Early detection of neutralizing antibodies against SARS-CoV-2 in COVID-19 patients in Thailand

Background The presence of neutralizing antibodies (NAbs) is an indicator of protective immunity for most viral infections. A newly developed surrogate viral neutralization assay (sVNT) offers the ability to detect total receptor binding domain-targeting NAbs in an isotype-independent manner, increasing the test sensitivity. Thus, specimens with low IgM/ IgG antibody levels showed strong neutralization activity in sVNT. Methods This study aimed to measure the %inhibition of NAbs measured by sVNT in PCR-confirmed COVID-19 patients. The sensitivity of sVNT for the diagnosis of SARS-CoV-2 infection and its kinetics were determined. Results Ninety-seven patients with PCR-confirmed SARS-CoV-2 infection were included in this study. Majority of the patients were 21–40 years old (67%) and 63% had mild symptoms. The sensitivity of sVNT for the diagnosis of SARS-CoV-2 infection was 99% (95% confidence interval (CI) 94.4–100%) and the specificity was 100% (95% CI 98.3–100%). The negative predictive value of sVNT from the samples collected before and after 7 days of symptom onset was 99.5% (95% CI 97.4–100%) and 100% (95% CI 93.8–100%), respectively. The level of inhibition at days 8–14 were significantly higher than days 0–7 (p<0.001). The median %inhibition values by severity of COVID-19 symptoms were 79.9% (interquartile range (IQR) 49.7–91.8%); 89.0% (IQR 71.2–92.4%); and 86.6% (IQR 69.5–92.8%), for mild, moderate and severe/critical symptoms respectively. The median level of sVNT %inhibition of severe was significantly higher than the mild group (p = 0.05). Conclusion The sVNT is a practical and robust serological test for SARS-CoV-2 infection and does not require specialized biosafety containment. It can be used clinically to aid diagnosis in both early and late infection especially in cases when the real-time RT-PCR results in weakly negative or weakly positive, and to determine the protective immune response from SARS-CoV-2 infection in patients.

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

10.21203/rs.3.rs-858163/v1 ◽

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.

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Long-Term Course of Humoral and Cellular Immune Responses in Outpatients After SARS-CoV-2 Infection

Frontiers in Public Health ◽

10.3389/fpubh.2021.732787 ◽

2021 ◽

Vol 9 ◽

Author(s):

Julia Schiffner ◽

Insa Backhaus ◽

Jens Rimmele ◽

Sören Schulz ◽

Till Möhlenkamp ◽

...

Keyword(s):

Immune Responses ◽

Peripheral Blood ◽

Neutralizing Antibodies ◽

Natural Infection ◽

Disease Course ◽

Igg Antibodies ◽

Igg Antibody ◽

Moderate Disease ◽

Ifn Γ ◽

Antibody Levels

Characterization of the naturally acquired B and T cell immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important for the development of public health and vaccination strategies to manage the burden of COVID-19 disease. We conducted a prospective, cross-sectional analysis in COVID-19 recovered patients at various time points over a 10-month period in order to investigate how circulating antibody levels and interferon-gamma (IFN-γ) release by peripheral blood cells change over time following natural infection. From March 2020 till January 2021, we enrolled 412 adults mostly with mild or moderate disease course. At each study visit, subjects donated peripheral blood for testing of anti-SARS-CoV-2 IgG antibodies and IFN-γ release after SARS-CoV-2 S-protein stimulation. Anti-SARS-CoV-2 immunoglobulin G (IgG) antibodies were positive in 316 of 412 (76.7%) and borderline in 31 of 412 (7.5%) patients. Our confirmation assay for the presence of neutralizing antibodies was positive in 215 of 412 (52.2%) and borderline in 88 of 412 (21.4%) patients. Likewise, in 274 of 412 (66.5%) positive IFN-γ release and IgG antibodies were detected. With respect to time after infection, both IgG antibody levels and IFN-γ concentrations decreased by about half within 300 days. Statistically, production of IgG and IFN-γ were closely associated, but on an individual basis, we observed patients with high-antibody titres but low IFN-γ levels and vice versa. Our data suggest that immunological reaction is acquired in most individuals after natural infection with SARS-CoV-2 and is sustained in the majority of patients for at least 10 months after infection after a mild or moderate disease course. Since, so far, no robust marker for protection against COVID-19 exists, we recommend utilizing both, IgG and IFN-γ release for an individual assessment of the immunity status.

