WO2001054701A9 - Vaccination de personnes infectees par le vih apres une therapie antiretrovirale a haute activite - Google Patents

Vaccination de personnes infectees par le vih apres une therapie antiretrovirale a haute activite

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Publication number
WO2001054701A9
WO2001054701A9 PCT/US2001/002766 US0102766W WO0154701A9 WO 2001054701 A9 WO2001054701 A9 WO 2001054701A9 US 0102766 W US0102766 W US 0102766W WO 0154701 A9 WO0154701 A9 WO 0154701A9
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WIPO (PCT)
Prior art keywords
hiv
virus
vaccine
cells
patient
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PCT/US2001/002766
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English (en)
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WO2001054701A1 (fr
Inventor
David Ho
Martin Markowitz
Michel Klein
Habib Raphaelle El
Original Assignee
Aventis Pasteur S A
Aaron Diamond Aids Res Ct
David Ho
Martin Markowitz
Michel Klein
Habib Raphaelle El
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Application filed by Aventis Pasteur S A, Aaron Diamond Aids Res Ct, David Ho, Martin Markowitz, Michel Klein, Habib Raphaelle El filed Critical Aventis Pasteur S A
Priority to US10/182,067 priority Critical patent/US20040034209A1/en
Priority to AU2001233063A priority patent/AU2001233063A1/en
Publication of WO2001054701A1 publication Critical patent/WO2001054701A1/fr
Publication of WO2001054701A9 publication Critical patent/WO2001054701A9/fr

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    • 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
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This invention relates to the field of methods of treating HIV-infected patients.
  • HIV infection is characterized by high levels of virus replication at all stages of infection. Virus replication causes increased levels of CD4 cell destruction and turnover, and when unchecked, immunodeficiency, AIDS and death. This model of pathogenesis has prompted a dramatic change in the treatment paradigm which has evolved from late intervention in symptomatic individuals to a "hit early, hit hard" strategy.
  • Perelson and co-workers developed a mathematical model based on the biphasic decay of plasma HIV RNA after initiating potent antiviral therapy.
  • the model hypothesized that two to three years of treatment with a completely suppressive regimen could result in a virologic remission or "eradication of infection" in HIV-infected individuals.
  • the two to three year estimate required complete suppression of virus replication, the absence of any additional slower decaying compartments and/or the absence of sequestered areas of virus replication.
  • the present invention provides a method of permitting cessation of antiviral therapy on such HIV-infected subjects without virus rebound, with a delayed viral rebound, or with decreased post-rebound set point.
  • the method comprises the re-induction of HIV-specific immune responses using a vaccination strategy to induce both humoral and cell-mediated immunity.
  • the present invention achieves an immunological control of persistent infectious virus after discontinuation of antiviral therapy.
  • the vaccine strategy according to the invention is safe and induces immune responses in the HTV-infected patient population.
  • the present invention is directed to a method of stimulating efficient CD4+ and CD 8+ responses in a human infected with an HIV retro virus who has a viral load of less than 10,000, preferably less than 5,000, viral copies per ml of plasma and a CD4+ T-cell count of above 300 cells/ml, preferably above 500 cells/ml, and who has been treated with a potent combination of antiviral agents that contributed to a lower viral copy number and equal or higher CD4+ cell count than before treatment.
  • the method comprises administering a nucleic acid-based vaccine that enters the cells and intracellularly produces HIV-specific immunogens for presentation on the cell's MHC class I and MHC class II molecules in an amount sufficient to stimulate HIV- specific CD4+ and CD8+ T-cell responses, thereby reversing the otherwise observed population decline of these cells during antiretroviral therapy.
  • the human has been treated with HAART therapy that 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 of above 500 cells/ml.
  • the method employs a vaccine that is a nucleic acid-based vaccine comprising naked or vectored nucleic acid.
  • the vaccine comprises an attenuated recombinant poxvirus, particularly NYVAC or ALVAC, that includes one or more nucleic acids encoding more or more HIV-specific immunogens.
  • the vaccine optionally further comprise an adjuvant and is administered one or multiple times.
  • the vaccine is optionally combined with an HIV antigen as well as immunostimulatory or co-stimulatory molecules such as interleukin 2 or CD40 ligand, respectively, in an amount that is sufficient to potentiate T-cell responses, in particular CD8+ responses.
  • the method of the mvention is particularly useful for people who have been infected by
  • HIV and who have demonstrated CD4+ and/or CD8+ T cell responses to HIV antigens such as people who have demonstrated prohferative T-cell responses to gpl20 envelope protein or p24 or both g ⁇ l20 envelope and p24 Gag antigen.
