Publications by authors named "Yoshimasa Takahashi"

103 Publications

Development of an Inflammatory CD14 Dendritic Cell Subset in Humanized Mice.

Front Immunol 2021 15;12:643040. Epub 2021 Mar 15.

Department of Immunology, National Institute of Infectious Diseases, Tokyo, Japan.

Humanized mouse models are attractive experimental models for analyzing the development and functions of human dendritic cells (DCs) . Although various types of DC subsets, including DC type 3 (DC3s), have been identified in humans, it remains unclear whether humanized mice can reproduce heterogeneous DC subsets. CD14, classically known as a monocyte/macrophage marker, is reported as an indicator of DC3s. We previously observed that some CD14 myeloid cells expressed CD1c, a pan marker for conventional DC2 (cDC2s), in humanized mouse models in which human and genes were transiently expressed using transfection (IVT). Here, we aimed to elucidate the identity of CD14CD1c DC-like cells in humanized mouse models. We found that CD14CD1c cells were phenotypically different from cDC2s; CD14CD1c cells expressed CD163 but not CD5, whereas cDC2s expressed CD5 but not CD163. Furthermore, CD14CD1c cells primed and polarized naïve CD4 T cells toward IFN-γ Th1 cells more profoundly than cDC2s. Transcriptional analysis revealed that CD14CD1c cells expressed several DC3-specific transcripts, such as CD163, S100A8, and S100A9, and were clearly segregated from cDC2s and monocytes. When lipopolysaccharide was administered to the humanized mice, the frequency of CD14CD1c cells producing IL-6 and TNF-α was elevated, indicating a pro-inflammatory signature. Thus, humanized mice are able to sustain development of functional CD14CD1c DCs, which are equivalent to DC3 subset observed in humans, and they could be useful for analyzing the development and function of DC3s .
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http://dx.doi.org/10.3389/fimmu.2021.643040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005643PMC
March 2021

Association of HLA-DRB1*09:01 with severe COVID-19.

HLA 2021 Mar 18. Epub 2021 Mar 18.

AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan.

HLA-A, -C, -B, and -DRB1 genotypes were analyzed in 178 Japanese COVID-19 patients to investigate the association of HLA with severe COVID-19. Analysis of 32 common HLA alleles at four loci revealed a significant association between HLA-DRB1*09:01 and severe COVID-19 (odds ratio [OR], 3.62; 95% CI, 1.57-8.35; p = 0.00251 [permutation p value = 0.0418]) when age, sex, and other common HLA alleles at the DRB1 locus were adjusted. The DRB1*09:01 allele was more significantly associated with risk for severe COVID-19 compared to preexisting medical conditions such as hypertension, diabetes, and cardiovascular diseases. These results indicate a potential role for HLA in predisposition to severe COVID-19.
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http://dx.doi.org/10.1111/tan.14256DOI Listing
March 2021

MRC5 cells engineered to express ACE2 serve as a model system for the discovery of antivirals targeting SARS-CoV-2.

Sci Rep 2021 03 8;11(1):5376. Epub 2021 Mar 8.

Drug Discovery and Disease Research Laboratory, Shionogi & Co., Ltd, Osaka, Japan.

Although the spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in a worldwide pandemic, there are currently no virus-specific drugs that are fully effective against SARS-CoV-2. Only a limited number of human-derived cells are capable of supporting SARS-CoV-2 replication and the infectivity of SARS-CoV-2 in these cells remains poor. In contrast, monkey-derived Vero cells are highly susceptibility to infection with SARS-CoV-2, although they are not suitable for the study of antiviral effects by small molecules due to their limited capacity to metabolize drugs compared to human-derived cells. In this study, our goal was to generate a virus-susceptible human cell line that would be useful for the identification and testing of candidate drugs. Towards this end, we stably transfected human lung-derived MRC5 cells with a lentiviral vector encoding angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2. Our results revealed that SARS-CoV-2 replicates efficiently in MRC5/ACE2 cells. Furthermore, viral RNA replication and progeny virus production were significantly reduced in response to administration of the replication inhibitor, remdesivir, in MRC5/ACE2 cells compared with Vero cells. We conclude that the MRC5/ACE2 cells will be important in developing specific anti-viral therapeutics and will assist in vaccine development to combat SARS-CoV-2 infections.
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http://dx.doi.org/10.1038/s41598-021-84882-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940632PMC
March 2021

Myeloid cell dynamics correlating with clinical outcomes of severe COVID-19 in Japan.

Int Immunol 2021 03;33(4):241-247

Department of Immunology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo, Japan.

An expanded myeloid cell compartment is a hallmark of severe coronavirus disease 2019 (COVID-19). However, data regarding myeloid cell expansion have been collected in Europe, where the mortality rate by COVID-19 is greater than those in other regions including Japan. Thus, characteristics of COVID-19-induced myeloid cell subsets remain largely unknown in the regions with low mortality rates. Here, we analyzed cellular dynamics of myeloid-derived suppressor cell (MDSC) subsets and examined whether any of them correlate with disease severity and prognosis, using blood samples from Japanese COVID-19 patients. We observed that polymorphonuclear (PMN)-MDSCs, but not other MDSC subsets, transiently expanded in severe cases but not in mild or moderate cases. Contrary to previous studies in Europe, this subset selectively expanded in survivors of severe cases and subsided before discharge, but such transient expansion was not observed in non-survivors in Japanese cohort. Analysis of plasma cytokine/chemokine levels revealed positive correlation of PMN-MDSC frequencies with IL-8 levels, indicating the involvement of IL-8 on recruitment of PMN-MDSCs to peripheral blood following the onset of severe COVID-19. Our data indicate that transient expansion of the PMN-MDSC subset results in improved clinical outcome. Thus, this myeloid cell subset may be a predictor of prognosis in cases of severe COVID-19 in Japan.
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http://dx.doi.org/10.1093/intimm/dxab005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928855PMC
March 2021

Incomplete humoral response including neutralizing antibodies in asymptomatic to mild COVID-19 patients in Japan.

