HLA-B27 is a Major Histocompatibility Complex (MHC) class I molecule which exhibits a strong association with the inflammatory arthritic disorder Ankylosing Spondylitis (AS). It has been postulated that the tendency for HLAB27 to misfold and form disulphide linked heavy chain homodimers may contribute to AS pathogenesis. However, it remains elusive how these aberrant forms occur and to which extent they participate in AS development. I have analysed the contribution of conserved cysteine residues at positions (p) 101, 164, 203 and 259 and unpaired cysteines at p67, 308 and 325, in the formation of ER resident heavy chain homodimers. I demonstrated that HLA-B27 dimerisation involves a hierarchy of disulphide bonding. Moreover, my study indicates that heavy chain-dimers are composed of multiple species, with each possibly exhibiting varying degrees of folding/unfolding. I found that HLA-B*27:05 adopts novel in vivo conformations within the ER lumen, which have yet be to identified for other HLA alleles. Detection of these conformations depends on cysteine residue exposure to the ER environment what can explain the propensity for heavy chains to misfold. Finally, I investigated the role of the MHC class I antigen presentation pathway in HLA-B27 dimerisation. I found that HLA-B27 retains its tendency to dimerise despite the presence of β2m and high affinity peptide and both β2m-free (unfolded) and β2m+peptide-associated (folded) homodimers can associate with the Peptide Loading Complex. Moreover, the tendency for HLA-B27 to dimerise is influenced by p116 within the peptide binding groove, which is a natural polymorphism within AS-associated and non-AS–associated HLA–B27 subtypes. These studies may have implications for inflammatory disease since they begin to provide a biochemical understanding for the ability of HLA-B27 to misfold.

The introduction of highly active antiretroviral therapy (HAART) has led to significant improvements in survival and morbidity for HIV-positive patients. Though HIV can now be well managed with treatment, interlinked barriers to successful treatment are still prevalent. In particular, low adherence to therapy, resistance to drugs and drug toxicity can have a considerable impact on the success of HAART. The potential of HAART is limited from the outset if patients are infected with a drug-resistant virus. Evidence suggests that a small minority of patients are starting HAART with drugs that the virus is resistant to, and consequently, are less likely to achieve virological suppression. A large proportion of resistance tests performed after patients‟ start HAART are not followed by a switch within 4 months of the result being received. This raises questions as to why the test was performed and whether limited future drug options are of concern. A single abnormal laboratory value may be the result of random fluctuations and may not necessarily be a reason for concern over drug toxicity. I derive a more stringent definition of an ALT flare and compare this definition with that commonly found in the literature. Treatment interruptions, perhaps due to drug toxicity, should not take place when the viral load is detectable. Patients who have achieved viral suppression after interrupting treatment and who have failed a higher number of HAART regimens are at a greater risk of viral rebound, though this risk is reduced substantially as duration of suppression increases. A score to characterise laboratory abnormalities is derived and used to predict mortality. Several methods were used; I felt the most appropriate was that based on the estimates from a regression model in which the current laboratory measurements were fitted; only three routinely measured laboratory measures were needed to calculate the score.

X-linked Severe Combined Immunodeficiency (X-SCID) caused by mutations in the common cytokine receptor γ chain (γc) is a fatal disease in which the immune system is severely compromised as T and NK cells are absent or profoundly diminished. Although dendritic cells (DCs) are key antigen presenting cells and are crucial for the induction of normal immune responses, little is known about their function in this disease. This is of specific importance as the myeloid compartment, including DCs, frequently remains of host origin, and therefore γc-deficient, after bone marrow transplant or gene therapy for this form of SCID. Curiously, even after curative treatment, patients with X-SCID continue to have a long-term susceptibility to human papilloma virus infection. We hypothesised that this could be due to persistent myeloid defects. Using a murine model of X-SCID, we demonstrated that, in the absence of functional γc, DCs are less efficient at priming antigen-specific responses in γc-expressing CD4+ T cells. Despite the widely held view that IL-15Rα is the chain responsible for IL-15 transpresentation to NK and CD8+ T cells, here we have shown that expression of the γc by DCs is necessary for effective transpresentation to CD4+ T cells. In a novel system of supported planar bilayers mimicking the T cell interface, we demonstrated that DC-expressed γc colocalises with MHC II clusters and mediates IL-15Rα recruitment to the immunological synapse (IS). This depended on simultaneous TCR engagement with MHC-bound antigen, suggesting that IL-15 transpresentation may have important costimulatory function for T cell activation. Altogether, these data indicate that γc-deficiency impairs DC functions and reveal a novel mechanism for recruitment or stabilization of DC IL-15/IL-15Rα complexes at the IS leading to localized transpresentation of IL-15. The contribution of myeloid γc-deficiency to persistent immunodeficiency in transplanted X-SCID patients warrants further investigation.

