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



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Table 31 Day 3 T-cell Proliferation Results

Antigen at", , rerage Vaccine Time : (da' s) Tssae : r : Anti eri--~ tration,'Prol'iferationi...,

1 3SpleenSTCF 10 22211 3645

3 Spleen STCF 10 22211 3645

3 5 46183 10494 a

3* 2. 5 35216 4243 a

1 3 Peyer's Patches STCF 10-

35 2788 2724

3 2. 5 1852 1565

Mesenteric Lymph

1 3 Nodes STCF 10 43747 8516 ab

3 5 6342i10534

3; 2. 5 3044+3631''

2 3 Spleen STCF 10 16971 2061 ab

3 5 26770+6074

3* 2. 5 27557 5034 b

2 3 Peyer's Patches STCF 10-

3 5 1203 574

3; 2. 5 1324+98

Mesenteric Lymph

2 3 Nodes STCF 10 10836 9228

35 12262 10266

3 2. 5 389 88

2 3 Spleen WTB 10 34607 9597

3 5 40528 4270

3* 2. 5 32887 4012

2 3 Peyer'sPatches WTB 10-

3 5 588+129

3 2. 5 32461

Mesenteric Lymph

2 3 Nodes WTB 10 1039140

3 5 970 102

3* 2. 5 979 98

2 3 Spleen STCF/WTB 10 20560 1847

35 22790 3553

3; 2. 5 35319 14319

2 3 Peyer's Patches STCF/WTB 10-

3 5 1106 362

3 2. 5 1014 211

Mesenteric Lymph

2 3 Nodes ~ STCFAMTB 10 1057 538

3 5 7272+11698

3* 2-5 382 65

3 3 Spleen STCF 10 22307 7309

3 5 19423 ~ 1105

3* 2. 5 19544 ~ 4182

3 3 Peyer's Patches STCF 10 25884 9838

3 5 10248 ~ 17341

3 2. 5 9533 ~ 9814

Mesenteric Lymph

3 3 Nodes STCF 10 -

3 5 -


3 2. 5 6039 ~ 4746

3 3 Spleen WTB 10 10006 ~ 1301a

3 5 24159 ~ 3743a

3* 2. 5 34319 ~ 2801a

3 3 Peyer's Patches WTB 10 573 ~ 210ab

3 5 1224 ~ 228a

3 2. 5 929 ~ 194b

Mesenteric Lymph

3 3 Nodes WTB 10-

3 5


3 2. 5 4075 ~ 4754

3 3 Spleen STCF/WTB 10 16661 4818

3 5 17678 ~ 4433

3; 2. 5 23106 ~ 7980

3 3 Peyer's Patches STCF/WTB 10 -

3 5 3528 ~ 1671

3; 2.5 8210 ~ 3860

Mesenteric Lymph

3 3 Nodes STCF/WTB 10 -

3 5 358 ~ 36

3 2. 5 42598

4 3 Spleen STCF 10 19068 ~ 5021a

3 5 12702 ~ 5101

3* 2. 5 7853 ~ 1894a

4 3 Peyer's Patches STCF 10

3 5 -


3; 2. 5 10810 2561

Mesenteric Lymph

4 3 Nodes STCF 10 469 28

3 5 430 106

3 2. 5 509 ~ 94

5 3 Spleen STCF 10 22309 731

35 27230 6176

3* 2. 5 23619 ~ 3499

5 3 Peyer's Patches STCF 10

3 5


3 2. 5 37729 ~ 4626

Mesenteric Lymph

5 3 Nodes STCE 10 625 ~ 71

3 5 833 ~ 972

3 2.5 337 ~ 86

5 3 Spleen WTB 10 20966 ~ 5345a

5 37563 ~ 3961a

3* 2. 5 48587 7123 a

5 3 Peyer'sPatches WTB 10

3 5 -


3; 2. 5 9870 1390

Mesenteric Lymph

5 3 Nodes WTB 10 416 ~ 100

35 520158

3 2. 5 393 125

5 3SpleenSTCF/WTB 10 25899 ~ 6288

3 5 29397 ~ 23319

3* 2. 5 45135 10877

5 3 Peyer's Patches STCF/WTB 10

35 30994 8780

3; 2. 5 55576 ~ 7169

Mesenteric Lymph

5 3 Nodes STCF/WTB 10 5716 ~ 10298

3 5 46181

3 2.5 20788

Values are mean SD (n=6-8) in counts per minute (CPM). Alphabetical prefix denotes significant differences (p < 0.05, student's t-test, two-tailed) between proliferation levels at different antigen concentrations within each vaccine group and respective stimulating antigen.