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Age-dependent immune response to the Biontech/Pfizer BNT162b2 COVID-19 vaccination

10.1101/2021.03.03.21251066 ◽

2021 ◽

Cited By ~ 4

Author(s):

Lisa Müller ◽

Marcel Andrée ◽

Wiebke Moskorz ◽

Ingo Drexler ◽

Lara Walotka ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Age Groups ◽

Real Life ◽

The Elderly ◽

Igg Antibody ◽

Real Life Data ◽

Antibody Titers ◽

Protection Against Infection ◽

Vaccination Campaigns ◽

Antibody Levels

AbstractBackgroundThe SARS-CoV-2 pandemic has led to the development of various vaccines. Real-life data on immune responses elicited in the most vulnerable group of vaccinees over 80 years old is still underrepresented despite the prioritization of the elderly in vaccination campaigns.MethodsWe conducted a cohort study with two age groups, young vaccinees below the age of 60 and elderly vaccinees over the age of 80, to compare their antibody responses to the first and second dose of the BNT162b2 COVID-19 vaccination.ResultsWhile the majority of participants in both groups produced specific IgG antibody titers against SARS-CoV-2 spike protein, titers were significantly lower in elderly participants. Although the increment of antibody levels after the second immunization was higher in elderly participants, the absolute mean titer of this group remained lower than the <60 group. After the second vaccination, 31.3 % of the elderly had no detectable neutralizing antibodies in contrast to the younger group, in which only 2.2% had no detectable neutralizing antibodies.ConclusionOur data suggests that lower frequencies of neutralizing antibodies after BNT162b2 vaccination in the elderly population may require earlier revaccination to ensure strong immunity and protection against infection.

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Development and utilization of a surrogate SARS-CoV-2 viral neutralization assay to assess mRNA vaccine responses.

10.1101/2021.08.05.21261616 ◽

2021 ◽

Author(s):

Adam V Wisnewski ◽

Jian Liu ◽

Carolina V Lucas ◽

Jon Klein ◽

Akiko Iwasaki ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralization Assay ◽

Competitive Elisa ◽

Prior History ◽

Viral Neutralization ◽

Neutralizing Activity ◽

Vaccine Responses ◽

Protein Receptor ◽

History Of ◽

High Level

Background: Tests for SARS-CoV-2 immunity are needed to help assess responses to vaccination, which can be heterogeneous and may wane over time. The plaque reduction neutralization test (PRNT) is considered the gold standard for measuring serum neutralizing antibodies but requires high level biosafety, live viral cultures and days to complete. We hypothesized that competitive enzyme linked immunoassays (ELISAs) based on SARS-CoV-2 spike protein receptor binding domain (RBD) attachment to its host receptor, the angiotensin converting enzyme 2 receptor (ACE2r), would correlate with PRNT, given the central role of RBD-ACE2r interactions in infection and published studies to date, and enable evaluation of vaccine responses. Methods and Findings: Configuration and development of a competitive ELISA with plate-bound RBD and soluble biotinylated ACE2r was accomplished using pairs of pre/post vaccine serum. When the competitive ELISA was used to evaluate N=32 samples from COVID-19 patients previously tested by PRNT, excellent correlation in IC50 results were observed (rs= .83, p < 0.0001). When the competitive ELISA was used to evaluate N=41 vaccinated individuals and an additional N=14 unvaccinated recovered COVID-19 patients, significant differences in RBD-ACE2r inhibitory activity were associated with prior history of COVID-19 and type of vaccine received. In longitudinal analyses pre and up to 200 days post vaccine, surrogate neutralizing activity increased markedly after primary and booster vaccine doses, but fell substantially, up to <12% maximal levels within 6 months. Conclusions: A competitive ELISA based on inhibition of RBD-ACE2r attachment correlates well with PRNT, quantifies significantly higher activity among vaccine recipients with prior COVID (vs. those without), and highlights marked declines in surrogate neutralizing activity over a 6 month period post vaccination. The findings raise concern about the duration of vaccine responses and potential need for booster shots.

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Reactogenicity Correlates Only Weakly with Humoral Immunogenicity after COVID-19 Vaccination with BNT162b2 mRNA (Comirnaty®)

Vaccines ◽

10.3390/vaccines9101063 ◽

2021 ◽

Vol 9 (10) ◽

pp. 1063

Author(s):

Jürgen Held ◽

Jan Esse ◽

Koray Tascilar ◽

Philipp Steininger ◽

Kilian Schober ◽

...

