Virus matrix interference on assessment of virucidal activity of high-touch surfaces designed to prevent hospital-acquired infections.

OBJECTIVES/PURPOSE: High-touch surfaces are a critical reservoir in the spread of nosocomial infections. Although disinfection and infection control protocols are well developed, they lack the ability to passively reduce the pathogenic load of high-touch surfaces. Copper and its alloys have been suggested as a surface that exhibit continuous biocidal effects. Antimicrobial studies on these surfaces are prevalent, while virucidal studies are not as well explored. The goal of this study was to first determine the virucidal activity of a copper-nickel-zinc alloy and to then examine the effect of soiling and virus preparation on virucidal activity. METHODS: A baculovirus vector was used as an easily quantifiable model of an infectious enveloped animal cell virus. Droplets containing virus were deposited on surfaces and allowed to stay wet using humidity control or were dried onto the surface. Virus was then recovered from the surface and assayed for infectivity. To examine how the composition of the droplet affected the survival of the virus, 3 different soiling conditions were tested. The first two were recommended by the United States Environmental Protection Agency and the third consisted of cell debris resulting from virus amplification. RESULTS: A copper-nickel-zinc alloy was shown to have strong virucidal effects for an enveloped virus. Copper, nickel, and zinc ions were all shown to leach from the alloy surface and are the likely cause of virucidal activity by this surface. Virucidal activity was achieved under moderate soiling but lost under high soiling generated by routine virus amplification procedures. The surface was able to repeatably inactivate dried virus droplets under moderate soiling conditions, but unable to do so for virus droplets kept wet using high humidity. CONCLUSION: Ion leaching was associated with virucidal activity in both wet and dried virus conditions. Soiling protected the virus by quenching metal ions, and not by inhibiting leaching. The composition of the solution containing virus plays a critical role in evaluating the virucidal activity of surfaces and surface coatings.

Critical assessment of influenza VLP production in Sf9 and HEK293 expression systems.

BACKGROUND: Each year, influenza is responsible for hundreds of thousand cases of illness and deaths worldwide. Due to the virus' fast mutation rate, the World Health Organization (WHO) is constantly on alert to rapidly respond to emerging pandemic strains. Although anti-viral therapies exist, the most proficient way to stop the spread of disease is through vaccination. The majority of influenza vaccines on the market are produced in embryonic hen's eggs and are composed of purified viral antigens from inactivated whole virus. This manufacturing system, however, is limited in its production capacity. Cell culture produced vaccines have been proposed for their potential to overcome the problems associated with egg-based production. Virus-like particles (VLPs) of influenza virus are promising candidate vaccines under consideration by both academic and industry researchers. METHODS: In this study, VLPs were produced in HEK293 suspension cells using the Bacmam transduction system and Sf9 cells using the baculovirus infection system. The proposed systems were assessed for their ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix Protein (M1) and compared through the lens of bioprocessing by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques, such as single radial immunodiffusion assay (SRID), HA assay, western blot and negative staining transmission electron microscopy (NSTEM) to quantify total particles. RESULTS: For the HEK293 production system, VLPs were found to be associated with the cell pellet in addition to those released in the supernatant. Sf9 cells produced 35 times more VLPs than HEK293 cells. Sf9-VLPs had higher total HA activity and were generally more homogeneous in morphology and size. However, Sf9 VLP samples contained 20 times more baculovirus than VLPs, whereas 293 VLPs were produced along with vesicles. CONCLUSIONS: This study highlights key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine.

Critical assessment of current adeno-associated viral vector production and quantification methods.

Adeno-associated viral vectors have emerged as one of the most studied vectors for gene therapy. Numerous production methods have been described, each with its advantages and disadvantages. A challenge in assessing the current state of the art exists in comparing yields from one production system to the next due to the wide variety of quantification techniques. In this review, AAV vector production methods are summarized and the yields of the different processes are standardized to the number of harvested cells. Titers are further streamlined into five categories: transduction units, enhanced transduction units, infectious particles, DNase-resistant particles and total particles, and the importance of each type of measure is discussed.