Walter Keller

Der p 2 and Blo t 5 are main allergens from house dust mites and play central roles in IgE-mediated hypersensitivity. Hybrid allergen molecules represent a promising strategy for component-resolved diagnostics. This study aimed to characterize a recombinant hybrid molecule composed of full-length Der p 2 and Blo t 5 (BP-2) and to evaluate its allergenic activity. Allergenicity and IgE-binding activity of BP-2 were assessed in asthma patients. Immunogenicity was examined in BALB/c mice sensitized and challenged with BP-2, followed by quantification of specific immunoglobulins, histopathological evaluation of airway inflammation and goblet cell hyperplasia, and assessment of airway hyperresponsiveness via methacholine challenge. BP-2 exhibited strong IgE reactivity in asthma patients and effectively discriminated between asthma patients and healthy controls (AUC = 0.806, 95% CI: 0.764–0.844). Cross-inhibition assays demonstrated that BP-2 retains functional epitopes shared with Blo t 5, Der p 2, and allergen extracts. In the murine model, the humoral and cellular immune responses induced by BP-2 suggest a profile with a predominant role of the T2 component characterized by IL-4 and IL-17A production and specific antibody generation. BP-2 is a biologically active hybrid allergen capable of detecting dual sensitization to Dermatophagoides pteronyssinus and Blomia tropicalis, supporting its potential application in component-resolved diagnostics for mite allergy.

Background: House dust mites (HDMs) are the most important respiratory allergen sources. In temperate regions, the genus Dermatophagoides predominates, whereas in sub‐tropical and tropical regions, the genus Blomia is also of high importance. There is only limited IgE cross‐reactivity between Dermatophagoides and Blomia . Objective: To produce a chip containing purified microarrayed Blomia tropicalis (Blo t) and Dermatophagoides pteronyssinus allergens (Der p) capable of identifying patients with a genuine Blo t or Der p IgE sensitisation, co‐sensitisation and/or cross‐sensitisation. Methods: Chips containing seven purified Blo t and thirteen Der p allergens were generated by microarray technology and tested for IgE and IgG reactivity in HDM ‐sensitised patients from Blo t‐endemic (group 1: n = 115) and Blo t‐non‐endemic (group 2: n = 33) regions. IgE cross‐reactivity was analysed by IgE inhibition studies. Results: IgE levels to Blo t 2, Blo t 5, Blo t 10, Blo t 12 and Blo t 21 were significantly higher in HDM‐sensitised patients from Blo t‐endemic as compared to patients from Blo t‐non‐endemic regions, whereas the opposite was observed for IgE to Der p 2 and Der p 21 in patients from Blo t‐non‐endemic regions. An algorithm based on IgE reactivity profiles and allergen‐specific IgE levels capable of discriminating genuine sensitisation to Blo t and Der p or co‐sensitisation was established. In HDM‐sensitised patients from Blo t‐endemic regions, each of the aforementioned sensitisation profiles was observed, whereas in HDM‐sensitised patients from Blo t‐non‐endemic regions, only genuine sensitisations to Der p and co‐sensitisations to Blo t and Der p were observed. Conclusion: The algorithm based on microarrayed Blo t and Der p allergens for discrimination of Blo t and Der p sensitisation may support prescription of allergen‐specific immunotherapy. At minimum, it will be helpful in understanding disease aetiology and for fine resolution mapping of allergic reactivities in HDM sensitisation.

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.

Type IV secretion systems (T4SS) are found in both monoderm and diderm bacteria. The broad-host-range conjugative plasmid pIP501 from Enterococcus faecalis harbors a T4SS encoding 15 tra genes responsible for the spread of antimicrobial resistance genes among diverse G+ pathogens. Eight Tra proteins (TraB, TraC_B3, TraF, TraH_B8, TraI, TraK, TraL_B6, and TraM_B8) are postulated to form the mating pair formation (MPF) complex representing the central DNA translocation pore. One of these proteins is TraF, a 52.8 kDa transmembrane protein which lacks any homologs in other well described T4SSs. In this study, TraF was proven to be an essential conjugative transfer protein. The TraF pulldown co-eluted all Tra proteins except TraG_B1 and TraN. Bacterial-two-hybrid assay showed a strong interaction between TraF and TraM_B8. We present a 1.25 Å resolution crystal structure of the N-terminal domain of TraF, which adopts a pseudokinase fold. AlphaFold predictions of full-length TraF with membrane mimetics show a transmembrane protein with two distinct soluble domains. FoldSeek revealed a strong similarity to YukC (EssB), a transmembrane pseudokinase from type VII secretion system (T7SS). YukC was shown to function as an interaction hub by mediating contacts between its pseudokinase domain and other T7SS proteins as part of the central membrane core complex. We postulate that TraF might play an important role in T4SS complex formation.

