-
Scientific Reports Sep 2022To monitor vulnerability of countries to poliovirus (PV) outbreaks, serosurveillance of anti-PV neutralization antibody is conducted by conventional PV neutralization...
To monitor vulnerability of countries to poliovirus (PV) outbreaks, serosurveillance of anti-PV neutralization antibody is conducted by conventional PV neutralization test (cPNT), which uses live PV strains. We previously developed a pseudovirus PV neutralization test (pPNT) as an alternative to cPNT, which uses PV pseudovirus that expresses luciferase as a reporter in the infection without producing infectious PV. In the present study, we established a high-throughput pPNT (HTpPNT) for a large-scale serosurveillance. The HTpPNT system was evaluated with 600 human serum samples obtained from a broad range of age groups of healthy volunteers (ages of 0-89 years). HTpPNT showed high correlation with cPNT (R for anti-type 1, 2, and 3 PV neutralization antibody titres are 0.90, 0.84, and 0.90, respectively). By using HTpPNT, we analyzed relative neutralizing antibody titre of the sera against a type 1 PV wild-type strain (Mahoney strain) to that against the type 1 Sabin strain. As a result, a correlation between the age (≥ 60 years) and the relative neutralizing antibody titre was observed (n = 15-16, P = 0.0000023-0.041), while the types of PV vaccine (i.e., oral PV vaccine and Sabin strain-based IPV) had no effect. HTpPNT would serve as a useful alternative to cPNT in a large-scale serosurveillance.
Topics: Antibodies, Neutralizing; Antibodies, Viral; Humans; Luciferases; Middle Aged; Neutralization Tests; Poliomyelitis; Poliovirus; Poliovirus Vaccine, Oral
PubMed: 36167892
DOI: 10.1038/s41598-022-20544-6 -
Clinical Chemistry and Laboratory... Sep 2023To 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...
Kinetics and ability of binding antibody and surrogate virus neutralization tests to predict neutralizing antibodies against the SARS-CoV-2 Omicron variant following BNT162b2 booster administration.
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.
Topics: Humans; Antibodies, Neutralizing; SARS-CoV-2; Neutralization Tests; BNT162 Vaccine; Kinetics; COVID-19; Immunoglobulin G; Antibodies, Viral
PubMed: 37078220
DOI: 10.1515/cclm-2022-1258 -
Journal of Medical Virology Jan 2022Fully automated immunoassays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies that are strongly correlated with neutralization...
Fully automated immunoassays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies that are strongly correlated with neutralization antibodies (nAbs) are clinically important because they enable the assessment of humoral immunity after infection and vaccination. Access SARS-CoV-2 immunoglobulin M (IgM) and immunoglobulin G (IgG) II antibody tests are semi-quantitative, fully automated immunoassays that detect anti-receptor-binding domain (RBD) antibodies and might reflect nAb levels in coronavirus disease 2019 (COVID-19). However, no studies have investigated the clinical utility of these tests in association with nAbs to date. To evaluate the clinical utility of Access SARS-CoV-2 IgM and IgG II antibody tests and their correlation with the SARS-CoV-2 surrogate virus neutralization test (sVNT) that measures nAbs in patients with COVID-19, we analyzed 54 convalescent serum samples from COVID-19 patients and 89 serum samples from non-COVID-19 patients. The presence of anti-RBD antibodies was detected using Access SARS-CoV-2 IgM and IgG II antibody tests, while nAbs were measured by sVNT. The sensitivity and specificity of sVNT were 94.4% and 98.9%, respectively. There were strong positive correlations between the inhibition values of sVNT and the results of the Access SARS-CoV-2 IgM (R = 0.95, R = 0.90, p < 0.001) and IgG II antibody tests (R = 0.96, R = 0.92, p < 0.001). In terms of the presence of nAbs, the sensitivity and specificity were 98.1% and 98.9% in the IgM assay and 100.0% and 100.0% in the IgG II assay, respectively. The Access SARS-CoV-2 IgM and IgG II antibody tests showed high sensitivity and specificity for the detection of nAbs in COVID-19 patients and might be alternatives for measuring nAbs.
