Abstract
Hepatitis E virus (HEV) is the aetiological agent of enterically transmitted hepatitis. The traditional methods for evaluating neutralizing antibody titres against HEV are real-time PCR and the immunofluorescence foci assay (IFA), which are poorly repeatable and operationally complicated, factors that limit their applicability to high-throughput assays. In this study, we developed a novel high-throughput neutralizing assay based on biotin-conjugated p239 (HEV recombinant capsid proteins, a.a. 368–606) and staining with allophycocyanin-conjugated streptavidin (streptavidin APC) to amplify the fluorescence signal. A linear regression analysis indicated that there was a high degree of correlation between IFA and the novel assay. Using this method, we quantitatively evaluated the neutralization of sera from HEV-infected and vaccinated macaques. The anti-HEV IgG level had good concordance with the neutralizing titres of macaque sera. However, the neutralization titres of the sera were also influenced by anti-HEV IgM responses. Further analysis also indicated that, although vaccination with HEV vaccine stimulated higher anti-HEV IgG and neutralization titres than infection with HEV in macaques, the proportions of neutralizing antibodies in the infected macaques’ sera were higher than in the vaccinated macaques with the same anti-HEV IgG levels. Thus, the infection more efficiently stimulated neutralizing antibody responses.
Highlights
Hepatitis E virus (HEV) is a non-enveloped virus with a worldwide distribution and may cause severe acute hepatitis[1]
We developed a high-throughput method to quantitatively evaluate the neutralization of anti-HEV monoclonal antibodies and sera based on the fluorescence signal of conjugated p239 (HEV recombinant capsid particle, assembled from a.a. 368–606 of pORF2)[10] instead of unstable HEV virions11. p239 presented the immune-dominant neutralization epitopes as native HEV particles[10] and could be used as a surrogate to study the HEV neutralization and infection process[12,13]
The fluorescein isothiocyanate (FITC) signal was not sufficiently strong, which resulted in a FITC-p239 input that was greater than or equal to 16.6 μ g/mL (Supplementary Fig. 2a)
Summary
Hepatitis E virus (HEV) is a non-enveloped virus with a worldwide distribution and may cause severe acute hepatitis[1]. There was previously no easy, high-throughput method for the evaluation of anti-HEV neutralization. The neutralization assay based on real-time PCR calculates the quantities of virus by detecting RNA. Real-time RT-PCR is an unstable method for high-throughput detection. IFA ensures that neutralization post-attachment can be tested because only replicating virus is detected. It is time-consuming (taking approximately 7 days) and labor-intensive. We developed a high-throughput method to quantitatively evaluate the neutralization of anti-HEV monoclonal antibodies (mAbs) and sera based on the fluorescence signal of conjugated p239 (HEV recombinant capsid particle, assembled from a.a. 368–606 of pORF2)[10] instead of unstable HEV virions. This report presents an ideal alternative method for measuring neutralization capacity of sera that it is adapted to high-throughput technology
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