ABSTRACT
COVID-19, caused by the novel SARS-CoV-2 virus, poses major challenges for global public health. The detection of antibodies in blood serum is one of the important methods for diagnostics of COVID-19 patients. The main aim was to study the dynamics of the appearance of neutralizing antibodies and antibodies to the SARS-CoV-2 proteins in COVID-19 patients sera. Material and methods. The blood sera of four groups of people were studied: "intact" donors (blood sera were collected in 2016-2019);patients with a laboratory-confirmed diagnosis of acute respiratory viral infection;patients with influenza (antibodies to the influenza virus have been identified) and patients with a PCR confirmed diagnosis of COVID-19. Blood sera were analyzed in ELISA with commercial kits for detection of IgG to SARS-CoV-2 (N, S) proteins and total antibodies to RBD of protein S and in neutralization test (NT). Results and discussion. Antibodies to SARS-CoV-2 were not detected in paired blood sera of people from groups 1-3 by ELISA and NT. At the time of hospitalization of patients with COVID-19 in the sera of 12 (19%) patients antibodies to SARS-CoV-2 were absent when they were determined by NT and ELISA. In blood sera taken 4-9 days after hospitalization, neutralizing antibodies and antibodies to at least one viral protein were detected in ELISA. Conclusion. At the time of hospitalization, the overwhelming majority of patients had a humoral immune response to the SARS-CoV-2. In the dynamics of observation, the levels of antibodies to SARS-CoV-2 proteins increased, to a greater extent to RBD.Copyright © 2022 Geotar Media Publishing Group
ABSTRACT
Intro: Different vaccines against COVID-19 have been approved by the World Health Organization (WHO) at different stages, however, limited data is available on long-term kinetics of antibodies induced by vaccines. This study was performed to investigate the persistence and dynamicity of BBV-152 (Covaxin)- and AZD1222 (Covishield)-induced immunoglobulin-G (IgG) antibodies over the year and neutralizing antibodies' status after the one-month post booster dose. Method(s): This 52-week longitudinal cohort study documented antibody persistence and neutralizing antibody status among 278 health-care workers (HCWs) from four different healthcare and research facilities in Odisha, enrolled in January 2021 and continued until March 2022. An automated chemiluminescence immune assay (CLIA) platform from Abbott Diagnostics was used to quantify IgG antibodies against SARS-CoV-2's spike receptor-binding domain (RBD) and a surrogate virus neutralization test (sVNT) was performed by enzyme-linked immunosorbent assay (ELISA). If any participants developed any symptoms of COVID-19, nasopharyngeal swabs were collected and sent to ICMR- RMRC, Bhubaneswar for RT-PCR confirmation. Finding(s): Among the 243 participants, 119 HCWs (48.97%) were Covaxin recipients and the remaining 124 (51.02%) were Covishield recipients. During the seven follow- ups, 104 participants (42.79%) were identified as vaccine breakthrough cases. In 139 non-infected HCWs, the median antibody titer significantly waned after ten months of double dose, both for Covaxin (342.7 AU/mL at DD1 vs 43.9 AU/mL at DD10) and Covishield (2325.8 AU/mL at DD3 vs 595.2 AU/mL at DD10). No statistically significant differences in antibody titers were observed based on age, gender, comorbidities, and blood groups. The median inhibition activity of sVNT was increased significantly for Covaxin and Covishield booster recipients. Among the booster dose recipients, 24 had breakthrough cases by the Omicron variant. Conclusion(s): Results of this longitudinal cohort study can be used to implement vaccination strategies and could also aid in tracking and designing vaccine mandates to minimize vaccine escape.Copyright © 2023
ABSTRACT
Intro: GBP510 contains the self-assembling recombinant nanoparticle displaying SARS-CoV-2 Spike protein receptor binding domain and is adjuvanted with AS03. We report interim Phase 3 study (NCT05007951) results up to 4 weeks post-dose 2 (Data-cut: March-18-2022), where immunogenicity to the D614G ancestral strain and safety of 25mug GBP510/AS03 candidate was compared to ChAdOx1-S (Vaxzevria). Method(s): This Phase 3 randomized, active-controlled, observer-blind, parallel- group study in adults was conducted in 6 countries. Cohort1: 1,895 subjects (naive to COVID-19 vaccination and infection) randomized at 2:1 ratio (GBP510/AS03:ChAdOx1-S) to assess immunogenicity and safety;Cohort 2: 2,141 subjects at 5:1 ratio, regardless of their serostatus at screening for safety assessment. Subjects were vaccinated twice at a 4-week interval with 0.5 mL of the test vaccine (GBP510/AS03) or