Most of what we know about adaptive immunity has come from inbred mouse studies, using methods that are often difficult or impossible to confirm in humans. In addition, vaccine responses in mice are ...often poorly predictive of responses to those same vaccines in humans. Here we use human tonsils, readily available lymphoid organs, to develop a functional organotypic system that recapitulates key germinal center features in vitro, including the production of antigen-specific antibodies, somatic hypermutation and affinity maturation, plasmablast differentiation and class-switch recombination. We use this system to define the essential cellular components necessary to produce an influenza vaccine response. We also show that it can be used to evaluate humoral immune responses to two priming antigens, rabies vaccine and an adenovirus-based severe acute respiratory syndrome coronavirus 2 vaccine, and to assess the effects of different adjuvants. This system should prove useful for studying critical mechanisms underlying adaptive immunity in much greater depth than previously possible and to rapidly test vaccine candidates and adjuvants in an entirely human system.
In this article, we discuss new insights into the distinct mechanisms for V(D)J recombination for different immunoglobulin loci. This follows the recent revelation that recombination signal sequences ...(RSS) within the IGKV locus have evolved to be more efficient mediators of recombination activating gene (RAG) recombination compared to the same elements in the IGH locus. This difference in RSS strength is proposed to be driven by different molecular mechanisms for RAG-mediated recombination between the two loci.In this article, we discuss new insights into the distinct mechanisms for V(D)J recombination for different immunoglobulin loci. This follows the recent revelation that recombination signal sequences (RSS) within the IGKV locus have evolved to be more efficient mediators of recombination activating gene (RAG) recombination compared to the same elements in the IGH locus. This difference in RSS strength is proposed to be driven by different molecular mechanisms for RAG-mediated recombination between the two loci.
Qualitative interview studies on sensitive topics often draw on principles of feminist methodologies which focus on developing and maintaining non-exploitative, caring relationships with ...participants. For early career researchers, who may have less research experience, managing relational ethical issues that arise in research relationships can be difficult. Additionally, they could experience further pressures because of their junior roles and precarious employment. In the context of health research, early career researchers working on qualitative studies may experience specific challenges because of the predominance of the biomedical paradigm in this discipline. In this article, I explore some of the relational ethical issues I deliberated as an early career researcher when working in a medical faculty on a semi-structured qualitative interview study about women’s alcohol drinking practices. I focus on two overlapping themes from my experience of ethics in practice “Trying to building and maintain relationships” and “Trying to stabilize inequalities in research relationships.” With a primary focus on how I negotiated differing responsibilities, I draw on examples from the community-based face-to-face and virtual recruitment, the fast-paced face-to-face interviews, and the process of returning interview transcripts to women to review. With this analysis, I contribute to existing literature about ethics in practice for early career researchers by indicating the types of relational ethics that will need to be navigated and the resources needed to support them. These resources include having adequate time, opportunities for reflection, and good supervisory support. I also contribute to scholarship which critiques the wider health research context by considering the challenges it can pose for early career researchers when managing relational ethical spaces in qualitative interview studies. This article will be of relevance to novice researchers and those who supervise and manage them.
A goal of HIV vaccine development is to elicit antibodies with neutralizing breadth. Broadly neutralizing antibodies (bNAbs) to HIV often have unusual sequences with long heavy-chain ...complementarity-determining region loops, high somatic mutation rates and polyreactivity. A subset of HIV-infected individuals develops such antibodies, but it is unclear whether this reflects systematic differences in their antibody repertoires or is a consequence of rare stochastic events involving individual clones. We sequenced antibody heavy-chain repertoires in a large cohort of HIV-infected individuals with bNAb responses or no neutralization breadth and uninfected controls, identifying consistent features of bNAb repertoires, encompassing thousands of B cell clones per individual, with correlated T cell phenotypes. These repertoire features were not observed during chronic cytomegalovirus infection in an independent cohort. Our data indicate that the development of numerous B cell lineages with antibody features associated with autoreactivity may be a key aspect in the development of HIV neutralizing antibody breadth.
Background The frequencies, cellular phenotypes, epitope specificity, and clonal diversity of allergen-specific B cells in patients with food allergy are not fully understood but are of major ...pathogenic and therapeutic significance. Objective We sought to characterize peanut allergen–specific B-cell populations and the sequences and binding activities of their antibodies before and during immunotherapy. Methods B cells binding fluorescently labeled Ara h 1 or Ara h 2 were phenotyped and isolated by means of flow cytometric sorting from 18 patients at baseline and 13 patients during therapy. Fifty-seven mAbs derived from allergen-binding single B cells were evaluated by using ELISA, Western blotting, and peptide epitope mapping. Deep sequencing of the B-cell repertoires identified additional members of the allergen-specific B-cell clones. Results Median allergen-binding B-cell frequencies were 0.0097% (Ara h 1) or 0.029% (Ara h 2) of B cells in baseline blood from allergic patients and approximately 3-fold higher during immunotherapy. Five of 57 allergen-specific cells belonged to clones containing IgE-expressing members. Almost all allergen-specific antibodies were mutated, and binding to both conformational and linear allergen epitopes was detected. Increasing somatic mutation of IgG4 members of a clone was seen in immunotherapy, whereas IgE mutation levels in the clone did not increase. Conclusion Most peanut allergen–binding B cells isolated by means of antigen-specific flow sorting express mutated and isotype-switched antibodies. Immunotherapy increases their frequency in the blood, and even narrowly defined allergen epitopes are recognized by numerous distinct B-cell clones in a patient. The results also suggest that oral immunotherapy can stimulate somatic mutation of allergen-specific IgG4.
