p-Methoxyphenol: A potent and effective scavenger for solid-phase peptide synthesis.

During the final step of t-Boc/Bzl, solid-phase peptide synthesis (SPPS)-protecting groups from amino acids (aa) side chains must be removed from the target peptides during cleavage from the solid support. These reaction steps involve hydrolysis with hydrogen fluoride (HF) in the presence of a nucleophile (scavenger), whose function is to trap the carbocations produced during SN 1-type reactions. Five peptide sequences were synthesised for evaluating p-methoxyphenol effectiveness as a potent scavenger. After the synthesis, the resin-peptide was then separated into two equal parts to be cleaved using two scavengers: conventional reactive p-cresol (reported in the literature as an effective acyl ion eliminator) and p-methoxyphenol (hypothesised as fulfilling the same functions as the routinely used scavenger). Detailed analysis of the electrostatic potential map (EPM) revealed similarities between these two nucleophiles, regarding net atomic charge, electron density distribution, and similar pKa values. Good scavenger efficacy was observed by chromatography and mass spectrometry results for the synthesised molecules, which revealed that p-methoxyphenol can be used as a potent scavenger during SPPS by t-Boc/Bzl strategy, as similar results were obtained using the conventional scavenger.

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.

Mycobacterium tuberculosis H37Rv LpqG Protein Peptides Can Inhibit Mycobacterial Entry through Specific Interactions.

Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease causing major mortality worldwide. As part of a systematic methodology for studying M. tuberculosis surface proteins which might be involved in host-pathogen interactions, our group found that LpqG surface protein (Rv3623) found in M. tuberculosis complex strains was located on the mycobacterial envelope and that peptide 16661 (21SGCDSHNSGSLGADPRQVTVY40) had high specific binding to U937 monocyte-derived macrophages and inhibited mycobacterial entry to such cells in a concentration-dependent way. A region having high specific binding to A549 alveolar epithelial cells was found which had low mycobacterial entry inhibition. As suggested in previous studies, relevant sequences in the host-pathogen interaction do not induce an immune response and peptides characterised as HABPs are poorly recognised by sera from individuals regardless of whether they have been in contact with M. tuberculosis. Our approach to designing a synthetic, multi-epitope anti-tuberculosis vaccine has been based on identifying sequences involved in different proteins' mycobacteria-target cell interaction and modifying their sequence to improve their immunogenic characteristics, meaning that peptide 16661 sequence should be considered in such design.

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 falciparumconserved 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.

A Large Size Chimeric Highly Immunogenic Peptide Presents Multistage Plasmodium Antigens as a Vaccine Candidate System against Malaria.

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.

Protecting capacity against malaria of chemically defined tetramer forms based on the Plasmodium falciparum apical sushi protein as potential vaccine components.

Developing novel generations of subunit-based antimalarial vaccines in the form of chemically-defined macromolecule systems for multiple antigen presentation represents a classical problem in the field of vaccine development. Many efforts involving synthesis strategies leading to macromolecule constructs have been based on dendrimer-like systems, the condensation of large building blocks and conventional asymmetric double dimer constructs, all based on lysine cores. This work describes novel symmetric double dimer and condensed linear constructs for presenting selected peptide multi-copies from the apical sushi protein expressed in Plasmodium falciparum. These molecules have been proved to be safe and innocuous, highly antigenic and have shown strong protective efficacy in rodents challenged with two Plasmodium species. Insights into systematic design, synthesis and characterisation have led to such novel antigen systems being used as potential platforms for developing new anti-malarial vaccine candidates.

Functional, biochemical and 3D studies of Mycobacterium tuberculosis protein peptides for an effective anti-tuberculosis vaccine.

Tuberculosis (TB) is an air-born, transmissible disease, having an estimated 9.4 million new TB cases worldwide in 2009. Eventual control of this disease by developing a safe and efficient new vaccine able to detain its spread will have an enormous impact on public health policy. Selecting potential antigens to be included in a multi-epitope, minimal subunit-based, chemically-synthesized vaccine containing the minimum sequences needed for blocking mycobacterial interaction with host cells is a complex task due to the multiple mechanisms involved in M. tuberculosis infection and the mycobacterium's immune evasion mechanisms. Our methodology, described here takes into account a highly robust, specific, sensitive and functional approach to the search for potential epitopes to be included in an anti-TB vaccine; it has been based on identifying short mycobacterial protein fragments using synthetic peptides having high affinity interaction with alveolar epithelial cells (A549) and monocyte-derived macrophages (U937) which are able to block the microorganism's entry to target cells in in vitro assays. This manuscript presents a review of the results obtained with some of the MTB H37Rv proteins studied to date, aimed at using these high activity binding peptides (HABPs) as platforms to be included in a minimal subunit-based, multiepitope, chemically-synthesized, antituberculosis vaccine.

