No description available.

Malaria is currently ranked as one of the most deadly human infectious diseases, causing an estimated three million deaths each year with half of these being children under the age of 5 years.  Approximately 500 million people are currently infected with one or more species of <i>Plasmodium,</i> with 90% of the infections occurring in sub-Saharan Africa.  In regions of the highest endemicity, a significant level of malaria transmission is vectored by the anthropophilic mosquito <i>An. gambiae sensu stricto</i> Giles.  Using molecular techniques, native <i>An. gambiae s.s.</i> carboxypeptidase A, a secondary digestive enzyme that breaks down bloodmeal proteins required for egg production, was isolated and two recombinant forms were produced.  These proteins were used as antigens in phage display panning to isolate humanised antibodies with high affinity to the enzyme.  Two of these phage antibodies, A7 and B10, were used in <i>in vitro</i> assays and <i>in vivo </i>feeding studies.  Neither antibody produced enzyme activity <i>in vitro</i>, but A7, which bind specifically to the active site region of the mosquito carboxypeptidase A, affected longevity, egg development, and F1 generation viability.  B10, binding outside of the active site region, generally responded in the same patterns as the PBS control.  A potential detrimental factor in the <i>in vivo</i> studies was the probable toxicity of imidazole, an eluate solution used in phage display, which may have contributed to the mortality rate of mosquitoes in the A7 and B10 treatment groups.  These results have contributed to the redesign of the phage display protocol for use in <i>n vivo</i> mosquito systems.

This thesis is a description of the effects of Sulphadoxine/Pyrimethamine (SP) selection on the genome of Plasmodium Jalciparum. Antimalarial resistance in P. Jalciparum to the antifolate combination of SP is conferred by a series of substitutions that alter the active sites of the target enzymes dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). Different combinations of substitutions result in differing levels of drug insensitivity as shown by both in vitro studies and association with treatment failure. In southeast Africa, where the samples taken for this study originate, the two most highly resistant dhfr and dhps alleles are the triple mutant (N51I+C59R+Sl08N) and the double mutant (A437G+K540E) ,respectively. A molecular population genetic analytic approach is applied to examine the emergence and spread of SP resistant mutations in Africa. We observe that differences in the frequencies of the resistance alleles between southeast African populations broadly reflect heterogeneity in drug selection history across the region. We find that this exists despite strongly homogenising gene flow. Selection for a favourable allele can have effects on neutral loci flanking the selected site. A selective sweep occurs when neutral flanking loci hitchhike with the selected allele as it increases in frequency, reducing genetic diversity along the chromosome in the population. The hitchhiking alleles indicate the ancestry of the selected allele and has shown that alleles at dhfr and dhps comprised of multiples of mutations have emerged rarely in east African parasite populations (Roper et al. 2003). The thesis is the first description of the full extent of the selective sweeps around three dhfr and dhps resistance alleles in southeast Africa. The thesis contains analysis of the changes that occur to a selective sweep over time and in populations with contrasting recombination rates and selection histories. Through use of a deterministic model we identify that gene flow plays an important role in establishing the frequency of the resistance allele at values greater than l/Ne, the frequency of a de novo mutation.

