Limited variation between SARS-CoV-2-infected individuals in domain specificity and relative potency of the antibody response against the spike glycoprotein

The spike protein of SARS-CoV-2 is arranged as a trimer on the virus surface, composed of three S1 and three S2 subunits. Infected and vaccinated individuals generate antibodies against spike, which can neutralize the virus. Most antibodies target the receptor-binding domain (RBD) and N-terminal domain (NTD) of S1; however, antibodies against other regions of spike have also been isolated. The variation between infected individuals in domain specificity of the antibodies and in their relative neutralization efficacy is still poorly characterized. To this end, we tested serum and plasma samples from 85 COVID-19 convalescent subjects using 7 immunoassays that employ different domains, subunits and oligomeric forms of spike to capture the antibodies. Samples were also tested for their neutralization of pseudovirus containing SARS-CoV-2 spike and of replication-competent SARS-CoV-2. We observed strong correlations between the levels of NTD- and RBD-specific antibodies, with a fixed ratio of each type to all anti-spike antibodies. The relative potency of the response (defined as the measured neutralization efficacy relative to the total level of spike-targeting antibodies) also exhibited limited variation between subjects, and was not associated with the overall amount of anti-spike antibodies produced. Accordingly, the ability of immunoassays that use RBD, NTD and different forms of S1 or S1/S2 as capture antigens to estimate the neutralizing efficacy of convalescent samples was largely similar. These studies suggest that host-to-host variation in the polyclonal response elicited against SARS-CoV-2 spike is primarily limited to the quantity of antibodies generated rather than their domain specificity or relative neutralization potency.

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SARS-CoV-2 Antibody Responses Correlate with Resolution of RNAemia But Are Short-Lived in Patients with Mild Illness

10.1101/2020.08.15.20175794 ◽

2020 ◽

Cited By ~ 2

Author(s):

Katharina Röltgen ◽

Oliver F Wirz ◽

Bryan A Stevens ◽

Abigail E Powell ◽

Catherine A Hogan ◽

...

Keyword(s):

Protective Immunity ◽

Antibody Responses ◽

Receptor Binding Domain ◽

Plasma Samples ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

Specific Antibodies ◽

Immune Clearance ◽

Humoral Immune ◽

Asymptomatic Individuals

SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, could offer protective immunity, and may affect clinical outcomes of COVID-19 patients. We analyzed 625 serial plasma samples from 40 hospitalized COVID-19 patients and 170 SARS-CoV-2-infected outpatients and asymptomatic individuals. Severely ill patients developed significantly higher SARS-CoV-2-specific antibody responses than outpatients and asymptomatic individuals. The development of plasma antibodies was correlated with decreases in viral RNAemia, consistent with potential humoral immune clearance of virus. Using a novel competition ELISA, we detected antibodies blocking RBD-ACE2 interactions in 68% of inpatients and 40% of outpatients tested. Cross-reactive antibodies recognizing SARS-CoV RBD were found almost exclusively in hospitalized patients. Outpatient and asymptomatic individuals' serological responses to SARS-CoV-2 decreased within 2 months, suggesting that humoral protection may be short-lived.

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Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals

10.1101/2020.05.13.092619 ◽

2020 ◽

Cited By ~ 43

Author(s):

Davide F. Robbiani ◽

Christian Gaebler ◽

Frauke Muecksch ◽

Julio C. C. Lorenzi ◽

Zijun Wang ◽

...

Keyword(s):

Virus Entry ◽

Protein S ◽

Memory B Cells ◽

Antibody Responses ◽

Spike Protein ◽

Human Antibody ◽

Receptor Binding Domain ◽

Single Digit ◽

Specific Antibodies ◽

Specific Memory

AbstractDuring the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21–5. Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal neutralizing titers ranging from undetectable in 33% to below 1:1000 in 79%, while only 1% showed titers >1:5000. Antibody cloning revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titers, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50s) as low as single digit ng/mL. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

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Homologous and heterologous serological response to the N-terminal domain of SARS-CoV-2

