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1.
Sci Transl Med ; 12(543)2020 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404508

RESUMO

Identifying immune correlates of protection and mechanisms of immunity accelerates and streamlines the development of vaccines. RTS,S/AS01E, the most clinically advanced malaria vaccine, has moderate efficacy in African children. In contrast, immunization with sporozoites under antimalarial chemoprophylaxis (CPS immunization) can provide 100% sterile protection in naïve adults. We used systems biology approaches to identifying correlates of vaccine-induced immunity based on transcriptomes of peripheral blood mononuclear cells from individuals immunized with RTS,S/AS01E or chemoattenuated sporozoites stimulated with parasite antigens in vitro. Specifically, we used samples of individuals from two age cohorts and three African countries participating in an RTS,S/AS01E pediatric phase 3 trial and malaria-naïve individuals participating in a CPS trial. We identified both preimmunization and postimmunization transcriptomic signatures correlating with protection. Signatures were validated in independent children and infants from the RTS,S/AS01E phase 3 trial and individuals from an independent CPS trial with high accuracies (>70%). Transcription modules revealed interferon, NF-κB, Toll-like receptor (TLR), and monocyte-related signatures associated with protection. Preimmunization signatures suggest that priming the immune system before vaccination could potentially improve vaccine immunogenicity and efficacy. Last, signatures of protection could be useful to determine efficacy in clinical trials, accelerating vaccine candidate testing. Nevertheless, signatures should be tested more extensively across multiple cohorts and trials to demonstrate their universal predictive capacity.

2.
Anal Chem ; 92(9): 6667-6675, 2020 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-32267675

RESUMO

Efforts to eradicate Plasmodium vivax malaria are hampered by the presence of hypnozoites, persisting stages in the liver that can reactivate after prolonged periods of time enabling further transmission and causing renewed disease. Large-scale drug screening is needed to identify compounds with antihypnozoite activity, but current platforms rely on time-consuming high-content fluorescence imaging as read-out, limiting assay throughput. We here report an ultrafast and sensitive dual-luciferase-based method to differentiate hypnozoites from liver stage schizonts using a transgenic P. cynomolgi parasite line that contains Nanoluc driven by the constitutive hsp70 promoter, as well as firefly luciferase driven by the schizont-specific lisp2 promoter. The transgenic parasite line showed similar fitness and drug sensitivity profiles of selected compounds to wild type. We demonstrate robust bioluminescence-based detection of hypnozoites in 96-well and 384-well plate formats, setting the stage for implementation in large scale drug screens.

3.
Exp Hematol ; 82: 8-23, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32007479

RESUMO

Establishing an in vitro "red blood cell matrix" that would allow uninterrupted access to a stable, homogeneous reticulocyte population would facilitate the establishment of continuous, long-term in vitro Plasmodium vivax blood stage cultures. In this study, we have explored the suitability of the erythroleukemia K562 cell line as a continuous source of such reticulocytes and have investigated regulatory factors behind the terminal differentiation (and enucleation, in particular) of this cell line that can be used to drive the reticulocyte production process. The Duffy blood group antigen receptor (Fy), essential for P. vivax invasion, was stably introduced into K562 cells by lentiviral gene transfer. miRNA-26a-5p and miRNA-30a-5p were downregulated to promote erythroid differentiation and enucleation, resulting in a tenfold increase in the production of reticulocytes after stimulation with an induction cocktail compared with controls. Our results suggest an interplay in the mechanisms of action of miRNA-26a-5p and miRNA-30a-5p, which makes it necessary to downregulate both miRNAs to achieve a stable enucleation rate and Fy receptor expression. In the context of establishing P. vivax-permissive, stable, and reproducible reticulocytes, a higher enucleation rate may be desirable, which may be achieved by the targeting of further regulatory mechanisms in Fy-K562 cells; promoting the shift in hemoglobin production from fetal to adult may also be necessary. Despite the fact that K562 erythroleukemia cell lines are of neoplastic origin, this cell line offers a versatile model system to research the regulatory mechanisms underlying erythropoiesis.

