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Tam, Hou Si. / 33 in title is subscript. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 174-187). / Abstracts in English and Chinese. / THESIS COMMITTEE --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.iii / 摘要 --- p.v / TABLE OF CONTENTS --- p.vii / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xvii / LIST OF ABBREVIATIONS --- p.xviii / CHAPTER / Chapter 1. --- INTRODUCTION / Chapter 1.1 --- Malaria --- p.1 / Chapter 1.2 --- Malaria is a public health problem --- p.1 / Chapter 1.3 --- Malarial parasite --- p.3 / Chapter 1.4 --- Life cycle of P. falciparum --- p.3 / Chapter 1.4.1 --- The pre-erythrocytic stage --- p.3 / Chapter 1.4.2 --- The asexual erythrocytic stage --- p.3 / Chapter 1.4.3 --- The sexual transmission stage --- p.6 / Chapter 1.5 --- Chemoprophylaxis and chemotherapy of malaria --- p.7 / Chapter 1.6 --- Drug resistance of malaria parasite --- p.7 / Chapter 1.7 --- The progress for malaria vaccine --- p.10 / Chapter 1.8 --- Vaccine candidates for asexual erythrocytic stage --- p.11 / Chapter 1.9 --- Merozoite Surface Protein-1 (MSP-1) --- p.13 / Chapter 1.9.1 --- Structure of MSP-1 --- p.13 / Chapter 1.9.2 --- The processing of MSP-1 --- p.17 / Chapter 1.9.3 --- MSP-1 as a blood-stage vaccine --- p.19 / Chapter 1.9.4 --- The vaccine potency of MSP-133 --- p.23 / Chapter 1.10 --- Merits of E. coli expression system --- p.25 / Chapter 1.11 --- Aim of study --- p.26 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.30 / Chapter 2.2 --- Methods --- p.39 / Chapter 3. --- EXPRESSION AND PURIFICATION OF RECOMBINANT MSP-l33kv+19 PROTEIN / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Results / Chapter 3.2.1 --- Construction of pET32a/MSP-l33kv+19 expression vector --- p.64 / Chapter 3.2.2 --- SDS-PAGE analysis of the expressed protein --- p.74 / Chapter 3.2.3 --- Western blot analysis of the expressed protein --- p.78 / Chapter 3.2.4 --- Modification of the expression conditions --- p.78 / Chapter 3.2.5 --- Protein purification by IMAC --- p.82 / Chapter 3.2.6 --- Cleavage of fusion partner from the rMSP-133kv+19 protein --- p.82 / Chapter 3.2.7 --- Verification of non-fused recombinant MSPl33kv+19 protein by N-terminal amino acid sequencing --- p.86 / Chapter 3.2.8 --- Separation of target protein from the fusion mixture by IMAC --- p.86 / Chapter 3.2.9 --- Separation of digestion product by Size Exclusion Chromatography --- p.89 / Chapter 3.2.10 --- Conformational test of the purified protein --- p.89 / Chapter 3.2.11 --- Separation of target protein from contaminants by Anion-Exchange Chromatography --- p.92 / Chapter 3.2.12 --- Separation of target protein from contaminants by Immuno-Affinity Chromatography --- p.95 / Chapter 3.3 --- Conclusion --- p.95 / Chapter 4. --- IMMUNOLOGICAL CHARACTERIZATION OF BACTERIAL EXPRESSED rMSP-l33kv+19 / Chapter 4.1 --- Introduction --- p.97 / Chapter 4.2 --- Results / Chapter 4.2.1 --- Immunogenicity of recombinant NfMSP-133kV+19 protein --- p.98 / Chapter 4.2.2 --- Specificity of anti-NfMSP-133kv+19 sera to MSP-l33kv. MSP-l33 and MSP-l19 --- p.98 / Chapter 4.2.3 --- Cross reactivity of