Towards Quantitative and Standardized Serological and Neutralization Assays for COVID-19.

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.

Quantifying the Vaccine-Induced Humoral Immune Response to Spike-Receptor Binding Domain as a Surrogate for Neutralization Testing Following mRNA-1273 (Spikevax) Vaccination Against COVID-19.

INTRODUCTION: There is a need for automated, high-throughput assays to quantify immune response after SARS-CoV-2 vaccination. This study assessed the combined utility of the Elecsys® Anti-SARS-CoV-2 S (ACOV2S) and the Elecsys Anti-SARS-CoV-2 (ACOV2N) assays using samples from the mRNA-1273 (Spikevax™) phase 2 trial (NCT04405076). METHODS: Samples from 593 healthy participants in two age cohorts (18-54 and ≥ 55 years), who received two injections with placebo (n = 198) or mRNA-1273 (50 µg [n = 197] or 100 µg [n = 198]), were collected at days 1 (first vaccination), 15, 29 (second vaccination), 43, and 57. ACOV2S results were used to assess humoral response to vaccination in different subgroups and were compared to live virus microneutralization assay. Samples from patients with either previous or concomitant infection (identified per ACOV2N) were analyzed separately. RESULTS: Receptor-binding domain-specific antibodies were readily detectable by ACOV2S for the vast majority of participants (174/189, 92.1% [50 µg dose] and 178/192, 92.7% [100 µg dose]) at the first post-vaccination assessment, with non-converters predominantly older in age. Seroconversion for all participants was observed at day 29 (before the second vaccine dose). Two weeks after the first dose, geometric mean concentration (GMC) of antibody levels was 1.37-fold higher in the 100 versus 50 µg group (p = 0.0098), reducing to 1.09-fold 2 weeks after the second dose (p = 0.0539, n.s.). In both dose groups, a more pronounced response was observed in the younger versus older age group on day 15 (50 µg, 2.49-fold [p < 0.0001]; 100 µg, 3.94-fold [p < 0.0001] higher GMC, respectively), and day 29 (1.93-fold, p = 0.0002, and 2.44-fold, p < 0.0001). Eight subjects had previous or concomitant SARS-CoV-2 infection; vaccination boosted their humoral response to very high ACOV2S results compared to infection-naïve recipients. ACOV2S strongly correlated with microneutralization (Pearson's r = 0.779; p < 0.0001), including good qualitative agreement. CONCLUSION: These results confirmed that ACOV2S is a highly valuable assay for tracking vaccine-related immune responses. Combined application with ACOV2N enables monitoring for breakthrough infection or stratification of previous natively infected individuals. The adaptive measuring range and high resolution of ACOV2S allow for early identification of seroconversion and resolution of very high titers and longitudinal differences between subgroups. Additionally, good correlation with live virus microneutralization suggests that ACOV2S is a reliable estimate of neutralization capacity in routine diagnostic settings.

Design and performance characteristics of the Elecsys anti-SARS-CoV-2 S assay.

Background: Automated, high throughput assays are required to quantify the immune response after infection with or vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study on the Roche Elecsys® Anti-SARS-CoV-2 S (ACOV2S) assay provides insights on the assay design and performance. Methods: The ACOV2S assay quantifies antibodies to the receptor-binding domain of the SARS-CoV-2 spike protein. The assigned units and the underlying standardization were compared to the international reference standard in BAU/mL. Assay specificity was assessed in samples (n=5981) collected prior to the COVID-19 pandemic and in samples from patients with non-COVID-19 respiratory infections (n=697) or other infectious diseases (n=771). Sensitivity was measured in 1313 samples from patients with mild COVID-19 and 297 samples from patients hospitalized with COVID-19. Comparison of results was performed to a comparator semi-quantitative anti-S1 assay of indirect detection format as well as a commercially available and an in-house version of a surrogate neutralization assay (ACE2-RBD). Results: The originally assigned units for the ACOV2S assay were shown to be congruent to the units of the First International WHO Standard for anti-SARS-CoV-2 immunoglobulins. Overall specificity was 99.98% with no geographical differences noted and no loss of specificity in samples containing potentially cross-reacting antibodies. High sensitivity was observed, with 98.8% of samples reported to be reactive >14 days after infection and sustained detection of antibodies over time. For all samples, ACOV2S titers and neutralization capacities developed with comparable dynamics. Robust standardization and assay setup enable excellent reproducibility of results, independent of lot or analyzer used. Conclusion: The results from this study confirmed that ACOV2S is a highly sensitive and specific assay and correlates well with surrogate neutralization assays. The units established for ACOV2S are also interchangeable with the units of the First International WHO Standard for anti-SARS-CoV-2 immunoglobulins. Worldwide availability of the assay and analyzers render ACOV2S a highly practical tool for population-wide assessment and monitoring of the humoral response to SARS-CoV-2 infection or vaccination.