4.
Commun Biol ; 3: 7, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31909199

RESUMO

Plasmodium vivax malaria is characterized by repeated episodes of blood stage infection (relapses) resulting from activation of dormant stages in the liver, so-called hypnozoites. Transition of hypnozoites into developing schizonts has never been observed. A barrier for studying this has been the lack of a system in which to monitor growth of liver stages. Here, exploiting the unique strengths of the simian hypnozoite model P. cynomolgi, we have developed green-fluorescent (GFP) hypnozoites that turn on red-fluorescent (mCherry) upon activation. The transgenic parasites show full liver stage development, including merozoite release and red blood cell infection. We demonstrate that individual hypnozoites actually can activate and resume development after prolonged culture, providing the last missing evidence of the hypnozoite theory of relapse. The few events identified indicate that hypnozoite activation in vitro is infrequent. This system will further our understanding of the mechanisms of hypnozoite activation and may facilitate drug discovery approaches.

5.
Front Immunol ; 10: 2479, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31736945

RESUMO

While tuberculosis continues to afflict mankind, the immunological mechanisms underlying TB disease development are still incompletely understood. Advanced preclinical models for TB research include both rhesus and cynomolgus macaques (Macaca mulatta and Macaca fascicularis, respectively), with rhesus typically being more susceptible to acute progressive TB disease than cynomolgus macaques. To determine which immune mechanisms are responsible for this dissimilar disease development, we profiled a broad range of innate and adaptive responses, both local and peripheral, following experimental pulmonary Mycobacterium tuberculosis (Mtb) infection of both species. While T-cell and antibody responses appeared indistinguishable, we identified anti-inflammatory skewing of peripheral monocytes in rhesus and a more prominent local pro-inflammatory cytokine release profile in cynomolgus macaques associated with divergent TB disease outcome. Importantly, these differences were detectable both before and early after infection. This work shows that inflammatory and innate immune status prior to and at early stages after infection, critically affects outcome of TB infection.

6.
Malar J ; 18(1): 344, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601222

RESUMO

Historically neglected, due to its biological peculiarities, the absence of a continuous long-term in vitro blood stage culture system and a propensity towards high morbidity rather than mortality, Plasmodium vivax was put back on the agenda during the last decade by the paradigm shift in the fight against malaria from malaria control to malaria eradication. While the incidence of the deadliest form of malaria, Plasmodium falciparum malaria, has declined since this paradigm shift took hold, the prospects of eradication are now threatened by the increase in the incidence of other human malaria parasite species. Plasmodium vivax is geographically the most widely distributed human malaria parasite, characterized by millions of clinical cases every year and responsible for a massive economic burden. The urgent need to tackle the unique biological challenges posed by this parasite led to renewed efforts aimed at establishing a continuous, long-term in vitro P. vivax blood stage culture. Based on recent discoveries on the role of nutrient sensing in Plasmodium's pathophysiology, this review article critically assesses the extensive body of literature concerning Plasmodium culture conditions with a specific focus on culture media used in attempts to culture different Plasmodium spp. Hereby, the effect of specific media components on the parasite's in vitro fitness and the maturation of the parasite's host cell, the reticulocyte, is analysed. Challenging the wide-held belief that it is sufficient to find the right parasite isolate and give it the right type of cells to invade for P. vivax to grow in vitro, this review contends that a healthy side-by-side maturation of both the parasite and its host cell, the reticulocyte, is necessary in the adaptation of P. vivax to in vitro growth and argues that culture conditions and the media in particular play an essential role in this maturation process.


Assuntos
Meios de Cultura/análise , Nutrientes/metabolismo , Plasmodium vivax/fisiologia
7.
Nat Commun ; 10(1): 3635, 2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31406175

RESUMO

The ability to culture pathogenic organisms substantially enhances the quest for fundamental knowledge and the development of vaccines and drugs. Thus, the elaboration of a protocol for the in vitro cultivation of the erythrocytic stages of Plasmodium falciparum revolutionized research on this important parasite. However, for P. vivax, the most widely distributed and difficult to treat malaria parasite, a strict preference for reticulocytes thwarts efforts to maintain it in vitro. Cultivation of P. cynomolgi, a macaque-infecting species phylogenetically close to P. vivax, was briefly reported in the early 1980s, but not pursued further. Here, we define the conditions under which P. cynomolgi can be adapted to long term in vitro culture to yield parasites that share many of the morphological and phenotypic features of P. vivax. We further validate the potential of this culture system for high-throughput screening to prime and accelerate anti-P. vivax drug discovery efforts.