10.1101/2021.02.17.431722 ◽

2021 ◽

Author(s):

Huibin Lv ◽

Owen Tak-Yin Tsang ◽

Ray T. Y. So ◽

Yiquan Wang ◽

Meng Yuan ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Global Economy ◽

Vaccine Development ◽

Serological Response ◽

Receptor Binding Domain ◽

Plasma Samples ◽

Mice Model ◽

Terminal Domain ◽

Major Antigen ◽

Serology Testing

SUMMARYThe increasing numbers of infected cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses serious threats to public health and the global economy. Most SARS-CoV-2 neutralizing antibodies target the receptor binding domain (RBD) and some the N-terminal domain (NTD) of the spike protein, which is the major antigen of SARS-CoV-2. While the antibody response to RBD has been extensively characterized, the antigenicity and immunogenicity of the NTD protein are less well studied. Using 227 plasma samples from COVID-19 patients, we showed that SARS-CoV-2 NTD-specific antibodies could be induced during infection. As compared to the serological response to SARS-CoV-2 RBD, the SARS-CoV-2 NTD response is less cross-reactive with SARS-CoV. Furthermore, neutralizing antibodies are rarely elicited in a mice model when NTD is used as an immunogen. We subsequently demonstrate that NTD has an altered antigenicity when expressed alone. Overall, our results suggest that while NTD offers an alternative strategy for serology testing, it may not be suitable as an immunogen for vaccine development.

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A dual antigen ELISA allows the assessment of SARS-CoV-2 antibody seroprevalence in a low transmission setting

10.1101/2020.09.09.20191031 ◽

2020 ◽

Cited By ~ 3

Author(s):

Sarah Hicks ◽

Kai Pohl ◽

Teresa Neeman ◽

Hayley McNamara ◽

Kate Parsons ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Receptor Binding ◽

Elective Surgery ◽

Receptor Binding Domain ◽

Assay Sensitivity ◽

Specific Antibodies ◽

Low Transmission ◽

Low Level ◽

Transmission Setting ◽

Antigen Elisa

Estimates of seroprevalence of SARS-CoV-2 antibodies have been hampered by inadequate assay sensitivity and specificity. Using an ELISA-based approach to that combines data about IgG responses to both the Nucleocapsid and Spike-receptor binding domain antigens, we show that near-optimal sensitivity and specificity can be achieved. We used this assay to assess the frequency of virus-specific antibodies in a cohort of elective surgery patients in Australia and estimated seroprevalence in Australia to be 0.28% (0 to 0.72%). These data confirm the low level of transmission of SARS-CoV-2 in Australia before July 2020 and validate the specificity of our assay.

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COVID 19 breakthrough infection risk: a simple physical model describing the dependence on antibody concentration

10.21203/rs.3.rs-1051588/v2 ◽

2021 ◽

Author(s):

David E Williams

Keyword(s):

Vaccine Efficacy ◽

Infection Risk ◽

Antibody Concentration ◽

Receptor Binding Domain ◽

Breakthrough Infection ◽

Virus Surface ◽

Domain Antibody ◽

Simple Physical Model ◽

Rational Explanation ◽

Spike Proteins

Abstract The empirically-observed dependence on blood IgG anti-receptor binding domain antibody concentration of SARS-CoV-2 vaccine efficacy against infection has a rational explanation in the statistics of binding of antibody to spike proteins on the virus surface: namely that the probability of protection is the probability of antibody binding to more than a critical number of the spike proteins protruding from the virus. The model is consistent with the observed antibody concentrations required to induce immunity and with the observed dependence of vaccine efficacy on antibody concentration and thus is a useful tool in the development of models to relate, for an individual person, risk of breakthrough infection given measured antibody concentration

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Microfluidic characterisation reveals broad range of SARS-CoV-2 antibody affinity in human plasma

Life Science Alliance ◽

10.26508/lsa.202101270 ◽

2021 ◽

Vol 5 (2) ◽

pp. e202101270

Author(s):

Matthias M Schneider ◽

Marc Emmenegger ◽

Catherine K Xu ◽

Itzel Condado Morales ◽

Georg Meisl ◽

...

