Delivery of viral agents

Abstract

Plant material has bacteriophage has been attached, wherein the bacteriophage retains infectivity. The plant material includes fruits, vegetables, leaves, stems, flowers, roots, tubers, seedlings and seeds. Plant diseases and animal diseases can be treated. A separate composition comprises a carrier selected from (i) a filament, (ii) a planar material, and (iii) particles and/or beads, and bacteriophage covalently attached thereto, wherein the bacteriophage retains infectivity, useful in treatment or prevention of bacterial infection in a deep wound.

Claims

1. Plant material, selected from seeds, roots and tubers, to which bacteriophage has been covalently attached, wherein the bacteriophage is attached to the plant material by electrical activation of the plant material and then attaching the bacteriophage to the activated plant material, and wherein the bacteriophage retains infectivity.

2. The plant material of claim 1, being seeds for consumption or seed stock.

3. The plant material of claim 1, being tomato seeds.

4. The plant material of claim 1, for prevention or treatment of a plant disease.

5. The plant material of claim 1, for prevention or treatment of bacterial infections in the gastrointestinal tract.

6. The plant material of claim 1, wherein the bacteriophage is covalently attached to a particle and the particle is covalently attached to the plant material.

7. A method of treating plant material selected from seeds, roots and tubers, comprising activating the plant material and then covalently attaching the bacteriophage to the activated plant material, wherein the bacteriophage retains infectivity.

8. The method of claim 7, wherein the plant material comprises seeds for consumption or seed stock.

9. The method of claim 7, wherein the plant material is for prevention or treatment of a plant disease.

10. The method of claim 7, comprising covalently attaching the bacteriophage to a particle or particles and covalently attaching the particle or particles to the plant material.

11. A composition comprising the plant material of claim 1.

Description

(1) The present invention will now be further described by way of example and with reference to the figures in which:

(2) FIG. 1 shows a schematic diagram of a wound dressing to which an LVCC (as one or more filaments or strips to which bacteriophage have been attached singly or in combination) is added;

(3) FIGS. 2a and 2b shows schematic diagrams of a dressing incorporating a LVCC;

(4) FIG. 3 shows a schematic diagram of a dressing bearing multiple bacteriophage in an identifiable pattern;

(5) FIG. 4a shows wound statistics for animals with deep wound infected with 70 l of 110.sup.5 cfu/ml E15 suspended in 5% hog gastric mucin using normal or bacteriophage treated LVCC-7 days post surgery;

(6) FIG. 4b shows bacterial load for animals with deep wound infected with 70 l of 110.sup.5 cfu/ml E15 suspended in 5% hog gastric mucin using normal or bacteriophage treated LVCC-7 days post surgery;

(7) FIG. 5 shows clearing of MRSA by bacteriophage immobilised onto sutures recovered from rats 14 days after insertion into infected wounds;

(8) FIG. 6a shows stability conferred by LVCC against desiccation;

(9) FIG. 6b shows stability conferred by LVCC against ultra violet radiation;

(10) FIG. 6c shows stability conferred by LVCC against temperature;

(11) FIG. 7 shows survival of immobilised bacteriophages in soil;

(12) FIG. 8 shows exposure of LVCC with two bacteriophages to two bacterial species each susceptible to one of the bacteriophages;

(13) FIG. 9 shows a lateral, external view of an infected control would site on day 14;

(14) FIG. 10 shows a dorsal, internal view of an infected control would site on day 14;

(15) FIG. 11 shows a lateral, external view of a wound site treated with a filament in accordance with the invention on day 14;

(16) FIG. 12 shows a dorsal, internal view of the site of FIG. 11, showing reduced swelling and reduced pus formation within the paraspinous muscle on day 14.

(17) FIG. 13 shows germinated tomato seeds (control);

(18) FIG. 14 shows germinated tomato seeds with phage immobilised on the seed surfaces in accordance with the invention;

(19) FIG. 15 shows germinated tomato seeds treated with corona discharge (but no phage);

(20) FIG. 16 shows germinated tomato seeds treated with free phage; and

(21) FIG. 17 shows clearing of bacteria around cellulose particles in accordance with the invention.

