EP0563323A1 - Impfstoff aus hiv-reverser transkriptase - Google Patents

Impfstoff aus hiv-reverser transkriptase

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Publication number
EP0563323A1
EP0563323A1 EP92904337A EP92904337A EP0563323A1 EP 0563323 A1 EP0563323 A1 EP 0563323A1 EP 92904337 A EP92904337 A EP 92904337A EP 92904337 A EP92904337 A EP 92904337A EP 0563323 A1 EP0563323 A1 EP 0563323A1
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EP
European Patent Office
Prior art keywords
hiv
reverse transcriptase
hiv reverse
virus
vaccine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP92904337A
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English (en)
French (fr)
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EP0563323A4 (en
Inventor
Adolph J. Ferro
Richard K. Bestwick
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Orasure Technologies Inc
Original Assignee
Epitope Inc
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Publication of EP0563323A1 publication Critical patent/EP0563323A1/de
Publication of EP0563323A4 publication Critical patent/EP0563323A4/en
Withdrawn legal-status Critical Current

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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
    • A61K39/275—Poxviridae, e.g. avipoxvirus
    • 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
    • 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
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • 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
    • C12N2710/10011—Adenoviridae
    • C12N2710/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—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
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to synthetic vaccines for immunizing against human immunodeficiency virus (HIV) . More particularly, the invention relates to recombinant vaccine vector-encoded HIV reverse transcriptase (RT) , combined with at least one booster inoculation of purified HIV RT in an adjuvant.
  • HIV human immunodeficiency virus
  • HIV a human retrovirus
  • AIDS acquired immune deficiency syndrome
  • ARC acquired immune deficiency related complex
  • HIV portion that elicits the most powerful natural immune response
  • HIV portion whose amino acid sequence is reasonably conserved
  • protection against any HIV isolate (4) a high enough level of immune response amplification to overcome HIV
  • an immunogen that produces humoral and cell-mediated immunity an immunogen that excludes sites which might induce deleterious side effects.
  • the present invention provides just such a vaccine using HIV RT to induce both a humoral and a cytotoxic T- ⁇ ell response.
  • this invention provides a synthetic vaccine. It comprises a recombinant vaccine vector that incorporates a nu ⁇ leotide sequence capable of being expressed as all or part of an HIV RT.
  • the vector is combined with all or part of a purified HIV RT in an adjuvant.
  • Both the HIV RT expressed by the recombinant vector and the purified HIV RT in the adjuvant can be a 66 Da subtthit Of the HlV RT.
  • the present invention provides a method for producing an immune response in an animal or human by the step of administering a synthetic vaccine, as broadly described above.
  • the present invention applies particularly to the augmentation of immune responses in HIV-infected asymptomatic, immunosuppressed, or immunodeficient animals or humans.
  • a synthetic vaccine is used in the treatment of HIV-infe ⁇ ted asymptomatic, immunosuppressed, or immunodeficient animals or humans.
  • the synthetic vaccine is made up of two parts:
  • a recombinant vaccine vector that incorporates a nucleotide sequence capable of being expressed as all or part of an HIV RT, and (2) at least one booster inoculation of purified HIV RT in adjuvant to enhance the immune response of the animal or human.
  • FIG. 1 schematically represents the construction of the vaccinia recombination vector
  • FIG. 2 shows a Western blot of W:RT-infected cell lysates ⁇
  • FIGS. 3a and 3b are Western blots showing a time course of expression study to detect HIV RT;
  • FIG.4 shows an antibody response in W:RT-infected mice and rabbits
  • FIG. 5 shows the longitudinal titers of anti-RT antibodies in rabbits
  • FIG. 6 shows antibody response against HIV RT in African green monkeysJ
  • FIG. 7 is a graph showing the results of an assay to determine the titer of the anti-RT antibody in African green monkey sera.
  • FIG. 8 is a graph showing the results of an assay to determine the titer of RTI antibodies in vaccinated African green monkeys.
  • the present invention relates to a synthetic vaccine containing HIV RT, which acts as an immunotherapeuti ⁇ or immunopreventive agent for the treatment of HIV infected individuals.
