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Altered signaling pathways, which are mediated by post-translational modifications and changes in protein expression levels, are key regulators of cancer initiation, progression, and therapeutic escape. Many aspects of cancer progression, including early carcinogenesis and immediate response to drug treatment, are beyond the scope of genetic profiling and non-invasive monitoring techniques. Global protein profiling of cancer cell line models, tumor tissues, and biofluids (e.g. serum or urine) using mass spectrometry-based proteomics produces novel biological insights, which support improved patient outcomes. Recent technological advances resulting in next-generation mass spectrometry instrumentation and improved bioinformatics workflows have led to unprecedented measurement reproducibility as well as increased depth and coverage of the human proteome. It is now possible to interrogate the cancer proteome with quantitative proteomics to identify prognostic cancer biomarkers, stratify patients for treatment, identify new therapeutic targets, and elucidate drug resistance mechanisms. There are, however, numerous challenges associated with protein measurements. Biological samples have a high level of complexity and wide dynamic range, which is even more pronounced in samples used for non-invasive disease monitoring, such as serum. Cancer biomarkers are generally found in low abundance compared to other serum proteins, particularly at early stages of disease where cancer detection would make the biggest impact on improving patient survival. Additionally, the large-scale datasets generally require bioinformatics expertise to produce useful biological insights. These difficulties converge to create obstacles for down-stream clinical translation. This dissertation research demonstrates how proteomics is applied to develop new resources and generate novel workflows to improve protein quantification in complex biosamples, which could improve translation of cancer research to benefit patient care. The studies described in this dissertation move from assessment of quantitative mass spectrometry platforms, to analytical assay development and validation, and ending with personalized biomarker development applied to patient samples. As an example, four different quantitative mass spectrometry acquisition platforms are explored and comparisons of their ability to quantify low abundance peptides in a complex background are explored. Lung cancers frequently have aberrant signaling resulting in increased kinase activity and targetable signaling hubs; kinase inhibitors have been successfully developed and implemented clinically. Therefore, changes in amounts of kinase peptides in the complex background of peptides from all ATP-utilizing enzymes in a lung cancer cell line model after kinase inhibitor treatment was selected as a model system. Traditional mass spectrometry platforms, data dependent acquisition and multiple reaction monitoring, are compared to the two newer methods, data independent acquisition and parallel reaction monitoring. Relative quantification is performed across the four methods and analytical performance as well as downstream applications, including drug target identification and elucidation of signaling changes. Liquid chromatography – multiple reaction monitoring (LC-MRM) was selected for development of multiplexed quantitative assays based on superior sensitivity and fast analysis times, allowing for larger peptide panels. Method comparison results also provide guidelines for quantitative proteomics platform selection for translational cancer researchers. Next, a multiplexed quantitative LC-MRM assay targeting a panel of 30 RAS signaling proteins was developed and described. Over 30% of all human cancers have a RAS mutation and these cancers are generally aggressive and limited treatment options, leading to poor patient prognosis. Many targeted inhibitors have successfully shut down RAS signaling, leading to tumor regression, however, acquired drug resistance is common. The multiplexed LC-MRM assays characterized and validated are a publically available resource for cancer researchers to interrogate the RAS signal transduction network. Feasibility has been demonstrated in cell line models in order to identify signaling changes that confer BRAF inhibitor resistance and biomarkers of sensitivity to treatment. This analytical LC-MRM panel could support meaningful development of new therapeutic options and identification of companion biomarkers, with the end goal of improving patient outcomes. Multiplexed LC-MRM assays developed for personalized disease biomarkers using an integrated multi-omics approach are described for Multiple Myeloma, an incurable malignancy with poor patient outcomes. This disease is characterized by clonal expansion of the plasma cells in the bone marrow, which secrete a monoclonal immunoglobulin, or M-protein. Clinical treatment decisions are based on multiple semi-quantitative assays that require manual evaluation. In the clinic, minimal residual disease quantification methods, including multi-parameter flow cytometry and immunohistochemistry, are applied to bone marrow aspirates, which is a highly invasive technique that does not provide a systemic evaluation of the disease. To address these issues, we hypothesized that unique variable region peptides could be identified and LC-MRM assays developed specific to each patient’s M-protein to improve specificity and sensitivity in non-invasive disease monitoring. A proteogenomics approach was used to design personalized assays for each patient to monitor their disease progression, which demonstrate improved specificity and up to a 500-fold increase in sensitivity compared to current clinical methods. Assays can be developed from marrow aspirates collected when the patient was at residual disease stage, which is useful if no sample with high disease burden is available. The patient-specific tests are also multiplexed with constant region peptide assays that monitor all immunoglobulin heavy and light chain classes, which could reduce analysis to a single test. In conclusion, highly sensitive patient-specific assays have been developed that could change the paradigm for patient evaluation and clinical decision-making, increasing the ability of clinicians to continue first line therapy in the hopes of achieving a cure, or to intervene at an earlier time point in disease recurrence. This study also provides a blueprint for future development of personalized diagnostics, which could be applied to biomarkers of other cancer types. Overall, these studies demonstrate how quantitative proteomics can be used to support translational cancer research, from the impact of different mass spectrometry platforms on elucidating signaling changes and drug targets to the characterization of multiplexed LC-MRM assays applied to cell line models for translational research purposes and in patient serum samples optimized for clinical translation. We believe that mass spectrometry-based proteomics is poised to play a pivotal role in personalized diagnostics to support implementation of precision medicine, an effort that will improve the quality and efficiency of patient care.

