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No description available.

No description available.

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No description available.

Cryptococcus neoformans is a basidiomycete fungus that is found throughout the world living as a saprophyte. It is an opportunistic pathogen in humans and prevalence has increased significantly with the spread of HIV-AIDS, particularly in sub-Saharan Africa. C. neoformans infects the body via inhalation into the lung and dissemination around the body, including the central nervous system. Infection of the brain is fatal if untreated. The virulence of this organism is dependent on the enzyme urease, which catalyses the degradation of urea to ammonia and carbamate. The molecular role of urease during infection is not clear, although there is evidence that it is involved in the ability of C. neoformans to cross the blood brain barrier. Urease enzymes require an iron or nickel cofactor to function, which suggests that acquisition and distribution of this cofactor is important for the virulence of this yeast. The homeostasis of transition metals in living organisms is tightly regulated by a number of mechanisms including; metallochaperones, selective metal permeases, metal-sensors and compartmentalisation. Cases of in vivo mis-population of metal proteins are extremely rare. In this project C. neoformans urease is identified as a nickel binding enzyme and regulation of urease activity in response to the available nitrogen source is characterised. The accumulation of nickel in C. neoformans is responsive to the available nitrogen source and a putative nickel-importer, CnNic1, is identified as the primary means of nickel accumulation. The urease accessory protein CnUreG is essential for urease maturation and binds two equivalents of nickel with high affinity. CnUreG is proposed to be a novel nickel chaperone for urease. Cobalt inhibits cryptococcal urease in vivo by binding to the protein and preventing nickel insertion into the active site. Urease inhibition by cobalt occurs at relatively low cobalt concentrations and therefore C. neoformans does not appear to have evolved effective mechanisms to protect against cobalt mis-population.

The tempo of HIV disease progression might rest with particular characteristics of certain HLA class I molecules, and the CD8+ T cell populations, which they dictate. Therefore we wanted to investigate if CD8+ T cell quality in HIV infection determines disease progression. To do this we used patients recruited from the UK arm of the SPARTAC clinical trial (n=155) that were acute (seroconverted within the previous 2 months, n=5) or early infected (seroconverted within the previous 6 months, n=150). These patients were followed into chronic infection (infected for 1 year, n= 117). To determine CD8+ T cell quality we developed novel HLA class I CD8null tetramers, which allow direct detection of high avidity CD8+ HIV -specific T cells ex vivo. Furthermore, by FACS analysis we analysed high and low avidity HIV -specific T cell T EMRA lineage differentiation (populations defined by CCR7, CD45RA and CD27) and T cell exhaustion (defined by PD-l, TIM-3 and LAG-3 expression). We show that high avidity HIV- specific T cells are present in both early and chronic HIV infection. A significantly greater proportion of CD8+ HIV -specific T cell populations restricted by HLA-B molecules are high avidity in comparison to HLA-A restricted populations (p<O.05). This is particularly prominent in HLA B*2705 KKlO specific populations (p-cfl. Ol ). The T EM RA differentiation of CD8+ HIV -specific T cells restricted by different HLA class I molecules differs, independent of T cell avidity. However, the T EMRA lineage differentiation of HIV -specific T cell populations fails to influence disease progression. HIV -specific T cell populations are significantly more exhausted than the total CD8+ T cell population (p<O.OOl), with high avidity HIV -specific T cell population being responsible for the majority of this exhaustion (p<O.Ol). Despite this the proportion of exhausted HIV -specific T cells does not correlate with HIV plasma viral loads. No clear relationship was found between CD8+ T cell quality, as measured by T cell avidity, T EMRA differentiation and T cell exhaustion and HIV disease progression.

