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1.
Parasitol Int ; 80: 102224, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33137499

RESUMO

Vaccines against infectious diseases have had great successes in the history of public health. Major breakthroughs have occurred in the development of vaccine-based interventions against viral and bacterial pathogens through the application of classical vaccine design strategies. In contrast the development of a malaria vaccine has been slow. Plasmodium falciparum malaria affects millions of people with nearly half of the world population at risk of infection. Decades of dedicated research has taught us that developing an effective vaccine will be time consuming, challenging, and expensive. Nevertheless, recent advancements such as the optimization of robust protein synthesis platforms, high-throughput immunoscreening approaches, reverse vaccinology, structural design of immunogens, lymphocyte repertoire sequencing, and the utilization of artificial intelligence, have renewed the prospects of an accelerated discovery of the key antigens in malaria. A deeper understanding of the major factors underlying the immunological and molecular mechanisms of malaria might provide a comprehensive approach to identifying novel and highly efficacious vaccines. In this review we discuss progress in novel antigen discoveries that leverage on the wheat germ cell-free protein synthesis system (WGCFS) to accelerate malaria vaccine development.

2.
Parasitol Int ; 80: 102240, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33147497

RESUMO

During intraerythrocytic development Plasmodium falciparum deploys numerous proteins to support erythrocyte invasion, intracellular growth and development, as well as host immune evasion. Since these proteins are key for parasite intraerythrocytic survival and propagation, they represent attractive targets for antimalarial vaccines. In this study we sought to characterize a member of the PHISTc family of proteins, PF3D7_0801000, as a potential vaccine target. Using the wheat germ cell-free system we expressed the N-terminal region of PF3D7_0801000 (G93-L494, PF3D7_0801000N) and generated specific immune sera. We observed that PF3D7_0801000 localizes in merozoites, and antibodies against PF3D7_0801000N modestly inhibit P. falciparum parasite growth in in vitro culture. Sliding window analysis of the coding sequence revealed that pf3d7_0801000n is relatively conserved among African parasite isolates. Antibody profiles in a malaria-exposed Ugandan population revealed that PF3D7_0801000N is strongly immunoreactive with antibody acquisition increasing with age. Taken together, these findings suggest the need for further evaluation of PF3D7_0801000 for its role in merozoite invasion and utility as an asexual blood-stage vaccine candidate antigen.

3.
PLoS One ; 15(12): e0238010, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33275613

RESUMO

Multiplexed bead-based assays that use Luminex® xMAP® technology have become popular for measuring antibodies against proteins of interest in many fields, including malaria and more recently SARS-CoV-2/COVID-19. There are currently two formats that are widely used: non-magnetic beads or magnetic beads. Data are lacking regarding the comparability of results obtained using these two types of beads, and for assays run on different instruments. Whilst non-magnetic beads can only be run on flow-based instruments (such as the Luminex® 100/200™ or Bio-Plex® 200), magnetic beads can be run on both these and the newer MAGPIX® instruments. In this study we utilized a panel of purified recombinant Plasmodium vivax proteins and samples from malaria-endemic areas to measure P. vivax-specific IgG responses using different combinations of beads and instruments. We directly compared: i) non-magnetic versus magnetic beads run on a Bio-Plex® 200, ii) magnetic beads run on the Bio-Plex® 200 versus MAGPIX® and iii) non-magnetic beads run on a Bio-Plex® 200 versus magnetic beads run on the MAGPIX®. We also performed an external comparison of our optimized assay. We observed that IgG antibody responses, measured against our panel of P. vivax proteins, were moderately-strongly correlated in all three of our comparisons (pearson r>0.5 for 18/19 proteins), however higher amounts of protein were required for coupling to magnetic beads. Our external comparison indicated that results generated in different laboratories using the same coupled beads are also highly comparable (pearson r>0.7), particularly if a reference standard curve is used.

