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

To determine the immunogenicity of MVTT HA-QH and MVTT HA-AH , two groups of mice were immunized twice with 3610 6 PFU of recombinant virus on day 0 and day 31, via the intranasal (I.N.) and intramuscular (I.M.) routes, respectively. Serum samples collected on day 0 and day 28 after the first immunization and 2 weeks after the second inoculation (day 45) were subjected to a neutralization (NT) assay by using a previously described pseudoviral assay [10] . We found that MVTT HA-QH was able to induce potent NT response against the autologous viral strain with a dilution factor of IC 50 over 1000 after the first immunization (Fig. 3A) . The I.N. route induced seemingly higher levels of NT responses than the I.M. route after the first immunization (IC 50 titer: 4076 vs. 1326). Moreover, the second immunization boosted primary responses by 2 (I.N.) to 8 (I.M.) fold for NT titers. Mice who received the control MVTT SIV did not generate any responses, as expected (Fig. 3A) . In contrast, after the 2 nd immunization, none of the mice immunized with MVTT HA-AH was able to induce any NT response against its homologous strain (Fig. 3B) . We, therefore, conducted a 3 rd immunization with the same dose of MVTT HA-AH three weeks after the 2 nd immunization, but only one in five mice was able to produce NT responses against its homologous strain (IC 50 titer: 70). However, MVTT HA-AH was able to induce potent NT responses with a dilution factor of IC 50 around 1000 after the 2nd immunization against the Qinghai strain (Fig. 3C) . Furthermore, compared to MVTT HA-QH , MVTT HA-AH induced seemingly lower levels of NT responses through the I.N. route than through the I.M. route after the 2nd immunization. The 3rd immunization boosted the primary responses for NT titers. These results suggest that immunization with MVTT HA-AH was successful, however, A/ Anhui/1/2005 is likely to be less sensitive to the neutralizing activity of vaccine-induced antibody. Moreover, HA from A/ Anhui/1/2005 is less antigenic compared to HA from the Qinghai strain.