Simple and robust downstream purification process for cell-derived influenza vaccines

New emerging influenza viruses with pandemic potentials were occurred in recent years, e.g. H5N1 in 1997, H1N1 in 2009, and H7N9 in 2013. The demand of producing pandemic influenza vaccines for human use with quick supply is high. For the cell-based pandemic influenza vaccines, we proposed a flow-through chromatography purification process. This process has only involved few purification steps and is easy to operate. Vero- and MDCK- cell derived avian influenza viruses including H5N1 and H7N9 were purified efficiently by the process proposed. The presented purification process consisted of clarification, inactivation, concentration, anion exchange chromatography (Capto Q), size exclusion and adsorption chromatography (Capto Core 700), diafiltration and sterile filtration. In the chromatography steps, cell DNA and protein were removed remarkably, and the virus were flowed through these columns. The flow rate was set as fast as 250 cm/min. The loading volume of virus solution was up to 50 times of column volume (CV).The DNA was removed over 90% after using Capto Q column, and was further removed by Capto Core 700 column. The overall removal rate of cellular DNA was more than 99%. The HA recovery rates of H5N1 and H7N9 influenza virus from Vero and MDCK cells were 20 to 40%. The DNA concentration of all purified bulks met the regulatory requirement of 10ng per dose. The developed purification process is simple and efficient, and it is suitable for purification of various influenza virus strains and can be used for the pandemic influenza vaccine production

Rapid and sensitive insulated isothermal PCR for point-of-need feline leukaemia virus detection

Objectives: Feline leukaemia virus (FeLV), a gamma retrovirus, causes diseases of the feline haematopoietic system that are invariably fatal. Rapid and accurate testing at the point-of-need (PON) supports prevention of virus spread and management of clinical disease. This study evaluated the performance of an insulated isothermal PCR (iiPCR) that detects proviral DNA, and a reverse transcription (RT)-iiPCR that detects both viral RNA and proviral DNA, for FeLV detection at the PON. Methods: Mycoplasma haemofelis, feline coronavirus, feline herpesvirus, feline calicivirus and feline immunodeficiency virus were used to test analytical specificity. In vitro transcribed RNA, artificial plasmid, FeLV strain American Type Culture Collection VR-719 and a clinical FeLV isolate were used in the analytical sensitivity assays. A retrospective study including 116 clinical plasma and serum samples that had been tested with virus isolation, real-time PCR and ELISA, and a prospective study including 150 clinical plasma and serum samples were implemented to evaluate the clinical performances of the iiPCR-based methods for FeLV detection. Results: Ninety-five percent assay limit of detection was calculated to be 16 RNA and five DNA copies for the RT-iiPCR, and six DNA copies for the iiPCR. Both reactions had analytical sensitivity comparable to a reference real-time PCR (qPCR) and did not detect five non-target feline pathogens. The clinical performance of the RT-iiPCR and iiPCR had 98.82% agreement (kappa[κ] = 0.97) and 100% agreement (κ = 1.0), respectively, with the qPCR (n = 85). The agreement between an automatic nucleic extraction/RT-iiPCR system and virus isolation to detect FeLV in plasma or serum was 95.69% (κ = 0.95) and 98.67% (κ = 0.85) in a retrospective (n = 116) and a prospective (n = 150) study, respectively. Conclusions and relevance: These results suggested that both RT-iiPCR and iiPCR assays can serve as reliable tools for PON FeLV detection

A Standardized Wedelia chinensis Extract Overcomes the Feedback Activation of HER2/3 Signaling upon Androgen-Ablation in Prostate Cancer

Crosstalk between the androgen receptor (AR) and other signaling pathways in prostate cancer (PCa) severely affects the therapeutic outcome of hormonal therapy. Although anti-androgen therapy prolongs overall survival in PCa patients, resistance rapidly develops and is often associated with increased AR expression and upregulation of the HER2/3-AKT signaling pathway. However, single agent therapy targeting AR, HER2/3 or AKT usually fails due to the reciprocal feedback loop. Previously, we reported that wedelolactone, apigenin, and luteolin are the active compounds in Wedelia chinensis herbal extract, and act synergistically to inhibit the AR activity in PCa. Here, we further demonstrated that an herbal extract of W. chinensis (WCE) effectively disrupted the AR, HER2/3, and AKT signaling networks and therefore enhanced the therapeutic efficacy of androgen ablation in PCa. Furthermore, WCE remained effective in suppressing AR and HER2/3 signaling in an in vivo adapted castration-resistant PCa (CRPC) LNCaP cell model that was insensitive to androgen withdrawal and second-line antiandrogen, enzalutamide. This study provides preclinical evidence that the use of a defined, single plant-derived extract can augment the therapeutic efficacy of castration with significantly prolonged progression-free survival. These data also establish a solid basis for using WCE as a candidate agent in clinical studies

Ample Pairs

We show that the ample degree of a stable theory with trivial forking is preserved when we consider the corresponding theory of belles paires, if it exists. This result also applies to the theory of $H$-structures of a trivial theory of rank $1$.Comment: Research partially supported by the program MTM2014-59178-P. The second author conducted research with support of the programme ANR-13-BS01-0006 Valcomo. The third author would like to thank the European Research Council grant 33882