RESUMEN
Plasmodium falciparum causes the most severe malaria and is exposed to various environmental and physiological stresses in the human host. Given that GCN5 plays a critical role in regulating stress responses in model organisms, we aimed to elucidate PfGCN5's function in stress responses in P. falciparum. The protein level of PfGCN5 was substantially induced under three stress conditions [heat shock, low glucose starvation, and dihydroartemisinin, the active metabolite of artemisinin (ART)]. With a TetR-DOZI conditional knockdown (KD) system, we successfully down-regulated PfGCN5 to ~50% and found that KD parasites became more sensitive to all three stress conditions. Transcriptomic analysis via RNA-seq identified ~1,000 up- and down-regulated genes in the wild-type (WT) and KD parasites under these stress conditions. Importantly, DHA induced transcriptional alteration of many genes involved in many aspects of stress responses, which were heavily shared among the altered genes under heat shock and low glucose conditions, including ART-resistance-related genes such as K13 and coronin. Based on the expression pattern between WT and KD parasites under three stress conditions, ~300-400 genes were identified to be involved in PfGCN5-dependent, general, and stress-condition-specific responses with high levels of overlaps among three stress conditions. Notably, using ring-stage survival assay, we found that KD or inhibition of PfGCN5 could sensitize the ART-resistant parasites to the DHA treatment. All these indicate that PfGCN5 is pivotal in regulating general and ART-resistance-related stress responses in malaria parasites, implicating PfGCN5 as a potential target for malaria intervention.
Asunto(s)
Antimaláricos , Artemisininas , Malaria Falciparum , Humanos , Plasmodium falciparum/metabolismo , Artemisininas/farmacología , Artemisininas/uso terapéutico , Malaria Falciparum/tratamiento farmacológico , Glucosa/metabolismo , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Resistencia a Medicamentos/genéticaRESUMEN
Plasmodium vivax is dependent on interaction with the Duffy antigen receptor for chemokines (DARC) for invasion of human erythrocytes. The P. vivax Duffy binding protein (PvDBP) mediates interaction of P. vivax merozoites with DARC. The DARC receptor-binding domain lies in a conserved N-terminal cysteine-rich region of PvDBP referred to as region II (PvDBPII). PvDBPII is an attractive vaccine candidate since antibodies raised against PvDBPII block erythrocyte invasion by P. vivax. Here, we describe methods to produce recombinant PvDBPII in its correctly folded conformation. A synthetic gene optimized for expression of PvDBPII in Escherichia coli and fed batch fermentation process based on exponential feeding strategy was used to achieve high levels of expression of recombinant PvDBPII. Recombinant PvDBPII was isolated from inclusion bodies, refolded by rapid dilution and purified by ion exchange chromatography. Purified recombinant PvDBPII was characterized for identity, purity and functional activity using standardized release assays. Recombinant PvDBPII formulated with various human compatible adjuvants including glycosylpyranosyl lipid A-stable emulsion (GLA-SE) and alhydrogel was used for immunogenicity studies in small animals to downselect a suitable formulation for clinical development. Sera collected from immunized animals were tested for recognition of PvDBPII and inhibition of PvDBPII-DARC binding. GLA-SE formulations of PvDBPII yielded higher ELISA and binding inhibition titres compared to PvDBPII formulated with alhydrogel. These data support further development of a recombinant vaccine for P. vivax based on PvDBPII formulated with GLA-SE.
