The hepatitis C virus (HCV) currently infects 3% of the world???s population, with chronic infection in 50-80% of exposed individuals. A small subset of individuals who are exposed to HCV do not develop anti-HCV antibodies, persistent viraemia or chronic hepatitis despite generating HCV-specific CD4+ and CD8+ T cells. These individuals are believed to develop an immune response which rapidly clears viraemia prior to the induction of an antibody response. Circumstantial evidence supports the likelihood that some of these individuals may generate these same responses and outcomes on repeated occasions of HCV infection. HCV-specific cellular immune responses in seronegative subjects have been the subject of only limited prior study, in part due to the lack of appropriate recombinant antigens and assay systems. Therefore, this thesis described the development and validation of an interferon-? (IFN-?) ELISPOT assay using overlapping peptides (n=441). Using this assay, HCV-specific cellular immune responses were detected in 5/10 (50%) of chronically infected subjects. Responses were identified more frequently, and were directed against more regions of the HCV genome, than with traditional assay systems. This IFN-? ELISPOT assay, a comparable interleukin (IL)-2 ELISPOT assay, and a multiplex in vitro cytokine production assay were then used to evaluate HCV-specific cellular immune responses in three cohorts of seronegative subjects at high-risk of exposure to HCV ??? babies born to infected mothers, multiply-transfused subjects with thalassaemia, and high risk injecting drug users. Cellular immune responses were evaluated in 23 infants born to HCV-antibody positive women. Responses were not detected in infants born to HCV-PCR negative mothers. IFN-? production was detected in 1/11 infants born to viraemic mothers using the ELISPOT assay, with cytokine production observed in an additional 3/5 infants studied using the in vitro cytokine production assay. HCV-specific cellular immune responses were assessed in a cohort of multiply transfused subjects with thalassaemia using assays for cytotoxic T lymphocyte activity, IFN-? and IL-2 ELISPOT, as well as lymphocyte proliferation and in vitro cytokine production. Responses were detected in 6/13 chronically infected subjects (46%), 4/7 subjects who had cleared infection (71%), and 14/17 seronegative subjects (82%). The seronegative subjects had responses which were broader and higher in magnitude than those with chronic HCV infection, although lower and narrower than in subjects who had cleared prior HCV infection. IFN-? and IL-2 ELISPOT assays, in additional to in vitro cytokine production assays, were performed on 41 injecting drug users (IDUs), with responses detected in 6 (15%). Seronegative IDUs with HCV-specific cellular immune responses had been injecting for a mean of 7.7 years, and reported multiple risk factors for exposure to HCV. The combined data from these three cohorts indicate that the HCV-specific cellular immune responses detected in seronegative subjects were generally broad in specificity. Cytokine production was generally Th1-biased, a pattern which has previously been associated with an increased likelihood of clearance in primary infection. The findings also suggest that responses can be maintained for decades after exposure, and may provide protection against repeated exposures. In summary, cellular immunity against HCV is evident in some seronegative high risk subjects, suggesting that the cellular immune responses may efficiently facilitate viral clearance. Understanding the mechanisms of this immune response pattern will allow better understanding of the host response to HCV and may provide key insights into vaccine design.