anti-MSP-133kv+19 and anti-BVp42 serum --- p.103 / Chapter 4.2.4 --- Competitive ELISA --- p.103 / Chapter 4.2.5 --- Test for the presence of inhibitory B-cell epitopes on rMSP-l33kv+19 --- p.111 / Chapter 4.2.6 --- In vitro parasitic growth inhibition assay --- p.113 / Chapter 4.3 --- Conclusion --- p.115 / Chapter 5. --- EXPRESSION AND PURIFICATION OF RECOMBINANT MSP-l33kc+19 PROTEIN / Chapter 5.1 --- Introduction --- p.116 / Chapter 5.2 --- Results / Chapter 5.2.1 --- Construction of pET32a/MSP-133kv+19 expression vector --- p.117 / Chapter 5.2.2 --- Expression of recombinant MSP-133kc+19 protien (rMSP-133kc+19) --- p.124 / Chapter 5.2.3 --- Purification of rMSP-l33kc+19 by IMAC --- p.127 / Chapter 5.2.4 --- Cleavage of fusion partner from target protein --- p.127 / Chapter 5.2.5 --- Construction of pRSETA/MSP-l3X33kc+19 expression vector --- p.135 / Chapter 5.2.6 --- SDS-PAGE analysis of the protein expression --- p.146 / Chapter 5.3 --- Conclusion --- p.153 / Chapter 6. --- DISCUSSION / Chapter 6.1 --- Expression of rMSP-l33kv+19 --- p.154 / Chapter 6.2 --- Purification of rMSP-l3.3kv+19 --- p.156 / Chapter 6.3 --- Conformational test of rMSP-133kv+19 --- p.157 / Chapter 6.4 --- Biological and immunological activity of NfMSP-133kv+19 --- p.158 / Chapter 6.5 --- Expression of rMSP-133kc+19 --- p.166 / Chapter 6.6 --- Future prospects --- p.167 / REFERENCES --- p.174 / APPENDICES / Chapter 1. --- HiTrap NHS-activated HP for ligand coupling procedure --- p.188 / Chapter 2. --- Reuse of Ni+-NTA Resin procedure --- p.190 / Chapter 3. --- Sequence alignment of MSP-133 (MAD20 & Welcome/Kl alleles) --- p.191 / Chapter 4. --- Nucleotide sequence and amino acid sequence of P. falciparum MSP-l33kv+19 --- p.192 / Chapter 5. --- Nucleotide sequence and amino acid sequence of P. falciparum MSP-l33kc+19 --- p.193 / Chapter 6. --- "Nucleotide sequence and amino acid sequence of P. falciparum MSP-142 (3D7 isolate, MAD20 allele)" --- p.194 / Chapter 7. --- Amino acid sequence of Plasmodium falciparum MSP-l42 --- p.195

Here we report the identification of 15 potential MSP-like proteins from the P. falciparum 3D7 genome using a bioinformatics-based approach. One candidate, renamed URF1, was further characterized by cloning into the Gateway system. We were able to demonstrate expression of URF1 during the blood stage, especially the trophozoite, early and late schizont phases, by immunofluorescence on infected RBC using antisera raised in mice with an URF1 DNA vaccine. URF1 expression in the merozoite stage could not be confirmed in this study. Future co-localization and immunosorbent electron microscopy (EM) experiments would help us determine the exact localization of URF1 on the parasite before officially categorizing URF1 as a merozoite surface protein. As a whole, this research project demonstrates the success of using bioinformatics in identifying potential new MSP-like proteins found in the malaria genome. Further characterization and sequence analysis of the other 15 candidates may reveal other novel antigens expressed during the erythrocytic stage, especially in the merozoite stage. Such antigens may prove to be good vaccine candidates.

