An antibody targeting the N-terminal domain of SARS-CoV-2 disrupts the spike trimer

The protective human antibody response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus focuses on the spike (S) protein which decorates the virion surface and mediates cell binding and entry. Most SARS-CoV-2 protective antibodies target the receptor-binding domain or a single dominant epitope (supersite) on the N terminal domain (NTD). Here, using the single B cell technology LIBRA-seq, we isolated a large panel of NTD-reactive and SARS-CoV-2 neutralizing antibodies from an individual who had recovered from COVID-19. We found that neutralizing antibodies to the NTD supersite commonly are encoded by the IGHV1-24 gene, forming a genetic cluster that represents a public B cell clonotype. However, we also discovered a rare human antibody, COV2-3434, that recognizes a site of vulnerability on the SARS-CoV-2 S protein in the trimer interface and possesses a distinct class of functional activity. COV2-3434 disrupted the integrity of S protein trimers, inhibited cell-to-cell spread of virus in culture, and conferred protection in human ACE2 transgenic mice against SARS-CoV-2 challenge. This study provides insight about antibody targeting of the S protein trimer interface region, suggesting this region may be a site of virus vulnerability.

Identification of TCR repertoires in functionally competent cytotoxic T cells cross-reactive to SARS-CoV-2

SARS-CoV-2-specific CD8+ T cells are scarce but detectable in unexposed healthy donors (UHDs). It remains unclear whether pre-existing human coronavirus (HCoV)-specific CD8+ T cells are converted to functionally competent T cells cross-reactive to SARS-CoV-2. Here, we identified the HLA-A24-high binding, immunodominant epitopes in SARS-CoV-2 spike region that can be recognized by seasonal coronavirus-specific CD8+ T cells from HLA-A24+ UHDs. Cross-reactive CD8+ T cells were clearly reduced in patients with hematological malignancy, who are usually immunosuppressed, compared to those in UHDs. Furthermore, we showed that CD8+ T cells in response to a selected dominant epitope display multifunctionality and cross-functionality across HCoVs in HLA-A24+ donors. Cross-reactivity of T-cell receptors isolated from them exhibited selective diversity at the single-cell level. Taken together, when stimulated well by immunodominant epitopes, selective pre-existing CD8+ T cells with high functional avidity may be cross-reactive against SARS-CoV-2.

High-resolution epitope mapping and characterization of SARS-CoV-2 antibodies in large cohorts of subjects with COVID-19

As Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to spread, characterization of its antibody epitopes, emerging strains, related coronaviruses, and even the human proteome in naturally infected patients can guide the development of effective vaccines and therapies. Since traditional epitope identification tools are dependent upon pre-defined peptide sequences, they are not readily adaptable to diverse viral proteomes. The Serum Epitope Repertoire Analysis (SERA) platform leverages a high diversity random bacterial display library to identify proteome-independent epitope binding specificities which are then analyzed in the context of organisms of interest. When evaluating immune response in the context of SARS-CoV-2, we identify dominant epitope regions and motifs which demonstrate potential to classify mild from severe disease and relate to neutralization activity. We highlight SARS-CoV-2 epitopes that are cross-reactive with other coronaviruses and demonstrate decreased epitope signal for mutant SARS-CoV-2 strains. Collectively, the evolution of SARS-CoV-2 mutants towards reduced antibody response highlight the importance of data-driven development of the vaccines and therapies to treat COVID-19.

21-Hydroxylase-Specific CD8+ T Cells in Autoimmune Addison’s Disease Are Restricted by HLA-A2 and HLA-C7 Molecules

