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WO1993001278A1 - Lignees cellulaires lymphoïdes felines susceptibles de produire le virus de l'immunodeficience feline (vif) - Google Patents

Lignees cellulaires lymphoïdes felines susceptibles de produire le virus de l'immunodeficience feline (vif) Download PDF

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WO1993001278A1
WO1993001278A1 PCT/US1992/005571 US9205571W WO9301278A1 WO 1993001278 A1 WO1993001278 A1 WO 1993001278A1 US 9205571 W US9205571 W US 9205571W WO 9301278 A1 WO9301278 A1 WO 9301278A1
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fiv
virus
cells
cats
cell
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PCT/US1992/005571
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Janet K. Yamamoto
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The Regents Of The University Of California
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    • 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
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/5555—Muramyl dipeptides
    • 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
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15034—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
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15051—Methods of production or purification of viral material

Definitions

  • the present invention relates generally to the detection and treatment of viral infection. More particularly, the invention relates to compositions and methods useful for the diagnosis of and vaccination against infection with a newly-discovered lymphotropic retrovirus, initially designated as feline T-lymphotropic lentivirus and presently designated feline immunodeficiency virus (FIV) .
  • a newly-discovered lymphotropic retrovirus initially designated as feline T-lymphotropic lentivirus and presently designated feline immunodeficiency virus (FIV) .
  • domestic cats may become infected with several retroviruses, including feline leukemia virus (FeLV) , feline sarcoma virus (FeSV) , endogenous type C oncorna- virus (RD-114) , and feline syncytia-forming virus (FeSFV) .
  • FeLV feline leukemia virus
  • FeSV feline sarcoma virus
  • RD-114 endogenous type C oncorna- virus
  • FeSFV feline
  • FeLV is the most significant pathogen, causing diverse symptoms, including lymphoreticular and myeloid neoplasms, anemias, immune- mediated disorders, and an immunodeficiency syndrome which is similar to human acquired immune deficiency syndrome (AIDS) .
  • AIDS human acquired immune deficiency syndrome
  • FeLV-AIDS a particular replication- defective FeLV mutant, designated FeLV-AIDS, has been more particularly associated with immunosuppressive properties.
  • feline T-lymphotropic lentivirus now designated feline immunodeficiency virus
  • AIDS 4:215-220 describes the serological response of cats infected with FIV.
  • a portion of the experimental data presented in this application was published in AIDS 1990 4 (Suppl. 1) :S163-S165.
  • the present invention comprises feline lymphoid cell lines chronically infected with FIV, specifically designated FL-4 and FL-6, both of which are IL-2 independent.
  • the live FIV-infected cell lines can be prolific sources of FIV, while inactivated or killed FIV- infected cell lines are useful as immunogens in vaccine compositions according to the present invention.
  • the present invention still further comprises non-infected feline T-lymphocyte cell lines which are designated FeT-lM and FeT-2D and which are susceptible to FIV infection.
  • FeT-lM and FeT-2D are IL-2 dependent.
  • the vaccine compositions are administered to susceptible hosts, usually cats, in amounts effective to afford immunity against subsequent challenge by FIV.
  • the vaccines may be administered by any conventional route, including subcutaneously, intramuscularly, and oranasally, and will usually be administered at least twice over intervals spaced-apart by one or more weeks to achieve the desired immunity.
  • FL-4 (A), FL-6 ( ⁇ ), FIV-FeTl ( ⁇ ) , and FIV-CRFK (D) cells were seeded at 5xl0 5 cells/ml and tested daily for the RT activity in their culture fluids.
  • a gradual increase in RT activity was observed over the four days of culture, with peak RT titers detected on Day 4 for all cell cultures except FIV-FeTl which had it on Day 3.
  • Peak viable cell counts (1.0-2.25x10 6 cells/ml) were observed on Day 3 for all cell cultures except for the FIV-FeTl culture ⁇ which had its peak viable cell count (1.3xl0 6 cells/ml) on Day 2.
  • the percent cell viabilities during trie four days of culturing were 75-90% for FIV-FL-4, 70-90% for FIV-FL-6, 70-80% for FIV-CRFK, and 55-65% for FIV-FeTl.
  • the FACS profiles of the surface phenotype of FL-4 (A,B,C) and FL-6 (D,E,F) were determined using characterized monoclonal antibodies to feline CD4 (Fel 7) , CD8 (FT2) , pan T-cell, and the feline light chain and ⁇ heavy chain specific (AC5) markers. Both cell lines had cell populations which were positive for CD4 (A,D) , CD8 (B,E), and pan T-cell (C,F) . Both FL-4 and FL-6 cells tested negative by FACS analysis for surface B cell markers using monoclonal antibodies (AC5) (data not shown) . The solid lines represent the FACS profiles of FL-4 and FL-6 cells and the dotted lines represent the FACS profiles of negative control cells.
  • the percentages of FL-4 cells that were positive for CD4, CD8 and pan T- cell markers were 10%, 20%, and 80% respectively.
  • the percentages of FL-6 cells that were positive for CD4, CD8 and pan T-cell markers were ⁇ 8%, 11%, and 76%, respectively.
  • expression of CD4 and CD8 on the cell membrane can be decreased or eliminated.
  • the abscissa represents fluorescence intensity and the ordinate represents relative cell number.
  • CRFK (C,F) cells were tested for their infectivity on different feline PBLs (A,B,C) and feline thymocytes (D,E,F) .
  • Uninfected feline lymphoid cells used in this study were FeTl.l ( ⁇ ) , FeT1.2 (D) , FeT1.3 (o) , Thyl ( ⁇ ) , and Thy2 ( ⁇ ). All of the FeTl cells were derived from uninfected PBLs and Thy cells were primary thymocytes obtained from FIV-free kittens.
  • FeTl.l, FeTl.2, and FeTl.3 were subclones of the uninfected FeTl line, which was the precursor line for FIV-FeTl cells.
  • the percentage of cells that was positive for CD4 and CD8 markers was ⁇ 2% and 5% for FeTl.l, ⁇ 2% and ⁇ 2% for FeTl.2, and ⁇ 2% and 4% for FeTl.3, 54% and 4% for Thy l, and 38% and ⁇ 2% for Thy2, respectively.
  • FIV from all cell lines were able to either transiently and persistently infect all lymphoid cells except for those from FeTl.2 cultures, whose cells also totally lacked the expression of both CD4 and CD8 markers.
