U.S. patent application number 12/845350 was filed with the patent office on 2010-11-18 for immunogenicity using a combination of dna and vaccinia virus vector vaccines.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Dept. of Health and Human Services. Invention is credited to Genoveffa Franchini, Zdenek Hel, George Pavlakis, James Tartaglia.
Application Number | 20100291037 12/845350 |
Document ID | / |
Family ID | 22741747 |
Filed Date | 2010-11-18 |
United States Patent
Application |
20100291037 |
Kind Code |
A1 |
Franchini; Genoveffa ; et
al. |
November 18, 2010 |
IMMUNOGENICITY USING A COMBINATION OF DNA AND VACCINIA VIRUS VECTOR
VACCINES
Abstract
This invention relates to improved methods of inducing an immune
response for the prevention or treatment of HIV-1 infection by
using a nucleic acid vaccine in conjunction with a recombinant
viral vaccine, e.g., a poxvirus vaccine, to potentiate and broaden
the immune response. The present invention further provides a
particularly effective vaccine regimen comprising a DNA vaccine
used in combination with a poxvirus virus, especially NYVAC or
ALVAC.
Inventors: |
Franchini; Genoveffa;
(Washington, DC) ; Hel; Zdenek; (Rockville,
MD) ; Pavlakis; George; (Rockville, MD) ;
Tartaglia; James; (Aurora, CA) |
Correspondence
Address: |
NIH-OTT;c/o Sheridan Ross P.C.
1560 Broadway, Suite 1200
Denver
CO
80202-5141
US
|
Assignee: |
The United States of America, as
represented by the Secretary, Dept. of Health and Human
Services
Bethesda
MD
|
Family ID: |
22741747 |
Appl. No.: |
12/845350 |
Filed: |
July 28, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11428815 |
Jul 5, 2006 |
7771729 |
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12845350 |
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10258570 |
Oct 25, 2002 |
7094408 |
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PCT/US2001/013968 |
Apr 28, 2001 |
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11428815 |
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60200444 |
Apr 28, 2000 |
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Current U.S.
Class: |
424/93.2 |
Current CPC
Class: |
A61K 2039/53 20130101;
A61P 37/04 20180101; A61K 2039/54 20130101; A61K 2039/545 20130101;
C12N 2740/15022 20130101; Y10S 530/826 20130101; C12N 2710/24143
20130101; C12N 2740/16222 20130101; C12N 2740/15034 20130101; A61K
2039/5256 20130101; A61K 39/21 20130101; C12N 2740/16034 20130101;
C12N 2710/24043 20130101; A61K 39/12 20130101; C12N 2740/16122
20130101; A61P 31/18 20180101; C07K 14/005 20130101; C12N 15/86
20130101; C12N 2740/16322 20130101 |
Class at
Publication: |
424/93.2 |
International
Class: |
A61K 35/76 20060101
A61K035/76; A61P 37/04 20060101 A61P037/04; A61P 31/18 20060101
A61P031/18 |
Claims
1-14. (canceled)
15. A method of potentiating a CD8+ response to human
immunodeficiency virus-1 (HIV-1) epitopes in a human comprising:
administering an expression vector encoding HIV-1 Gag, Pol, Pro,
Tat, Nef, Rev, Vif, Vpr or Env antigens; and administering a
recombinant Modified Vaccinia virus Ankara (MVA) encoding the same
antigens encoded by the expression vector; wherein the expression
vector and the recombinant MVA enter the cells of the human and
intracellularly produce HIV peptides that are presented on the
cell's MHC class I molecules in an amount sufficient to stimulate a
CD8+ response, and further, wherein administration of the
combination of the expression vector and the recombinant MVA
potentiates the immune response compared to administration of
either the expression vector or the recombinant MVA by itself.
16. The method of claim 15, wherein the expression vector is a DNA
expression vector.
17. The method of claim 15, wherein the HIV peptides are structural
viral peptides.
18. The method of claim 15, wherein the HIV peptides are
non-structural viral peptides.
19. The method of claim 15, wherein the expression vector or the
recombinant MVA is administered with an adjuvant.
20. The method of claim 15, further comprising two administrations
of the expression vector.
21. The method of claim 15, further comprising three
administrations of the expression vector.
22. The method of claim 15, wherein the expression vector is
administered before the recombinant MVA.
23. The method of claim 15, wherein the human is infected with
HIV-1.
24. The method of claim 23, wherein the human has a viral load of
less than 10,000 copies per milliliter.
25. The method of claim 15, wherein the human is not infected with
the HIV-1.
26. The method of claim 15, wherein the HIV peptides are HIV-1
envelope, gag or protease peptides.
27. A method of reducing viral load in a mammal that is infected
with an immunodeficiency virus, comprising: administering to the
mammal an expression vector that expresses encoded immunodeficiency
virus Gag, Pol and Env antigens; and later administering to the
mammal a recombinant MVA encoding said antigens; wherein the
expression vector and the recombinant MVA enter the cells of the
mammal and intracellularly produce said antigens, and wherein
administration of the combination of the expression vector and the
recombinant MVA reduces the viral load more than administration of
either the expression vector or the recombinant MVA by itself.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Continuation under 37 C.F.R.
1.53(b) of U.S. application Ser. No. 10/258,570, now U.S. Pat. No.
7,094,408, having a 371(c) date of Oct. 25, 2002; which is the
national phase application of PCT/US01/13968 filed Apr. 20, 2001;
which claims the benefit of priority of U.S. Provisional
Application 60/200,444, filed Apr. 28, 2000; which applications are
herein incorporated by reference.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] [Not Applicable]
FIELD OF THE INVENTION
[0003] This invention relates to improved methods of inducing an
immune response for the prevention or treatment of human
immunodeficiency virus-1 (HIV-1) infection by using a nucleic acid
vaccine in conjunction with a recombinant viral vaccine, e.g., a
poxvirus vaccine, to potentiate and broaden the immune response.
The present invention further provides a particularly effective
vaccine regimen comprising a DNA vaccine used in combination with a
recombinant poxvirus virus, especially NYVAC or ALVAC.
SUMMARY OF THE INVENTION
[0004] The present invention is directed to a method of stimulating
an immune response in a human at risk for infection, or infected
with, an HIV-1 retrovirus. The method comprises administering a
first vaccine, frequently a nucleic acid vaccine, which enters the
cells and intracellularly produces HIV-specific peptides for
presentation on the cell's MHC class I molecules in an amount
sufficient to stimulate a CD8.sup.+ immune response. The first
vaccine may be used in combination with a second vaccine comprising
another modality, e.g., a recombinant pox virus vector. The use of
the combination of vaccines potentiates the immune response
relative to the use of either of the vaccines alone.
