SARS-CoV-2 BA.2.86 enters lung cells and evades neutralizing antibodies with high efficiency.

BA.2.86, a recently identified descendant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.2 sublineage, contains ∼35 mutations in the spike (S) protein and spreads in multiple countries. Here, we investigated whether the virus exhibits altered biological traits, focusing on S protein-driven viral entry. Employing pseudotyped particles, we show that BA.2.86, unlike other Omicron sublineages, enters Calu-3 lung cells with high efficiency and in a serine- but not cysteine-protease-dependent manner. Robust lung cell infection was confirmed with authentic BA.2.86, but the virus exhibited low specific infectivity. Further, BA.2.86 was highly resistant against all therapeutic antibodies tested, efficiently evading neutralization by antibodies induced by non-adapted vaccines. In contrast, BA.2.86 and the currently circulating EG.5.1 sublineage were appreciably neutralized by antibodies induced by the XBB.1.5-adapted vaccine. Collectively, BA.2.86 has regained a trait characteristic of early SARS-CoV-2 lineages, robust lung cell entry, and evades neutralizing antibodies. However, BA.2.86 exhibits low specific infectivity, which might limit transmissibility.

The Omicron variant is highly resistant against antibody-mediated neutralization: Implications for control of the COVID-19 pandemic.

The rapid spread of the SARS-CoV-2 Omicron variant suggests that the virus might become globally dominant. Further, the high number of mutations in the viral spike protein raised concerns that the virus might evade antibodies induced by infection or vaccination. Here, we report that the Omicron spike was resistant against most therapeutic antibodies but remained susceptible to inhibition by sotrovimab. Similarly, the Omicron spike evaded neutralization by antibodies from convalescent patients or individuals vaccinated with the BioNTech-Pfizer vaccine (BNT162b2) with 12- to 44-fold higher efficiency than the spike of the Delta variant. Neutralization of the Omicron spike by antibodies induced upon heterologous ChAdOx1 (Astra Zeneca-Oxford)/BNT162b2 vaccination or vaccination with three doses of BNT162b2 was more efficient, but the Omicron spike still evaded neutralization more efficiently than the Delta spike. These findings indicate that most therapeutic antibodies will be ineffective against the Omicron variant and that double immunization with BNT162b2 might not adequately protect against severe disease induced by this variant.

SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies.

The global spread of SARS-CoV-2/COVID-19 is devastating health systems and economies worldwide. Recombinant or vaccine-induced neutralizing antibodies are used to combat the COVID-19 pandemic. However, the recently emerged SARS-CoV-2 variants B.1.1.7 (UK), B.1.351 (South Africa), and P.1 (Brazil) harbor mutations in the viral spike (S) protein that may alter virus-host cell interactions and confer resistance to inhibitors and antibodies. Here, using pseudoparticles, we show that entry of all variants into human cells is susceptible to blockade by the entry inhibitors soluble ACE2, Camostat, EK-1, and EK-1-C4. In contrast, entry of the B.1.351 and P.1 variant was partially (Casirivimab) or fully (Bamlanivimab) resistant to antibodies used for COVID-19 treatment. Moreover, entry of these variants was less efficiently inhibited by plasma from convalescent COVID-19 patients and sera from BNT162b2-vaccinated individuals. These results suggest that SARS-CoV-2 may escape neutralizing antibody responses, which has important implications for efforts to contain the pandemic.

Immunizations with diverse sarbecovirus receptor-binding domains elicit SARS-CoV-2 neutralizing antibodies against a conserved site of vulnerability.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.

Genetic barrier to resistance: a critical parameter for efficacy of neutralizing monoclonal antibodies against SARS-CoV-2 in a nonhuman primate model.