  • the immunogenicity of the FIV produced from our FIV-infected cell lines was evaluated in cats.
  • the reactivities of the antibodies produced in cats immunized with either inactivated FL-4 (A) or FL-6 (B) cells or with inactivated FL-4-produced virus (B) were determined by immunoblot analysis.
  • Cats were immunized six-times with inactivated FL-4 or uninfected FeTl cells and their serum immunoblot profiles were compared to those of serum from cats naturally (Cat #C12) or experimentally (Cat #H7) infected with FIV (A) .
  • Cats were also immunized four-times with inactivated FIV (produced by FL-4 cells) and with inactivated FL-6 cells (B) and evaluated similarly.
  • the molecular weights of the viral protein components correspond to the immunoblot patterns of 24-28 kD for major core, 15-17 kD for minor core, 10 kD for minor core, 54-55 kD for core precursor, 62 kD for RT, 32 kD for endonuclease, 37-44 kD (diffuse band) for transmembrane, and 100-120 kD for envelope (Yamamoto et al. (1988) supra. ,' Hosie et al. (1990) supra. ; and O'Connor et al. (1989) J. Clin. Micro. 27:474-479).
  • the FIV IgG antibody titer was measured by enzyme-linked immunosorbent assay (ELISA) using 250 ng/microwell of sucrose-gradient purified FIV as substrate and biotinylated goat anti-cat IgG (Vector Laboratories, BA-9000) as conjugating antibody (Pedersen et al. (1987) Science 235:790-793). Sera from the different bleeding dates of each cat were serially diluted and assayed simultaneously in a single test. The results are based on two separate ELISA testings. Part A gives the results from cats immunized with the fixed cell-virus vaccine and part B gives results from cats immunized with the inactivated whole-virus vaccine. Fig. 7
  • the neutralizing antibody titers to FIV were assayed the FIV-susceptible feline lymphoid cell line FeTl.
  • diluted samples of heat-inactivated serum 56°C for 30 min
  • TCID 50 tissue culture infective doses
  • FIV Fetaluma strain
  • the cells were washed once with Hank's balanced salt solution to remove residual virus from the culture and then resuspended in fresh culture media (RPMI 1640 containing 10% heat- inactivated fetal calf serum, 10 mM HEPES buffer, 50 ⁇ g/ml gentamicin, 1x10 s M 2-mercaptoethanol, and 100 U/ml human recombinant IL-2) .
  • Virus infection was monitored by Mg ++ -dependent RT assays of the culture fluid. The serum was considered positive for neutralizing antibodies when RT activity was ⁇ 50% of the infected control culture which had no serum exposure.
  • Nonspecific antiviral activity i.e., interferon activity
  • vesicular stomatitis virus Yamamoto et al. (1986) Vet. Immunol. Immunopathol. 11:1-19.
  • Part A gives the results from cats immunized with the fixed cell-virus vaccine and part B gives results from cats immunized with the inactivated whole-virus vaccine.
  • Figs. 9A and 9B
  • PBLs were isolated from blood harvested at 27 weeks pc from all challenged animals and at 14 weeks post-immunization from unchallenged cats.
  • the proliferation assay consisted of 3 H-thymidine incorporation by PBLs (lxlO 5 cells/microwell) upon stimulation with inactivated FIV (4.5 ⁇ m/microwell) for five days at 37°C.
  • IL-2 assay consisted of measuring the amount of 3 H-thymidine incorporation of the IL-2-dependent murine HT-2C cells in presence or absence of IL-2 containing samples (Gillis et al. (1978) J. Immunol. 120:2027-2032).
  • the vaccine protected cats and the vaccinated but unchallenged cats responded significantly (stimulation index > 2.0) to FIV antigenic stimulation in both proliferation (P ⁇ 0.001) and IL-2 induction (P ⁇ 0.001) assays.
  • the P value was derived by using two-tailed t-test. Part A presents the results from the proliferation assay and part B the results from the IL-2 induction assay.
  • feline immunodeficiency virus previously designated feline T-lymphotropic lentivirus (FTLV) has been discovered and isolated in substantially pure form.
  • the virus is infectious in cats, causing a wide variety of symptoms, including abortion, alopecia, anemia, chronic rhinitis, conjunctivitis, diarrhea, emaciation, enteritis, gingivitis, hematochezia, neurologic abnormalities, periodontitis, and seborrheic der ititis. The course of the disease is usually fatal.
  • FIV farnesoid fever
  • HAV human immunodeficiency virus
  • SAIDS simian T-lymphotropic virus III
  • FIV does not appear to be antigenically related to HIV or to SAIDS, but rather appears to be a species-adapted lentivirus that has existed in cats for some time.
  • Preliminary surveys conducted by the inventors herein indicate that FIV infection in cats may be widespread, possibly accounting for a significant proportion of the immunodeficiency symptoms found in cats who are free from FIV infection.
  • FIV is a feline immunodeficiency virus char ⁇ acterized as a retrovirus, more specifically as a lenti ⁇ virus, which is tropic for T-lymphocytes of the host which it infects.
  • the virus is also characterized by horizontal transmission, and may further be characterized by vertical transmission in at least some cases.
  • FIV is polymorphic, and reference to FIV in the present application is intended to encompass the entire FIV family, including a variety of strains which share substantial amino acid sequence and nucleotide sequence homology and which are immunologically related.
  • Substantial amino acid sequence homology means at least about 75% homology, usually at least about 80% homology, and frequently 90% homology and above in at least some of the viral genes and proteins.
  • the env, gag, or pol regions may display the requisite homology, while the genome as a whole does not. In such cases, so long as the viruses are immunologically related, the viruses will be considered to be FIV within the ambit of the present invention.
  • immunologically related it is meant that the various strains will display substantial serologic cross- reactivity with the newly-discovered strain which has 11 been deposited.
  • Serologic cross-reactivity is defined as the ability of an antiserum or antibodies specific for the deposited FIV strain to react with other FIV strains as well as the deposited strain.
  • immunologically related strains will cross-react with antibodies specific for more than one epitopic site, usually more than five epitopic sites, and frequently ten or more epitopic sites.
  • FIV strains may be identified by Western blot analysis where purified virus is disrupted with a suitable detergent, e.g., sodium dodecyl sulfate, and separated on a slab gel by electrophoresis. The separated polypeptide bands are transferred from the gel to nitrocellulose filter paper and visualized with labelled antibody. The molecular weights of the various resolved bands may then be determined by comparison to known molecular weight standards. Substantial similarity between the Western blot analysis of an unidentified virus and that of a known FIV virus indicates that the unknown virus is likely an FIV virus.
