VDJ Gene Research Articles

Characterization of a novel chicken γδ TCR-specific marker

Chickens are a species with a high number of γδ T cells in various tissues. Despite their abundance, γδ T cells are poorly characterized in chickens, partially due to a lack of specific reagents to characterize these cells. Up until now, the TCR1 clone has been the only γδ T cell-specific monoclonal antibody (mAb) in chickens and additional reagents for γδ T cell subsets are needed. In order to address this issue, new mAb were generated in our laboratory by immunizing mice with in vitro cultured γδ T cells. In an initial flow cytometric screen a new mAb, clone “8D2”, displayed an interesting staining pattern that mirrored γδ TCR up- and downregulation in the γδ T cell line D4 over time, prompting us to characterize this antibody further. We compared the expression of the unknown 8D2 epitope in combination with TCR1 staining across various primary cells. In splenocytes, peripheral blood lymphocytes and intestinal epithelial cells, 8D2 consistently labeled a subset of TCR1+ cells. To determine, whether specific γδ T cell receptors were recognized by 8D2, we sorted γδ T cells according to their 8D2 and TCR1 expression and analyzed their TCR V(D)J gene usage by TCR profiling. Strikingly, sorted 8D2+ cells preferentially expressed Vγ3 genes, whereas the TCR Vγ genes used by TCR1+ 8D2- cells were more variable. γδ TCR in 8D2+ cells were most frequently comprised of gamma chain VJ genes TRGV3-8 and TRGJ3, and delta chain VDJ genes TRDV1-2, TRDD2, TRDJ1. To confirm binding of 8D2 to specific γδ TCR, the preferentially utilized combination of TRG and TRD was expressed in HEK293 cells in combination with CD3, demonstrating surface binding of the 8D2 mAb to this Vγ3 γδ TCR-expressing cell line. Conversely, HEK293 cells expressing either Vγ1 or Vγ2 TCR did not react with 8D2. In conclusion, 8D2 is a novel tool for identifying specific Vγ3 bearing γδ T cells.

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.

Detection of disease-specific signatures in B cell repertoires of lymphomas using machine learning.

The classification of B cell lymphomas-mainly based on light microscopy evaluation by a pathologist-requires many years of training. Since the B cell receptor (BCR) of the lymphoma clonotype and the microenvironmental immune architecture are important features discriminating different lymphoma subsets, we asked whether BCR repertoire next-generation sequencing (NGS) of lymphoma-infiltrated tissues in conjunction with machine learning algorithms could have diagnostic utility in the subclassification of these cancers. We trained a random forest and a linear classifier via logistic regression based on patterns of clonal distribution, VDJ gene usage and physico-chemical properties of the top-n most frequently represented clonotypes in the BCR repertoires of 620 paradigmatic lymphoma samples-nodular lymphocyte predominant B cell lymphoma (NLPBL), diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL)-alongside with 291 control samples. With regard to DLBCL and CLL, the models demonstrated optimal performance when utilizing only the most prevalent clonotype for classification, while in NLPBL-that has a dominant background of non-malignant bystander cells-a broader array of clonotypes enhanced model accuracy. Surprisingly, the straightforward logistic regression model performed best in this seemingly complex classification problem, suggesting linear separability in our chosen dimensions. It achieved a weighted F1-score of 0.84 on a test cohort including 125 samples from all three lymphoma entities and 58 samples from healthy individuals. Together, we provide proof-of-concept that at least the 3 studied lymphoma entities can be differentiated from each other using BCR repertoire NGS on lymphoma-infiltrated tissues by a trained machine learning model.

Multimodal repertoire analysis unveils B cell biology in immune-mediated diseases

ObjectivesDespite the involvement of B cells in the pathogenesis of immune-mediated diseases (IMDs), biological mechanisms underlying their function are scarcely understood. To overcome this gap, here we constructed and investigated...

Evolution of immunogenetic components encoding ultralong CDR H3.

