Controlled infection with cryopreserved human hookworm induces CTLA-4 expression on Tregs and upregulates tryptophan metabolism

Abstract

Infecting humans with controlled doses of helminths, such as human hookworm (termed hookworm therapy), is proposed to prevent or treat various intestinal and extraintestinal diseases. However, full-scale clinical trials examining hookworm therapy are limited by the inability to scale-up the production of hookworm larvae to infect sufficient numbers of patients. With the aim of overcoming this challenge, this study infected four healthy individuals with hookworm larvae that had been reanimated from cryopreserved eggs to examine their viability and immunogenicity. We demonstrate that reanimated cryopreserved hookworm larvae establish a viable hookworm infection and elicit a similar immune response to larvae cultured from fresh stool. Furthermore, a refined understanding of the therapeutic mechanisms of hookworm is imperative to determine which diseases to target with hookworm therapy. To investigate potential therapeutic mechanisms, this study assessed changes in the immune cells, microbiome, and plasma metabolome in the four healthy individuals infected with cryopreserved hookworm larvae and another nine individuals infected with larvae cultured from freshly obtained stool. We identified potential immunoregulatory mechanisms by which hookworm may provide a beneficial effect on its host, including increased expression of CTLA-4 on regulatory T cells (Tregs) and upregulation of tryptophan metabolism. Furthermore, we found that a participant's baseline microbiome predicted the severity of symptoms and intestinal inflammation experienced during a controlled hookworm infection. In summary, our findings demonstrate the feasibility of full-scale clinical trials examining hookworm therapy by minimizing the reliance on human donors and optimizing the culturing process, thereby enabling viable hookworm larvae to be mass-produced and enabling on-demand inoculation of patients. Furthermore, this study provides insights into the complex interactions between helminths and their host, which could inform the development of novel therapeutic strategies.

Keywords: Hookworm; clinical trial; controlled infection; cryopreservation; eosinophilia; helminths; immune regulation; microbiome.

Figure 1.

A viable and durable controlled hookworm infection is established from reanimated cryopreserved larvae. (a) Schematic representation of the recruitment and eligibility process. Eligibility criteria described in data file S1. (b) Study design with sample collection. (c) Hookworm eggs count (eggs/gram) measured in fresh stool samples in participants that received fresh (red line) or cryopreserved (blue line) larvae. Mean ± SEM. (d) Adult hookworm counted in the gastrointestinal tract with PillCam^TM endoscopy in participants that received fresh (red) and cryopreserved (blue) larvae. Data were analyzed with the unpaired Student’s t-test.

Figure 2.

Hookworm infection induces a systemic and intestinal type 2 immune response (b) mean (±SEM) peripheral blood eosinophil count in participants infected with fresh (red line) or cryopreserved (blue line) larvae. (b) Mean (±SEM) serum IL-5 levels in all participants. (c) Fold change in type 2 cytokines, IL-6 and eotaxin-1 (CCL11) from baseline (day 0= pre-infection) in all participants. (d) Mean (±SEM) faecal eosinophilic cationic protein (ECP) in all participants. (e) mean (±SEM) faecal eosinophil derived neurotoxin (EDN) in all participants. f) mean (±SEM) faecal calprotectin (fCal – red line), neutrophils count (blue line) and human neutrophil lipocalin (HNL) (green line). Data were analysed with one-way ANOVA using Friedman test with Dunn’s multiple comparison test. p= * < 0.05, ** < 0.005, *** < 0.001, **** < 0.0001.

Figure 3.

Gastrointestinal symptomatology may depend on the baseline microbiome (a) changes in Bray-Curtis dissimilarity test for each single participant (gray lines) and mean (red line). (b) Changes in Shannon diversity for each participant (gray lines) and mean (red line). (c) PCA plot showing 16s analysis grouping participants according to symptoms score (no/mild symptoms (blue); moderate symptoms (red)). (d) Box plot showing Shannon diversity according to the severity of symptoms (no/mild symptoms (dark blue) and moderate symptoms (red)). (e) Comparison in the relative abundance of the top 100 most abundant taxa faceted by phylum in participants who experienced no or mild symptoms (symptom score 0) and participants who experience moderate symptoms (symptom score 1).

Figure 4.

Hookworm infection upregulates the immune checkpoint receptor CTLA-4 on tregs. PBMC samples were collected at baseline and weeks 4, 8, 12, 36, and 48. Two flow cytometry panels were used to analyze immune cells using OMIQ software to generate: (a) UMAPs showing the main PBMC populations identified using panel 1 overlaying all timepoints; (b) all timepoints from panel 1 were analysed using FlowSOM. Heatmap showing median FlowSOM cluster marker expression for panel 1 (red=high expression, blue= low expression). According to the marker expression, clusters were assigned a cell population as identified with the colored column on the right side of the heatmap; (c) UMAPs showing the main populations identified using panel 2 overlaying all timepoints; (d) all timepoints from panel 2 were analysed using FlowSOM. Heatmap showing the markers expression of each cluster identified with FlowSOM (red=high expression, blue= low expression). According to the marker expression, clusters were assigned a cell population as identified with the colored column on the right side of the heatmap. Clusters generated by FlowSOM in (b) were analysed for markers expression showing (e) cluster 1 median CTLA-4 expression comparison between week 0 (pre-infection) and week 4 post-infection. (f) UMAP showing the main CD4 T cell population overlapping FlowSOM cluster 1. Histograms showing FoxP3, CD25 and CTLA-4 expression for cluster 1. (g) CTLA-4 MFI of T_REG using expert gating during infection. (h) frequency of CTLA-4⁺ T_REG using expert gating. Analyzed using the paired t-test (e) or one-way ANOVA with mixed-effects analysis with Tukey’s multiple comparison test (g and h). p= *<0.05, **<0.005.

Figure 5.

Hookworm infection upregulates tryptophan metabolism. (a) Top six enriched metabolic pathways identified using MSEA comparing pre-infection vs acute phase (left panel) or chronic phase of infection (right panel). (b) Heatmap of five annotated plasma metabolites which significantly (p < 0.05) changed during the hookworm infection, as determined by mixed-mode, two-way analysis of variance (ANOVA), controlling for ‘participant ID’ and ‘sex’ as covariates.(c) changes in plasma kynurenine (c), melibiose (d), indoxyl sulphate (e), threonic acid (f), 5-methoxypsoralen (f), and tryptophan (h) levels during the hookworm infection. (i) changes in plasma kynurenine/tryptophan ratio, a marker of indoleamine 2,3-dioxygenase (IDO) activity. Analysed with mixed ANOVA with Dunnett’s. Values shown represent log₁₀ transformed (N = 12 participants). p = *< 0.05, **< 0.005, ****< 0.0001.