Safety and reactogenicity of a controlled human infection model of sand fly-transmitted cutaneous leishmaniasis

Leishmaniasis, a globally significant parasitic disease, lacks a human vaccine. Cutaneous leishmaniasis (CL), caused by various *Leishmania* species, affects over 200,000 individuals annually. CL caused by *L. major* typically self-resolves, but scarring can be lifelong. Other species cause more chronic or disseminated disease. Visceral leishmaniasis (VL) is life-threatening. Due to the limitations of vector control, vaccination is crucial. While the potential of a vaccine is recognized, few *Leishmania* vaccine candidates have progressed to clinical trials. Controlled human infection models (CHIMs) offer a direct way to address these challenges. CHIMs are established for other diseases, and while deliberate human infection with *Leishmania* (leishmanization) has historical precedent, it lacked consideration of vector-associated immune modulation. This study presents a CL CHIM incorporating natural sand fly transmission to determine the take rate and safety, providing insights into early human CL immune responses.

The literature extensively documents the global burden of leishmaniasis, highlighting its socio-economic impact and the urgent need for effective vaccines. Numerous animal studies have explored potential vaccine candidates, but few have advanced to clinical trials. The importance of vector-associated immune modulation in CL pathogenesis and the limitations of needle challenge models for vaccine development have been increasingly recognized. Existing literature emphasizes the lack of comprehensive understanding of human immune responses to *L. major* infection, especially during the early stages of lesion development. The present study addresses this gap by incorporating natural sand fly transmission in its CHIM approach.

This open-label observational study (LEISH_Challenge, NCT04512742) enrolled 14 healthy, *Leishmania*-naive volunteers (8 female, 6 male, median age 32). Participants were exposed to five *L. major*-infected *P. duboscqi* sand flies for 30 minutes using a biting chamber. Cohort 1 (n=6) used a 6-mm chamber aperture, while Cohort 2 (n=8) used smaller apertures (3-5 mm) for potentially reduced scarring. Participants were considered for therapeutic biopsy upon observing a 2-3 mm lesion. Lesions were confirmed by qPCR and/or IHC. Participants were followed for 12 months. Scarring was assessed. Safety was monitored using blood tests, physical examinations, and visual analogue scores (VAS) for symptoms. Lesion biopsies were analyzed by H&E staining, IHC for immune cell markers (CD4, CD8, CD68, CD14, CD20, CD66b), and qPCR for parasite load. Spatial transcriptomics (Visium) was used on selected biopsies to map immune cell distribution and cytokine/chemokine expression.

The study demonstrated a 64% overall take rate for CL development (9/14 participants), with higher rates in Cohort 1 (83%). Lesion size and parasite load varied considerably. Three participants experienced lesion recurrences (4-8 months post-biopsy), successfully treated with cryotherapy. No serious adverse events were reported. Scarring was evident in all biopsied participants; severity was influenced by biopsy type and wound infection. Immunohistochemical analysis revealed variable ratios of CD4+ and CD8+ T cells, with a positive correlation between the CD8:CD4 ratio and lesion duration. Macrophages were predominantly parasitized. Spatial transcriptomics identified distinct immune niches within the lesion, with the core enriched for myeloid cells, T cells, interferon-inducible genes, and effector/regulatory cytokines. The ulcer showed a different gene signature related to epidermal remodeling. The analysis revealed the functional compartmentalization and heterogeneity of immune responses within the lesion core.

This study established a safe and effective CHIM for sand fly-transmitted CL, with a take rate comparable to other CHIMs. The model's efficiency, coupled with the rapid lesion development, makes it cost-effective for vaccine evaluation. The high variability in parasite load highlights the complexity of vector transmission and inter-individual immune responses. The findings on scarring suggest that minimizing lesion size and early biopsy improves cosmesis. The detailed immunohistochemical and spatial transcriptomic analyses revealed distinct immune microenvironments within the lesion, providing novel insights into the pathogenesis of CL. These data offer a blueprint for identifying correlates of protection and pathology, informing the development of vaccines and therapies.

This study successfully established a safe and effective CHIM for sand fly-transmitted *L. major* CL. The model's high take rate and rapid lesion development make it suitable for efficient vaccine and therapeutic evaluations. Detailed immune profiling revealed distinct immune niches within the lesion, highlighting the complexity of host-parasite interactions. Future research should explore the potential of this model in diverse populations and investigate the mechanisms of scar formation.

The study population consisted predominantly of White individuals, limiting the generalizability of findings to other ethnic groups. The relatively small sample size and the focus on early lesion development restrict the comprehensive analysis of long-term outcomes. The observation of potential steroid-induced recurrence warrants further investigation. The absence of a control group makes it challenging to definitively distinguish scarring due to CL from that associated with the biopsy procedure.