scholarly journals Detecting and predicting neutralization of alemtuzumab responses in MS

2020 ◽  
Vol 7 (4) ◽  
pp. e767
Author(s):  
Gauri Saxena ◽  
James M. Moore ◽  
Meleri Jones ◽  
Gareth Pryce ◽  
Liaqat Ali ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Treatment Resistance ◽  
Single Chain ◽  
Serum Samples ◽  
Lymphocyte Depletion ◽  
Prognostic Feature ◽  
Number Of Treatment ◽  
Number Of Individuals ◽  
Poor Prognostic Feature ◽  
Binding Cell

ObjectiveTo test the hypothesis that antidrug antibodies (ADAs) against alemtuzumab could become relevant after repeated treatments for some individuals, possibly explaining occasional treatment resistance.MethodsRecombinant alemtuzumab single-chain variable fragment antibody with a dual tandem nanoluciferase reporter linker was made and used to detect binding ADAs. Alemtuzumab immunoglobulin G Alexa Fluor 488 conjugate was used in a competitive binding cell-based assay to detect neutralizing ADAs. The assays were used to retrospectively screen, blinded, banked serum samples from people with MS (n = 32) who had received 3 or more cycles of alemtuzumab. Lymphocyte depletion was measured between baseline and about 1 month postinfusion.ResultsThe number of individuals showing limited depletion of lymphocytes increased with the number of treatment cycles. Lack of depletion was also a poor prognostic feature for future disease activity. ADA responses were detected in 29/32 (90.6%) individuals. Neutralizing antibodies occurred before the development of limited depletion in 6/7 individuals (18.8% of the whole sample). Preinfusion, ADA levels predicted limited, postinfusion lymphocyte depletion.ConclusionsAlthough ADAs to alemtuzumab have been portrayed as being of no clinical significance, alemtuzumab-specific antibodies appear to be clinically relevant for some individuals, although causation remains to be established. Monitoring of lymphocyte depletion and the antidrug response may be of practical value in patients requiring additional cycles of alemtuzumab. ADA detection may help to inform on retreatment or switching to another treatment.

Low Immunogenicity to Romiplostim in Clinical Studies with ITP Subjects.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3425-3425 ◽  
Author(s):  
Vibha Jawa ◽  
Martha Hokom ◽  
Jenny Hu ◽  
Yao Zhuang ◽  
Dietmar Berger ◽  
...  
Keyword(s):  
Clinical Studies ◽  
Neutralizing Antibodies ◽  
Single Chain ◽  
Serum Samples ◽  
Binding Domains ◽  
C Terminus ◽  
Before And After ◽  
Immunogenicity Assessment ◽  
Covalently Linked

Abstract Romiplostim, a member of the thrombopoietin (TPO) mimetic class, is an Fc-peptide fusion protein (peptibody) that activates intracellular transcriptional pathways leading to increased platelet production via the TPO receptor (also known as cMpl). The peptibody molecule contains two identical single-chain subunits each consisting of human immunoglobulin IgG1 Fc domain, covalently linked at the C-terminus to a peptide containing two TPO receptor-binding domains. Due to the general concern regarding the immunogenic potential for all therapeutic proteins and the specific concern for monitoring antibodies capable of neutralizing thrombopoietin (TPO), an extensive immunogenicity assessment program was developed to support romiplostim. Romiplostim has been engineered to have no amino acid sequence homology to endogenous TPO. A low theoretical risk of developing conformational antibodies that cross-react against TPO exists. This risk was addressed by using an immunogenicity assessment strategy that relied upon a surface plasmon resonance based biosensor immunoassay using the Biacore 3000 capable of simultaneously monitoring antibodies that bind to romiplostim, TPO, or the active peptide portion of romiplostim (TMP). Samples that tested positive for binding antibodies in the Biacore immunoassay were then tested in the definitive functional biological assay to identify any antibodies capable of neutralizing the biological effect of romiplostim or TPO. Serum samples from 236 actively treated subjects were obtained both before and after exposure to romiplostim and were tested for romiplostim and TPO antibodies. In baseline samples, seventeen subjects (7.1%) tested romiplostim antibody positive and 12 subjects (5.1%) tested TPO antibody positive for pre-existing binding antibodies. After romiplostim exposure, twenty-five out of 236 (10.5%) subjects with ITP developed binding antibodies against romiplostim (inclusive of antibodies to both peptide and the whole molecule) and 12 out of 236 (5.1%) subjects with ITP developed binding antibodies against TPO. The antibodies that developed against romiplostim did not cross react with TPO and the antibodies that developed against TPO did not cross react with romiplostim. The incidence of anti-romiplostim neutralizing antibodies among 236 subjects with ITP who were treated with romiplostim across 10 clinical studies was 0.4% (1 out of 236). No cases of anti-TPO neutralizing antibodies were detected in romiplostim treated samples. In conclusion, after thorough immunogenicity assessment of all subjects treated with romiplostim using sensitive methods to detect binding and neutralizing antibodies, only one subject was found positive for the presence of antibodies capable of neutralizing romiplostim that was negative at the time of follow up 4 months later. As expected, none of the subjects treated were positive for antibodies capable of neutralizing TPO. No clinical sequelae were observed in association to the presence of antibodies.

