Development of a viral chemokine binding protein as a diagnostic tool for chemokine detection.

Abstract

Chemokines serve an integral role in the inflammatory response, specifically through the development of a chemotactic gradient which directs the trafficking of inflammatory leukocytes to the site of tissue damage or infection. Chemokines exert their effects through interactions with both chemokine receptors and glycosaminoglycans (GAGs). Targeting chemokines for therapeutic benefit has yielded limited success, likely due to the promiscuity of the system, where blocking individual chemokines does not necessarily inhibit chemotaxis. Alternately, the use of chemokines as diagnostics/prognostics for cancer, inflammatory disease, and autoimmune disease is a more promising area of research. The utilisation of chemokine targeting for both diagnostics and therapeutics has classically been undertaken utilising antibodies which are monospecific, hence issues may arise with regards to chemokine promiscuity. Viruses have developed numerous techniques to subvert the host inflammatory response, including the secretion of viral chemokine binding proteins (vCBPs). These proteins can exhibit binding specificity for multiple classes of chemokines, therefore offering a unique potential for use in clinical diagnostics. This study investigated the use of a vCBP derived from Orf virus stain NZ2, which binds across three classes of chemokines, as a diagnostic tool. The utility of CBP was investigated in assays for which antibodies are currently utilised, namely immunofluorescent cytology, western blotting, and enzyme-linked immunosorbent assays (ELISA). For use in immunofluorescent cytology, a protocol was developed to enable different levels of fluorophore conjugation to CBP. The fluorophore utilised was DyLight®594. Fluorophore conjugation to CBP was then assessed, indicating the extent of fluorophore conjugation did not impact binding to chemokines across different classes or species, including mouse CCL2 and CXCL2, and human CCL2 and CCL5. The ability of fluorophore-conjugated CBP to detect chemokines was then assessed in a cell-based inflammatory assay utilising the human monocyte cell line THP-1, expressing CCL2 and CCL5 in response to treatment with lipopolysaccharide (LPS). The ability of the fluorophore-conjugated CBP to detect chemokines in these cells was directly compared with a fluorophore-conjugated anti-CCL2 antibody using immuno-fluorescent microscopy. Whilst the antibody successfully detected CCL2 in activated THP-1 cells, the CBP exhibited reduced sensitivity and showed equivalent levels of detection irrespective of LPS stimulation. The specificity of chemokine binding exhibited by CBP and anti-CCL2 antibody was then assessed via pre-binding with soluble CCL2 and competitive inhibition with unlabelled CBP or antibody. Whilst chemokine binding by the fluorophore-conjugated antibody was affected following pre-binding and competition, neither had a substantial impact on CBP binding. The potential of a GAG-CBP interaction was then assessed through pre-binding with soluble heparin. The findings indicated that the observed THP-1/CBP interaction is mediated, at least in part, through cell-surface GAGs. Preliminary investigations into the use of fluorophore conjugated CBP in western blotting indicated that CBP is unable to detect denatured CCL2. The use of native CBP was also trialled within ELISA protocols, where CBP was shown to both detect capture-antibody bound CCL2, and when immobilised successfully captured CCL2 for antibody detection. This study suggests that fluorescent conjugation of CBP is easily achievable and does not impact protein function. Although CBP may not detect cellular chemokine expression, the findings here are the first to indicate that this specific CBP can bind heparin, and as such may have potential as a GAG targeting diagnostic. The use of the native CBP in ELISA protocols was also well supported, but its utility across a range of chemokine ELISA platforms needs to be further evaluated.