The invention relates to an antiviral device, a method of making the antiviral device, and uses of the antiviral device.

The use of activated carbon to capture and neutralise viruses is known. See for example US 2005/0183727. The use of 9 carbon monosaccharides having carboxylic acid substituents on the ring, such as sialic acid and its derivatives, to capture and neutralise viruses is also known, see for example US 2010/0330140. However sialic acid and its derivatives are expensive and cannot readily be attached to activated carbon, and therefore the effect of combining activated carbon and compounds such as sialic acid together has not been researched. It would therefore be desirable to explore ways to attach sialic acid and its derivatives to activated carbon, and also to alternative carbon substrates, and to explore the effect of such a combination of features on the neutralisation of viruses.

The present invention provides an antiviral device comprising carbon and a conjugated 9 carbon monosaccharide having a carboxylic acid substituent on the ring. The present invention further provides a substance able to bind conjugated 9 carbon monosaccharide having a carboxylic acid substituent on the ring.

The present invention yet further provides methods of making an antiviral device, the methods comprising the step of:

(i) exposing carbon to a conjugated monosaccharide having a carboxylic acid substituent on the ring; or of

(ii) Exposing carbon to a substance able to bind a conjugated 9 carbon monosaccharide having a carboxylic substituent on the ring The present invention also provides uses of an antiviral device: as a filter; in a facemask; in an air conditioning unit; in a blood filtration device; and in household, commercial or medical items suitable as any of furnishings, surface coverings or body coverings.

It has now been found that the effect of combining carbon and 9 carbon monosaccharides having carboxylic acid substituents on the ring (e.g. sialic acid) gives rise to a synergistic effect, i.e. a virus neutralisation effect that is greater than simply the sum of the individual neutralisation effects of carbon and sialic acid. This synergistic effect means that high virus retention is seen, and hence that less carbon is needed to effectively retain viruses. For example only a single layer of activated carbon cloth need be used in filters, in contrast to prior art systems which use multiple layers of activated carbon cloth.

As already mentioned, the antiviral device of the present invention comprises carbon. Suitable forms of carbon include activated carbon, which is a form of charcoal (carbon) processed to have a very high porosity and so a very large surface area, and can include for example activated carbon cloth. Alternative forms of carbon include meso porous carbon and synthetic carbons such as nanotubes and fullerenes. Activated carbon is preferable for use in this invention, and more preferably activated carbon cloth is used. Activated carbon is widely used to adsorb organic or inorganic molecules from gases or liquids. It can be manufactured in many different forms like powered, p-anular or bead activated carbon. Activated carbon cloth is a special form of activated charcoal, originally developed by the British Ministry of Defence for use in chemical warfare suits. Nowadays, however, it can be used in numerous applications for example in many filtration devices or in medicine. Preferably the carbon (be it activated carbon (cloth) or otherwise) is impregnated with silver nanoparticles which help to deactivate viruses.

In one embodiment, the antiviral device of the present invention also comprises a conjugated 9 carbon monosaccharide having a carboxylic acid substituent on the ring. Preferably this monosaccharide is sialic acid, and more preferably it is conjugated with an oligosaccharide to enable easy attachment of the monosaccharide to the carbon. Sialic acid is a generic term used for a family of 9-carbon monosaccarides structurally deduced from neuraminic acid. Currently more than 50 different sialic acids have been found in nature with a variety of structures. They are widely found in higher animals and to a lesser extent in other species ranging from plants and fungi to yeasts and bacteria. In humans, sialic acids are mainly derived from Neu5Ac (N-Acetyl neuraminic acid) and usually occur as terminal units on cell-surface conjugates. Due to the occurrence as terminal molecules on cell-surfaces, sialic acids take part in a variety of complex and important physiological and pathological events including cellular and molecular recognition processes.

Sialic acid plays an important role in viral infections such as human influenza infections. All influenza viruses contain hemagglutinin (HA), a protein that binds sialic acid found on the suiface of the cell that is being infected and so is responsible for entry of the virus into the cells usually in the upper respiratory tract. Free sialic acids are only very weak ligands for viral hemagglutinin. Generally, sialic acid is recognised when conjugated to an oligosaccharide with usually terminal galaclose in either 3- or 6-position, depending on the HA antigen. Thus 3- or 6- terminal galaclose is preferred as the oligosaccharide conjugated to 9 carbon monosaccharide. Whereas avian influenza HAs bind alpha 2,3- bound sialic acid, human influenza viruses bind alpha 2,6-sialic acid receptors and therefore from selectivity point of view, the ligand attached to carbon should display differently bound sialic acid in order to increase its efficiency towards flu viruses.

Similar mechanisms occur with viruses such as BK virus, adenovirus type 37, malaria, herpes simplex, HIV, cholera, sindbis, tumors, hepatitis, coxsackievirus A24 variant, rotavirus strains, and new and old world arenavirus (including ebola and rabies). Therefore the present invention is also envisaged for use with these viruses, and others like them.

