High Activity Binding Peptides Research Articles

Plasmodium falciparum rhoptry neck protein 4 has conserved regions mediating interactions with receptors on human erythrocytes and hepatocyte membrane

Plasmodium falciparum-related malaria represents a serious worldwide public health problem due to its high mortality rates. P. falciparum expresses rhoptry neck protein 4 (PfRON4) in merozoite and sporozoite rhoptries, it participates in tight junction-TJ formation via the AMA-1/RON complex and is refractory to complete genetic deletion. Despite this, which PfRON4 key regions interact with host cells remain unknown; such information would be useful for combating falciparum malaria. Thirty-two RON4 conserved region-derived peptides were chemically synthesised for determining and characterising PfRON4 regions having high host cell binding affinity (high activity binding peptides or HABPs). Receptor-ligand interaction/binding assays determined their specific binding capability, the nature of their receptors and their ability to inhibit in vitro parasite invasion. Peptides 42477, 42479, 42480, 42505 and 42513 had greater than 2% erythrocyte binding activity, whilst peptides 42477 and 42480 specifically bound to HepG2 membrane, both of them having micromolar and submicromolar range dissociation constants (Kd). Cell-peptide interaction was sensitive to treating erythrocytes with trypsin and/or chymotrypsin and HepG2 with heparinase I and chondroitinase ABC, suggesting protein-type (erythrocyte) and heparin and/or chondroitin sulphate proteoglycan receptors (HepG2) for PfRON4. Erythrocyte invasion inhibition assays confirmed HABPs’ importance during merozoite invasion. PfRON4 800–819 (42477) and 860–879 (42480) regions specifically interacted with host cells, thereby supporting their inclusion in a subunit-based, multi-antigen, multistage anti-malarial vaccine.

The molecular basis for peptide-based antimalarial vaccine development targeting erythrocyte invasion by P. falciparum

This work describes a methodology for developing a minimal, subunit-based, multi-epitope, multi-stage, chemically-synthesised, anti-Plasmodium falciparum malaria vaccine. Some modified high activity binding peptides (mHABPs) derived from functionally relevant P. falciparum MSP, RH5 and AMA-1 conserved amino acid regions (cHABPs) for parasite binding to and invasion of red blood cells (RBC) were selected. They were highly immunogenic as assessed by indirect immunofluorescence (IFA) and Western blot (WB) assays and protective immune response-inducers against malarial challenge in the Aotus monkey experimental model. NetMHCIIpan 4.0 was used for predicting peptide-Aotus/human major histocompatibility class II (MHCII) binding affinity in silico due to the similarity between Aotus and human immune system molecules; ∼50% of Aotus MHCII allele molecules have a counterpart in the human immune system, being Aotus-specific, whilst others enabled recognition of their human counterparts. Some peptides’ 1H-NMR-assessed structural conformation was determined to explain residue modifications in mHABPs inducing secondary structure changes. These directly influenced immunological behaviour, thereby highlighting the relationship with MHCII antigen presentation. The data obtained in such functional, immunological, structural and predictive approach suggested that some of these peptides could be excellent components of a fully-protective antimalarial vaccine.

Plasmodium falciparum Blood Stage Antimalarial Vaccines: An Analysis of Ongoing Clinical Trials and New Perspectives Related to Synthetic Vaccines.

