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Iron is essential for almost all biological systems and its metabolism is perturbed during infection. Clinical evidence suggests that elevated iron status correlates with a poor prognosis in HIV-1 infection. Four steps of the HIV-1 life cycle are thought to be iron dependent: reverse transcription, transcription, nuclear export of mRNA and virion assembly. The HIV protein Nef downregulates the haemochromatosis protein HFE in vitro, and iron chelators can inhibit HIV-1 replication. Given the evidence that iron can affect HIV -1 disease course and replication we investigated HIV -1 replication in monocyte-derived macrophages (MDMs) from both HFE wild type and C282Y-haemochromatosis patients. Our data from multiple patients suggests that the extent of HIV -1 replication is independent of HFE genotype in MDMs. Supplementation with the iron chelator DFO and the iron salt F AC both inhibited HIV-1 replication. Infecting differentiated U937 cells as a model system to avoid person to person variation gave inconsistent results when different viral strains and-experimental conditions were used. Changes in iron distribution during HIV-1 infection may be mediated by hepcidin. Hepcidin can be produced as part of the innate immune response which is mediated by the recognition of pathogen- associated molecular patterns (PAMPs) by a family of proteins including the Toll-like receptors (TLRs). We found that HIV-derived TLR7/8 ligands that stimulate the production ofTNFa. and IL6 in PBMCs and neutrophils does not result in a significant change in hepcidin expression whereas flagellin, a TLR5 ligand, induces TNFa., IL6 and hepcidin expression. This suggests that the changes in iron distribution may be more strongly influenced by IL6-mediated hepcidin induction in the liver or more directly by bacterial products that can cross the compromised gut mucosa during HIV-1 infection. Iron accumulation is an important eo-morbidity factor in Hepatitis C virus (HCV) infection. Chronic HCV patients have inappropriately low hepcidin expression which may explain their iron overload. Reduced hepcidin also causes the iron loading disorder hereditary haemochromatosis (HH). Genetic mutations underlying HH disrupt bone morphogenetic protein (BMP) dependent signalling pathways that control hepcidin synthesis. We investigated whether HCV was affecting the BMP/SMAD pathway, dampening down the signal for hepcidin induction, using an infectious HCV in vitro model. We found hepcidin induction by BMP6 but not BMP9 to be impaired by HCV infection. HJV, the BMP6 coreceptor, is downregulated and both SMAD6 and SMAD7 which suppress BMP signalling are upregulated in HCV infected cultures. HCV also increases expression of TNFa., and in vitro the inhibitory effect of HCV on BMP signalling can be mimicked by TNFa. In addition, supplementing cultures with an anti- TNFa. antibody restored hepcidin expression in response to BMP6. These observations were followed up in HCV patient liver biopsies from two separate cohorts. Both cohorts had lower hepcidin mRNA expression compared to a control group, consistent with other published clinical studies. HJV and ID1 mRNA was suppressed in biopsies from non-responders in the cohort with more severe disease whereas SMAD6 mRNA expression was raised in the biopsies from the cohort with mild disease. This demonstrates that the BMP/SMAD pathway can be disrupted by HCV infection and may explain the inappropriately low hepcidin expression found in vivo. Understanding the mechanism behind the hepcidin suppression found in HCV patients may suggest new therapies to prevent their iron overload thereby slowing their progression to cirrhosis and hepatocellular carcinoma.

Almost 30 years after the identification of HIV-I as the causative agent of AIDS, an effective prophylactic HIV-I vaccine has yet to be developed. Despite the design of a vast array of therapeutic agents and prevention strategies, which have helped to reduce AIDS mortality and the growth of the pandemic, HIV-I is still the fourth biggest killer worldwide and an effective vaccine will be needed to halt the pandemic. Over the past three decades, various HIV-I vaccination strategies have been explored leading to the key understanding that sterile protection against HIV-I infection will require a combination of cellular and humoral responses, particularly at the mucosal compartment, the common route of infection. Consequently, it has been postulated that this can only be achieved through a potent and effective immunisation strategy involving mucosal immunisation and the application of a heterologous prime-boost regimen. However, the mucosal route of immunisation, in particular for subunit vaccines, has not been well established as a mainstream mode of vaccination in human and results with experimental mucosal immunisation have generally been impeded by a lack of safe and effective mucosal adjuvants. In the work presented herein, I investigated whether the highly cationic polyethyleneimine (PEI), a gold standard DNA delivery agent, has adjuvant activity applied parenterally and mucosally in formulation with glycoprotein antigens, in particular HIV-I Env gp140, the sole target of neutralising antibodies (nAbs) and therefore the most attractive HIV-I sub unit vaccine candidate. I demonstrated that PEI is a potent inducer of Env-specific Ab responses in a mixed Ti.l and Th2 context. PEI is particularly potent via the mucosal route and superior to gold standard mucosal adjuvant Cholera Toxin Beta subunit (CTB) as measured by HIV-I specific IgA responses at the mucosal compartment. PEI showed direct immune stimulatory effects, marked by a significant recruitment of local leukocytes, including inflammatory monocytes and dendritic cells (DCs). Further it associated with antigen to form gpI40-PEI particles, which enhanced antigen targeting to, and uptake by, immune cells, in particular monocytes, macro phages, and DCs. The observed adjuvant activity was maintained with the use of various forms of PEI, including linear or branched, and durable or biodegradable, and under a range in molecular weight (MW), suggesting that immune potentiation is a shared attribute of this group of PEI polymers. This is the first demonstration of PEI as a mucosal adjuvant for protein immunisation. These results merit further exploration of PEI in protection models with the implication for clinical trials.

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HIV evolves very quickly permitting it to escape the immune system of an infected individual, limiting the effectiveness of drug treatment and making vaccine design extremely difficult. For these reasons identifying whether there are constraints on HIV evolution is of primary importance. In this thesis, I examined the relationships between sequence diversity and a number of factors including protein structure, co-evolution and RNA secondary structures, all of which contribute to evolutionary constraints. Firstly, I demonstrate that while there is an increase in sequence diversity over time, this variation has a tendency to be limited to specific structural regions. Relating the sequence variability of individual amino acid residues with three- dimensional protein structure, I find a significant difference between evolutionary rates in regions buried in the core of the protein as compared with those exposed on the surface. This result indicates that missense mutation is affected by structural constraints. Secondly, by relating recombinant breakpoint positions with the potential numbers of losses in amino acid interactions within the structure, I propose that as well as missense mutation, recombination is also affected by structural constraints, due to the need to maintain intra-molecular interactions. Thirdly, I demonstrate a relationship between conservation of RNA secondary structure and limited sequence variation in protein level, indicating RNA secondary structures are additional evolutionary constraint. This link between sequence and protein and RNA structures not only demonstrates the limits of recent HIV-1 evolution but also highlights the origins of evolutionary constraint on viral change. Lastly, I examined whether evolutionary constraints and co- evolution patterns of HIV genomes are applied in novel mosaic vaccine strategy and suggest that polyvalent mosaic vaccine sequence may generate proteins with stable structures and co-evolving units. Detailed understanding of the constraints that restrict HIV’s possible evolution will inform analysis of drug and immune escape.