In meeting the high demand for monoclonal antibodies, the chicken immunoglobulin system was exploited to generate recombinant antibodies against multiple target antigens. Following simultaneous immunisation of two chickens with a mixture of Plasmodium falciparum recombinant lactate dehydrogenase (LDH), histidine rich protein II (HRPII) and aldolase (ALDO), recombinant trypanosome variable surface glycoprotein (VSG) and malignant catarrhal fever virus (MCFV) each chicken produced egg yolk antibodies (IgY) against four of the five antigens. Using phage display technology, two single-chain variable fragment (scFv) antibody libraries, one with the immunoglobulin VH and VL chain regions joined by a single amino acid (G) and the other with a 15 amino acid flexible linker [(G4S) 3] were constructed using pooled splenic RNA. The single amino acid-linked scFv repertoire was evaluated as a source of highly specific diagnostic antibodies by panning against each of the five different antigens. After two rounds of panning, polyclonal phage ELISA showed the presence of antigen-specific phage antibodies against three (LDH, HRPII and VSG) of the five antigens. Five different anti-LDH and six different anti-HRPII scFvs were identified by sequence analysis. Evidence of high levels of antigen-driven gene conversion events was found in the framework and complementary determining regions and the VL chain pseudogene donors were identified. Stability of the selected scFvs was determined by incubation at different times and at different temperatures. The specificity and potential use of an LDH-specific scFv as a diagnostic reagent was shown in sandwich and competitive inhibition ELISAs. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2007. / Veterinary Tropical Diseases / unrestricted

Indiana University-Purdue University Indianapolis (IUPUI) / Plasmodium vivax malaria is geographically widespread and remains a significant public health burden in the Americas, Southeast Asia, and the western Pacific. In order to achieve the end goal of malaria eradication, a highly effective vaccine targeting P. vivax is urgently needed. Unlike pre-erythrocytic vaccines that aim to confer sterile immunity that prevents malaria infection altogether, Plasmodium vivax blood-stage vaccines aim to confer clinical immunity that protects against malarial disease by controlling parasitemia and mitigating the symptomatic manifestations of malaria after infection. To design an effective P. vivax blood-stage vaccine, it is essential to understand the acquisition and longevity of natural humoral immune responses against promising P. vivax blood-stage vaccine candidate antigens. We hypothesize that acute vivax malaria induces differential humoral immune responses against P. vivax antigens that exhibit antigen-specific kinetic and compositional profiles, which can be used to identify vaccine candidates that elicit durable humoral responses. Therefore, we compared the kinetic profiles and half-lives of naturally acquired IgG antibodies reactive against nine promising P. vivax blood-stage vaccine candidate antigens up to 180 days post-infection in Brazilians with acute vivax malaria. Naturally acquired IgG antibodies against these antigens have previously been associated with a reduced risk of vivax malaria. Among the P. vivax antigens evaluated, the merozoite antigen Pv12 elicited the most durable IgG antibodies, whereas the DBP-FL elicited the most short-lived responses. Neither patient age nor prior malaria exposure significantly correlated with the magnitude and durability of IgG responses to any P. vivax antigen. Seropositivity, against Pv12, was generally maintained for at least 30 days after acute vivax malaria. These findings suggest that a blood-stage vaccine targeting Pv12 may benefit from boosting IgG antibodies against this antigen after natural vivax “breakthrough” infections. Further studies will be needed to determine the Pv12-specific memory B cell response as well as the functional role for naturally acquired Pv12-specific antibodies in reducing parasitemia and/or clinical disease. In summary, the current study has provided insight into the longevity of IgG antibody responses to important P. vivax antigens after an acute malaria episode.

