Redefining an epitope of a malaria vaccine candidate, with antibodies against the N-terminal MSA-2 antigen of Plasmodium harboring non-natural peptide bonds.

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(21-40) 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 (30)FIN(32)-binding motif of MSA-2 were replaced with ψ-[CH2-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(25-32) 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.

Protection against malaria is conferred by passive transferring rabbit F(ab)(2)' antibody fragments, induced by Plasmodium falciparum MSP-1 site-directed designed pseudopeptide-BSA conjugates assessed in a rodent model.

F(ab)(2)'-immunoglobulin (Ig) fragments induced by site-directed designed immunogens are emerging as novel tools of potential utility in the treatment of clinical episodes of transmissible diseases such as malaria. Immunogens based on reduced amide pseudopeptides based on site-directed molecular modifications represent structural probes that could be considered as novel vaccine candidates, as we have previously demonstrated. We have obtained F(ab)(2)'-Ig rabbit antibodies induced against the N-terminal sequence of the native Merozoite Surface Protein-1 (MSP-1) of Plasmodium falciparum and a set of five MSP-1-derived reduced amide pseudopeptides. Pseudopeptides were designed for inducing functional neutralizing mono-specific polyclonal antibodies with potential applications in the control of malaria. Following a classical enzyme immunoglobulin fractionation, F(ab)(2)'-Ig fragments were tested for their ability to suppress blood-stage parasitemia by passive immunization in malaria-infected mice. Some of these fragments proved totally effective in suppressing a lethal blood-stage challenge infection and others reduced malarial parasitemia. These data suggest that protection against Plasmodium yoelii malaria following passive transfer of structurally well-defined ß-strand F(ab)(2)'-Ig fragments can be associated with specific immunoglobulins induced by site-directed designed MSP-1 reduced amide pseudopeptides.

Actividad funcional de anticuerpos inducidos por péptido-miméticos derivados del antígeno MSP-2 del Plasmodium, como potenciales agentes inmunoterapéuticos en malaria causada por Plasmodium yoelii y Plasmodium berghei en ratones BALB/c / Functional activity of antibodies induced by peptido-mimetics derived from the Plasmodium MSP-2 antigen, as potential immuno-therapeutic agents in rodent malaria induced by P. berghei and P. yoelii strains

Teniendo en cuenta el alto grado de conservación genética de los residuos críticos de la estructura primaria del péptido 4044 (21KNESKYSNTFINNAYNMSIR40) identificado como crucial en el antígeno MSP-2 para que el Plasmodium falciparum pueda unirse con alta capacidad específica a glóbulos rojos y causar malaria, se diseñaron y sintetizaron dos secuencias en formas monomérica y polimérica de péptido-miméticos denominados pseudopéptidos amida reducida en las cuales se sustituyó un enlace peptídico normal por su isóstero ψ[CH2-NH] entre los residuos fenilalanina-isoleucina y entre los residuos isoleucina-asparagina, para dar lugar a los pseudopéptidos codificados ψ-128 forma monomérica (ψ-129 forma polimérica) y ψ-130 forma monomérica (ψ-131 forma polimérica). Con estos péptido-miméticos se generaron anticuerpos monoclonales de isotipo IgM. Mediante experimentos controlados de inmunización in vitro se indujo el cambio isotipo de los clones reactivos a las subclases IgG1, IgG2a, IgG2b e IgG3. Estas inmunoglobulinas se ensayaron por su actividad funcional antimalárica in vivo mostrando una alta eficacia en el control de la infección por malaria al ser administradas por transferencia pasiva. El efecto neutralizador del desarrollo biológico del patógeno por parte de estos anticuerpos inducidos de manera sitio-dirigida los hacen potencialmente útiles, como una potencial herramienta para el control de la infección por malaria.

Bearing in mind the high degree of genetical conservation of critical binding residues from the primary structure of the peptide 4044 (21KNESKYSNTFINNAYNMSIR40), which was previously identified as being crucial for the MSP-2 antigen to lead Plasmodium falciparum to bind red blood cells with high specific capacity, and so causing malaria, two peptido-mimetics so-named reduced amide pseudopeptides, in which a nature-made amide bond was replaced with a ψ[CH2-NH] methylene amide isoster bond, one between the Phe-Ile aminoacid pair and the second between Ile-Asn, were designed and synthesized in a site-directed manner as monomer and polymer forms, and were coded as ψ-128 for the monomer (ψ-129 polymer) and ψ-130 for the monomer (ψ-131 for polymer) respectively. These peptido-mimetics were used to produce monoclonal antibodies which displayed in both cases IgM isotype. By controlled in vitro immunization experiments their parent reactive hybridomas were induced to a Ig isotype-switching to IgG1, IgG2a, IgG2b and Ig3 sub-classes. These immunoglobulins were tested for their in vivo functional activity against malaria, showing a high efficacy property for controlling the malaria infection when passively transferred into BALB/c mice. The neutralizing effect of these site-directed designed antibodies on the Plasmodium biological development, make them a potential tool for the control of malaria.

