Medicine (RMH) - Theses
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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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ItemEpidemiological and immunological studies of treatment for pregnancy associated malaria( 2010)Pregnant women are highly susceptible to malaria, and malaria in pregnancy causes a number of adverse outcomes such as maternal anaemia and delivery of low birth weight babies. Thus pregnant women are specifically targeted in malaria prevention efforts with control measures including IPTp and bed nets. Pregnant women are uniquely susceptible to malaria because Plasmodium falciparum infected erythrocytes can adhere to the placenta. This is mediated by the variant surface antigen (VSA) family Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Accumulation of infected erythrocytes in the placenta may subsequently result in acquisition of immunity targeting this protein especially among multigravid women. Longitudinal data collected over 9 years from Malawian pregnant women demonstrated decreased prevalence of peripheral and placental malaria, maternal anaemia and LBW. In the same time frame coverage with IPTp SP and bed nets increased. SP IPT doses were associated with protection against placental parasitaemia, maternal anaemia and LBW from 1997-2001, but not from 2002-2006. Bed net use was associated with protection from peripheral or placental parasitemia and LBW throughout the study, but not with anaemia. These results indicated decreased maternal malaria infection correlated with improved pregnancy outcomes. Increased bed net coverage explains more of this change than SP use. SP resistance may be compromising its effectiveness. In chapter 4 of this thesis, I explored the protective effect of immunity against pregnant associated malaria variant surface antigens (VSA-PAM) using sera from a group of Malawian pregnant women who undertook anti-malarial treatment. My results showed that the level of immunity against VSA-PAM was associated with improved anti-malarial treatment outcomes and decreased maternal anaemia at delivery. The finding presented in this thesis validated further studies investigating the role of antibodies against VSA-PAM in protecting against placental malaria infection. The decreased protective effect of SP-IPTp also urged the importance of replacing SP from the first line anti-malarial treatment drugs.