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    • 专利摘要:
    The present invention relates to novel methods and compositions for protecting cats against infection by a wide variety of FIV strains using a multi-subtype FIV vaccine. The present invention describes multi-subtype FIV vaccines comprising a cell glass full virus or a cell line infected with a virus. A method for vaccinating cats with the vaccine composition of the present invention is also disclosed. Cats vaccinated with the methods and compositions of the present invention exhibit protective body fluids and cellular immune response against FIV when attacked by allogeneic and heterologous strains of FIV. The present invention also relates to novel cat-cell lines susceptible to FIV and methods of using them.
    公开号:KR19990044093A
    申请号:KR1019980701326
    申请日:1996-08-23
    公开日:1999-06-25
    发明作者:제네트 케이 야마모토
    申请人:로날드 엠. 쿠들라；유니버시티 오브 플로리다；린다 에스. 스티븐슨；더 리전트 오브 더 유니버시티 오브 캘리포니아；
    IPC主号:
    专利说明:

    Multiple-Subtype FIV Vaccines
    The present invention was formulated with government support as a research project NIH AI 30904 sponsored by the National Institutes of Health (NIH). The Government has certain rights to the invention.
    The domestic cats are divided into three groups: feline leukemia virus (FeLV), feline sarcoma virus (FeSV), endogenous type of oncoronavirus (RD-114) ; feline syncytia-forming virus). Of these, FeLV is the most important pathogen and induces a variety of symptoms including lymphoid cephalad and myeloproliferative tumors, anemia, immune-mediated diseases, and immunodeficiency syndrome similar to human acquired immunodeficiency syndrome (AIDS). Recently, a special replication-defective FeLV mutant named FeLV-AIDS has been associated more specifically with immunosuppressive properties.
    T-lymphotropic lentivirus (now called cat immune deficiency virus, FIV) was first reported by Pedersen et al. (1987). The characteristics of FIV are Yamamoto et al. (1988a); Yamamoto et al. (1988b); And Ackley et al. (1990). Seroepidemiologic data demonstrates that infection by FIV is unique to domestic and wild cats worldwide. A wide variety of symptoms are associated with infection by FIV, including abortion, hair loss, anemia, conjunctivitis, chronic rhinitis, enteritis, gingivitis, excretion of blood, nervous system dysfunction, dermatomyositis, and seborrheic dermatitis. The immunological characteristics of FIV-infected domestic cats are chronic and gradual deficiency of cat CD4 + peripheral blood lymphocytes, reduction of CD4: CD8 cell ratio, and in some cases, increase of CD8-containing lymphocytes. Based on molecular, biochemical and immunopathological characteristics, current FIV infections of cats are recognized as a cat AIDS model rather than FeLV-FAIDS.
    Cloning and sequencing of FIV is described by Olmsted et al. (1989a); Omsted et al. (1989b); And Talbott et al. (1989). Hosie and Jarret (1990) described the serological response of cat infected with FIV. The FIV virus subtype can be classified according to the immunotype based on the concentration of cross-neutralizing antibodies induced by each strain (Murphy and Kingsbury, 1990). Recent viruses have been classified as subtypes according to genotypes based on nucleotide sequence homology. Although the subtyping of HIV and FIV is based on genotypes (Sodora et al. , 1994; Rigby et al. , 1993; and Louwagie et al. , 1993) There are few bars. FIV virus isolates are currently classified into four FIV subtypes: A, B, C, and D (Kakinuma et al. , 1995). Infectious isolates and infectious molecular clones have been described for all FIV subtypes except subtype C (Sodora et al., 1994). Subtype C FIV was identified only from the cellular DNA of cats in Canada (Sodora et al., 1994; Rigby et al. , 1993; and Kakinuma et al. , 1995).
    The main difficulty in developing the FIV vaccine was in identifying an effective vaccine approach to a wide range of FIV strains, including field isolates from different subtypes or clades. Although vaccine prevention against FIV has been achieved for homologous and heterologous strains using a single-strain vaccine, it has not been achieved for moderate to notably heterozygous strains (Johnson et al Yamamoto et al., 1993). Thus, the development of vaccines that provide protection against multiple FIV subtypes has been desired.
    [Summary of the Invention]
    The present invention relates to a vaccine that induces extensive protective immunity against FIV infection in host animals. Specifically, the present invention provides cell-free viral isolates from different FIV subtypes or a plurality of cell-free viral isolates prepared using a combination of cell lines infected with different prototype FIV viruses of different subtypes, - It concerns a multi-subtype FIV vaccine. The cats vaccinated with the FIV vaccine of the present invention exhibit body fluids and cellular immune responses against allogeneic and heterologous FIV strains.
    The present invention also relates to novel cat-cell lines susceptible to infection by multiple FIV subtypes. The cell lines of the present invention are useful for FIV vaccines according to the method of the present invention as well as for the proliferation and production of multiple FIV subtypes. The cell lines may also be used in place of feline peripheral blood mononuclear cells (PBMCs) in FIV virus neutralization assays of cat antisera.
    Figure 1 shows reverse transcriptase (RT) concentrations of these FIV strains produced after infection of FeT-1C and FeT-J cell lines with FIV Bang and FIV Shi strains.
    Figure 2 shows the immune response of anti-FIV antibodies from 2-subtype vaccinated cats by FIV proteins detected by immunoblot. The numbers on each blot represent the number of vaccinations administered to the animal during the relevant serum test.
    Figure 3 shows the immune response of anti-FIV antibodies from 3-subtype vaccinated cats by FIV proteins detected by immunoblot. The numbers on each blot represent the number of vaccinations administered to the animal during the relevant serum test.
    Figure 4 shows the immunoreactivity of anti-FIV antibodies from 3-subtype vaccinated cats by FIV SU-V3-2 peptide detected by ELISA.
    Figure 5 shows the immunoreactivity of anti-FIV antibodies from 3-subtype vaccinated cats by FIV TM-C1 peptide detected by ELISA.
    6 is FIV Pet (A P), FIV Dix (A D), FIV UK8 (A U), FIV Bang (B B), FIV Aom1 (B A), and cats infected with FIV Shi (D S) serum Cross-neutralizing antibody titer of < / RTI > Serum from pre-infection (column 1), 6 months post-infection (column 2), and 12 months post-infection (column 3) was detected in the FeT-1C-cell line as subtype A FIV Pet , subtype B FIV Bang , FIV Shi . At least three cats per strain were tested and the results showed the VN titers from representative cats of each strain. Similar results were obtained using primary PBMC in the VN assay.
    [A brief description of the sequence]
    SEQ ID NO: 1 is the amino acid sequence of the FIV surface envelope peptide designated SV-V3-2.
    Sequence 2 is the amino acid sequence of the FIV transmembrane peptide designated TM-Cl.
    Sequence 3 is the nucleotide sequence of one FIV PCR primer.
    Sequence 4 is the nucleotide sequence of one FIV PCR primer.
    Sequence 5 is the nucleotide sequence of one FIV PCR primer.
