Effect And Mechanism Of Human Papillomavirus Peptide Vaccine With CpG ODN As Adjuvant In Treating Cervical Cancer

Objective:Cervical cancer is the most common malignancy of the female reproductive system.Persistent infection with high-risk human papillomavirus(HPV)is closely related to cervical intraepithelial neoplasia and cervical cancer.Therefore,prophylactic and therapeutic vaccine against HPV infection can be used to prevent and treat cervical cancer.There are currently four types of prophylactic HPV vaccines approved for marketing in the world.Inoculation of prophylactic HPV vaccine in HPV-uninfected population showed good safety and efficacy,but prophylactic vaccine cannot clear existing HPV infection,and has no obvious therapeutic effect on cervical cancer caused by persistent HPV infection.Therapeutic HPV vaccine aim to clear HPV infected and malignantly transformed tumor cells by inducing robust HPV antigen-specific Th1 and cytotoxic T lymphocyte immune responses.Since there is no therapeutic HPV vaccine approved for marketing,it is particularly important to develop a safe and effective therapeutic HPV vaccine for the clinical treatment of HPV-related tumors.In this study,the HPV 16 E7 peptide vaccine adjuvanted with CpG ODN was inoculated by subcutaneous injection,and the effects of vaccination on the systemic immune response and local tumor immunity of mice were studied in the C57BL/6 mouse with TC-1 tumor.The effect of the vaccine on the tumor growth and the mechanism of inhibition of the tumor growth were clarified.The route of administration of tumor therapeutic vaccine is a key factor affecting vaccine-induced antitumor activity.Therefore,in the second part of the study,we continued to investigate the effect of three vaccination routes(subcutaneous,peritumoral and intratumoral injection)on the antitumor activity induced by the CpG ODN-adjuvanted HPV 16 E7 peptide therapeutic vaccine in TC-1 xenografts,and ultimately determined the optimal route of vaccination.Through the first two parts of the study,it was found that even with the same vaccine formulation and the same vaccination route,the antitumor immune responses induced by the therapeutic vaccine based on HPV 16 E7 peptide still vary widely among individuals.Consequently,in the third part of the study,the gut microbiota of two groups of mice with significant differences in vaccine-induced antitumor effects were systematically compared and analyzed,and the correlation between immune cells and gut microbiota was further explored.Methods:1.The therapeutic effect of HPV 16 E7 43-77 peptide vaccine adjuvanted with CpG ODN on early cervical cancer was studied in C57BL/6 mice bearing cervical cancer.C57BL/6 mice for 6-8 weeks were subcutaneously injected with TC-1 cells to prepare a tumor-bearing mouse model.Four days after inoculation with TC-1 cells,adjuvant CpG alone,E7 peptide alone,and a combination vaccine composed of CpG and HPV 16 E7 43-77 peptide were subcutaneously injected,and phosphate buffered saline(PBS)was used as a control.The general condition of the mice and the growth of the tumor were observed every day,and the data of the weight,tumor volume and survival time of the mice were recorded,and the inhibitory effect of the HPV 16 E7 43-77 peptide vaccine with CpG ODN as an adjuvant on the tumor growth of the mice was evaluated.2.To explore the effect of HPV 16 E7 43-77peptide vaccine adjuvanted with CpG ODN on the systemic immune response and tumor microenvironment in mice:flow cytometry was used to perceive the effect of vaccine on CD4~+IFN-γ~+T cells,CD8~+IFN-γ~+T cells,myeloid-derived suppressor cells(MDSC),regulatory T cells(Treg)in the spleen of C57BL/6 mice and the influence of immune cells such as CD4~+T cells,CD8~+T cells,MDSC,Treg and tumor-associated macrophage(TAM)in the tumor microenvironment;real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to appraise the effect of vaccine on the expression levels of cytokines,chemokines and matrix metalloproteinases(MMP)in tumor tissue;the changes of cell proliferation,apoptosis,and angiogenesis in tumor tissue were detected by immunohistochemistry.3.Using cervical cancer-bearing C57BL/6 mice to assess the therapeutic effect of HPV 16 E743-77 peptide vaccine adjuvanted with CpG ODN by different routes on middle-advanced cervical