Asunto(s)
Antígenos de Protozoos , Inmunogenicidad Vacunal , Vacunas contra la Malaria , Plasmodium vivax/genética , Proteínas Protozoarias , Receptores de Superficie Celular , Animales , Antígenos de Protozoos/biosíntesis , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/aislamiento & purificación , Humanos , Vacunas contra la Malaria/biosíntesis , Vacunas contra la Malaria/genética , Vacunas contra la Malaria/inmunología , Vacunas contra la Malaria/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Plasmodium vivax/inmunología , Dominios Proteicos , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/aislamiento & purificación , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Histone lysine acetyltransferase MYST-associated NuA4 complex is conserved from yeast to humans and plays key roles in cell cycle regulation, gene transcription, and DNA replication/repair. Here, we identified a Plasmodium falciparum MYST-associated complex, PfNuA4, which contains 11 of the 13 conserved NuA4 subunits. Reciprocal pulldowns using PfEAF2, a shared component between the NuA4 and SWR1 complexes, not only confirmed the PfNuA4 complex but also identified the PfSWR1 complex, a histone remodeling complex, although their identities are low compared to the homologs in yeast or humans. Notably, both H2A.Z/H2B.Z were associated with the PfSWR1 complex, indicating that this complex is involved in the deposition of H2A.Z/H2B.Z, the variant histone pair that is enriched in the activated promoters. Overexpression of PfMYST resulted in earlier expression of genes involved in cell cycle regulation, DNA replication, and merozoite invasion, and upregulation of the genes related to antigenic variation and DNA repair. Consistently, PfMYST overexpression led to high basal phosphorylated PfH2A (γ-PfH2A), the mark of DNA double-strand breaks, and conferred protection against genotoxic agent methyl methanesulfonate (MMS), X-rays, and artemisinin, the first-line antimalarial drug. In contrast, the knockdown of PfMYST caused a delayed parasite recovery upon MMS treatment. MMS induced the gradual disappearance of PfMYST in the cytoplasm and concomitant accumulation of PfMYST in the nucleus, suggesting cytoplasm-nucleus shuttling of PfMYST. Meanwhile, PfMYST colocalized with the γ-PfH2A, indicating PfMYST was recruited to the DNA damage sites. Collectively, PfMYST plays critical roles in cell cycle regulation, gene transcription, and DNA replication/DNA repair in this low-branching parasitic protist.IMPORTANCEUnderstanding gene regulation and DNA repair in malaria parasites is critical for identifying targets for antimalarials. This study found PfNuA4, a PfMYST-associated, histone modifier complex, and PfSWR1, a chromatin remodeling complex in malaria parasite Plasmodium falciparum. These complexes are divergent due to the low identities compared to their homologs from yeast and humans. Furthermore, overexpression of PfMYST resulted in substantial transcriptomic changes, indicating that PfMYST is involved in regulating the cell cycle, antigenic variation, and DNA replication/repair. Consistently, PfMYST was found to protect against DNA damage caused by the genotoxic agent methyl methanesulfonate, X-rays, and artemisinin, the first-line antimalarial drug. Additionally, DNA damage led to the relocation of cytoplasmic PfMYST to the nucleus and colocalization of PfMYST with γ-PfH2A, the mark of DNA damage. In summary, this study demonstrated that the PfMYST complex has critical functions in regulating cell cycle, antigenic variation, and DNA replication/DNA repair in P. falciparum.
Asunto(s)
Reparación del ADN , Plasmodium falciparum , Proteínas Protozoarias , Plasmodium falciparum/genética , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Replicación del ADN , Histonas/genética , Histonas/metabolismo , Regulación de la Expresión GénicaRESUMEN
Background: Despite decades of effort, Plasmodium falciparum malaria remains a leading killer of children. The absence of a highly effective vaccine and the emergence of parasites resistant to both diagnosis as well as treatment hamper effective public health interventions. Methods and results: To discover new vaccine candidates, we used our whole proteome differential screening method and identified PfGBP130 as a parasite protein uniquely recognized by antibodies from children who had developed resistance to P. falciparum infection but not from those who remained susceptible. We formulated PfGBP130 as lipid encapsulated mRNA, DNA plasmid, and recombinant protein-based immunogens and evaluated the efficacy of murine polyclonal anti-PfGBP130 antisera to inhibit parasite growth in vitro. Immunization of mice with PfGBP130-A (aa 111-374), the region identified in our differential screen, formulated as a DNA plasmid or lipid encapsulated mRNA, but not as a recombinant protein, induced antibodies that inhibited RBC invasion in vitro. mRNA encoding the full ectodomain of PfGBP130 (aa 89-824) also generated parasite growth-inhibitory antibodies. Conclusion: We are currently advancing PfGBP130-A formulated as a lipid-encapsulated mRNA for efficacy evaluation in non-human primates.
