Author ORCID Identifier

https://orcid.org/0000-0003-2897-204X

Date Available

8-12-2022

Year of Publication

2021

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Pharmacy

Department/School/Program

Pharmaceutical Sciences

First Advisor

Dr. Vincent J. Venditto

Second Advisor

Dr. David Feola

Abstract

Cardiovascular disease (CVD) is a major cause of morbidity and mortality around the world causing approximately 14% of total disease burden. A major factor in the progression to major CVD within patients is immune activation, which led to the study of various immune products as potential biomarkers for the prediction of cardiovascular events, including antigen-specific antibodies and immune complexes. One focus of this biomarker research is IgG autoantibodies targeting apolipoprotein A-I (ApoA-I), and several studies of these antibodies have found an association with increased CVD events. Based on the encouraging results from these studies and previous research which has identified the presence of ApoA-I/IgG immune complexes in humans and mice, we hypothesized that ApoA-I/IgG immune complexes may serve as a biomarker for cardiovascular disease risk in patients.

To study the potential association between ApoA-I/IgG immune complexes and CVD events, an immunoassay was designed to detect immune complexes by utilizing a capture antibody specific for ApoA-I and a detection antibody specific for human IgG. ELISA were completed in samples from 359 patients with a history of coronary artery disease who were followed for a median of 4.1 years. Responses in this assay are independently associated with CVD events after adjustment for 8 common CVD risk factors with an adjusted hazard ratio of 1.90 (95% CI, 1.03– 3.49; p=0.038) when comparing subjects in the lowest tertile ELISA values to subjects in the highest tertile.

Additional studies using samples from a cohort of blood donors sought to measure immune complexes containing a related protein, apolipoprotein B-100 (ApoB-100), by changing the capture antibody in the assay. The detected absorbance values are similar when utilizing a goat anti-ApoA-I IgG capture antibody or a goat anti-ApoB-100 IgG capture antibody, a possible indication of interference in our assay. Further studies find that the absorbance values in this ELISA depend on capture antibody species of origin and not the specificity of the antibody. Analyte identification occurs via the completion of competition ELISAs and plasma fractionation studies which indicate that the immunoassay results are caused by a human IgG antibody binding to goat-IgG. Characterization of these antibodies indicate that they bind with a high affinity to the heavy chain of goat IgG. Taken together, these results indicate that human anti-goat antibodies may serve as a biomarker for CVD events in patients.

Additional studies presented in this dissertation include development of high throughput ELISA methodology and modulation of epitope-specific immune responses via liposomal injections.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2021.330

Funding Information

V.J. Venditto received funding for this work including a funding from the College of Pharmacy as well as a Scientist Development Grant from the American Heart Association (17SDG32670001) from 2017 to 2019, and multiple grants from the National Institutes of Health (P30GM127211 in 2016, P20GM130456-01 in 2020, R01DA043938 from 2017-2021, R56HL145051 from 2019-2020, and R01HL152081 from 2020-2024) D. Henson was supported by a training grant through the National Center for Advancing Translational Sciences, at the National Institutes for Health (TL1TR001997) from 2016-2018, a Pharmaceutical Sciences Excellence in Graduate Achievement Fellowship from the University of Kentucky College of Pharmacy in 2018 and an American Heart Association predoctoral fellowship 19PRE34430120 from 2019-2020.

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