  • the approach is to produce a synthetic vaccine that consists of two parts.
  • the first part is in the form of a recombinant vaccine vector, such as a recombinant vaccinia virus (rW) .
  • the rW was constructed by inserting a nucleotide sequence from the HIV viral genome into a wild-type vaccinia virus (W) . That portion of the HIV genome thus becomes expressed by the recombinant virus.
  • the invention inserts a nucleotide sequence that encodes the HIV RT gene into W, so that the rW functions as a vaccine when inoculated into humans.
  • W the vaccine vector
  • other vaccine vectors such as other poxviruses, herpes virus, adenovirus, or bacteria
  • the second part of the vaccine consisting of purified HIV RT in adjuvant, provides a booster effect.
  • HIV RT is a suitable target for immunopreventative measures against HIV, as shown by the following: (1) a correlation between progression to acute AIDS and the loss of antibodies against HIV RT (Laurence, et al., Science , 2_i_> ⁇ 1501-1504, 1987), (2) the presence of cytotoxic T lymphocytes directed against HIV RT in infected individuals (Walker, et al., Science , 240:64-66, 1988), and (3) the expression of the HIV RT antigen on CD4- positive cells in the peripheral circulation of HIV- seropositive individuals. See U.S. Patent No. 4,904,581.
  • HIV infection or assists in therapy.
  • LTRs long terminal repeats
  • gag, pol r and env encode components of virus particles.
  • the other HIV genes regulate the expression of these three genes.
  • the amino acid horaology between at least 14 different HIV isolates has shown the pol gene of HIV to be the most highly conserved (Myers, et al., eds., Human Retroviru ⁇ es and AIDS, 1990, Los Alamos National Laboratory) .
  • the amino acid homology in the pol gene was 81% between isolates, compared with the gag gene, which was 71%, and the env gene, which was 44%. When the region encoding the RT polypeptide in pol is compared, an 83% homology is observed.
  • the conserved nature of RT supports its use as a vaccine, because, unlike the envelope gene, the degree of antigenic drift is minimal.
  • Previous efforts have focused on expression of the entire pol open reading frame in rW infected cells. This strategy required proteolyti ⁇ processing of the pol polyprotein into protease, HIV RT, and integrase in order to obtain native HIV RT (Farmerie, et al.. Science , 216:305-308,1987).
  • the native HIV RT underwent further pr ⁇ teolyti ⁇ processing by the HIV protease.
  • the HIV was partially cut an additional time, a small sequence at the c-terminus removed, and a heterodimer, containing 66 and 51 kDa stibunits, formed that had common N termini.
  • This further cleavage of the HIV RT may affect the characteristics of the enzyme.
  • this essential proteolysis is inefficient and results in low levels of native HIV RT in rW infected cells. Animals infected with these initial rW constructs developed low or undete ⁇ table immune responses to pol gene products.
  • rWs were constructed, using only the coding sequences for native HIV RT. This was accomplished using specially designed oligonu ⁇ leotide primers and the polymerase chain reaction (PCR) . The method allowed isolation of a segment of the pol region that encoded only a 66 kDa subunit of those amino acids comprising the native HIV RT. The resulting rW generated high levels of HIV RT in infected cells and significant antibody titers in vaccinated mice, rabbits, and monkeys.
  • PCR polymerase chain reaction
  • HIV-infe ⁇ ted individuals have been shown to present RT antigens on T-cells at early stages of HIV infection.
  • a monoclonal antibody against HIV RT specifically binds to CD4-positive cells in the peripheral circulation of HIV- seropositive individuals. See U.S. Patent No. 4,904,581. If these RT-presenting cells represent the HIV-infected cell population, a strong cytotoxic T- ⁇ ell response against HIV RT could protect an individual by clearing infected cells before the virus has a chance to spread.
  • HIV-infected individuals are known, to develop antibodies against HIV RT without this enzyme being presented to the immune system by viral particles. HIV is unique among retroviruses, including HTLV-I and HTLV-II, in its ability to induce anti-RT antibodies.
  • HIV is unique among retroviruses, including HTLV-I and HTLV-II, in its ability to induce anti-RT antibodies.
