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Apoptosis is considered to be a way of eliminating unwanted cells without causing major inflammation. Nevertheless, several lines of evidence show that apoptotic cell-derived antigens can be strong immunogens. The rabies virus glycoprotein G-ERA is an apoptotic molecule. We tested the ability of G-ERA to potentiate a B cell response against an exogenous antigen (influenza hemagglutinin, HA). We found that co-expression of G-ERA and HA in apoptotic bodies increased both the primary and memory HA-specific immune responses. The immunopotentiation of G-ERA is apoptosis-mediated but not necrosis-mediated. Our data indicate that G-ERA-mediated apoptosis might be useful to improve the immunogenicity of live vaccines.

This study sought to identify the RV protein that causes apoptosis. For this purpose, we first compared the ability of G and N proteins of a pathogenic and a nonpathogenic strain to trigger apoptosis of Jurkat rtTA by using an inducible Tet-on expression system. Then we analyzed apoptosis induced by a reverse genetic-engineered recombinant rabies virus in which the G gene from a nonpathogenic strain was replaced by its pathogenic strain counterpart. No other virus proteins than G of nonpathogenic RV strains induce apoptosis, and the G polypeptide of RV is a critical determinant for apoptosis in human cells.

We showed that, unlike pathogenic rabies virus (RV) strain CVS, attenuated RV strain ERA triggers the caspase-dependent apoptosis of human cells. Furthermore, we observed that the induction of apoptosis is correlated with a particular virus antigen distribution: the overexpression of the viral G protein on the cell surface, with continuous localization on the cytoplasmic membrane, and large cytoplasmic inclusions of the N protein. To determine whether one of these two major RV proteins (G and N proteins) triggers apoptosis, we constructed transgenic Jurkat T-cell lines that drive tetracycline-inducible gene expression to produce the G and N proteins of ERA and CVS individually. The induction of ERA G protein (G-ERA) expression but not of ERA N protein expression resulted in apoptosis, and G-ERA was more efficient at triggering apoptosis than was CVS G protein. To test whether other viral proteins participated in the induction of apoptosis, human cells were infected with recombinant RV in which the G protein gene from the attenuated strain had been replaced by its virulent strain counterpart (CVS). Only RV containing the G protein from the nonpathogenic RV strain was able to trigger the apoptosis of human cells. Thus, the ability of RV strains to induce apoptosis is largely determined by the viral G protein.

Viral superantigens bind several alleles and isotypes belonging to the MHC class II and subsequently activate particular T cell families via the variable portion of the beta chain of TCR. As a result, a superantigen bridges MHC and TCR molecules, leading to activation of T and B cells. The T expansion of various TCR V beta subsets is triggered on the basis of their V beta specificity, but not on their antigenic specificity. The best known superantigens are bacterial endotoxins produced by Staphylococcus aureus. However, viruses such as mouse mammary tumor or rabies viruses encode superantigens too. The ability of superantigens to break the barriers of MHC restriction and to activate large numbers of T and B cells has led to the hypothesis that superantigens may activate autoreactive T and B cells to initiate or worsen autoimmune diseases such as diabetes, multiple sclerosis, or psoriasis.

In this study, we designed a capture ELISA using a monoclonal antibody specific for the N protein, the major protein of the rabies virus nucleocapsid, to measure the N protein content in rabies vaccines. Free N protein content was compared in the two types of rabies vaccine currently used in humans: suckling mouse brain (SMB) vaccine prepared from rabies virus-infected brain tissue, and tissue culture (TC) vaccine prepared from supernatants of rabies virus-infected cells. It was found that SMB vaccines contained considerably higher amounts of N protein than most of the TC vaccines. Possible implications concerning the efficacy of rabies vaccines are discussed.

Superantigens bind class II major histocompatibility proteins and stimulate powerful proliferative responses of T lymphocytes bearing particular V beta sequences as part of their alpha beta antigen receptor. Exogenous bacterial superantigens are responsible for food poisoning and toxic shock syndrome. Murine virus-encoded self-superantigens induce clonal deletion of T lymphocytes. Although superantigen-like properties have been suggested for human immunodeficiency virus-1, no viral superantigen has been identified in humans. Here we report that the nucleocapsid of the rabies virus is an exogenous superantigen specific for V beta 8 human T lymphocytes which binds to HLA class II alpha-chains.

