The extensive allelic variability observed in several genes related to the immune response and its significance in transplantation, disease association studies and diversity in human populations has led the scientific community to analyse these variants among individuals. This thesis is focussed on the development of a database and software applications for the investigation of several immune genes and the frequencies of their corresponding alleles in worldwide human populations. The approach presented in this thesis includes the design of a relational database, a web interface, the design of models for data exchange and the development of online searching mechanisms for the analysis of allele, haplotype and genotype frequencies. At present, the database contains data from more than 1000 populations covering more than four million unrelated individuals. The repertory of datasets available in the database encompasses different polymorphic regions such as Human Leukocyte Antigens (HLA), Killer-cell Immunoglobulin-like Receptors (KIR), Major histocompatibility complex Class I chain-related (MIC) genes and a number of cytokine gene polymorphisms. The work presented in this document has been shown to be a valuable resource for the medical and scientific societies. Acting as a primary source for the consultation of immune gene frequencies in worldwide populations, the database has been widely used in a variety of contexts by scientists, including histocompatibility, immunology, epidemiology, pharmacogenetics and population genetics among many others. In the last year (August 2010 to August 2011), the website was accessed by 15,784 distinct users from 2,758 cities in 136 countries and has been cited in 168 peer-reviewed publications demonstrating its wide international use.

Background: Salmonella bacteraemia is an important public health problem in children from sub Saharan Africa (SSA). Understanding what constitutes natural acquired immunity to Salmonella is crucial for the development of Salmonella vaccine. It was hypothesized that natural Salmonella exposure within the GIT and peripheral blood induces the generation of specific-antibodies and T cells and these might provide protection to subsequent Salmonella infection. Methods: Natural acquisition of antibody and T cell immunity to Salmonella was investigated in healthy and Salmonella infected Malawian children. Acquisition of typhoid vaccine induced T cell immunity in healthy adults from the United Kingdom (UK) was investigated to model natural immunizing events occurring within the gut associated lymphoid tissues (GALTs) following Salmonella infection. Acquisition of immunity was examined using immunological tools including the intra-cellular cytokine staining assay (ICS), serum bactericidal activity (SBA) assay, ELISA and ELISpot. Exposure to Salmonella was examined using microbiological tools including standard culture and real-time PCR. Principal findings: CD4+ T cells and IgG antibodies to Salmonella develops sequentially in under-five children. Acquisition of Salmonella-specific CD4+ T cells and antibodies coincides with the decline in S. Typhimurium bacteraemia cases in older children. As much as 47% of Malawian children (aged 6-18 months) are exposed to Salmonella at least once within the gastrointestinal tract (GIT). Natural Salmonella exposure within the GIT is associated with development of potentially protective SBA in children. Invasive Salmonella infection elicits an increase in generation of Salmonella-specific CD4+T cells, IgG and IgA antibody secreting cells (ASC). Oral Ty21a vaccination (model of natural Salmonella infection) did not elicit an increase in generation of both CD4+Cytokine+ and CD8+Cytokine+ T cells in the peripheral blood and gut mucosa compartments at day 11, and day 18 post vaccination. Conclusion: Young children (<2 years of age) are more vulnerable to invasive Salmonella infection. Salmonella exposure within the GIT and peripheral blood compartments tissues facilitates acquisition of robust immunity (mediated by antibodies and T cells) in children and these might provide protection to subsequent Salmonella infection. Public health interventions are urgently required in SSA including vaccination with cross-protective Salmonella vaccine, improvements in sanitation, access to clean and safe water and food hygiene.

The recruitment of peripheral blood lymphocytes (PBL) to sites of inflammation and their subsequent traffic into the lymphatic circulation is important in host defense. However, surprisingly little is known about their recruitment from the blood vasculature into inflamed tissue, and almost nothing about their egress from inflamed tissue via the lymphatic circulation. We showed that both human macrovascular and microvascular endothelial cells stimulated by TNF\(\alpha\) and IFN\(\gamma\), preferentially recruited memory T-lymphocytes (CD45RO positive cells) from a mixed pool of PBL. T-cells that had migrated across vascular endothelial cells subsequently utilised a combination of \(\beta\)1 and \(\beta\)2 integrins to traverse cytokine activated lymphatic endothelium. In addition we provide evidence that PGD2 was critical for the transmigration of lymphocytes through vascular endothelium. The process of trans-lymphatic migration was also significantly retarded in the presence of a function neutralising antibody against CCR7. Most importantly, we observed that memory T-cells showed a markedly enhanced capacity to migrate across lymphatic endothelium if they had first traversed a vascular endothelial cell barrier. We have shown that addition of exogenous PGD2 to isolated lymphocytes is able to restore the enhanced migration capacity of lymphocytes that have previously migrated through a vascular monolayer. The nature of the priming signal delivered by the process of migration across blood vessel endothelium remains to be fully identified, but is likely to be important in regulating the dynamics of an inflammatory response.