  • the envelope protein gp4l the most frequently recognized viral antigen in seropositive individuals are the p51, p66 proteins which comprise RT.
  • RTI RT-inhibiting
  • RT-specific cytotoxic T-lympho ⁇ ytes is an important observation in infected individuals.
  • CTL cytotoxic T-lympho ⁇ ytes
  • VAC/ENV vaccinia recombinant virus expressing the HIV envelope glycoprotein, gpl60.
  • Such subjects demonstrated a significant boost in humoral, as well as cell-mediated immunity, following a boost with purified envelope glycoprotein (Hu, et al., Abstracts from VI International Conference on AIDS, Th.A.343, 1990).
  • Individuals with clinical and laboratory evidence of prior smallpox immunization demonstrated a low primary response to VAC/ENV but a subsequent boost effect with purified protein was observed.
  • ARC and AIDS patients were immunized against an HIV preparation to prevent worsening clinical status (Pi ⁇ ard, et al.. Lancet, 336:179, 1990).
  • the vaccine consisted of vac- pol, gag, and env constructs, as well as immunogenic synthetic peptides from the same proteins.
  • Autologous cells were isolated from each individual, infected with the vaccinia constructs in vitro, fixed with paraformaldehyde, and re-inno ⁇ ulated into the patient. In 19 vaccinated patients no opportunistic infection episodes were observed, and weight was maintained out to 9 months post-vaccination.
  • CD4 cells stabilized or increased significantly in the vaccinated individuals as compared with a control group.
  • Vaccination with the vaccinia ⁇ onstruct-infected cells may be an important part of the immunization protocol, because these cells are expressing HIV epitopes in association with class I MHC determinants. This is essential for the induction of CD8 positive cytotoxic cells.
  • soluble proteins found in conventional vaccines do not efficiently generate these types of effector cells.
  • the administration of the rW is generally by means of scratching the skin and application of infectious rW.
  • a localized lesion develops as a result of infection of cells at the site of administration.
  • Non-sterile conditions are often acceptable.
  • administration is slightly more complex.
  • the HIV RT can be prepared as an injectable, either as a liquid solution or suspension; a solid form suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified.
  • the HIV RT can be mixed with excipients which are pharmaceutically acceptable and compatible. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the HIV RT can be combined with minor amounts of auxiliary substances such as wetting or emulsifying agents, or pH buffering agents.
  • the invention encompasses the use of the purified HIV RT in adjuvant as a booster inoculation to enhance the immune response.
  • the adjuvant and the HIV RT are mixed prior to presentation to the immune system, or presented separately, but into the same site of the animal or human being immunized.
  • Adjuvants can be loosely divided into several groups based on their composition. These groups include water-ln-oil emulsion adjuvants, such as complete Freund's adjuvant, in which living or dead mycobacteria ar ⁇ i suspended. The intense, chronic inflammation around the deposits of emulsion of this adjuvant precludes it use in man.
  • incomplete Freund's adjuvant which has an emulsion without mycobacteria is less irritating and has been used clinically.
  • Other groups of adjuvants include mineral salts (for example, AlKfSO j , AlNa(S0 4 ) 2 , A1NH 4 (S0 4 ), silica, alum, Al(OH) 3 , Ca 3 (P0 4 ) j , kaolin, and carbon) , polynucleotides (for example, poly IC and poly AU acids), and certain natural substances (for example, wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, Bordetella Pertussis, and members of the genus Brucella) .
  • Another substance useful as an adjuvant is Quil A, a crude mixture of saponins that has detergent-like properties.
  • the purified HIV RT in adjuvant is conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration includesuppositories, intranasal aerosols, and, in some cases, oral formulations.
  • traditional binders and carriers may include, for example, polyalkylene gly ⁇ ols or trigly ⁇ erides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%- 2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain 10%-95% of active ingredient, preferably 25%-70%.
  • the HIV RT may be formulated into neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups) which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, and ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2- ethylamino ethanol, histidine, procaine, and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium, and ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2- ethylamino ethanol, histidine, procaine, and the like.
  • the HIV RT in adjuvant is administered in a manner compatabile with the dosage formulation, and in such amount as to be immunoeffective either as a therapeutic or preventitive.
