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Major histocompatibility class II molecule-peptide-T-cell receptor (MHCII-p-TCR) complex-mediated antigen presentation for a minimal subunit-based, multi-epitope, multistage, chemically-synthesised antimalarial vaccine is essential for inducing an appropriate immune response. Deep understanding of this MHCII-p-TCR complex's stereo-electronic characteristics is fundamental for vaccine development. This review encapsulates the main principles for achieving such epitopes' perfect fit into MHC-II human (HLADRβ̞1*) or Aotus (Aona DR) molecules. The enormous relevance of several amino acids' physico-chemical characteristics is analysed in-depth, as is data regarding a 26.5 ± 2.5Å distance between the farthest atoms fitting into HLA-DRβ1* structures' Pockets 1 to 9, the role of polyproline II-like (PPII L ) structures having their O and N backbone atoms orientated for establishing H-bonds with specific HLA-DRβ1*-peptide binding region (PBR) residues. The importance of residues having specific charge and orientation towards the TCR for inducing appropriate immune activation, amino acids' role and that of structures interfering with PPII L formation and other principles are demonstrated which have to be taken into account when designing immune, protection-inducing peptide structures (IMPIPS) against diseases scourging humankind, malaria being one of them., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Patarroyo, Bermudez, Alba, Patarroyo, Suarez, Aza-Conde, Moreno-Vranich and Vanegas.)

Fifty ~20-amino acid (aa)-long peptides were selected from functionally relevant SARS-CoV-2 S, M, and E proteins for trial B-21 and another 53 common ones, plus some new ones derived from the virus' main genetic variants for complementary trial C-21 . Peptide selection was based on tremendous SARS-CoV-2 genetic variability for analysing them concerning vast human immunogenetic polymorphism for developing the first supramutational, Colombian SARS-protection (SM-COLSARSPROT), peptide mixture. Specific physicochemical rules were followed, i.e., aa predilection for polyproline type II left-handed (PPII L ) formation, replacing β-branched, aromatic aa, short-chain backbone H-bond-forming residues, π-π interactions (n→π* and π-CH), aa interaction with π systems, and molecular fragments able to interact with them, disrupting PPII L propensity formation. All these modified structures had PPII L formation propensity to enable target peptide interaction with human leukocyte antigen-DRβ1* (HLA-DRβ1*) molecules to mediate antigen presentation and induce an appropriate immune response. Such modified peptides were designed for human use; however, they induced high antibody titres against S, M, and E parental mutant peptides and neutralising antibodies when suitably modified and chemically synthesised for immunising 61 major histocompatibility complex class II (MHCII) DNA genotyped Aotus monkeys (matched with their corresponding HLA-DRβ1* molecules), predicted to cover 77.5% to 83.1% of the world's population. Such chemically synthesised peptide mixture represents an extremely pure, stable, reliable, and cheap vaccine for COVID-19 pandemic control, providing a new approach for a logical, rational, and soundly established methodology for other vaccine development., Competing Interests: The authors declare that this study received funding from Greenstone International Foundation Limited. The funder was not involved in the study design and/or the collection, analysis and /or interpretation of data, the writing of the article or the decision to submit it for publication., (Copyright © 2022 Patarroyo, Patarroyo, Pabón, Alba, Bermudez, Rugeles, Díaz-Arevalo, Zapata-Builes, Zapata, Reyes, Suarez, Agudelo, López, Aza-Conde, Melo, Escamilla, Oviedo, Guzmán, Silva, Forero, Flórez-Álvarez, Aguilar-Jimenez, Moreno-Vranich, Garry and Avendaño.)

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' 1 H-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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Malaria remains a large-scale public health problem, killing more than 400,000 people and infecting up to 230 million worldwide, every year. Unfortunately, despite numerous efforts and research concerning vaccine development, results to date have been low and/or strain-specific. This work describes a strategy involving Plasmodium falciparum Duffy binding-like (DBL) and reticulocyte-binding protein homologue (RH) family-derived minimum functional peptides, netMHCIIpan3.2 parental and modified peptides' in silico binding prediction and modeling some Aotus major histocompatibility class II (MHCII) molecules based on known human molecules' structure to understand their differences. These are used to explain peptides' immunological behaviour when used as vaccine components in the Aotus model. Despite the great similarity between human and Aotus immune system molecules, around 50% of Aotus allele molecules lack a counterpart in the human immune system which could lead to an Aotus-specific vaccine. It was also confirmed that functional Plasmodium falciparum' conserved proteins are immunologically silent (in both the animal model and in-silico prediction); they must therefore be modified to elicit an appropriate immune response. Some peptides studied here had the desired behaviour and can thus be considered components of a fully-protective antimalarial vaccine., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Malaria continues being a high-impact disease regarding public health worldwide; the WHO report for malaria in 2018 estimated that ~219 million cases occurred in 2017, mostly caused by the parasite Plasmodium falciparum . The disease cost the lives of more than 400,000 people, mainly in Africa. In spite of great efforts aimed at developing better prevention (i.e., a highly effective vaccine), diagnosis, and treatment methods for malaria, no efficient solution to this disease has been advanced to date. The Fundación Instituto de Inmunología de Colombia (FIDIC) has been developing studies aimed at furthering the search for vaccine candidates for controlling P. falciparum malaria. However, vaccine development involves safety and immunogenicity studies regarding their formulation in animal models before proceeding to clinical studies. The present work has thus been aimed at evaluating the safety and immunogenicity of a mixture of 23 chemically synthesised, modified peptides (immune protection-inducing protein structure (IMPIPS)) derived from different P. falciparum proteins. Single and repeat dose assays were thus used with male and female BALB/c mice which were immunised with the IMPIPS mixture. It was found that single and repeat dose immunisation with the IMPIPS mixture was safe, both locally and systemically. It was observed that the antibodies so stimulated recognised the parasite's native proteins and inhibited merozoite invasion of red blood cells in vitro when evaluating the humoral immune response induced by the IMPIPS mixture. Such results suggested that the IMPIPS peptide mixture could be a safe candidate to be tested during the next stage involved in developing an antimalarial vaccine, evaluating local safety, immunogenicity, and protection in a nonhuman primate model., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2019 Jennifer Lambraño et al.)

