<p>Majority of new HIV-1 infections world-wide occur via the genital tract. Therefore, achieving effective mucosal immunity will be a critical component of vaccine strategies in the prevention and control of infection during early stages of transmission itself. Rigorous efforts have been made to identify conserved epitopes and develop rational design of immunogens, in order to elicit broadly-reactive protective Abs against HIV. Newly emerging data have highlighted the significance of Ab effector functions other than classical IgG-mediated neutralization in HIV infection. In the studies contributing towards this thesis, an optimized vaccine model is described that successfully elicits potent systemic and mucosal Abs against the highly conserved epitopes of the membrane-proximal external region and the coiled coil region of gp41. Intriguing observations are reported on the IgA-inducing capacity of the coiled coil epitope, QARVLAVERY, which highlight the potential of this epitope as an attractive candidate for mucosal vaccines. Most importantly, the epitope-specific Abs proved to be functional in neutralizing HIV in a standardized assay. With particular relevance to mucosal protection, epitope-specific IgA also effectively inhibited the transcytosis of HIV in an optimized transwell assay. The effect of gp41-specific Abs was also assessed against a novel panel of HIV-1 viral clones, and exhibited significant protection. These clones selectively express envelopes from viruses that would be desired targets of prophylactic immune responses, the earliest founder population in the mucosa after virus transmission.</p> / Doctor of Philosophy (PhD)

<p>NK cells play a pivotal role in tumor rejection; however, once present in the tumor microenvironment, they are characterized by decreased cytotoxicity and reduced expression of activating receptors. The mechanisms governing the inactivation of NK cells within tumors remain poorly understood. Since tumor associated macrophages (TAMs) are a highly abundant and suppressive cell type within tumors, we hypothesized that they are capable of altering the function of NK cells. Following the co-culture of alternatively activated macrophages (M2) or TAMs with NK cells we observed that the expression of the cytotoxic marker CD27 on NK cells was down-regulated as well as the ability of these cells to kill YAC-1 cells in a killing assay. We have demonstrated that the mechanism by which M2 cells inhibit NK cells is TGF-β dependent. Notably, the developmental stage of NK cells after interaction with TAMs was altered and the NK cells became phenoytpically mature and potentially exhausted (CD27^lowCD11b^high). This prompted our interest in examining the developmental stage of NK cells from polyoma MT antigen (pyMT) transgenic mouse (MMTV-pMT) breast tumors. Interestingly, in contrast to the <em>in vitro</em> results, we have shown that NK cells isolated from pyMT tumors are developmentally immature; however maintain their maturity within the spleen. Their immature phenotype correlates well with their decreased expression of perforin, granzyme, and NKp46. Both our <em>in vitro</em> studies with TAMs and our <em>in vivo</em> developmental studies using the pyMT model demonstrate that NK cells are altered by their surroundings. A better understanding of how NK cells are modified by the tumor microenvironment will help to develop strategies aimed at bolstering immune responses against tumors.</p> / Master of Science (MSc)

<p>Influenza is a highly contagious respiratory disease. Yearly epidemics and pandemics account for high morbidity and mortality worldwide. Lung immunopathology is a major factor causing death following influenza. In addition, secondary bacterial superinfections that occur after influenza further complicate the lung immunopathology and contribute to higher morbidity and mortality. The research presented in this thesis addressed important, understudied questions in the complicated field of tissue immunopathogenesis and host defense to influenza and pneumococcal infections. Firstly, in a model of acute respiratory influenza infection, we found that the classically proinflammatory cytokine TNF plays a dual and biphasic role at different times post-infection. While it does have pro-immune roles in the beginning stages, TNF acts as a negative type 1 immune regulator at later points of infection. TNF controls the level of immune activation and has a key role in preventing lung immunopathology and aberrant tissue remodeling. Secondly, to further investigate mechanisms of lung pathology, we elucidated the role of bacterial replication and over activated host immune responses during bacterial superinfection following influenza. In our model of pulmonary <em>Streptococcus pneumoniae</em> infection after influenza, we found that dual infected animals experience rapid weight loss and succumb to infection. Bacterial outgrowth, dysregulated cytokine and chemokine expression, and severe lung neutrophilia and immunopathology are linked to the poor clinical outcome. Combined treatment with both an antibiotic azithromycin and corticosteroid dexamethasone best improves clinical outcome, bacterial clearance, cellular and cytokine responses, and immunopathology. Thirdly, in our continuing interest for improved therapies during pulmonary infections, we tested the transgenic expression of type I IFN as a treatment during <em>S. pneumoniae</em> infection. We found that IFN-a controls bacterial outgrowth and improves clinical outcome. Together, our findings provide novel insights into the mechanisms of lung immunopathology and treatment protocols for pulmonary influenza and pneumococcal infections.</p> / Doctor of Philosophy (Medical Science)