4.
PLoS Negl Trop Dis ; 14(12): e0008998, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33370333

RESUMO

Clonorchiasis caused by Clonorchis sinensis is endemic in East Asia; approximately 15 million people have been infected thus far. To diagnose the infection, serodiagnostic tests with excellent functionality should be performed. First, 607 expressed sequence tags encoding polypeptides with a secretory signal were expressed into recombinant proteins using an in vitro translation system. By protein array-based screening using C. sinensis-infected sera, 18 antigen candidate proteins were selected and assayed for cross-reactivity against Opisthorchis viverrini-infected sera. Of the six antigenic proteins selected, four were synthesized on large scale in vitro and evaluated for antigenicity against the flukes-infected human sera using ELISA. CsAg17 antigen showed the highest sensitivity (77.1%) and specificity (71.2%). The sensitivity and specificity of the bacterially produced CsAg17-28GST fusion antigen was similar to those of CsAg17 antigen. CsAg17 antigen can be used to develop point-of-care serodiagnostic tests for clonorchiasis.


Assuntos
Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/imunologia , Clonorquíase/diagnóstico , Clonorchis sinensis/imunologia , Animais , Clonorchis sinensis/genética , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática , Peixes/parasitologia , Humanos , Imunoglobulina G/sangue , Opisthorchis/imunologia , Testes Imediatos , Proteogenômica , Alimentos Crus/parasitologia , Sensibilidade e Especificidade , Testes Sorológicos
5.
Cell Microbiol ; : e13294, 2020 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-33222390

RESUMO

Gametogenesis, the formation of gametes from gametocytes, an essential step for malaria parasite transmission, is targeted by transmission-blocking drugs and vaccines. We identified a conserved protein (PBANKA_0305900) in Plasmodium berghei, which encodes a protein of 22 kDa (thus named Pb22) and is expressed in both asexual stages and gametocytes. Its homologues are present in all Plasmodium species and its closely related, Hepatocystis, but not in other apicomplexans. Pb22 protein was localised in the cytosols of schizonts, as well as male and female gametocytes. During gamete-to-ookinete development, Pb22 became localised on the plasma membranes of gametes and ookinetes. Compared to the wild-type (WT) parasites, P. berghei with pb22 knockout (KO) showed a significant reduction in exflagellation (~89%) of male gametocytes and ookinete number (~97%) during in vitro ookinete culture. Mosquito feeding assays showed that ookinete and oocyst formation of the pb22-KO line in mosquito midguts was almost completely abolished. These defects were rescued in parasites where pb22 was restored. Cross-fertilisation experiments with parasite lines defective in either male or female gametes confirmed that the defects in the pb22-KO line were restricted to the male gametes, whereas female gametes in the pb22-KO line were fertile at the WT level. Detailed analysis of male gametogenesis showed that 30% of the male gametocytes in the pb22-KO line failed to assemble the axonemes, whereas ~48.9% of the male gametocytes formed flagella but failed to egress from the host erythrocyte. To explore its transmission-blocking potential, recombinant Pb22 (rPb22) was expressed and used to immunise mice. in vitro assays showed that the rPb22-antisera significantly inhibited exflagellation by ~64.8% and ookinete formation by ~93.4%. Mosquitoes after feeding on rPb22-immunised mice also showed significant decreases in infection prevalence (83.3-93.3%) and oocyst density (93.5-99.6%). Further studies of the Pb22 orthologues in human malaria parasites are warranted.

