In Vitro Antifungal Activity of LL-37 Analogue Peptides against Candida spp.

Fungal infections have increased in recent decades with considerable morbidity and mortality, mainly in immunosuppressed or admitted-to-the-ICU patients. The fungal resistance to conventional antifungal treatments has become a public health problem, especially with Candida that presents resistance to several antifungals. Therefore, generating new alternatives of antifungal therapy is fundamental. One of these possibilities is the use of antimicrobial peptides, such as LL-37, which acts on the disruption of the microorganism membrane and promotes immunomodulatory effects in the host. In this study, we evaluated the in vitro antifungal activity of the LL-37 analogue peptides (AC-1, LL37-1, AC-2, and D) against different Candida spp. and clinical isolates obtained from patients with vulvovaginal candidiasis. Our results suggest that the peptides with the best ranges of MICs were LL37-1 and AC-2 (0.07 µM) against the strains studied. This inhibitory effect was confirmed by analyzing the yeast growth curves that evidenced a significant decrease in the fungal growth after exposure to LL-37 peptides. By the XTT technique we observed a significant reduction in the biofilm formation process when compared to yeasts untreated with the analogue peptides. In conclusion, we suggest that LL-37 analogue peptides may play an important antimicrobial role against Candida spp.

COVID-19 Infection Detection and Prevention by SARS-CoV-2 Active Antigens: A Synthetic Vaccine Approach.

COVID-19, a global pandemic causing to date more than 50 million cases and more than a million deaths, has to be controlled. SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) was identified as the causative agent. Controversy about this virus origin and infectious mechanism for adapting to humans remains a matter for discussion. Among all strategies for obtaining safe and potent vaccines, approaches based on attenuated-killed virus and non-replicating RNA viral vectors are demonstrating promising results. However, specificity of viral components targeted by human antibodies so far has not been demonstrated. A consistent strategy for obtaining functional-active antigens from SARS-CoV-2 specific ligands lead us to propose and test a number of synthetic components. From hundreds of starting sequences only fifteen fulfilled the design requirements and were produced as monomer and polymer forms and immuno-chemically tested. The design was based on worldwide representative reported virus genomes. A bioinformatics scheme by conventional methods and knowledge on MHC-I and II antigen processing mechanisms and HLA haplotype-restriction was performed including sensitive and resistant human populations to virus infection. Covid-19 patients' sera reactivity for synthetic SARS-CoV-2-designed components have proven a high recognition of specific molecules, as well as some evidence for a long-lasting humoral immune response.

Quantifying intracellular Mycobacterium tuberculosis: An essential issue for in vitro assays.

Many studies about intracellular microorganisms which are important regarding diseases affecting public health have been focused on the recognition of host-pathogen interactions, thereby ascertaining the mechanisms by which the pathogen invades a cell and makes it become its host. Such knowledge enables understanding the immunological response triggered by these interactions for obtaining useful information for developing vaccines and drugs. Quantitative cell infection assay protocols are indispensable regarding studies involving Mycobacterium tuberculosis, which takes the lives of more than 2 million people worldwide every year; however, sometimes these are limited by the pathogen's slow growth. Concerning such limitation, a detailed review is presented here regarding the different methods for quantifying and differentiating an intracellular pathogen, the importance of mycobacteria aggregate dissociation and multiplicity of infection (MOI) in infection assays. The methods' differences, advantages, and disadvantages are discussed regarding intra and extracellular bacteria (on cell surface) differentiation, current problems are outlined, as are the solutions provided using fluorophores and projections made concerning quantitative infection assays.

The ATPase activity of the mycobacterial plasma membrane is inhibited by the LL37-analogous peptide LLAP.