Virology 2021 03 6;555:35-43. Epub 2021 Jan 6.

Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, 160-8582, Japan.

The pandemic of COVID-19 is still ongoing, and many studies on serum antibodies have been reported, however, there are few studies about asymptomatic and mild patients. In this study, we enrolled 44 COVID-19 patients with relatively mild disease and 48 pre-pandemic controls. We measured serum antibodies against extracellular domain, S1 domain, and receptor-binding domain of Spike and N protein, examined neutralization titers by authentic virus neutralization assay and newly-developed bead/cell-based Spike-ACE2 inhibition assay, and compared them with clinical features. Most of these antibodies, including neutralizing titers, were mutually correlated, and the production of antibodies were associated with low Ct values of PCR test, disease severity, symptoms especially pneumonia, lymphopenia, and serological test including CRP, LD, D-dimer, and procalcitonin. Notably, 87.5% of asymptomatic and 23.5% of mild patients did not have antibody against SARS-CoV-2. Our results revealed the inadequate acquisition of humoral immunity in patients with asymptomatic and mild COVID-19 patients.
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http://dx.doi.org/10.1016/j.virol.2020.12.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7787511PMC
March 2021

Tissue-resident CD4 T helper cells assist the development of protective respiratory B and CD8 T cell memory responses.

Sci Immunol 2021 Jan;6(55)

Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA.

Much remains unknown about the roles of CD4 T helper cells in shaping localized memory B cell and CD8 T cell immunity in the mucosal tissues. Here, we report that lung T helper cells provide local assistance for the optimal development of tissue-resident memory B and CD8 T cells after the resolution of primary influenza virus infection. We have identified a population of T cells in the lung that exhibit characteristics of both follicular T helper and T cells, and we have termed these cells as resident helper T (T) cells. Optimal T cell formation was dependent on transcription factors involved in T follicular helper and resident memory T cell development including BCL6 and Bhlhe40. We show that T cells deliver local help to CD8 T cells through IL-21-dependent mechanisms. Our data have uncovered the presence of a tissue-resident helper T cell population in the lung that plays a critical role in promoting the development of protective B cell and CD8 T cell responses.
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http://dx.doi.org/10.1126/sciimmunol.abb6852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056937PMC
January 2021

An influenza HA stalk reactive polymeric IgA antibody exhibits anti-viral function regulated by binary interaction between HA and the antibody.

PLoS One 2021 7;16(1):e0245244. Epub 2021 Jan 7.

Department of Pathology, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.

IgA antibodies, which are secreted onto the mucosal surface as secretory IgA antibodies (SIgAs), play an important role in preventing influenza virus infection. A recent study reported that anti-hemagglutinin (HA) head-targeting antibodies increase anti-viral functions such as hemagglutination inhibition (HI) and virus neutralization (NT), in addition to HA binding activity (reactivity) via IgA polymerization. However, the functional properties of anti-viral IgA antibodies with mechanisms of action distinct from those of anti-HA head-targeting antibodies remain elusive. Here, we characterized the functional properties of IgG, monomeric IgA, and polymeric IgA anti-HA stalk-binding clones F11 and FI6, and B12 (a low affinity anti-HA stalk clone), as well as Fab-deficient (ΔFab) IgA antibodies. We found that IgA polymerization impacts the functional properties of anti-HA stalk antibodies. Unlike anti-HA head antibodies, the anti-viral functions of anti-HA stalk antibodies were not simply enhanced by IgA polymerization. The data suggest that two modes of binding (Fab paratope-mediated binding to the HA stalk, and IgA Fc glycan-mediated binding to the HA receptor binding site (RBS)) occur during interaction between anti-stalk HA IgA antibodies and HA. In situations where Fab paratope-mediated binding to the HA stalk exceeded IgA Fc glycan-mediated binding to HA RBS, IgA polymerization increased anti-viral functions. By contrast, when IgA Fc glycan-mediated binding to the HA RBS was dominant, anti-viral activity will fall upon IgA polymerization. In summary, the results suggest that coordination between these two independent binding modules determines whether IgA polymerization has a negative or positive effect on the anti-viral functions of anti-HA stalk IgA antibodies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0245244PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790537PMC
January 2021

Identification of Two Critical Neutralizing Epitopes in the Receptor Binding Domain of Hepatitis B Virus preS1.

J Virol 2020 Dec 9. Epub 2020 Dec 9.

