Evidence for common, intertypic antigenic determinants on poliovirus capsid polypeptides

Polyclonal antibodies prepared against individualized type 1 poliovirus structural polypeptides VP1, VP2, and VP3 were used to analyze the presence of common antigenic determinants among the three poliovirus serotypes. Each anti-VP antiserum immunoprecipitated specifically the polypeptide against which it had been prepared, as well as the corresponding polypeptide of the heterotypic viruses. Anti-VP1 antisera also reacted with type 1, type 2, and type 3 heat-denaturated poliovirions (C particles), whereas anti-VP2 and anti-VP3 sera formed immune complexes with type 1 and type 3, but not with type 2, C particles. It was concluded that capsid polypeptides VP1, VP2, and VP3 of the three serotypes share common antigenic determinants which are masked in mature virions (D particles), but can be unmasked, at least partially, upon heat inactivation of the virus.     

Immunochemical studies of polioviruses: identification of immunoreactive virus capsid polypeptides

Investigation of the immunological reactions with individual poliovirus capsid polypeptides of antisera and monoclonal antibodies raised against poliovirus type 3 antigens are described. Virus polypeptides were separated by electrophoresis, transferred electrophoretically to nitrocellulose sheets and treated with antibody preparations. Antibody binding specifically to the virus polypeptides was then detected by application of 125I-labelled anti-immunoglobulin followed by autoradiography. The technique readily enabled the identification of the polypeptides recognized by the antibody. Antibodies present in polyclonal, type-specific neutralizing sera to poliovirus type 3 bound to the two largest capsid polypeptides (VP1 and VP2) of the homotypic poliovirus, and also to the VP1 of poliovirus type 1 and type 2. There was no obvious difference between the antibody binding patterns obtained with neutralizing and non-neutralizing antisera or between C-specific and D-specific antisera. VP1 appeared to be the immunodominant virus polypeptide. Among monoclonal antibodies specific for the C antigen of poliovirus type 3, a proportion reacted homotypically with the VP1 of poliovirus type 3. Other monoclonal antibodies of C antigen or D antigen specificity, or which reacted both with D and C antigens, some of which had potent virus-neutralizing activity, failed to give demonstrable binding reactions. The non-correlation of neutralization and immunoblot reactivity suggests that sequence determinants alone do not mediate virus neutralization which may depend on antigenic determinants specified by complex conformational arrangements of the virus capsid proteins.     

Binding site of neutralizing monoclonal antibodies obtained after in vivo priming with purified VP1 of poliovirus type 1 is located between amino acid residues 93 and 104 of VP1

Three hybridomas obtained after in vitro stimulation of spleen cells of mice primed in vivo with purified VP1 of poliovirus type 1 (Mahoney) with the homologous virus produced antibodies which reacted with VP1 and immunoprecipitated and neutralized only the homologous virus. Evidence for the location of their binding sites was obtained by inhibition of virus neutralization and virus binding by a synthetic peptide comprising the amino acid sequence 93-104 of VP1 of poliovirus type 1 (Mahoney).     

N-AgIB of poliovirus type 1: a discontinuous epitope formed by two loops of VP1 comprising residues 96-104 and 141-152

Analysis of resistant mutants to neutralizing monoclonal antibodies revealed a discontinuous neutralization epitope on VP1 of poliovirus type 1, Mahoney. The epitope has the unique property of being also part of a sequential epitope within neutralization antigenic site I (N-AgI). It is formed by residues in the loop 96-104 connecting the B and C strand and in the loop 141-152 connecting the D and E strand of VP1. Because of strong analogy to neutralization immunogen IB (NImIB) of human rhinovirus 14 (HRV-14) we have called this site N-AgIB of poliovirus type 1.     

Antigenic sites of poliovirus type 3 eliciting IgA monoclonal antibodies in orally immunized mice

A panel of neutralizing IgA monoclonal antibodies was produced from mice orally inoculated with poliovirus type 3 Sabin and cholera toxin as adjuvant. Low levels of neutralizing antibodies were elicited in mice after several boosts, but only in the presence of cholera toxin. Characterization of IgA MAbs by neutralization-escape virus mutants showed that all but one neutralizing MAbs against type 3 poliovirus were directed to antigenic site N-AgIII, which was previously found by us to be the major target of mucosal immune response to Sabin 1 in the mouse. Our data indicate that residue 236 of VP3, not previously reported, is also involved in forming site N-AgIII in addition to formerly described VP3 (aa 58-59) and VP1 (aa 286-290) residues. Unlike poliovirus type 1 IgA MAbs, all IgA MAbs herein described neutralized the wild-type parental poliovirus.     

