The transmission of some infectious diseases requires that pathogens can survive (i.e., remain infectious) in the environment, outside the host. The viability of pathogens that are immersed in aerosols and droplets is affected by factors such as relative humidity (RH) and the chemical composition of the liquid media, but the effects of these stressors on the viability of viruses have not been extensively studied. The overall objective of this work was to investigate the effects of RH and media composition on the viability of viruses in suspended aerosols and stationary droplets. We used a custom rotating drum to study the viability of airborne 2009 pandemic influenza A(H1N1) virus across a wide range of RHs. Viruses in culture medium supplemented with material from the apical surface of differentiated primary human airway epithelial cells remained equally infectious for 1 hour at all RH levels tested. We further investigated the viability of two model viruses, MS2 and Φ6, in suspended aerosols and stationary droplets consisting of culture media. Contrary to the results for influenza virus, we observed a U-shaped viability pattern against RH, where viruses retained their viability at low and extreme high RHs, but decayed significantly at intermediate to high RHs. By characterizing the droplet evaporation kinetics, we demonstrated that RH mediated the evaporation rate of droplets, induced changes in solute concentrations, and modulated the cumulative dose of solutes to which viruses were exposed as droplets evaporated. We proposed that the decay of viruses in droplets follows disinfection kinetics. Lastly, we manipulated the chemical composition of media to explore the stability of viruses as a function of pH and salt, protein, and surfactant concentrations. Results suggested that the effects of salt and surfactant were RH and strain-dependent. Acidic and basic media effectively inactivated enveloped virus. Protein had protective effect on both non-enveloped and enveloped viruses. Results from this work has advanced the understanding of virus viability in the environment and has significant implications for understanding infectious disease transmission. / Doctor of Philosophy / Pathogenic organisms, including bacteria, viruses, fungi, protozoa, and helminths, cause infections that are responsible for substantial morbidity and/or mortality. For example, it is estimated that influenza has caused 9 million to 45 million illnesses and 12,000 to 61,000 deaths annually since 2010 in the United States. The spread of certain diseases relies on people touching the pathogenic organism on surfaces or inhaling it from the air. Successful transmission requires that the pathogen survive, or maintain its infectivity, while it is in the environment. The survival of pathogens can be affected by temperature, humidity, composition of the respiratory fluid carrying them, and other factors. However, there is limited research investigating the effects of these factors on the survival of viruses in the environment. In this work, we studied the effect of relative humidity (RH) on the survival of viruses, including influenza virus and two other types of viruses, in inhalable aerosols and larger droplets. We found that influenza viruses survive well in aerosols across a wide range of RH levels for at least 1 h. Conversely, the two model viruses survived best at both low and very high RHs, such as found indoors in the wintertime or in tropical regions, respectively, but had a pronounced decay at intermediate RHs. By measuring how fast droplets evaporated, we found that RH affected their chemistry and determined the total amount of stress that viruses were exposed to. This explained why a "U-shaped" survival pattern was observed against RH. We also investigated the survival of viruses in droplets containing different components. Results indicated that the effects of salt, surfactant, protein, and droplet pH depended on RH and the type of virus. The outcomes of this work are meaningful in predicting the survival of viruses in aerosols and droplets of various compositions in the environment and could provide insight on developing strategies to minimize the spread of infectious diseases.

Yes / Traditional public health methods for detecting infectious disease transmission, such as contact tracing and molecular epidemiology, are time-consuming and costly. Information and communication technologies, such as global positioning systems, smartphones, and mobile phones, offer opportunities for novel approaches to identifying transmission hotspots. However, mapping the movements of potentially infected persons comes with ethical challenges. During an interdisciplinary meeting of researchers, ethicists, data security specialists, information and communication technology experts, epidemiologists, microbiologists, and others, we arrived at suggestions to mitigate the ethical concerns of movement mapping. These suggestions include a template Data Protection Impact Assessment that follows European Union General Data Protection Regulations. / European Research Council Proof of Concept “Enhanced Place Finding” (grant no. 727695).

