Antigenic diversity of the glycoprotein and nucleocapsid proteins of rabies and rabies-related viruses: implications for epidemiology and control of rabies.

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.

Epidemiology of rabies virus variants. Differentiation using monoclonal antibodies and discriminant analysis.

Rabies virus was isolated by cell culture from the brains of 104 confirmed rabies cases diagnosed by the fluorescent-antibody staining technique in the United States during 1974-1984. Eighty-seven isolates were obtained from wild-life species, 10 from humans, and seven from domestic animals. These isolates were tested in virus neutralization and immunofluorescence assays using a panel of 34 monoclonal antibodies specific for rabies virus nucleocapsid protein, 44 monoclonal antibodies specific for rabies virus glycoprotein, and two monoclonal antibodies specific for rabies virus nucleocapsid-associated phosphoprotein. Using discriminant analysis, a distinctly different reactivity pattern was revealed between virus isolates from terrestrial (raccoon, skunk) and nonterrestrial (bat) reservoir hosts. The usefulness of this approach for studying the epidemiology of rabies and for predicting the source of infection when this information is unknown is discussed.

Immune response in skunks to a vaccinia virus recombinant expressing the rabies virus glycoprotein.

Striped skunks (Mephitis mephitis) were vaccinated with a vaccinia virus recombinant expressing the rabies virus glycoprotein. Virus neutralizing antibodies to rabies virus were present at 14 days postvaccination by the following routes: scarification (6/6), intramuscular (4/4) and intestinal (5/8). Six out of seven skunks that ate vaccine filled baits had virus neutralizing antibodies at 28 days. When challenged intramuscularly with street virus, the survival rates were 5/7 for the bait-fed group, 4/8 for the intestinal group, 3/4 for the intramuscular group, 5/6 for the animals that were scarified, and 0/8 for controls. This is the first report of a high rate of immunization of skunks with a rabies vaccine administered orally.

Oral immunization and protection of raccoons (Procyon lotor) with a vaccinia-rabies glycoprotein recombinant virus vaccine.

Animal rabies control has been frustrated by the existence of multiple wildlife reservoirs and the lack of efficacious oral vaccines. In this investigation, raccoons fed a vaccinia-rabies glycoprotein recombinant virus in a sponge bait developed rabies virus-neutralizing antibody (0.6-54.0 units) and resisted street rabies virus infection 28 and 205 days after feeding. Additional raccoons immunized by oral infusion with attenuated antigenic variants of rabies virus strains CVS-11 and ERA failed to develop rabies virus-neutralizing antibody. This work demonstrates the feasibility of a recombinant virus vaccine containing the rabies glycoprotein gene for immunization of raccoons, and possibly other wildlife, to obtain long-term protection against rabies.

Molecular mimicry: frequency of reactivity of monoclonal antiviral antibodies with normal tissues.

More than 600 monoclonal antiviral antibodies made against 11 different viruses were screened against 14 different organs from normal uninfected mice. Of these antiviral antibodies, 21, or approximately 3.5%, reacted with specific cells in these organs. Several of these antibodies were of the multiple-organ-reactive type and recognized antigens in more than one organ. It was concluded that the reactivity of monoclonal antiviral antibodies with normal tissues is a common phenomenon.

Isolation and characterization of human T cell lines and clones reactive to rabies virus: antigen specificity and production of interferon-gamma.

By using a preparation of inactivated rabies virus, the blood mononuclear cells from five rabies vaccine recipients were stimulated in vitro in the presence of interleukin 2. T cell lines that displayed significant proliferative responses to whole rabies virus and to preparations of rabies glycoprotein and nucleocapsid were obtained from all the individuals. Other antigens, such as diphtheria and tetanus toxoids, influenza A virus, hepatitis B surface antigen, and serum albumin, failed to induce the proliferation of the T cell lines. One of these rabies-specific T cell lines was found to proliferate in response to rabies antigens only when the antigen-presenting cells expressed homologous HLA-DR antigens. The use of mouse monoclonal antibodies specific for human T cell surface markers revealed that most of the cells of these rabies-reactive lines were of the helper/inducer class of T lymphocytes. Stimulation of the T cell lines with the rabies antigens induced the production of interferon-gamma, a lymphokine with potent antiviral activity. Several T cell clones were isolated from two of these cell lines, and most of them appeared to be specific for the antigenic components of the viral nucleocapsid. Two T cell clones specific for the rabies glycoprotein were also isolated from one of these lymphocyte interleukin 2-dependent lines. Further in vitro studies with rabies-specific T cells could help us to understand in more depth the role of regulatory T cells in the human immune response to rabies virus.

