Background: Maternal-derived antibody (MDA) interferes with immune responses, leading to the failure of H9N2 avian influenza vaccinations in poultry. So far, none of the commercially available H9N2 avian influenza vaccines used in poultry have been able to overcome MDA interference.

Methods: To develop a vaccine that can overcome MDA interference, one or multiple copies of the minimum-binding domain (P29) from the complement protein C3d were inserted in between the signal peptide and the head domain of the hemagglutinin (HA) protein on a H9N2 avian influenza virus (A/Chicken/Shanghai/H514/2017, named H514).

Results: The HA proteins containing P29 stimulated stronger type I interferences than wild-type HA proteins in vitro. The modified viruses with the HA proteins containing one copy of P29 (rH514-P29.1) and two copies of P29.2 (rH514-P29.2) were successfully rescued using reverse genetics. The inactivated vaccines developed with rH514-P29.1 or rH514-P29.2 induced higher and faster humoral immune responses than the vaccine developed with rH514 in specific pathogen-free (SPF) chickens. To evaluate the vaccines´ efficacy in the presence of MDA and to ensure a uniform level of MDA, passively transferred antibody (PTA) was used as a model to mimic MDA in 1-day-old SPF chickens. Our results showed that the rH514-P29.2 inactivated vaccine induced significantly higher HI titers than the rH514 inactivated vaccine in the presence of PTA. More importantly, it reduced viral shedding after being challenged with H514 in the presence of PTA.

Conclusions: Our results suggest that vaccine antigens fused with two copies of P29 can decrease the interference of MDA on immunity in chickens. Overall, our results provide a new strategy for overcoming MDA interference.