The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID-19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2–specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1), and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies.
Herein, we describe pseudotyped and chimeric viruses that can evaluate the neutralizing activity of SARS-CoV-2 S-specific mAbs as well as convalescent sera or plasma. Many factors could, in principle, affect the apparent potency of neutralizing antibodies as measured using surrogate viruses. One key factor may be the density of spikes on the virion envelope. Spike density could affect the avidity of bivalent antibodies, particularly those that are unable to engage two S-protein monomers within a single trimer and whose potency is enhanced by engaging two adjacent trimers.Moreover, some antibodies do not compete for ACE2 binding, and their activity may be entirely dependent on bivalent interactions with adjacent trimers. Conversely, larger number of spikes per virion might increase the number of antibodies per virion required for neutralization. The net opposing effect s of spike density on neutralization is difficult to predict. We also note that IgG may not be the only immunoglobulin type that contributes to plasma neutralization in at least some instances, and the valency of various immunoglobulin types could further interact with spike density to modulate neutralization. Electron microscopic images of coronaviruses indicate a fairly high spike density, and the extent to which pseudotyped viruses mimic this property, might be important for determining the accuracy of neutralization assays. Notably, we found that truncation of the cytoplasmic tail of SARS-CoV-2 dramatically increased the infectious titer of SARS-CoV-2 pseudotypes, likely by facilitating incorporation of S protein into virions. During virion assembly, SARS-CoV-2 buds into secretory compartments, while HIV-1 and VSV assemble at the cell surface. Truncation of the S-protein cytoplasmic tail may increase cell surface levels and/or enable incorporation by alleviating structural incompatibility of the S-protein cytoplasmic tail and HIV-1 or VSV matrix proteins.
To the extent that RBD-specific antibodies may compete with target cell surface ACE2 for binding to virion spikes, the density of ACE2 molecules on the target cell surface could additionally affect antibody potency in neutralization assays. Moreover, the use of replication-competent, multicycle replication-based assays versus single-cycle infection with defective reporter viruses could additionally affect apparent neutralizing antibody potency. Specifically, partial neutralization at marginal antibody concentrations in a single replication cycle could be propagated and thus amplified over multiple rounds of replication, increasing the apparent level of neutralization at marginal antibody concentrations. Alternatively, the increase in viral dose during multiple replication cycles or the high multiplicity associated with direct cell-to-cell viral spread might overwhelm neutralizing antibodies at marginal concentrations in multicycle neutralization assays. Such a scenario would reduce apparent antibody potency compared with single-cycle assays. Finally, viruses may also generate defective or noninfectious particles to varying degrees which could be sufficient to sequester neutralizing antibodies and therefore affect neutralization potency. Despite the very different nature of the assays employed herein, as well as their different dynamic ranges, each of the surrogate virus-based assays generated quantitative measurements of neutralizing activity that correlated well with neutralization measured using authentic SARS-CoV-2. Naturally, the above considerations mean that these correlations are not precise; for example, both HIV-1– and VSV-based pseudotyped viruses were somewhat less sensitive to neutralization than authentic SARS-CoV-2, particularly by weakly neutralizing plasma. This finding may be because they are single-cycle assays, or perhaps because pseudotyped virions may have lower spike density than SARS-CoV-2. Notably, pseudoviruses employed by other investigators sometimes exhibit increased sensitivity to mAbs as compared with SARS-CoV-2, and sometimes their potencies against both virus types are closely matched.
In addition to replication-defective single-cycle pseudotyped viruses, we also developed a replication-competent rVSV/SARS-CoV-2/GFP chimeric virus. Notably, adaptation of rVSV/SARS-CoV-2/GFP in 293T/ACE2 cells led to the acquisition of mutations at the S-protein furin cleavage site. Adaptation of rVSV/SARS-CoV-2/GFP in different target cells that express different furin-like or other proteases may result in the acquisition of alternative adaptive mutations. Crucially, the sensitivity of the adapted rVSV/SARS-CoV-2/GFP to neutralization by mAbs mimicked that of authentic SARS-CoV-2. Interestingly, the rVSV/SARS-CoV-2/GFP appeared slightly more susceptible to plasma neutralization than SARS-CoV-2 for unknown reasons. Given that the design of rVSV/SARS-CoV-2/GFP is similar to that of the successful VSV/EboV ebolavirus vaccine, derivatives of the adapted 1D7 or 2E1 viruses could potentially be vaccine candidates. Additionally, these viruses could also be used in laboratory selection experiments to identify mutations that enable escape from inhibition by antibodies or other therapeutic agents that target the S protein.
Some of the pseudotype neutralization assays described herein can be executed in a standard BSL2 laboratory. Indeed, we have used the HIV-1 and VSV pseudotype approaches to conduct determine the neutralizing potencies of hundreds of plasma samples and mAbs in a BSL2 laboratory in a few weeks. Automation and additional miniaturization is certainly feasible to further increase throughput, a notable consideration given the sheer number of vaccine candidates in the development pipeline. We note, however, that miniaturization reduces the number of infected cells and the dynamic range of neutralization assays. We also note that the HT1080/ACE2cl.14 and Huh7.5 cell lines are significantly more adherent than 293T-derived cell lines and are recommended (for HIV-1 and VSV pseudotype assays, respectively) in high-throughput situations, as great care is necessary when using 293T-derived cells whose adhesive properties during washing steps are suboptimal. A key caveat associated with the measurement of neutralizing antibody activity, whether using pseudotype assays or authentic SARS-CoV-2 virions, is that the level of neutralizing activity required to protect against SARS-CoV-2 infection in a natural situation is unknown. Moreover, while neutralization assays measure the ability of antibodies to inhibit viral entry, they do not capture features of the antiviral activity of antibodies such as antibody-dependent cellular cytotoxicity that may be germane in vivo. Nevertheless, in vitro neutralizing activity has long been identified as a correlate of protection against infection in many viral infections, including coronaviruses. As such, we envisage that the techniques described herein might be of significant utility in curtailing the COVID-19 pandemic.
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