  • the method ofthe invention is also useful for people who have lost their CD4+ and/or CD8+ T cell responses to HIV antigens, such as people who have lost their prohferative T cell response to gpl20 or p24.
  • Figure 1 displays plasma RNA and CD4+ T-cell levels for HIV-infected patients undergoing HAART.
  • Figure 2 is a bar graph displaying the number of HIV-infected subjects undergoing HAART having plasma HIV RNA levels of less than 200, 50, and 25 copies/ml.
  • Figures 3 A and 3B display CTLp frequencies for two patients undergoing HAART.
  • Figures 4A-4D display the percent of CD8+ IFN- secreting cells to specific HIV antigens for four HAART patients receiving HIV vaccination according to the invention.
  • Figures 5A-5D display plasma viremia in four HAART patients receiving HIV vaccination according to the invention.
  • Figures 6A-6F display plasma HIV RNA and CD4 T-cell count levels as a function of days on therapy for several patients.
  • Figures 7A-7F display anti-gpl20 and anti-p24 antibody titers for several patients as a function of days post vaccination.
  • Figures 8A-8F displays intracellular cytokine staining.
  • Figure 9A-9F display data relating to various HIV antigens.
  • Figures 10A-10F display stimulation indexes as a function of days post vaccination.
  • FIGS 11 A-l 1-F display stimulation indexes as a function of days post vaccination.
  • the present invention provides a novel therapeutic modality for treating persons infected with a lymphotropic or immune-destroying retroviral infection.
  • a physician presented with a patient whose immune system is compromised by retroviral infection can select 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 fail to eliminate the virus; they merely hold the virus in check. If the patient is non- compliant, the viral count rebounds. Accordingly, for the vast majority of patients, a lifetime of drugs is advised.
  • the present invention comprises the discovery that after HIV infection, HAART treatment that decreases the viral load can be discontinued using an anti-HIV vaccine that induces an immune response.
  • This response effectively maintains a low titer of virus or controls the viral rebound when the antiretroviral therapy is discontinued,, pe ⁇ nitting significant reduction of the patient's dependency on antiretroviral therapy.
  • some such vaccines have been suggested as useful for seropositive patients (U.S. Patent No. 5,863,542 column 18, lines 60-63)
  • the art has not recognized that administration to seropositive patients receiving anti-viral treatment permits cessation of the anti-viral treatment without virus rebound, with delayed virus rebound, or with decreased post-rebound set point.
  • control of virus rebound we mean that after discontinuation of antiviral therapy the viral rebound that usually appears is delayed, the post-rebound set point is decreased, or there is no virus rebound.
  • Virus rebound appears usually within 1 to 3 weeks after discontinuation of the antiviral therapy.
  • virus rebound is "delayed” when it appears more than 1 month after discontinuation of the antiviral therapy.
  • the virus rebound appears more than 2 months and more preferably more than 6 months after discontinuation of the antiviral therapy.
  • the set point is defined as the plasmatic viral load that is maintained after viral rebound in the absence of antiviral treatment.
  • Viral rebound can be evaluated by various methods well known in the art. There are a variety of ways to measure viral titer in a patient. A review ofthe state ofthe art can be found in the "Report ofthe 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 HJV 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 target amplification methods (e.g., quantitative 13 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 HTV RNA bDNA assay, Chiron Diagnostics).
  • target amplification methods e.g., quantitative 13 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 [
  • 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.
  • the method of vaccination of the invention is useful for the treatment of HIV-infected patients undergoing an antiretroviral therapy and having a viral load of less than 10,000, preferably less than 5,000, and more preferably less than 1000 viral copies per ml of plasma and a CD4+ T-cell count of above 300 cells/ml, preferably above 500 cells/ml.
  • antiretroviral therapy or "antiviral therapy” we mean a treatment involving a potent combination of antiviral agents.
  • Antiviral retroviral treatment involves the use of two broad categories of therapeutics. They are reverse transcriptase inhibitors and protease inhibitors.
  • nucleoside analog reverse transcriptase 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 that 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, nucleotide analogs used are already partially phosphorylated and is one step closer to activation when it enters a cell.
  • NRTIs Non-nucleoside reverse transcriptase inhibitors
  • 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 HTV protease, which is a protease that is virally encoded and necessary for the infection process to proceed.
  • HIV infected persons have a number of clinically effective protease inhibitors to use on HIV infected persons. These include: SAQUINAVIR (Invirase); lNDINAVIR (Crixivan); and RITONAVIR (Norvir). Patients' viral load can be evaluated by various ways. Various methods which can be used have been disclosed above in relation with the virus rebound.
  • 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
  • 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 fluorocl rome (e.g., phycoerythrin [PE] or fluorescein isothiocyanate [FITC]).