    Sequence 6 is the nucleotide sequence of one FIV PCR primer.
    Sequence 7 is the nucleotide sequence of one FIV PCR primer.
    SEQ ID NO: 8 is the nucleotide sequence of one FIV PCR primer.
    SEQ ID NO: 9 is the nucleotide sequence of one FIV PCR primer.
    Sequence 10 is the nucleotide sequence of one FIV PCR primer.
    SEQ ID NO: 11 is the nucleotide sequence of one FIV PCR primer.
    SEQ ID NO: 12 is the nucleotide sequence of one FIV PCR primer.
    SEQ ID NO: 13 is the nucleotide sequence of one FIV PCR primer.
    Sequence 14 is the nucleotide sequence of one FIV PCR primer.
    Sequence 15 is the nucleotide sequence of one FIV PCR primer.
    Sequence 16 is the nucleotide sequence of one FIV PCR primer.
    The present invention relates to a method of inducing protective immunity against FIV infection in susceptible host animals and to vaccine compositions useful therefor. The vaccine compositions described herein, when administered to a host animal, are capable of providing protective humoral and cellular immune response against infection by homologous and heterologous strains of FIV cause. The vaccine compositions may comprise one of a cell-free FIV virus isolate or a FIV-infected cell line. In a preferred embodiment, the vaccine composition of the invention comprises FIV strains from two different FIV subtypes. Preferably, the vaccine composition may comprise three FIV subtypes, each of which is a different FIV subtype. More preferably, at least one FIV strain of FIV subtype A, subtype B, and subtype D is included in the vaccine composition.
    In a particular embodiment, the vaccine composition comprises FIV Pet < - > and FIV Shi -infected cell lines. In another embodiment, the vaccine composition comprises FIV Pet- , FIV Bang- , and FIV Shi -infected cell lines. The use of other FIV strains instead of all or part of the FIV subtypes is essentially anticipated by the present invention. For example, FIV Dix or FIV UK8 may be included in the vaccine composition in addition to or instead of FIV Pet for the purpose of providing a FIV subtype A round virus. Similar additions or substitutions to other FIV strains may also be made for FIV subtypes B and D circular viruses.
    As described herein, the vaccine compositions of the present invention may be used in combination with FIV-infected cell lines or combinations of cell-free viruses and infected cell lines, as well as cell-free whole FIV viruses or portions of viruses, FIV proteins and polypeptides. A vaccine composition comprising a FIV-infected cell line may comprise a plurality of cell lines each infected by a different FIV subtype. The vaccine compositions of the present invention may also include recombinant viral vector-based FIV constructs that may include, for example, FIV env , gag / pro , or env-gag / pro . It is anticipated that any suitable viral vector that may be used to make the recombinant vector / FIV constructs may be used in the present invention. For example, viral vectors derived from adenovirus, avipoxvirus, cats herpes virus, vaccinia, canary fox, entomovox, Svainfox, and other known viruses can be used in the compositions and methods of the invention have. Recombinant polynucleotide vectors encoding and expressing FIV components can be produced using standard genetic engineering techniques known in the art. In addition, the various vaccine compositions described herein may be used either individually or together. For example, primary immunization of animals can utilize recombinant vector-based FIV constructs with single or multiple subtype components, followed by vaccine compositions comprising inactivated FIV-infected cell lines Secondary boosts can be performed. Other vaccination protocols by vaccine compositions of the present invention will be apparent to those skilled in the art and are intended to be within the scope of the present invention.
    The multiple-subtype FIV vaccines specifically described herein have been tested for immunogenicity and efficacy in cats. Specific pathogen free (SPF) cats vaccinated with the composition of the present invention were monitored for humoral and cellular immune responses before and after challenge with allogeneic and heterologous FIV strains. Batch responses were monitored by measuring viral neutralization (VN) antibody activity and cell responses were monitored by measuring cytotoxic T lymphocyte (CTL) activity. Serum and immunocytes of vaccinated cats were tested for VN and CTL activity against allogeneic and heterologous FIV strains in vitro and as a result, vaccines could induce extensive protection against FIV infection Respectively. In accordance with the teachings of the present invention, effective multi-subtype FIV vaccines can be produced by combining circular virus isolates of different FIV subtypes or by binding individual cells infected by different subtypes of circular viruses.
    All FIV strains other than those specifically exemplified herein are expected to be utilized by the present invention. A number of FIV isolates are described in the literature and known to those skilled in the art. FIV Pet is described in U.S. Patent No. 5,037,753. Other FIV isolates described can be readily separated from infected cats by those skilled in the art using standard techniques. Methods for isolation and culture of FIV are described in U.S. Patent Nos. 5,037,753 and 5,118,602, which are incorporated herein by reference.
    The novel cell lines exemplified herein may be used in the vaccine methods and compositions of the present invention. Other cells or cell lines susceptible to FIV strains comprising peripheral blood mononuclear cells are also contemplated for use in the present invention.
    Natural, recombinant or synthetic polypeptides of FIV virus proteins and peptide fragments thereof may also be used as vaccine compositions according to the methods of the present invention. In a preferred embodiment, FIV polypeptides derived from a plurality of FIV subtypes are used to vaccinate a host animal in a vaccine composition. For example, polypeptides based on FIV envelope glycoproteins from at least two circular FIV strains of different subtypes may be included in the vaccine. Polypeptides may comprise "hybrid" or "chimeric" polypeptides that are homologous to a single strain or whose amino acid sequences are constructed by joining or joining polypeptides of at least two different FIV subtypes. Methods for producing FIV polypeptides are well known in the art. For example, FIV polypeptides can be synthesized using solid-phase synthesis methods (Merrifield, 1963). FIV polypeptides include recombinant DNA technology, which involves expressing a polynucleotide molecule encoding a FIV protein or peptide in a host cell such as a bacterial, yeast, or mammalian cell line and purifying the expressed protein according to standard techniques in the art . ≪ / RTI >
    The present invention also relates to a novel cat T-cell line susceptible to FIV. Both interleukin-2 (IL-2) dependent and independent cells are specifically exemplified. Cell lines represented by FeT-1C and FeT-J are described herein. The FeT-1C cell line is IL-2 dependent whereas the FeT-J cell line is IL-2 independent. Cell line of the present invention are useful for providing a vehicle for the growth and production as well as useful for FIV immunization of cats to FIV viral strains in vitro (in vitro). Both IL-2-dependent FeT-1C and IL-2-independent FeT-J non-infected cell lines were tested for reverse-electron enzyme (RT) activity in culture medium and 20 times for FIV promviral sequences by PCR , And FIV. The FeT-J cell line was highly infected by all tested FIV strains, including FIV Shi , FIV Dix , FIV UK8 , FIV Pet , and FIV Bang , but was more difficult to be directly infected by FIV Shi .
    The invention further relates to cell products produced by the cell lines of the invention. Cell products can be isolated and detected using methods known in the art. Antibodies to cell lines can also be produced by using known methods and are expected by the present invention.
