Modified merozoite surface protein-1 peptides with short alpha helical regions are associated with inducing protection against malaria Mary H. Torres 1 *, Luz M. Salazar 1 *, Magnolia Vanegas 1 , Fanny Guzman 1 , Raul Rodriguez 1 , Yolanda Silva 1 , Jaiver Rosas 1 and Manuel E. Patarroyo 1,2 1 Fundacion Instituto de Inmunologı ´ a de Colombia (FIDIC), Bogota ´ , Colombia; 2 Universidad Nacional de Colombia, Bogota ´ , Colombia The merozoite surface protein-1 represents a prime candi- date for development of a malaria vaccine. Merozoite sur- face protein-1 has been shown to demonstrate high-activity peptide binding to human red blood cells. One of the high-activity binding peptides, named 5501, located in the N-terminus (amino acid sequence MLNISQHQCVKKQ CPQNS) of the 19-kDa molecular mass fragment of mero- zoite surface protein-1, is conserved, nonimmunogenic and nonprotective. Its critical binding residues were identified and replaced with amino acids of similar mass but different charge, in order to modify their immunogenic and protective characteristics. Three analogues with positive or negative immunological results were studied by nuclear magnetic resonance to correlate their three-dimensional structure with their biological functions. The studied peptides presented a-helical fragments, but in different peptide regions and extensions, except for randomly structured 5501. We show that altering a few amino acids induced immunogenicity and protectivity against experimental malaria and changed the peptide three-dimensional structure, suggesting a better fit with immune-system molecules. Keywords: MSP-1 protein; peptide analogues; nuclear magnetic resonance; vaccine candidate. Multiple receptor–ligand type interactions are involved in the host-cell invasion process of the Plasmodium falciparum malaria parasite [1–3], allowing the entry and survival of this deadly parasite. More than 250 million people are infected with P. falciparum annually, and more than 2.5 million die; of these, the majority are children < 5 years-old, most of them in sub-Saharan Africa [4]. Our goal was to induce protective immune responses capable of blocking the receptor–ligand interactions, as a strategy for developing vaccines that could be used to impede invasion, inhibit infection and thus decrease the heavy burden that this disease imposes on mankind. One of the most studied proteins of P. falciparum (and a prime candidate for the development of a malarial vaccine) is the merozoite surface protein-1 (MSP-1) [5]. This molecule (synthesized as a 195–200 kDa molecular mass precursor in the parasite’s schizont) is enzymatically pro- cessed and cleaved into polypeptide fragments of 83 kDa, 38 kDa, 30 kDa and 42 kDa molecular mass. The 42 kDa molecular mass fragment is further cleaved (in a Ca 2+ - dependent process) into polypeptides of 33 kDa and 19 kDa molecular mass. The 19 kDa molecular mass fragment is the only one that enters red blood cells (RBCs) during invasion [6]. Urquiza et al. [1] and Rodriguez et al. [2] have developed a specific methodology for identifying microbial peptide receptor–ligand interactions with their host cells, via high- activity binding peptides (HABPs), to determine which MSP-1peptidesbindtoRBCs. One of these HABPs, the conserved 5501 HABP located in the N-terminus of the 19 kDa molecular mass fragment (amino acid sequence 1629 M LNISQHQCVKKQCPQNS 1646) has a 230-n M affinity constant (K d ), 1.18 Hill coefficient and a theoretical 11 800 ± 2300 number of RBC receptor sites [1]. Conserved malarial peptides are poorly or nonantigenic, and their respective HABPs are nonimmunogenic and nonprotective [7–10]. The critical RBC-binding residues (underlined above) were identified here by glycine-replace- ment analogue scanning, as previously described [2,3]. These critical residues were replaced with amino acids of similar mass, but different charge, to render them immunogenic andprotectiveintheAotus experimental model. The objective of this work was to identify a correlation between these modified HABPs and their 3D, as determined by 1 H-NMR. Materials and methods Peptide synthesis The peptides were synthesized by using standard t-Boc solid-phase peptide synthesis, previously described by Merrifield and modified by Houghten [11], in polypropylene bags with 150 mg of p-methylbenzhydrylamine resin HCl Correspondence to M. E. Patarroyo, Fundacio ´ n Instituto de Inmunologı ´ a de Colombia (FIDIC), Carrera 50, no. 26-00, Bogota ´ , Colombia. Fax: + 57 1 4815269, Tel.: + 57 1 4815219, E-mail: mepatarr@mail.com Abbreviations: FITC, fluorescein isothiocyanate; HABP, high-activity binding peptide; MSP-1, merozoite surface protein-1; RBC, red blood cell; RMSD, root mean square deviation. *Both authors contributed equally as first authors. (Received 22 May 2003, revised 23 July 2003, accepted 5 August 2003) Eur. J. Biochem. 