Therapeutic and vaccine-induced cross-reactive antibodies with effector function against emerging Omicron variants (preprint)

Currently circulating SARS-CoV-2 variants acquired convergent mutations at receptor-binding domain (RBD) hot spots. Their impact on viral infection, transmission, and efficacy of vaccines and therapeutics remains poorly understood. Here, we demonstrate that recently emerged BQ.1.1. and XBB.1 variants bind ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1 and XBB.1 RBDs bound to human ACE2 and S309 Fab (sotrovimab parent) explain the altered ACE2 recognition and preserved antibody binding through conformational selection. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1, the variant displaying the greatest loss of neutralization. Moreover, in several donors vaccine-elicited plasma antibodies cross-react with and trigger effector functions against Omicron variants despite reduced neutralizing activity. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring persistent immune imprinting. Our findings suggest that this previously overlooked class of cross-reactive antibodies, exemplified by S309, may contribute to protection against disease caused by emerging variants through elicitation of effector functions.

Maturation of SARS-CoV-2 Spike-specific memory B cells drives resilience to viral escape (preprint)

Memory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month timeframe. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both pre- and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sub-lineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly-reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants.

Imprinted antibody responses against SARS-CoV-2 Omicron sublineages (preprint)

SARS-CoV-2 Omicron sublineages carry distinct spike mutations and represent an antigenic shift resulting in escape from antibodies induced by previous infection or vaccination. We show that hybrid immunity or vaccine boosters result in potent plasma neutralizing activity against Omicron BA.1 and BA.2 and that breakthrough infections, but not vaccination-only, induce neutralizing activity in the nasal mucosa. Consistent with immunological imprinting, most antibodies derived from memory B cells or plasma cells of Omicron breakthrough cases cross-react with the Wuhan-Hu-1, BA.1 and BA.2 receptor-binding domains whereas Omicron primary infections elicit B cells of narrow specificity. While most clinical antibodies have reduced neutralization of Omicron, we identified an ultrapotent pan-variant antibody, that is unaffected by any Omicron lineage spike mutations and is a strong candidate for clinical development.

ACE2 engagement exposes the fusion peptide to pan-coronavirus neutralizing antibodies (preprint)

Coronaviruses use diverse Spike (S) glycoproteins to attach to host receptors and fuse with target cells. Using a broad screening approach, we isolated from SARS-CoV-2 immune donors seven monoclonal antibodies (mAbs) that bind to all human alpha and beta coronavirus S proteins. These mAbs recognize the fusion peptide and acquire high affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha and beta coronaviruses, including Omicron BA.1 variant and bat WIV-1, and reduce viral titers and pathology in vivo. Structural and functional analyses show that the fusion peptide-specific mAbs bind with different modalities to a cryptic epitope which is concealed by prefusion-stabilizing 2P mutations and becomes exposed upon binding of ACE2 or ACE2-mimicking mAbs. This study identifies a new class of pan-coronavirus neutralizing mAbs and reveals a receptor-induced conformational change in the S protein that exposes the fusion peptide region.

Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift (preprint)

The recently emerged SARS-CoV-2 Omicron variant harbors 37 amino acid substitutions in the spike (S) protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody therapeutics. Here, we show that the Omicron RBD binds to human ACE2 with enhanced affinity relative to the Wuhan-Hu-1 RBD and acquires binding to mouse ACE2. Severe reductions of plasma neutralizing activity were observed against Omicron compared to the ancestral pseudovirus for vaccinated and convalescent individuals. Most (26 out of 29) receptor-binding motif (RBM)-directed monoclonal antibodies (mAbs) lost in vitro neutralizing activity against Omicron, with only three mAbs, including the ACE2-mimicking S2K146 mAb, retaining unaltered potency. Furthermore, a fraction of broadly neutralizing sarbecovirus mAbs recognizing antigenic sites outside the RBM, including sotrovimab, S2X259 and S2H97, neutralized Omicron. The magnitude of Omicron-mediated immune evasion and the acquisition of binding to mouse ACE2 mark a major SARS-CoV-2 mutational shift. Broadly neutralizing sarbecovirus mAbs recognizing epitopes conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.

