Conformational study of a short Pertussis toxin T cell epitope incorporated in a multiple antigen peptide template by CD and two-dimensional NMR. Analysis of the structural effects on the activity of synthetic immunogens.

The immunogenic efficacy of multiple antigen peptides, MAPs, i.e. branched molecules in which multiple copies of a given immunogenic peptide are attached on a scaffold of lysine residues via both alpha and epsilon linkages, has been repeatedly demonstrated, but little is known about the structural arrangement of these peptide constructs. A conformational characterization was therefore performed for a known T cell epitope of the S1 subunit of Pertussis toxin, whose sequence is predicted to form alpha-helix. The peptide DNVLDHLTGR, its N-acetylated and C-amidated analogue and a tetrabranched MAP based on the N-acetylated peptide were prepared and studied by CD and two-dimensional 1H-NMR. No evidence of helical structure was obtained in water for the isolated peptides. In contrast, in triflouroethanol, the isolated epitopes fold into a helical structure spanning the segment Val3-Thr8 in the uncapped molecule and encompassing also the N-terminal region in the capped analogue. The mobile C-terminal region tends to adopt a distorted turn arrangement in both peptides due to the folding of Arg10 guanidinium over the backbone. No distortion of the helix structure was observed for the single-copy epitope in the four-branched MAP molecule in trifluoroethanol: each peptide chain is equivalent within the MAP and shows an even more regular helical pattern than the isolated end-blocked sequence. A slight difference was located at the junction with the lysine scaffold: the peptide bond to epsilon NH was found in a much more extended conformation than the corresponding link to alpha NH. These structural results correlate with in vitro T cell stimulatory activity of the three compounds examined and provide arguments supporting the previous suggestion that MAP tetramers are unlikely to elicit an immune response specific for the synthetic template assembly, a feature necessary to retain the advantage of the polymeric epitope presentation.

Side reactions in solid phase synthesis of histidine-containing peptides. Characterization of two major impurities by sample displacement chromatography and FAB-MS.

The flow-polyamide synthesis of a histidine-containing sequence (Ac-YDNVLDHLTGR) produced two major impurities which were isolated through Sample Displacement Chromatography and characterized by Fast Atom Bombardment-mass analysis. The impurities correspond to the sequence Ac-YDNVLDH, and to a peptide with the Asp residue cyclized to a succinimide. The latter side-reaction took place during the acetylation procedure, and demands attention where capping procedures are planned in the synthesis of Asp-His sequences.

Application of capillary zone electrophoresis to the characterization of multiple antigen peptides.

Capillary zone electrophoresis (CZE) was applied to the analysis of a recently described class of synthetic branched peptides, multiple antigen peptides (MAPs). In comparison with high-performance liquid chromatography (HPLC), CZE showed superior resolution of minor impurities in samples of MAPs obtained by preparative chromatography. MAPs that differed only in the substitution of uncharged residues (Ala-Ser) could be separated by the addition of organic solvents (acetonitrile, methanol, ethanol) to the aqueous background electrolyte.

Toward the elucidation of the mechanism of attachment and entry of malaria sporozoites into cells: synthetic polypeptides from the circumsporozoite protein of Plasmodium falciparum bind Ca2+ and interact with model phospholipid membranes.

Through the joint use of CD, Fourier transform ir (FTIR), and attenuated total reflectance FTIR we have found that synthetic polypeptide models of the Plasmodium falciparum circumsporozoite (CS) protein repeat domain bind calcium ions in helicogenic environments. Ca(2+)-(NANP)n complexes (n greater than or equal to 20) interact vectorially with model phospholipid membranes orienting their polypeptide axes preferentially along those of the lipid acyl chains. It is proposed that the P. falciparum CS protein central region, rather than acting as a molecular lure helping the parasite to evade host immune control, plays, as a specific Ca2+ macroligand, a critical functional role during attachment, invasion, and development of the malaria parasite in the hepatic cell.

Multiple antigen peptides (MAPs) as candidate vaccines against malaria.

Multiple Antigen Peptides (MAPs), branched molecules where multiple copies of a desired antigenic sequence are assembled on a small peptide core, have been recently described as an alternative approach to the synthesis of high molecular weight immunogens. In comparison with conventional peptide-carrier conjugates, the MAPs show several advantages, including chemical unambiguity and ease of synthesis. A MAP based on the sequence of the repetitive domain of P. malariae sporozoites was immunogenic in a large number of mouse strains. When covalently linked to the corresponding sequence of the P. falciparum circumsporozoite protein, [NANP]40, the resulting conjugate showed the properties of a multivalent vaccine, overcoming the severe genetic restriction of the [NANP] sequence. A second generation of MAPs including both sequences, with more desirable chemical properties, was equally effective. These compounds represent a promising step towards the development of synthetic, multivalent peptide vaccines against human malaria.

A multiple antigen peptide from the repetitive sequence of the Plasmodium malariae circumsporozoite protein induces a specific antibody response in mice of various H-2 haplotypes.

The major repetitive epitopes of the surface circumsporozoite (CS) protein of malaria sporozoites represent candidates for the development of subunit vaccines against malaria. However, previous experimental work has shown that repetitive peptides from the CS proteins of Plasmodium falciparum, P. vivax, P. yoelii and P. berghei are immunogenic only in mice with the H-2b or H-2k haplotype. This led to the conclusion that strong T helper epitopes from the non-repetitive CS sequences were required in the design of sporozoite vaccines. In the present study, we investigated the immunogenicity in mice of a octa-branched multiple antigen peptide (MAP) containing repeats of the CS protein of the human malaria parasite, P. malariae, [MAP8(NAAG)6], and found that mice with an H-2b, H-2d, H-2k, H-2f, H-2q, and H-2s haplotype produced anti-peptide antibodies after immunization and that only H-2r mice were nonresponsive. This antibody response, not induced in athymic H-2b nu/nu mice, was directed against the (NAAG) sequence, but not against the lysine core of the MAP construct. Finally, when covalently linked to a synthetic polymer of the repetitive (NANP) sequence of the P. falciparum CS protein, [MAP8(NAAG)6] behaved as a carrier molecule for the production of anti-(NANP)n antibodies in H-2d and H-2k mice, genetically nonresponder to the (NANP)n sequence. Should this wide immunogenicity of the P. malariae CS (NAAG) repetitive sequence also apply to humans, it might be considered for the design of multivalent subunit malaria vaccines.