Whole-genome sequencing of a Plasmodium vivax isolate from the China-Myanmar border area

Currently, there is a trend of an increasing number of Plasmodium vivaxmalaria cases in China that are imported across its Southeast Asia border, especially in the China-Myanmar border area (CMB). To date, little is known about the genetic diversity of P. vivaxin this region. In this paper, we report the first genome sequencing of a P. vivaxisolate (CMB-1) from a vivax malaria patient in CMB. The sequencing data were aligned onto 96.43% of the P. vivaxSalvador I reference strain (Sal I) genome with 7.84-fold coverage as well as onto 98.32% of 14 Sal I chromosomes. Using the de novoassembly approach, we generated 8,541 scaffolds and assembled a total of 27.1 Mb of sequence into CMB-1 scaffolds. Furthermore, we identified all 295 known virgenes, which is the largest subtelomeric multigene family in malaria parasites. These results provide an important foundation for further research onP. vivaxpopulation genetics.

Plasmodium vivax antigen discovery based on alpha-helical coiled coil protein motif.

Protein α-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with α-helical coiled coil motifs orthologous to Plasmodium falciparum were identified in the P. vivax genome. The peptides identified in silico were chemically synthesized; circular dichroism studies indicated partial or high α-helical content. Antigenicity was evaluated using human sera samples from malaria-endemic areas of Colombia and Papua New Guinea. Eight of these fragments were selected and used to assess immunogenicity in BALB/c mice. ELISA assays indicated strong reactivity of serum samples from individuals residing in malaria-endemic regions and sera of immunized mice, with the α-helical coiled coil structures. In addition, ex vivo production of IFN-γ by murine mononuclear cells confirmed the immunogenicity of these structures and the presence of T-cell epitopes in the peptide sequences. Moreover, sera of mice immunized with four of the eight antigens recognized native proteins on blood-stage P. vivax parasites, and antigenic cross-reactivity with three of the peptides was observed when reacted with both the P. falciparum orthologous fragments and whole parasites. Results here point to the α-helical coiled coil peptides as possible P. vivax malaria vaccine candidates as were observed for P. falciparum. Fragments selected here warrant further study in humans and non-human primate models to assess their protective efficacy as single components or assembled as hybrid linear epitopes.

Antigenicity and immunogenicity of a novel chimeric peptide antigen based on the P. vivax circumsporozoite protein.

BACKGROUND: Plasmodium vivax circumsporozoite (PvCS) protein is a major sporozoite surface antigen involved in parasite invasion of hepatocytes and is currently being considered as vaccine candidate. PvCS contains a dimorphic central repetitive fragment flanked by conserved regions that contain functional domains. METHODS: We have developed a chimeric 137-mer synthetic polypeptide (PvCS-NRC) that includes the conserved region I and region II-plus and the two natural repeat variants known as VK210 and VK247. The antigenicity of PvCS-NRC was tested using human sera from PNG and Colombia endemic areas and its immunogenicity was confirmed in mice with different genetic backgrounds, the polypeptide formulated either in Alum or GLA-SE adjuvants was assessed in inbred C3H, CB6F1 and outbred ICR mice, whereas a formulation in Montanide ISA51 was tested in C3H mice. RESULTS: Antigenicity studies indicated that the chimeric peptide is recognized by a high proportion (60-70%) of residents of malaria-endemic areas. Peptides formulated with either GLA-SE or Montanide ISA51 adjuvants induced stronger antibody responses as compared with the Alum formulation. Sera from immunized mice as well as antigen-specific affinity purified human IgG antibodies reacted with sporozoite preparations in immunofluorescence and Western blot assays, and displayed strong in vitro inhibition of sporozoite invasion (ISI) into hepatoma cells. CONCLUSIONS: The polypeptide was recognized at high prevalence when tested against naturally induced human antibodies and was able to induce significant immunogenicity in mice. Additionally, specific antibodies were able to recognize sporozoites and were able to block sporozoite invasion in vitro. Further evaluation of this chimeric protein construct in preclinical phase e.g. in Aotus monkeys in order to assess the humoral and cellular immune responses as well as protective efficacy against parasite challenge of the vaccine candidate must be conducted.