[0005] The vaccine combination may be administered prophylactically
to individuals at risk of HIV infection. To prevent infection,
individuals who are non vaccinia-naive may particularly benefit.
Alternatively, patients already infected with HIV may receive the
vaccine regimen therapeutically. Patients who are candidates for
treatment with the vaccine regimen of the invention include those
who have a viral load of less than 10,000 viral copies per ml of
plasma and a CD4.sup.+ cell count of above 500 cells/ml. Other
patients include those who have been treated with one or more
anti-viral agents.
[0006] The method frequently employs a nucleic acid vaccine that is
a DNA vaccine in combination with an attenuated recombinant virus.
A preferred virus is an attenuated pox virus, particularly NYVAC
and ALVAC, attenuated vaccinia and canarypox viruses respectively.
The DNA vaccine and/or the recombinant virus vaccine may be
administered one or more times. The DNA vaccine is preferably
administered prior to administration of the recombinant virus
vaccine, frequently in multiple doses. In one embodiment, a DNA
vaccine encoding various HIV antigens, or epitopes derived from the
antigens, is administered multiple times prior to administration of
a NYVAC-HIV vaccine.
[0007] The vaccine may also comprise interleukin-2 (IL-2) or CD40
ligand in an amount that is sufficient to further potentiate the
CD8.sup.+ and CD4.sup.+ T-cell responses.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIG. 1 shows the study design evaluating a combination DNA
vaccine/NYVAC vaccine in Rhesus macaques. Three groups of 8 rhesus
macaques each were included. The animals were immunized four times
with either mock NYVAC (group A) or NYVAC-SIV-gag-pol-env (group
B), or three times with DNA-SIV-gag-env followed by two
immunizations with NYVAC-SIV-gag-pol-env at the times
indicated.
[0009] FIG. 2 shows lymphoproliferative responses to gp120 (upper
panel) and p27 antigens (lower panel) in the three groups
inoculated in accordance with the study design set forth in FIG.
1.
[0010] FIG. 3 shows the frequency of Gag181-specific CD8.sup.+
T-cells in peripheral blood monocytes (PBMC) of vaccinated macaques
as measured by IFN-.gamma. ELISPOT assay at the times indicated.
"S.F.C." indicates spot-forming cells/per million; "N.D." indicates
not done. An asterisk above the bar marks the values obtained by
assaying frozen cells.
[0011] FIG. 4 shows Gag181-specific tetramer staining of fresh PBMC
at week 53 and 76. The cells depicted in the Figure were first
gated for CD3.sup.+ population.
[0012] FIG. 5 shows the T-cell responses to various SIV epitopes
measured using ELISPOT and .sup.51Cr-release assays.
[0013] FIG. 6 shows the average group viremia during the first 28
days following intrarectal challenge with SIVmac251. Points
represent group means with standard error indicated by the bar.
[0014] FIG. 7 shows the average value of Gag181 tetramer-specific
staining in all MAMU-A*01-positive animals in each group after
challenge. Points represent the mean of the percentage of Gag181
tetramer-positive cells of the total CD3.sup.+ CD8.sup.+ T-cell
population with standard errors indicated by bars.
[0015] FIG. 8 shows the average group viremia in MAMU A*01-positive
and MAMU A*01-negative animals of control and DNA/NYVAC-SIV
vaccinated (group C) animals. In the control group, the results
obtained in two studies were combined. Points represent group means
with standard errors indicated by the bars.
DEFINITIONS
[0016] "Attenuated recombinant virus" refers to a virus that has
been genetically altered by modern molecular biological methods,
e.g. restriction endonuclease and ligase treatment, and rendered
less virulent than wild type, typically by deletion of specific
genes or by serial passage in a non-natural host cell line or at
cold temperatures.
[0017] "Efficient CD8.sup.+ response" is referred to as the ability
of cytotoxic CD8.sup.+ T-cells to recognize and kill cells
expressing foreign peptides in the context of a major
histocompatibility complex (MHC) class I molecule.
[0018] "Nonstructural viral proteins" are those proteins that are
needed for viral production but are not necessarily found as
components of the viral particle. They include DNA binding proteins
and enzymes that are encoded by viral genes but which are not
present in the virions. Proteins are meant to include both the
intact proteins and fragments of the proteins or peptides which are
recognized by the immune cell as epitopes of the native
protein.
[0019] A "nucleic acid vaccine" or "naked DNA vaccine" refers to a
vaccine that includes one or more expression vectors that encodes
B-cell and/or T-cell epitopes and provides an immunoprotective
response in the person being vaccinated. As used herein, the term
does not include a recombinant pox viral vaccine.
[0020] "Plasma" refers to the fraction of whole blood resulting
from low speed centrifugation of EDTA- or heparin-treated
blood.
[0021] "Pox viruses" are large, enveloped viruses with
double-stranded DNA that is covalently closed at the ends. Pox
viruses replicate entirely in the cytoplasm, establishing discrete
centers of viral synthesis. Their use as vaccines has been known
since the early 1980's (see, e.g. Panicali, D. et al. "Construction
of live vaccines by using genetically engineered pox viruses:
biological activity of recombinant vaccinia virus expressing
influenza virus hemagglutinin", Proc. Natl. Acad. Sci. USA
80:5364-5368, 1983).
[0022] "Potentiating" or "enhancing" an immune response means
increasing the magnitude and/or the breadth of the immune response,
i.e., the number of cells induced by a particular epitope may be
increased and/or the numbers of epitopes that are recognized may be
increased ("breadth"). A 5-fold, often 10-fold or greater,
enhancement in both CD8.sup.+ and CD4.sup.+ T-cell responses is
obtained with administration of a combination of nucleic
acid/recombinant virus vaccines compared to administration of
either vaccine alone.
[0023] A "retrovirus" is a virus containing an RNA genome and an
enzyme, reverse transcriptase, which is an RNA-dependent DNA
polymerase that uses an RNA molecule as a template for the
synthesis of a complementary DNA strand. The DNA form of a
retrovirus commonly integrates into the host-cell chromosomes and
remains part of the host cell genome for the rest of the cell's
life.
[0024] "Structural viral proteins" are those proteins that are
physically present in the virus. They include the capsid proteins
and enzymes that are loaded into the capsid with the genetic
material. Because these proteins are exposed to the immune system
in high concentrations, they are considered to be the proteins most
likely to provide an antigenic and immunogenic response. Proteins
are meant to include both the intact proteins and fragments of the
proteins or peptides which are recognized by the immune cell as
epitopes of the native protein.
[0025] "Viral load" is the amount of virus present in the blood of
a patient. Viral load is also referred to as viral titer or
viremia. Viral load can be measured in variety of standard ways. In
preferred embodiments, the DNA/recombinant virus prime boost
protocol of the invention controls viremia and leads to a greater
reduction in viral load than that obtained when either vaccine is
used alone.
