No abstract

Human papillomaviruses (HPV)-related cervical cancer is the second leading cause of cancer death in women worldwide. Despite active development, HPV E6/E7 oncogene-specific therapeutic-vaccines have had limited clinical efficacy to date. Here we report that intravaginal instillation of CpG-ODN (TLR9-agonist) or poly-(I:C) (TLR3-agonist) after subcutaneous E7 vaccination increased approximately 5-fold the number of vaccine-specific IFN-γ-secreting CD8 T-cells in the genital mucosa (GM) of mice, without affecting the E7-specific systemic response. The intravaginal treatment locally increased both E7-specific and total CD8 T-cells, but not CD4 T-cells. This previously unreported selective recruitment of CD8 T-cells from the periphery by intravaginal CpG-ODN or poly-(I:C) was mediated by TLR9 and TLR3/Mda5 signaling-pathways, respectively. For CpG, this recruitment was associated with a higher proportion of GM-localized CD8 T-cells expressing both CCR5 and CXCR3 chemokine receptors and E-selectin ligands. Most interestingly, intravaginal CpG-ODN following vaccination led to complete regression of large genital HPV-tumors in 75% of mice, instead of 20% with vaccination alone. These findings suggest that mucosal application of immunostimulatory molecules might substantially increase the effectiveness of parenterally administered vaccines.

Cervical cancer results from infection with high-risk type human papillomaviruses (HPV). Therapeutic vaccines aiming at controlling existing genital HPV infections and associated lesions are usually tested in mice with HPV-expressing tumor cells subcutaneously implanted into their flank. However, effective vaccine-induced regression of these ectopic tumors strongly contrasts with the poor clinical results of these vaccines produced in patients with HPV-associated genital neoplasia. To assess HPV therapeutic vaccines in a more relevant setting, we have, here, established an orthotopic mouse model where tumors in the genital mucosa (GM) develop after an intravaginal instillation of HPV16 E6/E7-expressing tumor cells transduced with a luciferase-encoding lentiviral vector for in vivo imaging of tumor growth. Tumor take was 80-90% after nonoxynol-9 induced damage of the epithelium. Tumors remained localized in the genital tract, and histological analysis showed that most tumors grew within the squamous epithelium of the vaginal wall. Those tumors induced (i) E7-specific CD8 T cells restricted to the GM and draining lymph nodes, in agreement with their mucosal location and (ii) high Foxp31 CD41 infiltrates, similarly to those found in natural non-regressing HPV lesions. This novel genital HPV-tumor model by requiring GM homing of vaccine-induced immune responses able to overcome local immuno-suppression may be more representative of the situation occurring in patients upon therapeutic vaccination.Cervical cancer, the second most common cause of cancer death in women worldwide, is associated to high-risk types human papillomavirus (HPV) infections.1 The available prophylactic HPV virus like particles vaccines can efficiently prevent type-restricted cervical HPV infection and associated intraepithelial neoplasia, but they have no impact on established HPV lesions.2 Cell-mediated immune responses, targeting tumor-specific HPV E6 and/or E7 oncoproteins are likely necessary for regression of high-grade lesions. However, the therapeutic vaccines targeting E6/E7 tested to date have shown limited clinical efficacy, at best. 3,4 This strongly contrasts with the high preclinical efficacy of these vaccines when tested in mice with syngeneic tumor cells, engineered to express HPV proteins and subcutaneously (sc) implanted into their flank, suggesting that vaccine-induced regression of these ectopic HPV-tumors is not predictive of clinical outcome. HPV-associated neoplastic lesions in women are mostly restricted to the genital mucosa (GM), a mucosa that harbor a relatively immunosuppressed environment. Unfortunately, available animal models of natural papillomavirus infections do not lead to intraepithelial genital lesions, whereas transgenic mice expressing HPV oncogenes in their epithelium can develop cervical cancer but are of limited use for testing therapeutic vaccines, because expression is wide spread in the tissue and they exhibit tolerance to the transgenes. 3 To evaluate the importance of local cell-mediated immunity fo...

Endothelial connexin40 (Cx40) contributes to regulate the structure and function of vessels. We have examined whether the protein also modulates the altered growth of vessels in tumor models established in control mice (WT), mice lacking Cx40 (Cx40−/−), and mice expressing the protein solely in endothelial cells (Tie2-Cx40). Tumoral angiogenesis and growth were reduced, whereas vessel perfusion, smooth muscle cell (SMC) coverage and animal survival were increased in Cx40−/− but not Tie2-Cx40 mice, revealing a critical involvement of endothelial Cx40 in transformed tissues independently of the hypertensive status of Cx40−/− mice. As a result, Cx40−/− mice bearing tumors survived significantly longer than corresponding controls, including after a cytotoxic administration. Comparable observations were made in WT mice injected with a peptide targeting Cx40, supporting the Cx40 involvement. This involvement was further confirmed in the absence of Cx40 or by peptide-inhibition of this connexin in aorta-sprouting, matrigel plug and SMC migration assays, and associated with a decreased expression of the phosphorylated form of endothelial nitric oxide synthase. The data identify Cx40 as a potential novel target in cancer treatment.