Neutralization Epitopes on Poliovirus Type 3 Particles: an Analysis Using Monoclonal Antibodies

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1 J.-gen. Virol. (1984), 65, Printed in Great Britain 197 Key words: poliovirus type 3/monoclonal Abs/neutralization/immunoblot Neutralization Epitopes on Poliovirus Type 3 Particles: an Analysis Using Monoclonal Antibodies By MORAG FERGUSON,* P. D. MINOR, D. I. MAGRATH, QuI YI-HUA,t M. SPITZ AND G. C. SCHILD National Institute for Biological Standards and Control, Holly Hill, Hampstead, London NW3 6RB, U.K. (Accepted 10 October 1983) SUMMARY Monoclonal antibodies to poliovirus type 3 secreted by 51 hybridoma cell clones have been characterized in terms of (i) virus-neutralizing properties, (ii) reactivity in antigen-blocking tests with infectious, 155S ('D' antigen) and empty 80S ('C' antigen) poliovirus particles and (iii) reactivity in immunoblot tests with the isolated protein components of the poliovirus capsid. The antibodies could be separated into three groups on the basis of their reactivities with 'D' and 'C' antigens. All antibodies that reacted with both 'D' and 'C' antigen had potent neutralizing activity. Only a proportion of antibodies that reacted uniquely with 'D' antigen possessed neutralizing activity. Unexpectedly, one of 24 'C' antigen-specific antibodies inhibited virus growth. None of the antibodies that possessed virus-neutralizing activity reacted with isolated poliovirus capsid proteins, although the majority of these have been shown in previous studies to be specific for VP1 on intact virus particles. These findings suggest that antigenic determinants involved in virus neutralization do not survive the denaturing conditions required for the isolation of poliovirus capsid proteins and consequently are likely to be specified by the structural conformation of VP1 rather than by amino acid sequence alone. However, several of the antibodies which bound uniquely to 'C' antigen reacted in immunoblot tests, five with VPt and one with VP3. Some of these antibodies also possessed heterotypic reactivity with the corresponding capsid proteins separated from other poliovirus types. Although many details of the replication, assembly and structure of poliovirus have been reported, little is known about the antigenic composition of the virus (Putnak & Phillips, 1981). Serological examination of the virus present in tissue culture fluid has revealed two major populations of particles, infectious virus which sediments at 155S and empty, 80S, capsids (Mayer et al., 1957; Minor et al., 1980). These particles exist in distinct antigenic forms which have been termed 'D' and 'C' antigen respectively (Mayer et al., 1957). In order to characterize the antigenic determinants on infectious and empty particles we have developed hybridoma cell lines secreting monoclonal antibodies to poliovirus. The antibodies were tested for virusneutralizing activity and for their ability to bind to "D' and 'C' antigens in antigen-blocking tests. In addition, their reactivity with isolated poliovirus capsid proteins in immunoblot tests was examined. Mice and rats were immunized with several strains of poliovirus type 3 (Table 1) and hybridoma cell lines prepared by fusion of mouse spleen cells with P3. X63-Ag8 cells and rat spleen cells with rat Y3Agl.2.3 cells (Minor et al., 1982). Cell hybrids were screened for secretion of antibodies to poliovirus type 3 in radioimmunoassay or single radial diffusion (SRD) antigen-blocking tests (Ferguson et al., 1982) and secretor cell lines were cloned. Fifty-one stable, cloned cell lines secreting antibody to poliovirus type 3 were obtained. The antibody was characterized for immunoglobulin class by sedimentation in sucrose gradients and the migration t Present address: National Vaccine and Serum Institute, Beijing, China /84/ $ SGM