Clinical utility of Elecsys Anti-SARS-CoV-2 S assay in COVID-19 vaccination: An exploratory analysis of the mRNA-1273 phase 1 trial.

BACKGROUND: The ability to quantify an immune response after vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential. This study assessed the clinical utility of the quantitative Roche Elecsys® Anti-SARS-CoV-2 S assay (ACOV2S) using samples from the 2019-nCoV vaccine (mRNA-1273) phase 1 trial (NCT04283461). METHODS: Samples from 30 healthy participants, aged 18-55 years, who received two injections with mRNA-1273 at a dose of 25 µg (n=15) or 100 µg (n=15), were collected at Days 1 (first vaccination), 15, 29 (second vaccination), 43 and 57. ACOV2S results (shown in U/mL - equivalent to BAU/mL per the first WHO international standard) were compared with results from ELISAs specific to antibodies against the Spike protein (S-2P) and the receptor binding domain (RBD) as well as neutralization tests including nanoluciferase (nLUC80), live-virus (PRNT80), and a pseudovirus neutralizing antibody assay (PsVNA50). RESULTS: RBD-specific antibodies were already detectable by ACOV2S at the first time point of assessment (d15 after first vaccination), with seroconversion before in all but 2 participants (25 µg dose group); all had seroconverted by Day 29. Across all post-baseline visits, geometric mean concentration of antibody levels were 3.27-7.48-fold higher in the 100 µg compared with the 25 µg dose group. ACOV2S measurements were highly correlated with those from RBD ELISA (Pearson's r=0.938; p<0.0001) and S-2P ELISA (r=0.918; p<0.0001). For both ELISAs, heterogeneous baseline results and smaller increases in antibody levels following the second vs first vaccination compared with ACOV2S were observed. ACOV2S showed absence of any baseline noise indicating high specificity detecting vaccine-induced antibody response. Moderate-strong correlations were observed between ACOV2S and neutralization tests (nLUC80 r=0.933; PsVNA50, r=0.771; PRNT80, r=0.672; all p≤0.0001). CONCLUSION: The Elecsys Anti-SARS-CoV-2 S assay (ACOV2S) can be regarded as a highly valuable method to assess and quantify the presence of RBD-directed antibodies against SARS-CoV-2 following vaccination, and may indicate the presence of neutralizing antibodies. As a fully automated and standardized method, ACOV2S could qualify as the method of choice for consistent quantification of vaccine-induced humoral response.

Clinical Utility of Elecsys Anti-SARS-CoV-2 S Assay in COVID-19 Vaccination: An Exploratory Analysis of the mRNA-1273 Phase 1 Trial.

