We present a new strategy for self-adjuvanting vaccine development that has different types of covalently-linked immunostimulants as the carrier molecule.Using Tn antigen as the model,a three-component vaccine(MPLA-Tn-KRN7000)containing the TLR4 ligand MPLA and the iNKT cell agonist KRN7000 was designed and synthesized.This expands fully synthetic self-adjuvanting vaccine studies that use a single carrier to one with two different types of carriers.The corresponding two-component conjugate vaccines Tn-MPLA,Tn-KRN7000 and Tn-CRM197 were also synthesized,as controls.The immunological evaluation found that MPLA-Tn-KRN7000 elicits robust Tn-specific and T cell-dependent immunity.The antibodies specifically recognized,bound to and exhibited complement-dependent cytotoxicity against Tn-positive cancer cells.In addition,MPLA-Tn-KRN7000 increased the survival rate and survival time of tumor-challenged mice,and surviving mice reject further tumor attacks without any additional treatment.Compared to the glycoprotein vaccine Tn-CRM197,the two-component conjugate vaccines,Tn-MPLA and Tn-KRN7000,and the physical mixture of Tn-MPLA and Tn-KRN7000,MPLA-Tn-KRN7000 showed the most effect at combating tumor cells both in vitro and in vivo.The comparison of immunological studies in wild-type and TLR4 knockout mice,along with the test of binding affinity to CD 1d protein suggests that the covalently linked MPLA-KRN7000 immu-nostimulant induces a synergistic activation of TLR4 and iNKT cell that improves the immunogenicity of Tn.This work demonstrates that MPLA-Tn-KRN7000 has the potential to be a vaccine candidate and provides a new direction for fully synthetic vaccine design.

Deying Yang;Xiang Luo;Qinghai Lian;Lingqiang Gao;Chengxin Wang;Xiaoxiao Qi;Rong Zhang;Zhongqiu Liu;Guochao Liao

Joint Laboratory for Translational Cancer Research of Chinese Medicine of the Ministry of Education of the People's Republic of China,International Institute for Translational Chinese Medicine,Guangzhou University of Chinese Medicine,Guangzhou 510006,China;Guangdong-Hong Kong-Macau Joint Lab on Chinese Medicine and Immune Disease Research,Guangzhou 510120,China

药学学报（英文版）

[1]Deying Yang;Xiang Luo;Qinghai Lian;Lingqiang Gao;Chengxin Wang;Xiaoxiao Qi;Rong Zhang;Zhongqiu Liu;Guochao Liao-.Fully synthetic Tn-based three-component cancer vaccine using covalently linked TLR4 ligand MPLA and iNKT cell agonist KRN-7000 as built-in adjuvant effectively protects mice from tumor development)[J].药学学报（英文版）,2022(12):4432-4445

KRN,adjuvanting,immunostimulants,KRN7000,nostimulant

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