| Objective:Cervical cancer is the most common genital malignancy in female.High-risk human papillomavirus(HPV)persistent infection is closely associated with the occurrence of cervical cancer and its precursor lesions.Therefore,the prophylactic vaccine that prevent HPV infection and the therapeutic vaccine that eliminate pre-existing HPV-infection or HPV-associated diseases can be applied to ultimately achieves the goal of preventing and treating cervical cancer.Although the prophylactic HPV vaccine showed good safety and effectiveness in healthy people who are not infected with HPV,it has no therapeutic effect on patients with cervical cancer induced by persistent HPV infection.Based on the fact that no licensed therapeutic vaccine is available now,the development of safe and efficacious therapeutic vaccines to treat pre-existing HPV infections and HPV-associated malignancies is necessary.Therapeutic vaccine aims to eliminate HPV malignant transformed tumor cells by inducing powerful tumor-specific Th1 type and cytotoxic T lymphocyte immune responses.Though the promising results of a serial of candidate vaccines to inhibit or clear subcutaneous tumors in preclinical studies were reported,no satisfactory clinical outcomes in patients with HPV-associated genital neoplasia or cervical cancer was shown.This may be due to the immunosuppressive tumor microenvironment(TME)associated with tumor progression and inhibition on the function of vaccine-induced effector cells in the tumor.Another reason may be the discrepancies caused by the application of ectopic cervical cancer models in preclinical studies because HPV-associated neoplasia in patients commonly occurred in the genital mucosa where a relatively immunosuppressed environment may exist.HPV-related tumors usually occur in the genital mucosa,and the ectopic model cannot accurately simulate the conditions of cervical cancer patients.In this study,we establish orthotopic genital cancer model by implanting tumor cell suspensions into vaginal submucosa nearby the cervix in situ to mimic the mucous site and provide a clinically relevant setting as much as possible.Liposomes and liposome-derived nanovesicles,which elicit both antigen-specific humoral and cell-mediated immunity,have been considered as an excellent vaccine delivery carriers/adjuvant.Owing to their self-closed structures,liposomes can entrap hydrophobic agents in the lipid bilayer and hydrophilic agents in the aqueous compartment.Meanwhile,it protected the loaded drug,avoiding both degradation and undesirable exposure of the environment to the drug action.Mannose-modified liposomes specifically target to mannose receptors on DC,thus enhancing DC-mediated antitumor activities.To efficiently target and activate DC,mannose-modified liposome encapsulating HPV16 E7 peptide and Cp G ODN was constructed.Therapeutic efficiency after vaccination with this vaccine was evaluated in locally advanced cervical cancer mice with tumor volume over than 200 mm~3.In vivo studies performed to reveal the mechanisms governing the antitumor response induced by the formulation.Methods:1.Tumor-bearing mice were used to evaluate the therapeutic effect of HPV E7 peptide vaccine with Cp G ODN as an adjuvant on cervical cancer.Female C57BL/6 mice aged 6–8 weeks were randomly divided into 4 groups.On day 0,all mice were injected with 5×10~5 TC-1 cells in PBS into vaginal submucosa nearby the cervix.On day 4,four groups of mice were injected subcutaneously with 20?g Cp G ODN,50?g E7 peptide,or the vaccine containing 20?g Cp G ODN mixed with 50?g E7 peptide in 100?l PBS in the right flank,respectively.We observed the growth of mice and tumors daily,and recorded their body weight,tumor volume,and survival time to analyze the inhibitory effect of the vaccine on tumor growth in mice.2.To assess the effect of HPV16 E7 peptide vaccine with Cp G ODN as an adjuvant on the local tumor microenvironment of mouse cervix:flow cytometry was used to analyse the effect of the vaccine on CD4+T cells,CD8+T cells,IFN-?+T cells,Treg,MDSC and TAM in the tumor microenvironment;q RT-PCR was used to estimate the expression levels of cytokines,chemokines and MMPs in tumor tissues;immunohistochemistry was used to study apoptosis,invasion,angiogenesis and collagen distribution in tumor tissues.3.Preparation and characterization of liposome-based vaccine:the E7 peptide and Cp G ODN co-delivering liposome vaccine was prepared using the reverse-phase evaporation method.The morphologies of liposome-based vaccine were observed using transmission electron microscopy.Particle size distributions and zeta potential were measured using a Laser Particle size analyzer in water.4.Tumor growth inhibition induced by the liposome-based vaccine:Female C57BL/6 mice were inoculated subcutaneously with 5×10~5 TC-1 cells on their flank on day 0.On day 12,formulations were subcutaneously injected into the contralateral side of the inoculation side for the control,empty liposome,free E7/Cp G and liposome E7/Cp G groups.Thereafter,tumor size and body weight were monitored daily,and recorded their body weight,tumor volume,and survival time to analyze the inhibitory effect of the liposome-based vaccine on tumor growth.5.To evaluate the effect of the liposome-based vaccine on the systemic immune response of the mouse:flow cytometry was used to monitor the specific cellular immune response in mice including CD4+IFN-?