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SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.

Hantaan orthohantavirus (HTNV) is responsible for severe hemorrhagic fever with renal syndrome (HFRS), which has a case fatality rate of 1% to 10%. Currently, the inactive vaccine licensed in endemic areas elicit low levels of neutralizing antibodies (NAbs). Early NAbs administration is helpful for patients recovery from HFRS. Therefore, measuring NAbs is crucial for evaluating the immune response following infection or vaccination. The golden standard for HTNV NAbs measurement is the focus reduction neutralization test (FRNT), which typically requires skilled technicians and is performed under high biosafety containment facility. Here, we established a surrogate NAbs titration method with replication-competent vesicular stomatitis virus (VSV) bearing HTNV glycoprotein (rVSV-HTNV-GP) based plaque reduction neutralization test (PRNT). Then compared and correlated this method with the authentic HTNV based FRNT, and applied it to measure the NAbs level in 47 serum samples from HFRS patients, healthy donors and inactive vaccine recipients. We observed positive correlations between two neutralization assays among HFRS patients and inactive vaccine recipients (R 2  = 0.5994 and 0.3440, respectively) and confirmed the clear specificity with healthy donors without vaccinated and reproducibility with three more assays. Our results suggest that rVSV-HTNV-GP based PRNT is a reliable lower-biosafety level surrogate for HTNV NAbs evaluation, which is easy to perform with higher sensitivity. Graphical abstract

Vaccine-induced neutralizing antibodies (nAbs) are key biomarkers considered to be associated with vaccine efficacy. In United States government-sponsored phase 3 efficacy trials of COVID-19 vaccines, nAbs are measured by two different validated pseudovirus-based SARS-CoV-2 neutralization assays, with each trial using one of the two assays. Here we describe and compare the nAb titers obtained in the two assays. We observe that one assay consistently yielded higher nAb titers than the other when both assays were performed on the World Health Organization’s anti-SARS-CoV-2 immunoglobulin International Standard, COVID-19 convalescent sera, and mRNA-1273 vaccinee sera. To overcome the challenge this difference in readout poses in comparing/combining data from the two assays, we evaluate three calibration approaches and show that readouts from the two assays can be calibrated to a common scale. These results may aid decision-making based on data from these assays for the evaluation and licensure of new or adapted COVID-19 vaccines.

Respiratory Syncytial Virus (RSV), a leading cause of lower respiratory tract illness, has been a focus of vaccine development efforts in recent years. RSV neutralisation assays are particularly useful in the evaluation of immunogenicity of RSV vaccine candidates. Here we report a collaborative study that was conducted with the aim to establish the 1st International Standard for antiserum to RSV, to enable the standardisation of results across multiple assay formats. Two candidate standards were produced from serum samples donated by healthy adult individuals. 25 laboratories from 12 countries, including university laboratories, manufacturers/developers of RSV vaccines and public health laboratories, participated in the study. The study samples comprised the two candidate standards, NIBSC codes 16/284 and 16/322, naturally infected adult sera, age stratified naturally infected paediatric sera, sera from RSV vaccine clinical trials in maternal and elderly subjects, a monoclonal antibody to RSV (palivizumab), two cotton rat serum samples and samples from the BEI Resources panel of human antiserum and immune globulin to RSV. The collaborative study showed that between-laboratory variability in neutralisation titres was substantially reduced when values were expressed relative to those of either of the two candidate international standards. Stability of 16/284 and 16/322 maintained for 6 months at different temperatures showed no significant loss of activity (relative to that at −20 °C storage temperature) at temperatures of up to +20 °C. Based on these results, 16/284 was established as the 1st International Standard for antiserum to RSV, with an assigned unitage of 1000 International Units (IU) of anti-RSV neutralising antibodies per vial, by the WHO Expert Committee on Biological Standardisation, with 16/322 suitable as a possible replacement standard for 16/284.