6.
Front Immunol ; 11: 893, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32477363

RESUMO

Clinical immunity to malaria develops after repeated exposure to Plasmodium falciparum parasites. Broadly reactive antibodies against parasite antigens expressed on the surface of infected erythrocytes (variable surface antigens; VSAs) are candidates for anti-malaria therapeutics and vaccines. Among the VSAs, several RIFIN, STEVOR, and SURFIN family members have been demonstrated to be targets of naturally acquired immunity against malaria. For example, RIFIN family members are important ligands for opsonization of P. falciparum infected erythrocytes with specific immunoglobulins (IgG) acquiring broad protective reactivity. However, the global repertoire of human anti-VSAs IgG, its variation in children, and the key protective targets remain poorly understood. Here, we report wheat germ cell-free system-based production and serological profiling of a comprehensive library of A-RIFINs, B-RIFINs, STEVORs, and SURFINs derived from the P. falciparum 3D7 parasite strain. We observed that >98% of assayed proteins (n = 265) were immunogenic in malaria-exposed individuals in Uganda. The overall breadth of immune responses was significantly correlated with age but not with clinical malaria outcome among the study volunteers. However, children with high levels of antibodies to four RIFINs (PF3D7_0201000, PF3D7_1254500, PF3D7_1040600, PF3D7_1041100), STEVOR (PF3D7_0732000), and SURFIN 1.2 (PF3D7_0113600) had prospectively reduced the risk of developing febrile malaria, suggesting that the 5 antigens are important targets of protective immunity. Further studies on the significance of repeated exposure to malaria infection and maintenance of such high-level antibodies would contribute to a better understanding of susceptibility and naturally acquired immunity to malaria.

7.
Parasitol Int ; 79: 102160, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32574727

RESUMO

Members of the mitochondrial carrier (MC) family of membrane transporters play important roles in cellular metabolism. We previously established an in vitro reconstitution system for membrane transporters based on wheat germ cell-free translation system. We have now applied this reconstitution system to the comparative analysis of MC proteins from the malaria parasite Plasmodium falciparum and Saccharomyces cerevisiae. We synthesized twelve putative P. falciparum MCs and determined the transport activities of four of these proteins including PF3D7_1037300 protein (ADP/ATP translocator), PF3D7_1004800 protein (ADP/ATP translocator), PF3D7_1202200 protein (phosphate carrier), and PF3D7_1241600 protein (S-adenosylmethionine transporter). In addition, we tested the effect of cardiolipin on the activity of MC proteins. The transport activities of the yeast MCs, ScAac2p, ScGgc1p, ScDic1p, ScPic1p, and ScSam5p, which localize to the mitochondrial inner membrane, were increased by cardiolipin supplementation, whereas that of ScAnt1p, which localizes to the peroxisome membrane, was not significantly affected. Together, this indicates that the functional properties of the reconstituted MCs reflect the lipid content of their native membranes. Except for PF3D7_1241600 protein, these P. falciparum proteins manifested cardiolipin-dependent transport activities. Immunofluorescence analysis showed that PF3D7_1241600 protein is not mainly localized to the mitochondria of P. falciparum cells. We thus revealed the functions of four MC proteins of the malaria parasite and the effects of cardiolipin on their activities.

8.
Nat Med ; 26(5): 741-749, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32405064

RESUMO

A major gap in the Plasmodium vivax elimination toolkit is the identification of individuals carrying clinically silent and undetectable liver-stage parasites, called hypnozoites. This study developed a panel of serological exposure markers capable of classifying individuals with recent P. vivax infections who have a high likelihood of harboring hypnozoites. We measured IgG antibody responses to 342 P. vivax proteins in longitudinal clinical cohorts conducted in Thailand and Brazil and identified candidate serological markers of exposure. Candidate markers were validated using samples from year-long observational cohorts conducted in Thailand, Brazil and the Solomon Islands and antibody responses to eight P. vivax proteins classified P. vivax infections in the previous 9 months with 80% sensitivity and specificity. Mathematical models demonstrate that a serological testing and treatment strategy could reduce P. vivax prevalence by 59-69%. These eight antibody responses can serve as a biomarker, identifying individuals who should be targeted with anti-hypnozoite therapy.


Assuntos
Biomarcadores/sangue , Malária Vivax/diagnóstico , Testes Sorológicos/métodos , Adulto , Brasil/epidemiologia , Criança , Estudos de Coortes , Diagnóstico Precoce , Humanos , Imunoglobulina G/análise , Imunoglobulina G/sangue , Controle de Infecções/métodos , Estudos Longitudinais , Malária Vivax/sangue , Malária Vivax/epidemiologia , Melanesia/epidemiologia , Plasmodium vivax/fisiologia , Prevalência , Sensibilidade e Especificidade , Testes Sorológicos/normas , Tailândia/epidemiologia , Fatores de Tempo
9.
Malar J ; 19(1): 155, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32295584

