Correlation of the Gut Microbiota and Antitumor Immune Responses Induced by a Human Papillomavirus Therapeutic Vaccine.

Human papillomavirus (HPV) is the most common sexually transmitted pathogen worldwide and the major risk factor for cervical cancer. According to our previous study, antitumor immune responses induced by a therapeutic vaccine based on HPV E7 peptide are highly variable among individuals. Many studies have demonstrated that the discrepancy in the gut microbiota is an important factor in the development and regulation of the immune system. Therefore, we performed a systematic comparative analysis of gut microbiota in two groups of mice with significant differences in antitumor effects induced by the vaccine, as well as the correlation between immune cells and gut microbiota. We divided the mice into group A, in which the tumor continued growing, and group B, in which the tumor volume was significantly reduced. In group B mice, the vaccination induced a stronger antitumor activity with significantly enhanced IFN-γ-producing CD4+ and CD8+ T lymphocytes, as well as decreased immunosuppressive cells. A detailed gut microbiota analysis revealed a positive Spearman correlation between the percentage of CD8+ T cells and the relative abundance of Corynebacteriales, Parabacteroides, and Bacteroides_sp. Furthermore, the percentage of CD4+ and CD8+ T cells negatively correlated with the abundance of Proteobacteria and Bilophila. By contrast, the abundance of Proteobacteria, Desulfovibrio, and Bilophila positively correlated with the percentage of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and type 2-polarized tumor-associated macrophages (M2-TAMs). Overall, the composition of gut microbiota is related to vaccine-induced antitumor effects, and there is a correlation between some gut bacteria and vaccine-induced immune responses.

Intratumoral Injection of a Human Papillomavirus Therapeutic Vaccine-Induced Strong Anti-TC-1-Grafted Tumor Activity in Mice.

PURPOSE: The route of administration of a therapeutic tumor vaccine is a critical factor in inducing antitumor activity. In this study, we explored the effects of three vaccination routes (subcutaneous, peritumoral, and intratumoral injection) on antitumor activity induced by a human papillomavirus (HPV) therapeutic vaccine containing HPV16 E7 peptide combined with the adjuvant CpG ODN in established TC-1 grafted tumors. METHODS: We used flow cytometry to evaluate splenic and tumor-infiltrating immune cells. We also assessed transcriptional changes in a sequence of immune-related genes in tumors of different treatment groups using quantitative real-time polymerase chain reaction. Immunohistochemistry was used to determine the expression of molecules related to tumor infiltrating immune cells, angiogenesis, and cancer-associated fibroblasts in tumor tissues. RESULTS: Our results suggested that intratumoral and peritumoral vaccination generated enhanced antitumor activity compared to subcutaneous delivery. In particular, intratumoral vaccination elicited a stronger antitumor effect, with two of the six treated mice being nearly tumor-free at day 28. Three vaccination routes induced increases in splenic CD4+ and/or CD8+ T lymphocytes, and marked decreases in immunosuppressive cells. Peritumoral vaccination increased the tumor-infiltrating CD8+T cells in tumors, while intratumoral vaccination enhanced the tumor-infiltrating CD4+ and CD8+ T lymphocytes, as well as decreased the tumor-infiltrating of immunosuppressive cells, which may result in stronger inhibition of tumor growth and prolonged survival in mice bearing tumors. Furthermore, compared to the subcutaneous route, intratumoral vaccination led to a significant increase in antitumor cytokines and chemokines. In addition, our data showed marked downregulation of MMP-2, MMP-9, VEGF, CD31, and α-SMA in the intratumoral vaccination group, which might contribute to the suppression of tumor invasion, angiogenesis, and metastasis. CONCLUSION: Overall, intratumoral vaccination is superior to subcutaneous delivery and has the potential to inhibit tumor growth by improving the tumor microenvironment.

Inhibition of Orthotopic Genital Cancer Induced by Subcutaneous Administration of Human Papillomavirus Peptide Vaccine with CpG Oligodeoxynucleotides as an Adjuvant in Mice.