Malaria claims millions of lives annually. This global killer causes approximately 2.7 million annual deaths worldwide; addressing this problem has become more and more crucial. Due to pathogen evolution no efficient vaccine for treatment of malaria currently exists. As infection has developed as a field of study, it became ever more clear that infections could only be understood within the context of the host-pathogen community. This project aims to predict possible drug targets based on host-pathogen interactions rather than just protein-protein interactions within a single organism. Similar to Lee et al. (2008) pathogen-host interaction predictions are based on orthology, these interactions are then analysed to identify potential drug targets. This could potentially aid researchers in their continuous battle against malaria and the larger scale battle against pathogen evolution. To predict in vitro host-pathogen interactions DISCOVERY uses an ortholog clustering method called ORTHOMCL. ORTHOMCL is very suitable for ortholog clustering of malaria data for two reasons. Firstly, it is capable of distinguishing between recent paralogs and ancient paralogs, which enables the inclusion of recent paralogs together with orthologs. Secondly, ORTHOMCL was initially developed for the use of malaria data. Identification of in vitro interactions is followed by scoring methods to determine the possible in vivo interactions that might occur between the Plasmodium parasite and the human and mosquito hosts. Scoring measures and weights were applied to 5 different factors to calculate a final score. These final scores allow user input to define the preferred stringency when viewing possible interactions with a single protein. These different factors are sequence similarity, PEXEL/VTS motif presence, microarray expression, metabolic map sharing and sub-cellular locations boundaries. DISCOVERY’S results and results from two other (Dyer et al. and Lee et al.) in silico prediction methods were compared with Vignali et al’s experimental interactions which are based on a yeast two-hybrid approach. Similar to results shown by Doolittle and Gomez these comparisons had poor results. The next step was to compare the in silico results with each other. Dyer et al’s and Lee et al‘s results compared poorly with each other. Although DISCOVERY did not compare well with Dyer et al’s results, comparisons with Lee et al. showed more promise. Poor comparisons with Dyer et al. may be due to their unique approach to predict in vitro host-pathogen interactions. This project identified the lack of enough valid and reliable experimental data to evaluate in silico prediction methods as a definite challenge for host-pathogen interaction predictors. Although this is a major problem, DISCOVERY improved on older prediction methods with the use of a more applicable ortholog clustering technique and the use of more assessment methods during in vivo interaction predictions. DISCOVERY also used scoring methods rather than exclusion methods during the identification of in vivo interactions. This allows a user to specify a threshold of sensitivity when viewing interactions. The true potential of host-pathogen interaction predictions would only be realized when the gap between predictions and evaluation data is bridged. / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

The malaria-causing parasite Plasmodium falciparum 1s dependent on tightly regulated gene expression for its progression through the intra-erythrocytic life cycle, pathogenesis and establishment of persistent infection by evasion of the human host's immune system. Evidence points towards P. falciparum being unusually dependent on nuclear architecture and genomic organisation for the control of gene expression. Spatially defined nuclear regions of transcriptional activity have been detected and the spatial positioning of loci may determine their transcriptional potential. Additionally, a number of epigenetic markers have been shown to occupy spatially distinct subcompartments of the nuclear volume. Limitations of microscopic assays used until now have left us with a stereotyped and incomplete image of the organisation of the parasite nucleus and the transcriptional and epigenetic factors involved in the regulation of parasite gene expression, and the possible dynamics thereof. This work focused on the use of high-content and super-resolution fluorescent microscopy for the study and graphical representation of the spatial organisation of various nuclear factors involved in transcriptional regulation in P. falciparum parasites. The first objective (chapter 2) establishes P. falciparum parasite sample preparation and fluorescent labeling techniques for microscopy. Immunofluorescent labeling of var gene associated transcription repressive and permissive histone modifications, H3K9me3 and H3K9ac, respectively, as well as serine 2- phosphorylated RNA polymerase II and the putative transcription and splicing factor PfMyb2, was optimised. DNA fluorescent in situ hybridisation was also optimised for labeling of var gene exons. In the second objective (chapter 3), the assays established in the previous chapter are used for high-content combinatorial labeling in thousands of nuclei, followed by analysis using a bespoke computational algorithm for the detection and classification of different labeling patterns. This approach revealed a high level of diversity in the nuclear distributions of each assayed target. Superresolution stochastic optical reconstruction microscopy was used to further study the sub-diffraction organisation of selected labeling patterns. The data presented in this dissertation reveal that the complex spatial organisation of certain nuclear factors is subject to greater diversity within the nucleus of P. falciparum parasites than previously thought. / Dissertation (MSc)--University of Pretoria, 2012. / gm2013 / Biochemistry / unrestricted

