scholarly journals A bioluminescent and homogeneous SARS-CoV-2 spike RBD and hACE2 interaction assay for antiviral screening and monitoring patient neutralizing antibody levels

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Juliano Alves ◽  
Laurie Engel ◽  
Renata de Vasconcelos Cabral ◽  
Eduardo L. Rodrigues ◽  
Liane de Jesus Ribeiro ◽  
...  

AbstractHere we describe a homogeneous bioluminescent immunoassay based on the interaction between Fc-tagged SARS-CoV-2 Spike RBD and human ACE2, and its detection by secondary antibodies labeled with NanoLuc luciferase fragments LgBit and SmBit. The assay utility for the discovery of novel inhibitors was demonstrated with a panel of anti-RBD antibodies, ACE2-derived miniproteins and soluble ACE2. Studying the effect of RBD mutations on ACE2 binding showed that the N501Y mutation increased RBD apparent affinity toward ACE2 tenfold that resulted in escaping inhibition by some anti-RBD antibodies. In contrast, while E484K mutation did not highly change the binding affinity, it still escaped antibody inhibition likely due to changes in the epitope recognized by the antibody. Also, neutralizing antibodies (NAbs) from COVID-19 positive samples from two distinct regions (USA and Brazil) were successfully detected and the results further suggest the persistence of NAbs for at least 6 months post symptom onset. Finally, sera from vaccinated individuals were tested for NAbs and showed varying neutralizing activity after first and second doses, suggesting the assay can be used to assess immunity of vaccinated populations. Our results demonstrate the broad utility and ease of use of this methodology both for drug discovery and clinical research applications.

Author(s):  
Kai Wang ◽  
Quan-Xin Long ◽  
Hai-Jun Deng ◽  
Jie Hu ◽  
Qing-Zhu Gao ◽  
...  

Background Coronavirus disease 2019 (COVID-19) is a global pandemic with no licensed vaccine or specific antiviral agents for therapy. Little is known about the longitudinal dynamics of SARS-CoV-2-specific neutralizing antibodies (NAbs) in COVID-19 patients. Methods Blood samples (n=173) were collected from 30 COVID-19 patients over a 3-month period after symptom onset and analyzed for SARS-CoV-2-specific NAbs, using the lentiviral pseudotype assay, coincident with the levels of IgG and proinflammatory cytokines. Results SARS-CoV-2-specific NAb titers were low for the first 7-10 d after symtom onset and increased after 2-3 weeks. The median peak time for NAbs was 33 d (IQR 24-59 d) after symptom onset. NAb titers in 93.3% (28/30) of the patients declined gradually over the 3-month study period, with a median decrease of 34.8% (IQR 19.6-42.4%). NAb titers increased over time in parallel with the rise in IgG antibody levels, correlating well at week 3 (r = 0.41, p < 0.05). The NAb titers also demonstrated a significant positive correlation with levels of plasma proinflammatory cytokines, including SCF, TRAIL, and M-CSF. Conclusions These data provide useful information regarding dynamic changes in NAbs in COVID-19 patients during the acute and convalescent phases.


Author(s):  
Katharine HD Crawford ◽  
Adam S Dingens ◽  
Rachel Eguia ◽  
Caitlin R Wolf ◽  
Naomi Wilcox ◽  
...  

Most individuals infected with SARS-CoV-2 develop neutralizing antibodies that target the viral spike protein. Here we quantify how levels of these antibodies change in the months following SARS-CoV-2 infection by examining longitudinal samples collected between ≈30 and 152 days post-symptom onset from a prospective cohort of 34 recovered individuals with asymptomatic, mild, or moderate-severe disease. Neutralizing antibody titers declined an average of about four-fold from one to four months post-symptom onset. Importantly, our data are consistent with the expected early immune response to viral infection, where an initial peak in antibody levels is followed by a decline to a lower plateau. Additional studies of long-lived B-cells and antibody titers over longer time frames are necessary to determine the durability of immunity to SARS-CoV-2.


Author(s):  
Katharine H D Crawford ◽  
Adam S Dingens ◽  
Rachel Eguia ◽  
Caitlin R Wolf ◽  
Naomi Wilcox ◽  
...  