The hepatitis C virus (HCV) currently infects 3% of the world???s population, with chronic infection in 50-80% of exposed individuals. A small subset of individuals who are exposed to HCV do not develop anti-HCV antibodies, persistent viraemia or chronic hepatitis despite generating HCV-specific CD4+ and CD8+ T cells. These individuals are believed to develop an immune response which rapidly clears viraemia prior to the induction of an antibody response. Circumstantial evidence supports the likelihood that some of these individuals may generate these same responses and outcomes on repeated occasions of HCV infection. HCV-specific cellular immune responses in seronegative subjects have been the subject of only limited prior study, in part due to the lack of appropriate recombinant antigens and assay systems. Therefore, this thesis described the development and validation of an interferon-? (IFN-?) ELISPOT assay using overlapping peptides (n=441). Using this assay, HCV-specific cellular immune responses were detected in 5/10 (50%) of chronically infected subjects. Responses were identified more frequently, and were directed against more regions of the HCV genome, than with traditional assay systems. This IFN-? ELISPOT assay, a comparable interleukin (IL)-2 ELISPOT assay, and a multiplex in vitro cytokine production assay were then used to evaluate HCV-specific cellular immune responses in three cohorts of seronegative subjects at high-risk of exposure to HCV ??? babies born to infected mothers, multiply-transfused subjects with thalassaemia, and high risk injecting drug users. Cellular immune responses were evaluated in 23 infants born to HCV-antibody positive women. Responses were not detected in infants born to HCV-PCR negative mothers. IFN-? production was detected in 1/11 infants born to viraemic mothers using the ELISPOT assay, with cytokine production observed in an additional 3/5 infants studied using the in vitro cytokine production assay. HCV-specific cellular immune responses were assessed in a cohort of multiply transfused subjects with thalassaemia using assays for cytotoxic T lymphocyte activity, IFN-? and IL-2 ELISPOT, as well as lymphocyte proliferation and in vitro cytokine production. Responses were detected in 6/13 chronically infected subjects (46%), 4/7 subjects who had cleared infection (71%), and 14/17 seronegative subjects (82%). The seronegative subjects had responses which were broader and higher in magnitude than those with chronic HCV infection, although lower and narrower than in subjects who had cleared prior HCV infection. IFN-? and IL-2 ELISPOT assays, in additional to in vitro cytokine production assays, were performed on 41 injecting drug users (IDUs), with responses detected in 6 (15%). Seronegative IDUs with HCV-specific cellular immune responses had been injecting for a mean of 7.7 years, and reported multiple risk factors for exposure to HCV. The combined data from these three cohorts indicate that the HCV-specific cellular immune responses detected in seronegative subjects were generally broad in specificity. Cytokine production was generally Th1-biased, a pattern which has previously been associated with an increased likelihood of clearance in primary infection. The findings also suggest that responses can be maintained for decades after exposure, and may provide protection against repeated exposures. In summary, cellular immunity against HCV is evident in some seronegative high risk subjects, suggesting that the cellular immune responses may efficiently facilitate viral clearance. Understanding the mechanisms of this immune response pattern will allow better understanding of the host response to HCV and may provide key insights into vaccine design.

Abstract In order to determine whether antigens prepared from adult intestine of Angiostrongylus cantonensis have potential to induce a protective immunity in the rodent hosts, somatic antigens extracted from male and female adults as well as gut antigens isolated from female adults were used to immunize rats against A. cantonensis infection. A 14% reduction in L5 recorvery from brain as well as a 15% reduction in adult recovery from pulmonary artery were achieved in the immunized rats when compared with the control group after infection with 50 larvae. The length of worms recovered from immunized rats was shorter than that in other groups. The larvae recovered from fecal materials in immunized rats were also reduced. In cell proliferation test, the stimulation index of gut antigens increased with times of immunization and exhibited the highest values. However, serum IgG titers were not correlated with protective immune responses. A 84 kDa protein contained in all antigen preparations was recognized by immune serum against gut antigens. Strong positive reactions were detected by indirect immunofluorescent assay in the internal musculature of the body, gut and reproductive tract wall and gut lumen. The composition of gut antigens was similar to that of gut membrane proteins. Immune sera recognized several major gut proteins were also appeared on gut membrane proteins. Further studies are required to provide evidence that gut membrane proteins play in the protective immune response against A. cantonensis infections, including the 84 kDa protein.

