Evaluation of the immunoremodulatory effect of the chimeric protein Bet v 1- BanLec and its mutants on cells of the innate immune system

[I. Protić-Rosić]

100 str.

Hemijske nauke -Biohemija / Chemistry - Biochemistry

Kao jedna od najčešćih hroničnih bolesti, alergija predstavlja ozbiljan zdravstveni problem, koji osim toga utiče i na socioekonomski aspekt obolele osobe. Za razliku od farmakoterpije koja omogućava samo kratkoročno uklanjanje simptoma, alergen- specifična imunoterapija (AIT) je jedino dugoročno rešenje za problem alergija. U cilju poboljšanja AIT, istraživanja se usredsređuju na inovativne strategije koje obuhvataju zamenu konvencionalnih alergenih ekstrakata dobijenih iz prirodnih izvora. Ovaj pristup uključuje korišćenje rekombinantno proizvedenih alergena, alergenih peptida, ili hipoalergenih izoformi alergena, kao i primenu odgovarajućih adjuvanasa radi smanjenja potrebne doze alergena. Odabir idealnog adjuvansa u kombinaciji sa alergenom je ključan, te se teži pronalaženju adjuvanasa koji će efikasno aktivirati ćelije urođenog imunskog sistema, što bi dalje modulisalo adaptivni imunski odgovor. Na ovaj način bi se unapredila efikasnost i sigurnost AIT. Lektin banane (rBLwt) je protein koji specifično prepoznaje manozu i moduliše imuni odgovor mišijih makrofaga, najverovatnije kroz vezivanje za oligosaharidne komponente na površini TLR2 i CD14. U ovoj doktorskoj disertaciji je ispitivan imunomodulatorni efekat lektina banane kao potencijalnog adjuvansa na modelu glavnog alergena polena breze (rBet v 1 a; rBv1a). Rekombinantne himere koju čine rBLwt i rBv1a (rBv1a-BLwt; Cwt), kao i hipoalergena izoforma Bet v 1 (Bv1l) i mutant BLH84T (rBv1l-BLH84T (C1) i rBLH84T-Bv1l (C2)) su uspešno dizajnirana in silico nakon čega su eksprimirane u Escherichia coli ekspresionom sistemu. Nakon prečišćavanja, biohemijske i imunološke karakterizacije testiran je imunomodulatorni efekat proteina na peritonealnim mišjim makrofagima kao i na humanim ćelijskim linijama uključujući i ćelijske kokulture. Među testiranim himerama, Cwt je pokazala imunomodulatorni potencijal kroz stimulaciju sekrecije IL-10 i povećanjem odnosa izlučenih IFN-γ/IL-4. Ova studija ukazuje da Cwt himera ima najveći potencijal među analiziranim proteinima za primenu u sublingvalnoj terapiji pacijenata alergičnih na polen breze.

As one of the most common chronic diseases, allergy represents a serious health issue, which further affects the socio-economic aspect of allergic individuals. Unlike pharmacotherapy, which provides short-term relief of symptoms, allergen-specific immunotherapy (AIT) is currently the only long-term solution to the problem of allergies. To advance AIT, novel research focuses on innovative strategies involving the replacement of conventional allergenic extracts obtained from natural sources. This approach includes the use of recombinantly produced allergens, allergenic peptides, or hypoallergenic isoforms of allergens, as well as the use of appropriate adjuvants to reduce the therapeutic allergen dose. The selection of an ideal adjuvant in combination with the allergen is crucial. The aim is to find adjuvants that effectively activate cells of the innate immune system, thereby modulating the adaptive immune response. likely through binding to oligosaccharide components on the surface of TLR2 and CD14. In this doctoral dissertation, the immunomodulatory effect of banana lectin as a potential adjuvant was investigated on the model of the major birch pollen allergen (rBet v 1a; rBv1a). The recombinant chimeras consisting of rBLwt and rBv1a (rBv1a-BLwt; Cwt), as well as chimeras composed of a hypoallergenic isoform of Bet v 1 (rBv1l) and mutant banana lectin (BLH84T) (rBv1l-BLH84T (C1) and rBLH84T-Bv1l (C2)), were successfully designed in silico was successfully designed in silico, expressed in Escherichia coli expression system. After purification, biochemical, and immunological characterization the immunomodulatory ability of chimeras was examined on peritoneal murine macrophages and on human cell lines including the cell co-culture. Among tested chimeras, Cwt showed immunomodulatory potential through the stimulation of IL-10 secretion and increasment of IFN-γ/IL-4 ratio. This study suggests that among analysed chimeras, the Cwt chimera is the most suitable for further testing for the application in sublingual therapy for birch pollen allergic patients.

alergije, alergen-specifična imunoterapija, adjuvansi, lektin banane, Bet v 1, makrofagi, Caco-2 epitelne ćelije

allergy, allergen-specific immunotherapy, adjuvants, banana lectin, Bet v 1, macrophages, Caco-2 epithelial cells

547.9:577.112.8(043.3)

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