSF),
interleukin-2 (IL-2), interleukin-7 (IL-7), or tumor necrosis
factor a (TNFa). These and other immunopotentiating cytokines are
administered as discussed above for IL-12 and IFN.gamma..
Administration can be single or multiple (two, three, four, five,
six, eight or twelve administrations, for example). Where multiple,
they can be spaced from one day to one year apart. When using
peptides without a helper T-cell epitope, it can be necessary to
carry out such multiple administrations.
[0077] In preferred aspects of the invention formulations of
soluble antigen are prepared for intrarectal administration (i.e.,
delivery to the rectum, colon, sigmoid colon, or distal colon),
eg., by formulating the soluble antigen in a rectal enema, foam,
suppository, or topical gel. These rectal delivery formulations are
adapted for improved delivery, stability, and/or absorption in the
rectum, eg., by combining the soluble antigen with one or more
known intrarectal delivery base or carrier materials, intrarectal
absorption-promoting materials, and/or stabilizers.
[0078] Preferred base formulations for rectal delivery of soluble
antigen within the methods of the invention include hydrophilic and
hydrophobic vehicles or carriers such as those commonly used in
formulating suppositories and rectal emulsion or gel preparations.
Thus, emulsion vehicles are used which incorporate soluble antigen
in an oil/water emulsion suitable for intrarectal administration.
Alternatively, gel formulations are provided which incorporate the
soluble antigen in a homogenous gel carrier, for example a
polyoxyethylene gel such as polyoxyethylene (20) cetylether
(BC-20TX). When the antigen is formulated in a rectally compatible
foam, a non-CFC propellant foam is preferred.
[0079] Preferred carriers or delivery vehicles for use within the
invention are conventional suppository base materials that are
adapted for intrarectal use, such as polyethyleneglycols. Exemplary
polyethyleneglycols known and available in art include PEG 400, PEG
1500, PEG 2000, PEG 4000 or PEG 6000. Preferred bases in this
context include witepsol H15, witepsol W35, witepsol E85. These are
selected for their desired lipophilic and/or hydrophilic
properties, and are often selected to form a multiple layer
suppository with both lipophilic and hydrophilic base layers.
Preferred lipophilic base materials are macrogols of low molecular
weight. Particular examples of suitable lipophilic bases include
propyleneglycol dicaprylate. (Sefsol 228) and Miglyol810. A
preferred hydrophilic base is PEG, eg., PEG 400. In one formulation
useful within the invention, hydroxypropylcellulose-H (HPC) and/or
carbopol-934P (CP) are used as bases for an inner suppository
layer, and a witepsol base is used for the outer layer.
[0080] Crystalline cellulose or other common stabilizing agents can
be added in selected amounts (eg., 30-60% by weight) to the base to
promote sustained release.
[0081] Selection of base materials, stabilizers, and
absorption-promoting agents for formulating intrarectal delivery
compositions, particularly suppositories, is determined according
to conventional methods, eg., based on melting and drop rates,
breaking hardness, disintegration and special breaking times,
spreading properties, and diffusion rates. Preferred values of
these various properties are known and can be readily determined to
adjust antigen formulations to be suitable for intrarectal use. In
particular, appropriate values for formulating a time-release
suppository having appropriate structural and chemical properties
for rectal delivery are known or readily ascertained. In this
context, conventional additives, eg., softeners such as neutral
oils, Estasan, etc. may be included to optimize consistency, rate
of delivery and other characteristics of the formulation.
[0082] In formulating mucosal delivery compositions, it is also
desired to include absorption-promoting agents to enhance delivery
of the soluble antigen and, optionally the CTL-stimulatory
cytokine, to the mucosal surface. A variety of absorption promoting
agents (i.e., agents which enhance release or diffusion of the
antigen and/or CTL-stimulatory cytokine from the delivery vehicle
or base, or enhance delivery of the antigen and/or CTL-stimulatory
cytokine to the mucosal tissue or T-cells, for example by enhancing
membrane penetration) are known in the art and are useful in
mucosal delivery formulations of the invention, eg., for inclusion
in intrarectal delivery formulations, particularly suppositories.
These include, but are not limited to, surfactants (eg., tween 80),
mixed micelles, enamines, nitric oxide donors (eg.,
S-nitroso-N-acetyl-DL-penicillamine, NOR1, NOR4--which are
preferably co-administered with an NO scavenger such as
carboxy-PITO or doclofenac sodium), sodium salicylate, glycerol
esters of acetoacetic acid (eg., glyceryl-1,3-diacetoacetate or
1,2-isopropylideneglycerine-3-acetoacetate- ) and other
release-diffusion/absorption-promoting agents adapted for mucosal
delivery.
[0083] Absorption-promoting agents Useful within the invention
include a variety of compounds specifically adapted for intrarectal
use. In this context, the rate and extent of rectal drug absorption
are often lower than with oral absorption, possibly an inherent
factor owing to the relatively small surface area available for
drug uptake. In addition, the composition of rectal formulations
(solid vs liquid, nature of the suppository base) is an important
factor in the absorption process. This relationship between
formulation and drug uptake has been clearly demonstrated. Thus,
coadministration of absorption-promoting agents (eg., surfactants,
sodium salicylate, enamines) represents a key approach towards
optimizing rectal drug absorption.
[0084] Rectal drug delivery in a site- and rate-controlled manner
using suppositories, enemas, osmotic pumps, or hydrogel
formulations provides a range of options for manipulating mucosal
delivery, eg., controlling concentrations, time-release, and
immunogen-drug effects. Absorption from aqueous and alcoholic
solutions may occur very rapidly, but absorption from suppositories
is generally slower and very much dependent on the nature of the
suppository base, the use of surfactants or other additives,
particle size of the active ingredients, etc.
[0085] Accordingly, preferred formulations for administering
soluble antigens and CTL-stimulatory cytokines within the methods
of the invention are designed to optimize mucosal delivery. These
agents may thus include clyclodextrins and beta-cyclodextrin
derivatives (eg., 2-hydroxypropyl-beta-cyclodextrin and
heptakis(2,6-di-O-methyl-beta-cyclo- dextrin). These compounds,
preferably conjugated with one or more of the active ingredients
and formulated in an oleaginous base, are well documented to
enhance bioavailability in intrarectal formulations. Other
absorption-enhancing agents adapted for intrarectal delivery
include medium-chain fatty acids, including mono- and diglycerides
(eg., sodium caprate--extracts of coconut oil, Capmul), and
triglycerides (eg., amylodextrin, Estaram 299, Miglyol 810).
[0086] It is well known in the medical arts that dosages for any
one human subject depend on many factors, as well as the particular
compound to be administered, the time and route of administration
and other drugs being administered concurrently. Dosages for the
soluble antigens of the invention will vary, but can be
approximately 0.01 mg to 100 mg per administration. Dosages for the
mucosal adjuvants will be approximately 0.001 mg to 100 mg per
administration. Dosages for IL-12 will be approximately 25 .mu.g/kg
to 500 .mu.g/kg and for IFN.gamma. will be 300 KU to 30,000 KU per
administration--comparable dosages will be used for other
cytokines. For example, 3,000 KU of IFN.gamma. can be administered
to a human patient once per week. Methods of determining optimal
doses are well known to pharmacologists and physicians of ordinary
skill. Routes will be, as recited supra, mucosal, e.g., IR, IG,
IVG, IN, IPG or IT.
[0087] B.2.2 Administration of Soluble Antigens Utilizing
Expression Vectors Encoding the Soluble Antigens
[0088] An expression vector is composed of or contains a nucleic
acid in which a polynucleotide sequence encoding a peptide or
polypeptide of the invention is operatively linked to a promoter or
enhancer-promoter combination. A promoter is a trancriptional
regulatory element composed of a region of a DNA molecule typically
within 100 nucleotide pairs upstream of the point at which
transcription starts. Another transcriptional regulatory element is
an enhancer. An enhancer provides specificity in terms of time,
location and expression level. Unlike a promoter, an enhancer can
function when located at variable distances from the transcription
site, provided a promoter is present. An enhancer can also be
located downstream of the transcription initiation site. A coding
sequence of an expression vector is operatively linked to a
transcription terminating region. To bring a coding sequence under
control of a promoter, it is necessary to position the translation
initiation site of the translational reading frame of the peptide
or polypeptide between one and about fifty nucleotides downstream
(3') of the promoter. Examples of particular promoters are provided
infra. Expression vectors and methods for their construction are
known to those familiar with the art.
[0089] Suitable vectors include plasmids, and viral vectors such as
herpes viruses, retroviruses, vaccinia viruses, attenuated vaccinia
viruses, canary pox viruses, adenoviruses and adeno-associated
viruses, among others.
