Protective cellular immunity against P. falciparum malaria merozoites is associated with a different P7 and P8 residue orientation in the MHC-peptide-TCR complex

Developing a logical and rational methodology for obtaining vaccines, especially against the main parasite causing human malaria (P. falciparum), consists of blocking receptor-ligand interactions. Conserved peptides derived from proteins involved in invasion and having high red blood cell binding ability have thus been identified. Immunization studies using Aotus monkeys have revealed that these peptides were neither immunogenic nor protection inducing. When modified in their critical binding residues, previously identified by Glycine scanning, some of these peptides were immunogenic and non-protection inducers; others induced short-lived antibodies whilst a few were both immunogenic and protection inducing. However, very few of these modified high activity binding peptides (HABPs) reproducibly induced protection without inducing antibody production, but with high cytokine liberation, suggesting that cellular mechanisms had been activated in the protection process. The three-dimensional structure of these peptides inducing protection without producing antibodies was determined by 1H-NMR. Their HLA-DRbeta1* molecule binding ability was also determined to ascertain association between their 3D structure and ability to bind to Major Histocompatibility Complex Class-II molecules (MHC-II). 1H Nuclear Magnetic Resonance analysis and structure calculations clearly showed that these modified HABPs inducing protective cellular immune responses (but not producing antibodies against malaria) adopted special structural configuration to fit into the MHC II-peptide-TCR complex. A different orientation for P7 and P8 TCR contacting residues was clearly recognized when comparing their structure with modified peptides, which induced high antibody titers and protection, suggesting that these residues are involved in activating the immune system associated with antibody production and protection.     

High affinity interactions between red blood cell receptors and synthetic Plasmodium thrombospondin-related apical merozoite protein (PTRAMP) peptides

Plasmodium falciparum thrombospondin-related apical merozoite protein (PTRAMP) has a thrombospondin related (TSR) domain which in many proteins has been reported as a fragment involved in pathogen-host and cell-interactions. Receptor-ligand studies using eighteen non-overlapping 20-aminoacid-long synthetic peptides from this protein were carried out to determine regions involved in parasite invasion of red blood cells (RBC). Two high activity binding peptides (HABPs) were determined, 33405 (21YISSNDLTSTNLKVRNNWEH40) and 33413 (180LEGPIQFSLGKSSGAFRINY199), presenting high dissociation constants and positive cooperativity. One of the HABPs displayed a modified Plasmodium export element (PEXEL), suggesting that this protein could be involved in the merozoite cytoplasmic reticulum, parasitophorous vacuole, red blood cell (RBC) cytosol, and probably infected RBC (iRBC) membrane transport of some other molecules and nutrients. Enzymatic treatment of RBCs increased HABP 33405 binding to them whilst it decreased HABP 33413 binding. Merozoite invasion assays revealed that HABPs have around 57% ability to inhibit new RBC invasion. Circular dichroism revealed the presence of possible alpha-helical elements in both HABPs structures. RBC binding interaction specificity and the presence of a PEXEL motif make these 2 HABPs good candidates for being included in further studies to develop a new multi-antigenic, multi-stage, subunit-based, chemically-synthesised, anti-malarial vaccine.     

Synthetic peptides from two Pf sporozoite invasion-associated proteins specifically interact with HeLa and HepG2 cells

Two recently described molecules have been associated with sporozoite traversal ability and hepatocyte entry: sporozoite invasion-associated proteins (SIAP)-1 and -2. The HeLa and HepG2 cell binding ability of synthetic peptides spanning the whole SIAP-1 and -2 sequences has been studied in the search for identifying these proteins’ functionally active specific regions. Twelve HepG-2 and seventeen HeLa cell high-activity binding peptides (HABPs) have been identified in SIAP-1, 8 of them having high specific binding affinity for both cell lines. Four HepG2 HABPs and two HeLa HABPs have been identified in SIAP-2, one of them interacting with both HeLa and HepG2 cells. SIAP-1 and SIAP-2 HABPs bound specifically and saturably to heparin sulfate and chondroitin sulfate-type membrane receptors on host cells. Circular dichroism assays have shown high α-helix content in SIAP-1 and SIAP-2 HABP secondary structure. Immunofluorescence analysis has revealed that specific peptides against SIAP proteins are highly immunogenic in mice and that anti-SIAP-1 and -2 antibodies recognize the native protein in Plasmodium falciparum sporozoites. Polymorphism studies have shown that a most SIAP-1 and -2 HABPs are conserved among P. falciparum strains. Our results have suggested that SIAP-1 and -2 participate in host-pathogen interactions during cell-traversal and hepatocyte invasion and highlighted the relevance of the ongoing identification and study of potentially new molecules when designing a fully protective antimalarial vaccine.     