  • a fluorescent dye or fluorocl rome e.g., phycoerythrin [PE] or fluorescein isothiocyanate [FITC].
  • the fluorochiOme-labeled cells are analyzed by using a flow cytometer, which categorizes individual cells according to size, granularity, fluorochrome, and intensity of fluorescence.
  • WBCs Size and granularity, detected by light scattering, characterize the types of WBCs (i.e., granulocytes, monocytes, and lymphocytes). Fluorochrome-labeled antibodies distinguish C7 populations and subpopulations of WBCs.
  • Systems for measuring CD4+T-cells are commercially 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.
  • Patients that can be treated by the method of the invention thus include those newly infected with HIV who have undergone intense anti-retroviral therapy within a few months after infection resulting in a controlled viremia (who can be defined as individuals showing an incomplete Western Blot), as well as chronically-infected individuals undergoing an antiretroviral therapy.
  • a controlled viremia who can be defined as individuals showing an incomplete Western Blot
  • chronically-infected individuals undergoing an antiretroviral therapy we mean patients who have been infected 90 or fewer days.
  • controlled viremia we mean that the viral load is maintained at a level of less than 10,000 viral copies per ml of plasma.
  • a preferred population of retrovirally infected persons are those that exhibit CD4+ and CD8+ cell response to HIV antigens, such as those that exhibit prohferative T-cell responses to envelope epitopes, e.g., HIV gpl20.
  • PBMC peripheral blood monocytes
  • An alternative means is to use a skin test.
  • Skin tests involve the detection of a delayed type hypersensitive 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 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 a patient 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.
  • the method comprises administering to an HIV-infected patient as defined above a nucleic acid-based vaccine that enters the cells and intracellularly produces HIV-specific immunogens for presentation on the cell's MHC class I and MHC class II molecules in an amount sufficient to stimulate efficient HIV-specific CD4+ and CD8+ T-cell responses.
  • Efficient CD8+ responses 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.
  • CD8+ T-cell responses may be measured, for example, by using tetramer staining of fresh or cultured PBMC, INF- ⁇ Elispot assays, a combination of cell surface phenotyping and cytokine intracellular fluorescence staining intracellular INF- ⁇ or using functional cytotoxicity assays, which are well-known to those of skill in the art.
  • 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 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 maximal release with detergent (2% Triton X- 100) in all assays. "Efficient CD4+ responses" is referred to as the ability of CD4+ T-cells to be stimulated or activated by the vaccine of the invention. CD4+ T cell responses can be measured by various methods well-known in the art.
  • Nucleic acid-based vaccine means DNA and RNA-based vaccines and includes naked nucleic acids and vectored nucleic acids.
  • vectored nucleic acid we mean any kind of viral expression vectors such as DNA and RNA viruses or bacterial vectors such as BCG, salmonella or listeria or lactobacillus that delivers nucleic acid sequences coding for HIV specific immunogen into cells.
  • the vectored nucleic acid corresponds preferably to an attenuated recombinant DNA virus.
  • 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 permissive primary cells or at cold temperatures.
  • 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., Sarnulskl et al., J. Virol. 61:3096-3101 (1987); Samulski et al, J. Virol.
  • 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
  • suitable viral vectors include for example retroviruses (see, e.g., Miller, Human Gene Ther. 1:5-14 (1990); Ausubel et al, Current Protocols in Molecular Biology), coksackie viruses, vesicular stomatitis viruses (VSV) and poxviruses.
  • the poxviruses are preferred for use in this invention.
  • attenuated poxviruses that are available for use as a vaccine against HIV. These include attenuated vaccinia virus, fowlpox virus and canarypox virus. These recombinant virus can be easily constructed.
  • the basic technique of inserting foreign genes into live infectious poxvirus involves a recombination between poxvirus DNA sequences flanking a foreign genetic element in a donor plasmid and a homologous sequences present in the rescuing poxvirus as described in Piccini et al, Methods in Enzymology 153, 545-563 (1987).
  • 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 such as a known T-cell epitope is selected to be inserted into the virus and is placed into an E. coli plasmid construct into which DNA homologous to a section of DNA of the poxvirus has been inserted.
  • 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 poxvirus 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.
  • a cell culture e.g. chick embryo fibroblasts
  • Recombination between homologous pox DNA in the plasmid and the viral genome gives a poxvirus modified by the presence of foreign DNA sequences in a non-essential region of its genome.
  • Attenuated recombinant pox viruses are employed in a preferred vaccine.
  • Representative examples of recombinant pox viruses include recombinant ALVAC and NYVAC.
  • recombinant ALVAC is vCP205.
  • 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.
