RESUMO
BACKGROUND: To develop a plant-based vaccine against Plasmodium vivax, two P. vivax candidate proteins were chosen. First, the merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen that is currently considered a strong vaccine candidate. Second, the circumsporozoite protein (CSP), a component of sporozoites that contains a B-cell epitope. METHODS: A synthetic chimeric recombinant 516 bp gene encoding containing PvMSP-1, a Pro-Gly linker motif, and PvCSP was synthesized; the gene, named MLC, encoded a total of 172 amino acids. The recombinant gene was modified with regard to codon usage to optimize gene expression in Brassica napus. The Ti plasmid inducible gene transfer system was used for MLC chimeric recombinant gene expression in B. napus. Gene expression was confirmed by polymerase chain reaction (PCR), beta-glucuronidase reporter gene (GUS) assay, and Western blot. RESULTS: The MLC chimeric recombinant protein expressed in B. napus had a molecular weight of approximately 25 kDa. It exhibited a clinical sensitivity of 84.21% (n=38) and a clinical specificity of 100% (n=24) as assessed by enzyme-linked immunosorbent assay (ELISA). Oral immunization of BALB/c mice with MLC chimeric recombinant protein successfully induced antigen-specific IgG1 production. Additionally, the Th1-related cytokines IL-12 (p40), TNF, and IFN-γ were significantly increased in the spleens of the BALB/c mice. CONCLUSIONS: The chimeric MLC recombinant protein produced in B. napus has potential as both as an antigen for diagnosis and as a valuable vaccine candidate for oral immunization against vivax malaria.
Assuntos
Brassica napus/metabolismo , Vacinas Antimaláricas/imunologia , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Administração Oral , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Brassica napus/genética , Códon , Ensaio de Imunoadsorção Enzimática/métodos , Expressão Gênica , Vetores Genéticos , Imunoglobulina G/sangue , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/genética , Masculino , Proteína 1 de Superfície de Merozoito/biossíntese , Proteína 1 de Superfície de Merozoito/genética , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Plasmídeos Indutores de Tumores em Plantas , Plasmodium vivax/genética , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Oxygen dependent degradation of hypoxia-inducible factor (HIF)-1α is triggered with hydroxylation by proline hydroxylase domain 2 (PHD2) under normoxic conditions. Some of previously developed PHD2 inhibitors show a considerable potency against factor inhibiting HIF (FIH), the HIF asparagine hydroxylase. For specific inhibition of PHD2, we have synthesized peptides containing 556-575 residues of HIF-1α with modifications at the Pro-564 and examined their inhibitory effect against PHD2. Adopting fluorescence polarization-based assays, we evaluated inhibitory potency of the peptides and selected potent inhibitors. These PHD2 inhibitor peptides showed no significant potency against FIH, demonstrating their specific inhibitory effect on PHD2.
Assuntos
Inibidores Enzimáticos/química , Peptídeos/química , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Sequência de Aminoácidos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Prolina Dioxigenases do Fator Induzível por Hipóxia , Peptídeos/síntese química , Peptídeos/farmacologia , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Especificidade por SubstratoRESUMO
Merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen, and circumsporozoite protein (CSP), a component of sporozoites that includes a Plasmodium vivax B-cell epitope, are strong candidates for use in a malaria vaccine. A chimeric recombinant gene containing portions of both msp-1 and csp from P. vivax separated by Pro-Gly linker motif was generated. The construct gene was named mlc (msp-1, linker, and csp). The MLC chimeric recombinant protein had a molecular weight of approximately 25 kDa when expressed in Escherichia coli, as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The purified chimeric protein reacted with the sera of patients infected with P. vivax but not with the sera of uninfected patients according to western blot analysis. The chimeric protein reacted well with sera of malaria patients (109/115, 94.78%) as assessed with enzyme-linked immunosorbent assay (ELISA). BALB/c mice that were orally immunized with the MLC chimeric recombinant protein successfully produced antigen-specific antibodies. Additionally, levels of the Th1-associated cytokines IL-12(p40), TNF-α, and IFN-γ were significantly increased in the spleens of the BALB/c mice. Therefore, the E. coli-expressed MLC chimeric recombinant protein might be used as a valuable vaccine candidate for oral immunization against vivax malaria.