    FIV non-infective cell sets, designated FeT-1C (ATCC Accession No. CRL 11968) and FeT-J (ATCC Accession No. CRL 11967), both of which were deposited on August 24, 1995 in the American Type Culture Collection Lt; / RTI > FIV Bang - (ATCC Accession No. CRL 11975) and FIV Shi - (ATCC Accession No. CRL 11976). Cell lines infected were deposited with the ATCC on August 25, 1995.
    The cultured cells of the present invention were cultured in the presence of 37 CFR 1.14 and 35 U.S.C. 122 of the United States Patent and Trademark Office to the person who granted the right to a sale. Such a deposit may be obtained when the counterpart application of this application or its applicant is required by a foreign patent law in the country where the application was filed. However, it should be understood that the availability of such deposits does not constitute an enforcement permit of the present invention which would undermine the patent rights granted by government action.
    Moreover, the cultured cell deposits of the present invention will be placed in a state that can be stored and sold to the public in accordance with the provisions of the Budapest Treaty on the Deposit of Microorganisms. That is, the strains survive for at least five years after the recent request for the sale of the microbial sample to provide a sample of the deposit, in any case, for at least 30 years from the date of deposit, or for the duration of the patent, Will be kept with all the necessary care to keep it from becoming. The depositor recognizes that, due to the condition of the deposit, the depository has the obligation to replace the deposit (s) when it is not available for sale at the time of sale. Any restrictions on the availability of the public to the cultured cells of the present invention will be decisively withdrawn upon grant of the patent disclosing them.
    In accordance with the methods of the present invention, the FIV vaccine compositions described herein are administered in an amount and in a manner effective to induce a host susceptible host, typically a host, to a host challenge or infectious infection by FIV . Vaccines may generally be administered parenterally, for example, subcutaneously, intraperitoneally, or intramuscularly by injection. Other suitable methods of administration include oral or nasal administration. In general, vaccines are administered to the host more than once at intervals of one week or more between each administration. However, other methods for initial or booster administration of the vaccine may be contemplated and may depend on the particular host animal receiving the judgment and procedure of the practitioner.
    The vaccine compositions of the present invention can be prepared by methods well known in the art. For example, vaccines are generally prepared in an injectable form, e.g., a fluid, solution or suspension. Vaccines are administered in a manner applicable to the dosage formulation and administered in a therapeutically useful and immunogenic amount in the subject to be treated. Optimal dosages and dosage patterns for a particular vaccine formulation can be readily determined by one of ordinary skill in the art to which this invention belongs.
    The viruses and cells in the vaccine formulation can be inactivated or attenuated by methods known in the art. For example, whole viruses and infected cells can be inactivated or attenuated by exposure to paraformaldehyde, formalin, phenol, ultraviolet light, high temperature, and the like. The amount of cell-free complete FIV virus in the vaccine dose is usually in the range of from about 0.1 mg to about 5 mg, more usually from about 0.2 mg to about 2 mg. The dose of the vaccine formulation comprising FIV-infected cell lines will be from about 10 6 to about 10 8 cells / volume, more typically from about 5 × 10 6 to about 7.5 × 10 7 cells / volume.
    Viruses or cells are generally associated with the adjuvant just prior to administration. The adjuvants used in vaccine formulations were generally the combination of tryonyl muramyldipeptide (MDP) (Byars et al ., 1987) or the complete and incomplete adjuvant. A variety of other vesicles suitable for use in the methods and vaccines of the present invention, such as aluminum sulfate, are known in the art and are expected to be used in accordance with the present invention.
    The present invention further relates to a method for analyzing virus neutralization (VN) antibodies in a sample using uninfected cell lines of the present invention. Unlike PBMCs that die after a limited number of passages and do not proliferate as readily as FeT-1C or FeT-J cells, FeT-1C and FeT-J cells are established cell lines for future use It can be easily cryopreserved. The results obtained from the VN analysis using FeT-1C cells are more reproducible than the VN analysis using PBMC because PBMC from different SPF cats have individual variability in cell growth rate and FIV infectivity. Further, PBMC for VN analysis has to be obtained from the in vitro SPF cats which require germ-free housing and retention in order to rule out the possibility of in vivo infection which may affect the VN analysis using PBMC. Thus, cat cell lines such as FeT-1C, which can be easily infected by different subtypes of FIV in the VN assay, can preferably be used instead of PBMC in VN assays.
    The following abbreviations of FIV strains are used herein:
    Strain (Subtype)Abbreviation
    Petaluma (A) FIV Pet
    Dixon (A) FIV Dix
    UK8 (A) FIV UK8
    Bangston [Bangston] (B) FIV Bang
    Aomori-1 [Aomori-1] (B) FIV Aom1
    Aomori-2 [Aomori-2] (B) FIV Aom2
    Shizuoka (D) FIV Shi
    [Materials and Methods]
    Cell culture . All of the supernatant cell lines contained 10% heat-inactivated fetal bovine serum (FCS), 10 mM HEPES (N-2-hydroxyethylpiperazine-n'-2- ethanesulfonic acid), 2 mM L-glutamine, 50 Mu] g / ml gentamycin and 5 x 10 < -5 > M 2-mercaptoethanol. IL-2-dependent cells were supplemented with 100 U / ml recombinant human IL-2 (Cetus Corporation, Emeryville, Calif). The floating cells were transferred at a concentration of 0.5-4 × 10 6 cells / ml and re-cultured twice a week with a new culture medium. All monolayer cells were plated at an initial cell concentration of 2 x 10 6 cells / ml twice a week. The TCF (Tissue Culture Fluids) from FIV- infected cells were collected twice a week to rotate the cells at 3000 rpm for 1 hour to remove residual cells, and at -20 ° C or in the case of TCF to be used immediately in the test And stored at -70 < 0 > C. FIV-susceptible cells (1 x 106 cells / ml) were infected with FIV with approximately 30,000 cpm / ml of reverse transcriptase activity (RT).
    FIV purification . Tissue cultures from FIV-infected cell lines were individually centrifuged at 2000 to 3000 rpm for 1 hour to remove cells. The virus in TCF was centrifuged at 16,000 rpm for 2 hours to pelletize and first purified by ultracentrifugation on a 10/50% (w / v) discontinuous sucrose gradient followed by ultracentrifugation on a 10/50% continuous sucrose gradient (Pedersonet al, 1987; Yamamotoet al, 1988). Each virus isolate was inactivated with 1.25% sterile paraformaldehyde (0.22 μm sterile filtered) for 18 hours and then dialyzed against sterile PBS. Inactivated viruses were diluted with sterile PBS to a concentration of 500 μg / ml and each strain of 250 μg / 0.5 ml was placed in a sterile microfused tube and stored at -70 ° C. Inactivated FIV strains were thawed at room temperature and 250 ug of inactivated virus in 0.5 ml of sterile PBS and 0.5 ml of adjuvant were mixed immediately before vaccination. FIV-infected cell lines were individually inactivated with 1.25% sterile paraformaldehyde for 18 hours, washed three times with sterile PBS and then resuspended in fresh sterile PBS in sterile tubes at approximately 5.0 x 10 < RTI ID = 0.0 >7Cells / ml and stored at < RTI ID = 0.0 > 4 C. < / RTI > Typically, just prior to vaccination, 2.5 x 10 < RTI ID = 0.0 >7Inactivated infection Cells were mixed with 0.5 ml of adjuvant. 250 [mu] g / 0.5 ml of the threonyl muramyldipeptide (MDP MF75.2 adjuvant; Chiron Corporation, Emeryville, Calif.) Was used as the adjuvant.