Department of Virology II, National Institute of Infectious Diseases

Hepatitis B virus (HBV) infection is a major public health problem. Human hepatocytes are infected with HBV via binding between the preS1 region in the large envelope protein of HBV and sodium taurocholate cotransporting polypeptide. Although several monoclonal antibodies (MAbs) that recognize the receptor binding domain in preS1 and neutralize HBV infection have been isolated, details of neutralizing epitopes are not understood. In this study, we generated 13 MAbs targeting the preS1 receptor binding domain from preS1-specific memory B cells derived from DNA immunized mice. The MAbs were classified into three groups according to the epitope regions, designated epitopes I-III. A virus neutralization assay revealed that MAbs recognizing epitopes I and III neutralized HBV infection, suggesting that these domains are critical epitopes for viral neutralization. In addition, a neutralization assay against multiple genotypes of HBV revealed that epitope I is a semi-pangenotypic neutralizing epitope, whereas epitope III is a genotype-specific epitope. We also showed that neutralizing MAbs against preS1 could neutralize HBV bearing vaccine-induced escape mutation. These findings provide insight into novel immunoprophylaxis for the prevention and treatment of HBV infection. The HBV preS1 2-47 aa region (preS1/2-47) is essential for virus binding with sodium taurocholate cotransporting polypeptide. Several MAbs targeting preS1/2-47 have been reported to neutralize HBV infection; however, which region in preS1/2-47 contains the critical neutralizing epitope for HBV infection is unclear. Here, we generated several MAbs targeting preS1/2-47 and found that MAbs recognizing the N- or C-terminus of preS1/2-47 remarkably neutralized HBV infection. We further confirmed the neutralizing activity of anti-preS1 MAbs against HBV with vaccine escape mutation. These data clarified the relationship between the antibody epitope and the virus neutralizing activity and also suggested the potential ability of a vaccine antigen containing the preS1 region to overcome the weakness of current HB vaccines comprising the small S protein.
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http://dx.doi.org/10.1128/JVI.01680-20DOI Listing
December 2020

Majority of alpha2,6-sialylated glycans in adult mouse brain exist in O-glycans: SALSA-MS analysis for knockout mice of alpha2,6-sialyltransferase genes.

Glycobiology 2020 Nov 25. Epub 2020 Nov 25.

Department of Biomedical Sciences, Chubu University College of Life and Health Sciences, Kasugai, Aichi 487-8501, Japan.

Sialic acids are unique sugars with negative charge and exert various biological functions such as regulation of immune systems, maintenance of nerve tissues and expression of malignant properties of cancers. Alpha 2,6 silalylated N-glycans, one of representative sialylation forms, are synthesized by St6gal1 or St6gal2 gene products in human and mouse. Previously, it has been reported that St6gal1 gene is ubiquitously expressed in almost all tissues. On the other hand, St6gal2 gene is expressed mainly in the embryonic and perinatal stages of brain tissues. However, roles of St6gal2 gene have not been clarified. Expression profiles of N-glycans with terminal α2,6 sialic acid generated by St6gal gene products in the brain have never been directly studied. Using conventional lectin blotting and novel sialic acid linkage specific alkylamidation-mass spectrometry method, we investigated function and expression of St6gal genes and profiles of their products in adult mouse brain by establishing knockout mice lacking St6gal1 gene, St6gal2 gene, or both of them (double knockout). Consequently, α2,6-sialylated N-glycans were scarcely detected in adult mouse brain tissues, and majority of α2,6-sialylated glycans found in mouse brain were O-linked glycans. Majority of these α2,6-sialylated O-glycans were shown to be disialyl-T antigen and sialyl-(6)T antigen by mass spectrometry analysis. Moreover, it was revealed that a few α2,6-sialylated N-glycans were produced by the action of St6gal1 gene, despite that both St6gal1 and St6gal2 genes were expressed in the adult mouse brain. In the future, where and how sialylated O-linked glycoproteins function in the brain tissue remains to be clarified.
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http://dx.doi.org/10.1093/glycob/cwaa105DOI Listing
November 2020

Progress in environmental high-voltage transmission electron microscopy for nanomaterials.

Philos Trans A Math Phys Eng Sci 2020 Dec 26;378(2186):20190602. Epub 2020 Oct 26.

Institute of Materials and Systems for Sustainability (IMaSS), Nagoya University, Nagoya 464-8603, Japan.

A new environmental high-voltage transmission electron microscope (E-HVEM) was developed by Nagoya University in collaboration with JEOL Ltd. An open-type environmental cell was employed to enable in-situ observations of chemical reactions on catalyst particles as well as mechanical deformation in gaseous conditions. One of the reasons for success was the application of high-voltage transmission electron microscopy to environmental (in-situ) observations in the gas atmosphere because of high transmission of electrons through gas layers and thick samples. Knock-on damages to samples by high-energy electrons were carefully considered. In this paper, we describe the detailed design of the E-HVEM, recent developments and various applications. This article is part of a discussion meeting issue 'Dynamic microscopy relating structure and function'.
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http://dx.doi.org/10.1098/rsta.2019.0602DOI Listing
December 2020

Substantial induction of non-apoptotic CD4 T-cell death during the early phase of HIV-1 infection in a humanized mouse model.

Microbes Infect 2021 Jan-Feb;23(1):104767. Epub 2020 Oct 10.

Department of Immunology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan; Department of Medical Technology, School of Human Sciences, Tokyo University of Technology, 5-23-22 Nishikamata, Ota-ku, Tokyo, 144-8535, Japan.