DETAILED DESCRIPTION
Introduction
[0026] Recombinant pox viruses vaccines, e.g., NYVAC- and
ALVAC-based vaccines for HIV-1 have been tested in preclinical
trials using either HIV-2 or SIV Gag, Pol, and Env genes in
macaques (see, e.g., Benson et al., J. Virol. 72:4170-4182, 1998;
Abimiku et al., J. Acquir. Immune Defic. Synd. Hum. Retrovirol.
15:S78-S85, 1997; Myagkikh et al., AIDS Res. Hum. Retroviruses
12:985-991, 1996; and Hel et al., Nat. Med. 16:1140-1146, 2000).
Results from these early studies indicated that, while these
vaccines do not protect from infection, they significantly reduce
the viral replication within a few weeks from exposure in
approximately 50% of the animals. In the case of NYVAC-SIV
vaccination, the regimen changed the natural course of SIV.sub.251
infection.
[0027] In the macaque animal model, the addition of monomeric gp120
protein administered as a boost in conjunction with ALVAC-SIVgpe
did not appear to improve the level of protection. (see, e.g., Pal
et al., Abstract for "HIV/AIDS Vaccine Development Workshop,"
Paris, France, May 5-6, 2000). These studies also suggested that
more than three immunizations with NYVAC-SIV/ALVAC-SIV may not
further increase the pool of memory cells, and that the vector
immunity against vaccinia protein may blunt the response to SIV
antigens.
[0028] Various other prime boost immunization strategies against
HIV have also been proposed (see, e.g., Barnett et al., AIDS Res.
and Human Retroviruses Volume 14, Supplement 3, 1998, pp.
S-299-S-309 and Girard et al., C R Acad. Sci. III 322:959-966, 1999
for reviews). DNA immunization, when used in a boosting protocol
with modified vaccinia virus Ankara (MVA) or with a recombinant
fowl pox virus (rFPV) in the macaque model, has been shown to
induce CTL responses and antibody responses (see, e.g., Hanke et
al, J. Virol. 73:7524-7532, 1999; Hanke et al., Immunol. Letters
66:177-181; Robinson et al., Nat. Med. 5:526-534, 1999), but no
protection from a viral challenge was achieved in the immunized
animals. DNA immunization followed by administration of another
highly attenuated poxvirus has also been tested for the ability to
elicit IgG responses, but the interpretation of the results is
hampered by the fact that serial challenges were performed (see,
e.g., Fuller et al., Vaccine 15:924-926, 1997; Barnett et al.,
supra). In contrast, in a murine model of malaria, DNA vaccination
used in conjunction with a recombinant vaccinia virus was promising
in protecting from malaria infection (see, e.g., Sedegah et al.,
Proc. Natl. Acad. Sci. USA 95:7648-7653, 1998; Schneider et al.,
Nat. Med. 4:397-402, 1998).
[0029] The present invention provides for enhanced immunogenicity
of a recombinant poxvirus-based vaccine by administering a nucleic
acid, e.g., a DNA vaccine, to stimulate an immune response to the
HIV antigens provided in the poxvirus vaccine, and thereby increase
the ability of the recombinant pox virus, e.g., NYVAC or ALVAC, to
expand a population of immune cells.
[0030] Individuals who are treated with the vaccine regimen may be
at risk for infection with the virus or may have already been
infected.
Vaccines of Use in this Invention
[0031] Vaccines useful for the induction of CD8.sup.+ T-cell
responses comprise nucleic acid vaccines (preferably delivered as a
DNA vaccine) and recombinant pox virus vaccines that provide for
the intracellular production of viral-specific peptide epitopes
that are presented on MHC Class I molecules and subsequently induce
an immunoprotective cytotoxic T lymphocyte (CTL) response.
[0032] The invention contemplates single or multiple
administrations of the nucleic acid vaccine in combination with one
or more administrations of the recombinant virus vaccine. This
vaccination regimen may be complemented with administration of
recombinant protein vaccines, or may be used with additional
vaccine vehicles. Preferably, administration of the nucleic acid
vaccine precedes administration of the recombinant virus
vaccine.
[0033] In preferred embodiments, the DNA/recombinant virus prime
boost protocol controls viremia and reduces viral load as well as
potentiating a CD8.sup.+ response.
Attenuated Recombinant Viral Vaccines
[0034] Attenuated recombinant poxviruses that express
retrovirus-specific epitopes are of use in this invention.
Attenuated viruses are modified from their wildtype virulent form
to be either symptomless or weakened when infecting humans.
Typically, the genome of the virus is defective in respect of a
gene essential for the efficient production or essential for the
production of infectious virus. The mutant virus acts as a vector
for an immunogenic retroviral protein by virtue of the virus
encoding foreign DNA. This provokes or stimulates a cell-mediated
CD8.sup.+ response.
[0035] The virus is then introduced into a human vaccine by
standard methods for vaccination of live vaccines. A live vaccine
of the invention can be administered at, for example, about
10.sup.4-10.sup.8 organisms/dose, or 10.sup.6 to 10.sup.9 pfu per
dose. Actual dosages of such a vaccine can be readily determined by
one of ordinary skill in the field of vaccine technology.
[0036] The poxviruses are of preferred use in this invention. There
are a variety of attenuated poxviruses that are available for use
as a vaccine against HIV. These include attenuated vaccinia virus,
cowpox virus and canarypox virus. In brief, the basic technique of
inserting foreign genes into live infectious poxvirus involves a
recombination between pox DNA sequences flanking a foreign genetic
element in a donor plasmid and a homologous sequences present in
the rescuing poxvirus as described in Piccini et al., Methods in
Enzymology 153, 545-563 (1987). More specifically, the recombinant
poxviruses are constructed in two steps known in the art and
analogous to the methods for creating synthetic recombinants of
poxviruses such as the vaccinia virus and avipox virus described in
U.S. Pat. Nos. 4,769,330, 4,722,848, 4,603,112, 5,110,587, and
5,174,993, the disclosures of which are incorporated herein by
reference.
[0037] First, the DNA gene sequence encoding an antigenic sequence
such as a known T-cell epitope is selected to be inserted into the
virus and is placed into an E. coli plasmid construct into which
DNA homologous to a section of DNA of the poxvirus has been
inserted. Separately, the DNA gene sequence to be inserted is
ligated to a promoter. The promoter-gene linkage is positioned in
the plasmid construct so that the promoter-gene linkage is flanked
on both ends by DNA homologous to a DNA sequence flanking a region
of pox DNA containing a nonessential locus. The resulting plasmid
construct is then amplified by growth within E. coli bacteria.
