CHAPTER 1 INTRODUCTION
1.4 Vaccination against malaria
1.4.2 Merozoite stage vaccines
way to prevent this effect of genetic restriction of protection against malaria using DNA immunization with multi-gene representatives of the CSP..
Specific proteins have been shown to be required for sporozoite gliding motility and aspects of liver cell invasion and are part of a family of proteins found in the micronemes of invasive stages of apicomplexan parasites. These proteins are trans-membrane proteins and studies were performed investigating their role in cell invasion. Antibodies were effectively raised against these putative antigens, however they are not expressed in sufficient amounts on the cell surface for effective recognition and blocking by the immune system (Ganttet a!.,2000).
The advance in the technology of proteomics and genomics, along with more powerful methods of DNA sequencing, have allowed more detailed studies on the sporozoite stage, and the expression of proteins. cDNA Libraries were constructed, from the salivary gland sporozoites of theP. yoelii yoeliimodel (Kappe et al., 2001). A total of 1972 expressed sequence tags (ESTs) were generated from the construction of the library, enabling all expressed proteins in this stage to be evaluated.
Metabolic pathways and protein expression, thought not to occur during the sporozoite stages were identified, such as key enzymes in the shikimate pathway. Proteins were more effectively studied in the context of cell invasion. New insights into protein expression during the sporozoite stage were gained and various adhesion ligands were discovered which had potential for formulating a pre-erythrocytic based vaccine.
There are several proteins whose coding sequences belongingto the var gene family, including the gene for PfEMP1. This allows theP.falciparum parasite to express multiple forms of the protein, which contribute to the success of the parasite as a pathogen. These variant proteins have the ability to evade secondary disease, by the expression of different forms of proteins, avoiding detection by antibodies previously raised against proteins such as PfEMP1. This has been studied in several models including simian, murine and human malaria species indicating that anti genic variance in the surface of infected red blood cells has been linked to the lengthened persistence of infection, and the reestab1ishment of disease. Antibodies raised against specific forms of the PfEMP1 have shown to block adherence to CD36 in infection with the same isolates. This has paved the way for several important studies on vaccination against merozoite stage malaria (Baruch et al., 1995). These antibodies did not reduce the adherence to other receptors such as thrombospondin yet this study provided the molecular basis towards detailed studies on merozoite involvement in cerebral malaria.
The merozoite surface protein-1(MSP-1) is a 180 to 210kDa protein and has been found to elicit effective levels of immunity. This antigen is located on the surface of P. jalciparum merozoites and a successful vaccine against asexual blood stage malaria may be based on this protein.
Immunization with native or recombinant forms of MSP-1 has partially protected Aotus and Saimiri monkeys against P. jalciparum. Po1yclona1 and monoclona1 antibodies raised against MSP-1 have been shown to exhibit inhibitory effects on the growth of the parasites. Antibodies from human sera, affinity purified to MSP-1, inhibit parasite invasion in vitro and vaccinated mice elicits complete protection against a lethal challenge infection (Daly and Long, 1993). Vaccination of Aotus monkeys with two forms of MSP-1 provides protection against lethal challenge with P.
jalciparum malaria parasites (Egan et al.,2000).
Protein P126 has been associated with the release of merozoites from mature schizonts, and has been identified as possessing immunogenic properties. The protein is expressed by the parasite in the 32nd and 36thhour of a 42-hour erythrocytic cycle before being stored inside a parasitophorous vacuole. This antigen could be involved in a successful subunit vaccine due to its re1atedness to those antigens in several strains ofP.jalciparum and similarity in the amino acids in this region (Bhatia et al., 1987). Monoclona1 and polyc1onal antibodies specific for p126 are effective in the inhibition of the growth of the parasite in vitro (Chulay et al., 1987) and can induce partial protection against challenge in various species of monkeys (Banie et al., 1998).
A 155-kDa protein was identified on the surfaces of infected read blood cells due to the recognition of specific modifications of the erythrocyte in comparison to uninfected cells (Perlman
et al., 1984). The protein was termed Pfl55lRESA (ring-infected erythrocyte surface antigen) and is involved in merozoite invasion (Walinet al., 1992). Thesequence of the protein contains sets of blocks of tandemly repeated amino acid sequences mainly in the carboxy terminal (C-terminal) and central regions. These are repeats of the peptide (EENVEHDA) and (EENV) (Kabilanet al., 1988). Antibodies raised against RESA have been shown to specifically inhibit the rate of parasite growth in vitro, showing the potential of this protein as a possible vaccine candidate (Collins et al., 1986).Large proportions of human anti-Pfl55 antibodies are directed against epitopes formed by these sequences, and have shown to be conserved between different isolates ofP.falciparum.
Intact Pfl55 protein has lead to the in vitro proliferation, interleukin-2 (IL-2) release, and interferon-y production in T cells specific to this antigen in infection. This antigen,indicating that the protein contains T-helper cell stimulating epitopes, induced T cell-dependent anti-Pf155 antibodies. The identification of these epitopes would contribute towards the development of a subunit vaccine against the merozoite blood stage (Kabilan et al., 1988). Synthetic peptides corresponding to known epitopes ofPf155IRESA induce production of parasite-specific antibodies in vitro and were successful in verifying these epitopes (Chougnet et al., 1991). This protein was at first believed to be associated with the micronemes and rhoptries of merozoites but has now been localised to dense granules in the apical end of merozoites by immunofluorescent techniques.
During the process of erythrocyte invasion, the protein is expressed, released and transported to the red blood cell membrane due to interactions with the cytoskeleton structures, functioning in cell irivasion by the parasite (Ruangjirachupom, 1991).
A 96-kDa protein associated with the surface membrane of malaria infected mouse erythrocytes was identified to produce protective properties in P. chabaudi. Monoclonal antibodies against Pc96 identified cross-reacting antigens in P.falciparum (155 kDa), P. vivax (222kDa) and P.
cynomolgi (200 kDa). A specific monoclonal antibody against the protein Pch105 recognises Pc96 (Wanidworanun et al., 1989). Topographical location of Pc96 was further studied by immunofluorescent microscopy verifying that the protein is located in the surface membrane of infected erythrocytes. Further studies involved the elucidation of epitopes by mapping studies, identifying 3 distinct binding sites for the monoclonal antibodies. The fact that four different antigens in different species of malaria share the same, or similar topographical locations, along with at least one cross-reactive epitope indicates that the epitope is included in a conserved and functional region of the protein specific for the biology of the parasite.Pf155 and Pv222 share a cross-reacting epitope with Pc96, suggesting the potential of Pc96 as a vaccine candidate (Wanidworanun et al., 1989). The cross reactivity with Pf155IRESA suggests that the P. jalciparum protein may contain similar T cell epitopes to Pc96. In view of the protective T cell clones that Pc96 stimulated,·producing antibody responses against multiple parasite antigens
elicited in athymic mice challenged with P. chabaudi adami (Goldring, 1989), an investigation into similar functions inP.falciparum would be worthwhile.