Threonine phosphorylation of STAT1 restricts interferon signaling and promotes innate inflammatory responses.

Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and antiviral defense. Here, using genetic and biochemical assays, we unveil Thr748 as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically engineered mice expressing phospho-deficient threonine748-to-alanine (T748A) mutant Stat1 are resistant to LPS-induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of Stat1 following LPS-induced lethality, indicating that the Thr748 phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic IκB kinase-mediated Thr748 phosphorylation of Stat1, which promotes macrophage inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent modular functionality of Stat1 in innate immune responses: IFN phospho-tyrosine dependent and inflammatory phospho-threonine dependent. Better understanding of the Thr748 phosphorylation of Stat1 may uncover advanced pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually.

Rational in silico design identifies two mutations that restore UT28K SARS-CoV-2 monoclonal antibody activity against Omicron BA.1.

SARS-CoV-2 rapidly mutates and acquires resistance to neutralizing antibodies. We report an in-silico-designed antibody that restores the neutralizing activity of a neutralizing antibody. Our previously generated antibody, UT28K, exhibited broad neutralizing activity against mutant variants; however, its efficacy against Omicron BA.1 was compromised by the mutation. Using previously determined structural information, we designed a modified-UT28K (VH T28R/N57D), UT28K-RD targeting the mutation site. In vitro and in vivo experiments demonstrated the efficacy of UT28K-RD in neutralizing Omicron BA.1. Although the experimentally determined structure partially differed from the predicted model, our study serves as a successful case of antibody design, wherein the predicted amino acid substitution enhanced the recognition of the previously elusive Omicron BA.1. We anticipate that numerous similar cases will be reported, showcasing the potential of this approach for improving protein-protein interactions. Our findings will contribute to the development of novel therapeutic strategies for highly mutable viruses, such as SARS-CoV-2.

VNAR development through antigen immunization of Japanese topeshark (Hemitriakis japanica).

The VNAR (Variable New Antigen Receptor) is the smallest single-domain antibody derived from the variable domain of IgNAR of cartilaginous fishes. Despite its biomedical and diagnostic potential, research on VNAR has been limited due to the difficulties in obtaining and maintaining immune animals and the lack of research tools. In this study, we investigated the Japanese topeshark as a promising immune animal for the development of VNAR. This shark is an underutilized fishery resource readily available in East Asia coastal waters and can be safely handled without sharp teeth or venomous stingers. The administration of Venus fluorescent protein to Japanese topesharks markedly increased antigen-specific IgM and IgNAR antibodies in the blood. Both the phage-display library and the yeast-display library were constructed using RNA from immunized shark splenocytes. Each library was enriched by biopanning, and multiple antigen-specific VNARs were acquired. The obtained antibodies had affinities of 1 × 10-8 M order and showed high plasticity, retaining their binding activity even after high-temperature or reducing-agent treatment. The dissociation rate of a low-affinity VNAR was significantly improved via dimerization. These results demonstrate the potential utility of the Japanese topeshark for the development of VNAR. Furthermore, we conducted deep sequencing analysis to reveal the quantitative changes in the CDR3-coding sequences, revealing distinct enrichment bias between libraries. VNARs that were primarily enriched in the phage display had CDR3 coding sequences with fewer E. coli rare codons, suggesting translation machinery on the selection and enrichment process during biopanning.

Development of a Novel CD26-Targeted Chimeric Antigen Receptor T-Cell Therapy for CD26-Expressing T-Cell Malignancies.

Chimeric-antigen-receptor (CAR) T-cell therapy for CD19-expressing B-cell malignancies is already widely adopted in clinical practice. On the other hand, the development of CAR-T-cell therapy for T-cell malignancies is in its nascent stage. One of the potential targets is CD26, to which we have developed and evaluated the efficacy and safety of the humanized monoclonal antibody YS110. We generated second (CD28) and third (CD28/4-1BB) generation CD26-targeted CAR-T-cells (CD26-2G/3G) using YS110 as the single-chain variable fragment. When co-cultured with CD26-overexpressing target cells, CD26-2G/3G strongly expressed the activation marker CD69 and secreted IFNgamma. In vitro studies targeting the T-cell leukemia cell line HSB2 showed that CD26-2G/3G exhibited significant anti-leukemia effects with the secretion of granzymeB, TNFα, and IL-8, with 3G being superior to 2G. CD26-2G/3G was also highly effective against T-cell lymphoma cells derived from patients. In an in vivo mouse model in which a T-cell lymphoma cell line, KARPAS299, was transplanted subcutaneously, CD26-3G inhibited tumor growth, whereas 2G had no effect. Furthermore, in a systemic dissemination model in which HSB2 was administered intravenously, CD26-3G inhibited tumor growth more potently than 2G, resulting in greater survival benefit. The third-generation CD26-targeted CAR-T-cell therapy may be a promising treatment modality for T-cell malignancies.