Malaria continues to be a challenging problem in the developing world, and the burden of this life threatening disease continues to be borne by young children living in Sub Saharan Africa. One of the biggest challenges to the prevention and control of this problem lies in accurately diagnosing malaria, and distinguishing it from the many other febrile illnesses which present in children in this age group. Liberia is a West African country with a high burden of malaria. Very little is known about the presentation of severe malaria in children aged 5 years old and younger in Liberia. We undertook a prospective, hospital -based study of children 5 and under presenting to JKF Medical Center, the national referral hospital, with fever and signs and symptoms consistent with malaria. The aims of our study were to determine: 1) the frequency of confirmed malaria cases, 2) the frequency of non-malaria diagnoses, 3) the prevalence of anti-malarial drug resistance mutations, 4) the presence of other life threatening etiologies of febrile illness such as S. typhii and Dengue virus and 5) immunological profiling associated with severe malaria. We analyzed clinical and laboratory data from 462 children age 5 and under who presented to the national referral hospital in Monrovia, Liberia with signs and symptoms consistent with malaria over a one year period. Key findings included determining the demographic factors most closely associated with severe malaria in this population (age > 1yr and urban environment) and those that were negatively associated with the development of severe malaria (prior episodes of malaria, use of bednets and use of anti malarial medications prior to presentation). The clinical symptoms most closely associated with severe malaria in this population were found to be headache and vomiting. We found that 33% of children admitted and treated for severe malaria did not test positive for malaria by rapid diagnostic testing (RDT) or blood smear. These children had a case fatality rate that was 5 times higher than their RDT positive counter parts. Of the RDT negative children, 2 tested positive for salmonella typhii, but were not treated for this pathogen. Upon discharge from the hospital, 11% of children had resolved their symptoms, but had not cleared their malaria parasites. These findings will help to identify the children who present with true severe malaria in Liberia. They also underscore the need to expand diagnostic capabilities to determine which other types of pathogens cause febrile illness in this population, so that adequate treatment can be extended to these patients. The immunoprofiles of these children revealed 3 IgM antibodies (AMA-1, CSP and LSA-1) that were associated with the development of severe malaria. These antibodies also appear to be associated with initial infection with malaria. Such data will help to identify antigens could be potential targets for malaria vaccines, and which can play an important role in the development of new malaria diagnostics for this population.

IFNγ is an essential and pleiotropic activator of monocytes, but little is known about the effects IFNγ on cellular metabolism. Therefore, we sought to characterize and elucidate the mechanisms by which IFNγ reprograms monocyte metabolism to support its immunologic activities. First, we identified a critical role for IFNγ in the induction of immunoresponsive gene 1 (IRG1) and its product, itaconate. The immunometabolite, itaconate, has been reported to have antibacterial, anti-inflammatory and antioxidant activity. Irg1-/- mice, lacking itaconate, are highly susceptible and phenotypically similar to IFNγ knock out (GKO) mice upon infection with Mycobacterium tuberculosis. Therefore, we assessed the role of IRG1/itaconate in the context of non-tuberculous mycobacterial (NTM) infection, the most common type of infection in patients with immunodeficiencies caused by defects in IFNγ signaling. Our data suggest that impaired induction of itaconate in the context of mycobacterial infection may contribute to mycobacterial susceptibility and immune dysregulation in patients with defects in IFNγ signaling. Next, we evaluated the metabolic phenotype of IFNγ-stimulated human monocytes and found that IFNγ increased oxygen consumption rates (OCR), indicative of reactive oxygen species generation by both mitochondria and NADPH oxidase. Transcriptional profiling of human macrophages revealed that this oxidative phenotype was dependent on IFNγ-induced, nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD+ salvage to generate NADH and NADPH for oxidation by mitochondrial complex I and NADPH oxidase, respectively. These data identify an IFNγ-induced, NAMPT-dependent, NAD+ salvage pathway that is critical for complete induction of the respiratory burst in IFNγ stimulated human monocytes.

