Avian Influenza virus M2e protein: Epitope mapping, competitive ELISA and phage displayed scFv for DIVA in H5N1 serosurveillance

Abstract

Within the avian influenza virus (AIV) history, H5N1 subtype is the most alarming in terms of its spread rate throughout the globe with its demonstrated unusual pattern of evolution. Persistency and constant circulation of this subtype in poultry population in a number of countries have resulted its establishment and declaration as enzootic. The affected countries are commonly characterised by high poultry populations and productions. They are also developing countries which have minimal funding allocated for precaution on disease incursion. Past observations showed that a single AIV epizootic is capable of causing significant economic burden throughout the world. Although epizootic, it still resulted sporadic cases of human infection and mortality. Therefore, H5N1 enzootic countries opt for vaccination strategy (usually with inactivated whole virus) to evade AIV incursions. However, this interferes with the AIV surveillance effort. This is due to the lack of diagnostic tool with the ability to differentiate AIV infected animal from vaccinated animal (DIVA). Following this realisation, several options are made available. Diagnostic tool development which is capable of DIVA requires a highly sensitive and specific target which at the same time is economic, and pose ease of application. In recent years, growing interest on the AIV matrix 2 extracellular domain (M2e) protein has propelled its exploration as the target for AIV serosurveillance diagnostic tool development. It has been demonstrated to be highly sensitive and specific in detection for AIV infection in an indirect enzyme-linked immunosorbent assay (ELISA) setting. The factor which made it highly interesting is its ability for DIVA application. M2e protein can only be found in low concentration on an AIV particle which is used in an inactivated vaccination strategy, while present in high concentration if cells are AIV infected. Therefore, this study has further explores the AIV M2e protein potential for AIV serosurveillance diagnostic tool development and successfully demonstrated an M2e-based test in a competitive ELISA format for DIVA. This particular ELISA format was of interest as it can be potentially used in multiple species application, as AIV is a multispecies pathogen. To ensure the universality of the competitor antibody, comparative mapping of anti-M2e antibodies from chicken, mouse and rabbit was done. Findings highlighted slight variations in the epitope identified for the M2e antigen by antibodies from different species. Mouse anti-M2e antibodies are more suitable to be used as the competitor antibodies against anti-M2e chicken sera in the M2e-based competitive ELISA test. Consequently, application of the mouse anti-M2e antibodies in the M2e-based competitive ELISA has demonstrated specific and sensitive indication of AIV infection in the H5N1 challenged chicken sera. Biotechnology developments has also introduced the single chain variable fragment (scFv) antibodies as specific and stable bait for antibodies detection against targeted pathogen’s protein (antigen). Taking advantage of this knowledge, this study has also successfully isolated reactive and specific anti-M2e scFv antibodies from avian sources. This is critical as an avian sourced antibodies to be used as bait for the targeted pathogen’s protein is highly relevant in the setting for AIV serosurveillance application in the poultry industry. These findings are significant in the effort to provide a highly sensitive and specific diagnostic tool, which are also cost effective, easy to apply with high throughput ability. Such ideal diagnostic tool for AIV serosurveillance is highly valuable, as this may hold the key to break the AIV continuous circulation.