Figure 6.

Vaccine-induced NA-specific antibodies mediate protection against homologous influenza infection, inhibit NA enzymatic activity, and block viral replication

(A–D) BALB/c mice were immunized once i.m./EP with 50 μg each of A and B plasmids (100 μg total) as indicated. (A) Pooled sera (350 μL) from mice 5 weeks after vaccination were transferred i.p. to naive BALB/c mice. One day after transfer, mice were challenged with 2.5× LD₅₀ H1N1 PR8 virus. (B) 2 weeks after vaccination with the indicated DNA vaccines, mice were challenged with 5× LD₅₀ H1N1 PR8 virus. Starting on day 12 after vaccination, mice were treated with anti-CD4 and anti-CD8 mAbs or isotype-matched control mAbs every second day. n = 8 mice/group; weight (left, mean ± SEM) and survival (right) were monitored up to 12 days post challenge, and dead mice were assigned a weight of 75% (A and B). (C) Neutralization of influenza A H1N1 PR8 virus by serum from vaccinated mice (box) obtained 4 weeks after immunization was measured by microneutralization assay and is shown as IC₅₀. (D) The same sera were tested for ability to inhibit NA enzyme activity by ELLA, using a reassorted influenza A H6N1 virus containing NA from H1N1 PR8. IC₅₀ is indicated. (E and F) BALB/c mice were immunized once i.m./EP with 50 μg of each A and B plasmids (100 μg total, E) or 5 μg of each A and B plasmids (10 μg total, F) as indicated. Mice were challenged with homologous H1N1 PR8 virus 6 weeks after vaccination, and lungs were harvested on day 5 post challenge. Viral loads were determined by qRT-PCR (left), showing gene copy number per microgram total tissue RNA, and by measuring in vitro cell culture infectivity (right), presented as 50% tissue culture infectious dose (TCID₅₀) per milliliter of lung homogenates. Individual mice and mean ± SEM are shown; n = 4–6 mice/group (C–F). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; two-way ANOVA (weight curve), Mantel-Cox test (survival), Kruskal-Wallis multiple-comparisons test with Dunn’s correction (C and D, TCID₅₀), or multiple-comparisons one-way ANOVA with Tukey’s correction (copy number).