Maria Candida M. Mellado

Virus-like particles in vaccine development

Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome, potentially yielding safer and cheaper vaccine candidates. A handful of prophylactic VLP-based vaccines is currently commercialized worldwide: GlaxoSmithKline’s Engerix® (hepatitis B virus) and Cervarix® (human papillomavirus), and Merck and Co., Inc.’s Recombivax HB® (hepatitis B virus) and Gardasil® (human papillomavirus) are some examples. Other VLP-based vaccine candidates are in clinical trials or undergoing preclinical evaluation, such as, influenza virus, parvovirus, Norwalk and various chimeric VLPs. Many others are still restricted to small-scale fundamental research, despite their success in preclinical tests. This article focuses on the essential role of VLP technology in new-generation vaccines against prevalent and emergent diseases. The implications of large-scale VLP production are discussed in the context of process control, monitorization and optimization. The main up- and down-stream technical challenges are identified and discussed accordingly. Successful VLP-based vaccine blockbusters are briefly presented concomitantly with the latest results from clinical trials and the recent developments in chimeric VLP-based technology for either therapeutic or prophylactic vaccination.

Impact of Physicochemical Parameters on In Vitro Assembly and Disassembly Kinetics of Recombinant Triple-Layered Rotavirus-Like Particles

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°C. Aggregation occurred at 35–45°C and their disassembly became evident at 65°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. Biotechnol. Bioeng. 2009; 104: 674–686

Purification of recombinant rotavirus VP7 glycoprotein for the study of in vitro rotavirus-like particles assembly

Rotavirus VP7 is a glycoprotein that forms the viral capsid outerlayer and is essential to the correct assembly of triple-layered rotavirus-like particles (RLPs). In this work, a novel purification strategy was designed to allow obtaining highly pure monomeric VP7 required for the RLPs in vitro assembly. VP7 production kinetics in baculovirus-insect cells at cell concentration at infection (CCI) of 1x10(6)cellsmL(-1) was compared in terms of VP7/glycoprotein 64 (gp64) ratio at different multiplicity of infection (MOI). The best productivity was achieved at MOI of 0.1plaque forming unit (pfu)cell(-1) and time of harvest of 80h post-infection. After preliminary clarification steps, the proteins eluted from Concanavalin A were concentrated and loaded onto size exclusion chromatography. The polishing step was anion exchange chromatography with Mono Q. The high resolution of this column resulted in separation of monomers from dimers of VP7. Overall, the purification protocol yielded high level of purity (>90%). Purified VP7 was characterized by MALDI-TOF mass spectrometry and SDS-capillary gel electrophoresis. The MW and apparent MW were determined as 31.6 and 39kDa, respectively, confirming the efficacy of the proposed purification strategy that now enables RLPs assembly studies.

Sodium dodecyl sulfate-capillary gel electrophoresis analysis of rotavirus-like particles

This work describes the application of a sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) method for the analysis of triple 2/6/7 and double-layered 2/6 rotavirus-like particles (RLPs), candidate vaccines against rotavirus infection. SDS-CGE analysis of RLPs resulted in peaks that could be attributed to the viral proteins (VP2, VP6 and VP7) according to their apparent molecular mass (MWapp). Samples containing the glycoprotein VP7 were analysed after deglycosylation with PNGase F. Upon deglycosylation, VP7 MWapp decreased 4-7kDa consistent with a degree of glycosylation of approximately 12-21%. VP2 was eventually detected in the form of more than one related proteins, despite the small areas due to the relative low mass proportion of this protein in the particle (16%). The effect of analytical parameters such as capillary temperature on method performance was evaluated. MWapp values estimated by SDS-CGE were compared with values obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The method described in this work proved to be fast, consistent and reproducible, representing a feasible alternative to the laborious conventional electrophoresis for the characterization of RLPs.

On the effect of thermodynamic equilibrium on the assembly efficiency of complex multi-layered virus-like particles (VLP): the case of rotavirus VLP.

Previous studies have reported the production of malformed virus-like-particles (VLP) in recombinant host systems. Here we computationally investigate the case of a large triple-layered rotavirus VLP (RLP). In vitro assembly, disassembly and reassembly data provides strong evidence of microscopic reversibility of RLP assembly. Light scattering experimental data also evidences a slow and reversible assembly untypical of kinetic traps, thus further strengthening the fidelity of a thermodynamically controlled assembly. In silico analysis further reveals that under favourable conditions particles distribution is dominated by structural subunits and completely built icosahedra, while other intermediates are present only at residual concentrations. Except for harshly unfavourable conditions, assembly yield is maximised when proteins are provided in the same VLP protein mass composition. The assembly yield decreases abruptly due to thermodynamic equilibrium when the VLP protein mass composition is not obeyed. The latter effect is more pronounced the higher the Gibbs free energy of subunit association is and the more complex the particle is. Overall this study shows that the correct formation of complex multi-layered VLPs is restricted to a narrow range of association energies and protein concentrations, thus the choice of the host system is critical for successful assembly. Likewise, the dynamic control of intracellular protein expression rates becomes very important to minimize wasted proteins.