Immunologic evaluation and validation of methods using synthetic peptides derived from Mycobacterium tuberculosis for the diagnosis of tuberculosis infection.

The goal of this study was to demonstrate the usefulness of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB). This assay used 20 amino acid-long, non-overlapped synthetic peptides that spanned the complete Mycobacterium tuberculosis ESAT-6 and Ag85A sequences. The validation cohort consisted of 1,102 individuals who were grouped into the following five diagnostic groups: 455 patients with PTB, 60 patients with EPTB, 40 individuals with non-EPTB, 33 individuals with leprosy and 514 healthy controls. For the PTB group, two ESAT-6 peptides (12033 and 12034) had the highest sensitivity levels of 96.9% and 96.2%, respectively, and an Ag85A-peptide (29878) was the most specific (97.4%) in the PTB groups. For the EPTB group, two Ag85A peptides (11005 and 11006) were observed to have a sensitivity of 98.3% and an Ag85A-peptide (29878) was also the most specific (96.4%). When combinations of peptides were used, such as 12033 and 12034 or 11005 and 11006, 99.5% and 100% sensitivities in the PTB and EPTB groups were observed, respectively. In conclusion, for a cohort that consists entirely of individuals from Venezuela, a multi-antigen immunoassay using highly sensitive ESAT-6 and Ag85A peptides alone and in combination could be used to more rapidly diagnose PTB and EPTB infection.

Decreasing the configurational entropy and the hydrophobicity of EBV-derived peptide 11389 increased its antigenicity, immunogenicity and its ability of inducing IL-6.

Peptide 11389 from CD21-binding region of EBV-gp350/220 protein binds to PBMCs inducing IL-6 expression and inhibiting EBV-binding to PBMCs. In addition, anti-peptide 11389 antibodies recognize EBV-infected cells and inhibit both EBV infection and IL-6 production in PBMCs. We have postulated that native structure stabilization of peptide 11389 sequence can increase its biological activity. The strategy was to modify its sequence to restrict the number of structures that peptide 11389 could acquire in solution (decreasing peptide's configurational entropy) and to weaken the non-relevant intermolecular interactions (decreasing its hydrophobicity), preserving CD21-interacting residues and structure as displayed in the native protein. Thirteen analog peptides were designed and synthesized; most of them were monomers containing an intra-chain disulfide bridge. Analog peptides 34058, 34060, 34061, 34296, 34298, 34299 and 34300 inhibited EBV invasion of PBMCs. Peptides 34059, 34060, 34295 and 34297 induced IL-6 levels in PBMCs (EC50=3.4, 3.3, 0.5, 0.5 µM, respectively) at higher potency than peptide 11389 (EC50=5.8 µM). Peptides 34057, 34059, 34060, 34301 and 34302 interacted with anti-EBV antibodies with affinities from 3 to 50 times higher than peptide 11389. Most of analog peptides were highly immunogenic and elicited antibodies that cross-react with EBV. In conclusion, we have designed peptides displaying higher biological activity than peptide 11389.

Binding activity, structure, and immunogenicity of synthetic peptides derived from Plasmodium falciparum CelTOS and TRSP proteins.

Several sporozoite proteins have been associated with Plasmodium falciparum cell traversal and hepatocyte invasion, including the cell-traversal protein for ookinetes and sporozoites (CelTOS), and thrombospondin-related sporozoite protein (TRSP). CelTOS and TRSP amino acid sequences have been finely mapped to identify regions specifically binding to HeLa and HepG2 cells, respectively. Three high-activity binding peptides (HABPs) were found in CelTOS and one HABP was found in TRSP, all of them having high α-helical structure content. These HABPs' specific binding was sensitive to HeLa and HepG2 cells' pre-treatment with heparinase I and chondroitinase ABC. Despite their similarity at three-dimensional (3D) structural level, TRSP and TRAP HABPs located in the TSR domain did not compete for the same binding sites. CelTOS and TRSP HABPs were used as a template for designing modified sequences to then be assessed in the Aotus monkey experimental model. Antibodies directed against these modified HABPs were able to recognize both the native parasite protein by immunofluorescence assay and the recombinant protein (expressed in Escherichia coli) by Western blot and ELISA assays. The results suggested that these modified HABPs could be promising targets in designing a fully effective, antimalarial vaccine.

