Distinct features of B cell receptors in neuromyelitis optica spectrum disorder among CNS inflammatory demyelinating diseases.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) stands out among CNS inflammatory demyelinating diseases (CIDDs) due to its unique disease characteristics, including severe clinical attacks with extensive lesions and its association with systemic autoimmune diseases. We aimed to investigate whether characteristics of B cell receptors (BCRs) differ between NMOSD and other CIDDs using high-throughput sequencing. METHODS: From a prospective cohort, we recruited patients with CIDDs and categorized them based on the presence and type of autoantibodies: NMOSD with anti-aquaporin-4 antibodies, myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) with anti-myelin oligodendrocyte glycoprotein antibodies, double-seronegative demyelinating disease (DSN), and healthy controls (HCs). The BCR features, including isotype class, clonality, somatic hypermutation (SHM), and the third complementarity-determining region (CDR3) length, were analyzed and compared among the different disease groups. RESULTS: Blood samples from 33 patients with CIDDs (13 NMOSD, 12 MOGAD, and 8 DSN) and 34 HCs were investigated for BCR sequencing. Patients with NMOSD tended to have more activated BCR features compare to the other disease groups. They showed a lower proportion of unswitched isotypes (IgM and IgD) and a higher proportion of switched isotypes (IgG), increased clonality of BCRs, higher rates of SHM, and shorter lengths of CDR3. Notably, advanced age was identified as a clinical factor associated with these activated BCR features, including increased levels of clonality and SHM rates in the NMOSD group. Conversely, no such clinical factors were found to be associated with activated BCR features in the other CIDD groups. CONCLUSIONS: NMOSD patients, among those with CIDDs, displayed the most pronounced B cell activation, characterized by higher levels of isotype class switching, clonality, SHM rates, and shorter CDR3 lengths. These findings suggest that B cell-mediated humoral immune responses and characteristics in NMOSD patients are distinct from those observed in the other CIDDs, including MOGAD. Age was identified as a clinical factor associated with BCR activation specifically in NMOSD, implying the significance of persistent B cell activation attributed to anti-aquaporin-4 antibodies, even in the absence of clinical relapses throughout an individual's lifetime.

Accurate Diagnosis of COVID-19 from Self-Collectable Biospecimens Using Synthetic Apolipoprotein H Peptide-Coated Nanoparticle Assay.

A high-throughput, accurate screening is crucial for the prevention and control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current methods, which involve sampling from the nasopharyngeal (NP) area by medical staffs, constitute a fundamental bottleneck in expanding the testing capacity. To meet the scales required for population-level surveillance, self-collectable specimens can be used; however, its low viral load has hindered its clinical adoption. Here, we describe a magnetic nanoparticle functionalized with synthetic apolipoprotein H (ApoH) peptides to capture, concentrate, and purify viruses. The ApoH assay demonstrates a viral enrichment efficiency of >90% for both SARS-CoV-2 and its variants, leading to an order of magnitude improvement in analytical sensitivity. For validation, we apply the assay to a total of 84 clinical specimens including nasal, oral, and mouth gargles obtained from COVID-19 patients. As a result, a 100% positivity rate is achieved from the patient-collected nasal and gargle samples, which exceeds that of the traditional NP swab method. The simple 12 min pre-enrichment assay enabling the use of self-collectable samples will be a practical solution to overcome the overwhelming diagnostic capacity. Furthermore, the methodology can easily be built on various clinical protocols, allowing its broad applicability to various disease diagnoses.

High-throughput construction of multiple cas9 gene variants via assembly of high-depth tiled and sequence-verified oligonucleotides.

Selective retrieval of sequence-verified oligonucleotides (oligos) from next-generation sequencing (NGS) flow cells, termed megacloning, promises accurate and reliable gene synthesis. However, gene assembly requires a complete collection of overlapping sense and nonsense oligos, and megacloning does not typically guarantee the complete production of sequence-verified oligos. Therefore, missing oligos must be provided via repetitive rounds of megacloning, which introduces a bottleneck for scaled-up efforts at gene assembly. Here, we introduce the concept of high-depth tiled oligo design to successfully utilize megacloned oligos for gene synthesis. Using acquired oligos from a single round of the megacloning process, we assembled 72 of 81 target Cas9-coding gene variants. We further validated 62 of these cas9 constructs, and deposited the plasmids to Addgene for subsequent functional characterization by the scientific community. This study demonstrates the utility of using sequence-verified oligos for DNA assembly and provides a practical and reliable optimized method for high-throughput gene synthesis.