RAG1-/- mutant zebrafish lack T and B lymphocytes. However, when re-exposed to homologous bacteria, these fish mount a response that provides specific protection. To further define this response, we utilized microarray analyses to determine the mechanisms underlying innate immune system memory in zebrafish. We also analyzed interferon (IFN) gamma by qRT-PCR. It is produced by activated NK cells and could indicate if this cell mediates the protective response seen in lymphocyte deficient zebrafish. Pathological studies and in situ hybridizations were performed to observe tissue changes and location of the cells that produced IFN gamma. Following bacterial re-exposure, zebrafish transcripts in cell receptor activation, cell proliferation and cytotoxic function categories were differentially expressed. We found high expression of IFN gamma in the lymphocyte like cell population after bacterial exposure and this was induced to a higher level in fish that had been vaccinated. The phagocytic cell population showed no induction of INF gamma. Over-all, the pathological response was much less severe in the vaccinated (48 hps) fish. Our microarray and pathological findings indicate that the primary immune response of mutant zebrafish is not impaired, and they demonstrate an enhanced innate immune response following secondary bacteria exposure. Following homologous secondary exposure, mutant zebrafish have a cell population that is undergoing upregulated cell receptor activation, cell cytotoxic functions and cell proliferation. This cell population expresses INF gamma. Activated T cells, NK-T cells and NK cells express INF gamma. Since RAG1 deficient zebrafish do not have T or NK-T cells, this cell population is most likely NK cells.

Chlamydia trachomatis is a human pathogen of the genital tract and ocular epithelium. It is an obligate intracellular parasite with a unique biphasic development cycle. C.trachomatis infection is the most common bacterial sexually transmitted disease in industrialized nations. Its ability to cause chronic disease makes it a serious economic burden and health threat to developed and developing countries. Although treatable, approximately 70% of C.trachomatis infections are asymptomatic, potentially leading to the development of chronic sequelae. Furthermore, chlamydial genital tract infection has been associated with an increased risk of cervical cancer and human immunodeficiency virus infection. Consequently, the development of an efficacious vaccine is the most convenient, potentially reliable and cost effective option to control chlamydial infection and disease complications. Anti-chlamydial protective immunity is essentially mediated by a T helper, type 1 (Th1), response that is dependent upon the presentation of antigen via major histocompatibility (MHC) class II molecules. While antibody secreting cells are not critical components of the primary effector response, they have been shown to be important for clearance of re-infection. Thus an ideal vaccine would be one capable of inducing both a strong Th1 T cell response and a strong mucosal antibody response. Currently there are very few efficacious vaccine candidates that have been identified and characterized. More specifically, there is only a limited number of known T cell antigens processed and presented by the human leukocyte antigen (HLA) class II molecules. This type of antigen is going to be essential to the development of an efficacious chlamydial vaccine. In this study we have identified a number of unique vaccine candidates using a novel in silico approach. In an attempt to overcome HLA polymorphism the whole chlamydial genome was screened for proteins containing epitopes predicted to bind multiple HLA class II molecules (i.e. predicted ‘promiscuous’ T cell epitopes). A wide range of HLA class II molecules were used in this screen to identify vaccine antigens that could potentially offer broad and ethnically balanced population coverage. This analysis identified a number of novel targets and was validated by the identification of a known chlamydial T cell epitope. A selection of these target proteins was cloned, expressed and purified. Recombinant protein was screened against serum samples from patients with both acute and chronic chlamydial infections. Two novel targets, hypothetical protein CT425 and ribonucleotide reductase small chain protein (NrdB) were identified as being immunoreactive. The in vivo protective efficacy of NrdB was analyzed using a mouse model. CD4+ T cells were harvested from NrdB immunized mice and adoptively transferred to naïve mice, which were subsequently infected at the genital site. NrdB immunization was found to confer a CD4+ T cell driven degree of protection similar to that seen with CD4+ T cells primed from a live challenge. The adjuvants and route of immunization used ensured immunological responses were initiated at both the systemic and local sites of infection. Immunization elicited a predominant Th1 response with primed T cells producing high levels of interferon gamma, an essential requirement for the development of an efficacious chlamydial vaccine. Furthermore, high titres of antigen specific IgG and IgA were produced following immunization, with sera derived antibodies demonstrating neutralization properties. NrdB is a highly conserved chlamydial protein with an essential role in the replication of chlamydiae and could play a central role in a multi-subunit vaccine against chlamydial genital tract infections.

