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High-risk human papillomavirus (HPV) encoding E6/E7-HPV oncogenes are responsible for a subgroup of head and neck squamous-cell carcinoma (HNSCC) and thus therapeutic E7-vaccines may be used to control HPV + HNSCC tumors. Herein we investigated the effects of an optimized nanoparticle-conjugated E7 long-peptide vaccine adjuvanted with CpG (NP-E7LP) in an orthotopic immunocompetent mouse model of HPV + HNSCC which is based on injection of HPV16 E6/E7-expressing mEERL95-cells into the submental space. In absence of surgery, vaccination performed before or after tumor-cell injection decreased tumor growth or prolonged mice survival only marginally, despite the high numbers of vaccine-induced circulating E7-specific IFN-γ-secreting CD8 + T-cells. This contrasts with the high-efficacy of NP-E7LP-vaccination reported in the genital and subcutaneous HPV16-E6/E7-expressing TC-1 models. Our data show that in a direct comparison, NP-E7LP-vaccination fully controlled TC-1, but not mEERL95, tumors subcutaneously growing in the flanks. Immune-cell infiltration was 10-fold higher in TC-1-tumors, than in mEERL95-tumors, suggesting that vaccine-induced CD8 + T-cells can only poorly infiltrate mEERL95-tumors. Indeed, immunofluorescence staining of orthotopic mEERL95-tumors showed that CD3 + T-cells are preferentially located peritumorally. However, when NP-E7LP-vaccination was performed after mEERL95-cell injection, but before resection of primary tumors, no postsurgical recurrence was observed and 100% of the mice survived until the experimental endpoint (day 70) in the NP-E7LP-vaccinated group. In contrast, we observed a 60% recurrence rate and only 35% survival in PBS-vaccinated mice. This suggests that removal of the primary tumor modified the tumor microenvironment, allowing a therapeutic effect of the vaccine-induced anti-tumor response. E7-vaccination combined with surgery may thus benefit patients with HPV + HNSCC., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Intravesical Bacillus-Calmette-Guérin (BCG) immunotherapy can reduce recurrence/progression of non-muscle-invasive bladder cancer (NMIBC), although significant adverse events and treatment failure argue for alternative options. Here, we examined whether another attenuated live vaccine, Vivotif/Ty21a, used since more than 30 y against typhoid fever, may be safely used intravesically to improve bladder-tumor treatment. Mice-bearing MB49 orthotopic bladder-tumors treated with intravesical Ty21a or BCG were compared for survival and bacteria recovery. Both Ty21a and BCG enhanced mice survival when treating just after tumor implantation for 4 weeks ( p = 0.008 and 0.04, respectively), but only Ty21a was effective when treating once mice with larger already established bladder-tumors ( p = 0.0003). In contrast to BCG, no Ty21a bacteria survived in mouse bladder, human urothelial cell-lines or human peripheral blood mononuclear cells. However, Ty21a was as potent as BCG to induce tumor-cell death in vitro . In a human, 3D-bladder-tissue ex-vivo assay, Ty21a bacteria, still not surviving, induced a panel of cytokines associated with effective BCG-treatment in patient's urine. Overall, our pre-clinical data demonstrate that intravesical Ty21a is more effective than BCG for bladder-tumor treatment. Absence of surviving Ty21a bacteria and the excellent safety-record of the typhoid vaccine support its testing in NMIBC patients.

The efficacy of antitumoral responses can be increased using combinatorial vaccine strategies. We recently showed that vaccination could be optimized by local administration of diverse molecular or bacterial agents to target and augment antitumoral CD8 T cells in the genital mucosa (GM) and increase regression of cervical cancer in an animal model. Non muscle-invasive bladder cancer is another disease that is easily amenable to local therapies. In contrast to data obtained in the GM, in this study we show that intravesical (IVES) instillation of synthetic toll-like receptor (TLR) agonists only modestly induced recruitment of CD8 T cells to the bladder. However, IVES administration of Ty21a, a live bacterial vaccine against typhoid fever, was much more effective and increased the number of total and vaccine-specific CD8 T cells in the bladder approximately 10 fold. Comparison of chemokines induced in the bladder by either CpG (a TLR-9 agonist) or Ty21a highlighted the preferential increase in complement component 5a, CXCL5, CXCL2, CCL8, and CCL5 by Ty21a, suggesting their involvement in the attraction of T cells to the bladder. IVES treatment with Ty21a after vaccination also significantly increased tumor regression compared to vaccination alone, resulting in 90% survival in an orthotopic murine model of bladder cancer expressing a prototype tumor antigen. Our data demonstrate that combining vaccination with local immunostimulation may be an effective treatment strategy for different types of cancer and also highlight the great potential of the Ty21a vaccine, which is routinely used worldwide, in such combinatorial therapies.

We have recently reported that the intravaginal instillation of synthetic Toll-like receptor 3 (TLR3) or TLR9 agonists after a subcutaneous vaccination against human papillomavirus E7 highly increases (~5-fold) the number of vaccine-specific CD8 + T cells in the genital mucosa of mice, without affecting E7-specific systemic responses. Here, we show that the instillation of live attenuated Salmonella enterica serovar Typhimurium similarly, though more efficiently (~15- fold), increases both E7-specific and total CD8 + T cells in the genital mucosa. Cancer immunotherapeutic strategies combining vaccination with local immunostimulation with live bacteria deserve further investigations.