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DCs are critical to the early events of HIV-1 infection. They are the first cells that HIV-1 encounters at mucosal surfaces and as sentinel antigen-presenting cells of the immune system these should alarm the immune system and activate innate immune defences to recruit effective adaptive immunity and viral clearance. A peculiar characteristic of HIV – in contrast to other ssRNA viruses – is its ability to completely evade host innate recognition pathways. Additionally, it has the unique ability to manipulate the endo-lysosomal system of DCs and promote transmission via trans-infection to CD4+ T cells across virological synapses. However, it is largely unknown how HIV-1 is sensed by the innate immune system. Here, a multipronged experimental approach based on phosphoproteomics, transcriptomics and custom RNAi screen was developed to characterize the early signaling complex induced by HIV-1 in DCs. A novel method of phosphoproteomics to identify the HIV-1 phosphoproteome in DCs showed that 342 proteins were differentially phosphorylated following 10 min of HIV-1 infection compared to time-matched mock-infected DCs. Functional analysis of these phosphoproteins showed enrichments in several cellular pathways, including vesicular trafficking, cytoskeletal rearrangements and the secretory pathway and a relative paucity of signaling molecules involved in inflammatory pathways. Proteomics analysis of HIV-1 virions was undertaken to identify host molecules hijacked by HIV-1 during viral replication and revealed a close interaction between the virus and the endo-lysosomal system. Transcriptomics analysis of HIV-1 infected DCs showed a muted immune response with no detectable differentially regulated genes. The results of the phoshoproteomic screen provided the basis for a custom RNAi screen to identify host proteins that are differentially phosphorylated by the virus and required for efficient trans-infection from DCs to CD4+ lymphocytes. The results of this screen showed that 54 of the 120 host factors tested were required for efficient viral transfer to CD4+ T cells and characterize the compartment that HIV-1 is internalized in on a molecular level. Two host factors identified within the HIV-1 phosphoproteome were chosen for further studies. Studies of BLOC-1 (biogenesis of lysosome-related organelles complex-1) and its subunits identified a role for snapin in HIV-1 trans-infection and HIV-1 and TLR8 sensing. Snapin may act as determinant of sorting of HIV-1 intraluminal vesicles to non-degradative, non-immunogenic compartments by activating mammalian target of rapamycin, mTOR, and inhibiting autophagy. Furthermore, HIV-1 triggered dephosphorylation of the cytosolic tyrosine phosphatase possibly via the interaction of host CD47 incorporated in the virion and the transmembrane glycoprotein SIRPα expressed on DCs. Blocking of this interaction with an inhibitory CD47 antibody resulted in a reduction of HIV-1 replication. Taken together, this multipronged approach reveals the complexity of the interaction of HIV-1 with the host cell machinery and identifies novel mechanism of the immune evasion tactics usurped by HIV-1.

The HIV-1 surface glycoprotein, gp120, is made of a rapidly mutating protein core and an extensive carbohydrate shield which are, respectively, encoded by the viral genome and synthesised by the host cell. In contrast to host cell glycoproteins however, gp120 contains a population of unprocessed oligomannose-type glycans that interact with host lectins, promote HIV infection, and alter cell signalling. They also form the basis of the epitopes of several broadly neutralising antibodies isolated against HIV, making them a key feature for immunogen design. The mechanistic basis of how HIV glycans are differentially processed by the host cell was demonstrated on a recombinant gp120 model, suggesting that steric occlusion within the patch of densely packed glycans lead to lack of processing by ER and Golgi α-mannosidases. Furthermore, an elevated level of oligomannose-type glycans was evidenced on gp120 isolated from HIV-1JRCSF virions produced in PBMCs, compared to recombinant material (respectively ~79% and ~29% of total N-linked glycans), along with a subset of highly processed and sialylated, bi-, tri- and tetra-antennary complex-type glycans, which could be involved in direct interaction with key host cell immune receptors and strongly suppress both antibody and T-cell immune responses. The effect of variation in viral production systems was analysed, with envelope glycoprotein derived from pseudoviral particles produced in HEK 293T cells exhibiting predominantly an oligomannose population (98%), compared to gp120 isolated from a single-plasmid infectious molecular clone (56%). Finally, mutation of one or several glycosylation site(s), known to be required for oligomannose-restricted neutralizing antibodies, was shown to induce a subtle redistribution within the oligomannose series whilst maintaining overall oligomannose levels. The gp120 glycan profile is therefore robust to mutations and also remarkably similar across primary viral isolates from Africa, Asia and Europe and consequently represents an attractive target for vaccine development.