A vaccine candidate component must fit perfectly into the antigen presenting HLA-DRβ* molecule's groove (or canonical nonapeptide) peptide binding region (PBR) during antigen presentation to the T-cell receptor (TCR), conforming a specific and stable macromolecular complex and induce an appropriate immune response. Antigen's peripheral flanking residues (PFR, positions (p) -p2 and p10) must thus establish strong interactions with the HLA-DRβ* - TCR complex. These amino acids (aa) have specific physico-chemical characteristics enabling differentiation between non-protective but antibody-inducer (NPAI), short-lived protection inducer (SLPI) and long-lasting protection inducer (LLPI) peptides when used as an antimalarial vaccine component. Their identification (through 1 H-NMR and Aotus monkey immunization) and proper modification contributes to a logical and rational methodology for long-lasting and protective immunological memory., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The review covers the functional and structural approach followed by our group for more than 34 years in the search for a methodology that allows the rational design of chemically synthesised vaccines. An analogy between Odysseus, the cunning hero of the epic poem Odyssey by Homer, and the elusive Plasmodium parasite has been made, to review our research group's main considerations when developing a rational methodology for designing second generation, modified peptide-based, minimal subunit, multi-antigen, multi-stage, chemically synthesised vaccines against Plasmodium falciparum malaria., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: Obtaining an effective antimalarial vaccine has represented one of the biggest public health challenges over the last 50 years. Despite efforts by many laboratories around the world using whole-organism, recombinant proteins and genome-based approaches, the results have been disappointing. One of the main problems when designing an antimalarial vaccine is the poor immunogenicity induced by the functionally relevant and conserved protein regions of the parasite., Areas Covered: This review focuses on the logical and rational methodology followed to identify Plasmodium falciparum conserved functional regions with the ability to bind to target cells conserved high activity binding peptides (cHABPs) and the physicochemical and immunological characteristics that should be taken into account for modifying them into highly immunogenic and protection-inducing peptides (mHABPs) into highly immunogenic and protection-inducing in Aotus monkeys., Expert Opinion: The functional approach taken to develop a fully protective, minimal subunit-based, multiantigenic, multistage and synthetic peptide-based antimalarial vaccine has shown promising results. The clear relationship observed between mHABPs structure and their immunological properties highlights the challenges and opportunities arising from this methodology, as well as the universal principles and rules derived therefrom.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Plasmodium falciparum (Pf) malaria causes 200 million cases worldwide, 8 million being severe and complicated leading to ∼1 million deaths and ∼100,000 abortions annually. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) has been implicated in cytoadherence and infected erythrocyte rosette formation, associated with cerebral malaria; chondroitin sulphate-A attachment and infected erythrocyte sequestration related to pregnancy-associated malaria and other severe forms of disease. An endothelial cell high activity binding peptide is described in several of this ∼300 kDa hypervariable protein's domains displaying a conserved motif (GACxPxRRxxLC); it established H-bonds with other binding peptides to mediate red blood cell group A and chondroitin sulphate attachment. This motif (when properly modified) induced PfEMP1-specific strain-transcending, fully-protective immunity for the first time in experimental challenge in Aotus monkeys, opening the way forward for a long sought-after vaccine against severe malaria.

