Activity of antibody-derived peptides against protozoa of medical interest

The aim of the PhD research project was focused on the study of the potential activity of antibody-derived peptides against medically relevant protozoan parasites. In recent years, the potential antimicrobial and antiviral activity of antibody-derived peptides and their derivatives, synthesized from the sequence of variable and constant regions of Abs with different specificity, has been extensively studied. In particular, KP, a decapeptide engineered from the variable region of a single-chain recombinant anti-idiotypic antibody representing the internal image of a yeast killer toxin, displayed a wide spectrum of biological activity. KP proved to exert in vitro, ex vivo and/or in vivo activity, through different mechanisms of action, against different pathogens, including bacteria, yeasts, viruses and some protozoa. The aim of this project was to evaluate the potential activity and the possible mechanism of action of KP, and some KP-derivatives, against protozoa that cause globally widespread diseases: Toxoplasma gondii, Plasmodium falciparum, Leishmania infantum and L. tropica. In preliminary experiments, KP proved to be nontoxic for Vero cells used for Toxoplasma infection. KP effect on T. gondii tachyzoites invasion and intracellular proliferation in Vero cells was evaluated by microscopic observations and real-time PCR quantification of T. gondii DNA. The results showed that KP treatment of extracellular tachyzoites significantly reduced both the number of parasites able to invade the cells and their ability to replicate. KP potential mechanism of action was evaluated by the analysis of markers of apoptosis: phosphatidylserine externalization, DNA fragmentation, dissipation of mitochondrial membrane potential. The results showed that KP treatment is able to trigger apoptosis-like phenomena in T. gondii tachyzoites. This result was confirmed by tachyzoites morphological observation before and after KP treatment. In addition, the in vitro potential activity of KP and of five KP-derivatives (K10S, K10S-I, K10S-SS, K10T-TT e R10S-RR) was evaluated against P. falciparum (asexual stages of two different strains, one chloroquine-susceptible and one chloroquine-resistant, and sexual stages of a recombinant chloroquine-susceptible strain), L. infantum (promastigotes and amastigotes) and L. tropica (promastigotes). In particular, the ability of the selected peptides to inhibit P. falciparum viability was evaluated by measuring parasite lactate dehydrogenase activity (for asexual stages) and luciferase activity (for gametocytes). All peptides, with the exception of K10S-SS, showed activity against P. falciparum asexual stages, and KP was the most active against both tested strains. No peptide was active against P. falciparum gametocytes. Peptides activity against L. infantum and L. tropica promastigotes was assessed by a colorimetric MTT assay. In agreement with previous data, obtained against L. major and L. infantum, KP showed a significant activity in vitro against promastigotes of both strains, while KP-derivatives proved to be less active. Lastly, preliminary assays were carried out to evaluate the ability of peptides to inhibit L. infantum intracellular infection in THP-1 cells, differentiated in macrophages. All tested peptides showed a moderate inhibition of the infection index. A preliminary MTT assay, however, showed that all peptides exerted some cytotoxic effects against THP-1 cells. In conclusion, the proven in vitro antiprotozoan activity allow to look at KP and its derivatives as promising candidates for the development of novel agents for the treatment of the infections caused by such protozoa