Development of a recombinant hepatitis B immunoglobulin derived from B cells collected from healthy individuals administered with hepatitis B virus vaccines: A feasibility study.

BACKGROUND: In Japan, plasma with a high concentration of Hepatitis B Virus (HBV) antibodies for hepatitis B immunoglobulin (HBIG) is almost entirely imported. We aimed to produce recombinant HBIG by isolating immunoglobulin cDNAs against the HBV surface antigen (HBsAg). STUDY DESIGN AND METHODS: B cells expressing HBsAg antibodies were obtained from blood center personnel who had been administered HB vaccine booster and then isolated by either an Epstein-Barr virus hybridoma or an antigen-specific memory B cell sorting method. Each cDNA of the heavy and light chains of the target antibody was cloned into an IgG1 expression vector and transfected into Expi293F cells to produce a recombinant monoclonal antibody (mAb), which was screened by ELISA and in vitro HBV neutralizing assays. The cross-reactivity of the mAbs to normal human molecules was evaluated by ELISA and immunohistochemistry. RESULTS: Antibody cDNAs were cloned from 11 hybridoma cell lines and 204 HBsAg-bound memory B cells. Three of the resulting recombinant mAbs showed stronger neutralizing activity in vitro than the currently used HBIG. All three bind to the conformational epitope(s) of HBsAg but not to human DNA or cells. DISCUSSION: We successfully isolated HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV. To obtain an alternative source for HBIG, HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV may be useful.

Prohibitin-1 Contributes to Cell-to-Cell Transmission of Herpes Simplex Virus 1 via the MAPK/ERK Signaling Pathway.

Viral cell-to-cell spread, a method employed by several viral families for entrance via cell junctions, is highly relevant to the pathogenesis of various viral infections. Cell-to-cell spread of herpes simplex virus 1 (HSV-1) is known to depend greatly on envelope glycoprotein E (gE). However, the molecular mechanism by which gE acts in HSV-1 cell-to-cell spread and the mechanisms of cell-to-cell spread by other herpesviruses remain poorly understood. Here, we describe our identification of prohibitin-1 as a novel gE-interacting host cell protein. Ectopic expression of prohibitin-1 increased gE-dependent HSV-1 cell-to-cell spread. As observed with the gE-null mutation, decreased expression or pharmacological inhibition of prohibitin-1 reduced HSV-1 cell-to-cell spread without affecting the yield of virus progeny. Similar effects were produced by pharmacological inhibition of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, wherein prohibitin-1 acts as a protein scaffold and is required for induction of this pathway. Furthermore, artificial activation of the MAPK/ERK pathway restored HSV-1 cell-to-cell spread impaired by the gE-null mutation. Notably, pharmacological inhibition of prohibitins or the MAPK/ERK pathway reduced viral cell-to-cell spread of representative members in all herpesvirus subfamilies. Our results suggest that prohibitin-1 contributes to gE-dependent HSV-1 cell-to-cell spread via the MAPK/ERK pathway and that this mechanism is conserved throughout the Herpesviridae, whereas gE is conserved only in the Alphaherpesvirinae subfamily.IMPORTANCE Herpesviruses are ubiquitous pathogens of various animals, including humans. These viruses primarily pass through cell junctions to spread to uninfected cells. This method of cell-to-cell spread is an important pathogenic characteristic of these viruses. Here, we show that the host cell protein prohibitin-1 contributes to HSV-1 cell-to-cell spread via a downstream intracellular signaling cascade, the MAPK/ERK pathway. We also demonstrate that the role of the prohibitin-1-mediated MAPK/ERK pathway in viral cell-to-cell spread is conserved in representative members of every herpesvirus subfamily. This study has revealed a common molecular mechanism of the cell-to-cell spread of herpesviruses.

Self-reactive and polyreactive B cells are generated and selected in the germinal center during γ-herpesvirus infection.

