RESUMO
Respiratory syncytial virus (RSV) is an important viral pathogen that causes severe lower respiratory tract infection in infants, the elderly, and immunocompromised individuals. There are no licensed RSV vaccines to date. To prevent RSV infection, immune responses in both the upper and lower respiratory tracts are required. Previously, immunization with Venezuelan equine encephalitis virus replicon particles (VRPs) demonstrated effectiveness in inducing mucosal protection against various pathogens. In this study, we developed VRPs encoding RSV fusion (F) or attachment (G) glycoproteins and evaluated the immunogenicity and efficacy of these vaccine candidates in mice and cotton rats. VRPs, when administered intranasally, induced surface glycoprotein-specific virus neutralizing antibodies in serum and immunoglobulin A (IgA) antibodies in secretions at the respiratory mucosa. In addition, fusion protein-encoding VRPs induced gamma interferon (IFN-gamma)-secreting T cells in the lungs and spleen, as measured by reaction with an H-2K(d)-restricted CD8(+) T-cell epitope. In animals vaccinated with F protein VRPs, challenge virus replication was reduced below the level of detection in both the upper and lower respiratory tracts following intranasal RSV challenge, while in those vaccinated with G protein VRPs, challenge virus was detected in the upper but not the lower respiratory tract. Close examination of histopathology of the lungs of vaccinated animals following RSV challenge revealed no enhanced inflammation. Immunization with VRPs induced balanced Th1/Th2 immune responses, as measured by the cytokine profile in the lungs and antibody isotype of the humoral immune response. These results represent an important first step toward the use of VRPs encoding RSV proteins as a prophylactic vaccine for RSV.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vírus Sincicial Respiratório Humano/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Linhagem Celular , Cricetinae , Imunidade nas Mucosas , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Replicon/genética , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/patologia , Infecções por Vírus Respiratório Sincicial/virologia , Vacinas contra Vírus Sincicial Respiratório/genética , Vacinas contra Vírus Sincicial Respiratório/imunologia , Sigmodontinae , Vacinação , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Vírion/genéticaRESUMO
Venezuelan equine encephalitis virus replicon particles (VRP) were engineered to express different forms of SIV Gag to compare expression in vitro, formation of intra- and extracellular structures and induction of humoral and cellular immunity in mice. The three forms examined were full-length myristylated SIV Gag (Gagmyr+), full-length Gag lacking the myristylation signal (Gagmyr-) or a truncated form of Gagmyr- comprising only the matrix and capsid domains (MA/CA). Comparison of VRP-infected primary mouse embryo fibroblasts, mouse L929 cells and primate Vero cells showed comparable expression levels for each protein, as well as extracellular virus-like particles (VRP-Gagmyr+) and distinctive cytoplasmic aggregates (VRP-Gagmyr-) with each cell type. VRP were used to immunize BALB/c mice, and immune responses were compared using an interferon (IFN)-gamma ELISPOT assay and a serum antibody ELISA. Although all three VRP generated similar levels of IFN-gamma-producing cells at 1 week post-boost, at 10 weeks post-boost the MA/CA-VRP-induced response was maintained at a significantly higher level relative to that induced by Gagmyr+-VRP. Antibody responses to MA/CA-VRP and Gagmyr+-VRP were not significantly different.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Produtos do Gene gag/imunologia , Vetores Genéticos/genética , Vírus da Imunodeficiência Símia/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Embrião de Mamíferos/citologia , Ensaio de Imunoadsorção Enzimática , Feminino , Fibroblastos , Produtos do Gene gag/química , Antígenos H-2/imunologia , Interferon gama/biossíntese , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Microscopia Eletrônica de Transmissão , Modelos Animais , Gravidez , Replicon/genética , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Células Vero , Vacinas Virais/genéticaRESUMO
We investigated the immunogenicity of gonococcal transferrin binding protein B (TbpB) expressed with and without a eukaryotic secretion signal from a nonpropagating Venezuelan equine encephalitis virus replicon particle (VRP) delivery system. TbpB was successfully expressed in baby hamster kidney (BHK) cells, and the presence of the eukaryotic secretion signal not only apparently increased the protein's expression but also allowed for extracellular localization and glycosylation. Mice immunized with VRPs produced significant amounts of serum antibody although less than the amounts produced by mice immunized with recombinant protein. The response of mice immunized with VRPs encoding TbpB was consistently more Th1 biased than the response of mice immunized with recombinant protein alone. Boosting with recombinant protein following immunization with TbpB VRPs resulted in higher specific-antibody levels without altering the Th1/Th2 bias. Most of the immunization groups produced significant specific antibody binding to the intact surface of the homologous Neisseria gonorrhoeae strain. Immunization with TbpB VRPs without a eukaryotic secretion signal generated no measurable specific antibodies on the genital mucosal surface, but inclusion of a eukaryotic secretion signal or boosting with recombinant protein resulted in specific immunoglobulin G (IgG) and IgA in mucosal secretions after TbpB VRP immunization. The TbpB VRP system has potential for an N. gonorrhoeae vaccine.
