scholarly journals A human ex vivo dengue virus neutralization assay identifies priority antibodies and epitopes for vaccines and therapeutics

2019 ◽  
Author(s):  
Trung Tuan Vu ◽  
Hannah Clapham ◽  
Van Thi Thuy Huynh ◽  
Long Vo Thi ◽  
Dui Le Thi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Dengue Virus ◽  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Neutralization Assay ◽  
Virus Neutralization ◽  
Virus Infectivity ◽  
Virus Neutralization Assay ◽  
The Impact ◽  
Arboviral Disease

AbstractBackgroundDengue is the most prevalent arboviral disease, for which neither effective vaccines nor antivirals are available. Clinical trials with Dengvaxia, the first licensed dengue vaccine, show the conventional in vitro plaque reduction neutralization test (PRNT) failed to discriminate between neutralizing and non-neutralizing antibodies. A number of human monoclonal antibodies (mAbs) were characterized by PRNT as being neutralizers of virus infectivity for mammalian cells.Methodolody/Principle findingsWe developed a neutralization assay and tested the capacity of 12 mAbs to neutralize the infectiousness of dengue patient viremic blood in mosquitoes. We identified minimum concentrations of a subset of mAbs required to achieve dengue virus neutralization, and modelled the impact of a therapeutic mAb candidate on viremia.Five of the 12 mAbs (14c10, 2D22, 1L12, 747(4)B7, 753(3)C10), all of which target quaternary epitopes, potently inhibited dengue virus infection of Ae. aegypti. The potency of several mAbs was compromised in the context of patients with secondary serological profiles, possibly reflecting competition between the exogenously-added mAbs and the patient’s own antibody responses at or near the target epitopes. The minimum concentrations that mAbs neutralized DENV ranged from 0.1 – 5 µg/mL. An Fc-disabled variant of mAb (14c10-LALA) was as potent as its parent mAb. Within-host mathematical modelling suggests infusion of 14c10-LALA could bring about rapid acceleration of viremia resolution in a typical patient.Conclusions/SignificanceThese data delivered a unique assessment of anti-viral potency of a panel of human mAbs. Results support the advancement of dengue virus neutralization assays, and the development of therapeutics against flaviviruses, to which dengue virus and Zika virus belong.Author summaryDengue is the most prevalent arboviral disease affecting humans. There are no therapeutics for the disease. Antibody-mediated immunity against dengue is also not well-understood, as shown by the failure of the conventional neutralization assay used to predict the efficacy of Dengvaxia, the first licensed vaccine for the disease. It is likely that the neutralization assay targets non-neutralizing antibodies, but there are no validation assays available. To this end, we developed a novel virus neutralization assay, employing Aedes aegypti mosquitoes and viremic blood from dengue patients, to examine the virus-neutralizing potency of 12 human-derived monoclonal antibodies (mAbs). While all of these mAbs neutralized dengue virus using the conventional assay, seven of them failed to block dengue virus infections of mosquitoes using our assay. The remaining five mAbs neutralized at least one serotype of dengue virus and the minimum neutralizing concentrations of range from 0.1 – 5 µg/mL. Using the minimum neutralizing concentration of a therapeutic mAb candidate, we investigated the impact of the mAb on viremia using a mathematical model and found the mAb accelerated the reduction of viremia. The results support the advancement of dengue virus neutralization assays, and the development of therapeutics for dengue.

scholarly journals Inference of SARS-CoV-2 spike-binding neutralizing antibody titers in sera from hospitalized COVID-19 patients by using commercial enzyme and chemiluminescent immunoassays

2020 ◽  
Author(s):  
Arantxa Valdivia ◽  
Ignacio Torres ◽  
Victor Latorre ◽  
Carla Frances-Gomez ◽  
Eliseo Albert ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Neutralization Assay ◽  
Virus Neutralization ◽  
Pseudotyped Virus ◽  
S Protein ◽  
Neutralizing Activity ◽  
Virus Neutralization Assay ◽  
Degree Correlation ◽  
Antibody Titers

