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



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Th1 cytokine production is inhibited by IL-4, and the generation of a Th2 response is favoured by production of IL-4 and inhibited by IFN-y (Marth & Strober, 1997). IL-2 is precursor to a Thl response inducing proliferation of activated T and B cells (Roitt & Delves, 2001)

Cytokines were measured on day 3, which for IFN-y and IL-4 (Figure 18) is suited to their optimum presence, while for IL-2 (Figure 17), this time represents a decline phase because it is an early responding cytokine, found in high amounts in response to TB as soon as 24 hours after infection (Kaufmann & Andersen, 1998). Extended presence of IL-2, found in this study, indicates that a more prolonged immune response has been incurred, which is desirable for a vaccine. Any IL-2 response is significant. Clearly the strongest response came from vaccine 5 stimulated with WTB (Figure 17), once again suggesting there is some interaction of WTB with the lymphocytes.


A comparison of IFN-y and IL-4 results was used to determine the Thl or Th2 nature of the T-cell response (Marth & Strober, 1997). Current knowledge indicates that an optimal

adjuvant for a tuberculosis vaccine should skew the response in a Type 1 direction (Elhay & Andersen, 1997). All results across all vaccine groups showed that IFN-y was significantly higher than IL-4 levels (Figure 18). Previous studies have shown that oral vaccines typically induce a Th2 response (Doherty et al, 2002) and this is why oral vaccines are often overlooked as a method of vaccine administration. This study is unique, as it is the first to demonstrate a strong Thl T-cell response to an oral TB vaccine.


Although cellular immune responses are considered to be protective, the role that antibody responses play in controlling TB is currently under reevaluation (Attanasio, Pehler & McClure, 2000). Presence of Total IgG shows that a systemic immune response has been provoked (Roitt & , Delves, 2001). Clearly sero-conversion occurs with use of vaccine 1,2 and 5 (Table 11). Sero-conversion was also observed for the probiotic control group when tested against STCF-WTB. There is the chance that soluble proteins found in TB may be conserved and found in other bacteria, and it is possible that these conserved proteins may be immunogenic. It is possible that these proteins occur in P. jensenii 702.
It was anticipated that a strong IgA response may provide a first line of defense, responding to intracellular infectious pathogens, such as TB (Falero-Diaz, 2000). It was expected that vaccine 5 after inducing a strong mucosal immune response, would also produce a strong IgA response, however this did not occur, and in fact, vaccine 4 and 5 had significantly lower IgA levels at the end of the study (Table 11). It is possible that the low IgA levels could be attributed to a problem with the preparation of faeces samples to maintain IgA levels, or with the transportation of samples between campuses, in which temperature fluctuations may have had an adverse effect on immunoglobulin levels. These specimens were tested at 1: 10 dilution, which should be sufficient for IgA.
With vaccine 1 and 2, the fact that IgA levels did not increase (Table 35) is expected now that it is acknowledged that exposure time to P. jensenii 702 is critical. A final observation from this study involves an interesting pattern that can be noticed with regard to the deaths of five of the test animals during the vaccine trial. Autopsy revealed that the deaths of the mice were caused by presumptive mycoplasma infection. It is interesting to note that four of the five mice that died were from the vaccine groups receiving cholera toxin as the adjuvant, not the probiotic. The fact that the mice receiving P. jensenii 702 did not get sick, may be a further indication of the protective effect of this probiotic bacteria against respiratory infection. Literature shows that probiotics are known immune stimulating agents.
They also protect the host against potential pathogens by competitive exclusion and through the production of bacteriocins (Matsuzaki & Chin, 2000). Consequently results from this study, combined with this observation indicate that the probiotic bacteria P. jensenii 702 has immune stimulating and adjuvant effects.

A study of a similar nature was conducted this year by Doherty et al (2002) involving orally administering a subunit TB vaccine concluded that oral immunisation does not efficiently prime immune responses and that IFN-y levels in orally immunised test animals was no different to that of naive animals (Doherty et al, 2002). It was also found that oral vaccination failed to induce significant immunity against M. tuberculosis either systemically or at mucosal surfaces, with the best they could achieve was to boost previously existing immune responses. The results described here contrast these findings to conclude that oral vaccination of the soluble secreted and intracellular proteins of M. tuberculosis, stimulate a strong systemic and mucosal immune response, shown by high T-cell proliferations in spleen lymphocytes and Peyer's patches respectively. In addition to this the immune response was of the desired Thl type, indicated by high IFN-y and low IL-4 levels.


Conclusion

It is clear from this research that the oral administration of the soluble proteins from M. tuberculosis can induce an immune response. This is shown by the results from T-cell proliferations, cytokine analysis and immunoglobulin levels. From this array of immunological testing techniques it was found that vaccines 1,2 and 5 induced significant spleen cell T-cell proliferations. Only vaccine 5 produced significant proliferations in the Peyer's patches. These results indicate that the vaccines have potential to protect against tuberculosis infection.


The T-cell proliferation results highlighted the important role of the adjuvant in vaccine production. It is concluded that without P. jensenii 702, the likehood of successful oral vaccine production using the soluble M. tuberculoisis proteins would have been dubious.
This conclusion is clearly evident when spleen cell proliferation results between vaccine 1 and 4 are compared. Both vaccines contained the same protein, however only vaccine 1, which had P. jeiiseiiii 702 as the adjuvant, had a T-cell proliferation response higher than the control (vaccine 3).
A difference was observed in the ability of the two adjuvants to elicit a mucosal immune response. Vaccine 2 contained the same antigens as vaccine 5, but had P. jensenii 702 as the adjuvant. Both produced high proliferations in the spleen, however vaccine 2 failed to produce significant proliferation in lymphocytes isolated from the Peyer's patches. This failure to induce a mucosal immune response can be attributed to the need for P. jensenii 702 to colonise thoroughly before it can exert its effect as an adjuvant on the mucosal immune system.. The failure of P. je7lsenii 702 to adequately colonise within the short vaccine trial period was further verified by the low IgA levels in faeces and minimal colonisation in the first two weeks of the trial. The presence of P. jensenii 702 in the gastrointetinal tract of the mice was seen by day 7 of the study, however there was not a reduction of total anaerobe

counts. This indicates that a longer colonisation time is in fact necessary, and should a longer period be allowed in the future for establishment in the gut, P. jensenii 702 is likely to be quite an effective mucosal adjuvant.