    CTL analysis . Peripheral blood mononuclear cells (PBMC) were stimulated with concanavalin A (Con A) for 3 days and then infected with FIV for 10 days (Song et al ., 1992). These cells functioned as target cells for CTL analysis. CTL activity was generated by co-culturing Con A-stimulated PBMC with autologous UV- and radiation-inactivated FIV-infected PBMC for 5 days. These cells acted as stimulated effector cells. On the day of analysis, target cells were labeled with 50 μCi Na 51 CrO 4 for 1 hour to 3 hours, washed 3 times, and then a fixed number of labeled target cells (5 × 10 4 cells / well) Lt; / RTI > plate. Effector cells were added in triplicate at various effector / target cell ratios (i.e., 100: 1, 50: 1 and 10: 1). Plates were centrifuged at 400 rpm for 1 minute and incubated at 37 [deg.] C for 4 hours. Control 51 Cr-labeled target cells were lysed with detergent to obtain maximal release values. The supernatant of the test sample wells was collected and the radioactivity was measured with a gamma counter. Spontaneous release was measured by incubating 51 Cr-labeled target cells in the absence of effector cells. Percent of specific cytotoxicity was calculated as follows:
    Immunoblot and enzyme-linked immunosorbent assay (ELISA) . The sucrose gradient purified virus was used as a substrate for immunoblot analysis as described in Yamamoto et al., 1993. The FIV Pet from the tissue culture of the infected cells was purified by slow centrifugation (2000 rpm for 45 minutes), concentrated by ultracentrifugation (2 hours at 16,000 rpm), and eluted with 10/50% (w / v) And purified by ultracentrifugation on a sucrose gradient. The virus purified by this method was used as a substrate for immunoblot analysis.
    The previously described immunoblot technique was modified and used (Yamamoto et al., 1991a). Virus was dissolved in 0.1% SDS, electrophoresed on 10% SDS-polyacrylamide gel, and electrotransferred onto nitrocellulose membrane to prepare viral blot strips. Serum samples from vaccinated cats were diluted 1: 50 with buffer 3 (0.15 M sodium chloride, 0.001 M edetic acid, 0.05 M TRIS base, 0.05% tween 20, and 0.1% bovine serum albumin) Lt; RTI ID = 0.0 > 37 C < / RTI > for 18 hours. The blot strips were individually washed with a washing solution (0.15 M NaCl and 0.05% tween 20 in demineralized water) and incubated with biotinylated anti-cat IgG (Vector Laboratories, Burlingame, Calif.) For 1 hour at 37 & Solution three times. Strips were then individually incubated with horseradish peroxidase conjugate streptavidin (Vector Laboratories) for 30 minutes individually. After strong washing, each strip was incubated with fresh substrate solution (0.05% diaminobenzidine, 400 ug / ml NiCl 2 , and 0.01% H 2 O 2 / 0.1 M Tris buffer, pH 7.4) at room temperature. When an observable band was formed, the reaction was terminated by excess distilled water and the strips were blot dried. The molecular weights of the respective bands on the immunoblot stained with amido black in advance were then compared to the moving distances and bands of the molecular weight standards on the strip to determine the molecular weights of the respective bands on the immunoblot. Positive and negative control standards Serum was included in each immunoblot as an internal control standard for diagnostic evaluation.
    Virus-antigen-specific ELISA has been known for a long time (Yamamoto et al., 1991a; Yamamoto et al., 1993). The surface envelope (SU) and inter-membrane (TM) peptides of constant region (C) and variable region (V) of sucrose gradient purified FIV Pet and FIV Pet were incubated with 96 well Immunolon plates (Dynatech Laboratories, Inc., Chantilly, VA) at 250 ng / well for 12 to 18 hours at 37 < 0 > C and used as a substrate for ELISA. The amino acid sequence of the SU-V3-2 peptide is Gly Ser Trp Phe Arg Ala Ile Ser Ser Trp Lys Gln Arg Asn Arg Trp Glu Trp Arg Glu Trp Arg Pro Asp Phe (SEQ ID NO: 1); The amino acid sequence of the TM-C1 peptide is: Gln Glu Leu Gly Cys Asn Gln Asn Gln Phe Phe Cys Lys Ile (SEQ ID NO: 2). Synthetic peptides were synthesized by Biosearch 9500 peptide synthesizer (Biosearch, San Rafael, Calif.) Using FMOC peptide synthesis chemistry (Magazine et al., 1988). The purity of the synthesized peptides was determined by the presence of a single peak on reversed phase high performance liquid chromatography and confirmed by amino acid sequence analysis performed on the peak sample.
    Peptide coated plates were washed once with buffer 3 immediately prior to use. Serum samples were diluted 1: 200 in buffer 3 and incubated at 37 < 0 > C for 1 hour in a FIV antigen coated well and then washed 6 times. The wells were then washed with a washing solution and incubated with biotinylated anti-cat IgG (Vector Laboratories, Burlingame, Calif.) For 1 hour at 37 ° C, washed 6 times and then incubated with either horseradish peroxidase conjugated streptavidin Vector Laboratories) for 1 hour at < RTI ID = 0.0 > 37 C. < / RTI > The wells were then washed six times with the washing solution and then incubated with ELISA substrate solution (0.005% tetramethylbenzidine and 0.015% H 2 O 2 / 0.96% citrate solution) at room temperature. The reaction was terminated by the addition of 0.1 M hydrochloric acid when visible reaction color appeared in successively diluted standards consisting of known FIV-positive cats serum. Light absorption was measured with a Biorad ELISA reader (Bio-Rad Laboratories, Hercules, Calif.) At a light density of 414 nm.
    Polymerase chain reaction (PCR) . The proviral DNA concentration of infected cells was monitored by differential PCR recently developed to discriminate multiple FIV strains of the same or different subtypes (Okada et al., 1994). As a means for improving the sensitivity of PCR, the nested PCR primer set described in Table 1 was used. The PCR proceeded in a two-step reaction, with the first step performed with a pair of outer primers (common to all FIV strains) under the conditions described by Okada et al. (1994). In the second step PCR, 1/25 of the product of step 1 was amplified by an inner primer (specific for each FIV strain). Using nested PCR, the cells infected by FIV Pet , FIV UK8 , FIV Bang , FIV Aom1 , FIV Aom2 , and FIV Shi can be distinguished from each other.
