Cross-reactive and serotype-specific neutralization epitopes on VP7 of human rotavirus: nucleotide sequence analysis of antigenic mutants selected with monoclonal antibodies.

The neutralization epitopes of human and simian rotavirus protein VP7 were studied by producing six neutralizing monoclonal antibodies (N-MAbs) and using these N-MAbs to select antigenic mutants that resisted neutralization by the N-MAbs used for their selection. Cross-neutralization tests between the N-MAbs and the antibody-selected antigenic mutants identified one cross-reactive and five distinct serotype-specific neutralization epitopes which operationally overlapped one another and constituted a single antigenic site. In addition, the amino acid substitutions in human rotavirus VP7 that are responsible for the antigenic alterations in the mutants selected with anti-VP7 cross-reactive or serotype-specific N-MAbs were identified. All the amino acid substitutions in the antigenic mutants occurred in one of two variable regions: amino acids 87 to 101 and 208 to 221.     

Single point mutations may affect the serotype reactivity of serotype G11 porcine rotavirus strains: a widening spectrum?

A panel of single and double neutralization-resistant escape mutants of serotype G11 porcine rotavirus strains A253 and YM, selected with G11 monotype- and serotype-specific neutralizing monoclonal antibodies (MAbs) to VP7, was tested in neutralization assays with hyperimmune sera raised against rotavirus strains of different serotypes. Escape mutants with an amino acid substitution in antigenic region A (amino acids [aa] 87 to 101) resulting in a residue identical or chemically similar to those present at the same positions in serotype G3 strains, at positions 87 for strain A253 and 96 for strain YM, were significantly more sensitive than the parental strains to neutralization with sera against some serotype G3 strains. Also, one YM antigenic variant (YM-5E6.1) acquired reactivity by enzyme-linked immunosorbent assay with MAbs 159, 57/8, and YO-1E2, which react with G3 strains, but not with the serotype G11 parental strain YM. Cross-adsorption studies suggested that the observed cross-neutralization by the G3-specific sera was due to the sera containing antibodies reactive with the parental strain plus antibodies reactive with the epitope(s) on the antigenic variant that mimick the serotype G3 specific one(s). Moreover, antibodies reactive with antigenic region F (aa 235 to 242) of VP7 might also be involved since cross-reactivity to serotype G3 was decreased in double mutants carrying an additional mutation, which creates a potential glycosylation site at position 238. Thus, single point mutations can affect the serotype reactivity of G11 porcine rotavirus strains with both monoclonal and polyclonal antibodies and may explain the origin of rotavirus strains with dual serotype specificity based on sequence divergence of VP7.     

Cross-reactive neutralization epitopes on VP3 of human rotavirus: analysis with monoclonal antibodies and antigenic variants.

We analyzed cross-reactive neutralization epitopes on protein VP3 of human rotavirus (HRV) by the use of neutralizing monoclonal antibodies (N-MAbs), which showed a variety of interserotypic reactivity patterns when examined in a neutralization test and an enzyme-linked immunosorbent assay against 15 HRV and 2 animal RV strains. Serological study with the six cross-reactive N-MAbs revealed antigenic variations in some HRV strains within the same serotype as well as a marked antigenic difference between serotype 2 strains and serotype 1, 3, and 4 strains. Epitope analysis of the antigenic variants resistant to the six individual cross-reactive N-MAbs suggested the existence of at least three distinct cross-reactive neutralization epitopes on VP3 of HRV.     

Identification of VP7 epitopes associated with protection against human rotavirus illness or shedding in volunteers.