The genomes of most vertebrates contain many V, D, and J gene segments within their Ig loci to construct highly variable CDR3 sequences through combinatorial diversity. This nucleotide variability translates into an antibody population containing extensive paratope diversity. Cattle have relatively few functional VDJ gene segments, requiring innovative approaches for generating diversity like the use of ultralong-encoding IGHV and IGHD gene segments that yield dramatically elongated CDR H3. Unique knob and stalk microdomains create protracted paratopes, where the antigen-binding knob sits atop a long stalk, allowing the antibody to bind both surface and recessed antigen epitopes. We examined genomes of twelve species of Bovidae to determine when ultralong-encoding IGHV and IGHD gene segments evolved. We located the 8-bp duplication encoding the unique TTVHQ motif in ultralong IGHV segments in six Bovid species (cattle, zebu, wild yak, domestic yak, American bison, and domestic gayal), but we did not find evidence of the duplication in species beyond the Bos and Bison genera. Additionally, we analyzed mRNA from bison spleen and identified a rich repertoire of expressed ultralong CDR H3 antibody mRNA, suggesting that bison use ultralong IGHV transcripts in their host defense. We found ultralong-encoding IGHD gene segments in all the same species except domestic yak, but again not beyond the Bos and Bison clade. Thus, the duplication event leading to this ultralong-encoding IGHV gene segment and the emergence of the ultralong-encoding IGHD gene segment appears to have evolved in a common ancestor of the Bos and Bison genera 5-10 million years ago.

De novo design and Rosetta-based assessment of high-affinity antibody variable regions (Fv) against the SARS-CoV-2 spike receptor binding domain (RBD).

The continued emergence of new SARS‐CoV‐2 variants has accentuated the growing need for fast and reliable methods for the design of potentially neutralizing antibodies (Abs) to counter immune evasion by the virus. Here, we report on the de novo computational design of high affinity antibody variable regions (Fv) through the recombination of VDJ genes targeting the most solvent‐exposed hACE2‐binding residues of the SARS‐CoV‐2 spike receptor binding domain (RBD) protein using the software tool OptMAVEn‐2.0.2 Subsequently, we carried out computational affinity maturation of the designed variable regions through amino‐acid substitutions for improved binding with the target epitope. Immunogenicity of designs was restricted by preferring designs that match sequences from a 9‐mer library of “human antibodies” based on human string content score74. We generated 106 different designs and reported in detail on the top five that trade‐off the greatest computational binding affinity for the RBD with human string content scores. We further describe computational evaluation of the top five designs produced by OptMAVEn‐2.01 using a Rosetta‐based approach. We used Rosetta SnugDock 2,3 for local docking of the designs to evaluate their potential to bind the spike RBD and performed “forward folding” with DeepAb 4 to assess their potential to fold into the designed structures. Ultimately, our results identified one designed antibody variable region, P1.D1, as a particularly promising candidate for experimental testing. This effort puts forth a computational workflow for the de novo design and evaluation of antibodies that can quickly be adapted to target spike epitopes of emerging SARS‐CoV‐2 variants or other antigenic targets.

Novel skewed usage of B-cell receptors in COVID-19 patients with various clinical presentations

B cell-mediated immune responses play important roles in controlling SARS-CoV infection. Here, we performed the single-cell B cell receptor sequencing (scBCR-seq) of the PBMC samples from eleven healthy controls, five asymptomatic subjects and 33 symptomatic COVID-19 patients with various clinical presentations, and subsequently analyzed the abundance and diversity of the BCR repertoires in different groups, respectively. We revealed the skewed usage of the IGHV, IGLV and IGKV genes and identified a number of heavy or light chain VDJ gene pairs and combinational preference in each group, such as IGKV3–7 and IGKV2–24 enriched in the asymptomatic subjects, whereas IGHV3–13, IGHV3–23-IGHJ4, IGHV1–18–IGLV3–19, IGHV1–18–IGLV3–21, and IGHV1–18–IGLV3–25 enriched in the recovery patients with severe diseases. We also observed the differential expression of IGHV3–23 in various B cell clusters by analysis of the scRNA-seq data. Additional dock analysis indicated that IGHV3–13 could bind to the spike protein of SARS-CoV-2. These findings may advance our understanding of the humoral immune responses in COVID-19 patients and help develop novel vaccine candidates as well as therapeutical antibodies against SASR-CoV-2 infections.

