WO2001008702A2 - Immunotherapie traitant les personnes infectees par le vih utilisant des vaccins apres une multi-therapie - Google Patents

Immunotherapie traitant les personnes infectees par le vih utilisant des vaccins apres une multi-therapie Download PDF

Info

Publication number
WO2001008702A2
WO2001008702A2 PCT/US2000/020641 US0020641W WO0108702A2 WO 2001008702 A2 WO2001008702 A2 WO 2001008702A2 US 0020641 W US0020641 W US 0020641W WO 0108702 A2 WO0108702 A2 WO 0108702A2
Authority
WO
WIPO (PCT)
Prior art keywords
vaccine
viral
hiv
cell
siv
Prior art date
Application number
PCT/US2000/020641
Other languages
English (en)
Other versions
WO2001008702A3 (fr
WO2001008702A9 (fr
Inventor
Genoveffa Franchini
Zdenek Hel
Gene Shearer
James Tartaglia
Janos Nacsa
Original Assignee
The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services filed Critical The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services
Priority to EP00953728A priority Critical patent/EP1198248A2/fr
Priority to CA002380018A priority patent/CA2380018A1/fr
Priority to AU66128/00A priority patent/AU779494B2/en
Priority to JP2001513432A priority patent/JP2003505516A/ja
Publication of WO2001008702A2 publication Critical patent/WO2001008702A2/fr
Publication of WO2001008702A3 publication Critical patent/WO2001008702A3/fr
Publication of WO2001008702A9 publication Critical patent/WO2001008702A9/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
    • C12N2740/14022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • 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.
  • HAART highly active anti- retroviral therapy
  • 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 500 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.
  • 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 transcriptase.
  • 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.
  • IL-2 interleukin-2
  • 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 g l20 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 gpl20 envelope antigen.
  • Figure 1 shows the viral load and proliferative CD4 + T-helper responses to p27 gag and gpl20 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 shows the CD8 + response in the infected, treated, vaccinated animals.
  • Figure 3 shows p27-specific (Fig. 3a and 3c) and gpl20-specific (Fig. 3b and 3d) T-cell proliferation in SIV-infected HAART-treated Rhesus macaques following administration of a single dose of ALVAC-SIV gpe . (Fig. 3a and Fig. 3b) or ALVAC (Fig. 3c and 3d).
  • the arrows indicate time after SIV infection at which the animals were inoculated with vaccine.
  • 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-SIVgp e (Fig. 4a) or ALVAC (Fig. 4b).
  • Figure 5 shows the induction of CD8 + (Fig. 5a and 5b) and CD4 + (Fig. 5c and 5d) T-cell responses following administration of a DNA vaccine to na ⁇ ve macaques. DNA was administered at the times indicated by the arrow.
  • the plasmids employed were pCMV-g ⁇ g- and pCMV-env, which are CMV expression plasmids expressing the gag and env genes, respectively.
  • 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- STV-gag-pol-env to naive macaques.
  • the vaccine was at the times indicated by the arrows.
  • Figure 7 shows the administration schedule of NYVAC vaccine with and without interleukin-2 (IL-2) in HAART-treated macaques.
  • IL-2 interleukin-2
  • 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 -retro viral 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 ⁇ -interferon are shown by the black vertical bars.
  • Proliferative responses to p27 gag and gpl20 are indicated in the lower panel.
  • IL-2 was administered daily by subcutaneous injection at a dose of 120,000 units. 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.
  • Pulsma 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 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.
  • 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.
  • “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.
  • This invention is a novel therapeutic modality for treating persons infected with a lymphotropic or immune destroying retrovirus.
  • a physician presented with a 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).
  • HAART highly active anti-retroviral therapy
  • the conventional HAART protocols are complex and difficult for patients to follow.
  • the drugs also have a number of problematic side effects.
  • 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.
  • 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 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 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.
  • CTL cytotoxic T lymphocyte
  • the invention contemplates single or multiple administrations of a nucleic acid-based 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.
  • the recombinant viruses of use are adenoviruses, adeno-associated viruses, retro viruses 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.
