RESUMO
Venezuelan equine encephalitis virus (VEEV) is a known biological defense threat. A live-attenuated investigational vaccine, TC-83, is available, but it has a high non-response rate and can also cause severe reactogenicity. We generated two novel VEE vaccine candidates using self-amplifying mRNA (SAM). LAV-CNE is a live-attenuated VEE SAM vaccine formulated with synthetic cationic nanoemulsion (CNE) and carrying the RNA genome of TC-83. IAV-CNE is an irreversibly-attenuated VEE SAM vaccine formulated with CNE, delivering a TC-83 genome lacking the capsid gene. LAV-CNE launches a TC-83 infection cycle in vaccinated subjects but eliminates the need for live-attenuated vaccine production and potentially reduces manufacturing time and complexity. IAV-CNE produces a single cycle of RNA amplification and antigen expression without generating infectious viruses in subjects, thereby creating a potentially safer alternative to live-attenuated vaccine. Here, we demonstrated that mice vaccinated with LAV-CNE elicited immune responses similar to those of TC-83, providing 100% protection against aerosol VEEV challenge. IAV-CNE was also immunogenic, resulting in significant protection against VEEV challenge. These studies demonstrate the proof of concept for using the SAM platform to streamline the development of effective attenuated vaccines against VEEV and closely related alphavirus pathogens such as western and eastern equine encephalitis and Chikungunya viruses.
Assuntos
Vírus da Encefalite Equina Venezuelana/imunologia , Encefalomielite Equina Venezuelana/tratamento farmacológico , Amplificação de Genes , Imunogenicidade da Vacina , RNA Mensageiro/genética , Vacinas Atenuadas/uso terapêutico , Vacinas Virais/uso terapêutico , Células A549 , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Modelos Animais de Doenças , Emulsões/química , Encefalomielite Equina Venezuelana/virologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transfecção , Vacinas Virais/farmacologia , Replicação ViralRESUMO
Foot-and-mouth disease virus (FMDV) exhibits high mutation rates during replication. In this study, an isolate of FMDV serotype Asia-1 was serially passaged in a BHK-21 cell monolayer and then adapted to serum-free BHK-21 cell suspension culture to produce a seed virus for production of an inactivated vaccine. Analysis of the sequence encoding the structural proteins of the virus at various passages showed the presence of overlapping peaks in sequencing electropherograms after nucleotide 619 of VP1 in viruses recovered from the fourth passage in suspension culture, suggesting the possible introduction of an insertion or deletion into this portion of the viral genome of our seed virus stock. To evaluate this phenomenon, a virus designated "Vac-Asia1-VDLV", was isolated by plaque purification from the tenth passage in suspension culture. Sequencing results showed that a 12-nt-long exogenous sequence was inserted into the 3' end of the VP1 coding region at the position where the original overlapping peaks were identified. Analysis of the host cell transcriptome showed that the 12-nt sequence was identical to a highly expressed sequence in BHK-21 cells, strongly suggesting that recombination between the FMDV genome and host cell mRNA produced the recombinant virus. A growth curve showed that the virus with the 12-nt insertion reached a peak earlier than the parental strain and that this virus had acquired the ability to bind to the cell surface by a mechanism that was not dependent on integrin or the heparan sulfate receptor. This novel pathogen-host cell recombination event is discussed in terms of the mechanism of viral RNA replication and the phenotypic constraints of FMDV biology and evolution.
