Master of Agribusiness / Department of Agricultural Economics / Vincent R. Amanor-Boadu / Mycoplasma; it is not a household name for many Americans or people around the world, but for those in the livestock industry, it has been a major concern. Mycoplasma, a member of the class Mollicutes, has had and continues to have a major impact on the cattle, swine and poultry industry, causing conditions such as arthritis, otitis media, reduced growth rate and reduced egg production (Journal of Veterinary Internal Medicine 2011) (Okwara 2016). This class of bacteria is unlike other classes, as defined by the lack of a cell wall, and is considered by many to be the smallest self-replicating prokaryote (Jack Maniloff 1992). Due to its small size, it can reside within cells and even pass through some of the currently used sterilizing filters in the biological/pharmaceutical industry today (Pall Corporation n.d.). This creates a risk for Mycoplasma contamination for those facilities/research centers that use materials of animal origin, as Mycoplasma organisms have historically been a common contaminate of cell lines and laboratory cultures, affecting roughly 15-35% of cell cultures (Cara N. Wilder 2015). An added concern is the difficulty in treatment of infected animals once an infection is established. The Mollicutes class has been considered innately resistant to the antibiotic penicillin and other cephalosporins due to the lack of the cell wall (Jack Maniloff 1992). Due to the clinical significance and risk factors surrounding the Mollicutes class, it is a current regulatory requirement to test materials of animal origin for the presence or absence of Mycoplasma. The specific criteria for the presence or absence of Mycoplasma test is dependent upon the country in which the product is intended to be sold. For the purposes of this study, the required method and products will be for those intended for sale domestically in the United States, or countries accepting US methodologies. To test a material or product for the presence or absence of Mycoplasma according to the current USDA code of federal regulations (CFR), the method is not a rapid procedure or a simple traditional broth inoculation. The domestic method is a minimum 24 day test that requires complex broth and agar media for Mycoplasma recovery. The complex media requirement is due to the fact that Mycoplasma organisms have stringent nutritional requirements due to their simplified cell structure/genome, which often require materials of animal origin, such as serums for lipid supply/metabolism (Jack Maniloff 1992). The 24 day Mycoplasma test requires an initial inoculation into the aforementioned broth and agar media and then 4 subsequent subcultures from the broth media onto the agar media at specified time intervals. All of the broth and agar media plates are incubated at specific atmospheric conditions and temperature for the duration of the test. The initial inoculation and subcultures are all examined by a trained Microbiologist at specific time intervals to search for evidence of viable Mycoplasma growth. The examination by a trained Microbiologist/technician is a vital step as Mycoplasmas do not produce turbidity in media, such as in traditional bacterial growth, nor are they visible by traditional light microscopy (Farzaneh 2011). If a Mycoplasma contamination is found, a biological/pharmaceutical company can pay huge sums of money to investigate the cause of the contamination, initiate corrective action, decontaminate the facility and destroy impacted batches. As evidenced by the above description, Mycoplasma testing places a large burden on a biological/pharmaceutical production facility or even research institutions. The complex media and labor cost for the 24 day test is extensive, which must be repeated for each batch of new material received or produced. The cost skyrockets if any contamination event occurs or even appears to occur, as investigation and decontamination add cost due to delay of release or possible destruction.