  • the quantity to be administered depends on the animal or human to be treated, capacity of the individual's immune system to produce antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual.
  • the top line represents the genome of HIV-1 and the boxes below the line signify the known genes.
  • RF1, RF2, and RF3 in FIG. 1 indicate the three open reading frames. Each gene is aligned with one of these open reading frames.
  • the pol gene consists of a polyprotein encoding protease (prot) , reverse trans ⁇ riptase (RT) , and integrase (INT) .
  • the RT portion of the pol gene was isolated by PCR.
  • the PCR primers shown in FIG. 1 are aligned with the pBHIO sequence.
  • the PCR fragment produced was digested with Nco I and ligated to pUCK-19.
  • the pUCK-19 vector had been digested with Asp718, treated with Klenow, and digested finally with Nco I.
  • the resulting clone, pUCK-RT was used to generate the pW3-RT vaccinia recombination vector.
  • the remaining part of the example is a detailed description of the construction of the vaccinia recombination vector.
  • a full-length infectious clone of HIV-1, pBHIO (Ratner, et al., Nature, 313:277-284. 1985), was used as the source of cloned viral DNA.
  • Oligonucleotide primers were designed such that after PCR, an ATG start codon and a TAG stop codon are at the beginning and end, respectively, of the HIV RT coding region.
  • the oligonucleotide primers and the strategy used in the remainder of the recombinant DNA phase of this project are shown in FIG. 1.
  • the 5 1 primer was also designed to have a spe ⁇ fic restriction enzyme recognition site (Nco I) at the 5' end to facilitate recombinant DNA manipulations.
  • the amino terminal amino acid of native HIV RT is a proline at nucleotide position 1909.
  • the PCR- derived HIV RT required the addition of methionine and valine at the amino terminal end to allow translational initiation at the Nco I restriction site (see FIG. 1) .
  • PCR was performed using the two primers shown in FIG. 1 in a lOO ⁇ l reaction volume containing containing lO M Tris-HCl (pH 8.3), 50mM KCl, 1.5mM MgCl 2 , 0.1% gelatin, 100 pmoles of each primer, and 1 ng of pBHIO.
  • the reaction mixture was subjected to thermal cycling as follows: 94"C for 1.0 min. ,* 45*C for 1.0 min. ; and 72'C for 3 min. This cycle was repeated for 30 times and the amplification was terminated with the final 72*C incubation held for 10 minutes.
  • the 1691 base pair PCR fragment was purified by preparative agarose gel electrophoresis, digested with Nco I, and inserted between the Nco I site and Klenow- treated Asp 718 site of pUCK-19.
  • pUCK-19 is a vector specially engineered for insertion of eukaryoti ⁇ genes.
  • pUCK-19 was constructed by inserting a synthetic oligonucleotide containing the sequences shown in FIG. 1 between the Bam HI and Kpn I sites of pUC19 using standard procedures (Maniatis, et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982). The validity of the resulting pUCK-RT clone was verified by determination of the DNA sequence at the 5* and 3' ends of the HIV RT gene in this plasmid.
  • the DNA sequence confirmed the presence of the ATG start codon and the TAG stop codon in the proper reading frame. Furthermore, the 200bp of HIV RT coding sequences that were determined at the 5' and 3' ends of the gene matched exactly the published DNA sequence of the pBHIO clone (Ratner, et al. ,
  • pW3 is a vaccinia recombination vector developed by Dr. Denis Hruby at Oregon State University, Corvallis, OR (Thomas, et al., Science , 212:1641-1643, 1986).
  • Ltk Ltk cells obtained from Dr. Denis Hruby at Oregon State University, Corvallis, OR. Ltk " cells were seeded onto 100mm culture dishes at approximately 50% confluency on day one. On day two the cells were infected at a multiplicity of infection (MOI) of 0.05 with wtW. The infection was allowed to proceed for exactly 3 hours before the additon of the DNA.
  • DNA for transfe ⁇ ti ⁇ n was prepared using the Lipofectin reagent according to the manufacturer's instructions (Bethesda Research Laboratories) .