  • ALVAC is an attenuated canarypox virus-based vector that was a plaque-cloned derivative of the licensed canarypox vaccine, Kanapox (Tartaglia et al, 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.
  • canarypox virus replication is aborted early in the viral replication cycle prior to viral DNA synthesis.
  • 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 confers protection against challenge with the cognate pathogen.
  • NYVAC and ALVAC 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 safety containment of genetic material such as viruses and vectors, i.e., guidelines for safety procedures for the use of such viruses and vectors that 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 NTH has recognized that NYVAC and ALVAC have a lower pathogenicity than any other poxvirus.
  • Another attenuated poxvirus of preferred use in the invention is Modified Vaccinia virus
  • MVA Ankara
  • 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 or contagiousness for animals and humans.
  • expression of recombinant protein occurs during an abortive infection of human cells, thus providing a safe, yet effective, delivery system for foreign antigens.
  • the nucleic acid-based vaccine for use in the present invention further comprises sequences encoding HIV immunogens and intracellularly produces the HIV-specific immunogens.
  • the HIV antigen encoding DNA for insertion into the viral vectors of the invention or for use as naked nucleic acid are any that are known to be effective for protection against a retrovirus.
  • HIV-specific immunogens means any HIV protein, fragment, or epitope thereof that is recognized by an immune cell as an epitope of the native protein. HIV-specific immunogens are thus selected from both structural and non-structural proteins. Highly antigenic epitopes for provoking an immune response selective for a specific retroviral pathogen are known.
  • 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 ofthe native protein.
  • Structural viral proteins are those proteins that are physically present in the virus. They include the envelope, 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.
  • the envelope is a preferred source of epitopes and gp 160, 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 immunoprotective including gp41 and the Gag protein.
  • Gag protein we mean the whole Gag protein as well as proteins derived from Gag such as pl7 and p24.
  • Non-structural genes include the rev, tat, nef, vif, and vpr genes.
  • the nucleic acids include those that can code for at least one of- HIV-I Gag(+ pro)(LAI), g ⁇ l20(MN or another strain)(+ transmembrane), Nef(BRU)CTL, Pol( ⁇ iB)CTL, ELDKWA or LDKW epitopes, preferably HIV 1 Gag(+ pro)(IIIB), gpl20(MN) (+ transmembrane), two (2) Nef(BRU)CTL and three (3) Pol(III)CTL epitopes; or two ELDKWA in gpl20 V3 or another region of gpl60.
  • the two (2) Nef(BRU)CTL and three (3) Pol(IIIB)CTL epitopes are preferably Nefl, Ne£2, Poll, Pol2 and Pol3.
  • the corresponding sequences are given in U.S. 5,990,091.
  • sequences encoding Tat and/or Rev can advantageously be added.
  • the viral strains from which the antigens are derived are noted parenthetically.
  • the above-defined HIV antigen encoding DNA can be derived from any known HIV strain (HIV1, HIV2, preferably HIV 1), including laboratory strains and primary isolates.
  • the Pol and Nef epitopes have sequences presented in the following:
  • CTL epitope Pol-1 (49 aa)
  • Preferred viral vectors according to the invention include ALVAC HIV (vCP1452), which is a recombinant canarypox virus expressing Gag Ai, Protease L Ai, Env(120)M N ,
  • Env(41) A i, Nef, and Pol. vCP1452 is described in U.S. Patent Nos. 6,004,777 and 5,990,091.
  • vCP1433 which was deposited with the ATCC in accordance with the Budapest Treaty on March 6, 1997, under accession number VR-2556 and was also described in U.S. Patent Nos. 6,004,777 and 5,990,091.
  • Vaccine compositions e.g., compositions containing the poxvirus recombinants or DNA
  • Vaccine compositions can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art.
  • Vaccine compositions can comprise one or a plurality of vectors that effect HIV-antigen expression.
  • 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 ofthe particular patient, and the route of administration.
  • Vaccines may be delivered via a variety of routes of administration including, for example, a parenteral route (intradermal, intramuscular or subcutaneous, transdermal or epidermal). Other routes include oral administration, intranasal, intrarectal and intravaginal routes.
  • parenteral route intradermal, intramuscular or subcutaneous, transdermal or epidermal
  • Other routes include oral administration, intranasal, intrarectal and intravaginal routes.
  • 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.
  • the naked or vectored nucleic acid may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, Tris buffer or the like.
  • the vaccine of the invention may also comprise an adjuvant. Any adjuvant administrable to humans can be used.
  • Adjuvants useful in the invention include alum, calcium phosphate and, preferably PCPP (poly dicarboxylatopheoxylphosphazene), a synthetic hydrogel polymer developed for its adjuvant properties.