The emergence of multidrug-resistant Mycobacterium tuberculosis strains has led to the development of new antituberculous agents. In this context, antimicrobial targeting proteins to the cell membrane are interesting due to the avoidance of the plasma membrane permeation. Through this study, the antimicrobial activity, cellular toxicity, as well as the effect on the mycobacterial cell membrane ATPase activity of a cathelicidin-analogous peptide were assessed. By using bioinformatics analyses, a 15 amino acid LL37-analogous peptide called LLAP, which has the amino acid sequence: GRKSAKKIGKRAKRI, was designed to improve its helical structure and antibacterial activity compared to the native sequence. The LLAP peptide was synthesized, purified by RP-HPLC and its structural characteristics were determined by MALDI-TOF MS and circular dichroism. Compared to the native amino acid sequence, the minimum inhibitory concentration and cytotoxic activity of LLAP were 4.0 and 5.6-fold lower, respectively. In addition, the hemolytic activity of LLAP was lower than 1.1% and the cytotoxic activity of peptides was similar for both peptides. Interestingly, the LLAP peptide displayed approximately 50% inhibition of basal ATPase activity of the mycobacterial plasma membrane, which could in turn be associated with the impaired cell viability. The results suggest that LLAP could be considered as potential antimycobacterial compounds against cell membrane targeting ATPases. However, this antimycobacterial activity can be improved. It is expected further applications to be found for other antimicrobial peptides families based on the implemented methodology.

An unstable Th epitope of P. falciparum fosters central memory T cells and anti-CS antibody responses.

Malaria is transmitted by Plasmodium-infected anopheles mosquitoes. Widespread resistance of mosquitoes to insecticides and resistance of parasites to drugs highlight the urgent need for malaria vaccines. The most advanced malaria vaccines target sporozoites, the infective form of the parasite. A major target of the antibody response to sporozoites are the repeat epitopes of the circumsporozoite (CS) protein, which span almost one half of the protein. Antibodies to these repeats can neutralize sporozoite infectivity. Generation of protective antibody responses to the CS protein (anti-CS Ab) requires help by CD4 T cells. A CD4 T cell epitope from the CS protein designated T* was previously identified by screening T cells from volunteers immunized with irradiated P. falciparum sporozoites. The T* sequence spans twenty amino acids that contains multiple T cell epitopes restricted by various HLA alleles. Subunit malaria vaccines including T* are highly immunogenic in rodents, non-human primates and humans. In this study we characterized a highly conserved HLA-DRß1*04:01 (DR4) restricted T cell epitope (QNT-5) located at the C-terminus of T*. We found that a peptide containing QNT-5 was able to elicit long-term anti-CS Ab responses and prime CD4 T cells in HLA-DR4 transgenic mice despite forming relatively unstable MHC-peptide complexes highly susceptible to HLA-DM editing. We attempted to improve the immunogenicity of QNT-5 by replacing the P1 anchor position with an optimal tyrosine residue. The modified peptide QNT-Y formed stable MHC-peptide complexes highly resistant to HLA-DM editing. Contrary to expectations, a linear peptide containing QNT-Y elicited almost 10-fold lower long-term antibody and IFN-γ responses compared to the linear peptide containing the wild type QNT-5 sequence. Some possibilities regarding why QNT-5 is more effective than QNT-Y in inducing long-term T cell and anti-CS Ab when used as vaccine are discussed.

Structural features of the two-component systemLisR/LisK suggests multiple responses for the adaptation and survival of Listeria monocytogenes

Se caracterizó la estructura del sistema deregulación de dos componentes LisR/LisK de Listeriamonocytogenes. Se emplearon herramientas bioinformáticas ybases de datos para predecir la estructura e interacciones delas dos proteínas y se modelaron. Los resultados predicenque la proteína LisK está embebida en la membrana celulary su composición modular (dominios HAMP, histidinakinasa and ATPasa) está asociada a su autofosforilación(His-266). Un efecto estímulo respuesta determina lapropagación secuencial de la señal desde la membranacelular hacia componentes citoplasmáticos. A su vez, sepredice que LisR es una proteína citosólica con un dominiode receptor (homólogo a cheY) que incluye el residuofosfo-aceptor (Asp-52) y el dominio de unión a ADN,el cual puede permitir la transmisión de una respuestaespecífica a nivel transcripcional. Los componentes LisR/LisK han sido bioquímica y funcionalmente caracterizadosexperimentalmente en la patofisiología de otros bacilos. Espor ello, que la aproximación de los resultados basados enestructura-función podría facilitar el diseño de inhibidoresespecíficos...