Viruses and Virus-Like Particles in Biotechnology: Fundamentals and Applications

Although viruses are simple biological systems, they are capable of evolving highly efficient techniques for infecting cells, expressing their genomes, and generating new copies of themselves. It is possible to genetically manipulate most of the different classes of known viruses in order to produce recombinant viruses that express foreign proteins. Recombinant viruses have been used in gene therapy to deliver selected genes into higher organisms, in vaccinology and immunotherapy, and as important research tools to study the structure and function of these proteins. Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome. They have been applied not only as prophylactic and therapeutic vaccines but also as vehicles in drug and gene delivery and, more recently, as tools in nanobiotechnology. In this article, basic and advanced features of viruses and VLPs are presented and their major applications are discussed. The different production platforms based on animal cell technology are explained, and their main challenges and future perspectives are explored. The implications of large-scale production of viruses and VLPs are discussed in the context of process control, monitorization, and optimization. The main upstream and downstream technical challenges are identified and discussed accordingly. Abstract Although viruses are simple biological systems, they are capable of evolving highly efficient techniques for infecting cells, expressing their genomes, and generating new copies of themselves. It is possible to genetically manipulate most of the different classes of known viruses in order to produce recombinant viruses that express foreign proteins. Recombinant viruses have been used in gene therapy to deliver selected genes into higher organisms, in vaccinology and immunotherapy, and as important research tools to study the structure and function of these proteins. Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome. They have been applied not only as prophylactic and therapeutic vaccines but also as vehicles in drug and gene delivery and, more recently, as tools in nanobiotechnology. In this article, basic and advanced features of viruses and VLPs are presented and their major applications are discussed. The different production platforms based on animal cell technology are explained, and their main challenges and future perspectives are explored. The implications of large-scale production of viruses and VLPs are discussed in the context of process control, monitorization, and optimization. The main upstream and downstream technical challenges are identified and discussed accordingly.

Viruses and Virus-Like Particles in Biotechnology

Although viruses are simple biological systems, they are capable of evolving highly efficient techniques for infecting cells, expressing their genomes, and generating new copies of themselves. It is possible to genetically manipulate most of the different classes of known viruses in order to produce recombinant viruses that express foreign proteins. Recombinant viruses have been used in gene therapy to deliver selected genes into higher organisms, in vaccinology and immunotherapy, and as important research tools to study the structure and function of these proteins. Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome. They have been applied not only as prophylactic and therapeutic vaccines but also as vehicles in drug and gene delivery and, more recently, as tools in nanobiotechnology. In this article, basic and advanced features of viruses and VLPs are presented and their major applications are discussed. The different production platforms based on animal cell technology are explained, and their main challenges and future perspectives are explored. The implications of large-scale production of viruses and VLPs are discussed in the context of process control, monitorization, and optimization. The main upstream and downstream technical challenges are identified and discussed accordingly. Abstract Although viruses are simple biological systems, they are capable of evolving highly efficient techniques for infecting cells, expressing their genomes, and generating new copies of themselves. It is possible to genetically manipulate most of the different classes of known viruses in order to produce recombinant viruses that express foreign proteins. Recombinant viruses have been used in gene therapy to deliver selected genes into higher organisms, in vaccinology and immunotherapy, and as important research tools to study the structure and function of these proteins. Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome. They have been applied not only as prophylactic and therapeutic vaccines but also as vehicles in drug and gene delivery and, more recently, as tools in nanobiotechnology. In this article, basic and advanced features of viruses and VLPs are presented and their major applications are discussed. The different production platforms based on animal cell technology are explained, and their main challenges and future perspectives are explored. The implications of large-scale production of viruses and VLPs are discussed in the context of process control, monitorization, and optimization. The main upstream and downstream technical challenges are identified and discussed accordingly.

Erythropoietin Purification Employing Affinity Membrane Adsorbers

In the present work, rhEPO was purified from crude CHO cell culture supernatant either with Sartobind-Cibacron Blue (CB) or Sartobind-IDA-Cu +2 affinity membranes. Purity degrees were of 55 and 75%, respectively.

In Vitro Approaches for Improved Rotavirus VLP’s Quality

In vitro disassembly and reassembly of virus-like particles (VLPs) can be a powerful tool for optimization of particle quality and product homogeneity. In this work single-, double- and triple-layered rotavirus-like particles (SLP, DLP and TLP, respectively) were used as models to address this issue. They were produced by either single- or co-infection of insect cells with recombinant baculoviruses coding for rotavirus VP2, VP6 and/or VP7. Firstly, their characterization was accomplished using techniques such as dynamic light scattering. The second part of this work aimed at investigating the efficiency of in vitro TLP disassembly into DLP followed by its reassembly. Both the type of buffer and chelating agent affected the disassembly efficiency. Despite the polymorphism observed depending on the chaotropic agent used, DLP could be disassembled into SLP. Although further studies should be pursued, some preliminary insights into SLP disassembly into VP2 monomers were obtained. Overall, this work contributes to a better understanding of rotavirus TLP assembly with a bioprocessing alternative.

In Vitro Disassembly and Reassembly of Triple-Layered Rotavirus-Like Particles

Virus-like particles (VLPs) are of interest in vaccination, gene therapy and drug delivery. Their assembly process needs to be understood in order to improve their yield and quality. Triple-layered Rotavirus-like particle (TLP 2/6/7), a candidate vaccine against Rotavirus infection, is produced in insect cells by co-infection of baculoviruses coding for VP2, VP6 and VP7, the main structural proteins. Our earlier results have shown that the outer layer, constituted by VP7 monomers, is unstable, as VP7 can peel off resulting in the formation of an uncoated double-layered particle 2/6 (DLP). In this work, we investigated the parameters involved in the disassembly of TLP 2/6/7 into DLP 2/6. For this, purified TLP 2/6/7 was used for in vitro disassembly. Next, DLP 2/6 was assembled into TLP 2/6/7 by the addition of purified VP7 monomers. The kinetics of such disassembly and reassembly reactions, as monitored by light scattering spectroscopy, were found to be first and second order, respectively. The reaction constants were calculated at different temperatures, reactants and ionic strengths.