Note: Descriptions are shown in the official language in which they were submitted.
CA 02380018 2002-O1-28
WO 01/08702 PCT/US00/20641
IMMUNOTHERAPY IN HIV INFECTED PERSONS USING
VACCINES AFTER MULTI-DRUG TREATMENT
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application Serial Nos.
60/146,240, filed July 28, 1999; 60/178,989, filed January 28, 2000; and
60/200,445, filed
April 28, 2000, each of which is incorporated by reference herein.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY
SPONSORED RESEARCH AND DEVELOPMENT
[ Not Applicable
FIELD OF THE INVENTION
This invention relates to an improved method of maintaining an immuno-
protective response in persons infected with a retrovirus after highly active
anti-
retroviral therapy (HAART). Surpisingly, these patients demonstrate a CD8+
response following HAART treatment.
SUMMARY OF THE INVENTION
The present invention is directed to a method of stimulating an efficient CD8+
response in a human infected with an HIV or HTLV-1 retrovirus, who has a viral
load
of less than 10,000 viral copies, often 5,000 or 2,000 or less, per ml of
plasma and a
CD4+ cell count that is often above S00 cells/ml, but can be above 400
cells/ml or 300
cells/ml; and who has been treated with one or more anti-viral agents, which
contributed to a lower viral copy and higher CD4+ cell count than before
treatment. -
The method comprises administering a nucleic acid-based vaccine, which enters
the
cells and intracellularly produces HIV- or HTLV-1-specific peptides for
presentation
on the cell's MHC class I molecules in an amount sufficient to stimulate a
protective
CD8+ response.
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In a preferred embodiment the human has been treated with anti-viral agents,
which resulted in the human having a viral load of less than 1,000 viral
copies per ml
of blood serum and a CD4+ cell count that is above 500 cells/ml. The anti-
viral agents
can preferably comprise a combination of inhibitors of proteases and
inhibitors of
reverse transcnptase.
The method can use a vaccine that is a DNA based vaccine or that is an
attenuated recombinant virus. A preferred virus is an attenuated pox virus,
particularly NYVAC and ALVAC, attenuated vaccinia and canarypox viruses
respectively. Other attenuated pox viruses such as MVA can also be used.
The vaccine can further comprise an adjuvant and may be adiminstered a
second time. The vaccine can also comprise interleukin-2 (IL-2) and/or CD40
ligand
in an amount that is sufficient to potentiate the CD8+ response.
The method of the invention can be particularly useful for a person has who
been infected with HIV and has demonstrated repeated and sustained
proliferative T-
cell responses to gp120 envelope protein or both gp 120 envelope and p24 gag
antigen.
The person infected with HIV can be further tested by a skin test for a
hypersensitive response to the p24 gag antigen or to the gp120 envelope
antigen.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1: Figure 1 shows the viral load and proliferative CD4+ T-helper
responses to
p27 gag and gp 120 in the infected, treated vaccinated macaques. The top
panels
display the kinetics of virus load in the plasma animals from groups A, B, and
C. In
group A two animals (641 and 642) did not respond to therapy and were not
included
in the analyses.
Figure 2: Figure 2 shows the CD8+ response in the infected, treated,
vaccinated
animals.
Figure 3: Figure 3 shows p27-specific (Fig. 3a and 3c) and gp120-specific
(Fig. 3b
and 3d) T-cell proliferation in SIV-infected HAART-treated Rhesus macaques
following administration of a single dose of ALVAC-SIV~e. (Fig. 3a and Fig.
3b) or
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ALVAC (Fig. 3c and 3d). The arrows indicate time after SIV infection at which
the
animals were inoculated with vaccine.
Figure 4: Figure 4 shows the CD3+CD8+ T-cell responses detected in fresh
peripheral
blood mononuclear cells from SIV-infected, HAART-treated macaques inoculated
with ALVAC-SIV~e (Fig. 4a) or ALVAC (Fig. 4b).
Figure 5: Figure 5 shows the induction of CD8+ (Fig. 5a and Sb) and CD4+ (Fig.
5c
and Sd) T-cell responses following administration of a DNA vaccine to naive
macaques. DNA was administered at the times indicated by the arrow. The
plasmids
employed were pCMV-gag and pCMV-env, which are CMV expression plasmids
expressing the gag and env genes, respectively.
Figure 6: Figure 6 shows the induction of CD8+ (Fig. 6a and 6b) and CD4+ (Fig.
6c
and 6d) T-cell responses following administration of two inoculation of NYVAC-
SIV-gag pol-env to naive macaques. The vaccine was at the times indicated by
the
arrows.
Figure 7: Figure 7 shows the administration schedule of NYVAC vaccine with and
without interleukin-2 (IL-2) in HAART-treated macaques.
Figure 8: Figure 8 shows the amount of viral RNA (top panel) and the CD8+ and
CD4+ proliferative responses, top and lower panel respectively, in a NYVAC-SIV-
inoculated Rhesus macaque treated with IL-2. The horizontal bars indicate the
length
of treatment with anti-retroviral therapy (ART) and IL-2. Viral RNA levels in
the
plasmid (top panel) are indicated with open circles. The percent of CD8+CD3+
tetramer-binding cells is indicated in the top panel by the hatched vertical
bars.
ELISPOT assay results measuring the release of y-interferon are shown by the
black
vertical bars. Proliferative responses to p27 gag and gp120 are indicated in
the lower
panel. IL-2 was administered daily by subcutaneous injection at a dose of
120,000
amts.