Here, we characterized the structure of the two-component regulatory system, LisR/LisK, in Listeriamonocytogenes. To predict the structure of both proteins and the relationship between them, we employedseveral bioinformatic tools and databases. Based on our results, LisK protein is embedded in the cellmembrane and its modular composition (HAMP, histidine kinase and ATPase domains) is associatedwith its autophosphorylation (His-266). A stimulus-response likely determines the sequential signalpropagation from the bacterial cell surface to its cytoplasmic components. According to our results,LisR is a cytoplasmic protein with a receptor domain (homologous to CheY) that comprises a phosphoacceptorresidue (Asp-52) and a DNA-binding domain, which may allow the transmission of a specifictranscriptional response. LisR/LisK has been experimentally characterized both biochemically andfunctionally in other Bacilli pathophysiology; our structure-function approach may facilitate the design ofsuitable inhibitors...

O objetivo do estudo foi caracterizarestruturalmente o sistema de regulação de dois componentesLisR/LisK de Listeria monocytogenes. Foram utilizadasdiversas ferramentas de bioinformática e bancos de dadospara predizer a estrutura das duas proteínas, modelálase prever suas interações. Os resultados predizemque a proteína Lisk está incorporada na composição damembrana celular e sua composição modular (domíniosHAMP, histidina quinase e ATPase) está associada com asua autofosforilação (His-266). Um efeito de estímulo eresposta determina a propagação sequencial do sinal a partirda membrana celular em componentes citoplasmáticos. Osresultados predizem que LisR é uma proteína citosólicacom um domínio recetor (homólogo a CheY) que inclui oresíduo fosfo-aceitador (Asp-52) e o domínio de ligação aoADN, o que pode permitir a transmissão de uma respostaespecífica a nível transcricional. Como LisR/Lisk foi,química e funcionalmente, caracterizada experimentalmentena fisiopatologia de outros bacilos, esta abordagem baseadana estrutura-função pode facilitar a conceção de inibidoresespecíficos...

Estrategias metacognitivas y comprensión lectora en universitarios miembros de comunidades indígenas / Metacognitive strategies and reading comprehension in universitary students members of indigenous communities

Son escasos los estudios realizados sobre estrategias metacognitivas y compresión lectora en estudiantes indígenas, y menos aún en población universitaria. Este estudio descriptivo buscó identificar y analizar las estrategias metacognitivas usadas en relación con la comprensión lec-tora durante actividades estructuradas, con el fin de generar unas recomendaciones académicas generales para el programa institucional. El diseño metodológico incluyó informes verbales, en-trevistas, observación y cuestionarios para indagar sobre el uso de las estrategias metacognitivas de los universitarios. Participaron un grupo de 7 estudiantes miembros de comunidades indí-genas, 3 mujeres y 4 hombres, en edades comprendidas entre 17 y 22 años. Se encontró que los participantes conocen las estrategias metacognitivas que permiten mejorar el nivel de compren-sión lectora, aunque presentan dificultad en la planificación, supervisión y evaluación de estas. Se encontraron rasgos que denotan procesos de adaptación a una nueva cultura académica. Los resultados evidenciaron un cambio y una adaptación a nuevas culturas académicas, que traen consigo una serie de retos y dificultades que se reflejan en el rendimiento académico.

Studies of metacognitive strategies in relation to reading comprehension in college Indians stu-dents are very few. However, there are studies and research in basic secondary students. What drove this research in the university population to expand speech therapy practice. Identify and analyze students' metacognitive strategies in indigenous members of the Universidad Nacional de Colombia in relation to reading comprehension during structured activities. We conducted a descriptive study in which we used: verbal reports, interviews, observation and questionnaires to investigate the use of metacognitive strategies of Indian students. The study included a sample of 7 student members of indigenous communities, 3 women and 4 men, aged between 17 and 22. It was found that the participants know the metacognitive strategies that improve reading com-prehension level, but have difficulty in planning, monitoring and evaluation of these. Features were found which denote processes of adaptation to a new academic culture. The results are evidence of change and adaptation to new academic cultures, which brings a number of conse-quences that are reflected in academic performance.

In silico identification and characterization of the ion transport specificity for P-type ATPases in the Mycobacterium tuberculosis complex.

