Malaria causes a global burden in terms of both health and economy. With the emergence of drug resistant parasites and pesticide-resistant vectors, vaccination remains the most cost-effective and affordable method of malaria control and/or eradication. Effective vaccine development requires the identification and analysis of parasite antigens, demonstration of protection with the antigens and a thorough understanding of mechanisms of immunity induced by the protective antigens. Immunity to malaria is a function of both humoral and cellular immune responses and both T-cells and antibodies are crucial for protection against the blood stage parasite. The asexual blood stage is critical in the parasite life cycle because it is the only stage associated with disease leading to mortality.
Although much work has been done with surface membrane molecules as vaccine candidate antigens and particulate antigens accessible to humoral responses, little is known about soluble proteins especially the cytoplasmic molecules released during the asexual blood stage. Current malaria candidate vaccines have been identified by their ability to stimulate secretion of antibodies. However there is accumulating evidence of protection in studies with B-cell-deficient mice suggesting the existence of strong antibodyindependent protective responses induced by soluble proteins of the blood stage parasite. Despite accumulation of the knowledge of protective T cellmediated antibody-independent responses, no antigens have ever been isolated that define the specificity of the responses. This thesis aimed to isolate the soluble protein antigens of P. yoelii 17XNL and investigate protective T cell responses in mice as a means of determining the specificity of the T cells.
To demonstrate the relative role of T-cells in protection, parasite-specific CD4+ T-cells that showed strong lymphoproliferative responses to soluble and whole parasite antigens, and which secreted large amounts of IL-2, IFNγ and TNF-a and little or no IL-4, were generated. Following a challenge infection, B cell-deficient mice adoptively transfused with the parasite-specific T cells were protected as shown by delayed patent parasitaemia and suppressed parasite growth. The T-cells responded strongly to stimulation with soluble parasite antigen (sAg) indicating the presence of T cell-stimulating antigens in the fraction.
Following a sequential method of fractionation by isoelectric focussing (lEF) and SDS-PAGE, and vaccination and adoptive transfer of T-cells specific to fractionated antigens, a pool of fractions designated F (pH 5.4-5.8) that protected immunised normal BALB/c, was isolated. There was no significant difference in anti-malaria antibody titres in sera from F-immunised mice and PBS-immunised control littermates as tested by ELISA, suggesting that protection induced may not necessarily involve antibodies. Adoptive transfer of F-specific T cells also transferred immunity to P. yoelii infection in BALB/c nude and SCID recipients. The titres of anti-malaria antibody in sera from BALB/c nude mice transfused with F-specific T cells were similar to sera from control littermates receiving ovalbumin-specific T cells or no T cells, suggesting the transfused T-cells but not antibodies were responsible for induction of protection. Immunoblot analysis of sera from protected T cellreconstituted SCID mice revealed absolutely no antibodies to malaria parasite antigens, confirming the involvement of an antibody-independent T cell immune response.
A three-step method involving wide pH range lEF to isolate the protective fraction, narrow pH range lEF to separate F-fraction proteins into sub-fractions and SDS-PAGE to purify proteins, was used to obtain single band proteins. Two separate bands of very similar molecular weights (doublet band 16, approximately 26-28kDa) both associated with protection of SCID mice recipients of T-cells specific to band 16 polypeptides were purified. The amino terminal protein sequencing identified the amino acids as MKIPNNPGAGELGYEPVMI and MKIPN for the upper (PI) and lower (P2) bands respectively in the doublet, indicating that they are the same polypeptide but perhaps with different post-translation modifications. A Plasmodium protein database BLAST search using the nineteen-amino acid sequence as reference retrieved P. berghei and P. falciparum hypoxanthine-guanine- xanthine phosphoribosyltransferase (HGXPRT) enzyme, that showed 100% and 63% respective homology to the reference sequence. Lymph node cells primed in vivo to PI responded ex vivo to stimulation with both P2 and recombinant PfHGXPRT polypeptides, supporting the identity of PI, P2 and HGXPRT enzyme as the same molecules. At day 8, recombinant PfHGXPRTimmunised normal BALB/c mice demonstrated significantly lower parasitaemia than the ovalbumin- or PBS-immunised control littermate mice (p<0.0001 and p<0.001 respectively) and two of the five mice completely resolved their infection.
This study therefore has identified the Plasmodium purine salvage enzyme, HGXPRT, as a target antigen for induction of protective immunity mediated by Th1-like cells. The data here have demonstrated that, HGXPRT is a novel immunogen for consideration in designing a malaria vaccine against blood stage infection. Its protective efficacy is independent of antibody and therefore also represents a novel approach to malaria vaccine development.