Asunto(s)
Anticuerpos Antiprotozoarios , Eritrocitos , Vacunas contra la Malaria , Malaria Falciparum , Plasmodium falciparum , Proteínas Protozoarias , Animales , Plasmodium falciparum/inmunología , Anticuerpos Antiprotozoarios/inmunología , Ratones , Eritrocitos/parasitología , Eritrocitos/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Malaria Falciparum/parasitología , Humanos , Vacunas contra la Malaria/inmunología , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/genética , Antígenos de Protozoos/inmunología , Inmunización , FemeninoRESUMEN
Plasmodium falciparum causes the most severe malaria and is exposed to various environmental and physiological stresses in the human host. Given that GCN5 plays a critical role in regulating stress responses in model organisms, we aimed to elucidate PfGCN5's function in stress responses in P. falciparum . The protein level of PfGCN5 was substantially induced under three stress conditions (heat shock, low glucose starvation, and dihydroartemisinin, the active metabolite of artemisinin (ART)). With a TetR-DOZI conditional knockdown (KD) system, we successfully down-regulated PfGCN5 to â¼50% and found that KD parasites became more sensitive to all three stress conditions. Transcriptomic analysis via RNA-seq identified â¼1,000 up-and down-regulated genes in the wildtype (WT) and KD parasites under these stress conditions. Importantly, DHA induced transcriptional alteration of many genes involved in many aspects of stress responses, which were heavily shared among the altered genes under heat shock and low glucose conditions, including ART-resistance-related genes such as K13 and coronin . Based on the expression pattern between WT and KD parasites under three stress conditions, â¼300-400 genes were identified to be involved in PfGCN5-dependent, general and stress-condition-specific responses with high levels of overlaps among three stress conditions. Notably, using ring-stage survival assay (RSA), we found that KD or inhibition of PfGCN5 could sensitize the ART-resistant parasites to the DHA treatment. All these indicate that PfGCN5 is pivotal in regulating general and ART-resistance-related stress responses in malaria parasites, implicating PfGCN5 as a potential target for malaria intervention. IMPORTANCE: Malaria leads to about half a million deaths annually and these casualties were majorly caused by the infection of Plasmodium falciparum . This parasite strives to survive by defending against a variety of stress conditions, such as malaria cyclical fever (heat shock), starvation due to low blood sugar (glucose) levels (hypoglycemia), and drug treatment. Previous studies have revealed that P. falciparum has developed unique stress responses to different stresses including ART treatment, and ART-resistant parasites harbor elevated stress responses. In this study, we provide critical evidence on the role of PfGCN5, a histone modifier, and a chromatin coactivator, in regulating general and stress-specific responses in malaria parasites, indicating that PfGCN5 can be used as a potential target for anti-malaria intervention.
RESUMEN
Antibodies generated against Region II of Plasmodium vivax Duffy binding protein (PvRII) can block binding of this parasite ligand to its receptor, the Duffy antigen receptor for chemokines (DARC), and prevent erythrocyte infection by the parasite. An in vitro functional assay that can serve as an immune correlate of an antigen activity is an important tool to guide vaccine development. We describe here the development of a quantitative binding assay and its use to study immune responses against PvRII. The assay was used to test anti-PvRII mouse sera, and was found a useful tool for quantitative estimation of anti-PvRII blocking antibodies.
Asunto(s)
Anticuerpos Antiprotozoarios/análisis , Anticuerpos Antiprotozoarios/inmunología , Reacciones Antígeno-Anticuerpo/inmunología , Antígenos de Protozoos/inmunología , Proteínas Protozoarias/inmunología , Receptores de Superficie Celular/inmunología , Animales , Anticuerpos Bloqueadores/análisis , Anticuerpos Bloqueadores/inmunología , Antígenos de Protozoos/biosíntesis , Antígenos de Protozoos/aislamiento & purificación , Sitios de Unión , Células Cultivadas , Células HEK293 , Humanos , Sueros Inmunes/inmunología , Ligandos , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/aislamiento & purificación , Conejos , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib has significantly prolonged progression-free survival (PFS) in patients with EGFR-mutant lung cancer, including those with brain metastases. However, despite striking initial responses, osimertinib-treated patients eventually develop lethal metastatic relapse, often to the brain. Although osimertinib-refractory brain relapse is a major clinical challenge, its underlying mechanisms remain poorly understood. Using metastatic models of EGFR-mutant lung cancer, we show that cancer cells expressing high intracellular S100A9 escape osimertinib and initiate brain relapses. Mechanistically, S100A9 upregulates ALDH1A1 expression and activates the retinoic acid (RA) signaling pathway in osimertinib-refractory cancer