Background: Allergic sensitization to cats and other furry animals is a major cause of asthma and allergic rhinitis in more than 200 million people worldwide. According to the frequency of IgE recognition, allergen‐specific IgE levels, and allergenic activity, Fel d 4 is a major allergen in the cat ( Felis domesticus ). The lipocalin allergen Fel d 4 is highly homologous to dog (Can f 6) and major horse (Equ c 1) allergens. Accordingly, IgE cross‐reactivity to these allergens contributes to polysensitization and allergic responses upon exposure to different animals. Methods: Fel d 4 was recombinantly produced in two systems, E. coli and Expi293F mammalian cells. Recombinant forms were characterized by circular dichroism and mass spectrometry. The Fel d 4 3D structure was determined using X‐ray crystallography. Immunoreactivity, epitope analyses, and cross‐reactive properties were assessed by ELISA and basophil release assays using allergic patients’ sera. Results: We reveal the rFel d 4 crystal structures and demonstrate that mammalian cells produce an N‐glycosylated recombinant Fel d 4 allergen. The C‐terminal regions of Fel d 4, Can f 6, and Equ c 1 constitute conformational IgE ‐epitope‐containing areas responsible for cross‐reactivity. Conclusion: Uncovering the IgE ‐binding sites of Fel d 4 and cross‐reactive allergens contributes to future rational design of active and passive allergen‐specific treatment forms.

Der p 23 induces a high-frequency sensitization in allergic individuals. However, its allergenic activity and clinical impact are scarce. We aimed to evaluate the ability of rDer p 23 to induce allergic inflammation in a mouse model and to test IgE reactivity in humans. Female Balb/c mice were sensitized and challenged with rDer p 23 and Dermatophagoides pteronyssinus extract. Specific antibodies were determined by ELISA, inflammatory cell infiltration and goblet cells hyperplasia were evaluated by lung histology, and bronchial hyperreactivity (BHR) was assessed by the FinePoint RC System^TM and whole-body plethysmography (WBP). IgE reactivity was evaluated by ELISA, the basophils activation test (BAT) and the skin pick test (SPT) in humans. rDer p 23, produced in Escherichia coli, adopts a random coil structure, predominantly exists in a monomeric state, and exhibits high stability. rDer p 23-treated mice showed a significant increase in lung resistance and bronchial hyperreactivity, as well as in eosinophils, neutrophils, and T cell count in bronchoalveolar lavage fluid (BALF). Cytokine and antibodies profiles were biased to a Type-2 response. No significant difference was observed in group 2 Innate Lymphoid Cells (ILC-2s) in lung and regulatory T cells (Treg) in the spleen. In asthmatic individuals sensitized to D. pteronyssinus, serum IgE reactivity to rDer p 23 was 67.5%. BAT and SPT results were significantly higher in allergic patients. Our findings support the pro-allergenic role of rDer p 23 in the development of the pathological features of asthma.

Phospholipase A1 (Ves a 1), a major toxin from Vespa affinis venom, poses significant risks to allergic individuals. Nevertheless, the epitope determinants of Ves a 1 have not been characterized. Thus, identifying its linear B-cell epitopes is crucial for understanding envenomation mechanisms. In this study, we predicted and identified B-cell epitopes EP5 and EP6 as potential candidates. EP5 formed an α-helix at the active site of Ves a 1, whereas EP6 adopted an extended loop conformation. Both synthetic peptides were synthesized and evaluated for their inhibitory effects using immune-inhibitory assays with polyclonal antibodies (pAbs) targeting both native (nVes a 1) and recombinant (rVes a 1) forms. The Ves a 1 polyclonal antibodies (pAb-nVes a 1 and pAb-Ves a 1) were produced, and their specificity binding to Ves a 1 was confirmed by Western blot. Next, ELISA inhibition assays showed that EP5 and EP6 significantly blocked pAb binding to both nVes a 1 and rVes a 1. Dot blot and Western blot assays supported these findings, particularly with stronger inhibition toward rVes a 1. Furthermore, enzymatic assays indicated that nVes a 1 and rVes a 1 retained phospholipase activity. Immunoinformatics docking showed that EP5 and EP6 specifically bind to a single-chain variable fragment antibody (scFv) targeting Naja naja PLA2. Molecular analysis revealed similar amino acid interactions to the template, suggesting effective paratope–epitope binding. These results support the potential of EP5 and EP6 for future diagnosis and therapy of V. affinis venom allergy.

