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1.
PLoS Genet ; 15(10): e1008453, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31609965

RESUMO

Determining the genetic basis of fitness is central to understanding evolution and transmission of microbial pathogens. In human malaria parasites (Plasmodium falciparum), most experimental work on fitness has focused on asexual blood stage parasites, because this stage can be easily cultured, although the transmission of malaria requires both female Anopheles mosquitoes and vertebrate hosts. We explore a powerful approach to identify the genetic determinants of parasite fitness across both invertebrate and vertebrate life-cycle stages of P. falciparum. This combines experimental genetic crosses using humanized mice, with selective whole genome amplification and pooled sequencing to determine genome-wide allele frequencies and identify genomic regions under selection across multiple lifecycle stages. We applied this approach to genetic crosses between artemisinin resistant (ART-R, kelch13-C580Y) and ART-sensitive (ART-S, kelch13-WT) parasites, recently isolated from Southeast Asian patients. Two striking results emerge: we observed (i) a strong genome-wide skew (>80%) towards alleles from the ART-R parent in the mosquito stage, that dropped to ~50% in the blood stage as selfed ART-R parasites were selected against; and (ii) repeatable allele specific skews in blood stage parasites with particularly strong selection (selection coefficient (s) ≤ 0.18/asexual cycle) against alleles from the ART-R parent at loci on chromosome 12 containing MRP2 and chromosome 14 containing ARPS10. This approach robustly identifies selected loci and has strong potential for identifying parasite genes that interact with the mosquito vector or compensatory loci involved in drug resistance.


Assuntos
Interações Hospedeiro-Parasita/genética , Estágios do Ciclo de Vida/genética , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Animais , Anopheles/parasitologia , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Artemisininas/farmacologia , Artemisininas/uso terapêutico , Mapeamento Cromossômico , Modelos Animais de Doenças , Resistência a Medicamentos/genética , Feminino , Frequência do Gene , Loci Gênicos , Humanos , Malária Falciparum/tratamento farmacológico , Masculino , Camundongos , Mosquitos Vetores/parasitologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Polimorfismo de Nucleotídeo Único , Proteínas Ribossômicas/genética , Seleção Genética , Quimeras de Transplante
2.
Nat Commun ; 10(1): 3950, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477704

RESUMO

Immunization with attenuated whole Plasmodium sporozoites constitutes a promising vaccination strategy. Compared to replication-deficient parasites, immunization with replication-competent parasites confers better protection and also induces a type I IFN (IFN-1) response, but whether this IFN-1 response has beneficial or adverse effects on vaccine-induced adaptive immunity is not known. Here, we show that IFN-1 signaling-deficient mice immunized with replication-competent sporozoites exhibit superior protection against infection. This correlates with superior CD8 T cell memory including reduced expression of the exhaustion markers PD-1 and LAG-3 on these cells and increased numbers of memory CD8 T cells in the liver. Moreover, the adoptive transfer of memory CD8 T cells from the livers of previously immunized IFN-1 signaling-deficient mice confers greater protection against liver stage parasites. However, the detrimental role of IFN-1 signaling is not CD8 T cell intrinsic. Together, our data demonstrate that liver stage-engendered IFN-1 signaling impairs hepatic CD8 T cell memory via a CD8 T cell-extrinsic mechanism.


Assuntos
Imunidade Adaptativa/imunologia , Eritrócitos/imunologia , Imunidade Inata/imunologia , Malária/imunologia , Plasmodium yoelii/imunologia , Esporozoítos/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/parasitologia , Eritrócitos/parasitologia , Feminino , Imunização , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Fígado/imunologia , Fígado/metabolismo , Fígado/parasitologia , Malária/parasitologia , Malária/prevenção & controle , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmodium yoelii/fisiologia , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia
3.
Malar J ; 18(1): 295, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31462253