Diagnostic Potential of the Serological Response to Synthetic Peptides from Mycobacterium tuberculosis Antigens for Discrimination Between Active and Latent Tuberculosis Infections.

Diagnosis and treatment of active tuberculosis (ATB) as well as latent tuberculosis infection (LTBI) are required for effective tuberculosis (TB) control, especially in TB endemic area. The usefulness of conventional tests to distinguish between ATB and LTBI has remained challenging. The present study was aimed to demonstrate the usefulness of the serological response to synthetic peptides from Mycobacterium tuberculosis (Mtb) antigens for discrimination between ATB and LTBI in Warao Amerindians. Serum IgG antibody levels were measured by the indirect ELISA assay using 22 designed and synthesized peptides derived from immunogenic Mtb ESAT-6 and Ag85A proteins. A total of 211 adult Warao Amerindians were included; cases with active TB (ATB, n = 75), latent TB infection (LTBI, n = 85) and non-infected (NI, n = 51). The approach's diagnostic information was compared using receiver operating characteristic (ROC) curves. For ATB diagnostic performance between ATB and NI; ESAT-6; P-12037 had 100% of sensitivity (AUC = 0.812; 0.733 to 0.891 95% CI); and Ag85A; P-10997 had 100% of specificity (AUC = 0.691; 0.597 to 0.785 95% CI); and ATB and LTBI; Ag85A; P-29878 had 100% of sensitivity (AUC = 0.741; 0.666-0.817 95% CI), and P-29879 had 99% of specificity (AUC = 0.679; 0.593-0.765 95% CI). While that ESAT-6 P-12037 also allowed differentiation between LTBI and NI or healthy ones. It had 98.8% of sensitivity and 98.0% of specificity (AUC = 0.640; 0.545-0.735 95% CI). The potential of combination-antigen immunoassays with peptides could discriminate between Warao Amerindians with ATB, LTBI and NI. Further validation of this approach could lead to developing a complementary tool for rapid diagnosis of TB infections.

Stereo electronic principles for selecting fully-protective, chemically-synthesised malaria vaccines.

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 (HLADRß̞1*) 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-DRß1* structures' Pockets 1 to 9, the role of polyproline II-like (PPIIL) structures having their O and N backbone atoms orientated for establishing H-bonds with specific HLA-DRß1*-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 PPIIL 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.

Computational prediction and experimental assessment of secreted/surface proteins from Mycobacterium tuberculosis H37Rv.

The mycobacterial cell envelope has been implicated in the pathogenicity of tuberculosis and therefore has been a prime target for the identification and characterization of surface proteins with potential application in drug and vaccine development. In this study, the genome of Mycobacterium tuberculosis H37Rv was screened using Machine Learning tools that included feature-based predictors, general localizers and transmembrane topology predictors to identify proteins that are potentially secreted to the surface of M. tuberculosis, or to the extracellular milieu through different secretory pathways. The subcellular localization of a set of 8 hypothetically secreted/surface candidate proteins was experimentally assessed by cellular fractionation and immunoelectron microscopy (IEM) to determine the reliability of the computational methodology proposed here, using 4 secreted/surface proteins with experimental confirmation as positive controls and 2 cytoplasmic proteins as negative controls. Subcellular fractionation and IEM studies provided evidence that the candidate proteins Rv0403c, Rv3630, Rv1022, Rv0835, Rv0361 and Rv0178 are secreted either to the mycobacterial surface or to the extracellular milieu. Surface localization was also confirmed for the positive controls, whereas negative controls were located on the cytoplasm. Based on statistical learning methods, we obtained computational subcellular localization predictions that were experimentally assessed and allowed us to construct a computational protocol with experimental support that allowed us to identify a new set of secreted/surface proteins as potential vaccine candidates.

T Cell Peptides Derived from Invasive Stages of Schistosoma mansoni as Potential Schistosomiasis Vaccine.

Schistosomiasis is a parasitic disease that affects 143 million people in endemic countries. This work analyzed overexpressed sequences from the cercaria phase to the early schistosomulum phase using bioinformatics tools to predict host interaction and selected proteins for predicting T cell epitopes. The final peptides were chemically synthesized, and their toxicity was evaluated in vitro. Peptides were formulated in the Adjuvant Adaptation (ADAD) vaccination system and injected into BALB/c mice that were challenged with S. mansoni cercariae to assess protection and immunogenicity. A total of 39 highly expressed S.mansoni proteins were identified as being of potential interest. Three T cell peptides predicted to bind MHC mouse and human class II were synthesized and formulated for vaccination. SmGSP and SmIKE reduced the number of eggs trapped in the liver by more than 50% in challenged BALB/c mice. The liver of mice vaccinated with either SmGSP or SmTNP had a significantly reduced affected liver surface. Transcriptome-based T cell peptides elicit partial protection and could be candidates for a multiantigen vaccine.

