Construction of recombinant influenza virus carrying human metapneumovirus epitopes

Abstract

Objective To construct and rescue recombinant influenza virus strains expressing human metapneumovirus (hMPV) epitopes. Methods B cell, CTL and Th epitopes predicted by bioinformatics software were coupled together in different combinations. These different array genes were inserted into the NS1 gene of influenza virus strain A/PR/8/34(PR8), respectively. Recombinant PR8 influenza virus vectors expressing different hMPV antigenic epitopes were rescued by reverse genetics using eight-plasmid system. Sequencing analysis was conducted to verify whether the rescued viruses carried the chimeric hMPV epitopes. Hemagglutination (HA) titers, half tissue culture infection dose (TCID50) and growth curves were detected. Results Interval sequences GPGPG and KK were introduced into hMPV epitope combinations to construct multi-epitope antigens (MEA). These MEA were inserted into the PR8 NS gene, respectively. Using 8 plasmid system, three recombinant influenza virus strains were rescued successfully. After cultured for three passages in Madin-Darby canine kidney (MDCK) cells and one in eggs, these three recombinant strains could proliferate steadily. Whole genome sequencing verified that the three recombinant strains carried the chimeric MEA sequences, named as rFLU/hMPV/B, rFLU/hMPV/CTL-Th and rFLU/hMPV/B-Th. HA titers of the recombinant strains were 128, 128 and 256 using turkey erythrocyte, respectively. Their TCID50 were 107.0/ml, 106.8/ml and 107.0/ml, respectively. Growth curve tests also verified that the recombinant strains could proliferate steadily in MDCK cells. Conclusions Three recombinant influenza virus vector strains carrying the B cell, CTL and Th epitopes of hMPV were rescued successfully. This study lays the foundation for further evaluation of the immune effects of these recombinant viruses and their potential application value in vaccine development. Key words: Human metapneumovirus; Influenza virus vector; Virus rescue; Antigenic epitope