Accessory mutations are thought to arise alongside major or primary drug resistance mutations in order to augment resistance, restore viral fitness, or both. The H208Y mutation in the HIV-1 reverse transcriptase (RT) gene was hypothesised to be an accessory mutation. This thesis characterises the H208Y mutation in terms of linkage of H208Y to other major and accessory resistance mutations, and examines two phenotypic aspects, drug susceptibility and viral fitness. The HIV Resistance Database held at the Royal Free Hospital was searched for genotypes containing the H208Y mutation. The prevalence of H208Y in antiretroviral treatment naïve and treatment experienced patients was 5/3783 (0.1%) and 12/1304 (0.9%), respectively, indicating a high degree of conservation of position 208 in wild type virus and an increase in prevalence under selective drug pressure. Four patients were chosen to conduct further analysis of virus with H208Y, comprising a treatment naïve patient with subtype B virus, and three treatment experienced patients harbouring subtype A, subtype B and subtype C virus respectively. The RT gene from these patients was cloned and sequenced. H208Y was found to be associated with the thymidine analogue mutations (TAMs), particularly mutations at positions D67, T215 and K219. H208Y was always associated with accessory mutations at positions V35, K122 and T200. Recombinant viruses containing patient derived RT genes with and without H208Y were constructed to examine the impact of H208Y on drug susceptibility and viral fitness. A multiple cycle drug susceptibility assay showed that H208Y conferred a reduced susceptibility to the nucleotide RT inhibitor tenofovir in the context of subtype B wild type RT and subtype B RT containing TAMs. Growth competition assays were used to examine the fitness effects of H208Y using allele-specific PCR to differentiate between competing strains with and without H208Y. In the context of subtype B wild type RT, H208Y conferred a reduced viral fitness both in the presence and absence of drug. The effect may be proposed to contribute to the overall high degree of conservation of position 208. In contrast, H208Y did not appear to impact on viral fitness in the context of subtype B and subtype A RT containing TAMs.

The balance between signals delivered by positive and negative costimulatory molecules is crucial to the ultimate fate of cellular immune responses. Manipulation of T cell costimulatory pathways may offer a novel approach for reinvigorating exhausted T cell responses, especially in the context of chronic infections. T cells also display profound exhaustion in old age and this thesis investigates the hypothesis that T cell inhibitory receptor upregulation may define a reversible defect in age onset immune decline. Data presented here illustrate how T cells utilise different inhibitory receptors as they differentiate and that KLRG1 signalling is causative of dysfunctions in highly differentiated CD8+ T cells. The inhibitory receptors KLRG1 and CTLA-4 are revealed to undergo age-associated upregulations on CD8+ T cells but their blockade does not reverse the characteristic hypo-responsiveness of CD8+ T cells amongst old donors. The dysregulated immune response to lifelong chronic cytomegalovirus (CMV) infection is thought to play a major role in driving age related immune dysfunctions. We found that CMV accelerates age-associated telomere attrition amongst CD8+ and CD4+ T cells. CMV infection is also shown to drive CTLA-4, PD-1 and KLRG1 upregulation on both CD4+ and CD8+ T cells. Moreover, the PD-1/Ligand (L) inhibitory pathway defines a reversible proliferative dysfunction in the responses of CMV specific CD8+ T cells. Specifically, the CD45RA re-expressing memory subset exhibits a proliferative deficiency, relative to their central and effector memory counterparts, that is reversible upon PD-L blockade. However, this augmented proliferative response was not accompanied by increased telomerase function, suggesting this does not result in true reversal of exhaustion. In summary, the dysfunctions of highly differentiated and CMV specific CD8+ T cells can be at least partially reversed by perturbation of inhibitory receptor pathways, whose further manipulation may provide a therapeutic modality to combat age-associated immune decline.

