Probiotic composition based on the enterococcus strain and used as a treatment means and method for the production thereof

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Protein assay to determine protein concentration of vaccine

To determine the amount of protein in milligrams per millilitre that a sample may contain, a Bio-Rad protein Assay kit was used. The linear range for this assay is between 0. 05mg/ml and 0. 5mg/ml, consequently samples were tested in the neat form and prepared in the dilutions of 1: 5,1 : 10 and 1: 20. Then lOgI of blank (distilled water), standard or sample was pipetted into the assigned wells of a 96 well Nunc microtitre plate. Then 1001l1 of diluted dye reagent was added to each well and the plate was left to incubate at room temperature for a minimum of 5 minutes and a maximum of 1 hour.

A Bio-Rad Microplate Reader (Model 550) was used to read the plates at 595nm wavelength. Raw data was automatically processed through the Bio-Rad Microplate Manager computer program (Version 5.0. 1), producing a standard curve and calculating the protein concentration of the samples.
Production of probiotic adjuvant, P. ieiiseiiii 702
The strain of probiotic used as an adjuvant in this experiment is Propionibacterium jensenii 702. It is referred to as'P. jensenii 702'or'probiotic'in this example.
Preparation of P. jensenii 702 for drinking water

The Propionibacterium was resuscitated from frozen culture by placing 100PLI in 20 ml of Sodium Lactate Broth (SLB) and grown anaerobically at 30 C for 3 days. After the three days, 1001l1 was transferred into 20 new bottles each containing 20ml of SLB. These were also grown anaerobically at 30 C for 3 days. The bacterial cells were then collected by combining all of the culture bottles, except one and putting them into 2 sterile 250ml centrifuge bottles (Nalgene). The one extra bottle was kept aside for use in preparing the adjuvant for the vaccine. These were centrifuged at 10,000rpm for 7 minutes at 4 C and the supernatant was discarded. The cells were washed a further 2 times in 60mL of sterile 0.85% NaCl (Sigma). After the final wash the cells were resuspended in 30 ml of sterile 0. 85% NaCl (Sigma) and aliquoted in 0. 5ml lots in 5ml sterile containers (Sarstedt) and stored at 4 C until required for use in drinking water. This produced bacteria at a concentration of 108 cfu when added to the drinking water. Each week a fresh lot of bacteria was produced for use in the drinking water to ensure its viability.

Preparation of P. jensenii 702 for use as adjuvant i7l vaccine 10ml from the remaining 20ml bottle of P. jensenii 702 growth in SLB was centrifuged separately at 5000rpm for 10 minutes. The supernatant was removed and it was washed two times in sterile 0.85% NaCl (Sigma) at 5000RPM for 10 minutes. After the final wash, the supernatant was removed and the pellet of cells was resuspended in 200ut of sterile 0.85% NaCl (Sigma) in a sterile container. This was prepared the day prior to vaccination and stored at 4 C until used. The concentration of bacteria was approximately 109 cfu.
IMMUNISATION Ethics Approval

Ethics approval was gained from The University of Newcastle Animal Ethics

Committee. Consequently, this research was carried out under the ethics approval number of 712 0902.
Handling of Test Animals

The animals used for this experiment were between 8-12 week old female C57 mice.

The mice were separated into 5 groups, with 8 mice in each group, according to the vaccine that they were to be administered. Within each group the mice were further split into two more groups to accommodate 4 mice per cage with a total of 10 cages housing the total of 40 mice used in this experiment. All mice were checked daily and provided with fresh drinking water and feed. The 3 groups of mice that were given P. jeitsenii 702 in their vaccine were also given the probiotic in their drinking water. The 0. 5ml aliquots of 108 bacteria that were

prepared earlier were added to 50ml of drinking water. Fresh bacteria were added each day.

