Fig 1.
Vaccine Design and Immunization Schedule.
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.
Fig 2.
Hemagglutination Inhibition Assays after HA Vaccination.
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.
Fig 3.
Protection Against Lethal H1N1 Influenza Virus by a Centralized H1-Con Vaccine.
Mice were immunized and boosted with various doses of Ad4 and Ad5 expressing the H1-Con consensus HA gene. Four weeks post-boosting the mice were challenged with 100 MLD50 of mouse-adapted influenza A/Puerto Rico/8/34 (A), A/Fort Monmouth/1/47 (B), or A/WS/33 (C). The mice were monitored for weight loss, disease and survival. Mice that lost ≥25% of their baseline weight were humanely euthanized.
Fig 4.
Protection Against Lethal H3N1 and H3N2 Influenza Virus by a Centralized H3-Con Vaccine.
Mice were immunized and boosted with various doses of Ad4 and Ad5 expressing the H3-Con consensus HA gene. Four weeks post-boosting the mice were challenged with 100 MLD50 of mouse-adapted influenza A/Texas/1/77 (A) or 10 MLD50 of A/Mississippi/1/85 (B). The mice were monitored for weight loss, disease and survival. Mice that lost ≥25% of their baseline weight were humanely euthanized.
Fig 5.
Protection Against Lethal H5N1 Influenza Virus by a Centralized H5-Con Vaccine.
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.