Graduation date: 1995

Thesis (Ph. D.)--Oregon State University, 1995. / Typescript (photocopy). Includes bibliographical references (leaves 135-159). Also available online.

An oral Vibrio vaccine for salmonids was developed. The vaccine was produced by spray coating lyophilized formalin-killed whole cells of Vibrio anguillarum (VA LS 1- 74) onto non-pareil sugar beads. Then methacrylic acrylic acid copolymer (Eudragit L-30D) was applied as an enteric protective coating. Using x-ray radiographic techniques, it was found that large particles (> 1.1 mm) remain in the fish stomach for more than 2 hours before they would enter the pyloric caeca. The pyloric sphincter which has an opening of 0.94 mm, acts as barrier to prevent the passage of large food particles in the stomach to the pyloric caeca. Based on this information non-pareil sugar beads of 18-20 mesh or smaller should be used as the vaccine carriers. A 15% (w/w) Eudragit L-30D coating is needed to provide enteric protection of the vaccine loaded sugar beads of 18-20 mesh size. Lower levels of coating resulted in the bead breaking down in the stomach and releasing contents prior to entering the pyloric caeca. Since the lymphoid tissues are diffuse throughout the whole GI tract, it may not be necessary to target a vaccine to deliver antigens to a specific area of the intestinal tract, but only protect the antigens from gastric fluids. In vitro dissolution studies indicate that 10% VA LS 1- 74 loading was sufficient for rapid vaccine release (42% released in 30 minutes) and a 15% Eudragit L-30D coating was suitable for providing protection against stomach acid. The vaccine product used in vivo studies contained 10% VA LS 1- 74 and 15% Eudragit L-30D on non-pareil sugar seeds of 18-20 mesh size. Coho salmon were given the vaccine orally, and 30 days afterward a live challenge test was performed. There was no significant difference in the survival rates in a live bacteria challenge test with the positive control (83.3%) and test (80.3%) groups. Both had higher survival rates than the no vaccine fed control group. The serum and mucosal antibody levels to Vibrio were significantly higher (p<0.01) in the test group (19700 units/ml) than the other two groups (2530 units/ml in the positive control group and 617 units/ml in the negative control group). The antibody titer appears to be a better indicator for vaccine efficacy than survival rate of live bacteria challenge tests. The oral Vibrio vaccine developed is effective, and the technique to protect the antigen can be applied to other antigens or proteins for oral delivery producing an economical pathway for mass vaccination of fish. / Graduation date: 1991

Efficient delivery of vaccines and therapeutic agents in vivo is a critical aspect for ensuring a desired immunological or biological response is achieved. This work focuses on developing effective delivery systems for vaccines and therapeutics to achieve biological potency while maintaining patient friendly administration. Traditional vaccines are administered via parenteral injection, which requires skilled personnel for administration and does not elicit strong mucosal immune responses. An alternative approach is to develop an oral vaccine; however, this requires antigens to be protected during transit through the gastrointestinal tract and be transported across specialized intestinal sampling cells called M cells. These M cells are extremely rare, making them an important target for oral vaccines. The protein invasin, from Yersinia psuedotuberculosis, naturally binds [mathematical symbols] integrin, a receptor found exclusively on M cells within the gastrointestinal tract. Therefore, we generated combinatorial libraries of invasin, followed by a directed evolution and high-throughput screening strategy to identify invasin variants with increased affinity towards [mathematical symbols] integrin. This process led to the creation of an invasin variant exhibiting a nine-fold decrease in EC₅₀, which could be used for targeted oral vaccine systems. In order to test for increased vaccine efficacy due to the engineered invasin ligand, we developed a polymeric microparticle delivery system. These microparticles were formulated to encapsulate the model antigen ovalbumin and be decorated with the invasin targeting ligands. To measure physiological trafficking and intestinal retention, novel fluorescent nanocrystals were loaded into particles conjugated to invasin. These nanocrystals served as a contrast agent for in vivo imaging in mice. While these particles were unsuccessful in generating an antibody response toward ovalbumin when administered to mice, a response directed to the targeting ligand itself was observed. These findings provide insights for further optimizing a delivery system for oral vaccination. In addition to developing an oral vaccine delivery system, we created a high concentration therapeutic protein formulation, suitable for low-volume subcutaneous administration. By adding crowding agents, we were able to generate reversible protein nanoclusters with low viscosity. These nanoclusters were found to revert to monomer upon dilution and pharmacokinetic profiles similar to solutions. / text

