The development of a multiplexed Soluble Phage Array (SPAr) for the detection of zoonotic pathogens

Serosurveillance is a valuable approach to disease monitoring in animal populations to facilitate effective intervention and containment strategies. It has been suggested that effective serosurveillance of zoonotic diseases could provide an early-warning system for the potential emergence of a zoonotic pathogen in human populations. However, to date, serosurveillance is somewhat limited by a single-pathogen focus and lacks the ability to distinguish between very closely related pathogens. Therefore, there is a need for an affordable, accessible, multiplexed approach with very high specificity to monitor for a wide range of established and emerging pathogens.
The huge diversity of phage display peptide libraries combined with the screening power of next generation sequencing (NGS) in a process termed next generation phage display (NGPD) can be applied to discover large panels of peptide mimotopes recognised by antibody responses to disease. We propose that a phage-peptide sub-library enriched to contain multiple infection-specific mimotopes can itself be used as the assay 'antigen' to diagnose infection with a particular pathogen; an assay format we have termed Soluble Phage Array (SPAr). This assay would measure recognition of the panel of infection-specific phage-displayed mimotopes by antibodies in serum samples by NGS analysis of phage that are bound by the antibodies.
We hypothesise that NGPD can be used to develop SPAr assays that can accurately identify infection with zoonotic pathogens and that these can be multiplexed with high accuracy and reproducibility.
This proof-of-concept study will focus on four emergent zoonotic viruses that affect sheep or horses. These are louping ill (LIV), tick-borne encephalitis (TBEV) and Rift Valley fever (RVFV) viruses in sheep, and West Nile virus (WNV) in horses. The selected viruses represent the genera Flavivirus (LIV, TBEV and WNV) and Phlebovirus (RVFV). These viruses have been carefully chosen to represent the complexity of serosurveillance of emerging viral zoonoses in order to demonstrate the potential of the SPAr assay.
A multiplexed SPAr assay would have considerable advantages over existing serosurveillance approaches. It would not be limited to a single-pathogen approach and would have the ability to distinguish very closely related pathogens, a property vital to inform effective control strategies for zoonotic pathogens.

Phage display peptide libraries, where a short peptide is displayed on the surface of a bacteriophage, have been extensively used to epitope map monoclonal antibodies. Our previous work has combined the huge diversity of phage display peptide libraries with the screening power of next generation sequencing (NGS) in a process termed next generation phage display (NGPD) and applied this to discover large panels of peptide mimotopes recognised by antibody responses to infection. This has also included the identification of panels of peptides that can differentiate very closely related immune responses, for instance differentiating between infection with Salmonella enterica and vaccination with an attenuated or a killed vaccine of the same bacterium.
We have also generated preliminary data demonstrating that a phage-peptide sub-library enriched to contain multiple disease-specific peptides can itself be used as the assay 'antigen' in a format we have termed Soluble Phage Array (SPAr). This quantification of antibody recognition of a wide range of disease-specific phage-peptides from the polyclonal mix of phage using NGS as the assay readout produces an immunosignature analogous to that obtained with synthetic peptide solid-surface arrays. Importantly, the soluble phage arrays can be applied at a fraction of the cost, are readily scalable and should be highly reproducible. As the SPAr approach uses around 1 million phage as input, and within this only tens to hundreds of specific peptides form the assay for a particular infection, it therefore presents an ideal opportunity for the multiplexing of assays to numerous distinct infections. The proposed study would for the first time develop SPAr assays for infectious diseases and also look to multiplex the immunosignatures specific for a range of zoonotic pathogens into a single SPAr assay. This proof-of-concept study aims to develop a multiplexed SPAr assay to four zoonotic viral infections of sheep and horses.