IMMUNO-COV™ v2.0: Development and Validation of a High-Throughput Clinical Assay for Measuring SARS-CoV-2-Neutralizing Antibody Titers

Abstract

Neutralizing antibodies are key determinants of protection from future infection, yet well-validated high-throughput assays for measuring titers of SARS-CoV-2-neutralizing antibodies are not generally available. Here we describe the development and validation of IMMUNO-COV™ v2.0 a scalable surrogate virus assay, which titrates antibodies that block infection of Vero-ACE2 cells by a luciferase-encoding vesicular stomatitis virus displaying SARS-CoV-2 spike glycoproteins (VSV-SARS2-Fluc). Antibody titers, calculated using a standard curve consisting of stepped concentrations of SARS-CoV-2 spike monoclonal antibody, correlated closely (p < 0.0001) with titers obtained from a gold-standard PRNT50% assay performed using a clinical isolate of SARS-CoV-2. IMMUNO-COV™ v2.0 was comprehensively validated using data acquired from 242 assay runs performed over seven days by five analysts, utilizing two separate virus lots, and 176 blood samples. Assay performance was acceptable for clinical use in human serum and plasma based on parameters including linearity, dynamic range, limit of blank and limit of detection, dilutional linearity and parallelism, precision, clinical agreement, matrix equivalence, clinical specificity and sensitivity, and robustness. Sufficient VSV-SARS2-Fluc virus reagent has been banked to test 5 million clinical samples. Notably, a significant drop in IMMUNO-COV™ v2.0 neutralizing antibody titers was observed over a six-month period in people recovered from SARS-CoV-2 infection. Together, our results demonstrate the feasibility and utility of IMMUNO-COV™ v2.0 for measuring SARS-CoV-2-neutralizing antibodies in vaccinated individuals and those recovering from natural infections. Such monitoring can be used to better understand what levels of neutralizing antibodies are required for protection from SARS-CoV-2, and what booster dosing schedules are needed to sustain vaccine-induced immunity.

Boosting of SARS-CoV-2 immunity in nonhuman primates using an oral rhabdoviral vaccine

“…Vero-DSP1 and Vero-DSP2 co-monolayers, which produce functional Renilla luciferase following virus-mediated cell fusion, were seeded at 6×10 4 cells per well in 96-well black-walled plates the day before assay. Neutralizing antibody cocktails were prepared in OptiMEM as follows: α-SARS-CoV-2 spike cocktail (mAb10914 at 6 µg/mL combined with mAb10922 at 2 µg/mL, Genscript) [4], α-VSV-G cocktail (mAbVSV-G 8G5F11, Absolute Antibody #Ab01401-2.3) at 2 µg/mL combined with mAb VSV-G IE9F9 (Absolute Antibody #Ab01402-2.0) at 10 µg/mL [27], and α-SARS-CoV-2 spike and α-VSV-G cocktail (combination of all four antibodies). Three-fold serial dilutions of the antibody cocktails were prepared and incubated with an equal volume of VSV-SARS2 or VSV-SARS2(+G).…”

“…However, all currently approved SARS-CoV-2 vaccines are administered via intramuscular injection which increases the cost and complexity of vaccination programs, contributes to vaccine hesitancy and fails to stimulate mucosal immunity [2,3]. Also, protective antibody titers are known to wane after successful vaccination (or after natural infection), such that periodic booster immunizations will be needed to sustain immunity, particularly in vulnerable individuals, so long as the virus remains endemic in the human population [4][5][6].…”

“…Previous studies have shown that peak post-SARS-CoV-2 infection and post-vaccination neutralizing antibody titers to SARS-CoV-2 differ between individuals based on age, infection severity and vaccine identity [18]. Antibody titers have also been shown to fall quite rapidly over time with half-lives varying from 2.5 and 6 months, signifying 4-fold to 25-fold titer reductions in the first year [4, 6, 19]. Thus, while the appropriate timing of SARS-CoV-2 vaccine booster shots has not yet been determined for specific subgroups of individuals, booster vaccination will likely be an important part of the strategy for longer-term control of the pandemic.…”