RESUMO
Innovative vaccine platforms are needed to develop effective countermeasures against emerging and re-emerging diseases. These platforms should direct antigen internalization by antigen presenting cells and promote immunogenic responses. This work describes an innovative systems approach combining two novel platforms, αGalactose (αGal)-modification of antigens and amphiphilic polyanhydride nanoparticles as vaccine delivery vehicles, to rationally design vaccine formulations. Regimens comprising soluble αGal-modified antigen and nanoparticle-encapsulated unmodified antigen induced a high titer, high avidity antibody response with broader epitope recognition of antigenic peptides than other regimen. Proliferation of antigen-specific CD4(+) T cells was also enhanced compared to a traditional adjuvant. Combining the technology platforms and augmenting immune response studies with peptide arrays and informatics analysis provides a new paradigm for rational, systems-based design of next generation vaccine platforms against emerging and re-emerging pathogens.
Assuntos
Imunidade Inata , Nanopartículas/química , Vacinas/imunologia , alfa-Galactosidase/imunologia , Adjuvantes Imunológicos , Células Apresentadoras de Antígenos/imunologia , Antígenos/imunologia , Antígenos/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Epitopos/química , Epitopos/imunologia , Humanos , Nanopartículas/uso terapêutico , Peptídeos/química , Peptídeos/imunologia , Biologia de Sistemas , alfa-Galactosidase/uso terapêuticoRESUMO
Virus-like particles (VLPs) present viral antigens in a native conformation and are effectively recognized by the immune system and therefore are considered as suitable and safe vaccine candidates against many viral diseases. Here we demonstrate that chimeric VLPs containing Rift Valley fever virus (RVFV) glycoproteins G(N) and G(C), nucleoprotein N and the gag protein of Moloney murine leukemia virus represent an effective vaccine candidate against Rift Valley fever, a deadly disease in humans and livestock. Long-lasting humoral and cellular immune responses are demonstrated in a mouse model by the analysis of neutralizing antibody titers and cytokine secretion profiles. Vaccine efficacy studies were performed in mouse and rat lethal challenge models resulting in high protection rates. Taken together, these results demonstrate that replication-incompetent chimeric RVF VLPs are an efficient RVFV vaccine candidate.
Assuntos
Febre do Vale de Rift/imunologia , Vírus da Febre do Vale do Rift/imunologia , Vacinas Virossomais/imunologia , Replicação Viral , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Citocinas/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Vírus da Leucemia Murina de Moloney/genética , Ratos , Ratos Endogâmicos WF , Febre do Vale de Rift/prevenção & controle , Vírus da Febre do Vale do Rift/fisiologia , Análise de Sobrevida , Linfócitos T/imunologia , Vacinas Virossomais/genética , Proteínas Virais/genética , Virossomos/ultraestruturaRESUMO
To explore mechanisms of entry for Ebola virus (EBOV) glycoprotein (GP) pseudotyped virions, we used comparative gene analysis to identify genes whose expression correlated with viral transduction. Candidate genes were identified by using EBOV GP pseudotyped virions to transduce human tumor cell lines that had previously been characterized by cDNA microarray. Transduction profiles for each of these cell lines were generated, and a significant positive correlation was observed between RhoC expression and permissivity for EBOV vector transduction. This correlation was not specific for EBOV vector alone as RhoC also correlated highly with transduction of vesicular stomatitis virus GP (VSVG) pseudotyped vector. Levels of RhoC protein in EBOV and VSV permissive and nonpermissive cells were consistent with the cDNA gene array findings. Additionally, vector transduction was elevated in cells that expressed high levels of endogenous RhoC but not RhoA. RhoB and RhoC overexpression significantly increased EBOV GP and VSVG pseudotyped vector transduction but had minimal effect on human immunodeficiency virus (HIV) GP pseudotyped HIV or adeno-associated virus 2 vector entry, indicating that not all virus uptake was enhanced by expression of these molecules. RhoB and RhoC overexpression also significantly enhanced VSV infection. Similarly, overexpression of RhoC led to a significant increase in fusion of EBOV virus-like particles. Finally, ectopic expression of RhoC resulted in increased nonspecific endocytosis of fluorescent dextran and in formation of increased actin stress fibers compared to RhoA-transfected cells, suggesting that RhoC is enhancing macropinocytosis. In total, our studies implicate RhoB and RhoC in enhanced productive entry of some pseudovirions and suggest the involvement of actin-mediated macropinocytosis as a mechanism of uptake of EBOV GP and VSVG pseudotyped viral particles.