Background: The ability to quantify an immune response after vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential. This study assessed the clinical utility of the quantitative Roche Elecsys® Anti-SARS-CoV-2 S assay (ACOV2S) using samples from the 2019-nCoV vaccine (mRNA-1273) phase 1 trial (NCT04283461). Methods: Samples from 30 healthy participants, aged 18-55 years, who received two injections with mRNA-1273 at a dose of 25 µg (n=15) or 100 µg (n=15), were collected at Days 1 (first vaccination), 15, 29 (second vaccination), 43 and 57. ACOV2S results (shown in U/mL - equivalent to BAU/mL per the first WHO international standard) were compared with results from ELISAs specific to antibodies against the Spike protein (S-2P) and the receptor binding domain (RBD) as well as neutralization tests including nanoluciferase (nLUC80), live-virus (PRNT80), and a pseudovirus neutralizing antibody assay (PsVNA50). Results: RBD-specific antibodies were already detectable by ACOV2S at the first time point of assessment (d15 after first vaccination), with seroconversion before in all but two participants (25 µg dose group); all had seroconverted by Day 29. Across all post-baseline visits, geometric mean concentration of antibody levels was 3.27-7.48-fold higher in the 100 µg compared with the 25 µg dose group. ACOV2S measurements were highly correlated with those from RBD ELISA (Pearson's r=0.938; p<0.0001) and S-2P ELISA (r=0.918; p<0.0001). For both ELISAs, heterogeneous baseline results and smaller increases in antibody levels following the second vs first vaccination compared with ACOV2S were observed. ACOV2S showed absence of any baseline noise indicating high specificity detecting vaccine-induced antibody response. Moderate-strong correlations were observed between ACOV2S and neutralization tests (nLUC80 r=0.933; PsVNA50, r=0.771; PRNT80, r=0.672; all p ≤ 0.0001). Conclusion: The Elecsys Anti-SARS-CoV-2 S assay (ACOV2S) can be regarded as a highly valuable method to assess and quantify the presence of RBD-directed antibodies against SARS-CoV-2 following vaccination and may indicate the presence of neutralizing antibodies. As a fully automated and standardized method, ACOV2S could qualify as the method of choice for consistent quantification of vaccine-induced humoral response.

In Search of the SARS-CoV-2 Protection Correlate: Head-to-Head Comparison of Two Quantitative S1 Assays in Pre-characterized Oligo-/Asymptomatic Patients.

BACKGROUND: Quantitative serological assays detecting response to SARS-CoV-2 are needed to quantify immunity. This study analyzed the performance and correlation of two quantitative anti-S1 assays in oligo-/asymptomatic individuals from a population-based cohort. METHODS: In total, 362 plasma samples (108 with reverse transcription-polymerase chain reaction [RT-PCR]-positive pharyngeal swabs, 111 negative controls, and 143 with positive serology without confirmation by RT-PCR) were tested with quantitative assays (Euroimmun Anti-SARS-CoV-2 QuantiVac enzyme-linked immunosorbent assay [EI-S1-IgG-quant]) and Roche Elecsys® Anti-SARS-CoV-2 S [Ro-RBD-Ig-quant]), which were compared with each other and confirmatory tests, including wild-type virus micro-neutralization (NT) and GenScript®cPass™. Square roots R of coefficients of determination were calculated for continuous variables and non-parametric tests were used for paired comparisons. RESULTS: Quantitative anti-S1 serology correlated well with each other (true positives, 96%; true negatives, 97%). Antibody titers decreased over time (< 30 to > 240 days after initial positive RT-PCR). Agreement with GenScript-cPass was 96%/99% for true positives and true negatives, respectively, for Ro-RBD-Ig-quant and 93%/97% for EI-S1-IgG-quant. Ro-RBD-Ig-quant allowed distinct separation between positives and negatives, and less non-specific reactivity versus EI-S1-IgG-quant. Raw values (95% CI) ≥ 28.7 U/mL (22.6-36.4) for Ro-RBD-Ig-quant and ≥ 49.8 U/mL (43.4-57.1) for EI-S1-IgG-quant predicted NT > 1:5 in 95% of cases. CONCLUSIONS: Our findings suggest both quantitative anti-S1 assays (EI-S1-IgG-quant and Ro-RBD-Ig-quant) may replace direct neutralization assays in quantitative measurement of immune protection against SARS-CoV-2 in certain circumstances. However, although the mean antibody titers for both assays tended to decrease over time, a higher proportion of Ro-RBD-Ig-quant values remained positive after 240 days.

In Search of the SARS-CoV-2 Protection Correlate: Head-to-Head Comparison of Two Quantitative S1 Assays in Pre-characterized Oligo-/Asymptomatic Patients.

