De novo design of fibrils made of short alpha-helical coiled coil peptides.

BACKGROUND: The alpha-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 alpha-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 alpha-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 alpha-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.

Analysis of a bioactive synthetic analogue of tuftsin by tandem mass spectrometry: anomalous fast atom bombardment activated processes.

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.

Immunostimulation by a partially modified retro-inverso-tuftsin analogue containing Thr1 psi[NHCO](R,S)Lys2 modification.

The tuftsin retro-inverso analogue H-Thr psi[NHCO](R,S)Lys-Pro-Arg-OH was synthesized through a novel procedure for the high-yield incorporation of isolated retro-inverso bonds into peptide chains and the use of the new Meldrum's acid derivative (CH3)2C(OCO)2CH(CH2)4NHCOCF3 followed by its efficient coupling in solution to trimethylsilylated H-D-Thr(t-Bu)NH2. Closely related peptide impurities were eliminated both from the crude final peptide and the fully protected tetrapeptide amide precursor via ion-exchange and reversed-phase displacement chromatography, respectively. The tuftsin retro-inverso analogue proved to be completely resistant to enzymatic degradation in vitro, either against isolated aminopeptidases or human plasma proteolytic enzymes. When administered either orally or intravenously, it was significantly more active than normal tuftsin in increasing the number of specific antibody secreting cells in spleen of mice immunized with sheep erythrocytes. Furthermore, the analogue exerted an enhanced stimulatory effect on the cytotoxic activity of splenocytes against YAC-1 tumor cells. Finally, retro-inverso-tuftsin was about 10-fold more potent than the native peptide in reducing rat adjuvant arthritis. The resistance of the retro-inverso analogue to peptidases might explain the increased in vivo activities and allows its further immunopharmacological characterization.

Mycobacterial heat-shock proteins as carrier molecules.

We have previously shown that the priming of mice with live Mycobacterium tuberculosis var. bovis (Bacillus Calmette-Guérin, 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.

Characterization of murine monoclonal antibodies against a repetitive synthetic peptide from the circumsporozoite protein of the human malaria parasite, Plasmodium falciparum.

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.

Large-scale purification of the synthetic peptide fragment 163-171 of human interleukin-beta by multi-dimensional displacement chromatography.

Multi-dimensional chromatography has been used successfully in the displacement mode for the purification of the synthetic peptide H-Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys-OH, the fragment 163-171 of human interleukin-beta. This peptide can mimic several of the in vivo and in vitro immunostimulatory activities of the entire protein, except for the inflammatory effect. A large-scale procedure has been developed to purify the synthetic peptide by reversed-phase (RP) and ion-exchange (IE) displacement chromatography (DC) in a single run without any pretreatment. Masses from 100 mg to about 35 g of the unpurified compounds synthesized by a solid-phase technique on a Merrifield-type resin and obtained by acidolytic cleavage from the solid support, can be purified in this way. In the RP-DC mode the carrier and the displacer were aqueous solutions of 0.1% trifluoroacetic acid and 50 mM benzyltributylammonium chloride, respectively, whereas in the IE-DC mode the carrier was water and the displacer 50 mM ammonium citrate solution. RP-DC and IE-DC were also performed in series by directing the effluent of the RP column onto the IE column. Peptide purities and recoveries greater than 96 and 90%, respectively, were obtained.

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.

Synthetic peptides for Plasmodium vivax malaria sero-epidemiology. Application of Fmoc-polyamide and displacement chromatography.

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 x 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.

Use of a tuberculin purified protein derivative--Asn-Ala-Asn-Pro conjugate in bacillus Calmette-Guérin primed mice overcomes H-2 restriction of the antibody response and avoids the need for adjuvants.

Because of its immunodominancy, and because it is conserved in different geographical isolates of Plasmodium falciparum, the repetitive sequence of the circumsporozoite protein, (Asn-Ala-Asn-Pro)n [(NANP)n], has been envisaged for the development of an anti-falciparum malaria subunit vaccine. However, the murine immune response to (NANP)n peptides, either carrier-free or coupled to carrier proteins, was shown to be inducible only by using strong (e.g., Freund's) adjuvants. Furthermore, response to the carrier-free peptide, administered in adjuvant, is genetically restricted to I-Ab mice. In the present paper, we report that high titers of antibodies against the NANP repetitive epitope were obtained in responder C57BL/6 (H-2b) mice when they were primed with live BCG (bacillus Calmette-Guérin Mycobacterium tuberculosis var. bovis) and immunized once with the synthetic peptide (NANP)40 coupled to tuberculin purified protein derivative (PPD) without the use of any adjuvant. This approach also led to the production of high titers of anti-NANP antibodies in ASW (H-2s), B10.RIII (H-2r), BALB/c (H-2d), C3H/He (H-2k), and DBA/1 (H-2q) nonresponder mice after two injections of the conjugate. In both cases, BCG priming was obligatory for the induction of antibodies reacting with the synthetic peptide. The levels of anti-NANP antibodies in nonresponder BALB/c mice were demonstrated to be comparable to the levels induced after PPD-(NANP)40 immunization in Freund's complete or incomplete adjuvant. The antibodies induced were also capable of recognizing P. falciparum sporozoites in immunofluorescence assays and, furthermore, these antibodies inhibited the penetration of live sporozoites into human hepatocytes in vitro. This system functioned independently of the subjects' resistance or susceptibility to BCG infection. Given the widespread natural exposure to mycobacterial antigens and the extensive use of BCG and PPD in the human population, this approach might be envisaged for vaccination with malaria peptides.

Competitor analogs for defined T cell antigens: peptides incorporating a putative binding motif and polyproline or polyglycine spacers.

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.

A rapid inhibition micro ELISA for detecting antibodies to Plasmodium falciparum sporozoites in human blood.

A sensitive and specific micro ELISA, named MONOPLATE ELISA, for the detection of antibodies against P. falciparum sporozoites was developed. It can be applied to many kinds of samples including serum, plasma, whole blood, eluted bloodspot and mosquito bloodmeal as well. The method makes use of a single microtiter plate and the chemically synthesized (Asn-Ala-Asn-Pro)20 (NANP20) antigen both as coating material and as competitive (binding) inhibitor in the samples. The specific value of each sample is obtained as the absorbance difference between the uninhibited and the fully inhibited sample. Using appropriate conditions, the results can be evaluated by simple visual inspection of the plate, without any instrument. A rapid procedure, where the incubation times for sample and conjugate are just 15 minutes, is also described. When unknown samples from a P. falciparum endemic area were tested, a close correlation was found between our results and those obtained with the only commercial ELISA kit now available (Sclavo S.p.A). For screening purposes, as many as 48 samples per plate can be tested by this method.

Reduced antibody response to the repetitive sequence of the Plasmodium falciparum circumsporozoite protein in mice infected with Plasmodium yoelii blood forms.

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.

Antibodies to Plasmodium falciparum sporozoites following a malarial outbreak in a non-endemic area of Sri Lanka.

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.

Use of synthetic peptides in the study of the antibody response to Plasmodium vivax sporozoites.

Synthetic peptides reproducing 4 DRADGQPAG (D4) and a sequential array of DRADGQPAG and DRAAGQPAG repeats (DDAAD) of the Plasmodium vivax circumsporozoite (CS) protein were investigated for their potential use in the detection of P. vivax sporozoite antibodies in human sera. These peptides specifically inhibited the binding of monoclonal antibodies to the P. vivax CS protein in Western blots. However, when D4 and DDAAD peptides were used in an enzyme-linked immunosorbent assay (ELISA) for the detection of human antibodies, more sera bound to the DDAAD (61%) than to the D4 peptide (22%). This binding was specific, and suggested that the DDAAD peptide contained epitopes constituted by the sequential array of DRADGQPAG and DRAAGQPAG repeat variants and absent in the D4 peptide. The ELISA using the DDAAD peptide was applied to the detection of P. vivax CS protein antibodies in a large number of sera from Kataragama, an endemic area in Sri Lanka. The prevalence of these antibodies increased with age, reaching 40% in adults greater than 50 years old. The ELISA employing the DDAAD peptide represents a simple and useful tool for the analysis of the antibody response to P. vivax sporozoites in naturally exposed individuals.