Abstract Most individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop neutralizing antibodies that target the viral spike protein. In this study, we quantified how levels of these antibodies change in the months after SARS-CoV-2 infection by examining longitudinal samples collected approximately 30–152 days after symptom onset from a prospective cohort of 32 recovered individuals with asymptomatic, mild, or moderate-severe disease. Neutralizing antibody titers declined an average of about 4-fold from 1 to 4 months after symptom onset. This decline in neutralizing antibody titers was accompanied by a decline in total antibodies capable of binding the viral spike protein or its receptor-binding domain. Importantly, our data are consistent with the expected early immune response to viral infection, where an initial peak in antibody levels is followed by a decline to a lower plateau. Additional studies of long-lived B cells and antibody titers over longer time frames are necessary to determine the durability of immunity to SARS-CoV-2.


2022 ◽  
Author(s):  
Apoorva Munigela ◽  
Sasikala M ◽  
Gujjarlapudi Deepika ◽  
Anand V Kulkarni ◽  
Krishna Vemula ◽  
...  

Abstract Coronavirus disease (COVID-19) continues to be a major health concern leading to substantial mortality and morbidity across the world. Vaccination is effective in reducing the severity and associated mortality. Data pertaining to the duration of immunity, antibody waning and the optimal timing of booster dose administration is limited. In this cross-sectional study, we assessed the antibody levels in healthcare workers who were fully vaccinated after obtaining Institutional ethics committee approval and informed consent. Whole blood was collected and enumeration of S1/S2 neutralizing antibody levels was carried out using LIAISON SARS-COV-2 S1/S2 IgG assay. A total of 1636 individuals who were vaccinated with Covaxin or Covishield were included. Of these, 52% were males with a median age of 29 years. Diabetes and Hypertension was noted in 2.32% (38/1636) and 2.87% (47/1636) of the individuals. Spike neutralizing antibodies were below the detectable range (<15 AU/ml) in 6.0% (98/1636) of the individuals. Decline in neutralizing antibody was seen in 30% of the individuals above 40 years of age with comorbidities (diabetes and hypertension) after 6 months. These individuals may be prioritized for a booster dose at 6 months.


2021 ◽  
Author(s):  
Ernst J. Schaefer ◽  
Florence Comite ◽  
Latha Dulipsingh ◽  
Maxine Lang ◽  
Jessica Jimison ◽  
...  

AbstractMost deaths from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection occur in older subjects. We assessed age effects and clinical utility of serum SARS-CoV-2 immunoglobulin G (IgG), immunoglobulin M (IgM), and neutralizing antibodies and serum inflammatory markers. Serum IgG, IgM, and neutralizing antibody levels were measured using chemiluminescence assays from Diazyme (Poway, CA), while serum interleukin-6 (IL-6), C reactive protein (CRP), and ferritin were measured with immunoassays obtained from Roche (Indianapolis, IN). In 79,005 subjects, IgG and IgM levels were positive (≥1.0 arbitrary units [AU]/mL) in 5.29% and 3.25% of subjects, respectively. In antibody positive subjects, median IgG levels were 3.93 AU/mL if <45 years of age, 10.18 AU/mL if 45-64 years of age, and 10.85 AU/mL if ≥65 years of age (p<0.0001). In SARS-CoV-2 RNA positive cases, family members and exposed subjects (n=1,111), antibody testing was found to be valuable for case finding, and persistent IgM levels were associated with chronic symptoms. In non-hospitalized and hospitalized subjects assessed for SARS-CoV-2 RNA (n=278), median IgG levels in AU/mL were 0.05 in negative subjects (n=100), 14.83 in positive outpatients (n=129), and 30.61 in positive hospitalized patients (n=49, p<0.0001). Neutralizing antibody levels correlated significantly with IgG (r=0.875; p<0.0001). Two or more of the criteria of IL-6 ≥10 pg/mL, CRP ≥10 mg/L, and/or IgM >1.0 AU/mL occurred in 97.7% of inpatients versus 1.8% of outpatients (>50-fold relative risk, C statistic 0.986, p<0.0001). Our data indicate that: 1) IgG levels are significantly higher in positive older subjects, possibly to compensate for decreased cellular immunity with aging; 2) IgG levels are important for case finding in family clusters; 3) IgG levels are significantly correlated with neutralizing antibody levels; 4) persistently elevated IgM levels are associated with chronic disease; and 5) markedly elevated IL-6, hs-CRP, and/or positive IgM accurately identify SARS-CoV-2 RNA positive subjects requiring hospitalization.