2 198 Short communication Table 1. Characteristics of representative monoclonal antibodies to poliovirus type 3 SRD antigen Immunizing Hybridoma Particle r - virus* designation specificity 'D--antigen 'C' antigen titret specificity class:~ blocking titre - *- - a Virus neutralization Capsid protein Immunoglobulin NIBp 134 'D' < VPI IgM NIBp 135 'D' 1000 <2 < 10? IgM NIBp 139 'D' <2 < 10? IgM NIBp 165 'D' 300 <2 256 VP1 IgM Leon/USA/39 NIBp 194 "D' 1000 <2 150 VPt IgG 10/USA/57 Sabin vacc. NIBy NIBy 'D'+'C' 'D'+'C' VPI VPI IgM IgM Leon/USA/39 NIBp 175 'D'+'C' VPI IgG 10/USA/57 NIBy 'D'+'C' VPI IgG Sabin vacc. NIBy 'C' <2 50 <10 VP1H IgG NIBp 131 'C' < <10 9 IgM Leon/USA/39 NIBp 176 'C' <2 300 < 10 VP311 IgM Sabin vacc. NIBp 196 'C' <2 300 < 10 VP1 II IgG Sabin vacc. NIBp 198 'C' < VPI IgM * is a virulent revertant of Sabin type 3 vaccine (vacc.) virus isolated from a fatal case of poliomyelitis. For origins of viruses, see Ferguson et al. (1982). ~" Dilution of antibody just neutralizing 100 TCIDs0 virus in microtitre assays. :~ Determined by migration of 35S-labelled immunoglobulin chains in polyacrylamide gel electrophoresis. Determined by sequencing RNA from resistant mutants (Minor et al., 1983; Evans et al., 1983). II Immunoblot reactivity determined by the method of Thorpe et al. (1982). P. D. Minor, unpublished observations. rates of the immunoglobulin chains during polyacrylamide gel electrophoresis. Thirty-one of the antibodies were of the IgG class and 20 were IgM. All the antibodies were tested in SRD antigen-blocking tests to determine their reactivities with 'D' and 'C' antigens of poliovirus 3. The method used was a modification of the autoradiographic SRD method of Schild et al. (1980). Briefly, [3 s S]methionine-labelled 155S 'D' or 80S 'C' peaks of poliovirus antigen from sucrose gradients were mixed with the test monoclonal antibody before being added to wells in agarose gels containing low concentrations of hyperimmune anti-poliovirus type 3 serum. Diffusion of radiolabeued antigen in the gel after 24 to 48 h was detected by autoradiography. Test antibody that reacts with 'D' and/or 'C' antigen inhibits its diffusion into the gel compared with control antigen treated with phosphatebuffered saline (PBS). The antigen-blocking titre was assessed as the dilution of monoclonal antibody that reduced the zone size by 75 ~ in comparison with zones produced with control antigen mixed with PBS. Antigen-blocking titres of ascitic fluids prepared from representative hybridomas are shown in Table 1. Three categories of reactivity of the antibodies were seen: those with SRD blocking activity exclusively for 'D' antigen (e.g. NIBp 134, NIBp 194), those blocking exclusively 'C' antigen (e.g. NIBp 131, NIBp 198) and those blocking both 'D' and 'C' antigen (e.g. NIBp 175, NIBy ). Eight of the 51 monoclonal antibodies blocked 'D' and 'C' antigens, indicating that they reacted with determinant(s) common to both infectious and empty particles. Of the remaining 43 antibodies, 19 blocked exclusively 'D' antigen and 24 blocked exclusively 'C' antigen. Similar results have been obtained in immunoprecipitation studies by Brionen et al. (1982) with type 1 poliovirus. The monoclonal antibodies were assayed for neutralizing activity against a range of type 3 virus strains by the method of D6m6k & Magrath (1979). Microtitre assays were done in HEp-2c cells using 100 TCIDs0 as the challenge dose of virus. The neutralization titres of the representative group of viruses are shown in Table 1. Efficient neutralization was exhibited by antibodies of each class, IgG and IgM. In addition, antibodies of each immunoglobulin class exhibited 'D', 'C' or 'D' + 'C' antigen-blocking activity. A summary of the neutralization activities against poliovirus 3 (Sabin vaccine strain Leon 12al b) for all 51 antibodies is shown in Table 2.

