Medical Biology - Theses
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ItemA conserved molecular mechanism of erythrocyte invasion by malaria parasites( 2023-10)Malaria is major global health burden causing over 240 million cases every year and leading to over 600,000 deaths mostly in pregnant women and children under the age of five. There is an urgent need to development novel therapeutic interventions for the control and elimination of malaria. During infection, malaria parasites must invade host erythrocytes in order to live within them. Invasion is a complex multi-step process that involves many molecular host-parasite interactions. In Plasmodium falciparum, the deadliest species of the parasite, the invasion protein PfRh5 assembles into a complex to bind its receptor basigin on the erythrocyte surface. Recent work has revealed two novel members of this complex, PfPTRAMP and PfCSS, form a heterodimeric platform for PfRipr, PfCyRPA, and PfRh5 binding. The PfPTRAMP-PfCSS-PfRipr-PfCyRPA-PfRh5 (PCRCR) complex, and its engagement with basigin, is essential for P. falciparum invasion. PfRh5 does not have an orthologue in all species of malaria, however PTRAMP, CSS and Ripr orthologues are present across the entire Plasmodium genus. This thesis sought to investigate these conserved proteins in other malaria species to further dissect the essentiality of these proteins for Plasmodium invasion more broadly. Orthologues of PTRAMP, CSS and Ripr from two important species of malaria, P. vivax and P. knowlesi, were investigated using recombinant expression and biophysical analysis. Assessment of complex formation shows a conserved assembly of the three proteins in both species, with similarities to P. falciparum. Structural determination of part of the complex revealed the basis of heterodimer formation between PTRAMP and CSS. Antibodies and nanobodies were produced and exhibit a high degree of cross-reactivity between species. A novel protein was identified that may bind to the complex and impart an erythrocyte binding function. The function of the complex and its components in invasion was confirmed using ex vivo invasion assays in Cambodian P. vivax field isolates. Taken together, this thesis shows that a three-membered complex consisting of PTRAMP, CSS and Ripr is conserved in three species of Plasmodium, likely forming a common invasion scaffold in all species of the genus, suggesting a conserved invasion mechanism with implications for cross-species vaccine development for the control of both P. vivax and P. falciparum malaria.
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ItemDevelopment of antibody therapeutics for deadly infectious diseases: malaria and COVID-19( 2021)Monoclonal antibodies are one of the most powerful drugs used in the treatment of human diseases, particularly for autoimmune diseases and cancer. The development of therapeutic antibodies to combat infectious diseases is expanding, with successful treatments available against viral diseases such as COVID-19, Ebola virus, HIV and RSV. The use of monoclonal antibodies complements vaccines by providing immediate protection, preventing disease in immunocompromised people and in containing emerging disease outbreaks. In this thesis, I aimed to characterise neutralizing antibodies against two of the deadliest infectious diseases that affect global populations, malaria and COVID-19. The growth and replication of both these pathogens are critically dependent on their entry into host cells. Neutralizing antibodies that block pathogen entry into host cells can prevent infection and reduce severe disease. Plasmodium vivax is the most widespread relapsing human malaria, which invades reticulocytes through the critical interaction between P. vivax reticulocyte binding protein 2b (PvRBP2b) and human Transferrin receptor 1 (TfR1). I identified TfR1 residues that are critical for complex formation with PvRBP2b. In addition, I characterised naturally acquired human monoclonal antibodies to PvRBP2b, and using structural biology, revealed the epitopes of eight high affinity inhibitory antibodies that block complex formation through different mechanisms. The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which utilizes its spike protein to engage human angiotensin-converting enzyme 2 (ACE2) for host cell entry. I performed deep mutational scanning on a lead neutralising nanobody against SARS-CoV-2 to improve its potency, stability and affinity. These structural studies increase our understanding of host-pathogen interactions and the antibodies that block them, which can inform the development of antibody therapeutics or vaccines.