  • pW3-RT plasmid DNA Twenty ⁇ g of pW3-RT plasmid DNA was mixed with one ⁇ g of wtW DNA and used for the cotransfeetion step. After 24 hours, the infected cells were harvested by scraping the cell monolayer, and a crude W stock was prepared by three consecutive freeze thaw cycles, which lysed the infected cells. Further amplification of the W occurred in mouse Ltk " cells grown in BUdR (25 ⁇ g/ml) containing medium.
  • mice Ltk were selected for those vaccinia viruses that had lost the wt thymidine kinase (tk) gene as a result of recombination with the tk sequences that flank the HIV RT sequences in pW3-RT (see FIG. 1) . This was accomplished by infecting BudR-selected Ltk " cells with the crude stock at an MOI of 0.2 and by making a second crude stock by lysing the cells at 72 hours post-infection.
  • tk wt thymidine kinase
  • Viruses from the BudR selection were plated and screened for HIV RT gene sequences by plaque hybridization.
  • lOOmm dishes of confluent BSC40 cells (ATCC, Rockville, MD) were infected with 500 pla ue-forming units of W, and the infection was allowed to proceed for 36 hours.
  • the monolayers with vaccinia plaques were transfered to 87mm nitrocellulose filters by first draining the medium, then placing the filters directly onto the cells. The filters were lifted from the plates and placed cell side up on PBS-soaked filter paper. A replicate filter was made by placing a second nitrocellulose filter against the first filter.
  • the original filter was stored at 70 ⁇ C to preserve the viruses in each plaque, while the replica was screened for positive plaques using an approach identical to bacteriophage lambda plaque hybridization (Maniatis, et al., .Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, K.Y., 1982).
  • the plaque hybridization procedure used as a probe "P- labeled DNA, generated by random-priming DNA from the PCR reaction described above.
  • Several positive plaques were grown and screened for the correct genome structure using restriction mapping procedures of DNA isolated from partially purified W. Of the several W:RT recombinant clones resulting from this analysis, W:RT-3 and W:RT- 24 were selected.
  • W:RT-3 A sample of the vaccinia recombination vector herein designated W:RT-3 has been deposited with the American Type Culture Collection (ATCC) at 12301 Parklawn Drive, Rockville, MD 20852 USA and has been given ATCC accession number VR 2289.
  • ATCC American Type Culture Collection
  • FIG. 2 shows a Western blot of W:RT infected cell lysates using human anti-HIV sera, Human High Positive Sera (Epitope, Inc.), as the detecting antibody. HIV RT is expressed in the W:RT infected cells.
  • the Western Blot is performed on protein extracts from vaccinia-infected or mock infected cells. The polyacrylamide gel electrophoresis and electroblotting was performed using standard procedures. Lanes 1-3 are W:RT isolates #24 and #3, lane 4 is wild-type vaccinia virus (W:wt) ; and lane 5 is mock infected BSC40 cells.
  • FIGS. 3a and 3b are identical Western blots showing a time course of expression study of RT in W:RT-infected cells using two different anti-RT monoclonal antibodies (MAb) to detect HIV RT.
  • Lane 1 is a molecular weight marker
  • lane 2 is mock infected BSC40 cells
  • lanes 3-5 are pre- infection, T 0
  • lanes 6-8 are two hours post-infection, T 2
  • lanes 9-11 are seven hours post-infection, T 7
  • lanes 12- 14 are 24 hours post-infection, T 24
  • lane 15 is purified HIV.
  • the lane order within each time point is W:wt, W:RT3, and W:RT24.
  • the Western blot in FIG. 3a was developed using 1C11 MAb (Epitope, Inc.) and in FIG. 3b commercially available anti-RT MAb (Cellular Products, Inc.) . Both MAbs showed specific and strong reaction with a 66 kDa band that aligns exactly with the native p66 protein from purified HIV (lane 15) . It should be noted that W:RT does not express the HIV protease necessary for processing of HIV RT to the 51 kDa form. EZAMPLE 4
  • HIV RT was purified from W:RT-infected HeLa cells using a modification of a published protocol (Flexner, et al., Virology, X6£:339-349, 1988). Briefly, 5 x 10 9 HeLa cells grown in suspension were infected with 5 x 10 10 pfu of W:RT-3 and were allowed to grow for 24 hours. The cells were lysed in 25 mis of a hypotonic buffer, and the nuclei pelleted by centrifugation. The supernatant measured 25ml and contained the bulk of the HIV RT.