Assuntos
Anticorpos Antibacterianos/biossíntese , Vacinas Bacterianas/administração & dosagem , Gonorreia/imunologia , Neisseria gonorrhoeae/química , Proteínas Recombinantes/imunologia , Replicon/fisiologia , Proteína B de Ligação a Transferrina/administração & dosagem , Animais , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Vírus da Encefalite Equina Venezuelana/genética , Feminino , Imunoglobulina A/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Neisseria gonorrhoeae/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Replicon/genética , Proteína B de Ligação a Transferrina/genética , Proteína B de Ligação a Transferrina/imunologia , Vacinação , Vagina/imunologiaRESUMO
Porin (PorB) is a major outer membrane protein produced by all Neisseria gonorrhoeae strains and has been a focus of intense interest as a vaccine candidate. In this study, the immunogenicity of PorB in mice was investigated after several immunization regimens. Outer membrane vesicles (OMV), recombinant renatured PorB (rrPorB), and PorB-expressing Venezuelan equine encephalitis (VEE) virus replicon particles (PorB VRP) were delivered intranasally (i.n.) or subcutaneously (s.c.) into the dorsal area or the hind footpad in three-dose schedules; the PorB VRP-immunized mice were given a single additional booster dose of rrPorB in Ribi adjuvant. Different delivery systems and administration routes induced different immune responses. Mice immunized s.c. with rrPorB in Ribi had the highest levels of PorB-specific serum immunoglobulin G (IgG) by enzyme-linked immunosorbent assay. Surprisingly, there was an apparent Th1 bias, based on IgG1/IgG2a ratios, after immunization with rrPorB in Ribi in the footpad while the same vaccine given in the dorsal area gave a strongly Th2-biased response. PorB VRP-immunized mice produced a consistent Th1 response with a high gamma interferon response in stimulated splenic lymphocytes and very low IgG1/IgG2a ratios. Immunization by OMV delivered i.n. was the only regimen that resulted in a serum bactericidal response, and it generated an excellent mucosal IgA response. Serum from mice immunized with rrPorB preferentially recognized the surface of whole gonococci expressing a homologous PorB, whereas serum from PorB VRP-immunized mice had relatively low whole-cell binding activity but recognized both heterologous and homologous PorB equally. The data resulting from this direct comparison suggested that important aspects of the immune response can be manipulated by altering the form of the antigen and its delivery. This information coupled with an understanding of protective antigonococcal immune responses will enable the design of the optimal vaccine for N. gonorrhoeae.
Assuntos
Vacinas Bacterianas/imunologia , Vírus da Encefalite Equina Venezuelana/fisiologia , Gonorreia/imunologia , Porinas/administração & dosagem , Porinas/imunologia , Proteínas Recombinantes/imunologia , Replicon/fisiologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Linhagem Celular , Citocinas/biossíntese , Citocinas/metabolismo , Vírus da Encefalite Equina Venezuelana/genética , Feminino , Imunidade nas Mucosas/imunologia , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Porinas/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Replicon/genética , Vacinação , Replicação ViralRESUMO
VEE replicon particles (VRP), non-propagating vaccine vectors derived from Venezuelan equine encephalitis virus (VEE), were engineered to express immunogens from the cloned isolate SIVsmH-4, combined in a vaccine cocktail and inoculated subcutaneously to immunize rhesus macaques. The virulent, uncloned challenge stock, SIVsmE660, represented a type of heterologous challenge and the intrarectal challenge modeled infection across a mucosal surface. Prechallenge neutralizing antibodies against SIVsmH-4 were induced in all vaccinates, and a prechallenge cellular immune response could be detected in one of six. Post-challenge, virus loads were reduced at the peak, at set point and at termination (41 weeks post-challenge), although these differences did not reach statistical significance. Significantly elevated levels of CD4+ T cells were observed post-challenge. A strong correlation was noted between a net increase in CD4+ T cell count and lowered virus load at set point.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Contagem de Linfócito CD4 , Modelos Animais de Doenças , Vetores Genéticos , Imunidade Celular , Injeções Subcutâneas , Macaca mulatta , Testes de Neutralização , Replicon/genética , Vacinas contra a SAIDS/administração & dosagem , Vacinas contra a SAIDS/genética , Linfócitos T Citotóxicos/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Carga Viral , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
