RESUMEN
Molecular dispersions are a highly effective method of increasing bioavailability for a poorly soluble active pharmaceutical ingredient (API) and can be prepared on a large scale by hot melt extrusion (HME). Processing thermally labile active pharmaceutical ingredients (APIs) via HME is generally more difficult, with operating temperatures limited to below that of the API melting point. API melting is considered essential to facilitate the formation of a fully homogeneous amorphous system. Processing below the melting point renders the system much more susceptible to residual crystalline content; hence, HME is not suitable for APIs which degrade upon melting. In the following work, meloxicam (MEL) was used as a model API, possessing properties of high melting temperature and thermal lability. In this proof of concept work, a modified HME method, termed solvent-assisted HME, was used to overcome this issue and prepare an amorphous solid dispersion using HME, wherein a solvent was incorporated in the formulation blend during extrusion and removed post-processing. Formulations containing 10%wt meloxicam (MEL) and 90%wt polyvinylpyrrolidone vinyl acetate (PVPVA) copolymer were extruded using a twin-screw extruder at temperatures below the melting point of MEL. Dimethylformamide (DMF) solvent was added directly into the extruder barrel through a liquid addition port, resulting in extrudate products having a higher conversion of API to the amorphous form. The incorporation of solvent allowed a significant reduction in processing temperatures due to its increased mobility, while also driving the conversion of the API to its amorphous form. The solvent was successfully reduced through a secondary drying step using a vacuum oven. This advancement has demonstrated the potential for thermally labile APIs to be processed via HME expanding the applications of this technology.
Asunto(s)
Química Farmacéutica , Tecnología de Extrusión de Fusión en Caliente , Composición de Medicamentos , Calor , Povidona , Solubilidad , SolventesRESUMEN
Currently in the pharmaceutical industry, continuous manufacturing is an area of significant interest. In particular, hot-melt extrusion (HME) offers many advantages and has been shown to significantly reduce the number of processing steps relative to a conventional product manufacturing line. To control product quality during HME without process interruption, integration of inline analytical technology is critical. Vibrational spectroscopy (Raman, NIR and FT-IR) is often employed and used for real-time measurements because of the non-destructive and rapid nature of these analytical techniques. However, the establishment of reliable Process Analytical Technology (PAT) tools for HME of thermolabile drugs is challenging. Indeed, the Raman effect is inherently weak and might be subject to interference. Moreover, during HME, heating and photodecomposition can occur and disrupt spectra acquisition. The aim of this research article was to explore the use of inline Raman spectroscopy to characterise a thermolabile drug, ramipril (RMP), during continuous HME processing. Offline measurements by HPLC, LC-MS and Raman spectroscopy were used to characterise RMP and its main degradation product, ramipril-diketopiperazine (RMP-DKP, impurity K). A set of HME experiments together with inline Raman spectroscopic analyses were performed. The feasibility of implementing inline Raman spectroscopic analysis to quantify the level of RMP and RMP-DKP in the extrudate was addressed. Two regions in the Raman spectrum were selected to differentiate RMP and RMP-DKP. When regions were combined, a principle component analysis (PCA) model defined by these two main components (PC 1â¯=â¯50.1% and PC 2â¯=â¯45%) was established. Using HPLC analyses, we were able to confirm that the PC 1 score was attributed to the level of RMP-DKP, and the PC 2 score was related to the RMP drug content. Investigation of the PCA scatterplot indicated that HME processing temperature was not the only factor causing RMP degradation. Additionally, the plasticiser content, feeding speed and screw rotating speed contributed to RMP degradation during HME processing.