Acquired antibody responses against Plasmodium vivax infection vary with host genotype for duffy antigen receptor for chemokines (DARC).

BACKGROUND: Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC) is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are 'resistant' to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens. METHODOLOGY/FINDINGS: We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1) and Duffy binding protein (PvDBP) varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull) were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B). The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion. CONCLUSION/SIGNIFICANCE: Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the primary mechanisms by which P. vivax evades host immunity is through DARC indirectly down-regulating humoral responses against erythrocytic invasion and development.

Antigenicity and immunogenicity of a novel Plasmodium vivax circumsporozoite derived synthetic vaccine construct.

BACKGROUND: The circumsporozoite (CS) protein is a major malaria sporozoite surface antigen currently being considered as vaccine candidate. Plasmodium vivax CS (PvCS) protein comprises a dimorphic central repeat fragment flanked by conserved regions that contain functional domains involved in parasite invasion of host cells. The protein amino (N-terminal) flank has a cleavage region (region I), essential for proteolytic processing prior to parasite invasion of liver cells. METHODS: We have developed a 131-mer long synthetic polypeptide (LSP) named PvNR1R2 that includes the N-terminal flank and the two natural repeat variant regions known as VK210 and VK247. We studied the natural immune response to this region in human sera from different malaria-endemic areas and its immunogenicity in mice. RESULTS: PvNR1R2 was more frequently recognized by sera from Papua New Guinea (PNG) (83%) than by samples from Colombia (24%) when tested by ELISA. The polypeptide formulated in Montanide ISA51 adjuvant elicited strong antibody responses in both C3H and CB6F1 mice strains. Antibodies from immunized mice as well as affinity-purified human IgG reacted with native protein by IFA test. Moreover, mouse immune sera induced strong (90%) in vitro inhibition of sporozoite invasion (ISI) of hepatoma cell lines. CONCLUSIONS: These results encourage further studies in non-human primates to confirm the elicitation of sporozoite invasion blocking antibodies, to assess cell mediated immune responses and the protective efficacy of this polypeptide.

Phase I safety and immunogenicity trial of Plasmodium vivax CS derived long synthetic peptides adjuvanted with montanide ISA 720 or montanide ISA 51.

We assessed the safety, tolerability, and immunogenicity of a mixture of three synthetic peptides derived from the Plasmodium vivax circumsporozoite protein formulated in Montanide ISA 720 or Montanide ISA 51. Forty healthy malaria-naive volunteers were allocated to five experimental groups (A-E): four groups (A-D) were immunized intramuscularly with 50 and 100 μg/dose injections of a mixture of N, R, and C peptides formulated in the two different adjuvants at 0, 2, and 4 months and one group was administered placebo. Vaccines were immunogenic, safe, well tolerated, and no serious adverse events related to the vaccine occurred. Seroconversion occurred in > 90% of the vaccines and antibodies recognized the sporozoite protein on immunofluorescent antibody test. Vaccines in Montanide ISA 51 showed a higher sporozoite protein recognition and interferon production. Results encourage further testing of the vaccine protective efficacy.

Preclinical vaccine study of Plasmodium vivax circumsporozoite protein derived-synthetic polypeptides formulated in montanide ISA 720 and montanide ISA 51 adjuvants.

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate previously assessed in animals and humans. Here, combinations of three synthetic polypeptides corresponding to amino (N), central repeat (R), and carboxyl (C) regions of the CS protein formulated in Montanide ISA 720 or Montanide ISA 51 adjuvants were assessed for immunogenicity in rodents and primates. BALB/c mice and Aotus monkeys were divided into test and control groups and were immunized three times with doses of 50 and 100 μg of vaccine or placebo. Antigen-specific antimalarial antibodies were determined by enzyme-linked immunosorbent assay, immunofluorescent antibody test, and IFN-γ responses by enzyme-linked immunosorbent spot (ELIspot). Both vaccine formulations were highly immunogenic in both species. Mice developed better antibody responses against C and R polypeptides, whereas the N polypeptide was more immunogenic in monkeys. Anti-peptide antibodies remained detectable for several months and recognized native proteins on sporozoites. Differences between Montanide ISA 720 and Montanide ISA 51 formulations were not significant.

