`
`(19) World Intellectual Property Organization
`International Bureau
`
`
`
`(43) International Publication Date
`13 November 2003 (13.11.2003)
`
`(10) International Publication Number
`
`PCT
`
`WO 03/093462 A2
`
`(51)
`
`International Patent Classification7:
`
`C12N 7/00
`
`(81)
`
`(21)
`
`International Application Number:
`
`PCT/GB03/01797
`
`(22)
`
`International Filing Date:
`
`28 April 2003 (28.04.2003)
`
`(25)
`
`Filing Language:
`
`(26)
`
`Publication Language:
`
`(30)
`
`Priority Data:
`02096808
`
`English
`
`English
`
`27 April 2002 (27.04.2002)
`
`GB
`
`(71)
`
`(72)
`(75)
`
`Applicant (for all designated States except US): UNIVER-
`SITY OF STRATHCLYDE [GB/GB]; McCance Build—
`ing, 16 Richmond Street, Glasgow G1 1XQ (GB).
`
`Inventors; and
`SCOTT, Hugh
`Inventors/Applicants (for US only):
`[GB/GB]; 12 Glencoats Drive, Paisley PA3 lRW (GB).
`MATTEY, Michael [GB/GB]; 10 Ratho Gate, Cumber—
`nauld G68 OGG (GB).
`
`Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, FT, GB, GD, GE, GH,
`GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
`LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
`MX, MZ, NO, NZ, OM, PH, PL, PT, RO, RU, SC, SD, SE,
`SG, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ,
`VC, VN, YU, ZA, ZM, ZW.
`
`(84)
`
`Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, FI, FR, GB, GR, HU, IE, IT, LU, MC, NL, PT, RO,
`SE, SI, SK, TR), OAPI patent (BF, BJ, CF, CG, CI, CM,
`GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`
`without international search report and to be republished
`upon receipt of that report
`
`(74)
`
`Agents: MacDOUGALL, Donald, Carmichael et 211.;
`Cruikshank & Faiiweather, 19 Royal Exchange Square,
`Glasgow G1 3AE (GB).
`
`For two-letter codes and other abbreviations, refer to the "Guid-
`ance Notes on Codes and Abbreviations " appearing at the begin-
`ning ofeach regular issue ofthe PCT Gazette.
`
`W003/093462A2
`
`(54) Title: lMMOBlLlSATlON AND STABILISATION 0F VIRUS
`
`(57) Abstract: The present invention relates to a method for immobilization and optional stabilization of Viruses whilst retaining
`the Viral biological activity and the use of immobilized Virus in therapy. In particular, the immobilized Virus relates to immobilized
`bacteriophage and their use as an antibiotic or bacteriostatic agent and in the treatment of antibiotic—resistant infections.
`
`
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`
`IMMOBILISATION AND STABILISATION OF VIRUS
`
`The present
`
`invention relates
`
`to a method
`
`for
`
`immobilising and optionally stabilising viruses including
`
`for
`bacteriophage, preferably to a solid phase substrate,
`use
`in
`therapyfl
`in
`particular
`as
`an
`antibiotic
`
`(bactericide) or bacteriostatic agent,
`
`in the treatment of
`
`antibiotic—resistant
`
`superficial
`
`infections
`
`and use
`
`in
`
`vaccinations.
`
`Bacteria have proven adept at developing resistance to
`
`new anti—microbial agents and so—called "super—bugs" are a
`
`cause of rising costs spent on means to combat super—bug—
`
`related infections and fatalities in hospitals throughout
`the world. Foriexample,
`the use of antibiotics, whether in
`
`an individual patient or
`
`in a hospital with its special
`
`environment and catalogue of micro—organisms, will destroy
`
`antibiotic—susceptible
`
`bacteria
`
`but
`
`permit
`
`the
`
`proliferation of bacteria that are intrinsically resistant
`
`or that have acquired extra chromosomal resistance. Thus,
`
`the more antibiotics are used,
`
`the more resistant bacteria
`
`become.
`
`Bacteriat
`
`canu survive on common. hospital materials
`
`including cotton and/or polyester
`
`lab coats,
`
`privacy
`
`curtains and polyethylene splash aprons for anything up to
`
`seven weeks increasing the chance of spreading infection.
`
`Indeed,
`
`common disinfectants used to sterilise hospital
`
`rooms and equipment are not sufficient to curb the spread
`
`of "super—bugs".
`
`Furthermore, no fundamentally new antibiotic has been
`
`discovered for at least 30 years and there is no guarantee
`
`that new classes of antibiotics will be developed let alone
`
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`
`even discovered in the next decade.
`
`An alternative to antibiotics in the fight against
`
`"super—bugs" is the use of bacteriophage.
`
`A bacteriophage
`
`is a mater—borne virus that
`
`infects specific bacteria.
`
`Virus particles vary in shape and size,
`
`from 0.02 to 0.3um
`
`35
`
`and contain RNA or DNA, either double or single stranded,
`
`which forms the viral genome.
