*3.2. Opt-36*β*t and opt-36*γ*t Enhance Immune Responses against HIV Env DNA Vaccine at a Memory Time Point*

A major concern in the vaccine field is the generation of vaccine candidates that can provide durable, long-term immune responses, and so we examined whether immune responses following DNA vaccination would be maintained into memory. B6 mice (*n* = 5/group) were immunized using 2.5 μg of HIV Env DNA alone or a formulation with 11 μg of opt-36αt, opt-36βt, or opt-36γt three times at three-week intervals with CELLECTRA 3P electroporation (EP) (Figure 3A). Spleens were harvested 50 days post-final vaccination to analyze antigen specific responses at a memory time point. A quantitative ELISpot was performed to determine the number of Env-specific IFN-γ secreting T cells that responded to vaccination (Figure 3B). We observed that mice immunized with the HIV vaccine alone produced an average of 775 spot-forming units (SFU)/million splenocytes, while mice adjuvanted with opt-36αt, opt-36βt, and opt-36γt had an average of 1242, 1460, and 1610 SFU/million splenocytes, respectively, supporting an enhanced response to the vaccine was driven by the adjuvants. Similar to the results observed at an acute time point, we found that mice adjuvanted with opt-36βt showed a significant increase in the percent of CD4<sup>+</sup> T cells that expressed IFN-γ and TNF-α compared to vaccine only. Interestingly, mice adjuvanted with opt-36γt showed a 3-fold enhancement in the percent of antigen-specific CD8<sup>+</sup> T cells which expressed IFN-γ and TNF-α (Figure 3C). We further observed that mice vaccinated with vaccine and opt-36γt had a significant increase in the percent of CD107a<sup>+</sup> IFN-γ<sup>+</sup> CD8<sup>+</sup> T cells, suggesting the cytolytic potential of these cells (Figure 3C). We also examined the humoral response induced post vaccination, and observed that mice adjuvanted with opt-36αt and opt-36γt exhibited higher average antibody titers compared to mice immunized with Env alone, although this did not reach significance (Supplementary Materials Figure S1). Of note, mice adjuvanted with opt-36βt exhibited suppressed antibody binding compared to vaccine alone.

**Figure 3.** *Cont*.

**Figure 3.** Codelivery of truncated IL-36 beta and gamma enhance immune responses against HIV Env DNA vaccine. (**A**) Immunization delivery schedule. B6 mice were immunized three times 3 weeks apart with Env alone or Env adjuvanted with the opt-36αt, opt-36βt, or opt-36γt. Sera and spleens were harvested 50 days post final vaccination to analyze antigen specific immune responses. (**B**) The frequency of Env specific IFN-γ responses (spot forming units per million splenocytes) induced after vaccination was determined by IFN-γ ELISpot assay in response to pooled Env peptides. (**C**) Env specific CD4 and CD8 T cell responses by intracellular cytokine staining after peptide stimulation. \*, *p* < 0.05, \*\* *p*< 0.005, \*\*\* *p* < 0.0005, \*\*\*\* *p* < 0.0001.
