The increased presence of repetitive motifs in the KDDR-plus recombinant protein, a kinesin-derived antigen from Leishmania infantum, improves the diagnostic performance of serological tests for human and canine visceral leishmaniasis

Abstract

Visceral leishmaniasis (VL) is caused by protozoa belonging to the Leishmania donovani complex and is considered the most serious and fatal form among the different types of leishmaniasis, if not early diagnosed and treated. Among the measures of disease control stand out the management of infected dogs and the early diagnosis and appropriate treatment of human cases. Several antigens have been characterized for use in the VL diagnosis, among them are the recombinant kinesin-derived antigens from L. infantum, as rK39 and rKDDR. The main difference between these antigens is the size of the non-repetitive kinesin region and the number of repetitions of the 39 amino acid degenerate motif (6.5 and 8.5 repeats in rK39 and rKDDR, respectively). This repetitive region has a high antigenicity score. To evaluate the effect of increasing the number of repeats on diagnostic performance, we designed the rKDDR-plus antigen, containing 15.3 repeats of the 39 amino acid degenerate motif, besides the absence of the non-repetitive portion from L. infantum kinesin. Its performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and rapid immunochromatographic test (ICT), and compared with the kinesin-derived antigens (rKDDR and rK39). In ELISA with human sera, all recombinant antigens had a sensitivity of 98%, whereas the specificity for rKDDR-plus, rKDDR and rK39 was 100%, 96% and 71%, respectively. When evaluated canine sera, the ELISA sensitivity was 97% for all antigens, and the specificity for rKDDR-plus, rKDDR and rK39 was 98%, 91% and 83%, respectively. Evaluation of the ICT/rKDDR-plus, using human sera, showed greater diagnostic sensitivity (90%) and specificity (100%), when compared to the IT LEISH (79% and 98%, respectively), which is based on the rK39 antigen. These results suggest that the increased presence of repetitive motifs in the rKDDR-plus protein improves the diagnostic performance of serological tests by increasing the specificity and accuracy of the diagnosis.

Visceral leishmaniasis in a recent transmission region: 27.4% infectivity rate among seronegative dogs

“…Siqueira et al . (2021) also evaluated the effectiveness of an IEA with the rKDDR-plus protein in the diagnosis of CVL. The selected dogs were not evaluated for clinical status.…”

“…Better results were obtained in the group presenting with clinical signs; however, the agreement was still considered moderate. Siqueira et al (2021) also evaluated the effectiveness of an IEA with the rKDDR-plus protein in the diagnosis of CVL. The selected dogs were not evaluated for clinical status.…”

“…Currently, both involve the use of recombinant K28 proteins derived from proteins of the kinesin family (Souza-Filho et al ., 2016). In 2014, a recombinant protein, rKDDR (kinesin degenerated derived repeat), was developed; its optimized version, called rKDDR-plus, consists of approximately 15 repetitive motifs of 39 amino acids of the kinesin protein, increasing the immunogenic potential of the original (Dhom-Lemos et al ., 2019; Siqueira et al ., 2021).…”

Subcutaneous, Oral, and Intranasal Immunization of BALB/c Mice with Leishmania infantum K39 Antigen Induces Non-Protective Humoral Immune Response

“…The K39 antigen comprises a sequence of 39 amino acid residues arranged in tandem within the C-terminal region of this motor protein. The repetitive structure of this antigen renders it a potential immunogen capable of inciting an adaptive humoral response [ 14 ].…”

Use of Antigen Combinations to Address Complex Leishmania-Seropositivity Patterns in Dogs Living in Canine Leishmaniosis Endemic Regions of Portugal

“…Crude promastigote antigens, the SPLA, have been described as highly sensitive for subclinical and clinical infections, but with a lower specificity [ 25 , 26 ]. The rK39, rK28, and rKDDR are all kinesin-derived recombinant proteins with proven value for the serodiagnosis of CanL [ 27 , 28 ]. LicTXNPx is a recombinant protein that demonstrated an increased capacity to detect subclinical infections [ 20 , 29 ].…”