HIV -2 represents a natural model of attenuated HIV infection. Although capable of inducing AIDS, most patients maintain healthy CD4 counts and undetectable viral loads throughout their lives. However, once patients have progressed to AIDS, characterised by high viral loads, they show similar symptoms to those with HIV-l infection. We hypothesized that HIV-2 with low/undetectable viral load would not significantly affect Iymphocyte turnover and immune activation, compared to healthy controls, while HIV -2 with high viral load would accelerate turnover rate and increase immune activation in a pattern similar to HIV-l. Lymphocyte turnover was measured by in vivo deuterated glucose labelling, naive (CD45RA+) and memory (CD45RO+) CD4+ and CD8+ T Iymphocytes sorted magnetically. Proliferation rates were derived from modelling deuterium enrichment in DNA, measured by gas chromatography/mass spectrometry. Immune activation markers HLA-DR and CD38 were measured by ex vivo immunostaining and T cell receptor excision circle (TREC) content by real-time PCR. The pattern of accelerated turnover was similar in HIV -2 with high viral load and HIV - I and affected CD8+ more than CD4+ cells and memory more than naive cells. Turnover in HIV-2 low viral load subjects was comparable to controls. These kinetic findings paralleled markers of immune activation; CD4 lymphopenia, viral load 'and immune activation all correlated with turnover of both CD4+ and CD8+ memory cells but the strongest association was with immune activation. Reduced TREC content was also a feature in HIV -2 infection, especially in high viral load subjects. Mathematical modelling revealed that HIV -2 infection had different effect on CD4+CD45 RA + cells than did HIV - I infection. We can conclude that low levels of immune activation may be pivotal in determining the relatively benign outcome in HIV-2 infection. Kinetically, HIV-2 is dichotomous with high • viral load subjects showing a pattern of activation, accelerated turnover and progression similar to HIV - I infected subjects. 3

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

The contribution of intrinsic defects in B and/or T cell function or impaired T-B cell interaction towards poor recall and neo-antigen vaccine responses in HIV-1 infection are not fully understood. Using CVID as a model for B cell maturation, we show patients with untreated HIV-1 infection have increased transitional and tissue like B cells and reduced IgM memory and class switched memory B cell proportions. Loss of IgM memory B cells is associated with progressive HIV-1. Antiretroviral therapy reduces transitional and tissue like B cell percentages but does not restore IgM memory or class switched memory proportions. Most HIV-1 patients on ART have reduced antibody levels post tetanus and pneumococcal vaccination. IgM memory B cell depletion associates with poor post vaccine IgM pneumococcal titres in HIV-1 suggesting loss of IgM memory B cells may be a risk factor for invasive pneumococcal disease. CVID patients with lung disease had lower memory B cells and a trend towards a loss of IgM memory B cells. IgM memory B cell percentages were protective against bronchiectasis in CVID patients displaying extremely low class switched B cell percentages. Evaluation of proteins implicated in the pathogenesis of PID, autoimmunity and malignancy, showed increased expression of BAFF and APRIL in CVID and untreated HIV-1 and normalisation by ART. BAFF upregulation was associated with CD4 T cell decline without treatment. Expression of BAFF and APRIL ligands demonstrated decreased BAFF-R on class switched memory B cells in HIV-1 and increased TACI on tissue like and memory B cells. A loss of follicular helper T cells in untreated HIV-1 infection was reported, however this was not a selective depletion and numbers were normalised by ART. In conclusion, we identify multiple novel defects in B cell composition in HIV-1 and suggest these may have implications for the design of effective vaccination strategies.