Amplification of rabies virus-induced stimulation of human T-cell lines and clones by antigen-specific antibodies.

The effect of antigen-specific antibodies on the response of human T-cell lines and clones to rabies virus was studied. Plasmas from rabies-immune vaccine recipients, but not those from nonimmune individuals, enhanced the proliferative response of rabies-reactive T cells to whole inactivated virus or to the purified glycoprotein and nucleocapsid from the rabies virion. Rabies-immune plasma also increased the antigen-induced production of gamma interferon by the rabies-specific T-cell lines. Experiments performed on T-cell clones specific for either rabies glycoprotein or nucleocapsid showed that immune plasma as well as antiglycoprotein and antinucleoprotein murine monoclonal antibodies possessed the capacity to increase significantly the antigen-induced proliferative responses of these clones. The overall results indicate that this in vitro effect of antigen-specific antibodies on the response of regulatory T lymphocytes to rabies virus could be an important factor in the development of effective immune responses in vivo to rabies virus.

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.

Differences in cell-to-cell spread of pathogenic and apathogenic rabies virus in vivo and in vitro.

Pathogenic parental rabies virus and apathogenic variant virus were shown to differ in their ability to infect neurons in vivo and neuroblastoma cells in vitro. After intracerebral inoculation, the distribution of infected neurons in the brain was similar for both viruses, but the rate of spread throughout the brain, the number of infected neurons, and the degree of cellular necrosis were much lower in the case of apathogenic virus. After adsorption to mouse neuroblastoma cells, apathogenic virus was less rapidly internalized than pathogenic virus, and cell-to-cell spread of apathogenic variant virus was completely prevented by the addition of rabies virus-neutralizing antibody, whereas the spread of pathogenic virus was not affected.

Antigenic variants of rabies virus in isolates from eastern, central and northern Canada.

Street rabies virus isolated from 51 specimens from Ontario, Quebec, Manitoba and the Northwest Territories have been typed by a panel of 36 antinucleocapsid monoclonal antibodies. Three main groups were found. The first group comprised those terrestrial mammals originating in Ontario, Quebec and the Northwest Territories. The second group was found in terrestrial mammals from Manitoba. The third heterogenous group was made up of bats from Ontario.

Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene.

Inoculation of rabbits and mice with a vaccinia-rabies glycoprotein recombinant (V-RG) virus resulted in rapid induction of high concentrations of rabies virus-neutralizing antibodies and protection from severe intracerebral challenge with several strains of rabies virus. Protection from virus challenge also was achieved against the rabies-related Duvenhage virus but not against the Mokola virus. Effective immunization by V-RG depended on the expression of a rabies glycoprotein that registered proline rather than leucine as the eighth amino acid from its NH2 terminus (V-RGpro8). A minimum dose required for effective immunization of mice was 10(4) plaque-forming units of V-RGpro8 virus. beta-propiolactone-inactivated preparations of V-RGpro8 virus also induced high levels of rabies virus-neutralizing antibody and protected mice against intracerebral challenge with street rabies virus. V-RGpro8 virus was highly effective in priming mice to generate a secondary rabies virus-specific cytotoxic T-lymphocyte response following culture of lymphocytes with either ERA or PM strains of rabies virus.

T cell responses to cleaved rabies virus glycoprotein and to synthetic peptides.

