No description available.

The development of an efficacious P. falciparum malaria vaccine remains a top priority. Pre-erythrocytic vaccine efforts have traditionally focussed on two well- known antigens, CSP and TRAP, yet thousands of antigens are expressed throughout the liver-stage. The work described in this thesis aimed to assess the ability of other pre-erythrocytic antigens to induce an immune response and provide protective efficacy against transgenic parasites in a mouse model. Research undertaken in our laboratory has demonstrated the ability of prime-boost viral vectored sub-unit vaccination regimens to elicit high levels of antigen-specific T cells. Eight candidate antigens were therefore expressed individually in the viral vectors ChAd63 and MVA. Two antigens, PfLSA1 and PfLSAP2, were identified that confer greater protective efficacy in inbred mice than either CSP or TRAP. PfLSA1 was also able to induce almost complete sterile efficacy in outbred mice, suggesting this vaccine should be assessed in a clinical trial. Immune responses to the candidate antigens were also assessed in human volunteers following their first exposure to controlled malaria infection. The antigen TRAP was further characterised by epitope mapping in volunteers vaccinated with ChAd63-MVA ME-TRAP. However, no functional T cell assay exists to measure inhibition of P. falciparum liver-stage parasites. An improved murine in vitro T cell killing assay was developed, and preliminary experiments were conducted that demonstrate the potential and promise of a P. falciparum T cell killing assay. Such assays will not only allow mechanistic studies to be undertaken, but could also change the way we screen pre-clinical liver-stage vaccines.

A highly effective vaccine against malaria is urgently needed, with leading vaccination strategies involving the induction of protective antigen-specific CD8⁺ T-cells via heterologous prime-boost viral vector immunization, targeting primarily the pre- erythrocytic liver stages of the Plasmodium falciparum lifecycle. To date, the greatest immunogenicity has been obtained through a heterologous prime boost regimen, where vaccination with an Adenoviral vector is followed 8 weeks later by a Modified Vaccinia Ankara virus (MVA) boost. Experimental work directed at providing a greater understanding of CD8⁺ T-cell memory responses induced by Ad-MVA vaccinations lead to the development of a novel vaccine strategy aimed at priming CD8⁺ T-cells in the periphery and subsequently targeting them to hepatic tissue with protein loaded poly(lactic- co-glycolic acid) nanoparticles or recombinant viral vectors. Durable Ag-specific CD8+ T- cells exhibiting a phenotype of tissue-resident memory T-cells were generated in the liver, with a ten-fold increase over the conventional heterologous vector regimen. Importantly, in P. berghei sporozoite challenge models of liver-stage malaria, this strategy was found to result in unprecedented levels of sterile protection across multiple clinically relevant antigens and mouse strains. This prime and target immunization strategy for liver-stage malaria may provide a novel general approach for prevention or immunotherapy against other hepato-trophic pathogens.

No description available.

Naturally-occurring CD4+Foxp3+ regulatory T cells (nTreg) play a central role in maintaining immune self-tolerance as well as modulating immunity towards pathogens. Pathogens may establish chronic infections in immunocompetent hosts by engaging nT reg in order to promote immunosuppression. The goal of the research described here is to test the hypothesis that nTreg modulate protective immunity to malaria, and consequentially affect susceptibility to the parasite. To investigate this question, the functional dynamics of CD4+Foxp3 + nTreg cells were evaluated in mice infected with blood-stage Plasmodium chabaudi AS. Adoptive transfer of nTreg to infected wild-type C57BL/6 (B6) mice or infection of transgenic B6 mice over-expressing Foxp3 resulted in increased parasitemia and reduced survival compared to control mice. Moreover, while resistant B6 mice exhibited decreased splenic nT reg frequencies at day 7 post infection, susceptible A/J mice maintained high numbers of nTreg at this time. Investigation of the effects of nTreg regulation on immune cell function in P. chabaudi AS-infected mice revealed that increased nTreg frequencies led to decreased malaria-specific lymphoproliferation and increased systemic levels of IL-10. Unlike B6 mice, increased splenic nTreg frequencies in infected A/J mice correlated with decreased effector T cell proliferation and IFN-gamma secretion, decreased B cell and NK cell proliferation as well as deficient IFN-gamma secretion by NK cells. Finally, nTreg proliferated within infected sites in both B6 and A/J mice, albeit to a greater extent in susceptible A/J mice. Altogether, these results demonstrate that nTreg suppressed anti-malarial immunity, and in turn promoted parasite growth and persistence.

