The four serotypes of dengue virus (DENV 1-4) have a significant and growing impact on global health. Dengue disease encompasses a wide range of clinical symptoms, usually presenting as an uncomplicated febrile illness lasting 5-7 days; however, a small percentage of infections are associated with plasma leakage and bleeding tendency (called dengue hemorrhagic fever, DHF), which can result in shock. Epidemiological studies indicate that severe dengue disease most often occurs during secondary heterotypic DENV infection. Additionally, plasma leakage (the hallmark of DHF) coincides with defervescence and viral clearance, suggesting that severe disease arises from the immune response to infection rather than a direct effect of the virus. A number of studies have found increased levels of markers of immune cell activation in patients with DHF compared to patients with the less severe form of disease (DF). These markers include IFNγ, TNFα, soluble CD8, soluble IL-2 receptor, soluble TNF receptor, and CD69, which support a role for T cells in mediating immunopathology. Because of the high homology of DENV 1-4, some degree of serotype-cross-reactivity is seen for most T cell epitopes. A high percentage of DENV-specific T cells recognize multiple DENV serotypes, as demonstrated by peptide-MHC (pMHC) tetramer binding and in vitro functional assays performed on PBMC from subjects vaccinated with an experimental DENV vaccine or naturally-infected subjects with secondary (>1) DENV infection. This thesis sought to address several gaps in the literature, specifically whether T cell responses differ in primary versus secondary (natural) infection. We studied the frequency, phenotype, and function of DENV-specific T cells. We demonstrated substantial serotype-cross-reactivity of antigen-specific T cells generated in response to naturally-acquired primary as well as secondary DENV infection. The frequency of A11-NS3133 epitope-specific T cells during acute infection did not correlate with disease severity. However, the peak frequency occurred earlier in primary infection while the frequency of CD45RA+ T cells declined quicker in secondary infection, suggesting the expansion of DENV-specific memory T cells. DENV-immune T cells exhibited different functional capabilities that were dependent on the particular serotype of infection. Specifically, DENV-1 or -3 stimulation of A11-NS3133 epitope-specific T cell lines resulted in robust function that included IFNγ production, whereas DENV-2 stimulation resulted in limited function that often included MIP-1β but not IFNγ production. These data support a role for T cells in DENV infection and offer new insights into their potential contribution to dengue pathology.

The spread of pandemic influenza A (2009 H1N1 influenza) virus resulted in a global influenza pandemic in 2009. During the early stages of the pandemic, population surveillance was crucial. However, officials around the world realized that many of our surveillance and reporting systems were not prepared to respond in a coordinated, integrated way, which made informed public health decision-making very difficult. More accurate estimates of the total number of hospitalized 2009 H1N1 influenza cases were required to calculate population-based 2009 H1N1 influenza-associated mortality, morbidity and hospitalization rates. For instance, how many people were hospitalized with 2009 H1N1 influenza in Massachusetts? Of these, how many were admitted to the ICU and how many died? Compared to seasonal influenza, were some race/ethnic and age groups affected more than others, and what types of characteristics led to more severe manifestations of 2009 H1N1 influenza among these groups in Massachusetts? To address the above questions, I proposed a retrospective cohort study using data from the Hospital Discharge Database (HDD), which contains data for all inpatients discharged from 76 acute care hospitals in Massachusetts, as well as Census information to provide a measure of socioeconomic status (SES). My specific aims are as follows: 1. Develop methods to identify influenza cases precisely and describe characteristics of those hospitalized with ILI in MA between April 26-Sept 30, 2009; 2. Conduct analyses to identify race/ethnicity-related trends in reference to 2009 H1N1 influenza-related hospitalizations; 3. Conduct analyses to identify age-related trends in reference to 2009 H1N1 influenza-related hospitalizations. First, I established influenza case selection criteria using hospital discharge data. I addressed limitations in the published methods on defining cases of influenza using administrative databases, and evaluated ICD-9 codes that correspond with common and relatively serious respiratory infections and influenza using a ‘maximum’ and ‘minimum’ approach. Results confirmed that 2009 H1N1 influenza affected a younger population, and disproportionately affected racial minorities in Massachusetts. There were also higher rates of ICU admission compared to seasonal influenza. I then presented epidemiological data indicating race/ethnic disparity among 2009 H1N1 influenza cases in Massachusetts. I found that Hispanics had significantly lower odds of 2009 H1N1 influenza-related ICU stay. SES gradients calculated using five-digit zip code information did not account for these differences. Within race/ethnic strata, Hispanics Finally, I presented epidemiological data indicating differences among 2009 H1N1 influenza cases by age group in Massachusetts. I calculated measures of Diagnostic Cost Group (DxCG) comorbidity for the study population to provide a comorbidity measure at baseline. Main results indicate that although comorbidity scores were similar between the 2009 H1N1 influenza and seasonal influenza groups, 2009 H1N1 influenza caused more severe disease in younger age groups. This is the first study to report population-based statewide outcomes in all acute care centers in MA. In this dissertation I address challenges surrounding influenza surveillance to create case selection criteria within an administrative database. Using my case selection criteria, I then provide data related to fatality and severity of 2009 H1N1 influenza in Massachusetts in reference to sociodemographic variables such as racial/ethnicity and age groups, and provide evidence for patient-level interventions to those hardest hit by influenza. These findings provide valuable information about using large administrative databases to describe pandemic influenza cases and guide resource allocation to reduce disparities in relation to pandemic influenza preparedness.