270, 3946–3952 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03780.x (Bachem, Torrance CA, USA). The resin was deprotonated by adding 5% diisopropylethylamine (Merck) in methylene chloride before introducing the first amino acid. The coupling cycle was initiated by submerging the bags in a solution containing equimolecular amounts of t-Boc amino acid (Bachem-Peninsula, Torrance, CA, USA) and diiso- propylcarbodiimide (Merck), in a 10-fold molar excess over available amine in the bag. The reaction was allowed to proceed for 60 min and the product was washed with methylene chloride. The t-Boc groups of the newly coupled amino acids were removed with 55% trifluoracetic acid (Pierce, Rockford, IL, USA) in methylene chloride. Reac- tion products were washed and amino groups deprotonated with diisopropylethylamine. Asn and Gln coupling was carried out by adding 1-hydroxy-benzotriazole hydrate (Aldrich) in dimethylformamide. Protected amino acids were liberated from the resin by treatment with 2 mL of 10% anisole in anhydrous hydrogen fluoride (Air Products, Allentown, PA, USA) for 60 min at 0 °C. The hydrogen fluoride was distilled from the reaction and the product was washed five times with ethyl ether (Merck). The peptides were subsequently extracted in 5% acetic acid (Merck), and then analysed and purified by RP- HPLC on an analytical Vydac C-18 column and a Vydac preparative C-18 column by linear-gradient elution from 0 to 100% B with the following solvent system: A, H 2 Oand 0.05% trifluoroacetic acid; B, CH 3 CN and 0.05% trifluoro- acetic acid for 45 min (45–60 min for preparative process) at a1.0mLÆmin )1 flow rate (4.5 mLÆmin )1 for preparative process). The polypeptide molecularmasses were determined by MS (Bruker Protein MALDI-TOF spectrophotometer). Peptide analogues (called polymers) were synthesized with Cys-Gly in the C- and N-terminus of each peptide to allow polymerization forming disulfide bond by an oxida- tion reaction. This procedure has been carefully standard- ized to guarantee the inclusion of high-molecular-mass polymers for immunization purposes. The polymers were analysed by size-exclusion chromatography; their molecular masses ranged from 8 to 24 kDa. Competition-binding assay Critical amino acids were defined as being those amino acids which, upon replacement with glycine, diminished erythro- cyte-binding activity by > 50% of the original peptide activity throughout the concentration range used. The role of each HABP-5501 amino acid in erythrocyte binding was determined by competition-binding assays between the original radiolabelled peptide and nonlabelled original or glycine analogue peptides, as described previously [2,3]. In brief, 100 n M 125 I-labelled HABP-5501 was incubated with 10 8 erythrocytes, in the presence or absence of 100 n M or 800 n M nonlabelled peptides, for 60 min at room tempera- ture. Cells were then washed five times with isotonic NaCl/P i ; radioactivity associated with the cells was then determined. Animals and immunization Aotus nancymaae monkeys were immunized with the synthesized polymeric peptide analogues, shown in Table 1, to induce humoral immune responses as well as protection against experimental challenge with the P. falciparum malaria parasite. Spleen-intact Aotus monkeys, kept at our Primate Station in Leticia (Colombia) in the Amazon region, according to National Institutes of Health guidelines, were analysed by the IFA test for the presence of P. falciparum schizont parasite antibodies in their sera (a 1 : 20 dilution of sera was used). The few Aotus monkeys testing positive for the presence of P. falciparum schizont parasite anti- bodies were returned to the jungle. The Aotus monkeys testing negative for P. falciparum schizont parasite anti- bodies were distributed in random groups of five or six for immunization. Each monkey received, subcutaneously, 125 lg of the polymerized peptide homogenized in Fre- und’s Complete Adjuvant for the first dose on day 0, and homogenized in Freund’s Incomplete Adjuvant for the second dose on day 20; most also received a third immunization on day 40. Blood was drawn for immunological analysis on days 0 and 15, and 20 days after each immunization. Challenge and parasitemia assessment Both immunized and control A. nancymaae monkeys