* denotes a significant difference (p < 0.05, student's t-test, two-tailed) in proliferation between days 4 and 5 ; denotes when day 3 was not significantly different to days 4 or 5, however it did display the lowest variance, and hence was chosen on this basis - indicates there was insufficient cells to perform proliferations on Summary of T-cell proliferations using 2.5 g antigen on day 3

Three different antigen combinations were tested against each tissue lymphocyte source. In each case the stimulating antigen was only tested where it was a component of the original vaccine. On Day 3 the optimum concentration of stimulating antigen was 2. 5 g/ml based upon it producing overall, the highest proliferation readings (Table 31). This concentration was selected for the remaining analysis.


T-cell proliferations on day 3 with 2.5 g of STCF as stirraulating antigen

Table 32 shows the average proliferation results from day 3 against 2. 5gg of STCF for all vaccines tested in this study.


Table 32 Average Proliferation for each tissue with 2.5 Fg of STCF

Avexa e Proliferation-for 'issue {CP1VI) !

V'accine : Spleen Peyar's Patefie

Mesenterc Lyinpl c es,, '"1'35216 4243'1852 1565'3044363r

2 27557 5034ab 1324 98c 389 88e

3 19544+4182 953319814 6039 4746"

4 7853 10810 256 Id 509 94e

5 ' ? 3619 3499b 37729 4626 I 337 86e

Values are mean SD (n=6-8) in counts per minute (CPM) The same alphabetical prefix within a column indicates no significance between the marked vaccine groups for a given lymphocyte source (p > 0.05, student's t-test, two-tailed). Within the column, the remainder of the unmarked values, in addition to the values that do not share a similar letter indicator, are significantly different (p < 0.05 to p < 0.001, student's t-test, two- tailed)

Significance was only tested for across the vaccine groups within each tissue group.


The lymphocyte proliferation results using the different tissue reflects the type of immune response and is not being used to determine the best vaccine. This will be covered further in the discussion. Proliferation results for the spleen show that vaccine 1,2 and 5 had counts significantly higher than vaccine 3, the control. Vaccine 1 had the highest count for the spleen. In the Peyer's patches however vaccine 5 had the highest proliferation to 2. 5 g of STCF protein and was the only reading significantly higher than the control. There was no statistical significance between any of the vaccine groups for proliferation counts for the mesenteric lymph nodes.
T-cell proliferations on day 3 with 2. S, ug. of WTB as stimulating antigen

Table 33 shows the average proliferation values for each T-cell source when stimulated with 2. 511g of the WTB antigen.


Table 33 Average Proliferation for each tissue with 2.5 Zg of WTB

2 32887 + 3475 ¦ 324 + 50 979 i 80b 2"328873475'32450"97980" ; : .. E- Notes- . .