La famille des Arenaviridae comporte sept virus responsables de fièvres hémorragiques humaines. Ces virus représentent un risque naturel pour les populations vivant dans les zones endémiques, ou y séjournant comme les militaires français déployés. Ce risque peut également toucher des populations vivant en dehors des zones endémiques en raison du risque d'importation d'un patient infecté ou consécutivement à l'utilisation intentionnelle et malveillante de tels virus dans le cadre d'une attaque bioterroriste. Les fièvres hémorragiques humaines causées par les arénavirus sont relativement rares et les premiers symptômes, non spécifiques, sont souvent confondus avec ceux de maladies plus fréquentes dans ces régions, comme le paludisme ou les arboviroses. Par conséquent, le diagnostic clinique est souvent retardé, ce qui réduit l'efficacité du seul traitement étiologique actuellement préconisé, la ribavirine. Dans ce contexte, le développement de solutions prophylactiques similaires au vaccin Candid #1, protégeant contre l'arénavirus Junin, constituent une alternative intéressante. Dans le cadre du développement de candidats vaccins, la première stratégie utilisée dans ce travail a consisté à atténuer la pathogénicité du virus d'intérêt en ciblant une étape clé de la réplication des arénavirus. Nous avons choisi l'étape du bourgeonnement viral, dont l'acteur principal est la protéine Z. Une preuve de concept a été réalisée avec le virus de la chorioméningite lymphocytaire (LCMV). Pour cela, nous avons conçu un système de génétique inverse qui exprime un segment L viral où le gène de la protéine Z est remplacé par un gène d'intérêt. De manière surprenante, ce virus recombinant était capable de produire en culture cellulaire une progénie à un titre très faible sans l'apport en trans de la protéine Z. Nous avons identifié des domaines tardifs dans la séquence peptidique de la nucléoprotéine, motifs peptidiques permettant le détournement de la machinerie cellulaire impliquée dans la production d'exosomes et présents dans les protéines de matrices virales, comme la protéine Z des arénavirus. Nous avons observé que ces domaines pourraient partiellement compenser l'absence de la protéine Z. Des résultats similaires ont été obtenus avec deux autres arénavirus ayant une importance majeure en santé publique, les virus Lassa et Machupo, tous deux responsables de fièvres hémorragiques humaines. Cette suppression pourrait constituer une stratégie d'atténuation et semblerait prometteuse en vue du développement de candidats vaccins réplicatifs atténués. En effet, elle pourrait être utilisée sur plusieurs arénavirus responsables de pathologies humaines. Une approche complémentaire à cette stratégie vaccinale a été envisagée. Dans le but de développer un traitement d'urgence, utilisant des immunoglobulines équines hautement purifiées, les F(ab')2, selon la méthodologie de la société Fab'entech, deux études préliminaires ont été réalisées. La première a permis de vérifier la capcité des virus à se répliquer dans les cellules immunitaires circulantes de cheval. La seconde a permis l'évaluation du cahier des charges qualité de particules virales en vue de leur utilisation comme source d'antigène afin de produire les F(ab')2. Une seconde stratégie vaccinale a été envisagée, basée sur une modification du nombre de segments génomiques viraux. Des travaux précédents ont montré qu'un arénavirus à 3 segments, au lieu de 2, était viable et atténué, tout en pouvant exprimer 2 gènes d'intérêt supplémentaires. Cette stratégie a été utilisée sur le virus Machupo, responsable de fièvres hémorragiques en Bolivie. Ce virus recombinant devrait exprimer les glycoprotéiques tronquées des virus Chapare et Guanarito. Ce candidat vaccin a été caractérisé en culture cellulaire, et a induit une protection de 50% des animaux lors d'une administration en post-exposition [etc...] / The Arenaviridae family comprises seven viruses responsible for human hemorrhagic fevers. These viruses represent a natural threat to the local populations, healthcare workers and scientists, as well as to the French forces deployed in the regions where these viruses are endemic. This viral threat can also be intentional in case of a bioterrorist attack. Human hemorrhagic fevers caused by arenaviruses are relatively rare and the first symptoms, frequently non-specific, are often confused with more common diseases such as malaria. Therefore, their diagnosis is delayed, which reduces the efficacy of ribavirin, the only etiological treatment currently recommended. ln this context, the development of prophylactic treatments, such as the Candid #1 vaccine targeting the Junin arenavirus, are an interesting alternative. The first strategy developed in this work to produce a vaccine candidate relied on the attenuation of the virus of interest by targeting a key stage of its replication. We chose the egress step, in which the main actor is the Z protein. This work was conducted using the lymphocytic choriomeningitis virus (LCMV). We therefore designed a reverse genetic system, and replaced the Z gene by the fluorescent protein eGFP reporter gene. Surprisingly, during its cellular infection, a progeny was detected in absence of the Z protein trans-complementation although the titer remained very low. ln this infectious model, we further identified late motifs in the nucleoprotein genome, comparable to those known in the Z protein. These NP late motifs seemed to play an essential role in the compensation of the absence of the Z protein. Similar results were observed using two others arenaviruses of medical importance, the Lassa and Machupo viruses, responsible of human hemorrhagic fevers. The strong diminution of the resulting vaccine candidate replication suggests that this strategy would render safe enough BSL-4 viruses to be used as a multivalent vaccine platform in humans. A complementary approach has been studied in this work. ln order to develop an emergency treatment, based on the production of highly purified F(ab')2 equine immunoglobulins, according to the Fab'entech technology. Two preliminary studies were carried out. The first one consisted in the study of the replication of arenaviruses in circulating horse's white blood cells. The second tested the specifications of attenuated viral particles that could be used as an antigen source to produce the F(ab')2 under good manufacturing practices. Another vaccine strategy was developed using the previously described duplication of the LCMV S genomic small segment in order to produce a tri-segmented recombinant virus. This genetic modification, known to attenuate the LCMV virus pathogenicity, allows the expression of two genes of interest. This strategy has been applied onto the South American Machupo virus, responsible for hemorrhagic fevers in Bolivia. A recombinant Machupo virus was designed to express the truncated glycoproteins of the Chapare and Guanarito viruses, two other New World mammarenaviruses responsible of human hemorrhagic fevers. This vaccine candidate was characterized in cell culture, and showed a 50% post-exposure protective effect in the animal model used. Taken together this work led to the development of two vaccine strategies and to the identification of a promising source of antigens to be used to produce highly purified F(ab')2 polyclonal immunoglobulin, which is the first step to the development of an emergency treatment