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Malaria caused by Plasmodium vivax continues being a public health problem in tropical and subtropical areas throughout the whole world. In spite of this species' epidemiological importance, its biological complexity has hampered advances being made in the field of vaccine development. Few antigens have been described and analyzed to date in preclinical and clinical studies, thereby highlighting the great challenge facing groups currently working on this parasite species. This review summarizes the most representative work done during the last few years and discusses the approaches adopted in making progress towards an anti-Plasmodium vivax 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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This study describes the identification of the Plasmodium vivax rhoptry antigen Pv34 whose sequence was obtained based on homology comparison with the Plasmodium falciparum Pf34. The pv34 gene product was characterized by molecular biology and immunological techniques. Additionally, association of Pv34 to detergent-resistant microdomains (DRMs), expression in late blood-stage parasites and recognition of recombinant Pv34 (rPv34) by sera from P. vivax-infected Aotus monkeys and patients was assessed. Lymphoproliferation and cytokine secretion was also evaluated in individuals living in malaria endemic areas. Altogether, the data support carrying out further studies to assess the immunogenicity and protection-inducing ability of rPv34 as component of a multi-antigenic, multi-stage vaccine against vivax malaria.

Although largely considered benign, Plasmodium vivax causes disease in nearly 75 million people each year and the available strategies are not sufficient to reduce the burden of disease, therefore pointing to vaccine development as a cost-effective control measure. In this study, the P. vivax merozoite surface protein 10 (MSP-10) was expressed as a recombinant protein in Escherichia coli and purified by affinity chromatography. High antigenicity was observed since sera from P. vivax-infected patients strongly recognized rPvMSP10. The immunogenicity of rPvMSP10 was tested in Aotus monkeys, comparing responses induced by formulations with Freund's adjuvant, Montanide ISA720 or aluminum hydroxide. All formulations produced high antibody titers recognizing the native protein in late schizonts. Despite inducing strong antibody production, none of the formulations protected immunized Aotus monkeys upon experimental challenge.

Rhoptry proteins have been extensively shown to be important in invasion and parasitophorous vacuole (PV) formation. This work evaluates the immunogenicity and protective efficacy of Plasmodium vivax RAP2 in the non-human Aotus primate model, when expressed as a recombinant molecule in E. coli and formulated in Freund and Alum hydroxide adjuvants. Our results show that rPvRAP2 is immunogenic in both formulations, finding a trend of higher cytokine levels in immunized monkeys, specially in IL-4 levels (using Freund's adjuvant) and IL-5 (using Alum hydroxide). RAP2 is suggested as a P. vivax-vaccine candidate since immunized monkeys exhibited lower parasitemias than control groups after being experimentally challenged with the P. vivax VCG-I strain.

Seventeen million people die of transmittable diseases and 2/3 of the world's population suffer them annually. Malaria, tuberculosis, AIDS, hepatitis, and reemerging and new diseases are a great threat to humankind. A logical and rational approach for vaccine development is thus desperately needed. Protein chemistry provides the best tools for tackling these problems. The tremendous complexity of microbes, the different pathways they use for invading host cells, and the immune responses they induce can only be resolved by using the minimum subunit-based (chemically produced approximately 20-mer peptides), multiantigenic (most proteins involved in invasion), multistage (different invasion mechanisms) vaccine development approach. The most lethal form of malaria caused by Plasmodium falciparum (killing 3 million and affecting 500 million people worldwide annually) was used as target disease since many of its proteins, its invasion pathways, and its genome have been described recently. A New World primate (the Aotus monkey) is highly susceptibly to human malaria; its immune system molecules are 80-100% identical to those of its human counterpart, making it an excellent model for vaccine development. Chemically synthesized approximately 20-mer peptides, covering all the P. falciparum malaria proteins involved in red blood cell (RBC) invasion were synthesized by the classical t-Boc technology (based on synthetic SPf66 antimalarial vaccine information for identifying targets) and assayed in a highly sensitive, specific, and robust test for detecting receptor-ligand interactions between high-activity binding peptides (HABPs) and RBCs. HABPs were identified, some in which the molecule displays genetic variability (to be discarded due to their tremendous complexity) and elicits a strain-specific immune response and others that are conserved (no amino acid sequence variation). Conserved HABPs were synthesized in a polymeric form by adding cysteines at their N- and C-terminal ends to be used for monkey immunization. They became nonimmunogenic (no antibodies were induced) nonprotection inducers (monkeys were not protected against P. falciparum malaria challenge with a highly infective strain) suggesting a code of immunological silence or nonresponsiveness for these conserved HABPs. A large number of monkey trials involving a considerable number of Aotus monkeys were performed to break this code of immunological silence by replacing critical residues (determined by glycine peptide analogue scanning) to find that the following amino acid changes had to be made to render them antibody and protection inducing: F<-->R; W<-->Y; L<-->H; I<-->N; M<-->K; P<-->D; Q<-->E; C<-->T. The three-dimensional (3D) structure of >100 of these native modified HABPs (determined by (1)H NMR) revealed that the following structural changes had all to be achieved to allow a better fit into the major histocompatibility complex class II (MHC II)-peptide-TCR complex to properly activate the immune system: alpha-helix shortening, modifying their beta-turn, adopting segmental alpha-helix configuration, changing residue orientation, and increasing the distance of those residues fitting into the MHC II molecules from antigen-presenting cells. More than 100 such highly immunogenic, protection-inducing (against P. falciparum malaria) modified HABPs have been identified to date with this methodology, showing that it could lead to developing a highly effective subunit-based, multiantigenic, multistage synthetic vaccine against diseases scourging humankind, malaria being one of them.

