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Antibodies to the envelope protein (gp70) of endogenous xenotropic and ecotropic leukemia viruses: Induction by immunization with leukemia cells
VIROLOGY 96, 281-285 (1979)
Antibodies to the Envelope Protein (gp70) of Endogenous Xenotropic and Ecotropic Leukemia Viruses: Induction by Immunization with Leukemia Cells’ MAURO BOIOCCHI AND ROBERT C. NOWINSKP The Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington and The Divisione Oncologia Sperimentd A, In&it&o Nazionale Tumori, Via Venezian, Milano, Italia
98101,
Accepted March 6, 1979
Mice of different inbred strains varied in their immune response to endogenous leukemia viruses (MuLV). The ability of mice to produce antibodies against the gp70 proteins of xenotropic and ecotropic MuLV was to some extent dependent upon the level to which these mice naturally expressed their endogenous leukemia viruses. Three patterns of response were observed upon immunization with AKR leukemia cells: (1) Mice of low leukemic strains, which did not contain inducible ecotropic or xenotropic MuLV, produced antibodies that reacted with the gp70 proteins of both xenotropic and ecotropic MuLV; (2) mice of low leukemic strains, which contained low levels of ecotropic and inducible xenotropic MuLV, produced antibodies that reacted primarily with the gp70 proteins of ecotropic MuLV; and, (3) mice of high leukemic strains, which contained high levels of ecotropic and inducible xenotropic MuLV, failed to produce antibodies that reacted with the gp70 proteins of either xenotropic or ecotropic MuLV. Antibodies induced by immunization with leukemia cells reacted with typespecific antigenic determinants of the gp70 protein; by absorption analysis distinctive antigenie determinants could be identified on the gp70 proteins of BALB xenotropic, NZB xenotropic, and AKR ecotropic MuLV.
During the past several years extensive documentation (1-14) has been gathered concerning the immune response of mice to endogenous MuLV. These studies demonstrated that mice from a variety of inbred strains naturally produced antibodies against the g-p’70and p15(E) envelope proteins of ecotropic MuLV (2, 7). These antiviral antibodies neutralized the infectivity of the virion (4) and were cytotoxic for virus-producing cells in the presence of complement (5, 8). In addition, it was also found that normal serum from mice of virtually all inbred strains neutralized the infectivity of xenotropic MuLV (15). On the basis of the uniform presence of neutralizing factors in all mice it was thought that 1 These studies were funded in part by Grant CA 18074, Contract CP 61009 from the National Cancer Institute and Grant C.R. 77-00271-84 “Progetto Finalizzato Virus” C. N. R., Roma, Italia. 4 To whom reprint requests should be addressed. 281
the immune response to MuLV was directed primarily against xenotropic MuLV, and then secondarily to ecotropic MuLV. However, this concept was subsequently modified when it was found that the neutralization of xenotropic MuLV by normal mouse serum was mediated by proteins other than classical immunoglobulins (16, 17). As a result, the question of whether mice immunologically responded with antibody synthesis to their endogenous xenotropic viruses remained unanswered. During a routine survey of mouse antisera that were prepared against AKR leukemia cells we have observed strain-specific responses of mice to the proteins of MuLV. Unexpectedly, mice of some strains were found to produce antibody to the envelope proteins of both xenotropic and ecotropic MuLV, while mice of other strains produced antibody to the envelope proteins of only ecotropic MuLV. As described in this re0042-6822/79/090281-05$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
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port, these findings indicate that both xenotropic and ecotropic MuLV are immunogenic in mice, but that host genetic factors determine the range of immune responsiveness of mice to these viruses. For the analysis of anti-viral antibodies groups of 5 mice each from 15 different inbred strains were immunized with 8-12 challanges of viable AKR K36 cells. Antisera from these mice were tested in immune precipitation assays with NP40 lysates of lz51-labeled AKR ecotropic, BALB xenotropic, NZB xenotropie, and NIH xenotropic MuLV. Immune complexes were collected onStaphylococcus aureus (Staph A), eluted into SDS-containing sample buffer, and analyzed by polyacrylamide gel electrophoresis (PAGE). Mice of the C58, PL, C57BL/6 (B6), C57BL/lO (BlO), BALB, 129, A, NZB, C3H/He, I, and