active control (ChAdOx1-S) in deltoid muscle. The primary objective was to demonstrate the superiority of geometric mean titer (GMT) and non-inferiority in seroconversion rate (SCR: >=4-fold rise from baseline) of neutralizing antibodies over ChAdOx1-S by live-virus neutralization assay (FRNT). Finding(s): At 2 weeks post-dose 2, GMT ratio of the two groups (Test vaccine/Active control) was 2.93 [95% CI: 2.63, 3.27], satisfying the hypothesis of superiority (95% CI lower limit> 1). The SCR difference (Test vaccine - Active control) was 10.76% [95% CI: 7.68, 14.32], satisfying the hypothesis of non- inferiority (95% CI lower limit> -5%). Good cell-mediated immune responses for Th1 cytokines were also observed with the test vaccine (FluoroSpot). The AE incidence rate for the test vaccine was higher than the active control for solicited local AEs (56.69% vs 49.20%), and comparable for solicited systemic AEs (51.21% vs 53.51%) and unsolicited AEs (13.34% vs 14.66%) after any vaccination. Conclusion(s): Higher immune responses were observed with GBP510/AS03 compared to ChAdOx1-S against D614G strain after 2 weeks post-dose 2. GBP510/AS03 showed a clinically acceptable safety profile;no safety concerns were identified during the study period.Copyright © 2023
ABSTRACT
BACKGROUND: Since sensitivity and specificity vary widely between tests, SARS-CoV-2 serology results should be interpreted with caution. METHODS: The study included serum samples from patients who had recovered from COVID-19 (n = 71), individuals vaccinated against SARS-CoV-2 (n = 84), and asymptomatic individuals (n = 33). All samples were tested for the presence of binding antibodies (enzyme immunoassay; EIA), neutralizing (NT) antibodies (virus neutralization test; VNT), and surrogate NT (sNT) antibodies (surrogate virus neutralization test; sVNT) of SARS-CoV-2. RESULTS: SARS-CoV-2-binding antibodies were detected in 71 (100%) COVID-19 patients, 77 (91.6%) vaccinated individuals, and 4 (12.1%) control subjects. Among EIA-positive samples, VNT was positive (titer ≥ 8) in 100% of COVID-19 patients and 63 (75.0%) of the vaccinated individuals, while sVNT was positive (>30% inhibition) in 62 (87.3%) patients and 59 (70.2%) vaccinated individuals. The analysis of antibody levels showed a significant moderate positive correlation between EIA and VNT, a moderate positive correlation between EIA and sVNT, and a strong positive correlation between VNT and sVNT. The proportion of positive sVNT detection rate was associated with VNT titer. The lowest positivity (72.4%/70.8%) was detected in samples with low NT titers (8/16) and increased progressively from 88.2% in samples with titer 32 to 100% in samples with titer 256. CONCLUSIONS: sVNT appeared to be a reliable method for the assessment COVID-19 serology in patients with high antibody levels, while false-negative results were frequently observed in patients with low NT titers.
ABSTRACT
Towards the end of 2019 a novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) caused the ongoing global pandemic. The virus surface consists of spike proteins that mediate SARS-CoV-2 entry into cells through its receptor-binding domain (RBD) that attaches to the human receptor Angiotensin- Converting Enzyme 2 (ACE2). Upon infection with foreign material, like viruses and bacteria, the human immune system responds by producing a humoral response specific to the viral antigen. Cells from the innate immune system and antibodies generated in the humoral response work to destroy and block infectious antigens from causing damage to the human cells. The S protein of SARSCoV- 2 is the key protein that stimulates the immune system to generate neutralizing antibodies. To safely test and investigate SARS-CoV-2 in BSL-2 lab setting, we propagated a surrogate pseudo typed virus to evaluate the ability of antibodies to reduce viral cell entry and replication in SARS-CoV-2 infected mice model. Quantifying the functional ability of neutralizing antibodies would help us understand how they influence reinfection in recovered individuals. We hypothesize that antibodies generated in SARS-CoV-2 infected mice models will induce a protective immune response against the SARSCoV- 2 infection. To detect and quantify the protective immune response generated in mice, we performed two different serological assays and identified antibodies endpoint titers. Mice were infected with Delta and Beta at time points Day 3 and Day 4. We performed a SARS-CoV-2 Spike pseudo virus neutralization assay and measured luminescence to determine the percentage neutralization of functional antibodies induced in mice serum samples upon infection. Utilizing indirect ELISAs,' we measured absorbance for IgA antibodies in