Medicine (RMH) - Theses
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ItemUnderstanding mechanisms enabling long-lasting immunity to malaria( 2023-07)An efficacious vaccine that generates long-lasting immunity is critically needed to reduce the global burden of malaria and protect young children in high transmission areas, who are most at risk of severe disease and death. However, despite four decades of development, the most advanced malaria vaccine induces only moderate and short-lived protection and other candidate vaccines are yet to demonstrate more sustained efficacy. Advancement in malaria vaccine design is impeded by an absence of established correlates of immunity, limiting targets for directed vaccine improvements. Host-specific factors, such as age, previous malaria infection and nutritional status, may modulate immune function and impair vaccine responsiveness, highlighting the need for an extensive characterisation of immune function in children in malaria endemic regions. In this thesis, we explored correlates and determinants of sustained immunity to malaria and examined how vaccine modification may improve long-lasting protection. We first considered immune and host factors associated with antibody-mediated protection and antibody maintenance in clinical samples. We investigated the value of antibody avidity, previously reported as a marker of robust and high-quality immune induction, as a possible correlate of long-lasting malaria immunity following vaccination in children and adults. Our findings suggest that avidity is an unreliable marker of antibody maintenance and antibody functional properties. In malaria, monocytes may be key effector cells of antibody-mediated functions which have shown protective associations in vaccine trials. We found that monocytes in children with malaria infection expressed high levels of cell surface receptors relevant to functions in malaria immunity. Given the importance of potent antibody responses to generate protective and sustained immunity, we also explored the role of undernutrition in IgG responses to routine vaccinations in children living in regions of endemic malaria transmission and found that underweight and wasting were associated with reduced vaccine-induced antibody levels. Finally, we used mouse models to investigate vaccine strategies and dietary modifications that may alter the magnitude and longevity of antibodies induced by malaria vaccines. We demonstrated differences in antibody maintenance generated by different vaccine approaches, including varying adjuvants and antigens, and that deficiencies in critical micronutrients, such as zinc, may impair the antibody response to vaccination, an effect modulated by adjuvant selection. The work in this thesis supports the role of monocytes in immunity, shows that nutritional factors impact vaccine antibody levels, and demonstrates that vaccine formulation – including adjuvant and antigen selection – and host factors, such as zinc deficiency, interact to modulate the induction and maintenance of antibody responses to vaccination. In summary, we illustrate the challenges and complexity of inducing sustained immunity with malaria vaccines, providing foundations for future work characterising the interplay of host factors, the immune system and vaccine features to support vaccine-induced immunity to malaria.
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ItemIdentifying Antibody Responses Associated with Protection from Severe Malaria in Children( 2023-04)Malaria is a major global public health burden that affects individuals in 87 countries and causes 600,000 deaths annually. The majority of cases of severe malaria and malaria related deaths occur in children under the age of five, who lack protective immunity that is acquired by adulthood in endemic regions. Antibodies are a key component of protective immunity, however, the target antigens of antibodies and the functions of antibodies that confer protection are unclear. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) expressed on the surface of parasite infected erythrocytes is a highly variant, multi-domain protein that mediates adhesion to a range of vascular endothelial cell receptors including Intercellular Adhesion Molecule-1 (ICAM-1) and Endothelial Protein C Receptor (EPCR). PfEMP1 is an important virulence factor and is a strong candidate target of protective antibodies against severe malaria. Therefore, the overall objective of this thesis was to understand the antibody response to PfEMP1 antigens that may confer protection from severe malaria in children. This thesis aimed to: 1) characterize the antibody response to PfEMP1 antigens that are associated with protection from cerebral malaria (the most severe form of malaria) in a cohort of Malawian children; 2) characterize the antibody response to PfEMP1 antigens that are associated with protection from severe malaria in a cohort of Papua New Guinean children; 3) assess the association of disease severity with antibody dependent neutrophil and monocyte phagocytosis of infected erythrocytes expressing important PfEMP1 variants. For aims 1 and 2 we applied systems serology, which involves measuring multiple features of antibodies targeting multiple recombinant antigens and using machine learning with statistical methods to identify the best correlates of protection. For aim 3, we selected parasite-infected erythrocytes expressing two PfEMP1 variants (that bind to ICAM-1) and measured antibody dependent neutrophil phagocytosis (ADNP) and antibody dependent cell phagocytosis (ADCP) of opsonized infected erythrocytes by flow cytometry. The important findings of this thesis were: 1) in both Malawian children and Papua New Guinean children, a combination of a small subset of PfEMP1 specific antibody responses could be used to differentiate between children with severe and uncomplicated malaria with high accuracy; 2) in both cohorts, DBL-beta3 domains that bind to ICAM-1 receptor were targets of antibodies associated with protection from severe malaria. We also affirmed that 3) in addition to measuring the quantity of antigen specific antibodies, the biophysical properties and Fc interactions (or ‘Fc features’) of PfEMP1 specific antibodies are useful correlates of protection from severe malaria. A range of antibody Fc features were associated with protection, suggesting there are multiple pathways to protection from severe malaria. The antibody responses associated with protection included Fc-gamma-RIIIb binding to antibodies targeting DBL-beta3 domains that bind to ICAM-1, suggesting that antibodies to ICAM-1 binding PfEMP1 may induce killing mechanisms by neutrophils. In the final chapter, we found that 4) ADNP of ICAM-1+EPCR binding infected erythrocytes was associated with protection from cerebral malaria in Malawian children but was not associated with protection from severe malaria in Papua New Guinean children. Additionally, 5) ADCP of ICAM-1 binding infected erythrocytes was not associated with protection from severe malaria in either cohort of children. In considering the different methodological approaches used in this thesis, we concluded that systems serology is an effective approach to identify correlates of protective immunity to PfEMP1 but currently cannot replace more complex cell-based assays like ANDP and ADCP. Overall, this thesis highlights the important role of functional antibodies to PfEMP1 in protection from severe malaria in children from diverse geographic regions and supports the development of a DBL-beta domain based therapeutic.
