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1.
Int J Nanomedicine ; 7: 1575-86, 2012.
Article in English | MEDLINE | ID: mdl-22619514

ABSTRACT

BACKGROUND: Systemic delivery of small interfering RNA (siRNA) is limited by its poor stability and limited cell-penetrating properties. To overcome these limitations, we designed an efficient siRNA delivery system using polyethyleneimine-coated virus-like particles derived from adeno-associated virus type 2 (PEI-AAV2-VLPs). METHODS: AAV2-VLPs were produced in insect cells by infection with a baculovirus vector containing three AAV2 capsid genes. Using this method, we generated well dispersed AAV2-VLPs with an average diameter of 20 nm, similar to that of the wild-type AAV2 capsid. The nanoparticles were subsequently purified by chromatography and three viral capsid proteins were confirmed by Western blot. The negatively charged AAV2-VLPs were surface-coated with PEI to develop cationic nanoparticles, and the formulation was used for efficient siRNA delivery under optimized transfection conditions. RESULTS: PEI-AAV2-VLPs were able to condense siRNA and to protect it from degradation by nucleases, as confirmed by gel electrophoresis. siRNA delivery mediated by PEI-AAV2-VLPs resulted in a high transfection rate in MCF-7 breast cancer cells with no significant cytotoxicity. A cell death assay also confirmed the efficacy and functionality of this novel siRNA formulation towards MCF-7 cancer cells, in which more than 60% of cell death was induced within 72 hours of transfection. CONCLUSION: The present study explores the potential of virus-like particles as a new approach for gene delivery and confirms its potential for breast cancer therapy.


Subject(s)
Breast Neoplasms/therapy , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/therapeutic use , Animals , Breast Neoplasms/genetics , Capsid Proteins/chemistry , Capsid Proteins/genetics , Cell Line , Cell Line, Tumor , Dependovirus/chemistry , Dependovirus/genetics , Drug Carriers/chemistry , Drug Carriers/toxicity , Drug Delivery Systems , Female , Genetic Therapy , Humans , Nanomedicine , Nanoparticles/administration & dosage , Nanoparticles/therapeutic use , Polyethyleneimine/chemistry , Transfection
2.
Methods ; 56(3): 389-95, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22300754

ABSTRACT

The insect cell-baculovirus system (IC-BEVS) is widely used for the production of recombinant viral proteins for vaccine applications. It is especially suitable for the production of virus-like particles, which often require the simultaneous production of several recombinant proteins. Here, the available tools and process requirements for the simultaneous production of several recombinant proteins using the IC-BEVS are discussed. The production of double-layered rotavirus like particles is used as a specific example for the simultaneous production of two recombinant proteins. Methods to quantify VLP in small samples are described. The multiplicity and time of infection are presented as tools to manipulate protein concentration, and the effect on protein concentration ratios on the assembly efficiency of double-layered rotavirus like particles is discussed. It was found that not only the ratio between the recombinant proteins is determinant of VLP assembly efficiency, but also that assembly efficiency is related to the characteristics of the assembled proteins. This is the first time that kinetics of VLP production are followed during cultures, and that the assembly efficiency is quantitatively determined.


Subject(s)
Baculoviridae/genetics , Recombinant Proteins/biosynthesis , Rotavirus/physiology , Virion/physiology , Animals , Cells, Cultured , Spodoptera/cytology
3.
Expert Rev Vaccines ; 10(7): 1063-81, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21806400

ABSTRACT

Baculovirus and insect cell culture technologies have mostly been limited to research laboratories for the transient expression of target proteins for drug development purposes. With the renaissance of the vaccine field and the regulatory acceptance of recombinant DNA technology, the baculovirus expression system has been more broadly adopted for the development of subunit vaccines, including virus-like particles. In the numerous clinical trials extensively discussed and cross-referenced in this article, product quality, safety and efficacy have been demonstrated for many candidate vaccines targeting infectious diseases. The 2007 market authorization of Cervarix, a bivalent human papillomavirus virus-like particle vaccine against cervical cancer, was a critical milestone for the regulatory acceptance of insect cell technology in manufacturing human vaccines, opening the door to the approval of more baculovirus-derived vaccines. Insect cell technology is now a dominant platform for veterinary vaccines. This article covers the application of recombinant baculovirus as vectored vaccines to mediate systemic and mucosal immune responses through the display or expression of foreign antigens. We will probably observe increasingly more baculovirus-derived products and market licensing of safe and efficacious vaccines.


