In recent years the glycan shield of the HIV-1 envelope spike (Env) has emerged as a potential target for microbicide and vaccine design. The densely packed glycans on its surface include an intrinsic population of under-processed oligomannose structures, and a number of lectins and broadly neutralising antibodies (bnAbs) have been isolated which are reactive to these ‘non-self’ glycan structures. The potential value of these agents in therapeutic or vaccine contexts depends upon the prevalence of their glycan targets in nature and their resilience to sequence mutation. Here the prevalence of oligomannose-type glycans on recombinant gp120 was demonstrated across a panel of isolates, revealing subtle cross clade differences. Alanine scanning of all potential N-glycosylation sites (PNGSs) within a model gp120 demonstrated the overall stability of the oligomannose population, but highlighted regions of glycan clusters where individual glycans act to limit the processing of their neighbours. This was formally demonstrated for the N332 ‘site of vulnerability’, where deletion of nearby glycosylation sites led to altered glycan processing at the N332 site. A panel of N332-dependent bnAbs was screened for their ability to tolerate such changes in glycan processing, with differing results. While some displayed promiscuous binding, others were more sensitive to glycan microheterogeneity. Site-specific glycosylation analysis of the PGT135 epitope revealed that an intolerance of certain glycoforms may explain its limited breadth. While a greater understanding of Env glycan microheterogeneity and bnAb promiscuity is required, these findings reveal insights into the architecture of the HIV-1 glycan shield that suggest it is a conserved and robust target for drug and vaccine design.

Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis (M.tb), remains a global health problem and although BCG offers some protection against childhood disseminated disease and other mycobacterial infections, its efficacy against pulmonary TB varies between 0 and 80%. Modified Vaccinia virus Ankara expressing antigen 85A (MVA85A) is a novel TB vaccine designed to boost mycobacterium-specific CD4+ T cell response primed by BCG. MVA85A induces strong interferon (IFN)-γ responses, a cytokine known to be essential for protection following M.tb infection. A strong IFN-γ response is not a correlate of protection and in terms of the adaptive response, interleukin (IL)-17 is emerging as an important cytokine following vaccination as it is thought to help boost IFN-γ production by CD4+ T cells. This thesis shows that MVA85A induces IL-17 in PBMC and whole blood of human BCG – MVA85A vaccinees. This is replicated in mice receiving BCG – MVA85A intranasally. The administration of cholera toxin (CT) with BCG enhances IL-17 and confers improved protection following M.tb challenge, which is partially dependent on IL-17 and on the mucosal route of administration. Since CT is not a suitable adjuvant in humans, an alternative IL-17-inducing pathway was investigated. In human BCG – MVA85A-vaccinated volunteers, blocking the hydrolysing ability of the CD39, an apyrase responsible for hydrolysing pro-inflammatory ATP, enhances IL-17 production. Challenge of BCG – MVA85A-vaccinated CD39-/- mice with M.tb slightly improved the protective capacity of the vaccine, suggesting that a pathway dependent on ATP-driven inflammation may be a target for improving the immunogenicity of a vaccine against M.tb disease. Overall, this thesis has confirmed the important role of IL-17 in vaccine-induced protection against TB disease and identifies a possible target pathway for improvement of a novel vaccine.

Foot-and-mouth disease virus (FMDV) is an economically devastating and highly contagious livestock pathogen. It exists as seven serotypes, comprising numerous antigenically distinct subtypes. The large amount of antigenic heterogeneity has confounded attempts at developing broadly reactive vaccines. In order to overcome this issue the fundamentals of the interactions between the virus and the host humoral immune response must first be understood. Previous work in this area using monoclonal antibody (mAb) escape mutants has identified five antigenic sites for the O serotype and efforts have been made to quantify their relative importance. However, this does not represent a complete picture of serotype O antigenicity. The work conducted in this thesis demonstrates the role of a limited number of dominant substitutions in mediating the antigenic diversity of serotype O Foot-and-Mouth disease virus. Two alternative but complementary methods for identifying epitopes were developed. The first used a mathematical model to analyse newly generated serological and sequence data from 105 viruses, cultured for this purpose (and cross-reacted to 5 reference antisera), in the context of an existing crystallographic structure to identify and quantify the antigenic importance of sites on the surface of the virus. The second approach was purely structural, using existing B cell epitope prediction tools to develop a method for predicting FMDV epitopes using existing crystallographic structures of FMDV. These techniques were validated by the use of reverse genetics, which confirmed the impact on cross reactivity of two predicted novel serotype O antigenic residues, with a further four novel residues identified by looking in depth at the interactions between two genetically close, but antigenically distant viruses. This increased knowledge of the antigenic composition of serotype O FMDV contributes to our understanding of the nature of vaccine efficacy and the breadth of protection, which, in the longer term, will aid in the goal of developing vaccines to better protect livestock from such a highly antigenically variable disease.

