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 -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

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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 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-1_JRCSF 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.

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.

PhD (Microbiology) / Department of Microbiology

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.

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.