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

Sera from mice were treated with receptor-destroying enzyme (RDE, purchased from the Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention) in the ratio of 1:3 and heat-inactivated for 30 minutes at 56uC. Neutralization tests (NT) using the diverse H5N1 pseudoviruses was performed according to recently described methods [10] . Haemagglutination inhibition (HI) tests were performed with 1% chicken red blood cells according to a method recommended by the World Organization for Animal Health (http://www.oie.int/ Eng/Normes/Mmanual/A_00037.htm). Serum samples were also tested for neutralizing antibody against live A/BhG/QH1/05, A/ HA XJ /WSN and A/VN/1194/04 by a traditional micro-neutralization assay. Briefly, heat-inactivated sera were first two-fold diluted with DMEM, and then mixed with an equal volume of 100 TCID 50 of each of H5N1 influenza viruses. After an incubation period of 1 hour at 37uC, 200 ml of serum-virus mixtures were transferred onto MDCK cells in a 96-well plate, in triplicates. These cells were observed for any cytopathic effect (CPE), daily for 3 consecutive days. The titer of neutralizing antibody was defined as the highest serum dilution that inhibits the formation of 50% CPE. The cell-mediated immune response was determined by measuring H5-specific IFN-c released by splenocytes according to a previously published ELIspot assay [10] .