Antigen Extraction and B Cell Activation Enable Identification of Rare Membrane Antigen Specific Human B Cells

Determining antigen specificity is vital for understanding B cell biology and for producing human monoclonal antibodies. We describe here a powerful method for identifying B cells that recognize membrane antigens expressed on cells. The technique depends on two characteristics of the interaction between a B cell and an antigen-expressing cell: antigen-receptor-mediated extraction of antigen from the membrane of the target cell, and B cell activation. We developed the method using influenza hemagglutinin as a model viral membrane antigen, and tested it using acetylcholine receptor (AChR) as a model membrane autoantigen. The technique involves co-culturing B cells with adherent, bioorthogonally labeled cells expressing GFP-tagged antigen, and sorting GFP-capturing, newly activated B cells. Hemagglutinin-specific B cells isolated this way from vaccinated human donors expressed elevated CD20, CD27, CD71, and CD11c, and reduced CD21, and their secreted antibodies blocked hemagglutination and neutralized viral infection. Antibodies cloned from AChR-capturing B cells derived from patients with myasthenia gravis bound specifically to the receptor on cell membrane. The approach is sensitive enough to detect antigen-specific B cells at steady state, and can be adapted for any membrane antigen

Association of host factors with antibody response to seasonal influenza vaccination in allogeneic hematopoietic stem cell transplant (HSCT) patients

BACKGROUND Influenza vaccination efficacy is reduced after hematopoietic stem cell transplantation (HSCT) and patient factors determining vaccination outcomes are still poorly understood. METHODS We investigated the antibody response to seasonal influenza vaccination in 135 HSCT patients and 69 healthy volunteers (HVs) in a prospective observational multicenter cohort study. We identified patient factors associated with hemagglutination inhibition titers against A/California/2009/H1N1, A/Texas/2012/H3N2, and B/Massachusetts/2012 by multivariable regression on the observed titer levels and on seroconversion/seroprotection categories for comparison. RESULTS Both regression approaches yield consistent results but regression on titers estimated associations with higher precision. HSCT patients required two vaccine doses to achieve average responses comparable to a single dose in HVs. Pre-vaccination titers were positively associated with time after transplantation, confirming that HSCT patients can elicit potent antibody responses. However, an unrelated donor, absolute lymphocyte counts below the normal range and treatment with calcineurin inhibitors lower the odds of responding. CONCLUSIONS HSCT patients show a highly heterogeneous vaccine response, but overall, patients benefited from the booster shot and can acquire seroprotective antibodies over the years after transplantation. Several common patient factors lower the odds of responding, urging to identify additional preventive strategies in the poorly responding groups

IL-28B is a Key Regulator of B- and T-Cell Vaccine Responses against Influenza

Influenza is a major cause of morbidity and mortality in immunosuppressed persons, and vaccination often confers insufficient protection. IL-28B, a member of the interferon (IFN)-λ family, has variable expression due to single nucleotide polymorphisms (SNPs). While type-I IFNs are well known to modulate adaptive immunity, the impact of IL-28B on B- and T-cell vaccine responses is unclear. Here we demonstrate that the presence of the IL-28B TG/GG genotype (rs8099917, minor-allele) was associated with increased seroconversion following influenza vaccination (OR 1.99 p = 0.038). Also, influenza A (H1N1)-stimulated T- and B-cells from minor-allele carriers showed increased IL-4 production (4-fold) and HLA-DR expression, respectively. In vitro, recombinant IL-28B increased Th1-cytokines (e.g. IFN-γ), and suppressed Th2-cytokines (e.g. IL-4, IL-5, and IL-13), H1N1-stimulated B-cell proliferation (reduced 70%), and IgG-production (reduced>70%). Since IL-28B inhibited B-cell responses, we designed antagonistic peptides to block the IL-28 receptor α-subunit (IL28RA). In vitro, these peptides significantly suppressed binding of IFN-λs to IL28RA, increased H1N1-stimulated B-cell activation and IgG-production in samples from healthy volunteers (2-fold) and from transplant patients previously unresponsive to vaccination (1.4-fold). Together, these findings identify IL-28B as a key regulator of the Th1/Th2 balance during influenza vaccination. Blockade of IL28RA offers a novel strategy to augment vaccine responses

Identification of influenza urban transmission patterns by geographical, epidemiological and whole genome sequencing data: protocol for an observational study

Introduction Urban transmission patterns of influenza viruses are complex and poorly understood, and multiple factors may play a critical role in modifying transmission. Whole genome sequencing (WGS) allows the description of patient-to-patient transmissions at highest resolution. The aim of this study is to explore urban transmission patterns of influenza viruses in high detail by combining geographical, epidemiological and immunological data with WGS data. Methods and analysis The study is performed at the University Hospital Basel, University Children's Hospital Basel and a network of paediatricians and family doctors in the Canton of Basel-City, Switzerland. The retrospective study part includes an analysis of PCR-confirmed influenza cases from 2013 to 2018. The prospective study parts include (1) a household survey regarding influenza-like illness (ILI) and vaccination against influenza during the 2015/2016 season; (2) an analysis of influenza viruses collected during the 2016/2017 season using WGS-viral genomic sequences are compared with determine genetic relatedness and transmissions; and (3) measurement of influenza-specific antibody titres against all vaccinated and circulated strains during the 2016/2017 season from healthy individuals, allowing to monitor herd immunity across urban quarters. Survey data and PCR-confirmed cases are linked to data from the Statistics Office of the Canton Basel-City and visualised using geo-information system mapping. WGS data will be analysed in the context of patient epidemiological data using phylodynamic analyses, and the obtained herd immunity for each quarter. Profound knowledge on the key geographical, epidemiological and immunological factors influencing urban influenza transmission will help to develop effective counter measurements. Ethics and dissemination The study is registered and approved by the regional ethics committee as an observational study (EKNZ project ID 2015-363 and 2016-01735). It is planned to present the results at conferences and publish the data in scientific journals

Characterising the epidemic spread of influenza A/H3N2 within a city through phylogenetics

Infecting large portions of the global population, seasonal influenza is a major burden on societies around the globe. While the global source sink dynamics of the different seasonal influenza viruses have been studied intensively, its local spread remains less clear. In order to improve our understanding of how influenza is transmitted on a city scale, we collected an extremely densely sampled set of influenza sequences alongside patient metadata. To do so, we sequenced influenza viruses isolated from patients of two different hospitals, as well as private practitioners in Basel, Switzerland during the 2016/2017 influenza season. The genetic sequences reveal that repeated introductions into the city drove the influenza season. We then reconstruct how the effective reproduction number changed over the course of the season. While we did not find that transmission dynamics in Basel correlate with humidity or school closures, we did find some evidence that it may positively correlated with temperature. Alongside the genetic sequence data that allows us to see how individual cases are connected, we gathered patient information, such as the age or household status. Zooming into the local transmission outbreaks suggests that the elderly were to a large extent infected within their own transmission network. In the remaining transmission network, our analyses suggest that school-aged children likely play a more central role than pre-school aged children. These patterns will be valuable to plan interventions combating the spread of respiratory diseases within cities given that similar patterns are observed for other influenza seasons and cities

Identification of novel HPFH-like mutations by CRISPR baseediting that elevate the expression of fetal hemoglobin

Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T > C and -124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.ISSN:2050-084