Plasmodium vivax merozoites have been found to specifically invade immature red blood cells (reticulocytes) and this preference has been associated with two proteins named reticulocyte binding protein-1 and protein-2 (PvRBP1 and PvRBP2). Previous reticulocyte binding assays using 15-mer synthetic peptides spanning the entire PvRBP1 sequence have shown that 25 out of the 195 peptides synthesised (grouped into 4 different regions) displayed high affinity binding to reticulocytes. The PvRBP1 region III (amino acids 1998-2348), encompassing 9 of the previously described high-affinity reticulocyte binding peptides, was expressed as a recombinant protein in the present study. This protein has been shown to be antigenic in humans and it has also been able to induce good humoral and cellular immune responses in Aotus nancymaae monkeys. Despite its high immunogenicity, no protective efficacy was observed in the immunised animals.

Effector mechanisms responsible for providing protective immunity against Plasmodium vivax (Pv) infection were examined in Aotus monkeys vaccinated with two Pv Merozoite Surface Protein-1 (PvMSP-1) recombinant polypeptides that had previously been shown to protect vaccines against parasite challenge. Vaccine efficacy was reproducible in this trial, showing that one out of the five monkeys immunised with the recombinant protein mixture was partially protected while three others controlled parasitaemia. Antibodies reactive to the parasite's native proteins, the recombinant polypeptides and peptides spanning both recombinant fragments were detected in most vaccinees. Despite substantial Peripheral Blood Mononuclear Cell (PBMC) antigen-specific cellular proliferation not being detected, high rPvMSP-1(20) specific gamma interferon (IFN-gamma) production was found in the three animals that controlled parasitaemia. Altogether the results suggest that antibody titres and antigen-specific IFN-gamma production mediate protective immunity against P. vivax.

Parasite antigen genetic diversity represents a great obstacle when designing a vaccine against malaria caused by Plasmodium vivax. Selecting vaccine candidate antigens has been focused on those fulfilling a role in invasion and which are conserved, thus avoiding specific-allele immune responses. Most antigens described to date belong to the blood stage, thereby blocking parasite development within red blood cells, whilst studying antigens from other stages has been quite restricted. Antigens from different parasite stages are required for developing a completely effective vaccine; thus, pre-erythrocyte stage antigens able to block the first line of infection becoming established should also be taken into account. However, few antigens from this stage have been studied to date. Several P. falciparum sporozoite antigens are involved in invasion. Since 77% of genes are orthologous amongst Plasmodium parasites, P. vivax sporozoite antigen orthologs to those of P. falciparum might be present in its genome. Although these genes might have high genetic diversity, conserved functionally-relevant regions (ideal for vaccine development) could be predicted by comparing genetic diversity patterns and evolutionary rates. This study was thus aimed at searching for putative P. vivax sporozoite genes so as to analyse their genetic diversity for determining their potential as vaccine candidates. Several DNA sequence polymorphism estimators were computed at each locus. The evolutionary force (drift, selection and recombination) drawing the genetic diversity pattern observed was also determined by using tests based on polymorphism frequency spectrumas well as the type of intra- and inter-species substitutions. Likewise, recombination was assessed both indirectly and directly. The results showed that sporozoite genes weremore conserved thanmerozoite genes evaluated to date. Putative domains implied in cell traversal, glidingmotility and hepatocyte interaction had a negative selection signal, being conserved amongst different species in the genus. PvP52, PvP36, PvSPATR, PvPLP1, PvMCP1, PvTLP, PvCelTOS, and PvMB2 antigens or functionally restricted regions within them would thus seem promising vaccine candidates and could be used when designing a pre-erythrocyte and/or multi-stage vaccine against P. vivax to avoid allele-specific immune responses that could reduce vaccine efficacy. [ABSTRACT FROM AUTHOR]

Rational strategies for obtaining malaria vaccine candidates should include not only a proper selection of target antigens for antibody stimulation, but also a versatile molecular design based on ordering the right pieces from the complex pathogen molecular puzzle towards more active and functional immunogens. Classical Plasmodium falciparum antigens regarded as vaccine candidates have been selected as model targets in this study. Among all possibilities we have chosen epitopes of PfCSP, STARP; MSA1 and Pf155/RESA from pre- and erythrocyte stages respectively for designing a large 82-residue chimeric immunogen. A number of options aimed at diminishing steric hindrance for synthetic procedures were assessed based on standard Fmoc chemistry such as building block orthogonal ligation; pseudo-proline and microwave-assisted procedures, therefore the large-chimeric target was produced, characterized and immunologically tested. Antigenicity and functional in vivo efficacy tests of the large-chimera formulations administered alone or as antigen mixtures have proven the stimulation of high antibody titers, showing strong correlation with protection and parasite clearance of vaccinated BALB/c mice after being lethally challenged with both P. berghei-ANKA and P. yoelii 17XL malaria strains. Besides, 3D structure features shown by the large-chimera encouraged as to propose using these rational designed large synthetic molecules as reliable vaccine candidate-presenting systems. [ABSTRACT FROM AUTHOR]