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ItemCharacterizing functional antibody responses in malaria in pregnancy( 2019)Malaria is a major threat to the tropical regions around the world. The World Health Organization has reported Plasmodium falciparum as the most lethal malaria parasite species. It is also reported that a huge proportion of malaria-related morbidity and mortality affects the sub-Saharan African region. If malaria is caused during pregnancy, both the pregnant mother and her developing foetus are at greater risk of adverse outcomes, and this disease state is known as malaria in pregnancy (MiP). In MiP, the parasite-infected erythrocytes sequester in the intervillous spaces in the placenta affecting placental blood flow and nutrient transfer between the mother and the growing foetus. In response to this infection, active immune responses are triggered in the form of antibodies, infiltration of phagocytic cells (mainly monocytes and macrophages) in the placental blood spaces, and complement activation. Antibodies of the IgG class participate in MiP, which are further subdivided into four subclasses, namely, IgG1, IgG2, IgG3 and IgG4. IgG-opsonised infected erythrocytes are phagocytosed by phagocytic cells such as monocytes, and this opsonic phagocytosis is an important protective mechanism in MiP. On monocytes three main types of surface expressed Fcγ receptors (FcγRs) (FcγR III/CD16, FcγR II/CD32, FcγR I/CD64) interact with the opsonized antigenic particles and pull them inside the activated phagocytic cell. This PhD thesis aims to characterize functional roles of antibodies that protect against MiP using opsonic phagocytosis and ELISA based assays, and also examine other key participants of opsonic phagocytosis mechanism, namely, the monocytes and complement system. Different aims were divided into three results chapters as explained further. Aim 1 in this thesis examined differences in FcγR expression and function and opsonic phagocytosis variations using diverse monocyte cell models, namely, THP-1, Mono Mac 6 (MM6) and buffy coat purified human monocytes. On phenotyping the surface FcγR expression it was found that CD16 was not expressed on both THP-1 and MM6 cell lines. THP-1 cell line was observed to exhibit higher levels of phagocytosis than MM6. FcγR blocking assays were performed to assess which FcγR triggered phagocytosis in a particular cell model. It was observed that opsonic phagocytosis was driven synergistically from both CD32a and CD64 in THP-1, CD64 in isolated human monocytes and CD16 in monocytes of whole blood, while MM6 showed very low levels of opsonic phagocytosis and the outcomes could not be clearly interpreted. Aim 2 used plasma samples from a longitudinal study on a cohort of Malawian pregnant women to measure antibody levels and examine their correlations and associations with various risk factors and clinical outcomes. This study cohort included women randomized to different malaria prevention regimes, namely, intermittent preventive therapy in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) (control group) and intermittent screening and treatment in pregnancy with dihydroartemisinin-piperaquine (IST-DP) (intervention arm). The hypothesis for this aim was that antibodies to specific Duffy binding-like (DBL) domains are correlated with protection against adverse consequences of malaria in pregnancy. For the purpose of examining this hypothesis, antibodies were measured at study enrolment from the IPTp-SP control arm (578 women). Specifically, total IgG as well as IgG subclass responses were measured against Duffy binding-like domains (DBLs) 1, 3 and 5 of VAR2CSA. It was observed that out of the three antigenic domains assayed, most women’s plasma recognized DBL3 (DBL3 IgG1- 74.2%, DBL3 IgG2- 19.4%, DBL3 IgG3- 62.1%, DBL3 IgG4- 42.8%), while only few recognized DBL1 (DBL1 IgG1- 44.1%, DBL1 IgG3- 28.3%, DBL1 IgG2/4- all seronegative). Antibody responses to DBL3 and DBL5 were more strongly correlated with each other than DBL1 antibody responses, both for total IgG and IgG subclasses. Regression analyses of antigen-antibody outcomes showed that out of all maternal risk factors, significant positive associations with high antibody levels were observed for the presence of malaria infection at study enrolment and gravidity. Significant negative associations with high antibody levels were mainly observed for the increasing schooling years of women and very weakly with bed nets used before study enrolment. For the clinical outcomes at delivery, the presence of placental malaria at delivery was positively associated with high antibody levels measured at enrolment, while antibodies did not correlate with protection from any adverse outcomes. Aim 3 explored the role of complement system in MiP by analysing capacity of C1q complement protein to fix antibody against DBL1 and DBL3 domains, in the same study cohort as used in aim 2. It was observed that more of the samples were seropositive for DBL3-C1q measurements (71.21%), as compared to DBL1-C1q (17.09%). Regression analysis on maternal risk factors showed that presence of malaria infection at enrolment and increasing gravidity were significantly positively associated with high C1q fixation, while a significant negative association was observed between increasing schooling years of women and bed net usage and high C1q fixation. No significant association was observed for the clinical outcomes. Complement fixation process is activated through the antibodies binding specifically to their target antigens. If an antigen isn’t entirely immunogenic, leading to less antibody recruitment, the subsequent complement fixation process is also