A bactéria gram-positiva Streptococcus agalactiae do grupo B (GBS) faz parte da microbiota normal do trato geniturinário humano, sendo um organismo comensal do corpo da mulher. No entanto, em mulheres grávidas, quando há alterações na composição microbiana do canal vaginal, pode ocorrer a proliferação e a infecção pelo GBS. Este microrganismo, em sua forma patogênica oportunista, pode infectar o neonato durante o parto natural, assim como contribuir para infecções urinárias e uterinas durante a gestação. O GBS já foi identificado como um dos responsáveis pela alta taxa de mortalidade neonatal, sendo um dos principais agentes de infecção em recém-nascidos no mundo. Ele também pode ser a causa de infecções nas gestantes, levando a várias complicações, como corioamnionite, endometrite e infecções do trato urinário e do sítio cirúrgico. Pode haver comprometimento da gestação e do feto, com abortamento, morte fetal intrauterina e ruptura da membrana coriônica, levando a parto prematuro - que pode resultar em outras consequências graves. Este trabalho foi desenvolver um modelo vacinal para mucosa sublingual, utilizando o polissacarídeo capsular do Streptococcus agalactiae como antígeno, encapsulado em Nanopartículas de quitosana. Para o estudo de otimização dos parâmetros de fermentação, para aumentar a produtividade de cápsula polissacarídica (PS) presente na superfície celular, utilizou-se o Banco de Dados Kegg (Kyoto Encyclopedia of Genes and Genomes). A adição do suplemento L-Prolina foi o que propiciou a principio, maior relação entre crescimento bacteriano e formação de cápsula polissacarídica. A purificação e extração da cápsula polissacarídica foi realizada com etapas sucessivas de ultra filtração tangencial e precipitação alcóolica dos contaminantes. As caracterizações físico-químicas: difração de raios-X (DRX), cromatografia gasosa (CGMS), ressonância magnética (NMR) e determinação de açúcares pelo método fenol-sulfúrico, foram realizadas para identificação da composição e estrutura monossacarídica de açucares. O PS isolado apresenta ramificações de fucose, manose, glicose, galactose e N-acetil-glucosamina, apresentando estrutura amorfa. A liofilização do polissacarídeo foi realizada para fins de concentração e conservação. A encapsulação do polissacarídeo acoplada quimicamente com OVA, em uma Nanopartícula de quitosana, teve como finalidade aumentar a mucoadesividade e possibilitar maior absorção do antígeno entre as células da junção epitelial das mucosas sublinguais. A partir da análise de DLS (Espalhamento dinâmico de luz), as Nanopartículas apresentaram dimensões entre 200 a 400 nm e o Potencial Zeta acima de 20. O índice de polidispersão (PDI) está dentro do esperado (abaixo de 0.3). A capacidade de encapsulamento em relação à OVA foi de 92,8% dos grupos que continham PS. O teste IgG sérica total mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior reatividade no teste de ELISA, pela Análise de Variância (ANOVA) com ferramenta de Bonferrone. O teste sIgA mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior concentração de anticorpo sIgA total. Como resultado e conclusão, o polissacarídeo capsular do Streptococcus agalactiae é um bom candidato a antígeno vacinal. / Gram-positive bacteria Streptococcus agalactiae group B (GBS) is part of the normal microbiota of the human genitourinary tract, being a commensal organism of the female body. However, in pregnant women, when there are changes in the microbial composition of the vaginal canal, GBS proliferation and infection may occur. This microorganism, in its opportunistic pathogenic form, can infect the neonate during natural childbirth, as well as contribute to urinary and uterine infections during pregnancy. The GBS has already been identified as one of the responsible for the high neonatal mortality rate, being one of the main agents of infection in newborns in the world. It can also be the cause of infections in pregnant women, leading to various complications such as chorioamnionitis, endometritis, and urinary tract and surgical site infections. There may be pregnancy and fetal impairment, with abortion, fetal intrauterine death, and rupture of the chorionic membrane, leading to premature labor - which can result in other serious consequences. This work was to develop a vaccine model for sublingual mucosa using the capsular polysaccharide of Streptococcus agalactiae as antigen, encapsulated in chitosan nano particles. For the study of optimization of the fermentation parameters, the Kegg (Kyoto Encyclopedia of Genes and Genomes) database was used to increase the productivity of polysaccharide capsule (PS) present on the cell surface. The addition