RESUMO
A candidate vaccine against botulinum neurotoxin serotype A (BoNT/A) was developed by using a Venezuelan equine encephalitis (VEE) virus replicon vector. This vaccine vector is composed of a self-replicating RNA containing all of the VEE nonstructural genes and cis-acting elements and also a heterologous immunogen gene placed downstream of the subgenomic 26S promoter in place of the viral structural genes. In this study, the nontoxic 50-kDa carboxy-terminal fragment (H(C)) of the BoNT/A heavy chain was cloned into the replicon vector (H(C)-replicon). Cotransfection of BHK cells in vitro with the H(C)-replicon and two helper RNA molecules, the latter encoding all of the VEE structural proteins, resulted in the assembly and release of propagation-deficient, H(C) VEE replicon particles (H(C)-VRP). Cells infected with H(C)-VRP efficiently expressed this protein when analyzed by either immunofluorescence or by Western blot. To evaluate the immunogenicity of H(C)-VRP, mice were vaccinated with various doses of H(C)-VRP at different intervals. Mice inoculated subcutaneously with H(C)-VRP were protected from an intraperitoneal challenge of up to 100,000 50% lethal dose units of BoNT/A. Protection correlated directly with serum enzyme-linked immunosorbent assay titers to BoNT/A. The duration of the immunity achieved was tested at 6 months and at 1 year postvaccination, and mice challenged at these times remained refractory to challenge with BoNT/A.
Assuntos
Vacinas Bacterianas/imunologia , Toxinas Botulínicas Tipo A/imunologia , Botulismo/prevenção & controle , Encefalomielite Equina Venezuelana/genética , Replicon/genética , Animais , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Toxinas Botulínicas Tipo A/genética , Linhagem Celular , Clostridium botulinum/imunologia , Clostridium botulinum/metabolismo , Encefalomielite Equina Venezuelana/metabolismo , Vetores Genéticos , Camundongos , Camundongos Endogâmicos BALB C , Vacinação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
The complete nuleotide and predicted amino acid sequences of Venezuelan equine encephalitis (VEE) virus subtype IE (isolate 68U201) were determined and compared to those of other antigenic variants within the VEE complex, strains IAB-TrD, IC-P676, ID-3880, IE-Menall, and II-Fe3-7c. The 68U201 structural proteins were most closely related to their Menall counterparts (97--100% identity) and more distantly related to VEE strains of other antigenic varieties (83--93% identity). With the exception of nsP3, the 68U201 nonstructural proteins were 94--95% identical to those of TrD, P676, and 3880 (nonstructural gene sequences are not available for Menall and Fe3-7c). The amino-terminal region of nsP3 (aa 1--329), which is highly conserved among all alphaviruses, was 93--94% identical for all VEE strains. The nsP3 carboxyl region is highly divergent among alphaviruses in general, but well conserved among previously sequenced VEE strains (>90% identity). Surprisingly, the carboxyl region of 68U201 nsP3 (aa 330--563) was only 59--61% identical to that of subtype IAB, IC, and ID viruses, with large insertions and deletions in addition to numerous substitutions. The differences between the 68U201 and other VEE nsP3 carboxyl regions were not randomly distributed, as there were four domains of high similarity within the nonconserved region. To examine this divergence more closely, we sequenced a portion of the Menall ns3 gene. The 68U201 and Menall nsP3 nonconserved regions were 85.3% identical and had the same basic domain structure, which was distinct from the IAB, IC, and ID nsP3 proteins, suggesting that the domain structure of nsP3 may be subtype/variety-specific. VEE nsP3 sequence diversity may reflect ecological differences such as adaptation to different mosquito vectors or vertebrate hosts.