Supplementary MaterialsAdditional document 1: Flow cytometry data presented in tabular form. defined MHC class I epitope, and evaluate its immunogenicity and efficacy in combination with various adjuvant formulations. Methods E731C73 SLP was tested alone or in combination with toll-like receptor (TLR)3, TLR4, TLR7/8 and TLR9 agonists and formulated in oil-in-water (o/w) or water-in-oil (w/o) emulsions to determine a vaccine format inducing a strong CD8 T cell response in murine models. Once a lead vaccine format was decided, we examined its ability to inhibit tumor growth in the murine TC-1 model that expresses HPV16 E7 antigen. Results We identified the TLR9 agonist CpG formulated in a squalene-based o/w emulsion as the most potent adjuvant, inducing the growth of multifunctional antigen specific CD8 T cells with cytolytic potential. We also exhibited that SLP E731C73?+?CpG?+?o/w emulsion vaccine can provide prophylactic and more importantly, therapeutic benefit in the TC-1 murine tumor model. Conclusions Our results demonstrate that this novel vaccine format E7 SLP?+?CpG delivered in an o/w emulsion holds potential for the promotion of strong CTL responses and tumor eradication and encourages further development of peptide/adjuvant vaccines in cancer immunotherapy strategies. Electronic supplementary material The online version of this article (10.1186/s12885-019-5725-y) contains Docosapentaenoic acid 22n-3 supplementary material, which is available to authorized users. adenylate cyclase bearing E7 polypeptide . The presence of CpG has improved induction of cytolytic responses and/or tumor regression in each case. In a vaccine format more similar to that described herein, consisting of a 35-mer long peptide based on the HPV16 E7 RAHYNIVTF epitope formulated with CpG alone or with the w/o emulsion Montanide , investigators reported full tumor regression of TC-1 flank tumors after two s.c. opposite flank vaccinations at days 10 and 24. However, long-lasting protective responses were not fully evaluated as tumor growth was only recorded up to day 30. Since we observed tumor outgrowth occurring in some vaccinated mice post-day 30, it is unclear whether their vaccination regimen was truly curative. The authors also used 150?g E7 SLP, while we have found that a much lower dose of 20?g is very effective if delivered with an o/w squalene-based emulsion formulation, and that this formulation generates CD8 T cell responses of greatly increased magnitude when directly compared Docosapentaenoic acid 22n-3 to those induced with Montanide. W/o emulsions such as SEPPICs Montanide ISA51 and ISA720 have been used in multiple Phase I/II clinical trials and have Docosapentaenoic acid 22n-3 shown some degree of STK11 efficacy, but have shown safety signals at higher dose amounts [24C26] also. Furthermore, w/o emulsions like Montanide or IFA have already been found to become suboptimal delivery systems for peptides in types of tumor vaccination because they are in charge of an antigen depot impact which causes Compact disc8 T cells to house towards the vaccination site rather than the tumor and to convert to an exhaustion phenotype . On the other hand, pharmacology studies with o/w emulsions like MF59, the chemical composition of which the emulsion AddaVax used in our studies was based from, have exhibited that this emulsion is rapidly drained away from the injection site and that the half-life of the antigen is not affected (examined in ). MF59 has been shown to induce strong CD4 T cell responses and has been incorporated into some flu vaccines  and recently o/w emulsions have been shown in animal models to generate Docosapentaenoic acid 22n-3 strong cytotoxic CD8 T cell responses when paired with CpG against viral antigens . Combining TLR agonists with an emulsion delivery vehicle is thought to Docosapentaenoic acid 22n-3 increase the stability of the agonist as well as to improve antigen uptake and.