  • a suitable detergent e.g., sodium dodecyl sulfate
  • FIV isolates have been characterized, indicating that the nucleotide sequence of the envelope gene varies by no more than about 15% among isolates.
  • Such isolates from different regions, are described in Masashi et al. (1990) In: Proc. 6th Intnl. Conf. AIDS, June 20-24, San Francisco, Abstract Th.A. 284 (Japanese isolate); Phillips et al. (1990) J. Virol. 64:4605-4613 (San Diego, California) ; Olmsted et al. (1989) Proc. Natl. Acad. Sci. USA 86:2448-2452 (Petaluma, California) ; Talbot et al. (1989) Proc. Natl. Acad. Sci.
  • FIV encodes an RNA-dependent DNA polymerase (reverse transcriptase) which is Mg +2 -dependent with maximal activity occurring at a Mg "1*2 concentration of approximately 5 mM and pH of approximately 7.8.
  • FIV bands at a density of about 1.15 gem 3 in a continuous sucrose gradient.
  • Western blotting of FIV-infected cell lysate yields major bands at approximately 22 to 28 kD, usually about 26 kD; 50 to 60 kD, usually about 55 kD; and 28 to 36 kD, usually about 32 kD.
  • FIV may be isolated from the sera of infected cats by conventional techniques.
  • peripheral blood lymphocytes PBL
  • PBL peripheral blood lymphocytes
  • the cultures are incubated, with normal PBL's being periodically introduced to the culture in order to maintain its viability as the original cells are killed by the virus.
  • the infected cells should be placed in fresh culture medium periodically, and the virus may be recovered from the supernatant of the cell culture by sucrose-gradient separation, or other known separation techniques.
  • the FIV may also be obtained from other speci- mens, particularly from the lymph tissues of infected animals.
  • the lymph tissues are broken and then suspended in culture medium, and the procedures described above are then carried out.
  • compositions according to the present invention include the whole virus, as well as portions of the virus.
  • the whole virus may be maintained in in vitro culture, as described above, or may be viably frozen at a temperature at or below about -78°C (solid C0 2 -dry ice) , usually in the presence of agents which promote amorphous, vitreous solidification rather than crystallization. Suitable agents include glycerol and dimethylsulfoxide.
  • Portions of the FIV of particular interest include the structural and regulatory proteins encoded by the FIV genome, including the envelope and core proteins, and fragments thereof.
  • the FIV may also be maintained in chronically infected cell lines, particularly T-cell lines, as described in detail in the Experimental section hereinafter.
  • interleukin 2 (IL-2)-dependent T-cell lines can be infected with FIV and maintained in IL-2-supplemented culture media.
  • IL-2-independent cell lines can then be prepared by repeated subculturing with a gradual depletion of IL-2. Surviving cultures can then be maintained in culture free from IL-2.
  • the IL-2- independent FIV-infected cell lines have been found to possess enhanced viability and a reduced percentage of syncytial cells when compared to IL-2-dependent FIV- infected cell lines. See, Experimental section hereinafter.
  • the FIV used for infecting the cell lines may be isolated from infected cats, as described above, or may be obtained from the deposited Petaluma strain of the virus (A.T.C.C. VR 2186).
  • FeT-lM A.T.C.C. Accession No. CRL 10775
  • FeT-2 A.T.C.C. Accession No. CRL 10774
  • FIV-infected cell lines which have been established from FeT-lM are FL-4 (A.T.C.C. Accession No. CRL 10772 ) and FL-6 (A.T.C.C. Accession No. CRL 10773 ) , both deposited at the American Type Culture Collection on June _7_, 1991. Both these cell lines have been found to be prolific producers of FIV.
  • Polypeptides of the present invention will be either haptenic or antigenic, including at least six amino acids, usually at least nine amino acids, and more usually twelve or more amino acids found contiguously within one of the natural FIV proteins.
  • Polypeptides will generally correspond to at least one epitopic site which is characteristic of FIV. By characteristic, it is meant that the epitopic site will allow immunologic detection of the virus in a physiological sample with reasonable assurance. Usually, it will be desirable that the epitopic site be immunologically distinct from (i.e., not cross-reactive with antibodies which recognize) viruses other than FIV. In some cases, however, it may be desirable that the epitopic site be immunologically similar to other viruses.
  • the FIV polypeptides may be natural, i.e., including the entire FIV protein or fragments thereof isolated from a natural source, or may be synthetic.
  • the natural polypeptides may be isolated from the whole virus which is obtained as describe above by conventional techniques, such as affinity chromatography.
  • polyclonal or monoclonal antibodies obtained according to the present invention may be used to prepare a suitable affinity column by well-known techniques. Such techniques are taught, for example, in Hudson and Hay, Practical Immunology, Blackwell Scientific Publications, Oxford, United Kingdom, 1980, Chapter 8.
  • Synthetic polypeptides which are immunologi ⁇ cally cross-reactive with a natural FIV protein may be produced by either of two general approaches.
  • polypeptides having fewer than about 100 amino acids, more usually fewer than about 80 amino acids, and typi ⁇ cally fewer than about 50 amino acids may be synthesized by the well-known Merrifield solid-phase synthesis method where amino acids are sequentially added to a growing chain (Merrifield (1963) J. Am. Chem. Soc, 85:2149- 2156) .
  • the second and preferred method for synthesizing the polypeptides of the present invention involves the expression in cultured cells of recombinant DNA molecules encoding a desired portion of the FIV genome.
  • the portion of the FIV genome may itself be natural or synthetic, with natural genes obtainable from the isolated virus by conventional techniques.
  • the genome of FIV is RNA, and it will be necessary to transcribe the natural RNA into DNA by conventional techniques employing reverse transcriptase.
  • polynucleotides may be synthesized by well-known techniques. For example, short single- stranded DNA fragments may be prepared by the phosphoramidite method described by Beaucage and Carruthers (1981), Tett. Letters 22:1859-1862. Double- stranded fragments may then be obtained either by synthe ⁇ sizing the complementary strand and then annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • the natural or synthetic DNA fragments coding for the desired FIV protein or fragment may be incorporated in a DNA construct capable of introduction to and expression in in vitro cell culture.
