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A safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERS-CoV - PubMed

  • ️Tue Jan 01 2013

A safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERS-CoV

Guangyu Zhao et al. Virol J. 2013.

Abstract

Background: Evidence points to the emergence of a novel human coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), which causes a severe acute respiratory syndrome (SARS)-like disease. In response, the development of effective vaccines and therapeutics remains a clinical priority. To accomplish this, it is necessary to evaluate neutralizing antibodies and screen for MERS-CoV entry inhibitors.

Methods: In this study, we produced a pseudovirus bearing the full-length spike (S) protein of MERS-CoV in the Env-defective, luciferase-expressing HIV-1 backbone. We then established a pseudovirus-based inhibition assay to detect neutralizing antibodies and anti-MERS-CoV entry inhibitors.

Results: Our results demonstrated that the generated MERS-CoV pseudovirus allows for single-cycle infection of a variety of cells expressing dipeptidyl peptidase-4 (DPP4), the confirmed receptor for MERS-CoV. Consistent with the results from a live MERS-CoV-based inhibition assay, the antisera of mice vaccinated with a recombinant protein containing receptor-binding domain (RBD, residues 377-662) of MERS-CoV S fused with Fc of human IgG exhibited neutralizing antibody response against infection of MERS-CoV pseudovirus. Furthermore, one small molecule HIV entry inhibitor targeting gp41 (ADS-J1) and the 3-hydroxyphthalic anhydride-modified human serum albumin (HP-HSA) could significantly inhibit MERS-CoV pseudovirus infection.

Conclusion: Taken together, the established MERS-CoV inhibition assay is a safe and convenient pseudovirus-based alternative to BSL-3 live-virus restrictions and can be used to rapidly screen MERS-CoV entry inhibitors, as well as evaluate vaccine-induced neutralizing antibodies against the highly pathogenic MERS-CoV.

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Figures

Figure 1
Figure 1

Detection of MERS-CoV pseudovirus infectivity. Cell tropism of MERS-CoV pseudovirus in a variety of target cells from human and non-human hosts. VSV-G and Env- pseudoviruses were used as positive and negative controls, respectively. The data are expressed as mean relative luciferase units (RLU) ± standard deviation (SD) of 4 parallel wells in 96-well culture plates. The experiment was repeated three times, and similar results were obtained.

Figure 2
Figure 2

Detection of MERS-CoV S and HIV-1 p24 protein expression in the packaged MERS-CoV pseudovirus and DPP4 protein expression in target cells by Western blot. (A) Detection of p24 and MERS-CoV S in pseudotyped MERS-CoV. Anti-p24 (1:50) and MERS-CoV S-specific polyclonal antibodies (1:200) were used for the test. (B-C) Detection of DPP4 expression in different cell lines. Goat anti-DPP4 (1 μg/ml) was used for the test, and anti-β-actin monoclonal antibodies (1:5,000, Sigma) were applied as the internal control.

Figure 3
Figure 3

Detection of neutralizing antibodies of vaccinated sera against MERS-CoV infection. Mice were vaccinated with a recombinant protein expressing RBD of MERS-CoV S protein, and representative sera (Sera 1–3) collected from 10 days post-2nd vaccine were used for the test. Control sera were collected from mice injected with PBS. (A) MERS-CoV pseudovirus-based inhibition assay in DPP4-expreesing Huh-7 cells. The data are presented as mean percentages of inhibition ± SD of duplicate wells. (B) MERS-CoV live virus-based inhibition assay in Vero E6 cells. The titers were determined as the highest serum dilutions that completely prevent CPE in at least 50% of the wells (NT50) and are expressed as mean ± SD. The experiment was repeated three times, and similar results were obtained.

Figure 4
Figure 4

Detection of inhibitory ability of synthetic compounds against MERS-CoV pseudovirus infection. Compounds (HP-HSA and ADS-J1) and peptides (C34 and T20) were tested for the inhibition of MERS-CoV pseudovirus entry into target NBL-7 (A) and Huh-7 cells (B) at concentrations of 20 and 2.5 μM, respectively. VSV-G pseudotype was included as the negative control. (3) Detection of the potential cytotoxicity of the compounds to Huh-7 cells. The data are presented as mean percentages of inhibition (or cytotoxicity) ± SD of duplicate wells. The experiment was repeated three times, and similar results were obtained.

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References

    1. Chan JF, Li KS, To KK, Cheng VC, Chen H, Yuen KY. Is the discovery of the novel human betacoronavirus 2c EMC/2012 (HCoV-EMC) the beginning of another SARS-like pandemic? J Infect. 2012;10:477–489. doi: 10.1016/j.jinf.2012.10.002. - DOI - PMC - PubMed
    1. Zaki AM, Van BS, Bestebroer TM, Osterhaus AD, Fouchier RA. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med. 2012;10:1814–1820. doi: 10.1056/NEJMoa1211721. - DOI - PubMed
    1. Pollack MP, Pringle C, Madoff LC, Memish ZA. Latest outbreak news from ProMED-mail: Novel coronavirus - Middle East. Int J Infect Dis. 2013;10:e143–e144. doi: 10.1016/j.ijid.2012.12.001. - DOI - PMC - PubMed
    1. Memish ZA, Zumla AI, Al-Hakeem RF, Al-Rabeeah AA, Stephens GM. Family cluster of Middle East respiratory syndrome coronavirus infections. N Engl J Med. 2013;10:2487–2494. doi: 10.1056/NEJMoa1303729. - DOI - PubMed
    1. Du L, He Y, Zhou Y, Liu S, Zheng BJ, Jiang S. The spike protein of SARS-CoV–a target for vaccine and therapeutic development. Nat Rev Microbiol. 2009;10:226–236. doi: 10.1038/nrmicro2090. - DOI - PMC - PubMed

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