[0090] The application of antigen-encoding genes to the mucosal
induction of CTL in humans can utilize either in vivo or ex vivo
based approaches.
[0091] The ex vivo method includes the steps of harvesting cells
(e.g., dendritic cells) from a subject, culturing the cells,
transducing them with an expression vector, and maintaining the
cells under conditions suitable for expression of the soluble
antigen. These methods are well known in the art of molecular
biology. The transduction step is accomplished by any standard
means used for ex vivo gene therapy, including calcium phosphate,
lipofection, electroporation, viral infection, and biolistic gene
transfer. Cells that have been successfully transduced are then
selected, for example, for expression of a drug resistance gene.
The cells can then be lethally irradiated (if desired) and injected
or implanted into the subject. IL-12 and IFN.gamma. can be
administered either systemically or to the relevant mucosal surface
as discussed above (Section B.2.1).
[0092] The in viva approach requires delivery of a genetic
construct directly into the mucosa of a subject, preferably
targeting it to the cells or tissue of interest (e.g., dendritic
cells in PP). This can be achieved by administering it directly to
the relevant mucosa (e.g., IR in the case of PP). Tissue specific
targeting can also be achieved by the use of a molecular conjugate
composed of a plasmid or other vector attached to poly-L-lysine by
electrostatic or covalent forces. Poly-L-lysine binds to a ligand
that can bind to a receptor on target cells (Cristiano et al. J.
Mol. Med 73:479 (1995)). Similarly, cell specific antibodies of the
type described supra in Section B.2.1 can be bound to vectors and
thereby target them to the relevant cells of the mucosal immune
system. A promoter inducing relatively tissue or cell-specific
expression can be used to achieve a further level of targeting.
Appropriate tissue-specific promoters include, for example, the
inducible IL-2 (Thompson et al., Mol. Cell. Biol. 12: 1043 (1992)),
IL-4 (Todd et al., J. Exp. Med. 177:1663 (1993)) and IFN.gamma.
(Penix et al., J. Exp. Med. 178:483 (1993)) T-cell targeting
promoters. These promoters would allow production of the soluble
antigens in lymphoid tissue, including mucosal lymphoid tissue,
e.g., PP. Naturally, an ideal promoter would be a-dendritic cell
specific promoter.
[0093] Vectors can also be delivered by incorporation into
liposomes or other delivery vehicles either alone or
co-incorporated with cell specific antibodies, as described supra
in Section B.2.1.
[0094] DNA or transfected cells can be administered in a
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers are biologically compatible vehicles which are suitable
for administration to a human, e.g., physiological saline. A
therapeutically effective amount is an amount of the DNA of the
invention which is capable of producing a medically desirable
result in a treated animal. As is well known in the medical arts,
the dosage for any one patient depends upon many factors, including
the patient's size, body surface area, age, the particular compound
to be administered, sex, time and route of administration, general
health, and other drugs being administered concurrently. Dosages
will vary, but a preferred dosage for administration of DNA is from
approximately 10.sup.6 to 10.sup.12 copies of the DNA molecule.
This dose can be repeatedly administered, as needed. Routes of
administration will be the mucosal routes recited for soluble
antigens supra in Section B.2.1. The mucosal adjuvants, IL-12 and
IFN.gamma. can also be administered in the same combinations, by
the same routes and at the same dosages recited in Section
B.2.1.
[0095] B.3 Sources of Peptides and Polypeptides
[0096] Peptides and polypeptides used in the methods of the
invention can be obtained by a variety of means. Smaller peptides
(less than 100 amino acids long) can be conveniently synthesized by
standard chemical methods familiar to those skilled in the art
(e.g, see Creighton, Proteins: Structures and Molecular Principles,
W. H. Freeman and Co., N.Y. (1983)). Larger peptides (longer than
100 amino acids) can be produced by a number of methods including
recombinant DNA technology (see infra). Some polypeptides (e.g.,
HIV-1, gp160, CT, LT, IL-12, or IFN.gamma.) can be purchased from
commercial sources.
[0097] Polypeptides such as HIV-1, gp160, CT, IL-12, or IFN.gamma.
can be purified from biological sources by methods well-known to
those skilled in the art (Protein Purification, Principles and
Practice, second edition (1987) Scopes, Springer Verlag, N.Y.).
They can also be produced in their naturally occurring, truncated,
chimeric (as in the CLUVAC, for example), or fusion protein forms
by recombinant DNA technology using techniques well known in the
art. These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. See, for example, the techniques described in
Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Press, N.Y.,; and Ausubel et al., eds. (1989),
Current Protocols in Molecular Biology, Green Publishing
Associates, Inc., and John Wiley & Sons, Inc., N.Y.
Alternatively, RNA encoding the proteins can be chemically
synthesized. See, for example, the techniques described in
Oligonucleotide Synthesis, (1984) Gait, M. J. ed., IRL Press,
Oxford, which is incorporated by reference herein in its
entirety.
[0098] A variety of host-expression vector systems can be utilized
to express the nucleotide sequences. Where the peptide or
polypeptide is soluble, it can be recovered from: (a) the culture,
i.e., from the host cell in cases where the peptide or polypeptide
is not secreted; or (b) from the culture medium in cases where the
peptide or polypeptide is secreted by the cells, The expression
systems also encompass engineered host cells that express the
polypeptide in situ, i.e., anchored in the cell membrane.
Purification or enrichment of the polypeptide from such an
expression system can be accomplished using appropriate detergents
and lipid micelles and methods well known to those skilled in the
art. Alternatively, such engineered host cells themselves can be
used in situations where it is important not only to retain the
structural and functional characteristics of the protein, but also
to assess biological activity.
[0099] The expression systems that can be used for purposes of the
invention include but are not limited to microorganisms such as
bacteria (for example, E. coli and B. subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA
expression-vectors containing the nucleotide sequences; yeast
transformed with recombinant yeast expression vectors; insect cells
infected with recombinant viral expression vectors (baculovirus);
plant cell systems infected with recombinant viral expression
vectors (e.g., cauliflower mosaic virus, CAMV; tobacco mosaic
virus, TMV) or transformed with recombinant plasmid expression
vectors; or mammalian cells (e.g., COS, CHO, BHK, 293, 3T3)
harboring recombinant expression constructs containing promoters
derived from the genome of mammalian cells (e.g., metallothionein
promoter) or from mammalian viruses.
[0100] In bacterial systems, a number of expression vectors can be
advantageously selected depending upon the use intended for the
gene product being expressed. For example, when a large quantity of
such a protein is to be produced, e.g., for in vivo immunization,
vectors which direct the expression of high levels of fusion
protein products that are readily purified can be desirable. Such
vectors include, but are not limited to, the E. coli expression
vector pUR278, (Ruther et al., EMBO J. 2:1791 (1983)), in which the
coding sequence can be ligated individually into the vector in
frame with the lacZ coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
13:3101 (1985); Van Heeke & Schuster, J. Biol. Chem. 264:5503
(1989)); and the like. pGEX vectors can also be used to express
foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of
free glutathione. The PGEX vectors are designed to include thrombin
or factor Xa protease cleavage sites so that the cloned target gene
product can be released from the GST moiety.
[0101] In mammalian host cells, a number of viral-based expression
systems can be utilized. In cases where an adenovirus is used as an
expression vector, the nucleotide sequence of interest can be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene can then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the gene
product in infected hosts (e.g., See Logan and Shenk, Proc. Natl.
Acad. Sci. USA 81:3655 (1984)). Specific initiation signals can
also be required for efficient translation of inserted nucleotide
sequences. These signals include the ATG initiation codon and
adjacent sequences. In cases where an entire gene or cDNA,
including its own initiation codon and adjacent sequences, is
inserted into the appropriate expression vector, no additional
translational control signals may be needed. However, in cases
where only a portion of the coding sequence is inserted, exogenous
translational control signals, including, perhaps, the ATG
initiation codon, must be provided. Furthermore, the initiation
codon must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression can be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (Bittner et al., Methods in Enzymol. 153:516
(1987)).
[0102] In addition, a host cell strain can be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Appropriate cell lines or host systems can be chosen to
ensure the correct modification and processing of the foreign
protein expressed. Mammalian host cells include but are not limited
to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.
[0103] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the sequences described above can be
engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with
DNA controlled by appropriate expression control elements (e.g.,
promoter, enhancer sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells can be
allowed to grow for 1-2 days in an enriched medium, and then are
switched to a selective medium. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method can advantageously be used to engineer cell
lines which express the gene product. Such engineered cell lines
can be particularly useful in screening and evaluation of compounds
that affect the endogenous activity of the gene product.