  • the DNA constructs will be suitable for replication in a uni ⁇ cellular host, such as yeast or bacteria. They may also be intended for introduction and integration within the genome of cultured mammalian or other eukaryotic cells.
  • DNA constructs prepared for introduction into bacteria or yeast will include a replication system recognized by the host, the FIV DNA fragment encoding the desired polypeptide product, transcriptional and translational initiation regulatory sequences joined to the 5*-end of the FIV DNA fragment, and transcriptional and trans- lational termination regulatory sequences joined to the 3•-end of the fragment.
  • the transcriptional regulatory sequences will include a heterologous promoter which is recognized by the host.
  • a variety of suitable expression vectors are commercially available for a number of hosts.
  • the polypeptides are obtained in a substantially pure form, that is, typically from about 50% W/W or more purity, substantially free of interfering proteins and contaminants.
  • the FIV poly ⁇ peptides are isolated or synthesized in a purity of at least 80% W/W, and more preferably, in at least about 95% W/W purity.
  • homogeneous polypeptide compositions of at least about 99% W/W purity can be obtained.
  • the proteins may be purified by use of the antibodies described hereinafter using the immunoabsorbant affinity columns described hereinabove.
  • polyclonal antibodies specific for FIV may be produced by in vitro or in vivo techniques.
  • In vitro techniques involved in vitro exposure lymphocytes to the antigenic polypeptides while in vivo techniques require the injection of the polypeptides into a wide variety of vertebrates. Suitable vertebrates are non-human, including mice, rats, rabbits, sheep, goats, and the like.
  • Polypeptides having more than about thirty amino acids, usually more than about fifty amino acids, may serve directly as the immunogen.
  • the polypeptide is smaller than about lOkD, particularly less than about 6kD, however, it may be necessary to join the polypeptide to a larger molecule to elicit the desired immune response.
  • the immunogens are then injected into the animal according to a predetermined schedule, and the animals are bled periodically with successive bleeds having improved titer and specificity. Injections may be made intramuscularly, subcutaneously, or the like, and an adjuvant, such as a combination of complete and incomplete Freund's adjuvant, will usually be employed.
  • an adjuvant such as a combination of complete and incomplete Freund's adjuvant
  • the whole virus can also be used as the immunogen, although selection of antibodies specific for a particular determinant will be more difficult.
  • monoclonal antibodies can be obtained by preparing immortalized cell lines capable of producing antibodies having the desired specificity.
  • immortalized cell lines may be produced in a variety of ways. Conveniently, a small vertebrate, such as a mouse, is hyperimmunized with the desired antigen by the method just described. The vertebrate is then killed, usually several days after the final immunization, the spleen removed, and the spleen cells immortalized. The manner of immortalization is not critical. Presently, the most common technique is fusion with a myeloma cell fusion partner, as first described by Kohler and Milstein (1976) Eur. J. Immunol. 6:511-519. Other techniques include EBV transformation, transformation with oncogenes, retroviruses, etc. , or any other method which provides for stable maintenance of the cell line and production of monoclonal antibodies.
  • the manner of fusion is not critical and various tech ⁇ niques may be employed.
  • the spleen cells and myeloma cells are combined in the presence of a non- ionic detergent, usually polyethylene glycol, and other additives such as Dulbecco's Modified Eagle's medium, for a few minutes.
  • a non- ionic detergent usually polyethylene glycol
  • other additives such as Dulbecco's Modified Eagle's medium
  • the fused cells are promptly dispensed in small culture wells (usually in a microtiter plate at relatively low density, ranging from about one to 5x10 s cells/well) , in a selective medium chosen to support growth of the hybrid cells while being lethal to the myeloma cells.
  • a selective medium chosen to support growth of the hybrid cells while being lethal to the myeloma cells.
  • the myeloma cell line has been mutated to be sensitive to a lethal agent, typically being HAT sensitive, and the medium includes a HAT concentration sufficient to inhibit the proliferation of the unfused myeloma cells.
  • the cell line can be maintained as a viable culture and/or a quantity of the virus may be grown out, separated, and stored by lyophilization.
  • monoclonal antibodies may be isolated from supernatants of the growing colonies.
  • the yield of antibodies obtained however, is usually low.
  • the yield may be enhanced by various techniques, such as injection of the hybrido a cell line into the peritoneal cavity of a vertebrate host.
  • Monoclonal antibodies may then be harvested from the ascites fluid or the blood.
  • polypeptides and antibodies of the present invention may be used with or without modification for the detection of or vaccination against FIV infection. Frequently, the polypeptides and antibodies will be labelled by joining, either covalently or non-covalently, a substance which provides for detectable signal.
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Some of the labels include radio- nuclides, enzymes, substrates, cofactors, inhibitors, fluorescers, chemiluminescers, magnetic particles and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
  • Antibodies and polypeptides prepared as described above can be used in various immunological techniques for detecting FIV and anti-FIV antibodies in physiological specimens, particularly body fluid samples, including blood, plasma, serum, urine, and the like, and cell samples, such as lymphocytes. Depending on the nature of the sample, both immunoassays and immuno- histochemical staining techniques may find use. Liquid phase immunoassays and Western blot analysis will find use in detection of FIV in body fluids, particularly blood and urine. The use of anti ⁇ bodies in protein binding assays is well established. Numerous competitive and noncompetitive protein binding assays have been described in the scientific and patent literature, and a large number of such assays are commercially available. Detailed methods for detecting the presence of the viruses in serum samples are set forth in the Experimental section hereinafter. Additionally, enzyme linked immunosorbent assays (ELISA) for detecting presence of antibodies to FIV in blood are also set forth in the Experimental section.
  • ELISA enzyme linked immunosorbent assays
  • compositions of the present invention are also useful in preparing vaccines for protection against FIV infection.
  • the whole virus and/or FIV- infected cell lines may be wholly or partially inactivated and utilized as an immunogen in a vaccine composition.
  • Partial inactivation may be achieved by passage at elevated temperatures or by contact with mutagens, such as ultraviolet light, ethyl methanesulfonate, and the like.
  • Complete inactivation may be achieved by contact with other agents, including formalin, paraformaldehyde, phenol, ⁇ -lactopropionate, ultraviolet light, heat, psorlens, platinum complexes, ozone and other viricidal agents.
  • the source of whole FIV can be the FIV-infected cell lines which have been found to be prolific producers, such as FL-4 and FL-6.
  • Inactivated FL-4 and FL-6 are also suitable for preparing inactivated or attenuated whole cell vaccines.
