*3.4. Opt-36*γ*t Enhances Cellular Immune Responses Induced by a Zika DNA Vaccine Resulting in Enhanced Protection against Zika Challenge*

Based on the data generated in the two DNA vaccine models above, we now focused on studying opt-36γt in combination with a DNA vaccine against Zika and how vaccine-induced immune response impacted challenge outcome. This model allows us to confirm the relevance of the improved immunity and dose-sparing potential driven by the opt-36γt adjuvant. We immunized immunocompromised IFNAR−/<sup>−</sup> mice (*n* = 5–6 mice/group) once with an exceptionally low dose (0.5 μg) of Zika prME DNA vaccine alone (Figure 5A) or a combination of both. Two weeks following vaccination, we harvested spleens and blood (Figure 5B). We observed that mice immunized with vaccine only did not generate significant IFN-γ ELISpot responses, but the combination of the vaccine and opt-36γt resulted in a synergy resulting in 700 SFU/million splenocytes (Figure 5C). Immunization with opt-36γt alone did not generate significant cellular responses (Figure S3). Using intracellular cytokine staining, mice adjuvanted with opt-36γt exhibited increased IFN-γ and TNF-α expressing CD4<sup>+</sup> T cells as well as IFN-γ expressing CD8<sup>+</sup> T cells compared to the vaccine-only treated mice (Figure 5D). Overall antibody responses were very low in all groups (Supplementary Materials Figure S4). This suggests a need for an additional vaccine boost or using higher vaccine doses to further characterize the humoral immunity induced in this model.

We next repeated the study and this time performed a challenge using an immunocompromised mouse challenge model, IFNAR−/<sup>−</sup> mice (*n* = 12–14/ group), with a lethal dose of a validated Zika virus stock, strain PR 209. Challenge was performed two weeks after an immunization with either 0.5 μg of Zika prME alone or in combination with 11 μg of opt-36γt (Figure 6A). The animals were followed for two weeks post challenge. One of the side effects of ZIKV challenge typically observed in this mouse strain is weight loss [41]. Significant weight loss was observed in both the naïve and mice immunized with the suboptimal dose of the Zika prME vaccine alone, demonstrating substantial morbidity from the challenge (Figure 6B). Naïve mice appeared to be the most impacted, with many mice losing up to 20% of their starting body weight. The low-dose vaccine only group fared a bit better compared to the naive but still lost a considerable amount of weight. Strikingly, mice immunized with Zika prME in combination with opt-36γt were protected against weight loss, gaining weight during the course of the study. Additionally, mice were monitored for clinical symptoms during the challenge. Mice in both the naïve and vaccine-only groups became progressively sicker (i.e., hunched posture and paralysis of hind limbs) between days 5 and 7. However, the adjuvanted mice remain healthy and show no sign of disease following challenge (Figure 6C). As animals succumbed to disease they were sacrificed at predefined humane endpoints as described in the methods [41]. Mice immunized with Zika prME and opt-36γt exhibited a robust 92% survival rate, compared to 28% for mice immunized with the Zika prME only and 13% for naïve mice (Figure 6D). This data illustrates a significant benefit of the opt-36γt adjuvant in the context of this ZIKV IFNAR−/<sup>−</sup> challenge model. Study in additional models is important.

**Figure 5.** Codelivery of truncated IL-36 gamma enhances immune response to DNA prME vaccine. (**A**) Map of plasmid construct design. Consensus sequence of Zika precursor membrane and Envelope. (**B**) Immunization schedule for Zika vaccine immunization. IFNAR−/<sup>−</sup> mice were immunized once either vaccine alone or vaccine + opt-36γt (*n* = 5–6 per group). Spleens were harvested two weeks post vaccination to analyze antigen specific T cell responses. (**C**) The frequency of spot forming units per million splenocytes determined by IFN-γ ELISpot assay in response to pooled Zika prME peptides. (**D**) Zika prME specific CD4 and CD8 T cell responses by intracellular cytokine staining. \* *p* < 0.05, \*\* *p* < 0.005, \*\*\* *p* < 0.0005, \*\*\*\* *p* < 0.0001.

**Figure 6.** Truncated IL-36 gamma is able to protect against Zika challenge induced weight loss and mortality. (**A**) Immunization delivery schedule. IFNAR−/<sup>−</sup> mice were immunized with Zika prME plasmid or prME + opt-36γt once and challenged with Zika PR209 virus two weeks later. (**B**) Mouse body weight was tracked over two-week challenge period. (**C**) Clinical symptoms of immunized mice days 5–7 post challenge. (**D**) Survival curves of mice post Zika challenge over 14 days. \* *p* < 0.05, \*\* *p* < 0.005, \*\*\* *p* < 0.0005, \*\*\*\* *p* < 0.0001.
