Dengue is a global public health problem and is caused by four dengue virus serotypes (DENV1-4). A major challenge in dengue vaccine development is that cross-reactive anti-DENV antibodies can be protective or potentially enhance disease via antibody-dependent enhancement (ADE). DENV nonstructural protein 1 (NS1) has long been considered a vaccine candidate as it avoids ADE. Here, we evaluated survival to challenge in a lethal DENV vascular leak model in mice immunized with NS1 combined with alum, Monophosphoryl Lipid A + AddaVax (MA), or Sigma Adjuvant System + CpG DNA, compared to mice infected with a non-lethal dose of DENV2 and mice immunized with ovalbumin (negative control). We characterized antibody responses to DENV-1, -2 and -3 NS1 using an antigen microarray tiled with 20-mer peptides overlapping by 15 amino acids and identified 5 regions of DENV NS1 with significant levels of antibody reactivity in the NS1+MA group. Additionally, we profiled the antibody responses to NS1 of humans naturally infected with DENV2 or DENV3 in serum samples from Nicaragua collected at acute, convalescent and 12-month timepoints. One region in the “wing” domain of NS1 was immunodominant in both mouse vaccination and human infection studies, and two regions were identified only in NS1-immunized mice; thus, vaccination can generate antibodies to regions that are not targeted in natural infection and could provide additional protection against lethal DENV infection. Overall, we identified a small number of immunodominant regions, which were in functionally important locations on the DENV NS1 protein and are potential correlates of protection.

The rapid generation of strong T cell responses is highly desirable and viral vectors can have potent CD8+ T cell-inducing activity. Immunity to leishmaniasis requires selective T cell responses, with immunization schemes that raise either CD4 or CD8 T cell responses being protective in small animal models. We have defined the leishmaniasis vaccine candidate recombinant fusion antigens, LEISH-F2 and LEISH-F3+, that when formulated in a stable emulsion with a Toll-like receptor (TLR) 4 agonist, induce protective CD4+ T cell responses in animal models as well as providing therapeutic efficacy in canine leishmaniasis and in clinical trials in leishmaniasis patients. We used the genetic sequences of these validated vaccine antigens to design RNA vaccine constructs. Immunization of mice with the RNA replicons induced potent, local innate responses that were surprisingly independent of TLR7 and activated antigen-presenting cells (APC) to prime for extremely potent antigen-specific T helper 1 type responses upon heterologous boosting with either of the subunit vaccines (recombinant antigen with second generation glucopyranosyl lipid A in stable oil-in-water emulsion; SLA-SE). Inclusion of RNA in the immunization schedule also generated MHCI-restricted T cell responses. Immunization with LEISH-F2-expressing RNA vaccine followed later by subunit vaccine afforded protection against challenge with Leishmania donovani. Together, these data indicate the utility of heterologous prime-boost immunization schemes for the induction of potent antigen-specific CD4 and CD8 T cell responses for protection against intracellular pathogens.

Malaria vaccine development has been hampered by the limited availability of antigens identified through conventional discovery approaches, and improvements are needed to enhance the efficacy of the leading vaccine candidate RTS,S that targets the circumsporozoite protein (CSP) of the infective sporozoite. Here we report a transcriptome-based approach to identify novel pre-erythrocytic vaccine antigens that could potentially be used in combination with CSP. We hypothesized that stage-specific upregulated genes would enrich for protective vaccine targets, and used tiling microarray to identify P. falciparum genes transcribed at higher levels during liver stage versus sporozoite or blood stages of development. We prepared DNA vaccines for 21 genes using the predicted orthologues in P. yoelii and P. berghei and tested their efficacy using different delivery methods against pre-erythrocytic malaria in rodent models. In our primary screen using P. yoelii in BALB/c mice, we found that 16 antigens significantly reduced liver stage parasite burden. In our confirmatory screen using P. berghei in C57Bl/6 mice, we confirmed 6 antigens that were protective in both models. Two antigens, when combined with CSP, provided significantly greater protection than CSP alone in both models. Based on the observations reported here, transcriptional patterns of Plasmodium genes can be useful in identifying novel pre-erythrocytic antigens that induce protective immunity alone or in combination with CSP.

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A major challenge in dengue vaccine development is that cross-reactive anti-DENV antibodies (Abs) can be protective or potentially enhance disease via antibody-dependent enhancement (ADE). We recently showed that immunization with adjuvanted recombinant NS1 from all four DENV serotypes protects against DENV2 challenge in a mouse model of lethal vascular leak syndrome. Conversely, we found that DENV NS1 by itself triggers endothelial permeability in vitro and vascular leak in vivo. Here, we evaluated survival to lethal DENV challenge in mice immunized with (1) NS1+alum; (2) NS1+Monophosphoryl Lipid A and AddaVax (MA); (3) NS1+Sigma adjuvant system and CpG DNA (SCpG); (4) ovalbumin+adjuvant (OVA); or (5) a sublethal dose of DENV2 strain PLO46.NS1. OVA or NS1+alum immunized mice were not protected, whereas immunization with NS1+MA or NS1+SCpG or DENV2-PL046 resulted in 100% survival. We characterized Ab responses to NS1 from DENV1-3 using an antigen microarray tiled with 20-mer peptides overlapping by 15 amino acids and identified 5 regions with significant levels of antibody reactivity in the NS1+MA group. We also profiled Ab responses to NS1 in humans naturally infected with DENV-2 or -3 in longitudinal serum samples from Nicaragua. One region in the NS1 “wing” domain was immunodominant in both mouse and human studies, and two regions were identified only in NS1-immunized mice; thus, vaccination can generate Abs to regions that are not targeted in natural infection and could provide additional protection against a lethal DENV infection. Overall, we identified several immunodominant regions, which were in functionally important locations on the DENV NS1 protein and are potential correlates of protection.