U.S. patent application number 14/156105 was filed with the patent office on 2014-12-25 for recombinant vaccine viruses expressing il-15 and methods of using the same.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Dept. of Health & Human Services. The applicant listed for this patent is The United States of America, as represented by the Secretary, Dept. of Health & Human Services, The United States of America, as represented by the Secretary, Dept. of Health & Human Services. Invention is credited to Jay A. Berzofsky, Sang-Kon Oh, Liyanage P. Perera, Thomas A. Waldmann.
Application Number | 20140377306 14/156105 |
Document ID | / |
Family ID | 32681963 |
Filed Date | 2014-12-25 |
United States Patent
Application |
20140377306 |
Kind Code |
A1 |
Perera; Liyanage P. ; et
al. |
December 25, 2014 |
RECOMBINANT VACCINE VIRUSES EXPRESSING IL-15 AND METHODS OF USING
THE SAME
Abstract
The invention is directed to compositions capable of augmenting
the immunogenicity of a vaccine. The composition, or adjuvant, is
administered to a mammal in need thereof in sequential or
concurrent combination with a vaccine antigen. In one preferred
aspect, the adjuvant is provided in the form of a recombinant
poxvirus vector, such as a vaccinia virus vector, which comprises a
nucleic acid sequence encoding IL-15.
Inventors: |
Perera; Liyanage P.;
(Kensington, MD) ; Waldmann; Thomas A.; (Silver
Spring, MD) ; Oh; Sang-Kon; (Baltimore, MD) ;
Berzofsky; Jay A.; (Bethesda, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Dept. of Health & Human Services |
Rockville |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Dept. of Health & Human
Services
Rockville
MD
|
Family ID: |
32681963 |
Appl. No.: |
14/156105 |
Filed: |
January 15, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10538974 |
Nov 29, 2005 |
8663622 |
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PCT/US03/39967 |
Dec 15, 2003 |
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14156105 |
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60433703 |
Dec 16, 2002 |
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Current U.S.
Class: |
424/199.1 |
Current CPC
Class: |
A61K 2039/55527
20130101; A61K 39/0011 20130101; A61K 2039/5256 20130101; C12N
15/86 20130101; Y02A 50/412 20180101; A61K 39/001106 20180801; A61K
2039/545 20130101; A61K 39/39 20130101; C12N 2710/24143 20130101;
C12N 7/00 20130101; A61P 31/18 20180101; C12N 2740/16034 20130101;
C12N 2740/15034 20130101; Y02A 50/30 20180101; A61K 39/21 20130101;
A61K 2039/57 20130101; A61K 39/001194 20180801; A61K 39/12
20130101; A61K 2039/575 20130101; A61P 37/04 20180101 |
Class at
Publication: |
424/199.1 |
International
Class: |
A61K 39/39 20060101
A61K039/39; A61K 39/00 20060101 A61K039/00; C12N 7/00 20060101
C12N007/00; A61K 39/21 20060101 A61K039/21 |
Claims
1-38. (canceled)
39. A method for generating an immune response in an animal
comprising administering an attenuated or nonvirulent vaccine virus
vector comprising a nucleic acid sequence encoding mammalian IL-15
and an expression unit comprising a plurality of antigen encoding
sequences operably linked to a first expression control sequence to
an animal in an amount effective to stimulate the immune
response.
40. A method for generating an immune response in an animal
comprising administering a vaccine composition comprising a nucleic
acid sequence encoding mammalian IL-15 and an expression unit
comprising a plurality of antigen encoding sequences operably
linked to a first expression control sequence, to an animal in an
amount effective to stimulate the immune response.
41. The method of claim 39 or 40, wherein the immune response
comprises one or more of: the production of memory CD8.sup.+ T
cells specific for the at least one antigen, the production of
memory CD4.sup.+ T cells specific for the at least one antigen, and
the production of antibodies specific for the at least one
antigen.
42. The method of claim 39 or 40, wherein at least some of the
antibodies are neutralizing antibodies.
43.-46. (canceled)
47. The method of claim 39 or 40, further comprising
re-administering the nucleic acid encoding the antigen and nucleic
acid encoding IL-15 after an interval of time, wherein the interval
is at least about 6 months after the first administration.
48. The method of claim 39 or 40, comprising re-administering the
antigen after an interval of time, wherein the interval is at least
about 8 months after the first administration.
49. The method of claim 39 or 40, comprising re-administering the
antigen after an interval of time, wherein the interval is at least
about 10 months after the first administration.
50. The method of claim 39 or 40, comprising re-administering the
antigen after an interval of time, wherein the interval is at least
about 12 months after the first administration.
51. The method of claim 39 or 40, comprising re-administering the
antigen after an interval of time, wherein the interval is at least
about 14 months after the first administration.
52. The method of claim 39 or 40, wherein the at least one antigen
is a viral antigen.
53. The method of claim 52, wherein the viral antigen is from an
HIV virus.
54. The method of claim 39 or 40, wherein the animal is at high
risk of HIV infection.
55. The method of claim 39 or 40, wherein the animal is not HIV
positive at the time of first administration.
56. The method of claim 39 or 40, wherein the animal is HIV
positive at the time of first administration.
57. The method of claim 39 or 40, wherein the expression unit
comprises at least one CTL-recognized epitope, at least one T
helper cell-recognized epitope, and at least one B cell-recognized
epitope.
58. The method of claim 39 or 40, wherein the vaccine virus is a
poxvirus, wherein the expression control sequence comprises viral
regulatory elements, wherein at least two antigens are from two
different polypeptides, wherein the IL-15 encoding sequence is
operably linked to a second expression control sequence, and
wherein at least one antigen is an HIV, SIV, rabies, vaccinia,
influenza, avian influenza, papillomavirus, cancer specific
antigen, bacteria, M. tuberculosis, anthrax, or malaria peptide or
polypeptide.
59. The method of claim 39 or 40, wherein at least one antigen is
an HIV or SIV peptide or polypeptide.
60. The method of claim 39 or 40, wherein at least one antigen is a
rabies peptide or polypeptide.
61. The method of claim 39 or 40, wherein at least one antigen is a
vaccinia peptide or polypeptide and elicits a protective immune
response against smallpox.
62. The method of claim 39 or 40, wherein at least one antigen is a
cancer specific antigen peptide or polypeptide.
63. The method of claim 62, wherein the cancer specific antigen is
from a Her-2/neu or prostate specific antigen polypeptide.
64. The method of claim 39 or 40, wherein at least one antigen is a
bacterial antigen.
65. The method of claim 39 or 40, wherein at least two antigens are
from two different clades of HIV.
66. The method of claim 57, wherein the at least one antigen
comprising a CTL-recognized epitope, the at least one antigen
comprising a T helper cell-recognized epitope, and the at least one
antigen comprising a B cell-recognized epitope are from the same
HIV polypeptide.
67. The method of claim 39 or 40, wherein the vaccine virus vector
comprises one or more capsid polypeptides.
68. The method of claim 39 or 40, wherein the antigen encoding
sequence is expressed before the IL-15 encoding sequence.
69. The method of claim 39 or 40, wherein the antigen encoding
sequence is expressed after the IL-15 encoding sequence.
70. The method of claim 39 or 40, wherein at least one antigen is
selected from the group consisting of: an avian flu, human
papillomavirus, M. tuberculosis, anthrax or malaria.
71. The method of claim 39 or 40, wherein the nucleic acid encoding
at least one antigen is expressed by a second recombinant
attenuated or nonvirulent vaccine virus vector.
Description
RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. application Ser.
No. 10/538,974, filed Nov. 29, 2005 and allowed on Dec. 6, 2013,
which is a 35 U.S.C. .sctn.371 U.S. national entry of International
Application PCT/US2003/039967 (WO 2004/058278) having an
International filing date of Dec. 15, 2003, which claims priority
to U.S. Provisional Application Ser. No. 60/433,703, filed Dec. 16,
2002, all of which are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The invention relates to recombinant vaccine vectors capable
of expressing interleukin 15 (IL-15) and methods for modulating
immune responses using such viruses.
BACKGROUND OF THE INVENTION
[0003] With over 20 years into the AIDS pandemic, approximately 40
million people globally are estimated to be infected with human
immunodeficiency virus (HIV). AIDS has claimed more than 22 million
lives. Three million deaths occurred in 2001 alone. Despite the
knowledge gained over the past two decades as to the molecular
biology, pathogenesis and epidemiology of HIV, development of an
effective HIV vaccine still continues to be an unprecedented
challenge. Conventional strategies that have been successfully
exploited in the past in developing effective vaccines against
other human pathogens have proven ineffective in the case of
HIV.
[0004] Despite the lack of definitive immune correlates that confer
protection in humans, evidence from recent animal model experiments
have suggested that vaccine-induced immunity to HIV is possible
(Amara, et al., Science 292: 69-74, 2001; Rose, et al., Cell 106:
539-49, 2001; Barouch, et al., Science 290: 486-92, 2000; Shiver,
et al., Nature 415: 331-5, 2000; Chen, et al., Nat. Med. 7:
1225-31, 2001). Studies in rhesus macaques with pathogenic chimeric
SIV-HIV-1 (SHIV) have revealed that antibodies may play an
important role in protection against HIV. For example, passively
infused antibodies have been shown to prevent SHIV infection, not
only in oral or vaginal challenge, but even when the SHIV challenge
was provided intravenously (see, e.g., Shibata, et al., Nat. Med.
5: 204-10, 1999; Mascola, et al., Nat. Med. 6: 207-10, 2000).
Cell-mediated immune mechanisms are also likely to play a dominant
role in curtailing viral replication as well. This contention is
well supported by many studies of SIV in nonhuman primates and
studies in humans where cytotoxic T lymphocytes (CTLs) have been
shown to be pivotal in controlling viral replication in both acute
and chronic phases of the disease (Walker, et al., Nat. Med. 6:
1094-5, 2000).
[0005] As yet, no vaccine has been effective in conferring
protection against HIV infection. More significantly, current
candidate vaccines do not induce efficient cellular responses
against the infected cells, and therefore do not confer long term
protection.
[0006] While peptide or polypeptide vaccines have been used
successfully for certain pathologies, obstacles to using these
vaccines to generate protective immune responses against HIV
include the low immunogenicity of HIV peptides, the genetic
variability of HIV, and the dependence of T cell epitopes on the
patient's immunogenetic background.
[0007] Because of the low immunogenicity of peptides in general,
they are typically administered with adjuvants to stimulate immune
responses. Incomplete Freund's adjuvant and other oil-based
adjuvants appear to be more effective and favor the induction of
Th1 responses, while alum results in a preferentially Th2 response
(Grun and Maurer, Cell Immunol. 121(1): 134-45, 1989). Additional
adjuvant approaches to enhance the response to peptides include the
covalent association with lipopeptidic immunostimulants, or the
encapsidation of peptides into liposomes (Kim, et al., Int J.
Oncol. 21(5): 973-9, 2002; Martinon, et al., J Immunol. 149(10):
3416-22, 1992). Certain cytokines also have been demonstrated to be
useful. In experiments in mice, IL-12 and the active fragment of
IL-1 .beta. have been shown to enhance Th1 responses to peptides.
IL-2 has been approved by the FDA for use in the treatment of
patients with metastatic renal cell carcinoma or with metastatic
melanoma (Antonia, et al., J. Urol. 167(5): 1995-2000, 2002). It
has been used as a component of vaccines for cancer and as an
element of vaccine approaches in models of AIDS (see,
http://www.actis.org/vaccinetrials.asp). However, IL-2
administration is associated with the capillary leak syndrome and
due to its role in activation-induced cell death (AICD), IL-2 may
lead to death of T-cells that recognize the antigens expressed on
the tumor cells. See, e.g., Waldmann, et al., Immunity 14: 105-10,
2001. Furthermore, the IL-2 inhibits the survival of memory CD8+
T-cells, potentially reducing its utility as an adjuvant in HIV
vaccines.
SUMMARY OF THE INVENTION
[0008] The invention is directed to compositions capable of
augmenting the immunogenicity of a vaccine. The composition, or
adjuvant, is administered to a mammal in need thereof in sequential
or concurrent combination with a vaccine antigen. In one preferred
aspect, the adjuvant is provided in the form of a recombinant
poxvirus vector, such as a vaccinia virus vector, which comprises a
nucleic acid sequence encoding IL-15.
[0009] Both IL-15 and IL-2 utilize a receptor complex with shared
signaling components; however, the expression and interactions of
IL-15 and IL-2, while overlapping, are not identical. IL-15 mRNA is
widely distributed in fibroblasts, epithelial cells and monocytes
but not in resting or activated T cells, the predominant source of
IL-2. IL-15 stimulates the proliferation and differentiation of T
cells in vitro and can augment T cell mediated immune responses in
vivo. In addition, IL-15 has been shown to stimulate the induction
of B cell proliferation and differentiation. The proliferation and
differentiation of antigen specific T cells and B cells can augment
the protective immunity for a particular antigen. Armitage, et al.,
J. Immunology 154: 483-490 (1995), reported that IL-15 potently
induces IgM, IgG, and IgA secretion from activated human B cells.
Additionally, IL-15 can induce lymphokine-activated (LAK) activity
in Natural Killer (NK) cells.
[0010] The instant invention exploits the discovery that IL-15 can
potentiate a long-term immune response against an antigen that
significantly exceeds responses to IL-2. Accordingly, the invention
provides compositions and methods for generating a response against
acute, chronic and latent infections by pathogenic organisms and
against malignant cells.
[0011] In one aspect, the invention provides a recombinant vaccine
virus vector comprising a nucleic acid sequence encoding IL-15.
Preferably, the vector also comprises an expression unit comprising
a nucleic acid sequence encoding at least one antigen operably
linked to an expression control sequence.
[0012] The IL-15 encoding sequence and antigen encoding sequence
can be operably linked to expression control sequences with the
same or different temporal pattern of expression. In one aspect,
the antigen encoding sequence is expressed before the IL-15
encoding sequence. In another aspect, the antigen encoding sequence
is expressed after the IL-15 encoding sequence.
[0013] The vector may comprise one or more inactivated virulence
genes or may comprise a restricted host range, and preferably is
replication defective or incompetent or attenuated. Suitable
vectors include, but are not limited to those based on (i.e.,
comprising portions of viral genomes of): poxviruses, adenoviruses,
herpes viruses, alphaviruses, retroviruses, Epstein Barr viruses,
lentiviruses, and picornaviruses. In one preferred aspect, the
vector is a recombinant poxvirus vector. The recombinant
nonvirulent poxvirus is preferably a vaccinia virus but may also be
a fowlpox virus, such as a canarypox virus.
[0014] Preferably, the vector comprises one or more capsid
polypeptides. In one aspect the one or more polypeptides are linked
to a targeting molecule to facilitate selective infection of a cell
(e.g., an antigen presenting cell, such as a dendritic cell, or a
tumor cell).
[0015] Also preferably, the IL-15 encoding sequence comprises an
expression control sequence operably linked thereto.
[0016] In one aspect, at least one antigen is a cancer specific
antigen, such as an antigen from a Her-2/neu polypeptide.
[0017] In another aspect, at least one antigen is a bacterial
antigen, for example, such as an antigen from Borrelia burgdorferi,
Bartonella henselea, Yersinia pestis, and Bacillus anthracis.
[0018] In a further aspect, at least one antigen is a viral peptide
or polypeptide, for example a peptide/polypeptide expressed by a
rabies viral genome, canine distemper virus genome, Newcastle
disease virus genome, Ebola virus genome, West Nile virus genome,
Epstein Barr virus genome or a smallpox genome.
[0019] In a particularly preferred aspect, at least one antigen
comprises a viral peptide or polypeptide, such as a peptide or
polypeptide expressed by an HIV or SIV virus genome.
[0020] The antigen encoding expression unit in some embodiments is
a multivalent expression unit comprising a plurality of antigen
encoding sequences. In one aspect, at least two antigens are from
two different HIV polypeptides. In another aspect, at least two
antigens are from two different strains of HIV. In still another
aspect, at least two antigens are from two different isolates of
HIV, or are from two different clades of HIV.
