scholarly journals SARS-CoV-2 neutralizing antibodies: Longevity, breadth, and evasion by emerging viral variants

PLoS Medicine ◽  
2021 ◽  
Vol 18 (7) ◽  
pp. e1003656
Author(s):  
Fiona Tea ◽  
Alberto Ospina Stella ◽  
Anupriya Aggarwal ◽  
David Ross Darley ◽  
Deepti Pilli ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Rt Pcr ◽  
Breakthrough Infection ◽  
Nucleocapsid Proteins ◽  
Viral Neutralization ◽  
Polymerase Chain ◽  
Serial Samples ◽  
High Responders ◽  
Neutralization Response ◽  
Selection Of

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antibody neutralization response and its evasion by emerging viral variants and variant of concern (VOC) are unknown, but critical to understand reinfection risk and breakthrough infection following vaccination. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus–cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 reverse transcription polymerase chain reaction (RT-PCR)–confirmed Coronavirus Disease 2019 (COVID-19) individuals with detailed demographics and followed up to 7 months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization, was associated with COVID-19 severity. A subgroup of “high responders” maintained high neutralizing responses over time, representing ideal convalescent plasma donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants and VOC. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal responders and vaccine monitoring and design.

scholarly journals SARS-CoV-2 neutralizing antibodies; longevity, breadth, and evasion by emerging viral variants

2020 ◽  
Author(s):  
Fiona Tea ◽  
Alberto Ospina Stella ◽  
Anupriya Aggarwal ◽  
David Ross Darley ◽  
Deepti Pilli ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Antibody Responses ◽  
Plasma Therapy ◽  
Nucleocapsid Proteins ◽  
Viral Neutralization ◽  
Serial Samples ◽  
High Responders ◽  
Neutralization Response ◽  
Selection Of

AbstractThe SARS-CoV-2 antibody neutralization response and its evasion by emerging viral variants are unknown. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus-cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 RT-PCR-confirmed COVID-19 individuals with detailed demographics and followed up to seven months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization were associated with COVID-19 severity. A subgroup of ‘high responders’ maintained high neutralizing responses over time, representing ideal convalescent plasma therapy donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal plasma donors and vaccine monitoring and design.One Sentence SummaryNeutralizing antibody responses to SARS-CoV-2 are sustained, associated with COVID19 severity, and evaded by emerging viral variants

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 Neutralizing antibodies for SARS-CoV-2 in stray animals from Rio de Janeiro, Brazil

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248578
Author(s):  
Helver Gonçalves Dias ◽  
Maria Eduarda Barreto Resck ◽  
Gabriela Cardoso Caldas ◽  
Alessandro Fioretti Resck ◽  
Natália Valente da Silva ◽  
...  
Keyword(s):  
Rio De Janeiro ◽  
Neutralizing Antibodies ◽  
Neutralization Test ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Serum Samples ◽  
Stray Dog ◽  
Polymerase Chain ◽  
Stray Cat ◽  
Serological Data

The epidemic of coronavirus disease 2019 (COVID-19), caused by a novel Betacoronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a public health emergency worldwide. Few reports indicate that owned pets from households with at least one human resident that was diagnosed with COVID-19 can be infected by SARS-CoV-2. However, the exposure to SARS-CoV-2 of pets from households with no COVID-19 cases or stray animals remains less assessed. Using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and plaque reduction neutralization test (PRNT90), we investigated the infection and previous exposure of dogs and cats to SARS-CoV-2 during the ongoing COVID-19 epidemic in Rio de Janeiro, Brazil. From June to August 2020, 96 animals were sampled, including 49 cats (40 owned and 9 stray) and 47 dogs (42 owned and 5 stray). Regarding owned pets, 75.6% (62/82) belonged to households with no COVID-19 cases. Samples included serum, and rectal and oropharyngeal swabs. All swabs were negative for SARS-CoV-2 RNA, but serum samples of a stray cat and a stray dog presented neutralizing antibodies for SARS-CoV-2, with PRNT90 titer of 80 and 40, respectively. Serological data presented here suggest that not only owned pets from households with COVID19 cases, but also stray animals are being exposed to SARS-CoV-2 during the COVID-19 pandemic.

scholarly journals Characteristics of Anti-SARS-CoV-2 Antibodies in Recovered COVID-19 Subjects

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 697
Author(s):  
Angela Huynh ◽  
Donald M. Arnold ◽  
James W. Smith ◽  
Jane C. Moore ◽  
Ali Zhang ◽  
...  
Keyword(s):  
Population Level ◽  
Enzyme Linked Immunosorbent Assay ◽  
The Novel ◽  
Rt Pcr ◽  
S Protein ◽  
Viral Neutralization ◽  
Humoral Immune ◽  
Global Pandemic ◽  
Polymerase Chain ◽  
Ig G

