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OBJECTIVES: To investigate multi-dose and timings of COVID-19 vaccines in preventing antenatal infection. DESIGN: Prospective observational study investigating primary vaccinations, boosters, antenatal COVID-19 infections, neutralizing antibody (Nab) durability, and cross-reactivity to Delta and Omicron variants of concern (VOCs). RESULTS: Ninety-eight patients completed primary vaccination prepregnancy (29.6%) and antenatally (63.3%), 24.2% of whom had antenatal COVID-19, while 7.1% were unvaccinated (28.6% had antenatal COVID-19). None had severe COVID-19. Prepregnancy vaccination resulted in vaccination-to-infection delay of 23.3 weeks, which extended to 45.2 weeks with a booster, compared to 16.9 weeks following antenatal vaccination (P < 0.001). Infections occurred at 26.2 weeks gestation in women vaccinated prepregnancy compared to 36.2 weeks gestation in those vaccinated during pregnancy (P < 0.007). The risk of COVID-19 infection was higher without antenatal vaccination (hazard ratio [HR] 14.6, P = 0.05) and after prepregnancy vaccination without a booster (HR 10.4, P = 0.002). Antenatal vaccinations initially led to high Nab levels, with mild waning but subsequent rebound. Significant Nab enhancement occurred with a third-trimester booster. Maternal-neonatal Nab transfer was efficient (transfer ratio >1), and cross-reactivity to VOCs was observed. CONCLUSION: Completing vaccination during any trimester delays COVID-19 infection and maintains effective neutralizing activity throughout pregnancy, with robust cross-reactivity to VOCs and efficient maternal-neonatal transfer.

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BACKGROUND: Understanding the immune response to evolving viral strains is crucial for evidence-informed public health strategies. The main objective of this study is to assess the influence of vaccination on the neutralizing activity of SARS-CoV-2 delta and omicron infection against various SARS-CoV-2 variants. METHODS: A total of 97 laboratory-confirmed COVID-19 cases were included. To assess the influence of vaccination on neutralizing activity, we measured the neutralizing activity of SARS-CoV-2 delta or omicron (BA.1 or BA.2) infection against wild-type (WT), delta, BA.1, and BA.2, with the results stratified based on vaccination status. RESULTS: The neutralizing activity against the WT, delta, and omicron variants (BA.1 and BA.2) was significantly higher in the vaccinated patients than those in the unvaccinated patients. In the unvaccinated individuals infected with the delta variant, the decrease in binding to BA.1 and BA.2 was statistically significant (3.9- and 2.7-fold, respectively) compared to the binding to delta. In contrast, vaccination followed by delta breakthrough infection improved the cross-neutralizing activity against omicron variants, with only 1.3- and 1.2-fold decreases in BA.1 and BA.2, respectively. Vaccination followed by infection improved cross-neutralizing activity against WT, delta, and BA.2 variants in patients infected with the BA.1 variant, compared to that in unvaccinated patients. CONCLUSIONS: Vaccination followed by delta or BA.1 infection is associated with improved cross-neutralizing activity against different SARS-CoV-2 variants. The enhanced protection provided by breakthrough infections could have practical implications for optimizing vaccination strategies.