Keyword(s):

Clinical Outcome ◽

Cytopathic Effect ◽

Dissociation Constants ◽

Antibody Responses ◽

Receptor Binding Domain ◽

Plasma Samples ◽

Antibody Affinity ◽

Competition Assay ◽

Receptor Complexes ◽

Healthy Donors

The clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti–SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, Kd, of anti–receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect–based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations.

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RBD-Fc-based COVID-19 vaccine candidate induces highly potent SARS-CoV-2 neutralizing antibody response

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-020-00402-5 ◽

2020 ◽

Vol 5 (1) ◽

Author(s):

Zezhong Liu ◽

Wei Xu ◽

Shuai Xia ◽

Chenjian Gu ◽

Xinling Wang ◽

...

Keyword(s):

Subunit Vaccine ◽

Vaccine Candidate ◽

High Titer ◽

Neutralizing Antibody ◽

Spike Protein ◽

Receptor Binding Domain ◽

Angiotensin Converting Enzyme 2 ◽

Specific Antibodies ◽

Good Potential ◽

Neutralizing Epitopes

AbstractThe pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed serious threats to global health and economy, thus calling for the development of safe and effective vaccines. The receptor-binding domain (RBD) in the spike protein of SARS-CoV-2 is responsible for its binding to angiotensin-converting enzyme 2 (ACE2) receptor. It contains multiple dominant neutralizing epitopes and serves as an important antigen for the development of COVID-19 vaccines. Here, we showed that immunization of mice with a candidate subunit vaccine consisting of SARS-CoV-2 RBD and Fc fragment of human IgG, as an immunopotentiator, elicited high titer of RBD-specific antibodies with robust neutralizing activity against both pseudotyped and live SARS-CoV-2 infections. The mouse antisera could also effectively neutralize infection by pseudotyped SARS-CoV-2 with several natural mutations in RBD and the IgG extracted from the mouse antisera could also show neutralization against pseudotyped SARS-CoV and SARS-related coronavirus (SARSr-CoV). Vaccination of human ACE2 transgenic mice with RBD-Fc could effectively protect mice from the SARS-CoV-2 challenge. These results suggest that SARS-CoV-2 RBD-Fc has good potential to be further developed as an effective and broad-spectrum vaccine to prevent infection of the current SARS-CoV-2 and its mutants, as well as future emerging SARSr-CoVs and re-emerging SARS-CoV.

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Longitudinal and Functional Analysis of Spontaneous NY-ESO-1-Specific Antibody Responses in Multiple Myeloma Patients.

Blood ◽

10.1182/blood.v114.22.2831.2831 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2831-2831

Author(s):

Sebastian Kobold ◽

Yanran Cao ◽

Sinje Tams ◽

Britta Marlen Bartels ◽

Tim Lütkens ◽

...

Keyword(s):

