Publication Date

2015

Document Type

Thesis

Committee Members

Nancy Bigley (Advisor), Cheryl Conley (Committee Member), Barbara Hull (Committee Member)

Degree Name

Master of Science (MS)

Abstract

This study examines the effects of blocking the nectin-1 and Herpes Virus Entry Mediator (HVEM) receptors on Neuro-2a (N2a) cells in order to prevent HSV-1 infection. These two receptors have been identified as a primary and secondary mode of entry for HSV-1. Since there was some debate about the correct serum level to be used in growth media for healthy N2a cells, three concentrations (2%, 5%, 10%) were used to examine relative growth and neurite development, in addition to examining possible differences in infectivity levels in all other experiments. There was a difference in general morphology, with neurite abundance and length increasing as the serum concentration decreased. However, the three concentrations led to no visible differences in immunofluorescence staining of Nectin-1, HVEM or Nectin-1, or significant differences in the cell viability trials.

Immunofluorescence was performed to identify the presence of the nectin-1 and HVEM receptor on the N2a cells, as well as the general localization of the receptors on the cell and the relationship to each other. On the N2a cells, the nectin-1 seemed to occupy the non-contact regions of the cells and the HVEM was concentrated at the cell-to-cell contact regions. Based on this analysis, nectin-1 was also examined in conjunction with filamentous-actin (F-Actin). Nectin-1 was directly associated to the actin cytoskeleton, consistent with the literature. Immunofluorescence was also performed on the keratinocyte cell line PAM212 on which the nectin-1 and HVEM focused at the cell contact regions at the adherens junctions. These results illustrate that receptor location is dependent on cell type, but in general the receptors localize near each other, but do not directly overlap.

Cell viability and plaque formation studies were performed to determine the effectiveness of blocking the nectin-1 and HVEM, separately and together, in preventing HSV-1 entry into the cell. In all trials, the appropriate controls (control, HSV-1 only, and isotype) were employed to examine the efficiency of blocking the specific receptors against HSV-1 entry. Viabilities of the control and antibody treated cells were similar, and the isotype treated cells and the cells exposed to only HSV-1 were comparable at both the 0 and 24 hour observation times in all trials, regardless of singlet or multiple antibody blocking. However, the cells on which both the Nectin-1 and HVEM receptors were blocked showed a specific and significant increase in viability that did not deviate among the serum concentrations. Blocking both receptors was more successful at preventing virus entry than blocking only one receptor. Receptor blocking also led to inhibition of plaque formation. The cell lysates were collected, after the cell counts taken at 24 hours, and then used to infect a monolayer of Vero cells to demonstrate further that antibody blocking did protect the cells from viral entry through plaque formation. There was minimal to no plaque formation in the Vero cell monolayer treated with the lysate from the antibody blocked cells, compared to those that had been exposed to the isotype or HSV-1 only, suggesting that HSV-1 entry was reduced.

Page Count

104

Department or Program

Microbiology and Immunology

Year Degree Awarded

2015

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