Introduction: The surprising presence of P. vivax in West Africa and their ability to infect a Duffy negative population is one more threat to public health. In order to contribute to malaria elimination efforts, there is a need to investigate the origin and characteristics of P. vivax population isolates in Northern Mali. Next Generation Sequence Analysis (NGSA) can help us understand parasite genetic characteristics although low parasite density is a challenge for whole genome sequencing (WGS). In the present work, we investigated if selective whole genome amplification (sWGA) can enrich P. vivax DNA extracted from Rapid Diagnostic Tests (RDTs) for Whole Genome Sequencing. We also investigated the origin and the susceptibility to antimalarial drugs of the strains isolated in Northern Mali. Methods: Parasite DNA was extracted from 267 RDTs using the QIAamp DNA mini kit, then nested PCR and 7 samples were positive for P. vivax. After sWGA, the whole genomes were sequenced using the Illumina platform. Next Generation Sequences Analysis was done followed by population differentiation analyses. Twenty-two additional P. vivax whole genomes from other parts of the World were downloaded from the European Nucleotide Archive for further Neighbour Joining analysis. Results: The sequences extracted from RDTs showed high contamination with human DNA (80%). From the parasite DNA, in total 69529 SNPs were found in the seven P. vivax strains of Northern Mali. The most significant p-values per SNP were carried by the chromosomes 2, 3, 4, 5, 12, 13 and 14. With regard to variant effects, the Transition/Transversion ratio was 1.1. The density of variants with a high effect was 1.62%. There was no mutation associated with antimalarial drugs resistance on pvcrt-o or pvmdr-1 genes. Pairwise differentiation suggests a high degree of relatedness between P. vivax strains isolated in Northern Mali. The NeighboursJoining analysis shows clearly that strains from Mali cluster together and are genetically distinct from those from Mauritania, which shares a border with Mali. The strains isolated in Northern Mali are genetically closer to those from Madagascar, India and Latina America. Conclusion: We did not identify mutations associated to the resistance to antimalarial drugs in pvcrt-o and pvmdr-1 genes. This study confirms that P. vivax strains genetically distinct from those of Mauritania are circulating in Mali. Finally, we conclude that sWGA is a feasible approach for P. vivax DNA enrichment for WGS despite the high proportion of human contamination.

In spite of great effort aimed at eradication, the malaria epidemic still claims over 600,000 lives each year, and 50% of the world is at risk of contracting the disease. The most deadly form of malaria is caused by Plasmodium falciparum, which is spread from human to human via the female Anopheles mosquito. P. falciparum's lifecycle, which includes both sexual and asexual reproduction, facilitates rapid evolution in response to drug pressure, resulting in the emergence of resistant strains against every antimalarial medication that has been deployed. There is a great need for new antimalarial drugs. Chloroquine (CQ), an aminoquinoline drug deployed in the 1940s, was an inexpensive, effective and safe drug but now has been rendered ineffective throughout much of the tropical regions due to the emergence of CQ-resistant strains of P. falciparum. A new class of hybrid drugs, called Reversed-CQs, has been developed by linking a molecule with a CQ-like moiety to a molecule with a reversal agent (RA) moiety; an RA is a chemosensitizer that can reverse CQ-resistance. The prototype Reversed-CQ, PL01, was shown to be effective in vitro against sensitive and resistant P. falciparum cell cultures, with IC50 values of 2.9 and 5.3 nM, respectively, in comparison to IC50 values for CQ which were 6.9 and 102 nM, respectively. In the course of the Reversed-CQ research, PL74 was synthesized with a pyridine ring replacing the quinoline ring. It was expected that PL74 would display reversal agent activity but would not display antimalarial activity. However PL74 showed antimalarialactivity with IC50 values of 185 and 169 nM in vitro against CQ-sensitive and CQ-resistant strains, respectively. In the investigation of PL74 it has been found that this molecule has a pyridinium salt structure, novel to the Reversed-CQ compounds, and through a structure-activity relationship (SAR) study, it was shown to have activity that may indicate a mode of action different from the Reversed-CQ compounds. A study of the literature revealed that pyridinium salt compounds, with some similarity to PL74, were found to operate as choline analogs inhibiting the biosynthesis of phosphatidylcholine as their main antimalarial mode of action.