of the L-Proline supplement gave rise to a higher ratio between bacterial growth and polysaccharide capsule formation. The purification and extraction of the polysaccharide capsule was performed with successive stages of tangential ultrafiltration and alcoholic precipitation of the contaminants. The physicochemical characterization of X-ray diffraction (XRD), gas chromatography (CGMS), magnetic resonance (NMR) and determination of sugars by the phenol-sulfuric method were performed to identify the composition and monosaccharide structure of sugars. The isolated PS presents branches of fucose, mannose, glucose, galactose and N-acetyl-glucosamine, presenting amorphous structure. Lyophilization of the polysaccharide was performed for concentration and conservation purposes. The encapsulation of the polysaccharide coupled chemically with OVA in a chitosan nano particle was aimed at increasing mucoadhesiveness and allowing greater absorption of the antigen between the cells of the sublingual mucosal epithelial junction. From the analysis of DLS (dynamic light scattering), the nanoparticles presented dimensions between 200 to 400 nm and the Zeta potential above 20. The polydispersity index (PDI) is within the expected range (below 0.3). The encapsulation capacity for OVA was 92.8% of the groups containing PS. The total serum IgG test showed that the G2 group (Nano particle with Polysaccharide and Protein coupled) was the one that had the highest reactivity in the ELISA test, by Analysis of Variance (ANOVA) with Bonferrone tool. The sIgA test showed that the G2 group (Nanoparticle with Polysaccharide and Protein coupled) had the highest concentration of total sIgA antibody. As a result and conclusion, the capsular polysaccharide of Streptococcus agalactiae is a good candidate for vaccine antigen.

Este estudo buscou avaliar o grau de qualidade da conservação de vacina nas Unidades de Atenção Primária à Saúde - UAPS da região Centro-Oeste do Estado de Minas Gerais. Para tal foi realizada uma pesquisa avaliativa em duas etapas. Na primeira foram utilizados critérios das dimensões, estrutura e processo para mensurar o grau de qualidade das 261 salas de vacina distribuídas nos 55 municípios da região e analisar a relação com o porte populacional e a adesão ao Sistema Único de Saúde. O grau de qualidade foi definido por meio de um sistema de escores, com pesos diferenciados para indicadores em cada dimensão avaliada. Foram definidas as categorias para o grau de qualidade: \"adequado\", \"não adequado\" e \"crítico\". Para verificar a relação entre o grau de qualidade e o porte populacional e adesão ao SUS utilizou-se o teste do qui-quadrado de Pearson. Na segunda etapa do trabalho foram selecionadas as salas de vacina que obtiveram 100% dos critérios selecionados na dimensão estrutura. Assim, foram eleitas 12 salas de vacina para a realização de entrevista semiestruturada com o enfermeiro, técnico/auxiliar de enfermagem e referência técnica em imunização com o propósito de compreender os fatores que dificultam o processo de conservação de vacinas nas UAPS. Na análise dos resultados observou-se que o grau de qualidade de conservação de vacina não está adequado em 59,3% e 26,9% apresentaram grau crítico de qualidade. Os municípios de pequeno porte habilitados na Gestão Plena da Atenção Básica concentraram as piores salas de vacina. A inexistência de profissional atualizado em sala de vacina foi um dos principais achados do estudo na dimensão estrutura o que compromete uma assistência de qualidade em sala de vacina. Na dimensão do processo de conservação de vacinas, o componente organização do refrigerador, principalmente na organização das vacinas quanto à termoestabilidade, apresentou pior escore de qualidade. O estudo apontou um desconhecimento dos enfermeiros e técnicos/auxiliares de enfermagem sobre os efeitos da baixa temperatura sobre as vacinas. O componente condutas frente à alteração de temperatura não foi bem avaliado, grande parte dos trabalhadores em sala de vacina não cumpre as orientações em caso do imunobiológico sofrer alterações de temperatura. Limitações também foram encontradas em relação à supervisão das atividades nas salas de vacina. O excesso de demanda para o enfermeiro e a deficiência na organização do processo de trabalho acabam prejudicando o cumprimento da função primeira do enfermeiro que é o gerenciamento do cuidar, aqui representado pelo cuidar em sala de vacina. O cuidado com a conservação de vacinas, que perpassa por uma série de atividades presente na práxis da enfermagem, aponta para a necessidade de capacitação dos recursos humanos, de monitoramento e avaliação do processo de trabalho e de novos