Epidemiological observations have linked increased host iron with malaria susceptibility. At the same time, blood-stage malaria infection is associated with potentially life-threatening anemia. To improve our understanding of these relationships, this work presents an examination of the mechanisms controlling the upregulation of the hormone hepcidin, the master regulator of iron metabolism, in malaria infection. Chapter 2 presents data from a mouse model of malaria infection which indicate that hepcidin upregulation in malaria infection is associated with increased activity of the sons of mothers against decapentaplegic (Smad) signaling pathway. Although the canonical Smad pathway activators, bone morphogenetic proteins (Bmp) are not increased at the message level following infection, activin B, which has been recently shown to increase hepcidin through the Smad signaling pathway in conditions of inflammation and infection, is upregulated in the livers of malaria-infected mice. Chapter 3 shows that both activin B and the closely related protein activin A upregulate hepcidin in vitro and in vivo. Chapter 3 also explores the effects of the activin-binding protein follistatin in both systems and in the same malaria-infected mouse model as presented in Chapter 2. The work presented in Chapter 4 extends these studies to human infections by demonstrating that activin A protein co-increases with hepcidin in human serum during malaria infection. Taken together, these findings are consistent with a novel role for activin proteins in controlling hepcidin upregulation in the context of malaria infection. This work may form a basis for the development of novel therapeutics that speed recovery from malarial anemia by inhibiting activins’ actions. Chapter 5 examines the role of infected red blood cell-derived microparticles in the initial recognition of a P. falciparum malaria infection, and subsequent hepcidin upregulation. Microparticles stimulate production of cytokines from peripheral blood mononuclear cells (PBMC), which also upregulate activin A message in response to both microparticles and whole infected red blood cells. These data are consistent with a model in which malaria-derived stimuli such as microparticles trigger the systemic release of activin proteins, which then act on the liver to upregulate hepcidin. Evidence has shown that cytokine levels at birth are related to malaria risk. In Chapter 6, hepcidin is measured in cord blood samples from participants in a large-scale clinical study in a malaria-endemic area, and shown to be elevated in cord blood from neonates with a clinical history of placental malaria. Cord blood hepcidin is also compared to birth levels of iron markers and other cytokines, and future clinical outcomes. Finally, the contributions of DNA methylation levels to cord hepcidin and cytokine levels are assessed by comparison of CpG methylation, at sites in genes encoding hepcidin and cytokines, to the serum concentrations of the genes’ protein products. Several intriguing associations are noted which indicate a possible novel role for DNA methylation in the determination of birth cytokine and hepcidin levels. Chapter 7 synthesizes the data presented in this thesis, interprets the possible significance of the major findings, and offers suggestions for future work.

For both influenza viruses and hepatitis C viruses, T cell responses to conserved antigens are one strategy for the human host to control the spread of infection. Such T cell responses can be generated with the use of viral vectored vaccines. Initially I show that the viral vectored vaccine MVA-NP+M1 can boost memory T cell responses to influenza A virus in adults aged over 50 years old. However within this group, MVA-NP+M1 had reduced immunogenicity in adults who were aged over 70 years old. The influenza virus-specific T cell responses comprised both CD4 and CD8 T cells, and were capable of secreting multiple Th1 cytokines. I then show that MVA-NP+M1 can be safely co-administered alongside seasonal influenza vaccine. The combination does not interfere with the peak T cell response that normally occurs 1 week following MVA-NP+M1. There was a statistically significant increase in antibodies to the H3N2 strain when the vaccines were co-administered, suggesting that the MVA-NP+M1 can act as an adjuvant. The efficacy of MVA-NP+M1 in humans had been previously evaluated in an influenza virus challenge study. I used a whole blood transcriptome approach to improve the classification of outcomes following influenza virus challenge. For subjects with laboratory-confirmed influenza, individuals with moderate/severe symptoms were found to have a distinct transcriptional signature comprising over 2,000 genes. I used a machine learning algorithm to reduce this variation down to just six genes (CCL2, SEPT4, LAMP3, RTP4, MT1G and OAS3). I validated this finding using expression data from an independently conducted challenge experiment. Data from these six genes was successfully able to predict symptomatic and asymptomatic cases with 89% and 100% accuracy respectively. To induce T cell responses to hepatitis C virus, I used the vaccines ChAd3-NSmut and MVA-NSmut in a prime-boost regimen. While the combination was highly immunogenic in healthy young adults, MVA-NSmut alone was unable to prime immune responses. The magnitude of T cell responses to the vaccine immunogen was correlated with the breadth of the T cell responses to different epitopes. Re-administration of the same two vaccines after a short time interval (8 weeks) did not improve upon previous peaks in T cell response. However with a longer time interval (> 34 weeks), some individuals were able to achieve higher frequencies of virus-specific T cells compared to the first round of vaccines. A whole blood transcriptome approach was used to study gene expression in volunteers vaccinated with ChAd3-NSmut and MVA-NSmut. Vaccination with MVA-NSmut results in a very strong, but relatively short-lived host gene expression signature. In contrast, the transcriptional response seen following ChAd3-NSmut was much less pronounced. A comparison of the functional analysis of gene lists from both vaccines showed that similar pathways were being activated and repressed.

