TECHNICAL FIELD
The present invention relates to the filed of immunology and medicine and is directed to a method of immunization against Mycobacterium using recombinant BCG- Ag85C.
BACKGROUND
Mycobacterium tuberculosis continues to be a leading cause of human deaths due to an infectious agent (Global Tuberculosis Control: surveillance, planning, financing. In WHO report 2008. Geneva: World Health Organization). The situation has become even more precarious due to the emergence of multi drug resistant strains of M. tuberculosis and lethal combination of tuberculosis (TB) and HIV infections. Global trends in resistance to antituberculosis drugs.-. It has been indisputably accepted by TB experts that complete eradication of this disease may be difficult to achieve without the availability of an efficient vaccine. Mycobacterium bovis Bacille Calmette Gurein (BCG), the only vaccine currently in use against TB, despite its satisfactory performance against childhood TB, does not impart adequate protection against pulmonary TB in adults, with its efficacy ranging from 0-80%.
Development of recombinant BCG (rBCG) based vaccines over-expressing promising immuno-dominant antigens of M. tuberculosis represents one of the potential approaches to improve upon the performance of BCG. The proteins belonging to the antigen 85 complex (Ag85), a family of 30-32 kDa proteins (Ag85A, Ag85B and Ag85C) represent a group of the major secretory and immunodominant proteins of M bovis BCG and M. tuberculosis (Closs, 0., Harboe, M., Axelsen, N. H., Bunch-Christensen, K. and Magnusson, M. (1980). The antigens of Mycobacterium bovis, strain BCG, studied by crossed Immunoelectrophoresis: a reference system. Scand J Immunol 12, 249-63; Wiker, H. G., Harboe, M., Bennedsen, J. and Closs, O. (1988).

Evelyne Lozes et. al have described the immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex and concluded that plasmid encoding the Ag85A component was consistently found to be the most effective plasmid, however, DNA vaccine encoding Ag85C did not evoke any considerable immune responses in terms of Thl cytokine production and CTL activity thereby rendering this antigen non-protective and ineffective among the three members of the complex (Evelyne Lozes, Kris Huygen, Jean Content, Olivier Denis, Donna L. Montgomery, Anne M. Yawman, Paul Vandenbussche, Jean-Paul Van VoorenS, Annie Drowart, Jeffrey B. Ulmer and Margaret A. Liut, Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex. Vaccine, Vol. 15, No. 8, pp. 830433, 1997).
SUMMARY OF THE INVENTION
The present invention provides recombinant BCG over-expressing antigen 85C from Mycobacterium for immunization against Mycobacterium.
One aspect of the present invention relates to a recombinant BCG over-expressing antigen 85C from Mycobacterium for use in generating an immunogenic response in a subject against Mycobacterium, wherein said recombinant BCG having recombinant vector comprising nucleotide sequence coding for antigen 85C.
One aspect of the present invention relates to a vaccine formulation for generating an immunogenic response in a subject against Mycobacterium, said composition comprising an immunologically effective amount of the recombinant BCG as disclosed in the present invention.
One aspect of the present invention relates to a process for producing a recombinant BCG over-expressing antigen 85C from Mycobacterium as disclosed in the present invention, said process comprising, introducing a nucleotide sequence coding for antigen 85C from Mycobacterium into a suitable expression vector to obtain a

recombinant vector and transforming BCG with the recombinant vector to obtain a recombinant BCG over-expressing antigen 85C.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Figure 1 shows the effect of Ag85C based vaccination on lung and spleen bacillary load in guinea pigs. Guinea pigs (n = 6) immunized with various vaccines were challenged via aerosol route with 500 - 1000 bacilli and bacillary load was determined in the lung and spleen at (A, B) 10 weeks (Exp-I) and (C, D) 16 weeks (Exp-II) post-challenge, respectively. Each point in the graph represents the Log10 CFU value for an individual animal and the bar depicts mean (± SE) for each group. The lower limit of detection was 1.0 Log10/g of tissue and animals with undetectable bacillary count were allotted 1 logio. Missing data points represent the animals that succumbed to disease before the time of euthanasia. *, p < 0.05; **, p < 0.01 and ***, p < 0.001, when compared to the saline group, Φ p < 0.05; Φ,Φ, p < 0.01 and ΦΦΦ, < 0.001, when compared to the BCG group (One way ANOVA).
Figure 2 shows influence of vaccination on gross pathological lesions in guinea pigs. Gross pathological damage in lung, liver and spleen was scored and graded from 1-4 based on the extent of involvement, number and size of tubercles, areas of inflammation and necrosis. The gross scores are represented graphically for Exp-I (A-C) and Exp-II (D-F). Each point represents score for an individual animal and the bar depicts median (± inter quartile range) for each group. Missing data points represent the animals that succumbed to disease before the time of euthanasia. *,p < 0.05 and **, p <0.01, when compared to the saline group; Φ, p < 0.05, when compared to the BCG group (Mann-Whitney U test).
Figure 3 shows histo-pathological changes in lung and liver of immunized guinea pigs at 10 weeks post-infection with M. tuberculosis. The figure depicts the granuloma percent in (A) lung and (B) liver, respectively by box plot, wherein median values are denoted by horizontal lines, the mean is represented by '+', inter

