Development of a rapid neutralization testing system for Rhinovirus C15 based on the enzyme-linked immunospot assay

Abstract

Human Rhinoviruses (RVs) are dominant pathogens causing a wide range of respiratory tract diseases, posing a huge threat to public health worldwide. Viruses belonging to the RV-C species are more likely to cause severe illnesses and are strongly associated with asthma onset or exacerbations than RV-A or RV-B. Rapid and sensitive detection of neutralizing antibodies (NAbs) against RV-C can promote the development of vaccines and antiviral drugs and help in the diagnosis of viral infection. In this study, a rapid neutralization testing system for RV-C15, based on an enzyme-linked immunospot assay (Nt-ELISPOT) was developed. A monoclonal antibody (MAb), named 9F9, with high binding efficacy for RV-C15 conjugated to horseradish peroxidase (HRP), was used to detect RV-C15-infected cells at a concentration of 2 μg/ml. The optimal infectious dose of RV-C15 was set at 1 × 104 TCID50/well and the cells were fixed with 0.5% formaldehyde diluted in PBS after incubation for 20 h. Compared with the traditional cytopathic effect (CPE)-based neutralization assay (Nt-CPE), Nt-ELISPOT significantly shortened the detection period and showed good consistency with the detection of neutralizing titers of both sera and NAbs. Using Nt-ELISPOT, three anti-RV-C15 NAbs were obtained with IC50 values of 0.16, 0.27, and 11.8 μg/ml, respectively. Moreover, 64 human serum samples collected from a wide range of age groups were tested for NAb against RV-C15 by Nt-ELISPOT. The total seroprevalence was 48.4% (31/64) and the positive rate was lowest in the group under 6 years old. Thus, the Nt-ELISPOT established in this study can be used as a high-throughput and rapid neutralization assay for the screening of NAbs and for seroepidemiological investigation against RV-C15.

Rhinoviruses A and C elicit long‐lasting antibody responses with limited cross‐neutralization

“…Total immunoglobulin G responses to synthetic RV-C peptides were measured in several prior studies, 15,16 and assays for detection of nAbs to RV-C have recently been developed. 17,18 We reported that nAbs to RV-C in children develop earlier and are two to three times more prevalent than nAbs to RV-A throughout childhood. 17…”

“…Discovered in 2006, the RV‐C species is classified into 57 types based on the sequence identity thresholds in the VP1 capsid gene, 14 but there is limited information about their serological relationships. Total immunoglobulin G responses to synthetic RV‐C peptides were measured in several prior studies, 15,16 and assays for detection of nAbs to RV‐C have recently been developed 17,18 . We reported that nAbs to RV‐C in children develop earlier and are two to three times more prevalent than nAbs to RV‐A throughout childhood 17 .…”