Data from our group and others have demonstrated that tumor-derived factors directly skew T-cell differentiation from an effective tumor fighting Th1 state to a less effective Th2 state, allowing for tumor growth. Why the Th1 response is more effective is largely still unknown. The recently discovered microRNAs (miRNAs) are a large family of small regulatory RNAs that control diverse aspects of cell functions such as cell proliferation, apoptosis, development, differentiation and immune regulation. We thereby sought to examine miRNAs differentially expressed in Th1 and Th2 cells in an effort to better understand the enhanced ability of Th1 cells in tumor immunity. MicroRNA microarray analyses revealed that the miR-17-92 cluster of microRNAs (miR-17-92) is consistently over-expressed in murine Th1 cells compared to Th2 cells. Quantitative RT-PCR confirmed that the miR-17-92 cluster expression was consistently higher in Th1 cells than Th2 cells. Furthermore, disruption of IL-4 signaling through either IL-4 neutralizing antibody or knockout of STAT6 reversed the miR-17-92 cluster suppression in Th2 cells. MiR-17-92 expression correlated with differential proliferation capacity as Th1 cells proliferated at higher levels than Th2 cells, dependent on IL-4 and STAT6. Th1 cells consistently expressed lower levels of anti-proliferative transcription factors E2F1 and E2F2, which are the known targets of miR-17-92. Collectively, our data suggests that the Th2 skewing tumor microenvironment can induce the down-regulation of miR-17-92 expression in CD4+T cells, thereby diminishing the effective proliferation of tumor-specific T cells and tumor destruction. This has significant public health relevance as we propose that therapy targeting miR-17-92 cluster may provide enhanced T-cell function and prevent tumor growth.

K65R, a lysine to arginine change at codon 65 of HIV-1 reverse transcriptase (RT), has been selected in vitro by many NRTIs (nucleoside analog RT inhibitors), but until recently, was infrequently detected in patients. Located in the fingers subdomain of RT, K65R causes NRTI resistance through a discrimination mechanism by increasing selectivity for natural deoxynucleotide triphosphate substrates (dNTP) incorporation over triphosphorylated NRTI incorporation. The thymidine analog mutations (TAMs) include the following amino acid changes in RT: M41L, D67N, K70R, L210W, T215F/Y and K219Q. Different combinations of TAMs facilitate AZT resistance by a primer unblocking mechanism, also known as an excision mechanism, in which the chain-terminating NRTI is removed from the nascent DNA strand to allow polymerization to resume. From in vitro and clinical observations, we proposed that K65R and TAMs represent two different pathways of NRTI resistance that exhibit bi-directional phenotypic antagonism and counterselection in vivo. We have generated several lines of evidence in support of this hypothesis: (1) HIV encoding K65R has reduced susceptibility to all NRTIs tested except those with a 3 azido in the pseudosugar component (AZT and AZA); (2) in a large clinical database, K65R is increasing in prevalence in patient isolates, whereas TAMs are decreasing in prevalence; (3) a strong negative relation exists between the frequency of K65R and specific TAMs among HIV-1 isolates in a large clinical database; (4) K65R reverses viral resistance to AZT caused by TAMs and TAMs reverse resistance to abacavir and tenofovir caused by K65R; (5) K65R antagonizes the primer unblocking activity of TAMs and TAMs antagonize discrimination by K65R; and (6) in plasma samples in which both K65R and TAMs were detected by population sequencing, K65R was not found on the same genome with T215F/Y and 2 or more other TAMs, except when the Q151M multi-drug resistance complex was also present. HIV-1 drug resistance is a significant public health problem. This work contributes to the understanding of NRTI resistance and will help to optimize current and future therapy for HIV-1 infection.

