Plates were washed 3 times with serum-free MEM. later. Sera were collected 21 days after the boost and tested by ELISA against PR8 virus. Sera from mice that were boosted with PR8 or S12a had significantly higher PR8 titers than mice that were boosted with unfavorable controls PBS or B/Lee (p 0.05 Students t test of area under the curve). (C) Mice were immunized i.p. with 1000 HAU PR8 and then boosted i.v. with 1000 HAU PR8, S12a, J1, or A/California/7/2009 (CAL/09). Sera were collected 14 days later and HAI assays were completed with PR8. Data are mean +/- SEM. N = 4 mice per group. (* p 0.05 Students t test; ns = not significantly different)(EPS) ppat.1005806.s002.eps (1.4M) GUID:?A141449C-1B4B-4B88-9899-F21BCFF9F42A S2 Fig: S12a elicits protective Abs in mice pre-exposed to PR8. Sera were collected from naive mice, mice immunized with only Immethridine hydrobromide S12a, mice immunized sequentially with PR8 and S12a, and mice immunized sequentially with PR8 and B/Lee. Sera were collected 28 days after the last viral exposure. Sera were passively transferred into naive mice (25ul sera per mouse). 12 hours later, these mice were infected with 30 TCID50 S12a and weight loss was measured for 14 days. Mice that received sera from S12a and PR8-S12a immunized mice lost significantly less weight from days 6C14 post-infection compared to mice that received sera from naive or PR8-B/Lee immunized H3/l mice (p 0.05; Two way ANOVA). Shown are mean and SEM. n = 4 animals/group. Data are representative of 2 impartial experiments.(EPS) ppat.1005806.s003.eps (778K) GUID:?F06D72E2-1E2E-4431-9885-1584688CE140 S3 Fig: Hybridomas for these experiments were derived from many mice. Shown are the fraction of mAbs that were derived from individual mice. (A) 28 mAbs were isolated from mice exposed to PR8. (B) 125 mAbs were isolated from mice exposed to S12a. (C) 50 mAbs were isolated from mice sequentially exposed to PR8 twice. (D) 86 mAbs were isolated from mice sequentially exposed to PR8 and S12a. We did not use many mice for PR8 primary exposure since previous studies have characterized mAbs isolated from mice following a single PR8 exposure.(EPS) ppat.1005806.s004.eps (1.0M) GUID:?DF43D600-4412-4B45-BBC4-4B14A82F8A21 S4 Fig: Most cross-reactive mAbs bind better to PR8 compared to S12a. mAbs were tested by ELISA for reactivity against PR8, S12a, and J1 virus. (A-C) Shown are 3 mAbs that bound with a higher relative affinity to PR8 than to S12a. (D) As an ELISA coating control, we used the stalk-specific C179 mAb that bound with comparable affinities to both PR8 and S12a.(EPS) ppat.1005806.s005.eps (1.2M) GUID:?E74E5A42-5B82-413E-90D6-475A27CB7D6D S5 Fig: OAS mAbs bind very poorly to S12a. (A-C) mAbs were tested by ELISA for reactivity against PR8, S12a, and J1 virus. Shown are Immethridine hydrobromide three mAbs elicited in mice that received a PR8-S12a prime-boost vaccination that had measurable binding to PR8 but not to S12a. As an ELISA coating control, we used the stalk-specific C179 mAb that bound with comparable affinities to both PR8 and S12a (example shown in S4D Fig).(EPS) ppat.1005806.s006.eps (888K) GUID:?53E07F01-10CE-47C1-9AFB-5A9E8FEE8E0F S6 Fig: The HA Ab response is focused around the Sb antigenic site following sequential exposure. (A,B) Hybridomas derived from mice that were vaccinated with PR8 virus and boosted 28 days later with either PR8 (A) or S12a (B) were Immethridine hydrobromide predominantly specific for the Sb antigenic site. (C,D)This was in contrast to hybridomas derived from mice that were immunized only with PR8 (C) or S12a (D) (p 0.05; Fishers exact test).(EPS).