FIG 4.

Protective activity of stalk-reactive antibodies. (A) All participants with an 8-fold or higher induction (n = 27) of stalk-reactive antibody titers 42 days postpriming were selected. Their day 0 and day 42 sera were pooled and intraperitoneally injected into mice. After 2 h, mice were challenged intranasally with cH9/1 N3 virus. Lungs of 5 mice for each group were harvested 3 days and 6 days after challenge. (B) An ELISA against the H9 head domain confirmed that there was no significant increase in antibodies against the H9 head domain 42 days postpriming (1:1,131 on day 0 versus 1:1,275 on day 42; P = 0.4901). (C) Virus titers in the lung were assessed in a plaque assay 3 days and 6 days postchallenge. Virus titers in the lungs of mice that received postvaccination sera were significantly lower on day 3 postchallenge (9.6-fold difference; P = 0.0036). On day 6 after virus challenge, 3 of the mice that received postvaccination sera had lung virus titers below the detection limit of 10 PFU per ml; the other 2 showed titers of 0.5 × 10², which was significantly lower than the mean titer of 1.3 × 10⁴ for the mice that received prevaccination sera (P = 0.0243). (D) Mice that received postvaccination sera had significantly lower weight loss 3 days postchallenge (1.6% versus 3.7% weight loss; P = 0.0144) and even regained weight 6 days postchallenge, while mice that received prevaccination sera continued to lose weight (4.6% weight loss). (E) The neutralizing activity of stalk-reactive antibodies for the sera used in the passive-transfer experiment was assessed in a cH9/1 N3 microneutralization assay. The titer 42 days postpriming was significantly higher, at 1:1,225, than that on day 0, 1:582 (P = 0.0022).