Characteristics and functions of infection-enhancing antibodies to the N-terminal domain of SARS-CoV-2.

Characterization of functional antibody responses to the N-terminal domain (NTD) of the SARS-CoV-2 spike (S) protein has included identification of both potent neutralizing activity and putative enhancement of infection. Fcγ-receptor (FcγR)-independent enhancement of SARS-CoV-2 infection mediated by NTD-binding monoclonal antibodies (mAbs) has been observed in vitro , but the functional significance of these antibodies in vivo is not clear. Here we studied 1,213 S-binding mAbs derived from longitudinal sampling of B-cells collected from eight COVID-19 convalescent patients and identified 72 (5.9%) mAbs that enhanced infection in a VSV-SARS-CoV-2-S-Wuhan pseudovirus (PV) assay. The majority (68%) of these mAbs recognized the NTD, were identified in patients with mild and severe disease, and persisted for at least five months post-infection. Enhancement of PV infection by NTD-binding mAbs was not observed using intestinal (Caco-2) and respiratory (Calu-3) epithelial cells as infection targets and was diminished or lost against SARS-CoV-2 variants of concern (VOC). Proteomic deconvolution of the serum antibody repertoire from two of the convalescent subjects identified, for the first time, NTD-binding, infection-enhancing mAbs among the circulating immunoglobulins directly isolated from serum ( i.e ., functionally secreted antibody). Functional analysis of these mAbs demonstrated robust activation of FcγRIIIa associated with antibody binding to recombinant S proteins. Taken together, these findings suggest functionally active NTD-specific mAbs arise frequently during natural infection and can last as major serum clonotypes during convalescence. These antibodies display diverse attributes that include FcγR activation, and may be selected against by mutations in NTD associated with SARS-CoV-2 VOC.

Characterization of SARS-CoV-2 Convalescent Patients' Serological Repertoire Reveals High Prevalence of Iso-RBD Antibodies.

While our understanding of SARS-CoV-2 pathogenesis and antibody responses following infection and vaccination has improved tremendously since the outbreak in 2019, the sequence identities and relative abundances of the individual constituent antibody molecules in circulation remain understudied. Using Ig-Seq, we proteomically profiled the serological repertoire specific to the whole ectodomain of SARS-CoV-2 prefusion-stabilized spike (S) as well as to the receptor binding domain (RBD) over a 6-month period in four subjects following SARS-CoV-2 infection before SARS-CoV-2 vaccines were available. In each individual, we identified between 59 and 167 unique IgG clonotypes in serum. To our surprise, we discovered that ∼50% of serum IgG specific for RBD did not recognize prefusion-stabilized S (referred to as iso-RBD antibodies), suggesting that a significant fraction of serum IgG targets epitopes on RBD inaccessible on the prefusion-stabilized conformation of S. On the other hand, the abundance of iso-RBD antibodies in nine individuals who received mRNA-based COVID-19 vaccines encoding prefusion-stabilized S was significantly lower (∼8%). We expressed a panel of 12 monoclonal antibodies (mAbs) that were abundantly present in serum from two SARS-CoV-2 infected individuals, and their binding specificities to prefusion-stabilized S and RBD were all in agreement with the binding specificities assigned based on the proteomics data, including 1 iso-RBD mAb which bound to RBD but not to prefusion-stabilized S. 2 of 12 mAbs demonstrated neutralizing activity, while other mAbs were non-neutralizing. 11 of 12 mAbs also bound to S (B.1.351), but only 1 maintained binding to S (B.1.1.529). This particular mAb binding to S (B.1.1.529) 1) represented an antibody lineage that comprised 43% of the individual's total S-reactive serum IgG binding titer 6 months post-infection, 2) bound to the S from a related human coronavirus, HKU1, and 3) had a high somatic hypermutation level (10.9%), suggesting that this antibody lineage likely had been elicited previously by pre-pandemic coronavirus and was re-activated following the SARS-CoV-2 infection. All 12 mAbs demonstrated their ability to engage in Fc-mediated effector function activities. Collectively, our study provides a quantitative overview of the serological repertoire following SARS-CoV-2 infection and the significant contribution of iso-RBD antibodies, demonstrating how vaccination strategies involving prefusion-stabilized S may have reduced the elicitation of iso-RBD serum antibodies which are unlikely to contribute to protection.

Vaccination of SARS-CoV-2-infected individuals expands a broad range of clonally diverse affinity-matured B cell lineages.

