A potent pan-sarbecovirus neutralizing antibody resilient to epitope diversification.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution has resulted in viral escape from clinically authorized monoclonal antibodies (mAbs), creating a need for mAbs that are resilient to epitope diversification. Broadly neutralizing coronavirus mAbs that are sufficiently potent for clinical development and retain activity despite viral evolution remain elusive. We identified a human mAb, designated VIR-7229, which targets the viral receptor-binding motif (RBM) with unprecedented cross-reactivity to all sarbecovirus clades, including non-ACE2-utilizing bat sarbecoviruses, while potently neutralizing SARS-CoV-2 variants since 2019, including the recent EG.5, BA.2.86, and JN.1. VIR-7229 tolerates extraordinary epitope variability, partly attributed to its high binding affinity, receptor molecular mimicry, and interactions with RBM backbone atoms. Consequently, VIR-7229 features a high barrier for selection of escape mutants, which are rare and associated with reduced viral fitness, underscoring its potential to be resilient to future viral evolution. VIR-7229 is a strong candidate to become a next-generation medicine.

PET/CT Targeted Tissue Sampling Reveals Intravenously Administered HGN194 IgG1 Affects HIV Distribution after Rectal Exposure.

Neutralizing monoclonal antibodies hold great potential for prevention of human immunodeficiency virus (HIV) acquisition. IgG is the most abundant antibody in human serum, has a long half-life, and potent effector functions, making it a prime candidate for an HIV prevention therapeutic. We combined Positron Emission Tomography imaging and fluorescent microscopy of 64Cu-labeled, photoactivatable-green fluorescent protein HIV (PA-GFP-BaL) and fluorescently labeled HGN194 IgG1 to determine whether intravenously instilled IgG influences viral interaction with mucosal barriers and viral penetration in colorectal tissue 2 h after rectal viral challenge. Our results show that IgG1 did not alter the number of virions found throughout the colon or viral penetration into the epithelium of the rectum or descending colon. A minor increase in virions was observed in the transverse colon of IgG1 treated animals. Overall, the number of viral particles found in the mesenteric lymph nodes was low. However, IgG1 administration resulted in a significant reduction of virions found in mesenteric lymph nodes. Taken together, our results show that HGN194 IgG1 does not prevent virions from penetrating into the colorectal mucosa but may perturb HIV virion access to the lymphatic system.

Site-specific serology unveils cross-reactive monoclonal antibodies targeting influenza A hemagglutinin epitopes.

Efficient identification of human monoclonal antibodies targeting specific antigenic sites is pivotal for advancing vaccines and immunotherapies against infectious diseases and cancer. Existing screening techniques, however, limit our ability to discover monoclonal antibodies with desired specificity. In this study, we introduce a novel method, blocking of binding (BoB) enzyme-linked immunoassay (ELISA), enabling the detection of high-avidity human antibodies directed to defined epitopes. Leveraging BoB-ELISA, we analyzed the antibody response to known epitopes of influenza A hemagglutinin (HA) in the serum of vaccinated donors. Our findings revealed that serum antibodies targeting head epitopes were immunodominant, whereas antibodies against the stem epitope, although subdominant, were highly prevalent. Extending our analysis across multiple HA strains, we examined the cross-reactive antibody response targeting the stem epitope. Importantly, employing BoB-ELISA we identified donors harboring potent heterosubtypic antibodies targeting the HA stem. B-cell clonal analysis of these donors revealed three novel, genealogically independent monoclonal antibodies with broad cross-reactivity to multiple HAs. In summary, we demonstrated that BoB-ELISA is a sensitive technique for measuring B-cell epitope immunogenicity, enabling the identification of novel monoclonal antibodies with implications for enhanced vaccine development and immunotherapies.

A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

Antibodies Produced by CLL Phenotype B Cells in Patients With Myasthenia Gravis Are Not Directed Against Neuromuscular Endplates.

BACKGROUND AND OBJECTIVES: Myasthenia gravis (MG) can in rare cases be an autoimmune phenomenon associated with hematologic malignancies such as chronic lymphocytic leukemia (CLL). It is unclear whether in patients with MG and CLL, the leukemic B cells are the ones directly driving the autoimmune response against neuromuscular endplates. METHODS: We identified patients with acetylcholine receptor antibody-positive (AChR+) MG and CLL or monoclonal B-cell lymphocytosis (MBL), a precursor to CLL, and described their clinical features, including treatment responses. We generated recombinant monoclonal antibodies (mAbs) corresponding to the B-cell receptors of the CLL phenotype B cells and screened them for autoantigen binding. RESULTS: A computational immune cell screen revealed a subgroup of 5/38 patients with MG and 0/21 healthy controls who displayed a CLL-like B-cell phenotype. In follow-up hematologic flow cytometry, 2 of these 5 patients were diagnosed with an MBL. An additional patient with AChR+ MG as a complication of manifest CLL presented at our neuromuscular clinic and was successfully treated with the anti-CD20 therapy obinutuzumab plus chlorambucil. We investigated the specificities of expanding CLL-like B-cell clones to assess a direct causal link between the 2 diseases. However, we observed no reactivity of the clones against the AChR, antigens at the neuromuscular junction, or other common autoantigens. DISCUSSION: Our study suggests that AChR autoantibodies are produced by nonmalignant, polyclonal B cells The new anti-CD20 treatment obinutuzumab might be considered in effectively treating AChR+ MG. CLASSIFICATION OF EVIDENCE: This is a single case study and provides Class IV evidence that obinutuzumab is safe to use in patients with MG.