Steric-electronic effects in malarial peptides inducing sterile immunity

Conserved Plasmodium falciparum high activity binding peptides' (HABPs) most relevant proteins involved in malaria parasite invasion are immunologically silent; critical binding residues must therefore be specifically replaced to render them highly immunogenic and protection-inducing. Such changes have a tremendous impact on these peptides' steric-electronic effects, such as modifications to peptide length peptide bonds and electronic orbitals' disposition, to allow a better fit into immune system MHCII molecules and better interaction with the TCR which might account for the final immunological outcome.     

Binding activity, structure, and immunogenicity of synthetic peptides derived from Plasmodium falciparum CelTOS and TRSP proteins

Several sporozoite proteins have been associated with Plasmodium falciparum cell traversal and hepatocyte invasion, including the cell-traversal protein for ookinetes and sporozoites (CelTOS), and thrombospondin-related sporozoite protein (TRSP). CelTOS and TRSP amino acid sequences have been finely mapped to identify regions specifically binding to HeLa and HepG2 cells, respectively. Three high-activity binding peptides (HABPs) were found in CelTOS and one HABP was found in TRSP, all of them having high α-helical structure content. These HABPs' specific binding was sensitive to HeLa and HepG2 cells' pre-treatment with heparinase I and chondroitinase ABC. Despite their similarity at three-dimensional (3D) structural level, TRSP and TRAP HABPs located in the TSR domain did not compete for the same binding sites. CelTOS and TRSP HABPs were used as a template for designing modified sequences to then be assessed in the Aotus monkey experimental model. Antibodies directed against these modified HABPs were able to recognize both the native parasite protein by immunofluorescence assay and the recombinant protein (expressed in Escherichia coli) by Western blot and ELISA assays. The results suggested that these modified HABPs could be promising targets in designing a fully effective, antimalarial vaccine.     

P. falciparum: merozoite surface protein-8 peptides bind specifically to human erythrocytes

This work determined Plasmodium falciparum merozoite surface protein-8 (MSP-8) regions specifically binding to membrane surface receptors on human erythrocytes. Five high activity binding peptides (HABPs), whose binding to erythrocytes became saturable and sensitive on being treated with neuraminidase and chymotrypsin were identified from the MSP-8 protein. Those amino acids directly involved in interaction with erythrocytes were also determined for each one of the HABPs. Some of them specifically recognized 28, 46, and 73 kDa erythrocyte membrane proteins. Some HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by up to 98%, suggesting the MSP-8 protein's possible role in the invasion process.     

Biological and structural characteristics of the binding peptides from the sporozoite proteins essential for cell traversal (SPECT)-1 and -2

The sporozoite microneme proteins essential for cell traversal, SPECT-1 and SPECT-2, are considered attractive pre-erythrocytic immune targets due to the key role they play in crossing of the malaria parasite across the dermis and the liver sinusoidal wall, prior to invasion of hepatocytes. In this study, the sequences of SPECT-1 and SPECT-2 were mapped using 20 mer-long synthetic peptides to identify high-activity binding peptides (HABPs) to HeLa cells. 17 HABPs with enzyme sensitive bindings to HeLa cells were identified: 3 predominantly α-helical in SPECT-1, and 10 α-helical and 4 β-turns/random coils in SPECT-2. Immunofluorescence assays (IFA) with antibodies raised in rabbits against chemically synthesized B-cell epitopes suggests the presence of these two proteins in the micronemes and in sporozoite membrane. ¹H NMR studies showed that HABPs located in the membrane-attack complex/perforin (MACPF) domain of SPECT-2 share high similarity with the 3D structure of C8α. Altogether, the results highlight the potential of including HABPs from SPECT-1 and SPECT-2 as components of a fully effective multistage, multiepitopic, minimal subunit-based synthetic vaccine against Plasmodium falciparum malaria.     