GATS tag system is compatible with biotin labelling methods for protein analysis.

Polypeptide tags and biotin labelling technologies are widely used for protein analyses in biochemistry and cell biology. However, many peptide tag epitopes contain lysine residues (or amino acids) that are masked after biotinylation. Here, we propose the GATS tag system without a lysine residue and with high sensitivity and low non-specific binding using a rabbit monoclonal antibody against Plasmodium falciparum glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (PfGAMA). From 14 monoclonal clones, an Ra3 clone was selected as it recognized an epitope-TLSVGVQNTF-without a lysine residue; this antibody and epitope tag set was called the GATS tag system. Surface plasmon resonance analysis showed that the tag system had a high affinity of 8.71 × 10-9 M. GATS tag indicated a very low background with remarkably high sensitivity and specificity in immunoblotting using the lysates of mammalian cells. It also showed a high sensitivity for immunoprecipitation and immunostaining of cultured human cells. The tag system was highly sensitive in both biotin labelling methods for proteins using NHS-Sulfo-biotin and BioID (proximity-dependent biotin identification) in the human cells, as opposed to a commercially available tag system having lysine residues, which showed reduced sensitivity. These results showed that the GATS tag system is suitable for methods such as BioID involving labelling lysine residues.

DPP8 Selective Inhibitor Tominostat as a Novel and Broad-Spectrum Anticancer Agent against Hematological Malignancies.

DPP8/9 inhibition induces either pyroptotic or apoptotic cell death in hematological malignancies. We previously reported that treatment with the DPP8/9 inhibitor 1G244 resulted in apoptotic cell death in myeloma, and our current study further evaluates the mechanism of action of 1G244 in different blood cancer cell lines. Specifically, 1G244 inhibited DPP9 to induce GSDMD-mediated-pyroptosis at low concentrations and inhibited DPP8 to cause caspase-3-mediated-apoptosis at high concentrations. HCK expression is necessary to induce susceptibility to pyroptosis but does not participate in the induction of apoptosis. To further characterize this DPP8-dependent broad-spectrum apoptosis induction effect, we evaluated the potential antineoplastic role for an analog of 1G244 with higher DPP8 selectivity, tominostat (also known as 12 m). In vitro studies demonstrated that the cytotoxic effect of 1G244 at high concentrations was enhanced in tominostat. Meanwhile, in vivo work showed tominostat exhibited antitumor activity that was more effective on a cell line sensitive to 1G244, and at higher doses, it was also effective on a cell line resistant to 1G244. Importantly, the weight loss morbidity associated with increasing doses of 1G244 was not observed with tominostat. These results suggest the possible development of novel drugs with antineoplastic activity against selected hematological malignancies by refining and increasing the DPP8 selectivity of tominostat.

Epitope analysis of human monoclonal antibodies from a patient with autoimmune factor XIII deficiency reveals their inhibitory mechanisms.

Autoimmune coagulation factor XIII (FXIII) deficiency (AiF13D) is a bleeding disorder caused by anti-FXIII autoantibodies. Recently, we generated human monoclonal antibodies (mAbs) from the peripheral blood of an AiF13D patient and classified them into three groups: FXIII-dissociation inhibitor, FXIII-assembly inhibitor, and non-neutralizing/inhibitory mAbs. However, the epitope region and molecular inhibitory mechanism of each mAb remain unknown. Here, we localized the epitope regions of the representative inhibitory mAbs A69K (dissociation inhibitor) and A78L (assembly inhibitor) to the ß-barrel-2 domain and boundary of ß-barrel-1&2 domains, respectively, of the FXIII-A subunit, by combining a binding assay using its synthesized peptides and a protease-protection assay. Our findings suggest that A69K inhibits the activation-related conformational changes and dissociation of FXIII and that A78L competitively inhibits FXIII-assembly.

N-glycan in the variable region of monoclonal ACPA (CCP-Ab1) promotes the exacerbation of experimental arthritis.