scholarly journals Evaluation of a Pseudovirus Neutralization Assay for SARS-CoV-2 and Correlation with Live Virus-Based Micro Neutralization Assay

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 994
Author(s):  
Ahmed Majdi K. Tolah ◽  
Sayed S. Sohrab ◽  
Khaled Majdi K. Tolah ◽  
Ahmed M. Hassan ◽  
Sherif A. El-Kafrawy ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Epidemiological Studies ◽  
Neutralization Assay ◽  
Serum Samples ◽  
Live Virus ◽  
Spike Gene ◽  
Microneutralization Assay ◽  
Viral Particles ◽  
Reliable Performance ◽  
Prior Infection

The unusual cases of pneumonia outbreak were reported from Wuhan city in late December 2019. Serological testing provides a powerful tool for the identification of prior infection and for epidemiological studies. Pseudotype virus neutralization assays are widely used for many viruses and applications in the fields of serology. The accuracy of pseudotype neutralizing assay allows for its use in low biosafety lab and provides a safe and effective alternative to the use of wild-type viruses. In this study, we evaluated the performance of this assay compared to the standard microneutralization assay as a reference. The lentiviral pseudotype particles were generated harboring the Spike gene of SARS-CoV-2. The generated pseudotype particles assay was used to evaluate the activity of neutralizing antibodies in 300 human serum samples from a COVID-19 sero-epidemiological study. Testing of these samples resulted in 55 positive samples and 245 negative samples by pseudotype viral particles assay while microneutralization assay resulted in 64 positive and 236 negative by MN assay. Compared to the MN, the pseudotyped viral particles assay showed a sensitivity of 85.94% and a specificity of 100%. Based on the data generated from this study, the pseudotype-based neutralization assay showed a reliable performance for the detection of neutralizing antibodies against SARS-CoV-2 and can be used safely and efficiently as a diagnostic tool in a biosafety level 2 laboratory.

scholarly journals A longitudinal study of SARS-CoV-2-infected patients reveals a high correlation between neutralizing antibodies and COVID-19 severity

2021 ◽  
Author(s):  
Vincent Legros ◽  
Solène Denolly ◽  
Manon Vogrig ◽  
Bertrand Boson ◽  
Eglantine Siret ◽  
...  
Keyword(s):  
Intensive Care ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Surface Protein ◽  
Humoral Response ◽  
Neutralizing Antibody ◽  
Rapid Decline ◽  
Serum Samples ◽  
Immune Correlates ◽  
Human Coronaviruses

