Extraction of the CDRH3 sequence of the mouse antibody repertoire selected upon influenza virus infection by subtraction of the background antibody repertoire.

Historically, antibody reactivity to pathogens and vaccine antigens has been evaluated using serological measurements of antigen-specific antibodies. However, it is difficult to evaluate all antibodies that contribute to various functions in a single assay, such as the measurement of the neutralizing antibody titer. Bulk antibody repertoire analysis using next-generation sequencing is a comprehensive method for analyzing the overall antibody response; however, it is unreliable for estimating antigen-specific antibodies due to individual variation. To address this issue, we propose a method to subtract the background signal from the repertoire of data of interest. In this study, we analyzed changes in antibody diversity and inferred the heavy-chain complementarity-determining region 3 (CDRH3) sequences of antibody clones that were selected upon influenza virus infection in a mouse model using bulk repertoire analysis. A decrease in the diversity of the antibody repertoire was observed upon viral infection, along with an increase in neutralizing antibody titers. Using kernel density estimation of sequences in a high-dimensional sequence space with background signal subtraction, we identified several clusters of CDRH3 sequences induced upon influenza virus infection. Most of these repertoires were detected more frequently in infected mice than in uninfected control mice, suggesting that infection-specific antibody sequences can be extracted using this method. Such an accurate extraction of antigen- or infection-specific repertoire information will be a useful tool for vaccine evaluation in the future. IMPORTANCE: As specific interactions between antigens and cell-surface antibodies trigger the proliferation of B-cell clones, the frequency of each antibody sequence in the samples reflects the size of each clonal population. Nevertheless, it is extremely difficult to extract antigen-specific antibody sequences from the comprehensive bulk antibody sequences obtained from blood samples due to repertoire bias influenced by exposure to dietary antigens and other infectious agents. This issue can be addressed by subtracting the background noise from the post-immunization or post-infection repertoire data. In the present study, we propose a method to quantify repertoire data from comprehensive repertoire data. This method allowed subtraction of the background repertoire, resulting in more accurate extraction of expanded antibody repertoires upon influenza virus infection. This accurate extraction of antigen- or infection-specific repertoire information is a useful tool for vaccine evaluation.

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

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.

Distorted antibody repertoire developed in the absence of pre-B cell receptor formation.

The pre-B cell receptor (pre-BCR), consisting of the µ heavy chain (µHC) and the surrogate light chain (SLC, Vpre-B and λ5), plays important roles during B cell development. The formation of the pre-BCR, which enables the nascent immunoglobulin HC to associate with the SLC, is considered a prerequisite for B cell development. However, a significant number of peripheral mature (leaky) B cells exist in SLC-deficient mice. These leaky B cells develop in the absence of pre-BCR and do not undergo the pre-BCR checkpoint. The antibody repertoires of leaky B cells thus reflect the absence of pre-BCR function. To investigate how the absence of the pre-BCR is circumvented by these leaky-B cells and examine the effect of the pre-BCR checkpoint on the antibody system, we analyzed the antibody repertoires of λ5-deficient (λ5-/-) mice using next-generation sequencing. In λ5-/- mice, spleen B cells displayed different patterns of VDJ-usage, relative to those in wild-type (WT) mice. Moreover, leaky B cells were neither derived from unusual B2 cells, characterized by particular LC gene rearrangements in the absence of pre-BCR signaling, nor from B1 cells, originating from different B cell progenitors. Analysis of the CDR-H3 amino acid sequences of µ-chain repertoires revealed that certain bone marrow B cells with particular CDR-H3 profiles undergo clonal expansion in λ5-/- mice. Part of these CDR-H3s contain arginine(s) in the middle of the CDR-H3 loop in λ5-/- mice, whereas few arginine(s) exist in this middle loop in WT CDR-H3s in the absence of clonal expansion. This CDR-H3 feature in λ5-/- mice presumably reflects the role of the pre-BCR in autoantibody regulation, since arginine(s) are often found in the antigen-binding site of autoantibodies. Here, we present a unique viewpoint on the role of pre-BCR, by assessing the whole antibody repertoire formed in SLC-deficient mice.

Deciphering antigen-responding antibody repertoires by using next-generation sequencing and confirming them through antibody-gene synthesis.