The desired Thl type immune response was achieved by all vaccines tested in this study. This is a unique and valuable achievement as to date no other oral vaccines have been able to successfully induce a Thl response to TB. In addition to this the potential of P. jensenii 702 as an oral adjuvant was again highlighted by its ability to induce a Thl immune response. While Cholera toxin can induce a Th1 response, it along with its non-toxic subunits are not permitted for use in humans. P. jensenii 702 however has verified safety as a food borne/used organism.
Demonstration of P. jensenii 702 as an immune modulator

As described above in the vaccine experiment, P. jensenii 702 acts as an oral adjuvant. In so doing, it produces an immune response that is anti-allergy. In the immune system two types of T-cells exist; Thl and Th2. The presence of one prohibits the production of the other. In allergy, a Th2 immune system predominates, however as demonstrated above, P. jensenii 702 produces a Thl response, and in so doing reduces the Th2 and hence corrects the immunological cause of allergy.


EXAMPLE 5 SELECTION OF POTENTIAL PROTEINS FOR APPLICATION IN AN ORAL VACCINE USING PROPIONIBACTERIUM JENSENII 702 AS AN ADJUVANT AND CARRIER

The following is an example of the technology to be used to identify proteins for use in an oral vaccine. The technology is well defined in literature. Most infectious agents have well characterised immunogenic proteins (antigens), however in some cases an immunogenic response may vary according to the mode of delivery ie. oral versus parenteral, as well depending on the type of adjuvant used.. As the concept of an oral vaccine in particular applications, such as tuberculosis, is novel, the following describes an example of how immunogenic proteins can be isolated and screened for efficacy. It is not meant to describe the only approach, but demonstrates that known technology and can be used to produce an oral vaccine using both known and novel antigens, that could previously could not have been done with the existing adjuvant technology.


In this case the example is for tuberculosis, but application to any infectious agent can be extrapolated.
Method.
Isolation and identification of Immunogenic Proteins
Separation of protein fractions from sonicated whole M. tuberculosis or from short term culture filtrates can be used to provide antigens for the vaccines.
Sodium dodecyl sulfate-polyacrylafnide gel electrophoresis (SDS-PAGE) separation of sonicated whole M. tuberculosis cells SDS-PAGE Equipment

A Bio-Rad Protean II XI Cell electrophoresis unit is used for SDS-PAGE analysis in this experiment. A 16cm x 0.75mm gel, consisting of a 12% separating gel, overlaid with a 4% stacking gel, is prepared according to manufacturer's instructions. A 15 well comb defines the wells in the stacking gel. The gels and running buffer are mounted in the electrophoresis cell, according to manufacturer's instructions.


SDS-PAGE Molecular-Weight Standards

The choice of molecular weight standards depends on whether the gel is to be eluted or used for staining purposes. If the gel is to be eluted, a pre-stained low range SDS-PAGE standard (Bio-Rad) is used to indicate molecular weights. This standard does not require pre- treatment and 25u. l is added directly to the designated well. An unstained low range SDS- PAGE molecular weight standard (Bio-Rad) is used if the gel is to be stained for visualisation of protein bands. This standard is diluted 1 in 10 with sample buffer and heated at 95 C for 4 minutes and cooled. 1 0R1 of treated standard is added to the designated well of the prepared SDS-PAGE gel.


Both the pre-stained and unstained standards, each contain 6 different proteins ranging in size from approximately 20-110 kDa.
Preparation of Sonicated M. tuberculosis for SDS-PAGE Separation

Samples are diluted 1 in 3 with sample buffer and heated to 95 C for 4 minutes and cooled. 60RI of treated sample is added to the designated wells of the prepared gel prior to electrophoresis.


Rufarning Conditions for Separation of Sonicated M. tuberculosis

SDS-PAGE is performed at a constant current of 25 mA for approximately 8-12 hours, or until the dye front is approximately 1cm from the bottom of the gel. After the gel is run it may be eluted to harvest the separated protein fractions or it can be stained for visualisation of protein bands.


Electroelution of Separated Protein Fractiotis (Based on methods ofAndersen & Heron, 1993)

Following electrophoresis, the gel is soaked in three changes of 2mM phosphate buffer, pH6.8 on a Ratek rocker for a total of 20 minutes. This allowed the gel to swell prior to electro-elution and removes any excess SDS. The gel is then removed from the buffer,


placed on a clean flat surface and trimmed, recording the position of the molecular weight markers in the process. The gel is then placed in a Bio-Rad Whole Gel Eluter and assembled according to manufacturer's instructions. The gel is electroeluted at 40V for 20 minutes, and then the current is reversed for 15 seconds to dislodge any proteins that may have bound to the cellophane membrane.


Harvesting of Fractions, frofn the Bio-Rad Whole Gel Eluter

Following elution, the harvesting box is fitted to the Whole Gel Eluter (Bio-Rad) and thirty fractions harvested into separate test tubes. Individual fractions are pooled into the 5 designated size groups (Table 12) and stored at-80 C.