  • a live vaccine of the invention can be administered at, for example, about 10 4 -10 8 organisms/dose, or 10 6 to 10 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.
  • 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
  • EBV Epstein Barr virus
  • Rhinoviruses see, e.g., U.S. Patent No. 5,714,374.
  • 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.
  • HSV herpes simplex virus
  • Other suitable viral vectors include retro viruses (see, e.g., Miller, Human Gene Ther. 1 :5-14 (1990); Ausubel et al, Current Protocols in Molecular Biology).
  • the poxviruses are of preferred use in this invention.
  • attenuated poxviruses that are available for use as a vaccine against HIV. These include attenuated vaccinia virus, cowpox virus and canarypox virus.
  • 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.
  • the DNA gene sequence encoding an antigenic sequence 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.
  • 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.
  • 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.
  • Representative examples of recombinant pox viruses include ALVAC, TROVAC, NYV AC, 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: NYV AC 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.
  • NYV AC 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.
  • NYV AC 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)).
  • Kanapox a plaque- cloned derivative of the licensed canarypox vaccine
  • 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.
  • NYV AC, 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 NYV AC, 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 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 NYV AC 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.
  • these would include nucleic acid that can encode at least one of: HIVlgag( + pro)(IIIB), gpl20(MN)( + transmembrane), nef(BRU)CTL, pol(IIIB)CTL, ELDKWA or LDKW epitopes, preferably HIVlgag( + pro)(IIIB), gpl20(MN) ( + transmembrane), two (2) nef(BRU)CTL and three (3) pol(IIIB)CTL epitopes; or two ELDKWA in gpl20 V3 or another region or in gpl60.
  • the two (2) nef(BRU)CTL and three (3) pol(IIIB)CTL epitopes are preferably CTL1, CTL2, poll, pol2 and pol3.
  • the viral strains from which the antigens are derived are noted parenthetically.
  • 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.
  • 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).
  • 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.
  • 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.
  • exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of such promoters as the S V40 early promoter, SV40 later promoter, metallothionein promoter, human cytomegalo virus promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • promoters as the S V40 early promoter, SV40 later promoter, metallothionein promoter, human cytomegalo virus promoter, murine 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).
  • PBS sterile phosphate-buffer saline
  • This formulation is particularly suitable for intramuscular (IM) or intradermal (ID) administration.
  • IM intramuscular
  • ID intradermal
  • 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 Feigner, et al, Proc. Nat 'I Acad. Sci. USA 84:7413 (1987).
  • glycolipids, fusogenic liposomes, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
  • HlV-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 gpl60, 120 and 41 are sources of immunoprotective proteins. Both B and T cell epitopes have been described in the literature and can be used.
  • Non-structural genes include the rev, tat, nefi vifi and vpr genes.
  • a preferred patient population of retro virally infected persons are those that exhibit repeated and sustained proliferative T-cell responses to envelope epitopes, e.g., HIV gpl20. 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.
  • PBMC peripheral blood monocytes
  • 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.
  • DTH delayed type hypersenstive response
  • the reaction is measured by the ability or inability of a patient to exhibit hypersensitive response to an aqueous solution of a gp 120 or p24 antigen. Approximately, 1-20 ⁇ g is applied.
  • the reaction is determined by measuring wheal 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 wheal 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.
  • 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 transcriptase enzyme into newly synthesized viral DNA chains, resulting in chain termination.
  • NRTIs Nonnucleoside reverse transcriptase inhibitors
  • NRTIs 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.
  • 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.
  • 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: SAQULNAVIR (Invirase); INDLNAVIR (Crixivan); and RITONAVIR (Norvir).
  • 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.
  • WBC white blood cell
  • 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]).
  • 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.
