Table 1.
Design of study 1.
Table 2.
Design of study 2.
Fig 1.
The ISG and IFN responses in chickens vaccinated with LAIV or IIV at 1 day of age.
(A) Trachea tissue at 1 dpv. (B) Trachea tissue at 3 dpv. (C) Spleen tissue at 1 dpv. (D) Spleen tissue at 3 dpv. The bars represent Log2 fold change in transcription level of ISGs and IFN genes. Error bars represent mean±SD (n = 5 birds per group). Asterisks without horizontal bar indicate significant differences compared with unvaccinated group. Horizontal bar with asterisks indicates significant difference between LAIV and IIV (*p<0.05, **p<0.01). dpv = days post-vaccination.
Fig 2.
Serum HI antibody response in chickens vaccinated at 1 day of age.
(A) Homologous (TK/OR/71, H7N3) HI titers. (B) Heterologous (CK/NJ/02(H7N2)) HI titers. Sera were collected from birds vaccinated at 1 day of age at 14 days post-vaccination. Individual and median HI titers are indicated with symbols and horizontal lines, respectively. Different letters inside the plot indicate statistical significance among groups (p<0.05).
Fig 3.
Mucosal antibody responses in tears of 2 week-old chickens.
Birds were vaccinated at 1 day of age and tear samples collected at 14 days post-vaccination for antibody detection. (A) Total antibody against NP antigen. (B) Influenza virus-specific IgG. (C) Influenza virus-specific IgA. The NPRI and OD values shown on the y-axis were normalized to the mean value of the unvaccinated group. The asterisks indicate significant differences compared with unvaccinated group (*p<0.05, **p<0.01, and *** p<0.001).
Table 3.
The effect of antisera production method on the percent antigenic relatedness value between TK/OR/71 (H7N3) and CK/NJ/02 (H7N2) antigens.
Fig 4.
Replication level of heterologous challenge virus.
Birds were vaccinated at 1 day of age and challenged with heterologous virus (CK/NJ/02 (H7N2)) at 2 weeks post-vaccination. Following challenge, full tracheas were collected and washed with 2 ml of PBS and the replication level of challenge virus was determined by qRT-PCR. Virus titers are expressed as median egg infectious doses per ml of tracheal wash supernatant. (A) 3 days post-challenge. (B) 5 days post-challenge. Different letters inside the plot indicate statistical significance among groups (p<0.05).
Fig 5.
Serum antibody response to homologous (TK/OR/71 (H7N3)) and heterologous (CK/NJ/02 (H7N2)) strains at 4 and 5 weeks of age.
(A) Homologous HI titers at 4 weeks of age. (B) Homologous HI titers at 5 weeks of age. (C) Heterologous HI titers at 4 weeks of age. (D) Heterologous HI titers at 5 weeks of age. Four weeks of age corresponded with 4 wpv for 1d IIV and 1d LAIV; 1 wpv for 3w LAIV and 3w IIV. Five weeks of age corresponded with 5 wpv for 1d IIV and 1d LAIV; 2 wpv for 3w LAIV and 3w IIV. The arrangement of groups highlights the synergy of LAIV priming and IIV boosting—LAIV-IIV is placed in the middle while other LAIV and IIV vaccinations are placed on the left and right, respectively. The individual and median HI titers are illustrated as symbols and horizontal lines, respectively. Different letters inside the plot indicate statistical significance among groups (p<0.05).
Fig 6.
Differences in hemagglutination inhibition (HI) titers between homologous and heterologous strains.
The bars and lines represent the average and standard deviation of Log2 HI titers at 5 weeks of age (5 wpv for 1d IIV and 1d LAIV; 2 wpv for 3w LAIV and 3w IIV). Numbers indicated above the bars and lines show average HI titer differences obtained by subtracting heterologous titers from homologous titers. Groups are arranged as described in Fig 5.
Fig 7.
Mucosal antibody responses in tears of 5 weeks-old chickens.
Birds were vaccinated at 1 day (1d) or 3 weeks (3w) of age. Tear samples were collected at 5 weeks of age (5 wpv for 1d IIV and 1d LAIV; 2 wpv for 3w LAIV and 3w IIV) to measure antibody levels. (A) Total antibody against NP antigen. (B) Influenza virus-specific IgG. (C) Influenza virus-specific IgA. The NPRI and OD values shown on the y-axis were normalized to the mean value of the unvaccinated group. Groups are arranged as described in Fig 5. Different letters inside the plot indicate significant differences between groups (p<0.05).
Fig 8.
Reduction in heterologous challenge virus replication in vaccinated birds.
At 5 weeks of age (5 wpv for 1d IIV and 1d LAIV; 2 wpv for 3w LAIV and 3w IIV), chickens were challenged with heterologous virus and tracheal swabs were taken at 2 and 4 days post challenge to determine the level of challenge virus replication. Each swab was eluted in 2 ml of PBS. Virus titers are expressed as median egg infectious doses per ml of tracheal swab eluate. (A) 2 days post-challenge. (B) 4 days post-challenge. Groups are arranged as described in Fig 5. Different letters inside the plot indicate significant differences between groups (p<0.05).
Fig 9.
Effect of age and prime-boost vaccination on avidity index of serum HI antibodies.
Serum samples collected at 2 or 5 weeks post vaccination at 1 day of age or 2 weeks post boost-vaccination (5 weeks of age) were tested for their avidity as previously described [43]. (A) Age effect. (B) Prime-boost effect. Groups are arranged as described in Fig 5. Different letters inside the plot indicate significant differences between groups (p<0.05).