    The approximate amount of proviral DNA per cell was determined by semi-quantitative PCR, in which various dilutions of DNA extracted from known numbers of cells were made. For example, if 10 5 cells are used for DNA extraction, approximately 10 -5 dilutions of the DNA sample will correspond to the DNA present in one cell. PCR was performed on these various DNA dilutions, and the final dilution providing the positive PCR results was considered as end-point dilution. Using the number of cells corresponding to the end point dilution, the percentage of virus-infected cells in a given cell sample was calculated according to the following equation:
    Wherein Z = number of cells corresponding to the endpoint diluent
    Reverse transcriptase (RT) analysis . The presence of RNA-dependent DNA polymerase (RT) in cell culture supernatants was analyzed by the method described by Rey et al. RT analysis for FIV detection included poly (rA) -oligos (dT 12-18 ), four different deoxyribonucleotide triphosphates, 20 mM KCl and Mg ++ as cations and 5 μCi [ 3 H] - Labeled thymidine triphosphate (TTP) was used. When using a scintillation fluid mixture (1 part of xylene to 9 parts of Research Products International Biodegradable Counting Scintillant) on a Beckman LS250 scintillation counter (Beckman Instruments, Inc., Palo Alto, Calif.), 5 μCi [ 3 H] ≪ / RTI > Therefore, the RT value of the tested sample will be less than 1,200,000 cpm / ml.
    Virus neutralization analysis . Methods for the development of strain- and subtype-specific VN assays have been described (Okada et al., 1994). Heat-fire to the addition of the serial dilutions of the activated serum stained Cat peripheral blood mononuclear cells (PBMC) (s 2 × 10 5 cells / ml) (4 × 10 5 cells / ml) or FIV- infectious FeT-1C cells Previously incubated for 45 min at 37 [deg.] C with each FIV strain of 100 TCID 50 in a 24-well plate. After 3 days of incubation, the cells were washed with Hank's equilibrium salt solution to remove residual viruses from the culture and then cells were resuspended in fresh culture medium (10% heat-inactivated fetal bovine serum, 10 mM HEPES buffer, / Ml gentamycin, 5 x 10 -5 M 2-mercaptoethanol and RPMI-1640 containing 100 units / ml human recombinant IL-2). Viral infections of the cells were monitored by Mg ++ -dependent RT analysis of cultures recovered on day 9, day 12, day 15, and day 18 of culture. Serum was determined to be positive for VN antibodies when RT activity was less than 25% of the infected control cultures comprised of SPF serum.
    Hereinafter, a method for carrying out the present invention including the best embodiment will be described by way of examples. These embodiments are not to be construed in a limiting sense. Unless otherwise noted, all percentages are by weight and all solvent ratios are by volume.
    Example 1 - FIV-infected cell line
    A novel interleukin-2 (IL-2) dependent cat T-cell line, which is a mother line of an IL-2-dependent FeT-1M clone designated as FeT-1C, was used for FIV Pet , FIV Dix , FIV UK8 , FIV Bang , FIV Aom2 , or FIV Shi . The FeT-1M clone (also referred to as FIV-FeTlM) is described in U.S. Patent No. 5,275,823, which is incorporated herein by reference and is an IL-2-independent cell line that produces chronically FIV Pet , FL-4 No. 5,275,823). The FeT-1C cell line is highly susceptible to infection by different isolates from FIV subtypes A, B, Long-term passaging of the FeT-1C cell line reduces its infectivity, particularly infectivity against FIV subtype D; Thus, the passage number should be less than about 35 passages for optimal FIV infection rate or for use in its VN assay. Semi-quantitative PCR and viral core antigen analysis revealed that all cell lines exposed to FIV were significantly infected by individual FIV strains.
    All IL-2 independent cat cell lines susceptible to FIV were also made from FeT-1C cells. These cell lines, designated FeT-J, can be infected by FIV by co-culture with FIV infected cells or cells. For example, FIV Bang -infected FeT-1C cell lines were co-cultured with non-infected FeT-J cells in the absence of IL-2 to produce IL-2-independent FIV Bang -infected FeT-J cell lines (Bang / ). In the co-culture method of infection, Bang / FeT-1C cells were mixed with uninfected FeT-J cells at a ratio of about 2: 1 to about 10: 1 (infection: noninflammatory). The cell mixture was cultured in the absence of IL-2 for several weeks and the FeT-1C cells were allowed to die. The remaining cells consisted of FIV Bang -infected FeT-J cells. Thus, FIV-infected FeT-1C cells can be used to infect FeT-J cells and establish IL-2-independent FeT-J cell lines infected by different FIV subtypes. Co-culture methods with FIV infected FeT-1C cells resulted in IL-2-independent FeT-J cell lines producing moderate to high levels of different FIV subtypes.
    The FeT-1C cell line was also infected with FIV Shi and subcultured to make an IL-2-dependent cell line named Shi / FeT-1C cells. Then, the Shi / FeT-1C cell line was co-cultured with FeT-J in the absence of IL-2, and the resulting IL-2-independent FIV Shi -infected cell line was designated Shi / FeT-J. IL-2-independent Shi / FeT-J cell lines produce higher levels of FIV Shi than IL-2 dependent Shi / FeT-1C cell lines (Figure 1).
    FIV Bang -infected FeT-J cell lines were prepared without using FeT-1C cell lines. The FeT-J cell line was directly infected with the cell-free FIV Bang inoculum and subcultured in large quantities without IL-2. The resulting IL-2-independent FIV Bang production cell line was designated Bang / FeT-1C. Bang / FeT-1C cell line was producing IL-2- dependent FIV Bang in Bang / FeT-1C higher than cell lines made by infecting FeT-1C cell line with FIV Bang (Figure 1).
    Example 2 - Multiple-subtype FIV vaccine
    FIV-infected cells were separated from the supernatant by centrifugation and used as a vaccine. Likewise, the complete FIV virus was pelleted and inactivated by ultracentrifugation from infected cell-free supernatants. Both infected cells and virus were inactivated by treatment with 1.25% paraformaldehyde at 5 ° C for 24 hours and then washed well or dialyzed against PBS, respectively. This method effectively inactivates FIV without loss of immunogenicity. FIV immunogens produced according to the method of the present invention were very effective in inducing protective immunity (Yamamoto et al., 1993; Yamamoto et al., 1991a; Yamamoto et al., 1991b). It is anticipated that attenuated virus isolates may be used in the vaccine compositions of the present invention.
    FIV Shi -infected FeT-1C cell lines were over-infected with FIV Pet strains to produce a single cell line (i.e., a multiple-subtype A / D FeT-1C cell line) infected with the multiple-subtype FIV, Within two months of infection, the FIV Shi pro virus level was reduced from 50% to less than 5%, while the FIV Pet pro virus level was increased by about 50% simultaneously. Thus, maintaining a single cell line infected with the multiple-subtype of FIV for use as a FIV vaccine is not a preferred embodiment of the present invention.
