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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 P lasmodium e xport e lement (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 α-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.

Plasmodium falciparum rhoptry-associated proteins 1 (RAP1) and 2 (RAP2) are antigens presenting themselves as candidates for a subunit malaria vaccine. RAP2 protein, non-overlapping, consecutive peptides were synthesised and tested in red blood cell (RBC) binding assays to identify their receptor-ligand interaction in recognising RAP2 regions involved in the in vitro merozoite invasion process. Four high activity binding peptides (HABPs) were identified in the resulting 20 peptides. Peptides 26220 ((61)NHFSSADELIKYLEKTNINT(80)), 26225 ((161)IKKNPFLRVLNKASTTTHAT(180)) and 26229 ((241)RSVNNVISKNKTLGLRKRSS(260)) were located in the amino terminal and central part of the protein and HABP 26235 ((361)FLAEDFVELFDVTMDCYSRQ(380)) was located at the carboxy terminal. All these HABPs showed saturable binding and presented dissociation constants between 500 and 950 nM; the number of binding sites per RBC ranged from 48,000 to 160,000. High binding peptides' critical amino acids involved in RBC binding were determined by competition binding assays; their amino acids appear in bold in the sequences shown above. SDS-PAGE results showed that peptides 26220, 26225 and 26229 had at least two different sets of 62 and 42 kDa HABP receptors on RBCs and that peptide 26235 had at least two different sets of 77 and 62 kDa. HABPs inhibited in vitro merozoite invasion by between 54% and 94% at 200 microM, suggesting that these RAP2 peptides are involved in the in vitro P. falciparum invasion process.

Three Plasmodium falciparum serine repeat antigen (SERA) protein peptides were studied by NMR and structure calculations being done in 70:30 water:trifluoroethanol solution. Peptide 22834 was shown to be immunogenic and protective against malaria in Aotus monkeys, whilst native peptide 6737 and its analogue 14096 did not present protection against the disease in these monkeys. Results showed a relationship between these peptides' secondary structure and their function as immunogen against malaria.

Epstein–Barr virus (EBV) infects human target cells mainly through gp350/220-CD21 and gp42-MHCII interactions; however, it has been shown that these interactions are dispensable for EBV-invasion of susceptible cells, suggesting that other viral proteins are involved in this process. It is probable that tegument BNRF1/p140 protein is involved in EBV-invasion of target cells, since anti-p140 antibodies inhibit EBV-infection of B-lymphocytes and there is evidence that part of the protein is located on virus surface. Sixty-six peptides, covering the entire BNRF1/p140 sequence, were synthesised and tested in lymphoblastoid cell line binding assays. Peptides 11465 and 11521 bound with high affinity to Raji, Ramos and P3HR-1 cells but not to erythrocytes, showing cell-binding behaviour similar to EBV. These two peptides induced antibodies recognising live EBV-infected cells. Interestingly, peptide-11521 (YVLQNAHQIACHFHSNGTDA) or antibodies induced by this peptide inhibited EBV-binding to B-lymphocytes, suggesting that this p140-region could be involved in EBV and B-lymphocyte interaction.

Several EBA-175 paralogues (EBA-140, EBA-165, EBA-175, EBA-181, and EBL-1) have been described among the Plasmodium falciparum malaria parasite proteins, which are important in the red blood cell (RBC) invasion process. EBA-181/JESEBL is a 181 kDa protein expressed in the late schizont stage and located in the micronemes; it belongs to the Plasmodium Duffy binding-like family and is able to interact with the erythrocyte surface. Here, we describe the synthesis of 78, 20-mer synthetic peptides derived from the reported EBA-181/JESEBL sequence and their ability to bind RBCs in receptor-ligand assays. Five peptides (numbered 30030, 30031, 30045, 30051, and 30060) displayed high specific binding to erythrocytes; their equilibrium binding parameters were then determined. These peptides interacted with 53 and 33 kDa receptor proteins on the erythrocyte surface, this binding being altered when RBCs were pretreated with enzymes. They were able to inhibit P. falciparum merozoite invasion of RBCs when tested in in vitro assays. According to these results, these five EBA-181/JESEBL high specific erythrocyte binding peptides, as well as the entire protein, were seen to be involved in the molecular machinery used by the parasite for invading RBCs. They are thus suggested as potential candidates in designing a multi-sub-unit vaccine able to combat the P. falciparum malaria parasite.