The antigenic structure of the rabies virus glycoprotein has been studied. A limited number of fragments were obtained by cyanogen bromide (CNBr) cleavage of viral glycoprotein, and eight large peptides were isolated by using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These were tested for their capacity to stimulate the proliferation of nylon wool-purified T cells obtained from spleens of rabies-immune A/J mice. Three peptides (Cr1, Cr2 plus Cr2A, and Cr3) stimulated antigen-specific proliferation, indicating that at least three T cell determinants of the native molecule are sequential or continuous in nature. Stimulation was also obtained with 27-residue and 13-residue synthetic peptides (designated R21 and R20, respectively) that included sequences towards the carboxy terminal end of Cr1, but not with synthetic peptides that included sequences of Cr2 and Cr3 (which are both glycosylated in virus-derived material). The intact viral glycoprotein and synthetic peptide R21 stimulated T lymphocytes with surface characteristics of helper cells, and induced the production of interleukin 2 by these lymphocytes. Synthetic peptides R20 and R21 also stimulated a minor population of Lyt-2-positive cells, which were not yet identified as either suppressor or cytotoxic T lymphocytes.

Use of monoclonal antibodies to confirm vaccine-induced rabies in ten dogs, two cats, and one fox.

A panel of 8 monoclonal antibodies to rabies glycoprotein antigen was used to characterize the modified-live virus vaccines marketed in the United States during the last 10 years. Thirteen of 14 rabies virus isolates from 11 dogs, 2 cats, and 1 fox suspected of developing vaccine-induced rabies were shown to have reactivity patterns that were identical to the vaccine administered. Reactivity patterns for 20 rabies isolates from human beings, wild animals, or domestic animals with no history of recent vaccination with modified-live virus rabies vaccine were different from those obtained for vaccines.

Antigenic analysis of rabies and Mokola virus from Zimbabwe using monoclonal antibodies.

Eighteen strains of virus were recovered by tissue culture techniques from 20 samples of mouse brain received from Harare, Zimbabwe, and typed with monoclonal antibodies at The Wistar Institute. On the basis of reactivity with these monoclonal antibodies specific for rabies and rabies-related viruses, seven strains were identified as Mokola viruses, and the remaining 11, as rabies viruses. Seventeen of 36 monoclonal antibodies against the nucleocapsid antigen reacted with the Mokola strains, but none of 42 monoclonal antibodies against the glycoprotein that neutralized rabies virus was active against Mokola strains. Mokola virus was, however, neutralized by two monoclonal antibodies produced from mice immunized with Mokola, by a specific anti-Mokola serum prepared in rabbits, and to a lesser extent, by three polyclonal high titer antirabies sera of human or rabbit origin. Immunization of mice with a rabies vaccine (antigenic value, 10 international units) at a concentration 30-fold high than that necessary for complete protection against homologous challenge with rabies virus was not protective against Mokola infection. No cross-reactivity between Mokola and rabies viruses was seen with cytotoxic T lymphocytes.

Anti-idiotypic antibodies induce neutralizing antibodies to rabies virus glycoprotein.

Rabbit anti-idiotypic antibodies (alpha Id Ab) were prepared against five murine monoclonal antibodies (mAb) specific for the rabies virus glycoprotein. Four of the mAb were directed against three known, type-specific, neutralizing sites on the glycoprotein, and the other mAb was directed against a topographically uncharacterized, nonneutralizing epitope. An absence of significant cross-reactivity among the alpha Id Ab for heterologous mAb suggested that the alpha Id Ab were highly specific for unique variable region determinants. The binding of three of the five alpha Id Ab to their homologous mAb could be inhibited by rabies virus-soluble glycoprotein, suggesting that the alpha Id Ab possessed subpopulations similar or adjacent to the antigen-binding site of the mAb. Two of the five alpha Id Ab injected into mice elicited a specific virus-neutralizing antibody response. Mechanisms to account for the induction of the virus-neutralizing antibody by alpha Id Ab are discussed.

Epidemiologic analysis of antigenic variations of street rabies virus: detection by monoclonal antibodies.

The nucleocapsid antigen of 204 strains of street rabies virus, which originated in Europe, Africa and Asia, was analyzed by fluorescent antibody staining technique with a panel of 20 monoclonal antibodies specific for nucleocapsid of rabies and rabies-related viruses. A definite pattern of reactivity was observed with strains of the same geographic origin with the exception of strains originating from Madagascar, Thailand and Iran which were more diversified. Mice immunized with a vaccine prepared from a Pasteur PV-11 strain of virus were well protected against challenge with representative strains from Europe and Africa, and a partial protection was observed following challenge with strains from Madagascar and Thailand.

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.