Table 36 Fractions of Sonicated M. tuberculosis (kDa)
size , r ;

A 65


The fractions obtained are used as stimulating antigens in standard T-cell proliferation tests. Fractions yielding positive results according to this test may be further fractionated and the resulting subfractions used as stimulating antigens in standard T-cell proliferation tests to further refine the antigen.
Fractions may also be obtained using HPLC to generate greater yield.
Measurement of immune response Immune response can be measured by the method described in example 4. Once the best immune response is determined a challenge study would be performed.
Media Sodium Lactate Agar (SLA)
Reagent Quantity Supplier

Double distilled water 1 L

Tryptone 10 Oxoid

Yeast extract 10 g Oxoid

Sodium lactate (60%) 16.5 Ml Sigma

Di-potassium hydrogen phosphate 0.25 g AJAX

Manganes sulfate 0. 05 BDH

Bacteriological agar 15 g Oxoid

Preparation notes: Dissolve in water, adjust the pH to pH 7.0, then autoclave at 121 C for 15 min.

Store in cool room.


Sodium Lactate Broth (SLB)
Reagent Quantity Supplier

Double distilled water 1 L

Tryptone 10 g Oxoid

Yeast extract 10 Oxoid

Sodium lactate (60%) 16.5 Sigma

Di-potassium hydrogen phosphate 0. 25 g AJAX

Manganese sulphate 0. 05 BDH

Preparation notes: Dissolve in water, adjust the pH to pH 7.0, then autoclave at 121 C for 15 min.


Store in cool room.


Yeast Extract Lactate Agar (YELA)
Reagent Quantity ¦ Supplier

Double distilled water 1 L

Tryptone 30 g Oxoid

Yeast extract 30 g Oxoid

Sodium lactate (60%) 20 mL Sigma

Bacteriological agar 15 g Oxoid

Preparation notes: Dissolve in water, adjust the pH to pH 7.0, then autoclave at 121 C for 15 min.
Store in cool room.

*Sodium Lactate Base (SL)


Reagent Quantity Supplier

Double distilled water 1 L

Tryptone 10 g Oxoid

Yeast extract 10 g Oxoid

Potassium di-hydrogen phosphate 5 g BDH

Bromocresol purple 0. 025 g

Preparation notes: Dissolve in water, adjust the pH to pH 7.0, then autoclave at 121 C for 15 min.
Store in cool room.
Fermentation Medium (FM)

Reagent Quantity Supplier

Double distilled water 1 L

Glucose 20 Sigma

Yeast Extract 10 Oxoid

KH2PO4 1 g BDH

(NH4) 2HPO4 2 g Sigma

FeSO4 7H2O 5 mg Chem Supply

MgSO4, 7H20 10 mg Chem Supply

MnSO4 H20 2. 5 mg Chem Supply

CaCl2. 6H2O 10 mg Chem Suppl

NaCl 10 mg Chem Supply

CoCl2. 6H20 10 mg Sigma

Preparation notes: Dissolve in water, adjust the pH to pH 7.0, then autoclave at 121 C for 15 min.


Store in cool room.
Supplemented RPMI 1640
Quantity Supplier

RPMI 1640 75. 7 mL Trace Biosciences

200 mM L-glutamine 2. 5 mL-Sigma

45% glucose0. 8 mLSigma

100 mM Sodium Pyruvate1 mLSigma

20% fetal calf serum (heat inactivated) 20 mL HAPS

Preparation notes: Mix all of the reagent in Biohazard cabinet, store in cool room. Prewarmed to 37 C before use.
Buffer and Solutions 12% acrylamide gel
Reagent Quantity Supplier

Acrylamide/bis (30% T, 2.67% C) 40.0 mL Bio-Rad

Distilled Water 33. 5 mL Bio-Rad

1. 5 M Tris-HCl, pH 8.8 25. 0 mL Bio-Rad

10% (w/v) SDS 1 mL Bio-Rad

10% ammonium persulfate (fresh) 500 1 Bio-Rad

TEMED 50 y1 BioRad

Preparation notes: Prepare the monomer solution by combing all reagents except ammonium persulfate and TEMED. Deaerate the solution for 15 minutes. Add the two catalysts just prior to casting the gels.


4% acrylamide stacking gel
Reagent Quantity Supplier

Acrylamide/bis (30% T, 2. 67% C) 1.3 mL BioRad

Distilled Water 6. 1 mL Bio-Rad

0.5 M Tris-HCl, pH 6. 8 2. 5 mL Bio-Rad

10% (w/v) SDS 100 1 Bio-Rad

10% ammonium persulfate (fresh) 50 1 Bio-Rad

TEMED 10 l Bio-Rad

Preparation notes: Prepare the monomer solution by combing all reagents except ammonium persulfate and TEMED. Deaerate the solution for 15 minutes. Add the two catalysts just prior to casting the gels.


Acrylamide/Bis (30 % T, 2.67 % C)
Reagent Quantity Supplier

Acrylamide 146. 0 g Bio-Rad

N, N'-Methylene-bis Acrylamide 4.0 g Bio-Rad

Distilled water 500 mL

Preparation notes: Add together. Filter and store at 4 C in the dark. Maximum shelf life under these conditions is 30 days.

1.5 M Tris-HCI, pH 8.8


Reagent Quantity Supplier

Tris base 54. 45 Bio-Rad

Distilled water 300 mL

Preparation notes: Dissolve 54.45 g Tris base in 150 mL distilled water. Adjust to pH8. 8 with HCl.


Add distilled water to 300mL. Store at 4 C.
0.5 M Tris-HCI, pH 6.8
Reagent Quantity Supplier

Tris base 6 Bio-Rad

Distilled water 100 mL

Preparation notes: Dissolve 6 g Tris base in 60 mL distilled water. Adjust to pH6. 8 with HC1. Add

distilled water to 100mL. Store at 4 C.
10% (w/v) SDS
Reagent Quantity Supplier

SDS 10 g ICN

Distilled water 100 mL

Preparation notes: Dissolve 10 g SDS (ICN) in 60 mL water with gentle stirring. Add distilled water to 100mL. Store in room temperature

10% Ammonium persulfate (w/v)
Reagent Quantity Supplier

Ammonium Persulfate 100 mg Bio-Rad

Distilled water 1 mL

Preparation notes: Dissolve 100 mg ammonium persulfate (Bio-Rad) in 1 mL distilled water. Use immediately.


