Modifying RESA protein peptide 6671 to fit into HLA-DRbeta1* pockets induces protection against malaria

6671 is a non-immunogenic, conserved high activity red blood cell binding peptide located between residues 141 and 160 of the Plasmodium falciparum RESA protein. This peptide's critical red blood cell (RBC) binding residues have been replaced by amino acids having similar mass but different charge to change their immunologic properties. Three analogues (two of them immunogenic and protective and one immunogenic) were studied by purified HLA-DRbeta1* binding and NMR to correlate their structure with their immunological properties. Native peptide 6671 had a very flexible beta-sheet structure, whilst its immunogenic, protective, and non-protective peptide analogues presented an alpha-helical structure having different locations and lengths. These changes in peptide structure facilitated their fitting into HLA-DRbeta1* molecules. This paper shows for the first time how modifications performed on RESA protein non-immunogenic, non-protectogenic peptides impose a configuration allowing them to fit perfectly into the MHC II-TCR complex, in turn leading to appropriate activation of the immune system.     

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.     

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.     

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.     

Orientating peptide residues and increasing the distance between pockets to enable fitting into MHC-TCR complex determine protection against malaria

The erythrocyte binding antigen EBA-175 is a 175-kDa Plasmodium falciparum protein, which has been shown to be involved in the process of invasion of erythrocytes. It has been found that conserved peptide 1818 belonging to this protein has high red blood cell binding capacity and plays an important role in the invasion process. This peptide is neither immunogenic nor protective. Peptide 1818 analogues had some of their previously recognized critical red blood cell binding residues substituted for amino acids having similar volume or mass but different polarity to make them fit into HLA-DRbeta(1)*1101 molecules; these 1818 peptide analogues were then synthesized and inoculated into Aotus nancymaae monkeys, generating different immunogenic and/or protective immune responses. Short structures such as 3(10)-helix, classical, or distorted type-III beta-turns were found in the immunogenic and protective peptides once the secondary structure had been analyzed by NMR and its structure correlated with its immunological properties. These data suggest that peptide flexibility may lead to better fitting into immune system molecules, therefore making them excellent candidates for consideration as components of a subunit-based, multicomponent synthetic antimalarial vaccine.     

Fitting modified HRP-I peptide analogue 3D structure into HLA-DR molecules induces protection against Plasmodium falciparum malaria

Conserved, high-activity, red blood cell binding malaria peptide 6786, from the HRP-I protein, having a random 3D structure as determined by 1H-NMR, was non-immunogenic and non-protection inducing when used as an immunogen in Aotus monkeys. Modifications made in its amino acid sequence were thus performed to render it immunogenic and protection inducing. Non-immunogenic, non-protection inducing modified peptide 13852 presented A2-H8 and K14-L18 helix fragments. Immunogenic, non-protection inducing modified peptide 23428 presented a short, displaced helix in a different region, whilst immunogenic, protection inducing peptide 24224 had 2 displaced helical regions towards the central region giving more flexibility to its N- and C-terminals. Immunogenic and protection inducing peptides bound with high affinity to HLA-DRB1* 0301 whilst others did not bind to any HLA-DRB1* purified molecule. Structural modifications may thus lead to inducing immunogenicity and protection associated with their capacity to bind specifically to purified HLA-DRB1* molecules, suggesting a new way of developing multi-component, subunit-based malarial vaccines.     

Identification of immunogenic MHC class II Tyrosinase-derived peptides using HLA-DR1 and HLA-DR4 transgenic mice

The immunogenicity of "novel" MART-1 and Tyrosinase class-II peptides was assessed in transgenic mice. Tyrosinase(141-161) peptide was found to be immunogenic and endogenously processed in the HLA-DRbeta1*0101 and HLA-DRbeta1*0401 transgenic mice with peptide specific production of IFNgamma or IL-5 respectively. The MART-1(29-43) peptide was only found immunogenic in HLA-DRbeta1*0101 mice.     

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.     

NMR structure of Plasmodium falciparum malaria peptide correlates with protective immunity

Apical membrane antigen-1 is an integral Plasmodium falciparum malaria parasite membrane protein. High activity binding peptides (HABPs) to human red blood cells (RBCs) have been identified in this protein. One of them (peptide 4313), for which critical binding residues have already been defined, is conserved and nonimmunogenic. Its critical binding residues were changed for amino acids having similar mass but different charge to change such immunological properties; these changes generated peptide analogues. Some of these peptide analogues became immunogenic and protective in Aotus monkeys.Three-dimensional models of peptide 4313 and three analogues having different immune characteristics, were calculated from nuclear magnetic resonance (NMR) experiments with distance geometry and restrained molecular dynamic methods. All peptides contained a beta-turn structure spanning amino acids 7 to 10, except randomly structured 4313. When analysing dihedral angle phi and psi values, distorted type III or III' turns were identified in the protective and/or immunogenic peptides, whilst classical type III turns were found for the nonimmunogenic nonprotective peptides. This data shows that some structural modifications may lead to induction of immunogenicity and/or protection, suggesting a new way to develop multicomponent, subunit-based malarial vaccines.     