Evidence is accumulating that polymorphisms in plmdr 1 and plcrt are involved in resistance to the quinoline based blood schizontocides and possibly artemisinin derivatives; and mutations in the cytochrome b gene (cytb) are strongly associated with resistance to atovaquone. With this in mind, we studied sensitivity, drug interactions and uptake in a range of Plasmodium lalciparum lines, including pfmdr 1 transfectants, plcrt mutated lines 106/1 and K76I, and an atovaquone-resistant isolate (NGATVOl) with a unique point mutation (tyr268asn). In vitro susceptibility studies and genetic characterisation supported the role of plmdr 1 and plert polymorphisms in the sensitivity of parasite lines to a range of structurally diverse antimalarials. Mutations in plcrt conferred chloroquine resistance modulated by changes in plmdr 1, while the wild-type genes were associated with reduced sensitivity to mefloquine, halofantrine, lumefantrine and dihydroartemisinin. Drug interaction studies in vitro using a modified isobologram method showed that dihydroartemisinin in combination with chloroquine, amodiaquine or the new bisquinoline piperaquine was antagonistic in all parasite lines examined. The response was synergistic when the drug was combined with mefloquine, halofantrine or lumefantrine against chloroquine-sensitive (wild-type plmdrl and plcrt), but additive against chloroquine-resistant parasite lines. However, in the 7G8-m < fIP10 transfectant (wild-type plmdrl and mutated plert), synergy was shown between each of these three drugs and dihydroartemisinin. These results suggest that the interaction profile of dihydroartemisinin with arylaminoa1cohols depends on mutations in plmdrl in the presence of other chloroquine resistance mutations. In the D 1 0_mdr7G8 transfectant, the introduction of the mutated plmdr 1 converted the synergy between lumefantrine and dihydroartemisinin or artemether to addition; supporting the idea that interactions between artemisinin-derivatives and lumefantrine are dependent on mutations within pfmdr 1 alone. An exception was seen when dihydroartemisinin was combined with arylaminoa1cohol quinine. It showed a strain-specific effect not related to resistance polymorphisms, although a similar change to synergy in the 7G8-md~10 transfectant as for the other arylaminoalcohols was seen. A verapamil-reversible change in the quinine and dihydroartemisinin interaction from additive to synergistic was also noted with the introduction of a mutated PfCRT codon 76 in K76I. An antagonistic effect of dihydroartemisinin with pyrimethamine, was observed in all pyrimethamine-sensitive parasites, but the effect was additive in pyrimethamine-resistant parasites. In contrast to the quinolines, this was not associated with inhibition of [³H]-dihydroartemisinin uptake. The interaction between atovaquone and proguanil was strongly synergistic in atovaquone-sensitive lines Kl and T996 and moderately synergistic in the atovaquoneresistant NGATVOI isolate. While the interaction between atovaquone and dihydroartemisinin was additive in the NGATVOI isolate, the interaction was antagonistic in the atovaquone-sensitive strains tested. Mutations in plmdr 1 and plert had no effect on uptake of [³H]-dihydroartemisinin, although [³H]-chloroquine accumulation was strongly affected by these polymorphisms. Uptake studies in combination with other antimalarials indicated that some of the quinoline-related drugs and arternisinin-derivatives competed with the uptake of the two radiolabelled drugs. This work highlights the important role of drug resistance polymorphisms in sensitivity, interaction and uptake of structurally diverse antimalarials. The unusual pattern of antagonism between dihydroartemisinin and pyrimethamine seen in vitro is supported by observations in vivo with artemisinin in rodent parasites. These and other differing interactions between dihydroartemisinin and antimalarials could have particularly important implications for the design of drug combinations in the future.

Malaria is a tropical parasitic disease spread worldwide by protozoan parasites of the genus Plasmodium. Attempts to eradicate or control the disease have largely failed and the weapon-of-choice in combating malaria, a vaccine, still eludes us. Now that the Plasmodium falciparum (Pf ) genome has been revealed, it is essential to search for new potential antigens. On that basis, my project aims to select and produce putative vaccine candidates that together with protein microarray techniques will unravel possible correlates of protection against the human malaria parasite Pf, responsible for the majority (90%) of deaths from malaria. My PhD project forms part of FightMal a consortium-based study that first use bioinformatic analysis to identify those proteins within the Pf proteome that are known or anticipated to be either anchored on the parasite/infected host cell surface or secreted. Such in silico analysis produced 3 ‘candidate lists’ of interest, one for each of the 3 types of protein chips planned for production: i) current vaccine candidates (n.25), ii) putative immune targets (n.260), iii) variant surface antigens, VSA (n.74). Candidates belonging to CHIP 2 were cloned and expressed (in vivo) using customized high-throughput platforms. To date, 394 (88%) Chip 2 DNA targets have been successfully amplified by PCR, 325 (82%) of these have been inserted into the expression plasmid and sequenced. A total of 269 constructs resulted suitable for heterologous expression. Due to the complexity of soluble expression of plasmodial proteins a number of different expression conditions were tested. Both the strain of the E. coli host and the composition of the expression medium resulted crucial variables for satisfactory results. Via a multi-step optimisation phase we increased the initial number of soluble candidates from 18% to 44%, which eventually led to a 6-fold increase in the number of targets available for purification. During an early evaluation study, 77% (10/13) of the recombinant proteins produced in large-scale were purified using embedded affinity tags yielding a ≥90% purity level. Preliminary results, obtained spotting 40 in vitro expressed Pf candidates, successfully demonstrated the potentials of the protein microarray technology as an efficient serum-based multiplex assay capable of identifying new antigens. Using an optimised protocol, over 7000 antigen-antibody interactions were evaluated. 92% of the Pf protein panel was recognised by both total and cytophilic IgGs, with 6 novel candidates resulting as antigenic as some of the antigens evaluated in vaccine clinical trials for malaria. It is envisaged that my PhD will deliver multi-level technical know-how, which could improve the production and evaluation of novel plasmodial vaccine candidates. The final Pf microarray will be employed to screen clinical samples collected from protected and non-protected children enrolled in a longitudinal, case-control study in an area of Uganda where malaria is endemic. It is expected that the FightMal project overall will eventually generate novel immunological data and information pertaining to both the complexity of the plasmodial proteome and variability of the naturally-induced immune response against the malaria parasite.

