Journal of General Virology
(1992), 73, 2721-2724. Printed in Great Britain
2721
Identification of a gag protein epitope conserved among all four groups of primate immunodeficiency viruses by using monoclonal antibodies Aidert Otteken, l* Sigrid Nick, 2 Wolfgang Bergter, 1 Gerald Voss, 1 Arne-Christian Faisst, 1 Christiane Stahl-Hennig I and Gerhard Hunsmann 1 1Abteilung Virologie und Immunologie, Deutsches Primatenzentrum, Kellnerweg 4, D-W-3400 Gi~ttingen and 2institut Jar Klinische und Molekulare Virologic, Loschgestrasse 7, D-W-8520 Erlangen, Germany
Five monoclonal antibodies (MAbs) were raised against the gag proteins of simian immunodeficiency virus (SIV) from African green monkey (SIVagmTYO.7). Two MAbs reacted with the matrix protein pl 7 and the other three with the core protein p24. Studies on the cross-reactivity of the MAbs revealed that the anti-p24 MAbs detected an epitope shared by the viruses belonging to the human immunodeficiency virus type 2 (HIV-2)/SIV=a~ group and SIVayraT¥O.7 and SIV,g=TVO.5. The anti-p 17 MAbs recognized an epitope
present on all these viruses and on SlVa~mTYO_l, HIV-1 and SIVmna. This finding demonstrates for the first time that the matrix protein, p17 or p18, respectively, of all nine HIV and SIV isolates tested in this study expresses at least one conserved immunogenic epitope recognized serologically. By using synthetic peptides, this epitope was identified at the N terminus of p17. Furthermore, this epitope was analysed by multiple sequence alignments of the peptide with homologous sequences of HIV and SIV p17.
Since the discovery of human immunodeficiency virus (HIV) types 1 (Barr6-Sinoussi et al., 1983) and 2 (Clavel et al., 1986), an increasing number of related simian retroviruses (SIVs) has been isolated from non-human primates [SIVmae, SIVsmm and SlVagm (Daniel et al., 1985; Lowenstine et al., 1986; Ohta et al., 1988); SlVmnd (Tsujimoto et al., 1988); SIVcpz (Peeters et al., 1989)]. Monoclonal antibodies (MAbs) are needed to characterize immunodominant antigenic sites or functional domains on viral components. There has been only one report published about anti-SIVagm MAbs, which described four MAbs against the transmembrane protein of the S~IVagmTYO.1 isolate (Kodama et al., 1988). A small number of anti-p24 MAbs which cross-react with SIVaom have been produced against other immunodeficiency viruses (Minassian et al., 1988; Komatsu et al., 1990). Consequently, murine MAbs against the SIVagmTgo_7 isolate originating from Ethiopia (Ohta et al., 1988) were generated. MOLT-4 cells chronically infected with SIVagmTYO_l, SIVagmTgo.5 or SIVagmTYO.7 (Ohta et al., 1988), SlVmndGB (Tsujimoto et al., 1988) or HIV-1II m (Popovic et al., 1984) were cultured in RPMI 1640 medium supplemented with 10% foetal bovine serum, 100 international units/ml penicillin, 100 ktg/ml streptomycin and 2 mM-glutamine. HIV-2b~n (Schneider et al., 1990) and HIV-2roa (Clavel et al., 1986) were propagated on MOLT-4 clone 8 cells, SIVmac2Sl on HUT 78 cells (Daniel et al., 1985) and
SIVsmmH4 (Lowenstine et al., 1986) on H9 cells. To generate MAbs, BALB/c mice were immunized intraperitoneally with an initial dose of 50 ~tg of heat-inactivated SIVagmTvo.7, followed by booster injections 3 and 5 weeks later. Spleens were removed 3 days thereafter. The conventional technique (Fazekas de St Groth & Scheidegger, 1980) was used to obtain hybrids between P3X63-Ag8.653 myeloma cells and splenocytes. Antibodies were detected in hybridoma supernatants by ELISA using SIVagmTYO.7 antigen coupled to ELISA plates. Hybridoma cultures giving rise to absorbance values threefold greater than the background value were expanded and subcloned three times by limiting dilution. Antibody specificity was examined by Western blotting as described previously (Stahl-Hennig et al., 1990). Five hybridoma lines producing MAbs directed against gag proteins were generated. Antibodies 3B10 and 3Ell recognized p17, and the other three, 1.17.3, 1A7 and 1F6, were directed against p24 (Fig. 1). Analysis of antibody subclass revealed that all antibodies were of the IgG1 subtype. Ascitic fluid was then prepared in BALB/c mice. To investigate the level of conservation of the epitopes recognized by the five MAbs, their cross-reactivity with isolates from all four groups of primate immunodeficiency viruses was investigated. For this purpose, crossreactivity with SlVagmXVO.l,SIVaomTYO_5, SIVmac251, SIVsmmH4, HIV-2~n, HIV-2rod, HIV-1IIIB and SIVmndG8
0001-1020 © 1992 SGM
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l
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3
4
5
6
180K
--116K --84K
--58K --49K
--37K
--27K
Fig. 1. Reactivity of MAbs with SIVagmTVO-7 as determined by Western blot analysis. Virus proteins were separated by SDS-PAGE and assayed for reactivity with SIVag~-seropositive simian serum 5264 (Herchenr6der et al., 1989) diluted 1:50 (lane 1), or ascitic fluids containing MAbs 3BI 0, 3E 11, 1.17.3, 1A7 or 1F6 (lanes 2 to 6) diluted 1:100. Mr markers are indicated to the right and virus-specific polypeptides to the left.