RESUMO

BACKGROUND: Competitive outcomes between co-infecting malaria parasite lines can reveal fitness disparities in blood stage growth. Blood stage fitness costs often accompany the evolution of drug resistance, with the expectation that relatively fitter parasites will be more likely to spread in populations. With the recent emergence of artemisinin resistance, it is important to understand the relative competitive fitness of the metabolically active asexual blood stage parasites. Genetically distinct drug resistant parasite clones with independently evolved sets of mutations are likely to vary in asexual proliferation rate, contributing to their chance of transmission to the mosquito vector. METHODS: An optimized in vitro 96-well plate-based protocol was used to quantitatively measure-head-to-head competitive fitness during blood stage development between seven genetically distinct field isolates from a hotspot of emerging artemisinin resistance and the laboratory strain, NF54. These field isolates were isolated from patients in Southeast Asia carrying different alleles of kelch13 and included both artemisinin-sensitive and artemisinin-resistant isolates. Fluorescent labeled microsatellite markers were used to track the relative densities of each parasite throughout the co-growth period of 14-60 days. All-on-all competitions were conducted for the panel of eight parasite lines (28 pairwise competitions) to determine their quantitative competitive fitness relationships. RESULTS: Twenty-eight pairwise competitive growth outcomes allowed for an unambiguous ranking among a set of seven genetically distinct parasite lines isolated from patients in Southeast Asia displaying a range of both kelch13 alleles and clinical clearance times and a laboratory strain, NF54. This comprehensive series of assays established the growth relationships among the eight parasite lines. Interestingly, a clinically artemisinin resistant parasite line that carries the wild-type form of kelch13 outcompeted all other parasites in this study. Furthermore, a kelch13 mutant line (E252Q) was competitively more fit without drug than lines with other resistance-associated kelch13 alleles, including the C580Y allele that has expanded to high frequencies under drug pressure in Southeast Asian resistant populations. CONCLUSIONS: This optimized competitive growth assay can be employed for assessment of relative growth as an index of fitness during the asexual blood stage growth between natural lines carrying different genetic variants associated with artemisinin resistance. Improved understanding of the fitness costs of different parasites proliferating in human blood and the role different resistance mutations play in the context of specific genetic backgrounds will contribute to an understanding of the potential for specific mutations to spread in populations, with the potential to inform targeted strategies for malaria therapy.


Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Resistência a Medicamentos/genética , Evolução Molecular , Aptidão Genética , Plasmodium falciparum/crescimento & desenvolvimento , Genótipo , Técnicas de Genotipagem , Estágios do Ciclo de Vida/genética , Repetições de Microssatélites/genética , Mutação , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/genética
4.
Trends Parasitol ; 35(7): 583-584, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31176582
5.
Elife ; 82019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31094679

RESUMO

Plasmodium vivax hypnozoites persist in the liver, cause malaria relapse and represent a major challenge to malaria elimination. Our previous transcriptomic study provided a novel molecular framework to enhance our understanding of the hypnozoite biology (Voorberg-van der Wel A, et al., 2017). In this dataset, we identified and characterized the Liver-Specific Protein 2 (LISP2) protein as an early molecular marker of liver stage development. Immunofluorescence analysis of hepatocytes infected with relapsing malaria parasites, in vitro (P. cynomolgi) and in vivo (P. vivax), reveals that LISP2 expression discriminates between dormant hypnozoites and early developing parasites. We further demonstrate that prophylactic drugs selectively kill all LISP2-positive parasites, while LISP2-negative hypnozoites are only sensitive to anti-relapse drug tafenoquine. Our results provide novel biological insights in the initiation of liver stage schizogony and an early marker suitable for the development of drug discovery assays predictive of anti-relapse activity.