Bone Marrow ◽

Immune Responses ◽

Specific Antibody ◽

Antibody Responses ◽

Plasma Samples ◽

Specific Antibodies ◽

Functional Capabilities ◽

Bone Marrow Plasma ◽

Ct Antigen ◽

Antibody Titers

Abstract Abstract 2831 Poster Board II-807 Its tumor-restricted expression and its high immunogenicity render cancer-testis (CT) antigen NY-ESO-1 an exquisite target for antigen-specific immunotherapies. Spontaneous antibody responses against NY-ESO-1 are typically found in a subset of patients with solid tumors. However, little is known regarding serological immune responses against NY-ESO-1 in patients with hematological malignancies including multiple myeloma (MM). Furthermore, no consequent longitudinal analyses have been performed correlating NY-ESO-1-specific antibody titers with the clinical development of the given malignancy. Finally, nothing is known regarding the functional capabilities of spontaneously occurring anti-NY-ESO-1 antibodies in MM or other malignancies. Here, we performed the first longitudinal and functional investigation of NY-ESO-1-specific antibody responses in MM analyzing 1100 sera and 200 bone marrow plasma samples of 194 MM patients. Sera and BM plasma samples of 100 healthy donors served as controls. Screening sera and bone marrow plasma of our MM patients by Enzyme-linked Immunosorbent Assay (ELISA) using full length recombinant NY-ESO-1 protein, we found that 5/194 patients had high-titered antibody responses against this CT antigen. A quantitative B cell ELISPOT demonstrated NY-ESO-1-specific B cells in the peripheral blood as well as in the bone marrow of the respective MM patients. In a western blot analysis, spontaneous NY-ESO-1-specific immune responses in the patients were found to be highly specific for both native and recombinant protein. Epitope mapping in an ELISA using 18 overlapping NY-ESO-1 20mer peptides showed that antibody responses were restricted to the first 70 amino acids of the full-length protein. NY-ESO-1-specific antibodies consisted mainly of IgG1 and to a lower extent of IgG3 subtypes. No IgG2, IgG4, IgM or IgA antibodies against NY-ESO-1 were detected. Interestingly, antibody affinity increased over the course of the disease suggesting an affinity driven antibody maturation. Accordingly, NY-ESO-1-specific antibodies of MM patients were found to be potent complement activators in a western blot technique. On the other hand, despite the high functional capabilities of NY-ESO-1-specific antibodies, antibody titers increased with each NY-ESO-1-expressing (as indicated by reverse-transcriptase-polymerase-chain-reaction and immunohistochemistry) recurrence of the disease. In conclusion, we demonstrate here the spontaneous occurrence of high-titered NY-ESO-1-specific antibodies in MM patients. One reason for the relatively low frequency of antibody responses against NY-ESO-1 might be that most patients were in early stages of the disease or in remission at the time the analysis was performed. Antibodies were produced by NY-ESO-1-specific B cells detectable in the bone marrow as well as in the peripheral blood of the patients. NY-ESO-1-specific antibodies were evoked by a distinct and immunodominant fragment of NY-ESO-1. Affinity maturation of this response and complement activation by the spontaneously occurring NY-ESO-1-specific IgG1-type antibodies speak in favour of an effective serological immune response. However, positive correlation of antibody titers with tumor burden and recurrence of the disease suggest an inability of antibodies targeting intracellular protein NY-ESO-1 to control the course of the disease, at least in the long run. Antigen-specific immunotherapy might be necessary to shape NY-ESO-1-specific immunity in MM patients and, particularly, to mobilize tumor-specific T cell responses. Disclosures: No relevant conflicts of interest to declare.

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Experimental Antibody Filtration Inhibits Antibody-Mediated Neutrophil Priming and TRALI in An In Vivo model

Blood ◽

10.1182/blood.v118.21.41.41 ◽

2011 ◽

Vol 118 (21) ◽

pp. 41-41 ◽

Cited By ~ 1

Author(s):

Christopher C Silliman ◽

Marguerite R Kelher ◽

Samina Y Khan ◽

Samuel O. Sowemimo-Coker

Keyword(s):