  • CD4 + cells are commerically available.
  • 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. Viral titer
  • 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).
  • 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
  • signal amplification methods e.g., branched DNA [bDNA
  • 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.
  • 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 ⁇ -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.
  • 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 51 Cr-release assay using U-bottomed microtiter plates containing about 10 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 51 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.
  • Vaccine compositions e.g., compositions containing the poxvirus recombinants or DNA
  • 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.
  • NYVAC-HIV or other attenuated pox virus vaccines such as ALVAC-HIV or MVA-HIV
  • ALVAC-HIV or MVA-HIV is inoculated, more than once, by the intramuscular route at a dose of about 10 8 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.
  • 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 EnglJMed 1997 Apr 24;336(17):1260-1; and Smith, Cancer J Sci Am. 1997 Dec;3 Suppl 1. -SI 37-40).
  • 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.
  • 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).
  • 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)); Feigner et al. (U.S. Patent No. 5,580,859, issued December 3, 1996); Feigner (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.
  • a pharmaceutically acceptable carrier including a physiologically acceptable compound, depends, for example, on the route of administration of the expression vector.
  • 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.
  • 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., Feigner 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 administration involves mechanically or chemically irritating the outermost layer of epidermis to stimulate an immune response to the irritant (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 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.
  • 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.
  • the recombinant poxvirus, expression product, immunogen, DNA, or modified gpl20 or gpl60 can be in admixture with a suitable carrier, 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., Feigner 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 carriers 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 carriers can serve to target a particular tissue or infected cells, as well as increase the half- life of the vaccine.
  • 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.
  • 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.
  • 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.
  • Example 1 Administration of NYV AC-SI ⁇ gag - po i- e n V 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 SIV 5 ⁇ 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 SIV 251 (Pal). Following SIV 251 exposure, all twenty four animals became infected; the peak of plasma viremia occurred at approximately two weeks and ranged between 10 7 and 10 9 copies of viral RNA/ml of plasma in the twenty four animals.
  • HAART regimen 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 SIV 25 ⁇ .
  • 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).
  • HAART treatment was continued daily for 6 months.
  • the animals in group A received a placebo vaccine (non-recombinant NYV AC vector) and the animals in groups B and C received 10 8 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 gpl20 e «v SrV proteins.
  • LPR lymphoproliferativ responses
  • MAMU-A*01 Macaca mulata equivalent of HLA class I A*01 (Kuroda et ⁇ l, J. Exp. Med. 187:1373-1381, 1998) were included in each group.
  • MAMU-A*01 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 in vivo.
  • the tetramer formed by four identical MAMU-A*01 molecules conjugated to the peptide 1 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*01 animals.
  • NYVAC-SIV vaccination increases CD8 + /CD3 + MAMU-A *01-tetramer-positive cells only in HAART-treated animals.
  • Peptide 1 lc-m presented in the context of the MAMU-A*01 haplotype is recognized by CD8 + T- cells with cytolytic activity.
  • the top panel shows the results obtained in the three MAMU-A*01 animals from group A; middle panel, from group B; and bottom panel, from group C.
  • the percentage of MAMU-A* 01 /peptide- 1 lc-m- tetramer-staining cells within the first 4 weeks was evaluated using only ⁇ -CD8 + antibodies as a T cell marker whereas the data presented from weeks 19 through 29 were obtained using simultaneously -CD-3+ and ⁇ -CD8 + antibodies in conjunction with the MAMU-A*01 /peptide- 1 lc-m tetramers.
  • 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.
  • DTH Delayed T-cell hypersensitivity
  • Vaccines able to induce T-cell-mediated immunity are often able to induce DTH.
