Production, purification and structural characterisation of recombinant BanLec-Bet v 1

The sublingual route of allergens administration in allergen-specific immunotherapy (ASIT) is proven to be a successful way to treat patients with respiratory allergy. The trend of replacing natural extracts with purified recombinant allergens is growing. Although the purified allergens themselves are not good immunogens, the combined vector systems and adjuvans can improve their immunogenicity 1. Cell surfaces are decorated by different glycan structures, so the lectins specific for these glycans can be used to deliver particular therapeutic to target specific tissue 2. Banana lectin (BanLec) is mannose-specific protein which belongs to the subfamily of Jacalin related lectins 3. Apart from its characteristic to bind glycans, BanLec also modulates immune cells in vitro 4. On the other hand, Bet v 1 (Betula verrucosa) is the major birch pollen allergen. T-cell epitops are distributed over almost entire protein structure 5. In the study the recombinant BanLec-Bet v 1 construct is designed, produced by the recombinant DNA technology, purified and characterized by classical biochemical methods for the application in the ASIT of birch pollen allergy. The expression of newly designed BanLec-Bet v 1 was performed in E. coli BL21 (DE3). After expression the protein was found in the inclusion bodies from which it was extracted with 4 M urea solution. After renaturation, affinity chromatography (Sephadex G-75 superfine) was used for protein purification. Biochemical characterization of the chimera was performed by: SDS PAGE electrophoreses, CD spectroscopy and mass spectrometry. Biological activity of the construct was confirmed by binding of BanLecBet v 1 to a horseradish peroxidase glycoprotein in ELISA. Purified BanLec-Bet v 1 showed molecular mass of 32 kDa. CD spectra of the recombinant construct revealed well defined secondary structures with predominant beta sheets (41.2%). By mass spectrometry 51.8% of the BanLec-Bet v 1 primary structure was confirmed. Biologicaly active recombinant BanLec-Bet v 1 was produced by the recombinant DNA technology. Further in vitro and in vivo studies will evaluate immunomodulatory potential of BanLec-Bet v 1 for application in ASIT

Evaluation of the immunomodulatory potential of chimera Bv1a-BLwt and its mutants on the co-culture model system

Allergen immunotherapy (AIT) is currently the only disease-modifying treatment forallergies. Pre-clinical models for the evaluation of novel therapeutics are crucial forensuring their efficacy and safety. While cell culture models are cost-effective andefficient, they cannot fully replicate the cellular interactions in vivo. Therefore, it isessential to use more sophisticated model systems, such as co-cultures, to assess thepotential of new therapeutics more accurately. Immunomodulatory protein banana lectin(BLwt) is an attractive candidate for adjuvant in AIT. Its mutant BLH84T was developed toreduce its potential mitogenicity. The aim of this study was the development of the coculture model system for testing the immunomodulatory effect of chimeras composed ofthe major birch pollen allergen (Bv1a) and BLwt (Bv1a-BLwt, Cwt), the hypoallergenicisoform of Bv1a (Bv1l) and BLH84T (Bv1l-BLH84T, C1 and BLH84T-Bv1l, C2). Chimericstructures were designed in silico, fully minimized, and relaxed without van der Waalsatomic clashes. Afterward, proteins were successfully expressed in Escherichia coli andpurified by IMAC yielding around 0.4 mg per 1L of expression medium. The IgE bindingcapacity was assessed using ELISA inhibition with birch pollen allergic patients’ sera.Caco-2 intestinal epithelial cells and THP-1 differentiated macrophages were used for theco-culture model system development. After protein application on the apical side of theco-culture, the integrity of the epithelial monolayer was not disturbed. Theimmunomodulatory potential of antigens was tested by measuring the gene expressionlevels for pro- and anti-inflammatory cytokines in both cell lines from co-culture. Theobtained results indicate that the best anti-inflammatory response was favored aftertreatment with Cwt. Additionally, to further confirm the immunomodulatory effect of therecombinant chimeras, PBMCs obtained from individuals allergic to birch pollen wereemployed and treated with recombinant proteins. Only after treatment with Cwt, PBMCssecreted the anti-inflammatory cytokine IL-10. Obtained results suggest that Cwt chimeracould have a therapeutic effect in AIT in birch pollen allergy

Evaluation of the immunomodulatory potential of chimera Bv1a-BLwt and its mutants on the co-culture model system