Assuntos
Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/fisiologia , RNA Mensageiro/genética , Animais , Proteínas do Capsídeo , Linhagem Celular , Cricetinae , Febre Aftosa/virologia , Regulação Viral da Expressão Gênica , Genoma Viral , Mutação , RNA Viral/genética , Recombinação Genética , Cultura de Vírus , Replicação ViralRESUMO
UNLABELLED: Seasonal influenza is a vaccine-preventable disease that remains a major health problem worldwide, especially in immunocompromised populations. The impact of influenza disease is even greater when strains drift, and influenza pandemics can result when animal-derived influenza virus strains combine with seasonal strains. In this study, we used the SAM technology and characterized the immunogenicity and efficacy of a self-amplifying mRNA expressing influenza virus hemagglutinin (HA) antigen [SAM(HA)] formulated with a novel oil-in-water cationic nanoemulsion. We demonstrated that SAM(HA) was immunogenic in ferrets and facilitated containment of viral replication in the upper respiratory tract of influenza virus-infected animals. In mice, SAM(HA) induced potent functional neutralizing antibody and cellular immune responses, characterized by HA-specific CD4 T helper 1 and CD8 cytotoxic T cells. Furthermore, mice immunized with SAM(HA) derived from the influenza A virus A/California/7/2009 (H1N1) strain (Cal) were protected from a lethal challenge with the heterologous mouse-adapted A/PR/8/1934 (H1N1) virus strain (PR8). Sera derived from SAM(H1-Cal)-immunized animals were not cross-reactive with the PR8 virus, whereas cross-reactivity was observed for HA-specific CD4 and CD8 T cells. Finally, depletion of T cells demonstrated that T-cell responses were essential in mediating heterologous protection. If the SAM vaccine platform proves safe, well tolerated, and effective in humans, the fully synthetic SAM vaccine technology could provide a rapid response platform to control pandemic influenza. IMPORTANCE: In this study, we describe protective immune responses in mice and ferrets after vaccination with a novel HA-based influenza vaccine. This novel type of vaccine elicits both humoral and cellular immune responses. Although vaccine-specific antibodies are the key players in mediating protection from homologous influenza virus infections, vaccine-specific T cells contribute to the control of heterologous infections. The rapid production capacity and the synthetic origin of the vaccine antigen make the SAM platform particularly exploitable in case of influenza pandemic.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Vacinas de DNA/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteção Cruzada , Modelos Animais de Doenças , Feminino , Furões , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Procedimentos de Redução de Leucócitos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Sistema Respiratório/virologia , Análise de Sobrevida , Resultado do Tratamento , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Carga ViralRESUMO
Nucleic acid-based vaccines such as viral vectors, plasmid DNA, and mRNA are being developed as a means to address a number of unmet medical needs that current vaccine technologies have been unable to address. Here, we describe a cationic nanoemulsion (CNE) delivery system developed to deliver a self-amplifying mRNA vaccine. This nonviral delivery system is based on Novartis's proprietary adjuvant MF59, which has an established clinical safety profile and is well tolerated in children, adults, and the elderly. We show that nonviral delivery of a 9 kb self-amplifying mRNA elicits potent immune responses in mice, rats, rabbits, and nonhuman primates comparable to a viral delivery technology, and demonstrate that, relatively low doses (75 µg) induce antibody and T-cell responses in primates. We also show the CNE-delivered self-amplifying mRNA enhances the local immune environment through recruitment of immune cells similar to an MF59 adjuvanted subunit vaccine. Lastly, we show that the site of protein expression within the muscle and magnitude of protein expression is similar to a viral vector. Given the demonstration that self-amplifying mRNA delivered using a CNE is well tolerated and immunogenic in a variety of animal models, we are optimistic about the prospects for this technology.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Emulsões/administração & dosagem , Imunidade Celular , RNA Mensageiro/imunologia , RNA Viral/imunologia , Vacinas de DNA/administração & dosagem , Animais , Cátions , Emulsões/química , Feminino , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , RatosRESUMO