The central problem for researchers of HIV-1 evolution is explaining the apparent design of the virus for causing pandemic infection in humans: understanding how HIV-1 spreads is key to halting the pandemic. Current knowledge of how HIV-1 spreads from host to host is based upon experimental observation and indirect inferences informed by theory. The hypothesis of this thesis is that diversity of HIV-1 around the time of transmission is important for viral adaptation to a new human host, rather than intrinsic superiority of particular strains found in infectious fluids from human donor hosts, and that studying recombination is important for understanding this behaviour. To demonstrate the apparent randomness of transmission, I test the null-hypothesis that hard selection accounts for between-host viral divergence in a rare case study of contemporaneous infection. I explain how the experimental data that I have generated and the analyses I have carried out address certain basic assumptions and predictions about HIV-1 transmission and may inform current strategies for vaccine design. Specifically, my approach contributes to the current literature on HIV-1, by investigating an alternative hypothesis to the single virion theory of sexual transmission and by characterizing the role of recombination in a pseudodiploid virus following multiple-infection.

Despite 30 years of research, a prophylactic vaccine against HIV-1 is still lacking and is urgently needed in order to control the global AIDS pandemic. The discovery of broadly neutralising antibodies (BNAbs) was an important step for HIV-1 research but no vaccine candidate tested so far has been able to reproduce responses containing such antibodies, and it remains unclear how this could be achieved via immunisation. In this thesis, I attempted to explore this gap of knowledge in two ways. First, certain features (‘signatures’) of the Env protein that were associated with a broadly neutralising response were identified through machine learning. Further characterisation of these signatures revealed several ways by which these naturally-occurring mutations might alter the immunogenicity of the Env protein that could result in the elicitation of a broadly neutralising response. The incorporation of such signatures in future vaccine design could be useful as the Env protein might adopt a conformation that encourages the elicitation of a broadly neutralising response. Second, 3 novel vaccination approaches were proposed aiming to induce a BNAb antibody response. The development of 2 approaches proved to be difficult and was not continued. For the third approach, non-neutralising immunogen-derived antibodies were used to mask immunodominant epitopes on the Env protein (i.e. ‘antibody-shielding’), thus allowing the antibody response to be focused to the highly conserved CD4 binding site (CD4bs). Subsequent immunisation of the antibody-shielded gp120 proteins in mice and rabbits demonstrated that antibody-shielding was able to significantly dampen the V3-specific antibody response while retaining the CD4bs-specific response. However, the antibody response to the V1/V2 loop was enhanced upon V3-dampening which indicates that further optimisation of the antibody-shield is needed in order to eliminate any antibody response towards the immunodominant regions. In conclusion, these results are the first description of a number of novel vaccination ideas and provide valuable insights into how these approaches could be optimised to become effective HIV-1 vaccines that can lead to the elicitation of a broadly neutralising antibody response.

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.

Mucosal HIV infection is currently the leading cause of the global AIDS epidemic, with most infections occurring through sexual intercourse. A truly effective vaccine capable of protecting vaccines from HIV acquisition will likely require mucosal mediated immune protection. Previous non-human primate studies have provided the proof of concept that neutralising antibodies can prevent infection, however the levels of antibody at mucosal surfaces required for protection have not been fully characterised. Furthermore, to date no effective vaccination strategy has been shown to efficiently induce protective mucosal humoral responses. In spite of this, recent evidence suggests that directly targeting vaccines towards mucosal surfaces may enhance local immune responses. Thus this thesis has two hypotheses. First to determine the levels of mucosal antibody required to provide sterilising protection in the macaque model. Second using the murine model to develop strategies to enhance vaccine antigen delivery across mucosal surfaces, as an approach to induce mucosal humoral immune responses. We demonstrate that the presence of neutralising 2F5 IgG antibody at the time of vaginal challenge is able to give sterilising protection in the macaque model, and that the whole IgG molecule was essential for protection, while the Fab' portion was inefficient. Furthermore we show that permeation enhancer can play an important role in the delivery of vaccine antigens across mucosal surfaces, where the vaginal route of immunisation is the least effective one. In summary, we present supporting data for the role of humoral immune responses in protection against HIV infection and ways in which to increase the bioavailability of mucosally applied antigens to initiate the development of mucosal antibodies

