Pandemics are periodic events characterized by rapid and widespread transmission of infectious disease affecting a significant proportion of the population over a large geographical area. Zoonotic strains of influenza viruses and coronaviruses have both caused pandemics in the recent past. Although vaccination is the often the most effective way to prevent infection or serious outcomes of infection, vaccine development, production, distribution, and deployment are all time- consuming and logistically challenges processes. Alternative readily deployed approaches must be quickly executed to mitigate the toll of future pandemics, especially during the early phases. The work described in this thesis describes some of these approaches. Firstly, I describe the process by which I performed genome-wide CRISPR-Cas9 knockout screens using SARS-CoV-2 variants of concern to discover crucial host factors as targets for broad-acting antivirals. I found that all variants rely on similar host pathways to replicate in the glial cell line used for the screen. I identified BCL-xL, a regulator of apoptosis, as a potential target for a broad- acting antiviral. I show that chemical inhibition of BCL-xL results in accelerated cell death in infected cells in vitro, but improved clinical signs and disease mortality of SARS-CoV-2 in our murine infection model. Secondly, I describe a unique mechanism for cooperative antiviral combination therapy. I demonstrate that chemical inhibition of neuraminidase by oseltamivir improved immune effector cell activation by hemagglutinin stalk-binding antibodies. Combination therapy of oseltamivir and stalk-binding antibodies also improved clinical signs and disease mortality of influenza in our murine infection model compared to monotherapy in both prophylactic and therapeutic contexts. Finally, I show that non-pharmaceutical public health interventions used to restrict the spread of COVID-19 were also effective against several other infectious diseases. I used an interrupted time- series analysis on Ontario public health administrative data during the early COVID-19 pandemic period and found a drastic and sustained decline in outpatient visits for diseases that are typically caused by viruses that transmit by droplet or aerosol. The three projects described in this thesis outlines broadly-protective and distinct strategies to curb the spread of novel respiratory viruses. These new tools may be leveraged to improve the response and to mitigate the burden of future pandemics. / Thesis / Doctor of Philosophy (PhD) / Most pandemics in recent history have been caused by viruses that infect the respiratory tract. Vaccination is often the best way to prevent the spread of these pandemic viruses, but making these vaccines takes time. Vaccines also work less well in the very young, the elderly, and those with a compromised immune system. These people are often also the most vulnerable to severe disease. My work describes three novel approaches to help combat the next pandemic, especially during the early phases when vaccines are still being developed, or for the segments of the population that respond poorly to vaccination. These include discovering and using new drugs that work against a wide range of viruses, using combinations of previously-discovered antiviral drugs, and using non-pharmaceutical methods such as physical distancing and wearing masks.

The concept of creating a hybrid material is motivated by the development of an improved product with acquired properties by amalgamation of components with specific desirable traits. These new attributes can range from improvements upon existing properties, such as strength and durability, to the acquisition of new abilities, such as magnetism and conductivity. Currently, the concept of an organic-inorganic hybrid material typically describes the integration of an inorganic polymer with organically derived proteins. By building on this idea and applying the advanced technologies available today, it is possible to combine living and nonliving components to synthesize functional materials possessing unique abilities of living cells such as self-healing, evolvability, and adaptability. Furthermore, artificial gene regulation, achievable through synthetic biology, allows for an additional dimension of the control of hybrid material function. Here, I genetically engineer E. coli with a tightly controlled artificial protein construct, allowing for inducible expression of different amounts of the surface anchored protein by addition of varying concentrations of L-arabinose. The presence of the surface protein allows the cells to bind nonliving nanoparticle substrates, effectively turning the cells into living crosslinkers. By using the living crosslinker, I was able to successfully synthesize a robust, macroscale living-nonliving hybrid material with magnetic characteristics. Furthermore, by varying the particle size and inducer concentration, the resulting material exhibited alterations in structure and function. Finally, I was able to manipulate material kinetics within a PDMS channel by applying fluctuating magnetic fields and demonstrate material durability. These results demonstrate the ability to manipulate synthesis of living-nonliving hybrid materials, which demonstrate the potential for use in promising applications in areas such as environmental monitoring and micromachining. Additionally, this work serves as a foundational step toward the integration of synthetic biology with tissue engineering by exploiting the possibility of controlling material properties with genetic engineering. / Ph. D.

