Production optimization of rotavirus-like particles: a system biology approach

Dissertation presented to obtain a Ph.D. degree in Engineering and Technology Sciences, Systems Biology at the Instituto de Tecnologia Química e Biológica, Universidade Nova de LisboaRotavirus-like particles (RLPs), a vaccine candidate against rotavirus disease, were produced by infecting Spodoptera frugiperda Sf-9 cells with genetically engineered recombinant baculoviruses. RLPs are spherically shaped particles composed by three viral proteins (vp) of rotavirus, vp2, vp6 and vp7, arranged in a triple layered structure. A diversity of protein structures, other than the correctly assembled RLP, are observed at the end of a typical production run suggesting that the protein assembly process is rather inefficient. Contaminants such as trimers of vp6 and vp7, vp6 tube-like structures, single-layered vp2 particles, double layered particles of vp2 and vp6 or RLPs lacking one or more subunits represent almost 88% of the total mass of proteins expressed. Thus, optimal control of protein expression concomitant with efficient particle assembly are critical factors for economical RLP production in the baculovirus/insect cells system

Expression of hemagglutinin protein from the avian influenza virus H5N1 in a baculovirus/insect cell system significantly enhanced by suspension culture

10.1186/1471-2180-6-16BMC Microbiology

Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system

Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400–500 μg) was expressed from math formula infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig

Development of an influenza virus vaccine using the baculovirus-insect cell expression system : implications for pandemic preparedness

Key word Influenza, rHA, vaccine, baculovirus, insect cells, production, pandemic preparedness Influenza (or flu) is a highly contagious, acute viral respiratory disease that occurs seasonally in most parts of the world and is caused by influenza viruses. Influenza vaccination is an effective way to reduce the complications and the mortality rate following influenza infections. The currently available influenza vaccines are manufactured in embryonated chicken eggs, a 40-year old production technology. The research in this thesis was aimed at the design, validation and development of a production process for a recombinant hemagglutinin (rHA) influenza vaccine for the prevention of seasonal influenza. The viral surface protein HA is the key antigen in the host response to influenza virus since neutralizing antibodies directed against HA can mitigate or prevent infection. The baculovirus-insect cell system was selected for the synthesis of rHA molecules. The designed process was used to manufacture candidate trivalent rHA vaccines, which were tested in four clinical studies in a total of more than 3000 human subjects age 18 - 92 to support licensure of FluBlok under the “Accelerated Approval” procedure in the United States (U.S.). These studies demonstrated that the purified rHA protein was well tolerated and resulted in a strong and long lasting immune response. In addition, the novel vaccine provided cross protection against drifted influenza viruses. In response to the emergence of the new H1N1 A/California /04/2009 influenza strain, the outlined design was used to produce a rHA vaccine candidate and merely 6 weeks later, the first batches of vaccine were ready for human clinical testing. There are two especially important advantages to the use of this technology from a public health perspective: First, the insect cell-baculovirus system has demonstrated the potential to facilitate safe and expeditious responses to health care emergencies such as the one currently posed by the novel H1N1 virus pandemic and secondly, the rHA vaccine does not contain ovalbumin or other antigenic proteins that are present in eggs and may therefore be administered to people who are egg-allergic. <br/

Recombinant expression and purification of functional vascular endothelial growth factor-121 in the baculovirus expression system

AbstractObjectiveTo express human vascular endothelial growth factor121 (VEGF121) in insect cells.MethodsA gene construct containing VEGF was cloned in the pFastBac-HTA vector, followed by transformation in DH10BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF.ResultsOur results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells.ConclusionsResults from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes

Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS)

Glycolytic isozymes that are restricted to the male germline are potential targets for the development of reversible, non-hormonal male contraceptives. GAPDHS, the sperm-specific isoform of glyceraldehyde-3-phosphate dehydrogenase, is an essential enzyme for glycolysis making it an attractive target for rational drug design. Toward this goal, we have optimized and validated a high-throughput spectrophotometric assay for GAPDHS in 384-well format. The assay was stable over time and tolerant to DMSO. Whole plate validation experiments yielded Z’ values >0.8 indicating a robust assay for HTS. Two compounds were identified and confirmed from a test screen of the Prestwick collection. This assay was used to screen a diverse chemical library and identified fourteen small molecules that modulated the activity of recombinant purified GAPDHS with confirmed IC50 values ranging from 1.8 to 42 µM. These compounds may provide useful scaffolds as molecular tools to probe the role of GAPDHS in sperm motility and long term to develop potent and selective GAPDHS inhibitors leading to novel contraceptive agents

The neurotrophin receptor, gp75, forms a complex with the receptor tyrosine kinase TrkA

The high-affinity NGF receptor is thought to be a complex of two receptors , gp75 and the tyrosine kinase TrkA, but direct biochemical evidence for such an association had been lacking. In this report, we demonstrate the existence of such a gp75-TrkA complex by a copatching technique. Gp75 on the surface of intact cells is patched with an anti-gp75 antibody and fluorescent secondary antibody, the cells are then fixed to prevent further antibody-induced redistributions, and the distribution of TrkA is probed with and anti-TrkA antibody and fluorescent secondary antibody. We utilize a baculovirus-insect cell expression of wild-type and mutated NGF receptors. TrkA and gp75 copatch in both the absence and presence of NGF. The association is specific, since gp75 does not copatch with other tyrosine kinase receptors, including TrkB, platelet-derived growth factor receptor-beta, and Torso (Tor). To determine which domains of TrkA are required for copatching, we used a series of TrkA-Tor chimeric receptors and show that the extracellular domain of TrkA is sufficient for copatching with gp75. A chimeric receptor with TrkA transmembrane and intracellular domains show partial copatching with gp75. Deletion of the intracellular domain of gp75 decreases but does not eliminate copatching. A point mutation which inactivates the TrkA kinase has no effect on copatching, indicating that this enzymatic activity is not required for association with gp75. Hence, although interactions between the gp75 and TrkA extracellular domains are sufficient for complex formation, interactions involving other receptor domains also play a role