Magister Public Health - MPH / This study sought to explore and describe factors that influence parents’ decisions on childhood immunizations at Kumasi Metropolis in Ghana. Based on the Health Belief Model used as the theoretical framework guiding this study, immunization decision making is influenced by one’s knowledge on immunizations, perception on immunizations and sociodemographic factors. With an exploratory descriptive quantitative cross-sectional survey, a sample of 303 parents was obtained from five district hospitals in Kumasi metropolis. This was done through convenience sampling of participants at immunization sessions. Structured questionnaires were developed in line with the study’s objectives, literature review and theoretical framework. Data obtained from the survey were analysed with the computer-based facility of SPSS version 21 software. This enhanced the application of descriptive and inferential measures to present the results in graphs and tables. Findings from the study showed that most parents were aware of immunization but had limited knowledge on vaccines and immunization schedule. It also revealed that antenatal nurses constituted the most accessible source of information. Furthermore, the study established a high percentage of complete immunization, influenced by parents’ fear of their children contracting vaccine preventable diseases. However, the few parents who could not complete the immunization schedule for their children referred to challenges such as forgetfulness and lack of personnel or vaccine at the centre. Whereas the socio-demographic variables considered did not influence their decision on immunization, it was established that the percentage of complete immunization increased with increasing schooling level of parents. It was higher among Christians than Muslims. The study concluded that knowledge on immunization could not influence immunization decisions. However, the main factors that influence parents’ decision on childhood immunizations in Kumasi metropolis were parents’ fear of vaccine preventable diseases, awareness on the benefits of immunizations and sources of vaccine information

Comparison of poliovirus strains was carried out to determine the origin of the virus in two isolates obtained during the 1982 outbreak of poliomyelitis in Gazankulu. Comparisons of the outbreak isolates with vaccine and wild-type strains of the same poliovirus type were carried out using four biochemical techniques. SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional thin-layer chromatography (TLC) and reversed-phase high-performance liquid-chromatography (RP-HPLC) were used for comparing viral capsid proteins. Comparison of poliovirus strains at a genetic level was carried out using two-dimensional oligonucleotide mapping of viral RNA. Results showed the type 1 poliovirus isolate, 5061, to be a novel wild-type poliovirus. The type 2 isolate, 5068, was closely related to the poliovirus type 2 Sabin vaccine strain, P712. It was concluded that the intrinsic variability of poliovirus strains was responsible for the appearance of isolate 5068

Cancer vaccines can fail despite the induction of large numbers of CD8+ T cells. Two categories of memory CD8+ T cells have been defined; central memory (TCM, IL-7RαhighCD44highCD62Lhigh) and effector memory (TEM, IL-7RαhighCD44highCD62Llow). It is clear that the memory phenotype of CD8+ T cells can affect vaccine potential; however methods to augment a beneficial phenotype are not clear. I have compared three vaccine delivery systems: Listeria monocytogenes, Salmonella enterica serovar Typhimurium and the particulate liposomal adjuvant, archaeosomes, for their efficacy to protect against murine melanoma. My study revealed that the anti-tumour response is strongly influenced by the kinetics, phenotype, and lymph node homing potential of CD8+ T cells. Listeria monocytogenes-ovalbumin (LM-OVA) induced TCM cells were adept at long lasting protection against B16-OVA melanoma due to their increased homeostatic and antigen-induced proliferation, interleukin-2 production, and ability to extravasate into tumour draining lymph nodes. Conversely, although Salmonella Typhimurium-ovalbumin (ST-OVA) induced TEM, produced IFN-γ, and killed target cells, this was insufficient for long-term tumour protection. Selectin-ligand engagements of TCM cells influenced their homing potential and efficacy against murine melanoma. Fucosyltransferase deficient (FtDKO) mice, lacking functional selectin ligands, were vaccinated with LM-OVA; despite the activation of cytotoxic CD8+ T cells, there was a reduced protection against murine melanoma compared to wild-type. FtDKO CD8+ T cells exhibited reduced extravasation into FtDKO lymph nodes compared to wild-type. Additionally, fewer FtDKO CD8+ T cells compared to wild-type migrated into tumour sites. Archaeosome vaccination was used to compare the influence of CD8+ T cell quantity versus phenotype. Single or multiple therapeutic vaccinations with archaeosome-OVA yielded transient melanoma tumour protection, despite an increased frequency of circulating and tumour infiltrating CD8+ T cells. This correlated with increased expression of Program death receptor-1 (PD-1) on CD8+ T cells and induction of regulatory T cells. Prophylactic archaeosome-OVA vaccination resulted in a maximal frequency of antigen-specific CD8+ T cells of ~50-60 % with just three injections, and ~50 % of the mice were of mice were afforded long-term tumour protection (> 90 days). Overall, my study shows that the choice of vaccine adjuvant and/or vector can profoundly influence CD8+ T cell quality and cancer vaccine efficacy.