Antibody-mediated and cellular immune responses induced in naive volunteers by vaccination with long synthetic peptides derived from the Plasmodium vivax circumsporozoite protein.

Plasmodium vivax circumsporozoite (CS) protein is a leading malaria vaccine candidate. We describe the characterization of specific immune responses induced in 21 malaria-naive volunteers vaccinated with long synthetic peptides derived from the CS protein formulated in Montanide ISA 720. Both antibody- and cell-mediated immune responses were analyzed. Antibodies were predominantly of IgG1 and IgG3 isotypes, recognized parasite proteins on the immunofluorescent antibody test, and partially blocked sporozoite invasion of hepatoma cell lines in vitro. Peripheral blood mononuclear cells from most volunteers (94%) showed IFN-γ production in vitro upon stimulation with both long signal peptide and short peptides containing CD8+ T-cell epitopes. The relatively limited sample size did not allow conclusions about HLA associations with the immune responses observed. In summary, the inherent safety and tolerability together with strong antibody responses, invasion blocking activity, and the IFN-γ production induced by these vaccine candidates warrants further testing in a phase II clinical trial.

Leucograma en novillas y becerros (Holstein) infectados con una cepa venezolana de Trypanosoma vivax

Este trabajo tuvo como objetivo la evaluación del leucograma en novillas y becerros Holstein infectados experimentalmente con una cepa venezolana de Trypanosoma vivax. Los datos fueron analizados a través de la prueba no paramétrica de U de Mann-Whitney. La comparación de las medias de los numeros totales de los leucocitos (NTL) en las novillas, fue estadísticamente significativo (P < 0,05). Los becerros presentaron neutrofilia al compararse las medias totales del grupo infectado y control (P < 0,05). Los promedios totales de los linfocitos y monocitos en las novillas y becerros, fueron estadísticamente significativos (P < 0,05).

Valores de transaminasas en cabras criollas infectadas con Trypanosoma vivax

La presente comunicación reporta los valores de las enzimas transaminasas, Aspartatoaminotransferasa (AST) y Alaninaaminotransferasa (ALT) encontrados en sueros de cabras infectadas con la cepa de Trypanosoma vivax Stock (TvIIV) y sus controles. Las determinaciones se realizaron durante un lapso experimental de diez semanas, divididos en dos períodos iguales (pre y post-infección) por intermedio de un método colorimétrico, utilizando kits comerciales. Los datos fueron analizados mediante la prueba t Student's. En el caso de la AST, la comparación de las medias parciales de ambos grupos infectado y control, no indicó diferencias estadísticas. Con respecto a la ALT, la contrastación de las medias parciales de pre y post-infección del grupo de cabras infectadas, señaló diferencias significativas (P<0,01).

Functional analysis of Plasmodium vivax VIR proteins reveals different subcellular localizations and cytoadherence to the ICAM-1 endothelial receptor.

