Broad Neutralizing Research Articles

The SARS-CoV-2 neutralizing antibody response to SD1 and its evasion by BA.2.86

Under pressure from neutralising antibodies induced by vaccination or infection the SARS-CoV-2 spike gene has become a hotspot for evolutionary change, leading to the failure of all mAbs developed for clinical use. Most potent antibodies bind to the receptor binding domain which has become heavily mutated. Here we study responses to a conserved epitope in sub-domain-1 (SD1) of spike which have become more prominent because of mutational escape from antibodies directed to the receptor binding domain. Some SD1 reactive mAbs show potent and broad neutralization of SARS-CoV-2 variants. We structurally map the dominant SD1 epitope and provide a mechanism of action by blocking interaction with ACE2. Mutations in SD1 have not been sustained to date, but one, E554K, leads to escape from mAbs. This mutation has now emerged in several sublineages including BA.2.86, reflecting selection pressure on the virus exerted by the increasing prominence of the anti-SD1 response.

Rapid development of double-hit mRNA antibody cocktail against orthopoxviruses

The Orthopoxvirus genus, especially variola virus (VARV), monkeypox virus (MPXV), remains a significant public health threat worldwide. The development of therapeutic antibodies against orthopoxviruses is largely hampered by the high cost of antibody engineering and manufacturing processes. mRNA-encoded antibodies have emerged as a powerful and universal platform for rapid antibody production. Herein, by using the established lipid nanoparticle (LNP)-encapsulated mRNA platform, we constructed four mRNA combinations that encode monoclonal antibodies with broad neutralization activities against orthopoxviruses. In vivo characterization demonstrated that a single intravenous injection of each LNP-encapsulated mRNA antibody in mice resulted in the rapid production of neutralizing antibodies. More importantly, mRNA antibody treatments showed significant protection from weight loss and mortality in the vaccinia virus (VACV) lethal challenge mouse model, and a unique mRNA antibody cocktail, Mix2a, exhibited superior in vivo protection by targeting both intracellular mature virus (IMV)-form and extracellular enveloped virus (EEV)-form viruses. In summary, our results demonstrate the proof-of-concept production of orthopoxvirus antibodies via the LNP-mRNA platform, highlighting the great potential of tailored mRNA antibody combinations as a universal strategy to combat orthopoxvirus as well as other emerging viruses.

A panel of multivalent nanobodies broadly neutralizing Omicron subvariants and recombinant.

The emerging Omicron subvariants have a remarkable ability to spread and escape nearly all current monoclonal antibody (mAb) treatments. Although the virulence of SARS-CoV-2 has now diminished, it remains a significant threat to public health due to its high transmissibility and susceptibility to mutation. Therefore, it is urgent to develop broad-acting and potent therapeutics targeting current and emerging Omicron variants. Here, we identified a panel of Omicron BA.1 spike receptor-binding domain (RBD)-targeted nanobodies (Nbs) from a naive alpaca VHH library. This panel of Nbs exhibited high binding affinity to the spike RBD of wild-type, Alpha B.1.1.7, Beta B.1.351, Delta plus, Omicron BA.1, and BA.2. Through multivalent Nb construction, we obtained a subpanel of ultrapotent neutralizing Nbs against Omicron BA.1, BA.2, BF.7 and even emerging XBB.1.5, and XBB.1.16 pseudoviruses. Protein structure prediction and docking analysis showed that Nb trimer 2F2E5 targets two independent RBD epitopes, thus minimizing viral escape. Taken together, we obtained a panel of broad and ultrapotent neutralizing Nbs against Omicron BA.1, Omicron BA.2, BF.7, XBB.1.5, and XBB.1.16. These multivalent Nbs hold great promise for the treatment against SARS-CoV-2 infection and could possess a superwide neutralizing breadth against novel omicron mutants or recombinants.

A VRC13-like bNAb response is associated with complex escape pathways in HIV-1 envelope.