Allergen immunotherapy (AIT) is currently the only disease-modifying treatment for allergies. Pre-clinical models for the evaluation of novel therapeutics are crucial for ensuring their efficacy and safety. While cell culture models are cost-effective and efficient, they cannot fully replicate the cellular interactions in vivo. Therefore, it is essential to use more sophisticated model systems, such as co-cultures, to assess the potential of new therapeutics more accurately. Immunomodulatory protein banana lectin (BLwt) is an attractive candidate for adjuvant in AIT. Its mutant BLH84T was developed to reduce its potential mitogenicity. The aim of this study was the development of the coculture model system for testing the immunomodulatory effect of chimeras composed of the major birch pollen allergen (Bv1a) and BLwt (Bv1a-BLwt, Cwt), the hypoallergenic isoform of Bv1a (Bv1l) and BLH84T (Bv1l-BLH84T, C1 and BLH84T-Bv1l, C2). Chimeric structures were designed in silico, fully minimized, and relaxed without van der Waals atomic clashes. Afterward, proteins were successfully expressed in Escherichia coli and purified by IMAC yielding around 0.4 mg per 1L of expression medium. The IgE binding capacity was assessed using ELISA inhibition with birch pollen allergic patients’ sera. Caco-2 intestinal epithelial cells and THP-1 differentiated macrophages were used for the co-culture model system development. After protein application on the apical side of the co-culture, the integrity of the epithelial monolayer was not disturbed. The immunomodulatory potential of antigens was tested by measuring the gene expression levels for pro- and anti-inflammatory cytokines in both cell lines from co-culture. The obtained results indicate that the best anti-inflammatory response was favored after treatment with Cwt. Additionally, to further confirm the immunomodulatory effect of the recombinant chimeras, PBMCs obtained from individuals allergic to birch pollen were employed and treated with recombinant proteins. Only after treatment with Cwt, PBMCs secreted the anti-inflammatory cytokine IL-10. Obtained results suggest that Cwt chimera could have a therapeutic effect in AIT in birch pollen allergy

IgM and IgG Immunoreactivity of SARS-CoV-2 Recombinant M Protein

Diagnostic evaluation of specific antibodies against the SARS-CoV-2 virus is mainly based on spike (S) and nucleocapsid (N) proteins. Despite the critical functions in virus infection and contribution to the pattern of immunodominance in COVID-19, exploitation of the most abundant membrane (M) protein in the SARS-CoV-2 serology tests is minimal. This study investigated the recombinant M protein’s immunoreactivity with the sera from COVID-19 convalescents. In silico designed protein was created from the outer N-terminal part (19 aa) and internal C-terminal tail (101–222 aa) of the M protein (YP_009724393.1) and was recombinantly produced and purified. The designed M protein (16,498.74 Da, pI 8.79) revealed both IgM and IgG reactivity with serum samples from COVID-19 convalescents in Western blot. In ELISA, more than 93% (28/30) of COVID-19 sera were positive for IgM detection, and more than 96% (29/30) were positive for specific IgG detection to M protein. Based on the capacity to provoke an immune response and its strong antigenic properties, as shown here, and the fact that it is also involved in the virion entry into host cells, the M protein of the SARS-CoV-2 virus as a good antigen has the potential in diagnostic purposes and vaccine design

rBet v 1a-BanLec_wt induce upregulation of IL-10 and IFN-γ gene expression in Caco-2/THP-1 co-culture and secretion of IL-10 and IFN-γ/IL-4 levels in PBMCs of birch pollen allergic donors

Novel allergen immunotherapy (AIT) approaches necessitate the use of more effective and safe therapeutics, which can be accomplished by employing novel adjuvants for improved innate immune cell activation, as well as hypoallergenic allergen forms. In this study, we investigate the immunomodulatory effects of a chimera rBet v 1a-BanLecwt (rBv1a-BLwt; Cwt) composed of the major birch pollen allergen Bet v 1a and banana lectin (BanLecwt; BLwt) and two novel chimeras, rBv1l-BLH84T (rBet v 1l-BanLecH84T; C1) and rBLH84T-Bv1l (rBanLecH84T-Bet v 1l; C2), both composed of BLH84T and hypoallergenic birch pollen allergen Bv1l in the co-culture model Caco-2/THP-1, and PBMCs from donors with birch pollen allergy. The chimeric molecules rBv1l-BLH84T (C1) and rBLH84T-Bv1l (C2) were created in silico and then produced in E. coli using recombinant DNA technology. Real-time PCR analysis of gene expression following compound treatment in the co-culture model revealed that all three chimeras have the potential to induce the anti-inflammatory cytokine IL-10 gene expression in Caco-2 cells and IFN-γ gene expression in THP-1 cells. Sandwich ELISA revealed that Cwt increased IL-10 secretion and IFN-/IL-4 levels in PBMCs from birch pollen allergic donors, whereas C1 and C2 were less effective. The findings suggest that Cwt should be analyzed further due to its potential benefit in AIT