Assuntos
Malária Vivax/genética , Plasmodium cynomolgi/genética , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Aminoquinolinas/farmacologia , Animais , Antimaláricos/farmacologia , Biomarcadores/metabolismo , Biomarcadores Farmacológicos , Hepatócitos/metabolismo , Hepatócitos/parasitologia , Interações Hospedeiro-Parasita/genética , Humanos , Fígado/efeitos dos fármacos , Fígado/parasitologia , Macaca mulatta/genética , Macaca mulatta/parasitologia , Malária Vivax/tratamento farmacológico , Malária Vivax/parasitologia , Plasmodium cynomolgi/parasitologia , Plasmodium vivax/efeitos dos fármacos , Plasmodium vivax/patogenicidade , Proteínas de Protozoários/metabolismo , Esporozoítos/genética , Transcriptoma/efeitos dos fármacos
6.
Am J Trop Med Hyg ; 100(6): 1466-1476, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31017084

RESUMO

18S rRNA is a biomarker that provides an alternative to thick blood smears in controlled human malaria infection (CHMI) trials. We reviewed data from CHMI trials at non-endemic sites that used blood smears and Plasmodium 18S rRNA/rDNA biomarker nucleic acid tests (NATs) for time to positivity. We validated a multiplex quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for Plasmodium 18S rRNA, prospectively compared blood smears and qRT-PCR for three trials, and modeled treatment effects at different biomarker-defined parasite densities to assess the impact on infection detection, symptom reduction, and measured intervention efficacy. Literature review demonstrated accelerated NAT-based infection detection compared with blood smears (mean acceleration: 3.2-3.6 days). For prospectively tested trials, the validated Plasmodium 18S rRNA qRT-PCR positivity was earlier (7.6 days; 95% CI: 7.1-8.1 days) than blood smears (11.0 days; 95% CI: 10.3-11.8 days) and significantly preceded the onset of grade 2 malaria-related symptoms (12.2 days; 95% CI: 10.6-13.3 days). Discrepant analysis showed that the risk of a blood smear-positive, biomarker-negative result was negligible. Data modeling predicted that treatment triggered by specific biomarker-defined thresholds can differentiate complete, partial, and non-protective outcomes and eliminate many grade 2 and most grade 3 malaria-related symptoms post-CHMI. Plasmodium 18S rRNA is a sensitive and specific biomarker that can justifiably replace blood smears for infection detection in CHMI trials in non-endemic settings. This study led to biomarker qualification through the U.S. Food and Drug Administration for use in CHMI studies at non-endemic sites, which will facilitate biomarker use for the qualified context of use in drug and vaccine trials.


Assuntos
Malária/diagnóstico , Plasmodium/genética , RNA de Protozoário/genética , RNA Ribossômico 18S/sangue , Biomarcadores/sangue , Humanos , Reação em Cadeia da Polimerase Multiplex , Plasmodium/isolamento & purificação , RNA Ribossômico 18S/genética , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Artigo em Inglês | MEDLINE | ID: mdl-30547015

RESUMO

Within the liver, Plasmodium sporozoites traverse cells searching for a "suitable" hepatocyte, invading these cells through a process that results in the formation of a parasitophorous vacuole (PV), within which the parasite undergoes intracellular replication as a liver stage. It was previously established that two members of the Plasmodium s48/45 protein family, P36 and P52, are essential for productive invasion of host hepatocytes by sporozoites as their simultaneous deletion results in growth-arrested parasites that lack a PV. Recent studies point toward a pathway of entry possibly involving the interaction of P36 with hepatocyte receptors EphA2, CD81, and SR-B1. However, the relationship between P36 and P52 during sporozoite invasion remains unknown. Here we show that parasites with a single P52 or P36 gene deletion each lack a PV after hepatocyte invasion, thereby pheno-copying the lack of a PV observed for the P52/P36 dual gene deletion parasite line. This indicates that both proteins are equally important in the establishment of a PV and act in the same pathway. We created a Plasmodium yoelii P36mCherry tagged parasite line that allowed us to visualize the subcellular localization of P36 and found that it partially co-localizes with P52 in the sporozoite secretory microneme organelles. Furthermore, through co-immunoprecipitation studies in vivo, we determined that P36 and P52 form a protein complex in sporozoites, indicating a concerted function for both proteins within the PV formation pathway. However, upon sporozoite stimulation, only P36 was released as a secreted protein while P52 was not. Our results support a model in which the putatively glycosylphosphatidylinositol (GPI)-anchored P52 may serve as a scaffold to facilitate the interaction of secreted P36 with the host cell during sporozoite invasion of hepatocytes.