Paracoccina (PCN) é um constituinte de Paracoccidioides brasiliensis, um patógeno humano que causa a paracoccidioidomicose, micose sistêmica mais prevalente na América Latina. A PCN é uma proteína de função dual, com domínios de atividade lectínica e de N-acetilglicosaminidase. Análises proteômicas da preparação paracoccina revelaram a sua correspondência com uma proteína hipotética de P. brasiliensis do isolado 18 (Pb18), anotada como PADG-3347.1, que tem sequência polipeptídica semelhante a família das endoquitinases 18. Essas endoquitinases apresentam domínios distintos de atividade lectínica e enzimática. O conjunto de exons do gene correspondente, PADG-3347.1, foi clonado e expresso em E. coli, e as características físicas e biológicas da proteína recombinante foram comparadas com as da PCN. Além disso, a PADG-03347.1 recombinante (rPCN) foi avaliada por suas propriedades imunomoduladoras e sua capacidade em conferir proteção contra a infecção por P. brasiliensis. Nesse sentido, investigamos a interferência da administração profilática e terapêutica de rPCN no curso da infecção por P. brasiliensis em camundongos BALB/c. A histopatologia pulmonar dos camundongos tratados com a rPCN, mostrou menor ocorrência de granulomas, e estes também foram menores do que os observados nos animais controles. Consistente com a observação de poucas leveduras no centro dos granulomas, a contagem de UFC a partir do homogenato pulmonar dos camundongos tratados foi inferior ao observado nos animais controles. Além disso, a administração de rPCN, foi associada com altos níveis de IL-12, IFN-, TNF-, NO e IL-10 detectados no homogenato pulmonar. Os altos níveis de citocinas produzidos nos animais tratados com rPCN nos levou a investigar a ocorrência de interação da lectina com receptores presentes em células da imunidade inata, tais como TLR2 e TLR4. Verificamos que a rPCN ativa TLR2, nas formas homo ou heterodimérica, e TLR4, de modo independente dos correceptores CD14 e CD36. Estes dados revelam um possível mecanismo pelo qual rPCN gera proteção nos camundongos contra a PCM. rPCN, administrada terapêutica ou profilaticamente, induz a ativação de TLRs e imunidade Th1, conferindo proteção contra a infecção por P. brasiliensis. / Paracoccin is a constituent of Paracoccidioides brasiliensis, a human pathogen that causes paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. Paracoccin is a dual function protein exerting lectin and N-acetylglucosaminidase activities. Proteomic analysis of paracoccin preparation revealed its correspondence with a hypothetical protein from P. brasiliensis isolate Pb18 (Pb18), annotated as PADG-3347.1, which has a polypeptide sequence similar to the family 18 endochitinases. These endochitinases have distinct lectin and enzymatic domains. The multi-exon assembly of the correspondent gene (PADG-3347) was cloned and expressed in E. coli, and the physical and biological features of the recombinant protein were compared to those of the native paracoccin. Moreover, recombinant PADG-3347.1 (rPCN) was evaluated for its immunomodulatory properties and its ability to confer protection against murine P. brasiliensis infection. Thus, we investigated the interference of prophylactic and therapeutic administration of rPCN on the course of P. brasiliensis infection in BALB/c mice. The pulmonary histopathology of the treated mice showed lower incidence of granulomas, which were also smaller than those observed in the control animals. Consistently with the observation of few yeasts in the center of the granulomas, the CFU count provided by lung homogenates of treated mice was lower than the provided by control mice. Furthermore, administration of rPCN was associated with higher levels of IL-12, IFN-, TNF-, NO and IL-10, detected in the lung homogenates of animals. The high levels of cytokines produced in the rPCN treated mice prompted us to investigate the occurrence of interaction of the lectin with receptors present in innate immune cells, such as TLR2 and TLR4. We verified that rPCN activates TLR2,in homo or heterodimeric forms, and TLR4, in a manner that does not depend on CD14 and CD36 coreceptors. These data reveal a possible mechanism by which rPCN generates protection in mice against PCM. rPCN, administered therapeutic or prophylactically, induces TLRs activation and Th1 immunity, conferring protection against P. brasiliensis infection.

