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Journal of Clinical Microbiology Jun 2021Methods designed to measure severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) humoral response include virus neutralization tests to determine antibody...
Methods designed to measure severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) humoral response include virus neutralization tests to determine antibody neutralization activity. For ease of use and universal applicability, surrogate virus neutralization tests (sVNTs) based on antibody-mediated blockage of molecular interactions have been proposed. A surrogate virus neutralization test was established on a label-free immunoassay platform (LF-sVNT). The LF-sVNT analyzes the binding ability of SARS-CoV-2 spike protein receptor-binding domain (RBD) to angiotensin-converting enzyme 2 (ACE2) after neutralizing RBD with antibodies in serum. The LF-sVNT neutralizing antibody titers (50% inhibitory concentration [IC]) were determined from serum samples ( = 246) from coronavirus disease 2019 (COVID-19) patients ( = 113), as well as the IgG concentrations and the IgG avidity indices. Although there was variability in the kinetics of the IgG concentrations and neutralizing antibody titers between individuals, there was an initial rise, plateau, and then in some cases a gradual decline at later time points after 40 days after symptom onset. The IgG avidity indices, in the same cases, plateaued after an initial rise and did not show a decline. The LF-sVNT can be a valuable tool in research and clinical laboratories for the assessment of the presence of neutralizing antibodies to COVID-19. This study is the first to provide longitudinal neutralizing antibody titers beyond 200 days post-symptom onset. Despite the decline of IgG concentration and neutralizing antibody titer, IgG avidity index increases, reaches a plateau, and then remains constant up to 8 months postinfection. The decline of antibody neutralization activity can be attributed to the reduction in antibody quantity rather than the deterioration of antibody quality, as measured by antibody avidity.
Topics: Antibodies, Neutralizing; Antibodies, Viral; COVID-19; Humans; Longitudinal Studies; Neutralization Tests; SARS-CoV-2; Spike Glycoprotein, Coronavirus
PubMed: 33827900
DOI: 10.1128/JCM.00193-21 -
Current Medical Science Dec 2021The ongoing Coronavirus disease 19 pandemic has likely changed the world in ways not seen in the past. Neutralizing antibody (NAb) assays play an important role in the... (Review)
Review
The ongoing Coronavirus disease 19 pandemic has likely changed the world in ways not seen in the past. Neutralizing antibody (NAb) assays play an important role in the management of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak. Using these tools, we can assess the presence and duration of antibody-mediated protection in naturally infected individuals, screen convalescent plasma preparations for donation, test the efficacy of immunotherapy, and analyze NAb titers and persistence after vaccination to predict vaccine-induced protective effects. This review briefly summarizes the various methods used for the detection of SARS-CoV-2 NAbs and compares their advantages and disadvantages to facilitate their development and clinical application.
Topics: Antibodies, Neutralizing; Antibodies, Viral; COVID-19; COVID-19 Serological Testing; COVID-19 Vaccines; Humans; Immunization, Passive; Neutralization Tests; Pandemics; SARS-CoV-2; COVID-19 Serotherapy
PubMed: 34935114
DOI: 10.1007/s11596-021-2470-7 -
Microbiology Spectrum Jun 2022Quick and accurate detection of neutralizing antibodies (nAbs) against yellow fever is essential in serodiagnosis during outbreaks for surveillance and to evaluate...
