Novel recombinant multiepitope proteins for the diagnosis of asymptomatic leishmania infantum-infected dogs

Abstract

Background: Visceral 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 findings: Ten 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/significance: Our 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.

Figure 1. PQ10 and PQ20 amino acid sequences.

Predicted amino acid sequences of recombinant proteins PQ10 and PQ20, showing flexible linkers in red and 6XHIS tag in green.

Figure 2. Cell lysate fractions and purified multiepitope proteins.

Coomassie blue-stained 12% SDS-PAGE analysis of cell lysate insoluble fraction and purified fractions resulting from IPTG-induced bacterial cultures, showing bands in the expected sizes for the recombinant proteins. MM: molecular mass in kDa.

Figure 3. Comparison of ELISA reactivity of canine sera against PQ10.

ELISA was performed in different groups of dogs (N: negative group; P: positive group). Subgroup N1: samples with negative results in EIE-LVC kit and RIFI, from animals born in kennels with wire mesh in a non-endemic area (Ouro Preto, Minas Gerais state, Brazil). From an endemic area (Belo Horizonte, Minas Gerais state, Brazil), subgroup N2 shows samples with negative results in EIE-LVC kit, RIFI and PCR; subgroup N3 shows samples with negative results in EIE-LVC kit and RIFI. Subgroup P1: samples with positive results in EIE-LVC kit, RIFI and DPP, from endemic area animals (Teresina, Piauí state, Brazil). From other endemic area (Belo Horizonte, Minas Gerais state, Brazil), subgroup P2 shows samples with positive results in EIE-LVC kit, RIFI and PCR; subgroup P3 shows samples with positive results in EIE-LVC kit and RIFI.

Figure 4. Comparison of ELISA reactivity of canine sera against PQ20.

ELISA was performed in different groups of dogs (N: negative group; P: positive group) Subgroup N1: samples with negative results in EIE-LVC kit and RIFI, from animals born in kennels with wire mesh in a non-endemic area (Ouro Preto, Minas Gerais state, Brazil). From an endemic area (Belo Horizonte, Minas Gerais state, Brazil), subgroup N2 shows samples with negative results in EIE-LVC kit, RIFI and PCR; subgroup N3 shows samples with negative results in EIE-LVC kit and RIFI. Subgroup P1: samples with positive results in EIE-LVC kit, RIFI and DPP, from endemic area animals (Teresina, Piauí state, Brazil). From other endemic area (Belo Horizonte, Minas Gerais state, Brazil), subgroup P2 shows samples with positive results in EIE-LVC kit, RIFI and PCR; subgroup P3 shows samples with positive results in EIE-LVC kit and RIFI.

Figure 5. Positivities of canine sera against PCR, EIE-LVC kit and ELISA-PQs.

Canine sera was obtained in a first analysis (T0), after 12 months and after 18 months, and tested against PCR, EIE-LVC kit and ELISA-PQs.