*3.3. Opt-36*γ*t Enhances Humoral Immunity in Influenza DNA Vaccine Model*

We next sought to extend this finding to additional antigens with a different DNA vaccine antigen. We studied opt-36αt, opt-36βt, and opt-36γt's ability to impact immune responses driven by an HA1

Syncon influenza DNA vaccine [40]. Given the potency of the adjuvant response in the previous studies, we focused on a two-dose regimen to evaluate the vaccine-induced immune response in a dose-sparing model. BALB/c mice (*n* = 5/group) were immunized two times at two-week intervals with either 1 μg of HA1 DNA alone (Figure 4A) or 1 μg of HA1 and 11 μg of opt-36αt, opt-36βt, or opt-36γt followed by in vivo EP (Figure 4B). Ten days post final immunization, we observed both opt-36βt and opt-36γt significantly enhanced cellular responses compared to the low-dose vaccine alone (Figure 4C). We observed increased cellular responses in mice adjuvanted with opt-36αt; however, this was not as pronounced as the responses with the other two cytokines. As antibodies are known to be critical for prevention from influenza infection, we studied the binding antibodies generated post vaccination. Opt-36γt elicited significant higher endpoint binding titers compared naïve mice (Figure 4D). We further examined the quality of these antibodies by performing an ELISA based avidity test [51] to examine strength of binding to a HA1 influenza protein. Interestingly, we observed that antibodies from mice that received opt-36γt had greater antigen binding and maintained avidity compared to the antibodies from mice that received opt-36αt and opt-36βt, supporting the induction of improved magnitude of humoral responses (Figure 4E). We also examined the isotypes of the antibodies generated post vaccination, and while all immunized groups exhibited class switching, we did not observe a significant shift in IgG1 vs. IgG2a ratios among the different groups (Supplementary Materials Figure S2).

**Figure 4.** *Cont*.

**E** 

**Figure 4.** Codelivery of truncated IL-36 gamma enhances binding antibody while maintaining antibody integrity. (**A**) Map of plasmid construct design. Influenza hemagglutinin (HA) (from strain H1N1 A/PR/8/34) vaccine plasmid. Vaccine construct contains a cytomegalovirus (CMV) promoter followed by an IgE leader sequence. (**B**) Immunization delivery schedule. BALB/c mice were immunized two times two weeks apart with influenza HA1 alone or HA1 adjuvanted with opt-36αt, opt-36βt, or opt36γt. Sera and spleens were harvested two weeks post final vaccination to analyze antigen specific responses. (**C**) The frequency of HA specific IFN-γ responses (spot forming units per million splenocytes) induced after vaccination was determined by IFN-γ ELISpot assay in response to pooled HA peptides. (**D**) Endpoint binding titers post vaccination HA1 alone or HA1 + truncated IL-36 adjuvant. (**E**) The avidity of antibodies generated after vaccination at 1:50 dilution. \*, *p* < 0.05, \*\*, *p*< 0.005, \*\*\*, *p* < 0.0005, \*\*\*\*, *p* < 0.0001.