Despite more than two decades of research and development on nucleic acid vaccines, there is still no commercial product for human use. Taking advantage of the recent innovations in systemic delivery of short interfering RNA (siRNA) using lipid nanoparticles (LNPs), we developed a self-amplifying RNA vaccine. Here we show that nonviral delivery of a 9-kb self-amplifying RNA encapsulated within an LNP substantially increased immunogenicity compared with delivery of unformulated RNA. This unique vaccine technology was found to elicit broad, potent, and protective immune responses, that were comparable to a viral delivery technology, but without the inherent limitations of viral vectors. Given the many positive attributes of nucleic acid vaccines, our results suggest that a comprehensive evaluation of nonviral technologies to deliver self-amplifying RNA vaccines is warranted.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/administração & dosagem , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Alphavirus/genética , Análise de Variância , Animais , Eletroforese em Gel de Ágar , Escherichia coli , Feminino , Imunofluorescência , Humanos , Lipídeos/química , Nanopartículas/química , RNA Interferente Pequeno/química , Ratos , Estatísticas não ParamétricasRESUMO
Recognition of conserved pathogen-associated molecular patterns (PAMPs) by host pattern recognition receptors (PRRs) results in the activation of innate signaling pathways that drive the innate immune response and ultimately shape the adaptive immune response. RepliVAX WN, a single-cycle flavivirus (SCFV) vaccine candidate derived from West Nile virus (WNV), is intrinsically adjuvanted with multiple PAMPs and induces a vigorous anti-WNV humoral response. However, the innate mechanisms that link pattern recognition and development of vigorous antigen-specific B cell responses are not completely understood. Moreover, the roles of individual PRR signaling pathways in shaping the B cell response to this live attenuated SCFV vaccine have not been established. We examined and compared the role of TLR3- and MyD88-dependent signaling in the development of anti-WNV-specific antibody-secreting cell responses and memory B cell responses induced by RepliVAX WN. We found that MyD88 deficiency significantly diminished B cell responses by impairing B cell activation, development of germinal centers (GC), and the generation of long-lived plasma cells (LLPCs) and memory B cells (MBCs). In contrast, TLR3 deficiency had more effect on maintenance of GCs and development of LLPCs, whereas differentiation of MBCs was unaffected. Our data suggest that both TLR3- and MyD88-dependent signaling are involved in the intrinsic adjuvanting of RepliVAX WN and differentially contribute to the development of vigorous WNV-specific antibody and B cell memory responses following immunization with this novel SCFV vaccine.
Assuntos
Imunidade Adaptativa/imunologia , Linfócitos B/imunologia , Fator 88 de Diferenciação Mieloide/fisiologia , Receptor 3 Toll-Like/fisiologia , Febre do Nilo Ocidental/imunologia , Vacinas contra o Vírus do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/imunologia , Animais , Anticorpos Antivirais/sangue , Células Produtoras de Anticorpos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Citometria de Fluxo , Imunização , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Febre do Nilo Ocidental/prevenção & controle , Vacinas contra o Vírus do Nilo Ocidental/uso terapêuticoRESUMO
West Nile virus (WNV), and related flaviviruses such as tick-borne encephalitis, Japanese encephalitis, yellow fever and dengue viruses, constitute a significant global human health problem. However, our understanding of the molecular interaction of such flaviviruses with mammalian host cells is limited. WNV encodes only 10 proteins, implying that it may use many cellular proteins for infection. WNV enters the cytoplasm through pH-dependent endocytosis, undergoes cycles of translation and replication, assembles progeny virions in association with endoplasmic reticulum, and exits along the secretory pathway. RNA interference (RNAi) presents a powerful forward genetics approach to dissect virus-host cell interactions. Here we report the identification of 305 host proteins that affect WNV infection, using a human-genome-wide RNAi screen. Functional clustering of the genes revealed a complex dependence of this virus on host cell physiology, requiring a wide variety of molecules and cellular pathways for successful infection. We further demonstrate a requirement for the ubiquitin ligase CBLL1 in WNV internalization, a post-entry role for the endoplasmic-reticulum-associated degradation pathway in viral infection, and the monocarboxylic acid transporter MCT4 as a viral replication resistance factor. By extending this study to dengue virus, we show that flaviviruses have both overlapping and unique interaction strategies with host cells. This study provides a comprehensive molecular portrait of WNV-human cell interactions that forms a model for understanding single plus-stranded RNA virus infection, and reveals potential antiviral targets.
