**5. Conclusions**

As the next generation of vaccines are developed, they will likely benefit from the identification of novel adjuvants with unique immune modulating properties. Here we evaluated the adjuvant activity of 3 optimized versions IL-36 (opt-36αt, opt-36βt, and opt-36γt), novel members of the IL-1 gene family, previously reported to be involved in proinflammatory activity. We report that truncation of the IL-36 beta form (opt-36βt) enhanced immunization induced immune responses against a HIV Env DNA vaccine, compared to unadjuvanted HIV Env or the same vaccine adjuvanted by full length IL-36 beta (opt-36β). When memory responses were examined, the opt-36βt enhanced antigen specific CD4<sup>+</sup> T cell responses while opt-36γt more robustly enhanced antigen specific CD8<sup>+</sup> T cell responses. When these adjuvants were studied in an influenza vaccine model, opt-36γt codelivery increased antibody titers against the hemagglutinin protein. These antibody responses exhibited higher binding avidity compared to the control vaccine alone arm. We also evaluated opt-36γt's DNA vaccine dose sparing potency in a lethal Zika vaccine challenge model. Codelivery of opt-36γt with a very low dose Zika DNA vaccine was able to potently enhance IFN-γ T cell responses resulting in potent protection against the ZIKV challenge compared to vaccine only immunized or naïve mice. This study provides proof-of-concept that an optimized plasmid encoding truncated IL-36 gamma is an important new gene adjuvant which can simultaneously enhance both humoral and cellular immunity and positively impact challenge. Further study of this promising genetic adjuvant is warranted.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-393X/7/2/42/s1, Figure S1: (A) ELISA analysis measuring binding antibody production (measured by OD450 values) in immunized mice. The C57BL/6 mice (*n* = 5) were immunized intramuscularly three times three weeks apart with 2.5 μg of HIV Env plasmid or 2.5 μg of Env plasmid and 11 μg of opt-36αt, opt-36βt, or opt-36γt. Binding to consensus C gp120 was analyzed with sera from animals post final vaccination. (B) Average endpoint titers, Figure S2: (**A**) ELISA analysis measuring isotype binding antibody production (measured by OD 450 values) in immunized mice. BALB/c mice (*n* = 4–5) were immunized twice two weeks apart with 1 μg of HA1 DNA plasmid or HA1 DNA plasmid and 11 μg of opt-36αt, opt-36βt, or opt-36γt. Isotypes of antibodies generated were analyzed with sera from animals post final vaccination. (**B**) IgG2a/IgG1 antibody ratio was analyzed by dividing the OD450 values of IgG2a by the OD450 values of IgG1, Figure S3: Induction of Zika specific cellular immune responses following vaccination with either Zika prME DNA vaccine alone or opt-36γt alone. ELISpot analysis measuring IFN-γ secretion in splenocytes after one immunization, Figure S4: Induction of antigen specific antibody responses following immunization with either Zika prME DNA vaccine alone or Zika prME DNA vaccine and opt-36γt after one immunization.

**Author Contributions:** Conceptualization, L.L. and D.O.V.; methodology, L.L. and M.C.W.; validation, L.L.; formal analysis, L.L.; investigation, L.L., H.C.; resources, K.M. and D.B.W.; writing—original draft preparation, L.L.; writing—review and editing, L.L., M.C.W, H.C., D.O.V., and D.B.W.; visualization, L.L.; supervision, L.L., D.B.W.

**Funding:** This research was funded by an NIH Pharmacology training grant, grant number T32 GM008076, an NIH Training in HIV Pathogenesis grant, grant number T32 AI007632, and an NIAID grant, grant number U19 AI 109646.

**Acknowledgments:** We thank Sagar Kudchodkar, Piyush Borole, and Kanika Asija for their technical assistance in early Zika studies. We thank Imaging Facility Core at the Wistar Institute for assistance with confocal microscopy experiments. We thank Jeffery Faust and the Flow cytometry Core at the Wistar Institute for their advice on flow cytometry experiments.

**Conflicts of Interest:** M.C.W. is an employee of Inovio Pharmaceuticals and as such receives salary and benefits including ownership of stock and stock options from the company. K.M. receives grants and consulting fees from Inovio related to DNA vaccine development. D.B.W. has grant funding, participates in industry collaborations, has received speaking honoraria and fees for consulting. This service includes serving on scientific review committees and advisory boards. Remuneration includes direct payments and/or stock or stock options and in the interest of disclosure; therefore, he notes potential conflicts associated with this work with Inovio where he serves on the BOD, Merck, VGXI, OncoSec, Roche, Aldevron and possibly others. The remaining authors declare no conflict of interest.
