Tadeusz J. Wiktor

Antigenic sites on the CVS rabies virus glycoprotein: analysis with monoclonal antibodies.

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.

Use of Hybridoma Monoclonal Antibodies in the Detection of Antigenic Differences Between Rabies and Rabies-related Virus Proteins. II. The Glycoprotein

Twenty-five hybridoma cultures secreted monoclonal antibodies directed against the glycoprotein of rabies or rabies-related viruses. The antibodies had different specificities for the glycoproteins of eight rabies and rabies-related viruses. They could be classified into fourteen groups which probably correspond to different antigenic determinants on the glycoproteins. These hybridomas when used in either radioimmunoassay (RIA) or in neutralization tests allow differentiation of laboratory strains of rabies virus from each other as well as from the rabies-related viruses.

Use of monoclonal antibodies in diagnosis of rabies virus infection and differentiation of rabies and rabies-related viruses

Abstract A panel of selected monoclonal antibodies directed against the nucleocapsid antigens of rabies and rabies-related viruses allowed the rapid identification both of antigens of rabies virus origin present in impression smears of brains from infected animals and of three rabies-related viruses (Duvenhage, Lagos bat and Mokola). Within the rabies group, the CVS and Flury HEP strains could also be identified. Analysis of several fixed and street rabies viruses reveals great diversity in the reactivity of the viruses with a panel of monoclonal antibodies directed against glycoprotein antigens. Strains of the same geographical area and species origin displayed similar patterns of reactivity, suggesting an antigenic specificity related to animal species or area. The use of hybridoma monoclonal antibodies should provide a more sophisticated method than presently available for the diagnosis of rabies virus infection.

Chemical and immunological analysis of the rabies soluble glycoprotein.

Soluble glycoprotein (Gs), purified from virion-depleted, rabies-infected tissue culture fluid, was chemically and immunologically analyzed. A comparison of this antigen with the virion-associated glycoprotein showed that Gs lacks 58 amino acid residues from the carboxy terminus of the virion-associated glycoprotein. Analysis with monoclonal antibodies revealed that all the epitopes of the viral glycoprotein are also present in the soluble glycoprotein. However, when tested for its ability to protect mice against a lethal challenge infection with rabies virus, Gs in contrast to viral glycoprotein, showed no protective activity. These results suggest that the carboxy terminus of the rabies virus glycoprotein is necessary for its full protective activity even though this portion of the glycoprotein molecule does not contain any antigenic determinants.

RABIES SUBUNIT VACCINES

ConclusionsnThe secondary and tertiary structures of the rabies virus spike G protein are important for its ability to induce VN antibodies and confer immunity to the host. For a subunit peptide vaccine to be as effective as the native spike G protein, it would appear that the amino acid sequence comprising the antigenic determinant for VN antibody binding must be made to fold properly even when deprived of its native support structure. Since CNBr peptides have retained at least some of their antigenicity for binding antibodies from hyperimmune serum but not monoclonal VN antibodies, and their immunogenicity, then synthetic peptides containing corresponding sequences should show similar activities. Additionally, determinants that might be necessary for stimulating T lymphocytes would have to be built into the synthetic peptide preparation. It would also appear that a properly folded peptide might have to be aggregated into suitably large particles for it to achieve its full protective effect. Adjuvants may serve in this capacity to enhance the immune response to relevant peptides and thus improve the immunogenicity of a subunit vaccine that ultimately protects animals and humans against rabies virus infection.

Antigenic Sites on the ERA Rabies Virus Nucleoprotein and Non-structural Protein

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.

Immunogenic properties of vaccinia recombinant virus expressing the rabies glycoprotein

Summary The ability to construct recombinant viruses expressing foreign genes clearlyhas great potential for vaccine development. In the case of rabies, the V-RG virus has proved to be highly immunogenic, and has outperformed conventional rabies vaccines in every comparative protection test. The question remains, however, whether recombinant vaccines based on vaccinia viruses will ever be accepted for human or veterinary use. In this regard, it should be noted that the lessons learned in the construction and evaluation of V-RG virus can be rapidly applied for the development of other recombinant viruses (bases on adenoviruses, for example).

The inhibition of rabies virus by arabinosyl cytosine. Studies on the mechanism and specificity of action

Abstract Arabinosyl cytosine (ara-C) inhibits the replication of rabies virus strains in all tissue culture cell systems tested, including the BHK-21, RK 13 , and WI-38 cell lines. The growth of other RNA-containing viruses (Egtved, NDV, West Nile, Sindbis, WEE, LCM, Reo type 1, and Mengo) is not significantly affected by the antimetabolite, although the infective titers of both the Indiana and New Jersey serotypes of VSV and cocal virus are reduced 2- to 20-fold under the conditions used. When actinomycin D, nogalamycin, cycloheximide, or puromycin are added during the first 3 hours of rabies viral replication in BHK-21 cells in the presence of ara-C, the inhibitory effect of the pyrimidine nucleoside analog is partially or completely reversed. The data are consistent with the hypothesis that ara-C requires the induction of a cellular protein for its specific inhibitory action on rabies virus growth, and that this protein exerts its inhibitory effect at an early stage in viral replication.

Localization of immunogenic domainson the rabies virus glycoprotein

Resume Induire une reponse immunitaire a des virus rabiques est la fonction primordialedun vaccin antirabique, quil sagisse dun traitement preventif chez les animaux et les humains ou dun traitement en post-exposition pour lhomme. Le pic de surface de la glycoproteine des virus rabiques, principal responsable de cette induction, a ete etudie dans le but de definir les regions responsables de cette activite immunogenique et antigenique sur la molecule. Il a ete mis en evidence que la region N-terminale de la glycoproteine est capable de stimuler lactivite des lymphocytes T vis-a-vis du virus rabique, in vitro . Tandis que des peptides synthetiques etaient utilises dans le but de stimuler la multiplication des lymphocytes T in vitro , seules certaines sequences derivees de la region N-terminale de la glycoproteine se montraient actives. Ainsi la presence dun determinant essentiel des lymphocytes T a pu etre miseen evidence au niveau de lextremite C-terminale de 44 fractions situees dans la region N-terminale de la molecule. Des acides amines, capables dagir soit en tant que fractions de contact au niveau des epitopes (sites permettant la reaction virus-anticorps neutralisant) sur la glycoproteine, soit en provoquant des changements conformationnels, ont ete identifies chez des virus dont la partie antigenique de la glycoproteine est alteree. La region active du site antigenique III a tout particulierement ete etudiee.Ainsi des substitutions provoquant des charges locales et des changements conformationnels seront decrites en tant que variants antigeniques permettant de tracer le site antigenique III.

Rabies Virus Pathogenicity

Cell-mediated immunity plays a major role in pathogenicity of rabies virus infection, and is probably also involved in the mechanism of post-exposure rabies prophylaxis. Live-attenuated rabies virus vaccines (ERA, HEP, Kelev) induce a strong cytotoxic T lymphocyte (CTL)-mediated response following i.c. inoculation. This reaction is a result of virus replication, since it is not dose-dependent. Similar levels of CTL response are generated by a wide range of virus dilutions. Conversely, infection with pathogenic rabies viruses does not result in generation of CTL and induces a total suppression of this T cell subpopulation. Other immune reactions and induction of interferon production are the same in animals infected with pathogenic and non-pathogenic viruses.