**4. Discussion**

While the IL-36 cytokine family was first discovered nearly two decades ago, it is only recently that roles for these cytokines have begun to be elucidated. The IL-36 family, members of a larger proinflammatory IL-1 family, has been primarily implicated for their potential role in pustular psoriasis and inflammation of the skin and joints [29,30,52,53]. Dysregulation of the natural IL-36 receptor antagonist or overexpression of IL-36 in the skin has been implicated in a number of skin diseases and conditions [53–67]. However, some of these proinflammatory properties have also piqued the scientific community's interest regarding some of the other roles that these cytokines might play. Following reports that IL-36 beta could amplify Th1 responses in CD4<sup>+</sup> T cells [37], a number of studies have shown the induction of IL-36 cytokine expression, especially IL-36 gamma, in response to infections including pneumonia, herpes simplex virus (HSV), and candidiasis [68–73], suggesting that IL-36 cytokines may play a significant role in host immunity.

To our knowledge, this is the first study to compare the effects of all three truncated IL-36 cytokines in a vaccination model. In these studies we provide additional insight into the ability of truncated IL-36 gamma's (opt-36γt) ability to boost immune responses using three DNA vaccine antigens. As previously demonstrated by Towne et al. [42], we found that truncation of the IL-36 cytokines' nine amino acids at the N-terminal region was critical for their activity to enhance vaccine-induced immune responses. For future investigations of IL-36 cytokines in protective immunity studies, the truncated forms of these cytokines will almost certainly be necessary to exploit their full potential.

In the DNA vaccine models we tested, we found that mice immunized with opt-36βt and opt-36γt were both able to enhance vaccine-induced cellular immune responses. However, where opt-36βt was able to significantly increase the number of antigen-specific IFN-γ<sup>+</sup> and TNF-α<sup>+</sup> CD4<sup>+</sup> T cells, opt-36γt significantly increased the number of antigen-specific IFN-γ+, TNF-α+, and CD107a<sup>+</sup> CD8<sup>+</sup> T cells, suggesting an impact of opt-36γt to improve cytolytic activity of these cells. Further work must be done to understand the differences between the two cytokines' seemingly preferential action on various cell compartments. Regarding humoral immunity in the influenza DNA vaccination model, we found that opt-36γt was able to increase antibody-binding titers, while opt-36βt appeared to induce antibodies that have weaker avidity. Thus, in our models, opt-36γt can improve both arms of immune response, which is likely important for many of the challenging disease targets that remain. We also found that the synergy of a non-protective dose of Zika DNA vaccine with opt-36γt was able to protect mice against a lethal ZIKV challenge, highlighting the potential of opt-36γt to affect challenge outcome and drive protection. Furthermore, opt-36γt enhanced antibody binding in both the HIV and influenza DNA models, while overall humoral responses in the Zika DNA model were lower than the other models, possibly due to the low amount of plasmid used for immunization. Other differences among the models such as mouse genotype may also be relevant and could be examined in further studies.

There is still much work to be done to fully understand the roles that the IL-36 cytokines play under both homeostatic and pathologic conditions in the host immune system. Multiple studies in mice have shown that the IL-36 cytokines may have distinct functions in response to different inflammatory stimuli. Understanding how opt-36βt and opt-36γt may exert their activities on different cell populations and against additional vaccine targets will be important for further harnessing their potential. Given their ability to enhance CD4<sup>+</sup> and CD8<sup>+</sup> T cell responses, opt-36γt and opt-36βt look especially promising for disease models in which cellular responses are important, such as cancer where driving CD8<sup>+</sup> immunity is important to clear tumors. Studies examining the effects of opt-36γt on driving tumor-infiltrating lymphocytes (TILS) would be relevant. Work by Wang et al. has demonstrated that tumor growth was significantly inhibited in B16 melanoma IL-36 expressing cells compared to control B16 cells that did not express IL-36 gamma in mice [74]. Wang et al. also found that IL-36 gamma could promote early activation and expansion of naïve CD8<sup>+</sup> T cells, in line with what we have observed in our DNA vaccine models.

Furthermore, the induction of higher binding antibodies while maintaining avidity by opt-36γt as we observed in the influenza studies may have important implications in diseases in which high avidity and affinity antibody titers are important. As more emphasis is being focused to identify immunogens that can elicit broadly neutralizing antibodies (bNabs) for HIV and influenza, adjuvants that can further refine the antibody response may prove important.

Although there appears to be a deleterious effect on skin health when IL-36 signaling is left unchecked [55], localized controlled delivery of opt-36γt as an adjuvant during intradermal vaccination could enhance vaccine responses and recruitment of cells to the site of infection. This could be especially important for infectious diseases that breach the skin's natural barrier including herpes, malaria, and Leishmania, among others. As the largest organ in the human body, with a rich source of antigen-presenting cells (APCs) and Langerhans cells, as well as nearly 20 billion T cells, the skin is a particularly attractive site to administer an opt-36γt adjuvanted vaccine. Enhanced CTL responses in the skin can help control the spread of an infection before it is able to disseminate to other locations in the body, while greater antibody responses may help with prevention of infection. Studies that examine the delivery of opt-36γt in the skin compared to intramuscular delivery may shed light on another route to impact vaccine immune outcome as well as protection against infection.

As the global population and the demand for vaccines increase worldwide, the need to maximize immune responses while minimizing the effective dose necessary to induce protective responses will continue to grow. Here we describe the first study of an optimized plasmid encoding for a truncated form of IL-36 as a plasmid adjuvant, opt-36γt. We observed that opt-36γt exhibited a dose-sparing effect as well as enhancement of humoral and cellular immune responses to several antigens and improved challenge outcome in a well-studied mouse model system of viral challenge. Additional study of opt-36γt as a genetically encoded adjuvant is likely important.