Sample Buffer
Reagent Quantity Supplier

0. 5MTris-HCl, pH6. 81. 0 mL

Glycerol 1.6 mL Sigma

0. 5% (w/v) bromophenol blue (in water) 0.4 mL

10% SDS 1.6 mL

-Mercaptoethanol 0. 4 mL Bio-Rad

Distilled water 3 mL

Preparation notes: Mix together. Store at 4 C.


5x Running Buffer (SDS-PAGE Electrode Buffer), pH8.3
Reagent Quantity Supplier

Tris-base 45. 0 Bio-Rad

Glycine 216. 0 g Bio-Rad

SDS 15. 0 g ICN

Distilled water 3 L

Preparation notes: Added distilled water to 3L. Do not adjust the pH with acid or base. Store at 4 C. Warm to 37 C before use if precipitation occurs. Dilute 500 mL 5x stock with 2 L distilled water for one electrophoretic rum.


Destaining Solution
Reagent Quantity Supplier

Acetic acid 800 mL BDH

Methanol 200 mL Chem Supply

¦ Distilled water 1 L

Preparation notes: Mix together. Store at room temperature.
TE Buffer
Rea ent Quantit Su lier

Tris-HCL 10 mM Si a

EDTA 1 mM Sigma

Preparation notes: Adjust the pH to pH7.5 with 1M NaOH. Sterilized by filtering through 0. 20, um filter. Store at room temperature.


TES Buffer
Reagent Quantity Supplier

Tris-HCL 10 mM Si ma

EDTA 1 mM Sigma

NaCl 100 mM Chem Supply

Preparation notes: Adjust the pH to pH7.5 with 1M NaOH. Store at 4 C.
PBS Buffer
Reagent Quantity Supplier

kCl 0.2 g Chem Supply

Na2HPO4 1. 44 g Chem Supply

NaCl 8 g Chem Suppl

KH2PO4 0.24 g Chem Suppl

Distilled water 1 L

Preparation notes: Dissolve all the reagents in 800 mL distilled water. Adjust pH to pH7.0 with HC1. Add distilled water to 1 L, then autoclave at 121 C for 15 min. Store in cool room.
Simulated gastric juice (3g/L pepsin in 0.5% NaCl)
Reagent Quantity Supplier

Pepsin (1: 10000) 0. 12 g ICN

Sterile 0. 5% NaCl 40 rnL Chem Supply

Preparation notes: Dissolve the pepsin in sterile 0.5% NaCl in a sterile container. Adjust pH aseptically to pH2, pH3 or pH4 with concentrated HCl or sterile 0.1M NaoH.


Simulated intestinal juice without bile salt (lg/L pancreatin in 0.5% NaCI)
Reagent ¦ Quantity Supplier

Pancreatin 0.04 g Sigma

Sterile 0. 5% NaCl 40 mL Chem Supply

Preparation notes: Dissolve the pancreatin in sterile 0. 5% NaCl in a sterile container. Adjust pH aseptically to pH8 using sterile 0.1M NaoH.


Simulated intestinal juice with bile salt (Ig/L pancreatin, 0. 45% bile salt in 0. 5% NaCI)
Reagent Quantity Supplier

Pancreatin 0.04 g Sigma

Bile salt 0. 18 g Oxoid

Sterile 0. 5% NaCl 40 mL Chem Suppl


Preparation notes: Dissolve 0.18 g of bile salt in 40 mL of 0.5% NaCl, sterilized by autoclaving at 121C for 15 min. After cooling, add 0.04 g pancreatin aseptically. Adjust pH aseptically to pH8 using sterile 0. 1M NaoH.


Fixation Buffer (O. IM cacodylate buffer containing 2. 5% glutaraldehyde)
Reagent Quantity Supplier

0.4 M Cacodylic acid 7 mL Sigma

70% lutaraldehyde 1 mL Sigma

1M NaOH 1 mL Chem Supply

Distilled water 19 mL

Preparation notes: Store in cool room. Warm to room temperature before use.


0. 1M Cacodylate buffer (pH7.2)

Reagent Quantity Supplier

Cacodylic acid 2.77 g Sigma

Distilled water 200 mL

Preparation notes: Dissolve 2.77 g cacodylic acid in 100 mL distilled water. Adjust pH to pH7.2 using 1M NaOH, then add distilled water to 200 mL. Store in cool room. Warm to room temperature before use.
0.05M Sodium Phosphate buffer (pH 7.0)
Reagent Quantity Supplier

0.05 M Na2HP04500 mLChem Supply

0.05 M NaH2PO4 500 mL Chem Supply

Preparation notes: Add 0.05 M NaH2P04 solution to 0.05 M Na2HP04till pH7. Store in cool room.


B-Glucuronidase reaction mixture
Reagent Quantity Supplier

20mM p-nitrophenyl- -D-glucuronide 2 mL

1mM EDTA 4 mL Si ma

40mM potassium phosphate buffer 20 mL

Distilled water 14 mL

Preparation notes: Mix and store in cool room.