UA Open Access Publishing Fund / Background: Current understanding of tuberculosis (TB) genotype clustering in the US is based on individual risk factors. This study sought to identify whether area-based socioeconomic status (SES) was associated with genotypic clustering among culture-confirmed TB cases. Methods: A retrospective cohort analysis was performed on data collected on persons with incident TB in King County, Washington, 2004–2008. Multilevel models were used to identify the relationship between area-level SES at the block group level and clustering utilizing a socioeconomic position index (SEP). Results: Of 519 patients with a known genotyping result and block group, 212 (41%) of isolates clustered genotypically. Analyses suggested an association between lower area-based SES and increased recent TB transmission, particularly among US-born populations. Models in which community characteristics were measured at the block group level demonstrated that lower area-based SEP was positively associated with genotypic clustering after controlling for individual covariates. However, the trend in higher clustering odds with lower SEP index quartile diminished when additional block-group covariates. Conclusions: Results stress the need for TB control interventions that take area-based measures into account, with particular focus on poor neighborhoods. Interventions based on area-based characteristics, such as improving case finding strategies, utilizing location-based screening and addressing social inequalities, could reduce recent rates of transmission.

The British cattle population hosts a diverse community of endemic pathogens that impact the sustainability of beef and dairy production. As such, there has been a tremendous amount of ongoing research to develop more cost-effective strategies for controlling disease at the industry level. Cattle movements have come under particular scrutiny over the past decade both because of their role in spreading many economically important diseases and because the movements of individual cattle in Great Britain have been explicitly recorded in a centralized electronic database since 1998. Numerous studies have shown that these cattle movements organize into complex networks with key structural and temporal features that influence transmission dynamics. Building on previous work, this thesis used a variety of epidemiological and statistical models to highlight limitations in the current approaches to controlling disease as well as opportunities for reducing endemic disease prevalence through targeted interventions. Empirical disease data from the national bovine tuberculosis (bTB) control programme and from two seroprevalence studies of bovine viral diarrhoea virus (BVDV) in Scottish cattle herds were used in conjunction with movement data from the Cattle Tracing System (CTS) database. Endemic diseases are often challenging to control due to lack of affordable and accurate diagnostic tests as well as the presence of subclinically infected carriers that can easily escape detection. There was evidence that combined issues with the sensitivity and specificity of routine surveillance methods for bTB were contributing to a low level of disease transmission within and between Scottish cattle herds from 2002 to 2009. For BVDV, herds that purchased pregnant beef dams, beef dams with a calf at foot, and open dairy heifers were significantly more likely to be seropositive even though these movements were responsible for only a small number of network contacts. In both cases, targeting the subset of high risk movements with disease specific biosecurity measures may be a more cost-effective use of limited national disease control resources. Other researchers have suggested that control strategies should target multiple diseases simultaneously to reduce trade-offs in resource allocation. Using key indicators of herd reproductive performance derived from the CTS database, it was shown that improving the reproductive management of herds operating below industry standards could reduce endemic disease prevalence by reducing the movements of replacement breeding cattle. A series of network generation algorithms were also developed to study the effects of restricting contact formation based on key demographic and network characteristics of actively trading cattle farms. Strategies that increased network fragmentation either by forcing highly connected farms to form contacts with other highly connected farms or preventing the formation of movements with a high predicted betweenness centrality were found to be particularly effective in limiting disease transmission. For these models to be useful in guiding future policy decisions, it is important to incorporate financial and behavioural drivers of dynamic network change. Following the introduction of pre- and post-movement testing requirements for cattle imported into Scotland from endemic bTB regions, there was a significant decline in cross-border movements, which has likely contributed to the decreasing risk of bTB outbreaks as much as testing itself. Many endemic cattle diseases such as BVDV also spread through local transmission mechanisms, which may undermine the success of disease control programmes that exclusively target cattle movements. There was also evidence that in the absence of national animal legislation, few farmers were likely to adopt biosecurity measures against BVDV. This may be related to the perceived inefficacy of recommendations as well as general unawareness of farm disease status due to the non-specific clinical signs of BVDV outbreaks. Although the CTS database was originally intended for use in slaughter traceback investigations, results from this thesis show how the basic records of births, deaths, and movements can be used to generate valuable insights into the epidemiology of endemic cattle diseases. The findings also emphasize that the management decisions of individual herds can have a substantial impact on industry level transmission dynamics, which offers unique opportunities to develop novel and more cost-effective disease control programmes.

