PI3P signaling regulates receptor sorting but not transport in the endosomal pathway

While evidence is accumulating that phosphoinositide signaling plays a crucial role in growth factor and hormone receptor down-regulation, this signaling pathway has also been proposed to regulate endosomal membrane transport and multivesicular endosome biogenesis. Here, we have followed the fate of the down-regulated EGF receptor (EGFR) and bulk transport (fluid phase) markers in the endosomal pathway in vivo and in vitro. We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes. Similarly, we find that hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting. These observations thus show that transport and sorting can be uncoupled in the endosomal pathway. They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs

On-Line influenza virus quantification for viral production processes thanks to affinity-based surface plasmon resonance biosensor

Influenza virus seasonal epidemics, associated with the constant threat of new pandemic outbreak, challenge vaccine manufacturers to develop responsive processes that can outreach the limitations of traditional egg-based technology. Recent progress made regarding cell culture bioprocesses allowed for numerous alternative strategies to developed future vaccine candidates, as for example the recombinant HA or Virus—like Particles (VLP) vaccines. However, while cell culture allows for more versatility than ovoculture, regarding process development and monitoring, these alternatives still require optimization to seriously concurrence the traditional process. To drive these developments, WHO and regulatory agencies underlined the need for developing better influenza vaccine potency assays1,2. Actual influenza vaccine formulation and lot release rely on single-radial immunodiffusion (SRID) assay, which requires strain-specific reference sera and antigen reagents. However, the annual preparation of these reagents takes between 2 to 6 months and constitutes a critical bottleneck for the release of vaccine lots3. Additionally, SRID is not implementable for process development as such technique cannot handle in-process low concentrated and non-purified material. We developed an assay for rapid and label-free quantification of influenza hemagglutinin (HA) antigen and influenza virus based on surface plasmon resonance (SPR). The method is based on affinity capture of hemagglutinin antigen by sialic-acid terminated glycans present at the surface of the fetuin-functionalized sensor. Conditions were optimized for the regeneration of the surface, in order to run multiple sequential analyses on a unique sensor. Two types of purified standard were used during the development of the assay. Commercial trivalent inactivated vaccine (“TIV”) has been used for the determination of optimal analytical conditions, while a stock of split inactivated H1N1 virus has been produced and calibrated in our laboratory to study the specific response obtained toward this HA subtype. This assay offers a quantification of influenza hemagglutinin within minutes with a wide dynamic range (30 ng/mL-20 µg/mL). Also, the technique provides a limit of detection (LOD) 100 times lower than SRID, and a better reproducibility than SRID and its potential alternatives recently proposed (1,4,5. Additionally, the applicability of this assay for an on-line vaccine production monitoring has been validated by off-line measurement of influenza H1N1 virus particles derived from cell culture supernatant. Such a test allowed to achieve a LOD of 106 Infectious Viral Particles/mL Thus, our assay provides an innovative tool to evaluate influenza new vaccine bioprocesses, from viral production kinetics in mammalian cell culture to vaccine potency evaluation

Metabolic and Kinetic analyses of influenza production in perfusion HEK293 cell culture

<p>Abstract</p> <p>Background</p> <p>Cell culture-based production of influenza vaccine remains an attractive alternative to egg-based production. Short response time and high production yields are the key success factors for the broader adoption of cell culture technology for industrial manufacturing of pandemic and seasonal influenza vaccines. Recently, HEK293SF cells have been successfully used to produce influenza viruses, achieving hemagglutinin (HA) and infectious viral particle (IVP) titers in the highest ranges reported to date. In the same study, it was suggested that beyond 4 × 10⁶cells/mL, viral production was limited by a lack of nutrients or an accumulation of toxic products.</p> <p>Results</p> <p>To further improve viral titers at high cell densities, perfusion culture mode was evaluated. Productivities of both perfusion and batch culture modes were compared at an infection cell density of 6 × 10⁶cells/mL. The metabolism, including glycolysis, glutaminolysis and amino acids utilization as well as physiological indicators such as viability and apoptosis were extensively documented for the two modes of culture before and after viral infection to identify potential metabolic limitations. A 3 L bioreactor with a perfusion rate of 0.5 vol/day allowed us to reach maximal titers of 3.3 × 10¹¹IVP/mL and 4.0 logHA units/mL, corresponding to a total production of 1.0 × 10¹⁵IVP and 7.8 logHA units after 3 days post-infection. Overall, perfusion mode titers were higher by almost one order of magnitude over the batch culture mode of production. This improvement was associated with an activation of the cell metabolism as seen by a 1.5-fold and 4-fold higher consumption rates of glucose and glutamine respectively. A shift in the viral production kinetics was also observed leading to an accumulation of more viable cells with a higher specific production and causing an increase in the total volumetric production of infectious influenza particles.</p> <p>Conclusions</p> <p>These results confirm that the HEK293SF cell is an excellent substrate for high yield production of influenza virus. Furthermore, there is great potential in further improving the production yields through better control of the cell culture environment and viral production kinetics. Once accomplished, this cell line can be promoted as an industrial platform for cost-effective manufacturing of the influenza seasonal vaccine as well as for periods of peak demand during pandemics.</p

Perceptive study of an optimal trumpet using simulated sounds

International audienceDans le cadre de l'´ etude de la qualité des instruments de musique, nous avons optimisé, ` a partir de simulations sonores par modèle physique, la justesse de trompettes en faisant varier des paramètres géométriques de l'embouchure et de la branche d'embouchure. L'objectif de ce travail est de déterminer, par un test d'´ ecoute des sons simulés, dans quelles mesures les différences entre la géométrie optimale et des géométries courantes sont perceptibles. Trois trompettes d'intérêt sontétudiéessontétudiées : la trompette optimale, la trompette de référence (trompette initiale lors de la procédure d'optimisation), et une trompetté elémentaire (trompette admissible, mais dont la valeur de la justesse est faible). L'expérience perceptive est uné epreuve d'´ ecoute par paires de sons (same-different test) impliquant 32 participants (musiciens ou non). DeuxépreuvesDeuxépreuves sont proposées : l'´ evaluation des différences perceptives entre les 3 instrumentsétudiésinstrumentsétudiés, et l'´ evaluation des différences perceptives entre différents sons du même instrument. Les résultats de chaque participant pour chaque famille de sons sont représentés par la matrice de confusion de l'´ epreuve same-different, de laquelle différents indicateurs de performances issus de la théorie de détection du signal sont extraits. Les conclusions montrent que la trompette optimale et la trompette de référence présentent bien des différences perceptibles, les performances de détection des sujets musiciensétant par ailleurs significativement supérieuressupérieuresà celles des sujets non-musiciens

Pan-HA antibodies for influenza detection and quantification

The influenza virus imposes a heavy burden for society in terms of health and economy. Influenza is an elusive enveloped virus due to antigenic shift and drift of two surface proteins: neuraminidase (NA) and hemagglutinin (HA). As a result, new strains emerge every year which require seasonal vaccination for protection. Furthermore, large vaccine quantities are urgently needed in case of pandemics. Theoretically, vaccines against a new strain can be manufactured in as little as three weeks with certain platforms and technologies. However, vaccine quantification and release are still relying on the use of the Single Radial Immunodiffusion (SRID) assay using a strain-specific antibody to calculate HA concentration. This is a major limitation because it can take up to three months to generate the reagents necessary to run the SRID assay, including the strain-specific antibody. Hence, one of the major hurdles in the process of influenza vaccine production is the quantification of HA which is critical to establish proper dosing. To circumvent the need for strain-specific antibodies, we have produced two monoclonal antibodies (F211-11H12-3 and F211-10A9-2) against a highly conserved peptide sequence found within the HA molecule (1). Multiple strains belonging to 13 different influenza A subtypes, as well as 6 strains belonging to B lineages were detected by Western blot and dot blot. Overall, mAb F211-11H12-3 recognizes preferentially influenza A subtype 1, while the mAb F211-10A9-2 has a higher affinity for influenza A subtype 2. Therefore, all strains tested could be detected when both mAb are combined and used as a cocktail. Next, we performed quantitative dot blots by generating a standard curve ranging from 160ng/ml to 20µg/ml HA. This method is simple, easy to implement and highly reproducible. In-process samples as well as purified material can be quantified by dot blot after denaturation with urea. Even though the SRID is the only assay approved by regulatory agencies, quantitative dot blots can be used during manufacturing to optimize and monitor the production process. Finally, ELISA is widely used for quantification and preliminary data demonstrates that samples can be quantified with the pan-HA mAbs. In conclusion, a pan-HA antibody cocktail was generated against a highly conserved peptide sequence of influenza. Viruses produced in eggs and mammalian cells from 40 different strains were detected by Western blot. Reproducible quantification was achieved by dot blot using the two mAbs and an appropriate calibrating standard. The combination of pan-HA antibodies with an immunoassay such as the dot blot assay could accelerate process development and help establish new generation quantification methods for influenza. As the field is looking for flexible and versatile solutions to shift away from the SRID assay and strain-specific antibodies, the development of broad-spectrum antibodies offers a long-awaited alternative. 1) Chun et al, Universal antibodies and their applications to the quantitative determination of virtually all subtypes of the influenza A viral hemagglutinins, Vaccine (26), pp 6068-6076, 2008