2 32887-1-3475a 324-~1-50 979 80b

3 34319 2426"929 168 4075 3882

5 48587 6169 9870 983 393 -108b Values are mean SD (n=6-8) The same alphabetical prefix within a column indicates no significance between the marked vaccine groups (p > 0.05, student's t-test, two-tailed) for each tissue lymphocyte source. The remainder of the unmarked values, in addition to the values that do not share a similar letter indicator, are significantly different (p < 0.05 to p < 0.001, student's t-test, two-tailed)

Separate testing of the antigens STCF and WTB, was performed in an attempt to distinguish if one component was responsible for higher proliferations than the other. From Table 33 vaccine 5 once again displayed significantly higher proliferations than the control and vaccine 2, for both the spleen and Peyer's patches. The mesenteric lymph nodes had the highest scintillation count with the control.


T-cell proliferations on day 3 with 2. S8g o+STCF-WTB as stimulating antigen
Table 34 shows the average proliferation values for each T-cell source when stimulated with 2. 5ig of the STCF-WTB combination antigen.
Table 34 Average Proliferation for each tissue with 2. 5 pg of STCF-WTB
-for x

Patch 6 : Me C' en term

2 35319~ 14139ab 1014 211 382~65

3 23106+7980'8210 3860 425 98'

5 45135 ~10877 55576 +7169 207~gg Values are mean SD (n=6-8) The same alphabetical prefix within a column indicates no significance between the marked vaccine groups (p > 0.05, student's t-test, two-tailed) for each tissue lymphocyte source. The remainder of the unmarked values, in addition to the values that do not share a similar letter indicator, are significantly different (p < 0.05 to p < 0.001, student's t-test, two-tailed).
Using STCF-WTB as the stimulating antigen, and lymphocytes from the spleen cells, vaccine 5 was significantly different to the control, exhibiting a very high proliferation of 45,135 cpm. Vaccine 2 was also relatively high reading however it was not significantly different to the control. The Peyer's patches lymphocytes again showed a significantly higher proliferation (55,516cpm) for vaccine 5 compared to the control. The scintillation counts for the mesenteric lymph nodes were all low, and likely to be background rather than true readings.
Cytokine analysis using lynaphocyte culture supernatant

Cytokines are used to determine the type of immune response. Three cytokines were measured; IL-2, IL-4 and IFN-y. Only lymphocytes from spleen cells were cultured for cytokine production. As optimal proliferation was determined to be with a stimulating antigen concentration of 2. 5ug/ml, the results of the cytokines at 5ug/ml and lOug/ml are not shown. terleukin 2 Levels IL-2 is a general indicator of T-cell response. Cell supernatant was collected at day 3, which is not optimal for IL-2, however the results clearly indicate that all vaccines stimulated an IL-2 response (Figure 17).


IFN-y and IL-4 as a Measure of the Type of T-cell Response

Comparison of IFN-y and IL-4 (Figure 18) gives an indication of the type of T-cell response, that is, whether a Th1 or Th2 response is occurring.


In all cases except V I (STCF) the IFN-y response was significantly higher than the IL-4, indicating a Thl response. The results of Vaccine 1 (STCF) however are still significant. Vaccine 1 (STCF) produced an average IL-4 level of 7.58 pg/ml. Large difference between the duplicate in IFN-y readings (160. 5pg/ml and 743. 0pg/ml) caused a

large error, negating any measure of significance. It is evident however that even if the larger value is excluded, the IFN-y result is clearly much higher than the IL-4 result.


Measurement of immuonoglobulin levels in mice

ELISAs were performed to measure immunoglobulin. IgG and IgA were measured and compared. Table 35 shows the average absorbances of ELISA readings at a wavelength of 450nm. For IgG serum was tested at a 1: 100 dilution, while IgA faecal supernatant was measured at a 1: 10 dilution. Each vaccine group was tested for antibody against the antigen that each contained. The P. jensenii 702 control group was tested against both antigens, STCF and combination STCF-WTB, as it contained neither and was necessary for its role as a control.


