RESUMO
Replicon particles derived from Venezuelan equine encephalitis virus (VEE) are infectious non-propagating particles which act as a safe and potent systemic, mucosal, and cellular adjuvant when delivered with antigen. VEE and VEE replicon particles (VRP) can target multiple cell types including dendritic cells (DCs). The role of these cell types in VRP adjuvant activity has not been previously evaluated, and for these studies we focused on the contribution of DCs to the response to VRP. By analysis of VRP targeting in the draining lymph node, we found that VRP induced rapid recruitment of TNF-secreting monocyte-derived inflammatory dendritic cells. VRP preferentially infected these inflammatory DCs as well as classical DCs and macrophages, with less efficient infection of other cell types. DC depletion suggested that the interaction of VRP with classical DCs was required for recruitment of inflammatory DCs, induction of high levels of many cytokines, and for stable transport of VRP to the draining lymph node. Additionally, in vitro-infected DCs enhanced antigen-specific responses by CD4 and CD8 T cells. By transfer of VRP-infected DCs into mice we showed that these DCs generated an inflammatory state in the draining lymph node similar to that achieved by VRP injection. Most importantly, VRP-infected DCs were sufficient to establish robust adjuvant activity in mice comparable to that produced by VRP injection. These findings indicate that VRP infect, recruit and activate both classical and inflammatory DCs, and those DCs become mediators of the VRP adjuvant activity.
Assuntos
Adjuvantes Imunológicos/farmacologia , Células Dendríticas/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Replicon/imunologia , Animais , Anticorpos Antivirais/sangue , Citocinas/imunologia , Células Dendríticas/virologia , Feminino , Imunidade Celular , Imunidade nas Mucosas , Inflamação/imunologia , Linfonodos/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T/imunologia , Vírion/imunologiaRESUMO
Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne RNA virus of the genus Alphavirus, family Togaviridae, that is responsible for sporadic outbreaks in human and equid populations in Central and South America. In order to ascertain the role that complement plays in resolving VEEV-induced disease, complement-deficient C3(-/-) mice were infected with a VEEV mutant (V3533) that caused mild, transient disease in immunocompetent mice. In the absence of a functional complement system, peripheral inoculation with V3533 induced much more severe encephalitis. This enhanced pathology was associated with a delay in clearance of infectious virus from the serum and more rapid invasion of the central nervous system in C3(-/-) mice. If V3533 was inoculated directly into the brain, however, disease outcome in C3(-/-) and wild-type mice was identical. These findings indicate that complement-dependent enhancement of peripheral virus clearance is critical for protecting against the development of severe VEEV-induced encephalitis.
Assuntos
Infecções do Sistema Nervoso Central/virologia , Ativação do Complemento/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/imunologia , Imunidade Adaptativa/imunologia , Animais , Anticorpos Antivirais/imunologia , Encéfalo/imunologia , Encéfalo/virologia , Infecções do Sistema Nervoso Central/imunologia , Complemento C3/deficiência , Complemento C5/imunologia , Encefalomielite Equina Venezuelana/virologia , Imunidade Inata/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Carga Viral/imunologiaRESUMO
Venezuelan equine encephalitis (VEE) virus is a mosquito-borne alphavirus associated with sporadic outbreaks in human and equid populations in the Western Hemisphere. After the bite of an infected mosquito, the virus initiates a biphasic disease: a peripheral phase with viral replication in lymphoid and myeloid tissues, followed by a neurotropic phase with infection of central nervous system (CNS) neurons, causing neuropathology and in some cases fatal encephalitis. The mechanisms allowing VEE virus to enter the CNS are currently poorly understood. Previous data have shown that the virus gains access to the CNS by infecting olfactory sensory neurons in the nasal mucosa of mice. However, at day 5 after inoculation, the infection of the brain is multifocal, indicating that virus particles are able to cross the blood-brain barrier (BBB). To better understand the role of the BBB during VEE virus infection, we used a well-characterized mouse model system. Using VEE virus replicon particles (VRP), we modeled the early events of neuroinvasion, showing that the replication of VRP in the nasal mucosa induced the opening of the BBB, allowing peripherally administered VRP to invade the brain. Peripheral VEE virus infection was characterized by a biphasic opening of the BBB. Further, inhibition of BBB opening resulted in a delayed viral neuroinvasion and pathogenesis. Overall, these results suggest that VEE virus initially enters the CNS through the olfactory pathways and initiates viral replication in the brain, which induces the opening of the BBB, allowing a second wave of invading virus from the periphery to enter the brain.