RESUMO

BACKGROUND: The malaria parasite Plasmodium falciparum is a protozoan that develops in red blood cells (RBCs) and requires various host factors. For its development in RBCs, nutrients not only from the RBC cytosol but also from the extracellular milieu must be acquired. Although the utilization of host nutrients by P. falciparum has been extensively analysed, only a few studies have reported its utilization of host serum proteins. Hence, the aim of the current study was to comprehensively identify host serum proteins taken up by P. falciparum parasites and to elucidate their role in pathogenesis. METHODS: Plasmodium falciparum was cultured with human serum in vitro. Uptake of serum proteins by parasites was comprehensively determined via shotgun liquid chromatography-mass spectrometry/mass spectrometry and western blotting. The calcium ion concentration in serum was also evaluated, and coagulation activity of the parasite lysate was assessed. RESULTS: Three proteins, vitamin K-dependent protein S, prothrombin, and vitronectin, were selectively internalized under sufficient Ca2+ levels in the culture medium. The uptake of these proteins was initiated before DNA replication, and increased during the trophozoite and schizont stages, irrespective of the assembly/disassembly of actin filaments. Coagulation assay revealed that prothrombin was activated and thereby induced blood coagulation. CONCLUSIONS: Serum proteins were taken up by parasites under culture conditions with sufficient Ca2+ levels. This uptake phenomenon was associated with their pathogenicity.


Assuntos
Proteínas Sanguíneas/metabolismo , Eritrócitos/parasitologia , Malária Falciparum/parasitologia , Plasmodium falciparum/fisiologia , Western Blotting , Cromatografia Líquida , Plasmodium falciparum/patogenicidade , Esquizontes/fisiologia , Espectrometria de Massas em Tandem , Trofozoítos/fisiologia
10.
Sci Rep ; 10(1): 6573, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313230

RESUMO

Plasmodium falciparum merozoite invasion into erythrocytes is an essential step of the blood-stage cycle, survival of parasites, and malaria pathogenesis. P. falciparum merozoite Rh5 interacting protein (PfRipr) forms a complex with Rh5 and CyRPA in sequential molecular events leading to erythrocyte invasion. Recently we described PfRipr as a conserved protein that induces strain-transcending growth inhibitory antibodies in in vitro assays. However, being a large and complex protein of 1086 amino acids (aa) with 87 cysteine residues, PfRipr is difficult to express in conventional expression systems towards vaccine development. In this study we sought to identify the most potent region of PfRipr that could be developed to overcome difficulties related to protein expression, as well as to elucidate the invasion inhibitory mechanism of anti-PfRipr antibodies. Using the wheat germ cell-free system, Ecto- PfRipr and truncates of approximately 200 aa were expressed as soluble proteins. We demonstrate that antibodies against PfRipr truncate 5 (PfRipr_5: C720-D934), a region within the PfRipr C-terminal EGF-like domains, potently inhibit merozoite invasion. Furthermore, the antibodies strongly block PfRipr/Rh5 interaction, as well as that between PfRipr and its erythrocyte-surface receptor, SEMA7A. Taken together, PfRipr_5 is a potential candidate for further development as a blood-stage malaria vaccine.


Assuntos
Anticorpos/farmacologia , Antígenos CD/genética , Proteínas de Transporte/genética , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Semaforinas/genética , Anticorpos/genética , Anticorpos/imunologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Proteínas de Transporte/imunologia , Eritrócitos/parasitologia , Proteínas Ligadas por GPI/genética , Regulação da Expressão Gênica/genética , Humanos , Malária Falciparum/genética , Malária Falciparum/parasitologia , Merozoítos/genética , Merozoítos/patogenicidade , Plasmodium falciparum/patogenicidade , Ligação Proteica/imunologia , Proteínas de Protozoários/imunologia
11.
Vaccine ; 38(15): 3129-3136, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-32147299

