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Streptococcus pneumoniae (Spn) is a common cause of respiratory infection, but also frequently colonizes the nasopharynx in the absence of disease. We used mass cytometry to study immune cells from nasal biopsy samples collected following experimental human pneumococcal challenge in order to identify immunological mechanisms of control of Spn colonization. Using 37 markers, we characterized 293 nasal immune cell clusters, of which 7 were associated with Spn colonization. B cell and CD8+CD161+ T cell clusters were significantly lower in colonized than in non-colonized subjects. By following a second cohort before and after pneumococcal challenge we observed that B cells were depleted from the nasal mucosa upon Spn colonization. This associated with an expansion of Spn polysaccharide-specific and total plasmablasts in blood. Moreover, increased responses of blood mucosal associated invariant T (MAIT) cells against in vitro stimulation with pneumococcus prior to challenge associated with protection against establishment of Spn colonization and with increased mucosal MAIT cell populations. These results implicate MAIT cells in the protection against pneumococcal colonization and demonstrate that colonization affects mucosal and circulating B cell populations.

Naturally acquired clinical immunity to Plasmodium falciparum is partly mediated by antibodies directed at parasite-derived antigens expressed on the surface of red blood cells which mediate disease and are extremely diverse. Unlike children, adults recognize a broad range of variant surface antigens (VSAs) and are protected from severe disease. Though crucial to the design and feasibility of an effective malaria vaccine, it is not yet known whether immunity arises through cumulative exposure to each of many antigenic types, cross-reactivity\ud between antigenic types, or some other mechanism. In this study, we measured plasma antibody responses of 36 children with symptomatic malaria to a diverse panel of 36 recombinant proteins comprising part of the DBLα domain (the ‘DBLα-tag’) of PfEMP1, a major class of VSAs. We found that although plasma antibody responses were highly specific to individual antigens, serological profiles of responses across antigens fell into one of just two distinct types. One type was found almost exclusively in children that succumbed to severe disease (19 out of 20) while the other occurred in all children with mild disease (16 out of 16). Moreover, children with severe malaria had serological profiles that were narrower in antigen specificity and shorter-lived than those in children with mild malaria. Borrowing a novel technique used in influenza–antigenic cartography—we mapped these dichotomous serological profiles to amino acid sequence variation within a small sub-region of the PfEMP1 DBLα domain. By applying our methodology on a larger scale, it should be possible to identify epitopes responsible for eliciting the protective version of serological profiles to PfEMP1 thereby accelerating development of a broadly effective anti-disease malaria vaccine.

Malaria infections remain a serious global health problem in the world, particularly among children and pregnant women in Sub-Saharan Africa. Moreover, malaria control and elimination is hampered by rapid development of resistance by the parasite and the vector to commonly used antimalarial drugs and insecticides, respectively. Therefore, vaccine-based strategies are sorely needed, including those designed to interrupt disease transmission. However, a prerequisite for such a vaccine strategy is the understanding of both the human and vector immune responses to parasite developmental stages involved in parasite transmission in both man and mosquito. Here, we review the naturally acquired humoral and cellular responses to sexual stages of the parasite while in the human host and the Anopheles vector. In addition, updates on current anti-gametocyte, anti-gamete, and anti-mosquito transmission blocking vaccines are given. We conclude with our views on some important future directions of research into P. falciparum sexual stage immunity relevant to the search for the most appropriate transmission-blocking vaccine.

IgM is the first antibody to be produced in immune responses and plays an important role in the neutralization of bacteria and viruses. Human IgM is heavily glycosylated, featuring five N-linked glycan sites on the μ chain and one on the J-chain. Glycosylation of IgG is known to modulate the effector functions of Fcγ receptors. In contrast, little is known about the effect of glycosylation on IgM binding to the human Fcμ receptor (hFCMR). In this study, we identify the Cμ, omain of IgM as the target of hFCMR, and show that binding and internalization of IgM by hFCMR is glycan-independent. We generated a homology-based structure for hFCMR and used molecular dynamic simulations to show how this interaction with IgM may occur. Finally, we reveal an inhibitory function for IgM in the proliferation of T cells.

Protective immunity to the liver stage of the malaria parasite can be conferred by vaccine-induced T cells, but no subunit vaccination approach based on cellular immunity has shown efficacy in field studies. We randomly allocated 121 healthy adult male volunteers in Kilifi, Kenya, to vaccination with the recombinant viral vectors chimpanzee adenovirus 63 (ChAd63) and modified vaccinia Ankara (MVA), both encoding the malaria peptide sequence ME-TRAP (the multiple epitope string and thrombospondin-related adhesion protein), or to vaccination with rabies vaccine as a control. We gave antimalarials to clear parasitemia and conducted PCR (polymerase chain reaction) analysis on blood samples three times a week to identify infection with the malaria parasite Plasmodium falciparum. On Cox regression, vaccination reduced the risk of infection by 67% [95% confidence interval (CI), 33 to 83%; P = 0.002] during 8 weeks of monitoring. T cell responses to TRAP peptides 21 to 30 were significantly associated with protection (hazard ratio, 0.24; 95% CI, 0.08 to 0.75; P = 0.016).