The rational design of HIV-1 immunogens to trigger the development of broadly neutralizing antibodies (bNAbs) requires understanding the viral evolutionary pathways influencing this process. An acute HIV-1-infected individual exhibiting >50% plasma neutralization breadth developed neutralizing antibody specificities against the CD4-binding site (CD4bs) and V1V2 regions of Env gp120. Comparison of pseudoviruses derived from early and late autologous env sequences demonstrated the development of >2 log resistance to VRC13 but not to other CD4bs-specific bNAbs. Mapping studies indicated that the V3 and CD4-binding loops of Env gp120 contributed significantly to developing resistance to the autologous neutralizing response and that the CD4-binding loop (CD4BL) specifically was responsible for the developing resistance to VRC13. Tracking viral evolution during the development of this cross-neutralizing CD4bs response identified amino acid substitutions arising at only 4 of 11 known VRC13 contact sites (K282, T283, K421, and V471). However, each of these mutations was external to the V3 and CD4BL regions conferring resistance to VRC13 and was transient in nature. Rather, complete resistance to VRC13 was achieved through the cooperative expression of a cluster of single amino acid changes within and immediately adjacent to the CD4BL, including a T359I substitution, exchange of a potential N-linked glycosylation (PNLG) site to residue S362 from N363, and a P369L substitution. Collectively, our data characterize complex HIV-1 env evolution in an individual developing resistance to a VRC13-like neutralizing antibody response and identify novel VRC13-associated escape mutations that may be important to inducing VRC13-like bNAbs for lineage-based immunogens.IMPORTANCEThe pursuit of eliciting broadly neutralizing antibodies (bNAbs) through vaccination and their use as therapeutics remains a significant focus in the effort to eradicate HIV-1. Key to our understanding of this approach is a more extensive understanding of bNAb contact sites and susceptible escape mutations in HIV-1 envelope (env). We identified a broad neutralizer exhibiting VRC13-like responses, a non-germline restricted class of CD4-binding site antibody distinct from the well-studied VRC01-class. Through longitudinal envelope sequencing and Env-pseudotyped neutralization assays, we characterized a complex escape pathway requiring the cooperative evolution of four amino acid changes to confer complete resistance to VRC13. This suggests that VRC13-class bNAbs may be refractory to rapid escape and attractive for therapeutic applications. Furthermore, the identification of longitudinal viral changes concomitant with the development of neutralization breadth may help identify the viral intermediates needed for the maturation of VRC13-like responses and the design of lineage-based immunogens.

Strategy to develop broadly effective multivalent COVID-19 vaccines against emerging variants based on Ad5/35 platform

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron strain has evolved into highly divergent variants with several sub-lineages. These newly emerging variants threaten the efficacy of available COVID-19 vaccines. To mitigate the occurrence of breakthrough infections and re-infections, and more importantly, to reduce the disease burden, it is essential to develop a strategy for producing updated multivalent vaccines that can provide broad neutralization against both currently circulating and emerging variants. We developed bivalent vaccine AdCLD-CoV19-1 BA.5/BA.2.75 and trivalent vaccines AdCLD-CoV19-1 XBB/BN.1/BQ.1.1 and AdCLD-CoV19-1 XBB.1.5/BN.1/BQ.1.1 using an Ad5/35 platform-based non-replicating recombinant adenoviral vector. We compared immune responses elicited by the monovalent and multivalent vaccines in mice and macaques. We found that the BA.5/BA.2.75 bivalent and the XBB/BN.1/BQ.1.1 and XBB.1.5/BN.1/BQ.1.1 trivalent vaccines exhibited improved cross-neutralization ability compared to their respective monovalent vaccines. These data suggest that the developed multivalent vaccines enhance immunity against circulating Omicron subvariants and effectively elicit neutralizing antibodies across a broad spectrum of SARS-CoV-2 variants.