Plasmodium falciparum malaria is a disease causing high morbidity and mortality rates worldwide, mainly in sub-Saharan Africa. Candidates have been identified for vaccines targeting the parasite’s blood stage; this stage is important in the development of symptoms and clinical complications. However, no vaccine that can directly affect morbidity and mortality rates is currently available. This review analyzes the formulation, methodological design, and results of active clinical trials for merozoite-stage vaccines, regarding their safety profile, immunological response (phase Ia/Ib), and protective efficacy levels (phase II). Most vaccine candidates are in phase I trials and have had an acceptable safety profile. GMZ2 has made the greatest progress in clinical trials; its efficacy has been 14% in children aged less than 5 years in a phase IIb trial. Most of the available candidates that have shown strong immunogenicity and that have been tested for their protective efficacy have provided good results when challenged with a homologous parasite strain; however, their efficacy has dropped when they have been exposed to a heterologous strain. In view of these vaccines’ unpromising results, an alternative approach for selecting new candidates is needed; such line of work should be focused on how to increase an immune response induced against the highly conserved (i.e., common to all strains), functionally relevant, protein regions that the parasite uses to invade target cells. Despite binding regions tending to be conserved, they are usually poorly antigenic and/or immunogenic, being frequently discarded as vaccine candidates when the conventional immunological approach is followed. The Fundación Instituto de Inmunología de Colombia (FIDIC) has developed a logical and rational methodology based on including conserved high-activity binding peptides (cHABPs) from the main P. falciparum biologically functional proteins involved in red blood cell (RBC) invasion. Once appropriately modified (mHABPs), these minimal, subunit-based, chemically synthesized peptides can be used in a system covering the human immune system’s main genetic variables (the human leukocyte antigen HLA-DR isotype) inducing a suitable, immunogenic, and protective immune response in most of the world’s populations.

Towards designing a synthetic antituberculosis vaccine: The Rv3587c peptide inhibits mycobacterial entry to host cells

Mycobacterium tuberculosis is considered one of the most successful pathogens in the history of mankind, having caused 1.7 million deaths in 2016. The amount of resistant and extensively resistant strains has increased; BCG has been the only vaccine to be produced in more than 100 years though it is still unable to prevent the disease’s most disseminated form in adults; pulmonary tuberculosis. The search is thus still on-going for candidate antigens for an antituberculosis vaccine. This paper reports the use of a logical and rational methodology for finding such antigens, this time as peptides derived from the Rv3587c membrane protein. Bioinformatics tools were used for predicting mycobacterial surface location and Rv3587c protein structure whilst circular dichroism was used for determining its peptides’ secondary structure. Receptor-ligand assays identified 4 high activity binding peptides (HABPs) binding specifically to A549 alveolar epithelial cells and U937 monocyte-derived macrophages, covering the region between amino acids 116 and 193. Their capability for inhibiting Mtb H37Rv invasion was evaluated. The recognition of antibodies from individuals suffering active and latent tuberculosis and from healthy individuals was observed in HABPs capable of avoiding mycobacterial entry to host cells. The results showed that 8 HABPs inhibited such invasion, two of them being common for both cell lines: 39265 (155VLAAYVYSLDNKRLWSNLDT173) and 39266 (174APSNETLVKTFSPGEQVTTY192). Peptide 39265 was the least recognised by antibodies from the individuals’ sera evaluated in each group. According to the model proposed by FIDIC regarding synthetic vaccine development, peptide 39265 has become a candidate antigen for an antituberculosis vaccine.

Conserved Binding Regions Provide the Clue for Peptide-Based Vaccine Development: A Chemical Perspective.

Synthetic peptides have become invaluable biomedical research and medicinal chemistry tools for studying functional roles, i.e., binding or proteolytic activity, naturally-occurring regions’ immunogenicity in proteins and developing therapeutic agents and vaccines. Synthetic peptides can mimic protein sites; their structure and function can be easily modulated by specific amino acid replacement. They have major advantages, i.e., they are cheap, easily-produced and chemically stable, lack infectious and secondary adverse reactions and can induce immune responses via T- and B-cell epitopes. Our group has previously shown that using synthetic peptides and adopting a functional approach has led to identifying Plasmodium falciparum conserved regions binding to host cells. Conserved high activity binding peptides’ (cHABPs) physicochemical, structural and immunological characteristics have been taken into account for properly modifying and converting them into highly immunogenic, protection-inducing peptides (mHABPs) in the experimental Aotus monkey model. This article describes stereo–electron and topochemical characteristics regarding major histocompatibility complex (MHC)-mHABP-T-cell receptor (TCR) complex formation. Some mHABPs in this complex inducing long-lasting, protective immunity have been named immune protection-inducing protein structures (IMPIPS), forming the subunit components in chemically synthesized vaccines. This manuscript summarizes this particular field and adds our recent findings concerning intramolecular interactions (H-bonds or π-interactions) enabling proper IMPIPS structure as well as the peripheral flanking residues (PFR) to stabilize the MHCII-IMPIPS-TCR interaction, aimed at inducing long-lasting, protective immunological memory.

