Periodic Reporting for period 4 - DrySeasonPf (Dry season P. falciparum reservoir)
Periodo di rendicontazione: 2022-07-01 al 2022-12-31
Malaria parasites are transmitted by mosquitoes, causing yearly over 200 million malaria cases and almost half a million deaths of African children under five. Dependent on mosquito for transmission, the parasite faces a challenge during the dry season in the regions where rain seasonality limits vector availability for several months. While malaria cases are restricted to the wet season, clinically silent infections can persist through the dry season and are an important reservoir for transmission. Understanding the mechanisms allowing the parasite to remain undetectable in absence of mosquito vector, and to restart transmission in the ensuing rainy season will reveal complex interactions between malaria parasites and its host, and may inform public health strategies.
I proposed to: (i) Identify the signalling pathways and metabolic profile of malaria parasites associated with long-term maintenance of low parasitaemias during the dry season, (ii) Determine which PfEMP1 are expressed by parasites during the dry season and how effectively they are detected by the immune system, and (iii) Investigate the kinetics of P. falciparum sexual stages, their ability to transmit during the dry season, and uncover sensing molecules and mechanisms of the disappearance and return of the mosquito vector.
I proposed to: (i) Identify the signalling pathways and metabolic profile of malaria parasites associated with long-term maintenance of low parasitaemias during the dry season, (ii) Determine which PfEMP1 are expressed by parasites during the dry season and how effectively they are detected by the immune system, and (iii) Investigate the kinetics of P. falciparum sexual stages, their ability to transmit during the dry season, and uncover sensing molecules and mechanisms of the disappearance and return of the mosquito vector.
We confirmed that a proportion of children carries P. falciparum during the dry season without presenting symptoms, while clinical cases occur in the wet season.
Aim i) We profiled genetic diversity and gene expression of P. falciparum, as well as the parasite’s ability to replicate, and produce sexual forms. After excluding that host immunity is actively detecting and attacking the infected erythrocytes, we investigated how efficiently P. falciparum replication occurs in the dry vs transmission seasons. In Mali, we cultured parasites after blood collection form subclinical children carrying infections at the beginning, mid and end of the dry season, or at their first clinical malaria episode in the wet season. Parasites during the dry season were not impaired in their replicative ability, and seemed instead to be reaching the end of a 48h replicative cycle faster than parasites collected from malaria-causing infections in the transmission season. We then measured development of parasites collected straight from the arm of children in the dry season and in malaria cases in the transmission season.
We have analysed the metabolomic and proteomic analysis of plasma samples of the dry season vs malaria cases in the wet season and vs asymptomatic in the wet season and found that plasma composition is unrelated to time of year in non-infected children, and that carrying P. falciparum only minimally affects plasma constitution in asymptomatic hosts.
We have determined the ability of different antimalaria drugs to kill dry and wet season parasistes in vitro. We tested their in vitro susceptibility to existing and under-development antimalarials, and found that all parasites tested were susceptible to the tested drugs.
Aim ii) expression of var genes was analysed in blood samples collected in Mali during the dry and wet seasons. DNA and RNA was extracted and the samples were amplified by PCR using degenerate primers designed to target var gene sequences of interest. Amplified products were then sent for sequencing. We used a recently developed analytical pipeline to detect motifs identifying PfEMP1 coding genes in most of our analysed samples, and were able to annotate full-length var genes, but we did not see statistically significant enrichments.
Aim iii) with a multi-color qRT-PCR we showd that sexual commitment transcripts were unaffected throughout the year or clinical presentation, but clinical malaria cases in the wet season had higher gametocyte densities than infections maintained asymptomatic during the dry season, while the proportion of gametocytes relative to total parasite load was higher in dry season. Lysophosphatidylcholine (Lyso-PC) variations detected throughout the year were not sufficient to impose differences in gametocytogenesis in vitro.
We adapted a within-host mathematical model to infer the dynamics of gametocyte number and proportion as infections progress. We introduced increased clearance of infected erythrocytes in persisting parasitaemias compared to recently transmitted parasites. Keeping a constant sexual commitment rate throughout time, our model predicted higher number and lower proportion of gametocytes in the early acute phase of infection, but this trend was inverted in the chronic phase, when parasitaemias remain constant or are slowly decreasing.
Aim i) We profiled genetic diversity and gene expression of P. falciparum, as well as the parasite’s ability to replicate, and produce sexual forms. After excluding that host immunity is actively detecting and attacking the infected erythrocytes, we investigated how efficiently P. falciparum replication occurs in the dry vs transmission seasons. In Mali, we cultured parasites after blood collection form subclinical children carrying infections at the beginning, mid and end of the dry season, or at their first clinical malaria episode in the wet season. Parasites during the dry season were not impaired in their replicative ability, and seemed instead to be reaching the end of a 48h replicative cycle faster than parasites collected from malaria-causing infections in the transmission season. We then measured development of parasites collected straight from the arm of children in the dry season and in malaria cases in the transmission season.
We have analysed the metabolomic and proteomic analysis of plasma samples of the dry season vs malaria cases in the wet season and vs asymptomatic in the wet season and found that plasma composition is unrelated to time of year in non-infected children, and that carrying P. falciparum only minimally affects plasma constitution in asymptomatic hosts.
We have determined the ability of different antimalaria drugs to kill dry and wet season parasistes in vitro. We tested their in vitro susceptibility to existing and under-development antimalarials, and found that all parasites tested were susceptible to the tested drugs.
