The lack of an effective HIV vaccine calls for efforts to investigate the mechanism of protective immunity against AIDS viruses. It has been previously demonstrated that the live replication-competent modified vaccinia virus Tiantan (MVTT) is superior to non-replication vaccinia MVA in inducing high levels of neutralizing antibodies against SARS-CoV infection via mucosal vaccination. Therefore, the hypothesis was that MVTT could be a better HIV vaccine vector given its highly attenuated phenotypes such as no neurovirulence and safe in severe combined immunodeficiency disease (SCID) mice. Here, a recombinant MVTT expressing SIVmac239 Gag-Pol and Env (rMVTTSIVgpe) was constructed and its immunogenicity was assesed when administered via different routes using homologous prime-boost strategies or in heterologous regimens boosted with a recombinant adenovirus-based vaccine inserted matched SIVmac239 genes (rAd5SIVgpe). Results show that the heterologous prime-boost immunization with rMVTTSIVgpe and rAd5SIVgpe induces significantly greater humoral and T cell responses specific to SIV Gag, Pol and Env than homologous inoculations in mice with remarkable improvements in quality and quantity. The further study comparing different combinations of rMVTTSIVgpe and rAd5SIVgpe demonstrates that the rMVTTSIVgpe prime-rAd5SIVgpe boost regimens elicit systemic CD8+ T cell responses with augmented magnitude and polyfunctionality, as compared with rAd5SIVgpe-rMVTTSIVgpe and rAd5SIVgpe-rAd5SIVgpe regimens. Priming with rMVTTSIVgpe also increases frequencies of gut-homing Gag-specific CD8+ T cells (CCR9+47+ and CCR6+47+) and levels of CD8+ T cell ELISPOT responses against Gag, Pol and Env in mesenteric lymph nodes (MLNs) post-boost. The mucosal route of immunization is essential for rMVTTSIVgpe to induce rectal IgG with detectable neutralizing activity against SIVmac1A11. Furthermore, the regimen involving mucosal prime with rMVTTSIVgpe followed by systemic boost with rAd5SIVgpe proves to be efficient in protecting monkeys from mucosal challenge of a high dose of SIVmac239, a CCR5-tropic strain with high pathogenicity and neutralization-resistance. SIV-specific T cell ELISPOT responses specific to Gag and Pol but not Env and the frequency of Gag-specific IFN-+TNF-+CD8+ effector memory T cells (TEM) are likely associated with virological control after challenge. Mucosal immunity induced by this vaccination strategy also has important implications to the effectiveness of protection against disease progression. A hypothesis was generated that removal of non-protective but immune dominant determinant of SIVmac239 Env may drive antibody responses to protective domains. It was found that the neutralization-resistance of SIVmac239 could be partially explained by its high immunogenicity in eliciting CD4-induced neutralizing antibodies, which are unable to protect the CCR5-binding site due to the conformational masking and steric restriction. It was discovered that the immunodominance of CD4-induced neutralizing antibodies on SIV envelope is determined by a single highly conserved N-linked glycosylation site (N277) in the C2 domain. Substitution of this N-linked site abolishes viral entry and the immunogenicity of the CD4i domain while promotes V2-specific antibody responses, which have recently been identified as an important immunological correlate to HIV-1/SIV protection. Our findings demonstrate the concept that B cell immunodominance is relative and eliminating the dominant antigenic region can result in redirection of B cell recognition, which have critical implications for immunogen design and the development of protective antibody-based HIV vaccine. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