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ItemPlasmodium vivax naturally acquired immunity: patterns and influences( 2020)Malaria is caused by Plasmodium parasites and Plasmodium vivax is the dominant Plasmodium spp. in low-transmission regions outside of Africa. Due to the unique biological characteristics of this parasite, such regions often feature asymptomatic patients with sub-microscopic parasitaemia and relapses. Naturally acquired antibody responses are induced after Plasmodium infection, providing partial protection against high parasitaemia and clinical episodes. Serology is a promising tool for monitoring transmission levels, estimating past and recent exposure and identifying populations at risk of infections. However, due to key gaps in our knowledge of naturally acquired antibody responses to P. vivax, the full potential of serology has not yet been reached. This thesis aimed to establish antibody kinetics against a large panel of P. vivax antigens following infections in western Thailand, and investigate the factors potentially associated with the acquisition and development of antibody responses. A multiplexed bead-based assay was first established and antibody measurements against more than 50 antigens were taken in P. vivax-infected individuals from western Thailand following symptomatic and asymptomatic infections. I found that most P. vivax antigens followed a highly similar post-infection kinetic pattern in the absence of any boosting infections. The magnitude and longevity of antibody responses varied between antigens, antibody subclasses and subtypes. An assay quantifying the antigen-specific memory B cell responses was established and verified to determine the role of memory B cells on antibody kinetics for future experiments. Lastly, I reported that the genetic diversity of an antigen sequence had a significant impact on antigen-specific antibody responses, and such impact increased in individuals with more past exposure and mature immunity. The findings presented in this thesis provide novel insights into naturally acquired immunity development to P. vivax and support the decision of taking genetic diversity of antigen sequences into consideration for the development of highly efficacious sero-surveillance tools and vaccines.
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ItemExploring Plasmodium vivax transmission dynamics and population genetics through genetics and genomics( 2019)Globally, malaria remains one of the most life-threatening and highest burden infectious diseases. Amongst the six Plasmodium species that cause malaria in humans, Plasmodium vivax and Plasmodium falciparum are the predominant cause of morbidity and mortality. P. falciparum is the most virulent species; however, P. vivax has a wider geographic range and is more difficult to control and eliminate. The burden of malaria in Papua New Guinea (PNG) is among the highest in the Asia Pacific region with a hyperendemic transmission of P. falciparum and P. vivax. Since 2004, PNG has intensified malaria control efforts to alleviate this malaria burden and with the ultimate goal to eliminate malaria from the country by 2030. P. vivax is recognized as a major obstacle to achieve this elimination goal as evidenced by a smaller reduction in P. vivax prevalence relative to P. falciparum after intensified control efforts throughout the country. Indeed, in many areas where P. falciparum and P. vivax are co-endemic, P. vivax is now becoming the dominant malaria parasite. The main reason for this shift in malaria epidemiology may result from the use of malaria control measures primarily targeted at P. falciparum, and the fact that P. vivax is more difficult to treat and diagnose because of its distinct biology, in particular, its ability to form dormant liver-stage infections, known as hypnozoites. Therefore, approaches to eliminate P. vivax malaria need to address this unique parasite biology through the development and adoption of novel tools and strategies. To facilitate the discovery of novel strategies to contain Vivax malaria, this PhD project aimed to investigate P. vivax biology, molecular epidemiology and transmission dynamics using parasite population genetics, and through the development of a novel barcoding tool for high resolution genotyping. To address the existing knowledge gaps around the impact of the unique biology of P. vivax on malaria epidemiology, I compared the prevalence, multiplicity of infection (MOI) and genetic diversity of sympatric P. vivax and P. falciparum parasite populations throughout PNG (Chapter 3). A total of 892 P. vivax and 758 P. falciparum isolates from 16 Provinces (including 49 villages) were obtained from a 2008/2009 PNG national malaria survey completed after the nationwide intensification of control. All P. vivax and