RESUMO

Malaria transmission-blocking vaccines (TBVs) aim to inhibit parasite fertilization or further development within the mosquito midgut. Because TBV-immunized individuals reduce the transmission of malaria parasites to mosquito vectors, TBVs could serve as a promising strategy to eliminate malaria. We previously reported that a male specific protein, PyMiGS (Plasmodium yoelii microgamete surface protein), is localized to the surface of microgametes and anti-PyMiGS antibodies have strong transmission-blocking activity. In this study we determine a region of PyMiGS that contains epitopes inducing potent transmission-blocking antibodies. PyMiGS excluding the N-terminal signal sequence and C-terminal hydrophobic region (PyMiGS-full) was divided into five overlapping regions, named I through V, and corresponding truncated recombinant proteins were produced. Anti-region V antibody, affinity-purified from anti-PyMiGS-full rabbit antiserum, significantly reduced the number of oocysts in a mosquito membrane-feeding assay. Antibodies from mice immunized with PyMiGS-V recognized the microgamete surface and showed higher transmission-blocking efficacy than antibodies obtained by PyMiGS-full immunization. These results indicate that the major epitopes for transmission-blocking antibodies are within region V at the C-terminal region of PyMiGS. Therefore, region V of MiGS could be a promising pre-fertilization TBV candidate antigen.

12.
Malar J ; 19(1): 76, 2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-32070358

RESUMO

BACKGROUND: Usage of chloroquine was discontinued from the treatment of Plasmodium falciparum infection in almost all endemic regions because of global spread of resistant parasites. Since the first report in Malawi, numerous epidemiological studies have demonstrated that the discontinuance led to re-emergence of chloroquine-susceptible P. falciparum, suggesting a possible role in future malaria control. However, most studies were cross-sectional, with few studies looking at the persistence of chloroquine recovery in long term. This study fills the gap by providing, for a period of at least 6 years, proof of persistent re-emergence/stable recovery of susceptible parasite populations using both molecular and phenotypic methods. METHODS: Ex vivo drug-susceptibility assays to chloroquine (n = 319) and lumefantrine (n = 335) were performed from 2013 to 2018 in Gulu, Northern Uganda, where chloroquine had been removed from the official malaria treatment regimen since 2006. Genotyping of pfcrt and pfmdr1 was also performed. RESULTS: Chloroquine resistance (≥ 100 nM) was observed in only 3 (1.3%) samples. Average IC50 values for chloroquine were persistently low throughout the study period (17.4-24.9 nM). Parasites harbouring pfcrt K76 alleles showed significantly lower IC50s to chloroquine than the parasites harbouring K76T alleles (21.4 nM vs. 43.1 nM, p-value = 3.9 × 10-8). Prevalence of K76 alleles gradually increased from 71% in 2013 to 100% in 2018. CONCLUSION: This study found evidence of stable persistence of chloroquine susceptibility with the fixation of pfcrt K76 in Northern Uganda after discontinuation of chloroquine in the region. Accumulation of similar evidence in other endemic areas in Uganda could open channels for possible future re-use of chloroquine as an option for malaria treatment or prevention.


Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Resistência a Medicamentos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Uganda
13.
Parasitol Int ; 76: 102062, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31978597

RESUMO

A platyhelminth, Spirometra erinaceieuropaei, belonging to the class Cestoda, causes human sparganosis, and infection with its larva results in subtle inflammation in the body of its host. We previously reported the purification of a glycoprotein, plerocercoid-immunosuppressive factor (P-ISF) from the excretory/secretory products of S. erinaceieuropaei plerocercoids that may be involved in immuno-modification. We determined the sequence of P-ISF from the N-terminal and the internal 10 amino acids of P-ISF using degenerate PCR and 5'- and 3'-RACE methods. The putative gene encoding P-ISF was 1443 bp long and the gene contained 10 exons and 9 introns in a genomic DNA of size 5205 bp. P-ISF consists of 480 amino acids including the N-terminal signal peptide sequence, and has two unknown domains,-cestoda cysteine-rich domains (CCDs) and a fibronectin type III domain between the two CCDs. All cysteine residues were conserved in the two CCDs, which shared 62% amino acid identities. Homologous analysis revealed that the CCDs were homologous with an unknown protein of Diphyllobothrium latum. To produce specific antibodies, we expressed recombinant P-ISF (rP-ISF) using wheat germ protein synthetic system. P-ISF was localized in the sub-cutaneous tissues and the parenchymal tissues of plerocercoids. Transcription of P-ISF was detected only in plerocercoid stage, but not in adult stage. Western blotting also showed a band in plerocercoide stage but not in adult. The rP-ISF did not suppress nitrite production in RAW 264.7 cells stimulated with LPS, and this might be due to lack of carbohydrate chains in the recombinant protein.