Immune responses against certain viruses are accompanied by auto-antibody production although the origin of these infection-associated auto-antibodies is unclear. Here, we report that murine γ-herpesvirus 68 (MHV68)-induced auto-antibodies are derived from polyreactive B cells in the germinal center (GC) through the activity of short-lived plasmablasts. The analysis of recombinant antibodies from MHV68-infected mice revealed that about 40% of IgG+ GC B cells were self-reactive, with about half of them being polyreactive. On the other hand, virion-reactive clones accounted for only a minor proportion of IgG+ GC B cells, half of which also reacted with self-antigens. The self-reactivity of most polyreactive clones was dependent on somatic hypermutation (SHM), but this was dispensable for the reactivity of virus mono-specific clones. Furthermore, both virus-mono-specific and polyreactive clones were selected to differentiate to B220lo CD138+ plasma cells (PCs). However, the representation of GC-derived polyreactive clones was reduced and that of virus-mono-specific clones was markedly increased in terminally differentiated PCs as compared to transient plasmablasts. Collectively, our findings demonstrate that, during acute MHV68 infection, self-reactive B cells are generated through SHM and selected for further differentiation to short-lived plasmablasts but not terminally differentiated PCs.

Bone Marrow Mononuclear Cells Activate Angiogenesis via Gap Junction-Mediated Cell-Cell Interaction.

Background and Purpose- Bone marrow mononuclear cells (BM-MNCs) are a rich source of hematopoietic stem cells and have been widely used in experimental therapies for patients with ischemic diseases. Activation of angiogenesis is believed to be one of major BM-MNC mode of actions, but the essential mechanism by which BM-MNCs activate angiogenesis have hitherto been elusive. The objective of this study is to reveal the mechanism how BM-MNCs activate angiogenesis. Methods- We have evaluated the effect of direct cell-cell interaction between BM-MNC and endothelial cell on uptake of VEGF (vascular endothelial growth factor) into endothelial cells in vitro. Cerebral ischemia model was used to evaluate the effects of direct cell-cell interaction with transplanted BM-MNC on endothelial cell at ischemic tissue. Results- The uptake of VEGF into endothelial cells was increased by BM-MNC, while being inhibited by blockading the gap junction. Low-molecular-weight substance was transferred from BM-MNC into endothelial cells via gap junctions in vivo, followed by increased expression of hypoxia-inducible factor-1α and suppression of autophagy in endothelial cells. The concentration of glucose in BM-MNC cytoplasm was significantly higher than in endothelial cells, and transfer of glucose homologue from BM-MNC to endothelial cells was observed. Conclusions- Our findings demonstrated cell-cell interaction via gap junction is the prominent pathway for activation of angiogenesis at endothelial cells after ischemia and provided novel paradigm that energy source supply by stem cell to injured cell is one of the therapeutic mechanisms of cell-based therapy. Visual Overview- An online visual overview is available for this article.

The Kg-antigen, RhAG with a Lys164Gln mutation, gives rise to haemolytic disease of the newborn.

The Kg-antigen was first discovered in an investigation of a mother whose infant had haemolytic disease of the newborn (HDN). The antibody against the Kg-antigen is believed to be responsible for HDN. The Kg-antigen is provisionally registered under the number 700045, according to the Red Cell Immunogenetics and Blood Group Terminology. However, the molecular nature of the Kg-antigen has remained a mystery for over 30 years. In this study, a monoclonal antibody against the Kg-antigen and the recombinant protein were developed that allowed for the immunoprecipitation analysis. Immunoprecipitants from the propositus' red blood cell ghosts were subjected to mass spectrometry analysis, and DNA sequence analysis of the genes was also performed. A candidate for the Kg-antigen was molecularly isolated and confirmed to be a determinant of the Kg-antigen by cell transfection and flow cytometry analyses. The Kg-antigen and the genetic mutation were then screened for in a Japanese population. The molecular nature of the Kg-antigen was shown to be RhAG with a Lys164Gln mutation. Kg phenotyping further clarified that 0.22% of the Japanese population studied was positive for the Kg-antigen. These findings provide important information on the Kg-antigen, which has been clinically presumed to give rise to HDN.

Murine γ-Herpesvirus 68 Induces Severe Lung Inflammation in IL-27-Deficient Mice with Liver Dysfunction Preventable by Oral Neomycin.

