RESUMO
Plasmodium vivax is the most prevalent cause of malaria outside of Africa. P. vivax biology and pathogenesis are still poorly understood. The role of one highly occurring phenotype in particular where infected reticulocytes cytoadhere to noninfected normocytes, forming rosettes, remains unknown. Here, using a range of ex vivo approaches, we showed that P. vivax rosetting rates were enhanced by plasma of infected patients and that total immunoglobulin M levels correlated with rosetting frequency. Moreover, rosetting rates were also correlated with parasitemia, IL-6 and IL-10 levels in infected patients. Transcriptomic analysis of peripheral leukocytes from P. vivax-infected patients with low or moderated rosetting rates identified differentially expressed genes related to human host phagocytosis pathway. In addition, phagocytosis assay showed that rosetting parasites were less phagocyted. Collectively, these results showed that rosette formation plays a role in host immune response by hampering leukocyte phagocytosis. Thus, these findings suggest that rosetting could be an effective P. vivax immune evasion strategy.
Assuntos
Malária Vivax/parasitologia , Parasitemia/imunologia , Fagocitose/imunologia , Plasmodium vivax/imunologia , Formação de Roseta , Humanos , Imunoglobulina M/sangue , Interleucina-10/sangue , Interleucina-6/sangue , Malária Vivax/sangue , Malária Vivax/imunologia , Parasitemia/sangueRESUMO
Plasmodium vivax is the most widely distributed malaria species and the most prevalent species of malaria in America and Asia. Vaccine development against P. vivax is considered a priority in the global program for the eradication of malaria. Earlier studies have characterized the Apical Membrane Antigen 1 (AMA-1) ectodomain and the C-terminal region (19kDa) of the Merozoite Surface Protein 1 (MSP-1) of P. vivax as immunodominant antigens. Based on this characterization, we designed a chimeric recombinant protein containing both merozoite immunodominant domains (PvAMA166-MSP119). The recombinant PvAMA166-MSP119 was successfully expressed in Pichia pastoris and used to immunize two different mouse strains (BALB/c and C57BL/6) in the presence of the Poly (I:C) as an adjuvant. Immunization with the chimeric protein induced high antibody titers against both proteins in both strains of mice as detected by ELISA. Antisera also recognized the native proteins expressed on the merozoites of mature P. vivax schizonts. Moreover, this antigen was able to induce IFN-gamma-secreting cells in C57BL/6 mice. These findings indicate that this novel yeast recombinant protein containing PvAMA166 and PvMSP119 is advantageous, because of improved antibody titers and cellular immune response. Therefore, this formulation should be further developed for pre-clinical trials in non-human primates as a potential candidate for a P. vivax vaccine.
Assuntos
Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Proteínas de Membrana/imunologia , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Proteínas Recombinantes de Fusão/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/genética , Proteínas de Membrana/genética , Proteína 1 de Superfície de Merozoito/genética , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pichia/genética , Poli I-C/administração & dosagem , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Malaria remains a world-threatening disease largely because of the lack of a long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane molecule with a chimeric cysteine-rich ectodomain homologous to regions of the Duffy binding-like erythrocyte binding protein and apical membrane antigen 1 (AMA1) antigens. Although MAEBL does not appear to be essential for the survival of blood-stage forms, ectodomains M1 and M2, homologous to AMA1, seem to be involved in parasite attachment to erythrocytes, especially M2. MAEBL is necessary for sporozoite infection of mosquito salivary glands and is expressed in liver stages. Here, the Plasmodium yoelii MAEBL-M2 domain was expressed in a prokaryotic vector. C57BL/6J mice were immunized with doses of P. yoelii recombinant protein rPyM2-MAEBL. High levels of antibodies, with balanced IgG1 and IgG2c subclasses, were achieved. rPyM2-MAEBL antisera were capable of recognizing the native antigen. Anti-MAEBL antibodies recognized different MAEBL fragments expressed in CHO cells, showing stronger IgM and IgG responses to the M2 domain and repeat region, respectively. After a challenge with P. yoelii YM (lethal strain)-infected erythrocytes (IE), up to 90% of the immunized animals survived and a reduction of parasitemia was observed. Moreover, splenocytes harvested from immunized animals proliferated in a dose-dependent manner in the presence of rPyM2-MAEBL. Protection was highly dependent on CD4(+), but not CD8(+), T cells toward Th1. rPyM2-MAEBL antisera were also able to significantly inhibit parasite development, as observed in ex vivo P. yoelii erythrocyte invasion assays. Collectively, these findings support the use of MAEBL as a vaccine candidate and open perspectives to understand the mechanisms involved in protection.
