scholarly journals A Rapid Bead-Based Assay For Screening Of SARS-CoV-2 Neutralising Antibodies

2021 ◽  
Author(s):  
Santhik SL ◽  
Pramod Darvin ◽  
Aneesh Chandrasekharan ◽  
Shanakara Narayanan Varadarajan ◽  
Soumya Jaya Divakaran ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Neutralizing Antibody ◽  
Life Time ◽  
Cost Effective ◽  
Fluorescent Imaging ◽  
Antibody Detection ◽  
Screening Assay ◽  
Live Virus

Quantitative determination of neutralizing antibodies against SARS CoV2 is of paramount importance in immunodiagnostics, vaccine efficacy testing, and immune response profiling among the vaccinated population. Cost effective, rapid, easy-to-perform assays are essential to support the vaccine development process and immunosurveillance studies. Here, we describe a bead based screening assay for S1 neutralization using recombinant fluorescent proteins of hACE2 and SARS CoV2 S1, immobilized on solid beads employing nanobodies /metal-affinity tags. Nanobody-mediated capture of SARS CoV2 Spike (S1) on agarose beads served as the trap for soluble recombinant ACE2-GFPSpark, inhibited by neutralizing antibody. The first approach demonstrates single color fluorescent imaging of ACE2 GFPspark binding to His tagged S1 Receptor Binding Domain (RBD His) immobilized beads. The second approach is dual color imaging of soluble ACE2 GFPSpark to S1 Orange Fluorescent Protein (S1 OFPSpark) beads. Both methods showed a good correlation with the gold standard pseudovirion assay and can be adapted to any fluorescent platforms for screening. Life time imaging of the ACE2 GFPSpark confirmed the interaction of ACE2 and S1 OFPSpark on beads. The self-renewable source of secreted recombinant proteins from stable cells and its direct use without necessitating purification renders the platform a cost-effective and rapid one than the popular pseudovirion assay and live virus-based assays. Any laboratory with minimum expertise can rapidly perform this bead assay for neutralizing antibody detection using stable engineered cells.

scholarly journals SARS-CoV-2 RBD trimer protein adjuvanted with Alum-3M-052 protects from SARS-CoV-2 infection and immune pathology in the lung

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nanda Kishore Routhu ◽  
Narayanaiah Cheedarla ◽  
Venkata Satish Bollimpelli ◽  
Sailaja Gangadhara ◽  
Venkata Viswanadh Edara ◽  
...  
Keyword(s):  
Antibody Response ◽  
Antibody Titer ◽  
Neutralizing Antibodies ◽  
Rhesus Macaques ◽  
Neutralizing Antibody ◽  
Significant Loss ◽  
Lung Pathology ◽  
Live Virus ◽  
Suitable Candidate ◽  
Immune Pathology

AbstractThere is a great need for the development of vaccines that induce potent and long-lasting protective immunity against SARS-CoV-2. Multimeric display of the antigen combined with potent adjuvant can enhance the potency and longevity of the antibody response. The receptor binding domain (RBD) of the spike protein is a primary target of neutralizing antibodies. Here, we developed a trimeric form of the RBD and show that it induces a potent neutralizing antibody response against live virus with diverse effector functions and provides protection against SARS-CoV-2 challenge in mice and rhesus macaques. The trimeric form induces higher neutralizing antibody titer compared to monomer with as low as 1μg antigen dose. In mice, adjuvanting the protein with a TLR7/8 agonist formulation alum-3M-052 induces 100-fold higher neutralizing antibody titer and superior protection from infection compared to alum. SARS-CoV-2 infection causes significant loss of innate cells and pathology in the lung, and vaccination protects from changes in innate cells and lung pathology. These results demonstrate RBD trimer protein as a suitable candidate for vaccine against SARS-CoV-2.