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Definitions
"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.
"Efficient CD8+ response" is referred to as the ability of cytotoxic CD8+ T-
cells to recognize and kill cells expressing foreign peptides in the context
of a major
histocompatibility complex (MHC) class I molecule.
"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.
"Nucleic acid-based vaccines" include both naked DNA and vectored DNA
(within a viral capsid) where the nucleic acid encodes B-cell and T-cell
epitopes and
provides an immunoprotective response in the person being vaccinated.
"Plasma" refers to the fraction of whole blood resulting from low speed
centrifugation of EDTA- or heparin- treated blood.
"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).
A "retrovirus" is a virus containing an RNA genome and an enzyme, reverse
transcriptase, which is an RNA-dependent DNA polymerise 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.
"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
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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.
"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.
DETAILED DESCRIPTION
Introduction
This invention is a novel therapeutic modality for treating persons infected
with a lymphotropic or immune destroying retrovirus. A physician presented
with a
1 S patient whose immune system is compromised by retroviral infection can
elect to treat
that patient with a host of powerful antiviral agents including inhibitors of
viral
proteases and reverse transcriptase. This is known as highly active anti-
retroviral
therapy (HAART). The conventional HAART protocols are complex and difficult
for
patients to follow. The drugs also have a number of problematic side effects.
In
addition, these expensive and complicated treatments do not eliminate the
virus, but
merely hold the virus in check. If the patient is non-compliant, the viral
counts
rebound. Accordingly, for the vast majority of patients, a lifetime of drugs
is advised.
This invention is the discovery that after HIV infection, HAART treatment can
sufficiently restore a patient's immune system to effectively mount a CD8+
response
when a patient is provided with a CD8+-inducing vaccine. This response can
effectively maintain a low titer of virus and significantly reduce the patient
dependency on HAART when the CD8+-inducing vaccine is an HIV vaccine.. While
some such vaccines have been suggested as useful for seropositive patients
(U.S.
Patent No. 5,863,542 column 18, lines 60-63), they are surprisingly effective
for this
subpopulation of seropositive patients and not for other seropositive
patients.
Vaccines of use in this invention
Vaccines useful for the induction of CD8+ T-cell responses comprise nucleic
acid-based vaccines (delivered via a viral vector or directly as a DNA
vaccine) that
WO 01/08702 cA o23aooia 2oo2-oi-2a PCT/US00/20641
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.
The invention contemplates single or multiple administrations of a nucleic
acid-based
S vaccine as a direct DNA vaccine or as a recombinant virus vaccine, or both.
This vaccination
regimen may be complemented with administration of recombinant protein
vaccines (infra),
or may be used with additional vaccine vehicles.
Attenuated recombinant viral vaccines
Attenuated recombinant viruses 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. Among the recombinant
viruses
of use are adenoviruses, adeno-associated viruses, retroviruses and
poxviruses.
A recombinant, attenuated virus for use in this invention as a vaccine is a
virus
wherein the genome of the virus is defective with respect to a gene essential
for the efficient
production of, 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+response.
The virus is then introduced into a human vaccinee by standard methods for
vaccination of live vaccines. A live vaccine of the invention can be
administered at, for
example, about 104 -10g organisms/dose, or 106 to 109 pfu per dose. Actual
dosages of
such a vaccine can be readily determined by one of ordinary skill in the field
of vaccine
technology.
The selection of the virus is not critical. Examples of viral expression
vectors
include adenoviruses as described in M. Eloit et al, "Construction of a
Defective
Adenovirus Vector Expressing the Pseudorabies Virus Glycoprotein gp50 and its
Use as a
Live Vaccine", J. Gen. Virol., 71(10):2425-2431 (Oct., 1990).), adeno-
associated viruses
(see, e.g., Samulski et al., J. Virol. 61:3096-3101 (1987); Samulski et al.,
J. Virol. 63:3822-
3828 (1989)), papillomavirus, Epstein Barr virus (EBV) and Rhinoviruses (see,
e.g., U.S.
Patent No. 5,714,374). Human parainfluenza viruses are also reported to be
useful,
especially JS CP45 HPIV-3 strain. The viral vector may be derived from herpes
simplex
virus (HSV) in which, for example, the gene encoding glycoprotein H (gH) has
been
inactivated or deleted. Other suitable viral vectors include retroviruses
(see, e.g., Miller,
Human Gene Ther. 1:5-14 (1990); Ausubel et al., Current Protocols in Molecular
Biology).
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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 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.
First, the DNA gene sequence encoding an antigenic sequence, such as a
known T-cell epitope, is selected to be inserted into the virus. The sequence
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.
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.
Attenuated recombinant pox viruses are a preferred vaccine. A detailed
review of this technology is found in US Patent No. 5,863,542 which is
incorporated
by reference herein. 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;
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TROVAC under ATCC accession number VR-2553 on Feb. 6, 1997 and, ALVAC
under ATCC accession number VR-2547 on Nov. 14, 1996.
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+/nu+) 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.
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.
ALVAC is an attenuated canarypox virus-based vector that was a plaque-
cloned derivative of the licensed canarypox vaccine, Kanapox (Tartaglia et
al., AIDS
Res Hum Retroviruses 8:1445-7 (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. In 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.
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 BSLl physical
WO 01/08702 cA o23aooia 2oo2-oi-2a pCT/US00/20641
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.
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.