2021 ◽  
Author(s):  
Jira Chansaenroj ◽  
Ritthideach Yorsaeng ◽  
Nasamon Wanlapakorn ◽  
Chintana Chirathaworn ◽  
Natthinee Sudhinaraset ◽  
...  

Abstract Understanding antibody responses after natural severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can guide the coronavirus disease 2019 (COVID-19) vaccine schedule. This study aimed to assess the dynamics of SARS-CoV-2 antibodies, including anti-spike protein 1 (S1) immunoglobulin (Ig)G, anti-receptor-binding domain (RBD) total Ig, anti-S1 IgA, and neutralizing antibody against wild-type SARS-CoV-2 in a cohort of patients who were previously infected with SARS-CoV-2. Between March and May 2020, 531 individuals with virologically confirmed cases of SARS-CoV-2 infection were enrolled in our immunological study. The neutralizing titers against SARS-CoV-2 were detected in 95.2%, 86.7%, 85.0%, and 85.4% of recovered COVID-19 patients at 3, 6, 9, and 12 months after symptom onset, respectively. The seropositivity rate of anti-S1 IgG, anti-RBD total Ig, anti-S1 IgA, and neutralizing titers remained at 68.6%, 89.6%, 77.1%, and 85.4%, respectively, at 12 months after symptom onset. The half-life of neutralizing titers was estimated at 100.7 days (95% confidence interval = 44.5 – 327.4 days, R2 = 0.106). These results support that the decline in serum antibody levels over time depends on the symptom severity, and the individuals with high IgG antibody titers experienced a significantly longer persistence of SARS-CoV-2-specific antibody responses than those with lower titers.


2020 ◽  
Vol 223 (1) ◽  
pp. 47-55 ◽  
Author(s):  
William T Lee ◽  
Roxanne C Girardin ◽  
Alan P Dupuis ◽  
Karen E Kulas ◽  
Anne F Payne ◽  
...  

Abstract Passive transfer of antibodies from COVID-19 convalescent patients is being used as an experimental treatment for eligible patients with SARS-CoV-2 infections. The United States Food and Drug Administration’s (FDA) guidelines for convalescent plasma initially recommended target antibody titers of 160. We evaluated SARS-CoV-2 neutralizing antibodies in sera from recovered COVID-19 patients using plaque reduction neutralization tests (PRNT) at moderate (PRNT50) and high (PRNT90) stringency thresholds. We found that neutralizing activity significantly increased with time post symptom onset (PSO), reaching a peak at 31–35 days PSO. At this point, the number of sera having neutralizing titers of at least 160 was approximately 93% (PRNT50) and approximately 54% (PRNT90). Sera with high SARS-CoV-2 antibody levels (&gt;960 enzyme-linked immunosorbent assay titers) showed maximal activity, but not all high-titer sera contained neutralizing antibody at FDA recommended levels, particularly at high stringency. These results underscore the value of serum characterization for neutralization activity.


2021 ◽  
Author(s):  
Annika Fendler ◽  
Lewis Au ◽  
Scott Shepherd ◽  
Fiona Byrne ◽  
Maddalena Cerrone ◽  
...  

Abstract Patients with cancer have higher COVID-19 morbidity and mortality. Here we present the prospective CAPTURE study (NCT03226886) integrating longitudinal immune profiling with clinical annotation. Of 357 patients with cancer, 118 were SARS-CoV-2-positive, 94 were symptomatic and 2 patients died of COVID-19. In this cohort, 83% patients had S1-reactive antibodies, 82% had neutralizing antibodies against WT, whereas neutralizing antibody titers (NAbT) against the Alpha, Beta, and Delta variants were substantially reduced. Whereas S1-reactive antibody levels decreased in 13% of patients, NAbT remained stable up to 329 days. Patients also had detectable SARS-CoV-2-specific T cells and CD4+ responses correlating with S1-reactive antibody levels, although patients with hematological malignancies had impaired immune responses that were disease and treatment-specific, but presented compensatory cellular responses, further supported by clinical. Overall, these findings advance the understanding of the nature and duration of immune response to SARS-CoV-2 in patients with cancer.