[0021] In still a further aspect, at least two antigens are from
different subsequences of an HIV polypeptide. However, in another
aspect, at least two antigens are derived from the same subsequence
of an HIV polypeptide, but each subsequence differs by at least one
amino acid. In one aspect, the subsequence is from a high mutable
region in the HIV polypeptide.
[0022] In another aspect, the expression unit comprises a plurality
of antigen encoding sequences, at least one antigen comprising a
CTL-recognized epitope, at least one antigen comprising a T helper
cell-recognized epitope, at least one antigen comprising a B
cell-recognized epitope. In one aspect, the epitopes are from the
same HIV polypeptide.
[0023] In one aspect, at least one antigen is from an HIV
polypeptide while at least one other antigen is from an infectious
organism associated with an opportunistic infection in HIV positive
patients, e.g., such as Pneumocystis cariini.
[0024] The invention further provides a recombinant vaccine virus
vector comprising a nucleic acid encoding IL-15 and a nucleic acid
encoding at least one antigen, wherein the nucleic acid encoding at
least one antigen is expressed by a second recombinant vaccine
virus vector.
[0025] The invention also provides a method for generating an
immune response in an animal comprising administering any of the
recombinant vaccine virus vectors or compositions described above
to an animal in an amount effective to stimulate the immune
response. Preferably, the immune response comprises one or more of:
the production of memory CD8.sup.+ T cells specific for the at
least one antigen, the production of memory CD4.sup.+ T cells
specific for the at least one antigen, and the production of
antibodies specific for the at least one antigen. Also, preferably,
at least some of the antibodies are neutralizing antibodies.
[0026] In one aspect, the animal is a human being.
[0027] In another aspect, the animal is a domestic animal such as a
dog or cat. The animal may also be a feral or wild animal such as
foxes and raccoons. The animal may also be a non-human primate.
[0028] The method may be used to provide a prophylactic or
therapeutic vaccine to a patient at risk for being infected with or
already infected with a viral agent, such as smallpox or rabies. In
one aspect, the method is used to provide a prophylactic vaccine to
an individual at high risk of HIV infection and the vaccine may be
administered to an individual who is not HIV positive at the time
of first administration. However, the vaccine may also be
administered to an individual who is HIV positive at the time of
first administration.
[0029] Because the compositions according to the invention are able
to potentiate a long-term immune response, re-administration of the
composition may occur at longer intervals than vaccines of the
prior art. In one aspect, the interval between a primary
administration and re-administration of the recombinant vaccine
vector is at least about 6 months, at least about 8 months, at
least about 10 months, at least about 12 months, at least about 14
months, at least about 16 months, at least about 18 months, and at
least about 24 months.
BRIEF DESCRIPTION OF THE FIGURES
[0030] The objects and features of the invention can be better
understood with reference to the following detailed description and
accompanying drawings.
[0031] FIG. 1 is a graph showing percent specific lysis of HIV-1
gp120 peptide-pulsed cells by CD8.sup.+ T cells obtained from mice
2 months after receiving recombinant vaccinia viruses according to
the invention expressing both IL-15 and the gp160 antigen, both
IL-2 and gp160, or gp160 alone. "E/T" refers to effector/target
cell ratio.
[0032] FIG. 2 is a graph showing percent specific lysis of HIV-1
gp120 peptide-pulsed cells by CD8.sup.+ T cells obtained from mice
14 months after receiving recombinant vaccinia viruses according to
the invention expressing both IL-15 and the gp160 antigen, both
IL-2 and gp160, or gp160 alone. "E/T" refers to effector/target
cell ratio.
[0033] FIG. 3 is a bar graph showing percent of HIV gp120 V3 loop
peptide (P18-I10) tetramer positive CD8+ T cells in fresh spleen in
unimmunized mice, in mice receiving recombinant vaccinia virus
expressing HIV gp160, and in mice receiving vaccinia virus
co-expressing gp160 and IL-15 or IL-2 at various time periods after
immunization.
[0034] FIG. 4 is a bar graph showing percent of
.gamma.-IFN-producing CD8 T cells upon exposure to HIV gp120 V3
loop P18-I10 peptide pulsed cells at various time periods after
immunization in unimmunized mice, in mice receiving recombinant
vaccinia virus expressing HIV gp160, and in mice receiving vaccinia
virus co-expressing gp160 and IL-15 or IL-2.
[0035] FIG. 5 shows anti-gp120 antibody titer in unimmunized mice,
in mice receiving recombinant vaccinia virus expressing HIV gp160,
and in mice receiving vaccinia virus co-expressing gp160 and IL-15
or IL-2 at 8 months after immunization.
[0036] FIG. 6 is a graph showing numbers of tumor-bearing mice at
various time intervals after receiving a recombinant vaccinia virus
expressing Her-2/neu, both Her-2/neu and IL-15, or a control
vaccinia virus.
[0037] FIG. 7 is a graph showing numbers of tumors in tumor-bearing
mice after receiving a recombinant vaccinia virus expressing
Her-2/neu, both Her-2/neu and IL-15, or a control vaccinia
virus.
[0038] FIG. 8 shows levels of anti-Her-2/neu antibodies in serum in
mice receiving a recombinant vaccinia virus expressing Her-2/neu,
both Her-2/neu and IL-15, or a control vaccinia virus.
[0039] FIG. 9 illustrates that avidity of CD8+ CTLs determines the
effectiveness of CD8.sup.+ CTL-mediated immunity.
[0040] FIG. 10 is a graph showing that CD8.sup.+ CTLs induced with
IL-15 respond to lower density of antigen at 14 months after
boosting.
[0041] FIGS. 11A-D are graphs showing that CD8.sup.+ CTLs induced
with IL-15 respond to lower density of antigen.
[0042] FIGS. 12A-C are graphs showing cytolytic activity of
CD8.sup.+ CTLs expanded with high concentration of peptide (0.1
.mu.M).
[0043] FIGS. 13A-C are graphs showing cytolytic activity of
CD8.sup.+ CTLs expanded with low concentration of peptide (0.001
.mu.M).
[0044] FIGS. 14A-B are graphs showing cytolytic activity of
CD8.sup.+ CTLs expanded with low concentration of peptide (0.001
.mu.M).
DETAILED DESCRIPTION
[0045] The invention provides recombinant vaccine vectors,
comprising a nucleic acid sequence encoding IL-15. Preferably, the
vectors also comprise an expression unit comprising a nucleic acid
sequence encoding at least one antigen operably linked to an
expression control sequence; however, the antigen encoding sequence
may also be provided as part of a separate nucleic acid, e.g., as
part of a second recombinant vaccine vector.
[0046] The practice of the present invention employs, unless
otherwise indicated, conventional molecular biology, microbiology,
and recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Maniatis, Fritsch & Sambrook, In Molecular Cloning: A
Laboratory Manual (1982); DNA Cloning: A Practical Approach,
Volumes I and II (D. N. Glover, ed., 1985); Oligonucleotide
Synthesis (M. J. Gait, ed., 1984); Nucleic Acid Hybridization (B.
D. Hames & S. J. Higgins, eds., 1985); Transcription and
Translation (B. D. Hames & S. I. Higgins, eds., 1984); Animal
Cell Culture (R. I. Freshney, ed., 1986); Immobilized Cells and
Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to
Molecular Cloning (1984). All designations of vaccinia restriction
fragments, open reading frames and nucleotide positions are based
on the terminology reported in Goebel, et al., Virology 179:
247-266, 1990; Goebel, et al., Virology 179: 517-563, 1990.
DEFINITIONS
[0047] The following definitions are provided for specific terms
which are used in the following written description.
[0048] As used in the specification and claims, the singular form
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a cell" includes
a plurality of cells, including mixtures thereof. The term "a
nucleic acid molecule" includes a plurality of nucleic acid
molecules.
[0049] As used herein, the term "comprising" is intended to mean
that the compositions and methods include the recited elements, but
do not exclude other elements. "Consisting essentially of", when
used to define compositions and methods, shall mean excluding other
elements of any essential significance to the combination. Thus, a
composition consisting essentially of the elements as defined
herein would not exclude trace contaminants from the isolation and
purification method and pharmaceutically acceptable carriers, such
as phosphate buffered saline, preservatives, and the like.
"Consisting of" shall mean excluding more than trace elements of
other ingredients and substantial method steps for administering
the compositions of this invention. Embodiments defined by each of
these transition terms are within the scope of this invention. For
example, a composition consisting essentially of IL-15 would not
include other cytokines but could include non-cytokine adjuvants,
antigens, therapeutic agents and the like.
[0050] As used herein, "under transcriptional control" or "operably
linked" refers to expression (e.g., transcription or translation)
of a polynucleotide sequence which is controlled by an appropriate
juxtaposition of an expression control element and a coding
sequence. In one aspect, a DNA sequence is "operatively linked" or
"operably linked" to an expression control sequence when the
expression control sequence controls and regulates the
transcription of that DNA sequence. A construct comprising a
nucleic acid sequence operably linked to an expression control
sequence is referred to herein as an "expression unit" or
"expression cassette".
[0051] As used herein, "an expression control sequence" refers to
promoter sequences to bind RNA polymerase, enhancer sequences,
respectively, and/or translation initiation sequences for ribosome
binding. For example, a bacterial expression vector can include a
promoter such as the lac promoter and for transcription initiation,
the Shine-Dalgarno sequence and the start codon AUG (Sambrook, et
al., 1989, supra). Similarly, a eukaryotic expression vector
preferably includes a heterologous, homologous, or chimeric
promoter for RNA polymerase II, a downstream polyadenylation
signal, the start codon AUG, and a termination codon for detachment
of a ribosome.
[0052] As used herein, a "nucleic acid delivery vector" is a
nucleic acid molecule which can transport a polynucleotide of
interest into a cell. Preferably, such a vector comprises a coding
sequence operably linked to an expression control sequence.
[0053] As used herein, "nucleic acid delivery," or "nucleic acid
transfer," refers to the introduction of an exogenous
polynucleotide (e.g., such as an expression cassette) into a host
cell, irrespective of the method used for the introduction. The
introduced polynucleotide may be stably or transiently maintained
in the host cell. Stable maintenance typically requires that the
introduced polynucleotide either contains an origin of replication
compatible with the host cell or integrates into a replicon of the
host cell such as an extrachromosomal replicon (e.g., a plasmid) or
a nuclear or mitochondrial chromosome.
[0054] As used herein, a "a recombinant vaccine vector" refers to a
polynucleotide to be delivered into a host cell, either in vivo, ex
vivo or in vitro which comprises genomic sequences from a vaccine
virus and a heterologous nucleic acid sequence (e.g., such as an
expression unit for expressing IL-15 and/or an antigen).
Preferably, one or more virulence-associated sequences are
inactivated in the vector. A vector may be encapsulated by viral
capsid proteins or may comprise naked nucleic acids or may comprise
nucleic acids associated with one or more molecules for
facilitating entry into a cell (e.g., such as liposomes). However,
preferably, the vector is encapsulated with one or more viral
capsid proteins. Examples of vaccine viruses include, but are not
limited to, poxviruses as further defined below.
[0055] As used herein, "an attenuated virus" or a virus having one
or more "inactivated virulence associated genes" refers to a virus
that is replication deficient or which replicates less efficiently
than a wild type virus in a particular host.
[0056] As used herein, the term "administering a nucleic acid to a
cell" or "administering a vector to a cell" refers to infecting
(e.g., in the form of a virus), transducing, transfecting,
microinjecting, electroporating, or shooting the cell with the
nucleic acid/vector. In some aspects, molecules are introduced into
a target cell by contacting the target cell with a delivery cell
(e.g., by cell fusion or by lysing the delivery cell when it is in
proximity to the target cell).
[0057] A cell has been "transformed", "transduced", or
"transfected" by exogenous or heterologous nucleic acids when such
nucleic acids have been introduced inside the cell. Transforming
DNA may or may not be integrated (covalently linked) with
chromosomal DNA making up the genome of the cell. In prokaryotes,
yeast, and mammalian cells for example, the transforming DNA may be
maintained on an episomal element, such as a plasmid. In a
eukaryotic cell, a stably transformed cell is one in which the
transforming DNA has become integrated into a chromosome so that it
is inherited by daughter cells through chromosome replication. This
stability is demonstrated by the ability of the eukaryotic cell to
establish cell lines or clones comprised of a population of
daughter cells containing the transforming DNA. A "clone" is a
population of cells derived from a single cell or common ancestor
by mitosis. A "cell line" is a clone of a primary cell that is
capable of stable growth in vitro for many generations (e.g., at
least about 10).
[0058] As used herein, "IL-15 polypeptide" means a polypeptide
having at least about 70%, at least about 90%, at least about 97%
or about 100% homology to the wild type amino acid sequence
described in Grabstein, et al., Science 264: 96, 1994 and U.S. Pat.
No. 5,747,024. Variants, modified forms, biologically active
fragments and fusions of IL-15 are also encompassed within the
scope of this term so long as these have at least the following
properties: the nucleic acids encoding the variants, modified
forms, biologically active fragments and fusions of IL-15 bind to
the wild-type IL-15 sequence under conditions of moderate or high
stringency as defined below, and the polypeptides themselves are
able to stimulate the proliferation of CTLL-2 cells (see, e.g., as
described in Gillis and Smith, Nature 268:154, 1977; ATCC TIB
214).
[0059] As used herein, a "variant form of IL-15" refers to an IL-15
polypeptide which comprises conservatively substituted sequences,
meaning that one or more amino acid residues are replaced by
residues having similar physical and chemical characteristics.
Examples of conservative substitutions include substitution of one
aliphatic residue for another, such as Ile, Val, Leu, or Ala for
one another, or substitutions of one polar residue for another,
such as between Lys and Arg; Glu and Asp; or Gln and Asn. Guidance
concerning amino acid changes which are likely to be phenotypically
silent may be found in Bowie, et al., Science 247: 1306-1310, 1990,
for example. Other such conservative substitutions, for example,
substitutions of regions having similar hydrophobicity
characteristics, are encompassed within this definition.
Conservative substitution tables providing functionally similar
amino acids are well known in the art (see, e.g., Henikoff and
Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-10919, 1992).
Preferably, an IL-15 polypeptide retains the Asp.sup.56 and
Gln.sup.156 residues known to be important for signal transduction.
"Variants" also include forms of IL-15 which arise from alternative
RNA splicing events but which nevertheless are able to function in
a CTLL-2 proliferation assay and have activity substantially
similar to wild type IL-15.
[0060] As used herein, "modified forms" of an IL-15 polypeptide
refers to a post-translationally modified form of an IL-15
polypeptide (e.g., such as a glycosylated form or a proteolytically
processed form).
[0061] As used herein, "a nucleic acid sequence encoding an IL-15
polypeptide" includes nucleic acid sequences corresponding to the
wild type IL-15 nucleic acid sequence described in Grabstein, et
al., Science 264: 96, 1994 and U.S. Pat. No. 5,747,024 as well as
sequences which differ from the wild type IL-15 nucleic acid
sequence because of degenerate substitutions and nucleic acid
sequences encoding variants, modified forms, and biologically
active fragments of IL-15 polypeptides. Degenerate codon
substitutions can be achieved by generating sequences in which the
third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (See,
e.g., Batzer, et al., Nucleic Acid Res. 19: 5081, 1991; Ohtsuka, et
al., J: Biol. Chem. 20: 2605-2608, 1985; Rossolini, et al., 1994,
Mol. Cell. Probes 8: 91-98).
[0062] The term "biologically active fragment", "biologically
active form", "biologically active equivalent" of and "functional
derivative" of a wild-type protein, possesses a biological activity
that is at least substantially equal (e.g., not significantly
different from) the biological activity of the wild type protein as
measured using an assay suitable for detecting the activity (e.g.,
at least a CTLL-2 assay in the case of a biologically active
fragment of IL-15).