Background: Whether antibody levels measured by commercially-available enzyme or chemiluminescent immunoassays targeting the SARS-CoV-2 spike (S) protein can act as a proxy for serum neutralizing activity remains to be established for many of these assays. Objectives: To evaluate the degree of correlation between neutralizing antibodies (NtAb) binding the SARS-CoV-2 Spike (S) protein and SARS-CoV-2-S-IgG levels measured by four commercial immunoassays in sera drawn from hospitalized COVID-19 patients. Patients and Methods: Ninety sera from 51 hospitalized COVID-19 patients were assayed by a pseudotyped virus neutralization assay, the LIAISON SARS-CoV-2 S1/S2 IgG, the Euroimmun SARS-CoV-2 IgG ELISA, the MAGLUMI 2019-nCoV IgG and the COVID-19 ELISA IgG assays. Results: Overall, the results obtained with the COVID-19 ELISA IgG test showed the highest agreement with the NtAb assay (κ, 0.85; 95% CI, 0.63-1). The most sensitive tests were the pseudotyped virus NtAb assay and the COVID-19 ELISA IgG assay (92.2% for both). Overall, the degree correlation between antibody titers resulting in 50% virus neutralization (NtAb50) in the pseudotyped virus assay and SARS-CoV-2 IgG levels was strong for the Euroimmun SARS-CoV-2 IgG ELISA (Rho=0.73) and moderate for the remaining assays (Rho=0.48 to 0.59). The kinetic profile of serum NtAb50 titers could not be reliably predicted by any of the SARS-CoV-2 IgG immunoassays. Conclusions: the suitability of SARS-CoV-2-S-IgG commercial immunoassays for inferring neutralizing activity of sera from hospitalized COVID-19 patients varies widely across tests and is influenced by the time of sera collection after the onset of symptoms.

scholarly journals Quantifying absolute neutralization titers against SARS-CoV-2 by a standardized virus neutralization assay allows for cross-cohort comparisons of COVID-19 sera

2020 ◽  
Author(s):  
Kasopefoluwa Y. Oguntuyo ◽  
Christian S Stevens ◽  
Chuan-Tien Hung ◽  
Satoshi Ikegame ◽  
Joshua A. Acklin ◽  
...  
Keyword(s):  
Viral Entry ◽  
High Throughput Screening ◽  
Neutralizing Antibodies ◽  
Neutralization Assay ◽  
Virus Neutralization ◽  
Strong Positive Correlation ◽  
Plasma Therapy ◽  
Live Virus ◽  
Virus Neutralization Assay ◽  
Marked Variability

The global COVID-19 pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the SARS-CoV-2 spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous BSL3 conditions which limits high throughput screening of patient and vaccine sera. Myriad BSL-2 compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVdeltaG-based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale and generate accurate neutralizing titers within 18 hours post-infection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in confirmed convalescent patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n>120). Our data show that absolute (abs) IC50, IC80, and IC90 values can be legitimately compared across diverse cohorts, highlight the substantial but consistent variability in neutralization potency across these cohorts, and support the use of absIC80 as a more meaningful metric for assessing the neutralization potency of vaccine or convalescent sera. Lastly, we used our CoV2pp in a screen to identify ultra-permissive 293T clones that stably express ACE2 or ACE2+TMPRSS2. When used in combination with our CoV2pp, we can now produce CoV2pp sufficient for 150,000 standardized VNA/week.

scholarly journals Development of a Sensitive Detection Method for Alphaviruses and Its Use as a Virus Neutralization Assay

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1191
Author(s):  
Christin Schmidt ◽  
Mario Perkovic ◽  
Barbara S. Schnierle
Keyword(s):  
Neutralizing Antibodies ◽  
High Sensitivity ◽  
Neutralization Assay ◽  
Open Reading Frames ◽  
Virus Neutralization ◽  
Structural Genes ◽  
Virus Neutralization Assay ◽  
Subgenomic Promoter ◽  
Mayaro Virus ◽  
Reading Frames

Alphaviruses have a single-stranded, positive-sense RNA genome that contains two open reading frames encoding either the non-structural or the structural genes. Upon infection, the genomic RNA is translated into the non-structural proteins (nsPs). NsPs are required for viral RNA replication and transcription driven from the subgenomic promoter (sgP). Transfection of an RNA encoding the luciferase gene under the control of the sgP into cells enabled the detection of replication-competent chikungunya virus (CHIKV) or Mayaro virus (MAYV) with high sensitivity as a function of the induced luciferase activity. This assay principle was additionally used to analyze virus-neutralizing antibodies in sera and might be an alternative to standard virus neutralization assays based on virus titration or the use of genetically modified tagged viruses.

scholarly journals Infectivity and immune escape of the new SARS-CoV-2 variant of interest Lambda

2021 ◽  
Author(s):  
M&oacutenica L. Acevedo ◽  
Luis Alonso-Palomares ◽  
Andr&eacutes Bustamante ◽  
Aldo Gaggero ◽  
Fabio Paredes ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Immune Escape ◽  
Neutralization Assay ◽  
Spike Protein ◽  
Pseudotyped Virus ◽  
South American ◽  
Virus Neutralization Assay ◽  
Alpha Variant ◽  
Vaccination Campaigns ◽  
The Impact

