Gene design, optimization of protein expression and preliminary evaluation of a new chimeric protein for the serological diagnosis of both human and canine visceral leishmaniasis

Abstract

Background Visceral leishmaniasis (VL) is a major neglected disease, potentially fatal, whose control is still impaired by inefficient and/or expensive treatment and diagnostic methods. The most promising approach for VL diagnosis uses serological assays with recombinant proteins, since they are more efficient and easier to perform. Tests developed for the human form of the disease, however, have not been shown to be efficient for its diagnosis in the canine host, the major reservoir for the American VL. Methodology/Principal findings Here, we describe a systematic approach aimed at the production of a new chimeric protein potentially able to be used for both human and canine VL diagnosis and based both on in silico gene design and experimental data. Starting from the previous identification of Leishmania infantum recombinant antigens efficient for the diagnosis of either human or canine VL, three of the best performing antigens were selected (Lci2, Lci3 and Lci12). After a preliminary evaluation validating the chimeric approach, DNA fragments encoding predicted antigenic regions from each protein, enriched with repeats, were joined in various combinations to generate a total of seventeen chimeric genes optimized for prokaryotic expression. These were assessed for optimal expression and purification yield, with four chimeric proteins being efficiently produced. Their diagnostic potential was then evaluated through ELISA assays with sera from VL afflicted humans and dogs. After two rounds of gene design, the results showed high levels of sensitivity for the best chimeric protein, named Q5, in humans (82%) and dogs (100%) with 100% specificity in comparison with healthy a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Recombinant antigens used as diagnostic tools for lymphatic filariasis

“…Further technological advances, as reported in some of the most recent studies using phage display and the BmSXP and BmRI antigens [ 27 , 56 , 58 ], have the potential to facilitate the generation of more efficient antibodies that can be used for such new assays. Another possibility is the use of tests based on chimeric proteins which can potentially enhance the capabilities of the antibody capture assays, as has been done for other diseases [ 89 – 91 ]. Thus, along with what has been reported so far, it is expected that further progress (or advances in research) should facilitate the development of a test that combines the features of high sensitivity, high specificity (without cross-reactivity), and low cost, to assist GPELF.…”

ChimLeish, a new recombinant chimeric protein evaluated as a diagnostic and prognostic marker for visceral leishmaniasis and human immunodeficiency virus coinfection

“…In our study, ChimLeish showed 100% sensitivity and specificity for VL diagnosis. Recently, another recombinant chimeric protein called Q5 when used in ELISA showed sensitivity and specificity values of 82.0% and 100%, respectively, for VL diagnosis (Santos et al 2020). Figueiredo et al (2021) developed a recombinant chimeric protein called DTL-4 and when used in ELISA, this antigen presented sensitivity and specificity values of 94.6% and 99.4%, respectively.…”

Borrelia burgdorferi BmpA-BBK32 and BmpA-BBA64: New Recombinant Chimeric Proteins with Potential Diagnostic Value

“…These recombinant proteins combine regions of high immunogenicity derived from two (or more) different antigens into one, single polypeptide. Chimeric products are better at detecting the presence of specific antibodies than either single proteins or mixtures, as demonstrated in, for example, the diagnosis of toxoplasmosis or Chagas disease [ 41 , 42 , 43 , 44 , 45 , 46 , 47 ].…”