The apicomplexan parasite Plasmodium falciparum is the causative agent of the most severe and deadly form of human malaria. The production of an efficacious malaria vaccine is seen as one of the key steps towards the eradication of the disease, however to date only one candidate has progressed to application for licensure. Candidate malaria vaccines target the different stages of the P. falciparum lifecycle through induction of a functional immune response. Vaccines targeting the blood-stage parasite require induction of high titre neutralising antibodies. To achieve this, vaccine regimens have been designed specifically to maximise antibody induction and maintenance in humans. The ultimate test of any candidate vaccine is clinical efficacy and controlled human malaria infection (CHMI) is a powerful tool for measuring this. This model can also be used to study how vaccine induced antigen-specific components of the immune system respond to native antigen exposure in the context of parasitic infection In this Thesis I describe the induction and maintenance of B cell responses, including memory B cells (mBC) and antibody secreting cells (ASC) to the candidate blood-stage malaria antigens MSP1 and AMA1 following vaccination with a variety of regimens and CHMI. These B cell populations along with peripheral blood T follicular helper (Tfh) cells correlate strongly with antibody induction. Within these populations I have identified a number of phenotypically distinct subsets which contribute to a functional response to vaccine and/or parasite antigen. From single cell sorting of ASC at day seven post-boost I have managed to produce the first fully human monoclonal antibodies (hmAbs) specific for AMA1, one of which shows significant growth inhibitory activity (GIA). Despite promise the vaccine candidates MSP1 and AMA1 have been disappointing in terms of human efficacy. In this Thesis I have attempted to provide explanations on a cellular level as to why there is such disparity between pre-clinical and human data and ultimately why these candidates may have failed to provide efficacy. Such work will provide a strong basis for analysing future clinical trials of alternative candidate blood-stage vaccines and allow accurate characterisation of immune correlates and clinical efficacy when it is achieved.

Vaccines have had a profound influence on human health with no other health intervention rivalling their impact on the morbidity and mortality associated with infectious disease. However, the magnitude and persistence of vaccine immunity varies considerably between individuals, a phenomenon that is not well understood. Recent studies have used contemporary technologies to correlate variations in the genome and transcriptome to complex phenotypic traits, and these approaches have started to provide fresh insight into the intrinsic factors determining the generation and persistence of vaccine-induced immunity. This thesis aimed to describe the relationship between genomic and transcriptomic variations, and the immunogenicity of childhood immunisations. Candidate gene and genome-wide genotyping was conducted to evaluate the influence of genetic variants on vaccine-induced immunity following childhood immunisation. Furthermore, contemporary methodologies were used to assess non-coding and coding gene transcript profiles following vaccination, to further dissect the molecular systems involved in vaccine responses. Key findings from this thesis include the description of the first genome-wide association studies into the persistence of immunity to three routine childhood immunisations: capsular group C meningococcal (MenC) conjugate vaccine, Haemophilus influenzae type b (Hib) conjugate vaccine and tetanus toxoid (TT) vaccine. Genome-wide genotyping was completed on over 2000 participants, with an additional 1000 participants genotyped at selected genetic markers. Genome-wide significant associations (p<5×10⁻⁸) were described between single- nucleotide polymorphisms (SNPs) in two genes, CNTN6 and ENKUR, and the persistence of serological immunity to MenC following immunisation of children 6-15 years of age. In addition, genome-wide significant associations were described between SNPs within an intergenic region of chromosome 10 and the persistence of TT-specific IgG concentrations following childhood immunisations. Furthermore, a number of variants in loci with putative involvement in the immune system such as FOXP1, the human leukocyte antigen locus and the lambda light chain immunoglobulin locus, were shown to have suggestive associations (p<1×10⁻⁵) with the persistence of vaccine-induced serological immunity. The fundamental challenge will be to describe functional mechanisms associated with these findings, and to translate these into innovative and pragmatic strategies to develop new and more effective vaccines.

