WO1998050070A1 - Vaccins a base de virus vivants visant a proteger des primates contre l'infection et la maladie dues au vih-1 - Google Patents

Vaccins a base de virus vivants visant a proteger des primates contre l'infection et la maladie dues au vih-1 Download PDF

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WO1998050070A1
WO1998050070A1 PCT/US1998/008868 US9808868W WO9850070A1 WO 1998050070 A1 WO1998050070 A1 WO 1998050070A1 US 9808868 W US9808868 W US 9808868W WO 9850070 A1 WO9850070 A1 WO 9850070A1
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hiv
virus
shiv
infection
vaccine
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Opendra Narayan
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University Of Kansas Medical Center
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    • 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
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • 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
    • C12N2740/16061—Methods of inactivation or attenuation

Definitions

  • This invention relates to the field of prophylactic and therapeutic vaccines for generating protection from HIV-1 induced disease and infection, and compositions and methods for such vaccination, and compositions and methods for the therapeutic treatment of HIV- 1 infected individuals.
  • neutralizing antibodies to the virus pathogen, and cellular immunity against infected cells.
  • the generation of neutralizing antibodies and cellular immunity by the infected individual's immune system heralds recovery from infection.
  • neutralizing antibodies and cellular immunity appear very early during the infection, and have been associated with only a transient decrease in viral burden.
  • HIV-1 infection in spite of the generation of neutralizing antibodies and cellular immunity, viral replication rebounds and AIDS (acquired immune deficiency syndrome) develops.
  • neutralizing antibodies and cellular immunity have no curative effects on the infection (are not accurate measures of protective immunity).
  • induction and maintenance of these arms of the immune response by vaccine may prevent infection by the virus under natural conditions.
  • a further problem in developing an effective vaccine for HIV-1 which has concerned researchers is the antigenic diversity of the wild-type virus. It has been believed to be a strong possibility that vaccines generated via recombinant HIV-1 coat proteins will confer resistance to specific phenotypes of virus, and not broad spectrum immunity.
  • HIV-1 gpl20 peptide a viral coat protein of HIV- 1
  • phase-one clinical trials showing no toxicity.
  • recombinant HIV-1 gpl20 peptide vaccines may only act in the short-term, with reversion to susceptibility of infection occurring in the future.
  • live-virus vaccines induce better immunity against pathogenic viruses than isolated viral proteins (see for example Putkonen et al., Immuization with Live Attenuated SIVmac Can Protect Macaques against Mucosal Infection with SIVsm, Vaccines 96, pps. 20-210, 1996; Dimmock and Primrose Introduction to Modern Viroloev. 4 th Ed., Blackwell Science, 1994).
  • SIV mac Session Immunodeficiency Virus
  • macaque Session Immunodeficiency Virus
  • SIV macaque Session Immunodeficiency Virus
  • SIV mac is only an analogous model system for human HIV-1 infection, and does not replicate all of the salient features of HIV-1 infection/disease of humans (see generally Hu et al, Transmembrane Protein and Core Antigens in Protection against SIV Infection, Vaccines 95, pps.
  • the present invention teaches specific methods and virus constructs which are effective in generating an immune response in a vaccinated host to virus.
  • the present invention teaches constructs and methods for the effective in vivo therapeutic and/or prophylactic vaccination of mammals to inhibit or prevent HIV infection and/or related disease.
  • Figure 1 is a diagram of the genetic organization of HIV-l/HIV-2 virus for vaccine.
  • Figure 2 is a diagram of the genetic organization of SHIV, where the vpu gene does not encode for a functional protein (initiation ATG is ACG).
  • Figure 3 is a diagram of the genetic organization of SHIV-4 and ⁇ 2 SHIV, where a 60 bp Sspl-Bbsl fragment was deleted so that the vpu gene was permanently eliminated, and a 217 bp RsrII-Ncol fragment was deleted so that the we/gene was permanently eliminated.
  • Figure 4 is a diagram of the genetic organization of PPc SHIV and Avpu PPc SHIV, where a 60 bp Sspl-Bbsl fragment was deleted so that the vpu gene was permanently eliminated.
  • Figure 5 is a diagram of the generation of pathogenic SHIV: SHIV KU _, and SHIV KU _ 2 .
  • Figure 6 depicts the scheme used to construct recombinant pDSDvpu, where a 60 bp Sspl-Bbsl fragment was deleted, the details of the plasmid manipulations are described in the text.
  • Figure 8 is a graph of neutralizing antibody titers against SHIV KU ., in vaccinated macaques. Macaques were challenged with SH ⁇ V ⁇ lM at weeks 0 and 5. The * indicates terminal plasma obtained from macaque 42107, which died at week 24. Neither control animal developed neutralizing antibodies to SHIV KU _,.
