RESUMO
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.
Assuntos
Antígenos de Protozoários/química , Interações Hospedeiro-Parasita/imunologia , Vacinas Antimaláricas/química , Malária/imunologia , Malária/prevenção & controle , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Aotus trivirgatus , Cristalografia por Raios X , Ligação de Hidrogênio , Vacinas Antimaláricas/imunologia , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/síntese química , Peptídeos/química , Peptídeos/imunologia , Conformação ProteicaRESUMO
A fully effective antimalarial vaccine must contain multiple proteins from the different development stages of Plasmodium falciparum parasites involved in host-cell invasion or their biologically active fragments. It must therefore include sporozoite molecules able to induce protective immunity by blocking the parasite's access to hepatic cells, and/or proteins involved in the development of this stage, amongst which are included the Liver Stage Antigen-1 (LSA-1) and the Sporozoite and Liver Stage Antigen (SALSA). Our studies have focused on the search for an association between the structure of high activity binding peptides (HABPs), including both conserved native and their modified analogues, and their ability to bind to the MHC Class II HLA-DR molecules during formation of the MHCII-peptide-TCR complex leading to inducing the appropriate immune response. These studies are part of a logical and rational strategy for developing multi-stage, multi-component, minimal subunit-based vaccines, mainly against the P. falciparum malaria.
Assuntos
Antígenos de Protozoários/química , Fígado/parasitologia , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Antígenos HLA-DR/imunologia , Imunização , Vacinas Antimaláricas/química , Dados de Sequência Molecular , Plasmodium falciparum/crescimento & desenvolvimento , Estrutura Secundária de Proteína , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/isolamento & purificação , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/imunologiaAssuntos
Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Plasmodium falciparum/química , Proteínas de Protozoários/química , Animais , Eritrócitos/metabolismo , Humanos , Vacinas Antimaláricas/síntese química , Vacinas Antimaláricas/química , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/metabolismoRESUMO
Plasmodium falciparum thrombospondin-related apical merozoite protein (PTRAMP) has a thrombospondin related (TSR) domain which in many proteins has been reported as a fragment involved in pathogen-host and cell-interactions. Receptor-ligand studies using eighteen non-overlapping 20-aminoacid-long synthetic peptides from this protein were carried out to determine regions involved in parasite invasion of red blood cells (RBC). Two high activity binding peptides (HABPs) were determined, 33405 (21YISSNDLTSTNLKVRNNWEH40) and 33413 (180LEGPIQFSLGKSSGAFRINY199), presenting high dissociation constants and positive cooperativity. One of the HABPs displayed a modified Plasmodium export element (PEXEL), suggesting that this protein could be involved in the merozoite cytoplasmic reticulum, parasitophorous vacuole, red blood cell (RBC) cytosol, and probably infected RBC (iRBC) membrane transport of some other molecules and nutrients. Enzymatic treatment of RBCs increased HABP 33405 binding to them whilst it decreased HABP 33413 binding. Merozoite invasion assays revealed that HABPs have around 57% ability to inhibit new RBC invasion. Circular dichroism revealed the presence of possible alpha-helical elements in both HABPs structures. RBC binding interaction specificity and the presence of a PEXEL motif make these 2 HABPs good candidates for being included in further studies to develop a new multi-antigenic, multi-stage, subunit-based, chemically-synthesised, anti-malarial vaccine.