    Thus, in one embodiment of the invention, the vaccine compositions were made from two distinct cell lines, each infected by a different FIV subtype. In a particular embodiment, the 2-subtype FIV vaccine composition comprised a combination of FIV subtype A-infected cell line (Pet / FL-4) and FIV subtype D-infected cell line (Shi / FeT-1C). A-subtype and D-subtype infected cell lines were inactivated as described above and conjugated to equivalent cell numbers (2.5 x 10 7 cells in 250 μg MDP) to vaccinate cats. Three SPF cats were vaccinated with inactivated Pet / FL-4 cells and the other four cats were vaccinated with depressed Shi / FeT-1C cells (2.5 x 10 7 cells / dose) . After a series of 4 vaccinations, the 2-subtype (Pet / FL-4 and Shi / FeT-1C) vaccines induced anti-FIV antibodies containing significant VN antibody titers against the tested FIV strains 2 and Table 2, Test I). Four 2-subtype (Pet / FL-4 and Shi / FeT-1C) vaccinated cats were attacked with FIV Bang (50 CID 50 ). Pet / FL-4 vaccinated with all three of the two, and Shi / FeT-1C vaccinated cats with FIV Bang was an attack of 50 CID 50. The remaining two Shi / FeT-1C vaccinated cats were attacked with 50 CID 50 FIV Shi .
    All 2-subtype vaccinated cats were negative for FIV Bang when judged by virus isolation and PBMC PCR after 6 weeks of infection (pi), whereas all sham-immunized cats The virus was positive for FIV Bang or FIV Shi when judged by virus isolation and PCR 6 weeks after infection (Table 2, Test I). On the contrary, one of the cats from Pet / FL-4 vaccinated and the vaccine Shi / FeT-1C vaccinated groups each attack by FIV Bang was positive for FIV Bang. As expected, cats attacked by FIV Shi after being vaccinated by all FIV Shi were negative for FIV Shi at 6 weeks post infection. Thus, the specifically exemplified 2-subtype vaccines prevented or delayed infection to a heterologous FIV Bang attack as well as to an allogeneic FIV Shi attack.
    2-subtype vaccinated cats (Pet / FL-4 cells and Shi / FeT-1C cells) produced a FIV antibody specific for the viral core protein p25 (also called FIV p28) after the second vaccination (Fig. 2). Higher antibody titers to other viral antigens were identified after the third or fourth vaccination. VN antibodies against FIV Pet were made after the second vaccination, whereas VN antibodies against FIV Shi were made after the fourth vaccination (Table 4). The CTL response to FIV Pet and FIV Shi was detected as early as the third vaccination in all tested cats (Table 3) and a stronger CTL response to both strains occurred after the fourth vaccination. Furthermore, two of the three cats tested showed a CTL response to the FIV Bang after the fourth vaccination. The results show that after the fourth vaccination, the 2-subtype vaccine induced a strong CTL response to FIV Pet and FIV Shi (Table 3) and induces high FIV antibodies with a VN antibody titer against both FIV strains (Table 4).
    Cats vaccinated with inactivated Shi / FeT-1C cells produced a FIV antibody specific for viral core protein p25 after the second vaccination (Fig. 2), and after the third vaccination, Lt; / RTI > antibody. In these cats, the VN antibodies to FIV Shi were generated after the fourth vaccination, whereas the VN antibodies to FIV Pet were not detected during the vaccination period. Both Shi / FeT-1C vaccinated cats showed a CTL response only after the fourth vaccination for FIV Shi , but no CTL response after the fourth vaccination for FIV Pet (Table 3).
    Cats vaccinated with inactivated Pet / FL-4 cells produced antibodies against p25 after the second vaccination (FIG. 2), and VN antibodies against FIV Pet after the second or third inoculation To produce antibodies against other viral antigens (Table 4). The only CTL response detected in cats vaccinated with Pet / FL-4 cells was for FIV pet . Overall, the 2-subtype FIV vaccines induced faster and higher VN antibody titers and CTL responses to the FIV strains of both than the single-subtype vaccine. Simulated vaccinated SPF cats did not show viral antibodies, VN antibodies or anti-FIV CTL responses.

    In a preferred embodiment, the vaccine composition of the present invention comprises a 3-subtype FIV vaccine in which each cell line is produced with three cell lines infected with a virus strain of a different FIV subtype (A, B or D). Three SPF cats were vaccinated with a 3-subtype (FIV Pet + FIV Bang + FIV Shi ) vaccine. To evaluate the immunogenicity of macrophage-deficient FIV Bang as a component of the vaccine, other cats were vaccinated with a single-subtype FIV Bang vaccine. VN antibody titer results indicate that both the 3-subtype (FIV Pet + FIV Bang + FIV Shi ) and the single-subtype FIV Bang vaccine both induce high antiviral antibody titers even after the second vaccination (Table 2, Test II and Table 5). Thus, both lymphotropic and macrophage-tropic FIV can be used as components of the vaccine composition of the present invention.
    Three mice vaccinated with a combination of inactivated Pet / FL-4, inactivated Bang / FeT-J and inactivated Shi / FeT-1C cells (2.5 x 10 7 cells in 250 μg MDP) SPF cats have produced FIV antibodies specific for other viral antigens including viral core protein p25 and FIV SU and TM envelope proteins after the second vaccination (Figures 3, 4, 5). VN antibodies to FIV Pet , FIV Bang , and FIV Shi occurred in all cats immediately after the second vaccination and soon after the third vaccination (Table 5). One cat also included VN antibodies that interact with FIV UK8 after the third vaccination. Four SPF cats vaccinated only by Bang / FeT-J cells produced FIV antibodies specific for viral core protein p25 and other viral antigens after the second vaccination (Fig. 3). In these cats, VN antibodies to FIV Bang were made after the second vaccination (Table 5), whereas VN antibodies to FIV Pet and FIV UK8 were not detected throughout the course of the vaccination. CTL response to FIV A, B and D subtype target cells of cats immunized three times with 3-subtype FIV vaccine (Pet / FL-4, Bang / FeT-J and Shi / FeT-1C cells) Are shown in Table 6. CTL responses were detected for all three FIV subtypes tested. Thus, 3-subtype vaccines resulted in wider CTL responses and faster and higher VN and SU-envelope antibody titers than single-subtype vaccines. Both uninfected FeT-J and simulated vaccinated SPF cats did not produce viral or VN antibodies.

    Example 3 - VN Antibodies to FIV Subtypes
    Analysis of VN antibodies against FIV was performed using FeT-1C cells of the present invention. Serum of SPF cats vaccinated with FIV Pet -infected cat and inactivated Pet / FL-4 cells or with inactivated FIV Pet virus was assayed using FeT-1C cells or PBMC according to the VN assay described herein VN antibody titers. Serum from two SPF cats that were not vaccinated and not infected with FIV were used as control standard sera. Serum from vaccinated and FIV-infected cats had a high VN antibody titre of over 1000, whereas sera from unvaccinated SPF cats did not have detectable VN antibody titers. The FeT-1C-based VN assay results indicate that the VN antibody titer results are equivalent to those obtained using primary PBMCs from cats (Table 6). This finding demonstrates that the VN antibody titer in VN assay using FeT-1C cells correlates with the results obtained by VN analysis using PBMC. Thus, since FeT-1C cells can be infected by all FIV subtypes and can be easily proliferated by tissue culture, FeT-1C cells can be usefully used instead of PBMC in standard VN assays for FIV have.
