Changing ABRA protein peptide to fit into the HLA-DRbeta1*0301 molecule renders it protection-inducing

The Plasmodium falciparum acidic-basic repeat antigen represents a potential malarial vaccine candidate. One of this protein's high activity binding peptides, named 2150 ((161)KMNMLKENVDYIQKNQNLFK(180)), is conserved, non-immunogenic, and non-protection-inducing. Analogue peptides whose critical binding residues (in bold) were replaced by amino-acids having similar mass but different charge were synthesized and tested to try to modify such immunological properties. These analogues' HLA-DRbeta1* molecule binding ability were also studied in an attempt to explain their biological mechanisms and correlate binding capacity and immunological function with their three-dimensional structure determined by (1)H NMR. A 3(10) distorted helical structure was identified in protective and immunogenic peptide 24922 whilst alpha-helical structure was found for non-immunogenic, non-protective peptides having differences in alpha-helical position. The changes performed on immunogenic, protection-inducing peptide 24922 allowed it to bind specifically to the HLA-DRbeta1*0301 molecule, suggesting that these changes may lead to better interaction with the MHC Class II-peptide-TCR complex rendering it immunogenic and protective, thus suggesting a new way of developing multi-component, sub-unit-based anti-malarial vaccines.     

Shortening and modifying the 1513 MSP-1 peptide's alpha-helical region induces protection against malaria

Immunogenic and protective peptide sequences are of prime importance in the search for an anti-malarial vaccine. The MSP-1 conserved and semi-conserved sequences have been shown to contain red blood cell (RBC) membrane high affinity binding peptides (HABP). HABP 1513 sequence ((42)GYSLFQKEKMVLNEGTSGTA(61)), from this protein's N-terminal, has been shown to possess a T-epitope; however, it did not induce a humoral immune response or complete protection when evaluated in Aotus monkeys. Analogue peptides with critical binding residues replaced by amino acids with similar mass but different charge were synthesised and tested for immunogenicity and protectivity in monkey. NMR studies correlated structural behaviour with biological function. Non-immunogenic and non-protective 1513 native peptide presented a helical fragment between residues L(4) and E(14). C-terminal, 5-residue-shorter, non-immunogenic, non-protective peptide 17894 contained an alpha-helix from Q(6) to L(12) residues. Immunogenic and protective peptide 13946 presented a shorter alpha-helix between K(7) to N(13) residues. These data suggest that changing certain residues permits better peptide fit within the MHC class II-peptide-TCR complex, thus activating the immune system and inducing a protective immune response.     

High non-protective, long-lasting antibody levels in malaria are associated with haplotype shifting in MHC-peptide-TCR complex formation: a new mechanism for immune evasion

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.     

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.     

Protection against experimental P falciparum malaria is associated with short AMA-1 peptide analogue alpha-helical structures

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.     

Peptides inducing short-lived antibody responses against Plasmodium falciparum malaria have shorter structures and are read in a different MHC II functional register

The search for a rational method of developing an antimalarial vaccine (malaria caused by Plasmodium falciparum) consists of blocking receptor-ligand interaction. Conserved peptides derived from proteins involved in invasion and having strong red blood cell binding ability have thus been identified; immunization studies using Aotus monkeys revealed that these peptides were neither immunogenic nor protection-inducing. Some of these peptides induced long-lasting and very high antibody titers and protection when their critical red blood cell binding residues were replaced to change their immunological properties. Others induced short-lived antibodies that were not associated with inducing protection. The three-dimensional structure of the short-lived antibody-inducing peptide was determined by (1)H NMR. Their HLA-DRbeta1* molecule binding ability was also determined to ascertain the relationship among three-dimensional structure, their ability to bind to major histocompatibility complex class II molecules (MHC II), and possible short-lived antibody production. These short-lived antibody-inducing peptides were 6.8 +/- 0.5 A shorter between those residues theoretically coming into contact with pocket 1 and pocket 9 of HLA-DRbeta1* molecules to which they bind than immunogenic and protection-inducing peptides. These more compact alpha-helical structures suggest that these short-lived antibody-inducing peptides could have a structure more similar to those of native peptides than immunogenic and protective ones. Such shortening was associated with a shift in HLA-DRbeta1* molecule binding and a consequent shift in functional register reading, mainly by alleles of the same haplotype when compared with immunogenic protection-inducing HABPs, suggesting an imperfect and different conformation of the MHC II peptide-TCR complex.     

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.     

Modified merozoite surface protein-1 peptides with short alpha helical regions are associated with inducing protection against malaria.

The merozoite surface protein-1 represents a prime candidate for development of a malaria vaccine. Merozoite surface 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 MLNISQHQCVKKQCPQNS) of the 19-kDa molecular mass fragment of merozoite 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 alpha-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.     

