EP0662139A1 - Recombinant retroviral vector against felv and/or fiv - Google Patents

Recombinant retroviral vector against felv and/or fiv

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Publication number
EP0662139A1
EP0662139A1 EP93922364A EP93922364A EP0662139A1 EP 0662139 A1 EP0662139 A1 EP 0662139A1 EP 93922364 A EP93922364 A EP 93922364A EP 93922364 A EP93922364 A EP 93922364A EP 0662139 A1 EP0662139 A1 EP 0662139A1
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EP
European Patent Office
Prior art keywords
feline
vector construct
vims
expression
virus
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.)
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Application number
EP93922364A
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German (de)
English (en)
French (fr)
Inventor
William T.L. Lee
John J. Serbin
Douglas J. Jolly
Jack R. Barber
Sunil Chada
Stephen M.W. Chang
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Novartis Vaccines and Diagnostics Inc
Original Assignee
Chiron Viagene Inc
Chiron Corp
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Publication of EP0662139A1 publication Critical patent/EP0662139A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates generally to methods for treating felines, and more specifically, to methods and compositions for treating feline leukemia virus and feline immunodeficiency virus infections, as well as vaccines for preventing these infections.
  • Feline leukemia virus (“FeLV”) and feline immunodeficiency virus (“FIV”) are the two most common pathogenic retroviruses reported in feline populations.
  • FeLV Feline leukemia virus
  • FIV feline immunodeficiency virus
  • FeLV and FIV are acquired independently of each other, it has been reported that FeLV infected cats are 1.5 to 4 times more susceptible to FIV infection than are FeLV-negative cats (see Cohen et al., JAVMA 197:220:225, 1990; Moraillon, Vet Rec.
  • FeLV infection can generally be classified into three groups: acute infection, chronic viremia, and immunity. The outcome for any specific animal depends on a variety of viral, host, and environmental factors. In acute infection, FeLV first replicates in the lymphocytes and macrophages of the tonsils, and within 2 to 12 days is carried throughout the cat to the bone marrow, thymus, spleen, intestine, and lymph nodes. If the cat does not mount an adequate immune response, it will develop chronic viremia within 4 to 6 weeks after initial exposure.
  • V viral infectivity
  • IF A immunofluorescent antibody
  • ELISA enzyme-linked immunosorbent assay
  • FeLV-related diseases include lymphomas, non-lymphoid leukemias, myeloproliferative disorders, fibrosarcomas, cytosuppresive diseases, and myelosuppresion, anemia and leukopenia syndromes (see Hoover, JAVMA 799:1287-97, 1991). If the cat mounts an effective immune response, it will curtail viral replication and expression within 4 to 8 weeks after initial exposure. However, many cats do not completely eliminate the virus, and harbor a low-grade, latent, nonexpressed, FeLV infection of the bone marrow and lymph nodes for weeks to years. If such a cat is severely stressed, the latent virus may subsequently be reactivated, leading to FeLV-related diseases, and death of the cat.
  • a cat diagnosed with FeLV infection will generally die within 24 to 36 months.
  • FeLV-infected cats be isolated from other cats, regardless of whether or not the other cats have been vaccinated for FeLV.
  • a number of vaccines have been developed. More specifically, eight FeLV vaccines have been licensed by the USD A, all of which are based upon an inactivated virus or purified subunit. Most studies suggest that present commercial FeLV vaccines provide only a 60 to 90% efficacy in preventing cats from developing persistent infection (see Pollock et al., JAVMA 799:1406-1409, 1991). All of these vaccines require a second dose and annual boosts to maintain antibody persistance.
  • one difficulty with present vaccines is, that if the diagnostic test which was utilized to check for previous infection was inaccurate, not available, or too expensive to be utilized, vaccination of an infected animal may take place.
  • Conventional vaccines are not expected to be therapeutic, and therefore, infected animals which are subsequently vaccinated may nevertheless develop FeLV. This has led to the perception by some feline owners that vaccines are either ineffective, or may actually even lead to infection, and therefore are unnecessary.
  • AZT AZT-related hepatotoxicosis
  • undesirable side-effects including for example AZT- related hepatotoxicosis, and inability to eliminate viremia after incorporation of the virus into hematopoietic cells.
  • the virus is believed to be transmitted primarily through infected saliva during fighting, (see Wasmien et al., 'Transmission of feline immunodeficiency virus from infected queens to kittens (Abstract), First International Conference, supra), and therefore, cats which are most at risk are stray males over the age of 3 (see Yamamoto et al., JAVMA 194:213-220, 1989).
  • feline immunodeficiency virus in these high- risk animals ranges from 6 - 14% in the United States (see August, JAVMA 799:1472-1477, 1991; Macy et al., "The clinical findings and prevalence of FTV and FeLV in Colorado cats (Abstract), First International Conference, supra; O'Connor et al., JAVMA 799:11348-1359, 1991), 18% in Denmark (see Peterson et al. supra), and 44% in Japan (see Ishida et al, Jpn. J. Vet. Sci. 50:39-44, 1988). In addition, vertical transmission from infected queens to kittens has been observed (see Callahan et al, "Natural transmission of FIV in kittens.
  • feline immunodeficiency virus infection is estimated to be 1 - 3 % (see Peterson et al., supra). Unlike the human immunodeficiency virus (“HIV”), feline immunodeficiency virus infection does not seem to be spread by sexual contact (see Gardner and Luciw, FASEB J. 3:2593- 2606, 1989).
  • Feline immunodeficiency virus induces an immunodeficiency disease in its host by the gradual and persistent depletion of CD4 + lymphocytes with no apparent changes in the levels of CD8 + , CTL, and T suppressor cells (see Ackley et al., /. Virol. 64:5652-5655, 1990). This leads to an inversion of the CD4 + /CD8 + cell ratio which may be measured in order to determine the immunological status of a feline.
  • the course of disease caused by feline immunodeficiency virus is very similar to that caused by HIV.
  • Feline immunodeficiency virus positive cats also present a high incidence of neoplasms including lymphoma, squamous cell carcinoma, and myelodysplastic disease (see Hutson et al., JAVMA 199:1351-1362, 1991).
  • Three types of tests are currently available to determine if cats are infected with feline immunodeficiency virus. They are the enzyme-linked i munosorbent assay (ELISA), the immunofluorescent antibody (IFA) test, and the immunoblot test, the latter of which is considered to be the standard for methodology comparisons.
  • an ELISA or IFA can produce results with a sensitivity of 93-95%, and a specificity of 98% (see Barr et a ⁇ ., JAVMA 799:1377-1381, 1991).
  • the anti-retroviral human AIDS drugs AZT and 9-(2-phosphonomethoxyethyl)-adenine (PMEA) have been utihzed in cats with feline immunodeficiency virus. These drugs improve the clinical condition of infected cats by increasing their CD4 + /CD8 + ratio. However, both cause decreased hematocrit and hemoglobin levels which precludes their use as long term therapies (see Hartmann et al, "Use of two virustatica (AZT, PMEA) in the treatment of FIV- and FeLV-seropositive cats with clinical symptoms," (Abstract), First International Conference, supra).
