Construction of Lmdd-LMP2A, an attenuated Listeria vaccine strain expressing the Epstein-Barr virus latent membrane protein 2A (EBV-LMP2A) and evaluation of its anti-tumor effects against nasopharyngeal carcinoma

Objective To prepare an attenuated Listeria vaccine Lmdd-LMP2A expressing the Epstein-Barr virus latent membrane protein 2A (EBV-LMP2A) and evaluate its specific anti-tumor effects on nasopharyngeal carcinoma. Methods The gene fragment encoding EBV-LMP2A was amplified by PCR analysis and then subcloned into the shuttle vector p1565. PCR restriction enzyme digestion and DNA sequencing were performed to identify the recombinant shuttle vector p1565-LMP2A. The p1565-LMP2A vector was then transformed into competent strains of the attenuated Listeria monocytogenes (Lmdd). The recombinant attenuated Listeria vaccine strain Lmdd-LMP2A was verified by Western blot assay. The histological sections of spleen and liver tissues were stained by Haematoxylin and eosin (H&E) for analysis of inflammation. A tumor-bearing HLA-A2 transgenic mouse model was established by subcutaneous injection of CNE-1/HLA-A2/LMP2A nasopharyngeal carcinoma cell line. The prepared Lmdd-LMP2A vaccine was inoculated into the mice via tail intravenous injection for the evaluation of specific CTL induction and the in vivo anti-tumor effects. Results The shuttle vector p1565-LMP2A and the recombinant attenuated Listeria vaccine strain Lmdd-LMP2A with stable expression of LMP2A protein were successfully constructed. The immunized mice showed mild inflammations with no structural damage and necrosis as indicated by H&E staining. The growth of tumors in tumor-bearing HLA-A2 transgenic mice was significantly inhibited by the immunization of Lmdd-LMP2A vaccine as compared with mice without inoculation. The survival time of mice was prolonged with the immunization of Lmdd-LMP2A vaccine. Conclusion The prepared attenuated Listeria vaccine Lmdd-LMP2A showed specific anti-tumor effects with the safety advantage, suggesting the possibility of using it for anti-tumor therapy in clinic. Key words: Nasopharyngeal carcinoma; Eepstein-barr virus; Latent membrane protein 2A; Listeria monocytogenes; Tumor vaccine