Thierry Pelet, Jamie Paterson
Viroblock SA, Switzerland
There is growing public health concern over the spread of infectious diseases through airborne bacteria, viruses and fungi.1,2,3,4 Different technologies exist to protect against infection. One of the most widely used are face masks. However, current face masks are generally poor at removing particles in the range of 0.1mm to 0.5mm in diameter, which are small enough to escape the forces of mechanical filtration yet large enough to avoid being entrapped by electrostatic or Brownian motion. This particular gap in protection corresponds to the mean size of many viruses5 (see Figure 1).
Enveloped viruses are characterized by the presence of a lipidic membrane surrounding the viral core, which is derived from the host cells during the exit process (budding, see Figure 2). This envelope has been shown to be enriched in cholesterol molecules. Importantly, presence of cholesterol in the viral envelope is crucial for the infectivity of these viruses.6,7,8
Viroblock’s unique cholesterol depletion technology
Based on these observations, Viroblock developed a novel antiviral technology aimed at depleting significant amounts of viral cholesterol in order to reduce or suppress infectivity. This technology targets the entire class of enveloped viruses, which represents a significant part of all human viral pathogens (e.g., Influenza, SARS, Herpes, HIV, etc.).
Viroblock’s approach relies on a biphasic system. The first phase is composed of non-phospholipids, which, when properly formulated, form vesicles (termed NPV for non-phospholipid vesicles) mimicking the biological bilayer membranes. The second part is an aqueous phase containing a cyclodextrin derivative. Viroblock’s proprietary technology is coated onto non-woven materials and drastically improves the level of protection against viruses.
As illustrated in Figure 3, in this unique mode of action, the artificial lipid membranes act as a reservoir (storage) for the cholesterol, whereas the cyclodextrins contained in the mobile aqueous phase extract the cholesterol from the viral envelope and bring it to the vesicles in a cyclic mode (shuttle).
The transfer of non-covalently bound molecules, such as cholesterol, between the two membranes follows the Law of Mass Action so that, during an interaction, molecules will move from a region of high concentration (e.g. virus envelope) to one of lower concentration (e.g. NPV membrane).
Because cholesterol is present in most viral envelopes, the technology has a potentially broad-spectrum of antiviral activity. In vitro virucidal assays show that the technology is active on all enveloped viruses tested so far.
Application to air filtration systems
Because most air filtration systems are based on non-woven fabrics, the first step for a successful application was to develop a suitable coating process for these materials. As illustrated in Figure 4A, this was achieved by soaking the non-woven materials under mild pressure conditions. This process allows the vesicular form of NPV to convert to planar lamellar form, covering the non-woven fibers in a uniform way, as shown in Figures 4B, 4C and 4D, which are scanning electron micrographs (SEM) of non-woven material.
A number of tests were performed to ensure that the technology, when coated on non-woven fabrics, retains its whole efficacy. Initial testing using simple contact kill between Sendai virus and treated non-woven fabrics indicated that the antiviral technology was indeed still very efficient when coated onto solid substrates. The rapid kinetics of air filtration (≤ 1 sec range) require a more demanding assessment than a simple contact-kill test (usually ≥ 5min range). We therefore designed and built a prototype face mask composed of one external antiviral-coated layer, one thicker filtration layer and one thin internal comfort layer (schematically represented in Figure 5A). Figure 5B shows a picture of this horizontal foldable mask, which has been registered in Europe according to both the Medical Device Directive (surgical mask type 2R)9 and Personal Protective Equipment Directive (respirator type FFP2)10.
The overall respiratory protection of a face mask depends on both filtration efficiency and on face-fit, which, according to the Personal Protective Directive, should be maximized to prevent any inward leakage from the parts of the mask contacting the wearer’s face. We therefore performed a face fit assessment using the European standard EN149, a testing method that measures the total inward leakage (TIL) of a mask worn by a subject11. A TIL test performed on 10 subjects showed that the total inward leakage of the Viroblock mask is extremely low at a mean of <2% and very far from the upper allowed limit (>8%) for this category of masks. This indicates a very good face fit.
Aerosol challenges were then performed on the Viroblock antiviral facemask based on the ASTM F2101-01 Standard Test Method modified for mammalian viruses.12 The protocol, which uses aerosolized viral pathogens, is the method which is the most stringent and the closest to real-life conditions for viral filtration systems.
A high titer viral stock (≥ 108 TCID50/ml) was aerosolized using a nebulizer and pumped through the test mask at 28.3 L/min for 24 minutes. The residual pass-through viruses were then collected and quantified using Tissue Culture Infectious Dose 50 (TCID50).13 Figure 6 shows the results of the aerosol testing of three different viruses, which clearly demonstrates that the antiviral mask was able to decrease the infectious viral load by approximately 5 log (± 0.5 log). The results of these aerobiology tests show that in a very high viral setting (≥ 108 TCID50/ml), the Viroblock mask provides about 99,999% protection from H1N1, H5N1 and human coronaviruses in a total airflow volume corresponding to approximately 2-2.5 hours of normal human breathing. This means that in a typical hospital, agricultural or outdoor setting with much lower viral loads, the mask could be worn for much longer periods and still protect at the same levels.14 Current WHO/CDC guidelines do, however, advise wearers to change masks every 2 hours.
Studies are currently ongoing to determine the mask antiviral efficacy against more viruses such as Influenza H7N9, Respiratory Syncytial Virus (RSV), the Measles virus, etc., and preliminary results are encouraging.
Moreover, the addition of a small amount of a specific quaternary ammonium to the product confers anti-bacterial and anti-fungal properties to the Viroblock technology. Figure 7 shows the results of contact kill testing for two bacteria (one Gram- and one Gram+) and one fungus.
Based on its proprietary cholesterol sequestration technology, Viroblock has developed a unique antiviral fabric coating, which, once incorporated into a respiratory mask, is capable of inactivating Influenza viruses and other enveloped viruses on pass-through air.
The broad spectrum activity, coupled with the extreme rapidity of cholesterol depletion, opens the way to making antiviral fabrics that can inactivate airborne viruses during the very short time it takes for them to pass through a filter, hence providing an increased protection against bioaerosols of viral origin. Viroblock is also working to develop a number of other air filtration products including HME filters, air purifiers and industrial air conditioning systems.
About the Author
Thierry Pelet has worked for many years in the virology field on topics including molecular determinants of viral replication, viral pathogenesis and budding processes. He obtained his PhD in molecular virology in 1996 at the University of Geneva. He then carried out his post-doctoral studies at the Clinical Research Institute in Montreal, Canada. Once back in Switzerland, he worked as a senior scientist in several virology laboratories. His publications include more than a dozen scientific papers in peer-reviewed virology journals. Thierry Pelet co-founded Viroblock and is today the CSO of the company.
Dr. Jamie Paterson studied Molecular Biology at University of Edinburgh and completed a PhD at the Imperial College, University of London. Following his academic studies, he joined Procter and Gamble, initially in an R&D role, subsequently changing to a marketing and general management position, running the business in Italy, Spain and then Europe. After 18 years in Procter & Gamble, he left to become Chief Commercial Officer at biotech Neurim Pharmaceuticals, where he launched the sleep drug, Circadin, across Europe. In 2010, Jamie joined Viroblock SA as CEO and has led the development, registration and now commercialisation of their anti-viral face masks.
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