CD4+ lymphocytes (T helper cells) play a crucial role in orchestrating adaptive immune responses. This project aims to characterise the CD4+ cells in zebrafish (Danio rerio) for a better understanding of adaptive immunity in teleost fish. I cloned three CD4 homologues, termed zfCD4 and zfCD4 related (zfCD4rel1 and zfCD4rel2), with both reported previously in other bony fish species. The zfCD4 and zfCD4rels transcripts were detected in immune organs in zebrafish and were most highly expressed in the lymphocyte population. A moderate induction of the zfCD4 and zfCD4rels (and other immune related genes) was seen in kidney, spleen and intestine after poly (I:C) injection. Two antibodies against CD4 and IFN-γ, respectively, have been validated using various immunostaining approaches for further functional studies. The CD4 positive cells ranged from 20-30% of lymphocytes in zebrafish, similar to what is seen in other vertebrates. The expression level of IFN-γ and other Th cell related genes were analysed in immunised fish following re-stimulation with antigen, revealing that in zfCD4+ lymphocytes an increased expression of cytokines and master transcript factors was seen when the same antigen was used for boosting. This is the first demonstration of Th-type responses effected by CD4+ lymphocytes in a poikilotherm. Lastly, I studied an aspect of Treg function in zebrafish, focused on a master transcription factor of Tregs, the FoxP3 gene. Knocking down FoxP3 genes in zebrafish resulted in modulated gene expression of cytokines and transcription factors associated with Th and Treg cells, providing some evidence that the immune tolerance function of Treg cells may exist in teleost fish, with some sub-functionalisation of the two FoxP3 paralogues apparent. This thesis extends our knowledge into teleost adaptive immune responses mediated by CD4+ Th cells and putative FoxP3+ Treg cells and may aid future studies using zebrafish as a model of vertebrate immune function.

No description available.

Professional antigen presenting cells (pAPCs) process and present antigens on their cell surface in association with MHC class I molecules through two general pathways: direct or cross-presentation. The process of antigen presentation by pAPCs to naïve T cells resulting in their proliferation and differentiation into activated cytotoxic lymphocytes (CTLs) is called T cell priming. In these studies, we examine the cross-presentation of antigens from two non-replicating viruses: inactivated Lymphocytic Choriomeningitis virus (LCMV), and recombinant baculovirus encoding the LCMV nucleoprotein (NP). Since effective activation of pAPCs is essential for efficient priming of CD8+ T cells and CTL activation, and because infection with inactivated viruses generally induces an extremely poor level of CTL activation, we examined the activation state of pAPCs by measuring their cytokine profiles following infection to help further delineate their involvement in the CTL response to inactivated viruses. Our results indicate a pro-inflammatory cytokine mRNA upregulation in pAPCs in response to the inactivated virus, similar to the cytokine profiles subsequent to live LCMV infection, but to a lesser extent. In these studies, we also examined CTL activation following infection with inactivated LCMV and bAc-NP. We have demonstrated that the presentation of antigens from inactivated LCMV and bAC-NP results in a low level of CTL activation in vivo, though there is an undetectable level of CTL activation in vitro, in comparison to activation following infection with the live virus. Ultimately, the characterization of the cytokine profiles of pAPCs and the CD8+ T cell profiles induced in response to inactivated LCMV or the baculovirus derived NP may lead to a better understanding of how cross-presentation of these viral antigens may occur. This information may be applied to enhance the process of pAPC activation and T cell priming, for the induction of more effective cellular immune responses and the generation of stronger protective immunity. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-09-02 15:30:13.883

In this study we have reproduced the prolongation of graft survival by ITI in a rat heart transplant model in which an ITI of an optimal number of donor bone-marrow cells (BMC) was given together with 1ml ALS IP 14 days before transplant. The efficacy of this protocol was critically dependent on the donor-recipient haplotype and influenced by antigenic strength and MHC disparity but not by non-MHC background genes. In strain disparities where ITI was unsuccessful, this was caused by alloreactive recent thymic emigrant cells. In a high responder strain combination the effect was highly dependent on the dose of BMC in the intrathymic injection. Moreover it was readily reproduced with injection of antigen by the intravenous route, even at a lower dose than that required via the intrathymic route. This was in contrast to the other strain combinations tested in which the beneficial effect of donor antigen injection was specific to the intrathymic route, and it suggested that the effect in this group might be at least partly dependent on peripheral mechanisms. The polyclonal ALS can easily be demonstrated to be non-specific in its depletion of peripheral lymphocytes at the dose used in these studies, and we have shown that it also penetrates the thymus. Therefore treatment with ALS may have more effects that mere disablement of peripheral alloreactive T cells, thus complicating the interpretation of the experiments. We have therefore refined our model by recruiting in place of ALS a more specific agent, the partially depleting anti-CD4 monoclonal antibody MRC-OX38, which is equally effective as an adjunct to ITI. We have shown using flow cytometry and immunohistochemistry, that this and certain other monoclonal antibodies, at a therapeutic dose, do not cross the "blood-thymus barrier", and therefore do not complicate the model as ALS potentially does. We would recommend this approach in further studies.