Critical assessment of influenza VLP production in Sf9 and HEK293 expression systems

Background: Each year, influenza is responsible for hundreds of thousand cases of illness and deaths worldwide. Due to the virus' fast mutation rate, the World Health Organization (WHO) is constantly on alert to rapidly respond to emerging pandemic strains. Although anti-viral therapies exist, the most proficient way to stop the spread of disease is through vaccination. The majority of influenza vaccines on the market are produced in embryonic hen's eggs and are composed of purified viral antigens from inactivated whole virus. This manufacturing system, however, is limited in its production capacity. Cell culture produced vaccines have been proposed for their potential to overcome the problems associated with egg-based production. Virus-like particles (VLPs) of influenza virus are promising candidate vaccines under consideration by both academic and industry researchers. Methods: In this study, VLPs were produced in HEK293 suspension cells using the Bacmam transduction system and Sf9 cells using the baculovirus infection system. The proposed systems were assessed for their ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix Protein (M1) and compared through the lens of bioprocessing by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques, such as single radial immunodiffusion assay (SRID), HA assay, western blot and negative staining transmission electron microscopy (NSTEM) to quantify total particles. Results: For the HEK293 production system, VLPs were found to be associated with the cell pellet in addition to those released in the supernatant. Sf9 cells produced 35 times more VLPs than HEK293 cells. Sf9-VLPs had higher total HA activity and were generally more homogeneous in morphology and size. However, Sf9 VLP samples contained 20 times more baculovirus than VLPs, whereas 293 VLPs were produced along with vesicles. Conclusions: This study highlights key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine. © Thompson et al.; licensee BioMed Central

Charcot-Marie-Tooth disease type 2CC due to NEFH variants causes a progressive, non-length-dependent, motor-predominant phenotype

Objective: Neurofilaments are the major scaffolding proteins for the neuronal cytoskeleton, and variants in NEFH have recently been described to cause axonal Charcot-Marie-Tooth disease type 2CC (CMT2CC). Methods: In this large observational study, we present phenotype–genotype correlations on 30 affected and 3 asymptomatic mutation carriers from eight families. Results: The majority of patients presented in adulthood with motor-predominant and lower limb-predominant symptoms and the average age of onset was 31.0±15.1 years. A prominent feature was the development of proximal weakness early in the course of the disease. The disease progressed rapidly, unlike other Charcot-Marie-Tooth disease (CMT) subtypes, and half of the patients (53%) needed to use a wheelchair on average 24.1 years after symptom onset. Furthermore, 40% of patients had evidence of early ankle plantarflexion weakness, a feature which is observed in only a handful of CMT subtypes. Neurophysiological studies and MRI of the lower limbs confirmed the presence of a non-length-dependent neuropathy in the majority of patients. All families harboured heterozygous frameshift variants in the last exon of NEFH, resulting in a reading frameshift to an alternate open reading frame and the translation of approximately 42 additional amino acids from the 3' untranslated region (3′-UTR). Conclusions: This phenotype–genotype study highlights the unusual phenotype of CMT2CC, which is more akin to spinal muscular atrophy rather than classic CMT. Furthermore, the study will enable more informative discussions on the natural history of the disease and will aid in NEFH variant interpretation in the context of the disease’s unique molecular genetics