IL-27 is an immunoregulatory cytokine consisting of p28 and EBI3. Its receptor also has two subunits, WSX1 and gp130. Although IL-27 promotes Th1 differentiation in naive T cells, it also induces IL-10 expression in effector Th1 cells to curtail excessive immune responses. By using p28-deficient mice and WSX1-deficient mice (collectively called IL-27-deficient mice), we examined the role of IL-27 in primary infection by murine γ-herpesvirus 68 (MHV68), a murine model of EBV. Upon airway infection with MHV68, IL-27-deficient mice had more aggravated lung inflammation than wild-type mice, although MHV68 infection per se was better controlled in IL-27-deficient mice. Although epithelial cells and alveolar macrophages were primarily infected by MHV68, interstitial macrophages and dendritic cells were the major producers of IL-27. The lung inflammation of IL-27-deficient mice was characterized by more IFN-γ-producing CD8+ T cells and fewer IL-10-producing CD8+ T cells than that of wild-type mice. An infectious mononucleosis-like disease was also aggravated in IL-27-deficient mice, with prominent splenomegaly and severe hepatitis. Infiltration of IFN-γ-producing effector cells and upregulation of the CXCR3 ligand chemokines CXCL9, CXCL10, and CXCL11 were noted in the liver of MHV68-infected mice. Oral neomycin effectively ameliorated hepatitis, with decreased production of these chemokines in the liver, suggesting that the intestinal microbiota plays a role in liver inflammation through upregulation of these chemokines. Collectively, IL-27 is essential for the generation of IL-10-producing effector cells in primary infection by MHV68. Our findings may also provide new insight into the mechanism of hepatitis associated with infectious mononucleosis.

Mouse model of Epstein-Barr virus LMP1- and LMP2A-driven germinal center B-cell lymphoproliferative disease.

Epstein-Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 and LMP2A had limited effects in immunocompetent mice. However, upon T- and NK-cell depletion, LMP1/2A caused massive plasmablast outgrowth, organ damage, and death. RNA-sequencing analyses identified EBV oncoprotein effects on GC B-cell target genes, including up-regulation of multiple proinflammatory chemokines and master regulators of plasma cell differentiation. LMP1/2A coexpression also up-regulated key HL markers, including CD30 and mixed hematopoietic lineage markers. Collectively, our results highlight synergistic EBV membrane oncoprotein effects on GC B cells and provide a model for studies of their roles in immunosuppression-related lymphoproliferative diseases.

Bystander inhibition of humoral immune responses by Epstein-Barr virus LMP1.

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which mimics a constitutively active receptor, is required for viral transformation of primary B cells. LMP1 is expressed in EBV-infected germinal center (GC) B cells of immunocompetent individuals, suggesting that it may contribute to persistent EBV infection. In this study, we generated and analyzed mice that expressed LMP1 under the control of the CD19 or activation-induced cytidine deaminase (AID) promoter. Expression of LMP1 induced activation of B cells but severely inhibited their differentiation into antibody-secreting cells (ASCs) in vitro and GC B cells in vivo. LMP1-expressing (LMP1+) B cells not only suppressed the functions of wild-type (WT) B cells in in vitro co-culture, but also blocked differentiation of WT B cells into GC B cells and ASCs in immunized bone marrow chimeric mice. Microarray analysis revealed that the gene encoding indoleamine 2,3-dioxygenase 1 (IDO1), a major enzyme involved in the tryptophan metabolic process, was highly induced by LMP1. Either inhibition of IDO1 activity by methyl-l-tryptophan or knockout of Ido1 in LMP1+ B cells could rescue WT B cells from such suppression. IDO1-induced tryptophan consumption and production of tryptophan metabolites appeared to be responsible for inhibition of B-cell function. We conclude that LMP1 expression in antigen-committed B cells not only directly impairs GC B-cell differentiation, but also indirectly inhibits the functions of neighboring B cells, resulting in suppression of humoral immune responses. Such bystander inhibition by LMP1+ B cells may contribute to immune evasion by EBV.

Evasion of affinity-based selection in germinal centers by Epstein-Barr virus LMP2A.

