Receptor-binding Domain Epitopes Research Articles

Life Sciences Discovery and Technology Highlights

Life Sciences Discovery and Technology Highlights

Neutralization of SARS-CoV-2 requires antibodies against conformational receptor-binding domain epitopes.

BackgroundThe determinants of successful humoral immune response to the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) are of critical importance for the design of effective vaccines and the evaluation of the degree of protective immunity conferred by exposure to the virus. As novel variants emerge, understanding their likelihood of suppression by population antibody repertoires has become increasingly important.MethodsIn this study, we analyzed the SARS‐CoV‐2 polyclonal antibody response in a large population of clinically well‐characterized patients after mild and severe COVID‐19 using a panel of microarrayed structurally folded and unfolded SARS‐CoV‐2 proteins, as well as sequential peptides, spanning the surface spike protein (S) and the receptor‐binding domain (RBD) of the virus.ResultsS‐ and RBD‐specific antibody responses were dominated by immunoglobulin G (IgG), mainly IgG1, and directed against structurally folded S and RBD and three distinct peptide epitopes in S2. The virus neutralization activity of patients´ sera was highly correlated with IgG antibodies specific for conformational but not sequential RBD epitopes and their ability to prevent RBD binding to its human receptor angiotensin‐converting enzyme 2 (ACE2). Twenty percent of patients selectively lacked RBD‐specific IgG. Only immunization with folded, but not with unfolded RBD, induced antibodies against conformational epitopes with high virus‐neutralizing activity. Conformational RBD epitopes required for protection do not seem to be altered in the currently emerging virus variants.ConclusionThese results are fundamental for estimating the protective activity of antibody responses after natural infection or vaccination and for the design of vaccines, which can induce high levels of SARS‐CoV‐2–neutralizing antibodies conferring sterilizing immunity.

SARS-CoV-2 Vaccination, Immune Responses, and Antibody Testing in Immunosuppressed Populations: Tip of the Iceberg.

SARS-CoV-2 Vaccination, Immune Responses, and Antibody Testing in Immunosuppressed Populations: Tip of the Iceberg.

Identification of potent human neutralizing antibodies against SARS-CoV-2 implications for development of therapeutics and prophylactics

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that is spreading rapidly, which seriously impacts global public health and economy. Thus, developing effective drugs remains urgent. We identify two potent antibodies, nCoVmab1 and nCoVmab2, targeting the SARS-CoV-2 spike protein receptor-binding domain (RBD) with high affinities from a naïve human phage-displayed Fab library. nCoVmab1 and nCoVmab2 neutralize authentic SARS-CoV-2 with picomolar and nanomolar IC50 values, respectively. No detectable defects of nCoVmab1 and nCoVmab2 are found during the preliminary druggability evaluation. nCoVmab1 could reduce viral titer and lung injury when administered prophylactically and therapeutically in human angiotensin-converting enzyme II (hACE2)-transgenic mice. Therefore, phage display platform could be efficiently used for rapid development of neutralizing monoclonal antibodies (nmabs) with clinical potential against emerging infectious diseases. In addition, we determinate epitopes in RBD of these antibodies to elucidate the neutralizing mechanism. We also convert nCoVmab1 and nCoVmab2 to their germline formats for further analysis, which reveals the contribution of somatic hypermutation (SHM) during nCoVmab1 and nCoVmab2 maturation. Our findings not only provide two highly potent nmabs against SARS-CoV-2 as prophylactic and therapeutic candidates, but also give some clues for development of anti-SARS-CoV-2 agents (e.g., drugs and vaccines) targeting the RBD.

Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization.

The SARS-CoV-2 B.1.617 lineage was identified in October 2020 in India1-5. Since then, it has become dominant in some regions of India and in the UK, and has spread to many other countries6. The lineage includes three main subtypes (B1.617.1, B.1.617.2 and B.1.617.3), which contain diverse mutations in the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein that may increase the immune evasion potential of these variants. B.1.617.2-also termed the Delta variant-is believed to spread faster than other variants. Here we isolated an infectious strain of the Delta variant from an individual with COVID-19 who had returned to France from India. We examined the sensitivity of this strain to monoclonal antibodies and to antibodies present in sera from individuals who had recovered from COVID-19 (hereafter referred to as convalescent individuals) or who had received a COVID-19 vaccine, and then compared this strain with other strains of SARS-CoV-2. The Delta variant was resistant to neutralization by some anti-NTD and anti-RBD monoclonal antibodies, including bamlanivimab, and these antibodies showed impaired binding to the spike protein. Sera collected from convalescent individuals up to 12months after the onset of symptoms were fourfold less potent against the Delta variant relative to the Alpha variant (B.1.1.7). Sera from individuals who had received one dose of the Pfizer or the AstraZeneca vaccine had a barely discernible inhibitory effect on the Delta variant. Administration of two doses of the vaccine generated a neutralizing response in 95% of individuals, with titres three- to fivefold lower against the Delta variant than against the Alpha variant. Thus, the spread of the Delta variant is associated with an escape from antibodies that target non-RBD and RBD epitopes of the spike protein.