Sera from 17 of 18 adult volunteers challenged with a virulent serotype 1 rotavirus strain (D) were examined for prechallenge antibody levels against several well-defined rotavirus VP7 and VP4 neutralization epitopes by a competitive epitope-blocking immunoassay (EBA) in order to determine whether correlates of resistance to diarrheal illness could be identified. The presence of prechallenge serum antibody at a titer of greater than or equal to 1:20 that blocked the binding of a serotype 1 VP7-specific monoclonal antibody (designated 2C9) that maps to amino acid residue 94 in antigenic site A on the serotype 1 VP7 was significantly associated with resistance to illness or shedding (P less than 0.001) or illness and shedding (P less than 0.01) following challenge with the serotype 1 virus. In addition, an EBA antibody titer of greater than or equal to 1:20 in prechallenge serum against a serotype 3 VP7-specific epitope (defined by monoclonal antibody 954/159) that maps to amino acid 94 on the serotype 3 VP7 was also significantly associated with resistance to illness or shedding (P = 0.02), with a trend for protection against illness and shedding. A trend was also noted between the presence of EBA antibody against a cross-reactive VP4 epitope common to many human rotavirus strains, including the challenge virus, or a rhesus monkey rotavirus strain-specific VP4 antigenic site, and resistance to illness or shedding. These data confirm that the presence of serum antibody correlates with resistance to rotavirus illness or shedding but, in addition, demonstrate the association of antibody to a specific epitope with resistance to illness or shedding. These data also suggest that antigenic site A on the rotavirus VP7, composed of amino acids 87 to 96, may be involved in the formation of a major protective epitope. Further study of the role of this epitope in the development of homotypic and heterotypic immunity to rotaviruses following natural or vaccine-induced infection may be important in the development of strategies for control of rotavirus diarrheal disease.     

Neutralization epitopes on rotavirus SA11 4fM outer capsid proteins.

The VP7 and VP4 genes of seven antigenic mutants of simian rotavirus SA11 4fM (serotype 3) selected after 39 passages in the presence of SA11 4fM hyperimmune antiserum, were sequenced. Nucleotide sequence analysis indicated the following. (i) Twice as many amino acid substitutions occurred in the VP7 protein than in VP4, which has a molecular weight twice that of VP7. (ii) Most amino acid changes that occurred clustered in six variable regions of VP7 and in two variable regions of VP4; these variable regions may represent immunodominant epitopes. (iii) Most amino acid substitutions that occurred in VP7 and VP4 of these mutants were also observed in antigenic mutants selected with neutralizing monoclonal antibodies (NMAbs); however, some amino acid substitutions occurred that were not selected for NMAbs. (iv) On VP7, some of the neutralization epitopes appeared to be interrelated because amino acid substitution in one site affected binding of specific NMAbs to other sites, while other neutralization epitopes on VP7 appeared to be independent, in that amino acid substitution in one site did not affect the binding of NMAbs to another distant site.     

Complete nucleotide sequence of the gene encoding VP4 of a human rotavirus (strain K8) which has unique VP4 neutralization epitopes.

In our previous study (K. Taniguchi, Y. Morita, T. Urasawa, and S. Urasawa, J. Virol. 62:2421-2426, 1987) in which the cross-reactive neutralization epitopes on VP4 of human rotaviruses were analyzed, one strain, K8, was found to bear unique VP4 neutralization epitopes. This strain, which belongs to subgroup II and serotype 1, was not neutralized by any of six anti-VP4 neutralizing monoclonal antibodies which reacted with human rotavirus strains of serotypes 1, 3, and 4 or serotypes 1 through 4. We determined the complete nucleotide sequence of the gene encoding VP4 of strain K8 by primer extension. The VP4 gene is 2,359 base pairs in length, with 5' and 3' noncoding regions of 9 and 25 nucleotides, respectively. The gene contains a long open reading frame of 2,325 bases capable of coding for a protein of 775 amino acids. When compared with those of other human rotaviruses, VP4 of strain K8 had an insertion of one amino acid after residue 135, as found in simian rotavirus strains, and in addition, it had a deletion of one amino acid (residue 575). The amino acid homology of VP4 of strain K8 and those of other virulent human rotaviruses was only 60 to 70%. This was unusual, since over 90% VP4 homology has been found among the other virulent human rotavirus strains. In contrast, the VP7 amino acid sequence of the K8 strain was quite similar (over 98% homology) to those of other serotype 1 human rotaviruses. Thus, the K8 strain appears to have a unique VP4 gene previously not described.     