  • the present invention teaches an avirulent HIV-1 virus, wherein the vpu gene of the HIV-1 virus has been rendered non-functional or deleted.
  • the HIV-1 virus may have a non- functional or deleted nef gene.
  • the instant invention also encompasses a HIV-l/HIV-2 chimeric virus wherein the chimeric DNA comprises HIV-2 LTR, gag, pol, and «e/ ⁇ genes and HIV-1 env, tat, rev, vpu genes, where said vpu gene is rendered non-functional or is deleted.
  • the nef gene is optionally rendered non-functional or deleted.
  • the instant invention also encompasses an avirulent SIV/HIV-1 chimeric virus, having a SIV LTR, gag, pol, and nef genes and HIV-1 env, tat, rev, vpu genes, where said vpu gene is rendered non-functional or deleted.
  • the nef gene is optionally rendered non-functional or deleted.
  • the instant invention embodies vaccines which inhibit the infection of cells by HIV- 1, comprising a virus wherein the DNA of said virus comprises DNA consisting of HIV-2 LTR, gag, pol, and nef genes and HIV-1 env, tat, rev, vpu genes, where said vpu gene is rendered non-functional.
  • the vpu gene is rendered non-functional by deletion.
  • the nef gene is optionally rendered non-functional or deleted.
  • the instant invention teaches vaccines which inhibits the infection of cells by HIV-1 , comprising a virus wherein the DNA of said virus comprises DNA consisting of HIV-2 LTR, gag, pol, and nef genes and HIV-1 env, tat, rev genes, and no HIV-1 vpu gene.
  • a further embodiment of the invention encompasses vaccines which inhibit the infection of cells by HIV-1, comprising a virus wherein the DNA of said virus comprises HIV-1 DNA wherein the vpu gene of HIV- 1 has been rendered non- functional.
  • the vpu gene is rendered non- functional by deletion.
  • the instant invention teaches vaccines which inhibit the infection of cells by HIV-1 , comprising a virus wherein the DNA of said virus comprises HIV-1 DNA, and no vpu gene of HIV- 1.
  • Another embodiment of the invention encompasses a vaccine which inhibits infection of cells by such a SIV/HIV-1 chimeric virus such as SHIV KU ., which comprises said virus wherein the vpu gene is rendered non-functional or deleted, in a suitable carrier.
  • a vaccine contains said virus wherein the nef gene is optionally rendered non-functional or deleted.
  • the disclosure teaches methods for inoculating a subject to inhibit virus infection by chimeric SHIV/HIV-1 virus such as SHIV KU.
  • a vaccine contains said virus wherein the nef gene is optionally rendered non-functional or deleted.
  • the instant invention teaches methods for inoculating a subject to inhibit virus infection by HIV-1 comprising administering a vaccine containing a modified virus, the DNA of said virus comprising DNA consisting of HIV-2 LTR, gag, pol, and nef genes and HIV-1 env, tat, rev, vpu genes, where said vpu gene is non-functional.
  • the vpu gene is deleted.
  • such a vaccine contains said virus wherein the nef ' gene is optionally rendered non-functional or deleted.
  • Also encompassed by the instant invention are methods for inoculating a subject to inhibit virus infection by HIV-1 comprising administering a vaccine containing a modified virus, the DNA of said virus comprising DNA consisting of HIV- 1 DNA, where the HIV-1 vpu gene is non-functional.
  • the vpu gene is deleted.
  • such a vaccine contains said virus wherein the nef gene is optionally rendered non-functional or deleted.
  • the present invention also embodies the administration of viral DNA of the present invention directly to the subject to be treated. Such administration being made with DNA suspended in saline or other suitable carrier or packaging.
  • the instant invention provides for a general method for the creation of effective vaccines for conveying immunity to HIV-1 virus, wherein the virus is manipulated to impede its ability to effectively replicate and/or accumulate in the infected/inoculated host. Wherein one embodiment of this method is the perturbation of the activity of the vpu gene or gene product in the virus, or during host- virus interaction.
  • the instant invention embodies the treatment of currently infected HIV-1 positive patients to prevent further HIV-1 infection and/or continuing HIV- 1 infection.
  • the present invention teaches constructs and methods for the effective in vivo therapeutic and/or prophylactic vaccination of mammals to inhibit or prevent HIV infection and/or related disease.