Assuntos
Hepatócitos/parasitologia , Malária/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Esporozoítos/metabolismo , Animais , Antígenos CD36/metabolismo , Culicidae , Citoplasma/metabolismo , Feminino , Deleção de Genes , Técnicas de Inativação de Genes , Glicosilfosfatidilinositóis , Hepatócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium yoelii/metabolismo , Receptor EphA2/metabolismo , Glândulas Salivares/parasitologia , Glândulas Salivares/patologia
9.
Malar J ; 17(1): 135, 2018 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-29609625

RESUMO

BACKGROUND: Despite the importance of the Plasmodium berghei oocyst capsule protein (PbCap380) in parasite survival, very little is known about the orthologous Plasmodium falciparum capsule protein (PfCap380). The goal of this work was to study the growth of P. falciparum oocysts using PfCap380 as a developmental marker. METHODS: To study P. falciparum oocyst development using both in vivo (mosquito-derived) and in vitro (culture-derived) growth conditions, antibodies (polyclonal antisera) were raised against PfCap380. For studies on in vivo oocysts, mature P. falciparum gametocytes were fed to Anopheles stephensi mosquitoes. For studies on in vitro parasites, P. falciparum gametocytes were induced and matured for subsequent ookinete production. Ookinetes were purified and then tested for binding affinity to basal lamina components and transformation into early oocysts, which were grown on reconstituted basal lamia coated wells with novel oocyst media. To monitor in vivo oocyst development, immunofluorescence assays (IFA) were performed using anti-PfCap380 antisera on Pf-infected mosquito midguts. IFA were also performed on culture-derived oocysts to follow in vitro oocyst development. RESULTS: The anti-PfCap380 antisera allowed detection of early midgut oocysts starting at 2 days after gametocyte infection, while circumsporozoite protein was definitively observed on day 6. For in vitro culture, significant transformation of gametocytes to ookinetes (24%) and of ookinetes to early oocysts (85%) was observed. After screening several basal lamina components, collagen IV provided greatest binding of ookinetes and transformation into early oocysts. Finally, PfCap380 expression was observed on the surface of culture-derived oocysts but not on gametocytes or ookinetes. CONCLUSIONS: This study presents developmental monitoring of P. falciparum oocysts produced in vivo and in vitro. The anti-PfCap380 antisera serves as an important reagent for developmental studies of oocysts from the mosquito midgut and also from oocyst culture using in vitro methodology. The present data demonstrate that PfCap380 is a useful marker to follow the development and maturation of in vivo and in vitro produced oocysts as early as 2 days after zygote formation. Further in vitro studies focused on oocyst and sporozoite maturation will support the manufacturing of whole sporozoites for malaria vaccines.


Assuntos
DNA de Protozoário/genética , Marcadores Genéticos/genética , Malária Falciparum/parasitologia , Oocistos/genética , Plasmodium falciparum/genética , Humanos , Limite de Detecção , Malária Falciparum/diagnóstico , Tipagem Molecular , Parasitologia
10.
Infect Immun ; 86(5)2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29440367

RESUMO

Genetically attenuated malaria parasites (GAP) that arrest during liver stage development are powerful immunogens and afford complete and durable protection against sporozoite infection. Late liver stage-arresting GAP provide superior protection against sporozoite challenge in mice compared to early live stage-arresting attenuated parasites. However, very few late liver stage-arresting GAP have been generated to date. Therefore, identification of additional loci that are critical for late liver stage development and can be used to generate novel late liver stage-arresting GAPs is of importance. We further explored genetic attenuation in Plasmodium yoelii by combining two gene deletions, PlasMei2 and liver-specific protein 2 (LISP2), that each cause late liver stage arrest with various degrees of infrequent breakthrough to blood stage infection. The dual gene deletion resulted in a synthetic lethal phenotype that caused complete attenuation in a highly susceptible mouse strain. P. yoeliiplasmei2-lisp2- arrested late in liver stage development and did not persist in livers beyond 3 days after infection. Immunization with this GAP elicited robust protective antibody responses in outbred and inbred mice against sporozoites, liver stages, and blood stages as well as eliciting protective liver-resident T cells. The immunization afforded protection against both sporozoite challenge and blood stage challenge. These findings provide evidence that completely attenuated late liver stage-arresting GAP are achievable via the synthetic lethal approach and might enable a path forward for the creation of a completely attenuated late liver stage-arresting P. falciparum GAP.