ObjectivesCD8+ T cells targeting 21-hydroxylase (21OH) are presumed to play a central role in the destruction of adrenocortical cells in autoimmune Addison’s disease (AAD). Earlier reports have suggested two immunodominant CD8+ T cell epitopes within 21OH: LLNATIAEV (21OH342-350), restricted by HLA-A2, and EPLARLEL (21OH431-438), restricted by HLA-B8. We aimed to characterize polyclonal CD8+ T cell responses to the proposed epitopes in a larger patient cohort with AAD.MethodsRecombinant fluorescent HLA-peptide multimer reagents were used to quantify antigen-specific CD8+ T cells by flow cytometry. Interferon-gamma (IFNγ) Elispot and biochemical assays were used to functionally investigate the 21OH-specific T cells, and to map the exactly defined epitopes of 21OH.ResultsWe found a significantly higher frequency of HLA-A2 restricted LLNATIAEV-specific cells in patients with AAD than in controls. These cells could also be expanded in vitro in an antigen specific manner and displayed a robust antigen-specific IFNγ production. In contrast, only negligible frequencies of EPLARLEL-specific T cells were detected in both patients and controls with limited IFNγ response. However, significant IFNγ production was observed in response to a longer peptide encompassing EPLARLEL, 21OH430-447, suggesting alternative dominant epitopes. Accordingly, we discovered that the slightly offset ARLELFVVL (21OH434-442) peptide is a novel dominant epitope restricted by HLA-C7 and not by HLA-B8 as initially postulated.ConclusionWe have identified two dominant 21OH epitopes targeted by CD8+ T cells in AAD, restricted by HLA-A2 and HLA-C7, respectively. To our knowledge, this is the first HLA-C7 restricted epitope described for an autoimmune disease.

Identification of TCR repertoires in functionally competent cytotoxic T cells cross-reactive to SARS-CoV-2

SARS-CoV-2-specific CD8+ T cells are detectable in infected individuals but are low in unexposed healthy donors (UHD). Little is known about whether pre-existing human coronavirus (HCoV)-specific CD8+ T cells are converted to functionally competent T cells cross-reactive to SARS-CoV-2. Induction of cross-reactive immunity requires the recognition of multiple epitopes. Here, we show that SARS-CoV-2-specific T cells elicited in response to a selected dominant epitope are multifunctional and respond to various HCoVs in UHD. TCRαβ chains from each T cell clone were identified; TCRαβ-transduced T cells responded broadly to the relevant epitopes on several HCoVs, thus implying that TCRαβ may exhibit selective diversity at the single-cell level. We further defined four sets of optimal SARS-CoV-2-peptides and demonstrated the response of CD8+ T cells even in hematological malignant patients. Together, the proposed epitopes inducing pre-existing CD8+ T cells to cross-react with SARS-CoV-2 may be beneficial in vaccine development.

SARS-CoV-2 can recruit a haem metabolite to evade antibody immunity

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of haem metabolism, with nanomolar affinity. Using cryo-electron microscopy and X-ray crystallography, we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that SARS-CoV-2 spike NTD harbors a dominant epitope, access to which can be controlled by an allosteric mechanism that is regulated through the recruitment of a metabolite.

Impact of B.1.1.7 variant mutations on antibody recognition of linear SARS-CoV-2 epitopes

In 579 COVID patient samples collected between March and July of 2020, we examined the effects of non-synonymous mutations harbored by the circulating B.1.1.7 strain on linear antibody epitope signal for spike glycoprotein and nucleoprotein. At the antigen level, the mutations only substantially reduced signal in 0.5% of the population. Although some epitope mutations reduce measured signal in up to 6% of the population, these are not the dominant epitopes for their antigens. Given dominant epitope patterns observed, our data suggest that the mutations would not result in immune evasion of linear epitopes for a large majority of these COVID patients.

Structural characterization of a neutralizing mAb H16.001, a potent candidate for a common potency assay for various HPV16 VLPs

With more human papillomavirus (HPV) virus-like particle (VLP) vaccines to hit the market in future, a monoclonal antibody (mAb) with preferably comparable reactivity against vaccines from different expression systems and bioprocesses is urgently needed for the potency characterization. Among all mAbs against HPV16 collected, rabbit mAb H16.001 is potently neutralizing with the highest affinity, recognizes an immune-dominant epitope, and can comparably react with HPV16 vaccines from various sources. Cryo-electron microscopic (cryo-EM) structure demonstrated that 360 H16.001 Fabs could bind to HPV16 capsid in preferable binding manner without steric hindrance between neighboring Fabs, potentially supporting its identification for VLP structural integrity and utility in monitoring VLP structural probity. This structural analysis indicated that mAb H16.001 afforded unbiased potency characterization for various HPV16 vaccines and was potential for use in vaccine regulation practice. This study also showed a model process for selecting suitable mAbs for potency assays of other vaccines.