Research has demonstrated that porcine circovirus type 2 (PCV2) can be shed into boar semen, raising the possibility that artificial insemination may be an important route by which disease associated with PCV2 is transmitted. The objective of this experiment was to determine the effect of vaccination against PCV2 on ejaculate characteristics and PCV2-specific antibody titers in serum of PCV2-positive boars viremia and viral shedding in semen. Semen and blood samples were collected weekly from week 0 to week 8. After collections at week 0, boars were vaccinated with a commercial vaccine against PCV2 (n = 5) (Suvaxyn PCV2 One dose; Fort Dodge Animal Health, Fort Dodge, IA) or served as controls and received 2 ml 0.9% saline (n = 5). Sperm concentrations and characteristics of sperm motility were assessed using a computer-assisted sperm analysis system (Hamilton Thorne Research, Beverly, MA) and sperm morphology was evaluated after staining using light microscopy. The PCV2 antibody titers were determined in serum using an ELISA (Iowa State Veterinary Diagnostic Laboratory; Ames, IA). The genomic copy number of PCV2 DNA in serum and semen was determined by PCR (Iowa State Veterinary Diagnostic Laboratory; Ames, IA). There were no effects of treatment or treatment by week on semen characteristics (P > 0.05). An effect of treatment by week was detected for serum antibody titers (P < 0.01). Compared with controls, antibody titers in vaccinated boars tended to be greater at week 0 (1.13 ± 0.05 titer/ml vs 1.01 ± 0.05 titer/ml; P = 0.09) and were greater at week 2 (1.15 ± 0.05 titer/ml vs 1.01 ± 0.05 titer/ml; P < 0.05) but lesser at week 7 (1.01 ± 0.05 titer/ml vs 1.23 ± 0.05 titer/ml; P < 0.01) and tended to be lesser at week 8 (1.05 ± 0.05 titer/ml vs 1.17 ± 0.05 titer/ml; P = 0.07). There were no effects of treatment, week, or treatment by week for serum genomic copy number of PCV2 DNA (P > 0.1). An effect of week was detected for semen genomic copy number of PCV2 DNA (P < 0.04). During week 3, PCV2 genomic copy number was at its greatest numerical value, however, semen PCV2 genomic copy number was at its lowest point. This was followed by an increase in semen PCV2 genomic copy number during week 7. This increase could be related to the increase in viral shedding in the serum. In summary, vaccination against PCV2 can lower antibody titers when given post-infection and has no effect on indicators of semen fertility. Vaccination also can decrease the length of reoccurring infection by decreasing the length of viral shedding in serum. / Master of Science

No / Our objectives were to obtain monoclonal anti-endothelial cell antibodies (AECA) from systemic lupus erythematosus (SLE) patients, to characterize their antigen specificity, and their capability to induce a pro-inflammatory and pro-adhesive endothelial phenotype, and to investigate the mechanism of endothelial cell (EC) activation in vitro. Monoclonal IgG AECA were generated by hybridoma formation with human SLE B cells. Antigen specificity was characterized by immunoblotting with enriched cell membrane fractions, by cytofluorimetry and by cell solid-phase ELISA. Endothelial activation was evaluated by measuring increases in U937 cell adhesiveness, adhesion molecule (E-selectin and ICAM-1) expression and IL-6 production. In addition, mechanisms of endothelial activation were investigated by assessment of NF-B by measuring the loss of its inhibitor I-B. mAb E-3 bound live EC and recognized a 42 kDa EC membrane protein, it enhanced U937 adhesiveness, E-selectin and ICAM-1 expression and IL-6 production, and caused the loss of I-B. We conclude this is the first in vitro demonstration that a human monoclonal AECA from a SLE patient reacts with a constitutive endothelial membrane antigen and induces a pro-inflammatory endothelial phenotype through NF-B activation.