Mapping mutations to the SARS-CoV-2 RBD that escape binding by different classes of antibodies

Monoclonal antibodies targeting a variety of epitopes have been isolated from individuals previously infected with SARS-CoV-2, but the relative contributions of these different antibody classes to the polyclonal response remains unclear. Here we use a yeast-display system to map all mutations to the viral spike receptor-binding domain (RBD) that escape binding by representatives of three potently neutralizing classes of anti-RBD antibodies with high-resolution structures. We compare the antibody-escape maps to similar maps for convalescent polyclonal plasmas, including plasmas from individuals from whom some of the antibodies were isolated. While the binding of polyclonal plasma antibodies are affected by mutations across multiple RBD epitopes, the plasma-escape maps most resemble those of a single class of antibodies that target an epitope on the RBD that includes site E484. Therefore, although the human immune system can produce antibodies that target diverse RBD epitopes, in practice the polyclonal response to infection is skewed towards a single class of antibodies targeting an epitope that is already undergoing rapid evolution.

Bispecific IgG neutralizes SARS-CoV-2 variants and prevents escape in mice.

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.

A rapid, point-of-care red blood cell agglutination assay detecting antibodies against SARS-CoV-2

The COVID-19 pandemic has caused significant morbidity and mortality. There is an urgent need for serological tests to detect antibodies against SARS-CoV-2, which could be used to assess past infection, evaluate responses to vaccines in development, and determine individuals who may be protected from future infection. Current serological tests developed for SARS-CoV-2 rely on traditional technologies such as enzyme-linked immunosorbent assays (ELISA) and lateral flow assays, which have not scaled to meet the demand of hundreds of millions of antibody tests so far. Herein, we present an alternative method of antibody testing that depends on one protein reagent being added to patient serum/plasma or whole blood with direct, visual readout. Two novel fusion proteins, RBD-2E8 and B6–CH1-RBD, were designed to bind red blood cells (RBCs) via a single-chain variable fragment (scFv), thereby displaying the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on the surface of RBCs. Mixing mammalian-derived RBD-2E8 and B6–CH1-RBD with convalescent COVID-19 patient serum and RBCs led to visible hemagglutination, indicating the presence of antibodies against SARS-CoV-2 RBD. B6–CH1-RBD made in bacteria was not as effective in inducing agglutination, indicating better recognition of RBD epitopes from mammalian cells. Given that our hemagglutination test uses methods routinely used in hospital clinical labs across the world for blood typing, we anticipate the test can be rapidly deployed at minimal cost. We anticipate our hemagglutination assay may find extensive use in low-resource settings for detecting SARS-CoV-2 antibodies.

MRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants.

Here we report on the antibody and memory Bcell responses of a cohort of 20volunteers who received the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccine against SARS-CoV-21-4. Eight weeks after the second injection of vaccine, volunteers showed high levels of IgM and IgG anti-SARS-CoV-2 spike protein (S) and receptor-binding-domain (RBD) binding titre. Moreover, the plasma neutralizing activity and relative numbers of RBD-specific memory Bcells of vaccinated volunteers were equivalent to those of individuals who had recovered from natural infection5,6. However, activity against SARS-CoV-2 variants that encode E484K-, N501Y- or K417N/E484K/N501-mutant S was reduced by a small-but significant-margin. The monoclonal antibodies elicited by the vaccines potently neutralize SARS-CoV-2, and target a number of different RBD epitopes in common with monoclonal antibodies isolated from infected donors5-8. However, neutralization by 14 of the 17 most-potent monoclonal antibodies that we tested was reduced or abolished by the K417N, E484K or N501Y mutation. Notably, these mutations were selected when we cultured recombinant vesicular stomatitis virus expressing SARS-CoV-2 Sin the presence of the monoclonal antibodies elicited by the vaccines. Together, these results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid a potential loss of clinical efficacy.