The potency of antibody neutralization in cell culture has been used as the key criterion for selection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for clinical development. As other aspects may also influence the degree of protection in vivo, we compared the efficacy of two neutralizing monoclonal antibodies (TRES6 and 4C12) targeting different epitopes of the receptor binding domain (RBD) of SARS-CoV-2 in a prophylactic setting in rhesus monkeys. All four animals treated with TRES6 had reduced viral loads in the upper respiratory tract 2 days after naso-oropharyngeal challenge with the Alpha SARS-CoV-2 variant. Starting 2 days after challenge, mutations conferring resistance to TRES6 were dominant in two of the rhesus monkeys, with both animals failing to maintain reduced viral loads. Consistent with its lower serum neutralization titer at the day of challenge, prophylaxis with 4C12 tended to suppress viral load at day 2 less efficiently than TRES6. However, a week after challenge, mean viral loads in the lower respiratory tract in 4C12-treated animals were lower than in the TRES6 group and no mutations conferring resistance to 4C12 could be detected in viral isolates from nasal or throat swabs. Thus, genetic barrier to resistance seems to be a critical parameter for the efficacy of prophylaxis with monoclonal antibodies against SARS-CoV-2. Furthermore, comparison of antibody concentrations in respiratory secretions to those in serum shows reduced distribution of the 4C12 antibody into respiratory secretions and a delay in the appearance of antibodies in bronchoalveolar lavage fluid compared to their appearance in secretions of the upper respiratory tract.IMPORTANCEMonoclonal antibodies are a powerful tool for the prophylaxis and treatment of acute viral infections. Hence, they were one of the first therapeutic agents licensed for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Oftentimes, the main criterion for the selection of antibodies for clinical development is their potency of neutralization in cell culture. By comparing two antibodies targeting the Spike protein of SARS-CoV-2, we now observed that the antibody that neutralized SARS-CoV-2 more efficiently in cell culture suppressed viral load in challenged rhesus monkeys to a lesser extent. Extraordinary rapid emergence of mutants of the challenge virus, which had lost their sensitivity to the antibody, was identified as the major reason for the reduced efficacy of the antibody in rhesus monkeys. Therefore, the viral genetic barrier to resistance to antibodies also affects their efficacy.

Identification of TFG- and Autophagy-Regulated Proteins and Glycerophospholipids in B Cells.

Autophagy supervises the proteostasis and survival of B lymphocytic cells. Trk-fused gene (TFG) promotes autophagosome-lysosome flux in murine CH12 B cells, as well as their survival. Hence, quantitative proteomics of CH12tfgKO and WT B cells in combination with lysosomal inhibition should identify proteins that are prone to lysosomal degradation and contribute to autophagy and B cell survival. Lysosome inhibition via NH4Cl unexpectedly reduced a number of proteins but increased a large cluster of translational, ribosomal, and mitochondrial proteins, independent of TFG. Hence, we propose a role for lysosomes in ribophagy in B cells. TFG-regulated proteins include CD74, BCL10, or the immunoglobulin JCHAIN. Gene ontology (GO) analysis reveals that proteins regulated by TFG alone, or in concert with lysosomes, localize to mitochondria and membrane-bound organelles. Likewise, TFG regulates the abundance of metabolic enzymes, such as ALDOC and the fatty acid-activating enzyme ACOT9. To test consequently for a function of TFG in lipid metabolism, we performed shotgun lipidomics of glycerophospholipids. Total phosphatidylglycerol is more abundant in CH12tfgKO B cells. Several glycerophospholipid species with similar acyl side chains, such as 36:2 phosphatidylethanolamine and 36:2 phosphatidylinositol, show a dysequilibrium. We suggest a role for TFG in lipid homeostasis, mitochondrial functions, translation, and metabolism in B cells.

Single-cell resolution of plasma cell fate programming in health and disease.

Long considered a homogeneous population dedicated to antibody secretion, plasma cell phenotypic and functional heterogeneity is increasingly recognized. Plasma cells were first segregated based on their maturation level, but the complexity of this subset might well be underestimated by this simple dichotomy. Indeed, in the last decade new functions have been attributed to plasma cells including but not limited to cytokine secretion. However, a proper characterization of plasma cell heterogeneity has remained elusive partly due to technical issues and cellular features that are specific to this cell type. Cell intrinsic and cell extrinsic signals could be at the origin of this heterogeneity. Recent advances in technologies such as single cell RNA-seq, ATAC-seq, or ChIP-seq on low cell numbers helped to elucidate the fate decision in other cell lineages and similar approaches could be implemented to evaluate the heterogeneous fate of activated B cells in health and disease. Here, we summarized published work shedding some lights on the stimuli and genetic program shaping B-cell terminal differentiation at the single cell level in mice and men. We also discuss the fate and heterogeneity of plasma cells during immune responses, vaccination, and in the frame of human plasma cell disorders.

