Jimmy A. Mena

Population kinetics during simultaneous infection of insect cells with two different recombinant baculoviruses for the production of rotavirus-like particles

BackgroundThe 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.ResultsIn 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.ConclusionThe 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.

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

Simultaneous expression of recombinant proteins in the insect cell-baculovirus system: production of virus-like particles.

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.

Strategies for the purification and characterization of protein scaffolds for the production of hybrid nanobiomaterials

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.

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.

An integral process for the production of virus-like particles by insect cells

Virus-like particles (VLP) are produced when the structural proteins of a non-enveloped virus are expressed in a recombinant system. As a result, particles identical to the native virus, but devoid of the viral genetic material, are obtained. VLP are useful as vaccines, as carriers, and for basic research. Their production represents a challenge, as several proteins need to be simultaneously expressed and assembled into a complex structure. Other aspects that complicate process development include the difficulty of accurately quantifying complete VLP, as well as possible intermediates, and the difficulty of designing effective purification procedures.