Assuntos
Interferência de RNA , Febre do Nilo Ocidental/genética , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/fisiologia , Biologia Computacional , Vírus da Dengue/fisiologia , Retículo Endoplasmático/metabolismo , Perfilação da Expressão Gênica , Genoma Humano , HIV , Células HeLa , Humanos , Imunidade/genética , Transportadores de Ácidos Monocarboxílicos/deficiência , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Ligação Proteica , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética , Vesiculovirus , Replicação ViralRESUMO
Older adults exhibit higher morbidity and mortality from infectious diseases compared with those of the general population. The introduction and rapid spread of West Nile virus (WNV) throughout the continental United States since 1999 has highlighted the challenge of protecting older adults against emerging pathogens: to this day there is no therapy or vaccine approved for human use against West Nile encephalitis. In this study, we describe the characterization of T and B cell responses in old mice after vaccination with RepliVAX WN, a novel West Nile encephalitis vaccine based on single-cycle flavivirus particles. In adult mice, RepliVAX WN induced robust and long-lasting CD4(+) and CD8(+) T cell and Ab (B cell) responses against natural WNV epitopes, similar to those elicited by primary WNV infection. Primary and memory T and B cell responses in old mice against RepliVAX WN vaccination were significantly lower than those seen in younger mice, similar to the response of old mice to infection with WNV. Surprisingly, both the quality and the quantity of the recall Ab and T cell responses in vaccinated old mice were improved to equal or exceed those in adult animals. Moreover, these responses together (but not individually) were sufficient to protect both old and adult mice from severe WNV disease upon challenge. Therefore, at least two cycles of in vivo restimulation are needed for selection and expansion of protective lymphocytes in older populations, and live, single-cycle virus vaccines that stimulate both cellular and humoral immunity can protect older individuals against severe viral disease.
Assuntos
Imunidade Adaptativa , Subpopulações de Linfócitos B/imunologia , Senescência Celular/imunologia , Subpopulações de Linfócitos T/imunologia , Febre do Nilo Ocidental/mortalidade , Febre do Nilo Ocidental/prevenção & controle , Vacinas contra o Vírus do Nilo Ocidental/administração & dosagem , Vacinas contra o Vírus do Nilo Ocidental/imunologia , Animais , Subpopulações de Linfócitos B/patologia , Subpopulações de Linfócitos B/virologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Imunização Secundária/métodos , Memória Imunológica , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subpopulações de Linfócitos T/patologia , Subpopulações de Linfócitos T/virologia , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Febre do Nilo Ocidental/patologiaRESUMO
We have established a human RNA polymerase I (pol I)-driven influenza virus reverse genetics (RG) system in the Madin-Darby canine kidney 33016-PF cell line, which is approved for influenza vaccine manufacture. RNA pol I polymerases are generally active only in cells of species closely related to the species of origin of the polymerases. Nevertheless, we show that a nonendogenous RNA pol I promoter drives efficient rescue of influenza A viruses in a canine cell line. Application of this system allows efficient generation of virus strains and presents an alternative approach for influenza vaccine production.
Assuntos
Vírus da Influenza A/genética , RNA Polimerase I/fisiologia , Animais , Linhagem Celular , Cães , Humanos , Vacinas contra Influenza/biossíntese , Regiões Promotoras Genéticas , RNA Polimerase I/genéticaRESUMO
Flaviviruses transmitted by arthropods represent a tremendous disease burden for humans, causing millions of infections annually. All vector-borne flaviviruses studied to date suppress host innate responses to infection by inhibiting alpha/beta interferon (IFN-alpha/beta)-mediated JAK-STAT signal transduction. The viral nonstructural protein NS5 of some flaviviruses functions as the major IFN antagonist, associated with inhibition of IFN-dependent STAT1 phosphorylation (pY-STAT1) or with STAT2 degradation. West Nile virus (WNV) infection prevents pY-STAT1 although a role for WNV NS5 in IFN antagonism has not been fully explored. Here, we report that NS5 from the virulent NY99 strain of WNV prevented pY-STAT1 accumulation, suppressed IFN-dependent gene expression, and rescued the growth of a highly IFN-sensitive virus (Newcastle disease virus) in the presence of IFN, suggesting that this protein can function as an efficient IFN antagonist. In contrast, NS5 from Kunjin virus (KUN), a naturally attenuated subtype of WNV, was a poor suppressor of pY-STAT1. Mutation of a single residue in KUN NS5 to the analogous residue in WNV-NY99 NS5 (S653F) rendered KUN NS5 an efficient inhibitor of pY-STAT1. Incorporation of this mutation into recombinant KUN resulted in 30-fold greater inhibition of JAK-STAT signaling than with the wild-type virus and enhanced KUN replication in the presence of IFN. Thus, a naturally occurring mutation is associated with the function of NS5 in IFN antagonism and may influence virulence of WNV field isolates.
