[Advances in Experimental Medicine and Biology] Coronaviruses and their Diseases Volume 276 __ Background Paper Map

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[Advances in Experimental Medicine and Biology] Coronaviruses and their Diseases Volume 276 __ Background Paper Map

BACKGROUND PAPER MAPPING EPlTOPES ON CORONA VIRUS GLYCOPROTEINS Hubert Laude Institut National de la Recherche Agronomiq

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BACKGROUND PAPER MAPPING EPlTOPES ON CORONA VIRUS GLYCOPROTEINS Hubert Laude Institut National de la Recherche Agronomique Station de Recherches de Yirologie et d'Immunologie Moleculaires 78350 Jouy-en-Josas France INTRODUCTION The purpose of this paper is to summarise the state of coronavirus epitope research, with an emphasis laid on the most recent achievements, i.e. the correlation between antigenicity and polypeptide primary structure. The antigenic properties of coronaviruses reside mainly in their envelope glycoproteins: the S (spike) protein, a high molecular weight polypeptide which constitutes the petal-shaped peplomers and the M (membrane) protein, a smaller integral membrane polypeptide. Some coronaviruses possess an additional surface glycoprotein, the haemagglutinin-esterase (HE) protein. PEPLOMER PROTEIN S Analysis of the predicted amino acid sequences now established for 5 coronaviruses (IBY, MHY, TGEY, FIPY and BCY) has led to a model in which the protein is divided into 2 domains: the amino-terminal half, which is assumed to form the globular part of the peplomer, and the carboxy-terminal half, which is assumed to form an elongated structure, the stalk of the peplomer, and also contains a putative transmembrane anchoring region near the carboxy terminus. Both parts bear numerous asparagine-linked complex glycan sidechains, which may modulate their antigenicity. The sequences of the amino-terminal halves exhibit very little conservation, whereas homologies averaging 30% are observed in the carboxy-terminal halves. Several coronaviruses, including mY, MHY and BCY have a S protein which is cleaved approximately in the middle, thus defining an amino-terminal subunit (SI) and a carboxy-terminal subunit (S2). Several major antigenic sites have been identified on the S proteins of MHY, TGEY and my by competitive binding experiments; a number of these sites could be oriented, if not precisely localised, on the primary structure of the molecule. MHY S Protein Recombinant viruses generated in vivo by RNA-RNA recombination which contain approximately the 5' two-thirds of the MHY-JHM peplomer gene and the 3' one-third of the MHY-A59 peplomer gene have lost their reactivity to monoclonal antibodies (MAbs) defming the sites designated A and B on the JHM S protein. It was inferred that both sites are probably contained within the carboxy-terminal one-third of the protein (Makino et al., 1987). However the positions of the crossover sites have only been mapped by ologonucleotide finger-printing and require confirmation by nucleotide sequencing. Site A

Coronaviruses and Their Diseases Edited by D. Cavanagh and T.D.K. Brown Plenum Press, New York, 1990