Bronchoalveolar lavage fluid (BALF) serum and total IgG antibodies in cardiac bleeds. Our results showed we did not obtain neutralizing activity of antibodies in mice serum samples taken at early time points, 24 hrs and 4 days, after infection with the Delta variant of SARS CoV2 virus using both the pseudo viruses Omicron andWA spike.We obtained 100% neutralizing activity in mice serum samples taken at day 21 and infected with Beta variant of SARS CoV2 virus using both the pseudo viruses Omicron and WA spike demonstrating that there is cross-neutralization against various variants of concern. Antibodies (IgA, IgM, IgG) generated in mice 3 weeks post infection with SARS CoV2 (Beta) virus are capable of neutralizing and inhibiting the entry of WA spike and Omicron pseudo viruses in human HEK293 T Ace2 cells. Moving forward utilizing samples with timepoints surpassing 3 weeks could possibly yield higher concentrations of IgA and IgM antibodies that can neutralize the SARS-CoV-2 pseudo virus. Thank you to Dr. Rhea Coler, the entire Coler lab, National Institutes of Health (NIH), and Seattle Children's Research Institute.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
SARS-CoV-2 Spike harbors glycans which function as ligands for lectins. Therefore, it should be possible to exploit lectins to target SARS-CoV-2 and inhibit cellular entry by binding glycans on the Spike protein. Burkholderia oklahomensis agglutinin (BOA) is an antiviral lectin that interacts with viral glycoproteins via N-linked high mannose glycans. Here, we show that BOA binds to the Spike protein and is a potent inhibitor of SARS-CoV-2 viral entry at nanomolar concentrations. Using a variety of biophysical tools such as SEC chromatography, dynamics light scattering, fluorescence binding assays, and electron microscopy, we demonstrate that the interaction is avidity driven and that BOA crosslinks the Spike protein into soluble aggregates. Furthermore, using virus neutralization assays, we demonstrate that BOA effectively inhibits all tested variants of concern as well as SARS-CoV-1, establishing that glycan-targeting molecules have the potential to be pan-coronavirus inhibitors.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
Background: While remarkable and rapid progress was made in fighting the SARS-CoV-2 pandemic with vaccines and therapeutic antibodies, these approaches were quickly compromised by viral evolution. Therapeutic monoclonal antibodies (mAbs) that were once authorized for clinical use, which all target the receptor binding domain (RBD), are no longer effective against current variants of concern (VOCs) due to mutations in this region of Spike. Thus, to achieve durable protection against SARS-CoV-2, novel mAbs need to show breadth and potency across VOCs and target epitopes that are more constrained. Method(s): mAbs from an individual who had a breakthrough Delta VOC infection after vaccination were isolated from Spike-specific memory B cells. mAbs were assessed for binding affinity and neutralization potency using Spike-pseudotyped lentivirus (PSV) and live SARS-CoV-2 virus neutralization assays. Epitopes were mapped using deep mutational sequencing (DMS) and structural-based methods. Result(s): Three novel mAbs (C68.3, C68.13, C68.59) demonstrated binding breadth to Spikes from various VOCs including Omicron VOCs despite that C68 had not yet been exposed to Omicron. These mAbs potently neutralized the Wuhan-Hu-1 vaccine and Delta strains (IC50 = 9-61ng/mL), and early Omicron strains BA.1, BA.2, BA.5 (IC50 = 12-149 ng/mL). C68.3 and C68.59 retained potency against recent VOCs BQ.1.1 and XBB (IC50 = 121-122 ng/mL and 56-82 ng/mL, respectively) in the PSV assay. Similar neutralization activity was observed in the live virus assay. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs previously authorized for treatment and they showed greater breadth. The mAbs target distinct epitopes on the Spike glycoprotein, two in the RBD (C68.3, C68.13) and one in an invariant region downstream of RBD in subdomain 1 (SD1) (C68.59). Structural analysis of C68.59 Fab binding to Spike trimer revealed significant allosteric changes to regions of Spike outside of the epitope in the S2 unit. Finally, DMS escape pathways showed these mAbs target regions highly conserved across VOCs that are also functionally constrained, suggesting escape could incur a fitness cost. Conclusion(s): Overall, these mAbs are novel in their breadth across VOCs and include a potent mAb targeting a rare epitope outside of the RBD in SD1. These mAbs focus on diverse, functionally constrained regions in Spike making them candidates for development as combination therapeutics with good durability against future VOCs.
