NK cells are innate lymphocytes that are crucial for host immunity during infections. Discrimination between healthy and infected cells is facilitated through a sum of inhibitory and stimulatory signals. Host cells can modulate the expression of NK ligands in response to infection, transformation, and stress, while viruses can exploit these mechanisms to prevent the killing of the infected cells. This thesis focuses on the interaction between the NK receptor, NKR-P1B, and its ligand, Clr-b. First, we used a reporter cell-based protocol to establish hybridomas producing monoclonal antibodies against a putative viral immunoevasin, RCTL. We express CD3ζ-fusion proteins on the reporter cells, which we then use to immunize and screen hybridoma specificities. Our results demonstrate a rapid, efficient, and high-throughput method of monoclonal antibody screening, and provide the framework for our work on cytomegalovirus evasion of the NKR-P1B:Clr-b system. Next, we show that RCMV infection results in a notable downregulation of rClr-b at the protein and transcript levels. Conversely, RCTL is upregulated during infection, and binds to the same NK-inhibitory receptor as Clr-b, NKR-P1B. In the absence of RCTL, RCMV-mediated Clr-b loss leads to increased NK-killing of infected targets and an NK-dependent reduction of viral titers in vivo. Notably, NKR-P1B is highly polymorphic, and certain rat NKR-P1B alleles have lost binding to the viral RCTL but not to the host Clr-b molecule, suggesting co-evolution between the host and the virus. In the next chapter, we address some of the mechanisms responsible for CMV-mediated Clr-b downregulation, and show that Clr-b downregulation also occurs to mice in response to MCMV infection. Moreover, early gene expression (host or viral) appears to be required for Clr-b downregulation. Interestingly, engagement of any one conventional pattern recognition receptor is insufficient to mimic MCMV-mediated Clr-b downregulation. Similarly, fibroblasts lacking various intermediates for the interferon or inflammasome pathways still downregulate mClr-b, with the exception of the DNA sensor, Zbp-1. Lastly, a recently identified autocatalytic motif conserved in the rat and mouse Clr-b transcripts, the hammerhead ribozyme, appears to be involved in Clr-b regulation. Taken together, our results explore a novel and important role for NKR-P1B:Clr-b interactions in viral immunity.

First in 1997, and later re-emerging in 2003, highly pathogenic avian influenza A virus, subtype H5N1, has spread from wild bird reservoirs to domestic bird flocks. As a result, cross-transmission has been confirmed in people living or working in close contact with infected birds. H5N1 virus infection is associated with a high mortality rate (>60%) in humans and the rapid evolution of the virus suggests that it could potentially develop into a new, and possibly severe, pandemic influenza virus. To-date, conventional inactivated and live-attenuated vaccine strategies offers the best protection against influenza virus infection; however, poor immunogenicity and weaker efficacy have been observed against H5N1 viruses. It was hypothesized that experimental adenovirus-based vaccines based on human adenovirus serotype 5 (AdHu5) or porcine adenovirus serotype 3 (PAV3) can offer protection against a broad range of avian influenza, subtype H5N1, viruses. Ad vaccine vectors are highly immunogenic and have demonstrated protective efficacy against several disease models. However, natural immunity against AdHu5 can interfere with vector efficacy. The nonhuman PAV3 was not neutralized by pooled human serum from 10,000-60,000 individuals and offers a promising alternative to AdHu5-based vectors. Systematic antigen screening using DNA vaccines identified the hemagglutinin (HA) glycoprotein as the most immunogenic H5N1 antigen. HA was then inserted directly into PAV3 or AdHu5. Comparable immune responses were observed between both vectors but, interestingly, the PAV3-based vaccine generated stronger T-cell responses and better rapid protection 8 days following immunization. Additionally, better long-term protection 1 year following vaccination was observed with the PAV3-HA vaccine. The co-administration of multiple H5N1 antigens was also screened to improve protection against divergent H5N1 challenge. Combinations of DNA vaccines expressing (HA+NA) and (HA+NP) offered the best promise for enhancing protection against homologous and heterologous H5N1 challenges, respectively. However, addition of three or more antigens reduced overall protection possibly by antigen dilution, competition, or interference. Co-administration of PAV3 or AdHu5 vectors expressing both the HA and NP antigens reduced protection against homologous and heterologous H5N1 virus challenges. For all combination vaccines, T-cell responses were strong against HA but significantly decreased against additional antigens in each combination vaccine. Overall, the experimental porcine-based Ad-based vaccine offered better protection than the H5N1 conventional vaccine against a broad range of different H5N1 viruses. Understanding of the relationship between immune parameters and protection will be critical in future improvement of adenovirus-based and other vaccines against avian influenza H5N1.