quartile range by boxes, and the maximum and minimum values by whiskers. **,p < 0.01, when compared to the saline group; Φ, p < 0.05 and ΦΦ, p < 0.01, when compared to the BCG group (Mann-Whitney Utest).
Figure 4 shows histo-pathological changes in lung and liver of immunized guinea pigs at 16 weeks post-infection with M. tuberculosis. The figure depicts the granuloma percent in (A) lung and (B) liver respectively by box plot, wherein median values are denoted by horizontal line, the mean is represented by '+', inter quartile range by boxes, and the maximum and minimum values by whiskers. *,p < 0.05, when compared to the saline group; Φ, p < 0.05, when compared to the BCG group (Mann-Whitney f/test).
Figure 5 shows the influence of vaccination on pulmonary fibrosis. The figure depicts the graphic representation of the extent (quick score, Q) of pulmonary fibrosis (A) Exp-I and (B) Exp-II, as median (± inter quartile range). *, p < 0.05, when compared to the saline group; Φ, p < 0.05, when compared to BCG group (Mann-Whitney Utest).
Figure 6 shows the effect of Ag85 complex based vaccination on spleen bacillary load in guinea pigs. Guinea pigs (n - 6) immunized with various recombinant BCG vaccines were challenged via s.c. route with M. tuberculosis and bacillary load was determined in the spleen at 8 weeks post-challenge. Among the three recombinant strains of BCG, rBCG85C showed significant reduction in splenic bacillary load. Each bar in the graph depicts mean (± SE) CFU/g of tissue for each group. The lower limit of detection was 1.0 log10/g of tissue and animals with undetectable bacillary count were allotted 1 log10. *,p < 0.05 (One way ANOVA).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a recombinant BCG over-expressing antigen 85C for generating immunogenic response in a subject against tuberculosis.

The present invention further provides a recombinant vector comprising the polynucleotide having nucleotide sequence as set forth in SEQ ID NO:1 coding for Ag85C antigen having amino acid sequence as set forth in SEQ ID NO:2. The present invention also provides an expression system expressing high level of Ag85C antigen. The present invention also provides recombinant Mycobacterium bovis BCG strain that over expresses Ag85C antigen of Mycobacterium tuberculosis, the nucleotide sequence of the gene encoding Ag85C antigen is as set forth in SEQ ID NO: 1.
The present invention describes the importance of Ag85C (Rv0129c) of Mycobacterium tuberculosis as a potential vaccine candidate against TB. Further, the invention provides recombinant Mycobacterium bovis BCG- Ag85C based immunization against TB. The invention also provides a method for developing the recombinant Mycobacterium bovis BCG strain that over expresses Ag85C antigen of Mycobacterium tuberculosis under the transcriptional control of a strong mycobacterial promoter. Moreover, the invention provides a method to assess the protective efficacy of recombinant Mycobacterium bovis BCG-Ag85C against Mycobacterium tuberculosis infection.
In the present invention, the protective efficacy of rBCG85C was assessed in a highly susceptible guinea pig model against M. tuberculosis challenge by the aerosol route. Immunization of guinea pigs with rBCG85C resulted in a significantly enhanced protection characterized by a marked reduction in bacillary load in lungs and spleen along with a significantly reduced pathology in various organs, when compared to BCG immunization.
One embodiment of the present invention provides a recombinant BCG over-expressing antigen 85C from Mycobacterium for use in generating an immunogenic response in a subject against Mycobacterium, wherein said recombinant BCG having recombinant vector comprising nucleotide sequence coding for antigen 85C.

One embodiment of the present invention provides a recombinant BCG over-expressing antigen 85C, wherein the antigen 85C is from Mycobacterium tuberculosis.
One embodiment of the present invention provides a recombinant BCG over-expressing antigen 85C, wherein the antigen 85C is from Mycobacterium bovis.
One embodiment of the present invention provides a recombinant BCG, wherein the nucleotide sequence coding for antigen 85C is as set forth in SEQ ID NO: 1.
One embodiment of the present invention provides a recombinant BCG, wherein amino acid sequence of the antigen 85C is as set forth in SEQ ID NO: 2.
One embodiment of the present invention provides a recombinant BCG, wherein the BCG is selected from a group consisting of Danish strain, Glaxo strain, Pasteur strain, Tice strain and Connaught strain; preferably Danish strain.
One embodiment of the present invention provides a vaccine formulation for generating an immunogenic response in a subject against Mycobacterium, the composition comprising an immunologically effective amount of the recombinant BCG as described above.
One embodiment of the present invention provides a vaccine formulation as described above, wherein the formulation further comprises an adjuvant and a pharmaceutically effective carrier.
Another embodiment of the present invention provides a process for producing a recombinant BCG over-expressing antigen 85C from Mycobacterium, the process comprising, introducing a nucleotide sequence coding for antigen 85C from Mycobacterium into a suitable expression vector to obtain a recombinant vector and transforming BCG with the recombinant vector to obtain a recombinant BCG over-expressing antigen 85C.