Dengue viruses (DV) are re-emerging mosquito-borne pathogens for which four distinct lineages, grouped based on serology and referred to as serotypes 1-4 (DIV-D4V), have been described. Epidemiological data imply that re-infection with a "heterologous" serotype, i.e, one other than that to which the individual was originally exposed, enhances the risk for development of severe disease, dengue hemorrhagic fever (DHF). The hallmark of DHF is a transient capillary leakage syndrome of rapid onset, temporally associated with the resolution of fever and viremia. In its most grave form, the vascular permeability phenomenon in DHF may progress to dengue shock syndrome (DSS), which is often fatal in the absence of appropriate medical care. Despite the fulminant nature of vascular leakage during DHF/DSS, this phenomenon does not appear to be due to direct cytopathic effects of DV. Rather, inappropriate reactivation and/or regulation of dengue-specific memory are the prevailing theorized (immunopathological) etiologies. Traditional vaccine development techniques have proven insufficient for DV, since any vaccine must offer complete protection against all four serotypes to avoid enhanced pathology on natural viral challenge. Understanding the underlying mechanisms that contribute to dengue disease, particularly the development of dengue-specific memory, is therefore of critical importance. Dengue immunopathology and the specific aspects of immunological memory that determine disease severity are heatedly debated. Previous research in our lab has suggested that T cell responses contribute to the severity of dengue illness. Clinical data indicate enhanced immune activation in more grave cases of DV infection, and serotype cross-reactive T cells from multiple individuals are present after both primary and secondary dengue infections. However, little is known about the conditions under which T cells are primed and dengue-specific memory is generated. Dendritic cells (DCs) are bone marrow-derived cells that play a central role in directing activity within the immune system. DCs shape quantitative and qualitative aspects of adaptive immunity, and therefore the intrinsic characteristics of host memory to a pathogen. DCs are essential in generating primary immune responses, due to their particular effectiveness in stimulating naïve T cells. DCs also play important roles in the reactivation of memory to an infectious agent, and as reservoirs for the dissemination of invading microorganisms. Exposure to pathogens or their products initiates a series of phenotypic and functional changes in DCs, termed maturation. DC maturation involves a coordinated response of immunomodulatory surface molecule elaboration and cytokine production, culminating in antigen presentation to, and co-stimulation of, T cells specific for the invading agent. The DC response is ostensibly tailored to facilitate effective elimination by regulating effective downstream interactions of the DC with T cells. A number of viruses have evolved to infect DCs and alter their functional behavior, facilitating their own survival within the host, and the herd. DV readily infects DCs both in primary cell cultures and in vivo. However, reports on the effects of DV infection on DC maturation vary both with regard to some of the cytokines produced, and the phenotypes of infected versus bystander cells. Although DCs appear to be activated following DV exposure, responses on the single-cell level appear to depend on the infection state of the cell, hypothetically driven by intracellular virus-mediated effects. Therefore, downstream responses to these divergent populations - i.e., actively infected cells versus uninfected bystander cells - are likely to be the consequence of at least two modes of DC behavior. Because DCs play a pivotal role in adaptive immune development, and because the resulting memory response appears to be critical in affecting disease pathology after heterologous DV re-infection, I sought to explore the phenomena of DC maturation in response to dengue exposure, and to begin to answer the question of how active infection alters the functional capabilities of DCs. Notably, primary dengue infection is generally well-controlled with minimal pathology. Therefore, this thesis addresses the hypothesis that DV infection of DCs results in cellular activation and stimulation of antiviral immunity, despite virus-mediated alteration of DC maturation. In order to address this hypothesis, I examined both DV infection-dependent and independent effects on DC functional responses including surface molecule regulation secretory activity, and CD4 T cell allostimulatory priming. DCs derived from human peripheral blood monocytes were readily infected with multiple strains of DV. DV infection of DCs derived from separate donors was dose-dependent, with substantial variability in DC susceptibility to infection. Exposure to live DV activated surface molecule expression in DCs, similar to the effects of defined maturation stimuli including a combination of TNF-α and IFN-α, or LPS. In addition, UV-inactivated DV induced expression of cell surface molecules, albeit to a lesser extent than did live virus demonstrating inherent stimulatory properties of DV particles. Using intracellular staining for DV envelope (E) protein, I detected increased surface molecule expression on both infected DCs and uninfected bystander DCs from the same culture, as compared to mock-infected DCs. These data indicate that activation was not prevented in cells undergoing active viral replication. However, the degree of surface molecule induction depended on the infection state of the cell. Infected DCs had enhanced PD-L2 and MHC II expression relative to uninfected bystander cells, while PD-L1, CD80, CD86, and MHC I expression were suppressed with active infection. Therefore, intracellular DV replication altered the process of cell surface molecule regulation within these cells. DV infection of DCs also resulted in the secretion of a broad array of cytokines and chernokines. These included the antiviral cytokine IFN-α, inflammatory cytokines TNF-α, IL-6, and IL-1α, and inflammatory chemokines IP10, MCP-1, MIP-1α, and RANTES. DV infection did not induce DC production of the IL-12 p70 heterodimer, and secretion of the immunosuppressive cytokine IL-10 was low in most experiments. Similar to the results seen with surface molecule induction, UV inactivation of DV reduced, but did not eliminate, cytokine and chemokine responses. At the single-cell level, TNF-α and IP10 production profiles of infected DCs and uninfected bystander DCs were distinct. DV infection in DCs reduced production of IP10, but stimulated TNF-α as compared to uninfected bystander cells in the same culture. Blocking experiments demonstrated that IFN-α/β produced by DCs in response to infection actively inhibited viral protein expression