Three sets of molecular markes were used to investigate the population genetics of three populations of Plasmodium falciparum from Kenya; Mwea (low transmission), Tiwi (moderate transmission) and Bondo (high transmission). One set of markers codes for polymorphic antigens while the other two are microsatellite markers; one set located in non coding regions of the genome while the other set is located in the regions flanking two genes whose products are targets of the antimalarial drug sulphadoxine/pyrimethamine (SP). A comparison of the effectiveness of antigen-coding and the unlinked microsatellite loci in differentiating recrudescence from reinfection revealed that both sets of markers are equally effective. The microsatellite loci however, revealed more alleles per population than the antigen-coding loci possibly due to their different mutation rates. An analysis of the three populations using the neutral microsatellite loci revealed high levels of diversity, lack of linkage disequilibrium and virtually no population substructuring (FST < 0.008) in the Kenyan P. falciparum populations even with the geographical areas being as much as 800 km apart. This indicates a lot of gene flow among these populations a factor that can only be explained by movement of people between the areas studied. An analysis of the same samples from the three areas at the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) gene loci that code for targets of the antimalarial drug SP revealed high prevalence of the multiply substituted alleles associated with SP resistance in the three regions. An analysis of ~17 kb regions flanking both sides dhfr reveal a strong selective sweep of the 108N/51I/59R triple mutation alleles associated with pyrimethamine resistance. The work presented also demonstrates that alleles of the dhfr gene, especially the triple mutant allele, isolated from the three different areas are closely related to one another and probably share a common and very recent ancestor. Most notable is the finding that dhfr triple mutants seem to be imported into the country through immigration from elsewhere. An equivalent region flanking the dhps gene also revealed a strong selective sweep of the 437G/540E double mutation allele associated with sulphadoxine resistance in two of the three sites. However, double mutation dhps alleles from Mwea revealed no selection at all. While the three populations reveal no geographic substructuring using the results of the unlinked microsatellite loci, they seem to be highly structured in their drug resistance patterns. While it would be expected that these populations would have the same prevalence of drug resistance mutations (due to the apparent panmixia), the Mwea population appears quite different in regard to selection for drug resistance-associted alleles. This is possibly due to the diet, other drug interactions or the hosts' genetics in this area. A simplistic model on the rate of spread of drug resistance in the three populations reveals that the selection for drug resistance alleles is faster in the lower transmission area of Mwea (selection coefficient, s = 0.26) and slowest in Bondo (s = 0.10) indicating selection for drug resistant alleles is favoured by low transmission. These observations have implications for malaria drug resistance surveillance programs due to the fact that if treatment failure spreads faster in low transmission areas where almost all the population has low immunity, malaria epidemics are bound to occur resulting in huge morbidity and mortality.

Modified vaccinia virus Ankara (MVA) has been used extensively as a recombinant vector for delivery of antigens from diverse pathogens. Its ability to generate strong antigen specific CD8+ T cell responses in humans has been shown in clinical trials of novel vaccines against malaria, tuberculosis, HIV I AIDS, influenza and cancer. The work in this thesis describes the use of BAC recombineering technology to harness the endogenous regulatory signal (promoter) that drives the expression of non-essential open reading frames (ORFs) in MVA for immunogenic expression of a recombinant antigen. Replacement of the ORFs of four non-essential genes in MVA; C11R, F11l, A44L and B8R with an epitope tagged luciferase positioned to use the same endogenous promoter showed early transgene expression equal to or slightly higher than traditional p7.5 and short synthetic promoter (SSP) constructs. The frequency of antigen-specific CD8+ T cell induced in mice by single dose MVA or adenovirus-prime, rMVA-boost vaccination showed equivalent or slightly higher responses by the endogenous promoters compared to the traditional p7.5 and SSP constructs. Assessment of the growth rate of these viruses showed they were unimpaired and the insertions were genetically stable. Furthermore, the endogenous promoter driven insertion loci of B8R and C11R were used for the construction of a bivalent MVA expressing an epitope tagged luciferase (rLucPb9) and a Photinus pyralis (pLuc) luciferase. The frequency of antigen-specific CD8+ T cells induced in mice by bivalent MV A was equivalent to single-pLuc and single-Pb9 recombinants co-administered as a mixture, at separate sites or administered alone following single dose MV A vaccination but slightly lower for Pb9-specific CD8+ T cell following adenovirus-prime, rMVA-boost.

No description available.