Asunto(s)
Tecnología de Extrusión de Fusión en Caliente , Control de Calidad , Espectrometría Raman/métodos , Cromatografía Líquida de Alta Presión , Citratos/química , Combinación de Medicamentos , Plastificantes/química , Ácidos Polimetacrílicos/química , Ramipril/químicaRESUMEN
Hyperlipidaemia is considered as one of the main risk factors associated with cardiovascular diseases (CVDs). Among different lipid-lowering agents used to manage hyperlipidaemia, statins are highly prescribed for management of hyperlipidaemia with simvastatin being one of the most common. Simvastatin is susceptible to extensive metabolism by CYP450 3A4 and 3A5, which are expressed both in the liver and the gastrointestinal tract. Nevertheless, the localization of these enzymes is site-dependent with lower concentration at the distal/proximal regions of the small intestine/colon. In addition to statins, medications such as antihypertensive agents and anticoagulants are introduced as adjuvants, for the treatment of cardiovascular disease. The aim of this study was to design a bilayer delivery system capable of delivering biphasic release of simvastatin and aspirin, within a fixed dose combination. A delayed release platform based on a combination of anionic polymers prepared using hot-melt extrusion was developed to delay the release of simvastatin. An optimized formulation tested for dissolution performance clearly demonstrated an ability to delay the release of simvastatin. In addition, an immediate release layer based on Kollidon VA64 was successfully developed to deliver aspirin. Both formulations were then manufactured as a bilayer drug delivery system (tablets and coextrudates), and the release performance was examined. On the basis of the obtained results, these formulations may be used as a platform for delivering a wide range of medications in a biphasic manner.
Asunto(s)
Enfermedades Cardiovasculares/complicaciones , Enfermedades Cardiovasculares/tratamiento farmacológico , Composición de Medicamentos/métodos , Sistemas de Liberación de Medicamentos/métodos , Diseño de Fármacos , Hiperlipidemias/complicaciones , Hiperlipidemias/tratamiento farmacológico , Aspirina/administración & dosificación , Aspirina/química , Formas de Dosificación , Combinación de Medicamentos , Liberación de Fármacos , Tecnología de Extrusión de Fusión en Caliente/métodos , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Inhibidores de Hidroximetilglutaril-CoA Reductasas/química , Polietilenglicoles/química , Polivinilos/química , Pirrolidinas/química , Simvastatina/administración & dosificación , Simvastatina/química , Solubilidad , Compuestos de Vinilo/químicaRESUMEN
The purpose of this work was to investigate the application of different advanced continuous processing techniques (hot melt extrusion and spray drying) to the production of fixed-dose combination (FDC) monolithic systems comprising of hydrochlorothiazide and ramipril for the treatment of hypertension. Identical FDC formulations were manufactured by the two different methods and were characterised using powder X-ray diffraction (PXRD) and modulated differential scanning calorimetry (mDSC). Drug dissolution rates were investigated using a Wood's apparatus, while physical stability was assessed on storage under controlled temperature and humidity conditions. Interestingly both drugs were transformed into their amorphous forms when spray dried, however, hydrochlorothiazide was determined, by PXRD, to be partially crystalline when hot melt extruded with either polymer carrier (Kollidon® VA 64 or Soluplus®). Hot melt extrusion was found to result in significant degradation of ramipril, however, this could be mitigated by the inclusion of the plasticizer, polyethylene glycol 3350, in the formulation and appropriate adjustment of processing temperature. The results of intrinsic dissolution rate studies showed that hot-melt extruded samples were found to release both drugs faster than identical formulations produced via spray drying. However, the differences were attributable to the surface roughness of the compressed discs in the Wood's apparatus, rather than solid state differences between samples. After a 60-day stability study spray dried samples exhibited a greater physical stability than the equivalent hot melt extruded samples.