Keyword(s):

Linear Regression ◽

Regression Model ◽

Linear Regression Model ◽

Healthcare Workers ◽

Model Performance ◽

Neutralization Assay ◽

Spike Protein ◽

Igg Antibody ◽

Boost Vaccination ◽

Antibody Levels

mRNA vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), such as BNT162b2 (Comirnaty®), have proven to be highly immunogenic and efficient but also show marked reactogenicity, leading to adverse effects (AEs). Here, we analyzed whether the severity of AEs predicts the antibody response against the SARS-CoV-2 spike protein. Healthcare workers without prior SARS-CoV-2 infection, who received a prime-boost vaccination with BNT162b2, completed a standardized electronic questionnaire on the duration and severity of AEs. Serum specimens were collected two to four weeks after the boost vaccination and tested with the COVID-19 ELISA IgG (Vircell-IgG), the LIAISON® SARS-CoV-2 S1/S2 IgG CLIA (DiaSorin-IgG) and the iFlash-2019-nCoV NAb surrogate neutralization assay (Yhlo-NAb). A penalized linear regression model fitted by machine learning was used to correlate AEs with antibody levels. Eighty subjects were enrolled in the study. Systemic, but not local, AEs occurred more frequently after the boost vaccination. Elevated SARS-CoV-2 IgG antibody levels were measured in 92.5% of subjects with Vircell-IgG and in all subjects with DiaSorin-IgG and Yhlo-NAb. Gender, age and BMI showed no association with the antibody levels or with the AEs. The linear regression model identified headache, malaise and nausea as AEs with the greatest variable importance for higher antibody levels (Vircell-IgG and DiaSorin-IgG). However, the model performance for predicting antibody levels from AEs was very low for Vircell-IgG (squared correlation coefficient r2 = 0.04) and DiaSorin-IgG (r2 = 0.06). AEs did not predict the surrogate neutralization (Yhlo-NAb) results. In conclusion, AEs correlate only weakly with the SARS-CoV-2 spike protein antibody levels after COVID-19 vaccination with BNT162b2 mRNA.

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Convalescent Plasma in a Patient with Protracted COVID-19 and Secondary Hypogammaglobulinemia Due to Chronic Lymphocytic Leukemia: Buying Time to Develop Immunity?

Infectious Disease Reports ◽

10.3390/idr13040077 ◽

2021 ◽

Vol 13 (4) ◽

pp. 855-864

Author(s):

Jaap L. J. Hanssen ◽

Johan Stienstra ◽

Stefan A. Boers ◽

Cilia R. Pothast ◽

Hans L. Zaaijer ◽

...

Keyword(s):

Immune Response ◽

Chronic Lymphocytic Leukemia ◽

Neutralizing Antibodies ◽

Serological Test ◽

Viral Clearance ◽

Lymphocytic Leukemia ◽

Cell Immune Response ◽

Humoral And Cellular Immunity ◽

Immunological Responses ◽

Antibody Levels

It is not exactly clear yet which type of immune response prevails to accomplish viral clearance in coronavirus disease 2019 (COVID-19). Studying a patient with chronic lymphocytic leukemia and hypogammaglobulinemia who suffered from COVID-19 provided insight in the immunological responses after treatment with COVID-19 convalescent plasma (CCP). Treatment consisted of oxygen, repeated glucocorticosteroids and multiple dosages of CCP guided by antibody levels. Retrospectively performed humoral and cellular immunity analysis made clear that not every serological test for COVID-19 is appropriate for follow-up of sufficient neutralizing antibodies after CCP. In retrospect, we think that CCP merely bought time for this patient to develop an adequate cell immune response which led to viral clearance and ultimately clinical recovery.

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Distinct age-specific SARS-CoV-2 IgG decay kinetics following natural infection

10.1101/2021.08.05.21259465 ◽

2021 ◽

Author(s):

Calvin P Sjaarda ◽

Emily Moslinger ◽

Kyla Tozer ◽

Robert I Colautti ◽

Samira Kheitan ◽

...