C57L strains were purchased from the Jackson Laboratories (Bar Harbor, Maine). C3Hf/Fg, AKR.B6, 129.Gv-lb, and HSFS (inbred NIH Swiss) mice were obtained from our mouse colony. The initial breeding stocks for the AKR.BG and 129.Gv-lb strains were kindly provided by Dr. E. A. Boyse (Sloan-Kettering Institute). The AKR.BG strain was congenic at the major histocompatibility complex (MHC); the genetic background of this strain was AKR, with the MHC derived from B6. The 129.Gv-lb strain (also known as 129.GIx-) contained a substitution at the Gv-1 locus (1.2, 18); the genetic background of this strain was 129, with the Gv-lb locus derived from the B6 strain. The AKR.B6, C58, PL, and C3Hf/Fg strains were characterized by a high incidence of leukemia. The B6, BlO, BALB, 129, C3H/He, C57L, I, and A strains were characterized by a low incidence of leukemia. Mice of the NZB strain had a high incidence of autoimmune hemolytic anemia and associated autoimmune diseases, while mice of the C3H/He and A strains had a high incidence of spontaneous mammary tumors. The AKR leukemia K36 was originally derived from a spontaneous thymoma; during serial transplantation this tumor converted from a splenic form of leukemia to an ascites form (19). When adapted to growth in culture, cells of this leukemia produce primarily XC+ N-ecotropic MuLV
(unpublished data); however, the possibility that trace amounts of xenotropic and/or polytropic MuLV could also be produced by these cells has not been excluded. For the preparation of antisera, groups of five mice each were inoculated subcutaneously with lo5 viable AKR K36 cells. The mice were then boosted 2 weeks later with 106cells inoculated intraperitoneally. Subsequent immunizations were performed weekly with lo7 cells inoculated intraperitoneally. Goat antiserum against the gp70 protein of Rauscher leukemia virus was provided by the National Cancer Institute. AKR ecotropic, BALB xenotropic (BALBv.2), NZB xenotropic (clone 35), and NIH xenotropic (ATS 124) MuLV were obtained from the National Cancer Institute. These viruses were provided as 1000x concentrates of density-gradient purified viruses. Viruses were lz51 radiolabeled in lO+g amounts by the chloramine-T method (20). Immediately following the labeling the viruses were solubilized in phosphate-buffered saline (PBS, pH 7.4) containing 0.5% NP40. The viral lysates were then centrifuged at 100,000 g for 1 hr to remove nonsolubilized and aggregated viral components. Prior to use in immune precipitation assays each of the virus preparations were analyzed by PAGE to quantitate the labeling of gp70. Immune precipitation reactions were performed in 50+1 volumes consisting of: (a) 2 ~1 [lz51]labeled viral lysate containing 25,000 cpm of gp70, (b) 2 ~1 of antiserum, and (c) 46 ~1 of PBS containing 0.5% NP40. The reaction mixtures were incubated for 45 min on ice and the immune complexes then collected by the addition of2 mg Staph A (21). Immune complexes that bound to the Staph A were removed from the reaction mixture by centrifugation through a l-ml cushion of 5% sucrose in PBS containing 3% NP40. The Staph A was then washed four additional times and the immune complexes eluted into SDS-containing buffer for analysis of PAGE. PAGE was performed according to Laemmli (22) in 15% slab gels. Viral proteins precipitated by the antibody/Staph A complex were identified by autoradiography through the agency of X-ray intensifying screens (23). As shown in Fig. 1, mice of different
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FIG. 1. Immune precipitation of viral proteins by mouse antisera. Groups of 5 mice each were immunized with 8-12 challanges of viable AKR K36 cells. Pooled antisera were then tested in immune precipitation assays with NP40-lysates of ‘Y-labeled: (1) AKR ecotropic MuLV, (2) BALB xenotropic MuLV, (3) NZB xenotropic MuLV, and (4) NIH xenotropic MuLV. Immune precipitates were collected by the use of Staph A, eluted into SDS-containing sample buffer, and subjected to PAGE in 15% slab gels. Precipitated viral proteins were then identified by autoradiography with MuLV the aide of X-ray intensifying screens. Immune precipitates formed by goat anti-Rauscher gp70 serum are shown in the upper left panel for comparison. In other tests (data not shown) serum sample from each of the immunized mice were tested individually against the same panel of viruses; reactions with these individual sera were qualitatively similar to those observed with pooled antisera, although minor quantitative differences were observed between the sera of mice of the same group.