Vast diversity and high specificity of antigen recognition by antibodies are hallmarks of the acquired immune system. Although the molecular mechanisms that yield the extremely large antibody repertoires are precisely understood, comprehensive description of the global antibody repertoire generated in individual bodies has been hindered by the lack of powerful measures. To obtain holistic understanding of the antibody-repertoire space, we used next-generation sequencing (NGS) to analyze the deep profiles of naive and antigen-responding repertoires of the IgM, IgG1, and IgG2c classes formed in individual mice. The overall landscapes of naive IgM repertoires were almost the same for each mouse, whereas those of IgG1 and IgG2c differed considerably among naive individuals. Next, we immunized mice with a model antigen, nitrophenol (NP)-hapten linked to chicken γ-globulin (CGG) carrier, and compared the antigen-responding repertoires in individual mice. To extract the complete antigen response, we developed an intelligible method for detecting common components of antigen-responding repertoires. The major responding antibodies were IGHV1-72/IGHD1-1/IGHJ2 for NP-hapten and IGHV9-3/IGHD3-1/IGHJ2 for CGG-carrier protein. The antigen-binding specificities of the identified antibodies were confirmed through ELISA after antibody-gene synthesis and expression of the corresponding NGS reads. Thus, we deciphered antigen-responding antibody repertoires by inclusively analyzing the antibody-repertoire space generated in individual bodies by using NGS, which avoided inadvertent omission of key antibody repertoires.

Significant relationship between the extent of pleural plaques and pulmonary asbestos body concentration in lung cancer patients with occupational asbestos exposure.

BACKGROUND: The aim of this study was to elucidate whether there is a relationship between the extent of pleural plaques and pulmonary asbestos body concentration (PABC). METHODS: The subjects were 207 lung cancer patients with occupational asbestos exposure. We determined the plaque extent by findings on chest images using our own criteria. PABCs were measured in resected or autopsy lung specimens. RESULTS: There was a significant relationship between plaque extent and PABC. Seventy-five percent of the patients determined to have extensive plaques based on our criteria had a PABC of ≥5,000 asbestos bodies per gram of dry lung tissue, which is one of the certification criteria of lung cancer caused by asbestos for workers' compensation in Japan. CONCLUSIONS: In lung cancer patients, the plaque extent had a significant positive relationship with the PABC. The plaque extent would be useful as a proxy for PABC for lung cancer compensation purposes.

Development of a sensitive novel diagnostic kit for the highly pathogenic avian influenza A (H5N1) virus.

BACKGROUND: Sporadic emergence of the highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is a serious concern because of the potential for a pandemic. Conventional or quantitative RT-PCR is the standard laboratory test to detect viral influenza infections. However, this technology requires well-equipped laboratories and highly trained personnel. A rapid, sensitive, and specific alternative screening method is needed. METHODS: By a luminescence-linked enzyme immunoassay, we have developed a H5N1 HPAI virus detection kit using anti-H5 hemagglutinin monoclonal antibodies in combination with the detection of a universal NP antigen of the type A influenza virus. The process takes 15 minutes by use of the fully automated luminescence analyzer, POCube. RESUTLS: We tested this H5/A kit using 19 clinical specimens from 13 patients stored in Vietnam who were infected with clade 1.1 or clade 2.3.4 H5N1 HPAI virus. Approximately 80% of clinical specimens were H5-positive using the POCube system, whereas only 10% of the H5-positive samples were detected as influenza A-positive by an immunochromatography-based rapid diagnostic kit. CONCLUSIONS: This novel H5/A kit using POCube is served as a rapid and sensitive screening test for H5N1 HPAI virus infection in humans.

The non-Ig parts of the VpreB and λ5 proteins of the surrogate light chain play opposite roles in the surface representation of the precursor B cell receptor.

The VpreB and λ5 proteins, together with Igµ-H chains, form precursor BCRs (preBCRs). We established λ5(-/-)/VpreB1(-/-)/VpreB2(-/-) Abelson virus-transformed cell lines and reconstituted these cells with λ5 and VpreB in wild-type form or with a deleted non-Ig part. Whenever preBCRs had the non-Ig part of λ5 deleted, surface deposition was increased, whereas deletion of VpreB non-Ig part decreased it. The levels of phosphorylation of Syk, SLP65, or PLC-γ2, and of Ca(2+) mobilization from intracellular stores, stimulated by µH chain crosslinking Ab were dependent on the levels of surface-bound preBCRs. It appears that VpreB probes the fitness of newly generated VH domains of IgH chains for later pairing with IgL chains, and its non-Ig part fixes the preBCRs on the surface. By contrast, the non-Ig part of λ5 crosslinks preBCRs for downregulation and stimulation.