  • the lysate was adjusted to 75 M NaCl, 50mM Tris-HCl (pH 8.0), ImM DTT, and 0.01% NP40 (Buffer A) and applied to a 2.5 x 12.5 ⁇ m DEAE Sepharose (Pharmacia, Inc.) column equilibrated with Buffer A. Unbound protein was eluted with 100ml of Buffer A. Bound proteins were eluted with a two-step gradient of 150 and 3Q0mM NaCl. Column fractions were analyzed by Western blot for HIV RT using the 1C11 anti-RT mAb (Epitope, Inc.) as the detecting antibody.
  • the bulk of the HIV RT was in the flow through fractions and the 50ml that contained the HIV RT were loaded onto the same sized column containing phosphocellulose (Whatman, Inc.) equilibrated with buffer A. Unbound protein was eluted with 20ml of buffer A, and a 200ml NaCl gradient from 75mM to 300mM was used to elute the HIV RT that had bound to the column.
  • the collected fractions were analyzed for HIV RT on Western blots and for total protein on Coomassie blue-stained polyacrylamide gels.
  • the fractions containing pure RT were pooled and concentrated using a Centriprep 30 concentrator (Amicoh, Inc.). The protein concentration of the concentrated preparation was 133 ⁇ g/ml, as determined using the BCA Protein Assay Reagent (Pierce) .
  • mice were from days 14 and 32 post-infection and day 8 post-boost with 50 ⁇ g of purified RT.
  • NMS in FIG. 4 is normal mouse sera. Sera from all of the W:RT-infected mice developed a faint p66 band, at 14 days post- inoculation. In contrast, sera from mice immunized with a wild-type vaccinia preparation and normal mouse sera did not develop either p66 or p51 bands on the blots (FIG. 4) . At 28 days post-inoculation 3 of 4 rabbits were p66 and p51 seropositive. One rabbit designated CA. , which was positive at day 14, reverted to a seronegative status at day 42.
  • mice All four vac ⁇ inia-RT mice continued to be seropositive for p66, as well as for p51 at 32 days post- inoculation.
  • the three rabbits seropositive at day 28 continued to test positive against HIV RT (p66, p51) at both 35, 42, and 72 days post-inoculation.
  • the mice and rabbits were boosted with purified HIV RT in Complete Freund's adjuvant and both groups of animals responded by generating high titered antisera against HIV RT.
  • the boosted mice and rabbits were used to generate a panel of anti-RT monoclonal antibodies and high titered antisera, respectively.
  • Sera from two of the boosted rabbits were collected weekly for several months and were analyzed on EpiBlotTM strips as shown in FIG. 5.
  • W:RT-3 was Used to induce a humoral and cellular immune response against HIV-RT in African green monkeys (AGM) . Each AGM was inoculated by skin scarification with lO'PFU of vaccinia virus. Six animals were infected with W:RT and two with W:wt. As shown in FIG. 6, animals infected with the W:RT specifically induced antibodies against HIV-RT, as determined by running 0, 3, 4, and 5 week post- infection sera on HIV-1 Western blot strips. In FIG. 6, the top two sets of blots show the anti-RT response in monkeys following infection with W:wt or W:RT-3. Each set of 4 strips is from a single animal at days 0, 21, 28, and 35 days post-infection.
  • the bottom panel shows the boost response to RT in the vaccinated monkeys.
  • Each set of three strips is from a single animal and represents sera from 0, 7, and 17 days post-boost. Animals J261 and J571 were not boosted since they were the W:wt control animals.
  • the humoral anti-RT response was confirmed by running the sera against Western blot strips generated from either W:wt or W:RT infected cell lysates. Although the anti-RT reactivity is weak, a boost with purified RT significantly enhanced this response as was seen in both mice and rabbits.
  • a threonyl-MDP adjuvant (Syntex) .