  • the viral proteins and portions thereof, prepared as described above, may also be used in the preparation of subunit vaccines prepared by known tech- niques. Polypeptides displaying antigenic regions capable of eliciting protective immune response are selected and incorporated in an appropriate carrier. Alternatively, an antigenic portion of a viral protein or proteins may be incorporated into a larger protein by expression of fused proteins.
  • the preparation of subunit vaccines for other viruses is described in various references, including Lerner et al. (1981) Proc. Natl. Acad. Sci. USA 78:3403 and Bhatanagar et al. (1982) Proc. Natl. Acad. Sci. USA 79:4400. See also, U.S. Patent Nos.
  • 4,565,697 where a naturally-derived viral protein is incorporated into a vaccine composition
  • 4,528,217 and 4,575,495 where synthetic peptides forming a portion of a viral protein are incorporated into a vaccine composition
  • Other methods for forming vaccines employing only a portion of the viral proteins are described in U.S. Patent Nos. 4,552,757; 4,552,758; and 4,593,002. The relevant portions of each of these cited references and patents are incorporated herein by reference.
  • the vaccines prepared as described above may be administered in any conventional manner, including oranasally, subcutaneously, intraperitoneally or intramuscularly, except that oronasal administration will usually not be employed with a partially inactivated virus vaccine.
  • Adjuvants will also find use with subcutaneous and intramuscular injection of completely inactivated vaccines to enhance the immune response.
  • the preparation of viral vaccine compositions optionally employing adjuvants is described in numerous standard references, such as Remington ' s Pharmaceutical Sciences , Mack Publishing Co., Easton, PA, 16th ed. , 1982, the disclosure of which is incorporated herein by reference.
  • the dosage form and immunogen content of the vaccine will vary depending on the nature of the immunogen (i.e., whole virus, infected cell, or subunit) and the route of administration.
  • a single dose will have a total volume including carrier, adjuvant, and any other components, in the range from about 0.1 ml to about 5 ml, more usually being from about 0.5 ml, more usually being from about 0.5 ml to about 3 ml.
  • the amount of inactivated or attenuated whole FIV in each dose will usually be in the range from about 0.1 mg to about 5 mg, usually being from about 0.2 mg to 2 mg.
  • each dose will typically contain from about 10 6 to 10 8 cells, usually about 5xl0 6 to 5xl0 7 cells.
  • the number and temporal spacings of the inoculations will be sufficient to elicit the desired immunoprotective response against subsequent challenge by FIV.
  • a final inoculation may be administered at some longer interval following an initial series of administrations. The selection of optimum administration patterns for a particular vaccine formulation is well within the skill in the art.
  • Diagnostic tests for detecting the presence of FIV in biological samples may also be performed using polynucleotide probes.
  • polynucleotide probes may be prepared based on the sequence of the viral genome.
  • the length of the probe is not critical, but will usually comprise at least about 12 bases, more usually comprising at least about 16 bases, which are substantially complementary to a portion of the viral genome.
  • the probe itself may be DNA or RNA, and the probe need not have perfect complementarity with the FIV genome, with one or two mismatched pairs being acceptable for probes up to 20 bases in length and three to five mismatched pairs in probes from 20 to 35 bases.
  • the probes may be prepared synthetically, with suitable synthetic tech ⁇ niques having been described above, and will include a detectable label.
  • the synthetic sequences are expanded in commonly available cloning vectors and suitable hosts in order to obtain large quantities.
  • the expanded vectors may themselves be labelled for use as probes, or shorter fragments containing complementary strands may be excised and labelled.
  • Methods for the preparation and utilization of nucleotide probes for diagnostic testing are described in U.S. Patent No. 4,358,535 to Falkow et al. , the disclosure of which is incorporated herein by reference.
  • labels have been employed, including those which have been described above for use in immunoassays, particularly radionuclides. Suitable labels may be bound to the probe by a variety of techniques. Commonly employed is nick translation with ⁇ - 32 P-dNTP terminal phosphate hydrolysis with alkaline phosphatase followed by 5'-end labelling with radioactive 32 P employing ⁇ - 32 P-NTP and T4 polynucleotide kinase or 3•-end labelling with an ⁇ - 32 P-dNPT and terminal deoxynucleotidyl transferase.
  • nucleotides can be synthesized where one or more of the atoms present are replaced with a radioactive isotope, e.g., hydrogen with tritium.
  • a radioactive isotope e.g., hydrogen with tritium.
  • various linking groups can be employed.
  • the terminal hydroxol can be esterified with inorganic acids, e.g., 32 P phosphate or 14 C organic acids, or else esterified with bifunctional reagents to provide other reactive groups to which labels can be linked.
  • FIV- CRFK Crandell feline kidney cell line
  • FeTl feline mixed fresh PBLs
  • Both cell types were infected with the Petaluma strain of FIV (A.T.C.C. No. VR 2186; deposited on August 5, 1987, in connection with parent application serial number 07/089,700).
  • the FIV- CRFK line grows as a monolayer, morphologically similar to uninfected CRFK cells (Yamamoto et al. (1988) Am. J. Vet. Res. 49:1246-1258 and Fabricant et al. (1971) J. Am. Vet. Med. Assoc. 158:976-980).
  • FIV-FeTl cells like uninfected FeTl cells (mixed peripheral blood lymphocyte (PBL) cells from specific pathogen free (SPF) cats) , grow in suspension and require interleukin-2 (IL-2) .
  • the suspension cells were passaged at a cell concentration of 0.5-4xl0 6 cells/ml and recultured in fresh culture media twice a week.
  • FIV-CRFK cells were cultured in media consisting of equal volumes of L-15 and Eagle's minimum essential media, 10% heat- inactivated FCS, and 50 ⁇ g/ml gentamicin. All monolayer cells were passaged twice a week at an initial cell concentration of 2xl0 6 cells/ml.
  • the FIV-infected tissue culture fluids (TCF) were harvested twice a week, spun at 3000 rpm for 1 hr to remove residual cells, and stored at -20°C or -70°C or at 5°C for those scheduled to be used within 1-5 days.
  • RT reverse transcriptase
  • IL-2-independent FIV producing cell lines were developed from an IL-2-dependent FIV-infected feline PBL line (FIV-FeTl) .
  • the process of gradual IL-2 depletion from the FIV-FeTl cell line took extensive sub-culturing over a period of approximately three months.