Influence of pre-B cell receptor deficiency on the immunoglobulin repertoires in peripheral blood B cells before and after immunization.

During B cell development, pre-B cell receptor (pre-BCR), comprising the immunoglobulin heavy chain (HC) and surrogate light chain (SLC), plays a crucial role. The expression of pre-BCR serves as a certification of HC quality, confirming its ability to associate with the SLC and light chain (LC). In mice lacking SLC, the absence of this quality control mechanism leads to a distorted repertoire of HCs in the spleen and bone marrow. In this study, we conducted a comparative analysis of the immunoglobulin gene repertoire in peripheral blood cells of both wild-type mice and pre-BCR-deficient mice. Our findings reveal differences not only in the µ HC repertoire but also in the α HC and κ LC repertoires of the pre-BCR-deficient mice. These results suggest that the pre-BCR-mediated quality check of HC influences the selection of class-switched HC and LC repertoires. To further explore the impact of pre-BCR deficiency, we immunized these mice with thymus-dependent antigens and compared the antigen-responding repertoires. Our observations indicate that the affinity maturation pathways remain consistent between wild-type mice and pre-BCR-deficient mice, albeit with variations in the degree of maturation.

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing.

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5'-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird's-eye view of the antibody repertoire, and our computational methods.

A single amino acid substitution in the S1 and S2 Spike protein domains determines the neutralization escape phenotype of SARS-CoV.

In response to SARS-CoV infection, neutralizing antibodies are generated against the Spike (S) protein. Determination of the active regions that allow viral escape from neutralization would enable the use of these antibodies for future passive immunotherapy. We immunized mice with UV-inactivated SARS-CoV to generate three anti-S monoclonal antibodies, and established several neutralization escape mutants with S protein. We identified several amino acid substitutions, including Y442F and V601G in the S1 domain and D757N and A834V in the S2 region. In the presence of each neutralizing antibody, double mutants with substitutions in both domains exhibited a greater growth advantage than those with only one substitution. Importantly, combining two monoclonal antibodies that target different epitopes effected almost complete suppression of wild type virus replication. Thus, for effective passive immunotherapy, it is important to use neutralizing antibodies that recognize both the S1 and S2 regions.

Establishment and characterization of monoclonal antibodies against SARS coronavirus.

Immunological detection of viruses and their components by monoclonal antibodies is a powerful method for studying the structure and function of viral molecules. Here we describe detailed methods for establishing monoclonal antibodies against severe acute respiratory syndrome coronavirus (SARS-CoV). B cell hybridomas are generated from mice that are hyperimmunized with inactivated SARS-CoV virions. The hybridomas produce monoclonal antibodies that recognize viral component molecules, including the spike protein (S) and the nucleocapsid protein (N), enabling the immunological detection of SARS-CoV by immunofluorescence staining, immunoblot, or an antigen-capture ELISA system. In addition, several S protein-specific antibodies are shown to have in vitro neutralization activity. Thus the monoclonal antibody approach provides useful tools for rapid and specific diagnosis of SARS, as well as for possible antibody-based treatment of the disease.

Characterization of novel monoclonal antibodies against the MERS-coronavirus spike protein and their application in species-independent antibody detection by competitive ELISA.

Since discovering the Middle East respiratory syndrome coronavirus (MERS-CoV) as a causative agent of severe respiratory illness in the Middle East in 2012, serological testing has been conducted to assess antibody responses in patients and to investigate the zoonotic reservoir of the virus. Although the virus neutralization test is the gold standard assay for MERS diagnosis and for investigating the zoonotic reservoir, it uses live virus and so must be performed in high containment laboratories. Competitive ELISA (cELISA), in which a labeled monoclonal antibody (MAb) competes with test serum antibodies for target epitopes, may be a suitable alternative because it detects antibodies in a species-independent manner. In this study, novel MAbs against the spike protein of MERS-CoV were produced and characterized. One of these MAbs was used to develop a cELISA. The cELISA detected MERS-CoV-specific antibodies in sera from MERS-CoV-infected rats and rabbits immunized with the spike protein of MERS-CoV. The MAb-based cELISA was validated using sera from Ethiopian dromedary camels. Relative to the neutralization test, the cELISA detected MERS-CoV-specific antibodies in 66 Ethiopian dromedary camels with a sensitivity and specificity of 98% and 100%, respectively. The cELISA and neutralization test results correlated well (Pearson's correlation coefficients=0.71-0.76, depending on the cELISA serum dilution). This cELISA may be useful for MERS epidemiological investigations on MERS-CoV infection.