Fungal burden in the cerebrospinal fluid is an important determinant of mortality in cryptococcal meningitis but its use to aid clinical decision-making is hampered by the time involved to perform quantitative cultures. Here we demonstrate the potential of flow cytometry as a novel and rapid technique to address this.

Background. Cryptococcal meningitis remains a significant cause of death among human immunodeficiency virus type 1 (HIV)–infected persons in Africa. We aimed to better understand the pathogenesis and identify immune correlates of mortality, particularly the role of monocyte activation. Methods. A prospective cohort study was conducted in Cape Town, South Africa. Patients with a first episode of cryptococcal meningitis were enrolled, and their immune responses were assessed in unstimulated and stimulated blood specimens, using flow cytometry and cytokine analysis. Results. Sixty participants were enrolled (median CD4+ T-cell count, 34 cells/µL). Mortality was 23% (14 of 60 participants) at 14 days and 39% (22 of 57) at 12 weeks. Nonsurvivors were more likely to have an altered consciousness and higher cerebrospinal fluid fungal burden at presentation. Principal component analysis identified an immune signature associated with early mortality, characterized by monocyte deactivation (reduced HLA-DR expression and tumor necrosis factor α response to lipopolysaccharide); increased serum interleukin 6, CXCL10, and interleukin 10 levels; increased neutrophil counts; and decreased T-helper cell type 1 responses. This immune signature remained an independent predictor of early mortality after adjustment for consciousness level and fungal burden and was associated with higher serum titers of cryptococcal glucuronoxylomannan. Conclusions. Cryptococcal-related mortality is associated with monocyte deactivation and an antiinflammatory blood immune signature, possibly due to Cryptococcus modulation of the host immune response. Validation in other cohorts is required.

Background\ud In sub-Saharan Africa, where infectious diseases and nutritional deficiencies are common, severe anaemia is a common cause of paediatric hospital admission, yet the evidence to support current treatment recommendations is limited. To avert overuse of blood products, the World Health Organisation advocates a conservative transfusion policy and recommends iron, folate and anti-helminthics at discharge. Outcomes are unsatisfactory with high rates of in-hospital mortality (9–10 %), 6-month mortality and relapse (6 %). A definitive trial to establish best transfusion and treatment strategies to prevent both early and delayed mortality and relapse is warranted.\ud \ud Methods/Design\ud TRACT is a multicentre randomised controlled trial of 3954 children aged 2 months to 12 years admitted to hospital with severe anaemia (haemoglobin

The response of a leukocyte to immune complexes (ICs) is modulated by receptors for the Fc region of IgG (FcgammaRs), and alterations in their affinity or function have been associated with risk of autoimmune diseases, including systemic lupus erythematosus (SLE). The low-affinity FcgammaR genomic locus is complex, containing regions of copy number variation (CNV) which can alter receptor expression and leukocyte responses to IgG. Combined paralogue ratio tests (PRTs) were used to distinguish three intervals within the FCGR locus which undergo CNV, and to determine FCGR gene copy number (CN). There were significant differences in FCGR3B and FCGR3A CNV profiles between Caucasian, East Asian and Kenyan populations. A previously noted association of low FCGR3B CN with SLE in Caucasians was supported [OR = 1.57 (1.08-2.27), P = 0.018], and replicated in Chinese [OR = 1.65 (1.25-2.18), P = 4 x 10(-4)]. There was no association of FCGR3B CNV with vasculitis, nor with malarial or bacterial infection. Linkage disequilibrium (LD) between multi-allelic FCGR3B CNV and SLE-associated SNPs in the FCGR locus was defined for the first time. Despite LD between FCGR3B CNV and a variant in FcgammaRIIB (I232T) which abolishes inhibitory function, both reduced CN of FCGR3B and homozygosity of the FcgammaRIIB-232T allele were individually strongly associated with SLE risk. Thus CN of FCGR3B, which controls IC responses and uptake by neutrophils, and variations in FCGR2B, which controls factors such as antibody production and macrophage activation, are important in SLE pathogenesis. Further interpretations of contributions to pathogenesis by FcgammaRs must be made in the context of LD involving CNV regions.

