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Human fibroblast growth factor 8b (FGF8b) was expressed based on a baculovirus expression vector system (BEVS) and identified as having a protective effect on Parkinson's disease. Immunoblotting demonstrated that rhFGF8b proteins were recognized by a human anti-FGF8b antibody. The multiplicity of infection and timing of harvest had a significant effect on protein yield and protein quality. Our results indicated that the rhFGF8b was first detectable at 36 h postinfection and reached a maximum at 60 h. A multiplicity of infection (MOI) of 8 pfu/mL was suitable for harvest. The target protein was purified by heparin-affinity chromatography. In vitro methylthiazol tetrazolium (MTT) assays demonstrated that the purified rhFGF8b could significantly stimulate proliferation of NIH3T3 cells. Furthermore, to elucidate the effect of rhFGF8b on Parkinson's disease, we used FGF8b pretreatment on a cell model of Parkinson's disease. The results indicated that rhFGF8b prevented necrosis and apoptosis of 1-METHYL-4-phenyl pyridine (MPP super(+)) treated PC12 cells. Moreover, the effect of FGF8b on messenger RNA (mRNA) levels of apoptosis and ERS genes was investigated to clarify the molecular mechanisms of FGF8b. The results suggest that FGF8b exerts neuroprotective effects by alleviating endoplasmic reticulum (ER) stress during PD. These results suggest that FGF8b may be a promising candidate therapeutic drug for neurodegenerative diseases related to ER stress.

Aim: Dominant negative mutant G proteins have provided critical insight into the mechanisms of G protein-coupled receptor (GPCR) signaling, but the mechanisms underlying the dominant negative characteristics are not completely understood. The aim of this study was to determine the structure of the dominant negative G alpha sub(i1) beta sub(1) gamma sub(2) G203A/A326S complex (Gi-DN) and to reveal the structural basis of the mutation-induced phenotype of G alpha sub(i1) beta sub(1) gamma sub(2). Methods: The three subunits of the Gi-DN complex were co-expressed with a baculovirus expression system. The Gi-DN heterotrimer was purified, and the structure of its complex with GDP was determined through X-ray crystallography. Results: The Gi-DN heterotrimer structure revealed a dual mechanism underlying the dominant negative characteristics. The mutations weakened the hydrogen bonding network between GDP/GTP and the binding pocket residues, and increased the interactions in the G alpha -G beta gamma interface. Concomitantly, the Gi-DN heterotrimer adopted a conformation, in which the C-terminus of G alpha sub(i) and the N-termini of both the G beta and G gamma subunits were more similar to the GPCR-bound state compared with the wild type complex. From these structural observations, two additional mutations (T48F and D272F) were designed that completely abolish the GDP binding of the Gi-DN heterotrimer. Conclusion: Overall, the results suggest that the mutations impede guanine nucleotide binding and G alpha -G beta gamma protein dissociation and favor the formation of the G protein/GPCR complex, thus blocking signal propagation. In addition, the structure provides a rationale for the design of other mutations that cause dominant negative effects in the G protein, as exemplified by the T48F and D272F mutations.

Multiple different hemagglutinin (HA) protein antigens have been reproducibly manufactured at the 650L scale by Protein Sciences Corporation (PSC) based on an insect cell culture with baculovirus infection. Significantly, these HA protein antigens were produced by the same Universal Manufacturing process as described in the biological license application (BLA) for the first recombinant influenza vaccine approved by the FDA (Flublok registered ). The technology is uniquely designed so that a change in vaccine composition can be readily accommodated from one HA protein antigen to another one. Here we present a vaccine candidate to combat the recently emerged H7N9 virus as an example starting with the genetic sequence for the required HA, creation of the baculovirus and ending with purified protein antigen (or vaccine component) at the 10L scale accomplished within 38 days under GMP conditions. The same process performance is being achieved at the 2L, 10L, 100L, 650L and 2500L scale. An illustration is given of how the technology was transferred from the benchmark 650L scale facility to a retrofitted microbial facility at the 2500L scale within 100 days which includes the time for facility engineering changes. The successful development, technology transfer and scale-up of the Flublok registered process has major implications for being ready to make vaccine rapidly on a worldwide scale as a defense against pandemic influenza. The technology described does not have the same vulnerability to mutations in the egg adapted strain, and resulting loss in vaccine efficacy, faced by egg based manufacture.

