Aug 2026· Journal of Virology· 0 citations· 18 references
Medicine
TL;DR
Three commonly used types of neutralization assay were compared and strong correlations and significant differences in measured neutralization between the different assays were found, highlighting areas for more precise and harmonized terminology and particular assay elements that must be considered in efforts to harmonize neutralization assays.
Abstract
ABSTRACT SARS-CoV-2 antibody neutralization is a correlate of protection for COVID-19 and has been used to guide public health decisions. However, there is no standard neutralization assay, and absolute titers differ between assays. To investigate mechanisms causing these absolute titer differences, we compared 28 samples with three assays: a live virus cytopathic effect (CPE)-based microneutralization assay, a live virus focus reduction (immunospot, IS) assay, and a pseudotyped virus luciferase expression reduction (pseudovirus, PV) assay. We found significant correlations between all assays (ρ > 0.95 and P < 0.0001 for all pairwise comparisons), but also significant differences in absolute titers by assay (paired t-tests P < 0.001). IS and PV assays that assess neutralization as the titer required to reduce foci/luminescence by 50% had the smallest difference, with 1.2-fold higher IS assay titers (95% confidence interval, CI: 1.2–1.2). CPE assay titers were 5.5-fold lower (95% CI: 5.4–5.5) than in the IS assay. We show that these differences can be explained (i) by accounting for the size of inoculum being neutralized (half the infectious virions in the IS and PV assays, “per virion,” and the entire inoculum half the time in the CPE assay, “per well”) and (ii) by removing serum during the 5-day culture in the CPE assay (so serum can only neutralize the inoculum, rather than acting on the inoculum and slowing viral growth during culture). These findings highlight areas for more precise and harmonized terminology and particular assay elements that must be considered in efforts to harmonize neutralization assays. IMPORTANCE Protection against COVID-19 is related to how well serum antibodies neutralize SARS-CoV-2. This neutralization can be assessed using different assays, which often provide significantly different estimates. To better understand why assay results vary, we compared three commonly used types of neutralization assay. We found both strong correlations and significant differences in measured neutralization between the different assays. Differences in the type of virus (live SARS-CoV-2 virus compared to a pseudotyped virus with SARS-CoV-2 spike protein), how long cells, virus, and antibody were allowed to interact, and whether neutralization of a batch of virions (“per well” neutralization) or individual infectious virions (“per virion”) was measured, all impacted virus neutralization. Understanding which factors have the largest impact on the differences between neutralization assays is an important first step toward harmonization of neutralization assays across the field. Protection against COVID-19 is related to how well serum antibodies neutralize SARS-CoV-2. This neutralization can be assessed using different assays, which often provide significantly different estimates. To better understand why assay results vary, we compared three commonly used types of neutralization assay. We found both strong correlations and significant differences in measured neutralization between the different assays. Differences in the type of virus (live SARS-CoV-2 virus compared to a pseudotyped virus with SARS-CoV-2 spike protein), how long cells, virus, and antibody were allowed to interact, and whether neutralization of a batch of virions (“per well” neutralization) or individual infectious virions (“per virion”) was measured, all impacted virus neutralization. Understanding which factors have the largest impact on the differences between neutralization assays is an important first step toward harmonization of neutralization assays across the field.
Using systems serology, it is possible to create an assay that quantifies a human receptor’s binding levels to a virus receptor binding protein in the presence of neutralizing antibodies. For example, human influenza A virus predominantly binds to alpha 2’6-linked sialic acids, while avian influenza A prefers alpha 2’3-linked sialic acids. Modeling this binding in the presence of neutralizing antibodies using a systems serology approach would allow us to quantify neutralization breadth and capacity at a throughput and scale not previously known.
Using four parameter or Michaelis-Menton fitted models, we can quantify the complete binding and complete inhibition of binding for virus receptor binding proteins and human receptor pairs at the multiplex level. From this, percent inhibition can be quantified, and a systems-predicted neutralizing antibody (SNAb) titer can be calculated. This titer was compared against existing neutralization assays and readouts for validation. This was done using serum and lavage samples from vaccinated/boosted non-human primates.
Neutralizing antibody levels, whether they were measured by microneutralization assay, hemagglutinin inhibition assay, or SNAb were tightly correlated, and showed dependence on route of vaccination. The SNAb assay could successfully multiplex hemagglutinin molecules to quantify the predicted neutralization of twelve analytes simultaneously. The SNAb assay was performance-validated and did not show an overcounting or undercounting of neutralizing antibody titers.
Neutralizing antibody levels can be quantified in an efficient way using a systems-based approach. This allows for the multiplexing of readouts in a high-throughput assay. The assay itself is built on four-parameter or Michaelis-Menton binding modeling, which does not bias the titer compared to other commonly used methods.