The subcellular localization and function of variant subtelomeric multigene families in Plasmodium vivax remain vastly unknown. Among them, the vir superfamily is putatively involved in antigenic variation and in mediating adherence to endothelial receptors. In the absence of a continuous in vitro culture system for P. vivax, we have generated P. falciparum transgenic lines expressing VIR proteins to infer location and function. We chose three proteins pertaining to subfamilies A (VIR17), C (VIR14) and D (VIR10), with domains and secondary structures that predictably traffic these proteins to different subcellular compartments. Here, we showed that VIR17 remained inside the parasite and around merozoites, whereas VIR14 and VIR10 were exported to the membrane of infected red blood cells (iRBCs) in an apparent independent pathway of Maurer's clefts. Remarkably, VIR14 was exposed at the surface of iRBCs and mediated adherence to different endothelial receptors expressed in CHO cells under static conditions. Under physiological flow conditions, however, cytoadherence was only observed to ICAM-1, which was the only receptor whose adherence was specifically and significantly inhibited by antibodies against conserved motifs of VIR proteins. Immunofluorescence studies using these antibodies also showed different subcellular localizations of VIR proteins in P. vivax-infected reticulocytes from natural infections. These data suggest that VIR proteins are trafficked to different cellular compartments and functionally demonstrates that VIR proteins can specifically mediate cytoadherence to the ICAM-1 endothelial receptor.

Avaliação clínico-laboratorial de bovinos Nelore infectados experimentalmente com Trypanosoma vivax

Avaliaram-se as alterações clínico-laboratoriais de seis bezerros Nelore, de ambos os sexos, inoculados experimentalmente com 10(7) organismos viáveis de Trypanosoma vivax, isolados de bovinos da região de Poconé, Estado de Mato Grosso. Os animais foram observados diariamente, durante 30 dias, quanto aos parâmetros de temperatura retal, volume globular (VG), parasitemia, produção de anticorpos, coloração de mucosas, comportamento e apetite. Determinaram-se os níveis séricos de aspartato aminotransferase (AST), fosfatase alcalina (FA), gama glutamiltransferase (GGT), creatina kinase (CK), colesterol, uréia, creatinina, cálcio, fósforo e o perfil eletroforético das proteínas séricas aos 4, 8, 12, 16, 23 e 30 dias pós-inoculação (DPI). Durante os 6 meses seguintes, os animais foram observados semanalmente, avaliando-se a temperatura retal, o VG e a parasitemia. T. vivax foi evidenciado a partir do terceiro e quarto DPI em todos os bezerros e persistiu até o 30° DPI em cinco dos seis animais em estudo. Ocorreu um decréscimo significativo (p<0,05) do valor médio do VG (25%) aos dez DPI. Os animais não apresentaram qualquer alteração no quadro clínico, bem como na avaliação da bioquímica sérica durante o período experimental. A soroconversão ocorreu aos 6 e 8 DPI, permanecendo todos os animais soropositivos nos 30 dias experimentais. Bovinos nelores jovens, infectados experimentalmente com T. vivax, foram capazes de estabelecer um equilíbrio na relação hospedeiro-parasita.

Infecção experimental por Trypanosoma vivax em ovinos

O presente estudo teve por objetivo avaliar as alterações clínicas, hematológicas e patológicas em ovinos infectados experimentalmente com Trypanosoma vivax, utilizando-se um isolado proveniente de bovinos infectados naturalmente no município de Catolé do Rocha, Paraíba. Quatro ovinos da raça Santa Inês foram infectados por via intravenosa com 1ml de sangue contendo 1,85x10(5) tripomastigotas de T. vivax e outros quatro ovinos foram destinados ao grupo controle. A parasitemia e a temperatura foram determinadas diariamente durante 30 dias após a infecção (dpi) e quinzenalmente dos 31 aos 90 dias. A cada 15 dias os animais foram pesados e realizados coletas de sangue para hemograma. Um ovino morreu aos 75 dpi, os demais animais do grupo infectado e do grupo controle foram sacrificados 90 dias após o início do experimento. T. vivax foi evidenciado a partir do 4º dpi em todos os ovinos infectados. A parasitemia foi constante até os 15 dias e irregular entre os 16 e 30 dias. Após o 30º dia não foram observados parasitas no sangue. Foi observada correlação linear positiva entre temperatura retal e parasitemia [Y=0,027x + 38,515; R²=0,9444 (P<0,05)]. Diferença significativa do peso entre os grupos infectado e controle foi verificada a partir do 30º ao 90º dpi. Do 30º ao 90º os animais apresentaram anemia e leucopenia. As lesões macroscópicas encontradas na necropsia foram palidez da carcaça, aumento generalizado dos linfonodos e do baço, e discreta quantidade de líquido nas cavidades peritoneal e pericárdica. Histologicamente, em todos os animais infectados foi observada miocardite multifocal mononuclear. Concluiu-se que o isolado é patogênico para ovinos. Sugere-se que a região semi-árida onde ocorreu o surto, não é endêmica para a tripanossomíase e a doença pode ocorrer se o parasita for introduzido na presença de vetores.