  • the present invention provides compositions and methods for treating FeLV and FIV, vaccines for preventing FeLV and FIV, and additionally provides other related advantages.
  • the present invention provides methods for preventing or treating feline viral infections.
  • methods of treating or preventing feline leukemia virus infections comprising, administering to a feline a vector construct which directs the expression of at least one immunogenic portion of a feline leukemia virus antigen, such that a cellular immune response is generated.
  • vector constructs are provided which direct the expression of an antigen selected from the group consisting of pl5gag, pl2gag, p21gag, plOgag, pl4pol, p&Opol, p46pol, gp70env, and p!5env.
  • vector constructs are provided which direct the expression of gp85env.
  • methods of treating or preventing feline immunodeficiency virus infections comprising, administering to a feline a vector construct which directs the expression of at least one immunogenic portion of a feline immunodeficiency virus antigen, such that a cellular immune response is generated.
  • vector constructs are provided which direct the expression of an antigen selected from the group consisting of pl5gag, p24gag, plOgag, pl3pol, p62pol, pl5pol and p36po/.
  • vector constructs are provided with direct the expression of gp68em>, gp27env and rev.
  • methods of treating or preventing feline leukemia virus and feline immunodeficiency virus infections comprising, administering to a feline a vector construct which directs the co-expression of at least one immunogenic portion of a feline leukemia virus antigen, and at least one immunogenic portion of a feline immunodeficiency virus antigen, such that a cellular immune response is to said viruses is generated.
  • vector constructs which direct the expression of at least one immunogenic portion of a feline leukemia virus, vector constructs which direct the expression of at least one immunogenic portion of a feline immunodeficiency virus, and vector constructs which direct the co-expression of at least one immunogenic portion of a feline immunodeficiency virus, and at least one immunogenic portion of a feline leukemia virus.
  • the above-described vector constructs are carried by recombinant retroviruses, or by a recombinant virus selected from the group consisting of poliovirus, rhinovirus, pox virus (e.g., the canary pox virus or the vaccinia virus), influenza virus, adenovirus, parvovirus (e.g., the adeno- associated virus B19 or MVN), herpes virus, SV40, HIV, measles, and alpha viruses such as the Sindbis virus. Also provided are target cells infected with the above-described viruses.
  • pox virus e.g., the canary pox virus or the vaccinia virus
  • influenza virus e.g., the canary pox virus or the vaccinia virus
  • adenovirus e.g., the canary pox virus or the vaccinia virus
  • influenza virus e.g., the canary pox virus or the vaccinia virus
  • adenovirus e.g.,
  • compositions comprising the above described recombinant retroviral or recombinant viral constructs, in combination with a pharmaceutically acceptable carrier or diluent.
  • Figure 1 is a schematic illustration of the construction of a multivalent FIV - FeLV, retroviral vector.
  • Immunogenic portion refers to a portion of the respective antigen which is capable, under the appropriate conditions, of causing a cellular (Le., cell-mediated or humoral) immune response.
  • "Portions” may be of variable size, but generally should be at least 9 amino acids long, and may include the entire antigen. Representative assays which may be utilized to determine immunogenicity (e.g., cell-mediated immune response), are described in more detail below, as well as in Example 10A.
  • Cellular immune responses may be mediated through Major Histocompatibility ("MHC") Class I presentation, MHC Class II presentation, or both.
  • MHC Major Histocompatibility
  • Vector construct refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest.
  • the vector construct must include promoter element(s), as well as at least one sequence which, when transcribed, is operably linked to the sequence(s) or gene(s) of interest and acts as a translation initiation sequence.
  • the vector construct may also include a selectable marker such as Neo, SV2 Neo, TK, hygromycin, phleomycin, histidinol, or DHFR, a signal which directs polyadenylation, a translation termination sequence, and one or more restriction sites.
  • the vector construct if the vector construct is placed into a retrovirus* the vector construct must include a packaging signal and long terminal repeats (LTRs) appropriate to the retrovirus used (if these are not already present).
  • LTRs long terminal repeats
  • the present invention is generally directed towards compositions and methods for treating, as well as vaccines for preventing, various feline diseases, including for example feline leukemia virus and feline immunodeficiency virus infections.
  • various feline diseases including for example feline leukemia virus and feline immunodeficiency virus infections.
  • the ability to recognize and defend against foreign pathogens is central to the function of the immune system.
  • This system through immune recognition, is capable of distinguishing "self' from "nonself (foreign), and is essential to ensure that defensive mechanisms are directed towards invading entities rather than against host tissues.
  • the fundamental features of the immune system are highly polymorphic cell surface recognition structures (receptors), and effector mechanisms (antibodies and cytolytic cells), which act to destroy invading pathogens.
  • CTL Cytotoxic T Lymphocyte
  • MHC Cytotoxic T Lymphocyte
  • CTLs are normally induced by the display of processed pathogen-specific peptides in conjunction with MHC molecules along with molecules such as CD3, ICAM-1, ICAM-2, LFA-1, LFA-3, 3-microglobulin, chaperones, and analogs thereof (e.g., Altmann et al., Nature 338:512, 1989).
  • Other genes coding for proteins that enhance the stimulation or recognition of cell mediated responses may also be used in this context.
  • Antigenic peptide presentation in association with MHC Class I molecules leads to CD8 + CTL production.
  • Peptides presented in association with MHC Class II molecules leads to production of antibodies, helper cells and B-cell memory, and may induce CD4 + CTLs.
  • the methods which are described in greater detail below provide an effective means of inducing potent class I-restricted protective and therapeutic CTL responses, as well as humoral responses.
  • methods for treating or preventing feline leukemia virus infections comprising administering to a feline a vector construct which directs the expression of at least one immunogenic portion of a feline leukemia virus antigen, such that a cellular immune response is generated.
  • feline leukemia virus is a retrovirus of the oncornavirus subfamily. FeLV is presently believed to exist in three subgroups - A, B or C - which are differentiated by their envelope antigens gp70 and pl5E. FeLV is also comprised of a number of core antigens, including pl5, pl2, p27, and plO, which are highly conserved for all subgroups of FeLV (see Geering et al., Vir. 56:678-680, 1968; Hardy et al., JAVMA 755:1060-1069, 1971; Hardy et al., Science 766:1019-1021, 1969).
  • vector constructs which direct the expression of at least one portion of a feline leukemia virus antigen selected from the group consisting of pl5gag, pl2gag, p21gag, plOgag, pl4pol, p80pol, p46pol, gplOenv, and pl5env.
  • vector constructs are provided which direct the expression of gp85e ⁇ v. Sequences which encode these antigens may be readily obtained given the disclosure provided herein (see Donahue et al., /. Vir. 62(3):722-731, 1988; Stewart et al., /. Vir.
  • a method for treating or preventing feline immunodeficiency virus infections comprising administering to a feline a vector construct which directs the expression of at least one immunogenic portion of a feline immunodeficiency virus antigen, such that a cellular immune response is generated.