estudos na área. / This study sought to assess the quality of vaccine storage in Primary Healthcare Centers - PHC in the mid-west area of the state of Minas Gerais. To this end a two- stage assessment study was carried out. In the first stage, dimensions, structures and processes were the criteria used to measure quality grades in 261 vaccine rooms distributed over 55 municipalities in the area and to analyze its relation to population size and adherence to the Unified Health System. Quality grades were defined by means of a scoring system, with different weighted scores attributed to indicators for each dimension being rated. Categories for quality grades were then defined as: \"adequate\", \"inadequate\" and \"critical\". Pearson\'s chi-square test was used to verify the correlation between quality grade and population size and adherence to the UHS. In the second stage of the investigation, vaccine rooms meeting 100% of the selected criteria in the structure dimension were selected. Thus, 12 vaccine rooms were selected for the conduction of semi-structured interviews with nurses, nursing assistants and technical immunization coordinators with the aim of shedding light on the factors which render vaccine storage in PHCs difficult. On analysis of the results, it was observed that vaccine storage quality grades are inadequate in 59.3% of these centers, and critical in 26.9%. Small-sized municipalities qualified for Full Basic Healthcare Management aggregated the worst vaccine rooms. With respect to the structure dimension, one of the main findings was absence of professionals with up-to-date knowledge on immunization in the vaccine rooms, which jeopardizes the delivery of quality healthcare assistance in vaccine rooms. With reference to the vaccine storage process dimension, the refrigerator organization component achieved the worst quality evaluation, chiefly concerning the organization of the vaccines in a manner in which they are maintained at stabilized temperatures. The study points to an absence of knowledge on the parts of the nurses and nursing assistants as to the effects of low temperatures on vaccines. Additionally, breached cold chains did not score well; most vaccine room staff fail to follow guidelines when the immunobiologicals are subjected to temperature breaks. Limitations were also encountered in relation to vaccine-room task supervision. Excessive demands on the nurse\'s time and inadequate organization of work processes ultimately hamper the nurse\'s ability to exercise his/her primary role of healthcare management, represented here by vaccine room supervision. Vaccine storage supervision, which spans a series of activities present in nursing praxis, indicates the need to train human resources, monitor and assess work processes and conduct new studies in the field.

A bactéria gram-positiva Streptococcus agalactiae do grupo B (GBS) faz parte da microbiota normal do trato geniturinário humano, sendo um organismo comensal do corpo da mulher. No entanto, em mulheres grávidas, quando há alterações na composição microbiana do canal vaginal, pode ocorrer a proliferação e a infecção pelo GBS. Este microrganismo, em sua forma patogênica oportunista, pode infectar o neonato durante o parto natural, assim como contribuir para infecções urinárias e uterinas durante a gestação. O GBS já foi identificado como um dos responsáveis pela alta taxa de mortalidade neonatal, sendo um dos principais agentes de infecção em recém-nascidos no mundo. Ele também pode ser a causa de infecções nas gestantes, levando a várias complicações, como corioamnionite, endometrite e infecções do trato urinário e do sítio cirúrgico. Pode haver comprometimento da gestação e do feto, com abortamento, morte fetal intrauterina e ruptura da membrana coriônica, levando a parto prematuro - que pode resultar em outras consequências graves. Este trabalho foi desenvolver um modelo vacinal para mucosa sublingual, utilizando o polissacarídeo capsular do Streptococcus agalactiae como antígeno, encapsulado em Nanopartículas de quitosana. Para o estudo de otimização dos parâmetros de fermentação, para aumentar a produtividade de cápsula polissacarídica (PS) presente na superfície celular, utilizou-se o Banco de Dados Kegg (Kyoto Encyclopedia of Genes and Genomes). A adição do suplemento L-Prolina foi o que propiciou a principio, maior relação entre crescimento bacteriano e formação de cápsula polissacarídica. A purificação e extração da cápsula polissacarídica foi realizada com etapas sucessivas de ultra filtração tangencial e precipitação alcóolica dos contaminantes. As caracterizações físico-químicas: difração de raios-X (DRX), cromatografia gasosa (CGMS), ressonância magnética (NMR) e determinação de açúcares pelo método fenol-sulfúrico, foram realizadas para identificação da composição e estrutura monossacarídica de açucares. O PS isolado apresenta ramificações de fucose, manose, glicose, galactose e N-acetil-glucosamina, apresentando estrutura amorfa. A liofilização do polissacarídeo foi realizada para fins de concentração e conservação. A encapsulação do polissacarídeo acoplada quimicamente com OVA, em uma Nanopartícula de quitosana, teve como finalidade aumentar a mucoadesividade e possibilitar maior absorção do antígeno entre as células da junção epitelial das mucosas sublinguais. A partir da análise de DLS (Espalhamento dinâmico de luz), as Nanopartículas apresentaram dimensões entre 200 a 400 nm e o Potencial Zeta acima de 20. O índice de polidispersão (PDI) está dentro do esperado (abaixo de 0.3). A capacidade de encapsulamento em relação à OVA foi de 92,8% dos grupos que continham PS. O teste IgG sérica total mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior reatividade no teste de ELISA, pela Análise de Variância (ANOVA) com ferramenta de Bonferrone. O teste sIgA mostrou que o grupo G2 (Nanopartícula com Polissacarídeo e Proteína acoplados) foi o que teve maior concentração de anticorpo sIgA total. Como resultado e conclusão, o polissacarídeo capsular do Streptococcus agalactiae é um bom candidato a antígeno vacinal. / Gram-positive bacteria Streptococcus agalactiae group B (GBS) is part of the normal microbiota of the human genitourinary tract, being a commensal organism of the female body. However, in pregnant women, when there are changes in the microbial composition of the vaginal canal, GBS proliferation and infection may occur. This microorganism, in its opportunistic pathogenic form, can infect the neonate during natural childbirth, as well as contribute to urinary and uterine infections during pregnancy. The GBS has already been identified as one of the responsible for the high neonatal mortality rate, being one of the main agents of infection in newborns in the world. It can also be the cause of infections in pregnant women, leading to various complications such as chorioamnionitis, endometritis, and urinary tract and surgical site infections. There may be pregnancy and fetal impairment, with abortion, fetal intrauterine death, and rupture of the chorionic membrane, leading to premature labor - which can result in other serious consequences. This work was to develop a vaccine model for sublingual mucosa using the capsular polysaccharide of Streptococcus agalactiae as antigen, encapsulated in chitosan nano particles. For the study of optimization of the fermentation parameters, the Kegg (Kyoto Encyclopedia of Genes and Genomes) database was used to increase the productivity of polysaccharide capsule (PS) present on the cell surface. The addition of the L-Proline supplement gave rise to a higher ratio between bacterial growth and polysaccharide capsule formation. The purification and extraction of the polysaccharide capsule was performed with successive stages of tangential ultrafiltration and alcoholic precipitation of the contaminants. The physicochemical characterization of X-ray diffraction (XRD), gas chromatography (CGMS), magnetic resonance (NMR) and determination of sugars by the phenol-sulfuric method were performed to identify the composition and monosaccharide structure of sugars. The isolated PS presents branches of fucose, mannose, glucose, galactose and N-acetyl-glucosamine, presenting amorphous structure. Lyophilization of the polysaccharide was performed for concentration and conservation purposes. The encapsulation of the polysaccharide coupled chemically with OVA in a chitosan nano particle was aimed at increasing mucoadhesiveness and allowing greater absorption of the antigen between the cells of the sublingual mucosal epithelial junction. From the analysis of DLS (dynamic light scattering), the nanoparticles presented dimensions between 200 to 400 nm and the Zeta potential above 20. The polydispersity index (PDI) is within the expected range (below 0.3). The encapsulation capacity for OVA was 92.8% of the groups containing PS. The total serum IgG test showed that the G2 group (Nano particle with Polysaccharide and Protein coupled) was the one that had the highest reactivity in the ELISA test, by Analysis of Variance (ANOVA) with Bonferrone tool. The sIgA test showed that the G2 group (Nanoparticle with Polysaccharide and Protein coupled) had the highest concentration of total sIgA antibody. As a result and conclusion, the capsular polysaccharide of Streptococcus agalactiae is a good candidate for vaccine antigen.