B cells are critical for the production of antibodies and protective immunity to viruses. Here we show that patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) who ...develop coronavirus disease 2019 (COVID-19) display early recruitment of B cells expressing a limited subset of IGHV genes, progressing to a highly polyclonal response of B cells with broader IGHV gene usage and extensive class switching to IgG and IgA subclasses with limited somatic hypermutation in the initial weeks of infection. We identify convergence of antibody sequences across SARS-CoV-2-infected patients, highlighting stereotyped naive responses to this virus. Notably, sequence-based detection in COVID-19 patients of convergent B cell clonotypes previously reported in SARS-CoV infection predicts the presence of SARS-CoV/SARS-CoV-2 cross-reactive antibody titers specific for the receptor-binding domain. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and SARS-CoV.
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•Human IGH repertoire sequencing identifies SARS-CoV-2-specific B cell clones•Convergent virus-specific antibody sequences are shared between COVID-19 patients•SARS-CoV antibodies are detected by sequence in COVID-19 patients
B cells produce antibodies and provide protective immunity to viruses. In longitudinal data from COVID-19 patients, Nielsen et al. analyze the development of antibody gene repertoires responding to SARS-CoV-2. COVID-19 patients share subsets of similar antibody sequences that bind SARS-CoV-2 antigens, with rare antibodies that also recognize SARS-CoV.
Dengue is the most prevalent mosquito-borne viral disease in humans, and the lack of early prognostics, vaccines, and therapeutics contributes to immense disease burden. To identify patterns that ...could be used for sequence-based monitoring of the antibody response to dengue, we examined antibody heavy-chain gene rearrangements in longitudinal peripheral blood samples from 60 dengue patients. Comparing signatures between acute dengue, postrecovery, and healthy samples, we found increased expansion of B cell clones in acute dengue patients, with higher overall clonality in secondary infection. Additionally, we observed consistent antibody sequence features in acute dengue in the highly variable major antigen-binding determinant, complementarity-determining region 3 (CDR3), with specific CDR3 sequences highly enriched in acute samples compared to postrecovery, healthy, or non-dengue samples. Dengue thus provides a striking example of a human viral infection where convergent immune signatures can be identified in multiple individuals. Such signatures could facilitate surveillance of immunological memory in communities.
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•White blood cell DNA sequences reveal clonal B cell expansion in acute dengue patients•Multiple dengue cases exhibit convergent dengue-specific antibody (CDR3) signatures•Convergent CDR3s have diverse underlying nucleotide sequences•Dengue-specific CDR3s have unique amino acid physicochemical profiles
selection technology has transformed the development of therapeutic monoclonal antibodies. Using methods such as phage, ribosome, and yeast display, high affinity binders can be selected from diverse ...repertoires. Here, we review strategies for the next-generation sequencing (NGS) of phage- and other antibody-display libraries, as well as NGS platforms and analysis tools. Moreover, we discuss recent examples relating to the use of NGS to assess library diversity, clonal enrichment, and affinity maturation.
Antigen-specific B cells bifurcate into antibody-secreting cells (ASCs) and memory B cells (MBCs) after infection or vaccination. ASCs (plasmablasts) have been extensively studied in humans, but less ...is known about B cells that become activated but do not differentiate into plasmablasts. Here we have defined the phenotype and transcriptional program of a subset of antigen-specific B cells, which we have called 'activated B cells' (ABCs), that were distinct from ASCs and were committed to the MBC lineage. We detected ABCs in humans after infection with Ebola virus or influenza virus and also after vaccination. By simultaneously analyzing antigen-specific ASCs and ABCs in human blood after vaccination against influenza virus, we investigated the clonal overlap and extent of somatic hypermutation (SHM) in the ASC (effector) and ABC (memory) lineages. Longitudinal tracking of vaccination-induced hemagglutinin (HA)-specific clones revealed no overall increase in SHM over time, which suggested that repeated annual immunization might have limitations in enhancing the quality of influenza-virus-specific antibody.
Variation in the antibody response has been linked to differential outcomes in disease, and suboptimal vaccine and therapeutic responsiveness, the determinants of which have not been fully ...elucidated. Countering models that presume antibodies are generated largely by stochastic processes, we demonstrate that polymorphisms within the immunoglobulin heavy chain locus (IGH) impact the naive and antigen-experienced antibody repertoire, indicating that genetics predisposes individuals to mount qualitatively and quantitatively different antibody responses. We pair recently developed long-read genomic sequencing methods with antibody repertoire profiling to comprehensively resolve IGH genetic variation, including novel structural variants, single nucleotide variants, and genes and alleles. We show that IGH germline variants determine the presence and frequency of antibody genes in the expressed repertoire, including those enriched in functional elements linked to V(D)J recombination, and overlapping disease-associated variants. These results illuminate the power of leveraging IGH genetics to better understand the regulation, function, and dynamics of the antibody response in disease.
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