[0038] Second, the isolated plasmid containing the DNA gene
sequence to be inserted is transfected into a cell culture, e.g.
chick embryo fibroblasts, along with the poxvirus. Recombination
between homologous pox DNA in the plasmid and the viral genome
respectively gives a poxvirus modified by the presence, in a
nonessential region of its genome, of foreign DNA sequences.
[0039] Attenuated recombinant pox viruses are a preferred vaccine.
A detailed review of this technology is found in U.S. Pat. No.
5,863,542, which is incorporated by reference herein. These viruses
are modified recombinant viruses having inactivated virus-encoded
genetic functions so that the recombinant virus has attenuated
virulence and enhanced safety. The functions can be non-essential,
or associated with virulence. The poxvirus is generally a vaccinia
virus or an avipox virus, such as fowlpox virus and canarypox
virus. The viruses are generated using the general strategy
outlined above and in U.S. Pat. No. 5,863,542.
[0040] Representative examples of recombinant pox viruses include
ALVAC, TROVAC, NYVAC, and vCP205 (ALVAC-MN120TMG). These viruses
were deposited under the terms of the Budapest Treaty with the
American Type Culture Collection (ATCC), 12301 Parklawn Drive,
Rockville, Md., 20852, USA: NYVAC under ATCC accession number
VR-2559 on Mar. 6, 1997; vCP205 (ALVAC-MN120TMG) under ATCC
accession number VR-2557 on Mar. 6, 1997; TROVAC under ATCC
accession number VR-2553 on Feb. 6, 1997 and, ALVAC under ATCC
accession number VR-2547 on Nov. 14, 1996.
[0041] NYVAC is a genetically engineered vaccinia virus strain
generated by the specific deletion of eighteen open reading frames
encoding gene products associated with virulence and host range.
NYVAC is highly attenuated by a number of criteria including: i)
decreased virulence after intracerebral inoculation in newborn
mice, ii) inocuity in genetically (nu.sup.+/nu.sup.+) or chemically
(cyclophosphamide) immunocompromised mice, iii) failure to cause
disseminated infection in immunocompromised mice, iv) lack of
significant induration and ulceration on rabbit skin, v) rapid
clearance from the site of inoculation, and vi) greatly reduced
replication competency on a number of tissue culture cell lines
including those of human origin.
[0042] TROVAC refers to an attenuated fowlpox that was a
plaque-cloned isolate derived from the FP-1 vaccine strain of
fowlpoxvirus which is licensed for vaccination of 1 day old
chicks.
[0043] ALVAC is an attenuated canarypox virus-based vector that was
a plaque-cloned derivative of the licensed canarypox vaccine,
Kanapox (Tartaglia et al., 1992). ALVAC has some general properties
which are the same as some general properties of Kanapox.
ALVAC-based recombinant viruses expressing extrinsic immunogens
have also been demonstrated efficacious as vaccine vectors. This
avipox vector is restricted to avian species for productive
replication. On human cell cultures, canarypox virus replication is
aborted early in the viral replication cycle prior to viral DNA
synthesis. Nevertheless, when engineered to express extrinsic
immunogens, authentic expression and processing is observed in
vitro in mammalian cells and inoculation into numerous mammalian
species induces antibody and cellular immune responses to the
extrinsic immunogen and provides protection against challenge with
the cognate pathogen.
[0044] NYVAC, ALVAC and TROVAC have also been recognized as unique
among all poxviruses in that the National Institutes of Health
("NIH")(U.S. Public Health Service), Recombinant DNA Advisory
Committee, which issues guidelines for the physical containment of
genetic material such as viruses and vectors, i.e., guidelines for
safety procedures for the use of such viruses and vectors which are
based upon the pathogenicity of the particular virus or vector,
granted a reduction in physical containment level: from BSL2 to
BSL1. No other poxvirus has a BSL1 physical containment level. Even
the Copenhagen strain of vaccinia virus--the common smallpox
vaccine--has a higher physical containment level; namely, BSL2.
Accordingly, the art has recognized that NYVAC, ALVAC and TROVAC
have a lower pathogenicity than any other poxvirus.
[0045] Another attenuated poxvirus of preferred use for this
invention is Modified Vaccinia virus Ankara (MVA), which acquired
defects in its replication ability in humans as well as most
mammalian cells following over 500 serial passages in chicken
fibroblasts (see, e.g., Mayr et al., Infection 3:6-14 (1975);
Carrol, M. and Moss, B. Virology 238:198-211 (1997)). MVA retains
its original immunogenicity and its variola-protective effect and
no longer has any virulence and contagiousness for animals and
humans. As in the case of NYVAC or ALVAC, expression of recombinant
protein occurs during an abortive infection of human cells, thus
providing a safe, yet effective, delivery system for foreign
antigens.
[0046] The HIV antigen-encoding DNA for insertion into these
vectors are any that are known to be effective antigens for
protection against a retrovirus. These can include both structural
and non-structural proteins. The envelope, polymerase, gag, and
protease are preferred proteins or sources of epitopes, but other
proteins or epitopes can also be employed including those proteins
encoded by non-structural genes, e.g., rev, tat, nef, vif, and vpr.
For HIV, nucleic acids that can be inserted into the viral vector
includes, but are not limited to, nucleic acid that can code for at
least one of HIV1gag(+pro)(IIIB), gp120(MN)(+transmembrane),
nef(BRU)CTL, pol(IIIB)CTL, ELDKWA or LDKW epitopes, preferably
HIV1gag(+pro)(IIIB), gp120(MN) (+transmembrane), two (2)
nef(BRU)CTL and three (3) pol(IIIB)CTL epitopes; or two ELDKWA in
gp120 V3 or another region or in gp160. The two (2) nef(BRU)CTL and
three (3) pol(IIIB)CTL epitopes are preferably CTL1, CTL2, pol1,
pol2 and pol3. In the above listing, the viral strains from which
the antigens are derived are noted parenthetically.
Nucleic Acid Vaccines
[0047] The vaccine combination of the invention typically includes
as one of the vaccines a nucleic acid vaccine, preferably DNA.
Nucleic acid vaccines as defined herein, typically plasmid
expression vectors, are not encapsidated in a viral particle. The
nucleic acid vaccine is directly introduced into the cells of the
individual receiving the vaccine regimen. This approach is
described, for instance, in Wolff et. al., Science 247:1465 (1990)
as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566;
5,739,118; 5,736,524; 5,679,647; and WO 98/04720. Examples of
DNA-based delivery technologies include, "naked DNA", facilitated
(bupivacaine, polymers, peptide-mediated) delivery, and cationic
lipid complexes or liposomes. The nucleic acids can be administered
using ballistic delivery as described, for instance, in U.S. Pat.
No. 5,204,253 or pressure (see, e.g., U.S. Pat. No. 5,922,687).