Sporozoite antibodies and malaria in children in a rural area of The Gambia.

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.

Preparative purification of Plasmodium falciparum circumsporozoite protein synthetic polypeptides by displacement chromatography.

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.

Fast atom bombardment mass spectrometry and selective acid hydrolysis for the analysis of partially modified retro-inverso peptide analogues.

Fast atom bombardment (FAB) mass spectrometry has been successfully applied to the analysis of partially modified retro-inverso peptide isomers. The spectra are characterized by abundant protonated molecular ions and also by sequence ions due to fragmentation of the inverted bonds. Unambiguous information, as to the nature and the position in the backbone of the amino acids involved in the partial modification of the structure, are given by using a combination of FAB mass spectrometry and partial, selective acid hydrolysis, without separation of the resulting peptide mixtures.

Primary and secondary responses to (NANP) peptides by Plasmodium falciparum sporozoites in various strains of mice.

The repetitive epitope (Asn-Ala-Asn-Pro = NANP) of the Plasmodium falciparum circumsporozoite protein is considered as the basis for the development of a recombinant or synthetic subunit vaccine against malaria. Vaccines consisting of (NANP)n molecules coupled to carrier proteins have already been tested in trials in human volunteers with partial success. In this paper we show that C57BL/6 mice, genetically responsive to carrier-free (NANP)n molecules, exhibit a secondary antibody response to (NANP) if they are primed with carrier-free (NANP)40 synthetic peptide, and then challenged with P. falciparum sporozoites. However, such a sporozoite-mediated boosting effect is not observed if C57BL/6 and BALB/c mice were previously primed with (NANP)40 peptide conjugated to carrier proteins. The genetic restriction of the murine antibody response to (NANP)n is overcome when mice bearing seven different H-2 haplotypes are immunized with entire P. falciparum sporozoites. These results may have implications for the understanding of natural or induced anti-sporozoite immunity, and show that the use of T-cell epitopes from the plasmodial antigenic repertoire would be very likely to represent an efficient approach for the development of a subunit malaria vaccine.

Age-related prevalence of antibody response against three different, defined Plasmodium falciparum antigens in children from the Haut-Ogooué province in Gabon.

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.

1H-NMR studies of synthetic polypeptide models of Plasmodium falciparum circumsporozoite protein tandemly repeated sequence.

The major immunodominant region of the coating protein of Plasmodium falciparum sporozoites contains multiple tandem copies of the sequence Asn-Ala-Asn-Pro (NANP). Current efforts for the development of an antisporozoite vaccine are focused on the synthesis of polypeptides reproducing part of the circumsporozoite protein repeat sequence and, in an attempt to relate conformational properties and biological response, 1H-nmr one- and two-dimensional studies of the synthetic models (NANP)2NA and (NANP)6 were carried out in water and water/methanol mixtures, at 400 and 500 MHz. In water, (NANP)6 undergoes fast conformational averaging. At variance, in water/methanol, the molecule appears to adopt an extensive structure, but detailed analysis is impaired by high spectral degeneracy. Based on the results obtained with (NANP)2NA and from preliminary experiments in water/trifluoroethanol, an interpretation is suggested for the (NANP)6 data in water/methanol in terms of a mixed sequence of beta I-turns and half-turns (or/and gamma I-turns) around the positions Ni-1-Pi-Ni + 1.