    Example 4 - Immunotyping of FIV strains [
    In vitro studies were performed using FeT-1C cells to determine whether the FIV subtype reflects the FIV immunotype. Immunotyping is important for understanding the role of VN antibodies in vaccine protection. Anti-serum of cats infected by FIV subtype A strains (FIV Pet , FIV Dix , FIV UK8 ), FIV Bang , FIV Aom1 and Subtype D (FIV Shi ) ≪ / RTI > were tested for their ability to neutralize these strains in vitro (FIG. 6). All test antisera were capable of neutralizing the corresponding homologous FIV strain. Subtype A strain FIV Pet was significantly cross-neutralized by antisera from cats infected with FIV Dix . FIV Pet was about 9% different in FIV Dix and surface envelope glycoprotein (Env) sites. Antisera of cats infected by FIV subtype A strains mutually-neutralized subtype B FIV bang , but subtype D FIV Shi did not. Antisera from cats infected by subtypes B and D mutually-neutralized only the other FIV strains belonging to the homologous subtype. Furthermore, antisera of cats infected by FIV UK8 neutralized the FIV bang but did not neutralize FIV strains belonging to subtype A. Although FIV UK8 is classified as subtype A (Sodora et al ., 1994; Rigby et al ., 1993; Kakinuma et al , 1995), these results suggest that antisera for FIV UK8 recognize subtype B strains, Indicating that type A strains are unrecognized, which may explain why inactivated FIV Pet vaccines are not effective against FIV UK8 , and FIV Shi (Johnson et al ., 1994). Therefore, there is a loose correlation between the genotype and the immune type. Although genotype analysis permits FIV classification, the mutual-neutralizing antibody study reflects the immunogenicity of FIV strains, which are important parameters in a wide range of body fluids protection induced by vaccines.
    Example 5 - FIV Cell Tropism < RTI ID = 0.0 >
    Cellular excitability of FIV strains obtained from infected FeT-1C and infected FeT-J cell lines was compared to that of FIV strains obtained from primary PBMC (Table 8). The two FIV isolates, FIV UK8 and FIV Bang are all equally lymphotropic and macrophage-ovary, whereas FIV Shi is highly lymphotropic. FIV Pet was lymphoprotective rather than macrophage-reflex, and its cellular excitability was not significantly affected by its cellular source. The macrophage-excitation of the FIV Bang was not affected by the cell source of the virus. Because the cell excitability of FIV strains from infected FeT-1C cell lines is equivalent to that produced from primary PBMCs, viruses grown in FeT-1C cells can be used as an inoculum for VN analysis and a therapeutic and prophylactic approach Lt ; / RTI > in vivo for in vivo inoculation for the evaluation of < RTI ID = 0.0 >
    It is to be understood that the embodiments and implementations described herein are for illustrative purposes only and that various modifications and variations of the present invention will be apparent to those skilled in the art to which the invention pertains and that the spirit and scope of the present invention and the scope of the appended claims Lt; / RTI >
    [Sequence List]
    (1) General information:
    (I) Applicant Information:
    Name of Applicant: University of Florida
    Street Address: Greenter Hall 223
    City: Gainesville
    State / Province: Florida
    Postal Code: 32611
    Phone: (352) 392-8929 Fax: (510) 748-6600
    Name of Applicant: Regents of the University of California
    Street Address: Chute 520, Auckland 2150
    City: Berkeley
    State / Province: California
    Post Code: 94704
    Phone: (510) 748-6600 Fax: (510) 748-6639
    (Ii) Name of invention: Multiple-subtype FIV vaccine
    (Iii) Number of sequences: 16
    (Iv) Communication address:
    (A) Recipient: Salihan Rush and Salihwan
    (B) Distance: Suite A-1, 41 Street Northwest 2421
    (C) City: Gainesville
    (D) Note: Florida
    (E) Country: United States of America
    (F) Postal Code: 32606
    (V) Computer readable form:
    (A) Medium type: floppy disk
    (B) Computer: IBM PC compatible model
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    (D) Software: PatentIn Release # 1.0, Version # 1.30
    (Vi Current application data
    (A) Application number: US
    (B) Filing date:
    (C) Classification:
    (Ⅷ) Attorney / Agent Information:
    (A) Name: Face, Doran Al.
    (B) Registration number: 38,261
    (C): Reference / Gasket Number: UF152
    (Ⅸ) Telecommunication information:
    (A) Phone: (904) 375-8100
    (B) Fax number: (904) 372-5800
    (2) Information on SEQ ID NO: 1:
    (I) Characteristic of the sequence:
    (A) Length of sequence: 22 amino acids
    (B) Sequence type: Amino acid
    (C) Number of chains: 1 chain
    (D) Shape: Straight
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    (I) Characteristic of the sequence:
    (A) Sequence length: 14 amino acids
    (B) Sequence type: Amino acid
    (C) Number of chains: 1 chain
    (D) Shape: Straight
    (Ii) Molecular type: Peptide
    (2) Information on SEQ ID NO: 3:
    (I) Characteristic of the sequence:
    (A) Sequence length: 20 base pairs
    (B) Sequence type: nucleic acid
    (C) Number of chains: 1 chain
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    (I) Characteristic of the sequence:
    (A) Sequence length: 21 base pairs
    (B) Sequence type: nucleic acid
    (C) Number of chains: 1 chain
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    (2) Information on SEQ ID NO: 5:
    (I) Characteristic of the sequence:
    (A) Sequence length: 20 base pairs
    (B) Sequence type: nucleic acid
    (C) Number of chains: 1 chain
    (D) Shape: Straight
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    (I) Characteristic of the sequence:
    (A) Sequence length: 21 base pairs
    (B) Sequence type: nucleic acid
    (C) Number of chains: 1 chain
    (D) Shape: Straight
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    (2) Information on SEQ ID NO: 7:
    (I) Characteristic of the sequence:
    (A) Sequence length: 21 base pairs
    (B) Sequence type: nucleic acid
    (C) Number of chains: 1 chain
    (D) Shape: Straight
    (Ii) Molecular type: DNA (genomic)
    (2) Information on SEQ ID NO: 8:
    (I) Characteristic of the sequence:
    (A) Sequence length: 21 base pairs
    (B) Sequence type: nucleic acid
    (C) Number of chains: 1 chain
    (D) Shape: Straight
    (Ii) Molecular type: DNA (genomic)
    (2) Information on SEQ ID NO: 9:
    (I) Characteristic of the sequence:
    (A) Sequence length: 21 base pairs
    (B) Sequence type: nucleic acid
    (C) Number of chains: 1 chain
    (D) Shape: Straight
    (Ii) Molecular type: DNA (genomic)
    (2) Information on SEQ ID NO: 10:
    (I) Characteristic of the sequence:
    (A) Sequence length: 21 base pairs
    (B) Sequence type: nucleic acid
    (C) Number of chains: 1 chain
    (D) Shape: Straight
    (Ii) Molecular type: DNA (genomic)
    (2) Information on SEQ ID NO: 11:
    (I) Characteristic of the sequence:
    (A) Sequence length: 21 base pairs
    (B) Sequence type: nucleic acid
    (C) Number of chains: 1 chain
    (D) Shape: Straight
    (Ii) Molecular type: DNA (genomic)
    (2) Information on SEQ ID NO: 12:
    (I) Characteristic of the sequence:
    (A) Sequence length: 21 base pairs
    (B) Sequence type: nucleic acid
    (C) Number of chains: 1 chain
    (D) Shape: Straight
    (Ii) Molecular type: DNA (genomic)
    (2) Information on SEQ ID NO: 13:
    (I) Characteristic of the sequence:
    (A) Sequence length: 21 base pairs
    (B) Sequence type: nucleic acid
    (C) Number of chains: 1 chain
    (D) Shape: Straight
    (Ii) Molecular type: DNA (genomic)
    (2) Information on SEQ ID NO: 14:
    (I) Characteristic of the sequence:
    (A) Sequence length: 21 base pairs
    (B) Sequence type: nucleic acid
    (C) Number of chains: 1 chain
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    (2) Information on SEQ ID NO: 15:
    (I) Characteristic of the sequence:
    (A) Sequence length: 19 base pairs
    (B) Sequence type: nucleic acid
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    (2) Information on SEQ ID NO: 16:
    (I) Characteristic of the sequence:
    (A) Sequence length: 20 base pairs
    (B) Sequence type: nucleic acid
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    权利要求:
    Claims (18)
    [1" claim-type="Currently amended] A vaccine composition comprising an FIV immunogen capable of causing an immune response against a plurality of FIV subtypes in an animal susceptible to FIV.
    [2" claim-type="Currently amended] 2. The method of claim 1, wherein the vaccine composition comprises a recombinant viral vector FIV construct, a FIV polypeptide from a plurality of FIV subtypes, a plurality of cell lines infected with a FIV strain of multiple cell-free FIV viruses and a FIV subtype of a different FIV subtype ≪ / RTI > or a pharmaceutically acceptable salt thereof.
    [3" claim-type="Currently amended] 3. The vaccine composition of claim 2, wherein said FIV virus or FIV-infected cell line is treated by inactivating said virus or said cell line before said vaccine is administered to said host animal.
    [4" claim-type="Currently amended] 3. The vaccine composition of claim 2, wherein said FIV virus or FIV-infected cell line is treated by depressing said virus or said cell line before said vaccine is administered to said host animal.
    [5" claim-type="Currently amended] A method of inducing a protective immune response against FIV infection in an infectious host animal comprising administering to the susceptible host animal an effective amount of a vaccine composition capable of causing an immune response against a plurality of FIV subtypes.
    [6" claim-type="Currently amended] 6. The method of claim 5, wherein the vaccine composition comprises a recombinant viral vector FIV construct, a FIV polypeptide from a plurality of FIV subtypes, a plurality of cell lines infected with a FIV strain of multiple cell-free FIV viruses and a FIV subtype of a different FIV subtype ≪ / RTI >
    [7" claim-type="Currently amended] 7. The method of claim 6, wherein said FIV virus or FIV-infected cell is treated by inactivating said virus or said cell line before said vaccine is administered to said host animal.
    [8" claim-type="Currently amended] 7. The method of claim 6, wherein said FIV virus or FIV-infected cell line is treated with said virus or said cell line before said vaccine is administered to said host animal.
    [9" claim-type="Currently amended] 6. The method of claim 5, wherein the FIV subtype is selected from the group consisting of subtypes A, B, C,
    [10" claim-type="Currently amended] 6. The method of claim 5, wherein at least the first vaccination is selected from the group consisting of recombinant viral vector FIV constructs, FIV polypeptides, cell-free FIV viruses and FIV infected cell lines after administration of the recombinant viral vector FIV construct RTI ID = 0.0 > 1, < / RTI >
    [11" claim-type="Currently amended] A cat-derived T-cell line susceptible to infection by at least one FIV subtype selected from the group consisting of subtypes A, B, C,
    [12" claim-type="Currently amended] 12. The cell line according to claim 11, wherein the cell line is designated as FeT-1C.
    [13" claim-type="Currently amended] 12. The cell line of claim 11, wherein the cell line is infected by at least one of the FIV virus strains selected from the group consisting of FIV Dix , FIV UK8 , FIV Bang , FIV Aom1 , FIV Aom2 , FIV Pet and FIV Shi .
    [14" claim-type="Currently amended] 12. The cell line of claim 11, wherein the cell line is IL-2-independent.
    [15" claim-type="Currently amended] 15. The cell line of claim 14, wherein the cell line is infected by at least one of FIV virus strains selected from the group consisting of FIV Dix , FIV UK8 , FIV Bang , FIV Aom1 , FIV Aom2 , FIV Pet and FIV Shi .
    [16" claim-type="Currently amended] 15. The cell line of claim 14, wherein the cell line is designated FeT-J.
    [17" claim-type="Currently amended] Contacting the sample with FIV, culturing the cell line of claim 10 for an effective period of time in the sample, culturing the cells in a fresh medium, and then measuring the amount of reverse transcriptase activity in the culture medium. A method for detecting or quantifying a FIV virus neutralizing antibody.
    [18" claim-type="Currently amended] 18. The method of claim 17, wherein the cell line is selected from the group consisting of cell lines designated FeT-1C and FeT-J.
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    PL186706B1|2004-02-27|
    AU2004203358A1|2004-08-19|
    PL325376A1|1998-07-20|
    PL188041B1|2004-11-30|
    EP0848615B1|2005-06-08|
    AU1972501A|2001-05-03|
    JPH11513031A|1999-11-09|
    AU2004203358B2|2007-10-25|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1995-08-25|Priority to US51938695A
    1995-08-25|Priority to US8/519,386
    1995-08-25|Priority to US08/519386
    1996-08-23|Application filed by 로날드 엠. 쿠들라, 유니버시티 오브 플로리다, 린다 에스. 스티븐슨, 더 리전트 오브 더 유니버시티 오브 캘리포니아
    1999-06-25|Publication of KR19990044093A
    2005-09-02|Application granted
    2005-09-02|Publication of KR100482616B1
    优先权:
    申请号 | 申请日 | 专利标题
    US51938695A| true| 1995-08-25|1995-08-25|
    US8/519,386|1995-08-25|
    US08/519386|1995-08-25|
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