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.

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.

Macrophages are a natural target cell for HIV-1 infection, and they contribute to the development of disease as they are important for transmission, dissemination and persistence of the virus in an infected patient. Macrophages are less well-studied than T cells and cell lines in relation to HIV-1 infection, yet macrophages are highly specialised and key aspects of the HIV-1 life cycle in these cells are already known to differ compared to other cell types. HIV-1 entry into macrophages has been suggested to occur by macropinocytosis, however the entry route in these cells has not been fully characterised. In this thesis I have tested a panel of pharmacological inhibitors of cellular proteins and uptake pathways, in order to delineate the requirements for HIV-1 entry into macrophages and to determine the nature of the entry route. My findings suggest that the following host factors are important for entry; membrane cholesterol, actin rearrangements, dynamin, sodium-hydrogen exchange, Pak1, and Rac. Other factors including clathrin, PI-3 kinase, Rho kinase and some isoforms of PKC were found to be dispensable for infection or to inhibit infection. Macrophages are a heterogeneous group of cells, and tissue macrophages from different parts of the body differ in their morphology, phenotype and function. I have used the growth factors M-CSF and GM-CSF to direct monocytes to differentiate into distinct types of macrophage. This allowed me to determine that different macrophages differ in their susceptibility to infection and in their ability to support replication. This is likely to be due to variation in HIV-1 receptor expression and the levels of key HIV-1 transcription factors, respectively. Overall this thesis contributes to existing knowledge regarding HIV-1 infection of macrophages. These findings may assist with the design of entry inhibitors, and with therapies designed to eradicate HIV-1 from infected individuals.

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

The primary human immunodeficiency virus type 1 (HIV-1) - specific CD8+ T cell response plays an important role in control of early viral replication. The nature of this response may thus be among the factors determining the prognostically-important persisting viral load. Here, the primary HIV-specific CD8+ T cell response was characterised in a small cohort of patients, to gain insight into quantitative and qualitative features of the response and their relationship to the efficiency of control of virus replication. The majority of patients studied established intermediate or high persisting viral loads. In these individuals, no association was found between the persisting viral load established and the total magnitude of the early HIV- specific CD8+ T cell response, its epitope breadth or specificity. However the response was observed to be heavily biased towards the most immunodominant epitopes. Kinetic analyses of epitope-specific responses revealed that in most patients, responses expanded asynchronously, with rapidly-expanded responses being immunodominant in primary infection. Mechanisms that may account for the rapid initial expansion of selected responses in acute viral infection, including high avidity and pre-existence of cross-reactive memory cells, were explored using murine models. Notably, the most rapidly-expanded epitope-specific response(s) typically did not reach peak magnitude until several weeks after the peak in acute viral replication and the fastest response was observed in the one patient studied who established a low persisting viral load. Phenotypic studies suggested that HIV-specific CD8+ T cells exhibit an "immature" phenotype from primary infection onwards in all patients. Although these studies need to be extended to a larger number of patients, the results obtained suggest that the kinetics and synchronicity of expansion of epitope-specific CD8+ T cell responses in primary HIV-1 infection may be among the factors that impact on the efficiency of control of primary viraemia.

