FIG. 2.

Ability of the trimeric HAs from mammalian or insect cells to elicit neutralizing antibodies: higher neutralizing antibody responses were elicited by oligomers than by trimers, and the titers increased with a repeat immunization. (A) The neutralization by antisera from five mice immunized with insect-expressed HAs prepared from insect cells was assessed by the incubation of mouse sera with HA/NA-pseudotyped lentiviral reporter vectors encoding luciferase. The percent neutralization was calculated by the reduction of luciferase activity relative to the values achieved in the absence of sera. Among the insect cell-expressed proteins, oligomer, uncleaved trimer, and cleaved trimer from the VN1203 strain all elicited potent neutralizing antibodies against KAN-1 HA/NA pseudovirus, while the cleaved trimer induced only modest neutralizing activity. (B) Mammalian high-molecular-mass oligomers induced the highest titer of neutralizing antibody response, followed by cleaved trimer, uncleaved trimer, and cleaved monomer. Statistically significant differences were observed between oligomer and trimer (P < 0.0001), oligomer and cleaved trimer (P < 0.0001), and oligomer and cleaved monomer (P < 0.0001) forms. (C) Mice received a single injection of mammalian cell-expressed oligomer or a secondary boost, as indicated, 3 weeks after the initial injection. The neutralizing antibody responses were measured in serum samples collected 14 days after each vaccination using the HA/NA-pseudotyped lentivirus reporter assay. All animals were immunized with 20 μg/injection of HA protein in 50 μl PBS and an equal volume of Ribi as the adjuvant. (D) Total HA antibodies in mice immunized with insect- or mammalian cell-expressed HA proteins were measured by ELISA. (E) Antisera from mice immunized with insect- or mammalian cell-expressed HA proteins were characterized for IgG1, IgG2a, and IgM responses. Similarly to mice immunized with inactivated H5N1 subvirion vaccine (inact. H5 vaccine), both insect- and mammalian cell-expressed oligomeric and trimeric HA induced the highest levels of IgG2a, followed by IgG1. Naïve mouse serum was used as a negative control. IgG3 and IgA also were tested but were negative for all sera (not shown).