This study examines, from a modelling point of view, the dynamics of infectious diseases in wildlife caused by macroparasites and by tick-borne infections. The overall aim was to investigate the important role played by parasite aggregation in the dynamics of both systems. For macroparasites we first developed some deterministic models that incorporate explicit mechanisms for generating aggregation in parasite distribution, specifically multiple infections and host heterogeneity. We explored the role of aggregation in host regulation and in determining a threshold value for parasite establishment. A large aggregation makes it more difficult for parasites both to regulate hosts, and to get established in a population at carrying capacity. Furthermore, the stabilization yielded by aggregation strongly depends on the mechanism that produces the aggregation. We then introduced some uncertainties into the host-macroparasite system, presenting an individual-based stochastic model that incorporated the same assumptions as the deterministic model. Stochastic simulations, using parameter values based on some real case studies, preserved many features of the deterministic model, like the average value of the variables and the approximate length of the cycles. An important difference is that, even when deterministic models yield damped oscillations, stochastic simulations yield apparently sustained oscillations. The amplitude of such oscillations may be so large as to threaten the parasites’ persistence. With respect to tick-borne diseases we presented a general model framework that incorporated both viraemic and non-viraemic routes of infections. We compute the threshold for disease persistence and study its dependence on the parameters and on host densities. The effects of tick aggregation and correlation between different tick stages on the host have both an important effect on infection persistence, if non-viraemic transmission occurred. In the case of Lyme Disease and Tick-borne Encephalitis (TBE) in Trentino (northern Italy) we showed some numerical results, using parameter estimates based on a detailed field study, and explored the effects of uncertainty on the endemic equilibrium of both diseases assuming only viraemic transmission for Lyme Disease while for TBE we permitted only non-viraemic transmission through co-feeding ticks. In conclusion we have examined the patterns and changes of aggregation in a number of contrasting systems and believe that these studies highlight both the importance of considering heterogeneities in modelling host-parasite interactions and, more specifically, modelling the biological mechanisms that produce aggregation in parasite distributions.

In humans, protein-coding genes and most small nuclear (sn)RNA genes are transcribed by RNA polymerase II (pol II).The carboxy-terminal domain (CTD) of the largest subunit of pol II possesses multiple heptapetide repeats of the consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Phosphorylation of Ser2, Ser5 and Ser7 mediates the recruitment of transcription and RNA processing factors during the transcription cycle. There are notable differences between snRNA genes and protein-coding genes in terms of mechanisms controlling their expression. Pol II does not appear to make the transition to long-range productive elongation during transcription of snRNA genes, as happens during transcription of protein-coding genes. In addition, recognition of the snRNA gene-type specific 3' box RNA processing element requires initiation from an snRNA gene promoter. These characteristics may, at least in part, be driven by factors recruited to the promoter. Initiation of transcription of most human genes transcribed by pol II requires the formation of a preinitiation complex (PIC) comprising TFIIA, B, D, E, F and H and pol II. The general transcription factor, TFIID is composed of the TATA-binding protein and up to 13 TBP-associated factors (TAFs). Differences in the complement of TAFs might result in differential recruitment of elongation and RNA processing factors. It has already been shown that the promoters of some protein-coding genes do not recruit all the TAFs found in TFIID. Although TAF5, has been shown to be associated with pol II-transcribed snRNA genes, the full complement of TAFs associated with these genes remained unclear. Here I show, using a ChIP and siRNA-mediated knockdown approach, that the TBP/TAF complex on snRNA genes differs from that on protein-coding genes. Interestingly, the largest TAF, TAF1 and the core TAFs, TAF10 and TAF4 are not detected on snRNA genes. I propose that this snRNA gene-specific TAF subset plays a key role in gene-type-specific control of expression. In addition, in order to further understand the molecular mechanism underlying the differences between expression of protein-coding genes and snRNA genes, I have investigated the role of RNA pol II-associated protein 2 (RPAP2) in transcription of snRNA genes. Here I show that RPAP2 recognizes the phospho-Ser7 mark on the pol II CTD, siRNA mediated knockdown of RPAP2 causes defects in snRNA gene expression and that RPAP2 is a CTD Ser5 phosphatase. I also present my studies of the mechanism of inhibition of phospho-Ser2 by herpes simplex virus-1 (HSV-1) protein ICP22. Phosphorylation of Ser2 by the positive transcription elongation factor (P-TEFb) is associated with productive transcriptional elongation. However, P-TEFb is not required for elongation of transcription of snRNA genes, but functions only to activate 3' box-directed RNA processing. In addition, there are conflicting data as to whether Cdk9 is acting as a Ser2 kinase during transcription of pol II-transcribed snRNA genes. As ICP22 is thought to inhibit P-TEFb, this protein could provide an alternative means to study P-TEFb function in expression of snRNA genes.

