Objectives and expected results:
(1) To apply comparative genomics of leishmania for discovery of candidate improved diagnostic antigens and to synthesise peptides or clone recombinants for trials in serological assays.
(2) To examine immune response/metabolomic profiles in different clinical presentations as a potential means of providing staging or prognostic biomarkers.
Background: Highly sensitive, specific rapid diagnostics are required to indicate prognosis of asymptomatic Leishmania infections and distinguish cure from relapse. LSHTM and KAROLINSKA have a genomics strategy to select new candidate diagnostic antigens, in conjunction with new algorithms of APTUM Biologics to predict epitopes/antigenicity
Methodology: Genome sequences will be searched systematically in silico for motifs predictive of diagnostic antigens specific to the Leishmania donovani complex; conservation of candidate antigens will be confirmed by PCR amplification of multiple strains. Peptides corresponding with predicted epitopes/antigenicity will be synthesised, and/or regions of interest cloned to generate diagnostic recombinant proteins. The L. tarentolae expression system may be applied to produce glycosylated recombinants. Levels of antibody titre to the new diagnostic antigens in disparate endemic regions will be compared. Comparative analysis of antibody recognition profiles and mass spectrometry will be applied to sera from patients with different clinical status to search for biomarkers of clinical prognosis and post-treatment cure versus relapse. Selected diagnostic peptides, recombinant antigens, and serological biomarkers of clinical progression, will be tested in lateral flow rapid diagnostic tests (RDTs) with whole blood, plasma and serum.