Topics: Adult; Aged; Aged, 80 and over; Antibodies, Neutralizing; Antibodies, Viral; COVID-19; Female; Humans; Immunoassay; Immunoglobulin G; Immunoglobulin M; Male; Middle Aged; Neutralization Tests; SARS-CoV-2; Sensitivity and Specificity
PubMed: 34524695
DOI: 10.1002/jmv.27338 -
Scientific Reports Nov 2022Rapid and accurate measurement of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)-specific neutralizing antibodies (nAbs) is paramount for monitoring...
Rapid and accurate measurement of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)-specific neutralizing antibodies (nAbs) is paramount for monitoring immunity in infected and vaccinated subjects. The current gold standard relies on pseudovirus neutralization tests which require sophisticated skills and facilities. Alternatively, recent competitive immunoassays measuring anti-SARS-CoV-2 nAbs are proposed as a quick and commercially available surrogate virus neutralization test (sVNT). Here, we report the performance evaluation of three sVNTs, including two ELISA-based assays and an automated bead-based immunoassay for detecting nAbs against SARS-CoV-2. The performance of three sVNTs, including GenScript cPass, Dynamiker, and Mindray NTAb was assessed in samples collected from SARS-CoV-2 infected patients (n = 160), COVID-19 vaccinated individuals (n = 163), and pre-pandemic controls (n = 70). Samples were collected from infected patients and vaccinated individuals 2-24 weeks after symptoms onset or second dose administration. Correlation analysis with pseudovirus neutralization test (pVNT) and immunoassays detecting anti-SARS-CoV-2 binding antibodies was performed. Receiver operating characteristic (ROC) curve analysis was generated to assess the optimal threshold for detecting nAbs by each assay. All three sVNTs showed an excellent performance in terms of specificity (100%) and sensitivity (100%, 97.0%, and 97.1% for GenScript, Dynamiker, and Mindray, respectively) in samples collected from vaccinated subjects. GenScript demonstrated the strongest correlation with pVNT (r = 0.743, R = 0.552), followed by Mindray (r = 0.718, R = 0.515) and Dynamiker (r = 0.608, R = 0.369). Correlation with anti-SARS-CoV-2 binding antibodies was variable, but the strongest correlations were observed between anti-RBD IgG antibodies and Mindray (r = 0.952, R = 0.907). ROC curve analyses demonstrated excellent performance for all three sVNT assays in both groups, with an AUC ranging between 0.99 and 1.0 (p < 0.0001). Also, it was shown that the manufacturer's recommended cutoff values could be modified based on the tested cohort without significantly affecting the sVNT performance. The sVNT provides a rapid, low-cost, and scalable alternative to conventional neutralization assays for measuring and expanding nAbs testing across various research and clinical settings. Also, it could aid in evaluating actual protective immunity at the population level and assessing vaccine effectiveness to lay a foundation for boosters' requirements.
Topics: Humans; SARS-CoV-2; COVID-19; RNA, Viral; Antibodies, Viral; Neutralization Tests; Enzyme-Linked Immunosorbent Assay; Antibodies, Neutralizing
PubMed: 36347859
DOI: 10.1038/s41598-022-21317-x -
Human Vaccines & Immunotherapeutics 2019The evaluation of the immunogenicity of Sabin strain based Inactivated Poliovirus Vaccines (sIPV) necessitates the use of wild strains in neutralization assays to assess...
The evaluation of the immunogenicity of Sabin strain based Inactivated Poliovirus Vaccines (sIPV) necessitates the use of wild strains in neutralization assays to assess the potential cross-reactivity of antibodies. The live virus strains including wild and Sabin strains must be handled in level 3 biocontainment laboratories. To develop an alternative assay without the use of a live virus, we constructed Mahoney, MEF-1, and Saukett pseudovirions by inserting luciferase reporter genes into intact capsid proteins. Afterward, we developed a pseudovirus-based neutralization test (pNT) and evaluated for the specificity and reproducibility. We tested serum samples from a clinical trial on sIPV vaccines by pNT and compared the results with those obtained from conventional neutralization tests (cNT). A strong correlation was observed between two methods, with the correlation coefficients of all three types of IPV vaccines being greater than 0.82 (p < 0.0001). The Geometric Mean Titer (GMT) values obtained by pNT were approximately four times higher than that by cNT, revealing the better sensitivity of pNT. In conclusion, pNT is a safe, rapid and sensitive quantitative assay with the potential of being an alternative for the evaluation of the potency of polio vaccines.
