Meningococcal disease, caused by the bacterium Neisseria meningitidis, is an important cause of morbidity and mortality in young children and adolescents worldwide. There are 12 serogroups with most disease due to meningococci expressing one of five capsular polysaccharide antigens corresponding to serogroups A, B, C, Y and W135. In Europe, the majority of disease-causing strains are of serogroups B and C. No comprehensive vaccine is available against the bacterium due to the difficulty in producing serogroup B vaccines. A number of countries, e.g. UK and the Republic of Ireland have implemented routine meningococcal conjugate C (MCC) vaccine strategies. Due to the high proportion of disease accounted for by serogroup B in Europe and other developed countries, much research is currently being carried out to unearth vaccine candidates that would be protective and give as wide coverage as possible. Such candidates include the antigens PorA, FetA and factor H-binding protein. Potential drawbacks with antigens such as these which are under immune selection are high degrees of variability and lack of cross-immunity. Determination of the distribution, both geographically and temporally, of antigens and their association with clonal complex can aid in the formulation of novel vaccines and assess their potential coverage across Europe. Serological typing schemes involving characterisation of the polysaccharide capsule (serogroup) and outer membrane proteins such as PorA (serosubtype) and PorB (serotype) have been used for a number of years with some success. However, drawbacks associated with these methods include insufficient discrimination, limitations in panels of monoclonal antibodies used in the typing procedures and difficulty in comparison of results among labs. Consequently, in recent years genotypic methods such as multi-locus enzyme electrophoresis (MLEE) and subsequently multi-locus sequence typing (MLST) have been developed. These methods measure the variation in slowly evolving housekeeping genes whereas serological methods measure variation in antigens which are under immune pressure and are therefore more diverse. Combination of phenotypic and genotypic typing methods can offer high levels of discrimination. Molecular studies into meningococcal diversity have offered many important insights into its population biology, which have implications for prevention and control of meningococcal disease. These have included the identification of hyperinvasive lineages and the correlation of genetic type with antigenic type and disease epidemiology. The EU-MenNet programme was established as a pan-European infrastructure for the research and surveillance of European meningococcal disease. Its aim was to coordinate and disseminate the latest molecular isolate characterisation techniques (MLST) and electronic data transfer via the Internet to exploit epidemiological and population genetic studies. Within the EU-MenNet, the European Meningococcal MLST Centre (EMMC) was set up to carry out molecular typing — MLST, PorA and FetA — of European disease isolates from 18 countries over three years 2000, 2001 and 2002. The output of this project will be the largest representative molecular epidemiological study of meningococcal disease in Europe. Assessment of the data produced will give insights into the geographic and temporal distribution and structuring of disease-associated clonal complexes and antigens and their associations. This will give an indication of the meningococcal disease population in Europe and will be invaluable for the current, and ongoing, development and introduction of new meningococcal vaccines.

The past 10 years has seen mother to child transmission (MTCT) of HIV-1 shift from being one of the predominant forces in the global epidemic to a phenomenon that is largely preventable and envisioned as being on the path to elimination. This thesis is based on two cohorts of Kenyan infants recruited before and after the development of effective antiretroviral interventions to prevent MTCT. Two main lines of enquiry are pursued with the aim to contribute to improved health outcomes of infants affected by HIV-1. The first seeks to further our understanding of the capacity of the infant cytotoxic T lymphocyte (CTL) response to influence viral evolutionary dynamics in early infection. Chapter 3 presents a modern phylogenetic analysis of longitudinal viral sequences derived from infants following in utero or peripartum infection. The results indicate that despite high levels of viral replication, infant CTL selection pressure plays a significant role in shaping early viral evolution. The second stems from an accumulating body of evidence that suggests that infants born to HIV-1 infected mothers who themselves are free from infection, termed HIV-1 exposed uninfected (HEU) infants, nevertheless face significantly higher rates of infectious disease- associated morbidity and mortality than HIV-1 unexposed infants. This study therefore sought to characterise the immunological status of HEU infants with particular emphasis on the phenotypic and functional properties of the T cell compartment. Chapter 4 presents the immunological characterisation of a cohort of healthy Kenyan infants recruited as a control population at two time points in early life. Chapter 5 present a cross-sectional comparison of HEU and control infant cohorts. The results suggest a level of altered immunological reactivity with respect to the T helper type 1 (Th1) response to polyclonal stimulation. In addition a compromised memory Th1 response was observed following polyclonal stimulation and following stimulation with Bacillus Calmette-Guerin and tetanus toxoid vaccine antigens.

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.