PARV4 is a novel human parvovirus initially identified in an intravenous drug user at risk of HIV infection. PARV4 is a small single stranded DNA virus principally absent from the general population, but common in HCV- and HIV-infected individuals. Until 2009, most published PARV4 studies related to the prevalence of PARV4 in various risk groups. PARV4 has been detected in the liver of HCV-patients and the bone marrow of HIV-patients. Parvovirus B19, the closest related virus, elicits a strong immune response and can cause serious disease. Thus, this project was initiated to characterise the immune response to PARV4, and investigate the clinical significance of this virus. Cohorts of HCV-infected, HIV-infected, HCV-HIV co-infected, healthy and acute parvovirus B19-infected individuals were screened for humoral and cellular responses in both acute and chronic PARV4 infection. HCV- and HIV-related disease progression was also assessed relative to PARV4 infection. This study demonstrates that the highest prevalence of PARV4 infection is found in HCV-HIV co-infected intravenous drug users, and provides additional evidence for parenteral transmission. I present here the first data on the cellular immune response to PARV4 in acute and chronic infection and define PARV4 as a persistent virus. Although no clear correlation could be found between PARV4 and HCV or HIV disease progression, the high prevalence rates emphasize the importance of investigations into emerging infections such as PARV4.

HCV infects 170 million persons worldwide and is a serious global health problem. Genotype-3a is the dominant genotype in newly diagnosed infections within the UK and has a high response rate to interferon therapy, with up to 70% patients achieving a sustained virological response (SVR). The reason(s) for this are unknown; therefore the aim was to assess host and viral factors that determine treatment outcome of subtype-3a infection. Full-length subtype-3a viral sequence analysis identified 2 novel regions of hypervariability within E2 - HVR495 and HVR575, that are subject to positive selection pressure. A 5 amino-acid insertion found only in subtype-3a and a putative glycosylation site were contained within HVR575. These data suggest that HVR495 and HVR575 may serve as major antigenic sites in subtype-3a HCV infection. Successful treatment of chronic subtype-3a infection was not associated with pre-treatment quasispecies diversity and complexity, PePHD, HVR495 or HVR575 sequence. Different patterns of quasispecies variation were observed in patients that failed treatment. Subtype-3a specific CD8+ T-cell responses in chronic infection target non-structural proteins, in contrast to pre-dominant genotype-1 core-specific CD4+ T-cell responses. SVR was associated with a decline in subtype-3a specific and non-specific T-cell responses, and also total lymphocyte counts, which all recovered after treatment. These data do not support the theory that clearance of subtype-3a is associated with an enhancement of antiviral T-cell responses. Overlapping peptides detected a greater number of subtype-3a T-cell responses compared with peptides representing putative predicted CD8 epitopes. Therefore subtype-3a HCV is distinct from genotype-1 in terms of genome sequence, effect of treatment on quasispecies and subtype-3a specific T-cell responses, further emphasising the importance in understanding this distinct subtype.