No description available.

PhD (Biochemistry) / Department of Biochemistry / Malaria is a parasitic disease that accounts for more than 660 thousand deaths annually, mainly in children. Malaria is caused by five Plasmodium species P. ovale, P. vivax, P. malariae, P. falciparum and P. knowlesi. The most lethal cause of cerebral malaria is P. falciparum. The parasites have been shown to up-regulate some of their heat shock proteins (Hsp) in response to stress. Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is central to protein homeostasis and determines the fate of proteins. Six Hsp70 genes are represented on the genome of P. falciparum. The Hsp70 genes encode for proteins that are localised in different sub-cellular compartments. Of these two occur in the cytosol, PfHsp70-z and PfHsp70-1; two occur in the endoplasmic reticulum, PfHsp70-2 and PfHsp70-y; one in the mitochondria, PfHsp70-3 and one exported to the red blood cell cytosol, PfHsp70-x. PfHsp70-1 is a well characterized canonical Hsp70 involved in prevention of protein aggregation and facilitates protein folding. Little is known about PfHsp70-z. PfHsp70-z was previously shown to be an essential protein implicated in the folding of proteins possessing asparagine rich repeats. However, based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins and is thought to serve as a nucleotide exchange factor (NEF) of PfHsp70-1. The main aim of this study is to elucidate the functional roles of PfHsp70-z as a chaperone and its interaction with PfHsp70-1. In the current study, PfHsp70-z was cloned and expressed in E. coli JM109 cells. This was followed by its purification using nickel chromatography. The expression of PfHsp70-z in parasites cultured in vitro was investigated and its association with PfHsp70-1 was explored using a co-immuno precipitation assay. PfHsp70-z expression in malaria parasites is up regulated by heat stress and the protein is heat stable based on investigations conducted using Circular Dichroism. Furthermore, the direct interaction between recombinant forms of PfHsp70-z and PfHsp70-1 were investigated using slot blot and surface plasmon resonance assays. PfHsp70-z was observed to exhibit ATPase activity. In addition, the direct interaction between PfHsp70-z and PfHsp70-1 is promoted by ATP. Based on limited proteolysis and tryptophan fluorescence analyses, PfHsp70-z binds ATP to assume a unique structural conformation compared to the conformation of the protein bound to ADP or in nucleotide-free state. PfHsp70-z was able to suppress the heat-induced aggregation of malate dehydrogenase and luciferase in vitro. Interestingly, while ATP appears to modulate the conformation of PfHsp70-z, the chaperone function of PfHsp70-z was not influenced by ATP. Altogether, these findings suggest that Characterization of Heat Shock Protein 70-z (PfHsp70-z) from Plasmodium falciparum iii PfHsp70-z serves as an effective peptide substrate holding chaperone. In addition, PfHsp70-z may also serve as the sole nucleotide exchange factor of PfHsp70-1. The broad spectrum of functions of this protein, could explain this PfHsp70-z is an essential protein in malaria parasite survival. This is the first study to show that PfHsp70-z possess independent chaperone activity and that it interacts with its cytosolic counterpart, PfHsp70-1 in a nucleotide dependent fashion. Furthermore, the study shows that PfHsp70-z is a heat stable molecule and that it is capable of forming high order oligomers.

There is an urgent need for improvements to existing vaccine delivery technologies to run parallel with the development of new-generation vaccines. The burdens of needle-based immunisation strategies are exacerbated by poor resource provision in such areas as sub-Saharan Africa, where annual malaria mortality stands at 860,000. Needle-free delivery of vaccine to the skin holds promise for improved immunogenicity with lower doses of vaccine, in addition to significant logistical advantages. Various methods have been described for the transcutaneous delivery of vaccines, including the use of microneedles to overcome the outer stratum corneum of the skin for efficient delivery of liquid or solid, microneedle-coated vaccines into underlying strata rich in antigen-presenting cells. This thesis aims to evaluate two transcutaneous silicon microneedle and microprojection patch technologies for the delivery of live recombinant Adenovirus and Modified Vaccinia Ankara-vectored vaccines encoding pre-erythrocytic malaria antigens in mice. Cellular immunogenicity directed against a well-documented epitope of the Plasmodium berghei circumsporozoite protein is evaluated, as is protection against lethal P. berghei sporozoite challenge. Immunological and logistical benefits of each technology are assessed, as well as mechanisms underlying differences in the generation of a patch-induced immune response to vaccination. These data inform the future development of transcutaneous microneedle patches for the delivery of live vaccine.

