Problem: Herpes simplex virus type-2 (HSV-2) is one of the most prevalent sexually transmitted infections in the world, and rates of infection are higher in women compared to men. Furthermore, vaccines developed against HSV-2 have failed at various stages of clinical trials, due to their inability to induce protective mucosal immunity. In animal models, intranasal (IN) immunization with attenuated HSV-2 (TK−) virus has been shown to confer protection against wildtype HSV-2 challenge. Since IN immunization serves as a more practical and less intrusive vaccination strategy, further studies are warranted to characterize optimal immune responses following IN immunization. We have previously demonstrated that estradiol (E2) treatment promotes enhanced protection against HSV-2 through enhanced anti-viral T cells responses. However, the effect of E2 on B cell responses, which were recently shown to be critical in protecting the host following IN immunization, remain poorly understood. Therefore, in this study we aimed to examine if following IN immunization, E2 enhances the memory B cell (MBC) and antibody-secreting plasma cell populations within the secondary lymphoid tissues and nasal effector sites, and whether this enhancement leads to an overall better protection against intravaginal IVAG WT-HSV-2 challenge. Methodology: Ovariectomized (OVX) mouse model of HSV-2 were pre-treated with E2 or placebo pellets. Subsequently, both groups were immunized intranasally with TK- HSV-2. Four weeks later nasal associated lymphoid tissues, nasal mucosa, cervical and iliac lymph nodes, spleen and vaginal tract were collected and processed and MBC and antibody-secreting plasma cells were characterized by flow cytometric analysis. HSV-2 specific IgM and IgG antibody responses in serum and vaginal secretions were measured by ELISA. In parallel experiments, animals were IVAG challenged with WT-HSV-2 and the B cell subsets were characterized as above. Results: The formation of MBC subsets, as seen by the presence of CD19+ IgD- cells and the heterogenous expression of CD73, CD80, and PD-L2, were observed four-weeks post immunization within the cervical and iliac lymph nodes and spleen, which were further enhanced in the presence of E2. Additionally, E2-treated mice had increased number of B220- CD138+ IgG2c+ plasma cells within the nasal mucosa following immunization. These enhancements translated into increased levels of HSV-2 specific IgG2b and IgG2c antibodies within the serum and vaginal secretions of E2-treated mice at four-weeks post IN immunization. Upon IVAG challenge, E2-treated mice, but not control mice, were protected. Since the antibody isotypes that were enhanced in E2 treated mice are correlated with Th17 responses, E2 mediated antibody enhancement was tested in IL-17 knockout mice. E2 treatment in IL-17-knockout mice failed to induce similar responses observed in WT mice, indicating that the enhancement of B cells and antibodies seen following E2 treatment was mediated in an IL-17 dependent manner. Conclusion: This study highlights the importance of sex-dependent differences in vaccine-induced immunity. Specifically, the findings from this study will provide valuable information for the design of a potentially efficacious mucosal vaccine strategy, whereby immunization in the context of E2 could significantly enhance antigen-specific antibody responses in the genital tract. / Thesis / Master of Science (MSc)

HLA-DR1 transgenic (DR1 Tg) mice provide a model for evaluating the breadth and specificity of CD4 T cell responses that may develop in humans following influenza infection or vaccination. Recent studies identified a tremendously broad HLA-DR1-restricted CD4 T cell responses in DR1 Tg mice infected intranasally with influenza A/New Caledonia/20/99 (NC). In this study, our goals were to characterize B cell responses after NC infection in DR1 Tg mice and establish the correlation between B cell responses and CD4 T cell responses in this system. Influenza-specific B cell responses following virus administration were analyzed in DR1 Tg mice and in the genetically matched H-2b strain C57BL/10J (B10). Following intranasal (i.n.) NC infection, B cell responses in B10 mice featured strong IgG2b and IgG2c production and were typical of previously described B cell responses to a variety of mouse-adapted influenza strains. In contrast, B cell responses in DR1 Tg mice followed delayed kinetics and were strongly skewed to IgG1 production, suggesting the Th2 polarization of CD4 T cell responses. The different antibody isotype profile in DR1 Tg mice compared to B10 mice was evident in antibody secreting cells (ASCs) frequencies and in circulating Abs levels. Surprisingly, although DR1 Tg mice had lower influenza-specific Abs levels, they exhibited higher neutralizing Abs titers early in the response. B cell responses following intranasal infection of influenza A/Puerto Rico/8/1934 (PR8) or intramuscular vaccination of inactivated NC in DR1 Tg mice were different from the observed IgG1 bias after i.n. NC infection. After i.n. PR8 infection, B cell responses were similar in DR1 Tg mice and B10 mice, characterized by predominant IgM/IgG3 production. Additionally, following intramuscular administration of inactivated NC, B cell responses were skewed towards IgG2c production in both DR1 Tg mice and B10 mice, suggesting the Th1 polarization of CD4 T cell responses. A mechanistic understanding of IgG1/Th2 biased B cell responses and better neutralizing Abs production in DR1 Tg mice following i.n. NC infection may have implications for the optimal control of influenza infection.

HLA-DR1 transgenic (DR1 Tg) mice provide a model for evaluating the breadth and specificity of CD4 T cell responses that may develop in humans following influenza infection or vaccination. Recent studies identified a tremendously broad HLA-DR1-restricted CD4 T cell responses in DR1 Tg mice infected intranasally with influenza A/New Caledonia/20/99 (NC). In this study, our goals were to characterize B cell responses after NC infection in DR1 Tg mice and establish the correlation between B cell responses and CD4 T cell responses in this system. Influenza-specific B cell responses following virus administration were analyzed in DR1 Tg mice and in the genetically matched H-2b strain C57BL/10J (B10). Following intranasal (i.n.) NC infection, B cell responses in B10 mice featured strong IgG2b and IgG2c production and were typical of previously described B cell responses to a variety of mouse-adapted influenza strains. In contrast, B cell responses in DR1 Tg mice followed delayed kinetics and were strongly skewed to IgG1 production, suggesting the Th2 polarization of CD4 T cell responses. The different antibody isotype profile in DR1 Tg mice compared to B10 mice was evident in antibody secreting cells (ASCs) frequencies and in circulating Abs levels. Surprisingly, although DR1 Tg mice had lower influenza-specific Abs levels, they exhibited higher neutralizing Abs titers early in the response. B cell responses following intranasal infection of influenza A/Puerto Rico/8/1934 (PR8) or intramuscular vaccination of inactivated NC in DR1 Tg mice were different from the observed IgG1 bias after i.n. NC infection. After i.n. PR8 infection, B cell responses were similar in DR1 Tg mice and B10 mice, characterized by predominant IgM/IgG3 production. Additionally, following intramuscular administration of inactivated NC, B cell responses were skewed towards IgG2c production in both DR1 Tg mice and B10 mice, suggesting the Th1 polarization of CD4 T cell responses. A mechanistic understanding of IgG1/Th2 biased B cell responses and better neutralizing Abs production in DR1 Tg mice following i.n. NC infection may have implications for the optimal control of influenza infection.