inbred strains varied in their immune response to g~‘70.~Although young (2 months old) nonimmunized mice from each of the 3 These antisera also contained antibodies that reacted with the ~30, p15(E), and a yet unidentified 15,000-dalton viral protein (unpublished data). However, due to the serological complexity of these antisera, we have chosen to present in this report a study of only those antibodies that were directed against the viral envelope protein gp7’0. An analysis of the other reactions of these antisera will be presented in a future report.
strains did not produce anti-MuLV antibodies, three broad patterns of reaction were observed upon immunization with AKR K36 leukemia cells: (i) Antisera obtained from mice of the B6, BlO, C57L, I, C3H/He, BALB, A, and NZB strains reacted exclusively with the gp’70 of AKR ecotropic MuLV; these antisera did not react with the gp70 proteins of the BALB, NZB, or NIH xenotropic MuLV. (ii) Antisera obtained from mice of the 129, 129.Gv-lb, and NIH strains reacted with
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the gp’70 proteins of xenotropic, as well as ecotropic MuLV. In particular, antisera from 129 and 129.Gv-lb mice reacted with the gp70 of AKR ecotropic, BALI3 xenotropic, and NZB xenotropic MuLV; however, these antisera reacted only minimally with the gp’70 of NIH xenotropic MuLV. In contrast, antisera prepared in NIH mice reacted with the gp70 proteins of AKR ecotropic, NZB xenotropic, and NIH xenotropic MuLV, but not with the gp70 protein of BALB xenotropic MuLV. (iii) Antisera obtained from mice of the C58, PL, CSHf/Fg, and AKR.BG strains reacted only minimally with the gp70 proteins of both ecotropic or xenotropic MuLV. Differences observed in the reactions of mouse antisera with the gp70 proteins of ecotropic and xenotropic MuLV suggested that these sera contained antibodies that reacted primarily with type-specific antigenic determinants. The nature of these gp70 antigens was analyzed further by absorption tests. For this purpose, the antiserum l.%?.Gv-1b anti-AKR K36, which was known to react with the gp70 of multiple viruses, was absorbed with preparations of either AKR ecotropic or BALB xenotropic MuLV. The absorbed antisera were then tested for residual activity against lz51labeled ecotropic and xenotropic MuLV. As shown in Fig. 2, absorption of the 12$.Gv-1 b anti-AKR K36 serum with AKR ecotropic MuLV completely removed its precipitating
activity for AKR ecotropic gp70. This absorption also substantially reduced the precipitating activity of this antiserum against BALB xenotropic gp70 (although not completely), but did not affect the precipitating activity against NZB xenotropic gp70. In contrast, absorption of the 129.Gw1b anti-AKR K36 serum with BALB xenotropic MuLV completely removed precipitating activity against BALB xenotropic gp70, but only minimally affected the reaction of this antiserum with the gp70 proteins of AKR ecotropic and NZB xenotropic MuLV. On this basis we have concluded that the immune response of mice to gp70 proteins was directed primarily against type-specific antigenic determinants of gp70. Furthermore, the reactions of the 129.Gb-lb anti-AKR K36 serum with ecotropic and xenotropic MuLV was due to the presence of several different antibody populations, each of which reacted with a particular type-specific antigen of a gp70 protein. These findings confirmed other studies where antigenic polymorphism in g-p70 was demonstrated through the use of natural immune sera of mice (3) or hyperimmune antisera from mice (24) and animals of heterologous species (2.4). In fact, with the combined use of homologous and heterologous antisera it is now possible to distinguish the gp70 proteins of endogenous ecotropic MuLV from those of exogenous