No description available.

Prophylactic administration of the immunosuppressive drug cyclosporine A protects Balb/c mice from fatal Leishmania major infections. It is believed that distinct subpopulations of CD4+ T lymphocytes and their distinctive cytokines may determine susceptibility and resistance to leishmaniasis among inbred strains of mice. CsA may enhance disease resistance in Balb/c mice by modulating these T cell subsets and/or their cytokines. We have measured lymphoid cell production of IL-2, IL-4, and TNF-a in naturally resistant C57/Bl/6, CsA-treated Balb/c, and nontreated Balb/c mice during the course of L. major infection. CsA treatment inhibited IL-2 and IL-4 production for the first week of infection. Thereafter the cytokine production of all three groups of mice did not differ greatly except in week two when the treated mice produced significantly enhanced levels of IL-4. C57B1/6 mice did produce slightly more TNF-a than either group of Balb/c mice, but as the CsAprotected and diseased Balb/c mice produced similar amounts of this cytokine, the elevation in C57B1/6 animals probably reflects a strain-related difference rather than disease resistance. / Department of Biology

If used early in infection, prophylactic treatment with the immunomodulatory drug cyclosporin A of Leishmania ma'or infected Balb/c mice has been shown to enhance resistance of these mice to serious disease. It is thought that CsA treatment affects disease progression by altering the balance of specific T lymphocyte populations as well as the secretion of various cytokines. We have followed the levels of L3T4+ T cells, Ly-2+ T cells, and total T and B lymphocytes, as well as IL-4 in susceptible Balb/c mice, CsA-treated Balb/c mice, and naturally resistant C57B1/6 mice during the course of L. ma'or infection. The CsA-treated mice displayed a disease pattern similar to that of the C57B1/6 group throughout infection. Most importantly, CsA treatment appeared to inhibit IL-4 production early post infection in both spleen and lymph node, and also appeared to inhibit the dramatic early increase of L3T4+ (CD4+) T cells which is characteristic of the susceptible Balb/c mice. / Department of Biology

No description available.

The complement system is considered as the chief recognition and effector component of innate immunity; it is involved in inflammation and enhances the adaptive immune response. Factor I (fI) is a heterodimeric serine protease consisting of a heavy (HC) and a light-catalytic (LC) chain; it circulates in an active form regulating complement by selectively cleaving only C3b or C4b in the presence of a cofactor such as factor H (fH), CR1, MCP or C4bp. The cleavage of C3b occurs through a ternary complex formed between fI, C3b and a cofactor like fH and yields iC3b, a major opsonin. The structural and functional properties of fI were investigated. The interchain disulphide bond formed between C³⁰⁹-C⁴³⁵ tnat links the HC and LC of fI as well as the composition of the TV-linked carbohydrates of fI were determined. By using two independent assays, the proteolytic and amidolytic assays, the catalytic properties of human fI were characterised in detail. The catalytic subunit, the SP domain, was shown to have a native conformation that accommodates substrate recognition and cleavage, fI has specificity similar to thrombin, but exhibits lower catalytic activity. fI amidolytic activity reaches optimum at pH 8.25 and is insensitive to ionic strength. This is in contrast to its proteolytic activity within the fI-C3b-fH reaction, in which the pH optimum for C3b cleavage is <5.5 and the reaction rate is highly dependent on ionic strength. The rate of cleavage of tripeptide AMC substrates by fI was unaffected by fH or C3(NH₃) at optimum pH. fI and the isolated SP domain were found to have similar amidolytic activities, but strikingly different proteolytic activities on C3(NH 3 ). fl did not cleave C3(NH₃) in the absence of fH, but cleaved it rapidly at two sites in its presence. The SP domain however, cleaved C3(NH₃) slowly in the absence of fH, at more than two sites. Cleavage by the SP domain was inhibited, not stimulated, by fH. These results suggested that the HC domains and/or the cofactor must orient the natural substrates and restrict cleavage by fI to the two sites which yield iC3b. The implications of these findings are discussed.