Mats Ohlin

Allergy, characterised by antibody responses of the IgE isotype, is a major health concern. The set of monoclonal human IgE used for studying the molecular mechanisms of allergies is limited. Single-cell sequencing offers opportunities to establish novel antibodies for researching, diagnosis, and treatment of allergies. We describe and exploit a pipeline for generating recombinant IgE directly from the immune repertoires of allergic subjects. It uses single-cell sequencing of IgM ^– B cells of bone marrow and peripheral blood in an allergen-agnostic manner, combined with high-throughput transcriptome sequencing to identify clonotypes populating the IgE repertoire. Immunochemical and immunoprecipitation analyses are used to deconvolute the specificity of identified antibodies. High-affinity antibodies were raised against four grass pollen allergens, antibodies that illustrated aspects of the development of allergen-specific humoral immunity. The pipeline provides a streamlined approach for the development and characterisation of native allergen-specific antibodies as they occur in allergy and during allergy desensitisation.

Visualization. Numerous tools have been developed and are in widespread use. Recognizing the need for standardization, efforts to catalogue and define the genes involved in generating BCR/antibody and TCR-encoding sequences began more than three decades ago engaging both the international ImMunoGeneTics information system (IMGT) (1) and others (2). These standardized frameworks have greatly facilitated data analysis, interpretation, and communication within the research community. Developments in sequencing technology, in particular long-read sequencing and associated bioinformatics tools now enables valid reconstruction even of complex genomic loci such as those encoding AIRRs (3-7). However, as our understanding of the antibody and TCR loci has deepened-and as research has expanded to include previously understudied human populations and additional species-the challenges of developing complete, valid reference sets of germline genes participating in the generation of BCRs/antibodies and TCRs, while maintaining consistent nomenclature and naming standards, have become obvious (8). Some of these include:• Multiple Numbering and Naming Schemes: Various systems for definition of the most diverse, commonly antigen-binding parts, the complementarity determining regions (CDRs), of BCRs, antibodies and TCRs, and gene and sequence numbering have been developed (e.g. (9)(10)(11)(12)(13)(14)(15)) but also been volatile with respect to their standard definitions. Several of these definitions are still in concurrent use. Multiple systems for gene naming have been implemented over the years. Although naming authority ought to reside with the International Union of Immunological Societies (IUIS) and its Nomenclature Committee, this seems not to be universally accepted. This creates confusion and hinders the consistent interpretation of results. A single naming body ought to be universally accepted.• Position-Based Gene Naming: Gene names have traditionally been based on genomic position, aiding interpretation. However, growing evidence shows that genomic structures are far more complex than initially believed. Duplicated, inserted, and deleted genes and inverted segments (5,(16)(17)(18), absent from early maps, now challenge this approach. Moreover, AIRR loci in many species are significantly more diverse than in humans (19). Experimentally important model systems, like different mouse strains, differ substantially in their germline gene repertoire (20,21) and germline loci of individuals of rhesus and cynomolgus macaques are very different (22,23). Such complexity further complicates position-based naming-especially when diversity of the genomic loci structure is incompletely known. A cautious position-based practise of gene naming must be applied if we do not fully comprehend the diversity of these loci. It is even conceivable that the practice of a purely positionbased naming scheme must be avoided for some or even most species as it might imply relations and proximity that does not exist.• Evolving Nomenclature: Changes in gene names and numbering over time have not always been clearly traceable causing challenges in terms of understanding of analysis outcomes. In some cases, identical names have been reassigned to different sequences, introducing ambiguity. Such practices must be avoided; once assigned, a name should never be reused for a different sequence of a given species.• Unreliable Sequence Naming: Sequence names have sometimes been assigned based on presumed gene location, without solid genomic support. For example, the human allele originally named IGHV4-59*08 recently had to be renamed as an allele of germline gene IGHV4-61, as name assignment that initially was based on conventional sequencing data was challenged by additional analysis (24) and subsequently on an extended genomic analysis.• Reference sets are in some cases not open-source: Costly licensing agreements may prevent use of appropriate reference sets in commercial products aimed for analysis of AIRR sequence data. Although it is recognised that there is a need to retrieve costs associated to the generation and management of such reference sets, implementation of licencing fees that prevents the universal use of such sets for valid analysis negatively impacts the quality and reproducibility of AIRR research.To address these issues, we urge all stakeholders in the AIRR research communityincluding researchers, tool developers, database curators, journal editors, and standard-setting organizations-to agree on and adhere to a common set of principles. These may include: 23.pdf) as well as an overview of the challenges and likely best approaches to address them (25).• Universal adoption of a standard sequence numbering system-such as that developed by IMGT ( 14)-for representing AIRR sequence data. Databases containing AIRR data, including antibody and TCR protein structures, should convert existing entries to conform with this standard, if necessary.• Only well-documented genes and alleles should be recognised. These should be fulllength sequences, primarily those supported by long-read genomic data of sufficient coverage. Discovery by traditional Sanger sequencing, by computational inference of germline allele sequences from transcriptome data (26)(27)(28), and by high-throughput gene amplicon sequencing (29), may likely largely be replaced by modern long-read sequencing technologies, while inference may still aid to determine expression levels of inferred alleles. Independently of the methodology used to identify novel genes and alleles, the supporting data should be made fully available to the research community to judge the relevance of the discovery. Inferred sequences from short-read assemblies must not be added to reference sets due to inherent inaccuracies of such analytical processes (30,31).• Positional naming of germline genes should only be applied when the...