[0063] As used herein, the term "isolated" means separated from
constituents, cellular and otherwise, in which the polynucleotide,
peptide, polypeptide, protein, antibody, or fragments thereof, are
normally associated with in nature. For example, with respect to a
polynucleotide, an isolated polynucleotide is one that is separated
from the 5' and 3' sequences with which it is normally associated
in the chromosome. As is apparent to those of skill in the art, a
non-naturally occurring polynucleotide, peptide, polypeptide,
protein, antibody, or fragments thereof, does not require
"isolation" to distinguish it from its naturally occurring
counterpart.
[0064] As used herein, a "Poxvirus" includes any member of the
family Poxviridae, including the subfamilies Chordopoxviridae
(vertebrate poxviruses) and Entomopoxviridae (insect poxviruses).
See, for example, B. Moss in Virology, ed. Fields et al., Raven
Press p. 2080 (1990). The chordopoxviruses comprise the following
genera: Orthopoxvirus (e.g., vaccinia); Avipoxvirus (e.g.,
fowlpox); Capripoxvirus (e.g., sheeppox) Leporipoxvirus (e.g.,
rabbit (Shope) fibroma, myxoma); and Suipoxvirus (e.g.,
swinepox).
[0065] As used herein, a "target cell" or "recipient cell" refers
to an individual cell or cell which is desired to be, or has been,
a recipient of exogenous nucleic acid molecules, polynucleotides
and/or proteins. The term is also intended to include progeny of a
single cell, and the progeny may not necessarily be completely
identical (in morphology or in genomic or total DNA complement) to
the original parent cell due to natural, accidental, or deliberate
mutation. A target cell may be in contact with other cells (e.g.,
as in a tissue) or may be found circulating within the body of an
organism.
[0066] As used herein, a "patient" is a vertebrate, preferably a
mammal, more preferably a human. Mammals include, but are not
limited to, murines, non-human primates, humans, farm animals,
sport animals, pets, and feral or wild animals.
[0067] The terms "cancer," "neoplasm," and "tumor," are used
interchangeably and in either the singular or plural form, refer to
cells that have undergone a malignant transformation that makes
them pathological to the host organism. Primary cancer cells
transformation that makes them pathological to the host organism.
Primary cancer cells (that is, cells obtained from near the site of
malignant transformation) can be readily distinguished from
non-cancerous cells by well-established techniques, particularly
histological examination. The definition of a cancer cell, as used
herein, includes not only a primary cancer cell, but also any cell
derived from a cancer cell ancestor. This includes metastasized
cancer cells, and in vitro cultures and cell lines derived from
cancer cells. When referring to a type of cancer that normally
manifests as a solid tumor, a "clinically detectable" tumor is one
that is detectable on the basis of tumor mass; e.g., by procedures
such as CAT scan, MR imaging, X-ray, ultrasound or palpation,
and/or which is detectable because of the expression of one or more
cancer-specific antigens in a sample obtainable from a patient.
[0068] As used herein, the term "pharmaceutically acceptable
carrier" encompasses any of the standard pharmaceutical carriers,
such as a phosphate buffered saline solution, water, and emulsions,
such as an oil/water or water/oil emulsion, and various types of
wetting agents. The compositions also can include stabilizers and
preservatives. For examples of carriers, stabilizers and adjuvants,
see Martin Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co.,
Easton (1975).
[0069] The term "antigen source" as used herein covers any
substance that will elicit an innate or adaptive immune response.
An antigen source may require processing (e.g., such as
proteolysis) to produce an antigen. An antigen source may be a
polypeptide/protein, peptide, microorganism, tissue, oligo- or
polysaccharide, nucleic acid (encoding an antigen or a
polypeptide/protein comprising an antigen or itself serving as the
antigen).
[0070] As used herein, the terms "antigen", "antigenic determinant"
or "epitope" are used synonymously to refer to a short peptide
sequence or oligosaccharide, that is specifically recognized or
specifically bound by a component of the immune system. Generally,
antigens are recognized in the context of an MHC/HLA molecule to
which they are bound on an antigen presenting cell. Two antigens
"correspond" to each other if they can be specifically bound by the
same antibody, B cell, or T cell, and binding of the epitope to the
antibody, B cell, or T cell substantially prevents binding by the
other epitope (e.g., binding of a first epitope in the presence of
a second epitope is less than about 30%, preferably, less than
about 20%, and more preferably, less than about 10%, 5%, 1%, or
about 0.1% of binding observed in the absence of the second
epitope):
[0071] As used herein, a "vaccine composition" comprises a vaccine
vector encoding and IL-15 polypeptide and at least one antigen
source. The antigen source may comprise a nucleic acid encoding an
antigen included as part of the vaccine vector or which is provided
as a separate nucleic acid molecule. The components of a vaccine
composition may be administered together or sequentially.
[0072] As used herein, a "vaccine" refers to a material that
contains or encodes an antigen which will provide active immunity
to material comprising the antigen, but will not cause disease.
[0073] As used herein, a "subunit vaccine" refers to a vaccine that
contains only part of the virus or other microorganism, e.g.,
including viral or microbial polypeptides or nucleic acids encoding
such polypeptides capable of eliciting an immune response.
[0074] As used herein, a "therapeutic vaccine" is a vaccine
designed to boost the immune response to an antigen in a person
already exposed to the antigen.
[0075] As used herein, a "therapeutically effective amount" refers
to an amount sufficient to prevent, correct and/or normalize an
abnormal physiological response. In one aspect, a "therapeutically
effective amount" is an amount sufficient to reduce by at least
about 30 percent, more preferably by at least 50 percent, most
preferably by at least 90 percent, a clinically significant feature
of pathology, such as for example, e.g., suppression of CD4 cells
(i.e., resulting in an increase in CD4 cells by a least 30%, etc),
decrease in viral load; decrease in size of a tumor mass, and the
like. Preferably, a "therapeutically effective amount of a vaccine
composition" enhances a beneficial immune response to a vaccine
antigen by at least about 30%, more preferably by at least about
50% or at least about 90%, i.e., increasing CTL responses against
the antigen, increasing secretion of .gamma.-IFN by CD8.sup.+ T,
increasing production of antibodies specific for a vaccine antigen
and increasing the duration of these responses after administration
of a vaccine composition.
[0076] As used herein, a immune response with "increased duration"
refers to a significant response observed at at least about 4
months, about 6 months, about 8 months, about 10 months, about 12
months, about 16 months, about 18 months, or at least about 20
months after initial administration of an antigen.
[0077] An "antibody" is any immunoglobulin, including antibodies
and fragments thereof, that binds a specific antigen. The term
encompasses polyclonal, monoclonal, and chimeric antibodies (e.g.,
bispecific antibodies). An "antibody combining site" is that
structural portion of an antibody molecule comprised of heavy and
light chain variable and hypervariable regions that specifically
binds antigen. Exemplary antibody molecules are intact
immunoglobulin molecules, substantially intact immunoglobulin
molecules, and those portions of an immunoglobulin molecule that
contains the paratope, including Fab, Fab', F(ab').sub.2 and F(v)
portions, which portions are preferred for use in the therapeutic
methods described herein.
[0078] As used herein, the term "immune effector cells" refers to
cells capable of binding an antigen and which mediate an immune
response. These cells include, but are not limited to, T cells,
.beta. cells, monocytes, macrophages, dendritic cells, NK cells and
cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones,
and CTLs from tumor, inflammatory, or other infiltrates.
[0079] As used herein, the term "viral infection" describes a
disease state in which a virus invades healthy cells, uses the
cell's reproductive machinery to multiply or replicate and
ultimately lyse the cell resulting in cell death, release of viral
particles and the infection of other cells by the newly produced
progeny viruses. A "non-productive infection", i.e., by a vaccine
virus vector is an infection in which the vector is introduced into
a cell but does not replicate within the cell, either because of
inactivation of virulence associated gene(s) or because of a
restricted host-range.
[0080] As used herein, the term "treating or preventing viral
infections" means to inhibit the replication of the particular
virus, to inhibit viral transmission, or to prevent the virus from
establishing itself in its host, and to ameliorate or alleviate the
symptoms of the disease caused by the viral infection.
[0081] As used herein, an "adjuvant" refers to a substance that
enhances, augments or potentiates the host's immune response to a
vaccine antigen.
[0082] The term "immunogenicity" means relative effectiveness of an
immunogen or antigen to induce an immune response.
[0083] As used herein, "baseline" refers to the time point just
before administration of a vaccine when starting measurements are
taken.
[0084] As used herein, a "booster" refers to a second or later
vaccine dose given after the primary dose(s) to increase the immune
response to the original vaccine antigen(s). The vaccine given as
the booster dose may or may not be the same as the primary
vaccine.
[0085] As used herein, "challenge" refers to the deliberate
exposure of a host animal to a vaccine antigen.
[0086] As used herein, a "clade" refers to a group of related HIV
isolates classified according to their degree of genetic similarity
(e.g., such as measured by the similarity of their envelope
proteins). There are currently two groups of HIV-1 isolates, M and
O. The M group consists of at least nine clades, A through I. Group
O may consist of a similar number of clades. Clade B is commonly
found in North America and Europe, and includes isolates LAI b, MN,
and SF-2.
[0087] As used herein, "seroconversion" refers to the development
of antibodies to a particular antigen.
[0088] As used herein, "immunity" refers to natural or acquired
resistance provided by the immune system to a specific disease.
Immunity may be partial or complete, specific or nonspecific,
long-lasting or temporary.
[0089] As used herein, "sterilizing immunity" refers to an immune
response that completely prevents the establishment of an
infection.
[0090] As used herein, a "memory cell" refers to a subset of T
cells and B cells that have been exposed to specific antigens and
can then proliferate (recognize the antigen and divide) more
readily when the immune system re-encounters the same antigens.
Recombinant Vectors
[0091] The invention provides a vaccine vector comprising a nucleic
acid sequence encoding an IL-15 polypeptide for inducing or
enhancing an immune response to an antigen. Preferably, the vaccine
vector is a poxvirus vector such as a vaccinia vector. The
invention can generally be implemented to produce and/or enhance a
cellular or humoral immune response against a selected antigen ("a
vaccine antigen").
[0092] The cloning and sequencing of IL-15 is disclosed in
Grabstein, et al., Science 264: 96, 1994 and in U.S. Pat. No.
5,747,024. As used herein, an "IL-15 nucleic acid" hybridizes to
the wild-type IL-15 sequence under conditions of moderate or high
stringency and is able to stimulate the proliferation of CTLL-2
cells (Gillis and Smith, Nature 268: 154, 1977; ATCC TIB 214).
[0093] Examples of stringent hybridization conditions include:
incubation temperatures of about 25.degree. C. to about 37.degree.
C.; hybridization buffer concentrations of about 6.times.SSC to
about 10.times.SSC; formamide concentrations of about 0% to about
25%; and wash solutions of about 6.times.SSC. Examples of moderate
hybridization conditions include: incubation temperatures of about
40.degree. C. to about 50.degree. C.; buffer concentrations of
about 9.times.SSC to about 2.times.SSC; formamide concentrations of
about 30% to about 50%; and wash solutions of about 5.times.SSC to
about 2.times.SSC. Examples of high stringency conditions include:
incubation temperatures of about 55.degree. C. to about 68.degree.
C.; buffer concentrations of about 1.times.SSC to about
0.1.times.SSC; formamide concentrations of about 55% to about 75%;
and wash solutions of about 1.times.SSC, 0.1.times.SSC, or
deionized water. In general, hybridization incubation times are
from 5 minutes to 24 hours, with 1, 2, or more washing steps, and
wash incubation times are about 1, 2, or 15 minutes. SSC is 0.15 M
NaCl and 15 mM citrate buffer. It is understood that equivalents of
SSC using other buffer systems can be employed.
[0094] Preferably, the IL-15 nucleic acid sequence encodes an IL-15
polypeptide which comprises at least about 70% homology to the wild
type IL-15 polypeptide and more preferably, comprises at least
about 90% homology or about 100% homology to the wild type IL-15
polypeptide.
[0095] Percent identity and similarity between two sequences
(nucleic acid or polypeptide) can be determined using a
mathematical algorithm (see, e.g., Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991).
[0096] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps are introduced
in one or both of a first and a second amino acid or nucleic acid
sequence for optimal alignment and non-homologous sequences can be
disregarded for comparison purposes). The percent identity between
the two sequences is a function of the number of identical
positions shared by the sequences, taking into account the number
of gaps, and the length of each gap which need to be introduced for
optimal alignment of the two sequences. The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide
positions, respectively, are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology").
[0097] A "comparison window" refers to a segment of any one of the
number of contiguous positions selected from the group consisting
of from 25 to 600, usually about 50 to about 200, more usually
about 100 to about 150 in which a sequence may be compared to a
reference sequence of the same number of contiguous positions after
the two sequences are optimally aligned. Methods of alignment of
sequences for comparison are well-known in the art.
[0098] For example, the percent identity between two polypeptide
sequences can be determined using the Needleman and Wunsch
algorithm (J. Mol. Biol. (48): 444-453, 1970) which is part of the
GAP program in the GCG software package (available at
http://www.gcg.com), by the local homology algorithm of Smith &
Waterman (Adv. Appl. Math. 2: 482, 1981), by the search for
similarity methods of Pearson & Lipman (Proc. Natl. Acad, Sci.
USA 85: 2444, 1988) and Altschul, et al. (Nucleic Acids Res.
25(17): 3389-3402, 1997), by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and BLAST in the Wisconsin
Genetics Software Package (available from, Genetics Computer Group,
575 Science Dr., Madison, Wis.), or by manual alignment and visual
inspection (see, e.g., Ausubel et al., supra).
[0099] Gap parameters can be modified to suit a user's needs. For
example, when employing the GCG software package, a NWSgapdna.CMP
matrix and a gap weight of 40, 50, 60, 70, or 80 and a length
weight of 1, 2, 3, 4, 5, or 6 can be used. Exemplary gap weights
using a Blossom 62 matrix or a PAM250 matrix, are 16, 14, 12, 10,
8, 6, or 4, while exemplary length weights are 1, 2, 3, 4, 5, or 6.
The GCG software package can be used to determine percent identity
between nucleic acid sequences. The percent identity between two
amino acid or nucleotide sequences also can be determined using the
algorithm of E. Myers and W. Miller (CABIOS 4: 11-17, 1989) which
has been incorporated into the ALIGN program (version 2.0), using a
PAM120 weight residue table, a gap length penalty of 12 and a gap
penalty of 4.
[0100] In one aspect of the invention, a human cDNA of IL-15
encodes a 162-amino acid precursor, which contains a 316 bp 5'
noncoding region and a 489 bp open reading frame (including the
stop codon) and a 400 bp 3' noncoding region (see, e.g., Grabstein,
et al., Science 264: 965, 1994). Recombinant vectors can include
the full length cDNA or portions comprising the coding region or
biologically active fragments thereof (e.g., lacking one or more of
the 5' noncoding region and 3; noncoding region). Recombinant
vectors can also include sequences corresponding to sequences
encoding the 114 amino acid processed form of IL-15. Such sequences
may be fused to non-IL-15 secretory sequences, for example, the
signal sequence of an immunoglobulin kappa light chain.
[0101] Preferably, recombinant IL-15 polypeptides expressed in
vectors according to the invention have substantially the same
activity as wild type IL-15, as determined in a CTLL-2 assay. See,
Grabstein, et al., 1994, supra. Briefly, CTLL-2 cells (about
2.times.10.sup.4-10.sup.6) are added to serial dilutions of
recombinant IL-15 polypeptides in a 96-well plate or other suitable
container and incubated at 37.degree. C. and 5%, CO.sub.2 for 3
days. Subsequently, 0.5 .mu.Ci .sup.3H-thymidine is added to the
mixture. Thymidine is incorporated only if the cells proliferate.
After an overnight incubation, cells are harvested (e.g., on glass
fiber filters) and radioactivity is measured using a suitable
detector such as a beta counter (e.g., such as the Matrix 96 beta
counter, available from Packard Instrument Company, Meridien,
Conn.). The activity of recombinant IL-15 in the sample is
determined from a standard curve generated using known amounts of
IL-15. Recombinant human IL-15 for use as a standard can be
purchased commercially (e.g., from PeproTech, London, UK). A
recombinant IL-15 polypeptide according to the invention preferably
shows less than about 20% difference from the standard curve, more
preferably, less than about 10% difference, and still more
preferably, less than about 5% difference. In one aspect, the
recombinant IL-15 polypeptide has a specific activity of about
5.times.10.sup.5-2.times.10.sup.6 U/mg according to the
manufacturer. While a thymidine incorporation assay is described
above, it should be obvious to those of skill in the art that any
suitable assay for measuring cell proliferation may be used and is
encompassed within the scope of the instant invention.