`
`The viruses have a varied
`
`
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`
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`
`structure but the nucleic acid is always located within the
`
`virus particle surrounded by a pmotein coat
`
`(capsid or
`
`shell).
`
`The complex of nucleic acid and protein (the
`
`nucleocapsid) may be the whole structure of the virus (for
`
`example $6,
`
`an RNA bacterial virus, or ¢Xl74,. a DNA
`
`bacterial virus) but structures that are more complicated
`
`occur.
`
`The enveloped viruses may have lipid and protein
`
`membranes
`
`around.
`
`the capsid while the complex ‘viruses
`
`possess not only icosahedral heads but also helical tails
`
`with up to 20 proteins within the tail.
`
`Bacteriophage on infection of their hosts can multiply
`
`by either a lytic or lysogenic pathway. Bacteriophage that
`
`can integrate their DNA into bacterial
`
`chromosomes are
`
`known as lysogenic bacteriophage, with the integrated viral
`
`DNA replicating along with the host chromosome to produce
`
`new integrated‘viral DNA copies. Alternatively,
`
`the virus
`
`may replicate freely to produce several hundred progeny
`
`particles.
`
`Lysis of the cells then releases this large
`
`number‘ of
`
`free "viruses, which. are then. able to infect
`
`neighbouring bacteria. Although, bacteriophage were first
`
`identified in 1917,
`
`studies
`
`in the West
`
`into their
`
`application in medicine have been few and far between but
`
`studies have persisted and proved successful
`
`in Russia.
`
`Even
`
`so, various problems with bacteriophage
`
`therapy
`
`remain. For example, although bacteriophage are easy to
`
`grow they are particularly unstable and thus difficult to
`store.
`
`Bennett et al
`
`(1997) describe the immobilisation of a
`
`Salmonella—specific bacteriophage by adsorption.
`
`detail
`
`the passive
`
`adsorption of bacteriophage
`
`They
`
`onto
`
`polystyrene
`
`solid phases.
`
`However,
`
`this process
`
`is
`
`inefficient due to complex bacteriophages being immobilised
`
`via both "head"
`
`and "tail" groups.
`
`The
`
`tail group is
`
`required.
`
`to be
`
`free in order
`
`to recognise and infect
`
`specific bacteria. Further,
`
`the adsorption. process
`
`is
`
`reversible so that adsorbed bacteriophage will desorb and
`
`release free bacteriophage.
`
`The product of this process
`
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`
`is described for use as a separation system for the removal
`
`of specific bacteria from foods only and does not require
`
`the bacteriophage to be viable.
`
`It is an object of the present
`
`invention to obviate
`
`and/or mitigate at least some of the above disadvantages.
`
`Broadly speaking,
`
`the present invention describes for
`
`the first time a method for the immobilisation and optional
`
`stabilisation of viruses whilst
`
`retaining the viral
`
`biological activity. Furthermore, it documents its use in
`
`therapy,
`
`for example the manufacture of medical devices
`
`comprising immobilised virus,
`
`such as bacteriophage, with
`
`the ability to destroy specific resistant bacteria when,
`
`and only when,
`
`they are present.
`
`In a first aspect
`
`the present
`
`invention provides a
`
`device comprising"virus immobilised.
`
`to a substrate for
`
`medical application.
`
`In a further aspect of the present invention there is
`
`provided a device
`
`comprising virus
`
`immobilised to a
`
`substrate
`
`for use
`
`as
`
`an antibiotic
`
`(bactericide)
`
`or
`
`bacteriostatic
`
`agent.
`
`Preferably,
`
`said
`
`virus
`
`is
`
`a
`
`bacteriophage.
`
`Immobilisation is understood to relate to a specific
`
`physical immobilisaton, such as by chemical bonding and is
`
`therefore distinguished from any passive adherence of a
`
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`
`virus to a substrate.
`
`The term "virus" according to the present
`
`invention
`
`includes double—stranded. or
`
`single—stranded. RNA or DNA
`
`viruses, which infect cells of bacteria, plants and/or
`
`animals.
`
`These
`
`include viruses
`
`from the
`
`following
`
`families of viruses:
`
`Iridoviridae, African.