Augmented neutralization of SARS-CoV-2 Omicron variant by boost vaccination and monoclonal antibodies.

Effective vaccines and monoclonal antibodies have been developed against coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the appearance of virus variants with higher transmissibility and pathogenicity is a major concern because of their potential to escape vaccines and clinically approved SARS-CoV-2- antibodies. Here, we use flow cytometry-based binding and pseudotyped SARS-CoV-2 neutralization assays to determine the efficacy of boost immunization and therapeutic antibodies to neutralize the dominant Omicron variant. We provide compelling evidence that the third vaccination with BNT162b2 increases the amount of neutralizing serum antibodies against Delta and Omicron variants, albeit to a lower degree when compared to the parental Wuhan strain. Therefore, a third vaccination is warranted to increase titers of protective serum antibodies, especially in the case of the Omicron variant. We also found that most clinically approved and otherwise potent therapeutic antibodies against the Delta variant failed to recognize and neutralize the Omicron variant. In contrast, some antibodies under preclinical development potentially neutralized the Omicron variant. Our studies also support using a flow cytometry-based antibody binding assay to rapidly monitor therapeutic candidates and serum titers against emerging SARS-CoV-2 variants.

A pair of noncompeting neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model.

TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS-CoV-2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralize SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2-induced weight loss. The two clusters of potent noncompeting SARS-CoV-2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID-19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives.

miR-148a controls metabolic programming and survival of mature CD19-negative plasma cells in mice.

Long-lived antibody-secreting plasma cells are essential to establish humoral memory against pathogens. While a regulatory transcription factor network has been established in plasma cell differentiation, the regulatory role of miRNAs remains enigmatic. We have recently identified miR-148a as the most abundant miRNA in primary mouse and human plasma cells. To determine whether this plasma cell signature miRNA controls the in vivo development of B cells into long-lived plasma cells, we established mice with genomic, conditional, and inducible deletions of miR-148a. The analysis of miR-148a-deficient mice revealed reduced serum Ig, decreased numbers of newly formed plasmablasts and reduced CD19-negative, CD93-positive long-lived plasma cells. Transcriptome and metabolic analysis revealed an impaired glucose uptake, a reduced oxidative phosphorylation-based energy metabolism, and an altered abundance of homing receptors CXCR3 (increase) and CXCR4 (reduction) in miR-148a-deficient plasma cells. These findings support the role of miR-148a as a positive regulator of the maintenance of long-lived plasma cells.

A surrogate cell-based SARS-CoV-2 spike blocking assay.

To monitor infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and successful vaccination against coronavirus disease 2019 (COVID-19), the kinetics of neutralizing or blocking anti-SARS-CoV-2 antibody titers need to be assessed. Here, we report the development of a quick and inexpensive surrogate SARS-CoV-2 blocking assay (SUBA) using immobilized recombinant human angiotensin-converting enzyme 2 (hACE2) and human cells expressing the native form of surface SARS-CoV-2 spike protein. Spike protein-expressing cells bound to hACE2 in the absence or presence of blocking antibodies were quantified by measuring the optical density of cell-associated crystal violet in a spectrophotometer. The advantages are that SUBA is a fast and inexpensive assay, which does not require biosafety level 2- or 3-approved laboratories. Most importantly, SUBA detects blocking antibodies against the native trimeric cell-bound SARS-CoV-2 spike protein and can be rapidly adjusted to quickly pre-screen already approved therapeutic antibodies or sera from vaccinated individuals for their ACE2 blocking activities against any emerging SARS-CoV-2 variants.

Eosinophils are not essential for maintenance of murine plasma cells in the bone marrow.

