Mapping of a neutralizing antigenic site of Coxsackievirus B4 by construction of an antigen chimera

Abstract

A neutralizing antigenic site of coxsackievirus B4 (CVB4) was identified by construction of an antigen chimera between coxsackievirus B3 (CVB3) and CVB4. This chimera, designated CVB3/4, was constructed by inserting five amino acids of the putative BC loop of the structural protein VP1 of CVB4 into the corresponding loop of CVB3 by site-directed mutagenesis of infectious recombinant CVB3 cDNA. The chimeric cDNA was capable of inducing an infectious cycle upon transfection of permissive host cells. The resulting chimeric virus CVB3/4 was neutralized and precipitated by CVB4 and CVB3 serotype-specific polyclonal antisera, demonstrating that it unifies antigenic properties of both coxsackievirus serotypes. In addition, the chimera elicited antibodies in rabbits which were capable of neutralizing the two coxsackievirus serotypes CVB3 and CVB4. The insertion of the CVB4-specific antigenic site into the BC loop of CVB3 reduces the efficiency of viral replication, resulting in a small-plaque morphology of the virus chimera. In summary, these data give evidence for the presence of a serotype-specific neutralizing antigenic site in the BC loop of VP1 of CVB4 (amino acids 81 to 89). Our findings suggest that the construction of intertypic chimeras can be used as a tool for the identification of antigenic sites of coxsackieviruses. The retained immunogenicity of the mapped CVB4-specific antigenic epitope, when expressed in CVB3, indicates that CVB3 can be used as a RNA virus vector for heterologous antigenic sites.

Molecular Evolution of the Human Enteroviruses: Correlation of Serotype with VP1 Sequence and Application to Picornavirus Classification

“…For example, the B-C loop is one of five VP1 loops forming VP1 antigenic site 1. Replacement of the VP1 B-C loop of CB3 by that of CB4 through site-directed mutagenesis produced a viable virus with a mixed neutralization phenotype, demonstrating the presence of a serotypespecific antigenic neutralization site in the B-C loop (57). Our sequencing studies confirmed the presence of sequence domains that are conserved among all members of the Enterovirus genus, as well as intervening domains that vary in sequence between strains of different serotypes and in some cases within a serotype.…”

Molecular typing and epidemiology of enteroviruses identified from an outbreak of aseptic meningitis in Belgium during the summer of 2000

“…VP1 is the major surfaceaccessible protein in the mature picornavirus virion and contributes to all three major neutralization sites that have been identified on the poliovirus surface [Minor, 1990;Mateu, 1995]. The BC-loop, which participates to form the antigenic site 1, has proven to include a serotype-specific antigenic neutralization site [Minor, 1990;Norder et al, 2001;Reimann et al, 1991]. The BCloop covers the amino acids (AA) 82-93 of the 295-AA Fig.…”

Molecular Typing of Enteroviruses: Current Status and Future Requirements

“…Three or four neutralization determinants have been identified for each PV serotype by using monoclonal antibody neutralization escape mutants, and they have been mapped to VP1, VP2, and/or VP3 (reviewed in reference 278). Neutralization determinants have also been identified on VP1 for CBV 3 (149) and CBV 4 (266,359). Determinants of attenuation of virulence (52,278,355), virion thermostability (278), altered host range (82), in vitro cell tropism (245), persistent infection (53,331), and plaque morphology (354,488) have also been mapped to the capsid-encoding region.…”

“…Most linear epitopes identified to date are cross-reactive and nonneutralizing (58,168,386,387). However, important neutralization determinants formed from uninterrupted colinear amino acid residues have been identified for PV (278,280), CBV 3 (149), and CBV 4 (266,359), and it may be possible to identify "signature" neutralizing epitopes from the nucleotide sequence of the relevant genomic region which permit serotypic identification. It would be necessary to identify epitopes conserved among a wide range of field isolates for the development of molecular typing reagents.…”