`
`swine
`
`fever
`
`virus, Poxviridae, Parvoviridae, Reoviridae, Birnaviridae,
`
`Picornaviridae, Togaviridae, Flaviviridae, Rhabdoviridae,
`
`Bunyaviridae, Herpesviridae, Adenoviridae, Papovaviridae,
`
`Hepadnaviridae, Coronaviridae, Calicivirus, Arenaviridae,
`
`Paramyxoviridae,
`
`Orthomyxoviridae,
`
`Filoviridae,
`
`Retroviridae, Baculoviridae, Polydnaviridae, Nudaurelia B
`
`virus group, Nodaviridae, Caulimovirus, Geminivirus, Tomato
`
`3O
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`
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`
`spotted wilt
`
`virus
`
`group,
`
`Luteovirus, Machlovirus,
`
`Necrovirus,
`
`Sobemovirus,
`
`Tombusvirus,
`
`Tymovirus,
`
`Bromovirus, Cucumovirus,
`
`Ilarvirus, Alfafa. mosaic virus
`
`group, Comovirus, Dianthovirus, Nepovirus,
`
`Pea enation
`
`mosaic virus group, Tobamovirus, Tobravirus, Hordeivirus,
`
`Potexvirus,
`
`Potyvirus,
`
`Carlavirus,
`
`Closterovirus,
`
`Totiviridae, Partitiviridae, Myoviridae,
`
`Styloviridae,
`
`Podoviridae, Tectiviridae, Plasmaviridae, Corticoviridae,
`
`Microviridae, Inoviridae, Cystoviridae and Leviviridae.
`
`It
`
`should. be understood.
`
`that.
`
`a 'virus may'
`
`include
`
`viruses or infectious agents, which do not fall into the
`
`above mentioned families, e.g., plant satellite viruses,
`
`prions, baculoviruses and bacteriophage respectively.
`
`The
`
`term.
`
`"bacteriophage"
`
`according'
`
`to the present
`
`invention.
`
`is indicative of bacteriophage, which infect
`
`specific strains of bacteria e.g. salmonella, Escherichia
`
`coli, staphylococcus or pseudomonus bacteriophage.
`
`The term "medical" according to the present invention
`is understood to mean the treatment or prevention of viral,
`
`bacterial or prion infections and/or contamination in
`
`humans, animals or plants.
`
`For example,
`
`in the case of
`
`bacterial
`
`infections and/or contamination,
`
`treatment or
`
`prevention may be achieved by bacteriophage immobilised on
`
`a substrate.
`
`It will be understood to the skilled man that
`
`bacteriophage can recognise and infect specific strains of
`
`bacteria.
`
`Thus, bacteriophage immobilised to a substrate
`
`according to the present
`
`invention, may be utilised to
`
`fight
`
`strain—specific
`
`bacterial
`
`infections
`
`as
`
`a
`
`"bactericide" by inducing selective killing of bacteria
`
`through. cell
`
`lysis or
`
`as
`
`a "bacteriostatic agent"
`
`by
`
`inhibiting bacterial growth. Bacteriophage immobilised to
`
`a
`
`substrate may
`
`also be
`
`used
`
`as
`
`a
`
`antibacterial
`
`agent/disinfectant
`
`in. order
`
`to "sterilise" bacterially—
`
`contaminated material.
`
`The
`
`term "substrate"
`
`according to the present
`
`invention is understood to mean any solid phase material to
`
`which.
`
`a virus may be
`
`immobilised”
`
`For
`
`example,
`
`said
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`
`substrate may be a material which may be advantageously
`
`activated to allow head—group specific binding of a virus,
`
`such as complex bacteriophage.
`
`Said substrate may take
`
`many forms, for example, nylon and any other polymer with
`
`amino or
`
`carboxyl
`
`surface groups,
`
`cellulose or other
`
`hydroxyl—containing polymer, polystyrene or other similar
`
`polymer,
`
`various
`
`plastics
`
`or microbeads
`
`including
`
`magneticparticles, biological substances. More preferably,
`
`said. substrate is tnade of a Inaterial
`
`commonly" used in
`
`therapy/medicine.
`
`For example,
`
`nylon thread for use in
`
`surgery; plastics,
`
`lint or gauze material used to dress
`
`open wounds; microbeads, which can be ingested; adhesives
`
`such as cyanoacrylates; and/or biological substances such
`
`as collagen or hyaluronic acid.
`
`Immobilisation of virus
`
`to the substrate may be
`
`achieved in a number of ways. Preferably, viruses, such as
`
`bacteriophage,
`
`are
`
`immobilised via
`
`bonds,
`
`typically
`
`covalent
`
`bonds
`
`formed between the bacteriophage
`
`coat
`
`protein and the substrate.
`
`More preferably, bacteriophage are immobilised to the
`
`substrate via
`
`their head groups
`
`or nucleocapsid by
`
`activating the substrate before the addition and coupling
`
`of bacteriophage.
`
`The term "activated/activating/activation" according
`
`to the present
`
`invention is understood to mean
`
`the
`
`activation of a substrate by reacting said substrate with
`
`various chemical groups
`
`(leaving a surface chemistry able
`
`to bind viruses,
`
`sud: as bacteriophage head or capsid
`
`groups).
`
`Activation of said substrate may be achieved by,
`
`for
`
`example, preliminary hydrolysis with an acid, preferably
`
`HCl
`
`followed by a mash step of water and an alkali
`
`to
`
`remove
`
`the acid.
`
`Preferably,
`
`said alkali
`
`is
`
`sodium
`
`bicarbonate.