Epstein-Barr virus (EBV) infects germinal center (GC) B cells and establishes persistent infection in memory B cells. EBV-infected B cells can cause B-cell malignancies in humans with T- or natural killer-cell deficiency. We now find that EBV-encoded latent membrane protein 2A (LMP2A) mimics B-cell antigen receptor (BCR) signaling in murine GC B cells, causing altered humoral immune responses and autoimmune diseases. Investigation of the impact of LMP2A on B-cell differentiation in mice that conditionally express LMP2A in GC B cells or all B-lineage cells found LMP2A expression enhanced not only BCR signals but also plasma cell differentiation in vitro and in vivo. Conditional LMP2A expression in GC B cells resulted in preferential selection of low-affinity antibody-producing B cells despite apparently normal GC formation. GC B-cell-specific LMP2A expression led to systemic lupus erythematosus-like autoimmune phenotypes in an age-dependent manner. Epigenetic profiling of LMP2A B cells found increased H3K27ac and H3K4me1 signals at the zinc finger and bric-a-brac, tramtrack domain-containing protein 20 locus. We conclude that LMP2A reduces the stringency of GC B-cell selection and may contribute to persistent EBV infection and pathogenesis by providing GC B cells with excessive prosurvival effects.

Protein kinase N1, a cell inhibitor of Akt kinase, has a central role in quality control of germinal center formation.

Germinal centers (GCs) are specialized microenvironments in secondary lymphoid organs where high-affinity antibody-producing B cells are selected based on B-cell antigen receptor (BCR) signal strength. BCR signaling required for normal GC selection is uncertain. We have found that protein kinase N1 (PKN1, also known as PRK1) negatively regulates Akt kinase downstream of the BCR and that this regulation is necessary for normal GC development. PKN1 interacted with and inhibited Akt1 kinase and transforming activities. Pkn1(-/-) B cells were hyperresponsive and had increased phosphorylated Akt1 levels upon BCR stimulation. In the absence of immunization or infection, Pkn1(-/-) mice spontaneously formed GCs and developed an autoimmune-like disease with age, which was characterized by autoantibody production and glomerulonephritis. More B cells, with fewer somatic BCR gene V region hypermutations were selected in Pkn1(-/-) GCs. These results indicate that PKN1 down-regulation of BCR-activated Akt activity is critical for normal GC B-cell survival and selection.

Significance of glycosylphosphatidylinositol-anchored protein enrichment in lipid rafts for the control of autoimmunity.

Glycosylphosphatidylinositols (GPI) are complex glycolipids that are covalently linked to the C terminus of proteins as a post-translational modification and tether proteins to the plasma membrane. One of the most striking features of GPI-anchored proteins (APs) is their enrichment in lipid rafts. The biosynthesis of GPI and its attachment to proteins occur in the endoplasmic reticulum. In the Golgi, GPI-APs are subjected to fatty acid remodeling, which replaces an unsaturated fatty acid at the sn-2 position of the phosphatidylinositol moiety with a saturated fatty acid. We previously reported that fatty acid remodeling is critical for the enrichment of GPI-APs in lipid rafts. To investigate the biological significance of GPI-AP enrichment in lipid rafts, we generated a PGAP3 knock-out mouse (PGAP3(-/-)) in which fatty acid remodeling of GPI-APs does not occur. We report here that a significant number of aged PGAP3(-/-) mice developed autoimmune-like symptoms, such as increased anti-DNA antibodies, spontaneous germinal center formation, and enlarged renal glomeruli with deposition of immune complexes and matrix expansion. A possible cause for this was the impaired engulfment of apoptotic cells by resident peritoneal macrophages in PGAP3(-/-) mice. Mice with conditional targeting of PGAP3 in either B or T cells did not develop such autoimmune-like symptoms. In addition, PGAP3(-/-) mice exhibited the tendency of Th2 polarization. These data demonstrate that PGAP3-dependent fatty acid remodeling of GPI-APs has a significant role in the control of autoimmunity, possibly by the regulation of apoptotic cell clearance and Th1/Th2 balance.

Partial suppression of M1 microglia by Janus kinase 2 inhibitor does not protect against neurodegeneration in animal models of amyotrophic lateral sclerosis.

