Outbreaks of Ebolaviruses, such as Sudanvirus (SUDV) in Uganda in 2022, demonstrate that species other than the Zaire ebolavirus (EBOV), which is currently the sole virus represented in current licensed vaccines, remain a major threat to global health. There is a pressing need to develop effective pan-species vaccines and novel monoclonal antibody-based therapeutics for Ebolavirus disease. In response to recent outbreaks, the two dose, heterologous Ad26.ZEBOV/MVA-BN-Filo vaccine regimen was developed and was tested in a large phase II clinical trial (EBL2001) as part of the EBOVAC2 consortium. Here, we perform bulk sequencing of the variable heavy chain (VH) of B cell receptors (BCR) in forty participants from the EBL2001 trial in order to characterize the BCR repertoire in response to vaccination with Ad26.ZEBOV/MVA-BN-Filo. We develop a comprehensive database, EBOV-AbDab, of publicly available Ebolavirus-specific antibody sequences. We then use our database to predict the antigen-specific component of the vaccinee repertoires. Our results show striking convergence in VH germline gene usage across participants following the MVA-BN-Filo dose, and provide further evidence of the role of IGHV3–15 and IGHV3–13 antibodies in the B cell response to Ebolavirus glycoprotein. Furthermore, we found that previously described Ebola-specific mAb sequences present in EBOV-AbDab were sufficient to describe at least one of the ten most expanded BCR clonotypes in more than two thirds of our cohort of vaccinees following the boost, providing proof of principle for the utility of computational mining of immune repertoires.

BACKGROUND:Biallelic mutations in LIG4 encoding DNA-ligase 4 cause a rare immunodeficiency syndrome manifesting as infant-onset life-threatening and/or opportunistic infections, skeletal malformations, radiosensitivity and neoplasia. LIG4 is pivotal during DNA repair and during V(D)J recombination as it performs the final DNA-break sealing step. OBJECTIVES:This study explored whether monoallelic LIG4 missense mutations may underlie immunodeficiency and autoimmunity with autosomal dominant inheritance. METHODS:Extensive flow-cytometric immune-phenotyping was performed. Rare variants of immune system genes were analyzed by whole exome sequencing. DNA repair functionality and T-cell-intrinsic DNA damage tolerance was tested with an ensemble of in vitro and in silico tools. Antigen-receptor diversity and autoimmune features were characterized by high-throughput sequencing and autoantibody arrays. Reconstitution of wild-type versus mutant LIG4 were performed in LIG4 knockout Jurkat T cells, and DNA damage tolerance was subsequently assessed. RESULTS:A novel heterozygous LIG4 loss-of-function mutation (p.R580Q), associated with a dominantly inherited familial immune-dysregulation consisting of autoimmune cytopenias, and in the index patient with lymphoproliferation, agammaglobulinemia, and adaptive immune cell infiltration into nonlymphoid organs. Immunophenotyping revealed reduced naive CD4+ T cells and low TCR-Vα7.2+ T cells, while T-/B-cell receptor repertoires showed only mild alterations. Cohort screening identified 2 other nonrelated patients with the monoallelic LIG4 mutation p.A842D recapitulating clinical and immune-phenotypic dysregulations observed in the index family and displaying T-cell-intrinsic DNA damage intolerance. Reconstitution experiments and molecular dynamics simulations categorize both missense mutations as loss-of-function and haploinsufficient. CONCLUSIONS:This study provides evidence that certain monoallelic LIG4 mutations may cause human immune dysregulation via haploinsufficiency.

Immunoglobulin loci-transgenic animals are widely used in antibody discovery and increasingly in vaccine response modelling. In this study, we phenotypically characterised B-cell populations from the Intelliselect Transgenic mouse (Kymouse) demonstrating full B-cell development competence. Comparison of the naïve B-cell receptor (BCR) repertoires of Kymice BCRs, naïve human, and murine BCR repertoires revealed key differences in germline gene usage and junctional diversification. These differences result in Kymice having CDRH3 length and diversity intermediate between mice and humans. To compare the structural space explored by CDRH3s in each species’ repertoire, we used computational structure prediction to show that Kymouse naïve BCR repertoires are more human-like than mouse-like in their predicted distribution of CDRH3 shape. Our combined sequence and structural analysis indicates that the naïve Kymouse BCR repertoire is diverse with key similarities to human repertoires, while immunophenotyping confirms that selected naïve B cells are able to go through complete development.