  • feline immunodeficiency virus has been classified as a retrovirus of the lentivirus subfamily, based upon the magnesium requirement for reverse transcriptase (RT) and the morphology of viral particles (see Pederesen et al., Science 235:190-193, 1987).
  • feline immunodeficiency virus is morphologically and antigenically distinct from other feline retroviruses, including feline leukemia virus, type C oncorna virus (RD-114), and feline syncytium- forming virus (FeSFV) (see Yamamoto et al., "Efficacy of experimental FIV vaccines, (Abstract), First International Conference of Feline Immunodeficiency Virus researchers, University of California, Davis, CA, Sep. 4-7, 1991).
  • feline leukemia virus type C oncorna virus
  • FeSFV feline syncytium- forming virus
  • vector constructs are provided which direct the expression of at least one immunogenic portion of an feline immunodeficiency virus antigen selected from the group consisting of pl5gag, p24gag, plOgag, pl3pol, p62pol, p!5pol and p36po/.
  • vector constructs are provided which direct the expression of ⁇ p68env, gp21env and rev.
  • rev is understood to refer to the antigen corresponding to the rev open reading frame (see, Phillips et al., First International Conference, supra).
  • Sequences which encode the above-described feline leukemia virus and feline immunodeficiency virus antigens may be prepared as described within the references cited above, or obtained from a variety of sources.
  • sequences which encode the envelope protein of FeLV may be readily obtained from the American Type Culture Collection ("ATCC”; Rockville, Maryland) (see for example, ATCC Nos. 39528, 39529, and 39530).
  • ATCC American Type Culture Collection
  • AIDS Repository Disvision of AIDS, National Institute of Allergy and Infections Disease, Bethesda, Maryland; see NIH Publication No.
  • N2-1536 holds a deposit of a plasmid clone which contains a sequence encoding a full-length, replication competent FeLV (e.g., clone p61E-FeLV, Catalog No. 109), as well as a deposit of a plasmid clone which contains a sequence encoding a feline immunodeficiency virus (e.g., clone pFIV-14-Petaluma, Catalog No. 851).
  • a plasmid clone which contains a sequence encoding a full-length, replication competent FeLV e.g., clone p61E-FeLV, Catalog No. 109
  • a plasmid clone which contains a sequence encoding a feline immunodeficiency virus e.g., clone pFIV-14-Petaluma, Catalog No. 851).
  • sequences which encode the above-described feline viral antigens may be readily obtained from cells which express or contain sequences which encode these viruses (e.g., from cats which have been infected with FeLV or FIV).
  • primers are prepared on either side of the desired sequence, which is subsequently amplified by polymerase chain reaction ("PCR") (see U.S. Patent Nos. 4,683,202, 4,683,195 and 4,800,159) (see also, PCR Technology: Principles and Applications for DNA Amplification, Erlich (ed.), Stockton Press, 1989).
  • a double stranded DNA is denatured by heating in the presence of heat stable Taq polymerase, sequence specific DNA primers, ATP, CTP, GTP and TTP. Double-stranded DNA is produced when synthesis is complete. This cycle may be repeated many times, resulting in a factorial amplification of the desired DNA.
  • Sequences which encode the above-described feline viral antigens may also be synthesized, for example, on an Applied Biosystems Inc. DNA synthesizer (e.g., ABI DNA synthesizer model 392 (Foster City, California)). Such sequences may also be linked together through complementary ends, followed by PCR amplification, in order to form long double-stranded DNA molecules.
  • ABI DNA synthesizer model 392 Fraster City, California
  • At least one immunogenic portion of a feline viral antigen is incorporated into a vector construct.
  • the immunogenic portion(s) which are incorporated into the vector construct may be of varying length, although it is generally preferred that the portions be at least 9 amino acids * long, and preferably, include the entire antigen.
  • Immunogenicity of a particular sequence is often difficult to predict, although T cell epitopes may be predicted utilizing computer algorithms such as TSites (Medlmmune, Maryland), in order to scan coding regions of FeLV gag, env, FIV gag, env and rev for potential T-helper sites and CTL sites.
  • This analysis is primarily based upon 1) structural properties of the proteins (principally alpha-helical periodicity and amphipathicity), and 2) motifs found in sequences recognized by MHC Class I and Class II molecules. In general however, it is preferable to determine immunogenicity in an assay.
  • Representative assays include an ELISA which detects the presence of antibodies against a newly introduced vector, as well as assays which test for T helper cells, such as gamma-interferon assays, IL-2 production assays, and proliferation assays.
  • assays which test for T helper cells, such as gamma-interferon assays, IL-2 production assays, and proliferation assays.
  • a particularly preferred assay is described in more detail below in Example 10A.
  • Immunogenic proteins of the present invention may also be manipulated by a variety of methods known in the art, in order to render them more immunogenic.
  • Representative examples of such methods include: adding amino acid sequences that correspond to T helper epitopes; promoting cellular uptake by adding hydrophobic residues; by forming paniculate structures; or any combination of these (see generally, Hart, op. cit., Milich et al., Proc. Natl Acad. Set USA 55:1610-1614, 1988; Willis, Nature 340:323-324, 1989; Griffiths et al., /. Virol 65:450-456, 1991).
  • Particularly preferred immunogenic portions for incorporation into a vector construct include, for feline leukemia virus the gp85env antigen, and for feline immunodeficiency virus the gp68env, gp27env, and rev antigens.
  • a vector construct which expresses both FeLV and FIV envelope antigens (gp85e «v; and gp68tvzv, gp27 ⁇ nv, and rev, respectively) or both FeLV and FIV gag antigens (pl5g ⁇ g, p!2gag, p21gag, plOgag, and pUpol; and pl5g ⁇ j , p24gag, plOgag, and rev, respectively).
  • a vector construct may express (either separately or as one construct) all or portions of a feline leukemia virus antigen, a feline immunodeficiency virus antigen, as well as antigens which are associated with other feline diseases.
  • antigens include VP1 and VP2 for feline panleukopenia virus (see Martyn et al., "Nucleotide sequence of feline panleukopenia virus: comparison with canine parvovirus identifies host-specific differences", /. Gen. Vir.
  • an immunomodulatory cofactor refers to factors which, when manufactured by one or more of the cells involved in an immune response, or, which when added exogenously to the cells, causes the immune response to be different in quality or potency from that which would have occurred in the absence of the cofactor.
  • the quality or potency of a response may be measured by a variety of assays known to one of skill in the art, including for example, in vitro assays which measure cellular proliferation (e.g., 3 H thymidine uptake), and in vitro cytotoxic assays (e.g., which measure 51 Cr release) (see Warner et al., AIDS Res. and Human Retroviruses 7:645-655, 1991).
  • Immunomodulatory cofactors may be active both in vivo and ex vivo. Representative examples of such cofactors include alpha interferons (Finter et al., Drugs 42(5):149-165, 1991; U.S. Patent No. 4,892,743; U.S. Patent No.
  • IL-6 Interleukin-6
  • ICAM-1 Altaian et al., Nature 555:512-514, 1989
  • ICAM-2 LFA-1, LFA-3
  • MHC class I molecules MHC class II molecules
  • ⁇ -microglobulin, chaperones CD3, or analogs thereof.
