Undergraduate Honors Theses

Thesis Defended

Spring 2017

Document Type

Thesis

Type of Thesis

Departmental Honors

Department

Biochemistry

First Advisor

Dr. Robert Garcea

Second Advisor

Dr. Joseph Falke

Third Advisor

Dr. Robert Parson

Fourth Advisor

Dr. Theodore Randolph

Abstract

Cervical cancer is the second most common cancer among women worldwide, and the most common cancer among women in developing countries1. The causal role of human papillomavirus (HPV) infections in invasive cervical cancers is acknowledged beyond reasonable doubt, and the ability to vaccinate against HPV can drastically reduce the incidence of cervical cancer worldwide. There are currently marketed HPV vaccines that make use of HPV L1 virus-like particles (VLPs), and while these VLP vaccines confer immunogenicity, they are expensive, thermosensitive, and require multiple doses2. Expression of the HPV L1 protein in Escherichia coli (E. coli) bacteria has the potential to reduce production costs, while lyophilization creates a thermostable powdered vaccine formulation, and atomic layer deposition (ALD) generates a platform for a single-administration prime-boost vaccination.

By using E. coli cells for protein expression, endotoxin is therefore present and must be removed prior to vaccine injection. This is done through protein purification and further development into a powderized vaccine particle. The research project described in this thesis details the tracing of endotoxin levels throughout HPV16 L1 vaccine purification and particle production. Endotoxin is removed by the multi-step purification and it is hypothesized that it is removed in varying extents during the different steps. It is also hypothesized that endotoxin is not reintroduced during particle production. In order to test these hypotheses, and determine which stages yield the most removal or reintroduction of endotoxin, an endotoxin assay is run with samples at each stage of the protein purification, spray drying, and ALD coating processes. The endotoxin assay is crucial in determining where, if anywhere, endotoxin is reintroduced after purification. The results of the endotoxin assay are used to effectively fine-tune the vaccine development process in order to create a safe and effective second generation HPV vaccine.

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