A Replicating Modified Vaccinia Tiantan Strain Expressing an Avian-Derived Influenza H5N1 Hemagglutinin Induce Broadly Neutralizing Antibodies and Cross-Clade Protective Immunity in Mice

Xiao, Haixia; Liu, Li; Zhu, Qingyu; Tan, Zhiwu; Yu, Wenbo; Tang, Xian; Zhan, Dawei; Du, Yanhua; Wang, Haibo; Liu, Di; Li, Zhixin; Yuen, Kwok-Yung; Ho, David D.; Gao, George F.; Chen, Zhiwei

A modified shuttle vector pZCxz was constructed to target the H5N1 HA gene into the MVTT genome in the location of the MVTT HA gene. This shuttle vector contains dual promoters that allow the simultaneous expression of two target genes. Within this vector, the HA gene of A/BhG/QH/1/05 or A/Anhui/1/2005 was constructed under the strong synthetic promoter pSYN, whereas a reporter GFP or RFP gene was under a separate relatively weaker promoter pH 5 ( Fig.2A) . Since both genes were included within the same insertion frame, the reporter gene served as a surrogate marker for the selection of recombinant VTT carrying the HA gene. Using this technique, we were able to generate and to purify the recombinant virus MVTT HA-QH and MVTT HA-AH . The positive plaque was selected under a fluorescence microscope and subsequently confirmed by immunohistochemcal staining assay using a mouse anti-H5 serum (Fig. 2B) . To further confirm that the full length H5N1 H5 gene was inserted into the MVTT vector, the total DNA was extracted from Vero cells infected by either MVTT HA-QH or MVTT HA-AH . Total DNA was extracted from MVTT SIV infected Vero cells as negative control. The full-length H5N1 HA genes were amplified from the total DNA extracted from both MVTT HA-QH and MVTT HA-AH infected cells using specific primers, but not MVTT SIV infected Vero cells (Fig. 2C) . These results confirmed that we have successful constructed MVTT virus expressing H5 gene from A/ BhG/QH/1/05 and A/Anhui/1/2005 strains, respectively.