This thesis examined the evidence, policy decision-making, and implementation of HPV vaccination in Canada as a case study to explore normalization versus individualized decision making in public health programs. Mixed methods were used: a systematic review, content analyses and policy document analysis. Overall, the scientific evidence supported an effect of vaccination against HPV infection and precancerous cervical lesions, but evidence regarding cervical cancer incidence or mortality is lacking. Scientific and medical communities appeared optimistic about the vaccine, but cautious about its readiness for routine implementation. Policy decision-making was initially cautious, but shifted towards active program implementation, possibly related to the availability of federal funding. The educational materials and media coverage both sent clearly normalizing messages about HPV vaccination. The discussion suggests that HPV vaccination might be more suited to an individualized than population approach, but many factors coincided to promote its implementation, in Canada, within a traditional public health model.

Objectives: This thesis included 6 manuscripts which focused on the analysis of adverse events following immunization (AEFIs), including general health services utilization (emergency room (ER) visits and hospital admissions) and specific diagnoses (e.g. febrile convulsions). The main objectives of this research were: 1) To demonstrate the utility of the self-controlled case series (SCCS) design coupled with health administrative data for studying the safety of vaccines; 2) Introducing an innovative approach using relative incidence ratios (RIRs) within an SCCS analysis to identify risk factors for AEFIs and to overcome the healthy vaccinee bias; and 3) To demonstrate how SCCS and RIR analyses of health services outcomes in health administrative data can provide important insights into underlying physiological and behavioural mechanisms. Data Sources: This work utilized Ontario health administrative data housed at the Institute for Clinical Evaluative Sciences (ICES). The study included all children born in Ontario, Canada between 2002 and 2011 (over 1 million children). Vaccinations were identified using OHIP fee for service billing codes for general vaccination. Admissions and ER visits for any reason were identified in the Discharge Abstract Database (DAD) and National Ambulatory Care Reporting System (NACRS). Primary reasons for admissions and ER visits were investigated using ICD-10-CA codes reported in the DAD and NACRS databases. Statistical Methods: The self-controlled case series design (SCCS) was used to calculate the relative incidence of admissions, ER visits and other AEFIs. To investigate relative incidence for AEFIs across risk groups of interest, as well as addressing the healthy vaccinee effect bias, RIRs were calculated. RIRs are the ratio of incidence ratios in a subgroup of interest relative to a designated reference group. Results and Conclusions: The combined approach of using the SCCS design and RIRs to identify risk factors and overcome the healthy vaccinee bias proved to be a powerful approach to studying vaccine safety. Future work will be important to characterize the performance and validity of the SCCS + RIR approach in the presence of increasing levels of confounding and differing manifestations of the healthy vaccinee bias, as well as to elucidate the biological and behavioural mechanisms underlying our findings.

In view of the recent implementation of a publicly funded rotavirus vaccination program in Ontario, we undertook studies to help guide the design of a safety evaluation of the vaccine with respect to intussusception. We used administrative data to develop and validate an algorithm for intussusception, and quantified its incidence in Ontario. We also conducted a systematic review of study designs used to evaluate post-licensure vaccine safety, and discussed each design’s strengths and weaknesses. The validated algorithm for intussusception was sensitive (89.3%) and highly specific (>99.9%). We observed the highest mean incidence (34 / 100,000) in males <1 year of age. While other designs are more robust, the inability to ascertain individual vaccination status from Ontario’s administrative data dictated our selection of an ecological design for safety evaluation of rotavirus vaccine. Data assimilated from this thesis represent a critical step toward the timely evaluation of rotavirus vaccine safety in Ontario.