[0104] A fusion protein can be readily purified by utilizing an
antibody or a ligand that specifically binds to the fusion protein
being expressed. For example, a system described by Janknecht et
al., Proc. Natl. Acad. Sci. USA 88:8972 (1991) allows for the ready
purification of non-denatured fusion proteins expressed in human
cell lines. In this system, the gene of interest is subcloned into
a vaccinia recombination plasmid such that the gene's open reading
frame is translationally fused to an amino-terminal tag consisting
of six histidine residues. Extracts from cells infected with
recombinant vaccinia virus are loaded onto Ni.sub.2+ nitriloacetic
acid-agarose columns and histidine-tagged proteins are selectively
eluted with imidazole-containing buffers. If desired, the histidine
tag can be selectively cleaved with an appropriate enzyme.
[0105] The following examples are meant to illustrate the invention
and not to limit it.
EXAMPLES
[0106] Materials and Methods
[0107] Human IR immunization Soluble antigens of the invention are
dissolved in a pharmaceutically acceptable carrier, e.g.,
physiological saline and administered with and without adjuvant and
with or without cytokines, e.g., IL-12 and/or IFN.gamma.. The
latter can be administered systemically or together with the
antigen. Dosages of soluble antigen are approximately 0.001 mg to
100 mg per administration. Administration can be single, or
multiple. In the case of multiple immunizations, there can, for
example, be four weekly administrations, followed (if desired) by a
booster administration several months (e.g., two, three, four, six,
eight or twelve) or several years (e.g., two, three, four, five,
ten, twenty or thirty) thereafter. Administration of the antigen
(with and without adjuvant and with and without cytokine) can be by
IR insertion of a suppository into which the immunizing composition
has been incorporated, by enema or by flexible sigmoidoscope.
[0108] CTL activation cultures At various time points after
immunization (different for each experiment), CTL activity was
assessed. BALB/c mice were sacrificed and the intestines and SP
were surgically removed. Single cell suspensions were prepared from
the organs by methods familiar to those of average skill in the
art. In the case of PP and LP, the PP were first dissected from the
outer surface of the intestine. The rest of the intestine was cut
into small fragments 1 to 3 mm square which were suspended in
phosphate buffered saline (PBS). The tissue fragment suspension was
stirred for 20 minutes at room temperature and shaken under the
same conditions to liberate the intraepithelial lymphocytes. (IEL)
from the tissue. The suspended IEL were discarded and tissue
fragments were treated with collagenase type VIII (Sigma) (30
OU/ml) dissolved in PBS for 1 hour at room temperature to release
the LP cells. PP cells were extracted from the dissected PP by the
same collagenase treatment. Cells (PP or LP) were then placed on a
discontinuous gradient containing 75% and 40% of Percoll, followed
by centrifugation at 2,000 r.p.m. for 20 minutes. Lymphocytes were
harvested from the 75%/40% interface and washed two times. Immune
cells from SP, PP, LP were cultured with 1 .mu.M P18IIIB-I10
peptide at 5.times.10.sup.6 per/milliliter in 24-well culture
plates in complete T cell medium (CTM): RPMI 1640 containing 10%
fetal bovine serum, 2 mM L-glutamine, penicillin (100 U/ml),
streptomycin (100 .mu.g/ml), and 5.times.10.sup.-5 M
2-mercaptoethanol. P18IIIB-I10 is a peptide containing the minimal
essential CTL epitope of P18IIIB (SEQ ID NO:15) and has the
sequence: RGPGRAIVTI (SEQ ID NO:16). Three days later, 10%
supernatant from con canavalin A activated spleen cells was added
as a source of interleukin-2 (IL-2). After 7 days of culture, LP
lymphocytes (LPL) were stimulated in a second 7 day culture with 1
.mu.M P18IIIB-I10 peptide (SEQ ID NO:16) together with
4.times.10.sup.6 of 3300-rad irradiated syngeneic SP cells. Immune
SP and PP cells were similarly stimulated, in vitro for two 7-day
culture periods before assay. Cytolytic activity of CTL was
measured by a 4-hour assay with .sup.51Cr labeled P815 targets
using a method familiar to those of ordinary skill in the art. For
testing the peptide specificity of CTL, .sup.51Cr-labeled P815
targets were pulsed for 2 hours with peptide at the beginning of
the assay. The percent specific .sup.51Cr release was calculated as
100.times. (experimental release-spontaneous release)/(maximum
release-spontaneous release). Maximum release was determined from
supernatants of cells that were lysed by addition of 5% Triton-X
100. As a control, spontaneous release was determined from target
cells incubated without added effector cells. Standard errors of
the mean of triplicate cultures were all <5% of the mean.
[0109] Viral plague assay Six days after IR challenge with
recombinant vaccinia virus expressing HIV-1 IIIB gp160 (vPE16),
mice were sacrificed. Their ovaries were removed, dissociated into
single cell suspensions and assayed for vPE16 titer by plating
serial 10-fold dilutions on a plate of BSC-2 indicator cells,
staining with crystal violet and counting plaques at each dilution.
The minimal detectable level of virus was 70 plaque forming units
(pfu).
Example 1
Comparison of Mucosal and Systemic-CTL Responses After Mucosal and
Systemic Immunization
[0110] Mice were immunized IR with 4 doses (on days 0, 7, 14 and
21) of the HIV-1 CLUVAC PCLUS3-18IIIB (SEQ ID NO:2) (50
.mu.g/mouse). 5 weeks to 6 months after the first dose,
antigen-specific T cells were isolated from PP, LP and SP and
tested for the presence of HIV-1 P18IIIB peptide specific CTL (FIG.
1), as described above. Closed squares show killing of P18IIIB-I10
(SEQ ID NO:16) -pulsed targets and open diamonds show killing on
unpulsed targets. IR immunization induced long-lasting protective
immune responses: antigen-specific CTL were detected in mucosal
inductive (PP) and effector (LP) sites and in a systemic site (SP)
at least 6 months after immunization. In contrast, systemic
immunization (s.c. in incomplete Freund's adjuvant) induced CTL in
the spleen but not in the mucosal immune system (i.e., PP and
LP).
Example 2
Comparison of CTL Responses with and without a Mucosal Adjuvant
[0111] BALB/c mice were immunized IR with 4 doses of the synthetic
HIV-1 CLUVAC PCLUS3-18IIIB (SEQ ID NO:2) (50 .mu.g/mouse per
immunization) alone, i.e., in the absence of adjuvant or cytokine
on days 0, 7, 14 and 21. In parallel, another group of mice was
immunized IR with PCLUS3-P18IIIB (SEQ ID NO:2) HIV-1 peptide in
combination with CT (1 .mu.g/mouse). On day 35 antigen-specific T
cells were isolated from PP, LP and SP. Immune cells from SP, PP,
or LP were cultured and tested for antigen specific CTL (FIG. 2),
as described above. Closed squares show killing of P18IIIB-I10 (SEQ
ID NO:16) pulsed targets, and open diamonds show killing of
unpulsed targets. IR administration of peptide alone induced a
significant response. The response was enhanced by the
co-administration of CT.
Example 3
CTL Induced by Mucosal Immunization Lyse Targets Expressing HIV-1
gp160 Envelope Protein
[0112] Mice were immunized IR with 4 doses (on days 0, 7, 14 and
21) of the HIV-1 CLUVAC PCLUS3-18IIIB (SEQ ID NO:2) (5 .mu.g/mouse
per immunization) in the presence of CT (1 .mu.g/mouse). on day 35,
antigen-specific T cells were isolated from PP, LP and SP. Immune
cells from SP, PP, or LP were cultured as described above.
Cytolytic activity of CTL was measured using a standard .sup.51Cr
release assay (FIG. 3).
[0113] Three different cell lines were used as target cells: 15-12
cells, 3T3 18 Neo BALB/c cells and P815 cells. 15-12 cells are
BALB/c 3T3 fibroblasts transfected with a vector encoding HIV-1
gp160 (Takahashi et al. Proc. Natl. Acad. Sci. USA 85:3105 (1988));
3T3 18 Neo BALB/c cells are BALB/c 3T3 fibroblasts transfected with
an expression vector containing a Neor gene but no gp160 gene; and
P815 cells are untransfected cells that present antigenic peptides
added to the culture. CTL lysis of gp160 expressing 15-12 cells
(closed squares in right panels) was compared to that of control
gp160 non-expressing 3T3 18 Neo BALB/c cells (open diamonds right
panels). P815 target cells (left panel) were tested in the
presence.(closed squares) or absence (open diamonds) of P18IIIB-I10
peptide (SEQ ID NO:16) (1 .mu.M). The percent specific 51Cr release
was calculated as described above. CTL induced by IR immunization
killed target cells either endogenously expressing HIV-gp160 or
pulsed with P18IIIB peptide (SEQ ID NO:15).