Immunization remains one of the most successful and cost-effective public health interventions worldwide. The purpose of this study was to examine the individual and socioeconomic factors that influence childhood immunization coverage in Nigeria. The health belief model and the social ecological model were used as the theoretical framework for the study, which examined the effects of individual, parental, and socioeconomic factors on complete immunization among Nigerian children. Univariate, bivariate, and multivariate tests were conducted within a secondary analysis of 2013 Nigerian National Demographic and Health Survey was done. Of 27,571 children aged 0 to 59 months, 22.1% had full vaccination and 29% never received any vaccination. Immunization coverage was significantly related to the socioeconomic status of the child's parents, region, and marital status (p < 0.00). Similarly, child birth order, delivery place, child number, and presence or absence of child health card in the family were significantly related to the level of immunization (p < 0.00). Maternal age, geographical location, education, religion, literacy, wealth index, marital status, and occupation were significantly associated with immunization coverage. Respondent's age, educational attainment, and wealth index remained significantly related to immunization coverage at 95% confidence interval in multivariate analysis. Implications for positive social change include evidence on hindrances to successful immunization programs and relevant information for a more effective, efficient, sustainable and acceptable immunization program for the stakeholders in Nigeria.

Background: Although immunisation services are provided free at all public health facilities in South Africa, immunisation coverage remains variable and disease outbreaks still occur. The coverage rate in the Overberg district is recorded as 75.8%, below the national target of 90% (Western Cape Government Provincial Treasury 2013:2). The researcher wanted to understand what the barriers to accessing immunisation services were and how this might relate to other primary health care services. Methods: The researcher visited 22 households and interviewed nine mothers who had brought their children to Zwelihle Clinic to be immunised and nine community health workers servicing the Zwelihle community in the Overberg district, Western Cape Province. Findings: A key finding is that the data does not reflect the actual situation – children in the community either are immunised at other facilities or have left the catchment area, hence strong relationships between the facility and the community and an electronic patient tracking system become important. Findings impacting access to services include the attitude of administrative staff, waiting times and the impact of migratory communities. Recommendations are made to improve the quality of data, provide training to administrative staff, improve patient education, reduce waiting times and improve the relationship between the clinic and the community in order to better track patient migration / Health Studies / M.A. (Public Health)

Japanese encephalitis virus (JEV), a member of the family flaviviridae, is one of the most important pathogens of the developing countries, causing high mortality and morbidity amongst children. The present study is aimed at the development of a DNA vaccine for Japanese Encephalitis (JE). As a first step towards developing a DNA vaccine for JE, an eukaryotic expression plasmid encoding the envelope (E) glycoprotein of Japanese Encephalitis Virus (pCMXENV) was constructed. This plasmid expresses the E protein intracellularly, when transfected into Vero cells in culture. Several independent immunization and intracerebral (i.e.) JEV challenge experiments were carried out and the results indicate that 51% and 59% of the mice are protected from lethal i.e. JEV challenge, when immunized with pCMXENV via intramuscular (i.m.) and intranasal (i.n.) routes respectively. JEV-specific antibodies were not detected in pCMXENV-immunized mice either before or after challenge. JEV-specific T cells were observed in mice immunized with pCMXENV, which increased significantly after JEV challenge indicating the presence of vaccination-induced memory T cells. Enhanced production of interferon-y (EFN-y) and complete absence of interleukin-4 (IL-4) in splenocytes of pCMXENV-immunized mice on restimulation with JEV antigens