Assuntos
Barreira Hematoencefálica/fisiopatologia , Barreira Hematoencefálica/virologia , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/patologia , Encefalomielite Equina Venezuelana/virologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos BALB C , Neurônios Receptores Olfatórios/virologia , Doenças dos Roedores/patologia , Doenças dos Roedores/virologiaRESUMO
Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne RNA virus of the genus Alphavirus that is responsible for a significant disease burden in Central and South America through sporadic outbreaks into human and equid populations. For humans, 2 to 4% of cases are associated with encephalitis, and there is an overall case mortality rate of approximately 1%. In mice, replication of the virus within neurons of the central nervous system (CNS) leads to paralyzing, invariably lethal encephalomyelitis. However, mice infected with certain attenuated mutants of the virus are able to control the infection within the CNS and recover. To better define what role T cell responses might be playing in this process, we infected B cell-deficient microMT mice with a VEEV mutant that induces mild, sublethal illness in immune competent mice. Infected microMT mice rapidly developed the clinical signs of severe paralyzing encephalomyelitis but were eventually able to control the infection and recover fully from clinical illness. Recovery in this system was T cell dependent and associated with a dramatic reduction in viral titers within the CNS, followed by viral persistence in the brain. Further comparison of the relative roles of T cell subpopulations within this system revealed that CD4(+) T cells were better producers of gamma interferon (IFN-gamma) than CD8(+) T cells and were more effective at controlling VEEV within the CNS. Overall, these results suggest that T cells, especially CD4(+) T cells, can successfully control VEEV infection within the CNS and facilitate recovery from a severe viral encephalomyelitis.
Assuntos
Anticorpos Antivirais/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/imunologia , Linfócitos T/imunologia , Animais , Encéfalo/virologia , Encefalomielite Equina Venezuelana/patologia , Feminino , Humanos , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subpopulações de Linfócitos T/imunologia , Carga ViralRESUMO
The host innate immune response provides a critical first line of defense against invading pathogens, inducing an antiviral state to impede the spread of infection. While numerous studies have documented antiviral responses within actively infected tissues, few have described the earliest innate response induced systemically by infection. Here, utilizing Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) to limit infection to the initially infected cells in vivo, a rapid activation of the antiviral response was demonstrated not only within the murine draining lymph node, where replication was confined, but also within distal tissues. In the liver and brain, expression of interferon-stimulated genes was detected by 1 to 3 h following VRP footpad inoculation, reaching peak expression of >100-fold over that in mock-infected animals. Moreover, mice receiving a VRP footpad inoculation 6, 12, or 24 h prior to an otherwise lethal VEE footpad challenge were completely protected from death, including a drastic reduction in challenge virus titers. VRP pretreatment also provided protection from intranasal VEE challenge and extended the average survival time following intracranial challenge. Signaling through the interferon receptor was necessary for antiviral gene induction and protection from VEE challenge. However, VRP pretreatment failed to protect mice from a heterologous, lethal challenge with vesicular stomatitis virus, yet conferred protection following challenge with influenza virus. Collectively, these results document a rapid modulation of the host innate response within hours of infection, capable of rapidly alerting the entire animal to pathogen invasion and leading to protection from viral disease.
Assuntos
Encéfalo/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/prevenção & controle , Imunidade Inata , Fígado/imunologia , Linfonodos/imunologia , Animais , Encéfalo/virologia , Feminino , Perfilação da Expressão Gênica , Interferons/imunologia , Fígado/virologia , Linfonodos/virologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Rhabdoviridae/prevenção & controle , Análise de SobrevidaRESUMO
Venezuelan equine encephalitis virus (VEEV) is an important human and veterinary pathogen causing sporadic epizootic outbreaks of potentially fatal encephalitis. The type I interferon (IFN) system plays a central role in controlling VEEV and other alphavirus infections, and IFN evasion is likely an important determinant of whether these viruses disseminate and cause disease within their hosts. Alphaviruses are thought to limit the induction of type I IFNs and IFN-stimulated genes by shutting off host cell macromolecular synthesis, which in the case of VEEV is partially mediated by the viral capsid protein. However, more specific strategies by which alphaviruses inhibit type I IFN signaling have not been characterized. Analyses of cells infected with VEEV and VEEV replicon particles (VRP) demonstrate that viral infection rapidly disrupts tyrosine phosphorylation and nuclear translocation of the transcription factor STAT1 in response to both IFN-beta and IFN-gamma. This effect was independent of host shutoff and expression of viral capsid, suggesting that VEEV uses novel mechanisms to interfere with type I and type II IFN signaling. Furthermore, at times when STAT1 activation was efficiently inhibited, VRP infection did not limit tyrosine phosphorylation of Jak1, Tyk2, or STAT2 after IFN-beta treatment but did inhibit Jak1 and Jak2 activation in response to IFN-gamma, suggesting that VEEV interferes with STAT1 activation by the type I and II receptor complexes through distinct mechanisms. Identification of the viral requirements for this novel STAT1 inhibition will further our understanding of alphavirus molecular pathogenesis and may provide insights into effective alphavirus-based vaccine design.