Vaccines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1139
Author(s):  
Xiaoguang Li ◽  
Chao Liang ◽  
Xiumei Xiao

This study investigated the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies following inoculation with the coronavirus disease (COVID-19) vaccine. From June to July 2021, 127 participants who had completed COVID-19 vaccination (inactivated SARS-CoV-2 vaccine, 64; CoronaVac, 61; CanSino, 2) were recruited and tested using SARS-CoV-2 neutralizing antibody kits. The positive detection rate (inhibition of neutralizing antibodies ≥ 30%) was calculated and stratified according to population characteristics and inoculation time. The positive rate of neutralizing antibody was 47.22% (17/36) in men and 53.85% (49/91) in women, and 54.55% (24/44) in BMI ≥ 24 and 50.60% (42/83) in BMI < 24. Age was stratified as 20–29, 30–39, 40–49, and ≥50; positive detection rates of SARS-CoV-2 neutralizing antibodies were observed in 60.00% (24/40), 50.00% (21/42), 48.39% (15/31), and 42.86% (6/14), respectively, but with no significant difference (x2 = 1.724, p = 0.632). Among 127 vaccinated participants, 66 (51.97%) were positive. The positive detection rate was 63.93% (39/61) with CoronaVac and 42.19% (27/64) with the inactivated SARS-CoV-2 vaccine (significance x2 = 5.927, p = 0.015). Multivariate analysis revealed a significant difference in vaccination times, with average vaccination weeks in the positive and negative groups of 11.57 ± 6.48 and 17.87 ± 9.17, respectively (t= −4.501, p < 0.001). The positive neutralizing antibody rate was 100.00%, 60.00%, 58.33%, 55.56%, 43.14%, 28.57%, and 0.00% at 2–4, 5–8, 9–12, 13–16,17–20, 21–24, and >24 weeks, respectively (x2 = 18.030, p = 0.006). Neutralizing antibodies were detected after COVID-19 inoculation, with differences relating to inoculation timing. This study provides a reference for vaccine evaluation and follow-up immunization strengthening.


Author(s):  
Renata Varnaitė ◽  
Marina García ◽  
Hedvig Glans ◽  
Kimia T. Maleki ◽  
John Tyler Sandberg ◽  
...  

AbstractCoronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 and has since become a global pandemic. Pathogen-specific antibodies are typically a major predictor of protective immunity, yet B cell and antibody responses during COVID-19 are not fully understood. Here, we analyzed antibody-secreting cell (ASC) and antibody responses in twenty hospitalized COVID-19 patients. The patients exhibited typical symptoms of COVID-19, and presented with reduced lymphocyte numbers and increased T cell and B cell activation. Importantly, we detected an expansion of SARS-CoV-2 nucleocapsid protein-specific ASCs in all twenty COVID-19 patients using a multicolor FluoroSpot assay. Out of the 20 patients, 16 had developed SARS-CoV-2-neutralizing antibodies by the time of inclusion in the study. SARS-CoV-2-specific IgA, IgG and IgM antibody levels positively correlated with SARS-CoV-2-neutralizing antibody titers, suggesting that SARS-CoV-2-specific antibody levels may reflect the titers of neutralizing antibodies in COVID-19 patients during the acute phase of infection. Lastly, we showed that interleukin 6 (IL-6) and C-reactive protein (CRP) concentrations were higher in serum of patients who were hospitalized for longer, supporting the recent observations that IL-6 and CRP could be used to predict COVID-19 severity. Altogether, this study constitutes a detailed description of clinical and immunological parameters in twenty COVID-19 patients, with a focus on B cell and antibody responses, and provides tools to study immune responses to SARS-CoV-2 infection and vaccination.


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