Live attenuated Salmonella vectors provide a unique alternative in terms of antigen presentation by acting as a vector for heterologous antigens. The efficiency of any live bacterial vector rests with its ability to present sufficient foreign antigen to the human or animal immune system to initiate the desirable protective immune response. Salmonella vectors encoding heterologous protective antigens can elicit the relevant immune responses, be it humoral, mucosal or cell-mediated. STM-1 is a Salmonella mutant developed by RMIT, harbours a mutation in the aroA gene that renders it attenuated, and is a well characterised vaccine strain currently in use to protect livestock against Salmonella infection. In previous work in this laboratory, STM1 was shown to be capable of eliciting immune responses in mice to plasmid-borne antigens. In this study STM-1 was analysed for its ability to vector the model antigen chicken ovalbumin and test antigen C. jejuni major outer membrane protein using in vivo inducible promoters such as pagC and nirB from the plasmid location. The determination of the architecture around the lesion in STM-1 also allowed the development of constructs expressing heterologous antigen from the chromosome. The induction of immune responses, both humoral and cell mediated, was analysed. Another issue addressed in this study was effect of pre-existing immune responses in the animal host against the vector or related strains and the effects on generation of immune responses against the subsequently vectored antigen. Humoral and cellular immune responses to vectored ovalbumin and C. jejuni Momp antigens were observed following vaccination with STM-1, when antigens were expressed from either the plasmid or chromosomal location. Up-regulation of immune responses, both humoral and cell mediated, was observed against the vectored antigens in animals which were pre-exposed to either the bacterial vector or related strains. These results indicate that STM-1 has the potential to be used as a vector to deliver heterologous vaccine antigens from a single copy gene in the field. Lastly, the results from this study indicate that pre-existing immune responses against the bacterial vector or a related strain do in fact enhance both humoral and T cell responses against the heterologous antigen.

The area of self-esteem in people with learning disabilities has been largely neglected, and previous researchers have employed a variety of approaches. It is important to further our understanding in the context of providing appropriate clinical interventions and in monitoring the effect of social policy developments on the individuals at the receiving end of service provision. The study aimed to assess the reliability and validity of a set of measures devised specifically for use with learning disabled people, by Szivos-Bach (1993). The measures assess social comparisons, perception of stigma and aspirations and expectations. The study was carried out with 30 adults with mild and moderate learning disabilities between the ages of 18 and 65. The results provide initial support for the social comparisons test as a measure of self-esteem. Less evidence was found for the stigma questionnaire and the aspirations-expectations test. The results are discussed in the light of comparable research into self-esteem measures with non-learning disabled populations. Further research is required, and the most profitable way forward seems to be development of multi-dimensional measures of self-esteem.

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Bacterium Mycobacterium tuberculosis is an etiological agent of a deadly disease called tuberculosis that presents a global problem. According to The World Health Organization there are more than 2 billions people infected with latent tuberculosis all over the world. There is still need of specific, sensitive, quick and economic available method for identification of infected individuals. Currently in vitro blood tests are considered to be the best way of diagnosis. They are based on restimulation of specific T lymphocytes by mycobacterial antigens derived from virulent strains. There are several different approaches for enhancing of direct antigen delivery into antigen presenting cells and promising one is a genetically detoxified adenylate cyclase toxin (CyaA) of bacteria Bordetella pertussis. The main aim of the thesis includes construction and subsequent characterization of biological properties of CyaA protein carrying specific mycobacterial antigen TB7.7 in translocating domain. Here is shown that fusion protein CyaA-TB7.7 can form cation selective pores in target cell membranes and is able to deliver antigens into the cytosol of APC to be presented on surface with molecules MHC class II. Genetically detoxified CyaA- TB7.7 protein will be used to supplement current approaches such as also in vitro...