RNA exosome drives early B cell development via noncoding RNA processing mechanisms.

B cell development is linked to successful V(D)J recombination, allowing B cell receptor expression and ultimately antibody secretion for adaptive immunity. Germline noncoding RNAs (ncRNAs) are produced at immunoglobulin (Ig) loci during V(D)J recombination, but their function and posttranscriptional regulation are incompletely understood. Patients with trichohepatoenteric syndrome, characterized by RNA exosome pathway component mutations, exhibit lymphopenia, thus demonstrating the importance of ncRNA surveillance in B cell development in humans. To understand the role of RNA exosome in early B cell development in greater detail, we generated mouse models harboring a B cell-specific cre allele (Mb1cre), coupled to conditional inversion-deletion alleles of one RNA exosome core component (Exosc3) or RNase catalytic subunits (Exosc10 or Dis3). We noticed increased expression of RNA exosome subunits during V(D)J recombination, whereas a B cell developmental blockade at the pro-B cell stage was observed in the different knockout mice, overlapping with a lack of productive rearrangements of VDJ genes at the Ig heavy chain (Igh). This unsuccessful recombination prevented differentiation into pre-B cells, with accumulation of ncRNAs and up-regulation of the p53 pathway. Introduction of a prearranged Igh VDJ allele partly rescued the pre-B cell population in Dis3-deficient cells, although V-J recombination defects were observed at Ig light chain kappa (Igκ), preventing subsequent B cell development. These observations demonstrated that the RNA exosome complex is important for Igh and Igκ recombination and establish the relevance of RNA processing for optimal diversification at these loci during B cell development.

Single-cell TCR and mRNA sequencing of antigen-specific T cells reveal spatial trajectories from single time points with HLA and VDJ bias

Abstract Yellow fever (YF) vaccination induces massive CD8 expansion, comprising up to 10% of all circulating CD8 T cells. We hypothesize that HLA type and VDJ gene usage implicate the highly biased response towards certain immunodominant epitopes. PBMCs were collected from healthy human donors 3 weeks after vaccination with the yellow fever vaccine, Stamaril, and enriched for YF-CD8 T cells using HLA class I tetramers. We focused on the immunodominant epitopes NS4B and NS2A, and the less dominant epitope NS3. Cells were analyzed by single-cell T cell receptor (TCR) and mRNA sequencing, yielding detailed gene phenotypes for around 2000–3000 quality cells from each donor. Data was analyzed using SeqGeq, VDJ explorer, Seurat and Monocle2. From three donors, we performed in depth analyses on both TCR gene use bias across epitope specificity, and established pseudotime cell trajectories from a single sampling time point. We found that within the CD8 population from the same donor, VDJ usage was highly biased for the target antigen. Based on mRNA expression of a panel of 259 T-cell relevant genes, we performed unsupervised clustering, that shows cells with different epitope targets cluster interchangeably, indicating similar functional patterns. However, cells specific for the immunodominant epitopes had lower TCR clonal divergence between naïve-like and activated clusters, indicating a higher degree of clonal expansion compared to less immunodominant clones. Taken together, we show antigen-driven T cell responses are biased based on donor HLA-types, which could implicate development and success of T-cell driven immunotherapies. Supported by grants from Independent Research Fund Denmark (1029-00004B)

Characterization of human IgM and IgG repertoires in individuals with chronic HIV-1 infection.