Structure, receptor recognition and antigenicity of the human coronavirus CCoV-HuPn-2018 spike glycoprotein (preprint)

The recent isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. Here, we determined cryo-electron microscopy structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and identified that it binds canine, feline and porcine aminopeptidase N (APN encoded by ANPEP) orthologs which serve as entry receptors. Introduction of an oligosaccharide at position N739 of human APN renders cells susceptible to CCoV-HuPn-2018 spike-mediated entry, suggesting that single nucleotide polymorphisms could account for the detection of this virus in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 S-mediated entry, indicating elicitation of cross-neutralizing activity among -coronaviruses. These data provide a blueprint of the CCoV-HuPn-2018 infection machinery, unveil the viral entry receptor and pave the way for vaccine and therapeutic development targeting this zoonotic pathogen.

Defective neutralizing antibody response to SARS-CoV-2 in vaccinated dialysis patients (preprint)

Patients on dialysis are at risk of severe course of SARS-CoV-2 infection. Understanding the neutralizing activity and coverage of SARS-CoV-2 variants of vaccine-elicited antibodies is required to guide prophylactic and therapeutic COVID-19 interventions in this frail population. By analyzing plasma samples from 130 hemodialysis (HD) and 13 peritoneal dialysis patients after two doses of BNT162b2 or mRNA-1273 vaccines, we found that 35% of the patients had low-level or undetectable IgG antibodies to SARS-CoV-2 Spike (S). Neutralizing antibodies against the vaccine-matched SARS-CoV-2 and Delta variant were low or undetectable in 49% and 77% of patients, respectively, and were further reduced against other emerging variants. The fraction of non-responding patients was higher in SARS-CoV-2-naive HD patients immunized with BNT162b2 (66%) than those immunized with mRNA-1273 (23%). The reduced neutralizing activity correlated with low antibody avidity, consistent with a delayed affinity maturation of SARS-CoV-2 S-specific B cells. These data indicate that dialysis patients should be considered for an additional boost and other therapeutic strategies, including early immunotherapy with monoclonal antibodies.

Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies (preprint)

Investigating the mechanisms of SARS-CoV-2 cellular infection is key to better understand COVID-19 immunity and pathogenesis. Infection, which involves both cell attachment and membrane fusion, relies on the ACE2 receptor that is paradoxically found at low levels in the respiratory tract, suggesting that additional mechanisms facilitating infection may exist. Here we show that C-type lectin receptors, DC-SIGN, L-SIGN and the sialic acid-binding Ig-like lectin 1 (SIGLEC1) function as auxiliary receptors by enhancing ACE2-mediated infection and modulating the neutralizing activity of different classes of spike-specific antibodies. Antibodies to the N-terminal domain (NTD) or to the conserved proteoglycan site at the base of the Receptor Binding Domain (RBD), while poorly neutralizing infection of ACE2 over-expressing cells, effectively block lectin-facilitated infection. Conversely, antibodies to the Receptor Binding Motif (RBM), while potently neutralizing infection of ACE2 over-expressing cells, poorly neutralize infection of cells expressing DC-SIGN or L-SIGN and trigger fusogenic rearrangement of the spike promoting cell-to-cell fusion. Collectively, these findings identify a lectin-dependent pathway that enhances ACE2-dependent infection by SARS-CoV-2 and reveal distinct mechanisms of neutralization by different classes of spike-specific antibodies.