Assuntos
Eritrócitos/parasitologia , Macaca/parasitologia , Malária/veterinária , Doenças dos Macacos/parasitologia , Plasmodium cynomolgi/crescimento & desenvolvimento , Animais , Anopheles/parasitologia , Malária/parasitologia , Malária/transmissão
8.
BMC Immunol ; 20(1): 25, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31362695

RESUMO

BACKGROUND: In this study, seven adjuvants were compared for use with Plasmodium falciparum DiCo-Apical Membrane Antigen 1 (Pf-DiCo-AMA1), with the aim to identify an ideal adjuvant which yields high antibody titres and potentially broadens the responses in clinical trials. The following adjuvant formulations were evaluated: SE, SE-GLA, Liposomes, Liposomes-GLA, CoVaccine HT™, ImSaVac-P and ImSaVac-P o/w. The study was performed in rabbits, which were immunized with FVO-AMA1 in combination with one of the seven adjuvants. Antibody levels (humoral responses) and functional activity of the antibodies induced against malaria vaccine candidate AMA1 were evaluated. Thus, in this study the ideal adjuvant is expected to induce high functional antibody levels, a long-lived response, and a broad cross-strain activity. RESULTS: AMA1 formulated in all adjuvants was immunogenic. However, the magnitude of the immune responses differed between the seven adjuvants. The highest IgG levels were observed for the CoVaccine HT™ group, this was statistically significant for all four AMA1 variants versus all other adjuvant groups. No differences were observed in the breadth of the humoral response, i.e., increased recognition of AMA1 variants. Also, Growth Inhibition Activity (GIA) for both Plasmodium falciparum strains (FCR3 - homologous to FVO AMA1 protein and NF54 - heterologous to FVO AMA1 protein) were significantly higher in the CoVaccine HT™ group as compared to the other adjuvant groups. CONCLUSIONS: In brief, all seven vaccine - adjuvant formulations were immunogenic. The magnitude of the immune responses differed between the seven adjuvants. No statistically significant differences were observed in the breadth of the humoral response, nor in longevity of the response. Nevertheless, AMA1 formulated in CoVaccine HT™ appeared as the best adjuvant for use in clinical trials.


Assuntos
Adjuvantes Imunológicos , Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Proteínas de Membrana/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Formação de Anticorpos/imunologia , Modelos Animais de Doenças , Imunização , Imunoglobulina G/imunologia , Vacinas Antimaláricas/administração & dosagem , Coelhos
9.
Nat Med ; 25(2): 255-262, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30664782

RESUMO

Tuberculosis (TB) remains the deadliest infectious disease1, and the widely used Bacillus Calmette-Guérin (BCG) vaccine fails to curb the epidemic. An improved vaccination strategy could provide a cost-effective intervention to break the transmission cycle and prevent antimicrobial resistance2,3. Limited knowledge of the host responses critically involved in protective immunity hampers the development of improved TB vaccination regimens. Therefore, assessment of new strategies in preclinical models to select the best candidate vaccines before clinical vaccine testing remains indispensable. We have previously established in rhesus macaques (Macaca mulatta) that pulmonary mucosal BCG delivery reduces TB disease where standard intradermal injection fails4,5. Here, we show that pulmonary BCG prevents infection by using a repeated limiting-dose Mycobacterium tuberculosis challenge model and identify polyfunctional T-helper type 17 (TH17) cells, interleukin-10 and immunoglobulin A as correlates of local protective immunity. These findings warrant further research into mucosal immunization strategies and their translation to clinical application to more effectively prevent the spread of TB.