The aim of obtaining novel vaccine candidates against malaria and other transmissible diseases can be partly based on selecting non-polymorphic peptides from relevant antigens of pathogens, which have to be then precisely modified for inducing a protective immunity against the disease. Bearing in mind the high degree of the MSA-2 peptide primary structure's genetic conservation among malaria species, and its crucial role in the high RBC binding ability of Plasmodium falciparum (the main agent causing malaria), structurally defined probes based on non-natural peptide-bond isosteres were thus designed. Thus, two peptide mimetics were obtained (so-called reduced amide pseudopeptides), in which naturally made amide bonds of the FIN-binding motif of MSA-2 were replaced with ψ-[CH-NH] methylene amide isostere bonds, one between the F-I and the second between I-N amino acid pairs, respectively, coded as ψ-128 ψ-130. These peptide mimetics were used to produce poly- and monoclonal antibodies in Aotus monkeys and BALB/c mice. Parent reactive mice-derived IgM isotype cell clones were induced to Ig isotype switching to IgG sub-classes by controlled in vitro immunization experiments. These mature isotype immunoglobulins revealed a novel epitope in the MSA-2 antigen and two polypeptides of rodent malaria species. Also, these antibodies' functional activity against malaria was tested by in vitro assays, demonstrating high efficacy in controlling infection and evidencing neutralizing capacity for the rodent in vivo malaria infection. The neutralizing effect of antibodies induced by site-directed designed peptide mimetics on Plasmodium's biological development make these pseudopeptides a valuable tool for future development of immunoprophylactic strategies for controlling malarial infection. [ABSTRACT FROM AUTHOR]

Background: Plasmodium vivax continues to be the most widely distributed malarial parasite species in tropical and sub-tropical areas, causing high morbidity indices around the world. Better understanding of the proteins used by the parasite during the invasion of red blood cells is required to obtain an effective vaccine against this disease. This study describes characterizing the P. vivax asparagine-rich protein (PvARP) and examines its antigenicity in natural infection. Methods: The target gene in the study was selected according to a previous in silico analysis using profile hidden Markov models which identified P. vivax proteins that play a possible role in invasion. Transcription of the arp gene in the P. vivax VCG-1 strain was here evaluated by RT-PCR. Specific human antibodies against PvARP were used to confirm protein expression by Western blot as well as its subcellular localization by immunofluorescence. Recognition of recombinant PvARP by sera from P. vivax-infected individuals was evaluated by ELISA. Results: VCG-1 strain PvARP is a 281-residue-long molecule, which is encoded by a single exon and has an N-terminal secretion signal, as well as a tandem repeat region. This protein is expressed in mature schizonts and is located on the surface of merozoites, having an apparent accumulation towards their apical pole. Sera from P. vivax-infected patients recognized the recombinant, thereby suggesting that this protein is targeted by the immune response during infection. Conclusions: This study showed the characterization of PvARP and its antigenicity. Further assays orientated towards evaluating this antigen's functional importance during parasite invasion are being carried out. [ABSTRACT FROM AUTHOR]

A completely effective vaccine for malaria (one of the major infectious diseases worldwide) is not yet available; different membrane proteins involved in parasite-host interactions have been proposed as candidates for designing it. It has been found that proteins encoded by the merozoite surface protein (msp)-7 multigene family are antibody targets in natural infection; the nucleotide diversity of three Pvmsp-7genes was thus analyzed in a Colombian parasite population. By contrast with P. falciparum msp-7 loci and ancestral P. vivax msp-7 genes, specie-specific duplicates of the latter specie display high genetic variability, generated by single nucleotide polymorphisms, repeat regions, and recombination. At least three major allele types are present in Pvmsp-7C, Pvmsp-7H and Pvmsp-7I and positive selection seems to be operating on the central region of these msp-7 genes. Although this region has high genetic polymorphism, the C-terminus (Pfam domain ID: PF12948) is conserved and could be an important candidate when designing a subunit-based antimalarial vaccine. [ABSTRACT FROM AUTHOR]

The serine repeat antigen (SERA) protein is a leading candidate molecule for inclusion as a component in a multi-antigen, multi-stage, minimal subunit-based, chemically synthesised anti-malarial vaccine. Peptides having high red blood cell binding affinity (known as HABPs) have been identified in this protein. The 6733 HABP was located in the C-terminal portion of the 47-kDa fragment while HABP 6754 was located in the C-terminal region of the 56-kDa fragment. These conserved HABPs failed to induce an immune response. Critical red blood cell binding residues and/or their neighbours (assessed by glycine-analogue scanning) were replaced by others having the same mass, volume and surface but different polarity, rendering some of them highly immunogenic when assessed by antibody production against the parasite or its proteins and protection-inducers against experimental challenge with a highly infectious Aotus monkey-adapted Plasmodium falciparum strain. This manuscript presents some modified HABPs as vaccine candidate components for enriching our tailor-made anti-malarial vaccine repertoire, as well as their 3D structure obtained by H-NMR displaying a short-structured region, differently from the native ones having random structures. [ABSTRACT FROM AUTHOR]