Advancements in high-throughput sequencing (HTS) of antibody repertoires (Ig-Seq) have unprecedentedly improved our ability to characterize the antibody repertoires on a large scale. However, currently, only a few studies explored the influence of chronic HIV-1 infection on human antibody repertoires and many of them reached contradictory conclusions, possibly limited by inadequate sequencing depth and throughput. To better understand how HIV-1 infection would impact humoral immune system, in this study, we systematically analyzed the differences between the IgM (HIV-IgM) and IgG (HIV-IgG) heavy chain repertoires of HIV-1 infected patients, as well as between antibody repertoires of HIV-1 patients and healthy donors (HH). Notably, the public unique clones accounted for only a negligible proportion between the HIV-IgM and HIV-IgG repertoires libraries, and the diversity of unique clones in HIV-IgG remarkably reduced. In aspect of somatic mutation rates of CDR1 and CDR2, the HIV-IgG repertoire was higher than HIV-IgM. Besides, the average length of CDR3 region in HIV-IgM was significant longer than that in the HH repertoire, presumably caused by the great number of novel VDJ rearrangement patterns, especially a massive use of IGHJ6. Moreover, some of the B cell clonotypes had numerous clones, and somatic variants were detected within the clonotype lineage in HIV-IgG, indicating HIV-1 neutralizing activities. The in-depth characterization of HIV-IgG and HIV-IgM repertoires enriches our knowledge in the profound effect of HIV-1 infection on human antibody repertoires and may have practical value for the discovery of therapeutic antibodies.

VDJ Gene Usage in IgM Repertoires of Rhesus and Cynomolgus Macaques.

Macaques are frequently used to evaluate candidate vaccines and to study infection-induced antibody responses, requiring an improved understanding of their naïve immunoglobulin (IG) repertoires. Baseline gene usage frequencies contextualize studies of antigen-specific immune responses, providing information about how easily one may stimulate a response with a particular VDJ recombination. Studies of human IgM repertoires have shown that IG VDJ gene frequencies vary several orders of magnitude between the most and least utilized genes in a manner that is consistent across many individuals but to date similar analyses are lacking for macaque IgM repertoires. Here, we quantified VDJ gene usage levels in unmutated IgM repertoires of 45 macaques, belonging to two species and four commonly used subgroups: Indian and Chinese origin rhesus macaques and Indonesian and Mauritian origin cynomolgus macaques. We show that VDJ gene frequencies differed greatly between the most and least used genes, with similar overall patterns observed in macaque subgroups and individuals. However, there were also clear differences affecting the use of specific V, D and J genes. Furthermore, in contrast to humans, macaques of both species utilized IGHV4 family genes to a much higher extent and showed evidence of evolutionary expansion of genes of this family. Finally, we used the results to inform the analysis of a broadly neutralizing HIV-1 antibody elicited in SHIV-infected rhesus macaques, RHA1.V2.01, which binds the apex of the Env trimer in a manner that mimics the binding mode of PGT145. We discuss the likelihood that similar antibodies could be elicited in different macaque subgroups.

Targeted-Capture Sequencing Approach to V(D)J Rearrangement, Immunoglobulin (Ig) Translocation and Mutation Detection in Multiple Myeloma (MM): Potential Applications in Minimal Residual Disease (MRD) Analysis and Comparison to Multiparameter Flow Cytometry (MFC) in the Myeloma Canada Research Network (MCRN) 001 Study with Intravenous Busulfan and Melphalan (BuMel) Conditioning Followed By Lenalidomide Maintenance in Newly Diagnosed Multiple Myeloma (NDMM)

Targeted-Capture Sequencing Approach to V(D)J Rearrangement, Immunoglobulin (Ig) Translocation and Mutation Detection in Multiple Myeloma (MM): Potential Applications in Minimal Residual Disease (MRD) Analysis and Comparison to Multiparameter Flow Cytometry (MFC) in the Myeloma Canada Research Network (MCRN) 001 Study with Intravenous Busulfan and Melphalan (BuMel) Conditioning Followed By Lenalidomide Maintenance in Newly Diagnosed Multiple Myeloma (NDMM)

Single-Cell TCR Sequencing Reveals the Dynamics of T Cell Repertoire Profiling During Pneumocystis Infection.