BACKGROUND: Accumulating evidence has shown that the inflammatory process participates in the pathogenesis of amyotrophic lateral sclerosis (ALS), suggesting a therapeutic potential of anti-inflammatory agents. Janus kinase 2 (JAK2), one of the key molecules in inflammation, transduces signals downstream of various inflammatory cytokines, and some Janus kinase inhibitors have already been clinically applied to the treatment of inflammatory diseases. However, the efficacy of JAK2 inhibitors in treatment of ALS remains to be demonstrated. In this study, we examined the role of JAK2 in ALS by administering a selective JAK2 inhibitor, R723, to an animal model of ALS (mSOD1G93A mice). FINDINGS: Orally administered R723 had sufficient access to spinal cord tissue of mSOD1G93A mice and significantly reduced the number of Ly6c positive blood monocytes, as well as the expression levels of IFN-γ and nitric oxide synthase 2, inducible (iNOS) in the spinal cord tissue. R723 treatment did not alter the expression levels of Il-1ß, Il-6, TNF, and NADPH oxidase 2 (NOX2), and suppressed the expression of Retnla, which is one of the markers of neuroprotective M2 microglia. As a result, R723 did not alter disease progression or survival of mSOD1G93A mice. CONCLUSIONS: JAK2 inhibitor was not effective against ALS symptoms in mSOD1G93A mice, irrespective of suppression in several inflammatory molecules. Simultaneous suppression of anti-inflammatory microglia with a failure to inhibit critical other inflammatory molecules might explain this result.

An inhibitory role for Sema4A in antigen-specific allergic asthma.

PURPOSE: The class IV semaphorin Sema4A is critical for efficient Th1 differentiation and Sema4a (-/-) mice exhibit impaired Th1 immune responses. However, the role of Sema4A in Th2 cell-mediated allergic diseases has not been fully studied. The aim of this study was to clarify the regulatory role possessed by Sema4A in mouse models of allergic diseases, particularly allergic asthma. METHODS: Sema4a (-/-) mice on a BALB/c background were examined for the development of allergic diseases. To induce experimental asthma, mice were sensitized with ovalbumin (OVA) followed by intranasal challenges with OVA. After challenge, airway hyperreactivity (AHR) and airway inflammation were evaluated. The role of Sema4A in asthma was examined using Sema4a (-/-) mice and Sema4A-Fc fusion proteins. The direct effects of Sema4A-Fc on antigen-specific effector CD4(+) T cells were also examined. RESULTS: A fraction of Sema4a (-/-) BALB/c mice spontaneously developed skin lesions that resembled atopic dermatitis (AD) in humans. Furthermore, AHR, airway inflammation, and Th2-type immune responses were enhanced in Sema4a (-/-) mice compared to wild type (WT) mice when immunized and challenged with OVA. In vivo systemic administration of Sema4A-Fc during the challenge period ameliorated AHR and lung inflammation and reduced the production of Th2-type cytokines in WT mice. The inhibitory effects of Sema4A on airway inflammation were also observed in mice deficient in Tim-2, a Sema4A receptor. Finally, we showed that Sema4A-Fc directly inhibited IL-4-producing OVA-specific CD4(+) T cells. CONCLUSION: These results demonstrate that Sema4A plays an inhibitory role in Th2-type allergic diseases, such as allergic asthma.

Resting phase-administration of lemborexant ameliorates sleep and glucose tolerance in type 2 diabetic mice.

Sleep disorders are associated with increased risk of obesity and type 2 diabetes. Lemborexant, a dual orexin receptor antagonist (DORA), is clinically used to treat insomnia. However, the influence of lemborexant on sleep and glucose metabolism in type 2 diabetic state has remained unknown. In the present study, we investigated the effect of lemborexant in type 2 diabetic db/db mice exhibiting both sleep disruption and glucose intolerance. Single administration of lemborexant at the beginning of the light phase (i.e., resting phase) acutely increased total time spent in non-rapid eye movement (NREM) and REM sleep in db/db mice. Durations of NREM sleep-, REM sleep-, and wake-episodes were also increased by this administration. Daily resting-phase administration of lemborexant for 3-6 weeks improved glucose tolerance without changing body weight and glucose-stimulated insulin secretion in db/db mice. Similar improvement of glucose tolerance was caused by daily resting-phase administration of lemborexant in obese C57BL/6J mice fed high fat diet, whereas no such effect was observed in non-diabetic db/m+ mice. Diabetic db/db mice treated daily with lemborexant exhibited increased locomotor activity in the dark phase (i.e., awake phase), although they did not show any behavioral abnormality in the Y-maze, elevated plus maze, and forced swim tests. These results suggest that timely promotion of sleep by lemborexant improved the quality of wakefulness in association with increased physical activity during the awake phase, and these changes may underlie the amelioration of glucose metabolism under type 2 diabetic conditions.