BACKGROUND: P-type ATPases hydrolyze ATP and release energy that is used in the transport of ions against electrochemical gradients across plasma membranes, making these proteins essential for cell viability. Currently, the distribution and function of these ion transporters in mycobacteria are poorly understood. RESULTS: In this study, probabilistic profiles were constructed based on hidden Markov models to identify and classify P-type ATPases in the Mycobacterium tuberculosis complex (MTBC) according to the type of ion transported across the plasma membrane. Topology, hydrophobicity profiles and conserved motifs were analyzed to correlate amino acid sequences of P-type ATPases and ion transport specificity. Twelve candidate P-type ATPases annotated in the M. tuberculosis H37Rv proteome were identified in all members of the MTBC, and probabilistic profiles classified them into one of the following three groups: heavy metal cation transporters, alkaline and alkaline earth metal cation transporters, and the beta subunit of a prokaryotic potassium pump. Interestingly, counterparts of the non-catalytic beta subunits of Hydrogen/Potassium and Sodium/Potassium P-type ATPases were not found. CONCLUSIONS: The high content of heavy metal transporters found in the MTBC suggests that they could play an important role in the ability of M. tuberculosis to survive inside macrophages, where tubercle bacilli face high levels of toxic metals. Finally, the results obtained in this work provide a starting point for experimental studies that may elucidate the ion specificity of the MTBC P-type ATPases and their role in mycobacterial infections.

Effect of antimicrobial peptides on ATPase activity and proton pumping in plasma membrane vesicles obtained from mycobacteria.

The potential usefulness of antimicrobial peptides (AMPs) as antimycobacterial compounds has not been extensively explored. Although a myriad of studies on AMPs from different sources have been done, some of its mechanisms of action are still unknown. Maganins are of particular interest since they do not lyse non-dividing mammalian cells. In this work, AMPs with well-recognized activity against bacteria were synthesized, characterized, purified and their antimycobacterial activity and influence on ATPase activity in mycobacterial plasma membrane vesicles were assessed. Using bioinformatics tools, a magainin-I analog peptide (MIAP) with improved antimicrobial activity was designed. The influence of MIAP on proton (H(+)) pumping mediated by F(1)F(0)-ATPase in plasma membrane vesicles obtained from Mycobacterium tuberculosis was evaluated. We observed that the antimycobacterial activity of AMPs was low and variable. However, the activity of the designed peptide MIAP against M. tuberculosis was 2-fold higher in comparison to magainin-I. The basal ATPase activity of mycobacterial plasma membrane vesicles decreased approximately 24-30% in the presence of AMPs. On the other hand, the MIAP peptide completely abolished the F(1)F(0)-ATPase activity involved in H(+) pumping across M. tuberculosis plasma membranes vesicles at levels similar to the specific inhibitor N,N' dicyclohexylcarbodiimide. These finding suggest that AMPs can inhibit the H(+) pumping F(1)F(0)-ATPase of mycobacterial plasma membrane that potentially interferes the internal pH and viability of mycobacteria.

Characterizatio of the adsorption process analogous peptides on alumina gel / Carcterización ddelproceso de adsorción de péptidos análogos sobree gel de alúmina / Caracterização do processo de adsorção peptídeos análogos em gel alumnaa

Peptide antigen adsorption on aluminum hydroxide gel must be characterized when formulating vaccines. In this work a peptide belonging to the amino-terminal region of Plasmodium falciparum Merozoite Surface Protein and its analogues have been characterized. The adsorption of 17 analogues on aluminum hydroxide which had greater than 10 mmol/L solubility was quantified at 298 K. Adsorption capacity and affinity constant parameters were calculated by applying the Langmuir's adsorption model. The results have been presented in three groups according to adsorption isotherm trajectory. The first group consists of analogues where the first organization of peptide molecules was presented at low concentrations, followed by a rapid increase in adsorption to high concentrations. The second group consists of analogues having an adsorption pattern showing the formation of a first layer at low peptide concentrations and a second layer at greater concentrations. The third group contains analogues whose adsorption involved the formation of two simple layers, this being differentiated from the second group in that after the second layer had been completed, the amount adsorbed grew notably with increased concentration. The results revealed a more complex pattern that monolayer or bilayer formation. This work constitutes the first approach towards establishing an adsorbed layer structure model using a peptide system.