Assuntos
Interferon Tipo I/antagonistas & inibidores , Janus Quinases/antagonistas & inibidores , Fator de Transcrição STAT1/antagonistas & inibidores , Transdução de Sinais/fisiologia , Proteínas não Estruturais Virais/fisiologia , Animais , Chlorocebus aethiops , Humanos , Células Vero , Proteínas não Estruturais Virais/química , Vírus do Nilo Ocidental/fisiologiaRESUMO
We have previously described a novel flavivirus vaccine technology based on a single-cycle, capsid (C) gene-deleted flavivirus called RepliVAX. RepliVAX can be propagated in cells that express high levels of C but undergoes only a single cycle of infection in vaccinated hosts. Here we report that we have adapted our RepliVAX technology to produce a dengue vaccine by replacing the prM/E genes of RepliVAX WN (a West Nile virus [WNV] RepliVAX) with the same genes of dengue virus type 2 (DENV2). Our first RepliVAX construct for dengue virus (RepliVAX D2) replicated poorly in WNV C-expressing cells. However, addition of mutations in prM and E that were selected during blind passage of a RepliVAX D2 derivative was used to produce a second-generation RepliVAX D2 (designated D2.2) that displayed acceptable growth in WNV C-expressing cells. RepliVAX D2.2 grew better in DENV2 C-expressing cells than WNV C-expressing cells, but after several passages in DENV2 C-expressing cells it acquired further mutations that permitted efficient growth in WNV C-expressing cells. We tested the potency and efficacy of RepliVAX D2.2 in a well-described immunodeficient mouse model for dengue (strain AG129; lacking the receptors for both type I and type II interferons). These mice produced dose-dependent DENV2-neutralizing antibody responses when vaccinated with RepliVAX D2.2. When challenged with 240 50% lethal doses of DENV2, mice given a single inoculation of RepliVAX D2.2 survived significantly longer than sham-vaccinated animals, although some of these severely immunocompromised mice eventually died from the challenge. Taken together these studies indicate that the RepliVAX technology shows promise for use in the development of vaccines that can be used to prevent dengue.
Assuntos
Vacinas contra Dengue/imunologia , Dengue/prevenção & controle , Animais , Anticorpos Antivirais/sangue , Linhagem Celular , Cricetinae , Vacinas contra Dengue/genética , Vírus da Dengue/genética , Flavivirus/genética , Hospedeiro Imunocomprometido , Camundongos , Testes de Neutralização , Análise de Sobrevida , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vírus do Nilo OcidentalRESUMO
During acute infection, West Nile virus (WNV) has been reported to infect a variety of cell types in various tissues of both experimentally and naturally infected hosts. Virus infects epithelial cells in the skin, kidney, intestine, and testes, although the importance of these findings is unclear. In the current study, we have observed that WNV infection of kidney tubules in mice coincides with the loss of expression of several members of the claudin family. Proteins of this family are often involved in epithelial barrier formation and function. WNV infection of epithelial cells in culture resulted in a decrease in the transepithelial electrical resistance, an increase in the efflux of mannitol across the monolayer, and a loss of intracellular levels of claudin-1 to -4. WNV capsid alone was sufficient for the degradation event, which was mediated through lysosomal proteases. Since epithelial cells are frequent sites of WNV infection, these observations imply a potential mechanism for virus dissemination and extraneural pathogenesis.