Attaching histidine-tagged peptides and proteins to lipid-based carriers through use of metal-ion-chelating lipids

The therapeutic potential of selected peptides and proteins is enormous, with applications ranging from use as therapeutic vaccines, as modulators of intracellular signaling pathways and as highly selective agents capable of recognizing unique extracellular targets. We have been pursuing development of hybrid lipid-based carrier formulations designed to take advantage of the therapeutic benefits of peptides selected for their ability to act in a complementary fashion with the carrier system. In this regard, it is critical to have simple and versatile methods to promote and control the binding of diverse peptides to a broad range of carrier formulations. As demonstrated here, recombinant proteins and synthetic peptides containing poly-histidine residues (4 to 10) can be specifically bound to liposomes containing a metal-ion-chelating lipid, DOGS-NTA-Ni. The potential of this approach is demonstrated using two functional peptides, AntpHD-Cw3 (applications for vaccine production) and AHNP (specificity for Her-2 expressing cells).     

6746 SERA peptide analogues immunogenicity and protective efficacy against malaria is associated with short alpha helix formation: malaria protection associated with peptides alpha helix shortening

Erythrocyte high activity binding peptides (HABPs) have been identified for the Plasmodium falciparum serine repeat antigen (SERA). HABP 6746, located in this protein's 50 kDa fragment had its critical binding residues replaced by amino acids having similar mass but different charge to change their immunologic properties. This peptide analogues were used to immunize Aotus monkeys that were challenged later on with a virulent P. falciparum strain to determine their protective efficacy. A shortening in alpha helix structure was found in the immunogenic and protective ones when their secondary structure was analyzed by NMR, to correlate their structure with their immunologic properties. These data, together with results from previous studies, suggest that this shortening in HABP helical configuration may lead to better fitting with immune system molecules, rendering them immunogenic and protective and therefore making them excellent candidates for consideration as components of a subunit based multicomponent synthetic vaccine against malaria.     

Pooled-Peptide Epitope Mapping Strategies Are Efficient and Highly Sensitive: An Evaluation of Methods for Identifying Human T Cell Epitope Specificities in Large-Scale HIV Vaccine Efficacy Trials

The interferon gamma, enzyme-linked immunospot (IFN-γ ELISpot) assay is widely used to identify viral antigen-specific T cells is frequently employed to quantify T cell responses in HIV vaccine studies. It can be used to define T cell epitope specificities using panels of peptide antigens, but with sample and cost constraints there is a critical need to improve the efficiency of epitope mapping for large and variable pathogens. We evaluated two epitope mapping strategies, based on group testing, for their ability to identify vaccine-induced T-cells from participants in the Step HIV-1 vaccine efficacy trial, and compared the findings to an approach of assaying each peptide individually. The group testing strategies reduced the number of assays required by >7-fold without significantly altering the accuracy of T-cell breadth estimates. Assays of small pools containing 7–30 peptides were highly sensitive and effective at detecting single positive peptides as well as summating responses to multiple peptides. Also, assays with a single 15-mer peptide, containing an identified epitope, did not always elicit a response providing validation that 15-mer peptides are not optimal antigens for detecting CD8+ T cells. Our findings further validate pooling-based epitope mapping strategies, which are critical for characterizing vaccine-induced T-cell responses and more broadly for informing iterative vaccine design. We also show ways to improve their application with computational peptide:MHC binding predictors that can accurately identify the optimal epitope within a 15-mer peptide and within a pool of 15-mer peptides.     

Peptide inhibitors of the malaria surface protein, apical membrane antigen 1: identification of key binding residues

Apical membrane antigen 1 (AMA1) is essential for malaria parasite invasion of erythrocytes and is therefore an attractive target for drug development. Peptides that bind AMA1 have been identified from random peptide libraries expressed on the surface of phage. Of these, R1, which binds to a hydrophobic ligand binding site on AMA1, was a particularly potent inhibitor of parasite invasion of erythrocytes in vitro. The solution structure of R1 contains a turn-like conformation between residues 5-10. Here the importance of residues in this turn-like structure for binding to AMA1 was examined by site-directed mutagenesis and NMR spectroscopy. The peptide was expressed as a fusion protein following replacement of Met16 by Leu in order to accommodate cyanogen bromide cleavage. This modified peptide (R2) displayed the same affinity for AMA1 as R1, showing that the identity of the side chain at position 16 was not critical for binding. Substitution of Phe5, Pro7, Leu8, and Phe9 with alanine led to significant (7.5- to >350-fold) decreases in affinity for AMA1. Comparison of backbone amide and C(α) H chemical shifts for these R2 analogues with corresponding values for R2 showed no significant changes, with the exception of R2(P7A), where slightly larger differences were observed, particularly for residues flanking position 7. The absence of significant changes in the secondary chemical shifts suggests that these mutations had little effect on the solution conformation of R2. The identification of a nonpolar region of these peptides containing residues essential for AMA1 binding establishes a basis for the design of anti-malarial drugs based on R1 mimetics.     