20 mM p-nitrophenyl-B-D-glucuronide
Reagent Quantity Supplier

-nitrophenyl- -D-glucuronide 0. 012 g Sigma

20mM potassium phosphate buffer 2 mL

Distilled water 14 mL

Preparation notes: Store in cool room.
40mM potassium phosphate buffer (pH6.8)
Reagent Quantity Supplier

K2HP040. 544 g Chem supply

KH2PO4 0. 697 g Chem supply

Distilled water 200 mL

Preparation notes: Dissolve K2HPO4 or KH2PO4 in 100 ml distilled water respectively, mixed them together to pH6.8 0.23M glycine-NaOH-NaCI buffer (pH10. 4)
Reagent Quantity Supplier

Glycine 1. 7266 g Bio-Rad

NaCl 1. 344g Chem supply

Distilled water 100 mL

Preparation notes: Dissolve glycine and NaCl in 70 ml distilled water. Adjust the pH to pH10. 4 with 1M NaOH, add distilled water to 100 mL.
General reagents Phosphate Buffered Saline (PBS), pH 7.3
Reagent Quantity Supplier

Sodium chloride 8. 0g 1 Sigma

Potassium chloride 0. 2g

Disodium hydrogen phosphate 1. 15g 2 Sigma

Potassium dihydrogen phosphate 0.2g

Fisons


BDH

Dissolve all reagents in 1L distilled water. Autoclave 15 minutes at 121 C 70% Ethanol


Reagent Quantity Supplier

Ethanol 700mol 3 Fronine

Distilled Water 300ml

Mix well Mycobacterial culture Modified Sauton's Medium (MSM)


Reagent Quantity Supplier

Asparagine 8g Sigma

Tap Water 500ml

Dissolve asparagine in water (warm to 80 C in water bath if necessary)

Magnesium sulfate heptahydrate l. Og 4 Sigma

Citric acid 3.66g

Potassium orthophosphate l. Og Sigma

Ferric ammonium citrate O. lg Fronine

D-glucose 8.76g Sigma

Sodium pyruvate 9.64g Sigma

Glycerol 106ml Sigma

Tap water 1420ml Sigma

Add to dissolved asparagine. Adjust to pH 6.8 with concentrated ammonia (25-32%).
Dispense into desired containers. Autoclave at 127 C for 20 minutes. Allow to cool in autoclave for at least 2 hours. Store at 4 C for up to 6 weeks. Caution: Explosive

Protein analysis Protein Reagent


Reagent Quantity-Supplier

Dye reagent concentrate 4ml Bio-Rad

Distilled water 16ml

Mix well and filter through Whatman ;1 filter. Store at room temperature for 2 weeks.


Stock Standard (1. 43mg/ml protein)
Reagent Quantity Supplier

Lyophilized Bovine Serum 1 bottle Bio-Rad

Albumin (BSA)

Distilled Water 20ml

Aliquot 50A1 quantities and store at-80 C

Working Standards


Standard Protein Concentration Dilutions

(mg/ml)


Standard 1 (S1) 0.050 50A1 S2 + 50FL dH20

Standard 2 (S2) 0.100 50y1 S3 + 50 L dH2O

Standard 3 (S3) 0.200 50 l S4 + 50FL dH20

Standard 4 (S4) 0. 400 28R1 Stock Standard + 72 L dH20

Prepare the standards according to the above dilutions. Use immediately.
SDS-PAGE Pre-stained low molecular weight standards
Standard Size (kDa) Supplier

1 21. 4 Bio-Rad

2 29

3 36.2


4 51.2

5 90


zu

Acrylamide-Bis


Reagent Quantity Supplier

Acrylamide 146g Bio-Rad

N, N'-Methylene-bis Acrylamide 4g Bio-Rad

Add distilled water to 500ml. Filter and store at 4 C in the dark for up to 30 days.


1.5 M Tris-HCl, pH 8.8

Reagent Quantity Supplier

Tris base 54.45g Sigma

Distilled Water 150m1

Mix to dissolve. Adjust to pH 8. 8. Add distilled water to 300ml. Store at 4 C.

0.5M Tris-HCl, pH6.8


Reagent


Tris-HCl

Distilled water

Mix to dissolve. Adjust to pH 6.8. Add distilled water to 100ml. Store at 4 C.
10% (w/v) SDS
Reagent Quantity Supplier

SDS lOg ICN

Distilled water 60ml

I Dissolve with gentle stirring. Add distilled water to 100ml.


10 % (w/v) Ammonium persulfate
Reagent Quantit Supplier

Ammonium persulfate 100mg Bio-Rad

Distilled water 1.0ml

Dissolve and use immediately.


0. 5% (w/v) Bromophenol Blue
Reagent Quantity Supplier.

Bromophenol Blue 0. 05g Bio-Rad

Distilled Water l0. Oml

Mix and store in a dark place.


Sample Buffer, pH 6.8
Reagent Quantity Supplier

Distilled water 3. 0ml

0.5 M Tris-HCI, pH 6.8 1. Oml

Glycerol 1. 6ml Sigma

10% SDS 1. 6ml

2-mercaptoethanol 0. 4ml Bio-Rad

0.5% (w/v) bromophenol blue 0. 4ml

Mix well. Store 4 C 5x Electrode Buffer


Reagent Quantity Supplier

Tris base 45. Og Sigma

Glycine 216. 0g ICN

SDS 15.0 ICN

Add distilled water to 3. 0L. Do not adjust the pH. Store at 4 C. Warm to 37 C before use if precipitation occurs.
Running buffer
Reagent Quantity Supplier

5x Electrode buffer 0.5L

Distilled water 2.0L

Running buffer may be reused several times. Store at 4 C Separating Gel (12%, pH 8.8)


Reagent Quantity Supplier

Acrylamide-bis 40. Oml

Distilled water 33. 5ml

1.5 M Tris-HCI, pH8.8 25. Oml

10% (w/v) SDS l. Om1

10% (w/v) ammonium persulfate 500 l

TEMED 50 1 Bio-Rad

Combine all reagents without frothing, except ammonium persulfate and TEMED. Allow to stand 15 mins. Add the two catalysts immediately prior to casting gel.


Stacking Gel (4 %, pH 6. 8)
Reagent Quantity Supplier

Acrylamide-bis 1. 3ml

Distilled water 6. 1ml

0.5 M Tris-HCl, pH 6.8 2. 5ml

10% (w/v) SDS 100R1

10% (w/v) ammonium persulfate 50R1

TEMED 10R1 Bio-Rad

Combine all reagents without frothing, except ammonium persulfate and TEMED. Allow to stand 15 mins. Add the two catalysis immediately prior to casting gel.