Using this technique, particles comprised solely of DNA are
administered, or in an alternative embodiment, the DNA can be
adhered to particles, such as gold particles, for
administration.
[0048] As is well known in the art, a large number of factors can
influence the efficiency of expression of antigen genes and/or the
immunogenicity of DNA vaccines. Examples of such factors include
the reproducibility of inoculation, construction of the plasmid
vector, choice of the promoter used to drive antigen gene
expression and stability of the inserted gene in the plasmid.
[0049] Any of the conventional vectors used for expression in
eukaryotic cells may be used for directly introducing DNA into
tissue. Expression vectors containing regulatory elements from
eukaryotic viruses are typically used in eukaryotic expression
vectors, e.g., SV40 CMB vectors. Other exemplary eukaryotic vectors
include pMSG, pAV009/A+, pMT010/A+, pMAMneo-5, and any other vector
allowing expression of proteins under the direction of such
promoters as the SV40 early promoter, SV40 later promoter,
metallothionein promoter, human cytomegalovirus promoter, murine
mammary tumor virus promoter, Rous sarcoma virus promoter,
polyhedrin promoter, or other promoters shown effective for
expression in eukaryotic cells.
[0050] Therapeutic quantities of plasmid DNA can be produced for
example, by fermentation in E. coli, followed by purification.
Aliquots from the working cell bank are used to inoculate growth
medium, and grown to saturation in shaker flasks or a bioreactor
according to well known techniques. Plasmid DNA can be purified
using standard bioseparation technologies such as solid phase
anion-exchange resins. If required, supercoiled DNA can be isolated
from the open circular and linear forms using gel electrophoresis
or other methods.
[0051] Purified plasmid DNA can be prepared for injection using a
variety of formulations. The simplest of these is reconstitution of
lyophilized DNA in sterile phosphate-buffer saline (PBS). This
approach, known as "naked DNA," is particularly suitable for
intramuscular (IM) or intradermal (ID) administration.
[0052] To maximize the immunotherapeutic effects of minigene DNA
vaccines, alternative methods for formulating purified plasmid DNA
may be desirable. A variety of methods have been described, and new
techniques may become available. Cationic lipids can also be used
in the formulation (see, e.g., as described by WO 93/24640; Mannino
& Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No.
5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci.
USA 84:7413 (1987). In addition, glycolipids, fusogenic liposomes,
peptides and compounds referred to collectively as protective,
interactive, non-condensing compounds (PINC) could also be
complexed to purified plasmid DNA to influence variables such as
stability, intramuscular dispersion, or trafficking to specific
organs or cell types.
Selection of an HIV Specific Epitope.
[0053] Highly antigenic proteins or epitopes for provoking an
immune response selective for a specific retroviral pathogen are
known. Typically, HIV is the target retroviral pathogen. With minor
exceptions, the following discussion of HIV epitopes is applicable
to other retroviruses except for the differences in sizes of the
respective viral proteins. Nucleic acids for inclusion in the
expression constructs can can include sequences encoding either
structural or non-structural proteins or epitopes corresponding to
regions of the proteins. The envelope, gag, and protease genes are
preferred proteins or sources of epitopes for inclusion in the
nucleic acid expression vector, but other proteins can also be
used. Non-structural genes include the rev, tat, nef, vif, and vpr
genes and these may also be included as components of the nucleic
acid vaccines used in the invention.
Characterization of the Immune Response in Vaccinated
Individuals
[0054] The vaccine regimen can be delivered to individuals at risk
for infection with HIV or to patients who are infected with the
virus. In order to assess the efficacy of the vaccine, the immune
response can be assessed by measuring the induction of CD4.sup.+,
CD8.sup.+, and antibody responses to particular epitopes. Moreover,
viral titer can be measured in patients treated with the vaccine
who are already infected. These parameters can be measured using
techniques well known to those of skill in the art. Examples of
such techniques are described below.
CD4.sup.+ T Cell Counts
[0055] To assess the effectiveness of the vaccine combination in a
recipient and to monitor the immune system of a patient already
infected with the virus who is a candidate for treatment with the
vaccine regimen, it is important to measure CD4.sup.+ T cell
counts. A detailed description of this procedure was published by
Janet K. A. Nicholson, Ph.D et al. 1997 Revised Guidelines for
Performing CD4+ T-Cell Determinations in Persons Infected with
Human Immunodeficiency Virus (HIV) in The Morbidity and Mortality
Weekly Report, 46(RR-2):[inclusive page numbers], Feb. 14, 1997.
Centers for Disease Control.
[0056] In brief, most laboratories measure absolute CD4.sup.+
T-cell levels in whole blood by a multi-platform, three-stage
process. The CD4.sup.+ T-cell number is the product of three
laboratory techniques: the white blood cell (WBC) count; the
percentage of WBCs that are lymphocytes (differential); and the
percentage of lymphocytes that are CD4.sup.+ T-cells. The last
stage in the process of measuring the percentage of CD4.sup.+
T-lymphocytes in the whole-blood sample is referred to as
"immunophenotyping by flow cytometry.
[0057] Immunophenotyping refers to the detection of antigenic
determinants (which are unique to particular cell types) on the
surface of WBCs using antigen-specific monoclonal antibodies that
have been labeled with a fluorescent dye or fluorochrome (e.g.,
phycoerythrin [PE] or fluorescein isothiocyanate [FITC]). The
fluorochrome-labeled cells are analyzed by using a flow cytometer,
which categorizes individual cells according to size, granularity,
fluorochrome, and intensity of fluorescence. Size and granularity,
detected by light scattering, characterize the types of WBCs (i.e.,
granulocytes, monocytes, and lymphocytes). Fluorochrome-labeled
antibodies distinguish populations and subpopulations of WBCs.
[0058] Systems for measuring CD4.sup.+ cells are commercially
available. For example Becton Dickenson's FACSCount System
automatically measure absolutes CD4.sup.+, CD8.sup.+, and CD3.sup.+
T lymphocytes. It is a self-contained system, incorporating
instrument, reagents, and controls.
[0059] A successful increase of CD4.sup.+ cell counts would be a
2.times. or higher increase in the number of CD4.sup.+ cells.
Measurements of CD8.sup.+Responses
[0060] CD8.sup.+ T-cell responses may be measured, for example, by
using tetramer staining of fresh or cultured PBMC, ELISPOT assays
or by using functional cytotoxicity assays, which are well-known to
those of skill in the art. For example, a functional cytotoxicity
assay can be performed as follows. Briefly, peripheral blood
lymphocytes from patients are cultured with HIV peptide epitope at
a density of about five million cells/ml. Following three days of
culture, the medium is supplemented with human IL-2 at 20 units/ml
and the cultures are maintained for four additional days. PBLs are
centrifuged over Ficoll-Hypaque and assessed as effector cells in a
standard .sup.51Cr-release assay using U-bottomed microtiter plates
containing about 10.sup.4 target cells with varying effector cell
concentrations. All cells are assayed twice. Autologous B
lymphoblastoid cell lines are used as target cells and are loaded
with peptide by incubation overnight during .sup.51Cr labeling.