Estudio fitoquímico de hojas de Uncaria guianensis y evaluación de actividad antibacteriana / Phytochemical study of Uncaria guianensis leaves and antibacterial activity evaluation

Del extracto de éter de petróleo de hojas de Uncaria guianensis (Rubiaceae), se aisló un compuesto tipo clorina denominado éster etílico de feoforbida a y una mezcla de esteroles conocidos como ß-sitosterol y estigmasterol. Sus estructuras fueron elucidadas por análisis detallado de RMN, incluyendo técnicas bidimensionales, y por comparación con datos reportados en la literatura. Posteriormente, se evaluó la actividad antibacteriana al éster etílico de feoforbida a contra dos cepas Gram(+): S. aureus ATCC 6538 y E. faecalis ATCC 29212 y contra tres cepas Gram (-): E. coli ATCC 25922, S. typhimurium ATCC 14028s y S. typhimurium MS7953. Se encontró actividad significativa contra S. aureus, E. faecalis, E. coli y S. tiphymurium MS7953.

A chlorin compound, pheophorbide a ethyl ester and a mixture of sterols known as ß-sitosterol and stigmasterol, were isolated from the petroleum ether extract of Uncaria guianensis (Rubiaceae) leaves. Their structures were elucidated by detailed analysis of NMR spectra, including bidimensional techniques and by comparison with literature data. The antibacterial activity for the pheophorbide a ethyl ester was evaluated against two Gram (+) strains: S. aureus ATCC 6538 y E. faecalis ATCC 29212 and three Gram (-) strains: E. coli ATCC 25922, S. typhimurium ATCC 14028s y S. typhimurium MS7953S. aureus ATCC 6538 and E. fecalis ATCC 29212, finding significant activity against S. aureus 6538, E. faecalis 29212, S. tiphymurium MS7953 and E. coli 25922.

Development of designed site-directed pseudopeptide-peptido-mimetic immunogens as novel minimal subunit-vaccine candidates for malaria.

Synthetic vaccines constitute the most promising tools for controlling and preventing infectious diseases. When synthetic immunogens are designed from the pathogen native sequences, these are normally poorly immunogenic and do not induce protection, as demonstrated in our research. After attempting many synthetic strategies for improving the immunogenicity properties of these sequences, the approach consisting of identifying high binding motifs present in those, and then performing specific changes on amino-acids belonging to such motifs, has proven to be a workable strategy. In addition, other strategies consisting of chemically introducing non-natural constraints to the backbone topology of the molecule and modifying the α-carbon asymmetry are becoming valuable tools to be considered in this pursuit. Non-natural structural constraints to the peptide backbone can be achieved by introducing peptide bond isosters such as reduced amides, partially retro or retro-inverso modifications or even including urea motifs. The second can be obtained by strategically replacing L-amino-acids with their enantiomeric forms for obtaining both structurally site-directed designed immunogens as potential vaccine candidates and their Ig structural molecular images, both having immuno-therapeutic effects for preventing and controlling malaria.

Evaluación del efecto in vitro de anticuerpos inducidos por péptido-miméticos derivados del antígeno de superficie de merozoito-2 de plasmodium en malaria causada por plasmodium yoelii y plasmodium berghei en ratones BALB/c / Assessment of the in vitro effect of antibodies induced by peptido-mimetics derived from the Plasmodium merozoite surface protein-2 in malaria caused by P. berghei and P. yoelii in BALB/c mice

Con base en estudios realizados previamente en los cuales se identificaron los residuos críticos para la unión de la secuencia 21KNESKYSNTFINNAYNMSIR40 del antígeno MSP-2 del Plasmodium falciparum, se diseñaron y sintetizaron dos secuencias de pseudopéptidos amida reducida en las cuales se sustituyó un enlace peptídico normal por su isóstero ψ[CH2-NH] entre los residuos fenilalanina-isoleucina y entre los residuos isoleucina-asparagina, para dar lugar a los análogos codificados ψ-128 (forma monomérica), ψ-129 (forma polimérica), ψ-130 (forma monomérica) y ψ-131 (forma polimérica). Con los péptido-miméticos obtenidos en forma de polímero se inmunizaron ratones BALB/c para generar anticuerpos monoclonales que presentaron isotipo IgM. Mediante ensayos controlados de inmunización in vitro se indujo el cambio isotipo de los clones reactivos aislados de los hibridomas obtenidos de manera reproducible. Las inmunoglobulinas aisladas se ensayaron por su capacidad funcional neutralizadora de la infección controlada in vitro de cepas de Plasmodium de roedores a glóbulos rojos. Los resultados obtenidos evidencian el papel que pueden tener los anticuerpos inducidos por péptido-miméticos Plasmodium en ensayos de infección realizados en modelos animales de experimentación.

