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Vaccine thermostability is key to successful global immunization programs as it may have a significant impact on the continuous cold-chain maintenance logistics, as well as affect ...vaccine potency. Modern biological and biophysical techniques were combined to in-depth characterize the thermostability of a formulated rabies virus (RABV) in terms of antigenic and genomic titer, virus particle count and aggregation state. Tunable resistive pulse sensing (TRPS) and nanoparticle tracking analysis (NTA) were used to count virus particles while simultaneously determining their size distribution. RABV antigenicity was assessed by NTA using a monoclonal antibody that recognize a rabies glycoprotein (G protein) conformational epitope, enabling to specifically count antigenic rabies viruses. Agreement between antigenicity results from NTA and conventional method, as ELISA, was demonstrated. Additionally, NTA and ELISA showed mirrored loss of RABV antigenicity during forced degradation studies performed between 5 °C and 45 °C temperature exposure for one month. Concomitant with decreased antigenicity, emergence of RABV particle populations larger than those expected for rabies family viruses was observed, suggesting RABV aggregation induced by thermal stress. Finally, using a kinetic-based modeling approach to explore forced degradation antigenicity data (NTA, ELISA), a two-step model accurately describing antigenicity loss was identified. This model predicted a RABV shelf-life of more than 3 years at 5 °C; significant loss of antigenicity was predicted for samples maintained several months at ambient temperature. This thorough characterization of RABV forced degradation study originally provided a time-temperature mapping of RABV stability.
Hepatitis C virus (HCV), a major cause of chronic liver disease in humans, is the focus of intense research efforts worldwide. Yet structural data on the viral envelope glycoproteins E1 and E2 are ...scarce, in spite of their essential role in the viral life cycle. To obtain more information, we developed an efficient production system of recombinant E2 ectodomain (E2e), truncated immediately upstream its trans-membrane (TM) region, using Drosophila melanogaster cells. This system yields a majority of monomeric protein, which can be readily separated chromatographically from contaminating disulfide-linked aggregates. The isolated monomeric E2e reacts with a number of conformation-sensitive monoclonal antibodies, binds the soluble CD81 large external loop and efficiently inhibits infection of Huh7.5 cells by infectious HCV particles (HCVcc) in a dose-dependent manner, suggesting that it adopts a native conformation. These properties of E2e led us to experimentally determine the connectivity of its 9 disulfide bonds, which are strictly conserved across HCV genotypes. Furthermore, circular dichroism combined with infrared spectroscopy analyses revealed the secondary structure contents of E2e, indicating in particular about 28% beta-sheet, in agreement with the consensus secondary structure predictions. The disulfide connectivity pattern, together with data on the CD81 binding site and reported E2 deletion mutants, enabled the threading of the E2e polypeptide chain onto the structural template of class II fusion proteins of related flavi- and alphaviruses. The resulting model of the tertiary organization of E2 gives key information on the antigenicity determinants of the virus, maps the receptor binding site to the interface of domains I and III, and provides insight into the nature of a putative fusogenic conformational change.
During 2009, pandemic influenza A(H1N1)pdm09 virus affected humans on Réunion Island. Since then, the virus has sustained circulation among local swine herds, raising concerns about the potential for ...genetic evolution of the virus and possible retransmission back to humans of variants with increased virulence. Continuous surveillance of A(H1N1)pdm09 infection in pigs is recommended.
In spite of prevention measures enacted all over the world to control the COVID-19 pandemic outbreak, including mask wearing, social distancing, hand hygiene, vaccination, and other precautions, the ...SARS-CoV-2 virus continues to spread globally at an unabated rate of about 1 million cases per day. The specificities of superspreading events as well as evidence of human-to-human, human-to-animal and animal-to-human transmission, indoors or outdoors, raise questions about a possibly neglected viral transmission route. In addition to inhaled aerosols, which are already recognized as key contributors to transmission, the oral route represents a strong candidate, in particular when meals and drinks are shared. In this review, we intend to discuss that significant quantities of virus dispersed by large droplets during discussions at festive gatherings could explain group contamination either directly or indirectly after deposition on surfaces, food, drinks, cutlery, and several other soiled vectors. We suggest that hand hygiene and sanitary practices around objects brought to the mouth and food also need to be taken into account in order to curb transmission.
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•Coughing/sneezing/speaking while eating in a social venue appears to be a determining SARS-CoV-2 transmission factor.•The number of infectious viral particles contained in large droplets projected during the meal is a key consideration.•Indirect transmission by contaminated surfaces (drinks, food.) and hands brought to the mouth must be taken into account.•In addition to inhaled aerosols, the oral entry route (ingestion) may be relevant for SARS-CoV-2 transmission.
Numerous observational, epidemiologic data have suggested that the risk of COVID19 is related to shared meals or drinks. The presence of ACE2 receptors in the gastrointestinal tract supports this ...hypothesis. Furthermore, several patients experience gastrointestinal symptoms without any respiratory disease. The SARS-CoV-2 found on food and packaging in China and the epidemic resurgence attributed to foods are also strong indications of an oral transmission route. Unprecedented biopersistence on skin, food, and beverages supports this theory. Finally, animal models reproducing the disease by oral inoculation are additional arguments in favor of an oro-digestive route of infection.
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.
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