Hemagglutination Inhibition Assays after HA Vaccination.

<p>The HI titers were determined in mice immunized with Ad vaccines expressing either the H1-Con, H3-Con or H5-Con consensus HA gene. Ad vaccines were given at 3 different doses. The HI titers induced by the lowest dose of vaccine, 1 X 10⁷ vp/mouse are shown (A). A low intermediate dose vaccine of 5 X 10⁷ vp/mouse is shown (B). A high dose vaccine of 1 X 10¹⁰ vp/mouse is shown (C). Mice immunized with Ad vaccines expressing the H1-Con gene were assayed for HI antibodies against influenza A/Puerto Rico/8/34 (H1N1), A/Fort Monmouth/1/47 (H1N1) and A/California/04/09 (H1N1). Mice immunized with Ad vaccines expressing the H3-Con gene were assayed for HI antibodies against influenza A/Texas/1/77 (H3N1), A/Mississippi/1/85 (H3N2) and A/Aichi/2/68 (H3N2). Mice immunized with Ad vaccines expressing the H5-Con gene were assayed for HI antibodies against influenza A/Vietnam/1203/04 (H5N1), A/Japanese White Eye/Hong Kong/1038/2006 (H5N1) and A/BH Goose/Qinghai/A/05 (H5N1). The dotted line represents a theoretical titer indicative of protection.</p

Protection Against Lethal H5N1 Influenza Virus by a Centralized H5-Con Vaccine.

<p>Mice were immunized and boosted with various doses of Ad4 and Ad5 expressing the H5-Con consensus HA gene. Four weeks post-boosting the mice were challenged with 100 MLD₅₀ of mouse-adapted influenza A/Vietnam/1203/04 (A), A/BH Goose/Qinghai/A/05 (B) and A/Japanese White Eye/Hong Kong/1038/2006 (C). The mice were monitored for weight loss, disease and survival. Mice that lost ≥25% of their baseline weight were humanely euthanized.</p

Vaccine Design and Immunization Schedule.

<p>A schematic representation of the Ad4 shuttle plasmid is shown (A). The shuttle plasmid containing the CMV-HA-PolyA expression cassette is shown (B). The two Adenoviral types and the location of the HA expression transgenes used as vaccine vectors are shown (C). The Ad4 vaccine had an E1 deletion whereas the Ad5 vaccine had E1 and E3 deletions. The HA transgene expression cassette was recombined in place of the E1 genes in both vaccine vectors. Mice were immunized with the vaccine vectors using three different doses (D). The mice were primed with the Ad4 vaccine vector and boosted 4 weeks later with the Ad5 vaccine vector. Test bleeds were taken 2 weeks post-boosting and the mice were challenged with lethal doses of influenza 4 weeks post-boosting. The mice were monitored for weight loss, disease and death. Mice that lost ≥25% of the day 0 body weight were humanely euthanized.</p

Genetic stability of NA substitutions among recombinant influenza A and B viruses after three subsequent passages in MDCK cells.

<p>Genetic stability of NA substitutions among recombinant influenza A and B viruses after three subsequent passages in MDCK cells.</p

NAI susceptibility profiles for recombinant influenza A and B viruses carrying a single NA substitution.

<p>NAI susceptibility profiles for recombinant influenza A and B viruses carrying a single NA substitution.</p

The NA thermostability of the recombinant influenza A and B viruses carrying a single NA substitution.

<p>Influenza A/PR8 (A, B) and B/YAM (C, D) viruses were incubated for 15 or 30 min at the indicated temperatures. Residual NA enzyme activity was assayed and is presented as a percentage of the NA activity at 4°C (unheated). The bars represent the mean of the relative NA activity ± SD for different temperatures from three experiments. *<i>P</i> < 0.05.</p

Replication kinetics in MDCK cells of recombinant influenza A and B viruses carrying a single NA amino acid substitution.

<p>MDCK cells were infected with recombinant influenza A and B viruses at an MOI of 1 (A, C) or 0.001 (B, D). Virus titers were determined by TCID₅₀ assays in MDCK cells after incubation for 72 h at 35°C (for influenza A viruses) or 33°C (for influenza B viruses). Each data point represents the mean virus titer (log₁₀TCID₅₀/mL) ± the SD from three independent experiments. *<i>P</i> < 0.05.</p

NA activity and NA enzyme kinetics of recombinant influenza A and B viruses.

<p>NA activity and NA enzyme kinetics of recombinant influenza A and B viruses.</p

HA activation pH values for swine H1N1, H1N2, and H3N2 influenza viruses.

<p>Each data point represents the pH of activation of an individual virus, as measured by syncytium assay with 2 to 4 duplicates. Activation pH values of prepandemic (<2009) swine H1 viruses were previously reported [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006276#ppat.1006276.ref039" target="_blank">39</a>] and are used here for comparison. A list of contemporary swine H1 and H3 viruses is given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006276#ppat.1006276.s007" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006276#ppat.1006276.s008" target="_blank">S2</a> Tables.</p

H1N1 influenza viruses varying widely in hemagglutinin stability transmit efficiently from swine to swine and to ferrets

<div><p>A pandemic-capable influenza virus requires a hemagglutinin (HA) surface glycoprotein that is immunologically unseen by most people and is capable of supporting replication and transmission in humans. HA stabilization has been linked to 2009 pH1N1 pandemic potential in humans and H5N1 airborne transmissibility in the ferret model. Swine have served as an intermediate host for zoonotic influenza viruses, yet the evolutionary pressure exerted by this host on HA stability was unknown. For over 70 contemporary swine H1 and H3 isolates, we measured HA activation pH to range from pH 5.1 to 5.9 for H1 viruses and pH 5.3 to 5.8 for H3 viruses. Thus, contemporary swine isolates vary widely in HA stability, having values favored by both avian (pH >5.5) and human and ferret (pH ≤5.5) species. Using an early 2009 pandemic H1N1 (pH1N1) virus backbone, we generated three viruses differing by one HA residue that only altered HA stability: WT (pH 5.5), HA1-Y17H (pH 6.0), and HA2-R106K (pH 5.3). All three replicated in pigs and transmitted from pig-to-pig and pig-to-ferret. WT and R106 viruses maintained HA genotype and phenotype after transmission. Y17H (pH 6.0) acquired HA mutations that stabilized the HA protein to pH 5.8 after transmission to pigs and 5.5 after transmission to ferrets. Overall, we found swine support a broad range of HA activation pH for contact transmission and many recent swine H1N1 and H3N2 isolates have stabilized (human-like) HA proteins. This constitutes a heightened pandemic risk and underscores the importance of ongoing surveillance and control efforts for swine viruses.</p></div