Assuntos
Membrana Eritrocítica/metabolismo , Proteínas de Membrana/metabolismo , Plasmodium/metabolismo , Proteínas de Protozoários/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Proteínas de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Proteínas de Protozoários/química , Homologia de Sequência de AminoácidosRESUMO
A totally effective anti-malarial vaccine must contain epitopes derived from multiple proteins found in different stages of the particular parasite involved in invasion. It must therefore include sporozoite molecules able to induce protective immunity thereby blocking the parasite's access to hepatic cells; thrombospondin-related anonymous protein (TRAP) is one of them. Conserved high activity binding peptides (HABPs) attaching themselves to hepatic cells were used in immunisation studies with the highly malaria-susceptible Aotus monkey. However, they had to be modified to render them immunogenic. The changes induced in lead peptide 3D structure were analysed by correlating such substitutions with the induction of high anti-sporozoite antibody levels in the experimental monkey model. The modification induced structural changes in most modified HABPs, changing them from random-coil or distorted type III beta-turn structures to classical type III or III' beta-turn, thereby allowing a better fit into the MHC-II-peptide-TCR complex since they bound with high affinity to purified HLA-DRbeta1* molecules. These are the first (TRAP) conserved HABPs corresponding to functionally active amino acid sequences in sporozoite invasion and mobility which, when modified, were able to induce very high anti-sporozoite antibody responses, leading to suggesting them as components in the first line of defence of a fully-effective, subunit-based, multi-epitope, multi-stage, synthetic anti-malarial vaccine.
Assuntos
Vacinas Antimaláricas/imunologia , Peptídeos/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Esporozoítos/imunologia , Animais , Aotidae , Cristalografia por Raios X , Epitopos/imunologia , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Vacinas Antimaláricas/química , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Malária Falciparum/prevenção & controle , Peptídeos/síntese química , Peptídeos/química , Peptídeos/metabolismo , Plasmodium falciparum/citologia , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Esporozoítos/citologia , Esporozoítos/metabolismoRESUMO
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.
Assuntos
Malária Falciparum/imunologia , Proteína 1 de Superfície de Merozoito/imunologia , Peptídeos/imunologia , Plasmodium falciparum/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Eritrócitos/parasitologia , Haplorrinos , Proteína 1 de Superfície de Merozoito/química , Dados de Sequência Molecular , Testes de Neutralização , Ressonância Magnética Nuclear BiomolecularRESUMO
The Plasmodium falciparum malaria parasite produces several proteins characterised by an unusually high histidine content in infected red blood cells (iRBC). The histidine-rich protein II (HRP-II) is synthesised throughout the parasite's asexual and gametocyte stages, transported through the parasitophorous vacuole (PV) to iRBC cytosol and membrane and released to the bloodstream via a PEXEL motif. Immunogenicity and protection-inducing studies were begun with an RBC high activity binding peptide (HABP) from this protein named 6800 (preceding the PEXEL motif) in the experimental Aotus monkey model. Modifying critical residues (determined by glycine scanning in this HABP) induced immunogenicity and protection against experimental challenge. Native 6800 did not bind to any HLA-DRbeta(1)(*) molecule, but these modified HABPs acquired the ability to specifically bind to HLA-DRbeta(1)(*)0701. (1)H NMR studies revealed that whilst 6800 had a random structure, modified immunogenic and protection-inducing 24230 displayed very short alpha-helical segments allowing appropriate binding to the MHCII-pep-TCR complex. Modifications in conserved HABPs preceding PEXEL motifs thus open up new avenues for subunit-based, multi-component synthetic anti-malarial vaccine development.
Assuntos
Antígenos de Protozoários/administração & dosagem , Antígenos de Protozoários/química , Vacinas Antimaláricas/administração & dosagem , Malária Falciparum/prevenção & controle , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/química , Animais , Aotidae , Resultado do TratamentoRESUMO
A hepatitis C virus E(2) protein-derived sequence was selected for studying the effect of N-glycosylation on the peptide chain's conformational structure. The results suggested that the (534)TDVF(537) motif contained in peptide 33402 ((529)WGENDTDVFVLNNTRY(544)) had a type III beta-turn, relevant in antigen recognition of polyclonal antibodies, binding to human cells, and binding to HLA DRB1 *0401 molecules. N-Glycopeptides derived from this sequence contained monosaccharides in Asn(532). N-Glycopeptides presented differences in peptide chain structure compared to non-glycosylated peptides. Peptide 33402 specifically bound to human cells, specificity becoming lost when it was N-glycosylated. N-Glycosylation decreased antigen recognition of mouse polyclonal sera against this sequence. N-Glycopeptide binding to HLA DRB1 *0401 molecules was similar to that presented by non-glycosylated peptide, indicating that N-glycosylation did not affect binding to HLA DRB1 *0401 molecules. N-Glycosylation induced changes at structural and functional level which could be relevant for modulating human cell binding properties and antibody recognition.
