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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)

Mesenchymal stem cells are more and more widely used in tissue engineering due to their pluripotency and no relative ethical problems. Traditional characterization techniques to detect mesenchymal stem cell states include flow cytometry, gene expressing profiling and immunohistochemistry. However, these methods can only provide transient and low level information from current RNA or protein levels about mesenchymal stem cells, which may cause problems when predicting the possible downstream lineages they will commit into. We have developed chromatin immunoprecipitation (ChIP)-based epigenetic technique to detect mesenchymal stem cell states. For the systems we tested, this epigenetic assessor successfully characterized cell state changes and gave similar results obtained from gene expression profiling or protein expression assay. This epigenetic technique can provide information about mesenchymal stem cells states from a more fundamental chromatin level, which is promising for predicting future lineages from current states.

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.

Animals deficient in complement components C1q, C4, C3, and CR1/2 have severely impaired antibody responses. C1q is primarily activated by antibody-antigen complexes. Antigen-specific IgM in complex with an antigen is able to enhance the antibody response against that antigen. This is dependent on the ability of IgM to activate complement. Naïve mice have very low amounts of specific antibodies and therefore it is surprising that classical pathway activation plays a role for primary antibody responses. It was hypothesized that natural IgM, present in naïve mice, would bind an antigen with enough affinity to activate C1q. To test this, a knock-in mouse strain, Cm13, with a point mutation in m heavy chain, making its IgM unable to activate complement was constructed. Surprisingly, the antibody responses in Cm13 were normal. Puzzled by the finding that the ability of IgM to activate complement was required only for some effects, the immunization protocol was changed to mimic an infectious scenario. With this regime, Cm13 mice had an impaired antibody response compared to wildtype (WT) mice. The antibody response in WT mice to these repeated low-dose immunizations was also enhanced. These observations suggest that IgM-mediated enhancement indeed plays a physiological role in initiation of early antibody responses. IgM-mediated enhancement cannot however compensate for the dependecy of T-cell help. Although IgM from WT mice enhanced the antibody response, the T-cell response was not enhanced. The connection between classical pathway activation and CR1/2 is thought to be generation of ligands for CR1/2. In mice, CR1/2 are expressed on B cells and follicular dendritic cells (FDC). Although CR1/2 are crucial for a normal antibody response, the molecular mechanism(s) are not understood. To investigate whether CR1/2 must be expressed on B-cells or FDC to generate a normal antibody response, chimeric mice between WT and CR1/2-deficient mice were constructed. The results show that CR1/2+ FDC were crucial for the generation of antibody responses. In the presence of CR1/2+ FDC, both CR1/2+ and CR1/2- B cells were equally good antibody producers. However, for an optimally enhanced antibody response against IgM-antigen complexes, both B cells and FDC needed to express CR1/2.

Infections with the bacterial pathogen Chlamydia trachomatis are a critical public health problem. Chlamydia remains the number one cause of preventable blindness worldwide and the leading cause of bacterial sexually transmitted infections in the United States. In humans, repeat and persistent infections with Chlamydia result in severe inflammation. Inflammation in the conjunctiva can result in blindness, while inflammation in the genital tract can result in pelvic inflammatory disease, ectopic pregnancy or infertility. In order to curb the increasing incidence of Chlamydia infections worldwide it will be necessary to develop a protective vaccine that affords long-term protection and prevents pathologies. To better inform vaccine development we must understand the mechanisms that drive long-term immunity in the genital tract and elucidate critical interactions between Chlamydia and host cells to uncover potential mechanisms of immune evasion.

The Streptococcus Anginosus/Milleri Group (SMG) is made up of three closely related but distinct bacterial species: Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus. The SMG are recovered from about one-third of healthy, asymptomatic individuals. Despite this, the SMG cause more incidences of invasive streptococcal disease than Group A and Group B Streptococcus combined. Members of this group are somehow able to live a dual lifestyle. Little work has been conducted on the molecular pathogenicity of the SMG and host factors that contribute to host susceptibility to this group have been under-investigated. My research works towards discovering how the host recognizes the SMG as well as what enables the SMG to evade clearance by the immune system. I hypothesize that: 1) recognition of the SMG by toll-like receptor 2 (TLR2) plays a key role in triggering a cytokine response by the innate immune branch (which coordinates the immune response to the SMG), 2) the expression of cytolysins and extracellular polysaccharides by members of the SMG enables evasion of innate immune recognition and cytokine responses. hTLR2 reporter and monocyte-like cell lines as well as human blood samples from healthy donors were used to investigate the host factors that contribute to SMG infection. Five clinical reference SMG strains and a transposon mutant library were used to probe the contributing bacterial factors. It was found that TLR2 activation plays an important role in the cytokine response to the SMG, but there is heterogeneity between strains in their ability to activate TLR2. It was also found that intermedilysin expression by S. intermedius strains enables evasion of recognition; however, different hosts display varying susceptibility to this cytolysin. This study reveals that investigation of both host and microbial factors is essential to build an understanding of the mechanisms of SMG transition from commensalism to pathogenicity. / Thesis / Master of Science (MSc) / The Streptococcus Miller/Anginosus Group (SMG) is a group of bacteria comprised of three species. Members of this group are recovered from roughly one-third of healthy individuals. However, the SMG are also found in samples collected from patients with invasive disease. It is not well understood why some human-SMG relationships are pathogenic and others are not. However, it is likely that the combination of both human and SMG factors determine the nature of the relationship formed between the two. In this study, the human and SMG factors that contribute to infection were investigated. The ways by which human cells recognize members of the SMG and defend themselves from damage was explored. Additionally, SMG factors that potentially contribute to infection were probed to discover their effect on human cells. By investigating both the bacterial and host factors that lead to infection, disease treatments and preventative strategies can be tailored to individual cases.