Keyword(s):

Natural Infection ◽

Decay Rates ◽

Decay Kinetics ◽

Serum Samples ◽

Igg Antibody ◽

Multi Centre Study ◽

Viral Neutralization ◽

Antibody Levels ◽

Over Time

Background. Antibody responses to SARS-CoV-2 can be observed as early as 14 days post- infection, but little is known about the stability of antibody levels over time. Here we evaluate the long-term stability of anti-SARS-CoV-2 IgG antibodies following infection in 402 adult donors. Methods. We performed a multi-centre study carried out at Plasma Donor Centres in the city of Heidelberg (Plasmazentrum Heidelberg, Germany) and Munich (Plasmazentrum M&uumlnchen, Germany). We present anti-S/N and anti-N IgG antibody levels in prospective serum samples collected up to 403 days post recovery from SARS-CoV-2 infected individuals. Results: The cohort includes 402 adult donors (185 female, 217 male; 17 - 68 years of age) where anti-SARS-CoV-2 IgG levels were measured in plasma samples collected between 18- and 403-days post SARS-CoV-2 infection. A linear mixed effects model demonstrated IgG decay rates that decrease over time (χ2=176.8, p<0.00001) and an interaction of time*age (χ2=10.0, p<0.005)), with those over 60+ years showing the highest baseline IgG levels and the fastest rate of IgG decay. Baseline viral neutralization assays demonstrated that serum IgG levels correlated with in vitro neutralization capacity in 91% of our cohort. Conclusion. Long-term antibody levels and age-specific antibody decay rates suggest the potential need for age-specific vaccine booster guidelines to ensure long term vaccine protection against SARS-CoV-2 infection.

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Serology in Children with Multisystem Inflammatory Syndrome (MIS-C) associated with COVID-19

10.1101/2020.07.10.20150755 ◽

2020 ◽

Cited By ~ 1

Author(s):

Christina A. Rostad ◽

Ann Chahroudi ◽

Grace Mantus ◽

Stacey A. Lapp ◽

Mehgan Teherani ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Limit Of Detection ◽

Geometric Mean ◽

Neutralization Assay ◽

Adverse Outcomes ◽

Igg Antibody ◽

Live Virus ◽

Lengths Of Stay ◽

Antibody Titers ◽

Inflammatory Syndrome

Objectives: We aimed to measure SARS-CoV-2 serologic responses in children hospitalized with multisystem inflammatory syndrome (MIS-C) compared to COVID-19, Kawasaki Disease (KD) and other hospitalized pediatric controls. Methods: From March 17, 2020 - May 26, 2020, we prospectively identified hospitalized children at Children's Healthcare of Atlanta with MIS-C (n=10), symptomatic PCR-confirmed COVID-19 (n=10), KD (n=5), and hospitalized controls (n=4). With IRB approval, we obtained prospective and residual blood samples from these children and measured SARS-CoV-2 spike (S) receptor binding domain (RBD) IgM and IgG binding antibodies by quantitative ELISA and SARS-CoV-2 neutralizing antibodies by live-virus focus reduction neutralization assay. We statistically compared the log-transformed antibody titers among groups and performed correlation analyses using linear regression. Results: All children with MIS-C had high titers of SARS-CoV-2 RBD IgG antibodies, which correlated strongly with neutralizing antibodies (R2=0.667, P<0.001). Children with MIS-C had significantly higher SARS-CoV-2 RBD IgG antibody titers (geometric mean titer [GMT] 6800, 95%CI 3495-13231) than children with COVID-19 (GMT 626, 95%CI 251-1563, P<0.001), children with KD (GMT 124, 95%CI 91-170, P<0.001) and other hospitalized pediatric controls (GMT 85 [all below assay limit of detection], P<0.001). All children with MIS-C also had detectable RBD IgM antibodies, indicating recent SARS-CoV-2 infection. RBD IgG titers correlated with erythrocyte sedimentation rate (ESR) (R2=0.512, P<0.046) and with hospital and ICU lengths of stay (R2=0.590, P=0.010). Conclusion: Quantitative SARS-CoV-2 RBD antibody titers may have a role in establishing the diagnosis of MIS-C, distinguishing it from other similar clinical entities, and stratifying risk for adverse outcomes.