Example 4
CTL Induced by IR Immunization are IL-12 Dependent
[0114] BALB/c mice were immunized IR with 4 doses (on days 0, 7, 14
and 21) of CLUVAC PCLUS3-18IIIB (SEQ ID NO:2) (50 .mu.g/mouse per
immunization) in combination with CT (1 .mu.g/mouse). One day
before and one day after immunization with peptide mice were
treated intraperitoneally (i.p.) with anti-IL-12 antibody (0.5
mg/per injection: 4 mg/mouse total dose (FIG. 4, right panels) or
were untreated (FIG. 4, left panels). On day 35, antigen-specific T
cells were isolated from PP, LP and SP. Immune cells from SP, PP,
or LP were, cultured as described above. Cytolytic activity of CTL
was measured (FIG. 4) as described above. For testing the peptide
specificity of CTL, 51Cr labeled P815 targets were pulsed with
peptide P18IIIB-I10 (SEQ ID NO:16) at the beginning of the assay
(closed squares), or (as a control) left unpulsed i.e., without
peptide (open diamonds). Induction of both mucosal and systemic CTL
responses by IR immunization was IL-12-dependent, as shown by
inhibition of induction of CTL in mice treated i.p. with anti-IL-12
antibody.
[0115] BALB/c mice were immunized IR on days 0, 7, 14 and 21 with
either composition A containing the synthetic HIV-1 CLUVAC
PCLUS3-18IIIB (SEQ ID NO:2) ( 50 .mu.g/mouse per immunization), CT
(10.mu.g/mouse per immunization), and recombinant IL-12 (1
.mu.g/mouse per immunization), or composition B containing the
synthetic HIV-1 CLUVAC PCLUS3-18IIIB (SEQ ID NO:2) (50 .mu.g/mouse
per immunization), and CT (10 .mu.g/mouse per immunization), on day
35, antigen-specific T cells were isolated from PP and SP. Immune
cells from PP (FIG. 9A) and SP (FIG. 9B) were separately cultured
and tested for antigen specific CTL, as described above. FIG. 9A
shows killing of peptide P18IIIB-I10 (SEQ ID NO:16) pulsed target
cells by effector cells from mice immunized with composition A
(i.e., antigen with IL-12) (open squares), and FIG. 9B shows
killing of the P18IIIB-I10 (SEQ ID NO:16) pulsed target cells by
effector cells from mice immunized with composition B (i.e.,
antigen without IL-12) (open diamonds). In both FIGS. 9A and 9B,
control data was obtained using the two effector populations tested
on un pulsed targets. The enhanced CTL responses of both PP (FIG.
9A) and SP (FIG. 9B) effector cells from mice immunized IR with
composition A compared to the responses elicited by IR immunization
with composition B indicate that co-administration of IL-12
augments mucosal and systemic CTL responses. These data both
provide independent evidence for the role of IL-12 in eliciting
mucosal (and systemic) immunity by mucosal administration of
antigens and confirm the findings of the antibody inhibition data
described above.
Example 5
Intrarectal Peptide Immunization Protects Against Mucosal Challenge
with EIV-gp160 Expressing Recombinant Vaccinia Virus
[0116] Groups of 5 mice were immunized IR with 4 doses of the HIV-1
CLUVAC PCLUS3-18IIIB (SEQ ID NO:2) (50 .mu.g/mouse per
immunization) on days 0, 7, 14 and 21 in combination with CT. On
day 35, mice were challenged IR with 2.5.times.10.sup.7 pfU of
vaccinia virus expressing gp160IIIB (vPE16). After 6 days, the mice
were killed and their ovaries assayed for the presence of virus
(FIG. 5) as described above. The left bar of FIG. 5 shows virus
titer in the ovaries of unimmunized mice and the right bar shows
virus titer in the ovaries of immunized mice. IR immunization with
PLCUS3-18IIIB (SEQ ID NO:2) protected mice against IR challenge
with vPE16 as shown by a 4.5-log reduction in viral pfu in ovaries
compared to unimmunized animals (p<0.005).
Example 6
Comparison of Systemic CTL Responses Six Months After IR and IN
Immunization
[0117] Mice were immunized IR or IN with 4 doses (on days 0, 7, 14
and 21) of the HIV-1 CLUVAC PCLUS3-18IIIB (SEQ ID NO:2) (50
.mu.g/mouse). Six months after the first dose, antigen-specific T
cells were isolated from SP and tested for the presence of HIV-1
P18IIIB-I10 (SEQ ID NO:16) peptide specific CTL (FIG. 6A and 6B) as
described above. Closed squares show killing of P18IIIB-I10 (SEQ ID
NO:16)-pulsed targets and open diamonds show killing of unpulsed
targets. The level of CTL activity induced by IR immunization (FIG.
6A) was higher than that induced by IN immunization (FIG. 6B).
These data indicate that IR immunization induced potent,
long-lasting, antigen-specific splenic immune responses.
[0118] To evaluate different routes of immunization, mice were
immunized mucosally (IR, IN, IG) or systemically (SC) with 4 doses
(on days 0, 7, 14 and 21) of the HIV-1 CLUVAC PCLUS3-18IIIB (SEQ ID
NO:2) (50 .mu.g/mouse). Thirty-five days after the first dose,
antigen-specific T cells were isolated from PP and SP and tested
for the presence of HIV-1 P18IIIB-I10 (SEQ ID NO:16)
peptide-specific CTL as described above (FIG. 7). Closed bars show
killing of P18IIIR-I10 (SEQ ID NO:16) pulsed targets and open bars
show killing on unpulsed targets. The level of CTL activity induced
by IR immunization was significantly higher in inductive mucosal
tissue (PP) and much higher in systemic immunological tissue (SP)
than that induced by the other mucosal routes (IN and IG). CTL
activity was detected in both PP and SP after immunization via all
three mucosal routes. Systemic immunization (SC) only resulted in
significant CTL activity in SP.
Example 7
CTL Induced by IR Immunization are IFN.gamma. Dependent
[0119] Wild-type BALB/c mice and BALB/c mice lacking the ability to
produce functional IFN.gamma. (IFN.gamma..sup.-/- mice) (Dalton et
al., Science 259:1739 (1993); Tishon et al., Virology 212:244
(1995)) were immunized IR with 4 doses (on days 0, 7, 14 and 21) of
CLUVAC PCLUS3-18IIIB (SEQ ID NO:2) (50 .mu.g/mouse per
immunization) in combination with CT (1 .mu.g/mouse). On day 35,
antigen-specific T cells were isolated from PP, LP and SP. Immune
cells from SP, PP, or LP of wild type BALB/c and IFN.gamma..sup.-/-
mice were cultured as described above. Cytolytic activity of CTL
was measured (FIG. 8A and FIG. 8B) as described above. The peptide
specificity of CTL was tested as follows: .sup.51Cr labeled P815
targets were pulsed with peptide P18IIIB-I10 (SEQ ID NO:16) at the
beginning of the assay (solid bars), or (as a control) left
unpulsed i.e., without peptide (open bars). Induction of both
mucosal and systemic CTL responses by IR immunization was
IFN.gamma. dependent, as shown by the lack of detectable CTL
activity in SP, PP, and LP cells from IFN.gamma..sup.-/- mice (FIG.
8B) and potent CTL activity in SP, PP, and LP cells from wild-type
BALB/c mice (FIG. 8A).
[0120] Summarizing the foregoing results, mucosal, administration
of an antigenic peptide to mice results in the induction of a
protective CTL response detectable in both the inductive (Peyer's
patch (PP)) and effector (lamina propria (LP)) sites of the
intestinal mucosal immune system, as well as in systemic lymphoid
tissue, i.e., spleen (SP). The mucosal CTL response is enhanced by
co-administering the mucosal adjuvant, cholera toxin (CT) with the
antigenic peptide, is inhibited by antibody that specifically binds
to (thereby neutralizing the activity of) interleukin-12 (IL-12),
and is not detectable in mice lacking the ability to produce
functional interferon-.gamma. (IFN.gamma.). Furthermore, including
IL-12 in the composition of antigenic peptide and CT used for IR
immunization resulted in enhanced PP and SP CTL responses relative
to those obtained by IR immunization with antigenic peptide, CT and
no IL-12. IR immunization with the viral peptide resulted in
protection from viral infection upon subsequent IR challenge with
the appropriate virus.
Example 8
Comparison of Different HIV Vaccine Peptides for use in Eliciting
Viral Mucosal Protection
[0121] Because of the variability of the V3 loop of HIV, further
studies were conducted comparing two cluster peptide constructs
using the V3 loop and incorporating the CTL epitope P18 from strain
IIIB (Ratner et al., Nature 313:277-284, (1985)) or MN (Gurgo et
al., Virology 164:531-536, (1998)) of HIV-1. These studies were
conducted as an exemplary analysis to demonstrate that HIV vaccine
cluster peptide constructs can be prepared from different HIV
strains and screened in side-by-side assays to optimize induction
of mucosal CTL immunity.