<p>Vaccination with DNA encoding the encephalitogenic autoantigen myelin oligodendrocyte glycoprotein (MOG), pMOG91-108, induce a protective immunity against experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. By injection of a DNA vaccine that contains a DNA region encoding short interfering RNA specific for IFNβ (pMOG-IFNβ) the protective effect of the DNA vaccination is totally inhibited. This demonstrates that IFN-β is directly involved in the protective mechanism against EAE.</p><p>The objective of this project was to study how molecules involved in the inflammatory process in EAE are regulated by suppressive DNA vaccination. mRNA expression of IL-1β, TGF β, IL-23p40 and Axl receptor tyrosine kinas did not show any significant differences between the groups vaccinated with these DNA vaccines. IL-6 and IFNγ mRNA expression after MOG stimulation in rats treated with pCI, a control vaccine was significantly higher compared to the group vaccinated with vaccine containing pMOG-IFNβ. IL-17 m RNA expression after MOG stimulation in pCl-treated rats was significantly higher compared to the group vaccinated with vaccine containing pMOG-91-108. Of these results the mRNA expression of IL-17 and IL-6 were of interest for the project.</p><p>The immune system normally protects the body against infections and T-cells have an important role in this defence system. In MS and EAE, the immune system attacks the myelin and this process is caused by a dysregulation of the T-cells. IL-17-producing Th17 cells mediate EAE. Naïve CD4 T-cells in the presence of IL-6 and TGFβ are differentiated to Th17 cells instead of differentiating into T-helper or regulatory T-cells. These IL-17-producing T-cells are highly pathogenic and essential for the development of EAE. The results showed that pMOG IFNβ vaccine had an effect at the immune response, which resulted in an inhibition of the IL-6 production and that vaccination with pMOG91-108 impairs differentiation of IL-17-producing T-cells.</p>

Vaccination with DNA encoding the encephalitogenic autoantigen myelin oligodendrocyte glycoprotein (MOG), pMOG91-108, induce a protective immunity against experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. By injection of a DNA vaccine that contains a DNA region encoding short interfering RNA specific for IFNβ (pMOG-IFNβ) the protective effect of the DNA vaccination is totally inhibited. This demonstrates that IFN-β is directly involved in the protective mechanism against EAE. The objective of this project was to study how molecules involved in the inflammatory process in EAE are regulated by suppressive DNA vaccination. mRNA expression of IL-1β, TGF β, IL-23p40 and Axl receptor tyrosine kinas did not show any significant differences between the groups vaccinated with these DNA vaccines. IL-6 and IFNγ mRNA expression after MOG stimulation in rats treated with pCI, a control vaccine was significantly higher compared to the group vaccinated with vaccine containing pMOG-IFNβ. IL-17 m RNA expression after MOG stimulation in pCl-treated rats was significantly higher compared to the group vaccinated with vaccine containing pMOG-91-108. Of these results the mRNA expression of IL-17 and IL-6 were of interest for the project. The immune system normally protects the body against infections and T-cells have an important role in this defence system. In MS and EAE, the immune system attacks the myelin and this process is caused by a dysregulation of the T-cells. IL-17-producing Th17 cells mediate EAE. Naïve CD4 T-cells in the presence of IL-6 and TGFβ are differentiated to Th17 cells instead of differentiating into T-helper or regulatory T-cells. These IL-17-producing T-cells are highly pathogenic and essential for the development of EAE. The results showed that pMOG IFNβ vaccine had an effect at the immune response, which resulted in an inhibition of the IL-6 production and that vaccination with pMOG91-108 impairs differentiation of IL-17-producing T-cells.