was assessed by Western blotting. Interestingly, the pl7specific antibodies 3B10 and 3Ell bound to all the isolates included in the experiment. The three p24specific MAbs bound to SIVagmTVO.5, SIVmae, SIVsmm, HIV-2~n and HIV-2rod, but failed to recognize the core proteins of HIV-lnm, SIVagmTYO_1 and SIVmn d. Based on genetic sequence analysis, the HIV and SIV strains used in this study can be allocated to four discrete groups, SlVma¢, SIVsmm and HIV-2 constituting one, HIV-1 and SIVCpz constituting another, and SIV~nd and SIV~gm each representing a genetic group. Moreover, SIVagm isolates display an unusually high degree of
intragroup genetic diversity (Allan et al., 1991), greater even than that between different HIV-1 isolates or SIVma e and HIV-2. The anti-p24 MAbs recognized an epitope shared by the HIV-2/SIVsmm/SlVmae group and SIVagmTVO-5 and StVagmTVO.7, but not present o n SIVagrnTYO. 1. This shows the remarkable diversity of SIVagm isolates (Allan et al., 1991). The cross-reactivity of MAbs against p24 from isolates of the HIV-2/SIVma~ group with SIV~m p24 has been reported (Minassian et al., 1988; Komatsu et al., 1990). The cross-reactivity of MAbs against the matrix protein p17 of different HIV and SIV isolates has also been investigated, but only restricted cross-reactivity was demonstrated (Minassian et al., 1988; Niedrig et al., 1988; Komatsu et al., 1990; Sutjipto et al., 1990). In contrast, the anti-pl7 MAbs investigated here show unrestricted cross-reactivity with all four immunodeficiency virus groups mentioned. Since the anti-gag MAbs obviously recognized conserved epitopes, we tried to identify them. Among the SIV~gmTVO isolates only the SIV~gmTVO_I sequence is known and the anti-p24 MAbs did not cross-react with this isolate, therefore synthetic peptides homologous to the SIVmac251/32r t sequence were used for a peptide ELISA. Each peptide corresponded to a sequence of 18 to 21 amino acids (aa), resulting in 13 peptides for p17 and 22 peptides for p24. Each subsequent peptide overlapped with the previous one by 10 aa. Peptides were coated onto high-binding microtitre plates (Greiner) at a concentration of 1 ~tg/well and tested for reactivity with ascitic fluid containing MAbs diluted 1:1000. Two epitopes were defined. MAbs recognizing p17 were directed against p17 peptide 3 (aa 19 to 38) (data not shown). Therefore the epitope of the anti-p 17 MAbs was localized to the N terminus of the matrix protein. Previously, most epitopes have been mapped to the Cterminal half of p17 (Cogniaux et al., 1990; Hinkula et al., 1990; Shang et al., 1991). Only a few epitopes have been mapped to the N terminus of p17 (Papsidero et al., 1989), and the epitope identified by our anti-pl 7 MAbs had not been defined previously. The epitope recognized by the anti-p24 MAbs was localized to p24 peptide 16 (aa 152 to 172) (data not shown). The production of MAbs directed against epitopes overlapping the N or C terminus of peptide 16 has recently been reported (Hinkula et al., 1990; Carpio et al., 1991 ; Niedrig et al., 1991). Since all these MAbs cross-react with HIV-1, in contrast to the anti-p24 MAbs presented here, they probably recognize different epitopes. The position of the epitopes recognized by the anti-p 17 and anti-p24 MAbs could be further defined. The MAbs recognized no adjacent SIVmae peptides and, by common consensus, sequential epitopes are five to eight residues long. Therefore, the binding regions of the epitopes
Short communication
2723
Table 1. Comparison of homologous sequences of HIV and SIV pl7 and p24 with sequences of SIVma~peptides recognized by anti-p l 7 and anti-p24 MAbs p17
Strain SlVmac251 SlVsmmH* HIV-2,o~ HIV-2~n HIV'lmB
SlV~mTWl SlVmndGB
Sequence* IRLRPGGKKKYMLKHVVWAA V .... N .............. ........... R- -- I .... V .......... R--I .... ........... K--I---S ..... N ..... QI--LI--G ---KR .... C-R---LC-CK
p24 Identity Reactivity to SIVmac of anti-pl7 peptide (%) MAb
90 90 85 85 70 60
+ +
+ + + + +
Sequence* C V K Q G P K E P FQ S Y V D R F Y K SL D .................... DI ................... D I ................... DIR ....... RD . . . . . . . TDIR ....... KD . . . . . . . AI D-R ..... A-KD ...... NVM
Identity Reactivity to SIVmac of anti-p24 peptide (%) MAb
95 91 91 71 67 62
+ + + +
* Sequences are taken from the database by Myers et al. (1991). The sequence of SIVa0mxvo-7 is unknown. Areas with high probability for helical secondary structure are underlined.