The HIV antigen encoding DNA for insertion into these vectors is any that is
known to be an effective antigen for protection against a retrovirus. For HIV
these
would include nucleic acid that can encode at least one of: HIV 1 gag( +
pro)(IIIB),
gp120(MN)( + transmembrane), nef(BRU)CTL, pol(IIIB)CTL, ELDKWA or LDKW
epitopes, preferably HIV 1 gag( + pro)(IIIB), gp 120(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, poll, pol2 and pol3. In the above listing,
the
viral strains from which the antigens are derived are noted parenthetically.
Direct DNA delivery vaccines
As an alternative to a viral vaccine, the nucleic acid can also be directly
introduced into the cells of a patient. This approach is described, for
instance, in
Wolff et. al., Science 247:1465 (1990) as well as U.S. Patent 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
(bupivicaine, polymers, peptide-mediated) delivery, and cationic lipid
complexes or
liposomes. The nucleic acids can be administered using ballistic delivery as
described, for instance, in Fynan et al., Proc Natl Acad Sci USA. 90:11478-82
(1993)
and U.S. Patent No. 5,204,253 or pressure (see, e.g., U.S. Patent No.
5,922,687).
Using this technique, particles comprised solely of DNA are administered, or
in an
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WO 01!08702 cA o23aooia 2oo2-oi-2a pCT/US00/20641
alternative embodiment, the DNA can be adhered to particles, such as gold
particles,
for administration.
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.
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 vectors. Other exemplary eukaryotic vectors
include
pMSG, pAV009/A+, pMT010/A+, pMAMneo-5, baculovirus pDSVE, 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, marine mammary tumor virus promoter, Rous sarcoma
virus promoter, polyhedrin promoter, or other promoters shown effective for
expression in eukaryotic cells.
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.
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 formulation, known as "naked DNA," is
particularly suitable for intramuscular (IM) or intradermal (ID)
administration.
To maximize the immunotherapeutic effects of plasmid 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'! Acad. Sci. USA
84:7413
WO 01/08702 cA o23aooia 2oo2-oi-2a PCT/US00/20641
(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.
Highly antigenic epitopes for provoking an immune response selective for a
specific retroviral pathogen are known. The retrovirus, HIV is a major problem
in the
United States and in the world. 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. HIV-specific epitopes fall into two major
categories,
structural and non-structural proteins. Epitopes can be selected from either
or both
groups of proteins. Structural proteins are a physical part of the virion. Non-
structural proteins are regulatory proteins. The envelope is a preferred
source of
epitopes and gp160, 120 and 41 are sources of immunoprotective proteins. Both
B
and T cell epitopes have been described in the literature and can be used.
Peptides
selected from the V3 loop of the HIV envelope proteins are of preferred use.
In
addition other structural proteins have been reported to be immunoprotectme
including p41, p17 and the gag protein. Non-structural genes include the rev,
tat, nef,
vif, and vpr genes.
Patients
A preferred patient population of retrovirally infected persons are those that
exhibit
repeated and sustained proliferative T-cell responses to envelope epitopes,
e.g., HIV gp120.
More preferred are those patients that also respond to the gag epitopes, e.g.
HIV p24.
Typically these patients are identified by measuring the ability of their
lymphocytes to
proliferate in responses to highly purified antigen. In brief, peripheral
blood monocytes
(PBMC) are collected and cultured in the absence of IL-2 and in the presence
of 10 ~g of
highly purified antigen. After four days the cultures are harvested and
proliferation is
measured by uptake of radioactive thymidine.
An alternative means of identifying these patients is to use a skin test. Skin
tests
involve the detection of a delayed type hypersenstive response (DTH) by means
of injecting
or scratching antigen beneath the surface of the skin. The reaction is
measured by the ability
or inability of a patient to exhibit hypersensitive response to an aqueous
solution of a gp 120
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or p24 antigen. Approximately, 1-20 wg is applied. The reaction is determined
by measuring
wheat sizes from about 24 to about 72 hours after administration of a sample,
and more
preferably from about 48 hours to about 72 hours after administration of a
sample. Preferred
wheat sizes for evaluation of the hypersensitivity of an animal range from
about 16 mm to
about 8 mm, more preferably from about 15 mm to about 9 mm, and even more
preferably
from about 14 mm to about 10 mm in diameter.
Highly Active Anti-Retroviral Therapy (HAART)
Antiviral retroviral treatment involves the use of two broad categories of
therapeutics. They are reverse transcriptase inhibitors and protease
inhibitors. There
are two type of reverse transcriptase inhibitors: nucleoside analog reverse
transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors.
Both
types of inhibitors block infection by blocking the activity of the HIV
reverse
transcriptase, the viral enzyme that translates HIV RNA into DNA which can
later be
incorporated into the host cell chromosomes.
Nucleoside and nucleotide analogs mimic natural nucleotides, molecules that
act as the building blocks of DNA and RNA. Both nucleoside and nucleotide
analogs
must undergo phosphorylation by cellular enzymes to become active; however, a
nucleotide analog is already partially phosphorylated and is one step closer
to
activation when it enters a cell. Following phosphorylation, the compounds
compete
with the natural nucleotides for incorporation by HIV's reverse transcnptase
enzyme
into newly synthesized viral DNA chains, resulting in chain termination.
Examples of anti-retroviral nucleoside analogs are: AZT, ddI, ddC, d4T , and
3TC in combination with AZT and Combivir.
Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a structurally and
chemically dissimilar group of antiretroviral compounds. They are highly
selective inhibitors
of HIV-1 reverse transcriptase. At present these compounds do not affect other
retroviral
reverse transcriptase enzymes such as hepatitis viruses, herpes viruses, HIV-
2, and
mammalian enzyme systems. They are used effectively in triple-therapy regimes.
Examples
of NNRTIs are Delavirdine and Nevirapine which have been approved for clinical
use in
combination with nucleoside analogs for treatment of HIV-infected adults who
experience
clinical or immunologic deterioration. A detailed review can be found in
"Nonnucleoside
Reverse Transcriptase Inhibitors" AIDS Clinical Care (10/97) Vol. 9, No. 10,
p. 75.
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Proteases inhibitors are compositions that inhibit HIV protease, which is
virally encoded and necessary for the infection process to proceed. Clinicians
in the
United States have a number of clinically effective proteases to use for
treating HIV-
infected persons. These include: SAQUINAVIR (Invirase); INDINAVIR (Crixivan);
S and RITONAVIR (Norvir).
CD4+ T cell counts
To assess a patient's immune system before antiviral treatment and after
treatment as well as to determine if the claimed vaccine regimen is working it
is
important to measure CD4+ 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 Yirus (HIV) in The Morbidity and Mortality Weekly Report,
46(RR-2):[inclusive page numbers], Feb 14, 1997, Centers for Disease Control.
1 S In brief, most laboratories measure absolute CD4+ T-cell levels in whole
blood
by a multi-platform, three-stage process. The CD4+ 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+ T-cells. The last stage in the process of measuring the percentage of
CD4+ T
lymphocytes in the whole-blood sample is referred to as "immunophenotyping by
flow cytometry.
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.
Systems for measuring CD4+ cells are commerically available. For example
Becton
Dickenson's FACSCount System automatically measure absolutes CD4+, CD8+, and
CD3+ T
lymphocytes. It is a self contained system, incorporating instrument,
reagents, and controls.
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Viral titer
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,
April
24, 1998, Vol 47, No. RR-5, Revised 6/17/98. It is known that HIV replication
rates
in infected persons can be accurately gauged by measurement of plasma HIV
concentrations.
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~],
NucliSensTM HIV-1 QT assay, Organon Teknika) or signal amplification methods
(e.g., branched DNA [bDNA], QuantiplexTM 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~ 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.
Measurements of CD8+ Responses
CD8+ T-cell responses can be measured, for example, by using tetramer
staining of fresh or cultured PBMC (see, e.g., Altman, J. D. et al., Proc.
Natl. Acad.
Sci. USA 90:10330, 1993; Altman, J. D. et al., Science 274:94, 1996), or y-
interferon
release assays such as ELISPOT assays (see, e.g., 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), or by using functional cytotoxicity assays. Each
of these
assays are well-known to those of skill in the art. For example, a
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
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are maintained for four additional days. PBLs are centrifuged over Ficoll-
Hypaque
and assessed as effector cells in a standard 51 Cr-release assay using U-
bottomed
microtiter plates containing about 104 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
5'Cr labeling. Specific release is calculated in the following manner:
(experimental
release-spontaneous release)/(maximum release-spontaneous release) x 100.
Spontaneous release is generally less than 20% of maximual release with
detergent
(2% Triton X-100) in all assays.
Formulation of Vaccines and Administration
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
1 S 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.
For example, NYVAC-HIV or other attenuated pox virus vaccines such as
ALVAC-HIV or MVA-HIV, is inoculated, more than once, by the intramuscular
route
at a dose of about 10g pfu per inoculation, for a patient of 170 pounds. The
vaccine
can be delivered in a physiologically compatible solution such as sterile PBS
in a
volume of, e.g., one ml. The dose can be proportional to weight.
The compositions can be administered alone, or can be co-administered or
sequentially administered with other immunological, antigenic, vaccine, or
therapeutic compositions. Such compositions can include other agents to
potentiate or
broaden the immune response, e.g., IL-2 or CD40 ligand, which can be
administered
at specified intervals of time, or continuously administered (see, e.g., Smith
et al., N
Engl JMed 1997 Apr 24;336(17):1260-1; and Smith, Cancer JSci Am. 1997 Dec;3
Suppl 1:S137-40). For example, IL-2 can be administered in a broad range,
e.g., from
10,000 to 1,000,000 or more units. Administration can occur continuously
following
vaccination. Often, low doses, e.g. 100,000 to 200,000, often 120,000, 150,000
or
170,000, units of IL-2 can be particularly useful.
WO 01/08702 cA o23aooia 2oo2-oi-2a PCT/US00/20641
Other compositions that can be co-administered can include purified antigens
from immunodeficiency virus or antigens that are expressed by a second
recombinant
vector system which is able to produce other therapeutic compositions. Such
compositions can include a recombinant poxvirus which expresses other
immunodeficiency antigens or biological response modifiers (e.g. cytokines; co-
stimulating molecules). 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.
DNA expression vectors for direct introduction of DNA into the patient tissue
can additionally be complexed with other components such as peptides,
polypeptides and
carbohydrates. Expression vectors can also be complexed to particles or beads
that can be
administered to an individual, for example, using a vaccine gun.
The expression vectors 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.
Patent No. 5,580,859, issued December 3, 1996); Felgner (U.S. Patent No.
5,703,055, issued
December 30, 1997); and Carson et al. (U.S. Patent No. 5,679,647, issued
October 21, 1997),
each of which is incorporated herein by reference. One skilled in the art
would know that the
choice of a pharmaceutically acceptable Garner, including a physiologically
acceptable
compound, depends, for example, on the route of administration of the
expression vector.