Fixative I
Reagent Quantity Supplier

Methanol 800ml Fronine

Acetic Acid 200ml BDH

Distilled Water l. OL

Mix together. Store at room temperature.
Phosphate Buffer (2mM, pH 6.8)
Reagent Quantity Supplier

Sodium orthophosphate 0.48g BDH

Distilled Water 1.8L

Dissolve and adjust to pH 6.79. Make up to 2. 0L with distilled water and store at 4 C.


Immunisation Protease Inhibitor (Soybean Trypsin Inhibitor)
Reagent 4.1. 1.1 Quantity Supplier

Soybean Trypsin inhibitor (lOOmg/ml) 3. 0mg Sigma

PBS

lml


Autoclave 15 min at 121 C. Store at 4 C

PMSF Solution


Reagent T Quantity T Supplier

PMSF 20mg Sigma

Absolute ethanol lml RHONE-POULENC

Store at 4 C Lymphocyte culture and analysis RPMI Complete Medium


Reagent Quantity Supplier

DMEM/RPMI 500ml Trace

10,000 U/ml Penicillin, lOmg/ml 5ml Trace

Streptomycin

L-glutamine (200mM) 5ml Trace

2-Mercaptoethanol (5mM) 5ml Trace

HEPES buffer (1M) lOml Trace

Foetal Calf Serum (heat-inactivated*) 50ml Trace

Combine all reagents under sterile conditions. Store at 4 C.
(* To heat inactivate FCS : place in 56 C water bath for 45mins)

Mouse Red Blood Cell (RBC) Lysis Buffer


Reagent Quantity Supplier

NH4C1 4.15g BDH

NaHCO3 0. 5g BDH

Ethylene-diamine-tetra-acetic acid 0.0185g AJAX

(EDTA) di-sodium salt

Dissolve in approximately 400ml sterile distilled water. Adjust pH to 7.35 and make up to

500ml. Filter sterilise and store at 4 C

Trypan Blue, 0. 1%


Reagent Quantity Supplier

Trypan Blue 0. 05g Sigma

PBS 50ml


) Store at room temperature.
Working Thymidine

Reagent Quantity Supplier

[methyl- H] thymidme (1 mCi/ml) 250 l Amersham

RPMI complete medium 4.75ml

Store at 2 C.
Probiotic production

Sodium Lactate Broth (SLB)


Reagent ¦ Quantity ¦ Supplier

Double distilled water 1 L

Tryptone lOg Oxoid

Yeast extract lOg Oxoid

Sodium lactate (60%) 16. 5ml Sigma

Di-potassium hydrogen phosphate 0.25g AJAX

Manganese sulphate 0. 05 BDH

Make up to 1 L with distilled water. Autoclave at 121 C for 15 mins.


Sodium Lactate Agar (SLA)
Reagent Quantity Supplier

Double distilled water 1 L

Tryptone log Oxoid

Yeast extract lOg Oxoid

Sodium lactate (60%) 16. 5ml Sigma

Di-potassium hydrogen phosphate 0.25g AJAX

Manganese sulfate 0. 05g BDH

Bacteriological agar 15g Oxoid

Dissolve in water, adjust to pH 7.0, then autoclave at 121 C for 15 min. Store in cool room.
Diluted Wilken's Chalgren (WCAB) Broth
Reagent Quantity Supplier

WCAB 16.5g Oxoid

Distilled Water 1L

Dissolve in water, adjust to pH 7.0, then autoclave at 121 C for 15 minutes ELISA for IsG, IgG subclasses and IgA Bicarbonate Buffer


Reagent Quantity Supplier

15mM Na2CO3 1.59g/L Sigma

35mM NaHCO3 2.94g/L Sigma

Store at 4 C. I

Wash Buffer (PBS/0.05% Tween 20
Reagent Quantity Supplier

PBS lOL


Polyoxyethylenesorbitan Monolaurate 5ml Sigma

(Tween 20)

Store at 2-8 C for up to 7 days.
5% Foetal Calf Serum

Reagent Quantity Supplier

Foetal calf serum (*heat inactivated) 5ml Trace

PBS 9Sml


Store at 4 C for up to 7 days (* To heat inactivate FCS : place in 56 C water bath for 45mins) Streptavidin Horseradish Peroxidase (SA-HRP) Conjugate

Reagent Quantity Supplier

SA-HRP logi Amersham

PBS (0.05% tween) 9. 99ml

Use immediately.
TMB Substrate Solution
Reagent Quantity Supplier

Substrate A 5ml Pharmingen

Substrate B Sml Pharmingen

Warm reagents to room temperature prior to use. Use substrate solution immediately.


Stop Solution (2M)

Reagent Quantity Supplier

Sulphuric Acid (3M) 33ml BDH

Distilled Water 17ml

Mix together and store at room temperature.
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Zhou, J. S. , Shu, Q. , Rutherfund, K. J. , Prasad, J. , Gopal, P. K. and Gill, H. S. (2000) Food and Chemical Toxicology, 38,153-161.Claims:

CLAIMS: 1. An isolated Propionibacterium strain Propionibacteriunz jensenfi 702.


