Quantifying Absolute Neutralization Titers against SARS-CoV-2 by a Standardized Virus Neutralization Assay Allows for Cross-Cohort Comparisons of COVID-19 Sera

Abstract

The global coronavirus disease 2019 (COVID-19) pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent-phase plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous biosafety level 3 (BSL3) conditions, which limits high-throughput screening of patient and vaccine sera. Myriad BSL2-compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making intergroup comparisons difficult. To address these limitations, we developed a standardized VNA using CoV2-S pseudotyped particles (CoV2pp) based on vesicular stomatitis virus bearing the Renilla luciferase gene in place of its G glycoprotein (VSVΔG); this assay can be robustly produced at scale and generate accurate neutralizing titers within 18 h postinfection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S enzyme-linked immunosorbent assay (ELISA) results and live-virus neutralizations in confirmed convalescent-patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n > 120). Our data (i) show that absolute 50% inhibitory concentration (absIC50), absIC80, and absIC90 values can be legitimately compared across diverse cohorts, (ii) highlight the substantial but consistent variability in neutralization potency across these cohorts, and (iii) support the use of the absIC80 as a more meaningful metric for assessing the neutralization potency of a vaccine or convalescent-phase sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2 plus TMPRSS2. When these are used in combination with our CoV2pp, we can produce CoV2pp sufficient for 150,000 standardized VNAs/week. IMPORTANCE Vaccines and antibody-based therapeutics like convalescent-phase plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) are an essential part of determining vaccine or therapeutic efficacy. However, such efficacy testing is limited by the inherent dangers of working with the live virus, which requires specialized high-level biocontainment facilities. We therefore developed a standardized replication-defective pseudotyped particle system that mimics the entry of live SARS-CoV-2. This tool allows for the safe and efficient measurement of NATs, determination of other forms of entry inhibition, and thorough investigation of virus entry mechanisms. Four independent labs across the globe validated our standardized VNA using diverse cohorts. We argue that a standardized and scalable assay is necessary for meaningful comparisons of the myriad of vaccines and antibody-based therapeutics becoming available. Our data provide generalizable metrics for assessing their efficacy.

Anti-Spike Protein Assays to Determine SARS-CoV-2 Antibody Levels: a Head-to-Head Comparison of Five Quantitative Assays

“…The primary goal of active immunization is to induce many SARS-CoV-2-specific neutralizing antibodies that ideally prevent the pathogen's entry and thus infection or stop the systemic spread to prevent disease ( 25 ). The functional virus neutralization assays are not feasible everywhere: assays with live viruses require biosafety level 3, but variants such as pseudotyped neutralization assays are also labor-intensive and cannot be performed at high throughput ( 26 – 28 ). Classical antibody assays, which measure the reactivity of antibodies in serum/plasma with defined antigens, can be performed very rapidly and in high throughput, in contrast to neutralization tests.…”

Immunity Profiling of COVID-19 Infection, Dynamic Variations of Lymphocyte Subsets, a Comparative Analysis on Four Different Groups

“…Similarly, if we consider B-lymphocyte fluctuation between the different groups, a diminished B-cell modulatory activity would inhibit the conversion process of T cells to regulatory T cells (Tregs) generating a more inflammatory environment. This phenomenon was clearly seen in both PP and NP groups, where severe T cell depletion (regulatory phenotypes) was consistently associated with high neutrophil and NK levels, a significant sign that occurs during the first days of the onset of symptoms [30,31]. This phenomenon was clearly seen in both PP and NP groups in which, severe T-regs depletion was clinically concomitant with an increase in neutrophils and NK cells within the lungs' interstices and parenchyma.…”

“…However, dissimilarities, more than similarities would expose the unique tropism of SARS-CoV-2 that directly affects main lymphoid organs with the ability to hide in remote areas within tissues and cells, insistently triggering the release of inflammatory mediators in an equivalent manner of any chronic lung disease. Indeed, NP patients showed a clear quantitative increase in CD8+ (that was not seen in PP and NN), a stronger decrease in both CD4/CD8 naïve cells, a marked decrease in CD4+/CD8+ ratio and stronger increase in T CD3+DR+; a scenario that opens a debate on whether there is a stage-associated pattern of these markers expression on T-lymphocytes during SARS-CoV-2 infection, since specific phenotypic patterns may have functional links in the host response to the virus [1,18,20,30,31,[55][56][57][58][59][60][61][62][63][64][65].…”

“…However, regardless the quantity of circulating antibodies, the occurrence of memory T cells and B-lymphocytes due to SARS-CoV-2 stimulation is crucial for long-term immunity. In this scenario, the attendance of active T helper cells is suggestive of normal configurations of humoral immune response and the formation of specific memory B cell clusters capable of initiating responses against further reinfection [16,23,24,[29][30][31][32][33][34][35][36][37][38][39]. Indeed, the presence of neutralizing antibodies has been revealed to be a good resource to control SARS-CoV2 infection either in vitro or in vivo, though not all patients who recover from COVID-19 show enough neutralizing antibodies to be used as anti-COVID-19 therapy, suggesting a complex relationship between humoral and cellular response in COVID-19 pathogenesis [30][31][32][40][41][42].…”

Two courses of deconstructed coronavirus please

“…Endeavours like those pursued by Ju and colleagues highlight the need and potential for the democratisation of science in the face of significant societal challenges, if reagents and protocols are indeed adequately shared. There are numerous examples of this in the scientific community, such as the generation and sharing of SARS-CoV-2 pseudoparticles from the lab of Benhur Lee [8] and the construction of a Coronavirus Disease 2019 (COVID-19) toolbox from the MRC-University of Glasgow Centre for Virus Research [9]. It is hoped that efforts like this will enhance the efficiency of science, while not jeopardising the safety of researchers and their communities.…”