EXAMPLES

Example 1

Effectiveness of LVCC for Delivery of Bacteriophage to Counteract Deep Wound Infection

(22) FIG. 1 shows schematically an LVCC of the invention (1) pressed onto a wound (4) in a tissue (3) by dressing (2).

(23) FIG. 2a is an inverted dressing of the invention incorporating an LVCC, shown in cross section. A pouch (5) encloses an LVCC (6) adhered to dressing (7), having top dressing surface (8)hence it is inverted in the figure.

(24) FIG. 2b shows schematically an LVCC (9) woven into a dressing (10). The two filaments (9a and 9b) are of different colours and have different bacteriophage attached. FIG. 3 shows a further schematic of a dressing (11) into which a filament with phage attached (12) is incorporated.

(25) In a first example the effectiveness of an LVCC for delivering bacteriophage, to counteract bacterial infection within an infected deep wound environment using an in vivo model is demonstrated. Deep wounds penetrating several tissue layers and infected at all levels with Methycillin Resistant Staphylococcus aureus (MRSA) (E15) were treated with an LVCC comprised of bacteriophages active against the infecting MRSA strains.

(26) Bacteriophage were immobilised to a synthetic nylon polymer filament similar to that used in the manufacture of wound closure sutures. Infected wounds were closed using the LVCC as suture, at various levels. The filamentous LVCC had either anti-MRSA bacteriophages immobilised to their surface (the prototype LVCC to be tested) or did not (control).

(27) In all subjects treated with the LVCC, infection was controlled and decreased (as demonstrated visually and by bacterial count (see FIGS. 4a and 4b) with wound healing proceeding normally. By contrast MRSA growth in control subjects proceeded unchecked resulting in an inflamed suppurating wound that did not heal.

(28) The results show that, using LVCC's, bacteriophages can deliver a potent bactericidal effect for an extended period in an in vivo modelsee Tables 1A and 1B.

Example 2

Extended Stability

(29) LVCC's recovered from infected subjects were washed and stored at 4 C. and periodically evaluated for continuing antimicrobial activity against MRSA grown in vitro. In a series of experiments anti-MRSA activity was maintained as demonstrated by LVCC placed within a lawn of bacteria. Bacteriophage activity (as shown by the clearing seen around the recovered LVCCs) extended several weeks following storagesee FIG. 5.

Example 3

Stability Conferred by LVCC Against Desiccation, Ultra Violet Radiation and Temperature

(30) In these examples LVCC were generated using activated nylon polymer or cotton fibres as the carrier substrate. LVCC with attached bacteriophage were exposed to a series of environmental conditions, including dehydration, shown by the effect of relative humidity on bacteriophage survival (see FIG. 6a), ultraviolet light (see FIG. 6b) and elevated temperature (see FIG. 6c), all conditions known to denature free bacteriophages.

(31) Three 21 cm phage strips were placed on LBM agar and exposed to UV light in the sterile cabinet for 5, 15 and 30 min. The strips were then turned over and placed on a fresh area of agar and the UV exposure repeated. Control phage strips were treated similarly, but were shielded from UV. The strips were then transferred to 15 ml LB broth, inoculated with 50 ul Staphylococcus aureus suspension and incubated.

(32) Resultssee Table 2

(33) Conclusion: Attached phage can withstand up to 30 min exposure to cabinet UV. S. aureus bacterial cells are killed by 5 minutes exposure to the UV source.

Example 4

Sustained Viability in Extreme Environments

(34) In this example the enhanced stability and viability of LVCC bacteriophage was determined following exposure to the astringent conditions provided by soil. The study involved construction of two types of LVCC using bacteriophage immobilised to either a nylon polymer or cellulose. Each LVCC was then buried in non sterilised or sterilised soil and samples taken over a three week period to assess the antibacterial activity of the LVCC.

