Hepatitis E Virus Fluorescence Reduction Neutralization Test for Measuring Neutralizing Antibody Titers

Summary

There is no standard virus neutralization assay available for the Hepatitis E Virus (HEV), a non-cytopathic positive-strand RNA virus. Here,  a fluorescence reduction neutralization test (FRNT)-based on quantification of fluorescent labeled viral capsid protein is described for evaluating the efficacy of anti-HEV antibodies. 

Abstract

Virus neutralization assay measures the neutralization potential of an antibody or a mixture of antibodies against the corresponding virus. It is a valuable technique to evaluate the efficacy of vaccine candidates and neutralizing antibodies developed against the virus. The plaque reduction neutralization test (PRNT) is a popular method to evaluate the titer of virus-specific neutralizing antibodies. However, PRNT is not feasible in the case of non-cytopathic viruses. Hepatitis E virus (HEV) is a positive-sense single-stranded RNA virus, belonging to the family Hepeviridae. It is a major cause of acute viral hepatitis worldwide. It does not show any cytopathic effect in mammalian cell culture. A vaccine against HEV is available in China. However, vaccines or therapeutic antibodies against HEV are not available in most of the world. Several laboratories have been working on vaccine candidates against the HEV. A neutralization assay to measure the efficacy of HEV vaccines will be a very useful tool for researchers. Here, we describe a fluorescence reduction neutralization test (FRNT) to estimate the 50% neutralization titre (NT₅₀) of anti-HEV antibodies. Anti-HEV ORF2-neutralized, or rabbit IgG-neutralized HEV, was used to infect Huh7 cells, followed by immunofluorescent staining of the viral ORF2 protein. Fluorescent positive cells were quantified in the ImageJ application, and NT₅₀ was estimated to be 2.1 x 10³. This method may be applied to estimate the NT₅₀ of anti-HEV antibodies present in immunized-mice sera, HEV-recovered individual sera, and laboratory-produced anti-HEV antibodies.

Introduction

Hepatitis E virus (HEV) is a positive-sense single-stranded RNA virus that belongs to the Hepeviridae family^1,2. It usually causes self-limiting acute hepatitis but can cause chronic hepatitis in immunosuppressed individuals^3,4. Pregnant women are at high risk of developing fulminant hepatic failure, with the mortality rate increasing up to 30%⁵. The World Health Organization (WHO) reported about 19.47 million cases of acute hepatitis E (AHE) and 3450 deaths caused by HEV globally in 2021⁶. HEV i....

Protocol

Institutional ethics committee (IEC) approval was obtained to collect a fecal sample from an HEV-infected patient admitted to the Department of Gastroenterology, AIIMS, New Delhi. Informed consent was obtained from the patients participating in the study. The details of the reagents and the equipment used are listed in the Table of Materials.

1. Virus isolation and quantification

1. With informed consent, obtain the fecal sample from an HEV-infected patient for purification of the virus.
2. Resuspend the faecal sample in Dulbecco’s Phosphate buffered saline (DPBS) at 10% final concentr....

Results

Infection of Huh7 cells with g1-HEV and virus characterization

The g1-HEV was isolated from a faecal sample using a series of steps, as illustrated (Figure 1A). Genome copy number of the purified g1-HEV was estimated by RT-qPCR, followed by storage at -80 °C. To confirm viral infection and replication, Huh7 cells were infected with 1.8 × 10⁴ genome copy equivalents of the purified g1-HEV stock for 72 h, followed by RT-qPCR and IFA. Sign.......

Discussion

FRNT is an immunostaining-based method for estimating the neutralizing potential of antibodies. It has been used to estimate NT₅₀ values for antibodies against multiple viruses, including Dengue virus (DENV), Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), and Japanese encephalitis virus (JEV)^23,24,25. In this method, the virus is incubated with a test antibody (or sera), followed by infection of culture.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL microfuge tubes	Axygen	MCT-150-C
12-well cell culture dish	Corning	3512
37 degree incubator shaker	Thermo Scientific	50163013
4′,6-diamidino-2-phenylindole	Abcam	AB104139	DAPI
50mL centrifuge tube	Falcon	352070
6-well cell culture dish	Corning	3516
Alexa flour 594 secondary IgG Antibody	Thermo Scientific	A-11012
Anti-ORF2 antibody	Genscript	NA	Custom synthesized by Genscript and characterized in our laboratory14
Anti-rabbit IgG antibody	Genscript	NA
Bovine Serum Albumin	HIMEDIA	MB083	BSA
Confocal microscope	Olympus	FV3000
DMEM, high glucose	HIMEDIA	AL007A
Fetal Bovine Serum	GIBCO	16000-044	FBS
ImageJ 1.54g	ImageJ.org (Wayne Rasband and contributors, NIH, USA)
Normal Goat Serum	HIMEDIA	RM-10701
Paraformaldehyde	HIMEDIA	GRN3660	PFA
Phosphate Buffer Saline pH 7.4	HIMEDIA	TL1006	PBS
Polyethyleneglycol 6000	SIGMA	81255	PEG6000
SOLIScript 1-step Probe Kit	SOLIS BIODYNE	08-57-00250
TRI reagent	Molecular Research Centre	TR118
Triton X100	SIGMA	X100
Trypsin-EDTA	GIBCO	25300-54