Another embodiment of the present invention provides a process for producing a recombinant BCG over-expressing antigen 85C, wherein the transformation of the BCG strain with the recombinant vector results in over-expression of antigen 85C from an episomal plasmid.
Another embodiment of the present invention provides a process for producing a recombinant BCG over-expressing antigen 85C, wherein transformation of the BCG strain with the recombinant vector results in the integration of the DNA coding for the antigen 85C into the chromosome of said recombinant BCG.
Yet another embodiment of the present invention provides a process for producing a recombinant BCG over-expressing antigen 85C, wherein the BCG is selected from a group consisting of Danish strain, Glaxo strain, Pasteur strain, Tice strain and Connaught strain; preferably Danish strain.
Yet another embodiment of the present invention provides a process for producing a recombinant BCG over-expressing antigen 85C from Mycobacterium, wherein said Mycobacterium is Mycobacterium tuberculosis or Mycobacterium bovis.
A further embodiment of the present invention provides a process for producing a recombinant BCG over-expressing antigen 85C from Mycobacterium, wherein said nucleotide sequence coding for antigen 85C is as set forth in SEQ ID NO: 1.
One embodiment of the present invention provides a process for producing a recombinant BCG over-expressing antigen 85C from Mycobacterium, wherein amino acid sequence of said antigen 85C is as set forth in SEQ ID NO: 2.
One embodiment of the present invention provides a process for generating an immunogenic response in a subject against Mycobacterium, said process comprising administering to said subject vaccine formulation as claimed in claim 7.

One embodiment of the present invention provides a process for generating an immunogenic response in a subject against Mycobacterium, wherein said Mycobacterium is Mycobacterium tuberculosis or Mycobacterium bovis.
Another embodiment of the present invention provides a process for generating an immunogenic response in a subject against Mycobacterium, wherein said vaccine formulation is administered orally, subcutaneously, intraperitonealy, intravenously, intramuscularly, intradermaly or via respiratory route. '
A further embodiment of the present invention provides use of the recombinant BCG over-expressing antigen 85C for the preparation of pharmaceutical formulation for use as a vaccine against tuberculosis.
In the present invention it was found that recombinant BCG over expressing Ag85C antigen of Mycobacterium tuberculosis has excellent imuno-protective efficacy against tuberculosis, which is in striking contrast to the earlier findings of Evelyne Lozes et. al (Evelyne Lozes, Kris Huygen, Jean Content, Olivier Denis, Donna L. Montgomery, Anne M. Yawman, Paul Vandenbussche, Jean-Paul Van VoorenS, Annie Drowart, Jeffrey B. Ulmer and Margaret A. Liut, Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex. Vaccine, Vol. 15, No. 8, pp. 830433, 1997) that illustrates that Ag85C, when employed in DNA vaccine form was not effective in eliciting Thl cytokine production, CTL activity or protection. However, other members of this Ag85 complex (Ag85A, Ag85B) showed considerable promise as DNA vaccine.
Development of rBCG85C
Gene encoding for Ag85C {Rv 1029c) was amplified using polymerase chain reaction from M. tuberculosis genomic DNA and cloned in expression vector under the transcriptional control of promoters of varying strength known in the art (pSD5.hsp65, pSD5.S16, pSD5.acr, pSD5.T106, pSD5.T31 are some of the expression vectors employed for over-expression of these antigens under the

transcriptional control of strong mycobacterial promoters like hsp65, S16, acr, T106 and T31). Nucleotide sequence coding for Ag85C antigen is provided in SEQ ID No: 1 and amino acid sequence of Ag85C antigen is provided in SEQ ID No: 2. Detailed procedure of preparing recombinant Mycobacterium bovis BCG comprising the recombinant vectors carrying Ag85C gene is provided in Example 2.
Ag85C based vaccination regimens
Various immunization regimens based on Ag85C were evaluated for their protective potential against TB. Details are given in the Example. 11.
rBCG85C: A single dose of a recombinant M. bovis BCG strain over-expressing Ag85C of M. tuberculosis under the transcriptional control of hsp65 promoter derived from M. leprae by intradermal (i.d.) route.
rBCG85C/DNA: The regimen comprises injecting a single dose of rBCG85C by intradermal (i.d) route followed by a booster of DNA85C i.e. DNA vaccine by intramuscular route (100µg, i.m.) at 6 weeks. Details are provided in Example 11.
DNA/rBCG85C: The regimen comprises injecting a single dose of DNA vaccine i.e. DNA85C (100 µg, i.m.) followed by a booster of rBCG85C by i.d. route at 3 weeks.
DNA: The regimen comprises injecting only DNA vaccines i.e DNA85C (100 µg, i.m.) thrice at 3 weeks interval. Vector: The regimen comprises injecting plasmid DNA vector (100µg, i.m.) (devoid of the gene encoding for Ag85C) thrice at 3 weeks interval.
BCG: The regimen comprises injecting a single dose of BCG - an attenuated strain of M bovis by i.d. immunization.
Saline: The regimen comprises injecting a single dose of normal saline by i.d. injection.