cancer:6-8 weeks old C57BL/6 mice were selected and inoculated subcutaneously with 5×10~5TC-1 cells.When the tumor volume of the mice reached 150±50 mm~3,that is,9 days after the mice were inoculated with TC-1tumor cells,all mice were randomly divided into 4 groups.The mice in the control group were subcutaneously injected with 100μL of PBS buffer,and the mice in the experimental group were inoculated with 20μg adjuvant CpG ODN 1826 and 50μg HPV 16 E7 43-77 peptide in 100μL PBS buffer.The general condition of the mice in each group and the growth status of the tumor were observed every day,the data of the weight,tumor volume and survival time of the mice were recorded,and the differences in the inhibitory effects of different vaccination routes on the tumor growth of the mice were analyzed.4.To estimate the effects of different routes of inoculation of HPV 16 E7 43-77 peptide vaccine with CpG ODN as adjuvant on the systemic immune response and tumor microenvironment of mice:flow cytometry was used to monitor the effects of different vaccination routes on the spleen antigen-specific CD4~+IFN-γ~+T cells,CD8~+IFN-γ~+T cells,MDSC,Treg,M2-TAM of C57BL/6 mice and the effect of antigen-specific CD8~+IFN-γ~+T cells,MDSC,Treg and M2-TAM in tumor microenvironment;qRT-PCR was used to detect the effect of vaccine on the expression levels of cytokines,chemokines and MMP in tumor tissue;the changes of angiogenesis and collagen distribution in tumor tissue were evaluated by immunohistochemistry.5.To analyze the correlation between gut microbiota and antitumor immune response induced by HPV 16 E7 43-77 peptide vaccine adjuvanted with CpG ODN:according to the tumor growth curve,the mice were divided into group A with continuous tumor growth and group B with significantly reduced tumor volume.We collected fresh fecal samples from two groups of mice,extracted fecal genomicDNA,amplified the V3-V4 region of 16S rDNA,and used Illumina Miseq platform for sequencing;species components analysis,principal component analysis,LDA effect size analysis and other biological information analysis methods were used to compare the differences in the gut microbiota of the two groups of mice;flow cytometry was used to appraise spleen CD4~+T cells,CD8~+T cells,IFN-γ~+T cells,CD4~+IFN-γ~+T cells,CD8~+IFN-γ~+T cells,MDSC,Treg,and M2-TAM;correlations between immune cells and gut microbiota were assessed using Spearman correlation analysis.Results:1.A tumor-bearing mouse model was successfully established.Inoculation of HPV 16 E7 43-77 peptide vaccine adjuvanted with CpG ODN could significantly enhance the adaptive immune response level of mice.The proportion of CD4~+IFN-γ~+T cells(P=0.0314),CD8~+IFN-γ~+T cells(P=0.0003)in the spleen were significantly increased,and the percentages of immunosuppressive cells such as MDSC(P=0.0471)and Treg(P=0.0180)in the spleen were significantly decreased,which further enhanced the killing effect on tumor cells and effectively inhibiting tumor growth in mice.2.After tumor-bearing mice were vaccinated with HPV 16 E7 43-77 peptide vaccine adjuvanted with CpG ODN,the percentages of CD4~+T cells(P=0.0058)and CD8~+T cells(P=0.0018)in the tumor area were significantly increased,and the numbers of MDSC(P=0.0401),Treg(P=0.0004),and M2-TAM(P=0.0371)in the tumor microenvironment were significantly decreased.The results of qRT-PCR showed that the expression levels of cytokines such as interleukin(IL)-2(P=0.0134),IL-12(P<0.0001),tumor necrosis factor(TNF)-α(P<0.0001),IFN-γ(P<0.0001)and chemokines such as CCL-20(P=0.0159),CXCL-9(P<0.0001),CXCL-10(P=0.0188)and CXCL-14(P<0.0001)in the tumor tissue of the vaccine group were significantly increased.However,the expression levels of immunosuppressive cytokines such as IL-6(P=0.0063),IL-10(P=0.0002),and transforming growth factor(TGF)-β(P<0.0001)were significantly decreased.The mRNA expression levels of CCL-2(P=0.0007),CCL-3(P=0.0021),CCL-5(P=0.0154),CXCL-8(P=0.0001),MMP-2(P<0.0001),MMP-9(P=0.0225)and vascular endothelial growth factor(VEGF)(P=0.0460)were also significantly decreased in the tumor tissue of the vaccine group.The results of immunohistochemical analysis showed that the numbers of Ki67(P=0.0157)and p53(P=0.0018)positive cells and the number of angiogenesis(P<0.0001)in the tumor tissue of the mice in the vaccine group were