RESUMEN
Neonatal infections are a major cause of newborn mortality in low- and middle-income countries, particularly in areas without access to inpatient care. To address this, the World Health Organization developed guidelines for delivering simplified antibiotic regimens (oral amoxicillin and intramuscular gentamicin) in outpatient settings to young infants with suspected serious bacterial infection when referral is not feasible. However, there are still limitations to access, as the regimen requires a health care provider trained in giving intramuscular injections to infants. To provide a needle-free, simplified alternate to intramuscular delivery, PATH investigated the feasibility of the rectal administration of gentamicin. Potential formulations were screened by in vitro testing, and 2 liquid enema formulations and a cocoa butter suppository were developed and evaluated in a preclinical study of the rectal uptake of gentamicin in a neonatal minipig model. Sera samples from the control group, dosed by intramuscular injection, resulted in expected sera concentrations of gentamicin, but no gentamicin was detected in the sera of groups rectally dosed with the test formulations. The results of this study did not provide evidence to support the therapeutic feasibility of rectally absorbed gentamicin.
Asunto(s)
Amoxicilina , Gentamicinas , Administración Rectal , Animales , Antibacterianos/uso terapéutico , Estudios de Factibilidad , Humanos , Lactante , Inyecciones Intramusculares , Porcinos , Porcinos EnanosRESUMEN
BACKGROUND: Transmission-blocking vaccines (TBVs) have become a focus of strategies to control and eventually eliminate malaria as they target the entry of sexual stage into the Anopheles stephensi mosquito thereby preventing transmission, an essential component of the parasite life cycle. Such vaccines are envisioned as complements to vaccines that target human infection, such as RTS,S as well as drug treatment, and vector control strategies. A number of conserved proteins, including Pfs25, have been identified as promising TBV targets in research or early stage development. Pfs25 is a 25 kDa protein of Plasmodium falciparum expressed on the surface of zygotes and ookinetes. Its complex tertiary structure, including numerous cysteines, has led to difficulties in the expression of a recombinant protein that is homogeneous, with proper conformation, and free of glycosylation, a phenomenon not found in native parasite machinery. METHODS: While the expression and purification of Pfs25 in various systems, has been previously independently reported, here a parallel analysis of Pfs25 is presented to inform on the biochemical features of Pfs25 and their impact on functionality. Three scalable expression systems were used to express, purify, and evaluate Pfs25 both in vitro and in vivo, including the ability of each protein to produce functional antibodies through the standard membrane feeding assay. RESULTS: Through numerous attempts, soluble, monomeric Pfs25 derived from Escherichia coli was not achieved, while Pichia pastoris presented Pfs25 as an inhomogeneous product with glycosylation. In comparison, baculovirus produced a pure, monomeric protein free of glycosylation. The glycosylation present for Pichia produced Pfs25, showed no notable decrease in the ability to elicit transmission reducing antibodies in functional evaluation, while a reduced and alkylated Pfs25 (derived from plant and used as a control) was found to have significantly decreased transmission reducing activity, emphasizing the importance of ensuring correct disulfide stabilized conformation during vaccine design and production. CONCLUSIONS: In this study, the biochemical features of Pfs25, produced from different expression systems, are described along with their impact on the ability of the protein to elicit functional antibodies. Pfs25 expressed using baculovirus and Pichia showed promise as candidates for vaccine development.
Asunto(s)
Transmisión de Enfermedad Infecciosa/prevención & control , Vacunas contra la Malaria/inmunología , Malaria/prevención & control , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Baculoviridae/genética , Baculoviridae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/genética , Vacunas contra la Malaria/aislamiento & purificación , Ratones , Pichia/genética , Pichia/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/aislamiento & purificaciónRESUMEN
Carbon dioxide Assisted Nebulization with a Bubble Dryer((R)) (CAN-BD) processing allows particles to be made in the 3-5 mum size range, which is desirable for lung delivery, without destroying biological activity. In response to the Grand Challenge in Global Health Initiative #3, we have been developing an inhalable needle-free live-attenuated measles virus vaccine for use in developing countries. Measles was chosen because it is the number one vaccine preventable killer of children worldwide. Powders were processed by CAN-BD, where a solution containing excipients and live-attenuated measles virus in water was mixed intimately with supercritical or near superctitical carbon dioxide to form an emulsion. The emulsion was expanded to atmospheric pressure through a flow restrictor. The resulting plume was dried by heated nitrogen and the powders collected on a filter at the bottom of the drying chamber. Powders were analyzed using varying techniques including X-ray diffraction, scanning electron microscopy, Andersen cascade impaction, differential scanning calorimetery, Karl Fischer titration, and viral plaque assay. CAN-BD has been used to produce powders of live-attenuated measles virus vaccine with characteristics desirable for lung delivery. The powders retain viral activity through forming and drying the microparticles by CAN-BD, and have passed the WHO stability test for 1 week at 37 degrees C. The powders have an amorphous character and a glass transition temperature of around 60 degrees C. Lyophilization, the present standard commercial method of processing measles vaccine makes solids with a water content of less than 1%. By substituting myo-inositol for sorbitol and using the CAN-BD drying technique the water content can be lowered to 0.5%. The most successful formulations to date have been based conceptually on the current lyophilized formulation, but with modifications to the type and amounts of sugar. Of current interest are formulations containing myo-inositol, as they retain high viral activity and have low initial water content.