Assuntos
Vírus da Encefalite Equina Venezuelana/patogenicidade , Fator de Transcrição STAT1/antagonistas & inibidores , Transdução de Sinais , Animais , Chlorocebus aethiops , Cricetinae , Células HeLa , Humanos , Interferon beta/antagonistas & inibidores , Interferon beta/imunologia , Interferon gama/antagonistas & inibidores , Interferon gama/imunologia , Fosforilação , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/fisiologia , Tirosina/metabolismo , Células VeroRESUMO
Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) were used to model the initial phase of VEE-induced encephalitis in the mouse brain. VRP can target and infect cells as VEE, but VRP do not propagate beyond the first infected cell due to the absence of the structural genes. Direct intracranial inoculation of VRP into mice induced acute encephalitis with signs similar to the neuronal phase of wild-type VEE infection and other models of virus-induced encephalitis. Using the previously established VRP-mRNP tagging system, a new method to distinguish the host responses in infected cells from those in uninfected bystander cell populations, we detected a robust and rapid innate immune response in the central nervous system (CNS) by infected neurons and uninfected bystander cells. Moreover, this innate immune response in the CNS compromised blood-brain barrier integrity, created an inflammatory response, and directed an adaptive immune response characterized by proliferation and activation of microglia cells and infiltration of inflammatory monocytes, in addition to CD4(+) and CD8(+) T lymphocytes. Taken together, these data suggest that a naïve CNS has an intrinsic potential to induce an innate immune response that could be crucial to the outcome of the infection by determining the composition and dynamics of the adaptive immune response. Furthermore, these results establish a model for neurotropic virus infection to identify host and viral factors that contribute to invasion of the brain, the mechanism(s) whereby the adaptive immune response can clear the infection, and the role of the host innate response in these processes.
Assuntos
Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/crescimento & desenvolvimento , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/virologia , Vírion/crescimento & desenvolvimento , Animais , Proliferação de Células , Citocinas/metabolismo , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/metabolismo , Encefalomielite Equina Venezuelana/patologia , Feminino , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microglia/metabolismo , Microglia/patologia , RNA Viral/genética , Vírion/genéticaRESUMO
A novel genetic vaccine that is based on a Venezuelan equine encephalitis virus (VEE) replicon launched from plasmid DNA is described. The plasmid encodes a VEE replicon under the transcriptional control of the cytomegalovirus immediate-early promoter (VEE DNA). The VEE DNA consistently expressed 3- to 15-fold more green fluorescent protein in vitro than did a conventional DNA vaccine. Furthermore, transfection with the DNA-launched VEE replicon induced apoptosis and type I interferon production. Inoculation of mice with VEE DNA encoding human immunodeficiency virus type 1 gp160 significantly increased humoral responses by several orders of magnitude compared to an equal dose of a conventional DNA vaccine. These increases were also observed at 10- and 100-fold-lower doses of the VEE DNA. Cellular immune responses measured by gamma interferon and interleukin 2 enzyme-linked immunospot assay were significantly higher in mice immunized with the VEE DNA at decreased doses. The immune responses induced by the VEE DNA-encoded antigen, however, were independent of an intact type I interferon signaling pathway. Moreover, the DNA-launched VEE replicon induced an efficient prime to a VEE replicon particle (VRP) boost, increasing humoral and cellular immunity by at least 1 order of magnitude compared to VEE DNA only. Importantly, immunization with VEE DNA, as opposed to VRP, did not induce any anti-VRP neutralizing antibodies. Increased potency of DNA vaccines and reduced vector immunity may ultimately have an impact on the design of vaccination strategies in humans.