FIG. 2. Absorption analysis of mouse antisera prepared against AKR K36 cells. The antiserum KS6 was tested in immune precipitation assays with: (a) AKR ecotropic MuLV, (b) BALB xenotropic MuLV, and (c) NZB xenotropic MuLV. The antiserum (166 ~1) was also absorbed either with 1 mg AKR ecotropic MuLV or with 1 mg BALB xenotropic MuLV and retested for residual activity against the panel of three viruses. 129.Gw1 b anti-AKR
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ecotropic MuLV and endogenous xenotropic MuLV. These findings also provide a preview for the use of immunized mice as a source of lymphocytes that can be used for the preparation of hybrid cell lines that produce monoclonal antibodies against the proteins of MuLV (27, 28). The findings presented here suggest that the production of anti-gp’70 antibodies in mice is influenced by the viral status of the host. In this regard, mice from low leukemic strains have been classified (20) according to their endogenous content of inducible and noninducible xenotropic MuLV. Mice of certain strains (e.g., B6 and BALBlc) contained both types of xenotropic MuLV. These strains were found by us to be immunologically responsive to the gp70 proteins of only ecotropi!: MuLV. In contrast, mice of other strains that contained only the noninducible xenotropic MuLV (e.g., 129 and NIH) were found by us to be immunologically responsive to the gp70 proteins of both ecotropic and xenotropic MuLV. It should be further noted that mice of the NIH strain failed to respond to the gp70 of BALB xenotropic MuLV, while mice of the 129 strain responded poorly to the gp70 of NIH xenotropic virus. The question can be raised as to whether the inheritance of different xenotropic MuLV in these mice might result in specific immunological tolerance to certain gp70 proteins. As a final point, mice of high leukemic strains demonstrated still yet another type of immune response to MuLV. These mice were viremic with ecotropic MuLV and contained both the inducible and noninducible xenotropic viruses. Upon immunization with leukemia cells these mice were found to be essentially immunologically nonresponsive to the gp70 antigens of both ecotropic and xenotropic MuLV. REFERENCES 1. IHLE, J. N., and HANNA, M. G., JR., J. Exp. Med. 138, 194-208 (1973). 2. IHLE, J. N., HANNA, M. G., JR., ROBERTSON, L. E., and KENNEDY, F. T., J. Exp. Med. 136, 1568-1581 (1974). 3. IHLE, J. N., DOMOTOR, J. J., and BENGALI, K. M., J. Virot. 18, 124 (1976). 4. IHLE, J. N., and LAZOR, B., J. Viral. 21, 974980 (1977).