[0102] Preferably, IL-15 nucleic acids encode IL-15 polypeptides
which bind with specificity to the .alpha. subunit of the IL-15
receptor (IL-15R) and which can transduce a signal through either,
or both, the .beta.- or .gamma.-subunits of the IL-15 receptor
complex. In a further aspect, recombinant IL-15 polypeptides
according to the invention act as chemoattractant factors for human
peripheral blood T lymphocytes, as assayed by determining one or
more of induction of polarization, invasion of collagen gels and
redistribution of adhesion receptors (see, e.g., as described in
Wilkinson and Liew, J Exp Med. 181(3): 1255-9, 1995; Nieto, et al.,
Eur. J Immunol 26(6): 1302-7, 1996).
[0103] Administration of recombinant IL-15-expressing vaccine
vectors according to the invention results in the production of an
expanded population of memory cells which are primed to produce a
secondary response upon re-exposure to the antigen. This effect can
be monitored by the ability of such cells to expand rapidly in the
presence of the vaccine antigen presented by antigen presenting
cells (APCs), or their ability to display a rapid antigen-specific
cytolytic response even after the primary exposure to the
antigen.
[0104] Cell death can be monitored by assays known in the art, for
example, by counting trypan-blue dye excluding cells in a
hemocytometer. Apoptosis can be analyzed by addition of propidium
iodide (PI) (ICN) to harvested cells and determining the percentage
of cells taking up PI by flow cytometry. The percentage of
apoptotic cells can be determined by forward scatter analysis as is
known in the art and correlated with PI uptake. Preferably,
recombinant polypeptides protect CD8.sup.+ T cells from cell death,
causing less than about 20%, less than about 10%, and less than
about 5% change in the number of cells at concentrations of
recombinant IL-15 of about 0.1 ng/ml or higher, and less than 20%,
less than 10% and preferably, less than about 5% increase in the
number of apoptotic cells in the population.
[0105] The ability of recombinant IL-15 polypeptides to enhance a
memory response can be evaluated by infecting mice with vaccine
vectors according to the invention in the presence of a vaccine
antigen or a vaccine antigen-encoding sequence. Mice are sacrificed
at various days after antigen exposure and lymph node cells from
naive mice, treated mice, or mice treated with buffer, are isolated
and restimulated with vaccine antigen or an irrelevant antigen
(e.g., such as hen egg lysozyme or an influenza antigen). Cell
proliferation is monitored using methods routine in the art (e.g.,
by measuring the incorporation of .sup.3H-thymidine. A significant
increase in proliferation in memory cells as compared to buffer
treated or naive mice (as determined using statistical methods well
known in the art) is taken as an indication of an enhanced memory
response. Preferably, recombinant IL-15 expressing viral vectors
are capable of producing an at least about 25%, at least about 50%,
or at least about 100% increase in proliferation. Additionally, or
alternatively, the responses of memory cells are taken as indicia
of biologically active IL-15. Preferably, such responses include
cytolysis by vaccine-antigen specific CD8+ T cells and/or
.gamma.-IFN production by such cells. Preferably, administration of
biologically active IL-15 enhances the duration and magnitude of
responses by CD8.sup.+ T cells.
[0106] As discussed above, preferred vaccine vectors include
poxviruses. The large genome size of these viruses permits the
engineering of vectors capable of accepting at least 25,000 base
pairs of foreign. DNA (Smith, et al., Gene 25: 21, 1983).
Additionally, poxviruses can infect most eukaryotic cell types and
do not require specific receptors for entry into a cell. Unlike
other DNA viruses, poxviruses replicate exclusively in the
cytoplasm of infected cells, reducing the possibility of genetic
exchange of recombinant viral DNA with the host chromosome and
allowing heterologous genes to be expressed independent of host
cell regulation.
[0107] A poxvirus vector may be obtained from any member of the
poxviridae, in particular, a vaccinia virus or an avipox virus
(e.g., such as canarypox, fowlpox, etc.) provides a suitable
sequences for vaccine vectors.
[0108] In one aspect, the poxviral vector is a vaccinia virus
vector. Vaccinia virus has demonstrated physical and genetic
stability under field conditions, reducing problems and expense in
transport and storage. Recombinant vaccinia virus vectors have been
shown to confer cellular and humoral immunity against foreign gene
products and to protect against infectious diseases in several
animal models. Further, recombinant vaccinia viruses have also been
used in clinical trials to express the gp160 envelope gene of HIV
(see, e.g., Cooney, The Lancet 337: 567-572, 1991; Graham, et al.,
J. of Infectious Dis. 166: 244-252, 1992; Estin, et al., Proc.
Natl. Acad. Sci. USA 85: 1052-1056, 1988) and are thus clinically
accepted.
[0109] Suitable vaccinia viruses include, but are not limited to,
the Copenhagen (VC-2) strain (Goebel, et al., Virol. 179: 247-266,
1990; Johnson, et al., Virol. 196: 381-401, 1993), modified
Copenhagen strain (NYVAC) (U.S. Pat. No. 6,265,189), the WYETH
strain and the modified Ankara (MVA) strain (Antoine, et al.,
Virol. 244: 365-396, 1998). However, although the examples below
are directed to vaccinia viruses, other poxviruses suitable for use
as vaccines may be substituted and are also encompassed within the
scope of the invention. For example, fowlpox strains such as ALVAC
and TROVAC vectors also provide desirable properties and are highly
attenuated (See, e.g., U.S. Pat. No. 6,265,189, reviewed by
Tartaglia et al, In AIDS Research Reviews, Koff, et al., eds., Vol.
3, Marcel Dekker, N.Y., 1993; Tartaglia et al., 1990, Reviews in
Immunology 10: 13-30, 1990).
[0110] Methods and conditions for constructing recombinant poxvirus
virus vectors, such as vaccinia virus vectors, are known in the art
(see, e.g., Piccini, et al., Methods of Enzymology 153: 545-563,
1987; U.S. Pat. No. 4,769,330; U.S. Pat. No. 4,722,848; U.S. Pat.
No. 4,769,330; U.S. Pat. No. 4,603,112; U.S. Pat. No. 5,110,587;
U.S. Pat. No. 5,174,993; EP 83 286; EP 206 920; Mayr et al.,
Infection 3: 6-14, 1975; Sutter and Moss, Proc. Natl. Acad. Sci.
USA 89: 10847-10851, 1992). The preparation of fowlpox virus is
described in WO 96/11279, for example.
[0111] A vaccine vector is generally prepared as follows. In one
aspect, a donor plasmid comprising a nucleic acid sequence encoding
IL-15 is constructed, amplified by growth in E. coli and isolated
by conventional procedures. The donor plasmid comprises a nucleic
acid sequence homologous to vaccinia virus sequences. The nucleic
acid encoding IL-15 is operably linked to an expression control
element. Preferably, the expression control element comprises viral
regulatory elements, including upstream promoter sequences and,
where necessary, RNA processing signals. The expression control
sequences may be from a vaccinia virus, or other poxvirus (see,
e.g., Mackett, et al., J. Virol, 49: 857, 1982), and is operably
linked to the nucleic acid sequence encoding the IL-15 polypeptide.
The choice of promoter determines both the time (e.g., early or
late) and level of expression of the IL-15 sequence.
[0112] The expression unit comprising the expression control
sequence and IL-15 sequence is flanked on both ends by DNA
homologous to a vaccinia virus DNA sequence being targeted as a
recombination site. Preferably, the flanking sequences correspond
to a nonessential locus in the vaccinia viral genome. The resulting
plasmid construct is then amplified by replication in E. coli or
other suitable host and isolated using methods routine in the art
(see, e.g., Maniatis, T., Fritsch, B. F., and Sambrook, J., In
Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.) (1989)).
[0113] A suitable cell culture (e.g., chicken embryo fibroblasts,
CV-1 cells, BHK-21 cells, 143B tk-cells, vero cells, lung cells,
etc.) is transfected with the donor plasmid along with recipient
vaccinia virus sequences to select for recombinants that comprise
both donor and recipient sequences. Transfection may be facilitated
by providing one or more molecules for facilitating entry of a
nucleic acid into a cell. Suitable delivery vehicles include, but
are not limited to: liposomal formulations, polypeptides;
polysaccharides; lipopolysaccharides, cationic molecules, cell
delivery vehicles, vehicles for facilitating electroporation, and
the like.
[0114] Suitable recipient sequences are selected which will result
in the production of a recombinant virus that can induce and/or
enhance a protective immune response and which lacks any
significant pathogenic properties. Therefore, in one preferred
aspect, the recipient sequence comprises one or more genes which
are nonessential for growth of the virus in tissue culture and
whose deletion or inactivation reduces virulence in a host
organism, such as mammal (e.g., such as a mouse or a human
being).
[0115] Virulence associated sequences include, for example, the
thymidine kinase (TK) gene; hemagglutinin; 13.8 kD secreted
protein, ribonucleotide reductase genes; host range genes;
hemorrhagic gene, and A type inclusion body region. See, e.g.,
Hruby et al., J. Viral. 43: 403-409, 1982; Weir, et al., Proc.
Natl. Acad. Sci. USA 80: 3411-3415, 1983; Flexner, et al., Nature
330: 259, 1987; Buller, et al., Nature 317: 813, 1985; Buller, et
al., J. Virol, 62: 866, 1988; Shida et al., J. Virol, 62: 4474,
1988); Kotwal, et al., Virology 171: 579, 1989; Child, et al.,
Virology 174: 626, 1990). Inactivated virulence associated
sequences can comprise whole or partially deleted gene sequences,
substitutions, rearrangements, insertions, combinations thereof and
the like. Mutations can be engineered or selected for. For example,
attenuated viral strains can be selected for by repeated passages
in a suitable vertebrate host cell (e.g., such as chicken
embryonated eggs and/or chicken fibroblasts) and subsequent plaque
purification to identify plaques which are smaller, replicate more
slowly, or which display other indications of complete or partial
attenuation. Preferably, a poxvirus vector according to the
invention is replication incompetent and incapable of spreading
beyond initially infected cells or is attenuated.
[0116] Host restricted viruses such as avipox viruses can also be
used as these are nonvirulent in mammals, such as humans.
[0117] Recombination between a homologous vaccinia virus sequence
in the donor plasmid and the viral genome results in production of
vaccinia vector that comprises IL-15-encoding sequences.
[0118] Recombinants can be detected by including reporter gene
sequences in the donor plasmid and screening for recombinant
viruses that carry these sequences. For example, donor plasmids
that contain the E. coli .beta.-galactosidase gene provide a method
of distinguishing recombinant from parental viruses (Chakrabarti,
et al., Mol. Cell. Biol. 5: 3403, 1985). Plaques formed by such
recombinants can be positively identified by the blue color that
forms upon addition of an appropriate indicator. Alternatively, or
additionally, the recipient sequence comprises a reporter sequence
and recombinants are detected by loss of function of the reporter
sequence (i.e., resulting from insertion of donor sequences into
the recipient sequence). In one aspect, the recipient reporter
sequence is a virulence associated gene. For example, insertion
into the thymidine kinase gene will result in recombinants that
grow in the presence of bromo-deoxyuridine (BrdU) in thymidine
kinase negative 143B osteosarcoma cells.
[0119] However, in another aspect, the recipient reporter sequence
is a heterologous sequence (e.g., such as .beta.-galactosidase or
guanine-phosphoribosyl transferase). Additional strategies for
generating recombinant vaccinia virus are described in
Scheiflinger, et al., Proc. Natl. Acad. Sci, USA 89: 9977-9981,
1992; Merchlinsky and Moss, Virology 190: 522-526, 1992, for
example.
[0120] Viral particles can be recovered from the culture
supernatant or from the cultured cells after a lysis step (e.g.,
chemical lysis, freezing/thawing, osmotic shock, sonication and the
like). Consecutive rounds of plaque purification can be used to
remove contaminating wild type virus. Viral particles can then be
purified using the techniques known in the art (e.g.,
chromatographic methods or by ultracentrifugation on cesium
chloride or sucrose gradients).
[0121] Vectors according to the invention may additionally comprise
a detectable and/or selectable marker to verify that the vector has
been successfully introduced in a target cell. These markers can
encode an activity, such as, but not limited to, production of an
RNA, peptide, or protein, or can provide a binding site for RNA,
peptides, proteins, inorganic and organic compounds or compositions
and the like. In some cases the reporter sequence provided by the
donor plasmid is used as the marker to verify introduction into a
target cell.
[0122] Examples of detectable/selectable markers genes include, but
are not limited to: nucleic acid sequences which encode: products
providing resistance to otherwise toxic compounds (e.g., such as
antibiotics); products which are otherwise lacking in a recipient
cell (e.g., tRNA genes, auxotrophic markers, and the like);
products which suppress the activity of a gene product; enzymes
(e.g., such as .beta.-galactosidase or guanine-phosphoribosyl
transferase), fluorescent proteins (GFP, CFP, YFG, BFP, RFP, EGFP,
EYFP, EBFP, dsRed, mutated, modified, or enhanced forms thereof,
and the like); cell surface proteins (i.e., which can be detected
by an immunoassay); antisense oligonucleotides; and the like.
[0123] The marker gene can be used as a marker to confirm
successful IL-15 gene transfer by the vaccine vector and/or to
isolate recombinants expressing IL-15.
[0124] In one aspect, the vaccine vector comprises viral capsid
molecules to facilitate entry of the vaccine vector into a cell.
Additionally, viral capsid molecules may be engineered to include
targeting moieties to facilitate targeting and/or selective entry
into specific cell types. Suitable targeting molecules, include,
but are not limited to: chemical conjugates, lipids, glycolipids,
hormones, sugars, polymers (e.g. PEG, polylysine, PEI and the
like), peptides, polypeptides (see, e.g., WO 94/40958), vitamins,
lectins, antibodies and fragments thereof. Preferably, such
targeting molecules recognize and bind to cell-specific markers of
antigen presenting cells, such as dendritic cells (e.g., such as
CD44) or cancer cells.
Antigen Encoding Sequences
[0125] An antigenic source can be administered in the form of
polypeptides or peptides conjugated to a carrier or can be
administered in the form of nucleic acid sequences encoding the
antigen. Preferably, antigen source will contain regions that
stimulate one or more response of the immune system: e.g.,
including, but not limited to: immunoglobulin responses, MHC/HLA I
responses, MHC/HLA class II responses, NK responses and the like.
In the case of MHC/HLA class II mediated responses, the antigen
source will generally contain peptide segments that can be released
by lysosomal enzymes within a cell and, when released, correspond
to MHC/HLA class II epitopes.
[0126] Synthetic antigens and altered antigens also can be used in
the methods described herein. Synthetic antigens have modified
amino acid sequences relative to their natural counterparts (in
this embodiment, the antigen is provided in the form of a peptide
rather than a nucleic acid encoding the peptide). Also encompassed
within the scope of the invention multimers (concatamers) of the
antigen, optionally including intervening amino acid sequences or
linkers. Where a plurality of antigens are encoded by an antigen
expression unit forming a multivalent antigen expression unit, the
antigens may be the same or different.
[0127] In one aspect, a multivalent antigen expression unit
comprises at least one CTL epitope, at least one helper epitope,
and at least one B cell epitope from the same or different antigen
source. The different epitopes may be from the same or different
proteins. For example, a plurality of antigen sequences may be a
combination of antigens from at least two strains of infectious
organisms (e.g., from HIV and Pneumocystic cariini), from different
polypeptides encoded by the genome of a single infectious organism,
or from a single polypeptide, e.g., different antigenic regions or
representing variants of the same antigenic sequence. In the latter
embodiment, variants of the same antigenic sequence may be used to
induce or enhance an immune response against a particularly mutable
peptide sequence (e.g., within an infectious organism).
