Urinary Metabolic Profiling in Volunteers Undergoing Malaria Challenge in Gabon

The interaction of malaria parasites with their human host is extensively studied, yet only few studies reported how P. falciparum infection affects urinary metabolite profiles and how this is associated with immunity. We present a longitudinal study of the urinary metabolic profiles of twenty healthy Africans with lifelong exposure to malaria and five malaria-naïve Europeans, who were all challenged with direct venous inoculation of live P. falciparum sporozoïtes (PfSPZ) and followed up until they developed symptoms or became thick blood smear positive (TBS). Urine samples were collected before and at 2, 5, 9 and 11 days post challenge and were analysed. Upon infection, all Europeans became TBS positive, while Africans showed either a delay in time to parasitaemia or controlled infection. Our metabolic data showed that Europeans and Africans had distinct alterations in metabolite patterns, with changes mostly seen on days 5 and 9 post PfSPZ infection, and more prominently in Europeans. Within the African group, the levels of formate, urea, trimethylamine, threonine, choline, myo-inositol and acetate were significantly higher in TBS positive whereas the levels of pyruvate, 3-methylhistidine and dimethylglycine were significantly lower in individuals who remained TBS negative. Notably, before inoculation with PfSPZ, a group of metabolites including phenylacetylglutamine can potentially be used to predict parasitaemia control among Africans. Taken together, this study highlights the difference in urinary metabolic changes in response to malaria infection as a consequence of lifelong exposure to malaria and that change detectable before challenge might predict the control of parasitaemia in malaria-endemic areas

Investigation of urinary metabolomics in a phase I hookworm vaccine trial in Gabon

Metabolomics provides a powerful tool to study physiological changes in response to various perturbations such as vaccination. We explored whether metabolomic changes could be seen after vaccination in a phase I trial where Gabonese adults living either in rural or semi-urban areas received the subunit hookworm vaccine candidates (Na-GST-1 and Na-APR-1 (M74) adjuvanted with Alhydrogel plus GLA-AF (n = 24) or the hepatitis B vaccine (n = 8) as control. Urine samples were collected and assayed using targeted 1H NMR spectroscopy. At baseline, a set of metabolites significantly distinguished rural from semi-urban individuals. The pre- and post-vaccination comparisons indicated significant changes in few metabolites but only one day after the first vaccination. There was no relationship with immunogenicity. In conclusion, in a small phase 1 trial, urinary metabolomics could distinguish volunteers with different environmental exposures and reflected the safety of the vaccines but did not show a relationship to immunogenicity

Immunological profiles associated with distinct parasitemic states in volunteers undergoing malaria challenge in Gabon

Controlled human malaria infection (CHMI) using cryopreserved non-attenuated Plasmodium falciparum sporozoites (PfSPZ) offers a unique opportunity to investigate naturally acquired immunity (NAI). By analyzing blood samples from 5 malaria-naïve European and 20 African adults with lifelong exposure to malaria, before, 5, and 11 days after direct venous inoculation (DVI) with SanariaR PfSPZ Challenge, we assessed the immunological patterns associated with control of microscopic and submicroscopic parasitemia. All (5/5) European individuals developed parasitemia as defined by thick blood smear (TBS), but 40% (8/20) of the African individuals controlled their parasitemia, and therefore remained thick blood smear-negative (TBS− Africans). In the TBS− Africans, we observed higher baseline frequencies of CD4+ T cells producing interferon-gamma (IFNγ) that significantly decreased 5 days after PfSPZ DVI. The TBS− Africans, which represent individuals with either very strong and rapid blood-stage immunity or with immunity to liver stages, were stratified into subjects with sub-microscopic parasitemia (TBS-PCR+) or those with possibly sterilizing immunity (TBS−PCR−). Higher frequencies of IFNγ+TNF+CD8+ γδ T cells at baseline, which later decreased within five days after PfSPZ DVI, were associated with those who remained TBS−PCR−. These findings suggest that naturally acquired immunity is characterized by different cell types that show varying strengths of malaria parasite control. While the high frequencies of antigen responsive IFNγ+CD4+ T cells in peripheral blood keep the blood-stage parasites to a sub-microscopic level, it is the IFNγ+TNF+CD8+ γδ T cells that are associated with either immunity to the liver-stage, or rapid elimination of blood-stage parasites

An exploratory analysis of the entire dataset.

A, B C, D and E show the score plots of PCA model colored according to time of the sampling, type of vaccine used, area of residence (location = rural or semi-urban), gender (male and female) and anthelmintic pre-treatment (yes or no) respectively. PCA model built for the two first component cover 17% of the variance and 10 components was required to cover 50% of the variance. (TIF)</p

Metabolites that changed at day 1 post vaccination.

A, B, C, D and E show plots of metabolites that changed significantly at day 1 post vaccination. Y axis corresponds to metabolite log transformed concentration and X axis to timepoint. Dot and line are colored according to vaccine type (control = Hepatitis B, low = low dose HOOKVAC and high = high dose of HOOKVAC). Horizontal lines indicate 0.25, 0.50, 0.75 quantiles. HOOKVAC: Hookworm vaccine candidates comprising of Na-GST-1 and Na-APR-1 (M74).</p

Exploratory analysis of the urine samples of volunteers at baseline.

A, B and C show the score plots of PCA model according to sex (male and female), anthelmintic pre-treatment (yes or no) and area of residence (location = rural or semi-urban) respectively. PCA model built for the two first component cover 26% of the variance and 6 components were required to cover 50% of the variance. D- is a cross-validated score plot of 2 class PLS-DA model with location as class identity. Model metrics: R2X = 0.232, R2Y = 0.81, Q2 = 0.40.</p

An exploratory analysis of metabolic response to vaccination on the first (A, B) and the third (C, D) vaccination rounds.

The score plots of the PCA models are colored according to time of the sampling (A, C) type of vaccine used (B, D). 17% of the variance is covered by the first 2 components and 8 components are needed to explain 50% of the variance. (TIF)</p