My dissertation research focused on studying mechanisms of immunomodulation by probiotic lactobacilli on innate and T cell immune responses induced by rotavirus infection and vaccines in a gnotobiotic pig model of human rotavirus (HRV) infection and vaccination. We first studied the effects of probiotics on antigen-presenting cells (APCs) through TLR activation. We found that a mixture of Lactobacilli acidophilus strain NCFM (LA) and L. reuteri (ATCC# 23272) induced strong TLR2-expressing APC responses and virulent HRV induced a TLR3 response. Probiotics and HRV had an additive effect on TLR2- and TLR9-expressing APC responses, consistent with the adjuvant effect of lactobacilli. Dose effects of LA on T cell immune responses were investigated. We found that low dose LA significantly enhanced frequencies of HRV-specific IFN-γ producing CD4⁺ and CD8+ T cells whereas high dose LA reduced frequencies of HRV-specific IFN-γ producing CD4+ T cells. Low dose LA reduced frequencies of induced regulatory (iTreg) cells and TGF-β expression in the iTreg cells whereas high dose LA increased frequencies of iTreg cells and IL-10 expression in the iTreg cells. The dose effects of LA were independent of HRV infection/vaccination. In addition, we demonstrated that TCR-γδ T cells play an important role in modulating immune responses to rotavirus infections. All three γδ T cell subsets showed evidence of activation after HRV infection by increasing TLR2, TLR3, TLR9 expression and IFN-γ production during the acute phase of infection. There was an additive effect between lactobacilli and HRV in inducing total γδ T cell expansion in ileum and in recruiting the cells from blood. HRV infection induced a significant expansion of the CD2+CD8+ γδ T cell subset in the ileum. This subset mainly exerts regulatory functions as evident by expressing FoxP3, secreting TGF-β and IL-10 or increasing production of the anti-inflammatory cytokines by CD4+ and/or CD8+ αβ T cells in the co-cultures. CD2+CD8- and CD2-CD8- γδ T cell subsets have mainly pro-inflammatory and anti-viral functions as evident by secreting IFN-γ or promoting CD4+ αβ T cell proliferation and IFN-γ production. This knowledge will facilitate the development of more effective vaccination and therapeutic strategies to protect children and young animals against rotavirus gastroenteritis. / Ph. D.

Noroviruses (NoVs) are a cause of nonbacterial acute gastroenteritis affecting all ages. NoV infections result in over 200,000 pediatric deaths in developing countries annually. Vaccine development has been hindered by the lack of cell culture systems and small animal models; thus, vaccine development has relied upon recombinant VP1 capsid proteins, such as virus-like particles (VLPs) and P particles. P particles are a novel vaccine candidate derived from expression of the VP1 protruding (P) domain, while VLPs require expression of the full-length VP1. My studies utilize a gnotobiotic (Gn) pig model of human NoV infection and diarrhea to evaluate the protective efficacy and T cell responses induced by P particles and to compare them with prior NoV infection (NoVPO) and VLPs. Gn pigs received 100 µg of P particles (LoPP) or VLPs, 250 µg P particles (HiPP), or adjuvants only intranasally at post-inoculation day (PID) 0, 10, and 21. Monophosphoryl lipid A and chitosan were used as mucosal adjuvants. At PID 28, a subset of pigs were orally challenged with 10 median infectious doses (ID50) NoV. NoVPO, LoPP, HiPP, and VLPs provided partial protection from diarrhea (83%, 47%, 60%, and 60% protection rates, respectively). Only NoVPO and HiPP provided protection from shedding (49% and 60% protection rates, respectively) and also reduced the number of CD25- regulatory T cells (Tregs) in duodenum following challenge. NoV primary infection induced an overall pro-Treg and low, transient Th1 response. LoPP induced stronger overall T cell responses compared to VLPs, including activated CD4+ T cells and duodenal CD8+IFN-γ+ T cells, suggesting that P particles are more immunogenic than VLPs. I also evaluated the effects of simvastatin, a cholesterol-reducing drug that increases NoV infectivity, on P particle vaccine efficacy. Simvastatin abolished P particle-induced protection and significantly increased diarrhea severity. Simvastatin reduced total numbers of duodenal mononuclear cells, IFN-γ+ T cells pre-challenge, and Tregs post-challenge, indicating that simvastatin impairs development of immune system and immune responses. Findings from these studies elucidate potential mechanisms behind P particle-induced immunity and reveal the negative effects of simvastatin on NoV-induced protective immunity. The knowledge will facilitate the development of effective NoV vaccines. / Ph. D.

