Development of antibody therapeutic approaches for poultry diseases using avian influenza as a disease model
One of the main threats to poultry is avian influenza virus (AIV), causing significant economic losses worldwide and threatening human populations due to its zoonotic potential. To reduce disease impact, vaccination of poultry is carried out; however, most of the vaccines are insufficient to induce sterile immunity, leading to enzootic disease prevalence worldwide. In addition, due to virus evolution, new virus variants continuously arise, further compromising vaccine effectiveness. The aim of this study was to assess if monoclonal antibodies could be used as prophylactic treatment to reduce avian influenza disease impact as an alternative to vaccination in emergency situations, and to investigate approaches which could be employed for delivery of antibodies as antiviral therapeutics for poultry. A panel of monoclonal antibodies, specific to the AIV H9N2 subtype (A/chicken/Pakistan/UDL-01/2008) (UDL-1/08) major antigenic surface glycoprotein hemagglutinin (HA), generated from mouse hybridomas, were gene sequenced for their variable domains that were subsequently used for recombinant antibody production in cultured cell supernatants. Functional activities (HA binding affinity and AIV neutralizing activity) of the recombinant antibodies were evaluated against homologous and heterologous viruses. Three antibodies retained functional activity matching that of the natural antibody isotype after conversion to single chain variable fragment (scFv) format, suggesting the antibody fragment crystallizable (Fc) region did not mediate function for these antibodies, but that function was dependent on direct antigen recognition. Next, scFv antibodies were chosen for passive immunization purposes in vivo due to their small molecule size and potentially reduced immunogenicity. scFvs were administered to birds intranasally 24 h before challenge with H9N2 AIV representative UDL-1/08 and treatment was continued for seven days post-infection. Results indicated reduced morbidity and virus shedding in treated birds. Moreover, compared to non-treated birds, treated chickens also produced lower levels of IL-6, a known pro-inflammatory cytokine induced in response to virus infection. This data suggests treated birds experienced overall reduced impact of disease. Nevertheless, like in vivo vaccine induced antibodies, the antibody treatment also provoked the virus to generate HA antibody escape mutants likely to overcome the neutralizing activity of therapeutic antibodies. Finally, this study investigated if herpesvirus of turkeys (HVT), which is used as a viral vaccine vector in poultry, could act as a vector for therapeutic antibody delivery to poultry. A recombinant virus encoding a transgene of a broadly AIV-neutralizing antibody was generated using a CRISPR/Cas9 approach. It was found that antibody gene insertion into HVT altered recombinant virus growth kinetics, resulting in reduced replication when compared to a wildtype virus control. Antibody levels secreted in rHVT infected cell culture supernatant were retained after 20 virus passages. Next, to investigate antibody expression and tolerability in vivo, rHVT was administered to day-old birds; however, no detectable systemic antibody circulation was identified throughout 42-days post rHVT delivery. Instead, an anti-antibody response was generated, suggesting only a low level of expression occurred that was sufficient to act as an antigen. Taken together, this work has built a proof-of-concept suggesting that passive immunization for poultry can reduce weight loss in infected birds and overall disease burden, but selection of antibodies targeting different antigens and epitopes is crucial to avoid virus escape mutant formation. For the first time it was also demonstrated that HVT can act as an efficient vector for antigen but not antibody delivery. This information can be relevant not only to AIV but also other pathogens affecting poultry.