Adipose-derived stem cells (ASCs) hold promise for cartilage regeneration but their chondrogenesis potential is inferior. Here, we used a baculovirus (BV) system that exploited FLPo/Frt-mediated transgene recombination and episomal minicircle formation to genetically engineer rabbit ASCs (rASCs). The BV system conferred prolonged and robust TGF- beta 3/BMP-6 expression in rASCs cultured in porous scaffolds, which critically augmented rASCs chondrogenesis and suppressed osteogenesis/hypertrophy, leading to the formation of cartilaginous constructs with improved maturity and mechanical properties in 2-week culture. Twelve weeks after implantation into full-thickness articular cartilage defects in rabbits, these engineered constructs regenerated neocartilages that resembled native hyaline cartilages in cell morphology, matrix composition and mechanical properties. The neocartilages also displayed cartilage-specific zonal structures without signs of hypertrophy and degeneration, and eventually integrated with host cartilages. In contrast, rASCs that transiently expressed TGF- beta 3/BMP-6 underwent osteogenesis/hypertrophy and resulted in the formation of inferior cartilaginous constructs, which after implantation regenerated fibrocartilages. These data underscored the crucial role of TGF- beta 3/BMP-6 expression level and duration in rASCs in the cell differentiation, constructs properties and in vivo repair. The BV-engineered rASCs that persistently express TGF- beta 3/BMP-6 improved the chondrogenesis, in vitro cartilaginous constructs production and in vivo hyaline cartilage regeneration, thus representing a remarkable advance in cartilage engineering.

The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS.

Avian influenza A H7N7/NL/219/03 virus creates a serious pandemic threat to human health because it can transmit directly from domestic poultry to humans and from human to human. Our previous vaccine study reported that mice when immunized intranasally (i.n) with live Bac-HA were protected from lethal H7N7/NL/219/03 challenge, whereas incomplete protection was obtained when administered subcutaneously (s.c) due to the fact that H7N7 is a poor inducer of neutralizing antibodies. Interestingly, our recent vaccine studies reported that mice when vaccinated subcutaneously with Bac-HA (H7N9) was protected against both H7N9 (A/Sh2/2013) and H7N7 virus challenge. HA1 region of both H7N7 and H7N9 viruses are differ at 15 amino acid positions. Among those, we selected three amino acid positions (T143, T198 and I211) in HA1 region of H7N7. These amino acids are located within or near the receptor binding site. Following the selection, we substituted the amino acid at these three positions with amino acids found on H7N9HA wild-type. In this study, we evaluate the impact of amino acid substitutions in the H7N7 HA-protein on the immunogenicity. We generated six mutant constructs from wild-type influenza H7N7HA cDNA by site directed mutagenesis, and individually expressed mutant HA protein on the surface of baculovirus (Bac-HAm) and compared their protective efficacy of the vaccines with Bac-H7N7HA wild-type (Bac-HA) by lethal H7N7 viral challenge in a mouse model. We found that mice immunized subcutaneously with Bac-HAm constructs T143A or T198A-I211V or I211V-T143A serum showed significantly higher hemagglutination inhibition and neutralization titer against H7N7 and H7N9 viruses when compared to Bac-HA vaccinated mice groups. We also observed low level of lung viral titer, negligible weight loss and complete protection against lethal H7N7 viral challenge. Our results indicated that amino acid substitution at position 143 or 211 improve immunogenicity of H7N7HA vaccine against H7N7/NL/219/03 virus.

Baculoviruses generally produce two progeny phenotypes-the budded virus (BV) and the occlusion-derived virus (ODV)-and the intricate mechanisms that regulate the temporal synthesis of the two phenotypes are critical for the virus replication cycle, which are far from being clearly understood. FP25K was reported to be responsible for the regulation of BV/ODV, and the mutations within result in a decrease of normal ODVs formation and an increase of BVs production. In this study, we demonstrated that the increase of BV titer in an fp25k knockout recombinant (fp25k-negative) was a result of higher infectivity of BVs rather than an increased production of BVs. The constitution of the major structural proteins and genome of parental and fp25k-negative BVs were analyzed. The results showed that the integrity of the majority of DNA packaged into the fp25k-negative BVs was intact; i.e., the genomic DNA of fp25k-negative BV had better transformation and transfection efficiency than that of the parental virus, indicating more intact genomes in the virions. Although the analysis of proteins associated with BVs revealed that more envelope protein GP64 were incorporated into the fp25k-negative BVs, subsequent experiments suggested that overexpression of GP64 did not improve the titer of BVs. Thus, we conclude that the main reason for higher infectivity of BVs is due to better genome integrity, which benefits from the deletion of fp25k resulting in increased stability of the genome and produce a higher proportion of infectious BVs. FP25K acts as a negative factor for the infectivity of BV.