Malaria is a devastating disease caused by one of the world's most pathogenic parasites, Plasmodium. Five species of Plasmodium infect humans: P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. P. falciparum is the most pathogenic and causes the greatest numbers of deaths. To date, no licensed vaccine against malaria is available, although there are numerous vaccine candidates in various stages of development. Pca 96 is a 96 kDa Plasmodium chabaudi adami protein shown to have a protective property in mice challenged with P. chabaudi adami. Thus, a P. falciparum orthologue of Pca 96 may be useful in vaccine development. BLAST searches with the Pca 96 amino acid and nucleotide sequences revealed proteins with high sequence identity to Pca 96 including the hypothetical P. falciparum PFC0760c and P. yoelii yoelii PY05757 proteins. A peptide sequence FKLGSCYLYIINRNLKEI was found to be conserved in all homologues of Pca 96, including PFC0760c, PY05757 and in the sequences of proteins from 5 other Plasmodium species. Bioinformatic approaches were explored to attempt to find a possible role of the protein and the possible importance of the conserved sequence. The conserved sequence was predicted to be an alpha helix and to contain possible HLA-DRB1*1101 and HLA-DRB1*0401(Dr4Dw4) T-cell epitopes (GSCYLYIINRNLKEI) in addition to a possible H2-Kd T-cell epitope (CYLYIINRNL). Protein-protein interaction predictions revealed that PFC0760c was likely to interact with proteins involved with nucleic acid binding. PFC0760c was predicted to have a domain found in proteins involved in the structural maintenance of chromosomes, which may suggest the protein is involved in chromatid cohesion during mitotic chromosome condensation. PFC0760c was also predicted to be located in the nucleus by the sub cellular prediction program, SubLoc. Anti-peptide antibodies were raised against the conserved amino acid sequence and against a peptide specific for PY05757 (SDDDNRQIQDFE). Both antibodies detected native antigens with immunofluorescence microscopy. The fluorescent signal appeared throughout the parasite cytoplasm and as an intense signal in the parasite nucleus. These immunofluorescence data supports the predicted nuclear location of the protein. A 822 bp portion of PFC0760c gene was expressed as a maltose-binding protein fusion protein (Pf33-MBP). Pf33-MBP was expressed and purified. Reducing SDS-PAGE and western blotting analysis revealed the fusion protein to be expressed at low levels as four bands (79, 60, 45 and 37 kDa). The purified fusion protein was cleaved with Factor Xa. MBP and Pf33 were of similar molecular mass after cleavage. To attempt to obtain better expression and purification, the 822 bp insert from pTS822 was sub-cloned into pGEX4T1. A glutathione-S-transferase (GST)-fusion protein (Pf33-GST) was expressed. The level of expression was poor and therefore not pursued. To take the study further, potential proteins that interact with PFC0760c and Pf33 need to be identified. In addition, immunisation of mice with the protein and subsequent Plasmodium challenge needs to be performed to ascertain the protective potential of the protein. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

During the erythrocytic stage, the malaria parasite digests host cell haemoglobin into amino acids. Toxic haeme is released and is incorporated into an insoluble non-toxic crystal called haemozoin. Haemozoin is released into the blood stream along with the merozoites when the erythrocyte bursts and is phagocytosed by circulating monocytes and macrophages resident in tissues. Phagocytosed haemozoin impairs many functions of the monocytes, including antigen presentation and adhesion to T cells, differentiation and maturation to dendritic cells, erythropoiesis and thrombopoiesis, but stimulates the release of proinflammatory cytokines and activation of metalloproteinase 9 expression. In response to interferon-g secretion by T-helper cells subtype 1, monocytes secrete neopterin, which is used as a marker of a cell mediated immune response. Neopterin is an oxidation product of 7,8-dihydroneopterin, produced by the dephosphorylation of 7,8- dihydroneopterin triphosphate which results from the conversion of guanosine triphosphate that is catalysed by GTP-cyclohydrolase 1. Elevated plasma and urine neopterin levels have been detected in malaria infections and are associated with severe anaemia, respiratory distress, peak temperatures as well as fever- and parasite-clearance times. It has also been reported that monocytic U937 cells treated with P. falciparum-infected red blood cell lysate secrete elevated levels of neopterin. Antimalarial drugs are known to modulate the functions of monocytes, including inhibition of cytokine release, changes in phospholipid metabolism, decrease in expression of cytoadherance receptors as well as TNF receptors and MHC Class I and II molecules, changes in the production of reactive oxygen and nitrogen intermediates, and decreased phagocytosis. However, the effects of antimalarial drugs on haemozoin phagocytosis and GTP-cyclohydrolase 1 mRNA expression by monocytes are unknown. This study aimed to determine