Binding of neutralizing monoclonal antibodies to empty capsids of poliovirus can be blocked by monospecific antisera to structural polypeptides VP1 and VP2

Summary Binding of two neutralizing monoclonal antibodies (Nt-mAbs) to natural empty capsids (NEC) of poliovirus, type 1, was blocked to the extent of 83 per cent to 98 per cent by monospecific rabbit antisera directed against the structural polypeptides VP1 and VP2. Monospecific antisera against VP3 or VP4, however, did not show this blocking effect. It is therefore assumed that VP1 and VP2 are located close together at the antigenic sites for the two mAbs.With 1 Figure     

A new antigenic site of poliovirus recognized by an intertypic cross-neutralizing monoclonal antibody

A monoclonal antibody (mAb 7J6) neutralizing poliovirus type 2 (PV2) and poliovirus type 1 (PV1) was obtained after immunization of BALB/c mice with infectious PV2, strain MEF-1. Preincubation of mAb 7J6 with PV1 inhibited its binding to PV2 and vice versa. Neutralization-resistant variants of PV2 and PV1 were selected. Nucleotide sequencing of the RNAs of some variants revealed mutations in the loop of amino acid residues 239 to 245 in VP2 and in the loop of amino acid residues 195 to 207 in VP3. This is the first evidence that these two loops contribute to a neutralization antigenic site (N-Ag) for poliovirus. Moreover, this new site on PV2 induced intertypic cross-neutralizing antibodies.     

Isolation of a type 3 vaccine-derived poliovirus (VDPV) from an Iranian child with X-linked agammaglobulinemia

Type 3 immunodeficiency-associated vaccine-derived polioviruses (iVDPVs) were isolated from a 15-month-old Iranian boy with acute flaccid paralysis (AFP) who was subsequently diagnosed with X-linked agammaglobulinemia (XLA). VP1 nucleotide sequences of the two isolates differed from Sabin 3 by 2.0% and 2.1% and from each other by 0.6%. Although the key determinant of attenuation and temperature sensitivity in the 5'-untranslated region (U(472)-->C) had reverted, a second capsid-region determinant (VP3:Phe(091)) was unchanged, but a presumptive suppressor (VP1:Ala(054)-->Val) was found. The isolates were Sabin 3/Sabin 1 recombinants, sharing a single recombination breakpoint in the 2C region. Although the two isolates were antigenically distinct from Sabin 3, only one amino acid replacement was found in the neutralizing antigenic sites (VP3:Ser(059)-->Asn in site 3). The patient was placed on intravenous immunoglobulin (IVIG) therapy within 9 days of onset of AFP, and iVDPV excretion ceased thereafter, but the patient remained severely paralyzed until his death approximately 11 months after paralysis. No secondary AFP cases were found, and none of the seven tested contacts of the patient were found to be infected with poliovirus.     

Induction by synthetic peptides of broadly reactive, type-specific neutralizing antibody to poliovirus type 3

A region of virus capsid protein VP1 located 89-100 amino acids from the N-terminus has been proposed to comprise a major antigenic site involved in the neutralization of poliovirus type 3. Synthetic peptides 10-18 amino acids in length, containing all or part of this sequence, were tested for their ability to induce antiviral antibodies. Rabbits, but not guinea pigs or mice, immunized with the most active peptide, developed hightitered, type-specific, neutralizing antibodies for a wide range of poliovirus type 3 strains. Consistent with the broad type specificity of the antibody response was the observation that amino acids 89-100 of VP1 are highly conserved among different poliovirus type 3 strains. This sequence thus appears to provide, at least in part, a molecular basis for serotype antigenic specificity. Individual amino acids from 93 to 98 within this sequence were shown to be important for the neutralization of virus by antipeptide sera by examination of the ability of the sera to neutralize laboratory-derived poliovirus type 3 mutants with known single amino acid substitutions in the proposed antigenic site.     