  • Sequences which encode the above-described immunomodulatory cofactors may be readily obtained from a variety of sources, including for example, the American Type Culture Collection (ATCC, Rockville, Maryland), or from commercial sources such as British Bio-technology Limited (Cowley, Oxford England).
  • BBG 12 containing the GM-CSF gene coding for the mature protein of 127 amino acids
  • BBG 6 which contains sequences encoding gamma interferon
  • ATCC No. 39656 which contains sequences encoding TNF
  • ATCC No. 20663 which contains sequences encoding alpha interferon
  • ATCC Nos. 31902, 31902 and 39517 which contains sequences encoding beta interferon
  • ATCC Nos. 39405, 39452, 39516, 39626 and 39673 which contains sequences encoding Interleukin-2
  • ATCC No. 57592 which contains sequences encoding Interleukin-4
  • ATCC 67153 which contains sequences encoding Interleukin-6).
  • blood samples may be taken from a feline with a particular disease, for use in CTL assays. Briefly, peripheral blood lymphocytes (PBLs) are separated from the blood, and stimulated in vitro with concanavalin A, Interleukin-2, bovine T-cell growth factor, and autologous irradiated cells, followed by transduction with the above-described recombinant retroviruses which direct the expression of an immunogenic portion of an antigen which is associated with the above described diseases, and an immunomodulatory cofactor.
  • PBLs peripheral blood lymphocytes
  • Stimulated PBLs are used as effectors in a CTL assay with the autologous transduced cells as both restimulators and targets.
  • vector constructs may be constructed to include a promoter such as SV40 (see Kriegler et al., Cell 35:483, 1984), cytomegalovirus (“CMV”) (see Boshart et al., Cell 47:521-530, 1991), or an Internal Ribosomal Binding Site ("IRBS").
  • SV40 see Kriegler et al., Cell 35:483, 1984
  • CMV cytomegalovirus
  • IRBS Internal Ribosomal Binding Site
  • the five prime untranslated region of the immunoglobulin heavy chain binding protein has been shown to support the internal engagement of a bicistronic message (see Jacejak and Sarnow, Nature 353:90-94, 1991).
  • This sequence is small (300 bp), and may readily be incorporated into a retoviral vector in order to express multiple genes from a multi-cistronic message whose cistrons begin with this sequence.
  • a representative vector construct utilizing IRBS is set forth in more detail below in Example 4.
  • vector constructs may also be developed and utilized with other viral carriers including, for example, poliovirus (Evans et al, Nature 339:385-388, 1989, and Sabin, J. of Biol. Standardization 7:115-118, 1973); rhinovirus (Arnold, /. Cell Biochem.
  • pox viruses such as canary pox virus or vaccinia virus (Fisher-Hoch et al., PNAS 56:317-321, 1989; Flexner et ⁇ ., Ann. N.Y. Acad. Sc 569:86-103, 1989; Flexner et al., Vaccine 5:17-21, 1990; U.S. Patent Nos.
  • viral carriers may be homologous, non-pathogenic (defective), replication competent virus (e.g., Overbaugh et al., Science 239:906-910, 1988), and yet induce cellular immune responses, including CTL.
  • nude mice may be tested for tumorigenicity prior to administration to felines, for example, by determining the extent of tumor formation in nude mice, or by evaluating colony formation in soft agar.
  • tumor formation in nude mice is a particularly important and sensitive method for determining tumorigenicity.
  • Nude mice lack a functional cellular immune system, do not possess mature T- cells, and therefore provide a useful in vivo model in which to test the tumorigenic potential of cells. Normal non-tumorigenic cells do not display uncontrolled growth properties if injected into nude mice. However, transformed cells will rapidly proliferate and generate tumors in nude mice.
  • the vector construct is administered by injection into nude mice.
  • mice are visually examined for a period of 4 to 16 weeks after injection in order to determine tumor growth.
  • the mice may also be sacrificed and autopsied in order to determine whether tumors are present.
  • Giovanella et al., /. Natl Cancer Inst. 45:1531-1533, 1972; Furesz et al., 'Tumorigenicity testing of cell lines considered for production of biological drugs Abnormal Cells, New Products and Risk, Hopps and Petricciani (eds.), Tissue Culture Association, 1985; and Levenbook et al., /. Biol Std. 73:135-141, 1985).
  • Tumorigenicity may also be assessed by visualizing colony formation in soft agar (MacPherson and Montagnier, Vir. 23:291-294, 1964). Briefly, one property of normal non-tumorigenic cells is anchorage-dependent growth. More specifically, normal non-tumorigenic cells will stop proliferating when they are plated in a semi-solid agar medium, whereas tumorigenic cells will continue to proliferate and form colonies in soft agar.
  • a vector construct may also be administered to a feline in order to treat the above-described feline diseases. Similarly, the vector construct may be administered prophylactically, in order to prevent the above-described feline diseases.
  • Methods for administering a vector construct via a retroviral vector are described in greater detail in an application entitled "Recombinant Retroviruses" (U.S.S.N. 07/586,603).
  • methods for treating or preventing a feline disease comprising the steps of (a) removing cells from a feline, (b) administering to the removed cells a vector construct which directs the expression of at least one immunogenic portion of a feline leukemia virus antigen, a feline immunodeficiency virus antigen, or both, and (c) returning the cells to a feline, such that a cellular immune response is generated.
  • a vector construct which directs the expression of at least one immunogenic portion of a feline leukemia virus antigen, a feline immunodeficiency virus antigen, or both
  • returning the cells to a feline, such that a cellular immune response is generated.
  • the removed cells need not necessarily be returned to the same feline, but may be utilized within other cats as well. In such a case it is generally preferable to have histocompatibility matched felines (although not always, see e.g.,
  • Cells may be removed from a variety of locations, including for example from the skin (dermal fibroblasts) and the blood (peripheral blood leukocytes). If desired, particular fractions of cells such as a T cell subset or stem cells may also be removed from the blood for administration of the vector construct (e.g., PCT WO 91/16116, an application entitled "Immunoselection Device and Method"). Vector constructs may then be administered to the removed cells utilizing any of the above-described techniques, followed by the return of the cells to the feline.
  • a vector construct is provided which directs the expression of an immunogenic portion as described above, and directs the expression of a prodrug activator.
  • genes for an immunogenic portion and a prodrug activator such as Herpes Simplex Virus Thymidine Kinase (HSVTK) are incorporated into a vector construct.
  • This vector construct is then administered to cells, which can then be eliminated by administration of an exogenous substance, such as acyclovir, which kills cells that express the HSVTK.
  • an exogenous substance such as acyclovir
  • this vector construct may be utilized to ensure that even if the delivered genes contribute to a tumorigenic event in cells which have taken up the vector, these cells can be killed by, for example, exogenous application of acyclovir.
  • vector constructs of the present invention or nucleic acids which encode the immunogenic portion(s) discussed above, to felines, or to feline cells ex vivo.
