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      T Cell Responses Induced by DNA Vaccines Based on the DENV2 E and NS1 Proteins in Mice: Importance in Protection and Immunodominant Epitope Identification

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          Abstract

          The importance of the cellular immune response against DENV has been increasingly highlighted in the past few years, in particular for vaccine development. We have previously constructed two plasmids, pE1D2, and pcTPANS1, encoding the envelope (E) ectodomain (domains I, II, and III) and the non-structural 1 (NS1) protein of dengue virus serotype 2 (DENV2), respectively. In the present work, we analyzed the induction of the cellular response in mice immunized with these DNA vaccines and identified the immunogenic peptides. Vaccinated BALB/c mice became protected against a lethal challenge of DENV2. Depletion of CD4 + cells in vaccinated animals almost completely abolished protection elicited by both vaccines. In contrast, a significant number of pE1D2- and pcTPANS1-immunized mice survived virus challenge after depletion of CD8 + cells, although some animals presented morbidity. To identify immunogenic peptides recognized by T cells, we stimulated splenocytes with overlapping peptide libraries covering the E and NS1 proteins and evaluated the production of IFN-γ by ELISPOT. We detected two and three immunodominant epitopes in the E and NS1 proteins, respectively, and four additional NS1-derived peptides after virus challenge. Characterization by intracellular cytokine staining (ICS) revealed that both CD4 + and CD8 + T cells were involved in IFN-γ and TNF-α production. The IFN-γ ICS confirmed reaction of almost all E-derived peptides before challenge and identified other epitopes after infection. All NS1-derived peptides were able to elicit IFN-γ production in CD4 + cells, while only a few peptides induced expression of this cytokine in CD8 + T lymphocytes. Interestingly, we observed an increase in the frequency of either CD4 + or CD8 + T cells producing TNF-α after immunization with the pE1D2 and challenge with DENV2, while lymphocytes from pcTPANS1-vaccinated animals maintained ordinary TNF-α production after virus infection. We also assessed the recognition of E and NS1 immunogenic peptides in C57BL/6 mice due to the difference in MHC haplotype expression. Two NS1-derived epitopes featured prominently in the IFN-γ response with cells from both animal strains. Overall, our results emphasize the importance of the T cell response involved in protection against dengue induced by E and NS1 based DNA vaccines.

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          Efficacy and Long-Term Safety of a Dengue Vaccine in Regions of Endemic Disease.

          A candidate tetravalent dengue vaccine is being assessed in three clinical trials involving more than 35,000 children between the ages of 2 and 16 years in Asian-Pacific and Latin American countries. We report the results of long-term follow-up interim analyses and integrated efficacy analyses.
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            The flavivirus NS1 protein: molecular and structural biology, immunology, role in pathogenesis and application as a diagnostic biomarker.

            The flavivirus nonstructural glycoprotein NS1 is an enigmatic protein whose structure and mechanistic function have remained somewhat elusive ever since it was first reported in 1970 as a viral antigen circulating in the sera of dengue-infected patients. All flavivirus NS1 genes share a high degree of homology, encoding a 352-amino-acid polypeptide that has a molecular weight of 46-55 kDa, depending on its glycosylation status. NS1 exists in multiple oligomeric forms and is found in different cellular locations: a cell membrane-bound form in association with virus-induced intracellular vesicular compartments, on the cell surface and as a soluble secreted hexameric lipoparticle. Intracellular NS1 co-localizes with dsRNA and other components of the viral replication complex and plays an essential cofactor role in replication. Although this makes NS1 an ideal target for inhibitor design, the precise nature of its cofactor function has yet to be elucidated. A plethora of potential interacting partners have been identified, particularly for the secreted form of NS1, with many being implicated in immune evasion strategies. Secreted and cell-surface-associated NS1 are highly immunogenic and both the proteins themselves and the antibodies they elicit have been implicated in the seemingly contradictory roles of protection and pathogenesis in the infected host. Finally, NS1 is also an important biomarker for early diagnosis of disease. In this article, we provide an overview of these somewhat disparate areas of research, drawing together the wealth of data generated over more than 40 years of study of this fascinating protein. Copyright © 2013 Elsevier B.V. All rights reserved.
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              The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8+ T cell response.

              The live yellow fever vaccine (YF-17D) offers a unique opportunity to study memory CD8(+) T cell differentiation in humans following an acute viral infection. We have performed a comprehensive analysis of the virus-specific CD8(+) T cell response using overlapping peptides spanning the entire viral genome. Our results showed that the YF-17D vaccine induces a broad CD8(+) T cell response targeting several epitopes within each viral protein. We identified a dominant HLA-A2-restricted epitope in the NS4B protein and used tetramers specific for this epitope to track the CD8(+) T cell response over a 2 year period. This longitudinal analysis showed the following. 1) Memory CD8(+) T cells appear to pass through an effector phase and then gradually down-regulate expression of activation markers and effector molecules. 2) This effector phase was characterized by down-regulation of CD127, Bcl-2, CCR7, and CD45RA and was followed by a substantial contraction resulting in a pool of memory T cells that re-expressed CD127, Bcl-2, and CD45RA. 3) These memory cells were polyfunctional in terms of degranulation and production of the cytokines IFN-gamma, TNF-alpha, IL-2, and MIP-1beta. 4) The YF-17D-specific memory CD8(+) T cells had a phenotype (CCR7(-)CD45RA(+)) that is typically associated with terminally differentiated cells with limited proliferative capacity (T(EMRA)). However, these cells exhibited robust proliferative potential showing that expression of CD45RA may not always associate with terminal differentiation and, in fact, may be an indicator of highly functional memory CD8(+) T cells generated after acute viral infections.
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                Author and article information

                Contributors
                Journal
                Front Immunol
                Front Immunol
                Front. Immunol.
                Frontiers in Immunology
                Frontiers Media S.A.
                1664-3224
                03 July 2019
                2019
                : 10
                : 1522
                Affiliations
                [1] 1Laboratory of Biotechnology and Physiology of Viral Infections, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (Fiocruz) , Rio de Janeiro, Brazil
                [2] 2Laboratory of Immunopharmacology, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (Fiocruz) , Rio de Janeiro, Brazil
                Author notes

                Edited by: Gathsaurie Neelika Malavige, University of Sri Jayewardenepura, Sri Lanka

                Reviewed by: Bruno Mendes Roatt, Universidade Federal de Ouro Preto, Brazil; Alba Grifoni, La Jolla Institute for Immunology (LJI), United States; Arash Memarnejadian, Sernova Corp., Canada

                *Correspondence: Ada M. B. Alves ada@ 123456ioc.fiocruz.br

                This article was submitted to Viral Immunology, a section of the journal Frontiers in Immunology

                †These authors have contributed equally to this work

                Article
                10.3389/fimmu.2019.01522
                6617960
                31333657
                ecf44b38-1b27-4695-a878-6450a688ee6d
                Copyright © 2019 Pinto, Assis, Vallochi, Pacheco, Lima, Quaresma, Pereira, Costa and Alves.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 01 February 2019
                : 18 June 2019
                Page count
                Figures: 7, Tables: 2, Equations: 0, References: 67, Pages: 18, Words: 11726
                Funding
                Funded by: Conselho Nacional de Desenvolvimento Científico e Tecnológico 10.13039/501100003593
                Funded by: Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro 10.13039/501100004586
                Funded by: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior 10.13039/501100002322
                Categories
                Immunology
                Original Research

                Immunology
                dengue,dna vaccines,t cell response,ns1,envelope protein,mice
                Immunology
                dengue, dna vaccines, t cell response, ns1, envelope protein, mice

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