Identification of IgG antibody response to SARS-CoV-2 spike protein and its receptor binding domain does not predict rapid recovery from COVID-19

AbstractDiagnostic testing and evaluation of patient immunity against the novel severe acute respiratory syndrome (SARS) corona virus that emerged last year (SARS-CoV-2) are essential for health and economic crisis recovery of the world. It is suggested that potential acquired immunity against SARS-CoV-2 from prior exposure may be determined by detecting the presence of circulating IgG antibodies against viral antigens, such as the spike glycoprotein and its receptor binding domain (RBD). Testing our asymptomatic population for evidence of COVID-19 immunity would also offer valuable epidemiologic data to aid health care policies and health care management. Currently, there are over 100 antibody tests that are being used around the world without approval from the FDA or similar regulatory bodies, and they are mostly for rapid and qualitative assessment, with different degrees of error rates. ELISA-based testing for sensitive and rigorous quantitative assessment of SARS-CoV-2 antibodies can potentially offer mechanistic insights into the COVID-19 disease and aid communities uniquely challenged by limited financial resources and access to commercial testing products. Employing recombinant SARS-CoV-2 RBD and spike protein generated in the laboratory, we devised a quantitative ELISA for the detection of circulating serum antibodies. Serum from twenty SARS-CoV-2 RT-PCR confirmed COVID-19 hospitalized patients were used to detect circulating IgG titers against SARS-CoV-2 spike protein and RBD. Quantitative detection of IgG antibodies to the spike glycoprotein or the RBD in patient samples was not always associated with faster recovery, compared to patients with borderline antibody response to the RBD. One patient who did not develop antibodies to the RBD completely recovered from COVID-19. In surveying 99 healthy donor samples (procured between 2017-February 2020), we detected RBD antibodies in one donor from February 2020 collection with three others exhibiting antibodies to the spike protein but not the RBD. Collectively, our study suggests that more rigorous and quantitative analysis, employing large scale samples sets, is required to determine whether antibodies to SARS-CoV-2 spike protein or RBD is associated with protection from COVID-19 disease. It is also conceivable that humoral response to SARS-CoV-2 spike protein or RBD works in association with adaptive T cell response to determine clinical sequela and severity of COVID-19 disease.

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480 Preliminary evaluation of a novel coronavirus vaccine (CORVax) using electroporation of plasmid DNA encoding a stabilized prefusion SARS-CoV-2 spike protein alone or with transfection of plasmid IL-12

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0480 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A514-A514

Author(s):

Shawn Jensen ◽

Christopher Twitty ◽

Christopher Paustian ◽

Madelein Laws ◽

Glenna McDonnell ◽

...

Keyword(s):