Investigation of the structure of polio- and human rhinovirions through the use of selective chemical reactivity.

The configurations of poliovirus and human rhinovirus type 2 (HRV-2) virions and subviral particles were investigated by measuring the relative accessibility of the four virion polypeptides (VP1-4) to the labeling reagents [³H]acetic anhydride, ¹²⁵I, and [¹⁴C]iodoacetamide. The reaction of [³H]acetic anhydride with the intact virions of poliovirus or HRV-2 revealed that in both cases VP1 was labeled to a greater extent than the smaller polypeptides, VP2 and VP3. The smallest polypeptide, VP4, was not labeled at all, suggesting that it may be internal and may not contribute directly to the surface properties of native virions. Treatment of poliovirus at 47° or HRV-2 at pH 5 produces slower sedimenting A-particles which lack VP4 and resemble the particles produced during the early interaction of virus with host cells. The occurrence of a configurational change in the formation of A-particles was suggested by the observation that A-particles exhibited increased relative susceptibility to labeling of VP2 with [³H]acetic anhydride. Virions which had been disrupted by treatment with dodecyl sulfate at 100° were labeled with [³H]acetic anhydride approximately uniformly in all polypeptides including VP4. The labeling patterns of the polypeptides of poliovirions, A-particles, and disrupted virus with ¹²⁵I were qualitatively similar to those obtained with [³H]acetic anhydride. In contrast, VP1 in HRV-2 virions was relatively protected from labeling with ¹²⁵I and became accessible only in A-particles or disrupted virions. With both viruses, again, VP4 was not labeled with ¹²⁵I in intact virions but was labeled after virus disruption. Very little [¹⁴C]iodoacetamide was incorporated into native virions, although in a few experiments VP2 was labeled in HRV-2. Only the capsid polypeptides VP1, VP2, and VP3 were labeled with [¹⁴C]iodoacetamide following disruption with SDS, indicating that neither the poliovirus VP4 nor the HRV-2 VP4 contain free SH groups.     

Minor displacements in the insertion site provoke major differences in the induction of antibody responses by chimeric parvovirus-like particles.

An antigen-delivery system based on hybrid virus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of canine parvovirus (CPV) and expressing foreign peptides was investigated. In this report, we have studied the effects of inserting the poliovirus C3:B epitope in the four loops and the C terminus of the CPV VP2 on the particle structure and immunogenicity. Epitope insertions in the four loops allowed the recovery of capsids in all of the mutants. However, only insertions of the C3:B epitope in VP2 residue 225 of the loop 2 were able to elicit a significant anti-peptide antibody response, but not poliovirus-neutralizing antibodies, probably because residue 225 is located in an small depression of the surface. To fine modulate the insertion site in loop 2, a cassette-mutagenesis was carried out to insert the epitope in adjacent positions 226, 227, and 228. The epitope C3:B inserted into these positions was well recognized by the specific monoclonal antibody C3 by immunoelectron microscopy. BALB/c mice immunized with these chimeric C3:B CPV:VLPs were able to elicit an strong neutralizing antibody response (>3 log(10) units) against poliovirus type 1 (Mahoney strain). Therefore, minor displacements in the insertion place cause dramatic changes in the accessibility of the epitope and the induction of antibody responses.     

Antigenic conservation and divergence between the viral-specific proteins of poliovirus type 1 and various picornaviruses

Immuneprecipitation analyses of various picornavirus-infected cell lysates were performed using antisera to poliovirus type 1-specific structural and nonstructural proteins. The results showed differing patterns of antigenic conservation and divergence. However, the VP3 and 2C polypeptides were strongly antigenically conserved among the large majority of these viruses. This conservation was especially notable given the degree of divergence exhibited by the other viral proteins and may be due to environmental pressure exerted by interaction with the host cell. The results, furthermore, allowed for an analysis of the evolutionary relationship of the tested viruses. This analysis showed a particularly strong antigenic relationship between the proteins of the poliovirus group and coxsackievirus A21 as well as a weaker, but significant, relationship with coxsackieviruses B1 and B3.     