were infected with 200 000 P. falciparum FVO-strain infected RBC, via the femoral vein, for challenge 20 days after the last immunization [12]. Protection was defined as being the total absence of parasites in blood during the 15 days of the experiment. Nonprotected monkeys developed patent parasitemia on days 5 or 6, reaching levels of ‡ 6% between days 8 and 10. After receiving treatment with antimalarial drugs in paediatric doses, they were quarantined to ensure cure and subsequently returned to the jungle. The parasitemia in individual monkeys was measured daily, starting on day 5 after challenge. Immunofluorescence was used to evaluate the number of parasites, in terms of the percentage of parasitized RBC, on slides following Acridine Orange staining. IFA and Western blot Late-stage schizonts from a continuous P. falciparum culture (FCB-2 strain) were synchronized, according to the method of Lambros & Vandenberg [13]. They were washed and treated as described previously [12]. The slides with the dry parasites were blocked for 10 min with 1% nonfat milk and incubated for 30 min with appropriate dilutions of monkey sera (starting at a dilution of 1 : 40) for antibody analysis. Reactivity was observed by fluorescence microscopy using the F(ab¢) 2 fragments from a 1 : 100- diluted goat anti-(monkey IgG) fluorescein isothiocyanate (FITC) conjugate. Preimmune sera from all monkeys were used as negative controls. The20%late-parasitemiaRBCswerewashedwithNaCl/ P i (pH 7.2) and lysed with 0.2% saponin (Merck) for Western blot analysis. The parasite proteins were extracted by using lysis buffer (1 m M phenylmethanesulfonyl fluoride, 1m M EDTA and 5% SDS) and the lysate was centrifuged (25 000 g, 45 min). A 10% resolving gel was used for SDS/ PAGE. The ensuing product was transferred onto nitrocel- lulose paper and incubated with 1 : 100 diluted preimmune or immune sera for Western blot analysis. The reaction was Ó FEBS 2003 Modified MSP-1 peptides protect against malaria (Eur. J. Biochem. 270) 3947 revealed with affinity-purified goat anti-(Aotus IgG) alkaline phosphatase conjugate [14]. NMR Samples for NMR were prepared by dissolving 7 mg of peptide in 500 lL of dimethylsulfoxide-d 6 aprotic medium because these peptides were not soluble in aqueous solution or in trifluoroethanol, and recent studies have shown that preferential conformations of peptides dissolved dimethyl- sulfoxide are not destabilized [15,16]. All NMR spectra were recorded on a Bruker DRX-600 spectrometer at 295 K. The basic NMR structure determination protocol for all peptides can be described as follows: proton spectra were assigned by using DQF-COSY [17], TOCSY [18] and NOESY [19]. The TOCSY and NOESY (400 ms mixing time) spectra were first used to identify individual spin systems (amino acids) and then stretches of amino acids Table 1. Humoral immune response and protective efficacy induced by 5501-derived peptides in Aotus monkeys. 3948 M. H. Torres et al. (Eur. J. Biochem. 270) Ó FEBS 2003 within a given primary structure (sequential assignment) and 3D structure. Chemical shifts were referenced to the residual protonated dimethylsulfoxide signal, defining it as being 2.49 p.p.m. The 2D NMR data were processed using XWIN - NMR software. Structure calculations Peptide structure was determined by Molecular Simulations Inc. ( MSI ) software. Cross-peak volume was obtained by integration using FELIX software on the NOESY spectrum from which the interprotonic distances (constraints) had been obtained. NOESY peaks were classified as being strong, medium or weak signals, according to their relative intensity; these corresponded to 1.8–2.5 A ˚ , 2.5–3.5 A ˚ or 3.5–5.0 A ˚ interproton distances, respectively. Distance Geometry ( DGII ) software was used to generate a family of 50 structures. These structures were refined by using simulated annealing protocol with DISCOVER software in conditions restricting experimental distance and angular constraints. The calculations were repeated several times until a structure having a minimum of distance and angle restraint violations and the least root mean square deviation (RMSD), respecting consensus least energy structure, was obtained. Structures having reasonable geometry and few violations were then selected. Results and discussion Peptide analysis Peptide purity (as analyzed by HPLC with a C18 reverse- phase analytical column and MS analysis) showed that all synthesized monomers had one single peak and the expected theoretical mass and were used for the structural analysis (NMR and CD). CD spectra analysis of