  • a viral vector- based vaccine can be administered at about 10 3 -10 8 TCID50/dose or 10 4 to 10 9 pfu per dose.
  • ALVAC-HIV vaccine 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 may be delivered in a physiologically compatible solution such as sterile 0.4% NaCl in a volume of, e.g., one ml.
  • the vaccine ofthe invention is administered several times. Intervals between administrations and number of administration depend of the immune response of the patient. Vaccine doses have to be admimstered as long as it is necessary to re-induce the immune system. Actual dosages of such a vaccine can be readily determined by one of ordinary skill in the field of vaccine technology.
  • DNA may also be directly introduced into the cells of a patient.
  • This embodiment is defined in the present invention as naked-DNA vaccine.
  • This expression i.e., naked-DNA vaccine
  • naked-DNA vaccine thus encompasses naked DNA r»er se, including virus like particles, as well as formulated DNA-based vaccines as disclosed below.
  • 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, adjuvants) delivery, and cationic lipid complexes or liposomes and microspheres.
  • the nucleic acids can be administered using ballistic delivery as described, for instance, in 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.
  • DNA can be adhered to particles, such as gold particles.
  • a large number of factors can influence the efficiency of expression of antigen genes and/or the immunogenicity of DNA vaccines.
  • 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., CMV 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 the SV40 early promoter, SV40 later promoter, metallothionein promoter, human cytomegalovirus promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • 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 supplied by QIAG ⁇ N, Inc. (Valencia, California). 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 approach, known as "naked DNA,” is currently being used for intramuscular (JJVf) administration in clinical trials.
  • PBS sterile phosphate-buffer saline
  • Cationic lipids can also be used in the formulation (e.g., as described by WO 93/24640; Mannino & Gould-Fogen'te, BioTechniques 6(7): 682 (1988); U.S. Pat No. 5,279,833; WO 91/06309; and Felper, et al, Proc. Nat 'I Acad. Sci. USA 84:7413 (1987).
  • glycolipids, fusogenic liposomes, peptides targeting sequences 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.
  • DNA expression vectors for direct introduction of DNA into the patient tissue can additionally be complexed with other components such as peptides, polypeptides, lipopeptides, carbohydrates, microspheres, immunostimulants and adjuvants. 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, for example, 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 ofthe expression vector.
  • naked DNA or polynucleotide in an aqueous carrier can be injected into tissue, such as muscle or skin, in amounts of from 10 1 p er 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.
  • Actual dosages of the vaccine can be readily determined by one of ordinary skill in the field of vaccine technology
  • the expression vectors of use for the invention can be delivered to the interstitial spaces of tissues of an animal body (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 ionophoresis, 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 inlialation 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 ofthe active ingredient.
  • the vaccines can be incorporated, if desired, into liposomes, microspheres or other polymer matrices (Feigner et al, U.S. Patent No. 5,703,055; Gregoriadis, Liposome Technology, Vols. I to III (2nd ed. 1993), each of which is incorporated herein by reference).
  • Liposomes for example, which consist of phosphohpids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
  • Liposome carriers may serve to target a particular tissue or infected cells, as well as increase the half-life of the vaccine.
  • Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phosphohpid dispersions, lamellar layers and the like.
  • the vaccine to be delivered is incorporated as part of a liposome, alone or in conjunction with a targeting 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 targeting 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 immuno
  • Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phosphohpids 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.
  • Vaccines for use in the present invention can be administered alone or can advantageously be combined with an immunostimulating composition and/or another anti-HIV vaccine.
  • Vaccines for use in the invention can advantageously be combined with immunostimulatory or co-stimulatory molecules such as for example cytokines, interleukin 2 or CD40 ligand, which are used in an amount that is sufficient to potentiate the T-cell responses, in particular CD8+ responses.
  • immunostimulating compounds are used according to the recommendations ofthe manufacturer. Such compounds may be present as such or in the form of a recombinant virus expressing the same.
  • Vaccines for use in the invention can advantageously be combined with another anti-
  • HIV vaccine Such anti-HIV vaccine can be different from the first vaccine (for example, naked nucleic acid-based vaccine can be combined with a viral vector-based vaccine, naked DNA followed by a HIV immunogen-encoding poxvirus, or an HlV-immunogen encoding attenuated vaccinia virus followed by a HIV immunogen-encoding avipox virus), or can be a vaccine comprising a soluble antigen of HIV. Any soluble HIV antigen that is known to be an effective antigen for protection against HIV can be used.