Quick and accurate detection of neutralizing antibodies (nAbs) against yellow fever is essential in serodiagnosis during outbreaks for surveillance and to evaluate vaccine efficacy in population-wide studies. All of this requires serological assays that can process a large number of samples in a highly standardized format. Albeit being laborious, time-consuming, and limited in throughput, the classical plaque reduction neutralization test (PRNT) is still considered the gold standard for the detection and quantification of nAbs due to its sensitivity and specificity. Here, we report the development of an alternative fluorescence-based serological assay (SNT) with an equally high sensitivity and specificity that is fit for high-throughput testing with the potential for automation. Finally, our novel SNT was cross-validated in several reference laboratories and against international WHO standards, showing its potential to be implemented in clinical use. SNT assays with similar performance are available for the Japanese encephalitis, Zika, and dengue viruses amenable to differential diagnostics. Fast and accurate detection of neutralizing antibodies (nAbs) against yellow fever virus (YFV) is key in yellow fever serodiagnosis, outbreak surveillance, and monitoring of vaccine efficacy. Although classical PRNT remains the gold standard for measuring YFV nAbs, this methodology suffers from inherent limitations such as low throughput and overall high labor intensity. We present a novel fluorescence-based serum neutralization test (SNT) with equally high sensitivity and specificity that is fit for processing a large number of samples in a highly standardized manner and has the potential to be implemented for clinical use. In addition, we present SNT assays with similar performance for Japanese encephalitis, Zika, and dengue viruses, opening new avenues for differential diagnostics.
Topics: Antibodies, Neutralizing; Antibodies, Viral; Encephalitis, Japanese; Humans; Neutralization Tests; Yellow Fever; Yellow fever virus; Zika Virus; Zika Virus Infection
PubMed: 35670599
DOI: 10.1128/spectrum.02548-21 -
Viruses Jun 2022A robust serological test to measure neutralizing antibodies against SARS-CoV-2 in biosafety level-2 (BSL-2) laboratories is useful for monitoring antibody response...
A robust serological test to measure neutralizing antibodies against SARS-CoV-2 in biosafety level-2 (BSL-2) laboratories is useful for monitoring antibody response after vaccination or natural infection. The gold standard assay is the conventional plaque reduction neutralization test (PRNT) which requires extensive labor, live viruses, and BSL-3 facilities. Recently, we developed a novel single-round infection fluorescent SARS-CoV-2 virus (SFV) that can be safely used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. In this study, we evaluated the performance of the neutralization test using this SFV with 80 PRNT-positive and 92 PRNT-negative clinical serum or plasma specimens. The SFV neutralization test (SFVNT) has 100% sensitivity and specificity compared to the PRNT. Furthermore, the neutralizing titers generated by the SFVNT and PRNT are highly correlated, with R2 = 0.903 (p < 0.0001). Due to high sensitivity, specificity, accuracy, and reproducibility, the SFVNT can be deployed for the large-scale testing of COVID-19 patients or vaccinated people in general lab settings.
Topics: Antibodies, Neutralizing; Antibodies, Viral; COVID-19; COVID-19 Serological Testing; Containment of Biohazards; Humans; Laboratories; Neutralization Tests; Reproducibility of Results; SARS-CoV-2
PubMed: 35746682
DOI: 10.3390/v14061211 -
Viruses Apr 2023Since their first documentation in 1952, plaque reduction neutralization tests (PRNTs) have become the choice of test for the measurement of neutralizing antibodies... (Review)
Review
Since their first documentation in 1952, plaque reduction neutralization tests (PRNTs) have become the choice of test for the measurement of neutralizing antibodies against a particular virus. However, PRNTs can be performed only against viruses that cause cytopathic effects (CPE). PRNTs also require skilled personnel and can be time-consuming depending on the time required for the virus to cause CPE. Hence, their application limits large-scale studies or epidemiological and laboratory investigations. Since 1978, many surrogate PRNTs or immunocolorimetric assay (ICA)-based focus reduction neutralization tests (FRNT) have been developed. In this article, ICAs and their utility in FRNTs for the characterization of neutralizing antibodies, homologous or heterologous cross-neutralization, and laboratory diagnosis of viruses of public health importance have been discussed. Additionally, possible advancements and automations have been described that may help in the development and validation of novel surrogate tests for emerging viruses.
Topics: Animals; Chlorocebus aethiops; Neutralization Tests; Antibodies, Viral; Vero Cells; Public Health; Antibodies, Neutralizing
PubMed: 37112919
DOI: 10.3390/v15040939 -
Journal of Molecular Cell Biology Aug 2022The 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... (Review)
Review
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.