The assembly of enveloped viruses requires the correct trafficking of the viral envelope or membrane proteins to the site of virus assembly. To understand the molecular and cellular mechanisms in human immunodeficiency virus envelope protein (Env) trafficking, I analysed the activities of putative endocytosis signals in the cytoplasmic domain of Env. Morphological and biochemical analysis of HxB2 Env as well as CD4-Env chimeras revealed two functional endocytosis motifs, the membrane proximal GY712XX0 motif and the C-terminal dileucine motif. Both of these motifs work with equivalent efficiency and show no obvious additive effect. RNAi knock down experiments show that endocytosis mediated by both signals is at least partly dependent on the clathrin pathway and the clathrin adaptor AP-2. Motifs of the Yxx0 type have been implicated in transport to the late endosome and HIV has been reported to assemble on membranes of this compartment. I studied the intracellular itineraries of HIV Env and found clear evidence of trafficking through early endosomes. However, no obvious colocalisation with CD63 or LAMP-1 was observed. Further analysis suggested that Env is transported to an endosomal compartment positive for the tetraspanin CD81, a compartment that has recently been recognised as the site of HIV assembly in macrophages. Env was targeted to this compartment independently of other viral components in HeLa cells as well as in macrophages. In order to identify other cellular components interacting with Env and the machinery responsible for Env incorporation into virions I performed yeasts- hybrid assays. I found that Vps37B, a component of ESCRT-I, which is required for HIV assembly, can interact with both HIV and SIV Env cytoplasmic tails, and that the ubiquitin E3-ligase WWP2 binds SIV Env. These findings may enable us to establish a molecular mechanism for the incorporation of Env into budding HIV particles.

The nef gene of HIV is required for complete viral pathogenicity. As an accessory gene with no known paradigm its principle function has been enigmatic. Nef is capable of a variety of in vitro effects including CD4 downregulation, MHC I down regulation, alteration of T cell signalling activity and enhancement of viral infectivity. The effect of Nef on host CD4 T cell signalling paths is intriguing as it is the earliest expressed HIV gene and because HIV replicates to high levels in activated host cells. An effect of Nef on T cell activation state could dictate host T cell fate tipping the balance between productive viral infection and post-integration latency. Although Nef can form in vitro interactions with a variety of T cell signalling proteins in vivo evidence for signalling path activation has been inconclusive mainly due to the limitations of the experimental systems used to address the question. An in vivo role for Nef in alteration of T cell activation state should lead to gene expression changes in the host cell. Large scale gene expression profiling was used to monitor T cell gene expression after controlled expression of Nef using the tetracycline inducible system. Nef induces a widespread transcriptional program in T cells. Comparison of this profile with reference T ceil activation states reveals a strong identity to the gene expression profile of Jurkat activated by anti-CD3 cross-linking. Gene expression profiling was used to explore the mechanism by which Nef mediates this effect. After cyclosporin treatment the profiie was attenuated. A similar effect was observed after Nef expression in Jurkat T cells genetically deficient in ZAP-70 and functionally deficient in TCRÇ. Expression of a mutant defective in a functional SH3 binding domain had a stronger effect on attenuation of gene induction. Thus gene expression profiling demonstrates definitively Nef is capable of activating T cells at the transcriptional level and that this effect is initiated high in the T cell signalling path. Within the Nef expression profile differential expression of multiple genes with the ability to affect virai replication was noted. In a few instances it was possible to demonstrate the Nef mediated changes in mRNA levels were functionally significant. Proteomic analysis of Nef effect on T cells was initiated. 2D gel analysis of T cell lysates after Nef expression demonstrated a differential regulation of proteins in T cell lysates. The mechanism by which Nef initiates T cell signalling was explored using a similar approach. The proteins interacting with Nef in lipid rafts were identified after immunoprécipitation of HA tagged Nef from this subcellular compartment. 2D gel analysis reveals the presence of Nef changes the content of lipid rafts and that Nef co-immunoprecipitates with several proteins in the raft fraction. The identity of these interactors obtained by peptide microsequencing should give insight into the molecular mechanism by which Nef induces a CD4 T cell transcriptional programme.