For example, naked DNA or polynucleotide in an aqueous carrier can be injected
into
tissue, such as muscle, in amounts of from 10 ~l per site to about 1 ml per
site. The
concentration of polynucleotide in the formulation is from about 0.1 ~g/ml to
about 20
mg/ml.
Vaccines can be delivered via a variety of routes. Typical delivery routes
include parenteral administration, e.g., intradermal, intramuscular or
subcutaneous
routes. Other routes include oral administration, intranasal, and intravaginal
routes.
The expression vectors of use for the invention can be delivered to the
interstitial
spaces of tissues of a patient (see, e.g., Felgner et al., U.S. Patent Nos.
5,580,859, and
5,703,055). Administration of expression vectors of the invention to muscle is
a particularly
effective method of administration, including intradermal and subcutaneous
injections and
transdermal administration. Transdermal administration, such as by
iontophoresis, is also an
effective method to deliver expression vectors of the invention to muscle.
Epidermal
administration of expression vectors of the invention can also be employed.
Epidermal
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WO 01/08702 GA o23aooia 2oo2-oi-2a PCT/US00/20641
administration involves mechanically or chemically irntating the outermost
layer of
epidermis to stimulate an immune response to the irntant (Carson et al., U.S.
Patent No.
5,679,647).
The vaccines can also be formulated for administration via the nasal passages.
Formulations suitable for nasal administration, wherein the Garner 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 Garner 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, US 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.
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 can be in admixture with a suitable Garner, diluent, or excipient such
as sterile
water, physiological saline, glucose or the like.
The vaccines can be incorporated, if desired, into liposomes, microspheres or
other polymer matrices (see, e.g., Felgner et al., U.S. Patent No. 5,703,055;
Gregoriadis, Liposome Technology, Vols. I to III (2nd ed. 1993). Liposomes,
for
example, which consist of phospholipids or other lipids, are nontoxic,
physiologically
acceptable and metabolizable Garners that are relatively simple to make and
administer. Liposomes include emulsions, foams, micelles, insoluble
monolayers,
liquid crystals, phospholipid dispersions, lamellar layers and the like.
Liposome Garners can serve to target a particular tissue or infected cells, as
well
as increase the half life of the vaccine. 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
17
WO 01108702 cA o23aooia 2oo2-oi-2a pCT/US00/20641
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. Patent Nos. 4,235,871, 4,501,728,
4,837,028, and
5,019,369.
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.
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
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 NYVAC-SIVgag por-env to SIV-infected, HAART-
treated Rhesus macaques
The effectiveness of a highly attenuated Poxvirus vector as a therapeutic
vaccine to enhance host-immune responses was investigated in the SIV25~ Rhesus
macaque model, which models HIV-1 infection in humans. The vaccine used for
this
study was a highly attenuated NYVAC-SIV gag pol-env recombinant vaccine that
was demonstrated to have efficacy as a preventative vaccine in earlier studies
(Benson
et al., J. Virol. 72:4170-4182 (1998).
The study design included 24 animals which were divided into three groups,
A, B, and C. All the animals were infected intraveneously with ten infectious
doses
of highly pathogenic SIVzsi (Pal). Following SIV25~ exposure, all twenty four
18
WO 01/08702 CA 02380018 2002-O1-28 PCT/US00/20641
animals became infected; the peak of plasma viremia occurred at approximately
two
weeks and ranged between 10' and 109 copies of viral RNA/ml of plasma in the
twenty four animals.
Two and a half weeks after infection, sixteen animals, those in groups A and
B, received a HAART regimen that in a pilot study had reduced viremia to
undetectable levels in 80% of macaques chronically infected with SIV2si. The
HAART regimen included oral administration of two doses of Stavudine (1.2
mg/day), intravenous inoculation of DDI (10 mg/kg/day) and subcutaneous
inoculation of PMPA (20 mg/kg/day). Animals in group C were not treated with
drugs. Animals in groups A and B, but not C, experienced a significant
decrease in
viremia (Figure 1, top panels).
In the sixteen animals of groups A and B, HAART treatment was continued
daily for 6 months. At weeks 10, 19, and 23 post-infection, the animals in
group A
received a placebo vaccine (non-recombinant NYVAC vector) and the animals in
groups B and C received 108 pfu of NYVAC-SIV gag pol-env vaccine. All twenty-
four animals were monitored weekly for viral RNA copies/ml of plasma and
biweekly
for lymphoproliferativ responses (LPR) to highly purified native p27 gag and
gp 120
env SIV proteins.
In order to follow the CD8+ T-cell responses, three MAMU-A*O1 (Macaca
mulata equivalent of HLA class I A*O1 (Kuroda et al., J. Exp. Med. 187:1373-
1381,
1998) were included in each group. MAMU-A*Ol animals are generally able to
recognizes the immunodominant peptide 1 lc-m within the gag antigen of SIV. A
tetramer binding assay was therefore used to directly quantitate CD8+ T-cell
responses
tn vavo. The tetramer, formed by four identical MAMU-A*O1 molecules conjugated
to the peptide l lc-m, was linked to fluorescent-labeled streptavidin and used
to stain
the CD8+ T-cells in the blood of macaques that expressed the appropriate T
cell
receptor complex on their surface. The percentage of total CD8+/CD3+ staining
with
the peptide MAMU-A*-1 tetramer was measured in several consecutive time
intervals
in each of the MAMU-A*O1 animals.