cells. We demonstrate that the genetic repression of S100A9, ALDH1A1, or RA receptors (RAR) in cancer cells, or treatment with a pan-RAR antagonist, dramatically reduces brain metastasis. Importantly, S100A9 expression in cancer cells correlates with poor PFS in osimertinib-treated patients. Our study, therefore, identifies a novel, therapeutically targetable S100A9-ALDH1A1-RA axis that drives brain relapse. SIGNIFICANCE: Treatment with the EGFR TKI osimertinib prolongs the survival of patients with EGFR-mutant lung cancer; however, patients develop metastatic relapses, often to the brain. We identified a novel intracellular S100A9-ALDH1A1-RA signaling pathway that drives lethal brain relapse and can be targeted by pan-RAR antagonists to prevent cancer progression and prolong patient survival. This article is highlighted in the In This Issue feature, p. 873.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Familia de Aldehído Deshidrogenasa 1 , Compuestos de Anilina/farmacología , Compuestos de Anilina/uso terapéutico , Encéfalo/patología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mutación , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Retinal-Deshidrogenasa/genética , Transducción de Señal , Tretinoina/farmacologíaRESUMEN
Individuals residing in malaria-endemic regions acquire protective immunity after repeated infection with malaria parasites; however, mechanisms of protective immunity and their immune correlates are poorly understood. Blood-stage infection with Plasmodium vivax depends completely on interaction of P. vivax Duffy-binding protein (PvDBP) with the Duffy antigen on host erythrocytes. Here, we performed a prospective cohort treatment/reinfection study of children (5-14 years) residing in a P. vivax-endemic region of Papua New Guinea (PNG) in which children were cleared of blood-stage infection and then examined biweekly for reinfection for 25 weeks. To test the hypothesis that naturally acquired binding inhibitory antibodies (BIAbs) targeting PvDBP region II (PvDBPII) provide protection against P. vivax infection, we used a quantitative receptor-binding assay to distinguish between antibodies that merely recognize PvDBP and those that inhibit binding to Duffy. The presence of high-level BIAbs (>90% inhibition of PvDBPII-Duffy binding, n = 18) before treatment was associated with delayed time to P. vivax reinfection diagnosed by light microscopy (P = 0.02), 55% reduced risk of P. vivax reinfection (Hazard's ratio = 0.45, P = 0.04), and 48% reduction in geometric mean P. vivax parasitemia (P < 0.001) when compared with children with low-level BIAbs (n = 148). Further, we found that stable, high-level BIAbs displayed strain-transcending inhibition by reducing reinfection with similar efficiency of PNG P. vivax strains characterized by six diverse PvDBPII haplotypes. These observations demonstrate a functional correlate of protective immunity in vivo and provide support for developing a vaccine against P. vivax malaria based on PvDBPII.
Asunto(s)
Anticuerpos Bloqueadores/inmunología , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Malaria Vivax/inmunología , Malaria Vivax/prevención & control , Plasmodium vivax/inmunología , Proteínas Protozoarias/inmunología , Receptores de Superficie Celular/inmunología , Animales , Anticuerpos Bloqueadores/sangre , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/genética , Niño , Preescolar , Estudios de Cohortes , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Vacunas contra la Malaria/inmunología , Malaria Vivax/sangre , Masculino , Nueva Guinea , Plasmodium vivax/crecimiento & desarrollo , Estudios Prospectivos , Proteínas Protozoarias/genética , Receptores de Superficie Celular/genéticaRESUMEN
Nearly 80% of advanced cancer patients are afflicted with cachexia, a debilitating syndrome characterized by extensive loss of muscle mass and function. Cachectic cancer patients have a reduced tolerance to antineoplastic therapies and often succumb to premature death from the wasting of respiratory and cardiac muscles. Since there are no available treatments for cachexia, it is imperative to understand the mechanisms that drive cachexia in order to devise effective strategies to treat it. Although 25% of metastatic breast cancer patients develop symptoms of muscle wasting, mechanistic studies of breast cancer cachexia have been hampered by a lack of experimental models. Using tumor cells deficient for BARD1, a subunit of the BRCA1/BARD1 tumor suppressor complex, we have developed a new orthotopic model of triple-negative breast cancer that spontaneously metastasizes to the lung and leads to systemic muscle deterioration. We show that expression of the metal-ion transporter, Zip14, is markedly upregulated in cachectic muscles from these mice and is associated with elevated intramuscular zinc and iron levels. Aberrant Zip14 expression and altered metal-ion homeostasis could therefore represent an underlying mechanism of cachexia development in human patients with triple-negative breast cancer. Our study provides a unique model for studying breast cancer cachexia and identifies a potential therapeutic target for its treatment.