Plasmodium falciparum multi-stage proteins are involved in vital processes for parasite survival, which turns them into attractive targets for studies aimed at developing a fully effective antimalarial vaccine. MCP-1 and PfSPATR are both found in sporozoite and merozoite forms, and have been associated respectively with invasion of hepatocytes and red blood cells (RBCs). Binding assays with synthetic peptides derived from these two important proteins have enabled identifying those sequences binding with high specific activity (named High activity binding peptides-HABPs) to hepatoma-derived HepG2 cells and human RBCs. Twelve RBC HABPs were identified within the MCP-1 amino acid sequence, most of them in the C-terminal region. The MCP-1 HABPs 33387 and 33397 also presented high activity binding to HepG2 cells. PfSPATR presented four RBC HABPs and two HepG2 HABPs, but only one (32686) could bind to both cell types. RBC binding assays evidenced that binding of all HABPs was saturable and differentially affected by the enzymatic treatment of target cells. Moreover, all HABPs inhibited in vitro invasion of merozoites at 200 ?M and had particular structural features when analyzed by circular dichroism. The results suggest that these synthetic peptides capable of binding to the two P. falciparum target cells could be potentially included in the design of a multi-stage, subunit-based, chemically synthesized antimalarial vaccine. © 2008 Elsevier Masson SAS. All rights reserved.

An effective malarial vaccine must contain multiple immunogenic, protection-inducing epitopes able to block and destroy the P. falciparum malaria parasite, the most lethal form of this disease in the world. Our strategy has consisted in using conserved peptides blocking parasite binding to red blood cells; however, these peptides are non-immunogenic and non-protection-inducing. Modifying their critical residues can make them immunogenic. Such peptides induced antibody titers (determined by immunofluorescence antibody test, IFA) and made the latter reactive (determined by Western blot) and protection inducing against experimental challenge with a highly infective Aotus monkey adapted P. falciparum strain. Modified peptides also induce highly non-protective long-lasting antibody levels. Modifications performed might allow them to bind specifically to different HLA-DRbeta purified molecules. These immunological and biological activities are associated with modifications in their three-dimensional structure as determined by (1)H-NMR. It was found that modified, high non-protective long-lasting antibody level peptides bound to HLA-DR molecules from a different haplotype (to which immunogenic, protection-inducers bind) and had 4.6 +/- 1.4 A shorter distances between residues fitting into these molecules' Pocket 1 to Pocket 9, suggesting fitting into an inappropriate HLA-DR molecule. A multi-component, subunit-based, malarial vaccine is therefore feasible if modified peptides are suitably modified for an appropriate fit into the correct HLA-DRbeta1* molecule in order to form a proper MHC-II-peptide-TCR complex.

Interactions between the HLA-DRbeta1*0301 molecule and several occupying peptides obtained from computational substitutions made to the CLIP peptide are studied. The exploration was carried out using a vector composed of the first three terms of the multipolar expansion of the electrostatic field, namely, charge (q), dipole (d) and quadrupole (C). Comparisons between pocket-peptide interactions established that the binding pockets for this HLA molecule are ordered in terms of their importance for binding peptides, as follows: P1 >>> P4 > P6 > P7 > P9. A set of electrostatically distinct amino acids that determine interaction stability and specificity were identified for each pocket. The beta74R residue was especially identified as being the key amino acid mediating the occupying peptide binding for pocket 4; this residue has been recently associated with Graves' disease.

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.

Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine P. falciparum merozoite surface protein-10 (MSP-10) regions specifically binding to membrane surface receptors on human erythrocytes. Three MSP-10 protein High Activity Binding Peptides (HABPs) were identified, whose binding to erythrocytes became saturable and sensitive on being treated with neuraminidase, trypsin and chymotrypsin. Some of them specifically recognised a 50 kDa erythrocyte membrane protein. Some HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by 70%, suggesting that MSP-10 protein’s possible role in the invasion process probably functions by using similar mechanisms to those described for other MSP family antigens. In addition to above results, the high homology in amino-acid sequence and superimposition of both MSP-10, MSP-8 and MSP-1 EGF-like domains and HABPs 31132, 26373 and 5501 suggest that tridimensional structure could be playing an important role in the invasion process and in designing synthetic multi-stage anti-malarial vaccines.

Apical membrane antigen-1 (AMA-1) is an integral Plasmodium falciparum malaria parasite membrane protein. Peptides having high activity binding to human red blood cells have been identified in this protein. One of them, peptide 4325, with the amino acid sequence MIKSAFLPTGAFKADRYKSH, for which critical binding residues have already been defined (underlined), is conserved and non-immunogenic. Its critical binding residues were changed for amino acids having similar mass but different charge to change such immunological properties. These changes rendered some peptides immunogenic and protective against experimental challenge in Aotus monkeys. Three-dimensional models of peptide 4325 and its analogues, 20032 and 20034, were calculated from NMR experiments with distance geometry and restrained molecular dynamic methods. Non-immunogenic, non-protective peptide 4325 showed differences in its secondary structure with respect to protective, immunogenic peptides 20032 and 20034. Such data suggest that these modifications could have converted non-immunogenic peptides into immunogenic, protective ones, making them excellent candidates for a multi-component subunit synthetic malaria vaccine.