Broad Neutralizing Activity Research Articles

Engineering a SARS-CoV-2 Vaccine Targeting the Receptor-Binding Domain Cryptic-Face via Immunofocusing.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is the main target of neutralizing antibodies. Although they are infrequently elicited during infection or vaccination, antibodies that bind to the conformation-specific cryptic face of the RBD display remarkable breadth of binding and neutralization across Sarbecoviruses. Here, we employed the immunofocusing technique PMD (protect, modify, deprotect) to create RBD immunogens (PMD-RBD) specifically designed to focus the antibody response toward the cryptic-face epitope recognized by the broadly neutralizing antibody S2X259. Immunization with PMD-RBD antigens induced robust binding titers and broad neutralizing activity against homologous and heterologous Sarbecovirus strains. A serum-depletion assay provided direct evidence that PMD successfully skewed the polyclonal antibody response toward the cryptic face of the RBD. Our work demonstrates the ability of PMD to overcome immunodominance and refocus humoral immunity, with implications for the development of broader and more resilient vaccines against current and emerging viruses with pandemic potential.

Booster vaccination using bivalent DS-5670a/b is safe and immunogenic against SARS-CoV-2 variants in children aged 5-11 years: a phase 2/3, randomized, active-controlled study.

DS-5670 is a messenger ribonucleic acid (mRNA) vaccine platform targeting the receptor-binding domain (RBD) of the spike protein derived from severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Booster vaccination against coronavirus disease 2019 (COVID-19) with monovalent DS-5670a (incorporating mRNA encoding the RBD from the original SARS-CoV-2 strain) or bivalent DS-5670a/b (original and omicron BA.4-5 RBD antigens) is effective and safe in adults. Data from a phase 2/3 active-controlled, non-inferiority, pediatric study evaluating a third booster dose of DS-5670a/b are reported here. Children aged 5-11 years who had completed the two-dose primary vaccination series with monovalent BNT162b2 (original strain) at least 3 months prior to enrolment were randomly assigned to receive DS-5670a/b (20 µg of mRNA) or bivalent BNT1 62b2 (original/omicron BA.4-5; 10 µg of mRNA) on Day 1. The primary efficacy endpoint was blood neutralization geometric mean titer (GMT) against SARS-CoV-2 (omicron variant BA.5.2.1) and immune response rate (≥ 4-fold increase in post-vaccination circulating anti-SARS-CoV-2 neutralizing activity) on Day 29. Among evaluable participants (DS-5670a/b, n = 74; bivalent BNT162b2, n = 75), the adjusted GMT ratio of DS-5670a/b to bivalent BNT162b2 on Day 29 was 1.636 (95% CI, 1.221, 2.190). Immune response rates were ≥ 89% with both study vaccines; adjusted difference 2.6% (95% CI, -7.8, 13.8). The prespecified non-inferiority margins were exceeded, and the study met the primary endpoint. DS-5670a/b also demonstrated broad neutralization activity across recent omicron sublineages and no cases of COVID-19 between Days 8-29 post-administration were reported. There were no novel safety concerns in the pediatric population at data cut-off. Bivalent DS-5670a/b was non-inferior to bivalent BNT162b2 in terms of immunogenicity, and had a manageable safety profile, when administered as a heterologous booster in children aged 5-11 years. https://jrct.niph.go.jp/, identifier jRCT2031220665.

Evaluation of SARS-CoV-2-Specific IgY Antibodies: Production, Reactivity, and Neutralizing Capability against Virus Variants.

The emergence of SARS-CoV-2 in late 2019 initiated a global pandemic, which led to a need for effective therapeutics and diagnostic tools, including virus-specific antibodies. Here, we investigate different antigen preparations to produce SARS-CoV-2-specific and virus-neutralizing antibodies in chickens (n = 3/antigen) and rabbits (n = 2/antigen), exploring, in particular, egg yolk for large-scale production of immunoglobulin Y (IgY). Reactivity profiles of IgY preparations from chicken sera and yolk and rabbit sera were tested in parallel. We compared three types of antigens based on ancestral SARS-CoV-2: an inactivated whole-virus preparation, an S1 spike-protein subunit (S1 antigen) and a receptor-binding domain (RBD antigen, amino acids 319-519) coated on lumazine synthase (LS) particles using SpyCather/SpyTag technology. The RBD antigen proved to be the most efficient immunogen, and the resulting chicken IgY antibodies derived from serum or yolk, displayed strong reactivity with ELISA and indirect immunofluorescence and broad neutralizing activity against SARS-CoV-2 variants, including Omicron BA.1 and BA.5. Preliminary in vivo studies using RBD-lumazine synthase yolk preparations in a hamster model showed that local application was well tolerated and not harmful. However, despite the in vitro neutralizing capacity, this antibody preparation did not show protective effect. Further studies on galenic properties seem to be necessary. The RBD-lumazine antigen proved to be suitable for producing SARS-CoV-2 specific antibodies that can be applied to such therapeutic approaches and as reference reagents for SARS-CoV-2 diagnostics, including virus neutralization assays.