recognized by the MAbs should be located in the middle of peptide 3 of p17 and peptide 16 of p24, respectively. This conclusion is supported by multiple sequence alignments of the peptides with homologous sequences of HIV and SIV gag proteins. These alignments were performed to analyse sequence motifs conserved among the different isolates which might constitute the putative epitopes. Furthermore, the secondary structures of the homologous aa sequences were predicted by the method of Gamier et al. (1978). Viruses not recognized by the anti-p24 MAbs showed aa substitutions in the central area of the sequence homologous to peptide 16. These mutations caused an alteration of the predicted secondary structure, resulting in helical folding (Table 1). In the case of peptide 3, we found a surprisingly high rate of alterations distributed over the whole sequence of peptide 3. Following the method of Gamier et al. (1978), these aa exchanges should not influence the conformation substantially. The reason for the conformational stability might be that most mutations in the putative antigen-binding site lead to exchanges of similar aa; most mutations are exchanges of leucine, isoleucine and valine. The putative antigen-binding site should have a helical conformation. Thus, in this region, aa exchanges not involving similar residues may affect the other side of the helix. Therefore, they may still allow the MAbs to bind to the relevant residues. However, our study indicates that the matrix protein p17/p18 of all HIV and SIV isolates used in this study contains at least one conserved immunogenic epitope which can be defined serologically. The conserved p17 epitope has now to be characterized using smaller synthetic peptides. Moreover, whether the immunogenic and cross-reacting B cell epitopes on p24 and p17 bear some relevance for the humoral immunity of infected humans and monkeys should be examined. Most interestingly, it has been shown recently that the HIV-1
sequence homologous to peptide 3 contains an epitope recognized by human cytotoxic T lymphocytes (Phillips et al., 1991). We are indebted to Professor M. Hayami for providing SIVagmTYO-I-, TYO-5- and TYO-7-infected MOLT-4 cells, and Professor Dr J.-H. Peters for helpful advice on the immunization scheme and the fusion procedure. We are grateful to Dr F. Guillot for help in carrying out the fusions. Moreover, we thank the Medical Research Council AIDS Directed Programme and Programme EVA (European Vaccine Against AIDS) for kindly providing the synthetic peptides.
References ALLAN, J. S., SHORT, M., TAYLOR, M. E., SU, S., HIRSCH, V. M., JOHNSON, P. R., SHAW,G. M. 8£ HAHN, B. H. (1991). Species-specific diversity among simian immunodeficiency viruses from African green monkeys. Journal o f Virology 65, 2816-2828. BARRI~-SINOUSSI, F., CHERMANN, J. C., REY, F., NUGEYRE, i . T., CHAMARET, S., GRUEST, J., AXLER-BLIN, C., BRUN-VEZINET, F., ROUZIOUX,C., ROZENBAUM,W. & MONTAGNIER,L. (1983). Isolation of a T-lymphotropic retrovirus from a patient at risk for AIDS. Science 220, 868-871. CARPIO, E., DUARTE, C., HINKULA, J., BROLIOEN, P.-A., ROSEN, J., CAMPAL, A., GAVILONDO, J., WAHREN, B. 8£ JONDAL, M. (1991). Monoclonal antibodies to conserved regions of the major core protein (gag24) of HIV-1 and HIV-2. AIDS Research and Human Retroviruses 7, 97-101. CLAVEL, F., GUETARD, D., BRUN-VEZINET, F., CHAMARET,S., REY, M. A., SANTOS-FERREIRA, O., LAURENT, A. G., DAUQUET, C., KATLAMA, C., ROUZIOUX, C., KLATZMANN, D., CHAMPALIMAUD, J. L. d~ MONTAGNIER,L. (1986). Isolation of a new human retrovirus from West-African patients with AIDS. Science 223, 343-346. COGN1AUX, J., DE SCHEPPER, N., BLONDIAU, M.-L., CORNET, B., HORAL, P. & VAHLE, A. (1990). Characterization of monoclonal antibodies against the p17 core protein of the human immunodeficiency virus 1. Journal o f Immunological Methods 128, 165-175. DANIEL, M. D., LET'qlN, N. L., KING, N. W., KANNAGI,M., SEHGAL, P. K., HUNT, R. D., KANKI, P. J., ESSEX, i . & DESROSIERS,R. C. (1985). Isolation of a T-cell tropic HTLVIII-Iike retrovirus from macaques. Science 228, 1201-1204. FAZEKASDE ST GROTH, S. & SCHEIDEGGER, n . (1980). Production of monoclonal antibodies: strategy and tactics. Journal oflmmunalogical Methods 35, 1-21.
