Michael. Foley

MOLECULAR VARIATION IN A NOVEL POLYMORPHIC ANTIGEN ASSOCIATED WITH PLASMODIUM FALCIPARUM MEROZOITES

A cDNA clone encoding part of a novel polymorphic merozoite antigen from Plasmodium falciparum was isolated by screening a cDNA library with human immune serum from Papua New Guinea. Immunofluorescence microscopy and immunoblotting with affinity-purified antibodies recognized a highly polymorphic antigen, Ag956, present in schizonts and merozoites. Biosynthetic labeling and immunoprecipitation experiments demonstrated that Ag956 is proteolytically cleaved during merozoite maturation. The complete genomic sequence of Ag956 from the D10 clone of P. falciparum isolate FC27 encodes a secreted protein of calculated molecular mass 43,243 that is very hydrophilic and contains a region of unusual heptad repeats of the general structure AXXAXXX. This antigen has been named the secreted polymorphic antigen associated with merozoites (SPAM). The sequence of a second SPAM allele from the 3D7 clone of isolate NF54 reveals that the alanine heptad repeats and the hydrophilic C-terminal half of the protein are conserved. Variation among SPAM alleles is the result of deletions and amino acid substitutions in non-repetitive sequences within and flanking the alanine heptad-repeat domain. Heptad repeats in which the a and d position contain hydrophobic residues generate amphipathic alpha-helices which give rise to helical bundles or coiled-coil structures in proteins. Thus, SPAM is the first example of a P. falciparum antigen in which a repetitive sequence has features characteristic of a well-defined structural element.

The ring-infected erythrocyte surface antigen of Plasmodium falciparum associates with spectrin in the erythrocyte membrane

The malaria parasite Plasmodium falciparum synthesises a protein, RESA, which associates with the membrane of newly invaded erythrocytes. Using spent supernatants from P. falciparum growing in culture as a source of soluble RESA we have developed an assay to examine the characteristics of RESA binding to the erythrocyte membrane in vitro. RESA associated with the Triton X-100 insoluble proteins on the inner face of the host erythrocyte membrane but did not bind to the outer surface of intact erythrocytes. Other proteins present in culture supernatants did not bind to the erythrocyte membrane. RESA was co-sedimented with the ternary complex formed between actin, spectrin and band 4.1 and co-precipitated with spectrin precipitated with anti-spectrin antibodies. The extent of association between RESA and the inner face of the erythrocyte membrane was reduced by the inclusion of excess purified spectrin in the assay. Thus, RESA appears to be associated with spectrin in the erythrocyte membrane skeleton.

Structure of Domain III of the Blood-stage Malaria Vaccine Candidate, Plasmodium falciparum Apical Membrane Antigen 1 (AMA1)

Apical membrane antigen 1 of the malarial parasite Plasmodium falciparum (Pf AMA1) is a merozoite antigen that is considered a strong candidate for inclusion in a malaria vaccine. Antibodies reacting with disulphide bond-dependent epitopes in AMA1 block invasion of host erythrocytes by P.falciparum merozoites, and we show here that epitopes involving sites of mutations in domain III are targets of inhibitory human antibodies. The solution structure of AMA1 domain III, a 14kDa protein, has been determined using NMR spectroscopy on uniformly 15N and 13C/15N-labelled samples. The structure has a well-defined disulphide-stabilised core region separated by a disordered loop, and both the N and C-terminal regions of the molecule are unstructured. Within the disulphide-stabilised core, residues 443-447 form a turn of helix and residues 495-498 and 503-506 an anti-parallel beta-sheet with a distorted type I beta-turn centred on residues 500-501, producing a beta-hairpin-type structure. The structured region of the molecule includes all three disulphide bonds. The previously unassigned connectivities for two of these bonds could not be established with certainty from the NMR data and structure calculations, but were determined to be C490-C507 and C492-C509 from an antigenic analysis of mutated forms of this domain expressed using phage display. Naturally occurring mutations in domain III that are located far apart in the primary sequence tend to cluster in the region of the disulphide core in the three-dimensional structure of the molecule. The structure shows that nearly all the polymorphic sites have a high level of solvent accessibility, consistent with their location in epitopes recognised by protective antibodies. Even though domain III in solution contains significant regions of disorder in the structure, the disulphide-stabilised core that is structured is clearly an important element of the antigenic surface of AMA1 recognised by protective antibodies.

Rotational dynamics of the integral membrane protein, band 3, as a probe of the membrane events associated with Plasmodium falciparum infections of human erythrocytes.

Time-resolved phosphorescence anisotropy was used to study the molecular organisation of band 3 in the erythrocyte membrane. Three different rotational relaxation regimes of mobile band 3 were resolved. These populations may represent different aggregation states of band 3 within the membrane, or they may result from association of band 3 with other proteins at the cytoplasmic surface. The polycation spermine decreases the apparent mobility of band 3 by a mechanism that does not involve the underlying cytoskeleton. A monoclonal antibody directed against the cytoplasmic portion of band 3 can also cause an increase in the immobile fraction of band 3 molecules. This monoclonal antibody will inhibit invasion of erythrocytes by malaria parasites. Membranes prepared from erythrocytes infected with mature stages of the malaria parasite, Plasmodium falciparum, show altered dynamic properties corresponding to a marked restriction of band 3 mobility.

Isolation from Phage Display Libraries of Single Chain Variable Fragment Antibodies That Recognize Conformational Epitopes in the Malaria Vaccine Candidate, Apical Membrane Antigen-1

Phage display of single chain variable fragment (scFv) antibodies is a powerful tool for the selection of important and useful antibody specificities. We have constructed such a library from mice protected from malaria challenge by immunization with recombinantPlasmodium chabaudi DS apical membrane antigen (AMA-1). Panning on refolded AMA-1 enriched a population of scFvs which specifically bound the antigen. The single chain antibodies recognize conformational epitopes on AMA-1 from the P. chabaudi DS strain but not on AMA-1 of the 556KA strain of P. chabaudi. A subset of the antibody fragments recognized AMA-1 from the human malaria parasite Plasmodium falciparum. Nucleotide sequencing revealed that at least four unique scFv genes were selected by the panning procedure. These scFv antibodies are valuable reagents for probing the structure and function of AMA-1 and will be used to test the feasibility of using recombinant antibodies in a passive immunization therapy against malaria.