Study On Anti-infection And Anti-tumor Immunity Based On The Attenuated Listeria Monocytogenes

Listeria monocytogenes is a Gram-positive intracellular parasite.Listeria can migrate between cells and stimulate the body to promote the proliferation of antigen-specific CD4⁺T cells and CD8⁺T cells to generate adaptive cellular immunity,so it is a high-quality live vaccine vector.Since the wild-type strain of Listeria is highly virulent,research teams using Listeria as a vaccine carrier have been working on various strategies to reduce the virulence of Listeria.In the previous research in the laboratory,two attenuated Listeria strains,LADS and LAPD,constructed through genetic modification.The research found that the virulence of these two attenuated Listeria strains was significantly reduced,the hemolytic activity was significantly lower than that of the wild strain,and their intercellular activity was significantly lower,and maintained normal cell-to-cell migration.The purpose of this study was to investigate the immunogenicity and safety of these two attenuated Listeria to evaluate the ability of Listeria as a live vaccine vector,and used these two attenuated Listeria as background strains to deliver foreign source antigen,and evaluated its immune protection in animal models,laying a foundation for Listeria as a vaccine carrier in the future.1.To explore the immunogenicity of attenuated Listeria LADS and LAPDIn this study,the virulence of LAPD and the organ clearance test were investigated by infecting a mouse model,and the immunogenicity of LAPD and LADS was also investigated.The test results showed that the LD50 of LAPD was 10^9.19 CFU,the virulence was significantly lower than that of the wild strain,and the bacteria in the organs can be cleared by the body within a week,and it was preliminarily determined that LAPD was safe as a vaccine carrier.By exploring the immunogenicity of two attenuated Listeria strains,it was found that the immunogenicity of LAPD and LADS were well preserved,and the immunogenicity of LADS was better than that of LAPD.2.Construction of Aeromonas hydrophila anti-infective vaccine based on LADS as carrier and evaluation of its immune preventive effectIn this study,LADS was used as the vector,and the exogenous antigen of Aeromonas hydrophila was cloned downstream of hly by homologous recombination to construct LADS-OmpW.The test results showed that OmpW antigen did not affect the normal in vitro growth ability and hemolytic ability of LADS,and could be expressed in fusion with LLO.Through immune protection studies in mice and zebrafish,it was found that LADS and LADS-OmpW could provide 88.8%protection to mice infected with Aeromonas hydrophila higher than the control group.And provided32.5%immune protection higher than the control group in zebrafish,indicating that LADS and LADS-OmpW had a certain preventive effect on Aeromonas hydrophila infection.3.Construction of cervical cancer vaccine based on LAPD as carrier and evaluation of its therapeutic and preventive effect on cervical cancerUsing the same construction strategy as LADS-OmpW,LAPD was used as the background strain to present the cervical cancer antigen E7 gene,and constructed LAPD-E7.The experiment proved that E7 could be expressed in fusion with LLO without affecting the in vitro growth ability and hemolytic ability of LAPD.The cervical cancer model was constructed in C57BL/6 mice using the laboratory strategy of tumors construction and treated by tail vein injection of LAPD-E7.Evaluation of treatment effect by periodic measurement of tumor volume in mice,the results showed that after two treatments,The average tumor volume of the control group grew to 512.41 mm³,while the tumor volume of mice in the immunized LAPD-E7 group only grew to 161.12 mm³,indicating that LAPD-E7 immunization had a significant effect on tumor recession.In the immunoprophylaxis research for mice cervical cancer,the mice were immunized on the 1st day and the 14th day respectively.Seven days after the immunization was received,the tumor was inoculated into the abdomen of the mice subcutaneously,and the tumor size was measured regularly.The average tumor size of the control group grew to 57.38mm³,while the average tumor size of the mice in the vaccine group was 3.86mm³,and three mice in the vaccine group did not grow tumors,indicating that LAPD-E7 had a certain preventive effect on cervical cancer.In conclusion,this study verified the relatively complete immunogenicity of Listeria with two different attenuating strategies,and constructed vaccine strains successfully,confirming that LADS-OmpW and LADS had protective effects on Aeromonas hydrophila;LAPD-E7 had significant therapeutic and preventive effects on cervical cancer.This study further showed that the use of attenuated Listeria as a vector had great potential for anti-infection and anti-tumor applications.