  • methods include, for example, transfection by methods utilizing various physical methods, such as lipofection (Feigner et al., Proc. Natl Acad.
  • a cellular response may also be generated by administration of a bacteria which expresses the immunogenic portion(s) discussed above.
  • immunogenic portions carrying relevant epitopes can be produced in a number of known ways (Ellis and Gerety, /. Med. Virol 37:54-58, 1990), including chemical synthesis (Bergot et al., Applied Biosystems Peptide Synthesizer User Bulletin No.
  • the immunogenic proteins or peptides of the present invention may also be purified by conventional means, and delivered by a number of methods in order to induce cell-mediated responses, including class I and class II responses. These methods include the use of adjuvants of various types, such as ISCOMS (Morein, Immunology Letters 25:281-284, 1990; Takahashi et al, Nature 344:813- 875, 1990), squalene/Tween-80/pluronic L121 (Monow et al., Poster #32, "Advances in AIDS Vaccine Development, Proceedings of the Fifth Annual Meeting of the National Cooperative Vaccine Dev.
  • ISCOMS Immunology Letters 25:281-284, 1990
  • Takahashi et al, Nature 344:813- 875, 1990 Takahashi et al, Nature 344:813- 875, 1990
  • squalene/Tween-80/pluronic L121 Monow et al., Poster #32, "Adv
  • the proteins or peptides conesponding to the immunogenic portion(s) discussed above can be encapsidated for oral or rectal administration to elicit an immune response in enteric capsules (Channock et al., /. Amer. Med. Assoc. 795:445-452, 1966), or other suitable carriers, such as poly (DL-lactide-co-glycolate) spheres (Eldridge et al. in Proceedings of the International Conference on Advances in AIDS Vaccine Development, DAIDS, NIAID, U.S. Dept of Health & Human Services, 1991), for gastrointestinal release.
  • enteric capsules Chock et al., /. Amer. Med. Assoc. 795:445-452, 1966
  • suitable carriers such as poly (DL-lactide-co-glycolate) spheres (Eldridge et al. in Proceedings of the International Conference on Advances in AIDS Vaccine Development, DAIDS, NIAID, U.S.
  • compositions comprising one of the above described recombinant viruses, such as a recombinant retrovirus or recombinant virus selected from the group consisting of poliovirus, rhinovirus, pox virus, canary pox virus, vaccinia virus, influenza virus, adenovirus, parvovirus, adeno-associated virus herpes virus, SV40, HIV, measles and Sindbis virus, in combination with a pharmaceutically acceptable carrier or diluent.
  • the composition may be prepared either as a liquid solution, or as a solid form (e.g., lyophilized) which is suspended in a solution prior to administration.
  • the composition may be prepared with suitable carriers or diluents for either injection, oral, or rectal administration.
  • the recombinant virus is utilized at a concentration ranging from 0.25% to 25%, and preferably about 5% to 20% before formulation. Subsequently, after preparation of the composition, the recombinant virus will constitute about 1 ug of material per dose, with about 10 times this amount material (10 ug) as copurified contaminants.
  • the composition is prepared in 0.1-1.0 ml of aqueous solution formulated as described below.
  • the composition may contain an adjuvant such as, for example, aluminum hydroxide, saponin, and squalene.
  • compositions are nontoxic to recipients at the dosages and concentrations employed.
  • Representative examples of carriers or diluents for injectable solutions include water, isotonic saline solutions which are preferably buffered at a physiological pH (such as phosphate- buffered saline or Tris-buffered saline), mannitol, dextrose, glycerol, and ethanol, as well as polypeptides or proteins such as feline serum albumin.
  • the recombinant vector since the recombinant vector represents approximately 1 ⁇ g of material, it may be less than 1% of high molecular weight material, and less than 1/100,000 of the total material (including water).
  • This composition is ⁇ stable at - 70°C for at least six months.
  • the composition may be injected intravenously (i.v.) or subcutaneously (s.c), although it is generally preferable to inject it intramuscularly (i.m.), or by aerosol administration intranasally. These are administered at one to four week intervals for three or four doses initially. Subsequent booster shots may be given as one or two doses after 6-12 months, and thereafter annually.
  • Oral formulations may also be employed with carriers or diluents such as cellulose, lactose, mannitol, poly (DL-lactide-co-glycolate) spheres, and/or carbohydrates such as starch.
  • the composition may take the form of, for example, a tablet, gel capsule, pill, solution, or suspension, and additionally may be formulated for sustained release.
  • preparation of a suppository may be accomplished with traditional carriers such as polyalkalene glucose, or a triglyceride.
  • the N2R5 construct is mutated by site- directed in vitro mutagenesis to change the ATG start codon to ATT preventing gag expression.
  • This mutagenized fragment is 200 base pairs (bp) in length and flanked by Pst I restriction sites.
  • the Pst I-Pst I mutated fragment is purified from the SK+ plasmid and inserted into the Pst I site of N2 MoMLV 5' LTR: in plasmid pUC31 to replace the non-mutated 200 bp fragment.
  • the plasmid pUC31 is derived from pUC19 (Stratagene, Calif.) in which additional restriction sites Xho I, Bgl II, BssH II and Neo I are inserted between the EcoR I and Sac I sites of the polylinker. This construct is designated pUC31/N2R5gM.
  • N2 is cloned into plasmid SK+ resulting in a construct designated N2R3-.
  • a 1.0 Kb Cla I-Hind HI fragment is purified from this construct.
  • the Cla I-Cla I dominant selectable marker gene fragment from pAFVXM retroviral vector (Kriegler et al., Cell 38:483, 1984; St. Louis et al., PNAS 55:3150-3154, 1988), comprising a SV40 early promoter driving expression of the neomycin phosphotransf erase gene, is cloned into the SK+ plasmid.
  • a 1.3 Kb Cla I-BstB I gene fragment is purified from the SK+ plasmid.
  • the expression vector is constructed by a three part ligation in which the Xho I-Cla I fragment containing the gene of interest and the 1.0 Kb MoMLV 3' LTR Cla I-Hind III fragment are inserted into the Xho I-Hind III site of pUC31/N2R5gM plasmid.
  • the 1.3 Kb Cla I-BstB I neo gene fragment from the pAFVXM retroviral vector is then inserted into the Cla I site of this plasmid in the sense orientation.
  • the KT-1 retroviral backbone vector is constructed essentially as described for KT-3 in Example 1 A, with the exception that the dominant selectable marker gene, neo, is not inserted into the expression vector.
  • a plasmid containing a FeLV sequence (p61E-FeLV) is obtained from the NIH Research and Reference
  • a reaction mixture is then prepared according to procedures specified by Perkin Elmer Cetus (Emeryville, Calif.). More specifically, a reaction mixture is prepared containing 1 ⁇ g purified plasmid, 10 ⁇ l of 10X PCR reaction buffer, 2 ⁇ l 2.5 mM of each dATP, dCTP, dGTP, and dTTP, 0.5 2.5 units/lOO ⁇ l Taq polymerase, 10 ⁇ l of 10 mM MgCl2, and 0.5-1.0 ug of the primiers specificied below (Sequence ID No. 1 and Sequence ID No. 2).