Topics: Antibodies, Neutralizing; Antibodies, Viral; Cell Line; Clinical Trials, Phase II as Topic; Humans; Neutralization Tests; Poliovirus; Poliovirus Vaccine, Inactivated; Reproducibility of Results; Sensitivity and Specificity
PubMed: 30273512
DOI: 10.1080/21645515.2018.1526553 -
Transboundary and Emerging Diseases Sep 2022We monitored the severe acute respiratory syndrome coronavirus 2 antibody response in seven dogs and two cats by using two multispecies ELISA tests, plaque reduction...
We monitored the severe acute respiratory syndrome coronavirus 2 antibody response in seven dogs and two cats by using two multispecies ELISA tests, plaque reduction neutralisation test and virus neutralization. SARS-CoV-2 neutralizing antibodies in pets persisted up to 10 months since the first positive testing, thus replicating observations in COVID-19 human patients.
Topics: Animals; Antibodies, Neutralizing; Antibodies, Viral; COVID-19; Dog Diseases; Dogs; Humans; Neutralization Tests; SARS-CoV-2
PubMed: 34469620
DOI: 10.1111/tbed.14308 -
Journal of Virological Methods Feb 2020Foot-and-mouth disease virus (FMDV) is a highly variable RNA virus existing as seven different serotypes. The antigenic variability between and within serotypes can...
Foot-and-mouth disease virus (FMDV) is a highly variable RNA virus existing as seven different serotypes. The antigenic variability between and within serotypes can limit the cross-reactivity and therefore the in vivo cross-protection of vaccines. Selection of appropriate vaccine strains is crucial in the control of FMD. Determination of indirect relationships (r-value) between potential vaccine strains and field strains based on antibody responses against both are routinely used for vaccine matching purposes. Aiming at the investigation of the repeatability, reproducibility and comparability of r-value determination within and between laboratories and serological tests, a small scale vaccine matching ring test for FMDV serotype A was organized. Well-characterized serum pools from cattle vaccinated with a monovalent A24/Cruzeiro/Brazil/55 (A24) FMD vaccine with known in vivo protection status (homologous and heterologous) were distributed to four laboratories to determine r-values for the heterologous FMD strains A81/Argentina/87, A/Argentina/2000 and A/Argentina/2001 using the virus neutralization tests (VNT) and liquid phase blocking ELISA (LPBE). Within laboratories, the repeatability of r-value determination was high for both antibody assays. VNT resulted in reproducible and comparable r-values between laboratories, indicative of a lack of antigenic relatedness between the A24 strain and the heterologous strains tested in this work, thus corresponding to some of the in vivo findings with these strains. Using LPBE, similar trends in r-values were observed in all laboratories, but the overall reproducibility was lower than with VNT. Inconsistencies between laboratories may at least in part be attributed to differences in LPBE protocols as well as the in preexisting information generated in each laboratory (such as antibody titer-protection correlation curves). To gain more insight in the LPBE-derived r-values standard bovine control sera were included in the antibody assays performed in each laboratory and a standardization exercise was performed.
Topics: Animals; Cattle; Foot-and-Mouth Disease; Neutralization Tests; Observer Variation; Reproducibility of Results; Serologic Tests; Viral Vaccines
PubMed: 31765721
DOI: 10.1016/j.jviromet.2019.113786 -
Viruses May 2023The current gold standard assay for detecting neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the conventional...
The current gold standard assay for detecting neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the conventional virus neutralization test (cVNT), which requires infectious virus and a biosafety level 3 laboratory. Here, we report the development of a SARS-CoV-2 surrogate virus neutralization test (sVNT) that, with Luminex technology, detects NAbs. The assay was designed to mimic the virus-host interaction and is based on antibody blockage between the human angiotensin-converting enzyme 2 (hACE2) receptor and the spike (S) protein of the Wuhan, Delta, and Omicron (B.1.1.529) variants of SARS-CoV-2. The sVNT proved to have a 100% correlation with a SARS-CoV-2 cVNT regarding qualitative results. Binding between the hACE2 receptor and the S1 domain of the B.1.1.529 lineage of the Omicron variant was not observed in the assay but between the receptor and an S1 + S2 trimer and the receptor binding domain (RBD) in a reduced manner, suggesting less efficient receptor binding for the B.1.1.529 Omicron variant. The results indicate that the SARS-CoV-2 sVNT is a suitable tool for both the research community and the public health service, as it may serve as an efficient diagnostic alternative to the cVNT.