Assuntos
Glicoproteínas/genética , Proteínas de Helminto/genética , Spirometra/genética , Animais , Clonagem Molecular , Cisteína/análise , Cisteína/genética , Feminino , Fibronectinas/genética , Genoma Helmíntico , Camundongos , Camundongos Endogâmicos C57BL , Sinais Direcionadores de Proteínas , Células RAW 264.7 , Proteínas Recombinantes/genética , Organismos Livres de Patógenos Específicos
14.
Mol Biochem Parasitol ; 236: 111261, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31981605

RESUMO

Plasmodium parasites cause malaria in mammalian hosts and are transmitted by Anopheles mosquitoes. Gametocytes, which differentiate from asexual-stage parasites, are activated by environmental changes when ingested into the mosquito midgut, and are rapidly released from erythrocytes prior to fertilization. Secretory proteins localized to osmiophilic bodies (OBs), organelles unique to gametocytes, have been reported to be involved in female gametocyte egress. In this study, we investigate the dynamics of OBs in activated gametocytes of Plasmodium falciparum and Plasmodium yoelii using the female OB-specific marker protein, G377. After activation, female gametocyte OBs migrate to the parasite surface and fuse to form large vesicles beneath the parasite plasma membrane. At the marginal region of female gametocytes, fused vesicles secrete contents by exocytosis into the parasitophorous vacuole space, prior to parasite egress via the break-down of the erythrocyte membrane. This is the first detailed description of how proteins are transported through osmiophilic bodies.

15.
Parasitol Int ; 75: 102003, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31669509

RESUMO

Plasmodium falciparum proteins involved in vascular endothelial cell adherence are transported to the surface of infected erythrocytes. These proteins are exported through parasite-derived membrane structures within the erythrocyte cytoplasm called Maurer's clefts. Skeleton binding protein 1 (SBP1) is localized in the Maurer's clefts and plays an important role in transporting molecules to the surface of infected erythrocytes. Details of the translocation pathway are unclear and in this study we focused on the subcellular localization of SBP1 at an early intraerythrocytic stage. We performed immunoelectron microscopy using specific anti-SBP1 antibodies generated by immunization with recombinant SBP1 of P. falciparum. At the early trophozoite (ring form) stage, SBP1 was detected within an electron dense material (EDM) found in the parasite cytoplasm and in the parasitophorous vacuolar (PV) space. These findings demonstrate that SBP1 accumulates in EDM in the early trophozoite cytoplasm and is transported to the PV space before translocation to the Maurer's clefts formed in the erythrocyte cytoplasm.


Assuntos
Eritrócitos/parasitologia , Proteínas de Membrana/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Vacúolos/parasitologia , Animais , Membrana Celular/parasitologia , Citoplasma/parasitologia , Proteínas de Membrana/metabolismo , Microscopia Imunoeletrônica , Plasmodium falciparum/metabolismo , Transporte Proteico , Proteínas de Protozoários/metabolismo , Coelhos
16.
Front Cell Infect Microbiol ; 10: 605367, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33537242