The virulence of Plasmodium falciparum is largely attributed to the ability of asexual blood-stage parasites to cytoadhere to the microvascular endothelium of the human host. This pathogenic behavior is mediated by the primary parasite virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), an understanding of which is crucial to develop interventions to ameliorate the morbidity and mortality of P. falciparum malaria. The work presented in this thesis describes the application of a phenotype-to-genotype experimental approach to identify novel parasite proteins involved in the trafficking and display of PfEMP1. Guided by the overall hypothesis that the in vitro culture-adapted parasite line 3D7 harbors 1 or more genetic determinants of impaired PfEMP1 trafficking, surface PfEMP1 levels were first measured in 3D7, the presumably trafficking-competent parasite line HB3, and 16 unique progeny from an HB3 x 3D7 genetic cross (chapter 2). These phenotypes were then combined with genome-wide SNP data in QTL analysis to identify genetic polymorphisms potentially responsible for the impaired trafficking in 3D7 (chapter 3). A near-significant QTL containing a single protein-coding gene, the putative kinesin Pf3D7_1245600, was identified, characterized, and investigated in CRISPR-Cas9-driven allele-exchange parasite transfection experiments to establish a causal link between the gene and PfEMP1 trafficking (chapter 4). The parasite transfections were unsuccessful, but the potential role of Pf3D7_1245600 in PfEMP1 trafficking was indirectly assessed through the disruption of microtubules with colchicine (chapter 4), which significantly impacted the surface PfEMP1 levels of HB3 but not 3D7. The findings of this thesis suggest that kinesins and microtubules may play previously unconsidered roles in the regulation, production, or trafficking of PfEMP1.

Typhoid fever remains an important public health problem globally. Cluster randomized effectiveness trials with typhoid Vi polysaccharide vaccine were conducted in Kolkata, India and Karachi, Pakistan, to provide evidence for vaccine introduction. While efficacy trials are limited to estimate vaccine's performance on the vaccine recipients, effectiveness trials consider the public health impact, notably the herd protection, or indirect effect, which can only be seen when vaccines are administered to groups rather than to individuals. The observed total protection by the Vi polysaccharide vaccine in school-aged children was consistent in Kolkata and Karachi (61&percnt; and 56&percnt;, respectively), and was associated with minimal side-effects. The total protection in young children, however, was different (80&percnt; in Kolkata and no protection in Karachi). The Kolkata trial demonstrated significant herd protective effects, as demonstrated by indirect protection of non-vaccinees (45&percnt;), which was not shown in the Karachi trial. The difference in the effectiveness estimates between the trials may be due to the difference in study design and the population characteristics. Immunogenicity studies were undertaken for randomly selected persons from both sites at pre-vaccination, 6 weeks, and 2 years post-vaccination. Serum Vi antibody titres (IgG) were measured through ELISA. At baseline, the GMTs were below the protective level for both sites. At six weeks after vaccination, though there is a significant increase in the GMTs in children from both site, the level of GMTs were significantly lower from those in Karachi (2,307.0 ELU vs. 1,189.1 ELU). GMT declined from 6 week to 2 year testing points for both sites but maintained the protective level. These effectiveness trials gave a conclusive evidence of the protection conferred by the Vi polysaccharide vaccine in children older than 5 years of age. Targeted vaccination programme in high endemic areas, as stipulated in the WHO Position Paper, suggest the potential for effective control of typhoid fever in places like India and Pakistan with the school-based Vi vaccination.

Human CD161 is protein that is expressed by most Natural Killer (NK) cells, of T cells (including both CD4⁺ and CD8⁺ subsets), Natural Killer T (NKT) cells and immature thymocytes. CD161 is expressed on many of the T cell receptor (TCR) expressing cell types that “bridge” both the innate and the adaptive immune systems, including mucosal associated invariant T (MAIT) cells. Changes in expression levels of CD161 in peripheral blood or tissue have been seen in multiple disease states such as: HIV, tuberculosis, multiple sclerosis, rheumatoid arthritis and psoriasis. However, the functional role of CD161 on T cells was unclear. Therefore, this thesis explores the impact of CD161 ligation on CD8⁺ T cells. CD161 surface expression was seen to be significantly downregulated upon ligation with its receptor (LLT1) or cross-linked by anti-CD161 antibodies. Despite this, no clear functional impact of CD161 ligation was seen on resting CD8⁺ Va7.2⁺ cells. In contrast, ligation of CD161 on TCR stimulated CD8+ Va7.2+ cells resulted in increased IFNy and TNFa expression, cell activation and cytotoxicity. The increased cytotoxicity was potentially due to both an increase in the ability of the cells to degranulate and the expression of Granzyme B. Furthermore, ligation of CD161 induced an increase in activated Caspase 3 expression, indicating increased apoptosis. CD161 ligation over a prolonged period resulted in a decrease in proliferation. In total these results suggest that CD161 acts as a co-stimulatory molecule for T cells in the context of TCR activation.