Staining of fresh or cultured PBMC with the MAMU A*Ol/peptide l lc-m
tetramer was performed in all the nine MAMU A*O1 animals included in the study
(three in each group) following SIVZS~ infection and after the second NYVAC-
SIV
vaccination. MAMU-A*O1/peptide-l lc-m-tetramer-staining CD8+ T-cells (ranging
from 0.8 to 4.6%) were induced by SIV infection in all nine animal within the
first
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WO 01/08702 cA o23aooia 2oo2-oi-2a PCT/US00/20641
month after viral exposure, and the CD8+ T-cell population was expanded (up to
values of approximately 70%) in vitro following specific peptide 1 lc-m
stimulation.
At two months after SIV2si exposure, the infected macaques seroconverted to
SIV2s~
antigens, including the animals in groups A and B that were treated with
HAART.
Measurements of LPR to gp 120 and p27 gag was consistently negative in all
twenty
four animals within the first four weeks following viral infection.
The frequency and extent of CD4+ T helper responses in HAART treated animals
is
increased by NYVAC-SIV vaccination.
As stated above, acute infection by SIVZS~ was associated with the absence of
a proliferative response to both p27 gag and gp120 env. However, responses to
p27
gag appeared in HAART-treated animals at approximately 10 weeks after
infection
and, these responses were more frequent in animals of group A than in animals
of
group C. No difference in the LPR to gp120 was observed between these two
groups
(Figure 1, middle and lower panels).
This notion was further supported by the finding that two animals (647 and
655) in group B failed to respond to therapy, maintained high virus load, and
did not
develop CD4+ T-cell proliferative response following NYVAC-SIV vaccination.
Further corroborating this notion, two animals in group C that naturally
controlled viremia developed LPR following NYVAC-SIV vaccination. Thus, it
appears that CD4+ T-helper memory responses are induced inefficiently by NYVAC-
SIV animals with high viremia. Several factors may contribute to this finding:
CD4+
T-cells are already activated in vivo and do not further proliferate in vitro
in LPR
assays and/or the vaccine-induced memory cells become targets for SIV
infection and
die upon further antigen stimulation. These data provide the first evidence
that a
highly attenuated live recombinant poxvirus vector vaccine can induce and
boost
sustained CD4+ helper immune response in the context of a pharmacologically
controlled lentiviral infection.
NYVAC-SIV vaccination increases CD8+lCD3+ MAMU A *01-tetramer positive cells
only in HAART treated animals.
SIV infection induced a large number of CD8+ T-cells that bound the MAMU-
A*O1 tetramer. This response by week four was reduced in most animals.
Following
the second and third NYVAC-SIV vaccinations, a high percentage of CD8+/CD3+ T-
WO 01/08702 cA o23aooia 2oo2-oi-2a PCT/US00/20641
cells bound tetramers in the fresh PBMC of all MAMU-A*O1 animals in group B,
but
in none of the animals in groups C (Figure 2). The specificity of tetramer
staining
was shown in parallel experiments using PBMC from animals with a different
haplotype as well as by the expansion of the cells from the nine MAMU-A*O1
animals in vitro following peptide-11 c-m-specific stimulation. Peptide 11 c-m
presented in the context of the MAMU-A*O1 haplotype is recognized by CD8+ T-
cells with cytolytic activity. (In Figure 2, the top panel shows the results
obtained in
the three MAMU-A*O1 animals from group A; middle panel, from group B; and
bottom panel, from group C. The percentage of MAMU-A*O1/peptide-1 lc-m-
tetramer-staining cells within the first 4 weeks was evaluated using only a-
CD8+
antibodies as a T cell marker whereas the data presented from weeks 19 through
29
were obtained using simultaneously a-CD-3+ and a-CD8+ antibodies in
conjunction
with the MAMU-A*Ol/peptide-l lc-m tetramers. For some of the MAMU-A*Ol-
positive animals, CTL activity obtained in cultured PBMC [weeks 19 and 20] or
fresh
PBMC [week 23] is also presented. The numbers in the abscissa represent the
effector-target ratio of the CTL assay system.)
To assess whether the detection of MAMU-A*Ol/peptide-1 lc-m-tetramer-
staining CD8+/CD3+ T-cells in the PBL of macaques was associated with CTL
activity, cytotoxicity assays were performed using homologous B cells from
each
animal pulsed with peptide 11 c-m. It was shown that CTL activity was measured
after in vitro stimulation of CD8+ T-cells in all animals tested at weeks 19
and 20,
although the extent of killing did not correlate with the percentage of
tetramer-
staining cells (Figure 2). Most notably, CTL assays performed on fresh CD8+ T
cells
at day 23 demonstrated significant CTL activity in group B animals, confirming
the
CTL functional activity of the high number of circulating CD8+ tetramer-
staining cells
in the blood of these animals (Figure 2). Thus, NYVAC-SIV vaccination induced
high levels of CD8+ responses only in animals in which viral replication was
suppressed by therapy.
Delayed T cell hypersensitivity (DTH) to viral p27 gag
Vaccines able to induce T-cell-mediated immunity are often able to induce DTH.
To
assess whether any of the animals vaccinated with NYVAC-SIV developed this
response,
either 1 or 10 ug of highly purified SIV p27 or HTLV-I p24, as controls, were
inoculated
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intradermally in animals in groups B and C. DTH reactivity was considered
positive when a
thickness of more than 10 mm manifested at 72 h postinoculation. Only three
animals in
group B and two animals in group C fulfilled the requirement for DTH
positivity.