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/química , Antihipertensivos/química , Diuréticos/química , Calor , Hidroclorotiazida/química , Ramipril/química , Tecnología Farmacéutica/métodos , Rastreo Diferencial de Calorimetría , Cristalografía por Rayos X , Portadores de Fármacos , Combinación de Medicamentos , Composición de Medicamentos , Liberación de Fármacos , Estabilidad de Medicamentos , Cinética , Microscopía Electrónica de Rastreo , Tamaño de la Partícula , Plastificantes/química , Polietilenglicoles/química , Polivinilos/química , Difracción de Polvo , Pirrolidinas/química , Solubilidad , Propiedades de Superficie , Compuestos de Vinilo/químicaRESUMEN
The objective of this work was to investigate the feasibility of using a novel granulation technique, namely, fluidized hot melt granulation (FHMG), to prepare gastroretentive extended-release floating granules. In this study we have utilized FHMG, a solvent free process in which granulation is achieved with the aid of low melting point materials, using Compritol 888 ATO and Gelucire 50/13 as meltable binders, in place of conventional liquid binders. The physicochemical properties, morphology, floating properties, and drug release of the manufactured granules were investigated. Granules prepared by this method were spherical in shape and showed good flowability. The floating granules exhibited sustained release exceeding 10 h. Granule buoyancy (floating time and strength) and drug release properties were significantly influenced by formulation variables such as excipient type and concentration, and the physical characteristics (particle size, hydrophilicity) of the excipients. Drug release rate was increased by increasing the concentration of hydroxypropyl cellulose (HPC) and Gelucire 50/13, or by decreasing the particle size of HPC. Floating strength was improved through the incorporation of sodium bicarbonate and citric acid. Furthermore, floating strength was influenced by the concentration of HPC within the formulation. Granules prepared in this way show good physical characteristics, floating ability, and drug release properties when placed in simulated gastric fluid. Moreover, the drug release and floating properties can be controlled by modification of the ratio or physical characteristics of the excipients used in the formulation.
Asunto(s)
Excipientes/química , Rastreo Diferencial de Calorimetría , Celulosa/análogos & derivados , Celulosa/química , Química Farmacéutica , Composición de Medicamentos , Grasas/química , Ácidos Grasos/química , Metronidazol/química , Modelos Teóricos , Aceites/química , Bicarbonato de Sodio/química , TermogravimetríaRESUMEN
Sustained-release matrix tablets based on Eudragit RL and RS were manufactured by injection moulding. The influence of process temperature; matrix composition; drug load, plasticizer level; and salt form of metoprolol: tartrate (MPT), fumarate (MPF) and succinate (MPS) on ease of processing and drug release were evaluated. Formulations composed of 70/30% Eudragit RL/MPT showed the fastest drug release, substituting part of Eudragit RL by RS resulted in slower drug release, all following first-order release kinetics. Drug load only affected drug release of matrices composed of Eudragit RS: a higher MPT concentration yielded faster release rates. Adding triethyl citrate enhanced the processability, but was detrimental to long-term stability. The process temperature and plasticizer level had no effect on drug release, whereas metoprolol salt form significantly influenced release properties. The moulded tablets had a low porosity and a smooth surface morphology. A plasticizing effect of MPT, MPS and MPF on Eudragit RS and Eudragit RL was observed via DSC and DMA. Solubility parameter assessment, thermal analysis and X-ray diffraction demonstrated the formation of a solid solution immediately after production, in which H-bonds were formed between metoprolol and Eudragit as evidenced by near-infrared spectroscopy. However, high drug loadings of MPS and MPF showed a tendency to recrystallise during storage. The in vivo performance of injection-moulded tablets was strongly dependent upon drug loading.