and drove IP10, but not TNF-α, production. DV infection of DCs did not consistently suppress DC stimulation of allogeneic CD4 T cell proliferation. In cases where infection enhanced DC stimulatory function, T cell proliferation was less pronounced than that induced by DCs activated with exogenous TNF-α plus IFN-α. Increasing multiplicity of infection (MOI) of DCs with DV resulted in increasing DC infection rates, but a statistically significant trend at the highest MOIs for decreased T cell alloproliferation, suggesting that direct infection of DCs reduces their CD4 T cell priming function. MOI-dependent reduction in DC stimulatory function depended on replication-competent virus. Increased MOIs during DV infection of DCs did not cause an elevation in detectable IL-10 in supernatants derived from T-DC co-cultures. In addition, increased DV MOI of DCs was not associated with increased levels of either IL-13 or IFN-γ in supernatants from T-DC co-culture, suggesting that actively infected DC do not skew CD4 T cells towards a specific Th phenotype. These data demonstrate that DV infection induces functional maturation of DCs that is modified by the presence of virus through both IFN-dependent and independent mechanisms. However, the allostimulatory phenotype of DCs was not universally enhanced, nor was it skewed towards antiviral (Th1)-type responses. These data suggest a model whereby dengue infection during primary illness results in controlled immune stimulation through activation of bystander DCs, and the generation of mixed Th-type responses. Direct DV infection of DCs appears to attenuate activation of, and potentially clearance by, antiviral mechanisms. During secondary infection, reduced IP10 production and enhanced TNF-α secretion by infected cells coupled with MHC I downregulation and enhanced PD-L2 expression, would subvert both Th1 CD4 T cell recruitment and result in CD8 T cell suppression and death. Furthermore, DV-specific effects on DCs would allow for continued viral replication in the absence of effective clearance. These DV-mediated effects would modify T cell memory responses to infected DC, and potentially facilitate the expansion of pathologic T cell subsets. Contributing to this pathological cascade, antibody-dependent enhancement of infection in monocytic cells and macrophages would shift antigen presentation and cytokine production paradigms, increasing the risk of DHF.

Two decades after the discovery of the Hepatitis C Virus (HCV), Hepatitis C infection still persists to be a global health problem. With the recent approval of the first set of directly acting antivirals (DAAs), the rate of sustained viral response for HCV-infected patients increased significantly. However, a complete cure has not been found yet. Drug development efforts primarily target NS3/4A protease, bifunctional serine protease-RNA helicase of HCV. HCV NS3/4A is critical in viral function; protease domain processes the viral polyprotein and helicase domain aids replication of HCV genome by unwinding double stranded RNA transcripts produced by NS5B, RNA-dependent RNA polymerase of HCV. Protease and helicase domains can be isolated, expressed and purified separately while retaining function. Isolated domains of HCV NS3/4A have been extensively used in biochemical and biophysical studies for scientific and therapeutic purposes to evaluate functional capability and mechanism. However, these domains are highly interdependent and modulate the activities of each other bidirectionally. Interdomain dependence was demonstrated in comparative studies where activities of isolated domains versus the full length protein were evaluated. Nevertheless, specific factors affecting interdependence have not been thoroughly studied. Chapter II investigates the domain-domain interface formed between protease and helicase domains as a determinant in interdependence. Molecular dynamics simulations performed on single chain NS3/4A constructs demonstrated the importance of interface in the coupled dynamics of the two domains. The role of the interface in interdomain communication was experimentally probed by disrupting the domain-domain interface through Ala-scanning mutations in selected residues in the interface with significant buried surface areas. These interface mutants were assayed for both helicase and protease related activities. Instead of downregulating the activities of either domain, interface mutants caused enhancement of protease and helicase activities. In addition, the interface had minimal effect in RNA unwinding activity of the helicase domain, the mere presence of the protease domain was the main protagonist in elevated RNA unwinding activity. In conclusion, I suspect that the interface formed between the domains is transient in nature and plays a regulatory role more than a functional role. In addition, I found results supporting the suggestion that an alternate domain-domain arrangement other than what is observed in crystal structures is the active, biologically relevant conformation for both the helicase and the protease. Chapter III investigates structural features of HCV NS3/4A protease inhibitors in relation to effects on inhibitor potency, susceptibility to drug resistance and modulation of potency by the helicase domain. Nearly all NS3/4A protease inhibitors share common features, with major differences only in bulky P2 extension groups and macrocyclization statuses. Enzymatic inhibition profiles of different drugs were analyzed for wildtype isolated protease domain and single chain NS3/4A helicase-protease construct, their multi drug resistant variants, and additional helicase mutants. Inhibitor potency was mainly influenced by macrocyclization, where macrocyclic drugs were significantly more potent compared to acyclic variants. Potency loss with respect to resistance mutations primarily depended on the P2 extension, while macrocyclization had minimal effect except for P2-P4 macrocyclic compounds which were up to an order of magnitude more susceptible to mutations A156T and, in lesser extent, D168A. Modulation by helicase domain was also dependent on P2 extension, although opposite trends were observed for danoprevir analogs versus others. In conclusion, this study provides a basis for future inhibitor development in both avoiding drug resistance and exploitation of the helicase domain for additional efficacy. In this thesis, I have provided evidence further supporting and revealing the details of domain-domain dependency in HCV NS3/4A. Lessons learned here will aid future research for dissecting the interdependency to gain a better understanding of HCV NS3/4A function, which can possibly be extended to all Flaviviridae NS3 protease-helicase complexes. In addition, interdomain dependence can be exploited in future drug development efforts to create better drugs that will pave the way to an effective cure.