Critical role of IkappaB Kinase alpha in TLR7/9-induced type I IFN production by conventional dendritic cells.

A plasmacytoid dendritic cell (DC) can produce large amounts of type I IFNs after sensing nucleic acids through TLR7 and TLR9. IkappaB kinase alpha (IKKalpha) is critically involved in this type I IFN production through its interaction with IFN regulatory factor-7. In response to TLR7/9 signaling, conventional DCs can also produce IFN-beta but not IFN-alpha in a type I IFN-independent manner. In this study, we showed that IKKalpha was required for production of IFN-beta, but not of proinflammatory cytokines, by TLR7/9-stimulated conventional DCs. Importantly, IKKalpha was dispensable for IFN-beta gene upregulation by TLR4 signaling. Biochemical analyses indicated that IKKalpha exerted its effects through its interaction with IFN regulatory factor-1. Furthermore, IKKalpha was involved in TLR9-induced type I IFN-independent IFN-beta production in vivo. Our results show that IKKalpha is a unique molecule involved in TLR7/9-MyD88-dependent type I IFN production through DC subset-specific mechanisms.

Enhanced humoral immune responses against T-independent antigens in Fc alpha/muR-deficient mice.

IgM is an antibody class common to all vertebrates that plays a primary role in host defenses against infection. Binding of IgM with an antigen initiates the complement cascade, accelerating cellular and humoral immune responses. However, the functional role of the Fc receptor for IgM in such immune responses remains obscure. Here we show that mice deficient in Fc alpha/muR, an Fc receptor for IgM expressed on B cells and follicular dendritic cells (FDCs), have enhanced germinal center formation and affinity maturation and memory induction of IgG3(+) B cells after immunization with T-independent (TI) antigens. Moreover, Fc alpha/muR-deficient mice show prolonged antigen retention by marginal zone B (MZB) cells and FDCs. In vitro studies demonstrate that interaction of the IgM immune complex with Fc alpha/muR partly suppress TI antigen retention by MZB cells. We further show that downregulation of complement receptor (CR)1 and CR2 or complement deprivation by in vivo injection with anti-CR1/2 antibody or cobra venom factor attenuates antigen retention by MZB cells and germinal center formation after immunization with TI antigens in Fc alpha/muR(-/-) mice. Taken together, these results suggest that Fc alpha/muR negatively regulates TI antigen retention by MZB cells and FDCs, leading to suppression of humoral immune responses against T-independent antigens.

Monoclonal antibody therapeutics for infectious diseases: Beyond normal human immunoglobulin.

Antibody therapy is effective for treating infectious diseases. Due to the coronavirus disease 2019 (COVID-19) pandemic and the rise of drug-resistant bacteria, rapid development of neutralizing monoclonal antibodies (mAbs) to treat infectious diseases is urgently needed. Using a therapeutic human mAb with the lowest immunogenicity is recommended, because chimera and humanized mAbs are occasionally immunogenic. In order to directly obtain naïve human mAbs, there are three methods: phage display, B cell receptor (BCR) cDNA sequencing of a single cell, and antibody-encoding gene and amino acid sequencing of immortalized cells using memory B cells, which are isolated from human peripheral blood mononuclear cells of healthy, vaccinated, infected, or recovered individuals. After screening against the antigen and performing neutralization assays, a human neutralizing mAb is constructed from the antibody-encoding DNA sequences of these memory B cells. This review describes examples of obtaining human neutralizing mAbs against various infectious diseases using these methods. However, a few of these mAbs have been approved for therapy. Therefore, antigen characterization and evaluation of neutralization activity in vitro and in vivo are indispensable for the development of therapeutic mAbs. These results will accelerate the development of antibody drug as therapeutic agents.