Several mechanisms underline induction of CD4 T-cell death by human immunodeficiency virus (HIV) infection. For a long time, apoptosis was considered central to cell death involved in the depletion of CD4 T cells during HIV infection. However, which types of cell death are induced during the early phase of HIV infection in vivo remains unclear. In this study, CD4 T-cell death induced in early HIV infection was characterized using humanized mice challenged with CCR5-tropic (R5) or CXCR4-tropic (X4) HIV-1. Results showed that CD4 T-cell death was induced in the spleen 3 days post-challenge with both R5 and X4 HIV-1. Although cell death without caspase-1 and caspase-3/7 activation was preferentially observed, caspase-1 pyroptosis was also significantly induced within the memory subpopulation by R5 or X4 HIV-1 and the naïve subpopulation by X4 HIV-1. In contrast, caspase-3/7 apoptosis was not enhanced by either R5 or X4 HIV-1. Furthermore, phosphorylated mixed lineage kinase domain-like protein necroptosis was induced by only X4 HIV-1. These findings indicate that various types of non-apoptotic CD4 T-cell death, such as pyroptosis and necroptosis, are induced during the early phase of HIV infection in vivo.
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http://dx.doi.org/10.1016/j.micinf.2020.10.003DOI Listing
October 2020

Standardization of the first Korean national reference standard for snake () antivenom.

Toxicol Res 2020 Oct 14;36(4):407-413. Epub 2020 Apr 14.

Blood Products Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex 187, Osongsaengmyeong-2-ro, Heungdeok-gu, Cheongju, 28159 Korea.

In 2017, the second national reference standard (NRS) for Gloydius snake venom was established to replace the first NRS for Gloydius snake venom. In connection with the second venom NRS, a candidate for the first NRS for Gloydius snake antivenom was produced in 2017. In this study, the qualification of the candidate was estimated and the potency was determined by a collaborative study. The potency (anti-lethal titer and anti-hemorrhagic titer) of the candidate was determined by measuring the capability of the antivenom to neutralize the lethal and hemorrhagic effects of the second NRS for Gloydius snake venom, which was calibrated against the regional reference standard for Gloydius snake antivenom established in 2006. Two Korean facilities contributed data from 20 independent assays. Subsequently, one foreign national control research laboratories participated in this collaborative study. The general common potency of the anti-lethal and anti-hemorrhagic titers was obtained from the results of a total of 25 tests performed at three facilities. According to the results of the present study, the candidate preparation showed good quality and is judged to be suitable to serve as the first NRS for Gloydius snake antivenom with the following potency: an anti-lethal titer of 3100 unit (U) (95% confidence interval 2991-3276 U) and anti-hemorrhagic titer of 3000 U (95% confidence interval 2849-3159 U). In conclusion, the first NRS for Gloydius snake antivenom was established in this study. This reference standard will be used routinely for quality control of a snake antivenom product by manufacturer in Korea, which also can be used for national quality control, including a national lot-release test of the snake antivenom product.
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http://dx.doi.org/10.1007/s43188-020-00047-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494687PMC
October 2020

Frequent administration of abaloparatide shows greater gains in bone anabolic window and bone mineral density in mice: A comparison with teriparatide.

Bone 2021 Jan 18;142:115651. Epub 2020 Sep 18.

Developmental Biology of Hard Tissue, Graduate School of Dental Medicine, Hokkaido University, Sapporo, Japan.

Abaloparatide (ABL) is a novel 34-amino acid peptide analog of parathyroid hormone-related protein. In clinical studies, although ABL showed a greater bone mineral density (BMD) increase than teriparatide (TPTD, human parathyroid hormone 1-34), the responses of ABL to bone formation and resorption markers were weaker, making it difficult to understand the relationship between the bone anabolic window (increase in bone formation versus resorption) and bone mass. In the present study, the effects of ABL and TPTD were compared in mice. Given that the rate of bone turnover is higher in rodents than in humans, the comparison was made with several administration regimens providing equivalent daily dosages: once daily (QD, 30 μg/kg every 24 h), twice daily (BID, 15 μg/kg every 12 h), or three times a day (TID, 10 μg/kg every 8 h). Frequent administration of ABL showed higher BMD with enhancement of trabecular and cortical bone mass and structures than that of TPTD, consistent with the clinical results seen with once daily administration. ABL increased bone formation marker levels more than TPTD with more frequent regimens, while bone resorption marker levels were not different between ABL and TPTD in all regimens. Analysis of bone histomorphometry and gene expression also suggested that ABL increased bone formation more than TPTD, while the effect on bone resorption was almost comparable between ABL and TPTD. The bone anabolic windows calculated from bone turnover markers indicated that ABL enhanced the anabolic windows more than TPTD, leading to a robust increase in BMD. The mechanism by which ABL showed a better balance of bone turnover was suggested to be partly due to the enhanced remodeling-based bone formation involved in Ephb4. Taken together, our findings would help elucidate the mechanism by which ABL shows excellent BMD gain and reduction of fractures in patients with osteoporosis.
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http://dx.doi.org/10.1016/j.bone.2020.115651DOI Listing
January 2021

An anti-perfringolysin O monoclonal antibody cross-reactive with streptolysin O protects against streptococcal toxic shock syndrome.

BMC Res Notes 2020 Sep 5;13(1):419. Epub 2020 Sep 5.

Department of Immunology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan.

Objective: Streptococcus pyogenes (Group A Streptococcus; GAS) causes a variety of infections that include life-threatening, severe invasive GAS infections, such as streptococcal toxic shock syndrome (STSS), with > 30% mortality rate, despite effective antibiotics and treatment options. STSS clinical isolates highly express streptolysin O (SLO), a member of a large family of pore-forming toxins called cholesterol-dependent cytolysins (CDCs). SLO is an important toxic factor for GAS and may be an effective therapeutic target for the treatment of STSS. Our aim was to identify a monoclonal antibody (mAb) that reacts with SLO and has therapeutic potential for STSS treatment.

