Characterization of the CD8 T Lymphocyte Response to the Clec9A Polytope in vivo
Gigowski, Michelle R.
MetadataShow full item record
The Herpes Simplex Virus (HSV) affects one in five people at the age of 12 and older throughout the United States, causing blisters and sores throughout the surface of the body. Although there is not a current cure for the virus, effective treatments that limit outbreaks are available in developed nations (Bennington, 2006). Previous studies have implemented a novel technique in targeting the immune system of mammals by injecting polytopes specific to dendritic cell (DC) protein subsets. These polytopes allow peptides from the HSV protein to be presented to endogenous T-cells, also referred to as crosspresentation, in hopes of electing an endogenous immune response. If this technique were successful, it would result in protection from the virus and possibly serve as a vaccine against HSV (Caminschi, I. et al., 2008). In one of the many attempts in developing a possible vaccination against HSV, our current goal was to use a similar approach as Caminschi et al. as referred to above. We worked to characterize the Clec9A polytope in hopes of electing a helper (CD8) T-cell response in the C57BL/6 mouse strain (B6 mouse). The early proliferative and cytotoxic CD8 response to the Clec9A polytope was investigated using 5-(and-6-)- carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling and in vivo cytotoxic T-lymphocyte (CTL) assays. These experiments were quantified using flow cytometry (FACS) analysis. Post Clec9A priming, CFSE labeled T-cells did elicit a proliferative response when in the presence of double-stranded RNA polyriboinosinic polyribocytidylic acid (Poly I:C). To further characterize the Clec9A polytope, an in vivo CTL assay was used to target the delivery of antigen, either the gB or OVA peptides, to specific dendritic cells (DCs) efficient in cross-presentation. This was achieved by using the Clec9A polytope in the presence Poly I:C against the Clec9A protein subsets on DCs to induce CD8 T-cell proliferative responses and cytotoxicity in vivo. The GL117 polytope was used as a control in all trials since it contains the same protein peptide sequence following the antibody but lacks a specific target in the mouse system. The GL117 polytope in addition to Poly I:C provoked greater, but nonsignificant immune responses than expected. The Ovalbumen-Genetic Fusion (OVA-GF) was also used to quantify the CD8 response to the OVA peptide sequence contained within the Clec9A polytope. The OVA-GF was characterized to see whether the it would have an improved means of endogenously targeting DCs as the GF contains the entire OVA protein, unlike the polytope. The Clec9A polytope and GF did elicit significant CD8 proliferative and cytotoxic immune responses in vivo. However, Poly I:C was essential in all trials that elicited a significant response. This research has potential application for future protection from the HSV through this novel vaccination approach of targeting specific protein subsets on DCs, which results in endogenous antigen cross-presentation to Tcells, thereby electing a protective immune response.