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OBJECTIVES: To assess the long-term humoral immunity induced by booster administration, as well as the ability of binding antibody and surrogate virus neutralization tests (sVNT) to predict neutralizing antibodies (NAbs) against the SARS-CoV-2 Omicron variant. METHODS: A total of 269 sera samples were analyzed from 64 healthcare workers who had received a homologous booster dose of BNT162b2. Neutralizing antibodies assessed by sVNT and anti-RBD IgG measured with the sCOVG assay (Siemens Healthineers®) were analyzed at five timepoints; before and up to 6 months following the booster. Antibody titers were correlated with neutralizing antibodies against the Omicron BA.1 variant obtained by pseudovirus neutralization test (pVNT) as a reference method. RESULTS: While Wild-type sVNT percentage of inhibition (POI) remained above 98.6% throughout the follow-up period after booster administration, anti-RBD IgG and NAbs assessed by Omicron BA.1 pVNT showed respectively a 3.4-fold and 13.3-fold decrease after 6 months compared to the peak reached at day 14. NAbs assessed by Omicron sVNT followed a steady decline until reaching a POI of 53.4%. Anti-RBD IgG and Omicron sVNT assays were strongly correlated (r=0.90) and performed similarly to predict the presence of neutralizing antibodies with Omicron pVNT (area under the ROC: 0.82 for both assays). In addition, new adapted cut-off values of anti-RBD IgG (>1,276 BAU/mL) and Omicron sVNT (POI>46.6%) were found to be better predictors of neutralizing activity. CONCLUSIONS: This study showed a significant drop in humoral immunity 6 months after booster administration. Anti-RBD IgG and Omicron sVNT assays were highly correlated and could predict neutralizing activity with moderate performance.

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Detecting immunity against SARS-CoV-2 is vital for evaluating vaccine response and natural infection, but conventional virus neutralization test (cVNT) requires BSL3 and live viruses, and pseudo-virus neutralization test (pVNT) needs specialized equipment and trained professionals. The surrogate virus neutralization test (sVNT) was developed to overcome these limitations. This study explored the use of angiotensin converting enzyme 2 (ACE2) produced from Nicotiana benthamiana for the development of an affordable neutralizing antibodies detection assay. The results showed that the plant-produced ACE2 can bind to the receptor binding domain (RBD) of the SARS-CoV-2, and was used to develop sVNT with plant-produced RBD protein. The sVNT developed using plant-produced proteins showed high sensitivity and specificity when validated with a group of 30 RBD vaccinated mice sera and the results were correlated with cVNT titer. This preliminary finding suggests that the plants could offer a cost-effective platform for producing diagnostic reagents.

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Background: The pandemic unleashed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 500 million people worldwide and caused more than 6 million deaths. Cellular and humoral immunity induced by infection or immunization are key factors in controlling the viral burden and avoiding the recurrence of coronavirus disease. The duration and effectiveness of immunity after infection is relevant to pandemic policy interventions, including the timing of vaccine boosters. Objectives: We sought to evaluate longitudinal binding and functional antibodies against SARS-CoV-2 receptor-binding domain in police officers and health care workers with a history of coronavirus disease 2019 and compare with SARS-CoV-2-naive individuals after vaccination with adenovirus-based ChAdOx1 nCoV-19 (AstraZeneca-Fiocruz) or the inactivated CoronaVac vaccine (Sinovac-Butantan Institute). Methods: A total of 208 participants were vaccinated. Of these, 126 (60.57%) received the ChAdOx1 nCoV-19 vaccine and 82 (39.42%) received the CoronaVac vaccine. Prevaccination and postvaccination blood was collected, and the amount of anti-SARS-CoV-2 IgG and the neutralizing ability of the antibodies to block the interaction between angiotensin-converting enzyme 2 and receptor-binding domain were determined. Results: Subjects with preexisting SARS-CoV-2 immunity and who received a single dose of ChAdOx1 nCoV-19 or CoronaVac have similar or superior antibody levels when compared with levels in seronegative individuals even after 2 doses of the vaccine. Neutralizing antibody titers of seropositive individuals were higher with a single dose of either ChAdOx1 nCoV-19 or CoronaVac compared with those of seronegative individuals. After 2 doses, both groups reached a plateau response. Conclusions: Our data reinforce the importance of vaccine boosters to increase specific binding and neutralizing SARS-CoV-2 antibodies.