Development and evaluation of vaccination strategies for addressing the continuous evolution SARS-CoV-2 based on recombinant trimeric protein technology: Potential for cross-neutralizing activity and broad coronavirus response

Given the significant decline in vaccine efficacy against Omicron, the development of novel vaccines with specific or broad-spectrum effectiveness is paramount. In this study, we formulated four monovalent vaccines based on recombinant spike trimer proteins, along with three bivalent vaccines, and five monovalent vaccines based on recombinant spike proteins. We evaluated the efficacy of different vaccination regimens in eliciting neutralizing antibodies in mice through pseudovirus neutralization assays. Following two doses of primary immunization with D614G, mice received subsequent immunizations with Omicron (BA.1, BA.2, BA.4/5) boosters individually, which led to the generation of broader and more potent cross-neutralizing activity compared to D614G boosters. Notably, the BA.4/5 booster exhibited superior efficacy. Following two doses of primary immunization with Omicron (BA.1, BA.2, BA.4/5), mice were subsequently immunized with one dose of D614G booster which resulted in broader neutralizing activity compared to one dose of Omicron (BA.1, BA.2, or BA.4/5). In unvaccinated mice, full-course immunization with different bivalent vaccines induced broad neutralizing activity against Omicron and pre-Omicron variants, with D614G&BA.4/5 demonstrating superior efficacy. However, compared to other variants, the neutralizing activity against XBB.1.5/1.9.1 is notably reduced. This observation emphasizes the necessity of timely updates to the vaccine antigen composition. Based on these findings and existing studies, we propose a vaccination strategy aimed at preserving the epitope repertoire to its maximum potential: (1) Individuals previously vaccinated or infected with pre-Omicron variants should inoculate a monovalent vaccine containing Omicron components; (2) Individuals who have only been vaccinated or infected with Omicron should be inoculated a monovalent vaccine containing pre-Omicron variants components; (3) Individuals without SARS-CoV-2 infection and vaccination should inoculate a bivalent vaccine comprising both pre-Omicron and Omicron components for primary immunization. Additionally, through cross-inoculation of SARS-CoV-2 D614G spike trimer protein and SARS-CoV-1 spike protein in mice, we preliminarily demonstrated the possibility of cross-reaction between different coronavirus vaccines to produce resistance to the pan-coronavirus.

Engineering a SARS-CoV-2 vaccine targeting the RBD cryptic-face via immunofocusing.

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is the main target of neutralizing antibodies. Although they are infrequently elicited during infection or vaccination, antibodies that bind to the conformation-specific cryptic face of the RBD display remarkable breadth of binding and neutralization across Sarbecoviruses. Here, we employed the immunofocusing technique PMD (protect, modify, deprotect) to create RBD immunogens (PMD-RBD) specifically designed to focus the antibody response towards the cryptic-face epitope recognized by the broadly neutralizing antibody S2X259. Immunization with PMD-RBD antigens induced robust binding titers and broad neutralizing activity against homologous and heterologous Sarbecovirus strains. A serum-depletion assay provided direct evidence that PMD successfully skewed the polyclonal antibody response towards the cryptic face of the RBD. Our work demonstrates the ability of PMD to overcome immunodominance and refocus humoral immunity, with implications for the development of broader and more resilient vaccines against current and emerging viruses with pandemic potential.

Proteomics Platform Reveals Broad-Spectrum Nanobodies for SARS-CoV-2 Variant Neutralization.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the emergence of different variants of concerns with immune evasion that have been prevalent over the past three years. Nanobodies, the functional variable regions of camelid heavy-chain-only antibodies, have garnered interest in developing neutralizing antibodies due to their smaller size, structural stability, ease of production, high affinity, and low immunogenicity, among other characteristics. In this work, we describe an integrated proteomics platform for the high-throughput screening of nanobodies against different SARS-CoV-2 spike variants. To demonstrate this platform, we immunized a camel with subunit 1 (S1) of the wild-type spike protein and constructed a nanobody phage library. The binding and neutralizing activities of the nanobodies against 72 spike variants were then measured, resulting in the identification of two nanobodies (C-282 and C-39) with broad neutralizing activity against six non-Omicron variants (D614G, Alpha, Beta, Gamma, Delta, Kappa) and five Omicron variants (BA.1-5). Their neutralizing capability was validated using in vitro pseudovirus-based neutralization assays. All these results demonstrate the utility of our proteomics platform to identify new nanobodies with broad neutralizing capability and to develop a treatment for patients with SARS-CoV-2 variant infection in the future.

Balanced on the Biggest Wave: Nirsevimab for Newborns.