Immunotherapy has shown limited success in acute myeloid leukemia (AML), indicating an incomplete understanding of the underlying immunoregulatory mechanisms. Here we identify an immune evasion mechanism present in 60% of AML cases, wherein primitive AML cells aberrantly express the lymphoid surface protein SLAMF6 (signaling lymphocyte activation molecule family member 6). Knockout of SLAMF6 in AML cells enables T cell activation and highly efficient killing of leukemia cells in coculture systems, demonstrating that SLAMF6 protects AML cells from recognition and elimination by the immune system in a mode analogous to the programmed cell death protein–ligand (PDL1/PD1) axis. Targeting SLAMF6 with an antibody against the SLAMF6 dimerization site inhibits the SLAMF6–SLAMF6 interaction and induces T cell activation and killing of AML cells both in vitro and in humanized in vivo models. In conclusion, we show that aberrant expression of SLAMF6 is a common and targetable immune escape mechanism that could pave the way for immunotherapy in AML.

Background: Immunoglobulin (Ig)E can mediate food allergy and anaphylaxis, which can be inhibited by clonally related IgG. It is unclear whether differential hypermutation of clonally related IgE and IgG limits IgG suppression of IgE-mediated disease. Methods: A murine model of hen’s egg allergy with the major allergens ovalbumin (OVA) and ovomucoid (OVO) was employed to investigate allergen-induced induced anaphylaxis (detected as hypothermia). Deep sequencing of IgE and IgG1 was used to identify IgE clones with divergent hypermutation profiles compared to their clonally related IgG progenitors. Binding to OVA and OVO was predicted by in silico analysis. IgG-mediated blockade of IgE binding to OVA and OVO was tested by ELISA. Results: A small subset of IgE clones acquired somatic CDR3 hypermutations that were absent in their clonally related IgG1 progenitors, these clones were called ”divergent” IgE. The presence and clonal expansion of highly mutated divergent IgE clonotypes correlated with the development of allergen-induced severe hypothermia. In contrast and independent of their hypermutation rate, the presence of other IgE clones did not correlate with allergic hypothermia. Predictive analysis suggests that the relevant divergent IgE clonotypes predominantly reacted to OVO rather than OVA. Serum IgG from allergic mice could block binding of OVA-specific but not OVO-specific IgE. Divergent IgE was also observed in the bone marrow (BM) of allergic patients. Conclusion: This study identifies a small subset of highly hypermutated “divergent IgE clonotypes” associated with severe anaphylaxis and provides a possible mechanism by which allergy-promoting IgE escape from competition by IgG progenitor clones.

Background: Group 5 allergens, such as Phl p 5 of timothy grass, are major contributors to grass pollen allergy. Antibody 212597 specific for this allergen was recently isolated by single cell sequencing of bone marrow B cells of a grass pollen-allergic subject. This antibody, although subjected only to a low level of hypermutation resulting in six amino acid substitution across the heavy and light chain variable domains, has achieved sub-nM affinity for the allergen, suggesting that antibodies specific for this major group of allergens can be of high affinity even at the naïve, unmutated stage. The present study was designed to assess affinity and biophysical characters of the antibody, its inferred unmutated ancestor, and other intermediate and allelic variants thereof.Methods: Site-directed mutagenesis was used to revert substitutions of antibody 212579. Mutants, including its inferred unmutated common ancestor were characterized with respect to allergen affinity, thermostability, and hydrodynamic radius.Results: We demonstrate that even the antibody’s inferred unmutated common ancestor shows high affinity for the allergen in the low-nM range. Glutamate at heavy chain position 38, a residue unique to allele IGHV3-48*03, the germline gene origin of the heavy chain of antibody 212579, was critical for high affinity binding. Substitution to serine as found in other alleles of IGHV3–48 reduced the affinity about 20-fold. A substitution, N40_HT in the heavy/light chain variable domain interface, introduced into the antibody through somatic hypermutation, did not impact its affinity for the allergen but reduced its thermal stability and increased its hydrodynamic radius.Conclusion: Unmutated, high affinity (low-nM) antibodies specific for a major allergen (Phl p 5) can be generated directly in naïve B cells and are, given an appropriate rearrangement, imprinted into the repertoire through rearrangements involving immunoglobulin germline gene alleles IGHV3-48*03 and IGKV3-20*01. This specificity depends on an allele-unique residue encoded by the immunoglobulin germline repertoire. Substitutions in the heavy/light chain variable domain interface, such as N40_HT in a heavy chain variable domain, might negatively impact biophysical properties of the antibody and should be considered as targets for further evolution or reversion if they negatively impact an antibody’s developability properties.