O Antígeno-1 de Membrana Apical (AMA-1) de merozoítas de Plasmodium é um dos principais candidatos a compor uma vacina contra a malária. A função biológica de AMA-1 não é totalmente conhecida, entretanto, existem evidências que sugerem a participação dessa proteína na ligação a eritrócitos de diferentes espécies de Plasmodium. O objetivo deste estudo foi investigar a participação de AMA-1 de P. vivax (PvAMA-1) na ligação a eritrócitos humanos utilizando células COS-7 transfectadas com plasmídios recombinantes codificando diferentes regiões do ectodomínio de PvAMA-1. Para isso, os genes que codificam os domínios I-II ou III de PvAMA-1 foram inseridos no vetor pDisplay-EGFP, que permite a expressão das proteínas recombinantes em fusão com a porção N-terminal da Proteína Fluorescente Verde (GFP). Em paralelo, utilizamos construções contendo os genes que codificam a região C-terminal de 19 kDa da Proteína 1 de Superfície do Merozoíta (PvMSP119) e a região II da Duffy Binding Protein (PvDBP-RII). As quatro construções foram utilizadas para transfectar células COS-7 na presença de lipofectamina. A eficiência das transfecções transientes foi confirmada por imunofluorescência utilizando anticorpos específicos. Em seguida, estudamos a participação dos diferentes domínios de PvAMA-1 na ligação aos eritrócitos. Nossos resultados mostraram que os domínios contíguos I-II, ao contrário do domínio III, foram capazes de se ligar a eritrócitos in vitro. Essa ligação foi específica, pois soros de indivíduos infectados por P. vivax e soros policlonais de camundongos contendo anticorpos anti-PvAMA-1 foram capazes de inibir essa ligação em 82,0% e 79,8%, respectivamente. Além disso, anticorpos monoclonais dirigidos contra o domínio II dessa proteína foram capazes de inibir parcialmente essa ligação. Após o tratamento de eritrócitos com tripsina ou quimiotripsina, estas células perderam grande parte de sua capacidade de ligação, sugerindo que o receptor para PvAMA-1 tenha constituição predominantemente protéica. Em conjunto, nossos resultados podem servir de base para futuros estudos visando um melhor entendimento da função de anticorpos gerados durante a infecção natural ou induzidos após vacinação com PvAMA-1. / The Apical Membrane Antigen (AMA-1) of Plasmodium merozoites is one of the main candidates to be part of a vaccine against malaria. The biological function of AMA-1 is unknown. However, there are evidences that suggest the participation of this protein in the interaction with erythrocytes (RBC) of different Plasmodium species. Using transfected COS-7 cells with recombinant plasmids encoding different portions of the PvAMA-1 ectodomain, our aim was to identify possible domains of PvAMA-1 able to interact with human RBC. The genes that encoded domains I and II in combination or domain III of PvAMA-1 were cloned into the pDisplay-EGFP vector. This vector allows expression of the protein fused to the N-terminus of enhanced green fluorescent protein (GFP). In parallel, we also used constructions containing the genes that encoded the 19 kDa C-terminal region of Merozoite Surface Protein 1 (PvMSP119) and region II of the Duffy Binding Protein (PvDBP-RII). Constructions were used to transiently transfect COS-7 cells. The efficiency of expression of all constructs was confirmed by immunofluorescence assay using specific antibodies. After that, we studied the participation of the different domains of PvAMA-1 in the binding to human RBC. We found that COS-7 cells expressing domains I-II, but not domain III, bound to human RBC in vitro. This binding was specific, because sera from malaria-infected patients and mouse polyclonal sera containing antibodies to PvAMA-1 were able to block the adhesion by 82.0% and 79.8%, respectively. Moreover, monoclonal antibodies directed against domain II were partially inhibitory in the cytoadherence assays. The receptor recognized on the surface of COS-7 cells expressing the domains I and II was partially removed after human RBC were treated with trypsin or chymotrypsin, suggesting that its composition is predominantly protein. In conclusion, our results can be used as basis for future studies aimed at better understating the function of the antibodies generated during the natural infection or after vaccination with PVAMA-1.

