| Cervical cancer is the second malignant tumor that threatens global women's health,in which human papillomavirus(HPV)can be detected in nearly 100%of cervical cancer patients.Except for cervical cancer,HPV can also cause head and neck cancer,anal and genital cancer and so on.Currently,despite the availability of preventive HPV vaccines that have been approved listing,HPV remains a huge threat to human health due to the vaccines' limited vaccination coverage area,limited cross protection between viral genotypes and no significant treatment for established HPV long-term infections and cancer patients.Therefore,developing the therapeutic HPV vaccines for cervical intraepithelial neoplasia(CIN)and cancer cases by immunotherapy has a broad application prospect for the control of HPV-related diseases.However,most of the current therapeutic vaccines lack significant treatment effects in the clinical trial stage.It is reported that the failure of stimulating a high level of antigen-specific cellular immune response becomes a remarkble problem in anti-tumor vaccine design.As a result,it is an important strategy to explore a novel antigen displaying system for enhancing the antigen delivery to antigen-presenting cells(APCs),aming at increasing the intensity of tumor antigen-specific cellular immune response induced by vaccine and the clinical potency of anti-tumor vaccine.Outer membrane vesicles(OMVs)are proteoliposomes,with the diameter of 20-250 nm.These particles form as the gram-negative baterial outer membrane blebs outwards and pinches off during growth.OMVs have various characteristics and advantages to be a vaccine vehicle,such as the feasibility of genetic engineering modification,the ability of displaying macromolecular proteins and multiple proteins,containing many receptors promoting APCs' recognition,self-adjuvant activity and tropism to lymph nodes.In addition,the successful application in clinical of OMVs vaccine against Neisseria meningitidis serogroup B provides a hint to develop OMVs as a protential vaccine vehicle.Recently,researchers have attempted to integrate exogenous proteins into OMVs,for example,to display PspA protein from pneumococcus on salmonela-derived OMVs against pneumococcus infection and to display Omp22 protein on E.coli-derived OMVs against Acinetobacter baumannii infection in our previous study.Genetic engineering OMVs can bear exogenous antigen protein,provoke antigen-specific humoral immune response and combat pathogenic bacteria infections.However,the technology,immune characteristics and intervention effects of E.coli-derived OMVs against tumor as a therapeutic vaccine vector inducing antigen-specific cellular immune response remain unknown.This research aims at discussing the features and potency of bacterial OMVs as a kind of anti-cancer vaccine vector.Firstly,the tumoral antigen HPV16E7 was presented in E.coli-derived OMVs by genetic engineering methods,acquiring the recombinant OMVs vaccine.Secondly,the ability of recombinant OMVs delivering its components and the model antigen GFP to APCs was investigated in the macrophage Raw264.7 cells in vitro.It was also investigated in bone marrow-derived dendritic cells(BMDCs)that recombinant OMVs was uptaken efficiently,the significance of vesicular structure in the process of uptake and the maturation of DCs.Thirdly,it was evaluated in TC-1 graft tumor model in mice that the recombinant OMVs displaying HPV16E7 stimulated specific cellular immune response and intervened the growth of established tumor.Results showed that HPV16E7 protein could be embedded on the surface as well as in the lumen of OMVs through the guidance of cytolysin A(ClyA)and thioredoxin(trx).OMVs could be taken up efficiently by Raw264.7 and dendritic cells(DCs),for which vesicle structure had been proven to be very important.OMVs significantly stimulated the expression of DCs' surface co-stimulatory molecules CD80,CD86,CD83 and CD40,coupled with the secretion of TNFa,which means the functional maturation of BMDCs.Without combination with conventional adjuvants,engineered OMVs induced E7 antigen-specific cellular and humoral immune responses and biased to Thl/CTL response.Therapeutic immunization with recombinant OMVs significantly surpressed tumor growth.Meanwhile,OMVs presented with E7 induced stronger immune effects compared with E7 protein mixed with wild type OMVs(wt OMVs)and Freund's adjuvant.This study demonstrated that the nano-grade structure of E.coli-derived OMVs could be used as a novel vaccine delivery vector with the potency of stimulating tumoral antigen-specific cellular immune response effectively.It provided a brand new strategy for the breakthrough of anti-tumor therapeutic vaccine. |
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