Avian influenza vaccination in chickens and pigs with replication-competent adenovirus-free human recombinant adenovirus 5

Abstract

Protective immunity to avian influenza (AI) virus can be elicited in chickens by in ovo or intramuscular vaccination with replication-competent adenovirus (RCA)-free human recombinant adenovirus serotype 5 (Ad5) encoding AI virus H5 (AdTW68.H5) or H7 (AdCN94.H7) hemagglutinins. We evaluated bivalent in ovo vaccination with AdTW68.H5 and AdCN94.H7 and determined that vaccinated chickens developed robust hemagglutination inhibition (HI) antibody levels to both H5 and H7 AI strains. Additionally, we evaluated immune responses of 1-day-old chickens vaccinated via spray with AdCN94.H7. These birds showed increased immunoglobulin A responses in lachrymal fluids and increased interleukin-6 expression in Harderian gland-derived lymphocytes. However, specific HI antibodies were not detected in the sera of these birds. Because pigs might play a role as a "mixing vessel" for the generation of pandemic influenza viruses we explored the use of RCA-free adenovirus technology to immunize pigs against AI virus. Weanling piglets vaccinated intramuscularly with a single dose of RCA-free AdTW68.H5 developed strong systemic antibody responses 3 wk postvaccination. Intranasal application of AdTW68.H5 in piglets resulted in reduced vaccine coverage, i.e., 33% of pigs (2/6) developed an antibody response, but serum antibody levels in those successfully immunized animals were similar to intramuscularly vaccinated animals.

Fig. 1

In ovo vaccination (day 18 of embryonation) with AdTW68.H5 and AdChNY.H7 elicited serum HI antibody responses against both expressed AI HAs. (A) HI (H5) antibodies (mean ± SD) to A/tk/WI/68 (H5N9) in chickens (n = 12) vaccinated with AdTW68.H5 alone (200 µl/egg containing 5.0 × 10⁹ ifu) or in chickens (n = 24) vaccinated with AdTW68.H5 (same dose as above) in combination with AdCN94.H7 (200 µl/egg containing 5.0 × 10⁷ ifu). (B) HI (H7) antibodies (mean ± SD) to A/Tk/OR/71 (H7N3) in chickens (n = 12) vaccinated with AdCN94.H7 alone or in chickens (n = 24) vaccinated with AdTW68.H5 in combination with AdCN94.H7 (same doses as above). All control birds produced no measurable AI antibody titers.

Fig. 2

ELISA IgA levels in chickens vaccinated with AdCN94.H7 by spray at 1 day of age using a commercial spray cabinet. Group “Single dose” (n = 15) was sprayed once with 8 ml of virus suspension containing 1.1 × 10¹⁰ ifu/ml. Group “Primed & booster” (n = 15) received three times the dose of group A (24 ml) and a boost (8 ml) on day 16 of age. Spray-vaccinated chickens showed higher specific IgA levels in tear fluids on day 10 of age (9 days postvaccination). IgA levels declined with time in singly vaccinated chickens. Chickens receiving a booster vaccination on day 16 of age showed an increase of specific IgA on day 30 of age. Unvaccinated controls (n = 15) were negative for specific IgA throughout the experimental period.

Fig. 3

IFN-γ and IL-6 RNA by RT-PCR on lymphocytes derived from the HGs 4 days after spray booster vaccination with AdCN94.H7. IFN-γ was detected in both controls (n = 2) and vaccinated birds without significant differences. Chickens initially receiving a low (n = 4) or high (n = 4) dose of vaccine displayed similar levels of IL-6 in the HG as determined by ImageQuant analysis. IL-6 was not detected in unvaccinated control chickens. β-actin gene was used as the housekeeping gene.

Fig. 4

Serum H5 antibodies induced by RCA-free recombinant Ad-vector encoding AI H5 (AdTW68.H5) vaccination in pure-breed Yorkshire piglet weaners. (A) Intramuscular vaccination (n = 6) performed at 5 wk of age with 1 ml of virus suspension containing 10⁹ ifu of AdTW68.H5 in the neck muscle (~70 mm behind the base of the ear). (B) Intranasal vaccination (n = 6) performed at 5 and 7 wk of age with 1 ml of virus suspension containing 10¹⁰ ifu of AdTW68.H5 in each nostril. HI antibody titers were measured in chickens on day 7 and at 7 day intervals through 42 days of age. All naïve control piglets (n = 4) produced no measurable AI antibody titers.

Fig. 5

Serum ELISA antibodies in Yorkshire piglet weaners. (A) H5 antibodies in pigs (n = 6) subjected to intranasal vaccination at 5 and 7 wk of age with 1 ml of virus suspension containing 10¹⁰ ifu of AdTW68.H5 in each nostril. Positive control serum (+) is a pool of sera from two pigs vaccinated intramuscularly in experiment 3. Negative control (−) is the mean OD value of unvaccinated controls. The baseline was established from the mean of unvaccinated controls plus three times their SD. (B) Antibodies reacting with Ad5 in each group of pigs prenasal (Pre-N) and pre-intramuscular (Pre-IM) immunizations: unvaccinated controls (n = 4), Pre-N (n = 6), and Pre-IM (n = 6). Two piglets within the Pre-N group showed ELISA antibody levels higher than the mean + 3 SD of all other animals.

Fig. 6

Specific antibody levels determined by ELISA in Yorkshire piglet weaners vaccinated intranasally with AdTW68.H5 at 5 and 7 wk of age. (A) IgA in individual unvaccinated controls (n = 4) in tear fluid (Ctr-Tear) or nasal secretion (Ctr-N) and in individual intranasally vaccinated piglets (N-Tear; N-N) on week 10. (B) IgA in tear or nasal secretions on week 11. Baselines obtained from the mean OD values of unvaccinated controls plus three times the SD. IgA OD values in tear and nasal secretions were low compared to values for serum IgG (Fig. 5). Even though a few animals show OD values above the baseline, only one pig on week 11 seem to have developed a significant IgA response.