Reductive acetogenesis via the acetyl-CoA pathway is a hydrogenotrophic pathway that has the potential to reduce methanogenesis from ruminant livestock. However our understanding of the organisms capable of this transformation (acetogens) is hindered by a lack of specific molecular tools for this group. In the present thesis, a PCR primer set specific for a wide range of acetogens was developed, targeting the acetyl-CoA synthase (ACS) gene which is unique to the acetyl-CoA pathway. ACS was found to be useful marker for potential acetogens and ACS sequences could be used to infer family-level phylogeny for many acetogens. ACS gene specific primers were used in combination with existing molecular tools targeting the gene encoding formyltetrahydrofolate synthetase (FTHFS, present in the acetyl-CoA pathway but not unique to it) and 16S rRNA genes, as well as cultivation techniques, to investigate acetogen diversity in the rumen and two analogous gut systems where microbial hydrogenotrophy differs: the forestomach of a native Australian marsupial, the tammar wallaby Macropus eugenii; and the developing rumen of young lambs. Novel potential acetogens present naturally in the rumen of pasture fed and grain fed cattle affiliated with the Ruminococcaceae/Blautia group and distantly with the Lachnospiraceae. A large diversity of potential acetogens with functional genes affiliating broadly between the Lachnospiraceae and Clostridiaceae though without a close sequence from a cultured relative were also detected. Rumen acetogen enrichment cultures revealed the presence of a known acetogen, Eubacterium limosum, in grain fed cattle, as well as novel acetogens affiliating with the Lachnospiraceae and Ruminococcaceae/Blautia group. The novel potential acetogen population detected in this study may represent an important hydrogenotrophic group in the rumen that we understand very little about and that requires further investigation. The tammar wallaby, which exhibits foregut fermentation analogous to that of the rumen but resulting in lower methane emissions, housed a different acetogen population to that of the bovine rumen (LIBSHUFF, p <0.0001) though novel potential acetogens in the tammar wallaby forestomach affiliated broadly in the same family groups (Blautia group, Lachnospiraceae and between Lachnospiraceae and Clostridiaceae without a close cultured isolate). Acetogen enrichment cultures from the tammar wallaby forestomach facilitated isolation of a novel acetogen, which was closely related to potent reductive acetogens from kangaroos. The differences between the acetogen population of the tammar wallaby forestomach and the bovine rumen may be a factor in explaining lower methane emissions and methanogen numbers in tammar wallabies relative to ruminants. Using a gnotobiotically reared lamb model, the unique acetogen population present in the developing rumen was identified and it’s response to methanogen colonisation examined. The acetogen E. limosum and potential acetogen Ruminococcus obeum were identified as well as a small diversity of novel potential acetogens affiliating with the Blautia group and the Lachnospiraceae. A small but diverse population of naturally resident methanogens were also identified in gnotobiotically reared lambs that had been isolated at 17 hours of age. After inoculation with Methanobrevibacter sp. 87.7, methanogen numbers in gnotobiotically reared lambs significantly increased but acetogen diversity was not altered, indicating that this population is resilient to methanogen colonisation to some degree. The potential acetogen population in gnotobiotically reared lambs was significantly different (LIBSHUFF, p < 0.0001) to that in conventionally reared sheep, which indicates that factors other than methanogen establishment alone, probably relating to other microbes and associated hydrogen concentrations in the rumen, affect acetogens during rumen development.

La relation de la microflore lactique avec l'homme n'a été que très peu étudiée dans le contexte des aliments amylacés fermentés tropicaux. La plupart des recherches dans ce domaine sont réalisées par des combinaisons de tests phénotypiques (modèles cellulaires et animaux) et des essais cliniques. Cependant, la disponibilité des données génomiques permettent d'envisager de nouvelles stratégies. L'objectif de ce travail est de rechercher la présence d'une cinquantaine de gènes impliqués dans des fonctions probiotiques dans une collection de 152 bactéries lactiques isolées d'un aliment fermenté africain à base de mil, le ben-saalga, ainsi que dans le métagénome de différents aliments amylacés fermentés. Plusieurs couples d'amorces ont été dessinés par nos soins et ont permis de détecter par PCR la présence de ces gènes. Le criblage génétique est efficace pour déterminer le potentiel lié à certaines fonctions « simples » (synthèse de vitamines B et caroténoïdes, métabolisme de l'amidon, etc.), puisqu'il permet le plus souvent de réduire le nombre de tests phénotypiques à réaliser aux souches porteuses des gènes d'intérêt. Au contraire, des tests in vitro complémentaires (résistance au pH acide et aux sels biliaires, adhésion sur des modèles cellulaires, imagerie à résonnance plasmonique de surface) montrent les limites de l'approche moléculaire appliquée à la détection de fonctions plus complexes que sont l'adhésion et la survie des bactéries. Par ailleurs, les profils d'expression des gènes impliqués dans la fonction d'adhésion par PCR en temps réel sont fonction du modèle utilisé (cellules ou rats). Nous avons montré qu'un mélange des trois souches les plus prometteuses