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Rotavirus is the leading cause of severe infantile diarrhea worldwide. Most related deaths occur in infants from developing countries. Current vaccines are expensive and not readily available throughout the world. Chloroplast transformation technology can be utilized to generate genetically modified plants that produce large quantities of therapeutic proteins and vaccine antigens within their leaves. Plants that are used as bioreactors for vaccine antigens are economically advantageous because they eliminate the need for purification steps and are cheaper to transport. A genetically modified crop could potentially be grown near an endemic area and harvested as needed. There are many influencing factors for transgene expression levels within plant leaves that must be taken into account prior to their harvest. In this work, we seek to determine the optimal expression of CTB-NSP4 in two different cultivars of tobacco plant that have been previously generated by the Daniell lab. The fusion protein, CTBNSP4, is hoped to confer resistance to both rotavirus and cholera. We will determine how the expression of the protein is affected by different variables such as the lighting conditions during harvest and the relative age of leaf at the time of harvest. This knowledge can be used to raise the productivity of the genetically modified plants, further decreasing the cost. Additionally, as unprocessed leaf cannot be used directly for oral delivery due to an unknown concentration of the vaccine antigen, quantification is an important barrier to overcome. Low cost vaccines can be prepared after optimization of dosage and stability. This project seeks to substantiate and quantify genetically modified tobacco plants producing the rotavirus and cholera vaccine antigens.

A toxoplasmose, causadas por protozoário Apicomplexa, Toxoplasma gondii, é amplamente disseminada e pouco sintomática. A infecção crônica mantém cistos residuais por toda a vida, mas protege da reinfecção. Neste cenário complexo, vacinas com cistos residuais não são factíveis e vacinas de subcomponentes resultam em baixa proteção. A radiação ionizante foi usada para aprimoramento de imunógenos tanto vivos ou de subcomponentes. O uso da radiação gama em extratos solúveis de taquizoítos de T. gondii foi eficiente na proteção de camundongos contra a infecção utilizando diferentes cepas, sem adjuvantes e de fácil conservação. O antígeno irradiado passa por alterações físicas sem adição de novas moléculas, com agregação de proteínas, quebras de cadeias e reações oxidativas, que podem melhorar seu direcionamento a receptores celulares em células apresentadoras de antígeno (APC). Os extratos irradiados apresentaram alterações estruturais mínimas afetando 60% das proteínas, mas com manutenção das características antigênicas e imunogênicas. O extrato irradiado a 1500Gy (STag 1500Gy) induziu maior proteção e maior resposta humoral que o extrato nativo (p<0.05), mais evidente na dose de 10?g/animal, com altos índices de anticorpos IgG específicos e maior maturação da afinidade de IgG específica, com eficiência similar ou inferior em doses maiores. Animais imunizados com STag 1500Gy apresentaram maiores proporções de linfócitos B e T CD4+ de memória, enquanto que a imunização com taquizoítos íntegros irradiados mostrou aumento de linfócitos T CD8+, ambas muito maiores que a induzida por STag nativo. Construímos STag marcados por via biossintética ou por acoplamento a diferentes marcadores não-oxidativos. STag3H 1500Gy apresentou captação maior e mais duradoura por macrófagos, sem degradação como STag3H nativo. O uso de STags fluorescentes, mostrou que a maior ligação do STag 1500Gy não é relacionada a susceptibilidade a proteases, dada a mesma sensibilidade dos extratos para as peptidases testadas, além de permanecer na célula por muito mais tempo. Na presença de bloqueadores de receptores Scavengers Dextran sulfato (SRA) e Probucol (CD36), a ligação e captação por macrófagos do STag 1500Gy foi mais afetada por inibidores de radicais oxidados (CD36) do que por inibidores de radicais negativos (SRA). Em macrófagos peritoneais de camundongos deficientes do receptor Scavenger CD36 (KOCD36-/-), observamos uma cinética inversa a que foi obtida em macrófagos normais, com menor incorporação do STag 1500Gy, fato comprovado tanto por ensaios quantitativos como por ensaios em células individuais por citometria de fluxo. Os animais KOCD36-/- não mostram produção significativa de IgG específica em todos os imunógenos usados, e foram altamente suscetíveis ao desafio com cepas viáveis de T. gondii agressivas ou cistogênicas. O transplante de macrófagos peritoneais normais \"primados\" com STag 1500Gy em animais KOCD36-/- mostrou aumento de IgG específica em soro de animais recipientes. A melhor imunogenicidade dos antígenos irradiados deve ser relacionada a captação de proteínas oxidadas via CD36, que dirige estes antígenos para via intracelular favorável à sua apresentação para resposta imune adaptativa. Nossos resultados mostram que a radiação ionizante foi capaz de modificar proteínas dos STag tornando seu processamento por células imunes mais eficiente, sem a adição de adjuvantes ao processo. / Toxoplasmosis, caused by the protozoan Apicomplexa, Toxoplasma gondii, is widely disseminated and little symptomatic. Chronic infection maintains residual cysts throughout life, but protects from reinfection. In this complex scenario, vaccines with residual cysts are not feasible and vaccines of subcomponents result in low protection. Ionizing radiation was used for enhancement of either live or subcomponent immunogens. The use of gamma radiation in soluble extracts of T. gondii tachyzoites was efficient in protecting mice against infection using different strains, without adjuvants and easy management. The irradiated antigen undergoes physical changes without addition of new molecules, with protein aggregation, chain breaks and oxidative reactions, which can improve its targeting to cellular receptors in antigen-presenting cells (APCs). The irradiated extracts showed minimal structural alterations affecting 60% of the proteins, but with maintenance of the antigenic and immunogenic characteristics. The extracts irradiated at 1500Gy (STag 1500Gy) induced greater protection and higher humoral response than the native extract (p <0.05), more evident at a dose of 10?g/animal, with high specific IgG antibody levels and increased maturation of specific IgG affinity, with similar or lower efficiency at higher doses. Animals immunized with STag 1500Gy presented higher proportions of memory lymphocytes B and CD4+ while immunization with irradiated intact tachyzoites showed an increase in CD8+ lymphocytes, both much larger than that induced by native STag. We construct STag labeled by biosynthetic pathway or by coupling to different non-oxidative markers. STag3H 1500Gy showed greater and longer uptake by macrophages, with no degradation as STag3H native. The use of fluorescent STags showed that the greater binding of the STag 1500Gy is not related to the loss of protease susceptibility, given the same sensitivity of the extracts for peptidases, besides remaining in the cell for much longer time by fluorescence. In the presence of Scavengers receptor blockers Dextran sulfate (SRA) and Probucol (CD36), binding and uptake of STag 1500Gy in macrophages was more affected by oxidized radical (CD36) inhibitors than by negative radical inhibitors (SRA). In peritoneal macrophages of Scavenger receptor CD36 deficient mice (KOCD36-/-), we observed an inverse kinetics, with less incorporation of STag 1500Gy, as evidenced by both quantitative assays and individual cells by cytometry flow, compared to wild type macrophages. KOCD36-/- animals did not show significant production of specific IgG in all immunogens used, and were highly susceptible to challenge with viable strains of aggressive or cistogenic T. gondii strains. Transplantation of normal peritoneal macrophages \"primed\" with STag 1500Gy induced increase of specific IgG in sera from CD36-/-recipient animals. The best immunogenicity of the irradiated antigens should be related to the uptake of oxidized proteins via CD36 in APCs, which directs these antigens to the intracellular route favorable to their presentation for adaptive immune response. Our results show that ionizing radiation was able to modify STag proteins, making its processing by immune cells more efficient without the addition of adjuvants to the process.