Assuntos
Hepacivirus/metabolismo , Monossacarídeos/farmacologia , Peptídeos/farmacologia , Proteínas Virais/farmacologia , Sequência de Aminoácidos , Dicroísmo Circular , Ensaio de Imunoadsorção Enzimática , Glicosilação , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/metabolismo , Ligação Proteica , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
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.
Assuntos
Antígenos de Histocompatibilidade Classe II , Imunidade Celular/fisiologia , Complexo Principal de Histocompatibilidade , Vacinas Antimaláricas , Peptídeos/imunologia , Plasmodium falciparum , Receptores de Antígenos de Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Aotus trivirgatus , Citocinas/imunologia , Humanos , Imunização , Malária Falciparum , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/genética , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Plasmodium falciparum/metabolismo , Conformação Proteica , Alinhamento de SequênciaRESUMO
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.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Aotus trivirgatus , Antígenos HLA-DR/imunologia , Malária/imunologia , Malária/prevenção & controle , Fragmentos de Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/genética , Anticorpos Antiprotozoários/metabolismo , Sítios de Ligação de Anticorpos , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Cadeias HLA-DRB1 , Malária/genética , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Plasmodium falciparum/imunologiaAssuntos
Complexo Principal de Histocompatibilidade/imunologia , Malária/imunologia , Malária/prevenção & controle , Peptídeos/química , Sequência de Aminoácidos , Sequência Conservada/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/imunologia , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
EBA-175 protein is used as a ligand in the binding of P. falciparum to red blood cells (RBCs). Evidence shows that the conserved peptide 1779 from this protein (with high red blood cell binding ability and known critical erythrocyte binding residues) plays an important role in the invasion process. This peptide is neither immunogenic nor protective; analogs having critical residues replaced by amino acids with similar volume or mass but different polarity were synthesized and inoculated into Aotus monkeys, and elicited different immunogenic and protective responses. Nuclear Magnetic Resonance (1H-NMR) studies revealed that peptide analog 21696 (non-immunogenic and non-protective) presents a large helical fragment, that the peptide 14012 (immunogenic and non-protective) helical fragment is smaller, while the peptide 22812 (immunogenic and protective) alpha-helix is shorter in a different region and possesses greater flexibility at its N-terminus. The presence of methionine residues could affect the structural stability of peptide 22812 and ultimately its immunological response. Our results suggest a new strategy for designing a new malaria multi-component subunit-based vaccine.
Assuntos
Antígenos de Protozoários/imunologia , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/administração & dosagem , Antígenos de Protozoários/química , Haplorrinos , Ligantes , Espectroscopia de Ressonância Magnética , Vacinas Antimaláricas/administração & dosagem , Malária Falciparum/sangue , Metionina/química , Dados de Sequência Molecular , Peptídeos/administração & dosagem , Peptídeos/síntese química , Peptídeos/imunologia , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/síntese química , Proteínas de Protozoários/químicaRESUMO
The conserved, nonantigenic, nonimmunogenic malaria Merozoite Surface Protein-2 peptide 1, having high affinity for red blood cells, was rendered immunogenic and protective in Aotus monkeys by specifically changing some critical residues. The NMR structure revealed a switch from classical type III' into distorted III' and III beta turns in the protective peptides. These changes may lead to a better fit into the Aotus MHC class II human HLA-DRbeta1 12 molecule equivalent, thus activating the immune system.