Molecular and antigenic characterization of porcine rotavirus YM, a possible new rotavirus serotype.

In 1983, we isolated a porcine rotavirus (strain YM) that was prevalent in several regions of Mexico, as judged by the frequency of its characteristic electropherotype. By a focus reduction neutralization test, rotavirus YM was clearly distinguished from prototype rotavirus strains belonging to serotypes 1 (Wa), 2 (S2), 3 (SA11), 4 (ST3), 5 (OSU), and 6 (NCDV). Minor, one-way cross-neutralization (1 to 5%) was observed when antisera to the various rotavirus strains were incubated with rotavirus YM. In addition, the YM virus was not neutralized by neutralizing monoclonal antibodies with specificity to serotypes 1, 2, 3, and 5. The subgroup of the virus was determined to be I by enzyme-linked immunosorbent assay. To characterize the serotype-specific glycoprotein of the virus at the molecular level, we cloned and sequenced the gene coding for VP7. Comparison of the deduced amino acid sequence with reported homologous sequences from human and animal rotavirus strains belonging to six different serotypes further supported the distinct immunological identity of the YM VP7 protein.     

Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure.

We previously characterized three neutralization-positive epitopes (NP1 [1a and 1b], NP2, and NP3) and three neutralization-negative epitopes on the simian rotavirus SA11 VP4 with 13 monoclonal antibodies (MAbs). Conformational changes occurred as a result of the binding of NP1 MAbs to the SA11 spike VP4, and enhanced binding of all neutralization-negative MAbs was observed when NP1 MAbs bound VP4 in a competitive MAb capture enzyme-linked immunosorbent assay. To further understand the structure and function of VP4, we have continued studies with these MAbs. Electron microscopic and sucrose gradient analyses of SA11-MAb complexes showed that triple-layered viral particles disassembled following treatment with NP1b MAbs 10G6 and 7G6 but not following treatment with NP1a MAb 9F6, NP2 MAb 2G4, and NP3 MAb 23. Virus infectivity was reduced approximately 3 to 5 logs by the NP1b MAbs. These results suggest that NP1b MAb neutralization occurs by a novel mechanism. We selected four neutralization escape mutants of SA11 with these VP4 MAbs and characterized them by using plaque reduction neutralization assays, hemagglutination inhibition assays, and an antigen capture enzyme-linked immunosorbent assay. These analyses support the previous assignment of the NP1a, NP1b, NP2, and NP3 MAbs into separate epitopes and confirmed that the viruses were truly neutralization escape mutants. Nucleotide sequence analyses found 1 amino acid (aa) substitution in VP8* of VP4 at (i) aa 136 for NP1a MAb mutant 9F6R, (ii) aa 180 and 183 for NP1b MAb mutants 7G6R and 10G6R, respectively, and (iii) aa 194 for NP3 MAb mutant 23R. The NP1b MAb mutants showed an unexpected enhanced binding with heterologous nonneutralization MAb to VP7 compared with parental SA11 and the other mutants. Taken together, these results suggest that the NP1b epitope is a critical site for VP4 and VP7 interactions and for virus stability.     

Serotypic analysis of VP3 and VP7 neutralization escape mutants of rhesus rotavirus.

Neutralization escape mutants of simian rotaviruses (rhesus rotavirus and SA11) were tested in hemagglutination inhibition and neutralization assays against hyperimmune and infection sera to determine if mutation in an immunodominant epitope could enable neutralization escape. An SA11 mutant with a new glycosylation site at amino acid 211 of VP7 was shown to escape neutralization by hyperimmune but not infection sera.     

Identification of amino acids involved in recognition by dengue virus NS3-specific, HLA-DR15-restricted cytotoxic CD4+ T-cell clones.