P. falciparum isolates were genotyped using the size polymorphic markers Pvms16/Pvmsp1F3, and Pfmsp2, respectively. The comparative analyses of within host multiplicity of infection (MOI) and population level genetic diversity showed striking differences between these sympatric Plasmodium species. Approximately 70% of P. vivax infected individuals carry more than one clone (i.e. MOI>1, known as polyclonal infections) compared to 20% for P. falciparum. There was a strong association between MOI and infection prevalence for P. falciparum but not for P. vivax, consistent with the maintenance of high infection complexity for P. vivax despite the reduction in new infections resulting from control interventions, and therefore suggesting a higher effective transmission potential for this species. The results showed that both species have high genetic diversity in populations throughout PNG. However, while immune selection on the antigen loci used (Pfmsp2 and Pvmsp1F3) and unusually high diversity of Pvms16, facilitates the detection of unique clones, it may limit the resolution of the comparative genetic diversity analyses. To further explore P. vivax genetic diversity and population structure throughout PNG, I then conducted a detailed population genetic analyses of P. vivax using a panel of 10 well-validated and putatively neutral microsatellite markers (Chapter 4). I genotyped a total of 230 P. vivax isolates with low complexity infections (maximum MOI=2) from eight geographically and ecologically distinct regions (“geographic populations”) throughout PNG. Population genetic analyses of microsatellite haplotypes/genotypes revealed a spectrum of genetic diversity, which was associated with prevalence. Multilocus linkage disequilibrium (mLD) was identified in six of the eight parasite populations indicating significant inbreeding. Strong geographic population structure (clustering of isolates according to their geographic origins) was identified among regions, dividing the Mainland (lowlands), Highlands and Islands, and the genetic divergence was significantly correlated with geographic distance. Sources and sinks of vivax transmission and parasite population connectivity between regions were defined. Patterns of P. vivax gene flow appear to follow major human migration routes, implying that the human host could be the main driver for the spread of infections and potentially, drug resistance. This find suggests assessing the contribution of the P. vivax asymptomatic reservoir and imported infections for sustainable local transmission are necessary to design targeted control interventions. Small panels (~10) of microsatellite markers used have been the “gold standard” genotyping tool for population genetic analyses of P. vivax. However, because of their small number, extremely high diversity and propensity for genotyping error they may have limited resolution for identifying local population structure in high transmission areas (such as the north coast of PNG). In addition, several other technical challenges make them unsuitable for large scale malaria surveillance. Therefore, I developed a novel genotyping tool known as a ‘single nucleotide polymorphism (SNP) barcode’ for high resolution monitoring of P. vivax parasite populations in PNG (Chapter 5). The barcode was specifically designed to capture the genetic diversity of PNG parasite populations by using SNPs identified amongst 20 high quality published PNG genomes. A total of 24283 SNPs with minor allele frequencies (MAF) of more than 10% were identified in putatively neutral regions of the P. vivax parasite genome. A filtered subset of 178 evenly spaced informative SNPs were selected for development of a high throughput parallel targeted amplicon sequencing (PTAS) assay using a multiplex PCR and Illumina MiSeq platform. To validate the developed barcode, I tested the genotyping success of 20 SNPs using small number of mono- and polyclonal infections, followed by genotyping of 96 P. vivax isolates from four catchment areas on the north coast of PNG collected in 2012-2014. Compared to the available microsatellite data for the same samples (E. Kattenberg, unpublished), the SNP barcode revealed more variable genetic diversity and stronger population structure with greater resolution to assign genotypes according to their geographic origins than the microsatellite panel. The developed SNP barcode has low genotyping error, is relatively cheap (~$-26-30/isolate) and transfer of the technology to field settings like PNG is technically feasible. As transmission declines, measuring changes in parasite population structure is important to assess the impact of control on malaria transmission, and to guide future control and elimination of malaria. To explore P. vivax population genetic signals with changing transmission due to intensified