Assuntos
Capsídeo/metabolismo , Túbulos Renais/virologia , Proteínas de Membrana/metabolismo , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/patogenicidade , Animais , Células CACO-2 , Permeabilidade da Membrana Celular , Chlorocebus aethiops , Claudina-1 , Claudina-3 , Claudinas , Células Epiteliais/virologia , Humanos , Manitol/metabolismo , Camundongos , RNA Mensageiro/metabolismo , Junções Íntimas/virologia , Células VeroRESUMO
Subunit antigens are attractive candidates for vaccine development, as they are safe, cost-effective, and rapidly produced. Nevertheless, subunit antigens often need to be adjuvanted and/or formulated to produce products with acceptable potency and efficacy. Here, we describe a simple method for improving the potency and efficacy of a recombinant subunit antigen by its immobilization on nickel-chelating nanolipoprotein particles (NiNLPs). NiNLPs are membrane mimetic nanoparticles that provide a delivery and presentation platform amenable to binding any recombinant subunit immunogens featuring a polyhistidine tag. A His-tagged, soluble truncated form of the West Nile virus (WNV) envelope protein (trE-His) was immobilized on NiNLPs. Single inoculations of the NiNLP-trE-His produced superior anti-WNV immune responses and provided significantly improved protection against a live WNV challenge compared to mice inoculated with trE-His alone. These results have broad implications in vaccine development and optimization, as NiNLP technology is well-suited to many types of vaccines, providing a universal platform for enhancing the potency and efficacy of recombinant subunit immunogens.
Assuntos
Quelantes/química , Encefalite Viral/prevenção & controle , Lipoproteínas/química , Nanopartículas/química , Níquel/química , Vacinas de Subunidades Antigênicas/imunologia , Febre do Nilo Ocidental/prevenção & controle , Vacinas contra o Vírus do Nilo Ocidental/imunologia , Animais , Quelantes/administração & dosagem , Encefalite Viral/imunologia , Ensaio de Imunoadsorção Enzimática , Camundongos , Fatores de Tempo , Vacinas de Subunidades Antigênicas/química , Proteínas do Envelope Viral/imunologia , Febre do Nilo Ocidental/imunologia , Vacinas contra o Vírus do Nilo Ocidental/administração & dosagem , Vacinas contra o Vírus do Nilo Ocidental/químicaRESUMO
Obtaining suitable seed viruses for influenza vaccines poses a challenge for public health authorities and manufacturers. We used reverse genetics to generate vaccine seed-compatible viruses from the 2009 pandemic swine-origin influenza virus. Comparison of viruses recovered with variations in residues 186 and 194 (based on the H3 numbering system) of the viral hemagglutinin showed that these viruses differed with respect to their ability to grow in eggs and cultured cells. Thus, we have demonstrated that molecular cloning of members of a quasispecies can help in selection of seed viruses for vaccine manufacture.
Assuntos
Surtos de Doenças , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/virologia , Mutação Puntual , Replicação Viral , Sequência de Aminoácidos , Animais , Linhagem Celular , Embrião de Galinha , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/epidemiologia , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Avian influenza (AI) surveillance in commercial poultry is accomplished by detecting the presence of antibodies to two group-specific antigens, NP and M1, using the agar gel immunodiffusion test. In order to determine the viral subtype responsible for the infection, positive samples must be further subtyped using the hemagglutination inhibition and neuraminidase inhibition tests. These tests are labor intensive and may take up to 4 days, thus slowing down responses to outbreaks. To expedite the subtyping of chicken sera we have developed a multiplex fluorescence microsphere immunoassay (FMIA), which allows for the simultaneous detection and subtyping of chicken sera to H5 influenza viruses. The FMIA was developed using NP (full length) and H5 (HA1 region) proteins expressed in baby hamster kidney cells using a Venezuela equine encephalitis virus replicon system. Both proteins were tagged with 6xHis at the carboxy-end and purified using cobalt-coated agarose beads. Purified H5 protein showed minimal cross-reactivity with anti-H2 serum, while no cross-reactivity was observed with sera to other AI virus (AIV) subtypes and other important poultry viral pathogens. In addition, and as expected, all the AIV sera tested reacted strongly with purified NP protein. Our results indicate that FMIA can be used for rapid subtyping of chicken sera.