Specific release is calculated in the following manner:
(experimental release-spontaneous release)/(maximum
release-spontaneous release).times.100. Spontaneous release is
generally less than 20% of maximal release with detergent (2%
Triton X-100) in all assays. A successful CD8.sup.+ response occurs
when the induced cytolytic activity is above 10% of controls.
[0061] Another measure of CD8.sup.+ responses provides direct
quantification of antigen-specific T cells by staining with
Fluorescein-labeled HLA tetrameric complexes (Altman, J. D. et al.,
Proc. Natl. Acad. Sci. USA 90:10330, 1993; Altman, J. D. et al.,
Science 274:94, 1996). Other assays include staining for
intracellular lymphokines, and .gamma.-interferon release assays or
ELISPOT assays. Tetramer staining, intracellular lymphokine
staining and ELISPOT assays all are sensitive measures of T cell
response (Lalvani, A. et al., J. Exp. Med. 186:859, 1997; Dunbar,
P. R. et al., Curr. Biol. 8:413, 1998; Murali-Krishna, K. et al.,
Immunity 8:177, 1998).
Viral Titer
[0062] There are a variety of ways to measure viral titer in a
patient. A review of the state of this art can be found in the
Report of the NIH To Define Principles of Therapy of HIV Infection
as published in the; Morbidity and Mortality Weekly Reports, Apr.
24, 1998, Vol 47, No. RR-5, Revised Jun. 17, 1998. It is known that
HIV replication rates in infected persons can be accurately gauged
by measurement of plasma HIV concentrations.
[0063] HIV RNA in plasma is contained within circulating virus
particles or virions, with each virion containing two copies of HIV
genomic RNA. Plasma HIV RNA concentrations can be quantified by
either target amplification methods (e.g., quantitative RT
polymerase chain reaction [RT-PCR], Amplicor HIV Monitor assay,
Roche Molecular Systems; or nucleic acid sequence-based
amplification, [NASBA.RTM.], NucliSens.TM. HIV-1 QT assay, Organon
Teknika) or signal amplification methods (e.g., branched DNA
[bDNA], Quantiplex.TM. HIV RNA bDNA assay, Chiron Diagnostics). The
bDNA signal amplification method amplifies the signal obtained from
a captured HIV RNA target by using sequential oligonucleotide
hybridization steps, whereas the RT-PCR and NASBA.RTM. assays use
enzymatic methods to amplify the target HIV RNA into measurable
amounts of nucleic acid product. Target HIV RNA sequences are
quantitated by comparison with internal or external reference
standards, depending upon the assay used.
Formulation of Vaccines and Administration
[0064] The administration procedure for recombinant virus or DNA is
not critical. Vaccine compositions (e.g., compositions containing
the poxvirus recombinants or DNA) can be formulated in accordance
with standard techniques well known to those skilled in the
pharmaceutical art. Such compositions can be administered in
dosages and by techniques well known to those skilled in the
medical arts taking into consideration such factors as the age,
sex, weight, and condition of the particular patient, and the route
of administration.
[0065] The vaccines can be administered prophylactically or
therapeutically. In prophylactic administration, the vaccines are
administered in an amount sufficient to induce CD8.sup.+ and
CD4.sup.+, or antibody, responses. In therapeutic applications, the
vaccines are administered to a patient in an amount sufficient to
elicit a therapeutic effect, i.e., a CD8.sup.+, CD4.sup.+, and/or
antibody response to the HIV-1 antigens or epitopes encoded by the
vaccines that cures or at least partially arrests or slows symptoms
and/or complications of HIV infection. An amount adequate to
accomplish this is defined as "therapeutically effective dose."
Amounts effective for this use will depend on, e.g., the particular
composition of the vaccine regimen administered, the manner of
administration, the stage and severity of the disease, the general
state of health of the patient, and the judgment of the prescribing
physician.
[0066] The vaccine can be administered in any combination, the
order is not critical. In some instances, for example, a DNA HIV
vaccine is administered to a patient more than once followed by
delivery of one or more administrations of the recombinant pox
virus vaccine. The recombinant viruses are typically administered
in an amount of about 10.sup.4 to about 10.sup.9 pfu per
inoculation; often about 10.sup.4 pfu to about 10.sup.6 pfu. Higher
dosages such as about 10.sup.4 pfu to about 10.sup.10 pfu, e.g.,
about 10.sup.5 pfu to about 10.sup.9 pfu, or about 10.sup.6 pfu to
about 10.sup.8 pfu, can also be employed. For example, a NYVAC-HIV
vaccine can be inoculated by the intramuscular route at a dose of
about 10.sup.8 pfu per inoculation, for a patient of 170
pounds.
[0067] Suitable quantities of DNA vaccine, e.g., plasmid or naked
DNA can be about 1 .mu.g to about 100 mg, preferably 0.1 to 10 mg,
but lower levels such as 0.1 to 2 mg or 1-10 .mu.g can be employed.
For example, an HIV DNA vaccine, e.g., naked DNA or polynucleotide
in an aqueous carrier, can be injected into tissue, e.g.,
intramuscularly or intradermally, in amounts of from 10 .mu.l per
site to about 1 ml per site. The concentration of polynucleotide in
the formulation is from about 0.1 .mu.g/ml to about 20 mg/ml.
[0068] The vaccines may be delivered in a physiologically
compatible solution such as sterile PBS in a volume of, e.g., one
ml. The vaccines can also be lyophilized prior to delivery. As well
known to those in the art, the dose may be proportional to
weight.
[0069] The compositions included in the vaccine regimen of the
invention can be co-administered or sequentially administered with
other immunological, antigenic or vaccine or therapeutic
compositions, including an adjuvant, a chemical or biological agent
given in combination with or recombinantly fused to an antigen to
enhance immunogenicity of the antigen. Additional therapeutic
products can include, e.g., interleukin-2 (IL-2) or CD40 ligand in
an amount that is sufficient to further potentiate the CD8.sup.+
and CD4.sup.+ T-cell responses. Such other compositions can also
include purified antigens from the immunodeficiency virus or from
the expression of such antigens by a second recombinant vector
system which is able to produce additional therapeutic
compositions. For examples, these compositions can include a
recombinant poxvirus which expresses other immunodeficiency
antigens or biological response modifiers (e.g., cytokines or
co-stimulating molecules). Examples of adjuvants which also may be
employed include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
Again, co-administration is performed by taking into consideration
such known factors as the age, sex, weight, and condition of the
particular patient, and, the route of administration.