Neutralising antibodies and antigen-specific B cells are important for protection against influenza A virus. However, the antigenic evolution of influenza A viruses has made a continuing challenge to the design of vaccine and the public health. The ability to generate cross-reactive response against influenza remains unclear in human. It is important to explore the antibody and B cell repertoire at single cell level. The pandemic H1N1 and seasonal influenza vaccine induced robust antibody response in adults. However, pre- or co-vaccination with the seasonal vaccine led to a significantly reduced antibody response to pandemic H1N1 virus. Whether this interference has impact on subsequent infection rates remains undetermined. There observed substantial cross-reactive antibody response upon vaccination, as measured by HI, MN and B cell ELISpot assays. The antibody recognizing conserved proteins could be the main component of cross-reactivity against influenza A strains and subtypes. A significant expansion of influenza-specific MBC was observed after infection. Crossreactive response was also noted in the MBC response. Importantly, a robust early-phase ASC response was detected in the peripheral blood upon influenza vaccination or infection. The size of ASC response significantly correlated with serum HI, MN and anti-HA IgG titre three weeks after vaccination. The sequence analysis revealed that early-phase ASC accumulated high level of somatic mutations on Ig variable region and affinity maturation, as well as anti-influenza mAb, which suggested their origin from pre-existing MBC. Eight anti-influenza mAb were made from early-phase ASC, including one high-titre virus-neutralising HA1-specific, two other HA1-specific, one cross-reactive HA2-specific, and four cross-reactive NP-specific antibodies, indicating of the broad diversity of ASC repertoire. In conclusion, this study demonstrated the properties of antibody and B cell responses to influenza A virus at serological, cellular and sequence level. The virus-neutralising and cross-reactive mAb derived from ASC could have therapeutic potential and their analysis might direct the vaccine design in the near future.

Protective immunity against influenza virus infection is believed to be mediated by neutralising antibodies. Despite substantial evidence in animal models which suggests critical roles for T cells in viral clearance, the precise role of cellular immunity in human influenza immunity remains uncertain. The first aim of this project was to determine cellular immune responses in seronegative human volunteers following nasal challenge with live seasonal H3N2 or H1N1 viruses. T cell responses before and during infection were mapped. A large increase in both breadth and magnitude in influenza-specific CD4⁺ T cell responses was seen in the blood by day 7, when virus was completely cleared from nasal samples and serum antibodies were still undetectable. These acutely expanded T cells were shown to be highly activated (CD38⁺) and proliferating (Ki-67⁺). Pre-existing CD4⁺ T cells, but not CD8⁺, specific to internal proteins nucleoprotein and matrix proteins, were associated with lower virus shedding and less severe illness. These influenza-specific T cells also responded to A/California/07/2009 (H1N1) peptides, were able to kill antigen-loaded autologous B cell lines in vitro and were polyfunctional in cytokine production. The second aim was to assess the cellular immune responses to unadjuvanted, inactivated seasonal and pandemic A/California/07/2009 (H1N1) influenza vaccines. 151 healthy adult volunteers were vaccinated: modest influenza-specific T cell responses were induced, and specific responses to HA and NA peptide pools were found to be mediated by CD4⁺ T cells. Activated and proliferating cells induced during vaccination were found to be of a central memory phenotype. Lastly, I explored the link between antibody production and CD4⁺ T cells. The ability of CXCR5⁺ CD4⁺ T cells from peripheral blood to support antibody production was examined and antigen-specific CD4⁺ T cells with helper functions could be found in the peripheral blood of healthy volunteers with memory influenza-specific T cells. This thesis suggests that influenzaspecific CD4⁺ T cells have an important role in limiting the severity of influenza infection in the absence of specific antibody responses through a number of mechanisms. This work provides information on how cellular immunity can be targeted in conferring broad protection against different subtypes of influenza A viruses in the development of universal flu vaccine.