Innate sensing of nucleic acids lies at the heart of antiviral host defense. However, aberrant activation of innate sensors by host nucleic acids can also lead to the development of autoimmune diseases. Such host nucleic acids can also be released from stressed, damaged or dying cells into the tissue microenvironment. It however remains unclear how the extracellular nucleic acids impacts the quality of the host immune responses against viral infections. Using a mouse model of influenza A virus (IAV) infection, we uncovered an important immune-regulatory pathway that tempers the intensity of the host-response to infection. We found that host-derived DNA from necrotic cells accumulates in the lung microenvironment during IAV infection, and is sensed by the DNA receptor Absent in Melanoma 2 (AIM2). AIM2-deficiency resulted in severe immune pathology highlighted by enhanced recruitments of immune cells, and excessive systemic inflammation after IAV challenge, which led to increased morbidity and lethality in IAV-infected mice. Interestingly, these effects of AIM2 were largely independent of its ability to mediate IL-1β maturation through inflammasome complexes. Finally, ablation of accumulated DNA in the lung by transgenic expression of DNaseI in vivo had similar effects. Collectively, our results identify a novel mechanism of cross talk between PRR pathways, where sensing of hostderived nucleic acids limits immune mediated damage to virus infected tissues.

Chronic hepatitis C virus (HCV) infection exhibits persistent high viral load, inducing T cells differentiation and dysfunction in chronically infected individuals. Recent longitude studies in both HCV specific- and bulk T cells reveal that chronic immune stimulation is the driving force for the impaired T cell functions, however, the underlying mechanisms remain elusive. Here, we show that peripheral CD4+ T cells from chronically HCV-infected patients exhibit lymphopenia with the reduction of naïve population and expansion of effector memory T cells. CD4+ T cells from HCV patient also display elevated activation markers. including HLA-DR, GLUT1, Granzyme B, and short-lived effector marker CD127- KLRG1+, whereas stem cell-liked transcription factor TCF1 and telomere sheterin subunit TRF2 are significant reduced, comparing to age- and gender-matched healthy controls. Mechanistically, ex vivo T cell differentiation revealed that CD4+ T cells from HCV patients exhibit PI3K/Akt/mTOR signaling hyperactivation upon TCR stimulation, favoring pro-inflammatory effector differentiation with TRF2 downregulation, rendering telomere dysfunction induced foci (TIFs) accumulation, resulting in telomeric DNA damage and cellular apoptosis. Importantly, exacerbation of telomere deprotection by knockdown of TRF2 expression in healthy T cells resulted in an increase in telomeric DNA damage and T cell apoptosis; whereas overexpression of TRF2 in HCV-T cells led to an alleviation of telomeric DNA damage and T cell death. Additionally, inhibition of Akt signaling during T cell activation can preserve precursor memory population, while limiting inflammatory effector expansion, DNA damage, and cell death. Taken together, these results suggest that modulation of immune activation by inhibiting Akt signaling and protection of telomeres by enforcing TRF2 expression could open new therapeutic strategies to balance adaptive immune responses in the setting of chronic immune activation and inflammatory in vulnerable populations such as chronically viral infected individuals.