  • the depletion process entailed a gradual reduction of the percentage of IL-2 containing media from the culture in the following weekly sequence: 75%, 50%, 25%, 5% and 0% IL-2-containing media. During this period over 80% of the starting cultures which were depleted of IL-2 did not survive the procedure.
  • Surviving cultures were placed in individual 2-cm 2 multiwells at a viable cell concentration of 2xl0 6 cells/ml/well.
  • the clarified infectious TCF from FL-4, FL-6, FIV-FeTl, and FIV-CRFK cells was filtered individually with 0.45 ⁇ m sterile filters to remove residual cells.
  • FIV inocula were aliquoted into 8-ml samples, stored at -70 ⁇ C and samples of these frozen inocula were retested for RT activity prior to in vitro infectivity studies. In all studies, the frozen inocula were thawed at room temperature immediately prior to use.
  • FIV- susceptible feline cells (lxlO 6 cells/ml) were infected with FIV at RT activity of 30,000 cpm/ml.
  • IP intraperitoneally
  • Virus isolation consisted of co- cultivating 2-lOxlO 5 cells/ml with equal number of FIV- susceptible uninfected FeTl cells and monitoring the TCF from these cultures for six weeks by RT assay.
  • the PBL were considered positive for FIV isolation when RT activity of >10,000 cpm/ml were detected in TCF from at least two consecutive harvest days.
  • the RT activity of the TCF from co-culturing PBL from SPF cats with FeTl cells was ⁇ 2,500 cpm/ml.
  • FIV from infected TCF was concentrated and purified by ultracentrifugation, first on a 10/50% (w,v) discontinuous sucrose gradient and then on a 10/50% continuous sucrose gradient (Pedersen et al. (1987) Science 235:790-793 and Yamamoto et al. (1988) Leukemia, December Supplement 2:204S-215S) .
  • the virus purified by this procedure was used for comparing the biochemical properties of FIV derived from different culture preparations and as the viral substrate for the immunoblot assay.
  • the strips were incubated individually in wells with biotinylated anti-cat IgG (Vector Laboratories, Burlingame, CA) for 30 min and washed three times with wash solution. The strips were then incubated individually with horseradish peroxidase Avidin D (Vector Laboratories) for 30 min.
  • the FIV core protein p28 was detected by an enzyme-linked immunoadsorbent assay (ELISA) using two different monoclonal antibodies. Al and BI Abs, to FIV p28 as either capture or substrate-reactive antibodies, respectively. Reactivity of both mAbs to FIV p28 antigen was confirmed by immunoblot analysis.
  • the capture antibody (mAb Al) was coated on the plate overnight with bicarbonate buffer (pH 9.6) and washed once before its use. Serum samples to be tested were diluted in Buffer 3 and then incubated in the coated wells for 30 min at 37°C.
  • the phenotypic profiles of the feline cells were determined by fluorescence activated cell sorter
  • the second procedure involved passaging test cells onto indicator cells which were then DNA/fluorochrome stained for mycoplasma. Detection of FeLV p27 core antigen was performed using the p27 antigen ELISA assay (Lutz et al. (1983) J. Immunol. Methods 56:209-220). Polymerase chain reaction (PCR) was used to test for the presence of FeLV provirus DNA. Briefly, a pair of primer sequences from the U3 region of the FeLV LTR were chosen so as to avoid the possibility of overlap with endogenous sequences of FeLV. The sequences of the two oligonucleotides primers used for PCR were 14 base pairs (bp 24 to 37) and 17 base pairs (bp 239 to 255) long.
  • the pelleted virus and the infected cells were each inactivated with 1.25% paraformaldehyde, dialyzed against PBS, and then combined with adjuvant just prior to immunization.
  • the adjuvants used were either threonyl muramyl dipeptide (MDP) (Byars et al. (1987) Vaccine 5:223-228) or a combination of Freund's complete and incomplete adjuvants.
  • MDP threonyl muramyl dipeptide
  • Control cats were immunized with either uninfected FeTl cells with adjuvant or diluent with adjuvant. All cats were immunized at two week intervals for a total of four or eight immunizations, unless stated otherwise.
  • IL-2-Independent FIV-Producin ⁇ Cell Lines The development of IL-2-independent cell lines from FIV infected mixed PBLs (FIV-FeTl cells) entailed the gradual depletion of IL-2 from the cultures. Only two out of 20 cultures, FL-4 and FL-6, survived the depletion process. Significant RT titers (100,000-400,000 cpm/ml) , Mg ++ cation-dependent, were detected in these cultures during the expansion and large scale-production stage. Electron microscopy demonstrated numerous typical lentivirus particles in these cultures (data not shown) .
  • the growth rates of these cell lines were compared to those of FIV-FeTl and FIV-CRFK.
  • the viable cell doubling time for FL-6 was found to be approximately 24 hours, whereas the doubling time for FL-6 was found to be approximately 24 hrs, whereas the doubling time for FL-4 was approximately 48 hrs. Both cell lines grew at an exponential rate. From a starting cell concentration of 5xl0 5 cells/ml, peak viable cell counts wee observed after 3-4 days of culturing. Viability of the cells present in these cultures ranged from 70 to 90% over the four day culturing period. The number of syncytial cells in the FL-4 and FL-6 cultures was less than 0.1%.
  • the phenotypic profiles of FL-4 and FL-6 cells were determined by flow cytometric analysis using monoclonal antibodies (mAb) to feline CD4 (fel 7) , CD8 (FT2), pan T-cell (42) markers (Ackley et al. (1990) J. Virol. 64:5652-5655; Carlson et al. (1985) supra. ; Ackley et al. (1990) supra . ; and Klotz et al. (1986) supra . ) and mAb that detect both feline immunoglobulin light chains and ⁇ heavy chain (AC5) (Klotz et al. (1985) supra . ) (Fig. 2) .
  • mAb monoclonal antibodies
  • feline CD4 feline CD4
  • CD8 FT2
  • pan T-cell (42) markers Ackley et al. (1990) J. Virol. 64:5652-5655; Carlson et al. (1985) supra. ; Ackley et
  • FL-4 cells were CD4 ⁇ , CD8 + , and Pan-T + whereas FL-6 cells were CD4", CDS*, and Pan-T + . Both cell lines were negative for surface IgM and ⁇ and K light chains. It should be noted that both CD4 and CD8 antigens were lost in cultures maintained for several months. FL-4 and FL-6 cells were >95% positive by IFA for surface FIV antigen expression using polyclonal antibodies to FIV (Table 1) . Additional tests were performed to ensure that these cells were free of known contaminants which could limit their use. The results are summarized in Table 1. The two cell lines were mycoplasma-free both by direct DNA/fluorochrome stain and indirectly by passaging onto indicator cells prior to staining.