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.     

The cell adhesion and proliferation activities of a peptide derived from human tenascin-C are dependent on two Ile residues

A tenascin-C derived peptide (TNIIIA2 peptide, 1) stimulated β1 integrin-mediated cell adhesion via binding to syndecan-4. Ala-substituted peptides were synthesized to understand the structure-activity relationship. Peptides in which basic amino acids were substituted showed reduced cell adhesion activity, but their proliferation activities were similar to or higher than those mediated by peptide 1. In contrast, peptides in which the Ile residues of peptide 1 were replaced were inactive, indicating that the Ile residues are critical for the peptide's activity. CD analysis suggested that the Ile residues are necessary for the formation of a specific conformation required for binding to syndecan-4.     

Functional fine-mapping and molecular modeling of a conserved loop epitope of the measles virus hemagglutinin protein.

Neutralizing and protective monoclonal antibodies (mAbs) were used to fine-map the highly conserved hemagglutinin noose epitope (H379-410, HNE) of the measles virus. Short peptides mimicking this epitope were previously shown to induce virus-neutralizing antibodies [El Kasmi et al. (2000) J. Gen. Virol.81, 729-735]. The epitope contains three cysteine residues, two of which (Cys386 and Cys394) form a disulfide bridge critical for antibody binding. Substitution and truncation analogues revealed four residues critical for binding (Lys387, Gly388, Gln391 and Glu395) and suggested the binding motif X7C[KR]GX[AINQ]QX2CEX5 for three distinct protective mAbs. This motif was found in more than 90% of the wild-type viruses. An independent molecular model of the core epitope predicted an amphiphilic loop displaying a remarkably stable and rigid loop conformation. The three hydrophilic contact residues Lys387, Gln391 and Glu395 pointed on the virus towards the solvent-exposed side of the planar loop and the permissive hydrophobic residues Ile390, Ala392 and Leu393 towards the solvent-hidden side of the loop, precluding antibody binding. The high affinity (Kd = 7.60 nm) of the mAb BH216 for the peptide suggests a high structural resemblance of the peptide with the natural epitope and indicates that most interactions with the protein are also contributed by the peptide. Improved peptides designed on the basis of these findings induced sera that crossreacted with the native measles virus hemagglutinin protein, providing important information about a lead structure for the design of more stable antigens of a synthetic or recombinant subunit vaccine.     

Binding modes of Bcl-2 homology 3 (BH3) peptides with anti-apoptotic protein A1 and redesign of peptide inhibitors: a computational study

The interaction between protein and peptide ligand is a challenging problem in molecular biology and drug design. The binding of the Bcl-2 homology 3 (BH3) peptide to the anti-apoptotic protein A1 was revealed as a critical step in the regulation of apoptosis. These BH3 peptides hold high structural similarity, but are diverse in their regulation abilities. Based on molecular simulations and MM-P(G)BSA methods, this work presented a detailed analysis on binding mechanism of the BH3 peptides derived from PUMA and BMF. Residue-level energy decomposition showed that the core regions of BH3 peptides maintain in stable helical conformations and the four conserved hydrophobic residues together with an invariant aspartic acid contribute the major driving force for binding, whereas their two terminal segments exhibit obvious flexibility and various binding modes. Such kind of behavior was suggested as the reason for binding diversity and selectivity of BH3 peptides. As a further step, several BH3-mimetic peptides have been redesigned by computational mutation. Those new peptides showed not only stronger affinities when binding to protein A1, as well transferable binding patterns at some specific positions. A long-range coupling effect was disclosed for BH3 peptides, side-chain orientation and binding contribution of terminal residues were even affected by mutations at large sequence interval. Overall, this work reports that the binding modes of BH3 peptides are primarily dependent on its two terminal segments. The computational methods applied herein are also demonstrated to be of great assistance in the rational design of peptide inhibitors.     

Among all human T-cell leukemia virus type 1 proteins, tax, polymerase, and envelope proteins are predicted as preferential targets for the HLA-A2-restricted cytotoxic T-cell response.