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

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

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

scholarly journals Antibody titers measured by commercial assays are correlated with neutralizing antibody titers calibrated by international standards

2021 ◽  
Author(s):  
Yu-An Kung ◽  
Chung-Guei Huang ◽  
Sheng-Yu Huang ◽  
Kuan-Ting Liu ◽  
Peng-Nien Huang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Geometric Mean ◽  
Neutralizing Antibody ◽  
Diagnostic Techniques ◽  
International Standards ◽  
World Health ◽  
Serum Samples ◽  
Antibody Titers ◽  
Health Organization

The World Health Organization (WHO) has highlighted the importance of an international standard (IS) for SARS-CoV-2 neutralizing antibody titer detection, with the aim of calibrating different diagnostic techniques. In this study, IS was applied to calibrate neutralizing antibody titers (IU/mL) and binding antibody titers (BAU/mL) in response to SARS-CoV-2 vaccines. Serum samples were collected from participants receiving the Moderna (n = 20) and Pfizer (n = 20) vaccines at three time points: pre-vaccination, after one dose, and after two doses. We obtained geometric mean titers of 1404.16 and 928.75 IU/mL for neutralizing antibodies after two doses of the Moderna and Pfizer vaccines, respectively. These values provide an important baseline for vaccine development and the implementation of non-inferiority trials. We also compared three commercially available kits from Roche, Abbott, and MeDiPro for the detection of COVID-19 antibodies based on binding affinity to S1 and/or RBD. Our results demonstrated that antibody titers measured by commercial assays are highly correlated with neutralizing antibody titers calibrated by IS.

scholarly journals Safety and Immunogenicity Evaluation of Inactivated whole-virus-SARS-COV-2 As Emerging Vaccine Development In Egypt

2021 ◽  
Author(s):  
Amani A. Saleh ◽  
Mohamed A. Saad ◽  
Islam Ryan ◽  
Magdy Amin ◽  
Mohamed I. Shindy ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Clinical Symptoms ◽  
Neutralizing Antibody ◽  
Positive Development ◽  
Virus Challenge ◽  
Cell Monolayers ◽  
Transmission Electron ◽  
Dose Concentration ◽  
Antibody Titers

AbstractThe current worldwide pandemic COVID-19 is causing severe human health problems, with high numbers of mortality rates and huge economic burdens that require an urgent demand for safe, and effective and vaccine development. Our study was the first trail to development and evaluation of safety and immune response to inactivated whole SARS-COV-2 virus vaccine adjuvanted with aluminium hydroxide. We used characterized SARS-COV-2 strain, severe acute respiratory syndrome coronavirus 2 isolates (SARS-CoV-2/human/EGY/Egy-SERVAC/2020) with accession numbers; MT981440; MT981439; MT981441; MT974071; MT974069 and MW250352 at GenBank that isolated from Egyptian patients SARS-CoV-2-positive. Development of the vaccine was carried out in a BSL - 3 facilities and the immunogenicity was determined in mice at two doses (55µg and 100µg per dose). All vaccinated mice were received a booster dose 14 days post first immunization. Our results demonstrated distinct cytopathic effect on the vero cell monolayers induced through SARS-COV-2 propagation and the viral particles were identified as Coronaviridae by transmission electron microscopy. SARS-CoV-2 was identified by RT-PCR performed on the cell culture. Immunogenicity of the developed vaccine indicated the high antigen-binding and neutralizing antibody titers, regardless the dose concentration, with excellent safety profiles.However, no deaths or clinical symptoms in mice groups. The efficacy of the inactivated vaccine formulation was tested by wild virus challenge the vaccinated mice and detection of viral replication in lung tissues. Vaccinated mice recorded complete protection from challenge infection three weeks post second dose. SARS-COV-2 replication was not observed in the lungs of mice following SARS-CoV-2 challenge, regardless of the level of serum neutralizing antibodies. This finding will support the future trials for evaluation an applicable SARS-CoV-2 vaccine candidate.

