ABVIC: A NOVEL FLOW CYTOMETRY-BASED ASSAY FOR THE DETECTION OF HSV-SPECIFIC ANTIBODIES

Author

Gavin Michael Sowa, Southern Illinois University CarbondaleFollow

Date of Award

12-1-2016

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

Halford, William

Abstract

Herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) are closely related viruses that establish lifelong infection in their hosts and can periodically reactivate to cause painful lesions. Approximately 50 million Americans are infected with HSV-2, the primary cause of genital ulcerative disease. In addition to the pain of the physical symptoms, genital herpes is highly stigmatized and can cause significant reductions in quality of life. HSV-2 has been demonstrated to have a synergistic relationship with HIV, where it enhances the transmissibility, susceptibility and severity of disease. In addition, potentially life-threatening neonatal herpes occurs in about 1 in 10,000 live births. While there are known means of reducing the risk of transmission, the overwhelming majority of infected persons are unaware of their status, and they remain the driving force behind new infections. Virological tests, such as PCR, are ill suited for asymptomatic infections since the detectable virus is only shed on <10% of days. Currently available type-specific serological tests based on glycoprotein G are prone to false-positive results, lack the sensitivity to detect new infections and may be affected by cross-reactivity between HSV-1 and HSV-2. Due to the known limitations of HSV serological testing, it is not recommended for the general population or pregnant women. In the first half of my thesis, I evaluate a new flow-cytometry-based method that measures serum antibody-binding to virus-infected cells (ABVIC). We obtained a panel of human control sera from Westover Heights Clinic (WHC) and determined if the ABVIC could measure HSV-specific antibody binding to test-cells. We found that the assay was sensitive, semi-quantitative, and could be made type-specific with the addition of a pre-adsorption step. The ABVIC offers an advantage over the standard of care single-antigen tests as the result measuring antibody binding to all viral proteins fixed in their native conformation. In the second half of my thesis, I used the ABVIC assay test n=34 blinded test-sera. Of these, n=17 had previously tested as “indeterminate” by bother Herpeselect ELISA as well as Western Blot. Following unblinding, we found that the ABVIC properly identified all n=17 patient sera of an unambiguous serostatus. Of the indeterminate sera, all were found to be seronegative for HSV-2. Based on these surprising results, we requested an additional n=11 indeterminate samples, which were also found to be HSV-2 seronegative. Most of the "Indeterminate" serum samples exhibited high background, which produced weak reactivity in HerpeSelect and Western blot assays, but did not confound the internally controlled ABVIC test.