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Previously, we constructed the pE1D2 and pcTPANS1 DNA vaccines encoding the DENV2 envelope (E) and non-structural 1 (NS1) proteins, respectively. To decrease the number of plasmids in a tetravalent candidate vaccine, we constructed a bicistronic plasmid, pNS1\/E\/D2, encoding these two proteins simultaneously. We evaluated the protective immunity induced in mice vaccinated with the pNS1\/E\/D2 candidate and compared to the responses elicited by immunization with the former vaccines isolated or in combination. We transfected BHK-21 cells with the different plasmids and detected recombinant proteins by immunofluorescence and mass spectrometry assays to confirm antigen expression. BALB\/c mice were inoculated with the DNA vaccines followed by a lethal DENV2 challenge. ELISA, PRNT50, and IFN-gamma ELISPOT assays were performed for the investigation of the humoral and cellular responses. We observed the concomitant expression of NS1 and E proteins in pNS1\/E\/D2-transfected cells. All E-based vaccines induced anti-E and neutralizing antibodies. However, anti-NS1 antibodies were only observed after immunization with the pcTPANS1 administered alone or combined with pE1D2. In contrast, splenocytes from pNS1\/E\/D2- or pcTPANS1 + pE1D2-vaccinated animals responded to NS1- and E-derived synthetic peptides. All the DNA vaccines conferred protection against DENV2.<\/jats:p>","DOI":"10.3390\/v14071452","type":"journal-article","created":{"date-parts":[[2022,7,1]],"date-time":"2022-07-01T01:40:36Z","timestamp":1656639636000},"page":"1452","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Combination of E- and NS1-Derived DNA Vaccines: The Immune Response and Protection Elicited in Mice against DENV2"],"prefix":"10.3390","volume":"14","author":[{"given":"Paolla Beatriz A.","family":"Pinto","sequence":"first","affiliation":[{"name":"Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro 21040-900, RJ, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4047-2091","authenticated-orcid":false,"given":"Tamiris A. C.","family":"Barros","sequence":"additional","affiliation":[{"name":"Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro 21040-900, RJ, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lauro M.","family":"Lima","sequence":"additional","affiliation":[{"name":"Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro 21040-900, RJ, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Agatha R.","family":"Pacheco","sequence":"additional","affiliation":[{"name":"Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro 21040-900, RJ, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maysa L.","family":"Assis","sequence":"additional","affiliation":[{"name":"Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro 21040-900, RJ, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7825-5164","authenticated-orcid":false,"given":"Bernardo A. S.","family":"Pereira","sequence":"additional","affiliation":[{"name":"Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro 21040-900, RJ, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ant\u00f4nio J. S.","family":"Gon\u00e7alves","sequence":"additional","affiliation":[{"name":"Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro 21040-900, RJ, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7401-5107","authenticated-orcid":false,"given":"Adriana S.","family":"Azevedo","sequence":"additional","affiliation":[{"name":"Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro 21040-900, RJ, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7863-9512","authenticated-orcid":false,"given":"Ana Gisele C.","family":"Neves-Ferreira","sequence":"additional","affiliation":[{"name":"Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro 21040-900, RJ, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simone M.","family":"Costa","sequence":"additional","affiliation":[{"name":"Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro 21040-900, RJ, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ada M. B.","family":"Alves","sequence":"additional","affiliation":[{"name":"Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro 21040-900, RJ, Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,6,30]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2009). Special Programme for Research and Training in Tropical Diseases. Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control, New ed., World Health Organization."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Brady, O.J., Gething, P.W., Bhatt, S., Messina, J.P., Brownstein, J.S., Hoen, A.G., Moyes, C.L., Farlow, A.W., Scott, T.W., and Hay, S.I. (2012). Refining the Global Spatial Limits of Dengue Virus Transmission by Evidence-Based Consensus. PLoS Negl. Trop. 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