• The application of transcriptional profiling to uncovering of the biology of the pre-erythrocytic stages of Plasmodium and the identification of protective pre-erythrocytic stage antigens

      Williams, Calvin; Azad, Abdu F. (2009)
      Malaria, caused by infection with parasites of the Plasmodium genus, remains a serious public health threat. Plasmodium sporozoites, injected into humans by mosquitoes, migrate to the liver, invade hepatocytes and transition into merozoites. Radiation-attenuated sporozoites (RAS) are incapable of forming merozoites and shown to elicit sterile protective immune responses in humans but technical reasons prevent large-scale human immunizations using RAS. T cells, elicited by sporozoites in lymph nodes draining the site of infection that target Plasmodium infected hepatocytes, mediate this immune response. Similarly, wild-type sporozoites (wtSPZ) given to mice or humans under chloroquine treatment can illicit protection from wtSPZ challenge, suggesting that wtSPZ can be immunogenic. Together these observations imply that protective Plasmodium antigens are expressed by RAS, wtSPZ, and liver stage parasites. Unfortunately, the molecular biology of the clinically silent pre-erythrocytic stages, composed of the sporozoite and liver stages, remains largely uncharacterized. Improved understanding of the biological processes required for progression through the pre-erythrocytic stages could lead to the identification of new drug and vaccine targets. The hypothesis of this project is three part: one, the biological processes important for progression through the pre-erythrocytic stage are reflected in the transcriptional profile of the constituents of the pre-erythrocytic stages; two, RAS developmental arrest is mediated by radiation induced DNA damage that results in transcriptional changes; and three, the protective pre-erythrocytic antigens are contained within a set of commonly expressed pre-erythrocytic stage genes. To test these hypothesis, transcriptional analysis of liver stage parasites, RAS, and wtSPZ was performed, resulting in the identification of 1100 genes significantly differentially expressed during the pre-erythrocytic stages, represented within this data set are many genes involved in a variety of cellular functions. Next, the microarray results present above were combined with other Plasmodium sporozoite and liver stage proteomic and transcriptional data sets to yield a list of ~4000 P. yoelii proteins, with P. falciparum homologs and evidence of pre-erythrocytic stage expression. Application of bioinformatics tools to this combined data set yielded over 3300 Plasmodium proteins potentially involved in the protective immune response observed after immunization with Plasmodium RAS.
    • Deciphering relatedness and population demographics in diverse population structures by leveraging haplotype and rare variant sharing detected from whole genome sequencing

      Shetty, Amol Carl; O'Connor, Timothy D; 0000-0001-8790-7649 (2019)
      Genealogical analysis using genomic variants is essential for a variety of applications in human genetics such as estimating population structure, migration events and evolutionary history. The 1000 Genomes Project is an example of a study of human genomic diversity and continental population structure. Multiple studies illustrate the utility of genetic variation for the reconstruction of human migratory patterns within and between continental populations and the demographic events influencing evolutionary history. Current methods for assessment of population structure and genetic relatedness use individual genetic loci and do not take full advantage of the large number of markers provided by whole genome sequencing techniques. More recently, haplotype sharing or identity by descent (IBD) estimates have been used as a promising method to elucidate demographic admixture/migratory events. This dissertation focuses on the application of IBD sharing to decipher genetic relatedness and demographic events that influence population substructure. Knowledge of genome-wide patterns of IBD sharing among individuals helped distinguish between ancient and recent demographic events and detect fine-structure among the recently expanded and admixed New World populations from Peru. This addresses the gap in knowledge regarding the population fine-structure of indigenous and admixed communities from geographically distinct regions of Peru. IBD sharing, primarily utilized to study human demography, was applied to study fine-structure and demography of haploid malarial parasite populations in Southeast Asia which helped elucidate the migratory patterns of the parasite and guide the elimination strategies of the World Health Organization (WHO). Current IBD methods accurately detect long segments based on information from common variants. However, cohorts involving cryptic relatedness mostly share short IBD segments. In light of this limitation, rare variants arising from recent dramatic events of population expansion convey more information on short IBD segments than common variants. This knowledge of IBD sharing leveraged by rare variants influences the timescales at which familial relatedness and population structure can be assessed. In sum, this dissertation illustrates the utility of IBD segments of variable lengths and the accumulation of rare variants within these segments to detect fine-scale population structure at different evolutionary timescales and fills the gaps in knowledge in both human and non-human populations.
    • Designing Next Generation Genomics and Serological Tools for Surveillance of Plasmodium vivax Malaria to Guide Elimination Efforts in Southeast Asia