lower. Complement fixing antibodies have been shown to be functionally better at being protective as opposed to non-complement fixing antibodies (Kurtovic, Behet et al. 2018). A subsection in this result chapter also detailed outcomes from IgG3 depletion impact on opsonic phagocytosis and complement fixation in a select number of plasma samples. It was observed that for both these functional assays, there were no significant differences observed between the intact plasma versus the IgG3-depleted version, except only for the high responders for complement fixation outcomes. Nonetheless, the complement fixation assays did show a visible reduction in the overall complement fixation activity in almost all of the IgG3-depleted samples. Overall, this PhD thesis provides insight into diverse aspects of the opsonic phagocytosis of P. falciparum-infected erythrocytes and individual functional participants, namely, the opsonizing IgG immunoglobulin, antigenic particles (pregnancy-associated), phagocytes and the helper complement system, by studying functional traits of different FcγRs, and examining multiple aspects of antibody response to MiP, such as total IgG, subclass and C1q fixation. Longitudinal studies like these are an ideal tool to assess the potential protective functions of antibody to targets such as VAR2CSA, and to identify maternal social and demographic factors that may influence levels of these antibodies. In this thesis for the Malawian women cohort, the antibodies were not found to be protective, rather the findings suggest they are primarily markers of parasite exposure. Future studies relating to some of the technical issues and knowledge gaps are covered in detail in individual results chapters.
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ItemA customisable biosensor platform for point-of-care applications in precision medicine and immunodiagnostics( 2018)Point-of-care diagnostics play an indispensable role in clinical decision-making pathways as rapid and cost-effective alternatives or complementation to laboratory- based testing. The overarching aim of this thesis was to develop a point-of-care biosensor platform for precision medicine and immunodiagnostic applications. Four sub-studies were undertaken to demonstrate sensor development within the two themes. The interdigitated electrode (IDE) biosensor was used as the core technology, based on its rapid and label-free potential, low-cost fabrication, and amenability towards integration into a compact lab-on-chip device. The first theme focused on biosensor development for pharmacogenetic testing. Associations between HLA variations and medication-specific adverse drug reactions (ADRs) have led to screening recommendations for the HLA-B*15:02 allele before carbamazepine therapy and the HLA-B*57:01 allele before abacavir therapy. However, implementation is hindered by the high-cost, complexity, and lengthy turnaround times of current gold standards. Sub-study 1 aimed to develop the IDE sensor for rapid genotyping of HLA-B*15:02, which is strongly associated with severe cutaneous reactions to the antiepileptic drug carbamazepine in broad Asian populations. A two-step detection platform was established, combining loop-mediated isothermal amplification (LAMP) of HLA-B alleles with HLA-B*15:02 LAMP amplicon hybridisation on the IDE sensor. Validation was performed on purified extracted DNA samples and 27 whole-blood samples from Hong-Kong, where HLA-B*15:02 is prevalent. Impedance characterisation of the LAMP-IDE platform resulted in 92.86% sensitivity and 84.63% specificity towards ii HLA-B*15:02, with a short turnaround time of ~1 hour and 20 minutes without additional DNA staining. Sub-study 2 aimed to develop a biosensor platform for typing of HLA-B*57:01, which is strongly associated with abacavir hypersensitivity A cell capture approach was established using a monoclonal antibody (3E12) specific towards the HLA-B17 group (encompassing HLA-B57 and HLA-B58). Detection of HLA-B*57:01 cell line and HLA-B57 peripheral blood mononuclear cell (PBMC) capture was achieved within 15 minutes. Impedance characterisation demonstrated differentiation between sensors incubated in HLA-B57 PBMCs with non-HLA-B57 PBMCs (p < 0.0001) and blank media (p = 0.02). The platform is also potentially applicable for prevention of allopurinol hypersensitivity through HLA-B*58:01 typing. The second theme explored the use of IDE sensors as an alternative method to bridge the gap between highly-sensitive benchtop immunoassays and less-sensitive and qualitative first-generation point-of-care immunoassays. Sub-study 3 demonstrated the development of a quantitative immunodiagnostic platform for colorectal cancer (CRC) screening. Using anti-haemoglobin (Hb) antibodies, the IDE sensor showed successful quantification of faecal occult blood within ~1 hour, with a sensitivity of 10 μg haemoglobin per gram (Hb/g) of faeces, comparable to laboratory-based quantitative faecal immunochemical testing (FIT) systems. In sub-study 4, an ultrasensitive IDE detection platform was developed for malaria elimination purposes, targeting the Plasmodium falciparum histidine-rich protein II (PfHRP2). Using anti-PfHRP2 antibodies, the IDE sensor successfully detected PfHRP2 at 2.5 pg/mL in PBS and 25 pg/mL in saliva within ~2 hours without labelling. iii Collectively, results of the four sub-studies demonstrate the IDE sensor’s versatility as a relatively-rapid, label-free, and customisable diagnostic platform, comparable to each application’s laboratory-based gold standard methods. The platform has potential to be integrated into a low-cost and compact lab-on-chip device for multiple diagnostic applications.
