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The kinetics of the humoral response to defined Plasmodium falciparum antigens was studied in 543 children, 1 month to 15 years old, living in an area endemic for malaria. The antigens used for enzyme-linked immunosorbent assay were (i) the synthetic peptide (NANP)40 representing the immunodominant repeated region of the circumsporozoite protein, and (ii) the fusion peptide 31.1, representing the N-terminal portion of the 83 kDa polypeptide expressed at the surface of merozoites which is a processed product of the 190-200 kDa glycoprotein. In addition, glutaraldehyde-fixed infected red blood cells (RBC) were used to detect ring-infected erythrocyte surface antigen (RESA) and unfixed infected RBC to detect intra-erythrocytic asexual form (IEF) antigens by immunofluorescence. In the 1 to 2 months age group, 50%, 26% and 21% of the children had antibodies for IEF, (NANP)40 and 31.1 respectively, but none had anti-RESA antibodies. The proportions of positive subjects decreased until 3 to 6 months and then increased progressively for the 4 antigens, approaching, but not reaching, adult values by the age of 15 years. Antibodies against specific antigens were acquired concomitantly. Children born from (NANP)40-positive mothers showed enhanced anti-(NANP)40 IgG responses.

An enzyme-linked immunosorbent assay (ELISA) based on the synthetic peptide (NANP)40 was used to characterize the sporozoite antibodies in an unusual Plasmodium falciparum outbreak in a non-malarious area in Sri Lanka. A positive antibody response was seen in 62% of patients with their first P. falciparum illness. There was no correlation between sporozoite antibodies and the antibody against blood stages, determined by immunofluorescence assay. The majority (91%) of the patients lost the antibodies to circumsporozoite (CS) protein within one year (in the absence of re-exposure). Three patients had high levels of CS antibodies even after one year, and this persistence was related to the level of the initial antibody response. In the area of the outbreak 10% of schoolchildren had antibodies to the (NANP)40 peptide. 21% of the 42 children with present or past overt malaria were antibody positive. Of the children with no such background, 8% were antibody positive. The corresponding seropositivity rates for asexual blood stages were 31% and 1% for the 2 groups respectively. It is concluded that (NANP)40 ELISA is potentially a valuable tool in sero-epidemiology, particularly in situations of seasonal transmission and recurrences due to drug resistance.

The solid phase synthesis of a partially modified retro-inverso analogue of the bradykinin potentiating peptide BPP9a, [gLys6, (R,S)-mPhe7, Ala8] BPP9a is described. The analogue, which is active in vitro and in vivo, displays prolonged resistance towards cleavage by angiotensin converting enzyme (ACE).

A new method for oxidative folding of synthetic polypeptides assembled by stepwise solid phase synthesis is introduced. Folding is obtained in excellent yields by reacting S-tert-butylthiolated polypeptides with a 100-fold molar excess of cysteine at 37 °C in a slightly alkaline buffer containing chaotropic salts, and in the presence of air-oxygen. This novel protocol has been applied to the folding of S-tert-butylthiolated human thymus and activation-regulated chemokine (hu-TARC) derivatives as well as to larger segments of Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins. Folded P. falciparum polypeptides have been used as substrates of endoproteinase Glu-C (Glu-C) and endoproteinase Asp-N (Asp-N) in an attempt to identify their disulfide connectivities. Particular practical advantages of the present method are (i) easy purification and storage of the S-protected peptide derivatives, (ii) elimination of the risk of cysteine alkylation during the acidolytic cleavage deprotection and resin cleavage steps, (iii) possibility to precisely evaluate the extent of folding and disulfide bond formation by mass spectrometry, and (iv) facile recovery of the final folded product. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.

Background: The α-helical coiled coil structures formed by 25–50 residues long peptides are recognized as one of Nature's favorite ways of creating an oligomerization motif. Known de novo designed and natural coiled coils use the lateral dimension for oligomerization but not the axial one. Previous attempts to design α-helical peptides with a potential for axial growth led to fibrous aggregates which have an unexpectedly big and irregular thickness. These facts encouraged us to design a coiled coil peptide which self-assembles into soluble oligomers with a fixed lateral dimension and whose α-helices associate in a staggered manner and trigger axial growth of the coiled coil. Designing the coiled coil with a large number of subunits, we also pursue the practical goal of obtaining a valuable scaffold for the construction of multivalent fusion proteins. Results: The designed 34-residue peptide self-assembles into long fibrils at slightly acid pH and into spherical aggregates at neutral pH. The fibrillogenesis is completely reversible upon pH change. The fibrils were characterized using circular dichroism spectroscopy, sedimentation diffusion, electron microscopy, differential scanning calorimetry and X-ray fiber diffraction. The peptide was deliberately engineered to adopt the structure of a five-stranded coiled coil rope with adjacent α-helices, staggered along the fibril axis. As shown experimentally, the most likely structure matches the predicted five-stranded arrangement. Conclusions: The fact that the peptide assembles in an expected fibril arrangement demonstrates the credibility of our conception of design. The discovery of a short peptide with fibril-forming ability and stimulus-sensitive behavior opens new opportunities for a number of applications.

Gating of an artificial receptor was monitored for the first time by impedance spectroscopy in tethered lipid bilayer. Subnanomolar concns. of antibody were detected by modulation of the channel activity. The principle of gating by an antibody is particularly attractive because of its general character, and thus opens new possibility of immunosensing. [on SciFinder (R)]

Long synthetic peptides (LSPs) have a variety of important clinical uses as synthetic vaccines and drugs. Techniques for peptide synthesis were revolutionized in the 1960s and 1980s, after which efficient techniques for purification and characterization of the product were developed. These improved techniques allowed the stepwise synthesis of increasingly longer products at a faster rate, greater purity, and lower cost for clinical use. A synthetic peptide approach, coupled with bioinformatics analysis of genomes, can tremendously expand the search for clinically relevant products. In this Review, we discuss efforts to develop a malaria vaccine from LSPs, among other clinically directed work.

Monoclonal antibodies (mAbs) were raised in mice against the synthetic peptide (NANP)40, consisting of 40 (NANP) repeats of the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium falciparum, and characterized. (i) Five of these mAbs recognized the P. falciparum CS protein in western blot experiments and in immunofluorescence assays using different preparations of sporozoites. The remaining two mAbs (CT3.2 and CT3.3, both IgG1) gave negative results by both techniques. (ii) When the anti-(NANP)40 peptide mAbs were functionally tested in vitro to assess their ability to inhibit the attachment and penetration of the parasites into cultured human liver cells, six of them exhibited inhibitory activities ranging between 66 and 90%. CT3.2 mAbs, also, inhibited sporozoite attachment and penetration, despite the negative results by immunofluorescence and western blot experiments. However, when immunofluorescence was repeated in the presence of calcium, CT3.2 did reveal a positive recognition of P. falciparum sporozoites, suggesting that this mAb could recognize the (NANP) sequence when calcium was bound to the repetitive peptide. (iii) Furthermore, the binding of an anti-(NANP)40 IgM mAb (CT1) to the solid-phase peptide was not inhibited by preincubation of the peptide with a mAb against the P. falciparum CS protein. (iv) Finally, one anti-(NANP)40 IgG1 mAb (CT3.1) was unable to bind to the shorter (NANP)3 peptide, although it recognized the (NANP)40 peptide and the P. falciparum CS protein. The results presented here suggest that heterogeneous antibody populations are produced upon immunization of mice with (NANP)40 synthetic peptide and that epitopes different from those simply related to the linear (NANP) amino acid sequence are likely to be present in long (NANP)n constructs as well as in the repetitive domain of the P. falciparum CS protein.

The immunogenicity of the carrier-free synthetic peptide, (NANP)40, from the repetitive region of the Plasmodium falciparum circumsporozoite (CS) protein was investigated in genetically responder mice (C57BL/6, H-2b) acutely infected with blood forms of the non-lethal murine malaria parasite, P. yoelii. As compared to non-infected mice, P. yoelii-infected C57BL/6 mice produced significantly lower titers of anti-(NANP)40 IgG antibodies. This decrease in the anti-(NANP)40 antibody response peaked with the peak of parasitemia, and involved all the IgG subclasses. Interestingly, this P. yoelii-mediated effect was evident both on the development of the antibody response to the (NANP)40 peptide, and on an already established anti-(NANP)40 antibody titer, as seen in mice immunized with the peptide 1 month before the infection. Since (NANP)n-based constructs are strongly envisaged as potential vaccines against falciparum malaria, these results might be important in the evaluation of the efficacy of these vaccine candidates, when they will be used in individuals living in endemic areas.