  • This pathogenic virus is the first virus bearing the envelope of HIV- 1 which can cause AIDS in a non-human primate, as indicated by a) all animals lost CD4 + T cells during the first three weeks after inoculation with the virus (an excellent marker for virus pathogenicity); b) the virus is predictably pathogenic, with 70% of inoculated animals developing AIDS within six months (and thus vaccine efficacy can be evaluated in a short time using this monkey model system); and c) the virus invades across mucosal surfaces and causes AIDS after deposition in the mouth or in the vagina (thus allowing for evaluation of testing for efficacy against sexual transmission).
  • SHIV KU _ (originally isolated as PPc and PNb) is the first virus bearing the envelope of HIV- 1 which can cause AIDS in a non-human primate, as indicated by a) all animals lost CD4 + T cells during the first three weeks after inoculation with the virus (an excellent marker for virus pathogenicity); b) the virus is predictably pathogenic, with 70%
  • the DNA of the virus was constructed of SIV LTR, gag, pol, and «e genes and HIV-1 env, tat, rev, vpu genes.
  • vpu gene present in the original SHIV as a non-functional 5 gene, became functional in the SHIV KL .
  • the vpu gene is an accessory gene found only in HIV-1. (for review see Vaishnav and Wong-Staal, The Biochemistry of AIDS, Annual Review of Biochemistry 60:577-630, 1991). It has been found that the env gene of HIV-1 in the pathogenic SHIV contained ten consensus mutations, and the nef gene of SIV in the SHIV had five mutations.
  • the vaccine vims of the invention are somehow impeded in the ability to replicate or accumulate efficiently, and as such the immune system of the inoculated host is able to respond to this viral challenge as if to any other virus, by mounting an appropriate, effective and thus immunity-conferring response.
  • agents that will interfere with the HIV-1 vpu gene or gene product can be effective therapeutics for modulating the effects of HIV- 1 infection and/or continuing HIV-1 infection.
  • agents can be either chemical, antibody based, or some other form of bioactive molecule.
  • the vpu gene of HIV- 1 and the nef gene of SIV mac are thought to encode for nonstructural regulatory proteins. Deletion of both these genes from the SHIV KL molecular construct resulted in a first vaccine virus called ⁇ 2 SHIV ( ⁇ 2 SHIV —4).
  • a second vaccine was made using a virus which incorporated the env and nef genes taken from the SHIV KL virus, and deleted only the vpu gene, this virus construct being named Avpu SHIV KU _, (Avpu SHIV-PPc) It has been found that Avpu SHrV ⁇ u ., (Avpu SHIV-PPc) replicates more vigorously than ⁇ 2 SHIV in macaque tissue cell cultures and in macaques.
  • the vpu gene has not been considered as an important gene for pathogenesis because of lack of experimental evidence. Examination of the genetic mutations which occurred during the course of successive passages of non-pathogenic SHIV through monkey bone marrow, and the onset of pathogenicity reveals important information as to the genetic changes which lead to pathogenic SHIV such as SHIV KU .,. This examination has shown that while a functional vpu is most likely not the sole determinant of viral pathogenicity/virulence, it is however required that the vpu gene must become functional before other mutations could occur in either the env or nef genes and the subsequent development of pathogenic SHIV.
  • vpu gene product may play an important role in down-regulation of the expression of MHC class I molecules, and thereby contribute to the inability of CD8 + T cells to eradicate HIV-1 from infected individuals (Kerkau et al., J. Exp. Medicine 185(7): 1295-1305, 1997).
  • Test post infection Intravenous route Intravaginal route Assay
  • Plasma and culture fluids were assayed for p27 using a capture ELISA kit from Coulter Laboratories (Hialeah, FL). For each assay, a standard curve was prepared according to the kit instructions, and p27 concentrations were calculated form the OD 45() using linear regression analysis. The sensitivity for detection of p27 was approximately 30 pg/ml.
  • Example 3 ⁇ 2 SHIV Vaccine
  • the ⁇ 2 SHIV virus was constructed (with the nef and vpu genes deleted from SHIV-
  • nef and vpu genes could be completely deleted or only partially deleted, or rendered non-functional by insertion of DNA into the gene, with the objective being that no functional nef or vpu gene product is produced.
  • the ⁇ 2 SHIV virus was inoculated subcutaneously into six adult female macaques. Two months later, the animals were re-exposed by oral innoculation with ⁇ 2 SHIV virus. Infectious virus was recovered from the animals during the first three weeks following initial inoculation, and viral DNA was tested via PCR (Polymerase Chain Reaction) of DNA isolated from blood mononuclear cells for another month after the re-exposure.
  • All six animals produced antibodies detectable by immunoprecipitation with SHIV KU ., virus by three months after initial inoculation. By five months after initial inoculation, all six animals were producing neutralizing antibodies to SHIV KU _, virus measuring with a response of 1 in 10 to 1 in 20. At six months after initial inoculation, all six animals were healthy and sho_wed no manifestations of AIDS pathology.