T cell receptor repertoire sequencing (TCRseq) has become one of the major omic tools to study the immune system in health and disease. Multiple commercial solutions are currently available, greatly facilitating the implementation of this complex method into translational studies. However, the flexibility of these methods to react to suboptimal sample material is still limited. In a clinical research context, limited sample availability and/or unbalanced sample material can negatively impact the feasibility and quality of such analyses. We sequenced the T cell receptor repertoires of three healthy controls and four patients with GATA2 deficiency using a commercially available TCRseq kit and thereby (1) assessed the impact of suboptimal sample quality and (2) implemented a subsampling strategy to react to biased sample input quantity. Applying these strategies, we did not find significant differences in the global T cell receptor repertoire characteristics such as V and J gene usage, CDR3 junction length and repertoire diversity of GATA2-deficient patients compared with healthy control samples. Our results prove the adaptability of this TCRseq protocol to the analysis of unbalanced sample material and provide encouraging evidence for use of this method in future studies despite suboptimal patient samples.

Background and Objectives Autologous hematopoietic stem cell transplantation (aHSCT) is increasingly used to treat aggressive forms of multiple sclerosis (MS). This procedure is believed to result in an immune reset and restoration of a self-tolerant immune system. Immune reconstitution has been extensively studied for T cells, but only to a limited extent for B cells. As increasing evidence suggests an important role of B cells in MS pathogenesis, we sought here to better understand reconstitution and the extent of renewal of the B-cell system after aHSCT in MS. Methods Using longitudinal multidimensional flow cytometry and immunoglobulin heavy chain (IgH) repertoire sequencing following aHSCT with BCNU + Etoposide + Ara-C + Melphalan anti-thymocyte globulin, we analyzed the B-cell compartment in a cohort of 20 patients with MS in defined intervals before and up to 1 year after aHSCT and compared these findings with data from healthy controls. Results Total B-cell numbers recovered within 3 months and increased above normal levels 1 year after transplantation, successively shifting from a predominantly transitional to a naive immune phenotype. Memory subpopulations recovered slowly and remained below normal levels with reduced repertoire diversity 1 year after transplantation. Isotype subclass analysis revealed a proportional shift toward IgG1-expressing cells and a reduction in IgG2 cells. Mutation analysis of IgH sequences showed that highly mutated memory B cells and plasma cells may transiently survive conditioning while the analysis of sequence cluster overlap, variable (IGHV) and joining (IGHJ) gene usage and repertoire diversity suggested a renewal of the late posttransplant repertoire. In patients with early cytomegalovirus reactivation, reconstitution of naive and memory B cells was delayed. Discussion Our detailed characterization of B-cell reconstitution after aHSCT in MS indicates a reduced reactivation potential of memory B cells up to 1 year after transplantation, which may leave patients susceptible to infection, but may also be an important aspect of its mechanism of action.

Several human B cell subpopulations are recognised in the peripheral blood, which play distinct roles in the humoral immune response. These cells undergo developmental and maturational changes involving VDJ recombination, somatic hypermutation and class switch recombination, altogether shaping their immunoglobulin heavy chain (IgH) repertoire. Here, we sequenced the IgH repertoire of naïve, marginal zone, switched and plasma cells from 10 healthy adults along with matched unsorted and in silico separated CD19+ bulk B cells. Using advanced bioinformatic analysis and machine learning, we show that sorted B cell subpopulations are characterised by distinct repertoire characteristics on both the individual sequence and the repertoire level. Sorted subpopulations shared similar repertoire characteristics with their corresponding in silico separated subsets. Furthermore, certain IgH repertoire characteristics correlated with the position of the constant region on the IgH locus. Overall, this study provides unprecedented insight over mechanisms of B cell repertoire control in peripherally circulating B cell subpopulations.