After arrival at the animal holding facilities, the mice were allowed a week of rest to adjust to their new surroundings.
Oral Vaccine Administration 'The vaccine was administered orally by a 21-gavage lavage needle. Each mouse was anaesthetised by inhalation (CIG TM41 Anaesthetic Apparatus, Nova Medical Vaporiser) in an anaesthetic chamber, using Halothane BP (lml/ml, Laser Animal Health) at a rate of 5L/min and 4L/min oxygen prior to having the needle inserted into the mouse's stomach. A total of 100, ul of vaccine (Table 26) was administered to each animal. The vaccines were made up in PBS. The animals were vaccinated a total of three times, with a 7day period elapsing between each vaccination.
Table 26 Five vaccines tested in this study and their contents
S I I t ,,, re o., - ages ; : I STCF 200gg/looftl 5-6 aAd-'uvanf

1 STCF 200) ig/100) il 5-6

P SOwI 108 cfu l 100w1

2 STCF loogg/looftl 7-8

WTB 100wg/100w1

P 50 1 10$ cfu/100 1

3 P100110'cfu/1009-10

4 STCF 200Rg/100ul 1-2

CT 10R1

5 STCF loogg/loogI 3-4

WTB 1001lg/100ll1

CT 10, ul STCF= Short Term Culture Filtrate; WTB= Sonicated Whole Tuberculosis; P= P. jensellfi 702, CT= Cholera Toxin (Sigma, lmg/ml in PBS) Blood Sa zples

Blood was taken on two occasions. On the same day prior to the first vaccination and on the last day of the animal study, blood was taken from the saphenous vein of each mouse.
The blood was then pumped into 1. 5ml eppendorf tubes and centrifuged at 5000rpm for 8 minutes. The serum was then transferred to a new eppendorf tube and stored at-80 C until required for immunoglobulin testing.
Faeces Collection

Two lots of faeces had to be collected for analysis in two separate tests. Both faecal samples were collected at the same time, on day 0 (first day of vaccination) and day 25 (final day of experiment). The two tests were to test for the antibody IgA and the other sample was to be used for testing for viable P. jensenii 702 cells.

Preparation of Faecal pellets for IgA Analysis

The faecal pellets were collected from each mouse and placed into an eppendorf tube containing 0. 5ml of Protease Inhibitor. The samples were then vortexed for 15 minutes at room temperature. Once the pellets had dissolved, the samples were centrifuged (Eppendorf Centrifuge 5415C) at full speed for 15 minutes at 4 C. Then 4001l1 of the supernatant from each sample was transferred to a new eppendorf tube containing 100F1 of glycerol (Sigma) and then 10u. l ofPMSF solution was added. The tubes were vortexed again briefly and then stored at-80 C until required for IgA testing.

Faecal Plate Counts

The faeces were also tested for viable Propionibacterium jensenii 702. This was performed to see if the bacteria survived the gastrointestinal tract. The faeces samples collected for this test were contained from each animal and placed in 2ml of faeces buffer.

Dilutions of 10 4, 10-5, and 10-6 Diluted Wilkins Chalgren Anaerobe Broth (WCAB) (Oxoid).
The samples were vortexed and Iml was placed into the dilute WCAB and the serial dilutions were made, vortexing the samples between each dilution. Then 100F1 of each dilution were put onto plates containing Sodium Lactate Agar (SLA). The plates were spread with a glass rod, left to dry and then inverted and incubated anaerobically at 30 C for 6 days.
Removal of Tissue Samples

The mice were euthanased by firstly anaesthetising with Halothane and then submersion in a tank containing excess carbon dioxide. The abdomen area was then sterilised with 70% ethanol. The abdomen was cut open with sterile dissection scissors and the spleen, Peyer's patches and mesenteric lymph nodes were placed in 40ml of cold sterile PBS and kept on crushed ice. Organs from each vaccine group were pooled to ensure an adequate number of cells.

Preparation of lymphocyte proliferations and cultures

A sterile environment was set up in a Clemco Laminar flow hood by subjecting the cabinet to UV light overnight prior to use. Anything that entered the hood was sprayed with 70% alcohol to retain the sterile environment.

