MSACL 2022 Abstract

Introduction: Artemisinin resistance is a primary challenge in the control of malaria. The primary goal of this study is to determine the proteomic signature that is correlative to parasite burden. To address this question, the investigators use the induced blood-stage malaria (IBSM) model, whereby healthy participants are intravenously inoculated with blood-stage parasites either P. falciparum ART-R or artemisinin-sensitive (ART-S) parasites.

Objective: The two aims of this study are to identify the underlying plasma proteomic signatures between 1) individuals infected with either 3D7 (drug responsive malaria) or K13 (artemisinin-resistant parasite line, drug resistant) Malaria and 2) any differential response to drug treatment over time.

Methods: The plasma samples were analyzed by liquid chromatography-mass spectrometry (LC-MS) using the Data Independent Acquisition (DIA) strategy that efficiently provides deep plasma protein coverage. Mid-throughput analysis scheme was used for this study using 60-minute gradient separation following DIA method with 50 isolation windows (12Da) which was previously optimized for blood and plasma samples. Sample preparation was automated using a Beckman i7 robot. QC samples were included in each batch of sample analysis to track performance of the workflow. The MS spectra were analyzed using OpenSwath and MapDIA pipelines, running on our in-house ProEpicTM software platform to identify human host protein changes during different stages of infection.

Results: The plasma samples obtained from 22 individual at the 4 times were analyzed by LC-MS DIA method. The time points were baseline (day 0), five and seven days after infection (day5, day7) height of infection and start of treatment (day 9 +0h),16 hours after first round of treatment, completion of first round of drug treatment (day 12 +72h) and the last time point (washout) for each volunteer. Gene ontology analysis for both strains points out to IGF-1 transport, platelet activation & signaling and complement coagulation pathways. Drug responsive 3D7 strain shows changes in plasma lipoprotein remodeling and fibrin clotting pathways. Drug resistant K13 strain shows changes in extracellular matrix organization and fibrin clotting pathways. The most significant changes were between drug responsive and drug resistant groups after 84 hours of drug treatment with lipopolysaccharide binding protein (LBP) upregulated in drug resistant group. Contrary to the expectation that there will be more differentially expressed proteins in day 9 after infection which is the height of infection there were significantly more differentially expressed proteins at Day7 compared to baseline. DEAD-box helicase 6 protein (DDX6) was the highly expressed one in Day7 compared to baseline. Comparison of day9 post infection proteome analysis reveals that the Lympocyte cytosolic protein 1 (PLSL) and mannosidase alpha class2A member 1(MA2A1) proteins are upregulated in drug resistant strain infected individuals. More analysis is underway to look at different pathways and protein networks playing a role in drug resistance in malaria. Initial analysis of the acquired data against a Malaria spectral library is very promising with some parasite proteins detected in human plasma. The results from this study might lead way to biomarker discovery for identification of drug resistant malaria diagnosis in vulnerable population.