Margarete Focke-Tejkl

The crosslinking of IgE-bound FcεRI by multivalent allergens initiates mast cell and basophil signaling underlying Type 1 allergy. Yet, how allergen properties and IgE occupancy impact receptor aggregation and downstream signaling remains unclear. We used Phl p 1-specific IgE and recombinant fusion proteins presenting a Phl p 1-derived peptide in defined valencies and positions (MB1N, MB1N1C, MB2N, and MB4N) to probe antigen-dependent signaling. Tetravalent MB4N evoked stronger degranulation and Ca2+ response than bivalent antigens MB1N1C and MB2N. MB4N was also capable of signaling at low IgE occupancy and in Lyn-deficient cells. Monte Carlo simulations predicted that MB4N forms larger, complex receptor aggregates, while MB1N1C and MB2N produce linear chains. Consistently, the addition of MB4N showed larger aggregates by electron microscopy and slower mobility by single-particle tracking, compared to bivalent antigens. Thus, allergen valency and epitope spatial arrangement dictate FcεRI aggregate organization and subsequent effector cell activation.

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.

Background: Approximately 10–20% of subjects vaccinated with HBsAg-based hepatitis B virus (HBV) vaccines are non-responders. BM32 is a recombinant grass pollen allergy vaccine containing the HBV-derived preS surface antigen as an immunological carrier protein. PreS includes the binding site of HBV to its receptor on hepatocytes. We investigated whether immunological non-responsiveness to HBV after repeated HBsAg-based vaccinations could be overcome by immunization with VVX001 (i.e., alum-adsorbed BM325, a component of BM32). Methods: A subject failing to develop protective HBV-specific immunity after HBsAg-based vaccination received five monthly injections of 20 µg VVX001. PreS-specific antibody responses were measured by enzyme-linked immunosorbent assay (ELISA) and micro-array technology. Serum reactivity to subviral particles of different HBV genotypes was determined by sandwich ELISA. PreS-specific T cell responses were monitored by carboxyfluorescein diacetate succinimidyl ester (CFSE) staining and subsequent flow cytometry. HBV neutralization was assessed using cultured HBV-infected HepG2 cells. Results: Vaccination with VVX001 induced a strong and sustained preS-specific antibody response composed mainly of the IgG₁ subclass. PreS-specific IgG antibodies were primarily directed to the N-terminal part of preS containing the sodium taurocholate co-transporting polypeptide (NTCP) attachment site. IgG reactivity to subviral particles as well as to the N-terminal preS-derived peptides was comparable for HBV genotypes A–H. A pronounced reactivity of CD3⁺CD4⁺ lymphocytes specific for preS after the complete injection course remaining up to one year after the last injection was found. Maximal HBV neutralization (98.4%) in vitro was achieved 1 month after the last injection, which correlated with the maximal IgG reactivity to the N-terminal part of preS. Conclusions: Our data suggest that VVX001 may be used as a preventive vaccination against HBV even in non-responders to HBsAg-based HBV vaccines.

Background: Approximately 10-20% of subjects vaccinated with HBsAg-based hepatitis B virus (HBV) vaccines are non-responders. BM32 is a recombinant grass pollen allergy vaccine containing the HBV-derived preS surface antigen as immunological carrier protein. PreS includes the binding site of HBV to its receptor on hepatocytes. We investigated if immunological non-responsiveness to HBV after repeated HBsAg-based vaccinations can be overcome by immunization with VVX001 (i.e., Alum-adsorbed BM325, a component of BM32). Methods: A subject failing to develop protective HBV-specific immunity after HBsAg-based vaccination received five monthly injections of 20 µg VVX001. PreS-specific antibody responses were measured by ELISA and micro-array technology. Serum reactivity to subviral particles of different HBV genotypes was determined by sandwich ELISA. PreS-specific T cell responses were monitored by CFSE staining and subsequent FACS analysis. HBV neutralization was assessed using cultured HBV-infected HepG2 cells. Results: Vaccination with VVX001 induced a strong and sustained preS-specific antibody response composed mainly of the IgG1 subclass. PreS-specific IgG antibodies were primarily directed to the N-terminal part of preS containing the NTCP attachment site. IgG reactivity to sub-viral particles as well as to the N-terminal preS-derived peptides was comparable for HBV genotypes A-H. A pronounced reactivity of CD3+CD4+ lymphocytes specific for preS after the complete injection course remaining up to one year after the last injection was found. Maximal HBV neutralization (98.4%) in vitro was achieved 1 month after the last injection which correlated with the maximal IgG reactivity to the N-terminal part of preS. Conclusion: Our data suggest that VVX001 may be used as a preventive vaccination against HBV even in non-responders to HBsAg-based HBV vaccines.

Up to a third of the world’s population suffers from allergies, yet the effectiveness of available preventative measures remains, at large, poor. Consequently, the development of successful prophylactic strategies for the induction of tolerance against allergens is crucial. In proof-of-concept studies, our laboratory has previously shown that the transfer of autologous hematopoietic stem cells (HSC) or autologous B cells expressing a major grass pollen allergen, Phl p 5, induces robust tolerance in mice. However, eventual clinical translation would require safe allergen expression without the need for retroviral transduction. Therefore, we aimed to chemically couple Phl p 5 to the surface of leukocytes and tested their ability to induce tolerance. Phl p 5 was coupled by two separate techniques, either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or by linkage via a lipophilic anchor, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-maleimide (DSPE-PEG-Mal). The effectiveness was assessed in fresh and cultured Phl p 5-coupled cells by flow cytometry, image cytometry, and immunofluorescence microscopy. Chemical coupling of Phl p 5 using EDC was robust but was followed by rapid apoptosis. DSPE-PEG-Mal-mediated linkage was also strong, but antigen levels declined due to antigen internalization. Cells coupled with Phl p 5 by either method were transferred into autologous mice. While administration of EDC-coupled splenocytes together with short course immunosuppression initially reduced Phl p 5-specific antibody levels to a moderate degree, both methods did not induce sustained tolerance towards Phl p 5 upon several subcutaneous immunizations with the allergen. Overall, our results demonstrate the successful chemical linkage of an allergen to leukocytes using two separate techniques, eliminating the risks of genetic modifications. More durable surface expression still needs to be achieved for use in prophylactic cell therapy protocols.