in vitro indicated that the protection is likely to be mediated by T helper (Th) lymphocytes of the Thl sub type. These results demonstrated that immunization with a plasmid DNA expressing intracellular form of JEV E protein confers significant protection against i.e. JEV challenge even in the absence of detectable antiviral antibodies. We then examined the potency of JEV DNA vaccines as well as that of the inactivated mouse brain derived BIKEN vaccine in the i.e. challenge model. The results indicate that all the mice immunized with BIKEN JE vaccine were protected against i.e. JEV challenge while 50% protection was observed in case of mice immunized with pJME or pJNSl and 38% protection was observed in pCMXENV-immunized mice. Immunization with both pJME and pJNSl resulted in 66% protection. These results indicate that the BIKEN JE vaccine confers better protection against i.e. JEV challenge than DNA vaccines. The fact that the BIKEN vaccine conferred better protection against i.e. JEV challenge than DNA vaccines indicated that the i.e. JEV challenge model can be exploited further to examine the potency of different DNA vaccine constructs. Towards this goal, we constructed plasmids that encode secretory or nonsecretory forms of JEV E protein and examined their potency in the i.e. JEV challenge model. Our results indicate that i.m. immunization of mice with plasmid encoding secretory form of JEV E protein confers higher level (75%-80%) protection than those encoding nonsecretory forms. Cytokine analysis of splenocytes isolated from DNA immunized mice after stimulation in vitro with JEV revealed that immunization with plasmid encoding secretory form of JEV E protein induces both Thl and Th2 responses while those encoding nonsecretory forms induce only Thl type of response. Thus, synthesis of secretory form of JEV E protein results in an altered immune response leading better protection against i.e. JEV challenge. Based on our studies, we propose that both cellular and humoral immune responses play a key role in protective immunity against i.e. JEV challenge and DNA vaccines that can induce higher levels of neutralizing antibodies will be as efficient as the BIKEN vaccine in conferring protection against i.e. JEV challenge.

<p>This thesis consists of five papers. In the first two papers we consider a general approach to cash flow valuation, focusing on dynamic properties of the value of a stream of cash flows. The third paper discusses immunization theory, where old results are shown to hold in general deterministic models, but often fail to be true in stochastic models. In the fourth paper we comment on the connection between arbitrage opportunities and an immunized position. Finally, in the last paper we study coherent and convex measure of risk applied to portfolio optimization and insurance.</p>

Thesis (Ph. D.)--University of Missouri--Columbia, 2005. / "May 2005." Typescript. Vita. Includes bibliographical references (leaves 139-157). Also issued on the Internet.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Lectins are proteins that bind specifically to carbohydrates and have important role in modulation of the immune response. KM+ and Jacalin (JAC) are lectins from the seeds from jackfruit (Artocarpus integrifolia) and ScLL is a lectin from the Synadenium carinatum latex. Neospora caninum is an apicomplexan parasite that causes neuromuscular disease in dogs and reproductive disorders in cattle, with serious economic impact on the livestock industry. The immunestimulatory role of plant lectins has been investigated in several parasitic infections, but not in neosporosis. This study aimed to evaluate the adjuvant effect and the immunestimulatory potential of KM+, JAC and ScLL in immunization of mice against neosporosis. C57BL 6 mouse groups were subcutaneously immunized three times at 15-day intervals with Neospora lysate antigen (NLA) associated with lectins (NLA+KM, NLA+JAC and NLA+ScLL groups), NLA alone, lectins alone (KM, JAC and ScLL groups), and PBS group (infection control). Animals were challenged with Nc-1 isolate (2x107 tachyzoites) and evaluated for morbidity, mortality, specific antibody response, cytokine production by spleen cells, cerebral parasite burden and histopathological lesions. Serological assays demonstrated higher levels of IgG to N. caninum for NLA+KM and NLA+ScLL than NLA+JAC and NLA groups. NLA+KM group induced higher levels of IgG2a isotype whereas NLA+ScLL