Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 139-166.

As formulações vacinais contra o Vírus Respiratório Sincicial Humano, HRSV, estão associadas à indução de eosinofilia pulmonar mediada por uma resposta de células TCD4+ Th2, após exposição ao HRSV selvagem. Foi identificado um peptídeo da proteína viral G, que modificado perde a capacidade de predispor à eosinofilia, tornando-o um imunógeno atraente. Células T CD8+ específicas para HRSV reduzem a resposta Th2, mediam resistência a desafio com o vírus, e estão relacionadas à redução dos sintomas. Assim, neste trabalho buscamos e identificamos epítopos de células TCD8+ na polimerase viral, utilizando programas de predição, imunização com peptídeos e avaliação da resposta celular. Também construímos vacinas de DNA contendo a seqüência nucleotídica do peptídeo da proteína G modificado. A caracterização da resposta imune estimulada por essas vacinas e por peptídeos purificados revelou que o plasmídio pTGMCTB, bem como os peptídeos GM e GMCTB, foram capazes de induzir anticorpos que, porém, não se mostraram neutralizantes de HRSV e protetores frente a desafio. / Vaccines against human respiratory syncytial virus (HRSV) are associated with pulmonary eosinophilia induction mediated by a TCD4+ Th2 response, after exposition to wild HRSV. A peptide from the viral protein G was identified to predispose to eosinophilia and loses this ability when mutated, making it an interesting immunogen. CD8+ T cells specific to HRSV reduce the Th2 response, mediate resistance to virus challenge, and are related to symptom-reduction. Thus, in the present work, we searched for and identified CD8+ T cell epitopes in the viral polymerase; using prediction programs, peptide immunization and evaluation of the cellular response. We also constructed DNA vaccines containing the nucleotide sequence of the mutated peptide from G protein mentioned above. The characterization of the immune response elicited by these vaccines and purified peptides showed that the pTGMCTB plasmid, as well as GM and GMCTB peptides were able to induce antibody response; however they are not neutralizing and protective against HRSV challenge.

Abstract The characteristics of humoral immunity have been proposed to reflect the bias between two T helper (Th) lymphocyte subsets: Th1 cells, which activate cell-mediated immunity, and Th2 cells, which mediate humoral immunity. The present study aimed to characterize the humoral immunity to beta-cell autoantigens insulin and glutamic acid decarboxylase (GAD65) in preclinical and clinical type 1 diabetes. Insulin antibodies were analyzed in pregnant women with or without type 1 diabetes and their newborn infants and in prediabetic children. Epitope or/and isotype-specific GAD65 antibodies (GAD65Abs) were analyzed in prediabetic children, in children and adolescents diagnosed with type 1 diabetes, and in patients with the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome. Antibodies were determined by radioligand immunoassays. The humoral immune response to insulin and GAD65 was observed to be a highly dynamic process, comprising mainly the IgG1 subclass and, less frequently, other IgG subclasses. GAD65Abs were directed primarily to the middle region and secondarily to the C-terminal region of GAD65 as a consequence of epitope spreading. Young children who progressed to overt type 1 diabetes were characterized by a broad initial isotype response to insulin and GAD65 and by a strong IgG1 and IgG3 response to insulin. Children who did not progress to clinical type 1 diabetes were characterized by an emerging IgG4 response to GAD65. Rising levels of GAD65Abs targeted to the middle region of GAD65 were associated with high titers of islet cell antibodies and a decreased requirement for exogenous insulin, probably reflecting a persistent residual beta-cell mass, in patients with manifest type 1 diabetes. Non-immunoglobulin insulin-binding activity was observed to be induced by pregnancy. APECED-associated humoral autoimmunity to GAD65 did not differ markedly from that observed in subjects with type 1 diabetes alone. In conclusion, isotype-specific GAD65 and especially insulin antibodies are valuable markers of the risk of progression to type 1 diabetes in young children. The appearance of an initial IgG3 subclass response and a strong IgG3 response to insulin in children who progressed to overt type 1 diabetes may reflect the role of cytotoxic Th1-biased immunity in the disease process leading to clinical presentation of type 1 diabetes.

Introduction: Fungi are common aeroallergens responsible for at least 3% – 10% of allergic diseases worldwide, with the proportion hugely variable in different populations. Treatment is complicated by viable nature and disease causing ability of the allergen and is often only palliative. Thus, this study aimed to serve as a pilot investigation to design novel anti-allergy therapeutics to cure allergy at the molecular level. It investigates the effect of wild type fungal peptides and corresponding variant peptides on allergy associated immunological responses – cellular and cytokine based – to use such variant peptides to cause the delicate shift from an allergic to a normal immune response. Further, the study explores the role of bioinformatics in investigating allergy and designing novel therapeutics. Methods: This study used ProPred, a bioinformatics software, to predict wild type peptides from selected allergens of Aspergillus fumigatus and Alternata alternaria for a target population. These were then modified to generate single amino acid variants. Both these peptide sets were tested to compare the cellular and cytokine patterns they generated in sensitised (n = 3) and healthy volunteers (n = 3) to check for anti-allergy responses that may be exerted by certain variants. The recruited population was also subjected to skin prick testing (SPT, n = 46) to check for co-sensitisations patterns and HLA typing (n = 40) to evaluate ProPred accuracy for peptide prediction. This study also attempted an in silico search for unknown Penicillium chrysogenum allergens by comparing known Penicillium and A. fumigatus allergens to identify probable agents of co-sensitization. Results: Of the wild type and variant peptides tested in this study, one variant peptide – peptide 1.1v from Asp f 2 – was successfully identified to change the cellular and cytokine profile to promote an anti-allergic response when compared to its corresponding wild type form (1.1o). This candidate is a good target for further investigation for use in peptide immunotherapy. Further, 8 shared allergens between A. fumigatus and P. chrysogenum were identified that may possibly be agents of co-sensitization between these species. SPT results indicated maximum subject co-sensitization between A. fumigatus and Candida albicans and P. chrysogenum. HLA typing results demonstrated the efficiency of ProPred to be 96.29%, thus implying that bioinformatics can effectively be used to study allergy in this novel manner. Conclusion: This study has demonstrated that variant peptides with a single amino acid change can cause the delicate shift from an allergic to a healthy immune response in sensitised subjects. This approach – in combination with other allergy associated factors such as epitope specificity for HLA types and inherent co-sensitization patterns in a population – can effectively be used to design peptide candidates for immunotherapy to target allergy at the molecular level. With promising results obtained in this pilot study, this approach guarantees further investigation in immunotherapy. This study has also demonstrated that bioinformatics can be effectively used to design and execute allergy studies in a targeted and inexpensive manner.

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