The present Special Research Topic "Allergen-specific Antibodies: From Basic Science to Clinical Application" offers a current snapshot about the latest findings concerning antibodybased approaches to study the development of allergic disorders, to establish efficient standardisation of allergen extracts and to employ antibodies for allergy treatment. This collection includes three review articles covering antibody isotype diversity (Platts-Mills et al.) in the context of allergy, the importance of microbial influence during pregnancy and first year of life (Pirker et al.), and the significance of IgE epitope recognition for both developing blocking antibodies and peptide-based immunotherapies (Fernandes et al.). Furthermore this edition emcompasses one original article describing the establishment of a highly efficient ELISA using human IgG4 antibodies to standardize commercially available allergen extracts used for diagnosis and therapy of type I allergy in the US (Rabin et al.) and a narrative mini review article summarizing recent news on the sucessful treatment with omalizumab to manage various food allergies (Ghouri et al.). Over the last 40 years two developments have been important in the allergy field: 1) the improvement of techniques for measuring isotype specific antibodies to allergens, and 2) the identification of allergic diseases or phenomena involving isotype-diversity of antibodies. The manuscript by Dr. Platts-Mills et al. highlights the importance of assessing allergen specific antibodies of diverse isotypes, not only IgE, for the investigation of allergic diseases. The review shows the relevance of antibody isotypes in certain allergic diseases with a historical perspective. Compelling evidence is presented for the role of IgG4 in the development of eosinophilic esophagitis and tolerance to cat, and for the presence of IgA and IgG, in addition to IgE, in nasal secretions of pollen allergic patients. On the other hand, IgE recognition of the oligosaccharide galactose alpha, 1-3-galactose is essential for the alpha-gal syndrome, primarily induced by tick bites, but the role of IgG isotypes on the inflammatory response to the sugar needs further investigation. Microbial colonization of human mucosal surfaces is an essential component of host immune system development. It's widely recognized that a critical window of time extending from pregnancy through the first year of life exists in which early microbial encounters influence and shape the immune response. However, mechanisms underlying the impact of such interactions remain incompletely understood. Development of asthma and allergic diseases has been associated with microbial dysbiosis, and recent research suggests that both preand postnatal antibody production plays a role in shaping the microbiome. In their review, Pirker et al. discuss early life factors influencing the composition of the gut microbiome, including delivery and feeding modes, medication and maternal-, geographical-and social factors. They provide a comprehensive review of recent work aimed to better understand innate and adaptive immune mechanisms underlying the microbiota-immune axis and how it may relate to the onset of allergic disease in early life.The diversity and complexity of B and T cell epitope recognition profiles of adaptive immune responses in relation to allergic disease is an underexplored domain of allergy research and their impact on disease, diagnosis and immunological intervention is poorly understood. In their review, Fernandes et al. thoroughly outline our understanding of epitopes of major Hymenoptera (bee, wasp, and ant) venom allergens. Such proteins display very substantial potential to cause severe, life-threatening allergic disease in sensitized individuals. They furthermore exemplify epitope-based strategies that have been explored for treatment of allergic disease through passive and active immunotherapy in other allergies, and advocate for the importance of an epitope-based strategy in the development of diagnostic and therapeutic approaches for the treatment and prevention of Hymenoptera venom allergies. Between 10% and 20% of the population suffer from IgE-mediated cat allergy. Cat allergen extracts standardized for the major cat allergen, Fel d 1 are used to diagnose cat allergy but also applied as immunotherapy to prevent allergic symptoms. So far, a radial immunodiffusion assay and polyclonal serum have been utilized to determine the concentration of Fel d 1 in commercially available cat dander and pelt extracts in the US. However, this technology comes along with some limitations including difficulties with interpreting precipitation results and insufficient homogeneity of polyclonal serum. Within their original article Robert Hamilton and his team describe the generation and evaluation of human Fel d 1-specific IgE-derived IgG4. These antibodies guarantee IgE epitope binding clinically relevant for allergen recognition and the elaboration of a highly reproducible and precise assay based on a selected pair of these human antibodies binding to non-overlapping epitopes. Emphasizing that measurement of Fel d 1 levels is sufficient to detect the potency of cat allergen extracts, they prove their quantitative two-site assay to be equivalent to the radial immunodiffusion currently applied for standardization in the US. Food allergy is a serious threat implying considerable restriction in daily life for those who are impacted. Injections with omalizumab have been approved by the FDA for the treatment of food allergic adults and children aged one year or older. The narrative Mini review written by Ghouri and coauthors provides a current overview on the efficacy, safety and clinical implications of omalizumab in most frequent food allergies including peanut, milk, shellfish but also fruits. While their summary clearly demonstrated that administration of omalizumab is a major progress in the management of food...