Coronavirus Disease 2019 (COVID-19) is a global pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While detection of SARS-CoV-2 by polymerase chain reaction with reverse transcription (RT-PCR) is currently used to diagnose acute COVID-19 infection, serological assays are needed to study the humoral immune response to SARS-CoV-2. Anti-SARS-CoV-2 immunoglobulin (Ig)G/A/M antibodies against spike (S) protein and its receptor-binding domain (RBD) were characterized in recovered subjects who were RT-PCR-positive (n = 153) and RT-PCR-negative (n = 55) using an enzyme-linked immunosorbent assay (ELISA). These antibodies were also further assessed for their ability to neutralize live SARS-CoV-2 virus. Anti-SARS-CoV-2 antibodies were detected in 90.9% of resolved subjects up to 180 days post-symptom onset. Anti-S protein and anti-RBD IgG titers correlated (r = 0.5157 and r = 0.6010, respectively) with viral neutralization. Of the RT-PCR-positive subjects, 22 (14.3%) did not have anti-SARS-CoV-2 antibodies; and of those, 17 had RT-PCR cycle threshold (Ct) values > 27. These high Ct values raise the possibility that these indeterminate results are from individuals who were not infected or had mild infection that failed to elicit an antibody response. This study highlights the importance of serological surveys to determine population-level immunity based on infection numbers as determined by RT-PCR.

scholarly journals B.1.617.3 SARS CoV-2 spike E156G/Δ157-158 mutations contribute to reduced neutralization sensitivity and increased infectivity

2021 ◽  
Author(s):  
Tarun Mishra ◽  
Garima Joshi ◽  
Atul Kumar ◽  
Rishikesh Dalavi ◽  
Pankaj Pandey ◽  
...  
Keyword(s):  
Immune Evasion ◽  
Neutralizing Antibodies ◽  
Immune Escape ◽  
Coding Region ◽  
Rt Pcr ◽  
Breakthrough Infection ◽  
Neutralization Sensitivity ◽  
Spike Gene ◽  
Spike Coding ◽  
The Usa

SARS CoV-2 variants raise significant concerns due to their ability to cause vaccine breakthrough infections. Here, we sequence-characterized the spike gene, isolated from a breakthrough infection, that corresponded to B.1.617.3 lineage. Delineating the functional impact of spike mutations using reporter pseudoviruses (PV) revealed that N-terminal domain (NTD)-specific E156G/Δ157-158 contributed to increased infectivity and reduced sensitivity to ChAdOx1 nCoV-19 vaccine (CovishieldTM)-elicited neutralizing antibodies. A six-nucleotide deletion (467-472) in the spike coding region introduced this change in the NTD. We confirmed the presence of E156G/Δ157-158 in the RT-PCR-positive cases concurrently screened, in addition to other circulating spike (S1) mutations like T19R, T95I, L452R, E484Q, and D614G. Notably, E156G/Δ157-158 was present in more than 85% of the sequences reported from the USA, UK, and India in August 2021. The spike PV bearing combination of E156G/Δ157-158 and L452R further promoted infectivity and conferred immune evasion. Additionally, increased cell-to-cell fusion was observed when spike harbored E156G/Δ157-158, L452R, and E484Q, suggesting a combinatorial effect of these mutations. Notwithstanding, the plasma from a recovered individual robustly inhibited mutant spike PV, indicating the increased breadth of neutralization post-recovery. Our data highlights the importance of spike NTD-specific changes in determining infectivity and immune escape of variants.

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

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0259551
Author(s):  
Syed Hani Abidi ◽  
Kehkashan 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

Background Individuals recovering from COVID-19 are known 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 although they may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays for 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 a cytopathic effect, lysed and RNA RT-PCR conducted for SARS-CoV-2. PCR target 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, 1 sample showed neutralization at the first dilution, 4 samples showed neutralization at lower dilutions, while one sample did not demonstrate any neutralization. The RBD ODs and neutralization potential percentages were found to be positively correlated. Conclusion We describe a rapid RT-PCR-based SARS-CoV-2 microneutralization assay for the detection of neutralizing antibodies. This can effectively be used to test the antiviral activity of serum antibodies for the investigation of both disease-driven and vaccine-induced responses.

scholarly journals Monitoring of Tumor Cell Purging After Highly Efficient Immunomagnetic Selection of CD34 Cells From Leukapheresis Products in Breast Cancer Patients: Comparison of Immunocytochemical Tumor Cell Staining and Reverse Transcriptase–Polymerase Chain Reaction