Topics: Antibodies, Neutralizing; Antibodies, Viral; COVID-19; Humans; Neutralization Tests; SARS-CoV-2; Technology
PubMed: 35416249
DOI: 10.1093/jmcb/mjac023 -
Emerging Microbes & Infections Dec 2021Neutralizing antibodies to SARS-CoV-2 have been shown to correlate with protection in animals and humans, disease severity, survival, and vaccine efficacy. With the...
Neutralizing antibodies to SARS-CoV-2 have been shown to correlate with protection in animals and humans, disease severity, survival, and vaccine efficacy. With the ongoing large-scale vaccination in different countries and continuous surge of new variants of global concerns, a convenient, cost-effective and high-throughput neutralization test is urgently needed. Conventional SARS-CoV-2 neutralization test is tedious, time-consuming and requires a biosafety level 3 laboratory. Despite recent reports of neutralizations using different pseudoviruses with a luciferase or green fluorescent protein reporter, the laborious steps, inter-assay variability or high background limit their high-throughput potential. In this study we generated lentivirus-based pseudoviruses containing a monomeric infrared fluorescent protein reporter to develop neutralization assays. Similar tropism, infection kinetics and mechanism of entry through receptor-mediated endocytosis were found in the three pseudoviruses generated. Compared with pseudovirus D614, pseudovirus with D614G mutation had decreased shedding and higher density of S1 protein present on particles. The 50% neutralization titers to pseudoviruses D614 or D614G correlated with the plaque reduction neutralization titers to live SARS-CoV-2. The turn-around time of 48-72 h, minimal autofluorescence, one-step image quantification, expandable to 384-well, sequential readouts and dual quantifications by flow cytometry support its high-throughput and versatile applications at a non-reference and biosafety level 2 laboratory, in particular for assessing the neutralization sensitivity of new variants by sera from natural infection or different vaccinations during our fight against the pandemic.
Topics: Ammonium Chloride; Animals; Antibodies, Viral; Antigen-Antibody Reactions; Blotting, Western; COVID-19; Chlorocebus aethiops; Convalescence; Defective Viruses; Genes, Reporter; Genetic Vectors; HEK293 Cells; HIV-1; Humans; Immunoglobulin G; Lentivirus; Mutagenesis, Site-Directed; Neutralization Tests; Pandemics; Point Mutation; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Vero Cells
PubMed: 33929934
DOI: 10.1080/22221751.2021.1925163 -
The Journal of General Virology Dec 2011Dengue viruses (DENV) cause countless human deaths each year, whilst West Nile virus (WNV) has re-emerged as an important human pathogen. There are currently no WNV or...
Dengue viruses (DENV) cause countless human deaths each year, whilst West Nile virus (WNV) has re-emerged as an important human pathogen. There are currently no WNV or DENV vaccines licensed for human use, yet vaccines exist against other flaviviruses. To investigate flavivirus cross-reactivity, sera from a human cohort with a history of vaccination against tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV) and yellow fever virus (YFV) were tested for antibodies by plaque reduction neutralization test. Neutralization of louping ill virus (LIV) occurred, but no significant neutralization of Murray Valley encephalitis virus was observed. Sera from some individuals vaccinated against TBEV and JEV neutralized WNV, which was enhanced by YFV vaccination in some recipients. Similarly, some individuals neutralized DENV-2, but this was not significantly influenced by YFV vaccination. Antigenic cartography techniques were used to generate a geometric illustration of the neutralization titres of selected sera against WNV, TBEV, JEV, LIV, YFV and DENV-2. This demonstrated the individual variation in antibody responses. Most sera had detectable titres against LIV and some had titres against WNV and DENV-2. Generally, LIV titres were similar to titres against TBEV, confirming the close antigenic relationship between TBEV and LIV. JEV was also antigenically closer to TBEV than WNV, using these sera. The use of sera from individuals vaccinated against multiple pathogens is unique relative to previous applications of antigenic cartography techniques. It is evident from these data that notable differences exist between amino acid sequence identity and mapped antigenic relationships within the family Flaviviridae.