La adsorción de un antígeno peptídico sobre gel de hidróxido de aluminio debe ser caracterizada para la formulación de vacunas. En este trabajo se caracterizó la adsorción de un péptido que pertenece a la región amino-terminal de la proteína de superficie del merozoite de Plasmodium falciparum y sus análogos. Se cuantificó la adsorción a 298 K sobre hidróxido de aluminio de 17 análogos con una solubilidad mayor de 10 mmol/L. Los parámetros de capacidad de adsorción y constante de afinidad se calcularon aplicando el modelo de adsorción de Langmuir. Los resultados se presentan en tres grupos, de acuerdo con la trayectoria de la isoterma de adsorción. El primer grupo consta de los análogos que presentaron la primera organización de las moléculas de péptido en concentraciones bajas, seguida de un rápido incremento de la adsorción a altas concentraciones. El segundo grupo de análogos tiene un patrón de adsorción que muestra la formación de una primera capa a concentraciones bajas de péptido y una segunda capa a concentraciones mayores. El tercer grupo contiene los análogos cuya adsorción muestra la formación de dos capas simples y se diferencia del segundo grupo en que después de la segunda capa, la cantidad adsorbida crece notablemente con el aumento de la concentración. Los resultados revelaron un patrón de adsorción más complejo que la formación de monocapa o bicapa. Este trabajo constituye la primera aproximación hacia el establecimiento de un modelo de estructura de la capa adsorbida en un sistema peptídico.

A adsorção de um antígeno peptídico sobre um gel de hidróxido de alumíniodeve de ser caracterizado para a formulação de vacinas. Neste estudo foi caracterizada a adsorção de um peptídeo que pertence á região amino-terminal da proteína de superfície do merozoito de Plasmodium falciparum e seus análogos. Foi quantificada a adsorção a 298 K sobre hidróxido de alumínio de 17 análogos com uma solubilidade maior de 10 mmol/L. Os parâmetros de capacidade de adsorção e constante de afinidade foram calculados aplicando o modelo de adsorção de Langmuir. Os resultados sãoapresentados em três grupos de acordo á trajectória da isoterma de adsorção. O primeiro grupo consta dos análogos que apresentaram a primeira organização das moléculas de peptídeoem concentraçõesbaixas, seguido de um rápido incremento da adsorção a altas concentrações. O segundo grupo de análogos tem um padrão de adsorção que mostra a formação de uma primeira camada a concentraçõesbaixas de peptídeo e uma segunda camada a concentraçõesmaiores. O terceiro grupo contém os análogos cuja adsorçãomostra a formação de duas camadas simples e é diferenciado do segundo grupo em que depois da segunda camada, a quantidade adsorvida cresce notavelmente com o aumento da concentração. Os resultados revelaram um padrão de adsorçãomais complexo que a formação de monocamada ou bicamada. Este trabalho constitui a primeira aproximaçãoao estabelecimento de um modelo de estrutura da camada adsorvida num sistema peptídico.

Construcción de una filogenia molecular para las especies de los géneros Klebsiella y Raoultella basada en los genes ARNr 16S y ARN polimerasa subunidad / Construction of a molecular phylogeny for klebsiella and Raoultella SP based on rRNA 165 and RNA polimarase subunit genes

Las bacterias de los géneros Raoultella y Klebsiella son patógenos oportunistas para las cuales no existe un sistema uniforme de clasificación taxonómica internacional. En el presente estudio se propone una filogenia molecular basada en el gen ribosomal 16S (ADNr 16S) y el gen codificante de la subunidad de la ARN polimerasa (rpoB) de los géneros Klebsiella y Raoultella con el fin de establecer relaciones evolutivas entre dichos géneros. Los resultados evidencian una agrupación acorde con la taxonomía y las propiedades bioquímicas características, reportadas en el Genbank. Se estableció una bifurcación en los árboles, lo cual confirma la separación de los géneros Klebsiella y Raoultella. Adicionalmente, se confirmó el carácter polifilético de K. aerogenes por el gen ADNr 16S y la agrupación de R. terrigena y K. oxytoca de acuerdo con el gen rpoB. La comparación entre los árboles obtenidos permitió determinar relaciones evolutivas entre las especies, a partir de los genes evaluados, lo cual refleja cambios aparentes a nivel taxonómico y corrobora la importancia del análisis a nivel de multilocus. Este tipo de estudios permite monitorear la estabilidad de los genotipos microbianos sobre la escala temporal y espacial, mejorar la precisión de las anotaciones taxonómicas (mejor descripción de taxones o subdivisiones genéticas) y evaluar la diversidad genética y adaptabilidad en términos de virulencia o resistencia a drogas.