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GARNIER,J., OSGUTHORPE,D. J. & ROBSON,B. (1978). Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. Journalof Molecular Biology 120, 97-120. HERCHENRfDER, O., STAHL-HENNIG,C., LOKE, W., SCHNEIDER,J., SCHULZE, G., HARTMA~Cr~,H., SCHMIDT, H., TENNER-RACz, C., RACZ, P., HAYAm, M., KELLmEX, J. C. & HUNSMANN,G. (1989). Experimental infection of rhesus monkeys with SIV isolated from African green monkeys (SIVagm). lntervirology 30, 66-72." HINKULA,1., ROSEN,J., SUNDQVIST,V.-A., STIGBRAND,T. & WAHREN, B. (1990). Epitope mapping of the HIV-1 gag region with monoclonal antibodies. Molecular Immunology 27, 395-403. KODAMA, T., OHTA, Y., MASUDA,T., ISHIKAWA,K., TSUJIMOTO,H., ISAHAKt~,M. & HAYAMI,M. (1988). Production and characterization of monoclonal antibodies specific for the transmembrane protein of simian immunodeficiency virus from the African green monkey. Journal of Virology 62, 4782-4785. KOMATSU,H., TOZAWA,H., KAWAMURA,M., KODAMA,T. & HAYAMI, M. (1990). Multiple antigenic epitopes expressed on gag proteins, p26 and plS, of a human immunodeficiency virus (HIV) type 2 as defined with a libary of monoclonal antibodies. AIDS Research and Human Retrovirt~es 6, 871-881. LOWENSTINE, L. J., PEDERSEN, N. C., HIGGIN$, J., PALLIS, K. C., UYEDA, A., MARX, P., LERCHE, N. W., MUNN, R. J. & GARDNER, M. B. (1986). Seroepidemiologic survey of captive Old-World primates for antibodies to human and simian retroviruses, and isolation of a lentivirus from sooty mangabeys (Cercocebus atys). International Journal of Cancer 38, 563-574. MINASSIAN,A. A., KALYANARAMAN,V. S., GALLO, R. C. & POPOVIC, M. (1988). Monoclonal antibodies against human immunodeficiency virus (HIV) type 2 core proteins: cross-reactivities with HIV type 1 and simian immunodeficiency virus. Proceedings of the National Academy of Sciences, U.S.A. 85, 6939-6943. MYERS, G., BERZOFSKY,J. A., RABSON,A. B., SMITH,T. F. & WONGSTAAL, F. (editors) (1991). Database of Human Retroviruses and AIDS. Los Alamos: Los Alamos National Laboratory. NIEDRIG, M., RABANUS,J. P., L'AGE STEHR,J., GELDERBLOM,H. R. & PAULI, G. (1988). Monoclonal antibodies directed against human immunodeficiency virus (HIV) gag proteins with specificity for conserved epitopes in HIV-1, HIV-2 and simian immunodeficiency virus. Journal of General Virology 69, 2109-2114. N1EDRIG, M., HINKULA,J., HARTHUS,H.-P., BR6KER, M., HOPP, L., PAULI, G. & WAHXEN, B. (1991). Characterization of routine monoclonal antibodies against the core proteins of human immunodeficiency virus types 1 and 2. Journal of Virology 65, 4529-4533. OHTA, Y., MASUDA,T., TSUJIMOTO,H., ISHIKAWA,K., KODAMA,T., MORIKAWA, S., NAKAI, M., HONJO, S. & HAYAMI, M. (1988). Isolation of simian immunodeficiency virus from African green
monkeys and seroepidemiologicai survey of the virus in various nonhuman primates. International Journal of Cancer 41, 115-122. PAPSIDERO, L. D., SHEU, M. & RUSCEa'n, F. W. 0989). Human immunodeficiency virus type l-neutralizing monoclonal antibodies which react with pl7 core protein: characterization and epitope mapping. Journal of Virology 63, 267-272. PEETERS,M., HONORE,C., HUET, T., BEDJABAGA,L., OSSARI,S., BUSSI, P., COOPER, R. W. & DELXPOXTE,E. (1989). Isolation and partial characterization of an HIV-related virus occurring naturally in chimpanzees in Gabon. AIDS 3, 625-630. PmLLIPS, R. E., ROWLAND-JoNEs,S., NlXON, D. F., GOTCH, F. M., EDWARDS,J. P., OGUNLESI,A. O., ELVlN, J. G., ROTHBARD,J. A., BANGHAM,C. R. M., RlZZA, C. R. & McMICHAEL, A. J. (1991). Human immunodeficiency virus genetic variation that can escape cytotoxic T cell recognition. Nature, london 354, 453-459. POPOVlC,M., SARNGADHARAN,M. G., READ,E. & GALLO,R. C. (1984). Detection, isolation, and continous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224, 497-499. SCHNEIDER,J., LOKE, W., KIRCHHOFF,F., JUNG, R., JURKIEWlCZ,E., STAHL-HENNIG, S., NICK, S., KLEMM, E., JENTSCH, K.-D. & HUNSMANN,G. (1990). Isolation and characterization of HiV-2ben obtained from a patient with predominantly neurological defects. AIDS 4, 455-457. SHANG,F., HUANG,H., REVESZ,K., CHEN, H.-C., HERZ, R. & PINTER, A. (1991). Characterization of monoclonal antibodies against the human immunodeficiency virus matrix protein, pl70a0: identification ofepitopes exposed at the surfaces of infected cells. Journalof Virology 65, 4798-4804. STAHL-HENNIG,C., HERCHENRODER,O., NICK, S., EVERS,M., STILLESIEGENER, i . , JENTSCH,K.-D., KIRCHHOFF,F., TOLLE, T., GATESMA~, T. J., LOKE, W. & HUNSlANN, G. (1990). Experimental infection of macaques with HIV-2ben, a novel HIV-2 isolate. AIDS 4, 611-617. SUTJIPTO, S., KODAMA, T., YEE, J., GETTIE, A., JENNINGS, M., DESROSIERS, R. C. & MARX, P. A. (1990). Characterization of monocional antibodies that distinguish simian immunodeficiency virus isolates from each other and from human immunodeficiency virus types 1 and 2. Journal of General Virology 71, 247-249. TSUJIMOTO,H., COOPER, R. W., KODAMA,T., FUKASAWA,M., MIURA, T., OtrrA, Y., ISmKAWA,K., NAKALM., FROST,E., ROELANTS,G. E., ROFFI, J. & HAYAMI,M. (1988). Isolation and characterization of simian immunodeficiency virus from mandrills in Africa and its relationship to other human and simian immunodeficiency viruses. Journal of Virology 62, 4044-4050.
