Derechos fundamentales

In general, both humoral and cellular responses to the P. falciparum MSP-1 (MSP-1) appear to be acquired in an age-dependent manner with peak prevalence, or maximum intensity of the response, in late childhood or early adolescence (Riley et al,

1992). This suggests that repeated exposure to infections is required to induce responses in the majority of the population, and may reflect a cumulative response to different polymorphic forms of MSP-1. High titres of antibody to the C-terminal MSP-142 domain

are significantly associated with resistance to both clinical malaria and high parasitaemia, indicating that T- and B- cell epitopes within the C-terminal region of MSP-1 are associated with the induction of protective cellular and humoral responses (Riley et al,

1992, 1993; Egan et a l, 1997). Studies in human populations in malaria endemic areas have revealed an association between the presence of antibodies against epitopes within the MSP-119 and the lack of clinical disease (Riley et a l, 1991; Egan et a l, 1995;

al-Yaman et a l, 1996).

1.9 M SP-l-based m alaria vaccine candidates.

The P. falciparum MSP-1 is the major candidate for a blood-stage malaria vaccine. Data from epidemiological studies, discussed above, suggest that MSP-119 is an

important target of antimalarial immunity. Additionally, epidemiological studies on naturally acquired immune responses indicate a significant association between anti-MSP- li9 antibody titer and reduction in malaria morbidity (Egan et al., 1996). Siddiqui et al.

(1987) and Etingler et al. (1991) have shown that vaccination with MSP-1 isolated from cultured parasites can protect monkeys against P. falciparum infection. Vaccination experiments with the C-terminal domain of MSP-1 from P. yoelii expressed in E. coli

have shown that mice immunised with this antigen are protected (Ling et al., 1994; Daley and Long, 1993). Currently, efforts are being made in the development of a recombinant MSP-1-based vaccine that can induce a protective level of immunity. Three of the major candidates will be briefly discussed.

1.9.1 G ST-M SP-Iiq (Burghaus and Holder, 1993; Burghaus et a l, 1996).

GST-M SP-I19 is a recombinant fusion protein expressed in E. coli, based on the

sequence of the C-terminal domain of MSP-1 (Wellcome allelic type). The protein appears to have similar folding and antigenicity to the native MSP-119 (Burghaus and Holder,

1993). However, immunisation-challenge experiments in which Aotus nancymai were vaccinated with GST-MSP-I19 in liposomes and alum adjuvant prior to blood-stage

P30P2 MSP-119 is a fusion protein secreted in S. cerevisiae. The protein contains

two universal T-helper-cell epitopes from tetanus toxoid (P30 and P2) fused to the amino terminus of the Wellcome-type P. falciparum MSP-119 (Kumar et a l, 1995). Kumar et a l

have shown in immunisation-challenge experiments, in which Aotus nancymai were vaccinated with P30P2 MSP-119 in complete Freund’s adjuvant prior to challenge with P.

falciparum F VO strain, that the animals self-resolved an otherwise lethal infection.

1.9.3 BVp42 rChang e t a l , 1992; 1996).

BVp42 is a baculovirus-derived recombinant polypeptide corresponding to the 42 kDa C-terminal fragment of MSP-1 (MAD20 allelic type). The protein appears to conserve the disulphide-dependent conformation of the C-terminal region of MSP-1 critical for its immunogenicity. The efficacy and immunogenicity of BVp42 has been evaluated. Rabbit sera raised against BVp42 inhibited P. falciparum growth in vitro

(Chang et a l, 1992). Recently, BVp42 was tested in immunisation-challenge experiments (Chang et a l, 1996). It was shown that all the animals immunised with BVp42 in complete Freund’s adjuvant, produced antibodies that inhibited parasite growth in vitro

and the animals were protected against blood-stage challenge with P. falciparum.

1.10 Aims of this project.

The project has three main aims; to study the role of inhibition of MSP-1 processing in antibody-mediated inhibition of erythrocyte invasion; to investigate the location, within the merozoite surface MSP-1 complex, o f epitopes recognised by processing inhibitory and “blocking” antibodies; to establish whether there is any correlation between the immune status of primates vaccinated with MSP-1 based vaccine, and serum levels of processing inhibitory anti-MSP-1 antibodies; and to investigate further the structure of the C-terminal domain of MSP-1. In the first part of the project, the mechanism by which processing inhibitory antibodies interfere with erythrocyte invasion by the merozoite was meticulously studied. For this purpose an assay capable of measuring quantitatively the processing-inhibitory activity o f antibodies was developed. Also, the prevalence of processing-inhibitory antibodies in sera from primates vaccinated with MSP-1 based vaccines have been measured, and the association between the level of such antibodies and clinical status of the immunised animals has been investigated. In the second part of the project, antibodies to various regions of MSP-1 were tested for their ability to either inhibit processing of MSP-1, or to act as blocking antibodies preventing the binding of inhibitory antibodies. This part of the project also allowed the identification of domains of MSP-1 which are targets for inhibitory or blocking antibodies. In the third part of the project, structural studies were carried out on the native and recombinant MSP-119.

Figure 1.

Life cycle of Plasmodium in mosquito and human.

There are two distinct phases; sexual stages and asexual stages. An infected mosquito feeds and injects sporozoites into the bloodstream. Sporozoites invade hepatocytes and undergo growth and asexual division to form exoerythrocytic schizonts. Infected liver cells burst and release 10 to 40,000 merozoites. Merozoites invade red blood cells, develop through ring and schizonts to form the multinucleate mature erythrocytic schizonts. Erythrocytic schizonts burst and merozoites are released to invade new red blood cells. Some merozoites undergo sexual differentiation to form gametocytes and these are taken up by feeding mosquitoes and produce female and male gametes in the mosquito gut. After fertilisation, the motile zygote (ookinete) penetrates the stomach wall and an oocyst develops. Inside the oocyst repeated divisions (sporogony) produce many sporozoites. Sporozoites migrate to and mature in the insect salivary glands from where they can be transmitted to a host during feeding.

ookinee Gamete Mosquito gut Sporozoites Merozoites Schizont Erythrocyte Gametocytes

Figure 2.

Processing of M SPl.

The membrane-bound intact M S P l (A) is processed into small fragments which are found on the surface of the free merozoite (B). At or just before erythrocyte invasion a secondary processing step cleaves M S P l42 to produce MSPI33 and M S P l 19 (C). M S P l 19

is carried into the newly invaded erythrocyte. M S P l33 is shed in a soluble form from the merozoite surface as part o f a complex with the other M S P l-derived polypeptides.