Abstract: 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-DRβ purified molecules. These immunological and biological activities are associated with modifications in their three-dimensional structure as determined by 1H-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 Å 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-DRβ1* molecule in order to form a proper MHC-II–peptide–TCR complex. [Copyright &y& Elsevier]

The Aotus monkey has been of great value in the pre-clinical study of malaria vaccine candidates. Several components of this primate’s immune system have been studied and they display great similarity to their human counterparts. Cloning and sequencing studies have revealed extensive sequence polymorphisms in Aotus MHC-DRB with very high similarities to several human allelic lineages, grouping at least nine distinct MHC-DRB lineages. As the efficacy of peptide vaccines in this animal model may be strongly influenced by exon 2 MHC-DRB polymorphism, the availability of a reliable and rapid MHC-DRB typing method for three species of Aotus ( Aotus nancymaae, Aotus vociferans and Aotus nigriceps) is necessary. Reference strand conformational analysis (RSCA) was used here for differentiating the distinctive Aotus MHC-DRB sequences’ mobility using five fluorescently labelled references proved to be very useful for resolving closely related sequences, establishing the number of sequences transcribed in a particular monkey and their identity. The RSCA method’s reliability in terms of identifying Aotus MHC-DRB sequences will facilitate evaluating individual responsiveness to vaccines and prompt studies associating susceptibility/resistance to infectious agents or auto-immune disease, for which Aotus monkeys may be considered to be an appropriate animal model. [ABSTRACT FROM AUTHOR]

Merozoite Surface Protein-1 (MSP-1) has been considered as a malaria vaccine candidate. It is processed during the Plasmodium falciparum invasion process of red blood cells (RBCs). A conserved MSP-1 C-terminal peptide was identified as a high-activity erythrocytebinding peptide (HAEBP) termed 1585. Since conserved HAEBPs are neither antigenic nor immunogenic we decided to assess the significance of a single peptide bond replacement in 1585. Thus, two pseudopeptides were obtained by introducing a Y[CH[sub2]-NH] reduced amide isoster into the 1585 critical binding motif. The pseudopeptides bound to different HLA-DR alleles, suggesting that backbone modifications affect MHC-II binding patterns. Pseudopeptideantibodies inhibit in vitro parasite RBC invasion by recognizing MSP-1. Each pseudopeptide-induced antibody shows distinct recognition patterns. ¹H-NMR studies demonstrated that isoster bonds modulate the pseudopeptides' structure and thus their immunological properties, therefore representing a possible subunit malaria vaccine component. [ABSTRACT FROM AUTHOR]

The New World primate Aotus nancymaae has been recommended by the World Health Organization (WHO) as a model for evaluation of malaria vaccine candidates, given its susceptibility to experimental infection with the human malaria parasites Plasmodium falciparum and Plasmodium vivax. We present here the nucleotide sequences of the complete cDNA of MHC-DQA1 and of the polymorphic exon 2 segments of MHC-DQB1/DQB2. In a group of three nonrelated animals captured in the wild, five alleles of MHC-DQA1 could be identified. They all belong to one lineage, namely Aona-DQA1*27. This lineage has not been described in any other New World monkey species studied. In a group of 19 unrelated animals, 14 Aona-DQB1 alleles could be identified which are grouped into the two lineages Aona-DQB1*22 and Aona-DQB1*23. These lineages have been described previously in the common marmoset and cotton-top tamarin. In addition, two Aona-DQB2 sequences could be identified which are highly similar to HLA-DQB2 sequences. Essential amino acid residues contributing to MHC DQ peptide binding pockets number 1 and 4 are conserved or semi-conserved between HLA-DQ and Aona-DQ molecules, indicating a capacity to bind similar peptide repertoires. These results fully support the use of Aotus monkeys as an animal model for evaluation of future subunit vaccine candidates. [ABSTRACT FROM AUTHOR]

Analysis of our Plasmodium falciparum malaria parasite peptides' 1 H-NMR database in the search for H-bonds and π-interactions led us to correlate their presence or absence with a peptide's particular immunological behavior. It was concluded that a 26.5 ± 1.5 Å between positions 1 to 9 of the HLA-DRβ1* interacting region was necessary for proper docking of 20mer-long peptides and these MHC Class II molecules for full-protective immunity. Presence of intramolecular H-bonds or π-interactions leading to righ-handed α-helix or β-turn conformation in this peptide's region induces different immune responses or none. PPII L conformation and the absence of any intramolecular interaction thus became the first feature characterising our immune protection-inducing structures as malaria vaccine candidates. [ABSTRACT FROM AUTHOR]