The hypotheses of this study are that reduction and rational usage of antibiotics reduces development of antibiotic resistance. In Sweden, the trends do not follow this pattern. Despite a decrease in prescriptions of antibiotics, there is an increase in the number of patients infected with Methicillin-resistant Staphylococcus Aureus (MRSA), Extended Spectrum Beta-Lactamases (ESBL) and ESBL selecting for carbapenem-resistance (ESBLCARBA). This study aims to study factors affecting antibiotic resistance management. An additional aim is to use a multidisciplinary approach for a subject that has mostly been studied with quantitative methods. First, linear regressions investigated any possible significant changes of prescription rates in outpatient care, hospital usage of antibiotic groups and antibiotic resistance. After this, nine interviews were conducted with physicians in outpatient care, hospital care and with representatives from the Swedish Strategic Programme for the Rational Use of Antimicrobial Agents and Surveillance of Resistance (Strama), a network working for Swedish prevention against antibiotics resistance. There was a significant decrease in the number of prescriptions of antibiotics in outpatient care among all Swedish counties and a small, but significant increase of antibiotics used in hospitals. The number of patients infected with multidrug resistant bacteria also show a significant increase. The interviews revealed that health care workers in all counties follow the same guidelines and try to be as specific as possible in choosing antibiotics to hit specific bacteria. The respondents suggested migration and extended travelling as explanations to the growing number of cases of multidrug resistant bacteria. Further, two major factors emerged as important for an efficient antibiotic resistance management; Education/knowledge and Discussion. The results indicate a need for further research on rational usage of antibiotics and the use of broad-spectrum antibiotics in hospital care, rather than the reduction through prescriptions. The results indicate that rational usage has a bigger impact than reduction. Using a multidisciplinary approach gave a broader perspective on the issue and future studies should see the possibilities of mixing quantitative and qualitative studies.

This thesis presents the development and optimization of a high-throughput fluorescence microbead based approach for multiplexed, large scale medical diagnostics of biological fluids. Specifically, different sizes of semiconductor nanocrystals, called quantum dots, are infused into polystyrene microspheres, yielding a set of spectrally unique optical barcodes. The surface of these barcodes are then used for sandwich assays with target molecules and fluorophore-conjugated detection antibodies, changing the optical spectra of beads that have associated with (or captured) biomolecular targets. These assayed microbeads are analyzed at a single bead level in a high-throughput manner using an electrokinetic microfluidic system and laser induced fluorescence. Optical signals collected by solid state photodetectors are then processed using novel signal processing algorithms. This document will discuss developments made in each area of the platform as well as optimization of the platform for improved future performance.

Public health planning for influenza is based on morbidity and mortality estimates derived from statistical models. Lower than anticipated 2009 H1N1 pandemic death estimates have raised questions about the method. Examining the statistical method is important for future policy and program development. We compared the main methods of estimating influenza burden through a systematic literature review and by comparing statistical estimates of influenza-attributable burden at the Ottawa Hospital (TOH) to clinical estimates validated through chart review. We identified heterogeneity in methods used to estimate influenza-attributable mortality in the literature which resulted in within-season estimate variation by study. We found statistical estimates of influenza burden at TOH to be 4-8 times greater than clinically validated data. We also found no significant association between the outcomes examined and epidemic periods at TOH. The findings of this study suggest discordance between model estimates by model approach and between model estimates and validated findings. Examining reasons for these discordances should be pursued.