ESAT-6 and Ag85A Synthetic Peptides as Candidates for an Immunodiagnostic Test in Children with a Clinical Suspicion of Tuberculosis.

BACKGROUND: Tuberculosis (TB) is being underdetected in children as most are smear-negative. This work was aimed at evaluating ESAT-6 and Ag85A synthetic peptides' serodiagnostic potential for diagnosing children having a clinical suspicion of TB. METHODS: The study involved 438 children: 77 Creole nonindigenous (13 suspected of having TB and 64 healthy ones) and 361 Warao indigenous children (39 suspected of TB and 322 healthy children). The approach's diagnostic information was compared using operational characteristics and receiver-operating characteristic (ROC) curves. RESULTS: Ag85A P-29879 had 94.6% sensitivity (AUC = 0.741: 0.651 to 0.819 95% CI) in indigenous children. ESAT-6 P-12036 and P-12037 had 100% and 92.3% of sensitivity (AUC = 0.929: 0.929: 0.846 to 0.975 95% CI and 0.791: 63.9 to 98.7 95% CI, respectively) in Creole children. ESAT-6 peptides also allowed a differentiation between children with TB and healthy ones. CONCLUSIONS: Further validation of this approach could lead to developing a complementary tool for rapid TB diagnosis in children.

Conserved regions from Plasmodium falciparum MSP11 specifically interact with host cells and have a potential role during merozoite invasion of red blood cells.

Despite significant global efforts, a completely effective vaccine against Plasmodium falciparum, the species responsible for the most serious form of malaria, has not been yet obtained. One of the most promising approaches consists in combining chemically synthesized minimal subunits of parasite proteins involved in host cell invasion, which has led to the identification of peptides with high binding activity (named HABPs) to hepatocyte and red blood cell (RBC) surface receptors in a large number of sporozoite and merozoite proteins, respectively. Among these proteins is the merozoite surface protein 11 (MSP11), which shares important structural and immunological features with the antimalarial vaccine candidates MSP1, MSP3, and MSP6. In this study, 20-mer-long synthetic peptides spanning the complete sequence of MSP11 were assessed for their ability to bind specifically to RBCs. Two HABPs with high ability to inhibit invasion of RBCs in vitro were identified (namely HABPs 33595 and 33606). HABP-RBC bindings were characterized by means of saturation assays and Hill analysis, finding cooperative interactions of high affinity for both HABPs (n(H) of 1.5 and 1.2, K(d) of 800 and 600 nM for HABPs 33595 and 33606, respectively). The nature of the possible RBC receptors for MSP11 HABPs was studied in binding assays to enzyme-treated RBCs and cross-linking assays, finding that both HABPs use mainly a sialic acid-dependent receptor. An analysis of the immunological, structural and polymorphic characteristics of MSP11 HABPs supports including these peptides in further studies with the aim of designing a fully effective protection-inducing vaccine against malaria.

Atomic evidence that modification of H-bonds established with amino acids critical for host-cell binding induces sterile immunity against malaria.

Based on the 3D X-ray crystallographic structures of relevant proteins of the malaria parasite involved in invasion to host cells and 3D NMR structures of High Activity Binding Peptides (HABPs) and their respective analogues, it was found that HABPs are rendered into highly immunogenic and sterile immunity inducers in the Aotus experimental model by modifying those amino acids that establish H-bonds with other HABPs or binding to host's cells. This finding adds striking and novel physicochemical principles, at the atomic level, for a logical and rational vaccine development methodology against infectious disease, among them malaria.

Mycobacterium tuberculosis Rv0679c protein sequences involved in host-cell infection: potential TB vaccine candidate antigen.