Assuntos
Eritrócitos/imunologia , Fígado/imunologia , Vacinas Antimaláricas/imunologia , Malária/imunologia , Plasmodium yoelii/imunologia , Proteínas de Protozoários/imunologia , Esporozoítos/imunologia , Animais , Imunização/métodos , Malária/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium yoelii/genética , Proteínas de Protozoários/genética , Esporozoítos/genética
11.
Front Immunol ; 9: 2748, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30619241

RESUMO

Each year malaria kills hundreds of thousands of people and infects hundreds of millions of people despite current control measures. An effective malaria vaccine will likely be necessary to aid in malaria eradication. Vaccination using whole sporozoites provides an increased repertoire of immunogens compared to subunit vaccines across at least two life cycle stages of the parasite, the extracellular sporozoite, and intracellular liver stage. Three potential whole sporozoite vaccine approaches are under development and include genetically attenuated parasites, radiation attenuated sporozoites, and wild-type sporozoites administered in combination with chemoprophylaxis. Pre-clinical and clinical studies have demonstrated whole sporozoite vaccine immunogenicity, including humoral and cellular immunity and a range of vaccine efficacy that depends on the pre-exposure of vaccinated individuals. While whole sporozoite vaccines can provide protection against malaria in some cases, more recent studies in malaria-endemic regions demonstrate the need for improvements. Moreover, challenges remain in manufacturing large quantities of sporozoites for vaccine commercialization. A promising solution to the whole sporozoite manufacturing challenge is in vitro culturing methodology, which has been described for several Plasmodium species, including the major disease-causing human malaria parasite, Plasmodium falciparum. Here, we review whole sporozoite vaccine immunogenicity and in vitro culturing platforms for sporozoite production.


Assuntos
Imunogenicidade da Vacina , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Esporozoítos/imunologia , Humanos , Malária Falciparum/prevenção & controle
12.
Cell Microbiol ; 20(4)2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29253313

RESUMO

Gliding motility and cell traversal by the Plasmodium ookinete and sporozoite invasive stages allow penetration of cellular barriers to establish infection of the mosquito vector and mammalian host, respectively. Motility and traversal are not observed in red cell infectious merozoites, and we have previously classified genes that are expressed in sporozoites but not merozoites (S genes) in order to identify proteins involved in these processes. The S4 gene has been described as criticaly involved in Cell Traversal for Ookinetes and Sporozoites (CelTOS), yet knockout parasites (s4/celtos¯) do not generate robust salivary gland sporozoite numbers, precluding a thorough analysis of S4/CelTOS function during host infection. We show here that a failure of oocysts to develop or survive in the midgut contributes to the poor mosquito infection by Plasmodium yoelii (Py) s4/celtos¯ rodent malaria parasites. We rescued this phenotype by expressing S4/CelTOS under the ookinete-specific circumsporozoite protein and thrombospondin-related anonymous protein-related protein (CTRP) promoter (S4/CelTOSCTRP ), generating robust numbers of salivary gland sporozoites lacking S4/CelTOS that were suitable for phenotypic analysis. Py S4/CelTOSCTRP sporozoites showed reduced infectivity in BALB/c mice when compared to wild-type sporozoites, although they appeared more infectious than sporozoites deficient in the related traversal protein PLP1/SPECT2 (Py plp1/spect2¯). Using in vitro assays, we substantiate the role of S4/CelTOS in sporozoite cell traversal, but also uncover a previously unappreciated role for this protein for sporozoite gliding motility.


