De Souza, Joseph Brian; (1992) Blood-stage malaria: Identification of protective antigens for vaccination. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A Triton X-100 soluble lysate prepared from heavily parasitized erythrocytes of blood-stage P.yoelii malaria has been shown to be an effective vaccine against this fatal infection in mice. The large number of antigens present within this lysate were fractionated and tested for their ability to protect against live challenge and for their ability to stimulate parasite-specific T-helper cells and T cells responsible for delayed hypersensitivity (DTH). Enzymatic digestion of the protective fractions with either proteases or lipases suggested that the protective moiety of the antigens is mainly protein. Amino acid sequence analysis of the most protective antigens was attempted. A two-step physical separation procedure was established for fractionation of the lysate. Isoelectric focussing (IEF) or Sephacryl S.200 gel filtration chromatography (S.200) were used as first step purification procedures. This was essential for the removal of impurities such as lipoprotein complexes and excess detergent from the lysate, thereby making it more suitable for final purification by High (HPLC) or Fast (FPLC) Performance Liquid Chromatography. Several fractions with varying degrees of protection were isolated. The most consistently protective fractions appeared around the pH4, 6 and 8 regions on IEF. The pH4 fraction contained six protective FPLC purified components. The pH6 and 8 fractions contained one and six semi-protective HPLC purified components respectively. Other consistently protective fractions included Peaks I and II (S.200) and an HPLC purified fraction (fraction h) derived from Peak I. A partial amino acid sequence of fraction h has been obtained; weight for weight this is the most protective preparation available. There was a significant correlation between protection and T- helper cell priming with the most protective antigens. However S.200 Peak I and its HPLC purified fraction h were relatively weaker stimulators of DTH. Thus although T-helper cell priming may be an essential requirement for the induction of protective immunity against malaria, strong priming for DTH may not always be necessary. Selecting antigens that are strong stimulators of T-helper cell responses for antibody ("TH2" type of T cell) and to a lesser extent for DTH ("TH1" type), may therefore be important in terms of vaccine development.

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