The AIDS epidemic is a major global health problem with an estimated 33.2 million infected with HIV, mainly in Sub-Saharan Africa. Effective drugs have been developed that are targeted against selected viral proteins but HIV mutates very rapidly and invariably becomes resistant to available drugs, even when they are used in combination. A complementary, yet little explored possibility is to develop new drugs that target and block cellular factors necessary for virus replication. Host factors mutate less rapidly and hence HIV-1 drug resistance may be less likely to emerge. During my Ph.D. project I have performed a small chemical screen of inhibitors of ATP-dependent DNA motors to test their ability to inhibit HIV infection. The approach called “chemical genetics” consists in screening a library of small compounds for their ability to inhibit HIV infection, the identification of the step of the viral cycle inhibited, the identification and validation of the target and the improvement of the compound potency by chemical modification and, where possible, rational drug design. The screen revealed that Coumermycin A1 (C-AI), a gyrase B inhibitor coumarin antibiotic, potently suppressed HIV-1 infection. CA1 impaired HIV-l integration and early gene expression in a Tat-independent way by targeting two host factors. A search for conserved ATPase domains capable of binding coumarin antibiotics revealed several potential targets, including DNA Topoisomerase II and Hsp9O. Topoisomerase II played no significant role in HIV-1 infection, however Hsp9O was required for early gene expression. Docking studies revealed two C-A1 binding pockets in the C-terminus of Hsp9O. The sensitivity to C-Al-mediated inhibition of viral gene expression mapped to p24 CA protein, suggesting that CA may affect Hsp90 function to prime cells for infection. Coumermycin Al, developed by Roche in the early 1970s shows a good pharmacological profile but is not efficiently adsorbed by the gastrointestinal tract needing therefore structural improvement to be considered as anti-HIV drug. Hsp90 inhibitors might be used as antiretrovirals in selected AIDS patients.

It is estimated that one third of the world‘s population are infected with the bacterium M. tuberculosis and approximately 9.4 million new cases of tuberculosis were diagnosed globally in 2008. Molecular tools, developed over the previous two decades, have allowed further in-depth study of this historic disease. Genotyping M. tuberculosis allows the study of evolutionary relationships and well as the routes of transmission of the organism between hosts. The pairing of genotyping with demographic data allows the analysis of the current trends of disease within a given patient population. Two genotyping methodologies (IS6110 RFLP and Mycobacterial Interspersed Repetitive Units – Variable Number Tandem Repeats (MIRU-VNTR)), alongside patient demographic data have been utilised for the TB population at the Royal Free Hospital, London. The data shows a patient population that is largely concordant with that seen in London as a whole; with the majority of TB patients being born outside of the UK and drug resistance rates that are higher than the national average. The acquisition of drug resistance-conferring mutations by M. tuberculosis is often presumed to be associated with a fitness cost. Here we investigate the fitness of isolates from two outbreaks involving large numbers of drug resistant strains. The first group of strains was found to be part of the ongoing north London isoniazid resistant outbreak. The data suggests that this outbreak consists of successful, closely related, circulating strains with heterogeneous resistance profiles and mutations and little or no associated fitness cost. The relationship between the biology of the organisms isolated from a cohort of TB patients who underwent a novel diagnostic interferon gamma release assay was investigated. Genotyping, ESAT-6 gene sequencing and gene expression assays suggest that varying immune response between individuals is driven by host factors rather than a characteristic of the strain of M. tuberculosis. This study shows that this geographic location in London is home to a diverse population of M. tuberculosis with a low rate of transmission. The demographic characteristics of the TB patients are largely concordant with the UK as a whole. These findings show that M. tuberculosis genotyping is critically important in the identification of suspected outbreaks and contamination events as well as the investigation of successful related strains and characteristic disease phenotypes. Genotyping can augment classic epidemiology and clinical practice to provide a holistic approach in the investigation, treatment and control of the global health problem. Understanding the biology and genomics of M. tuberculosis is how the enormous disease burden will be tackled.