The data presented in this example demonstrated that inoculations of NYVAC-SIV
following HAART greatly increased the frequency, extent, and duration of those
responses in
animals in which viremia was efficiently suppressed, indicating that the
ability to detect
vaccine-induced CD4+ T-cell helper responses was strictly dependent on the
level of viral
replication in the host. Similarly, NYVAC-SIV vaccination induced significant
expansion of
the number of CD8+/CD3+ MAMU-A*O1 cells specific for an immunodominant SIV gag
peptide only in animals treated effectively with antiviral therapy. Following
therapy
suspension, NYVAC-SIV-vaccinated animals were able to control viremia better
than
animals treated with antiviral therapy alone. These data demonstrate that
vaccination can
further induce both CD4+ and CD8+ T-cell responses in SIV-infected macaques.
Example 2. Immunization of a person infected with HIV.
A 35 year old male patient is seropositive for HIV and has a viral count in
his
plasma of 15000 copies per milliliter and a CD4+ count of 300. The patient is
treated
with a cocktail of antiviral agents which consist of: two nucleotide
inhibitors and one
protease inhibitor (Zaduvidine [three time a day], Lamivudine [two times a
day], and
Nesinavir [three times day] at preformulated doses. Typically, antiretroviral
therapy
is considered effective if a decrease of at least one log of the plasma viral
RNA is
observed. The plasma RNA load as well as the CD4+ T-cell count in the blood
will be
measured monthly.
After six months the patient is re-evaluated and is determined to have a viral
count in his plasma of 1500 copies per milliliter or less and a CD4+ count of
500. He
is then injected with an attenuated pox virus vector NYVAC carrying the
following
viral peptides: gag, pro, gp120-TM, pol and nef string of CTL epitopes. The
injection
comprises 10g pfu of the pox virus.
The patient's immune responses is evaluated (CD4+ proliferative response;
cytotoxic CD8+ T-cell activity, etc.) and a decision is made as to whether and
when to
immunize again. Typically, a maximum of three to four immunizations with
NYVAC-HIV-1 is considered. This regimen could be followed by three to four
immunizations with ALVAC-HIV-1 (carrying a similar HIV-I genetic content) and,
if
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necessary, an additional regimen of DNA-only i.e., DNA that is not in a viral
vector,
immunization could follow. NYVAC-HIV-1 followed by ALVAC-HIV-1 is effective
in inducing immunoresponses in chimpanzees. Similarly, immunization regimens
as
follows have been shown to be effective: NYVAC followed by DNA and ALVAC
followed by DNA. Thus, a vaccination regimen including all of the above
vaccines
may be used.
Often, the vaccine regimen is administered with IL-2, preferably at low doses
such as 100,000 to 200,000 units of IL-2 administered daily. CD40+ ligand can
also
be included in the treatment protocol, either by itself or administerd in
conjunction
with the IL-2 treatment.
Example 3. Administration of ALVAC-SIVgPe to SIV-infected, HAART-treated
Rhesus
macaques
The effectiveness of a highly attenuated ALVAC vector as a therapeutic
1 S vaccine to enhance host-immune responses in HAART-treated animals was
investigated in
the SIV25~ Rhesus macaque model, which models HIV-1 infection in humans. The
study
includes 16 macaques inoculated with the SIV25, virus and treated with the
HAART regimen
as described in Example 1 at day 15 and thereafter. Of those macaques, 8 are
immunized
with a total of 3 doses of 10g pfu of the mock-vaccine ALVAC vector (group D)
and the
remaining 8 (group E) with a recombinant ALVAC-SIV-gag pol-env vector (ALVAC-
SIV~), which is analogous to the NYVAC vector of Example 1.
The data obtained after a single dose of the ALVAC-SIV~,e vaccine indicated
that the ALVAC-SIV~e vaccine is able to boost CD4+ T-cell responses against
the p27 Gag
protein as well as to the gp120 Env forefront of the vaccine (see Figure 3).
In addition, a
specific CD8+ T-cell response detected using the MAMU A*O1/peptide l lc-m
tetramer
reagent (see, e.g., Example 1) was also boosted by the ALVAC-SIV~,e (Figure
4), and
further, those CD8+ T-cells could be expanded in culture. The mock-vaccinated
animals did
not experience an expansion of these immune responses.
Thus, ALVAC-SIV~e is immunogenic in animals undergoing HAART
therapy. An ALVAC vaccine analogous to the NYVAC vaccine of Example 2 can
similarly
be employed to treat HIV-infected individuals as described in Example 2 who
are undergoing
HAART.
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w0 01/08702 CA 02380018 2002-0l-28 PCT/~500/20641
Example 4. MVA-SIV gag pol-env immunization of SIV-infected macaques.
The effectiveness of an MVA-SIV vaccine was evaluated using methodology
analogous to that used to evaluate NYVAC and ALVAC SIV gag pol-env vaccines.
The
CD8+ and CD4+ responses were determined in SIVZSi-infected macaques that were
able to
control viremia, i.e., the CD4+ T-cell counts were above 500, following
administrations of a
single dose of 10g pfu of MVA-SIV-gag pol-env recombinant vaccine. The results
showed
that both CD4+ and CD8+ responses could be expanded in infected animals.
Thus, MVA-SIV-gag pol-env is also immunogenic in animals and can be used
in patients undergoing HAART therapy.