Respiratory syncytial virus (RSV) is the leading cause of hospitalization in infancy in the United States. Nearly all infants are infected by 2 years of age, with bronchiolitis requiring hospitalization often occurring in previously healthy children and long-term consequences of severe disease including delayed speech development and asthma. Incomplete passage of maternal immunity and a high degree of genetic variability within the virus contribute to morbidity and have also prevented successful neonatal vaccine development. Monoclonal antibodies reduce the risk of hospitalization from severe RSV disease, with palivizumab protecting high-risk newborns with comorbidities including chronic lung disease and congenital heart disease. Unfortunately, palivizumab is costly and requires monthly administration of up to five doses during the RSV season for optimal protection.Rapid advances in the past two decades have facilitated the identification of antibodies with broad neutralizing activity and allowed manipulation of their genetic code to extend half-life. These advances have culminated with nirsevimab, a monoclonal antibody targeting the Ø antigenic site on the RSV prefusion protein and protecting infants from severe disease for an entire 5-month season with a single dose. Four landmark randomized controlled trials, the first published in July 2020, have documented the efficacy and safety of nirsevimab in healthy late-preterm and term infants, healthy preterm infants, and high-risk preterm infants and those with congenital heart disease. Nirsevimab reduces the risk of RSV disease requiring medical attention (number needed to treat [NNT] 14-24) and hospitalization (NNT 33-63) with rare mild rash and injection site reactions. Consequently, the Centers for Disease Control and Prevention has recently recommended nirsevimab for all infants younger than 8 months of age entering or born during the RSV season and high-risk infants 8-19 months of age entering their second season. Implementing this novel therapy in this large population will require close multidisciplinary collaboration. Equitable distribution through minimizing barriers and maximizing uptake must be prioritized.

A VHH single-domain platform enabling discovery and development of monospecific antibodies and modular neutralizing bispecifics against SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, escape coronavirus disease 2019 therapeutics and vaccines, and jeopardize public health. To combat SARS-CoV-2 antigenic escape, we developed a rapid, high-throughput pipeline to discover monospecific VHH antibodies and iteratively develop VHH-Fc-VHH bispecifics capable of neutralizing emerging SARS-CoV-2 variants. By panning VHH single-domain phage libraries against ancestral or beta spike proteins, we discovered high-affinity VHH antibodies with unique target epitopes. Combining two VHHs into a tetravalent bispecific construct conferred broad neutralization activity against multiple variants and was more resistant to antigenic escape than the monospecific antibody alone. Following the rise of the Omicron variant, a VHH in the original bispecific construct was replaced with another VHH discovered against the Omicron BA.1 receptor binding domain; the resulting bispecific exhibited neutralization against both BA.1 and BA.5 sublineage variants. A heavy chain-only tetravalent VHH-Fc-VHH bispecific platform derived from humanized synthetic libraries held a myriad of unique advantages: (i) synthetic preconstructed libraries minimized risk of liabilities and maximized discovery speed, (ii) VHH scaffolds allowed for a modular "plug-and-play" format that could be rapidly iterated upon as variants of concern arose, (iii) natural dimerization of single VHH-Fc-VHH polypeptides allowed for straightforward bispecific production and purification methods, and (iv) multivalent approaches enhanced avidity boosting effects and neutralization potency, and conferred more robust resistance to antigenic escape than monovalent approaches against specific variants. This iterative platform of rapid VHH discovery combined with modular bispecific design holds promise for long-term viral control efforts.

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.

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.

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.

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.

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.

Broad and potent neutralizing antibodies are elicited in vaccinated individuals following Delta/BA.1 breakthrough infection.

With the emergence of SARS-CoV-2 viral variants, there has been an increase in infections in vaccinated individuals. Here, we isolated monoclonal antibodies (mAbs) from individuals experiencing a breakthrough infection (Delta or BA.1) to determine how exposure to a heterologous Spike broadens the neutralizing antibody response at the monoclonal level. All mAbs isolated had reactivity to the Spike of the vaccine and infection variant. While many mAbs showed reduced neutralization of current circulating variants, we identified mAbs with broad and potent neutralization of BA.2.75.2, XBB, XBB.1.5, and BQ.1.1 indicating the presence of conserved epitopes on Spike. These results indicate that variant-based vaccine boosters have the potential to broaden the vaccine response.

Longitudinal kinetics of neutralizing antibodies against circulating SARS-CoV-2 variants and estimated level of group immunity of booster-vaccinated individuals during omicron-dominated COVID-19 outbreaks in the Republic of Korea, 2022.

