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<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>

Brachyspira hyodysenteriae is an anaerobic intestinal spirochaete and the aetiological agent of swine dysentery (SD). Throughout the UK and Europe, pathogenic and potential non-pathogenic isolates of B. hyodysenteriae have been recovered from pig herds, creating major obstacles for the detection and control of this economically important pathogen. Therefore, the main aim of this research was to compare one representative of virulent (P8544) and one representative of avirulent (P7455) strains of B. hyodysenteriae using genomic and proteomics approaches with a view to identify distinctive genes or proteins. The B. hyodysenteriae draft genomes of P8544 and P7455 consisted of a circular 3.0 Mb chromosome and a 31,469-34,822 bp circular plasmid that is also present in the only published B. hyodysenteriae genome, WA1. A considerable number of genes (~27-35) were identified in both the virulent and avirulent strains that shared high sequence homology with genes found in other spirochaetes, such as B. murdochii and B. intermedia, as well as in other species of bacteria; these may have been acquired via horizontal gene transfer. Comparative genomics of the two pathogenic genomes P8544 and WA1 versus the non-pathogenic genome P7455 revealed that the gene encoding for the methyltransferase type 11 (Bhyoa7455_20) was identified as being unique to the P7455 plasmid sequence and was successfully PCR amplified in a greater number of avirulent than virulent strains. However, as this was only just statistically significant (P=0.049), screening of a much larger strain set would clearly be required to support this gene as a suitable genetic marker to distinguish virulent and avirulent B. hyodysenteriae strains. Bacterial acquisition of iron in-vivo is crucial for successful colonisation and persistence in the host. A further aim of this study was to compare the growth phenotype of B. hyodysenteriae isolates P8544 and P7455 grown under iron-limiting conditions; such as would be found in-vivo in the large intestine of the host. Analysis of P8544 and P7455 growth rate in iron-sequestered media (containing 0.1 mM of the iron-chelator dipyridyl) demonstrated that both these isolates could replicate in this media although with an extended lag-phase of approximately 32-34 hrs; growth rate was on par with the iron-replete conditions. qRT-PCR analysis of eight putative iron-acquisition genes under iron-sequestered and iron-replete conditions revealed a difference in transcription for a number of ABC-transporter genes in P8544 and P7455, however, none of these were classified as statistically significant. Non-quantitative shotgun proteomic based approach was used to analyse outer-membrane protein (OMPs) expression of P8544 and P7455 under low-iron and iron-replete growth conditions and revealed alteration in the OM expression profiles between the isolates and conditions using KEGG analysis. The majority of expressed proteins under iron-replete conditions were categorized in membrane transport (11%) and carbohydrate metabolism (7%). Under iron-restriction the OM profile changed most obviously in a decreased percentage of proteins particularly assigned in the categories energy metabolism and membrane transport. The percentage of proteins assigned no predicted function increased by 19% under iron-limited conditions highlighting the fact that biological functions of the majority of these expressed proteins in such an environment remains to be determined. Two-dimensional gel-electrophoresis (2-DGE) of whole cell fraction indicated that the alkyl-hydrogen peroxide reductase protein (AhpC) in P7455 and the non-haem iron-containing ferritin (Bhyov8544_1528) in P8544 were significantly (P<0.05; 1.5-fold) more expressed under iron-restricted conditions than under iron-replete conditions. These data confirmed the importance of iron to virulent and avirulent B. hyodysenteriae. The so far identified significantly expressed proteins may serve as a potential biomarker for global diagnostic purposes for B. hyodysenteriae infections rather than a tool for differentiation for virulent and avirulent isolates. However, further work is required to prove if these candidates are expressed in-vivo and conserved in a wider panel of field isolates. In conclusion, this research has contributed to the scientific knowledge regarding B. hyodysenteriae stress responses induced by iron-starvation and has provided further insight into the genetic and proteomic make up of this spirochaete. This work should also aid future investigations concerning the biology and pathogenicity of this important and grossly understudied swine pathogen.

<p>It is known that antibiotics play a role in clearing bacterial infections, and sometimes yeast infections. Antibiotics can either kill or inhibit bacteria in many different ways, but they also effect the role white blood cells play in an infection. Some will enhance white blood cell effects, while other will inhibit. How antibiotics do this is not well understood. Azithromycin and daptomycin are two different antibiotics. Very little is known on how they affect the immune response, including phagocytosis, delayed type hypersensitivity (DTH), and cytokine release. Murine macrophages and neutrophils were treated with azithromycin or daptomycin, along with yeast, Candida lusitaniae, and plated to see the effects of the antibiotic on macrophage and neutrophil phagocytosis. Mice were also sensitized to dinitrofluorobenzene and given azithromycin or daptomycin to see how the antibiotics effect DTH. Lastly, an ELISA was done to see how much of the cytokine, IL-6, the macrophages treated with the antibiotics produced compared to the untreated macrophages. It was seen that both azithromycin and daptomycin have significant effects on phagocytosis and DTH. More research is needed to see the effects of the antibiotics on IL-6 release, as well as their effects on other aspects of the immune response.

<p>The complex interactions between diet and the microbiota that influence mucosal inflammation and inflammatory bowel disease (IBD) are poorly understood. Experimental colitis models provide the opportunity to control and systematically perturb diet and the microbiota in parallel to quantify the contributions between multiple dietary ingredients and the microbiota on host physiology and colitis. To examine the interplay of diet and the gut microbiota on host health and colitis, we fed over 40 different diets with varied macronutrient sources and concentrations to specific pathogen free or germ-free mice either in the context of healthy, unchallenged animals or the dextran sodium sulfate (DSS) colitis model with follow-up studies for 4 diets in the T cell transfer colitis model. Diet influenced physiology in both health and colitis across all models, with the concentration of protein and psyllium fiber having the most profound effects. Increasing dietary protein elevated gut microbial density and worsened DSS colitis severity. Depleting gut microbial density by using germ-free animals or antibiotics negated the effect of a high protein diet. Psyllium fiber influenced host physiology and attenuated colitis severity through microbiota-dependent and microbiota-independent mechanisms. Combinatorial perturbations to dietary protein and psyllium fiber in parallel explain most variation in gut microbial density, intestinal permeability, and DSS colitis severity, and changes in one ingredient can be offset by changes in the other. Our results demonstrate the importance of examining complex mixtures of nutrients to understand the role of diet in intestinal inflammation.

<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>