Human Plasma ◽

Antibody Detection ◽

In Vivo Model ◽

Plasma Samples ◽

Specific Antibodies ◽

Plasma Filtration ◽

The Mean ◽

Experimental Filter ◽

Priming Activity

Abstract Abstract 41 Transfusion-related acute lung injury (TRALI) is a leading cause of transfusion-related death with a majority of the reported cases secondary to the infusion of antibodies (Abs) contained within the plasma/blood component. An experimental filter that removes IgG was developed. We hypothesize that filtration of plasma with antibodies to leukocyte antigens will decrease both antibody-mediated priming of PMNs and antibody-mediated TRALI in a two-event in vivo model. Methods: Human plasma was drawn from healthy volunteers and IgG concentrations were measured before and after filtration. Plasma was obtained from two multiparous female donors: one with antibodies to HLA-A2 and to DR7 and the other with antibodies against HNA-3a. These plasma samples were filtered (F-Plas) or left as an unmodified control (Plas) and the anti-leukocyte antibodies were measured in a blinded fashion in referral labs using flow cytometry and Luminex™ beads or standard granulocyte antibody detection assays. These plasma samples were then used to prime the fMLP-activated respiratory burst, measured as the SOD-inhibitable reduction of cytochrome c (nmol O2−/min), of PMNs from HNA-3a+ donors or donors homozygous donors for HLA-A2, respectively. For the two-event in vivo modeling rats were incubated with 2 μg/ml endotoxin (LPS, S. enteritides) or saline (NS) for 2 hours (first event) and then were transfused with heat-treated human plasma that contained 25 μg/ml of an antibody against the MHC class I antigen OX27 that was either filtered (or left unmodified) prior to infusion (second event) followed by Evans Blue dye (EBD). ALI was measured as %EBD leak from the plasma into the bronchoalveolar lavage fluid. Statistical differences were measured via paired (PMN priming) or independent (in vivo TRALI) ANOVA, and data are reported as the mean ± the standard error of the mean. *=p<.05 vs. all groups (Table). Results: Plasma filtration removed 98±2.1% of IgG from normal plasma and both the antibodies to HNA-3a and HLA-A2 such that they were no longer detected (HLA-A2: 94 Luminex™ units (LU) pre-filtration and 0 LU units post-filtration and DR7: 30 LU pre-filtration and 0 LU post-filtration). In addition, filtration also inhibited the priming activity of the plasma containing antibodies to HNA-3a and HLA-A2 on HNA-3A+ PMNs and HLA-A2+ PMNs, respectively. Moreover, the plasma spiked with antibodies to OX27 caused ALI in LPS-treated rats, but not NS treated animals, which was inhibited by filtration. We conclude that this experimental filter removes IgG and detectable amounts of specific antibodies to HLA and HNA ligands as well as obviating the priming activity of these antibodies in PMNs which express the cognate antigens. Filtration of plasma spiked with specific antibodies to MHC ligands also abrogated the antibody-induced TRALI in a two-event, in vivo model. Such a filtration step could mitigate antibody-mediated TRALI.Abs/Tx'sfMLP (O2− nmol/min)Plas+ fMLPF-plas+ fMLPNS/plas (% EBD)NS/F-PlasLPS/PlasLPS/F-PlasHNA-3a0.7 ± .31.1 ± .2*0.6 ± .2HLA-A21.6 ± .63.4 ± .4*1.5 ± .4OX270.09 ± .020.13 ± .030.4 ± .14*0.13 ± .03 Disclosures: Silliman: Pall Corporation: Honoraria. Sowemimo-Coker:Pall Medical Corporation: Employment.

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A Dual-Antigen Enzyme-Linked Immunosorbent Assay Allows the Assessment of Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Seroprevalence in a Low-Transmission Setting

The Journal of Infectious Diseases ◽

10.1093/infdis/jiaa623 ◽

2020 ◽

Vol 223 (1) ◽

pp. 10-14 ◽

Cited By ~ 1

Author(s):

Sarah M Hicks ◽

Kai Pohl ◽

Teresa Neeman ◽

Hayley A McNamara ◽

Kate M Parsons ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Confidence Interval ◽

Sensitivity And Specificity ◽

Immunosorbent Assay ◽

Elective Surgery ◽

Enzyme Linked Immunosorbent Assay ◽

Receptor Binding Domain ◽

Assay Sensitivity ◽

Specific Antibodies ◽

Transmission Setting

Abstract Estimates of seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies have been hampered by inadequate assay sensitivity and specificity. Using an enzyme-linked immunosorbent assay–based approach that combines data about immunoglobulin G responses to both the nucleocapsid and spike receptor binding domain antigens, we show that excellent sensitivity and specificity can be achieved. We used this assay to assess the frequency of virus-specific antibodies in a cohort of elective surgery patients in Australia and estimated seroprevalence in Australia to be 0.28% (95% Confidence Interval, 0–1.15%). These data confirm the low level of transmission of SARS-CoV-2 in Australia before July 2020 and validate the specificity of our assay.

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