Comparison of Four SARS-CoV-2 Neutralization Assays

Abstract

Neutralizing antibodies are a major correlate of protection for many viruses including the novel coronavirus SARS-CoV-2. Thus, vaccine candidates should potently induce neutralizing antibodies to render effective protection from infection. A variety of in vitro assays for the detection of SARS-CoV-2 neutralizing antibodies has been described. However, validation of the different assays against each other is important to allow comparison of different studies. Here, we compared four different SARS-CoV-2 neutralization assays using the same set of patient samples. Two assays used replication competent SARS-CoV-2, a focus forming assay and a TCID50-based assay, while the other two assays used replication defective lentiviral or vesicular stomatitis virus (VSV)-based particles pseudotyped with SARS-CoV-2 spike. All assays were robust and produced highly reproducible neutralization titers. Titers of neutralizing antibodies correlated well between the different assays and with the titers of SARS-CoV-2 S-protein binding antibodies detected in an ELISA. Our study showed that commonly used SARS-CoV-2 neutralization assays are robust and that results obtained with different assays are comparable.

Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity

“…Importantly, our studies rely on pseudoviruses, which are only capable of modeling the ACE2-dependent entry step of the SARS-CoV-2 life cycle. While numerous studies have now demonstrated a close correlation between neutralization titers measured against pseudovirus and live SARS-CoV-2 cultures (Wang et al, 2020;Ju et al, 2020;Pinto et al, 2020;Yang et al, 2020;Moore et al, 2004;Crawford et al, 2020;Riepler et al, 2020), it is unclear what impact additional mutations located outside of the spike may have on immunological escape, virulence, infectivity, or pathogenesis. Several recent reports and preprints, including studies conducted by Pfizer as well as Moderna, have produced similar findings in terms of vaccine potency against B.1.1.7 and B.1.1.298 variants but substantially less neutralization resistance by B.1.351 than we measured.…”

Characterization of Immune Responses to SARS-CoV-2 and Other Human Pathogenic Coronaviruses Using a Multiplex Bead-Based Immunoassay

“…Samples testing positive in one or both of the assays above were additionally analyzed using an in-house neutralization assay based on a replication-defective vesicular stomatitis virus (VSV) vector, which was pseudotyped with a C-terminally truncated version of the spike protein of SARS-CoV-2 (Wuhan isolate), [11] VSV∆G-S, as previously described [12]. Briefly, serial 1:4 dilutions of heat-inactivated plasma (starting at a 1:16 dilution) were pre-incubated with the virus for 1 h at 37 • C. Subsequently, they were used to infect 293 T-cells seeded the previous day.…”

Scalable, Micro-Neutralization Assay for Assessment of SARS-CoV-2 (COVID-19) Virus-Neutralizing Antibodies in Human Clinical Samples

“…These trials are evaluating the potential therapeutic benefit of IVIg for treatment of SARS-CoV-2 infection in both hospitalized and ambulatory patients. Further, considerable effort by the World Health Organization (WHO) and others has focused on correlating data from a range of SARS-CoV-2-specific ELISAs to neutralization assays with the objective of understanding both the diagnostic and predictive value of these and other point-of-care diagnostic tools [ 24 , 25 , 26 , 27 , 28 ]. Although neutralization assays, including the one described here, are not typically used as diagnostic tools, we showed that our assay correlates well with a commonly used diagnostic ELISA.…”