Engineered heat-stable variants of Trypanosoma cruzi flagellar protein Tc24 enable serological detection of Chagas disease

Chagas disease, caused by the protozoan Trypanosoma cruzi, is one of the major neglected diseases globally, killing 12,000 people per year and silently infecting an estimated 7 million people worldwide. Current diagnostic methods are limited by cost, complexity and cold-chain requirements. The T. cruzi flagellar protein Tc24 is a promising antigen for serological tests but suffers of poor solubility and heat stability. Here, we computationally engineered and produced three variants of Tc24, which exhibit remarkable heat stability, up to 69{degrees}C, and express with higher solubility in E. coli compared to the wild-type protein, reducing production costs, eliminating the need for a cold chain and therefore facilitating cost-effective production and storage without refrigeration and even in lyophilised form. These variants remained remarkably stable for 70 days in solution at 25{degrees}C and successfully detected antibodies in human sera samples from Chagas disease patients from Northern and Southern regions of Latin America, demonstrating the preservation of their antigenicity. The best-performing engineered variant was incorporated into a prototype of lateral flow test, demonstrating potential for rapid, affordable and accessible Chagas disease diagnostics in resource-limited settings.