3D structure and immunogenicity of Plasmodium falciparum sporozoite induced associated protein peptides as components of fully-protective anti-malarial vaccine

SIAP-1 and SIAP-2 are proteins which are implicated in early events involving Plasmodium falciparum infection of the Anopheles mosquito vector and the human host. High affinity HeLa and HepG2 cell binding conserved peptides have been previously identified in these proteins, i.e. SIAP-1 34893 ((421)KVQGLSYLLRRKNGTKHPVY(440)) and SIAP-1 34899 ((541)YVLNSKLLNSRSFDKFKWIQ(560)) and SIAP-2 36879 ((181)LLLYSTNSEDNLDISFGELQ(200)). When amino acid sequences have been properly modified (replacements shown in bold) they have induced high antibody titres against sporozoites in Aotus monkeys (assessed by IFA) and in the corresponding recombinant proteins (determined by ELISA and Western blot). (1)H NMR studies of these conserved native and modified high activity binding peptides (HABPs) revealed that all had α-helical structures in different locations and lengths. Conserved and corresponding modified HABPs displayed different lengths between the residues fitting into MHCII molecule pockets 1-9 and different amino acid orientation based on their different HLA-DRβ1(?) binding motifs and binding registers, suggesting that such modifications were associated with making them immunogenic. The results suggested that these modified HAPBs could be potential targets for inclusion as components of a fully-effective, minimal sub-unit based, multi-epitope, and multistage anti-malarial vaccine.     

Atomic evidence that modification of H-bonds established with amino acids critical for host-cell binding induces sterile immunity against malaria

Based on the 3D X-ray crystallographic structures of relevant proteins of the malaria parasite involved in invasion to host cells and 3D NMR structures of High Activity Binding Peptides (HABPs) and their respective analogues, it was found that HABPs are rendered into highly immunogenic and sterile immunity inducers in the Aotus experimental model by modifying those amino acids that establish H-bonds with other HABPs or binding to host’s cells. This finding adds striking and novel physicochemical principles, at the atomic level, for a logical and rational vaccine development methodology against infectious disease, among them malaria.     

Identifying Plasmodium falciparum EBA-175 homologue sequences that specifically bind to human erythrocytes

Erythrocyte binding antigen-160 (EBA-160) protein is a Plasmodium falciparum antigen homologue from the erythrocyte binding protein family (EBP). It has been shown that the EBP family plays a role in parasite binding to the erythrocyte surface. The EBA-160 sequence has been chemically synthesised in seventy 20-mer sequential peptides covering the entire 3D7 protein strain, each of which was tested in erythrocyte binding assays to identify possible EBA-160 functional regions. Five EBA-160 high activity binding peptides (HABPs) specifically binding to erythrocytes with high affinity were identified. Dissociation constants lay between 200 and 460 nM and Hill coefficients between 1.5 and 2.3. Erythrocyte membrane protein binding peptide cross-linking assays using SDS-PAGE showed that these peptides bound specifically to 12, 28, and 44 kDa erythrocyte membrane proteins. The nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. HABPs were able to block merozoite in vitro invasion of erythrocytes. HABPs’ potential as anti-malarial vaccine candidates is also discussed.     

Identifying Plasmodium falciparum merozoite surface antigen 3 (MSP3) protein peptides that bind specifically to erythrocytes and inhibit merozoite invasion

Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine Plasmodium falciparum merozoite surface protein-3 (MSP-3) FC27 strain regions that specifically bind to membrane surface receptors on human erythrocytes. Three MSP-3 protein high activity binding peptides (HABPs) were identified; their binding to erythrocytes became saturable, had nanomolar affinity constants, and became sensitive on being treated with neuraminidase and trypsin but were resistant to chymotrypsin treatment. All of them specifically recognized 45-, 55-, and 72-kDa erythrocyte membrane proteins. They all presented alpha-helix structural elements. All HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by ~55%-85%, suggesting that MSP-3 protein's role in the invasion process probably functions by using mechanisms similar to those described for other MSP family antigens.     

Peptides from the Plasmodium falciparum STEVOR putative protein bind with high affinity to normal human red blood cells