National Institutes of Health Grant CA260476, PO1AI165072, and U19AI135995, and National Institutes of Health Contract 75N93021C00029
Technological Innovations in Immunology (TECH)
Lindsay R. McManus, Qixin Wang, Kate S. Levine et al.· Journal of Immunology· 0 citations
ABSTRACT To establish a laboratory detection assay for a human neutralizing antibody (NAb) against rabies virus and to evaluate its efficiency in human serum samples. A pseudovirus pXN2‑RABV‑G harboring the RABV G gene was applied. The precision, robustness, linearity, stability, inclusivity, and limit of detection of the assay were evaluated. The consistency and correlation of the assay with ELISA and rapid fluorescent focus inhibition test (RFFIT) were analyzed. A pseudovirus-based neutralizing antibody (pVNA) detection system based on the pXN2‑RABV‑G was successfully developed and showed broad robustness (culture media: χ2 = 4.00, P > 0.05, cell passages: χ2 = 0.25, P > 0.05), precision (independent detection: P > 0.05), lower/upper limit of linearity (0.09 to 93.73 IU/mL, R2 > 0.95), and stability (P > 0.05). The limit of detection (LOD) was 2.63 IU/mL (95% CI: 1.81–3.84), expressed as the lowest human sera antibody level reliably distinguishable from background signals, and the coefficient of variation was less than 20%. Compared with the commercial ELISA and the gold standard RFFIT assay, the kappa values were 0.74 and 0.82, respectively, and the results were highly consistent. Linear correlation analysis revealed a strong correlation between these variables (R2 > 0.90). A total of 103 recipients from 2019 to 2023 were included in the serological testing and the following analysis. NAb titers were significantly correlated with vaccine dose (P < 0.001) and the most recent vaccination time (P < 0.001). The geometric mean titer (GMT) of the 5-dose vaccination group was greater than that of the 1-dose, 2-dose, 3-dose, and 4-dose groups (3.15 vs 3.19 vs 6.18 vs 12.21 vs 13.40 IU/mL). The established pVNA assay for human rabies NAb detection has a detection capability similar to that of the gold standard RFFIT, but it has the advantages of reduced biosafety requirements, laboratory activity, and time, which highlight its safety, high efficiency, and feasibility for screening and monitoring rabies virus antibodies after vaccination. IMPORTANCE Rabies is a lethal disease with no effective clinical treatment. Vaccination is the only way to prevent it routinely, which makes post-vaccination antibody testing essential. The current standard test requires a highly equipped laboratory, biosafety measures, and is time-consuming. This study develops a safe, efficient test for detecting rabies neutralizing antibodies using pseudovirus technology. It is comparable to the gold standard test in accuracy but easier to implement in routine labs, which enables broader use in screening and monitoring antibodies post-vaccination. This advantage addresses a key gap in rabies prevention, reducing the uncertainty for individuals with immunodeficiency who are nonresponsive to vaccination and at risk of rabies exposure. Rabies is a lethal disease with no effective clinical treatment. Vaccination is the only way to prevent it routinely, which makes post-vaccination antibody testing essential. The current standard test requires a highly equipped laboratory, biosafety measures, and is time-consuming. This study develops a safe, efficient test for detecting rabies neutralizing antibodies using pseudovirus technology. It is comparable to the gold standard test in accuracy but easier to implement in routine labs, which enables broader use in screening and monitoring antibodies post-vaccination. This advantage addresses a key gap in rabies prevention, reducing the uncertainty for individuals with immunodeficiency who are nonresponsive to vaccination and at risk of rabies exposure.
Xinxin Li, Pei Hu, Yueling Chen et al.· Journal of Clinical Microbio...· 0 citations
Coronavirus disease 2019 (COVID-19) presents with diverse symptoms ranging from mild to severe. Patients with severe COVID-19 can show impaired humoral and cellular immunity and excessive pro-inflammatory cytokine production. Antibodies are generated against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) due to the activation of the humoral immune system. Molecular-based testing is currently used to diagnose COVID-19. Determining antibody and CD4+ T cell counts can help monitor the antibody response to assess the risk of reinfection and vaccine response. This study aimed to determine the relationships between Aanti-SARS-CoV-2 titer, CD4+ T cell count, and COVID-19 severity. This analytic observational study used a cross-sectional design with consecutive sampling and was conducted at RSUP Prof. Dr. R. D. Kandou Manado Hospital from April to October 2021. It comprised 48 patients with COVID-19. The data were compared using Spearman’s rank correlation coefficient (rs). Anti-SARS-CoV-2 titers were nonsignificantly correlated with COVID-19 severity (rs = 0.081, p = 0.582) and CD4+ T cell counts (rs = 0.214, p = 0.145). In contrast, CD4+ T cell counts were significantly negatively correlated with COVID-19 severity (rs = -0.454, p = 0.001). Anti-SARS-Cov-2 titers cannot be used as an indicator of COVID-19 severity. Moreover, CD4+ T cell counts cannot be used as a marker of anti-SARS-CoV-2 titers. However, COVID-19 severity correlates negatively with CD4+ T cell counts, indicating that CD4+ T cell counts can be used as an indicator of COVID-19 severity.