significantly reduced.In conclusion,vaccination with HPV 16 E7 peptide vaccine adjuvanted with CpG ODN reduced systemic and local tumor immunosuppression,and induced a strong antitumor immune response,which ultimately inhibited the growth of transplanted tumors.3.There are differences in the antitumor responses induced by different routes of vaccination in tumor-bearing mice.In the middle-advanced tumor-bearing mice treated with intratumoral injection of the vaccine,the tumor volume was significantly reduced(P<0.0001).The proportion of HPV 16 E7-specific CD4~+IFN-γ~+T cells(P=0.0453)and CD8~+IFN-γ~+T cells(P=0.0281)in the spleen cells of the mice in the intratumoral injection group increased significantly.The percentages of immunosuppressive cells in the spleen such as MDSC(P=0.0211),Treg(P=0.0040),and M2-TAM(P=0.0015)were significantly decreased in the intratumoral injection group.4.Intratumoral injection of vaccine promoted the recruitment and activation of HPV 16 E7 antigen-specific CD8~+IFN-γ~+T cells in the tumor area(P=0.0231),reduced infiltration of immunosuppressive cells including MDSC(P=0.0002),Treg(P=0.0167)and M2-TAM(P=0.0024),thereby effectively enhancing the level of antitumor immune response.The results of qRT-PCR showed that compared with the control group,the levels of pro-inflammatory and antitumor cytokines such as IL-2(P=0.0005),IL-12(P<0.0001),TNF-α(P<0.0001),IFN-γ(P=0.0001),and chemokines CXCL-9(P<0.0001)and CXCL-10(P=0.0325),which can promote the migration of effector T cells and natural killer(NK)cells to the local tumor,were significantly increased in the intratumoral group.The expression level of IL-10(P=0.0030),TGF-β(P<0.0001),CXCL-1(P=0.0014),MMP-2(P=0.0254),MMP-9(P=0.0373),and VEGF(P<0.0001)was significantly decreased.The results of immunohistochemical staining analysis showed that intratumoral injection of the vaccine significantly increased the density of CD4~+(P=0.0080)and CD8~+(P=0.0006)T cells in tumor tissue,and significantly decreased the number of angiogenesis(P=0.0124)and the expression level of alpha-smooth muscle actin(α-SMA)molecule(P=0.0108).5.Compared with mice in group A,vaccination induced stronger antitumor activity in mice in group B,and the CD4~+IFN-γ~+(P=0.0385)and CD8~+IFN-γ~+(P=0.0102)T lymphocytes were significantly increased,immunosuppressive cells such as MDSC(P=0.0089),Treg(P=0.0005)and M2-TAM(P=0.0005)were significantly reduced.6.Similar levels of bacterial richness and diversity in the gut microbiota in the two groups of mice were shown.A detailed gut microbiota analysis revealed a positive Spearman correlation between the percentage of CD8~+T cells and the relative abundance of Corynebacteriales(P=0.0069),Parabacteroides(P=0.0206)and Bacteroides＿sp.(P=0.0141).Furthermore,the percentage of CD4~+T cells and CD8~+T cells was negatively correlated with the abundance of Proteobacteria(P=0.0188,P=0.0104)and Bilophila(P=0.0223,P=0.0081).In contrast,the abundance of Proteobacteria(P=0.0055,P=0.0147,P=0.0017),Desulfovibrio(P=0.0266,P=0.0110,P=0.0178)and Bilophila(P=0.0063,P=0.0037,P=0.0075)was positively correlated with the percentage of MDSC,Treg and M2-TAM.Additionally,it was showed a negative Spearman correlation between the relative abundance of Bacteroides＿sp.(P=0.0475,P=0.0123,P=0.0003)and the percentage of the three immunosuppressive cells.Conclusion:1.Subcutaneous injection of HPV 16 E7 43-77 peptide vaccine adjuvanted with CpG ODN can effectively inhibit the tumor growth of tumor-bearing mice by increasing CD4~+IFN-γ~+and CD8~+IFN-γ~+T cells in mouse spleen and the number of CD4~+and CD8~+T cells infiltrated locally in the tumor,reducing the number of immunosuppressive cells such as MDSC and Treg in the spleen and tumor microenvironment,promoting the levels of IL-2,IL-12,TNF-α,IFN-γin tumor-bearing mice,and inhibiting immunosuppressive cytokines such as IL-10 and TGF-βand the expression of matrix metalloproteinases such as MMP-2 and MMP-9.2.Intratumoral injection of the vaccine can significantly inhibit the growth of advanced cervical cancer by enhancing the HPV antigen-specific antitumor immune response and improving the immunosuppressive microenvironment of the tumor,which is a better vaccination route than subcutaneous injection and peritumoral injection.3.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.