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 337-344 ◽  
Author(s):  
Markus Y. Mapara ◽  
Ida J. Körner ◽  
Martin Hildebrandt ◽  
Ralf Bargou ◽  
Dorothea Krahl ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Cell ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Cell Depletion ◽  
Breast Cancer Patients ◽  
Rt Pcr ◽  
Cd34 Cells ◽  
Polymerase Chain ◽  
Selection Of

We studied the efficiency of indirect tumor cell purging via enrichment of CD34+ hematopoietic progenitor cells from leukapheresis products (LP) in breast cancer patients based on immunomagnetic selection of CD34+ cells. Detection of tumor cells was made by immunocytochemical staining. In addition, we evaluated the capacity of cytokeratin 19 (CK19)- and a novel epidermal growth factor receptor (EGF-R)-specific reverse transcriptase–polymerase chain reaction (RT-PCR) for monitoring tumor cell depletion. LP from 13 breast cancer patients were analyzed. Twenty-three CD34 selection procedures were performed. A median of 1.4 × 1010 total nucleated cells ([TNC] range, 0.88 to 3.5 × 1010) with a median CD34 purity of 2.5% (range, 0.4% to 6.3%) were entered into the selection procedure. Immunomagnetic CD34 enrichment resulted in a median purity of 83.3% (range, 45% to 95.4%) and a median recovery of 73.2% (range, 22% to 95%). Retransfusion of CD34-selected cells after high-dose chemotherapy resulted in a rapid and sustained hematologic recovery, reaching an absolute neutrophil count of 500/μL at day +10 and platelet count of 20,000/μL at day +11. Tumor cell depletion was quantified by immunocytochemical detection of CK19-positive cells. By this method, a median tumor cell depletion of 1.9 log (range, 0.7 to <3 log) could be demonstrated. Immunocytochemical detection of tumor cells was more sensitive than RT-PCR, yielding positive results in 81% of LP (17 to 21) versus 58% positive LP (10 of 17). However, EGF-R–based RT-PCR was much more sensitive than CK19-based RT-PCR (10 of 17 v 1 of 17). Despite highly efficient CD34 selection, tumor cells were still detectable after CD34 enrichment using immunocytochemistry and EGF-R–specific RT-PCR. Thus, this novel EGF-R–specific RT-PCR appears to be of value as an additional method to detect contaminating breast cancer cells within LP.

scholarly journals Monitoring of Tumor Cell Purging After Highly Efficient Immunomagnetic Selection of CD34 Cells From Leukapheresis Products in Breast Cancer Patients: Comparison of Immunocytochemical Tumor Cell Staining and Reverse Transcriptase–Polymerase Chain Reaction

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 337-344 ◽  
Author(s):  
Markus Y. Mapara ◽  
Ida J. Körner ◽  
Martin Hildebrandt ◽  
Ralf Bargou ◽  
Dorothea Krahl ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Cell ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Cell Depletion ◽  
Breast Cancer Patients ◽  
Rt Pcr ◽  
Cd34 Cells ◽  
Polymerase Chain ◽  
Selection Of

Abstract We studied the efficiency of indirect tumor cell purging via enrichment of CD34+ hematopoietic progenitor cells from leukapheresis products (LP) in breast cancer patients based on immunomagnetic selection of CD34+ cells. Detection of tumor cells was made by immunocytochemical staining. In addition, we evaluated the capacity of cytokeratin 19 (CK19)- and a novel epidermal growth factor receptor (EGF-R)-specific reverse transcriptase–polymerase chain reaction (RT-PCR) for monitoring tumor cell depletion. LP from 13 breast cancer patients were analyzed. Twenty-three CD34 selection procedures were performed. A median of 1.4 × 1010 total nucleated cells ([TNC] range, 0.88 to 3.5 × 1010) with a median CD34 purity of 2.5% (range, 0.4% to 6.3%) were entered into the selection procedure. Immunomagnetic CD34 enrichment resulted in a median purity of 83.3% (range, 45% to 95.4%) and a median recovery of 73.2% (range, 22% to 95%). Retransfusion of CD34-selected cells after high-dose chemotherapy resulted in a rapid and sustained hematologic recovery, reaching an absolute neutrophil count of 500/μL at day +10 and platelet count of 20,000/μL at day +11. Tumor cell depletion was quantified by immunocytochemical detection of CK19-positive cells. By this method, a median tumor cell depletion of 1.9 log (range, 0.7 to <3 log) could be demonstrated. Immunocytochemical detection of tumor cells was more sensitive than RT-PCR, yielding positive results in 81% of LP (17 to 21) versus 58% positive LP (10 of 17). However, EGF-R–based RT-PCR was much more sensitive than CK19-based RT-PCR (10 of 17 v 1 of 17). Despite highly efficient CD34 selection, tumor cells were still detectable after CD34 enrichment using immunocytochemistry and EGF-R–specific RT-PCR. Thus, this novel EGF-R–specific RT-PCR appears to be of value as an additional method to detect contaminating breast cancer cells within LP.