Yes / Recombinant human erythropoietin (rHuEPO) is an effective treatment for anaemia but concerns that it causes disease progression in cancer patients by activation of EPO receptors (EPOR) in tumour tissue have been contro- versial and have restricted its clinical use. Initial clinical studies were flawed because they used polyclonal antibodies, later shown to lack specificity for EPOR. Moreover, multiple isoforms of EPOR caused by differential splicing have been reported in cancer cell lines at the mRNA level but investigations of these variants and their potential impact on tumour progression, have been hampered by lack of suitable antibodies. The EpoCan consortium seeks to promote improved pathological testing of EPOR, leading to safer clinical use of rHuEPO, by producing well characterized EPOR antibodies. Using novel genetic and traditional peptide immunization protocols, we have produced mouse and rat monoclonal antibodies, and show that sev- eral of these specifically recognize EPOR by Western blot, immunoprecipi- tation, immunofluorescence, flow cytometry and immunohistochemistry in cell lines and clinical material. Widespread availability of these antibodies should enable the research community to gain a better understanding of the role of EPOR in cancer, and eventually to distinguish patients who can be treated safely by rHuEPO from those at increased risk from treatment. / Study was supported by the FP7-Health European commission EpoCan grant (282551).

No / The duocarmycins belong to a class of agent that has fascinated scientists for over four decades. Their exquisite potency, unique mechanism of action, and efficacy in multidrug-resistant tumour models makes them attractive to medicinal chemists and drug hunters. However, despite great advances in fine-tuning biological activity through structure-activity relationship studies (SARS), no duocarmycin-based therapeutic has reached clinical approval. In this review, we provide an overview of the most promising strategies currently used and include both tumour-targeted prodrug approaches and antibody-directed technologies.

Disease outbreaks have overwhelmed the aquaculture industry as a whole and have been catastrophic for many single operations. To minimize disease outbreaks, efforts are underway to enhance animal health and disease resistance to pathogens without the use of antibiotics. The overall purpose of this study was to explore a potential prophylactic, a novel antibody diet, for the bacterial pathogen, Aeromonas hydrophila, in tilapia. The tilapia were on an anti-interleukin-10 antibody diet as a neutralization of interleukin-10 (IL-10), an anti-inflammatory cytokine. The anti-inflammatory function of IL-10 has been shown to allow persistence of gastrointestinal pathogens. Tilapia were fed the novel diet and were challenged via bath immersion or oral gavage with A. hydrophila. Four trials of challenge studies were conducted. Clinical signs of the disease and survival were monitored post-challenge of the bacteria. Out of the 4 trials, one bath immersion trial showed significantly lower survival in the group fed the novel antibody diet (p=0.044) compared to the control fed group, after challenged with A. hydrophila. The other trials tested showed no significant differences in survival between diets. Among the survival percentages collected as a whole, it cannot be determined from in vivo results whether this anti-IL-10 diet is effective in preventing mortality from A. hydrophila in tilapia. Therefore, an in vitro study using an enzyme-linked immunosorbent assay (ELISA) was used to determine the neutralization capability of anti-IL-10 on IL-10 using tilapia splenocytes. Interferon-γ, a pro-inflammatory cytokine, was quantified in order to find a trend in expression of IL-10 in vitro in various tilapia cell treatments. The protocol for the ELISA study is under development being that the use of this antibody is novel and has never before been done in fish. / Master of Science in Life Sciences / Aquaculture is the fastest growing animal-production sector for food in the world. Health and safety issues are prevalent among aquatic animals during massive growth and production. Disease outbreaks within aquaculture facilities can cause losses worth billions of dollars. Antibiotics are currently in use in aquaculture as a therapeutant for treating disease. However, over time the use of antibiotics has brought up a new set of issues; antibiotic resistant bacteria/genes and transfer of these to the environment and to humans via consumption. A novel antibody feed, containing an antibody to interleukin-10, with potential as a preventative disease measure was used to study disease development after exposure with a bacterial pathogen. Thus, tilapia were exposed to the bacteria, Aeromonas hydrophila, and signs of disease and survival were monitored to see if the novel antibody feed would prevent disease onset. Further research is necessary on the antibody before confirming effectiveness on disease prevention.