Fetuin (from for example foetal bovine serum or bovine milk is a heavily glycosylated protein which contains up to 8.7% sialic acid on its surface and has been found to bind influenza and other viruses. Its oligosaccharides contain both 2,3- as well as 2,6- bound NeuSAc. Therefore fetuin is a preferred source of sialic acid. It can be bought directly from chemical suppliers such as Sigma Aldrich or can be extracted from bovine serum or milk using chromatography.

In another embodiment of the invention, the antiviral device comprises a substance able to bind a conjugated 9 carbon monosaccharide having a carboxylic acid substituent on the ring. The substance can be asialofetuin, and the monosaccharide can be sialic acid.

Many pathogens have evolved to decorate their cell surfaces with sialic acid, which results in their ability to resist the host's innate immune response and also their ability to interact specifically with different host-cell surfaces. Asialofetuin is the desialylated form of fetuin and binds equally well to an activated carbon substracture, and binding is not dependent on the presence of sialic acid residues. Thus, the use of asialofetuin in a filter would bind a pathogen having sialic acid on its surface via the sialic acid in the pathogen. Asialofetuin would also be useful in binding to the sialic acid in inflammatory IgG antibodies specific for self-antigens (autoantibodies) which significantly participate in chronic inflarnmatory processes that lead to the destruction of healthy tissues.

Asialofetuin can also be found in for example foetal bovine serum and bovine milk. Again it can be bought from a chemical supplier or extracted using chromatography.

The antiviral device of the present invention can be used to trap and neutralise viruses present in airflow or in blood. Specific embodiments include use of the antiviral device as a filter, for example a filter in an article such as a facemask, an air conditioning unit, or a blood filtration device (such as a hemofilter). Use as a facemask isspecifically envisaged.

When used as a hemofilter it can be used as part of a dialysis apparatus, or as an external filter through which the heart itself pumps blood. Recent research into H1N1 and H5N1 viruses has implicated high death rate and systemic failure in patients to the virus being found in the blood. A CVYR (continuous venovenous hemofiitration) hemofilter can therefore be used to clear viruses from the blood which would have a beneficial effect on the patient by restricting the transmission of the virus to other sites in the body, and would be of use in combating the high death rate associated with pandemic influenza.

Existing methods of hemofiitration using membranes or nano carbon tubes require pumps because of the pressure drops across the membrane. There are also methods of continuous hemofiitration that do not require pumps or expensive equipment such as continuous arteriovenous haemofiltration (CAVH) and continuous venovenous hemofiitration (CVVH). These are recognized treatment methods consisting of hemofiitration with arterial venous access and venous to venous access powered by the patient's own arterial pressure, without the necessity of a mechanical pump. With the discovery of attaching sialic acid to carbon using fetuin there is provided a hemofilter for use in CAVH and CVVH systems using activated carbon granules. The simplicity of these hemofilters enable the treatment of large numbers of patients infected with a pandemic virus such as influenza.

Percutaneous cannulation of an artery and a vein, or vein to vein, is adequate for access to the circulatory system. The ease of establishing access and the lack of need for special equipment allows rapid institution of this treatment.

There are major considerations regarding treatment of a pandemic vims:

1. A new strain of virus acting on an uneducated immune system allows the virus to replicate without an immune response.

2. Depending on the virus the immune system could take a number of days before it recognizes the invading virus and responds.

3. The huge number of viruses in the system elicits an overreaciion of the

immune system generating a cytokine storm that the body cannot cope with.

If the proposed hemofilter is used before the immune response then it reduces the viral load whilst the immune system is inactive thereby giving the immune system time to recognize the invading virus and reduce the resulting cytokine response.

If treatment is needed after immune system recognition it is envisaged that a dual hemofilter is used with a membrane to filter the cytokines and also reduce viruses in the blood.

As explained an autoimmune response to viral infection produces cytokines and in the case of pandemic infection this immune system response is disproportionate and may be the cause of death. There is therefore provided a dual hemofilter with an orthodox method of cytokine removal using nanotubes in a chamber of the filter prior to the blood entering a second chamber containing the fetuin treated carbon granules.

Further new research suggests that trans placental acquired antibodies to infectious agents, for example those with molecular mimicry to nervous system sialic acid epitopes, can cause neuromental disorders in the later life of a child who acquires such antibodies. Such neuromental disorders include obsessive compulsive disorder (OCD), autism spectrum disorder (ASD), and schizophrenia (SZ). Indeed, modeling this risk factor in mice through maternal immune activation (MIA) causes ASD- and SZ-like neuropathologies and behaviors in the offspring. A possible progression of such infectious agents is:

(i) Infectious agents with molecules - primarily sialic acids - common to those of particular components of the nervous system.

(ii) Stimulation of IgG antibodies to the common molecules in sera of women prior to, or during, pregnancy.

(iii) Transplacental transmission of cross-reacting antibodies to fetus/infant (from 4 months gestation, persisting for up to about 6 months postnatally).

(iv) Passage of antibodies across Blood-Nervous system Barriers.