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

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 time frame. 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 prefusion 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 sublineages, 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.

Clonal composition and persistence of antigen-specific circulating T follicular helper cells.

T follicular helper (TFH ) cells play an essential role in promoting B cell responses and antibody affinity maturation in germinal centers (GC). A subset of memory CD4+ T cells expressing the chemokine receptor CXCR5 has been described in human blood as phenotypically and clonally related to GC TFH cells. However, the antigen specificity and relationship of these circulating TFH (cTFH ) cells with other memory CD4+ T cells remain poorly defined. Combining antigenic stimulation and T cell receptor (TCR) Vß sequencing, we found T cells specific to tetanus toxoid (TT), influenza vaccine (Flu), or Candida albicans (C.alb) in both cTFH and non-cTFH subsets, although with different frequencies and effector functions. Interestingly, cTFH and non-cTFH cells specific for C.alb or TT had a largely overlapping TCR Vß repertoire while the repertoire of Flu-specific cTFH and non-cTFH cells was distinct. Furthermore, Flu-specific but not C.alb-specific PD-1+ cTFH cells had a "GC TFH -like" phenotype, with overexpression of IL21, CXCL13, and BCL6. Longitudinal analysis of serial blood donations showed that Flu-specific cTFH and non-cTFH cells persisted as stable repertoires for years. Collectively, our study provides insights on the relationship of cTFH with non-cTFH cells and on the heterogeneity and persistence of antigen-specific human cTFH cells.

Imprinted antibody responses against SARS-CoV-2 Omicron sublineages.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages carry distinct spike mutations resulting in escape from antibodies induced by previous infection or vaccination. We show that hybrid immunity or vaccine boosters elicit plasma-neutralizing antibodies against Omicron BA.1, BA.2, BA.2.12.1, and BA.4/5, and that breakthrough infections, but not vaccination alone, induce neutralizing antibodies 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, BA.2, and BA.4/5 receptor-binding domains, whereas Omicron primary infections elicit B cells of narrow specificity up to 6 months after infection. Although most clinical antibodies have reduced neutralization of Omicron, we identified an ultrapotent pan-variant-neutralizing antibody that is a strong candidate for clinical development.

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

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.

Different classes of genomic inserts contribute to human antibody diversity.

Recombination of antibody genes in B cells can involve distant genomic loci and contribute a foreign antigen-binding element to form hybrid antibodies with broad reactivity for Plasmodium falciparum. So far, antibodies containing the extracellular domain of the LAIR1 and LILRB1 receptors represent unique examples of cross-chromosomal antibody diversification. Here, we devise a technique to profile non-VDJ elements from distant genes in antibody transcripts. Independent of the preexposure of donors to malaria parasites, non-VDJ inserts were detected in 80% of individuals at frequencies of 1 in 104 to 105 B cells. We detected insertions in heavy, but not in light chain or T cell receptor transcripts. We classify the insertions into four types depending on the insert origin and destination: 1) mitochondrial and 2) nuclear DNA inserts integrated at VDJ junctions; 3) inserts originating from telomere proximal genes; and 4) fragile sites incorporated between J-to-constant junctions. The latter class of inserts was exclusively found in memory and in in vitro activated B cells, while all other classes were already detected in naïve B cells. More than 10% of inserts preserved the reading frame, including transcripts with signs of antigen-driven affinity maturation. Collectively, our study unravels a mechanism of antibody diversification that is layered on the classical V(D)J and switch recombination.

ACE2-binding exposes the SARS-CoV-2 fusion peptide to broadly neutralizing coronavirus antibodies.

The coronavirus spike glycoprotein attaches to host receptors and mediates viral fusion. Using a broad screening approach, we isolated seven monoclonal antibodies (mAbs) that bind to all human-infecting coronavirus spike proteins from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune donors. These mAbs recognize the fusion peptide and acquire affinity and breadth through somatic mutations. Despite targeting a conserved motif, only some mAbs show broad neutralizing activity in vitro against alpha- and betacoronaviruses, including animal coronaviruses WIV-1 and PDF-2180. Two selected mAbs also neutralize Omicron BA.1 and BA.2 authentic viruses and reduce viral burden and pathology in vivo. Structural and functional analyses showed that the fusion peptide-specific mAbs bound with different modalities to a cryptic epitope hidden in prefusion stabilized spike, which became exposed upon binding of angiotensin-converting enzyme 2 (ACE2) or ACE2-mimicking mAbs.

Clonal structure, stability and dynamics of human memory B cells and circulating plasmablasts.