(Received 6 April 1992; Accepted 26 June 1992)
(1992), 73, 2721-2724. Printed in Great Britain
2721
Identification of a gag protein epitope conserved among all four groups of primate immunodeficiency viruses by using monoclonal antibodies Aidert Otteken, l* Sigrid Nick, 2 Wolfgang Bergter, 1 Gerald Voss, 1 Arne-Christian Faisst, 1 Christiane Stahl-Hennig I and Gerhard Hunsmann 1 1Abteilung Virologie und Immunologie, Deutsches Primatenzentrum, Kellnerweg 4, D-W-3400 Gi~ttingen and 2institut Jar Klinische und Molekulare Virologic, Loschgestrasse 7, D-W-8520 Erlangen, Germany
Five monoclonal antibodies (MAbs) were raised against the gag proteins of simian immunodeficiency virus (SIV) from African green monkey (SIVagmTYO.7). Two MAbs reacted with the matrix protein pl 7 and the other three with the core protein p24. Studies on the cross-reactivity of the MAbs revealed that the anti-p24 MAbs detected an epitope shared by the viruses belonging to the human immunodeficiency virus type 2 (HIV-2)/SIV=a~ group and SIVayraT¥O.7 and SIV,g=TVO.5. The anti-p 17 MAbs recognized an epitope
present on all these viruses and on SlVa~mTYO_l, HIV-1 and SIVmna. This finding demonstrates for the first time that the matrix protein, p17 or p18, respectively, of all nine HIV and SIV isolates tested in this study expresses at least one conserved immunogenic epitope recognized serologically. By using synthetic peptides, this epitope was identified at the N terminus of p17. Furthermore, this epitope was analysed by multiple sequence alignments of the peptide with homologous sequences of HIV and SIV p17.
Since the discovery of human immunodeficiency virus (HIV) types 1 (Barr6-Sinoussi et al., 1983) and 2 (Clavel et al., 1986), an increasing number of related simian retroviruses (SIVs) has been isolated from non-human primates [SIVmae, SIVsmm and SlVagm (Daniel et al., 1985; Lowenstine et al., 1986; Ohta et al., 1988); SlVmnd (Tsujimoto et al., 1988); SIVcpz (Peeters et al., 1989)]. Monoclonal antibodies (MAbs) are needed to characterize immunodominant antigenic sites or functional domains on viral components. There has been only one report published about anti-SIVagm MAbs, which described four MAbs against the transmembrane protein of the S~IVagmTYO.1 isolate (Kodama et al., 1988). A small number of anti-p24 MAbs which cross-react with SIVaom have been produced against other immunodeficiency viruses (Minassian et al., 1988; Komatsu et al., 1990). Consequently, murine MAbs against the SIVagmTgo_7 isolate originating from Ethiopia (Ohta et al., 1988) were generated. MOLT-4 cells chronically infected with SIVagmTYO_l, SIVagmTgo.5 or SIVagmTYO.7 (Ohta et al., 1988), SlVmndGB (Tsujimoto et al., 1988) or HIV-1II m (Popovic et al., 1984) were cultured in RPMI 1640 medium supplemented with 10% foetal bovine serum, 100 international units/ml penicillin, 100 ktg/ml streptomycin and 2 mM-glutamine. HIV-2b~n (Schneider et al., 1990) and HIV-2roa (Clavel et al., 1986) were propagated on MOLT-4 clone 8 cells, SIVmac2Sl on HUT 78 cells (Daniel et al., 1985) and
SIVsmmH4 (Lowenstine et al., 1986) on H9 cells. To generate MAbs, BALB/c mice were immunized intraperitoneally with an initial dose of 50 ~tg of heat-inactivated SIVagmTvo.7, followed by booster injections 3 and 5 weeks later. Spleens were removed 3 days thereafter. The conventional technique (Fazekas de St Groth & Scheidegger, 1980) was used to obtain hybrids between P3X63-Ag8.653 myeloma cells and splenocytes. Antibodies were detected in hybridoma supernatants by ELISA using SIVagmTYO.7 antigen coupled to ELISA plates. Hybridoma cultures giving rise to absorbance values threefold greater than the background value were expanded and subcloned three times by limiting dilution. Antibody specificity was examined by Western blotting as described previously (Stahl-Hennig et al., 1990). Five hybridoma lines producing MAbs directed against gag proteins were generated. Antibodies 3B10 and 3Ell recognized p17, and the other three, 1.17.3, 1A7 and 1F6, were directed against p24 (Fig. 1). Analysis of antibody subclass revealed that all antibodies were of the IgG1 subtype. Ascitic fluid was then prepared in BALB/c mice. To investigate the level of conservation of the epitopes recognized by the five MAbs, their cross-reactivity with isolates from all four groups of primate immunodeficiency viruses was investigated. For this purpose, crossreactivity with SlVagmXVO.l,SIVaomTYO_5, SIVmac251, SIVsmmH4, HIV-2~n, HIV-2rod, HIV-1IIIB and SIVmndG8
0001-1020 © 1992 SGM
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Short communication
l
2
3
4
5
6
180K
--116K --84K
--58K --49K
--37K
--27K
Fig. 1. Reactivity of MAbs with SIVagmTVO-7 as determined by Western blot analysis. Virus proteins were separated by SDS-PAGE and assayed for reactivity with SIVag~-seropositive simian serum 5264 (Herchenr6der et al., 1989) diluted 1:50 (lane 1), or ascitic fluids containing MAbs 3BI 0, 3E 11, 1.17.3, 1A7 or 1F6 (lanes 2 to 6) diluted 1:100. Mr markers are indicated to the right and virus-specific polypeptides to the left.