The human T-cell leukemia virus type 1 (HTLV-1) is a human retrovirus associated with two diseases for which no successful treatment is yet available; the development of a vaccine is therefore an important issue. Since HTLV-1 is a persistent virus, an efficient vaccine will probably require a cytotoxic T-lymphocyte (CTL) response in addition to the production of antibodies. To identify potential CTL epitopes, we have selected, within all of the HTLV-1 proteins, nonapeptides containing anchor residues required for association with HLA-A2 molecules (residues at positions 2 and 9), which is the most frequently occurring A allele in all human populations. A set of 111 peptides was synthetized and tested in vitro in two assembly assays using processing-defective T2 cells. Anchor motifs selected were those containing two major anchor residues (L2/M2/12-V9/L9/I9) (one letter amino-acid code) and those including tolerated anchor residues (V2/A2/T2 and/or A9/M9/T9). The analysis of the binding capacity of the peptides confirms the high efficiency of the L2-V9 anchor motif and shows that a systematic research of potential binding peptides should exclude peptides containing known detrimental residues rather than select only peptides with known favored residues. We show that 39 peptides representative of all the HTLV-1 proteins are able to bind to HLA-A2 molecules. Strong binder peptides which are very likely good CTL epitopes were identified in three HTLV-1 proteins, Tax, envelope, and polymerase. Three of the strong binder peptides correspond to previously described HLA-A2-restricted CTL epitopes in the Tax protein, and two others are localized in a domain of the viral envelope recognized by natural neutralizing antibodies. This latter result has important implications for the development of an anti-HTLV-1 vaccine.     

Identification of cell-binding sites on the Laminin alpha 5 N-terminal domain by site-directed mutagenesis

The newly discovered laminin alpha(5) chain is a multidomain, extracellular matrix protein implicated in various biological functions such as the development of blood vessels and nerves. The N-terminal globular domain of the laminin alpha chains has an important role for biological activities through interactions with cell surface receptors. In this study, we identified residues that are critical for cell binding within the laminin alpha(5) N-terminal globular domain VI (approximately 270 residues) using site-directed mutagenesis and synthetic peptides. A recombinant protein of domain VI and the first four epidermal growth factor-like repeats of domain V, generated in a mammalian expression system, was highly active for HT-1080 cell binding, while a recombinant protein consisting of only the epidermal growth factor-like repeats showed no cell binding. By competition analysis with synthetic peptides for cell binding, we identified two sequences: S2, (123)GQVFHVAYVLIKF(135) and S6, (225)RDFTKATNIRLRFLR(239), within domain VI that inhibited cell binding to domain VI. Alanine substitution mutagenesis indicated that four residues (Tyr(130), Arg(225), Lys(229), and Arg(239)) within these two sequences are crucial for cell binding. Real-time heparin-binding kinetics of the domain VI mutants analyzed by surface plasmon resonance indicated that Arg(239) of S6 was critical for both heparin and cell binding. In addition, cell binding to domain VI was inhibited by heparin/heparan sulfate, which suggests an overlap of cell and heparin-binding sites. Furthermore, inhibition studies using integrin subunit monoclonal antibodies showed that integrin alpha(3)beta(1) was a major receptor for domain VI binding. Our results provide evidence that two sites spaced about 90 residues apart within the laminin alpha(5) chain N-terminal globular domain VI are critical for cell surface receptor binding.     

Goodpasture syndrome. Localization of the epitope for the autoantibodies to the carboxyl-terminal region of the alpha 3(IV) chain of basement membrane collagen.

The autoantibodies of patients with Goodpasture syndrome are primarily targeted to the noncollagenous (NC1) domain of the alpha 3(IV) chain of basement membrane collagen (Saus, J., Wieslander, J., Langeveld, J. P. M., Quinones, S., and Hudson, B. G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the location of the Goodpasture epitope in human alpha 3NC1 was determined, and its structure was partially characterized. This was achieved by identification of regions of alpha 3NC1 which are candidates for the epitope and which are structurally unique among the five known homologous NC1 domains (alpha 1-alpha 5); amino acids that are critical for Goodpasture antibody binding, by selective chemical modifications; and regions that are critical for Goodpasture antibody binding, by synthesis of 12 alpha 3NC1 peptides and measurement of their antibody binding capacity. The carboxyl-terminal region, residues 198-233, was identified as the most likely region for the epitope. By experiment, lysine and cysteine were identified as critical amino acids for antibody binding. Three synthetic peptides were found to inhibit Goodpasture antibody binding to alpha 3NC1 markedly: a 36-mer (residues 198-233), a 12-mer (residues 222-233), and a 5-mer (residues 229-233). Together, these results strongly indicate that the Goodpasture epitope is localized to the carboxyl-terminal region of alpha 3NC1, encompassing residues 198-233 as the primary antibody interaction site and that its structure is discontinuous. These findings provide a conceptual framework for future studies to elucidate a more complete epitope structure by sequential replacement of residues encompassing the epitope using cDNA expression products and peptides synthesized chemically.