Immune senescence is associated with a predisposition to infections, poor vaccination responses and early mortality in older individuals. Furthermore, evidence that chronic cytomegalovirus (CMV) infection is a key driver of immune senescence is becoming increasingly recognised. This thesis aimed to investigate the hypothesis that large CD8+ T cell expansions (TCEs) caused by chronic CMV infection during ageing may be instrumental in this association. Data presented here shows CMV-specific CD8+ TCEs that accumulate during ageing are predominately of the CD45RA+ memory phenotype. However, these cells exhibit low Ki-67 positivity and low Bcl-2 levels directly ex-vivo, in addition to poor proliferation and low telomerase activity in response to activation. This indicates they are not accumulating through increased proliferation or resistance to cell-death, and may represent a population close to senescence. These CMV-specific CD8+CD45RA+ memory T cells were also found to display a lower functional avidity for peptide, with higher activation threshold compared with CMV-specific CD8+CD45RO+ T cells. Furthermore, IL-15 was shown to cause CMV-specific CD8+CD45RO+ memory T cells to proliferate and re-express CD45RA in-vitro; adding to existing evidence indicating a role for IL-15 in the homeostatic, rather than antigenic, driven generation of CD8+CD45RA+ memory T cells from a CD45RO+ memory T cell pool. The possible impact of these CMV-specific TCEs during ageing is highlighted by the finding that old CMV positive individuals had significantly shorter T cell telomere lengths than old CMV negative individuals. Therefore, the accumulation of TCEs is likely to impact the CD8 compartment of healthy individuals in two ways; by restricting immune space and also lowering the overall telomere length of the compartment through the accumulation of highly differentiated CD8+ TCEs. Both of these have been shown to increase susceptibility to infection. This study therefore provides further evidence for the detrimental effects of CMV infection and its role in driving immune senescence.

Subtype C HIV-1 is currently responsible for the majority of new infections in the world, particularly in parts of Africa where the adult prevalence rate is as high as 15%. In the absence of a viable vaccine in the near future, the study of new neutralising antibodies that can inhibit virus entry is urgently needed. To understand the subtype C HIV-1 envelopes, the env gene was cloned directly from 15 patient plasma samples obtained from a few countries in Africa and in the UK, and 18 replication-competent chimeric viruses were created. These envelopes were then characterised and compared with other envelopes in standard reference panels. We then exploited the unique properties of llama heavy-chain antibodies to create antibody fragments (VHH) that can recognise HIV-1 envelopes and prevent infection. Four VHH that recognise a conformation dependent epitope on gp41 were isolated from a llama that was immunised with recombinant gp140 derived from a subtype B’/C isolate after panning of the phage libraries on recombinant gp41. These VHH were more potent in neutralising subtype C isolates than subtype B isolates. Based on the success of an earlier study on VHH that recognise an epitope overlapping the CD4 binding site on gp120, a novel strategy was used to isolate variants of the VHH to create a family-specific VHH library. Thirty-one new VHH were characterised and grouped according to their neutralisation breadth against 3 subtype C viruses. The neutralisation breadth of the VHH correlated with its dissociation rate with gp120, and was found to be dependent on 3 amino acid residues in the third complementarity determining region of the VHH. These VHH may have further use in applications such as HIV-1 microbicides development and immunogen design through reverse immunology.

Lentiviral vectors (LVs) are promising tools for in vivo gene delivery, either to correct genetic defects or for vaccination. Intravenous administration of LVs results in stable transduction and expression of the transgene in antigen presenting cells (APCs) from the spleen. Therefore, it was decided to investigate the reasons for and the consequences of sustained antigen expression in these cells after systemic in vivo administration of LVs. Intravenous injection of a LV encoding green fluorescent protein (GFP) resulted in transduction of lymphocytes, macrophages and all subsets of dendritic cells (DCs) in the spleen, detected 5 days later. In the case of macrophages and DCs, the percentage of transduced cells increased between 5 and 30 days after injection. The transduction of dividing precursors contributes to the persistence of the transgene-expressing DCs, as shown by BrdU incorporation. Expression of ovalbumin (OVA) resulted in a reduced number of transgeneexpressing cells after 30 days. However, the remaining transduced cells stimulated proliferation and activation of OVA-specific CD8+ T cells up to 3 months after LV administration, in spite of a reduction in the activation status of transduced DCs over time. Mice also maintained cytolytic activity against OVA-pulsed targets following a single immunisation. In conclusion, this thesis shows that LVs can transduce DCs and macrophages, leading to persistent antigen expression. These modified APCs are functional and capable of activating T cells. Therefore, LVs can be used as tools for persistent genetic modification of APCs, opening the opportunity for their use in long-term immunomodulation.