F ully- p rotective, l ong- l asting, i mmunological (FPLLI) memory against Plasmodium falciparum malaria regarding im mune p rotection- i nducing p rotein s tructures (IMPIPS) vaccinated into monkeys previously challenged and re-challenged 60 days later with a lethal Aotus monkey-adapted P. falciparum strain was found to be associated with preferential high binding capacity to HLA-DRβ1* allelic molecules of the m ajor h istocompatibility c lass II (MHC-II), rather than HLA-DRβ3*, β4*, β5* alleles. Complete PPII L 3D structure, a longer distance (26.5 Å ± 1.5 Å) between residues perfectly fitting into HLA-DRβ1*PBR pockets 1 and 9, a gauche − rotamer orientation in p8 TCR-contacting polar residue and a larger volume of polar p2 residues was also found. This data, in association with previously-described p3 and p7 apolar residues having gauche + orientation to form a perfect MHC-II-peptide-TCR complex, determines the stereo-electronic and topochemical characteristics associated with FPLLI immunological memory. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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

Abstract: 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. [Copyright &y& Elsevier]

Abstract: In this study, we synthesized the complete sequence of the CLAG-9 protein as 67 20-mer-long non-overlapped peptides and assessed their ability to bind to erythrocytes in receptor–ligand assays. Twenty CLAG-9 peptides were found to have specific high-affinity binding ability to erythrocytes (thereby named as HABPs), with nanomolar dissociation constants. CLAG-9 HABPs interacted with different erythrocyte surface receptors having apparent molecular weights of 85, 63 and 34kDa. CLAG-9 HABPs binding was also affected by pre-treatment of RBCs with enzymes and inhibited erythrocyte invasion in vitro by up to 72% at 200μM. These results suggest that some protein fragments of CLAG-9 may be part of the molecular machinery used by malaria parasites to invade erythrocytes, hence supporting their study as possible vaccine candidates. [Copyright &y& Elsevier]

Abstract: Severe malaria pathology is directly associated with cytoadherence of infected red blood cells (iRBCs) to healthy RBCs and/or endothelial cells occurring during the intraerythrocytic development of Plasmodium falciparum. We synthesized, as 20-mer long peptides, the members of the ring exported (REX) protein family encoded in chromosome 9, as well as the early transcribed membrane proteins (E-TRAMP) 10.2 and 4, to identify specific RBC binding regions in these proteins. Twelve binding peptides were identified (designated as HABPs): three were identified in REX1, two in REX2, one in REX3, two in REX4 and four in E-TRAMP 10.2. The majority of these HABPs was conserved among different P. falciparum strains, according to sequence analysis. No HABPs were found in E-TRAMP 4. Bindings of HABPs were saturable and sensitive to the enzymatic treatment of RBCs and HABPs had different structural features, according to circular dichroism studies. Our results suggest that the REX and E-TRAMP families participate in relevant interactions with RBC membrane proteins, which highlight these proteins as potential targets for the development of fully effective immunoprophylactic methods. [Copyright &y& Elsevier]

Abstract: The Plasmodium falciparum circumsporozoite protein is considered a major antimalarial-vaccine target due to its involvement in sporozoite invasion of mosquito’s salivary glands and human hepatocytes. The 4383, 4388 and 4389 CSP-conserved high activity hepatocyte binding peptides and their modified analogues were synthesised and their immunogenicity was tested in Aotus monkeys. Peptide 4388 modified analogues induced higher and more permanent antibody titers against sporozoites in ∼40% of immunised monkeys; whilst peptides 4383 and 4389 modified analogues elicited high, long-lasting antibody titers as well as short-lived antibodies. 1H NMR studies showed that native peptides displayed random conformations, whereas most modified immunogenic HABPs contained type I, II and IV β-turn structures. HLA-DRβ1* molecule binding assays revealed that 4383 modified HABPs bound to HLA-DRβ1*0701/HLA-DRβ1*0401 molecules, whilst 4388 and 4389 modified HABPs bound to HLA-DRβ1*0401/HLA-DRβ1*0101, respectively. The results support these high-immunogenic CSP-derived modified peptides’ inclusion in a multi-antigenic, multistage, minimal subunit-based synthetic antimalarial vaccine. [Copyright &y& Elsevier]

Abstract: The merozoite surface protein (MSP) family is involved in the initial interaction between merozoites and erythrocytes in Plasmodium species, its members are therefore becoming major targets for vaccine development. Considering that antigens included in a subunit malaria vaccine should be both accessible to the immune system and lack genetic diversity or have very limited polymorphism, we have analyzed the genetic diversity of three msp genes (msp-7A, msp-7K and msp-10) in different geographical regions of Colombia. The results showed that these genes follow the neutral model of evolution and also display low genetic diversity. The strong conservation found for msp-7 haplotypes in isolates from geographically different regions further suggests that these proteins could be good components of a vaccine against Plasmodium vivax malaria, thereby avoiding strain-specific immune responses. [Copyright &y& Elsevier]