B cells play a central role in adaptive immune processes, mainly through the production of antibodies. The maturation of the B cell system with age is poorly studied. We extensively investigated age-related alterations of naïve and antigen-experienced immunoglobulin heavy chain (IgH) repertoires. The most significant changes were observed in the first 10 years of life, and were characterized by altered immunoglobulin gene usage and an increased frequency of mutated antibodies structurally diverging from their germline precursors. Older age was associated with an increased usage of downstream IgH constant region genes and fewer antibodies with self-reactive properties. As mutations accumulated with age, the frequency of germline-encoded self-reactive antibodies decreased, indicating a possible beneficial role of self-reactive B cells in the developing immune system. Our results suggest a continuous process of change through childhood across a broad range of parameters characterizing IgH repertoires and stress the importance of using well-selected, age-appropriate controls in IgH studies.

B cells play a central role in adaptive immune processes, mainly through the production of antibodies. The maturation of the B-cell system with age is poorly studied. We extensively investigated age-related alterations of naïve and antigen-experienced B-cell receptor (BCR) repertoires. The most significant changes were observed in the first 10 years of life, and were characterized by altered immunoglobulin gene usage and an increased frequency of mutated antibodies structurally diverging from their germline precursors. Older age was associated with an increased usage of downstream constant region genes and fewer antibodies with self-reactive properties. As mutations accumulated with age, the frequency of germline-encoded self-reactive antibodies decreased, indicating a possible beneficial role of self-reactive B-cells in the developing immune system. Our results suggest a continuous process of change through childhood across a broad range of parameters characterizing BCR repertoires and stress the importance of using well-selected, age-appropriate controls in BCR studies.

Nowadays, due to the advance of experimental techniques in glycomics, large collections of glycan profiles are regularly published. The rapid growth of available glycan data accentuates the lack of innovative tools for visualizing and exploring large amount of information. Scientists resort to using general-purpose spreadsheet applications to create ad hoc data visualization. Thus, results end up being encoded in publication images and text, while valuable curated data is stored in files as supplementary information. To tackle this problem, we have built an interactive pipeline composed with three tools: Glynsight, EpitopeXtractor and Glydin'. Glycan profile data can be imported in Glynsight, which generates a custom interactive glycan profile. Several profiles can be compared and glycan composition is integrated with structural data stored in databases. Glycan structures of interest can then be sent to EpitopeXtractor to perform a glycoepitope extraction. EpitopeXtractor results can be superimposed on the Glydin' glycoepitope network. The network visualization allows fast detection of clusters of glycoepitopes and discovery of potential new targets. Each of these tools is standalone or can be used in conjunction with the others, depending on the data and the specific interest of the user. All the tools composing this pipeline are part of the Glycomics@ExPASy initiative and are available at https://www.expasy.org/glycomics.

The advent of next-generation sequencing (NGS) now allows a detailed assessment of the adaptive immune system in health and disease. In particular, high-throughput B-cell receptor (BCR) repertoire sequencing provides detailed information about the functionality and abnormalities of the B-cell system. However, it is mostly unknown how the BCR repertoire is altered in the context of primary immunodeficiencies (PID) and whether findings are consistent throughout phenotypes and genotypes. We have performed an extensive literature search of the published work on BCR repertoire sequencing in PID patients, including several forms of predominantly antibody disorders and combined immunodeficiencies. It is somewhat surprising that BCR repertoires, even from severe clinical phenotypes, often show only mild abnormalities and that diversity or immunoglobulin gene segment usage is generally preserved to some extent. Despite the great variety of wet laboratory and analytical methods that were used in the different studies, several findings are common to most investigated PIDs, such as the increased usage of gene segments that are associated with self-reactivity. These findings suggest that BCR repertoire characteristics may be used to assess the functionality of the B-cell compartment irrespective of the underlying defect. With the use of NGS approaches, there is now the opportunity to apply BCR repertoire sequencing to multiple patients and explore the PID BCR repertoire in more detail. Ultimately, using BCR repertoire sequencing in translational research could aid the management of PID patients by improving diagnosis, estimating functionality of the immune system and improving assessment of prognosis.