  • the soluble antigen corresponds to the gpl60 HIV-1 envelope glycoprotein and, in particular, the gpl60MN/LAI-2, corresponding to an envelope glycoprotein from HIV-1 virus expressed by vaccinia virus VV.TG.9150 on BHKi cells wherein the gpl20 portion is derived from HIV M N and the gp41 transmembrane portion from HIV LAI -
  • the soluble antigen can be readily determined by one of ordinary skill in the field of vaccine technology
  • the vaccine comprises a nucleic acid vector (e.g., a viral vector) comprising genes encoding and expressing a plurality of HIV antigens and is co- administered with an HIV antigen.
  • a vector comprising the ALVAC canarypox vector expressing the HIV Gag, Protease, Env(120), Env (41), Nef, and Pol antigens is co-administered with the gpl60 HIV-1 envelope glycoprotein.
  • the clinical program divided study subjects into two groups, those newly infected and those infected for greater than 90 days on entry into the screening phase.
  • New infections were diagnosed on the basis of a positive plasma HIV-1 RNA in the setting of one of the following three criteria: absence of HIV-antibody by ELISA, progression ofthe antibody response as determined by the appearance of at least two new bands on Western blot and a clinical syndrome consistent with acute infection within 90 days of screening, and a documented negative test within the previous 120 days.
  • Routine laboratory determinations include plasma HIV-RNA levels using either bDNA signal amplification or PCR technology, safety laboratory studies including routine hematology and chemistry, and assessments of immunologic status including a variety of cell surface markers used to define naive and memory cell subsets.
  • FIG. 1 Representative longitudinal plasma HIV-RNA and CD4 cell data of a chronically infected cohort participating in study MMA-197 is shown in Figure 1.
  • suppression of virus replication is accompanied by a 2 log drop in HIV RNA during the early weeks.
  • Further suppression of the productive infection of new susceptible cells results in a continued drop in the plasma HIV-1 RNA reflecting the loss of cells continuing to produce non- infectious virus particles.
  • the antiviral effect is dramatic and results in a nearly 4 log reduction as the nadir is reached at week 24.
  • MC mononuclear cells
  • Viral load of CD8+ T cell-depleted PHA-stimulated co-cultures after 19 to 24 months of therapy were less than 0.1 TCIDso/lO 6 CD4 in Subjects 2, 3, 7, and 8. Cultures were strongly positive in subjects 6, 9 and 11 and borderline positive in Subject 5. Quantitative PCR detected both MS and US-mRNA in PBMC from subject 11.
  • Table 3 These are compared to a control with high levels of virus replication in blood and lymphatic tissue.
  • a lumbar puncture was performed in subjects 3 and 9 at months 24 and 15, respectively.
  • the fluid was acellular and had less than 25 HIV-RNA copies/ml as determined by ultra-sensitive RNA PCR (Roche).
  • MC mononuclear cells
  • Proviral DNA was detected at low levels, between 10 and 100 copies/10 6 MC in all but one subject (#9).
  • intensive virologic measurement were performed in early infected HAART treated subjects. New infections were diagnosed on the basis of a positive plasma HIV-1 RNA in the setting of one of the following three criteria: absence of HIV- antibody by ELISA, progression ofthe antibody response as determined by the appearance of at least two new bands on Western blot and a clinical syndrome consistent with acute infection within 90 days of screening, and a documented negative test within the previous 120 days.
  • lymphoid tissue including tonsil and/or lymph node(Study #MMA-189), semen
  • Peripheral blood mononuclear cells PBMC are isolated by Ficoll-Hypaque gradient using standard techniques. Aliquots of a minimum of 10 7 cells were prepared and stored at -150°C for future use. Cells were CD8 depleted using magnetized-antibody-coated polystyrene beads (Dynal).
  • l-2xl0 7 CD8-depleted MC were stimulated with PHA and irradiated feeder cells and co-cultured in IL-2 containing medium with HIV-negative donor CD4+ T-cells. Cultures were maintained for three weeks and culture supernatants assayed weekly for levels of p24. A positive culture requires a p24 concentration of at least 100 pg/ml in the culture supernatant.
  • lymphoid system As the lymphoid system is the preferred site of virus replication in an infected host, a comprehensive surgical program was established at Rockefeller University Hospital to meet the specific needs of the AD ARC clinical program.
  • ENT otolaryngologist
  • a board-eligible gasfroenterologist obtained gastrointestinal-associated lymphoid tissue (GALT). These procedures were done under separate protocols MMA-189 and ATA-207.
  • Consenting subjects were well-known to the clinical staff, but screening for coagulopathy with measurements of prothrombin time (PT) and partial thromboplastin time (PTT) was included prior to procedure. A careful surgical history was also required to screen for rarer causes of hemostatic dysfunction. Biopsies were performed using local anesthesia without the need for conscious sedation. Lymphoid tissue was divided into three sections, a portion immediately frozen in liquid nitrogen for PCR analysis, a portion formalin-fixed and subsequently paraffin embedded for in situ hybridization and immunohistochemistry, and a portion transported in culture medium from which MC were mechanically disrupted and cultured using standard co-culture techniques.