Asunto(s)
Preparaciones de Acción Retardada/química , Portadores de Fármacos/química , Metoprolol/química , Comprimidos/química , Resinas Acrílicas/química , Disponibilidad Biológica , Química Farmacéutica/métodos , Citratos/química , Composición de Medicamentos/métodos , Estabilidad de Medicamentos , Cinética , Plastificantes/química , Polímeros/química , Porosidad , Solubilidad , TemperaturaRESUMEN
Although Yersinia pestis is classified as a "high-virulence" pathogen, some host species are variably susceptible to disease. Coyotes (Canis latrans) exhibit mild, if any, symptoms during infection, but antibody production occurs postinfection. This immune response has been reported to be against the F1 capsule, although little subsequent characterization has been conducted. To further define the nature of coyote humoral immunity to plague, qualitative serology was conducted to assess the antiplague antibody repertoire. Humoral responses to six plasmid-encoded Y. pestis virulence factors were first examined. Of 20 individual immune coyotes, 90% were reactive to at least one other antigen in the panel other than F1. The frequency of reactivity to low calcium response plasmid (pLcr)-encoded Yersinia protein kinase A (YpkA) and Yersinia outer protein D (YopD) was significantly greater than that previously observed in a murine model for plague. Additionally, both V antigen and plasminogen activator were reactive with over half of the serum samples tested. Reactivity to F1 was markedly less frequent in coyotes (35%). Twenty previously tested antibody-negative samples were also examined. While the majority were negative across the panel, 15% were positive for 1-3 non-F1 antigens. In vivo-induced antigen technology employed to identify novel chromosomal genes of Y. pestis that are up-regulated during infection resulted in the identification of five proteins, including a flagellar component (FliP) that was uniquely reactive with the coyote serum compared with immune serum from two other host species. Collectively, these data suggest that humoral immunity to pLcr-encoded antigens and the pesticin plasmid (pPst)-encoded Pla antigen may be relevant to plague resistance in coyotes. The serologic profile of Y. pestis chromosomal antigens up-regulated in vivo specific to C. latrans may provide insight into the differences in the pathogen-host responses during Y. pestis infection.
Asunto(s)
Antígenos Bacterianos/inmunología , Coyotes , Inmunidad Humoral , Peste/veterinaria , Yersinia pestis/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/inmunología , Coyotes/inmunología , Coyotes/microbiología , Femenino , Masculino , Peste/sangre , Peste/diagnóstico , Peste/inmunología , Wyoming , Yersinia pestis/patogenicidadRESUMEN
Elk in the Greater Yellowstone Area are a major reservoir for brucellosis, which represents an obstacle to eradication of the disease in domestic livestock. Furthermore, immune responses to Brucella abortus infection in the wild host are not well-understood. In this regard, in vivo-induced antigen technology (IVIAT) was employed to identify novel B. abortus antigens expressed during infection in elk. Sera collected from sero-positive Wyoming elk were pooled and absorbed against in vitro-grown cultures of B. abortus. Approximately 35,000 E. coli clones, expressing B. abortus DNA, were then screened by colony immunoblot, yielding ten genes with immuno-reactive products, to include seven proteins secreted beyond the inner membrane. Three products, an outer membrane protein (D15), malate dehydrogenase (Mdh), and an ion transporter (AfuA), were examined by Western blot against individual elk serum samples. Sero-reactivity was significantly more frequent for both Mdh and D15 in naturally infected animals, compared to vaccinated and uninfected elk, indicating that antibody to these two antigens is a predictor of natural infection. Cross-reactivity of all three proteins was next examined with serum samples from confirmed brucellosis-positive cattle. While variable patterns of reactivity were seen with the antigens, the sample group was equivalently reactive to AfuA and Mdh, compared to elk, suggesting that these antigens are commonly expressed during infection in both hosts. We conclude that the application of IVIAT to B. abortus may not only facilitate the identification of serologic markers for brucellosis in elk, but may provide further insight into biological processes of the pathogen in different hosts.