Edwin Hadinata, E. Surachmanto, Paul N. Harijanto· Biology, Medicine, & Nat...· 0 citations
PURPOSE
The COVID-19 pandemic has witnessed the rapid evolution of the SARS-CoV-2 virus, resulting in the emergence of multiple variants with mutations in critical regions of the spike protein, notably in the receptor binding domain. These mutations can lead to immune evasion and breakthrough (BT) infections, even in the vaccinated individuals. While previous studies have documented various mutations, there is a limited understanding of these mutations in different epitopes and their influence on the vaccine elicited immune response.
METHODS
Neutralizing antibodies were detected using Plaque reduction neutralization test and peptide enzyme linked immunosorbent assay was standardized to identify possible epitopes contributing to immune escape. We used GraphPad Prism 9.5.0 (525) for analysis. Results were significant at p < 0.05.
RESULTS
In this study, we observed that individuals with hybrid immunity, defined as immunity derived from both the natural infection and vaccination, exhibit significantly higher levels of neutralizing antibodies than individuals who had been solely vaccinated with COVISHIELD. Consequently, we highlight the substantial differences in the reactivity of neutralizing antibodies against the Wuhan, Delta, and Omicron variants. Our findings indicate that individuals who have recovered from COVID-19 and /or who experienced BT infections, showed stronger reactivity toward the conserved peptides than individuals vaccinated with no prior exposure.
CONCLUSIONS
COVISHIELD induced antibodies effectively neutralized original Wuhan strain but showed reduced neutralization against Delta and Omicron due to mutations in specific epitopes within their spike proteins. Mutations in variant specific epitopes can lead to evasion of the virus, emphasizing the importance of targeting linear region epitopes as critical binding sites.
P. Das, G. Sapkal, Pragya D. Yadav et al.· Indian Journal of Medical Mi...· 0 citations
Coronavirus disease 2019 (COVID-19) that broke out at end of 2019 caused a global damage to both lives and economy. Although the U.S. Food and Drug Administration (FDA) has approved one antiviral treatment and authorized others for emergency use, there is no fully effective antiviral therapy for COVID-19, which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Assays detecting virus-specific immuno-globulins (Ig) or nucleic acids in large-scale epidemiological, vaccine, and drug development studies remain limited due to high costs, reagent accessibility, and cumbersome protocols.
A multiplex bead-based assay was developed to simultaneously detect human IgM, IgG, and IgA antibodies against the SARS-CoV-2 spike receptor binding domain (RBD) in serum using flow cytometry. Assay performance was evaluated for sensitivity, specificity, reproducibility, and cross-reactivity and compared to another immuno-assay platform.
The assay enabled simultaneous measurement of three antibody isotypes across 624 samples within 2 h. Intra-plate coefficients of variation (CVs) ranged from 3.16 to 6.71%, and inter-plate CVs ranged from 3.33 to 5.49%, demonstrating high re-producibility. The platform also quantified background noise from nonspecific binding, facilitating straightforward data interpretation.
This novel, flexible multiplex bead-based assay utilizing a well-established platform provides a rapid and reproducible approach for detecting SARS-CoV-2-specific antibodies. Its high throughput capacity and low variability make it well suited for large-scale epidemiological, vaccine, and therapeutic studies. The platform’s adaptability further supports application to other infectious diseases, offering an ideal tool for broad immunological surveillance.
n/a
Technological Innovations in Immunology (TECH)
Ruo-Pan Huang, Benyue Zhang, Zhuo Zhang et al.· Journal of Immunology· 0 citations
Primary infection with one of the four dengue viruses (DENV-1—4) induces serotype-specific antibodies (Abs) that confer durable protection and cross-reactive Abs that offer transient immunity. While neutralizing Ab titers are commonly used to assess protection, they do not reliably predict disease outcome, and the role of Abs that mediate non-neutralizing effector functions remains poorly understood. We present an approach to evaluate the non-neutralizing Ab (nNAb) response to experimental DENV infection in humans.
In a phase 1 open-label study, 14 dengue—naïve subjects were subcutaneously inoculated with a slightly attenuated, live DENV-4 strain, H-241. Plasma was collected from pre- and 6 months post-infection to measure Ab-dependent complement deposition, Ab-dependent cellular phagocytosis, and Ab-dependent cellular cytotoxicity (ADCC). All assays used target cells infected with DENV or transfected to express DENV-derived surface-bound NS1 protein.
Infected subjects generated DENV-4-specific complement-fixing Abs and anti-NS1 Abs that facilitated monocyte-mediated phagocytosis of target cells. DENV-4-specific Abs also mediated NK cell activation in a subset of subjects.
Future work will focus on extending this functional study to recipients of a tetravalent vaccine followed by DENV-1 challenge to investigate how nNAbs correlate with disease protection/risk, and broaden our understanding of immune defense against dengue beyond neutralizing Abs.
DOD
Viral Immunology (VIR)
Y. Zheng, J. Currier, Heather Fribert et al.· Journal of Immunology· 0 citations
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