ABSTRACT
Background: Individuals living with HIV are at increased risk of morbidity and mortality from COVID-19. Furthermore, SARS-CoV-2 infection in immunocompromised HIV infected individuals poses a risk to prolonged infection and viral shedding and the emergence of new variants of concern (VOCs). Using the SIV macaque model for AIDS, we are investigating the hypothesis that immune dysfunction during HIV infection will prolong SARSCoV- 2 viral infection, promote enhanced COVID-19 disease, and accelerate viral evolution. Here, we report the impact of SIV-CoV-2 co-infection on immune responses and pathogenesis. Method(s): Eight female rhesus macaques (aged 7-15 years, 5.5-9.9kg) were infected with SIVmac251 via low dose intravaginal challenge and then inoculated with 6.5x105 TCID50/mL SARS-CoV-2 (WA-1) at 17-34 weeks post-SIV infection via combined intranasal and intratracheal routes. Blood, bronchoalveolar lavage (BAL), stool, and nasal, oral, and rectal swabs were collected pre-infection through 14 days post-infection (DPI) to measure immune responses and viremia. ELISAs, ELISPOT, qRT-PCR, lung pathology, cytokine multiplex, and virus neutralization assays were performed to measure viral loads, pathogenesis, and immune responses. Result(s): Three days post-SARS-CoV-2 infection, we observed a transient decrease in CD4 counts, but there were no changes in clinical symptoms or plasma SIV viral loads. However, SARS-CoV-2 replication persisted in the upper respiratory tract, but not the lower respiratory tract. In addition, SARS-CoV-2 IgG seroconversion was delayed and antigen-specific T-cell responses were dampened. Notably, viral RNA levels in nasal swabs were significantly higher 7-14 DPI in SIV+ compared to previously published results using the same SARS-CoV-2 challenge virus in SIV- rhesus (PMCID: PMC8462335, PMC8829873). In addition, SIV/CoV-2 co-infected animals exhibited elevated levels of myeloperoxidase (MPO), a marker of neutrophil activation and increased lung inflammation. Conclusion(s): Here we provide evidence for the utility of the rhesus macaque in modeling human HIV-SARS-CoV-2 co-infection. Our results suggest that immunosuppression during SIV infection impairs de novo generation of anti-SARS-CoV-2 immunity, that may contribute to prolonged SARS-CoV-2 viral shedding, increased transmission windows, altered disease pathogenesis, and lower protection against subsequent SARS-CoV-2 exposures. Studies in progress will determine if SARS-CoV-2 viral evolution is accelerated in SIV-infected macaques.
ABSTRACT
Background: Serum virus-neutralization (VNT)capacity is an important parameter of immunological response in COVID-19 recovered individuals and it can also be used to predict the effectiveness of therapy with COVID-19 convalescent plasma. The most commonly used tests to assess virus-neutralization are those that use native SARS-CoV- 2 (cVNT), SARS-CoV- 2 spike pseudotyped lentivirus-like particles (pVNT), and also inhibition of recombinant RBD binding to ACE2 receptor in vitro (surrogate VNT, sVNT). The aim of this study was to determine the interchangeability of different approaches in the evaluation of the virus-neutralization activity of convalescent serum samples. Method(s): Serum samples (n = 111) were collected 10-36 days after recovery between May and September 2020. The SARS-CoV- 2 strain PMVL-12/ 2020 was used in cVNT at 100 doses of TCID50. In pVNT SARS-CoV- 2 pseudotyped HIV-1 based virus-like particles with GFP reporter gene were used. sVNT was performed with a kit from Xema Co., Russia. Result(s): A very strong correlation was observed between cVNT and pVNT results (Spearman's r = 0.841). The correlation of cVNT and pVNT with the sVNT was only moderate (r = 0.674 and 0.696, respectively). This is consistent with the fact that sVNT detects only RBD blocking antibodies, which are the main but not the only inhibitors of viral infection. Serum samples were also tested for RBD-specific IgG, IgM, IgA antibodies by ELISA. A good correlation was found between the cVNT, pVNT, sVNT results and the RBD-specific IgG (r = 0.669, 0.620, and 0.643, respectively), that confirms a crucial role of specific IgG antibodies in virus-neutralization. RBD-specific IgA showed a moderate correlation with the neutralization capacity of sera from recovered individuals (r = 0.563, 0.583, 0.544). Correlation of approximately the same level was observed between cVNT, pVNT, and RBD-specific IgM (r = 0.663, 0.615), but not sVNT and RBD-specific IgM (r = 0.395). Conclusion(s): This study demonstrated the possibility of using the safe and relatively simple pseudotyped virus-neutralization test instead of cVNT to assess the sera virus-neutralizing capacity. sVNT may be efficiently used in screening studies.