Subject(s)
Insecta/cytology , Vaccination , Vaccines, Subunit/metabolism , Vaccines, Virus-Like Particle/immunology , Adjuvants, Immunologic , Animals , Baculoviridae/immunology , Baculoviridae/metabolism , Drug Compounding , Humans , Insecta/metabolism , Insecta/virology , Vaccines, Virus-Like Particle/metabolism
4.
J Chromatogr B Analyt Technol Biomed Life Sci ; 879(15-16): 1105-11, 2011 May 01.
Article in English | MEDLINE | ID: mdl-21474396

ABSTRACT

Rotavirus VP6 self-assembles into high order macrostructures useful as novel scaffolds for the construction of multifunctional hybrid nanobiomaterials. This application requires large quantities of high quality pure material with strict structural consistency. Strategies for obtaining high quality recombinant VP6 and different characterization techniques are explored and compared in this work. VP6 was expressed in the insect cell-baculovirus system. VP6 assemblies were selectively purified utilizing an ion exchange and size exclusion (SE) chromatography. Purification steps were monitored and characterized by dynamic light scattering (DLS), ELISA, SDS-PAGE, HPLC and Western blot. DLS showed that the initial ultrafiltration step removed small particles, the intermediate anion exchange chromatographic step completely removed the baculovirus, whereas the final size exclusion chromatography permitted the selective recovery of correctly assembled VP6 nanotubes and discrimination of non-assembled VP6, as confirmed by transmission electron microscopy. VP6 assembled into tubular structures with diameter of 75 nm and several nanometers in length. The purification yield was 20% of multimeric assemblies with a purity >98%. The resulting material was suitable for the production of functionalized hybrid nanobiomaterials through in situ synthesis of metallic nanoparticles.


Subject(s)
Antigens, Viral/chemistry , Biotechnology/methods , Capsid Proteins/chemistry , Nanotubes/chemistry , Animals , Antigens, Viral/genetics , Antigens, Viral/isolation & purification , Antigens, Viral/metabolism , Baculoviridae/genetics , Biotechnology/instrumentation , Capsid Proteins/genetics , Capsid Proteins/isolation & purification , Capsid Proteins/metabolism , Cell Line , Chromatography, Gel/methods , Microscopy, Electron, Transmission , Nanotubes/ultrastructure , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spodoptera/virology
5.
Article in English | MEDLINE | ID: mdl-21123120

ABSTRACT

A HPLC method using an anion exchange column was developed for the quantification of baculovirus particles. To properly detect the virus eluting from the column, a nucleic acid dye was used to amplify the signal projected by the virus. The viral genome was labeled by incubating the virus with SYBR Green I at 37°C for a minimum of 1h. The virus was specifically eluted from the contaminants in 8.9 min at a NaCl concentration of 480 mM NaCl (in 20 mM Tris-HCl, pH 7.5). The total run time of the method was 25 min. The method resulted in a linear response from 1×10(8) to 5.0×10(10)viral particles (VP/ml). The detection limit was 3.0×10(7) and the quantification limit was 1×10(8)VP/ml. The intra-assay precision was <10% for both purified and crude virus preparations whereas the inter-assay precisions were <5% and <10% for purified and crude virus preparations, respectively. The recovery/accuracy of the method ranged from 78 to 101%. This method is a robust monitoring tool to facilitate research activities with baculovirus vector and accelerate development of baculovirus-based processes for manufacturing of biologics.


Subject(s)
Baculoviridae/isolation & purification , Chromatography, High Pressure Liquid/methods , DNA, Viral/analysis , Virion/isolation & purification , Virology/methods , Animals , Baculoviridae/chemistry , Baculoviridae/metabolism , Benzothiazoles , Blotting, Western , Cells, Cultured , Chromatography, Ion Exchange , DNA, Viral/chemistry , DNA, Viral/metabolism , Diamines , Electrophoresis, Polyacrylamide Gel , Genome, Viral , Linear Models , Organic Chemicals/chemistry , Organic Chemicals/metabolism , Quinolines , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, Fluorescence , Spodoptera , Virion/chemistry , Virion/metabolism , Virus Cultivation
6.
ACS Appl Mater Interfaces ; 2(10): 2924-32, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20919683

ABSTRACT

Probing of cellular uptake and cytotoxicity was conducted for two fluorescent cellulose nanocrystals (CNCs): CNC-fluorescein isothiocyanate (FITC) and newly synthesized CNC-rhodamine B isothiocyanate (RBITC). The positively charged CNC-RBITC was uptaken by human embryonic kidney 293 (HEK 293) and Spodoptera frugiperda (Sf9) cells without affecting the cell membrane integrity. The cell viability assay and cell-based impedance spectroscopy revealed no noticeably cytotoxic effect of the CNC-RBITC conjugate. However, no significant internalization of negatively charged CNC-FITC was observed at physiological pH. Indeed, the effector cells were surrounded by CNC-FITC, leading to eventual cell rupture. As the surface charge of CNC played an important role in cellular uptake and cytotoxicity, facile surface functionalization together with observed noncytotoxicity rendered modified CNC as a promising candidate for bioimaging and drug delivery systems.