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A severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) surrogate virus neutralization test (sVNT) was used to determine the degree of inhibition of binding between human angiotensin converting enzyme 2 (hACE2) and the receptor binding domain (RBD) of spike protein by neutralizing antibodies in a biosafety level 2 facility. Here, to improve the sensitivity and specificity of the commercial sVNT, we developed a new biotin based sVNT using biotinylated RBD and HRP conjugated streptavidin instead of HRP conjugated RBD for direct detection in an ELISA assay that strongly correlated to the FDA approved cPass sVNT commercial kit (R2 = 0.8521) and pseudo virus neutralization test (R2 = 0.9006) (pVNT). The biotin based sVNT was evaluated in 535 postvaccination serum samples corresponding to second and third boosts of AZD1222 and BNT162b2 vaccines of the wild type strain. We confirmed that the neutralizing antibodies against SARS-CoV-2 variants in second vaccination sera decreased after a median of 141.5 days. Furthermore, vaccination sera from BNT162b2-BNT162b2 vaccines maintained neutralizing antibodies for longer than those of AZD1222 only vaccination. In addition, both vaccines maintained high neutralizing antibodies in third vaccination sera against Omicron BA.2 after a median of 27 days, but neutralizing antibodies significantly decreased after a median of 141.5 days. Along with the cPass sVNT commercial kit, biotin based sVNTs may also be suitable for specifically detecting neutralizing antibodies against multiple SARS-CoV-2 variants; however, to initially monitor the neutralizing antibodies in vaccinated sera using high throughput screening, conventional PRNT could be replaced by sVNT to circumvent the inconvenience of a long test time.

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The receptor binding domain (RBD) plays a pivotal role in the viral entry as it enables the engagement of severe acute respiratory syndrome 2 (SARS-CoV-2) with the human angiotensin-converting enzyme 2 (ACE2) receptor for host cell entry. RBD is the major target for developing viral inhibitors and vaccines. Expression of recombinant RBD in E.coli is highly scalable with a low-cost procedure despite its high expression level compared to expression in mammalian and yeast cells. Using an alternative natural adjuvant system instead of alum adjuvant, increased immunogenicity of RBD antigen in serological assay including direct ELISA and surrogate Virus Neutralization Test (sVNT) was demonstrated with high levels of IgGs and neutralizing antibodies in mice sera immunized with RBD:AlSa (Alum and Sodium alginate) formulation. The sVNT is a simple and fast test that can be used instead of the conventional virus neutralization test requiring live virus and BSL3 laboratory to detect total neutralizing antibodies against RBD. Additionally, results showed a safety profile for sodium alginate which supported using it as an alternative natural adjuvant.

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Background: Heterologous prime-boost SARS-CoV-2 vaccination is a widely accepted strategy during the COVID-19 pandemic, which generated a superior immune response than homologous vaccination strategy. Objective: To describe immunogenicity of heterologous prime-boost vaccination with inactivated vaccine, CoronaVac, followed by BNT162b2 and 5-month booster dose with BNT162b2 in healthy Thai adolescents. Methods: Adolescents aged 12-18 years were randomized 1:1:1:1 to receive CoronaVac (SV) followed by BNT162b2 (PZ) 30 or 20 µg at either 3- or 6-week interval (SV3w/PZ30µg, SV3w/PZ20µg, SV6w/PZ30µg or SV6w/PZ20µg). During the Omicron-predominant period, participants were offered a BNT162b2 booster dose 30, 15, or 10 µg. Immunogenicity was determined using IgG antibody against spike-receptor-binding domain of wild type(anti-S-RBD IgG) and surrogate virus neutralization test(sVNT) against Delta variant at 14 days and 5 months after the 2nd dose. Neutralization tests(sVNT and pseudovirus neutralization test; pVNT) against Omicron strain were tested pre- and 14 days post-booster dose. Results: In October 2021, 76 adolescents with a median age of 14.3 years (IQR 12.7-16.0) were enrolled: 20 in SV3w/PZ30µg; 17 in SV3w/PZ20µg; 20 in SV6w/PZ30µg; 19 in SV6w/PZ20µg. At day 14, the geometric mean(GM) of anti-S-RBD IgG in SV3w/PZ30µg was 4713 (95 %CI 4127-5382) binding-antibody unit (BAU)/ml, while geometric mean ratio(GMR) was 1.28 (1.09-1.51) in SV6w/PZ30µg. The GMs of sVNT against Delta variants at day 14 among participants in SV3w/PZ30µg and SV6wk/PZ30µg arm were 95.3 % and 99.7 %inhibition, respectively. At 5 months, GMs of sVNT against Delta variants in SV3w/PZ30µg were significantly declined to 47.8 % but remained at 89.0 % inhibition among SV6w/PZ30µg arm. In April 2022, 52 adolescents received a BNT162b2 booster dose. Proportion of participants with sVNT against Omicron strain > 80 %inhibition was significantly increased from 3.8 % pre-booster to 67 % post-booster. Proportion of participants with pVNT ID50 > 185 was 42 % at 14 days post 2nd dose and 88 % post booster, respectively. Conclusions: Heterologous prime-boost vaccination with CoronaVac followed by BNT162b2 induced high neutralizing titer against SARS-CoV-2 Delta strain. After 5-month interval, booster with BNT162b2 induced high neutralizing titer against Omicron strain.Thai Clinical Trials Registry (thaiclinicaltrials.org): TCTR20210923012.