Asunto(s)
Brucella abortus/genética , Brucelosis/veterinaria , Ciervos/microbiología , Genes Bacterianos/genética , Técnicas Inmunológicas/veterinaria , Animales , Antígenos Bacterianos/inmunología , Biomarcadores/sangre , Western Blotting , Brucella abortus/inmunología , Brucelosis/inmunología , Brucelosis/microbiología , Reacciones Cruzadas , Ciervos/inmunología , WyomingRESUMEN
The serological response and efficacy of Bacillus anthracis recombinant protective antigen (rPA) vaccines formulated with aluminum hydroxide adjuvant, either with or without formaldehyde, were evaluated in rabbits. Rabbits that had been injected with a single dose of 25 microg of rPA adsorbed to 500 microg of aluminum in aluminum hydroxide gel (Alhydrogel) had a significantly higher quantitative anti-rPA IgG ELISA titers (p<0.0001) and toxin neutralizing antibody (TNA) assay titers (p<0.0001) than rabbits tested at the next lowest concentration of aluminum (158 microg). Rabbits injected with two doses of 50 microg of rPA formulated with 500 microg of aluminum also had significantly higher serological responses, as measured by a quantitative anti-rPA IgG ELISA (p<0.0001) and TNA assay (p<0.0001), than sera from rabbits injected with a rPA vaccine formulated without adjuvant. Short-term protection against an aerosol spore challenge (448 LD(50)), however, was not significantly different between the two groups (12/12 and 11/12, respectively). Rabbits injected with a single dose of 50 microg of rPA formulated with 500 microg of aluminum and 0.2% formaldehyde had significantly higher ELISA (p<0.0001) and TNA assay (p<0.0001) titers than rabbits that had been injected with a rPA vaccine formulated with adjuvant but without formaldehyde. Short-term protection against a 125 LD(50) parenteral spore challenge, however, was not significantly different between the two groups (14/24 and 9/24, respectively; p=0.2476). Under the conditions tested in the rabbit animal model, significantly higher serological responses were observed in rabbits that had been injected with rPA formulated with aluminum hydroxide gel adjuvant and formaldehyde. However, differences in short-term efficacy were not observed.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Hidróxido de Aluminio/farmacología , Vacunas contra el Carbunco/química , Vacunas contra el Carbunco/inmunología , Formaldehído/farmacología , Adyuvantes Inmunológicos/química , Hidróxido de Aluminio/química , Hidróxido de Aluminio/inmunología , Animales , Carbunco/inmunología , Carbunco/prevención & control , Vacunas contra el Carbunco/farmacología , Vacunas contra el Carbunco/uso terapéutico , Ensayo de Inmunoadsorción Enzimática , Femenino , Formaldehído/química , Formaldehído/inmunología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Conejos , Vacunas Sintéticas/química , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
The next-generation human anthrax vaccine developed by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) is based upon purified Bacillus anthracis recombinant protective antigen (rPA) adsorbed to aluminum hydroxide adjuvant (Alhydrogel). In addition to being safe, and effective, it is important that such a vaccine be fully characterized. Four major protein isoforms detected in purified rPA by native PAGE during research and development were reduced to two primary isoforms in bulk material produced by an improved process performed under Good Manufacturing Practices (GMP). Analysis of both rPA preparations by a protein-isoaspartyl-methyl-transferase assay (PIMT) revealed the presence of increasing amounts of iso-aspartic acid correlating with isoform content and suggesting deamidation as the source of rPA charge heterogeneity. Additional purification of GMP rPA by anion exchange chromatography separated and enriched the two principal isoforms. The in vitro and in vivo biological activities of each isoform were measured in comparison to the whole GMP preparation. There was no significant difference in the biological activity of each isoform compared to GMP rPA when analyzed in the presence of lethal factor using a macrophage lysis assay. Vaccination with the two individual isoforms revealed no differences in cytotoxicity neutralization antibody titers when compared to the GMP preparation although one isoform induced more anti-PA IgG antibody than the GMP material. Most importantly, each of the two isoforms as well as the whole GMP preparation protected 90-100% of rabbits challenged parenterally with 129 LD50 of B. anthracis Ames spores. The equivalent biological activity and vaccine efficacy of the two isoforms suggests that further processing to separate isoforms is unnecessary for continued testing of this next-generation anthrax vaccine.