Synthetic 20-mer long non-overlapped peptides, from STEVOR protein, were tested in RBC binding assays for identifying STEVOR protein regions having high RBC binding activity and evaluating whether these regions inhibit Plasmodium falciparum in vitro invasion. Affinity constants, binding site number per cell and Hill coefficients were determined by saturation assay with high activity binding peptides (HABPs). HABP binding assays using RBCs previously treated with enzymes were carried out to study the nature of the receptor. The molecular weight of RBC surface proteins interacting with HABPs was determined by cross-linking assays and SDS-PAGE analysis. RBC binding assays revealed that peptides 30561 (41MKSRRLAEIQLPKCPHYNND60), 30562 (61PELKKIIDKLNEERIKKYIE80) and 30567 (161ASCCKVHDNYLDNLKKGCFG180) bound saturably and with high binding activity, presenting nanomolar affinity constants. HABP binding activity to RBCs previously treated with neuraminidase and trypsin decreased, suggesting that these peptides bound to RBC surface proteins and that such binding could be sialic acid dependent. Cross-linking and SDS-PAGE assays showed that the three HABPs specifically bound to 30 and 40 kDa molecular weight RBC membrane proteins. Peptides 30561, 30562 and 30567 inhibited P. falciparum in vitro invasion of red blood cells in a concentration-dependent way. Goat sera having STEVOR protein polymeric peptides antibodies inhibit parasite in vitro invasion depending on concentration. Three peptides localized in STEVOR N-terminal and central regions had high, saturable, binding activity to 30 and 40 kDa RBC membrane proteins. These peptides inhibited the parasite's in vitro invasion, suggesting that STEVOR protein regions are involved in P. falciparum invasion processes during intra-erythrocyte stage.     

Structural characterisation of sporozoite components for a multistage, multi-epitope, anti-malarial vaccine

A totally effective anti-malarial vaccine must contain epitopes derived from multiple proteins found in different stages of the particular parasite involved in invasion. It must therefore include sporozoite molecules able to induce protective immunity thereby blocking the parasite's access to hepatic cells; thrombospondin-related anonymous protein (TRAP) is one of them. Conserved high activity binding peptides (HABPs) attaching themselves to hepatic cells were used in immunisation studies with the highly malaria-susceptible Aotus monkey. However, they had to be modified to render them immunogenic. The changes induced in lead peptide 3D structure were analysed by correlating such substitutions with the induction of high anti-sporozoite antibody levels in the experimental monkey model. The modification induced structural changes in most modified HABPs, changing them from random-coil or distorted type III beta-turn structures to classical type III or III' beta-turn, thereby allowing a better fit into the MHC-II-peptide-TCR complex since they bound with high affinity to purified HLA-DRbeta1* molecules. These are the first (TRAP) conserved HABPs corresponding to functionally active amino acid sequences in sporozoite invasion and mobility which, when modified, were able to induce very high anti-sporozoite antibody responses, leading to suggesting them as components in the first line of defence of a fully-effective, subunit-based, multi-epitope, multi-stage, synthetic anti-malarial vaccine.     

Specific erythrocyte binding capacity and biological activity of Plasmodium falciparum erythrocyte binding ligand 1 (EBL-1)-derived peptides

Erythrocyte binding ligand 1 (EBL-1) is a member of the ebl multigene family involved in Plasmodium falciparum invasion of erythrocytes. We found that five EBL-1 high-activity binding peptides (HABPs) bound specifically to erythrocytes: 29895 ((41)HKKKSGELNNNKSGILRSTY(60)), 29903 ((201)LYECGK-KIKEMKWICTDNQF(220)), 29923 ((601)CNAILGSYADIGDIVRGLDV(620)), 29924((621)WRDINTNKLSEK-FQKIFMGGY(640)), and 30018 ((2481)LEDIINLSKKKKKSINDTSFY(2500)). We also show that binding was saturable, not sialic acid-dependent, and that all peptides specifically bound to a 36-kDa protein on the erythrocyte membrane. The five HABPs inhibited in vitro merozoite invasion depending on the peptide concentration used, suggesting their possible role in the invasion process.     

Characterization of hyaluronan-binding proteins on guinea pig polymorphonuclear leukocytes: possible involvement of complement receptor type 3 (CR3, CD11b/CD18) in the hyaluronan-leukocyte interaction

Hyaluronan (HA), a high-molecular-weight glycosaminoglycan ubiquitously present in the extracellular matrices (ECMs) of animals, plays important roles in ECM organization and cell behavior through binding to hyaluronan-binding proteins (HABPs). We previously reported that HA has anti-inflammatory effects on guinea pig phagocytes, although the nature of guinea pig HABPs was unknown. In this study, we characterized guinea pig HABPs on peritoneal polymorphonuclear leukocytes (PMNs) and blood neutrophils by flow cytometry and affinity chromatography. It was found that PMNs express diverse HABPs with different molecular weights. These HABPs maximally bound with HA over a wide pH range (6-8), and recognized HAs as small as the pentadisaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine. Furthermore, they could be divided into Mg(2+)-dependent and Ca(2+)/Mg(2+)-independent groups. Interestingly, two proteins in the Mg(2+)-dependent group were found to be the two subunits of complement receptor type 3 (CR3, CD11b/CD18). Unlike PMNs, blood neutrophils expressed several functionally inactive HABPs. Among these inactive HABPs, Mg(2+)-dependent proteins including CR3 but not Ca(2+)/Mg(2+)-independent proteins were activated on phorbol ester-stimulation. These results show the existence of diverse HABPs on guinea pig neutrophils and the cell activation-dependent activation of HABPs. It is also suggested that the CR3-HA interaction is possibly involved in the regulation of neutrophil function.     