Despite the development of various systems to generate live recombinant Salmonella Typhimurium vaccine strains, little work has been performed to systematically evaluate and compare their relative immunogenicity. Such information would provide invaluable guidance for the future rational design of live recombinant Salmonella oral vaccines. Here, a series of recombinant Salmonella Typhimurium strains were constructed to express either the enhanced green fluorescent protein (EGFP) or a fragment of the hemagglutinin (HA) protein from the H5N1 influenza virus, as model antigens. To investigate different delivery and expression methods, antigens were expressed from the chromosome, from high or low-copy plasmids, or encoded on a eukaryotic expression plasmid. Antigens were targeted for expression in the cytoplasm, or the outer membrane. In addition, combinations of two expression strategies were employed to evaluate the efficacy of combined delivery approaches. After investigating in vitro and in vivo antigen expression, growth and infection abilities, the immunogenicity of the constructed recombinant Salmonella strains was evaluated and compared in mice. Using soluble model antigen EGFP, my results indicated that vaccine strains with high and stable antigen expression exhibited high B cell responses, while eukaryotic expression or colonization with good construct stability is critical for T cell responses. For insoluble antigen model HA, the outer membrane strategy induced better B cell and T cell responses than cytoplasmic strategy. Most notably, the combination of two different expression strategies did not increase the immune response elicited as initially expected. Based on the advantages, deleterious or synergistic effects of different strategies identified in this study, I conclude that different construction strategies of recombinant Salmonella vaccine strains are needed for different forms of antigens (soluble or insoluble antigens). If the antigen (such as EGFP) is soluble and easily expressed in Salmonella, a low-copy plasmid-based strategy should be employed, as it can provoke both strong B cell and T cell responses with better plasmid stability. If a T cell response is preferred, a eukaryotic plasmid, or chromosome-based, cytoplasmic-expression strategy may achieve better results. For heterologous antigens that are likely to be expressed in an insoluble form inside Salmonella (such as HA), an outer membrane-targeting approach is recommended. In addition, I found that the combination of two expression strategies did not enhance the immune response, and hence I caution the use of such an approach. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

An in vitro model of the follicle-associated epithelia that overlie the Peyer's patches of the small intestine was developed and validated to examine the mechanisms of mucosal antigen sampling. This model displays many phenotypic and physiological characteristics of M cells including apical expression of [alpha]5[beta]l integrin and enhanced energy dependent participate transport. CD4+ T-cells were shown to be an important influence on the development of Mlike cells. The model was used to examine the M cell mediated uptake of several putative whole-cell killed bacterial vaccines. Greater numbers of non-typeable Haemophilus influenzae NTHi 289, NTHi 2019, Escherichia coli 075 HMN and Streptococcus pneumoniae were transported by model M cells compared to control Caco-2 enterocyte-like cells. Studies in isolated murine intestine segments confirmed the selective uptake of NTHi 289 and Escherichia coli demonstrating that intestinal mucosal sampling of these antigens is performed by M cells. Pseudomonas aeruginosa was not absorbed as whole cell bacteria but as soluble antigen, as indicated by the presence of bacterial DNA in the cytoplasm of epithelial cells. These results suggest that bacteria such as NTHi and E. coli are sampled by the mucosal immune system in a different manner to that of bacteria such as Pseudomonas. A number of potential cell surface receptors were investigated to identify which molecules are responsible for intestinal uptake whole-cell killed bacteria. Immunofluorescence studies detected the presence of toll-like receptor-2, toll-like receptor-4, PAF-R and [alpha]5[beta]l integrin on in vitro M-like cell cultures. Examinations of murine intestine confirmed the presence of TLR-4 and PAF-R. TLR-4 was found in small quantities and on M cells. In contrast to the M cell model, TLR-2 expression in the murine intestine was sparse. Receptor inhibition experiments provided evidence for the involvement of TLR-4, PAF-R and [alpha]5[beta]l integrin in M cell uptake of killed bacteria both in vitro and in vivo. This thesis has contributed valuable information regarding the mechanisms of uptake of whole-cell killed bacteria by the intestinal mucosal immune system. For the first time, M cell sampling of whole-cell killed bacteria has been demonstrated. Furthermore, the receptors involved in these processes have been identified. This information will be of great use in the development and optimisation of new oral vaccines.

Copy of one of the author's previously published articles inserted. / Bibliography: leaves 357-404. / 405 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Describes a methodology for the detailed evaluation of the processes involved in the assessment of recombinant orally administrable vaccines against mucosal pathogens (a bivalent vaccine against diarrhoeal disease in this case) / Thesis (M.D.)--University of Adelaide, Dept. of Medicine 1991