Enhancement versus neutralization by SARS-CoV-2 antibodies from a convalescent donor associates with distinct epitopes on the RBD

Several potent neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus have been identified. However, antibody-dependent enhancement (ADE) has not been comprehensively studied for SARS-CoV-2, and the relationship between enhancing versus neutralizing activities and antibody epitopes remains unknown. Here, we select a convalescent individual with potent IgG neutralizing activity and characterize his antibody response. Monoclonal antibodies isolated from memory B cells target four groups of five non-overlapping receptor-binding domain (RBD) epitopes. Antibodies to one group of these RBD epitopes mediate ADE of entry in Raji cells via an Fcγ receptor-dependent mechanism. In contrast, antibodies targeting two other distinct epitope groups neutralize SARS-CoV-2 without ADE, while antibodies against the fourth epitope group are poorly neutralizing. One antibody, XG014, potently cross-neutralizes SARS-CoV-2 variants, as well as SARS-CoV-1, with respective IC50 (50% inhibitory concentration) values as low as 5.1 and 23.7 ng/mL, while not exhibiting ADE. Therefore, neutralization and ADE of human SARS-CoV-2 antibodies correlate with non-overlapping RBD epitopes.

Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology

Analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. In a cohort of 647 SARS-CoV-2-infected subjects, we found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49 days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. We structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Our results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics.

Human-IgG-Neutralizing Monoclonal Antibodies Block the SARS-CoV-2 Infection

Coronavirus disease 2019 (COVID-19) has become a worldwide threat to humans, and neutralizing antibodies have therapeutic potential. We have purified more than 1,000 memory B cells specific to SARS-CoV-2 S1 or its RBD (receptor binding domain) and obtain 729 paired heavy- and light-chain fragments. Among these, 178 antibodies test positive for antigen binding, and the majority of the top 17 binders with EC50 below 1 nM are RBD binders. Furthermore, we identify 11 neutralizing antibodies, eight of which show IC50 within 10 nM, and the best one, 414-1, with IC50 of 1.75 nM. Through epitope mapping, we find three main epitopes in RBD recognized by these antibodies, and epitope-B antibody 553-15 could substantially enhance the neutralizing abilities of most of the other antibodies. We also find that 515-5 could cross neutralize the SARS-CoV pseudovirus. Altogether, our study provides 11 potent human neutralizing antibodies for COVID-19 as therapeutic candidates.

Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies

SummaryNeutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

Single-dose treatment with a humanized neutralizing antibody affords full protection of a human transgenic mouse model from lethal Middle East respiratory syndrome (MERS)-coronavirus infection

Middle East respiratory syndrome coronavirus (MERS-CoV) is continuously spreading and causing severe and fatal acute respiratory disease in humans. Prophylactic and therapeutic strategies are therefore urgently needed to control MERS-CoV infection. Here, we generated a humanized monoclonal antibody (mAb), designated hMS-1, which targeted the MERS-CoV receptor-binding domain (RBD) with high affinity. hMS-1 significantly blocked MERS-CoV RBD binding to its viral receptor, human dipeptidyl peptidase 4 (hDPP4), potently neutralized infection by a prototype MERS-CoV, and effectively cross-neutralized evolved MERS-CoV isolates through recognizing highly conserved RBD epitopes. Notably, single-dose treatment with hMS-1 completely protected hDPP4 transgenic (hDPP4-Tg) mice from lethal infection with MERS-CoV. Taken together, our data suggest that hMS-1 might be developed as an effective immunotherapeutic agent to treat patients infected with MERS-CoV, particularly in emergent cases.

Antigenicity and immunogenicity of SARS-CoV S protein receptor-binding domain stably expressed in CHO cells

The receptor-binding domain (RBD) of SARS coronavirus (SARS-CoV) spike (S) protein contains multiple conformation-dependent epitopes that induce neutralizing antibody responses. Here we used CHO-K1 cells to establish a cell line for stable expression of a 193-mer (residues 318–510) RBD (RBD193-CHO) and determined its antigenicity and immunogenicity. We found that RBD193-CHO reacted strongly with a panel of six monoclonal antibodies recognizing various conformational and linear epitopes in RBD, suggesting that this recombinant protein maintains intact conformation and good antigenicity. Immunization of mice with RBD193-CHO resulted in induction of high titers of RBD-specific neutralizing antibodies and potent IL-4-expressing T cell responses. RBD193-CHO induced immunity that protected a majority of the vaccinated mice from SARS-CoV challenge. These results suggest that the recombinant RBD produced in an established stable cell line maintains strong immunogenicity with high potential for use as an effective and economic subunit SARS vaccine.