To identify malaria antigens for vaccine development, we selected α-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. The corresponding synthetic peptides are expected to mimic structurally “native” epitopes. Indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. Peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. These antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. Circular dichroism studies indicated that the selected peptides assumed partial or high α-helical content. Thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. This strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens.

The review surveys potential “structural antigens” which represent small protein domains that can be chemically synthesized and, isolated from the context of the whole protein, can fold in the same native structure. They include natively unfolded protein regions, small globular domains, α-helical coiled coils and regions with tandem repeats forming structures ranging from the collagen triple helices to solenoid-like arrangements. We also describe and compare new strategies for development of vaccine that use the concept of structural epitopes. One type of approach is based on engineering artificial mini-proteins able to mimic structural epitopes of natural proteins. The review compares the “engineering” methodologies with “bioinformatics” approaches that became possible recently, after the sequencing of the genomes of many pathogens, and involve genome-wide bioinformatics searches for “structural antigens”. In particular, based on the known P. falciparum genome, we identified putative α-helical coiled coil regions, 30-40 amino acids long, in proteins presented in asexual malaria blood stages. Peptides of such regions frequently fold into the “native” structure. A hundred such peptides were synthesized and all of them were recognized at various degrees (5-80%) by a panel of sera from donors living in malaria-endemic areas. The results obtained demonstrate that a bioinformatics/chemical synthesis strategy can rapidly lead to the identification of new proteins that can be targets of potential vaccines and/or drugs against malaria and other infectious organisms.

We have identified new malaria vaccine candidates through the combination of bioinformatics prediction of stable protein domains in the Plasmodium falciparum genome, chemical synthesis of polypeptides, in vitro biological functional assays, and association of an antigen-specific antibody response with protection against clinical malaria. Within the predicted open reading frame of P. falciparum hypothetical protein PFF0165c, several segments with low hydrophobic amino acid content, which are likely to be intrinsically unstructured, were identified. The synthetic peptide corresponding to one such segment (P27A) was well recognized by sera and peripheral blood mononuclear cells of adults living in different regions where malaria is endemic. High antibody titers were induced in different strains of mice and in rabbits immunized with the polypeptide formulated with different adjuvants. These antibodies recognized native epitopes in P. falciparum -infected erythrocytes, formed distinct bands in Western blots, and were inhibitory in an in vitro antibody-dependent cellular inhibition parasite-growth assay. The immunological properties of P27A, together with its low polymorphism and association with clinical protection from malaria in humans, warrant its further development as a malaria vaccine candidate.

Background: Synthetic contiguous overlapping peptides (COPs) may represent an alternative to allergen extracts or recombinant allergens for allergen specific immunotherapy. In combination, COPs encompass the entire allergen sequence, providing all potential T cell epitopes, while preventing IgE conformational epitopes of the native allergen.Methods: Individual COPs were derived from the sequence of Bet v 1, the major allergen of birch pollen, and its known crystal structure, and designed to avoid IgE binding. Three sets of COPs were tested in vitro in competition ELISA and basophil degranulation assays. Their in vivo reactivity was determined by intraperitoneal challenge in rBet v 1 sensitized mice as well as by skin prick tests in volunteers with allergic rhinoconjunctivitis to birch pollen.Results: The combination, named AllerT, of three COPs selected for undetectable IgE binding in competition assays and for the absence of basophil activation in vitro was unable to induce anaphylaxis in sensitized mice in contrast to rBet v 1. In addition no positive reactivity to AllerT was observed in skin prick tests in human volunteers allergic to birch pollen. In contrast, a second set of COPs, AllerT4‐T5 displayed some residual IgE binding in competition ELISA and a weak subliminal reactivity to skin prick testing.Conclusions: The hypoallergenicity of contiguous overlapping peptides was confirmed by low, if any, IgE binding activity in vitro, by the absence of basophil activation and the absence of in vivo induction of allergic reactions in mouse and human.Trial registration: ClinicalTrials.gov NCT01719133