[0122] Animals For each of the following Examples, female BALB/c
mice were purchased from Frederick Cancer Research Center
(Frederick, Md.). IFN-.gamma..sup.-/- mice were purchased from
Jackson Laboratories (Bar Harbor, Me.). Mice used in this study
were 6-12 weeks old. The IFN-.gamma..sup.-/- mice were maintained
in a specific pathogen-free microisolator environment.
[0123] Immunization Mice were immunized with 4 doses of the
synthetic HIV peptide vaccine construct PCLUS3-18IIIB (Ahlers et
al., J. Immunol. 150:5647-5665, (1993)) (50.COPYRGT..mu.g/mouse for
each immunization) on days 0, 7, 14 and 21 in combination with
cholera toxin (CT) (10 .mu.g/mouse) (List Biological Laboratories,
Campbell, Calif.) by intrarectal administration. For subcutaneous
immunization, incomplete Freund's adjuvant was used. rm IL,-12 (a
generous gift of Genetics Institute, Inc., Cambridge, Mass.) was
delivered either intraperitoneally (IP) (1 .mu.g) or intrarectally
(1 .mu.g) mixed with DOTAP (Boehringer Mannheim), a cationic
lipofection agent, along with the peptide vaccine.
[0124] Cell purification Five weeks to 6 months after the first
dose, antigen-specific T cells were isolated from Peyer's patches
(PP), lamina propria (LP) and the spleen (SP). The Peyer's patches
were carefully excised from the intestinal, wall and dissociated
into single cells by use of collagenase type VIII, 300 U/ml (Sigma)
as described, Mega et al., Int. Immunol. 3:793-805, (1991). Our
data showed that most PP CD3.sup.+ T cells isolated from normal
mice were CD4.sup.+, while CD3.sup.+CD8.sup.+ T cells were less
frequent. Further, collagenase did not alter expression of CD3,
CD4, or CD8 on splenic T cells treated with this enzyme. Lamina
propria lymphocyte (LPL) isolation was performed as described, Mega
et al., Int. Immunol. 3:793-805, (1991). The small intestines were
dissected from individual mice and the mesenteric and connective
tissues carefully removed. Fecal material was flushed from the
lumen with un-supplemented medium (RPMI 1640). After the PP were
identified and removed from the intestinal wall, the intestines
were opened longitudinally, cut into short segments, and washed
extensively in RPMI containing 2% fetal bovine serum (FBS). To
remove the epithelial cell layer, tissues were placed into 100 ml
of 1 mM EDTA and incubated twice (first for 40 min and then for 20
min) at 37.degree. C. with stirring. After the EDTA treatment,
tissues were washed in complete RPMI medium for 10 min at room
temperature and then placed into 50 ml of RPMI containing 10% FCS
and incubated for 15 min at 37.degree. with stirring. The tissues
and medium were transferred to a 50 ml tube and shaken vigorously
for 15 seconds, and then the medium containing epithelial cells was
removed. This mechanical removal of cells was repeated twice more,
using fresh medium each time, in order to completely remove the
epithelial cell layer. Histologic examination revealed that the
structure of the villi and lamina propria were preserved. To
isolate LPL, tissues were cut into small pieces and incubated in
RPMI 1640 containing collagenase type VIII 300 U/ml (Sigma) for 50
min at 37.degree. C. with stirring. Supernatants containing cells
were collected, washed and then re-suspended in complete RPMI 1640.
This collagenase dissociation procedure was repeated two times and
the isolated cells pooled and washed again. Cells were passed
through a cotton-glass wool column to remove dead cells and tissue
debris and then layered onto a discontinuous gradient containing
75% and 40% Percoll (Pharmacia Fine Chemicals, Pharmacia Inc.,
Sweden). After centrifugation (4.degree. C., 600 g, 20 min), the
interface layer between the 75% and 40% Percoll was carefully
removed and washed with incomplete medium. This procedure provided
>90% viable lymphocytes with a cell yield of
1.5-2.times.10.sup.6 lymphocytes/mouse. The SP were aseptically
removed and single cell suspensions prepared by gently teasing them
through sterile screens. The erythrocytes were lysed in
Tris-buffered ammonium chloride and the remaining cells washed
extensively in RPMI 1640 containing 20/o FBS.
[0125] Cytotoxic T lymphocyte assay Immune cells from SP, PP, LP
were cultured at 5.times.10.sup.6 per/milliliter in 24-well culture
plates in complete T cell medium (CTM): RPMI 1640 containing 10%
FBS, 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100
.mu.g/ml), and 5.times.10.sup.-5 M 2-mercaptoethanol. Three days
later we added 10% concanavalin A supernatant-containing medium as
a source of IL-2. LPL were studied after 7 days stimulation with
I.mu.M P18IIIB-I10 peptide together with 4.times.10.sup.6 of
3300-rad irradiated syngeneic spleen cells. SP and PP cells were
stimulated in vitro similarly for one or two 7-day culture periods
before assay. Cytolytic activity of CTL lines was measured by a
4-hour assay with .sup.51Cr labeled targets. Two different cell
lines were used as a target cells: 1) 15-12 cells, Takahashi et
al., Proc. Natl. Acad. Sci. USA 85:3105-3109 (1988) (BALB/c 3T3
fibroblasts transfected with HIV-1IIIB gp160 and endogenously
expressing HIV gp106), compared with 18 Neo BALB/c 3T3 fibroblasts
transfected with Neo.sup.R alone as a control, and 2) P815 targets
tested in the presence or absence of I10 peptide (I.mu.M). For
testing the peptide specificity of CTL, .sup.51Cr labeled P815
targets were pulsed for 2 hours with peptide at the beginning of
the assay. The percent specific .sup.5Cr release was calculated as
100.times.(experimental release-spontaneous release)/(Maximum
release-spontaneous release). Maximum release was determined from
supernatants of cells that were lysed by addition of 5% Triton-X
100. Spontaneous release was determined from target cells incubated
without added effector cells.
[0126] Vaccinia virus Recombinant vaccinia virus vPE16 expresses
the HIV-1 gp160 gene from isolate IIIB (BH8) (Earl et al., J.
Virol. 64:2448-2451 (1990)). Expression is directed by the compound
early/late P7.5 promoter. Two copies of the sequence T5NT, which
serves as a transcription termination signal for early vaccinia
virus genes, are present in the IIIB gp 160 gene. Both of these
have been altered in vPE16, so as to retain the original coding
sequence and allow early transcription of the gene. The virus,
vSC8, is used as a negative control without gp160 (Chakrabarti et
al., Mol. Cell Biol. 5:3403-3409 (1985)). Both vPE16 and VSC8
express beta-galactosidase.
[0127] Determination of virus titer in the ovary On day 35 or 6
months after cluster peptide HIV vaccine immunization, mice were
challenged intrarectally with 2.5.times.10.sup.7 or
5.times.10.sup.7 pfu of vaccinia virus expressing gp160IIIB
(vPE16). Six days after the challenge with recombinant vaccinia
virus expressing HIV-gp160, the mice were killed and ovaries were
removed, homogenized, sonicated, and assayed for vPE16 titer by
plating serial 10-fold dilutions on a plate of BSC-2 indicator
cells staining with crystal violet and counting plaques at each
dilution. The minimal detectable level of virus was 100 pfu.
[0128] To compare different HIV vaccine cluster peptide constructs,
BALB/c mice were immunized intrarectally with peptide
(PCLUS3-18IIIB or PCLUS3-18MN) in the presence of CT as a mucosal
adjuvant weekly for four weeks (on days 0, 7, 14, and 21). Mice
were studied either two weeks later (day 35) or at six months for
memory CTL responses in the Peyer's Patches (PP) or spleen (SP). IR
immunization with both peptides PCLUS3-18IIIB or PCLUS3-18MN in
combination with CT induces a P18-specific CTL response in the
intestinal PP (FIG. 10, panel A) and in the spleen (FIG. 10, panel
B).
[0129] However, the level of CTL response after IR immunization
with PCLUS3-18MN was significantly lower than after IR immunization
with PCLUS3-18IIIB. The difference may reflect a higher affinity of
the minimal 10-mer P18IIIB-I10, compared to P18MN-T10, for
H-2D.sup.d (Takahashi et al., Science 246:118-121 (1989); Takeshita
et al., J. Immunol. 154:1973-1986 (1995)). Also, much higher
production of IFN-.gamma. by mucosal P18IIIB-specific CD8.sup.+ CTL
was observed compared to P18-MN-specific CTL after stimulation with
specific peptide in vitro for 48 hours. When tested on 15-12
gp160IIIB-transfected fibroblast targets endogenously expressing gp
160IIIB, the CTL elicited by immunization with PCLUS3-18IIIB also
killed these targets. On the basis of these observations, the
PCLUS3-18IIIB construct was used in, the protection experiments
described below.