[0128] Also included within the scope of the invention are
antigenic peptides that are differentially modified during or after
translation, e.g., by phosphorylation, glycosylation, crosslinking,
acylation, proteolytic cleavage, linkage to an antibody molecule,
membrane molecule or other ligand (see, e.g., Ferguson et al., Ann.
Rev. Biochem. 57: 285-320, 1998).
[0129] Suitable antigens include, but are not limited to:
cancer/tumor antigens, autoantigens (e.g., such as antigens
recognized in transplant rejection); allergens; antigens associated
with hypersensitivity; prion antigens; viral antigens; bacterial
antigens; antigens from protozoa or fungi; and parasitic antigens,
including especially proteins found in the cell walls or cell
membranes of these organisms.
[0130] In particular, suitable antigens are antigens encoded by the
genomes of organisms associated with rabies (e.g., GenBank
Accession No. M34678), malaria (Shetty, Lancet Infect Dis. 2(11):
648, 2002), parasitic infections (e.g., such as schistosomiasis),
hantavirus (Meissner, et al., Virus Res. 89(1): 131, 2002; Padula,
et al., J Gen Virol. 83(Pt 9): 2117-22, 2002; Hoffacker, et al.,
Nucleic Acids Res. 26(16): 3825-36, 1998); yellow fever (Pugachev,
et al., Vaccine 20(7-8): 996-9, 2002); West Nile fever (Lanciotti,
et al., Virology 298(1): 96-105, 2002); measles (Crowley, et al.,
Intervirology 28(2): 65-77, 1987); mumps (Jin, et al., Virus Res.
70(1-2): 75-83, 2000); rubella Dominguez, et al., Virology 177(1):
225-38, 1990); poliomyelitis (see, e.g., Kinnunen, et al., J Gen
Virol. 72 (Pt 10): 2483-9, 1991); smallpox (Massung, et al.,
Virology 201(2): 215-40, 1994; Mayr, et al., Zentralbl. Bakteriol.
[B]. 167(5-6): 375-90, 1978; Aguado, et al., J Gen Virol. 73(Pt
11): 2887-902, 1992; Slchelkunov, et al., Virus Res. 36(1): 107-18,
1995); anthrax; Ebola (Sanchez, et al., Virus Res, 29(3): 215-40,
1993); equine encephalitis (Kinney, et al., Virology 170(1):19-30,
1989); Rift valley fever (Muller, et al., Nucleic Acids Res.
19(19): 5433, 1991); cat scratch fever (see, e.g., Renesto, et al.,
Res Microbiol. 151(10): 831-6, 2000); viral meningitis; Marburg
virus (Sanchez, et at, 1993, supra); hepatitis A, B, C, D, and E
(see, generally GenBank); Japanese encephalitis (e.g., GenBank
Accession No. E07883); dengue (e.g., GenBank Accession No, M24444);
plague (Parkhill, et al., Nature 413(6855): 523-7, 2001), tularemia
(Karlsson, et al., Microb. Comp. Genomics 5(1): 25-39, 2000); and
diseases caused by other pathogenic organisms including Chlamydial
and Rickettsial agents.
[0131] Particularly preferred antigens are virally-encoded proteins
encoded by the genome of viruses pathogenic to man or domestic
animals. Non-limiting examples include peptides from the influenza
nucleoprotein composed of residues 365-80 (NP365-80), NP50-63, and
NP147-58 and peptides from influenza hemagglutinin HA202-21 and
HA523-45, defined previously in class I restricted cytotoxicity
assays (Perkins et at, 1989, J. Exp. Med. 170: 279-289). Relevant
protozoan antigens include peptides representing epitopes displayed
by the malarial parasite Plasmodium falciparum have been described
(see, e.g., U.S. Pat. No. 5,609,872). Papilloma virus core antigen,
HCV structural and non-structural proteins; and CMV structural and
non-structural proteins, Ebola GP1 or GP2 protein (see, e.g.,
Feldmann, et al., Arch Virol Suppl. 15: 159-69, 1999; Sanchez, et
al., J. Viral 72(8): 6442-7, 1998; Volchkov, V. E., et al., FEBS
Lett 305(3): 181-4, 1992), or nucleocapsid protein (Vanderzanden,
et al., Virology, 246(1): 134-44, 1998) also provide sources of
antigens. In another aspect, antigens are derived from a
respiratory syncytial virus (RSV). For example, the RSV viral
antigen may be the glycoprotein (G-protein) or the fusion protein
(F-protein). In a further aspect, antigens are derived from the
herpes simplex virus (HSV), such as HSV-1 and HSV-2. For example,
the HSV viral antigen may be the glycoprotein D from HSV-2.
[0132] In one preferred aspect, a rabies virus provides a source of
antigen. Currently, multivalent rabies antigens demonstrate
"efficacy interference" in dogs, namely a failure of one or more
antigens (canine distemper virus antigens, canine adenovirus
antigens, canine coronavirus antigens, canine parainfluenza
antigens, canine parvovirus antigens, and Leptospira bacterin
antigens), when used in combination with a rabies antigen, to
maintain a satisfactory immune response. See, e.g., U.S. Pat. No.
5,843,456. Therefore, the recombinant vaccinia viruses according to
the invention are particularly useful because of their ability to
maintain a significant long-term immune response (greater than a
year).
[0133] Thus, in one preferred aspect of the invention, vaccine
compositions according to the invention comprise a source of rabies
antigen (i.e., a nucleic acid encoding a rabies antigen) and a
recombinant IL-15 expressing sequence. Suitable sources of rabies
antigens include, but are not limited to glycoprotein G. See, e.g.,
Wiktor, et al., Imunol. 110: 269-276, 1973. Vectors comprising
encoding rabies antigens are described in U.S. Pat. No. 6,210,663,
for example. Such compositions may include additional antigen
sources (e.g., including different portions of glycoprotein G) to
provide multivalent vaccines as described above.
[0134] In aspect, a preferred antigen is a small pox antigen. In
this embodiment, a vaccinia virus itself provides a suitable source
of antigens because of its close antigenic and genetic relatedness
to small pox strains. Currently, vaccinia vaccines, such as the
Wyeth strain, can give rise to adverse effects in a small number of
vaccines, including eczema vaccinatum and encephalitis. During the
smallpox eradication era, about 1250 in every million people
vaccinated suffered these side effects and about one in a million
died. Young children under two years of age were especially
vulnerable. The numbers of deaths would likely increase if a
smallpox eradication program commenced again because of the higher
percentage of immunosuppressed individuals in the population (e.g.,
individuals with AIDS or taking immunosuppressive drugs). Further,
the prevalence of eczema in the general population has risen for
unknown reasons (see, Science 296: 1592-1595, 2002; Smith, et al.,
Nature Reviews/Immunology 2: 521-526, 2002). Because of these
reasons, there is a real need for a safer vaccine and
recombinant-IL-15 expressing vaccines generated from strains such
as Wyeth and MVA can meet this need in at least three ways. First,
by incorporating IL-15 expressing sequences into the vaccine, both
humoral and cell-mediated immune responses will be augmented.
Second, insertional inactivation by IL-15 sequences of a virulence
associated gene such as the hemagluttinin will result in
attenuation of the virus. Third, expressed IL-15 itself will lead
to reduction in virulence due to activation of NK cells, as well as
enhanced interferon gamma and chemokine production.
[0135] In another particularly preferred aspect, an antigen is
selected which is associated with a chronic pathology, such as AIDS
(e.g., GenBank Accession No. U18552). The causative agent of AIDS
is the human immunodeficiency virus (HIV), a pathogenic retrovirus
(see, e.g., Barre-Sinossi, et al., Science 220: 868-870, 1983;
Gallo, et al., Science 224: 500-503, 1984). There are at least two
distinct types of HIV: HIV-1 (Barre-Sinossi, et al., 1983, supra;
Gallo, et al., 1984, supra) and HIV-2 (Clavel, et al., Science 223:
343-346, 1986; Guyader, et al., Nature 326: 662-669, 1987). Types
of HIV are further divisible into strains, isolates and clades.
[0136] Therefore, preferably, in one aspect according to the
invention, the antigen is from a pathogenic virus which comprises
HIV, including various types of HIV (e.g., HIV-1 and HIV-2),
strains (e.g., strain BH10 and pNL4-3 of HIV-1), isolates, clades
(e.g., clade A, B, C, D, E, F, and G of group M) and the like.
[0137] The viral antigen may be an HIV envelope protein, such as
HIV envelope protein Env, either full-length (gp160), truncated
(e.g., gp120 and gp41), and can be modified with insertions,
deletions or substitutions. The HIV envelope proteins have been
shown to be the major antigens for anti-HIV antibodies present in
AIDS patients (see, e.g., Barin, et al., Science 228: 1094-1096,
1985). The amino acid and RNA sequences encoding HIV envelope from
a number of HIV strains are known. See Myers, G. et al., Human
Retroviruses and AIDS: A compilation and analysis of nucleic acid
and amino acid sequences, Los Alamos National Laboratory, Los
Alamos, N.M. (1992).
[0138] In one preferred aspect, an epitope is selected from the V3
region of the gp120 polypeptide, the V1-V2 region, the CD4 binding
site, the C4 region, a CCR binding region, such as the binding
region for CCR5 or CCR3. The location of neutralizing epitopes in
the V3 domain is well known. It has been found that neutralizing
epitopes in the V2 and C4 domains are located between residues 163
and 200 and between about 420 and 440, respectively. In addition,
residues for antibody binding also include residues 171, 174, 177,
181, 183, 187, 188 in the V2 domain and residues 429 and 432 in the
C4 domains. See, e.g., Berman, et al. Virology 265: 1-9, 1999; and
Berman, AIDS Res. Human Retroviruses 15: 115-132.
[0139] In another embodiment, the HIV antigen expressed by the
recombinant virus of the present invention is a modified Env
protein that contains deletions and/or mutations in the
glycosylation sites. The gp120 of HIV-1 contains 24 potential sites
for N-linked glycosylation (Asn-X-Ser/Thr). Approximately 13 of the
24 glycosylation sites are conserved in the different viral
isolates. Analysis of HIV-1 Env proteins has demonstrated that 17
of 24 potential glycosylation sites are modified with carbohydrate
side chains and therefore, because of the extensive glycosylation
of Env gp proteins, very few regions of the peptide backbone of
gp120 may protrude from the carbohydrate mass. Some of the
glycosylation sites have been found in non-neutralizing epitopes
that dilute the immunity against true neutralizing epitopes or
serve as decoy epitopes. Thus, deletion or mutation of these
glycosylation sites may enhance immunity of the antigen by
unmasking the true neutralizing epitopes. See, e.g., Mizuochi, et
al. J. Biol. Chem., 265: 8519-8524, 1990; and Leonard et al., J.
Biol. Chem. 265:10373-10382, 1990.
[0140] Other viral antigens include either full length wild type,
modified, or protease-processed products or fragments, including,
but not limited to: capsid proteins such as HIV (gp17); or HIV
regulatory proteins, such as Tat, Vif, Vpr, Nef, and Rev.
[0141] Another preferred antigen source is the HIV matrix protein,
or gag. Gag (gp24) is relatively conserved among diverse HIV
strains and subtypes and broad cross-class anti-Gag CTL responses
have been demonstrated in 111V-infected patents. Studies of exposed
but sero-negative subjects indicate that Gag-specific CTL may be
involved in protection against the establishment of a persistent
HIV infection. Additionally, Gag-specific CD8.sup.+ cytotoxic T
lymphocytes are important in controlling virus load during acute
infection as well as during the asymptomatic stages of the
infection. Multiple discrete Gag epitopes have been described and
shown to mediate cytotoxic activities. Moreover, levels of
p24-specific CTL proliferative responses of infected untreated
persons have been positively correlated with levels of Gag-specific
CTL and negatively correlated with levels of plasma HIV-1 RNA. See,
e.g., U.S. Published Application No. 20020160430.
[0142] Other characterized HIV epitopes are included in the HIV
Molecular Immunology Database published yearly by Los Alamos. See,
e.g., HIV Molecular Immunology 2001, Editors: Bette T. M. Korber,
Christian Brander, Barton F. Haynes, Richard Koup, Carla Kuiken,
John P. Moore, Bruce D. Walker, and David I. Watkins. Publisher:
Los Alamos National Laboratory, Theoretical Biology and Biophysics,
Los Alamos, N. Mex. LA-UR 02-4663, at
http://hiv-web.lanl.gov/content/immunology/maps/maps.html.
[0143] In one aspect, HIV antigenic peptides matched to particular
clades provide the vaccine antigen. The sequences of different
clade strains are known in the art and include clade A (Accession
No: HIV-1 92UG037WHO.0108HED), B (Accession No: pNL4-3), C
(Accession No: HIV-1 92BR025WHO.109HED), D (Accession No: HIV-1
92UG024.2), E (Accession No: HIV-1 93TH976.17), F (Accession No:
HIV-1 93BR020.17), and G (Accession No: HIV-1 92RU131.9). For
example, the following peptides can be used: from p17--SLFNTVATL
(clade A), SLYNTVATL (clade B); from pol A--ILKDPVHG (clade A);
ILKEPVHGV (clade B); from p24--DRFFKTLRA (clade A), DLNNMLNI (clade
A), PPIPVGDIY (clade A), DLNTMLNTV (clade B), DRFYKTLRA (clade B),
PPIPVGEIY (clade B); from nef--YPLTGWCY (clade B), YPLTFGWCF (clade
D). These are epitopes particularly prevalent in African clades.
See, e.g., Rowland-Jones, et al. J Clin. Invest. 102(9): 1758-65,
1998.
[0144] Corresponding SIV sequences are also encompassed within the
scope of the present invention.
[0145] As discussed above, a vaccine composition according to the
invention may provide a plurality of different types of vaccine
antigens. For example, for an HIV vaccine, the composition may
provide a source of vaccine antigens comprising at least two of any
of the HIV antigens described above. Antigens may be from different
HIV polypeptides, different strains, different clades, or different
regions of the same HIV polypeptide. In one aspect, a panel of
peptides representing a mutable region of an HIV polypeptide is
provided. The peptides may represent known variants of the region
and/or may comprise randomly generated variants.
[0146] In another aspect, a vaccine composition according to the
invention may provide at least one HIV antigen source and an
antigen that enhances a protective immune response against the HIV
virus. For example, a universal T cell epitope-containing peptide
from hepatitis B surface antigen (amino acids 19-33) is reported to
enhance the production of antibodies specific for gp120. See, e.g.,
Greenstein, et al., J Immunol. 148(12): 3970-7, 1992. In yet
another aspect, at least one HIV antigen source is provided with an
antigen source of an infectious microorganism associated with HIV
infection, such as Pneumocystis cariini or Bartonella henselea.
[0147] One preferred multivalent combinations comprises gag, pol
and env. Another preferred multivalent combination comprises a gag,
pol and reverse transcriptase (RT). Yet another multivalent
combination comprises gag, tat and nef.
[0148] In another aspect of the invention, the antigen source
comprises a bacterial antigen. Bacterial sources of antigens
include, but are not limited to, Bacillus tuberculoses, Bacillus
anthracis, the spirochete Borrelia burgdorferi that causes the Lyme
disease in animals, and Bartonella henselea, the causative agent of
cat scratch fever (see, e.g., Renesto, et al., Res Microbiol.
151(10): 831-6, 2000; Regnery, et al., Clin. Infect. Dis. 21:94-98,
1995).
[0149] Parasites also serve as sources of antigens and include, but
are not limited to: Cryptosporidium; Eimeria; Histomonas;
Leucocytozoon, Toxoplasma; Trichomonas; Leishmania; Trypanosoma;
Giardia (e.g., GenBank Accession No. M33641); Plasmodium (e.g.,
GenBank Accession No. X53832); hookworm; onchocerciasis (e.g.,
GenBank Accession No. M27807); schistosomiasis (e.g., GenBank
Accession No. L08198); trypanosomiasis; amoebiasis, filariasis
(e.g., GenBank Accession No. J03266), borreliosis; Babesia, and
Theileria. Antigenic polypeptides of such organisms include coat
proteins, proteins of the pathogenic parasites, and the like.