      Agrawal, Sonia; Plowe, Christopher V.; 0000-0003-4484-7433 (2019)
      Malaria is a major global health problem caused by mosquito-borne, protozoan parasites belonging to the genus Plasmodium. Plasmodium vivax, the human malaria parasite with the widest global distribution, accounts for majority of the total malaria cases outside sub-Saharan Africa. The inability to establish long term in vitro culture system and low parasite densities, combined with high levels of human genomic DNA isolated from patient samples with P. vivax infections, makes it difficult to obtain sufficient amounts of parasite DNA for whole genome sequencing (WGS). New, reliable, highly sensitive and specific methods are needed to produce high quality P. vivax WGS data. Genome-wide analyses of the parasite, using WGS, have the potential to improve our understanding of parasite population dynamics and help identifying locations that serve as possible transmission sources and sinks. Additionally, protein microarrays that can simultaneously measure human antibody responses to a large number of Plasmodium antigens have the potential to identify P. vivax specific biomarkers to detect not only current but also past malaria infections, providing a more sensitive surveillance tool for identifying human populations at risk. To address these needs, using Roche/NimbleGen SeqCap EZ whole genome capture technology, high quality WGS data was generated from P. vivax clinical samples collected from the China-Myanmar border. This new genome-wide data along with publicly available WGS from circulating isolates in Southeast Asia were utilized to characterize parasite genetic diversity and relatedness, population structure, complexity of infection, and distinguish locally transmitted infections from imported P. vivax infections revealing clonal parasite population on the China-Myanmar border. Using protein microarray analyses, several P. vivax specific serologic markers during active infection were identified that may serve as useful biomarkers of current or recent P. vivax infection supporting the possibility of serology as a tool for estimating species-specific malaria exposure to P. vivax in heterogeneous malaria transmission settings. The combination of next generation tools attempted to be designed as part of this dissertation will help improve the understanding of the genomic epidemiology and estimates of transmission patterns of this human malaria parasite, thus, guiding rational P. vivax malaria control and elimination policies in Southeast Asia.
    • The Effect of Drug Pressure and Transmission Setting on Sulfadoxine-Pyrimethamine Resistant Plasmodium falciparum Haplotype Prevalence and Selective Sweep Characteristics, in Malawi

      Artimovich, Elena M.; Takala-Harrison, Shannon (2014)
      Background: The continued expansion of resistance to anti-malarial chemotherapies is a threat to public health, and to malaria control and elimination. The reexpansion of drug sensitive parasites after the removal of drug pressure has renewed interest in epidemiological factors affecting resistance haplotype dynamics, in the hopes that previously abandoned drugs might once again find clinical utility. Objectives: Estimate the effect of changes in drug pressure and different malaria transmission settings on sulfadoxine-pyrimethamine (SP)-resistant haplotype prevalence and characteristics of selective sweeps. Methods: DNA was extracted from dried blood spots representing malaria infections from three time periods (high-SP use 1999-2001, transition-period 2007-2008, low-SP use 2012) of drug pressure in Malawi and three transmission settings (urban-low, rural-moderate, rural-high). Pyrosequencing and microsatellite genotyping were performed on all samples to determine haplotype prevalence and sweep characteristics. Changes in haplotype prevalence were assessed via Chi-squared tests and changes in sweep characteristics via permutation. Results: We observed the persistence of the DHFR 51I/59R/108N and DHPS 437G/540E haplotypes, five years after reduction in SP pressure as well as an increase in the prevalence of DHPS 437G/540E/581G haplotype. Selective sweeps indicated little to no fitness cost to the DHFR 51I/59R/108N and DHPS 437G/540E haplotypes in the absence of strong SP pressure. A decline in polyclonal infections was found across the three time periods. No significant difference in haplotype prevalence was found between transmission settings. Sweep characteristics could suggest divergent evolutionary history in the rural-moderate transmission setting. Conclusions: There is little to no fitness cost of SP-resistance in the absence of strong SP pressure in these three transmission settings within Malawi. The reexpansion of SP sensitive parasites in the region is not expected under current epidemiological conditions. Reduction in the amount of malaria in the region could further reduce the likelihood of reexpansion through the elimination of rare haplotypes due to genetic drift.
    • The Effect of Malaria During Pregnancy on Infant Susceptibility to Malaria

