Desenvolvimento de moléculas com potencial teranóstico para os vírus Zika e Dengue

Abstract

Arboviruses caused by the Dengue virus (DENV) and Zika (ZIKV), are currently considered diseases that represent a potential challenge to public health, resulting in intense and limiting clinical manifestations, which can culminate in microcephaly (ZIKV) and death. There are still no vaccines in the public network or any other preventive drug, the diagnosis is inaccurate due to the cross reactions between the antibodies of DENV, ZIKV and other flaviviruses. Given this context, we aim to develop new molecules based on small peptides (phage) that bind to the ZIKV and DENV-2 virus using the Phage-Display methodology. Thus, the intention of the present study was to select, characterize and validate ZIKV and DENV-2 ligands, assessing their ability to inhibit and also differentiate ZIKV and DENV. The ELISA immunoassay, ZIKV and DENV-2 entry inhibition assay in vitro by flow cytometry using Vero cells and 4G2 antibody and silica analysis were used for the validation of the selected phages. ELISA was also performed with a phage that did not show significant viral inhibition and another that was effective for inhibition using samples from patients infected with DENV and ZIKV. Phage D4, selected for ZIKV, was able to inhibit the entry of ZIKV and DENV-2. F12 phage, also selected for ZIKV, was not able to inhibit ZIKV entry, however it was able to discriminate DENV patients from ZIKV patients and healthy individuals (p <0.0001) with high accuracy, presenting 94.9% specificity and 81.2% sensitivity for DENV infection. The G8 phage selected for DENV-2 was able to inhibit the entry of DENV-2 and also discriminated against DENV from ZIKV and healthy patients in a preliminary test that will be optimized with a larger number of samples. The silica study showed that the possible interaction sites of the peptides expressed by the D4 phage, which inhibit the entry of ZIKV and DENV, were located in domain III of the envelope protein (EDIII) or very close to the fusion loop. Both EDIII and the fusion loop, which is located in EDII, are important target epitopes for antibodies already described in the literature with wide neutralizing action for flavivirus. In the study of phages F12 and G8, which differentiated DENV patients from ZIKV and healthy individuals, we observed their interactions with amino acid residues from the ZIKV and DENV-2 envelope protein, which comprise the kl loop, which is discussed in comparative studies of Zika and Dengue viruses. Through these studies we will seek the integration of important methodologies, covering low cost, speed and high efficiency, in order to fill possible gaps and demands that we still find in the diagnosis of DENV and ZIKV in different stages of the infection, as well as to contribute to future complementary studies, in order to produce possible therapeutic and preventive interventions. In general, this study demonstrated that Phage-Display technology is a strong tool for the development of molecules with antiviral and diagnostic potential. With this, the next step will be to synthesize the peptides expressed in the phages, carrying out tests that can contribute to the development of vaccines for ZIKV, DENV or even for other infectious agents from synthetic molecules.