Phylogenetic Reconstruction and Evolutionary Analysis of Vaccine Candidate Genes in Clinical Plasmodium falciparum Isolates using Nextstrain

Sydney Tola

Mentor: Dr. Alexander Pichugin, Ph.D., M.D., Chief, Genetics and Parasite Biology Lab, BR&D, WRAIR.

Date/Time: August 22nd, 2025 at 10:00 AM.

Abstract: Malaria remains a significant global health challenge, particularly in endemic regions and among deployed military personnel. Controlled Human Malaria Infection (CHMI) studies are the gold standard for testing malaria vaccines and therapeutics, yet the parasite strains currently used in CHMI trials do not reflect the full scope of Plasmodium falciparum diversity circulating worldwide. This gap limits the ability of CHMI studies to predict real-world performance of anti-malaria interventions. The need for diversity in CHMI strains is underscored by efficacy variability observed with World Health Organization–recommended vaccines such as RTS,S and R21, whose protective performance differs geographically in part due to underlying parasite genetic variation. To address this, we analyzed the sequences of 62 monoclonal clinical isolates of the circumsporozoite protein (CSP), the primary surface antigen on sporozoites and a malaria vaccine target of interest. These sequences were sourced from clinical samples collected over a 4 year period from different countries. Using the Nextstrain Augur pipeline and its Auspice visualization platform, we implemented a phylogenetic workflow that included sequence alignment, maximum-likelihood tree construction, and ancestral state reconstruction. This framework enabled us to identify lineage diversification, geographic clustering, clade-defining mutations, and evolutionary events. We further applied entropy-based analyses to quantify site-specific diversity across CSP, highlighting regions of elevated variability that coincide with immune-relevant epitopes. Importantly, domain-specific analyses of the CSP C-terminus revealed sharper geographic clustering compared to full-length sequences, reflecting epitope-focused evolutionary pressures. By integrating phylogenetic reconstruction with entropy metrics in an interactive, reproducible bioinformatics workflow, we provide a scalable approach for evaluating antigen diversity in clinical isolates. These analyses offer a framework for selecting CHMI strains that more accurately reflect circulating P. falciparum diversity, ultimately supporting CHMI trials for development of anti-malaria biologics. Beyond malaria, this work demonstrates the utility of modern phylogenetic platforms, like Nextstrain Augur and Auspice, for linking pathogen evolution to translational research needs.