Assuntos
Anticorpos Antivirais/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunoensaio/métodos , Influenza Aviária/imunologia , Nucleoproteínas/imunologia , Animais , Linhagem Celular , Galinhas , Clonagem Molecular , Cricetinae , FluorescênciaRESUMO
Existing live-attenuated flavivirus vaccines (LAV) could be improved by reducing their potential to recombine with naturally circulating viruses in the field. Since the highly conserved cyclization sequences (CS) found in the termini of flavivirus genomes must be complementary to each other to support genome replication, we set out to identify paired mutant CS that could support the efficient replication of LAV but would be unable to support replication in recombinant viruses harboring one wild-type (WT) CS. By systematic evaluation of paired mutated CS encoded in West Nile virus (WNV) replicons, we identified variants having single and double mutations in the 5'- and 3'-CS components that could support genome replication at WT levels. Replicons containing only the double-mutated CS in the 5' or the 3' ends of the genome were incapable of replication, indicating that mutated CS could be useful for constructing safer LAV. Despite the identity of the central portion of the CS in all mosquito-borne flaviviruses, viruses carrying complementary the double mutations in both the 5'- and the 3'-CS were indistinguishable from WT WNV in their replication in insect and mammalian cell lines. In addition to the utility of our novel CS pair in constructing safer LAV, we demonstrated that introduction of these mutated CS into one component of a recently described two-component genome system (A. V. Shustov, P. W. Mason, and I. Frolov, J. Virol. 81:11737-11748, 2007) enabled us to engineer a safer single-cycle WNV vaccine candidate with reduced potential for recombination during its propagation.
Assuntos
Mutação Puntual , RNA Viral/genética , Montagem de Vírus/fisiologia , Replicação Viral/fisiologia , Vacinas contra o Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/fisiologia , Animais , Linhagem Celular , Cricetinae , Culicidae , Vacinas Atenuadas/genética , Montagem de Vírus/genética , Replicação Viral/genética , Vírus do Nilo Ocidental/genéticaRESUMO
Nanolipoprotein particles (NLPs) are nanometer-sized, discoidal particles that self-assemble from purified apolipoprotein and phospholipid. Their size and facile functionalization suggest potential application of NLPs as platforms for the presentation and delivery of recombinant proteins. To this end, we investigated incorporation of nickel-chelating lipids into NLPs (NiNLPs) and subsequent sequestration of polyhistidine (His)-tagged proteins. From initial lipid screens for NLP formation, the two phospholipids DMPC and DOPC were identified as suitable bulk lipids for incorporation of the nickel-chelating lipid DOGS-NTA-Ni into NLPs, and NiNLPs were successfully formed with varying amounts of DOGS-NTA-Ni. NiNLPs consisting of 10% DOGS-NTA-Ni with 90% bulk lipid (either DMPC or DOPC) were thoroughly characterized by size exclusion chromatography (SEC), non-denaturing gradient gel electrophoresis (NDGGE), and atomic force microscopy (AFM). Three different His-tagged proteins were sequestered on NiNLPs in a nickel-dependent manner, and the amount of immobilized protein was contingent on the size and composition of the NiNLP.