(35) Results show enhanced survival of bacteriophage for both LVCC bacteriophage types compared to free bacteriophage (see FIG. 7).

Example 5

LVCC Composed of Multiple Bacteriophage

(36) Evaluation of LVCC performance using two bacteriophages active against two different target bacteria following immobilisation onto a common carrier substrate and tested for antibacterial activity. Percentage clearing on bacterial lawn is based upon the average clearing by each phage on their host strain measured in mm).

(37) Results show no diminution in antibacterial activity, with LVCC bacteriophages remaining active against both target bacteria (see FIG. 8). Exposure of treated material to 2 bacterial species (1 host, 1 non-host) still results in clearing of susceptible hosts.

Example 6

Deep Wound Treatment

(38) In 10 control animals (rats), a deep incision into the paraspinous muscle was infected with 100 l of 510.sup.7 cfu/ml E15 MRSA suspended in 5% hog gastric mucin and the wound was closed with normal sutures. The 10 test animals with the same incision received 100 l of 510.sup.7 cfu/ml E15 MRSA suspended in 5% hog gastric mucin and a bacteriophage treated filament was inserted into the deep wound. The bacteriophage immobilised was phage K.

(39) All animals were treated with analgesics following surgery and observed at hourly intervals for the first 12 hours and at 3 hour intervals for the next 12 hours, then at 6 hourly intervals for the next 2 days post infection and twice daily thereafter.

(40) Wound sites were examined at these time intervals for signs of inflammation and infection (pus formation) up to 14 days. Visual results are presented in Table 3.

(41) Referring to FIG. 9, showing the infected control: as seen, the site remained large and swollen and was poorly if at all healed, with a raised incision site on day 14. When opened, as shown in FIG. 10, a pus-filled incision site was revealed.

(42) The use of a test filament with bacteriophages attached is shown in FIG. 11. This was a well healed incision site with minimal swelling on day 14. The internal site exhibited reduced swelling to the wound site and reduced pus formation within the paraspinous muscle on day 14.

Example 7

Immobilisation of Bacteriophages onto Tomato Seeds

(43) Bacteriophage Preparation

(44) We grew Pectobacterium carotovorum strains on 20% tryptic soy broth (other suitable medium are also known) and infected the culture with a lytic bacteriophage in the logarithmic phase of growth. When lysis had occurred we purified the bacteriophages by centrifugation and washed the bacteriophage pellet three times by re-suspending in distilled water and concentrating by centrifugation.

(45) Corona Treatment

(46) Tomato seeds were treated with a corona field at 75 kV for 1 second, then immersed in the bacteriophage suspension (an alternative is to spray the treated seeds with the bacteriophage suspension). We washed the seeds with water three times to remove unbound bacteriophages (an alternative is that the bacteriophage suspension be applied to the seeds prior to corona treatment). Note that other field strengths are also effective and washing is only needed to demonstrate that the observed anti-bacterial effects are produced by covalently bound bacteriophages rather than adsorbed free bacteriophages.

(47) Germination

(48) We thus prepared seeds that were (i) untreated, (ii) treated with corona discharge, (iii) treated with corona discharge in the presence of bacteriophage and (iv) treated with free bacteriophage, and germinated the seeds on paper moistened with water in a dish at room temperature (an alternative it to germinate on tryptic soy agar (broth with 2% agar) with and without Pectobacterium).

(49) Results

(50) Tomato seeds showed enhanced germination with corona (FIG. 15) and corona with anti-Pectobacterium phages (FIG. 14) compared with the control (FIG. 13, no corona) and with no phage (FIG. 16). Clearing zones will be seen around the seeds treated with corona and anti-Pectobacterium phages on agar inoculated with suitable Pectobacterium strains (susceptible to the bacteriophages used). Tomato seed surface is largely cellulose and we then separately tested the viability of bacteriophage immobilised onto cellulose particlessee below.

Example 8

In Vivo Plant Pathogen Assay

(51) We immobilised Pectobacterium phages onto cellulose powder (size range approximately 100 microns-1 mm) following the corona discharge methods of WO2007/072049.

