HIV-1 has claimed the lives of millions of people globally and continues to spread despite development of highly active antiretroviral therapy. In 2013, 2.1 million new infections occurred and over 35 million people were living with HIV-1 infection. A prophylactic HIV-1 vaccine that can prevent infection or reduce viremia and subsequent transmission will always be an important part of the solution to bring this epidemic under control. In this thesis, the first HIV-1 vaccine candidate to focus on conserved regions of the virus (HIVconsv) was assessed in a phase I clinical trial conducted in healthy HIV-1/2 negative volunteers in Oxford. The HIVconsv T-cell immunogen was delivered using three leading vaccine modalities (DNA (D), modified vaccinia virus Ankara (M) and chimpanzee adenovirus serotype 63 (C)), in several novel heterologous prime-boost regimens. The frequency of T cells elicited through HIVconsv vaccination in the CM and DDDCM regimens surpassed that of previous HIV-1 cell-mediated vaccines. A large proportion of these T cells produced multiple cytokines and proliferated in response to recall peptides. The breadth of T-cell responses were also greater than the non-efficacious STEP study vaccine, with an average of 10 T-cell epitopes per vaccine recipient recognised across CM and DDDCM regimens. In vitro HIV-1 control mediated by CD8⁺ T cells was demonstrated for all vaccinees receiving the CM regimen, mainly against clade A (U455) and clade B (IIIB) isolates. Two vaccinees, demonstrated superior control of 6/8 and 7/8 viruses from the panel. The CM regimen induced significantly higher magnitudes of viral inhibition compared to the DDDCM or DDDMC regimens, with this regimen showing potential to overcome the disadvantage for subjects of carrying non-protective HLA alleles. Investigation of T-cell specificities revealed that the frequencies of T cells specific for conserved Gag but more so Pol regions significantly correlated with in vitro virus control. Direct examination of peptide expanded T-cell lines showed that all Pol pool- and limited Gag pool-specific cell lines reduced HIV-1 replication in vitro. In most individuals, targeting multiple HIV-1 epitopes concomitantly resulted in higher levels of virus inhibition than targeting a single viral epitope and two T-cell specificities showed enhanced control of HIV-1; the first within Pol (TAFTIPSI) and second from Gag (TERQANFL). These data support further development of the conserved region strategy for T-cell vaccines against HIV-1.

The HIV-1 reservoir is the major barrier to eradication and cure of HIV-1 infection. The aim of this thesis was to characterise the HIV-1 reservoir in PHI and to investigate two potential reservoir clearance strategies. Sensitive Q-PCR assays that quantify HIV-1 DNA reservoir level were developed and applied to characterise the HIV-1 reservoir in acutely infected patients enrolled in the SPARTAC trial. HIV-1 DNA levels were found to be a useful biomarker for predicting clinical progression and the time of viral remission in patients, after HAART interruption. Since low HIV-1 DNA reservoir levels were clinically beneficial, potential therapies aimed at decreasing the reservoir burden were investigated. Thermotherapy was employed as a possible method for increasing CTL killing ability of HIV-1-infected cells. This may increase the likelihood of virological control and functional cure. SPION uptake impacted negatively on the natural killing ability of CTL that expressed the high affinity 868-TCR. However, localised thermotherapeutic heat generation was insufficient to cause direct thermal ablation of bound target cells or to enhance the natural killing ability of CTL. 868-TCR transduced CTL were employed directly as a means to target the latent viral reservoir by the ‘kick and kill’ hypothesis. Latent HIV-1 was reactivated in a variety of latent cell line models, but inconclusively in a primary model. 868-TCR transduced CTL killing of latently infected cells was observed. However, reactivation of latent virus did not necessarily relate to increased antigenicity of latent cells, perhaps in part due to anti-latency drug-induced alterations in MHC class I expression. Despite recent and sustained interest in HIV-1 cure strategies, the prospect still remains elusive. Only through the development of sensitive assays that measure the HIV-1 reservoir and their application to novel and innovative cure strategies will HIV-1 ever be functionally cured or eradicated in patients on a large scale.

Natural exposure to Plasmodium falciparum’s asexual blood-stage results in protection against severe disease, but no vaccine using the widely-studied blood-stage antigens apical membrane antigen 1 (AMA1) or merozoite surface protein 1 (MSP1) has proven convincingly protective in clinical trials. Challenges include antigenic polymorphism, the apparent requirement for exceptionally high antibody concentrations for protection, and clinical-grade production of conformationally-accurate recombinant protein antigens followed by formulation with a human-compatible adjuvant. This thesis describes the generation of viral-vectored vaccines targeting ten less-studied blood-stage antigens, focusing upon antigens implicated in erythrocyte invasion. These vaccines were immunogenic in mice and rabbits. The rabbit antibodies raised were functionally active in the in vitro assay of parasite growth inhibitory activity (GIA). GIA with antibodies against one antigen, RH5, exceeded that achieved with antibodies against the ‘gold standard’ AMA1 or MSP1 antigens. This antigen’s amino acid sequence is relatively conserved between parasite strains. Importantly, and unlike anti-AMA1 and MSP1 antibodies, the GIA effects transcend genetically diverse strains. It was hypothesised that blockade of the interaction of RH5 with its receptor basigin was likely to be a mechanism of action of anti-RH5 antibodies. Vaccine-induced polyclonal anti-RH5 serum was found to be capable of blocking this interaction, as well as merozoite attachment to erythrocytes. A panel of RH5-specific monoclonal antibodies were raised: those which block the RH5-receptor interaction were capable of neutralising parasites. Minimal linear epitopes recognised by these antibodies were mapped, and are likely to be within or close to RH5’s receptor binding site. These data support prompt clinical testing of RH5-based vaccines, and shed light upon the mechanism of action of anti-RH5 antibodies. However substantial challenges remain in establishing whether this antigen, selected on the basis of the in vitro assay of GIA, will be capable of achieving in vivo protection against P. falciparum. Further work presented in this thesis addresses the use of quantitative PCR data to assess blood-stage vaccine efficacy in experimental human challenge with P. falciparum, and the use of surface plasmon resonance to establish more detailed characterisation of vaccine-induced antibody responses. Finally, the results of P. falciparum challenge of RH5-vaccinated Aotus nancymaae non-human primates are presented.

Replication-deficient adenovirus (Ad) and modified vaccinia virus Ankara (MVA) vectors expressing single Plasmodium falciparum antigens can induce potent T cell and antibody responses and have entered clinical testing using a heterologous prime-boost immunisation approach (Ad_MVA). This thesis describes a number of pre-clinical approaches aimed at enhancing the efficacy of these viral vectored vaccines targeting the blood-stage of malaria. First, the development of a highly efficacious malaria vaccine is likely to require a multi-antigen and/or multi-stage subunit vaccine. The utility of an Ad_MVA immunisation regime combining vaccines expressing the 42kDa C-terminus of the blood- stage antigen merozoite surface protein 1 (MSP142) and the pre-erythrocytic antigen circumsporozoite protein (CSP) in the P. yoelii mouse model was investigated. It was found that vaccine co- administration leads to maintained antibody responses and efficacy against blood-stage infection, but reduced secondary CD8+ T cell responses and efficacy against liver-stage infection. CD8+ T cell interference can be minimised by co-administering the MVA vaccines at separate sites, resulting in enhanced liver-stage efficacy. The mechanisms of CD8+ T cell interference were explored. Second, Ad_MVA regimes expressing blood-stage antigens that can protect against P. chabaudi and P. yoelii blood-stage infection were tested against P. berghei, but did not confer protection. Similarly, IgG from rabbits immunised against P. falciparum MSP1 (PfMSP1) could not protect mice from a chimeric P. berghei parasite expressing PfMSP1. Third, two molecular adjuvants, the C4bp α-chain oligomerisation domain (IMX108/313) and the Fc fragment of murine IgG2a, were tested for their ability to enhance immunogenicity of recombinant adenoviruses when fused at the C-terminus of a blood-stage antigen. IMX108/313 was found to adjuvant T cell responses of small (< 80kDa) antigens and this was associated with antigen oligomerisation. However, the Fc fragment did not adjuvant responses. Finally, it was found that using a strong early promoter to drive antigen expression enhances the immunogenicity of single administration MVA vaccines, but that this did not enhance post-boost immunogenicity in an Ad_MVA regime.

During its intraerythrocytic stages (IE), Plasmodium falciparum, the causative agent of the deadliest human malaria, remodels the host red cell membrane with a poorly defined assortment of parasite-­encoded proteins that undergo antigenic variation. Despite the requirement for immunologic stealth, exported parasite proteins also mediate strain-independent functions such as endothelial sequestration that are critical for parasite survival and pathogenesis. This thesis explores the hypothesis that P. falciparum displays novel structurally conserved proteins on the IE surface and these proteins may serve as useful antigens for a broadly effective anti-­malarial vaccine. In order to test this hypothesis, we developed an in vitro selection technique that sequentially incorporates unique P. falciparum isolates as the targets for Systematic Evolution of Ligands by EXponential enrichment (Serial-SELEX) to generate nucleic acid molecular probes, aptamers, capable of recognizing conserved cell surface determinants. Ten of 11 enriched aptamers were -parasite selective and three of these aptamers demonstrated strain-independent binding to P. falciparum. Aptamer recognition extended beyond the parasites used in Serial-SELEX to other laboratory and recent field isolates. Surprisingly the same three broadly binding aptamer selected against P. falciparum also recognized all laboratory-adapted and clinical isolates of P. vivax and P. knowlesi tested, strongly supporting our hypothesis that structurally conserved molecules are present on the surface IEs. Competition studies showed that the aptamers bound a single target which was confirmed as an IE membrane protein. Aptamer­‐mediated affinity purification and tandem mass spectrometry enabled identification of the aptamer target as parasite-encoded protein. Discovery of a protein conserved between the major human malarias may have implications for vaccine development and validates the Serial‑SELEX technique as a powerful tool for antigen discovery.

The efficacy of Bacille-Calmette Guerin (BCG) vaccination in protection against pulmonary tuberculosis (TB) is highly variable between populations. One possible explanation is increased exposure of certain populations to non-tuberculous mycobacteria (NTM). Given the variable efficacy of BCG an improved vaccine against TB is required. The novel TB vaccine MVA85A has shown promising results, however, the immunogenicity of the vaccine is reduced when it is administered in the Expanded Programme on Immunisation (EPI) schedule. This thesis aims to explore: (A) the effect of exposure to NTM on the level of protection afforded by BCG vaccination against Mycobacterium tuberculosis (M. tb) and (B) the immunological mechanisms behind EPI interference with MVA85A. The effect of M. avium (MA) exposure via systemic and oral routes on the efficacy of BCG was tested using M. tb aerosol infection in a mouse model. The adaptive immune response was profiled in BCG vaccinated mice with and without exposure to MA pre- and post- M. tb infection. The results showed BCG efficacy could be enhanced by exposure to dead MA by a systemic route; T helper 1 and T helper 17 responses were associated with increased protection. In contrast, BCG efficacy may have been reduced by exposure to live MA by the oral route; T helper 2 and regulatory T cells were associated with reduced protection. To answer the second aim MVA85A was co-administered to mice with aluminium adjuvants or aluminium-containing vaccines to replicate the effect of co-administration in the EPI schedule; the adaptive immune response was profiled. T helper 2 and regulatory T cell responses induced by aluminium-containing vaccines were associated with a reduction in the immunogenicity of MVA85A.

Neisseria meningitidis causes 500,000 cases of meningitis and septicaemia annually worldwide, with a mortality rate of approximately 10%. Most disease in developed countries is caused by serogroup B infection, against which there is no universal vaccine. Opa proteins are major meningococcal outer membrane proteins, and a limited number of Opa variants have been associated with hyperinvasive serogroup B meningococci, suggesting their use as a potential novel vaccine. Immunisation of mice with recombinant Opa elicited high levels of meningococcal-specific serum bactericidal antibody (SBA), demonstrating proof in principle of this approach. Opa proteins mediate bacterial adherence to host cells and modulate human cellular immunity, and there are conflicting data regarding their effects on CD4⁺ T cells. opa genes from N. meningitidis strain H44/76 were cloned into the plasmid vector pBluescript, disrupted using antibiotic resistance cassettes and transformed into H44/76 to sequentially disrupt the four opa genes. This produced a unique panel of 15 isogenic Opa-deficient strains, including an Opa-negative strain, which enabled investigation of the immunomodulatory role of surface-expressed Opa proteins. There was no consistent effect of Opa expressed on the surface of OMVs and inactivated bacteria on CD4⁺ T cells, with significant heterogeneity of responses between individuals. The rate of Opa phase variation was between 10^-3 and 10^-4, and increased 180-fold following transformation of bacteria with unrelated DNA. These data support further investigation of Opa as a potential meningococcal vaccine component, and highlight the importance of host and bacterial factors in the development of OMV vaccines.

The bacteria Streptococcus pyogenes produces a multitude of proteins which interact with and alter the functions of the host immune system. Two such proteins, Endoglycosidase S (EndoS) and Immunoglobulin G-degrading enzyme from S. pyogenes (IdeS) are able to specifically alter the effector functions of immunoglobulin G (IgG). EndoS is a glycoside hydrolase which removes the conserved <i>N</i>-linked glycan from IgG Fc whereas IdeS is a cysteine protease that cleaves the exible protein hinge of IgG. The activity of both proteins results in the reduced ability of IgG to elicit immune responses through Fc receptor binding and complement activation. Amongst other applications, both EndoS and IdeS are actively being explored as new therapeutics for IgG-mediated autoimmune diseases. Given the therapeutic potential of EndoS and IdeS, experiments were designed to investigate the structural and functional characteristics of these enzymes in an effort to understand their specficity for and activity against IgG. Here, bioinformatic and biophysical characterisation of EndoS identified subdomains outside of the catalytic domain which contribute to glycoside hydrolase activity. The substrate specificity of EndoS was also explored and showed that EndoS hydrolyses a broad range of glycans from the IgG scaffold. EndoS was also shown to have activity against alternative glycoprotein substrates, however, this non-specific activity was negligible in the context of whole serum. The effect of EndoS-mediated deglycosylation on the structure of the IgG Fc domain was explored using both X-ray crystallography and small-angle X-ray scattering. Small angle X-ray scattering was also used to characterise both EndoS and IdeS in complex with IgG Fc. Solution-state models of each complex were produced providing preliminary data towards how these enzymes interact with IgG. Overall, the results presented here contribute to our understanding of these enzymes which is of importance as they go forward into clinical applications.

Streptococcus pneumoniae is a significant cause of mortality and morbidity in both children and older adults, with infection resulting in invasive disease, pneumonia and otitis media. The inclusion of pneumococcal conjugate vaccines in routine infant immunisation programmes has had a major impact on disease rates. Vaccine-induced protection against pneumococcal infection is thought to be mediated by the generation of persistent serotype-specific functional antibodies and antigen-specific memory B cells, the latter capable of generating a rapid secondary antibody response on re-exposure to antigen. Although many studies have investigated the immunogenicity of pneumococcal vaccines in different age groups by measuring serotype-specific antibodies, there is more limited information about the B cells underlying such an immune response. Important areas to investigate include the identity of the B cell subsets involved in antibody production and the potential link between memory B cells (B_MEM) and persistent antibody production by long-lived plasma cells. In this thesis I have investigated in detail the immune response to pneumococcal vaccines given to children and adults by a variety of different methods. By examining the variability of a B_MEM ELISpot method, it was shown that this assay is robust and reproducible and can be performed on fresh or frozen samples and in different laboratories. Using this technique, in a study of pre-school children, it was demonstrated for the first time that the level of pre-existing serotype 3-specific antibody is negatively correlated with, and may directly impair the B_MEM response to a booster dose of 13-valent pneumococcal conjugate vaccine (PCV-13) containing serotype 3 glycoconjugate. In the same study, it was shown that antibody persistence against most vaccine serotypes can be expected until the age of 3.5 years. A novel antigen-labelling technique was used in a detailed kinetics study of antigen-specific B cell subsets in response to either PCV-13 or 23-valent pneumococcal polysaccharide vaccine in adults. The results of this study revealed distinct B cell subset response patterns that were observed in all study participants indicating that IgM B_MEM seem to play a major role in the immune response to pneumococcal vaccines. In addition, in the same study, genome wide analysis of gene expression was performed and it was shown that vaccination with either a pneumococcal conjugate or polysaccharide vaccine results in a marked difference in numbers of differentially expressed genes 8 days following vaccination. A further tool likely to be of use in investigating B cell responses is the analysis of the antibody repertoire using next-generation sequencing techniques. In order to test the ability of these methods to detect vaccine responses, a large dataset of high-throughput B cell receptor sequences was analysed and revealed convergence of antigen-specific complementary-determining region (CDR)₃ amino acid (AA) sequences following vaccination and identified antigen-specific sequences. It was further demonstrated that for sequences directed against the H. influenzae type b (Hib) polysaccharide, diversity of immunoglobulin gene rearrangements is much greater than previously recognised. Frequencies of Hib-specific CDR₃ AA sequences were linked with anti-Hib avidity indices highlighting the potential of this method as an alternative (functional) measure of vaccine immunogenicity. These data suggest that studying the B cells and antibody repertoire post-vaccination can give novel insights into the biology that underlies the immune responses.

Leishmania spp. undergo substantial adaptations from being promastigotes, found in sandflies, to being amastigotes, residing in parasitophorous vacuoles within mammalian macrophages. In the past, microarray studies have sought to elucidate these adaptations using axenic amastigote systems or amastigotes purified from host-cells, raising the question whether the observed transcriptomic signatures were a true reflection of intracellular amastigotes. Moreover, with ever-improving genome annotations being available, it is clear that these studies failed to address the transcriptomic behaviour of a considerable number of transcripts. In the work presented herein, I employed RNA-sequencing to obtain transcriptomic profiles of Leishmania mexicana axenic promastigotes (PRO), axenic amastigotes (AXA) and intracellular amastigotes (AMA) in murine bone-marrow derived macrophages. The intracellular amastigotes were not purified from host cells, but instead sequencing reads assigned to a hybrid L. mexicana - Mus musculus genome and the transcriptomes separated in silico. We were able to map pre-mRNA processing sites, thereby defining transcript boundaries, proposing 184 truncations and 1253 extensions of existing gene models as well as discovering 936 novel genes. Mass-spectrometric evidence was obtained for both proposed extended and novel proteins. Using this improved genome annotation, we generated gene expression profiles for AMA, AXA and PRO, identifying 3832 differentially expressed transcripts between PRO and AMA as well as 2176 between PRO and AXA and 1234 between AXA and AMA. Transcripts differentially expressed between AMA and PRO correlated well with previous reports, were enriched for novel transcripts identified in this study and contained an unprecedented wealth of yet uncharacterised transcripts. Guided by these data, I performed a GFP-tagging screen identifying two proteins which may play an important role in L. mexicana biology, LmxM.16.0500, a member of a small, divergent, amastin-derived gene family, which appears to be released from the cell body of PRO, and LmxM.09.1330 a specific marker of the amastigote flagellar pocket.