Aplicações da Tecnologia do Phage Display na seleção de anticorpos e ligantes para fins terapêuticos em infecções, inflamação e ofidismo

Abstract

Phage Display (PD) technique is widely used for drug discovery and development. One of its great applications is to improve immunological studies. The objective of this work was to select by PD molecules with antivenom and anti-inflammatory activities, besides proposing an in vivo model for validations. For this, we selected neutralizing antibodies from B. pauloensis venom by PD technology that was subsequently expressed in plants to evaluate the enzymatic activity of the antibody. To propose a model for inflammation and infection useful for testing phages and peptides selected by PD technology, we standardized an in vivo model of systemic inflammation and infection from chicken embryo so that phages selected by PD can be used in preliminary tests. And finally, from the PD technology a phospholipase A2 (PLA2) inhibitor selected from B. pauloensis venom, named as F7, was tested for anti-inflammatory action in peripheral blood mononuclear cells (PBMC) and chicken embryo model. Proteolytic assays showed the ability of single chain variable fragment (scFv) molecules to inhibit the damaging actions of B. pauloensis venom and we found that the antibody neutralized the toxic effects of the envenomation, mainly those related to systemic processes, by interacting with one of the predominant enzyme classes, the metalloproteinases. A model for systemic infection and inflammation has been standardized in chicken embryos infected by Salmonella Pullorum (SP). This is an interesting model for infection and systemic inflammation and phages selected by PD can be tested in this model provided they are purified without the presence of Escherichia coli (E.coli). The selected PLA2 inhibitor (F7) was able to modulate the secretion of inflammatory cytokines (IL-1β and TNF-α) in lipopolysaccharide (LPS)-treated PBMCs showing the anti-inflammatory effects of F7. In the embryo the phage F7 also showed the same properties for the cytokines IL-1β and IFN-γ although it did not contain embryo mortality. This work presents two potential molecules for antiviral and anti-inflammatory therapy, being a scFv and F7, respectively. In particular, the F7 peptide should be tested in other models of mild and localized inflammation to enable its application in vivo.