Results: We focused on mAbs that had originally been established as neutralizing reagents to perfringolysin O (PFO), another member of the CDC family, as some cross-reactivity with SLO had been reported. Here, we confirmed cross-reactivity of an anti-PFO mAb named HS1 with SLO. In vitro analysis revealed that HS1 mAb sufficiently prevented human neutrophils from being killed by STSS clinical isolates. Furthermore, prophylactic and therapeutic injection of HS1 mAb into C57BL/6 mice significantly improved the survival rate following lethal infection with an STSS clinical isolate. These results highlight the therapeutic potential of HS1 mAb for STSS treatment.
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http://dx.doi.org/10.1186/s13104-020-05264-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7487723PMC
September 2020

Bim establishes the B cell repertoire from early to late in the immune response.

Int Immunol 2020 Sep 5. Epub 2020 Sep 5.

Drug Discovery Antibody Platform Unit, RIKEN Research Center for Integrative Medical Sciences, Suehiro-cho, Tsurumi, Yokohama, Kanagawa, Japan.

In T cell-dependent antibody responses, some of the activated B cells differentiate along extrafollicular pathways into low-affinity memory and plasma cells, whereas others are involved in subsequent GC formation in follicular pathways, in which somatic hypermutation and affinity maturation occur. The present study demonstrated that Bim, a proapoptotic BH3-only member of the Bcl-2 family, contributes to the establishment of the B cell repertoire from early to late stages of immune responses to T-cell dependent antigens. Extrafollicular plasma cells grew in the spleen during the early immune response, but their numbers rapidly declined with the appearance of GC-derived progeny in wild type mice. By contrast, conditional Bim deficiency in B cells resulted in expansion of extrafollicular IgG1 + antibody-forming cells (AFCs) and this expansion was sustained during the late response, which hampered the formation of GC-derived high-affinity plasma cells in the spleen. Approximately 10% of AFCs in mutant mice contained mutated VH genes, thus Bim deficiency appears not to impede the selection of high-affinity AFC precursor cells. These results suggest that Bim contributes to the replacement of low affinity antibody by high affinity antibody as the immune response progresses.
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http://dx.doi.org/10.1093/intimm/dxaa060DOI Listing
September 2020

Stereotyped B-cell response that counteracts antigenic variation of influenza viruses.

Int Immunol 2020 09;32(9):613-621

Department of Immunology, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.

Influenza A subtypes are categorized into group 1 and group 2 based on the hemagglutinin (HA) sequence. Owing to the phylogenetic distance of HAs in different groups, antibodies that bind multiple HA subtypes across different groups are extremely rare. In this study, we demonstrated that an immunization with acid-treated HA antigen elicits germinal center (GC) B cells that bind multiple HA subtypes in both group 1 and group 2. The cross-group GC B cells utilized mostly one VH gene (1S56) and exhibited a sign of clonal evolution within GCs. The 1S56-lineage IgGs derived from GC B cells were able to bind to HA protein on the infected cell surface but not to the native form of HA protein, suggesting the cryptic nature of the 1S56 epitope and its exposure in infected cells. Finally, the 1S56-lineage IgGs provided protection against lethal infection in an Fc-dependent manner, independent of the virus-neutralizing activity. Thus, we identified 1S56-lineage antibodies as a unique stereotype for achieving cross-group influenza specificity. The antigens exposing the 1S56 epitope may be good candidates for broadly protective immunogens.
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http://dx.doi.org/10.1093/intimm/dxaa038DOI Listing
September 2020

Diagnosis of NTM active infection in lymphadenopathy patients with anti-interferon-gamma auto-antibody using inhibitory ELISA vs. indirect ELISA.

Sci Rep 2020 06 2;10(1):8968. Epub 2020 Jun 2.

Cellular and Molecular Immunology Unit, Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand.

The anti-interferon-gamma (IFN-gamma) autoantibody is a known cause of opportunistic non-tuberculous mycobacterial (NTM) infection in adults. Diagnosis of those patients is difficult due to the low sensitivity of bacterial culture, and because detection of the neutralizing autoantibody needs special laboratory devices. We conducted a retrospective review of indirect and inhibitory ELISA, both used for detection of anti-IFN-gamma auto-antibody in 102 patients with lymphadenopathies. We assessed hospital records of NTM isolation and/or diagnosis of NTM infection. The review revealed the compatible sensitivity and superior specificity and predictive values for inhibitory ELISA over against indirect ELISA-the latter achieving 100% specificity and positive predictive value for diagnosis of NTM infection in patients with lymphadenopathies. The results confirm functional assays that show plasma samples from NTM-infected patients with positive results by either indirect and/or inhibitory ELISA are IFN-gamma neutralizing autoantibodies. The inhibitory titer of anti-IFN-gamma auto-antibody can be used to distinguish patients with active from inactive NTM infection. Inhibitory ELISA is thus a practical, rapid, high performance tool for routine detection of anti-IFN-gamma autoantibody and NTM infection diagnosis before confirmation, enabling a timely therapeutic strategy for active infection treatment.
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http://dx.doi.org/10.1038/s41598-020-65933-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265449PMC
June 2020

Respiratory syncytial virus infection exacerbates pneumococcal pneumonia via Gas6/Axl-mediated macrophage polarization.

