Development of a Stable Respiratory Syncytial Virus Pre-Fusion Protein Powder Suitable for a Core-Shell Implant with a Delayed Release in Mice:A Proof of Concept Study

Currently, there is an increasing interest to apply pre-fusion (pre-F) protein of respiratory syncytial virus (RSV) as antigen for the development of a subunit vaccine. A pre-F-containing powder would increase the flexibility regarding the route of administration. For instance, a pre-F-containing powder could be incorporated into a single-injection system releasing a primer, and after a lag time, a booster. The most challenging aspect, obtaining the booster after a lag time, may be achieved by incorporating the powder into a core encapsulated by a nonporous poly(dl-lactic-co-glycolic acid) (PLGA) shell. We intended to develop a stable freeze-dried pre-F-containing powder. Furthermore, we investigated whether incorporation of this powder into the core-shell implant was feasible and whether this system would induce a delayed RSV virus-neutralizing antibody (VNA) response in mice. The developed pre-F-containing powder, consisting of pre-F in a matrix of inulin, HEPES, sodium chloride, and Tween 80, was stable during freeze-drying and storage for at least 28 days at 60 degrees C. Incorporation of this powder into the core-shell implant was feasible and the core-shell production process did not affect the stability of pre-F. An in vitro release study showed that pre-F was incompletely released from the core-shell implant after a lag time of 4 weeks. The incomplete release may be the result of pre-F instability within the core-shell implant during the lag time and requires further research. Mice subcutaneously immunized with a pre-F-containing core-shell implant showed a delayed RSV VNA response that corresponded with pre-F release from the core-shell implant after a lag time of approximately 4 weeks. Moreover, pre-F-containing core-shell implants were able to boost RSV VNA titers of primed mice after a lag time of 4 weeks. These findings could contribute to the development of a single-injection pre-F-based vaccine containing a primer and a booster

Cloning and expression of a full-length cDNA encoding human inositol 1,4,5-trisphosphate 3-kinase B

Inositol 1,4,5-trisphosphate (InsP(3)) 3-kinase catalyzes the phosphorylation of InsP(3) to inositol 1,3,4,5-tetrakisphosphate (InsP(4)). cDNAs encoding three isoenzymes of InsP(3) 3-kinase (3-kinases A, B, and C) have been previously reported; however, a demonstrably full-length cDNA encoding human InsP(3) 3-kinase B was still lacking. Here we report the cloning of a full-length 2841-bp cDNA encoding human InsP(3) 3-kinase B. Northern blot analysis shows the presence of an ubiquitous transcript of approximately 7.2 kb in a large number of human tissues. InsP(3) 3-kinase activity measured in COS-7 cells transfected with InsP(3) 3-kinase B shows an activity that was 8-fold increased upon the addition of Ca(2+)/calmodulin in the assay mixture.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Inhibition of nucleoside diphosphate kinase (NDPK/nm23) by cAMP analogues

AbstractNucleoside diphosphate kinase (NDPK/nm23) ATP/GDP phosphotransferase activity and serine autophosphorylation is inhibited by N6-mbcAMP, 8-ClcAMP and 8-BrcAMP. Inhibition of the enzymatic activity largely depends on the concentration of ATP and becomes significant at ATP concentrations up to 0.5 mM and at effector concentrations measured in C6 cells stimulated with 1 mM cAMP analogue. N6-mbcAMP is a substrate of the enzyme. DbcAMP and O′2-mbcAMP, cAMP analogues with a modified O′2-ribose, did not affect the NDPK activity. Cyclic AMP is only a moderate inhibitor of NDPK even at low ATP concentrations. Possible inhibitory effects of cAMP and cAMP analogues on reported extra- and intracellular functions of NDPK/nm23 are discussed

Cell-based models to study hepatic drug metabolism and enzyme induction in humans

Cell-based in vitro models are invaluable tools in elucidating the pharmacokinetic profile of a drug candidate during its drug discovery and development process. As biotransformation is one of the key determinants of a drug's disposition in the body, many in vitro models to study drug metabolism have been established, and others are still being developed and validated. This review is aimed at providing the reader with a concise overview of the characteristics and optimal application of established and emerging in vitro cell-based models to study human drug metabolism and induction of drug metabolising enzymes in the liver. The strengths and weaknesses of liver-derived models, such as primary hepatocytes, either freshly isolated or cryopreserved, and from adult or fetal donors, precision-cut liver slices, and cell lines, including immortalised cells, reporter cell lines, hepatocarcinoma-derived cell lines and recombinant cell lines, are discussed. Relevant cell culture configuration aspects as well as other models such as stem cell-derived hepatocyte-like cells and humanised animal models are also reviewed. The status of model development, their acceptance by health authorities and recommendations for the most appropriate use of the models are presented.status: publishe

A comparison of RSV and influenza in vitro kinetic parameters reveals differences in infecting time.

Influenza and respiratory syncytial virus (RSV) cause acute infections of the respiratory tract. Since the viruses both cause illnesses with similar symptoms, researchers often try to apply knowledge gleaned from study of one virus to the other virus. This can be an effective and efficient strategy for understanding viral dynamics or developing treatment strategies, but only if we have a full understanding of the similarities and differences between the two viruses. This study used mathematical modeling to quantitatively compare the viral kinetics of in vitro RSV and influenza virus infections. Specifically, we determined the viral kinetics parameters for RSV A2 and three strains of influenza virus, A/WSN/33 (H1N1), A/Puerto Rico/8/1934 (H1N1), and pandemic H1N1 influenza virus. We found that RSV viral titer increases at a slower rate and reaches its peak value later than influenza virus. Our analysis indicated that the slower increase of RSV viral titer is caused by slower spreading of the virus from one cell to another. These results provide estimates of dynamical differences between influenza virus and RSV and help provide insight into the virus-host interactions that cause observed differences in the time courses of the two illnesses in patients