Developing Methods to Study Pathogenesis and Immune Response to HSV-2 in the Guinea Pig Model

In order to develop an effective vaccine for HSV-2 it is important to adequately understand the immune response in the proper animal model. Guinea pigs fully recapitulate human HSV-2 disease, but reagents and assays available for this animal are limited. Consequently, many reagents and assays used must be developed and optimized in lab. We utilized several different hybidoma lines received from a partnering lab which produced guinea pig IFN detecting antibodies. To evaluate the sensitivity and assay applicability of these antibodies, I generated and purified antibody from two Hybridoma lines; NG3.5 and VE-4. To generate enough antibody for immunological assays, I grew large quantities of antibody-producing hybridoma cells and collected the supernatant. From this, I isolated and purified the antibody for future assays. We evaluated the ability of the antibodies to detect intracellular IFN production for flow cytometry (FC) analysis. The purified antibody was conjugated to a fluorochrome and applied to IFN producing guinea pig (gp) spleen cells post stimulation with PMA. FC data analysis showed this antibody-fluorochrome conjugation was not suitable for flow cytometry. Upon changing the fluorochrome, cell stimulant, and IFN antibody (NG3.55 to VE-4) we still were unable to develop a working FC intracellular cytokine staining protocol. Contrasting the disappointing results with FC, we developed an ELISPOT which used the NG3.5 antibody to detect IFN secretion from stimulated guinea pig cells. We also used a modification of this assay to quantify antibody secreting cells in immunized guinea pig cells. Tissues were isolated from the spleen, inguinal lymph node, and bone marrow of infected and uninfected male and female guinea pigs. Antibody secreting cell numbers were highest in the spleen and bone marrow of both male and female guinea pigs. Interestingly, we also found the magnitude of ASC response to total HSV-2 glycoprotein was similar to that of gD alone. This was true in all three tissues. The assays optimized here will provide future tools with which to better understand which protective immune responses are necessary for an effective therapeutic/prophylactic vaccine. In short, here we outline the development and optimization of assays to quantify 2 important immune responses to HSV-2 infection in guinea pigs. These studies will provide a platform from which other immune assays can be developed to better understand immunity against HSV-2. For example, there is still a need to better understand the protection provided by effector CD4 and CD8 T cells as well as important sites of their localization in a prophylactic and therapeutic setting. Further immune assay development will expand our knowledge of necessary immune correlates of protection for an effective vaccine against genital HSV-2 infection.