Sieve Methodfor Preparing Cell Suspefasions frorn Tissue Samples

Single cell suspensions were created by macerating the tissue through a sterile sieve using the flat end of a sterile 20ml syringe plunger. The cells were washed through the sieve with sterile PBS and tissue debris, fat and connective tissue remained on the sieve surface.

The cell suspension was collected in a 50ml centrifuge tube (FALCON, Becton Dickinson).
Isolation of Lymphocytes

Spleen Cells

The cell suspension in PBS was placed on ice for several minutes. Large pieces of debris were removed and then suspension was centrifuged (Heraeus Megafuge 1. OR) at 1200rpm for 10 minutes at 4 C. The supernatant was removed and then the spleen cell pellet was resuspended in 5ml of Red Blood Cell Lysis Buffer and placed on ice for 5 minutes. Then 5ml of RPMI complete medium was added and centrifuged for a further 10 minutes at 1200rpm at 4 C. The spleen cell pellet was washed 3 times with 10 ml RPMI complete medium and finally resuspended in 10ml of RPMI complete medium and stored on ice until required.
Peyer's Patches and Meserzteric Lvmph Nodes

The cell suspension in PBS was centrifuged (Heraeus Megafuge 1. OR) at 1200rpm for 10 minutes at 4 C. The supernatant was discarded and the pellet was washed a further 3 times with 10 ml RPMI complete medium, and finally resuspended in 10ml RPMI complete medium and stored on ice until required.

Cell Counts of Spleen, Mesenterc Lymph Nodes a7Id Peyer's Patches

Cell counts were made by using trypan blue dye exclusion. The spleen cells were diluted 1 in 5 with trypan blue (Sigma), and the Peyer's patches and Mesenteric lymph nodes were diluted 1 in 2. The cell count was obtained using a counting chamber (WEBER) and an Olympus inverted microscope. The number of cells per ml was then calculated.

Lynzplzocyte Proliferations

The lymphocyte proliferations were set up in Nunclon Surface tissue culture plates.

A total of eighteen plates were set up. Six plates were needed to accommodate the several different proliferation combinations for each day of harvest. The plates were set up in a manner, as to test for the optimum antigen concentration, the best stimulating antigen, WTB, STCF, or a combination of WTB/STCF and the best vaccine. The optimum day for T-cell proliferation was determined by setting the plates up in replicate and harvesting the plates on day 3,4 and 5, to determine which gave the highest T-cell proliferations.
100. 1 of each respective antigen was added to the plates. The antigens were incorporated at different concentrations in RPMI complete medium. The final antigen concentrations tested were 2.5, 5 and lOjug/ml of each STCF, WTB or the combination WTB/STCF. Four wells were designated as blank control wells and were filled with 100wl of RPMI complete medium.
100tel of the cell suspensions were added to the plates at a concentration of 2 x 106 cells/ml. Tissue samples were run in quadruplicate where possible, however in some cases where low cell counts were obtained, particularly in the case of Peyer's patches, it was only possible to test some samples in duplicate or singlicate. The plates were incubated at 5% carbon dioxide (CO2) at 37 C for 3-5 days.

Concanavalin A Control Plate

A plate was set up containing 100ss1 of Lectin Concanavalin A (Con A) (Boehringer Mannheim). The Con A was made up to 2. 5) ig/ml. To the wells of the plate, 100u. l of tissue cells were added at concentrations of 2 x 106 cells/ml in RPMI complete medium. The plate was then incubated for 48 hours in 5% CO2 at 37 C.
Lymphocyte Cultures for Cytokisze Analysis

Spleen lymphocyte cultures were set up for use in testing for the presence of the cytokines, Interleukin 2 (IL-2), Interleukin 4 (IL-4) and Interferon Gamma (IFN-Y). The cultures were set up in duplicate in Multiwell tissue culture plates (Falcon, Becton Dickinson) using spleen cell tissue. Then 0. 5ml of 2 x 106 lymphocyte cells/ml in RPMI complete medium were added to each well. Different concentrations of the stimulating antigens, WTB, STCF and WTB/STCF were made up in RPMI complete medium in the following concentrations 2. 5 g, 5 g and 101lg. 0. 5ml of each of these antigens were added to the plate.