Memory B cells persist for a lifetime and rapidly differentiate into antibody-producing plasmablasts and plasma cells upon antigen re-encounter. The clonal relationship and evolution of memory B cells and circulating plasmablasts is not well understood. Using single-cell sequencing combined with isolation of specific antibodies, we found that in two healthy donors, the memory B cell repertoire was dominated by large IgM, IgA and IgG2 clonal families, whereas IgG1 families, including those specific for recall antigens, were of small size. Analysis of multiyear samples demonstrated stability of memory B cell clonal families and revealed that a large fraction of recently generated plasmablasts was derived from long-term memory B cell families and was found recurrently. Collectively, this study provides a systematic description of the structure, stability and dynamics of the human memory B cell pool and suggests that memory B cells may be active at any time point in the generation of plasmablasts.

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

The isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. We determined the structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and showed that its domain 0 recognizes sialosides. We identified that the CCoV-HuPn-2018 spike binds canine, feline, and porcine aminopeptidase N (APN) orthologs, which serve as entry receptors, and determined the structure of the receptor-binding B domain in complex with canine APN. The 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 might account for viral detection in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 spike-mediated entry, underscoring the cross-neutralizing activity among ɑ-coronaviruses. These data pave the way for vaccine and therapeutic development targeting this zoonotic pathogen representing the eighth human-infecting coronavirus.

Imprinted antibody responses against SARS-CoV-2 Omicron sublineages.

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.

Poor neutralization and rapid decay of antibodies to SARS-CoV-2 variants in vaccinated dialysis patients.

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 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-naïve hemodialysis patients immunized with BNT162b2 (66%) than those immunized with mRNA-1273 (23%). The reduced neutralizing activity correlated with low antibody avidity. Patients followed up to 7 months after vaccination showed a rapid decay of the antibody response with an average 21- and 10-fold reduction of neutralizing antibodies to vaccine-matched SARS-CoV-2 and Delta variant, which increased the fraction of non-responders to 84% and 90%, respectively. These data indicate that dialysis patients should be prioritized for additional vaccination boosts. Nevertheless, their antibody response to SARS-CoV-2 must be continuously monitored to adopt the best prophylactic and therapeutic strategy.

Exceptionally potent human monoclonal antibodies are effective for prophylaxis and treatment of tetanus in mice.

We used human monoclonal antibodies (humAbs) to study the mechanism of neuron intoxication by tetanus neurotoxin and to evaluate these antibodies as a safe preventive and therapeutic substitute for hyperimmune sera to treat tetanus in mice. By screening memory B cells from immune donors, we selected 2 tetanus neurotoxin-specific mAbs with exceptionally high neutralizing activities and extensively characterized them both structurally and functionally. We found that these antibodies interfered with the binding and translocation of the neurotoxin into neurons by interacting with 2 epitopes, whose identification pinpoints crucial events in the cellular pathogenesis of tetanus. Our observations explain the neutralization ability of these antibodies, which we found to be exceptionally potent in preventing experimental tetanus when injected into mice long before the toxin. Moreover, their Fab derivatives neutralized tetanus neurotoxin in post-exposure experiments, suggesting their potential for therapeutic use via intrathecal injection. As such, we believe these humAbs, as well as their Fab derivatives, meet the requirements to be considered for prophylactic and therapeutic use in human tetanus and are ready for clinical trials.

Interprotomer disulfide-stabilized variants of the human metapneumovirus fusion glycoprotein induce high titer-neutralizing responses.

Human metapneumovirus (HMPV) is a major cause of respiratory disease worldwide, particularly among children and the elderly. Although there is no licensed HMPV vaccine, promising candidates have been identified for related pneumoviruses based on the structure-based stabilization of the fusion (F) glycoprotein trimer, with prefusion-stabilized F glycoprotein trimers eliciting significantly higher neutralizing responses than their postfusion F counterparts. However, immunization with HMPV F trimers in either prefusion or postfusion conformations has been reported to elicit equivalent neutralization responses. Here we investigate the impact of stabilizing disulfides, especially interprotomer disulfides (IP-DSs) linking protomers of the F trimer, on the elicitation of HMPV-neutralizing responses. We designed F trimer disulfides, screened for their expression, and used electron microscopy (EM) to confirm their formation, including that of an unexpected postfusion variant. In mice, IP-DS-stabilized prefusion and postfusion HMPV F elicited significantly higher neutralizing responses than non-IP-DS-stabilized HMPV Fs. In macaques, the impact of IP-DS stabilization was more measured, although IP-DS-stabilized variants of either prefusion or postfusion HMPV F induced neutralizing responses many times the average titers observed in a healthy human cohort. Serological and absorption-based analyses of macaque responses revealed elicited HMPV-neutralizing responses to be absorbed differently by IP-DS-containing and by non-IP-DS-containing postfusion Fs, suggesting IP-DS stabilization to alter not only the immunogenicity of select epitopes but their antigenicity as well. We speculate the observed increase in immunogenicity by IP-DS trimers to be related to reduced interprotomer flexibility within the HMPV F trimer.