Topics: Antibodies, Neutralizing; Antibodies, Viral; Cross Reactions; Dengue Vaccines; Dengue Virus; Encephalitis Virus, Japanese; Encephalitis Viruses, Tick-Borne; Humans; Neutralization Tests; Viral Plaque Assay; West Nile virus; Yellow Fever Vaccine; Yellow fever virus
PubMed: 21900425
DOI: 10.1099/vir.0.031641-0 -
Journal of Clinical Virology : the... Jul 2022The virus neutralization test (VNT) is the reference for the assessment of the functional ability of neutralizing antibodies (NAb) to block SARS-CoV-2 entry into cells....
The virus neutralization test (VNT) is the reference for the assessment of the functional ability of neutralizing antibodies (NAb) to block SARS-CoV-2 entry into cells. New competitive immunoassays measuring antibodies preventing interaction between the spike protein and its cellular receptor are proposed as surrogate VNT (sVNT). We tested three commercial sVNT (a qualitative immunochromatographic test and two quantitative immunoassays named YHLO and TECO) together with a conventional anti-spike IgG assay (bioMérieux) in comparison with an in-house plaque reduction neutralization test (PRNT) using the original 19A strain and different variants of concern (VOC), on a panel of 306 sera from naturally-infected or vaccinated patients. The qualitative test was rapidly discarded because of poor sensitivity and specificity. Areas under the curve of YHLO and TECO assays were, respectively, 85.83 and 84.07 (p-value >0.05) using a positivity threshold of 20 for PRNT, and 95.63 and 90.35 (p-value =0.02) using a threshold of 80. However, the performances of YHLO and bioMérieux were very close for both thresholds, demonstrating the absence of added value of sVNT compared to a conventional assay for the evaluation of the presence of NAb in seropositive subjects. In addition, the PRNT assay showed a reduction of NAb titers towards different VOC in comparison to the 19A strain that could not be appreciated by the commercial tests. Despite the good correlation between the anti-spike antibody titer and the titer of NAb by PRNT, our results highlight the difficulty to distinguish true NAb among the anti-RBD antibodies with commercial user-friendly immunoassays.
Topics: Antibodies, Neutralizing; Antibodies, Viral; COVID-19; Humans; Neutralization Tests; SARS-CoV-2
PubMed: 35568003
DOI: 10.1016/j.jcv.2022.105169 -
Nature Communications May 2022The Omicron SARS-CoV-2 has several distinct sublineages, among which sublineage BA.1 is responsible for the initial Omicron surge and is now being replaced by BA.2...
The Omicron SARS-CoV-2 has several distinct sublineages, among which sublineage BA.1 is responsible for the initial Omicron surge and is now being replaced by BA.2 worldwide, whereas BA.3 is currently at a low frequency. The ongoing BA.1-to-BA.2 replacement underscores the importance to understand the cross-neutralization among the three Omicron sublineages. Here we test the neutralization of BA.1-infected human sera against BA.2, BA.3, and USA/WA1-2020 (a strain isolated in late January 2020). The BA.1-infected sera neutralize BA.1, BA.2, BA.3, and USA/WA1-2020 SARS-CoV-2s with geometric mean titers (GMTs) of 445, 107, 102, and 16, respectively. Thus, the neutralizing GMTs against heterologous BA.2, BA.3, and USA/WA1-2020 are 4.2-, 4.4-, and 28.4-fold lower than the GMT against homologous BA.1, respectively. These findings have implications in COVID-19 vaccine strategy.
Topics: Antibodies, Viral; COVID-19; COVID-19 Vaccines; Humans; Neutralization Tests; SARS-CoV-2
PubMed: 35618703
DOI: 10.1038/s41467-022-30580-5