  • FL-4 and FL-6 cells were shown to be negative for FeLV core protein p27 expression by ELISA and for exogenous FeLV DNA by PCR.
  • the cells were determined to be negative by IFA for feline syncytial-forming virus (FeSFV) .
  • DNA/fluorochrome staining for mycoplasma Indirectly by staining indicator cells which were passaged with FL-4 and FL-6 cells.
  • Purified virus was disrupted with 0.1% SDS prior to its use in immunoblot production, as described in Methods.
  • the amount of FIV produced from the FL-4, FL-6, FIV-FeTl and FIV-CRFK cell lines was determined by comparing the total protein and RT levels of FIV in different fractions from sucrose gradient preparations (data not shown) . High titers of both RT activity and total protein were observed in FIV preparations from FL-4, FL-6, and FIV-FeTl cells.
  • the FIV-CRFK produced low titers of FIV as demonstrated by the low levels of both protein concentration and RT activity in the fractions.
  • the three peak fractions of the gradient purified virus from each cell line were pooled and measured for total protein concentration, RT titer, and viral core protein (p28) concentration.
  • the FIV produced from FL-4 and FL-6 cells was tested for its ability to infect FIV-susceptible cell lines (Fig. 3) .
  • Cell-free TCF from different infected cell lines was inoculated into various feline cell cultures at a set RT concentration of 30,000 cpm/ml.
  • FIV from FIV-CRFK cells did not readily infect certain feline lymphoid cells, in particular thymus-derived cultures, as compared to the FIV from FL-4 and FIV-FeTl cells.
  • the FIV from FL-6 cells was also highly infectious to FIV- susceptible cell lines (data not shown) .
  • the FIV preparations produced from FL-4 and FL-6 cells were tested for their ability to infect SPF cats (Fig. 4).
  • the fixed cell-virus vaccine consisted of FIV- FeTl and FIV-FL-4 inactivated with paraformaldehyde. In each culture (which was subsequently inactivated) essentially 100% of the cells were productively infected with FIV and 5x10 7 cells were required to obtain 100 ⁇ of total viral protein. Analysis of the FIV-infected cells in both T-cell lines by immunoblot using serum from an FIV immunized cat and by Coomassie stain, showed that the vaccine preparations contained the env, gag and pol virion proteins and their precursors as well as some regulatory proteins and cellular proteins (data not shown) . The adjuvant used was threonylmuramyl dipeptide (MDP) (Syntex SAF-A) .
  • MDP threonylmuramyl dipeptide
  • the infected cells were inactivated with 1.25% paraformaldehyde for 24 hrs and washed three times with phosphate buffered saline (PBS) .
  • the vaccine consisted of IxlO 7 inactivated FIV-FeTl cells (Group IA) or FIV-FL-4 cells (Group IB) mixed with 250 ⁇ g of MDP. All cats in Group 1 were specific pathogen free (SPF) cats of 4-6 months of age, which were previously exposed to feline herpes virus (FHV C-27 strain) and were free of FHV symptoms two weeks prior to and during immunization. Ten control cats were immunized with either uninfected FeTl cells with MDP (Group 1C) or MDP alone (Group ID) .
  • Virus was isolated from PBL and bone marrow cells by co-culturing with FIV-susceptible FeTl cells. PCR analysis was performed using the method previously described (Pedersen et al. (1989) J. Virol. 64:598-606).
  • Number indicates positive result from a specific cat with corresponding identification number. ALL indicates that all cats in the specific group are positive. ND indicates not done.
  • virus was recovered from PBLs only one occasion, at 5 weeks pc. after which it was no longer detectable in either the PBLs (by virus isolation and PCR) or the bone marrow cells (by virus isolation) . Antibody levels decreased steadily in this animal.
  • this animal may also be protected.
  • Virus was recovered persistently after 5 weeks pc from the PBLs of one vaccinated cat (#178) and after 21 weeks pc from another vaccinated cat (#138) .
  • the PBLs of both animals were PCR positive at 21 weeks pc at which time infectious virus was isolated from their bone marrow.
  • These two persistently infected cats showed a sudden rise in antibodies by ELISA at the time virus was recovered, and the antibodies remained high thereafter (Fig. 6A) .
  • both core and envelope antibodies persisted longer in these cats than in the protected cats.
  • the seven vaccine protected cats showed FIV specific cell mediated response (CMR) as measured by positive lymphocyte proliferation and IL-2 induction assays (Figs. 9A and 9B) as well as a positive response to non-specific mitogens (data not shown) .
  • CMR FIV specific cell mediated response
  • the two persistently infected vaccinates and all infected control cats showed a lack of cellular response to FIV while the non-specific mitogen response remained intact. Since these cats were not tested for CMR before challenge we do not know if they were genetically poor responders and therefore vulnerable to infection or whether these defects in CMR were the result of infection.
  • the cell-free whole virus vaccine was prepared from FIV-FL-4. Virus released from this cell line in high titer (5xl0 8 cells produced 1 mg viral protein per litter) was pelleted, filtered (0.45 ⁇ m) , inactivated with paraformaldehyde, and given with a combination of Freund's complete and incomplete adjuvants. Analysis of the cell-free pelleted FIV preparation from the whole- virus vaccine by immunoblot using serum from an FIV immunized cat showed that this vaccine contained all of the viral antigens, although a lesser amount of env glycoproteins than was present in the fixed infected cell vaccine, and also a trace amount of cellular antigens (data not shown) .
  • PBL cultures became virus and PCR positive by seven weeks pc from the three controls (Table 2), whereas five of six vaccinated cats remained uninfected up to 14 weeks.
  • the PBLs of one vaccinated cat (#551) were transiently infected at 7 weeks pc but were negative by virus isolation and PCR at 17 weeks pc. After challenge, gradual decreases in antibody titers were observed in all immunized and protected cats including the single transiently infected cat.
  • the cell-virus vaccine may also have elicited an allogenic effect from the inclusion of other cellular antigens.
  • a mixture of uninfected allogeneic (FeTl) cells and inactivated whole virus did not enhance the ELISA and neutralizing antibodies to FIV as compared to whole virus alone. This indicates that the expression of viral antigens on the infected cell apparently provides the most effective immunogenicity.