Formalin-treated UV-inactivated SARS coronavirus vaccine retains its immunogenicity and promotes Th2-type immune responses.

The demand for rapid and simple development of a vaccine against a newly emerging infectious disease is increasing worldwide. We previously revealed that UV-inactivated severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) virions (UV-V) elicited high levels of humoral immunity and a weak Th0 response in mice immunized subcutaneously. To ensure the safety of such a whole inactivated SARS-CoV vaccine, we additionally treated the UV-V vaccine with formalin, resulting in the UV-F-V vaccine. Analysis of the immunogenicity of the UV-F-V+alum vaccine in mice revealed that it generated comparable neutralizing serum anti-SARS-CoV IgG antibody levels as the UV-V+alum vaccine. Moreover, both vaccines induced similar frequencies of anti-SARS-CoV IgG antibody-producing cells in bone marrow. Interestingly, the UV-F-V vaccine induced fewer IgG(2a) subtype antibodies and higher interleukin-4 production in vaccinated mice than did UV-V. Thus, UV-F-V imposes a Th2-type bias on the immune response, unlike UV-V. We propose here that doubly-inactivated SARS-CoV virions by UV and formalin constitute a safe vaccine that may effectively induce neutralizing antibodies in humans.

Immunological detection of severe acute respiratory syndrome coronavirus by monoclonal antibodies.

In order to establish immunological detection methods for severe acute respiratory syndrome coronavirus (SARS-CoV), we established monoclonal antibodies directed against structural components of the virus. B cell hybridomas were generated from mice that were hyper-immunized with inactivated SARS-CoV virion. By screening 2,880 generated hybridomas, we established three hybridoma clones that secreted antibodies specific for nucleocapsid protein (N) and 27 clones that secreted antibodies specific for spike protein (S). Among these, four S-protein specific antibodies had in vitro neutralization activity against SARS-CoV infection. These monoclonal antibodies enabled the immunological detection of SARS-CoV by immunofluorescence staining, Western blot or immunohistology. Furthermore, a combination of monoclonal antibodies with different specificities allowed the establishment of a highly sensitive antigen-capture sandwich ELISA system. These monoclonal antibodies would be a useful tool for rapid and specific diagnosis of SARS and also for possible antibody-based treatment of the disease.

BILL-cadherin/cadherin-17 contributes to the survival of memory B cells.

Memory B cells (MBCs) and long-lived plasma cells (LLPCs) are responsible for immunological "memory", which can last for many years. The long-term survival niche for LLPCs in the bone marrow is well characterized; however, the corresponding niche for MBCs is unclear. BILL-cadherin/cadherin-17 (CDH17) is the only member of the cadherin superfamily that is expressed on mouse B lymphocytes in a spatiotemporally regulated manner. Here, we show that half of all MBCs regain expression of CDH17 during the later stage of development. The maintenance of high affinity antigen-specific serum antibodies was impaired in CDH17(-/-) mice and the number of antigen-specific MBCs was reduced as compared to wild-type mice (WT). Also, specific responses to secondary antigens were ablated in CDH17(-/-) mice, whereas primary antibody responses were the same as those in WT mice. Cell cycle analysis revealed a decline in the proliferation of CDH17(-) MBCs as compared to CDH17(+) MBCs. In addition, we identified a subpopulation of splenic stromal cells, MAdCAM-1(+) blood endothelial cells (BEC), which was CDH17(+). Taken together, these results suggest that CDH17 plays a role in the long-term survival of MBCs, presumably via an "MBC niche" comprising, at least in part, BEC in the spleen.

Clinical study of asbestos-related lung cancer.