Human malaria caused by Plasmodium falciparum is a major global burden killing between 700,000 and 2.7 million people every year. Africa bears the greatest portion of this burden, with over three quarters of deaths occurring in African children, accounting for 18% of all child deaths in sub-Saharan Africa. Synthesis of tetrahydrofolate through the folate biosynthetic pathway is vital for the survival of P. falciparum parasites and is lacking in the human host. As such, enzymes of this pathway have long presented attractive targets for drug therapy and although increasingly being compromised by resistance, anti-folates such as pyrimethamine and sulfadoxine are still very valuable drugs in many malaria-endemic regions.In this project, further investigation of the enzymes of the folate biosynthetic pathway has been attempted by developing protocols to quantify these proteins and others through proteomic techniques. Two quantification techniques were pursued. The first was quantification using whole, heterologously expressed, stable-isotope labelled forms of P. falciparum proteins for use as heavy standards in mass spectrometry. Great difficulty was experienced in the effort to express and purify P. falciparum enzymes in E. coli expression systems, with only one enzyme successfully expressed and purified in a 13C-labelled form. This one protein was taken forward into quantification experiments. The second quantification technique used a stable-isotope labelled ‘QConcat’ protein, consisting of a number of peptides from 12 P. falciparum proteins of interest, as a heavy standard in mass spectrometry. This was successfully expressed and purified in a 13C-labelled form from an artificial gene using an E. coli expression system. This too was taken forward into quantification experiments.Quantification experiments using the QConcat-based quantification technique were successfully performed on whole P. falciparum extract. Among the proteins quantified were SHMT and DHFR, two proteins of great interest from the folate biosynthetic pathway. Consistent with results from different expression analysis techniques in the literature, the folate enzymes were found to be of lower abundance than housekeeping enzymes and SHMT was found to be more abundant than DHFR.For deep quantitative analysis of the P. falciparum proteome, it was found that fractionation was necessary. Fractionation in this project was performed using a ZOOM™ IEF fractionator (Invitrogen), an OFFGEL™ IEF fractionator (Agilent) and 1D SDS-PAGE. It was found that by using these fractionation techniques, more proteins could be identified within the P. falciparum proteome, with all but one of the enzymes of the folate biosynthetic pathway being identified. Significant advances in the sensitivity of mass spectrometers during this project have also greatly facilitated the investigation of the proteome. In some cases, this meant that proteins which were only previously accessible by prefractionation of the proteome could be seen in whole P. falciparum extract. Unfortunately, QConcat-based quantification using both fractionation and sensitive mass spectrometry could not be successfully achieved in the time available. However, the promising results obtained suggest that, after careful optimisation, such an approach will be valuable.

Early diagnosis and prompt treatment is one of the malaria control strategies used to minimize malaria morbidity and mortality. One of the mechanisms to implement early diagnosis and prompt treatment is community access to diagnostic services and effective antimalarial drugs. However, in Ethiopia the health system is underdeveloped and much of the rural population has limited access to modern health services. Therefore, the Ethiopian government introduced the Health Extension Programme(HEP) which is a community-based health care delivery system aimed at accessing essential health services through its health extension workers (HEWs). Involvement of the HEWs in prescribing and dispensing antimalarial drugs is shown to have improved community access to antimalarial drugs. However, there is insufficient knowledge of HEWs compliance to malaria treatment guidelines and patient adherence of patients treated by HEWs. The objectives of this study has been to describe the HEWs practice in malaria treatment, to evaluate adherence of patients to antimalarial drugs, to explore the factors influencing the HEWs malaria treatment practice and patient adherence, and to develop the guidelines to support the HEWs in malaria treatment practice. A qualitative study design was used to study the HEWs practice in malaria treatment along with patient adherence. Data were collected using in-depth face-to-face interviews, focus group discussion and patient medical record review and were analysed according to Tesch’s steps. The study revealed that the HEWs adequately comply with malaria treatment guidelines during diagnosis of malaria, as well as during the prescribing and dispensing of antimalarial drugs. However, there are some factors influencing the performance of HEWs. These are: shortage of diagnostic kit/RDT, shortage of antimalarial drugs, patient pressure to obtain coartem, work load, and community beliefs with regard to antimalarial drugs effectiveness. This study also revealed that the HEWs follow up after treatment of patients and good community support systems improved patient adherence to antimalarial drug use. Factors negatively influencing patient adherence were identified to include: forgetfulness, fear of shortage of drugs, adverse drug effects, duration of treatment, rapid relief of malaria symptoms and inadequate awareness of the consequence of incomplete dosage. Guidelines were developed to support the HEWs in malaria treatment practice with the aim to improve patient adherence to antimalarial drugs / Health Studies / D. Litt. et Phil. (Health Studies)