  • Monthly analysis of blood cells via FACS (Fluorescence activated cell sorting) showed that the six animals were free from AIDS pathology. Virus was no longer detectable in blood mononuclear cells by six months after initial inoculation, as detectable by infectivity assays and by PCR analysis.
  • the six inoculated animals and two un-inoculated control animals were challenged by intravaginal administration of SHIV KL virus.
  • the animals were evaluated for presence of infectious cells, and for their CD4 + cell count.
  • PBMC peripheral bone marrow cells, in particular blood mononuclear cells
  • CPE cytopathic effects
  • Cell counts were generally determined using FACS (fluorescence-activated cell sorting) where cells were reacted with mAb to CD4 (SIM.4, NIH AIDS research and Reagents repository), or CD8 (Dako T8, Dako). After washing, the cells were stained with FITC-conjugated goat anti-mouse IgG (Dako), fixed in 1% buffered formalin, and analyzed on a fluorescence-activated cell counter. Evaluation of the efficacy of the vaccine is summarized by the data in Table 3 below.
  • the immune response of the host monkeys to the inoculation was monitored via standard immunological assays for immune response to the vaccine virus, including PHA, CD8 depleted PHA and assay for neutralizing antibody titer (see Joag et al., supra).
  • PBMC cultured with PHA and IL-2 For virus recovery from PBMC cultured with PHA and IL-2, a total of 2 x 10 6 PBMC were cultured in medium containing l ⁇ g of PHA-P (Wellcome) for 2 days. Cultures were then rinsed once, the cell pellets resuspended in medium with 100 U of recombinant human IL-2 (Cetus) per ml, cultured for 5 days, and then centrifuged again, and the cell-free supernatant fluids were assayed for virus infectivity as above.
  • PHA-P Wellcome
  • PBMC For virus recovery from CD8 + -depleted PBMC, a total of 2 x 10 6 PBMC were incubated with a monoclonal antibody to CD8 (Dako-T8; Dako) for lhr at 4°C and washed once, and sheep anti-mouse immunoglobulin G-coated paramagnetic beads (Dynal
  • SHIV PPc is a non-pathogemc SHIV generated from animal PPc during the course of sequential passage through bone marrow as descibed previously (see Figure 5) Modification of the parental SIV virus, was done to incorporate HIV-1 vpu, nef and env to create a SHIV It was found that this modified parental SHIV was actually not pathogenic Inoculation of this modified parental SHIV into two macaques demonstrated that the virus was avirulent, and could result in the generation of immunity to the virus.
  • deletion of the vpu gene was made which resulted in Avpu PPc SHIV
  • the inactivation of the vpu gene can be accomplished by complete deletion of the vpu gene, partial deletion of portions of the vpu gene, or it could be rendered non-functional by insertion of DNA, the objective being to inactivate the gene such that no functional vpu gene product is present
  • complete deletion of the vpu gene may be preferred
  • the Avpu PPc SHIV was inoculated into six adult pigtailed macaques, and a summary of the data evaluating the vaccine is given below in Table 5.
  • Avpu PPc SHIV and ⁇ 2 SHIV do not replicate efficiently in human cells, and thus while useful as vaccines in humans, may not generate complete immunity.
  • a human HIV-1 vaccine be constructed by using HIV-2 as the substitute for the SHIV component of the validated model vaccines.
  • construction of an effective human vaccine would involve combining the LTR, gag, pol, and «e/genes of HIV-2 in the same arrangement as in SIV ma , with the genes of HIV- 1 to construct a purely human HIV counterpart to the Avpu SHIV KU _, and ⁇ 2 SHIV virus vaccines.
  • the genes of HIV-2 is combined in the same arrangement as in SIV mac , of course it is also possible to combine the genes in the natural order as found in HIV-2, or in different order or orientations. However, the genes should be expressed in a fashion as to allow for the generation of an effective immune response in the inoculated subject.
  • a HIV-2 virus is used to provide the background of genes, and the HIV-1 env, tat, rev, vpu genes are substituted into this background of HIV-2 genes.
  • the human HIV vaccines can undergo preliminary testing in human cell cultures of lymphocytes and macrophages for evidence of infection and any retained pathogenicity. These virus constructs can also be tested in monkeys, such as chimpanzees and macaques, for evidence of replication.
  • construction of an effective human vaccine would involve rendering non-functional the vpu gene in an HIV-1 virus.
  • the nef gene can also be rendered non-functional or deleted.
  • the vpu gene in many HIV-1 cultured virus is inactivated (Vaishnav & Wong-Staal, 1991, supra.), but as shown by the results here, activation of vpu in these HIV-1 culture stocks will create pathogenic virulent HIV-1.