Clonal dissection of immunodominance and cross-reactivity of the CD4+ T cell response to SARS-CoV-2 (preprint)

The identification of CD4+ T cell epitopes is essential for the design of effective vaccines capable of inducing neutralizing antibodies and long-term immunity. Here we demonstrate in COVID-19 patients a robust CD4+ T cell response to naturally processed SARS-CoV-2 Spike and Nucleoprotein, including effector, helper and memory T cells. By characterizing 2,943 Spike-reactive T cell clones, we found that 34% of the clones and 93% of the patients recognized a conserved immunodominant region encompassing residues S346-365 in the RBD and comprising three nested HLA-DR and HLA-DP restricted epitopes. By using pre- and post-COVID-19 samples and Spike proteins from alpha and beta coronaviruses, we provide in vivo evidence of cross-reactive T cell responses targeting multiple sites in the SARS-CoV-2 Spike protein. The possibility of leveraging immunodominant and cross-reactive T helper epitopes is instrumental for vaccination strategies that can be rapidly adapted to counteract emerging SARS-CoV-2 variants.

SARS-CoV-2 B.1.1.7 escape from mRNA vaccine-elicited neutralizing antibodies (preprint)

SARS-CoV-2 transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 mutations found in the B.1.1.7 Spike protein. The vaccine sera exhibited a broad range of neutralizing titres against the wild-type pseudoviruses (<1:4 to 3449) that were reduced against B.1.1.7 variant by 3.85 fold (IQR 2.68-5.28). This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralization was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the Receptor Binding Motif (RBM) (5 outof 29), but not in neutralizing mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background led to a further loss of neutralizing activity by vaccine-elicited antibodies over that conferred by the B.1.1.7 mutations alone. Further work is needed to establish the impact of these observations on protective vaccine efficacy in the context of the evolving B.1.1.7 lineage that will likely acquire E484K.

Impact of SARS-CoV-2 B.1.1.7 Spike variant on neutralisation potency of sera from individuals vaccinated with Pfizer vaccine BNT162b2 (preprint)

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) transmission is uncontrolled in many parts of the world, compounded in some areas by higher transmission potential of the B1.1.7 variant now seen in 50 countries. It is unclear whether responses to SARS-CoV-2 vaccines based on the prototypic strain will be impacted by mutations found in B.1.1.7. Here we assessed immune responses following vaccination with mRNA-based vaccine BNT162b2. We measured neutralising antibody responses following a single immunization using pseudoviruses expressing the wild-type Spike protein or the 8 amino acid mutations found in the B.1.1.7 spike protein. The vaccine sera exhibited a broad range of neutralising titres against the wild-type pseudoviruses that were modestly reduced against B.1.1.7 variant. This reduction was also evident in sera from some convalescent patients. Decreased B.1.1.7 neutralisation was also observed with monoclonal antibodies targeting the N-terminal domain (9 out of 10), the Receptor Binding Motif (RBM) (5 out of 31), but not in neutralising mAbs binding outside the RBM. Introduction of the E484K mutation in a B.1.1.7 background to reflect newly emerging viruses in the UK led to a more substantial loss of neutralising activity by vaccine-elicited antibodies and mAbs (19 out of 31) over that conferred by the B.1.1.7 mutations alone. E484K emergence on a B.1.1.7 background represents a threat to the vaccine BNT162b.

Structural and functional analysis of a potent sarbecovirus neutralizing antibody (preprint)

SARS-CoV-2 is a newly emerged coronavirus responsible for the current COVID-19 pandemic that has resulted in more than one million infections and 73,000 deaths1,2. Vaccine and therapeutic discovery efforts are paramount to curb the pandemic spread of this zoonotic virus. The SARS-CoV-2 spike (S) glycoprotein promotes entry into host cells and is the main target of neutralizing antibodies. Here we describe multiple monoclonal antibodies targeting SARS-CoV-2 S identified from memory B cells of a SARS survivor infected in 2003. One antibody, named S309, potently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as authentic SARS-CoV-2 by engaging the S receptor-binding domain. Using cryo-electron microscopy and binding assays, we show that S309 recognizes a glycan-containing epitope that is conserved within the sarbecovirus subgenus, without competing with receptor attachment. Antibody cocktails including S309 along with other antibodies identified here further enhanced SARS-CoV-2 neutralization and may limit the emergence of neutralization-escape mutants. These results pave the way for using S309 and S309-containing antibody cocktails for prophylaxis in individuals at high risk of exposure or as a post-exposure therapy to limit or treat severe disease.