Medicine (RMH) - Theses
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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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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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ItemPregnancy-malaria: immunity and a mechanism for low birth weight( 2010)Introduction: Pregnant women are more susceptible to Plasmodium falciparum malaria than their non-pregnant peers and the consequences for the mother and child can include maternal anaemia and low birth weight (LBW). The development of immunity to P. falciparum in pregnancy is of importance for vaccine development and identifying women at risk of disease. Knowledge of the mechanisms of pathogenesis, such as how malaria results in LBW, is needed to effectively identify therapeutic targets and prevent adverse outcomes. Methods: In this thesis, the immune response towards P. falciparum was assessed in a cohort of women receiving intermittent preventive treatment in pregnancy (IPTp) by measuring antibody towards the surface of placental-binding parasitised red blood cell using flow cytometry. The dynamics of these antibody levels, from 14-26gw until six months post partum, were described, and the protective relationships between antibody and clinical outcomes in the women were examined. A mechanism for LBW in pregnancy-malaria was also investigated. This involved looking at the relationship between placental levels of proinflammatory cytokine interleukin-11β (IL-1β) (as measured by ELISA) and birth weight. And, modelling nutrient transport across the placenta in pregnancy-malaria using radiolabelled amino acids, placental cell lines and various malaria-associated stimuli, such as pro-inflammatory cytokines and monocyte:parasite products. Results: Among women receiving IPTp who are at low to moderate risk of parasitemia, development of immunity to the placental-binding parasite was dynamic. Many women in their first pregnancy did not develop an immune response. However, there was no evidence that this leads to lower immunity in successive pregnancies. In addition, it was found that immunity measured mid pregnancy did not predict protection against clinical outcomes such as birth weight, maternal haemoglobin or gestation length later in pregnancy. Regarding a mechanism for LBW, the proinflammatory cytokine IL-1β was increased in placentas of women with placental malaria and inflammation. This cytokine was negatively associated with birth weight and was shown to be able to decrease amino acid transport in an in-vitro model. Monocyte:parasite products in the supernatant of an inflammatory cell and parasite co-culture were also able to decrease placental amino acid transport in an in-vitro model. Interestingly pro-inflammatory cytokines TNF-α, IL-18 and chemokine IL-8 did not alter amino acid transport in the in-vitro model. Conclusions: Antibody dynamics and the relationships between antibody and outcome were described. Lower than expected parasitemia prevalence and a lack of association between antibody early pregnancy and outcome suggests that evaluating the protective efficacy of pregnancy-malaria vaccines may be difficult in the contexts of routine IPTp administration and falling malaria prevalence’s. Results from the nutrient transport models support a possible pathogenic mechanism for foetal growth restriction in placental malaria where monocyte:parasite products, such as IL-1b, decrease the activity of placental amino-acid transporters. These products may be therapeutic targets for the prevention of LBW in placental malaria.
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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.