`
`Binding
`
`of
`
`viruses,
`
`for
`
`example
`
`bacteriophage, via their head groups is important.
`
`In the
`
`case of complex bacteriophage for example, binding via head
`
`groups
`
`leaves the tail groups, which are necessary for
`
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`
`bacteria—specific recognition,
`
`free to infect, i.e., bind
`
`and penetrate a host bacterial cell.
`
`It will be understood
`
`that this mechanism of infection of a host cell is similar
`
`for many other viruses other than viruses that infect and
`
`multiply only in bacteria.
`
`A plurality of viruses, e.g.,
`
`various strain-specific bacteriophage, may be immobilised
`
`to a substrate at any one time.
`
`Coupling of viruses to a substrate is as a result of
`
`the formation of covalent
`
`bonds between the viral coat
`
`protein and the substrate such.as through an amino group on
`
`a peptide,
`for example a peptide bond.
`"Coupling Agents"
`that aid this process vary, dependent on the substrate
`
`used.
`
`For example, for coupling to the substrate nylon or
`
`other polymer with amino or carboxy surface groups
`
`the
`
`coupling agents carbodiimide or glutaraldehyde may be used.
`
`For coupling to the substrate cellulose or other hydroxyl—
`
`containing
`
`polymer
`
`the
`
`coupling
`
`agents
`
`vinylsulfonylethylene ether or
`
`triazine may be used.
`
`Coupling agents for the coupling of virus to the substrate
`
`polythene or other similar polymer include corona discharge
`
`or permanganate oxidation.
`
`Generally speaking, coupling
`
`agents for the coupling of bacteriophage to a substrate
`
`include: S—Acetylmercaptosuccinic anhydride;
`
`S—Acetylthioglycolic
`
`acid. N—hydroxysuccinimide
`
`ester;
`
`Adipic
`
`acid
`
`dihydrazide;
`
`4—Azidobenzoic
`
`acid
`
`N—
`
`h}deWDX§7SLIC<3i11iTniCie
`
`est:er‘;
`
`IJ— (5—ZXZicio-2—
`
`nituroberlzlebxyfi succ:inixnide;
`
`6— (4~Zkzidx>—2—
`
`nitrophenylamino)hexanoic acid N—hydroxysuccinimide ester;
`
`p—Azidophenacyl bromide; 4—Azidosalicylic acid
`
`N—hydroxysuccinimide ester; Bromoacetic acid
`
`N—hydroxysuccinimide
`
`ester;
`
`1,4~Butanediol
`
`diglycidyl
`
`ether;
`
`2~Diazo—3,3,3—trifluoroproprionic
`
`acid
`
`p—
`
`nitrophenyl
`
`ester;
`
`Diethyl
`
`malonimidate;
`
`4,4'~Diisothiocyanatostilbener2,2’—disulfonic acid; Dimethyl
`
`adipimidate;
`
`Dimethyl
`
`3,3'—dithiobispropionimidate;
`
`Dimethyl
`
`pimelimidate;
`
`Dimethyl
`
`suberimidate;
`
`4,4’—
`
`Dithiobisphenyl
`
`azide;
`
`Dithiobis<propionic
`
`acid
`
`N—
`
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`
`hydroxysuccinimide ester); Ethylene Glycol bis—(succinic
`
`acid N—hydroxysuccinimide ester); 4—Fluoro—3—nitrophenyl
`
`azide;
`
`p—Formylbenzoic acid N—hydroxysuccinimide ester;
`
`Glutaraldehyde; 2—Iminothiolane; 6—(Iodoacetamide)caproic
`
`acid N—hydroxysuccinimide ester;
`
`lodoacetic acid
`
`N—hydroxysuccinimide ester; 3-Maleimidoacetic acid
`
`N—hydroxysuccinimide ester; 3—Maleimidobenzoic acid
`
`N—hydroxysuccinimide ester;
`
`4—(N.Ma1eimido)benzophenone;
`
`v—Maleimidobutyric acid N—hydroxysuccinimide ester;
`
`‘
`
`s—Maleimidocaproic acid N—hydroxysuccinimide ester;
`
`4—(N—Maleimidomethyl)cyclohexenecarboxylic acid
`
`N—hydroxysuccinimide
`
`ester;
`
`4—(N—
`
`Maleimidomethyl)cyclohexanecarboxylic
`
`acid
`
`3—sulfo—N—
`
`hydroxysuccinimide ester; B—Maleimidopropionic acid
`
`N—hydroxysuccinimide estery N,N’—bis(3—Maleimidopropionyl)—
`
`2—hydroxy—l,3—propanediamine;
`
`1,4—Phenylene
`
`diisothiocyanete;
`
`N,N’~o—Phenylenedimaleimide;
`
`Polyoxyethylene bis(glycidyl ether); bis(Polyoxyethylene
`
`bi_s (g lyrci_d}rl
`
`et:heer ); Pc>l}703<y"et:h}rl eI1e
`
`bis(imidazolylcarbonyl)l;
`
`Bis(Polyoxyethylene
`
`bis[Imidoly1carbonyl]); Polyoxyethylene bis(p—nitropheny)
`
`carbonate);
`
`3—(2—Pyridyldithio)propionic acid
`
`N—hydroxysuccinimide
`
`ester;
`
`Suberic
`
`acid
`
`bis(N—
`
`hydroxysuccinimide) ester; Succinic Acid Maleimidoethyl—N—
`
`h y d r o x y s u c c i n i m i d e
`
`e s t e r ;
`
`l
`
`, 5 -
`
`bis(succinimidooxycarbonyloxy)—pentene;
`
`bis(N—
`
`succinimidyl)carbonate.