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ItemDissecting human IgG subclass responses in natural and vaccine acquired immunity to malaria( 2017)Naturally-acquired antibody responses against malaria antigens play an important role in protecting individuals in malaria endemic areas from symptomatic malaria. Merozoites are the extracellular form of blood-stage parasites, and it is likely that some antibodies can target merozoites and inhibit erythrocyte invasion. Naturally-acquired antibodies to different merozoite antigens are typically skewed towards IgG1 or IgG3 responses, and IgG3 responses have generally been more strongly associated with protection. Even though both subclasses are considered to be cytophilic antibodies, the specific differences in their effector responses against merozoite antigens are unclear. It is therefore unknown whether optimal vaccine induced responses need to induce IgG1 or IgG3 responses, or if there are no significant functional differences. This thesis focused on two goals: (i) to compare antibody responses between natural and vaccine induced immunity against merozoite antigens, and (ii) to dissect how these IgG subclasses contribute to natural immunity to merozoite antigens. The first aim focused on comparing naturally acquired and vaccine induced immunity (chapter 3). Erythrocyte Binding Antigen-175 (EBA-175) Region II is a leading vaccine candidate that plays a key role in merozoite invasion through its binding to glycophorin A on the erythrocyte surface. EBA- 175 RII was recently assessed in a dose escalation Phase Ia trial; vaccinated individuals (n=71) developed high titers of IgG but relatively low growth inhibitory responses. In this study, we further assessed the properties and functional activity of these vaccine-induced antibodies and related the magnitude of these responses to antibodies acquired following natural infection in a cohort of children from Papua New Guinea (n=206). Vaccine induced antibodies were able to inhibit EBA-175 binding to its receptor, at similar levels to the naturally exposed cohort. Furthermore, both cohorts were able to elicit antibodies that targeted two inhibitory epitopes. However, the IgG subclass profiles differed and we observed that the vaccine cohort mainly induced IgG1 to EBA-175 RII, while the naturally exposed cohort showed high levels of IgG1 and IgG3. This chapter demonstrates that a recombinant EBA-175 RII vaccine can induce high level binding inhibitory antibodies that target key functional epitopes on EBA-175 Region II however the observable differences in subclass responses may impact on protective function. he second goal of this work was to better understand how these IgG subclasses contribute to immunity against merozoites. To assess this, we made IgG subclass-switch variants (chapter 4) and compared them using in vitro functional assays (chapter 5). IgG subclass-switch antibodies are chimeric recombinant antibodies with the same variable region (and same target epitope) were 2engineered with different human IgG subclass backbones. We chose well-characterised invasion inhibitory monoclonal antibodies to merozoite proteins that are leading vaccine candidates (mAb R217 against EBA-175; mAb QA1 against Rh5; and mAb 1F9 against AMA-1). As non-blood stage controls, monoclonal antibodies to a sporozoite protein (mAb 2A10 to CSP) and an ookinete protein (mAb 4B7 to Pfs25) were also developed. These IgG subclass-switch antibodies were expressed recombinantly in HEK293F cells and showed high yield expression. Characterisation experiments confirmed that the IgG subclass-switch mAbs demonstrated the expected size, covalent structure, subclass and N-glycosylation pattern; therefore, they could be progressed for assessment in functional assays. The IgG subclass-switch mAbs to EBA-175, Rh5 and AMA-1 were assessed using in vitro functional assays to understand how IgG subclasses contribute to merozoite immunity (chapter 5). To assess for neutralisation, the mAbs were tested for merozoite invasion inhibition and binding inhibition. The IgG3 subclass-switch variants consistently showed more potent inhibition of merozoite invasion, compared to other subclasses. In contrast, all subclasses showed similar levels of receptor binding inhibition. For complement fixation, the IgG3 subclass-switch variants also showed consistently higher ability to fix C1q and deposit downstream complement components. IgG subclass-switch mAbs were also assessed for interactions with Fcᵞ receptors, and IgG3 still induced a strong response, while IgG1 responses varied according to the antigen. The superior Fab flexibility of IgG3 could mediate more effective antigen binding leading to these effector functions. These results provide a potential mechanism of action for the high levels of IgG1 and IgG3 responses observed against merozoite antigens in field studies. This work is discussed in the context of vaccine development to suggest approaches to induce an IgG subclass specific response, and to highlight key properties of IgG3 that are likely to impact vaccine induced immunity. Consideration is given to the other uses of these developed IgG subclass-switch antibodies, including as therapeutic antibodies or as standards in functional assays. This work provides insights into the different functional roles of IgG subclasses that mediate effective immunity to merozoites, and paves the way for future studies on their role in combination and for other stages of the malaria life cycle.