The bacteria belonging to Raoultella and Klebsiella genera are opportunistic pathogens for which there is no consensus about a unique internationally taxonomic system. In this study, we suggest a molecular phylogeny based on 16S (rDNA 16S) ribosomal gene and the beta subunit RNA polymerase (rpoB) encoding gene of Klebsiella and Raoultella in order to set up evolutionary relationships among these genera. Results showed a cluster with similar biochemical and taxonomy characteristics in agreement to Genbank description, and a tree bifurcation which confirms the Klebsiella and Raoultella genera separation. Moreover, we verified the polyphyletic character of K. aerogenes by rDNA 16S and particularly the clustering for both R. terrigena and K. oxytoca based on rpoB gene. The evolutionary relationships recognition was obtained by comparison among the corresponding trees for both genes unraveling significant changes at taxonomic level and stressing the importance of multilocus analysis approach. These studies are useful for tracking the microbial genotype stability over time and space scale; as well as on improving taxonomic annotations (taxa descriptions and genetic subdivisions) and evaluation of genetic diversity in terms of virulence and drug resistance.

HLA-DR allele reading register shifting is associated with immunity induced by SERA peptide analogues.

SERA protein is a leading candidate molecule to be included in an antimalarial vaccine. Conserved high activity binding peptides (HABP) binding to red blood cells (RBC) have been identified in this protein. One of them (6762) localising in the 18-kDa C-terminal fragment was used to induce protective immunity with negative result. Critical RBC binding residues (assessed by glycine-analogue scanning) were replaced by others having the same mass, volume and surface but different polarity, rendering some of them immunogenic as assessed by antibody production against the parasite or its proteins and protection-inducing against challenge with a highly infectious Aotus monkey-adapted Plasmodium falciparum strain. A shift in binding to purified HLA-DR allelic molecules from the same haplotype and in their reading register was found, suggesting that modified molecules had adopted a different (1)H NMR 3D structure allowing a better fit into the MHCII-pept-TCR complex, thereby representing a new mechanism for inducing immune protection.

BTM-P1 polycationic peptide biological activity and 3D-dimensional structure.

The novel BTM-P1 peptide interferes with energetic processes in mitochondria; its antimicrobial activity against Gram-positive and Gram-negative bacteria is described here. BTM-P1 three-dimensional structure was determined by 1H NMR to explain its biological mechanisms and membrane activity. Structural data indicated that BTM-P1 can form an alpha-helix; circular dichroism analysis confirmed the peptide's propensity to behave as a typical transmembrane helix in a lipidic environment. According to the structural characteristics of the polycationic BTM-P1 peptide so revealed, its biological activity can be explained by a mechanism involving the formation of ion-permeable channels in biomembranes.

Major histocompatibility complex and T cell interactions of a universal T cell epitope from Plasmodium falciparum circumsporozoite protein.