Assuntos
Proteína 1 de Superfície de Merozoito/genética , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/sangue , Sequência de Bases , Feminino , Expressão Gênica , Humanos , Vacinas Antimaláricas , Malária Vivax/prevenção & controle , Proteína 1 de Superfície de Merozoito/biossíntese , Proteína 1 de Superfície de Merozoito/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plasmodium vivax/imunologia , Plasmodium vivax/metabolismo , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologiaRESUMO
BACKGROUND: To use pyrimethamine as an alternative anti-malarial drug for chloroquine-resistant malaria parasites, it was necessary to determine the enzyme's genetic variation in dihydrofolate reductase-thymidylate syntase (DHFR-TS) among Korean strains. METHODS: Genetic variation of dhfr-ts genes of Plasmodium vivax clinical isolates from patients who did not respond to drug treatment (n = 11) in Korea were analysed. The genes were amplified using the polymerase chain reaction (PCR) with genomic DNA as a template. RESULTS: Sequence analysis showed that the open reading frame (ORF) of 1,857 nucleotides encoded a deduced protein of 618 amino acids (aa). Alignment with the DHFR-TS genes of other malaria parasites showed that a 231-residue DHFR domain and a 286-residue TS domain were seperated by a 101-aa linker region. This ORF shows 98.7% homology with the P. vivax Sal I strain (XM001615032) in the DHFR domain, 100% in the linker region and 99% in the TS domain. Comparison of the DHFR sequences from pyrimethamine-sensitive and pyrimethamine-resistant P. vivax isolates revealed that nine isolates belonged to the sensitive strain, whereas two isolates met the criteria for resistance. In these two isolates, the amino acid at position 117 is changed from serine to asparagine (S117N). Additionally, all Korean isolates showed a deletion mutant of THGGDN in short tandem repetitive sequences between 88 and 106 amino acid. CONCLUSIONS: These results suggest that sequence variations in the DHFR-TS represent the prevalence of antifolate-resistant P. vivax in Korea. Two of 11 isolates have the Ser to Asn mutation in codon 117, which is the major determinant of pyrimethamine resistance in P. vivax. Therefore, the introduction of pyrimethamine for the treatment of chloroquine-resistant vivax malaria as alternative drug in Korea should be seriously considered.
Assuntos
Antimaláricos/uso terapêutico , Cloroquina/uso terapêutico , Malária Vivax/tratamento farmacológico , Plasmodium vivax/efeitos dos fármacos , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase/genética , Análise Mutacional de DNA , DNA de Protozoário/genética , Humanos , Malária Vivax/parasitologia , Mutação de Sentido Incorreto , Plasmodium vivax/isolamento & purificação , Reação em Cadeia da Polimerase , Proteínas de Protozoários/genética , República da Coreia , Análise de Sequência de DNA , Falha de TratamentoRESUMO
There has been a great increase of Plasmodium vivax incidences in the Republic of Korea and the genetic diversity of the parasite became more complex with the rapid dissemination of newly introduced genotypes. Surveillance of imported malaria is very important, but there is no good way to determine imported vs. internal cases. In this study, we characterized imported vivax cases, analyzed the genetic sequence of three imported vivax malaria cases for the merozoite surface protein-1 (MSP-1) and circumsporozoite protein (CSP) genes, and clearly discriminated an imported vivax case that was misdiagnosed as indigenous by genetic analysis. PCR reaction for the merozoite surface protein-1 (MSP-1) and circumsporozoite protein (CSP) genes from three imported vivax cases were amplified and sequenced. The genetic variations were compared with a previously constructed database of South Korean isolates. The imported vivax cases showed various patterns on incubation period before onset. Most cases were from other parts of Asia. The MSP-1 gene sequence analysis of three imported cases showed that the imported cases had completely different sequences from any subtypes from Korean isolates. Case-1 and Case-2 exact match with an Indian isolate, and Case-3 had great similarity with isolates from countries neighboring Indonesia. CSP gene analysis based on the repeat patterns showed similar results that the sequences from the imported cases well matched with the patient's traveled countries and completely discriminated with indigenous cases. AMA-1 gene analysis also supported these results. We were able to clearly distinguish three imported vivax cases from indigenous by using a genetic database of Korean isolates and were able to suspect its origin by genotyping. This study demonstrated the usefulness of genetic survey on imported malaria cases.
Assuntos
Malária Vivax/parasitologia , Proteína 1 de Superfície de Merozoito/genética , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Viagem , Adulto , Sequência de Aminoácidos , Humanos , Índia , Indonésia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Plasmodium vivax/classificação , Reação em Cadeia da Polimerase , República da Coreia , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Reemerged Plasmodium vivax malaria in South Korea has not yet been eradicated despite continuous governmental efforts. It has rather become an endemic disease. Our study aimed to determine the genetic diversity in P. vivax merozoite surface protein-1 (PvMSP-1) and circumsporozoite protein (PvCSP) genes over an extended period after its reemergence to its current status. Sequence analysis of PvMSP-1 gene sequences from the 632 P. vivax isolates during 1996-2007 indicates that most isolates recently obtained were different from isolates obtained in the initial reemergence period. There was initially only one subtype (recombinant) present but its subtypes have varied since 2000; six MSP-1 subtypes were recently found. A similar variation was observed by CSP gene analysis; a new CSP subtype was found. Understanding genetic variation patterns of the parasite may help to analyze trends and assess extent of endemic malaria in South Korea.