Inhibitory Effect Of Live-Attenuated Listeria Monocytogenes-based Vaccines Carrying MART-1 Gene On Malignant Melanoma

Objective: To investigate the inhibitory effects and tumor immunogenicity of Live-Attenuated Listeria Monocytogenes-based vaccines expressing Melanoma differentiation antigen MART-1 gene on malignant melanoma.Method: The plasmid pERL3-MART-1 was constructed and used to transform live-attenuated Listeria monocytogenes by electroporation. The attenuated listeria strains'LD₅₀were determined by concentration gradient dilution method. C57BL/6 mice were randomly divided into three groups, namely PBS group,â–³inlB LM-MART-1 group andâ–³actA/â–³inlB LM-MART-1 group. Mice were inoculated i.p. with 0.1LD₅₀of each rLM strain or PBS only. One week later, mice were injected s.c. with 1×105 tumor cell in 200μl of PBS. Reimmunization was preformed on days 14 and 21. Tumor growth and survival of tumor bearing mice were observed. MART-1 expressing in tumor were detected by real-time quantitative PCR. Mouse spleen cells by flow cytometry in CD4 + CD25 + T cells positive rate.Result: The results showed thatâ–³inlB LM-MART-1, andâ–³actA/â–³inlB LM-MART-1 were constructed successfully. The experiment of LD₅₀show that LD₅₀ofâ–³inlB LM andâ–³actA/â–³inlB LM than the LM-EGD strain virulence decreased 100 times and 10,000 times. In vivo anti-tumor trials showâ–³inlB LM-MART-1 group andâ–³actA /â–³inlB LM-MART-1 group than in PBS group tumors significantly reduced. Compared with the PBS group,â–³inlB LM-MART-1 group of tumor inhibition rate was 46.95% (F = 6.3, P <0.05),â–³actA /â–³inlB LM-MART-1 tumor inhibition rate was 83.96% (F = 37.8, P <0.001), the differences were statistically significant。PBS group,â–³inlB LM-MART-1 group andâ–³actA /â–³inlB LM-MART-1 group of tumor tissue expression of MART-1 increments, in order to express the amount of PBS as a group, the latter two groups is 8.988±0.207 and 11.315±0.445, among all groups were statistically significant differences (P <0.05)。And the three groups of mouse spleen cells, CD⁴⁺ CD²⁵⁺ T cells, followed by the positive rate of 2.52±0.20%, 1.14±0.13% and 0.44±0.15% (2.52±0.20% vs 1.14±0.13%, F = 271.7, P <0.001 ; 2.52±0.20% vs 0.44±0.15%, F = 564, P <0.001; 2.52±0.20% vs 0.44±0.15%, F = 95.7, P <0.001). The differences were statistically significant.Conclusion: MART-1 gene carrying attenuated Listeria monocytogenes bacteria toxicity was significantly lower than the standard strains of Listeria monocytogenes, using recombinant attenuated LM immune mice can inhibit melanoma growth and prolong survival.