Vaccination of SARS-CoV-2 convalescent individuals generates broad and potent antibody responses. Here, we isolate 459 spike-specific monoclonal antibodies (mAbs) from two individuals who were infected with the index variant of SARS-CoV-2 and later boosted with mRNA-1273. We characterize mAb genetic features by sequence assignments to the donors' personal immunoglobulin genotypes and assess antibody neutralizing activities against index SARS-CoV-2, Beta, Delta, and Omicron variants. The mAbs used a broad range of immunoglobulin heavy chain (IGH) V genes in the response to all sub-determinants of the spike examined, with similar characteristics observed in both donors. IGH repertoire sequencing and B cell lineage tracing at longitudinal time points reveals extensive evolution of SARS-CoV-2 spike-binding antibodies from acute infection until vaccination five months later. These results demonstrate that highly polyclonal repertoires of affinity-matured memory B cells are efficiently recalled by vaccination, providing a basis for the potent antibody responses observed in convalescent persons following vaccination.

Potently neutralizing and protective anti-human metapneumovirus antibodies target diverse sites on the fusion glycoprotein.

Human metapneumovirus (hMPV) is a leading cause of acute lower respiratory tract infections in high-risk populations, yet there are no vaccines or anti-viral therapies approved for the prevention or treatment of hMPV-associated disease. Here, we used a high-throughput single-cell technology to interrogate memory B cell responses to the hMPV fusion (F) glycoprotein in young adult and elderly donors. Across all donors, the neutralizing antibody response was primarily directed to epitopes expressed on both pre- and post-fusion F conformations. However, we identified rare, highly potent broadly neutralizing antibodies that recognize pre-fusion-specific epitopes and structurally characterized an antibody that targets a site of vulnerability at the pre-fusion F trimer apex. Additionally, monotherapy with neutralizing antibodies targeting three distinct antigenic sites provided robust protection against lower respiratory tract infection in a small animal model. This study provides promising monoclonal antibody candidates for passive immunoprophylaxis and informs the rational design of hMPV vaccine immunogens.

Recall of preexisting cross-reactive B cell memory after Omicron BA.1 breakthrough infection.

Understanding immune responses after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) breakthrough infection will facilitate the development of next-generation vaccines. Here, we profiled spike (S)-specific B cell responses after Omicron/BA.1 infection in messenger RNA-vaccinated donors. The acute antibody response was characterized by high levels of somatic hypermutation and a bias toward recognition of ancestral SARS-CoV-2 strains, suggesting the early activation of vaccine-induced memory B cells. BA.1 breakthrough infection induced a shift in B cell immunodominance hierarchy from the S2 subunit, which is highly conserved across SARS-CoV-2 variants of concern (VOCs), and toward the antigenically variable receptor binding domain (RBD). A large proportion of RBD-directed neutralizing antibodies isolated from BA.1 breakthrough infection donors displayed convergent sequence features and broadly recognized SARS-CoV-2 VOCs. Together, these findings provide insights into the role of preexisting immunity in shaping the B cell response to heterologous SARS-CoV-2 variant exposure.

Human antibody recognizing a quaternary epitope in the Puumala virus glycoprotein provides broad protection against orthohantaviruses.

The rodent-borne hantavirus Puumala virus (PUUV) and related agents cause hemorrhagic fever with renal syndrome (HFRS) in humans. Other hantaviruses, including Andes virus (ANDV) and Sin Nombre virus, cause a distinct zoonotic disease, hantavirus cardiopulmonary syndrome (HCPS). Although these infections are severe and have substantial case fatality rates, no FDA-approved hantavirus countermeasures are available. Recent work suggests that monoclonal antibodies may have therapeutic utility. We describe here the isolation of human neutralizing antibodies (nAbs) against tetrameric Gn/Gc glycoprotein spikes from PUUV-experienced donors. We define a dominant class of nAbs recognizing the "capping loop" of Gn that masks the hydrophobic fusion loops in Gc. A subset of nAbs in this class, including ADI-42898, bound Gn/Gc complexes but not Gn alone, strongly suggesting that they recognize a quaternary epitope encompassing both Gn and Gc. ADI-42898 blocked the cell entry of seven HCPS- and HFRS-associated hantaviruses, and single doses of this nAb could protect Syrian hamsters and bank voles challenged with the highly virulent HCPS-causing ANDV and HFRS-causing PUUV, respectively. ADI-42898 is a promising candidate for clinical development as a countermeasure for both HCPS and HFRS, and its mode of Gn/Gc recognition informs the development of broadly protective hantavirus vaccines.

Broad anti-SARS-CoV-2 antibody immunity induced by heterologous ChAdOx1/mRNA-1273 vaccination.

Heterologous prime-boost immunization strategies have the potential to augment COVID-19 vaccine efficacy. We longitudinally profiled severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-specific serological and memory B cell (MBC) responses in individuals who received either homologous (ChAdOx1:ChAdOx1) or heterologous (ChAdOx1:mRNA-1273) prime-boost vaccination. Heterologous messenger RNA (mRNA) booster immunization induced higher serum neutralizing antibody and MBC responses against SARS-CoV-2 variants of concern (VOCs) compared with that of homologous ChAdOx1 boosting. Specificity mapping of circulating B cells revealed that mRNA-1273 boost immunofocused ChAdOx1-primed responses onto epitopes expressed on prefusion-stabilized S. Monoclonal antibodies isolated from mRNA-1273-boosted participants displayed overall higher binding affinities and increased breadth of reactivity against VOCs relative to those isolated from ChAdOx1-boosted individuals. Overall, the results provide molecular insight into the enhanced quality of the B cell response induced after heterologous mRNA booster vaccination.