Computational design and engineering of self-assembling multivalent microproteins with therapeutic potential against SARS-CoV-2

Multivalent drugs targeting homo-oligomeric viral surface proteins, such as the SARS-CoV-2 trimeric spike (S) protein, have the potential to elicit more potent and broad-spectrum therapeutic responses than monovalent drugs by synergistically engaging multiple binding sites on viral targets. However, rational design and engineering of nanoscale multivalent protein drugs are still lacking. Here, we developed a computational approach to engineer self-assembling trivalent microproteins that simultaneously bind to the three receptor binding domains (RBDs) of the S protein. This approach involves four steps: structure-guided linker design, molecular simulation evaluation of self-assembly, experimental validation of self-assembly state, and functional testing. Using this approach, we first designed trivalent constructs of the microprotein miniACE2 (MP) with different trimerization scaffolds and linkers, and found that one of the constructs (MP-5ff) showed high trimerization efficiency, good conformational homogeneity, and strong antiviral neutralizing activity. With its trimerization unit (5ff), we then engineered a trivalent nanobody (Tr67) that exhibited potent and broad neutralizing activity against the dominant Omicron variants, including XBB.1 and XBB.1.5. Cryo-EM complex structure confirmed that Tr67 stably binds to all three RBDs of the Omicron S protein in a synergistic form, locking them in the “3-RBD-up” conformation that could block human receptor (ACE2) binding and potentially facilitate immune clearance. Therefore, our approach provides an effective strategy for engineering potent protein drugs against SARS-CoV-2 and other deadly coronaviruses.Graphical

A monoclonal antibody targeting a large surface of the receptor binding motif shows pan-neutralizing SARS-CoV-2 activity

Here we report the characterization of 17T2, a SARS-CoV-2 pan-neutralizing human monoclonal antibody isolated from a COVID-19 convalescent individual infected during the first pandemic wave. 17T2 is a class 1 VH1-58/κ3-20 antibody, derived from a receptor binding domain (RBD)-specific IgA+ memory B cell, with a broad neutralizing activity against former and new SARS-CoV-2 variants, including XBB.1.16 and BA.2.86 Omicron subvariants. Consistently, 17T2 demonstrates in vivo prophylactic and therapeutic activity against Omicron BA.1.1 infection in K18-hACE2 mice. Cryo-electron microscopy reconstruction shows that 17T2 binds the BA.1 spike with the RBD in “up” position and blocks the receptor binding motif, as other structurally similar antibodies do, including S2E12. Yet, unlike S2E12, 17T2 retains its neutralizing activity against all variants tested, probably due to a larger RBD contact area. These results highlight the impact of small structural antibody changes on neutralizing performance and identify 17T2 as a potential candidate for future clinical interventions.

NSP6 inhibits the production of ACE2-containing exosomes to promote SARS-CoV-2 infectivity.

The outbreak of coronavirus disease 2019 (COVID-19) severely endangers global public health. The efficacy of vaccines and antibodies declined with the rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants. Angiotensin-converting enzyme 2-containing exosomes (ACE2-exos) therapy exhibits a broad neutralizing activity, which could be used against various viral mutations. Our study here revealed that SARS-CoV-2 nonstructural protein 6 inhibited the production of ACE2-exos, thereby promoting viral infection to the adjacent bystander cells. The identification of a new target for blocking SARS-CoV-2 depends on fully understanding the virus-host interaction networks. Our study sheds light on the mechanism by which the virus resists the host exosome defenses, which would facilitate the study and design of ACE2-exos-based therapeutics for COVID-19.

Heterologous Sarbecovirus Receptor Binding Domains as Scaffolds for SARS-CoV-2 Receptor Binding Motif Presentation.

Structure-guided rational immunogen design can generate optimized immunogens that elicit a desired humoral response. Design strategies often center on targeting conserved sites on viral glycoproteins that will ultimately confer potent neutralization. For SARS-CoV-2 (SARS-2), the surface-exposed spike glycoprotein includes a broadly conserved portion, the receptor binding motif (RBM), that is required to engage the host cellular receptor, ACE2. Expanding humoral responses to this site may result in a more potent neutralizing antibody response against diverse sarbecoviruses. Here, we used a "resurfacing" approach and iterative design cycles to graft the SARS-2 RBM onto heterologous sarbecovirus scaffolds. The scaffolds were selected to vary the antigenic distance relative to SARS-2 to potentially focus responses to RBM. Multimerized versions of these immunogens elicited broad neutralization against sarbecoviruses in the context of preexisting SARS-2 immunity. These validated engineering approaches can help inform future immunogen design efforts for sarbecoviruses and are generally applicable to other viruses.