control, I applied the developed SNP barcode assay to a total of 376 P. vivax isolates obtained from serial cross-sectional surveys in two north coast Provinces of PNG (East Sepik and Madang) (Chapter 6). Samples were collected between 2005 and 2016, a period of initially declining transmission and later rebound. This investigation revealed interesting spatio-temporal patterns with declining transmission including a reduction in polyclonal infections, genetic diversity and population connectedness as indicated by an increase in population structure. Significant multilocus linkage disequilibrium (mLD) and a large cluster of parasites with high genomic relatedness (identity by descent, IBD) was also detected at low transmission. These population genetic signatures signify a strong population bottleneck, highly clonal transmission and inbreeding resulted from significant reductions in prevalence. For Madang Province, I also observed some genetic signatures related to the sources of malaria rebound providing insight into malaria resurgence through increasing migration of parasites from other endemic areas in addition to expansion of the residual parasite population. Containment of P. vivax infections and drug resistance may be facilitated by the existence of distinct parasite populations at low transmission yet will be undermined by increased parasite migration due to frequent human movement between endemic areas. Overall, this thesis demonstrates how genotyping a relatively small number of parasite isolates from representative geographic areas can provide surrogate signals of malaria transmission dynamics and infection spread. Moreover, it reveals the power of molecular epidemiology and population genetics and genomics for malaria surveillance that in combination with epidemiological data will allow the prioritisation and optimisation of malaria control and elimination strategies. I also discuss potential approaches to integrate population genetics and genomics into current malaria surveillance systems and how the control efforts can be benefited from this approach.
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ItemCharacterizing targets of naturally acquired immunity and correlates of clinical protection against Plasmodium vivax( 2018)Plasmodium vivax is the most widely distributed human malaria parasite and accounts for 13 million clinical cases per year. Although it has significant impact on global health systems and can cause severe illness and mortality, only two P. vivax candidate vaccines have reached Phase I clinical trials. Passive transfer of sera from clinically immune individuals significantly protects non-immune recipients from high parasite burden and clinical symptoms. Such observations have established that antibodies are critical for naturally acquired immunity to malaria infection. Therefore, the characterization of antigenic targets of naturally acquired immunity remains a key step towards the identification of novel vaccine candidates. This thesis investigates the breadth of antibody responses to a panel of P. vivax proteins involved in parasite invasion into red blood cells in three longitudinal cohorts from Brazil, Thailand and Papua New Guinea with different transmission intensities. Collectively, the main findings of this thesis have showed several strong associations of antibody responses towards several P. vivax antigens with protection from clinical malaria. Additional protective effects are identified for combinations of P. vivax ligands which supports the inclusion of different P. vivax parasite ligands in a multi-component vaccine.
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ItemThe molecular epidemiology of Plasmodium spp. in Solomon Islands( 2018)Malaria is a major global disease burden with significant mortality rates amongst young children. The renewed commitment to eradicate malaria along with intensified control efforts has drastically reduced malaria transmission worldwide. In Solomon Islands, the malaria burden had steadily reduced over the last 20 years, achieving 90% reduction in malaria cases, and now the country is aiming to eliminate malaria by 2030. The journey towards malaria elimination requires knowledge of the local malaria epidemiology to identify the population at risk, as well as to understand the underlying risk of infection. In 2013, a longitudinal cohort study was conducted in Ngella. This study was a follow up from the 2012 cross-sectional survey of all ages in Ngella, which previously identified with a relatively higher prevalence of P. vivax infections (13.4% or 468 P. vivax infections) compared to only five P. falciparum infections, with majority of infections asymptomatic and submicroscopic (Waltmann et al., 2015). However, the underlying Plasmodium spp. infection dynamics in Ngella is unclear and