  • either 1 or 10 ug of highly purified SIV p27 or HTLV-I p24, as controls, were inoculated 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.
  • 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.
  • 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.
  • the patient 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 NYV AC carrying the following viral peptides: gag,pro, gpl20-TM, pol and «ef string of CTL epitopes. The injection comprises 10 8 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.
  • a maximum of three to four immunizations with NYV AC -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 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.
  • immunization regimens as follows have been shown to be effective: NYV AC followed by DNA and ALVAC followed by DNA.
  • a vaccination regimen including all of the above vaccines may be used.
  • 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-SIV gpe to SIV-infected, HAART-treated Rhesus macaques
  • the effectiveness of a highly attenuated ALVAC vector as a therapeutic vaccine to enhance host-immune responses in HAART-treated animals was investigated in the SIV 25 ⁇ Rhesus macaque model, which models HIV-1 infection in humans.
  • the study includes 16 macaques inoculated with the SIV 251 virus and treated with the HAART regimen as described in Example 1 at day 15 and thereafter.
  • the data obtained after a single dose of the ALVAC-SIVgpe vaccine indicated that the ALVAC-SIV g p e vaccine is able to boost CD4 + T-cell responses against the p27 Gag protein as well as to the gpl20 Env forefront of the vaccine (see Figure 3).
  • a specific CD8 + T-cell response detected using the MAMU A*01/peptide 1 lc-m tetramer reagent was also boosted by the ALVAC-SIVgpe ( 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.
  • ALVAC-SIV gpe is immunogenic in animals undergoing HAART therapy.
  • An ALVAC vaccine analogous to the NYV AC vaccine of Example 2 can similarly be employed to treat HIV-infected individuals as described in Example 2 who are undergoing HAART.
  • the effectiveness of an MVA-SIV vaccine was evaluated using methodology analogous to that used to evaluate NYV AC and ALVAC SIV gag-pol-env vaccines.
  • the CD8+ and CD4 + responses were determined in SIV 251 -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 10 8 pfu of MVA-SIV-g ⁇ g-po/-ewv recombinant vaccine.
  • the results showed that both CD4 + and CD8 + responses could be expanded in infected animals.
  • MVA-SIV-g ⁇ g- oZ-env is also immunogenic in animals and can be used in patients undergoing HAART therapy.
  • 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 SI V 25 ⁇ -infected animals.
  • a study conducted in parallel compared 2 inoculations of 10 pfu of NYVAC- SIV gpe 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-SIVgpe vaccine ( Figures 5 and 6).
  • ALVAC-SIV gpe was at least as immunogenic as NYVAC-SIVgpe (see, e.g., Example 1) and NYVAC-SIVg pe was as immunogenic as DNA.
  • all three vaccines either alone or in various combinations can be used in HIV-I-infected individuals.
  • ALVAC-SIV gpe 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 gpe -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.
  • Viremia in the remaining 2 animals became undetectable by weeks 6 and 8 of treatment.
  • Proliferative responses to p27 Gag and gpl20 were increased by NYVAC-SrV gag-p0/-e , ! v 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.
  • IL-2 can be used with the vaccine to control viremia after antiretroviral therapy interruption
  • a HAART-treated macaque was inoculated with NYVAC-SIVg ⁇ g- 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.
  • transient viral rebound occurred following interruption of HAART treatment and after suspension of the IL2 treatment.
  • administration of IL-2 in conjunction with vaccination can contribute to the control of viremia after interruption of antiretroviral therapy.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • AIDS & HIV (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un procédé amélioré dont l'objectif est de maintenir une réponse immuno-protectrice chez les personnes infectées par un rétro-virus après une thérapie anti-rétrovirale de haute efficacité.
PCT/US2000/020641 1999-07-28 2000-07-27 Immunotherapie traitant les personnes infectees par le vih utilisant des vaccins apres une multi-therapie WO2001008702A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP00953728A EP1198248A2 (fr) 1999-07-28 2000-07-27 Immunotherapie traitant les personnes infectees par le vih utilisant des vaccins apres une multi-therapie
CA002380018A CA2380018A1 (fr) 1999-07-28 2000-07-27 Immunotherapie traitant les personnes infectees par le vih utilisant des vaccins apres une multi-therapie
AU66128/00A AU779494B2 (en) 1999-07-28 2000-07-27 Immunotherapy in HIV infected persons using vaccines after multi-drug treatment
JP2001513432A JP2003505516A (ja) 1999-07-28 2000-07-27 Ｈｉｖ感染者における多薬物療法後のワクチンを用いた免疫療法

