Application Of Built-In Adjuvant And Antigen Modification Strategies In Antitumor And Antiviral Vaccines

With the advent of vaccines,human health has improved significantly.Vaccines work primarily by stimulating the immune system to recognize target antigens and subsequently induce a specific immune response.Subunit vaccines use specific immunogenic components of pathogens as antigen and have great biosafety.However,these components generally are weakly immunogenic.Therefore,how to effectively enhance the immunogenicity of antigens is a key scientific problem in the development of subunit vaccines.In this paper,antitumor and antiviral subunit vaccines were designed with built-in adjuvant and protein modification strategies.The research is divided into the following three parts:a three-component fully synthetic antitumor vaccine with built-in Toll-like receptor（TLR7）agonist,a COVID-19 protein vaccine with built-in TLR7agonist,and an adjuvant-free COVID-19 protein vaccine with oxidized glycoprotein antigen.In Chapter 1,the history and mechanism of action of vaccines are briefly introduced.The development of antitumor glycopeptide vaccines targeting Mucin 1（MUC1）and SARS-CoV-2 subunit vaccines are highlighted.The current status of research on the application of TLR7 small molecule agonists as adjuvants in vaccines is summarized.In Chapter 2,to improve the immunogenicity of the tumor-associated antigen MUC1 glycopeptide,we prepared a fully synthetic anti-tumor glycopeptide vaccine by covalently coupling the Th epitope peptide PADRE to the MUC1 peptide via solid-phase peptide synthesis,and employing a TLR7 agonist（TLR7a）as a built-in adjuvant.The vaccine is facilely prepared and facilitates the large-scale preparation of vaccines.The results showed that the fully synthetic anti-tumor vaccine TLR7a-PADRE-MUC1 could effectively enhance the immunogenicity of MUC1 glycopeptide and induced high titers of specific antibodies.More importantly,tumor-bearing mice inoculated with TLR7a-PADRE-MUC1 showed that the vaccine effectively inhibited tumor growth.In Chapter 3,we designed an adjuvant-protein conjugate vaccine candidate,in which the TLR7a was conjugated to S1 subunit of SARS-CoV-2 spike protein via active ester chemistry,and systematically compared the effect of different numbers of built-in TLR7a on the immune activity for the first time.This adjuvant-protein conjugate strategy allows the built-in adjuvant to provide cluster effects and reduce the systemic toxicity caused by adjuvant diffusion to a certain extent and facilitates the co-delivery of adjuvant and antigen.Immunization results showed that TLR7a（10）-S1（with around 10 built-in adjuvant molecules on one S1 protein）triggered robust humoral and cellular immunity and a balanced Th1/Th2 immune response.In addition,the vaccine induces effective neutralizing antibodies against SARS-CoV-2 wild-type and variants of concern.In Chapter 4,we designed adjuvant-free COVID-19 vaccines for the first time in which the N-glycan of SARS-CoV-2 receptor binding domain（RBD）glycoprotein was oxidized by sodium periodate.This strategy only minimally modifies the glycans and does not interfere with the epitope peptides.Meanwhile,we prepared RBD_LO（oxidation with low concentration of periodate）and RBD_HO（oxidation with high concentration of periodate）by differential oxidation of glycoproteins with different concentrations of periodate.The RBD_HO significantly enhanced antigen uptake and promoted the activation of antigen-presenting cells without any external adjuvant.Compared to unmodified RBD antigen,RBD_HO induced 324-fold and 27-fold increases in IgG antibody titers and neutralizing antibody titers.Meanwhile,the RBD_HO vaccine was able to cross-neutralize all the SARS-CoV-2 variants of concern,demonstrating broad-spectrum antiviral activity.In addition,RBD_HO effectively enhanced cellular immune responses and exihibited great biosafety at both cellular and animal levels.