There is an urgent need for improvements to existing vaccine delivery technologies to run parallel with the development of new-generation vaccines. The burdens of needle-based immunisation strategies are exacerbated by poor resource provision in such areas as sub-Saharan Africa, where annual malaria mortality stands at 860,000. Needle-free delivery of vaccine to the skin holds promise for improved immunogenicity with lower doses of vaccine, in addition to significant logistical advantages. Various methods have been described for the transcutaneous delivery of vaccines, including the use of microneedles to overcome the outer stratum corneum of the skin for efficient delivery of liquid or solid, microneedle-coated vaccines into underlying strata rich in antigen-presenting cells. This thesis aims to evaluate two transcutaneous silicon microneedle and microprojection patch technologies for the delivery of live recombinant Adenovirus and Modified Vaccinia Ankara-vectored vaccines encoding pre-erythrocytic malaria antigens in mice. Cellular immunogenicity directed against a well-documented epitope of the Plasmodium berghei circumsporozoite protein is evaluated, as is protection against lethal P. berghei sporozoite challenge. Immunological and logistical benefits of each technology are assessed, as well as mechanisms underlying differences in the generation of a patch-induced immune response to vaccination. These data inform the future development of transcutaneous microneedle patches for the delivery of live vaccine.

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.

L’entérite nécrotique aviaire causée par Clostridium perfringens est une maladie économiquement dévastatrice et celle-ci est en émergence dans les troupeaux de poulets de chair éliminant l’usage des antibiotiques. À ce jour, aucune alternative en élevage ne permet de prévenir efficacement la maladie et un contrôle par une stratégie vaccinale serait des plus prisé. Une approche par génomique comparative jumelée à la vaccinologie réverse soustractive et comparative identifiant des protéines bactériennes de surface immunogènes figure parmi les approches méthodologiques des plus prometteuses pour le développement rapide d’un vaccin efficace. Une étude génomique comparative réalisée sur 48 souches de C. perfringens provenant de poulets de chair en santé ou affectés par l’entérite nécrotique a permis d’établir que les génomes analysés étaient composés de 155 700 protéines distinctes, où 13% étaient extracellulaires, 65% cytoplasmiques et 22% membranaires. L’évaluation du pouvoir immunogène de ces protéines à l’aide de l’outil de prédiction VaxiJen v.2.0 a permis d’identifier 4 catégories de scores pour les protéines identifiées, allant de 0,5 (seuil minimal recommandé) à 1,5. Les protéines présentant les scores les plus élevés ont été majoritairement associées à des localisations extracellulaires. La combinaison du score d’immunogénicité et de la localisation cellulaire des protéines analysées a mené à la sélection de 12 protéines candidates vaccinales, la plupart d’entre elles étant de fonction hypothétique. Une description plus approfondie de ces protéines permettra de mieux définir leur fonction, d’évaluer leur potentiel antigénique réel en caractérisant leur interaction avec le système immunitaire de la volaille et ultimement, d’évaluer leur rôle probable dans la pathogénie de l’entérite nécrotique. / Avian necrotic enteritis caused by Clostridium perfringens is a disease with a major economical impact, generating losses up to 6 billion dollars for the poultry industry worldwide. This disease appears in broiler chicken flocks that no longer employ the use of antibiotics. To date, no alternative method allows for the efficient prevention of necrotic enteritis (NE) and a control by a vaccinal strategy would be mostly prized. A comparative genomics approach as well as comparative and subtractive reverse vaccinology identifying immunogenic bacterial surface proteins is one of the most promising methodologies for the rapid development of an efficient vaccine. A comparative genomic study was performed on 48 C. perfringens strains isolated from healthy broiler chickens and from broilers affected by necrotic enteritis. From this study, it was established that the genomes analyzed were composed of 155 700 distinct proteins where 13% were predicted to have an extracellular expression, 65% at the cytoplasma level and 22% within the plasma membrane. The evaluation of the immunogenic potential of these proteins was established with the prediction software VaxiJen v2.0 for which a 0.5 threshold score allowed for the identification of four score categories among the identified proteins, from 0.5 to 1.5. For the most part, proteins with the highest scores were associated with an extracellular localisation. The combination of the immunogenicity score and localisation of the analysed proteins led to the selection of 12 vaccinal candidate proteins that were mostly identified as hypothetical. A more in-depth description of these proteins would allow the assessment of their function, the evaluation of their true immunogenic potential by characterizing their interaction with the avian immune system and ultimately, evaluate their probable role in the pathogenesis of necrotic enteritis.