Toxicity of keratinocytes, the major cell type in the epidermis, is found in several skin inflammatory diseases. Environmental influences such as chemicals, UV light, mechanical insult, or extreme temperatures can cause necrosis of keratinocytes leading to failure of local homeostasis, breaching of the skin barrier, and the potential for infection. In such situations dermal dendritic cells (DCs) may be the important sentinels by responding to danger signals released by the dying cells and potentially activating the adaptive immune system. The aim of this project is the development of a human two cell model to study the influence of skin sensitisers and irritants on DCs in the presence of keratinocytes and test the hypothesis that DCs can respond directly to keratinocyte damage via the release of Interleukin-1 alpha (IL-1α) from the dying cells. The coculture of sensitiser/irritant treated HaCaT cells with human monocytederived DCs led to activation (upregulation of cell surface markers and cytokine/chemokine production), and an increase in T cell stimulatory activity for the sensitisers and the irritants. The supernatant produced by sensitiser and irritant-labelled HaCaT cells was sufficient to induce DC maturation, again showing no difference between sensitisers and irritants. Both HaCaT cells and primary keratinocytes (EpiDermTM) released IL-1α after chemical exposure. IL-1α neutralising antibodies attenuated DC maturation induced by chemical-treated HaCaT supernatants. Furthermore the effect of IL-1α on DCs was studied. Our in vitro model studying the interaction between keratinocytes and DCs suggests that both sensitisers and irritants can lead to cell damage in the skin which is associated with the release of IL-1α and possibly other danger signals. These can activate DCs in the underlying dermis and lead to activation of the adaptive immune system causing the clearance of infections or resulting in allergic reactions.

A study of the sequence immediately upstream of the Fv1 ORF predicted several transcription factor binding motifs and multiple transcription start sites, suggesting the presence of a core promoter. This was confirmed using in vitro assays with a Luciferase reporter, which demonstrated that this sequence could drive transcription. Promoter activity could also be detected in the antisense direction, prompting the hypothesis that Fv1, which is derived from an endogenous retrovirus, was formed by insertion of an open reading frame into the mouse genome, ‘hijacking’ the promoter of the upstream gene, Miip, to form a novel bidirectional gene pair. The SC-1 and 3T3-FL cell lines were described in the 1970s as being dually sensitive to both N- and B-tropic MLV. An analysis of these cell lines revealed that they both encode a functional Fv1n protein but that transcription of Fv1 was repressed. It was hypothesized that this was due to inhibitory epigenetic marks on the Fv1 promoter. It was also shown that Fv1 is constitutively expressed at very low levels, not requiring up-regulation by viral infection or interferon in order to display antiretroviral restriction. To study the relationship between Fv1 expression levels and virus restriction, a number of constructs were developed that expressed Fv1 under the control of the natural promoter. In addition, a tetracycline-inducible Fv1 system was also developed. With these systems the low concentration requirements for Fv1 were confirmed. Moreover, an interesting difference between the n and b alleles was revealed. Whereas Fv1n appears only to repress B-tropic MLV, Fv1b restricts a broader range of viruses when expressed at higher concentrations.

The pir genes comprise the largest multi-gene family in Plasmodium, with members found in P. vivax, P. knowlesi and rodent malaria species. Despite their almost universal presence, little is known about the functions of the PIR proteins. To investigate the role PIR proteins play during the erythrocytic stages of infection, the P. chabaudi model was chosen, where this gene family is termed cir. 198 cir genes were identified in the P. chabaudi genome, 86% of which clustered to form two major sub-families on the basis of sequence similarity. Quantitative RT-PCR and Illumina RNA sequencing were used to investigate cir transcription during P. chabaudi infection. Both methods detected many cir genes transcribed at low levels, as shown previously for other pirs. Three of the transcribed cir genes were selected for recombinant protein expression in the yeast Pichia pastoris: PCHAS_000100, PCHAS_070130 and PCHAS_040110. Soluble PCHAS_000100 was used for measurements of CIR secondary structure. Conserved and sub-family specific peptides were also synthesized. Antibodies present in the sera of P. chabaudi immune mice recognized all CIR proteins and peptides. Polyclonal antibodies were used to determine CIR localization by confocal microscopy and flow cytometry. Whilst most CIRs were located within the parasites, some CIRs were also found on the infected erythrocyte surface of mature trophozoites. In addition, CIRs were detected at the apical end of merozoites. These results imply that CIR proteins are exposed to the immune system during P. chabaudi infection and are antigenic, yet immunization with most CIR proteins and peptides did not protect mice from P. chabaudi infection. Upon P. chabaudi challenge of mice immunized with CIR sub-family specific reagents, increased levels of cir transcripts belonging to the other major sub-family were detected. This may explain why few differences in parasitaemia were observed. The exception was observed during P. chabaudi challenge of mice immunized with PCHAS_000100, which were able to clear parasitaemia earlier than controls.