Asunto(s)
Vacunas contra el Carbunco/inmunología , Carbunco/prevención & control , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Vacunas Sintéticas/inmunología , Animales , Antígenos Bacterianos/análisis , Antígenos Bacterianos/aislamiento & purificación , Toxinas Bacterianas/análisis , Toxinas Bacterianas/aislamiento & purificación , Isoformas de Proteínas , Conejos , Proteínas Recombinantes/inmunología , Esporas BacterianasRESUMEN
Long-term protection of rabbits that had been vaccinated with two doses of a recombinant protective antigen (rPA) vaccine was examined against an aerosol spore challenge with the Ames isolate of Bacillus anthracis at 6 and 12 months. At 6 months after the primary injection, survival was 74.1% (20/27) with quantitative ELISA titer of 22.3 microg of anti-rPA IgG per millilitre and toxin neutralizing antibody (TNA) assay titer of 332. At 12 months after the primary injection, only 37.5% (9/24) of the rabbits were protected with quantitative ELISA titer of 19.8 microg of anti-rPA IgG per millilitre and TNA assay titer of 286. There was a significant loss of protection (p = 0.0117) and a significant difference in survival curves (p = 0.0157) between the 6- and 12-month groups. When ELISA or TNA assay titer, gender, and challenge dose were entered into a forward logistic regression model, week 26 ELISA titer (p = 0.0236) and week 13 TNA assay titer (p = 0.0147) for the 6-month group, and week 26 ELISA titer (p = 0.0326) and week 8 TNA assay titer (p = 0.0190) for the 12-month group, were significant predictors of survival. Neither gender nor challenge dose were identified as having a statistically significant effect on survival. Booster vaccinations with rPA may be required for the long-term protection of rabbits against anthrax.
Asunto(s)
Vacunas contra el Carbunco/administración & dosificación , Carbunco/prevención & control , Anticuerpos Antibacterianos/biosíntesis , Bacillus anthracis/química , Animales , Carbunco/inmunología , Vacunas contra el Carbunco/inmunología , Anticuerpos Antibacterianos/metabolismo , Antígenos Bacterianos/inmunología , Bacillus anthracis/inmunología , Ensayo de Inmunoadsorción Enzimática , Conejos , Factores de Tiempo , Vacunación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
A recombinant protective antigen (rPA)-based enzyme-linked immunosorbent assay (ELISA) was developed to measure the serological response of female A/J mice after inoculation with the new rPA-based anthrax vaccine. Several fundamental parameters of the ELISA were evaluated: specificity, precision, accuracy, linearity, and stability. Experimental results suggested that the quantitative anti-rPA IgG ELISA could be used to measure antibody levels in female A/J mice and may be useful as a potency assay to monitor consistency of manufacture of a rPA-based vaccine for planned clinical trials.
Asunto(s)
Vacunas contra el Carbunco/farmacología , Ensayo de Inmunoadsorción Enzimática/métodos , Animales , Anticuerpos Monoclonales/química , Formación de Anticuerpos , Unión Competitiva , Ensayos Clínicos como Asunto , Relación Dosis-Respuesta a Droga , Estudios de Evaluación como Asunto , Femenino , Inmunoglobulina G/química , Modelos Lineales , Modelos Logísticos , Ratones , Unión Proteica , Sensibilidad y EspecificidadRESUMEN
The potency assay currently used to evaluate consistency of manufacture for the anthrax vaccine is contingent upon meeting specified parameters after statistical analysis of the percent survival and time to death of vaccinated guinea pigs after challenge with spores of a virulent strain of Bacillus anthracis. During the development of a new anthrax vaccine based upon recombinant protective antigen (rPA) adsorbed to aluminum hydroxide gel (Alhydrogel), we found that the serological response of female A/J mice, as measured by a quantitative anti-rPA IgG ELISA, may be an effective method to monitor a manufacturer's consistency for rPA-based vaccines. An advantage of the proposed in vitro-based potency assay is that it will not need stringent biosafety containment measures as required by the current guinea pig potency assay.
