Surface-modified measles vaccines encoding oligomeric, fusion-stabilized SARS-CoV-2 spike glycoproteins bypass measles seropositivity, boosting neutralizing antibody responses to omicron and historical variants. (preprint)

Serum titers of SARS-CoV-2 neutralizing antibodies (nAb) correlate well with protection from symptomatic COVID-19, but decay rapidly in the months following vaccination or infection. In contrast, measles-protective nAb titers are life-long after measles vaccination, possibly due to persistence of the live-attenuated virus in lymphoid tissues. We therefore sought to generate a live recombinant measles vaccine capable of driving high SARS-CoV-2 nAb responses. Since previous clinical testing of a live measles vaccine encoding a SARS-CoV-2 spike glycoprotein resulted in suboptimal anti-spike antibody titers, our new vectors were designed to encode prefusion-stabilized SARS-CoV-2 spike glycoproteins, trimerized via an inserted peptide domain and displayed on a dodecahedral miniferritin scaffold. Additionally, to circumvent the blunting of vaccine efficacy by preformed anti-measles antibodies, we extensively modified the measles surface glycoproteins. Comprehensive in vivo mouse testing demonstrated potent induction of high titer nAb in measles-immune mice and confirmed the significant incremental contributions to overall potency afforded by prefusion stabilization, trimerization, and miniferritin-display of the SARS-CoV-2 spike glycoprotein, and vaccine resurfacing. In animals primed and boosted with a MeV vaccine encoding the ancestral SARS-CoV-2 spike, high titer nAb responses against ancestral virus strains were only weakly cross-reactive with the omicron variant. However, in primed animals that were boosted with a MeV vaccine encoding the omicron BA.1 spike, antibody titers to both ancestral and omicron strains were robustly elevated and the passive transfer of serum from these animals protected K18-ACE2 mice from infection and morbidity after exposure to BA.1 and WA1/2020 strains. Our results demonstrate that antigen engineering can enable the development of potent measles-based SARS-CoV-2 vaccine candidates.

Longitudinal monitoring of SARS-CoV-2 neutralizing antibody titers and its impact on employee personal wellness decisions (preprint)

Virus neutralizing antibody (vnAb) titers are the strongest laboratory correlate of protection from SARS-CoV-2. Providing individuals with real-time measures of their vnAb titers is predicted to improve their ability to make personal wellness decisions. Yet, widespread commercial testing of SARS-CoV-2 vnAbs does not currently occur. Here, we examined whether knowing their vnAb titer impacted wellness decision-making among individuals. To this end, starting on January 1, 2021, we offered all employees from two companies free IMMUNO-COV testing and conducted a survey to assess their behaviors and decisions regarding booster vaccination. IMMUNO-COV is a clinically validated, surrogate virus assay that quantitates serum titers of SARS-CoV-2 vnAbs. To help participants gauge their level of protection based on their vnAb titer, we calibrated IMMUNO-COV titers to the World Health Organization (WHO) International Standard (IU/mL), making them comparable to published reports of correlates of protection, and we fit historical IMMUNO-COV vnAb titer values into predictive models of immune protection from COVID-19. As expected, data for the 56 program participants showed variability in vnAb titers post vaccination, rates vnAb decay, and fold-increases in vnAb titers after booster vaccination. Based on the participant survey, the majority (66%) of participants indicated that knowing their vnAb titer impacted their social behaviors and/or their decision on the timing of a booster vaccination. Several participants indicated that knowing their vnAb titer contributed to their peace of mind regarding their high level of protection from COVID-19. Together, these data demonstrate that regular determination of SARS-CoV-2 neutralizing antibody titers can significantly impact decisions regarding social interactions and timing of booster vaccinations.

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

An orally active vaccine capable of boosting SARS-CoV-2 immune responses in previously infected or vaccinated individuals would help efforts to achieve and sustain herd immunity. Unlike mRNA-loaded lipid nanoparticles and recombinant replication-defective adenoviruses, replicating vesicular stomatitis viruses with SARS-CoV-2 spike glycoproteins (VSV-SARS2) were poorly immunogenic after intramuscular administration in clinical trials. Here, by G protein trans-complementation, we generated VSV-SARS2(+G) virions with expanded target cell tropism. Compared to parental VSV-SARS2, G-supplemented viruses were orally active in virus-naive and vaccine-primed cynomolgus macaques, powerfully boosting SARS-CoV-2 neutralizing antibody titers. Clinical testing of this oral VSV-SARS2(+G) vaccine is planned.