Assuntos
Ebolavirus/enzimologia , Vetores Genéticos , Vesiculovirus/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Clostridioides difficile , Humanos , Microscopia de Fluorescência/métodos , Modelos Biológicos , Plasmídeos/metabolismo , Células VeroRESUMO
The advent of reverse genetics technology has revolutionized the field of RNA viruses. It is now possible to manipulate even negative-stranded RNA viruses at will, and evaluate the effects of these changes on the biology and pathogenesis of these viruses. The fundamental insights gleaned from the reverse genetics-based studies over the last several years have provided a new momentum for the development of designed therapies for the control and prevention of these viral pathogens. The recombinant viruses have been exploited also as vectors for devising targeted therapies for non-viral diseases such as malignancies, and in gene therapy for inherited disorders. This review provides a brief summary of the stumbling blocks and the successes in the development of the technology for the negative-stranded RNA viruses. The many and varied applications of the recombinant vectors are also outlined.
Assuntos
Vírus de RNA/genética , Animais , DNA Complementar/genética , DNA Viral/genética , Técnicas Genéticas , Vetores Genéticos , Humanos , Vírus de RNA/crescimento & desenvolvimento , Vírus de RNA/fisiologia , RNA Viral/biossíntese , RNA Viral/genética , Recombinação Genética , Vacinas Atenuadas/genética , Vacinas Virais/genética , Replicação ViralRESUMO
Crimean-Congo hemorrhagic fever (CCHF) virus is highly pathogenic for humans and remains the only Category A virus for which full sequence information is currently unavailable. In this study we completed CCHF genome characterization by determining the L segment sequence using Dugbe and CCHF virus-specific oligonucleotides. Sequence alignments revealed the presence of four previously described conserved regions in all Bunyaviridae polymerases. Interestingly, additional regions containing putative Ovarian Tumor (OTU)-like cysteine protease and helicase domains were identified in the L segments of CCHF and Dugbe viruses, suggesting an autoproteolytic cleavage process for nairovirus L proteins.
Assuntos
Genoma Viral , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , RNA Polimerase Dependente de RNA/genética , Sequência de Aminoácidos , Clonagem Molecular , Cisteína Endopeptidases/genética , Vírus da Febre Hemorrágica da Crimeia-Congo/classificação , Vírus da Febre Hemorrágica da Crimeia-Congo/enzimologia , Dados de Sequência Molecular , Fases de Leitura Aberta , Estrutura Terciária de Proteína/genética , RNA Helicases/genética , Alinhamento de SequênciaRESUMO
We sequenced the 29,751-base genome of the severe acute respiratory syndrome (SARS)-associated coronavirus known as the Tor2 isolate. The genome sequence reveals that this coronavirus is only moderately related to other known coronaviruses, including two human coronaviruses, HCoV-OC43 and HCoV-229E. Phylogenetic analysis of the predicted viral proteins indicates that the virus does not closely resemble any of the three previously known groups of coronaviruses. The genome sequence will aid in the diagnosis of SARS virus infection in humans and potential animal hosts (using polymerase chain reaction and immunological tests), in the development of antivirals (including neutralizing antibodies), and in the identification of putative epitopes for vaccine development.
Assuntos
Genoma Viral , RNA Viral/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Proteínas Virais/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Sequência Conservada , Coronavirus/classificação , Coronavirus/genética , Proteínas M de Coronavírus , Proteínas do Nucleocapsídeo de Coronavírus , DNA Complementar , Mudança da Fase de Leitura do Gene Ribossômico , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Fases de Leitura Aberta , Filogenia , RNA Viral/isolamento & purificação , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Sequências Reguladoras de Ácido Nucleico , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/classificação , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/isolamento & purificação , Análise de Sequência de DNA , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas Virais/químicaRESUMO
A new coronavirus has been implicated as the causative agent of severe acute respiratory syndrome (SARS). We have used convalescent sera from several SARS patients to detect proteins in the culture supernatants from cells exposed to lavage another SARS patient. The most prominent protein in the supernatant was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a approximately 46-kDa species. This was found to be a novel nucleocapsid protein that matched almost exactly one predicted by an open reading frame in the recently published nucleotide sequence of the same virus isolate (>96% coverage). A second viral protein corresponding to the predicted approximately 139-kDa spike glycoprotein has also been examined by MALDI-TOF MS (42% coverage). After peptide N-glycosidase F digestion, 12 glycosylation sites in this protein were confirmed. The sugars attached to four of the sites were also identified. These results suggest that the nucleocapsid protein is a major immunogen that may be useful for early diagnostics, and that the spike glycoprotein may present a particularly attractive target for prophylactic intervention in combating SARS.