Purpose
The goal of this research is to develop stable formulations for live attenuated influenza vaccines (LAIV) by employing the drying methods freeze drying, spray drying, and foam drying.
Methods
...Formulated live attenuated Type-A H1N1 and B-strain influenza vaccines with a variety of excipient combinations were dried using one of the three drying methods. Process and storage stability at 4, 25 and 37°C of the LAIV in these formulations was monitored using a TCID
50
potency assay. Their immunogenicity was also evaluated in a ferret model.
Results
The thermal stability of H1N1 vaccine was significantly enhanced through application of unique formulation combinations and drying processes. Foam dried formulations were as much as an order of magnitude more stable than either spray dried or freeze dried formulations, while exhibiting low process loss and full retention of immunogenicity. Based on long-term stability data, foam dried formulations exhibited a shelf life at 4, 25 and 37°C of >2, 1.5 years and 4.5 months, respectively. Foam dried LAIV Type-B manufactured using the same formulation and process parameters as H1N1 were imparted with a similar level of stability.
Conclusion
Foam drying processing methods with appropriate selection of formulation components can produce an order of magnitude improvement in LAIV stability over other drying methods.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The filoviruses, Ebola virus and Marburg virus, cause severe hemorrhagic fever with up to 90% human mortality. Virus‐like particles of EBOV (eVLPs) and MARV (mVLPs) are attractive vaccine candidates. ...For the development of stable vaccines, the conformational stability of these two enveloped VLPs produced in insect cells was characterized by various spectroscopic techniques over a wide pH and temperature range. Temperature‐induced aggregation of the VLPs at various pH values was monitored by light scattering. Temperature/pH empirical phase diagrams (EPDs) of the two VLPs were constructed to summarize the large volume of data generated. The EPDs show that both VLPs lose their conformational integrity above about 50°C–60°C, depending on solution pH. The VLPs were maximally thermal stable in solution at pH 7–8, with a significant reduction in stability at pH 5 and 6. They were much less stable in solution at pH 3–4 due to increased susceptibility of the VLPs to aggregation. The characterization data and conformational stability profiles from these studies provide a basis for selection of optimized solution conditions for further vaccine formulation and long‐term stability studies of eVLPs and mVLPs.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Abstract A combination of unique stabilizers and mild spray drying process conditions was employed to produce heat-stable measles vaccine powder. Live attenuated measles vaccine from Serum Institute ...of India was formulated with pharmaceutically approved stabilizers, including sugars, proteins, amino acids, polymers, surfactants, and plasticizers, as well as charged ions. In addition, the effects of buffer salt and pH on the storage stability of measles virus were examined. The potency of the dried vaccine stored at several temperatures was quantified by TCID50 assay on Vero cells. As a comparison to other process methods, lead formulations were also subjected to freeze drying and foam drying. The optimized measles vaccine formulation tested at 37 °C was stable for approximately 8 weeks (i.e. time for 1 log TCID50 loss). The measles titer decreased in a bi-phasic manner, with initial rapid loss within the first week but relative stability thereafter. Key stabilizers identified during the formulation screening processes were l -arginine, human serum albumin, and a combination of divalent cations. Spray drying was identified as the optimal processing method for the preparation of dried vaccine, as it generally resulted in negligible process loss and comparable, if not better storage stability, with respect to the other processes. Processing methods and formulation components were developed that produced a measles vaccine stable for up to 8 weeks at 37 °C, which surpassed the WHO requirement for heat stability of 1 week at that temperature.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The stability of attenuated virus vaccines has traditionally been assessed by a plaque assay to measure the virus's loss of replication competency in response to a variety of environmental ...perturbations. Although this method provides information regarding the impact of the vaccine formulation, it involves an empirical approach to evaluate stability. Biophysical studies on the other hand have the potential to provide insight into the mechanisms of inactivation of a viral vaccine in response to a variety of stressed conditions. Herein, we have employed a variety of spectroscopic techniques (i.e., circular dichroism, fluorescence spectroscopy, and dynamic light scattering) for a comprehensive examination of the thermal stability of three live-attenuated human-bovine reassortant rotavirus strains (G1, G3, and G4) in the 5-8 pH range. The spectroscopic methods employed are not specific and response changes reflect an average change over the entire virus structure. The present work, however, suggests the utility of these methods in early formulation of rotaviral vaccines due to their ability to identify regions of marginal stability over which high throughput excipient screening assays can be designed. We have further shown that these methods are sufficiently sensitive to differentiate the stability of the three homologous G-subtypes differing only in the composition of their surface antigenic proteins. The data from these spectroscopic methods are also compared to biological activity using a tissue culture viral infectivity assay. Partial correlation between the structural alterations and losses in activity are observed, further suggesting the utility of biophysical studies in early formulation studies of rotavirus vaccines.
Abstract
Background
Gallium nitrate citrate exhibits strong antibacterial activity and was recently shown to be safe and efficacious when intravenously administered to cystic fibrosis patients in a ...Phase 2 clinical study conducted by the University of Washington. We are developing an inhaled formulation of gallium citrate (AR-501), which is being tested in a Phase 1/2a clinical study. The in vitro antimicrobial activities, drug resistance profile, activities in combination with selected antibiotics, and in vivo animal efficacy if the inhaled vs. IV formulation is being presented.
Methods
MIC tests were performed on strains using the CLSI susceptibility test standards. Resistance testing exposed bacteria to 20 cycles at ranges above and below the MIC level of the drug used.SPF mice (C57BL/6J, 7–9 weeks) were inoculated intranasally with P. aeruginosa under ketamine/xylazine anesthesia. Inhalation of AR-501 used an Aeroneb Solo nebulizer. Gallium levels were determined by elemental analysis using atomic absorption spectroscopy. CFU levels were measured by enumeration of bacterial colonies following serial dilution of tissue homogenates.