  • Viral envelope appears an essential determinant because, in another trial, cats immunized with an FIV Iscom vaccine that was deficient in envelope antigen failed to make gpl20 antibody and were not protected against challenge infection with 20 ID 50 of homologous virus.
  • the vaccines of the present invention probably achieved a minimal threshold of protection because, using a similar fixed cell-virus vaccine we were previously unable to protect against a higher challenge dose (5xl0 3 ID) of virus (data not shown) .
  • neutralizing antibody would seem a logical mechanism, other means of vaccine protection, such as antibody dependent complement lysis or cellular cytotoxicity (ADCC) against cell-free virus or infected cells, may also contribute.
  • ADCC antibody dependent complement lysis or cellular cytotoxicity

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Abstract

Des compositions dérivées d'un nouvel isolat viral désigné comme virus de l'immunodéficience féline (VIF) contiennent le virus entier, des protéines, des polypeptides et des séquences polynucléotidiques dérivées du virus; ainsi que des anticorps dirigés contre les sites antigéniques du virus. Ces compositions peuvent être utilisées dans différentes techniques de détection de VIF et de vaccination contre celui-ci. Les procédés de dépistage décrits comprennent des dosages immunologiques destinés à la fois au virus et aux anticorps dirigés à la fois au virus et aux anticorps dirigés contre le virus, et ainsi que l'utilisation de sondes polynucléotidiques permettant de détecter le génome viral. Des vaccins comprennent à la fois des virus entièrement ou partiellement inactivés, des lignées cellulaires inactivées exprimant les antigènes de VIF, et des vaccins à sous-unités. Le virus entier et vivant peut aussi être utilisé comme un système de modèle pour prédire le comportement du virus de l'immunodéficience humaine (VIH).
PCT/US1992/005571 1991-07-05 1992-07-01 Lignees cellulaires lymphoïdes felines susceptibles de produire le virus de l'immunodeficience feline (vif) WO1993001278A1 (fr)

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WO1996030045A1 (fr) * 1995-03-31 1996-10-03 Istituto Superiore Di Sanita' Vaccin preventif contre le virus de l'immunodeficience feline chez le chat domestique
WO1996040953A1 (fr) * 1995-06-07 1996-12-19 American Home Products Corporation Virus de l'immunodeficience feline modifies par genie genetique et leur utilisation en tant que vaccin efficace contre l'infection due au virus de l'immunodeficience feline
WO1997007817A1 (fr) * 1995-08-25 1997-03-06 University Of Florida Vaccins contre le virus de l'immunodeficience feline (fiv) a sous-classes multiples
US5820869A (en) * 1995-06-07 1998-10-13 American Home Products Corporation Recombinant raccoon pox viruses and their use as an effective vaccine against feline immunodeficiency virus infection
US6254872B1 (en) 1995-08-25 2001-07-03 University Of Florida Multi-subtype FIV vaccines
US7658927B2 (en) 2003-05-12 2010-02-09 University Of Florida Research Foundation, Inc. Materials and methods for immunizing against FIV infection
US8703145B2 (en) 2003-05-12 2014-04-22 University Of Florida Research Foundation, Inc. Materials and methods for immunizing against FIV infection

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Title
AMERICAN JOURNAL OF VETERINARY RESEARCH, Volume 49, No. 8, issued August 1988, J.K. YAMAMOTO et al., "Pathogenesis of Experimentally Induced Feline Immunodeficiency Virus Infection in Cats", pages 1246-1258. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Volume 86, issued August 1989, R.C. DESROSIERS et al., "Vaccine Protection Against Simian Immunodeficiency Virus Infection", pages 6353-6357. *
SCIENCE, Volume 224, issued 22 June 1984, D.A. CANTRELL et al., "The Interleukin-2 T-Cell System: A New Cell Growth Model", pages 1312-1316. *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996030045A1 (fr) * 1995-03-31 1996-10-03 Istituto Superiore Di Sanita' Vaccin preventif contre le virus de l'immunodeficience feline chez le chat domestique
US6300118B1 (en) 1995-06-07 2001-10-09 American Home Products Corporation Plasmids comprising a genetically altered feline immunodeficiency virus genome
WO1996040953A1 (fr) * 1995-06-07 1996-12-19 American Home Products Corporation Virus de l'immunodeficience feline modifies par genie genetique et leur utilisation en tant que vaccin efficace contre l'infection due au virus de l'immunodeficience feline
US5820869A (en) * 1995-06-07 1998-10-13 American Home Products Corporation Recombinant raccoon pox viruses and their use as an effective vaccine against feline immunodeficiency virus infection
US5989562A (en) * 1995-06-07 1999-11-23 American Home Products Corporation Recombinant raccoon pox viruses and their use as an effective vaccine against feline immunodeficiency virus infection
AU728750B2 (en) * 1995-08-25 2001-01-18 Regents Of The University Of California, The Multi-subtype FIV vaccines
EP1090985A1 (fr) * 1995-08-25 2001-04-11 University Of Florida Cellules T derivées d'un félin
WO1997007817A1 (fr) * 1995-08-25 1997-03-06 University Of Florida Vaccins contre le virus de l'immunodeficience feline (fiv) a sous-classes multiples
US6605282B2 (en) 1995-08-25 2003-08-12 University Of Florida Research Foundation, Inc. Multi-subtype FIV vaccines
US6254872B1 (en) 1995-08-25 2001-07-03 University Of Florida Multi-subtype FIV vaccines
US6544528B1 (en) 1995-08-25 2003-04-08 University Of Florida Research Foundation, Inc. Multi-subtype FIV vaccines
US6447993B1 (en) * 1995-08-25 2002-09-10 University Of Florida Research Foundation, Inc., Multi-subtype FIV vaccines
US7267824B2 (en) 1995-08-25 2007-09-11 University Of Florida Research Foundation, Inc. Multi-subtype FIV vaccines
US7311921B2 (en) 1995-08-25 2007-12-25 University Of Florida Research Foundation, Inc. Multi-subtype FIV vaccines
US7658927B2 (en) 2003-05-12 2010-02-09 University Of Florida Research Foundation, Inc. Materials and methods for immunizing against FIV infection
US8703145B2 (en) 2003-05-12 2014-04-22 University Of Florida Research Foundation, Inc. Materials and methods for immunizing against FIV infection

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