Vaccines that induce T cells which recognize conserved viral proteins could confer cross-strain protection against pathogens with fast-mutating B cell epitopes. Influenza is an example of such a pathogen for which there is a pressing need for a universal vaccine. Lentiviral vectors are a counterintuitive choice as vaccines since they have low inherent immunogenicity. However, their efficient transduction of non-dividing cells and high capacity permits transduction of antigen presenting cells with not only antigen but also molecular adjuvants that directly or indirectly enhance the T cell response. We therefore investigated the potential of two such adjuvants: viral flice-like inhibitor protein, which activates dendritic cells through nuclear factor kappa-B, and 4-1BB ligand, which activates T cells directly through 4-1BB. By co-encoding these with influenza nucleoprotein, we have shown that the influenza-specific T cell response to lentiviral vector vaccination is significantly enhanced in mice. Furthermore, we have demonstrated that intranasally delivered lentiviral vectors transduce alveolar macrophages with high efficiency, recalling and expanding large and sustained populations of nucleoprotein-specific CD8+ T cells in the lung and airway in mice that have been primed subcutaneously or previously exposed to influenza. These lung-resident T cell populations persist for at least 4 months and are sufficiently abundant to rapidly control a mouse-adapted lethal influenza challenge without invocation of a secondary cytokine response, weight loss or lung injury. Furthermore, dendritic cells expressing 4-1BBL potently trans-activate bystander dendritic cells, both in vitro and in vivo, demonstrating an indirect mechanism by which the 4-1BBL:4-1BB signaling axis can enhance T cell responses.

Background: In the UK, men who have sex with men (MSM) continue to be disproportionately affected with HIV and sexually transmitted infections (STI). Due to the increasing emphasis on using biomedical strategies like cART for prevention of sexual transmission of HIV, I examined HIV positive MSM’s sexual partnerships and behaviours; their attitudes towards biomedical and behavioural HIV transmission risk reduction strategies, and their association with sexual behaviour. I also examined their attitudes towards partner notification for STI, willingness and preferred methods to notify partners of STI in the future. Methods: 429 HIV positive MSM attending a central London clinic completed a computer assisted self-interview on sexual partnerships and behaviours, attitudes, preferences and willingness to notify partners for STI in the future. 24 purposively selected men participated in in-depth interviews. Results: Of 429 men, 380 men had been sexually active in the last year. The survey data showed that the prevalence of unprotected anal intercourse (UAI) with a serodiscordant (i.e., HIV negative or unknown status) primary partner and the most recent non-primary partner was high, 18.3% and 16.9% respectively. A substantial minority of men had positive attitudes towards biomedical and various behavioural HIV transmission risk reduction strategies. Duration of partnership, recreational drug use, and belief that undetectable viral load reduces the risk of HIV transmission during UAI were all independently associated with HIV transmission risk behaviours with a serodiscordant primary partner. Disclosure of HIV status and recreational drug use during sex were independently associated with UAI with the most recent serodiscordant non-primary partner. Of the 258 men who had new partners, 53% had engaged in UAI with new partners. Prevalence of anonymous partnerships was high. Stigma associated with HIV/STI diagnosis, and the venues for meeting sexual partners also influenced men’s sexual partnerships and behaviour, and disclosure of HIV status. Approximately one in five sexually active men had not tested for STI and 25% of men had been diagnosed with STI in the last year. Young age; self-reported detectable viral load status; greater number of new anal sex partners; UAI with new and concurrent partners; having a seroconcordant primary partner; frequency of engagement in group sex were independently associated with STI diagnosis in the last year. The qualitative data highlighted that the majority of men felt an emotional responsibility towards and acknowledged the personal health benefits of notifying primary and regular partners of STI. A greater proportion of men would be less willing to notify casual partners of STI in the future (21%) compared to a primary partner (5.3%) and regular partners (7.5%). Attitudes such as ‘it is not my responsibility to notify partners of STI’, and the lack of previous experience of notification were independently associated with unwillingness to notify casual partners of STI in the future. The qualitative study indicates that the lack of emotional responsibility; fear of stigma and breach of HIV-related confidentiality due to partner notification; and fear of criminalisation for HIV/STI transmission were barriers to notifying sexual partners of STI, especially casual and group sex partners. Patient-referral was the most preferred method of notifying partners of STI in the future, particularly a primary partner; whereas there was greater willingness for notifying regular, casual, and group sex partners using remote self-led methods, provider referral or an anonymous e-card. The acceptability of sending an anonymous e-card and taking a home sampling kit for partners, and telephone assessment of partners for STI by clinic staff was low to moderate. Conclusion: The findings of this study underscore the need for sustained interventions to ensure sexual health of HIV positive MSM and prevent HIV/STI in MSM. They highlight that cART should be offered to sexually active HIV positive MSM, especially those in serodiscordant partnerships irrespective of CD4 cell count to minimize the risk of onward HIV transmission in this population. Frequent STI testing of sexually active men should be integral part of routine HIV care. Various partner notification choices should be offered to those diagnosed with STI. Interventions to reduce stigma associated with homosexuality and HIV continue to remain vital in this population. Research examining the feasibility, acceptability, effectiveness, and cost effectiveness of integrating brief behavioural interventions to enhance regular STI testing, adherence to cART, address recreational drug use and mental health needs, and promote safer sex with routine HIV care is urgently needed.