Asunto(s)
Caquexia/metabolismo , Proteínas de Transporte de Catión/metabolismo , Neoplasias Pulmonares/metabolismo , Músculo Esquelético/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Ubiquitina-Proteína Ligasas/deficiencia , Animales , Proteína BRCA1/metabolismo , Caquexia/genética , Caquexia/patología , Proteínas de Transporte de Catión/genética , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Ratones , Músculo Esquelético/patología , Norisoprenoides/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Regulación hacia Arriba , Zinc/metabolismoRESUMEN
BACKGROUND: The Plasmodium vivax Duffy Binding Protein (PvDBP) is a key target of naturally acquired immunity. However, region II of PvDBP, which contains the receptor-binding site, is highly polymorphic. The natural acquisition of antibodies to different variants of PvDBP region II (PvDBPII), including the AH, O, P and Sal1 alleles, the central region III-V (PvDBPIII-V), and P. vivax Erythrocyte Binding Protein region II (PvEBPII) and their associations with risk of clinical P. vivax malaria are not well understood. METHODOLOGY: Total IgG and IgG subclasses 1, 2, and 3 that recognize four alleles of PvDBPII (AH, O, P, and Sal1), PvDBPIII-V and PvEBPII were measured in samples collected from a cohort of 1 to 3 year old Papua New Guinean (PNG) children living in a highly endemic area of PNG. The levels of binding inhibitory antibodies (BIAbs) to PvDBPII (AH, O, and Sal1) were also tested in a subset of children. The association of presence of IgG with age, cumulative exposure (measured as the product of age and malaria infections during follow-up) and prospective risk of clinical malaria were evaluated. RESULTS: The increase in antigen-specific total IgG, IgG1, and IgG3 with age and cumulative exposure was only observed for PvDBPII AH and PvEBPII. High levels of total IgG and predominant subclass IgG3 specific for PvDBPII AH were associated with decreased incidence of clinical P. vivax episodes (aIRR = 0.56-0.68, P≤0.001-0.021). High levels of total IgG and IgG1 to PvEBPII correlated strongly with protection against clinical vivax malaria compared with IgGs against all PvDBPII variants (aIRR = 0.38, P<0.001). Antibodies to PvDBPII AH and PvEBPII showed evidence of an additive effect, with a joint protective association of 70%. CONCLUSION: Antibodies to the key parasite invasion ligands PvDBPII and PvEBPII are good correlates of protection against P. vivax malaria in PNG. This further strengthens the rationale for inclusion of PvDBPII in a recombinant subunit vaccine for P. vivax malaria and highlights the need for further functional studies to determine the potential of PvEBPII as a component of a subunit vaccine for P. vivax malaria.
Asunto(s)
Antígenos de Protozoos/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/fisiología , Malaria Vivax/inmunología , Proteínas Protozoarias/inmunología , Receptores de Superficie Celular/inmunología , Especificidad de Anticuerpos , Preescolar , Femenino , Humanos , Lactante , Malaria Vivax/epidemiología , Masculino , Papúa Nueva Guinea/epidemiología , ParasitemiaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer type in which the mortality rate approaches the incidence rate. More than 85% of PDAC patients experience a profound loss of muscle mass and function, known as cachexia. PDAC patients with this condition suffer from decreased tolerance to anti-cancer therapies and often succumb to premature death due to respiratory and cardiac muscle wasting. Yet, there are no approved therapies available to alleviate cachexia. We previously found that upregulation of the metal ion transporter, Zip14, and altered zinc homeostasis are critical mediators of cachexia in metastatic colon, lung, and breast cancer models. Here, we show that a similar mechanism is likely driving the development of cachexia in PDAC. In two independent experimental metastasis models generated from the murine PDAC cell lines, Pan02 and FC1242, we observed aberrant Zip14 expression and increased zinc ion levels in cachectic muscles. Moreover, in advanced PDAC patients, high levels of ZIP14 in muscles correlated with the presence of cachexia. These studies underscore the importance of altered ZIP14 function in PDAC-associated cachexia development and highlight a potential therapeutic opportunity for improving the quality of life and prolonging survival in PDAC patients.
