Design and pre-clinical profiling of a Plasmodium falciparum MSP-3 derived component for a multi-valent virosomal malaria vaccine.

BACKGROUND: Clinical profiling of two components for a synthetic peptide-based virosomal malaria vaccine has yielded promising results, encouraging the search for additional components for inclusion in a final multi-valent vaccine formulation. This report describes the immunological characterization of linear and cyclized synthetic peptides comprising amino acids 211-237 of Plasmodium falciparum merozoite surface protein (MSP-3). METHODS: These peptides were coupled to phosphatidylethanolamine (PE); the conjugates were intercalated into immunopotentiating reconstituted influenza virosomes (IRIVs) and then used for immunizations in mice to evaluate their capacity to elicit P. falciparum cross-reactive antibodies. RESULTS: While all MSP-3-derived peptides were able to elicit parasite-binding antibodies, stabilization of turn structures by cyclization had no immune-enhancing effect. Therefore, further pre-clinical profiling was focused on FB-12, a PE conjugate of the linear peptide. Consistent with the immunological results obtained in mice, all FB-12 immunized rabbits tested seroconverted and consistently elicited antibodies that interacted with blood stage parasites. It was observed that a dose of 50 microg was superior to a dose of 10 microg and that influenza pre-existing immunity improved the immunogenicity of FB-12 in rabbits. FB-12 production was successfully up-scaled and the immunogenicity of a vaccine formulation, produced according to the rules of Good Manufacturing Practice (GMP), was tested in mice and rabbits. All animals tested developed parasite-binding antibodies. Comparison of ELISA and IFA titers as well as the characterization of a panel of anti-FB-12 monoclonal antibodies indicated that at least the majority of antibodies specific for the virosomally formulated synthetic peptide were parasite cross-reactive. CONCLUSION: These results reconfirm the suitability of IRIVs as a carrier/adjuvant system for the induction of strong humoral immune responses against a wide range of synthetic peptide antigens. The virosomal formulation of the FB-12 peptidomimetic is suitable for use in humans and represents a candidate component for a virosomal multi-valent malaria subunit vaccine.

Antibodies elicited by a virosomally formulated Plasmodium falciparum serine repeat antigen-5 derived peptide detect the processed 47 kDa fragment both in sporozoites and merozoites.

Serine repeat antigen-5 (SERA5) is a candidate antigen for inclusion into a malaria subunit vaccine. During merozoite release and reinvasion the 120 kDa SERA5 precursor protein (P120) is processed, and a complex consisting of an N-terminal 47 kDa (P47) and a C-terminal 18kDa (P18) processing product associates with the surface of merozoites. This complex is thought to be involved in merozoite invasion of and/or egress from host erythrocytes. Here we describe the synthesis and immunogenic properties of virosomally formulated synthetic phosphatidylethanolamine (PE)-peptide conjugates, incorporating amino acid sequence stretches from the N-terminus of Plasmodium falciparum SERA5. Choosing an appropriate sequence was crucial for the development of a peptide that elicited high titers of parasite cross-reactive antibodies in mice. Monoclonal antibodies (mAbs) raised against the optimized peptide FB-23 incorporating amino acids 57-94 of SERA5 bound to both P120 and to P47. Western blotting analysis proved for the first time the presence of SERA5 P47 in sporozoites. In immunofluorescence assays, the mAbs stained SERA5 in all its predicted localizations. The virosomal formulation of peptide FB-23 is suitable for use in humans and represents a candidate component for a multi-valent malaria subunit vaccine targeting both sporozoites and blood stage parasites.

Induction of parasite growth-inhibitory antibodies by a virosomal formulation of a peptidomimetic of loop I from domain III of Plasmodium falciparum apical membrane antigen 1.

Apical membrane antigen 1 (AMA-1) of Plasmodium falciparum is a leading candidate antigen for inclusion in a malaria subunit vaccine. Its ectodomain can be divided into three subdomains, each with disulfide bond-stabilized structures. Since the majority of antibodies raised against the ectodomain appear to recognize strain-specific epitopes in domain I, we attempted to develop a vaccine formulation which directs the immune response to a region that contains more conserved epitopes. Here we demonstrate that a virosomal formulation of a peptide that mimics the semiconserved loop I of domain III elicits parasite growth-inhibitory antibodies. A synthetic peptide comprising residues 446 to 490 of AMA-1 (AMA-1(446-490)) was conjugated through the N terminus to a derivative of phosphatidylethanolamine and the phosphatidylethanolamine-peptide conjugate was incorporated into immunopotentiating reconstituted influenza virosomes as a human-compatible antigen delivery system. Both cyclized and linear versions of the peptide antigen elicited antibodies which specifically bound to parasite-expressed AMA-1 in Western blotting with parasite lysates as well as in immunofluorescence assays with blood stage parasites. All 11 peptidomimetic-specific monoclonal antibodies generated were cross-reactive with parasite-expressed AMA-1. Antigen binding assays with a library of overlapping cyclic peptides covering the target sequence revealed differences in the fine specificity of these monoclonal antibodies and provided evidence that at least some of them recognized discontinuous epitopes. The two immunodominant epitopes comprised the conserved linear sequences K(459)RIKLN(464) and D(467)DEGNKKII(475). A key feature of the synthetic vaccine formulation proposed here is the display of the peptide antigen in a native-like state on the surface of the virosome.

Red grape juice inhibits iron availability: application of an in vitro digestion/caco-2 cell model.

Adequate bioavailable Fe intake is essential for optimal growth and intellectual development of infants and children. Fruit juices are nutritious and popular drinks for infants and children and are known to contain Fe uptake inhibitors (e.g., polyphenolic compounds) and a dominant promoter, ascorbic acid. Ascorbic acid is naturally present in fruit juices and is added during processing to almost all juices found in supermarkets. With these facts taken into account, an in vitro digestion/Caco-2 cell culture model was developed to compare the effects of apple, pear, white grape, red grape, prune, grapefruir, and orange juices on iron bioavailability. In two series of experiments, juices from a local supermarket were combined with FeCl(3) or commercial infant cereal fortified with elemental iron and subject to simulated gastric and intestinal digestion. Caco-2 cell ferritin formation in response to exposure to the digests served as the measure of Fe uptake. The pear, apple, grapefruit, orange, and white grape juice significantly increased Fe bioavailability from FeCl(3). For the infant cereal studies, the apple, orange, pear, and white grape juices increased the Fe bioavailability of the infant cereal. In contrast, the red grape juice and prune juice had profound inhibitory effects on iron bioavailability. These inhibitory effects were likely due to high levels of polyphenolic compounds that bind and thereby prevent absorption of soluble Fe. These inhibitory compounds appeared to counteract the promotional effects of ascorbic acid as they were in considerable molar excess relative to ascorbic acid and Fe in the digest. From a nutritional standpoint, the results suggest that individuals in need of optimal Fe absorption should avoid red grape and prune juice or at least vary the types of juices consumed. Alternatively, individuals seeking to limit Fe uptake (e.g., hemochromatitics and astronauts) may be able to utilize red grape or prune juice as effective inhibitors of Fe uptake. Consumers should be aware that the compounds that inhibit Fe availability are also linked to anticancer benefits; thus, a dietary balance of the above juices may be optimal.