Results
In vitro efficacy: MIC testing demonstrates the efficacy of AR-501 against gram (−), gram (+) and several species of mycobacteria of clinical isolates and the comparative antibacterial response with antibiotics. Resistance testing showed that AR-501 exhibited lower propensity to develop resistance than the antibiotics tested. In vivo efficacy: AR-501 Inhalation also increased the median survival time compared with IV dosing in the murine model. Bacterial clearance was increased when Tobramycin and AR-501 are co-administered. Comparative analysis of AR-501 after IH route demonstrate increased gallium levels in BAL and reduced levels in the kidney in contrast to IV route.
Conclusion
In vitro studies demonstrate the susceptibility of gram (−), gram (+) and mycobacteria pathogens and the dose range of AR-501 compared with SOC antibiotics. In vivo studies confirm the therapeutic efficacy of AR-501 in bacterial pneumonia by IH delivery and demonstrate that bacterial clearance is enhanced when SOC antibiotics are used in combination with AR-501.
Disclosures
All authors: No reported disclosures.
Abstract
Background
Anti-bacterial monoclonal antibodies can serve as a new treatment modality for difficult to treat infections. AR-105 is a fully human IgG1 monoclonal antibody (mAb) that binds to ...an extracellular polysaccharide epitope of Pseudomonas aeruginosa (PA) and was shown to mediate in vitro complement-dependent opsonophagocytic killing. AR-105 is currently being tested in a global Phase 2 clinical trial as an adjunctive treatment to standard of care antibiotics in ventilator-associated pneumonia patients. Here we present pre-clinical efficacy and clinical safety data for AR-105.
Methods
Efficacy in nonclinical studies against PA pneumonia was tested in prophylactic and therapeutic mouse models, either as a stand-alone therapy or in combination with antibiotics. Mice were dosed intranasally or by intravenous infusion with AR-105 post or prior to infection with PA and survival or lung bacteriology were monitored. In a clinical Phase 1 open-label study, 16 healthy volunteers received 2, 8, or 20 mg/kg of AR-105. Adverse events, immunogenicity, and pharmacokinetic (PK) profiles were evaluated for up to 84 days following administration.
Results
In the animal models, AR-105 reduced lung bacterial counts in a dose-dependent manner, and improved survival (80% in the treated group vs. 0% in the control group). Combination of AR-105 with antibiotics was more effective than monotherapy. In the Phase I study, no serious adverse events (AE) were observed in any cohort. Few AE were deemed related to the investigational drug, and all were mild and transient. AR-105 was found to be well tolerated in healthy volunteers with no anti-drug antibodies (ADA) detected. The PK profile was comparable with other human IgG1 mAbs, exhibiting a serum half-life of approximately 20 days.
Conclusion
AR-105 was confirmed to be effective in PA pneumonia animal models, either as stand-alone therapeutic or in combination with antibiotics. In the Phase 1 clinical study, AR-105 was shown to be safe and well-tolerated, with a PK profile similar to that of other IgG1 mAbs. AR-105 is a promising drug candidate for therapy of PA pneumonia.
Disclosures
All authors: No reported disclosures.
To investigate the impact of drying method on the storage stability of dried vaccine formulations.
A sucrose-based formulation of a live attenuated virus vaccine of a parainfluenza strain, with and ...without surfactant, was dried from by different methods; freeze drying, spray drying and foam drying. Dried powders were characterized by differential scanning calorimetry, specific surface area (SSA) analysis and by electron spectroscopy for chemical analysis (ESCA) to evaluate vaccine surface coverage in the dried formulations. Dried formulations were subjected to storage stability studies at 4, 25 and 37 degrees C. The vaccine was assayed initially and at different time points to measure virus-cell infectivity, and the degradation rate constant of the vaccine in different dried preparations was determined.
SSA was highest with the spray dried preparation without surfactant (approximately 2.8 m(2)/g) and lowest in the foam dried preparations (with or without surfactant) (approximately 0.1 m(2)/g). Vaccine surface coverage was estimated based on ESCA measurements of nitrogen content. It was predicted to be highest in the spray dried preparation without surfactant and lowest in the foam with surfactant. Stability studies conducted at 25 degrees C and 37 degrees C showed that the vaccine was most stable in the foam dried preparation with surfactant and least stable in spray dried preparations without surfactant and in all freeze dried preparations regardless of the presence of surfactant. Addition of surfactant did lower the SSA and vaccine surface coverage in freeze dried preparations but still did not improve storage stability.
In drying methods that did not involve a freezing step, good storage stability of Medi 534 vaccine in the dried form was found with low SSA and low vaccine surface accumulation, both of which integrate into low fraction of vaccine at the surface. Ice appears to be a major destabilizing influence.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Giardia duodenalis is a parasite of great medical interest due to the number of infections it causes worldwide each year. Although research on epigenetic mechanisms in this protist has only begun ...recently, epigenetic regulation has already been shown to have important roles in encystation, antigenic variation, and resistance to antibiotics in Giardia. In this work, we show that a Giardia ortholog of Sir2, GdSir2.4, is involved in the silencing of rRNA expression. Our results demonstrate that GdSir2.4 localizes to the nucleolus, and its binding to the intergenic spacer region of the rDNA is associated with the deacetylation of the chromatin in this region. Given the importance of the regulation of rRNA expression to maintain adequate levels of ribosomes and genomic stability within the cells, GdSir2.4 can be considered a target to create new therapeutic agents against this parasite.
Sirtuin GdSir2.4 participates in the regulation of rRNA transcription in the Giardia duodenalis parasite by removing acetyl groups in histones placed on rDNA.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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