Assuntos
Plasmodium yoelii/fisiologia , Proteínas de Protozoários/metabolismo , Esporozoítos/metabolismo , Animais , Movimento Celular , Interações Hospedeiro-Parasita , Malária/parasitologia , Mosquitos Vetores , Plasmodium yoelii/genética , Proteínas de Protozoários/genética
13.
NPJ Vaccines ; 2: 27, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29263882

RESUMO

A malaria vaccine that prevents infection will be an important new tool in continued efforts of malaria elimination, and such vaccines are under intense development for the major human malaria parasite Plasmodium falciparum (Pf). Antibodies elicited by vaccines can block the initial phases of parasite infection when sporozoites are deposited into the skin by mosquito bite and then target the liver for further development. However, there are currently no standardized in vivo preclinical models that can measure the inhibitory activity of antibody specificities against Pf sporozoite infection via mosquito bite. Here, we use human liver-chimeric mice as a challenge model to assess prevention of natural Pf sporozoite infection by antibodies. We demonstrate that these mice are consistently infected with Pf by mosquito bite and that this challenge can be combined with passive transfer of either monoclonal antibodies or polyclonal human IgG from immune serum to measure antibody-mediated blocking of parasite infection using bioluminescent imaging. This methodology is useful to down-select functional antibodies and to investigate mechanisms or immune correlates of protection in clinical trials, thereby informing rational vaccine optimization.

14.
Cell Rep ; 21(7): 1839-1852, 2017 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-29141217

RESUMO

Effector T cells exhibiting features of either T helper 1 (Th1) or T follicular helper (Tfh) populations are essential to control experimental Plasmodium infection and are believed to be critical for resistance to clinical malaria. To determine whether Plasmodium-specific Th1- and Tfh-like effector cells generate memory populations that contribute to protection, we developed transgenic parasites that enable high-resolution study of anti-malarial memory CD4 T cells in experimental models. We found that populations of both Th1- and Tfh-like Plasmodium-specific memory CD4 T cells persist. Unexpectedly, Th1-like memory cells exhibit phenotypic and functional features of Tfh cells during recall and provide potent B cell help and protection following transfer, characteristics that are enhanced following ligation of the T cell co-stimulatory receptor OX40. Our findings delineate critical functional attributes of Plasmodium-specific memory CD4 T cells and identify a host-specific factor that can be targeted to improve resolution of acute malaria and provide durable, long-term protection against Plasmodium parasite re-exposure.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Imunidade Humoral , Memória Imunológica , Malária/imunologia , Plasmodium/imunologia , Células Th1/imunologia , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores OX40/metabolismo
15.
PLoS Negl Trop Dis ; 11(7): e0005791, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28759593