AIM: This work identifies recent HIV infection (RHI) in men who have sex with men (MSM) and the specific phylogenetically-linked transmission events during which they were infected. The overall aim is to identify the clinical characteristics of each infection source individual and evaluate their proportional contribution to transmission. OBJECTIVES: 1. Construct a dataset to capture cohort characteristics; 2. Identify RHI in subtype B infection using the Serological Testing Algorithm for Recent HIV Seroconversion (STARHS); 3. Identify the most-likely infection source individual for each RHI by phylogenetic analysis; 4. Observe the clinical characteristics of each infection source individual at the time of infection and estimate the proportional contribution to transmission of unknown infection, disease stage and HAART status METHODOLOGY: The study population was a cohort of mainly MSM attending the single HIV clinic in Brighton, UK. The study involved phylogenetic analysis of HIV genotypic sequences obtained for routine clinical care. Clinical data were also collected. Written, informed consent was obtained and every study described in this thesis had formal ethical approval. RESULTS: 1. STARHS confirmed RHI in 71/74 (96%) individuals identified as RHI by conventional methods; 2. Pol sequence data were obtained for 859/1144 (75%), of whom 159/859 (19%) were RHI at diagnosis; 3. For only 41/159 (26%) RHI could an infection source individual be identified; 4. RHI contributed 3% of follow-up time but 41/59 (27%) transmissions (RR 4.44, 95% CI 2.11-9.33, p=0.0001); 5. 39/41 (95%) transmissions were from untreated individuals or those interrupting antiretroviral treatment (ART). CONCLUSIONS: Three-quarters of infections came from undiagnosed infections or from outside Brighton. Of those from diagnosed infections, transmission was significantly associated with RHI, STI and viral load, and reduced by effectively-taken ART. These results demonstrate that effective behavioural interventions to increase HIV testing and improve ART uptake and adherence will reduce onward transmission.

The activation of B cells, a type of lymphocytes whose main function is to produce antibodies during an immune response, is tightly controlled by antigen binding to the cell surface B-cell receptor (BCR). The present thesis investigates the early cellular and molecular events that regulate B cell activation by membrane-bound antigen, which likely represents the predominant form of antigen encountered by B cells in vivo. Upon recognition of such membrane-bound antigen, B cells undergo a spreading and contraction response that serves to aggregate antigen for subsequent extraction and ultimately determines the extent of B-cell activation. Using planar lipid bilayers and advanced microscopy in combination with genetic and pharmacological approaches, I have characterised the key events and the molecular mechanism of this cellular response. I show that B-cell spreading is propagated through successive rounds of phosphotyrosine signalling and actin polymerization, proportionally to antigen density and affinity. Downstream of the BCR, I identify Lyn, Syk, phospholipase C g2 (PLCγ2) and Vav as the key intracellular players involved in this process. I have visualized, with high definition, the rapid recruitment of signalling molecules such as PLCγ2 to antigen micro-clusters formed across the contact site and demonstrate that they are incorporated into spatially defined ‘microsignalosomes’. The assembly of these micro-signalosomes is sequential and highly coordinated and the presence and correct functioning of all component parts is required for the spreading response to proceed. Results also show that PLCγ2 and Vav cooperate from within micro-signalosomes and they further synergize with the co-receptor CD19 to amplify the spreading response. Finally, the thesis addresses the functioning of cytoskeletal effector pathways downstream of the BCR and I identify a partial role for Rap and Wiskott Aldrich syndrom protein (WASp) in the spreading response, as well as their critical requirement for inside-out integrin activation and immunological synapse (IS) formation.

The ability to manipulate the immune system to induce protection against tumour, is one of the most fascinating challenges in immunology. In this regard, TCR gene transfer is an attractive and powerful strategy to generate high numbers of tumour antigen-specific T cells for adoptive transfer treatment. This thesis describes the optimization of lentiviral vectors for TCR gene transfer in the absence of polyclonal activation of the transduced T cells, which may improve subsequent adoptive T cell therapy. The murinised and codon optimised chains of an HLA-A*0201-restricted TCR specific for Wilms` tumour antigen 1 were cloned in lentiviral vector constructs improved with a Leader sequence and the WPRE elements for redirecting T cells specificity. The effects of common gamma chain receptor cytokines IL-2, IL-7, IL-15 and IL-21 were investigated using WT1 TCR-transduced T cells for transduction efficiency, proliferative potential, phenotype and functional activity in response to cognate antigen. Although all cytokines tested allowed transduction, stimulations with IL-15 and IL-15 with IL-7 or with IL-21 promoted a higher efficiency. Expression analysis of CD28 and CD62L showed an important role of IL-21 in maintenance of a naïve phenotype. In addition, all cytokines promoted maintenance of “quality” of T cells as shown by co-expression of IL- 2, IFN-γ and TFN-α after specific stimulation. To further sustain the in vitro results, several in vivo models were tested. Consistently, using F5 transgenic mice recognizing the NP peptide presented on EL4-NP cell line, IL-15 with IL- 21 exposed CD8+ T cells were able to efficiently protect against tumour and to persist longer in tumour bearing mice compared to IL-2 treated T cells. Because previous reports demonstrated that efficient LN homing of T cell correlates with efficient tumour protection in vivo, an imaging approach to study the molecular signalling in vivo during T cell activation in the LN has been developed. In conclusion, in this thesis, it is demonstrated that lentiviral transduction of cytokine exposed T cell can improve gene therapy approach of adoptive therapy.