Abstract: To block the different stages of Plasmodium falciparum invasion into human hepatocytes and red blood cells, we have focused on those proteins belonging to the pre-erythrocytic stage. One of these proteins is Sporozoite Threonine and Asparagine Rich Protein (STARP), which is a ligand used by P. falciparum parasites to bind Hepatic cells (HepG2). Previous studies on this protein identified two conserved peptides binding with high activity to HepG2 cells (namely 20546 and 20570) with corresponding critical hepatic-cell binding residues and determined an important role for these two peptides in the invasion process. This study shows the results of immunization trials in Aotus monkeys with native STARP peptides and analogues modified in critical hepatic-cell binding residues. The results show that native peptides are not immunogenic but can induce high-antibody titers when their critical residues are replaced by other with similar volume and mass but different polarity. Nuclear Magnetic Resonance (1H NMR) studies revealed that native peptides (non-immunogenic) displayed shorter α-helical regions compared to their highly immunogenic modified analogues. Binding assays with HLA-DRβ1* molecules showed that 20546 modified peptides inducing high-antibody titers (24972, 24320 and 24486) bound to HLA-DRβ1*0301 molecules, while the 20570 modified analogue (24322) bound to HLA-DRβ1*0101. The results support including these high-immunogenic STARP-derived modified peptides as pre-erythrocytic candidates to be included in the design of a synthetic antimalarial vaccine. [Copyright &y& Elsevier]

Abstract: Pseudopeptide chemistry is gaining ground in the field of synthetic vaccine development. We have previously demonstrated the potential scope of introducing reduced amide peptide bond isosters in a site-directed design for obtaining structurally modified probes able to induce malaria infection-neutralizing antibodies derived from the MSP-1 antigen. This work reports the functional properties of polyclonal and monoclonal antibodies induced by site-directed designed MSP-2 N-terminus pseudopeptides and their capacity for antibody isotype switching in in vitro immunization. Structural properties of the native peptide and its pseudopeptide analogs are discussed within the context of these novel pseudopeptides’ induced monoclonal antibody functional and physical–chemical properties. [Copyright &y& Elsevier]

Abstract: Plasmodium vivax merozoite surface protein 4 (PvMSP4) has been considered to be a promising malarial vaccine candidate. The antigenic diversity displayed by parasite populations is one of the main factors limiting the efficacy of asexual-stage anti-malarial vaccine candidates. The present study is the first characterising PvMSP4 polymorphism. P. vivax isolates were collected from endemic areas in Colombia and diversity and selection pattern studies were carried out. Overall conservation in this protein was remarkable. Changes were only found in exons I and II, the former only having single nucleotide polymorphisms (SNPs) whilst the latter showed variations in tandem repeat number caused by exon II slippage. The remaining regions (EGF-like domain, GPI anchor and intron) were completely conserved. Selection and neutrality tests carried out over variant exons indicated negative selective forces acting on them. No evidence of intragenic recombination was found. The strong conservation displayed in this molecule by isolates from geographically different regions (Colombia, Salvador and Thailand) stresses its potential importance as a candidate for a vaccine against P. vivax malaria. [Copyright &y& Elsevier]

The New World primate Aotus nancymaae is susceptible to infection by the human malaria parasite Plasmodium vivax and has therefore been recommended by the World Health Organization as a model for malaria vaccine candidate evaluation. We report the isolation, adaptation, titration and genetic characterization of a P. vivax wild strain in splenectomized A. nancymaae monkeys. Parasitemia remained high after 22 passages, reaching 7.88% by Giemsa and Acridine Orange staining and Real-Time PCR determination, making this P. vivax strain a highly infective and reliable asset to be used in P. vivax biological studies and vaccine development. [Copyright &y& Elsevier]

SPf66 is a synthetic malaria peptide vaccine, which has been widely tested in combination with aluminium hydroxide (alum) as the adjuvant. Since this formulation is weakly immunogenic, we sought to improve its immunogenicity by using the saponin adjuvant QS-21. SPf66/QS-21 vaccines were evaluated for safety, tolerability and immunogenicity in healthy adults. The vaccines were found to be safe in 87/89 (97.8%) volunteers studied. However, two individuals developed severe vaccine allergy following the third dose of 1/3 SPf66/QS-21 formulations tested. Vaccine formulations containing QS-21 induced a 45- to over 200-fold increase in anti-SPf66 IgG titres over the alum formulation after the second and third doses, respectively. Anti-SPf66 antibody from some subjects reacted against asexual blood stage parasites, as demonstrated by immunofluorescence and immunoblotting. Antibody responses generated by the QS-21 formulations were of longer duration compared to those evoked by the alum formulation. While SPf66/alum has been found to induce only CD4+ T cell response, the QS-21 formulations exhibited the potential to also elicit SPf66-specific CD8+ responses. These observations demonstrate that the use of QS-21 can substantially enhance the immunogenicity of peptide vaccines, such as SPf66. [Copyright &y& Elsevier]