Virus Instability Research Articles

Stabilization of an Infectious Enveloped Virus by Spray-Drying and Lyophilization

Enveloped viruses are attractive candidates for use as gene- and immunotherapeutic agents due to their efficacy at infecting host cells and delivering genetic information. They have also been used in vaccines as potent antigens to generate strong immune responses, often requiring fewer doses than other vaccine platforms as well as eliminating the need for adjuvants. However, virus instability in liquid formulations may limit their shelf life and require that these products be transported and stored under stringently controlled temperature conditions, contributing to high cost and limiting patient access. In this work, spray-drying and lyophilization were used to embed an infectious enveloped virus within dry, glassy polysaccharide matrices. No loss of viral titer was observed following either spray-drying (at multiple drying gas temperatures) or lyophilization. Furthermore, viruses embedded in the glassy formulations showed enhanced thermal stability, retaining infectivity after exposure to elevated temperatures as high as 85 °C for up to one hour, and for up to 10 weeks at temperatures as high as 30 °C. In comparison, viruses in liquid formulations lost infectivity within an hour at temperatures above 40 °C, or after incubation at 25 °C for longer periods of time.

A new single-step protocol for rapid baculovirus-driven protein production in insect cells

BackgroundIn the last three decades, the Baculovirus expression vector system (BEV) has evolved to one of the most widely used eukaryotic systems for heterologous protein expression including approved vaccines and therapies. Despite the significant improvements introduced during the past years, the BEV system still has major drawbacks, primarily the time required to generate recombinant virus and virus instability for certain target proteins. In this study we show that the conventional method to generate recombinant Baculovirus using a Tn7 transposition based system can be shortened to a single-step transfection-only procedure without further amplification.MethodsIn a first step we have adapted a recently published protocol that replaces the standard liposome-based transfection procedure of adherent insect cells by transfecting insect cells in suspension with a preformed DNA-PEI complex generating P0 virus. We have then expressed and purified six different target proteins, among them four intracellular and two secreted proteins, by infecting insect cells either with P0 or P1 virus.ResultsWe demonstrate that transfection in suspension is as efficient as the standard protocol, but in addition allows generation of high amounts of P0 virus early in the process. To test if this P0 virus generated by bacmid transfection can be used directly for protein expression in either the screening or production process, we compared P0 versus amplified P1 virus-mediated protein expression. We show that protein expression levels, purity and yield of the purified proteins are equally high for P0 and P1.ConclusionThe standard protocol for generating recombinant baculovirus comprises transfection of the bacmid followed by one or two subsequent virus amplification steps. In this study we show that Baculovirus generated by transfection-only is equally efficient in driving protein expression. This reduces the time from bacmid DNA to protein to eight days and reduces the risk of virus decay. In contrast to transient gene expression protocols, the required amount of DNA is minimal: 100 µg bacmid DNA is sufficient for a production scale of 10 L.

Importance of virus–medium interactions on the biological activity of wild-type Heliothine nucleopolyhedroviruses propagated via suspension insect cell cultures

In vitro serial passaging of nucleopolyhedroviruses often results in virus instability, leading to reduction of both yield and biological activity of polyhedra (virus occlusion bodies). In this study, uncloned Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) and cloned Helicoverpa zea single-nucleocapsid nucleopolyhedrovirus (HzSNPV) were each serially passaged five times in both low cost (VPM) and commercial (Excell 401) media using H. zea cell cultures. When the experimental data was analysed as a 2 3 full factorial design (testing two levels of virus, medium and passage number), the passage number was shown to have the most significant effect on polyhedra yield, while the virus–medium interaction had the most important effect on polyhedra biological activity. This interaction was most pronounced for HzSNPV, which experienced a dramatic decline in biological activity when switched from its original Excell 401 medium to the in-house VPM medium. This suggests that genetically homogeneous viruses are less able to adapt to perturbations in the nutrient environment, hence medium changes should be avoided. Therefore, it is important to standardise the scale-up process of nucleopolyhedrovirus biopesticides at the earliest stage of development, especially when it requires both a low cost medium and a plaque purified virus.

Investigation on the infection of cucumber mesophyll protoplasts with cucumber mosaic virus.

Isolated protoplasts from the first leaf mesophyll of cucumber plants have been successfully infected in vitro with cucumber mosaic virus (CMV). Virus instability before, during and after inoculation of the protoplasts resulted in low infectivities when extracts were assayed on cowpea; however, viral RNA extraction improved the bioassay technique. Attempts to optimize inoculation and incubation of protoplasts are outlined, incorporating the improved assay.