AbstractUnderstanding the immune responses elicited by SARS-CoV-2 infection is critical in terms of protection against reinfection and, thus, for public health policy and vaccine development for COVID-19. In this study, using either live SARS-CoV-2 particles or retroviruses pseudotyped with the SARS-CoV-2 S viral surface protein (Spike), we studied the neutralizing antibody (nAb) response in serum samples from a cohort of 140 SARS-CoV-2 qPCR-confirmed infections, including patients with mild symptoms and also more severe forms, including those that required intensive care. We show that nAb titers correlated strongly with disease severity and with anti-spike IgG levels. Indeed, patients from intensive care units exhibited high nAb titers; conversely, patients with milder disease symptoms had heterogeneous nAb titers, and asymptomatic or exclusive outpatient-care patients had no or low nAbs. We found that nAb activity in SARS-CoV-2-infected patients displayed a relatively rapid decline after recovery compared to individuals infected with other coronaviruses. Moreover, we found an absence of cross-neutralization between endemic coronaviruses and SARS-CoV-2, indicating that previous infection by human coronaviruses may not generate protective nAbs against SARS-CoV-2. Finally, we found that the D614G mutation in the spike protein, which has recently been identified as the current major variant in Europe, does not allow neutralization escape. Altogether, our results contribute to our understanding of the immune correlates of SARS-CoV-2-induced disease, and rapid evaluation of the role of the humoral response in the pathogenesis of SARS-CoV-2 is warranted.

scholarly journals Antibodies against Bovine herpesvirus 1 in dairy herds in the state of Espirito Santo, Brasil

Revista CERES ◽  
2014 ◽  
Vol 61 (2) ◽  
pp. 280-283 ◽  
Author(s):  
Marcus Rebouças Santos ◽  
Hanna Carolina Campos Ferreira ◽  
Marcos Antônio dos Santos ◽  
Giuliana Loreto Saraiva ◽  
Natália Filardi Tafuri ◽  
...  
Keyword(s):  
Dairy Cattle ◽  
Neutralizing Antibodies ◽  
Bovine Herpesvirus ◽  
The State ◽  
Serum Samples ◽  
Bovine Herpesvirus 1 ◽  
Espírito Santo ◽  
Herpesvirus 1 ◽  
Cattle Herds ◽  
Espirito Santo

Bovine herpesvirus 1 (BoHV-1) causes major losses in worldwide livestock, affecting the respiratory and reproductive tracts of bovine. In the past decades, the number of cases in Brazil has been gradually increasing. Therefore, it is important to assess the distribution of infection in different regions of the country. In the state of Espírito Santo (ES) the BoHV 1 infection rate in dairy cattle herds is unknown. Thus, the aim of this study was to detect neutralizing antibodies against BoHV-1 in serum samples from 1,161 non-vaccinated cows from 59 dairy cattle herds in 23 municipalities of the Metropolitan, North, Northwest and South macro-regions. The identification of seropositive cows was evaluated by the virus neutralization test. The results showed that of all serum samples evaluated 775 (66.75%) had neutralizing antibodies against BoHV-1. Moreover, all herds were found positive; however, the percentage of positive cows varied among regions; 49.06%, 62.15%, 67.21% and 80.04% for the Metropolitan, South, North and Northwest macro-regions, respectively. In this study, the results clearly indicate the dissemination of the viral agent in dairy cattle in the ES state, requiring the monitoring and control of diseases related to BoHV-1 infection.

scholarly journals Serological response to rabies virus induced by commercial vaccines in cattle

2017 ◽  
Vol 47 (10) ◽  
Author(s):  
Mathias Martins ◽  
João Motta de Quadros ◽  
Eduardo Furtado Flores ◽  
Rudi Weiblen
Keyword(s):  
Rabies Virus ◽  
Neutralizing Antibodies ◽  
Vaccine Dose ◽  
Booster Vaccination ◽  
Serological Response ◽  
Serum Samples ◽  
Anamnestic Response ◽  
Post Vaccination ◽  
Antibody Titers ◽  
One Year

ABSTRACT: The antibody response to rabies virus (RABV) induced by commercial vaccines in heifers was investigated. For this, 84 heifers were vaccinated twice (30 days interval) with each of four vaccines (G1 = 14 animals; G2 = 24; G3 = 22 and G4 = 24) and received a booster vaccination 360 days later. Serum samples collected at different intervals after vaccination and 30 days after booster were submitted to a virus neutralizing (VN) assay for RABV antibodies. Thirty days after the second vaccine dose, 92% of the immunized animals presented VN titers ≥0.5UI/mL (geometric medium titers [GMT] 1.7 to 3.8UI/mL). At the day of the booster (360 days post-vaccination); however, the percentage of animals harboring antibody titers ≥0.5UI/mL had dropped to 31% (0-80% of the animals, depending on the vaccine), resulting in lower GMT (0.1 to 0.6UI/mL). Booster vaccination at day 360 resulted in a detectable anamnestic response in all groups, resulting in 83% of animals (65 to 100%) harboring VN titers ≥0.5UI/mL thirty days later (GMT 0.6 to 4.3UI/mL). These results indicated that these vaccines were able to induce an adequate anti-RABV response in all animals after prime vaccination (and after booster as well). However, the titers decreased, reaching titers <0.5UI/mL in approximately 70% of animals within the interval before the recommended booster. Thus, booster vaccination for rabies in cattle using the current vaccines should be performed before the recommended one-year interval, as to maintain neutralizing antibodies levels in most vaccinated animals.

