RESUMO
Phylogenetic relatedness and cocirculation of several major human pathogen flaviviruses are recognized as a possible cause of deleterious immune responses to mixed infection or immunization and call for a greater understanding of the inter-Flavivirus protein homologies. This study focused on the identification of human leukocyte antigen (HLA)-restricted West Nile virus (WNV) T-cell ligands and characterization of their distribution in reported sequence data of WNV and other flaviviruses. H-2-deficient mice transgenic for either A2, A24, B7, DR2, DR3, or DR4 HLA alleles were immunized with overlapping peptides of the WNV proteome, and peptide-specific T-cell activation was measured by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assays. Approximately 30% (137) of the WNV proteome peptides were identified as HLA-restricted T-cell ligands. The majority of these ligands were conserved in â¼≥88% of analyzed WNV sequences. Notably, only 51 were WNV specific, and the remaining 86, chiefly of E, NS3, and NS5, shared an identity of nine or more consecutive amino acids with sequences of 64 other flaviviruses, including several major human pathogens. Many of the shared ligands had an incidence of >50% in the analyzed sequences of one or more of six major flaviviruses. The multitude of WNV sequences shared with other flaviviruses as interspecies variants highlights the possible hazard of defective T-cell activation by altered peptide ligands in the event of dual exposure to WNV and other flaviviruses, by either infection or immunization. The data suggest the possible preferred use of sequences that are pathogen specific with minimum interspecies sequence homology for the design of Flavivirus vaccines.
Assuntos
Antígenos Virais/imunologia , Flavivirus/imunologia , Antígenos de Histocompatibilidade/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Proteínas Virais/imunologia , Vírus do Nilo Ocidental/imunologia , Sequência de Aminoácidos , Animais , ELISPOT , Variação Genética , Antígenos de Histocompatibilidade/genética , Interferon gama , Ligantes , Camundongos , Camundongos Transgênicos , Proteoma , Linfócitos T/metabolismo , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/metabolismoRESUMO
Increasing levels of plasmid vector-mediated activation of innate immune signaling pathways is an approach to improve DNA vaccine-induced adaptive immunity for infectious disease and cancer applications. Retinoic acid-inducible gene I (RIG-I) is a critical cytoplasmic double-stranded RNA (dsRNA) pattern receptor required for innate immune activation in response to viral infection. Activation of RIG-I leads to type I interferon (IFN) and inflammatory cytokine production through interferon promoter stimulator 1 (IPS-1)-mediated activation of interferon regulatory factor 3 (IRF3) and NF-κB signaling. DNA vaccines coexpressing antigen and an expressed RNA (eRNA) RIG-I agonist were made, and the effect of RIG-I activation on antigen-specific immune responses to the encoded antigen was determined. Plasmid vector backbones expressing various RIG-I ligands from RNA polymerase III promoters were screened in a cell culture assay for RIG-I agonist activity, and optimized, potent RIG-I ligands were developed. One of these, eRNA41H, combines (i) eRNA11a, an immunostimulatory dsRNA expressed by convergent transcription, with (ii) adenovirus VA RNAI. eRNA41H was integrated into the backbone of DNA vaccine vectors expressing H5N1 influenza virus hemagglutinin (HA). The resultant eRNA vectors potently induced type 1 IFN production in cell culture through RIG-I activation and combined high-level HA antigen expression with RNA-mediated type I IFN activation in a single plasmid vector. The eRNA vectors induced increased HA-specific serum antibody binding avidity after naked DNA intramuscular prime and boost delivery in mice. This demonstrates that DNA vaccine potency may be augmented by the incorporation of RIG-I-activating immunostimulatory RNA into the vector backbone.
Assuntos
Anticorpos Antivirais/sangue , RNA Helicases DEAD-box/imunologia , Vacinas contra Influenza/imunologia , RNA de Cadeia Dupla/imunologia , Vacinas de DNA/imunologia , Adenoviridae/genética , Animais , Proteína DEAD-box 58 , Hemaglutininas Virais/biossíntese , Imunidade Humoral , Imunização Secundária/métodos , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Injeções Intramusculares , Interferon Tipo I/biossíntese , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA de Cadeia Dupla/genética , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genéticaRESUMO
The neglected tropical diseases (NTDs) are a group of 17 lesser known chronic infections which predominantly affect poor and disenfranchised communities. There are a number of NTDs that cause significant global morbidity in children, including the three major soil transmitted helminth (STH) infections (ascariasis, trichuriasis and hookworm infection), schistosomiasis and trachoma. These NTDs, together with lymphatic filariasis and onchocerciasis, are currently being targeted for global control and elimination through mass drug administration (MDA) campaigns. They represent the most common NTDs and share significant geographical overlap. Additionally, many individuals are polyparasitised with more than a single NTD. Integrated NTD control and elimination MDA programmes offer safe and efficacious treatments for all seven NTDs. However, the current global level of MDA coverage for the leading childhood NTDs, that is, STH infections, schistosomiasis and trachoma, remains well under 50%. Limiting factors for global coverage include insufficient global financial support, drug donation capacity of pharmaceutical companies and targeting school age children to the exclusion of other age groups in need of treatment, such as preschool age children. There is also a need for development of novel prevention and treatment modalities, such as next-generation small molecule drugs and vaccines. Efforts are underway to harness the momentum of a 2012 London Declaration on NTDs and a 2013 World Health Assembly (WHA) resolution as a means to control or in some cases eliminate by 2020 these NTDs that affect children worldwide.