Efficient Selection of Antibodies Reactive to Homologous Epitopes on Human and Mouse Hepatocyte Growth Factors by Next-Generation Sequencing-Based Analysis of the B Cell Repertoire.

: YYB-101 is a humanized rabbit anti-human hepatocyte growth factor (HGF)-neutralizing antibody currently in clinical trial. To test the effect of HGF neutralization with antibody on anti-cancer T cell immunity, we generated surrogate antibodies that are reactive to the mouse homologue of the epitope targeted by YYB-101. First, we immunized a chicken with human HGF and monitored changes in the B cell repertoire by next-generation sequencing (NGS). We then extracted the VH gene repertoire from the NGS data, clustered it into components by sequence homology, and classified the components by the change in the number of unique VH sequences and the frequencies of the VH sequences within each component following immunization. Those changes should accompany the preferential proliferation and somatic hypermutation or gene conversion of B cells encoding HGF-reactive antibodies. One component showed significant increases in the number and frequencies of unique VH sequences and harbored genes encoding antibodies that were reactive to human HGF and competitive with YYB-101 for HGF binding. Some of the antibodies also reacted to mouse HGF. The selected VH sequences shared 98.3% identity and 98.9% amino acid similarity. It is therefore likely that the antibodies encoded by them all react to the epitope targeted by YYB-101.

Preparation of VCSM13 Helper Phage for Display Library Reamplification and Bio-Panning.

Phage-displayed antibody libraries can be constructed using any species that is easily immunized. The pComb3XSS phagemid vector is commonly used for library cloning and phage display. This phagemid encodes the origin of replication of the filamentous bacteriophage f1 but lacks all the genes required for replication and assembly of phage particles. The replication and the assembly of phage from these phagemids thus requires a "helper" phage that provides the genes essential for those steps during library production and bio-panning. One of those helper phages is VCSM13. In this protocol, we describe the preparation of VCSM13 helper phage. Users should prepare VCSM13 helper phage for library reamplification and for bio-panning.

Generation of Chicken Antibody Libraries and Selection of Antigen Binders.

Chicken antibodies have been widely used for research and diagnostic purposes. Chicken antibodies are often cross-reactive to epitopes shared by humans, nonhuman primates, and other mammals, and can be tested in many mouse disease models, which provides an advantage for their preclinical study and evaluation. In addition, the variable region of chicken antibodies has unique structural characteristics, including noncanonical cysteine residues in the heavy chain complementarity-determining region (CDR)3 and a long heavy chain CDR3, which together with a short light chain CDR enable the formation of unconventional antibody paratopes. As chickens have single functional copies of the V H and J H genes, and the somatic gene conversion process usually involves D H genes, all functional VDJ gene fragments can be obtained from the B-cell repertoire using a single PCR primer set, without any primer bias. As for the light chain, chickens only have a V λ light chain, composed of a single V λ and J λ gene pair. Therefore, the chicken light chain repertoire can also be accurately amplified using a single primer set. This unbiased reconstitution of the chicken B-cell repertoire provides a great advantage not only in the construction of phage display libraries but also for the in silico selection of antigen binders from a virtual B-cell receptor repertoire. Here, we introduce the use of chicken antibodies in research, diagnostic, and therapeutic fields. In addition, the chromosomal organization of chicken immunoglobulin genes and its diversification mechanisms for shaping the antibody repertoire are also discussed.

Chicken Immunization followed by RNA Extraction and cDNA Synthesis for Antibody Library Preparation.