monomers and polymers, at a concentration where these peptides were soluble in water, showed similar structural conformation (data not shown). The peptides’ polymer form was used for immunization studies. Critical binding residue Critical binding residues were considered as being those where analogue peptide-binding activity decreased by > 50% of native peptide-binding activity; these were: L2, K11, Q13, C14, P15 and S18, as shown in Fig. 1. According to these results, the interaction was located mainly at the C-terminus of peptide 5501. Fine erythrocyte-binding specificity was observed, as Q13 and C14 were critical residues for erythrocyte binding, but Q8 and C9 were not. Immunological and protection studies Table 1 shows lead, nonimmunogenic, nonprotective pep- tide 5501 becoming immunogenic and protective, or only immunogenic, as a result of specific changes made in some of its critical binding residues. As reported previously [7–10], three groups of peptide modifications were observed. These modifications induced high antibody titres against experimental challenge in group A (as assessed by IFA) and protected monkeys with three or two doses. The experiment was repeated once more using one peptide (24148) and eight monkeys, giving complete protection in three of 16 monkeys (18% protective efficacy). Group B (including peptide 23754) had modifications that induced high antibody titres without protection. The largest panel of modified peptides, grouped in C (including peptide 24326), did not induce either antibodies or protection. A Western blot (Fig. 2) of immunized monkey sera, taken 15 days after the second immunization, showed monkeys with high antibody titres, but not protected (such as sera 13466 and 23754), owing to the fact that their antibodies had disappeared by day 20 and did not reappear following the third immunization, perhaps as a consequence of an anti-idiotypic phenomenon or short- lived antibodies. These sera reacted strongly with 195 kDa, 140 kDa and 83 kDa molecular mass molecules and weakly with the 42 kDa molecular mass molecule. By contrast, monkeys with antibodies that persisted for longer than 20 days (data not shown) and that were protected with only two immunizations (such as those immunized with peptide 24148), reacted strongly with 70 kDa and 42 kDa molecular mass molecules. This data suggests that immunization with peptides having different modifications induces antibodies that recognize different protein structural configurations in the same molecule during MSP-1 protein cleavage and processing. Only the latter peptide analogue was associated with inducing protection. NMR structure determinations were performed on immunogenic and completely protective peptide 24148, immunogenic but not protective 23754, and nonimmuno- genic, nonprotective 24326, to establish the relationship Fig. 1. Identification of critical residues for erythrocyte binding. The height of the bars is proportional to the erythrocyte-binding activity. Analogue peptides with erythrocyte-binding activity that were < 50% of the original peptide-binding activity were considered to be peptides containing modified critical residues. Ó FEBS 2003 Modified MSP-1 peptides protect against malaria (Eur. J. Biochem. 270) 3949 between relevant substitution analogue 3D structure and the immunogenicity and protective efficacy elicited in the experimental monkeys. NMR assignments Completed d aN sequential NOEs were generally found for all peptides. Intraresidual signal intensity (which was greater than that of the d aN sequential signal) and the presence of strong d NN cross-peaks, indicated that there was a signifi- cant population of conformations in the a region of the /w space. The presence of NOEs between P15 d protons and T14 a proton for peptides 24148 and 24326, and V14 a proton for peptide 23754, indicated that these peptides were trans isomers. The NOESY spectra of all peptides showed aN(i,i+1) sequence signals to be stronger than intraresidue cross- peaks. In addition to these sequential cross-peaks, some medium-range d NN (i,i +1),d ab (i,i +3),d aN (i,i +3), and d aN (i,i + 4) cross-peaks were found, indicating the presence of typical helical fragments in all peptides included in this study (except for 5501 that had a totally extended form owing to the absence of medium-range signals, thus making it impossible to determine its 3D structure). Peptides 24148, 23754 and 24326 sequential medium-range NOEs are summarized in Table 2. Molecular dynamics calculations A set of 50 independently generated structures were obtained, satisfying