  • the vpu gene be rendered inactive by deletion of some or all of the gene from the virus DNA, preferably where the vpu gene has been deleted completely.
  • SHIV KU. bearing the envelope of HIV-1, causes fulminant infection with sub-total loss of CD4 + T cells and AIDS in intravaginally-inoculated macaques.
  • This virus and route of inoculation was used to challenge six adult macaques immunized seven months previously by subcutaneous injection with attenuated Avp « ⁇ «e/ " SHIV-4.
  • the animals developed neutralizing antibodies to SHIV, but only minimally-detectable cell mediated immune responses. Two unvaccinated controls and one vaccinate succumbed to challenge, but five of six vaccinates developed only transient infection with the virulent virus and have remained healthy, without CD4 ⁇ T cell loss.
  • HIV-1 is primarily a sexually transmitted virus that causes persistent systemic infection and loss of CD4 + T cells. The infection culminates in loss of immunocompetence and development of AIDS.
  • effective anti-HIV drugs notably viral protease inhibitors
  • their widespread use is hampered by their high cost and the need for multiple treatments daily for an indefinite period. Their use is thus not feasible in less developed countries where the bulk of HIV infections occur. Under these conditions, development of a safe and effective vaccine is a priority. Moreover, such a vaccine should protect against the sexually transmitted virus.
  • the inability of HIV- 1 to infect animals other than chimpanzees has meant that macaque models using non-human primate lentiviruses have become the only practical alternative for testing proof-of-concept approaches to vaccines against HIV-1.
  • SIV mac which is closely related to HIV-2 and SIVsm, causes AIDS in macaques and has been extensively used in vaccine studies. Macaques vaccinated with attenuated strains of SIV mac , produced by deleting auxiliary genes, particularly nef, resisted infection after challenge with virulent strains of SIVmac (Daniel, M. D., Kirchhoff, F., Czajak, S. C, Sehgal, P. K. & Desrosiers, R. C. Science 258, 1938-1941 (1992); Almond, N., Kent, K., Cranage, M., Rud, E., Clarke, B. & Stott, E. J.
  • the new virus is virulent after intravenous, oral, or intravaginal infusion. This virus thus proved ideal for evaluating efficacy of vaccines.
  • the choice of a live-virus vaccine was based on observations of genetic changes occurring in the virus during animal passages which yielded SHrV ⁇ l l .
  • the original molecularly cloned SHIV-4 is avirulent and the vpu gene does not encode a functional protein.
  • SHIV KU _ which is extremely virulent, encodes a functional Vpu and has amino acid substitutions in other viral proteins, including gpl60, Nef, Tat, Rev and Vpr (Stephens, E. B., Joag, S.
  • Viruses SHIV-4 DNA encoding the env, tat, rev, and vpu genes of HIV- 1 HXBc2 on a background of SIV mac 239 (Li, J., Lord, C. I., Haseltine, W., Letvin, N. L. & Sodroski, J. J. AIDS 5, 639-646 (1992)) was obtained from Dr. Joseph Sodroski, Harvard University. Viral DNA was transfected into CEMxl74 cells to produce a virus that was used to initiate passage in macaques.
  • Virus from passage 4 which was associated with AIDS and death of macaque PNb at 6 months was amplified in culture of peripheral blood mononuclear cells (PBMC) from a normal macaque, as described previously.
  • PBMC peripheral blood mononuclear cells
  • SHIV KU. Supernatant fluid from this culture
  • PBMC peripheral blood mononuclear cells
  • SHIV-4 Construction of ⁇ v > « ⁇ ie SHIV-4 .
  • the original SHIV-4 DNA consisted of 2 plasmids with the 5 ' and 3 ' regions, respectively. All manipulations were performed with the p3 ' SHIV-4.
  • plasmid pUC19 was digested with Sspl, blunt-ended by Klenow fragment of DNA polymerase and ligated to generate pDS.
  • the Sphl-Kpnl fragment (nt 6450 to 6985) of p3 ' SHIV-4 was subcloned into pDS to generate pDSvpu.
  • Plasmid pDSvpu was digested with Sspl and Bbsl, blunt-ended, and religated to delete a 60 bp Sspl-Bbsl fragment and generate pDSDvpu.
  • AvpuAnef SHIV-4 the Sphl-Kpnl fragment of 3 ' SHIV-4 ( Figure 7A) was replaced with the corresponding fragment of pDSDvpu to yield p3 ' Avp wSHIV-4 ( Figure 7B).