[0150] It is contemplated that suitable microorganisms and
parasitic antigens will be derived from known causative agents
responsible for diseases including, but not limited to, diphtheria,
pertussis (e.g., GenBank Accession No. M35274), tetanus (e.g.,
GenBank Accession No. M64353), tuberculosis, bacterial and fungal
pneumonias (e.g., Haemophilus influenzae, Pneumocystis carinii,
etc.), cholera, typhoid, plague, shigellosis, salmonellosis (e.g.,
GenBank Accession No. L03833), Legionnaire's Disease, Lyme disease
(e.g.) GenBank Accession No. U59487), hepatitis B antigen such as
HBeAg, HBcAg and HBsAg (e.g., S, pre-S1, or pre-S2), ORF 5, ORF 6
(see, e.g., see Blum, et al., TIG 5(5): 154-158, 1989); the HBV poi
antigen, or a hepatitis C antigen such as the core antigen C, E1,
E2/NS1, NS2, NS3, NS4 and NS5
[0151] Cancer-specific antigens are also encompassed within the
scope of the instant invention. These include, but are not limited
to; a polypeptide comprising an epitope derived from Her-2/neu; gp
100; MAGE proteins (MAGE 1, e.g., GenBank Accession No. M77481;
MAGE 2, e.g., GenBank Accession No. U03735; MAGE 3, MAGE 4);
TAG-72; CEA; MART; tyrosinase-related-protein 1 and 2 (TRP-1,
TRP-2) (see, e.g., Boon et al., Immunol. Today 16: 334-336, 1998);
CD20; Mucin 1 (e.g., GenBank Accession No. J03651) and Mucin 2; p53
(see e.g., Harris, et al., Mol. Cell. Biol., 6: 4650-4656, 1986)
and is deposited with GenBank under Accession No. M14694); mutant
p53 (e.g., GenBank Accession No. X54156 and AA494311); p21/ras,
p210/bcr-abl fusion polypeptides; c-myc; p97 melanoma antigen
(e.g., GenBank Accession No. M12154); blood antigens T, Tn and
sialyl-Tn; tuncated form of EGF; Lewis-Y antigen; squamous cell
carcinoma antigens (see, e.g., sequences described in U.S. Pat. No.
5,763,164); prostate specific antigen (PSA; Osterling, J. Urol.
145: 907-923, 1991; GenBank Accession No. X14810); epithelial
membrane antigen (Pinkus et al., Am. J. Clin. Pathol 85: 269-277,
1986); CYFRA 21-1 (Lai et al., 199Jpn. J. Clin. Oncol. 29: 421-421,
1999) and Ep-CAM (Chaubal et al., Anticancer Res. 19: 2237-2242,
1999). Epstein-Barr virus gene products also encode antigenic
polypeptides which are expressed in Hodgkin's lymphomas as well as
Burkits and other lymphomas. Products of the HTLV-1 genome have
been found in adult T cell leukemia cells, while human
papillomavirus (HPV) E6 and E7 gene products are found in cervical
carcinoma cells. In addition, Human herpesvirus 8 (HHV8) genomic
products have been found in Kaposi sarcomas.
[0152] Examples of transplant antigens include the CD3 receptor on
T cells. Treatment with an antibody to CD3 receptor has been shown
to rapidly clear circulating T cells and reverse most rejection
episodes.
[0153] Examples of autoimmune antigens include IAS chain.
Vaccination of mice with an 18 amino acid peptide from IAS chain
has been demonstrated to provide protection and treatment to mice
with experimental autoimmune encephalomyelitis. Autoimmune
polypeptides found in patients with system lupus are also
encompassed within the scope of the invention (e.g., GenBank
Accession No. D28394; Bruggen et al., Ann. Med Interne 147:
485-489, 1996). Additional antigens include .beta.-amyloid
antigens.
[0154] Antigenic peptides can also include allergens such as the
Der p I allergen (Hoyne, et al., Immunol. 83: 190-195, 1994); bee
venom phospholipase A2 (PLA) (Akdis, et al., J. Clin. Invest,
98:1676-1683, 1996); birch pollen allergen Bet v 1 (Bauer, et al.,
Clin. Exp. Immunol, 107: 536-541, 1997), and the multi-epitopic
recombinant grass allergen rKBG8.3 (Cao, et al., Immunol. 90:
46-51, 1997).
[0155] New antigens also can be identified using methods well known
in the art. Any conventional method, e.g., subtractive library,
comparative Northern and/or Western blot analysis of normal and
tumor cells, Serial Analysis of Gene Expression (U.S. Pat. No.
5,695,937) and SPHERE (described in PCT WO 97/3 5 03 5), can be
used to identify putative antigens for use.
[0156] Differential screening of nucleic acid sequences expressed
by the two cell lines can be used to select sequences encoding
antigens specific to cancer cells, and even specific stages of
cancer cells. When the non-target cell is a normal cell,
differential screening eliminates or reduces the nucleic acid
sequences common to normal cells, thereby avoiding an immune
response directed at antigens present on normal cells. When the
non-target cell is a normal cell, differential screening eliminates
or reduces sequences common to normal cells, thereby avoiding an
immune response directed at antigens present on normal cells.
[0157] For example, expression cloning as described in Kawakami et
al., 1994, Proc. Natl. Acad. Sci. 91: 3515-19, also can be used to
identify a novel tumor-associated antigen. Briefly, in this method,
a library of cDNAs corresponding to mRNAs derived from tumor cells
is cloned into an expression vector and introduced into target
cells which are subsequently incubated with cytotoxic T cells.
Pools of cDNAs that are able to stimulate T Cell responses are
identified and through a process of sequential dilution and
re-testing of less complex pools of cDNAs, unique cDNA sequences
that are able to stimulate the T cells and thus encode a tumor
antigen are identified. The tumor-specificity of the corresponding
mRNAs can be confirmed by comparative Northern and/or Western blot
analysis of normal and tumor cells.
[0158] SAGE analysis can be employed to identify the antigens
recognized by expanded immune effector cells such as CTLs, by
identifying nucleotide sequences expressed in the
antigen-expressing cells. SAGE analysis begins with providing
complementary deoxyribonucleic acid (cDNA) from an
antigen-expressing population and cells not expressing the antigen.
Both cDNAs can be linked to primer sites. Sequence tags are then
created, for example, using appropriate primers to amplify the DNA.
By measuring the differences in these tag sets between the two cell
types, sequences which are aberrantly expressed in the
antigen-expressing cell population can be identified.
[0159] Another method to identify optimal epitopes and new
antigenic peptides is a technique known as Solid PHase Epitope
REcovery ("SPHERE"). This method is described in detail in PCT WO
97/35035. Although used to screen for MHC class I-restricted CTL
epitopes, the method can be modified to screen for class II
epitopes by screening for the stimulation of antigen-specific MHC
class U specific T cell lines, for example, rather than CTL. In
SPHERE, peptide libraries are synthesized on beads where each bead
contains a unique peptide that can be released in a controlled
manner. Eluted peptides can be pooled to yield wells with any
desired complexity. After cleaving a percentage of the peptides
from the beads, these are assayed for their ability to stimulate a
Class I or Class II response, as described above. Positive
individual beads are then be decoded, identifying the
reactive-amino acid sequence. Analysis of all positives will give a
partial profile of conservatively substituted epitopes which
stimulate the T cell response being tested. The peptide can be
resynthesized and retested to verify the response. Also, a second
library (of minimal complexity) can be synthesized with
representations of all conservative substitutions in order to
enumerate the complete spectrum of derivatives tolerated by a
particular response. By screening multiple T cell lines
simultaneously, the search for crossreacting epitopes can be
facilitated.
[0160] Suitable antigen portions of polypeptides portions may be
readily identified by synthesis of relevant epitopes, and analysis
using methods routine in the art (see, e.g., Manca et al. Eur. J.
Immunol. 25:1217-1223, 1995; Sarobe, et al., J. Acquir. Immune
Defic. Syndr. 7: 635-40, 1994; Shirai, et al., J. Immunol. 152:
549-56, 1994; Manca, et al., Int. Immunol. 5: 1109-1117, 1993;
Ahlers, et al., J. Immunol. 150: 5647-65, 1993; Kundu and Merigan,
AIDS 6: 643-9, 1992; Lasarte, et al. Cell Immunol. 141: 211-8,
1992; and Hosmalin et al., J. Immunol. 146: 1667-73, 1991).
[0161] Isolated peptides can be synthesized using an appropriate
solid state synthetic procedure (Steward and Young, Solid Phase
Peptide Synthesis, Freemantle, San Francisco, Calif. 1968). A
preferred method is the Merrifield process (Merrifield, Recent
Progress in Hormone Res. 23: 451, 1967). Once an isolated peptide
is obtained, it may be purified by standard methods including
chromatography (e.g., ion exchange, affinity, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for protein purification. For
immunoaffinity chromatography, an epitope may be isolated by
binding it to an affinity column comprising antibodies that were
raised against that peptide, or a related peptide, and were affixed
to a stationary support. Alternatively, affinity tags such as
hexa-His (Invitrogen), Maltose binding domain (New England
Biolabs), influenza coat sequence (Kolodziej, et al., Methods
Enzymol. 194: 508-509, 1991), and glutathione-S-transferase can be
attached to the peptides to allow easy purification by passage over
an appropriate affinity column.
[0162] Isolated peptides also can be physically characterized using
such techniques as proteolysis, nuclear magnetic resonance, and ray
crystallography. Antigen mimitopes may be selected for that mimic
conformationally dependent antigenic epitopes as determined by
their ability to bind to conformationally dependent, neutralizing
antibodies.
[0163] Selection of the most appropriate portion of the desired
antigen protein for use as the antigenic domain can be done by
functional screening. The particular screening procedure depends
upon the type of antigen and the assays for its antigenic activity.
Antigenicity may be measured by stimulation of antigen-specific
MHC/HLA class I or MHC/HLA class II specific T cell line or clone.
Alternatively, antigenicity may be determined by measurement of the
ability to generate antibodies or T cells specific for the antigen
in vivo.
[0164] In one aspect, epitopes are identified which are recognized
by specific HLA haplotypes. For example, peripheral blood monocytes
(PBMCs) obtained from HIV-infected donors and expanded cultures are
obtained by restimulation with autologous PHA-stimulated
lymphoblasts for about a week. CTLs are cultured in a suitable
culture medium, e.g., such as RPMI 1640 (Gibco Life Technologies,
Glasgow, Scotland); 10% FCS (Gibco Life Technologies), antibiotics,
and 10% Lymphocult T (Biotest, Solihull, England) (IL-2) for
another week.
[0165] Standard .sup.51-Chromium release assays are performed using
HLA-matched or mismatched target B-lymphoblastoid cell lines
labeled with .sup.51-chromium (Amersham, Buckinghamshire, England)
and pulsed with a pool of epitope peptides predicted to bind to the
HLA molecule or a control peptide (e.g., such as an influenza
antigen) at about 50 .mu.M in multiple different wells of a
microliter plate. Individual peptides from a pool which reacts with
the HLA molecule to stimulate a CTL response are then tested to
identify the specific reactive peptide epitope. Chromium is counted
in a scintillation counter (e.g., such as available at Wallac,
Gaithersburg, Md.) and percent lysis calculated from the formula
100.times.(E-M/T-M), where E is the experimental release of
chromium, M is release in the presence of medium without detergent
(i.e., release which occurs because of a CTL response), and T is
release in the presence of 5% Triton X-100 detergent. Results are
regarded as positive if recognition of the HIV peptide is >10%
above that of a control peptide in at least two separate
assays.
[0166] Immunogenic portions may also be selected or validated in
animal models. For example, the HLA A2. 1/Kb transgenic mouse has
been shown to be useful as a model for human T-cell recognition of
viral antigens. In both the influenza and hepatitis B viral
systems, the murine T-cell receptor repertoire recognizes the same
antigenic determinants recognized by human T-cells. In both
systems, the CTL response generated in the HLA A2.1/Kb transgenic
mouse is directed toward virtually the same epitope as those
recognized by human CTLs of the HLA A2.1 haplotype (Vitiello et
al., J. Exp. Med. 173: 10074015, 1991; Vitiello et al., Abstract of
Molecular Biology of Hepatitis B Virus Symposia, 1992). In
particular, CTL induction in mice may be utilized to predict
cellular immunogenicity in humans (see, Warner et al., AIDS Res.
and Human Retroviruses 7: 645-655, 1991).
[0167] Alternatively, or additionally, immunogenic regions of a
polypeptide may be identified using computer programs for
identifying conserved regions amongst different pathogenic strains
of an organism or polypeptides associated with a pathology (e.g.,
such as an autoimmune disease) and/or to identify regions that bind
to particular MHC haplotypes (see, e.g., Falk, et al., Nature 351:
290, 1991). A number of software programs are known in the art. For
example, Peptgen generates maps of overlapping peptides. Motifscan
scans polypeptide sequences for possible epitopes based on HLA
binding motifs, while ELF (Epitope Location Finder Tool) may be
used to identify potential CTL epitopes. From this analysis,
peptides can be validated in appropriate in vitro or in viva
assays.
[0168] Having isolated and identified the peptide sequence of a
desired epitope, nucleic acids comprising sequences encoding these
epitopes can be sequenced readily.
[0169] The immunogenic portion(s) which are incorporated into an
expression construct (i.e., a nucleic acid molecule comprising an
expression control sequence operably linked to the immunogenic
portion or antigen-encoding sequence) may be of varying length,
although it is generally preferred that nucleic acid encode a
portion at least about 8-30 amino acids long, more preferably, 8-24
amino acids long. As discussed above, in some cases it is desirable
to provide repeating units of antigens optionally separated by
linker sequences or linked to helper sequences to facilitate
recognition by an appropriate MHC/HLA molecule.
Antigen-Encoding Vector Constructs
[0170] Antigenic material can be administered to a host organism
simultaneously along with the IL-15-encoding viral vector or
shortly before or after administration of the vector.
[0171] When isolated polypeptide vaccines are injected into a host,
the antigen is presented from the outside of the host cell and
often does not generate strong, long-lasting immune response, since
the polypeptide is not processed appropriately and is susceptible
to rapid clearance. Therefore, preferably, antigenic material is
provided by administering nucleic acids encoding the antigen.
[0172] Expression constructs comprising a nucleic acid sequence
encoding at least one antigen operably linked to a promoter can be
administered as linear fragments or circular molecules along with
the recombinant IL-15 vector. The constructs may further comprise
at least one origin of replication for replicating in at least one
type of host cell for amplification of the vector (e.g., such as E.
coli). Antigen encoding expression constructs can be administered
to the host organism as naked DNA or in a delivery vehicle
associated with one or more molecules for facilitating entry of the
expression construct into the cell. Suitable molecules include, but
are not limited to: liposomes; polypeptides; polysaccharides;
lipopolysaccharides; cationic molecules; viral particles, and the
like.
[0173] The antigen encoding expression unit may also be provided in
the form of a viral vector and the vector may be the same or
different as the recombinant IL-15-expressing vector. In one
aspect, the vector is a poxvirus vector, such as a vaccinia virus
vector. In another aspect, the antigen-encoding expression unit is
part of the same viral vector encoding IL-15. In one aspect,
expression of both the antigen and IL-15 is under the control of
individual poxvirus promoters.
[0174] The temporal course of expression of IL-15 and antigen may
be the same or different. For example, both IL-15- and
antigen-encoding sequences may be under the control of an early,
intermediate, or late promoter. However, in another aspect, the
sequences are under the control of promoters such that IL-15 and
antigen show different temporal expression patterns, e.g., IL-15 is
expressed first or antigen is expressed first. Preferably, the
antigen encoding sequence is under the control of a strong
promoter, such as an early/late hybrid promoter p7.5 to ensure its
expression throughout the replicative cycle of the virus. In still
another aspect, the nucleic acid sequence encoding IL-15 and the
nucleic acid encoding the vaccine antigen are expressed
coordinately on a single polycistronic mRNA (i.e., by including one
or more IRES sequences in the vector). See, for example, as
described in EP 0 803 573.
