[CANCER RESEARCH 42. 3995-3999, October 1982] 0008-5472/82/0042-OOOOS02.00 Neutralization of Feline Leukemia Virus with Feline Antisera to Leukocyte Alloantigens1 T.H.Lee,M.Essex,2F.deNoronha,andJ.Azocar Department of Microbiology. Harvard University School of Public Health. Boston, Massachusetts 02115 [T. H. L., M. £..J. A.¡.and Department of Microbiology, New York State College of Veterinary Medicine. Cornell University, Ithaca. New York 14853 [F. N.¡ ABSTRACT could be lysed by antisera with specificities to selected human leukocyte alloantigens expressed by those cells (1). The possibility that normal cellular antigens might serve as Since FeLV is transmitted horizontally among outbred cats, targets for antibody neutralization of the feline leukemia virus it is theoretically possible that perturbation of the immune was investigated. Xenospecific antiserum directed to normal system of the host might occur in the early phase of a FeLV feline cells was shown to inactivate feline leukemia virus grown infection if antigens such as the FLA are associated with FeLV in fibroblasts. Cat antisera to normal feline leukocyte alloanti- in vivo. In the present study, we addressed the question of gens were then prepared, after which persistent viremia was whether FeLV grown in cells of feline origin could be lysed by induced in the donor cats. Such alloantisera neutralized virus rabbit antiserum directed to antigens present in normal unin- taken from plasma of the appropriate cat but did not neutralize fected cells. Subsequently, we examined FeLV derived from virus from a different cat. The virus neutralization was depend viremic cats to determine if it could be lysed by feline antiserum ent on the presence of complement. These results indicate that directed to the alloantigens expressed by the same viremic alloantigens are present at the virus surface and raise the cats. possibility that such antigens may play a role in the natural immune response directed to retrovirus infections. MATERIALS AND METHODS INTRODUCTION Cells. The feline embryo cell line FEA, both uninfected and infected with FeLV, was kindly supplied by Dr. O. Jarrett, Department of In the replication of enveloped viruses, assembly occurs at Veterinary Pathology, University of Glasgow, Glasgow, Scotland. The cell line CCC-81 was described before (10). FEA cells were grown in the surface of infected cells where progeny virions mature by McCoy's Medium 5A supplemented with 1% antibiotic:antimycotic budding (2). The possible incorporation of cell surface com mixture and 15% FBS. CCC-81 was grown in the same media except ponents into virus envelopes was suggested by de Harven (5), that 5% heat-inactivated FBS was used. FBS, McCoy's Medium 5A, as continuity was observed ultrastructurally between plasma and the antibiotic:antimycotic mixture were purchased from Grand membranes and viral envelopes at the time of budding. More Island Biological Co. (Grand Island, N. Y.). direct evidence came from subsequent studies in which the Cats. Three specific-pathogen-free domestic, 7-week-old orange lipid or carbohydrate composition of several viruses was shown tiger cats designated OR6, OR7, and OR8 were from the established to reflect that of the plasma membrane of the cells in which the colony at Cornell University (Ithaca, N. Y.) (6). PBL from these cats, virus was grown (4, 15-18). Similar findings were reported by which were shown to lack FeLV structural proteins (12), were used as de Théef al. (7) using AMV,3 where the presence or absence donor cells to raise antisera directed to FLA. The same cats were later of ATPase on AMV was shown to be dependent upon the challenged with FeLV so that virus could be derived directly from viremic cats. Three other specific-pathogen-free domestic 6-week-old presence or absence of ATPase on the cells in which AMV was grown. black cats designated B1, B2, and B3 and purchased from Liberty Laboratory, Inc. (Liberty Corner, N. J.), were used as recipients for Interactions between enveloped viruses and cytoplasmic membrane antigens may also involve molecules of immunolog- FLA immunization. Separation of PBL. For each immunization or live cell immunofluo- ical importance such as histocompatibility antigens. It was rescence assay, 3 to 5 ml of heparinized blood were collected from the reported by Hecht ef al. (13), for example, that vesicular jugular veins of Cats OR6, OR7, and OR8. PBL were separated with stomatitis virus grown in L-cells (H-2*) could inhibit cytolysis lymphocyte separation medium (Litton Bionetics, Kensington, Md.) and mediated by anti-/-/-2/< antisera. On the basis of a similar inhi washed 3 to 5 times with cold Dulbecco's phosphate-buffered saline bition assay, Bubbers and Lilly (3) demonstrated that Friend (Grand Island Biological Co.) before use. The yield of PBL was approx imately 1 to 2 x 106 cells/ml