•A wide variety of RSV neutralization assay formats are currently used by the field.•This study assessed the current level of agreement between twelve assay formats.•Results showed precision was consistently high, whereas agreement varied widely.•Results indicated harmonization could be improved with an international standard.•Results provided information on samples that may be appropriate for IS development. A current barrier to the standardized evaluation of respiratory syncytial virus (RSV) vaccine candidates is the wide variety of virus neutralization assay formats currently in use for assessing immunogenicity. Assay formats vary widely in labor intensiveness, duration, and sample throughput. Furthermore, the cell lines and virus strains used are not consistent among formats. The purpose of this multi-laboratory study was to assess the variability across a diverse array of assay formats that quantitate RSV neutralizing antibodies. Using a common specimen panel, the degree of overall agreement among existing assays was evaluated to inform on the need for harmonization of assay results. A total of 12 laboratories participated in the blinded survey study by testing a panel comprised of 57 samples chosen to span the reportable titer range of the assays. An independent statistical analysis was conducted to measure overall agreement of assay results. This analysis showed that precision was consistently high, whereas agreement varied widely among assays. To examine whether agreement could be improved, we conducted a harmonization exercise using a variety of sample types as pseudo standards. The results showed that the level of agreement could be improved, and provided information on the suitability of samples for developing an international standard.

Background Dengue is a global public health challenge which requires accurate diagnostic methods for surveillance and control. The gold standard for detecting dengue neutralizing antibodies (nAbs) is the plaque reduction neutralization test (PRNT), which is both labor-intensive and time-consuming. This study aims to evaluate three alternative approaches, namely, the MTT-based (or (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) microneutralization assay, the xCELLigence real-time cell analysis (RTCA), and the immuno-plaque assay-focus reduction neutralization test (iPA-FRNT). Methods Twenty-two residual serum samples were tested for DENV-2 nAbs using all four assays at three neutralization endpoints of 50%, 70% and 90% inhibition in virus growth. For each neutralization endpoint, results were compared using linear regression and correlation analyses. Test performance characteristics were further obtained for iPA-FRNT using 38 additional serum samples. Results Positive correlation of DENV-2 neutralization titers for the MTT-based microneutralization assay and the PRNT assay was only observed at the neutralization endpoint of 50% ( r  = 0.690). In contrast, at all three neutralization end points, a linear trend and positive correlation of DENV-2 neutralization titers for the xCELLigence RTCA and the PRNT assays were observed, yielding strong or very strong correlation ( r  = 0.829 to 0.967). This was similarly observed for the iPA-FRNT assay ( r  = 0.821 to 0.916), which also offered the added advantage of measuring neutralizing titers to non-plaque forming viruses. Conclusion The xCELLigence RTCA and iPA-FRNT assays could serve as suitable alternatives to PRNT for dengue serological testing. The decision to adopt these methods may depend on the laboratory setting, and the utility of additional applications offered by these technologies.

Standard precautions to minimize the risk of SARS-CoV-2 transmission implies that infected cell cultures and clinical specimens may undergo some sort of inactivation to reduce or abolish infectivity. We evaluated three heat inactivation protocols (56 °C-30 min, 60 °C-60 min and 92 °C-15 min) on SARS-CoV-2 using (i) infected cell culture supernatant, (ii) virus-spiked human sera (iii) and nasopharyngeal samples according to the recommendations of the European norm NF EN 14476-A2. Regardless of the protocol and the type of samples, a 4 Log10 TCID50 reduction was observed. However, samples containing viral loads > 6 Log10 TCID50 were still infectious after 56 °C-30 min and 60 °C-60 min, although infectivity was < 10 TCID50. The protocols 56 °C-30 min and 60 °C-60 min had little influence on the RNA copies detection, whereas 92 °C-15 min drastically reduced the limit of detection, which suggests that this protocol should be avoided for inactivation ahead of molecular diagnostics. Lastly, 56 °C-30 min treatment of serum specimens had a negligible influence on the results of IgG detection using a commercial ELISA test, whereas a drastic decrease in neutralizing titers was observed.