the experimental constraints when using 162 distance restraints (including short-range and medium- range) and 16 x-dihedral angle restraints. A family of 28 low-energy conformers, which did not have a distance violation larger than 0.40 A ˚ or dihedral angle violation greater than 2°, were accepted. These structures had a 0.45-A ˚ RMSD superimposition value for the backbone atoms. Structures were helical between residues S5 and V10 for peptide 24148. New calculations were performed for this peptide, using 1.8-A ˚ lower-distance limits on all bins; 22 overlapped structures were found to have 0.19 A ˚ RMSD and lesser violation (0.30 A ˚ ), confirming previous results (this and other analogue results are presented in Table 3). According to Kabasch & Sander [20], all peptides have well-defined helical structures. Results of structure calculations for peptides 24326 and 23754 are also shown in Table 3. It can be seen in Fig. 3 that immunogenic, protective peptide 24148 presents a helical fragment between residues 5 and 10, maintaining great flexibility in the rest of the molecule. Immunogenic, nonprotective peptide 23754 Fig. 2. Western blot analysis of solubilized antigens obtained from late Plasmodium falc iparum schizonts. Table 2. Summary of sequential medium range NOE connectivities represented by different line thickness for peptides 24148, 24326 and 23754. 3950 M. H. Torres et al. (Eur. J. Biochem. 270) Ó FEBS 2003 presents a helical fragment between residues 6 and 12. Nonimmunogenic, nonprotective peptide 24326 also pre- sents a longer helical structural motif between residues 5 and 12. It can be observed that changes in critical amino acids lead to conformational changes in the native peptide random structure. Our results also suggested that the presence of a short helical region between amino acids 5 and 10 (24148), and greater flexibility in the rest of the molecule, lead to greater recognition of immune-system molecules generating antibodies inducing protection against P. falci- parum. This was not seen in peptide 24326, as its a-helical motif was two residues larger, limiting the flexibility of the rest of the molecule and thus perhaps preventing antibody production and the induction of protection. It has been shown that peptides fitting properly into major histocompatibility complex (MHC) class II molecule grooves have a polyproline II conformation [21]. Helical peptides may not fit well into these grooves as a consequence of such a characteristic structure. The reduction in a Table 3. Summary of structure calculation results. RMSD, root mean square deviation. Peptide no. Peptide sequence (helical segments shaded) NOEs used RMSD (A ˚ ) Maximum NOE A ˚ violations Maximum angular violations Immuno- genicity Protection 5501 MLNISQHQCVKKQCPQNS –– 24148 MLNISMLQTVMMMTPQK 162 0.19 0.30 2 + + 23754 MHNISQLQVVKKMVPQK 141 0.45 0.40 3 + – 24326 MLNISMLQTVMKMTPQK 151 0.51 0.30 2 – – Fig. 3. Families of structures selected from nonimmunogenic, nonprotective peptide 24326, immunogenic, nonprotective peptide 23754 and immuno- genic, protective peptide 24148. Left: backbone representation of analogue peptides; the core where the main modifications were made is shown in red. Right: ribbon representation of the same fragment. Colour code: I4 fuchsia; S5 red; M6 (24148, 24326), Q6 (23754) pale blue; L7 dark blue; Q8 pink; T9 (24148, 24326), V9 (23754) orange; V10 grey (24148, 23754 y 24324); M11 (24148, 24326), K11 (23754) yellow; M12 (24148), K12 (23754, 24326) green. Ó FEBS 2003 Modified MSP-1 peptides protect against malaria (Eur. J. Biochem. 270) 3951 peptide’s helical proportion may allow for a better fit with MHC class II molecules, thereby activating the immune system, producing antibodies and inducing protection. A protection of 18%, conferred exclusively by modified peptide 24148, as a part of a multicomponent malaria vaccine that may require as many as 50–100 different epitopes, is a major achievement. The new recombinant or synthetic antimalarial vaccines tested by different groups have included six to 24 different recombinant proteins or peptides. Others under study include many DNA fragments in a DNA vector and hundreds of epitopes [22]. Therefore, 18% protection represents very high protective immunity induced by one single peptide. It has also been shown that a minimal structural modification in haemoglobin 64–76 (E73D) can reduce the potency of this peptide by 1000-fold, highlighting the role of subtle variations in inducing the appropriate immune response [23]. 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