T cell responses play critical roles in host adaptive immunity against Pneumocystis. However, the dynamics and diversity of the T cell immune repertoire in human immunodeficiency virus (HIV)-negative Pneumocystis pneumonia (PCP) remains unclear. In this study, single-cell RNA and single-cell T cell receptor (TCR) sequencing were applied to cells sorted from lung tissues of mice infected with Pneumocystis. Our findings demonstrated the clonal cells were mainly composed of CD4+ T cells in response to Pneumocystis, which were marked by highly expressed genes associated with T cell activation. Mice infected with Pneumocystis showed reduced TCR diversity in CD4+ T cells and increased diversity in CD8+ T cells compared with uninfected controls. Furthermore, Th17 cells were mostly clonal CD4+ T cells, which exhibited the phenotype of tissue-resident memory-like Th17 cells. In addition, Pneumocystis-infected mice showed biased usage of TCRβ VDJ genes. Taken together, we characterized the transcriptome and TCR immune repertoires profiles of expanded T cell clones, which demonstrate a skewed TCR repertoire after Pneumocystis infection.

Decrease of Clone Diversity in IgM Repertoires of HBV Chronically Infected Individuals With High Level of Viral Replication.

High-throughput antibody sequencing allows in-depth insights into human antibody repertoires. To investigate the characteristics of antibody repertoires in patients with chronic HBV infection, we performed Illumina sequencing and IMGT/HighV-QUEST analysis of B lymphocytes from healthy adults and the HBV carriers with high or low level of viral replication. The comparative study revealed high levels of similarity between the IgM and IgG repertoires of the HBV carriers and the healthy adults, including the somatic mutations in V regions, the average CDR3 length, and the occurrence of junctional modifications. Nevertheless, the diversity of the unique clones decreased and some clusters of unique clones expanded in the IgM repertoire of chronic HBV carriers (CHB) compared with healthy adults (HH) and inactive HBV carriers (IHB). Such difference in clone diversity and expansion was not observed in the IgG repertoires of the three populations. More shared antibody clones were found between the IgM repertoires of IHB and HH than that found between CHB and HH (7079 clones vs. 2304 clones). Besides, the biased used IGHD genes were IGHD2-2 and IGHD3-3 in CHB library but were IGHD3-10 and IGHD3-22 in IHB and HH library. In contrast, for IgG repertories, the preferred used VDJ genes were similar in all the three populations. These results indicated that low level of serum HBV might not induce significant changes in BCR repertoires, and high level of HBV replication could have more impacts on IgM repertories than IgG repertoires. Taken together, our findings provide a better understanding of the antibody repertoires of HBV chronically infected individuals.

Genomic analysis of a second rainbow trout line (Arlee) leads to an extended description of the IGH VDJ gene repertoire

High-throughput sequencing technologies brought a renewed interest for immune repertoires. Fish Ab and B cell repertoires are no exception, and their comprehensive analysis can both provide new insights into poorly understood immune mechanisms, and identify markers of protection after vaccination. However, the lack of genomic description and standardized nomenclature of IG genes hampers accurate annotation of Ig mRNA deep sequencing data. Complete genome sequences of Atlantic salmon and rainbow trout (Swanson line) recently allowed us to establish a comprehensive and coherent annotation of Salmonid IGH genes following IMGT standards. Here we analyzed the IGHV, D, and J genes from the newly released genome of a second rainbow trout line (Arlee). We confirmed the validity of salmonid IGHV subgroups, and extended the description of the rainbow trout IGH gene repertoire with novel sequences, while keeping nomenclature continuity. This work provides an important resource for annotation of high-throughput Ab repertoire sequencing data.

Evidence of B Cell Clonality and Investigation Into Properties of the IgM in Patients With Schnitzler Syndrome.