Table 35 Average Absorbance Values of each Immunoglobulin for vaccine groups
Immunoglobuli Vaccine

n

Da 3 STCF/5



STCF WTB

Total IgG 0 0. 180. 05* 0. 260. 07* 0. 180. 06 0. 280. 07* 0. 160. 05 0. 170. 05*

25 0. 410. 17* 0. 430. 12* 0. 220. 08 0. 410. 08* 0. 480. 54 0. 610. 38*

IgA 0 0. 580. 03 0. 310. 03 0. 510. 04 0. 310. 03 0. 580. 06* 0. 320. 03*

25 0. 550. 06 0. 270. 07 0. 55+0. 08 0. 340. 05 0. 460. 1 1 * 0. 260. 06*

Values are mean plus standard deviation (n=6-8) in Absorbance (nm), measured at 450nm (Bio-Rad Microplate Reader (Model 550) *denotes a significant difference between immunoglobulin levels (p < 0.05, student's t-test, two-tailed) within each vaccine group, between days 0 and 25.


The total IgG levels increased for each vaccine from day 0 to day 25, but significant increases were seen only in Vaccine 1, 2,3 (STCF/WTB) and 5.
There was no significant difference in the IgA levels for Vaccine 1,2 and 3. Vaccine 4 and 5 showed a significant reduction in IgA. Preparation of the faeces may have had a detrimental effect on preservation of the IgA, and will be addressed further in the Discussion.
DISCUSSION

The efficacy of four oral vaccines against M. tuberculosis was tested. In addition to finding the most immune stimulating components of each vaccine, the effectiveness of adjuvant containing the probiotic bacteria P. jensenii 702 was tested against the commonly used mucosal adjuvant, Cholera Toxin, for its ability stimulate an immune response to the given antigens. It is clear from the results that the vaccines effectively induced an immune response in vitro.


The importance of a strong Th1 T-cell response to TB immunity is recognised (Kaufmann & Andersen, 1998). The intracellular nature of M. tuberculosis makes the role of T-cells vital in the immune response to this bacterium. Consequently in vitro T-cell proliferations provide a good indicator for the success of a potential vaccine. An optimal

adjuvant for a TB vaccine should favour the development of a cell-mediated immune response and preferentially stimulate a strong IFN-y response (Agger & Andersen, 2001). The demonstrated efficacy of P. jetiseiiii 702 as an adjuvant isnoteworthy, particularly in light of the fact that currently, none of the adjuvants generally approved for human vaccination promote the development of the type 1 immune responses that are crucial for the establishment of protective immunity to M. tuberculosis (Medina & Guzman, 2000).


Since it is notoriously difficult to generate an adequate immune response against soluble peptides and proteins, immunisation with an appropriate adjuvant is essential (Mahairas et al, 1995). Consequently the results obtained across all tests for the different vaccines, containing the two tested adjuvants, provide valuable information and insight into the effectiveness of the adjuvants.
Data was collected in this study to determine the optimum antigen concentration and day of T-cell proliferation (Table 31). It is important to determine the optimum antigen concentration for T-cell proliferation. This is due to the fact that if the antigen is present in too higher concentrations it may exert a toxic effect on the lymphocytes, too low a concentration may be insufficient to stimulate any response at all. Three antigen concentrations were tested in this research, 2. 5, ug, Szg and 10pLg/ml. From statistical analysis it was found that Day 3 with a stimulating antigen concentration of 2. 5, ug/1nl gave the highest T-cell proliferations in most situations (Table 31).
The three tissue samples that were chosen to perform T-cell proliferations, were selected for their ability to represent the different aspects of the immune response. Spleen T- cell proliferations are indicative of a systemic immune response, whereas the Peyer's patches were tested for evidence of a mucosal immune response (Bouvet, Decroix & Pamonsinlapatham, 2002). Mesenteric lymph nodes, which are also a part of the mucosal associated lymphoid tissue (MALT), were tested (Roitt & Delves, 1997). However since the Peyer's patches have first contact with an antigen, and play a role in stimulating antigen- sensitised lymphocytes, they were chosen as the primary indicator of a mucosal immune response. These stimulated lymphocytes from the Peyer's patches enter the lymph and drain through the mesenteric lymph nodes (Roitt & Delves, 1997). Consequently the mesenteric lymph nodes were used as a secondary indicator of a mucosal immune response, in the instance that an insufficient quantity of Peyer's patches was obtained. Since the Peyer's patches gave a sufficient indication of T-cell proliferations, the mesenteric lymph nodes will not be further discussed.
In general the systemic immune response was better than the mucosal, shown by higher proliferation of spleen cells, compared to that of the Peyer's patches and mesenteric lymph nodes. The preferred aim of orally administering the vaccines is to induce a mucosal immune response. It has long been hypothesised that immunisation via one mucosal surface