Assuntos
Antígenos de Protozoários/química , Antígenos de Histocompatibilidade Classe II/imunologia , Peptídeos/química , Proteínas de Protozoários/química , Animais , Antígenos de Protozoários/imunologia , Aotidae , Espectroscopia de Ressonância Magnética , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Modelos Moleculares , Peptídeos/imunologia , Plasmodium falciparum/imunologia , Conformação Proteica , Proteínas de Protozoários/imunologia , VacinaçãoRESUMO
A 175-erythrocyte-binding protein (EBA-175) conserved high-activity binding peptide (HABP), called 1783 (nonimmunogenic, nonprotective against Plasmodium falciparum malaria), was analyzed for antigenic and protective activity in Aotus monkeys, together with several of its analogues. 1H NMR studies of peptides 17912, 14016, and 22814 allowed their structure to be related to their biological function. These peptides showed helical regions having differences in their position and length. Nonimmunogenic, nonprotective peptides 1783 and 17912 showed an extensive helical region, while the 22814 immunogenic protective peptide's alpha-helix was found in the N-terminal region. This suggests that the more flexible C-terminal region will allow better interaction between these peptides and immune system molecules as well as relating these peptides' three-dimensional structure to their immunogenicity and protective activity, thus leading to a more rational development of the new malaria multicomponent vaccine.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Plasmodium falciparum/metabolismo , Sequência de Aminoácidos , Animais , Aotus trivirgatus , Western Blotting , Modelos Moleculares , Dados de Sequência Molecular , Biossíntese Peptídica , Peptídeos/química , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
La completa remoción de la dentina cariada es un proceso complejo, por la escasa definición de criterios objetivos, para diferenciar la dentina infectada y la afectada. Una de las formas de reducir la subjetividad es la utilización de colorantes, aunque existe controversia respecto a las indicaciones, ventajas y desventajas de su uso. Este estudio comparó la efectividad de la técnica convencional óptica y táctil con la utilización de rojo ácido al 1 por ciento, en la remoción de la dentina cariada y microbiológicamente cuantificó y clasificó los microorganismos de la dentina remanente, en 34 pacientes con caries moderada. Se encontró que el 97 por ciento de las cavidades presentó tinción de la dentina, siendo más frecuente en la pared pulpar y unión amelodentinaria, pero el colorante no siempre tiñó bacterias, por lo cual llevó a remoción innecesaria la dentina y varias exposiciones pulpares. El 61.81 por ciento del total de los microorganismos cultivalbes no se logró identificar y su papel en el progreso de la caries no se conoce. Estreptococos del grupo "mutans" y "Lactobacillus" spp. representaron sólo el 12.79 por ciento del total de los microorganismos. No se encontró correlación entre el frente de decoloración, el sitio de tinción y el frente bacteriano
Assuntos
Humanos , Masculino , Feminino , Testes de Atividade de Cárie Dentária , Cárie Dentária/diagnóstico , Cárie Dentária/microbiologia , Cárie Dentária/terapia , Dentina/microbiologia , Distribuição por Idade , Bactérias Anaeróbias/isolamento & purificação , Contagem de Colônia Microbiana , Corantes , Meios de Cultura , Lactobacillus/isolamento & purificação , Interpretação Estatística de Dados , Streptococcus mutans/isolamento & purificaçãoRESUMO
La remoción completa de la dentina cariada es un proceso complejo, por la escasa definición de criterios objetivos para diferenciar la dentina infectada de la afectada. Una de las formas de reducir la subjetividad es la utilización de colorantes, aunque existe controversia respecto a las indicaciones, ventajas y desventajas de su uso. Este estudio comparó la efectividad de la técnica convencional óptica y táctil con la utilización de rojo ácido al 1 por ciento en la remoción de la dentina cariada, y microbiológicamente cuantificó y clasificó los microorganismos de la dentina remanente, en 34 pacientes con caries moderada. Se encontró que el 97 por ciento de las cavidades presentaron tinción de la dentina, siendo más frecuente ésta en la pared pulpar y unión amelodentinaria, pero el colorante no siempre tiñó bacterias, por lo cual llevó a remoción innecesaria de dentina y a varias exposiciones pulpares. El 61.81 por ciento del total de los microorganismos cultivables no se logró indentificar y su papel en el progreso de la caries no se conoce. Los S. mutans y Lactobacillus representaron sólo el 12.79 por ciento del total de los microorganismos. No se encontró correlación entre el frente de decoloración, el sitio de tinción y el frente bacteriano