RESUMO

Plasmodium falciparum and Plasmodium vivax cause the majority of human malaria cases. Research efforts predominantly focus on P. falciparum because of the clinical severity of infection and associated mortality rates. However, P. vivax malaria affects more people in a wider global range. Furthermore, unlike P. falciparum, P. vivax can persist in the liver as dormant hypnozoites that can be activated weeks to years after primary infection, causing relapse of symptomatic blood stages. This feature makes P. vivax unique and difficult to eliminate with the standard tools of vector control and treatment of symptomatic blood stage infection with antimalarial drugs. Infection by Plasmodium is initiated by the mosquito-transmitted sporozoite stage, a highly motile invasive cell that targets hepatocytes in the liver. The most advanced malaria vaccine for P. falciparum (RTS,S, a subunit vaccine containing of a portion of the major sporozoite surface protein) conferred limited protection in Phase III trials, falling short of WHO-established vaccine efficacy goals. However, blocking the sporozoite stage of infection in P. vivax, before the establishment of the chronic liver infection, might be an effective malaria vaccine strategy to reduce the occurrence of relapsing blood stages. It is also thought that a multivalent vaccine comprising multiple sporozoite surface antigens will provide better protection, but a comprehensive analysis of proteins in P. vivax sporozoites is not available. To inform sporozoite-based vaccine development, we employed mass spectrometry-based proteomics to identify nearly 2,000 proteins present in P. vivax salivary gland sporozoites. Analysis of protein post-translational modifications revealed extensive phosphorylation of glideosome proteins as well as regulators of transcription and translation. Additionally, the sporozoite surface proteins CSP and TRAP, which were recently discovered to be glycosylated in P. falciparum salivary gland sporozoites, were also observed to be similarly modified in P. vivax sporozoites. Quantitative comparison of the P. vivax and P. falciparum salivary gland sporozoite proteomes revealed a high degree of similarity in protein expression levels, including among invasion-related proteins. Nevertheless, orthologs with significantly different expression levels between the two species could be identified, as well as highly abundant, species-specific proteins with no known orthologs. Finally, we employed chemical labeling of live sporozoites to isolate and identify 36 proteins that are putatively surface-exposed on P. vivax salivary gland sporozoites. In addition to identifying conserved sporozoite surface proteins identified by similar analyses of other Plasmodium species, our analysis identified several as-yet uncharacterized proteins, including a putative 6-Cys protein with no known ortholog in P. falciparum.


Assuntos
Proteínas de Membrana/análise , Plasmodium vivax/isolamento & purificação , Processamento de Proteína Pós-Traducional , Proteoma/análise , Proteínas de Protozoários/análise , Animais , Anopheles/parasitologia , Malária Vivax/metabolismo , Espectrometria de Massas , Proteogenômica , Glândulas Salivares/parasitologia , Esporozoítos/metabolismo
17.
Artigo em Inglês | MEDLINE | ID: mdl-28242785

RESUMO

In their infection cycle, malaria parasites undergo replication and population expansions within the vertebrate host and the mosquito vector. Host infection initiates with sporozoite invasion of hepatocytes, followed by a dramatic parasite amplification event during liver stage parasite growth and replication within hepatocytes. Each liver stage forms up to 90,000 exoerythrocytic merozoites, which are in turn capable of initiating a blood stage infection. Liver stages not only exploit host hepatocyte resources for nutritional needs but also endeavor to prevent hepatocyte cell death and detection by the host's immune system. Research over the past decade has identified numerous parasite factors that play a critical role during liver infection and has started to delineate a complex web of parasite-host interactions that sustain successful parasite colonization of the mammalian host. Targeting the parasites' obligatory infection of the liver as a gateway to the blood, with drugs and vaccines, constitutes the most effective strategy for malaria eradication, as it would prevent clinical disease and onward transmission of the parasite.


Assuntos
Hepatócitos/parasitologia , Hepatopatias Parasitárias/parasitologia , Malária/parasitologia , Plasmodium/fisiologia , Esporozoítos/imunologia , Animais , Culicidae , Interações Hospedeiro-Parasita , Humanos , Malária/complicações , Camundongos , Esporozoítos/crescimento & desenvolvimento , Esporozoítos/metabolismo
18.
Sci Transl Med ; 9(371)2017 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-28053159

RESUMO

Immunization of humans with whole sporozoites confers complete, sterilizing immunity against malaria infection. However, achieving consistent safety while maintaining immunogenicity of whole parasite vaccines remains a formidable challenge. We generated a genetically attenuated Plasmodium falciparum (Pf) malaria parasite by deleting three genes expressed in the pre-erythrocytic stage (Pf p52-/p36-/sap1-). We then tested the safety and immunogenicity of the genetically engineered (Pf GAP3KO) sporozoites in human volunteers. Pf GAP3KO sporozoites were delivered to 10 volunteers using infected mosquito bites with a single exposure consisting of 150 to 200 bites per subject. All subjects remained blood stage-negative and developed inhibitory antibodies to sporozoites. GAP3KO rodent malaria parasites engendered complete, protracted immunity against infectious sporozoite challenge in mice. The results warrant further clinical testing of Pf GAP3KO and its potential development into a vaccine strain.