Asunto(s)
Vacunas contra el Carbunco/inmunología , Animales , Vacunas contra el Carbunco/administración & dosificación , Química Farmacéutica , Relación Dosis-Respuesta Inmunológica , Ensayo de Inmunoadsorción Enzimática , Femenino , Hidrogeles , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos A , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Pruebas de Neutralización , Estándares de Referencia , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
The F1 capsule of Yersinia pestis, encoded by the 100 kb plasmid pFra, is often assumed to be essential for full virulence of Y. pestis. However, virulent strains of Y. pestis that are F1- and either pFra+ or pFra- have been reported. To assess the role of pFra-encoded factors in virulence, mutants in pFra with insertions of the defective transposing bacteriophage Mu dl(Ap lac) were obtained, by using the wild type (wt) and the pLcr-cured derivative of strain C092. Mutants that exhibited temperature regulation of lactose fermentation and retarded electrophoretic mobility of pFra were selected. A total of 15 insertion mutants were isolated in the wt strain (12 of which had a single insertion in the genome, in pFra); and 24 mutants in the isogenic pLcr- derivative. Four of the pLcr+ mutants, and none of the pLcr- mutants, were F1-. All F1- mutants were decreased in virulence for mice compared to the wt parent; and five of the F1+ mutants also were significantly attenuated in mice. Fusion end-joints of insert DNA were cloned into Escherichia coli by using pMLB524, a vector for rescuing operon fusions of lacZ. Recombinants were obtained which contained pFra inserts ranging from < 2kb to approximately 36 kb, and the insertions occurred at several sites on pFra. All of the four F1- mutants tested mapped within the F1 capsule operon (caf1). The remaining five attenuated mutants sequenced were F1+ and mapped outside of but near the operon. Sequencing and complete analysis of the pFra insertions mutants could facilitate identification of new potential virulence factors.
Asunto(s)
Plásmidos , Temperatura , Yersinia pestis/genética , Animales , Cápsulas Bacterianas/genética , Humanos , Ratones , Mutación , Virulencia/genética , Yersinia pestis/patogenicidadRESUMEN
In these studies, a serological correlate of protection against anthrax was identified in New Zealand white (NZW) rabbits that had been given one or two injections of various amounts of recombinant protective antigen (rPA) combined with aluminum hydroxide adjuvant (Alhydrogel). Rabbits were subsequently challenged by the aerosol route with spores of the Ames isolate of Bacillus anthracis. Results suggested that the antibody response, as determined by the quantitative anti-rPA IgG ELISA and toxin neutralizing antibody (TNA) assay, were significant predictors (P<0.0015) of protection against a B. anthracis aerosol spore challenge in rabbits.
Asunto(s)
Vacunas contra el Carbunco/inmunología , Carbunco/prevención & control , Adyuvantes Inmunológicos , Aerosoles , Hidróxido de Aluminio , Animales , Carbunco/inmunología , Vacunas contra el Carbunco/administración & dosificación , Bacillus anthracis/inmunología , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina G/biosíntesis , Cinética , Pruebas de Neutralización , Conejos , Esporas Bacterianas/inmunología , Análisis de Supervivencia , Vacunación , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
A whole-body mouse model of pneumonic melioidosis was established for future evaluation of biodefense vaccine candidates. The aerosol 50% lethal doses of Burkholderia pseudomallei strain 1026b for BALB/c and C57BL/6 mice and the times to death, dissemination in organs, and tissue loads after exposure of the mice to low- and high-dose aerosols are reported. In addition, rpsL mutant backgrounds were attenuated in this acute model of disease.