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.     

Immunogenicity and protectivity of Plasmodium falciparum EBA-175 peptide and its analog is associated with alpha-helical region shortening and displacement

EBA-175 protein is used as a ligand in the binding of P. falciparum to red blood cells (RBCs). Evidence shows that the conserved peptide 1779 from this protein (with high red blood cell binding ability and known critical erythrocyte binding residues) plays an important role in the invasion process. This peptide is neither immunogenic nor protective; analogs having critical residues replaced by amino acids with similar volume or mass but different polarity were synthesized and inoculated into Aotus monkeys, and elicited different immunogenic and protective responses. Nuclear Magnetic Resonance (1H-NMR) studies revealed that peptide analog 21696 (non-immunogenic and non-protective) presents a large helical fragment, that the peptide 14012 (immunogenic and non-protective) helical fragment is smaller, while the peptide 22812 (immunogenic and protective) alpha-helix is shorter in a different region and possesses greater flexibility at its N-terminus. The presence of methionine residues could affect the structural stability of peptide 22812 and ultimately its immunological response. Our results suggest a new strategy for designing a new malaria multi-component subunit-based vaccine.     

Alpha helix shortening in 1522 MSP-1 conserved peptide analogs is associated with immunogenicity and protection against P. falciparum malaria

1522 is a nonimmunogenic conserved high-activity binding peptide (HABP) belonging to Plasmodium falciparum MSP-1 protein N-terminal fragment. The key amino acids in binding to red blood cells (RBC) were identified and replaced by others having similar mass but different charge. Because conserved HABPs are not antigenic nor immunogenic, immunogenicity and protectivity studies were then conducted on them in the Aotus monkey. 1H-NMR studies included the lead peptide 1522 as well as the analogs 9782, 13446, 13448, and 13442 to relate their structure to biological function. All the peptides presented alpha-helical structure, with differences observed in helix location and extension. The nonprotective 1522 peptide was totally helical from the N- to the C-terminus, very similar to nonprotective 13442 and 13448 peptides whose extension was almost totally helical. The 9782 and 13446 protective peptides, however, possessed a shorter helical region where modified critical binding residues were not included. A more flexible region was generated at the C-terminus in those peptides with a shorter helical region, leading to a greater number of conformers. These data suggest that peptide flexibility results in increased interaction with immune system molecules, generating protective immunity.     

Human leukocyte antigen (HLA) class I restricted epitope discovery in yellow fewer and dengue viruses: importance of HLA binding strength

Epitopes from all available full-length sequences of yellow fever virus (YFV) and dengue fever virus (DENV) restricted by Human Leukocyte Antigen class I (HLA-I) alleles covering 12 HLA-I supertypes were predicted using the NetCTL algorithm. A subset of 179 predicted YFV and 158 predicted DENV epitopes were selected using the EpiSelect algorithm to allow for optimal coverage of viral strains. The selected predicted epitopes were synthesized and approximately 75% were found to bind the predicted restricting HLA molecule with an affinity, K(D), stronger than 500 nM. The immunogenicity of 25 HLA-A*02:01, 28 HLA-A*24:02 and 28 HLA-B*07:02 binding peptides was tested in three HLA-transgenic mice models and led to the identification of 17 HLA-A*02:01, 4 HLA-A*2402 and 4 HLA-B*07:02 immunogenic peptides. The immunogenic peptides bound HLA significantly stronger than the non-immunogenic peptides. All except one of the immunogenic peptides had K(D) below 100 nM and the peptides with K(D) below 5 nM were more likely to be immunogenic. In addition, all the immunogenic peptides that were identified as having a high functional avidity had K(D) below 20 nM. A*02:01 transgenic mice were also inoculated twice with the 17DD YFV vaccine strain. Three of the YFV A*02:01 restricted peptides activated T-cells from the infected mice in vitro. All three peptides that elicited responses had an HLA binding affinity of 2 nM or less. The results indicate the importance of the strength of HLA binding in shaping the immune response.     