Assuntos
Proteção da Criança , Controle de Doenças Transmissíveis , Saúde Global , Doenças Negligenciadas/epidemiologia , Medicina Tropical/métodos , Criança , Humanos , Doenças Negligenciadas/prevenção & controleRESUMO
Multidetector coronary computed tomography angiography (CTA) is a promising modality for widespread clinical application because of its noninvasive nature and high diagnostic accuracy as found in previous studies using 64 to 320 simultaneous detector rows. It is, however, limited in its ability to detect myocardial ischemia. In this article, we describe the design of the CORE320 study ("Combined coronary atherosclerosis and myocardial perfusion evaluation using 320 detector row computed tomography"). This prospective, multicenter, multinational study is unique in that it is designed to assess the diagnostic performance of combined 320-row CTA and myocardial CT perfusion imaging (CTP) in comparison with the combination of invasive coronary angiography and single-photon emission computed tomography myocardial perfusion imaging (SPECT-MPI). The trial is being performed at 16 medical centers located in 8 countries worldwide. CT has the potential to assess both anatomy and physiology in a single imaging session. The co-primary aim of the CORE320 study is to define the per-patient diagnostic accuracy of the combination of coronary CTA and myocardial CTP to detect physiologically significant coronary artery disease compared with (1) the combination of conventional coronary angiography and SPECT-MPI and (2) conventional coronary angiography alone. If successful, the technology could revolutionize the management of patients with symptomatic CAD.
Assuntos
Angiografia Coronária/métodos , Doença da Artéria Coronariana/diagnóstico por imagem , Circulação Coronária , Imagem de Perfusão do Miocárdio/métodos , Projetos de Pesquisa , Tomografia Computadorizada por Raios X , Idoso , Idoso de 80 Anos ou mais , Brasil , Canadá , Doença da Artéria Coronariana/fisiopatologia , Europa (Continente) , Feminino , Hemodinâmica , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Interpretação de Imagem Radiográfica Assistida por Computador , Índice de Gravidade de Doença , Singapura , Estados UnidosRESUMO
This report describes the identification and bioinformatics analysis of HLA-DR4-restricted HIV-1 Gag epitope peptides, and the application of dendritic cell mediated immunization of DNA plasmid constructs. BALB/c (H-2d) and HLA-DR4 (DRA1*0101, DRB1*0401) transgenic mice were immunized with immature dendritic cells transfected by a recombinant DNA plasmid encoding the lysosome-associated membrane protein-1/HIV-1 Gag (pLAMP/gag) chimera antigen. Three immunization protocols were compared: 1) primary subcutaneous immunization with 1x10(5) immature dendritic cells transfected by electroporation with the pLAMP/gag DNA plasmid, and a second subcutaneous immunization with the naked pLAMP/gag DNA plasmid; 2) primary immunization as above, and a second subcutaneous immunization with a pool of overlapping peptides spanning the HIV-1 Gag sequence; and 3) immunization twice by subcutaneous injection of the pLAMP/gag DNA plasmid. Primary immunization with pLAMP/gag-transfected dendritic cells elicited the greatest number of peptide specific T-cell responses, as measured by ex vivo IFN-gamma ELISpot assay, both in BALB/c and HLA-DR4 transgenic mice. The pLAMP/gag-transfected dendritic cells prime and naked DNA boost immunization protocol also resulted in an increased apparent avidity of peptide in the ELISpot assay. Strikingly, 20 of 25 peptide-specific T-cell responses in the HLA-DR4 transgenic mice contained sequences that corresponded, entirely or partially to 18 of the 19 human HLA-DR4 epitopes listed in the HIV molecular immunology database. Selection of the most conserved epitope peptides as vaccine targets was facilitated by analysis of their representation and variability in all reported sequences. These data provide a model system that demonstrates a) the superiority of immunization with dendritic cells transfected with LAMP/gag plasmid DNA, as compared to naked DNA, b) the value of HLA transgenic mice as a model system for the identification and evaluation of epitope-based vaccine strategies, and c) the application of variability analysis across reported sequences in public databases for selection of historically conserved HIV epitopes as vaccine targets.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , DNA/administração & dosagem , Células Dendríticas/metabolismo , Epitopos/imunologia , Produtos do Gene gag/genética , Antígeno HLA-DR4/imunologia , Plasmídeos , Sequência de Aminoácidos , Animais , Células Cultivadas , Eletroporação , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Proteínas Ligadas por GPI , HIV-1/imunologia , Antígeno HLA-DR4/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Dados de Sequência MolecularRESUMO
We have previously established a model inducing hematopoietic stem cell (HSC) production of circulating endothelial progenitor cells (EPCs) to revascularize ischemic injury in adult mouse retina. The unique vascular environment of the retina results in new blood vessel formation primarily from HSC-derived EPCs. Using mice deficient (-/-) in inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS), we show that vessel phenotype resulting from hemangioblast activity can be altered by modulation of the NO/NOS pathway. iNOS-/- or eNOS-/- animals were engrafted with wild-type (WT) HSCs expressing green fluorescence protein (gfp+) and subjected to our adult retinal ischemia model. WT hemangioblast activity in adult iNOS-/- recipients resulted in the formation of highly branched blood vessels of donor origin, which were readily perfused indicating functionality. In contrast, eNOS-/- recipients produced relatively unbranched blood vessels with significant donor contribution that were difficult to perfuse, indicating poor functionality. Furthermore, eNOS-/- chimeras had extensive gfp+ HSC contribution throughout their vasculature without additional injury. This neovascularization, via EPCs derived from the transplanted HSCs, reveals that the NO pathway can modulate EPC activity and plays a critical role in both blood vessel formation in response to injury and normal endothelial cell maintenance.