The central objective of this research was to test molecularly defined, live attenuated Venezuelan equine encephalitis virus (VEEV) vaccine candidates that were produced through precise genetic manipulation of rationally selected viral nucleotide sequences. Molecular clones of vaccine candidates were constructed by inserting either three independently attenuating mutations or a PE2 cleavage-signal mutation with a second-site resuscitating mutation into full-length cDNA clones. Vaccine candidate viruses were recovered through DNA transcription and RNA transfection of cultured cells, and assessed in rodent and non-human primate models. Based on results from this assessment, one of the PE2 cleavage-signal mutants, V3526, was determined to be the best vaccine candidate for further evaluation for human use.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/biossíntese , Clonagem Molecular , Cricetinae , DNA Complementar/imunologia , Vírus da Encefalite Equina Venezuelana/patogenicidade , Feminino , Macaca fascicularis , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Mutação/imunologia , Engenharia de Proteínas , Vacinas Atenuadas/imunologia , Vacinas Virais/genéticaRESUMO
Venezuelan Equine Encephalitis (VEE) virus replicon particles (VRPs) encoding Borrelia burgdorferi Outer surface protein A (OspA) were evaluated for their ability to induce an immune response and provide protection from tick-borne spirochetes. VRPs expressing ospA that accumulated intracellularly (VRP OspA) or that was secreted from host cells (VRP tPA-OspA) were tested. Both VRP OspA and VRP tPA-OspA expressed ospA in immunized mice. Mice vaccinated with VRPs expressing secreted OspA produced significant amounts of anti-OspA antibodies, whereas VRPs expressing intracellular OspA were less immunogenic. The VRP method of delivery induced a Th1 type immune response unlike the recombinant OspA protein in Freund's adjuvant, which induced a mixed (Th1 and Th2) immune response. The VRP tPA-OspA construct induced an immune response that reduced the bacterial load in feeding Ixodes scapularis and blocked transmission to the host. These results indicate that VRPs are capable of providing protection against tick-borne B. burgdorferi, and potentially can be used for developing improved vaccines against Lyme disease.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos de Superfície/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Lipoproteínas , Vacinas contra Doença de Lyme/imunologia , Doença de Lyme/imunologia , Doença de Lyme/prevenção & controle , Replicon/imunologia , Carrapatos/microbiologia , Animais , Antígenos de Superfície/genética , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas , Células Cultivadas , Cricetinae , Vírus da Encefalite Equina Venezuelana/genética , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/biossíntese , Imuno-Histoquímica , Doença de Lyme/microbiologia , Camundongos , Camundongos Endogâmicos C3H , Plasmídeos/genética , RNA Viral/biossíntese , RNA Viral/genética , Replicon/genéticaRESUMO
The Norwalk virus (NV) capsid protein was expressed using Venezuelan equine encephalitis virus replicon particles (VRP-NV1). VRP-NV1 infection resulted in large numbers of recombinant NV-like particles that were primarily cell associated and were indistinguishable from NV particles produced from baculoviruses. Mutations located in the N-terminal and P1 domains of the NV capsid protein ablated capsid self-assembly in mammalian cells.
Assuntos
Capsídeo/metabolismo , Vírus da Encefalite Equina Venezuelana/genética , Vírus Norwalk/metabolismo , Replicon , Montagem de Vírus , Animais , Células CACO-2 , Capsídeo/genética , Linhagem Celular , Vírus da Encefalite Equina Venezuelana/fisiologia , Vetores Genéticos , HumanosRESUMO
Norwalk-like viruses (NLVs) are a diverse group of single-stranded, nonenveloped, positive-polarity RNA viruses and are the leading cause of epidemic acute gastroenteritis in the United States. In this study, the major capsid gene of Norwalk virus, the prototype NLV, has been cloned and expressed in mammalian cells using a Venezuelan equine encephalitis (VEE) replicon expression system. Upon infection of baby hamster kidney (BHK) cells with VEE replicon particles (VRPs), the Norwalk virus capsid proteins self-assemble to generate high titers of Norwalk virus-like particles (VLPs) that are morphologically and antigenically analogous to wild-type Norwalk virus. Mice inoculated subcutaneously with VRPs expressing the Norwalk virus capsid protein (VRP-NV1) developed systemic and mucosal immune responses to Norwalk VLPs, as well as heterotypic antibody responses to the major capsid protein from another genogroup I NLV strain (NCFL) isolated from a recent outbreak. A second Norwalk virus capsid clone (NV2) containing three amino acid codon mutations from the NV1 clone was also expressed using VEE replicons (VRP-NV2), but upon infection of BHK cells failed to confer VLP self-assembly. Mice inoculated with VRP-NV2 elicited reduced systemic and mucosal immune responses to Norwalk VLPs, demonstrating the importance and potential utility of endogenous VLP presentation for maximum immune induction. Inoculation with either VRP-NV1 or VRP-NV2 resulted in serum antibody responses far superior to the induction in mice dosed orally with VLPs that were prepared using the VEE-NV1 replicon construct, a regimen similar to current models for NLV vaccination. Expression of NLV VLPs in mammalian cells offers a powerful approach for the design of novel NLV vaccines, either alone or in combination with current vaccination models.