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J., Sustained specific and cross-reactive T cell responses to Zika and dengue virus NS3 in West Africa. mice using an alphavirus-based replicon RNA vaccine expressing ZIKV nonstructural protein NS3, a potent T cell antigen. The NS3 vaccine did not induce a neutralizing antibody response but elicited polyfunctional CD8+ T cells that were necessary and sufficient for preventing death in lethally infected adult mice and fetal growth restriction in infected pregnant mice. These data identify CD8+ T cells as the major mediators of ZIKV NS3 vaccineCinduced protection and suggest a new strategy to develop safe and effective anti-flavivirus vaccines. INTRODUCTION Zika computer virus (ZIKV) is usually a reemerging member of the flaviviridae family, which includes dengue (DENV), yellow fever, Japanese encephalitis, and West Nile viruses. ZIKV was first isolated in Uganda in 1947, and since then, outbreaks have occurred in several regions of the globe. The major route of ZIKV transmission is usually through the bite of spp. mosquitoes, but it is also transmitted through sexual contact and blood transfusions, as well as transplacentally. ZIKV has been found to persist in the semen, testes, and female reproductive tract of humans and animal models for up to 6 months after contamination (promoter to drive expression of a heterologous gene of interest. All viral structural genes are deleted and can be replaced by a gene or antigen of interest. To express ZIKV NS3 and prM-E proteins, we used the Synthetically Modified Alpha RNA Replicon Technology (SMARRT) platform (Synthetic Genomics Inc.), which is an alphavirus (Venezuelan equine encephalitis computer virus)Cbased replicon designed to evade the antiviral immune response (Fig. 1A). A ZIKV prM-ECbased vaccine lacking the immunodominant EDII fusion loop (FL) epitope has previously been shown to induce protection against ZIKV while minimizing the production of Abs that mediate DENV-ADE (promoter. BHK-21 cells were EX 527 (Selisistat) electroporated with water (Mock), an irrelevant RNA (bridging control), or RNA encoding either ZIKV prM-E or NS3 and then analyzed 20 hours after EX 527 (Selisistat) electroporation. UTR, untranslated region. (B) Western blot analysis of whole-cell lysates probed with anti-ZIKV E or NS3 Abdominal muscles. FLNA MW, molecular excess weight. (C) Quantification of launch efficiency by intracellular staining of BHK-21 cells with an anti-dsRNA (J2) Ab. Data are offered as the mean SD of triplicates from one experiment, EX 527 (Selisistat) representative of two impartial experiments. The nonparametric Mann-Whitney test was used to compare Mock versus each group; ****< 0.0001. To confirm the ability of these constructs to launch and produce protein efficiently, baby hamster kidney (BHK)C21 cells were electroporated with the replicon RNA and analyzed 20 hours later. Western blot analysis of cell lysates with Abs against ZIKV NS3 or E revealed expression of proteins with the predicted molecular weights (Fig. 1B), and circulation cytometry of cells stained with an antiCdouble-stranded RNA (dsRNA) Ab indicated that approximately 80% of cells were dsRNA positive (Fig. 1C). These data confirm efficient launch and protein production by the alphavirus-derived replicon in mammalian cells. For the mouse experiments, the replicon constructs formulated in lipid nanoparticles are referred to as vaccines. Immunogenicity of ZIKV NS3 and prM-E vaccines in C57BL/6 mice To assess the immunogenicity of the ZIKV vaccines, we first examined T and B cell responses in wild-type C57BL/6 mice. Age- and gender-matched groups of na?ve 4- to 5-week-old female and male mice were intramuscularly injected with 10 g of NS3 or prM-E vaccine or saline alone (control) and EX 527 (Selisistat) boosted 28 days later in the same manner (Fig. 2A). Three weeks later (day 49), splenocytes were prepared and stimulated for 20 hours in vitro with a pool of H-2bCrestricted peptides derived from ZIKV NS3 or prM-E, which were previously identified as epitopes for CD8+ and CD4+ T cells in C57BL/6 mice (Table 1) (= 10) and prM-E (= 11) vaccines or with saline (= 4) and boosted in the same manner on day 28. (B and C) On day 49, EX 527 (Selisistat) splenocytes were stimulated with pooled ZIKV prM-EC or NS3-derived peptides. IFN-producing CD8+ T cells (B) and CD4+ T cells (C) were quantified as SFCs per 106 of splenocytes. (D) U937-DC-SIGN cell-based circulation cytometric assay of ZIKV neutralizing activity (NT50) of sera collected on days 27 and 49. (E and F) Sera from ZIKV NS3- and prM-ECvaccinated C57BL/6 wild-type mice were collected on day 49, and 1 to 10 l were intraperitoneally injected into 5- to 6-week-old AG129 mice (= 6). Mice (= 6) were injected with 15 g of antiCDENV-prM Ab (2H2) or PBS as positive and negative controls, respectively. One day later, all AG129 mice were intravenously infected with 105 focus-forming models (FFU) of DENV2 S221..