Genotype-specific features reduce the susceptibility of South American yellow fever virus strains to vaccine-induced antibodies.

The resurgence of yellow fever in South America has prompted vaccination against the etiologic agent, yellow fever virus (YFV). Current vaccines are based on a live-attenuated YF-17D virus derived from a virulent African isolate. The capacity of these vaccines to induce neutralizing antibodies against the vaccine strain is used as a surrogate for protection. However, the sensitivity of genetically distinct South American strains to vaccine-induced antibodies is unknown. We show that antiviral potency of the polyclonal antibody response in vaccinees is attenuated against an emergent Brazilian strain. This reduction was attributable to amino acid changes at two sites in central domain II of the glycoprotein E, including multiple changes at the domain I-domain II hinge, which are unique to and shared among most South American YFV strains. Our findings call for a reevaluation of current approaches to YFV immunological surveillance in South America and suggest approaches for updating vaccines.

A Combination of Receptor-Binding Domain and N-Terminal Domain Neutralizing Antibodies Limits the Generation of SARS-CoV-2 Spike Neutralization-Escape Mutants.

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150, and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent-phase sera were reduced by 4- to 16-fold against rVSV-SARS2 bearing Y145D, K150E, or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs did not enhance their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. IMPORTANCE The U.S. FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (MAb) therapies for the treatment of mild to moderate COVID-19. These MAb therapeutics are solely targeting the receptor-binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent-phase COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor-binding and N-terminal domains may be beneficial to combat the emergence of virus variants.

Prolonged evolution of the human B cell response to SARS-CoV-2 infection.

A comprehensive understanding of the kinetics and evolution of the human B cell response to SARS-CoV-2 infection will facilitate the development of next-generation vaccines and therapies. Here, we longitudinally profiled this response in mild and severe COVID-19 patients over a period of five months. Serum neutralizing antibody (nAb) responses waned rapidly but spike (S)-specific IgG+ memory B cells (MBCs) remained stable or increased over time. Analysis of 1,213 monoclonal antibodies (mAbs) isolated from S-specific MBCs revealed a primarily de novo response that displayed increased somatic hypermutation, binding affinity, and neutralization potency over time, providing evidence for prolonged antibody affinity maturation. B cell immunodominance hierarchies were similar across donor repertoires and remained relatively stable as the immune response progressed. Cross-reactive B cell populations, likely re-called from prior endemic beta-coronavirus exposures, comprised a small but stable fraction of the repertoires and did not contribute to the neutralizing response. The neutralizing antibody response was dominated by public clonotypes that displayed significantly reduced activity against SARS-CoV-2 variants emerging in Brazil and South Africa that harbor mutations at positions 501, 484 and 417 in the S protein. Overall, the results provide insight into the dynamics, durability, and functional properties of the human B cell response to SARS-CoV-2 infection and have implications for the design of immunogens that preferentially stimulate protective B cell responses.

Broad and potent activity against SARS-like viruses by an engineered human monoclonal antibody.

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope that overlaps the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses.

An Engineered Antibody with Broad Protective Efficacy in Murine Models of SARS and COVID-19.

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. Here, we employed a directed evolution approach to engineer three SARS-CoV-2 antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains (RBDs) and neutralizes representative epidemic sarbecoviruses with remarkable potency. Structural and biochemical studies demonstrate that ADG-2 employs a unique angle of approach to recognize a highly conserved epitope overlapping the receptor binding site. In murine models of SARS-CoV and SARS-CoV-2 infection, passive transfer of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate for the treatment and prevention of SARS-CoV-2 and future emerging SARS-like CoVs.

Broad sarbecovirus neutralizing antibodies define a key site of vulnerability on the SARS-CoV-2 spike protein.

Broadly protective vaccines against known and pre-emergent coronaviruses are urgently needed. Critical to their development is a deeper understanding of cross-neutralizing antibody responses induced by natural human coronavirus (HCoV) infections. Here, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a new target for the rational design of pan-sarbecovirus vaccines.

Broad neutralization of SARS-related viruses by human monoclonal antibodies.

Broadly protective vaccines against known and preemergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent severe acute respiratory syndrome (SARS) donor and identified 200 SARS coronavirus 2 (SARS-CoV-2) binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of preexisting memory B cells elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a target for the rational design of pan-sarbecovirus vaccines.