PURPOSE: Persistent high-risk human papillomavirus (HPV) infection is the most common cause of cervical cancer and its precursor lesions. Although prophylactic HPV vaccines have been applied in the general population for the prevention of HPV infections, no licensed therapeutic HPV vaccine is currently available to treat preexisting HPV infections or HPV-associated diseases, including cervical cancer. MATERIALS AND METHODS: The most common murine cervical cancer model used for the evaluation of the efficacy of a therapeutic HPV vaccine in preclinical studies is the ectopic model, which is established by the subcutaneous inoculation of tumor cells, such as TC-1 cells, into the flank of an animal. We have previously demonstrated the efficacy of a therapeutic HPV peptide vaccine adjuvanted with unmethylated cytosine-phosphate-guanosine oligodeoxynucleotide in the clearance of ectopic subcutaneous tumors in C57BL/6 mice after vaccination. In the current study, we established orthotopic genital tumors by injecting TC-1 cells into the vaginal submucosa close to the cervix and assessed whether the subcutaneous administration of the therapeutic vaccine could inhibit the growth of genital tumors. Additionally, we evaluated the effect of the vaccination on the tumor microenvironment. RESULTS: The results showed that the vaccination induced an increase in infiltrating CD4+ and CD8+ T cells, a decrease in myeloid-derived suppressor cells and cancer-associated fibroblasts, as well as the differential expression of a panel of cytokines, chemokines, and matrix metalloproteinases within the tumor microenvironment. CONCLUSION: The administration of the vaccine resulted in the inhibition of established implanted orthotopic genital tumors by inducing strong antitumor immune responses and reversed tolerogenic local immunosuppression in a mouse model of orthotopic genital cancer.

Anti-pulmonary metastases from cervical cancer responses induced by a human papillomavirus peptide vaccine adjuvanted with CpG-oligodeoxynucleotides in vivo.

Metastasis, particularly hematogenous metastasis, is associated with poor prognosis in patients with cervical cancer. The lungs are the most common site for hematogenous metastasis of cervical cancer. The currently available therapeutic modalities, including surgery, radiotherapy, or chemotherapy do not provide satisfactory clinical outcome for patients with pulmonary metastases. Therefore, it is necessary to investigate an alternative efficacious treatment modality. Therapeutic vaccines may evoke tumor-specific immune responses in patients to attack tumor cells, representing an attractive treatment option for controlling metastatic tumors. Our previous study demonstrated that a single administration of a human papillomavirus 16 E7 peptide vaccine, adjuvanted with unmethylated CpG-oligodeoxynucleotides, induced the clearance of subcutaneous xenograft cervical cancer. In this study, we investigated the anti-metastases responses induced by this vaccine using a murine model of pulmonary metastases from cervical cancer. The results showed that subcutaneous administration of the vaccine inhibited the growth of pulmonary metastases, which may be attributed to the increased infiltration of CD4 + and CD8 + T cells, and decreased number of immunosuppressive cells (including myeloid-derived suppressive cells and tumor-associated macrophages) in the lungs. Meanwhile, the alteration in a panel of cytokines, chemokines, and matrix metalloproteinases induced by the vaccination may contribute to the re-modulation of the local suppressive environment and inhibition of pulmonary metastases. To the best of our knowledge, this is the first report on the efficacy of the vaccine formula against murine pulmonary metastases from cervical cancer.

Induction of systemic immune responses and reversion of immunosuppression in the tumor microenvironment by a therapeutic vaccine for cervical cancer.

Cervical cancer is the most common malignant tumor of the genital tract in females worldwide. Persistent human papillomavirus (HPV) infection is closely associated with the occurrence of cervical cancer. No licensed therapeutic HPV vaccines for cervical cancer are currently available. In our previous study, we demonstrated that the vaccine containing the HPV16 E7 43-77 peptide and the adjuvant unmethylated cytosine-phosphate-guanosine oligodeoxynucleotide elicited significant prophylactic and therapeutic effects on cervical cancer. In the current study, we comprehensively evaluated the effect of the vaccine on systemic immune responses and the tumor microenvironment (TME) in a mouse model of cervical cancer. The results showed that the administration of the vaccine induced a significant increase in splenic IFN-γ-producing CD4 and CD8 T cells as well as tumor infiltrating CD4 and CD8 T cells. Moreover, marked decreases in splenic MDSCs and Tregs as well as intratumoral MDSCs, Tregs and type 2-polarized tumor-associated macrophages were observed in the vaccine group. The profile of cytokines, chemokines and matrix metalloproteinases (MMPs) in the TME revealed significantly increased expression of IL-2, IL-12, TNF-α, IFN-γ, CCL-20, CXCL-9, CXCL-10 and CXCL-14 and decreased expression of IL-6, IL-10, TGF-ß, CCL-2, CCL-3, CCL-5, CXCL-8, MMP-2, MMP-9 and VEGF in the vaccine group. The expression of the cell proliferation indicator Ki67, apoptosis regulatory protein p53 and angiogenesis marker CD31 was significantly decreased in the vaccine group. In conclusion, the vaccine reversed tolerogenic systemic and local TME immunosuppression and induced robust antitumor immune responses, which resulted in the inhibition of established implanted tumors.