was assessed by Western blotting. Interestingly, the pl7specific antibodies 3B10 and 3Ell bound to all the isolates included in the experiment. The three p24specific MAbs bound to SIVagmTVO.5, SIVmae, SIVsmm, HIV-2~n and HIV-2rod, but failed to recognize the core proteins of HIV-lnm, SIVagmTYO_1 and SIVmn d. Based on genetic sequence analysis, the HIV and SIV strains used in this study can be allocated to four discrete groups, SlVma¢, SIVsmm and HIV-2 constituting one, HIV-1 and SIVCpz constituting another, and SIV~nd and SIV~gm each representing a genetic group. Moreover, SIVagm isolates display an unusually high degree of
intragroup genetic diversity (Allan et al., 1991), greater even than that between different HIV-1 isolates or SIVma e and HIV-2. The anti-p24 MAbs recognized an epitope shared by the HIV-2/SIVsmm/SlVmae group and SIVagmTVO-5 and StVagmTVO.7, but not present o n SIVagrnTYO. 1. This shows the remarkable diversity of SIVagm isolates (Allan et al., 1991). The cross-reactivity of MAbs against p24 from isolates of the HIV-2/SIVma~ group with SIV~m p24 has been reported (Minassian et al., 1988; Komatsu et al., 1990). The cross-reactivity of MAbs against the matrix protein p17 of different HIV and SIV isolates has also been investigated, but only restricted cross-reactivity was demonstrated (Minassian et al., 1988; Niedrig et al., 1988; Komatsu et al., 1990; Sutjipto et al., 1990). In contrast, the anti-pl7 MAbs investigated here show unrestricted cross-reactivity with all four immunodeficiency virus groups mentioned. Since the anti-gag MAbs obviously recognized conserved epitopes, we tried to identify them. Among the SIV~gmTVO isolates only the SIV~gmTVO_I sequence is known and the anti-p24 MAbs did not cross-react with this isolate, therefore synthetic peptides homologous to the SIVmac251/32r t sequence were used for a peptide ELISA. Each peptide corresponded to a sequence of 18 to 21 amino acids (aa), resulting in 13 peptides for p17 and 22 peptides for p24. Each subsequent peptide overlapped with the previous one by 10 aa. Peptides were coated onto high-binding microtitre plates (Greiner) at a concentration of 1 ~tg/well and tested for reactivity with ascitic fluid containing MAbs diluted 1:1000. Two epitopes were defined. MAbs recognizing p17 were directed against p17 peptide 3 (aa 19 to 38) (data not shown). Therefore the epitope of the anti-p 17 MAbs was localized to the N terminus of the matrix protein. Previously, most epitopes have been mapped to the Cterminal half of p17 (Cogniaux et al., 1990; Hinkula et al., 1990; Shang et al., 1991). Only a few epitopes have been mapped to the N terminus of p17 (Papsidero et al., 1989), and the epitope identified by our anti-pl 7 MAbs had not been defined previously. The epitope recognized by the anti-p24 MAbs was localized to p24 peptide 16 (aa 152 to 172) (data not shown). The production of MAbs directed against epitopes overlapping the N or C terminus of peptide 16 has recently been reported (Hinkula et al., 1990; Carpio et al., 1991 ; Niedrig et al., 1991). Since all these MAbs cross-react with HIV-1, in contrast to the anti-p24 MAbs presented here, they probably recognize different epitopes. The position of the epitopes recognized by the anti-p 17 and anti-p24 MAbs could be further defined. The MAbs recognized no adjacent SIVmae peptides and, by common consensus, sequential epitopes are five to eight residues long. Therefore, the binding regions of the epitopes
Short communication
2723
Table 1. Comparison of homologous sequences of HIV and SIV pl7 and p24 with sequences of SIVma~peptides recognized by anti-p l 7 and anti-p24 MAbs p17
Strain SlVmac251 SlVsmmH* HIV-2,o~ HIV-2~n HIV'lmB
SlV~mTWl SlVmndGB
Sequence* IRLRPGGKKKYMLKHVVWAA V .... N .............. ........... R- -- I .... V .......... R--I .... ........... K--I---S ..... N ..... QI--LI--G ---KR .... C-R---LC-CK
p24 Identity Reactivity to SIVmac of anti-pl7 peptide (%) MAb
90 90 85 85 70 60
+ +
+ + + + +
Sequence* C V K Q G P K E P FQ S Y V D R F Y K SL D .................... DI ................... D I ................... DIR ....... RD . . . . . . . TDIR ....... KD . . . . . . . AI D-R ..... A-KD ...... NVM
Identity Reactivity to SIVmac of anti-p24 peptide (%) MAb
95 91 91 71 67 62
+ + + +
* Sequences are taken from the database by Myers et al. (1991). The sequence of SIVa0mxvo-7 is unknown. Areas with high probability for helical secondary structure are underlined.