ABSTRACT
Introduction: Kidney transplant recipients (KTRs) are at risk for substantial morbidity and mortality during COVID-19 infection. Vaccination for this group of patients is reccommended. However, immunogenicity and safety data after COVID-19 vaccination among KTRs remains limited. Method(s): We conducted an observational prospective trial involving KTRs at Chiang Mai University hospital, Chiang Mai, Thailand. The participants were received homologous ChAdOx1 nCoV-19 (AZ-AZ), or the heterologous prime-boost of CoronaVac,followed by AZ (SV-AZ). The immunogenicity was assessed by measuring antibodies against the S1 receptor-binding domain (anti-RBD), and SARS-CoV-2 surrogate virus neutralization test (sVNT) at specific timepoints. The primary outcome was the seroconversion rate of sVNT at day 28 after complete vaccination. The secondary outcomes were the seroconversion rate of sVNT at day 28 after the first dose of vaccination, the level of sVNT and anti-RBD at specific timepoints, and the adverse events of each vaccine regimen. Result(s): A total of 18 KTRs were recruited. Among those, 13 (72.2%), and 5 (27.8%) patients were received AZ-AZ, and SV-AZ regimen, respectively. The seroconversion rate of sVNT at day 28 after the second dose were 23.1%, and 20.0% for AZ-AZ, and SV-AZ, respectively (P>0.99). The level of sVNT and the level of anti-RBD at day 28 after the first and at day 28 after the second dose were not different between groups (Figure 1). There were no serious adverse events reported in any vaccine groups. However, AZ-AZ showed sign of tubular dysfunction demonstrated by increasing of fractional excretion of magnesium after complete course of vaccination which correlated to the trend of urine albumin and urine protein creatinine ratio (r=0.720, P=0.013;and r=0.726, P=0.011, respectively). [Formula presented] [Formula presented] Figure 1 Percentage of neutralization inhibition (a) and level of anti-RBD antibody (b) at each visit of homologous ChAdOx1 nCoV-19 (AZ-AZ), and heterologous prime-boost of CoronaVac, followed by AZ (SV-AZ) regimen Conclusion(s): Immunogenicity after COVID-19 vaccination with either homologous or heterologous prime-boost regimen among KTRs was compromised. Homologous replication-defective viral vectors vaccine regimen seemed to affect renal tubular function, and further follow-up should be warranted. No conflict of interestCopyright © 2023
ABSTRACT
Detection and diagnosis platforms play key roles in early warning, outbreak control and exit strategy for any pandemic, and they are especially pertinent for the Coronavirus disease 2019 (COVID-19) pandemic. The challenges posed by the speed and extent of severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) spread around the globe also offered unprecedented opportunities for the development and deployment of novel strategies and products - not only vaccines and therapeutics, but also diagnostics. This chapter provides a brief summary of the vast array of molecular, serological, cell-based and other diagnostic tools for the specific detection of SARS-CoV-2 infections and immune responses. The focus is on the principles and applications of each platform, while detailed protocols can be found in the cited references. © 2023 by World Scientific Publishing Co. Pte. Ltd.
ABSTRACT
The development of COVID-globulin, a COVID-19-specific human immunoglobulin preparation, involved choosing a method to quantify antibodies to SARS-CoV-2. Antibody titre determination by virus neutralisation (VN) is labour-intensive and unsuitable for large-scale application. To enable routine testing, it was necessary to develop a less demanding method;the enzyme-linked immunosorbent assay (ELISA) was the most appropriate of solutions. The lack of international and industry reference standards (RS) prompted the preparation and certification of an RS for COVID-globulin potency control. The aim of the study was to examine the possibility of substituting ELISA for VN and to develop an RS for SARS-CoV-2 antibody quantification in immunoglobulin preparations. Material(s) and Method(s): the authors used commercial ELISA kits by several manufacturers, COVID-globulin by Microgen (48 batches), and human plasma samples from multiple sources (1499 samples). The tests were performed by VN, ELISA, and chemiluminescent microparticle immunoassay. Result(s): the authors validated an ELISA method for SARSCoV-2 antibody quantification with the selected reagent kits by the National Medical Research Center for Hematology (NMRC for Hematology) and Euroimmun AG. The authors demonstrated the possibility of using ELISA instead of VN (with a correlation coefficient of more than 0.9). They developed and characterised an in-house RS for SARS-CoV-2 antibody content in human immunoglobulin preparations. The RS was certified in newly introduced anti-COVID units (ACU) and in international binding antibody units (BAU) using the World Health Organisation (WHO) international reference panel (NIBSC code: 20/268). The RS's potency was measured in terms of its neutralising activity in ACU (320 ACU/mL) and BAU (2234.8 BAU/mL). The authors established the relationship between ACU and BAU units. For the selected ELISA reagent kits, the conversion factors were 6.4 (NMRC for Hematology) and 7.0 (Euroimmun AG). Conclusion(s): the ELISA method for SARS-CoV-2 antibody quantification and the RS for SARS-CoV-2 antibody content can be applied to determine the potency of human anti-COVID-19 immunoglobulins.Copyright © 2023 Safety and Risk of Pharmacotherapy. All rights reserved.
