Characterization of novel antibodies against human rhinoviruses
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Human rhinoviruses (RV) are the most common cause of the common cold. Most of the upper respiratory tract infections are caused by RVs. Although the symptoms of RV infections are typically mild including rhinorrhea and pharyngitis, they can also lead to more severe complications. Increasing evidence suggests that RV infections during the first year of life can lead to the development of asthma. In addition, the exacerbations of chronic lung illnesses, such as asthma or chronic obstructive pulmonary disease, are strongly associated with RV infections. Despite the overall prevalence and the significant impact on economics by direct and indirect costs of respiratory infections, RVs are highly under-diagnosed. This is partially related to the lack of antibodies available for rapid immunodiagnostics.
Rhinoviruses are small, positive-stranded RNA viruses, which are classified by their genetic features into three species: RV-A, RV-B and RV-C, and in total these species include 169 rhinovirus types. Due to high antigenic variation of RV types, there are currently no conserved antibodies available for immunodiagnostics. The aim of this study is to examine the ability of novel antibodies to recognize a highly conserved rhinoviral protein to facilitate the development of specific point-of-care immunoassay for the detection of RV infections. In addition, such antibodies could be used in studies related to RV life cycle and potentiate antiviral development.
Three monoclonal antibodies (mAbs) and two sets of polyclonal antisera (pAbs) were tested against specific RV protein and conserved RV peptide using enzyme-linked immunosorbent assay (ELISA). Monoclonal cell lines were cultured, and antibodies were purified in TuProtCore using ProtG columns. Two of the tested polyclonal antisera were designed to recognize the RV protein (PRO-pAbs) while four of them were designed to recognize the conserved peptide in the target protein (PEP-pAbs). Different RV subtypes were inoculated to HeLa cells and virus replication was verified by RT-qPCR from cell lysates. Virus samples from infected cells were also used as antigens in ELISA to determine whether mAbs or pAbs could recognize the target protein from native RVs. Immunofluorescence assay (IFA) was also used to analyze antibody functionality in RV infection.
The monoclonal crude antibodies were collected and tested successfully in ELISA assay. However, further purification in TuProtCore failed which is why prior purified antibody preparations were mostly used during this study. All three purified monoclonal antibodies recognized both RV protein and RV peptide in ELISA assay. In contrast, the results from ELISA for pAbs were somewhat disparate. One of the pAbs targeting RV protein could not recognize neither of the targets while the other PRO-pAb was able to recognize RV protein. Three of the PEP-pAbs could recognize both RV protein and the RV peptide indicating different binding sites. One of the PEP-pAbs did not recognize either of the targets. The establishment of virus infection in HeLa cells proved to be cumbersome. While virus replication was detectable using RT-qPCR, no viral protein was detected in ELISA or in IFA assays.
This study confirmed that antibodies developed against conserved viral protein or amino acid motif within, detected its target specifically and with high sensitivity indicating that the 3C protein could potentially be used as the target protein in a point-of-care immunoassay development. Further analysis indicated that while RV infection in HeLa cells was successful, target protein was not detected in cell lysates or using IFA. This suggests either low detection limit, possibly due to low target protein production during infection and/or wrong timing of sample collection, or low sensitivity of antibodies. Further analysis is required to determine whether these antibodies could recognize their target from clinical samples.