To evaluate the efficacy of various vaccine regimens comprising of rBCG85C and DNA85C vaccines to restrict the growth of M. tuberculosis in guinea pigs, the number of bacilli was measured in lung and spleen.
In Exp-I, immunization with both BCG as well as with rBCG85C resulted in a significant reduction in lung and spleen bacillary load, when compared to the saline treated animals at 10 weeks post infection. However, the extent of reduction in case of rBCG85C immunization was markedly greater (by 1.87 log10 in lung and 2.36 log10 in spleen), when compared to BCG immunized animals (Fig. 1 A, B). Employing DNA vaccine expressing Ag85C in homologous or heterologous prime boost approach with rBCGSSC did not provide any significant protection, when compared to saline and vector treated animals (Table I)
Based on the results of Exp-I, vaccine candidates showing efficacy greater than BCG were reevaluated by increasing both the pre- and post-challenge period. The results obtained from Exp-II also substantiated the data of Exp-I. On extending the time of euthanasia to 16 weeks, wherein BCG showed a considerable reduction in its ability to impede bacillary multiplication, rBCG85C immunization successfully restricted the bacillary growth in both lung and spleen (Fig. 1 C, D). The reduction in bacillary load in rBCG85C group was found to be significantly greater, when compared to BCG group (by 0.87 logio in lung and 1.99 logio in spleen). These observations clearly indicated enhanced protective efficacy of rBCGSSC, when compared to BCG.
Table I: Effect of Ag85C based prime boost vaccination regimens on lung and spleen bacillary load in guinea pigs
(Table Removed)

To evaluate the effect of Ag85C based vaccination regimens on gross pathological outcome various organs were scored. The trend in gross pathological damage supported the findings of bacteriological evaluation. In Exp-I, severe pathological damage was observed in case of saline treated animals as characterized by extensive involvement and numerous large tubercles with scattered areas of necrosis in both lung and liver. (Fig. 2 A-C). In addition, a marked enlargement of spleen with numerous large and small size tubercles with occasional attrition of capsular structure was also observed. Immunization of animals with BCG or rBCG85C showed a significant reduction in gross pathological damage with fewer and smaller tubercles in lung along with none or occasional lesions in liver. Lesions were scanty and extremely small in the organs of rBCG85C-immunized animals, when compared to BCG group. In response to immunization with DNA85C alone, no significant improvement in the pathological damage was observed when compared to the vector treated animals. A booster dose of DNA85C subsequent to rBCG85C priming (R/D) resulted in a significantly greater pathological damage in lung, when compared to rBCG85C indicating the detrimental influence of DNA85C boosting, although such a deleterious effect was not noted incase of liver and spleen, which exhibited pathological damage that was comparable to rBCG85C vaccination. On reverting the order of immunization (D/R), the pathological damage in all the organs was more or less similar to R/D regimen. Thus, it appeared that DNA85C when used alone or in a heterologous prime boost approach did not provide expected improvement, if at all; it reduced the protective efficacy of rBCG85C when used in conjunction with the latter.
In Exp-II, due to uncontrolled bacillary multiplication and extensive inflammatory response, 50% of the non-vaccinated animals succumbed to disease by 16 weeks;