[0070] The viral and DNA vaccines can additionally be complexed
with other components such as lipids, peptides, polypeptides and
carbohydrates for delivery.
[0071] The DNA vaccines are administered by methods well known in
the art as described in Donnelly et al. (Ann. Rev. Immunol.
15:617-648 (1997)); Felgner et al. (U.S. Pat. No. 5,580,859, issued
Dec. 3, 1996); Felgner (U.S. Pat. No. 5,703,055, issued Dec. 30,
1997); and Carson et al. (U.S. Pat. No. 5,679,647, issued Oct. 21,
1997). The vectors can also be complexed to particles or beads that
can be administered to an individual, for example, using a vaccine
gun. One skilled in the art would know that the choice of a
pharmaceutically acceptable carrier, including a physiologically
acceptable compound, depends, for example, on the route of
administration of the expression vector.
[0072] Vaccines may be delivered via a variety of routes. Typical
delivery routes include parenteral administration, e.g.,
intradermal, intramuscular or subcutaneous delivery. Other routes
include oral administration, intranasal, and intravaginal routes.
For DNA vaccines in particular, the vaccines can be delivered to
the interstitial spaces of tissues of an individual (Felgner et
al., U.S. Pat. Nos. 5,580,859 and 5,703,055). Administration of DNA
vaccines to muscle is also a frequently used method of
administration, as is intradermal and subcutaneous injections and
transdermal administration. Transdermal administration, such as by
iontophoresis, is also an effective method to deliver nucleic acid
vaccines to muscle. Epidermal administration of expression vectors
of the invention can also be employed. Epidermal administration
involves mechanically or chemically irritating the outermost layer
of epidermis to stimulate an immune response to the irritant
(Carson et al., U.S. Pat. No. 5,679,647).
[0073] The vaccines can also be formulated for administration via
the nasal passages. Formulations suitable for nasal administration,
wherein the carrier is a solid, include a coarse powder having a
particle size, for example, in the range of about 10 to about 500
microns which is administered in the manner in which snuff is
taken, i.e., by rapid inhalation through the nasal passage from a
container of the powder held close up to the nose. Suitable
formulations wherein the carrier is a liquid for administration as,
for example, nasal spray, nasal drops, or by aerosol administration
by nebulizer, include aqueous or oily solutions of the active
ingredient. For further discussions of nasal administration of
AIDS-related vaccines, references are made to the following
patents, U.S. Pat. Nos. 5,846,978, 5,663,169, 5,578,597, 5,502,060,
5,476,874, 5,413,999, 5,308,854, 5,192,668, and 5,187,074.
[0074] Examples of vaccine compositions of use for the invention
include liquid preparations, for orifice, e.g., oral, nasal, anal,
vaginal, etc. administration, such as suspensions, syrups or
elixirs; and, preparations for parenteral, subcutaneous,
intradermal, intramuscular or intravenous administration (e.g.,
injectable administration) such as sterile suspensions or
emulsions. In such compositions the recombinant poxvirus,
expression product, immunogen, DNA, or modified gp120 or gp160 may
be in admixture with a suitable carrier, diluent, or excipient such
as sterile water, physiological saline, glucose or the like.
[0075] The vaccines can be incorporated, if desired, into
liposomes, microspheres or other polymer matrices (Felgner et al.,
U.S. Pat. No. 5,703,055; Gregoriadis, Liposome Technology, Vols.
Ito III (2nd ed. 1993), each of which is incorporated herein by
reference). Liposomes, for example, which consist of phospholipids
or other lipids, are nontoxic, physiologically acceptable and
metabolizable carriers that are relatively simple to make and
administer.
[0076] Liposome carriers may serve to target a particular tissue or
infected cells, as well as increase the half-life of the vaccine.
Liposomes include emulsions, foams, micelles, insoluble monolayers,
liquid crystals, phospholipid dispersions, lamellar layers and the
like. In these preparations the vaccine to be delivered is
incorporated as part of a liposome, alone or in conjunction with a
molecule which binds to, e.g., a receptor prevalent among lymphoid
cells, such as monoclonal antibodies which bind to the CD45
antigen, or with other therapeutic or immunogenic compositions.
Thus, liposomes either filled or decorated with a desired immunogen
of the invention can be directed to the site of lymphoid cells,
where the liposomes then deliver the immunogen(s). Liposomes for
use in the invention are formed from standard vesicle-forming
lipids, which generally include neutral and negatively charged
phospholipids and a sterol, such as cholesterol. The selection of
lipids is generally guided by consideration of, e.g., liposome
size, acid lability and stability of the liposomes in the blood
stream. A variety of methods are available for preparing liposomes,
as described in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng.
9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and
5,019,369.
[0077] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0078] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
EXAMPLES
[0079] The following examples are provided by way of illustration
only and not by way of limitation. Those of skill will readily
recognize a variety of noncritical parameters which could be
changed or modified to yield essentially similar results.
Example 1
Administration of DNA Priming Vaccines in Combination with
NYVAC-SIV.sub.gag-pol-env Rhesus Macaques
[0080] The study design included 24 animals which were divided into
three groups, A, B, and C as follows:
[0081] Group A: eight animals vaccinated with the nonrecombinant
NYVAC control
[0082] Group B: eight animals vaccinated with
NYVAC-SIV.sub.gag-pol-env
[0083] Group C: eight animals vaccinated with 3 DNA immunizations
with 2 constructs expressing the Gag and Env proteins of
SIV.sub.239. followed by inoculation NYVAC-SIV.sub.gag-pol-env at
the indicated time.
[0084] In each group, macaques carrying the MHC class I molecule
MAMU-A*01 were included to quantitate the CD3.sup.+CD8.sup.+ T-cell
immune response in the blood. Animals were immunized with either 4
inoculation of 10.sup.8 pfu of NYVAC or NYVAC-SIV or with 3
inoculations of DNA (4 mg intramuscularly and 1 mg subcutaneously
of each plasmid) followed by two inoculation of NYVAC-SIV at the
indicated times (FIG. 1).
[0085] The following measure of immune response were obtained:
[0086] (1) in vitro lymphoproliferative responses to gp120 and p27
Gag
[0087] (2) ex vivo percentage of CD3.sup.+CD8.sup.+ T-cells
staining the p11C, C.fwdarw.M-Mamu-A*01 tetramer in peripheral
blood monocytes.
[0088] (3) expansion of the CD3.sup.+CD8.sup.+ tetramer-positive
population in vitro in the presence of a specific peptide (p11C,
C.fwdarw.M)
[0089] (4) ELISPOT for .gamma.-IFN secretion following stimulation
of CD8.sup.+ T-cells with virus-specific nonamers
[0090] (5) Serum Ab response to the Gag and gp120 env protein
antigens can also be measured.
