Novel Recombinant Multiepitope Proteins for the Diagnosis of Asymptomatic Leishmania infantum-Infected Dogs

Abstract

BackgroundVisceral leishmaniasis is the most severe form of leishmaniasis. Worldwide, approximately 20% of zoonotic human visceral leishmaniasis is caused by Leishmania infantum, also known as Leishmania chagasi in Latin America. Current diagnostic methods are not accurate enough to identify Leishmania-infected animals and may compromise the effectiveness of disease control. Therefore, we aimed to produce and test two recombinant multiepitope proteins as a means to improve and increase accuracy in the diagnosis of canine visceral leishmaniasis (CVL).Methodology/Principal FindingsTen antigenic peptides were identified by CVL ELISA in previous work. In the current proposal, the coding sequences of these ten peptides were assembled into a synthetic gene. Furthermore, other twenty peptides were selected from work by our group where good B and T cell epitopes were mapped. The coding sequences of these peptides were also assembled into a synthetic gene. Both genes have been cloned and expressed in Escherichia coli, producing two multiepitope recombinant proteins, PQ10 and PQ20. These antigens have been used in CVL ELISA and were able to identify asymptomatic dogs (80%) more effectively than EIE-LVC kit, produced by Bio-Manguinhos (0%) and DPP kit (10%). Moreover, our recombinant proteins presented an early detection (before PCR) of infected dogs, with positivities ranging from 23% to 65%, depending on the phase of infection in which sera were acquired.Conclusions/SignificanceOur study shows that ELISA using the multiepitope proteins PQ10 and PQ20 has great potential in early CVL diagnosis. The use of these proteins in other methodologies, such as immunochromatographic tests, could be beneficial mainly for the detection of asymptomatic dogs.

Evaluation of a new set of recombinant antigens for the serological diagnosis of human and canine visceral leishmaniasis

“…An alternative serological method that could be used to deal with the limitations presented by the protein mixture is the development of chimeric proteins, containing the regions of the proteins that presented the best performance in serological evaluations. Some studies in this area have already been done and they showed a significant improvement in the sensitivity of the serological test [ 46 – 49 ].…”

A Novel Structurally Stable Multiepitope Protein for Detection of HCV

“…The urgent need for a diagnostic test which offers increased degrees of sensibility and specificity prompted us to develop a recombinant multiepitope protein bearing HCV-specific immunodominant epitopes. Several studies have reported the successful use of multiepitope protein for diagnosis of infectious diseases such as leishmaniasis [ 22 ], hepatitis B [ 23 ], hepatitis C [ 12 ], toxoplasmosis [ 24 ], tuberculosis [ 25 ], leprosy [ 26 ], leptospirosis [ 27 ], dengue [ 28 ], and Chagas disease [ 29 ]. A multiepitope protein (r-HCV-F-MEP) for hepatitis C diagnosis has been previously developed bearing 5 immunodominant regions comprising genotypes circulating worldwide and one Indian isolate [ 12 ].…”

Canine visceral leishmaniasis: Diagnosis and management of the reservoir living among us

“…The combination of these two antigens exhibited higher sensitivity (85%) and specificity (93%) compared with their individual sensitivity and specificity (KLO8, 68% and 92%, respectively; rK26, 77% and 91%, respectively). Also, chimeric multi-epitope proteins are being engineered to improve the detection of asymptomatic infected dogs [ 64 ]. Novel approaches like immunoproteomics identified an uncharacterized hypothetical protein with high specificity and sensitivity for CVL [ 65 ].…”