Immune Responses ◽

Receptor Binding ◽

Binding Domain ◽

Tumor Rejection ◽

Spike Protein ◽

Preliminary Evaluation ◽

List Type ◽

Receptor Binding Domain ◽

Igg Antibodies ◽

Viral Neutralization

BackgroundSARS-CoV-2 (CoV2) has precipitated a global pandemic and the effectiveness of standard vaccine strategies to induce potent and persistent immunity to CoV2 is in question, particularly for the elderly. This problem is not dissimilar to what we have struggled with in our quest to induce immunity to cancer antigens, where vaccine-induced anti-cancer immune responses can be weak. Here, we describe a novel vaccine approach which leverages electroporation (EP) of a plasmid encoding a prefusion stabilized CoV2 spike protein (CORVax). As IL-12 has been shown to augment the efficacy of immunotherapy in aged mice,1 we have initiated studies to evaluate if plasmid IL-12 (TAVO™) can similarly augment anti-CoV2 immune responses in young mice and have planned studies in aged animals.MethodsA prefusion stabilized CoV2 spike plasmid expression vector was constructed, a master cell bank generated and clinical-grade plasmid manufactured. C57BL/6 and BALB/c were vaccinated via intramuscular (IM) and/or intradermal (ID) injection followed immediately by EP of plasmids encoding the CoV2 spike protein with or without plasmid-encoded murine IL-12 on days 1 and 14 or 21. Mice were followed for >120 days to assess safety. Splenocytes and serum were harvested at different time points to interrogate virus-specific cellular responses as well anti-spike IgG1/IgG2 antibody titers. A surrogate viral neutralization test (sVNT) assessed serum blockade of soluble hACE2R binding to immobilized CoV2 spike.ResultsPreliminary data shows that EP of CORVax alone or combined with IL-12 was safe. EP of CORVax was able to elicit anti-Spike IgG antibodies (IC50 = 1/2112), as well as IgG antibodies targeting the receptor binding domain of the Spike protein (IC50 = 1/965) approximately 40 days after the booster vaccination. In 2 of 2 experiments, CORVax combined with IL-12 significantly (P<0.0001) increased the sVNT titers at 2 months, but this benefit was lost by 3 months.ConclusionsEarly preclinical data shows that EP of CORVax can induce IgG responses to CoV2 Spike and the receptor binding domain (RBD) as well as apparent viral neutralizing activity. The addition of IL-12, at least transiently, increased sVNT titer. We plan to investigate alternate vaccine boosting strategies while extending these studies into aged animals and initiate a clinical trial in the near future.ReferencesRuby CE, Weinberg AD. OX40-Enhanced tumor rejection and effector T cell differentiation decreases with age. J Immunol2009;182:1481–9. https://doi.org/10.4049/jimmunol.182.3.1481.

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Bacterial expression and purification of functional recombinant SARS-CoV-2 spike receptor binding domain

10.1101/2021.02.03.429601 ◽

2021 ◽

Author(s):

Janani Prahlad ◽

Lucas R. Struble ◽

William E. Lutz ◽

Savanna A. Wallin ◽

Surender Khurana ◽

...

Keyword(s):

Receptor Binding ◽

Disulfide Bonds ◽

Rapid Diagnosis ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Expression And Purification ◽

Protein Receptor ◽

The World ◽

Active Antigen

AbstractThe COVID-19 pandemic caused by SARS-CoV-2 has applied significant pressure on overtaxed healthcare around the world, underscoring the urgent need for rapid diagnosis and treatment. We have developed a bacterial strategy for the expression and purification of the SARS-CoV-2 spike protein receptor binding domain using the CyDisCo system to create and maintain the correct disulfide bonds for protein integrity and functionality. We show that it is possible to quickly and inexpensively produce functional, active antigen in bacteria capable of recognizing and binding to the ACE2 (angiotensin-converting enzyme) receptor as well as antibodies in COVID-19 patient sera.

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High seroprevalence for SARS-CoV-2 among household members of essential workers detected using a dried blood spot assay

10.1101/2020.06.01.20119602 ◽

2020 ◽

Cited By ~ 1

Author(s):

Thomas W. McDade ◽

Elizabeth M. McNally ◽

Aaron S. Zelikovich ◽

Richard D’Aquila ◽

Brian Mustanski ◽

...

Keyword(s):

Receptor Binding ◽

Venous Blood ◽

Binding Domain ◽

Spike Protein ◽

Dried Blood Spot ◽

Receptor Binding Domain ◽

Serological Testing ◽

Igg Antibodies ◽

Household Members ◽

Blood Spot

AbstractObjectiveSerological testing is needed to investigate the extent of transmission of SARS-CoV-2 from front-line essential workers to their household members. However, the requirement for serum/plasma limits serological testing to clinical settings where it is feasible to collect and process venous blood. To address this problem we developed a serological test for SARS-CoV-2 IgG antibodies that requires only a single drop of finger stick capillary whole blood, collected in the home and dried on filter paper (dried blood spot, DBS).MethodsAn ELISA to the receptor binding domain of the SARS-CoV-2 spike protein was optimized to quantify IgG antibodies in DBS. Samples were self-collected from a community sample of 232 participants enriched with health care workers, including 30 known COVID-19 cases and their household members.ResultsAmong 30 individuals sharing a household with a virus-confirmed case of COVID-19, 80% were seropositive. Of 202 community individuals without prior confirmed acute COVID-19 diagnoses, 36% were seropositive. Of documented convalescent COVID-19 cases from the community, 29 of 30 (97%) were seropositive for IgG antibodies to the receptor binding domain.ConclusionDBS ELISA provides a minimally-invasive alternative to venous blood collection. Early analysis suggests a high rate of transmission among household members. High rates of seroconversion were also noted following recovery from infection. Serological testing for SARSCoV-2 IgG antibodies in DBS samples can facilitate seroprevalence assessment in community settings to address epidemiological questions, monitor duration of antibody responses, and assess if antibodies against the spike protein correlate with protection from reinfection.