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5. MARTIN, S. E., and MARTIN, W. J., Nature (London) 256, 498-499 (1975). 6. NOWINSKI, R. C., and KAEHLER, S. L., Science 185, 869 (1974). 7. NOWINSKI, R. C., KAEHLER, S. L., and BURGESS, R. R., C.S.H.S. Q.B. 39, 1123-1128(1974). 8. NOWINSKI, R. C., and KLEIN, P. A., J. Immunol. 115, 1261-1268 (1975). 9. NOWINSKI, R. C., and WATSON, A., J. Immunol. 117, 693-696 (1976). 10. NOWINSKI, R. C., and DOYLE, T., J. Immunol. 117, 350-351 (1976). Immun. 13, 1098Il. NOWINSKI, R. C., Infect. 1102 (1976). 12. OBATA, Y., STOCKERT, E., BOYSE, E. A., TUNG, J.-S., and LITMAN, G. W., J. Exp. Med. 144, 533 (1976). 13. OBATA, Y., STOCKERT, E., O’DONNELL, P. V., OKUBO, S., SNYDER, H. W., JR., and OLD, L. J., J. Exp. Med., in press. 14. STEPHENSON, J. R., PETERS, R. L., HINO, S., DONAHOE, R. M., LONG, L. K., AARONSON, S. A., and KELLOF, G. J., J. Viral. 19, SW898 (1976). 15. AARONSON, S. A., and STEPHENSON, J. R., Proc. Nat. Acad. Sci. USA 71, 1957 (1974). 16. FISCHINGER, P. J., IHLE, J. N., BOLOGNESI, D. P., and SCHAFER, W., Virology 71, 346351 (1976). 17. LEVY, J. A., IHLE, J. N., OLESZXO, O., and BARNES, R. D., Proc. Nat. Acad. Sci. USA 72, 5071-5075 (1975). 18. STOCKERT, E., OLD, L. J., and BOYSE, E. A., J. Exp. Med. 133, 1334-1345 (1971). 19. OLD, L. J., BOYSE, E. A., and STOCKERT, E., Cancer Res. 25, 813-819 (1965). 20. GREENWOOD, F. C., HUNTER, W. M., and GLOVER, J. S., Biochem. J. 89, 114-123 (1963). 115, 1617-1624 21. KESSLER, S. W., J. Immunol. (1975). 22. LAEMMLI, U. K., Nature (London) 227, 680685 (1970). 23. SWANSTROM, R., and SHANK, P. R., Anal. Biothem. 86, 184-192 (1978). 24. BARBACID, K., ROBBINS, K. C., HINO, S., and AARONSON, S. A., Proc. Nat. Acad. Sci. USA 75, 923-927 (1978). 25. HIRSCH, M. S., In “Tumor Virus and Infections and Immunity” (R. L. Crowell et al., eds.) University Park Press, Baltimore, 1976, pp. 175- 185. 26. PHILIPS, S. M., STEPHENSON, J. R., GREENBERGER, J. S., LANE, P. E., and AARONSON, S. A., J. Zmmunol. 116, 1123-1128 (1976). 27. KOHLER, G., and MILSTEIN, C., Nature (London) 256, 495-497 (1975). 28. NOWINSKI, R. C., LOSTROM, M. E., TAM, M. R., STONE, M. R., and BURNETTE, W. N., Virology 93, 111-126 (1979).
Antibodies to the Envelope Protein (gp70) of Endogenous Xenotropic and Ecotropic Leukemia Viruses: Induction by Immunization with Leukemia Cells’ MAURO BOIOCCHI AND ROBERT C. NOWINSKP The Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington and The Divisione Oncologia Sperimentd A, In&it&o Nazionale Tumori, Via Venezian, Milano, Italia
98101,
Accepted March 6, 1979
Mice of different inbred strains varied in their immune response to endogenous leukemia viruses (MuLV). The ability of mice to produce antibodies against the gp70 proteins of xenotropic and ecotropic MuLV was to some extent dependent upon the level to which these mice naturally expressed their endogenous leukemia viruses. Three patterns of response were observed upon immunization with AKR leukemia cells: (1) Mice of low leukemic strains, which did not contain inducible ecotropic or xenotropic MuLV, produced antibodies that reacted with the gp70 proteins of both xenotropic and ecotropic MuLV; (2) mice of low leukemic strains, which contained low levels of ecotropic and inducible xenotropic MuLV, produced antibodies that reacted primarily with the gp70 proteins of ecotropic MuLV; and, (3) mice of high leukemic strains, which contained high levels of ecotropic and inducible xenotropic MuLV, failed to produce antibodies that reacted with the gp70 proteins of either xenotropic or ecotropic MuLV. Antibodies induced by immunization with leukemia cells reacted with typespecific antigenic determinants of the gp70 protein; by absorption analysis distinctive antigenie determinants could be identified on the gp70 proteins of BALB xenotropic, NZB xenotropic, and AKR ecotropic MuLV.
