Antibody fragments as a possible therapeutic treatment for infectious bronchitis in poultry

[摘要] Infectious bronchitis virus (IBV), a coronavirus, is the etiological agent for infectious bronchitis (IB), an acute respiratory disease of poultry. Infectious bronchitis is a notifiable disease, and taking in consideration that poultry is the second most consumed meat within South Africa, it highlights the importance of monitoring IBV outbreaks. Recombinant single chain variable fragments (scFv) have been used in therapeutic treatments for various human and veterinarian viruses, including other coronaviruses, such as SARS-CoV and MERS-CoV. The study aims to select scFv against the IBV antigen, with the use of phage display technology and commercial ELISA plates coated with the most prevalent IBV strains namely, H120 and M41. It has been proven that the S1 protein induces the binding of neutralising antibodies which provides protection against lethal CoV infections, thus, indicating a possible application for a therapeutic treatment. In this study, phage clones were selected from a human domain (dAb) library (Source BioScience, Australia). Panning was repeated three times using commercial ELISA plates coated with M41 and H120 IBV strains. Positive monoclonal phage clones were retrieved from the polyclonal mix by a sandwich ELISA and sequenced. The selected scFvs were expressed by Isopropyl β-D-1 thiogalactopyranoside induction and purified by immunoprecipitation with the Pierce Anti-c-Myc Agarose kit (Thermo Scientific, USA). After purification, binding ability of the scFvs were determined by means of a direct competitive ELISA. The neutralising ability of the scFvs was then determined by a virus neutralisation assay in ovo with the Avipro IBV H120 strain (Lohmann Animal Health Gmbh, Germany). This was performed in 9-day old SPF eggs over a time-period of six days. One set of eggs were injected with a dilution range of the IBV H120 strain and another set by a mixture of 2 μg/ml scFv with the IBV H120 strain. The end-point titres were determined and compared by the Spearman-Karber method (Spearman, 1908). Statistical analysis was performed using the student t-test with a p-value of 0.05. Round 1 of panning resulted in a total of 1.2 x 106 phages/ml and after round 3 a total of 3.0 x 1010 phages/ml were obtained. A total of 96 phage clones were manually selected from which only 12.5% showed a positive result during the sandwich ELISA. The 12 positive clones were sequenced and analysed based on nucleotide and amino acid composition. A total of five scFvs contained a complete variable heavy (VH) chain sequence, two of which was identical. This resulted in four unique and complete scFv sequences. These sequences showed a high variance in the nucleotide composition through- out the sequence. However, variation in the amino acid composition was only observed in the third complementary determining region. The scFvs were expressed and resulted in concentrations ranging from 204.38 μg/ml to 265.07 μg/ml. Detection of IBV antigen binding ability of the purified scFvs was conducted by a direct competitive ELISA. However, no statistical difference in absorbance values were observed, indicating insufficient binding of the scFvs. The in ovo virus neutralisation assay resulted in a one log reduction of the end-point titres. Statistical analysis proved one of the reductions to be statistically significant with a p- value less than 0.05, resulting in a partial neutralisation effect from the scFv. In conclusion, the selection process showed a progressive enrichment of antigen specific clones from the dAb library. The scFvs were successfully expressed, purified and characterised in terms of binding ability.

[发布机构] University of the Free State