+T cells,CD8+IFN-?+T cells,Treg,MDSC and TAM,as well as the CTL response in vivo induced by liposome vaccination.6.To estimate the effect of the liposome-based vaccine on mouse tumor microenvironment:q RT-PCR was used to detect the level of cytokines,chemokines and MMPs in tumor tissues;immunohistochemistry was used to analyse apoptosis,invasion,angiogenesis,and collagen distribution in tumor tissues.Results:1.Successfully established the mouse orthotopic cervical cancer model.HPV E7 peptide vaccine with Cp G ODN as an adjuvant can significantly enhance the adaptive immune response of mice,especially the HPV antigen-specific cellular immune response,thereby effectively inhibit the growth of cervical cancer in situ and prolong the survival time of mice.2.After mice vaccinated with HPV E7 peptide adjuvanted with Cp G ODN,the percentage of CD4+T cells(P<0.05)and CD8+T cells(P<0.001)in mice tumors increased significantly.Furthermore,the administration of the vaccine significantly reduced the percentage of MDSC(P<0.05).The q RT-PCR results showed that the expression level of IL-2(P<0.05)was significantly increased,while the expression levels of immunosuppressive cytokines such as IL-4(P<0.05)and TGF-?(P<0.05)were significantly reduced.The expression levels of chemokines such as CCL-1(P<0.001),CCL-2(P<0.001),CCL-3(P<0.001),CCL-5(P<0.001),CCL-12(P<0.001)and CCL-21(P<0.001)were significantly reduced,and the expression levels of matrix metalloproteinases such as MMP-2(P<0.001),MMP-3(P<0.001),and MMP-9(P<0.001)were also significantly reduced.The results of immunohistochemical analysis showed that when mice were immunized with HPV E7 peptide adjuvanted by Cp G ODN,the expression levels of?-SMA,Ki67 and p53 in tumor tissues were significantly reduced.In short,vaccination of HPV E7 peptide with Cp G ODN as an adjuvant helps to enhance the anti-tumor immune response,reverse tumor immunosuppression,and improve the efficacy of tumor immunotherapy.3.Successfully prepared the mannose-modified liposome co-delivering human papillomavirus type 16 E7 peptide and Cp G ODN adjuvant.The liposome has a particle size in the range of 60-120 nm,spherical in shape and negatively charged on the surface.4.The mannose-modified liposome co-delivering human papillomavirus type 16 E7 peptide and Cp G ODN adjuvant can specifically target to mannose receptors on DC and promote DC maturation,thus effectively inhibited the growth of mouse TC-1 transplanted tumors and prolonged their survival time.5.After mice were vaccinated with the liposome-based vaccine,the percentage of CD4+IFN-?+T cells(P<0.01)and CD8+IFN-?+T cells(P<0.05)in the spleen increased significantly.Splenocytes of immunized mice with Lip E7/Cp G showed significant reduction in the percentages of MDSC(P<0.001)and TAM(P<0.01).In addition,Lip E7/Cp G vaccine elicited potent E7-specific in vivo CTL response.6.The results of q RT-PCR showed that the expression levels of anti-tumor cytokines such as IL-2(P<0.001),IL-12(P<0.001),TNF-?(P<0.001)and IFN-?(P<0.001)in the tumor tissues when mice were treated with Lip E7/Cp G were significantly increased,while the expression level of immunosuppressive cytokines such as IL-4(P<0.001)and TGF-?(P<0.001)were significantly reduced.In terms of chemokines,it was detected that the expression levels of CCL-3(P<0.001),CXCL-9(P<0.01)and CXCL-10(P<0.001)in the Lip E7/Cp G group increased significantly.The results of immunohistochemical analysis showed that when mice were immunized with mannose-modified liposome co-delivering human papillomavirus type 16 E7 peptide and Cp G ODN adjuvant,the number of angiogenesis in tumor tissue was significantly reduced(P<0.001),and the expression of Ki67(P<0.001)and p53(P<0.01)was also significantly reduced.Conclusion:1.The vaccination of HPV E7 peptide with Cp G ODN as adjuvant induced an increase of infiltrating CD4 and CD8 T cells in tumors,as well as the expression variation in a panel of cytokines,chemokines and matrix metalloproteinases in tumor microenvironment,which may result in the inhibition of established implanted orthotopic genital tumors,and enhanced the ability of tumor-bearing mice to resist cervical cancer.2.A novel vaccine Lip E7/Cp G was established using mannose-modified liposomes as a delivering carrier which was loaded with Cp G ODN and HPV16 E7 peptide.The vaccination of Lip E7/Cp G inhibited the growth of local advanced cervical cancer by inducing enhanced cellular immunity and improving immunosuppressive environment in tumors.In a word,mannose-modified liposomes is a promising therapeutic vaccine delivery strategy. |
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