During the past several years extensive documentation (1-14) has been gathered concerning the immune response of mice to endogenous MuLV. These studies demonstrated that mice from a variety of inbred strains naturally produced antibodies against the g-p’70and p15(E) envelope proteins of ecotropic MuLV (2, 7). These antiviral antibodies neutralized the infectivity of the virion (4) and were cytotoxic for virus-producing cells in the presence of complement (5, 8). In addition, it was also found that normal serum from mice of virtually all inbred strains neutralized the infectivity of xenotropic MuLV (15). On the basis of the uniform presence of neutralizing factors in all mice it was thought that 1 These studies were funded in part by Grant CA 18074, Contract CP 61009 from the National Cancer Institute and Grant C.R. 77-00271-84 “Progetto Finalizzato Virus” C. N. R., Roma, Italia. 4 To whom reprint requests should be addressed. 281
the immune response to MuLV was directed primarily against xenotropic MuLV, and then secondarily to ecotropic MuLV. However, this concept was subsequently modified when it was found that the neutralization of xenotropic MuLV by normal mouse serum was mediated by proteins other than classical immunoglobulins (16, 17). As a result, the question of whether mice immunologically responded with antibody synthesis to their endogenous xenotropic viruses remained unanswered. During a routine survey of mouse antisera that were prepared against AKR leukemia cells we have observed strain-specific responses of mice to the proteins of MuLV. Unexpectedly, mice of some strains were found to produce antibody to the envelope proteins of both xenotropic and ecotropic MuLV, while mice of other strains produced antibody to the envelope proteins of only ecotropic MuLV. As described in this re0042-6822/79/090281-05$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
282
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port, these findings indicate that both xenotropic and ecotropic MuLV are immunogenic in mice, but that host genetic factors determine the range of immune responsiveness of mice to these viruses. For the analysis of anti-viral antibodies groups of 5 mice each from 15 different inbred strains were immunized with 8-12 challanges of viable AKR K36 cells. Antisera from these mice were tested in immune precipitation assays with NP40 lysates of lz51-labeled AKR ecotropic, BALB xenotropic, NZB xenotropie, and NIH xenotropic MuLV. Immune complexes were collected onStaphylococcus aureus (Staph A), eluted into SDS-containing sample buffer, and analyzed by polyacrylamide gel electrophoresis (PAGE). Mice of the C58, PL, C57BL/6 (B6), C57BL/lO (BlO), BALB, 129, A, NZB, C3H/He, I, and C57L strains were purchased from the Jackson Laboratories (Bar Harbor, Maine). C3Hf/Fg, AKR.B6, 129.Gv-lb, and HSFS (inbred NIH Swiss) mice were obtained from our mouse colony. The initial breeding stocks for the AKR.BG and 129.Gv-lb strains were kindly provided by Dr. E. A. Boyse (Sloan-Kettering Institute). The AKR.BG strain was congenic at the major histocompatibility complex (MHC); the genetic background of this strain was AKR, with the MHC derived from B6. The 129.Gv-lb strain (also known as 129.GIx-) contained a substitution at the Gv-1 locus (1.2, 18); the genetic background of this strain was 129, with the Gv-lb locus derived from the B6 strain. The AKR.B6, C58, PL, and C3Hf/Fg strains were characterized by a high incidence of leukemia. The B6, BlO, BALB, 129, C3H/He, C57L, I, and A strains were characterized by a low incidence of leukemia. Mice of the NZB strain had a high incidence of autoimmune hemolytic anemia and associated autoimmune diseases, while mice of the C3H/He and A strains had a high incidence of spontaneous mammary tumors. The AKR leukemia K36 was originally derived from a spontaneous thymoma; during serial transplantation this tumor converted from a splenic form of leukemia to an ascites form (19). When adapted to growth in culture, cells of this leukemia produce primarily XC+ N-ecotropic MuLV