<p>Recent evidence suggests that vitamin D may be important for immune function. Canadian studies have reported varying prevalences of low levels of vitamin D. Whether these low vitamin D levels are associated with susceptibility to respiratory tract infections (RTIs) and infection severity remains unclear given the inconsistent association in recent studies. Influenza virus as a cause of RTI is of particular interest given its prevalence, morbidity and economic burden. Vaccination is a key strategy in prevention, but little is known about the effect of vitamin D on influenza vaccine response.</p> <p>A prospective cohort study of children 3 to 15 years old living in Hutterite communities in Alberta, Saskatchewan and Manitoba was conducted to assess the prevalence and predictors of low vitamin D levels and evaluate the association between vitamin D and the incidence and severity of laboratory proven respiratory tract infections. In those who received influenza vaccination, the relationship between vitamin D and influenza vaccine immunogenicity was examined.</p> <p>A total of 743 children were included in the study. The median serum 25-hydroxyvitamin D level (25[OH]D) was 62.0 nmol/L (interquartile range 51.0, 74.0). Levels lower than 50 nmol/L were present in 152 children (20.5%) and lower than 75 nmol/L in 565 children (76%). Lower serum 25(OH)D levels were associated with increased risk of RTI. No association was found between serum 25(OH)D level and disease severity. There was also no relationship found between serum 25(OH)D level and seroprotection or seroconversion from inactivated influenza vaccine.</p> <p>In conclusion, low serum 25(OH)D levels are a significant problem in Canadian Hutterite communities. Furthermore, low serum 25(OH)D levels were associated with increased risk of proven upper RTIs. Studies evaluating the role of vitamin D supplementation to reduce the burden of disease are warranted, and strategies to improve vitamin D status in rural communities in Canada are needed.</p> / Master of Science (MSc)

This study proposes to directly test the hypothesis that antibody-dependent enhancement (ADE) is the critical factor in the development of dengue hemorrhagic fever (DHF) in infants. DHF occurs in two distinct clinical settings: a) in children and adults with secondary DENV infection, and b) in infants with primary DENV infection born to mothers with prior DENV infection. The ADE hypothesis proposes that pre-existing serotype-cross-reactive non-neutralizing anti-DENV antibodies bind the heterotypic DENV during secondary infection and enhance its uptake into immune cells, leading to increased viral load and DHF. This model suggests that DHF in DENV-infected infants is caused by the enhancing effect of waning maternal anti-DENV antibodies, thus causing a “physiologic secondary infection” during an infant’s primary infection and thereby increasing the infant’s risk for DHF. The effect of maternal immunity on DHF in infants has been studied exclusively in Southeast Asia. However, the maternal DENV seroprevalence approaches 100% in this part of the world. As a consequence, the ADE model of infant DHF cannot truly be tested in Southeast Asia, because all infants possess anti-DENV antibody at birth. In the Western Hemisphere, by contrast, women may have experienced either a single DENV infection, more than one DENV infection, or no DENV infection at all. The ability to include DENV-seronegative mothers as controls allows for the ADE hypothesis to be directly tested in a clinical study. To our knowledge, no such study has been previously conducted. This thesis presents a case-control study designed to evaluate the influence of positive maternal dengue seroprevalence on the risk of DHF in infants. As the MSCI program provides instruction in study design, this thesis does not present findings. The clinical trial described herein began in May 2010 and enrollment is expected to continue through May 2012 (see Table 4).

