**1. Introduction**

The most successful approach to controlling infectious diseases on a global scale has been through vaccination. Vaccines have led to control, eradication, or near eradication of several infectious diseases, positively impacting both human longevity and the quality of life. However, much work remains in this area. For many targets, current studies have suggested the need for adjuvants which can provide a number of benefits, including improved vaccine effectiveness, as discussed in several papers and reviews [1–9]. Adjuvants can boost overall immune responses to a specific vaccine, thereby requiring either a lower dose or fewer immunizations, improving protection and compliance as well as increasing the global vaccine supply for a particular product [3]. Adjuvants can also help skew and tailor the immune response, which may be useful in scenarios where specific correlates of protection are understood [10–12]. Furthermore, adjuvants can boost immunity and shorten the time to induce a protective vaccine response in populations that traditionally have a difficult time mounting protective responses, including the elderly and immunocompromised patients [3]. Alum, the most widely used adjuvant among current licensed vaccines, is well documented to enhance humoral immunity [9,13]. Newer vaccine adjuvants including MF59 and the Adjuvant Systems group 03 and 04 (AS03, AS04, respectively) have also been licensed and shown to improve antibody responses to antigens as well as provide dose-sparing effects (among other benefits) for humoral responses [14–18]. Shingrix, the latest vaccine approved to protect against reactivation of herpes zoster and postherpetic neuralgia (shingles), is a recombinant vaccine

made of glycoprotein E and AS01B adjuvant [5,19,20]. This vaccine demonstrated an efficacy of over 95% against herpes zoster, with greater efficacy compared to a live attenuated vaccine, ZostaVax, highlighting the impact that adjuvants can have on vaccine outcomes. However, in spite of this success, there is still a major need in the clinic for adjuvants that can improve cytotoxic T lymphocyte (CTL) responses [7], and there is a lot of exciting work being done in this field. Some of this work includes nontraditional adjuvants such as pathogen-recognition receptor agonists, liposomes, nanoparticles, and gene-encoded adjuvants that can potentially jumpstart the innate immune system and work in concert with the adaptive immune arm to drive lasting memory against antigen [6]. One cytokine, Interleukin-12 (IL-12), has garnered much attention in the field for its adjuvant properties in a number of preclinical models [21–27]. In addition, data from a clinical study showed that inclusion of plasmid IL-12 as part of a human immunodeficiency virus (HIV) synthetic DNA vaccine increased T cell magnitude and response rates in people [28]. These data encourage further investigation of additional less well-studied cytokines as DNA or other potential adjuvants to further broaden immunity and improve cellular as well as humoral immunity for DNA-encoded antigens.

The IL-36 family is made up of the pro-inflammatory mediators alpha, beta, and gamma, as well as antagonist IL-36Ra [29,30]. This relatively novel cytokine family remains poorly understood, although recent important studies have begun to shed light on their mechanism of action. The IL-36 family is a part of the IL-1 superfamily, of which alpha, beta, and gamma are agonists. Upon binding to the IL-36 receptor (IL-36R) and recruitment of the interleukin-1 receptor accessory protein (IL-1RAcP), these cytokines activate the nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPK) pathway, resulting in the stimulation of pro-inflammatory intracellular responses, whereas binding of the antagonist, IL-36Ra, prevents recruitment of IL-1RAcP and does not lead to intracellular response [31–33]. IL-36R is primarily expressed on naïve CD4<sup>+</sup> T cells, but is also found on dendritic cells, while the cytokines are expressed mainly in skin keratinocytes and epithelium, although they are also expressed at low levels in the lung, kidneys, and intestine [29,34–36]. Given reports of IL-36 beta's ability to amplify Th1 responses [37,38], we sought to understand whether these cytokines could act as adjuvants for DNA vaccination models. Here, we describe that a novel designed truncated IL-36 gamma (opt-36γt), as a co-formulated adjuvant plasmid, boosts humoral as well as CD4<sup>+</sup> and CD8<sup>+</sup> T cell immunity against three model synthetic DNA antigens including HIV envelope (Env), Influenza hemagglutinin 1 (HA1), and Zika premembrane and envelope (prME). Furthermore, opt-36γt enhanced protection by improving both clinical symptoms and mortality against a Zika virus (ZIKV) challenge and provided significant dose sparing for the Zika vaccine as studied using a suboptimal vaccine dose model. This not only supports the potential of opt-36γt as a gene adjuvant, but also highlights an underappreciated area of importance for protective cellular immune responses in Zika virus pathogenesis. Further investigation into opt-36γt as a potential new adjuvant for enhancing immunity against vaccine antigens is warranted.