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was defined by neutralising or non-neutralising MAbs and site B by neutralising MAbs; site A MAbs also inhibited cell fusion. Both sites contained epitopes which exhibited a natural variation from strain to strain and allowed the generation of escape mutants. In addition site B escape mutants showed markedly altered neurovirulence when inoculated into mice. Reactivity analysis of selected or spontaneous variants suggested that sites A and and B are topologically related (Fleming et al., 1986; Taguchi and Fleming, 1989). Three antigenic sites delineated by other groups on the JHM S protein, designated B and C (Talbot et al., 1984) and Ba (Wege et al., 1984), apparently possess the same characteristics with respect to neutralisation, fusion inhibition, variation and generation of attenuated mutants (Dalziel et al., 1986; Wege et al., 1988). It is therefore tempting to speculate that sites B, B-C and Ba are topographically related to the above-mentioned site A. At least part of the latter sites might be conformation-dependent, as was the case for sites B and C (Talbot et al., 1984); this would explain why they could not be mapped using a bacterial expression system. Expression of fragments of a protein as a fusion polypeptide using a procaryotic vector such as pEX is a classical approach to the mapping of antigenic determinants. Most probably, however, this method mainly detects epitopes that are conformation-independent. Fine mapping of a linear epitope on JHM S protein has been achieved by combining pEX expression and peptide scanning (Lutyes et al., 1989). This epitope belongs to the site designated A (Talbot et al., 1984); this site is: i) recognised by strongly neutralising and fusion-inhibiting MAbs, ii) resistant to denaturation, iii) highly conserved among MHV strains, and iv)possibly crucial for virus infectivity (failure to select escape mutants). The antibody binding site stretched from residue 848 to 856, i.e. about 130 residues from the Nterminus of subunit S2. This 9 amino acid sequence is markedly hydrophobic and located immediately upstream of a region largely conserved in the coronavirus peplomer protein. Finally, a synthetic decapeptide corresponding to the sequence 993-1002 of the JHM S protein, which was delineated using a surface probability algorithm, elicited a high level of neutralising antibodies and protected mice against a lethal challenge (Talbot et al., 1989). Its position is near the middle of the S2 subunit, between two potentially alpha helical regions which have been suggested to frame the peplomer stalk. Whether this region corresponds to a natural antigenic site remains to be determined. The above observations support the proposal that the carboxy-terminal S2 subunit of MHV i) contains distinct major antigenic sites, ii) bears neutralisation epitopes, strain-specific epitopes and essential virulence determinants. Surprisingly, there is date, no published evidence of MAb binding to the SI subunit of MHV. However studies based on the localisation of point mutants or deletions conferring neutralisation-resistance have clearly established the existence of major neutralisation sites on S 1 (see articles by Gallagher et al and Parker et al in Chapter of this volume).

mv SProtein In striking contrast, consistent evidence has been obtained that the S 1 subunit of IBV is both the major inducer of neutralising MAbs and the major site of antigenic variation. Isolated SI reacted with strongly neutralising MAbs (Mockett et al., 1984) and elicited neutralising antisera (Cavanagh et aI., 1986). By comparing the amino acid sequences of different mv strains it was found that most substitutions had occurred in the SI subunit; SI proteins can differ in upto 50% of their amino acids (Niesters et al., 1986, Binns et aI., 1986, Kusters et al., 1989). In particular, sequence alignments allowed the identification in Massachusetts serotype strains of two regions of clustered substitutions, the hypervariable regions, HVRI (56-69) and HVR2 (117-133), which were suggested to contain neutralisation epitopes (Niesters et al., 1986). Indeed a mutation which prevented neutralisation by two MAbs was localised in HVRI by direct sequencing of the genomic RNA of the relevant escape mutants (Cavanagh et al., 1988). Whereas most neutralising MAbs investigated so far are reported to be directed against SI, the S2 subunit has been shown to react with MAbs having a weak neutralising activity (Koch et al., 1986) Several overlapping conformation-independent epitopes recognised by such MAbs have been precisely localised in the 30 N-terminal residues of S2 through expression of random fragments of S using the pEX system (Lenstra et al., 1989). At least 140