Background SARS-CoV-2 is a novel coronavirus that emerged in 2019 and is now classified in the genus Coronavirus with closely related SARS-CoV. SARS-CoV-2 is highly pathogenic in humans and is classified as a biosafety level (BSL)-3 pathogen, which makes manipulating it relatively difficult due to its infectious nature. Methods To circumvent the need for BSL-3 laboratories, an alternative assay was developed that avoids live virus and instead uses a recombinant VSV expressing luciferase and possesses the full length or truncated spike proteins of SARS-CoV-2. Furthermore, to measure SARS-CoV-2 neutralizing antibodies under BSL2 conditions, a chemiluminescence reduction neutralization test (CRNT) for SARS-CoV-2 was developed. The neutralization values of the serum samples collected from hospitalized patients with COVID-19 or SARS-CoV-2 PCR-negative donors against the pseudotyped virus infection evaluated by the CRNT were compared with antibody titers determined from an enzyme-linked immunosorbent assay (ELISA) or an immunofluorescence assay (IFA). Results The CRNT, which used whole blood collected from hospitalized patients with COVID-19, was also examined. As a result, the inhibition of pseudotyped virus infection was specifically observed in both serum and whole blood and was also correlated with the results of the IFA. Conclusions In conclusion, the CRNT for COVID-19 is a convenient assay system that can be performed in a BSL-2 laboratory with high specificity and sensitivity for evaluating the occurrence of neutralizing antibodies against SARS-CoV-2.

Neutralizing antibodies can block the entry of viruses into host cells. Next-generation influenza vaccines should stimulate the production of high levels of neutralizing antibodies capable of preventing influenza infection or reducing the severity of disease. Currently, multiple assays and protocols are used to measure influenza neutralization and there is a critical need to compare antibody responses elicited by different candidate vaccines tested in different populations to advance the most promising vaccine candidates to efficacy studies and licensure. A standardized approach for safely testing human sera against influenza viruses is to employ replication restricted reporter influenza viruses in which an essential viral gene (i.e., polymerase basic 1, PB1, gene) is replaced with a reporter gene. The replication restricted reporter viruses can be propagated in specific cell lines expressing the deleted viral gene, which make them suitable for biosafety level 2 (BSL-2) laboratory. Qualification of this method is needed for standardized testing in human clinical trials. In compliance with Good Clinical Laboratory Practice (GCLP) guidelines, we qualified a reporter virus microneutralization method by evaluating linearity, precision, accuracy, range, limits of detection and quantitation, specificity and robustness for A/New Caledonia/20/1999 (H1N1). We utilized unique sets of human clinical trial samples positive for influenza specific antibodies to enable development and testing. We compared the reporter virus microneutralization assay to the standard microneutralization assay with three additional viruses: A/California/07/2009 (H1N1), A/Singapore/INFIMH-16-0019/2016 (H3N2) and A/Texas/71/2017 (H3N2). The qualified reporter virus microneutralization assay was linear (R2 = 0.99, p-value <0.0001), precise (0–8 % GCV repeatability), accurate (%RE −18 to −12 %) with LLOQ of 19.4, robust for laboratory testing conditions (i.e. incubation times, temperature) and correlated with the traditional microneutralization assay (Spearman's r = 0.84–0.98, p < 0.001 for four virus strains). This qualified method will enable defining correlates of protection and contribute to meta-analyses of future human clinical trials. •The reporter virus assay is a robust BSL-2 method to measure neutralizing antibodies for pandemic influenza viruses.•We qualified a reporter virus assay showing linearity, precision, accuracy, specificity for human neutralizing antibodies.•This qualified method can assess countermeasures that aim to induce broad and potent heterologous influenza neutralization.

Anti-SARS-CoV-2-specific serological responses are a topic of ongoing evaluation studies. In the study presented here, the anti-SARS-CoV-2 surrogate neutralization assays by TECOmedical and DiaPROPH -Med were assessed in a head-to-head comparison with serum samples of individuals after vaccination as well as after previous infection with SARS-CoV-2. In case of discordant results, a cell culture-based neutralization assay was applied as a reference standard. The TECOmedical assay showed sensitivity and specificity of 100% and 61.3%, respectively, the DiaPROPH-Med assay 95.0% and 48.4%, respectively. As a side finding of the study, differences in the likelihood of expressing neutralizing antibodies could be shown for different exposition types. So, 60 of 81 (74.07%) of the samples with only one vaccination showed an expression of neutralizing antibodies in contrast to 85.71% (60 of 70 samples) of the samples with two vaccinations and 100% (40 of 40) of the samples from previously infected individuals. In conclusion, the both assays showed results similar to previous assessments. While the measured diagnostic accuracy of both assays requires further technical improvement of this diagnostic approach, as the calculated specificity values of 61.3% and 48.4%, respectively, appear acceptable for diagnostic use only in populations with a high percentage of positive subjects, but not at expectedly low positivity rates.