Antibody mediated immunity against malaria and application of phage display technology

Vukovic, Peter (2002). Antibody mediated immunity against malaria and application of phage display technology PhD Thesis, The School of Population Health, The University of Queensland.

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Author Vukovic, Peter
Thesis Title Antibody mediated immunity against malaria and application of phage display technology
School, Centre or Institute The School of Population Health
Institution The University of Queensland
Publication date 2002
Thesis type PhD Thesis
Supervisor Prof. Michael Good
Prof. Alan Saul
Dr. Michael Foley
Total pages 179
Language eng
Subjects 1107 Immunology
Formatted abstract Malaria is a major disease of the 21st century, being the cause of significant morbidity and mortality throughout the world - particularly in the tropics, sub-tropics and sub-Saharan regions. The disease is caused by protozoan parasites of the genus Plasmodium and various species of this genus infect most land animals. There are 4 main species infecting man: falciparum, vivax, ovale and malariae. P. falciparum is by far the most virulent, and is the main cause of death, especially in children under the age of 5 years. Mosquitoes, generally of the genus Anopheles, spread the parasite from person to person. The clinical presentations of disease symptoms are caused by the asexual replication of the parasite inside the red blood cell (RBC) of the host. It is for this reason that the bulk of vaccine research against malaria has focused on this asexual life-cycle stage. Nevertheless, other stages of the life cycle are important and are also researched, such as transmission blocking vaccines and liver stage vaccines. Indeed, the development of new chemotherapeutics and effective vaccines has now become imperative, with the evolution of drug resistant strains of malaria parasites. Vaccine research concentrates upon the use of protein subunit vaccines. Merozoite Surface Protein-1 (MSP1) is a leading vaccine candidate and research is focussing on the 19-kilodalton-remnant fragment (MSP119) on the surface of merozoites during the asexual blood stage of the parasite. The mechanism of immunity to the malaria parasite has long been the subject of intense speculation and research. With regards to MSP119, the main mechanisms of protection against challenge have been thought to be mediated by antibody (adaptive responses), with possible roles played by the cell-mediated immunity (CMI) or innate arm of the immune system.

In order to study these mechanisms, experiments were carried out in murine experimental systems to determine: firstly, the systems needed for the development of immunity by immunisation with MSP119 and the effects of passively transferring protective antibody to naïve mice; secondly, the spectrum of action with regards to species specifictiy of immunisation with MSP119; and finally, experiments in Fc-gamma receptor 1 (FcγR1) knockout mice to further explore the mechanisms of anti-MSP119 antibody-mediated protection against malaria. Additionally, the new technique of phage display technology has been utilised to produce new tools in the form of antibody fragments: single chain variable fragments (scFv). These scFv were used to probe the mechanism of antibody -mediated protection against malaria challenge with respect to immunity afforded by MSP119. Specific scFv against MSP119 were isolated from this novel phage display library and analysed in their properties.

It is reported here that it is vitally important for the development of an effective immunity against malaria, with respect to immunisation with MSP119, for an active immune response involving B cells and helper T cells, to produce sufficient antibody levels to protect an individual against further infection. Additionally, the passive transfer of high levels protective antibody can sufficiently delay the onset of parasite patency possibly to allow for the development of said immunity. Of course, the actions of the innate immune system are not to be completely precluded from a role in early responses to the parasite. Secondly, mice transferred with protective anti-MSP119 antibody develop immunity due to the delay in the onset of patency, and the same is true for mice who do not posses Fc receptors (FcγR1) for the monoclonal antibody (class lgG3) used in these studies, thus ruling out Antibody Dependent Cell Cytotoxicity (ADCC) as a mechanism of protection in this mouse model involving anti-MSP119 antibodies. Thirdly, the spectrum of action of protection afforded by anti-MSP119 antibodies unfortunately appears to be species specific, although it is still unknown whether the protection is strain specific. It would also appear that antibody-mediated protection could be mediated by Fab fragments alone, showing promise for the use of scFv fragments. Finally, the construction of a novel scFv phage display library and the enrichment for- and isolation of- MSP119-specific scFv and subsequent analysis have shown apparently favourable comparison with protective monoclonal antibodies as studied by surface plasmon resonance on a BIACORE. In vivo mouse protection experiments using MSP119 specific scFv have also shown significant delays in the onset of parasite growth (the hallmark of antibody effectiveness). The fact that these scFv apparently work in vivo in a similar manner as normal antibody confirms the data that Fab are effective and indicates that the mechanism of protection mediated by antibody is not that of ADCC (confirming the FcyRI knockout mouse experiments); opsonisation or activation of complement cascades (both mediated by Fc); or agglutination/precipitation of the parasite (effect of divalency of the antibody). Therefore, the main action of the antibody would appear to be blocking antibodies or steric hindrance of the malaria parasite. This could be either in the form of inhibiting the parasite derived serine protease processing of the precursor MSP1 molecule or interference of the parasite interaction with the surface of fresh RBC: both mechanisms prevent the parasite invading fresh RBC and may therefore allow bystander immune cells to capture the exposed merozoites.

MSP119 specific antibody, present at the time of infection with malaria parasites, from either active immunisation or passive adoptive transfer, has been shown to be effective in the delay of parasite growth. In conjunction with an active immune system, this allows the mobilisation of the immune system to mount a defence against the parasite and form a long lasting immunity to challenge. Although the protection afforded by this is seemingly species specific, this could be easily overcome by the addition of different species MSP119 proteins or protective antibody to a final vaccine product. Phage display technology has been useful in the examination of the mechanisms of antibody-mediated protection against MSP119. Products of this technology may even be used as a therapeutic reagent themselves, or as an adjunct to other vaccines or chemotherapeutic regimes.
Keyword Malaria -- Immunological aspects

Document type: Thesis
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