The Schnitzler Syndrome (SchS) is an acquired, autoinflammatory condition successfully treated with IL-1 inhibition. The two main defining features of this late-onset condition are neutrophilic urticarial dermatoses (NUD) and the presence of an IgM monoclonal component. While the former aspect has been extensively studied in this disease setting, the enigmatic paraproteinaemia and its potential consequential effects within SchS, has not previously been thoroughly addressed. Previous studies analyzing clonal B cell repertoires have largely focused on autoimmune disorders such as Systemic Lupus Erythematous (SLE) and hematological malignancies such as Chronic Lymphocytic Leukaemia (CLL), where B-cell clonality is central to disease pathology. The present study uses next-generation sequencing to provide detailed insight into aspects of B cell VDJ recombination and properties of the resulting immunoglobulin chains. An overview of IgH regional dynamics in 10 SchS patients, with a particular focus on CDR3 sequences and VDJ gene usage is reported, highlighting the presence of specific B cell expansions. Protein microarray detected a substantial proportion of autoreactive IgM to nuclear target proteins, though a single universal target was not identified. Together, these genetic and functional findings impart new understanding into this rare disorder.

The respiratory syncytial virus fusion protein-specific B cell receptor repertoire reshaped by post-fusion subunit vaccination

Respiratory syncytial virus (RSV) is the major cause of acute lower respiratory illness in children of less than 5 years of age which usually results in hospitalization or even in death. Vaccine development is hampered in consequence of a failed vaccine trial with fatalities in the 1960s. Even though research has been more focused on the RSV fusion protein in its pre-fusion conformation, maternal vaccination with post-fusion protein (post F) was considered as a promising vaccine strategy for passive immunization of babies, because post F preserves very potent neutralizing epitopes. We extensively analyzed post F-binding B cell receptor (BCR) repertoires of three vaccinees who received a post F-subunit vaccine in the context of a first-in-human, Phase 1, randomized, observer-blind, placebo-controlled clinical trial (ClinicalTrials.gov Identifier: NCT02298179). In order to compare the vaccine-induced BCR repertoires with BCR repertoires induced by natural infection, we also analyzed pre F- and post F-binding BCRs isolated from a healthy blood donor with relatively high F-binding memory B cell (MBC) frequencies. Analysis of the vaccine-induced repertoires revealed that preferentially VH4-encoded BCRs were expanded in response to vaccination. Estimation of antigen-driven selection further demonstrated that expanded BCRs accumulated positively selected replacement mutations which substantiated the hypothesis that post F-vaccination induces diversification of VH4-encoded BCRs in germinal centers. Comparison of the vaccine-induced BCR repertoires with clonally related pre and post F-binding BCRs of the healthy blood donor suggested that the vaccine expanded pre/post F cross-reactive MBCs. Interestingly, several vaccine-induced BCRs shared stereotypic VDJ gene junctions with known neutralizing Abs. Once expressed for functional characterization, the selected monoclonal Abs demonstrated the predicted neutralization activities in plaque reduction neutralization assays indicating that the post F-vaccine induced expansion of neutralizing BCRs.