of heparinized blood. leukemia virus isolated directly from viremic mice also incor porated H-2 antigens. These observations were recently ex Anti-FLA Sera. Preimmune sera were collected 3 to 7 days before Cats B1, B2, and B3 were immunized. These sera were inactivated by tended to another retrovirus that occurs naturally in outbred heat at 56°for 30 min and then filtered through a 45-nm filter. Aliquots mammals, the FeLV. It was shown that Subgroups B and C of were prepared and stored at -20° until use. Cat B1 was 11 weeks old FeLV derived from infected human lymphoblastoid cell lines when it received the first i.v. inoculation of 5 to 10 x 106 PBL of Cat 1 This work was supported by Grants CA-13885, CA-18216, and CA-30520 OR6. The second immunization was done 10 days later by the same from the National Cancer Institute and Grant PDT-36 from the American Cancer route. Four more booster shots were given i.v. at weekly intervals. Society. Serum samples were collected at Days 3,7, 11, and 15 after the last 2 To whom requests for reprints should be addressed. immunization. These 4 serum preparations were pooled and heat 3 The abbreviations used are: AMV, avian myeloblastosis virus; FeLV, feline inactivated for 30 min at 56°. Before aliquots were made, the pooled leukemia virus; FLA, feline leukocyte alloantigens; FBS, fetal bovine serum; PBL, peripheral blood lymphocytes; FFU, focus-forming units; gp70, envelope glyco- serum, designated anti-FLA Serum B1, was filtered through a 45-nm filter to ensure sterility. Aliquots were then stored at -20° until use. protein of feline leukemia virus. Received February 17, 1982; accepted July 1, 1982. The immunization protocol for raising anti-FLA Serum B2 was the same OCTOBER 1982 3995 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1982 American Association for Cancer Research. T. H. Lee et al. as thai for anti-FLA serum B1 except that PBL of Cat OR8 was the incubated for 1 hr at room temperature with intermittent shaking. The source of antigen. Cat B3 was 16 weeks old when it received the first infectious virus titer was then determined on CCC-81 cells. For neu i.v. inoculation of PBL from Cat OR7. Four more booster shots were tralization by anti-FLA sera, aliquots of plasma samples derived from given at weekly intervals. Serum samples were collected 3, 7, 11, and viremic cats were incubated with anti-FLA sera in the presence of 1:10 15 days after the last immunization. The remaining procedures for diluted Low-Tox-M rabbit complement (Accurate Chemical and Scien preparation of antiserum, designated as anti-FLA Serum B3, were the tific Corp., Westbury, N. Y.) for 30 min at room temperature with same as those described above. intermittent shaking. This concentration of complement showed no Rabbit Anti-FEA Serum. Antiserum against uninfected FEA cells, toxicity for either the target cell or the virus preparations. Titers of designated anti-FEA serum, was prepared in adult rabbits by inocula residual infectious virus were scored on CCC-81 cells. Virus neutrali tion with 1 x 10e phosphate-buffered saline-washed cells in the ear zation by anti-gp70 serum was done without adding complement. vein every 7 days for 3 weeks. The rabbits were subsequently bled by Procedures for this assay have been described before (23). Briefly, cardiac puncture 1 week after the last inoculation. The sera obtained virus derived from viremic cats were first incubated with 30 ¡L\ofserially were filtered using a 45-nm filter, aliquoted, and kept at —80°until diluted goat anti-gp70 serum at 37°for 1 hr with intermittent shaking use. As a source of complement, fresh serum from an unimmunized before virus titers were scored on CCC-81 cells. In all these assays, rabbit was frozen at -80°. Preimmunization serum was also obtained samples were run in triplicate. from the ear vein of the inoculated rabbits and subsequently filtered Statistical Analysis. The Student f test was applied to evaluate the and aliquoted as described above. One ml of undiluted, heat-inactivated significance of virus titer reduction. rabbit anti-FEA serum was incubated 3 consecutive times at 25°for 1 hr with approximately 1 x 108 fresh uninfected FEA cells each time. RESULTS The absorbed antiserum was centrifuged at 10,000 x g for 15 min, filtered through a 45-nm filter, aliquoted, and kept at -20°. Virus Neutralization by Rabbit Anti-FEA Serum. As shown Immunofluorescence Assays. Live-cell membrane immunofluores- in Table 1, when rabbit anti-FEA serum was incubated with cence was used to titrate anti-FLA Sera B1, B2, and B3 according to FeLV derived from FEA cells, a statistically significant reduction the procedures published previously (1, 14) with minor modifications. Briefly, 1 x 106 PBL were incubated with 30 n\ of each serially diluted in the virus titer was observed as compared to preimmune rabbit serum.