Graduation Year
2018
Document Type
Dissertation
Degree
Ph.D.
Degree Name
Doctor of Philosophy (Ph.D.)
Degree Granting Department
Global Health
Major Professor
John H. Adams, Ph.D.
Committee Member
Dennis E. Kyle, Ph.D.
Committee Member
Rays H.Y. Jiang, Ph.D.
Committee Member
Photini Sinnis, M.D.
Keywords
Liver, Plasmodium, RNA-seq, Screening, Sporozoite
Abstract
The malaria pre-erythrocytic stages have been identified as an ideal therapeutic target, but complex in vitro models for Plasmodium vivax and Plasmodium falciparum lack the efficiency needed for rapid screening and evaluation of new vaccines and drugs, especially targeting the P. vivax hypnozoite. To address this challenge, we employed a multi-parameter approach using “omics’” to identify pre-erythrocytic targets and biomarkers, guide phenotypic therapeutic screening, and study parasite functionality with innovative bioassays using highcontent screening. Herein, we discuss three novel bioassays formatted in 384-well plate systems with utilization of commercially-available materials and application of high-content imaging for rapid bio-image analysis. To refine functional assessment of pre-erythrocytic targets in early infection phases, we developed a real-time, ‘live’ sporozoite motility assay and a live sporozoite hepatocyte cell traversal assay to examine chemotherapeutic and immunoprophylactic interventions in biologically relevant environments. Furthermore, our 384-well primary hepatocyte culture system and methodology maintains stable hepatocyte physiology of cryopreserved primary human hepatocytes in addition to primary non-human primate hepatocytes for greater than 30 days, thus ideal for robust liver parasite development following infection with P. vivax, P. falciparum or P. cynomolgi sporozoites. We report antimalarial drug and vaccine studies performed in all bioassays with identification of novel anti-LS inhibition mechanisms. Additionally, this research discusses the discovery of potential sporozoite and liver stage targets identified through transcriptomic profiling of freshly isolated P. vivax and P. cynomolgi sporozoites using a candid approach of recapitulating the pivotal transition period from mosquito to human through microenvironment reconstruction and exposure to biological stimuli. We further characterize sporozoite invasive phenotypes through the application of the bioassays. Together, these novel functional assays enable us to rapidly evaluate potential preerythrocytic therapeutic candidates and analyze complex Plasmodium sporozoite phenotypes.
Scholar Commons Citation
Roth, Alison E., "A Multivariate Approach for an Improved Assessment of Pre-erythrocytic Stage Therapies Targeting Plasmodium vivax and Plasmodium falciparum" (2018). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/7641