induced higher levels of IgG1 isotype. In all groups, IgG1 response was higher than IgG2a response before and after challenge, but the IgG2a/IgG1 ratio increased after challenge in NLA+KM, NLA+ScLL and KM groups. Cytokine production after in vitro antigenic stimulation showed that NLA+KM induced high levels of IFN-g and IL-10, presenting the highest IFN-g/IL-10 ratio, followed by NLA+ScLL group, indicating a pattern of immune response toward Th1 type. NLA+JAC induced low levels of these cytokines and the lowest IFN-g/IL-10 ratio in relation to other groups, indicating a profile of Th2 type immune response. After parasite challenge, NLA+KM mice showed the highest survival with low brain parasite burden and moderate tissue inflammation, whereas NLA+ScLL mice presented intermediate survival with low brain parasite burden and low scores of morbidity and inflammation. NLA+JAC group exhibited intermediate survival, but with the highest brain parasite burden and mild inflammation. In conclusion, KM+ and ScLL lectins showed suitable adjuvant effect by increasing NLA immunogenicity and immunostimulatory role by conferring partial protection of mice immunized and challenged with lethal dose of N. caninum, while the JAC lectin showed no adequate adjuvant effect in the immunization against neosporosis. / Lectinas são proteínas que se ligam especificamente a carboidratos e possuem importantes papéis na modulação da resposta imune. KM+ e Jacalina (JAC) são lectinas da semente da jaca (Artocarpus integrifolia) e ScLL é uma lectina do látex da planta Synadenium carinatum. Neospora caninum é um parasito do filo Apicomplexa que causa doença neuromuscular em cães e desordens reprodutivas em bovinos, causando sério impacto econômico na indústria agropecuária. O papel imunoestimulador de lectinas de plantas tem sido investigado em diversas infecções parasitárias, mas não na neosporose. Esse estudo teve como objetivo avaliar o efeito adjuvante e o potencial imunoestimulador de KM+, JAC e ScLL na imunização de camundongos contra a neosporose. Grupos de camundongos C57BL/6 foram imunizados subcutaneamente por três vezes, em intervalos de quinze dias, com o antígeno de lisado total de Neospora (NLA) associado com as lectinas (grupos NLA+KM, NLA+JAC, NLA+ScLL), NLA somente, lectinas somente (KM, JAC e ScLL), além do grupo PBS (controle da infecção). Os animais foram desafiados com isolado Nc-1 (2x107 taquizoítas) e avaliados quanto aos escores de morbidade, mortalidade, resposta imune humoral específica, produção de citocinas por células do baço, carga parasitária cerebral e lesões histopatológicas. Resultados sorológicos demonstraram maiores níveis de IgG anti N. caninum produzidos pelos animais dos grupos NLA+KM e NLA+ScLL que NLA+JAC e NLA somente. O grupo NLA+KM induziu maiores níveis do isotipo IgG2a, enquanto NLA+ScLL induziu maiores níveis do isotipo IgG1. Em todos os grupos, a resposta de IgG1 foi maior do que de IgG2a antes e após o desafio, porém a razão IgG2a/IgG1 aumentou após o desafio nos grupos NLA+KM, NLA+ScLL e KM. A produção de citocinas após o estímulo antigênico in vitro demonstrou que NLA+KM induziu altos níveis de IFN-g e IL-10, apresentando a maior razão IFN-g/IL-10, seguido pelo grupo NLA+ScLL, indicando um padrão de resposta imune direcionado ao perfil Th1. NLA+JAC induziu baixos níveis destas citocinas e menor razão IFN-g/IL-10 em relação aos demais grupos, indicando um padrão de resposta imune do tipo Th2. Após o desafio com o parasito, camundongos do grupo NLA+KM apresentaram a maior sobrevida com baixa carga parasitária cerebral e moderada inflamação tecidual, enquanto animais imunizados com NLA+ScLL apresentaram sobrevida intermediária com baixa carga parasitária cerebral e baixos escores de morbidade e inflamação. O grupo NLA+JAC exibiu sobrevida intermediária, mas com alta carga parasitária cerebral e suave inflamação. Em conclusão, as lectinas KM+ e ScLL mostraram ser adjuvantes satisfatórios por aumentar a imunogenicidade do NLA e apresentaram efeito imunoestimulador por conferir proteção parcial dos camundongos imunizados e desafiados com dose letal de N. caninum, enquanto a lectina Jacalina não produziu efeito adjuvante suficiente na imunização contra a neosporose. / Mestre em Imunologia e Parasitologia Aplicadas