Made available in DSpace on 2014-12-17T14:13:42Z (GMT). No. of bitstreams: 1 HenioGL_TESE.pdf: 1861952 bytes, checksum: 59df83a02a682e8397eb8b241531d77b (MD5) Previous issue date: 2011-11-16 / Visceral leishmaniasis (VL) is a disease caused by protozoa of the Leishmania donovani complex, whose infection has clinical spectrum ranging from asymptomatic infection to active disease characterized by fever, cachexia, hepatosplenomegaly, and immunosuppression. The healing or protective immunity require an antigen-specific type 1. The Montenegro skin test (DTH) has been interpreted as a marker of protective immunity. However, there is no known correlation between the DTH response to type 1 and DTH and immunity of type 1 are maintained in the long term. Thus, a longitudinal study of 8 years, nested in a cohort family held in Brazil, documented the status of DTH and cytokine production by peripheral blood mononuclear cells in response to antigen-specific stimulation. This study was the interdisciplinary approach of physicians, biochemists, nutritionists, veterinary medicine, biology and statistics. The results show that 46.2% of subjects were analyzed DTH positive at baseline. The prevalence of positive and DTH induration size increased with age (p = 0.0021). 15.7% of individuals positive DTH "retro-converted" the negative and 50.4% (64) of individuals negative DTH became positive. The size of DTH induration was correlated significantly with the antigen-induced production of IFN-γ (r = 0.6186, p = 0.0001). IL-6 was secreted at higher levels in peripheral blood mononuclear cells of individuals who "retro-converted" DTH positive to negative than individuals who remained stable DTH status (p = 0.005). Thus, IFN-γ produced by peripheral blood mononuclear cells, may be a surrogate marker for protective immunity instead of the DTH response. In addition, differences in innate immune response may determine whether individuals maintain or eliminate the infection by Leishmania infantum chagasi in asymptomatic patients / A leishmaniose visceral (LV) ? uma doen?a ocasionada por protozo?rios do complexo Leishmania donovani, cuja infec??o possui espectro cl?nico variando desde infec??o assintom?tica a doen?a ativa caracterizada por febre, caquexia, hepatoesplenomegalia e imunossupress?o. A cura ou prote??o exigem uma imunidade ant?geno espec?fica do tipo 1. O teste cut?neo de Montenegro (DTH) tem sido interpretado como um marcador de imunidade protetora. No entanto, n?o se sabe a correla??o do DTH com a resposta tipo 1 e se o DTH e a imunidade do tipo 1 s?o mantidos a longo prazo. Assim, um estudo longitudinal de 8 anos, aninhado a uma coorte familiar realizada no Brasil, documentou o status do DTH e a produ??o de citocinas por c?lulas mononucleares do sangue perif?rico em resposta a estimulo ant?geno-espec?fico. Os resultados obtidos mostram que 46,2% dos indiv?duos analisados foram DTH positivos no in?cio do estudo. A preval?ncia do DTH positivo e o tamanho da endura??o aumentaram com a idade (p = 0,0021). 15,7% dos indiv?duos DTH positivos retroconverteram a negativos e 50,4% (64) dos indiv?duos DTH negativos tornaram-se positivos. O tamanho da endura??o do DTH correlacionou-se significativamente com a produ??o ant?geno induzida de IFN-γ (r=0,6186, p=0,0001). IL-6 foi secretado em n?veis mais elevados por c?lulas mononucleares do sangue perif?rico dos indiv?duos que retroconverteram de DTH positivo para negativo do que os indiv?duos que mantiveram o status de DTH est?vel (p=0,005). Assim, o IFN-γ, produzido por c?lulas mononucleares do sangue perif?rico, pode ser um marcador substituto para a imunidade protetora em vez da resposta DTH. Al?m disso, as diferen?as na resposta imune inata podem determinar se os indiv?duos mant?m ou eliminam a infec??o por L. infantum chagasi em assintom?ticos