Identifying and characterising PPE7 (Rv0354c) high activity binding peptides and their role in inhibiting cell invasion.

This study was aimed at characterising the PPE7 protein from the PE/PPE protein family. The presence and transcription of the rv0354c gene in the Mycobacterium tuberculosis complex was determined and the subcellular localisation of the PPE7 protein on mycobacterial membrane was confirmed by immunoelectron microscope. Two peptides were identified as having high binding activity (HABPs) and were tested in vitro regarding the invasion of Mycobacterium tuberculosis H37Rv. HABP 39224 inhibited invasion in A549 epithelial cells and U937 macrophages by more than 50%, whilst HABP 39225 inhibited invasion by 40% in U937 cells. HABP 39224, located in the protein's C-terminal region, has a completely conserved amino acid sequence in M. tuberculosis complex species and could be selected as a base peptide when designing a subunit-based, anti-tuberculosis vaccine.

Functionally relevant proteins in Plasmodium falciparum host cell invasion.

A totally effective, antimalarial vaccine must involve sporozoite and merozoite proteins (or their fragments) to ensure complete parasite blocking during critical invasion stages. This Special Report examines proteins involved in critical biological functions for parasite survival and highlights the conserved amino acid sequences of the most important proteins involved in sporozoite invasion of hepatocytes and merozoite invasion of red blood cells. Conserved high activity binding peptides are located in such proteins' functionally strategic sites, whose functions are related to receptor binding, nutrient and protein transport, enzyme activity and molecule-molecule interactions. They are thus excellent targets for vaccine development as they block proteins binding function involved in invasion and also their biological function.

Immunoglobulin response to the low polymorphic Pf113 antigen in children from Lastoursville, South-East of Gabon

Pf113 is a P. falciparum putatively GPI-anchored protein that has been so far localized at the surface of merozoites, suggesting it could interact with RBC surface during merozoite invasion. Previous studies conducted in Papua New Guinea and in Kenya have revealed that this protein is recognized by natural antibodies in individuals living in malaria-endemic areas and is associated with protective immunity in malaria, further supporting the potential of Pf113 for the development of anti-malaria vaccines. However, in Central Africa, no study on the immunogenicity of this protein has been conducted. Here, we report the characterization of the Pf113 immune response in 103 children by Enzyme-Linked Immunoabsorbent Assay (ELISA), using a recombinant form of Pf113 expressed in Escherichia coli, together with the study of the Pf113 polymorphism, after amplification and sequencing of 40 field isolates. Data showed that almost 51% of the studied individuals had positive antibody responses to the recombinant Pf113 protein, and that IgG subclass response was dominated by IgG3 (84%) followed by IgG1 (50%). Surprisingly the prevalence of IgG4 was 92%. In addition, gene analysis in field isolates from this region indicated that Pf113 was not highly polymorphic, in particular regarding high-activity binding peptides (HABPs). Our data reinforce the idea that Pf113 may be considered for inclusion in multicomponent blood-stage vaccines.