Absence of subviral particles and assembly activity in HeLa cells infected with defective-interfering (DI) particles of poliovirus

HeLa cells infected with defective-interfering (DI) particles of poliovirus were examined for their capacity to synthesize viral proteins, viral-related particles, and the viral factor(s) that promotes the assembly of 14 S particles into empty capsidsin vitro. As reported by C. N. Cole and D. Baltimore (1973,J. Mol. Biol.76, 325–343) cells infected with purified DI particles failed to synthesize the capsid precursor NCVP 1a or any of the capsid polypeptides VP 0, VP 1, VP 2, or VP 3. Consequently, no 14 S particles, empty capsids, or virions were formed at low to moderate multiplicities. Cytoplasmic extracts prepared from cells infected with purified DI particles do not promote the assembly of viral 14 S particles into empty capsids. We conclude that the presence of assembly activity is dependent on the formation of the capsid precursor protein (NCVP-1a) or its cleavage products.     

Isolation of recombinant type 2 vaccine-derived poliovirus (VDPV) from a Nigerian child

A type 2 vaccine-derived poliovirus (VDPV), differing from Sabin 2 at 2.5% (22/903) of VP1 nucleotide (nt) positions, was isolated from an incompletely immunized 21-month-old Nigerian child who developed acute flaccid paralysis in 2002. Sequences upstream of nt position 620 (within the 5'-untranslated region [5'-UTR]) and downstream of nt position 5840 (in the 3C(pro) region) were derived from species C enteroviruses unrelated to the oral poliovirus vaccine (OPV) strains. The two substitutions associated with the attenuated phenotype had either recombined out (A(481)-->G in the 5'-UTR) or reverted (Ile(143)-->Thr in VP1). The VDPV isolate had lost the temperature sensitive phenotype of Sabin 2 and it was antigenically distinct from the parental OPV strain, having amino acid substitutions in or near neutralizing antigenic sites 1 and 3. The date of the initiating OPV dose, calculated from the number of synonymous substitutions in the capsid region, was estimated to be approximately 16 to 18 months before onset of paralysis, a finding inconsistent with the most recent mass OPV campaign (conducted 12 days before onset of paralysis) as being the source of infection. Although no related type 2 VDPVs were detected in Nigeria or elsewhere, the VDPV was found in an area where conditions favor VDPV emergence and spread.     

Isolation and characterization of defective-interfering particles of poliovirus Sabin 1 strain

Defective-interfering (DI) particles of the Sabin strain of type 1 poliovirus were generated on serial high m.o.i. passaging. The deletions, measured by agarose gel electrophoresis, appeared to comprise approximately 10% of the total genome. Analysis of the RNAs, after digestion with RNase T1, by two-dimensional polyacrylamide gel electrophoresis revealed that the locations of the deleted genome regions were similar to those of the DI particles of the Mahoney strain of type 1 poliovirus (A. Nomoto, A. Jacobson, Y. F. Lee, J. Dunn, and E. Wimmer, (1979), J. Mol. Biol. 128, 179-196). Taking the known nucleotide sequences of the total genome and large RNase T1-resistant oligonucleotides into account, the deletions of almost all DI RNAs were found to exist between nucleotide positions 1307 and 2630, a genome region encoding capsid polypeptides VP2, VP3, and VP1. In cells coinfected with the purified DI particles and the Sabin strain of type 2 or type 3 poliovirus, particles containing the DI genomes were effectively produced. These results suggest that encapsidation signals are conserved in all three serotypes of polioviruses. However, only a very small amount of similar DI particles appeared to be produced in cells coinfected with coxsackie virus B1, although the genomes of polioviruses and coxsackie viruses have common sequences and therefore these viruses are considered to have arisen from a common ancestor. These data may suggest differences in encapsidation signals between polioviruses and coxsackie viruses.     

Comparison of the antibodies elicited by the individual structural polypeptides of foot-and mouth disease and polio viruses.

Antibody produced against preparations of VP1, one of the four structural polypeptides of foot-and-mouth disease virus, neutralized the virus and reacted with both full and empty particles in radioimmunoassays (RIA). Antiserum against VP2 reacted with artificial empty particles of the virus but not with full particles. In contrast, none of the individual polypeptides of poliovirus produced antisera which neutralized the virus nor reacted with it in RIA. However, antisera produced with VP1 and VP2 reacted with artificial empty particles in RIA.     