The surface glycocalyx of Leishmania parasites consists of a complex mix of glycoconjugates, with varied roles in cell invasion and protection from host immune responses. Most abundant amongst these is a family of glycoinositolphospholipids (GIPLs), the glycan components of which vary between Leishmania species. Type-II GIPLs are abundantly expressed by L. major parasites and contain terminal αGalactosyl residues that are recognised by serum from infected patients. Since the oligosaccharidic portion of the main L. major GIPLs terminate with the linear sequences Galα(1,3)Galfβ(1,4)-R (GIPL-2) or Galα(1,6)Galα(1,3)Galfβ(1,4)-R (GIPL-3), which differs to the main epitope (Galα(1,3)Galβ(1,4)GlcNAcβ1-R) recognised by normal anti-Gal antibodies, I hypothesised that the specificity of the anti-Gal produced during a course of a Leishmania infection will be different to that of normal anti-Gal. In this thesis, I set out to characterise the human anti-Gal response that is triggered during Leishmania infection, aiming to exploit this knowledge for the development of a novel diagnostic test. The utility of native GIPLs extracted from both L. major and L. tropica cells as diagnostic antigens was assessed first through a combination of Thin Layer Chromatography (TLC) immuno- and lectin-staining, mass spectrometry analysis, and chemiluminescent ELISA (CL-ELISA). GIPLs extracted from L. major promastigotes were strongly recognised by sera from L. major-positive patients, but not from L. tropica-positive patients, or that from healthy controls. In addition, it was confirmed that L. tropica does not express αGalactosylated GIPLs, suggesting that this residue must be decorating an uncharacterised glycoconjugate. To determine the specificity of the Leishmania spp. anti-Gal, a bespoke panel of neoglycoproteins (NGPs) was used to investigate anti-Gal produced in response to leishmaniasis infection in three patient cohorts. These sample collections encompassed the spectrum of the leishmaniases, from systemic visceral disease to localised cutaneous lesions. Antibody detection in Leishmania patients has potential in screening of suspect cases, but also in monitoring response to treatment. NGPs with Galα(1,3)Galfβ-R and Galα(1,6)Galα(1,3)Galfβ-R terminating glycans were the best discriminators of L. major infections and also showed potential in the diagnosis of American cutaneous leishmaniasis caused by L. braziliensis. In addition, the presence of a terminal β-galactofuranose residue in these NGPs dramatically improved their recognition in CL-ELISA, and their value in the diagnosis of Old World CL. Combining these two glycotopes maximises antibody detection, through capture of two anti-Gal populations. Fluorescent microscopy analysis confirmed L. major cells express αGalactosylated epitopes throughout the parasite lifecycle, whilst in L. tropica the same epitope was only detected in promastigote cells. While the nature of this epitope remains unknown, future work should focus on the analysis of L. tropica glycoconjugates. Results in this thesis are discussed in relation to the variability of αGalactosyl epitopes recognised by sera from patients with different Leishmania infections, and their possible exploitation in the development of novel disease biomarkers.

The burden of pneumococcal pneumonia remains high worldwide. The disease disproportionately affects infants, the elderly and the immunocompromised. Nasopharyngeal colonisation with S. pneumoniae is the pre-requisite for disease but also, paradoxically, an immunising event. Current pneumococcal vaccines offer limited protection against pneumonia and do not offer serotype-independent coverage. Serotype replacement is particularly concerning. Identification of serotype-independent correlates of protection against S. pneumoniae infection will accelerate development of vaccines with broad coverage. Protein vaccines are a particularly promising way to confer serotype-independent protection.\ud The experimental human pneumococcal challenge (EHPC) model enables us to investigate correlates of protection against experimental colonisation with S. pneumoniae and study protein-directed immune responses to nasopharyngeal colonisation with the bacteria. A multiplex Luminex assay was developed and used to measure serum IgG against 75 conserved pneumococcal antigens pre- and post-intranasal inoculation with two serotypes of pneumococcus (6B and 15B) in EHPC volunteers. Peripheral blood mononuclear cells taken pre- and post-challenge with serotype 6B pneumococcus were used to evaluate correlation between protein-specific IgG+ memory B-cell responses and protection against experimental colonisation as well as memory B-cell responses to pneumococcal challenge by ELISpot. Memory B-cell responses to six selected proteins and the novel PnuBioVax vaccine were investigated. The results of this work showed that colonisation with serotype 6B pneumococcus induces significant systemic protein-mediated antibody and memory B-cell responses. No single protein antigen correlated with protection against experimental colonisation with S. pneumoniae. However, whilst not significant, there was a trend that PnuBioVax-specific memory B-cells conferred protection against experimental pneumococcal colonisation. Five novel nanoparticle-based vaccines were also investigated to determine their effects on dendritic cell maturation and support of T-cell activation. One of these nanoparticles was subsequently loaded with pneumococcal protein PspA to investigate antigen-specific response. Further work is needed to ascertain the immune response to these novel vaccines, though they are a promising method of serotype-independent vaccine delivery.\ud It is likely that for a novel pneumococcal protein vaccine to confer serotype-independent protection against pneumococcal pneumonia, a combination of protein antigens will need to be included in vaccine formulations. Mucosal vaccination in particular will likely be needed to induce robust immunity