Aim ii) expression of var genes was analysed in blood samples collected in Mali during the dry and wet seasons. DNA and RNA was extracted and the samples were amplified by PCR using degenerate primers designed to target var gene sequences of interest. Amplified products were then sent for sequencing. We used a recently developed analytical pipeline to detect motifs identifying PfEMP1 coding genes in most of our analysed samples, and were able to annotate full-length var genes, but we did not see statistically significant enrichments.
Aim iii) with a multi-color qRT-PCR we showd that sexual commitment transcripts were unaffected throughout the year or clinical presentation, but clinical malaria cases in the wet season had higher gametocyte densities than infections maintained asymptomatic during the dry season, while the proportion of gametocytes relative to total parasite load was higher in dry season. Lysophosphatidylcholine (Lyso-PC) variations detected throughout the year were not sufficient to impose differences in gametocytogenesis in vitro.
We adapted a within-host mathematical model to infer the dynamics of gametocyte number and proportion as infections progress. We introduced increased clearance of infected erythrocytes in persisting parasitaemias compared to recently transmitted parasites. Keeping a constant sexual commitment rate throughout time, our model predicted higher number and lower proportion of gametocytes in the early acute phase of infection, but this trend was inverted in the chronic phase, when parasitaemias remain constant or are slowly decreasing.
With samples from Malians exposed to alternating 6-month dry and wet seasons, we show that parasites persist in the blood of asymptomatic individuals during the dry season, rarely causing symptoms and minimally affecting host immunity. Parasites isolated during the dry season were transcriptionally distinct from those of subjects with febrile malaria in the wet season, and that the differences reflected longer circulation within each 48h intra-erythrocytic cycle.
We determined antibody reactivity to PfEMP1, and the expression of var genes coding for PfEMP1. More contigs of var gene fragments were seen in the wet season. Although we did not see statistically significant enrichment, we observed a trend that the top expressed var gene in the wet season are more highly expressed, and we plan further studies with larger samples sizes.
Circulation of young forms of the parasite is a hallmark of falciparum malaria, as older infected erythrocytes adhere to vascular endothelium to evade clearance by the spleen. We hypothesized that longer circulation of parasites in blood during the dry season enhances splenic clearance of infected erythrocytes, enabling infections to persist. We show using a spleen-mimicking filtration system that circulating parasites in the dry season are at higher risk of clearance by the spleen. When modelled mathematically, the removal of poorly adherent parasites by the spleen limits parasite growth within the host, thereby promoting the low-level persistent parasitaemia observed during the dry season. (Andrade et al. 2020 PMID: 33106664).
We bioinformatically re-analysed published studies comparing parasite transcription of lower vs. higher parasitaemias and milder vs. severe malaria and found signatures of more developed parasites, linked to lower parasite burdens and milder malaria forms. This highlights for the first time cytoadhesion efficiency of infected erythrocytes as a major force driving prognosis of malaria. (Thomson-Luque et al. 2021 PMID: 34330920).
We questioned whether sensing the time of year, or duration of time since infection drives persistence of P. falciparum infection. We tested the response of parasites collected longitudinally to potential cues present in plasmas obtained during the dry and the wet seasons. We tracked individual parasite clones over time and related them with clinical presentation. Plasma composition did not vary significantly between uninfected and asymptomatic individuals. Parasites from the dry or the wet season were not differentially affected by dry or wet season plasma supplementation in vitro. Instead, we found that length of infection determines the reservoir composition, and that time of transmission determined whether parasites will survive the dry season. (Andrade & Carrasquilla et. al under review).
We determined antibody reactivity to PfEMP1, and the expression of var genes coding for PfEMP1. More contigs of var gene fragments were seen in the wet season. Although we did not see statistically significant enrichment, we observed a trend that the top expressed var gene in the wet season are more highly expressed, and we plan further studies with larger samples sizes.
Circulation of young forms of the parasite is a hallmark of falciparum malaria, as older infected erythrocytes adhere to vascular endothelium to evade clearance by the spleen. We hypothesized that longer circulation of parasites in blood during the dry season enhances splenic clearance of infected erythrocytes, enabling infections to persist. We show using a spleen-mimicking filtration system that circulating parasites in the dry season are at higher risk of clearance by the spleen. When modelled mathematically, the removal of poorly adherent parasites by the spleen limits parasite growth within the host, thereby promoting the low-level persistent parasitaemia observed during the dry season. (Andrade et al. 2020 PMID: 33106664).
We bioinformatically re-analysed published studies comparing parasite transcription of lower vs. higher parasitaemias and milder vs. severe malaria and found signatures of more developed parasites, linked to lower parasite burdens and milder malaria forms. This highlights for the first time cytoadhesion efficiency of infected erythrocytes as a major force driving prognosis of malaria. (Thomson-Luque et al. 2021 PMID: 34330920).
We questioned whether sensing the time of year, or duration of time since infection drives persistence of P. falciparum infection. We tested the response of parasites collected longitudinally to potential cues present in plasmas obtained during the dry and the wet seasons. We tracked individual parasite clones over time and related them with clinical presentation. Plasma composition did not vary significantly between uninfected and asymptomatic individuals. Parasites from the dry or the wet season were not differentially affected by dry or wet season plasma supplementation in vitro. Instead, we found that length of infection determines the reservoir composition, and that time of transmission determined whether parasites will survive the dry season. (Andrade & Carrasquilla et. al under review).