Eosinophils were reported to serve as an essential component of the plasma cell niche within the bone marrow. As the potential contribution of eosinophils to humoral immunity has remained incompletely understood, we aimed to further characterize their role during antibody responses and to additionally investigate their role in autoimmune disease. Contrary to our expectations and the currently prevailing paradigm, we found that eosinophils are fully dispensable for the survival of murine bone marrow plasma cells and accordingly do not contribute to antibody production and autoantibody-mediated disease. Littermate wild type and eosinophil-deficient ΔdblGATA-1 animals showed similar numbers and frequencies of plasma cells and did not differ in steady state levels of immunoglobulins or their ability to raise antigen-specific antibody responses. Eosinophils were likewise dispensable for autoantibody production or autoantibody-induced disease in a mouse model of systemic lupus erythematosus. Our findings thus argue against a role of eosinophils during the maintenance of the plasma cell pool and challenge the hitherto postulated concept of an eosinophil-sustained bone marrow niche.

miRNA meets plasma cells "How tiny RNAs control antibody responses".

We review the importance of small non-coding microRNAs for the generation of germinal center B cells and their differentiation in antibody-secreting plasma cells. In the last part, we briefly elucidate the role of microRNAs in some plasma cell disorders.

The role of the miR-148/-152 family in physiology and disease.

MicroRNAs (miRNAs) are endogenously encoded ∼22 nt small non-coding RNAs. They function as key players of many cellular processes by base pairing with target mRNAs and thereby impairing gene expression at the post-transcriptional level. Recent findings demonstrate a critical role of many miRNAs in immune cell differentiation and immune responses, which is also associated with the development and progression of many tumor and non-tumor diseases. Here we review the multifaceted miRNA-148/-152 family members consisting of miR-148a, miR-148b and miR-152. Next to regulation mechanisms that control the expression of this miRNA family, we will focus on (i) the role of miR-148a in regulating B and T lymphocyte function and its role in associated diseases and (ii) the importance of miR-148/-152 family members for cancer initiation, tumor growth and metastasis formation. In addition, this review aims to highlight some selected targets of the miRNA-148/-152 family members, which are involved in the biology of cancer and maintenance of epigenetic patterns. In conclusion, members of the miR-148/-152 family might represent prognostic markers and/or potential therapeutic targets for treatment of autoimmune disorders, chronic inflammatory diseases and multiple types of cancer.

A new staining protocol for detection of murine antibody-secreting plasma cell subsets by flow cytometry.

We provide a robust four-color fluorescence-based flow cytometry protocol that distinguishes viable dividing plasmablasts from nondividing plasma cells and, based on CD19 surface abundance, identifies two mature plasma cell populations in the spleen and the bone marrow of mice.

Interleukin-36 receptor mediates the crosstalk between plasma cells and synovial fibroblasts.

The IL-1 family member IL-36α has proinflammatory and pathogenic properties in psoriasis. IL-36α binds to the IL-36 receptor leading to nuclear factor kappa B/mitogen activated protein kinase mediated cytokine release. The IL-36R antagonist prevents recruitment of IL-1 receptor accessory protein and therefore IL-36-dependent cell activation. In inflamed human tissue, we previously could show that resident B cells and plasma cells (PC) express IL-36α. Further, fibroblast-like synoviocytes (FLS) produced proinflammatory cytokines upon IL-36α-stimulation. We hypothesize an IL-36-specific crosstalk between B cells/PCs and FLS permitting a proinflammatory B cell niche. Here, we firstly demonstrated that B cell lines and B cells from healthy donors express IL-36α and stimulation increased IL-36α in B cells and primary plasmablasts/PCs. Moreover, FLS respond specifically to IL-36α by proliferation and production of matrix metalloproteinases via p38/HSP27 signaling. Importantly, IL-36R-deficiency abrogated IL-36α-induced production of inflammatory mediators in FLS and changed the intrinsic FLS-phenotype. Using an in vitro co-culture system, we could show that IL-36R-deficient FLS had a limited capacity to support PC survival compared to wild-type FLS. Hence, we demonstrated an IL-36R-dependent crosstalk between B cells/PCs and FLS. Our data support the concept of initiation and maintenance of a proinflammatory niche by B cells in the joints.

A web platform for the network analysis of high-throughput data in melanoma and its use to investigate mechanisms of resistance to anti-PD1 immunotherapy.