Assuntos
Vacinas Antimaláricas/imunologia , Malária/prevenção & controle , Plasmodium yoelii/imunologia , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Eritrócitos/parasitologia , Feminino , Humanos , Imunização , Malária/imunologia , Malária/mortalidade , Malária/parasitologia , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/química , Vacinas Antimaláricas/genética , Masculino , Merozoítos/química , Merozoítos/crescimento & desenvolvimento , Merozoítos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmodium yoelii/química , Plasmodium yoelii/genética , Plasmodium yoelii/crescimento & desenvolvimento , Estrutura Terciária de Proteína , Proteínas de Protozoários/administração & dosagem , Proteínas de Protozoários/genética , Esporozoítos/química , Esporozoítos/crescimento & desenvolvimento , Esporozoítos/imunologiaRESUMO
There is now a growing body of evidence that challenges the current view that Plasmodium vivax-infected erythrocyte (Pv-iE) are unable to sequester. Here we used ex vivo adhesion assays with Pv-iE before and after maturation to demonstrate a higher binding potential of schizonts compared to other asexual stages. These experimental results are correlated with our observations in a panel of 50 vivax malaria patients where schizonts were completely absent in 27 isolates, and few schizonts were observed in the remaining patients. These observations prompt a paradigm shift in P. vivax biology and open avenues to investigate the role of Pv-iE sequestration.
Assuntos
Adesão Celular/fisiologia , Eritrócitos/parasitologia , Malária Vivax/sangue , Malária Vivax/parasitologia , Plasmodium vivax/fisiologia , Antimaláricos/uso terapêutico , Cloroquina/uso terapêutico , Humanos , Malária Vivax/tratamento farmacológico , Parasitemia/sangue , Parasitemia/parasitologia , Plasmodium vivax/crescimento & desenvolvimento , Primaquina/uso terapêutico , Esquizontes/fisiologia , Estatísticas não ParamétricasRESUMO
In a recent vaccine trial performed with African children, immunization with a recombinant protein based on Plasmodium falciparum apical membrane antigen 1 (AMA-1) conferred a significant degree of strain-specific resistance against malaria. To contribute to the efforts of generating a vaccine against Plasmodium vivax malaria, we expressed the ectodomain of P. vivax AMA-1 (PvAMA-1) as a secreted soluble protein in the methylotrophic yeast Pichia pastoris. Recognized by a high percentage of sera from individuals infected by P. vivax, this recombinant protein was found to have maintained its antigenicity. The immunogenicity of this protein was evaluated in mice using immunization protocols that included homologous and heterologous prime-boost strategies with plasmid DNA and recombinant protein. We used the following formulations containing different adjuvants: aluminum salts (Alum), Bordetella pertussis monophosphoryl lipid A (MPLA), flagellin FliC from Salmonella enterica serovar Typhimurium, saponin Quil A, or incomplete Freund's adjuvant (IFA). The formulations containing the adjuvants Quil A or IFA elicited the highest IgG antibody titers. Significant antibody titers were also obtained using a formulation developed for human use containing MPLA or Alum plus MPLA. Recombinant PvAMA-1 produced under "conditions of good laboratory practice" provided a good yield, high purity, low endotoxin levels, and no microbial contaminants and reproduced the experimental immunizations. Most relevant for vaccine development was the fact that immunization with PvAMA-1 elicited invasion-inhibitory antibodies against different Asian isolates of P. vivax. Our results show that AMA-1 expressed in P. pastoris is a promising antigen for use in future preclinical and clinical studies.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Proteínas de Membrana/imunologia , Pichia/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Leveduras/imunologia , Adjuvantes Imunológicos/genética , Animais , Formação de Anticorpos/imunologia , Antígenos de Protozoários/genética , Feminino , Humanos , Imunização/métodos , Imunoglobulina G/imunologia , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/imunologia , Malária Vivax/genética , Malária Vivax/imunologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Pichia/genética , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Leveduras/genéticaRESUMO
A recent clinical trial in African children demonstrated the potential utility of merozoite surface protein (MSP)-3 as a vaccine against Plasmodium falciparum malaria. The present study evaluated the use of Plasmodium vivax MSP-3 (PvMSP-3) as a target antigen in vaccine formulations against malaria caused by P. vivax. Recombinant proteins representing MSP-3α and MSP-3ß of P. vivax were expressed as soluble histidine-tagged bacterial fusions. Antigenicity during natural infection was evaluated by detecting specific antibodies using sera from individuals living in endemic areas of Brazil. A large proportion of infected individuals presented IgG antibodies to PvMSP-3α (68.2%) and at least 1 recombinant protein representing PvMSP-3ß (79.1%). In spite of the large responder frequency, reactivity to both antigens was significantly lower than was observed for the immunodominant epitope present on the 19-kDa C-terminal region of PvMSP-1. Immunogenicity of the recombinant proteins was studied in mice in the absence or presence of different adjuvant formulations. PvMSP-3ß, but not PvMSP-3α, induced a TLR4-independent humoral immune response in the absence of any adjuvant formulation. The immunogenicity of the recombinant antigens were also tested in formulations containing different adjuvants (Alum, Salmonella enterica flagellin, CpG, Quil A,TiterMax® and incomplete Freunds adjuvant) and combinations of two adjuvants (Alum plus flagellin, and CpG plus flagellin). Recombinant PvMSP-3α and PvMSP-3ß elicited higher antibody titers capable of recognizing P. vivax-infected erythrocytes harvested from malaria patients. Our results confirm that P. vivax MSP-3 antigens are immunogenic during natural infection, and the corresponding recombinant proteins may be useful in elucidating their vaccine potential.
