Analysis of the high molecular weight rhoptry complex ofPlasmodium falciparumusing monoclonal antibodies

Abstract

SUMMARYTwenty-one monoclonal antibodies, obtained after immunization of mice with erythrocytic stages ofPlasmodium falciparum, produced a double dot image in IFA. Immunoelectronmicroscopy indicated that the mAbs reacted with the rhoptries. Rhoptries are pear-shaped apical organelles, believed to be involved in invasion of the host cell by the parasite. The mAbs all immunoprecipitated the high molecular weight antigen complex. Some mAbs recognized on immunoblots only 1 protein of this complex, whereas others reacted with RhopH1 and RhopH3, or RhopH2 and RhopH3 or with the 3 proteins. An additional antigen of 52 kDa was also recognized by some of the mAbs. The epitopes defined by the mAbs were present in most of the 40P. falciparumstrains or isolates studied by IFA. Interestingly, the mAbs also reacted with high titres onP. vivaxandP. ovale, but produced images that did not indicate an apical location. The mAbs failed to react on the non-human malaria parasites studied,P. cynomolgiandP. inui. OnP. bergheiorP. chabaudiparasites, only 5 mAbs gave a positive reaction, labelling a large network outside the parasite. Finally, the mAbs did not react withP. falciparumsporozoites, indicating that the rhoptries of merozoites and sporozoites, the two invasive stages of the malaria life-cycle are equipped with distinct sets of proteins.

The Plasmodium falciparum rhoptry protein RhopH3 plays essential roles in host cell invasion and nutrient uptake

“…This appears to occur even in the absence of other members of the RhopH complex, indicating that, for its role in invasion, RhopH3 may not require the function of the other proteins of the complex. Further supporting a role for RhopH3 in invasion is the finding that anti-RhopH3 antibodies can block invasion (Doury et al, 1994; Sam-Yellowe and Perkins, 1991). Nonetheless it is curious that ~50% of the parasites still enter the erythrocyte in the absence of full-length RhopH3.…”

“…There is no report of attempted disruption of the rhopH2 gene, but the rhopH3 gene is refractory to deletion in the haploid blood stages (Cowman et al, 2000), suggesting an essential role. Hints that this might include a function in invasion derive from studies showing that antibodies to RhopH3 can inhibit invasion (Cooper et al, 1988; Doury et al, 1994). However, whether RhopH3 plays other essential roles that involve all forms of the RhopH complex is unknown.…”

Diversity and evolution of the rhoph1/clag multigene family of Plasmodium falciparum

“…Strong diversifying selections on microneme proteins have been detected (e.g., AMA-1 and EBA-175), suggesting that polymorphism of these proteins has been maintained to evade host immunity in parasite populations [17,18]. Antibodies against the PfRhopH complex partially inhibit the growth of P. falciparum in vitro and in vivo, consistent with its potential as a vaccine target [19][20][21]. Although the RhopH complex has been shown to induce host protective immunity and is likely to be under host immune pressure, the genetic diversity and immunologic characteristics of this complex are not fully understood.…”

Associations between Responses to the Rhoptry-Associated Membrane Antigen of Plasmodium falciparum and Immunity to Malaria Infection

“…Our results suggest that RAMA-E epitopes may be important for inducing more efficient antimalarial responses which develop over a Ag44 represents the C-terminal portion of RhopH3, and a previous seroepidemiological study indicated that this region is strongly immunogenic and that ongoing P. falciparum infection results in significant increases in antibody responses to the antigen (23). Monoclonal antibodies directed against epitopes within Ag44 showed growth-inhibitory properties in in vitro assays (6,7). As in vitro RBC-binding assays indicated that Ag44 harbors an RBC-binding domain (21), there appears to be a link between protection and responses to RBC-binding domains for both RAMA and RhopH3.…”

“…Although their contents are only transiently accessible to antibodies, seroepidemiological studies have demonstrated the development of antibody responses to the rhoptry proteins RhopH3, RAP1, and RAP2 following infection (8,12,20,23). In vitro growth inhibition assays have indicated that antibodies directed against the RAP1 and RAP2 proteins have inhibitory effects on P. falciparum growth in in vitro culture (6,9,14,18). Moreover, immunization of Saimiri monkeys with RAP1 protected the animals from a lethal malaria parasite infection (17), suggesting that interference with the rhoptry protein function by immune responses can prevent RBC invasion.…”