An observational study to test the potency of GLP-grade phenol-free lyophilised leishmanin antigen formulation.

Abstract

Leishmaniasis is a vector-borne disease transmitted through sand fly vectors carrying protozoan parasites from the genus Leishmania. The leishmanin skin test (LST), used to detect exposure to Leishmania parasites, is no longer available due to a lack of well-characterised antigens. Towards reintroducing the LST, we previously developed a preparation of the leishmanin antigens using a freeze-thaw (FT) process, which induced a delayed-type hypersensitivity (DTH) response in hosts exposed to Leishmania parasites. The storage of FT preparation antigens requires refrigeration, which is not practical for use in field settings. We developed a safe, simple and scalable antigen extraction process and a phenol-free lyophilised-LST antigen formulation that obviates the need for a cold-chain under Good Laboratory Practice conditions (GLP-LST^Lyo). The immunogenicity of GLP-LST^Lyo was evaluated in murine models of Leishmania infections in support of two distinct use cases: (i) to detect latent infections of Leishmania in endemic areas and (ii) as a surrogate of vaccine-induced immunogenicity. Towards this goal, leishmanisation with wild-type Leishmania major and immunisation of mice with the live-attenuated L. major vaccine strain lacking the centrin gene (LmCen^-/-) were used as animal models. The GLP-LST^Lyo antigen was administered, and its ability to induce a DTH response was monitored for 48 h. Additionally, the potency of the GLP-LST^Lyo was assessed by immune phenotyping of the cells isolated from the DTH tissue via flow cytometry. Only the DTH sites from pre-exposed animals, but not the excipient controls, showed enrichment for predominantly CD8⁺ T cells, along with CD4⁺ T cells and macrophages, consistent with previous studies. Additionally, in an interferon gamma release assay (IGRA), stimulation of whole blood collected from healed, active cutaneous leishmaniasis (CL) cases in a Leishmania endemic area in Brazil with GLP-LST^Lyo induced IFN-γ and IL-10. These results demonstrated the potency of our formulation in IGRA studies, which could aid immunogenicity studies in vaccine trials, and surveillance studies in both endemic and emerging areas of Leishmania infection. These studies are supported by funding from GHIT Fund, Japan, CIHR, Canada, intramural funding from the FDA, and FIOCRUZ and INCT-DT Brazil.