ABSTRACT
Bovine respiratory diseases (BRD) are associated with various predisposing factors, such as physical and physiological stress factors, and bacterial and viral pathogens. These stressors and viruses suppress immune defenses, leading to bacterial growth in the upper respiratory tract and invasion of pathogens into the lower respiratory tract. Therefore, continuous monitoring of the causative pathogens would contribute to the early detection of BRD. Nasal swabs and sera from 63 clinically healthy calves were continuously collected from seven farms in Iwate prefecture from 2019 to 2021. We attempted to monitor dynamics of BRD-associated pathogens by multiplex real-time RT-PCR (RT-qPCR) using their nasal swab samples. In addition, we attempted to monitor fluctuation of antibody titers against each BRD-associated pathogen by virus neutralization test (VNT) using their sera. In contrast, nasal swabs from 89 calves infected with BRD were collected from 28 farms in Iwate prefecture from 2019 to 2021. We attempted to analyze their nasal swab samples by multiplex RT-qPCR aim to detect BRD-associated pathogens that are dominant in this region. As a result, our analyses using samples from clinically healthy calves showed that positive results by multiplex RT-qPCR were closely related to a significant increase of antibody titers by VNT in bovine coronavirus (BCoV), bovine torovirus (BToV), and bovine respiratory syncytial virus (BRSV). In addition, our data exhibited that BCoV, BToV, BRSV, bovine parainfluenza virus 3, and Mycoplasma bovis have been more frequently detected in calves infected with BRD compared to those detected in clinically healthy calves. Moreover, the data presented herein revealed co-infections by combination multiple viral pathogens with bacterial pathogens are closely involved in the onset of BRD. Taken together, our study demonstrates multiplex RT-qPCR which can simultaneously analyze multiple pathogens, including viruses and bacteria, and is useful for the early detection of BRD.
Subject(s)
Cattle Diseases , Coronavirus, Bovine , Respiratory Syncytial Virus, Bovine , Respiratory Tract Diseases , Animals , Cattle , Cattle Diseases/diagnosis , Respiratory Tract Diseases/veterinary , Nose , TracheaABSTRACT
Background: The pandemic unleashed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 500 million people worldwide and caused more than 6 million deaths. Cellular and humoral immunity induced by infection or immunization are key factors in controlling the viral burden and avoiding the recurrence of coronavirus disease. The duration and effectiveness of immunity after infection is relevant to pandemic policy interventions, including the timing of vaccine boosters. Objectives: We sought to evaluate longitudinal binding and functional antibodies against SARS-CoV-2 receptor-binding domain in police officers and health care workers with a history of coronavirus disease 2019 and compare with SARS-CoV-2-naive individuals after vaccination with adenovirus-based ChAdOx1 nCoV-19 (AstraZeneca-Fiocruz) or the inactivated CoronaVac vaccine (Sinovac-Butantan Institute). Methods: A total of 208 participants were vaccinated. Of these, 126 (60.57%) received the ChAdOx1 nCoV-19 vaccine and 82 (39.42%) received the CoronaVac vaccine. Prevaccination and postvaccination blood was collected, and the amount of anti-SARS-CoV-2 IgG and the neutralizing ability of the antibodies to block the interaction between angiotensin-converting enzyme 2 and receptor-binding domain were determined. Results: Subjects with preexisting SARS-CoV-2 immunity and who received a single dose of ChAdOx1 nCoV-19 or CoronaVac have similar or superior antibody levels when compared with levels in seronegative individuals even after 2 doses of the vaccine. Neutralizing antibody titers of seropositive individuals were higher with a single dose of either ChAdOx1 nCoV-19 or CoronaVac compared with those of seronegative individuals. After 2 doses, both groups reached a plateau response. Conclusions: Our data reinforce the importance of vaccine boosters to increase specific binding and neutralizing SARS-CoV-2 antibodies.