(unpublished data); however, the possibility that trace amounts of xenotropic and/or polytropic MuLV could also be produced by these cells has not been excluded. For the preparation of antisera, groups of five mice each were inoculated subcutaneously with lo5 viable AKR K36 cells. The mice were then boosted 2 weeks later with 106cells inoculated intraperitoneally. Subsequent immunizations were performed weekly with lo7 cells inoculated intraperitoneally. Goat antiserum against the gp70 protein of Rauscher leukemia virus was provided by the National Cancer Institute. AKR ecotropic, BALB xenotropic (BALBv.2), NZB xenotropic (clone 35), and NIH xenotropic (ATS 124) MuLV were obtained from the National Cancer Institute. These viruses were provided as 1000x concentrates of density-gradient purified viruses. Viruses were lz51 radiolabeled in lO+g amounts by the chloramine-T method (20). Immediately following the labeling the viruses were solubilized in phosphate-buffered saline (PBS, pH 7.4) containing 0.5% NP40. The viral lysates were then centrifuged at 100,000 g for 1 hr to remove nonsolubilized and aggregated viral components. Prior to use in immune precipitation assays each of the virus preparations were analyzed by PAGE to quantitate the labeling of gp70. Immune precipitation reactions were performed in 50+1 volumes consisting of: (a) 2 ~1 [lz51]labeled viral lysate containing 25,000 cpm of gp70, (b) 2 ~1 of antiserum, and (c) 46 ~1 of PBS containing 0.5% NP40. The reaction mixtures were incubated for 45 min on ice and the immune complexes then collected by the addition of2 mg Staph A (21). Immune complexes that bound to the Staph A were removed from the reaction mixture by centrifugation through a l-ml cushion of 5% sucrose in PBS containing 3% NP40. The Staph A was then washed four additional times and the immune complexes eluted into SDS-containing buffer for analysis of PAGE. PAGE was performed according to Laemmli (22) in 15% slab gels. Viral proteins precipitated by the antibody/Staph A complex were identified by autoradiography through the agency of X-ray intensifying screens (23). As shown in Fig. 1, mice of different
SHORT COMMUNICATIONS G anti- gp70 1234 P“; “:
283
I234
9P70
1 2 3 4
I2
i 234
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34
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FIG. 1. Immune precipitation of viral proteins by mouse antisera. Groups of 5 mice each were immunized with 8-12 challanges of viable AKR K36 cells. Pooled antisera were then tested in immune precipitation assays with NP40-lysates of ‘Y-labeled: (1) AKR ecotropic MuLV, (2) BALB xenotropic MuLV, (3) NZB xenotropic MuLV, and (4) NIH xenotropic MuLV. Immune precipitates were collected by the use of Staph A, eluted into SDS-containing sample buffer, and subjected to PAGE in 15% slab gels. Precipitated viral proteins were then identified by autoradiography with MuLV the aide of X-ray intensifying screens. Immune precipitates formed by goat anti-Rauscher gp70 serum are shown in the upper left panel for comparison. In other tests (data not shown) serum sample from each of the immunized mice were tested individually against the same panel of viruses; reactions with these individual sera were qualitatively similar to those observed with pooled antisera, although minor quantitative differences were observed between the sera of mice of the same group.
inbred strains varied in their immune response to g~‘70.~Although young (2 months old) nonimmunized mice from each of the 3 These antisera also contained antibodies that reacted with the ~30, p15(E), and a yet unidentified 15,000-dalton viral protein (unpublished data). However, due to the serological complexity of these antisera, we have chosen to present in this report a study of only those antibodies that were directed against the viral envelope protein gp7’0. An analysis of the other reactions of these antisera will be presented in a future report.
strains did not produce anti-MuLV antibodies, three broad patterns of reaction were observed upon immunization with AKR K36 leukemia cells: (i) Antisera obtained from mice of the B6, BlO, C57L, I, C3H/He, BALB, A, and NZB strains reacted exclusively with the gp’70 of AKR ecotropic MuLV; these antisera did not react with the gp70 proteins of the BALB, NZB, or NIH xenotropic MuLV. (ii) Antisera obtained from mice of the 129, 129.Gv-lb, and NIH strains reacted with
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the gp’70 proteins of xenotropic, as well as ecotropic MuLV. In particular, antisera from 129 and 129.Gv-lb mice reacted with the gp70 of AKR ecotropic, BALI3 xenotropic, and NZB xenotropic MuLV; however, these antisera reacted only minimally with the gp’70 of NIH xenotropic MuLV. In contrast, antisera prepared in NIH mice reacted with the gp70 proteins of AKR ecotropic, NZB xenotropic, and NIH xenotropic MuLV, but not with the gp70 protein of BALB xenotropic MuLV. (iii) Antisera obtained from mice of the C58, PL, CSHf/Fg, and AKR.BG strains reacted only minimally with the gp70 proteins of both ecotropic or xenotropic MuLV. Differences observed in the reactions of mouse antisera with the gp70 proteins of ecotropic and xenotropic MuLV suggested that these sera contained antibodies that reacted primarily with type-specific antigenic determinants. The nature of these gp70 antigens was analyzed further by absorption tests. For this purpose, the antiserum l.%?.Gv-1b anti-AKR K36, which was known to react with the gp70 of multiple viruses, was absorbed with preparations of either AKR ecotropic or BALB xenotropic MuLV. The absorbed antisera were then tested for residual activity against lz51labeled ecotropic and xenotropic MuLV. As shown in Fig. 2, absorption of the 12$.Gv-1 b anti-AKR K36 serum with AKR ecotropic MuLV completely removed its precipitating
