FIG 3.

Immunization of mice with the hyperglycosylated HA induces high titers of stalk-directed antibodies against distinct seasonal H1N1 and pandemic H5N1 viruses. Sera originating from mice vaccinated with the wild-type or hyperglycosylated HAs were assayed by ELISA for reactivity against purified viruses: A/Puerto Rico/8/34, cH5/1, A/USSR/92/77, A/Brisbane/59/07, and A/Vietnam/1203/04. Sera originating from mice vaccinated with the hyperglycosylated HA were less reactive to virus containing the full-length PR/8 HA compared to mice vaccinated with the wild-type HA after two (A) and three (B) immunizations. In contrast, seroreactivity to a virus bearing the cH5/1 HA, which consists of a subtype H5 head domain and the PR/8 stalk domain, was higher for sera originating from mice vaccinated with the hyperglycosylated HA than in mice vaccinated with the wild-type HA. This difference in reactivity was observed after two immunizations (C) and became more apparent after three immunizations (D). The seroreactivity to distinct H1N1 viruses (E, F) and an H5N1 virus (G) was also enhanced in mice vaccinated with the hyperglycosylated HA compared to that in mice immunized with the wild-type HA.