Assuntos
Proteínas de Bactérias/metabolismo , Quelantes/química , Lipídeos/química , Lipoproteínas/química , Nanopartículas/química , Níquel/química , Proteínas de Bactérias/química , Quelantes/metabolismo , Histidina/química , Histidina/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Níquel/metabolismo , Tamanho da Partícula , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Yersinia pestis/químicaRESUMO
Dendritic cells (DCs) play a central role in innate immunity and antiviral responses. In this study, we investigated the production of alpha interferon (IFN-alpha) and inducible chemokines by human monocyte-derived dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) infected with West Nile virus (WNV), an emergent pathogen whose infection can lead to severe cases of encephalitis in the elderly, children, and immunocompromised individuals. Our experiments demonstrated that WNV grown in mammalian cells (WNV(Vero)) was a potent inducer of IFN-alpha secretion in pDCs and, to a lesser degree, in mDCs. The ability of WNV(Vero) to induce IFN-alpha in pDCs did not require viral replication and was prevented by the treatment of cells with bafilomycin A1 and chloroquine, suggesting that it was dependent on endosomal Toll-like receptor recognition. On the other hand, IFN-alpha production in mDCs required viral replication and was associated with the nuclear translocation of IRF3 and viral antigen expression. Strikingly, pDCs failed to produce IFN-alpha when stimulated with WNV grown in mosquito cells (WNV(C7/10)), while mDCs responded similarly to WNV(Vero) or WNV(C7/10). Moreover, the IFN-dependent chemokine IP-10 was produced in substantial amounts by pDCs in response to WNV(Vero) but not WNV(C7/10), while interleukin-8 was produced in greater amounts by mDCs infected with WNV(C7/10) than in those infected with WNV(Vero). These findings suggest that cell-specific mechanisms of WNV recognition leading to the production of type I IFN and inflammatory chemokines by DCs may contribute to both the innate immune response and disease pathogenesis in human infections.
Assuntos
Quimiocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/virologia , Interferon-alfa/metabolismo , Monócitos/virologia , Vírus do Nilo Ocidental/patogenicidade , Aedes/virologia , Animais , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Células Dendríticas/classificação , Humanos , Monócitos/citologia , Especificidade da Espécie , Células Vero , Replicação Viral , Vírus do Nilo Ocidental/fisiologiaRESUMO
The effect of long-term West Nile virus (WNV) infection on Culex salivary gland morphology and viability was evaluated by transmission electron microscopy during a four week period post-blood feeding. These studies showed that apoptosis and other cytopathologic changes occurred more frequently in WNV-infected mosquitoes compared with uninfected controls. The effect of long-term infection on WNV transmission was evaluated by titering virus in saliva over the same time period. Although the mean titer of WNV in mosquito saliva did not change significantly over time, the percentage of saliva samples containing WNV decreased. Because of the importance of saliva in blood meal acquisition and virus delivery, salivary gland pathology has the potential to affect mosquito feeding behavior and virus transmission. Results from this study add to a growing body of evidence that arbovirus infections in mosquito vectors can be cytopathic, and offer a potential mechanism for virus-induced cell death in mosquitoes.
Assuntos
Culex/virologia , Efeito Citopatogênico Viral , Glândulas Salivares/citologia , Glândulas Salivares/virologia , Febre do Nilo Ocidental/transmissão , Vírus do Nilo Ocidental/fisiologia , Animais , Morte Celular , Feminino , Febre do Nilo Ocidental/patologia , Febre do Nilo Ocidental/veterináriaRESUMO
Alphavirus-based replicons are a promising nucleic acid vaccine platform characterized by robust gene expression and immune responses. To further explore their use in vaccination, replicons were engineered to allow conditional control over their gene expression. Riboswitches, comprising a ribozyme actuator and RNA aptamer sensor, were engineered into the replicon 3' UTR. Binding of ligand to aptamer modulates ribozyme activity and, therefore, gene expression. Expression from DNA-launched and VRP-packaged replicons containing riboswitches was successfully regulated, achieving a 47-fold change in expression and modulation of the resulting type I interferon response. Moreover, we developed a novel control architecture where riboswitches were integrated into the 3' and 5' UTR of the subgenomic RNA region of the TC-83 virus, leading to an 1160-fold regulation of viral replication. Our studies demonstrate that the use of riboswitches for control of RNA replicon expression and viral replication holds promise for development of novel and safer vaccination strategies.