(v) 111 effects on the developing nervous system, causing a proportion of certain neuro-mental disorders, that may not be manifest for years or decades.

The herein-described filtration system can reduce the infectious agents and antibodies in the bloodstream prior to their trans placental transmission to a foetus. In particular, the asialofetuin attached to the carbon granules in the filter captures the antibodies via the antibodies' sialic acid molecules.

When the antiviral device is used in air conditioning units, it can for example be used in veliicles such as cars or aircraft (for example in the form of granules or powder to be placed in pipes of the air conditioning unit). HEP A filters are currently used in aircraft, however these can still transmit viruses when they become wet. Additionally, the antiviral device could be used to make commercial/household/medical items suitable for use as fuiiiishings (such as curtains), surface coverings (such as tablecloths), or body coverings (such as gloves), to absorb viruses in the home, commercial or medical environment, or even to make cloths for the wiping of potentially viras-infected surfaces.

Carbon for use in the present invention can be acquired commercially. Mesoporous carbon, carbon nanotubes and fullerenes are all commercially available, and activated carbon, in the form of cloth or otherwise, can also be acquired commercially either ready impregnated with silver nanoparticles or without silver impregnation. It is also possible to impregnate unimpregnated activated carbon cloth, e.g. by introducting nano silver into a fibre manufactured from modified cellulose, then knitting or weaving a textile, and then carbonising it at e.g. around 350°C.

Devices according to the present invention can be made by exposing carbon to a conjugated monosaccharide having a carboxylic acid substituent on the ring, e.g. sialic acid as present in Fetuin. This can be achieved by dipping the carbon into aqueous Fetuin solution, e.g. for at least 20 minutes, or by filtering Fetuin solution through carbon, or by spraying aqueous Fetuin onto carbon. Fetuin adsorption onto carbon can generally be improved by using a detergent (e.g. cationic detergents, preferably hexadecyltrimethyl ammonium bromide or sodium dodecyl sulphate), by using a phosphate buffered saline (PBS), or by drying the carbon cloth prior to treatment with Fetuin. Heating Fetuin solution (e.g. to around 100°C) also seems to affect adsorption of Fetuin onto carbon as Fetuin initially adsorbs onto the cloth faster, but is then denatured. Denatured Fetuin actually appears to adsorb better than regular Fetuin, however, and therefore the end result is that more Fetuin is adsorbed. Typically, Fetuin is adsorbed onto carbon in an amount of about 0.1 -lmg of sialic acid per gram of carbon end product.

The invention will now be illustrated by reference to the following figures and an example.

Figure 1 : A simple hemofilter CVVH system for viral removal.

Figure 2: A first enhanced hemofilter with pump for dual virus and cytokine removal.

Figure 3: A second enhanced hemofilter with pump for dual virus and cytokine removal.

Figures 1, 2 and 3 show hemofilters according to the invention being used to filter viruses from a patient's blood. In all Figures, blood flows out of the patient (1) via a line out (2) through a hemofilter (3) and then back to the patient (1) via a line in (4). In Figure 1 the hemofilter (3) is a granulated activated carbon with attached fetuin, and in Figure 2 the hemofilter is enhanced by comprising a cytokine chamber (5) (with a tube for cytokine removal (17)) and a membrane filter (6) as well as the chamber comprising activated carbon granules with fetuin (7). Figure 3 is broadly similar to Figure 2 save that it shows a nanofilter (16) (ie a carbon nanotube filter) instead of a membrane filter, such that the cytokines are collected before the nanofilter rather than through a membrane filter. Figures 2 and 3 both additionally show an access pressure (8), a blood pump (9), an anticoagulant (10), a filter pressure (11), a return pressure (12), an air detector (13), a return clamp (14) and an infusion (15). Example 1: Aerosolised flu virus particles passing through an device according to the invention.

Influenza virus strain A/England/ 195/2009 (H1N1) was loaded into a nebuliser in a PBS buffer at an amount of 500μ1 of 1.275x10 pfu/ml. A flow rate of 2.5 1/min was selected and programmed to run for 10 mins. The airflow passes through a filter comprising either a device according to the invention or a reference device, of diameter 20mm, and then into an air sampler filled with PBS buffer to collect the filtered phase for viral analysis. Results:

Empty filter (reference device): 77500 pfu

One layer of Zorflex VB carbon cloth (reference device): 7875 pfu

0.6mg Fetuin/lOOmg single layer Zorflex VB carbon cloth (device of invention): O pfu

2.5mg fetuin/lOOmg single layer Zorflex VB carbon cloth (device of invention): 625 pfu.

As can be seen, Zorflex VB carbon cloth alone has approximately 90% virus retention, but Fetuin treated Zorflex VB carbon cloth according to the invention has over 99% virus retention in the 2.5mg sample, and 100% vims retention in the 0.6mg sample. Prior art systems only exhibit virus retention of 99% when multiple layers of cloth are used, whereas the above example uses only one layer of cloth.