The normal physiological activation of naive T cells requires the engagement of both the T cell receptor (TCR) and the co-stimulatory molecule, CD28. However, a group of monoclonal antibodies (mAbs) have been identified that are able to activate T cells in vitro and in vivo via CD28 engagement alone. Two defining characteristics found in all CD28 superagonist mAbs are their membrane proximal CD28 epitopes and the requirement for mAb immobilisation. To investigate whether agonistic mAbs to similar cell molecules could be identified based on epitope position alone, mAbs to the inhibitory receptor PD-1 were generated and characterised. Using a drastic mutation-based epitope mapping technique, one mAb was identified with a membrane proximal epitope along with two other mAbs with membrane distal epitopes. These mAbs were tested for triggering activity in a hybridoma stimulation assay. mAb stimulation was observed with all three mAbs but only in cells expressing a PD-1 chimera that associated with the TCR and the strength of activation was dependent on epitope location. Cross-linking of a monomeric PD-1/CD28 chimera with a pair of anti-PD-1 mAbs resulted in signalling in this system, however, suggesting a role for ligand aggregation in addition to epitope position in mAb signalling. To further investigate the role of epitope position in CD28 superagonism, a cell line expressing a chimeric form of CD28 was created wherein the superagonistic mAb epitope was moved to a membrane distal position. When stimulated with a CD28 superagonist mAb signalling was no longer observed. However stimulation with another mAb that had an epitope to a membrane proximal location on the chimera resulted in superagonistic effect. These results show that epitope location is the dominant cause of T cell stimulation observed by CD28 superagonist mAbs and that epitope dependent mAb signalling is possible in other T cell surface molecules. The work described in this thesis has implications for both the development of immune modulating mAb therapeutics and for the general mechanism of triggering of cell surface receptors dependent on extrinsic tyrosine kinases.

In this work experimental design and tests were established to determine whether antibody production in guinea pigs injected with a bacterial antigen is Influenced by the environment of simulated high altitude with normoxic conditions. Hematological and electrophoretic studies were simultaneously run with the antibody determinations as a check on related responses of the animals.