Vectors based on human immunodeficiency virus type 1 (HIV-1) offer a means for the delivery of therapeutic transgenes into a wide variety of cell types, both dividing and non-dividing. Virus entry is mediated through interactions between the viral envelope and its cellular receptor(s). For the benefit of gene transfer efficiency and safety it is practical and necessary to complement the vector particle with an envelope protein from another virus (vector pseudotyping) rather than using an HIV-1 envelope that has a limited host-range and cell tropism. The ability of HIV-1 based vectors to be pseudotyped with gammaretroviral envelope glycoproteins was examined. Modifications to the cytoplasmic tails of the envelope glycoproteins from gibbon ape leukaemia virus (GALV) and the feline endogenous retrovirus RD114 resulted in an increase in HIV-1 vector titre compared to pseudotyping with the wild-type envelope glycoproteins. Attempts were made to understand how these modifications affect vector titre. Pseudotyping of HIV-1 based vectors with alphaviral envelopes glycoproteins was also investigated, and it was found that the envelope glycoproteins of Ross River virus (RRV) and Semliki Forest virus (SFV) could pseudotype HIV-1 vectors. Stable HIV-1 vector producer cells were established by introducing envelopes suitable for HIV-1 pseudotyping into a novel HIV-1 stable pre-packaging cell line, STAR, which continuously produces HIV-1 Gag-Pol proteins. Pseudotyped vectors harvested from these producer cells were characterised with regard to future ex vivo and in vivo use as the envelope borne by the vector can dictate certain properties of the virion. These data include the stability of each pseudotype in human sera and at 31°C. Finally, attempts were made to transduce primary human cell cultures, particularly dendritic cells, with different HIV-1 pseudotypes. Although pseudotyped HIV-1 vectors could transduce human dendritic cells to some degree, transduction of dendritic cells with virus from STAR cells appeared to be limited by some facet of the codon-optimised packaging construct used in this cell line.

Highly active anti-retroviral therapy (HAART) has been very effective in reducing viral loads in HIV+ patients. However, current therapies carry detrimental side effects, require complex drug regimes for administration and are threatened by the emergence of resistant variants. The need for new effective therapies targeted to different stages in the HIV life cycle is urgent. The 7-transmembrane G protein-coupled chemokine receptors CCR5 and CXCR4 are the major coreceptors for HIV and SIV. The majority of transmitted viruses are R5-tropic, yet variants able to exploit CXCR4 (R5X4- or X4-tropic) emerge in the late stages of disease in up to 50% of individuals. As R5 strains remain present throughout disease, CCR5 is an ideal target for novel therapeutic intervention. At least a dozen other chemokine receptors or close relatives also support infection by particular HIV/SIV strains on CD4+ indicator cell lines in vitro. Despite the expression of many of these different receptors on primary CD4+ cells, their role during in vivo infection is currently thought to be insignificant. However, in the advent of CCR5 inhibitors, minority populations of variants able to use such coreceptors may become predominant and thus escape inhibition by CCR5-specific drugs. Here I have analysed the sensitivity of R5 and R5X4 strains of HIV and SIV to a series of six novel small molecule inhibitors of CCR5 on a diverse range of cell types, including lymphocytes and macrophages, the main cell types targeted by HIV-1 in vivo. In order to better evaluate the contribution of alternative coreceptors in vivo in the event of CCR5 being blocked, several primary untransformed cell cultures, including peripheral blood mononuclear cells (PBMCs), brain microvascular endothelial cells (BMVECs) and cells from immunoprivileged sites such as astrocytes and Leydig cells, were tested for expression of functional coreceptors able to support infection by HIV and SIV. A coreceptor of unknown identity has been discovered that is expressed on these primary cells and that supports infection by a subset of HIV and SIV isolates, including some from both subtypes B and C. The potential in vivo roles of CCR5 inhibitors and of alternative coreceptors will be discussed.