it is unknown if P. falciparum infections has disappeared. This follow up study therefore aimed to investigate the local epidemiology of asymptomatic and submicroscopic Plasmodium spp. infections among a cohort of children aged between six months to twelve years old, which will be presented in this thesis. During the 2013 paediatric cohort study, monthly active case detection (ACD) and passive case detection (PCD) were conducted from May 2013 until April 2014. Plasmodium spp. infections were detected by light microscopy, rapid diagnostics test (RDT) and quantitative polymerase chain reaction (qPCR). The qPCR-detectable infection prevalence for P. vivax infections among the 860 children that attended at least six ACD visits was 11.9% (989 episodes), while P. falciparum infections was 0.3% (27 episodes). Most infections detected were submicroscopic (68%) and/or asymptomatic (92.8%). Of children who had at least one P. vivax infection, two thirds had subsequent infections. Genotyping of these P. vivax infections revealed that the subsequent infections have similar genotypes as the initial infections. Since the recorded primaquine (PQ) treatment among the cohort children was uncommon, this indicated that relapses from hypnozoites contributed a majority of P. vivax infections and anti-relapse treatment is important. Heterogeneous transmission and strong individual variation in risk were observed, suggesting that future malaria control program should tailor made accordingly to the varying risk of infections within Ngella and individuals. Drug treatment with PQ among P. vivax infected individuals is nonetheless challenging. It requires the individual to be glucose-6-phosphate dehydrogenase (G6PD) normal to avoid haemolysis during treatment and have a normal cytochrome P450 2D6 (CYP2D6) phenotype for an efficient treatment. All cohort children were screened for G6PD deficiency prior to the start of the study using the BinaxNOW G6PD Test (Alere Inc., USA), a qualitative rapid diagnosis test for G6PD enzyme activity in human whole blood. If any children were identified with P. vivax infections during the study, only G6PD normal children were administered with treatment for both liver (PQ) and blood (artemether-lumefantrine) stages. Meanwhile, P. vivax infected G6PD deficient children were treated with blood stage drug only. Without PQ treatment, these G6PD deficient children were observed to have significantly higher risk of P. vivax infection. This highlights G6PD deficiency is an obstacle for hypnozoite treatment. As such, it is important to screen individuals for G6PD and CYP2D6 prior to PQ drug treatment. This leads to one of the main aims of this thesis, which is to develop a high throughput population genetic screening assay with long read sequencing capability of both G6PD and CYP2D6 genes. A G6PD and CYP2D6 population based genotyping assay can serve to identify populations at risk of unsafe or ineffective treatment with PQ, thus improving the safety and efficacy of PQ treatment among P. vivax infected individuals in future. The occurrence of P. falciparum infections in Ngella was observed to be sporadic yet persisting throughout the year. By employing ten microsatellite markers and 192 SNP barcodes, the genetic diversity, structure and connectivity of the P. falciparum population in Ngella was studied. Comparative examination of the 27 P. falciparum infections from the cohort along with the five P. falciparum infections from previous cross-sectional survey in Ngella, against other villages (Auki and Tetere) of neighbouring islands of P. falciparum infections revealed that the P. falciparum population in Ngella is genetically related and inbred. Evidence of bottleneck event among the Ngella’s P. falciparum population was observed as well. The data presented in this thesis highlighted the risk factors associating with P. vivax infections, as well as the genetic structure of residual P. falciparum in Ngella. The development of large-scale genotyping assay for G6PD and CYP2D6 with long read sequencing allows population level genetic risk assessment for future radical cure of P. vivax infections. Collectively, these findings may support follow up elimination strategies in Solomon Islands or in other low malaria transmission setting with both P. falciparum and P. vivax coexist. For instance, a safe and efficient PQ mass drug administration can be organised among residents after genetic risk evaluation and high P. vivax transmission pockets identification. Evidence from population genetic approaches such as the identified population bottleneck event and low genetic diversity among the P. falciparum population can be useful knowledge that malaria control program has work effectively and further strengthening of the local control program could possibly eliminate P. falciparum.