will lead to enhanced immunity at other mucosal surfaces (Famularo, 1997). Doherty (et al, 2002) found that orally administering a booster vaccine, produced high levels of protection in the lungs of previously primed animals, displaying the ability to confer immunity across mucosal surfaces. Induction of an immune response at the mucosal surface is of major interest because of its ability to modulate colonisation by commensals, as well as increase defenses against penetration of pathogens through the epithelium, reducing their concentration to a harmless level (Bouvet, Decroix & Pamonsinlapatham, 2002). The ability to prevent establishment of M. tuberculosis in the body would be an extremely beneficial achievement.


Results from immunoglobulin testing further reinforce that a strong systemic immune response was obtained, shown through an increase in Total IgG levels in serum (Table 35).
Low IgA levels were observed (Table 35). Secretory IgA is the predominant form of antibody that mediates specific immunological defense at mucosal surfaces, and has been found to have a role in limiting respiratory infections (Gleeson et al, 1999). The low IgA response to vaccines 1 and 2 containing the adjuvant P. jensenii 702, could be due to the need for a longer exposure time to the bacteria to obtain the desired mucosal immune response.
Bouvet et al (2002) found that the extent of the immune response is associated with the degree of colonisation of the vaccine microorganism. Subsequently this suggests that allowing sufficient time for the colonisation of P. jensenii 702 would further improve its activity as a vaccine vector.
From the faecal plate counts for P. jensenii 702 (Table 28) it can be seen that at least 7 days were required to obtain viable P. jensenii 702 counts from the mice faeces, however at the same time there was not a corresponding reduction in total anaerobes (Table 29). In previous research by Huang (2002) in male Wistar rats it took one month for P. jensenii 702 to colonise, and it was not until a competitive colonisation of P. jensenii 702 occurred that a reduction of anaerobes was evident. Further measures in this study, such as beneficial production of Vitamin B 12 and reduction of serum cholesterol levels, also revealed that it took a month for the bacteria to establish, before it could exert any beneficial effect on the host (Huang, 2002). For P. jensenii 702 to work as an efficient adjuvant in producing a mucosal response, exposure time may be critical. It is also likely that this colonisation period is species specific.
Results from Peyer's patches indicate that the vaccine can stimulate a mucosal immune response when cholera toxin is the adjuvant (Tables 32-34). Therefore there is no doubt that the vaccine itself is effective as an oral vaccine in stimulating a mucosal and systemic immune response. This is an important finding since oral vaccination to facilitate effective mucosal and systemic immunity has to date been largely ineffective (Russell-Jones, 2000 ; Doherty et al, 2002).

It is worthwhile noting that the proliferation results varied depending on the type of stimulating antigen. Vaccine 2 and 5 contained equal amounts of STCF and WTB. When stimulated by STCF alone (Table 32), the proliferation results for spleens were much lower than when stimulated with the combination of STCF-WTB (Table 34). When stimulated by WTB (Table 33), vaccine 2 had a lower result than with the combination (Table 34), but vaccine 5 had a higher result. In the case of vaccine 5, there was a significant increase in proliferation when the stimulating antigen was either STCF-WTB or WTB, when compared to STCF alone. The fact that the same significant differences were not observed for vaccine 2 indicates that the mitogenic effect of WTB is more pronounced when Cholera toxin is used as the adjuvant. Significant difference between vaccine 2 and 5 was only observed when the stimulating antigen was WTB, which could also be attributed to this mitogenic effect. For protective immunity, a specific immune response is desired, not a mitogenic response.