Dendritic cells (DC) play a key role in immunity and represent a great target for modulation, because of their ability to prime T cells and direct their polarisation into effector subsets. Ticks release immunomodulatory compounds in their saliva, possibly in order to evade host immune responses during feeding. We have recently reported that Rhipicephalus appendiculatus ticks produce ‘Japanin’, a secretory lipocalin that arrests differentiation of monocytes into DC and reprogrammes maturation of DC in response to various stimuli towards a tolerogenic phenotype . Japanin was cloned and recombinantly expressed in a baculovirus system for subsequent immunological and biochemical analysis. This study was set out to further investigate the immunomodulatory activity of Japanin as well as the underlying mechanism of action. We have discovered that Japanin prevents DC-mediated proliferation and polarisation of allogeneic T cells. Experiments with labelled Japanin have demonstrated that it binds predominantly to ex vivo generated human monocyte-derived DC (moDC) and to a reduced degree to monocyte and DC populations in peripheral blood, yet to no other blood leucocytes. We have identified CD206, also known as the mannose receptor, as a Japanin-binding receptor on moDC. This identification has been achieved by crosslinking and subsequent pull-down of Japanin-receptor complexes from moDC. Affinity studies with recombinant CD206 constructs have confirmed the binding to Japanin. Moreover, the binding has been verified by specific siRNA knock-down of CD206 in moDC, which resulted in significantly decreased binding of Japanin. Unexpectedly, CD206 has appeared to be dispensable for at least most of the DC-modulatory activity of Japanin. Therefore, attempts were made to determine other factors in the mode of action of Japanin, through which we have found that IL-10 is not essentially involved. Further results have suggested that the activity of Japanin demands cell contact. Collectively, we have come to the conclusion that the mechanism of action of Japanin might require internalisation by DC, potentially enabling modulation of intracellular pathways involved in the regulation of DC maturation.

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.

There is currently a need for vaccine adjuvants that are effective for eliciting Th1-type CD4 and CD8 T cell responses when formulated with protein and peptide-based subunit vaccines. Some of the most promising adjuvants in this regard are combined small molecule Toll-like receptor-7/8 agonists (TLR-7/8a). However, poor pharmacokinetic properties have precluded TLR-7/8a for use in vaccines. In this thesis, polymer carriers were used to control pharmacokinetics and to modulate activity of TLR-7/8a for use as vaccine adjuvants. Combinatorial synthesis and in vivo structure-activity studies were used to evaluate how properties of Polymer-TLR-7/8a conjugates (Poly-7/8a) influence innate immune activation in lymph nodes that drain the site of vaccine administration. The most striking finding was that particle formation by Poly-7/8a strongly enhances the magnitude and duration (>14 days) of innate immune activation in lymph nodes by restricting agonist biodistribution and promoting uptake by dendritic cells. Particle-forming Poly-7/8a optimized for activity were found to induce only local innate immune activation (not systemic) and were effective for eliciting Th1-type CD4 and CD8 T cells that mediated protection against infectious challenge. Based on the importance of particle formation for activity of Poly-7/8a, thermo-responsive Poly-7/8a were developed that exist as single water-soluble macromolecules in solution but undergo temperature-driven particle formation in vivo. In conclusion, polymer carriers of TLR-7/8a represent a versatile and effective platform for modulating innate immune activity and warrant further investigation as a class of adjuvants for vaccines.

The envelope glycoprotein (Env) of Human Immunodeficiency Virus 1 (HIV-1) has developed several immune-evasion mechanisms to avoid the induction of neutralising antibodies, including immunodominant non-neutralising epitopes, conformational flexibility of conserved epitopes, and spontaneous subunit dissociation, thus impeding vaccine development. Here, chemical modification of Env-based vaccine antigens is explored to overcome these obstacles. Firstly, covalent fixation of Env by chemical cross-linking was used to stabilise the conformationally flexible structure and prevent subunit dissociation. Cross-linked Env constructs showed reduced binding of many non-neutralising antibodies whilst largely maintaining antibody recognition by broadly neutralising antibodies. Compared to unmodified material, immunisation with some of these cross-linked proteins led to the induction of significantly increased antibody titres targeting the conserved CD4 binding site of Env despite similar overall antibody titres. These refocused antibody responses resulted in increased serum neutralising titres compared to animals receiving unmodified protein. Secondly, an epitope masking strategy was developed to reduce or eliminate the immunogenicity of neutralisation-irrelevant surfaces. This was achieved using site-selective addition of theoretically immunosilent glycoconjugates to lysine residues. Masking of model protein hen egg lysozyme (HEL) led to site-selective loss of antibody binding to the modification sites in vitro, which translated into refocusing of antibody responses from masked to unmasked epitopes in vivo. Mutant HIV-1 and influenza virus surface glycoproteins were designed that had lysine residues removed from close proximity to the respective broadly neutralising epitopes, but added throughout the remaining surface. Masking of these mutant proteins with second-generation glycoconjugates led to predictable perturbations of antibody binding in vitro. However, administration of these modified glycoproteins revealed unexpectedly that the masking glycans were highly immunogenic in vivo. Thus, this strategy may well prove useful if truly non-immunogenic glycoconjugates can be identified. Taken together, these chemical modifications of vaccine antigens may allow focused targeting of specific antigenic regions for increased B cell recognition, and may thus be a valuable tool for vaccine antigen design.