modifie le profil de protéines impliquées dans la maturation de l'épithélium intestinal de rats initialement axéniques. Nous pouvons conclure que le criblage génétique des métagénomes d'aliments amylacés fermentés tropicaux permet de mettre en évidence un potentiel probiotique et nutritionnel prometteur. / The relationship between the lactic acid bacteria composing the microbiota of tropical starchy fermented foods and humans has been poorly investigated. Most of the studies focus on a combination of phenotypical (cells models, animals) and clinical trials. However the increasing numbers of genomic data allow new strategies. The objective of this work was to screen the presence of around 50 genes involved in probiotic functions in a collection of 152 lactic acid bacteria isolated from an African fermented cereal based food called ben-saalga, and in the metagenome of various starchy fermented foods. In this study, several primers have been designed allowing the detection of genes of interest by PCR. The genetic screening is efficient for determining the potential linked to simple functions (B vitamins and carotenoids synthesis, starch metabolism, tannin degradation) as in most cases it allows to limit the number of phenotypical tests to the strain harbouring the genes of interest. On the opposite, more complex functions such as cell binding or bacterial survival, estimated in vitro (low pH, bile salts, cell models, surface plasmonic resonance imagery) revealed the limit of the approach. The expression of genes involved in cell adhesion measured by real time PCR vary depending on the model used (cells or animal).We showed that a cocktail of three potentially probiotic strains modifies the profile of proteins involved in the maturation of the intestinal epithelium of initially germ free rats. The genetic screening of the metagenomes shows that the traditional starchy fermented foods harbour a promising probiotic and nutritional potential.

Human rotavirus (HRV) is a major causative agent of acute gastroenteritis (AGE), which causes severe dehydrating diarrhea in children under the age of five and results in up to 215,000 deaths worldwide each year. There are two live oral attenuated vaccines licensed for use in the United States that are highly effective in high-income countries but much less so in low-and middle-income countries (LMICs). Several factors contributing to decreased efficacy in these areas include chronic malnutrition, gut dysbiosis, and concurrent viral infection. Along with this, currently used vaccines require constant cold-chain storage to maintain vaccine stability, and those resources can be scarce in LMICs. These areas continue to maintain a high burden of HRV morbidity and mortality, and more efficacious vaccines are needed. The gnotobiotic (Gn) pig model of HRV infection and diarrhea has long been used in the evaluation of novel HRV vaccines due to Gn pigs' susceptibility to HRV infection, development of clinical signs, histopathological changes in the intestine, and the infection kinetics that mimic those seen in human infants. The first project in this dissertation used the Gn pig model to evaluate a thermostable live oral attenuated vaccine administered as a dissolvable film. Two doses of the tetravalent dissolvable film vaccine conferred significant protection from virus shedding by delaying its onset and reducing peak titers in feces. It also significantly delayed the onset of diarrhea and reduced the duration and area under the curve (AUC) of diarrhea. The dissolvable film was highly immunogenic, inducing high titers of serum virus neutralizing (VN) antibodies specific to each of the four G-types included in the vaccine formulation, HRV-specific serum IgA and IgG, and intestinal IgA. These data confirm the thermostable platform as a useful alternative to liquid vaccines that require cold-chain. The second project evaluated three mRNA-based nonreplicating vaccine candidates in the Gn pig model. All three mRNA candidates encoded a universal CD4+ T cell epitope, P2, derived from tetanus toxoid, fused with the encoded VP8* from P[4], P[6], and P[8] HRVs. Two candidates also encoded for a lumazine synthase (LS) domain fused with the P2-VP8*. A dose response study of the LS-P2-VP8* candidates was conducted simultaneously. Significant protection against virus shedding was induced by all three candidates, with LS-P2-VP8* candidates inducing significantly higher VP8*-specific serum IgG. All three candidates induced significantly higher numbers of P[8]-VP8*-specific IgG antibody-secreting cells (ASCs) and IFN-γ-producing T cells in the ileum, spleen and blood. These data provide guidance for further development of the relatively new mRNA-based technology for use in HRV vaccine development. In the final study of this dissertation, we used the Gn pig model of both P[8] and P[6] HRV infection to evaluate a cocktail nanoparticle-based HRV vaccine. This vaccine was made up of an S60 nanoparticle, self-assembled from the S domain of the human norovirus capsid protein. The exposed C-termini on the S60 nanoparticle were utilized as an antigen display platform, where VP8* from P[4], P[6] and P[8] HRVs was fused. This vaccine was tested as both a two-dose intramuscular (IM) regimen, or as an IM booster preceded by an oral priming immunization with commercial monovalent Rotarix®. Pigs were challenged with either P[6] or P[8] HRV to evaluate cross-protection of the nanoparticle vaccine. Both regimens were highly immunogenic, inducing high titers of serum VN, IgG and IgA antibodies. Furthermore, the prime-boost regimen conferred significant