`
`Advantageously the present inventor has observed that
`where virus
`is
`immobilised
`to said substrate,
`said
`
`immobilization.
`
`confers
`
`stability:
`
`For
`
`example,
`
`'the
`
`immobilised virus is stabilised in such 51 way that
`
`maintains
`
`its viability‘
`
`and infectivity' even when
`
`it
`
`in
`
`contact with agents,
`
`for
`
`example proteases, which. may
`
`otherwise inactivate the virus and similarly, when exposed
`
`to physical stress, such as dehydration,
`
`temperature or pH
`
`which. would otherwise inactivate the virus.
`
`Further
`
`stability is conferred to the immobilised virus using known
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`compounds
`
`that protect proteins
`
`against
`
`dehydration,
`
`prolonged storage and other stresses.
`
`An example of such
`
`a compound is trehalose.
`
`Trehalose
`
`andh other'
`
`similar
`
`agents
`
`including
`
`functional analogues are known as stabilizing agents for a
`
`number of chemicals,
`
`living tissues and even organisms,
`
`including viruses (Colaco et al., 1992;
`
`Crowe and Crowe
`
`2000). Trehalose, a disaccharide,
`
`has been documented to
`
`be involved in the stabilisation of membranes and proteins
`
`in dry animals and other anhydrobiotic organisms.
`
`Such
`
`organisms
`
`include,
`
`for
`
`example,
`
`dry
`
`baker's
`
`yeast
`
`Sacchoromyces cerevisiae,
`
`resurrection plants, cysts of
`
`certain crustaceans
`
`(including the brine shrimp Artemia)
`
`and many bacteria (Crowe and Crowe, 2000, Nature Biotech.,
`
`18, pp 145—146). Trehalose has also been shown to preserve
`
`mammalian. cells during freezing‘
`
`(Beattie et al.,
`
`1997,
`
`Diabetes, 46, pp519—523) and proteins (Colaco et al., 1992,
`Biotechnology, 10, pplOO7—1011) during drying. Trehalose
`
`has
`
`also
`
`been
`
`documented
`
`to
`
`be
`
`involved
`
`in
`
`the
`
`stabilisation.of viruses in their native state by Bieganski
`
`et
`
`a1. ,
`
`1998,
`
`Biotechnol.
`
`Prog.,
`
`14,
`
`615—620.
`
`”Stabilization of active recombinant
`
`retroviruses in an
`
`amorphos dry state with trehalose"
`
`The present
`
`invention not only shows that
`
`trehalose
`
`may be used to stabilise viruses in their native state but
`
`for the first time shows
`
`that further stability results
`
`from trehalose treatment of viruses immobilised.by covalent
`
`attachment to a substrate. Thus, in a further aspect of the
`
`present
`
`invention there is provided use of
`
`trehalose for
`
`the further stabilisation of
`
`21 device comprising virus
`
`immobilised to a
`
`substrate according to the present
`
`invention and
`
`as
`
`hereinbefore
`
`described.
`
`Covalent
`
`immobilization (forming of a chemical bond) of proteins as
`
`known, results in a substantial increase in stability; The
`present
`inventors have also shown that this is true for
`
`bacteriophage 13f describing' herein.
`
`the stability‘ of
`
`an
`
`insoluble nylon/bacteriophage co—polymer of considerable
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`molecular weight.
`
`Thus,
`
`according
`
`to
`
`the
`
`present
`
`invention
`
`said
`
`virus(es) immobilised to said substrate may be for example,
`
`coated with.
`
`trehalose by,
`
`for example, dipping into a
`
`solution of trehalose before drying and storage such that
`
`the virus(es) maintain their viability and infectivity.
`
`Further applications of
`
`the present
`
`invention. may
`
`include the treatment of MRSA;
`
`food poisoning, wherein
`
`bacteriophage may be immobilised on a substrate such as
`
`microbead suspension, which can be ingested; or
`
`in the
`
`decontamination of hospital equipment/surfaces; Prevention
`
`of infection.byInethicillin—resistant Staphylococcus‘aureus
`
`or
`
`VISA
`
`(vancomycin
`
`insensitive
`
`S.
