Selection of binding targets in parasites using phage-display and aptamer libraries in vivo and in vitro

Abstract

Parasite infections are largely dependent on interactions between pathogen and different host cell populations to guarantee a successful infectious process. This is particularly true for obligatory intracellular parasites as Plasmodium, Toxoplasma, and Leishmania, to name a few. Adhesion to and entry into the cell are essential steps requiring specific parasite and host cell molecules. The large amount of possible involved molecules poses additional difficulties for their identification by the classical biochemical approaches. In this respect, the search for alternative techniques should be pursued. Among them two powerful methodologies can be employed, both relying upon the construction of highly diverse combinatorial libraries of peptides or oligonucleotides that randomly bind with high affinity to targets on the cell surface and are selectively displaced by putative ligands. These are, respectively, the peptide-based phage display and the oligonucleotide-based aptamer techniques. The phage display technique has been extensively employed for the identification of novel ligands in vitro and in vivo in different areas such as cancer, vaccine development, and epitope mapping. Particularly, phage display has been employed in the investigation of pathogen–host interactions. Although this methodology has been used for some parasites with encouraging results, in trypanosomatids its use is, as yet, scanty. RNA and DNA aptamers, developed by the SELEX process (Systematic Evolution of Ligands by Exponential Enrichment), were described over two decades ago and since then contributed to a large number of structured nucleic acids for diagnostic or therapeutic purposes or for the understanding of the cell biology. Similarly to the phage display technique scarce use of the SELEX process has been used in the probing of parasite–host interaction. In this review, an overall survey on the use of both phage display and aptamer technologies in different pathogenic organisms will be discussed. Using these techniques, recent results on the interaction of Trypanosoma cruzi with the host will be highlighted focusing on members of the 85 kDa protein family, a subset of the gp85/TS superfamily.

Antibody Engineering for Pursuing a Healthier Future

“…Numerous screening cycles are essential in biopanning to attain the preferred binding activity of the acquired monoclonal phage antibodies. Several tests including ELISA, fluorometric microvolume assay technology (FMAT), and chromophoreassisted laser inactivation (CALI) are used to analyze this activity (Tonelli et al, 2012).…”

“…Antigen specific scFv can be easily generated by phage display. These fusion proteins have extensive applications in cancer therapeutics such as in lymphatic invasion vessels, colon cancer, hepatocarcinoma, and diagnostics of human disease (Tonelli et al, 2012). Moreover, scFv have been widely used with phage display panning i.e., an affinity selection technique, to construct ligands to detect toxins produced by various pathogenic entities in vitro or in vivo.…”

“…Similarly, coexpression vector systems have been used to prevent and cure diseases by scFv. Currently, various scFv fragments have been constructed against toxins and virulence factors of pathogens (Tonelli et al, 2012;Wang et al, 2013Wang et al, , 2016. Additionally, a method for rapid and effective high-affinity GFP-based antibody production corresponding to scFv was developed.…”

Theranostic applications of phage display to control leishmaniasis: selection of biomarkers for serodiagnostics, vaccination, and immunotherapy

“…species express a variety of surface and secreted molecules used by the parasite to attach and enter mammalian cells. These factors are key determinants of the disease progression, and most studies on host-pathogen interactions are focused on the identifi cation of Leishmania ligands and related host receptors using classical biochemical approaches such as affi nity purifi cation, crosslinking, immunoprecipitation, and fractionation (12) . However, these techniques are not intended for high-throughput screening of multiple candidate molecules.…”

Synthetic, Switchable Enzymes

“…Several systems could be used to construct the 1-binders, 2-binders and the 1,2-binders, i.e., the candidate swenzymes [Tonelli et al, 2012]. Phage display [Smith, 1985] could be used with, for example, bacterial plaques (in which bacteriophages are concentrated) on a lawn of bacteria being transferred to nitrocellulose and probed with the product-binder to select swenzymes.…”