Human antibody recognition and neutralization mode on the NTD and RBD domains of SARS-CoV-2 spike protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Variants of concern (VOCs) such as Delta and Omicron have developed, which continue to spread the pandemic. It has been reported that these VOCs reduce vaccine efficacy and evade many neutralizing monoclonal antibodies (mAbs) that target the receptor binding domain (RBD) of the glycosylated spike (S) protein, which consists of the S1 and S2 subunits. Therefore, identification of optimal target regions is required to obtain neutralizing antibodies that can counter VOCs. Such regions have not been identified to date. We obtained 2 mAbs, NIBIC-71 and 7G7, using peripheral blood mononuclear cells derived from volunteers who recovered from COVID-19. Both mAbs had neutralizing activity against wild-type SARS-CoV-2 and Delta, but not Omicron. NIBIC-71 binds to the RBD, whereas 7G7 recognizes the N-terminal domain of the S1. In particular, 7G7 inhibited S1/S2 cleavage but not the interaction between the S protein and angiotensin-converting enzyme 2; it suppressed viral entry. Thus, the efficacy of a neutralizing mAb targeting inhibition of S1/2 cleavage was demonstrated. These results suggest that neutralizing mAbs targeting blockade of S1/S2 cleavage are likely to be cross-reactive against various VOCs.

Food odor perception promotes systemic lipid utilization.

Food cues during fasting elicit Pavlovian conditioning to adapt for anticipated food intake. However, whether the olfactory system is involved in metabolic adaptations remains elusive. Here we show that food-odor perception promotes lipid metabolism in male mice. During fasting, food-odor stimulation is sufficient to increase serum free fatty acids via adipose tissue lipolysis in an olfactory-memory-dependent manner, which is mediated by the central melanocortin and sympathetic nervous systems. Additionally, stimulation with a food odor prior to refeeding leads to enhanced whole-body lipid utilization, which is associated with increased sensitivity of the central agouti-related peptide system, reduced sympathetic activity and peripheral tissue-specific metabolic alterations, such as an increase in gastrointestinal lipid absorption and hepatic cholesterol turnover. Finally, we show that intermittent fasting coupled with food-odor stimulation improves glycemic control and prevents insulin resistance in diet-induced obese mice. Thus, olfactory regulation is required for maintaining metabolic homeostasis in environments with either an energy deficit or energy surplus, which could be considered as part of dietary interventions against metabolic disorders.

Deleterious effects of lymphocytes at the early stage of neurodegeneration in an animal model of amyotrophic lateral sclerosis.

BACKGROUND: Non-neuronal cells, such as microglia and lymphocytes, are thought to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Previous studies have demonstrated neuroprotective effects of lymphocytes at the end stage of ALS, partly through induction of alternatively activated microglia (M2 microglia), which are neuroprotective. In this study, we investigated the role of lymphocytes in the early stage of the disease using an animal model of inherited ALS. METHODS: We established a transgenic mouse line overexpressing the familial ALS-associated G93A-SOD1 mutation (harboring a single amino acid substitution of glycine to alanine at codon 93) with depletion of the Rag2 gene (mSOD1/RAG2-/- mice), an animal model of inherited ALS lacking mature lymphocytes. Body weights, clinical scores and motor performance (hanging wire test) of mSOD1/RAG2-/- mice were compared to those of mutant human SOD1 transgenic mice (mSOD1/RAG2+/+ mice). Activation of glial cells in the spinal cords of these mice was determined immunohistochemically, and the expression of mRNA for various inflammatory and anti-inflammatory molecules was evaluated. RESULTS: Clinical onset in mSOD1/RAG2-/- mice was significantly delayed, and the number of lectin-positive cells in spinal cord was increased at the early stage of disease when compared to mSOD1/RAG2+/+ mice. Quantitative RT-PCR confirmed that mRNA for Ym1, an M2 microglial-related molecule, was significantly increased in mSOD1/RAG2-/- mouse spinal cords at the early disease stage. CONCLUSIONS: Compared with mSOD1/RAG2+/+ mice, mSOD1/RAG2-/- mice displayed delayed onset and increased M2 microglial activation at the early stage of disease. Thus, lymphocytes at the early pathological phase of ALS display a deleterious effect via inhibition of M2 microglial activation.