*3.1. Opt-36*β*t Co-Formulation Leads to Enhanced Immune Responses against HIV Env DNA Vaccine Compared to Opt-36*β

While the IL-36 family was discovered in 1999 [29,30,34,35,45], members of this family remain poorly understood and continue to be investigated. In the initial studies of their biology, large quantities of IL-36 ligands were needed, in greater excess than those traditionally used for cytokines, to observe their activity [32,46]. With recent reports of IL-36 cytokines gaining activity after N-terminal residue truncation [42,47,48], we studied whether truncation was important for an IL-36 in vivo produced gene adjuvant to impact immune profile of DNA vaccine antigens in an HIV Env (Figure 1A) in vivo DNA vaccine model system. We chose to initially start our studies with IL-36 beta, as IL-36 beta has been reported to amplify Th1 responses [37], making it a potential cellular adjuvant candidate. We designed two DNA constructs encoding either full-length (opt-36β) or truncated (opt-36βt) IL-36 beta (Figure 1B) for these comparative studies. We added a highly efficient IgE leader sequence to both of the sequences as well as RNA and codon optimized them in order to enhance protein expression. We then immunized C57BL/6 (B6) (*n* = 5) mice with 2.5 μg of HIV Env DNA alone or with 2.5 μg of HIV Env and 11 μg opt-36β or opt-36βt, three times at three-week intervals using the 3P electrode driven by an adaptive electroporation CELLECTRA (EP) device (Figure 1C). Spleens were harvested 10 days post-final vaccination for analysis of antigen-specific responses. We observed a significant increase in the number of antigen-specific CD4<sup>+</sup> T cells that secreted IFN-γ and TNF-α in the animals whose vaccine included opt-36βt compared to opt-36β (Figure 1D). There was a trend towards a similar pattern of enhancement for the antigen-induced CD8<sup>+</sup> T cell responses, but in contrast to the CD4<sup>+</sup> T cell responses, this did not reach significance. A dosing study was next performed, focusing primarily on T cell induction to determine the optimal dose of opt-36βt. We found no significant difference in T cell response with higher doses and, in fact, there appeared to be a trend towards decreased immune response at the 30-μg dose of opt-36βt (Figure 1E). Going forward, we maintained our established dose of 11 μg for adjuvant plasmids for the remainder of the studies.

**Figure 1.** *Cont*.

**Figure 1.** Truncation of IL-36 beta enhances immune responses to HIV Env DNA vaccine. (**A** and **B**) Map of plasmid construct design. HIV Consensus Clade C vaccine plasmid, full length IL-36 beta plasmid and IL-36 beta truncated 9 amino acids N-terminal to anchoring residue. Each construct contains a cytomegalovirus (CMV) promoter followed by an IgE leader sequence. (**C**) Immunization delivery schedule. C57BL/6 mice were immunized three times at three week intervals. (**D**) Env specific CD4<sup>+</sup> and CD8<sup>+</sup> T cell responses by intracellular cytokine staining after peptide stimulation. E Opt-36βt dosing study of Env specific CD4<sup>+</sup> and CD8<sup>+</sup> T cell responses by intracellular cytokine staining after peptide stimulation. \* *p* < 0.05, \*\* *p* < 0.005, \*\*\* *p* < 0.0005, \*\*\*\* *p* < 0.0001.

Given these results, we next examined the rest of the IL-36 family as truncated cytokines. In this regard, even less is known about IL-36 alpha or gamma compared to beta, so we wanted to evaluate the immune responses in mice adjuvanted with each of the three cytokines in comparative studies. We also assessed the durability of the immune responses elicited by each of the IL-36 ligands post vaccination at a memory time point. Truncated IL-36 alpha (opt-36αt) and IL-36 gamma (opt-36γt) were designed and modified as illustrated (Figure 2A) [49,50]. An HA tag was added to the C-terminus of the sequences to facilitate in vitro detection. Construct expression in vitro was confirmed using Western blot and IFA (Figure 2B,C).

**Figure 2.** Expression of truncated IL-36 constructs. (**A**) Map of plasmid construct design for IL-36 sequences. Each sequence was truncated 9 amino acids N-terminal to conserved A-X-Asp residue. Each construct contains a cytomegalovirus (CMV) promoter followed by an IgE leader sequence besides the IL-36 sequence, and a HA tag at the C-terminus. (**B**) U2OS cells were transfected with each truncated IL-36 plasmids that contain a HA tag for detection. Lysates from these cells were used in Western blot for detection of plasmid expression. (**C**) Immunofluorescence (IFA) was performed on U2OS cells transfected with truncated IL-36 plasmids to verify plasmid expression.