recognized by the MAbs should be located in the middle of peptide 3 of p17 and peptide 16 of p24, respectively. This conclusion is supported by multiple sequence alignments of the peptides with homologous sequences of HIV and SIV gag proteins. These alignments were performed to analyse sequence motifs conserved among the different isolates which might constitute the putative epitopes. Furthermore, the secondary structures of the homologous aa sequences were predicted by the method of Gamier et al. (1978). Viruses not recognized by the anti-p24 MAbs showed aa substitutions in the central area of the sequence homologous to peptide 16. These mutations caused an alteration of the predicted secondary structure, resulting in helical folding (Table 1). In the case of peptide 3, we found a surprisingly high rate of alterations distributed over the whole sequence of peptide 3. Following the method of Gamier et al. (1978), these aa exchanges should not influence the conformation substantially. The reason for the conformational stability might be that most mutations in the putative antigen-binding site lead to exchanges of similar aa; most mutations are exchanges of leucine, isoleucine and valine. The putative antigen-binding site should have a helical conformation. Thus, in this region, aa exchanges not involving similar residues may affect the other side of the helix. Therefore, they may still allow the MAbs to bind to the relevant residues. However, our study indicates that the matrix protein p17/p18 of all HIV and SIV isolates used in this study contains at least one conserved immunogenic epitope which can be defined serologically. The conserved p17 epitope has now to be characterized using smaller synthetic peptides. Moreover, whether the immunogenic and cross-reacting B cell epitopes on p24 and p17 bear some relevance for the humoral immunity of infected humans and monkeys should be examined. Most interestingly, it has been shown recently that the HIV-1
sequence homologous to peptide 3 contains an epitope recognized by human cytotoxic T lymphocytes (Phillips et al., 1991). We are indebted to Professor M. Hayami for providing SIVagmTYO-I-, TYO-5- and TYO-7-infected MOLT-4 cells, and Professor Dr J.-H. Peters for helpful advice on the immunization scheme and the fusion procedure. We are grateful to Dr F. Guillot for help in carrying out the fusions. Moreover, we thank the Medical Research Council AIDS Directed Programme and Programme EVA (European Vaccine Against AIDS) for kindly providing the synthetic peptides.
References ALLAN, J. S., SHORT, M., TAYLOR, M. E., SU, S., HIRSCH, V. M., JOHNSON, P. R., SHAW,G. M. 8£ HAHN, B. H. (1991). Species-specific diversity among simian immunodeficiency viruses from African green monkeys. Journal o f Virology 65, 2816-2828. BARRI~-SINOUSSI, F., CHERMANN, J. C., REY, F., NUGEYRE, i . T., CHAMARET, S., GRUEST, J., AXLER-BLIN, C., BRUN-VEZINET, F., ROUZIOUX,C., ROZENBAUM,W. & MONTAGNIER,L. (1983). Isolation of a T-lymphotropic retrovirus from a patient at risk for AIDS. Science 220, 868-871. CARPIO, E., DUARTE, C., HINKULA, J., BROLIOEN, P.-A., ROSEN, J., CAMPAL, A., GAVILONDO, J., WAHREN, B. 8£ JONDAL, M. (1991). Monoclonal antibodies to conserved regions of the major core protein (gag24) of HIV-1 and HIV-2. AIDS Research and Human Retroviruses 7, 97-101. CLAVEL, F., GUETARD, D., BRUN-VEZINET, F., CHAMARET,S., REY, M. A., SANTOS-FERREIRA, O., LAURENT, A. G., DAUQUET, C., KATLAMA, C., ROUZIOUX, C., KLATZMANN, D., CHAMPALIMAUD, J. L. d~ MONTAGNIER,L. (1986). Isolation of a new human retrovirus from West-African patients with AIDS. Science 223, 343-346. COGN1AUX, J., DE SCHEPPER, N., BLONDIAU, M.-L., CORNET, B., HORAL, P. & VAHLE, A. (1990). Characterization of monoclonal antibodies against the p17 core protein of the human immunodeficiency virus 1. Journal o f Immunological Methods 128, 165-175. DANIEL, M. D., LET'qlN, N. L., KING, N. W., KANNAGI,M., SEHGAL, P. K., HUNT, R. D., KANKI, P. J., ESSEX, i . & DESROSIERS,R. C. (1985). Isolation of a T-cell tropic HTLVIII-Iike retrovirus from macaques. Science 228, 1201-1204. FAZEKASDE ST GROTH, S. & SCHEIDEGGER, n . (1980). Production of monoclonal antibodies: strategy and tactics. Journal oflmmunalogical Methods 35, 1-21.