  • p3 ' ⁇ vpuSHIV-4 was digested with RsrII and Ncol, blunt-ended, and ligated, resulting in the deletion of a 216 bp Rsrll-Ncol fragment (including 205 bp of the nef gene, encoding the first 69 amino acids of Nef).
  • This plasmid was designated as p ⁇ vpw ⁇ «e SHIV-4 (figure 7C).
  • the p ⁇ v/? w ⁇ «e SHIV-4 and p5 ' SHIV-4 plasmids were digested with SphI, ligated with T4 DNA ligase and the ligated DNA used to transfect C8166 cells as described previously (Stephens, E.
  • Virus stocks were prepared and aliquots were stored at -80°C. The virus stock had a titer of 10,000 TCID 50 /ml in C8166 cells.
  • Human T cell line C8166 was used as indicator cells to measure virus infectivity.
  • Cells were cultured at a concentration of 1 x 10 6 /ml in RPMI medium (RPMI supplemented with 10 mM Hepes buffer pH 7.3, 50 ug/ml gentamicin, 5 x 10 "5 M 2-mercaptoethanol, and 2 mM glutamine) with 10% fetal bovine serum (FBS).
  • RPMI medium RPMI supplemented with 10 mM Hepes buffer pH 7.3, 50 ug/ml gentamicin, 5 x 10 "5 M 2-mercaptoethanol, and 2 mM glutamine
  • FBS fetal bovine serum
  • Plasma obtained from the femoral vein was centrifuged to separate plasma and buffy coats. Plasma was assayed for p27 using a capture ELISA kit (Coulter Laboratories, Hialeah, FL), and for infectivity in C8166 cells. Peripheral blood mononuclear cells (PBMC) were separated from buffy coats by centrifugation through a Ficoll-Paque (Pharmacia) density gradient.
  • PBMC Peripheral blood mononuclear cells
  • Infectious cell frequency was measured by inoculation of serial ten- fold dilutions of PBMC, starting with 1 x 10 6 cells into 24-well tissue culture plates containing 1 x 10 5 indicator C8166 T cells which were observed for development of syncytial cytopathic effects (CPE) during a 7 day period, after which cells and supernatant fluid in 100 ⁇ l from each well were transferred to another plate, fresh indicator cells added, and these observed for a further 7 days (Joag, S. V., Stephens, E. B., Adams, R. J., Foresman, L. & Narayan, O. Virology 200, 436-446 (1994)). Results were expressed as the number of infectious cells/10 6 PBMC. Mesenteric lymph nodes were obtained at 19 weeks post-challenge by biopsy. A single cell suspension was prepared and infectious cell frequency assessed as for PBMC. Other portions of the biopsy material were used for analysis of DNA or formalin-fixed for histological examination.
  • PBMC or lymph node cells were reacted with mAb to CD4 or CD8. After washing, the cells were stained with FITC-conjugated goat anti-mouse IgG , fixed in 1% buffered formalin, and analyzed on a fluorescence-activated cell counter.
  • Neutralizing antibody assays This test is performed as described previously. Briefly, serial doubling dilutions of plasma in RPMI were prepared in quadruplicate in 96-well plates, 10 to 20 TCID 50 of the virus were added to each well, plates incubated 1 h at 37°C and 1 x 10 4 indicator C8166 cells added to each well. Plates were observed for CPE 7 days later, wells scored individually, and the 50% neutralization end point was calculated using the Karber method (Lennette, E. H. in Diagnostic procedures for viral and rickettsial infections (eds Lennette, E.H. & Schmidt, N.J.) Vol.4th, 1 -65 ( American Public Health Association, New York, 1969)).
  • PCR PCR-specific oligonucleotides and conditions of amplification as described earlier.
  • PCR of the vpu gene was used distinguish between vaccine and challenge viruses in PBMC and mesenteric lymph nodes of the six vaccinates and the non-vaccinated control animals after challenge.
  • GTACCTCTGTATCATATGCTTTAGCAT-3' antisense
  • nucleotides 5845 to 5870 and 6393 to 6420, respectively of the HIV-1 (HXb2) genome were used.
  • One ug of genomic DNA was used in the PCR containing 4.0 mM MgC12, 200 uM each of the four deoxynucleotide triphosphates, 100 pM each oligonucleotide primer and 2.5 U of Taq polymerase (Perkin-Elmer Cetus, Norwalk, Conn.).
  • the template was denatured at 95°C for 3 min and PCR amplification performed with an automated DNA Thermal Cycler (Perkin-Elmer Cetus) for 35 cycles using the following profile: denaturation at 92°C for 1 min, annealing at 55°C for 1 min and primer extension at 72°C for 3 min. Amplification was completed by incubation of the PCR for 10 minutes at 72°C.