Lower respiratory tract infections (LRTIs) are one of the most common infectious diseases in Europe and worldwide. Very little is known about the genetic factors associated with susceptibility to LRTI and so far studies have only analysed candidate gene loci. This work aimed to find genetic loci associated with susceptibility to severe and mild LRTI. To analyse severe LRTI, a European cohort of community acquired pneumonia (CAP) sepsis patients was used. To analyse mild LRTI, patients attending primary care with a cough were recruited across Europe. Two important candidate genes for susceptibility to severe and mild LRTI were studied. None of the four functional polymorphisms analysed in MBL2 were associated individually or when combined with susceptibility to LRTI or survival from severe LRTI. Rare homozygotes for rs12252 in the IFITM3 gene were found to associate with susceptibility to mild and severe viral LRTI, particularly influenza infection. Using a combination of genome-wide and candidate gene methods, 93 single nucleotide polymorphisms (SNPs) were chosen to genotype and analyse for susceptibility to mild LRTI. A gene region containing the MPHOSPH8, TPTE2, PSPC1 and ZMYM5 genes was found to associate with susceptibility to rhinovirus infection. A functional promoter SNP in the IL6 gene and a gene region containing the SAP18, MRP63 and SKA3 genes were associated with susceptibility to mild LRTI in smokers. A GWAS was performed for susceptibility to CAP sepsis in Europe. Genotypes were imputed for eight GWAS data sets comprising over 1000 CAP sepsis cases and over 4000 controls. Preliminary analysis results suggest an association in a chromosome 1 region containing the genes VASH2, ANGEL2 and RPS6KC1. This thesis presents results of the largest known genetic association study for susceptibility to CAP sepsis, and the only known genetic association study for mild LRTI. These novel findings will facilitate further research which may lead to better treatment and outcome for LRTI.

Mastitis represents a major issue within the dairy industry responsible for economic loss via decreased animal productivity and associated veterinary costs. Currently, there is a push to identify a phenotypic innate immune response that will yield dairy cows with an enhanced resistance to mastitis. Bovine dermal fibroblasts were used as a cell model to measure the response of individuals to Gram-negative bacterial stimuli through the TLR4 signaling pathway. Fibroblast cultures were isolated from 15 dairy heifers at 5, 11, and 16 months of age in order to determine the variability in responsiveness to LPS as well as to monitor the development of the innate immune response in calves. These individuals displayed a large range in response to LPS as measured by IL-8 production. In addition, response within individuals increased dramatically with age. To determine the cause behind this, DNA methylation was investigated as a potential player in the variation in response described both within an individual over time as well as across individuals. Fibroblast exposure to 5-aza-2'-deoxycytidine, a DNA demethylating agent, increased the cellular response to LPS, but more so in cultures that had previously displayed low responding phenotypes. This suggested that DNA methylation acted as an inhibitor of the innate immune response, and may be responsible for some degree of the variation seen in the LPS response. To determine the effect of epigenetic factors on this response, microarray analysis was conducted on RNA isolated from cells either having been epigenetically modified (DNA demethylation and histone hyperacetylation) or without undergoing any epigenetic treatment. This analysis identified 1,758 genes with altered expression due to epigenetic modification. To focus on DNA methylation's role, methylated CpG island recovery assay (MIRA-Seq) libraries were created from fibroblasts to investigate differential methylation from a group of the same individuals sampled at 5 and 16 months of age. In addition, transcriptomic data were generated by RNA-Seq from fibroblasts collected from the young and older samples and exposed to LPS for 0, 2, and 8 hours to characterize age-associated changes in the innate immune response. Cultures from older animals were much more responsive to LPS as indicated by greater expression of IL-8, IL-6, TNF-α, and CCL20 at various times in response to LPS. TLR4 and CD14 were more highly expressed in older cultures, suggesting these fibroblasts are more able to detect the presence of LPS. Analysis of the bovine fibroblast methylome revealed methylation with remarkable stability except for 20 regions along the genome undergoing major shifts due to age. Similar data were collected from fibroblasts isolated from different individuals displaying either a low or high responding phenotype resulting in 843 regions with differential methylation between groups. This suggests that DNA methylation may be playing a role in both the age-dependent and between animal differential responses to LPS, and also gives the first in depth look at the bovine fibroblast methylome and its stability over time.