Assuntos
Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/genética , Esporozoítos/genética , Adulto , Animais , Anticorpos Antiprotozoários/sangue , Feminino , Deleção de Genes , Engenharia Genética , Humanos , Imunoglobulina G/sangue , Vacinas Antimaláricas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Plasmodium falciparum/imunologia , Proteínas de Protozoários/genética , Esporozoítos/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Adulto Jovem
19.
Cell Rep ; 17(12): 3193-3205, 2016 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-28009289

RESUMO

Many current malaria vaccines target the pre-erythrocytic stage of infection in the liver. However, in malaria-endemic regions, increased blood stage exposure is associated with decreased vaccine efficacy, thereby challenging current vaccine efforts. We hypothesized that pre-erythrocytic humoral immunity is directly disrupted by blood stage infection. To investigate this possibility, we used Plasmodium-antigen tetramers to analyze B cells after infection with either late liver stage arresting parasites or wild-type parasites that progress to the blood stage. Our data demonstrate that immunoglobulin G (IgG) antibodies against the pre-erythrocytic antigen, circumsporozoite protein (CSP), are generated only in response to the attenuated, but not the wild-type, infection. Further analyses revealed that blood stage malaria inhibits CSP-specific germinal center B cell differentiation and modulates chemokine expression. This results in aberrant memory formation and the loss of a rapid secondary B cell response. These data highlight how immunization with attenuated parasites may drive optimal immunity to malaria.


Assuntos
Imunidade Humoral , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Animais , Antígenos de Protozoários/imunologia , Linfócitos B/imunologia , Linfócitos B/parasitologia , Eritrócitos/imunologia , Eritrócitos/parasitologia , Humanos , Imunoglobulina G/imunologia , Fígado/imunologia , Fígado/parasitologia , Vacinas Antimaláricas/uso terapêutico , Malária Falciparum/genética , Malária Falciparum/parasitologia , Plasmodium falciparum/patogenicidade , Vacinação
20.
PLoS Pathog ; 12(4): e1005606, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-27128092

RESUMO

Malaria parasite infection is initiated by the mosquito-transmitted sporozoite stage, a highly motile invasive cell that targets hepatocytes in the liver for infection. A promising approach to developing a malaria vaccine is the use of proteins located on the sporozoite surface as antigens to elicit humoral immune responses that prevent the establishment of infection. Very little of the P. falciparum genome has been considered as potential vaccine targets, and candidate vaccines have been almost exclusively based on single antigens, generating the need for novel target identification. The most advanced malaria vaccine to date, RTS,S, a subunit vaccine consisting of a portion of the major surface protein circumsporozoite protein (CSP), conferred limited protection in Phase III trials, falling short of community-established vaccine efficacy goals. In striking contrast to the limited protection seen in current vaccine trials, sterilizing immunity can be achieved by immunization with radiation-attenuated sporozoites, suggesting that more potent protection may be achievable with a multivalent protein vaccine. Here, we provide the most comprehensive analysis to date of proteins located on the surface of or secreted by Plasmodium falciparum salivary gland sporozoites. We used chemical labeling to isolate surface-exposed proteins on sporozoites and identified these proteins by mass spectrometry. We validated several of these targets and also provide evidence that components of the inner membrane complex are in fact surface-exposed and accessible to antibodies in live sporozoites. Finally, our mass spectrometry data provide the first direct evidence that the Plasmodium surface proteins CSP and TRAP are glycosylated in sporozoites, a finding that could impact the selection of vaccine antigens.


Assuntos
Malária Falciparum/metabolismo , Proteômica/métodos , Proteínas de Protozoários/análise , Proteínas de Protozoários/metabolismo , Esporozoítos/metabolismo , Animais , Culicidae , Imunofluorescência , Glicosilação , Espectrometria de Massas , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Organismos Geneticamente Modificados , Esporozoítos/química
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