2. A formulation comprising Propionibacterium jensenii 702 together with a culture medium, storage medium excipient, carrier or diluent.
3. Use of Propionibacterium. jensenii 702 as a probiotic strain.
4. A Vitamin B12 supplement comprising Propionibacterium jensenii 702 together with a delivery agent.
5. The supplement of claim 4 which is in the form of a capsule, tablet or powder (loose or in capsules), granule or paste or spray.
6. The supplement of claim 4 which is provided in the form of a vitamin B12 fortified food product, such as a breakfast cereal, soy milk product, or vegetarian burger patty.
7. A method for preventing, treating or ameliorating the adverse effects of Vitamin B12 deficiency in a host which method comprises administering a supplement or food of claim 4 or 12 to the host.
8. A growth promotor for animal feed comprising Propionibacterium jensenii 702 together with a suitable carrier, excipient or diluent.
9. A method of improving the growth and/or vitamin supply and/or inhibiting harmful intestinal bacteria in a host animal comprising administering an effective dose of the growth promoter of claim 8 to the animal.
10. A method of preventing food spoilage and/or extending the shelf life of food products comprising preparing the food by including a fermentation step involving use of Propionibacterium jensenii 702.
11. A method of preventing food spoilage and/or extending the shelf life of food products comprising preparing the food by including a fermentation step involving use of Propionibacterium jenseni. i 702 in mixed culture with another food fermentation microorganism.
12. A food using Propionibacterium jensenii 702 or parts thereof 13. A food according to claim 12 wherein the food is a cultured milk product such as Propioni-acidophilus milk.
14. A method of preparing a food comprising using Propionibacteriurra jensenii 702 or parts thereof in the preparation.
15. A method of treating gastrointestinal disease and/or lactose intolerance in a patient comprising administering a food or supplement according to claim 4 or claim 12 to the patient.

16. A method for enhancing the growth of bifidobacteria in the gut of a host comprising administering Propionibacterium jensenii 702 or a food or supplement of the invention to the host.


17. A mixed culture of Propionibacterium jensenii 702 with one or more other probiotic bacteria.
18. A method for affecting the lipid metabolism of a host comprising administering Propionibacterium jensenii 702 or a food or supplement according to claim 4 or 12 to the host.
19. A method for affecting the immune system of a host which method comprises administering Propionibacterium jensenii 702 or a food or supplement according to claim 4 or claim 12 to the host, and wherein affecting the immune system includes providing adjuvanting effects, generalised immune stimulation or alleviating allergy.
20. A method for protecting a probiotic microorganism during passage through the gastrointestinal tract which method comprises administering the probiotic in the presence of a soy or cereal based product.
21. A method for preventing, treating or ameliorating the adverse effects of high homocysteine in a host which method comprises administering a supplement or food of claim 4 or 12 to the host.
22. A method for preventing or ameliorating the adverse effects of (3-glucuronidase in a host which method comprises administering a supplement or food of claim 4 or 12 to the host.
23. A method to reduce the risk of cardiovascular disease in a host which method comprises administering a supplement or food of claim 4 or 12 to the host.
24. A method for preventing, treating or ameliorating the adverse effects of an immune imbalance in a host which method comprises administering P. jensenii 702, a supplement or food of claim 4 or 12 to the host.
25. A method for modulating the immune response in a host which method comprises administering P. jensenii 702, a supplement or food of claim 4 or 12 to the host.
26. A method for increasing infectious disease resistance in a host which method comprises administering a supplement or food of claim 4 or 12 to the host.
27. A mucosal vaccine for tuberculosis comprising an antigen comprising at least one cellular or secreted protein of Mycobacterium tuberculosis or at least one immunologically active part thereof together with a mucosal adjuvant.
28. A vaccine according to claim 27 wherein the vaccine is an oral vaccine.
29. A vaccine according to claim 28 wherein the oral adjuvant is a bacterial vector.
30. A vaccine according to claim 29 wherein the oral adjuvant is P. jensenii 702, or part thereof.

31. A vaccine according to any one of claims 27 to 30 wherein the antigen is selected from the group consisting of whole M. tuberculosis cells, disrupted M. tuberculosis cells or parts thereof, and soluble M. tuberculosis proteins which are either secreted or cellular or a combination of secreted and cellular soluble proteins, collected from actively multiplying M tuberculosis cultures.


32. A vaccine according to claim 31 wherein the antigen is provided as a short term culture filtrate.
33. A vaccine according to claim 31 wherein the antigen is a combination of whole cell sonicate and short term culture filtrate.
34. A vaccine according to claim 31 wherein the antigen preparation comprises soluble proteins fractionated into different size ranges.
35. A vaccine according to claim 34 wherein fractions of < 20kDa, 20-30kDa, 30-35kDa, 35-65kDa, and > 65kDa are employed.
36. A vaccine according to claim 34 wherein the antigen comprises an effective amount of a combination of secreted and cellular soluble proteins present at an effective ratio.
37. A vaccine according to claim 27 wherein the vaccine is formulated as a liquid, tablet, powder, as part of a food or in an aerosol.
38. A vaccine according to claim 27 wherein the vaccine provides a systemic or a mucosal immune response or both in the host to Mycobacterium tuberculosis.
39. A method for producing an immune response in a patient to Mycobacterium tuberculosis which method comprises delivering a vaccine of the according to claim 27 to a patient via a mucosal surface.
40. A method for vaccinating a patient against Mycobacterium tuberculosis which method comprises delivering a vaccine according to claim 27 to a patient via a mucosal surface.
41. A method for protecting a patient against Mycobacterium tuberculosis infection which method comprises delivering a vaccine according to claim 27 to a patient via a mucosal surface.
42. A method for protecting a patient against Mycobacterium tuberculosis reactivation which method comprises delivering a vaccine according to claim 27 to a patient via a mucosal surface.
43. A method for protecting a patient against tuberculosis which method comprises delivering a vaccine according to claim 27 to a patient via a mucosal surface.
44. Use of an antigen comprising at least one cellular or secreted protein of Mycobacteriuna tuberculosis or at least one immunologically active part thereof in the manufacture of a vaccine for use in a method for producing an immune response in a patient

to Mycobacterium tuberculosis which method comprises delivering a vaccine according to claim 27 to a patient via a mucosal surface.