Therefore, although the vaccine containing STCF and WTB proteins is effective in inducing an immune response, for protective immunity, P. jensenii 702 may be the better adjuvant.
A T-cell proliferation response was observed for the control, vaccine 3, which contained only P. jensenii 702. This may have been due to shared immunogenic proteins with M. tuberculosis, and is discussed further below. Regardless of this, proliferation results significantly higher than the control were found with vaccine 1,2 and 5, using STCF as the stimulating antigen in spleen cells (Table 32). When spleen lymphocytes were stimulated with WTB (Table 33) or the combination of WTB/STCF (Table 34), vaccine 5 was significantly higher than the control. There was no significant difference between vaccine 1 and vaccine 5 when stimulated by any of the antigen combinations (Tables 32-34). Therefore both vaccine 1 and vaccine 5 demonstrate a significant systemic T-cell response. In consideration of the proposed mitogenic effect of the WTB, it is concluded that vaccine 1 was the better vaccine.
There was however, no significant difference in proliferation response between vaccine 1 and vaccine 2, indicating that when the adjuvant is the same, this data cannot identify what protein composition produces the better vaccine. Previous data on parenteral vaccines demonstrated that secreted M. tuberculosis proteins are more important than whole cell protein in inducing an immune response (Weldingh and Anderson, 1999). This study suggests that soluble cellular proteins of the M. tuberculosis may in fact play a role in this response. It hypothesized that the percentage of these proteins in the vaccine may be a critical factor in it efficacy.
Furthermore, vaccine 1 produced significantly higher proliferations than vaccine 4.
As these two vaccines contained the same proteins, this indicates that the adjuvant is a critical indicator for vaccine success. Vaccine 4 failed to induce T-cell responses higher than the control, vaccine 3 (Table 32). This demonstrates that P. jensenii 702 is an effective oral

adjuvant. Few vaccines administered via the mucosal route have actually been able to stimulate effective cell-meditated immune responses (Doherty et al, 2002), so the significant spleen cell proliferations, indicative of systemic immunity, obtained in this study are unique and important.


In the case of the Peyer's patches, only vaccine 5 when stimulated with all 3 combinations of antigen, STCF, WTB and STCF-WTB (Tables 32-34), showed a significant difference to the P. jensenii 702 control. As Peyer's patches are a measure of mucosal immunity, this indicates that the proposed vaccine will induce a significant mucosal immune response. It is probable that the reason that vaccine 5 produced this response, and vaccine 2, which contained the same protein components, did not, is because cholera toxin acts more immediately as an adjuvant than P. jensenii 702. Cholera toxin however cannot be used in humans due to its toxic nature, and although non-toxic subunits of cholera toxin are available, they are still yet to be permitted for use in humans (Pizza et al, 2001). P. jensenii 702 is a safe food-based bacteria. Studies by Yang (2002) suggest that increasing the colonisation period of P. jensenii 702 would likely enhance the adjuvant effect, such that it demonstrates similar or better activity than cholera toxin, with the advantage that it is suitable for use in humans.
Testing for cytokines is extremely important to this study, as cytokines are a key indicator of the type of immune response (Kaufmann & Andersen, 1998). The type of immune response induced by a vaccine is crucial to TB immunity, as the incorrect immune response (Th2) has been found to be ineffective in controlling TB and actually elicits a disease exacerbating effect (Linblad et al, 1997). Thl responses are characterised by secretion of IL-2 and IFN-y (Kaufmann & Andersen, 1998), and efficient protection from TB requires the induction of a potent Thl response with high levels of IFN-y (Agger & Andersen, 2001).
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