The virion-associated glycoprotein of the CVS-11 strain of rabies virus exists in two forms, GI and GII, which differ in their carbohydrate content. The structural relationship between GI and GII is investigated in order to account for the difference in glycosylation. Partial sequence analysis and mapping of tryptic glycopeptides isolated from the parent CVS-11 GI and GII glycoprotein forms revealed that two of the three predicted glycosylation sites (at Asn-204 and Asn-319) were utilized in the GI form whereas only one of these two sites (at Asn-319) was utilized in the GII form. One predicted glycosylation site (at Asn-37) was not utilized in either species. In the variant virus, RV231-22, a single glycoprotein species was detected which corresponded in electrophoretic mobility to the GI form of the parent virus. Nucleotide sequence analysis of the variant glycoprotein gene revealed a base mutation which specifies an amino acid change six residues upstream from the predicted glycosylation site at Asn-204. This single amino acid substitution apparently results in utilization of the signal at Asn-204 in the GII form of RV231-22 virus. The amino acid substitution is discussed in relation to altered conformation. In the variant virus RV194-2 (F3), both GI and GII glycoprotein forms were present, but each revealed slower electrophoretic mobilities compared with the corresponding parent glycoprotein forms in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An extra glycosylation site was identified in both glycoprotein forms of this variant virus.

Antigenic variation in the glycoprotein of rabies (CVS-11) virus was studied. Neutralization-resistant variant viruses were isolated in vitro at high frequency (10(-4) to 10(-5)) in the presence of anti-glycoprotein monoclonal antibody. Analysis of these variants identified at least three functionally independent antigenic sites, based on the grouping of variants that were no longer neutralized by one or more of a panel of 24 monoclonal antibodies. Competition radioimmunoassay suggested that one of these three antigenic sites was topologically distinct, with the other two in close proximity. In addition, it was shown that most (but not all) neutralization-resistant variants failed to bind the relevant monoclonal antibody. Viruses with altered antigenicity were shown to accumulate in virus stocks following several passages in vitro in the absence of antibody. In addition, variants were isolated in vivo following treatment of mice with monoclonal antibody.

Rabies virus-specific monoclonal antibodies (MAbs) have served to describe operationally the topography of the antigenic structure of the glycoprotein and nucleocapsid proteins of rabies virus. With the use of nucleocapsid protein-specific MAbs and cleavage fragments of the nucleoprotein and phosphoprotein, it has been possible to identify the chemical structure of two antigenic sites of the nucleoprotein and one antigenic site of the phosphoprotein. Antisera produced to synthetic peptides that make up the structure of these antigenic sites exhibited reactivities similar to those of MAbs. Analysis of a large number of isolates of rabies virus from different animal species and from different geographic locations revealed that rabies viruses differ considerably in their antigenic structure and can be identified according to their characteristic reactivity patterns with MAbs. Analysis of field virus isolates has also revealed that strains of rabies virus generally are associated with only one or a few major mammalian hosts within any given geographic area. Protection experiments in mice have not demonstrated correlations between protective activity and degree of antigenic difference between the vaccine strain and the challenge virus. Therefore, changes in antigenic structure, as determined by analysis with rabies virus-specific MAbs, cannot predict whether a given rabies vaccine will protect against a particular field virus.

To locate epitopes on internal antigens of rabies virus, purified N and NS proteins of the nucleocapsid were cleaved at methionine, tryptophan or glutamic acid residues, transferred to nitrocellulose and immunostained using monoclonal antibodies (MAbs) specific for N and NS proteins, respectively. Five MAb-positive fragments of N protein and one fragment of NS protein were located after NH2-terminal amino acid sequence analysis within the deduced amino acid sequences of N and NS proteins. Antigenic analysis of synthetic overlapping peptides corresponding to the amino acid sequences of these fragments localized two major antigenic sites of N protein and one antigenic site of NS protein. Like the N- and NS-specific MAbs, anti-peptide antisera produced against the different synthetic antigens either reacted in a type-common fashion with all rabies virus strains, or in a type-specific manner with a restricted number of strains. The synthetic peptides corresponding to the three antigenic regions of the N and NS proteins also stimulated proliferation of human T lymphocytes derived from vaccinees who received inactivated rabies virus vaccine. This suggested that the antigenic regions of N and NS proteins are recognized by both B and T cells.

Thirty-one monoclonal antibodies, specific for either the nucleoprotein (N) or the non-structural protein (NS; nucleocapsid-associated protein) of the nucleocapsid of the ERA strain of rabies virus, were used to investigate the topography of antigenic sites on the nucleocapsid complex. Based on the results of a competitive enzyme immunoabsorbent assay using these antibodies, five spatially distinct antigenic sites were defined: three on the N protein (groups N I, N II and N III) and two on the NS protein (groups NS I and NS II). Antigenic variations among various street and laboratory strains of rabies virus were analysed by indirect immunofluorescence assay with the monoclonal antibodies specific for the nucleocapsid. Some correlation between the natural nucleocapsid variation and the antigenic topographical map was observed.