  • the sense primer sequence is from the 5' region of the FeLV gag/prot gene upstream from the ATG start codon at position 609 of the FGA provirus.
  • the 5' end of the primer contains two consecutive Xho I restriction sites.:
  • the anti-sense primer sequence is complementary to a sequence at position 2800 of the FGA provirus and contains two consecutive stop codons in frame with FeLV gag/prot gene. The 5' end of the primer contains two consecutive
  • reaction mixture is the brought to 100 ⁇ l with DI H2O, and each tube is placed into a PCR machine (Gene Amp PCR System 9600, Perkin- Elmer, Cetus, Calif.).
  • the PCR program regulates the temperature of the reaction vessel first at 94°C for 2 minutes, next at 56°C for 30 seconds, 72°C for 30 seconds, and finally, 94°C for 30 seconds. This cycle is repeated 35 times. After the 35th cycle, the reactions are held at 4°C.
  • the PCR reaction is transfened into a 1.5 ml microfuge tube, and
  • the 2.0 Kb Pst I fragment from FeLV-A-Gardner-Arnstein [FGA] provirus (Donahue et al., /. Vir. 62:122-131, 1988) is subcloned into the Pst I site of the psp72 vector (Promega Biotech, Wise). Subclones containing FeLV env in the sense orientation with respect to the 5' Xho I and 3' Cla I sites are selected by restriction enzyme analysis. This construct is designated psp72 FeLV env.
  • the Xho I-Cla I fragment is then excised and inserted into the KT-3 backbone.
  • DNA encoding the FeLV gag/prot gene is prepared as described above in Example 2, and placed into the Xho I and Cla I sites of the pBluescript KS 11+ plasmid (Stratagene, Calif.) and verified by DNA sequencing. This construct is designated pBluescript KS 11+ FeLV gag/prot. The Xho I-Cla I fragment is then excised and inserted into the KT-3 backbone.
  • Sequences encoding the FIV env/rev/RRE gene are amplified and isolated from plasmid pFIV-14-Petaluma (NIH Research and Reference Reagent
  • the sense primer sequence has two consecutive Xho I restriction sites that are placed at the 5' end at position 6020 of clone 34F10 (Talbott et al.,
  • the antisense primer sequence is complementary to a sequence at position 9387 of clone 34F10.
  • the 5' end of the primer has two consecutive Cla I sites: (Sequence ID No. 4)
  • the PCR product is placed in the pBluescript KS 11+ plasmid (Stratagene, Calif.) and verified by DNA sequencing. This construct is designated pBluescript KS 11+ FIV env/rev/RRE.
  • the Xho I-Cla I fragment is then excised and inserted into the KT-3 backbone.
  • the sense primer sequence is from position 612 of the clone 34F10. Two consecutive Sph I restriction sites are placed at the 5' end of the primer: (Sequence ID No. 5)
  • the antisense primer sequence is complementary to a sequence at position 1959 of the 34F10 clone. Two consecutive Bgl II sites are placed at the 5' end of the primer. This oligonucleotide contains two consecutive in-frame stop codons with the FIV gag open reading frame: (Sequencing ID No. 6)
  • the resultant PCR product is designated Sph I-Bgl II/FIV gag.
  • Sph I-Bgl II/FIV gag PCR product is ligated into the
  • CLP calf intestinal phosphatase, New England Biolabs, Mass.
  • Bgl II site of the re-engineered psp72 vector without the Cla I site. The insert is verified by
  • This construct is designated psp72 BIP-FTV gag.
  • the Bgl II fragment containing BIP-FIVg ⁇ j is excised and used in the ligation below, ii.
  • Construction of pBluescript KSII+ /FIV rev/RRE The FIV rev/RRE is constructed by PCR site directed mutagenesis (Ho et al., Gene 77:51-59, 1989) with the sense primer sequence ID No. 3 and antisense sequence ID No.4 used in generating the FIV env/rev/RRE with two additional oligonucleotides: the sense primer sequence
  • primers Sequence ID Nos. 7 and 8 Embedded within primers Sequence ID Nos. 7 and 8 are two consecutive stop codons in frame with the FIV env gene at position 6798 of the 34F10 clone.
  • the primers Sequence ID Nos. 3 and 8 are used in the first PCR reaction to generate the amino terminal region of FIV env with two in-frame stop codons. This double stranded DNA is designated FIV env /amino /stop.
  • the primers Sequence ID Nos. 7 and 4 are used in the second PCR reaction to generate the carboxyl terminal region of FIV env with a complementary region encompassing both stop codons of FIV env/amino/stop.
  • This double stranded DNA is designated FIV env/carboxyl/stop.
  • the PCR products, FIV env/amino/stop and FIV env/carboxyl/stop are denatured, reannealed, and allowed to undergo a third PCR reaction with primers Sequence ID Nos. 3 and 4.
  • This double stranded DNA is designated FIV rev/RRE.
  • the FIV rev/RRE DNA is digested with Xho I and Cla I, and subcloned into the Xho I and Cla I sites of the pBluescript KS 11+ plasmid and verified by DNA sequencing. This intermediate construct was designated pBluescript KSII + /FTV rev/RRE.
  • KT-3 FIV gag/rev/RRE pBluescript KSII + /FIV rev/RRE is digested with Bel I at position 7249 (Talbott et al, PNAS 56:5743-5747, 1989) and CIP treated.
  • the Bgl II BIP- FTV gag fragment is excised from psp72 BIP-FIV gag and inserted in the Bel I site in the sense orientation.
  • This construct is designated pBluescript KSII+/BIP-FTV gag/rev/RRE.
  • the construct is cleaved at the Apa I site blunted by Klenow fragment followed by cleavage with Cla I.
  • the Xho I site of the KT-3 backbone is cleaved by Xho I and blunted with Klenow fragment followed by cleavage with Cla I.
  • the BIP-FTV gag/rev/RRE fragment from the blunted Apa I to the Cla I site is inserted into the blunted Xho I site and Cla I sites of the KT-3 backbone.
  • the Cla I-Hind III fragment containing the IRBS (BIP) from psp72 is first inserted in the respective sites within the pBluescript KS 11+ plasmid.
  • the FIV env/rev/RRE is first excised from pBluescript KS 11+ /FTV env/rev/RRE plasmid by Xho I and Cla I digestion and inserted into the Xho I-Cla I sites of the KT-1 backbone. This construct is cleaved at the Cla I site and blunted by Klenow fragment.
  • KSII+ /BIP-FeLV env is then isolated, blunted by Klenow fragment, and inserted in the sense orientation at the blunted Cla I sites of the KT-1 retroviral backbone.
  • the FIV env/rev/RRE is first excised from pBluescript KS 11+ /FTV env/rev/RRE plasmid by Xho I and Cla I digestion and inserted into the Xho I-Cla I sites of the KT-1 backbone. This construct is cleaved at the Cla I site and blunted by Klenow fragment.