The primary approach to the control of meningococcal disease remains effective vaccination programmes in susceptible populations. Vaccines against serogroups A, C, W and Y offer broad protection against meningococci and both polysaccharide and conjugate quadrivalent vaccines are licensed for use in the UK. Previous studies have assessed the antibody response to meningococcal polysaccharide and conjugate vaccines, but there is limited information on the nature of the B cell response to these antigens. As part of a clinical trial using both polysaccharide (MenACWY-PS) and conjugate (MenACWY-CRM) vaccines in adult volunteers, this DPhil reports the analysis of subsets of antigen specific B-cells produced in response to either vaccine. Prior MenACWY-PS impaired the response to a subsequent dose of MenACWY-CRM. This may be due to MenACWY-PS driving terminal differentiation of antigen specific cells into plasma cells, without replenishment of the memory B cell pool. In addition, despite prior data indicating that it may act as a thymus dependent antigen, the serogroup A polysaccharide component of MenACWY-PS appears to behave in the same way as serogroup C, W & Y polysaccharide components. Antibody molecules recognise and bind to a multitude of conformational epitopes. This variability is enabled by the complexities of immunoglobulin variable domain gene recombination which can generate a vast potential repertoire of unique antibody molecules. However, the diversity of the antibody repertoire is more restricted against specific antigens and within defined B cell subsets. In this DPhil, ‘next generation’ sequencing technologies were used to investigate the diversity of the B cell variable domain before and after vaccination of adult volunteers. Individuals at baseline were found to have distinct antibody repertoires. Vaccination with a Haemophilus influenzae type b (Hib) conjugate vaccine resulted in an oligoclonal antibody response, with enrichment for Hib specific canonical antibody sequences.

Human T lymphotropic virus type -1 (HTLV-1) infections are important causes of mortality and morbidity in endemic areas worldwide. There is neither a vaccine specific for the virus nor satisfactory treatment for the associated malignancy or inflammatory syndromes. HTLV-1 associated infective dermatitis (IDH) is a chronic dermatitis that has been observed in a variable proportion of HTLV-1 infected children. IDH may serve as an early clinical marker for HTLV-1 and an indicator of increased risk for developing other HTLV-1 associated conditions such as adult T cell leukaemia/lymphoma (ATLL) and HTLV-1-associated myelopathy or transient spastic paraparesis (HAM/TSP). However the mechanisms underlying IDH and the relationships with HAM/TSP and ATLL are poorly understood. We undertook skin biopsies from 14 cases with IDH, and controls which included five asymptomatic carriers (ACs) and 18 healthy uninfected individuals from South Africa. We conducted clinical assessments, proviral load, allergen-specific IgE, peripheral blood and cutaneous T cell and virological analyses. We obtained relevant clinical history and examined all cases and controls based on a pre-formed questionnaire. Despite the partial clinical similarities with atopic dermatitis, the individuals with IDH did not have an increased incidence of atopic disease including asthma or rhinitis. Furthermore house dust mite-specific IgE levels were not elevated in the cases compared to the controls, suggesting that atopy is not a predisposing factor for the development of IDH in HTLV-1 infected individuals. Circulating proviral load was significantly higher in those with IDH compared to asymptomatic carriers and skin biopsy revealed acanthosis, and lymphocytic epidermotropism associated with a superficial perivascular and periadnexal lymphocytic infiltration of CD8+, and CD4+ T cells. Furthermore IDH associated with an infiltrate of epidermal and dermal FoxP3+ T cells and lesional down-regulation of filaggrin expression compared to non-lesional skin. We did not observe an elevation of pro-inflammatory cytokines in the sera of individuals with IDH compared to the controls. We investigated integration patterns in the skin and blood of 10 cases with IDH, and two asymptomatic carrier (AC) individuals from South Africa. We first showed that the virus is present in the skin at high levels (total mean levels of 47.09 proviral copies per 1000 cells) as comparable to that which has been observed in blood (total mean levels 137 proviral copies per 1000 cells). Using a high throughput Illumina sequencing system in collaboration with Professor Bangham, we mapped and quantified the relationship between oligoclonal proliferation of HTLV-1 infected T cells in the skin and blood of IDH patients. It was found that in IDH, a selective outgrowth of certain clones is favoured, supporting the possibility of skin-specific factors exerting positive selection on proliferation. In IDH, there was not a preferential integration of the provirus in transcriptionally active regions of the gene sites, as had been observed in other HTLV-1 associated conditions. These observations imply that the selection forces that favour oligoclonal proliferation of HTLV-1+ T cells differ fundamentally between simple HTLV-1 infection and other events associated with the dermatitis. In conclusion, these data show that HTLV-1 is not associated with an atopic diathesis. Given the lack of elevated pro-inflammatory cytokines and presence of a cutaneous infiltrate of FoxP3+ T cells, the findings suggest that high levels of HTLV-1 replication promotes a regulatory environment leading to filaggrin down-regulation, cutaneous susceptibility to infection, and secondary inflammatory skin disease. Viral integration patterns would support the presence of skin-specific positive selection, perhaps eventually leading to expansion of particular clones with the potential to develop towards ATLL. It remains to be explained whether the high viral load in IDH changes over time, more specifically in the steps leading to ATLL.