Effective isolation of specific antibodies from immunological repertoires requires the generation of a diverse library against a specific antigen of interest, as well as efficient selection procedures, such as bio-panning and phage ELISA. Key to this is the generation of a good immune response in the host, followed by preparation of high-quality RNA and cDNA from which a library can be constructed by the amplification and cloning of immunoglobulin heavy and light chain genes. The first step in the construction of such an "immune library" is a successful course of immunization. Detection of a strong serum antibody titer will theoretically then result in a pool of extracted RNA that is enriched for transcripts of genes encoding the antibody of interest. Chicken antibodies have been widely used for research and diagnostic purposes, largely because of both their cross-reactivity to epitopes shared by humans, mice, primates, and other mammals, and their simple characteristics, with chickens featuring single functional copies of V H /J H and V λ /J λ gene pairs. In chickens, antibodies against an antigen of interest can be detected in the serum as soon as 5-7 d after immunization. Once the antibody titer reaches an appropriate level in the serum, the spleen, bursa of Fabricius, and bone marrow are then harvested, and antibody libraries can be prepared from extracted RNA. Here, we describe a protocol for chicken immunization with an antigen of interest, followed by RNA extraction from the relevant tissues and cDNA synthesis, which users can use for antibody library construction.

Generation of a Phage Display Chicken Single-Chain Variable Fragment Library.

Phage-displayed antibody fragment libraries can be constructed using essentially any species that is easily immunized, as long as the immunoglobulin variable region gene sequences are known. This protocol describes the procedures for the generation of a phage-displayed chicken single-chain variable fragment (scFv) library after immunization with a target antigen. Briefly, the rearranged heavy chain variable region (V H ) genes and the λ light chain variable region (V λ ) genes are amplified separately and are linked through two separate PCR steps to give the final scFv genes. The genes are then cloned into pComb3XSS to generate the phage display chicken scFv library, which can then be used for test and final library ligations.

Selection of Antigen Binders from a Chicken Single-Chain Variable Fragment Library.

Antibody production against an antigen of interest is highly efficient in chickens, and the use of chicken antibody libraries in phage display can result in high-affinity single-chain variable fragments (scFvs) for multiple applications. After library preparation from an animal immunized with the antigen of interest, the next step involves the identification of antigen binders. Here, we describe a process for the screening of a phage display chicken library using a technique called bio-panning. It consists of several rounds of binding scFv-displaying phage to antigens, followed by washing, elution, and reamplification. We also describe the steps for assessing clone pools obtained after bio-panning via an ELISA-based procedure known as "phage ELISA" to identify single clones. Last, we provide the steps for using high-throughput sequencing to analyze the pool of selected clones.

An ancestral SARS-CoV-2 vaccine induces anti-Omicron variants antibodies by hypermutation.

The immune escape of Omicron variants significantly subsides by the third dose of an mRNA vaccine. However, it is unclear how Omicron variant-neutralizing antibodies develop under repeated vaccination. We analyze blood samples from 41 BNT162b2 vaccinees following the course of three injections and analyze their B-cell receptor (BCR) repertoires at six time points in total. The concomitant reactivity to both ancestral and Omicron receptor-binding domain (RBD) is achieved by a limited number of BCR clonotypes depending on the accumulation of somatic hypermutation (SHM) after the third dose. Our findings suggest that SHM accumulation in the BCR space to broaden its specificity for unseen antigens is a counterprotective mechanism against virus variant immune escape.

Optimization of peripheral blood volume for in silico reconstitution of the human B-cell receptor repertoire.

B cells recognize antigens via membrane-expressed B-cell receptors (BCR) and antibodies. Similar human BCR sequences are frequently found at a significantly higher frequency than that theoretically calculated. Patients infected with SARS-CoV2 and HIV or with autoimmune diseases share very similar BCRs. Therefore, in silico reconstitution of BCR repertoires and identification of stereotypical BCR sequences related to human pathology have diagnostic potential. Furthermore, monitoring changes of clinically significant BCR sequences and isotype conversion has prognostic potential. For BCR repertoire analysis, peripheral blood (PB) is the most convenient source. However, the optimal human PB volume for in silico reconstitution of the BCR repertoire has not been studied in detail. Here, we sampled 5, 10, and 20 mL PB from the left arm and 40 mL PB from the right arm of two volunteers, reconstituted in silico PB BCR repertoires, and compared their composition. In both volunteers, PB sampling over 20 mL resulted in slight increases in functional unique sequences (FUSs) or almost no increase in repertoire diversity. All FUSs with a frequency above 0.08% or 0.03% in the 40 mL PB BCR repertoire were detected even in the 5 mL PB BCR repertoire from each volunteer. FUSs with a higher frequency were more likely to be found in BCR repertoires from reduced PB volume, and those coexisting in two repertoires showed a statistically significant correlation in frequency irrespective of sampled anatomical site. The correlation was more significant in higher-frequency FUSs. These observations support the potential of BCR repertoire analysis for diagnosis.