The liver has a unique connection with the immune system; harbouring vast numbers of lymphocytes, able to instigate secondary lymphoid organ-independent naive T cell activation, and promoting potent immune tolerance. We set out to determine the effect of this unique microenvironment on the biology of CD4+ T cells at three key interaction points: following migration into the parenchyma, after short-term hepatocyte contact, and at long-term tissue-residency. Modelling transmigration through hepatocytes revealed intrinsic, disease-specific cytokine responses in blood-derived CD4+ T cells, not discernible through static co-culture. However, short-term co-culture did induce activation-independent CD69 upregulation, reliant upon cell-cell contact. This phenotype mimicked the similar hepatic CD4+ CD69INT cells that we discovered in liver tissue. Unlike CD69HI cells which represented the tissue-resident memory T cells (TRM) of the liver, CD69INT cells were the most activated population, likely able to migrate to many liver and gut niches, and singularly able to produce IL-4 and IL-10. By contrast, CD69HI TRM displayed a resting phenotype, marked for more restricted movement, and produced the best multifunctional TH1 responses following stimulation. These data demonstrate the importance of studying migration, and provide detailed characterisation of CD69HI TRM and novel CD69INT cells, along with their proposed roles and generation pathways.

Invasive non-typhoidal Salmonellosis is a major cause of bloodstream infections in Sub-Saharan Africa, mainly caused by Salmonella enterica serovars Typhimurium and Enteritidis. Naturally shed outer membrane vesicles from Gram-negative bacteria are being explored to generate cost-effective vaccines against many infections, since antigens within vesicles maintain their natural conformation and orientation. Shedding can be enhanced through genetic modification and the resulting vesicles, Generalized Modules for Membrane Antigens (GMMA) offer potential as vaccines. We explored the potential of expressing a known immunogenic antigen of S. Typhimurium, OmpD, in GMMA derived from E. coli as a vaccine. Further, we showed that immunization with GMMA derived from S. Typhimurium (STm-GMMA) induced protection in mice. The response to STm-GMMA immunization included the generation of antibodies to two immunodominant antigens, lipopolysaccharide and porins. Strikingly, the IgG response towards these two antigens was induced with different rates during first week, with a dramatic induction of IgG targeting porins, and not LPS, in a T-cell independent manner. Nevertheless, the antibody response against both antigens persisted for over 200 days in sites including the bone marrow. Results from this thesis shows that STm-GMMA is both attractive as a vaccine and as a tool to facilitate investigations of B-cell responses.

This study examined the relationship between Mycobacterium tuberculosis global clades and patient origin. In the UK, the rate of tuberculosis is higher amongst patients who originated from the Indian subcontinent (ISC), where two dominant lineages are present, the Central Asian Strain (CAS) and the East African Indian (EAI) lineage. Mycobacterial interspersed units containing variable number of tandem repeats DNA fingerprinting of M. tuberculosis strains isolated from UK patients who originated from the ISC, as defined by novel software, identified that CAS was the most prevalent clade (39%) and EAI was the third most prevalent clade (15%). To further elucidate the relationship between host origin and strain lineage, two rigorous new models of infection were developed which assessed mycobacterial growth and host cell response. The monocyte-derived macrophage model was more appropriate for measuring cytototoxicity than the THP-1 cell model as in the absence of infection, 50% of THP-1 cells died compared to 2% of macrophages. CAS strains caused 1.5 fold more cell necrosis and their growth was four fold higher than EAI strains in the monocyte-derived macrophage model. Finally, the response of polarised monocyte-derived macrophages from Asian and Caucasian donors to different M. tuberculosis lineages was compared. CAS strains grew preferentially better in M2 macrophages from Asian donors. The prevalence of CAS in the Midlands is likely to be due to a combination of specific strain importation and increased ability of this strain to transmit to the population present in the Midlands.

Mycobacterium tuberculosis (Mtb) is the causative agent of the disease tuberculosis (TB). While approximately one third of the world’s population is infected with this pathogen, only a small minority of these individuals has active TB infection, where these individuals are able to transmit the pathogen to others. In previous microarrays performed in our lab from lung tissue of non-human primates (NHPs), it was noted that animals undergoing the activation of TB showed greatly increased expression of lymphocyte activation gene 3 (LAG-3). This protein performs immunomodulatory roles, which include: increased function of regulatory T cells, decreased function of Th1 effector T cells, and decreased monocyte differentiation. When studied in rhesus macaques infected with Mtb, RNA expression and protein levels of LAG-3 in lung tissue of active TB animals was found to be greatly increased when compared to lung from animals with latent TB. Interestingly enough, there was a bimodal distribution of LAG-3 expression in animals undergoing reactivation of the disease; the animals with greater levels of LAG-3 were the fast reactivators. LAG-3 expression in the lung tissue of animals with Mtb infections was mainly isolated to the outer periphery of the Mtb induced lung granuloma, where predictably, LAG-3 was expressed by lymphocyte populations of immune cells; mainly NK cells and various populations of T cells. To gain a greater understanding of the function of LAG-3, we created a co-culture system where CD4 T cells derived from blood and lung of Mtb infected NHPs were supplemented to Mtb infected differentiated monocytes. With this co-culture model, we utilized short interfering RNA (siRNA) to silence LAG-3. We observed a decreased bacterial burden, as well as decreased frequencies of IL-10 and IFN-γ producing CD4 T cells. This illustrates that the silencing of LAG-3 in CD4 T cells resulted in increased bacterial clearance, not due to up-regulation of IFN-γ. We believe that the bacterial reduction may be due to increased T cell proliferation, along with production of another proinflammatory cytokines. In the near future, we will utilize cytokine assays and microarrays to better understand the mechanism of action through which increased bacterial killing is occurring. / acase@tulane.edu

