Middle East respiratory coronavirus (MERS-CoV) spike (S) protein vesicular stomatitis virus pseudoparticle neutralization assays offer a reliable alternative to the conventional neutralization assay in human seroepidemiological studies

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel zoonotic coronavirus that was identified in 2012. MERS-CoV infection in humans can result in an acute, severe respiratory disease and in some cases multi-organ failure; the global mortality rate is approximately 35 %. The MERS-CoV spike (S) protein is a major target for neutralizing antibodies in infected patients. The MERS-CoV microneutralization test (MNt) is the gold standard method for demonstrating prior infection. However, this method requires the use of live MERS-CoV in biosafety level 3 (BSL-3) containment. The present work describes the generation and validation of S protein-bearing vesicular stomatitis virus (VSV) pseudotype particles (VSV-MERS-CoV-S) in which the VSV glycoprotein G gene has been replaced by the luciferase reporter gene, followed by the establishment of a pseudoparticle-based neutralization test to detect MERS-CoV neutralizing antibodies under BSL-2 conditions. Using a panel of human sera from confirmed MERS-CoV patients, the VSV-MERS-CoV particle neutralization assay produced results that were highly comparable to those of the microneutralization test using live MERS-CoV. The results suggest that the VSV-MERS-CoV-S pseudotype neutralization assay offers a highly specific, sensitive and safer alternative method to detect MERS-CoV neutralizing antibodies in human sera.

Keywords: Middle East respiratory syndrome coronavirus (MERS-CoV); luciferase; microneutralization test (MNt); neutralizing antibodies; pseudoparticle; vesicular stomatitis virus.

Fig. 1.

Generation and characterization of VSV-MERS-CoV-S pseudoparticles. (a) IFA analysis of the expression of MERS-CoV-S protein. HEK-293T cells were transiently transfected with pCAGGS-MERS-CoV-S plasmid DNA or mock transfected. The expression of MERS-CoV-S was detected by convalescent sera from a MERS-CoV patient with a known neutralizing titre against live virus as a positive control (MERS immune sera). (b, c) Titration of VSV-based pseudoparticles on Vero cells (b) and (c) BHK-21 cells. VSV pseudotyped with MERS-S (VSV-MERS-S) or negative control pCAGGS empty vector containing no glycoprotein (VSV-EV) was used to infect Vero cells. The results are expressed as the average relative luciferase units (RLU) ±standard deviation (sd). The error bars indicate the sd.

Fig. 2.

Neutralization of VSV-based pseudoparticles. (a) VSV-MERS-CoV-S pseudoparticles or (b) VSV-G pseudoparticles were preincubated with equal volumes of two fold serially diluted negative control pooled normal human serum (pNHS), positive control MERS-CoV immune serum or anti-VSV-G monoclonal antibody. The results are expressed as the average relative luciferase units (RLU) ±sd. The error bars indicate the sd.

Fig. 3.

Percentage neutralization of RLU of VSV-MERS-CoV-S pseudoparticles in comparison to pNHS. VSV-MERS-CoV-S pseudoparticles were preincubated with equal volumes of two fold serial dilutions of MERS-CoV immune serum and then inoculated onto Vero cells. The results are normalized to the negative control pNHS serum. The results are expressed as percentage neutralization ±sd. The error bars indicate sd.

Fig. 4.

Correlation analysis between VSV-MERS-CoV-S pseudoparticle and live virus neutralization tests. VSV-MERS-CoV-S pseudoparticles were preincubated with equal volumes of 52 human serum samples in two fold serial dilutions and then inoculated in triplicate onto Vero cells. (a) Pearson correlation analysis of anti-MERS-CoV neutralizing antibody titres measured both by MNt and VSV-MERS-CoV-S pseudoparticle assays. The results on the x-axis are expressed as the log₂ inverse dilution that resulted in 80 % or greater reduction in RLU as compared to the negative control pNHS serum. The results on the y-axis are expressed as the log₂ inverse dilution that blocked cell death by live MERS-CoV infection. The Pearson’s correlation coefficient (R) value and the P value are depicted for the comparison. (b) Bland–Altman method comparative analysis of anti-MERS-CoV neutralizing antibody titres measured both by the MNt and VSV-MERS-CoV-S pseudoparticle assays. The solid line represents the bias, while the dashed lines represent the upper and lower 95 % confidence limits.

Fig. 5.

Cross-neutralization of VSV-MERS-CoV-S by sera from patients with confirmed human coronavirus infections. VSV-MERS-CoV pseudoparticles were preincubated with equal volumes of two fold serially diluted MERS-CoV immune serum or antisera from the human coronaviruses 229E, HKU1, OC43, NL63 and SARS-CoV and then inoculated in triplicate onto Vero cells for each dilution of serum samples. The results are normalized to the negative control pNHS serum. The results are expressed as percentage neutralization and are the average of two independent experiments.