J Clin Invest 2020 06;130(6):3021-3037

Department of Immunology, National Institute of Infectious Diseases, Tokyo, Japan.

Patients with respiratory syncytial virus (RSV) infection exhibit enhanced susceptibility to subsequent pneumococcal infections. However, the underlying mechanisms involved in this increased susceptibility remain unclear. Here, we identified potentially novel cellular and molecular cascades triggered by RSV infection to exacerbate secondary pneumococcal pneumonia. RSV infection stimulated the local production of growth arrest-specific 6 (Gas6). The Gas6 receptor Axl was crucial for attenuating pneumococcal immunity in that the Gas6/Axl blockade fully restored antibacterial immunity. Mechanistically, Gas6/Axl interaction regulated the conversion of alveolar macrophages from an antibacterial phenotype to an M2-like phenotype that did not exhibit antibacterial activity, and the attenuation of caspase-1 activation and IL-18 production in response to pneumococcal infection. The attenuated IL-18 production failed to drive both NK cell-mediated IFN-γ production and local NO and TNF-α production, which impair the control of bacterial infection. Hence, the RSV-mediated Gas6/Axl activity attenuates the macrophage-mediated protection against pneumococcal infection. The Gas6/Axl axis could be a potentially novel therapeutic target for RSV-associated secondary bacterial infection.
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http://dx.doi.org/10.1172/JCI125505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260035PMC
June 2020

Memory B Cells in Local and Systemic Sites.

Adv Exp Med Biol 2020 ;1254:55-62

Department of Immunology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo, 162-8640, Japan.

Memory B cells are a key cellular component of the protective humoral responses to infectious pathogens. Most of our knowledge of memory B-cell responses comes from studies using mono-epitopic model antigens that elicit systemic humoral responses dominated by canonical B-cell antigen receptors. This approach successfully dissected the systemic responses of memory B cells and greatly advanced our understanding of memory B-cell formation, maintenance, and reactivation to re-invading antigens in the secondary lymphoid organs. However, the canonical memory B-cell responses fail to fully recapitulate the heterogeneity of the protective memory responses. Indeed, accumulating studies using "natural" antigens and live pathogens have uncovered new aspects of memory B-cell responses, which are achieved by memory B cells with different phenotypes, tissue residence, and responsiveness to antigen stimulation. Such non-canonical memory B-cell responses are frequently observed in local sites where live pathogens initially infect and replicate. Importantly, the local memory B-cell responses often serve as the first line of defense against re-infecting pathogens, thereby playing an essential role in controlling the pathogens. Here, we provide a comprehensive overview of the systemic and local memory B-cell responses in the humoral protective immunity against pathogens.
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http://dx.doi.org/10.1007/978-981-15-3532-1_5DOI Listing
July 2020

Better Epitope Discovery, Precision Immune Engineering, and Accelerated Vaccine Design Using Immunoinformatics Tools.

Front Immunol 2020 7;11:442. Epub 2020 Apr 7.

EpiVax, Inc., Providence, RI, United States.

Computational vaccinology includes epitope mapping, antigen selection, and immunogen design using computational tools. Tools that facilitate the prediction of immune response to biothreats, emerging infectious diseases, and cancers can accelerate the design of novel and next generation vaccines and their delivery to the clinic. Over the past 20 years, vaccinologists, bioinformatics experts, and advanced programmers based in Providence, Rhode Island, USA have advanced the development of an integrated toolkit for vaccine design called iVAX, that is secure and user-accessible by internet. This integrated set of immunoinformatic tools comprises algorithms for scoring and triaging candidate antigens, selecting immunogenic and conserved T cell epitopes, re-engineering or eliminating regulatory T cell epitopes, and re-designing antigens to induce immunogenicity and protection against disease for humans and livestock. Commercial and academic applications of iVAX have included identifying immunogenic T cell epitopes in the development of a T-cell based human multi-epitope Q fever vaccine, designing novel influenza vaccines, identifying cross-conserved T cell epitopes for a malaria vaccine, and analyzing immune responses in clinical vaccine studies. Animal vaccine applications to date have included viral infections of pigs such as swine influenza A, PCV2, and African Swine Fever. "Rapid-Fire" applications for biodefense have included a demonstration project for Lassa Fever and Q fever. As recent infectious disease outbreaks underscore the significance of vaccine-driven preparedness, the integrated set of tools available on the iVAX toolkit stand ready to help vaccine developers deliver genome-derived, epitope-driven vaccines.
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http://dx.doi.org/10.3389/fimmu.2020.00442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7154102PMC
March 2021

Hide and seek: interplay between influenza viruses and B cells.

Int Immunol 2020 09;32(9):605-611

Department of Immunology, National Institute of Infectious Diseases, Tokyo, Japan.

Influenza virus constantly acquires genetic mutations/reassortment in the major surface protein, hemagglutinin (HA), resulting in the generation of strains with antigenic variations. There are, however, HA epitopes that are conserved across influenza viruses and are targeted by broadly protective antibodies. A goal for the next-generation influenza vaccines is to stimulate B-cell responses against such conserved epitopes in order to provide broad protection against divergent influenza viruses. Broadly protective B cells, however, are not easily activated by HA antigens with native structure, because the virus has multiple strategies to escape from the humoral immune responses directed to the conserved epitopes. One such strategy is to hide the conserved epitopes from the B-cell surveillance by steric hindrance. Technical advancement in the analysis of the human B-cell antigen receptor (BCR) repertoire has dissected the BCRs to HA epitopes that are hidden in the native structure but are targeted by broadly protective antibodies. We describe here the characterization and function of broadly protective antibodies and strategies that enable B cells to seek these hidden epitopes, with potential implications for the development of universal influenza vaccines.
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http://dx.doi.org/10.1093/intimm/dxaa028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7478158PMC
September 2020

Metformin-induced suppression of IFN-α via mTORC1 signalling following seasonal vaccination is associated with impaired antibody responses in type 2 diabetes.