OBJECTIVES: The variable region of most ACPA IgG molecules in the serum of RA patients carries N-glycan (N-glycanV). To analyse the pathogenicity of N-glycanV of ACPAs, we analysed the pathogenicity of a monoclonal ACPA, CCP-Ab1, with or without N-glycanV, which had been isolated from a patient with RA. METHODS: CCP-Ab1 with no N-glycosylation site in the variable region (CCP-Ab1 N-rev) was generated, and antigen binding, the effect on in vitro differentiation of osteoclasts from bone marrow mononuclear cells of autoimmune arthritis-prone SKG mice (the cell size of TRAP+ cells and bone resorption capacity) and the in vivo effect on the onset or exacerbation of autoimmune arthritis in SKG mice were evaluated in comparison with glycosylated CCP-Ab1. RESULTS: Amino acid residues in citrullinated peptide (cfc1), which are essential for binding to CCP-Ab1 N-rev and original CCP-Ab1, were almost identical. The size of TRAP+ cells was significantly larger and osteoclast bone resorption capacity was enhanced in the presence of CCP-Ab1, but not with CCP-Ab1 N-rev. This enhancing activity required the sialic acid of the N-glycan and Fc region of CCP-Ab1. CCP-Ab1, but not CCP-Ab1 N-rev, induced the exacerbation of experimental arthritis in the SKG mouse model. CONCLUSIONS: These data showed that N-glycanV was required for promoting osteoclast differentiation and bone resorption activity in both in vitro and in vivo assays. The present study demonstrated the important role of N-glycanV in the exacerbation of experimental arthritis by ACPAs.

Selection of highly responsive T cell receptors by an analysis combining the expression of multiple markers.

The clinical success of T cell receptor (TCR) gene-transduced T (TCR-T) cell therapy is expected as one of the next-generation immunotherapies for cancer, in which the selection of TCRs with high functional avidity (high-functional TCRs) is important. One widely used approach to select high-functional TCRs is a comparison of the EC50 values of TCRs, which involves laborious experiments. Therefore, the establishment of a simpler method to select high-functional TCRs is desired. We herein attempted to establish a simple method to select high-functional TCRs based on the expression of T cell activation markers using the mouse T cell line BW5147.3 (BW). We examined relationships between the EC50 values of TCRs in interleukin-2 production and the expression levels of TCR activation markers on BW cells. In TCR-expressing BW cells stimulated with antigenic peptides, the CD69, CD137, and PD-1 expression was differentially induced by various doses of peptides. An analysis of TCRs derived from the tumor-infiltrating lymphocytes of murine melanoma and peripheral blood T cells of hepatocellular carcinoma patients treated with a peptide vaccination revealed that an analysis combining CD69, CD137, and PD-1 expression levels in BW cells stimulated with a single dose of an antigenic peptide selected high-functional TCRs with functional avidity assessed by EC50 values. Our method facilitates the section of high-functional TCRs among tumor-reacting TCRs, which will promote TCR-T cell therapy. The stimulation of BW cells expressing objective TCRs with a single dose of antigenic peptides and analysis combining the expression of CD69, CD137, and PD-1 allows us to select highly responsive TCRs.

High-throughput droplet-based analysis of influenza A virus genetic reassortment by single-virus RNA sequencing.

The segmented RNA genome of influenza A viruses (IAVs) enables viral evolution through genetic reassortment after multiple IAVs coinfect the same cell, leading to viruses harboring combinations of eight genomic segments from distinct parental viruses. Existing data indicate that reassortant genotypes are not equiprobable; however, the low throughput of available virology techniques does not allow quantitative analysis. Here, we have developed a high-throughput single-cell droplet microfluidic system allowing encapsulation of IAV-infected cells, each cell being infected by a single progeny virion resulting from a coinfection process. Customized barcoded primers for targeted viral RNA sequencing enabled the analysis of 18,422 viral genotypes resulting from coinfection with two circulating human H1N1pdm09 and H3N2 IAVs. Results were highly reproducible, confirmed that genetic reassortment is far from random, and allowed accurate quantification of reassortants including rare events. In total, 159 out of the 254 possible reassortant genotypes were observed but with widely varied prevalence (from 0.038 to 8.45%). In cells where eight segments were detected, all 112 possible pairwise combinations of segments were observed. The inclusion of data from single cells where less than eight segments were detected allowed analysis of pairwise cosegregation between segments with very high confidence. Direct coupling analysis accurately predicted the fraction of pairwise segments and full genotypes. Overall, our results indicate that a large proportion of reassortant genotypes can emerge upon coinfection and be detected over a wide range of frequencies, highlighting the power of our tool for systematic and exhaustive monitoring of the reassortment potential of IAVs.