Subject(s)
Cell Membrane Permeability/drug effects , Cell Membrane/metabolism , Cellulose/pharmacokinetics , Fluorescein-5-isothiocyanate/pharmacology , Fluorescent Dyes/pharmacology , Nanoparticles , Rhodamines/pharmacology , Animals , Cell Survival/drug effects , Cellulose/chemistry , Fluorescein-5-isothiocyanate/chemistry , Fluorescent Dyes/chemistry , HEK293 Cells , Humans , Rhodamines/chemistry , Spodoptera
7.
Curr Gene Ther ; 10(3): 174-86, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20380645

ABSTRACT

The baculovirus/insect cell expression system is best known, and used, as a research tool for the production of recombinant proteins often requiring post-translational modifications. Although studies on the larger scale use of the system have been reported on for three decades, the recognition of this system as an industrially relevant platform for the production of biologics has only been recently achieved with the approvals of baculovirus-derived human and veterinary vaccines for commercialization. The full utility of baculoviruses, however, does not end with the production of recombinant proteins; baculoviruses are increasingly being studied for their ability to transduce mammalian cells and use as a therapeutic themselves. There is, therefore, a need to revisit the current state of the art in the bioprocessing of baculoviruses as the product. This paper is an extensive review of what is currently known about the amplification and recovery of baculoviruses and highlights the gaps that have not been focused on in the midst of optimizing this system for protein production.


Subject(s)
Baculoviridae/genetics , Genetic Vectors/genetics , Animals , Baculoviridae/metabolism , Humans , Insecta/genetics , Insecta/metabolism , Insecta/virology , Protein Processing, Post-Translational , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism
8.
J Gene Med ; 12(2): 157-67, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20101623

ABSTRACT

BACKGROUND: Recombinant adeno-associated virus (rAAV) are the most promising vectors for gene therapy. However, large-scale rAAV production remains a challenge for the translation of rAAV-based therapeutic strategies to the clinic. The baculovirus expression vector system (BEVS) has been engineered to produce high rAAV titers in serum-free suspension cultures of insect cells. METHODS: The typical approach of rAAV production in BEVS has been based on a synchronous infection with three baculoviruses at high multiplicity of infection (MOI) [>3 plaque forming units (pfu)/cell]. An alternative approach is to co-infect at low MOI (0.1 pfu/cell). Both strategies (high and low MOI) were compared at a cell density of 1.0 x 10(6) cells/ml in shake-flask experiments. To increase the rAAV titer, a low MOI combined with an initial cell density at infection of 5.0 x 10(6) cells/ml, in fed-batch mode, was evaluated. Subsequently, the production strategy was validated in 3-l bioreactor runs. RESULTS: An increase of 210% in the rAAV titer (4.7 x 10(11) enhanced transduction units/l) was observed when using low MOI, an effect primarily caused by the increase in cell density. The fed-batch approach resulted in a seven-fold increase of rAAV yield. Controlled operations in bioreactor contributed to further increase the rAAV yield (2.8 x 10(14) vector genomes/l) by 25% in comparison to the shake flask results. CONCLUSIONS: This high yield production process using low MOIs and a feeding strategy successfully addresses several limitations of current rAAV production in insect cells and contributes to position the BEVS system as one of the most efficient for large-scale manufacturing of rAAV vectors.


Subject(s)
Cell Culture Techniques/methods , Dependovirus/genetics , Genetic Vectors/biosynthesis , Insecta/cytology , Animals , Bioreactors , Cell Count , Cells, Cultured
9.
Biotechnol Bioeng ; 104(4): 674-86, 2009 Nov 01.
Article in English | MEDLINE | ID: mdl-19623564