The cultures were incubated for 72 hours in 5% carbon dioxide at 37 C. The supernatants from each well were then collected and stored at-80 C until required for testing.
Harvesting of T-Cell Proliferations

On day 3 of the proliferation the first set of 6 plates were removed from the incubator and in a sterile bio-safety cabinet, 10, ul of [methyl-3H] thymidine tracer (Amersham) diluted in complete RPMI was added to each well (0. 51lCi/well). The plates were incubated for a further 6 hours before harvesting and reading. This process was repeated on the next two consecutive days for the day 4 and 5 sets of plates.

After the 6 hour incubation, the lymphocytes were harvested on a Packard Filtermate 196 onto glass fibre filters (Packard) and dried in a 60 C incubator. Once dry, each filter was placed on a plastic film on a hotplate and a sheet of Meltilex A scintillator wax (Wallac) was placed on top and melted through the filter. Once the filter cooled and the wax hardened, the filter was trimmed and placed in a sample bag (Wallac) and sealed on a Wallac 1295-012 Heat Sealer. The trimmed and sealed sample bag was placed in a cassette and read on a Liquid Scintillation and Luminescence Counter (TRILUX 1450 MicroBeta).
ELISA assays ELISA testing for IL-2, IL-4 and IFN-y

Pharmingen OptEIA Kits were used in cytokine testing. The ELISAs and required buffers and reagents were prepared according to manufacturer's instructions.

NUNC Immunosorbent multi-well plates were coated with 106RI of dilute capture antibody (Pharmingen), sealed and incubated overnight at 4 C. The wells were then washed 5 times with wash buffer. The plates were then blocked with Assay Diluent (Pharmingen)

and incubated at room temperature for 1 hour. Following that the plates were washed 5 times with wash buffer. Then 100111 of standard or sample was added to each well and incubated for 2 hours at room temperature. The plates were once again washed 5 times with wash buffer, and then 100, ut of Working Detector (Pharmingen) was added to each well, and the plate was incubated for a further hour at room temperature.

The plates were then washed 10 times with 30 second soaks. lOOgI of TMB substrate solution (Pharmingen) was added to each well, and was incubated at room temperature for 30 minutes in the dark. After this time had elapsed, 50gel of stop solution was added to each well. The resulting plates were immediately read on a Bio-Rad Microplate Reader (Model 550) at an absorbance of 450nm and 570nm. The 570nm reading gave the background reading that could be subtracted from the 450nm readings to give the final absorbance. The microplate reader adjusted results automatically, according to the standards run in each plate and gave final cytokine concentrations in picograms/ml (pg/ml).
ELISA testing of Seruf z and Faecal Sarnples for IgG afad IgA

The appropriate wells of NUNC Immunosorbent multi-well plates were coated with lOO, ul of 2511g/ml of antigen, STCF or combination STCF/WTB diluted in Bicarbonate Buffer. The plates were incubated overnight at 4 C. The plates were then washed twice with wash buffer. The plates were then blocked with 5% heat inactivated Foetal calf Serum (FCS) and incubated for 1 hour at 37 C. The samples were diluted. The serum was titrated 10 fold from 1 in 10 to 1 in 10000. Doubling dilutions were performed on the faeces from linIO to lin 80. Control serum was diluted 1 in 100 and added to each plate as a control. Two wells were also designated as blank wells, to act as a control for the presence of background staining. When the appropriate wells were filled with sample the plates were incubated for 1 hour at 37 C. The plates were then washed 3 times with wash buffer.