BACKGROUND: To date, the function of many hypothetical membrane proteins of Mycobacterium tuberculosis is still unknown and their involvement in pathogen-host interactions has not been yet clearly defined. In this study, the biological activity of peptides derived from the hypothetical membrane protein Rv0679c of M. tuberculosis and their involvement in pathogen-host interactions was assessed. Transcription of the Rv0679c gene was studied in 26 Mycobacterium spp. Strains. Antibodies raised against putative B-cell epitopes of Rv0679c were used in Western blot and immunoelectron microscopy assays. Synthetic peptides spanning the entire length of the protein were tested for their ability to bind to A549 and U937 cells. High-activity binding peptides (HABPs) identified in Rv0679c were tested for their ability to inhibit mycobacterial invasion into cells. RESULTS: The gene encoding Rv0679c was detected in all strains of the M. tuberculosis complex (MTC), but was only transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra and M. africanum. Anti-Rv0679c antibodies specifically recognized the protein in M. tuberculosis H37Rv sonicate and showed its localization on mycobacterial surface. Four HABPs inhibited invasion of M. tuberculosis to target cells by up to 75%. CONCLUSIONS: The results indicate that Rv0679c HABPs and in particular HABP 30979 could be playing an important role during M. tuberculosis invasion of host cells, and therefore could be interesting research targets for studies aimed at developing strategies to control tuberculosis.

α-Helix peptides designed from EBV-gH protein display higher antigenicity and induction of monocyte apoptosis than the native peptide.

We tested the hypothesis that stabilizing α-helix of Epstein-Barr virus gH-derived peptide 11438 used for binding human cells will increase its biological activity. Non-stable α-helix of peptide 11438 was unfolded in an entropy-driven process, despite the opposing effect of the enthalpy factor. Adding and/or changing amino acids in peptide 11438 allowed the designing of peptides 33207, 33208 and 33210; peptides 33208 and 33210 displayed higher helical content due to a decreased unfolding entropy change as was determined by AGADIR, molecular dynamics and circular dichroism analysis. Peptides 33207, 33208 and 33210 inhibited EBV invasion of peripheral blood mononuclear cells and displayed epitopes more similar to native protein than peptide 11438; these peptides could be useful for detecting antibodies induced by native gH protein since they displayed high reactivity with anti-EBV antibodies. Anti-peptide 33207 antibodies showed higher reactivity with EBV than anti-peptide 11438 antibodies being useful for inducing antibodies against EBV. Anti-peptide 33210 antibodies inhibit EBV invasion of epithelial cells better than anti-peptide 11438 antibodies. Peptide 33210 bound to normal T lymphocytes and Raji cells stronger than peptide 11438 and also induced apoptosis of monocytes and Raji cells but not of normal T cells in a similar way to EBV-gH. Peptide 33210 inhibited the monocytes' development toward dendritic cells better than EBV and peptide 11438. In conclusion, stabilizing the α-helix in peptides 33208 and 33210 designed from peptide 11438 increased the antigenicity and the ability of the antibodies induced by peptides of inhibiting EBV invasion of host cells.

Identification and characterization of the Plasmodium vivax thrombospondin-related apical merozoite protein.

BACKGROUND: Malaria caused by Plasmodium vivax is a major public health problem worldwide that affects 70-80 million people in the Middle East, Asia, Western Pacific, South America and the Caribbean. Despite its epidemiological importance, few antigens from this parasite species have been characterized to date compared to Plasmodium falciparum, due in part to the difficulties of maintaining an in vitro culture of P. vivax. This study describes the identification of the P. falciparum thrombospondin-related apical merozoite protein homologue in P. vivax (PvTRAMP) and examines its potential to be further evaluated as vaccine candidate. METHODS: The gene encoding PvTRAMP was identified through an extensive search of the databases hosting the genome sequence of P. vivax. Genes adjacent to pvtramp were identified in silico to determine the degree of similarity between the protein sequences encoded by equivalent chromosomic fragments in P. falciparum and Plasmodium knowlesi. The pvtramp gene was amplified from cDNA of P. vivax schizont stages, cloned and expressed in Escherichia coli. Anti-PvTRAMP antisera was obtained by inoculating rabbits with PvTRAMP B cell epitopes produced as synthetic peptides in order to assess its recognition in parasite lysates by Western blot and in intact parasites by indirect immunofluorescence. The recognition of recombinant PvTRAMP by sera from P. vivax-infected individuals living in endemic areas was also assessed by ELISA. RESULTS: The PfTRAMP homologue in P. vivax, here denoted as PvTRAMP, is a 340-amino-acid long antigen encoded by a single exon that could have a potential role in cytoadherence, as indicated by the presence of a thrombospondin structural homology repeat (TSR) domain. According to its transcription and expression profile, PvTRAMP is initially located at the parasite's apical end and later on the parasite surface. Recombinant PvTRAMP is recognized by sera from infected patients, therefore, indicating that it is targeted by the immune system during a natural infection with P. vivax. CONCLUSIONS: The results of this work support conducting further studies with PvTRAMP to evaluate its immunogenicity and protection-inducing ability in the Aotus animal model.