Example 9
Mucosal Immunization of Mice with Cluster Peptide Construct
Provides Longlasting Protection from Infection with Recombinant
Vaccinia Virus Expressing HIVg160
[0130] In the foregoing Examples, the ability of the mucosal immune
responses induced by the HIV cluster peptide vaccine to protect
against virus challenge via a mucosal route is demonstrated. To
determine the specificity of this protection for recombinant
protein HIV-1 IIIB gp160, IR immunized mice were challenged on day
35 after the start of immunization by IR infusion with vaccinia
virus expressing HIV-1 IIIB gp 160 (vPE16), or with control
vaccinia virus expressing .beta.-galactosidase (vSC8). Unimmunized
animals challenged with vPE16 or vSC8 served as controls. Six days
after the challenge, mice were sacrificed and the ovaries were
removed and assayed for vaccinia titer (6 days after infection with
vaccinia, the ovaries contain the highest titer of virus).
[0131] IR immunization with the synthetic HIV peptide vaccine
protected mice against an IR challenge with vaccinia virus
expressing HIV-1 IIIB gp160 compared to unimmunized controls, but
did not protect against IR challenge with vaccinia virus expressing
only an unrelated protein, .beta.-galactosidase (FIG. 11). Thus,
the protection was specific for virus expressing HIV-1 gp160, and
any nonspecific inflammatory response induced by the peptide
infusion intrarectally was not sufficient to protect against viral
challenge two weeks after the last dose of the immunization.
[0132] Although the presence of mucosal memory CTL precursors was
observed, requiring restimulation in vitro for activity 6 months
after IR immunization (Belyakov et al., Proc. Nati. Acad. Sci.
95:1709-1714 (1998)), the strength and duration of protection
remained unclear. To resolve this question, the IR immunized mice
were challenged 6 months after the start of immunization with
PCLUS3-18IIIB by IR administration with vaccinia virus expressing
HIV-1 IIIB (vPE16). This study showed that, even 6 months after HIV
cluster peptide immunization, BALB/c mice exhibit protection
against recombinant HIV-vaccinia challenge (FIG. 12).
Example 10
Protection of Mice Against Mucosal Viral Challenge is Mediated by
CD8.sup.+ CTL in the Mucosal Site
[0133] To determine the immune mechanism responsible for protection
against mucosal challenge with virus expressing HIV gp160, mice
were treated IP with 0.5 mg monoclonal anti-CD8 antibody (clone
2.43, NIH, Frederick, Md.) one day before and after each of the
four immunizations and also two days before and three days after
the challenge with vPE16. This treatment led to a significant
inhibition of the protection against mucosal challenge with vPE16
(FIG. 13). Thus, protection in the mucosal site against the virus
expressing HIV-1 gp160 is mediated by CD8.sup.+ lymphocytes.
[0134] Because the HIV peptide constructs disclosed herein elicit
both strong mucosal and systemic MHC class I restricted CD8.sup.+
CTL responses (Belyakov et al., Proc. Nati. Acad. Sci. 95:1709-1714
(1998)), the role of these responses in mediating protection were
further investigated. Because SC immunization with peptide vaccine
elicits splenic but not mucosal CTL, whereas IR immunization
elicits both (FIG. 14), SC and IR immunizations can be compared to
determine whether systemic CTL are sufficient to protect against
mucosal challenge, or whether local mucosal CTL are necessary.
Accordingly, mice were immunized with PCLUS3-18IIIB plus IFA by the
SC route or with PCLUS3-18IIIB and CT by the IR route on days 0, 7,
14 and 21, and compared these. On day 35 after the start of
immunization, these groups of mice as well as unimmunized control
mice were challenged by IR administration of vaccinia virus
expressing-HIV-1 gp160 (vPE16). Finally, six days after the
challenge, mice were sacrificed and their ovaries assayed for viral
titer.
[0135] SC immunization with PCLUS3-18IIIB did not protect mice
against mucosal challenge with vPE16, whereas IR immunization with
the same peptide did protect (FIG. 14B). Thus, protection against
mucosal challenge with virus expressing HIV-1 gp160 can be induced
only by mucosal immunization of mice, and correlates with local
mucosal CTL activity, not with splenic CTL activity. On this basis
one can conclude that the CD8.sup.+ CTL-mediated protection from
mucosal challenge with recombinant vaccinia expressing HIV-1 gp160
requires local mucosal CD8.sup.+ CTL, whereas a systemic CTL
response is not sufficient.
Example 11
Cytokine Dependence and Enhancement of Protection by Local
Administration of IL-12 with the Vaccine
[0136] Induction of mucosal CTL by peptide vaccine is dependent on
endogenous IL-12 in that it can be blocked by in vivo treatment of
mice with anti-IL-12 (Belyakov et al., Proc. Nati. Acad. Sci.
95:1709-1714, (1998)). To further define the role of IL-12 in the
CTL response and protection, BALB/c (H-2D.sup.d) mice were treated
by the IP route with 1 .mu.g of the rmIL-12 each day of the IR
immunization with PCLUS3-18IIIB (50 .mu.g/mice). This treatment did
not lead to significant changes in the HIV-specific CTL activity in
either mucosal or systemic sites (FIG. 15). However, when the mice
were treated with the rmIL-12 (1 .mu.g)+DOTAP intrarectally
together with peptide, we found a significant increase in the CTL
level in both mucosal and systemic sites 35 days after the start of
immunization (FIG. 15).
[0137] In view of the above results, the possibility that rmIL-12
administered at the local site and time of mucosal immunization
might increase protection against mucosal challenge with vaccinia
virus expressing HIV-1 gp160 was investigated. To address this
question, BALB/c mice were immunized with the rmIL-12+DOTAP
intrarectally together with peptide. The mice were then challenged
mice on day 35 after the start of immunization by IR administration
of vaccinia virus expressing HIV-1IIIB gp160. In this study, twice
the dose of challenge virus was used, whereby the unimmunized mice
had a titer of several times 10.sup.10 rather than several times
10.sup.8 seen in the previous Examples using a lower challenge
dose. Nevertheless, the immunized mice showed a reduction of
greater than 4 logs in virus titer, as had been seen in the earlier
experiments (FIG. 16, bar 2).
[0138] Importantly, IR immunization with the synthetic HIV peptide
vaccine plus rmIL-12 protected mice against an IR challenge with
this gp-160-recombinant vaccinia virus even more effectively than
after the IR immunization with peptide alone (6-log reduction in
viral pfu versus 4-log reduction, p<0.05) (FIG. 16, bar 3 versus
bar 2).
[0139] As the induction of mucosal CD8.sup.+ CTL is strongly
dependent on IL-12 and IFN-.gamma. (Belyakov et al., Proc. Nati.
Acad. Sci. 95:1709-1714 (1998)), further studies were undertaken
herein to determine which cytokine acts directly for the generation
of mucosal CTL, and which acts through a secondary mechanism. To
address this question, IFN.gamma..sup.-/- mice (BALB/c background)
and conventional BALB/c mice were treated with the rmIL-12 (I
.mu.g/mouse)+DOTAP IR together with peptide. Mucosal treatment of
IR-immunized IFN.gamma..sup.-/- mice with rmIL-12 did not lead to
the induction of mucosal or systemic CTL (FIG. 17). It thus appears
that IL-12 cannot act directly in the induction of mucosal
CD8.sup.+ CTL in the absence of IFN.gamma..
[0140] In summary, the foregoing Examples incorporate a novel viral
challenge system in which recombinant vaccinia virus expressing
HIV-1 gp160 s used as a surrogate for HIV-1, since we cannot infect
the mice with HIV-1. Importantly, in this system, neutralizing
antibodies to gp160 cannot protect against recombinant vaccinia
expressing gp160, because the virus does not incorporate gp160 in
the virus particle but expresses it only in the infected cell.
Thus, the protective immune response must be directed at the
infected cell.
[0141] The results herein demonstrate that the protective response
is completely dependent on CD8.sup.+ cells., by the abrogation of
protection after in vivo depletion of CD8.sup.+ cells. Thus, the
results show unequivocally that it is CD8.sup.+ CTL (whether via
lytic activity or via secretion of cytokines or other soluble
factors) that protect. Since it has been shown that protection
against vaccinia infection can be mediated by interferon-.gamma.
which is secreted by CD8.sup.+ CTL in response to antigen
stimulation (Harris et al., J. Virol. 69:910-915 (1995)), it is
possible that the mechanism involves local secretion of this
cytokine by the CTL rather than lysis of infected cell. By either
mechanism, the CTL are responsible for mediating protection.