Background: The newly described SARS-CoV-2 lineage C.37 was recently classified as a variant of interest by the WHO (Lambda variant) based on its high circulation rates in South American countries and the presence of critical mutations in the spike protein. The impact of such mutations in infectivity and immune escape from neutralizing antibodies are entirely unknown. Methods: We performed a pseudotyped virus neutralization assay and determined the impact of the Lambda variant on infectivity and immune escape using plasma samples from healthcare workers (HCW) from two centers in Santiago, Chile who received the two-doses scheme of the inactivated virus vaccine CoronaVac. Results: We observed an increased infectivity mediated by the Lambda spike protein that was even higher than that of the D614G (lineage B) or the Alpha and Gamma variants. Compared to the Wild type (lineage A), neutralization was decreased by 3.05-fold for the Lambda variant while it was 2.33-fold for the Gamma variant and 2.03-fold for the Alpha variant. Conclusions: Our results indicate that mutations present in the spike protein of the Lambda variant of interest confer increased infectivity and immune escape from neutralizing antibodies elicited by CoronaVac. These data reinforce the idea that massive vaccination campaigns in countries with high SARS-CoV-2 circulation must be accompanied by strict genomic surveillance allowing the identification of new isolates carrying spike mutations and immunology studies aimed to determine the impact of these mutations in immune escape and vaccines breakthrough.

scholarly journals The Impact on Infectivity and Neutralization Efficiency of SARS-CoV-2 Lineage B.1.351 Pseudovirus

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 633
Author(s):  
Yeong Jun Kim ◽  
Ui Soon Jang ◽  
Sandrine M. Soh ◽  
Joo-Youn Lee ◽  
Hye-Ra Lee
Keyword(s):  
South Africa ◽  
Monoclonal Antibodies ◽  
Cell Fusion ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Receptor Binding Domain ◽  
Viral Spread ◽  
New Variant ◽  
Global Concern ◽  
The Impact

A new variant of SARS-CoV-2 B.1.351 lineage (first found in South Africa) has been raising global concern due to its harboring of multiple mutations in the spike that potentially increase transmissibility and yield resistance to neutralizing antibodies. We here tested infectivity and neutralization efficiency of SARS-CoV-2 spike pseudoviruses bearing particular mutations of the receptor-binding domain (RBD) derived either from the Wuhan strains (referred to as D614G or with other sites) or the B.1.351 lineage (referred to as N501Y, K417N, and E484K). The three different pseudoviruses B.1.351 lineage related significantly increased infectivity compared with other mutants that indicated Wuhan strains. Interestingly, K417N and E484K mutations dramatically enhanced cell–cell fusion than N501Y even though their infectivity were similar, suggesting that K417N and E484K mutations harboring SARS-CoV-2 variant might be more transmissible than N501Y mutation containing SARS-CoV-2 variant. We also investigated the efficacy of two different monoclonal antibodies, Casirivimab and Imdevimab that neutralized SARS-CoV-2, against several kinds of pseudoviruses which indicated Wuhan or B.1.351 lineage. Remarkably, Imdevimab effectively neutralized B.1.351 lineage pseudoviruses containing N501Y, K417N, and E484K mutations, while Casirivimab partially affected them. Overall, our results underscore the importance of B.1.351 lineage SARS-CoV-2 in the viral spread and its implication for antibody efficacy.

scholarly journals Neutralization of Virus Infectivity by Antibodies: Old Problems in New Perspectives

2014 ◽  
Vol 2014 ◽  
pp. 1-24 ◽  
Author(s):  
P. J. Klasse
Keyword(s):  
Cellular Immunity ◽  
Neutralizing Antibodies ◽  
Viral Infections ◽  
Viral Proteins ◽  
Virus Neutralization ◽  
Virus Infectivity ◽  
Viral Pathogens ◽  
Enveloped Viruses ◽  
Vaccine Candidates ◽  
Hiv 1

Neutralizing antibodies (NAbs) can be both sufficient and necessary for protection against viral infections, although they sometimes act in concert with cellular immunity. Successful vaccines against viruses induce NAbs but vaccine candidates against some major viral pathogens, including HIV-1, have failed to induce potent and effective such responses. Theories of how antibodies neutralize virus infectivity have been formulated and experimentally tested since the 1930s; and controversies about the mechanistic and quantitative bases for neutralization have continually arisen. Soluble versions of native oligomeric viral proteins that mimic the functional targets of neutralizing antibodies now allow the measurement of the relevant affinities of NAbs. Thereby the neutralizing occupancies on virions can be estimated and related to the potency of the NAbs. Furthermore, the kinetics and stoichiometry of NAb binding can be compared with neutralizing efficacy. Recently, the fundamental discovery that the intracellular factor TRIM21 determines the degree of neutralization of adenovirus has provided new mechanistic and quantitative insights. Since TRIM21 resides in the cytoplasm, it would not affect the neutralization of enveloped viruses, but its range of activity against naked viruses will be important to uncover. These developments bring together the old problems of virus neutralization—mechanism, stoichiometry, kinetics, and efficacy—from surprising new angles.

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