The majority of T-cell clones derived from a donor who experienced dengue illness following receipt of a live experimental dengue virus type 3 (DEN3) vaccine cross-reacted with all four serotypes of dengue virus, but some were serotype specific or only partially cross-reactive. The nonstructural protein, NS3, was immuno-dominant in the CD4+ T-cell response of this donor. The epitopes of four NS3-specific T-cell clones were analyzed. JK15 and JK13 recognized only DEN3 NS3, while JK44 recognized DEN1, DEN2, and DEN3 NS3 and JK5 recognized DEN1, DEN3, and West Nile virus NS3. The epitopes recognized by these clones on the DEN3 NS3 protein were localized with recombinant vaccinia viruses expressing truncated regions of the NS3 gene, and then the minimal recognition sequence was mapped with synthetic peptides. Amino acids critical for T-cell recognition were assessed by using peptides with amino acid substitutions. One of the serotype-specific clones (JK13) and the subcomplex- and flavivirus-cross-reactive clone (JK5) recognized the same core epitope, WITDFVGKTVW. The amino acid at the sixth position of this epitope is critical for recognition by both clones. Sequence analysis of the T-cell receptors of these two clones showed that they utilize different VP chains. The core epitopes for the four HLA-DR15-restricted CD4+ CTL clones studied do not contain motifs similar to those proposed by previous studies on endogenous peptides eluted from HLA-DR15 molecules. However, the majority of these dengue virus NS3 core epitopes have a positive amino acid (K or R) at position 8 or 9. Our results indicate that a single epitope can induce T cells with different virus specificities despite the restriction of these T cells by the same HLA-DR15 allele. This finding suggests a previously unappreciated level of complexity for interactions between human T-cell receptors and viral epitopes with very similar sequences on infected cells.     

Similarity of the outer capsid protein VP4 of the Gottfried strain of porcine rotavirus to that of asymptomatic human rotavirus strains.

Genomic segment 4 of the porcine Gottfried strain (serotype 4) of porcine rotavirus, which encodes the outer capsid protein VP4, was sequences, and its deduced amino acid sequence was analyzed. Amino acid homology of the porcine rotavirus VP4 to the corresponding protein of asymptomatic or symptomatic human rotaviruses representing serotypes 1 to 4 ranged from 87.1 to 88.1% for asymptomatic strains and from 77.5 to 77.8% for symptomatic strains. Amino acid homology of the Gottfried strain to simian rhesus rotavirus, simian SA11 virus, bovine Nebraska calf diarrhea virus, and porcine OSU strains ranged from 71.5 to 74.3%. Antigenic similarities of VP4 epitopes between the Gottfried strain and human rotaviruses were detected by a plaque reduction neutralization test with hyperimmune antisera produced against the Gottfried strain or a Gottfried (10 genes) x human DS-1 rotavirus (VP7 gene) reassortant which exhibited serotype 2 neutralization specificity. In addition, a panel of six anti-VP4 monoclonal antibodies capable of neutralizing human rotaviruses belonging to serotype 1, 3, or 4 was able to neutralize the Gottfried strain. These observations suggest that the VP4 outer capsid protein of the Gottfried rotavirus is more closely related to human rotaviruses than to animal rotaviruses.     

Generation of recombinant rotavirus with an antigenic mosaic of cross-reactive neutralization epitopes on VP4

Recombinant rotavirus (RV) with cDNA-derived chimeric VP4 was generated using recently developed reverse genetics for RV. The rescued virus, KU//rVP4(SA11)-II(DS-1), contains SA11 (simian RV strain, G3P[2])-based VP4, in which a cross-reactive neutralization epitope (amino acids 381 to 401) on VP5* is replaced by the corresponding sequence of a different P-type DS-1 (human RV strain, G2P[4]). Serological analyses with a panel of anti-VP4- and -VP7-neutralizing monoclonal antibodies revealed that the rescued virus carries a novel antigenic mosaic of cross-reactive neutralization epitopes on its VP4 surface. This is the first report of the generation of a recombinant RV with artificial amino acid substitutions.     