A total of 338 infants with suspected clinical sepsis were investigated, 115 of whom were found to be clinically infected. Twenty-one healthy term neonates were recruited as control subjects. The expression of CD64 on neutrophils in infected infants was significantly elevated at both 0 h and 24 h, compared with those of noninfected infants or controls (both p &lt; 0.0005). The calculated optimal cutoff value for CD64 was 6136 antibody-phycoerythrin molecules bound/cell. CD64 has a very high sensitivity (96%) and NPV (97%) at 24 h. The use of CRP in combination with CD64 as predictive markers only marginally enhanced the sensitivity and NPV (97% and 98%, respectively). There was no statistical difference in the expression of monocyte HLA-DR among infected, noninfected, and control subjects. As a result, the optimal cutoff value for HLA-DR could not be determined. The technology of flow cytometry has potential applications for use in the diagnosis of neonatal sepsis because the measurement is quantitative, requiring only a minimal amount of whole blood and a short duration (within 3 h) for the provision of results. (Abstract shortened by UMI.) / Term newborns in whom infection was suspected when they were &lt;72 h of age were recruited into the study. The expressions of CD64 on neutrophils and HLA-DR on monocytes were measured by flow cytometry at 0 h (the time of sepsis evaluation) and 24 h after the onset of presentation. A full sepsis screen, including complete blood count, serial C-reactive protein (CRP), blood culture, cerebrospinal fluid culture, and chest radiograph were performed. The demographic and clinical data were documented. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of neutrophil CD64, monocyte HLA-DR and the combination of markers for predicting neonatal sepsis were determined. / This prospective study aimed to evaluate the diagnostic utilities of two cell surface markers, neutrophil CD64 and monocyte HLA-DR, for the identification of early-onset clinical infection and pneumonia in term infants. The optimal cutoff value of each marker was defined according to the Receiver Operating Characteristic curve so that it could be used as a reference with which future studies can be compared. / Li Geng. / "May 2005." / Adviser: Pak Cheung Ng. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0174. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 129-150). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

by Ling Lin. / "November 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 149-168). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.