We describe a new approach for modeling antigenic peptides recognized by T cells. Peptide A24 170-182 can compete with other antigenic peptides that are recognized by H-2kd-restricted cytolytic T cells, presumably by binding to the Kd molecule. By comparing substituted A24 peptides as competitors in a functional competition assay, the A24 residues Tyr-171, Thr-178, and Leu-179 were identified as possible contact residues for Kd. A highly active competitor peptide analog was synthesized in which Tyr was separated from the Thr-Leu pair by a pentaproline spacer. The choice of proline allowed the prediction of a probable conformation for the analog when bound to the Kd molecule. The simplest conformation of the A24 peptide that allows the same spacing and orientation of the motif as in the analog would be a nearly extended polypeptide chain incorporating a single 3(10) helical turn or similar structural kink.

This review covers the latest developments of long synthetic peptide technology for the rapid identification and development of malaria vaccine candidates and immunological modulators. A brief description of the two most common solid-phase synthetic procedures, together with the latest advances in optimisation of peptide chain assembly and analytical instrumentation, is given, with special attention to non-specialists. Several examples of vaccine candidates developed in the authors’ or their collaborators’ laboratories are also provided.

Fast atom bombardment (FAB) tandem mass spectrometry has been used to analyse the biologically potent, partially modified retro-inverso (PMRI) synthetic isomer of tuftsin: this compound represents the active peptide of the fraction of gamma-globulin (leukokinin) which binds specifically to blood neutrophilic leukocytes and monocytes. Protonated molecules and fragment ions were collisionally dissociated at low energies in a triple-quadrupole mass spectrometer to yield a complete picture of the reactions that occur in the condensed and in the gas phase. The study shows that, when retro-inversion is within the N-terminal amino acid, charge localization at the basic sites (possibly at the N-terminus) induces a marked decomposition of the molecule, the loss of ammonia being the most favourable fragmentation process. Also, artifacts are formed in the liquid phase via bimolecular reactions promoted by the high-energy beam. The findings indicate that despite the fact that PMRI isomers of this type are stable against exo-peptidases and also stable under acidic conditions, they appear to be labile under conditions where the energy deposition, due to FAB is necessarily high.

We have previously shown that the priming of mice with live Mycobacterium tuberculosis var. bovis (Bacillus Calmette-Guerin, BCG) and immunization with the repetitive malaria synthetic peptide (NANP)40 conjugated to purified protein derivative (PPD), led to the induction of high and long-lasting titers of anti-peptide IgG antibodies, overcoming the requirement of adjuvants and the genetic restriction of the antibody response to the peptide (Lussow et al., Proc. Natl. Acad. Sci. USA 1990. 87:2960). This initial work led us to the following observations. BCG had to be live for priming to lead to the induction of anti-peptide antibodies. Surprisingly, priming with other living microorganisms which chronically infect the macrophage (e.g. Salmonella typhimurium and Leishmania major) also induced anti-peptide antibodies in mice immunized with PPD-(NANP)40 conjugate. It was, thus, hypothesized that molecules expressed during active infection and also known to be highly conserved between species, namely the heat-shock proteins (hsp), could mediate the T cell sensitization required for the production of anti-peptide antibodies. In fact, when the PPD protion of the conjugate was replaced by a highly purified recombinant protein corresponding to the 65-kDa (GroEL-type) hsp of M. bovis, this resulted in the production of anti-(NANP) IgG antibodies in BCG-primed mice, irrespective of the major histocompatibility complex-controlled responsiveness to the (NANP) sequence itself. Further, similar induction of anti-peptide antibody response was also obtained with a recombinant 70-kDa (DnaK-type) hsp of M. tuberculosis, but not with a small molecular mass (18 kDa) of M. leprae. Finally, an adjuvant-free carrier effect for anti-peptide IgG antibody production in BCG-primed mice, was also exerted by the GroEL hsp of Escherichia coli. This finding that hsp can act as carrier molecules without requiring conventional adjuvants is of potential importance in the development of vaccine strategies.