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ItemCharacterisation of dendritic cell responses to blood-stage Plasmodium falciparum( 2017)Malaria is a major public health burden in tropical and subtropical regions and a malaria vaccine will be crucial for achieving long term-malaria eradication. The RTS,S vaccine is the only malaria vaccine candidate to reach Phase III trials but has only 29-36% efficacy. A better understanding of antimalarial immunity will aid development of more efficacious vaccines. Specifically, it is important to improve our understanding of dendritic cells (DCs) in malaria. DCs are crucial in the adaptive immune response and DC activation is required for high-quality vaccine responses. Studies of DCs in malaria-infected individuals indicate that malaria impairs key DC functions such as cytokine production, antigen uptake, and T-cell stimulation. Little is known about how DCs respond directly to Plasmodium falciparum as the majority of work has been done using monocyte-derived DCs (moDCs), which do not necessarily represent bona fide DCs. We aimed to determine how DCs upregulate co-stimulatory markers and secrete cytokines in response to blood-stage P. falciparum in vitro. We examined how this response can be affected by four factors: i) the ratio of parasites to DCs, ii) whether the parasite stimulus is merozoites or pigmented trophozoites (pRBCs), iii) the DC subset, and iv) the tissue of origin of DCs (blood, spleen, liver, and bone marrow). To address these questions we used three models: murine Flt3L-induced DCs (FL-DCs), a culture model that produces large quantities of DCs equivalent to those of murine spleen; human peripheral blood DCs; and DCs from a humanised mouse model. Merozoites and low doses of pRBCs consistently activated FL-DCs whereas high doses of pRBCs selectively suppressed CD40 and CD86 expression on conventional DCs (cDCs) as well as TNFα and IL-10 production. Furthermore, we demonstrated that high dose of pRBCs could suppress specific aspects of DC activation induced by potent TLR agonists, confirming a suppressive effect of pRBCs. Thus merozoites activated FL-DCs, whereas pRBCs dose-dependently suppressed certain parameters of FL-DC activation. We demonstrated that PfEMP1 and the host red cell membrane were not required for FL-cDC suppression, suggesting a novel mechanism of suppression. Human DC responses were much lower than those observed in mice. Notably, human peripheral blood DCs did not upregulate any co-stimulatory markers or cytokines in response to pRBCs or merozoites. Humanised mouse splenic DCs upregulated MHCII and CD40 in response to merozoites, and upregulated all co-stimulatory markers in response to pRBCs. Plasmacytoid DCs (pDCs) upregulated low levels of all co-stimulatory markers in response to either stage. Similar to what we observed in FL-DCs, high doses of pRBC specifically suppressed CD40 expression even in the presence of TLR agonists. We also found that opsonising pRBCs with IgG from naturally immune individuals did not enhance responses of peripheral blood DCs. Human DCs show much lower levels of activation to P. falciparum than their murine counterparts. We also examined how sorted FL-DC subsets responded to P. falciparum. In general merozoites induced higher upregulation of co-stimulatory markers from each subset, while pRBCs induced more cytokine production, particularly of pro-inflammatory cytokines such as TNFα and IL-6. Sorted human DCs were less responsive than their murine counterparts. However, we observed that, as with FL-DCs, pRBCs induced production of a broader range of cytokines. Merozoites generally induced lower activation from human DCs. We examined three anatomical sites and found that splenic DCs were most responsive to P. falciparum, while BM and liver DCs had very low responsiveness. We have shown that P. falciparum pRBCs dose-dependently suppressed CD40 expression in all the bona fide DC models we tested. Importantly, our FL-DC findings indicate a novel non-PfEMP1 dependent, non-host-RBC dependent mechanism of suppression. Merozoites and pRBCs induced different responses, with pRBCs generally inducing production of a broader range of cytokines. Human DCs from all tissues show much lower levels of activation than murine FL-DCs in response to P. falciparum. Our work has described the diversity of DC responses to P. falciparum, and provides novel insights into the acquisition of antimalarial immunity in humans. The generally low or limited activation of human DCs by P. falciparum may be part of the reason why immunity to malaria is typically slow to develop and immune memory is not optimal. These findings will be valuable in guiding the development of future vaccines which achieve highly effective and long-lived immunity to malaria.
