SUMMARYThe antigenic sites recognized by monoclonal antibodies with neutralizing activity for the Sabin vaccine strains of poliovirus of serotypes 1, 2 and 3 have been studied by the isolation and characterization of mutants resistant to neutralization by antibody. Three distinct sites have been identified which are designated site 1, site 2 and site 3. Site 1 includes a region of 12 amino acids of VP1, from residues 89 to 100, and a corresponding region of VP1 has been identified as an antigenic site for poliovirus 2. This site was strongly immunodominant in type 2 and type 3 but was not detected for poliovirus 1. Site 2 is a complex site including residues 220 to 222 from VP1 (site 2a) with residues including 169 and 170 and others of VP2 (site 2b). Both site 2a and site 2b have been detected in type 1 poliovirus, while as yet only site 2b has been detected in type 3 poliovirus. Site 3 is a complex site including residues 286 to 290 from VP1 (site 3a) with residues including 58 and 59 and others of VP3 (site 3b). Both sites 3a and 3b have been detected in type 3 poliovirus, while as yet only site 3b has been detected in type 1 poliovirus. INTRODUCTIONPoliovirus is a picornavirus of the enterovirus genus occurring in three distinct serotypes. The virion consists of a single strand of messenger-sense RNA enclosed in a capsid made up of 60 copies of each of the four structural proteins VP1, VP2, VP3 and VP4. Nucleotide sequences of the genomic RNA of several strains of poliovirus have been published (Kitamura et al., 1981 ;Toyoda et al., 1984;Cann et al., 1984) and the X-ray crystallographic structure of the Mahoney strain of type 1 poliovirus has been solved to 2.9 A (0-29 nm) resolution .

SUMMARY A 4 month old child was immunized with a vaccine containing the Sabin live attenuated vaccine strains of all three serotypes of poliovirus. The antigenic and molecular evolution of the Sabin strain of poliovirus type 3 was then followed throughout the entire period of virus excretion. Novel strains appeared at 8, 42 and 52 days post-vaccination and were the products of both intertypic recombination between type 2 and type 3 poliovirus in regions of the genome coding for non-structural proteins and of point mutations in the region coding for the structural proteins. Excretion of virus continued for 73 days. All strains examined reacted with all monoclonal antibodies specific for the main immunodominant antigenic site of type 3 poliovirus, but variation was observed at other, immunorecessive sites. These findings have possible implications for the evolution of the virus in vaccinees or in epidemics and are consistent with the known antigenic stability of the virus. INTRODUCTIONPoliovirus is a picornavirus of the enterovirus genus. The virion consists of a single strand of messenger-sense RNA enclosed in an icosahedral capsid composed of 60 copies each of the coat proteins VP1, VP2, VP3 and VP4. The sequence of the genomic RNA is known for at least one strain of each of the three serotypes (Toyoda et al., 1984;Kitamura et al., 1981 ;Cann et al., 1984) and the precise structure of the capsid proteins in infectious virus of the type 1 serotype has now been established by X-ray crystallography .Studies of the molecular basis of such biological properties as the attenuation of neurovirulence of specific virus strains are in progress (Omata et al., 1985;Evans et al., 1985) and antigenically important structural features of the virus have been identified Hogle et al., 1985; P. D. Minor, unpublished results). However, many details of the biology of the virus and its relationship with its human host remain unexplored in the light of the current detailed understanding of the molecular biology of the virus. Thus vaccines containing one strain of each serotype have proved extremely effective in controlling poliomyelitis, implying that immunization with one virus confers immunity to all strains of the same serotype. This strongly suggests that polioviruses are not able to evade host immunity by antigenic drift of

The Sabin type 1 vaccine strain of poliovirus is probably the safest and most successful live-attenuated vaccine virus used in humans. Its widespread use since the early 1960s has contributed significantly to the virtual eradication of poliomyelitis in developed countries. We have reported previously the construction of an intertypic antigen chimaera of poliovirus, based on the Sabin 1 strain, and proposed that this virus could be modified to express on its surface antigenic determinants from other pathogens. We describe here the construction and characterization of a poliovirus antigen chimaera containing an epitope from the transmembrane glycoprotein (gp41) of human immunodeficiency virus type 1 (HIV-1). In antibody absorption experiments, the virus chimaera inhibited neutralization of HIV-1 by antipeptide monoclonal antibodies specific for the gp41 epitope and significantly reduced the group specific neutralizing activity of HIV-1-positive human sera. Rabbit antisera raised by subcutaneous injection of the polio/HIV chimaera in adjuvant was shown to be specific for HIV-1 gp41 in peptide-binding assays and by western blotting. Moreover, the antisera neutralized a wide range of American and African HIV-1 isolates and also inhibited virus-induced cell fusion. Monoclonal antibodies against the HIV-1 derived regions of the chimaera also neutralized HIV-1. These results establish the potential of using poliovirus for the presentation of foreign antigens and suggest that Sabin 1 poliovirus/HIV chimaeras could offer an approach to the development of an HIV vaccine.

We have determined a major antigenic site for virus neutralization on the capsid protein VP1 of poliovirus type 3. Antigenic mutant viruses selected for resistance to individual monoclonal antibodies had point mutations concentrated in a region 277-294 bases downstream from the start of the region of viral RNA coding for VP1. These findings provide the basis for an improved understanding of the molecular basis of virus neutralization.

Polioviruses occur as three distinct serotypes, 1, 2 and 3, and are composed of a single-stranded positive-sense RNA genome of approximately 7,450 nucleotides enclosed in an icosahedral particle of diameter 27 nm. The three-dimensional crystallographic structure of poliovirus type 1 has been determined at 2.9 A resolution, providing a detailed knowledge of the folding and arrangement of the individual virus proteins, VP1-VP4. From this and the characterization of monoclonal antibody-resistant mutants, the amino acids contributing to antigenic sites have been identified and located on the surface of the virus particle. Here we describe the construction and characterization of a poliovirus chimaera having a defined region of type 3 inserted into type 1. This virus has composite antigenicity and the substitute site is immunogenic in small animals and primates. The ability to construct such viruses has implications for the design of improved poliovirus vaccine strains or vaccines against other picornaviruses, such as hepatitis A.