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US14624099P 1999-07-28 1999-07-28
US60/146,240 1999-07-28
US17898900P 2000-01-28 2000-01-28
US60/178,989 2000-01-28
US20044500P 2000-04-28 2000-04-28
US60/200,445 2000-04-28

Publications (3)

Publication Number Publication Date
WO2001008702A2 true WO2001008702A2 (fr) 2001-02-08
WO2001008702A3 WO2001008702A3 (fr) 2001-08-30
WO2001008702A9 WO2001008702A9 (fr) 2001-09-20

Family

ID=27386369

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/020641 WO2001008702A2 (fr) 1999-07-28 2000-07-27 Immunotherapie traitant les personnes infectees par le vih utilisant des vaccins apres une multi-therapie

Country Status (5)

Country Link
EP (1) EP1198248A2 (fr)
JP (1) JP2003505516A (fr)
AU (1) AU779494B2 (fr)
CA (1) CA2380018A1 (fr)
WO (1) WO2001008702A2 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001054701A1 (fr) * 2000-01-31 2001-08-02 Aventis Pasteur, S.A. Vaccination de personnes infectees par le vih apres une therapie antiretrovirale a haute activite
WO2005040816A1 (fr) * 2003-10-24 2005-05-06 Immunaid Pty Ltd Methode therapeutique
WO2006010106A2 (fr) * 2004-07-09 2006-01-26 The Government Of The United States, As Represented By The Secretary Of The Department Of Health And Human Services Vaccination par adn de sujets atteints d'un retrovirus et suivant un traitement
WO2008089144A2 (fr) * 2007-01-12 2008-07-24 The Government Of The United States, As Represented By The Secretary Of Health And Human Services Protocoles améliorés de vaccination par adn
US9122778B2 (en) 2009-05-27 2015-09-01 Biotempus Limited Methods of treating diseases
CN109300529A (zh) * 2018-09-12 2019-02-01 阿呆科技（北京）有限公司 基于人工智能和虚拟现实/增强现实的毒瘾渴求靶向干预矫治系统
US11013791B2 (en) 2017-06-22 2021-05-25 The Government Of The United States, As Represented By The Secretary Of The Army NYVAC-based plasmodium malaria vaccine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998008539A1 (fr) * 1996-08-26 1998-03-05 Chiron Corporation Therapie de vaccination apres l'infection par le virus de l'immunodeficience humaine (vih)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998008539A1 (fr) * 1996-08-26 1998-03-05 Chiron Corporation Therapie de vaccination apres l'infection par le virus de l'immunodeficience humaine (vih)

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
F. GOTCH ET AL.: "Therapeutic Vaccines in HIV 1 infection" IMMUNOLOGICAL REVIEWS, vol. 170, 1999, pages 173-182, XP000982295 *
M. JOHN ET AL.: "Control of HIV replication by cytoxic T-lymphocyte responses" J. HIV THERAPY, vol. 4, no. 4, 1999, pages 91-87, XP000982254 *
R. MACGREGOR ET AL.: "First human trial of a DNA-based vaccine for treatment of HIV1 infection: safety and host response" J. INF. DIS., vol. 178, 1998, pages 92-100, XP000982247 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001054701A1 (fr) * 2000-01-31 2001-08-02 Aventis Pasteur, S.A. Vaccination de personnes infectees par le vih apres une therapie antiretrovirale a haute activite
WO2005040816A1 (fr) * 2003-10-24 2005-05-06 Immunaid Pty Ltd Methode therapeutique
US8524651B2 (en) 2003-10-24 2013-09-03 Immunaid Pty Ltd. Method of therapy
WO2006010106A3 (fr) * 2004-07-09 2006-06-01 Human Services D By The Secret Vaccination par adn de sujets atteints d'un retrovirus et suivant un traitement
WO2006010106A2 (fr) * 2004-07-09 2006-01-26 The Government Of The United States, As Represented By The Secretary Of The Department Of Health And Human Services Vaccination par adn de sujets atteints d'un retrovirus et suivant un traitement
WO2008089144A2 (fr) * 2007-01-12 2008-07-24 The Government Of The United States, As Represented By The Secretary Of Health And Human Services Protocoles améliorés de vaccination par adn
WO2008089144A3 (fr) * 2007-01-12 2009-02-26 Us Gov Health & Human Serv Protocoles améliorés de vaccination par adn
US8586055B2 (en) 2007-01-12 2013-11-19 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services DNA immunization protocols
US9122778B2 (en) 2009-05-27 2015-09-01 Biotempus Limited Methods of treating diseases
US9239904B2 (en) 2009-05-27 2016-01-19 Biotempus Limited Computer systems for treating diseases
US9268908B2 (en) 2009-05-27 2016-02-23 Biotempus Limited Computer systems for treating diseases
US10714208B2 (en) 2009-05-27 2020-07-14 Biotempus Pty Ltd Computer systems for treating diseases
US11013791B2 (en) 2017-06-22 2021-05-25 The Government Of The United States, As Represented By The Secretary Of The Army NYVAC-based plasmodium malaria vaccine
CN109300529A (zh) * 2018-09-12 2019-02-01 阿呆科技（北京）有限公司 基于人工智能和虚拟现实/增强现实的毒瘾渴求靶向干预矫治系统
CN109300529B (zh) * 2018-09-12 2021-09-21 阿呆科技（北京）有限公司 基于人工智能和虚拟现实/增强现实的毒瘾渴求靶向干预矫治系统