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Polymersomes decorated with SARS-CoV-2 spike protein receptor binding domain elicit robust humoral and cellular immunity

10.1101/2021.04.08.438884 ◽

2021 ◽

Author(s):

Lisa R Volpatti ◽

Rachel P Wallace ◽

Shijie Cao ◽

Michal Raczy ◽

Ruyi Wang ◽

...

Keyword(s):

T Cell ◽

Antibody Response ◽

Receptor Binding ◽

Neutralizing Antibody ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Vaccination Site ◽

Monophosphoryl Lipid A ◽

Protein Receptor

A diverse portfolio of SARS-CoV-2 vaccine candidates is needed to combat the evolving COVID-19 pandemic. Here, we developed a subunit nanovaccine by conjugating SARS-CoV-2 Spike protein receptor binding domain (RBD) to the surface of oxidation-sensitive polymersomes. We evaluated the humoral and cellular responses of mice immunized with these surface-decorated polymersomes (RBDsurf) compared to RBD-encapsulated polymersomes (RBDencap) and unformulated RBD (RBDfree), using monophosphoryl lipid A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBDsurf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBDsurf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBDsurf and RBDencap drove similarly robust CD4+ and CD8+ T cell responses that produced multiple Th1-type cytokines. We conclude that multivalent surface display of Spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity.

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Binding Profile Assessment of N501Y: a More Infectious Mutation on the Receptor Binding Domain of SARS-CoV-2 Spike Protein

10.26434/chemrxiv.13710961.v1 ◽

2021 ◽

Author(s):

Yuzhao Zhang ◽

Xibing He ◽

Viet Hoang Man ◽

Jingchen Zhai ◽

Beihong Ji ◽

...

Keyword(s):

Receptor Binding ◽

Rna Virus ◽

Md Simulations ◽

Binding Domain ◽

Spike Protein ◽

World Health ◽

Receptor Binding Domain ◽

Binding Profile ◽

New Variant ◽

The World

<p>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in December 2019 and has accumulated nearly a hundred million reported infections thereafter. This highly transmissible and pathogenic coronavirus has caused a pandemic of acute respiratory disease, coronavirus disease 2019 (COVID-19), which has caught extensive attention and greatly changed people’s lifestyles all over the world. As an RNA virus, SARS-CoV-2 mutates rapidly as the virus replicates. The world health organization is now closely monitoring the emergence of a new variant, N501Y, on the spike protein. This N501Y variant is found to have higher transmission ability and infectivity, and is believed to be related to the rapid increase of COVID-19 cases in December 2020 in the UK. It was recently reported that the N501Y variants reduce neutralization sensitivity to convalescent sera and monoclonal antibodies. The Tyr mutation at 501 is located at the receptor binding domain (RBD) of the spike protein, the area that directly contacts human ACE2 (hACE2). It’s urgent to figure out the driving force of the new mutant’s enhanced infectivity. Thus, a computational aided binding profile prediction is made to investigate the binding affinity alteration and potential structural change of the N501Y mutant. <a>The resulting structures of N501Y mutant from MD simulations could be used to develop drug inhibitors against hACE2/RBD binding. </a></p>

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SARS-CoV-2 Variant of Concern Substitutions Alter Spike Glycoprotein Receptor Binding Domain Structure and Stability

10.1101/2021.05.11.443443 ◽

2021 ◽

Author(s):

Daniel L Moss ◽

Jay Rappaport

Keyword(s):