The coronavirus disease 2019 pandemic persisted for 3 years and is now transitioning to endemicity. We illustrated the change in group immunity induced by vaccination (monovalent vaccines) and breakthrough infections (BIs) in a healthcare worker (HCW) cohort. Five sampling points were analyzed: before the third dose and 1, 3, 5, and 8 months after the vaccination. The last two points corresponded roughly to 1 and 4 months after omicron BA.1/BA.2 BI. A semi-quantitative anti-spike binding antibody (Sab) assay and plaque reduction neutralization test (PRNT) against circulating variants were conducted. A linear regression model was utilized to deduce correlation equations. Baseline characteristics and antibody titers after the third dose were not different between 106 HCWs with or without BI (54/52). One month after the third dose, BA.1 PRNT increased with wild-type (WT), but 3 months after the third dose, it decreased more rapidly than WT PRNT. After BI, BA.1 PRNT increased robustly and waned slower than WT. A linear equation of waning kinetics was deduced between log10Sab and months, and the slope became gradual after BI. The estimated BA.5 PRNT titers at the beginning of the BA.5 outbreak were significantly higher than the BA.1 PRNT titers of the initial BA.1/BA.2 wave, which might be associated with the smaller size of the BA.5 wave. BA.1/BA.2 BI after the third dose elicited robust and broad neutralizing activity, preferentially maintaining cross-neutralizing longevity against BA.1 and BA.5. The estimated kinetics provide an overview of group immunity through the third vaccination and BA.1/BA.2 BI, correlating with the actual outbreaks. IMPORTANCE This study analyzed changes in group immunity induced by coronavirus disease 2019 (COVID-19) vaccination and BA.1/BA.2 breakthrough infections (BIs) in a healthcare worker cohort. We investigated the longitudinal kinetics of neutralizing antibodies against circulating variants and confirmed that BA.1/BA.2 BIs enhance the magnitude and durability of cross-neutralization against BA.1 and BA.5. Correlation equations between semi-quantitative anti-spike antibody and plaque reduction neutralization test titers were deduced from the measured values using a linear regression model. Based on the equations, group immunity was estimated to last up to 11 months following the third dose of the COVID-19 vaccine. The estimated group immunity suggests that the augmented immunity and flattened waning slope through BI could correlate with the overall outbreak size. Our findings could provide a better understanding to establish public health strategies against future endemicity.

Spike protein 1-77: a potential “magic bullet” for coronavirus disease 2019? Single antibody with broad neutralizing activity against all known coronavirus disease 2019 variants—a commentary

Spike protein 1-77: a potential “magic bullet” for coronavirus disease 2019? Single antibody with broad neutralizing activity against all known coronavirus disease 2019 variants—a commentary

Multimodal, broadly neutralizing antibodies against SARS-CoV-2 identified by high-throughput native pairing of BCRs from bulk B cells

TruAB Discovery is an approach that integrates cellular immunology, high-throughput immunosequencing, bioinformatics, and computational biology in order to discover naturally occurring human antibodies for prophylactic or therapeutic use. We adapted our previously described pairSEQ technology to pair B cell receptor heavy and light chains of SARS-CoV-2 spike protein-binding antibodies derived from enriched antigen-specific memory B cells and bulk antibody-secreting cells. We identified approximately 60,000 productive, in-frame, paired antibody sequences, from which 2,093 antibodies were selected for functional evaluation based on abundance, isotype and patterns of somatic hypermutation. The exceptionally diverse antibodies included RBD-binders with broad neutralizing activity against SARS-CoV-2 variants, and S2-binders with broad specificity against betacoronaviruses and the ability to block membrane fusion. A subset of these RBD- and S2-binding antibodies demonstrated robust protection against challenge in hamster and mouse models. This high-throughput approach can accelerate discovery of diverse, multifunctional antibodies against any target of interest.

Broad-spectrum humanized monoclonal neutralizing antibody against SARS-CoV-2 variants, including the Omicron variant.

Therapeutic monoclonal antibodies (mAbs) against the SARS-CoV-2 spike protein have been shown to improve the outcome of severe COVID-19 patients in clinical trials. However, novel variants with spike protein mutations can render many currently available mAbs ineffective. We produced mAbs by using hybridoma cells that generated from mice immunized with spike protein trimer and receptor binding domain (RBD). The panel of mAbs were screened for binding and neutralizing activity against different SARS-CoV-2 variants. The in vivo effectiveness of WKS13 was evaluated in a hamster model. Out of 960 clones, we identified 18 mAbs that could bind spike protein. Ten of the mAbs could attach to RBD, among which five had neutralizing activity against the ancestral strain and could block the binding between the spike protein and human ACE2. One of these mAbs, WKS13, had broad neutralizing activity against all Variants of Concern (VOCs), including the Omicron variant. Both murine or humanized versions of WKS13 could reduce the lung viral load in hamsters infected with the Delta variant. Our data showed that broad-spectrum high potency mAbs can be produced from immunized mice, which can be used in humans after humanization of the Fc region. Our method represents a versatile and rapid strategy for generating therapeutic mAbs for upcoming novel variants.