Cellular phenotypes are established and controlled by complex and precisely orchestrated molecular networks. In cancer, mutations and dysregulations of multiple molecular factors perturb the regulation of these networks and lead to malignant transformation. High-throughput technologies are a valuable source of information to establish the complex molecular relationships behind the emergence of malignancy, but full exploitation of this massive amount of data requires bioinformatics tools that rely on network-based analyses. In this report we present the Virtual Melanoma Cell, an online tool developed to facilitate the mining and interpretation of high-throughput data on melanoma by biomedical researches. The platform is based on a comprehensive, manually generated and expert-validated regulatory map composed of signaling pathways important in malignant melanoma. The Virtual Melanoma Cell is a tool designed to accept, visualize and analyze user-generated datasets. It is available at: https://www.vcells.net/melanoma. To illustrate the utilization of the web platform and the regulatory map, we have analyzed a large publicly available dataset accounting for anti-PD1 immunotherapy treatment of malignant melanoma patients.

Essential control of early B-cell development by Mef2 transcription factors.

The sequential activation of distinct developmental gene networks governs the ultimate identity of a cell, but the mechanisms involved in initiating downstream programs are incompletely understood. The pre-B-cell receptor (pre-BCR) is an important checkpoint of B-cell development and is essential for a pre-B cell to traverse into an immature B cell. Here, we show that activation of myocyte enhancer factor 2 (Mef2) transcription factors (TFs) by the pre-BCR is necessary for initiating the subsequent genetic network. We demonstrate that B-cell development is blocked at the pre-B-cell stage in mice deficient for Mef2c and Mef2d TFs and that pre-BCR signaling enhances the transcriptional activity of Mef2c/d through phosphorylation by the Erk5 mitogen-activating kinase. This activation is instrumental in inducing Krüppel-like factor 2 and several immediate early genes of the AP1 and Egr family. Finally, we show that Mef2 proteins cooperate with the products of their target genes (Irf4 and Egr2) to induce secondary waves of transcriptional regulation. Our findings uncover a novel role for Mef2c/d in coordinating the transcriptional network that promotes early B-cell development.

Regulation of Energy Metabolism during Early B Lymphocyte Development.

The most important feature of humoral immunity is the adaptation of the diversity of newly generated B cell receptors, that is, the antigen receptor repertoire, to the body's own and foreign structures. This includes the transient propagation of B progenitor cells and B cells, which possess receptors that are positively selected via anabolic signalling pathways under highly competitive conditions. The metabolic regulation of early B-cell development thus has important consequences for the expansion of normal or malignant pre-B cell clones. In addition, cellular senescence programs based on the expression of B cell identity factors, such as Pax5, act to prevent excessive proliferation and cellular deviation. Here, we review the basic mechanisms underlying the regulation of glycolysis and oxidative phosphorylation during early B cell development in bone marrow. We focus on the regulation of glycolysis and mitochondrial oxidative phosphorylation at the transition from non-transformed pro- to pre-B cells and discuss some ongoing issues. We introduce Swiprosin-2/EFhd1 as a potential regulator of glycolysis in pro-B cells that has also been linked to Ca2+-mediated mitoflashes. Mitoflashes are bioenergetic mitochondrial events that control mitochondrial metabolism and signalling in both healthy and disease states. We discuss how Ca2+ fluctuations in pro- and pre-B cells may translate into mitoflashes in early B cells and speculate about the consequences of these changes.

The microprocessor component, DGCR8, is essential for early B-cell development in mice.

microRNAs (miRNAs) are important posttranscriptional regulators during hematopoietic lineage commitment and lymphocyte development. Mature miRNAs are processed from primary miRNA transcripts in two steps by the microprocessor complex, consisting of Drosha and its partner DiGeorge Critical Region 8 (DGCR8), and the RNAse III enzyme, Dicer. Conditional ablations of Drosha and Dicer have established the importance of both RNAses in B- and T-cell development. Here, we show that a cre-mediated B-cell specific deletion of DGCR8 in mice results in a nearly complete maturation block at the transition from the pro-B to the pre-B cell stage, and a failure to upregulate Ig µ heavy chain expression in pro-B cells. Furthermore, we found that the death of freshly isolated DGCR8-deficient pro-B cells could be partially prevented by enforced Bcl2 expression. We conclude from these findings that the microprocessor component DGCR8 is essential for survival and differentiation of early B-cell progenitors.