Vaccinia virus (VAC) is the prototypical member of the orthopoxvirus genus of the poxvirus family and the virus used for smallpox vaccinations. The following describes the testing of VAC variants designed to have similar immuno-protective profiles with decreased pathogenicity, examines the immune response to VAC after lethal infection in wild type and lupus-prone mice, and describes a method that allows for the enumeration of VAC-specific CD8+ T in naïve and VAC-immune mice. The first part describes work examining VAC Wyeth (VAC-Wy) variants engineered to be less pathogenic in vivo. VAC-Wy variants included genes that code for three immunomodulatory proteins, an interferon-γ (IFNγ) binding protein (B8R), an interleukin 18 (IL-18) binding protein (C12L), and a complement binding protein (C3L, or C21L) or various combinations of the three knockouts, and a triple knockout (VAC-Wy -/-/-) in which all three genes were knocked out of a variant virus. The immunomodulatory effects of other IFNγ binding proteins on VAC-Wy pathogenesis in the mouse were also examined. Virus recombinants where the B8R gene was replaced with a truncated mouse IFNγ receptor gene or a gene that encodes a B8R/IFNγ fusion that allows for dimerization of the secreted IFNγ receptor were studied. As the knockouts and variants were made in the current vaccine VAC-Wy strain, only high dose (1x107 PFU’s) intra nasal (I.N.) infection of mice reliably resulted in detectable virus in the lungs. Further testing revealed that all knockout and variant viruses grew to similar levels after high dose I.N. infections. Protection induced by vaccination with the VAC-Wy variants was studied in comparison to immunizations with the VAC-Wy parental strain. Mice were immunized by tail skin scarification to mimic human immunizations, and this was followed months later by I.N. challenge with 20 LD50’s of VAC-WR. All VAC-Wy recombinants tested, including the VAC-Wy -/-/-, provided similar levels of protection as the parental VAC-Wy strain from a lethal VAC-WR I.N. infection. Mice immunized with the VAC-Wy -/-/- induced similar amounts of neutralizing antibody and similar numbers of CD8+ T cells specific to a subdominant determinant as VAC-Wy. While examining high dose, normally lethal, VAC-WR I.N. infections, a profound splenic CD8+ T cell immune suppression was noted that might have been caused by Fas dependent activation induced cell death (AICD). Using high dose intra-peritoneal (I.P.) and I.N. models of VAC-WR infection, decreased weight loss, decreased virus titers, and increased T cell numbers were found in Fas mutant (B6.MRL-Faslpr/J) mice in comparison to B6 wild type mice on day 6. It would be expected that Fas-deficient CD8+ T cells from B6.MRL-Faslpr/J mice (B6-lpr) would survive a high dose VAC-WR infection better than CD8+ T cells that could express Fas if T cells were being eliminated by Fas-dependent AICD, but co-adoptive transfer experiments using splenocytes from B6-lpr and B6.Cg- IgHaThy-1aGPi-1a/J (IgHa) wild type counterparts found no difference in the numbers or proliferation of donor CD8+ T cells at day 6. As the B6-lpr mice were better protected from VAC-induced weight loss early after lethal VAC-WR infections, it was possible that B6-lpr mice might be protected early in infection. In fact, Fas mutant mice had decreased virus loads in the fat pads, livers, and spleens in comparison to B6 wild type mice at days 2 and 3. In addition to the decreased virus titers, the severe splenic lymphocyte deficiency noted in B6 wild type mice as early as day 2 after high dose I.P. infection was ameliorated in B6-lpr mice. Further experiments demonstrated that uninfected B6-lpr mice had increased numbers of memory phenotype (CD44+) CD4+, CD8+ and γδ+ T cells, with an increased number of γδ+ T cells and NK cells in splenic lymphocytes in comparison to wild type B6 