3 Short communication Table 2. Neutralization titres of monoelonal antibodies against poliovirus type 3 No. of antibodies with virus-neutralizing 'D'+'C' antigen No. of activity at stated titre (log,0)$ specificity* antibodies r ~ ~ Immunoblot of antibody tested# <1 1 <2 2 <3 3 <4 >~4 reactivity 'D' + 'C-reactive 'D'-specific l l 0 'C'-specific VPI 1 VP3 Total * Based on autoradiographic SRD antigen-blocking assays. t Each antibody was obtained from an independent hybridoma clone in fusions involving the lymphocytes of mice or rats immunized with poliovirus type 3 strains (Minor et al., 1982). $ Dilution of antibody just neutralizing 100 TCIDs0 of virus in microtitre assays. 199 All antibodies that reacted with the determinant(s) common to 'D' and 'C' particles possessed neutralizing activity at titres >t 1:100, whereas only 12 of the 19 'D'-specific antibodies had detectable neutralizing activity. Where neutralizing activity was detected it was often at low titre. It is of interest that not all antibodies which bind to infectious virus, as indicated by their blocking titres with 'D' antigen, possessed neutralizing activity. This suggests that virus neutralization involves critical antigenic sites on the infectious particle and is not a necessary consequence of binding of antibodies to any antigenic site on the virus surface. All neutralization was poliovirus type 3-specific, suggesting that antigenic sites common to the other poliovirus serotypes are not involved in neutralization. Surprisingly, one antibody that reacted exclusively with 'C' antigen in SRD antigen-blocking tests appeared to neutralize virus infectivity in the standard neutralization test. The mechanism of neutralization of this antibody is being investigated further. Antibodies with virus neutralization activity which do not react with free infectious virus may nevertheless react with particles after attachment to cells or at a late stage in replication, during maturation or release. It is of interest that antibodies reacting with poliovirus 'C' but not 'D' are the most abundant antibodies to poliovirus detected in some collections of human sera (G. C. Schild, J. M. Wood & D. I. Magrath, unpublished data) and their relevance to immunity requires further study. Antibodies that bound to the determinants common to 'D' and 'C' particles possessed neutralizing activity against a wide range of poliovirus type 3 strains and, in some cases, against all of the 30 strains tested. In contrast, 'D'-specific antibodies often reacted with a narrow range of strains in neutralization and antigen-blocking tests. Thus, some antigenic determinants involved in neutralization appear to be highly conserved within a serotype whereas others are unique to individual strains. Several antibodies reacted only with Sabin vaccine virus or with strains closely related to Sabin vaccine virus as evidenced by their T1 oligonucleotide maps (Ferguson et al., 1982). Such monoclonal antibodies will be of use as diagnostic reagents for the identification of vaccine-derived viruses. The reactivity of the 51 monoclonal antibodies with separated poliovirus capsid proteins was investigated using the method described by Thorpe et al. (1982). Briefly, virus was treated with 2~ (w/v) SDS, 2~ (w/v) 2-mercaptoethanol, and 2 M-urea for 1 rain at 100 C. The denatured proteins were separated by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) and then transferred electrophoretically onto nitrocellulose paper. Nitrocellulose strips were treated with monoclonal antibody and antibodies binding specifically to the virus polypeptides were detected by application of 125 I-labelled anti-mouse F(ab')z fragments followed by autoradiography. The results are shown in Table 2. None of the antibodies that were specific for 'D' antigen, or those which reacted with antigenic determinants common to 'D' and 'C' antigen, were found to bind to separated polypeptides. This suggests that the antigenic sites present on infectious particles, including those responsible for virus neutralization, are complex and dependent on the threedimensional conformation of viral polypeptide (VP 1) at the virus surface. In contrast, six of the 'C'-specific antibodies reacted with denatured proteins, five with VP 1 and one with VP3. These