  • One ul of the PCR product from above was used in a nested PCR using the same reaction conditions described above.
  • the second round of amplification used oligonucleotide primers 5'-TTAGGCATCTCCTATGGCAGGAAGAAG-3' (sense) and 5'-CACAAAATAGAGTGGTGGTTGCTTCCT-3', which are complementary to nucleotides 5956 to 5984 and 6386 to 6413, respectively of the HIV-1 (HXb2) genome.
  • oligonucleotide primers 5'-TTAGGCATCTCCTATGGCAGGAAGAAG-3' (sense) and 5'-CACAAAATAGAGTGGTGGTTGCTTCCT-3' which are complementary to nucleotides 5956 to 5984 and 6386 to 6413, respectively of the HIV-1 (HXb2) genome.
  • the result of this PCR was the amplification of a 397 base pair fragment if the deleted vpu was present (i.e., the vaccine virus) or a 457 base pair fragment if the intact vpu was present (i.e., the challenge virus).
  • T cell proliferation assays PBMC were tested for T cell proliferative responses against equal concentrations of extracts of SHIV-infected and control cells. Additionally, a panel of 6 synthetic peptides was used (peptides 61, 63, 104, 1 1 1 , 113, 116) representing T cell epitopes present in the gpl ⁇ O of HIV-1, and against a control peptide. The sequences and synthesis of the gpl ⁇ O and control peptides have been previously described (Nehete, P. N., Satterfield, W. C, Matherne, C. M., Arlinghaus, R. B. & Sastry, K. J. AIDS Res. Hum.
  • Retroviruses 9, 235-240 (1993) Cells were suspended at 2 x lOVml in medium and 0.1 ml aliquots dispensed into 96-well U bottom microtiter plates and incubated with different concentrations of peptides or medium alone. All treatments were done in triplicate and cells were incubated for 96 hr at 37°C. During the final 16 to 18 hr, 1 ⁇ Ci of [ 3 H]thymidine (6-7 Ci/mmol; ICN Biochemicals, Costa Mesa, CA) added. Cells were harvested on filter strips, the amount of [ 3 H]thymidine incorporated was measured, and specific radioactivity for each addition calculated as described (Nehete, P. N., et al, supra).
  • PBMC peripheral blood mononuclear cells
  • Concanavalin A 10 mg/ml
  • RPMI medium containing 10%FCS and 10 U/ml rIL-2 for 7 days and used as effector cells.
  • Autologous B lymphoblastoid cell lines generated by transformation with He ⁇ esvirus papio were infected with recombinant vaccinia viruses for expressing HIV-Env (vPE16) or SIV-Gag (vgag), SIV-pol (vpol) or with control vaccinia virus (vSC8) and radiolabeled with 51 Cr for use as target cells as described.
  • control and recombinant vaccinia vectors were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. CTL activity was detected in standard 4 hour 5l Cr release assays using various effector : target cell ratios.
  • PBMC depleted of CD8 + T cells, and treated with phytohemagglutinin for two days followed by cultivation in medium containing IL-2 for five days, yielded virus, but only at the week four time point and from only three of the six macaques, 42105, PNa and PDj.
  • the six vaccinated and two control animals were challenged intravaginal ly with
  • PCR was used to detect not only gag but also detect vpu sequences in order to discriminate between the vaccine virus which has a vpu gene containing a 60 bp deletion, and SHIV KU , which has non-deleted and a functional vpu PCR analysis of vpu sequences on DNA from PBMC of the eight animals at three weeks after challenge showed that seven of eight animals had become infected with SHIV KU , virus, the exception being a control animal, PFy (Table 6) Virus recovery data coming two weeks later after the initial post-challenge blood sample showed that infectious virus was recoverable from only one control animal, PLy, and three vaccinates, 42105, 42107 and PNa (Table 7) The remaining animals were therefore re- challenged mtravaginally with SHIV KU , at week 5 PCR analysis of gag
  • Macaques were vaccinated at week -29 and challenged twice with SHIV KU , at weeks 0 and 5.
  • Vaccinate 42107 which succumbed to AIDS had an unanticipated high virus burden, similar to the control animals. It is of interest that both of the vaccinates, 42105 and 42107, that became productively infected with SHIV KL developed partial loss of CD4+ T cells shortly after challenge. Infections in these two animals had diametrically opposite outcomes, since macaque 42105 gradually gained control over the virus with restoration of CD4+ T cell values, whereas in 42107 there was a slowly progressive loss of the CD4+ T cells that ended in development of AIDS. Thus, only one of the six animals developed disease following exposure to the challenge virus.
  • Methods using the DNA constructs of the present invention to confer protective immunity and/or other therapeutic benefits on non-infected or HIV infected individuals can be accomplished by the direct administration of DNA.