Association of B cell profile and receptor repertoire with the progression of Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent type of dementia. Reports have revealed that the peripheral immune system is linked to neuropathology; however, little is known about the contribution of B lymphocytes in AD. For this longitudinal study, 133 participants are included at baseline and second-year follow-up. Also, we analyze B cell receptor (BCR) repertoire data generated from a public dataset of three normal and 10 AD samples and perform BCR repertoire profiling and pairwise sharing analysis. As a result, longitudinal increase in B lymphocytes is associated with increased cerebral amyloid deposition and hyperactivates induced pluripotent stem cell-derived microglia with loss-of-function for beta-amyloid clearance. Patients with AD share similar class-switched BCR sequences with identical isotypes, despite the high somatic hypermutation rate. Thus, BCR repertoire profiling can lead to the development of individualized immune-based therapeutics and treatment. We provide evidence of both quantitative and qualitative changes in B lymphocytes during AD pathogenesis.

Spatial epitranscriptomics reveals A-to-I editome specific to cancer stem cell microniches.

Epitranscriptomic features, such as single-base RNA editing, are sources of transcript diversity in cancer, but little is understood in terms of their spatial context in the tumour microenvironment. Here, we introduce spatial-histopathological examination-linked epitranscriptomics converged to transcriptomics with sequencing (Select-seq), which isolates regions of interest from immunofluorescence-stained tissue and obtains transcriptomic and epitranscriptomic data. With Select-seq, we analyse the cancer stem cell-like microniches in relation to the tumour microenvironment of triple-negative breast cancer patients. We identify alternative splice variants, perform complementarity-determining region analysis of infiltrating T cells and B cells, and assess adenosine-to-inosine base editing in tumour tissue sections. Especially, in triple-negative breast cancer microniches, adenosine-to-inosine editome specific to different microniche groups is identified.

Amplification of a minimally biased antibody repertoire for in vitro display using a universal primer-based amplification method.

Immune hosts are valuable sources for antibody discovery. To construct in vitro display antibody libraries from immune repertoires, singleplex or multiplex PCR amplification were employed using primers targeting multiple immunoglobulin genes. However, during this process, the B cell receptor repertoire is distorted due to interactions between multiple target genes and primers. To minimize this alternation, we devised a new method for harvesting immunoglobulin genes and tested its performance in rabbit variable heavy chain (VH) and variable kappa light chain (VK) genes. Double-stranded cDNA was synthesized using primers containing V/J gene-specific regions and universal sequence parts for in vitro display. VH and VK gene libraries were obtained through subsequent PCR amplification using primers with universal sequences. Next-generation sequencing analysis confirmed that universal PCR libraries had more diverse VH and VK clonotypes, and a less biased clonal distribution, than conventional singleplex or multiplex gene-specific PCR libraries.

Induction of Anti-Aquaporin 5 Autoantibody Production by Immunization with a Peptide Derived from the Aquaporin of Prevotella melaninogenica Leads to Reduced Salivary Flow in Mice.