      Boudova, Sarah Elizabeth; Laufer, Miriam K. (2016)
      Malaria is a major cause of morbidity and mortality. Malaria during pregnancy threatens the health of both the mother and the child, with long-lasting consequences on infant health that may be attributable to the impact of maternal malaria on the fetal immune system. It is unknown what effect different manifestations of pregnancy-associated malaria have on child immunity and health. Moreover, the timing during pregnancy when malaria needs to be prevented to maximize mother-infant health is not known. In a longitudinal study in Malawi we examined the effect of maternal peripheral and placental malaria on infant risk of malaria, by following mother-infant pairs from early in pregnancy through the first two years of the child's life. We conducted active and passive surveillance for malaria infection and disease. We assessed the concentrations of serum cytokines (IFNγ, IL13, IL12p70, IL10, IL1β, IL2, IL4, IL6, TNFα, CRP and TGFβ) in cord blood, as well as peripheral blood drawn at one year of age. One in five women was infected with malaria at the first antenatal visit and this frequency decreased immediately following a universal bed net campaign. Children born to mothers with placental malaria, but not children born to mothers with peripheral malaria, were at increased risk of malaria during infancy as compared to those born to mothers with no malaria. Most cases of placental malaria were cleared by delivery. Children born to mothers with chronic placental malaria had elevated levels of TNFα, CRP and IL10 and a significantly decreased TNFα:IL10 ratio as compared to those born to mothers with peripheral malaria or no malaria. These differences in cytokine profile at birth disappeared by one year of age. We found no association between cytokine concentrations at birth and increased risk of malaria in infancy. We hypothesize that placental malaria, even early in pregnancy, causes cytokine dysregulation and induction of long-lived cellular alterations that are maintained in infancy, leading to increased risk of malaria. Our findings have direct public health significance. Interventions need to target all women of childbearing age and prevent all placental malaria in order to maximize the health of mother and child.
    • The Epidemiology and Control of Malaria in a Low Malaria Transmission Setting along the Thai-Myanmar Border

      Lawpoolsri, Saranath; Hungerford, Laura; Maguire, James H. (2009)
      Background: Thailand is considered to be a good candidate for malaria elimination. Currently, malaria transmission is concentrated only in areas along the international borders where additional specific interventions are required. In an area along the Thai-Myanmar border, we have studied the epidemiology of malaria, in terms of the disease distribution and its determinants. In addition, we have developed mathematical models to critically evaluate malaria transmission related to different malaria control strategies. Methods: We conducted a retrospective cohort study using data of a community-cohort of residents living in seven hamlets along the Thai-Myanmar border between 1999 and 2006. Spatial and temporal clusters of P. falciparum and P. vivax cases were identified using scan statistics. Malaria determinants that characterize people (individual-level), place (hamlet-level), and time (temporal change) were examined using the generalized linear mixed model (GLMM). Mathematical models were developed to simulate the P. falciparum transmission in the area; an additional model was constructed to consider P. falciparum gametocyte dynamics related to primaquine (gametocytocidal agent) pharmacokinetics. Results: Malaria incidence in the area along the Thai-Myanmar border was clustered across space and time. The seasonal variation of malaria cases was best explained by temperature rather than rainfall. The inter-annual reduction of malaria incidence was significantly associated with the deployments of active surveillance and artemisinin-mefloquine combination therapy (ACT). The spatial clustering patterns of malaria cases in this small area could not be solely explained by the variation of geographical characteristics. An individual risk of contracting malaria was largely affected by the density of malaria cases occurring within a hamlet in the previous month, relative to the topographical characteristics of individuals' houses. In the transmission modeling, early detection and treatment with ACT and primaquine had a substantial impact on reducing malaria transmission; however, primaquine was most beneficial when the administration was delayed about 8 days following initial treatment. Conclusions: The knowledge of spatial-temporal clustering provides guidance for targeted intervention to be conducted more efficiently. Transmission from nearby human was the most significant factor for continuation of malaria transmission in the area. Reducing the gametocyte carriage rate through a modified treatment regimen should have a potential impact on malaria transmission.
    • Informing the role of RIFINs in malaria pathogenesis, natural immunity, and design of a severe malaria vaccine