[0142] However, since the mucosal immunization induces CTL in both
the local mucosal site and the spleen, this result does not
distinguish which CTL are responsible for protection. To address
this important question, the present Examples take advantage of the
fact that subcutaneous immunization with the peptide vaccine
induces systemic CTL in the spleen at a level at least as high as
that induced by mucosal immunization, but does not induce mucosal
CTL. Splenic CTL resulting from both immunization routes kill
target cells endogenously expressing HIV-1 gp160. Thus, if systemic
CTL against this epitope protected against mucosal challenge, then
the subcutaneously immunized mice would have been expected to be
protected.
[0143] However, the subcutaneously immunized mice showed no
evidence of protection against mucosal challenge. Thus, the
protection correlated with CTL activity in the local mucosal sites,
Peyer's patches and lamina propria, not with CTL activity in the
spleen. The protection was not only mediated by CTL, but also
required CTL in the local mucosal site of challenge. Systemic CTL
were not sufficient.
[0144] Protection mediated by local mucosal CTL appears
independently sufficient to mediate a protective immune response.
This conclusion is supported by the observation that a two-log
reduction in viral pfu occurs in the ovary even at day 2 after
mucosal viral challenge (from 2.3.times.10.sup.6 pfu in unimmunized
mice to 3.34.times.10.sup.4 pfu in IR immunized mice), before much
replication could have occurred in the ovary. This finding
indicates that the reduction in titer in the ovary reflects a
reduction in the amount of virus that can escape the initial
mucosal site of infection. In addition, the enhancement of CTL
activity and protection by rmIL-12 depended on local mucosal
administration of the cytokine, not systemic administration.
[0145] One possible difference between CTL induced by mucosal
versus systemic immunization is that the CTL resulting from the SC
immunization do not have homing receptors for the GI mucosa, as
evidenced by the fact that they are note detected in the lamina
propria or Peyer's patches.
[0146] The present disclosure represents the first demonstration of
protection against GI mucosal challenge requiring local mucosal CTL
at the site of challenge. These results have important implications
for the development of protective vaccines against mucosal exposure
to viruses.
[0147] In addition to these results, the Examples provided herein
also demonstrate a surprising persistence, not only of memory CTL
in the mucosa, but also of protective immunity against mucosal
viral challenge. Factors controlling CTL memory, and the role of
persistent antigen in maintaining memory CTL, represent another
issue that has been of interest for some time (Ahmed and Gray,
Science 272:54-60 (1996); Kundig et al., Proc. Natl. Acad. Sci.
U.S.A. 93:9716-9723 (1996); Ehl et al., Eur. J. Immunol.
27:3404-3413 (1997)), but has been little studied in the context of
mucosal immune responses and protection. In studies of systemic
immunity, it was shown (Slifka et al., Blood 90:2103-2108, (1997))
that after infection with lymphocytic choriomeningitis virus
(LCMV), CTL memory responses were present in the bone marrow for at
least 325 days, indicating long-term persistence of antiviral T
cells at this site. While the antigen-specific CD8.sup.+ T cell
number, dropped precipitously following viral clearance,
substantial numbers persisted for the life of the mouse
(Muraii-Krishna et al., Immunity 8:177-187, (1998)). Upon
rechallenge with LCMV, there was rapid expansion of memory
CD8.sup.+ T cells.
[0148] These results indicate that systemic infection with virus
can lead to long-term memory and protection. In the case of mucosal
CTL memory, it was shown that memory CTL remained at the mucosal
site longer if the immunization was via the mucosal route
(Gallichan and Rosenthal, J. Exp. Med. 184:1879-1890 (1996)), but
the duration of protection by such mucosal CTL was not studied. The
ability of mucosal memory CTL to protect will depend in part on the
rapidity by which they can expand and be activated after virus
exposure, which may relate to the level of virus replication in the
mucosal site. For these reasons, it is important to determine the
duration of mucosal protection dependent on local mucosal CD8.sup.+
CTL. The results herein show persistence of CTL even six months
after mucosal immunization with the peptide vaccine construct,
without additional reimmunization beyond the initial three-week
course. This response is accompanied by protection against mucosal
challenge with vaccinia virus expressing gp160.
[0149] This latter result is particularly striking because the
exemplary immunogen is a peptide administered without any depot
form of adjuvant that would maintain the presence of antigen for
extended periods. It would be expected that free peptide delivered
to the lumen of the gut, or even after transport by mucosal cells,
would have a very short half-life. Therefore, either memory CTL can
persist in the mucosa at levels sufficient to mediate protection in
the absence of persistent antigen, or antigen must persist locally
in some cell-bound form, perhaps on N4HC molecules of dendritic
cells. Yet another possibility is that the peptide crossreacts with
one of the antigens in the mucosal flora and that these
crossreactive antigens maintain the memory CTL.
[0150] Protection against viral infection by CTL involves more than
just the number of CTL induced. Quality of CTLs is as important for
in vivo protection as quantity. Previous reports have shown that
high-avidity P18IIIB-specific CTLs adoptively transferred into
severe combined immunodeficient (SCID) mice were 100- to 1000-fold
more effective at viral clearance than the low-avidity CTLs
specific for the same peptide-MHC complex, despite the fact that
all-CTL lines lysed virus infected targets in vitro
(Alexander-Miller et al., Proc. Natl. Acad. Sci. U.S.A.
93:4102-4107 (1996)). Thus, the CTL induced by mucosal immunization
with the synthetic peptide vaccine must be present not only in
sufficient quantity to protect, but also must be of high enough
avidity to protect.
[0151] Other aspects of the present disclosure enable optimization
of induction of the CTL response and protective immunity. Delivery
of certain cytokines such as IL-12 at the site of antigen
immunization systemically have been shown to enhance systemic CTL
responses (Ahlers et al., J. Immunol. 159:3947-3958 (1997); Iwasaki
et al., J. Immunol. 159:4591-4601 (1997); Xiang and Ertl, Immunity
2:129-135 (1995); Irvine et al., J. Immunol. 156:238-245 (1996)).
However, no comparable studies have been conducted for mucosal CTL
responses.
[0152] In the model system disclosed herein, induction of mucosal
CTL is dependent on endogenous production of IL-12 by the mouse,
because it could be inhibited by anti-IL-12 antibody given in vivo
before and after each immunization. In the present Examples, IL-12
co-administered with the antigen intrarectally significantly
enhanced CTL induction, and also increased protection against
intrarectal vaccinia viral challenge. However, it was striking that
IL-12 delivered systemically (i.p.) did not enhance CTL induction
either in the systemic sites (eg. spleen) or in the mucosa. This
difference may be due to the short half life of IL-12 delivered
systemically, which prevented it from surviving long enough to get
to the sites of CTL induction. Therefore, for mucosal CTL induction
as well as for systemic CTL induction, it is important to deliver
the cytokine directly to the site of antigen administration and CTL
induction.
[0153] Enhancement of CTL induction in the mucosa with recombinant
IL-12 is a useful strategy for mucosal vaccine development. In this
context, small doses given locally in the mucosal sites are not
likely to have the global toxicity that has been associated with
systemic administration of this cytokine.
[0154] The results herein further show that enhancement of the CTL
response in vivo by rmIL-12 is dependent on interferon-.gamma., as
no enhancement was observed in IFN.gamma..sup.-/- mice. At least
two mechanisms can explain this:result. First, IL-12 may be acting
through its well-defined ability to induce production of
IFN-.gamma. (Trinchieri, G., Blood 94:4008-4027 (1994)), which then
acts directly on CTL precursors. Alternatively, since IFN-.gamma.
is important for expression of the IL-12 receptor (Szabo, et al.,
J. Exp. Med. 185:817-824 (1997)) IL-12 may act directly on CTL, but
may not be able to act in IFN.gamma..sup.-/- mice because of the
lack of IL-12R expression.
[0155] There is evidence that CTL activity may play a role in
protective immunity in humans against HIV-1 (reviewed in
(Rowland-Jones et al., Adv. Immunol. 65:277-346 (1997); Yang, O. O.
and B. D. Walker, Adv. Immunol. 66:273-311 (1997); Berzofsky and
Berkower, AIDS 9(A):S 143-S 157 (1995)). Cell-mediated immunity to
HIV-1 has been demonstrated in uninfected high risk adults (Pinto
et al., J. Clin. Invest. 96:867-876, (1995); Rowland-Jones et al.,
Nature Medicine 1:59-64, (1995)) and in uninfected children born to
infected mothers (Clerici et al., AIDS 7:1427-1433 (1993);
Luzuriaga and Sullivan, J. Cell. Blochem. Supplement
17E:98.(Abstract) (1993)). CTL activity has been correlated with
low viral load and long-term non-progressor status in some infected
individuals (Cao et al., N. Engl. J. Med. 332:201-208 (1995)). CTL
have also been associated with recovery from acute HIV or SIV
infection (Yasutomi et al., J. Virol. 67:1707-1711 (1993); Reimann
et al., J. Virol. 68:2362-2370 (1994); Koup et al. J. Virol.