First in 1997, and later re-emerging in 2003, highly pathogenic avian influenza A virus, subtype H5N1, has spread from wild bird reservoirs to domestic bird flocks. As a result, cross-transmission has been confirmed in people living or working in close contact with infected birds. H5N1 virus infection is associated with a high mortality rate (>60%) in humans and the rapid evolution of the virus suggests that it could potentially develop into a new, and possibly severe, pandemic influenza virus. To-date, conventional inactivated and live-attenuated vaccine strategies offers the best protection against influenza virus infection; however, poor immunogenicity and weaker efficacy have been observed against H5N1 viruses. It was hypothesized that experimental adenovirus-based vaccines based on human adenovirus serotype 5 (AdHu5) or porcine adenovirus serotype 3 (PAV3) can offer protection against a broad range of avian influenza, subtype H5N1, viruses. Ad vaccine vectors are highly immunogenic and have demonstrated protective efficacy against several disease models. However, natural immunity against AdHu5 can interfere with vector efficacy. The nonhuman PAV3 was not neutralized by pooled human serum from 10,000-60,000 individuals and offers a promising alternative to AdHu5-based vectors. Systematic antigen screening using DNA vaccines identified the hemagglutinin (HA) glycoprotein as the most immunogenic H5N1 antigen. HA was then inserted directly into PAV3 or AdHu5. Comparable immune responses were observed between both vectors but, interestingly, the PAV3-based vaccine generated stronger T-cell responses and better rapid protection 8 days following immunization. Additionally, better long-term protection 1 year following vaccination was observed with the PAV3-HA vaccine. The co-administration of multiple H5N1 antigens was also screened to improve protection against divergent H5N1 challenge. Combinations of DNA vaccines expressing (HA+NA) and (HA+NP) offered the best promise for enhancing protection against homologous and heterologous H5N1 challenges, respectively. However, addition of three or more antigens reduced overall protection possibly by antigen dilution, competition, or interference. Co-administration of PAV3 or AdHu5 vectors expressing both the HA and NP antigens reduced protection against homologous and heterologous H5N1 virus challenges. For all combination vaccines, T-cell responses were strong against HA but significantly decreased against additional antigens in each combination vaccine. Overall, the experimental porcine-based Ad-based vaccine offered better protection than the H5N1 conventional vaccine against a broad range of different H5N1 viruses. Understanding of the relationship between immune parameters and protection will be critical in future improvement of adenovirus-based and other vaccines against avian influenza H5N1.

No description available.

Three potential driver genes were identified by use of retroviral mutagenesis in the newly developed iMyc model of B cell malignancy. To do so, iMyc mice, which bear an inserted copy of c-Myc in the IgH locus, were treated with MOL4070LTR to favor the development of B cell malignancies. After tumor development, B cell origin tumors were identified by use of immunohistochemistry and selected for downstream analysis. Three genes were chosen as potential driver genes and validated in mouse or human disease as involved in disease or directly in malignancy. These genes are CD82, IRAK2 and DNMT3a.

In early life the immature immune system has a reduced ability to control infection. This susceptibility is offset by transfer of protective immune components from the mother. Helminth infections are widespread and can have a long lasting influence on host immunity. Children of mothers exposed to helminth infections may display T cell sensitization to endemic helminth infections and associations have been made between maternal helminth infection and altered immune responses to childhood diseases and vaccinations. This shows that helminth-modified maternal immunity may imprint on early offspring immune development in-utero or through breast milk in the form of transfer of, for example, antibodies, cytokines and lymphocytes. Our study shows that, in mice, maternal infection with the helminth Nippostrongylus brasiliensis is not only associated with a passive transfer of antigen specific antibody(IgG1) but also inherently alters offspring immunity, increasing offspring cytokine production, alveolar macrophages, lung neutrophils and B cell population development and proliferation. Pups born to N. brasiliensis exposed mothers also had increased populations of lung and spleen CD4+ cells and higher subpopulations of central memory and effector CD4+ cells compared to pups born to naive mothers.

Includes abstract. / Includes bibliographical references (leaves 170-210). / Human immunodeficiency virus (HIV) infected children are at greater risk of developing tuberculosis disease, and might benefit from vaccination with novel TB vaccines. However, little is known about the effect of HIV-infection on function and phenotype of T cell responses to mycobacterial antigens in children. This study compares both CD4 and CD8 T cell cytokine expression and memory phenotype in children, following in vitro stimulation with mycobacterial antigens, also contained in novel anti-TB vaccines that are currently undergoing clinical trials.