This thesis is concerned with exploring how cell heterogeneity and drug resistance can cause long term persistence of HIV. We examine models of multiple viral strains to assess the impact of drug resistance on viral persistence and extend our cell heterogeneity models to include multiple strains. Chapter 1 summarises the nature of HIV infection within host. The key barriers to HIV eradication within host and the role of mathematical models to help understand these issues are discussed. In Chapter 2 we analyse models that include cell heterogeneity. We find robust long term viral persistence is possible on therapy and differences in viral load between body compartments explained by cell heterogeneity. The inclusion of a drug sanctuary also allows low level viral load on treatment. Competition and evolutionary models of wildtype and drug resistant strains of virus are described in Chapter 3. We analyse two models containing three strains of virus with different mutation mechanisms. We find that the proportion of the minority strains of virus is determined by the number of mutations away from the dominant strain. In Chapter 4 we extend our cell heterogeneity models from Chapter 2 to include a drug resistant strain of virus. We find that when a drug sanctuary is present coexistence is possible in the absence of an evolutionary mechanism. The two compartment model also shows differential dominance whereby a different strain is dominant in each compartment. within the host. We find the latent cell reservoir acts as an archive for previously dominant viral strains when there is a mechanism for latent cell maintenance and that the balance between ongoing viral and latent cell replication determines the longevity of the archive.

HIV -l-specific T -cell responses are preserved in HIV -1 infected individuals with non- progressing HIV -1 disease. Evaluation of HIV -I-specific T -cell functionality; T -cell phenotype; HLA class; subtype, RNA and DNA levels of HIV -1; and other factors in long- term non-progressors (LTNP) in comparison with rapid progressors (RP) could reveal novel correlates of protection against disease progression. We show that HIV -1 RNA viral load, proviral DNA load and lipopolysaccharide (LPS) were positively correlated with each other and negatively correlated with CD4 count. We show that IFN-y Gag-specific responses were higher in LTNP, whereas IFN-y Nef-specific responses were higher in RP. Nef-specific responses positively correlated with HIV-I RNA viral load, proviral DNA load and LPS. Flow cytometric analysis of HIV -l-specific responses showed that IFN -y +, CD I 07 a +IFN -y +, and IFN-y+TNF-a+ dual responses to Gag-p24 were higher in LTNP and correlated positively with CD4 count. Nef-specific responses in LTNP were predominantly polyfunctional. Among HIV-I-specific responses to Gag-pI7, Gag-p24 and Nef, only production of IFN- y+TNF-a+ in response to the Nef-specific pep tides correlated negatively with both RNA viral load and proviral DNA load and was a good correlate of protection. In analysis of perforin and degranulation, Nef-specific responses showed higher monofunctional CD8+CD 1 07a +PF in RP and CD8+CD107a+PF+ in LTNP. CD8+CD107a+PF positively correlated with RNA viral load, proviral DNA load and LPS and negatively correlated with CD4 T-cell count. IL- 21 levels in the CD8 T-cells of RP were higher than in the LTNP. CD4+CD45RA +CD38+HLADR+ T-celllevels, PD-l levels and CD8+ naive T-cells were higher in the LTNP. PD-1 levels and CD8+ naive T-cells correlated negatively with LPS and positively with CD4 count, PD-l also correlated negatively with RNA viral load. High CD8 naive T -cells and PD-I expression were good correlates of protection. T -cell regulatory levels were not significantly different in LTNP and RP. Correlates of rapid disease progression included high IFN-y Nef-specific responses and high monofunctional CD8+CDI07a+PF Nef-specific responses.

We have characterized an assay measuring CD8+ T cell-mediated inhibition of human immunodeficiency virus (HIV) type 1 replication, demonstrating specificity and reproducibility and employing a panel of HIV -1 isolates. The assay uses relatively simple autologous cell culture and enzyme-linked immunosorbent assay (ELlSA), avoids generation of T cell clones and can be performed with <2 million peripheral blood mononuclear cells. Efficient CD8+ T cell-mediated cross-clade inhibition of HIV-I replication in vitro was demonstrated in antiretroviral therapy-naive HIV -l-infected subjects with controlled viral replication in vivo but not in viremic subjects. The observed HIV-I inhibition is reversed by blockade of MHC I and physical separation of effector CD8+ and target CD4+ T cells by transwell chambers. An HIV-I vaccine candidate, consisting of adenovirus (Ad) serotype 35 vectors tested in a phase I clinical trial, induced CD8+ T cells that efficiently inhibited HIV -1. Assessment of direct antiviral T cell function by this assay provides additional information to guide vaccine design and the prioritizing of candidates for further clinical trials. 2