BACKGROUND: Quantitative serological assays detecting response to SARS-CoV-2 are needed to quantify immunity. This study analyzed the performance and correlation of two quantitative anti-S1 assays in oligo-/asymptomatic individuals from a population-based cohort. METHODS: In total, 362 plasma samples (108 with reverse transcription-polymerase chain reaction [RT-PCR]-positive pharyngeal swabs, 111 negative controls, and 143 with positive serology without confirmation by RT-PCR) were tested with quantitative assays (Euroimmun Anti-SARS-CoV-2 QuantiVac enzyme-linked immunosorbent assay [EI-S1-IgG-quant]) and Roche Elecsys® Anti-SARS-CoV-2 S [Ro-RBD-Ig-quant]), which were compared with each other and confirmatory tests, including wild-type virus micro-neutralization (NT) and GenScript®cPass™. Square roots R of coefficients of determination were calculated for continuous variables and non-parametric tests were used for paired comparisons. RESULTS: Quantitative anti-S1 serology correlated well with each other (true positives, 96%; true negatives, 97%). Antibody titers decreased over time (< 30 to > 240 days after initial positive RT-PCR). Agreement with GenScript-cPass was 96%/99% for true positives and true negatives, respectively, for Ro-RBD-Ig-quant and 93%/97% for EI-S1-IgG-quant. Ro-RBD-Ig-quant allowed distinct separation between positives and negatives, and less non-specific reactivity versus EI-S1-IgG-quant. Raw values (95% CI) ≥ 28.7 U/mL (22.6-36.4) for Ro-RBD-Ig-quant and ≥ 49.8 U/mL (43.4-57.1) for EI-S1-IgG-quant predicted NT > 1:5 in 95% of cases. CONCLUSIONS: Our findings suggest both quantitative anti-S1 assays (EI-S1-IgG-quant and Ro-RBD-Ig-quant) may replace direct neutralization assays in quantitative measurement of immune protection against SARS-CoV-2 in certain circumstances. However, although the mean antibody titers for both assays tended to decrease over time, a higher proportion of Ro-RBD-Ig-quant values remained positive after 240 days. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40121-021-00475-x.

HBsAg, HBeAg and HBV DNA level changes and precore/basal core promoter mutations in the natural history of chronic hepatitis B in Indonesian patients.

INTRODUCTION: Chronic hepatitis B (CHB) is a state of complex interactions between the hepatitis B virus (HBV) and host. We studied the changes in hepatitis B surface antigen (HBsAg), hepatitis B 'e' antigen (HBeAg) and HBV DNA levels, considering the implications of HBV genotype, basal core promoter (BCP) A1762T/G1764A and precore G1896A mutations in CHB. METHODS: One hundred fifty-two treatment-naïve CHB patients were classified into immune-tolerant (IT), immune-clearance (IC), low/non-replicative (LR) and 'e'-negative hepatitis B (ENH) phases, based on HBeAg status, HBV DNA and ALT levels. HBV DNA was detected and quantified by polymerase chain reaction, then analyzed by sequencing. HBsAg and HBeAg levels were measured serologically. RESULTS: HBsAg and HBV DNA levels varied between CHB phases, with HBsAg highest in IT and lowest in LR, and HBV DNA high in IT and IC, and lowest in LR. Both markers increased in ENH. Correlation between HBsAg and HBV DNA was significant in IT and IC, modest in ENH, but missing in LR. HBeAg and HBV DNA levels were dissociated in HBeAg-positive patients. Genotypes B and C were similarly distributed, with precore mutations higher in HBeAg-negative patients and BCP mutations comparable in all phases. Temporal association between HBeAg seroconversion and an increase of BCP/precore mutations was observed. CONCLUSION: HBsAg and HBV DNA levels were high and correlated in early CHB phases and dissociated after HBeAg seroconversion, indicating different controls affecting HBV replication and HBsAg production. Selection of BCP/precore mutants may affect disease course and explain the HBeAg-HBV DNA dissociation, a precaution for clinical application of quantitative HBeAg.