Les vaccins représentent l’un des progrès majeurs de l’Histoire pour la santé publique, concrétisant notamment l’éradication de la variole en 1980. Les vaccins historiques, à base de pathogènes entiers atténués ou inactivés et donc très immunogènes ont été progressivement remplacés par des vaccins à sous-unités, beaucoup plus sûrs mais en contrepartie moins immunogènes. Des adjuvants tels que des vecteurs et des molécules immunostimulantes ont donc été incorporés dans les formulations vaccinales dans le but de générer des réponses immunitaires de grande amplitude. Cependant, les principaux adjuvants actuellement autorisés chez l’homme induisent exclusivement une réponse immunitaire humorale, à savoir la production d’anticorps permettant de neutraliser les pathogènes extracellulaires. Or, certains pathogènes comme le VIH requièrent une immunité cellulaire, indispensable à l’élimination du virus persistant dans les cellules infectées. Dans ce contexte, les adjuvants de vaccin sont en plein essor dans le but d’identifier de nouveaux candidats plus performants et sûrs. Nous décrivons ici la démarche suivie afin de proposer un vecteur lipidique nanoparticulaire (LNP), dont la stabilité, l’innocuité et la versatilité en font un outil idéal pour la délivrance d’antigènes. Nous avons dans un premier temps réalisé la preuve de concept sur la base de l’antigène modèle ovalbumine, dont la délivrance aux cellules immunitaires a permis d’augmenter significativement la réponse humorale in vivo chez la souris. D’autre part, l’induction d’une réponse cellulaire a été observée par la double délivrance de l’antigène et d’un immunostimulant. Plusieurs combinaisons et stratégies de vectorisations ont été évaluées, dans le but d’identifier la formulation la plus performante en vue d’une étude de protection anti-tumorale. Finalement, nous avons appliqué ces technologies au cas concret du VIH avec l’antigène de capside p24, ce qui s’est conclu par une étude d’immunogénicité chez le primate non-humain. L’ensemble de ces résultats met en lumière la versatilité des LNP et leur capacité à induire des réponses immunitaires de grande magnitude, à médiation humorale et cellulaire. / The development of vaccines was one of the major health advances of the last century, with the success of smallpox eradication in 1980. Historical vaccines, based on attenuated or killed pathogens thus strongly immunogenic were finally replaced by subunit candidates, much safer but also poorly immunogenic. Therefore, adjuvants such as vectors and immunostimulants were incorporated in vaccine formulations in order to generate immune responses of high magnitude. However, actual adjuvants authorized in human vaccines only trigger humoral immune responses, with the production of antibodies which neutralize extracellular pathogens. Yet, some pathogens such as HIV require the induction of a cell-mediated immunity, necessary to eliminate viral reservoirs in infected cells. In this context, new adjuvant systems are being developed in order to identify the most efficient and safe candidates. Here we describe the approach followed to prepare a stable, safe and versatile vector consisting in lipid nanoparticles (LNP), for the delivery of antigens. We first report the proof of concept of antigen delivery based on the model ovalbumin, leading to the significant enhancement of humoral responses in vivo in mice. Thereafter, we focused on the induction of cell-mediated immune responses through the vectorization of both antigens and immunostimulants. Several combinations and vectorization strategies were assessed in the aim to identify the best prototype for a study of protection against tumor challenge. Finally, we applied these systems to HIV and its capsid antigen p24, which allowed us to conduct an immunogenicity study on a non-human primate model. Altogether, these results highlight the versatility of LNP and their ability to induce potent humoral and cell-mediated immune responses.

<p>Cancer vaccine therapy aims at harnessing effective antigen specific immune responses to treat tumor. In particular, CD8+ T cells have the unique capacity to recognize and destroy tumor cell throughout the body. One potential approach to elicit high numbers of effector CD8+ T cells to control tumor growth is through repeated vaccination, a strategy called prime-boost vaccination. However, booster immunization is relatively inefficient during primary immune response because pre-activated effector T cells tend to impair robust antigen presentation. This phenomenon has been interpreted as a negative feedback mechanism where recently activated CD8+ T cells clear the antigen-bearing dendritic cells (DCs) and prevent memory T cells from the access of the boosting antigen. Interestingly, however, using in vitro activated B cell as a viral vector delivery system, we can boost T cell responses with the minimum viral input at a very short interval between immunizations. This B cell carrier is capable of delivering different viral vectors expressing different antigens, displaying a potential for broad application. The mechanisms behind B cell carrier-mediated efficient secondary responses are three fold: 1. Without the engagement of MHC molecules and antigen presentation, B cell carrying viral vector can bypass the killing by pre-existing effector T cells 2. B cells can delivery viruses to B cell follicles, a place separated from effector T cells, and mediate memory T cell expansion. 3. B cells can deliver antigen to both spleen and lymph node and induce antigen specific T cell expansion in multiple lymphoid organs. Our studies provide a novel boosting platform to accelerate CTL responses that has important clinical implications.</p> / Master of Science (MSc)

Genetically modified organisms (GMOs) are confined physically in order to contain their spread in nature and to minimise chances of horizontal gene transfer. However, with the potential that GMOs hold as cheap, reliable and efficient micro-machines, their eventual uncontrolled release into the wider space is becoming more likely. Indeed, their application as environmental sensors is largely increasing. Nevertheless, the field of synthetic biology may also afford solutions to the problem. A major potential application of GMOs is the delivery of antigens to human and animal hosts, through the utilization of live, engineered microbes. Recombinant technology is promising for several reasons including their capacity to be less reactogenic, more potent, safer and genetically definable. Also, they have the potential to provide protection against multiple targets simultaneously, are relatively inexpensive and can be eradicated with antibiotics, as the need arises. Besides, delivery of vaccines to mucosal surfaces is more efficient. Mutant Salmonella expressing heterologous antigens have been shown to induce protection against a variety of pathogens. Nevertheless, limited containment systems are available that can be applicable for bacterial antigen carriers. This project aims to design safeguards for the bacterial antigen delivery systems that limit ORF translatability and self-inactivates/destructs upon exit from the host. In this work, double quadruplet codons were suppressed by orthogonal tRNAs, providing a barrier for gene translation in the recipient cells when antigen is horizontally transferred. Furthermore, three kill switches were designed that are activated by a decrease in temperature from 37 °C. First, Sau3AI endonuclease was activated by protein self-splicing at low temperature mediated by Mtu recA intein. The activation of the endonuclease led to three-fold logarithmic decrease in the number of viable cells within two hours of gene expression. Second, RNA-dependent activation of RNase 7 showed a reduction in the number of viable cells at low temperature of three logarithmic folds. RNase 7 was controlled by the cspA 5’UTR, which sequesters ribosome binding site at 37 °C and allows translation at low temperature. Third, CspA 5’UTR was shown to regulate expression of TEV protease at 37 °C and low temperature. This led to bacterial cellular inhibition within two hours of TEV induction and five-fold logarithmic reduction in the number of viable cells at low temperature. In addition, for the first time and contrary to previous studies, the TEV protease was shown to inhibit cellular growth. It was also shown that biofilm formation was drastically impaired by the TEV activity. The three killing switches and the quadruplet translation system are poised to function as robust safeguards for bacterial antigen delivery systems.