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Serological assays are useful in investigating the development of humoral immunity against SARS-CoV-2 in the context of epidemiological studies focusing on the spread of protective immunity. The plaque reduction neutralization test (PRNT) is the gold standard method to assess the titer of protective antibodies in serum samples. However, to provide a result, the PRNT requires several days, skilled operators, and biosafety level 3 laboratories. Therefore, alternative methods are being assessed to establish a relationship between their outcomes and PRNT results. In this work, four different immunoassays (Roche Elecsys® Anti SARS-CoV-2 S, Snibe MAGLUMI® SARS-CoV-2 S-RBD IgG, Snibe MAGLUMI® 2019-nCoV IgG, and EUROIMMUN® SARS-CoV-2 NeutraLISA assays, respectively) have been performed on individuals healed after SARS-CoV-2 infection. The correlation between each assay and the reference method has been explored through linear regression modeling, as well as through the calculation of Pearson's and Spearman's coefficients. Furthermore, the ability of serological tests to discriminate samples with high titers of neutralizing antibodies (>160) has been assessed by ROC curve analyses, Cohen's Kappa coefficient, and positive predictive agreement. The EUROIMMUN® NeutraLISA assay displayed the best correlation with PRNT results (Pearson and Spearman coefficients equal to 0.660 and 0.784, respectively), as well as the ROC curve with the highest accuracy, sensitivity, and specificity (0.857, 0.889, and 0.829, respectively).

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Background: Limited commercial LFA assays are available to provide a reliable quantitative measurement of the total binding antibody units (BAU/mL) against the receptor-binding domain of the SARS-CoV-2 spike protein (S-RBD). Aim: This study aimed to evaluate the performance of the fluorescence LFA FinecareTM 2019-nCoV S-RBD test along with its reader (Model No.: FS-113) against the following reference methods: (i) the FDA-approved GenScript surrogate virus-neutralizing assay (sVNT); and (ii) three highly performing automated immunoassays: BioMérieux VIDAS®3, Ortho VITROS®, and Mindray CL-900i®. Methods: Plasma from 488 vaccinees was tested by all aforementioned assays. Fingerstick whole-blood samples from 156 vaccinees were also tested by FinecareTM. Results and conclusions: FinecareTM showed 100% specificity, as none of the pre-pandemic samples tested positive. Equivalent FinecareTM results were observed among the samples taken from fingerstick or plasma (Pearson correlation r = 0.9, p < 0.0001), suggesting that fingerstick samples are sufficient to quantitate the S-RBD BAU/mL. A moderate correlation was observed between FinecareTM and sVNT (r = 0.5, p < 0.0001), indicating that FinecareTM can be used for rapid prediction of the neutralizing antibody (nAb) post-vaccination. FinecareTM BAU results showed strong correlation with VIDAS®3 (r = 0.6, p < 0.0001) and moderate correlation with VITROS® (r = 0.5, p < 0.0001) and CL-900i® (r = 0.4, p < 0.0001), suggesting that FinecareTM can be used as a surrogate for the advanced automated assays to measure S-RBD BAU/mL.