Prevalence and Persistence of Maternal Dengue Neutralizing Antibodies in Infants From Central and Southern Thailand: A Retrospective Cohort Study

2019 ◽  
Vol 31 (4) ◽  
pp. 288-295 ◽  
Author(s):  
Adrienne Guignard ◽  
François Haguinet ◽  
Stéphanie Wéry ◽  
Phirangkul Kerdpanich
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Geometric Mean ◽  
Neutralizing Antibody ◽  
Maternal Antibodies ◽  
Childhood Immunization ◽  
Serum Samples ◽  
Denv Serotypes ◽  
Antibody Titers ◽  
Antibody Kinetics

Understanding maternal dengue virus (DENV) neutralizing antibody kinetics in infants remains timely to develop a safe and effective childhood immunization. This retrospective study evaluated the prevalence and persistence of maternal antibody titers against DENV serotypes 1 to 4 in 139 Thai infants at 2, 6, and 7 months of age, using serum samples collected in a vaccination trial ( http://clinicaltrials.gov ; NCT00197275). Neutralizing antibodies against all 4 DENV serotypes were detected in 87.8% and 22.9% of infants at 2 and 7 months, respectively. At 2 months, DENV-4 neutralizing antibody geometric mean titers were notably lower (80) compared with DENV-1 to DENV-3 (277-471). Our results corroborate previous findings that DENV-1 to DENV-4 maternal antibodies persist at 7 months despite titers decrease from 2 months onwards. As persisting maternal antibodies may inhibit immune responses in DENV-vaccinated infants, a comprehensive understanding of DENV antibody kinetics is required in the perspective of vaccine development for infants.

scholarly journals Human Immunodeficiency Virus Env-Independent Infection of Human CD4− Cells

2000 ◽  
Vol 74 (23) ◽  
pp. 10994-11000 ◽  
Author(s):  
Shen Pang ◽  
Duan Yu ◽  
Dong Sung An ◽  
Gayle C. Baldwin ◽  
Yiming Xie ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Alternative Pathway ◽  
Neutralizing Antibody ◽  
Green Fluorescent Protein Gene ◽  
Immunodeficiency Virus ◽  
Green Fluorescent ◽  
Hiv 1

ABSTRACT CD4− epithelial cells covering mucosal surfaces serve as the primary barrier to prevent human immunodeficiency virus type 1 (HIV-1) infection. We used HIV-1 vectors carrying the enhanced green fluorescent protein gene as a reporter gene to demonstrate that HIV-1 can infect some CD4− human epithelial cell lines with low but significant efficiencies. Importantly, HIV-1 infection of these cell lines is independent of HIV-1 envelope proteins. The Env-independent infection of CD4− cells by HIV-1 suggests an alternative pathway for HIV-1 transmission. Even on virions bearing Env, a neutralizing antibody directed against gp120 is incapable of neutralizing the infection of these cells, thus raising potential implications for HIV-1 vaccine development.

scholarly journals Comparative Immunogenicity of Subtype A Human Immunodeficiency Virus Type 1 Envelope Exhibiting Differential Exposure of Conserved Neutralization Epitopes

2009 ◽  
Vol 84 (5) ◽  
pp. 2573-2584 ◽  
Author(s):  
Catherine A. Blish ◽  
D. Noah Sather ◽  
George Sellhorn ◽  
Leonidas Stamatatos ◽  
Yide Sun ◽  
...  
Keyword(s):  
Antibody Response ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Neutralizing Antibody ◽  
Immunodeficiency Virus ◽  
Cd4 Binding Site ◽  
Variable Loops ◽  
Linear Epitopes ◽  
Subtype A ◽  
Hiv 1