L’apoptose, mort cellulaire observée suite à l’activation des caspases effectrices, est un moyen de défense contre les pathogènes, en particulier les virus. Le virus de la vaccine (VACV) est un virus contenant un grand génome à ADN codant pour environ 200 protéines, dont plusieurs inhibent l’apoptose. Cette apparente redondance fonctionnelle complique l’étude des protéines antiapoptotiques du virus dans un contexte d’infection virale. Dans ce travail, nous comparerons les propriétés antiapoptotiques des protéines B13, F1, GAAP et N1 de VACV. Cette comparaison sera établie dans un premier temps en dehors de toute infection virale. En utilisant des vecteurs lentiviraux, nous avons obtenu des lignées cellulaires stables (U2-OS) exprimant ces protéines en isolation. Nous avons alors pu tester les capacités antiapoptotiques de ces protéines en réponse à des stimuli provoquant l’apoptose extrinsèque et intrinsèque. Les résultats ont montré que B13 était la plus puissante molécule inhibitrice de l’apoptose intrinsèque et qu’elle était la seule à inhiber l’apoptose extrinsèque. Ensuite nous avons tiré avantage d’un virus de la vaccine déficient (vv811) qui ne possède aucune de ces protéines antiapoptotiques, capable à lui seul d’induire l’apoptose, en l’absence de toute autre stimulus. En infectant nos lignées cellulaires exprimant les molécules in trans avec vv811, nous avons pu montrer que B13 inhibait cette apoptose induite par le virus beaucoup plus efficacement que F1. GAAP et N1 dans ce contexte n’ont pas démontré de propriétés antiapoptotiques. Enfin, nous avons construit par mutagénèse des virus vv811 recombinants exprimant les molécules étudiées in cis. Suite à l’infection par ces virus de cellules U2-OS et Hela, B13, de nouveau, et F1 ont montré des capacités d’inhibition importantes de l’apoptose. L’action de GAAP s’est révélée dépendante du type cellulaire et N1 n’a pas pu inhiber l’apoptose induite par ce virus déficient dans aucune des cellules testées. En utilisant ces différentes approches, nous avons pu nous affranchir des problèmes de redondance et comparer 4 molécules antiapoptotiques du virus de la vaccine, y compris dans un contexte d’infection virale. Les résultats ont confirmé que toutes les protéines étudiées possédaient des propriétés antiapoptiques et ont clairement montré que B13 était la plus puissante / Apoptosis, which occurs following activation of effector caspases, can restrict the replication of intracellular pathogens, especially viruses. Vaccinia virus (VACV) is a large dsDNA virus encoding approximately 200 proteins, several of which inhibit apoptosis. This redundancy of viral anti-apoptotic proteins complicates the study of these proteins in the context of viral infection. Here a comparative study of the anti-apoptotic proteins B13, F1, GAAP and N1 with and without virus infection is presented. Firstly, using lentiviral constructs, we generated transduced cell lines expressing the anti-apoptotic proteins in isolation and we analysed their ability to protect against extrinsic and intrinsic apoptosis induced by different drugs. In that context B13 was the most potent inhibitor of intrinsic apoptosis and the only protein to inhibit both extrinsic and intrinsic apoptosis. We then used a deficient VACV strain, vv811, that lacks the genes coding for the four anti-apoptotic proteins. Infection with vv811 can induce apoptosis without the need for any other stimulus. After vv811 infection of cell lines expressing the anti-apoptotic proteins in trans, B13 and to a lesser extent F1, inhibited apoptosis. Finally, we introduced each gene separately into vv811 by genetic recombination. Using these recombinant viruses to induce apoptosis, B13 and F1 were very potent inhibitors. The protection conferred by GAAP was cell type dependant and N1 failed to protect any of the tested cells from the virus induced apoptosis. Using these different approaches, we have been able to overcome the redundancy issue to compare 4 anti-apoptotic proteins from VACV, including in the context of viral infection. The results illustrate that vv811 is a useful tool to determine the role of VACV anti-apoptotic proteins during infection and that whilst all of these proteins have some anti-apoptotic activity, B13 is most potent.

In the early 1970s, a trial was conducted in Beagles comparing an irradiated spherule vaccine for Valley Fever against a control vaccine. The results did not show a significant difference between the vaccinated and the control dogs. Reactions to the vaccine were significant.