Mycobacterium tuberculosis PE9 protein has high activity binding peptides which inhibit target cell invasion

PE/PPE proteins are involved in several processes during Mycobacterium tuberculosis (Mtb) infection of target cells; studying them is extremely interesting as they are the only ones from the Mycobacterium genus, they abound in pathogenic species such as Mtb and their function remains yet unknown. The PE9 protein (Rv1088) was characterised, the rv1088 gene was identified by PCR in Mtb complex strains and its expression and localisation on mycobacterial surface was confirmed by Western blot and immunoelectron microscopy. Bioinformatics tools were used for predicting PE9 protein structural aspects and experimental study involved the circular dichroism of synthetic peptides. The peptides were tested in binding assays involving U937 and A549 cells; two high activity binding peptides (HABPs) were found for both cell lines (39226-1MSYMIATPAALTAAATDIDGI21 and 39232-125YQRHFGTGGQPEFRQHSEHRR144), one for U937 (39231-104YAGAGRRQRRRRSGDGQWRLRQ124) and one for A549 (39230-83YGTGVFRRRRGRQTVTAAEHRA103). HABP 39232 inhibited mycobacterial entry to A549 cells (∼70%) and U937 cells (∼50%), peptides 39226 and 39231 inhibited entry to U937 cells (∼60% and 80%, respectively) and peptide 39230 inhibited entry to A549 cells (∼60%). This emphasised HABPs’ functional importance in recognition between Mtb H37Rv and target cell receptors. These peptide sequences could be involved in invasion and were recognised by the host’s immune system, thereby highlighting their use when designing an efficient anti-tuberculosis multiantigenic vaccine.

IMPIPS: the immune protection-inducing protein structure concept in the search for steric-electron and topochemical principles for complete fully-protective chemically synthesised vaccine development.

Determining immune protection-inducing protein structures (IMPIPS) involves defining the stereo-electron and topochemical characteristics which are essential in MHC-p-TCR complex formation. Modified high activity binding peptides (mHABP) were thus synthesised to produce a large panel of IMPIPS measuring 26.5 ±3.5Å between the farthest atoms fitting into Pockets 1 to 9 of HLA-DRβ1* structures. They displayed a polyproline II-like (PPIIL) structure with their backbone O and N atoms orientated to establish H-bonds with specific residues from HLA-DRβ1*-peptide binding regions (PBR). Residues having specific charge and gauche+ orientation regarding p3χ1, p5χ2, and p7χ1 angles determined appropriate rotamer orientation for perfectly fitting into the TCR to induce an appropriate immune response. Immunological assays in Aotus monkeys involving IMPIPS mixtures led to promising results; taken together with the aforementioned physicochemical principles, non-interfering, long-lasting, protection-inducing, multi-epitope, multistage, minimal subunit-based chemically-synthesised peptides can be designed against diseases scourging humankind.

The Malaria Parasite's Achilles' Heel: Functionally-relevant Invasion Structures

Malaria parasites have their Achilles' heel; they are vulnerable in small parts of their relevant molecules where they can be wounded and killed. These are sporozoite and merozoite protein conserved high activity binding peptides (cHABPs), playing a critical role in binding to and invasion of host cells (hepatocytes and erythrocytes, respectively). cHABPs can be modified by specific amino acid replacement, according to previously published physicochemical rules, to produce analogues (mHABPs) having left-handed polyproline II (PPIIL)-like structures which can modulate an immune response due to fitting perfectly into the HLA-DRβ1* peptide binding region (PBR) and having an appropriate presentation to the T-cell receptor (TCR).

Mce4F Mycobacterium tuberculosis protein peptides can inhibit invasion of human cell lines.

This work was aimed at studying the Mycobacterium tuberculosis H37Rv Rv3494c protein, taking into account that it belongs to the mammalian cell entry family (mce) which is thought to have important functions in the disease's pathogenesis. The protein was characterized in silico; its presence on mycobacterial surface was confirmed by immunoelectron microscopy. High-activity binding peptides (HABPs) were identified by binding assays with (125)I; their ability to inhibit mycobacterial entry to two cell lines (U937 alveolar macrophages and A549 epithelial cells) was ascertained and their role in bacterial entry was confirmed by fluorescent microsphere internalization assay. This protein's predicted alpha-helix structure was confirmed by circular dichroism of its peptides. All HABPs inhibited mycobacterial entry to cells and that the 38379 peptide ((201)IDQAGPFLQAQIRAGGDIKSY(220)) had high binding ability and inhibited the mycobacterial entry to both cell lines assayed here. Rv3494c peptides 38370 ((21)LSVMAIFYLRLPATFGIGTY(40)), 38373 ((81)HMRLNSGTAIPSNVTATVRSY(100)) and 38379 ((201)IDQAGPFLQAQIRAGGDIKSY(220)) showed to be HABP and inhibited mycobacterial entry to A549 cells and peptide 38382 ((261)RPSFPALAASLANLGRVGVIY(280)) bind to U937 and inhibited the mycobacterial entry to this cell line; all of these sequences play an important role in cell line recognition and invasion, and may thus be considered in the search for prophylactic candidates against tuberculosis.