Sci Rep 2020 02 24;10(1):3229. Epub 2020 Feb 24.

Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.

Diabetes mellitus (DM) patients are at an increased risk of complications following influenza-virus infection, seasonal vaccination (SV) is recommended. However, SV with trivalent influenza vaccine (TIV) can induce antibody and type-I interferon (IFN) responses, and the effect of anti-DM treatment on these responses is incompletely understood. We evaluated the antibody response and IFN-α expression in individuals with and without type 2 DM (T2DM) following SV, and examined the effects on anti-DM treatment. TIV elicited sero-protection in all groups, but antibody persistency was <8 months, except for the antibody response to B-antigens in non-DM. T2DM impaired the IgG avidity index, and T2DM showed a significantly decreased response against H1N1 and H3N2, in addition to delaying and reducing haemagglutination-inhibition persistency against influenza B-antigens in DM groups treated with metformin (Met-DM) or glibenclamide (GB-DM). Following TIV, the Met-DM and GB-DM groups exhibited reduced IFN-α expression upon stimulation with whole- and split-virion influenza vaccines. Suppression of IFN-α expression in the Met-DM group was associated with a reduction in the mechanistic target of rapamycin complex-1 pathway and impaired IgG avidity index. Thus, single-dose TIV each year might not be suitable for T2DM. Our data could aid the development of an efficacious influenza vaccine for T2DM.
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http://dx.doi.org/10.1038/s41598-020-60213-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039947PMC
February 2020

Immune-Focusing Properties of Virus-like Particles Improve Protective IgA Responses.

J Immunol 2019 12 8;203(12):3282-3292. Epub 2019 Nov 8.

Department of Immunology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan;

Virus-like particles (VLPs) provide a well-established vaccine platform; however, the immunogenic properties acquired by VLP structure remain poorly understood. In this study, we showed that systemic vaccination with norovirus VLP recalls human IgA responses at higher magnitudes than IgG responses under a humanized mouse model that was established by introducing human PBMCs in severely immunodeficient mice. The recall responses elicited by VLP vaccines depended on VLP structure and the disruption of VLP attenuated recall responses, with a more profound reduction being observed in IgA responses. The IgA-focusing property was also conserved in a murine norovirus-primed model under which murine IgA responses were recalled in a manner dependent on VLP structure. Importantly, the VLP-driven IgA response preferentially targeted virus-neutralizing epitopes located in the receptor-binding domain. Consequently, VLP-driven IgA responses were qualitatively superior to IgG responses in terms of the virus-neutralizing activity in vitro. Furthermore, the IgA in mucosa obtained remarkable protective function toward orally administrated virus in vivo. Thus, our results indicate the immune-focusing properties of the VLP vaccine that improve the quality/quantity of mucosal IgA responses, a finding with important implications for developing mucosal vaccines.
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http://dx.doi.org/10.4049/jimmunol.1900481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900486PMC
December 2019

Publisher Correction: A humanized mouse model identifies key amino acids for low immunogenicity of H7N9 vaccines.

Sci Rep 2019 Oct 8;9(1):14730. Epub 2019 Oct 8.

Department of Immunology, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-019-50828-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783480PMC
October 2019

Exposure of an occluded hemagglutinin epitope drives selection of a class of cross-protective influenza antibodies.

Nat Commun 2019 08 28;10(1):3883. Epub 2019 Aug 28.

Department of Immunology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo, 162-8640, Japan.

Germinal center (GC) B cells at viral replication sites acquire specificity to poorly immunogenic but conserved influenza hemagglutinin (HA) epitopes. Here, high-throughput epitope mapping of local GC B cells is used to identify conserved HA epitope selecting cross-reactive antibodies that mediate heterosubtypic protection. A distinct feature of this epitope is an occlusion in the naive trimeric HA structure that is exposed in the post-fusion HA structure to occur under low pH conditions during viral replication. Importantly, systemic immunization by the post-fusion HA antigen results in GC B cells targeting the occluded epitope, and induces a class of protective antibodies that have cross-group specificity and afford protection independent of virus neutralization activity. Furthermore, this class of broadly protective antibodies develops at late time points and persists. Our results identify a class of cross-protective antibodies that are selected at the viral replication site, and provide insights into vaccine strategies using the occluded epitope.
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http://dx.doi.org/10.1038/s41467-019-11821-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713747PMC
August 2019

Requirement for memory B-cell activation in protection from heterologous influenza virus reinfection.

Int Immunol 2019 11;31(12):771-779

Graduate School of Frontier Biosciences, WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan.