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

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.

Seroprevalence of Anti-SARS-CoV-2 Antibodies in a Cohort of New York City Metro Blood Donors using Multiple SARS-CoV-2 Serological Assays: Implications for Controlling the Epidemic and Reopening. (preprint)

Projections of the stage of the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) pandemic and local, regional and national public health policies designed to limit the spread of the epidemic as well as reopen cities and states, are best informed by reproducible, high throughput, and statically credible antibody (Ab) assays. To date, a myriad of Ab tests, both available and authorized for emergency use by the FDA, has led to confusion rather than insight per se. The present study reports the results of a rapid, point-in-time 1,000-person cohort study using serial blood donors in the New York City metropolitan area (NYC) using multiple serological tests, including enzyme-linked immunosorbent assays (ELISAs) and high throughput serological assays (HTSAs). These were then tested and associated with assays for neutralizing Ab (NAb). Of the 1,000 NYC blood donor samples in late June and early July 2020, 12.1% and 10.9% were seropositive using the Ortho Total Ig and the Abbott IgG HTSA assays, respectively. These serological assays correlated with neutralization activity specific to SARS-CoV-2. The data reported herein suggest that seroconversion in this population occurred in approximately 1 in 8 blood donors from the beginning of the pandemic in NYC (considered March 1, 2020). These findings deviate with an earlier seroprevalence study in NYC showing 13.7% positivity. Collectively however, these data demonstrate that a low number of individuals have serologic evidence of infection during this first wave and suggest that the notion of herd immunity at rates of ~60% or higher are not near. Furthermore, the data presented herein show that the nature of the Ab-based immunity is not invariably associated with the development of NAb. While the blood donor population may not mimic precisely the NYC population as a whole, rapid assessment of seroprevalence in this cohort and serial reassessment could aid public health decision making.

Development and validation of IMMUNO-COV: a high-throughput clinical assay for detecting antibodies that neutralize SARS-CoV-2 (preprint)

We here describe the development and validation of IMMUNO-COV, a high-throughput clinical test to quantitatively measure SARS-CoV-2-neutralizing antibodies, the specific subset of anti-SARS-CoV-2 antibodies that block viral infection. The test measures the capacity of serum or purified antibodies to neutralize a recombinant Vesicular Stomatitis Virus (VSV) encoding the SARS-CoV-2 spike glycoprotein. This recombinant virus (VSV-SARS-CoV-2-S-{Delta}19CT) induces fusion in Vero cell monolayers, which is detected as luciferase signal using a dual split protein (DSP) reporter system. VSV-SARS-CoV-2-S-{Delta}19CT infection was blocked by monoclonal -SARS-CoV-2-spike antibodies and by plasma or serum from SARS-CoV-2 convalescing individuals. The assay exhibited 100% specificity in validation tests, and across all tests zero false positives were detected. In blinded analyses of 230 serum samples, only two unexpected results were observed based on available clinical data. We observed a perfect correlation between results from our assay and 80 samples that were also assayed using a commercially available ELISA. To quantify the magnitude of the anti-viral response, we generated a calibration curve by adding stepped concentrations of -SARS-CoV-2-spike monoclonal antibody to pooled SARS-CoV-2 seronegative serum. Using the calibration curve and a single optimal 1:100 serum test dilution, we reliably measured neutralizing antibody levels in each test sample. Virus neutralization units (VNUs) calculated from the assay correlated closely (p < 0.0001) with PRNTEC50 values determined by plaque reduction neutralization test against a clinical isolate of SARS-CoV-2. Taken together, these results demonstrate that the IMMUNO-COV assay accurately quantitates SARS-CoV-2 neutralizing antibodies in human sera and therefore is a potentially valuable addition to the currently available serological tests. The assay can provide vital information for comparing immune responses to the various SARS-CoV-2 vaccines that are currently in development, or for evaluating donor eligibility in convalescent plasma therapy studies.