animals that survived showed characteristic signs of end stage tuberculosis. Although, the number of animals that survived till the time of euthanasia was similar in case of both BCG and rBCG85C immunized animals, a remarkable improvement in the gross pathological damage was observed in the latter group, as was evident from the minimal involvement in lung and no evident signs of tissue damage in both liver and spleen (Fig. 2 D-E). However, 75% of the animals in BCG group showed extensive pulmonary damage with several large and small size tubercles distributed throughout the lung, together with progressive splenic and hepatic tissue destruction.
To evaluate the histopathological damage in lung and liver of immunized and saline treated animals, the tissue sections were stained with H&E and granuloma percent was measured. At 10 weeks post infection, the type of granuloma observed in lungs of the saline treated guinea pigs typically represented an advanced stage granuloma, with extensive coalescence of multiple granulomas covering 67.5% area of the lung sections (Fig. 3) and the lung granulomas were predominantly composed of a central necrotic core with a dense aggregate of macrophages encircled by thin layer of lymphocytes. This extensive necrosis resulted in the loss of micro-architecture of lung and erosion of airway epithelial lining with alveolar collapse. The extent of granulomatous response observed in case of BCG vaccinated animals was only marginally lower (60%), when compared to that observed in the saline treated animals. However, the extent of necrosis in this case was relatively less, when compared to saline treated animals. In comparison to animals treated with BCG or saline, immunization with rBCG85C resulted in a significant reduction in the granulomatous inflammation (7.5%)) with relatively compact and discrete granulomas. The alveolar and bronchiolar structure in the surrounding areas were well preserved in this group. Immunization of animals with DNA vaccine alone resulted in extensive granulomatous inflammation in lung (55%)), which was similar to that observed in the case of vector treatment. Commensurate to observations made with respect to gross pathology, heterologous prime boost regimens involving

DNA85C (R/D and D/R) resulted in far more pathological damage in lung (22.5%) as compared to rBCG85C vaccination, further substantiating its detrimental role in heterologous prime boost approach.
As depicted in Fig. 3, in comparison to the extent of hepatic damage and percentage of granuloma in saline group (25.8%), a significant reduction was observed in case of BCG immunized animals (12.2%). However, vaccination with rBCG85C resulted in a significantly greater protection, resulting only in a negligible granulomatous response and no evident sign of necrosis. Immunization with DNA85C could not prevent hepatic damage thus resulting in extensive granulomatous lesions spreading over 60% area of hepatic parenchyma, which was comparable with the vector treated animals. However, unlike the observations made in case of lung and spleen, DNA85C, when used in heterologous prime boost regimens did not show any detrimental influence on liver and the histopathological damage in both R/D and D/R regimens was comparable to rBCG85C immunization.
In Exp-II, in saline treated animals, majority of the pulmonary parenchyma was effaced by extensive granulomatous inflammation (80%) with coalescing necrotic granulomas. Granulomas observed in case of BCG immunized animals also represented a situation similar to the one observed in case of saline treated animals, with percent granuloma being 65% (Fig. 4). In contrast, immunization with rBCG85C preserved the pulmonary microstructure with a significantly less granulomatous area (10%) and no evident signs of necrosis. On comparing the pathological damage in liver, a substantial increase was observed in case of saline treated animals with multiple coalescing foci of necrotic granulomas (50%). Immunization with rBCG85C resulted in significantly reduced hepatic damage with only a negligible granulomatous inflammation (3.5%)) (Fig. 4). However, the extent of hepatic granulomatous inflammation in case of BCG immunization was relatively higher (30%)). Since DNA85C vaccination showed no significant protection when used either alone or in heterologous prime boost regimens, further studies, which are

described below, were carried out only with rBCG85C vaccination and its comparison to BCG immunization and saline treatment.
To evaluate the effect of Ag85C based vaccination on collagen deposition lung sections were evaluated for extent of fibrosis. In case of saline treated animals at 10 weeks time point (Exp-I), extensive areas of collagen deposition in and around the granulomas were observed (Fig. 5). In addition to the surrounding of the necrotized core, collagen was also detected as widespread irregular thick bands within the granuloma. However, in case of BCG and rBCG85C groups, a marked reduction in collagen deposition was observed, wherein, thin and diffused bands of collagen were primarily restricted to the periphery of granulomatous areas. In Exp-II, a marked increase in collagen deposits was observed in case of both BCG and saline treated animals (Fig. 5). Immunization with rBCG85C resulted in only a negligible collagen staining, which was significantly less, when compared to both the control groups.
The Applicants have tested the protective efficacy of other two members of Ag85 complex, i.e. Ag85A and Ag85B as rBCG strain over expressing these antigens as described in Example 17. However surprisingly only Ag85C showed substantial protective potential against tuberculosis among the three members of the antigen 85 complex (Figure 6).
Moreover, the applicants found that the magnitude of protection conferred by rBCG85C vaccine in comparison to the BCG vaccine has not been reported in the art earlier in any guinea pig study (Table II).
In the present invention it was found that recombinant BCG over expressing Ag85C antigen of Mycobacterium tuberculosis has enhanced immuno-protective efficacy against tuberculosis. The present invention provides an expression system that show high level of expression of the Ag85C antigen

