Dose response of PIV5-H7.

<p>BALB/c mice in groups of 15 were infected IN with PIV5-H7 at a dose of 10⁴, 10⁵ and 10⁶ PFU, or PBS or intramuscularly injected with 256 HAU of iH7N9. Mice were rested for 3 weeks then challenged with 10 LD₅₀ of A/Anhui/1/2013 (H7N9) and monitored for (A) survival and (B) weight loss. (<i>P</i><0.05, ANOVA, *compared to PBS on days 2–8; †compared to PBS on days 2, 6, and 8; ^‡compared to PBS on day 8 (10⁴ only) (C) Titers of H7N9 in the lungs of mice Day 3 post H7N9 challenge. (<i>P</i><0.05, Kruskal-Wallis)</p

PIV5-H7 and PIV5-NP protected mice against H7N9 challenge.

<p>BALB/c mice in groups of 9–10 were infected IN with PIV5-H7 at a dose of 10⁶ PFU, 10⁶ PFU of PIV5-NP, 10⁶ PFU of PIV5, or PBS. Mice were rested for 8 weeks and were then challenged with 10 50% lethal doses (LD₅₀) of A/Anhui/1/2013 (H7N9) and monitored for (A) survival and (B) weight loss. (*<i>P</i><0.05, log-Rank, compared to PBS or PIV5)</p

Antibody responses in immunized mice.

<p>96-well plates were coated with inactivated H7N9 and incubated overnight. After blocking, serial dilutions of serum, nasal wash or bronchoalveolar lavage (BAL) samples were added to the coated plates. After wash, alkaline phosphatase-labeled goat anti-mouse IgG (A-C) or IgA (D, E) were added and plates were developed using phosphatase substrates. Optical density (OD) was measured at 405 nm on a Bio-Tek Powerwave XS plate reader.</p

Generation and analysis of PIV5 expressing HA of H7N9 (PIV5-H7).

<p>(A) Schematic of PIV5-H7. The PIV5 genome contains seven known transcriptional units and transcribes eight known viral mRNAs. The V and P mRNAs both originate from the same V/P gene by a RNA editing process called pseudo-templated transcription. Leader and trailer sequences are important for viral RNA synthesis and transcription initiation. (B) Expression of H7 in PIV5-H7-infected cells. MDBK cells were mock infected or infected with PIV5-H7. At 2 dpi, the cells were fixed and stained with anti-PIV5-V/P or anti-H7N9 serum. (C) Growth of PIV5-H7. MDBK cells were infected with PIV5 or PIV5-H7 at a moi of 0.1. The media were collected at the indicated intervals and plaque assays were performed on BHK cells.</p

Protection of guinea pigs against H7N9 challenge.

<p>(A) Scheme of immunization and infection. Guinea pigs were immunized IN with PIV5 or PIV5-H7 (10⁷ PFU), or PIV5-H7 + PIV5-NP (5x10⁶ PFU of each). Six guinea pigs were immunized IM with 512 HAUs of iH7N9. At 21 days post immunization (dpi), guinea pigs were bled and HAI titers were measured. The guinea pigs were then infected IN with 10 ID₅₀ of A/Anhui/1/13 (H7N9). One dpi, one naïve guinea pig was co-housed with each infected guinea pig in a single cage. Nasal washes were obtained from guinea pigs at day 2, 4, 6 and 8 after challenge (day post-challenge). Titers of H7N9 in nasal washes were determined by TCID₅₀ assay. (B—E) Nasal wash titers of individual animals. (F) Mean nasal wash titers (+ SEM). (*<i>P</i><0.05 compared to PIV5-vaccinated; Kruskal-Wallis test) (G) Hemagglutination inhibition titers of individual vaccinated animals. (*P < 0.05; Kruskal-Wallis test) Order and shading of bars matches individual guinea pigs for panels B—E, Symbols match for panels F and G.</p

Protection of anti-H7 serum antibodies.

<p>Sera from PIV5-H5 or PIV5-H7 immunized guinea pigs were pooled and assayed for HAI titers. 200 μl of anti-H7 (HAI titer of 80) diluted 1:2 or naïve serum was injected IP into naïve mice and the mice were challenged at 1 day post-injection with 10 LD₅₀ of H7N9. 200 μl of anti-H5 serum (HAI titer of 80) serially diluted, 1:8, 1:16, and 1:32 was injected IP into mice and the mice were challenged with 10 LD₅₀ of H5N1 the next day. (A) Mortality of H7N9 challenge after antibody transfer. (B) Weight loss after H7N9 challenge. (C) Virus titers in the lungs of mice at 3 days after H7N9 challenge. (D) Mortality of H5N1 challenge after antibody transfer.</p