RESUMEN
Reticulocyte invasion by Plasmodium vivax requires interaction of the Duffy-binding protein (PvDBP) with host Duffy antigen receptor for chemokines (DARCs). The binding domain of PvDBP maps to a cysteine-rich region referred to as region II (PvDBPII). Blocking this interaction offers a potential path to prevent P. vivax blood-stage growth and P. vivax malaria. This forms the rationale for development of a vaccine based on PvDBPII. Here we report results of a Phase I randomized trial to evaluate the safety and immunogenicity of recombinant PvDBPII formulated with glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE). Thirty-six malaria-naive, healthy Indian male subjects aged 18-45 years were assigned into three cohorts corresponding to doses of 10, 25 and 50 µg of PvDBPII formulated with 5 µg of GLA-SE. Each cohort included nine PvDBPII/GLA-SE vaccinees and three hepatitis B control vaccine recipients. Each subject received the assigned vaccine intramuscularly on days 0, 28 and 56, and was followed up till day 180. No serious AE was reported and PvDBPII/GLA-SE was well-tolerated and safe. Analysis by ELISA showed that all three doses of PvDBPII elicited antigen-specific binding-inhibitory antibodies. The 50 µg dose elicited antibodies against PvDBPII that had the highest binding-inhibitory titres and were most persistent. Importantly, the antibody responses were strain transcending and blocked receptor binding of diverse PvDBP alleles. These results support further clinical development of PvDBPII/GLA-SE to evaluate efficacy against sporozoite or blood-stage challenge in controlled human malaria infection (CHMI) models and against natural P. vivax challenge in malaria endemic areas.
RESUMEN
Invasion of human erythrocytes by Plasmodium vivax requires interaction between Duffy binding protein (PvDBP) and the Duffy blood group antigen. The receptor-binding domain of PvDBP lies in a conserved N-terminal, cysteine-rich region, region II (PvRII). PvRII is a valuable malaria subunit vaccine candidate for asexual blood stages. We have evaluated in Aotus monkeys the immunogenicity and protective efficacy of recombinant PvRII formulated in Freund's and Montanide ISA720 adjuvants. Specific antibody titers were determined by an enzyme-linked immunosorbent assay after each of three doses of 50 microg of protein administered by the subcutaneous route. Immunization with PvRII formulated in Freund's adjuvant yielded higher antibody titers than immunization with the Montanide ISA720 formulation and offered partial protection. Although the Montanide ISA720 formulation was immunogenic, it did not provide any protection. Given the immunogenicity and partial protection observed, further studies are needed to optimize the PvRII vaccine formulation with adjuvants suitable for human use.
Asunto(s)
Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Cebidae , Modelos Animales de Enfermedad , Sistema del Grupo Sanguíneo Duffy/metabolismo , Vacunas contra la Malaria/inmunología , Malaria Vivax/prevención & control , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/inmunología , Vacunas Sintéticas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/genética , Femenino , Adyuvante de Freund/administración & dosificación , Humanos , Inmunización , Vacunas contra la Malaria/administración & dosificación , Malaria Vivax/parasitología , Masculino , Manitol/administración & dosificación , Manitol/análogos & derivados , Ácidos Oléicos/administración & dosificación , Plasmodium vivax/inmunología , Plasmodium vivax/patogenicidad , Proteínas Protozoarias/genética , Receptores de Superficie Celular/genética , Vacunas Sintéticas/administración & dosificaciónRESUMEN
Plasmodium vivax continues to cause significant morbidity outside Africa with more than 50% of malaria cases in many parts of South and South-east Asia, Pacific islands, Central and South America being attributed to P. vivax infections. The unique biology of P. vivax, including its ability to form latent hypnozoites that emerge months to years later to cause blood stage infections, early appearance of gametocytes before clinical symptoms are apparent and a shorter development cycle in the vector makes elimination of P. vivax using standard control tools difficult. The availability of an effective vaccine that provides protection and prevents transmission would be a valuable tool in efforts to eliminate P. vivax. Here, we review the latest developments related to P. vivax malaria vaccines and discuss the challenges as well as directions toward the goal of developing highly efficacious vaccines against P. vivax malaria.
Asunto(s)
Vacunas contra la Malaria/inmunología , Malaria Vivax/prevención & control , Plasmodium vivax/inmunología , África , Investigación Biomédica , Humanos , Estadios del Ciclo de Vida/inmunología , Malaria Vivax/inmunología , Malaria Vivax/transmisión , Islas del Pacífico , Plasmodium vivax/fisiología , América del SurRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0117820.].