Gauche+ side-chain orientation as a key factor in the search for an immunogenic peptide mixture leading to a complete fully protective vaccine

Topological and stereo-electron characteristics are essential in major histocompability class II–peptide–T–cell receptor (MHC–p–TCR) complex formation for inducing an appropriate immune response. Modified high activity binding peptides (mHABPs) were synthesised for complete full protection antimalarial vaccine development producing a large panel of individually fully protection-inducing protein structures (FPIPS) and very high long-lasting antibody-inducing (VHLLAI) mHABPs. Most of those which did not interfere, compete, inhibit or suppress their individual VHLLAI or FPIPS activity contained or displayed a polyproline II-like (PPIIL) structure when mixed. Here we show that amino acid side-chains located in peptide binding region (PBR) positions p3 and p7 displayed specific electron charges and side-chain gauche+ orientation for interacting with the TCR. Based on the above, and previously described physicochemical principles, non-interfering, long-lasting, full protection-inducing, multi-epitope, multistage, minimal subunit-based chemically synthesised mHABP mixtures can be designed for developing vaccines against diseases scourging humankind, malaria being one of them.

Rh1 high activity binding peptides inhibit high percentages of Plasmodium falciparum FVO strain invasion

Identifying the minimal functional regions of the proteins which the malaria parasite uses when invading its host cells constitutes the first and most important approach in an effective design for a chemically synthesised, multi-antigen, multi-stage, subunit-based vaccine. This work has been aimed at identifying the PfRh1 protein binding regions (residues 1–2580) belonging to the reticulocyte binding-like (RBL or P. falciparum Rh [PfRh]) family implicated in the parasite's alternative target cell invasion routes. Eighteen peptide regions (called high activity binding peptides – HABPs) binding to red blood cells (RBC) were identified in peptides mapped in a highly robust, specific and sensitive receptor–ligand assay. These HABPs were saturable in the experimental conditions assayed here and most had an alpha helix structure. Polymorphism studies revealed that only six of the eighteen HABPs identified had changes at amino acid level amongst the seven P. falciparum strains evaluated. Most HABPs’ specific binding became altered when RBC were treated with neuraminidase, chymotrypsin and trypsin, suggesting differing sensitivity for RBC membrane receptors. After ascertaining that the Rh1 gene was transcribed and expressed in late-stage schizonts of the FCB-2 strain, invasion inhibition assays were carried out. When most of these HABPs were assayed in P. falciparum in vitro culture they were able to inhibit high percentages of FVO strain invasion compared to low inhibition percentages observed with the FCB-2 strain. This data shows small Rh1 regions’ participation during invasion and suggests that these units should be included in further immunological and structural studies.

Mycobacterium tuberculosis surface protein Rv0227c contains high activity binding peptides which inhibit cell invasion

Mycobacterium tuberculosis surface proteins involved in target cell invasion may be identified as a strategy for developing subunit-based, chemically-synthesized vaccines. The Rv0227c protein was thus selected to assess its role in the invasion and infection of Mycobacterium tuberculosis target cells. Results revealed Rv0227c localization on mycobacterial surface by immunoelectron microscopy and Western blot. Receptor–ligand assays using 20-mer, non-overlapping peptides covering the complete Rv0227c protein sequence revealed three high activity binding peptides for U937 phagocytic cells and seven for A549 cells. Peptide 16944 significantly inhibited mycobacterial entry to both cell lines while 16943 and 16949 only managed to inhibit entrance to U937 cells and 16951 to A549 cells. The Jnet bioinformatics tool predicted secondary structure elements for the complete protein, agreeing with elements determined for such chemically-synthesized peptides. It was thus concluded that high activity binding peptides which were able to inhibit mycobacterial entry to target cells are of great importance when selecting peptide candidates for inclusion in an anti-tuberculosis vaccine.