`
`aureus)
`
`by
`
`immobilization of
`
`appropriate bacteriophage onto,
`
`for
`
`example, sutures or wound dressings; prevention of specific
`
`pathogen entry through catheters and similar devices by
`
`immobilization of
`
`appropriate bacteriophage
`
`onto
`
`the
`
`treatment of pulmonary infection
`the device;
`surface of
`such as tuberculosis with, for example, micro—particles of
`
`about 10 microns diameter with appropriate bacteriophage
`
`immobilized
`
`onto
`
`them,
`
`by
`
`inhalation;
`
`treatment
`
`of
`
`infections such.as meningitis, for example, by injection of
`
`micro particles with. appropriate bacteriophage strains
`
`immobilized;
`
`treatment of gastrointestinal infections, for
`
`example,
`
`by particles or gels
`
`containing appropriate
`
`immobilized bacteriophage;
`
`treatment of bacterial plant
`
`diseases; elimination of, for example, E.coli in cattle by
`
`incorporation into the diet of
`
`immobilized bacteriophage;
`
`incorporation of
`
`appropriate
`
`bacteriophage
`
`into food
`
`wrapping materials to prevent or eliminate contamination by
`organisms
`causing
`food.
`poisoning;
`incorporation.
`of
`
`prevent
`to
`paints
`into
`bacteriophage
`appropriate
`contamination of surfaces in; for example, hospitals, or
`
`farms; treatment of surfaces of air—conditioning units with
`
`appropriate
`
`immobilized bacteriophage
`
`to prevent,
`
`for
`
`example, Legionella contamination.
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`The present
`
`invention may also be used for
`
`the
`
`purposes of vaccination.
`
`For
`
`example,
`
`the present
`
`invention may be used to immobilize any live,
`
`infectious
`
`virus, which could be then used to vaccinate populations at
`
`risk.
`
`This may be particularly useful where no vaccine
`
`exists for a particular virus and any form of prevention of
`
`viral
`
`infection. would. be useful.
`
`Thus,
`
`a 'virus as
`
`hereinbefore described may be immobilised and used directly
`
`to vaccinate reducing the time taken to develop a more
`
`standard attenuated virus.
`
`It will be understood that such
`
`vaccinations may only be conducted, especially with regard
`
`to humans,
`
`in extreme cases. Without wishing to be bound
`
`by theory,
`
`the action of
`
`the immobilised virus used to
`
`vaccinate would.be such that the immobilized virus would be
`
`unable to reach its target cells from the vaccination site;
`
`preventing the immobilised virus infecting the patient.
`
`It will be understood,
`
`therefore,
`
`that
`
`immobilised HIV
`
`virus would not be able to be used for vaccination in this
`
`regard.
`
`Preferably,
`
`the immobilised virus is kept at the
`
`inoculation. site for
`
`longer
`
`than conventional
`
`‘vaccines
`
`allowing
`
`a better
`
`immune
`
`response
`
`to be
`
`raised.
`
`Advantageously,
`
`the present
`
`inventor has
`
`shown that
`
`the
`
`present
`
`invention of
`
`immobilising viruses substantially
`
`reduces or eliminates free, unimmobilised viruses.
`
`In a further aspect,
`
`the present invention provides a
`
`method of preparing a device comprising a substrate having
`
`virus immobilised thereon, said method comprising the steps
`
`Of:
`
`a) activating the substrate so as to enable virus to
`
`bind thereto;
`
`b) mixing the modified substrate with virus and a
`
`coupling agent
`substrate.
`
`to aid the binding of virus to the
`
`D1 a yet
`
`further aspect said method comprises
`
`the
`
`further step of:
`
`c) mixing the device with a a stabilising agent that
`
`maintains the viability and infectivity of the virus
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`
`bound to the modified substrate when said modified
`
`substrate is exposed.to dehydration, prolonged storage
`
`and/or other stresses.
`
`It should be understood that the term "activating" is
`
`as hereinbefore defined.
`
`Preferably,
`
`the stabilising agent according to the
`
`present invention is trehalose or other agent such as known
`
`heat shock proteins known in the art that protect proteins
`
`or viruses against dehydration, prolonged storage and other
`
`10
`
`stresses.
`
`In a preferred embodiment of the present
`
`invention,
`
`said device mixed with a stabilising agent may be dried,
`
`allowing prolonged storage of said device whilst
`
`maintaining the infectivity and viability of the virus.
`
`The present invention will now be further described by
`
`way of example, with reference to the following methods and
`
`figures in which:
`
`Figure l— Graphical Representation of Wound Model 1
`
`The graph depicts the activity over
`
`three days of
`
`nylon strips with. or without bacteriophage in. killing
`
`Staphylococcus aureus bacterium present on the surface of
`
`raw pork to simulate a surgical wound.
`
`The activity
`
`represents the average of three replicates, score + or —,
`
`wherein + depicts the clearing of bacterium and — depicts
`
`the bacterium remaining cloudy.
