A Study On The Attenuated Listeria Monocytogenes-based Tumor Immunotherapy

Listeria monocytogenes(L.monocytogenes,LM)is a Gram-positive intracellular pathogen which can proliferate in a variety of cells.L.monocytogenes can activate the body’s adaptive cellular immune response through two different major histocompatibility complexes(MHC I and MHC II)after infect host cells.L.monocytogenes,which has unique intracellular infection mechanism,has been widely used in tumor immunotherapy research as a novel antigen delivery live carrier through genetic modification.Previous research found that the virulence of L.monocytogenes was greatly reduced after changed two key amino acid sites on the important virulence factor LLO(encoded by the hly gene),but it still retained the ability to proliferate in the cell.This research is mainly based on attenuated Listeria antigen delivery vector which is modificated the two key active sites of the LLO,and used for the evaluation of the immunotherapy effects of the mouse cervical cancer model.In this study,the basic strain EGD-e of L.monocytogenes was used as the background strain.We used amino acid mutation and homologous recombination technology on the L.monocytogenes hly gene to obtain attenuated L.monocytogenes(LADS).Molecular biology,cell biology and infection biology were used to evaluate the biological characteristics and safety of LADS.We found that LADS can grow normally in BHI medium,and can stably synthesize and secrete LLO mutant proteins,but the activity of secreted LLO was influenced due to mutations in key active sites.LADS can proliferate and migrate in fibroblasts(L929)normally.Although LADS cannot proliferate in macrophages,they can still stimulate macrophages to produce tumor immune-related inflammatory factors(IL-1β,IL-6,TNF-α and IL-10).Furthermore,the virulence of LADS is highly weak.The lethal dose(LD50)in the mouse infection model is nearly 10,000 times higher than the wild strain,which meets the biological safety of the attenuated vaccine carrier.Then we used homologous recombination to seamlessly clone the tumor-associated antigen E7,which is the main oncogene of human cervical cancer,into the C-terminus of the hly gene to construct a vaccine strain(LADS-E7).We found that LADS-E7 successfully composited and secreted recombinant fusion mutant protein LLO-E7,and grew normally in BHI medium.Then,C57 BL / 6 mice were subcutaneous injectied with cervical cancer tumor cells TC-1 to construct a murine tumor model,and LADS-E7 was injected into the tail vein to evaluate the tumor immunotherapy effect.The results show that LADSE7 can significantly inhibit the growth of tumor after two immunizations,and some tumers have completely disappeared.The ratio of CD4+ and CD8+ T cells in the splenocytes of the LADS-E7 treatment group was significantly higher than that of the control group(PBS or LADS)and the Treg cells were significantly reduced by flow cytometry.It is shows that LADS-E7 successfully causes mouse T cell immune response and then inhibits and kills tumors.In summary,for the first time,we construct an attenuated L.monocytogenes LADS by irreversible mutation of key amino acid sites of virulence factors.And it remained infection characteristics and immunogenicity greatly with a significant attenuation effect and no resistance,which is in line with the biological safety of vaccine vectors.Furthermore,LADS-E7 showed good therapeutic effect in mouse HPV tumor model.This study is expected to provide innovative and efficient strategies for tumor immunotherapy,and has great application prospects.