RESUMO
We have previously shown that delivery of the porcine type I interferon gene (poIFN-α/ß) with a replication-defective human adenovirus vector (adenovirus 5 [Ad5]) can sterilely protect swine challenged with foot-and-mouth disease virus (FMDV) 1 day later. However, the need of relatively high doses of Ad5 limits the applicability of such a control strategy in the livestock industry. Venezuelan equine encephalitis virus (VEE) empty replicon particles (VRPs) can induce rapid protection of mice against either homologous or, in some cases, heterologous virus challenge. As an alternative approach to induce rapid protection against FMDV, we have examined the ability of VRPs containing either the gene for green fluorescent protein (VRP-GFP) or poIFN-α (VRP-poIFN-α) to block FMDV replication in vitro and in vivo. Pretreatment of swine or bovine cell lines with either VRP significantly inhibited subsequent infection with FMDV as early as 6 h after treatment and for at least 120 h posttreatment. Furthermore, mice pretreated with either 10(7) or 10(8) infectious units of VRP-GFP and challenged with a lethal dose of FMDV 24 h later were protected from death. Protection was induced as early as 6 h after treatment and lasted for at least 48 h and correlated with induction of an antiviral response and production of IFN-α. By 6 h after treatment several genes were upregulated, and the number of genes and the level of induction increased at 24 h. Finally, we demonstrated that the chemokine IP-10, which is induced by IFN-α and VRP-GFP, is directly involved in protection against FMDV.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Vírus da Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Terapia Genética/métodos , Vetores Genéticos , Interferon-alfa/genética , Interferon-alfa/imunologia , Animais , Modelos Animais de Doenças , Febre Aftosa/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de SobrevidaRESUMO
There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine.
Assuntos
Ebolavirus/imunologia , Vírus da Encefalite Equina Venezuelana/genética , Doença pelo Vírus Ebola/prevenção & controle , Replicon , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Ebolavirus/genética , Vírus da Encefalite Equina Venezuelana/fisiologia , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Macaca fascicularis , Vacinação , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
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
Dendritic cells (DCs) consist of heterogeneous phenotypic populations and have diverse immunostimulatory functions dependent on both lineage and functional phenotype, but as exceptionally potent antigen-presenting cells, they are targets for generating effective antigen-specific immune responses. A promising replicon particle vector derived from Venezuelan equine encephalitis virus (VEE) has been reported to transduce murine footpad DCs. However, the receptive DC subset, the degree of restriction for this tropism, and the extent of conservation between rodents and humans have not been well characterized. Using fresh peripheral blood DCs, mononuclear cells, monocyte-derived macrophages, and monocyte-derived DCs, our results demonstrate conservation of VEE replicon particle (VRP) tropism for DCs between humans and rodents. We observed that a subset of immature myeloid DCs is the target population, and that VRP-transduced immature DCs retain intact functional capacity, for example, the ability to resist the cytopathic effects of VRP transduction and the capacity to acquire the mature phenotype. These studies support the demonstration of selective VRP tropism for human DCs and provide further insight into the biology of the VRP vector, its parent virus, and human DCs.
Assuntos
Células Dendríticas/virologia , Vírus da Encefalite Equina Venezuelana/genética , Vetores Genéticos/genética , Replicon , Células Dendríticas/fisiologia , Vírus da Encefalite Equina Venezuelana/imunologia , Humanos , Transdução Genética , TropismoRESUMO
Many tumor-associated antigens (TAAs) represent 'self' antigens and as such, are subject to the constraints of immunologic tolerance. There are significant barriers to eliciting anti-tumor immune responses of sufficient magnitude. We have taken advantage of a Venezuelan equine encephalitis-derived alphavirus replicon vector system with documented in vivo tropism for immune system dendritic cells. We have overcome the intrinsic tolerance to the 'self' TAA rat neu and elicited an effective anti-tumor immune response using this alphavirus replicon vector system and a designed target antigen in a rigorous rat mammary tumor model. We have demonstrated the capacity to generate 50% protection in tumor challenge experiments (p = 0.004) and we have confirmed the establishment of immunologic memory by both second tumor challenge and Winn Assay (p = 0.009). Minor antibody responses were identified and supported the establishment of T helper type 1 (Th1) anti-tumor immune responses by isotype. Animals surviving in excess of 300 days with established effective anti-tumor immunity showed no signs of autoimmune phenomena. Together these experiments support the establishment of T lymphocyte dependent, Th1-biased anti-tumor immune responses to a non-mutated 'self' TAA in an aggressive tumor model. Importantly, this tumor model is subject to the constraints of immunologic tolerance present in animals with normal developmental, temporal, and anatomical expression of a non-mutated TAA. These data support the continued development and potential clinical application of this alphaviral replicon vector system and the use of appropriately designed target antigen sequences for anti-tumor immunotherapy.