Sequence of the fourth gene of human rotaviruses recovered from asymptomatic or symptomatic infections.

The complete nucleotide sequence of the fourth gene of symptomatic (Wa, DS-1, P, and VA70) and asymptomatic (M37, 1076, McN13, and ST3) rotaviruses of serotype 1, 2, 3, or 4 was determined by the dideoxy chain termination method. In each strain, the fourth gene, which encodes the outer capsid protein VP3, is 2,359 base pairs in length and has 5'- and 3'-noncoding regions of 9 and 25 nucleotides, respectively. The gene has a single long open reading frame of 2,325 base pairs that is capable of coding for a protein of 775 amino acids. A total of 14 N-terminal and 12 C-terminal amino acids are completely conserved or almost completely conserved, respectively, among nine human rotavirus VP3 genes that have been sequenced. In addition, there is conservation of arginine at the two trypsin cleavage sites as well as conservation of clusters of amino acids in different regions of the two VP3 cleavage products, VP8 and VP5. Three distinct forms of VP3 were identified among the nine human rotavirus strains analyzed. Three symptomatic rotaviruses (serotypes 1, 3, and 4) possess highly related VP3 genes (92.2 to 97% nucleotide identity). Two symptomatic serotype 2 rotaviruses possess VP3 genes which are even more closely related to each other (98.6% nucleotide identity) and only moderately related to the aforementioned VP3 genes of serotypes 1, 3, and 4 (87.4 to 88.2% nucleotide identity). The four asymptomatic rotaviruses, which constitute the third group, possess highly related VP3 genes (95.5 to 97.5% nucleotide identity) which are distinct from those of the virulent rotaviruses (73 to 74.8% nucleotide identity). At 91 positions in the protein sequence of VP3, an amino acid is conserved among the asymptomatic rotaviruses, while a different amino acid is conserved among the symptomatic rotaviruses. Notably, five regions are conserved among the symptomatic rotaviruses, while a different set of sequences are conserved among the asymptomatic rotaviruses. It is possible that some or all of these regions of sequence dimorphism may be responsible for the difference in virulence of these two groups of human rotaviruses. There are 13 regions in the VP3 protein sequence which exhibit the greatest variability; the majority of these variable regions are observed between amino acids 106 to 192. These regions may represent potential antigenic sites related to heterotypic rotavirus neutralization.     

A Novel Universal Neutralizing Monoclonal Antibody against Enterovirus 71 That Targets the Highly Conserved “Knob” Region of VP3 Protein

Hand, foot and mouth disease caused by enterovirus 71(EV71) leads to the majority of neurological complications and death in young children. While putative inactivated vaccines are only now undergoing clinical trials, no specific treatment options exist yet. Ideally, EV71 specific intravenous immunoglobulins could be developed for targeted treatment of severe cases. To date, only a single universally neutralizing monoclonal antibody against a conserved linear epitope of VP1 has been identified. Other enteroviruses have been shown to possess major conformational neutralizing epitopes on both the VP2 and VP3 capsid proteins. Hence, we attempted to isolate such neutralizing antibodies against conformational epitopes for their potential in the treatment of infection as well as differential diagnosis and vaccine optimization. Here we describe a universal neutralizing monoclonal antibody that recognizes a conserved conformational epitope of EV71 which was mapped using escape mutants. Eight escape mutants from different subgenogroups (A, B2, B4, C2, C4) were rescued; they harbored three essential mutations either at amino acid positions 59, 62 or 67 of the VP3 protein which are all situated in the “knob” region. The escape mutant phenotype could be mimicked by incorporating these mutations into reverse genetically engineered viruses showing that P59L, A62D, A62P and E67D abolish both monoclonal antibody binding and neutralization activity. This is the first conformational neutralization epitope mapped on VP3 for EV71.     