4 200 Short communication Table 3. Polypeptide specificity of non-neutralizing, "C" antigen-specific monoclonal antibodies Type 3 : Type 2: Type 1 : immunoblot immunoblot immunoblot 'C' antigen- reactivity 'C' antigen- reactivity 'C' antigen- reactivity Monoclonal antibody blocking titre z, rvpi VP2 VP3 ~ blocking titre "~ rvp1 VP2 VP3 ~ blockingtitre CVPl VP2~' VP; NIBy < < NIBy < < NIBp < < NIBp < NIBp < NIBp < < determinants are therefore likely to be specified simply by amino acid sequence rather than by tertiary configuration of the protein. The six antibodies found positive in immunoblot tests with poliovirus 3 were also examined in tests with poliovirus I and 2 capsid proteins and the results are shown in Table 3. Monoclonal antibodies NIBp 196 and NIBp 203 reacted with VP1 of poliovirus types 2 and 3, whereas NIBp 118, NIBy and NIBy reacted only with VP 1 of the homotypic virus. Similar intertypic reactions were found in antigen-blocking tests with these monoclonal antibodies. Although antibody NIBp 176 reacted with VP3 of all three poliovirus types in immunoblot experiments, it reacted only with poliovirus type 3 'C' antigen in antigen-blocking tests, suggesting that the antigenic determinant against which it is directed is in a different conformation in poliovirus types 1 and 2. Polyclonal antisera to poliovirus that cross-react with all three poliovirus types in immunobtot (Thorpe et al., 1982) and immunoprecipitation studies (Blondel et al., 1982) have been described. This is, however, the first report of monoclonal antibodies recognizing common determinants in different poliovirus serotypes. The finding of shared, sequence-specified antigenic determinants between poliovirus types is consistent with the observation of high levels (approx. 90~) of predicted amino acid sequence homology between polioviruses of types 1 and 3 (J. W. Almond & G. Stanway, unpublished results). Monoclonal antibodies are potent reagents for use in studies to locate and identify the antigenic sites on virus capsid proteins which are involved in virus neutralization (Minor et al., 1983; Evans et al., 1983). None of the 21 monoclonal antibodies described here which neutralized virus infectivity bound to isolated VP1 which had been denatured during its preparation. However, genetic and molecular studies of a large collection of poliovirus 3 mutants selected for resistance to individual neutralizing antibodies, 16 of which are described in this report, indicate that all 16 antibodies bind to the same eight amino acid region of VP1 (Minor et al., 1983; Evans et al., 1983). Examples of such antibodies are indicated in Table 1. Resistant mutant viruses with identical point mutations within this antigenic site could be isolated by selection in the presence of either 'D'-specific or 'D'- and 'C'-reactive monoclonal antibodies (Evans et al., 1983). Thus, neutralizing monoclonal antibodies specific for 'D' and 'C' antigen, or 'D' antigen appear to react with the same antigenic region of VP1. In addition, some of the viruses which have point mutations in this region are resistant to NIBp 198, the 'C'-specific antibody that neutralizes the parental virus, suggesting that this antibody also reacts with the same antigenic site on VP1 as the other neutralizing antibodies. Non-neutralizing, 'C'- or 'D'- specific antibodies may be directed against other sites, as for example NIBp 176 which reacts with VP3. Emini et al. (1982) have shown that neutralizing monoclonal antibodies for poliovirus"type 1 react with epitopes in VP1. Blondel et al. (1983) have described a neutralizing monoclonal antibody which immunoprecipitates both 'D' and 'C' antigen of poliovirus type 1 and also isolated VP1. Nevertheless, a further 'D'-specific antigen-neutralizing antibody against poliovirus type 1 with virus-neutralizing activity failed to react with separated homologous poliovirus type 1 capsid polypeptides. Similarly, several neutralizing monoclonal antibodies against foot-and-mouth disease virus failed to react with the separated capsid polypeptides of