The vectors of avian malaria (Haemosporida) are an understudied component of wildlife disease ecology. Most studies of avian malaria have focused on the secondary bird hosts. This imbalance leaves a significant gap in our knowledge and understanding of the insect hosts. This study investigates the diversity of malaria parasites carried by mosquitoes (Diptera, Culicidae) in the state of Mississippi. Using PCR techniques, haemosporidian infection rates were determined and parasites were identified in a phylogenetic context to those previously annotated. A total of 27,157 female mosquitoes representing 15 species were captured. Five of those species tested positive for malaria parasites with an overall infection rate of 4 per 1000 mosquitoes infected. Mosquitoes were shown to harbor Plasmodium and Haemoproteus parasites. Surprisingly, a unique lineage of parasites was discovered in Anopheles mosquitoes potentially representing a new genus of haemosporidian parasites, reinforcing the need to continue investigating this diverse group of parasites.

Haemosporidian parasites are the agents of malaria. Countless vertebrates are affected by haemosporidians each year. Haemosporidians have been shown to be evolving at rapid rates; leading to new species of haemosporidians being discovered and new host associations being made. Adaptive molecular evolution was detected in an important hemoglobin degradation gene, falcilysin. At multiple sites across multiple genes involved in important functions signatures of negative selection were detected. The signatures of selection across non-hemoglobin degradation genes were indicative of evolutionary conservation when compared to the more variable hemoglobin degradation genes. This is probably due to the important role the hemoglobin degradation genes play in haemosporidian metabolism. A survey of local passerines detected a parasite prevalence rate of 57%. This included three genera of haemosporidians detected across six lineages and two more distantly related sequences. Leucocytozoon was detected for the first time in Mississippi songbirds, indicating the importance of surveying for understanding haemosporidian evolution and range.

Malaria is one of the worlds most deadly infectious diseases and results in almost a million deaths each year, largely in children under the age of five in Sub-Saharan Africa. Outside Africa, malaria is responsible for a large number of cases in the Amazon rainforest of Brazil, Middle East, and in some areas of Asia [37]. According to the World Health Organization, there was an estimated 655, 000 deaths from malaria in 2012. Malaria is caused by a eukaryotic Apicomplexan parasite, Plasmodium, which has three distinct life cycles occurring in the midgut of the female Anopheles mosquito, the liver of the human host, and human erythrocytes. When the parasite infects the erythrocyte, some induced cell host modifications are made in order to accommodate growth. During its intra-erythrocytic life cycle, the malaria parasite traffics numerous proteins to a set of unique destinations within its own plasma membrane including the digestive vacuole, the apicoplast, rhoptries, and micronemes. Vesicular transport is an essential process in eukaryotic cells. This coordinated process is responsible for moving thousands of proteins between compartments within the cell. Essential to the targeting and fusion of protein transport vesicles in eukaryotes are SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors), a family of fusogenic proteins that are localized to distinct intracellular compartments [11]. Studies performed in our laboratory have identified 18 proteins putatively belonging to the PfSNARE family [2]. To date the exact role of PfSNAREs in the unique trafficking pathways of malaria is undetermined. Of particular interest to our study is PfVAMP8. In model eukaryotic organisms, VAMP8 containing vesicles deliver cargo to lysosomes and are involved in endocytosis. The food vacuole of the parasite is very similar to that of lysosomes and is essential to parasite survival. The study aims to identify the organelle(s) to which PfVAMP8 is localized and characterize membrane-association properties of this parasite’s R-SNARE protein. We believe that PfVAMP8 would localize to unique compartments in the parasite protein network flow. An in depth understanding of its mechanisms and localizations could be a key in developing novel anti-malarials. This study aims to identify the organelle(s) to which PfVAMP8 are localized, determine the trafficking determinants of this protein and determine this proteins’ expression and membrane association during the intra-erythrocytic stages of Plasmodium falciparum. Our immunofluorescence studies with known biological markers reveals that, PfVAMP8 passes through the endoplasmic reticulum, Golgi, and localizes to the food vacuole during trophozoite and schizont stage. Further characterization of the membrane association properties of the protein in this study reveals that PfVAMP8 is a soluble integral membrane protein with amphipathic characteristics.

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