Serotype-specific epitope(s) present on the VP8 subunit of rotavirus VP4 protein.

cDNA clones representing the VP8 and VP5 subunits of VP4 of symptomatic human rotavirus strain KU (VP7 serotype 1 and VP4 serotype 1A) or DS-1 (VP7 serotype 2 and VP4 serotype 1B) or asymptomatic human rotavirus strain 1076 (VP7 serotype 2 and VP4 serotype 2) were constructed and inserted into the pGEMEX-1 plasmid and expressed in Escherichia coli. Immunization of guinea pigs with the VP8 or VP5 protein of each strain induced antibodies that neutralized the rotavirus from which the VP4 subunits were derived. In a previous study (M. Gorziglia, G. Larralde, A.Z. Kapikian, and R. M. Chanock, Proc. Natl. Acad. Sci. USA 87:7155-7159, 1990), three distinct serotypes and one subtype of VP4 outer capsid protein were identified among 17 human rotavirus strains that had previously been assigned to five distinct VP7 serotypes. The results obtained by cross-immunoprecipitation and by neutralization assay with antisera to the VP8- and VP5-expressed proteins suggest that the VP8 subunit of VP4 contains the major antigenic site(s) responsible for serotype-specific neutralization of rotavirus via VP4, whereas the VP5 subunit of VP4 is responsible for much of the cross-reactivity observed among strains that belong to different VP4 serotypes.     

Mutations conferring resistance to neutralization with monoclonal antibodies in type 1 poliovirus can be located outside or inside the antibody-binding site.

Antigenic variants resistant to eight neutralizing monoclonal antibodies were selected from wild (Mahoney) and attenuated (Sabin) type 1 infectious poliovirions. Cross-immunoprecipitation revealed interrelationships between epitopes which were not detected by cross-neutralization. Operational analysis of antigenic variants showed that seven of eight neutralization epitopes studied were interrelated. Only one neutralization epitope, named Kc, varied independently from all the others. This latter, recognized by C3 neutralizing monoclonal antibody, was present not only on infectious virions but also on heat-denatured (C-antigenic) particles and on isolated capsid protein VP1. Loss of the neutralization function of an epitope did not necessary result from the loss of its antibody-binding capacity. Such potential, but not functional, neutralization epitopes exist naturally on Mahoney and Sabin 1 viruses. Their antibody-binding property could be disrupted by isolating antigenic variants in the presence of the nonneutralizing monoclonal antibody and anti-mouse immunoglobulin antibodies. Single-point mutations responsible for the acquisition of resistance to neutralization in the antigenic variants were located by sequence analyses of their genomes. Mutants selected in the presence of C3 neutralizing monoclonal antibody always had the mutation located inside the antibody-binding site (residues 93 through 103 of VP1) at the amino acid position 100 of VP1. On the contrary, antigenic variants selected in the presence of neutralizing monoclonal antibodies reacting only with D-antigenic particles had mutations situated in VP3, outside the antibody-binding site (residues 93 through 103 of VP1). The complete conversion of the Mahoney to the Sabin 1 epitope map resulted from a threonine-to-lysine substitution at position 60 of VP3.     

Distribution of conserved and specific epitopes on the VP8 subunit of rotavirus VP4.