Antibodies from dengue patients with prior exposure to Japanese encephalitis virus are broadly neutralizing against Zika virus

Exposure to multiple mosquito-borne flaviviruses within a lifetime is not uncommon; however, how sequential exposures to different flaviviruses shape the cross-reactive humoral response against an antigen from a different serocomplex has yet to be explored. Here, we report that dengue-infected individuals initially primed with the Japanese encephalitis virus (JEV) showed broad, highly neutralizing potencies against Zika virus (ZIKV). We also identified a rare class of ZIKV-cross-reactive human monoclonal antibodies with increased somatic hypermutation and broad neutralization against multiple flaviviruses. One huMAb, K8b, binds quaternary epitopes with heavy and light chains separately interacting with overlapping envelope protein dimer units spanning domains I, II, and III through cryo-electron microscopy and structure-based mutagenesis. JEV virus-like particle immunization in mice further confirmed that such cross-reactive antibodies, mainly IgG3 isotype, can be induced and proliferate through heterologous dengue virus (DENV) serotype 2 virus-like particle stimulation. Our findings highlight the role of prior immunity in JEV and DENV in shaping the breadth of humoral response and provide insights for future vaccination strategies in flavivirus-endemic countries.

Structural and biochemical rationale for Beta variant protein booster vaccine broad cross-neutralization of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, uses a surface expressed trimeric spike glycoprotein for cell entry. This trimer is the primary target for neutralizing antibodies making it a key candidate for vaccine development. During the global pandemic circulating variants of concern (VOC) caused several waves of infection, severe disease, and death. The reduced efficacy of the ancestral trimer-based vaccines against emerging VOC led to the need for booster vaccines. Here we present a detailed characterization of the Sanofi Beta trimer, utilizing cryo-EM for structural elucidation. We investigate the conformational dynamics and stabilizing features using orthogonal SPR, SEC, nanoDSF, and HDX-MS techniques to better understand how this antigen elicits superior broad neutralizing antibodies as a variant booster vaccine. This structural analysis confirms the Beta trimer preference for canonical quaternary structure with two RBD in the up position and the reversible equilibrium between the canonical spike and open trimer conformations. Moreover, this report provides a better understanding of structural differences between spike antigens contributing to differential vaccine efficacy.

Identification of a broad sarbecovirus neutralizing antibody targeting a conserved epitope on the receptor-binding domain

Omicron, as the emerging variant with enhanced vaccine tolerance, has sharply disrupted most therapeutic antibodies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the subgenus Sarbecovirus, members of which share high sequence similarity. Herein, we report one sarbecovirus antibody, 5817, which has broad-spectrum neutralization capacity against SARS-CoV-2 variants of concern (VOCs) and SARS-CoV, as well as related bat and pangolin viruses. 5817 can hardly compete with six classes of receptor-binding-domain-targeted antibodies grouped by structural classifications. No obvious impairment in the potency is detected against SARS-CoV-2 Omicron and subvariants. The cryoelectron microscopy (cryo-EM) structure of neutralizing antibody 5817 in complex with Omicron spike reveals a highly conserved epitope, only existing at the receptor-binding domain (RBD) open state. Prophylactic and therapeutic administration of 5817 potently protects mice from SARS-CoV-2 Beta, Delta, Omicron, and SARS-CoV infection. This study reveals a highly conserved cryptic epitope targeted by a broad sarbecovirus neutralizing antibody, which would be beneficial to meet the potential threat of pre-emergent SARS-CoV-2 VOCs.

A Universal Aptamer for Influenza A Viruses: Selection, Recognition, and Infection Inhibition.