Hepatitis B virus (HBV), which belongs to the family Hepadnaviridae, is responsible for acute and chronic hepatitis. The vaccines presently used to immunize patients against HBV are recombinant subunit vaccines consisting of viral surface antigens (S protein). However, they are expensive and their use is limited in poor countries. For that reason, HBV remains an important worldwide health problem. Of the 2 billion people who have been infected with the HBV, more than 350 million have chronic (lifelong) infections, who face increased risk of developing cirrhosis and hepatocellular carcinoma. In this study, high-level expression of recombinant Hepatitis B surface Antigen (rHBsAg), PreS2-S was achieved in the methylotrophic yeast, Pichia pastoris. For this aim, a single copy of HBV M gene (PreS2-S) was inserted at the downstream of the alcohol oxidase (AOX1) promoter of the pPICZA vector. rHBsAg protein could then be expressed intracellularly by induction with methanol. High cell density fermentation was followed by chromatographic separation to obtain pure rHBsAg. Humoral response after immunization with the purified protein was observed in mice using commercial Hepatitis B surface antigen kits. It was verified by the atomic force microscopy that rHBsAg has been produced in the desired conformation.

Group A streptococcus (GAS) is a strictly human pathogen that causes infections ranging from asymptomatic carriage to the highly lethal streptococcal toxic shock syndrome (STSS). GAS are classified according to the sequence of the variable 5’ end of the emm-gene that encodes the surface associated M-protein. In the late 1980s, outbreaks of GAS infections with high rates of STSS were reported in several parts of the world, including Sweden. Superantigens (SAgs), a group of exotoxins, have been described as key mediators of STSS due to their capacity to polyclonally activate T-cells and induce a massive release of inflammatory cytokines. Previous reports have revealed that sera from STSS patients have lower capacity to neutralize this SAg-mediated immune stimulation and a higher prevalence of GAS isolates with specific emm-genotypes during disease outbreaks. The aims of this thesis were to analyse the protective antibody response mounted by the host against SAgs produced by the infecting GAS isolate and to characterise the isolates emm-genotypes and SAg gene profiles. The clinical material examined was collected from patients with STSS, sepsis, erysipelas, or tonsillitis in Sweden between 1986 and 2001. Both acute- and convalescence-phase sera were analyzed, along with the infecting GAS isolates. The 92 clinical GAS isolates examined were found to exhibit a high degree of genetic diversity in terms of the number and identity of their SAg genes. Isolates with a given emm-genotype could be divided into subgroups on the basis of their SAg gene profiles. Ten different SAg gene profiles were identified in the 45 emm1 isolates examined; one of these ten was highly persistent, being observed in 22 isolates collected over 14 years. Two of the 11 known SAg genes in GAS, smeZ-1 and speA, were more prevalent in the emm1 associated profiles than in the SAg gene profiles of isolates with other emm-genotypes. Patients infected by GAS with the emm1-genotype were less likely to produce acute-phase sera that could effectively neutralize the T-cell mitogenicity induced by the infecting isolate’s extracellular products (EP). Sepsis patients whose sera exhibited this lack of neutralizing ability were more prone to developing STSS. Most patients whose acute-phase sera did not effectively neutralize the EP from the infecting isolate lacked protective antibodies in their convalescent-phase sera despite having elevated ELISA titers. The results reported herein show that combining SAg gene profiling with emm-genotyping may be useful for tracking the spread of GAS clones in the community. It was also shown that a lack of neutralizing activity in convalescence-phase sera might be due to an inability of those patients to mount a protective immune response against SAgs produced by the infecting GAS isolate.

