Characterization of a novel Leishmania antigen containing a repetitive domain and its potential use as a prophylactic and therapeutic vaccine.

Human visceral leishmaniasis (HVL) is the second most lethal tropical parasitic disease. Currently, no prophylactic or therapeutic vaccines exist for HVL. Thus, the development of an efficacious vaccine is still needed. We previously performed an immunoproteomics analysis on Leishmania amazonensis parasite extracts to identify immunodominant antigens recognized by the sera of vaccinated and protected mice. Among the identified antigens, we discovered a novel, previously unstudied repetitive protein, initially annotated in Leishmania genomes as a kinetoplast-associated protein-like protein from Leishmania infantum (LinKAP), containing conserved domains (trichohyalin-plectin-homology [TPH] and TolA) that are associated with other mitochondrial proteins. LinKAP sequences are conserved across trypanosomatids, including Endotrypanum, Leishmania, and Trypanosoma species. Using differential centrifugation of Leishmania subcellular structures, we showed that LinKAP was enriched in fractions colocalizing with other mitochondrial proteins. mNeonGreen labeling at the endogenous locus using CRISPR-Cas9 and confocal microscopy confirmed that LinKAP is a mitochondrial-associated protein in Leishmania but not specifically colocalized with kDNA. We cloned and expressed a truncated version of LinKAP (rLinKAP), containing part (15) of the several LinKAP amino acid repeats, demonstrating over 85% homology across L. infantum, L. amazonensis, L. braziliensis, and L. mexicana species. An adjuvanted formulation of LinKAP with Poly ICLC, a polyinosinic-polycytidylic acid (Poly I:C) stabilized with carboxymethylcellulose and polylysine, was used to vaccinate mice and hamsters as a prophylactic vaccine for visceral leishmaniasis. Animals immunized with rLinKAP showed a potent cellular and humoral response and a significant decrease in tissue parasitism when challenged with L. infantum. We also tested rLinKAP as a therapeutic vaccine in mice. Following therapeutic vaccination, antibody responses were enhanced, and cellular responses became apparent. Our treatment protocol inhibited splenic parasite burden by 75% in treated mice. In conclusion, our antigen discovery strategy and the observed protective effect highlight rLinKAP as a promising vaccine candidate for leishmaniasis. IMPORTANCE: A previous reverse vaccinology study identified kinetoplast-associated protein-like protein from Leishmania infantum (LinKAP) as a potential new vaccine target, as this protein was recognized by the sera of protected mice in extracts of Leishmania promastigotes. Interestingly, LinKAP is a repetitive protein containing trichohyalin-plectin-homology (TPH) and TolA domains and was initially annotated as a kinetoplast-associated protein. We further characterized LinKAP as a mitochondrial-associated protein highly conserved among trypanosomatids. We also validated LinKAP as a promising vaccine antigen by using a truncated version of LinKAP (rLinKAP) as both a prophylactic and therapeutic vaccine, adjuvanted with Poly ICLC, to immunize animals against visceral leishmaniasis (VL). This disease, caused by the Leishmania parasite, affects several populations globally and still lacks highly effective vaccines. Identifying LinKAP and its preliminary characterization also provides new perspectives for studying its role in the parasite's biology.