It is crucial to develop universal inhibitors for viral inhibition due to the rapid mutation of viruses. Herein, a universal aptamer inhibitor was developed that enabled a single DNA molecule to recognize several hemeagglutinin (HA) protein subtypes, inducing broad neutralization against influenza A viruses (IAVs). Through a multi-channel enrichment (MCE) strategy, a high-affinity aptamer named UHA-2 was obtained, with its dissociation constants (Kd) for three different HA proteins being 1.5 ± 0.2 nM (H5N1), 3.7 ± 0.4 nM (H7N9), and 10.1 ± 1.1 nM (H9N2). The UHA-2 aptamer had a universal inhibition effect, by which it could broadly neutralize influenza A H5N1, H7N9, H9N2, H1N1, and H3N2 viruses. Universal aptamer inhibitors have the advantages of acquisition in vitro, stability, simple structure, small size, etc. This study not only develops a novel universal aptamer to achieve a broad inhibition effect on various IAVs, but also opens up an efficient strategy for the development of universal inhibitors against viruses.

Defining a de novo non-RBM antibody as RBD-8 and its synergistic rescue of immune-evaded antibodies to neutralize Omicron SARS-CoV-2.

Currently, monoclonal antibodies (MAbs) targeting the SARS-CoV-2 receptor binding domain (RBD) of spike (S) protein are classified into seven classes based on their binding epitopes. However, most of these antibodies are seriously impaired by SARS-CoV-2 Omicron and its subvariants, especially the recent BQ.1.1, XBB and its derivatives. Identification of broadly neutralizing MAbs against currently circulating variants is imperative. In this study, we identified a "breathing" cryptic epitope in the S protein, named as RBD-8. Two human MAbs, BIOLS56 and IMCAS74, were isolated recognizing this epitope with broad neutralization abilities against tested sarbecoviruses, including SARS-CoV, pangolin-origin coronaviruses, and all the SARS-CoV-2 variants tested (Omicron BA.4/BA.5, BQ.1.1, and XBB subvariants). Searching through the literature, some more RBD-8 MAbs were defined. More importantly, BIOLS56 rescues the immune-evaded antibody, RBD-5 MAb IMCAS-L4.65, by making a bispecific MAb, to neutralize BQ.1 and BQ.1.1, thereby producing an MAb to cover all the currently circulating Omicron subvariants. Structural analysis reveals that the neutralization effect of RBD-8 antibodies depends on the extent of epitope exposure, which is affected by the angle of antibody binding and the number of up-RBDs induced by angiotensin-converting enzyme 2 binding. This cryptic epitope which recognizes non- receptor binding motif (non-RBM) provides guidance for the development of universal therapeutic antibodies and vaccines against COVID-19.

A novel nanobody broadly neutralizes SARS‐CoV‐2 via induction of spike trimer dimers conformation

AbstractThe ongoing mutations of the SARS‐CoV‐2 pose serious challenges to the efficacy of the available antiviral drugs, and new drugs with fantastic efficacy are always deserved investigation. Here, a nanobody called IBT‐CoV144 is reported, which exhibits broad neutralizing activity against SARS‐CoV‐2 by inducing the conformation of spike trimer dimers. IBT‐CoV144 was isolated from an immunized alpaca using the RBD of wild‐type SARS‐CoV‐2, and it showed strong cross‐reactive binding and neutralizing potency against diverse SARS‐CoV‐2 variants, including Omicron subvariants. Moreover, the prophylactically and therapeutically intranasal administration of IBT‐CoV144 confers fantastic protective efficacy against the challenge of Omicron BA.1 variant in BALB/c mice model. The structure analysis of the complex between spike (S) protein, conducted using Cryo‐EM, revealed a special conformation known as the trimer dimers. This conformation is formed by two trimers, with six RBDs in the “up” state and bound by six VHHs. IBT‐CoV144 binds to the lateral region of the RBD on the S protein, facilitating the aggregation of S proteins. This aggregation results in steric hindrance, which disrupts the recognition of the virus by ACE2 on host cells. The discovery of IBT‐CoV144 will provide valuable insights for the development of advanced therapeutics and the design of next‐generation vaccines.

