RESUMO
BACKGROUND: The Venezuelan equine encephalitis (VEE) virus replicon system was used to produce virus-like replicon particles (VRP) packaged with a number of different VEE-derived glycoprotein (GP) coats. The GP coat is believed to be responsible for the cellular tropism noted for VRP and it is possible that different VEE GP coats may have different affinities for cells. We examined VRP packaged in four different VEE GP coats for their ability to infect cells in vitro and to induce both humoral and cellular immune responses in vivo. METHODOLOGY/PRINCIPAL FINDINGS: The VRP preparations were characterized to determine both infectious units (IU) and genome equivalents (GE) prior to in vivo analysis. VRP packaged with different VEE GP coats demonstrated widely varying GE/IU ratios based on Vero cell infectivity. BALB/c mice were immunized with the different VRP based on equal GE titers and the humoral and cellular responses to the expressed HIV gag gene measured. The magnitude of the immune responses measured in mice revealed small but significant differences between different GP coats when immunization was based on GE titers. CONCLUSIONS/SIGNIFICANCE: We suggest that care should be taken when alternative coat proteins are used to package vector-based systems as the titers determined by cell culture infection may not represent accurate particle numbers and in turn may not accurately represent actual in vivo dose.
Assuntos
Vírus da Encefalite Equina Venezuelana/metabolismo , Replicon , Animais , Encefalomielite Equina Venezuelana/virologia , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos , Genoma , Glicoproteínas/química , Sistema Imunitário , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We used a propagation-defective, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the hemagglutinin (HA) and neuraminidase (NA) proteins from influenza A/Wyoming/03/2003 (H3N2). Efficient production methods were scaled to produce pilot lots of HA VRP and NA VRP and clinical lots of HA VRP. HA VRP-induced high-titered antibody responses in mice, rabbits and rhesus macaques, as measured by ELISA or hemagglutination inhibition (HI) assays, and robust cellular immune responses in mice and rhesus macaques, as measured by IFN-gamma ELISPOT. NA VRP also induced cellular immune responses in mice. A toxicology study with HA VRP and NA VRP in rabbits showed no adverse effects in any parameter. These studies support clinical testing of alphavirus replicon vaccines for influenza.
Assuntos
Alphavirus/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/imunologia , Neuraminidase/imunologia , Animais , Anticorpos Antivirais , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/genética , Imunidade Celular , Vacinas contra Influenza/genética , Macaca mulatta , Camundongos , Coelhos , RepliconRESUMO
We used a replication-incompetent, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the extracellular domain of human cytomegalovirus (CMV) glycoprotein B or a pp65/IE1 fusion protein. Efficient production methods were scaled to produce pilot lots and clinical lots of each alphavirus replicon vaccine component. The vaccine induced high-titered antibody responses in mice and rabbits, as measured by ELISA and CMV neutralization assays, and robust T-cell responses in mice, as measured by IFN-gamma ELISPOT assay. A toxicity study in rabbits showed no adverse effects in any toxicology parameter. These studies support clinical testing of this novel CMV alphavirus replicon vaccine in humans.
Assuntos
Alphavirus/genética , Vacinas contra Citomegalovirus/genética , Citomegalovirus/genética , Citomegalovirus/imunologia , Animais , Anticorpos Antivirais/sangue , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/prevenção & controle , Vacinas contra Citomegalovirus/imunologia , Vacinas contra Citomegalovirus/toxicidade , Feminino , Vetores Genéticos , Humanos , Imunidade Celular , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Coelhos , Replicon , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/toxicidadeRESUMO
PURPOSE: Prostate-specific membrane antigen (PSMA) is an attractive target for active immunotherapy. Alphavirus vaccines have shown promise in eliciting immunity to tumor antigens. This study investigated the immunogenicity of alphavirus vaccine replicon particles (VRP) that encode PSMA (PSMA-VRP). EXPERIMENTAL DESIGN: Cells were infected with PSMA-VRP and evaluated for PSMA expression and folate hydrolase activity. Mice were immunized s.c. with PSMA-VRP or purified PSMA protein. Sera, splenocytes, and purified T cells were evaluated for the magnitude, durability, and epitope specificity of the anti-PSMA response. Antibodies were measured by flow cytometry, and cellular responses were measured by IFN-gamma enzyme-linked immunospot and chromium release assays. Cellular responses in BALB/c and C57BL/6 mice were mapped using overlapping 15-mer PSMA peptides. A Good Laboratory Practice-compliant toxicology study was conducted in rabbits. RESULTS: PSMA-VRP directed high-level expression of active PSMA. Robust T-cell and B-cell responses were elicited by a single injection of 2 x 10(5) infectious units, and responses were boosted following repeat immunizations. Anti-PSMA responses were detected following three immunizations with 10(2) infectious units and increased with increasing dose. PSMA-VRP was more immunogenic than adjuvanted PSMA protein. Responses to PSMA-VRP were characterized by Th-1 cytokines, potent CTL activity, and IgG2a/IgG2b antibodies. T-cell responses in BALB/c and C57BL/6 mice were directed toward different PSMA peptides. Immunogenic doses of PSMA-VRP were well tolerated in mice and rabbits. CONCLUSIONS: PSMA-VRP elicited potent cellular and humoral immunity in mice, and specific anti-PSMA responses were boosted on repeat dosing. PSMA-VRP represents a promising approach for immunotherapy of prostate cancer.
