RESUMEN
Malaria transmission blocking vaccines (TBV) directed against proteins expressed on sexual stages of Plasmodium falciparum in the mosquito midgut are considered an effective means to reduce malaria transmission. Antibodies induced by TBV block sporogonic development in the mosquito, and thus transmission to the next human host. The Pfs25 protein, expressed on the surface of gametes, zygotes and ookinetes, is one of the primary targets for TBV development. Using a plant virus-based transient expression system, we have successfully produced Pfs25 fused to a modified lichenase (LicKM) carrier in Nicotiana benthamiana, purified and characterized the protein (Pfs25-FhCMB), and evaluated this vaccine candidate in animal models for the induction of transmission blocking antibodies. Soluble Pfs25-FhCMB was expressed in plants at a high level, and induced transmission blocking antibodies that persisted for up to 6 months post immunization in mice and rabbits. These data demonstrate the potential of the new malaria vaccine candidate and also support feasibility of expressing Plasmodium antigens in a plant-based system.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Transmisión de Enfermedad Infecciosa/prevención & control , Vacunas contra la Malaria/inmunología , Malaria/prevención & control , Proteínas Protozoarias/inmunología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Femenino , Expresión Génica , Vectores Genéticos , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/genética , Ratones Endogámicos BALB C , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/inmunología , Potyvirus/genética , Proteínas Protozoarias/genética , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Factores de Tiempo , Nicotiana/genética , Nicotiana/metabolismo , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Platelet microaggregates have been demonstrated in the systemic circulation of patients with atherosclerotic cardiovascular diseases. Glycoprotein IIb/IIIa antagonists have been reported to play roles in platelet activation, which may be associated with pro-thrombotic events. We report the effect of the orally active GPIIb/IIIa antagonist, orbofiban, on human platelet microaggregate formation in vitro. Laser light scatter (LSS) technology was used to monitor small, medium and large platelet aggregates formed in platelet-rich plasma in response to ADP or thrombin receptor activating peptide (TRAP)-6. ADP at 0.5 microM induced only small platelet aggregates that were prevented by orbofiban in a concentration-dependent manner. Likewise, orbofiban dissolved small aggregates after their formation. In marked contrast, in the presence of strong agonist stimulation (20 microM ADP or 3 microM TRAP-6) which generated primarily large platelet aggregates, orbofiban blocked the large aggregates while significantly augmenting small aggregates by three- to sixfold. The data suggest that GPIIb/IIIa antagonists do not induce platelet microaggregation directly but may augment small platelet microaggregate formation indirectly at conditions of strong stimuli. The percentage of the microaggregate population expressing P-selectin remained the same in the presence of orbofiban, indicating that these small microaggregates remain activated, although the mean intensity of expression was diminished, possibly reflecting the reduced size of the particles or density of P-selectin molecules. In summary, an increase in small platelet microaggregates might have contributed to pro-thrombotic events in orbofiban-treated patients.
Asunto(s)
Alanina/farmacología , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Pirrolidinas/farmacología , Adenosina Difosfato/farmacología , Plaquetas/efectos de los fármacos , Humanos , Técnicas In Vitro , Selectina-P/farmacología , Fragmentos de Péptidos/farmacología , Factores de TiempoRESUMEN
Malaria transmission blocking vaccines (TBVs) are considered an effective means to control and eventually eliminate malaria. The Pfs25 protein, expressed predominantly on the surface of the sexual and sporogonic stages of Plasmodium falciparum including gametes, zygotes and ookinetes, is one of the primary targets for TBV. It has been demonstrated that plants are an effective, highly scalable system for the production of recombinant proteins, including virus-like particles (VLPs). We engineered VLPs (Pfs25-CP VLP) comprising Pfs25 fused to the Alfalfa mosaic virus coat protein (CP) and produced these non-enveloped hybrid VLPs in Nicotiana benthamiana plants using a Tobacco mosaic virus-based 'launch' vector. Purified Pfs25-CP VLPs were highly consistent in size (19.3±2.4 nm in diameter) with an estimated 20-30% incorporation of Pfs25 onto the VLP surface. Immunization of mice with one or two doses of Pfs25-CP VLPs plus Alhydrogel® induced serum antibodies with complete transmission blocking activity through the 6 month study period. These results support the evaluation of Pfs25-CP VLP as a potential TBV candidate and the feasibility of the 'launch' vector technology for the production of VLP-based recombinant vaccines against infectious diseases.