Neonatal mice have immature immune systems with defects in several components of inflammatory, innate, and specific immune responses and develop a preferential T helper type 2 (Th2) response following immunization with many vaccine antigens. Although maternal antibody is the major form of protection from disease in early life when the neonatal immune system is still immature, the presence of maternal antibody also interferes with active immunization, placing infants at risk for severe bacterial and viral infection. Recent studies have suggested that immunizing with DNA plasmids encoding the vaccine antigen of interest is highly efficacious in a variety of adult animal models. However, similar extensive studies have not been conducted in infants. In this dissertation, we examine both the quantitative and qualitative differences between neonatal and adult humoral and cell-mediated immune responses in the presence or absence of maternal antibody. First, we wished to determine if one-day-old neonatal mice immunized with plasmid DNA expressing influenza A/PR/8/34 hemagglutinin (HA) by either intramuscular (i.m.) or gene gun (g.g.) inoculation were capable of generating humoral responses comparable to those in mice immunized as adults. We found that newborn mice developed stable, long-lived, protective anti-HA-specific IgG responses similar in titer to those of adult DNA-immunized mice. However, unlike the adult i.m. and g.g. DNA immunizations, which develop polarized IgG2a and IgG1 responses, respectively, mice immunized as neonates developed a variety of IgG1-, IgG2a-, and mixed IgG1/IgG2a responses regardless of the inoculation method. Boosting increased, but did not change these antibody profiles. We also found that, in contrast to the DNA immunizations, inoculations of newborn mice with an A/PR/8/34 viral protein subunit preparation failed to elicit an antibody response. Further, temporal studies revealed that both responsiveness to protein vaccination and development of polarized patterns of T help following DNA immunization appeared by 2 weeks of age. To determine if the disparity of polarized IgG responses between neonatal and adult DNA vaccinated mice was due to deficiencies in Th1 promoting cytokines, we addressed the ability of DNA encoding Th1 cytokines to bias the isotype of antibody raised by neonatal DNA immunization. We found that neonatal mice coimmunized with HA and either IL-12 or IFNγ-expressing DNAs developed IgG2a-biased immune responses, regardless of inoculation method, whereas these DNAs had no effect on IgG subtype patterns in adult DNA immunized mice. Consistent with the Th1-promoting effects of these cytokines, we also observed that codelivery of IL-12 or IFNγ DNAs raised T helper responses toward Th1 in mice immunized both as neonates or adults. Thus, codelivery of cytokine DNAs may be effective at tailoring immune responses depending on the required correlates of protection for a given pathogen. Finally, we addressed the effect of maternal antibody on the elicitation of humoral and cell-mediated immune responses. We tested the ability of i.m. and g.g. immunization with DNA expressing influenza HA and/or nucleoprotein (NP) to raise protective humoral and cellular responses in the presence and absence of maternal antibody. We found that neonatal mice born to influenza-immune mothers raised full antibody responses to NP but failed to generate antibody responses to HA. In contrast, the presence of maternal antibody did not affect the generation of long-lived CD4+ and CD8+ T cell responses to both HA and NP. Thus, maternal antibody did not affect cell-mediated responses, but rather it limited humoral responses, with the ability to limit the antibody response correlating with whether the DNA-expressed immunogen was localized in the plasma membrane or within the cell. We further observed that protection from influenza virus challenge was dependent on the presence of anti-HA IgG and was independent of the presence T cell responses. Taken together with other published studies, the data presented in this dissertation help better characterize the responses elicited by DNA vaccines at birth. This dissertation presents several novel observations including the temporal development of polarized IgG subtype responses, the ability of codelivered Th1 cytokine DNA to affect both antibody and T cell responses in the neonate, and the ability to generate humoral responses to intracellular, but not plasma membrane proteins, in the presence of maternal antibody. Furthermore, the data provides rationale for further development of DNA vaccines in the neonate.