The present invention further provides the expression system comprising the gene coding for Ag85C antigen, wherein the gene is present under the control of hsp65 promoter.
The expression system disclosed in the present invention produces the Ag85C antigen ~ 6.0 fold in extra cellular and ~ 3.0 fold in intracellular fraction.
The present invention provides the comparative protective values depicted in Table II in regard with the improvements/ modifications that have been made in the approach of developing a recombinant BCG vaccine.
The comparative protective values depicted in Table II relates to the improvements/ modifications that have been made in approach of developing a recombinant BCG vaccine disclosed in the present invention.
None of the TB vaccines depicted in Table II result's from the same approach as disclosed in the present invention. Vaccine composition no.l namely recombinant BCG85B (Table II) is based on the expression of Ag85B gene under its native promoter. Vaccine composition no.2 namely, fusion protein of ESAT-6 and Ag85B is based on the injection of a purified fusion protein, hence in this case there is no continual production of protein as provided in the case of a live vaccine. The vaccine regimen no.3 in Table II is based on provision of BCG as a primary vaccine followed by booster dose of recombinant Modified Vaccinia Ankara virus expressing AgSSA and then a second booster in the form of a recombinant Fowl Pox Virus expressing Ag85A.Regimen no. 4 is based on recombinant BCG expressing Listeriolysin gene from Listeria monocytogenes, which supposedly helps, in improved presentation of antigens by MHC class I pathway by allowing the BCG to escape phagolysosomal fusion and thereby come out in the cytosol. Thus this recombinant BCG approach is not based on the over expression of an M tuberculosis antigen. Regimen No. 5 is based on the administration of a combination of BCG and a purified poly protein (72f fusion protein) comprising of domains from two different proteins stitched

together. In the present invention, the Ag85C antigen regimen is based on the administration of recombinant BCG strain which expresses high levels of Ag85C under one of the very strong promoters of Mycobacteria, namely hsp65. The vector is also provided with the facility of secreting high levels of Ag85C outside the mycobacterial cell. Thus, it can be concluded that the present invention provides unexpected and surprisingly high levels of Ag85C protein expression which have not been achieved by any of the earlier approaches and results.
In accordance with the present invention, one embodiment provides the polynucleotide having nucleotide sequence as set forth in the SEQ ID NO: 1, the polynucleotide encodes Ag85C antigen of Mycobacterium tuberculosis.
In another embodiment the present invention.provides a polypeptide sequence of Ag85c antigen having amino acid sequence as set forth in SEQ ID NO: 2.
In yet another embodiment of the present invention there is provided a recombinant vector comprising the polynucleotide having nucleotide sequence as set forth in the SEQ ID NO: 1 encoding Ag85C antigen.
In still yet another embodiment of the present invention there is provided a recombinant Mycobacterium bovis BCG strain that over expresses Ag85C gene of Mycobacterium tuberculosis under the transcriptional control of a strong mycobacterial promoter.
In another embodiment there is provided a regimen for immunization against tuberculosis comprising injecting a single dose of rBCG85C by i.d. route followed by a booster of DNA85C i.e. DNA vaccine by intramuscular (i.m.) route.
In yet another embodiment there is provided a regimen for immunization against tuberculosis comprising injecting a single dose of DNA vaccine i.e. DNA85C by i.m. route followed by a booster of rBCG85C by i.d. route.
Further in another embodiment, the present invention provides a regimen for

Table II: A comparative depiction of the protective efficacy of rBCG85C and the vaccine candidates in clinical trials existing in the art
(Table Removed)

immunization against tuberculosis comprising injecting only DNA vaccines i.e DNA85C by i.m. route thrice at 3 weeks interval.
In one embodiment, the present invention provides a regimen for immunization against tuberculosis comprising injecting plasmid DNA vector by i.m. route thrice at 3 weeks interval.
In another embodiment, the present invention provides a regimen for .immunization against tuberculosis comprising injecting a single dose of M. bovis BCG by i.d. immunization.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete invention and the description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all and only experiments performed.
Example 1: Microbiological techniques
Various recombinant strains of E. coli were grown either in LB mediurn or in 2XYT medium containing appropriate antibiotic(s) by the conventional techniques and high efficiency competent cells were prepared by the standard CaC12 method (Sambrook, J., Fritsch, E.F, and Maniatis, T. (1989), Molecular cloning. A laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory 'Press.). Mycobacteria were cultured and electrocompetent cells prepared. Stocks of mycobacteria were then prepared for immunization and infection (Dhar, N., Rao, V. and Tyagi, A. K. (2003). Skewing of the Thl/Th2 responses in mice due to variation in the level of expression of an antigen in a recombinant BCG system. Immunol Lett 88, 175-84).
Example 2: E. coli transformation