Based on previous studies in which those residues being critical for Plasmodium falciparum binding to red blood cells (RBCs) through the antigenic sequence (21KNESKYSNTFINNAYNMSIR40) from the merozoite surface antigen-2 (MSP-2) were identified, we have designed and synthesized two reduced amide pseudopeptide sequences based on the 31IN32 binding motif. Synthesized peptidomimetics, possess each one a modified peptide bond that presented as a ψ[CH2-NH] reduced amide isoster bond, to allowing the Phe-Ile modified aminoacid pair allowing pseudopeptides coded ψ-128 (monomer form) and ψ-129 (polymer form) and the Ile-Asn modified aminoacid pair for pseudopeptides coded ψ-130 (monomer form) and ψ-131 (polymer form). By using the polymer forms of both peptido-mimetics as immunogens, monoclonal antibodies were produced in BALB/c mice. These Ig showed an IgM isotype. The isotype antibody switching was lead by in vitro immunization of the original hybridomas. Isolated immunoglobulins were tested for their functional in vitro activity, on a infection controlled experiment of rodent Plasmodium strains infecting red blood cells. Obtained results reveal the role played by antibodies to peptido-mimetic in infection assays performed further on animal experimental models.

A C-terminal cationic fragment derived from an arginine-rich peptide exhibits in vitro antibacterial and anti-plasmodial activities governed by its secondary structure properties.

The differential in vitro antimicrobial activity of a 12-residue-long arginine-rich peptide derived from protamine was examined against bacterial and parasite microbes. A design of discrete peptide fragments based on the thermolysin-digestion map allowed us to propose three peptide fragments to be further assessed regarding their biological and secondary structural properties. Peptide structure allowed designing three arginine-rich fragments. All peptide fragments were assessed regarding their antimicrobial activity against Gram-positive and Gram-negative bacteria and a human malaria strain. Qualitative and quantitative assays carried out for determining all peptides' antibacterial activity at different concentration levels included radial diffusion and a time-controlled technique. Tests demonstrated that all assessed molecules inhibited invasion of Plasmodium falciparum parasites to human red blood cells. Cytolytic activity of the parent protamine peptide was completely abolished by strategically fragmenting its aminoacid sequence. Remarkably, the cationic C-fragment exhibited stronger biological activity than its parent peptide. Interestingly, the peptide fragment denoted as 2077 displays a typical alpha-helix profile according to its CD spectrum. The results support proposing the protamine C-terminal fragment as a potential new antimicrobial peptide.

Passive transfer of Plasmodium falciparum MSP-2 pseudopeptide-induced antibodies efficiently controlled parasitemia in Plasmodium berghei-infected mice.

We have developed monoclonal antibodies directed against the pseudopeptide psi-130, derived from the highly conserved malarial antigen Plasmodium falciparum merozoite surface protein 2 (MSP-2), for obtaining novel molecular tools with potential applications in the control of malaria. Following isotype switching, these antibodies were tested for their ability to suppress blood-stage parasitemia through passive immunization in malaria-infected mice. Some proved totally effective in suppressing a lethal blood-stage challenge infection and others reduced malarial parasitemia. Protection against P. berghei malaria following Ig passive immunization can be associated with specific immunoglobulins induced by a site-directed designed MSP-2 reduced amide pseudopeptide.

Protection against malaria induced by chirally modified Plasmodium falciparum's MSP-1 42 pseudopeptides.

The C-terminal portion of the Plasmodium falciparum blood stage MSP-1 antigen plays a key role in invasion of human erythrocytes. The MSP-1(1282-1301) non-polymorphic 1585 peptide, from the processed MSP-1(42) fragment, is poorly immunogenic and highly alpha-helical [Angew. Chem. Int. Ed. 40 (2001) 4654]. Assessing the alpha-carbon asymmetry and its implication in the host immune response is proposed in this work to overcome the 1585 peptide's immunological properties. Accordingly, the effect of incorporating single D-amino acids and psi-[CH(2)-NH] isoster bonds into the 1585 peptide was examined both at the immunogenic and 3D-structure levels. Therefore, specific binding to RBCs is promoted by site-directed chiral modifications on the native peptide as well as by simultaneously combining specific D-substitutions with psi-[CH(2)-NH] isoster bonds transforming this molecule into a high specific HLAbeta1*1101 allele binder. D-analog pseudopeptide immunized animals induced antibodies selectively recognizing a recombinant as well as native MSP-1(42) and MSP-1(33) fragments. Protection and low parasitemia levels were induced in Aotus monkeys immunized with the EVLYL(dK)PLAGVYRSLKKQLE analog. Peptide alpha-carbon chiral transformation is therefore an important target for structural modulation and, consequently, represents a novel approach towards designing multi-component subunit-based malarial vaccines.