Trypanosoma vivax infection dynamics in a cattle herd maintained in a transition area between Pantanal lowlands and highlands of Mato Grosso do Sul, Brazil

Trypanosoma vivax outbreaks in beef cattle in the Pantanal region of Mato Grosso do Sul state, Brazil, causes relevant economical impact due to weight loss, abortion and mortality. Cattle moved from the Pantanal to adjacent areas of this ecosystem for breeding and fattening is a common feature. Therefore an epidemiological study on breeding cows in the transition area between Pantanal lowland and adjacent highlands of Mato Grosso do Sul was performed to determine the T. vivax infection dynamics and outbreak risk. Three experimental groups were formed: Group 1 consisted of cows parasitologically negative by the Woo test and in the enzyme-linked immunosorbent assay for T. vivax antibody detection (Tv-ELISA-Ab); Group 2 parasitologically negative and positive in the Tv-ELISA-Ab; and in Group 3 cows were parasitologically positive and with positive reactions in the Tv-ELISA-Ab. During 24 months, the cows' dislodgment between the above established groups was monitored by Woo test and Tv-ELISA-Ab exams. The tabanid population was also monitored and the highest number occurred during the rainy season. Although parasitemias were detected only in the first four samplings of the experimental period, the cows could be considered as trypanotolerant, because no clinical signs were observed. Despite the higher T. vivax incidence during the dry season, no disease symptoms were seen. Even though T. vivax epidemiological situation in the herd was characterized as endemic with seasonal variation, the probability of outbreaks was null within the conditions of the study.

Aspectos clínicos, epidemiológicos e patológicos da infecção natural em bovinos por Trypanosoma vivax na Paraíba

Descrevem-se dois surtos de tripanossomíase por Trypanosoma vivax em bovinos, ocorridos em dois estabelecimentos do alto sertão da Paraíba. Os sinais clínicos, a patologia e a epidemiologia da doença foram estudados no período de maio de 2005 a novembro de 2006.T. vivax foi identificado em esfregaços da capa leucocitária e mediante a reação em cadeia da polimerase (PCR). Os animais afetados apresentaram anorexia, depressão, febre, anemia, perda de peso, redução da produção leiteira, cegueira transitória, aborto e sinais nervosos caracterizados por incoordenação motora, salivação, opistótono, nistagno, tetania e bruxismo. Todos os animais que apresentaram sintomatologia nervosa morreram. As alterações macroscópicas observadas em um bovino submetido à necropsia foram aumento de volume dos linfonodos, atrofia serosa dos depósitos de gordura, aumento de volume do baço com evidência da polpa branca, hidropericárdio, além de petéquias e equimoses no epicárdio. Histologicamente havia meningoencefalite. O controle da doença na propriedade com tratamento específico dos casos clínicos com aceturato de diminazene foi eficiente, pois após o tratamento não se verificou mais a presença do parasita em esfregaços sanguíneos nem evidência clínica da enfermidade em até 2 meses após o início do surto. Os fatores epidemiológicos favoráveis à ocorrência dos surtos foram a abundância de vetores mecânicos, como tabanídeos e Stomozys spp., e a entrada, no rebanho, de animais oriundos de propriedades onde ocorreu a doença em questão. Sugere-se que o semi-árido do Nordeste, devido a períodos prolongados de secas e altas temperaturas, é região de instabilidade enzoótica para a tripanossomíase, em conseqüência, provavelmente, ao ambiente desfavorável para o desenvolvimento de vetores durante a maior parte do ano.