protection against virus shedding in P[8] HRV-challenged pigs as evidenced by the shortened duration of fecal virus shedding. There was also significant protection in P[6] HRV-challenged pigs vaccinated with the prime-boost regimen, as evidenced by the shortened duration, reduced mean peak titer and AUC of virus shedding. Prime-boost-vaccinated pigs challenged with P[8] HRV had significantly higher P[8]-specific IgG ASCs in the spleen post-challenge. Prime-boost-vaccinated pigs challenged with P[6] HRV had significantly higher numbers of P[6] and P[8]-specific IgG ASCs in the ileum, as well as significantly higher numbers of P[8]-specific IgA ASCs in the spleen post-challenge. Oral priming followed by parenteral boosting appears to be a promising vaccination strategy for HRV and these data warrant further investigation into this regimen. Through these studies, we improved our understanding of the effect of different vaccination routes and formulations in the effectiveness of conferring protection against an enteric virus. The knowledge will facilitate the development of more effective vaccination strategies against HRV, the leading cause of infantile diarrhea in LMICs, as well as other enteric viruses. / Doctor of Philosophy / Human rotavirus (HRV) is a major causative agent of acute gastroenteritis (AGE) in children under the age of five. Acute gastroenteritis is characterized by nausea, vomiting, and potentially deadly dehydrating diarrhea. There are two highly effective vaccines licensed for use in the United States; however, these vaccines are much less effective in low- and middle-income countries (LMICs), where HRV disease burden is the highest. There are several reasons thought to be responsible for the decrease in effectiveness seen in these areas, including chronic malnutrition and gut dysbiosis. Non-biological reasons for decreased efficacy may include the breakdown of cold-chain storage for these vaccines, which require constant low temperature storage that is often unavailable in LMICs. Thermostable vaccines are necessary for increasing vaccine distribution and efficacy in these areas. Because many of the biologic factors thought to interfere with the effectiveness of these vaccines appear to be confined to the gastrointestinal tract, development of next generation HRV vaccines has focused on the parenteral route of administration. The gnotobiotic (Gn) pig model is a highly relevant animal model that has been used for decades to evaluate novel HRV vaccine efficacy. Our first study evaluated a thermostable, dissolvable live oral vaccine administered as a dissolvable film in our Gn pig model. Two doses of this vaccine significantly reduced the severity of diarrhea and virus shedding in the stool. Our second study evaluated three mRNA-based intramuscular (IM) vaccines in the Gn pig model. Three doses of all mRNA candidates provided significant protection from virus shedding in the stool, as well as inducing the production of strong HRV-specific antibodies in the serum and high numbers of virus-specific T cells in the tissues. In our final study, we evaluated a nanoparticle-based vaccine as a two-dose IM regimen or as an IM booster preceded by an oral immunization using the commercially available Rotarix® vaccine. The prime-boost regimen significantly shortened the duration and severity of virus shedding in the stool. We also detected more cross-strain HRV-specific antibody-secreting cells in the tissues. All three vaccines evaluated in this dissertation offer differing novelty in the field of HRV vaccine development, and the Gn pig model has been instrumental in the evaluation of these vaccines.

β-Glucans have long been used as an immunostimulant in aquaculture. However, the relationship of its structure to its immunomodulatory properties are poorly understood. In this study, the particle size and chemical structure of β-glucans extracted from wild-type strain of baker's yeast (Saccharomyces cerevisiae) and its null-mutant yeasts Gas1 were characterised. Using Sigma β-glucan as a reference, the immunomodulatory properties of these polysaccharides in the germ-free Artemia franciscana model system in the presence of Vibrio harveyi bacterial challenge were investigated. The survival of the A. franciscana nauplii, upon challenge with V. harveyi, was significantly higher in all three glucan-treated groups compared to the control. The glucan Gas1 with a lower degree of branching and shorter side chain length had the most prominent V. harveyi-protective effects. The particle size did not affect the nauplii survival when challenged with V. harveyi. Results also showed that the salutary effect of the tested glucans was associated with the upregulation of innate immune genes such as lipopolysaccharide and β-1,3-glucan-binding protein (lgbp), high mobility group box protein (hmgb), and prophenoloxidase (proPO). Interestingly, the up-regulation of superoxidase dismutase (sod) and glutathione-s-transferase (gst) was only observed in Gas1 treated group, indicating that Gas1 could function to induce higher reactive oxygen species and stronger immunomodulatory function in A. franciscana, and therefore higher survival rate. The expression of heat shock protein 70 (hsp70), peroxinectin (pxn), and down syndrome cell adhesion molecule (dscam) remain unaltered in response to glucan treatment. Taken together, this study provides insights into the structure-function relationship of β-glucan and the results confirmed that β-glucan can be an effective immunostimulant in aquaculture, especially the Gas1 glucan.

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