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Determination of neutralizing antibody titers is still considered the gold standard for infection protection. A full virus neutralization test (VNT) with replication-competent, infectious SARS-CoV-2, is labor-intensive and requires Biosafety Level 3 certified laboratories. Therefore, several commercial SARS-CoV-2 surrogate virus neutralization tests (sVNTs) have been developed that aim to detect neutralizing antibodies targeting the receptor binding domain (RBD) of the viral spike glycoprotein (S). Neutralizing antibodies to the RBD block its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor protein. Here, we compared a full virus neutralization test (VNT) with two SARS-CoV-2 surrogate virus neutralization tests (sVNT) and validated them in two cohorts of i) convalescent SARS-CoV-2-infected individuals and ii) COVID vaccinated individuals. The sVNTs showed highly different results both, compared to the VNT-titers and also between the two cohorts. This indicates that currently, sVNT provide a qualitative instead of a quantitative measurement of neutralizing antibodies. The findings in this work show that the cutoff levels for sVNTs might need to be readjusted for convalescent and vaccinated individuals.

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The coronavirus disease 2019 (COVID-19) pandemic has progressed over 2 years since its onset causing significant health concerns all over the world and is currently curtailed by mass vaccination. Immunity acquired against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be following either infection or vaccination. However, one can never be sure whether the acquired immunity is adequate to protect the individual from subsequent infection because of three important factors: individual variations in humoral response dynamics, waning of protective antibodies over time, and the emergence of immune escape mutants. Therefore, a test that can accurately differentiate the protected from the vulnerable is the need of the hour. The plaque reduction neutralization assay is the conventional gold standard test for estimating the titers of neutralizing antibodies that confer protection. However, it has got several drawbacks, which hinder the practical application of this test for wide-scale usage. Hence, various tests have been developed to detect protective immunity against SARS-CoV-2 that directly or indirectly assess the presence of neutralizing antibodies to SARS-CoV-2 in a lower biosafety setting. In this review, the pros and cons of the currently available assays are elaborated in detail and special focus is put on the scope of the novel split nanoluciferase technology for detecting SARS-CoV-2 neutralizing antibodies.

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OBJECTIVES: Evaluating anti-SARS-CoV-2 antibody levels is a current priority to drive immunization, as well as to predict when a vaccine booster dose may be required and for which priority groups. The aim of our study was to investigate the kinetics of anti-SARS-CoV-2 Spike S1 protein IgG (anti-S1 IgG) antibodies and neutralizing antibodies (NAbs) in an Italian cohort of healthcare workers (HCWs), following the Pfizer/BNT162b2 mRNA vaccine, over a period of up to six months after the second dose. METHODS: We enrolled 57 HCWs, without clinical history of COVID-19 infection. Fluoroenzyme-immunoassay was used for the quantitative anti-S1 IgG antibodies at different time points T1 (one month), T3 (three months) and T6 (six months) following the second vaccine shot. Simultaneously, a commercial surrogate virus neutralization test (sVNT) was used for the determination of NAbs, expressed as inhibition percentage (% IH). RESULTS: Median values of anti-S1 IgG antibodies decreased from T1 (1,452 BAU/mL) to T6 (104 BAU/mL) with a percent variation of 92.8% while the sVNT showed a percent variation of 34.3% for the same time frame. The decline in anti-S1 IgG antibodies from T1 to T6 was not accompanied by a loss of the neutralizing capacity of antibodies. In fact at T6 a neutralization percentage <20% IH was observed only in 3.51% of HCWs. CONCLUSIONS: Our findings reveal that the decrease of anti-S1 IgG levels do not correspond in parallel to a decrease of NAbs over time, which highlights the necessity of using both assays to assess vaccination effectiveness.