RESUMEN
BACKGROUND: A phase I randomised, controlled, single blind, dose escalation trial was conducted to evaluate safety and immunogenicity of JAIVAC-1, a recombinant blood stage vaccine candidate against Plasmodium falciparum malaria, composed of a physical mixture of two recombinant proteins, PfMSP-1(19), the 19 kD conserved, C-terminal region of PfMSP-1 and PfF2 the receptor-binding F2 domain of EBA175. METHOD: Healthy malaria naïve Indian male subjects aged 18-45 years were recruited from the volunteer database of study site. Fifteen subjects in each cohort, randomised in a ratio of 2:1 and meeting the protocol specific eligibility criteria, were vaccinated either with three doses (10 µg, 25 µg and 50 µg of each antigen) of JAIVAC-1 formulated with adjuvant Montanide ISA 720 or with standard dosage of Hepatitis B vaccine. Each subject received the assigned vaccine in the deltoid muscle of the upper arms on Day 0, Day 28 and Day 180. RESULTS: JAIVAC-1 was well tolerated and no serious adverse event was observed. All JAIVAC-1 subjects sero-converted for PfF2 but elicited poor immune response to PfMSP-1(19). Dose-response relationship was observed between vaccine dose of PfF2 and antibody response. The antibodies against PfF2 were predominantly of IgG1 and IgG3 isotype. Sera from JAIVAC-1 subjects reacted with late schizonts in a punctate pattern in immunofluorescence assays. Purified IgG from JAIVAC-1 sera displayed significant growth inhibitory activity against Plasmodium falciparum CAMP strain. CONCLUSION: Antigen PfF2 should be retained as a component of a recombinant malaria vaccine but PfMSP-1(19) construct needs to be optimised to improve its immunogenicity. TRIAL REGISTRATION: Clinical Trial Registry, India CTRI/2010/091/000301.
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Antígenos de Protozoos/administración & dosificación , Vacunas contra la Malaria/administración & dosificación , Malaria Falciparum/prevención & control , Proteína 1 de Superficie de Merozoito/administración & dosificación , Plasmodium falciparum/inmunología , Proteínas Protozoarias/administración & dosificación , Adolescente , Adulto , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/efectos adversos , Antígenos de Protozoos/inmunología , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta Inmunológica , Femenino , Vacunas contra Hepatitis B/administración & dosificación , Vacunas contra Hepatitis B/efectos adversos , Vacunas contra Hepatitis B/inmunología , Humanos , Inmunoglobulina G/inmunología , India , Vacunas contra la Malaria/efectos adversos , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Masculino , Manitol/administración & dosificación , Manitol/efectos adversos , Manitol/análogos & derivados , Proteína 1 de Superficie de Merozoito/efectos adversos , Proteína 1 de Superficie de Merozoito/inmunología , Persona de Mediana Edad , Ácidos Oléicos/administración & dosificación , Ácidos Oléicos/efectos adversos , Proteínas Protozoarias/efectos adversos , Proteínas Protozoarias/inmunologíaRESUMEN
Malaria parasites require specific receptor-ligand interactions to invade host erythrocytes. The 175 kDa Plasmodium falciparum erythrocyte binding antigen (EBA-175) binds sialic acid residues on glycophorin A to mediate erythrocyte invasion. The amino-terminal, conserved, cysteine-rich region of EBA-175, referred to as F2, contains receptor-binding sites. We propose to develop a recombinant malaria vaccine based on region F2. Recombinant P. falciparum region F2 (PfF2) was expressed in Escherichia coli, purified from inclusion bodies under denaturing conditions by metal affinity chromatography, renatured by oxidative refolding and purified further by ion-exchange and gel filtration chromatography. Recombinant PfF2 was characterized and shown to be pure, homogeneous and functionally active in that it binds human erythrocytes with specificity. The immunogenicity of recombinant PfF2 formulated with three human compatible adjuvants, namely, Montanide ISA720, AS02A and alum was tested in mice. All the formulations tested elicited high titer antibodies that block erythrocyte invasion in vitro. The AS02 formulation yielded sera with the highest end-point ELISA titers followed by Montanide ISA720 and alum. Analysis of cellular immune responses indicated that all formulations resulted in significant splenocyte proliferation. Analysis of cytokines secreted by proliferating splenocytes indicated that all the adjuvant formulations tested induced Th1 type responses. These results suggest that recombinant PfF2 formulated with human compatible adjuvants is immunogenic and can elicit high titer invasion inhibitory antibodies providing support for further clinical development of this promising vaccine candidate.
