Pathogen surface antigens are at the forefront of the viral strategy when invading host organisms. These antigens, including membrane proteins (MPs), are broadly targeted by the host immune response. ...Obtaining these MPs in a soluble and stable form constitutes a real challenge, regardless of the application purposes (e.g. quantification/characterization assays, diagnosis, and preventive and curative strategies). A rapid process to obtain a native‐like antigen by solubilization of a full‐length MP directly from a pathogen is reported herein. Rabies virus (RABV) was used as a model for this demonstration and its full‐length G glycoprotein (RABV‐G) was stabilized with amphipathic polymers, named amphipols (APols). The stability of RABV‐G trapped in APol A8‐35 (RABV‐G/A8‐35) was evaluated under different stress conditions (temperature, agitation, and light exposure). RABV‐G/A8‐35 in liquid form exhibited higher unfolding temperature (+6°C) than in detergent and was demonstrated to be antigenically stable over 1 month at 5°C and 25°C. Kinetic modeling of antigenicity data predicted antigenic stability of RABV‐G/A8‐35 in a solution of up to 1 year at 5°C. The RABV‐G/A8‐35 complex formulated in an optimized buffer composition and subsequently freeze‐dried displayed long‐term stability for 2‐years at 5, 25, and 37°C. This study reports for the first time that a natural full‐length MP extracted from a virus, complexed to APols and subsequently freeze‐dried, displayed long‐term antigenic stability, without requiring storage under refrigerated conditions.
A rapid process to obtain a native‐like antigen by solubilization of a full‐length membrane protein (MP) directly from a pathogen is reported herein, using rabies virus (RABV) as a model. Its full‐length G glycoprotein (RABV‐G) was stabilized with amphipols (APols), confirming the ability of such amphipathic polymers to stabilize pathogen surface MPs. RABV‐G/APol exhibited a high thermal stability of G specific antigenicity in a defined freeze‐dried formulation for at least 2 years under storage conditions from 5°C to 37°C.
Integral membrane proteins (MP) exhibit specific tridimensional conformation and topology that define their various functions. Pathogen surface antigens, encompassing many MP, are at the forefront of ...the viral strategy which is broadly targeted by the host immune response. These antigens are present in equilibrium under different oligomeric forms with distinctive epitopes, and to obtain them in a soluble form and/or stable constitutes a real risk. The solubilization of a full-length MP directly from a pathogen to rapidly obtain a native antigen mimicking the original conformation of the MP at the pathogen surface is the process development reported in this work. Rabies virus (RABV) was used as a model for this demonstration and its full-length glycoprotein (G) was stabilized in amphiphatic polymers (A8-35 amphipols). The stability of the soluble RABV-G was evaluated under various stress conditions (temperatures, agitation and light exposures) and a long-term stable RABV-G formulation, suitable for the freeze-drying process, was defined using a design of experiment approach. RABV-G/A8-35 in liquid form was shown to be antigenically stable at 5 °C and 25 °C for one month, and a dedicated kinetic model predicted its stability up to 1 year at 5 °C. To mitigate the RABV-G/A8-35 sensitivity to mechanical stress, a solid form of RABV-G/A8-35 and a freeze-drying process were considered, resulting in a 2-year thermally stable product at 5 °C, 25 °C and 37 °C. To the best of our knowledge, this is the first time that a natural full-length MP, extracted from a virus and trapped in amphipols, was kept antigenically stable in the long term, in a defined freeze-dried form out of any refrigerated storage conditions. These results described an easy process to obtain a pure, well conformed native-like antigen of interest, from a circulating pathogen which is of concern for diagnostic (quantification/characterization assays), therapeutic and vaccine strategies. After the physical characterization of the protein, the identification of RABV G/A8-35 neutralizing epitopes has been underway before in vivo testing.
•Two methodologies for modeling individual customer flexibility behavior are proposed.•Models and analysis use real-world data from a field trial with smart appliances.•Customer flexibility behavior ...is best modeled with finite mixture models.•Presented models can be used for data generation in simulations.
Challenges that smart grids aim to address include the increasing fraction of supply by renewable energy sources, as well as plain rise of demand, e.g., by increased electrification of transportation. Part of the solution to these challenges lies in exploiting the opportunity to steer residential electricity consumption (e.g., for flattening the peak load or balancing the supply and demand in presence of the renewable energy production). To optimally exploit this opportunity, it is crucial to have insights on how flexible the residential demand is. Load flexibility is characterized by the amount of power, time of availability and duration of deferrable consumption. Residential flexibility however, is challenging to exploit due to the variation in types of customer loads and differences in appliance usage habits from one household to the other. Existing analyses of individual customer flexibility behavior in terms of timing are often based on inferences from surveys or customer load patterns (e.g., as observed through smart meter data): there is a high level of uncertainty about customer habits in offering the flexibility. Even though some of these studies rely on real world data, only few of them have quantitative data on actual flexible appliance usage, and none of them characterizes individual user behavior. In this paper, we address this gap and contribute with: (1) a new quantitative specification of flexibility, (2) two systematic methodologies for modeling individual customer behavior, (3) evaluation of the proposed models in terms of how accurately the data they generate corresponds with real world customer behavior, and (4) a basic analysis of factors influencing the flexibility behavior based on statistical tests. Experimental results for (2)–(4) are based on a unique data set from a real-life field trial.
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