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Commercial Serology Assays Predict Neutralization Activity Against SARS-CoV-2

10.1101/2020.07.10.20150946 ◽

2020 ◽

Cited By ~ 1

Author(s):

Raymond T Suhandynata ◽

Melissa A Hoffman ◽

Deli Huang ◽

Jenny T Tran ◽

Michael J Kelner ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Neutralization Assay ◽

Positive Serology ◽

Neutralization Activity ◽

Antibody Titers ◽

Negative Controls ◽

First Time ◽

The Relationship ◽

Positive Results

Background. Currently it is unknown whether a positive serology results correlates with protective immunity against SARS-CoV-2. There are also concerns regarding the low positive predictive value of SARS-CoV-2 serology tests, especially when testing populations with low disease prevalence. Methods. A neutralization assay was validated in a set of PCR confirmed positive specimens and in a negative cohort. 9,530 specimens were screened using the Diazyme SARS-CoV-2 IgG serology assay and all positive results (N=164) were reanalyzed using the neutralization assay, the Roche total immunoglobin assay, and the Abbott IgG assay. The relationship between the magnitude of a positive SARS-CoV-2 serology result and the levels of neutralizing antibodies detected was correlated. Neutralizing antibody titers (ID50) were also longitudinally monitored in SARS-CoV-2 PCR confirmed patients. Results. The SARS-CoV-2 neutralization assay had a PPA of 96.6% with a SARS-CoV-2 PCR test and a NPA of 98.0% across 100 negative controls. ID50 neutralization titers positively correlated with all three clinical serology platforms. Longitudinal monitoring of hospitalized PCR confirmed COVID-19 patients demonstrates they made high neutralization titers against SARS-CoV-2. PPA between the Diazyme IgG assay alone and the neutralization assay was 50.6%, while combining the Diazyme IgG assay with either the Roche or Abbott platforms increased the PPA to 79.2% and 78.4%, respectively. Conclusions. For the first time, we demonstrate that three widely available clinical serology assays positively correlate with SARS-CoV-2 neutralization activity observed in COVID-19 patients. When a two-platform screen and confirm approach was used for SARS-CoV-2 serology, nearly 80% of two-platform positive specimens had neutralization titers (ID50 >50).

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Can individuals with low antibody responses to vaccines against other viruses acquire adequate SARS-CoV-2 antibody after vaccination with the BNT162b2 mRNA vaccine?

10.1101/2021.12.26.21268358 ◽

2021 ◽

Author(s):

Wataru Ogura ◽

Kouki Ohtsuka ◽

Sachiko Matsuura ◽

Takahiro Okuyama ◽

Satsuki Matsushima ◽

...

Keyword(s):

Cohort Study ◽

Neutralizing Antibodies ◽

Healthcare Workers ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Antibody Activity ◽

Low Responders ◽

Before And After ◽

Positive Threshold ◽

Antibody Levels

Objective In Japan, healthcare workers (HCWs) are vaccinated against coronavirus disease (COVID-19) and other contagious viruses (measles, rubella, chickenpox, mumps, and hepatitis B) to prevent nosocomial infection. However, some do not produce sufficient antibodies after vaccination (low responders). This study investigated changes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody levels among HCWs after SARS-CoV-2 vaccination and assessed whether low responders produced adequate SARS-CoV-2 anti-spike and neutralizing antibodies. Methods We conducted a prospective cohort study of HCWs before and after vaccination with the BNT162b2 mRNA vaccine in a hospital in Tokyo, Japan. The HCWs received two doses of BNT162b2 vaccine, 3 weeks apart. Those whose antibody levels against previous antiviral vaccines did not reach protective antibody levels after receiving two doses were defined as low responders, whereas those who produced adequate antibodies were defined as normal responders. SARS-CoV-2 anti-spike antibodies were measured 11 times from before the first BNT162b2 vaccination to 5 months after the second vaccination. SARS-CoV-2 neutralizing antibody activity was measured twice in low responders, 1 week to 1 month and 5 months after the second vaccination. Results Fifty HCWs were included in the analytic cohort. After vaccination, SARS-CoV-2 anti-spike antibody was detectable in the samples from both responders at each timepoint, but the level was lower at 5 months than at 1 week after the second vaccination. Low responders had SARS-CoV-2 neutralizing antibody activity 1 week to 1 month after the second vaccination, which exceeded the positive threshold after 5 months. Conclusion After BNT162b2 vaccination, low responders acquired adequate SARS-CoV-2 anti-spike and SARS-CoV-2 neutralizing antibodies to prevent SARS-CoV-2. However, SARS-CoV-2 anti-spike antibody levels were lower at 5 months than at 1 week after the second dose of BNT162b2 vaccine in low and normal responders. Therefore, low responders should also receive a third dose of BNT162b2 vaccine.

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