RESUMO
Humans exposed to rabies virus must be promptly treated by passive immunization with anti-rabies antibody and active immunization with rabies vaccine. Currently, antibody prepared from pooled human serum or from immunized horses is utilized. However, neither of these reagents are readily available, entirely safe, or consistent in their biological activity. An ideal reagent would consist of a panel of human monoclonal antibodies. Such antibodies are now available, their only drawback being the cost of production. Using recombinant technology, we constructed a rabies virus-based vector which expresses high levels (approximately 60 pg/cell) of rabies virus-neutralizing human monoclonal antibody. The vector is a modified vaccine strain of rabies virus in which the rabies virus glycoprotein has been replaced with a chimeric vesicular stomatitis virus glycoprotein, and both heavy and light chain genes encoding a human monoclonal antibody have been inserted. This recombinant virus can infect a variety of mammalian cell lines and is non-cytolytic, allowing the use of cell culture technology routinely employed to produce rabies vaccines.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Vetores Genéticos , Vírus da Raiva/imunologia , Rhabdoviridae , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Expressão Gênica , Humanos , Testes de Neutralização , Plasmídeos , Vírus da Raiva/genética , Células Tumorais CultivadasRESUMO
Rabies virus is not a single entity but consists of a wide array of variants that are each associated with different host species. These viruses differ greatly in the antigenic makeup of their G proteins, the primary determinant of pathogenicity and major inducer of protective immunity. Due to this diversity, existing rabies vaccines have largely been targeted to individual animal species. In this report, a novel approach to the development of rabies vaccines using genetically modified, reverse-engineered live attenuated rabies viruses is described. This approach entails the engineering of vaccine rabies virus containing G proteins from virulent strains and modification of the G protein to further reduce pathogenicity. Strategies employed included exchange of the arginine at position 333 for glutamine and modification of the cytoplasmic domain. The recombinant viruses obtained were non-neuroinvasive when administered via a peripheral route. The ability to confer protective immunity depended largely upon conservation of the G protein antigenic structure between the vaccine and challenge virus, as well as on the route of immunization.
Assuntos
Vacina Antirrábica/genética , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/genética , Sequência de Bases , Primers do DNA/genética , Feminino , Engenharia Genética , Glicoproteínas/genética , Glicoproteínas/imunologia , Injeções Intramusculares , Camundongos , Testes de Neutralização , Raiva/imunologia , Raiva/prevenção & controle , Raiva/veterinária , Vacina Antirrábica/administração & dosagem , Vírus da Raiva/genética , Vírus da Raiva/imunologia , Vírus da Raiva/patogenicidade , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Virulência/genética , Virulência/imunologiaRESUMO
Novel viral vectors that are able to induce both strong and long-lasting immune responses may be required as effective vaccines for human immunodeficiency virus type 1 (HIV-1) infection. Our previous experiments with a replication-competent vaccine strain-based rabies virus (RV) expressing HIV-1 envelope protein from a laboratory-adapted HIV-1 strain (NL4-3) and a primary HIV-1 isolate (89.6) showed that RV-based vectors are excellent for B-cell priming. Here we report that cytotoxic T-lymphocyte (CTL) responses against HIV-1 gp160 are induced by recombinant RVs. Our results indicated that a single inoculation of mice with an RV expressing HIV-1 gp160 induced a solid and long-lasting memory CTL response specific for HIV-1 envelope protein. Moreover, CTLs from immunized mice were not restricted to the homologous HIV-1 envelope protein and were able to cross-kill target cells expressing HIV-1 gp160 from heterologous HIV-1 strains. These studies further suggest promise for RV-based vectors to elicit a persistent immune response against HIV-1 and their potential utility as efficacious anti-HIV-1 vaccines.
Assuntos
Vetores Genéticos , Proteína gp160 do Envelope de HIV/imunologia , HIV-1/imunologia , Vírus da Raiva , Linfócitos T Citotóxicos/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Reações Cruzadas , Citotoxicidade Imunológica , Feminino , Proteína gp160 do Envelope de HIV/genética , HIV-1/isolamento & purificação , Humanos , Camundongos , Camundongos Endogâmicos BALB C , VacinaçãoRESUMO
To investigate the importance of the rabies virus (RV) glycoprotein (G) in protection against rabies, we constructed a recombinant RV (rRV) in which the RV G ecto- and transmembrane domains were replaced with the corresponding regions of vesicular stomatitis virus (VSV) glycoprotein (rRV-VSV-G). We were able to recover rRV-VSV-G and found that particle production was equal to rRV. However, the budding of the chimeric virus was delayed and infectious titers were reduced 10-fold compared with the parental rRV strain containing RV G. Biochemical analysis showed equal replication rates of both viruses, and similar amounts of wild-type and chimeric G were present in the respective viral particles. Additional studies were performed to determine whether the immune response against rRV-VSV-G was sufficient to protect against rabies. Mice were primed with rRV or rRV-VSV-G and challenged with a pathogenic strain of RV 12 days later. Similar immune responses against the internal viral proteins of both viruses indicated successful infection. All mice receiving the rRV vaccine survived the challenge, whereas immunization with rRV-VSV-G did not induce protection. The results confirm the crucial role of RV G in an RV vaccine.
Assuntos
Antígenos Virais , Glicoproteínas/imunologia , Glicoproteínas de Membrana , Vacina Antirrábica/imunologia , Vírus da Raiva/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Feminino , Expressão Gênica , Glicoproteínas/metabolismo , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Processamento de Proteína Pós-Traducional , Raiva/prevenção & controle , Vírus da Raiva/genética , Vírus da Raiva/fisiologia , Recombinação Genética , Vacinação , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vírion , Replicação ViralRESUMO
The rabies virus glycoprotein (G) gene of the highly neuroinvasive and neurotropic strains SHBRV-18, CVS-N2c, and CVS-B2c was introduced into the non-neuroinvasive and less neurotropic SN-10 strain to provide further insight into the role of G in the pathogenesis of rabies. Phenotypic analyses of the recombinant viruses revealed, as expected, that the neurotropism of a particular rabies virus strain was a function of its G. Nevertheless, the pathogenicity of the recombinant viruses was, in every case, markedly lower than that of the wild-type viruses suggesting that while the G dictates neurotropism, other viral attributes are also important in pathogenesis. The low pathogenicity of the recombinant viruses is at least in part due to a strong increase in transcription activity. On the other hand, the production of infectious virus by the R-SHB18 recombinant virus-infected cells was significantly delayed by comparison with SHBRV-18 wild-type virus infected-cells. Replacement of the R-SHB18 G cytoplasmic domain, transmembrane domain, and stem region with its SN-10 G counterparts neither results in a significant increase in budding efficiency nor an increase in pathogenicity. These results suggest that an optimal match of the cytoplasmic domain of G with the matrix protein may not be sufficient for maximal virus budding efficiency, which is evidently a major factor of virus pathogenicity. Our studies indicate that to maintain pathogenicity, the interactions between various structural elements of rabies virus must be highly conserved and the expression of viral proteins, in particular the G protein, must be strictly controlled.