Generation of MERS-CoV Pseudotyped Viral Particles for the Evaluation of Neutralizing Antibodies in Mammalian Sera

Abstract

Pseudotyped viral particle production has been used extensively and broadly for many viruses to evaluate levels of neutralizing antibodies, viral entry inhibitors and vaccine immunogenicity. This assay is extremely safe and useful alternative to live virus-based assay without the need for high containment facilities. In this chapter, we describe the generation of MERS-CoV pseudotyped viral particles (MERSpp) expressing full-length spike protein using second-generation lentiviral packaging system. This platform is optimized to generate high titer of MERSpp and to test sera from different mammalian species.

Keywords: MERS-CoV; Neutralization assay; Pseudotype viral particles; Pseudovirus; Transfection.

Fig. 1

Plate layout for MERSpp titration. The sequential steps of plate preparation are indicated by numbered boxes. The color intensity indicates the expected values of luciferase readout

Fig. 2

An example readout of MERSpp titration plate

Fig. 3

Plate preparation for MERSpp neutralization assay. The preparation steps for MERSpp neutralization assay plate are indicated by numbered boxes. The color intensity indicates the expected values of luciferase readout

Fig. 4

MERSpp neutralization assay for naïve serum samples from different species. Serum samples from different species, including humans, camels, rats, and mice, were tested in a MERSpp NA with two different concentrations of pseudotyped viruses (200,000 and 550,000 RLU per well). All samples were negative for Anti-MERS-CoV antibodies by standard commercial ELISA before conducting the MERSpp NA. The results showed levels of background neutralization that varied between species. This background neutralization is expected based on our previous observation (unpublished data)