Towards the development of a fully protective Plasmodium falciparum antimalarial vaccine

If ever there were a truism then it would be that a completely protective Plasmodium falciparum malaria vaccine is desperately needed. Our institute has devoted all its efforts during the last 30 years to developing a fully protective, minimal subunit-based, multiepitope, multistage (targeting sporozoite and merozoite proteins), chemically synthesized antimalarial vaccine, given that peptides with high binding activity to their corresponding host cells (liver cells or red blood cells) form the springboard for vaccine design. However, such conserved high activity binding peptides have to be specifically modified to render them into highly immunogenic and protection-inducing peptides since they are immunologically silent. These modifications, analyzed at the 3D structural level by 1H-NMR, allow them a better fit into the MHC II–peptide–T-cell receptor complex to induce an appropriate immune response, providing a rational and logical approach (analyzed at the single atom level) for vaccine development, particularly in the field of malaria.

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.

The role of Mycobacterium tuberculosis Rv3166c protein-derived high-activity binding peptides in inhibiting invasion of human cell lines

Given the urgent need for designing a new antituberculosis vaccine conferring total protection on patients of all ages, following the line of research adopted by our institute, this work has identified Mycobacterium tuberculosis (Mtb) Rv3166c protein high-activity binding peptides (HABPs) which are able to inhibit bacterial invasion of U937 (monocyte-derived macrophages) and A549 (type II alveolar epithelial cells) cell lines. The presence and transcription of the rv3166c gene in the Mtb species complex was confirmed by polymerase chain reaction (PCR) and reverse transcriptase-PCR; Rv3166c expression was evaluated by western blot and cellular localisation confirmed by immunoelectron microscopy. Its presence was mainly determined on cell surface. Sixteen peptides covering its entire length were chemically synthesised and tested for their ability to bind to U937 and A549 cells. Two U937 HABPs were identified and three for A549, one of them being shared by both cell lines. The four HABPs found inhibited Mtb entry by 15.07-94.06%. These results led us to including Rv3166c HABPs as candidates for further studies contributing towards the search for a multiepitope, chemically synthesised, subunit-based antituberculosis vaccine.

A single amino acid change in the Plasmodium falciparum RH5 (PfRH5) human RBC binding sequence modifies its structure and determines species-specific binding activity

Identifying the ligands or regions derived from them which parasites use to invade their target cells has proved to be an excellent strategy for identifying targets for vaccine development. Members of the reticulocyte-binding homologue family (PfRH), including RH5, have been implicated in invasion as adhesins binding to specific receptors on erythrocyte surface. The regions mediating PfRH5-RBC specific interactions have been identified here by fine mapping the whole PfRH5 protein sequence. These regions, called high activity binding peptides (HABPs), bind to a receptor which is sensitive to trypsin treatment and inhibit merozoite invasion of RBCs by up to 80%, as has been found for HABP 36727. Our results show that a single amino acid change in the HABP 36727 sequence modifies a peptide's 3D structure, thereby resulting in a loss of specific binding to human RBCs and its inhibition ability, while binding to Aotus RBC remains unmodified. Such invasion differences and binding ability produced by replacing a single amino acid in an essential molecule, such as PfRH5, highlight the inherent difficulties associated with developing a fully effective vaccine against 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 (421KVQGLSYLLRRKNGTKHPVY440) and SIAP-1 34899 (541YVLNSKLLNSRSFDKFKWIQ560) and SIAP-2 36879 (181LLLYSTNSEDNLDISFGELQ200). 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). 1H 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.