Anti-poliovirus IgG subclass antibodies in cytomegalovirus infected mice

A considerable strain difference was noted in BALB/c and C57BL/6 mice with regard to the impairment of antibody responses to poliovirus antigens in the course of infection with murine cytomegalovirus (MCMV): a long lasting reduction in antibody formation in BALB/c mice contrasted with an only moderate depression observed in C57BL/6 animals. Analysis of antibody classes and IgG subclasses revealed that anti-poliovirus VP1 antibodies in BALB/c mice were predominantly of the IgG3 subclass, a subclass most drastically affected by MCMV infection, while C57BL/6 mice produced antibodies of the IgM class and of IgG1 and IgG2 subclasses which were reduced to a lesser extent by the infection with MCMV. It is concluded that the strain difference observed may be explained on the basis of differences in the handling of poliovirus antigenic determinants by BALB/c and C57BL/6 mice.     

Resolution of the major poliovirus capsid polypeptides into doublets.

Using the pH gradient electrophoretic technique of Vrijsen and Boeyé, the coat protein of poliovirus types 1 and 2 was resolved into six components, Cl to C6, instead of the classical VP1-2-3 (the much smaller polypeptide VP4 was excluded from this study). The multiple components ran true upon reelectrophoresis, and there were no technical artifacts. Their resolution did not depend on a particular method for the preparation or disruption of the virion. The C1–C6 components of pH gradient electrophoresis and the classical VP1–VP3 polypeptides were examined with regard to (i) their migration in normal and pH gradient reelectrophoresis and (ii) their kinetics of leucine incorporation in emetine-stopped pulses. It is concluded that C1 and C2 were both derived from VPl, C3 and C4 from VP2, and C5 and C6 from VP3.     

Antigenic regions of poliovirus type 3/Sabin capsid proteins recognized by human sera in the peptide scanning technique.

We used the peptide scanning technique to identify regions of poliovirus type 3/Sabin capsid proteins that bind antibodies from human immune sera. Several reactive regions were seen in VP1, VP2, and VP3 while peptides resembling VP4 did not bind antibodies. Peptides derived from sequences of the previously known antigenic sites 1 and 3 were recognized to a moderate degree. Peptides imitating the four loops in the closed ends of the beta barrels or the alpha helical CD insertions of VP1, VP2 or VP3, whether exposed in the crystal structure or not, all represented major reactivity in the scans. In VP1 several additional reactive regions were found in the amino terminal quarter of the protein, which is buried in the crystal structure, and in a partially exposed region close to but separated from the carboxy terminus. In VP2 the nonexposed peak activities clustered in a bridge-like structure spanning from the outer to the inner surface of the capsid shell. Likewise, most of the novel antigenic regions of VP3 clustered in an internal location and partially composed of beta sheets with a conserved amino acid sequence. Whether any of the novel antigenic sites is capable of inducing neutralizing antibodies is not known.     

Identification of precursors of structural proteins VP1 and VP2 of hepatitis A virus.

The morphogenetic pathway of hepatitis A virus (HAV), classified as a member of the enteroviruses within the Picornaviridae, still remains obscure and seems to differ considerably from that of poliovirus, the most studied representative of this genus. In order to elucidate the precursor/product relationship of HAV structural proteins, subviral particles, which represent more than 50% of the viral antigen produced in infected cells, were separated from mature virions and their polypeptide pattern was analyzed by polyacrylamide gel electrophoresis and immunoblotting using monospecific antisera. Whereas mature virions are composed of viral proteins VP1, VP2, and VP3, subviral particles contained VP0 and smaller polypeptides instead of VP2. Comparison of proteins of different strains of HAV showed that VP0 of strain HAS-15 migrated slower than that of strains MBB or GBM. During the course of the infectious cycle, VP0 accumulated and only small portions were converted to VP2 supporting earlier observations that encapsidation of RNA with concomitant cleavage of VP0 is rate-limiting, leaving a large amount of viral antigen in premature particles. Similar to VP0, accumulation of VP1 was observed and two immunologically related precursor proteins, p38 and p36, were found during the course of infection. Immunological characterization of p38 using antisera directed to the N-terminus of VP1 and to synthetic peptides located at the presumptive C- and N-termini of 2A suggests that p38 is VP1 delta 2A carrying 45 N-terminal amino acids of the P2-region.