Assuntos
Vacina BCG/imunologia , Tuberculose/imunologia , Tuberculose/prevenção & controle , Animais , Carga Bacteriana , Relação Dose-Resposta Imunológica , Imunidade Humoral , Interferon gama/metabolismo , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Macaca mulatta , Masculino , Membrana Mucosa/imunologia , Vacinação
10.
Parasitology ; 145(1): 56-70, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-27938428

RESUMO

The primate malaria Plasmodium knowlesi has a long-standing history as an experimental malaria model. Studies using this model parasite in combination with its various natural and experimental non-human primate hosts have led to important advances in vaccine development and in our understanding of malaria invasion, immunology and parasite-host interactions. The adaptation to long-term in vitro continuous blood stage culture in rhesus monkey, Macaca fascicularis and human red blood cells, as well as the development of various transfection methodologies has resulted in a highly versatile experimental malaria model, further increasing the potential of what was already a very powerful model. The growing evidence that P. knowlesi is an important human zoonosis in South-East Asia has added relevance to former and future studies of this parasite species.


Assuntos
Modelos Animais de Doenças , Haplorrinos , Interações Hospedeiro-Parasita , Malária/parasitologia , Plasmodium knowlesi/fisiologia , Adaptação Biológica , Animais , Eritrócitos/parasitologia , Humanos , Macaca fascicularis , Macaca mulatta , Malária/imunologia , Malária/prevenção & controle , Malária/veterinária , Vacinas Antimaláricas/análise , Vacinas Antimaláricas/farmacologia , Doenças dos Macacos/imunologia , Doenças dos Macacos/parasitologia , Doenças dos Macacos/prevenção & controle , Plasmodium knowlesi/imunologia , Zoonoses/imunologia , Zoonoses/parasitologia , Zoonoses/prevenção & controle
11.
Elife ; 62017 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-29215331

RESUMO

Plasmodium liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relapsing parasite Plasmodium cynomolgi. Hypnozoites express only 34% of Plasmodium physiological pathways, while 91% are expressed in replicating schizonts. Few known malaria drug targets are expressed in quiescent parasites, but pathways involved in microbial dormancy, maintenance of genome integrity and ATP homeostasis were robustly expressed. Several transcripts encoding heavy metal transporters were expressed in hypnozoites and the copper chelator neocuproine was cidal to all liver stage parasites. This transcriptomic dataset is a valuable resource for the discovery of vaccines and effective treatments to combat vivax malaria.


Assuntos
Perfilação da Expressão Gênica , Fígado/parasitologia , Macaca mulatta/parasitologia , Plasmodium cynomolgi/crescimento & desenvolvimento , Plasmodium cynomolgi/genética , Esquizontes/crescimento & desenvolvimento , Esquizontes/genética , Animais , Feminino , Masculino
12.
PLoS One ; 12(9): e0185303, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28945794

RESUMO

INTRODUCTION: Plasmodium falciparum induced antibodies are key components of anti-malarial immunity in malaria endemic areas, but their antigen targets can be polymorphic. Induction of a high proportion of strain-specific antibodies will limit the recognition of a broad diversity of parasite strains by these responses. There are indications that circulating parasite diversity varies with malaria transmission intensity, and this may affect the specificity of elicited anti-malarial antibodies. This study therefore assessed the effect of varying malaria transmission patterns on the specificity of elicited antibody responses and to identify possible antibody correlates of naturally acquired immunity to malaria in children in an area of Ghana with seasonal malaria transmission. METHODS: This retrospective study utilized plasma samples collected longitudinally at six time points from children aged one to five years. Multiplex assays were used to measure antibody levels against four P. falciparum AMA 1 variants (from the 3D7, FVO, HB3 and CAMP parasite strains) and the 3D7 variant of the EBA 175 region II antigen and the levels compared between symptomatic and asymptomatic children. The relative proportions of cross-reactive and strain-specific antibodies against the four AMA 1 variants per sampling time point were assessed by Bland-Altman plots. The levels of antibodies against allelic AMA1 variants, measured by singleplex and multiplex luminex assays, were also compared. RESULTS: The data show that increased transmission intensity is associated with higher levels of cross-reactive antibody responses, most likely a result of a greater proportion of multiple parasite clone infections during the high transmission period. Anti-AMA1 antibodies were however associated with a history of infection rather than protection in this age group. CONCLUSION: The data contribute to understanding the underlying mechanism of the acquisition of strain-transcending antibody immunity following repeated exposure to diverse parasite strains.