the effects of seven antimalarial drugs, amodiaquine, artemisinin, chloroquine, doxycycline, primaquine, pyrimethamine and quinine, on the phagocytosis of latex beads and b-haematin, a synthetic equivalent of haemozoin. Phagocytosis of b-haematin and latex beads by two monocytic cell lines, J774A.1 and U937, as well as peripheral blood mononuclear cells were monitored by enumeration and a novel spectrophotometric method. Patterns of inhibition and activation differed with each cell type investigated, due to the differing stages of cell differentiation. In general, artemisinin, primaquine, pyrimethamine and quinine activated the phagocytosis of b-haematin, whereas amodiaquine and chloroquine inhibited b-haematin phagocytosis. Doxycycline had different effects on each cell type investigated. Artemisinin, chloroquine, primaquine and quinine inhibited latex bead phagocytosis. The remaining drugs had minimal effects on latex bead phagocytosis. Thus, the effects of antimalarial drugs on monocyte phagocytosis appear to be dependent on the substance being phagocytosed. The effects of antimalarial drugs, b-haematin, latex beads, non-infected- and P. falciparuminfected cell lysates on interferon-g-induced neopterin secretion by U937 cells was monitored by GTP-cyclohydrolase 1 mRNA expression using quantitative PCR. Artemisinin, primaquine and quinine down-regulated the interferon-g-induced expression of GTPcyclohydrolase 1 mRNA, but by no greater than 1.7-fold. b-haematin up-regulated mRNA expression by 1.2-fold whereas P. falciparum-infected red blood cell lysate down-regulated the mRNA expression of GTP-cyclohydrolase 1 by 1.6-fold. Quinine and artemisinin, currently used to treat malaria, increased b-haematin phagocytosis suggesting that quinine and artemisinin might promote increased phagocytosis of infected red blood cells and enhance clearance of the parasite from circulation. Increased b- haematin phagocytosis also reduces ICAM-1 expression on the monocyte surface, thereby leading to reduced cytoadherance and sequestration, thus increasing the number of circulating monocytes to phagocytose infected red blood cells. Down regulation of GTPcyclohydrolase 1 mRNA expression by quinine and artemisinin suggested that the drugs reduce the responsiveness of the monocyte to interferon-g. Thus, quinine and artemisinin might also decrease the production of interferon-g-induced proinflammatory cytokines by monocytes, and potentially play a role in maintaining the balance between the pro- and antiinflammatory cytokines that determines the progression from acute to severe malaria. Therefore, in addition to the drug’s ability to kill the malaria parasite, the immunomodulatory effects of the antimalarial drugs may play a role in controlling the pathophysiology associated with the malaria infection. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

The kinetics of production and role of tumor necrosis factor-alpha (TNF-$ alpha$) and nitric oxide (NO) during the early phase of blood-stage infection with Plasmodium chabaudi AS were investigated using two inbred strains of mice which differ in the level of resistance to this parasite. Analysis of the in vivo expression of TNF-$ alpha$ and inducible nitric oxide synthase (iNOS) revealed that, early during infection, resistant C57BL/6 mice, which clear the infection by 4 weeks, have higher levels of TNF-$ alpha$ and iNOS mRNA in the spleen and TNF-$ alpha$ mRNA in the liver than susceptible A/J mice which succumb to the disease 10 days after initiation of infection. Moreover, resistant mice expressed high levels of IFN-$ gamma$ (a Th1 marker) and low levels of IL-4 (a Th2 marker) mRNA in the spleen, whereas susceptible A/J mice had low levels of IFN-$ gamma$ but high levels of IL-4 mRNA in the spleen early during infection. Increased levels of NO$ sb3 sp-$ were detected in serum of resistant C57BL/6 mice only at the time of peak parasitemia. Furthermore, treatment of resistant C57BL/6 mice with anti-IFN-$ gamma$ and anti-TNF-$ alpha$ monoclonal antibody demonstrated that TNF-$ alpha$, either alone or in synergy with IFN-$ gamma$, plays a major role in the up-regulation of NO production during P. chabaudi AS malaria. Moreover, treatment with the iNOS inhibitor aminoguanidine, eliminated resistance of these mice to infection with P. chabaudi AS without affecting parasitemia, suggesting that NO may not be involved in parasite killing in vivo. Taken together, these results demonstrate that a Th1-associated increase in TNF-$ alpha$ early during infection, as occurs in resistant mice, leads to the up-regulation of NO production which is crucial for survival of the host. On the other hand, our results also suggest that a Th2 response, as occurs in susceptible mice, does not result in protective levels of TNF-$ alpha$ and NO. However, susceptible A/J mice were found to