5 Short communication 201 the homologous virus (Meloen et al., 1983). With the exception of the single 'D'- and 'C'-specific antibody of Blondel et al. (1983), these findings are in accordance with the studies on poliovirus type 3 reported here. The relative infrequency with which picornavirus-neutralizing monoclonal antibodies have been reported which react with separated capsid proteins implies that the antigenic sites which are involved in neutralization are determined largely by the conformational arrangement of the capsid proteins in the intact virion. Although studies with monoclonal antibodies are of value in locating and identifying the antigenic sites involved in virus neutralization, critical information on the three-dimensional structure of such sites at the virus surface must await the application of more precise analytical methods, such as X-ray crystallography. We are grateful for the excellent technical assistance of Susan Fraser, Ann John, Philip Yates and to Phyllis Young for secretarial assistance. REFERENCES BLONDEL, B., CRAINIC, R., AKACEM, O., BRUNEAU, P., GIRARD, M. & HORODNICEANU, F. (]982). Evidence for common intertypic antigenic determinants on poliovirus capsid polypeptides. Virology 123, BLONDEL, B., AKACEM, O., CRAINIC, R., COUILLIN, P. & HORODNICEANU, F. (1983). Detection by monoclonal antibodies of an antigenic determinant critical for poliovirus neutralization present on VP1 and on heat inactivated virions. Virology 126, BRIONEN, P., SIJENS, R. J., VRIJSEN, R., ROMBAUT, B., THOMAS, A. A., JACKERS, A. & BOEYE, A. (1982). Hybridoma antibodies to poliovirus N and H antigens. Archives of Virology 74, D6M6K, I. & MAGRATtt, D. I. (1979). Guide to poliovirus isolation and serological techniques for poliomyelitis surveillance. WHO Offset Publication No. 46. EMINI, E. A., JAMESON, B. A., LEWIS, A. J., LARSEN, G. R. & WIMMER, E. (1982). Poliovirus neutralization epitopes: analysis and localization with monoclonal antibodies. Journal of Virology 43, EVANS, D. M. A., MINOR, P. D., SCHILD, G. C. & ALMOND, J. W. (1983). Critical role of an eight amino acid sequence of VP1 in the neutralization of poliovirus type 3. Nature, London 304, , FEROUSON, M., QUI YI-HUA, MINOR, P. D., MAGRATH, D. I., SPITZ, M. & SCHILD, G. C. (1982). Monoclonal antibodies specific for the Sabin vaccine strain of poliovirus 3. Lancet ii, LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, London 227, MAYER, M. M., RAPP, H. J., ROIZMAN, B., KLEIN, S. W., COWAN, K. M., LUKERY, D., SCHWERDT, C. E., SCHAFFER, F. L. & CHARNEY, J. J. (1957). The purification of poliomyelitis virus as studied by complement fixation. Journal of Immunology 78, MELOEN, R. H., BRIAIRE, J., WOORTMEYER, R. J. & VAN ZAANE, D. (1983). The main antigenic determinant detected by neutralizing monoclonal antibodies on the intact foot-and-mouth disease particle is absent from isolated VP 1. Journal of General Virology 64, 3-8. MINOR, P. D., SCHILD, G. C., WOOD, J. M. & DANDAWATE, C. N. (1980). The preparation of specific immune sera against type 3 poliovirus D-antigen and C-antigen and the demonstration of two C-antigenic components in vaccine strain populations. Journal of General Virology 51, MINOR, P. D., SCHILD, G. C., FERGUSON, M., MACKAY, A., MAGRATH, D. I., JOHN, A., YATES, P. J. & SPITZ, M. (1982). Genetic and antigenic variation in type 3 polioviruses: characterization of strains by monoclonal antibodies and T1 oligonucleotide mapping. Journal of General Virology 61, M/NOR, P. D., SCHILD, G. C., BOOTMAN, J., EVANS, D. M. A., FERGUSON, M., REEVE, P., SPITZ, M., STANWAY, G,, CANN, A. J., HAUPTMANN, R., CLARKE, L. D., MOUNTFORD, R. C. & ALMOND, J. W. (1983). Location and primary structure of a major antigenic site for poliovirus neutralisation. Nature, London 301, PUTNAK, J. g. & PHILLIPS, B. A. (1981). Picornaviral structure and assembly. Microbiological Reviews 45, SCHILD, G. C., WOOD, J. M., MINOR, P. D.~ DANDAWATE, C. N. & MAGRATH, D. I. (1980). Immunoassay of poliovirus antigens by single-radial-diffusion : development and characteristics of a sensitive autoradiographic zone size enhancement (ZE) technique. Journal of General Virology 51, THORPE, R., MINOR, P. D., MACKAY, A., SCHILD, G. C. & SPITZ, M. (1982). Immunochemical studies of polioviruses: identification of immunoreactive virus capsid polypeptides. Journal of General Virology 63, (Received 22 July 1983)