  • Purified HIV RT was dialyzed against PBS (pH 7.2) without Mg ** and Ca* * and the concentration was determined to be 133 ⁇ g/ml.
  • the threonyl-MDP was administered at lOO ⁇ g/kg body weight.
  • the adjuvant was mixed with an equal volume of SAF carrier and administered at lOO ⁇ g/kg body weight and then injected intramuscularly.
  • Sera were obtained at 7, 17, 21, and 28 days post-boost and analyzed as in FIG. 4 on EpiBlotTM HIV- 1 Western blot strips.
  • RT-enzyme-1inked immunoabsorbent assay (ELISA) was performed using serial dilutions of the sera from the 28 day bleeds. This assay was performed by coating the wells of a 96-well mi ⁇ rotiter plate with lOOng of purified HIV RT and reacting the wells with serial two-fold dilutions of the antisera. RT- specific antibody was detected using peroxidase conjugated to antibody that recognizes AGM antibodies. As seen in FIG. 1 , the anti-RT titer of the animals exceeded a 10*- fold dilution at two weeks post-boost.
  • the antibody titer was measured by determining the dilution the sera could withstand and still have reactivity to purified RT.
  • the titer is represented as the dilution factor of each sera that resulted in a signal two-times background.
  • Each line represents one animal.
  • Animal J261 and J571 were the W:wt control animals.
  • RT-inhibitory antibodies that inhibit the enzymatic actvity of HIV RT are of clinical relevance.
  • an enzymatic assay was developed, and serial dilutions of the AGM antisera were tested for the ability to inhibit HIV RT. As shown in FIG. 8, the titer of RTI antibodies in four of the AGMs exceeded a 12,000 dilution. The assay was conducted using detergent disrupted HIV-1 from actively infected cells and a standard HIV-1 RT assay (Goff, et al., J. Virol . , 18.:239-248, 1981).
  • This assay was performed using HIV strain 111b from Hut78-infected cells cultured at Epitope.
  • a standard HIV reverse transcriptase assay (Technicmes in HIV Research. Eds. A. Aldovini and, B.D. Walker, Stockton Press, 1990, and Goff et al., J. Virol . , 8.:239-248, 1981) was modified to incorporate a preincubation with monkey sera as follows. Sera were diluted in PBS and 5 ⁇ l of each dilution was incubated with 5 ⁇ l of detergent-disrupted cell-free supernatant from the Hut78-infected cultures. The detergent disruption was performed by making the HIV containing culture medium 0.05% NP40.
  • the incubation of virus with sera was performed at 4°C for 30 minutes.
  • the 10 ⁇ l samples were then added to a 50 ⁇ l RT reaction mixture and assayed as described in the above cited assays. Data are represented as that dilution at which there is 20% ina ⁇ tivation of the RT activity when compared to negative control values.
  • the two wild-type vaccinia control monkeys are J261 and J571 and are slhown as having an inhibition titer of 25. Since the lowest dilution measured was a 25-fold dilution and these two sera allowed 100% RT activity at this dilution, their actual inhibition titer is probably much lower.
  • the cell mediated response of the immunized animals to both HIV RT and W was assessed by stimulating peripheral blood lymphocytes with the antigen and measuring tritiated thymidine uptake at six days. As shown in Table 1 below, two of the six RT-vac ⁇ ina ed animals had lymphocyte stimulation indices (LSI) that exceeded five. In Table 1, the numbers under the headings of No Antigen and [RT] represent the counts per minute of tritriated thymidine incorporated into cellular DNA. Although only two of six animals demonstrated cellular proliferation in the presence of RT, it is clear that the vaccine described herein is fully capable of eliciting an RT-specifi ⁇ cellular response.
  • RT-specific Lymphocyte Proli eration in Vaccinated African Green Monkeys Fresh whole blood from each monkey was used as the source of peripheral blood lymphocytes used in the assay results shown in Table 1. The amount of RT in each well is shown at the top of Table 1. The lymphocytes stimulation was determined by measuring tritriated thymidine uptake at six days. The degree of stimulation is shown as the LSI and is the ratio of the incorporated counts from the antigen- containing wells divided by the counts from the no antigen wells. Table 1. RT- specific Lymphocyte Proli eration in Vaccinated African Green Monkeys
  • the starting virus for the adenovirus construct is adenoviru type 5 deletion mutant dl 327 that lacks the Xba fragment fro 78.5 map units to 84.7 map units in early region 3 (Cutt, e al . , J. Virol., 61:543, 1987).