      Zhou, Albert; Travassos, Mark A.; Laufer, Miriam K. (2020)
      Plasmodium falciparum is a eukaryotic parasite that causes severe malaria and contributed to 405,000 deaths worldwide in 2018. Victims of severe malaria are predominantly sub-Saharan African children, who typically present with symptoms of severe anemia or unarousable coma. The pathogenesis of severe malaria is poorly understood but mediated by the expression of adhesive variant surface antigens (VSAs) on infected red blood cells. VSAs are involved in sequestration and rosetting, unique virulence processes that allow the parasite to evade host immune responses and prevent clearance in the spleen. A relatively unstudied family of VSAs, the repetitive interspersed family (RIFIN) proteins, have recently been found to be important in rosetting and host immune suppression. RIFINs also appear to be targets for protective immunity; humoral immune responses against RIFINs have been correlated with asymptomatic infections. In this dissertation, I applied a multi-faceted approach using protein and peptide microarrays, transcriptomics, and reverse vaccinology to identify appealing RIFIN candidates for inclusion in a future severe malaria vaccine. I show that serological responses against epitopes within the semi-conserved domain of RIFINs associated with severe malaria reflected age-related malaria exposure. Sequencing and identifying specific rif genes expressed in clinical infections have not been feasible. I have addressed these challenges by adapting a novel bioinformatic pipeline and developing an HMM-based tool to process, assemble, classify, and subtype RIFIN sequences from peripheral blood samples. This takes advantage of a targeted probe capture method that I determined yields more abundant, full-length RIFIN sequences than other library enrichment approaches. Finally, I performed a comprehensive genomic survey of RIFIN gene repertoires using publicly available whole genome data of sixteen P. falciparum isolates to identify highly conserved, strain-transcendent sequences. Together, these results provide insights and powerful tools that can advance our understanding of the role RIFINs play in severe malaria pathogenesis and the development of naturally-acquired immunity to severe malaria. This work will aid efforts to determine targets for vaccines to protect children from the deadliest consequences of malaria.
    • Malaria Transmission in Households in Blantyre, Malawi

      Cassin, Jessica Bailey Walters; Laufer, Miriam K. (2012)
      Although great strides have been made in the reduction of the worldwide burden of malaria disease, a better understanding of the epidemiology of malaria is needed to continue the fight against the disease. Specifically, insight into the transmission of malaria within households might offer new targets of malaria intervention, and policy changes aimed at the control of the spreading antimalarial resistance. To this end, this study examines the relationship between malaria infection and household exposure in Blantyre, Malawi. Blood samples were collected from children and their caregivers for analysis using six neutral, unlinked microsatellite markers. Parasites within infections were genotyped and the infections in children were compared to infections in their caregivers to determine the number of microsatellites shared between the two infections, a marker for the genetic relationship of the two infections. The comparison of genotype between infections, allows a specific infection to be tracked through the human-vector-human transmission cycle. The mean proportion of parasite alleles shared for individuals residing in the same house was compared to that of individuals residing in different households. Overall, children had infections that were more similar to their parents than to that of other caregiver in the population (p value =.036). This indicates that intra-household malaria infections are more similar than inter-household malaria infection and suggests that individuals in the household are a source of malaria infection within the household. For half of the occurrences of shared infections, the parasite was found in both caregiver and child at the same time, a synchronous exposure indicating a shared exposure either within the area or travel outside of the Blantyre area. The results provide encouraging indications that future research may yield new information that will be influential in reducing the burden of malaria disease worldwide through policy decisions regarding parasite control and the prevention of the spread of antimalarial resistance. Further research is needed to assess the intra-household source of infection, and validate the study in other populations.
    • New tools for var gene assembly and analysis of genetic diversity of var2csa, the locus encoding a placental malaria vaccine candidate antigen: vaccine development implications