Cloning of human anti-factor XIII monoclonal antibody dissects mechanisms of polyclonal antibodies in a single patient.

BACKGROUND: Coagulation factor XIII (FXIII) consists of 2 A (FXIII-A) and 2 B (FXIII-B) subunits that cross-link and strengthen the hemostatic fibrin thrombus; thus, abnormal bleeding occurs when FXIII is significantly reduced. Autoimmune-acquired FXIII deficiency (AiF13D) is characterized by lethal bleeding secondary to the development of autoantibodies against FXIII. However, since anti-FXIII autoantibodies are polyclonal, the mechanism underlying FXIII dysfunction is unclear. OBJECTIVES: The objective of this study was to dissect the inhibitory mechanisms of polyclonal anti-FXIII autoantibodies. METHODS: In this study, we prepared the human monoclonal antibodies (hmAbs) from the peripheral blood of an 86-year-old man with AiF13D by using a new complementary DNA cloning method and analyzed the properties of each autoantibody. RESULTS: Seventeen clones obtained from hmAbs were divided into the following 3 groups: dissociation inhibitors of FXIII-A2B2 (6 clones), assembly inhibitors of FXIII-A2B2 (3 clones), and nonneutralizing/inhibitory hmAbs (8 clones). Dissociation inhibitors strongly inhibited fibrin cross-linking and amine incorporation. Assembly inhibitors extracted FXIII-A from FXIII-A2B2, strongly inhibited binding of FXIII-A to FXIII-B, and activation peptide cleavage. However, the patient's plasma presented a strong inhibition of A2B2 heterodimer assembly but only a slight inhibition of thrombin-Ca2+-dependent dissociation, suggesting that the assembly inhibitors concealed the effect of dissociation inhibitors in plasma. By contrast, nonneutralizing antibodies had little effect on the function of FXIII, suggesting that nonneutralizing hmAbs (and/or dissociation inhibitors and/or assembly inhibitors) promoted the clearance of FXIII-A from the blood. CONCLUSION: Cloning of anti-FXIII autoantibodies enabled us to not only elucidate the mechanism and pathophysiology of AiF13D but also develop a completely new type of anticoagulant.

Gene modified NK cell line as a powerful tool for evaluation of cloned TCRs for TCR-T cell therapy.

T cell receptor-engineered T cell (TCR-T) therapy is anticipated as a next generation-immunotherapy for cancer and recent advances of TCR isolation technology have enabled patient's T cells to express TCRs recognizing multiple combinations of specific peptides and human leukocyte antigens (HLA). However, evaluation processes for the TCR-induced cytotoxicity activity using primary T cells are laborious and time-consuming. In this study, we established a cell line that do not express endogenous TCRs, enabling to generate large numbers of homogeneous cells, and can measure the cytotoxic activity of the isolated TCRs. To this end, we transduced a Natural Killer (NK) cell line with human CD3 molecules and interleukin (IL)-2. The TCR expressing NK cells killed target cells as similarly to TCR-transduced primary T cells and secreted various cytokines/chemokines including IL-2. Thus, the gene-modified NK cell can be a powerful tool to rapidly and efficiently evaluate the functions of isolated TCRs.

Hematopoietic stem progenitor cells with malignancy-related gene mutations in patients with acquired aplastic anemia are characterized by the increased expression of CXCR4.

The phenotypic changes in hematopoietic stem progenitor cells (HSPCs) with somatic mutations of malignancy-related genes in patients with acquired aplastic anemia (AA) are poorly understood. As our initial study showed increased CXCR4 expression on HLA allele-lacking (HLA[-]) HSPCs that solely support hematopoiesis in comparison to redundant HLA(+) HSPCs in AA patients, we screened the HSPCs of patients with various types of bone marrow (BM) failure to investigate their CXCR4 expression. In comparison to healthy individuals (n = 15, 12.3%-49.9%, median 43.2%), the median CXCR4+ cell percentages in the HSPCs of patients without somatic mutations were low: 29.3% (14.3%-37.3%) in the eight patients without HLA(-) granulocytes, 8.8% (4.1%-9.8%) in the five patients with HLA(-) cells accounting for >90% of granulocytes, and 7.8 (2.1%-8.7%) in the six patients with paroxysmal nocturnal hemoglobinuria. In contrast, the median percentage was much higher (78% [61.4%-88.7%]) in the five AA patients without HLA(-) granulocytes possessing somatic mutations (c-kit, t[8;21], monosomy 7 [one for each], ASXL1 [n = 2]), findings that were comparable to those (66.5%, 63.1%-88.9%) in the four patients with advanced myelodysplastic syndromes. The increased expression of CXCR4 may therefore reflect intrinsic abnormalities of HSPCs caused by somatic mutations that allow them to evade restriction by BM stromal cells.