activity for AKR ecotropic gp70. This absorption also substantially reduced the precipitating activity of this antiserum against BALB xenotropic gp70 (although not completely), but did not affect the precipitating activity against NZB xenotropic gp70. In contrast, absorption of the 129.Gw1b anti-AKR K36 serum with BALB xenotropic MuLV completely removed precipitating activity against BALB xenotropic gp70, but only minimally affected the reaction of this antiserum with the gp70 proteins of AKR ecotropic and NZB xenotropic MuLV. On this basis we have concluded that the immune response of mice to gp70 proteins was directed primarily against type-specific antigenic determinants of gp70. Furthermore, the reactions of the 129.Gb-lb anti-AKR K36 serum with ecotropic and xenotropic MuLV was due to the presence of several different antibody populations, each of which reacted with a particular type-specific antigen of a gp70 protein. These findings confirmed other studies where antigenic polymorphism in g-p70 was demonstrated through the use of natural immune sera of mice (3) or hyperimmune antisera from mice (24) and animals of heterologous species (2.4). In fact, with the combined use of homologous and heterologous antisera it is now possible to distinguish the gp70 proteins of endogenous ecotropic MuLV from those of exogenous
FIG. 2. Absorption analysis of mouse antisera prepared against AKR K36 cells. The antiserum KS6 was tested in immune precipitation assays with: (a) AKR ecotropic MuLV, (b) BALB xenotropic MuLV, and (c) NZB xenotropic MuLV. The antiserum (166 ~1) was also absorbed either with 1 mg AKR ecotropic MuLV or with 1 mg BALB xenotropic MuLV and retested for residual activity against the panel of three viruses. 129.Gw1 b anti-AKR
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ecotropic MuLV and endogenous xenotropic MuLV. These findings also provide a preview for the use of immunized mice as a source of lymphocytes that can be used for the preparation of hybrid cell lines that produce monoclonal antibodies against the proteins of MuLV (27, 28). The findings presented here suggest that the production of anti-gp’70 antibodies in mice is influenced by the viral status of the host. In this regard, mice from low leukemic strains have been classified (20) according to their endogenous content of inducible and noninducible xenotropic MuLV. Mice of certain strains (e.g., B6 and BALBlc) contained both types of xenotropic MuLV. These strains were found by us to be immunologically responsive to the gp70 proteins of only ecotropi!: MuLV. In contrast, mice of other strains that contained only the noninducible xenotropic MuLV (e.g., 129 and NIH) were found by us to be immunologically responsive to the gp70 proteins of both ecotropic and xenotropic MuLV. It should be further noted that mice of the NIH strain failed to respond to the gp70 of BALB xenotropic MuLV, while mice of the 129 strain responded poorly to the gp70 of NIH xenotropic virus. The question can be raised as to whether the inheritance of different xenotropic MuLV in these mice might result in specific immunological tolerance to certain gp70 proteins. As a final point, mice of high leukemic strains demonstrated still yet another type of immune response to MuLV. These mice were viremic with ecotropic MuLV and contained both the inducible and noninducible xenotropic viruses. Upon immunization with leukemia cells these mice were found to be essentially immunologically nonresponsive to the gp70 antigens of both ecotropic and xenotropic MuLV. REFERENCES 1. IHLE, J. N., and HANNA, M. G., JR., J. Exp. Med. 138, 194-208 (1973). 2. IHLE, J. N., HANNA, M. G., JR., ROBERTSON, L. E., and KENNEDY, F. T., J. Exp. Med. 136, 1568-1581 (1974). 3. IHLE, J. N., DOMOTOR, J. J., and BENGALI, K. M., J. Virot. 18, 124 (1976). 4. IHLE, J. N., and LAZOR, B., J. Viral. 21, 974980 (1977).
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