RESUMO

Malaria causes a half a million deaths annually. The parasite intraerythrocytic lifecycle in the human bloodstream is the major cause of morbidity and mortality. Apical organelles of merozoite stage parasites are involved in the invasion of erythrocytes. A limited number of apical organellar proteins have been identified and characterized for their roles during erythrocyte invasion or subsequent intraerythrocytic parasite development. To expand the repertoire of identified apical organellar proteins we generated a panel of monoclonal antibodies against Plasmodium falciparum schizont-rich parasites and screened the antibodies using immunofluorescence assays. Out of 164 hybridoma lines, 12 clones produced monoclonal antibodies yielding punctate immunofluorescence staining patterns in individual merozoites in late schizonts, suggesting recognition of merozoite apical organelles. Five of the monoclonal antibodies were used to immuno-affinity purify their target antigens and these antigens were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two known apical organelle protein complexes were identified, the high-molecular mass rhoptry protein complex (PfRhopH1/Clags, PfRhopH2, and PfRhopH3) and the low-molecular mass rhoptry protein complex (rhoptry-associated proteins complex, PfRAP1, and PfRAP2). A novel complex was additionally identified by immunoprecipitation, composed of rhoptry-associated membrane antigen (PfRAMA) and rhoptry neck protein 3 (PfRON3) of P. falciparum. We further identified a region spanning amino acids Q221-E481 within the PfRAMA that may associate with PfRON3 in immature schizonts. Further investigation will be required as to whether PfRAMA and PfRON3 interact directly or indirectly.

17.
Front Immunol ; 10: 2669, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824483

RESUMO

Clinical manifestation of malaria is mainly due to intra-erythrocytic development of Plasmodium parasites. Plasmodium falciparum merozoites, the invasive form of the blood-stage parasite, invade human erythrocytes in a complex but rapid process. This multi-step progression involves interactions between parasite and human host proteins. Here we show that antibodies against a vaccine antigen, PfGAMA, co-immunoprecipitate with PfMSP10. This interaction was validated as direct by surface plasmon resonance analysis. We then demonstrate that antibodies against PfMSP10 have growth inhibitory activity against cultured parasites, with the region PfMSP10 R1 that is critical for its interaction with PfGAMA being the key target. We also observe that the PfMSP10 R1 region is highly conserved among African field isolates. Lastly, we show that high levels of antibodies against PfMSP10 R1 associate with reduced risk to clinical malaria in children resident in a malaria endemic region in northern Uganda. Put together, these findings provide for the first time the functional context of the important role of PfGAMA/PfMSP10 interaction in erythrocyte invasion and unveil a novel asexual blood-stage malaria vaccine target for attenuating P. falciparum merozoite invasion.


Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita/fisiologia , Malária Falciparum/imunologia , Humanos , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/imunologia
18.
Expert Rev Vaccines ; 18(10): 1017-1027, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31566026

RESUMO

Introduction: Highly effective malaria vaccines are essential component toward malaria elimination. Although the leading malaria vaccine, RTS,S/AS01, with modest efficacy is being evaluated in a pilot feasibility trial, development of a malaria transmission-blocking vaccine (TBV) could make a major contribution toward malaria elimination. Only a few TBV antigens have reached pre-clinical or clinical development but with several challenges including difficulties in the expression of malaria recombinant proteins and low immunogenicity in humans. Therefore, novel approaches to accelerate TBV research to preclinical development are critical to generate an efficacious TBV.Areas covered: PubMed was searched to review the progress and future prospects of malaria TBV research and development. We also reviewed registered trials at ClinicalTrials.gov as well as post-genome TBV candidate discovery research including our efforts.Expert opinion: Wheat germ cell-free protein synthesis technology can accelerate TBV development by overcoming some current challenges of TBV research.


Assuntos
Sistema Livre de Células , Vacinas Antimaláricas/imunologia , Malária/prevenção & controle , Bases de Dados Factuais , Células Germinativas , Humanos , Imunogenicidade da Vacina , Malária/transmissão , Malária Falciparum/prevenção & controle , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Proteínas Recombinantes , Triticum
19.
Artigo em Inglês | MEDLINE | ID: mdl-31552198