A 20-residue sequence from the C-terminal region of the circumsporozoite protein of the malaria parasite Plasmodium falciparum is considered a universal helper T cell epitope because it is immunogenic in individuals of many major histocompatibility complex (MHC) haplotypes. Subunit vaccines containing T* and the major B cell epitope of the circumsporozoite protein induce high antibody titers to the malaria parasite and significant T cell responses in humans. In this study we have evaluated the specificity of the T* sequence with regard to its binding to the human class II MHC protein DR4 (HLA-DRB1*0401), its interactions with antigen receptors on T cells, and the effect of natural variants of this sequence on its immunogenicity. Computational approaches identified multiple potential DR4-binding epitopes within T*, and experimental binding studies confirmed the following two tight binding epitopes: one located toward the N terminus (the T*-1 epitope) and one at the C terminus (the T*-5 epitope). Immunization of a human DR4 volunteer with a peptide-based vaccine containing the T* sequence elicited CD4+ T cells that recognize each of these epitopes. Here we present an analysis of the immunodominant N-terminal epitope T*-1. T*-1 residues important for interaction with DR4 and with antigen receptors on T*-specific T cells were mapped. MHC tetramers carrying DR4/T*-1 MHC-peptide complexes stained and efficiently stimulated these cells in vitro. T*-1 overlaps a region of the protein that has been described as highly polymorphic; however, the particular T*-1 residues required for anchoring to DR4 were highly conserved in Plasmodium sequences described to date.

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

An effective malarial vaccine must contain multiple immunogenic, protection-inducing epitopes able to block and destroy the P. falciparum malaria parasite, the most lethal form of this disease in the world. Our strategy has consisted in using conserved peptides blocking parasite binding to red blood cells; however, these peptides are non-immunogenic and non-protection-inducing. Modifying their critical residues can make them immunogenic. Such peptides induced antibody titers (determined by immunofluorescence antibody test, IFA) and made the latter reactive (determined by Western blot) and protection inducing against experimental challenge with a highly infective Aotus monkey adapted P. falciparum strain. Modified peptides also induce highly non-protective long-lasting antibody levels. Modifications performed might allow them to bind specifically to different HLA-DRbeta purified molecules. These immunological and biological activities are associated with modifications in their three-dimensional structure as determined by (1)H-NMR. It was found that modified, high non-protective long-lasting antibody level peptides bound to HLA-DR molecules from a different haplotype (to which immunogenic, protection-inducers bind) and had 4.6 +/- 1.4 A shorter distances between residues fitting into these molecules' Pocket 1 to Pocket 9, suggesting fitting into an inappropriate HLA-DR molecule. A multi-component, subunit-based, malarial vaccine is therefore feasible if modified peptides are suitably modified for an appropriate fit into the correct HLA-DRbeta1* molecule in order to form a proper MHC-II-peptide-TCR complex.

Protective cellular immunity against P. falciparum malaria merozoites is associated with a different P7 and P8 residue orientation in the MHC-peptide-TCR complex.

Developing a logical and rational methodology for obtaining vaccines, especially against the main parasite causing human malaria (P. falciparum), consists of blocking receptor-ligand interactions. Conserved peptides derived from proteins involved in invasion and having high red blood cell binding ability have thus been identified. Immunization studies using Aotus monkeys have revealed that these peptides were neither immunogenic nor protection inducing. When modified in their critical binding residues, previously identified by Glycine scanning, some of these peptides were immunogenic and non-protection inducers; others induced short-lived antibodies whilst a few were both immunogenic and protection inducing. However, very few of these modified high activity binding peptides (HABPs) reproducibly induced protection without inducing antibody production, but with high cytokine liberation, suggesting that cellular mechanisms had been activated in the protection process. The three-dimensional structure of these peptides inducing protection without producing antibodies was determined by 1H-NMR. Their HLA-DRbeta1* molecule binding ability was also determined to ascertain association between their 3D structure and ability to bind to Major Histocompatibility Complex Class-II molecules (MHC-II). 1H Nuclear Magnetic Resonance analysis and structure calculations clearly showed that these modified HABPs inducing protective cellular immune responses (but not producing antibodies against malaria) adopted special structural configuration to fit into the MHC II-peptide-TCR complex. A different orientation for P7 and P8 TCR contacting residues was clearly recognized when comparing their structure with modified peptides, which induced high antibody titers and protection, suggesting that these residues are involved in activating the immune system associated with antibody production and protection.

Elongating modified conserved peptides eliminates their immunogenicity and protective efficacy against P. falciparum malaria.