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.

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 are dominating. 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 by protein chemical methods, mass-spectrometry, 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 specific antibody and cytokine responses, safety and Omicron neutralization. Results: Defined and pure immunogens could be produced in large amounts as secreted and folded proteins in mammalian cells. 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, RBD-specific IgG levels induced by the different vaccines were comparable but the W-PreS-O-induced virus neutralization titers against Omicron (median VNT50: 5000) were 7- and 2-fold higher than the W-PreS-W- and O-PreS-O-specific ones, respectively and 6-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.

Molecular crowding of agonist peptide/MHC class II complexes (pMHCIIs) with structurally similar, yet per se non‐stimulatory endogenous pMHCIIs is postulated to sensitize T‐cells for the recognition of single antigens on the surface of dendritic cells and B‐cells. When testing this premise with the use of advanced live cell microscopy, we observe pMHCIIs as monomeric, randomly distributed entities diffusing rapidly after entering the APC surface. Synaptic TCR engagement of highly abundant endogenous pMHCIIs is low or non‐existent and affects neither TCR engagement of rare agonist pMHCII in early and advanced synapses nor agonist‐induced TCR‐proximal signaling. Our findings highlight the capacity of single freely diffusing agonist pMHCIIs to elicit the full T‐cell response in an autonomous and peptide‐specific fashion with consequences for adaptive immunity and immunotherapeutic approaches.

Background: The nature of epitopes on Bet v 1 recognized by natural IgG antibodies of birch pollen allergic patients and birch pollen‐exposed but non‐sensitized subjects has not been studied in detail.Objective: To investigate IgE and IgG recognition of Bet v 1 and to study the effects of natural Bet v 1‐specific IgG antibodies on IgE recognition of Bet v 1 and Bet v 1‐induced basophil activation.Methods: Sera from birch pollen allergic patients (BPA, n = 76), allergic patients without birch pollen allergy (NBPA, n = 40) and non‐allergic individuals (NA, n = 48) were tested for IgE, IgG as well as IgG₁ and IgG₄ reactivity to folded recombinant Bet v 1, two unfolded recombinant Bet v 1 fragments comprising the N‐terminal (F1) and C‐terminal half of Bet v 1 (F2) and unfolded peptides spanning the corresponding sequences of Bet v 1 and the apple allergen Mal d 1 by ELISA or micro‐array analysis. The ability of Bet v 1‐specific serum antibodies from non‐allergic subjects to inhibit allergic patients IgE or IgG binding to rBet v 1 or to unfolded Bet v 1‐derivatives was assessed by competition ELISAs. Furthermore, the ability of serum antibodies from allergic and non‐allergic subjects to modulate Bet v 1‐induced basophil activation was investigated using rat basophilic leukaemia cells expressing the human FcεRI which had been loaded with IgE from BPA patients.Results: IgE antibodies from BPA patients react almost exclusively with conformational epitopes whereas IgG, IgG₁ and IgG₄ antibodies from BPA, NBPA and NA subjects recognize mainly unfolded and sequential epitopes. IgG competition studies show that IgG specific for unfolded/sequential Bet v 1 epitopes is not inhibited by folded Bet v 1 and hence the latter seem to represent cryptic epitopes. IgG reactivity to Bet v 1 peptides did not correlate with IgG reactivity to the corresponding Mal d 1 peptides and therefore does not seem to be a result of primary sensitization to PR10 allergen‐containing food. Natural Bet v 1‐specific IgG antibodies inhibited IgE binding to Bet v 1 only poorly and could even enhance Bet v 1‐specific basophil activation.Conclusion: IgE and IgG antibodies from BPA patients and birch pollen‐exposed non‐sensitized subjects recognize different epitopes. These findings explain why natural allergen‐specific IgG do not protect against allergic symptoms and suggest that allergen‐specific IgE and IgG have different clonal origin.

Scope: Red meat, a staple food of Western diets, can also induce IgE‐mediated allergic reactions. Yet, apart from the heat‐labile protein serum albumin and the carbohydrate α‐Gal, the molecules causing allergic reactions to red meat remain unknown.Methods and results: IgE reactivity profiles of beef‐sensitized individuals are analyzed by IgE‐immunoblotting with protein extracts from raw and cooked beef. Two IgE‐reactive proteins are identified by peptide mass fingerprinting as myosinlight chain 1 (MYL1) and myosin light chain 3 (MYL3) in cooked beef extract and are designated Bos d 13 isoallergens. MYL1 and MYL3 are produced recombinantly in Escherichia coli. ELISAs proved their IgE reactivity and circular dichroism analysis showed that they represent folded molecules with remarkable thermal stability. In vitro gastrointestinal digestion experiments showed the higher stability of rMYL1 as compared to rMYL3. Exposure of a monolayer of Caco–2 cells to rMYL1 indicated that the molecule is able to cross intestinal epithelial cells without disturbing the integrity of the tight junctions, suggesting the sensitizing capacity of MYL1.Conclusion: MYLs are identified as novel heat‐stable bovine meat allergens.