Robust in vitro assay for analyzing the neutralization activity of serum specimens against hepatitis B virus

Ya-Li Zhang; Ying Gao; Jia-Li Cao; Jing-Hua Zhao; Tian-Ying Zhang; Chuan-Lai Yang; Hua-Long Xiong; Ying-Bin Wang; Shan-Hai Ou; Tong Cheng; Chang-Rong Chen; Quan Yuan; Ning-Shao Xia

doi:10.1080/22221751.2019.1619485

Robust in vitro assay for analyzing the neutralization activity of serum specimens against hepatitis B virus

Emerg Microbes Infect. 2019;8(1):724-733. doi: 10.1080/22221751.2019.1619485.

Authors

Ya-Li Zhang^{1

2}, Ying Gao^{1

3}, Jia-Li Cao^{1

2}, Jing-Hua Zhao^{2

4}, Tian-Ying Zhang^{1

2}, Chuan-Lai Yang^{1

2}, Hua-Long Xiong^{1

2}, Ying-Bin Wang^{1

2}, Shan-Hai Ou⁵, Tong Cheng^{1

2}, Chang-Rong Chen⁶, Quan Yuan^{1

2}, Ning-Shao Xia^{1

2}

Affiliations

¹ a State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics , School of Public Health, Xiamen University , Xiamen , People's Republic of China.
² b School of Life Science , National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University , Xiamen , People's Republic of China.
³ d Hainan Health Disseminate Centre , Haikou , People's Republic of China.
⁴ e Natural Medicine Institute of Zhejiang Yangshengtang , Hangzhou , People's Republic of China.
⁵ c Xiamen Blood Service , Xiamen , People's Republic of China.
⁶ f Xiamen Haicang Hospital , Xiamen , People's Republic of China.

Abstract

Anti-HBs is a well-known marker of protective capability against HBV. However, little is known about the association between the qAnti-HBs determined by immunoassays and the neutralization activity (NAT) derived from functional assays. We developed an in vitro assay for direct measurement of the NAT of human sera. The new assay was highly sensitive, with an analytical sensitivity of 9.6 ± 1.3 mIU/mL for the HBIG standard. For serum detection, the maximum fold dilution required to produce ≥50% inhibition (MDF₅₀) of HBV infection was used as the quantitative index. In vitro NAT evaluations were conducted for a cohort of 164 HBV-free healthy individuals. The results demonstrated that the NAT positively correlated with the qAnti-HBs (R² = 0.473, p < 0.001). ROC analysis indicated that the optimal cutoff value of the qAnti-HBs to discriminate significant NAT (MDF₅₀ ≥ 8) was 62.9 mIU/mL, with an AUROC of 0.920. Additionally, we found that the qAnti-HBc was another independent parameter positively associated with the NAT (R² = 0.300, p < 0.001), which suggested that antibodies against other HBV proteins generated by previous HBV exposure possibly also contribute to the NAT. In summary, the new cell-based assay provides a robust tool to analyse the anti-HBV NAT. Abbreviations: HBV: Hepatitis B virus; HBsAg: Hepatitis B surface antigen; Anti-HBs: Hepatitis B surface antibody; HBeAg: Hepatitis B e antigen; Anti-HBc: Hepatitis B core antibody; qAnti-HBs: quantitative hepatitis B surface antibody; qAnti-HBc: quantitative hepatitis B core antibody; qHBeAg: quantitative hepatitis B e antigen; NAT: neutralization activity; HBIG: hepatitis B immune globulin; NTCP: Na⁺-taurocholate cotransporting polypeptide; IRES: internal ribosome entry site; ccHBV: cell culture derived hepatitis B virus; GE/cell: genome equivalent per cell; MOI: multiplicity of infection; Dpi: day post infection; HepG2-TetOn: a HepG2-derived cell line that expresses the doxycycline-regulated transactivator; ROC: receiver operating characteristic curve; AUROC: area under receiver operating characteristic curve; LLOQ: the lower limits of quantification; MDF₅₀: the maximum fold dilution required to produce ≥50% inhibition; IC50: half maximal inhibitory concentration.

Keywords: Hepatitis B virus; cell-based assay for neutralization activity against HBV; hepatitis B core antibody; hepatitis B neutralizing antibodies; hepatitis B surface antibody.

Publication types

Evaluation Study

MeSH terms

Antibodies, Neutralizing / blood*
Hep G2 Cells
Hepatitis B Antibodies / blood*
Hepatitis B virus / immunology*
Humans
Neutralization Tests / methods*
ROC Curve
Sensitivity and Specificity
Serum / immunology*

Substances

Antibodies, Neutralizing
Hepatitis B Antibodies

Grants and funding

This work was supported by the National Natural Science Foundation of China [grant number 81672023, 81871316] (Q.Y.), Young Researchers Foundation of the Health and Family Planning Commission of Fujian Province [grant numbet 2015-2-58] (Y.L.Z.), and Medical Innovation Grant of Fujian Province [grant number 2016-CXB-15] (S.H.O.). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.