A Modified Recombinant DNA-Based SARS-CoV-2 Vaccine Expressing Stabilized Uncleavable Spike Protein Elicits Humoral and Cellular Immunity against Various SARS-CoV-2 Variants of Concern

The appearance of several variants of concern (VOCs) of SARS-CoV-2 affects the efficacy of currently available vaccines and causes continuous spread and reinfection between humans. These variants possess different spike (S) protein mutations, which could affect viral pathogenicity, transmission, and immune escape. Herein, we develop a synthetic codon-optimized DNA vaccine (VIU-1007) expressing full-length S protein. The developed vaccine is stabilized by two K986P and V987P proline substitutions and resistant to cleavage by proteases such as furin by deletion of arginine residues (R682, R683, and R685) in multibasic furin cleavage site (RRAR). Additionally, it carries K417N, E484K, N501Y, and D614G substitutions in the receptor binding domain (RBD) derived from the beta VOC. Following the validation and characterization of the in vitro S protein expression, the humoral and cellular immunogenicity of VIU-1007 was assessed in immunized Balb/c mice. While both regimens elicited a Th-1-biased immune response based on S1-specific binding IgG isotypes, three vaccine doses significantly enhanced IgG levels. Furthermore, CD4+ and CD8+ memory T cell responses in spleens and draining inguinal lymph nodes were significantly higher in mice received three doses of VIU-1007 when compared to those received two doses only. Importantly, sera from mice immunized with three doses showed broad neutralization breadth against several SARS-CoV-2 variants, including alpha, beta, gamma, delta, and omicron VOCs. Moreover, the sera showed limited neutralization capacity against SARS-CoV-1, Bat SARS-like coronavirus WIV1, and MERS-CoV. Together, while these data suggest the presence of common neutralizing-rich epitopes between SARS-CoV-2 variants and some other betacoronaviruses, the ongoing evolution of SARS-CoV-2 could result in escape from vaccine-induced immunity, which requires a continuous update of vaccines.

Novel S2 subunit-specific antibody with broad neutralizing activity against SARS-CoV-2 variants of concern.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), had a major impact on both the global health and economy. Numerous virus-neutralizing antibodies were developed against the S1 subunit of SARS-CoV-2 spike (S) protein to block viral binding to host cells and were authorized for control of the COVID-19 pandemic. However, frequent mutations in the S1 subunit of SARS-CoV-2 enabled the emergence of immune evasive variants. To address these challenges, broadly neutralizing antibodies targeting the relatively conserved S2 subunit and its epitopes have been investigated as antibody therapeutics and universal vaccines. We initiated this study by immunizing BALB/c mice with β-propiolactone-inactivated SARS-CoV-2 (IAV) to generate B-cell hybridomas. These hybridomas were subsequently screened using HEK293T cells expressing the S2-ECD domain. Hybridomas that produced anti-S2 antibodies were selected, and we conducted a comprehensive evaluation of the potential of these anti-S2 antibodies as antiviral agents and versatile tools for research and diagnostics. In this study, we present a novel S2-specific antibody, 4A5, isolated from BALB/c mice immunized with inactivated SARS-CoV-2. 4A5 exhibited specific affinity to SARS-CoV-2 S2 subunits compared with those of other β-CoVs. 4A5 bound to epitope segment F1109-V1133 between the heptad-repeat1 (HR1) and the stem-helix (SH) region. The 4A5 epitope is highly conserved in SARS-CoV-2 variants, with a significant conformational feature in both pre- and postfusion S proteins. Notably, 4A5 exhibited broad neutralizing activity against variants and triggered Fc-enhanced antibody-dependent cellular phagocytosis. These findings offer a promising avenue for novel antibody therapeutics and insights for next-generation vaccine design. The identification of 4A5, with its unique binding properties and broad neutralizing capacity, offers a potential solution to the challenge posed by SARS-CoV-2 variants and highlights the importance of targeting the conserved S2 subunit in combating the COVID-19.