Rhesus macaques (RMs) are a vital model for studying human disease and invaluable to pre-clinical vaccine research, particularly for the study of broadly neutralizing antibody responses. Such studies require robust genetic resources for antibody-encoding genes within the immunoglobulin (IG) loci. The complexity of the IG loci has historically made them challenging to characterize accurately. To address this, we developed novel experimental and computational methodologies to generate the largest collection to date of integrated antibody repertoire and long-read genomic sequencing data in 106 Indian origin RMs. We created a comprehensive resource of IG heavy and light chain variable (V), diversity (D), and joining (J) alleles, as well as leader, intronic, and recombination signal sequences (RSSs), including the curation of 1474 novel alleles, unveiling tremendous diversity, and expanding existing IG allele sets by 60%. This publicly available, continually updated resource ( https://vdjbase.org/reference_book/Rhesus_Macaque ) provides the foundation for advancing RM immunogenomics, vaccine discovery, and translational research.

Substantial evidence supports that Fc-mediated effector functions of anti-spike Abs contribute to anti–SARS-Cov-2 protection. We have previously shown that two non-neutralizing but opsonic mAbs targeting the receptor-binding domain and N-terminal domain (NTD), Ab81 and Ab94, respectively, are protective against lethal Wuhan SARS-CoV-2 infection in K18-hACE2 mice. In this article, we investigated whether these protective non-neutralizing Abs maintain Fc-mediated function and Ag binding against mutated SARS-CoV-2 variants. Ab81 and Ab94 retained their nanomolar affinity and Fc-mediated function toward Omicron and its subvariants, such as BA.2, BA.4, BA.5, XBB, XBB1.5, and BQ1.1. However, when encountering the more heavily mutated BA.2.86, Ab81 lost its function, whereas the 10 new mutations in the NTD did not affect Ab94. In vivo experiments with Ab94 in K18-hACE2 mice inoculated with a stringent dose of 100,000 PFU of the JN.1 variant revealed unexpected results. Surprisingly, this variant exhibited low disease manifestation in this animal model with no weight loss or death in the control group. Still, assessment of mice using a clinical scoring system showed better protection for Ab94-treated mice, indicating that Fc-mediated functions are still beneficial. Our work shows that a protective anti–receptor-binding domain non-neutralizing mAb lost reactivity when BA.2.86 emerged, whereas the anti-NTD mAb was still functional. Finally, this work adds new insight into the evolution of the SARS-CoV-2 virus by reporting that JN.1 is substantially less virulent in vivo than previous strains.

Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens.

Streptococcus pyogenes can cause invasive disease with high mortality despite adequate antibiotic treatments. To address this unmet need, we have previously generated an opsonic IgG1 monoclonal antibody, Ab25, targeting the bacterial M protein. Here, we engineer the IgG2-4 subclasses of Ab25. Despite having reduced binding, the IgG3 version promotes stronger phagocytosis of bacteria. Using atomic simulations, we show that IgG3’s Fc tail has extensive movement in 3D space due to its extended hinge region, possibly facilitating interactions with immune cells. We replaced the hinge of IgG1 with four different IgG3-hinge segment subclasses, IgGh_xx. Hinge-engineering does not diminish binding as with IgG3 but enhances opsonic function, where a 47 amino acid hinge is comparable to IgG3 in function. IgGh₄₇ shows improved protection against S. pyogenes in a systemic infection mouse model, suggesting that IgGh₄₇ has promise as a preclinical therapeutic candidate. Importantly, the enhanced opsonic function of IgGh₄₇ is generalizable to diverse S. pyogenes strains from clinical isolates. We generated IgGh₄₇ versions of anti-SARS-CoV-2 mAbs to broaden the biological applicability, and these also exhibit strongly enhanced opsonic function compared to the IgG1 subclass. The improved function of the IgGh₄₇ subclass in two distant biological systems provides new insights into antibody function.