`
`Figure 2 — Graphical Representation of Wound Model 2
`
`Activity is measure over 9 days and is scored as in Figure
`
`1.
`
`Nylon +/— bacteriophage is inserted into a wound in
`
`fresh raw pork, which is replaced with fresh tissue every
`
`three days.
`
`Figure 3
`— Graphical Representation of Resistnce of
`
`Immobilised Bacteriophage to proteolytic activity
`
`Series 1 — control;
`
`Series 2V— 0.1 g/l trypsin;
`
`Series 3
`
`— 0.5 g/l trypsin;
`
`Series 4 — 2.5 g/l trypsin
`
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`Figure 4
`
`12
`
`Graphical representation of numbers of bacteriophage
`
`immobilised on activated nylon.
`
`The reduction of phage
`
`numbers with time is depicted.
`
`Lisilre—S
`
`As
`
`for Figure 4,
`
`repeated. with larger strips of
`
`activated nylon (5 x 1 cm)
`
`Figure 6
`
`Schematic drawing of Wound Models 1 and 2 (depicted in
`
`10
`
`Figures 1 and 2 respectively).
`
`Figure 7
`
`— Graphical Representation of Infection of
`
`Animal Cells by Adenovirus.
`
`The graph depicts the infectivity of free adenovirus,
`
`and adenovirus immobilised onto nylon on HEK 293 cells.
`
`15
`
`Methods
`
`Propagation: An overnight subculture of bacteria was
`
`adjusted to a cell concentration of 1.5x109 cells/ml. 0.1ml
`
`of this was mixed with 1x1§ pfu (plaque forming units) of
`
`bacteriophage. After incubation at 37°C for 2Q minutes the
`
`mixture was poured onto 1.5% LB agar,
`
`0.7% LB agar was
`
`layered over
`
`this
`
`and allowed to set.
`
`Plates were
`
`incubated at
`
`37°C for
`
`12
`
`hours.
`
`Almost
`
`confluent
`
`bacteriophage plaques were formed.
`
`Bacteriophage were harvested by adding 5ml of sterile
`
`bacteriophage
`
`suspension
`
`buffer
`
`and
`
`shaking.
`
`The
`
`bacteriophage suspended in the
`
`buffer were purified by
`
`centrifugation. and filtration through. a 100kDa cut—off
`
`filter to remove bacterial protein” Yield was 1xl0919fu/ml.
`
`Plaque assays: for the presence of bacteriophage were
`
`carried out by the two layer plate assay as described for
`
`propagation.
`
`Immobilisation: Nylon strip 8x1 cm was used.
`
`Activation: Nylon was
`
`activated by preliminary
`
`hydrolysis with 4M HCl for 2.5 minutes at 70°C, washed in
`
`distilled water and 0.1M sodium bicarbonate to remove acid.
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`
`Coupling to nylon or other polymer with amino or carboxyl
`
`surface groups.
`
`i)
`
`Carbodiimide as a coupling agent.
`
`After this brief acid hydrolysis of the nylon surface the
`
`sample is washed with dimethylformamide (DMF) and 20mM l—
`
`cyclohexyl—B—[2—morpholinoethyl]—carbodiimidemethyl—p—
`
`toluene sulphonate is added.
`
`The solution is stirred for
`
`90 minutes, and then the nylon is washed with DMF.
`
`The
`
`activated nylon is stirred overnight with bacterophage in
`
`suitable buffer,
`
`and
`
`then washed
`
`to remove
`
`unbound
`
`bacteriophage.
`
`ii)
`
`Glutaraldehyde as a coupling agent.
`
`After a brief acid hydrolysis of
`
`the nylon surface the
`
`sample is washed with 0.1M bicarbonate buffer pH9.4 and
`
`incubated. with 10% glutaraldehyde in 0.1M bicarbonate
`
`buffer.
`
`The surface was then washed in bicarbonate buffer
`
`and distilled water before being incubated overnight with
`
`bacteriophage in a suitable buffer.
`
`Coupling to cellulose or other hydroxyl—containing polymer.
`
`i)
`
`Vinylsulfonylethylene ether
`
`Vinylsulfonyl groups
`
`can. be
`
`introduced into hydroxyl—
`
`containing polymers by treatment of the polymer with vinyl
`
`sulfone
`
`at
`
`lel.
`
`The activated polymer
`
`is
`
`stirred
`
`overnight with bacteriophage in suitable buffer, and then
`
`washed to remove unbound bacteriophage.
`
`ii)
`
`Triazine addition
`
`Cellulose or a modified cellulose (about 109)
`
`is added to
`
`50ml of acetone/water
`
`(1:1) containing lg 2—amino—4,
`
`6—
`
`dichloro—s—triazine at 50° and stirred for 5 minutes. Then
`
`20ml of
`
`15% (w/v) aqueous sodium carbonate to which 0.6
`
`vol. of 1M HCl has been added is poured into the reaction
`
`mixture.