A sílica mesoporosa SBA-15 é uma forte candidata para uso como adjuvante por apresentar propriedades vantajosas, como a capacidade de integração com moléculas, baixa toxicidade, boa estabilidade química, térmica e hidrotérmica. Seu potencial adjuvante foi demonstrado em trabalhos onde a SBA-15 associada a antígenos de naturezas distintas foi capaz de induzir uma alta produção de anticorpos específicos quando administrados por via parenteral. Além disso, a SBA-15 diminuiu a toxicidade da toxina diftérica na imunização de cavalos. Os efeitos da SBA-15 na produção de anticorpos com imunizações exclusivamente administradas pela via oral não havia sido testada. Assim, verificou-se a produção de anticorpos em camundongos heterogênicos selecionados para alta (HIII) e baixa (LIII) produção de anticorpos e em animais de fundo genético conhecido (BALB/c), após imunização oral com dois antígenos de naturezas distintas: LPS (antígeno T-independente) e flagelina (antígeno Tdependente), associados a sílica SBA-15. Os camundongos foram inoculados com duas doses de LPS ou flagelina de Salmonella typhimurium, associados ou não a SBA-15, pela via oral ou por via subcutânea. Foram pesquisados anticorpos séricos IgM, IgG e IgA, S-IgA no lavado intestinal e citocinas no soro. Células do linfonodo mesentérico foram coletadas após a imunização oral para caracterização fenotípica. Houve IgM anti-flagelina detectável em BALB/c no período de 37 dias no grupos Fla+SBA-15. Os animais de ambas as linhagens produziram IgG anti-LPS e anti-flagelina em todos os períodos avaliados. Foi encontrada uma pequena diferença significativa em BALB/c, entre os grupos Fla+SBA-15 e Fla após reforço por via oral. Não foi possível detectar IgA sérica nem secretória nos lavados intestinais. Não houve níveis detectáveis de citocinas nos soros do período avaliado. Foram encontrados linfócitos B1, T CD4+ e T CD8+, células dendríticas e moléculas coestimulatórias CD80 e CD86. Houve diferença estatística nas células dendríticas de HIII entre os grupos LPS+SBA-15 e LPS. / Ordered mesoporous sílica SBA-15 is a vaccine adjuvant candidate due to its advantageous properties such as the ability to associate with molecules, low toxicity, and also good chemical, thermal and hydrothermal stability. It has been demonstrated that the association of SBA-15 with several antigens, including toxins, induce high production of specific antibodies when injected parenterally.Since the oral route is natural for most infections, oral vaccination would be suitable for immunization against several diseases. Thus, we verified the antibody production of the non-isogenic mice selected for high (HIII) and low (LIII)antibody response after oral immunization with two antigens with distinct nature: LPS (T-independent antigen) and flagellin (Tdependent antigen), associated with silica SBA-15 Three mice per groupwere orally inoculated (gavage) with twodoses of LPS (75&#956;g) or Salmonella typhimurium flagellin (50&#956;g) adsorbed/encapsulated or not to SBA-15 at a 1:10 ratio on days 0 and 30. Blood sampleswere collectedon days 7 and 37andIgG serum levels were determined by ELISA.There was an increasing in antibody levels in HIII animalsi mmunized with both antigens associated with silica when compared to those immunized with antigen alone. We observed highest antibody levels in two of three HIII mice immunized with both antigens, although, due to the small sample, no significant differences were found between groups treated with silica or not. No significant difference was observed in LIII animals. The oral adjuvant effect of silica is encouraging by these preliminary results but must be confirmed by further experiments with a larger number of animals.

Since diffusion models of social network are widely used in studying epidemiology, in this thesis, we apply diffusion models to study the contact immunity generated by attenuated vaccines.Oral polio vaccine (OPV) is a typical attenuated vaccine for polio that can produce contact immunity and therefore help protect more individuals than vaccinees. / To better capture the utilization of OPV’s contact immunity, we model the community as a social network, and formulate the task of maximizing the contact immunity effect as an optimization problem on graphs, which is to find a sequence of vertices to be “vaccinated” to maximize the total number of vertices “infected” by the attenuated virus. Furthermore, as immune defiicient patients may suffer from the live attenuated virus in the vaccine, we develop models in consideration of this restriction, and study related problems. / We present polynomial-time algorithms for these problems on trees, and show the intractability of problems on general graphs. / 社交網絡的擴散模型被廣泛運用于對流行病學的研究，在本文中，我們使用擴散模型對減毒活疫苗產生的接觸性免疫進行研究。口服脊髓灰質炎疫苗（OPV）是一種典型的減毒活疫苗，它可以在人群中產生接觸性免疫，使得更多未接種疫苗的人獲得免疫力。 / 爲了更好的刻畫OPV 產生的接觸性免疫，我們將社區模型化為社交網絡，從而將接觸性免疫效應最大化的任務轉化爲圖優化問題，即通過發現頂點的一個「接種」序列來最大化被減活病毒「感染」的頂點數量。此外，因爲減毒疫苗中的活病毒會使患有免疫缺陷的病人患病，我們考慮在此因素限制下的模型，并研究相關的問題。 / 我們給出這些問題在樹上的多項式時間算法，并證明其在一般圖上的複雜性。 / Ma, Chenglong. / Thesis M.Phil. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 40-47). / Abstracts also in Chinese. / Title from PDF title page (viewed on 12, September, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.