A DNA sequence coding for the immunogenic capsid protein VP3 of foot-and-mouth disease virus A12, prepared from the virion RNA, was ligated to a plasmid designed to express a chimeric protein from the Escherichia coli tryptophan promoter-operator system. When Escherichia coli transformed with this plasmid was grown in tryptophan-depleted media, approximately 17 percent of the total cellular protein was found to be an insoluble and stable chimeric protein. The purified chimeric protein competed equally on a molar basis with VP3 for specific antibodies to foot-and-mouth disease virus. When inoculated into six cattle and two swine, this protein elicited high levels of neutralizing antibody and protection against challenge with foot-and-mouth disease virus.

A series of seven neutralizing monoclonal antibodies (nMAbs) directed against type A12 foot-and-mouth disease virus was used to generate neutralization-resistant variants. Both plaque reduction neutralization and microneutralization assays showed that the variants were no longer neutralized by. the nMAbs used to generate them, although some of the variants still reacted with the nMAbs at high antibody concentrations. Results of cross-neutralization studies by both plaque reduction neutralization and microneutralization assays suggested the presence of at least one immunodominant antigenic site on the surface of type A12 foot-and-mouth disease virus, along with evidence of a second antigenic site on the viral surface. Two of the variants had reduced virulence in tissue culture as evidenced by their inability to inhibit cellular protein synthesis and a marked reduction in virus-induced cellular morphological alterations. Nucleotide sequencing of the variant genomes placed three epitopes of the major antigenic site on VP1 and the fourth epitope on VP3 and VP1. The one epitope of the minor site appears to reside only on VP1.

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SUMMARYNeutralizing monoclonal antibodies raised against type 0 foot-and-mouth disease virus have been characterized on the basis of their reactivity with a panel of single site monoclonal antibody-resistant mutants which had defined three antigenic sites. Five antibodies neutralized all these mutants, but by selecting further single site mutants with one of these antibodies it was possible to define a fourth site involved in virus neutralization. Two monoclonal antibodies still neutralized these mutants and all multiple site resistant mutants. One multiple site resistant mutant was resistant to neutralization at each of four antigenic sites but was still efficiently neutralized by type 0 convalescent cattle sera. The relationship between sites recognized by different monoclonal antibodies generated in different laboratories is discussed. INTRODUCTIONStudies on the antigenic structure of two picornaviruses, poliovirus and rhinovirus, have been considerably enhanced by the solving of their three-dimensional crystal structure (Hogle et al., 1985;Rossmann et al., 1985) and the isolation and sequence analysis of monoclonal antibody (MAb) escape mutants (Minor et al., 1986;Sherry et al., 1986). Crystallographic studies on footand-mouth disease virus (FMDV), another picornavirus, are also in progress (Fox et al., 1987) and a number of laboratories have generated MAbs capable of neutralizing type O virus. characterized some 30 MAb-resistant mutants of the O1 Kaufbeuren strain of FMDV isolated using five different neutralizing MAbs. Three distinct antigenic sites were involved in virus neutralization. In this context the term antigenic site is used to describe an area of the virus surface which may contain several MAb epitopes; if one epitope is changed and this affects the ability of a second MAb to neutralize the virus then it is considered that the second MAb recognizes an epitope in the same antigenic site as that changed.Recently Stave et al. (1988) have used different MAbs to define three sites of neutralization. In this communication we now use the panel of mutants generated by Xie et al. (1987) to map the binding sites of MAbs generated in different laboratories and then use these antibodies to show the existence of further sites involved in the neutralization of type O FMDV. We have also generated multiple site mutants which are resistant to neutralization at each of two, three or four sites.