Evaluation of chimeric antigen receptor of humanized rabbit-derived T cell receptor-like antibody.

T-cell receptor (TCR)-like Abs that specifically recognize antigenic peptides presented on MHC molecules have been developed for next-generation cancer immunotherapy. Recently, we reported a rapid and efficient method to generate TCR-like Abs using a rabbit system. We humanized previously generated rabbit-derived TCR-like Abs reacting Epstein-Barr virus peptide (BRLF1p, TYPVLEEMF) in the context of HLA-A24 molecules, produced chimeric antigen receptor (CAR)-T cells, and evaluated their antitumor effects using in vitro and in vivo tumor models. Humanization of the rabbit-derived TCR-like Abs using the complementarity-determining region grafting technology maintained their specificity and affinity. We prepared a second-generation CAR using single-chain variable fragment of the humanized TCR-like Abs and then transduced them into human T cells. The CAR-T cells specifically recognized BRLF1p/MHC molecules and lysed the target cells in an antigen-specific manner in vitro. They also demonstrated antitumor activity in a mouse xenograft model. We report the generation of CAR-T cells using humanized rabbit-derived TCR-like Abs. Together with our established and efficient generation procedure for TCR-like Abs using rabbits, our platform for the clinical application of humanized rabbit-derived TCR-like Abs to CAR-T cells will help improve next-generation cancer immunotherapy.

Novel super-neutralizing antibody UT28K is capable of protecting against infection from a wide variety of SARS-CoV-2 variants.

Many potent neutralizing SARS-CoV-2 antibodies have been developed and used for therapies. However, the effectiveness of many antibodies has been reduced against recently emerging SARS-CoV-2 variants, especially the Omicron variant. We identified a highly potent SARS-CoV-2 neutralizing antibody, UT28K, in COVID-19 convalescent individuals who recovered from a severe condition. UT28K showed efficacy in neutralizing SARS-CoV-2 in an in vitro assay and in vivo prophylactic treatment, and the reactivity to the Omicron strain was reduced. The structural analyses revealed that antibody UT28K Fab and SARS-CoV-2 RBD protein interactions were mainly chain-dominated antigen-antibody interactions. In addition, a mutation analysis suggested that the emergence of a UT28K neutralization-resistant SARS-CoV-2 variant was unlikely, as this variant would likely lose its competitive advantage over circulating SARS-CoV-2. Our data suggest that UT28K offers potent protection against SARS-CoV-2, including newly emerging variants.

Frequent HLA-DR loss on hematopoietic stem progenitor cells in patients with cyclosporine-dependent aplastic anemia carrying HLA-DR15.

To determine whether antigen presentation by HLA-DR on hematopoietic stem progenitor cells (HSPCs) is involved in the development of acquired aplastic anemia (AA), we studied the HLA-DR expression on CD45dimCD34+CD38+ cells in the peripheral blood of 61 AA patients including 23 patients possessing HLA-class I allele-lacking (HLA-class I[-]) leukocytes. HLA-DR-lacking (DR[-]) cells accounted for 13.0-57.1% of the total HSPCs in seven (11.5%) patients with HLA-DR15 who did not possess HLA-class I(-) leukocytes. The incubation of sorted DR(-) HSPCs in the presence of IFN-γ for 72 h resulted in the full restoration of the DR expression. A comparison of the transcriptome profile between DR(-) and DR(+) HSPCs revealed the lower expression of immune response-related genes including co-stimulatory molecules (e.g., CD48, CD74, and CD86) in DR(-) cells, which was not evident in HLA-class I(-) HSPCs. DR(-) cells were exclusively detected in GPI(+) HSPCs in four patients whose HSPCs could be analyzed separately for GPI(+) and GPI(-) HSPCs. These findings suggest that CD4+ T cells specific to antigens presented by HLA-DR15 on HSPCs may contribute to the development of AA as well as the immune escape of GPI(-) HSPCs in a distinct way from CD8+ T cells recognizing HLA-class I-restricted antigens.