RESUMO

In the Plasmodium lifecycle two infectious stages of parasites, merozoites, and sporozoites, efficiently infect mammalian host cells, erythrocytes, and hepatocytes, respectively. The apical structure of merozoites and sporozoites contains rhoptry and microneme secretory organelles, which are conserved with other infective forms of apicomplexan parasites. During merozoite invasion of erythrocytes, some rhoptry proteins are secreted to form a tight junction between the parasite and target cell, while others are discharged to maintain subsequent infection inside the parasitophorous vacuole. It has been questioned whether the invasion mechanisms mediated by rhoptry proteins are also involved in sporozoite invasion of two distinct target cells, mosquito salivary glands and mammalian hepatocytes. Recently we demonstrated that rhoptry neck protein 2 (RON2), which is crucial for tight junction formation in merozoites, is also important for sporozoite invasion of both target cells. With the aim of comprehensively describing the mechanisms of sporozoite invasion, the expression and localization profiles of rhoptry proteins were investigated in Plasmodium berghei sporozoites. Of 12 genes representing merozoite rhoptry molecules, nine are transcribed in oocyst-derived sporozoites at a similar or higher level compared to those in blood-stage schizonts. Immuno-electron microscopy demonstrates that eight proteins, namely RON2, RON4, RON5, ASP/RON1, RALP1, RON3, RAP1, and RAMA, localize to rhoptries in sporozoites. It is noteworthy that most rhoptry neck proteins in merozoites are localized throughout rhoptries in sporozoites. This study demonstrates that most rhoptry proteins, except components of the high-molecular mass rhoptry protein complex, are commonly expressed in merozoites and sporozoites in Plasmodium spp., which suggests that components of the invasion mechanisms are basically conserved between infective forms independently of their target cells. Combined with sporozoite-stage specific gene silencing strategies, the contribution of rhoptry proteins in invasion mechanisms can be described.


Assuntos
Perfilação da Expressão Gênica , Merozoítos/química , Plasmodium berghei/química , Proteínas de Protozoários/análise , Esporozoítos/química , Animais , Anopheles , Western Blotting , Células Cultivadas , Células Epiteliais/parasitologia , Hepatócitos/parasitologia , Mamíferos , Merozoítos/genética , Microscopia Imunoeletrônica , Organelas/química , Plasmodium berghei/genética , Transporte Proteico , Reação em Cadeia da Polimerase em Tempo Real , Esporozoítos/genética
20.
PLoS One ; 14(9): e0221733, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31504038

RESUMO

The development of effective malaria vaccines remains a global health priority. Currently, the most advanced vaccine, known as RTS,S, has only shown modest efficacy in clinical trials. Thus, the development of more efficacious vaccines by improving the formulation of RTS,S for increased efficacy or to interrupt malaria transmission are urgently needed. The RTS,S vaccine is based on the presentation of a fragment of the sporozoite antigen on the surface of virus-like particles (VLPs) based on human hepatitis B virus (HBV). In this study, we have developed and evaluated a novel VLP platform based on duck HBV (known as Metavax) for malaria vaccine development. This platform can incorporate large and complex proteins into VLPs and is produced in a Hansenula cell line compatible with cGMP vaccine production. Here, we have established the expression of leading P. falciparum malaria vaccine candidates as VLPs. This includes Pfs230 and Pfs25, which are candidate transmission-blocking vaccine antigens. We demonstrated that the VLPs effectively induce antibodies to malaria vaccine candidates with minimal induction of antibodies to the duck-HBV scaffold antigen. Antibodies to Pfs230 also recognised native protein on the surface of gametocytes, and antibodies to both Pfs230 and Pfs25 demonstrated transmission-reducing activity in standard membrane feeding assays. These results establish the potential utility of this VLP platform for malaria vaccines, which may be suitable for the development of multi-component vaccines that achieve high vaccine efficacy and transmission-blocking immunity.


Assuntos
Vacinas Antimaláricas/imunologia , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anopheles/parasitologia , Afinidade de Anticorpos , Células HEK293 , Vírus da Hepatite B/genética , Humanos , Vacinas Antimaláricas/genética , Mosquitos Vetores/parasitologia , Pichia/genética , Pichia/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/genética , Coelhos , Proteínas Recombinantes/genética , Vacinas de Partículas Semelhantes a Vírus/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo
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