Paracoccina (PCN) é um constituinte de Paracoccidioides brasiliensis, um patógeno humano que causa a paracoccidioidomicose, micose sistêmica mais prevalente na América Latina. A PCN é uma proteína de função dual, com domínios de atividade lectínica e de N-acetilglicosaminidase. Análises proteômicas da preparação paracoccina revelaram a sua correspondência com uma proteína hipotética de P. brasiliensis do isolado 18 (Pb18), anotada como PADG-3347.1, que tem sequência polipeptídica semelhante a família das endoquitinases 18. Essas endoquitinases apresentam domínios distintos de atividade lectínica e enzimática. O conjunto de exons do gene correspondente, PADG-3347.1, foi clonado e expresso em E. coli, e as características físicas e biológicas da proteína recombinante foram comparadas com as da PCN. Além disso, a PADG-03347.1 recombinante (rPCN) foi avaliada por suas propriedades imunomoduladoras e sua capacidade em conferir proteção contra a infecção por P. brasiliensis. Nesse sentido, investigamos a interferência da administração profilática e terapêutica de rPCN no curso da infecção por P. brasiliensis em camundongos BALB/c. A histopatologia pulmonar dos camundongos tratados com a rPCN, mostrou menor ocorrência de granulomas, e estes também foram menores do que os observados nos animais controles. Consistente com a observação de poucas leveduras no centro dos granulomas, a contagem de UFC a partir do homogenato pulmonar dos camundongos tratados foi inferior ao observado nos animais controles. Além disso, a administração de rPCN, foi associada com altos níveis de IL-12, IFN-, TNF-, NO e IL-10 detectados no homogenato pulmonar. Os altos níveis de citocinas produzidos nos animais tratados com rPCN nos levou a investigar a ocorrência de interação da lectina com receptores presentes em células da imunidade inata, tais como TLR2 e TLR4. Verificamos que a rPCN ativa TLR2, nas formas homo ou heterodimérica, e TLR4, de modo independente dos correceptores CD14 e CD36. Estes dados revelam um possível mecanismo pelo qual rPCN gera proteção nos camundongos contra a PCM. rPCN, administrada terapêutica ou profilaticamente, induz a ativação de TLRs e imunidade Th1, conferindo proteção contra a infecção por P. brasiliensis. / Paracoccin is a constituent of Paracoccidioides brasiliensis, a human pathogen that causes paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America. Paracoccin is a dual function protein exerting lectin and N-acetylglucosaminidase activities. Proteomic analysis of paracoccin preparation revealed its correspondence with a hypothetical protein from P. brasiliensis isolate Pb18 (Pb18), annotated as PADG-3347.1, which has a polypeptide sequence similar to the family 18 endochitinases. These endochitinases have distinct lectin and enzymatic domains. The multi-exon assembly of the correspondent gene (PADG-3347) was cloned and expressed in E. coli, and the physical and biological features of the recombinant protein were compared to those of the native paracoccin. Moreover, recombinant PADG-3347.1 (rPCN) was evaluated for its immunomodulatory properties and its ability to confer protection against murine P. brasiliensis infection. Thus, we investigated the interference of prophylactic and therapeutic administration of rPCN on the course of P. brasiliensis infection in BALB/c mice. The pulmonary histopathology of the treated mice showed lower incidence of granulomas, which were also smaller than those observed in the control animals. Consistently with the observation of few yeasts in the center of the granulomas, the CFU count provided by lung homogenates of treated mice was lower than the provided by control mice. Furthermore, administration of rPCN was associated with higher levels of IL-12, IFN-, TNF-, NO and IL-10, detected in the lung homogenates of animals. The high levels of cytokines produced in the rPCN treated mice prompted us to investigate the occurrence of interaction of the lectin with receptors present in innate immune cells, such as TLR2 and TLR4. We verified that rPCN activates TLR2,in homo or heterodimeric forms, and TLR4, in a manner that does not depend on CD14 and CD36 coreceptors. These data reveal a possible mechanism by which rPCN generates protection in mice against PCM. rPCN, administered therapeutic or prophylactically, induces TLRs activation and Th1 immunity, conferring protection against P. brasiliensis infection.

No description available.

No description available.