Then 100FL1 of labeled anti-mouse IgG Biotinylated (l. Omg/ml) was tested for serum samples or anti mouse IgA biotinylated (0. Smg/ml) (Southern Biotechnology Associates) for faeces samples was added to each plate. They were then returned to the incubator for 2 hours at 37 C. After this time elapsed, the plates were washed 3 times with wash buffer and then 100 l of Streptavidin Horse Radish Peroxidase (SA-HRP) was added to each plate. The plates were incubated for a further 1 hour at 37 C.
Then lOOul of the colour reagent TMB substrate solution (Pharmingen) was added to each well and the reaction was checked after 5 minutes for a change in colour. After 5 to 7 minutes, 50gel of stop solution was added to each well. The plates were then read at 450nm on a Bio-Rad Microplate Reader (Model 550).
Statistical analysis
Statistical analysis of the results were performed using Microsoft Excel 2000.
Student t-test, two-tailed, assuming equal variance was used. A'p'value of less than 0.05 was considered indicative of statistical significance between sets of data. Mean, standard deviation and all graphs were also generated in Microsoft Excel 2000.
RESULTS Vaccine preparation

Vaccine production for this study involved producing and concentrating sufficient quantities of STCF and sonicated WTB VACCINE ADMINISTRATION Animal Model : Mouse Monitoring

Mice were monitored daily by examination of their general appearance and behaviour. The mice were weighed prior to every procedure. Following any procedure mice were monitored regularly for one to two hours.
Table 27 Average weight of mice over study period of 25 days
t 6,-, E <

S., L t d k 0 E m X w ; S g-

1 18. 6 0. 9 19. 40. 8 18. 42. 0 18. 51. 3

2 18. 60. 9 19. 3 + 0. 8 19. 1 0. 8 18. 6 1. 3

3 18. 20. 9 19. 01. 0 19. 2 0. 9 19. 1 0. 6

4 18. 01. 5 18. 81. 1 19. 3 + 1. 1 18. 1 0. 8

5 18. 00. 7ab 18. 7 + 0. 6 19. 4 + 0. 6au 19. 00. 7b Values are mean SD (n=6-8). The same alphabetical prefix on values within a row indicates significant statistical difference (p < 0.05, student's t-test, two-tailed) between the weights on the indicated days.
The weight of mice in the study group remained relatively constant across the study period (Table 27). No statistical difference was observed in the weights between the mice groups on each weighing day. A statistically significant difference (p < 0.05) was identified between the weights of the mice in vaccine group 5 over the four week period. However this is not of any significance to the vaccine trial, nor did it affect results in other areas either.
In the last week of the vaccine trial, several of the test animals became sick and either died, or were euthanased. An autopsy on the deceased animals revealed that pneumonia caused by a presumptive mycoplasma infection was the cause of death. The source of the infection was not identified. The deaths were spread out over the vaccine groups, with one death in vaccine group 2, and two deaths in both vaccine groups 4 and 5. The overall loss of five of the forty mice did not affect the statistical validity of mice numbers in each group.
Interestingly only one death occurred within the vaccine groups receiving P. jensenii 702,

suggesting a possible protective effect of the probiotic bacteria, on the animals'overall health and immune system functioning.

Changes in the gut microbiology of mice during the study period

Anaerobic and probiotic counts were performed on the mice faeces.

Recovery of P. jensenii 702 from mice faeces

Plate counts were performed on faecal samples from the test animals, to ensure that P. jensenii 702 both survived and colonised the gastrointestinal tract of the mice, and that the mice that were not receiving the probiotic were not cross contaminated or naturally contain P. jensenii 702 within their gastrointestinal tract.

