Ex Vivo Immunogenicity Screening of Vaccine Candidate Antigen Combinations in Ethiopian Patients With Leishmaniasis

Ex Vivo Immunogenicity Screening of Vaccine Candidate Antigen Combinations in Ethiopian Patients With Visceral and Cutaneous Leishmaniasis

This study assesses the immunoprevalence (presence of the induced T-cell response across different patients (and thus HLA types) of six prioritized IPX-derived Leishmania antigens, using Good Laboratory Practice-grade soluble Leishmania antigen (GLP-SLA) as a positive control. Longitudinal GLP-SLA stimulation validates its IFN-γ release assay performance to support licensing of a QuantiFERON-like test (Leish-IGRA) for treatment monitoring. IPX-derived Leishmania antigen screening focuses on day 0 and EOT, while GLP-SLA includes all timepoints. Induced T-cell responses by ex vivo stimulation of blood and tissue samples (lesion, bone marrow, or spleen) from patients with cutaneous and visceral leishmaniasis (two-centre cohort) before and after treatment will be verified. Samples from Ethiopian 'non-infected' healthy volunteers will be included in parallel to differentiate from Leishmania antigen-specific versus aspecific T-cell activation. The induced T-cell response will mainly be determined by the expressed IFN-γ levels after stimulation. Additional analyses are included to further characterize the activation and cytokine profiles of these IPX-derived Leishmania antigen-specific T-cells, while the breadth of the T-cell response will be determined by mapping the response across different patients (and thus different HLA types).

1. VL and CL patients

   INCLUSION CRITERIA:

   • Aged 18-65 years To minimize variability within this first study to verify induced T-cell responses specifically towards vaccine candidate target antigens/peptide pools, we focus on more robust adult immune responses. Children and elderly are vulnerable populations with divergent immune responses and are therefore excluded.

   • Suspected diagnosis of VL or CL, defined as:

     • VL: Clinically suspected VL presentation (e.g., prolonged fever, splenomegaly)
     • CL: Clinically suspected lesions (e.g., nodular, ulcerative, or plaque-like lesions) Including suspected VL and CL patients was done in earlier VL and CL studies (Clinicaltrials.gov Identifier: NCT05602610 and NCT05332093, >95% of suspected cohort confirmed to have VL or CL, respectively) to facilitate efficient recruitment flow for both the patient as the study team.

   • Willing and able to provide informed consent Ensures autonomy and understanding, allowing participants to fully understand their risks and benefits, and the possibility to withdraw at any time without affecting care.

   EXCLUSION CRITERIA:

   • Are currently enrolled in another interventional clinical study

   • Have known severe comorbidities (e.g., autoimmune disease, HIV, tuberculosis, leprosy, or malaria)

   • Have known pregnancy

   • Cognitively impaired individuals

   • Have received immunosuppressive drugs in the past month

   • Have received a vaccine in the past month

   • Have received modern antileishmanial treatment in the past month

   • Are on anticoagulation medication or have bleeding disorders that would contraindicate safe blood or tissue sampling

   • For CL patients:

     • Primary lesion size < 2 cm
     • Too difficult or too painful sampling zone (e.g., close to the mucosa or lymphatic system, on joints, eyelid or ear)
     • Not eligible for systemic SSG treatment

2. Ethiopian 'non-infected' healthy volunteers

INCLUSION CRITERIA:

• Aged 18-65 years Matching the age range of VL and CL patients

• Resides in Addis Ababa An uninfected control group is included to distinguish true antigen-specific T-cell responses from non-specific (background) T-cell activation. This control group should ideally consist of healthy individuals without prior symptomatic or asymptomatic exposure to Leishmania, as previous (unknown) asymptomatic infections could lead to detectable Leishmania-specific T-cell responses and thus confound background baseline measurements. To minimize this risk, we will recruit healthy volunteers from 'non-infected' areas, specifically the Addis Ababa region, and apply strict (retrospective) exclusion criteria to ensure absence of prior Leishmania exposure.

• Generally healthy Ensures volunteers who are not in a state of severe medical nor socioeconomic vulnerability, to ensure that participation is not unduly influenced by financial compensation.

• Willing and able to provide informed consent. Ensures autonomy and understanding, allowing participants to fully understand their risks and benefits, and the possibility to withdraw at any time without affecting care.

• Passed autonomy and voluntariness assessment (questions listed below) to further ensure autonomy and understanding.

  • Can you explain in your own words what participation involves and that it is voluntary?
  • Would saying no affect your access to care in any way?
  • Is the compensation offered influencing your decision in a way that makes you feel you must participate?
  • Would not receiving this compensation cause you significant difficulty?

EXCLUSION CRITERIA:

• Have history of contracting VL or CL

• Cohabitate with a person treated for CL/VL within the past 12 months

• Residence or overnight stays in any of these areas within the past 5 years should be considered potentially exposed, even if they reside in the capital.

  • Kebeles in Addis: Saris, Kality, and Akaki
  • Ethiopian highlands: widespread foci for CL in Amhara (South Gondar, North Gondar, South Wollo, North Wollo), Tigray (Wukro, Mekelle surroundings), Oromia (Sebeta, Bale, Jimma), and SNNPR (Silte, Gurage, Sidama highlands).
  • VL endemic lowland/hotspot areas: Afar, Somali Region, Benishangul-Gumuz, Metema-Humera lowlands (Amhara/Tigray border), and South Omo (SNNPR).

• Have known severe comorbidities (e.g., autoimmune disease, HIV, tuberculosis, leprosy, and malaria)

• Have known pregnancy

• Cognitively impaired individuals

• Have received immunosuppressive drugs in the past month

• Have received a vaccine in the past month

• Are on anticoagulation medication or have bleeding disorders that would contraindicate safe blood or tissue sampling

• If their Leish-IGRA demonstrate positive values, the participant will be retrospectively excluded as this indicates the presence of a past Leishmania infection.