Transformation of E. coli was carried out by the method described by Mandel and Higa (Mandel, M. and Higa, A. (1970), Calcium-dependent bacteriophage DNA infection. J Mol Biol 53, 159-62). Transformation of M. bovis was performed by electroporation method. 1 µg of plasmid DNA was mixed with 20 µg of electrocompetent cells, the cells were revived and the transformants were selected on MB7H11 agar plates supplemented with appropriate antibiotic(s) ( Dhar, N., Rao, V. and Tyagi, A. K. (2003), Skewing of the Thl/Th2 responses in mice due to variation in the level of expression of an antigen in a recombinant BCG system. Immunol Lett 88, 175-8).
Example 3: Preparation of plasmid DNA from E. coli transformants
Mini-preparation of plasmid DNA: A mini preparation of plasmid DNA was performed by Alkaline lysis method or Boiling lysis method and maxi preparation of plasmid DNA: Plasmid DNA was isolated on a large scale by the alkaline SDS method ( Sambrook, J., Fritsch, E.F, and Maniatis, T. (1989), Molecular cloning, A laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).
Large-scale preparation of plasmid DNA vaccines: For immunization of mice and guinea pigs with DNA vaccines, recombinant DNA plasmids were purified on large scale by using the QIAGEN endofree giga kit as per the manufacturer's recommendation (Qiagen GmbH, Germany). Bacterial cells (2 liter culture) were harvested at 6000 g at 4°C for 15 min and resuspended in buffer PI (containing RNase, 0.1 mg/ml), followed by successive addition of buffer P2 and P3 (pre-chilled) at an interval of 5 min. The cell lysate was clarified by centrifugation at 6000 g at 4°C for 15 min, followed by filtration through QIA filter cartridge. Clarified cell lysate was then incubated with Endotoxin removal solution on ice for 30 min. prior to loading on to the Qiagen tip-1000 (pre-equilibrated with buffer QBT), cell lysate was clarified again by centrifugation at 6000 g at 4°C for 15 min and allowed to enter the resin by gravity flow. Contaminating proteins and low molecular weight impurities were

removed by washing the resin with medium salt buffer (Buffer QC). Plasmid DNA was eluted with a high salt buffer (Buffer QN). After elution, plasmid DNA was desalted and precipitated with 0.7 v/v of isopropanol in sterile endotoxin free corex tubes and recovered by centrifugation at 17000 g at 4°C for 30 min. The pellet was washed with 70% ethanol, air-dried and resuspended in appropriate volume of endotoxin free water.
Example 4: Isolation of chromosomal DNA from Mycobacteria
M. tuberculosis culture (100 ml) was grown to an A600nm of 1.5 in MB7H9 medium at 37°C in an orbital shaker at 200 rpm followed by incubation with glycine (1%) at 37°C for 24 hr. Spheroplasts were harvested by centrifugation at 3,076 g at room temperature for 10 min and were lysed by incubating first with lysis buffer (TEG containing lysozyme 2 mg/ml) at 37°C for 16 hr followed by incubation with 1% SDS and Proteinase K (0.71 mg/ml) at 55°C for 40 min with intermittent gentle swirling. The lysate was incubated with NaCl (1 M) and CTAB (1.5%) at 65°C for 10 min. Genomic DNA was extracted twice with phenol-chloroform (1:1) followed by chloroform extraction two times. DNA in the aqueous phase was precipitated by incubation with 0.6 v/v isopropanol at room temperature for 15 min. The genomic DNA spool was removed by using a sterile microtip washed with 70% ethanol, air-dried and resuspended in 100 µl autoclaved double distilled water. Agarose gel electrophoresis was carried out essentially as described by Sambrook et al (1989). DNA fragments were recovered from agarose gel by using Amersham gel elution GFX columns as per the manufacturer's instructions.
Example 5: Cloning of the gene encoding Ag85C into pAK4
The vector pQE30.85C carrying the gene encoding Ag85C was digested with EcoK I and Hind III. DNA fragments encoding the gene were end repaired with Klenow DNA polymerase, ligated with EcoR V digested pLITMUS38 and the ligation mixture was

used to transform E. coli XL-IB cells. The transformants were selected by blue white selection on LB plates containing Xgal, IPTG, ampicillin and tetracyclin. The plasmid DNA isolated from the transformants were analysed by restriction enzyme digestion. DNA fragments encoding Ag85C gene were excised out by restriction digestion of recombinant plasmid, pLITMUS38.85C with BamH I, gel purified and ligated with vector pAK4 (linearized by Bgl II and dephosphorylated by CIP treatment). The ligation mixture was used to transform HBl0l cells, transformants were selected on LB plates containing kanamycin and ampicillin and the plasmids isolated from the transformants were analysed by restriction enzyme digestion to confirm the presence and orientation of the gene (Fig. 1, Section I in Results & Discussion). Nucleotide sequence of the cloned gene was confirmed by sequencing (ABI Prism 3100 sequencer, version 3.0) by using dye terminator chemistry cycle sequencing kit (Applied Biosystems, CA, USA).
Example 6: Methods for gene expression in E. coli and protein purification
Expression of genes in E. coli: For expression and purification of recombinant proteins, pQE30 vector was employed which allowed the expression of a protein as fusion protein with a Histidine tag (containing 6 His residues) towards N terminus of the expressed protein. Escherichia coli Ml5 cells were transformed with the Ag85C derivative of pQE30 and the transformants were selected on LB plates containing ampicillin and kanamycin. Under conditions of maximal expression, Ag85C, was found to be aggregated resulting in insoluble inclusion bodies as described above. In order to purify these His-tagged proteins sequestered in inclusion bodies, proteins were solublized with 8M urea as denaturant and purified by employing Ni-NTA based affinity chromatography. The protein purification was carried out by using Ni-NTA superflow resin as per the manufacturer's recommendations.
Example 7: Expression of antigens in Mycobacteria

