Date of Award

Fall 2019

Document Type

Open Access Dissertation

Department

Pharmacology, Physiology and Neuroscience

First Advisor

Kim E. Creek

Abstract

Human papillomavirus (HPV) is the etiologic agent for cervical cancer with HPV type 16 (HPV16) being found in about 50% of cervical cancers worldwide. HPVs vary in genomic (DNA) sequence not only between types, but also within types, known as variants. Variants of a particular HPV type may differ by up to 2% in nucleotide sequence in the coding region and up to 5% in the noncoding region. Variants within HPV16 have been identified and grouped into six distinct phylogenetic branches, which segregate geographically: European (E), Asian-American (As.A), African-1 (Af-1), African-2 (Af- 2), Asian (As), and North American (NA). However, there remains an unclear relationship of HPV variants as they pertain to viral infection, HPV persistence, and the risk of developing cervical cancer. Thus, we studied HPV16 variants from exfoliated cervical cells obtained from women enrolled in the Carolina Women’s Care Study (CWCS).

The CWCS followed the HPV status of freshman female students attending the University of South Carolina (UofSC) from enrollment at UofSC until graduation. Exfoliated cervical cells were collected from CWCS participants every 6-months and analyzed for the presence and type of HPV using real-time PCR and a commercial line- blot assay (INNO-LiPA). HPV16 positive cervical DNA samples were amplified by PCR using specific primers for the Long Control Region (LCR) of the HPV16 genome, the DNA sequence of the PCR product was determined, and the HPV16 variant identified.

Of the 467 CWCS participants, 160 participants (365 samples) were HPV16 positive at some point during the study. We obtained informative HPV16 variant results on

74 participants (160 samples). Our results determined that the E variant is by far the most common amongst the participants (77%). However, the Af-2 variant was much more common in African American CWCS participants (24% of HPV16 positive samples) than in European American CWCS participants (4% of HPV positive samples). Unfortunately, due to small sample size and the predominance of the E variant, it was not possible to determine what role, if any; the different variants play in HPV persistence.

In the second part of this thesis we present data from microarray studies comparing the gene expression profiles in RNA isolated from exfoliated cervical cells from CWCS participants who either clear an HPV16 infection or have a persistent HPV16 infection. HPV infection is very common but HPV infections in most women are cleared by the immune system in a period of 12-24 months. However, about 10% of HPV infections persist, leading to cytological abnormalities of the squamous epithelium of the cervix, lesions which can ultimately progress to cervical cancer. Most cervical premalignant lesions are currently treated since there are no diagnostic tools available to predict which lesions will progress or regress. This leads to expensive and painful procedures and over treatment. Thus, the identification of potential biomarkers that could predict which women are more likely to have a persistent HPV infection would we extremely useful, as these would be the women most likely to experience cervical disease.

RNA was isolated from exfoliated cervical cells collected as part of the CWCS and gene expression profiles determined using Human Clariom S arrays (Affymetrix/Thermo Fisher Scientific). These arrays contain probes for over 20,000 well-annotated genes. We compared gene expression profiles in HPV16 negative samples (N=10) to cervical cell samples collected from women that were HPV16 positive and cleared (N=10) or did not clear (persistor, N=10) the HPV16 infection.

Differentially expressed genes were determined and the data subjected to Ingenuity Pathway Analysis (IPA, Qiagen). The IPA results determined that pathways involved in cellular immune response were activated in women who cleared the HPV infection but not in women who were HPV persistent. Thus, no immune response to the HPV16 infection is elicited in women with a persistent HPV16 infection and somehow the HPV “hides” from the immune system in these women.

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