some of these epitopes are conserved in several serotypes as judged by reactivity of the fusion protein with different antisera. In contrast none of the pEX expression products containing fragments of S 1 reacted with MAbs suggesting that this subunit contains conformation-dependent epitopes. The above findings lend support to the view that the major neutralisation domain ofIBV resides on the amino-terminal SI subunit and is composed mainly of serotype specific and conformation-dependent epitopes. Whether distinct regions of S 1 are involved remains to be established. In addition at least one immunodominant site, defined by weakly-neutralising and broadly reactive MAbs, is present on the carboxy-terminal S2 subunit. TGEV S Protein Detailed epitope maps have defined 4 to 5 major antigenic sites on the TGEV S protein (Delmas et at .. , 1986, Correa et at., 1988). Most of the determinants critical for neutralisation were highly conserved among the strains and susceptible to denaturation (Laude et at., 1986, Jimenez et at., 1986). Recent data indicate that all of these sites are located in the amino-terminal half of the protein (see articles by Delmas et at. and Enjuanes et at. in this volume). BCV S Protein In the case of the BCV S protein all the neutralising antibodies characterised so far have been directed against gpl00 which has recently been found to correspond to the SI subunit of the S protein (Deregt & Babiuk, 1987, Vautherot et at., this volume). These data are consistent with recent observations on the role of S 1 in the neutralisation of MHV (see above) to which BCV is closely related (see Chapter 10 of this volume). OTHER GLYCOPR01EINS MProtein The M glycoprotein is largely buried within the virus membrane or closely associated with its inner surface. Two adjacent epitopes recognised by polyclonal antibodies are located in the 15 carboxy-terminal residues of the MHV M protein as revealed by studies of fragments expressed in the pEX system. Proteolytic digestion abolished the binding of one MAb, presumably directed against the C-terminal region (Tooze & Stanley, 1986). Three overlapping epitopes have been mapped in the first 30 N-terminal residues of TGEV M protein, through localisation of amino acid substitutions in mutants resistant to complementmediated neutralisation (Laude et ai., to be published). Thus in agreement with its predicted relationship with the virus envelope, the major antigenic sites of the M protein correspond to the short protruding hydrophilic domains located at each extremity of the molecule. HE Protein The HE protein, associated with a coronavirus subgroup including BCV, HEV and HCV -OC43, is responsible for the strong haemagglutinating activity of these viruses and is also able to elicit neutralising antibodies. Competition experiments between anti-BCV MAbs have defined at at least one major neutralisation site, not yet correlated with HE primary structure (Deregt & Babiuk, 1987). CONCLUDING REMARKS Despite significant advances in the physical mapping of B cell epitopes on coronavirus glycoproteins, the emerging picture is still incomplete. The published data mainly concern the peplomer proteins ofMHV and IBV. However substantial information about the TGEV peplomer protein has been presented during the symposium. Moreover additional data reported on the MHV peplomer protein, which have established the presence of neutralisation sites on the S 1 subunit, have reconciled the apparent discrepancy between MHV and other coronavirues. The difficulties inherent in the elucidation of antigenic structures on a polypeptide which is both very large and highly glycosylated such as the coronavirus peplomer protein should not be underestimated. Clearly, a single linear epitope is less 141

difficult to map than a highly conformation-dependent antigenic site. In this context, methods other than bacterial expression of antigen fragments need to be employed. Moreover, the use of complementary approaches should be preferred whenever possible, since each of them contains its own pitfall. Finally, it should be noted that the role of carbohydrate sidechains in the expression of antigenicity has received very little attention so far. Continuing investigation in this area is of obvious importance for coronavirologists. First, the resulting information is most helpful for the development of recombinant or synthetic vaccines. In this respect, T cell-recognised structures deserve increased attention in future research. Second, as epitopes often coincide with functional determinants, these studies provide a unique opportunity to explain in terms of molecular structure, fundamental virus processes such as neutralisation, cell receptor recognition and antigenic variation.

SELECIED LITERATURE Cavanagh, D., Davis, P.J. and Mockett, AP.A (1988) Amino acids within hypervariable region 1 of avian coronavirus IBV (Massachusetts serotype) spike glycoprotein are associated with neutralisation epitopes. Virus Res., 11: 141-150. Lenstra, J.A, Kusters, J.G., Koch, G. and van der Zeijst, B.A.M. (1989) Antigenicity of the peplomer protein of infectious bronchitis virus. Mol. Immunol., 26: 7-15. Lutyes, W., Geerts, D., Posthumus, W., Meloen, R. and Spaan, W. (1989) Amino acid sequence of a conserved neutralising epitope of murine coronaviruses. J. Virol., 63: 1408-1412. Makino, S., Fleming, J.O., Keck, J.G., Stohlman, S.A and Lai, M.M.C. (1987) RNA recombination of coronaviruses: localisation of neutralising epitopes and neuropathogenic determinants on the carboxy terminus of peplomers. Proc. Natl. Acad. Sci. USA 84: 6567-6571. Talbot, P.J., Dionne, G. and Lacroix, M. (1988) Vaccination against lethal coronavirusinduced encephalitis with a synthetic decapeptide homologous to a domain in the predicted peplomer stalk. 1. Virol., 62: 3032-3036. Tooze, S.A, and Stanley, K.K. (1986) Identification of two epitopes in the carboxyterminal 15 amino acids of the El glycoprotein of mouse hepatitis virus A %9 by using hybrid proteins. J. Virol. 60: 928-934.

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