Asunto(s)
Bioterrorismo/prevención & control , Burkholderia pseudomallei/patogenicidad , Modelos Animales de Enfermedad , Melioidosis/patología , Neumonía Bacteriana/patología , Animales , Humanos , Melioidosis/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neumonía Bacteriana/microbiología , Proteínas Ribosómicas/genética , VirulenciaRESUMEN
There is limited information concerning the nature and extent of the immune response to the virulence determinants of Yersinia pestis during the course of plague infection. In this study, we evaluated the humoral immune response of mice that survived lethal Y. pestis aerosol challenge after antibiotic treatment. Such a model may replicate the clinical situation in humans and indicate which virulence determinants are expressed in vivo. Immunoglobulin G enzyme-linked immunosorbent assay and immunoblotting were performed by using purified, recombinant antigens including F1, V antigen, YpkA, YopH, YopM, YopB, YopD, YopN, YopE, YopK, plasminogen activator protease (Pla), and pH 6 antigen as well as purified lipopolysaccharide. The major antigens recognized by murine convalescent sera were F1, V antigen, YopH, YopM, YopD, and Pla. Early treatment with antibiotics tended to reduce the immune response and differences between antibiotic treatment regimens were noted. These results may indicate that only some virulence factors are expressed and/or immunogenic during infection. This information may prove useful for selecting potential vaccine candidates and for developing improved serologic diagnostic assays.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Peste/inmunología , Yersinia pestis/inmunología , Animales , Antiinfecciosos/uso terapéutico , Anticuerpos Antibacterianos/inmunología , Modelos Animales de Enfermedad , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Immunoblotting , Inmunoglobulina G/inmunología , Ratones , Ofloxacino/uso terapéutico , Peste/tratamiento farmacológico , Dodecil Sulfato de Sodio , Factores de TiempoRESUMEN
To evaluate the role of Yersinia outer proteins (Yops) in conferring protective immunity against plague, six yop loci from Yersinia pestis were individually amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant proteins were purified and injected into mice. Most Yop-vaccinated animals succumbed to infection with either wild-type encapsulated Y. pestis or a virulent, nonencapsulated isogenic variant. Vaccination with YpkA significantly prolonged mean survival time but did not increase overall survival of mice infected with the nonencapsulated strain. The only significant protection against death was observed in YopD-vaccinated mice challenged with the nonencapsulated strain.
Asunto(s)
Cápsulas Bacterianas/fisiología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Peste/prevención & control , Vacunas Sintéticas/inmunología , Yersinia pestis/inmunología , Animales , Femenino , Ratones , Embarazo , Proteínas Recombinantes/inmunología , VacunaciónRESUMEN
Although Campylobacter spp. are perhaps the most ubiquitous bacterial pathogens known to man, laboratory diagnosis of Campylobacter enteritis is not particularly problematic. The organism's ability to thrive at elevated temperatures facilitates its isolation directly from stool. Enteric infection with Campylobacter spp., despite its invasive nature, is usually self-limiting in the otherwise healthy individual. Management of enteric Campylobacter infection, therefore, is not complex and for most cases simply involves an appropriate rehydration strategy. In addition to the 'tried and true' approaches of good sanitation and thorough cooking of meats to prevent Campylobacter infection, recent advances in vaccine development appear promising.
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Infecciones por Campylobacter/etiología , Campylobacter/patogenicidad , Gastroenteritis/etiología , Animales , Campylobacter/aislamiento & purificación , Infecciones por Campylobacter/diagnóstico , Infecciones por Campylobacter/epidemiología , Campylobacter jejuni/aislamiento & purificación , Campylobacter jejuni/patogenicidad , Microbiología de Alimentos , Gastroenteritis/diagnóstico , Gastroenteritis/epidemiología , Humanos , Microbiología del AguaRESUMEN
The current human whole-cell vaccine is ineffective against pneumonic plague caused by typical F1 capsule positive (F1+) strains of Yersinia pestis. The authors found this vaccine to also be ineffective against F1-negative (F1-) Y. pestis strains, which have been isolated from a human case and from rodents. For these reasons, the authors developed a recombinant vaccine composed of a fusion protein of F1 with a second protective immunogen, V antigen. This vaccine protected experimental mice against pneumonic as well as bubonic plague produced by either an F1+ or F1- strain of Y. pestis, gave better protection than F1 or V alone against the F1+ strain, and may provide the basis for an improved human plague vaccine.