Receptor Binding ◽

Rapid Development ◽

Structure And Function ◽

Binding Domain ◽

Amino Acid Substitutions ◽

Receptor Binding Domain ◽

Spike Glycoprotein ◽

Structure And Stability ◽

The World ◽

And Function

The emergence of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and the subsequent COVID-19 pandemic has significantly impacted the world not just with disease and death but also economic turmoil. The rapid development and deployment of extremely effective vaccines against SARS-CoV-2 has made the end of the pandemic a reality within reach. However, as the virus spreads it has acquired mutations; and thus, variants of concern have emerged that are more infectious and reduce the efficacy of existing vaccines. While promising efforts are underway to combat these variants, the evolutionary pressures leading to these variants are poorly understood. To that end, here we have studied the effects of three amino-acid substitutions on the structure and function of the SARS-CoV-2 spike glycoprotein receptor-binding domain found in several variants of concern such as B.1.1.7, B.1.351 and P.1 that are now circulating. We found that these substitutions alter the RBD structure and stability, as well as its ability to bind to ACE2, which may have opposing and compensatory effects. These findings provide new insights into how these Variants of Concern (VOC) may have been selected to optimize infectivity while maintaining the structure and stability of the receptor binding domain.

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Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in patients with COVID-19

Science Immunology ◽

10.1126/sciimmunol.abe5511 ◽

2020 ◽

Vol 5 (52) ◽

pp. eabe5511

Author(s):

Baweleta Isho ◽

Kento T. Abe ◽

Michelle Zuo ◽

Alainna J. Jamal ◽

Bhavisha Rathod ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Antibody Response ◽

Receptor Binding ◽

Antibody Responses ◽

Spike Protein ◽

Receptor Binding Domain ◽

Igg Antibodies ◽

Surrogate Measure ◽

Antibody Levels ◽

Negative Controls

Although the antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been extensively studied in blood, relatively little is known about the antibody response in saliva and its relationship to systemic antibody levels. Here, we profiled by enzyme-linked immunosorbent assays (ELISAs) immunoglobulin G (IgG), IgA, and IgM responses to the SARS-CoV-2 spike protein (full-length trimer) and its receptor binding domain (RBD) in serum and saliva of acute and convalescent patients with laboratory-diagnosed coronavirus disease 2019 (COVID-19) ranging from 3 to 115 days postsymptom onset (PSO), compared with negative controls. Anti–SARS-CoV-2 antibody responses were readily detected in serum and saliva, with peak IgG levels attained by 16 to 30 days PSO. Longitudinal analysis revealed that anti–SARS-CoV-2 IgA and IgM antibodies rapidly decayed, whereas IgG antibodies remained relatively stable up to 105 days PSO in both biofluids. Last, IgG, IgM, and, to a lesser extent, IgA responses to spike and RBD in the serum positively correlated with matched saliva samples. This study confirms that serum and saliva IgG antibodies to SARS-CoV-2 are maintained in most of the patients with COVID-19 for at least 3 months PSO. IgG responses in saliva may serve as a surrogate measure of systemic immunity to SARS-CoV-2 based on their correlation with serum IgG responses.

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Structure and binding properties of Pangolin-CoV spike glycoprotein inform the evolution of SARS-CoV-2

Nature Communications ◽

10.1038/s41467-021-21006-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Antoni G. Wrobel ◽

Donald J. Benton ◽

Pengqi Xu ◽

Lesley J. Calder ◽

Annabel Borg ◽

...

Keyword(s):

Receptor Binding ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Spike Glycoprotein ◽

Binding Properties ◽

Origin And Evolution ◽

Closed Conformation ◽

Bat Coronavirus

AbstractCoronaviruses of bats and pangolins have been implicated in the origin and evolution of the pandemic SARS-CoV-2. We show that spikes from Guangdong Pangolin-CoVs, closely related to SARS-CoV-2, bind strongly to human and pangolin ACE2 receptors. We also report the cryo-EM structure of a Pangolin-CoV spike protein and show it adopts a fully-closed conformation and that, aside from the Receptor-Binding Domain, it resembles the spike of a bat coronavirus RaTG13 more than that of SARS-CoV-2.

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Identification of the Receptor-Binding Domain of the Spike Glycoprotein of Human Betacoronavirus HKU1

Journal of Virology ◽

10.1128/jvi.03737-14 ◽

2015 ◽

Vol 89 (17) ◽

pp. 8816-8827 ◽

Cited By ~ 19

Author(s):

Zhaohui Qian ◽

Xiuyuan Ou ◽

Luiz Gustavo Bentim Góes ◽

Christina Osborne ◽

Anna Castano ◽

...