Assuntos
Anticorpos Antiprotozoários/sangue , Malária Falciparum/imunologia , Malária Falciparum/transmissão , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Criança , Pré-Escolar , Estudos de Coortes , Reações Cruzadas , Feminino , Gana/epidemiologia , Humanos , Imunoensaio/métodos , Lactente , Estudos Longitudinais , Malária Falciparum/epidemiologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Estações do Ano
13.
Trends Parasitol ; 33(12): 921-924, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28780020

RESUMO

The recent research efforts to establish a Plasmodium vivax continuous, long-term blood-stage culture have focused on the ideal host cell type. However, this is only part of the story, as the P. vivax intraerythrocytic life cycle is complex. A successful, long-term, robust culture system will depend on a multifaceted approach combining the ideal cell type and parasite isolates, and the culture conditions.


Assuntos
Plasmodium vivax/crescimento & desenvolvimento , Animais , Técnicas de Cultura de Células , Células Cultivadas , Eritrócitos/parasitologia , Humanos , Estágios do Ciclo de Vida
14.
Nat Commun ; 8: 15159, 2017 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-28537265

RESUMO

K13 gene mutations are a primary marker of artemisinin resistance in Plasmodium falciparum malaria that threatens the long-term clinical utility of artemisinin-based combination therapies, the cornerstone of modern day malaria treatment. Here we describe a multinational drug discovery programme that has delivered a synthetic tetraoxane-based molecule, E209, which meets key requirements of the Medicines for Malaria Venture drug candidate profiles. E209 has potent nanomolar inhibitory activity against multiple strains of P. falciparum and P. vivax in vitro, is efficacious against P. falciparum in in vivo rodent models, produces parasite reduction ratios equivalent to dihydroartemisinin and has pharmacokinetic and pharmacodynamic characteristics compatible with a single-dose cure. In vitro studies with transgenic parasites expressing variant forms of K13 show no cross-resistance with the C580Y mutation, the primary variant observed in Southeast Asia. E209 is a superior next generation endoperoxide with combined pharmacokinetic and pharmacodynamic features that overcome the liabilities of artemisinin derivatives.


Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium vivax/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Tetraoxanos/química , Tetraoxanos/farmacologia , Animais , Antimaláricos/química , Cães , Relação Dose-Resposta a Droga , Resistência a Medicamentos/genética , Eritrócitos/parasitologia , Feminino , Meia-Vida , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Plasmodium falciparum/genética , Plasmodium vivax/genética , Ratos , Ratos Sprague-Dawley , Tetraoxanos/farmacocinética , Transgenes
15.
Tuberculosis (Edinb) ; 104: 46-57, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28454649

RESUMO

M.bovis BCG vaccination against tuberculosis (TB) notoriously displays variable protective efficacy in different human populations. In non-human primate studies using rhesus macaques, despite efforts to standardise the model, we have also observed variable efficacy of BCG upon subsequent experimental M. tuberculosis challenge. In the present head-to-head study, we establish that the protective efficacy of standard parenteral BCG immunisation varies among different rhesus cohorts. This provides different dynamic ranges for evaluation of investigational vaccines, opportunities for identifying possible correlates of protective immunity and for determining why parenteral BCG immunisation sometimes fails. We also show that pulmonary mucosal BCG vaccination confers reduced local pathology and improves haematological and immunological parameters post-infection in animals that are not responsive to induction of protection by standard intra-dermal BCG. These results have important implications for pulmonary TB vaccination strategies in the future.


Assuntos
Vacina BCG/administração & dosagem , Imunogenicidade da Vacina , Mycobacterium tuberculosis/imunologia , Tuberculose/prevenção & controle , Vacinação , Administração por Inalação , Animais , Vacina BCG/toxicidade , Modelos Animais de Doenças , Feminino , Imunidade nas Mucosas , Injeções Intradérmicas , Macaca mulatta , Masculino , Mycobacterium tuberculosis/patogenicidade , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologia , Fatores de Tempo , Tuberculose/imunologia , Tuberculose/microbiologia
16.
Sci Transl Med ; 9(387)2017 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-28446690

RESUMO

As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all Plasmodium life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized Plasmodium falciparum mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the Plasmodium berghei mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapse in a Plasmodium cynomolgi monkey model. Both genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment.


Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Aminopiridinas/uso terapêutico , Antimaláricos/uso terapêutico , Sulfonas/uso terapêutico , Aminopiridinas/farmacologia , Animais , Antimaláricos/farmacologia , Feminino , Malária/tratamento farmacológico , Malária/enzimologia , Masculino , Camundongos , Camundongos SCID , Testes de Sensibilidade Parasitária , Plasmodium/efeitos dos fármacos , Plasmodium/patogenicidade , Sulfonas/farmacologia
19.
PLoS Pathog ; 12(11): e1005917, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27851824

RESUMO

Many variant proteins encoded by Plasmodium-specific multigene families are exported into red blood cells (RBC). P. falciparum-specific variant proteins encoded by the var, stevor and rifin multigene families are exported onto the surface of infected red blood cells (iRBC) and mediate interactions between iRBC and host cells resulting in tissue sequestration and rosetting. However, the precise function of most other Plasmodium multigene families encoding exported proteins is unknown. To understand the role of RBC-exported proteins of rodent malaria parasites (RMP) we analysed the expression and cellular location by fluorescent-tagging of members of the pir, fam-a and fam-b multigene families. Furthermore, we performed phylogenetic analyses of the fam-a and fam-b multigene families, which indicate that both families have a history of functional differentiation unique to RMP. We demonstrate for all three families that expression of family members in iRBC is not mutually exclusive. Most tagged proteins were transported into the iRBC cytoplasm but not onto the iRBC plasma membrane, indicating that they are unlikely to play a direct role in iRBC-host cell interactions. Unexpectedly, most family members are also expressed during the liver stage, where they are transported into the parasitophorous vacuole. This suggests that these protein families promote parasite development in both the liver and blood, either by supporting parasite development within hepatocytes and erythrocytes and/or by manipulating the host immune response. Indeed, in the case of Fam-A, which have a steroidogenic acute regulatory-related lipid transfer (START) domain, we found that several family members can transfer phosphatidylcholine in vitro. These observations indicate that these proteins may transport (host) phosphatidylcholine for membrane synthesis. This is the first demonstration of a biological function of any exported variant protein family of rodent malaria parasites.


Assuntos
Hepatócitos/virologia , Malária Falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Modelos Animais de Doenças , Eritrócitos/parasitologia , Imunofluorescência , Humanos , Fígado , Malária Falciparum/virologia , Camundongos , Família Multigênica , Organismos Geneticamente Modificados , Filogenia , Plasmodium falciparum , Transporte Proteico , Vacúolos/virologia
20.
PLoS One ; 11(10): e0164053, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27695087

RESUMO

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, three Diversity Covering (DiCo) PfAMA1 ectodomain proteins, designed to overcome the intrinsic polymorphism that is present in PfAMA1, were produced under Good Manufacturing Practice (GMP) in Pichia pastoris. Using identical methodology, the 3 strains were cultivated in 70-L scale fed-batch fermentations and PfAMA1-DiCos were purified by two chromatography steps, an ultrafiltration/diafiltration procedure and size exclusion chromatography, resulting in highly pure (>95%) PfAMA1-DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3, with final yields of 1.8, 1.9 and 1.3 gram, respectively. N-terminal determinations showed that approximately 50% of each of the proteins lost 12 residues from their N-terminus, in accordance with SDS-PAGE (2 main bands) and MS-data. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident. The three proteins quantitatively bound to the mAb 4G2 that recognizes a conformational epitope, suggesting proper folding of the proteins. The lyophilized Drug Product (1:1:1 mixture of PfAMA1-DiCo1, DiCo2, DiCo3) fulfilled all pre-set release criteria (appearance, dissolution rate, identity, purity, protein content, moisture content, sub-visible particles, immuno-potency (after reconstitution with adjuvant), abnormal toxicity, sterility and endotoxin), was stable in accelerated and real-time stability studies at -20°C for over 24 months. When formulated with adjuvants selected for clinical phase I evaluation, the Drug Product did not show adverse effect in a repeated-dose toxicity study in rabbits. The Drug Product has entered a phase Ia/Ib clinical trial.


Assuntos
Variação Antigênica , Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/imunologia , Proteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Feminino , Fermentação , Humanos , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/efeitos adversos , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Estabilidade Proteica , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Controle de Qualidade , Coelhos , Proteínas Recombinantes
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