The immunodominant epitope of Plasmodium vivax, one of the major causative agents of malaria in man, consists of the tandem repetitions of a nonapeptide sequence, AspArgAlaAsp/AlaGlyGlnProAlaGly, with Asp (variant d) or Ala (variant a), in the fourth position. Synthetic peptides corresponding to the P. vivax epitope, containing a different number of nonapeptide sequences, were prepared by solid-phase synthesis according to the Fmoc-polyamide method. Three peptides, containing 1, 2, and 4 copies of the d variant, were assembled on the gel polymer; none of these peptides, however, was suitable for P. vivax sero-epidemiology. A 45-peptide containing both the d and a variants, ddaad, was prepared by continuous-flow Fmoc-polyamide (flow-polyamide). Among the cleavage procedures evaluated for the removal of the five Mtr groups only TFMSA/TFA/ 1,2-ethanedithiol (1:89:10 by vol.) brought deblocking to completion; a substantial level of impurities originated, however, from these procedures. The product was purified by reversed-phase displacement chromatography, a technique only recently applied to peptides, which shows distinct advantages over conventional, linear elution chromatography. In a single experiment, 107 mg of the crude mixture were loaded onto an analytical column (250 × 4 mm), obtaining in purified form 85% of the desired material present in the sample. An ELISA test base on the ddaad peptide was developed and is being applied to the sero-epidemiology of P. vivax malaria.

The synthetic octapeptide lophyrotomin (PhCO-D-Ala-D-Phe-L-Val-L-lle-D-Asp-L-Asp-D-Glu-L-Gln), the toxic factor of the larvae of an Australian species of sawfly, Lophyrotoma interrupta, has been prepared by solid-phase synthesis and proved to be identical with the natural material.

A large peptide consisting of about 40 (Asn-Ala-Asn-Pro) repeats of Plasmodium falciparum circumsporozoite protein, (NANP)40, was synthesized. It was recognized specifically by monoclonal antibodies produced against P. falciparum sporozoites. Moreover, this peptide strongly inhibited the binding of such monoclonal antibodies to antigens present in a sporozoite extract. The (NANP)40 peptide was employed without any carrier to develop an enzyme-linked immunosorbent assay to detect sporozoite-specific serum antibodies arising after natural malaria infections. Antibodies were detected in a high percentage (43.1%) of European patients suffering from acute P. falciparum malaria and in Africans living in an area of Gabon endemic for malaria. In the latter group, the frequency of antisporozoite antibodies increased with age, reaching 65.9% in individuals more than 40 years old. There was a significant correlation between the results obtained with an immunofluorescence assay with glutaraldehyde-fixed sporozoites and those obtained by enzyme-linked immunosorbent assay with (NANP)40. Therefore, such synthetic peptides representing the repetitive epitope of P. falciparum circumsporozoite protein can be used for the detection of antisporozoite antibodies and for the epidemiological studies required to obtain base-line data concerning the immune status of individuals before their participation in a sporozoite vaccine trial.

Displacement chromatography was used for the preparative purification of a synthetic polypeptide that is a promising malaria vaccine. It was prepared by solid-phase synthesis and contains two important epitopes of circumsporozoite (CS) protein of Plasmodium falciparum sporozoite. With apparatus typically employed in analytical high-performance liquid chromatography (HPLC) and on a 250 x 4.6 mm I.D. reversed-phase column, up to 50 mg of crude polypeptide were purified in a single run and with a yield higher than 95%. The results demonstrate that displacement chromatography is suitable for the isolation of several milligrams of a pure polypeptide from a complex mixture that is difficult to separate even by analytical HPLC. In such a preparative application, displacement appears to be superior to elution chromatography as used traditionally.

The complete solid phase synthesis of a partially modified retro-inverso analogue of Substance P, [Glp6, gPhe8, mGly9]SP 6–11, [gPhe =–NHCH(CH2Ph)NH–, mGly =–OCCH2CO–] has been performed using a polydimethylacrylamide-based solid support and [bis(trifluoroacetoxy)iodo]benzene.

The major antigenic protein of the P. falciparum circumsporozoite (CS) is comprised of about 400 amino acids (1,2). The main feature of this protein is the presence of repeats consisting of four amino acids, ASN-ALA-ASN-PRO (NANP; Figure 1).

Sporozoite antibody levels were measured in a group of children aged one to nine years resident in a rural area of The Gambia, using an ELISA to the repeat peptide (NANP)40. The prevalence and titre of antibodies varied with age but not with sex or ethnic group. Significant variations in prevalence were recorded within a group of adjacent villages. Children who were seropositive at the beginning of the dry season had higher spleen and parasite rates both at this time and at the end of the subsequent rainy season than did seronegative children, suggesting that they were exposed more frequently to infection. However, seropositive children had fewer episodes of fever accompanied by high levels of parasitaemia than did seronegative children, suggesting that they had a greater degree of clinical immunity. No differences were found in seroprevalence rates or in mean antibody titres between children who slept under conventional or Permethrin treated bed nets and those who did not, even though bed nets significantly reduced the number of bites by vector mosquitoes.