Discovery and fine epitope mapping of novel serology-based markers for diagnosis of Congenital Chagas Disease using high-density peptide chips

BRACCO L; JUAN MUCCI,; MOSCATELLI G; MORONI S; BALLERING G; BUSCAGLIA CA; CAMPETELLA O; NIELSEN M; TEKIEL V; ALTCHEH J; AGÜERO F

New Orleans, LA

Conferencia; 67th ASTMH Annual Meeting; 2018

American Society of Tropical Medicine and Hygiene

Chagas Disease, caused by ​ Trypanosoma cruzi, ​ affects 6-8 million people in the Americas andat least 2 million women of fertile age are estimated to be chronically infected with ​ T. cruzi ​ .Congenital infections with ​ T. cruzi ​ represent a global problem, occurring on average in 5% ofchildren born from chronically infected mothers, both in endemic and non-endemic areas.Current diagnostic protocols and techniques for Congenital Chagas Disease (CCD) arecumbersome, have poor performance or require re-testing of newborns and infants during thefirst year of age. Therefore, new and improved diagnostics that can be deployed in primaryhealth centers are urgently needed. Serology-based assays are preferred due to their low costand simplicity, but currently suffer of poor performance in newborns due to the presence ofconfounding maternal antibodies. Furthermore, there is a very limited set of ​ T. cruzi ​ antigenscharacterized from congenital infections. Here we present a set of defined antigens andepitopes with an excellent potential for diagnosis of CCD. The antigens were identified using ahighly parallelized screening platform based on high-density peptide-arrays displaying >175,000short peptides derived from 457 ​ T. cruzi proteins (Carmona SJ et al 2015). These peptide chipswere screened with 2 pools of sera from 10 infected newborns, and with 2 paired pools of serafrom their mothers. Each microarray slide was assayed with anti-human IgG (Cy3 labeled), andanti-human IgM (Cy5 labeled) secondary antibodies, and imaged in a fluorescence scanner.After data normalization, we reconstructed the antibody-binding profiles for each protein in thearray and validated the assay by profiling known antigens. Comparative analysis of IgGreactivity between paired sera pools of newborns and their mothers allowed the identification ofcandidate antigens for diagnosis of CCD. These display high IgG reactivity in infectednewborns, and low or null reactivity in the paired samples from their mothers. Analysis of IgMantibody-binding also led to the identification of candidate antigens with strong IgM reactivity inthe samples from infected newborns. From these data we prioritized 125 highly reactivepeptides from the top 5% of both IgG and IgM sets, and selected 48 short peptides for furthervalidation and assessment of their seroprevalence in standard ELISA format in 96-well plates(currently underway). This study represents the largest screening so far for discovery ofcongenital markers of infection with T. cruzi.