Assuntos
Anticorpos Antivirais/sangue , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/imunologia , Interferon Tipo I/biossíntese , Interleucina-2/biossíntese , Plasmídeos/genética , Replicon/imunologia , Vacinas de DNA/imunologia , Animais , Apoptose , Linhagem Celular , Chlorocebus aethiops , Vetores Genéticos , Proteína gp160 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV/imunologia , HIV-1/genética , Humanos , Imunização , Imunoglobulina G/sangue , Células L , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Células NIH 3T3 , Regiões Promotoras Genéticas , Replicon/genética , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Células VeroRESUMO
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
Many RNA viruses, which replicate predominantly in the cytoplasm, have nuclear components that contribute to their life cycle or pathogenesis. We investigated the intracellular localization of the multifunctional nonstructural protein 2 (nsP2) in mammalian cells infected with Venezuelan equine encephalitis virus (VEE), an important, naturally emerging zoonotic alphavirus. VEE nsP2 localizes to both the cytoplasm and the nucleus of mammalian cells in the context of infection and also when expressed alone. Through the analysis of a series of enhanced green fluorescent protein fusions, a segment of nsP2 that completely localizes to the nucleus of mammalian cells was identified. Within this region, mutation of the putative nuclear localization signal (NLS) PGKMV diminished, but did not obliterate, the ability of the protein to localize to the nucleus, suggesting that this sequence contributes to the nuclear localization of VEE nsP2. Furthermore, VEE nsP2 specifically interacted with the nuclear import protein karyopherin-alpha1 but not with karyopherin-alpha2, -3, or -4, suggesting that karyopherin-alpha1 transports nsP2 to the nucleus during infection. Additionally, a novel nuclear export signal (NES) was identified, which included residues L526 and L528 of VEE nsP2. Leptomycin B treatment resulted in nuclear accumulation of nsP2, demonstrating that nuclear export of nsP2 is mediated via the CRM1 nuclear export pathway. Disruption of either the NLS or the NES in nsP2 compromised essential viral functions. Taken together, these results establish the bidirectional transport of nsP2 across the nuclear membrane, suggesting that a critical function of nsP2 during infection involves its shuttling between the cytoplasm and the nucleus.
Assuntos
Núcleo Celular/metabolismo , Vírus da Encefalite Equina Venezuelana/metabolismo , Sinais de Localização Nuclear/genética , Proteínas não Estruturais Virais/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Núcleo Celular/química , Citoplasma/química , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Sinais de Localização Nuclear/metabolismo , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas não Estruturais Virais/análise , Proteínas não Estruturais Virais/genética , alfa Carioferinas/metabolismoRESUMO
An anti-poxvirus vaccine based on replicon particles of Venezuelan equine encephalitis virus (VRP) is being developed. The cowpox virus genes encoding structural proteins corresponding to vaccinia virus proteins A33, B5, and A27 were each expressed from VRP. High serum IgG titers against these proteins were generated in BALB/c mice vaccinated with each of these VRP. VRP induced both IgG1 and IgG2a with a strong predominance of IgG2a production. The response is long-lasting, as evidenced by the retention of high anti-B5 serum IgG titers through at least 50 weeks after priming immunization. Mice vaccinated with B5-, A33- or A27-VRP individually or together survived intranasal challenge with cowpox virus, with the multivalent vaccine formulation providing more effective protection from weight loss and clinical signs of illness than the monovalent vaccines. These results demonstrate that VRP may provide an effective alternative to vaccinia virus vaccines against poxvirus infection.
Assuntos
Vírus da Varíola Bovina/imunologia , Varíola Bovina/prevenção & controle , Vírus da Encefalite Equina Venezuelana/genética , Vacinas Sintéticas/imunologia , Proteínas Estruturais Virais/imunologia , Vacinas Virais/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Peso Corporal , Varíola Bovina/imunologia , Varíola Bovina/fisiopatologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos/genética , Imunoglobulina G/sangue , Memória Imunológica , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Replicon/genética , Homologia de Sequência de Aminoácidos , Análise de Sobrevida , Fatores de Tempo , Vacinas Sintéticas/genética , Proteínas Estruturais Virais/genética , 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.