Table 28 Viable counts of P. jensefzii 702 in the faeces of mice during the study period
ca ami) ti . , ~ ine D"0 1 0 1. 25 xlO'-+ 5. 0 xIO'1. 60 x106-+ 5. 47 x 10'

1 0 1. 25 X106 + 5. 0 xlOs 1. 60 X106 + 5. 47 x105

3 0 0 3. 50 X106 +-2. 59 X106

3003. 50x1012. 59x10

4 0 0 0

5000 Values are mean SD (n=6-8)

Faeces samples were collected on day 0 (prior to P. jensenii 702 administration), day 7 and day 25 (Table 28). No P. jensenii 702 were found in the faeces of any mouse prior to administration of the bacteria. By day 7 the probiotic was present in faecal samples of mice in vaccine group 1 and 2, and by day 25, all vaccine groups receiving P. jensenii 702 (1,2 and 3) had high counts of the probiotic bacteria. No P. jensenii 702 was isolated from any mouse not receiving the bacteria.
Anaerobic plate counts of mice faeces

Total anaerobe plate counts were performed to detect if the presence of the probiotic caused any reduction in anaerobes in the gastrointestinal tract (Table 29). This was used as a measure of colonization of P. jeiiseiiii 702 in the gastrointestinal tract.

Table 29 Anaerobic plate counts on mice faeces during the 25 day study
Faecal Plae Count cfu),

X,. f,, j,}, w7XD v ; 25Xe Sa p Vaccine : Day 0, x10). Day 7X10 : =) Day 25 (x10)

Grou . . : ~ -~

1 3. 983. 56 14. 781. 15 5. 792. 41

2 6. 143. 84 18. 684. 99 8. 33 ~ 9. 61

3 6. 255. 74 5. 783. 93 12. 08~8. 1S

4 7. 1310. 5 4. 64+1. 94 7. 18+3. 11

5 10. 2 5. 59ab 13. 04 1. 47 a 3. 42 1. 96b

Values are mean ~ SD (n=6-8). The same alphabetical prefix across rows indicates a significant difference between anaerobic plate counts over the three sampling occasions of day 0, day 7 and day 25. The same numerical prefix down a column indicates significant difference (p < 0.05) between vaccine groups on a given day

There was no significant difference in faecal anaerobe counts within vaccines groups 1,2, 3 and 4 over the three sampling occasions (Table 29). Vaccine group 5 had a significant reduction in plate count between day 0 and days 7 and 25 (Table 29). On day 0 and 25 there was no statistical difference between faecal anaerobic counts between vaccine groups. At day 7 there was a statistical difference (p < 0.05) between vaccine groups 1, 2, and 5.

T-cell proliferations Concanavalin A

The Concanavalin A (Con A) control plate was used as a general indicator of T-cell reactivity and proliferative ability to the mitogen Con A. Table 30 displays the proliferations in counts per minute (CPM) for the three lymphocyte sources, for each vaccine group. Higher proliferations were seen from all tissues to vaccine 1 and 3, however statistical significance was not tested on these results, as they were a general indicator of proliferation only and do not specifically indicate the effectiveness of one vaccine over another.

Table 30 Concanavalin A T-cell Proliferations

0 et

E ; "Vacexne'Sleen Peyex s. Patehes. eenitxic Lymph,

G j, G. w r =3 a r. 3 .. , 4 : Nodes... F. x

1 1326 72 5230 664"8257713242

2 517 203 434 260 367 196

3 1315 112 3058 294 67324 949

4 649 i 301 140 + 16 24 + 15

5 2697 529 94 53 478 0 Values are mean ~ SD (N=6-8) in counts per minute (CPM) T-cell Proliferation to Administered Vaccine

For each vaccine, T-cell proliferation was measured using the variables: antigen concentration, type of stimulating antigen, time of incubation, and type of tissue used for lymphocyte collection.

Table 31 indicates the average T-cell proliferation results for Day 3. Data for day 4 and 5 is not shown. Day 3 was selected as the optimum day for T-cell proliferations based

upon higher T-cell proliferations overall, but also because it had the greatest amount of statistically higher proliferations. Where the T-cell response was significantly higher for Day 3 compared to days 4 and 5, this is indicated on Table 31.

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