A lung-selective delivery of mRNA encoding broadly neutralizing antibody against SARS-CoV-2 infection

The respiratory system, especially the lung, is the key site of pathological injury induced by SARS-CoV-2 infection. Given the low feasibility of targeted delivery of antibodies into the lungs by intravenous administration and the short half-life period of antibodies in the lungs by intranasal or aerosolized immunization, mRNA encoding broadly neutralizing antibodies with lung-targeting capability can perfectly provide high-titer antibodies in lungs to prevent the SARS-CoV-2 infection. Here, we firstly identify a human monoclonal antibody, 8-9D, with broad neutralizing potency against SARS-CoV-2 variants. The neutralization mechanism of this antibody is explained by the structural characteristics of 8-9D Fabs in complex with the Omicron BA.5 spike. In addition, we evaluate the efficacy of 8-9D using a safe and robust mRNA delivery platform and compare the performance of 8-9D when its mRNA is and is not selectively delivered to the lungs. The lung-selective delivery of the 8-9D mRNA enables the expression of neutralizing antibodies in the lungs which blocks the invasion of the virus, thus effectively protecting female K18-hACE2 transgenic mice from challenge with the Beta or Omicron BA.1 variant. Our work underscores the potential application of lung-selective mRNA antibodies in the prevention and treatment of infections caused by circulating SARS-CoV-2 variants.

Breakthrough infection elicits hypermutated IGHV3-53/3-66 public antibodies with broad and potent neutralizing activity against SARS-CoV-2 variants including the emerging EG.5 lineages.

The rapid emergence of SARS-CoV-2 variants of concern (VOCs) calls for efforts to study broadly neutralizing antibodies elicited by infection or vaccination so as to inform the development of vaccines and antibody therapeutics with broad protection. Here, we identified two convalescents of breakthrough infection with relatively high neutralizing titers against all tested viruses. Among 50 spike-specific monoclonal antibodies (mAbs) cloned from their B cells, the top 6 neutralizing mAbs (KXD01-06) belong to previously defined IGHV3-53/3-66 public antibodies. Although most antibodies in this class are dramatically escaped by VOCs, KXD01-06 all exhibit broad neutralizing capacity, particularly KXD01-03, which neutralize SARS-CoV-2 from prototype to the emerging EG.5.1 and FL.1.5.1. Deep mutational scanning reveals that KXD01-06 can be escaped by current and prospective variants with mutations on D420, Y421, L455, F456, N460, A475 and N487. Genetic and functional analysis further indicates that the extent of somatic hypermutation is critical for the breadth of KXD01-06 and other IGHV3-53/3-66 public antibodies. Overall, the prevalence of broadly neutralizing IGHV3-53/3-66 public antibodies in these two convalescents provides rationale for novel vaccines based on this class of antibodies. Meanwhile, KXD01-06 can be developed as candidates of therapeutics against SARS-CoV-2 through further affinity maturation.

Development of a neutralization monoclonal antibody with a broad neutralizing effect against SARS-CoV-2 variants

BackgroundThe emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has challenged the effectiveness of current therapeutic regimens. Here, we aimed to develop a potent SARS-CoV-2 antibody with broad neutralizing effect by screening a scFv library with the spike protein receptor-binding domain (RBD) via phage display.MethodsSKAI-DS84 was identified through phage display, and we performed pseudovirus neutralization assays, authentic virus neutralization assays, and in vivo neutralization efficacy evaluations. Furthermore, surface plasmon resonance (SPR) analysis was conducted to assess the physical characteristics of the antibody, including binding kinetics and measure its affinity for variant RBDs.ResultsThe selected clones were converted to human IgG, and among them, SKAI-DS84 was selected for further analyses based on its binding affinity with the variant RBDs. Using pseudoviruses, we confirmed that SKAI-DS84 was strongly neutralizing against wild-type, B.1.617.2, B.1.1.529, and subvariants of SARS-CoV-2. We also tested the neutralizing effect of SKAI-DS84 on authentic viruses, in vivo and observed a reduction in viral replication and improved lung pathology. We performed binding and epitope mapping experiments to understand the mechanisms underlying neutralization and identified quaternary epitopes formed by the interaction between RBDs as the target of SKAI-DS84.ConclusionsWe identified, produced, and tested the neutralizing effect of SKAI-DS84 antibody. Our results highlight that SKAI-DS84 could be a potential neutralizing antibody against SARS-CoV-2 and its variants.