ABSTRACT

Virus-like particles constitute potentially relevant vaccine candidates. Nevertheless, their behavior in vitro and assembly process needs to be understood in order to improve their yield and quality. In this study we aimed at addressing these issues and for that purpose triple- and double-layered rotavirus-like particles (TLP 2/6/7 and DLP 2/6, respectively) size and zeta potential were measured using dynamic light scattering at different physicochemical conditions, namely pH, ionic strength, and temperature. Both TLP and DLP were stable within a pH range of 3-7 and at 5-25 degrees C. Aggregation occurred at 35-45 degrees C and their disassembly became evident at 65 degrees C. The isoelectric points of TLP and DLP were 3.0 and 3.8, respectively. In vitro kinetics of TLP disassembly was monitored. Ionic strength, temperature, and the chelating agent employed determined disassembly kinetics. Glycerol (10%) stabilized TLP by preventing its disassembly. Disassembled TLP was able to reassemble by dialysis at high calcium conditions. VP7 monomers were added to DLP in the presence of calcium to follow in vitro TLP assembly kinetics; its assembly rate being mostly affected by pH. Finally, DLP and TLP were found to coexist under certain conditions as determined from all reaction products analyzed by capillary electrophoresis. Overall, these results contribute to the design of new strategies for the improvement of TLP yield and quality by reducing the VP7 detachment from TLP.


Subject(s)
Rotavirus/metabolism , Viral Proteins/metabolism , Virosomes/metabolism , Hydrogen-Ion Concentration , Isoelectric Point , Kinetics , Macromolecular Substances/metabolism , Osmolar Concentration , Protein Binding , Temperature , Viral Proteins/chemistry
10.
BMC Biotechnol ; 7: 39, 2007 Jul 04.
Article in English | MEDLINE | ID: mdl-17610729

ABSTRACT

BACKGROUND: The simultaneous production of various recombinant proteins in every cell of a culture is often needed for the production of virus-like particles (VLP) or vectors for gene therapy. A common approach for such a purpose is the coinfection of insect cell cultures with different recombinant baculoviruses, each containing one or more recombinant genes. However, scarce information exists regarding kinetics during multiple infections, and to our knowledge, no studies are available on the behavior of the different populations that arise during coinfections. Such information is useful for designing infection strategies that maximize VLP or vector yield. In this work, kinetics of cell populations expressing rotavirus GFPVP2 (infected with bacGFPVP2), VP6 (infected with bacVP6), or both proteins simultaneously (coinfected with both baculoviruses) were followed by flow cytometry. RESULTS: In single infections, the population infected with any of the recombinant baculoviruses followed the Poisson distribution, as the population expressing a recombinant protein exhibited a hyperbolic-type function with respect to the multiplicity of infection (MOI) up to 5 pfu/cell. In coinfections, the population fraction expressing each recombinant protein could not be anticipated from results of single infections, as in some cases interference and synergistic effects were found. Only cultures with a total MOI below 5 pfu/cell followed the Poisson distribution. For cultures with a MOI of bacGFPVP2 above that of bacVP6 (overall MOI above 5 pfu/cell), the total population expressing one or both recombinant proteins was as low as 50% below that predicted by Poisson. In contrast, the population fraction expressing VP6 increased in coinfections, compared to that in single infections. The largest population fraction simultaneously expressing both recombinant proteins was 58%, and corresponded to cultures infected at a MOI of 5 and 1 pfu/cell of bacGFPVP2 and bacVP6, respectively. CONCLUSION: The infection conditions that maximize the cell population simultaneously expressing two recombinant proteins were determined. Such conditions could not have been anticipated from population kinetics in individual infections. This information should be taken into account for improved simultaneous production of various recombinant proteins in any work dealing with coinfections.


Subject(s)
Baculoviridae/genetics , Insecta/metabolism , Protein Engineering/methods , Recombinant Proteins/biosynthesis , Rotavirus/genetics , Transfection/methods , Virion/metabolism , Animals , Cell Line , Computer Simulation , Flow Cytometry/methods , Genetic Vectors/genetics , Insecta/genetics , Insecta/virology , Kinetics , Metabolic Clearance Rate , Models, Biological , Virion/genetics
11.
Article in English | MEDLINE | ID: mdl-16725390