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ItemMalarial antibodies to Plasmodium falciparum during pregnancy in times of decreasing malaria prevalence( 2015)Malaria remains entrenched in tropical and sub-tropical countries with the highest burden in sub-Saharan Africa. Plasmodium falciparum infection during pregnancy, commonly termed as malaria in pregnancy (MiP), contributes a significant health risk to both pregnant women and their infants. Malaria exposure during pregnancy results in the development of pregnancy-associated antimalarial antibody, which is thought to protect against MiP and its severe consequences. Malaria preventive therapies such as intermittent preventive therapy during pregnancy (IPTp) with antimalarials and insecticide treated bed nets are protective against MiP, but the interventions may lead to reduced malaria exposure, and thus may affect the development of protective malarial antibodies. The present study investigated the impact of decreasing parasite prevalence on acquisition and maintenance of malarial antibodies in pregnant women from Malawi and Papua New Guinea (PNG). Secondary analyses evaluated whether pregnancy-associated malarial immunity is protective against the consequences of MiP. To address the research questions, enzyme-linked immunosorbent assays and flow cytometry based assays were performed on plasma samples from pregnant women residing in Malawi and PNG, to measure malarial antibodies to a range of P. falciparum antigens including recombinant proteins and whole parasites. In the context of decreasing parasite prevalence during pregnancy in Malawi and PNG, plasma samples collected at various time points (Malawi, from 1999-2006; PNG, from 2005-2012) were tested. Total antibody levels to recombinant merozoite antigens and to schizont extracts declined over time in both sample sets. Similarly, total antibody levels to variant surface antigens (VSA) of endothelial-binding infected erythrocytes (IEs) declined. However, total antibody to VSA of placental-binding IEs declined in PNG setting but not in Malawi, possibly due to differences in transmission setting. On the other hand, opsonising antibody to both whole merozoites (only tested in PNG) and whole IEs did not differ over time. Malaria prevention therapies for pregnant women may affect the acquisition of pregnancy-associated malaria antibody, and this was found in studies conducted in sub-Saharan Africa. Whether this observation will be seen in the Asia Pacific region is unclear. A subset of plasma samples from pregnant PNG women recruited for a clinical trial study was used. They were enrolled and randomised to receive either multiple courses of sulphadoxine-pyrimethamine (SP) with azithromycin or a single course of SP with chloroquine. Antibody levels to recombinant merozoite antigens, schizont extract and opsonising antibody to IEs at term were comparable between both interventions, suggesting minimal impact of malarial antibody development in both groups. Antibodies measured to recombinant antigens but not opsonising antibody to IEs declined over a course of pregnancy in both groups. Critically, multiple courses of antimalarials did not affect development of malarial antibodies after adjusting for confounders. SP-resistant parasites are prevalent in Malawi, and since resistance threatens the effectiveness of IPTp-SP, alternatives are needed. Plasma samples from a subset of Malawian women involved in a clinical trial were used. Women were recruited and randomly allocated to receive either IPTp-SP or intermittent screening and treatment with dihydroartemisinin-piperaquine. Plasma samples were collected at enrolment and at delivery and tested for malarial antibodies. At delivery, total antibody levels to recombinant antigens and IEs, and opsonising antibodies to IEs were comparable between both groups. Malarial antibodies to recombinant antigens, but not to VSA of IEs declined between enrolment and delivery in both groups. After adjusting for confounders, there were no significant differences in levels of malarial antibody between the two interventions. Pregnancy-associated malarial antibodies at enrolment from women enrolled to receive IPTp-SP were assayed and analysed to determine if they were predictive of good outcomes at delivery, on the presumption that SP was a sub-optimal drug. Pregnancy-associated malarial antibodies at enrolment were not protective of pregnancy and malaria clinical outcomes, instead, they were positively associated with malaria exposure during pregnancy. A subset of plasma collected at delivery from these women was tested for pregnancy-associated malarial antibody. This sub-study revealed that opsonising antibodies to placental-binding IEs were protective against placental malaria and maternal anaemia at delivery. Overall, this research provided several important pieces of new data: 1) the impact of falling parasite prevalence on anti-P. falciparum serological indicators differs in different settings; 2) different malaria interventions in both Malawi and PNG did not affect the development of malarial antibodies, however, over the course of pregnancy, antibodies to individual antigens were more likely to decline, and antibody levels may decline in the absence of boosters; 3) malarial antibodies targeting whole parasites were more stable over pregnancy than antibodies to recombinant antigens; 4) and opsonising antibody to placental-binding IEs may be a good correlate of protection from MiP.