Ovalbumin - Two Sides of the Same Coin

Ovalbumin (OVA) is the most abundant egg white protein. It is a globular, acidic phosphorylated glycoprotein of the serpin family with a molecular weight of 45 kDa. OVA is rich in essential amino acids and upon proteolytic digestion yields bioactive peptides (BAPs), recognized nutraceuticals with hypotensive, antimicrobial, antioxidant, and anticancer properties that contribute to the overall nutritional and health benefits of eggs. OVA is a common choice in the food, biomedical, and pharmaceutical industries due to its useful properties during food processing, capacity to form biocompatible gels, and special properties as an effective transporter for a variety of nutraceuticals and pharmaceuticals. Cellular agriculture is an innovative interdisciplinary approach that bypasses conventional animal husbandry in the production of animal proteins. OVA expressed in Trichoderma reesei (T. reesei) most closely mimics the structural and functional properties of its natural homolog and is therefore considered a sustainable alternative to chicken egg white protein powder. Egg allergy poses serious concerns for food safety and an important socioeconomic burden to the food sector and public health. OVA has been extensively studied as an important egg allergen in mice and in vitro experimental models, providing fundamental insights into the molecular mechanisms of allergy and identifying new therapeutic targets. This chapter focuses on providing a comprehensive overview of the state-of-the-art of OVA in human nutrition and the food industry. After presenting the structure underlying the functional properties of OVA, we provide a critical perspective on cellular agriculture as a non-poultry production of OVA. Additionally, the detailed nutritional and biotechnological significance of OVA is elaborated. The final part of this chapter provides a comprehensive insight into OVA as a model antigen and food allergen from a food safety perspective

Identification of S-adenosyl-L-homocysteine hydrolase from banana fruit as a novel plant panallergen

Banana allergy is often associated with the pollen and latex allergies, which led us to the hypothesis that some yet unidentified banana allergen could provide a basis of the latex-pollen-fruit syndrome. S-adenosyl-L-homocysteine hydrolase (SAHH) was recently identified in the literature as a novel plant allergen. This study aimed to assess the allergenic potential of the naturally occurring banana SAHH (nSAHH) and its recombinant homolog produced in E. coli (rSAHH). nSAHH showed IgE reactivity with a serum pool of twelve banana-allergic persons, while rSAHH displayed IgE reactivity in ten out of the twelve tested patients. Five linear B-cell epitopes were identified on the rSAHH surface, exhibiting ≥ 90 % sequence homology with relevant plant SAHH allergens. Our findings have elucidated SAHH as a novel plant panallergen, underlying the cross-reactivity between plant derived food and respiratory allergens, confirming our initial hypothesis

Recombinant Bet v 1-BanLec chimera modulates functional characteristics of peritoneal murine macrophages by promoting IL-10 secretion

Allergen-specific immunotherapy (AIT) is a desensitizing treatment for allergic diseases that corrects the underlined pathological immune response to innocuous protein antigens, called allergens. Recombinant allergens employed in the AIT allowed the production of well-defined formulations that possessed consistent quality but were often less efficient than natural allergen extracts. Combining recombinant allergens with an adjuvant or immunomodulatory agent could improve AIT efficacy. This study aimed to perform structural and functional characterization of newly designed recombinant chimera composed of the Bet v 1, the major birch pollen allergen, and Banana Lectin (BanLec), TLR2, and CD14 binding protein, for the application in AIT. rBet v 1-BanLec chimera was designed in silico and expressed as a soluble fraction in Escherichia coli. Purified rBet v 1-BanLec (33.4 kDa) retained BanLec-associated biological activity of carbohydrate-binding and preserved IgE reactive epitopes of Bet v 1. The chimera revealed secondary structures with predominant β sheets. The immunomodulatory capacity of rBet v 1-BanLec tested on macrophages showed changes in myeloperoxidase activity, reduced NO production, and significant alterations in the production of cytokines when compared to both rBanLec and rBet v 1. Comparing to rBet v 1, rBet v 1-BanLec was demonstrated to be more efficient promoter of IL-10 production as well as weaker inducer of NO production and secretion of pro-inflammatory cytokines TNFα, and IL-6. The ability of rBet v 1-BanLec to promote IL-10 in together with the preserved 3D structure of Bet v 1 part implies that the construct might exert a beneficial effect in the allergen-specific immunotherapy