Since emerging in Europe and the US, PRRS has spread globally and become the most significant infectious disease currently devastating the swine industry. In the US alone, the economic losses caused by this disease amount to more than 560 million US dollars every year. Modified-live PRRSV vaccines (MLV) are the most effective option currently available for the control of the disease. MLVs can confer solid protection against homologous re-infection and have significant effects in reducing viral shedding. But the vaccine efficacy varies upon heterologous challenge. None of the current vaccines are able to completely prevent respiratory infection, transplacental transmission, as well as pig-to-pig transmission of the virus. More importantly, the intrinsic risk of MLV vaccine to revert to virulent virus under farm conditions poses a great safety concern. The unsatisfactory efficacy and safety of current PRRSV vaccines drives the continuous efforts of developing a new generation of vaccines. The strategy we focus on for novel PRRSV vaccine development is subunit vaccine. The reasons for choosing this strategy are: 1) subunit vaccines only contain the immunogenic fragments of a pathogen. Administration of such pathogen fragments eliminates the risk of pathogens reverting back to their virulent form as in the case of modified live vaccines. 2) Subunit vaccines have advantages in terms of vaccine production since a well-defined pathogen fragment can more easily be produced consistently. To achieve of our goal of developing safe and efficacious subunit vaccines against PRRSV, three projects were completed. First, a scalable process for purification of PRRSV particles from cell culture was developed. This process produced purified viral particles for ELISA and cell-based assays used in vaccine development. Second, a plant-made oral subunit vaccine against PRRSV was developed. Administration of the plant-made vaccine, the vaccinated animals produced virus-specific serum and intestine mucosal antibodies with neutralization activity, as well as cellular immune responses with a preference of virus-specific IFN-γ production. Since neutralization antibodies and virus-specific IFN-γ response are the crucial factors contributing to protection against PRRSV infection, the plant-made oral subunit vaccine strategy is an attractive strategy for developing a new generation of the vaccine to control PRRS disease. Third, a chimeric protein consisting of the ectodomains of viral M and GP5 proteins was expressed and purified. The protein product showed a single band on a silver-stained gel and contained an endotoxin level of less than 10 EU/mg protein. In addition, the purified protein showed expected bioactivities. It was antigenic, could bind to a cellular receptor for the virus (heparan sulfate), and could block virus infection of susceptible cells. Therefore, the chimeric protein is a promising subunit vaccine candidate against PRRSV. / Ph. D.

Strangles is considered the main respiratory disease in horses. The etiologic agent is the bacterium Streptococcus equi subsp. equi (S. equi), responsible for approximately 30% of horse diseases worldwide notifications. The clinical signs of strangles are fever, nasal secretion and lymph node enlargement. The last one occurs due the incomplete phagocytosis of S. equi by defense cells because bacterial hyaluronic acid capsule and M protein (SeM). The epidemiology, the risk factors and the strangles control are poorly understood. The 5' end of the seM gene sequence has been used for isolate differentiation by characterization of the alleles. Therefore, this thesis aimed to study the prevalence of Streptococcus equi subsp. equi (S. equi) in healthy horses, the alleles frequency and the risk factors involved on equine adenitis. One thousand and ten nasal swabs were obtained from healthy horses from 341 farms. Twenty four horses were positive for S. equi, confirmed by PCR and DNA sequencing. The prevalence of S. equi per equine was 2.37%, and 20 farms were positive (5.86%). Risk factor analysis showed by confirming and quantifying statistically that: the number of agglomeration events that horses participate (RR: 1.6), the situation with food container shared (RR: 3.74) and the positive diagnosis for adenitis (RR: 3.20) are significant risk factors for Strangles. These results provide important epidemiological contribution to the equine industry and can give support for the disease control. In addition, the 5 'end of the gene seM was amplified by PCR and sequenced and allele characterized. It was found the same allele (SEM-61) in all the samples. These results support the hypothesis of natural selection of alleles apparently more suited to survive, persist and perpetuate in the population studied. / A adenite equina é uma doença infecto-contagiosa que acomete o trato respiratório superior, sendo uma das principais doenças respiratórias de equinos. O agente etiológico dessa enfermidade é o Streptococcus equi subespécie equi (S. equi), responsável por aproximadamente 30% das notificações em todo o mundo. Os principais sinais clínicos da adenite são febre, secreção nasal e enfartamento de linfonodos, que ocorre pela dificuldade de fagocitose do S. equi por células de defesa devido a presença da cápsula de ácido hialurônico e proteína M. O entendimento sobre a epidemiologia, a análise de fatores de risco para adenite equina e o controle dessa enfermidade ainda são limitados. Estudos moleculares demonstram diferenças na extremidade 5 da sequência do gene (seM) codificador da proteína M de S. equi. Esta região do gene já foi utilizada na diferenciação de isolados por meio da caracterização de diferentes alelos. Por tudo isso, essa tese objetivou obter resultados de prevalência, e também análise de fatores de risco para adenite equina através de um desenho experimental para coleta de suabes nasais. Foram obtidos 1.010 suabes nasais de equinos sadios em 341 fazendas, de onde foram identificados 24 equinos positivos para S. equi em isolamento, que posteriormente foram confirmados por PCR e sequenciamento de DNA. A prevalência estimada por equino foi de 2.37%, e 20 fazendas foram consideradas positivas (5.86%). Na análise de fatores de risco, foi comprovado e quantificado estatisticamente que: número de eventos de aglomeração que os equinos participam (RR:1.06), o ato de compartilhar recipiente de alimento (RR:3.74) e ter tido diagnóstico positivo para adenite (RR:3.20) são fatores de risco relevantes para adenite equina. Estes resultados oferecem contribuições epidemiológicas importantes para a indústria de equinos e pode apoiar o controle da doença. Em paralelo, a região 5 terminal do gene seM das 24 amostras positivas foi amplificada por PCR e sequenciada para caracterização de alelos, sendo identificado o mesmo alelo (seM-61) em todas as amostras. Esses resultados evidenciam a hipótese de seleção natural de alelos aparentemente mais adaptados a sobreviver, persistir e se perpetuar na população estudada.

