A set of monoclonal antibodies was used to isolate nonneutralizable foot-and-mouth disease virus variants, and the RNAs of the variants were sequenced. Cross-neutralization studies and mapping of the amino acid changes indicated two major antigenic sites. The first site was trypsin sensitive and included the VP1 140 to 160 sequence. The second site was trypsin insensitive and included mainly VP3 residues. Two minor sites were located near VP1 169 and on the C terminus of VP1. Comparison with poliovirus type 1 and human rhinovirus 14 showed a similarity in the immunogenicity of comparable sites on the viruses.

The neutralization of type 1 poliovirus by monoclonal antibody 35-1f4 was studied. The virions were rapidly linked by antibody into oligomers and larger aggregates, followed by slow redistribution of antibody between the immune complexes. The antibody content and infectivity of immune complexes were determined. Remaining single virions were fully infectious and free of antibody. The oligomers and larger aggregates did not signfficantly contribute to the residual infectivity, which therefore correlated with the number of remaining single virions. Papain digestion of neutralized poliovirus released fully infectious, antibody-free virions from the immune complexes. Anti-immunoglobulin antibodies reneutralized these virions. Polymerization was shown to occur even at virus concentrations of less than 103 PFU per ml. * Corresponding author. that papain depolymerized virus-35-1f4 complexes and caused the reappearance of free 160S particles. MATERIALS AND METHODS Virus cultivation and infectivity titration. Unlabeled or

The interaction of mono-and polyclonal neutralizing antibodies with poliovirus was studied. In all cases, neutralization was due to antibody-mediated virus aggregation, and the unpolymerized virions accounted for the residual infectivity. The effect of papain on previously neutralized virus was to deaggregate the virus to fully infective single virions. With some antibodies, the amount of aggregated virus regressed in the region of greatest antibody excess, even though the virus remained fully neutralized. Under these conditions, noninfective, unaggregated immune complexes were formed. A mutant resistant to one of the monoclonal antibodies was selected. The mutant virions were still bound but no longer aggregated or neutralized by the selecting antibodies.

SummaryA set of 13 hybridoma antibodies to type 1 poliovirus has been studied with regard to neutralization and bindi_ng to N antigen, H antigen and capsid proteins. Two hybridomas produced heterogeneous antibodies, even after repeated attempts to reelone them. ,A set of thirteen mouse hybridoma antibodies to type 1 poliovirus was developed. The antibodies were studied with regard to a) neutralization of type 1, 2 and 3 poliovirus; b) reaction with N antigen, i.e. native, mature virions; c) reaction with H antigen, i.e., heat-denatured virions (6); d) reaction with individual capsid polypeptides prepared by detergent disruption of the virions.Two subsets of antibodies are tentatively distinguished: a) neutralizing monoelones recognizing N antigen only, and b) non-neutralizing monoctones, which recognized H antigen; some of these were also able to bind to the capsid polypeptides. The case of two hybridomas which produced antibodies sharing both sets of properties will be discussed.To prepare N antigen, type 1 (strain I a/S 3) poliovirus was grown and purified as described (10), and freed of empty eapsids by sucrose gradient eentrifugation and collection of the virions as 160 S particles. One microgram of this antigen emulsified in Freund's complete adjuvant was used for the initial immunization of BALB/c mice by intraperitoneal injection. A single booster of 1 ~zg N antigen was given intravenously after 1 month, and the animals were sacrificed 4 to 6 days later. The spleen cells were fused with SP2/0 mouse myeloma cells in the ratio of 10:1. Fusion and hybrid selection were done by conventional methods using PEG 1500 and ttAT medium (3).tIybmdomas secreting polio-specific antibodies were screened for by a doublesandwich ELISA method. The capture layer was formed by coating polystyrene 0304-8608/82/0074/0325/$ 01.20

SUMMARYThe effect of mono-and polyclonal antibodies on the infectivity and pI (isoelectric pH) of type 1 poliovirus was studied. According to Mandel's hypothesis, the isoelectric pH of poliovirus should change to about pI 4 upon neutralization. However, several antibodies did not follow this rule. Moreover, when antibodies did shift the pI, no quantitative correlation existed between the extent of neutralization and the amount of poliovirus shifted to low pI. Native poliovirus focuses at approximately pH 7. The p] (isoelectric pH) is lowered to 4-0 to 5.5 (B zone of the pH gradient) after acidification before focusing, after mild heating or u.v. inactivation (Mandel, 1971), and after treatment with a neutralizing antiserum (Mandel, 1976). Based on the latter finding, Mandel (1976Mandel ( , 1978 hypothesized that the neutralization of poliovirus occurred by means of stabilization of the virions in a biologically inactive conformation focusing in the B zone.According to this model, three predictions can be made. When partly neutralized virus is submitted to isoelectric focusing, (i) virus recovered from the A zone should be fully infectious, (ii) virus recovered from the B zone should be non-infectious, and (iii) the fraction of virus focusing in the A zone should be equal to the virus survival ratio. Mandel (1976) reported an excellent correlation between the amount of virus focusing in the A zone and the survival ratio, in agreement with prediction (iii). However, the quantitative data concerned only a single serum.Recently, it was reported (Emini et al., 1983a, b;Icenogle et al., 1983) that the pI shift could also be induced by monoclonal antibodies (MoAbs); however, no quantitative data were presented to test for Mandel's hypothesis. Moreover, these reports also mentioned cases of neutralization by both mono-and polyclonal antibodies without concomitant shift in the isoelectric pH of the virus.The apparent absence of a pI shift with some neutralizing antibodies prompted us to investigate the quantitative correlation between the antibody-mediated pl shift and the neutralization of poliovirus. Five different antibody preparations will be discussed. We used Mandel's original focusing technique, slightly modified by prelbrmation of the pH gradient before introduction of the virus (Vrijsen et aL, 1983).The Nl-specific IgG2a-~c antibody, MoAb 35-1f4 Brioen et al., 1982), was shown to neutralize poliovirus by antibody-mediated aggregation (Brioen et al., 1983). Immunoglobulins were purified from ascitic fluids by ammonium sulphate precipitation. Part of this preparation was 14C-labelled by reductive methylation (A. A. M. Thomas et al., unpublished), and the fraction of poliovirus-specific immunoglobulin was determined by ultracentrifugation of a mixture of antibody and excess poliovirus. As 58 ~ of the 1"C co-sedimented with the viral material, this fraction of the immunoglobulin was considered to represent poliovirus-specific antibody.At the antibody/virus ratio of 12:1, the virus was neutralized by 95~, but its pI...