ABSTRACT Development of broadly cross-reactive neutralizing antibodies (NAbs) remains a major goal of HIV-1 vaccine development, but most candidate envelope immunogens have had limited ability to cross-neutralize heterologous strains. To evaluate the immunogenicity of subtype A variants of HIV-1, rabbits were immunized with pairs of closely related subtype A envelopes from the same individual. In each immunogen pair, one variant was readily neutralized by a variety of monoclonal antibodies and plasma antibodies, while the other was neutralization resistant, suggesting differences in the exposures of key epitopes. The breadth of the antibody response was evaluated against subtype A, B, C, and D variants of HIV-1. The specificity of the immunogen-derived neutralizing antibody response was also compared to that of the infected individuals from whom these variants were cloned. None of the immunogens produced broad neutralizing antibodies in immunized animals, and most of the neutralizing antibodies were directed to the variable loops, particularly the V3 loop. No detectable antibodies to either of the potentially exposed conserved epitopes, the membrane proximal external region, or the CD4 binding site were found with immunized rabbits. In contrast, relatively little of the neutralizing activity within the plasma samples of the infected individuals was directed to linear epitopes within the variable loops. These data indicate that immunogens designed to expose conserved regions did not enhance generation of broadly neutralizing antibodies in comparison with the immunogens that failed to expose those regions using this immunization approach.

scholarly journals Receptor Binding, Fusion Inhibition, and Induction of Cross-Reactive Neutralizing Antibodies by a Soluble G Glycoprotein of Hendra Virus

2005 ◽  
Vol 79 (11) ◽  
pp. 6690-6702 ◽  
Author(s):  
Katharine N. Bossart ◽  
Gary Crameri ◽  
Antony S. Dimitrov ◽  
Bruce A. Mungall ◽  
Yan-Ru Feng ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Gradient Centrifugation ◽  
Hendra Virus ◽  
Host Cells ◽  
Cell Surface Receptor ◽  
Size Exclusion ◽  
Emerging Viruses ◽  
Fusion Event ◽  
Live Virus

ABSTRACT Hendra virus (HeV) and Nipah virus (NiV) are closely related emerging viruses comprising the Henipavirus genus of the Paramyxovirinae, which are distinguished by their ability to cause fatal disease in both animal and human hosts. These viruses infect cells by a pH-independent membrane fusion event mediated by their attachment (G) and fusion (F) glycoproteins. Previously, we reported on HeV- and NiV-mediated fusion activities and detailed their host-cell tropism characteristics. These studies also suggested that a common cell surface receptor, which could be destroyed by protease, was utilized by both viruses. To further characterize the G glycoprotein and its unknown receptor, soluble forms of HeV G (sG) were constructed by replacing its cytoplasmic tail and transmembrane domains with an immunoglobulin κ leader sequence coupled to either an S-peptide tag (sGS-tag) or myc-epitope tag (sGmyc-tag) to facilitate purification and detection. Expression of sG was verified in cell lysates and culture supernatants by specific affinity precipitation. Analysis of sG by size exclusion chromatography and sucrose gradient centrifugation demonstrated tetrameric, dimeric, and monomeric species, with the majority of the sG released as a disulfide-linked dimer. Immunofluorescence staining revealed that sG specifically bound to HeV and NiV infection-permissive cells but not to a nonpermissive HeLa cell line clone, suggesting that it binds to virus receptor on host cells. Preincubation of host cells with sG resulted in dose-dependent inhibition of both HeV and NiV cell fusion as well as infection by live virus. Taken together, these data indicate that sG retains important native structural features, and we further demonstrate that administration of sG to rabbits can elicit a potent cross-reactive neutralizing antibody response against infectious HeV and NiV. This HeV sG glycoprotein will be exceedingly useful for structural studies, receptor identification strategies, and vaccine development goals for these important emerging viral agents.

scholarly journals Neutralization of SARS-CoV-2 Omicron variant by sera from BNT162b2 or Coronavac vaccine recipients