Example 5. Comparison of the Immunogenicity of DNA and NYVAC vaccines.
A vaccine regimen comprising administration of DNA alone or DNA in
combination with NYVAC-SIV or ALVAC-SIV, or administration of a combination of
NYVAC-SIV and ALVAC-SIV is also effective in continuous boost of the immune
response
in SIV25~-infected animals.
A study conducted in parallel compared 2 inoculations of 10g pfu of NYVAC-
SIV~e and 3 inoculations of DNA, which was administered using 4mg
intramuscularly and 1
mg intradermally of each plasmid, in naive animals. The DNA vaccine induced
CD4+ and
CD8+ T-cell responses that were equivalent to those induced by a NYVAC-SIV~e
vaccine
(Figures 5 and 6). ALVAC-SIV~e was at least as immunogenic as NYVAC-SIV~,e
(see, e.g.,
Example 1) and NYVAC-SIV~e was as immunogenic as DNA. Thus, all three vaccines
either alone or in various combinations can be used in HIV-I-infected
individuals.
ALVAC-SIV~,e was also able to induce both CD4+ and CD8+ T-cell response
in chronically infected animals (CD4+ T-cell range 50-900) and was able to
suppress viremia
in the absence of HAART. These animals, which had been previously vaccinated
with a
NYVAC-SIV~,e-based vaccine, appear to model long-term progressor HIV-I-
infected
individuals. The vaccines of the invention can therefore be used alone or in
various
interchangeable combinations in early infection as well as late infection in
individuals in
which viremia is controlled pharmacologically or otherwise.
Example 6. Therapeutic Vaccination with NYVAC and IL-2.
This examples demonstrates the ability of immunomodulatory
molecules, e.g. IL-2, to further expand both CD4+ and CD8+ T-cell responses
induced
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WO 01/08702 cA o23aooia 2oo2-oi-2a PCT/US00/20641
by the NYVAC-SIVgag pot-env vaccine and increase the breadth of the host
response to
the virus.
SIV2si-infected HAART-treated Rhesus macaques (Figure 7) were
vaccinated intramuscularly with NYVAC-SIVgag_po~-en~ with or without
simultaneous
and continued daily treatment with IL-2 (120,000 ItJ) administered
subcutaneously.
A control group of animals was treated with IL-2 and mock-vaccinated (NYVAC
nonrecombinant vector). All 15 macaques in the study responded to HAART (10
mg/kg/day DDI, intravenously; 2.4 mg/kg/day Stavudine, orally; 10 mg/kg/day
PMPA, subcutaneously) and in 13 animals viremia was suppressed below 5 x 103
copies/ml within the first 4 weeks of treatment. Viremia in the remaining 2
animals
became undetectable by weeks 6 and 8 of treatment. Proliferative responses to
p27
Gag and gp120 were increased by NYVAC-SIVgag pot_e"~. vaccination up to three-
and
twelve-fold, respectively, regardless of IL-2 treatment, indicating that
either IL-2 does
not increase bulk proliferative response or that the assay was not sensitive
enough to
measure subtle variation in the antigen-specific CD4+ T-helper response.
Approximately half of the animals in the study were genetically
selected as carriers of the Mamu-A*Ol molecules. CTL CD8+ T-cell responses to
SIVZS~ were therefore measured by ELISPOT using several purified SIVmac239
nonamer peptides and their corresponding Mamu-A*O1 tetramers. The ex vivo PBMC
of all SIVZSi-infected Mamu-A*O1 animals recognized the immunodominant
p1 IC,C-~M peptide and produced y-interferon (y-INF) following in vitro
stimulation.
This response was further expanded following immunization with NYVAC-
SIVgag_Pot-
e"". While IL-2 did not expand the number of y-INF-producing cells in response
to the
p1 lc, C-~M peptide in mock-vaccinated animals, the expansion of this response
was
higher in the NYVAC-SIVgag pot-enV treated macaques that also received IL-2
than in
those that received NYVAC-SIVgag_pot-env alone. In contrast, IL-2 per se
appeared to
expand the immune response to two other immunodominant epitopes within the SIV
tat and vif proteins in treated animals. (Vaccination with NYVAC-SIVgag
pot_env did
not further expand these responses as these antigens are not included in the
vaccine.)
Moreover, the CD8+ T-cell responses to 2 subdominant epitopes within the Gag
and
Env proteins of SIV were clearly expanded following NYVAC-SIVgag pot-env
vaccination in macaques that received simultaneous and continuous IL-2
treatment.
Thus, the administration of low-dose IL-2 in conjunction with vaccination with
the
WO 01/08702 cA o23aooia 2oo2-oi-2a PCT/US00/20641
highly attenuated NYVAC-SIVgag por-env vaccine potentiated and broadened CD8+
T-
cell functional responses to SIVZSi.
IL-2 can be used with the vaccine to control viremia after antiretroviral
therapy
interruption
A HAART-treated macaque was inoculated with NYVAC-SIVgag-
pol-env at the intervals shown in Figure 8. The macaque also recived low dose
IL-2,
i.e., 120,000 units daily administered subcutaneously. This animal exhibited
expanded CD8+ (top panel, Figure 8) and CD4+ proliferative (lower panel,
Figure 8)
responses. Furthermore, as shown in the top panel of Figure 8, transient viral
rebound
occurred following interruption of HAART treatment and after suspension of the
IL2
treatment. Thus, administration of IL-2 in conjunction with vaccination can
contribute to the control of viremia after interruption of antiretroviral
therapy.
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