(52) We inoculated an agar plate with suitable Pectobacterium strains (susceptible to the bacteriophages used) and added treated cellulose particles in three locations.

(53) Referring to the results shown in FIG. 17, clearing around the powder particles can be seen showing the activity against the soft rot bacteria of the immobilised bacteriophages. This confirmed viable bacteriophage were attached to the cellulose material.

Example 9

Bacteriophage on Cellulose Particles

(54) We immobilised Pectobacterium phages onto cellulose powder (size range approximately 100 microns-1 mm) following the corona discharge methods of WO2007/072049.

(55) We inoculated agar plates with suitable Pectobacterium strains (susceptible to the bacteriophages used) and added treated cellulose particles in three locations.

(56) We treated plates with (i) nothing=control, (ii) free phage in solution and (iii) phage attached to the cellulose particles in solution, and titrated (ii) and (iii) until similar activity in clearing bacteria was observed. Similar activity occurred when approximately 100 free phage were used compared with 1 phage attached to a cellulose particle. This confirmed improved properties of immobilised phage compared with free phage.

Example 10

Treatment of Potatoes

(57) Potato tubers were treated with (i) sterile water=control, (ii) free phage, and (iii) phage specific for the bacterium responsible for potato blackleg immobilised onto cellulose particles as per examples 8 and 9. Tubers were sprayed with solution and allowed to dry.

(58) Tubers were planted and growth was recorded as number of plants, total number of stems, and number of plants infected with Blackleg. The results are shown in Table 4.

(59) Hence, we found that treating with Pectobacterium and spraying with water (+ve control) had the lowest number of emerging plants and stems with greater incidence of blackleg, comparing to the other treatments.

(60) The treatment with free phage had little effect on the total number of emerged plants.

(61) There was an increase in the number of stems per plant when compared to the +ve control.

(62) Only one plant treatment with free phage had symptoms of blackleg.

(63) The treatment with immobilised phage increased the number of stems per plant when compared to the control, as well as the number of plants emerged. At the last observations, 8 weeks after planting, no symptoms of blackleg had been observed in the tubers treated with immobilised phage, showing persistence of the anti-bacterial activity.

(64) TABLE-US-00001 TABLE 1A Visual results from wounds treated with untreated LVCC or untreated LVCC infected with 100 l 10.sup.8 cfu/ml E15 suspended in 5% hog gastric mucin Untreated sutures No. of animals Procedure Visual observations 3 Incision only (negative No swelling or inflammation. control) Slightly raised appearance with presence of clear fluid internally 4 Incision infected with Significantly raised hard lump 100 l 10.sup.7 cfu/ml E15 & with presence of dark thick hog gastric mucin necrotic pus internally (positive control)

(65) TABLE-US-00002 TABLE 1B Visual results from wounds treated with either untreated or bacteriophage-treated LVCC & 100 l of 5 10.sup.7 cfu/ml E15 suspended in 5% hog gastric mucin No. of Appearance of animals wound Internal observation Untreated sutures 10 Large, hard swollen Dark, pus formation lump Bacteriophage-treated LVCC 10 Small, raised, Clear, fluid-filled incision site swelling

(66) TABLE-US-00003 TABLE 2 (Phage activity (P) indicated by clear broth. Bacterial growth (+) by cloudy broth) Exposed strips Shielded strips UV (min) r1 r2 r3 r1 r2 r3 5 P P P P P P 15 P P P P P P 30 P P P P P P

(67) TABLE-US-00004 TABLE 3 Visual results from wounds treated with either untreated or bacteriophage-treated filaments No. of Appearance of animals wound Internal observation Untreated filaments 10 Large, hard swollen Dark, pus formation lump Bacteriophage treated filaments 10 Small, raised, Clear, fluid-filled incision site swelling

(68) TABLE-US-00005 TABLE 4 No. of Group plants No. of stems No infected with Blackleg Control 20 52 3 Free Phage 21 82 1 Immobilised Phage 26 110 0