2021 ◽  
Author(s):  
Lu Lu ◽  
Bobo Mok ◽  
Linlei Chen ◽  
Jacky Chan ◽  
Owen Tsang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Geometric Mean ◽  
Neutralizing Antibody ◽  
World Health ◽  
Whole Genome Sequence ◽  
Live Virus ◽  
Significant Difference ◽  
Antibody Titers ◽  
Ancestral Virus ◽  
Health Organization

Background The SARS-CoV-2 Omicron variant, designated as a Variant of Concern(VOC) by the World Health Organization, carries numerous spike protein mutations which have been found to evade neutralizing antibodies elicited by COVID-19 vaccines. The susceptibility of Omicron variant by vaccine-induced neutralizing antibodies are urgently needed for risk assessment. Methods Omicron variant strains HKU691 and HKU344-R346K were isolated from patients using TMPRSS2-overexpressing VeroE6 cells. Whole genome sequence was determined using nanopore sequencing. Neutralization susceptibility of ancestral lineage A virus and the Omicron, Delta and Beta variants to sera from 25 BNT162b2 and 25 Coronavac vaccine recipients was determined using a live virus microneutralization assay. Results The Omicron variant strain HKU344-R346K has an additional spike R346K mutation, which is present in 8.5% of strains in GISAID database. Only 20% and 24% of BNT162b2 recipients had detectable neutralizing antibody against the Omicron variant HKU691 and HKU344-R346K, respectively, while none of the Coronavac recipients had detectable neutralizing antibody titer against either Omicron isolates. For BNT162b2 recipients, the geometric mean neutralization antibody titers(GMT) of the Omicron variant isolates(5.43 and 6.42) were 35.7-39.9-fold lower than that of the ancestral virus(229.4), and the GMT of both omicron isolates were significantly lower than those of the beta and delta variants. There was no significant difference in the GMT between HKU691 and HKU344-R346K. Conclusions Omicron variant escapes neutralizing antibodies elicited by BNT162b2 or CoronaVac. The additional R346K mutation did not affect the neutralization susceptibility. Our data suggest that the Omicron variant may be associated with lower COVID-19 vaccine effectiveness.

scholarly journals Persistence of Neutralizing Antibodies to SARS-CoV-2 in First Wave Infected Individuals at Ten Months Post-Infection: The UnIRSA Cohort Study

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2270
Author(s):  
Gloria Griffante ◽  
Shikha Chandel ◽  
Daniela Ferrante ◽  
Valeria Caneparo ◽  
Daniela Capello ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Natural Infection ◽  
Large Fraction ◽  
Geometric Mean ◽  
Neutralizing Antibody ◽  
Northern Italy ◽  
Serum Samples ◽  
Neutralization Titer ◽  
Igg Antibody

Longitudinal mapping of antibody-based SARS-CoV-2 immunity is critical for public health control of the pandemic and vaccine development. We performed a longitudinal analysis of the antibody-based immune response in a cohort of 100 COVID-19 individuals who were infected during the first wave of infection in northern Italy. The SARS-CoV-2 humoral response was tested using the COVID-SeroIndex, Kantaro Quantitative SARS-CoV-2 IgG Antibody RUO Kit (R&D Systems, Bio-Techne, Minneapolis, USA) and pseudotype-based neutralizing antibody assay. Using sequential serum samples collected from 100 COVID-19 recovered individuals from northern Italy—mostly with mild disease—at 2 and 10 months after their first positive PCR test, we show that 93% of them seroconverted at 2 months, with a geometric mean (GeoMean) half-maximal neutralization titer (NT50) of 387.9. Among the 35 unvaccinated subjects retested at 10 months, 7 resulted seronegative, with an 80% drop in seropositivity, while 28 showed decreased anti-receptor binding domain (RBD) and anti-spike (S) IgG titers, with a GeoMean NT50 neutralization titer dropping to 163.5. As an NT50 > 100 is known to confer protection from SARS-CoV-2 re-infection, our data show that the neutralizing activity elicited by the natural infection has lasted for at least 10 months in a large fraction of subjects.

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