Despite 30 years of intensive research，an effective human immunodeficiency virus (HIV) vaccine still remains elusive. The desirable immune response capable of providing protection against HIV acquisition is still not clear. The accumulating evidence learned from a recent vaccine efficacy correlate study not only confirmed the importance of antibody responses, but also highlighted potential protective functions of antibodies with a broad repertoire of HIV-1 epitope specificities and a wide range of different antiviral mechanisms. This necessitates a deep understanding of the complexity and diversity of antibody responses elicited by HIV-1 vaccines. My dissertation characterizes antibody response profiles of HIV-1 Env antibodies elicited by several novel immunogens or different immunization regimens, in terms of magnitude, persistence, epitope specificity, binding affinity, and biological function. First, to overcome the challenge of studying polyclonal sera without established assays, we expanded a novel platform to isolate Env-specific Rabbit mAbs (RmAb) elicited by DNA prime-protein boost immunization. These RmAbs revealed diverse epitope specificity and cross-reactivity against multiple gp120 antigens from more than one subtype, and several had potent and broad neutralizing activities against sensitive Tier 1 viruses. Further, structural analysis of two V3 mAbs demonstrated that a slight shift of the V3 epitope might have a dramatic impact on their neutralization activity. All of these observations provide a useful tool to study the induction of a desired type of antibody by different immunogens or different immunization regimens. Since heavily glycosylated HIV Env protein is a critical component of an HIV vaccine, we wanted to determine the impact of the HIV Env-associated glycan shield on antibody responses. We were able to produce Env proteins with a selective and homogeneous pattern of N-glycosylation using a glycoengineered yeast cell line. Antigenicity of these novel Env proteins was examined by well-characterized human mAbs. Immunogenicity studies showed that they were immunogenic and elicited gp120- specific antibody responses. More significantly, sera elicited by glycan-modified gp120 protein immunogens revealed better neutralizing activities and increased diversity of epitopes compared to sera elicited by traditional gp120 produced in Chinese Hamster Ovary (CHO) cells. Further, we examined the impact of the delivery order of DNA and protein immunization on antibody responses. We found that DNA prime-protein boost induced a comparable level of Env-specific binding Abs at the peak immunogenicity point to codelivery of DNA. However, antibody responses from DNA prime-protein boost had high avidity and diverse specificities, which improved potency and breadth of neutralizing Abs against Tier 1 viruses. Our data indicate that DNA vaccine priming of the immune system is essential for generation of high-quality antibodies. Additionally, we determined the relative immunogenicity of gp120 and gp160 Env in the context of DNA prime-protein boost vaccination to induce high-quality antibody responses. Immunized sera from gp120 DNA primed animals, but not those primed with gp160 DNA, presented with distinct antibody repertoire specificities, a high magnitude of CD4 binding site-directed binding capabilities as well as neutralizing activities. We confirmed the importance of using the gp120 Env form at the DNA priming phase, which directly determined the quality of antibody response.

Sepsis is the most frequent cause of mortality in most intensive care units. Cardiovascular dysfunction is a major complication associated with sepsis, with high mortality rates up to 70%. Currently, there is no effective treatment approach for sepsis. The integrity of the endothelium is fundamental for the homeostasis of the cardiovascular system. Sepsis induces endothelial cell injury which is the key factor for multiple organ failure. The increased expression of adhesion molecules and chemokines in endothelial cell promotes leukocytes infiltration into the tissue. The loss of tight junction proteins and increased permeability of the endothelial cells will provoke tissue hypoxia and subsequent organ failure. Therefore, preservation of endothelial function is a critical approach for improving sepsis-induced outcome. Here, we showed that endothelial specific protein HSPA12B plays a critical role in the preservation of cardiovascular function in polymicrobial sepsis. HSPA12B is the newest member of HSP70 family which predominantly expresses in endothelial cells. We observed that HSPA12B deficiency (HSPA12B-/-) exaggerated polymicrobial sepsis-induced endothelial dysfunction, leading to worse cardiac dysfunction. HSPA12B-/- significantly increases the expression of adhesion molecules, decreases tight junction protein levels and enhances vascular permeability. HSPA12B-/- alsomarkedly promotes the infiltration of inflammatory cells into the myocardium and inflammatory cytokine production. We investigated the cardioprotective mechanisms of HSPA12B in sepsis induced cardiovascular dysfunction. Exosomes play a critical role in intercellular communication. Exosome is a natural vehicle of microRNAs. We found that exosomes isolated from HSPA12B-/- septic mice induced more expression of adhesion molecules in endothelial cells and inflammation in macrophages. Interestingly, the levels of miR-126 in serum exosomes isolated from HSPA12B-/- septic mice were significantly lowers than in WT septic mice. Importantly, delivery of miR-126 carried exosomes significantly improved cardiac function, suppressed the expression of adhesion molecules, reduced immune cell infiltration in the myocardium, and improved vascular permeability in HSPA12B-/- septic mice. The data suggests that HSPA12B is essential for endothelial function in sepsis and that miR-126 containing exosomes plays a critical role in cardiovascular-protective mechanisms of endothelial HSPA12B in polymicrobial sepsis.