<p> Mucosal surfaces act as functional barriers against the perpetual bombardment of foreign antigens and pathogens to the body. This barrier is maintained by homeostatic interactions between the microbiome and cells of the innate and adaptive immune system, interactions that mucosal vaccines can exploit to yield both mucosal and systemic amnestic responses to foreign antigen. The commensal lactic acid <i>Lactobacillus</i> spp. represent one constituent of this microbiome that has been utilized as both a homeostatic promoting probiotic and as a vaccine vector. The immune modulatory capacity of <i>Lactobacillus</i> spp. has been demonstrated in proof-of-principle studies utilizing lactobacilli-based vaccine vectors against several pathogens. Our laboratory has focused on the development of <i>Lactobacillus gasseri </i> and <i>Lactobacillus acidophilus</i> NCFM (NCFM) as mucosal vaccine vectors for human immunodeficiency virus-1 (HIV-1), a mucosal pathogen affecting more than 35 million people worldwide and for which no current licensed vaccine exists. As activation of innate immune receptors, including toll-like receptor (TLR), NOD-like receptor (NLR), and C-type lectin receptor (CLR), by lactobacilli have been shown to be species and strain specific, characterizing the innate receptors specific to our vectors is important for rationale vaccine design. </p><p> We first demonstrate that in addition to the previously characterized TLR2/6 activating capacity of lactobacilli, <i>L. gasseri </i>and NCFM activate intracellular NOD2 receptor. Co-culture of murine macrophages with <i>L.gasseri</i>, NCFM, or NCFM-derived mutants NCK2025 and NCK2031 elicited an M2b-like phenotype, a phenotype associated with TH2 skewing and immune regulatory function. For NCFM, this M2b phenotype was dependent on expression of lipoteichoic acid and S layer proteins, as demonstrated by the use of respective mutants, NCK2025 and NCK2031. Through the use of macrophage genetic knockouts, we identified TLR2, NOD2, and inflammasome associated caspase 1 as contributors to macrophage activation to varying degrees, with NOD2 cooperating with caspase 1 for inflammasome derived IL-1&beta; in a pyroptosis-independent fashion. Finally, utilizing an NCFM-based mucosal vaccine with surface expression of HIV-1 Gag, we show that NOD2 signaling and the presence of an intact microbiome is required for HIV-specific IgG. We show that lactobacilli differentially utilize innate immune pathways and highlight NOD2 as a key mediator of macrophage function and antigen-specific humoral responses to a NCFM-based mucosal vaccine vector.</p>

<p> Viral invasion of the central nervous system (CNS) is a significant cause of morbidity and mortlity worldwide, particularly in young children (1). The nervous system presents a challenging site for viruses to access, with multiple physical and immunological barriers that limit pathogen invasion. To invade the CNS, viruses must access cell-surface receptors for binding and entry events. Virus-receptor interactions also govern tropism and often control disease type and severity. For many viruses, the identities of receptors and other cellular determinants of viral tropism remain elusive. Understanding where and how viral capsid components engage neural receptors and the effect of these interactions on tropism and disease may illuminate targets to prevent viral neuroinvasion.</p><p> Mammalian orthoreoviruses (reoviruses) provide a highly tractable and well-established system to identify mechanisms of viral entry into the CNS. Reoviruses are non-enveloped particles containing a 10-segmented, double-stranded (ds) RNA genome that replicate well in culture and can be altered via a robust reverse-genetics system (2, 3). While reovirus causes similar age-restricted disease in many young mammals (4-6), most studies employ newborn mice. Following peroral or intracranial inoculation of newborn mice, reovirus displays serotype-specific patterns of tropism in the brain and concomitant disease (<b>Fig. I-1</b>). Serotype 1 (T1) strains infect ependymal cells lining the ventricles of the brain and cause a non-lethal hydrocephalus (7). In contrast, serotype 3 (T3) strains infect specific neuron populations in the CNS and produce a fulminant, and often lethal, encephalitis (8). These differences in tropism and disease have been genetically mapped to the reovirus S1 gene using single-gene reassortant viruses (9). However, viral and host gene sequences that mediate either T1 or T3 tropism have not been defined. </p><p> In Chapter I of my dissertation, I introduce key themes about mechanisms of neuroinvasion and the disease consequences of CNS infection. I describe fundamental knowledge and open areas of research pertaining to reovirus infection in the CNS and expand on reovirus-receptor interactions. I conclude Chapter I with a summary of viral oncolytic therapies and highlight strengths and opportunities for improvement of reovirus oncolytics. In Chapter II, I describe the design and implementation of &sigma;1- chimeric reoviruses to identify sequences in the S1 gene that dictate neurotropism and virulence in the CNS. In these studies, I found that homologous sequences at the viriondistal end of the viral attachment protein are responsible for neuron and ependymal cell targeting. In Chapter III, I identify sequences of the NgR1 reovirus receptor that are required for binding and post-binding functions and elucidate the viral ligand for NgR1, which is the &sigma;3 outer-capsid protein, using a combination of genetic, biochemical, and structural approaches. Finally, in Chapter IV, I review conclusions from results presented in Chapters II and III, examine new questions raised by these studies, and discuss future directions of this work. Collectively, my dissertation research has unveiled viral and host sequences that contribute to neural cell targeting and will improve strategies and knowledge to design targeted oncolytic therapies.</p><p>

<p> Flaviviruses are a diverse group of emerging arboviruses capable of infecting an extraordinarily broad range of vertebrate and invertebrate hosts. Nearly half of the viruses in this rapidly expanding genus have been reported to be pathogenic for humans, as well as other vertebrates. The spectrum of human disease includes asymptomatic and febrile illnesses, rash, arthralgia, encephalitis and hemorrhagic fever. The recent outbreak of Zika virus (ZIKV) has uncovered pathology in the form of microcephaly and Guillain-Barr&eacute; syndrome, cementing the importance of flaviviruses as emerging human pathogens. All vector-borne flaviviruses studied thus far have to overcome type I interferon (IFN) antiviral responses in order to replicate and cause disease in vertebrates. The non-structural protein NS5 is a potent and specific antagonist of IFN signaling for human pathogenic flaviviruses such as dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and tick-borne encephalitis viruses (TBEVs). Intriguingly, each of these viruses exhibits different mechanisms of IFN antagonism, highlighting the complicated evolutionary nature of flaviviruses. This thesis work presents novel insights into the NS5-mediated antagonism of IFN signaling for several underexamined flaviviruses. Notably, all NS5 proteins examined were able to inhibit IFN-induced gene expression in a mammalian system, indicating a functional conservation of IFN antagonism for flavivirus NS5 proteins. However, mechanistically NS5 function was diverse. Of great interest, ZIKV NS5 bound to the human, but not mouse, IFN-regulated transcriptional activator STAT2 and targeted it for proteasomal degradation. This phenomenon may explain the requirement for IFN deficiency in order to observe ZIKV pathogenesis in mice. Furthermore, the mechanism of ZIKV NS5 resembles that of DENV NS5, but not that of its closer relative Spondweni virus (SPOV). However, unlike DENV NS5, ZIKV NS5 did not require the E3 ubiquitin ligase UBR4 to induce STAT2 degradation. Consequently, flavivirus NS5 proteins exhibit a remarkable functional convergence in IFN antagonism, albeit by virus-specific mechanisms. The potent antagonism of human IFN responses by neglected flaviviruses such as SPOV and Usutu virus (USUV), coupled with similar ecologies to that of known human flavivirus pathogens, suggests their potential for broad emergence into the human population.</p><p>