  • the Xho I-Cla I FeLV env fragment is isolated from the cloning intermediate psp72-FeLV env vector and substituted for Xho I - Cla I insert into pUC 18 CMV gag/pol/CAR.
  • the CMV FeLV env is then excised as a Pst I fragment, blunted by T4 DNA polymerase (New England Biolabs, Mass.) and inserted in the sense orientation at the blunted Cla I sites of the KT-1 retroviral backbone.
  • the pUC 18 CMV gag/pol/CAR is constructed essentially as follows. Briefly, from pAF/CMV/Env R (U.S. Patent Application No. 07/395,932), the 4.7 Kb CMV Env R Pst-RI fragment is isolated, and inserted into pUC 18 (New England Biolabs, Mass.) at the Pst I and RI sites. This construct is designated pUC 18 CMV Env R . To generate CMV gag/pol/CAR, HIV-1 IIIB CAR is subcloned as a Sau 3A fragment into the Bam HI site of pBluescript II KS + /CAR.
  • the CAR fragment is excised from pBluescript II KS + /CAR as a Xba I-Cla I fragment.
  • the Xho I- Xba I HJV-1 IIIB gag/pol fragment is excised from SK + gag/pol SD delta (U.S. Patent Application No. 07/395,932).
  • the plasmid backbone containing the CMV promoter is excised from pUC18 CMV/Env R with Xho I and Cla I.
  • the Xho I-Xba I HIV lug 8 a g ⁇ P°l fragment, the Xba I-Cla I CAR fragment is inserted into the Xho I- Cla I sites of the pUC 18 CMV/Env R backbone to generate pUC 18 CMV . gag/pol/CAR.
  • the sense primer sequence is from the 5' region of the FeLV gag/prot gene upstream from the ATG start codon at position 609 of the FGA provirus.
  • the 5' end of the primer contains two consecutive Xho I restriction sites: (Sequence ID No. 1)
  • the antisense primer sequence is complementary to a sequence at position 2800 of the FGA provirus and contains two consecutive stop codons in frame with FeLV gag/prot gene. The 5' end of the primer contains two consecutive
  • the resultant PCR product is inserted at the Xho I site in the sense orientation with respect to BIP of the sp72 BIP plasmid, and is designated psp72
  • the BIP-FIV gag/rev/RRE fragment is isolated from pBluescript KSII + /BIP-FIV gag/rev/RRE, cleaved at the Apa I site, blunted by Klenow fragment, followed by cleavage with Cla I. This fragment is inserted into the Xho
  • KT-l/BIP-FTV gag/rev/RRE I blunted-Cla I sites of the KT-1 backbone and is designated KT-l/BIP-FTV gag/rev/RRE.
  • KT-1/BIP-FIV gag/rev/RRE is then cleaved at the Cla I site and blunted by Klenow fragment.
  • the Cla I-Nde I BIP-FeLV gag/prot fragment from psp72 BIP-FeLV gag/prot is blunted by Klenow fragment, and inserted in the sense orientation at the blunted Cla I sites of the KT-1 retroviral backbone.
  • the pBluescript KSII + /BIP-FIV gag/rev/RRE is cleaved at the Apa
  • DX cells (W092/05266) are seeded at 5 x 10 5 confluence on a 6 cm tissue culture dish on Day 1. On Day 2, the media is replaced with a 4 ml fresh media 4 hours prior to transfection.
  • a standard calcium phosphate-DNA coprecipitation is performed by mixing 25 ⁇ l 2.0 M CaCl2, 10 ⁇ g plasmid DNA (in 10 mM Tris-Cl, pH 7.5) and water to make 200 ⁇ l total.
  • Precipitation buffer is freshly prepared by mixing 100 ⁇ l 500 mM HEPES-NaOH (pH 7.1), 125 ⁇ l 2.0 M NaCl, 10 ⁇ l 150 mM Na2HP ⁇ 4-NaH2P ⁇ 4 (pH 7.0) and water to make 1 ml total.
  • DNA-CaCl2 solution 200 ⁇ l is added dropwise with constant agitation to 200 ⁇ l precipitation buffer. After 30 min. at room temperature the resultant fine precipitate is added to a dish of cells. Cells are exposed to the DNA precipitate until Day 3 when the medium is aspirated and fresh medium is added. On Day 4 the virus-containing medium is removed, and passed through a 0.45 ⁇ m filter.
  • DA WO92/05266 cells are seeded at 1 x 10 5 cells/6 cm dish. Half a ml of the freshly collected virus-containing DX medium is added to the DA cells with fresh medium containing 4 ⁇ g/ml Polybrene (Sigma, Missouri). The following day, G418 (800 ⁇ g/ml) is added to these cells and a drug resistant pool is generated over the following week. The pool of cells is dilution cloned by adding 0.8-1.0 cells to each well of 96 well plates. Forty-eight clones are expanded to 24 well plates, then to 6 well plates, at which time cell supernatants are collected for titer.
  • DA producer cell expressing neo- multivalent vector are dilution cloned 3 days after transduction of 0.5 ml of freshly collected virus-containing DX medium with fresh medium containing 4 ⁇ g/ml Polybrene. Forty-eight clones are expanded to 24 well plates and cell supernatants titered in Example 6.
  • the multivalent vectors do not contain a selectable marker such as the neomycin gene, another way of titering the vector is described. More specifically, 1.0 ml of vector supernatant is diluted five fold until 10 '9 dilution, and each dilution is then used to transduce 5 x 10-* CRFK cells. One week later, DNA is extracted from each dish (Willis et al, /. Biol Chem. 259:7842-7849, 1984). The
  • FTV gag or FTV env is amplified by PCR using the following PCR primers.
  • the sense primer sequence (Sequence ID No. 10)
  • GAGATTCTACAGCAACATGGGGAATGGACAG is from position 612 of the clone 34F10.
  • GTATCTGTGGGAGCCTCAAGGGAAC is from position 9387 of clone 34F10.
  • the PCR products are analyzed by Southern blot analysis with the appropriate probes (Sambrook et al. Molecular Cloning, a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). Signal is expected to be seen in all the lower dilutions and fall off at a certain dilution with all higher dilutions not having signal. The last dilution where a signal is visible yields the infectious U/ml of the vector.
  • the murine fibroblast cell line BC10ME (Patek et al, Cell Imm.
  • BC10ME cell line is transduced with the retroviral vector and clones are selected using 800 ⁇ gm/ml G418 for 14 days as described in Example 5B. Cells are lysed to assay for protein expression using Western blots When the BC10ME cell line is transduced with a multivalent vector, these cells are transduced at a multiplicity of infection of 20 U/cell. One week after transduction, cells are lysed and assayed for protein expression using Western blot analysis.
  • DMEM Dulbecco's modified eagle medium
  • FBS fetal bovine serum
  • the feline kidney cell line (CRFK) is grown in DMEM containing
  • CRFK cells are transduced with the vector construct as described in Example 5B, above and used to show vector expression in feline cells using Western blot analysis.
  • Established autologous feline T-cell (as described in Example 10B, below) lines are transduced by cocultivation with the vector producer cell line.