ABSTRACT

Virus-like particles have a wide range of applications, including vaccination, gene therapy, and even as nanomaterials. Their successful utilization depends on the availability of selective and scalable methods of product recovery and purification that integrate effectively with upstream operations. In this work, a strategy based on aqueous two phase system (ATPS) was developed for the recovery of double-layered rotavirus-like particles (dlRLP) produced by the insect cell-baculovirus expression system. Polyethylene glycol (PEG) molecular mass, PEG and salt concentrations, and volume ratio (Vr, volume of top phase/volume of bottom phase) were evaluated in order to determine the conditions where dlRLP and contaminants concentrated to opposite phases. Two-stage ATPS consisting of PEG 400-phosphate with a Vr of 13.0 and a tie-line length (TLL) of 35% (w/w) at pH 7.0 provided the best conditions for processing highly concentrated crude extract from disrupted cells (dlRLP concentration of 5 microg/mL). In such conditions intracellular dlRLP accumulated in the top phase (recovery of 90%), whereas cell debris remained in the interface. Furthermore, dlRLP from culture supernatants accumulated preferentially in the interface (recovery of 82%) using ATPS with a Vr of 1.0, pH of 7.0, PEG 3350 (10.1%, w/w) and phosphate (10.9%, w/w). The purity of dlRLP from culture supernatant increased up to 55 times after ATPS. The use of ATPS resulted in a recovery process that produced dlRLP with a purity between 6 and 11% and an overall product yield of 85% (w/w), considering purification from intracellular and extracellular dlRLP. Overall, the strategy proposed in this study is simpler than traditional methods for recovering dlRLP, and represents a scalable and economically viable alternative for production processes of vaccines against rotavirus infection with significant scope for generic commercial application.


Subject(s)
Chromatography, Gel/methods , Chromatography, High Pressure Liquid/methods , Insecta/virology , Rotavirus/isolation & purification , Animals , Centrifugation , Insecta/cytology , Microscopy, Electron , Water
12.
J Biotechnol ; 122(4): 443-52, 2006 Apr 20.
Article in English | MEDLINE | ID: mdl-16290307

ABSTRACT

The production of virus-like particles (VLP) is of interest to several fields. However, little is known about their assembly when they are expressed in insect cells, as it occurs in conditions different to those of native virus. Knowledge of the localization of recombinant proteins and of the site of accumulation of VLP can increase the understanding of VLP assembly and be useful for proposing production strategies. In this work, the rotavirus proteins VP6 and the fusion protein GFPVP2 were expressed in High Five insect cells. Recombinant proteins and rotavirus-like particles (RLP) were located and visualized by confocal, epifluorescence and electron microscopy. Single-layered (sl) RLP (conformed by GFPVP2) accumulated in the cytoplasm as highly ordered aggregates. In contrast, VP6 formed fibrillar structures composed of various tubes of VP6 that were not associated to microtubules. Coexpression of GFPVP2 and VP6 altered the distribution of both proteins. VP6 formed aggregates, even when all other conditions of individual protein expression remained unchanged. Double-layered (dl) RLP were observed in dense zones of the cytoplasm, but were not in ordered aggregates. It was determined that the assembly of both slRLP and dlRLP occurs intracellularly. Accordingly, strategies for the optimum assembly of dlRLP should guarantee that each cell produces both recombinant proteins.


Subject(s)
Antigens, Viral/metabolism , Baculoviridae/metabolism , Capsid Proteins/metabolism , Cell Nucleus/metabolism , Rotavirus/metabolism , Animals , Baculoviridae/genetics , Fluorescent Antibody Technique , Genetic Vectors/metabolism , Insecta/cytology , Insecta/metabolism , Insecta/virology , Microscopy, Electron , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rotavirus/enzymology
13.
J Chromatogr B Analyt Technol Biomed Life Sci ; 824(1-2): 267-76, 2005 Sep 25.
Article in English | MEDLINE | ID: mdl-16095985

ABSTRACT

There is a lack of accurate and practical methods that require only small amounts of sample for quantifying virus-like particles (VLP). In this work, gel permeation (GP) HPLC was used to quantify double-layered rotavirus-like particles (dlRLP) produced in insect cells. The proposed methodology utilized two columns in series (pore sizes of 200 and 50 nm) and had a high precision (relative standard deviation below 5%). GP-HPLC not only allowed the routine quantification of dlRLP, but also of assembly intermediaries and other viral structures present in the samples. For the first time, kinetics of dlRLP accumulation could be followed. This methodology is valuable for designing new production processes and for optimizing dlRLP monitoring.


Subject(s)
Chromatography, High Pressure Liquid/methods , Rotavirus/isolation & purification , Animals , Antigens, Viral/genetics , Antigens, Viral/metabolism , Blotting, Western , Capsid Proteins/genetics , Capsid Proteins/metabolism , Cell Survival , Cells, Cultured , Centrifugation, Density Gradient/methods , Cesium , Chlorides , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hydrogen-Ion Concentration , Insecta/cytology , Insecta/metabolism , Insecta/virology , Kinetics , Microscopy, Electron , Particle Size , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Reproducibility of Results , Rotavirus/genetics , Rotavirus/ultrastructure , Temperature
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