Topics: Humans; Neutralization Tests; Angiotensin-Converting Enzyme 2; SARS-CoV-2; COVID-19; Antibodies, Neutralizing; Spike Glycoprotein, Coronavirus; Antibodies, Viral
PubMed: 37376580
DOI: 10.3390/v15061280 -
Journal of Immunological Methods Dec 2021In response to the SARS-CoV-2 pandemic many vaccines have been developed and evaluated in human clinical trials. The humoral immune response magnitude, composition and...
In response to the SARS-CoV-2 pandemic many vaccines have been developed and evaluated in human clinical trials. The humoral immune response magnitude, composition and efficacy of neutralizing SARS-CoV-2 are essential endpoints for these trials. Robust assays that are reproducibly precise, linear, and specific for SARS-CoV-2 antigens would be beneficial for the vaccine pipeline. In this work we describe the methodologies and clinical qualification of three SARS-CoV-2 endpoint assays. We developed and qualified Endpoint titer ELISAs for total IgG, IgG1, IgG3, IgG4, IgM and IgA to evaluate the magnitude of specific responses to the trimeric spike (S) antigen and total IgG specific to the spike receptor binding domain (RBD) of SARS-CoV-2. We also qualified a pseudovirus neutralization assay which evaluates functional antibody titers capable of inhibiting the entry and replication of a lentivirus containing the Spike antigen of SARS-CoV-2. To complete the suite of assays we qualified a plaque reduction neutralization test (PRNT) methodology using the 2019-nCoV/USA-WA1/2020 isolate of SARS-CoV-2 to assess neutralizing titers of antibodies in plasma from normal healthy donors and convalescent COVID-19 individuals.
Topics: Enzyme-Linked Immunosorbent Assay; Humans; Immunity, Humoral; Neutralization Tests; Pandemics; SARS-CoV-2
PubMed: 34599915
DOI: 10.1016/j.jim.2021.113160 -
Journal of Virological Methods Dec 2020Feline coronaviruses (FCoV) are members of the alphacoronavirus genus that are further characterized by serotype (types I and II) based on the antigenicity of the spike...
Feline coronaviruses (FCoV) are members of the alphacoronavirus genus that are further characterized by serotype (types I and II) based on the antigenicity of the spike (S) protein and by pathotype based on the associated clinical conditions. Feline enteric coronaviruses (FECV) are associated with the vast majority of infections and are typically asymptomatic. Within individual animals, FECV can mutate and cause a severe and usually fatal disease called feline infectious peritonitis (FIP), the leading infectious cause of death in domestic cat populations. There are no approved antiviral drugs or recommended vaccines to treat or prevent FCoV infection. The plaque reduction neutralization test (PRNT) traditionally employed to assess immune responses and to screen therapeutic and vaccine candidates is time-consuming, low-throughput, and typically requires 2-3 days for the formation and manual counting of cytolytic plaques. Host cells are capable of carrying heavy viral burden in the absence of visible cytolytic effects, thereby reducing the sensitivity of the assay. In addition, operator-to-operator variation can generate uncertainty in the results and digital records are not automatically created. To address these challenges we developed a novel high-throughput viral microneutralization assay, with quantification of virus-infected cells performed in a plate-based image cytometer. Host cell seeding density, microplate surface coating, virus concentration and incubation time, wash buffer and fluorescent labeling were optimized. Subsequently, this FCoV viral neutralization assay was used to explore immune correlates of protection using plasma from naturally FECV-infected cats. We demonstrate that the high-throughput viral neutralization assay using the Celigo Image Cytometer provides a robust and efficient method for the rapid screening of therapeutic antibodies, antiviral compounds, and vaccines. This method can be applied to various viral infectious diseases to accelerate vaccine and antiviral drug discovery and development.
Topics: Animals; Cat Diseases; Cats; Cell Line; Coronavirus Infections; Coronavirus, Feline; Feline Infectious Peritonitis; High-Throughput Screening Assays; Image Cytometry; Neutralization Tests; Viral Load
PubMed: 32979406
DOI: 10.1016/j.jviromet.2020.113979