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Previous studies on immune responses following COVID-19 vaccination in patients with common variable immunodeficiency (CVID) were inconclusive with respect to the ability of the patients to produce vaccine-specific IgG antibodies, while patients with milder forms of primary antibody deficiency such as immunoglobulin isotype deficiency or selective antibody deficiency have not been studied at all. In this study we examined antigen-specific activation of CXCR5-positive and CXCR5-negative CD4+ memory cells and also isotype-specific and functional antibody responses in patients with CVID as compared to other milder forms of primary antibody deficiency and healthy controls six weeks after the second dose of BNT162b2 vaccine against SARS-CoV-2. Expression of the activation markers CD25 and CD134 was examined by multi-color flow cytometry on CD4+ T cell subsets stimulated with SARS-CoV-2 spike peptides, while in parallel IgG and IgA antibodies and surrogate virus neutralization antibodies against SARS-CoV-2 spike protein were measured by ELISA. The results show that in CVID and patients with other milder forms of antibody deficiency normal IgG responses (titers of spike protein-specific IgG three times the detection limit or more) were associated with intact vaccine-specific activation of CXCR5-negative CD4+ memory T cells, despite defective activation of circulating T follicular helper cells. In contrast, CVID IgG nonresponders showed defective vaccine-specific and superantigen-induced activation of both CD4+T cell subsets. In conclusion, impaired TCR-mediated activation of CXCR5-negative CD4+ memory T cells following stimulation with vaccine antigen or superantigen identifies patients with primary antibody deficiency and impaired IgG responses after BNT162b2 vaccination.

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BACKGROUND: Several commercial surrogate Virus Neutralization Tests (sVNTs) have been developed in the last year. Neutralizing anti-SARS-CoV-2 antibodies through interaction with Spike protein Receptor Binding Domain (S-RBD) can block the virus from entering and infecting host cells. However, there is a lack of information about the functional activity of SARS-CoV-2 antibodies that may be associated with protective responses. For these reasons, to counteract viral infection, the conventional virus neutralization test (VNT) is still considered the gold standard. The aim of this study was to contribute more and detailed information about sVNTs' performance, by determining in vitro the anti-SARS-CoV-2 neutralizing antibody concentration using four different commercial assays and then comparing the obtained data to VNT. METHODS: Eighty-eight samples were tested using two chemiluminescence assays (Snibe and Mindray) and two ELISA assays (Euroimmun and Diesse). The antibody titers were subsequently detected and quantified by VNT. RESULTS: The overall agreement between each sVNT and VNT was 95.45% for Euroimmun and 98.86% for Diesse, Mindray and Snibe. Additionally, we investigated whether the sVNTs were closer to the gold standard than traditional anti-SARS-CoV-2 antibody assays S-RBD or S1 based, finding a higher agreement mean value for sVNTs (98.01 ± 1.705% vs 95.45 ± 1.921%; p < 0.05). Furthermore, Spearman's statistical analysis for the correlation of sVNT versus VNT showed r = 0.666 for Mindray; r = 0.696 for Diesse; r = 0.779 for Mindray and r = 0.810 for Euroimmun. CONCLUSIONS: Our data revealed a good agreement between VNT and sVNTs. Despite the VNT still remains the gold standard, the sVNT might be a valuable tool for screening wider populations.