Keyword(s):

Receptor Binding ◽

Hepatitis Virus ◽

Mouse Hepatitis Virus ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Spike Glycoprotein ◽

S Protein ◽

Receptor Proteins ◽

Group A

ABSTRACTCoronavirus spike (S) glycoproteins mediate receptor binding, membrane fusion, and virus entry and determine host range. Murine betacoronavirus (β-CoV) in group A uses the N-terminal domain (NTD) of S protein to bind to its receptor, whereas the β-CoVs severe acute respiratory syndrome CoV in group B and Middle East respiratory syndrome CoV in group C and several α-CoVs use the downstream C domain in their S proteins to recognize their receptor proteins. To identify the receptor-binding domain in the spike of human β-CoV HKU1 in group A, we generated and mapped a panel of monoclonal antibodies (MAbs) to the ectodomain of HKU1 spike protein. They did not cross-react with S proteins of any other CoV tested. Most of the HKU1 spike MAbs recognized epitopes in the C domain between amino acids 535 and 673, indicating that this region is immunodominant. Two of the MAbs blocked HKU1 virus infection of primary human tracheal-bronchial epithelial (HTBE) cells. Preincubation of HTBE cells with a truncated HKU1 S protein that includes the C domain blocked infection with HKU1 virus, but preincubation of cells with truncated S protein containing only the NTD did not block infection. These data suggest that the receptor-binding domain (RBD) of HKU1 spike protein is located in the C domain, where the spike proteins of α-CoVs and β-CoVs in groups B and C bind to their specific receptor proteins. Thus, two β-CoVs in group A, HKU1 and murine CoV, have evolved to use different regions of their spike glycoproteins to recognize their respective receptor proteins.IMPORTANCEMouse hepatitis virus, a β-CoV in group A, uses the galectin-like NTD in its spike protein to bind its receptor protein, while HCoV-OC43, another β-CoV in group A, uses the NTD to bind to its sialic-acid containing receptor. In marked contrast, the NTD of the spike glycoprotein of human respiratory β-CoV HKU1, which is also in group A, does not bind sugar. In this study, we showed that for the spike protein of HKU1, the purified C domain, downstream of the NTD, could block HKU1 virus infection of human respiratory epithelial cells, and that several monoclonal antibodies that mapped to the C domain neutralized virus infectivity. Thus, the receptor-binding domain of HKU1 spike glycoprotein is located in the C domain. Surprisingly, two β-CoVs in group A, mouse hepatitis virus and HKU1, have evolved to use different regions of their spike glycoproteins to recognize their respective receptors.

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Sequence signatures of two IGHV3-53/3-66 public clonotypes to SARS-CoV-2 receptor binding domain

10.1101/2021.01.26.428356 ◽

2021 ◽

Author(s):

Timothy J.C. Tan ◽

Meng Yuan ◽

Kaylee Kuzelka ◽

Gilberto C. Padron ◽

Jacob R. Beal ◽

...

Keyword(s):

Antibody Response ◽

Receptor Binding ◽

Somatic Hypermutation ◽

Binding Domain ◽

Spike Protein ◽

Light Chains ◽

Receptor Binding Domain ◽

Sequence Motifs ◽

Fundamental Understanding ◽

Distinct Sequence

AbstractSince the COVID-19 pandemic onset, the antibody response to SARS-CoV-2 has been extensively characterized. Antibodies to the receptor binding domain (RBD) on the spike protein are frequently encoded by IGHV3-53/3-66 with a short CDR H3. Germline-encoded sequence motifs in CDRs H1 and H2 play a major role, but whether any common motifs are present in CDR H3, which is often critical for binding specificity, have not been elucidated. Here, we identify two public clonotypes of IGHV3-53/3-66 RBD antibodies with a 9-residue CDR H3 that pair with different light chains. Distinct sequence motifs on CDR H3 are present in the two public clonotypes that appear to be related to differential light chain pairing. Additionally, we show that Y58F is a common somatic hypermutation that results in increased binding affinity of IGHV3-53/3-66 RBD antibodies with a short CDR H3. Overall, our results advance fundamental understanding of the antibody response to SARS-CoV-2.

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