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A memory response is established following primary antigen exposure that stays more or less constant. It appears to adopt a set-point in magnitude but upon re-exposure the response is quicker and better and there is an upward shift in memory frequency that varies with individuals based on the exposure pattern to other microbes or its components. Our investigations were designed to test such differences of non-specific stimulation by PAMPs in lowering the threshold of activation. Neonatal mice were pre-exposed to TLR-ligands intermittently and later analyzed for its resilience to challenge with virus during adult-life. Secondly, adult mice with pre-existing memory to virus were exposed to various TLR-ligands and analyzed for their quality of memory response. The TLR-ligands exposed animals were better responders to a new agent exposure compared to the animals kept in sterile surroundings. Moreover, immune memory recall and the viral specific CD8+ T cells response with TLR-ligands were comparable to the recall response with the cognate antigen. The results provide insights into the role of hyper-sanitized environment versus PAMPs mediated signaling in adaptive immunity and long-term immune memory.

Background: Herpes simplex virus type 2 (HSV-2), the primary cause of genital herpes, is one of the most widespread, lifelong sexually transmitted infections (STIs). Incidence is disproportionately higher in women compared to men, so a better understanding of vaginal transmission, the primary mode for HSV-2 infection in women, is crucial for developing preventative strategies. Female sex hormone, estrogen (E2), has been shown to play a protective role against sexually transmitted viral infections and previous studies have shown that vaginal epithelial cells treated with E2 are protected against HSV-2 infection; however, the underlying mechanism of E2 protection remains unclear, so a transcriptome analysis followed by functional studies was performed. Method of Study: In this study, VK2/E6E7 (vaginal epithelial) cells were used to study HSV-2 entry, infection and replication. VK2s were grown in Air-Liquid-Interface (ALI) cultures, allowing for their proliferation and stratified layer formation in transwells; closely mimicking physiological conditions. Media was supplemented with no hormone (NH) or physiological concentrations of E2, P4 and MPA for 7 days. After 24 hours of HSV-2 infection in these cultures, VK2 cells were lysed and processed for RNA isolation. We performed a comprehensive genome-wide microarray to profile gene expression of VK2 cells pre-treated with and without E2, prior to, and following HSV-2 infection. For data analysis, “R” software was used to perform all pre-processing steps and normalization. Gene Set Enrichment Analysis (GSEA) was performed to identify potential cellular pathways regulated by E2 after infection using the Hallmark database, relative to NH conditions. Immunofluorescence staining was used for functional analysis to confirm transcriptomic data. After selecting a pathway for investigation, small-molecule inhibitors and activators of this pathway were used in combination with NH or E2. Vero plaque assay and HSV-2-GFP infection were used to identify examine the protective effects of E2 and the selected pathway. In addition, we also used siRNA to specifically knockdown proteins part of the pathway and investigate the specific effects on protection against HSV-2. Results: Microarray analysis indicated that exposure to HSV-2 in the presence of E2 resulted in differential transcriptional profile compared to NH and P4. GSEA assigned one of the highest enrichment scores to the p53 pathway compared to other pathways under the influence of E2 compared to NH, following HSV-2 infection. Studies to correlate bioinformatic results with functional analysis showed significant increase in p53 protein expression after E2 treatment compared to NH. Vero plaque assay demonstrated 10-fold decrease in viral replication following E2 treatment as well as by direct activation of p53 in absence of E2. In contrast, p53 inhibition even in the presence of E2 resulted in 100-fold increased viral replication compared to E2 alone, suggesting that the p53 is involved in E2-mediated protection. We deduced that E2 particularly affects HSV-2 replication and not entry into VK2 cells. We also found that BST2 is strongly regulated by E2-mediated p53 and also contributes to protection against HSV-2. Lastly, we demonstrated that E2 demonstrates anti-inflammatory effects that correlate with its increase in barrier integrity seen with VK2s. Conclusions: With bioinformatic and functional analysis, we found that E2 provides protection through the p53 pathway, as well as through downstream BST2. Our data provides the first comprehensive overview of host cellular responses to HSV-2 and female sex hormones at a transcriptional level and highlights the protective role of E2-mediated p53 pathway. This study is the first to deduce the antiviral mechanism of E2 against HSV-2 infection in human vaginal epithelial cells. / Thesis / Master of Science (MSc)

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