The current worldwide epidemic of Mycobacterium tuberculosis infection is a huge global health problem. Widespread BCG vaccination remains a useful tool in combating this epidemic; however, its variable efficacy requires urgent development of novel vaccines against Mycobacterium tuberculosis. Such a candidate vaccine is a serotype 5 adenovirus expressing antigen 85A from M. tuberculosis (Ad85A). In animal models Ad85A confers significant protection when administered intra-nasally. The work in this thesis demonstrates that intra-nasal immunisation with Ad85A results in inhibition of M. tuberculosis growth in the lung early after infection, in contrast to the late inhibition induced by parenterally administered vaccines. Early inhibition correlates with the presence in the lung of a highly activated population of antigen-specific CD8 T cells, maintained for at least 6 months post-immunisation by persistent antigen. For intra-nasal Ad85A to be effective, the vaccine must be delivered into the lower respiratory tract, as immunisation targeting only the nasal-associated lymphoid tissue (NALT) does not result in protection. Following a change of animal facility, the lung immune response to intra-dermal immunisation with Ad85A increased and this route of immunisation now induced protection, though growth of M. tuberculosis was inhibited only late after infection. However, this response and protection can be altered by exposure to environmental mycobacteria. Further experiments showed that simultaneous respiratory and parenteral immunisations (SIM) act additively, where local lung immunity inhibits the growth of M. tuberculosis early after infection and systemic immunity protects later. SIM regimes generate greatly improved protection over either immunisation alone and do not depend on priming and boosting.

Background: Persistent cough following an acute respiratory tract infection is a challenging and frequently encountered problem in primary care. Mycoplasma pneumoniae (M. pneumoniae) and Bordetella pertussis (pertussis) particularly predispose patients to persistent cough. Whilst the incidence of M. pneumoniae is highest in children, pertussis may also occur in adults. Method: Four studies were conducted for this thesis. First, a systematic review to assess the diagnostic accuracy of symptoms and signs in the clinical recognition of M. pneumoniae. Second, a retrospective analysis of a cohort of children with persistent cough to assess the prognostic value of diagnosing M. pneumoniae. Third, a prospective cohort study to estimate the prevalence of M. pneumoniae and pertussis in children with persistent cough following recent changes in vaccination policy. Fourth, a double-blind randomised placebo-controlled trial to determine the effectiveness of montelukast in the treatment of persistent cough and pertussis-induced cough in adults. Results: M. pneumoniae and pertussis can each be found in one-sixth of children who present in primary care with persistent cough. Although coverage with the preschool pertussis booster vaccine is high, its efficacy wanes rapidly, with the likelihood of pertussis increasing by 30% per year after vaccination. Montelukast is not an effective treatment for persistent cough, but may be an effective treatment for pertussis-induced cough. Median duration of cough in children with M. pneumoniae is only one-third of that in children with pertussis (39 days versus 118 days). However, the diagnostic accuracy of symptoms and signs in the clinical recognition of M. pneumoniae is limited. Since M. pneumoniae occurs in cyclical epidemics, clinicians should consider current prevalence of M. pneumoniae when making a clinical diagnosis. Conclusions: Diagnosing M. pneumoniae and pertussis can help clinicians give patients an explanation for their cough and inform them about its likely prognosis. At the moment, clinicians should adopt a conservative approach to managing postinfectious persistent cough. A further trial is needed to assess the efficacy of montelukast for the treatment of pertussis-induced cough.