  • PT prothrombin time
  • PTT partial thromboplastin time
  • Subjects eligible for vaccination had to meet the following virologic criteria: 1. Undetectable levels of MS-mRNA in blood and/or tissue 2. Rare to no HIV expressing cells by in-situ hybridization (tissue sampling is optional) 3. Viral cultures from blood and/or tissue either negative for culturable virus or yielding drug-sensitive virus by genotype and phenotype Subjects failing to meet these virologic criteria could be re-evaluated at 6 month intervals.
  • Direct CTL effector activity was measured from freshly isolated PBMC using autologous B-lymphoblastoid cell targets infected with recombinant vaccinia virus expressing HIV-1 specific genes (gag, pol, env, nef). 4
  • HIV-specific CTL precursor frequencies were similarly performed in selected subjects.
  • 49 Patient PBMC were seeded at varying concentrations in 200 ⁇ l of IL-2-containing medium in 24 replicate-wells of a 96-well tissue culture plate. Irradiated donor PBMC and anti-CD-3 antibody were added to each well and incubated at 37°C for 14 days. Wells were split into four and assayed for the ability to lyse an autologous chromium-labeled B- lymphoblastoid cell line infected with a vaccima- virus expressing HIV-1 env, gag, pol, and nef genes as well as an antigen negative control.
  • CTLp with a given specificity were determined by plotting the log of the fraction of negative wells (less than 3 S.D. above the mean for the 24 control wells or below 10% specific lysis) versus the number of input cells. 4 Patients with detectable fresh CTL activity above 30% specific lysis to one or more antigens at an effector to target ratio of 25:1 were not eligible for participation in the vaccination protocol. Subjects with levels of CTL precursors above 1 in 100,000 to one or more specific antigens including Env, Gag, Pol, or Nef were similarly excluded.
  • HIV-RNA RT-PCR
  • HIV-specific proliferation assays to HIV antigens HIV-specific antibody levels (p24 and gpl20) *Blood was drawn at 2 weeks, then monthly for virology and immunology. Assays other than HIV-RNA were performed at the discretion of the investigators, but no less than every three months. HIV-RNA was performed at each visit.
  • ALVAC HIV (vCP1452) is a recombinant canarypox virus expressing the gag LA i, proteaseLAi, env(120)MN, env(41) L Ai 5 nef, and pol genes.
  • VCP1452 is described in U.S. Patent Nos. 6,004,777 and 5,990,091.
  • vCP1452 is modified to include 2 vaccinia virus coding sequences to enhance expression in mammalian cells. The pol and nef sequences are scrambled such that no functional proteins can be expressed., Approximately 10 7 TCID 50 in 1.0 ml were given with each dose.
  • Recombinant gpl60MN/LAI-2 is an envelope glycoprotein from HIV-1 virus expressed by vaccinia virus VV.TG.9150 on BHK cells.
  • the gpl20 portion is derived from HIV M N and the transmembrane gp41 portion from HIV LAI -
  • the adjuvant, PCPP is a synthetic soluble polymer developed for its adjuvant properties.
  • the vaccine contained 50 ⁇ g of recombinant gpl60 in 500 ⁇ g PCPP (1.0 ml).
  • the vaccines being used in this study as well as the adjuvant are novel.
  • each vaccination dose was 1.0 mli.m. [approximately 10 7 TCID 50 ]; for gpl60 MN/LAI-2, each vaccination dose was 50 ⁇ g in 500 ⁇ g PCPP (1.0 ml).
  • the ALVAC portion given with the gpl60 portion together has caused at least one of the following side effects in at least 75% of the subjects: pain and redness at the site of injection, weakness, muscle aches, joint aches, headache, and fever above 38°C.
  • ALVAC is an avian virus (canarypox) that cannot replicate in man and therefore undergoes only one abortive cycle of replication. Over 1800 subjects received an ALVAC construct without significant serious adverse events. Additionally, over 700 subjects received ALVAC/soluble Env vaccine regimens with no severe reactions (unpublished data).
  • Antibody titers, prohferative responses and CTL activity to HIV specific antigens were measured at baseline and post-vaccination as indicated using standard techniques. Blood was drawn and cells and plasma stored for immunologic and virologic studies on days 0, 15, 30, 60, 90 120, 180, and 210 and the above assays performed. Criteria for response included: a twofold increase in antibody titer to env and/or gag, a measurable increase in level and/or broadening of detectable fresh CTL activity and/or CTLp, and a three fold increase in proliferation index to HIV specific antigens measured in vitro.