Restriction factors interfere with retroviral infection, blocking the viral life cycle after cell entry, but prior to integration into the chromosomal DNA. Fv1 restricts murine leukaemia virus (MLV), whereas TRIM5α (T5) restricts human immunodeficiency virus (HIV), and other retroviruses. The mechanism of restriction is still poorly understood, but a key factor is the interaction between restriction factors and the viral capsid (CA). Development of reliable and efficient binding assays would greatly enhance our understanding of this process. This thesis describes the development of a novel MLV binding assay. Such an assay has been described for HIV, using in vitro polymerised CA-NC and lysate from cells expressing T5 (Stremlau et al., 2006), however, initial attempts to use this method to explore binding of T5 found the assay to be somewhat unreliable. The interaction between T5 and HIV CA is thought to be one of low affinity, and so binding could be aided by using a higher concentration of T5. Following the purification of T21T5 fusion protein from baculovirus, it was possible to demonstrate an interaction with HIV CA-NC. In contrast to T5, binding of CypA, Fv1Cyp and T5Cyp to HIV CA-NC could be consistently demonstrated, regardless of whether cell lysate or purified protein was used. To generate an MLV binding assay, it was first necessary to develop a novel method for the in vitro polymerisation of CA. By incubating HIS-tagged CA protein with Ni-chelating lipid nanotubes it was possible generate CA-coated tubes. Analysis of these tubes by electron microscopy confirmed that the CA molecules were arranged in a regular lattice, and using these CA-coated tubes, it was possible to show an interaction with Fv1. Investigation of a number of Fv1 and CA variants revealed a good correlation between restriction and binding specificity. It was also possible to confirm that multimerisation of both Fv1 and CA were necessary requirements for a binding interaction to occur. With further development, it should be possible to use this assay to investigate the binding for other retroviral CAs.

Standard marginal structural models (MSMs) are commonly applied to estimate causal effects in the presence of time-dependent confounding; these may be extended to history-adjusted MSMs to estimate effects conditional on time-updated covariates, and dynamic MSMs to estimate e¤ects of pre-speci…ed dynamic regimes (Cain et al., 2010). We address methods to assess the optimal time for treatment initiation with respect to CD4 count in HIV-infected persons, and apply these to CASCADE cohort data. We advocate the application of all three types of MSM to address such causal questions and investigate gaps in the literature concerning their application. Of importance is the construction of suitable inverse probability weights. We have structured this process as four key decisions, de…fining a range of strategies; all demonstrated a bene…ficial effect of ART in CASCADE. We found a trend towards greater treatment bene…fit at lower CD4 across a range of models. Via large simulated randomised trials based on CASCADE data, longer grace periods (permitted delay in treatment initiation) and in particular less-frequently observed CD4 indicated higher optimal regimes (earlier treatment initiation at higher CD4), although similar AIDS-free survival rates may be achieved at these higher optimal regimes. In realistically-sized observational simulations, the optimal regime estimates lacked precision, mainly due to broadly constant AIDS-free survival rates at higher CD4. Optimal regimes estimated from dynamic MSMs should be interpreted with regard to the shape of the outcome-by-regime curve and the precision. In our clinical setting, we found that allowing a 3-month grace period may increase precision with little bias under the interpretation of no grace period; under longer grace periods, the bias outweighed the efficiency gain. In our CASCADE population, immediate treatment was preferable to delay, although estimation was limited by relatively short follow-up. Comparison across the MSM approaches offers additional insights into the methodology and clinical results.