Στην παρούσα μελέτη, διερευνήθηκε η αντιλεϊσμανιακή δράση της LeIF του
στελέχους L. infantum (LieIF) και αποδείχθηκε η προστατευτική ή/και
ανοσοθεραπευτική δράση της, σε in vitro σύστημα L. donovani - μολυσμένων
μακροφάγων. Προσδιορίστηκαν οι δραστικοί μηχανισμοί που επιστρατεύουν τα
μακροφάγα και επιβεβαιώθηκε πως η λεϊσμανιοκτόνος δράση της LieIF,
διαμεσολαβείται από την παραγωγή των NO και ROS. Η προστατευτική της δράση
συσχετίστηκε με την ενεργοποίηση ενός TNF-α εξαρτώμενου μονοπατιού και με την
παραγωγή της IL-12. Η ανοσοθεραπευτική της δράση, αποδείχθηκε πως δεν
ρυθμίζεται από μηχανισμούς που περιλαμβάνουν τον TNF-α.
Επιπλέον, μελετήθηκαν οι ανοσοτροποποιητικές ιδιότητές της, σε in vitro σύστημα
δενδριτικών κυττάρων (ΔΚ). Πραγματοποιήθηκε επίσης, in silico ανάλυση της
πρωτεΐνης. Οι ανοσοτροποποιητικές ιδιότητες συνθετικών πεπτιδίων και
μεγαλύτερων αλληλοεπικαλυπτόμενων ανασυνδυασμένων τμημάτων της LieIF,
ελέγχθηκαν σε in vitro συστήματα μακροφάγων και ΔΚ. Τα αποτελέσματα έδειξαν την
ύπαρξη πολυεπιτοπικών τμημάτων με ισχυρές ανοσοτροποποιητικές ιδιότητες.
Ακολούθησε αξιολόγηση της in vivo δράσης της LieIF σε πειραματικά μοντέλα
ευαίσθητων έναντι της λεϊσμανίασης, ποντικών. Μελετήθηκε η δράση της LieIF ως
ανοσοενισχυτικό και αναπτύχθηκαν πειραματικά πρωτόκολλα ανοσοθεραπείας και
πρωτόκολλα προστατευτικού εμβολιασμού.
Η LieIF επέδειξε δραστικότητα ανοσοενισχυτικού καθώς και ισχυρές
ανοσοτροποποιητικές ιδιότητες σε in vitro και in vivo συστήματα, που
αποδείχθηκαν ικανές να προσανατολίσουν την πόλωση μιας Th1–διαμεσολαβούμενης
προστατευτικής ανοσολογικής απόκρισης.
(EL)
Th1 immune responses contribute in an essential way to the development of a
protective immune response, against Leishmania species. Over the past years,
several Leishmania recombinant antigens have been identified and demonstrated
to have a promising vaccine potential to leishmaniasis, since they induce the
desired Th1 immune response.
Eukaryotic initiation factors of Leishmania spp. (LeIF), are highly conserved
antigens among Leishmania species and studies with eIF proteins from L.
braziliensis and L. major, revealed the capacity of these proteins to induce
effective protection in murine models of disease.
In the present study, the in vitro antileishmanial activity of the eukaryotic
initiation factor eIF from L. infantum (LieIF) was investigated and the
protective and/or immunotherapeutic activity of this protein in an in vitro
system based on L. Donovani - infected macrophages, was demonstrated for the
first time. It was also determined the mechanisms activated by macrophages in
order to suppress the intracellular multiplication of the parasite, thus was
confirmed that the leishmanicidal effect of LieIF was mediated by the
production of antimicrobial agents such as NO and ROS. This protective effect
of LieIF was associated with activation of TNF-α - dependent pathway and the
production of the IL-12 cytokine. In contrary, the corresponding output of
antimicrobial molecules induced by the immunotherapeutic activity of LieIF,
proved not to be regulated by mechanisms that involve TNF-α production.
Then, the immunomodulatory properties of LieIF were studied in an in vitro
system of myeloid dendritic cells (DCs), by determining both maturation and
functional differentiation of these cells, as well as its synergy with the
Toll-like receptors (TLRs). Confirmation of amplification of the
immunomodulatory properties of the recombinant protein LieIF, in synergy with
the TLRs, enhances the future perspectives of exploiting this protein in
various experimental protocols of immunotherapy with enhanced therapeutic
effect.
Following the methodology of reverse vaccinology, an in silico analysis of
protein LieIF was performed in order to identify short peptides that are
recognized by T cell receptors (TCR) and have immunogenic properties. The in
silico analysis of proteins with the use of epitope prediction algorithms,
accelerates the process of finding antigenic epitopes and amplifies the use of
these peptides as promising synthetic vaccine candidates. Synthetic peptides
derived from such process and larger overlapping recombinant protein fragments
of LieIF, were further tested for their immunomodulating properties in in vitro
systems, including macrophage and dendritic cells. The results showed for the
first time, the existence of polyepitopic fragments of the protein with potent
immunomodulatory properties.
The data obtained by the in vitro studies, led to the evaluation of the in vivo
activity of LieIF in experimental models of susceptible mice. Specifically, the
effect of LieIF as adjuvant was studied as well as the use of the recombinant
protein in experimental protocols of immunotherapy and of protective
vaccination against the disease.
In brief, the obtained data showed that LieIF acts as adjuvant that is capable
of inducing adaptive immunity, as well as a strong immunomodulator in in vitro
and in vivo experimental systems. This effect has proved capable to direct the
polarization towards a Th1 - mediated immune response in experimental animal
models. These findings provide a better understanding of the role of this
cytoplasmic protein of the parasite, showing its potential for exploitation in
effective disease control strategies.
(EN)
