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Evaluating Humoral Immunity against SARS-CoV-2: Validation of a Plaque-Reduction Neutralization Test and a Multilaboratory Comparison of Conventional and Surrogate Neutralization Assays.

ABSTRACT: The evaluation of humoral protective immunity against SARS-CoV-2 remains crucial in understanding both natural immunity and protective immunity conferred by the several vaccines implemented in the fight against COVID-19. The reference standard for the quantification of antibodies capable of neutralizing SARS-CoV-2 is the plaque-reduction neutralization test (PRNT). However, given that it is a laboratory-developed assay, validation is crucial in order to ensure sufficient specificity and intra- and interassay precision. In addition, a multitude of other serological assays have been developed, including enzyme-linked immunosorbent assay (ELISA), flow cytometry-based assays, luciferase-based lentiviral pseudotype assays, and commercially available human ACE2 receptor-blocking antibody tests, which offer practical advantages in the evaluation of the protective humoral response against SARS-CoV-2. In this study, we validated a SARS-CoV-2 PRNT to assess both 50% and 90% neutralization of SARS-CoV-2 according to guidelines outlined by the World Health Organization. Upon validation, the reference-standard PRNT demonstrated excellent specificity and both intra- and interassay precision. Using the validated assay as a reference standard, we characterized the neutralizing antibody response in specimens from patients with laboratory-confirmed COVID-19. Finally, we conducted a small-scale multilaboratory comparison of alternate SARS-CoV-2 PRNTs and surrogate neutralization tests. These assays demonstrated substantial to perfect interrater agreement with the reference-standard PRNT and offer useful alternatives to assess humoral immunity against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the causal agent of COVID-19, has infected over 246 million people and led to over 5 million deaths as of October 2021. With the approval of several efficacious COVID-19 vaccines, methods to evaluate protective immune responses will be crucial for the understanding of long-term immunity in the rapidly growing vaccinated population. The PRNT, which quantifies SARS-CoV-2-neutralizing antibodies, is used widely as a reference standard to validate new platforms but has not undergone substantial validation to ensure excellent inter- and intraassay precision and specificity. Our work is significant, as it describes the thorough validation of a PRNT, which we then used as a reference standard for the comparison of several alternative serological methods to measure SARS-CoV-2-neutralizing antibodies. These assays demonstrated excellent agreement with the reference-standard PRNT and include high-throughput platforms, which can greatly enhance capacity to assess both natural and vaccine-induced protective immunity against SARS-CoV-2.

SUBMITTER: Valcourt EJ

PROVIDER: S-EPMC8597631 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Determinants of protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using 40 plasma samples from convalescent individuals with mild to moderate coronavirus disease 2019 (COVID-19): four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate enzyme-linked immunosorbent assay (ELISA)-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor human angiotensin converting enzyme 2 (hACE2). Vero cells, Vero E6 cells, HEK293T cells expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND50) geometric mean titers (GMTs) that were highly correlated (Pearson r = 0.81 to 0.89) and ranged within 3.4-fold. The live virus assay and LV pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers, 141 and 178, respectively. ND50 titers positively correlated with plasma IgG targeting SARS-CoV-2 spike protein and RBD (r = 0.63 to 0.89), but moderately correlated with nucleoprotein IgG (r = 0.46 to 0.73). ND80 GMTs mirrored ND50 data and showed similar correlation between assays and with IgG concentrations. The VSV pseudovirus assay and LV pseudovirus assay with HEK293T/hACE2 cells in low- and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms.

Project description:BackgroundThe plaque reduction neutralization test (PRNT) remains the gold standard for the detection of serologic immune responses to dengue virus (DENV). While the basic concept of the PRNT remains constant, this test has evolved in multiple laboratories, introducing variation in materials and methods. Despite the importance of laboratory-to-laboratory comparability in DENV vaccine development, the effects of differing PRNT techniques on assay results, particularly the use of different dengue strains within a serotype, have not been fully characterized.MethodsWe conducted a systematic review and pooled analysis of published literature reporting individual-level PRNT titers to identify factors associated with heterogeneity in PRNT results and compared variation between strains within DENV serotypes and between articles using hierarchical models.ResultsThe literature search and selection criteria identified 8 vaccine trials and 25 natural exposure studies reporting 4,411 titers from 605 individuals using 4 different neutralization percentages, 3 cell lines, 12 virus concentrations and 51 strains. Of 1,057 titers from primary DENV exposure, titers to the exposure serotype were consistently higher than titers to non-exposure serotypes. In contrast, titers from secondary DENV exposures (n = 628) demonstrated high titers to exposure and non-exposure serotypes. Additionally, PRNT titers from different strains within a serotype varied substantially. A pooled analysis of 1,689 titers demonstrated strain choice accounted for 8.04% (90% credible interval [CrI]: 3.05%, 15.7%) of between-titer variation after adjusting for secondary exposure, time since DENV exposure, vaccination and neutralization percentage. Differences between articles (a proxy for inter-laboratory differences) accounted for 50.7% (90% CrI: 30.8%, 71.6%) of between-titer variance.ConclusionsAs promising vaccine candidates arise, the lack of standardized assays among diagnostic and research laboratories make unbiased inferences about vaccine-induced protection difficult. Clearly defined, widely accessible reference reagents, proficiency testing or algorithms to adjust for protocol differences would be a useful first step in improving dengue PRNT comparability and quality assurance.

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Evaluating Humoral Immunity against SARS-CoV-2: Validation of a Plaque-Reduction Neutralization Test and a Multilaboratory Comparison of Conventional and Surrogate Neutralization Assays.

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