The development of a drug able to distinguish between tumor and host cells has been long sought, but the solid tumor microenvironment (TME) confounds many current therapeutics. Solid tumors present several challenges for oncotherapeutics, primarily, (1) aberrant vascularization, resulting in hypoxia, necrosis, abnormally high pH, and (2) tumor immune suppression. Oncolytic microbes are drawn to this microenvironment by an innate ability to selectively penetrate, colonize, and eradicate solid tumors as well as reactivate tumor associated immune components. To consider oncolytic bacteria deployment into this microenvironment, Chapter 1 dives into the background of oncolytic microbes. A discussion of the oncolytic bacterial field state, identifying Clostridium novyi? as a promising species, and details genetic engineering techniques to develop customized bacteria. Despite the promise of C.novyi in preclinical/clinical trials when administered intratumorally, the genetic and biochemical uniqueness of C.novyi necessitated the development of new methodologies to facilitate more widespread acceptance. Chapter 2 reports the development of methods that facilitate experimental work and therapeutic translation of C.novyi, including the ability to work with this obligate micro-anaerobe aerobically on the benchtop. While methods development is a necessary step in the clinical translation of C.novyi so too is choosing the correct model of the TME within which to test a potential anti-cancer therapy. While the typical solid TME includes both phenotypic and genotypic heterogeneity, the methods used to model this disease state often do not reflect this complexity. This simplistic approach may have contributed to stagnant five-year survival rates over the past four decades. Nevertheless, simplistic models are a necessary first step in clinical translation. Chapter 3 explores the impact of cancer cell lines co-cultured with C. novyi to establish the efficacy of this oncolytic bacteria in a monolayer culture. Chapter 4 extends this analysis adding not only a level of complexity by using an in vivo model, but also using CRISPR/Cas9 to modify the genome of C.novyi to encode a tumor targeting peptide, RGD, for expression within the spore coat. The combination of these studies indicates that C. novyi is uniquely poised to accomplish the long sought after selective tumor localization via intravenous delivery.

A bovinocultura brasileira possui grande ênfase no mercado nacional. Doenças que acometem rebanhos comprometem o mercado além de gerarem grandes prejuízos econômicos. O Clostridium novyi tipo B provoca necrose hepática em bovinos através da produção da alfa toxina, uma potente exotoxina que reduz a produtividade através de alterações como hemoglobinúria, redução do apetite, febre, letargia, diminuição da produção de leite e sangue nas fezes. Conter o microrganismo causador torna-se uma busca necessária tanto do ponto de vista econômico quanto social. Entretanto, o controle da doença ainda é realizado por vacinas formuladas com múltiplos antígenos. A emulsão pode ser uma alternativa promissora para a melhoria da adsorção de antígenos nas formulações vacinais. Camundongos da linhagem Swiss foram utilizados a fim de se avaliar aspectos clínicos e validar resultados referentes a composição de uma nova formulação vacinal contendo adjuvante Montanide ISA 61 VG e antígeno inativado de C. novyi. Os testes de caracterização e antigenicidade indicaram a presença da proteína alfa toxina na composição avaliada. A imunogenicidade do antígeno inativado em emulsão W/O (água/óleo) foi verificada e a proporção empregada (40/60) mostrou ser ideal no uso de múltiplos antígenos, apresentando inocuidade, estabilidade do produto, liberação controlada e estímulo da resposta imune. A determinação da concentração de antígeno foi averiguada pela relação antígeno ativo e inativado com soros de animais doentes, visto a eficácia vacinal de 40%. A adequação da concentração de alfa toxina inativada na emulsão mostrou ser necessária para atingir melhores valores de proteção animal. Análises de hemograma, bioquímicas e morfologia de fígado, baço e coxa contribuíram para elucidar os efeitos da emulsão e comprovar necrose hepática nos grupos não imunizados, além de sugerir avanços na adsorção de vacinas. Os resultados possibilitaram o estabelecimento de um modelo murino de infecção de C. novyi com futuras aplicações relacionadas à produção vacinal com múltiplos antígenos emulsificados para controle das clostridioses. / Brazilian cattle breeding has great emphasis on the national market. Diseases that affect herds compromise the market as well as generate great economic losses. Clostridium novyi type B causes hepatic necrosis in cattle through the production of alpha toxin, a potent exotoxin that reduces productivity through changes such as hemoglobinuria, reduced appetite, fever, lethargy, decreased milk and stool production. Containing the causative micro-organism becomes a necessary quest both economically and socially. However, control of the disease is still performed by vaccines formulated with multiple antigens. The emulsion may be a promising alternative for the improvement of antigen adsorption in vaccine formulations. Swiss strain mice were used to evaluate clinical aspects and validate results regarding the composition of a new vaccine formulation containing Montanide ISA 61 VG adjuvant and C. novyi inactivated antigen. Characterization and antigenicity tests indicated the presence of the alpha toxin protein in the evaluated composition. The immunogenicity of antigen inactivated in W / O emulsion (water / oil) was verified and the ratio employed (40/60) showed to be ideal in the use of multiple antigens, presenting innocuousness, product stability, controlled release and stimulation of the immune response. The determination of the antigen concentration was investigated by the active antigen ratio and inactivated with sera from sick animals, since the vaccine efficacy was 40%. The suitability of the inactivated alpha toxin concentration in the emulsion was shown to be necessary to achieve better animal protection values. Hemogram, biochemical and liver, spleen and thigh morphology contributed to elucidate the effects of the emulsion and to verify hepatic necrosis in the nonimmunized groups, in addition to suggesting advances in the adsorption of vaccines. The results allowed the establishment of a murine model of C. novyi infection with future applications related to the vaccine production with multiple emulsified antigens to control clostridia.