Titer on Chip (Flu-ToC) is a new technique for quantification of influenza hemagglutinin (HA) concentration. In order to evaluate the potential of this new technique, a comparison of Flu-ToC to more conventional methods was conducted using recombinant HA produced in a baculovirus expression system as a test case. Samples from current vaccine strains were collected from four different steps in the manufacturing process. A total of 19 samples were analysed by Flu-ToC (blinded), single radial immunodiffusion (SRID), an enzyme-linked immunosorbent assay (ELISA), and the purity adjusted bicinchoninic acid assay (paBCA). The results indicated reasonable linear correlation between Flu-ToC and SRID, ELISA, and paBCA, with regression slopes of log-log plots being 0.91, 1.03, and 0.91, respectively. The average ratio for HA content measured by Flu-ToC relative to SRID, ELISA, and paBCA was 83%, 147%, and 81%, respectively; indicating nearly equivalent potency determination for Flu-ToC relative to SRID and paBCA. These results, combined with demonstrated multiplexed analysis of all components within a quadrivalent formulation and robust response to HA strains over a wide time period, support the conclusion that Flu-ToC can be used as a reliable and time-saving alternative potency assay for influenza vaccines.

Background Both tumor cells and their supporting endothelial cells should be considered for targeted cell killing when designing cancer treatments. Here we investigated the feasibility of combining radioiodide and antiangiogenic therapies after baculovirus-mediated transfer of genes encoding the sodium iodide symporter (NIS) and plasminogen kringle 5 (K5). Methods A recombinant baculovirus containing the NIS gene under control of the human telomerase reverse transcriptase (hTERT) promoter and the K5 gene driven by the early growth response 1 (Egr1) promoter was developed. Dual-luciferase reporter assay was performed to confirm the activation of hTERT transcription. NIS and K5 gene expression were identified by Western blot and Real-Time PCR. Functional NIS activity in baculovirus-infected Hela cells was confirmed by the uptake of 125I and cytotoxicity of 131I. The apoptotic effect of 131I-induced K5 on baculovirus-infected human umbilical vein endothelial cells (HUVECs) was analyzed by a flow cytometry-based assay. In vivo, NIS reporter gene imaging and therapeutic experiments with 131I were performed. Finally, the microvessel density (MVD) in tumors after treatment was determined by CD31 immunostaining. Results The activation of hTERT transcription was specifically up-regulated in tumor cells. NIS gene expression markedly increased in baculovirus-infected HeLa cells, but not in MRC5 cells. The Hela cells showed a significant increase of 125I uptake, which was inhibited by NaClO4, and a notably decreased cell survival rate by 131I treatment. Expression of the K5 gene induced by 131I was elevated in a dose- and time-dependent manner and resulted in the apoptosis of HUVECs. Furthermore, 131I SPECT imaging clearly showed cervical tumor xenografts infected with recombinant baculovirus. Following therapy, tumor growth was significantly retarded. CD31 immunostaining confirmed a significant decrease of MVD. Conclusion The recombinant baculovirus supports a promising strategy of NIS-based raidoiodide therapy combined with K5-based antiangiogenic therapy by targeting both the tumor and its supporting vessels.

Analyses of protein complexes are facilitated by methods that enable the generation of recombinant complexes via coexpression of their subunits from multigene DNA constructs. However, low experimental throughput limits the generation of such constructs in parallel. Here we describe a method that allows up to 25 cDNAs to be assembled into a single baculoviral expression vector in only two steps. This method, called biGBac, uses computationally optimized DNA linker sequences that enable the efficient assembly of linear DNA fragments, using reactions developed by Gibson for the generation of synthetic genomes. The biGBac method uses a flexible and modular “mix and match” approach and enables the generation of baculoviruses from DNA constructs at any assembly stage. Importantly, it is simple, efficient, and fast enough to allow the manual generation of many multigene expression constructs in parallel. We have used this method to generate and characterize recombinant forms of the anaphase-promoting complex/cyclosome, cohesin, and kinetochore complexes.