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ItemIntermittent preventive treatment in pregnancy with azithromycin plus sulphadoxine-pyrimethamine and maternal undernutrition in Papua New Guinea – impact on newborn and maternal health( 2015)Low birthweight (LBW) is common in developing countries. It is largely the result of fetal growth restriction (FGR) and/or preterm birth (PTB). LBW is associated with increased infant mortality rates and has been linked to ill-health in adult life. Principal factors associated with LBW in low-income environments include malaria and maternal undernutrition, primarily through affecting fetal growth, and sexually transmitted infections (STI), via processes that predispose to PTB. Intermittent preventive treatment in pregnancy (IPTp) with sulphadoxine-pyrimethamine (SP) prevents FGR due to mechanisms associated with sequestration of Plasmodium falciparum-infected erythrocytes in the placental intervillous space. However, drug resistance has become a concern, and this strategy has not been evaluated in malaria-endemic areas where Plasmodium falciparum and P. vivax are sympatric and endemic. Azithromyin (AZ), a broad-spectrum macrolide antibiotic frequently used to treat STIs, has favourable antimalarial properties and is deemed safe in pregnancy. The present research evaluated the safety and impact of IPTp with SP plus azithromycin (AZ) compared against a single course of SP and chloroquine at first antenatal visit on birthweight, malaria at delivery, carriage of STIs at second antenatal visit and maternal nasopharyngeal carriage of pathogenic bacteria at delivery in women residing in rural coastal Papua New Guinea (PNG). Secondary analyses included an evaluation of the effect of maternal macronutritient undernutrition on birth outcomes and potential modification of gestational weight gain by SPAZ. Fetal growth and risk factors for growth restriction were additionally evaluated using ultrasound technology in a subset of pregnancies. In the context of decreasing malaria prevalence in pregnant women in PNG, SPAZ-IPTp significantly reduced the risk of LBW and PTB, increased mean birthweight, reduced the risk of malaria but not anaemia at delivery, and reduced the risk of N. gonorrhoeae infection at second antenatal visit. SPAZ-IPTp was additionally associated with a temporary reduction of maternal nasopharyngeal carriage of Streptococcus pneumoniae and Haemophilus influenze at delivery, whilst simultaneously increasing the proportion of macrolide-resistant pneumococcal isolates. Maternal undernutrition (short stature, low mid-upper arm circumference at first antenatal visit, low pregnancy weight gain) was associated with an increased risk of LBW and reduced mean birthweight. Gestational weight gain was significantly increased amongst women who received SPAZ-IPTp, who also had a lower risk of postpartum maternal undernutrition. In a subset of women with ultrasound assessment of fetal growth, factors significantly associated with indicators of FGR were maternal undernutrition and anaemia. Comparison of fetal size parameters derived from low-risk PNG pregnancies with published fetal size standards suggest there may be differences in fetal growth according to ethnicity. This research suggests that the reduction of LBW by SPAZ-IPTp is achieved through several mechanisms. In addition to its antimalarial effect SPAZ may prevent LBW by clearing STIs and preventing and treating STI-associated chorioamnionitis. SPAZ also significantly increased gestational weight gain, which in turn was associated with increased birthweight: this suggests that SPAZ affects processes involved in nutrient uptake and storage. Further evaluations of azithromycin-based IPTp as an intervention to reduce the high burden of LBW and PTB in low-resource settings are warranted.
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ItemCharacterisation of novel epigenetic pathways in the malaria parasite Plasmodium falciparum( 2014)The protozoan parasite Plasmodium falciparum is the main agent responsible for the severe and potentially fatal form of human malaria, which is predominantly prevalent in tropical and sub-tropical countries. Malaria is transmitted through the bite of an infected female Anopheles mosquito and the parasites eventually invade and multiply asexually in red blood cells. Malaria pathogenesis is associated with the expression of clonally variant proteins on the surface of the infected erythrocyte which mediate cytoadhesion of the infected red blood cell to the endothelium of blood vessels. Thereby the infected erythrocytes sequester in the vasculature and avoid clearance by the spleen. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is the main variant antigen expressed on the surface of the infected erythrocyte that is responsible for cytoadhesion and immune evasion. PfEMP1 is encoded by the var multi-gene family and var genes are expressed in a mutually exclusive manner. To escape the human immune system, the parasite switches expression of the single expressed var gene. These pathogenic processes of invasion and cytoadhesion are tightly regulated by epigenetic mechanisms. Epigenetics refers to heritable changes in gene activity that are caused by changes in chromatin structure rather than changes in the DNA sequence. Nucleosomes are the basic repeat elements that make up chromatin and are formed by DNA wrapping around a histone octamer. Chromatin can assume two principle states; the open and transcriptionally competent euchromatin or the tightly packed and silent heterochromatin. The majority of the parasite’s genome exists in a euchromatic state. Genes in the euchromatin compartment are not necessarily active; those that are not are maintained in a readily inducible state and can be transcribed later in the parasite’s life cycle. Some of the gene families involved in pathogenic processes such as invasion and cytoadhesion are largely restricted to the subtelomeric and central clusters of heterochromatin and are transcriptionally silent but single genes can assume a euchromatic structure and be activated. One of the epigenetic mechanisms of