ABSTRACT
The development of COVID-globulin, a COVID-19-specific human immunoglobulin preparation, involved choosing a method to quantify antibodies to SARS-CoV-2. Antibody titre determination by virus neutralisation (VN) is labour-intensive and unsuitable for large-scale application. To enable routine testing, it was necessary to develop a less demanding method;the enzyme-linked immunosorbent assay (ELISA) was the most appropriate of solutions. The lack of international and industry reference standards (RS) prompted the preparation and certification of an RS for COVID-globulin potency control. The aim of the study was to examine the possibility of substituting ELISA for VN and to develop an RS for SARS-CoV-2 antibody quantification in immunoglobulin preparations. Materials and methods: the authors used commercial ELISA kits by several manufacturers, COVID-globulin by Microgen (48 batches), and human plasma samples from multiple sources (1499 samples). The tests were performed by VN, ELISA, and chemiluminescent microparticle immunoassay. Results: the authors validated an ELISA method for SARSCoV-2 antibody quantification with the selected reagent kits by the National Medical Research Center for Hematology (NMRC for Hematology) and Euroimmun AG. The authors demonstrated the possibility of using ELISA instead of VN (with a correlation coefficient of more than 0.9). They developed and characterised an in-house RS for SARS-CoV-2 antibody content in human immunoglobulin preparations. The RS was certified in newly introduced anti-COVID units (ACU) and in international binding antibody units (BAU) using the World Health Organisation (WHO) international reference panel (NIBSC code: 20/268). The RS's potency was measured in terms of its neutralising activity in ACU (320 ACU/mL) and BAU (2234.8 BAU/mL). The authors established the relationship between ACU and BAU units. For the selected ELISA reagent kits, the conversion factors were 6.4 (NMRC for Hematology) and 7.0 (Euroimmun AG). Conclusions: the ELISA method for SARS-CoV-2 antibody quantification and the RS for SARS-CoV-2 antibody content can be applied to determine the potency of human anti-COVID-19 immunoglobulins.
ABSTRACT
Background: Cancer patients are at high risk of severe COVID infection and recommended at least three doses of SARS-CoV2 mRNA vaccines. Various anti-neoplastic treatments may affect long-term vaccine immunogenicity. Methods: Patients with solid or haematological cancer were recruited from two Singapore hospitals between July 2021 and March 2022. GenScript cPASS surrogate virus neutralisation assays measured antibody responses, which were correlated with clinical outcomes obtained from medical records and national mandatory-reporting databases. Results: In total, 273 patients were recruited (40 with haematological malignancies and the rest solid tumours). Two-hundred and four patients (74.7%) were receiving active cancer therapy: 98 (35.9%) receiving systemic chemotherapy and the rest targeted or immunotherapy. All patients were seronegative at baseline. After receiving one, two and three doses of SARS-CoV-2-mRNA vaccination, seroconversion rate was 35.2%, 79.4% and 92.4% respectively. After three doses, patients on active treatment for haematological malignancies had lower antibodies (57.3%±46.2) as compared to patients on immunotherapy (94.1%±9.56, p<0.05) and chemotherapy (92.8%±18.1, p<0.05). SARS-CoV-2 infection was reported in 77 (28.2%) patients of which 18 were severe. Conclusion: This study demonstrates high immunogenicity of three doses of vaccines and protection against severe infection in cancer patients.
ABSTRACT
Background: Impaired generation of antibody responses define 'predominantly antibody immuno-deficiencies' (PAD) with reduced IgG and impaired vaccination responses. However, the antibody repertoire defects underpinning PAD are unknown. Here, we examine the antibody repertoire using mass spectrometry-based proteomics (MS-proteomics) in PAD and healthy controls (HC). Method(s): Following SARS-CoV-2 vaccination, anti -S1 ELISA, and live-virus neutralisation assays were assessed. Purified anti-S1 IgG and IgM was sequenced by MS-Proteomics to define immunoglobulin heavy chain variable region subfamily (IGHVsf) usage and somatic hypermutation (SHM). Result(s): 12 vaccine responsive PAD subjects were included, matched to 11 HC. Neutralisation and anti-S1 titres were reduced in PAD. Strikingly, all PAD subjects demonstrated restricted IgG IGHVsf utilisation, [median 3, (range 2-4), vs 6 (5-11) in HC, p<0.001], irrespective neutralisation or total antibody response. IgG SHM and IgM repertoire did not differ but IgG IGHV 3-7 utilisation was less frequent in PAD. Conclusion(s): MS proteomics uncovers stereotyped anti-S1 IgG IGHVsf restriction in PAD subjects following vaccination. Our results suggest that a relatively pauci-clonal antibody repertoire can produce a functional immune response, otherwise masked by traditional serology measures. Further studies to uncover the determinants of antibody repertoire breadth and elaborate on this novel approach to assessing serological responses are required. Copyright © 2022
ABSTRACT
The receptor binding domain (RBD) plays a pivotal role in the viral entry as it enables the engagement of severe acute respiratory syndrome 2 (SARS-CoV-2) with the human angiotensin-converting enzyme 2 (ACE2) receptor for host cell entry. RBD is the major target for developing viral inhibitors and vaccines. Expression of recombinant RBD in E.coli is highly scalable with a low-cost procedure despite its high expression level compared to expression in mammalian and yeast cells. Using an alternative natural adjuvant system instead of alum adjuvant, increased immunogenicity of RBD antigen in serological assay including direct ELISA and surrogate Virus Neutralization Test (sVNT) was demonstrated with high levels of IgGs and neutralizing antibodies in mice sera immunized with RBD:AlSa (Alum and Sodium alginate) formulation. The sVNT is a simple and fast test that can be used instead of the conventional virus neutralization test requiring live virus and BSL3 laboratory to detect total neutralizing antibodies against RBD. Additionally, results showed a safety profile for sodium alginate which supported using it as an alternative natural adjuvant.
Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Mice , Humans , Antibodies, Blocking , Antibodies, Viral , Antibodies, Neutralizing , Spike Glycoprotein, Coronavirus/chemistry , MammalsABSTRACT
A severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) surrogate virus neutralization test (sVNT) was used to determine the degree of inhibition of binding between human angiotensin converting enzyme 2 (hACE2) and the receptor binding domain (RBD) of spike protein by neutralizing antibodies in a biosafety level 2 facility. Here, to improve the sensitivity and specificity of the commercial sVNT, we developed a new biotin based sVNT using biotinylated RBD and HRP conjugated streptavidin instead of HRP conjugated RBD for direct detection in an ELISA assay that strongly correlated to the FDA approved cPass sVNT commercial kit (R2 = 0.8521) and pseudo virus neutralization test (R2 = 0.9006) (pVNT). The biotin based sVNT was evaluated in 535 postvaccination serum samples corresponding to second and third boosts of AZD1222 and BNT162b2 vaccines of the wild type strain. We confirmed that the neutralizing antibodies against SARS-CoV-2 variants in second vaccination sera decreased after a median of 141.5 days. Furthermore, vaccination sera from BNT162b2-BNT162b2 vaccines maintained neutralizing antibodies for longer than those of AZD1222 only vaccination. In addition, both vaccines maintained high neutralizing antibodies in third vaccination sera against Omicron BA.2 after a median of 27 days, but neutralizing antibodies significantly decreased after a median of 141.5 days. Along with the cPass sVNT commercial kit, biotin based sVNTs may also be suitable for specifically detecting neutralizing antibodies against multiple SARS-CoV-2 variants; however, to initially monitor the neutralizing antibodies in vaccinated sera using high throughput screening, conventional PRNT could be replaced by sVNT to circumvent the inconvenience of a long test time.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Biotin , BNT162 Vaccine , ChAdOx1 nCoV-19 , Neutralization Tests , COVID-19/prevention & control , Antibodies, Neutralizing , Antibodies, Viral , Spike Glycoprotein, CoronavirusABSTRACT
Background. We compared neutralizing antibodies in healthy healthcare workers against two different coronavirus 2019 disease (Covid-19) vaccines, a messenger RNA (mRNA)-based vaccine (BNT162b2) and a genetically modified organism (virus vector) vaccine (ChAdOx1), to obtain immunity. In our cohort, we tried to compare the level of neutralizing antibody production after a BNT162b2 vaccine booster and to analyze the level of neutralizing antibody titers for delta and omicron variant. Methods. On November 23 to 25, 2021 and December 23 to 24, 2021, a total of 2,133 HCWs at Soonchunhyang University Bucheon Hospital had been vaccinated with BNT162b2 (Pfizer-BioNTech). Among the 115 HCWs who participated in the preceding study, all participants had no history of Covid-19 infection or suspected symptoms at the time of registration. We collected blood samples from participants four weeks after a third dose. All blood samples were analyzed using the commercial virus neutralization test kit (Genscript Biotech Corporation, Piscataway, NJ, USA). Results. Titers were measured 2 months after the initial vaccination and before the booster of the BNT162b2 vaccine (i.e. 6 months after the initial vaccination), and 4 weeks after the booster. Neutralizing antibody level measured by percentage inhibition of surrogate virus neutralization test (sVNT) readings at negative control (unvaccinated) and 4 weeks after booster vaccination. BNT/BNT/BNT group was defined as a booster dose with BNT162b2 (Pfizer) after two doses of BNT162b2 vaccine (Pfizer) 3 weeks apart. ChA/ChA/BNT group was defined as a booster dose with BNT162b2 (Pfizer) after two doses of ChAdOx1 vaccine (AstraZeneca/Oxford) 12 weeks apart. Conclusion. We found that the neutralizing antibody levels against omicron variant were higher among those who received a booster dose of the BNT162b2 vaccine.