Date of Award

5-31-2017

Document Type

Campus Access Thesis

Degree Name

Master of Science (MS)

Department

Biotechnology and Biomedical Science

First Advisor

Greg Beck

Second Advisor

Colby A. Souders

Third Advisor

Kellee Siegfried-Harris

Abstract

Even more than a century after the discovery of the most severe malaria-causing parasite, Plasmodium falciparum, the disease still kills nearly 1 million people every year, many of which are children. Attempts to prevent infection and curb the prevalence of the disease have reduced infection rates in recent years; however, these interventions don’t prove to be reliable, long-term, prophylactic strategies. Resistance is developing to antimalarial drugs and a recently developed vaccine, RTS,S, only exhibited modest efficacy in clinical trials. Disease burden is enormous in endemic regions of the world. Consequently, a need for a pre-exposure prophylaxis still exists for travelers and residents in these areas of the world. Data from vaccine trials and individuals with acquired immunity suggest that a monoclonal antibody could prove to be a potent protection strategy against malaria. Circumsporozoite Protein (CSP), a plasmodium surface protein expressed during initial infection, was selected as a target for monoclonal antibody discovery and produced in a soluble form. Here I describe a flow cytometry-based methodology for identifying, isolating and genotyping CSP-specific memory B cells from a peripheral blood mononuclear cell (PBMC) population.

The PBMC samples were obtained from naturally infected children in Kenya and enriched for B cells. Heavy and light chain immunoglobulin (Ig) genes were PCR amplified from sorted CSP-specific memory B cells and cloned into expression vectors to produce recombinant antibodies in a mammalian expression cell line. Screening for CSP specificity and epitope mapping revealed a cross-reactive, CSP binding monoclonal antibody. Supporting data suggests that this antibody could prove to be a viable resource in the fight against malaria. I established a platform process for discovering and identifying CSP-specific antibodies from PBMC populations of previously infected individuals.

Comments

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Available for download on Sunday, June 30, 2019

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