68:4650-4655 (1994); Borrow et al., Nature Medicine 3:205-211,
(1997)). Induction of escape mutations by CTL implies that the CTL
are eliminating the bulk of the wild type virus (Borrow et al.,
Nature Medicine 3:205-211 (1997); Phillips et al., Nature
354:453-459 (1991); Nowak et al., Nature 375:606-611 (1995);
Goulder et al., Nature Medicine 3:212-217 (1997); Couillin et al.,
J. Exp. Med. 180:1129-1134 (1994); Koenig et al., Nature Medicine
1:330-336 (1995)). In addition, in an SIV study, protection was
associated with a particular simian class I MHC molecule (Heeney et
al., J. Exp. Med. 180:769-774, (1994)). CD8.sup.+ cells have been
demonstrated to suppress replication of human and simian
lentiviruses in autologous CD4 cells by a non-lytic mechanism
involving soluble factors synthesized and released by activated
CD8.sup.+ cells (Walker et al., Science 234:1563-1566 (1986);
Tsubota et al., J. Exp. Med. 169:1421-1434 (1989); Walker and Levy,
Immunology 66:628-630 (1989); Mackewicz et al., J. Clin. Invest.
87:1462-1466, (1991); van Kuyk et al., J. Immunol. 153:4826-4833
(1994)). Such soluble factors include the chemokines RANTES,
MIP-1.alpha., and MIP-1.beta. (Cocchi et al., Science 270:1811-1815
(1995)).
[0156] The present disclosure provides the first demonstration of
protection against mucosal viral challenge mediated by CTL, which
must be present in the local mucosa. Also provided are methods and
compositions for enhancing the induction of such local mucosal CTL
by a peptide vaccine given together with recombinant IL-12 at the
mucosal site. These methods and compositions for stimulating CTL
immunity at local sites of mucosal exposure represent important
tools for vaccine development to prevent HIV infection or disease
in humans. Because the gastrointestinal tract appears to be a major
site of early SIV and HIV replication, among other pathogens, and
because this and other mucosal sites are frequent sites of entry
for these pathogens, it is particularly critical to achieve
protection at mucosal sites. The present disclosure satisfies these
objects and provides other advantages as set forth above.
[0157] Although the invention has been described with reference to
the presently preferred embodiment, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
20 1 35 PRT Human immunodeficiency virus type 1 1 Glu Gln Met His
Glu Asp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys 1 5 10 15 Pro Cys
Val Lys Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr 20 25 30
Ile Gly Lys 35 2 39 PRT Human immunodeficiency virus type 1 2 Lys
Gln Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala 1 5 10
15 Pro Pro Ile Ser Gly Gln Ile Arg Arg Ile Gln Arg Gly Pro Gly Arg
20 25 30 Ala Phe Val Thr Ile Gly Lys 35 3 39 PRT Human
immunodeficiency virus type 1 3 Arg Asp Asn Trp Arg Ser Glu Leu Tyr
Lys Tyr Lys Val Val Lys Ile 1 5 10 15 Glu Pro Leu Gly Val Ala Pro
Thr Arg Ile Gln Arg Gly Pro Gly Arg 20 25 30 Ala Phe Val Thr Ile
Gly Lys 35 4 48 PRT Human immunodeficiency virus type 1 4 Ala Val
Ala Glu Gly Thr Asp Arg Val Ile Glu Val Val Gln Gly Ala 1 5 10 15
Tyr Arg Ala Ile Arg His Ile Pro Arg Arg Ile Arg Gln Gly Leu Glu 20
25 30 Arg Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly
Lys 35 40 45 5 42 PRT Human immunodeficiency virus type 1 5 Asp Arg
Val Ile Glu Val Val Gln Gly Ala Tyr Arg Ala Ile Arg His 1 5 10 15
Ile Pro Arg Arg Ile Arg Gln Gly Leu Glu Arg Arg Ile Gln Arg Gly 20
25 30 Pro Gly Arg Ala Phe Val Thr Ile Gly Lys 35 40 6 30 PRT Human
immunodeficiency virus type 1 6 Asp Arg Val Ile Glu Val Val Gln Gly
Ala Tyr Arg Ala Ile Arg Arg 1 5 10 15 Ile Gln Arg Gly Pro Gly Arg
Ala Phe Val Thr Ile Gly Lys 20 25 30 7 32 PRT Human
immunodeficiency virus type 1 7 Ala Gln Gly Ala Tyr Arg Ala Ile Arg
His Ile Pro Arg Arg Ile Arg 1 5 10 15 Arg Ile Gln Arg Gly Pro Gly
Pro Arg Ala Phe Val Thr Ile Gly Lys 20 25 30 8 35 PRT Human
immunodeficiency virus type 1 8 Glu Gln Met His Glu Asp Ile Ile Ser
Leu Trp Asp Gln Ser Leu Lys 1 5 10 15 Pro Cys Val Lys Arg Ile His
Ile Gly Pro Gly Arg Ala Phe Tyr Thr 20 25 30 Thr Lys Asn 35 9 39
PRT Human immunodeficiency virus type 1 9 Lys Gln Ile Ile Asn Met
Trp Gln Glu Val Gly Lys Ala Met Tyr Ala 1 5 10 15 Pro Pro Ile Ser
Gly Gln Ile Arg Arg Ile His Ile Gly Pro Gly Arg 20 25 30 Ala Phe
Tyr Thr Thr Lys Asn 35 10 39 PRT Human immunodeficiency virus type
1 10 Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys
Ile 1 5 10 15 Glu Pro Leu Gly Val Ala Pro Thr Arg Ile His Ile Gly
Pro Gly Arg 20 25 30 Ala Phe Tyr Thr Thr Lys Asn 35 11 48 PRT Human
immunodeficiency virus type 1 11 Ala Val Ala Glu Gly Thr Asp Arg
Val Ile Glu Val Val Gln Gly Ala 1 5 10 15 Tyr Arg Ala Ile Arg His
Ile Pro Arg Arg Ile Arg Gln Gly Leu Glu 20 25 30 Arg Arg Ile His
Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Lys Asn 35 40 45 12 42 PRT
Human immunodeficiency virus type 1 12 Asp Arg Val Ile Glu Val Val
Gln Gly Ala Tyr Arg Ala Ile Arg His 1 5 10 15 Ile Pro Arg Arg Ile
Arg Gln Gly Leu Glu Arg Arg Ile His Ile Gly 20 25 30 Pro Gly Arg
Ala Phe Tyr Thr Thr Lys Asn 35 40 13 30 PRT Human immunodeficiency
virus type 1 13 Asp Arg Val Ile Glu Val Val Gln Gly Ala Tyr Arg Ala
Ile Arg Arg 1 5 10 15 Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr
Thr Lys Asn 20 25 30 14 31 PRT Human immunodeficiency virus type 1
14 Ala Gln Gly Ala Tyr Arg Ala Ile Arg His Ile Pro Arg Arg Ile Arg
1 5 10 15 Arg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Lys
Asn 20 25 30 15 15 PRT Human immunodeficiency virus type 1 15 Arg
Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Lys 1 5 10 15
16 10 PRT Human immunodeficiency virus type 1 16 Arg Gly Pro Gly
Arg Ala Phe Val Thr Ile 1 5 10 17 20 PRT Human immunodeficiency
virus type 1 17 Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp Gln
Ser Leu Lys 1 5 10 15 Pro Cys Val Lys 20 18 24 PRT Human
immunodeficiency virus type 1 18 Lys Gln Ile Ile Asn Met Trp Gln
Glu Val Gly Lys Ala Met Tyr Ala 1 5 10 15 Pro Pro Ile Ser Gly Gln
Ile Arg 20 19 24 PRT Human immunodeficiency virus type 1 19 Arg Asp
Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile 1 5 10 15
Glu Pro Leu Gly Val Ala Pro Thr 20 20 33 PRT Human immunodeficiency
virus type 1 20 Ala Val Ala Glu Gly Thr Asp Arg Val Ile Glu Val Val
Gln Gly Ala 1 5 10 15 Tyr Arg Ala Ile Arg His Ile Pro Arg Arg Ile
Arg Gln Gly Leu Glu 20 25 30 Arg
* * * * *