O Antígeno-1 de Membrana Apical (AMA-1) de merozoítas de Plasmodium é um dos principais candidatos a compor uma vacina contra a malária. A função biológica de AMA-1 não é totalmente conhecida, entretanto, existem evidências que sugerem a participação dessa proteína na ligação a eritrócitos de diferentes espécies de Plasmodium. O objetivo deste estudo foi investigar a participação de AMA-1 de P. vivax (PvAMA-1) na ligação a eritrócitos humanos utilizando células COS-7 transfectadas com plasmídios recombinantes codificando diferentes regiões do ectodomínio de PvAMA-1. Para isso, os genes que codificam os domínios I-II ou III de PvAMA-1 foram inseridos no vetor pDisplay-EGFP, que permite a expressão das proteínas recombinantes em fusão com a porção N-terminal da Proteína Fluorescente Verde (GFP). Em paralelo, utilizamos construções contendo os genes que codificam a região C-terminal de 19 kDa da Proteína 1 de Superfície do Merozoíta (PvMSP119) e a região II da Duffy Binding Protein (PvDBP-RII). As quatro construções foram utilizadas para transfectar células COS-7 na presença de lipofectamina. A eficiência das transfecções transientes foi confirmada por imunofluorescência utilizando anticorpos específicos. Em seguida, estudamos a participação dos diferentes domínios de PvAMA-1 na ligação aos eritrócitos. Nossos resultados mostraram que os domínios contíguos I-II, ao contrário do domínio III, foram capazes de se ligar a eritrócitos in vitro. Essa ligação foi específica, pois soros de indivíduos infectados por P. vivax e soros policlonais de camundongos contendo anticorpos anti-PvAMA-1 foram capazes de inibir essa ligação em 82,0% e 79,8%, respectivamente. Além disso, anticorpos monoclonais dirigidos contra o domínio II dessa proteína foram capazes de inibir parcialmente essa ligação. Após o tratamento de eritrócitos com tripsina ou quimiotripsina, estas células perderam grande parte de sua capacidade de ligação, sugerindo que o receptor para PvAMA-1 tenha constituição predominantemente protéica. Em conjunto, nossos resultados podem servir de base para futuros estudos visando um melhor entendimento da função de anticorpos gerados durante a infecção natural ou induzidos após vacinação com PvAMA-1. / The Apical Membrane Antigen (AMA-1) of Plasmodium merozoites is one of the main candidates to be part of a vaccine against malaria. The biological function of AMA-1 is unknown. However, there are evidences that suggest the participation of this protein in the interaction with erythrocytes (RBC) of different Plasmodium species. Using transfected COS-7 cells with recombinant plasmids encoding different portions of the PvAMA-1 ectodomain, our aim was to identify possible domains of PvAMA-1 able to interact with human RBC. The genes that encoded domains I and II in combination or domain III of PvAMA-1 were cloned into the pDisplay-EGFP vector. This vector allows expression of the protein fused to the N-terminus of enhanced green fluorescent protein (GFP). In parallel, we also used constructions containing the genes that encoded the 19 kDa C-terminal region of Merozoite Surface Protein 1 (PvMSP119) and region II of the Duffy Binding Protein (PvDBP-RII). Constructions were used to transiently transfect COS-7 cells. The efficiency of expression of all constructs was confirmed by immunofluorescence assay using specific antibodies. After that, we studied the participation of the different domains of PvAMA-1 in the binding to human RBC. We found that COS-7 cells expressing domains I-II, but not domain III, bound to human RBC in vitro. This binding was specific, because sera from malaria-infected patients and mouse polyclonal sera containing antibodies to PvAMA-1 were able to block the adhesion by 82.0% and 79.8%, respectively. Moreover, monoclonal antibodies directed against domain II were partially inhibitory in the cytoadherence assays. The receptor recognized on the surface of COS-7 cells expressing the domains I and II was partially removed after human RBC were treated with trypsin or chymotrypsin, suggesting that its composition is predominantly protein. In conclusion, our results can be used as basis for future studies aimed at better understating the function of the antibodies generated during the natural infection or after vaccination with PVAMA-1.