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Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Proteínas Protozoarias/inmunología , Vacunas Sintéticas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Antígenos de Protozoos/química , Sitios de Unión , Citocinas/biosíntesis , Vacunas contra la Malaria/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Pliegue de Proteína , Proteínas Protozoarias/química , Vacunación , Vacunas Sintéticas/administración & dosificaciónRESUMEN
An effective malaria vaccine will probably require the delivery of multiple antigens that induce several layers of immunity. Malaria antigens expressed on the surface and in apical organelles of blood-stage merozoites are potential vaccine candidates given their importance in the invasion of erythrocytes. The present study examined the kinetics of humoral response in BALB/c mice following immunization with combination of two blood-stage Plasmodium vivax invasion related molecules, the N-terminal, cysteine-rich region II of P. vivax Duffy binding protein (PvRII) and the 19kDa C-terminal region of merozoite surface protein 1 (PvMSP1(19)) formulated with Montanide ISA 720 and alhydrogel. Immunization with combination of recombinant PvRII and PvMSP1(19) formulated with the Montanide ISA 720 elicited higher antibody titer compared to the alhydrogel formulation. In case of both the adjuvants tested, combination of PvRII and PvMSP1(19) did not result in suppression of antibody response against either antigen when compared to immunization with individual antigens alone. Analysis of IgG subclasses showed that combination of both the recombinant proteins induced a mixed Th1/Th2-type response with almost all IgG subtypes being expressed in equivalent amount. Antibodies elicited against PvRII showed significant inhibitory effect on the binding of PvRII to recombinant Duffy antigen receptor for chemokines (DARC) in an in vitro binding assay. The results of the present study provide a rationale for a combination vaccine against P. vivax malaria based on PvMSP1(19) and PvRII.
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Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/inmunología , Plasmodium vivax/inmunología , Proteínas Protozoarias/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antiprotozoarios/sangre , Formación de Anticuerpos/inmunología , Antígenos de Protozoos/genética , Western Blotting , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática , Inmunización/métodos , Inmunoglobulina G/sangre , Vacunas contra la Malaria/administración & dosificación , Malaria Vivax/inmunología , Malaria Vivax/prevención & control , Masculino , Proteína 1 de Superficie de Merozoito/genética , Proteína 1 de Superficie de Merozoito/inmunología , Ratones , Ratones Endogámicos BALB C , Plasmodium vivax/genética , Proteínas Protozoarias/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Proteínas Recombinantes/inmunología , Factores de TiempoRESUMEN
A recombinant blood-stage vaccine for Plasmodium vivax malaria based on the functional receptor-binding domain of PvDBP (PvRII) has been developed. A synthetic gene coding for PvRII was expressed in Escherichia coli using codon optimization. Expression level of recombinant PvRII was 10% of the total cellular proteins. Truncated PvRII products, seen when the native PvRII gene was expressed, were absent in case of synthetic gene.
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Antígenos de Protozoos/análisis , Antígenos de Protozoos/metabolismo , Mejoramiento Genético/métodos , Vacunas contra la Malaria/análisis , Vacunas contra la Malaria/metabolismo , Plasmodium vivax/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Protozoarias/análisis , Proteínas Protozoarias/metabolismo , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/metabolismo , Animales , Antígenos de Protozoos/genética , Codón/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Vacunas contra la Malaria/genética , Plasmodium vivax/genética , Estructura Terciaria de Proteína , Proteínas Protozoarias/genética , Receptores de Superficie Celular/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección/métodosRESUMEN
Summary Plasmodium vivax depends on interaction with the Duffy antigen/receptor for chemokines (DARC) for invasion of human erythrocytes. The 140 kDa P. vivax Duffy-binding protein (PvDBP) mediates interaction with DARC. The receptor-binding domain of PvDBP maps to its N-terminal, cysteine-rich region, region II (PvRII), which contains approximately 300 amino acid residues including 12 conserved cysteines. Using surface plasmon resonance, we show that binding of PvRII to DARC is a high-affinity interaction with a binding constant (K(D)) of 8.7 nM. The minimal binding domain of PvRII has been previously mapped to a central 170-amino-acid stretch that includes cysteines 5-8. Here, we have used site-directed mutagenesis and quantitative binding assays to map amino acid residues within PvRII that make contact with DARC. Of the seven alanine replacement mutations that had an effect on binding, five were mutations in hydrophobic residues suggesting that hydrophobic interactions play a major role in the interaction of PvDBP with DARC. Genetic diversity studies have shown that six of the seven binding residues identified in PvRII are conserved in P. vivax field isolates, which provides support for their role in interaction with DARC.