`
`Concentrated HCl
`
`is then added to bring the
`
`mixture
`
`pH
`
`below 7.
`
`The
`
`amino—chloro—s—triazine
`
`substituted cellulose is washed with acetone/water,
`
`then
`
`water and finally with 0.05M phosphate buffer as pH7.0.
`
`The COupling reaction with the bacteriophage is carried out
`
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`at pH8.0 in 0.05M phosphate buffer by stirring for 12 to 18
`hours.
`
`14
`
`Coupling to Polythene or other similar polymer
`
`1. Corona discharge
`
`Polythene was exposed to a corona discharge for about
`
`1
`
`second;
`
`bacteriophage dehydrated in the presence
`
`of
`
`trehalose was dusted onto the treated surface immediately.
`
`2.
`
`Permanganate oxidation.
`
`10
`
`Polythene
`
`was
`
`exposed
`
`to
`
`concentrated
`
`potassium
`
`permanganate
`
`solution, for
`
`several hours, washed with
`
`distilled water and immediately treated with bacteriophage
`
`in trehalose or other stabilizing agent.
`
`15
`
`Example 1
`
`1. Bacteriophage P1 with Escherichia coli 11291
`
`The nylon/bacteriophage preparation was challenged with
`
`50ml of bacterial culture at about 1x1f cells/ml.
`
`After incubation the culture was assayed by the two layer
`
`20
`
`plaque assay.
`
`Example 2 Bacteriophage A against E.coli.
`
`In this experiment
`
`the number of pfu's used in the
`
`preparation of the immobilised system is compared with the
`
`number of pfu's observed when the immobilised system is
`
`challenged with the bacteria.
`
`Immobilised bacteriophage
`
`Free bacteriophage
`
`no bacteriophage
`
`The numbers of bacteriophage plaques arising from the
`
`immobilised
`
`systems with
`
`this
`
`bacteriophage—bacteria
`
`combination shows:
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`15
`
`0 That
`
`immobilised bacteriophage are viable and infective.
`
`O a relationship between free bacteriophage numbers used.in
`
`the preparation and the number of bacteriophage produced.by
`
`immobilised systems.
`
`Example 3
`
`« Unknown bacteriophage against
`
`Staphylococcus.aureus.
`
`Bacteriophage was isolated by incubating a lawn of S.
`
`aureus with contaminated water which had been filtered
`
`through a 0.18u filter to remove bacteria. Where a plaque
`
`formed indicated the presence of a lytic bacteriophage.
`
`This was isolated and grown as previously described.
`
`Plaques expected
`
`Plate count
`
` _
`
`n
`
`Example 4 Effect of trehalose on viability
`
`The experimental system was as previously described
`
`except
`
`that
`
`the nylon/bacteriophage preparations were
`
`dipped into a trehalose solution of various concentrations
`
`and dried before assay (Nylon/bacteriophage preparations
`
`previously described were stored in buffer for 24 to 48
`
`hours before use) dried preparations were used 72 hours
`later.
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`
`Free bacteriophage
`
`no bacteriophage
`
`Immobilised bacteriophage
`
`Trehalose 0.1% Trehalose 0.05%
`
`Trehalose 1%
`
`Trehalose 0.5%
`
`The data indicate that trehalose enables immobilised
`
`bacteriophage to withstand dessication and storage for at
`
`least 72 hours without significant
`
`loss of viability and
`
`infectivity. The free bacteriophage, no bacteriophage and
`
`immobilised.bacteriophage samples were controls not treated
`
`to dessication and storage.
`
`Dilution
`
`Plaques expected
`
`Plate count
`
`1
`
`1
`
`100000
`
`1000
`
`10000
`
`The data show that significant numbers of viable and
`
`infective bacteriophage have been immobilised on the nylon
`
`sheet. There is also a dose/response relationship between
`
`the estimated numbers of bacteriophage immobilised and the
`
`plaques formed from the immobilised system.
`
`Validation of washings:
`
`The washings
`
`from.
`
`the nylon/bacteriophage reaction
`
`were assayed by the two layer plaque assay to determine the
`
`rate and efficiency of removal of free bacteriophage (those
`
`that did not
`
`form covalent attachment as a result of the
`
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`
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`chemistry).
`
`17
`
`pm
`
`10000
`
`1
`
`5
`
`10
`
`Example 5
`
`Bacteriophage NCIMB 9563(ATCC6538—B) against
`
`Staphylococcus aureus in the presence of tissue.
`
`Bacteriophage 9563 was grown as previously described,
`
`immobilized onto nylon membrane, washed to remove unbound
`
`15
`
`bacteriophage and tested against a strain of S. aureus in
`
`two experimental situations.
`
`1.
`
`Does the presence of animal tissue affect the response
`
`of the immobilised bacteriophage?
`
`The nylon membrane with immobilized bacteriophage was
`
`20
`
`placed in a flask with 50m
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