Development of chimeric multivalent proteins for serological diagnosis of African animal trypanosomosis

The control of African animal trypanosomosis (AAT) relies on accurate diagnostic tools. Serological diagnosis using ELISA is well-suited for surveillance due to its high-throughput capacity, low cost, and adaptability to rapid formats. However, the WOAH-recommended antibody ELISA for AAT, based on trypanosome lysates purified from rodent blood, can lack specificity and presents standardization challenges as well as ethical concerns. Recombinant proteins offer a solution to standardization, often improving specificity, though potentially at the expense of sensitivity. Combining multiple recombinant proteins can enhance sensitivity while maintaining specificity. Therefore, this study developed chimeric proteins for serological diagnosis of AAT, composed of highly immunoreactive regions from multiple known antigens using genetic engineering. Following an inventory of immunodominant antigens, we selected candidates and, using bioinformatics, designed five chimeric constructs in silico: three species-specific and two pan-trypanosome. The coding sequences for these chimeras were synthesized, cloned into expression vectors, and expressed in Escherichia coli. Purification was achieved through a series of chromatographic steps, including Ni-NTA affinity chromatography, Q Sepharose ion-exchange chromatography, and Sephadex 200 size-exclusion chromatography. Preliminary assessment of their reactivity with sera from cattle experimentally infected with Trypanosoma vivax, T. congolense or T. brucei yielded promising results. Longitudinal analysis comparing their reactivity with trypanosome lysates revealed that those specific to T. congolense, and T. vivax, as well as one pan-trypanosome, can discriminate pre- and post-infection sera. These observations confirm the potential of our chimeric constructs. Future work will involve evaluating these chimeras against a broader panel of sera.