  • 1 x 10" DA vector producer cell line
  • 1 x 10" T-cells from the established autologous T-cell line.
  • These cells are then cocultivated two more times with the vector producer line in the same method as above. After the third cocultivation, these cells are placed under G418 selection. These cells are then assayed for expression of the desired protein by Western blot analysis.
  • Proteins are separated according to their molecular weight (MW) by means of SDS polyacrylamide gel electrophoresis. Proteins are then transfened from the gel to a IPVH Immobilon-P membrane (Millipore Corp, Bedford, Mass.). The Hoefer HSI TTE transfer apparatus (Hoefer Scientific Instruments, Calif.) is used to transfer proteins from the gel to the membrane. The membrane is then probed with polyclonal antibodies that react specifically with the expressed protein. The bound antibody is detected using 125j-labeled protein A, which allows visualization of the transduced protein by autoradiography.
  • MW molecular weight
  • Tumor formation in nude mice is a particularly important and sensitive method for determining tumorigenicity.
  • Nude mice do not possess mature T-cells, and therefore lack a functional cellular immune system, providing a useful in vivo model in which to test the tumorigenic potential of cells.
  • Normal non-tumorigenic cells do not display uncontrolled growth properties if injected into nude mice. However, transformed cells will rapidly proliferate and generate tumors in nude mice.
  • the vector construct is administered by injection into nude mice. The mice are visually examined for a period of 4 to 16 weeks after injection in order to determine tumor growth. The mice may also be sacrificed and autopsied in order to determine whether tumors are present. (Giovanella et al, /. Natl Cancer Inst.
  • Tumorigenicity may also be assessed by visualizing colony formation in soft agar (MacPherson etal, Vir. 23:291-294, 1964). Briefly, one property of normal non-tumorigenic cells is anchorage dependent growth. Normal non- tumorigenic cells will stop proliferating when they are in semi-solid agar support medium, whereas tumorigenic cells will continue to proliferate and form colonies in soft agar.
  • HT1080, (ATCC CCL 121) a neoplastic cell line derived from human fibrosarcoma and known to cause tumors in 100% of nude mice is used as the assay positive control.
  • WI-38 (ATCC CCL 75) a diploid embryonic human lung cell line which is not tumorigenic in nude mice, is used as the assay negative control.
  • Primary feline fibroblasts or WI-38 cell lines are tranduced with the vector construct as described in Example 6B.
  • Duplicate samples of each of the transduced cell lines, HT1080, and WI-38 are cultured in agar. Briefly, a lower layer of 5.0 ml 0.8% Bactoagar (Difco, Michigan) in DMEM 17% FBS is set on 60 mm tissue culture plates. This is overlaid with 2.0 ml 0.3% Bactoagar in the same medium with the cells suspended at a concentration of 5 x 10-* cells per ml.
  • each cell line is strained through a 70 ⁇ m nylon mesh before suspending in the agar solution.
  • the plates are incubated at 37°C in a humidified atmosphere of 5% CO2 for 14 days.
  • representative plates of each cell line are examined for cell clumps present at the time of plating.
  • the plates are stained with 1.0 ml INT viral stain (Sigma, Missouri) and on day 14, they are scanned for colonies of >150 ⁇ m in diameter using a 1 mm eyepiece reticle.
  • mice Six- to eight-week- old female BALB/c mice (Harlan Sprague- Dawley, Indianapolis, Indiana) are injected twice intraperitoneally (i.p.) with 1 x 10' inadiated (10,000 rads at room temperature) vector transduced cells. Animals are sacrificed 7 days later and the splenocytes (3 x 10"/ml) cultured in vitro with inadiated syngeneic transduced cells (6 x 10 /ml) in flasks (T-25, Corning, Corning, New York).
  • Culture medium consists of RPMI 1640 (Irvine Scientific, Santa Ana, Calif, heat-inactivated fetal bovine serum (5%, Hyclone, Logan, Utah), sodium pyruvate (1 mM), gentamicin (50 ug/ml) and 2- mercaptoethanol (10 "5 M, Sigma Chemical, St. Louis, Missouri.). Effector cells are harvested 4-7 days later and tested using various Effecto ⁇ Target cell ratios in 96 well microtiter plates (Corning, Corning, New York) in a standard 4-6 hour assay.
  • the assay employs Na2 Cr ⁇ 4-labeled (Amersham, Arlington Heights, Illinois) (100 uCi, 1 hr at 37°C) target cells (1 x 10 4 cells/well) in a final volume of 200 ul. Following incubation, 100 ul of culture medium is removed and analyzed in a Beckman gamma spectrometer. Spontaneous release (SR) is determined as CPM from targets plus medium and maximum release (MR) is determined as CPM from targets plus 1M HC1. Percent target cell lysis is calculated as: [(Effector cell + target CPM) - (SR)/(MR) - (SR)] x 100. Spontaneous release values of targets are typically 10%-20% of the MR.
  • PBMC Peripheral blood mononuclear cells
  • PBMCs are stimulated by 5 ⁇ g/ml concanavalin A (Con A, Sigma) for three days, and maintenance in medium containing 25 U/ml human recombinant interleukin- 2 (IL-2) (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and 10% bovine T-cell growth factor (TCGF).
  • IL-2 human recombinant interleukin- 2
  • TCGF bovine T-cell growth factor
  • RPMI Complete RPMI consisted of RPMI 1640 medium containing 10% FBS, 2 mM L-glutamine, 5 x 10 " ⁇ M 2-mercaptoethanol, and 50 ⁇ g of gentamicin per ml. Clones are expanded sequentially to 48-well and 24-well plates. After several weeks, cells are transduced with retroviral vectors expressing either FeLV or FIV gag or env genes as in Example 7B. Expression of these cell lines are monitored by Western blot analysis as in Example 8. Cell lines expressing high levels of the desired protein function as restimulators and targets in a standard 51 Cr release assay as in Example 10A.

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EP93922364A 1992-09-21 1993-09-21 Recombinant retroviral vector against felv and/or fiv Withdrawn EP0662139A1 (en)

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US94835892A 1992-09-21 1992-09-21
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US5833993A (en) * 1994-04-29 1998-11-10 Pharmacia & Upjohn Company Feline immunodeficiency virus vaccine
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
US6300118B1 (en) 1995-06-07 2001-10-09 American Home Products Corporation Plasmids comprising a genetically altered feline immunodeficiency virus genome
FR2751223B1 (fr) 1996-07-19 1998-12-04 Rhone Merieux Formule de vaccin polynucleotidique felin
ES2303726T3 (es) 1997-04-10 2008-08-16 University Of Southern California Proteinas modificadas que se fijan a componentes de la matriz extracelular.

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DE10399031I1 (de) * 1987-08-28 2004-01-29 Health Research Inc Rekombinante Viren.
WO1990001547A1 (en) * 1988-08-08 1990-02-22 The Upjohn Company Methods for isolating herpes virus thymidine kinase-encoding dna
GB8901778D0 (en) * 1989-01-27 1989-03-15 Univ Court Of The University O Manipulatory technique

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