45. Use of an antigen comprising at least one cellular or secreted protein of Mycobacterium tuberculosis or at least one immunologically active part thereof in the manufacture of a vaccine for use in a method for vaccinating a patient against Mycobacterium tuberculosis which method comprises delivering a vaccine according to claim 27 to a patient via a mucosal surface.
46. Use of an antigen comprising at least one cellular or secreted protein of Mycobacterium tuberculosis or at least one immunologically active part thereof in the manufacture of a vaccine for use in a method for protecting a patient against Mycobacterium tuberculosis infection which method comprises delivering a vaccine according to claim 27 to a patient via a mucosal surface.
47. Use of an antigen comprising at least one cellular or secreted protein of Mycobacterium tuberculosis or at least one immunologically active part thereof in the manufacture of a vaccine for use in a method for protecting a patient against Mycobacterium tuberculosis reactivation which method comprises delivering a vaccine according to claim 27 to a patient via a mucosal surface.
48. Use of an antigen comprising at least one cellular or secreted protein of Mycobacterium tuberculosis or at least one immunologically active part thereof in the manufacture of a vaccine for use in a method for protecting a patient against tuberculosis which method comprises delivering a vaccine according to claim 27 to a patient via a mucosal surface.
49. Use according to any one of claims 44 to 48 wherein the mucosal surface is the mouth or nose.
50. Use according to any one of claims 44 to 49 wherein the vaccine provides a systemic or a mucosal immune response or both in the patient to Mycobacterium tuberculosis.
51. Use according to claim 50 wherein the immune response prevents TB disease from occurring and/or initial infection with TB from occurring.
52. A method for preparing a vaccine according to claim 27 comprising combining an antigen comprising at least one cellular or secreted protein of Mycobacterium tuberculosis or at least one immunologically active part thereof, a mucosal adjuvant and a suitable carrier wherein the antigen is selected to provide an immune response against Mycobacterium tuberculosis.
53. An isolated Mycobacterium tuberculosis antigen comprising at least one cellular or secreted protein of Mycobacterium tuberculosis or at least one immunologically active part thereof.

54. An antigen according to claim 53 selected from the group consisting of soluble M. tuberculosis proteins which are either secreted or cellular or a combination of secreted and cellular soluble proteins, collected from actively multiplying M. tuberculosis cultures.


55. An antigen according to claim 54 wherein the antigen is provided as a short term culture filtrate.
56. An antigen according to claim 54 wherein the antigen preparation comprises soluble proteins fractionated into different size ranges.
57. An antigen according to claim 56 wherein an initial size separation of < 20kDa, 20- 30kDa, 30-35kDa, 35-65kDa and > 65kDa is employed.
58. An antigen according to claim 54 wherein an effective amount of a combination of secreted and cellular soluble proteins is present at an effective ratio.
59. A method for producing an immune response in an animal host to a Mycobacterium species which is capable of causing tuberculosis in the host which method comprises delivering a vaccine according to claim 27 to the animal host via a mucosal surface wherein the vaccine comprises an antigen derived from a Mycobacterium species which produces TB in the relevant host or one which can provide effective protection against the Mycobacterium species which causes TB in the relevant host.
60. A method for vaccinating an animal host against a Mycobacterium species which is capable of causing tuberculosis in the host which method comprises delivering a vaccine according to claim 27 to the animal host via a mucosal surface wherein the vaccine comprises an antigen derived from a Mycobacterium species which produces TB in the relevant host or one which can provide effective protection against the Mycobacterium species which causes TB in the relevant host.
61. A method for protecting an animal host against a Mycobacterium species which is capable of causing tuberculosis in the host which method comprises delivering a vaccine according to claim 27 to the animal host via a mucosal surface wherein the vaccine comprises an antigen derived from a Mycobacterium species which produces TB in the relevant host or one which can provide effective protection against the Mycobacterium species which causes TB in the relevant host.
62. A method for protecting an animal host against tuberculosis reactivation which method comprises delivering a vaccine according to claim 27 to the animal host via a mucosal surface wherein the vaccine comprises an antigen derived from a Mycobacterium species which produces TB in the relevant host or one which can provide effective protection against the Mycobacterium species which causes TB in the relevant host.
63. A method for protecting an animal host against a Mycobacteriunt species which is capable of causing tuberculosis in the host which method comprises delivering a vaccine according to claim 27 to the animal host via a mucosal surface wherein the vaccine comprises

an antigen derived from a Mycobacterium species which produces TB in the relevant host or one which can provide effective protection against the Mycobacteriunt species which causes TB in the relevant host.


64. Use of an antigen comprising at least one cellular or secreted protein of a Mycobacterium species or at least one immunologically active part thereof in the manufacture of a vaccine for use in a method according to any one of claims 59 to 63.
65. A method for preparing a vaccine according to claim 27 comprising combining an antigen comprising at least one cellular or secreted protein of a Mycobacterium species or at least one immunologically active part thereof, a mucosal adjuvant and a suitable carrier wherein the antigen is selected to provide an immune response against a Mycobacterium species.
66. An isolated Mycobacteriuna antigen comprising at least one cellular or secreted protein of a Mycobacterium species or at least one immunologically active part thereof.
67. An isolated antigen according to claim 66 wherein the antigen is selected from the group consisting of soluble proteins which are either secreted or cellular or a combination of secreted and cellular soluble proteins, collected from actively multiplying cultures.
68. An isolated antigen according to claim 67 wherein the antigen is provided as a short term culture filtrate.
69. An isolated antigen according to claim 67 wherein the antigen preparation comprises soluble proteins can be fractionated into different size ranges.
70. An isolated antigen according to claim 69 wherein an initial size separation of < 20kDa, 20-30kDa, 30-35kDa, 35-65kDa and > 65kDa is employed.
71. An isolated antigen according to claim 66 wherein a combination of selected fractions of secreted and cellular proteins is utilised with the proportion of each fraction optimised to produce the desired immune response.

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