Regulatory T cells (Tregs) suppress effector immune responses and have been implicated in promoting chronic viral infections. Their role during influenza infection and vaccination, however, is still unclear. Influenza is a major public health concern, claiming over 49,000 lives annually in the U.S. alone. Vaccination is the best approach for preventing disease but frequent mutations of immunogenic epitopes requires a new vaccine to be formulated and administered annually. This poses a challenge for vaccine manufacturing and may strain patient compliance. A universal influenza vaccine, which targets the highly conserved extracellular domain of the influenza matrix protein 2 (M2e), may circumvent this problem by generating cross-protective immunity. In this study, we tested the efficacy of the M2e universal vaccine in the ferret, and determined whether vaccination induces a Treg response after influenza infection. We found that vaccination promotes the development of M2e specific IgM and IgG antibodies after boosting. Upon challenge with A/Memphis H1N1, vaccinated ferrets exhibited a lower body temperature and reduced virus titer compared to non-vaccinated animals. Together these findings suggest that the M2e vaccine protects ferrets against influenza infection. In order to determine whether Tregs increase after vaccination in ferrets, we had to first clone and characterize genes involved with Treg phenotype and function including CD25, Foxp3, and IL-10. The reciprocal nature between Tregs and Th17 cells and their involvement during influenza infection prompted us to also clone ferret IL-17F. Using these sequences, we designed a qRT-PCR array to measure the expression of Foxp3, IL-10, and IL-17F in ferret tissue. We also identified cross-reactive antibodies against ferret CD8, CD25, and Foxp3 for use in FACS, western blot, and ICC. Using these tools, we found that vaccination significantly increased the expression of Foxp3 in the spleen. An increased percentage of Foxp3+ lymphocytes was detected in both the PBMCs and splenocytes of immunized animals. In contrast, IL-10 and IL-17F expression decreased significantly in both immunized and non-immunized ferrets compared with naïve animals. These studies suggest that the M2e influenza vaccine induces a regulatory T cell response in ferrets and protects against influenza infection. / acase@tulane.edu

Despite the development of a vaccine and several antibiotics, tuberculosis continues to be one of the leading causes in mortality in the world. The pathogenesis of the main causative agent, Mycobacterium tuberculosis, has puzzled many researchers for over a century. Research on the origin of M. tuberculosis can provide new knowledge on how the organism has evolved into the dangerous pathogen it is today. This thesis reviews recent literature on how the evolution of tuberculosis has contributed to the genetic diversity and positive control of select genes in the tuberculosis genome and how this can impact future development of therapeutic agents. / acase@tulane.edu

Mycobacterium tuberculosis (Mtb) remains a pathogen of significant importance with respect to global health. Although approximately one third of the world is infected with TB, only 5-10% develop clinical manifestations of active TB within 2 years post exposure. Infection with Mtb can cause active tuberculosis (ATB), inactive latent infection (LTBI) and be reactivated. The immune response is contained within the formation of a collection of immune cells, a granuloma, in the infected individual’s lungs, which is seen in all disease states. The granuloma functions both as an immune response to contain the bacterium from surrounding lung parenchyma as well as a site for the bacterium to remain in the individual; therefore, providing an environment in which the bacterium maintains the ability to reactivate. We currently lack a complete understanding of the physiology and the metabolic state of Mtb in this granulomatous environment during different states of infection. Leveraging a novel technique known as mesodisection, we microdissect TB granulomas from various infective stages as well as from different sections of the granuloma from non-human primate (NHP) derived formalin fixed paraffin embedded (FFPE) lung tissue. From these extracted tissue sections, RNA is extracted, amplified and subsequent microarray and nCounter analysis is performed; consequently, allowing us to uncover the Mtb specific transcriptomic profiles. First, our findings reveal statistically significant Mtb genes induced in ATB and LTBI in various granuloma types. Of the genes induced, many belong to the following categories: PE/ PPE, sigma factors, toxin-antitoxin complexes and the DosR regulon. In addition, our findings reveal a core group of genes commonly identified in both ATB and LTBI induced in ATB and LTBI in various granuloma types. Of the genes induced, many belong to the following categories: PE/ PPE, sigma factors, toxin-antitoxin complexes and the DosR regulon. In addition, our findings reveal a core group of genes commonly identified in both ATB and LTBI in all lesion types. Further, we propose that these findings improve our understanding of the physiology of the pathogen as well as its virulence which in turn can be used for the development of improved therapeutics, diagnostics and vaccines. / acase@tulane.edu