Also Published As

Publication number Publication date
AU779494B2 (en) 2005-01-27
WO2001008702A3 (fr) 2001-08-30
JP2003505516A (ja) 2003-02-12
CA2380018A1 (fr) 2001-02-08
AU6612800A (en) 2001-02-19
EP1198248A2 (fr) 2002-04-24
WO2001008702A9 (fr) 2001-09-20

Similar Documents

Publication Publication Date Title
US7771729B2 (en) Methods of potentiating HIV-1-specific CD8+ immune responses involving the concomitant administration of DNA and ALVAC expression vectors
KR100571479B1 (ko) Hiv에 대한 폴리엔브백신으로서 재조합 백시니아벡터의 혼합물
US20140377306A1 (en) Recombinant vaccine viruses expressing il-15 and methods of using the same
AU2001259291A1 (en) Improved immunogenicity using a combination of DNA and vaccinia virus vector vaccines
JP2000506727A (ja) 組換え生ネコ免疫不全ウイルスおよびプロウイルスｄｎａワクチン
Hel et al. Equivalent immunogenicity of the highly attenuated poxvirus-based ALVAC-SIV and NYVAC-SIV vaccine candidates in SIVmac251-infected macaques
US20090169503A1 (en) Dna-based vaccination of retroviral-infected individuals undergoing treatment
AU779494B2 (en) Immunotherapy in HIV infected persons using vaccines after multi-drug treatment
US20050019752A1 (en) Novel chimeric rev, tat, and nef antigens
EP2021356B1 (fr) Vaccin contre le vih
KENT et al. Analysis of cytotoxic T lymphocyte responses to SIV proteins in SIV-infected macaques using antigen-specific stimulation with recombinant vaccinia and fowl poxviruses
US20040034209A1 (en) Vaccination of hiv infected persons following highly active antiretrovial therapy
WO2001054701A9 (fr) Vaccination de personnes infectees par le vih apres une therapie antiretrovirale a haute activite
US20060094006A1 (en) Immunotherapy regimens in hiv-infected patients
US6723558B1 (en) Preparation and use of viral vectors for mixed envelope protein vaccines against human immunodeficiency viruses
WO2004043490A1 (fr) Schema posologique pour vaccin antivariolique

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

AK Designated states

Kind code of ref document: C2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: C2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

COP Corrected version of pamphlet

Free format text: PAGES 1/8, 2/8 AND 8/8, DRAWINGS, REPLACED BY NEW PAGES 1/8, 2/8 AND 8/8; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

WWE Wipo information: entry into national phase

Ref document number: 66128/00

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2000953728

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 10048072

Country of ref document: US

ENP Entry into the national phase in:

Ref country code: JP

Ref document number: 2001 513432

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 2380018

Country of ref document: CA

WWP Wipo information: published in national office

Ref document number: 2000953728

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWG Wipo information: grant in national office

Ref document number: 66128/00

Country of ref document: AU