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The humoral response through neutralizing antibodies (NAbs) is a key component of the immune response to COVID-19. However, the plaque reduction neutralization test (PRNT), the gold standard for determining NAbs, is technically demanding, time-consuming and requires BSL-3 conditions. Correlating the NAbs and total antibodies levels, assessed by generalized and automated serological tests, is crucial. Through a commercial surrogate virus neutralization test (sVNT), we aimed to evaluate the production of SARS-CoV-2 NAbs in a set of vaccinated healthcare workers and to correlate these NAbs with the SARS-CoV-2 IgG anti-S1, anti-RBD and anti-S2 serological titers. We found that 6 months after vaccination, only 74% maintain NAbs for the Wuhan strain/UK variant (V1) and 47% maintain NAbs for the South African and Brazil variants (V2). Through Spearman's correlation, we found the following correlations between the percentage of inhibition of NAbs and the SARS-CoV-2 IgG II Quant (Abbott Laboratories, Chicago, IL, USA) and BioPlex 2200 SARS-CoV-2 IgG Panel (Bio-Rad, Hercules, CA, USA) immunoassays: rho = 0.87 (V1) and rho = 0.73 (V2) for anti-S1 assessed by Abbott assay; rho = 0.77 (V1) and rho = 0.72 (V2) for anti-S1, rho = 0.88 (V1) and rho = 0.82 (V2) for anti-RBD, and rho = 0.68 (V1) and rho = 0.60 (V2) for anti-S2 assessed by BioPlex assay (p < 0.001 for all). In conclusion, we found a strong correlation between this fast, user-friendly, mobile and bio-safe sVNT and the serological immunoassays.

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BACKGROUND: The SARS-CoV-2 virus has mutated and evolved since the inception of the COVID-19 pandemic bringing into question the future effectiveness of current vaccines and antibody therapeutics. With evolution of the virus updated methods for the evaluation of the immune response in infected and vaccinated individuals are required to determine the durability of the immune response to SARS-CoV-2 variants. METHODS: We developed a multiplexed surrogate virus neutralization test (plex-sVNT) that simultaneously measures the ability of antibodies in serum to inhibit binding between angiotensin converting enzyme-2 (ACE2) and 7 SARS-CoV-2 trimeric spike protein variants, including wild type, B.1.1.7(α), B.1.351(ß), P.1(γ), B.1.617.2(δ), B.1.617.1(κ), and B.1.429(ε). The assay was validated against a plaque reduction neutralization test (PRNT).We evaluated 170 samples from 97 COVID-19 patients and 281 samples from 188 individuals that received the Pfizer-BioNTech or Moderna mRNA vaccines. RESULTS: The plex-sVNT demonstrated >96% concordance with PRNT. Antibody neutralization activity was significantly reduced for all SARS-CoV-2 variants compared to wild type in both the infected and vaccinated cohorts. There was a decline in overall antibody neutralization activity, within both cohorts, out to 5 months post infection or vaccination, with the rate of decline being more significant for the vaccinated. CONCLUSIONS: The plex-sVNT provides a correlative measure to PRNT and a convenient approach for evaluating antibody neutralization against SARS-CoV-2 variants. Neutralization of SARS-CoV-2 variants is reduced compared to wild type and declines over the ensuing months after exposure or vaccination within each cohort, however it is still unknown what degree of neutralizing capacity is protective.

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BACKGROUND: Due to large vaccination efforts with novel vaccines there is an increasing need for laboratory tests assessing successful immunizations with SARS-CoV-2 vaccines. Unfortunately classical neutralization assays are laborious, time-consuming and require an adequate biosafety level laboratory. Recently, convenient ELISA-based surrogate neutralization assays (sVNTs) for determination of neutralizing SARS-CoV-2 antibodies have been developed. STUDY DESIGN: Our study compares the two novel ELISA-based SARS-CoV-2 surrogate neutralization assays "cPass SARS-CoV-2 Surrogate Virus Neutralization Test Kit" (GenScript Biotech, USA) and the "TECO SARS-CoV-2 Neutralization Antibody Assay" (TECOmedical, Switzerland) using 93 sera drawn from health care workers (HCVs) 2-3 weeks following the second vaccination with mRNA-1273 and 40 control sera from the pre-SARS-CoV-2 era before 2019. RESULTS: We found a sensitivity of 100% and 91,4% and a specificity of 100% and 100% for the GenScript assay and the TECO assay, respectively. Both sVNTs show a high correlation with anti-S IgG. Moreover, both sVNTs correlate well with each other. CONCLUSIONS: Surrogate neutralization assays based on the RBD as bait feature a high specificity and sensitivity for identifying humoral neutralizing activity in individuals vaccinated with the spike-based vaccine mRNA-1273. Although these assays appear well-suited for confirming successful vaccinations with spike-based vaccines, additional studies should compare both assays regarding other purposes such as screening COVID-recovered patients or individuals vaccinated with inactivated whole virus vaccines.