P. falciparum pro-histoaspartic protease (proHAP) protein peptides bind specifically to erythrocytes and inhibit the invasion process in vitro

Plasmodium falciparum histoaspartic protease (HAP) is an active enzyme involved in haemoglobin degradation. HAP is expressed as an inactive 51-kDa zymogen and is cleaved into an active 37-kDa enzyme. It has been proposed that this kind of protease might be implicated in the parasite's invasion of erythrocytes; however, this protein's role during invasion has still to be determined. Synthetic peptides derived from the HAP precursor (proHAP) were tested in erythrocyte binding assays to identify their possible function in the invasion process. Two proHAP high-activity binding peptides (HABPs) specifically bound to erythrocytes; these peptides were numbered 30609 (101LKNYIKESVKLFNKGLTKKS120) and 30610 (121YLGSEFDNVELKDLANVLSF140 ). The binding of these two peptides was saturable, presenting nanomolar affinity constants. These peptides interacted with 26- and 45-kDa proteins on the erythrocyte surface; the nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. The HABPs showed greater than 90% merozoite invasion inhibition in in vitro assays. Goat serum containing proHAP polymeric peptide antibodies inhibited parasite invasion in vitro .     

Conserved regions from Plasmodium falciparum MSP11 specifically interact with host cells and have a potential role during merozoite invasion of red blood cells

Despite significant global efforts, a completely effective vaccine against Plasmodium falciparum, the species responsible for the most serious form of malaria, has not been yet obtained. One of the most promising approaches consists in combining chemically synthesized minimal subunits of parasite proteins involved in host cell invasion, which has led to the identification of peptides with high binding activity (named HABPs) to hepatocyte and red blood cell (RBC) surface receptors in a large number of sporozoite and merozoite proteins, respectively. Among these proteins is the merozoite surface protein 11 (MSP11), which shares important structural and immunological features with the antimalarial vaccine candidates MSP1, MSP3, and MSP6. In this study, 20‐mer‐long synthetic peptides spanning the complete sequence of MSP11 were assessed for their ability to bind specifically to RBCs. Two HABPs with high ability to inhibit invasion of RBCs in vitro were identified (namely HABPs 33595 and 33606). HABP‐RBC bindings were characterized by means of saturation assays and Hill analysis, finding cooperative interactions of high affinity for both HABPs (n_H of 1.5 and 1.2, K_d of 800 and 600 nM for HABPs 33595 and 33606, respectively). The nature of the possible RBC receptors for MSP11 HABPs was studied in binding assays to enzyme‐treated RBCs and cross‐linking assays, finding that both HABPs use mainly a sialic acid‐dependent receptor. An analysis of the immunological, structural and polymorphic characteristics of MSP11 HABPs supports including these peptides in further studies with the aim of designing a fully effective protection‐inducing vaccine against malaria. J. Cell. Biochem. 110: 882–892, 2010. © 2010 Wiley‐Liss, Inc.     

The role of amino acid electron-donor/acceptor atoms in host-cell binding peptides is associated with their 3D structure and HLA-binding capacity in sterile malarial immunity induction

Plasmodium falciparum malaria continues being one of the parasitic diseases causing the highest worldwide mortality due to the parasite’s multiple evasion mechanisms, such as immunological silence. Membrane and organelle proteins are used during invasion for interactions mediated by high binding ability peptides (HABPs); these have amino acids which establish hydrogen bonds between them in some of their critical binding residues. Immunisation assays in the Aotus model using HABPs whose critical residues had been modified have revealed a conformational change thereby enabling a protection-inducing response. This has improved fitting within HLA-DRβ1¹ molecules where amino acid electron-donor atoms present in β-turn, random or distorted α-helix structures preferentially bound to HLA-DR53 molecules, whilst HABPs having amino acid electron-acceptor atoms present in regular α-helix structure bound to HLA-DR52. This data has great implications for vaccine development.