Significance The protooncogene c-Myc (Myc) is an oncogenic driver in many cancers, but is difficult to target directly with drugs. An alternative strategy is to use drugs that inhibit factors that regulate Myc expression. Notch drives Myc expression in most T-cell leukemias, but clinical trials of Notch inhibitors have been disappointing, possibly because cells emerge that express Myc in a Notch-independent fashion. Here we identify the genomic switches that regulate Myc expression in the Notch-inhibitor–sensitive and –resistant states. Our findings suggest that Notch inhibitor resistance occurs through a “switch swap” that relieves Notch dependency while increasing dependency on a different factor, bromodomain containing 4 (Brd4). These studies provide a rationale for targeting Myc in T cell leukemias with combinations of Notch and Brd4 inhibitors.

Vaccination remains one of the greatest medical breakthroughs in human history and has resulted in the near eradication of many formerly lethal diseases in many countries, including the complete eradication of smallpox. However, there remain a number of diseases for which there are no or only partially effective vaccines. There are numerous hurdles in vaccine development, of which knowing the appropriate immune response to target is one of them. Recently, tissue-resident T cells have been shown to mediate high levels of protection for several infections, although the best way to induce these cells is still unclear. Here we compare the ability to generate skin-resident T cells in sites distant from the immunization site following intramuscular and intradermal injection using optimized synthetic DNA vaccines. We found that mice immunized intradermally with a synthetic consensus DNA HIV envelope vaccine by electroporation (EP) are better able to maintain durable antigen-specific cellular responses in the skin than mice immunized by the intramuscular route. We extended these studies by delivering a synthetic DNA vaccine encoding Leishmania glycosomal phosphoenolpyruvate carboxykinase (PEPCK) by EP and again found that the intradermal route was superior to the intramuscular route for generating skin-resident PEPCK-specific T cells. We observed that when challenged with Leishmania major parasites, mice immunized intradermally exhibited significant protection, while mice immunized intramuscularly did not. The protection seen in intradermally vaccinated mice supports the viability of this platform not only to generate skin-resident T cells but also to promote durable protective immune responses at relevant tissue sites.

Identification of novel molecular adjuvants which can boost and enhance vaccine-mediated immunity and provide dose-sparing potential against complex infectious diseases and for immunotherapy in cancer is likely to play a critical role in the next generation of vaccines. Given the number of challenging targets for which no or only partial vaccine options exist, adjuvants that can address some of these concerns are in high demand. Here, we report that a designed truncated Interleukin-36 gamma (IL-36 gamma) encoded plasmid can act as a potent adjuvant for several DNA-encoded vaccine targets including human immunodeficiency virus (HIV), influenza, and Zika in immunization models. We further show that the truncated IL-36 gamma (opt-36γt) plasmid provides improved dose sparing as it boosts immunity to a suboptimal dose of a Zika DNA vaccine, resulting in potent protection against a lethal Zika challenge.

The use of plasmid adjuvants encoding cytokine, chemokine or immune modulators to tailor vaccine-induced response is a strength of the DNA vaccine platform. Due to its role in both innate and adaptive immunity, we hypothesis that the co-delivery of plasmid encoded CD40 ligand (CD40L) could increase DNA vaccine responses. In its natural form, CD40L can occur as either a surface bound form or a cleaved/solubilized form. Thus, we sought to determine if different forms of pCD40L can influence cellular and humoral responses when co-delivered with a HPV16 DNA vaccine expressing the oncogenic proteins E6 and E7. Mice were immunized with HPV DNA with or without synthetic optimized plasmids expressing various forms of CD40L followed by electroporation (EP). Mice that received the soluble form of CD40L (sCD40L) exhibited significantly higher antigen specific CD8+ T cell responses including IFN-γ, IL-2 and TNF-α expression as well as slight increases in CD4+T cells and antibody responses. These responses were also maintained into memory. Conversely, the surface bound as well as the wild-type form of CD40L blunted vaccine-induced responses compared to vaccine alone. Time course analysis revealed that 11 days after primary immunization, CD8+ tetramer specific (H-2Db HPV16 E7 (RAHYNIVTF)) T cells in mice immunized with sCD40L averaged around 18% compared to vaccine alone at 4%. These responses were partially dependent on CD4+ T cell help and were functionally similar to responses observed post final immunization. We have also observed similar vaccine-induced immune responses when sCD40L was combined with other cancer vaccines including mouse telomerase reverse transcriptase (mTERT). Upon therapeutic tumor challenge, mice immunized with HPV + sCD40L displayed significant tumor regression compared to vaccine alone or naïve animals. Additionally, when sCD40L was included the ratio of tetramer specific CD8+ T cells to CD4+ Tregs infiltrating the tumor was significantly higher compared to vaccine alone or naïve group. These results demonstrate the power of using a plasmid adjuvant encoding a synthetic optimized sCD40L in a DNA vaccine. Additional studies in other animal cancer models are important for determining the possible broad importance of this approach for cancer immune therapy. Citation Format: Megan C. Wise, Elizabeth K. Duperret, Daniel O. Villarreal, Lumena Louis, Jian Yan, Matthew P. Morrow, Laurent M. Humeau, Niranjan Y. Sardesai, David B. Weiner. A novel synthetic CD40L plasmid adjuvant generates unique anti-HPV DNA vaccine induced responses that impact tumor growth [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A020.

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