      Dara, Antoine; Plowe, Christopher V. (2017)
      Encoded by the var gene family, highly variable Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) proteins mediate tissue-specific cytoadherence of infected erythrocytes (iEs), resulting in immune evasion and severe malaria disease. Sequencing and assembling the 40-60 var gene complement for individual infections has been notoriously difficult, impeding molecular epidemiological studies and the assessment of var-encoded proteins as subunit vaccine candidates. VAR2CSA, a member of this family, mediates the binding of iEs to chondroitin sulfate A, a surface-associated molecule expressed in placental cells, and plays a central role in the pathogenesis of placental malaria. VAR2CSA is a target of naturally-acquired immunity and, as such, is a leading vaccine candidate against placental malaria. The gene encoding the VAR2CSA is technically challenging to sequence. Published var2csa sequences, mostly limited to specific domains, have been generated through the sequencing of cloned PCR amplicons using capillary electrophoresis, a method that is both time-consuming and costly. We developed and validated novel tools to assemble var gene sequences from clinical samples, with a focus on var2csa. These tools include Exon-Targeted Hybrid Assembly (ETHA), which is based on a combination of Pacific Biosciences (PacBio) and Illumina data, an amplicon sequencing of var2csa using PacBio, and a pipeline to reconstruct var2csa from short sequence reads. Using ETHA, we characterized the repertoire of var genes in samples from uncomplicated malaria infections in children from a single Malian village and showed them to be as genetically diverse as vars from isolates from around the globe. The amplicon sequencing allowed us to recover a segment encoding the first four domains in the 5' end of VAR2CSA (~5kb), and the full-length of the VAR2CSA's extracellular region from clinical malaria samples. The reconstruction of var2csa from short reads permitted us to generate a worldwide set of var2csa sequences. We used the VAR2CSA sequences to show that despite the extreme sequence variation, VAR2CSA from around the globe shared conserved motifs supporting the feasibility of VAR2CSA-based vaccines. The findings presented in this work will aid the understanding of malaria pathogenesis and inform the design of malaria vaccines based on PfEMP1, in particular VAR2CSA-based vaccines.
    • Plasmodium falciparum malaria in the Greater Mekong Sub-region: Elucidating parasite migration and genomic signatures of selection

      Jacob, Christopher George; Plowe, Christopher V. (2015)
      Current estimates place over one third of the world's population at risk of contracting malaria, with approximately 300-500 million cases of clinical illness each year. Among these cases it is estimated more than 600k end in death, mostly among children less than 5 years of age in Africa, making malaria one of the leading causes of infectious disease death in the world. There is currently widespread drug resistance in Plasmodium falciparum to most anti-malaria medications, with some areas harboring multidrug resistant parasites, including those resistant to the current first line treatment artemisinin. Single nucleotide polymorphisms (SNPs) were typed from over 2000 field samples by DNA microarray and whole-genome sequencing. Genotypes were used to group parasites into putative subpopulations and bidirectional migration rates between geographic locations were estimated using model-based ADMIXTURE and LAMARC. We detected thirteen subpopulations within Southeast Asian samples including a core of six sub-populations within West Cambodia, the region with the highest prevalence of artemisinin resistance. We find evidence of parasite gene flow across Southeast Asia and between proximal and distant populations. Analysis investigating genes under positive selection using long-haplotype methods (iHS and XP-EHH) as well as population differentiation found multiple shared loci across populations including drug resistance genes, vaccine antigens, and genes possibly involved in local natural adaptation. Our study highlights the complex genetic structure within Southeast Asia and patterns of parasite migration that identify areas most susceptible to the import of resistant parasites, as well as genes under selection that could be future drug targets or vaccine antigens.
    • Polyclonal Antibody Recognition of Plasmodium falciparum Apical Membrane Antigen 1 Fragments Expressed in an Escherichia coli Autotransporter