We analyzed the characteristics of 120 patients of primary lung cancer supposed to be induced by exposure to asbestos. Most of 120 patients were male and the age ranged from 47 to 87 years with a median of 70 years. No particular tendency was observed in the histological types of the lung cancer in 120 patients. Forty of the 120 patients were heavy smokers. When the occupational history was analyzed, most of the patients had been exposed to asbestos in former Japanese naval shipyard, commercial shipyards, construction industry and ironworks. The term of asbestos exposure was 2 to 60 years with a median 27 years. Lung cancers appeared after 15 to 69 years with a median 43 years from the initial exposure to asbestos. Lung cancer was accompanied by asbestosis in 35 patients and by pleural plaques in 77. Twenty-two patients had both asbestosis and pleural plaques. The number of asbestos bodies per 5 g wet lung tissue for 72 patients whose lung tissues obtained from autopsy or surgery was more than 150 bodies which meant the number of occupational asbestos exposure. As for the kinds of asbestos fibers of 32 patients, 14 patients exposed to crocidolite, 10 patients to amosite and 8 patients to chrysotile.

Broad cross-reactive epitopes of the H5N1 influenza virus identified by murine antibodies against the A/Vietnam/1194/2004 hemagglutinin.

There is an urgent need for a rapid diagnostic system to detect the H5 subtype of the influenza A virus. We previously developed monoclonal antibodies (mAbs) against the H5 hemagglutinin (HA) for use in a rapid diagnostic kit. In this study, we determined the epitopes of the anti-H5 HA murine mAbs OM-b, AY-2C2, and YH-1A1. Binding assays of the mAbs to different strains of H5 HAs indicated that OM-b and AY-2C2 cross-reacted with HAs from clades 1, 2.1.3.2, 2.2, and 2.3.4, whereas YH-1A1 failed to bind to those of clades 2.1.3.2 and 2.3.4. HA chimeras revealed that the epitopes for each of the mAbs were in the HA1 region. Analysis of escape mutants revealed that OM-b and AY-2C2 mAbs interacted mainly with amino acid residues D43 and G46, and the YH-1A1 mAb interacted with G139 and K or R140 of H5 HA. Multiple alignments of H5 HA protein sequences showed that D43 and G46 were very conserved among H5N1 HAs, except those in clade 2.2.1 and clade 7 (88.7%). The epitope for YH-1A1 mAb was highly variable in the HAs of H5N1, although it was well conserved in those of H5N2-N9. The OM-b and AY-2C2 mAbs could bind to the HAs of clades 1.1 and 2.3.2.1 that are currently epidemic in Asia, and we conclude that these would be effective for the detection of H5N1 infections in this region.

Reprint of: Environments of B cell development.

B lymphocyte development in the mouse begins with the generation of long-term reconstituting, pluripotent hematopoietic stem cells, over multipotent myeloid/lymphoid progenitors and common lymphoid progenitors to B-lineage committed pro/pre B and pre B cells, which first express pre B cell receptors and then immunoglobulins, B cell receptors, to generate the repertoires of peripheral B cells. This development is influenced and guided by cells of non-hematopoietic and hematopoietic origins. We review here some of the recent developments, and our contributions in this fascinating field of developmental immunology.

Environments of B cell development.

B lymphocyte development in the mouse begins with the generation of long-term reconstituting, pluripotent hematopoietic stem cells, over multipotent myeloid/lymphoid progenitors and common lymphoid progenitors to B-lineage committed pro/pre B and pre B cells, which first express pre B cell receptors and then immunoglobulins, B cell receptors, to generate the repertoires of peripheral B cells. This development is influenced and guided by cells of non-hematopoietic and hematopoietic origins. We review here some of the recent developments, and our contributions in this fascinating field of developmental immunology.

Newly established monoclonal antibodies for immunological detection of H5N1 influenza virus.

The H5N1 subtype of the highly pathogenic (HP) avian influenza virus has been recognized for its ability to cause serious pandemics among humans. In the present study, new monoclonal antibodies (mAbs) against viral proteins were established for the immunological detection of H5N1 influenza virus for research and diagnostic purposes. B-cell hybridomas were generated from mice that had been hyperimmunized with purified A/Vietnam/1194/2004 (NIBRG-14) virion that had been inactivated by UV-irradiation or formaldehyde. After screening over 4,000 hybridomas, eight H5N1-specific clones were selected. Six were specific for hemagglutinin (HA) and had in vitro neutralization activity. Of these, four were able to broadly detect all tested clades of the H5N1 strains. Five HA-specific mAbs detected denatured HA epitope(s) in Western blot analysis, and two detected HP influenza virus by immunofluorescence and immunohistochemistry. A highly sensitive antigen-capture sandwich ELISA system was established by combining mAbs with different specificities. In conclusion, these mAbs may be useful for rapid and specific diagnosis of H5N1 influenza. Therapeutically, they may have a role in antibody-based treatment of the disease.