While two memory compartments, memory B cells and long-lived plasma cells, are thought to contribute to the successful establishment of memory recall responses, the unique roles of each cellular compartment are still unclear. Herein, by tracing influenza anti-hemagglutinin (HA)-specific antibodies in mice, we demonstrate that pre-existing antibodies secreted by long-lived plasma cells are essential for protection from reinfection with the same influenza virus, whereas protection from secondary infection with an antigenically distinct influenza virus requires memory B-cell activation. These activated memory B cells were largely specific for the conserved HA stem region, and generated sufficient levels of antibodies for protection from heterologous reinfection. Given that the anti-stem plasmablasts derived from the memory B cells were higher affinity than those from naive B cells, our results suggest that maturation of anti-stem memory B cells during primary influenza infection and their subsequent activation are required for protection from reinfection by mutant viruses.
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http://dx.doi.org/10.1093/intimm/dxz049DOI Listing
November 2019

Correction to: A CCR5 memory subset within HIV-1-infected primary resting CD4 T cells is permissive for replication-competent, latently infected viruses in vitro.

BMC Res Notes 2019 06 10;12(1):322. Epub 2019 Jun 10.

Department of Immunology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan.

After publication of the original article [1], the authors became aware of a miscalculation in the original Fig. 2d.
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http://dx.doi.org/10.1186/s13104-019-4357-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6556951PMC
June 2019

Artificial immunoglobulin light chain with potential to associate with a wide variety of immunoglobulin heavy chains.

Biochem Biophys Res Commun 2019 07 2;515(3):481-486. Epub 2019 Jun 2.

Department of Immunology, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan. Electronic address:

Immunoglobulins play important roles in antigen recognition during the immune response, and the complementarity-determining region (CDR) 3 of the heavy chain is considered as the critical antigen-binding site. We previously developed a statistical protocol for the extensive analysis of heavy chain variable region repertoires and the dynamics of their immune response using next-generation sequencing (NGS). The properties of important antibody heavy chains predicted in silico by the protocol were examined by gene synthesis and antibody protein expression; however, the corresponding light chain that matches with the heavy chain could not be predicted by our protocol. To understand the dynamics of the heavy chain and the effect of light chain pairing on it, we firstly tried to obtain an artificial light chain that pairs with a broad range of heavy chains and then analyzed its effect on the antigen binding of heavy chains upon pairing. During the pre-B cell stage, the surrogate light chain (SLC) could pair with the nascent immunoglobulin μ heavy chains (Ig-μH) and promote them to function in the periphery. On the basis of this property, we designed several versions of genetically engineered "common light chain" prototypes by modifying the SLC structure. Among them, the mouse-derived VpreB1λ5Cκ light chain showed acceptable matching property with several different heavy chains without losing specificity of the original heavy chains, though the antigen affinities were variable. The extent of matching depended on the heavy chain; surprisingly, a specific heavy chain (IGHV9-3) could match with two different conventional Vκs (IGKV3-2*01 and IGKV10-96*01) without losing the antigen affinities, whereas another heavy chain (IGHV1-72) completely lost its antigen affinities by the same matching. Thus, the results suggested that the antigen recognition of the heavy chain is variably affected by the paired light chain, and that the artificial light chain, Mm_VpreB1λ5Cκ, has the potential to be a "common light chain", providing a novel system to analyze the effects of light chains in antigen recognition of heavy chains.
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http://dx.doi.org/10.1016/j.bbrc.2019.05.149DOI Listing
July 2019

Influenza Antigen Engineering Focuses Immune Responses to a Subdominant but Broadly Protective Viral Epitope.

Cell Host Microbe 2019 Jun 16;25(6):827-835.e6. Epub 2019 May 16.

Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA; Department of Microbiology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Viral glycoproteins are under constant immune surveillance by a host's adaptive immune responses. Antigenic variation including glycan introduction or removal is among the mechanisms viruses have evolved to escape host immunity. Understanding how glycosylation affects immunodominance on complex protein antigens may help decipher underlying B cell biology. To determine how B cell responses can be altered by such modifications, we engineered glycans onto the influenza virus hemagglutinin (HA) and characterized the molecular features of the elicited humoral immunity in mice. We found that glycan addition changed the initially diverse antibody repertoire into an epitope-focused, genetically restricted response. Structural analyses showed that one antibody gene family targeted a previously subdominant, occluded epitope at the head interface. Passive transfer of this antibody conferred Fc-dependent protection to influenza virus-challenged mice. These results have potential implications for next-generation viral vaccines aimed at directing B cell responses to preferred epitope(s).
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http://dx.doi.org/10.1016/j.chom.2019.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748655PMC
June 2019

Antibodies to a Conserved Influenza Head Interface Epitope Protect by an IgG Subtype-Dependent Mechanism.

Cell 2019 05;177(5):1124-1135.e16

Department of Immunology, Duke University, Durham, NC 27710, USA; Duke Human Vaccine Institute, Duke University, Durham, NC 27710, USA. Electronic address:

Vaccines to generate durable humoral immunity against antigenically evolving pathogens such as the influenza virus must elicit antibodies that recognize conserved epitopes. Analysis of single memory B cells from immunized human donors has led us to characterize a previously unrecognized epitope of influenza hemagglutinin (HA) that is immunogenic in humans and conserved among influenza subtypes. Structures show that an unrelated antibody from a participant in an experimental infection protocol recognized the epitope as well. IgGs specific for this antigenic determinant do not block viral infection in vitro, but passive administration to mice affords robust IgG subtype-dependent protection against influenza infection. The epitope, occluded in the pre-fusion form of HA, is at the contact surface between HA head domains; reversible molecular "breathing" of the HA trimer can expose the interface to antibody and B cells. Antigens that present this broadly immunogenic HA epitope may be good candidates for inclusion in "universal" flu vaccines.
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http://dx.doi.org/10.1016/j.cell.2019.03.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825805PMC
May 2019