  • Subjects demonstrating an immune response to the vaccines without significant adverse events that is, no Grade 3 or 4 nor significant local reactions, were offered the opportunity to participate in an extension that provides for vaccination every three months with identical follow-up, that is, observation in clinic for 30 minutes, telephone follow-up within 72 hours, diary cards to record temperature and adverse events, clinic visits 2 weeks after vaccination, and careful virologic monitoring, all as described above.
  • Immunogenicity was determined by baseline and post vaccination measurement of: CTL activity using bulk CTL assays, CTLp frequencies, CTLe frequencies by tetramers if available, proliferation to HIV-specific antigens in vitro, and levels of HIV specific antibodies to gpl20 and p24.
  • CD4+cell-associated proviral DNA were interpreted as being the result of exposure to vaccine antigens as opposed to the result of activation of virus replication.
  • Subjects were allowed, if desired, to participate in this vaccine protocol without consenting to collection of tissue and or fluid other than blood. These were optional procedures and serve to establish the absence of virus replication as completely as possible. 3. Recruitment of subjects
  • Plasma HIV-1 RNA levels were monitored with the Ultrasensitive RT PCR Assay (Roche) and the Bayer signal amplification assay (version 3.0) as per manufacturer's instructions.
  • Subjects who discontinued therapy include 1306, 1308, 1309, and 1310 and
  • Subjects 1308 and 3002 did not respond to vaccination with an increase in the level of CD8+ IFN ⁇ secreting cells to HIV specific antigens presented in the context of vaccinia.
  • Subject 1310 did respond with an increase in levels of CD8+ IFN ⁇ -secreting cells specific for Gag.
  • Subjects 1306 and 1309 responded with an increase in CD8+ IFN- ⁇ -secreting cells specific for more than 1 HIV-1 specific antigen (see Figure 4).
  • Day 0 refers to the day that subjects discontinued therapy. Period of vaccination occurred during days -217 to 0. Post- discontinuation levels of CTLe are similarly displayed.
  • Post-therapy discontinuation subjects 1310 and 1306 rebounded after 68 and 85 days respectively.
  • the subjects 1308 and 1310 rebounded within 23 and 13 days of therapy cessation.
  • the initial doubling times (t 2 ) of plasma viremia post therapy cessation were 4.5 and 3.2 days respectively, whereas the subjects who rebounded rapidly had a t 2 of approximately 1.5 days.
  • the virology data for the 4 subjects are shown in Figure 5. It is clear that Subjects 1309 and 1306 not only exhibit a delayed rebound but the mean HIV-1 RNA levels post rebound are also significantly lower than in rapidly rebounding individuals.
  • Daar ES Moudgil T, Meyer RD, Ho DD. Transient high levels of viremia in patients with primary human immunodeficiency virus type 1 infection. N. Engl. J. Med. 1991; 324:961-964.
  • HIV infection is active and progressive in lymphoid tissue during the clinically latent stage of disease. Nature 1993; 362:355-358. 8. Embretson J, Zupacic M, Ribas JL, et al. Massive covert infection of helper T lymphocytes and macrophages by HIV during the incubation period of AIDS. Nature 1993; 362:359-362. 9. Ho DD, Neumann AU, Perelson AS, Chen W, Leonard JM, Markowitz M. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 1995; 373:123-126. 10. Mellors JW, Rinaldo Jr. CR, Gupta P, White RM, Todd JA, Kingsley LA.
  • Kempf D, Marsh K, Denissen J, al. e. ABT-538 is a potent inhibitor of human immunodeficiency virus protease and has high oral bioavailability in humans. Proc. Natl. Acad. Sci. USA 1995; 92:2484-2488.
  • Pantaleo G Demarest JF, Schacker T, et al.
  • the qualitative nature of the primary immune response to HIV infection is a prognosticator of disease progression independent ofthe inital level of plasma viremia.

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Abstract

La présente invention concerne un procédé qui permet d'arrêter la thérapie antirétrovirale chez des sujets infectés par le virus VIH sans provoquer de rebond viral ou au moins un rebond viral retardé ou un point de consigne post-rebond réduit. Ce procédé consiste à réinduire les réponses immunitaires spécifiques au VIH à l'aide d'une stratégie vaccinale permettant d'induire à la fois l'immunité à médiation humorale et l'immunité à médiation cellulaire. La présente invention réalise un contrôle immunologique du virus infectieux persistant après l'interruption d'une thérapie antivirale. La stratégie vaccinale de cette invention est à la fois sans danger et immunogène chez la population infectée par le virus VIH étudiée.
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