scholarly journals A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serum

2021 ◽  
Author(s):  
Syed Hani Abidi ◽  
Kehkeshan Imtiaz ◽  
Akbar Kanji ◽  
Shama Qaiser ◽  
Erum Khan ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vero Cells ◽  
Neutralization Assay ◽  
Dilution Method ◽  
Rt Pcr ◽  
Serum Samples ◽  
Live Virus ◽  
Viral Neutralization ◽  
Microneutralization Assay ◽  
Infected Cells

Abstract Background Individuals recovering from COVID-19 are shown to have antibodies against the Spike and other structural proteins. Antibodies against Spike have been shown to display viral neutralization. However, not all antibodies against Spike have neutralizing ability and some may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays that allow the determination of virus neutralizing activity in sera of individuals. Here we describe a PCR-based micro-neutralization assay that can be used to evaluate the viral neutralization titers of serum from SARS-CoV-2 infected individuals. Methods The SARS-CoV-2 strain used was isolated from a nasopharyngeal specimen of a COVID-19 case. The limiting dilution method was used to obtain a 50% tissue culture infective dose (TCID50) of Vero cells. For the micro‐neutralization assay, 19 serum samples, with positive IgG titers against Spike receptor binding domain (RBD) were tested. After 24 hours, infected cells were inspected for the presence of the cytopathic effect, then lysed and RNA RT-PCR of SARS-CoV-2. The Ct values were used to calculate percent neutralization/inhibition of SARS-CoV-2. Results Out of 19 samples, 13 samples gave 100% neutralization at all dilutions, while 4 samples gave neutralization at lower dilution, while one sample did not give any neutralization. The correlation between RBD OD and neutralization potential was found to be statistically correlated. Conclusion We describe a rapid RT-PCR based SARS-CoV-2 microneutralization assay for detection of neutralizing antibodies. This can effectively be used to test anti-viral activity of serum antibodies for investigation of both disease-driven and vaccine-induced responses.

scholarly journals Combinations of Single Chain Variable Fragments From HIV Broadly Neutralizing Antibodies Demonstrate High Potency and Breadth

2021 ◽  
Vol 12 ◽  
Author(s):  
Rebecca T. van Dorsten ◽  
Kshitij Wagh ◽  
Penny L. Moore ◽  
Lynn Morris
Keyword(s):  
Neutralizing Antibodies ◽  
Passive Immunization ◽  
Neutralization Assay ◽  
Single Chain ◽  
Broadly Neutralizing Antibodies ◽  
High Potency ◽  
Hill Model ◽  
Mucosal Tissues ◽  
Action Model ◽  
Single Chain Variable Fragments

Broadly neutralizing antibodies (bNAbs) are currently being assessed in clinical trials for their ability to prevent HIV infection. Single chain variable fragments (scFv) of bNAbs have advantages over full antibodies as their smaller size permits improved diffusion into mucosal tissues and facilitates vector-driven gene expression. We have previously shown that scFv of bNAbs individually retain significant breadth and potency. Here we tested combinations of five scFv derived from bNAbs CAP256-VRC26.25 (V2-apex), PGT121 (N332-supersite), 3BNC117 (CD4bs), 8ANC195 (gp120-gp41 interface) and 10E8v4 (MPER). Either two or three scFv were combined in equimolar amounts and tested in the TZM-bl neutralization assay against a multiclade panel of 17 viruses. Experimental IC50 and IC80 data were compared to predicted neutralization titers based on single scFv titers using the Loewe additive and the Bliss-Hill model. Like full-sized antibodies, combinations of scFv showed significantly improved potency and breadth compared to single scFv. Combinations of two or three scFv generally followed an independent action model for breadth and potency with no significant synergy or antagonism observed overall although some exceptions were noted. The Loewe model underestimated potency for some dual and triple combinations while the Bliss-Hill model was better at predicting IC80 titers of triple combinations. Given this, we used the Bliss-Hill model to predict the coverage of scFv against a 45-virus panel at concentrations that correlated with protection in the AMP trials. Using IC80 titers and concentrations of 1μg/mL, there was 93% coverage for one dual scFv combination (3BNC117+10E8v4), and 96% coverage for two of the triple combinations (CAP256.25+3BNC117+10E8v4 and PGT121+3BNC117+10E8v4). Combinations of scFv, therefore, show significantly improved breadth and potency over individual scFv and given their size advantage, have potential for use in passive immunization.