Sjögren's syndrome (SS) is an autoimmune disease characterized by dryness of the mouth and eyes. The glandular dysfunction in SS involves not only T cell-mediated destruction of the glands but also autoantibodies against the type 3 muscarinic acetylcholine receptor or aquaporin 5 (AQP5) that interfere with the secretion process. Studies on the breakage of tolerance and induction of autoantibodies to these autoantigens could benefit SS patients. To break tolerance, we utilized a PmE-L peptide derived from the AQP5-homologous aquaporin of Prevotella melaninogenica (PmAqp) that contained both a B cell "E" epitope and a T cell epitope. Repeated subcutaneous immunization of C57BL/6 mice with the PmE-L peptide efficiently induced the production of Abs against the "E" epitope of mouse/human AQP5 (AQP5E), and we aimed to characterize the antigen specificity, the sequences of AQP5E-specific B cell receptors, and salivary gland phenotypes of these mice. Sera containing anti-AQP5E IgG not only stained mouse Aqp5 expressed in the submandibular glands but also detected PmApq and PmE-L by immunoblotting, suggesting molecular mimicry. Characterization of the AQP5E-specific autoantibodies selected from the screening of phage display Ab libraries and mapping of the B cell receptor repertoires revealed that the AQP5E-specific B cells acquired the ability to bind to the Ag through cumulative somatic hypermutation. Importantly, animals with anti-AQP5E Abs had decreased salivary flow rates without immune cell infiltration into the salivary glands. This model will be useful for investigating the role of anti-AQP5 autoantibodies in glandular dysfunction in SS and testing new therapeutics targeting autoantibody production.

Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals.

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were composed of immunoglobulin heavy variable 3-53 (IGHV3-53) or IGHV3-66 and immunoglobulin heavy joining 6 (IGHJ6) genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different IGHV chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in 6 of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

Cell-Free Bacteriophage Genome Synthesis Using Low-Cost Sequence-Verified Array-Synthesized Oligonucleotides.

Synthesizing engineered bacteriophages (phages) for human use has potential in various applications ranging from drug screening using a phage display to clinical use using phage therapy. However, the engineering of phages conventionally involves the use of an in vivo system that has low production efficiency because of high virulence against the host and low transformation efficiency. To circumvent these issues, de novo phage genome synthesis using chemically synthesized oligonucleotides (oligos) has increased the potential for engineering phages in a cell-free system. Here, we present a cell-free, low-cost, de novo gene synthesis technology called Sniper assembly for phage genome construction. With massively parallel sequencing of microarray-synthesized oligos, we generated and identified approximately 100 000 clonal DNA clusters in vitro and 5000 error-free ones in a cell-free environment. To demonstrate its practical application, we synthesized the Acinetobacter phage AP205 genome (4268 bp) using 65 sequence-verified DNA clones. Compared to previous reports, Sniper assembly lowered the genome synthesis cost ($0.0137/bp) by producing low-cost sequence-verified DNA.

Barcode-free next-generation sequencing error validation for ultra-rare variant detection.

The advent of next-generation sequencing (NGS) has accelerated biomedical research by enabling the high-throughput analysis of DNA sequences at a very low cost. However, NGS has limitations in detecting rare-frequency variants (< 1%) because of high sequencing errors (> 0.1~1%). NGS errors could be filtered out using molecular barcodes, by comparing read replicates among those with the same barcodes. Accordingly, these barcoding methods require redundant reads of non-target sequences, resulting in high sequencing cost. Here, we present a cost-effective NGS error validation method in a barcode-free manner. By physically extracting and individually amplifying the DNA clones of erroneous reads, we distinguish true variants of frequency > 0.003% from the systematic NGS error and selectively validate NGS error after NGS. We achieve a PCR-induced error rate of 2.5×10-6 per base per doubling event, using 10 times less sequencing reads compared to those from previous studies.

High-throughput retrieval of physical DNA for NGS-identifiable clones in phage display library.

In antibody discovery, in-depth analysis of an antibody library and high-throughput retrieval of clones in the library are crucial to identifying and exploiting rare clones with different properties. However, existing methods have technical limitations, such as low process throughput from the laborious cloning process and waste of the phenotypic screening capacity from unnecessary repetitive tests on the dominant clones. To overcome the limitations, we developed a new high-throughput platform for the identification and retrieval of clones in the library, TrueRepertoire™. This new platform provides highly accurate sequences of the clones with linkage information between heavy and light chains of the antibody fragment. Additionally, the physical DNA of clones can be retrieved in high throughput based on the sequence information. We validated the high accuracy of the sequences and demonstrated that there is no platform-specific bias. Moreover, the applicability of TrueRepertoire™ was demonstrated by a phage-displayed single-chain variable fragment library targeting human hepatocyte growth factor protein.