Immunotherapy of Epstein-Barr Virus Associated Malignancies Using Mycobacterial HSP70 and LMP2A_356–364 Epitope Fusion Protein

Abstract

Epstein-Barr virus infection is strongly associated with a number of malignancies. The EBV latent membrane protein 2A has been implicated as one of the most attractive candidates for immunotherapy of related malignancies. In previous studies, the T cell epitopes of LMP2A have been identified systematically. However, the epitope-based vaccine generally meets inefficient immunogenicity when used in vivo directly, which could be overcome by combination with appropriate adjuvants. Heat shock protein is a natural chaperon, which is able to activate the classical major histocompatibility complex class I antigen-processing pathway (cross-presentation). In this study, a minigene encoding LMP2A_356–364 (FLYALALLL) was genetically fused to the carboxy-terminal of mycobacterial heat shock protein 70. The epitope fusion protein was expressed and purified, and the cross-presentation of LMP2A_356–364 by monocyte-derived dendritic cells pulsed with the epitope fusion protein was evaluated. Results showed that the epitope fusion protein-pulsed mDCs were much more efficient than the single peptide-pulsed mDCs on CTL activation. Immunization of HLA-A2.1 transgenic mice with MtHsp70-LMP2A_356–364 generated peptide specific CTL more effectively than a single peptide plus incomplete Freund's adjuvant (IFA). Growth of LMP2A expressing B16 melanoma tumor cells was suppressed in the vaccinated groups. Our results suggested that MtHsp70-LMP2A_356–364 fusion protein was more effective than the CD8₊ T cell epitope alone on anti-tumor immunity. As a result, the MtHsp70-LMP2A_356–364 fusion protein is considered to be a promising candidate vaccine for EBV related malignancies.

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Glossary

EBV

Epstein-Barr virus

LMP2A

latent membrane protein 2A

NPC

nasopharyngeal carcinoma

MtHsp70

mycobacterium tuberculosis heat shock protein 70

CTL

cytotoxic T lymphocyte

IFA

incomplete Freund's adjuvant

mDCs

Monocyte-derived dendritic cells

HSP

heat shock protein.