1H-NMR structures of the Plasmodium falciparum 1758 erythrocyte binding peptide analogues and protection against malaria

A conserved high activity erythrocyte binding peptide (HAEBP) derived from the 175-erythrocyte binding antigen (EBA-175), coded 1758, was synthesized and analyzed for antigenic and protective activities in Aotus monkeys, together with several of its analogues. Conformational analysis by 1H Nuclear Magnetic Resonance in TFE-solution was done for some of them, as well as the 1758 parent peptide. We show that the conserved 1758 HAEBP (being neither immunogenic nor protective) has an alpha helical structure, whilst its analogues contain beta-turn structures. The 13790 peptide (highly immunogenic and protective for some monkeys) shows a type I beta-turn structure distorted in psi(i + 1) psi(i + 2) angles, whilst immunogenic and non-protective (as well as the non-immunogenic and non-protective peptides) have type III' beta-turns. An understanding of native peptide's correlation with altered peptide three-dimensional structure and resulting immunogenicity and protective activity may lead to a more rational design of multi-antigenic, multi-stage P. falciparum subunit based malaria vaccines.     

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.     

A theoretical analysis of HLA-DRbeta1*0301-CLIP complex using the first three multipolar moments of the electrostatic field

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.     

Electrostatic potential as a tool to understand interactions between malaria vaccine candidate peptides and MHC II molecules

One of the most important problems in vaccine development consists in understanding receptor–ligand interactions between Class II Major Histocompatibility Complex molecules (MHC II) and antigenic peptides involved in inducing an appropriate immune response. In this study, we used X-ray crystallography structural data provided by the HLA-DRβ1*0301–CLIP peptide interaction to compare native non-immunogenic and specifically-modified immunogenic peptides derived from the malarial SALSA protein, by analyzing molecular electrostatic potential surfaces on the most important regions of the peptide binding groove (Pockets 1, 4, 6 and 9). Important differences were found on the electrostatic potential induced by these peptides, particularly in MHC II conserved residues: Qα9, Sα53, Nα62, Nα69, Yβ30, Yβ60, Wβ61, Qβ70, Kβ71 and Vβ86, the same ones involved in establishing hydrogen bonds between Class II molecule-peptide and the recognition by T cell receptor, it correlating well with the change in their immunological properties.The results clearly suggest that modifications done on the electrostatic potential of these amino acids could favor the induction of different immune responses and therefore, their identification could allow modifying peptides a priori and in silico, so as to render them into immunogenic and protection-inducers and hence suitable components of a chemically-synthesized, multi-antigenic, minimal subunit based vaccine.     

A comparative study of MHC Class-II HLA-DRbeta1*0401-Col II and HLA-DRbeta1*0101-HA complexes: a theoretical point of view

A study was performed on the HLA-DRbeta1*0401-collagen II peptide complex using the computation of electronic multipolar variables proposed by us previously. Furthermore, these results were compared with those obtained for the HLA-DRbeta1*0101-haemaglutinin peptide complex studied by us with the same tools, confirming that Pocket 1 for this new complex is also the most important pocket for the interaction between the presenting molecule and the presented peptide. The pocket hierarchy established for HLA-DRbeta1*0401 allele was P1 > P9 approximately P7 > P6 > P4, whilst a P1 > P4 > P9 approximately P7>P6 pocket hierarchy was found for HLA-DRbeta1*0101, showing how the relative importance of the pockets distinguishes the two alleles. There are high correlation levels with experimental results (when possible), again confirming the validity of using calculated values for electronic multipolar variables as a useful tool for studying interactions between immune system molecules and peptides.     

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.     

Analysis of a Plasmodium falciparum EBA-175 peptide with high binding capacity to erythrocytes and their analogues using 1H NMR

A 175-erythrocyte-binding protein (EBA-175) conserved high-activity binding peptide (HABP), called 1783 (nonimmunogenic, nonprotective against Plasmodium falciparum malaria), was analyzed for antigenic and protective activity in Aotus monkeys, together with several of its analogues. 1H NMR studies of peptides 17912, 14016, and 22814 allowed their structure to be related to their biological function. These peptides showed helical regions having differences in their position and length. Nonimmunogenic, nonprotective peptides 1783 and 17912 showed an extensive helical region, while the 22814 immunogenic protective peptide's alpha-helix was found in the N-terminal region. This suggests that the more flexible C-terminal region will allow better interaction between these peptides and immune system molecules as well as relating these peptides' three-dimensional structure to their immunogenicity and protective activity, thus leading to a more rational development of the new malaria multicomponent vaccine.