scholarly journals Genetic Diversity of Sapoviruses among Inpatients in Germany, 2008−2018

Viruses ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 726 ◽  
Author(s):  
Mann ◽  
Pietsch ◽  
Liebert
Keyword(s):  
Genetic Diversity ◽  
Age Groups ◽  
Structural Protein ◽  
Rt Pcr ◽  
Sporadic Cases ◽  
Stool Samples ◽  
Gastrointestinal Complaints ◽  
Polymerase Chain ◽  
Infants And Young Children ◽  
Selection Of

Sapovirus enteric disease affects people of all ages across the globe, in both sporadic cases and outbreak settings. Sapovirus is seldom assessed in Germany and its epidemiology in the country is essentially unknown. Thus, sapovirus occurrence and genetic diversity were studied by real-time reverse transcription polymerase chain reaction (RT-PCR) and partial sequencing of major viral structural protein (VP1) gene in two different sets of stool samples: 1) a selection of 342 diarrheal stools collected from inpatient children during 2008−2009, and 2) 5555 stool samples collected during 2010–2018 from inpatients of all age groups with gastrointestinal complaints. Results showed year-round circulation of sapoviruses, with peaks during cooler months. In total, 30 samples (8.8%) of the first and 112 samples of the second set of samples (2.0%) were sapovirus positive. Capsid gene sequencing was successful in 134/142 samples (94.4%) and showed circulation of all known human pathogenic genogroups. Genotype GI.1 predominated (31.8%), followed by GII.1 (16.7%), GII.3 (14.5%), GI.2 (13.8%) and GV.1 (12.3%). Additionally, minor circulation of GI.3, GI.6, GII.2, GII.4, GII.6 and GIV.1 was shown. Consequently, sapovirus diagnostics need broadly reactive RT-PCR protocols and should particularly be considered in infants and young children. Further studies from other sampling sites are essential to extend our knowledge on sapovirus epidemiology in Germany.

scholarly journals Development of a reverse transcription polymerase chain reaction for the detection of severe fever with thrombocytopenia syndrome virus from suspected infected animals

2020 ◽  
Author(s):  
Eun-sil Park ◽  
Osamu Fujita ◽  
Masanobu Kimura ◽  
Akitoyo Hotta ◽  
Koichi Imaoka ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Neutralizing Antibodies ◽  
Clinical Symptoms ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Primer Sets ◽  
Severe Fever

AbstractBackgroundSevere fever with thrombocytopenia syndrome virus (SFTSV) causes severe hemorrhagic fever in humans and cats. Clinical symptoms of SFTS-infected cats resemble to those of SFTS patients and SFTS-contracted cats shows high levels of viral RNA loads in the serum and body fluids. Due to the risk of direct infection from SFTS-infected cats to human, it is important to diagnose SFTS-suspected animals.Methodology/Principle findingsFour primer sets were newly designed from consensus sequences constructed by 108 strains of SFTSV. A reverse transcription polymerase chain reaction (RT-PCR) with these four primer sets were successfully and specifically detected several clades of SFTSV. Their limits of detection are 1-10 copies/reaction. By this RT-PCR, 5 cat cases among 56 SFTS-suspected animal cases were diagnosed as SFTS. From these cats, IgM or IgG against SFTSV were detected by enzyme-linked immunosorbent assay (ELISA), but not neutralizing antibodies by plaque reduction neutralization titer (PRNT) test. This phenomenon is similar to those of fatal SFTS patients.Conclusion/SignificanceThis newly developed RT-PCR could detect SFTSV RNA of several clades from SFTS-suspected animals. In addition to ELISA and PRNT test, the useful laboratory diagnosis systems of SFTS-suspected animals has been made in this study.Author summaryThis study developed RT-PCR to detect SFTS animal cases. This assay could detect SFTSV RNA belonging to different clades. Cats diagnosed as SFTS had IgM or IgG, but not neutralizing antibodies. SFTS cat cases were distributed in the area where SFTS patients have been reported highly, indicating the establishment of the circulation of SFTSV in the environment. These diagnostic assays could be helpful tools to detect and not to miss SFTS animal cases.

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