Rapid cloning of antigen-specific T-cell receptors by leveraging the cis activation of T cells.

It is commonly understood that T cells are activated via trans interactions between antigen-specific T-cell receptors (TCRs) and antigenic peptides presented on major histocompatibility complex (MHC) molecules on antigen-presenting cells. By analysing a large number of T cells at the single-cell level on a microwell array, we show that T-cell activation can occur via cis interactions (where TCRs on the T cell interact with the antigenic peptides presented on MHC class-I molecules on the same cell), and that such cis activation can be used to detect antigen-specific T cells and clone their TCR within 4 d. We used the detection-and-cloning system to clone a tumour-antigen-specific TCR from peripheral blood mononuclear cells of healthy donors. TCR cloning by leveraging the cis activation of T cells may facilitate the development of TCR-engineered T cells for cancer therapy.

The Lipopolysaccharide Mutant Re-LPS Is a Useful Tool for Detecting LPS Contamination in Rheumatoid Synovial Cell Cultures.

INTRODUCTION: Lipopolysaccharide (LPS) contamination of commercially available proteins has seriously impeded research on citrullinated fibrinogen (cit-Fb) in rheumatoid synovial cells (RSCs). METHODS: RSCs obtained from 4 rheumatoid arthritis patients who underwent full knee arthroplasty were cultured, stimulated with cit-Fb, and cytokine expression levels were measured. We then evaluated polymyxin-B (PMB), heat inactivation, and rough (R)-type LPS mutants for rapid detection of LPS contamination. RESULTS: cit-Fb induced expression of CXCL10 and IFNB in RSCs via the toll-like receptor. PMB inhibited cit-Fb-mediated CXCL10 gene expression but not protein expression induced by 20 µg/mL cit-Fb. Heat inactivation did not affect LPS-mediated CXCL10 or IL-6 induction; however, cit-Fb-mediated CXCL10expression was inhibited. Wild-type LPS from Escherichia coli (WT-LPS) strongly induces CXCL10 expression, but induction by Ra-LPS was weak, and induction by Rc- and Re-LPS was minimal. Re-LPS suppression of WT-LPS-mediated CXCL10 induction in RSCs and peripheral blood monocytes (PBMs) was dose dependent. Furthermore, Re-LPS completely suppressed cit-Fb-mediated CXCL10 induction in RSCs and PBMs. CONCLUSION: To easily identify LPS contamination during routine experiments, our results suggest that Re-LPS is a better tool for rapid detection of LPS contamination compared to PMB and heat treatment.

O-glycosylated HBsAg peptide can induce specific antibody neutralizing HBV infection.

BACKGROUND: Hepatitis B virus (HBV), which causes hepatitis, liver cirrhosis, and hepatocellular carcinoma, is a global human health problem. HBV contains three envelope proteins, S-, M-, and L-hepatitis B surface antigen (HBsAg). We recently found that O-glycosylated M-HBsAg, reactive with jacalin lectin, is one of the primary components of HBV DNA-containing virus particles. Thus, we aimed to analyze and target the glycosylation of HBsAg. METHODS: HBsAg prepared from the serum of Japanese patients with HBV were analyzed using mass spectrometry. The glycopeptide modified with O-glycan was generated and used for immunization. The specificity of the generated antibody and the HBV infection inhibition activity was examined. RESULTS: Mass spectrometry analysis revealed that T37 and/or T38 on M-HBsAg of genotype C were modulated by ±NeuAc(α2,3)Gal(ß1,3)GalNAc. Chemically and enzymatically synthesized O-glycosylated peptide (Glyco-PS2) induced antibodies that recognize mainly PreS2 in M-HBsAg not in L-HBsAg, whereas the non-glycosylated peptide (PS2) induced antisera recognizing L-HBsAg but not O-glycosylated M-HBsAg. The removal of O-glycan from M-HBsAg partly decreased the reactivity of the Glyco-PS2 antibody, suggesting that peptide part was also recognized by the antibody. The antibody further demonstrated the inhibition of HBV infection in human hepatic cells in vitro. CONCLUSIONS: Glycosylation of HBsAg occurs differently in different HBsAgs in a site-specific manner. The new Glyco-PS2 antibody, recognizing O-glycosylated M-HBsAg of genotype C, could inhibit HBV infection. GENERAL SIGNIFICANCE: The detailed analysis of HBsAg identified different glycosylations of HBV surface. The glycosylated peptide based on mass spectrometry analysis showed higher potential to induce functional antibody against HBV.