      Sedegah, Mary; Berry, Andrea A. (2013)
      Introduction: Plasmodium falciparum apical membrane antigen 1 (AMA1) is a blood stage protein involved with erythrocyte invasion. Because anti-AMA1 antibodies inhibit parasite growth and are associated with clinical immunity, AMA1 is a leading malaria vaccine candidate in clinical testing. However, AMA1 is highly polymorphic and antibodies elicited by one variant of AMA1 may not confer protection against others; therefore, an effective vaccine might need to include multiple variants. Studies of antibody cross-reactivity to AMA1 variants would aid in the rational design of an AMA1 vaccine. The current study used an autotransporter expression system to express several variant AMA1 fragments. We expected that antibodies raised against one P. falciparum AMA1 variant would recognize homologous AMA1 fragments but not highly dissimilar variants. Methods: Fragments of the AMA1 protein corresponding to AMA1 sequences from six P. falciparum strains (3D7, FVO, DD2, 7G8, Fab9, and M5) were genetically engineered and incorporated into an EspP autotransporter. Proteins were expressed, purified and used in western blotting assays to assess reactivity of polyclonal rabbit antibodies that were raised against recombinant 3D7-AMA1 and FVO-AMA1. Results: Anti-3D7 AMA1 polyclonal rabbit antibodies preferentially recognized the 3D7 AMA1 fragments, and cross-reacted with the heterologous Fab9 AMA1 fragments via western blotting. Polyclonal rabbit antibodies raised against FVO AMA1 preferentially recognized FVO AMA1 fragments but also had a higher level of cross-reactivity, strongly recognizing heterologous M5, and 7G8 AMA1 fragments. Conclusions: The autotransporter expression system allowed for characterization of polypeptide fragments from different AMA1 strains based on antibody recognition. It was evident that antibody recognition became more specific when larger fragments were studied. The identification of residues that form the basis of cross-reactivity between strains could yield useful information to guide the design of future AMA1 vaccines.
    • Potential for School-based Malaria Treatment to Reduce P. falciparum Transmission

      Cohee, Lauren; Laufer, Miriam K.; 0000-0002-7575-630X (2019)
      School-age children bear an under-appreciated burden of malaria and are a key reservoir for the spread of P. falciparum. We conducted school-based cohort studies to measure the impact of treating students with positive malaria rapid diagnostic tests on subsequent gametocyte, the parasite stage required for human-to-mosquito transmission, prevalence and density. We concomitantly quantified the proportion of gametocyte burden in school-age children and compared it to that of other age groups in household-based surveys in the school catchment area. Treatment reduced the prevalence and density of gametocytes by 79% and 89%, respectively. Half of all gametocyte-containing infections were detected in school-age children. We estimated that school-based malaria treatment could reduce overall gametocyte prevalence in the community by 26% and 34% in the rainy and dry seasons, respectively. These results suggest that school-based malaria treatment could further decrease the burden of malaria in areas where malaria has remained entrenched despite current control measures.
    • Whole-genome analysis of Plasmodium falciparum isolates to understand allele-specific immunity to malaria

      Shah, Zalak; Takala-Harrison, Shannon (2020)
      After repeated P. falciparum infections, individuals in high-transmission areas acquire clinical immunity to malaria. However, the genes important in determining allele-specific immunity are not entirely known. Previous genome-wide approaches explored signatures of selection in the parasite genome to identify targets of clinical immunity; however, these approaches did not account for individual level allele-specific immunity. Here we take a whole-genome approach to identify genes that may be involved in acquisition of allele-specific immunity to malaria by analyzing parasite genomes collected from infected individuals in Malawi. However, obtaining whole genome sequence data from clinical samples is one of the major hurdles in the field of malaria genomics. In order to obtain whole genome sequence data from non-leukocyte depleted, low parasitemia samples, we optimized a selective-whole genome amplification (sWGA) by filtering the DNA prior to sWGA, to generate high coverage, whole genome sequence data from P. falciparum clinical samples with low amounts of parasite DNA. Using this optimized approach, we successfully performed whole-genome sequencing on 202 parasite isolates. We compared parasite genomes from individuals with varying levels of clinical immunity, defined using an individual’s proportion of symptomatic infections during the course of the study, hypothesizing that individuals with higher immunity become symptomatically ill due to infection with parasites with less common alleles. Using FST, we identified 161 SNPs to be genetically differentiated between the two groups and the median allele frequency was significantly lower at these sites in individuals in higher immunity group compared to the lower immunity group. We also examined pairs of parasites collected at different time points from the same individuals and identified 225 loci in 174 genes that vary within same individuals more often than expected by chance. Using both of these approaches, we identified 25 genes that encode likely targets of immunity, including a known antigen, CLAG8. Further analysis of clag8 global diversity showed evidence of immune selection in the C-terminal region, supporting the use of this approach in identification of new vaccine targets. Identifying and further analyzing these genomic regions will provide insights into mechanisms involved in allele-specific acquired immunity.