1876. A Hepacivirus-Like Protein Is Targeted by the Antibody Response to Kawasaki Disease (KD)

BackgroundClinical and epidemiologic data support a viral cause of KD, but the etiology has eluded 50 years of study. We previously identified virus-like intracytoplasmic inclusion bodies (ICI) in ciliated bronchial epithelium of KD children but not infant controls, but the antigens within the ICI were unknown. At 1–2 weeks following infection, 75% of peripheral blood plasmablasts (PB) specifically target the infectious agent. We cloned the PB response to KD to identify KD-specific antibodies and their target antigens.MethodsWe isolated single PB from children with KD 1–3 weeks after fever onset by flow cytometry, and amplified immunoglobulin VDJ and VJ genes from each PB by RT-PCR. We sequenced the products and made monoclonal antibodies (Mab) from clonally expanded PB in individual patients. Mab were tested for binding to KD tissues and to a viral peptide array containing 29,939 peptides from known B cell epitopes of animal viruses (www.iedb.org).ResultsWe sequenced 1156 PB from 11 KD patients, and identified 44 clonally expanded sets of PB. We prepared 61 Mab from clonally expanded and highly mutated IgA PB, and found that 33/61 bind to KD ICI, 10 strongly and 23 weakly. Of 10 Mab that strongly bind, 2 were VH3-33 (single patient), 2 VH3-23 (single patient), 1 VH3-15, 1 VH3-74, 3 VH1-46 (2 patients), and 1 VH4-59. These Mab CDR3s varied from 11 to 20 aacids, with 4–28 aacid mutations. Mab KD4-2H4 recognized multiple similar peptides from nonstructural protein 4A of hepacivirus C; pt KD4 sera was negative for hepatitis C by fourth-generation ELISA. Amino acid substitution analysis yielded an optimized peptide, and 6 KD Mab recognized this peptide by ELISA. These 6 Mab derived from 3 KD patients, all of whom had coronary aneurysms, and were VH3-74 (n = 1), VH3-33 (n = 2, single patient), VH1-45 (n = 1), and VH3-72 (n = 2, single patient). Strong binding of KD Mab KD4-2H4 and KD6-2B2 to ICI was totally blocked by pre-incubation with optimized peptide. KD but not control sera react with optimized peptide expressed as a glutathione S-transferase fusion protein by western blot.ConclusionChildren with KD make antibodies to a hepacivirus-like protein, and KD ICI contain this protein. These results strongly suggest that a previously unidentified hepacivirus with a respiratory portal of entry is etiologically related to KD.Disclosures All Authors: No reported Disclosures.

Benchmark datasets of immune receptor-epitope structural complexes

BackgroundThe development of accurate epitope prediction tools is important in facilitating disease diagnostics, treatment and vaccine development. The advent of new approaches making use of antibody and TCR sequence information to predict receptor-specific epitopes have the potential to transform the epitope prediction field. Development and validation of these new generation of epitope prediction methods would benefit from regularly updated high-quality receptor-antigen complex datasets.ResultsTo address the need for high-quality datasets to benchmark performance of these new generation of receptor-specific epitope prediction tools, a webserver called SCEptRe (Structural Complexes of Epitope-Receptor) was created. SCEptRe extracts weekly updated 3D complexes of antibody-antigen, TCR-pMHC and MHC-ligand from the Immune Epitope Database and clusters them based on antigen, receptor and epitope features to generate benchmark datasets. SCEptRe also provides annotated information such as CDR sequences and VDJ genes on the receptors. Users can generate custom datasets based by selecting thresholds for structural quality and clustering parameters (e.g. resolution, R-free factor, antigen or epitope sequence identity) based on their need.ConclusionsSCEptRe provides weekly updated, user-customized comprehensive benchmark datasets of immune receptor-epitope structural complexes. These datasets can be used to develop and benchmark performance of receptor-specific epitope prediction tools in the future. SCEptRe is freely accessible at http://tools.iedb.org/sceptre.

Sequencing of VDJ genes in Lepus americanus confirms a correlation between VHn expression and the leporid species continent of origin

Leporid VH genes used in the generation of their primary antibody repertoire exhibit highly divergent lineages. For the European rabbit (Oryctolagus cuniculus) four VHa lineages have been described, the a1, a2, a3 and a4. Hares (Lepus spp.) and cottontail (Sylvilagus floridanus) express one VHa lineage each, the a2L and the a5, respectively, along with a more ancient lineage, the Lepus spp. sL and S. floridanus sS. Both the European rabbit and the Lepus europaeus use a third lineage, VHn, in a low proportion of their VDJ rearrangements. The VHn genes are a conserved ancestral polymorphism that is being maintained in the leporid genome.Their usage in a low proportion of VDJ rearrangements by both European rabbit and L. europaeus but not S. floridanus has been argued to be a remnant of an ancient European leporid immunologic response to pathogens. To address this hypothesis, in this study we sequenced VDJ rearranged genes for another North American leporid, L. americanus. Our results show that L. americanus expressed these genes less frequently and in a highly modified fashion compared to the European Lepus species. Our results suggest that the American leporid species use a different VH repertoire than the European species which may be related with an immune adaptation to different environmental conditions, such as different pathogenic agents.