Lyme borreliosis (LB), the most common tick-borne disease in Europe and North America, is caused by spirochetes of the Borrelia burgdorferi sensu lato complex. The spirochetes can invade several different organs, thereby causing many different symptoms and signs. Diagnosis of LB relies on patient history, physical examination, and detection of anti-Borrelia antibodies. However, anti-Borrelia antibodies are not always detectable, and they commonly persist even after LB is successfully treated or spontaneously healed. The aim of my work was to study diagnostic aspects on clinical cases of LB and control subjects in an area endemic to LB, with a focus on newly developed anti-Borrelia antibody tests. A total of 617 patients with symptoms and/or signs consistent with LB, as well as 255 control subjects, were studied. The diagnostic panel included the following new LB tests: Immunetics Quick ELISA C6 Borrelia assay kit (C6), invariable region 6 peptide antibody assays (IR6), Liaison Borrelia CLIA (Li) and the chemokine CXCL13. Results were compared with the older Virotech Borrelia burgdorferi ELISA (VT) and with a Western blot method, the Virotech Borrelia Ecoline IgG/IgM Line Immunoblot (WB EL), when appropriate. In general, no significant differences were noted between the C6, VT and Li tests regarding serosensitivity in various LB manifestations. However, the seropositivity rate was lower for the C6 test compared with the VT and Li tests 2–3 and 6 months after diagnosis of erythema migrans (EM), indicating normalization of antibody levels. In addition, EM patients reporting a previous LB episode had a C6 seropositivity rate similar to that of patients without a previous LB episode, and seroprevalence in healthy blood donors was lower in the C6 test than the VT and Li tests. Taken together, these results support the recommendation of the serum C6 test as a Borrelia serological test due to its ability to reflect ongoing or recent infection. Although the majority of EM patients at presentation showed concordant serological responses to IR6 peptides representing the three main Borrelia species and the C6 peptide, there were also clinical EM cases that were C6-negative and could be detected mainly by a seroresponse to a B. burgdorferi sensu stricto-derived IR6 peptide. Thus, an antibody test combining antigens could be of value in the serodiagnosis of LB in Europe. The serosensitivity of the C6 test in cases of Lyme neuroborreliosis (LNB) was shown to be associated with symptom duration. A serosensitivity rate of 93% was found in LNB patients ³ 12 years of age with a symptom duration of more than 30 days. Therefore, a negative C6 test in serum in such a patient argues against an LNB diagnosis. The presence of chemokine CXCL13 in cerebrospinal fluid was confirmed to be a reliable marker of LNB. CXCL13 differentiated LNB from other conditions and also indicated a high probability of LNB in children with short symptom duration where anti-Borrelia antibodies were still lacking in the cerebrospinal fluid. A two-tiered approach (C6 test in combination with WB EL) showed no significant improvement in specificity over the C6 test alone. However, WB EL may be useful in diagnosing suspected cases of acrodermatitis chronicum atrophicans and Lyme arthritis, usually displaying multiple IgG bands. In conclusion, although the serodiagnosis of LB remains to be settled, this thesis provides some practical tools regarding the use and interpretation of Borrelia serology including proposed diagnostic routines.

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.

Perinatal infections are associated with significant morbidity and mortality for both pregnant women and their infants, including while in utero. Prenatal screening for potential infectious diseases can effectively prevent MTCT infections. It allows both timely and suitable medical interventions when required. In Manitoba, prenatal screening for Rubella, Hepatitis B Virus (HBV), Human Immunodeficiency Virus (HIV), Treponema pallidum, Chlamydia Trachomatis (CT) and Neisseria gonorrhoeae (GC) is recommended for all pregnant women and in each pregnancy. The research described in this thesis assesses the current adherence to the Manitoba prenatal screening guidelines. Data consisted of prenatal screening tests conducted at Cadham Provincial Lab (CPL) for the time period of 2006 to 2011. Approximately one fifth of pregnant women did not receive any form of recommended prenatal testings’. Adherence to prenatal screening guidelines varied by type of infection, age of women and area of residence. Overall, Rubella, HBV and syphilis prenatal screening were requested more frequently than HIV, CT and GC. From year to year, a significant improvement of HIV prenatal screening uptake was observed. Rubella, HBV and syphilis screening declined while GC and CT screening remained stable. Among screened women, HIV testing was requested significantly less frequently in the youngest <15 and oldest >45 age groups versus other age groups. Women >45 years old also received less GC and CT screening. A year- II to-year increase in HIV and GC screening was observed in pregnant women aged 15-25, 26-35, and 36-45 years old. Although HIV screening uptake increased over time among residents of Brandon and rural areas, the overall HIV screening test was still higher among residents of Winnipeg versus other areas. Similarly, residents of Brandon and rural areas were tested less frequently for CT infection. A significant improvement in GC screening among residents of Winnipeg and rural areas was observed. The results described in this thesis demonstrates inconsistent adherence to provincial guidelines – creates higher risk areas and population subsets for congenital infections.. The results also demonstrate the importance of promoting testing of this type among pregnant women. Improvement and enhancement of current practice is required to reach standard, satisfactory and appropriate adherence to screening guidelines. Ongoing periodic assessments are suggested to continually document and monitor uptake and adherence to recommended prenatal screening in Manitoba.

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.

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