scholarly journals Scalable, Micro-Neutralization Assay for Qualitative Assessment of SARS-CoV-2 (COVID-19) Virus-Neutralizing Antibodies in Human Clinical Samples

2021 ◽  
Author(s):  
Richard S. Bennett ◽  
Elena N. Postnikova ◽  
Janie Liang ◽  
Robin Gross ◽  
Steven Mazur ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Imaging System ◽  
Neutralization Assay ◽  
Qualitative Assessment ◽  
Clinical Samples ◽  
Therapeutic Approaches ◽  
Serum Samples ◽  
Specific Test ◽  
Multiple Virus ◽  
Infected Cells

AbstractAs the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic was expanding, it was clear that effective testing for the presence of neutralizing antibodies in the blood of convalescent patients would be critical for development of plasma-based therapeutic approaches. To address the need for a high-quality neutralization assay against SARS-CoV-2, a previously established fluorescence reduction neutralization assay (FRNA) against Middle East respiratory syndrome coronavirus (MERS-CoV) was modified and optimized. The SARS-CoV-2 FRNA provides a quantitative assessment of a large number of infected cells through use of a high-content imaging system. Because of this approach, and the fact that it does not involve subjective interpretation, this assay is more efficient and more accurate than other neutralization assays. In addition, the ability to set robust acceptance criteria for individual plates and specific test wells provided further rigor to this assay. Such agile adaptability avails use with multiple virus variants. By February 2021, the SARS-CoV-2 FRNA had been used to screen over 5,000 samples, including acute and convalescent plasma or serum samples and therapeutic antibody treatments, for SARS-CoV-2 neutralizing titers.

scholarly journals Neutralizing activity of Sputnik V vaccine sera against SARS-CoV-2 variants

2021 ◽  
Author(s):  
Satoshi Ikegame ◽  
Mohammed Siddiquey ◽  
Chuan-Tien Hung ◽  
Griffin Haas ◽  
Luca Brambilla ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Cell Line ◽  
Vesicular Stomatitis Virus ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
The Novel ◽  
Serum Samples ◽  
Egfp Reporter ◽  
Vesicular Stomatitis ◽  
Reduced Activity

Abstract The novel pandemic betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected at least 120 million people since its identification as the cause of a December 2019 viral pneumonia outbreak in Wuhan, China1,2. Despite the unprecedented pace of vaccine development, with six vaccines already in use worldwide, the emergence of SARS-CoV-2 ‘variants of concern’ (VOC) across diverse geographic locales have prompted re-evaluation of strategies to achieve universal vaccination3. All three officially designated VOC carry Spike (S) polymorphisms thought to enable escape from neutralizing antibodies elicited during initial waves of the pandemic4–8. Here, we characterize the biological consequences of the ensemble of S mutations present in VOC lineages B.1.1.7 (501Y.V1) and B.1.351 (501Y.V2). Using a replication-competent EGFP-reporter vesicular stomatitis virus (VSV) system, rcVSV-CoV2-S, which encodes S from SARS coronavirus 2 in place of VSV-G, and coupled with a clonal HEK-293T ACE2 TMPRSS2 cell line optimized for highly efficient S-mediated infection, we determined that only 1 out of 12 serum samples from a cohort of recipients of the Gamaleya Sputnik V Ad26 / Ad5 vaccine showed effective neutralization (IC90) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralized S from B.1.1.7 and showed only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines.

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