Three cDNA clones comprising the VP8 subunit of the VP4 of human rotavirus strain KU (VP7 serotype G1; VP4 serotype P1A) G1 were constructed. The corresponding encoded peptides were designated according to their locations in the VP8 subunit as A (amino acids 1 to 102), B (amino acids 84 to 180), and C (amino acids 150 to 246 plus amino acids 247 to 251 from VP5). In addition, cDNA clones encoding peptide B of the VP8 subunit of the VP4 gene from human rotavirus strains DS-1 (G2; P1B) and 1076 (G2; P2) were also constructed. These DNA fragments were inserted into plasmid pGEMEX-1 and expressed in Escherichia coli. Western immunoblot analysis using antisera to rotavirus strains KU (P1A), Wa (P1A), DS-1 (P1B), 1076 (P2), and M37 (P2) demonstrated that peptides A and C cross-reacted with heterotypic human rotavirus VP4 antisera, suggesting that these two peptides represent conserved epitopes in the VP8 subunit. In contrast, peptide B appears to be involved in the VP4 serotype and subtype specificities, because it reacted only with the corresponding serotype- and subtype-specific antiserum. Antiserum raised against peptide A, B, or C of strain KU contained a lower level of neutralizing activity than did that induced by the entire VP8 subunit. In addition, the serotype-specific neutralizing activity of anti-KU VP8 serum was ablated after adsorption with the KU VP8 protein but not with a mixture of peptides A, B, and C of strain KU, suggesting that most of the serotype-specific epitopes in the VP8 subunit are conformational and are dependent on the entire amino acid sequence of VP8.     

Identification of neutralizing antigenic sites on VP1 and VP2 of type A5 foot-and-mouth disease virus, defined by neutralization-resistant variants.

Five neutralizing monoclonal antibodies (nMAbs) obtained against type A5 Spain-86 foot-and-mouth disease virus were used to generate a series of neutralization-resistant variants. In vitro and in vivo assays showed that the variants were fully refractory to neutralization by the selecting nMAb. On the basis of cross-neutralization and binding assays, two neutralizing antigenic sites have been located on the virus surface; one, located near the C-terminus of VP1, displayed a linear epitope, and the second, located on VP2, displayed two conformational epitopes. Nucleotide sequencing of RNA of the parental and variant capsid protein-coding region P1 has placed the amino acid changes at position 198 of VP1 for the first site and at positions 72 and 79 of VP2 for the related epitopes in the second site. The relative importance of these two sites in the biological properties of foot-and-mouth disease virus is discussed.     

Antigenic sequences of poliovirus recognized by T cells: serotype-specific epitopes on VP1 and VP3 and cross-reactive epitopes on VP4 defined by using CD4+ T-cell clones.

A panel of poliovirus-specific murine CD4+ T-cell clones has been established from both BALB/c (H-2d) and CBA (H-2k) mice immunized with Sabin vaccine strains of poliovirus serotype 1, 2, or 3. T-cell clones were found to be either serotype specific or cross-reactive between two or all three serotypes. Specificity analysis against purified poliovirus proteins demonstrated that T-cell clones recognized determinants on the surface capsid proteins VP1, VP2, and VP3 and the internal capsid protein VP4. Panels of overlapping synthetic peptides were used to identify eight distinct T-cell epitopes. One type 3-specific T-cell clone recognized an epitope within amino acids 257 and 264 of VP1. Three T-cell epitopes corresponding to residues 14 to 28, 189 to 203, and 196 to 210 were identified on VP3 of poliovirus type 2. The remaining four T-cell epitopes were mapped to an immunodominant region of VP4, encompassed within residues 6 and 35 and recognized by both H-2d and H-2k mice. The epitopes on VP4 were conserved between serotypes, and this may account for the predominantly cross-reactive poliovirus-specific T-cell response observed with polyclonal T-cell populations. In contrast, T-cell clones that recognize epitopes on VP1 or VP3 were largely serotype specific; single or multiple amino acid substitutions were found to be critical for T-cell recognition.     

Evidence for a complex structure of neutralization antigenic site I of poliovirus type 1 Mahoney

We have selected neutralization escape mutants by using a monoclonal antibody (nt-MAb) against a sequential epitope between amino acids 93 through 104 (neutralization antigenic site I) of poliovirus type 1 Mahoney. The majority of mutants were also resistant against five strain-specific nt-MAbs which recognized conformation-dependent epitopes, suggesting that the neutralization antigenic site I must be involved in the formation of such epitopes. An analysis of all mutants by the binding of nt-MAbs and by isoelectric focusing of VP1 allowed discrimination of five classes of mutants. Sequence analysis of mutant RNAs revealed point mutations and deletions in the antibody-binding site.