Strangles is an equine infectious disease that affects the upper respiratory tract, being considered the main respiratory disease in horses. The etiologic agent is Streptococcus equi subsp. equi (S. equi), responsible for approximately 30% of horse diseases worldwide notifications. The clinical signs of strangles are fever, nasal secretion and lymph node enlargement. The last one occurs due the incomplete phagocytosis of S. equi by defense cells because the presence of hyaluronic acid capsule and M protein (SeM) on the bacteria. The understanding of strangles epidemiology and its control is still limited. Molecular studies demonstrate differences in the gene sequence that codify the N-terminal region of the M protein (SeM) of S. equi. This gene region was already used in the differentiation of isolates by characterization of different alleles. This thesis aims to analyze and differentiate 47 S. equi isolates from equine clinical specimens from southern Brazil (15 Thoroughbred horses, 29 animals from the Crioula breed and three Brasileiro de Hipismo) through phylogenetic analysis and differentiation of alleles based on sequencing of the N-terminal region of the SeM protein. Samples were obtained from 31 outbreaks in 20 premises. Fifteen alleles were identified being only one (allele 9), with 7 isolates (14.9%), was already available in the PubMLST-SeM database (allele 61). Among the new identified alleles, the number 1 was the most prevalent with 13 isolates (27.7%), followed by allele 3 with 10 isolates (21.3%). The results demonstrate the great diversity of the amino acid sequence among the S. equi isolates from the studied equine population. Therefore the N-terminal sequence of SeM gene of the S. equi isolates is a useful tool in epidemiological investigation to differentiate isolates in strangles outbreaks with the identification of alleles in horses population, and may represent an alternative for to control the illness with guidance in selecting strains for production of commercial and autogenous vaccines. / A adenite equina é uma doença infecto-contagiosa que acomete o trato respiratório superior, sendo uma das principais doenças respiratórias de equinos. O agente etiológico dessa enfermidade é o Streptococcus equi subespécie equi (S. equi), responsável por aproximadamente 30% das notificações em todo o mundo. Os principais sinais clínicos da adenite são febre, secreção nasal e enfartamento de linfonodos, que ocorre pela dificuldade de fagocitose do S. equi por células de defesa devido a presença da cápsula de ácido hialurônico e proteína M. Somado a isso, o entendimento sobre a epidemiologia e o controle da adenite equina ainda é limitado. Estudos moleculares demonstram diferenças na região N-terminal na sequência do gene codificador da proteína M (SeM) de S. equi. Esta região do gene já foi utilizada na diferenciação de isolados por meio da caracterização de diferentes alelos. Esta dissertação objetivou analisar e diferenciar 47 isolados bacterianos de S. equi provenientes de amostras clínicas de equinos da região sul do Brasil, oriundas de 15 animais Puro Sangue de Corrida (PSC), 29 da raça Crioula e três da raça Brasileiro de Hipismo (BH), por meio de análise filogenética e diferenciação de alelos, com base no sequenciamento da região Nterminal do gene SeM. As amostras foram oriundas de 31 surtos em 20 estabelecimentos de criação. Foram encontrados 15 alelos de SeM, dentre os quais apenas um (alelo 9) já disponível no banco de dados PubMLST-SeM, como alelo 61, com sete isolados (14,9%). Entre os novos alelos identificados, o alelo 1 foi o mais prevalente com 13 isolados (27,7%), seguido pelo alelo 3 com 10 isolados (21,3%). Os resultados demonstram a grande diversidade da proteína M entre os isolados de S. equi na população equina estudada. Portanto, o sequenciamento parcial do gene da proteína M do S. equi é uma ferramenta útil na investigação epidemiológica para a diferenciação de isolados em surtos de adenite equina, com a identificação de alelos em populações de equinos. Além disso, pode representar uma perspectiva para o controle da enfermidade com a orientação na escolha de cepas para confecção de vacinas comerciais e autógenas.