altering this chromatin state is through the exchange of core histones with variant histones. P. falciparum expresses the histone variants H2A.Z, a conserved histone variant present in the promoters of genes in many eukaryotes, and H2B.Z, a unique apicomplexan specific histone variant. Pf H2A.Z is enriched at promoters of euchromatic genes in an expression independent manner. In var genes however, Pf H2A.Z is enriched at promoters of only active var genes and not silent var genes. Here I explore the interaction between Pf H2A.Z and Pf H2B.Z and their correlation with gene expression. Through co-immunoprecipitation assays on isolated mononucleosomes I found that in the malaria parasite Pf H2A.Z and Pf H2B.Z are present in the same nucleosomes. These double variant nucleosomes also harbour the active euchromatin marks H3K4me3, H3K9ac and H4K12ac but not the repressive mark H4K20me3. To further investigate at which sequences in the genome Pf H2B.Z is present, I performed chromatin immunoprecipitation. I found that Pf H2A.Z and Pf H2B.Z are significantly enriched at the transcriptional start sites of most genes, but this enrichment does not correlate with gene activity. However, in var genes, Pf H2B.Z together with Pf H2A.Z is enriched near the TSS of only the transcriptionally active var gene, suggesting that nucleosomes containing Pf H2A.Z and Pf H2B.Z may have a distinct function in var gene regulation. One mechanism by which Pf H2A.Z and Pf H2B.Z may play a role in gene expression is through the acetylation of their N-terminal tails, and H2A.Z acetylation correlates with transcriptional activity in yeast and humans. I tested for the presence of N-terminally acetylated Pf H2A.Z and Pf H2B.Z in active and inactive genes, but variant histone acetylation did not correlate with gene activity in the set of genes examined. Instead, acetylated Pf H2A.Z and Pf H2B.Z correlated well with total Pf H2A.Z and Pf H2B.Z occupancy, indicating that variant histone acetylation at the residues investigated may have a role that is distinct from gene regulation in P. falciparum. Epigenetic marks have been studied widely in the asexual stages of the parasite but have not yet been investigated in detail in the sexual stage gametocytes. Cellular differentiation in eukaryotes involves changes in gene expression that are regulated by modifications in chromatin composition, and recent evidence suggests that this might also be important in P. falciparum gametocytogenesis. Consistent with a role of epigenetic mechanisms in sexual differentiation, I found that the total levels of some active and inactive chromatin marks slightly fluctuate during gametocyte development. Chromatin immunoprecipitation in mid stage female gametocytes revealed that similar to the asexual stages, Pf H2A.Z is generally enriched at the TSS of active and inactive genes, however the levels of enrichment did not correlate between asexual and sexual stages revealing a novel stage specific plasticity in double-variant nucleosome occupancy in gene regulatory regions. In yeast the multi-subunit chromatin remodelling SWR1 complex exchanges the H2A.Z/H2B dimer for the H2A/H2B dimer in a replication independent manner. The Swr1 protein is the catalytic subunit of this complex, so to determine if a similar histone deposition mechanism occurs in P. falciparum, I characterised PfSwr1. Cellular fractionation showed that throughout the asexual life cycle PfSwr1 is predominantly found in the nucleus. To investigate if PfSwr1 is involved in the Pf H2A.Z/Pf H2B.Z deposition, co-immunoprecipitation assays were carried out. I found that PfSwr1 associates with both core and variant histones consistent with a multistep process for depositing the Pf H2A.Z/Pf H2B.Z dimer into nucleosomes. These studies give key insight into the epigenetic mechanisms and their possible role in gene expression in the malaria parasite.
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ItemHLA-G and malaria in pregnancy( 2011)HLA-G is an atypical class I MHC molecule that promotes tolerance and attenuates the immune response. It is highly expressed on the placental cells that invade the maternal tissues, and is one of the ways in which the immune system is modified in pregnancy to allow maternal tolerance of the foetus. The modification of the maternal immune response may also inadvertently promote tolerance of infections in pregnancy, and HLA-G may contribute to this tolerance of infection. Malaria is an infectious disease with high rates of morbidity and mortality globally. The most severe form is caused by the parasite Plasmodium falciparum. In endemic areas it is primarily a disease of children. Immunity is gradually acquired and disease amongst adults is uncommon. However during pregnancy, the high rates of infection return. Malaria in pregnancy is associated with morbidity and mortality for the mother and the foetus. This study is based on the hypothesis that the immune changes in pregnancy that allow maternal tolerance of the foetus may also contribute to the increased rate of malaria in pregnancy. It contributes to the understanding of the pathology of malaria in pregnancy, but also to the knowledge of the immunological response to malaria, of the role of HLA-G in response to infection and how the changes in placental immunology may contribute to other infections in the placenta. This study shows there is an association between HLA-G genotype and Plasmodium falciparum infection in the placenta, that genotype only corresponds with mRNA for a specific pattern of malaria infection in the placenta, and that HLA-G expression correlates with an important consequence of malaria in pregnancy, low birth weight. These findings suggest the hypothesis that placentae that can quickly reduce their HLA-G expression are able to mount a more efficient immune response to the pathogen. Those who have a persistently elevated HLA-G are unable to mount an efficient immune response, and develop a chronic infection. The study also demonstrates a reduction in cytokine response to Plasmodium falciparum in the presence of placental cells, although this is not mediated by HLA-G.