Background: Molecular forms of allergen‐specific immunotherapy (AIT) for cat allergy are needed. Fel d 1, Fel d 4, and Fel d 7 are the most important cat allergens.Methods: IgE epitopes of Fel d 4 and Fel d 7 were mapped by blocking allergic patients' IgE binding with allergen peptide‐specific antisera. Five recombinant fusion proteins (PreS‐Cat 1‐PreS‐Cat 5) each containing hepatitis B virus (HBV)‐derived PreS as an immunological carrier and non‐allergenic peptides from the IgE binding sites of Fel d 1, Fel d 4, and Fel d 7 were expressed in Escherichia coli, purified, and characterized by mass spectrometry, circular dichroism (CD), and size exclusion chromatography. ImmunoCAP and basophil activation experiments demonstrated the hypoallergenic activity of PreS‐Cat 1–5. The ability of PreS‐Cat 1–5 to induce IgE‐blocking antibodies in rabbits was compared to three licensed allergen extract‐based AIT vaccines. PreS‐Cat 1‐5‐specific IgG antibodies were tested for inhibition of allergen‐specific IgE binding and specific basophil activation. T cell activation and induction of specific cytokine secretion by PreS‐Cat proteins were compared with cat allergens in PBMC cultures.Results: Recombinant hypoallergenic, biochemically and structurally defined PreS‐Cat 1–5 were obtained. Two subcutaneous immunizations of rabbits with PreS‐Cat 1–5 induced equal (Fel d 1) or better (Fel d 4 and Fel d 7) antibodies (PreS‐Cat 5 > PreS‐Cat 1 > PreS‐Cat 3) blocking allergic patients' IgE binding to cat allergens than six to fifteen immunizations with allergen extract‐based vaccines. PreS‐Cat‐specific antibodies strongly inhibited specific basophil activation. PreS‐Cat 5 > PreS‐Cat 1 induced significantly more IL‐10 in cultured PBMCs from cat allergic patients than cat allergens.Conclusions: PreS‐Cat 5 and PreS‐Cat 1 are highly promising molecular vaccine candidates for AIT of cat allergy, combining Fel d 1‐, Fel d 4‐, and Fel d 7‐peptides in single PreS fusion proteins.

S-layers are crystalline arrays found on bacterial and archaeal cells. Lactobacillus is a diverse family of bacteria known especially for potential gut health benefits. This study focuses on the S-layer proteins from Lactobacillus acidophilus and Lactobacillus amylovorus common in the mammalian gut. Atomic resolution structures of Lactobacillus S-layer proteins SlpA and SlpX exhibit domain swapping, and the obtained assembly model of the main S-layer protein SlpA aligns well with prior electron microscopy and mutagenesis data. The S-layer’s pore size suggests a protective role, with charged areas aiding adhesion. A highly similar domain organization and interaction network are observed across the Lactobacillus genus. Interaction studies revealed conserved binding areas specific for attachment to teichoic acids. The structure of the SlpA S-layer and the suggested incorporation of SlpX as well as its interaction with teichoic acids lay the foundation for deciphering its role in immune responses and for developing effective treatments for a variety of infectious and bacteria-mediated inflammation processes, opening opportunities for targeted engineering of the S-layer or lactobacilli bacteria in general.

Background: COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a recurrent endemic disease affecting the whole world. Since November 2021, Omicron and its subvariants have dominated in the spread of the disease. In order to prevent severe courses of disease, vaccines are needed to boost and maintain antibody levels capable of neutralizing Omicron. Recently, we produced and characterized a SARS-CoV-2 vaccine based on a recombinant fusion protein consisting of hepatitis B virus (HBV)-derived PreS and two SARS-CoV-2 wild-type RBDs. Objectives: To develop a PreS-RBD vaccine which induces high levels of Omicron-specific neutralizing antibodies. Methods: We designed, produced, characterized and compared strain-specific (wild-type: W-PreS-W; Omicron: O-PreS-O), bivalent (mix of W-PreS-W and O-PreS-O) and chimeric (i.e., W-PreS-O) SARS-CoV-2 protein subunit vaccines. Immunogens were characterized in vitro using protein chemical methods, mass spectrometry, and circular dichroism in combination with thermal denaturation and immunological methods. In addition, BALB/c mice were immunized with aluminum–hydroxide-adsorbed proteins and aluminum hydroxide alone (i.e., placebo) to study the specific antibody and cytokine responses, safety and Omicron neutralization. Results: Defined and pure immunogens could be produced in significant quantities as secreted and folded proteins in mammalian cells. The antibodies induced after vaccination with different doses of strain-specific, bivalent and chimeric PreS-RBD fusion proteins reacted with wild-type and Omicron RBD in a dose-dependent manner and resulted in a mixed Th1/Th2 immune response. Interestingly, the RBD-specific IgG levels induced with the different vaccines were comparable, but the W-PreS-O-induced virus neutralization titers against Omicron (median VNT50: 5000) were seven- and twofold higher than the W-PreS-W- and O-PreS-O-specific ones, respectively, and they were six-fold higher than those of the bivalent vaccine. Conclusion: Among the tested immunogens, the chimeric PreS-RBD subunit vaccine, W-PreS-O, induced the highest neutralizing antibody titers against Omicron. Thus, W-PreS-O seems to be a highly promising COVID-19 vaccine candidate for further preclinical and clinical evaluation.