mice. Uninfected B6-lpr mice also had increased numbers of IFNγ+ CD8+ T cells after polyclonal stimulation with an antibody against CD3ε. In lymphocyte depletion experiments performed at day 3, antibody depletion of CD4, CD8, or NK or treatment with an antibody that was specific to the γδ+ TCR did not significantly alter virus loads in B6-lpr mice. In co-adoptive transfer experiments, splenocytes from wild type or B6-lpr mice survived high dose VAC-WR challenge similarly suggesting that B6- lpr splenocytes were not intrinsically better protected from lymphocyte depletion by lack of the Fas protein. On day 2 after high dose I.P. VAC-WR infection, B6- lpr mice had increased numbers of IFNγ+ NK cells, IFNγ+ CD8+ T cells, and IFNγ+ CD4+ T cells. B6-lpr and B6 mice treated with an antibody against IFNγ had significantly increased virus titers in the spleens and livers. Interestingly, there was no significant difference in liver or spleen virus titers when comparing anti- IFNγ antibody treated B6 mice or anti-IFNγ antibody treated B6-lpr mice. These results suggest that multiple leukocyte populations co-operatively or redundantly provide B6-lpr mice with increased protection from high dose VAC-WR infections through increased production of IFNγ. The third part of this work describes the enumeration of total numbers of pathogen-specific CD8+ T cells in a mouse through use of an in vivo limiting dilution assay (LDA). The extensive proliferation of virus-specific CD8+ T cells that occurs after virus infection was used to enumerate numbers of virus-specific CD8+ T cells in a naïve mouse. By transferring limiting amounts of carboxyfluorescein succinimidyl ester (CFSE)-labeled Thy1.1+Ly5.2+ heterogeneous CD8+ T cells into Thy1.2+Ly5.1+ hosts, CD8+ T cell precursor frequencies to whole viruses can be calculated. The calculations are based on finding the number of donor CD8+ T cells that results in CFSElo (i.e. proliferated) donor CD8 T cells in 50% of the hosts. Using probit or Reed and Muench 50% endpoint calculations, CD8+ T cell precursor determinations were made for naïve and immune states to a virus challenge. It was found that in naïve B6 mice, 1 in 1444 CD8+ T cells proliferated in response to VAC-WR (~13,852 VAC-WR-specific CD8+ T cells per mouse) and 1 in 2956 proliferated in response to lymphocytic choriomeningitis virus (LCMV) (~6,761 LCMV-specific CD8+ T cells per mouse). In mice immune to VAC-WR, the number of VAC-WR-specific LDA precursors, not surprisingly, dramatically increased to 1 in 13 (~1,538,462 VAC-WR- specific CD8+ T cells per mouse) consistent with estimates of VAC-WR-specific memory T cells. In contrast, precursor numbers to LCMV did not increase in VAC-WR-immune mice (1 in 4562, ~4384 LCMV-specific CD8+ T cells in a VAC-WR-immune mouse) consistent with the fact that VAC-WR provides no heterologous immunity to LCMV. Using H-2Db-restricted LCMV GP33-specific P14 transgenic T cells it was found that, after accounting for take of donor T cells, approximately every T cell transferred underwent a full proliferative expansion in response to an LCMV infection and a high efficiency was also seen in memory populations. This suggests that most antigen-specific T cells will proliferate in response to infections at limiting dilution. These results, which are discussed in comparison to other methods, show that naïve and memory CD8+ T cell precursor frequencies to whole viruses can be remarkably high. In total this work further advances knowledge of the immunity, pathogenesis, and prevention of poxvirus infections. This was accomplished by studying VAC-Wy recombinants as improved vaccines, by examining the mechanisms and cell types important in early protection from high dose poxvirus infections in B6 and B6-lpr mice, and by describing a method to enumerate total numbers of virus-specific CD8+ T cells in a mouse.