  • This deletion mutant allow the insertion of DNA without exceeding the amount of DNA tha can be included in the virus particle.
  • the removal of the E region also prevents production of a virus protein that complexes with the major histocompatibility heavy chain protein and reduces the eel1-mediated immune response to th virus.
  • the Bam fragment from 60 map units to the right han end of the viral DNA is cloned in plasmid.
  • the plasmid DN is cut downstream of the E3 promoter with a suitable restriction enzyme, and the HIV RT gene inserted in place o the original E3 gene, under the control of the natural
  • the resulting plasmid containing the HIV RT gene in the 60 to 100 map unit fragment of dl 327 is cut with the appropriate restriction enzyme to separate viral and plasmid DNA, and transfected into cells together with the overlapping E ⁇ oRl A fragment (0 to 76 map units) of wild type virus. Recombination between the two overlapping DNA fragments will reconstitute viable adenovirus in which the E3 gene is replaced by the HIV RT gene.

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US8367315B2 (en) 2010-03-05 2013-02-05 Adan Rios Inactivation of reverse transcriptases by azido-diarylpyrimidines

Citations (4)

* Cited by examiner, † Cited by third party
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WO1987007296A1 (en) * 1986-05-20 1987-12-03 The Trustees Of Columbia University In The City Of Expression of human t-cell lymphotropic virus (htlv-iii) reverse transcriptase and uses thereof
EP0372904A2 (de) * 1988-12-07 1990-06-13 The Research Foundation for Microbial Diseases of Osaka University Protease, reverse Transcriptase und Endonuklease von Retroviren und Verfahren zur Herstellung dieser Enzyme
WO1990010230A1 (en) * 1989-02-23 1990-09-07 University Of Ottawa Polypeptide having immunological activity for use as diagnostic reagent and/or vaccine
WO1991013910A1 (en) * 1990-03-09 1991-09-19 The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce Peptides stimulating cytotoxic t cells immune to hiv rt

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987007296A1 (en) * 1986-05-20 1987-12-03 The Trustees Of Columbia University In The City Of Expression of human t-cell lymphotropic virus (htlv-iii) reverse transcriptase and uses thereof
EP0372904A2 (de) * 1988-12-07 1990-06-13 The Research Foundation for Microbial Diseases of Osaka University Protease, reverse Transcriptase und Endonuklease von Retroviren und Verfahren zur Herstellung dieser Enzyme
WO1990010230A1 (en) * 1989-02-23 1990-09-07 University Of Ottawa Polypeptide having immunological activity for use as diagnostic reagent and/or vaccine
WO1991013910A1 (en) * 1990-03-09 1991-09-19 The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce Peptides stimulating cytotoxic t cells immune to hiv rt

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, vol. 27, no. 25, 13 December 1988 EASTON, PA US, pages 8884-8889, LOWE, D.M. ET AL. 'HIV-1 Reverse Transcriptase: crystallization and analysis domain structure by limited proteolysis' *
JOURNAL OF BIOLOGICAL CHEMISTRY (MICROFILMS), vol. 264, no. 24, 25 August 1989 MD US, pages 13975-13978, M]LLER, B. ET AL. 'Co-expression of the subunits of the heterodimer of HIV-1 Reverse Transcriptase in Escherichia coli' *
JOURNAL OF MEDICAL VIROLOGY, vol. 39, no. 3, March 1993 pages 251-259, LI, X. ET AL. 'Generation and characterization of murine monoclonal antibodies reactive against N-terminaland other regions of HIV-1 Reverse Transcriptase' *
See also references of WO9211028A1 *
VIROLOGY, vol. 166, no. 2, October 1988 pages 339-349, FLEXNER, C. ET AL. 'Characterization of human Immunodeficiency Virus gag-pol gene products expressed by recombinant vaccinia viruses' *

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