Enhancing and/or Inducing Immune Responses
[0175] Vertebrates exploit two basic strategies to mount immune
responses against antigens. Humoral immunity involves the direct
recognition of antigens by antibodies. Cellular immunity relies on
special cells which recognize and kill other cells which are
producing foreign antigens. Humoral immunity is mainly directed at
antigens which are exogenous (e.g., extracellular) whereas the
cellular system generally provides a response to intracellular
antigens.
[0176] The humoral system protects a vaccinated individual from
subsequent challenge from a pathogen and can prevent the spread of
an intracellular infection if the pathogen goes through an
extracellular phase during its life cycle; however, it can do
relatively little to eliminate intracellular pathogens. Cytotoxic
immunity complements the humoral system by eliminating the infected
cells.
[0177] In a natural process of viral infection, virus-infected
cells display viral antigens on their surface in the context of the
MHC-I or HLA class I receptor, while viral particles are digested
by professional antigen-presenting cells and display antigens in
association with MHC-II or HLA class II receptors. Both humoral and
cellular responses are necessary to prevent new infection. While
cytotoxic T cells (recognizing antigens presented in the context of
MHC I/HLA-I molecules) and antibodies are required to remove
extracellular pathogens, memory cells (i.e., CD4+ cells recognizing
antigens presented in the context of MHC II) are required to
prevent re-infection. However, cytotoxic T cells (CD8.sup.+ T
cells) can also provide a source of memory T cells, providing long
lasting CTL responses after an initial exposure to antigen. Thus,
effective vaccination should activate all of these types of immune
responses
[0178] The vaccine compositions according to the invention are used
to induce and/or enhance one or more immune responses. Such immune
responses generally involve one or more of production of antibodies
specific for a selected antigen; and production of antigen specific
T cells (e.g., helper cells, suppressor cells, and/or cytotoxic
cells). Preferably, the immune response comprises a memory response
that results in the production of memory CD8.sup.+ T cells and
vaccine antigen-specific antibodies.
[0179] Preferably, the compositions according to the invention
induce sustained polyclonal crossreactive CTL responses. In one
aspect, individuals vaccinated with compositions according to the
invention have a circulating frequency of CTL to vaccine antigens
of between about 1:2000 and 1:50,000 as determined by Elispot
analysis up to at least about 4 months, at least about 8 months, at
least about 10 months, at least about 12 months, at least about 14
months, at least about 16 months, at least about 18 months, at
least about 20 months, at least about 22 months, and at least about
24 months, Methods for performing Elispot analysis are known in the
art and described in Vogel, J. Clin. Invest. 102: 1758-1765, for
example.
[0180] The assay detects peptide-specific CTL-mediated IFN-.gamma.
release from peripheral blood mononuclear cells (PBMCs). In one
aspect, to perform the assay, 96-well nitrocellulose plates are
coated with an antibody to IFN-.gamma. (e.g., available from
Mabtech, Stockholm, Sweden) and PBMC are placed in wells at varying
concentrations (e.g., 2.times.10.sup.5, 10.sup.5 and
5.times.10.sup.4), preferably in duplicate. Suitable autologous
target cells are pulsed with either no peptide or one of a panel of
peptides selected according to the class I HLA-type of a particular
donor, at a final concentration of 10 .mu.M, and the plate is
incubated at 37.degree. C., 5% CO.sub.2 for about 16 hours allowing
IFN-.gamma. released by PBMCs to be detected by monitoring antibody
binding. Binding can be visualized using detector and conjugate
antibodies followed by chromagen to detect a color change as is
well known in the art. The assays are regarded as positive if there
is greater than about 10% specific lysis of peptide-pulsed target
cells at two different effector/target (E/T) ratios in at least two
separate experiments. Control assays are carried out under
identical conditions by using cells from uninfected patients with
the same class I alleles.
[0181] In one aspect, compositions according to the invention are
screened to identify those which elicit the production of
neutralizing antibodies. The presence of neutralizing antibodies
can be detected by premixing a virus sample and antibody sample
(e.g., obtained from sera of a vaccinated patient), inoculating
PBMCs or a cell line, culturing for a suitable time period (e.g.,
up to about two weeks) and measuring viral replication (e.g., via a
p24 or reverse transcriptase assay) or by detecting a lack of
cytopathic effect in the cells (e.g., by trypan blue or by
monitoring syncytia formation). A decrease in infectivity or cell
death indicates that the antibody sample is capable of neutralizing
the virus. A decrease in infectivity is reflected by decreased
levels of p24 or reverse transcriptase with respect to controls
which have not been exposed to antibody while a decrease in cell
death is indicated by significantly fewer dead cells and/or
syncytia.
[0182] PCR assays may also be used to detect the presence of
neutralizing antibodies. In one aspect, antibody and virus samples
are mixed with cells. After a suitable incubation period (e.g., 2-5
hours), cells are collected and PCR is performed to detect viral
sequences (such as the LTR or gag sequences). A decrease in
amplified products compared to controls lacking antibody is an
indicia of neutralization (i.e., fewer infection events). Antibody
and virus samples are mixed and applied to cells. After several
hours, cells are collected, DNA isolated and PCR performed to
detect LTR or gag region DNA. The PCR products are quantitated and
compared to controls lacking antibody. Neutralization is defined as
a decrease in the PCR signal, which correlates with fewer infection
events.
[0183] In still another aspect, a MAGI assay is performed to
measure virus infectivity. In this assay, HeLa cells stably
transfected with CD4 and a .beta.-galactosidase gene modified to
localize to the nucleus operably linked to a truncated HIV-1 LTR
which is activated by Tat. Thus, expression of .beta.-galactosidase
is dependent on the level of Tat which in turn is dependent on the
presence of virus. The Hela cells will stain blue and the number of
blue cells in a population will be proportional to the number of
infectious particles in an inoculum.
[0184] Efficacy of a vaccine composition also can be evaluated by
monitoring the level of the selected antigen and/or the presence of
antibodies in sera which specifically cross-react with the antigen.
For example, the efficacy of HIV vaccines can be monitored by
measuring viral titer at selected time intervals such as by
performing an immunoassay using an antibody specific for the HIV.
Highly sensitive nucleic acid-based tests may also be employed as
described in EP 617, 132, WO 94/20640, WO 92/02526 and U.S. Pat.
Nos. 5,451,503 and 4,775,619 for example. Viral load can be
monitored by measuring an amount of HIV RNA in plasma, cells or
tissue from a patient. Subsequent monitoring of the patient can
include periodic diagnostic tests following administration of the
vaccination therapy.
[0185] Vaccines may be tested initially in a non-human mammal
(e.g., a mouse or primate) as described further below in Examples
1-3. For example, assays of the immune responses of inoculated mice
can be used to demonstrate greater antibody production, T cell
proliferation, and cytotoxic T cell responses to the vaccine
compositions according to the invention. Vaccines can be evaluated
in Rhesus monkeys to determine whether a vaccine formulation that
is highly effective in mice will also elicit an appropriate monkey
immune response.
Dosage and Routes of Administration
[0186] The invention further provides pharmaceutical compositions
comprising recombinant IL-15 expressing poxvirus vectors and at
least one antigen source (i.e., the vaccine antigen). Preferably,
the antigen source is an expression construct comprising a nucleic
acid sequence encoding at least one antigen operably linked to an
expression control sequence. Also, preferably, the composition
comprises a pharmaceutically acceptable diluent, carrier, or
excipient carrier. Additionally the vaccine may also contain an
aqueous medium or a water-containing suspension, to increase the
activity and/or the shelf life of the vaccine. The
medium/suspension can include salt, glucose, pH buffers,
stabilizers, emulsifiers, and preservatives.
[0187] In addition to IL-15, other adjuvants may be included, e.g.,
including, but not limited to: muramyl dipeptide; aluminum
hydroxide; saponin; polyanions; anamphipatic substances; bacillus
Calmette-Guerin (BCG); endotoxin lipopolysaccharides; keyhole
limpet hemocyanin (GKLH); and cytoxan. However, it is a discovery
of the instant invention that IL-15 can potentiate a long-term
immune response without necessitating the use of other
adjuvants.
[0188] The invention also encompasses a kit including a recombinant
poxvirus encoding IL-15 and a nucleic acid encoding an antigen. The
recombinant poxvirus can be provided in lyophilized form for
reconstituting, for instance, in an isotonic aqueous, saline
buffer. The kit can include a separate contain containing a
suitable carrier, diluent or excipient. The kit can also include an
additional therapeutic agents, such as anti-cancer agents; agents
for ameliorating symptoms of a viral infection (e.g., such as a
protease inhibitor, Cimetidine (Smith/Kline, Pa.), low-dose
cyclophosphamide (Johnson/Mead, N.J.); and the like); and genes
encoding proteins providing immune helper functions (such as B-7);
and the like. In one aspect, the kit alternatively, or
additionally, includes an antigen presenting cell. Additionally,
the kit can include instructions for mixing or combining
ingredients and/or administering the kit components.
[0189] In one aspect, the invention provides a method of
administering a therapeutically effective vaccine compositions
according to the invention. The desired therapeutic effect
comprises one or more of reducing or eliminating viral load,
increasing numbers of CD4.sup.+ and/or CD8.sup.+ T cells or
antibodies which recognize the vaccine antigen; increasing overall
levels of CD4.sup.+ T cells; increasing levels of neutralizing
antibodies which recognize the antigen; decreasing the number of or
severity of symptoms of a disease; decreasing the expression of a
cancer specific marker; decreasing size or rate of growth of a
tumor; preventing metastasis of a tumor; preventing infection by a
pathogenic organism; and the like. The therapeutic effect may be
monitored by evaluating biological markers and/or abnormal
physiological responses. Generally, an effective dose of a
composition according to the invention comprises a viral titer that
can modulate an immune response against the vaccine antigen such
that memory T cells are generated which are specific for the
vaccine antigen.
[0190] Both the dose and the administration means can be determined
based on the condition of the patient (e.g., age, weight, general
health), risk for developing a disease, or the state of progression
of a disease. A preferred route of administration is by intradermal
scarification when the delivery vaccine vector is a poxvirus.
[0191] In one aspect, an effective amount of recombinant virus
ranges from about 10 .mu.l to about 25 .mu.l of saline solution
containing concentrations, preferably, of from about
1.times.10.sup.10 to 1.times.10.sup.11 plaque forming units (pfu)
virus/ml.
[0192] In a preferred aspect of the invention, a priming
immunization is performed, followed, optionally, by a booster
immunization at about 3-4 weeks after the priming immunization.
However, subsequent immunizations need not be provided until at
least about 4 months, about 6 months, about 8 months, about 12
months, about 10 months, about 16 months, about 18 months, or about
24 months after the priming boost. In one aspect, the vaccine is a
prophylactic vaccine, administered to a patient who has not been
exposed to the vaccine antigen, e.g., such as to an individual who
is HIV negative. In another aspect, the vaccine is administered
therapeutically, to a person who is seropositive for the vaccine
antigen (although not necessarily displaying symptoms) (i.e., such
as to an HIV positive individual). In a further aspect, the vaccine
is administered to an immunodeficient individual. In this aspect,
the vaccine preferably is derived from a replication defective
virus such as MVA or AVIPOX.
EXAMPLES
[0193] The invention will now be further illustrated with reference
to the following examples. It will be appreciated that what follows
is by way of example only and that modifications to detail may be
made while still falling within the scope of the invention.
Example 1
Dual Recombinant HIV gp160/IL-15 Increases the Magnitude and
Duration of Cellular Immune Responses
[0194] To demonstrate that co-delivery of immunostimulatory
cytokines with HIV antigens would result in a more robust immune
response, two recombinant vaccinia viruses that express HIV-1 gp160
in tandem with either human IL-2 or human IL-15 were generated. The
coding region of the human IL-15 gene was cloned into transfer
vector pSC11 for recombination with the WR vaccinia strain,
commercially available from the American Type Culture Collection
(ATCC No. VR-119).
[0195] To create recombinant vaccinia expressing human IL-15, the
coding region of IL-15 including 3 nucleotides upstream of the
start codon, ATG, and the entire coding region of IL-15 including
the TGA terminator codon and 4 nucleotides downstream of the
terminator codon, was used. The plasmid from which IL-15 sequences
were obtained is described in Burton, et al., Proc. Natl. Acad.
Sci. USA 91:4935-4939.
[0196] To create recombinant vaccinia expressing human IL-2, the
coding region of the human IL-2 gene was derived from the pTCGF-11
plasmid obtained from ATCC catalog number 39673 as a 0.8 kb Pst-1
fragment including in addition to the coding sequence, 17
nucleotides upstream of the start codon, ATG, and the entire coding
region of IL-2, including the terminator codon and 250 nucleotides
downstream of the terminator codon.
[0197] The pSC11 transfer vector (see, e.g., Toth, et al., Vet.
Microbiol. 45(2-3):171-83, 1995) used for integration of IL-2 or
IL-15 into the thymidine kinase ("tk") gene locus of the vaccinia
genome. In recombinants carrying only the cytokine (e.g., either
IL-15 vaccinia or IL-2 vaccinia), the respective cytokine was
integrated into the tk locus with insertional inactivation of the
viral tk gene.
[0198] The original pVOTE1 vector has been described in Ward, et
al., Proc. Natl. Acad. Sci. USA 92: 6773. In modifying the pVOTE1,
the DNA sequence between the Apa-I site and Sma-I site was removed
and replaced by a sequence having an early/late vaccinal promoter:
5'-CACCCATAAATAATAAATACAATAATTAATTTCTCGTAAAAGTA
GAAAATATATTCTAATTTATTGCACGGTAAGGAAGTAGAATCATAAAG
AACAGTGACGGATCCC-3' (SEQ ID NO: 1). This modified plasmid was then
used for integration of IL-2 or IL-15 into the hemagglutinin gene
locus of vaccinia genome. In the dual recombinants which express
both HIV gp160 and either IL-2 or IL-15, a HIV vaccinia viral
vector comprising gp160 was intergrated into the tk locus as
described in Earl, et al., J. Virol. 65: 31-41, 1991) used to
integrate either human IL-2 or IL-15-encoding sequences using the
modified pVOTE transfer vector comprising either IL-2 or IL-15
inserted into the hemagglutinin locus.
[0199] The immune responses of mice virus inoculated with virus
recombinants over a period of 14 months, were strikingly different
hi terms of CTL activity, both in magnitude and duration. Inbred
Balb/c female mice were immunized subcutaneously (at the base of
the tail) with 6.times.10.sup.6 pfu in 100 41 volume. After 3-4
weeks, animals were boosted with the same dose of the respective
virus as the primary immunization.
[0200] The data shown in FIGS. 1 and 2 depict CD8.sup.+ cells
displaying cytolytic activity towards HIV-1 gp120 peptide-pulsed
target cells. Splenic CD8.sup.+ T cells from each group of animals
were stimulated in vitro with 1.0 nM of immunodominant HIV gp120 V3
loop peptide P18-I10 for 7 days. These in vitro stimulated cells
were used as effector cells to lyse cognate peptide-pulsed P815
fibroblasts (H-2D.sup.d haplotype) target cells in a 5 hour
.sup.51Cr release assay. While the early response was superior in
the IL-2 group, in the late phase IL-15 group displayed a more
robust response.
[0201] As can be seen from the Figures, antigen-specific CTL
responses in mice receiving the recombinant IL-15 vector lasted
much longer than CTL responses observed in mice receiving
recombinant IL-2, e.g., to at least 14 months after the initial
injection.
[0202] The presence of antigen-specific CD8.sup.+ was quantitated
using labeled H2D.sup.d-p18-I10 tetramer using an assay as
described in Klenerman, et al., Nature Reviews/Immunology 2:
263-272, 2002. As shown in FIG. 3, CD8.sup.+ T cells positive for
an immunodominant gp120 were present in both groups of mice as
determined by tetramer staining. Mice receiving recombinant IL-15
comprised approximately four times as many gp120 specific CD8.sup.+
T cells as mice receiving recombinant IL-2.