[0091] The results showed that low levels of lymphoproliferative
responses to p27 Gag and gp120 were observed in animals in group B
(FIG. 2) immunized with NYVAC-SIV.sub.gag-pol-env alone. A marked
lymphoproliferative response was observed in Group C, however.
These animals received 3 inoculations of DNA prior to vaccination
with NYVAC-SIV.sub.gag-pol-env. High lymphoproliferative responses
to p27 Gag and Env occurred in seven of the eight animals (FIG. 2)
and overall, a difference of approximately ten-fold was observed in
comparison to group B animals. Additionally, in all of the
MAMU-A*01 animals, expansion of the ex vivo and cultured
tetramer-positive cells from the blood was observed.
[0092] To further assess whether DNA priming resulted in
potentiation and an increase in breadth of the immune response,
ELISPOT analysis of .gamma.-INF-producing cells following a
specific peptide stimulation was performed. The results are shown
in FIG. 3. The peptides used to stimulate the responses are shown
in the X-axes of the middle and bottom panels. An asterisk above
the bar (top panel) indicates values obtained by assaying frozen
cells (in control experiments, cell freezing decreased the number
of peptide-specific spots by 0-20%). Controls include
NYVAC-SIV-gpe-vaccinated, MAMU A*01-negative animal 17M, mock
NYVAC-vaccinated, MAMU A*01-negative animal 11 M, and mock
NYVAC-vaccinated, MAMU A*01-positive animal 671.
[0093] The combination of DNA vaccination with NYVAC-SIV
vaccination expanded the immunodominant response (p181) in all
animals in group C more than 10-fold compared to the animals in
group B (FIG. 3, upper panel). These responses were not only of a
greater magnitude, but also of longer duration. Moreover, the
animals in group C responded to more SIV epitopes and, again, the
responses were higher at 2 weeks following immunization (FIG. 3,
lower panels).
[0094] Gag181-specific tetramer staining of fresh PBMC at week 53
and week 76 was also performed. The results (FIG. 4) showed that
DNA vaccination also increased the frequency of memory T-cells
recognizing the p11C, C.fwdarw.M tetramer, as exemplified by the
detection of a clear population of these CD8.sup.+ T-cells in the
blood of five of five animals of group C and one of four in group B
at week 76.
[0095] Similarly, the functional activity of these cells in
cytolytic assays indicated that five of five macaques in group C
had CTL against viral epitopes whereas only two of five in group B
did (FIG. 5). The data in FIG. 5 show T-cell responses to various
SW epitopes measured using the ELISPOT and .sup.51Cr-release assay.
The bar charts represent the results of an IFN-.gamma. ELISPOT
assays with a specific MAMU A*01-restricted peptide indicated for
each set of bars at the indicated times. The values exceeding the
chart scale are indicated by number at the top of the bar.
Asterisks indicate the values obtained using frozen cells; all
other assays were performed using fresh PBMC. "Ctrl" indicates
unrelated control peptide; "N.D." indicates not done. Line charts
represent the percentage of a specific killing of unpulsed control
cells or cells pulsed with a specific MAMU A*01-restricted peptide.
All assays were performed using the cells from 7 day cultures with
a specific peptide at week 53 or 56, as indicated. "E:T" represents
the effector to target cell ratio. The percentage value in the top
right corner indicates the percentage of Gag181 tetramer-staining
CD3.sup.+CD8.sup.+ cells in culture PBMC.
[0096] Following intrarectal challenge with the highly pathogenic
SIV.sub.mac251(561) strain, most animals became viremic except one
of the animals in group C. The ability of vaccinated animals to
suppress viremia was assessed within the first 28 days and, as
demonstrated in FIG. 6, macaques immunized with DNA at first were
better able to control viremia than control macaques.
Interestingly, quantification of the anamnestic response in
vaccinated animals using the Gag 181 tetramer indicated that in DNA
primed animals, the response was higher and sustained (FIG. 7).
[0097] Because it has been previously demonstrated that
Mamu-A*01-positive animals are genetically advantaged and better
control viremia than Mamu-A*01-negative animals, virus load and
anamnestic response were investigated independently in
Mamu-A*01-positive and -negative macaques. As demonstrated in FIG.
8, analysis of virus load in Mamu-A*01-positive control animals
(including data from historical control animals challenged with the
same virus stock by the same route to increase the statistical
power, of our analyses) and vaccinated animals in group C
demonstrated a vaccine effect, and a similar effect was observed in
Mamu-A*01-negative macaques (bottom panel). Thus, DNA vaccination
ameliorated the virological outcome even in animals with an
inherent genetic predisposition to control viremia.
[0098] Data in the literature indicated that DNA priming followed
by MVA boost was not so effective in reducing virus load (see,
e.g., Hanke et al., J. Virol. 73:7524-7532, 1999). The
demonstration that DNA in combination with NYVAC significantly
improved viral outcome was surprising and may be dependent on
inherent features of this poxvirus vector.
[0099] These data demonstrate that DNA vaccination greatly
potentiates and increases the breadth of the immune response
induced by a NYVAC-based vaccine and shows that this vaccine
combination increases the immunogenicity and efficacy of the highly
attenuated poxvirus vectors.
[0100] ALVAC-based vaccine are similarly analyzed demonstrating
that they also potentiate the immune response when used in
conjunction with DNA vaccines.
Example 2
Administration to a Person
[0101] A vaccine regimen of a DNA priming vaccine followed by
inoculation with a vaccine such as NYVAC or ALVAC, is used
prophylactically in individuals at risk for HIV infection. (Such a
vaccine regimen can also be used therapeutically for HIV-infected
patients).
[0102] The individual is injected with a DNA priming vaccine that,
e.g, expresses the HIV-1 gag, pro, tat, nef, rev, and env genes.
Multiple priming inoculations are typically administered. The
amount of DNA administered is typically 800 .mu.g intramuscularly
or 200 intradermally. After an interval determined by the
physician, the patient is subsequently injected with a vaccine
comprising about 10.sup.8 pfu of a recombinant pox virus, e.g.
NYVAC, expressing HIV-1 gag, pro, tat, nef, rev, and env
epitopes.
[0103] The patient's immune response is evaluated (CD4.sup.+
proliferative response, cytotoxic CD8.sup.+ T-cell activity, etc.)
and a decision is made as to whether and when to immunize
again.
[0104] The combination of administration of the DNA vaccine
followed by immunization with the recombinant NYVAC vaccine
provides a protective immune response in uninfected patients and a
therapeutic effect in those individuals already infected with
HIV-1.
* * * * *