Plasmodium falciparum malaria protein peptides were synthesised in the search for more effective routes for inducing a protective immune response against this deadly parasite and this information has been associated with such molecules' three-dimensional structure. These peptides had high red blood cell binding activity and their carboxy- and amino-terminal extremes were elongated for determining their immunogenic and protection-inducing activity against this disease in the Aotus monkey experimental model. 1H-NMR was used for analysing their three-dimensional structure; FAST ELISA, immunofluorescence antibody test, and Western blot were used for identifying their antibody inducing capacity and these previously immunised Aotus were inoculated with a highly infective P. falciparum strain to determine whether these elongated peptides were able to induce protection. This was aimed at establishing an association or correlation between long peptides' three-dimensional structure and their immunogenic and protection-inducing response in these monkeys. Peptides 20026 (25 residue), 20028 (30 residue), and 20030 (35 residues) were synthesised based on elongating the amino-terminal region of the 10022 highly immunogenic and protection-inducing modified peptide. 1H-NMR studies revealed that the first three had Classical type III beta-turn structures, different from the 20-amino acid long modified peptide 10022 which had a distorted type III beta-turn. Humoral immune response analysis showed that even when some antibodies could be generated against the parasite, none of the immunised Aotus could be protected with elongated peptides suggesting that elongating them eliminated modified peptide 10022 immunogenic and protection-inducing capacity.

Evidence supporting the hypothesis that specifically modifying a malaria peptide to fit into HLA-DRbeta1*03 molecules induces antibody production and protection.

EBA-175 protein is used as ligand in Plasmodium falciparum binding to erythrocytes. Evidence shows that conserved peptide 1815 from this protein having high red blood cell binding ability plays an important role in the invasion process. This peptide is neither immunogenic nor protective. Residues were substituted by amino acids having similar volume or mass but different polarity in 1815 analogues had to make them fit into HLA-DRbeta1*03 molecules; these were synthesised and inoculated into Aotus monkeys, generating different immunogenic and/or protective immune responses. A shortening in alpha-helix structure was found in the immunogenic and protective ones when their secondary structure was analyzed by NMR to correlate their structure with their immunological properties. This data, together with results from previous studies, suggests that this shortening in high-activity binding peptide (HABP) helical configuration may lead to better fitting into immune system molecules as shown by binding to purified HLA-DRbeta1* molecules rendering them immunogenic and protective and therefore, excellent candidates for consideration as components of a subunit based multi-component synthetic vaccine against malaria.

Mapping the anatomy of a Plasmodium falciparum MSP-1 epitope using pseudopeptide-induced mono- and polyclonal antibodies and CD and NMR conformation analysis.

Antigen structure modulation represents an approach towards designing subunit malaria vaccines. A specific epitope's alpha carbon stereochemistry, as well as its backbone topochemistry, was assessed for obtaining novel malarial immunogens. A variety of MSP-1(38-61) Plasmodium falciparum epitope pseudopeptides derived were synthesised, based on solid-phase pseudopeptide chemistry strategies; these included all-L, all-D, partially-D substituted, all-Psi-[NH-CO]-Retro, all-Psi-[NH-CO]-Retro-inverso, and Psi-[CH2NH] reduced amide surrogates. We demonstrate that specific recombinant MSP-1(34-469) fragment binding to red blood cells (RBCs) is specifically inhibited by non-modified MSP-1(42-61), as well as by its V52-L53, M51-V52 reduced amide surrogates and partial-D substitutions in K48 and E49. In vivo tests revealed that reduced amide pseudopeptide-immunised Aotus monkeys induced neutralising antibodies specifically recognising the MSP-1 N-terminus region. These findings support the role of molecular conformation in malaria vaccine development.

Induction and displacement of an helix in the 6725 SERA peptide analogue confers protection against P. falciparum malaria.

The protein called serine repeat antigen (SERA) is a Plasmodium falciparum malaria antigen; high activity erythrocyte binding peptides have been identified in this protein. One of these, the 6725 peptide (non-immunogenic and non-protective), was analyzed for immunogenicity and protective activity in Aotus monkeys, together with several of its analogues. These peptides were studied by 1H NMR to try to correlate their structure with their biological function. These peptides showed helical regions having differences in their position, except for randomly structured 6725. It is shown that replacing some amino acids induced immunogenicity and protectivity against experimental malaria and changed their three-dimensional (3D) structure, suggesting that such modifications may allow a better fit with immune system molecules.