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Malaria is a devastating parasitic disease in humans caused by species in the genus Plasmodium. With over 100 million cases and at least 1.5 million fatalities each year, the disease accounts for 4-5% of all fatalities in the world. A recent increase in the number of malaria cases in South Africa has imposed severe costs on the economy and public health. Immunity to malaria is a multi-component system involving both B and T celllymphocytes. Pc96 is a 96 kDa antigen identified in the mouse malaria model Plasmodium chabaudi adami. It is known to be associated with the outer membrane of mouse erythrocytes infected with the parasite and has shown protective roles in mice challenged with P. chabaudi adami. A specific T cell clone has been identified that adoptively provides protection to athymic mice infected with P. chabaudi adami. Antibodies raised against Pc96 identified proteins that induced the proliferation of the protective T cell clones. At least four other antigens of different species of. malaria share at least one cross-reactive epitope. In an attempt to identify a Plasmodiumfalciparum homologue ofPc96, the amino-acid sequence was used in a BLAST search of the P. falciparum genome database, identifying a 403 kDa protein with a high degree of homology to Pc96. Sequence alignments indicated a region spanning 90 amino acids in Pf403 that overlaps the Pc96 amino acid sequence. A 178 kDa protein in P. yoelii yoelii (Pyy178) was shown to be highly similar to Pc96. Tvcell epitope prediction programs identified putative T cell epitopes in Pc96 which appear to be conserved in Pf403 and Pyy178. A casein kinase IT phosphorylation site was also identified in this region and is conserved in both sequences. PCR primers were designed to amplify regions of the MAL3P6.11 gene coding for Pf403 from P.falciparum genomic DNA. An 817 bp region in the MAL3P6.11 gene was amplified. This codes for the region ofPf403 that shows high homology to Pc96 and contains the conserved T cell epitopes and casein kinase phophorylation site. A BamHI site was incorporated into the forward primer to facilitate in-frame ligation with cloning vectors. The PCRproduct obtained was verified by restriction analysis using HindIII and EcoRI sites within the fragment. The 817 bp peR product was cloned into the pMOSBlue vector using a blunt-endedPCR cloning kit, and transformed into MOSBlue competent cells. Recombinants were identified using the uIV complementation system, and verified by PCR, plasmid DNA isolation, and restriction digestion analysis. The insertDNA in pMOSBlue was cut out with BamHI and sub-cloned into the BamHI site in the pMAL-C2x expression vector. Sequencing ofthe construct confirmed the identity of the cloned insert and showed the sequence to be in frame with the malE gene coding for maltose binding protein (MBP). The fusion protein, MBP-Pf32 .5, was induced and expressed as a 75 kDa protein comprising ofthe 32.5 kDa region ofPf403, and MBP (42.5 kDa) and was detected by anti-MBP antibodies, by western blotting. This recombinant protein has many applications for further studies involving the characterisation of the Pf403 protein, and the determination of possible roles that the protein may have in stimulating an immune response during human malaria infections. / Thesis (M.Sc.) - University of Natal, Pietermaritzburg, 2003.

Heat Shock 40 kDa proteins (Hsp40s) partner with heat shock 70 kDa proteins (Hsp70s) in facilitating, among other chaperone activities; correct protein transport, productive protein folding and assembly within the cells; under both normal and stressful conditions. Hsp40 proteins regulate the ATPase activity of Hsp70 through interaction with the J-domain. Plasmodium falciparum Hsp70s (PfHsp70s) do not contain a Plasmodium export element (PEXEL) sequence although PfHsp70-1 and PfHsp70-3 have been located outside of the parasitophorous vacuole. Studies reveal that a type I P. falciparum (PfHsp40) chaperone (PF14_0359) stimulates the rate of ATP hydrolysis of the cytosolic PfHsp70 (PfHsp70-1) and that of human Hsp70A1A. PFE0055c is a PEXEL-bearing type II Hsp40 that is exported into the cytosol of P. falciparum-infected erythrocytes; where it potentially interacts with human Hsp70. Studies reveal that PFE0055c associates with structures found in the erythrocyte cytosol termed “J-dots” which are believed to be involved in trafficking parasite-encoded proteins through the erythrocyte cytosol. If P. falciparum exports PFE0055c into the host cytosol, it may be proposed that it interacts with human Hsp70, making it a possible drug target. The effect of PFE0055c on the ATPase activity of human Hsp70A1A has not been previously characterised. Central to this study was bioinformatic analysis and biochemical characterisation PFE0055c using an in vitro (ATPase assay) approach. Structural domains that classify PFE0055c as a type II Hsp40 were identified with similarity to two other exported type II PfHsp40s. Plasmids encoding the hexahistidine-tagged versions of PFE0055c and human Hsp70A1A were used for the expression and purification of these proteins from Escherichia coli. Purification was achieved using nickel affinity chromatography. The urea-denaturing method was used to obtain the purified PFE0055c whilst human Hsp70A1A was purified using the native method. PFE0055c could stimulate the ATPase activity of alfalfa Hsp70, although such was not the case for human Hsp70A1A in vitro.