Long-term antiviral antibody responses provide protection from re-infection and recurrence of persistent viruses. Using a polyomavirus (PyV) mouse model, our lab has shown that MyD88-deficient mice generate low levels of virus-specific IgG after the acute phase of infection and that these IgG responses have a skewed isotype distribution with low levels of IgG2a/c. Moreover MyD88-deficient mice have reduced numbers of long-lived plasma cells in the bone marrow. These studies suggest an important role of MyD88-mediated signaling in long-term antiviral responses. Our lab has shown that T cell-deficient mice can also maintain long-term virus-specific IgG responses following PyV infection. The goal of this thesis is to evaluate the role of innate signaling pathways in maintaining serological memory to persistent virus infection and to elaborate on how long-term antiviral responses can be maintained in an immunocompetent or partially immune compromised, T cell-deficient host. Regarding T cell-dependent B cell responses, I set out to investigate the upstream and downstream components of the MyD88-mediated pathways required for normal antibody isotype and long-term humoral responses. IgG2a is a predominant immunoglobulin isotype in most virus infections. Wild type mice, in response to PyV infection, primarily induce antiviral IgG2a with some IgG1. MyD88-deficient mice in response to PyV infection display attenuated levels of virus-specific IgG2a, but normal levels of IgG1. Using Unc93B1 mutant mice (3d mice), which are defective in TLRs 3, 7 and 9 signaling, I show that 3d mice also generated low levels of virus-specific IgG2a following PyV infection. Studies in individual TLR3-/-, TLR7-/- or TLR9-/- mice displayed PyV-specific IgG2a responses similar to wild type responses. TLR7 and TLR9 double deficient mice generated similar skewed antibody isotype responses, where virus-specific IgG2a was reduced compared to wild type mice. This shows that TLR7 and TLR9-MyD88 mediated pathways are important in regulating IgG2a responses during a PyV infection. To investigate what components downstream of MyD88 are involved in mediating IgG2a responses, I worked with IRF5-deficient mice. IRF5 is a transcription factor that is activated upon stimulation of TLR7 or TLR9-MyD88-mediated pathways. Moreover, IRF5-deficient mice cannot generate autoantibodies specifically of the IgG2a isotype in a mouse lupus model, suggesting that IRF5 plays an important function in mediating class switching to IgG2a. In vitro studies where IRF5-/- B cells were stimulated with TLR7 or TLR9 ligands also generated low levels of γ2a germ-line transcripts, suggesting a B cell-intrinsic role for IRF5 in regulating γ2a germ-line transcription. PyV infection of IRF5-deficient mice resulted in similar skewed isotypes as observed in MyD88-deficient and 3d mice. To investigate a B cell-intrinsic role for IRF5 in regulating IgG2a responses in vivo upon PyV infection, I transferred IRF5-/- B cells and WT T cells into RAG KO mice prior to infection and compared the responses of these mice with mice reconstituted with wild type B6 B and T cells. Diminished numbers of IgG2a+ B cells and reduced levels of virus-specific IgG in mice reconstituted with IRF5-/- B cells were seen compared to mice reconstituted with wild type B cells. Regarding the defect in long-term IgG production in MyD88-/- mice upon PyV infection, I conducted studies in IRF5-/-, 3d, single TLR3-/-, TLR7-/-, TLR9-/- and TLR7/9 double deficient mice. These studies reveal an important and redundant role for TLR7- and TLR9-MyD88 signaling in maintaining long-term anti-PyV IgG responses. To determine how MyD88 signaling affects the generation of long-lived plasma cells and memory B cells, I investigated germinal center (GC) responses in MyD88-deficient mice. A defect in GC B cell numbers is observed in MyD88-deficient mice after the acute phase of infection. The GC reaction is essential for the generation and maintenance of long-lived plasma cells and memory B cells. T follicular helper (TFH) cells are absolutely required to generate normal GC. l found reduced numbers of TFH cells in MyD88-deficient mice. Lower numbers of T FH cells suggests that poor T cell help may contribute to the diminished number of GC B cells. However, interaction with B cells is required for the formation of fully differentiated TFH cells. Along with B cell function, MyD88 signaling can affect T cell and dendritic cell function as well. Thus, it is not clear at this point whether the requirement for intact MyD88 signaling for the formation and maintenance of long-term B cell populations is completely B cell-intrinsic. Some viruses can induce T cell-independent B cell responses, perhaps due to their complex arrays of repetitive antigenic epitopes on virions, coupled with the induction of innate cytokines. Nevertheless, T cell help is usually necessary for generating long-term antibody responses in the form of long-lived plasma cells and memory B cells. In contrast, our lab has found that T cell-deficient mice infected with PyV develop long-lasting, protective antiviral IgG responses. I questioned whether these mice could generate TI B cell memory cells or long-lived plasma cells. I show that long-lasting anti-PyV antibody in T cell-deficient mice was not due to the presence of long-lived plasma cells or memory B cell responses. TCRβδ deficient mice, which lack both CD4 and CD8 T cells, had ~10 a times higher virus load persisting in various organs. Therefore, I hypothesized that the high level of persistent PyV antigen, in completely T cell-deficient mice, may activate naïve B cell populations continuously, thereby maintaining the long-lasting IgG responses. Prior to PyV infection, T cell-deficient mice received wild type CD8 T cells, which reduced PyV loads, and this was associated with decreased levels of antiviral serum IgG over time. As in TCRβδ deficient mice, high PyV loads were detected in the bone marrow, which is the site for B cell lymphopoiesis, I questioned how B cells develop in the presence of PyV antigen and still stay responsive to PyV, generating long-term antiviral IgG responses in the periphery. Studies have shown that self-antigens that trigger both B cell receptor signaling and TLR-MyD88 signaling pathways in the bone marrow lead to the breaking of B cell tolerance and production of autoantibody in the periphery. Thus, we hypothesized that high PyV levels in the bone marrow signal through both B cell-receptors and TLRs, allowing continuous antiviral antibody production by B cells. Using mice that are deficient in T cells and MyD88 signaling, I found that PyV-specific TI IgG levels gradually decreased, supporting this hypothesis. Thus, high PyV loads and innate signaling together can break B cell tolerance. During a persistent virus infection this can result in sustaining long-term protective T cell-independent IgG responses.