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Short communication
GARNIER,J., OSGUTHORPE,D. J. & ROBSON,B. (1978). Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. Journalof Molecular Biology 120, 97-120. HERCHENRfDER, O., STAHL-HENNIG,C., LOKE, W., SCHNEIDER,J., SCHULZE, G., HARTMA~Cr~,H., SCHMIDT, H., TENNER-RACz, C., RACZ, P., HAYAm, M., KELLmEX, J. C. & HUNSMANN,G. (1989). Experimental infection of rhesus monkeys with SIV isolated from African green monkeys (SIVagm). lntervirology 30, 66-72." HINKULA,1., ROSEN,J., SUNDQVIST,V.-A., STIGBRAND,T. & WAHREN, B. (1990). Epitope mapping of the HIV-1 gag region with monoclonal antibodies. Molecular Immunology 27, 395-403. KODAMA, T., OHTA, Y., MASUDA,T., ISHIKAWA,K., TSUJIMOTO,H., ISAHAKt~,M. & HAYAMI,M. (1988). Production and characterization of monoclonal antibodies specific for the transmembrane protein of simian immunodeficiency virus from the African green monkey. Journal of Virology 62, 4782-4785. KOMATSU,H., TOZAWA,H., KAWAMURA,M., KODAMA,T. & HAYAMI, M. (1990). Multiple antigenic epitopes expressed on gag proteins, p26 and plS, of a human immunodeficiency virus (HIV) type 2 as defined with a libary of monoclonal antibodies. AIDS Research and Human Retrovirt~es 6, 871-881. LOWENSTINE, L. J., PEDERSEN, N. C., HIGGIN$, J., PALLIS, K. C., UYEDA, A., MARX, P., LERCHE, N. W., MUNN, R. J. & GARDNER, M. B. (1986). Seroepidemiologic survey of captive Old-World primates for antibodies to human and simian retroviruses, and isolation of a lentivirus from sooty mangabeys (Cercocebus atys). International Journal of Cancer 38, 563-574. MINASSIAN,A. A., KALYANARAMAN,V. S., GALLO, R. C. & POPOVIC, M. (1988). Monoclonal antibodies against human immunodeficiency virus (HIV) type 2 core proteins: cross-reactivities with HIV type 1 and simian immunodeficiency virus. Proceedings of the National Academy of Sciences, U.S.A. 85, 6939-6943. MYERS, G., BERZOFSKY,J. A., RABSON,A. B., SMITH,T. F. & WONGSTAAL, F. (editors) (1991). Database of Human Retroviruses and AIDS. Los Alamos: Los Alamos National Laboratory. NIEDRIG, M., RABANUS,J. P., L'AGE STEHR,J., GELDERBLOM,H. R. & PAULI, G. (1988). Monoclonal antibodies directed against human immunodeficiency virus (HIV) gag proteins with specificity for conserved epitopes in HIV-1, HIV-2 and simian immunodeficiency virus. Journal of General Virology 69, 2109-2114. N1EDRIG, M., HINKULA,J., HARTHUS,H.-P., BR6KER, M., HOPP, L., PAULI, G. & WAHXEN, B. (1991). Characterization of routine monoclonal antibodies against the core proteins of human immunodeficiency virus types 1 and 2. Journal of Virology 65, 4529-4533. OHTA, Y., MASUDA,T., TSUJIMOTO,H., ISHIKAWA,K., KODAMA,T., MORIKAWA, S., NAKAI, M., HONJO, S. & HAYAMI, M. (1988). Isolation of simian immunodeficiency virus from African green
monkeys and seroepidemiologicai survey of the virus in various nonhuman primates. International Journal of Cancer 41, 115-122. PAPSIDERO, L. D., SHEU, M. & RUSCEa'n, F. W. 0989). Human immunodeficiency virus type l-neutralizing monoclonal antibodies which react with pl7 core protein: characterization and epitope mapping. Journal of Virology 63, 267-272. PEETERS,M., HONORE,C., HUET, T., BEDJABAGA,L., OSSARI,S., BUSSI, P., COOPER, R. W. & DELXPOXTE,E. (1989). Isolation and partial characterization of an HIV-related virus occurring naturally in chimpanzees in Gabon. AIDS 3, 625-630. PmLLIPS, R. E., ROWLAND-JoNEs,S., NlXON, D. F., GOTCH, F. M., EDWARDS,J. P., OGUNLESI,A. O., ELVlN, J. G., ROTHBARD,J. A., BANGHAM,C. R. M., RlZZA, C. R. & McMICHAEL, A. J. (1991). Human immunodeficiency virus genetic variation that can escape cytotoxic T cell recognition. Nature, london 354, 453-459. POPOVlC,M., SARNGADHARAN,M. G., READ,E. & GALLO,R. C. (1984). Detection, isolation, and continous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224, 497-499. SCHNEIDER,J., LOKE, W., KIRCHHOFF,F., JUNG, R., JURKIEWlCZ,E., STAHL-HENNIG, S., NICK, S., KLEMM, E., JENTSCH, K.-D. & HUNSMANN,G. (1990). Isolation and characterization of HiV-2ben obtained from a patient with predominantly neurological defects. AIDS 4, 455-457. SHANG,F., HUANG,H., REVESZ,K., CHEN, H.-C., HERZ, R. & PINTER, A. (1991). Characterization of monoclonal antibodies against the human immunodeficiency virus matrix protein, pl70a0: identification ofepitopes exposed at the surfaces of infected cells. Journalof Virology 65, 4798-4804. STAHL-HENNIG,C., HERCHENRODER,O., NICK, S., EVERS,M., STILLESIEGENER, i . , JENTSCH,K.-D., KIRCHHOFF,F., TOLLE, T., GATESMA~, T. J., LOKE, W. & HUNSlANN, G. (1990). Experimental infection of macaques with HIV-2ben, a novel HIV-2 isolate. AIDS 4, 611-617. SUTJIPTO, S., KODAMA, T., YEE, J., GETTIE, A., JENNINGS, M., DESROSIERS, R. C. & MARX, P. A. (1990). Characterization of monocional antibodies that distinguish simian immunodeficiency virus isolates from each other and from human immunodeficiency virus types 1 and 2. Journal of General Virology 71, 247-249. TSUJIMOTO,H., COOPER, R. W., KODAMA,T., FUKASAWA,M., MIURA, T., OtrrA, Y., ISmKAWA,K., NAKALM., FROST,E., ROELANTS,G. E., ROFFI, J. & HAYAMI,M. (1988). Isolation and characterization of simian immunodeficiency virus from mandrills in Africa and its relationship to other human and simian immunodeficiency viruses. Journal of Virology 62, 4044-4050.
(Received 6 April 1992; Accepted 26 June 1992)