We proposed that the development of a new high capacity polymer microsphere technology, termed ReSyn, could be developed as viable detection reagents for lateral flow technology. This body of work outlines the development of this new high capacity polymer microsphere technology for suitability to flow on lateral flow membranes, and highly specific biomarker detection for immunoassay development. Proof-of-concept was achieved using hCG (pregnancy biomarker) and validated for detection of pLDH and HRP2 as biomarkers of malaria. The sensitivity, stability and multiplex capability of these microspheres were further explored and compared against the current ‘gold' standard detection agent for lateral flow, colloidal gold. Malaria was selected as a suitable target for evaluation of the microsphere technology since it is considered to be a global epidemic that can benefit greatly from improved point-of-care diagnostics. Malaria affects almost half of the world's population and is responsible for causing approximately 655 000 deaths per annum in 2010, with 90% of these deaths occurring in Africa and 85% of these deaths occurring in children under 5 years of age (del Prado et al., 2014; Kokwaro, 2009; White et al., 2014; WHO, 2009). Febrile disease diagnosis at point-of-care is often based on symptomatic diagnosis rather than on the use of validated diagnostic technologies, and is considered one of the major contributing factors for the high morbidity and mortality rate of malaria (Chandler et al., 2008; Kain et al., 1998; Kokwaro, 2009). Improved diagnostic technologies, allowing for sensitive and accurate diagnosis at the point-of-care, could assist alleviating these problems through the improved management of disease (Bell et al., 2006). Lateral flow rapid diagnostic tests are the preferred method for point-of-care diagnostics in resource constrained areas but have several limitations including sensitivity and stability in resource constrained settings (Bell et al., 2006). Improvements in detection agents are seen as a viable approach to improving these features of diagnostic assays. The results of this study show that the polymer microspheres provide improved stability to immobilised antibodies, with potential for translation into improved stability for diagnostic assays in tropical malaria endemic regions. The polymer microspheres offered high specificity and comparable visual sensitivity to the market leader colloidal gold and is therefore considered as alternate detector agents in lateral flow assays. Additionally, the microspheres can be dyed various colours (red and blue in this study), allowing for specific and sensitive multiplex detection of multiple analytes in a single sample. This increases the versatility of the microspheres for lateral flow diagnostic application, and improves the interpretation of lateral flow diagnostic technology at the point-of-care.

Includes abstract. / Includes bibliographical references. / Finding and treating cases [of tuberculosis] in the community before they present to health facilities, a strategy known as active-case-finding is gaining momentum as a way to decrease the infectious pool. This can be achieved through door-to-door community surveys using a TB symptom-screening questionnaire, and is an economical and practical tool to employ in poor, high burden areas. However, unlike for the high risk group of people infected with HIV, there is a lack of evidence supporting the adaptation of a symptom screening tool in the other high risk groups. In 2010, a TB prevalence survey was conduceted in 24 high TB and HIV burden communities in Zambia and the Western Cape, South Africa. This prevalence survey served as the endpoint for the Zambia and South Africa TB and AIDS Reduction study (ZAMSTAR). This survey made use of a questionnaire the collected, among other information, data regarding individual TB symptom reporting, HIV status, diabetes mellitus status and cigarette smoking.

Tuberculosis disease is currently a global health emergency, causing the most deaths worldwide due a single infectious agent. Eradication of TB is hampered by lack of an effective vaccine and poor treatment options. During infection, host-derived cues such as hypoxia and starvation induce Mycobacterium tuberculosis to halt replication and become dormant, which leads to tolerance to front-line antibiotics used in the TB treatment. This dormant phenotype causes delayed clearance of M. tuberculosis, therefore a long treatment time is required for stable cure without relapse. Poor patient compliance increases the emergence of drug resistant strains, posing yet another challenge for the eradication of TB. There is dire need for novel compounds targeting not only drug-resistant, but also dormant bacteria so as to effectively eliminate drug-resistant strains and also shorten treatment time. This requires compounds with novel modes of action and novel drug screening approaches which focus on dormant M. tuberculosis. In the current work a method was optimized which induces the dormant phenotype of M. tuberculosis in vitro allowing large scale screening of compounds against these tolerant bacteria. The high chemical diversity of marine natural products was explored to increase the chances of finding novel compounds with novel mechanisms of action. Additionally, gold-complexed scaffolds were examined for their putative ability to inhibit topoisomerase 1, which is a highly conserved and essential protein of mycobacteria, not currently targeted in classical treatment regimens. Several marine natural products were identified with selective bactericidal activity against dormant bacteria, emphasizing the powerful tool that was developed for drug screening. Moreover, the gold-complexes were also bactericidal against not only replicating and dormant bacilli, but also mycobacteria resistant to front-line TB drugs. Compounds characterized in this study represent a promising starting point for the development of novel TB therapeutics and discovery of new conditionally essential pathways of dormant bacteria.