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Assays to measure SARS-CoV-2-specific neutralizing antibodies are important to monitor seroprevalence, to study asymptomatic infections and to reveal (intermediate) hosts. A recently developed assay, the surrogate virus-neutralization test (sVNT) is a quick and commercially available alternative to the "gold standard" virus neutralization assay using authentic virus, and does not require processing at BSL-3 level. The assay relies on the inhibition of binding of the receptor binding domain (RBD) on the spike (S) protein to human angiotensin-converting enzyme 2 (hACE2) by antibodies present in sera. As the sVNT does not require species- or isotype-specific conjugates, it can be similarly used for antibody detection in human and animal sera. In this study, we used 298 sera from PCR-confirmed COVID-19 patients and 151 sera from patients confirmed with other coronavirus or other (respiratory) infections, to evaluate the performance of the sVNT. To analyze the use of the assay in a One Health setting, we studied the presence of RBD-binding antibodies in 154 sera from nine animal species (cynomolgus and rhesus macaques, ferrets, rabbits, hamsters, cats, cattle, mink and dromedary camels). The sVNT showed a moderate to high sensitivity and a high specificity using sera from confirmed COVID-19 patients (91.3% and 100%, respectively) and animal sera (93.9% and 100%), however it lacked sensitivity to detect low titers. Significant correlations were found between the sVNT outcomes and PRNT50 and the Wantai total Ig and IgM ELISAs. While species-specific validation will be essential, our results show that the sVNT holds promise in detecting RBD-binding antibodies in multiple species.

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Background: High-throughput assays that can infer neutralizing activity against SARS-CoV-2 are of great importance for assessing the immunity induced by natural infection and COVID-19 vaccines. We aimed to evaluate the performance and degree of correlation of three fully automated anti-SARS-CoV-2 immunoassays with neutralization activity using a surrogate virus-neutralizing test (sVNT) from GenScript, targeting the receptor-binding domain. Methods: 110 sera collected from PCR-confirmed asymptomatic COVID-19 individuals were tested for neutralizing antibodies (nAbs) using the sVNT. Positive samples were tested on three automated immunoassays targeting different viral antigens: Mindray CL-900i®, Abbott Architect, and Ortho VITROS®. The diagnostic sensitivity, specificity, agreement, and correlation with the sVNT were assessed. Receiver operating characteristic (ROC) curve analysis was performed to determine optimal thresholds for predicting the presence of neutralizing activity by each assay. Results: All three assays showed 100% specificities. The highest sensitivity was 99.0%, demonstrated by VITROS®, followed by 94.3%, for CL-900i®, and 81.0%, for Architect. Both VITROS® and CL-900i® had the strongest correlation with the sVNT (ρ = 0.718 and ρ = 0.712, respectively), while Architect showed a moderate correlation (ρ = 0.618). ROC curve analysis indicated that the manufacturer's recommended cutoff values are adequate for predicting the presence of nAbs and providing a strong correlation with the sVNT. Conclusion: VITROS® and CL-900i® serological assays, which detect antibodies against SARS-CoV-2 spike protein, could serve as reliable assays to predict neutralization activity after infection or vaccination.

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