Immune Characteristics And Anti-tumor Effects Of Nanoparticle Vaccines Using ATP As A Novel Adjuvant

Background Cancer seriously harms human health.Cervical cancer is the second most frequent tumor that endangers women’s health,and persistent high-risk human papillomavirus(HPV,human papillomavirus)infection is a key factor leading to in cervical cancer.There are currently three types of preventive HPV vaccines(two-,four-,and nine-valent)approved for marketing.Although they can trigger the production of neutralizing antibodies that bind to the virus particles and prevent them from entering the host cell,they lack therapeutic effects on established HPV infections or HPV-related tumors.Therefore,there is an urgent need to develop effective therapeutic HPV vaccines.Synthetic long peptide(SLP)vaccines based on HPV oncoprotein E7 or E6 have shown encouraging results in preclinical studies and clinical trials for the treatment of HPV-associated malignancies.However,the clinical efficacy of peptide vaccines may be limited by the disadvantages of vulnerability to enzymatic degradation and low immunogenicity.It is not enough to stimulate the body’s cellular immune response to clear the virus or virus-infected cells.In addition,adjuvant is an important component of tumor vaccines.However,there is currently no safe and effective adjuvant to promote the antigen-specific immune response of the HPV peptide vaccine.As a result,the clinical application of the HPV peptide vaccine is greatly restricted.The widely used aluminum adjuvant mainly stimulates the humoral immune response,which is not conducive to stimulating the cellular immune response.Therefore,it is very important to solve the shortcomings of peptide vaccines and find safe and effective new vaccine adjuvants to enhance the role of peptide-based vaccines in tumor immunotherapy.ATP is one of the most important intracellular metabolites and an endogenous extracellular danger signal for the immune system.ATP plays an important role in the immune regulation when tumor cells undergo immunogenic cell death(ICD).It is a potential vaccine adjuvant.Objective Solve the shortcomings of systemic diffusion,easy enzymatic degradation and low immunogenicity of peptide vaccine.Looking for safe and effective new tumor vaccine adjuvants to promote the effect of peptide vaccines in tumor immunotherapy,and provide new ideas and strategies for tumor immunotherapy.Methods To further improve vaccine potency,we developed a poly(lactide-co-glycolide)-acid(PLGA)nanoparticle,which encapsulated the antigenic peptide HPV16 E744-62,and used ATP,as a new adjuvant component.Transwell experiment was used to evaluate the effect of ATP adjuvant on the migration or recruitment of bone marrow-derived dendritic cells(BMDCs),and nasal drip was used to evaluate the effect of ATP on immune cell infiltration in animal models.In addition,the morphology,particle size and polymer dispersity index(PDI)of nanovaccine were detected by transmission electron microscopy(TEM)and dynamic light scattering(DLS).The release kinetics properties of E7 peptide and ATP in nanosystem were evaluated by dialysis technique.In vivo imaging system was used to monitor the retention time of nano vaccine at the injection site to evaluate its stability in vivo.To establish the grafted tumor model,TC-1 tumor cells were injected s.c.into the right flank of the mice.For the preventive studies,C57BL/6 mice were immunized with ATP-adjuvanted nanovaccine(ATP+E7-NPs).The tumor-free mice were rechallenged with TC-1 cells to assess whether ATP+E7-NPs stimulate long-term immune protection.For the therapeutic studies,the mice were first challenged with TC-1 cells.When the tumor diameters reached approximately 5-6 mm,the mice were immunized three times at one-week intervals.Tumor growth was monitored periodically using a slide caliper.Seven days after the third immunization,the mice were sacrificed,and the spleens and tumors were collected for immunological tests.Results New adjuvant ATP can effectively promote the migration and maturation of BMDCs,and increase the local infiltration of inflammatory cells in the body.The prepared PLGA nanoparticles are spherical,with a particle size in the range of 100-500 nm,and sustained release of antigen in the form of "antigen storage" for up to 150 h.Compared with the free E7 peptide,encapsulation of E7 with PLGA nanoparticles can improve the stability of E7 in vivo,increase the residence time of antigen at the injection site,and improve lymph node accumulation and DC uptake.More importantly,compared with E7-NPs,ATP as a nanovaccine adjuvant further increased DC migration,nanoparticle uptake and maturation.In the animal tumor model,preventive immune intervention with ATP+E7-NPs can completely eliminate the growth of TC-1 tumors,and 100%of the mice reach tumor-free.After the second tumor attack,ATP+E7-NPs provided persistent immune protection against the second tumor attack,significantly inhibiting tumor growth.Immunization with ATP+E7-NPs not only significantly increased the frequencies of CD8+and CD4+ effector cells in the spleen of mice,but also significantly increased the frequencies of CTLs(CD8+IFN-γ+)in CD8+ cells and Th1(CD4+IFN-γ+)in CD4+ cells.When tumors were fully established,therapeutic immunization with ATP-adjuvanted nanoparticles still significantly inhibited tumor progression.Mechanistically,ATP-adjuvanted nanoparticles significantly improved the systemic generation of antitumor effector cells,boosted the local functional status of these cells in tumor tissue and suppressed the generation and tumor infiltration of immunosuppressive Tregs and myeloid-derived suppressor cells(MDSCs).Conclusion These findings indicate that ATP is an effective vaccine adjuvant and that nanoparticles adjuvanted with ATP were able to elicit robust antitumor cellular immunity,which may provide a promising therapeutic vaccine candidate for the treatment of clinical malignancies,such as cervical cancer.