The innate immune system provides an essential first line of defense against infection. Innate immune cells detect pathogens through several classes of Pattern Recognition Receptors (PRR) allowing rapid response to a broad spectrum of infectious agents. Activated receptors initiate signaling cascades that lead to the production of cytokines, chemokines and type I interferons all of which are vital for controlling pathogen load and coordinating the adaptive immune response. Detection of nucleic acids by the innate immune system has emerged as a mechanism by which infection is recognized. Recognition of DNA is complex, influenced by sequence, structure, covalent modification and subcellular localization. Interestingly certain synthetic oligodeoxynucleotides comprised of the TTAGGG motif inhibit proinflammatory responses in a variety of disease models. These suppressive oligodeoxynucleotides (sup ODN) have been shown to directly block TLR9 signaling as well as prevent STAT1 and STAT4 phosphorylation. Recently AIM2 has been shown to engage ASC and assemble an inflammasome complex leading to the caspase-1-dependent maturation of IL-1β and IL-18. The AIM2 inflammasome is activated in response to cytosolic dsDNA and plays an important role in controlling replication of murine cytomegalovirus (MCMV). In the second chapter of this thesis, a novel role for the sup ODN A151 in inhibiting cytosolic nucleic acid sensing pathways is described. Treatment of dendritic cells and macrophages with the A151 abrogated type I IFN, TNF-α and ISG induction in response to cytosolic dsDNA. A151 also reduced INF-β and TNF-α induction in BMDC and BMDM responding to the herpesviruses HSV-1 and MCMV but had no effect on the responses to LPS or Sendai virus. In addition, A151 abrogated caspase-1-dependent IL-1β and IL-18 maturation in dendritic cells stimulated with dsDNA and MCMV. Although inhibition of interferon-inducing pathways and inflammasome assembly was dependent on backbone composition, sequence differentially affected these pathways. While A151 more potently suppressed the AIM2 inflammasome, a related construct C151, proved to be a more potent inhibitor of interferon induction. A151 suppressed inflammasome signaling by binding to AIM2 and competing with immune-stimulatory DNA. The interaction of A151 and AIM2 prevented recruitment of the adapter ASC and assembly of the macromolecular inflammasome complex. Collectively, these findings reveal a new route by which suppressive ODNs modulate the immune system and unveil novel applications for suppressive ODNs in the treatment of infectious and autoimmune diseases. The innate immune response to HSV-1 infection is critical for controlling early viral replication and coordinating the adaptive immune response. The cytokines IL-1β and IL-18 are important effector molecules in the innate response to HSV-1 in vivo. However, the PRRs responsible for the production and maturation of these cytokines have not been fully defined. In the third chapter of this thesis, The TLR2-MyD88 pathway is shown to be essential for the induction of pro-IL-1β transcription in dendritic cells and macrophages responding to HSV-1. The HSV-1 immediate-early protein ICP0 has previously been shown to block TLR2 responses and in keeping with this finding, ICP0 blocked pro-IL-1β expression. Following translation, pro-IL-1β exists as an inactive precursor that must be proteolytically cleaved by a multiprotein complex known as the inflammasome to yield its active form. Inflammasomes are composed of cytoplasmic receptors such as NLRP3 or AIM2, the adapter molecule ASC, and pro-caspase-1. In the present study we found that the NLRP3 inflammasome is important for maturation of IL-1β in macrophages and dendritic cells responding to HSV-1. In contrast the related NLRP12 protein controls IL-1β production in neutrophils. These data indicate that sensing of HSV-1 by TLR2 drives pro-IL-1β transcription and infection activates the inflammasome to mature this cytokine. Moreover, these studies reveal cell type-specific roles for NLRP3 and NLRP12 in inflammasome assembly.

HIV/AIDS is a disease of grave global importance with over 33 million people infected world-wide and nearly 2 million deaths each year. The rapid emergence of drug resistance, due to viral mutation, renders anti-retroviral drug candidates ineffective with alarming speed and regularity. Instead of targeting mutation prone viral proteins, an alternative approach is to target host proteins that interact with viral proteins and are critical for the HIV life-cycle. APOBEC3G is a host anti-HIV restriction factor that can exert tremendous negative pressure by hypermutating the viral genome and has the potential to be a promising candidate for anti-retroviral therapeutic research. The work presented in this thesis is focused on investigating the A3G catalytic domain structure and implications of various observed structural features for biological function. High-resolution crystal structures of the A3G catalytic domain were solved using data from macromolecular X-ray crystallographic experiments, revealing a novel intermolecular zinc coordinating motif unique to A3G. Major intermolecular interfaces observed in the crystal structure were investigated for relevance to biochemical activity and biological function. Co-crystallization with a small-molecule A3G inhibitor, discovered using high-throughput screening assays, revealed a cysteine residue near the active site that is critical for inhibition of catalytic activity by catechol moieties. The serendipitous discovery of covalent interactions between this inhibitor and a surface cysteine residue led to further biochemical experiments that revealed the other cysteine, near the active site, to be critical for inhibition. Computational modeling was used to propose a steric-hinderance based mechanism of action that was supported by mutational experiments. Structures of other human APOBEC3 homologs were modeled using in-silico methods examined for similarities and differences with A3G catalytic domain crystal structures. Comparisons based on these homology models suggest putative structural features that may endow substrate specificity and other characteristics to the APOBEC3 family members.

