PROPUESTA PARA LA INSTRUMENTACION DE LOS PROYECTOS ESTRATEGICOS

During the development of clinical immunity, especially during the first few years of life, production of strain-specific antibodies to PfEMP1 domains is essential for preventing infection with previously challenged isolates (Newbold et al., 1992, Marsh and Howard, 1986, Bull et al., 1998, Iqbal et al., 1993). This type of immune response is important during and after infections with isolates causing severe disease (Miller et al., 2002). Several published studies that measured the agglutination responses to P. falciparum isolates have demonstrated that children acquired strain-specific anti-VSA antibodies rapidly following malaria infection (Bull et al., 1998, Bull et al., 1999). This suggests that immune response acquired following an infection comprises antibodies targeting erythrocytes infected by the homologous isolate, and supports the essential role of strain-specific antibodies in protection against malaria infection and severe disease (Bull et al., 1998, Newbold et al., 1992).

Despite the fact that the dominant immune response following an infection is variant-specific (Newbold et al., 1992), evidence for the existence of at least some degree of cross-reactive response to distinct parasite isolates has been observed in some studies (Marsh and Howard, 1986, Newbold et al., 1992, Giha

et al., 1999, Ofori et al., 2002). Antibodies in convalescent sera collected from adults agglutinated distinct P. falciparum isolates (Chattopadhyay et al., 2003), and immune sera obtained from people living in diverse African countries agglutinated parasitized erythrocytes from different geographical areas, suggesting the presence of cross-reactive epitopes expressed by different parasite isolates (Aguiar et al., 1992).

Marsh and Howard (1986) investigated parasites isolated from ten Gambian children with acute and convalescent sera from each child, and showed that most acute sera did not react with homologous isolates whereas each convalescent serum was highly reactive with homologous but not heterologous isolates. This study suggested that infection by P. falciparum results in a great diversity of antigenic phenotypes expressed on the surface of IEs, which was evident from the variant-specific nature of antibody response. Moreover, semi-immune sera from Gambian adults agglutinated erythrocytes infected by the ten children, suggesting that by adulthood most individuals acquire a broader cross-reactive response that protects against heterologous isolates (Marsh and Howard, 1986). However, it should be noted that the exposure to cross-reactive, conserved determinants on the surface of IEs was the key factor for induction of a broadly reactive response in adult semi-immune sera in this study but not the breadth of response to recognise distinct important targets on the surface of the parasitized erythrocyte. It was suggested that the parasite evades strong immune responses directed to conserved sites on PfEMP1, such as sites involved in cytoadhesion, by ''drawing the attention'' of the host humoral immune response to extremely diverse and at the same time more immunogenic epitopes on the protein (Staalso et al., 1998). If the immune response to conserved regions is significant for protection and essential for the parasite pathogenesis, such conserved regions might useful for vaccine development.

Bull et al. (1999, 2000) have provided evidence for the presence of rare and prevalent parasite isolates, and suggested that isolates causing severe disease express a unique subset of VSAs associated with severe disease in younger children with relatively lower immunity. This was evident from the findings that parasites isolated from children with severe malaria had a significantly higher agglutination frequency than those isolated from patients with mild malaria

infection (Bull et al., 2000). Consistent with these findings, Nielsen et al. (2002) showed that parasites isolated from children with severe malaria were broadly and more frequently recognised by VSA-specific antibodies in plasma samples of local healthy individuals than parasites isolated from children with non-severe disease (Nielsen et al., 2002). Thus, P. falciparum isolates that cause clinical disease to semi-immune young children appear to express VSAs that are not identified by previously existed strain-specific antibodies, which supports the ''hole in the antibody repertoire'' hypothesis (Nielsen et al., 2002, Ofori et al., 2002, Bull et al., 1998). Malaria exposure may increase the levels of specific antibodies that recognise VSAs expressed by parasite isolates causing current infections (Bull et al., 1998, Giha et al., 1999), and acquisition of VSA-specific antibodies after repeated exposure appears to gradually limit VSA variants causing severe disease and help to close the ''holes'' in antibody repertoire (Nielsen et al., 2002).

Aims of study

The main aim of this thesis was to develop monoclonal and polyclonal antibodies in mice against recombinant PfEMP1ICAM-1-DBLβ domains and characterise the immune response of these antibodies. One of the existing approaches to overcome the polymorphism of PfEMP1 in the development of therapeutic interventions is to target PfEMP1 subdomains involved in parasite binding (Gratepanche et al., 2003). Recombinant proteins corresponding to different PfEMP1 domains have been synthesised and used for the induction of specific immune responses in standard immunoassays. A benefit of this approach is the ability to direct a specific immune response to anticipated regions or epitopes for extending the protective response whilst excluding undesirable responses. Another advantage is the production of a response with an estensive broad-range of antigen coverage maintained under correct protein expression and production (Richards and Beeson, 2009). Individual DBLβ domains were used rather than the full-length PfEMP1 protein to map the minimal receptor-adhesion parts for ICAM-1 binding parasites and to understand the functional requirements for receptor adhesion. The specific aims of the different chapters were as follows: