Functional characterization of antibodies against rhinoviral 3C protease

Abstract

Rhinovirus (RV) is the most frequent cause of upper respiratory tract illness in humans and the causal agent of common cold, which is the most common human disease. In addition, rhinovirus is associated with acute otitis media (ear infection), and asthma exacerbation in children. Most children under three years of age are infected with one of the rhinovirus types for one to four times per year. Due to high genetic variation, rhinoviruses are classified into three different species: RV-A, RV-B and RV-C. Currently 169 virus types have so far been recognized, which makes the development of diagnostic tools challenging. As of now, no commercial antibodies against rhinoviruses are available, which would recognize a broad range of virus types and which would thus enable a development of a point-of-care assay for rhinovirus detection. The aim of this study was to characterize antibodies that bind to rhinoviral 3C protease. 3C protease is highly conserved between virus types making it a potentially good broad range target in diagnostics.

In total, six different antibody forms were analysed: one phage display Fab antibody (Fab-2D02), three rat monoclonal IgG antibodies (ratmab-3, ratmab-16 and ratmab-43), and two polyclonal antisera produced in rabbits (Pab-9282-1 and Pab-9312-2). Functional characterization of the antibodies was done using indirect enzyme-linked immunosorbent assay (ELISA), Western blot, and Immunofluorescence assay (IFA). The antibodies were first tested using a recombinant (bacterial) 3C protease, and then with virus infected samples assuming they contain virally-expressed 3C protein, and finally with samples transfected with a plasmid encoding 3C protease.

All the antibodies recognized the purified 3C protease in indirect ELISA and Western blot. As for the virus infected samples, two of the antibodies, one of the rat monoclonal antibodies (ratmab-4), and one of the polyclonal antisera (Pab-9282-1) recognized the 3C protease in Western blot, but no notable signal was detected in IFA with any of the antibodies. With transfected samples, ratmab-4 and Pab-9282-1 recognized the 3C protease in Western blot. In IFA, Fab-2D02, ratmab-4, ratmab-43 and Pab-9282-1, recognized the 3C protease from the transfected samples. Fab-2D02 produced the lowest background out of all antibodies in IFA. The differences in antibody functions are possibly due to insufficient amounts of the target protein in the samples, poor affinity of the antibodies, and/or different conformation of the virus-produced 3C compared to bacterial 3C protease or target peptide used in antibody generation. In conclusion, ratmab-4 and Pab-9282-1 bound to all the different forms of 3C protease used in this study and should be subject to further evaluation when optimized samples for 3C protease are available. Even though no signal was received for viral samples using Fab-2D02, it showed to be the most specific and sensitive against recombinant 3C protease. Its functionality could be increased by IgG conversion.