A contaminação da água para consumo humano por toxinas produzidas por cianobactérias é um problema de saúde pública e das autoridades em todo o mundo. Microcistina-LR (MCLR) é uma cianotoxina heptapeptídica cíclica que inibe as proteínas fosfatases PP1 E PP2A nos hepatócitos. Microcistinas são produzidas por diversos gêneros de cianobactérias e mais de 70 variações estruturais têm sido caracterizadas em florações naturais. Por serem haptenos, as microcistinas são incapazes de induzir uma resposta imune em animais. Conseqüentemente, foi necessário aplicar métodos de conjugação envolvendo a adição de uma proteína carreadora, mcKLH (cationized Keyhole Limpet Hemocyanin). Portanto, o objetivo inicial desta tese foi o de obter anticorpos monoclonal (em camundongos) e policlonal (em coelho) anti- MCLR. Com relação ao anticorpo monoclonal foram obtidos 9 hibridomas (k29, k210, k317, k248, k284, k290, k2161, k2226, k2232), sendo que apenas 5 se mostraram estáveis (k29, k317, k248, k284, k2232). Estes foram selecionados para serem isotipados, expandidos em líquido ascítico, purificados em coluna cromatográfica de proteína-A e titulados. Dentre estes cinco hibridomas secretores de anticorpos, o clone k317 foi o que melhor reconheceu (mais específico) a toxina MCLR. Os anticorpos do sobrenadante de meio de cultura do hibridoma e o fluido ascítico purificado foram identificados pelo ensaio ELISA (Enzyme Linked Immunosorbent Assay) previamente padronizado. Mesmo sensibilizando a placa de ELISA com diferentes antígenos, tais como MCLR-cBSA, MCLR, MCLR, MCRR, MCYR e MCLA, o clone 17 foi o que apresentou melhor linearidade frente às variantes de microcistina. Portanto, o clone 17 (isótipo IgG1) obtido é muito promissor e será usado para detecção de MCLR na água para consumo humano através do desenvolvimento de um kit de ELISA competição. Com relação ao anticorpo policlonal, o antígeno de imunização foi MCLR-mcKLH, enquanto que o antígeno de sensibilização foi MCLR-cBSA para o ensaio de titulação de anticorpos de classe IgG por ELISA indireto. Na seqüencia, foi padronizado um ensaio ELISA competição utilizando somente a toxina MCLR como antígeno de sensibilização. Este método Caseína foi padronizado, validado e comparado com o kit comercial Abraxis®. O kit ELISA competição que utiliza anticorpo policlonal, nomeado como método Caseína, foi avaliado quanto Limite Inferior de Quantificação, Especificidade, Seletividade, influência do metanol no ensaio, Recuperação, Linearidade, Precisão, Exatidão e Robustez. Este método de triagem apresentou excelente resultado quando comparado ao kit comercial Abraxis®, pois foi capaz de detectar tanto variantes de microcistinas como nodularinas no ambiente aquático. O ensaio ELISA competição utilizando anticorpo policlonal anti-MCLR foi submetido à patente pela Agência USP de Inovação (I.N.P.I. 018090046230). / The contamination of drinking water by cyanobacterial toxins is a public health issue and a concern for water authorities throughout the world. Microcystin-LR (MCLR) is a hazardous cyclic heptapeptide cyanotoxin, which inhibits protein phosphatase PP1 and PP2A in hepatocytes. Microcystins are produced by several genera of cyanobacteria and presents more than 70 structural variations characterized in natural blooms. As haptens, microcystins are unable to invoke an immune response in animals. Consequently, the application of conjugation methods with an additional carrier protein, the KLH (Keyhole Limpet Hemocyanin) was necessary. The main objective of this study was to obtain monoclonal (in mice) and polyclonal (in rabbits) antibodies for reacting against MCLR. In what refers to monoclonal antibodies, 9 hybridomas (k29, k210, k317, k248, k284, k290, k2161, k2226, k2232) were obtained; however only 5 were stables (k29, k317, k248, k284, k2232). These were selected to be isotyped, expanded in ascitic fluid, purified by protein-A column chromatography and then, they were titrated. Out of these five antibody-secretor hybridomas, clone k317 was the best to recognize (more specific) the MCLR toxins. Antibodies in hybridoma cell culture supernatant and purified ascites fluid were identified by ELISA assay (Enzyme Linked Immunosorbent Assay) as prior standardized. Even when sensitizing ELISA plate with different antigens, as MCLR-cBSA, MCLR, MCLR, MCRR, MCYR and MCLA, clone 17 presented the best linearity against microcystin variants. Therefore, the obtained clone 17 (isotype IgG1) is a promising clone and shall be used for detecting MCLR in drinking water through the development of a competitive ELISA immunoassay kit. In what refers to the polyclonal antibody, MCLR-mcKLH was used as immunization antigen, while MCLR-cBSA was used as sensitizing antigen for the IgG titration assay by indirect ELISA. In the sequence, a competition ELISA assay was standardized using the MCLR toxin as sensitizing antigen. This Casein method was standardized, validated and compared to the commercial kit Abraxis®. The competition ELISA kit using polyclonal antibody, known as Casein method, was analyzed concerning its Quantification Inferior Limit, Specificity, Selectivity, methanol influence of the assay, Recuperation, Linearity, Precision, Accuracy and Robustness. This screening method reached excellent results if compared to the commercial kit Abraxis®, for being able to detect both the microcystins variants and the nodularins in aquatic environmental. The competition ELISA assay using anti-MCLR polyclonal antibody was submitted to the grant of a patent by USP Innovation Agency (INPI 018090046230).