Telomere length has been shown to be a critical determinant of T cell replicative capacity and in vivo persistence in humans. We evaluated telomere lengths in virus-specific T cells to understand how they may both shape and be changed by the maintenance of memory T cells during a subsequent virus re-infection or reactivation. We used longitudinal peripheral blood samples from healthy donors and samples from a long-term HCV clinical interferon therapy trial to test our hypotheses. To assess T cell telomere lengths, I developed novel modifications to the flow cytometry fluorescence in situ hybridization (flowFISH) assay. These flowFISH modifications were necessary to enable quantification of telomere length in activated, proliferating T cells. Adoption of a fixation-permeabilization protocol with RNA nuclease treatment prior to telomere probe hybridization were required to produce telomere length estimates that were consistent with a conventional telomere restriction fragment length Southern blot assay. We hypothesized that exposure to a non-recurring, acute virus infection would produce memory T cells with longer telomeres than those specific for recurring or reactivating virus infections. We used two acute viruses, vaccinia virus (VACV) and influenza A virus (IAV) and two latent-reactivating herpesviruses, cytomegalovirus (CMV) and varicella zoster virus (VZV) for these studies. Combining a proliferation assay with flowFISH, I found telomeres in VACV-specific CD4 + T cells were longer than those specific for the recurring exposure IAV; data which support my hypothesis. Counter to my hypothesis, CMV-specific CD4 + T cells had longer telomeres than IAV-specific CD4 + T cells. We assessed virus-specific CD4 + T cell telomere length in five donors over a period of 8-10 years which allowed us to develop a linear model of average virus-specific telomere length changes. These studies also found evidence of long telomere, virus-specific CD45RA + T cell populations whose depletion may precede an increased susceptibility to latent virus reactivation. I tested the hypothesis that type I interferon therapy would accelerate T cell telomere loss using PBMC samples from a cohort of chronic hepatitis C virus patients who either did or did not receive an extended course of treatment with interferon-alpha. Accelerated telomere losses occurred in naïve T cells in the interferon therapy group and were concentrated in the first half of 48 months of interferon therapy. Steady accumulation of CD57 + memory T cells in the control group, but not the therapy group, suggested that interferon also accelerated memory turnover. Based on our data, I present proposed models of memory T cell maintenance and impacts of T cell telomere length loss as we age.

The Hepatitis C Virus (HCV) is a global health problem as it afflicts an estimated 170 million people worldwide and is the major cause of viral hepatitis, cirrhosis and liver cancer. HCV is a rapidly evolving virus, with 6 major genotypes and multiple subtypes. Over the past 20 years, HCV therapeutic efforts have focused on identifying the best-in-class direct acting antiviral (DAA) targeting crucial components of the viral lifecycle, The NS3/4A protease is responsible for processing the viral polyprotein, a crucial step in viral maturation, and for cleaving host factors involved in activating immunity. Thus targeting the NS3/4A constitutes a dual strategy of restoring the immune response and halting viral maturation. This high priority target has 4 FDA approved inhibitors as well as several others in clinical development. Unfortunately, the heterogeneity of the virus causes seriously therapeutic challenges, particularly the NS3/4A protease inhibitors (PIs), which suffer from both the rapid emergence of drug resistant mutants as well as a lack of pan-genotypic activity. My thesis research focused on filling two critical gaps in our structural understanding of inhibitor binding modes. The first gap in knowledge is the molecular basis by which macrocyclization of PIs improves antiviral activity. Macrocycles are hydrophobic chains used to link neighboring chemical moieties within an inhibitor and create a structurally pre-organized ligand. In HCV PIs, macrocycle come in two forms: a P1 - P3 and P2 - P4 strategy. I investigated the structural and thermodynamic basis of the role of macrocyclization in reducing resistance susceptibility. For a rigorous comparison, we designed and synthesized both a P1 - P3 and a linear analog of grazoprevir, a P2 - P4 inhibitor. I found that, while the P2 - P4 strategy is more favorable for achieving potency, it does not allow the inhibitor sufficient flexibility to accommodate resistance mutations. On the other hand, the P1 - P3 strategy strikes a better balance between potency and resistance barrier. The second gap my thesis addresses is elucidating the structural basis by which highly potent protease inhibitors function in genotype 1 but not in genotype 3, despite having an 87% sequence similarity. After mapping the amino acids responsible for this differential efficacy in genotypes 1 and 3, I engineered a 1a3a chimeric protease for crystallographic studies. My structural characterization of three PIs in complex with both the 1a3a and genotype 1 protease revealed that the loss of inhibitor efficacy in the 1a3a and GT-3 proteases is a consequence of disrupted electrostatic interactions between amino acids 168 and 155, which is critical for potent binding of quinoline and isoindoline based PIs. Here, I have revealed details of molecular and structural basis for the lack of PI efficacy against GT-3, which are needed for design of pan-genotypic inhibitors.