  • Week -7 to -1 were after Cornea inoculation; Week 1 to 4 after Intra-dermal inoculation.
  • Samples taken at time points 1 to 1 1 were during active PMPA therapy. Samples marked with an * correspond to the samples tested and illustrated in table 11 below which shows the results of a more sensitive assay to detect virus RNA in the blood via QCPCR.
  • the Q-PCR technique is currently performed at the NIH, and can detect as little as 300 copies of RNA ml of blood.
  • Table 1 1 illustrates the relatively rapid decline of virus RNA in the blood during PMPA treatment.
  • Table 11 QCPCR of Samples during PMPA Inhibition of SHIV ⁇ Pathogenesis

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Abstract

L'invention concerne le virus pathogène simien et humain VISH (SHIV en anglais) provoquant le sida chez le singe, la construction du virus destiné à être utilisé dans des vaccins contre les VISH et VIH-1 pathogènes ainsi que des procédés de vaccination prophylactique et thérapeutique visant la prévention ou l'inhibition de l'infection et/ou de la maladie dues au VIH-1. L'invention concerne également la construction d'un virus vivant modifié, utilisé dans des vaccins contre le VIH-1, et des procédés de vaccination prophylactique et thérapeutique visant la prévention ou l'inhibition de l'infection et/ou de la maladie dues au VIH-1. Enfin, l'invention concerne l'utilisation de vaccins à base d'ADN viral dans la prévention et le traitement de l'infection et/ou de la maladie dues à VIH-1.
PCT/US1998/008868 1997-05-02 1998-05-01 Vaccins a base de virus vivants visant a proteger des primates contre l'infection et la maladie dues au vih-1 WO1998050070A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2000018430A2 (fr) * 1998-09-29 2000-04-06 University Of Kansas Medical Center Vaccins a virus actif destines a la protection de primates contre des infections et maladies dues au vih-1

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992000987A1 (fr) * 1990-07-12 1992-01-23 President And Fellows Of Harvard College Vaccins prepares a partir d'un lentivirus de primate
WO1993024632A1 (fr) * 1992-05-22 1993-12-09 Dana Farber Cancer Institute Vecteurs viraux hybrides siv/hiv-1 et modele simien du sida
WO1995021912A1 (fr) * 1994-02-14 1995-08-17 The Macfarlane Burnet Centre For Medical Research Limited Souches non pathogenes de vih-1

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992000987A1 (fr) * 1990-07-12 1992-01-23 President And Fellows Of Harvard College Vaccins prepares a partir d'un lentivirus de primate
WO1993024632A1 (fr) * 1992-05-22 1993-12-09 Dana Farber Cancer Institute Vecteurs viraux hybrides siv/hiv-1 et modele simien du sida
WO1995021912A1 (fr) * 1994-02-14 1995-08-17 The Macfarlane Burnet Centre For Medical Research Limited Souches non pathogenes de vih-1

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
B. CHAKRABARTI ET AL.: "A candidate live inactivatable attenuated vaccine for AIDS.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE U.S.A., vol. 93, no. 18, 3 September 1996 (1996-09-03), Washington, DC, USA, pages 9810 - 9815, XP002077810 *
G. ALDROVANDI ET AL.: "Replication and pathogenicity of human immunodeficiency virus type 1 accessory gene mutants in SCID-hu mice.", JOURNAL OF VIROLOGY, vol. 70, no. 3, March 1996 (1996-03-01), Baltimore, MD, USA, pages 1505 - 1511, XP002077808 *
R. DESROSIERS: "HIV with multiple gene deletion as a live attenuated vaccine for AIDS.", AIDS RESEARCH AND HUMAN RETROVIRUSES, vol. 8, no. 8, August 1992 (1992-08-01), New York, NY, USA, pages 1457, XP002077807 *
S. JOAG ET AL.: "Animal model of mucosally transmitted human immunodeficiency virus type 1 disease: Intravaginal and oral deposition of simian/human immunodeficiency virus in macaques results in systemic infection, elimination of CD4+ T cells, and AIDS.", JOURNAL OF VIROLOGY, vol. 71, no. 5, May 1997 (1997-05-01), Baltimore, MD, USA, pages 4016 - 4023, XP002077809 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000018430A2 (fr) * 1998-09-29 2000-04-06 University Of Kansas Medical Center Vaccins a virus actif destines a la protection de primates contre des infections et maladies dues au vih-1
WO2000018430A3 (fr) * 1998-09-29 2000-07-06 Univ Kansas Medical Center Vaccins a virus actif destines a la protection de primates contre des infections et maladies dues au vih-1

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