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ItemNaturally acquired humoral responses to Plasmodium vivax and Plasmodium falciparum: identification of antigenic targets to inform rational biomarker and vaccine development( 2016)Malaria is an infectious disease caused by Plasmodium spp. parasites, transmitted by the bite of infected Anopheles mosquitoes. Among the five species that can cause disease in humans, P. falciparum and P. vivax are responsible for the majority of the cases and deaths. Due to increased political commitment and funding, the last decades have experienced a dramatic reduction in the burden of malaria, with several countries now attempting to permanently eliminate this disease. Achieving the goal of malaria elimination would be greatly facilitated by the development of biomarkers that can identify the remaining populations at-risk, as well as an effective vaccine. However, while it is clear that individuals living in endemic areas become gradually protected against malaria disease, the targets and mechanisms underlying the acquisition of natural immunity are complex and still poorly understood, hindering the development of such tools. This thesis aimed to investigate comprehensive panel of P. vivax and P. falciparum proteins as targets of natural immunity in Asia Pacific populations, and how this information can be used to inform rational vaccine and biomarker development. Strong associations of antibody responses to both novel and known P. vivax antigens with protection against clinical malaria were identified, as well as optimal antigenic combinations with predicted protective efficacy above 90%. By comparing humoral responses to P. vivax and P. falciparum, this thesis shows that early immune responses are markers of exposure and thus increased risk, whereas prolonged exposure and higher antibody titers are required to achieved clinical protection. The findings of this study support the development of a highly efficacious multicomponent malaria vaccine, and the use of serology as a surveillance tool.
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ItemThe molecular epidemiology of malaria in Solomon Islands( 2016)Historically, Solomon Islands in the Southwest Pacific has endured considerable P. falciparum and P. vivax burden. In the last 20 years, it has achieved 90% reduction in malaria cases through sustained, intensified malaria interventions (long lasting insecticide nets, indoor residual sprays and artemisinin-combination therapy) and is aiming for elimination by 2030. In 2012 and 2013, we conducted two cross-sectional surveys (study 1, all age, n=3501; study 2, age 0.5-12 years, n=1078) in Ngella, an area of low to moderate transmission. We aimed to investigate the natural reservoir and local epidemiology of P. vivax and P. falciparum. The contrast was striking. In the 2012 survey, only five clonal P. falciparum infections were identified from a single village and had the same msp2 genotpye. P. vivax prevalence was found to be moderately high (PCR, 13.4%), with predominantly afebrile, submicroscopic infections. The P. vivax infections displayed high genetic complexity (by genotyping with msp1F3 and MS16) and considerable spatial heterogeneity among and within different Ngella regions, and even at sub-village level with some households disproportionately harboring more infected co-inhabitants than others. In the 2013 study, a further seven P. falciparum infections were found in multiple locations, indicating that transmission of this species is continuing but at very low levels and infections are predominantly asymptomatic. To investigate the transmission scenario of the two species in more detail, we undertook population genetics analyses. We typed the five 2012 P. falciparum infections at 10 polymorphic microsatellite loci and 323 P. vivax infections at nine microsatellite loci. The five P. falciparum infections also clonal by this panel of 10 markers. Subsequent analyses of diversity (FST, GST, Jost’s D) and structure (Bayesian clustering) for P. vivax, revealed a genetically diverse population, but spatially fragmented, even among villages 6-15km apart. This indicates that whilst P. vivax may be more difficult to eliminate than P. falciparum, local parasite populations of both species have been affected by control interventions. A noteworthy epidemiological result from the 2012 survey was that living in a household with at least one other P. vivax carrier increased the risk of P. vivax infection, suggesting possible intra-household transmission. Subsequent analysis of genetic relatedness of P. vivax infections within households vs. among households indicated supported this hypothesis. Isolates from the same household were more genetically related than isolates from different households, and a high level of genetic kinship was retained among households located up to 100 meters of each other. Associations of P. vivax infection with human genetic factors known to confer protection against infection (α-thalassemia and Southeast Asian ovalocytosis, SAO) have been investigated in a second cross-sectional study conducted in 2013 in children aged 6 months to 12 years of age. SAO was not found in Ngella, whereas approximately a third of 1078 subjects were found to harbor the α-thalassemia alleles. The findings presented in this thesis will be discussed in the context of factors which may impact on follow-up elimination strategies in Solomon Islands, the Southwest Pacific and elsewhere in the endemic world where both P. falciparum and P. vivax are co-endemic.