[0203] As shown in FIG. 4, CD8.sup.+ T cells that produce
.gamma.-interferon upon exposure to an immunodominant HIV-1 gp120
peptide (i.e., memory CD8.sup.+ cells) are present at much higher
levels (approximately 4-fold) in mice vaccinated with vaccinia
expressing gp160 with IL-15 compared to mice vaccinated with
vaccinia expressing gp160 with IL-2 at 14 months, indicating long
term potentiation of memory CD8.sup.+ cells.
[0204] Thus, in all tests used, while the group of mice that
received the HIV gp160/IL-2 formulation responded vigorously, by
generating a gp120-specific CTL activity initially, the duration
and the magnitude of this CTL response was relatively short-lived.
The level of CTL activity in this group dropped to that of the
baseline unvaccinated control group by about 120 days post
vaccination. In contrast, the group of mice that received the
gp160/IL-15 formulation maintained high levels of gp120-specific
CTL activity beyond 14 months post vaccination (compare FIG. 1 vs
FIG. 2),
Example 2
Dual Recombinant Antigen/IL-15 Constructs Enhance Humoral Immune
Responses
[0205] Levels of antibodies specific for HIV gp120 in the sera of
vaccinated animals were evaluated by ELISA, using methods routine
in the art. Levels of gp120 specific antibody in mice vaccinated
with vaccinia that expressed HIV gp160 alone were undetectable at 8
months after vaccination (i.e., no different from unvaccinated
animals). However, mice that were vaccinated with the dual
recombinant vaccinia expressing HIV gp160 in tandem with IL-15
displayed high levels of gp120-specific antibodies even at this
late time point (see, FIG. 5).
Example 3
Recombinant Her-2/neu Vaccine Vectors
[0206] A plasmid comprising a human Her-2/neu encoding sequence as
described in Ye, et al., Mol. Cell. Biol. 16: 6178-6189, 1996 was
used to obtain the coding region of Her-2/neu which was
subsequently cloned into pSC11 and integrated into the tk locus of
WR strain of vaccinia to generate a Her-2/neu vaccinia virus
vector. The Her-2/neu/IL-15 dual recombinant was generated by
integrating IL-15 into the hemagglutinin locus of the recombinant
Her-2/neu vaccinia vector using the modified pVOTE vector
comprising
[0207] Recombinants were created in WR strain (ATCC#VR-119), Wyeth
Strain (ATCC#VR-1536) and MVA strain (ATCC-VR1508) backbones and
animals were vaccinated as described in the above Examples.
[0208] Animals vaccinated with recombinant vaccinia expressing the
Her-2/neu oncogene in tandem with IL-15 also demonstrated enhanced
cellular and humoral responses in a mouse model of breast cancer.
The specific over-expression of Her-2/neu oncogene in mammary
tissues in this model leads to spontaneous tumor formation in
mammary glands of the transgenic animals (see, e.g., Guy, et al.,
Proc. Natl. Acad. Sci. 89: 10578-10582, 1992). Animals vaccinated
with a recombinant vaccinia virus expressing the Her-2/neu oncogene
in tandem with IL-15 displayed a reduced tumor burden (see, FIGS. 6
and 7) and higher levels of Her-2/neu-specific antibodies (see,
FIG. 8) in their sera than animals vaccinated with a recombinant
vaccinia virus expressing Her-2/neu oncogene alone.
[0209] Thus it is clear that incorporating IL-15 in vaccine
virus-based vaccine formulations, augments both cell-mediated
immunity as well as antibody mediated humoral immunity against a
vaccine antigen, whether it be a viral antigen or a cancer-specific
antigen. In developing a vaccinia-based vaccine for either
infectious diseases or cancer, if the desired response is to
achieve a long lasting cell mediated and antibody mediated humoral
response in the vaccinees, for example as in the case of HIV or
smallpox, then incorporating IL-15 results in a superior vaccine.
It can also be beneficial in the case of subunit vaccines such as
rabies vaccine where the required response is to achieve a solid
antibody, response, to incorporate IL-15. As has been shown in
post-exposure rabies infections, administration of anti-rabies
antibody can effectively prevent the development of rabies. See,
e.g., Recommendations of the Advisory Committee on Immunization
Practices, MMWR 1999 48(No RR-1): 1-21 (available at
http://www.cdc.gov/mmwr). This illustrates that sufficiently
effective antibody levels alone can provide protection against a
vaccine antigen, such as rabies, without any involvement of
cell-mediated immune responses.
Example 4
Enhancing Immune Responses in Non-Human Primates
[0210] The above examples reinforce the notion that incorporation
of immunostimulatory cytokines in vaccine virus-based vectors can
significantly enhance the immune responses in vaccinees. To
demonstrate the production of a long term protective immune
response against a vaccine antigen in non-human primates, an MVA
(modified-virus Ankara) recombinant 89.6 env-gag-pol virus which
carries the HIV-1 envelope gene with SW gag and pol genes (see,
Amara, et al., Science 292: 6974, 2001) was used to generate dual
recombinants expressing IL-15 by homologous recombination and
insertion into the hemagglutinin locus with a pVOTE-modified vector
comprising IL-15 sequences. Construction of multivalent MVA vectors
is generally described in Amara, et al., Science, 2001, supra. For
comparison purposes, dual recombinant strains expressing the same
antigen(s) together with IL-2 (using a pVOTE modified vector to
integrate at the hemagluttin locus) were also generated.
[0211] A cohort of 23 juvenile rhesus monkeys (Macaca mulatta) is
used for immunization studies with the dual recombinant MVA viruses
generated as described above. Animals that express the major
histocompatibility complex (MHC) class I allele Mamu-A*01 are used,
or, alternatively, animals are typed to identify those which
express this allele using polymerase-chain reaction (PCR) analyses.
Primers A*-01/R (5'-GAC AGC GAC GCC GCG AGC CAA-3') (SEQ ID NO: 2)
and A*01/R (5'-GCT GCA GCG TCT CCT TCC CC-3') (SEQ ID NO: 3) are
used to identify monkeys with Mamu-A*01
[0212] It is currently accepted that the tetramer technology is a
reliable technique in quantitatively assessing CD8.sup.+ cells
specific for a particular antigen. In rhesus monkeys, the most
reliable and best-studied CTL epitopes for SIV mac gag polypeptide
is restricted by the HLA-A homologue molecule Mamu-A*01. Thus,
having monkeys with Mamu-A*01 haplotype permits use of tetrameric
Mamu-A*01/SIV gag epitopes to precisely quantify the CD8.sup.+ T
cell response in vaccinated animals See, e.g., Juroda, et al., J.
Exp. Med. 187: 1373-1381, 1998.
[0213] Animals are divided into 5 groups to be vaccinated
intradermally with 10.sup.9 pfu of recombinant MVA vaccinia virus
according to the following protocol: [0214] i) 5 animals receive
MVA vaccinia expressing the SHIV antigens; [0215] ii) 5 animals
receive dual recombinant MVA vaccinia expressing SHIV antigens with
IL-2; [0216] iii) 5 animals receive dual recombinant MVA vaccinia
expressing SHIV antigens with IL-15; [0217] iv) 4 animals remain as
unvaccinated controls but will be infected with the pathogenic
SHIV89.6P virus when vaccinated animals are challenged with this
virus; and [0218] v) 4 animals remain unvaccinated and uninfected
throughout the study and serve as healthy controls.
[0219] Following initial vaccination, animals are boosted twice in
4-week intervals with the respective viruses and their
immunological parameters are monitored for 8 months after the final
booster inoculation. Blood is collected from each individual animal
every 4 weeks and the following assays are done to monitor cellular
and humoral immune responses: Once the animals are challenged,
monthly assessments of immunological parameters are continue in the
infected animals until the termination of the study or
euthanization of the diseased animal.
[0220] Sera is tested for antibodies to oligomeric HIV envelope
gp140 using an ELISA assay initially as a screening test. See, as
described in Chen, et al., Nature Medicine 7: 1225-1231, 2001.
Virus neutralization assays on selected serum specimens from
sequentially collected serum samples from each animal are
performed, to determine the titer of neutralizing antibodies using
MT-2 cells infected with SHIV 89.6 virus using the assay as
described in Rose, et al., Cell 106: 539-549, 2001. Antibody titers
are measured to ascertain the effect of IL-2 and IL-15 on the
magnitude and the duration of the humoral response. However,
neutralizing antibodies to SHIV89.6 virus will not likely influence
the containment of challenge virus SHIV89.6P since these antibodies
are non-cross-neutralizing.
[0221] For animals expressing the Mamu-A*01 histocompatibility
type, the frequency of CD8.sup.+ T cells recognizing the dominant
Gag-CM9 epitope is determined by means of Mamu-A*01 tetramers. For
animals other than of Mamu-A*01 type, frequency of T cells
recognizing epitopes throughout the Gag polypeptide using pools of
overlapping peptides. See, e.g., as described in Amara, et al.,
2001, supra. Peripheral blood mononuclear cells (PBMC) are pulsed
with pools of overlapping gag peptides followed by dual labeling
for intracellular .gamma.-interferon expression and expression of
surface CD4 or CD8. Suitable monoclonal antibodies are commercially
available, e.g., from Biosource International, Camarillo, Calif.
Assay methodology is described in Rose, et al., Cell 106: 539-549,
2001.
[0222] To establish correlations between immune parameters ad
disease progression in the vaccinated animals, animals treated as
above are challenged with a pathogenic derivative of SHIV89.6,
i.e., SHIV89.6P. SHIV89.6P carries an R5/X4 envelope and induces a
rapid decline of CD4.sup.+ T cells. In unvaccinated animals, this
is associated with rapid onset of an AIDS-like disease. Animals are
challenged 8 months post-vaccination, when vaccine-induced effector
immune responses are not at peak levels, but when the memory phase
is more important in dictating immune responses to a virus
challenge. Thirty mucosal infectious doses of cell-free SHIV89.6P
virus are administered intrarectally since most HIV-1 infections
are transmitted across mucosal surfaces.
[0223] In addition to the assays described above, the following
assays are performed.
[0224] Clinical Assessment Of Infected Animals
[0225] Monkeys are monitored clinically by routine hematological
testing and blood chemistry analysis regularly (once a month)
following virus challenge.
[0226] Post-Challenge Viral Load Quantitation
[0227] Viral loads in plasma are assessed weekly by detecting SHIV
RNA using a real time PCR with a detection limit of 200 virus RNA
copy equivalents per ml. If plasma viremia turns out to be
negative, growth of virus from the PBMC of infected animals will be
attempted by a co-culture technique.
[0228] CD4.sup.+ T Lymphocyte Counts
[0229] Peripheral blood CD4 cell counts are done weekly following
virus challenge to detect and monitor any depletion of CD4 cells in
the infected animals. Rose, et al., Cell 106: 539-549, 2001.
[0230] By monitoring immunological parameters separately in each
individual animal sequentially, reliable and meaningful information
is maximized despite the small sample size and any genetic
heterogeneity in the study cohort.
[0231] Lymph Node Biopsies
[0232] Lymph node biopsies are taken from infected animals at
3-month intervals and cytomorphological analyses are performed on
them to evaluate the integrity of follicular architecture,
expansion or atrophy of germinal centers, and to identify the
presence and/or amount of follicular and paracortical depletions or
hyperplasia. In addition, the presence of SHIV89.6P viral RNA is
assessed by in situ hybridization in the lymph node material.
[0233] SHIV89.6P Virus Neutralizing Antibodies
[0234] ELISA antibody titers for oligomeric HIV envelope gp140 and
the presence of neutralizing antibodies against SHIV 89.6 and
SHIV89.6P are determined on a monthly basis, following challenge
with the SHIV89.6P. See, e.g., Chen, et al., Nat Med. 7: 1225-31,
2001; Shibata, et al., Nat. Med. 5: 204-10, 1995.
Example 5
Enhancement of CTL Avidity by IL-15
[0235] An ideal vaccine against viral pathogens and cancers must
induce high avidity CD8.sup.+ cytotoxic T lymophocytes (CTLs) and
the immunity provided by the vaccine should be long lasting. As
illustrated in FIG. 9, high avidity CD8.sup.+ CTLs provides more
effective and protective immunity by recognition of lower densities
of antigen in virus infection and tumor models. In contrast, low
avidity CD8+ CTLs provide less effective or no immunity. See, e.g.,
Ansari, et al., Cell. Immunol. 210(2): 125-42, 2001; Gray, et al.,
J. Virol. 75(21): 10065-72, 2001; Oh, et al. J. Immunol. 170(5):
2523-30, 2003; Pittet, et al., J. Immunol. 171 (4):1844-9 (2003);
Romieu, et al., J. Immunol, 161(10): 5133-7, 1998.
[0236] High avidity CD8.sup.+ CTLs can be selectively induced by
increasing costimulatory signals and by boosting appropriately. As
disclosed herein, long-lasting immunity can also be achieved by
using IL-15 as a molecular vaccine adjuvant.
[0237] As demonstrated in the Examples above, IL-15 can be used as
an adjuvant for an HIV vaccine because IL-15 results in
long-lasting immunity. IL-15 is also associated with increased
expression of anti-apoptotic proteins that can block high avidity
CD8.sup.+ CTL death. Animals immunized with recombinant vaccinia
expressing gp160 (vPE16), gp160 with IL-15 (vPE16/IL-15), or gp160
with IL-2 (vPE16/IL-2) induced a broad range of functional avidity
in CD8.sup.+ CTsL. The range of avidity of CD8.sup.+ CTLs narrowed
depending on the period after immunization. Surprisingly, however,
CD8.sup.+ CTL induced with IL-15 responded to 10-fold lower and
100-fold lower amounts of antigen than CD8.sup.+ CTLs induced with
vPE16 and vPE16/IL-2 did. As shown in FIG. 10, CD8.sup.+ CTLs
induced with IL-15 respond to lower density of antigen at 14 months
after boosting. As shown in FIGS. 11A-D, this difference increased
with time after immunization. As shown in FIGS. 12A-C and FIGS.
13A-C, increased avidity over time correlates with increased
specific cytolytic activity of CD8+ CTLs. This effect was more
pronounced for CD8.sup.+ CTLs expanded with low concentrations of
peptide (0.001 .mu.M) (FIGS. 13A-C). Similarly, high avidity
CD8.sup.+ CTLs induced with IL-15 persisted for longer periods of
time in vivo, up to 14 months after boosting (FIGS. 14A and B).
[0238] Further, IL-15R.alpha. expression levels on CD8.sup.+ T
cells bearing different avidities were found to be different (not
shown). High avidity CD8.sup.+ T cells expressed higher levels of
IL-15R.alpha., but not IL-2R.beta. or IL-7R.alpha., suggesting that
IL-15R.alpha. on CD8.sup.+ CTLs and IL-15 in vivo control the life
of CD8.sup.+ CTLs with different avidities. Consistent with this
result, data showed that high avidity CD8.sup.+ CTLs expressed
increased levels of apoptotic proteins and proliferated better than
low avidity CD8.sup.+ CTLs in response to IL-15. These findings
support a mechanism in which IL-15 preferentially contributes to
the maintenance of high avidity CD8.sup.+ CTLs. In addition, high
avidity CD8.sup.+ CTLs express higher levels of CD8.beta..
[0239] These results indicate that IL-15, at the time of priming,
selects or induces a different phenotype CTLs with greater avidity
and longevity, providing more effective CTL immunity when IL-15 is
included as an adjuvant.
[0240] Variations, modifications, and other implementations of what
is described herein will occur to those of ordinary skill in the
art without departing from the spirit and scope of the
invention.
[0241] All of the references, patents, and patent applications
identified above, are expressly incorporated herein by reference in
their entireties.
Sequence CWU 1
1
31108DNAArtificial SequenceDescription of Artificial Sequence
Synthetic ologonucleotide 1cacccataaa taataaatac aataattaat
ttctcgtaaa agtagaaaat atattctaat 60ttattgcacg gtaaggaagt agaatcataa
agaacagtga cggatccc 108221DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 2gacagcgacg ccgcgagcca a
21320DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 3gctgcagcgt ctccttcccc 20
* * * * *
References