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
RNA replicons derived from an attenuated strain of Venezuelan equine encephalitis virus (VEE), an alphavirus, were configured as candidate vaccines for Ebola hemorrhagic fever. The Ebola nucleoprotein (NP) or glycoprotein (GP) genes were introduced into the VEE RNA downstream from the VEE 26S promoter in place of the VEE structural protein genes. The resulting recombinant replicons, expressing the NP or GP genes, were packaged into VEE replicon particles (NP-VRP and GP-VRP, respectively) using a bipartite helper system that provided the VEE structural proteins in trans and prevented the regeneration of replication-competent VEE during packaging. The immunogenicity of NP-VRP and GP-VRP and their ability to protect against lethal Ebola infection were evaluated in BALB/c mice and in two strains of guinea pigs. The GP-VRP alone, or in combination with NP-VRP, protected both strains of guinea pigs and BALB/c mice, while immunization with NP-VRP alone protected BALB/c mice, but neither strain of guinea pig. Passive transfer of sera from VRP-immunized animals did not confer protection against lethal challenge. However, the complete protection achieved with active immunization with VRP, as well as the unique characteristics of the VEE replicon vector, warrant further testing of the safety and efficacy of NP-VRP and GP-VRP in primates as candidate vaccines against Ebola hemorrhagic fever.
Assuntos
Vírus da Encefalite Equina Venezuelana/imunologia , Doença pelo Vírus Ebola/prevenção & controle , RNA Viral/administração & dosagem , Replicon/imunologia , Animais , Chlorocebus aethiops , Ebolavirus/genética , Ebolavirus/imunologia , Ebolavirus/metabolismo , Vírus da Encefalite Equina Venezuelana/genética , Glicoproteínas/biossíntese , Glicoproteínas/genética , Glicoproteínas/imunologia , Cobaias , Doença pelo Vírus Ebola/imunologia , Imunização Passiva , Camundongos , Nucleoproteínas/biossíntese , Nucleoproteínas/genética , Nucleoproteínas/imunologia , RNA/administração & dosagem , Replicon/genética , Vacinas Atenuadas/administração & dosagem , Células VeroRESUMO
A replicon vaccine vector system was developed from an attenuated strain of Venezuelan equine encephalitis virus (VEE). The replicon RNA consists of the cis-acting 5' and 3' ends of the VEE genome, the complete nonstructural protein gene region, and the subgenomic 26S promoter. The genes encoding the VEE structural proteins were replaced with the influenza virus hemagglutinin (HA) or the Lassa virus nucleocapsid (N) gene, and upon transfection into eukaryotic cells by electroporation, these replicon RNAs directed the efficient, high-level synthesis of the HA or N proteins. For packaging of replicon RNAs into VEE replicon particles (VRP), the VEE capsid and glycoproteins were supplied in trans by expression from helper RNA(s) coelectroporated with the replicon. A number of different helper constructs, expressing the VEE structural proteins from a single or two separate helper RNAs, were derived from attenuated VEE strains Regeneration of infectious virus was not detected when replicons were packaged using a bipartite helper system encoding the VEE capsid protein and glycoproteins on two separate RNAs. Subcutaneous immunization of BALB/c mice with VRP expressing the influenza HA or Lassa virus N gene (HA-VRP or N-VRP, respectively) induced antibody responses to the expressed protein. After two inoculations of HA-VRP, complete protection against intranasal challenge with influenza was observed. Furthermore, sequential immunization of mice with two inoculations of N-VRP prior to two inoculations of HA-VRP induced an immune response to both HA and N equivalent to immunization with either VRP construct alone. Protection against influenza challenge was unaffected by previous N-VRP immunization. Therefore, the VEE replicon system was characterized by high-level expression of heterologous genes in cultured cells, little or no regeneration of plaque-forming virus particles, the capability for sequential immunization to multiple pathogens in the same host, and induction of protective immunity against a mucosal pathogen.
Assuntos
Proteínas do Capsídeo , Capsídeo/imunologia , Vírus Defeituosos/fisiologia , Vírus da Encefalite Equina Venezuelana/genética , Vetores Genéticos/genética , Vírus Auxiliares/fisiologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/imunologia , Vírus Lassa/imunologia , Replicon , Vacinas Combinadas/genética , Vacinas Sintéticas/genética , Vacinas Virais/genética , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Capsídeo/biossíntese , Capsídeo/genética , Linhagem Celular , Embrião de Galinha , Chlorocebus aethiops , Cricetinae , Patos , Vírus da Encefalite Equina Venezuelana/imunologia , Vírus da Encefalite Equina Venezuelana/fisiologia , Fibroblastos , Regulação Viral da Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/biossíntese , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , Vírus Lassa/genética , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/prevenção & controle , RNA/genética , RNA Viral/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinação , Vacinas Atenuadas/imunologia , Vacinas Combinadas/imunologia , Vacinas Sintéticas/imunologia , Células Vero , Proteínas Estruturais Virais/biossíntese , Proteínas Estruturais Virais/genética , Vacinas Virais/imunologiaRESUMO
We have cloned and sequenced the L1 and L2 genes from human papillomavirus type 16 (HPV16) DNA-containing cervical cytology samples collected from the U.K. and Trinidad. Samples containing high copy numbers of HPV16 DNA were selected as being likely to contain fully functional virus DNA molecules in an episomal state, rather than in an integrated and possibly altered state. In comparison with the previously published sequence of HPV16 isolated from an invasive cancer a variety of differences were detected in both L1 and L2. The pattern of changes appears to be different in samples from the two geographic regions. One of the differences (resulting in D at position 202 of the L1 protein) reported recently to be functionally important for virus particle assembly was found to occur in all the samples examined. Variations in L1 found within known immunoreactive regions or hydrophobic domains should be taken into account in design of prophylactic vaccines for HPV16 based on virus-like particles. All variations within L2 protein were found in hydrophilic domains in the carboxy-terminal half of L2. These positions were highly variable among other types of papillomavirus and are located outside the known L2 immunoreactive region.
Assuntos
Proteínas do Capsídeo , Capsídeo/genética , Variação Genética/genética , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Infecções Tumorais por Vírus/virologia , Aminoácidos/análise , Capsídeo/química , Clonagem Molecular , Sequência Consenso/genética , DNA Viral/isolamento & purificação , Feminino , Genes Virais , Humanos , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Mutação Puntual/genética , Análise de Sequência de DNA , Trinidad e Tobago , Reino Unido , Neoplasias do Colo do Útero/virologia , Displasia do Colo do Útero/virologiaRESUMO
We have cloned and sequenced the L1 and L2 genes from human papillomavirus type 16 (HPV16) DNA-containing cervical cytology samples collected from the U.K. and Trinidad. Samples containing high copy numbers of HPV16 DNA were selected as being likely to contain fully functional virus DNA molecules in an episomal state, rather than in an integrated and possibly altered state. In comparison with the perviously published sequence of HPV16 isolated from an invasive cancer a variety of differences were detected in both L1 and L2. The pattern of changes appears to be different in samples from the two geographic regions. One of the differences (resulting in D at position 202 of the L1 protein) reported recently to be functionally important for virus particle assembly was found to occur in all the samples examined. Variations in L1 found within known immunoreactive regions or hydrophobic domains should be taken into account in design of prophylactic vaccines for HPV16 based on virus-like particles. All variations within L2 protein were found in hydrophilic domains in the carboxy-terminal half of L2. These positions were highly variable among other types of papillomavirus and are located outside the known L2 immunoreactive region. (AU)