Assuntos
Antígenos de Protozoários/uso terapêutico , Vacinas Antimaláricas/uso terapêutico , Malária Vivax/prevenção & controle , Plasmodium vivax/imunologia , Proteínas de Protozoários/uso terapêutico , Animais , Formação de Anticorpos , Antígenos de Protozoários/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Vacinas Antimaláricas/imunologia , Malária Vivax/sangue , Malária Vivax/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêuticoRESUMO
Life-threatening Plasmodium vivax malaria cases, while uncommon, have been reported since the early 20th century. Unfortunately, the pathogenesis of these severe vivax malaria cases is still poorly understood. In Brazil, the proportion of vivax malaria cases has been steadily increasing, as have the number of cases presenting serious clinical complications. The most frequent syndromes associated with severe vivax malaria in Brazil are severe anaemia and acute respiratory distress. Additionally, P. vivax infection may also result in complications associated with pregnancy. Here, we review the latest findings on severe vivax malaria in Brazil. We also discuss how the development of targeted field research infrastructure in Brazil is providing clinical and ex vivo experimental data that benefits local and international efforts to understand the pathogenesis of P. vivax.
Assuntos
Malária Vivax/patologia , Malária Vivax/parasitologia , Plasmodium vivax , Brasil/epidemiologia , Adesão Celular , Feminino , Humanos , Malária Vivax/epidemiologia , Plasmodium vivax/patogenicidade , Plasmodium vivax/fisiologia , Gravidez , Complicações Parasitárias na Gravidez/epidemiologia , Complicações Parasitárias na Gravidez/parasitologia , Complicações Parasitárias na Gravidez/patologia , Medicina Tropical/organização & administraçãoRESUMO
Apical membrane antigen 1 (AMA-1) is an invasion-related Plasmodium antigen that is expressed during both intracellular and extracellular asexual stages of the parasite's life cycle, making it an ideal target for induction of humoral and cellular immune responses that can protect against malaria. We show here that when it is administered as a recombinant protein (P) in Montanide ISA720 adjuvant, followed by a recombinant human type 5 adenovirus (Ad), intense and long-lasting Plasmodium vivax AMA-1-specific antibody responses (including both IgG1 and IgG2a), as well as proliferative memory T cell responses, can be detected in immunized mice. Memory T cells displayed both central (CD44(hi) CD62L(hi)) and effector (CD44(hi) CD62L(lo)) phenotypes, with the central memory phenotype prevailing (56% of AMA-1-specific proliferating cells). Considering the main traits of the memory immune responses induced against AMA-1, this particular sequence of immunogens (P followed by Ad), but no others (Ad/Ad, Ad/P, or P/P), displayed an optimal synergistic effect. These results give further support to the need for preclinical studies of P. vivax vaccine candidate AMA-1 administered in prime/boost protocols that include recombinant proteins and adenoviral vectors.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Plasmodium vivax/imunologia , Adenoviridae , Adjuvantes Imunológicos , Animais , Anticorpos Antiprotozoários/biossíntese , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Feminino , Receptores de Hialuronatos/biossíntese , Imunidade Celular , Imunidade Humoral , Imunização , Imunização Secundária , Memória Imunológica , Selectina L/biossíntese , Vacinas Antimaláricas/administração & dosagem , Malária Vivax/imunologia , Malária Vivax/prevenção & controle , Manitol/administração & dosagem , Manitol/análogos & derivados , Manitol/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ácidos Oleicos/administração & dosagem , Ácidos Oleicos/imunologia , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia , Vacinas Sintéticas/imunologiaRESUMO
BACKGROUND: Plasmodium falciparum and Plasmodium vivax are responsible for most of the global burden of malaria. Although the accentuated pathogenicity of P. falciparum occurs because of sequestration of the mature erythrocytic forms in the microvasculature, this phenomenon has not yet been noted in P. vivax. The increasing number of severe manifestations of P. vivax infections, similar to those observed for severe falciparum malaria, suggests that key pathogenic mechanisms (eg, cytoadherence) might be shared by the 2 parasites. METHODS: Mature P. vivax-infected erythrocytes (Pv-iEs) were isolated from blood samples collected from 34 infected patients. Pv-iEs enriched on Percoll gradients were used in cytoadhesion assays with human lung endothelial cells, Saimiri brain endothelial cells, and placental cryosections. RESULTS: Pv-iEs were able to cytoadhere under static and flow conditions to cells expressing endothelial receptors known to mediate the cytoadhesion of P. falciparum. Although Pv-iE cytoadhesion levels were 10-fold lower than those observed for P. falciparum-infected erythrocytes, the strength of the interaction was similar. Cytoadhesion of Pv-iEs was in part mediated by VIR proteins, encoded by P. vivax variant genes (vir), given that specific antisera inhibited the Pv-iE-endothelial cell interaction. CONCLUSIONS: These observations prompt a modification of the current paradigms of the pathogenesis of malaria and clear the way to investigate the pathophysiology of P. vivax infections.