HIGH-THROUGHPUT MUTATIONAL ANALYSIS OF TRYPANOSOMA CRUZI ANTIGENIC EPITOPES REVEAL CONSISTENT CONSERVATION OF KEY RESIDUES ACROSS HUMAN CHAGAS DISEASE POPULATIONS.

NOLAN MELISA; AGUERO FERNAN; RAMSEY JANINE; VILLAR JUAN CARLOS; BRACCO LEONEL; ALTCHEH JAIME

Congreso; Reunion anual Sociedades de Biociencia, SAIC; 2019

Soc Argentina de Investigacion Clinica

Chagas Disease is a major health problem for which no vaccine for public health interventions are yet available. Diagnosis is essential for the identification of infection and ecoepidemiological surveillance. Diagnosis is routinely performed using serological methods, which require well characterized antigens. Although available tests give satisfactory results in most cases, discordant results remain as a possible cause of undetected infected patients. We have previously conducted a large-scale screening of T. cruzi linear B-cell epitopes using high-density peptide arrays, leading to the development of a new proof-of-principle multiepitope diagnostic test that has excellent diagnostic performance1. However, understanding which residues in an epitope are important for antibody binding can lead to improved reagents. To further characterize known antigens, we performed Alanine scans of 649 different proteins (881 antibody-binding peaks/epitopes, represented by 2,913 peptides). This experiment was based on replacing each amino acid residue in each peptide for an Alanine (or a Glycine if the original residue was itself an Alanine), and assessing the impact on reactivity of each modified epitope. Using this peptide array design, we have assayed 45,492 peptide variants against 108 Chagas Disease serum samples (from 6 different countries). We developed an algorithm to integrate and analyze the effect of these epitope mutations, and to visualize key residues for each antigen and sample. We identified precise residue positions in epitopes that play a fundamental role in the seroreactivity. In summary, we observe an average of ~6 key residues per epitope. As an example, Ag2/CA-2, a known antigen, displays the same 7 key residues in all reactive sera. In contrast, SAPA, Ag36, and B12 display different degrees of reactivity conservation of their key residues. This variable responses for different antigens, can be used in the design of improved antigens for diagnostic applications.