Expression of antigen Ag85C in recombinant M bovis BCG vaccine strain was analyzed as described in (Dhar et al, 2003; Rao, V., Dhar, N. and Tyagi, A. K. (2003). Modulation of host immune responses by overexpression of immunodominant antigens of Mycobacterium tuberculosis in bacille Calmette-Guerin. Scand J Immunol 58, 449-61). Briefly, M bovis BCG was separately transformed with pSD5.hsp65.85C. The amount of protein in the different cell free extracts and culture supernatants was estimated by Bradford's method ( Bradford, M. M. (1976), A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-54). Equal amounts of various protein samples (50 µg) were subjected to SDS - PAGE and immunoblot analysis.
Example 8: Bacteria
M. bovis BCG (Danish strain) was procured from BCG laboratories, Chennai, India. M. tuberculosis H37Rv was kindly provided by Dr. J. S. Tyagi, All India institute of medical sciences, New Delhi, India. BCG, rBCG85C and M. tuberculosis strains were grown to mid-log phase in Middle Brook (MB) 7H9 media and stocks were prepared as described earlier ( Singh, R., Rao, V., Shakila, H., Gupta, R., Khera, A., Dhar, N., Singh, A., Koul, A., Singh, Y., Naseema, M. et al. (2003). Disruption of mptpB impairs the ability of Mycobacterium tuberculosis to survive in guinea pigs. Mol Microbiol 50,751-62).
Example 9: Preparation of antigens for immunization
For preparation of rBCG85C, a Mycobacteria - Escherichia coli shuttle plasmid pSD5.pro was used as described earlier (Dhar, N., Rao, V. and Tyagi, A. K. (2000). Recombinant BCG approach for development of vaccines: cloning and expression of immunodominant antigens of M. tuberculosis, FEMS Microbiol Lett 190, 309-16; Jain, S., Kaushal, D., DasGupta, S. K. and Tyagi, A. K. (1997). Construction of shuttle

vectors for genetic manipulation and molecular analysis of mycobacteria. Gene 190, 37-44). Briefly, the plasmid (pSD5.pro) was engineered to over-express Ag85C along with its native signal sequence under transcriptional control of the promoter of M. leprae gene encoding heat shock protein 65 (hsp65). The plasmid was electroporated into M. bovis BCG and selected on MB7H11 plates containing Kanamycin (25)µg/ml).
Example 10: Experimental animals
Pathogen free 200-300g female outbred guinea pigs (Dunkin Hartley strain) used for the protective efficacy studies were procured from Disease Free Small Animal House Facility, Haryana Agricultural University, Hissar, India. The animals were housed in stainless steel cages and were provided with ad libitum food and water in a BSLIII facility (National JALMA Institute of Leprosy and Other Mycobacterial Diseases, Agra, India). All the experimental protocols were reviewed and approved by the animal ethics committee of the institute.
Example 11: Immunization and aerosol challenge of guinea pigs with M, tuberculosis
For protective efficacy studies, pathogen free 200-300 g female guinea pigs (outbred Dunkin Hartley strain) were employed. Groups of 6 or 7 guinea pigs were immunized with one of the following: (i) 5X105 CFU of either BCG (Danish strain) or rBCG85C strain in 100 µ1 of saline by i.d. route, (ii) 100µg of either vector or plasmid DNA vaccine in 100 µl of saline by i.m. route thrice at 3 weeks interval (V/V/V and D/D/D), (Hi) 5X105 CFU of rBCG85C once, followed by a booster of 100 µg of DNA at 6 weeks (R/D), (iv) 5X105 CFU of rBCG85C booster 3 weeks after primary immunization with 100 µg of DNA (D/R), and (v) 100 µl of saline by i.d. route as a negative control.
For protective efficacy studies, two different experimental protocols (Protocol I and II) were employed. In protocol I, guinea pigs were challenged at 6 weeks after the last

immunization with -500 bacilli of virulent Mtb H37Rv via the respiratory route in an aerosol chamber (Inhalation exposure system, Glas-col, IN, USA) and were euthanized at 10 weeks post-infection. Since the schedules of boosting and infection for various vaccination regimens were variable in protocol I, in order to dissect out the role of the heterologous prime boost regimens per say versus the differences in the time interval between primary immunization and infection in different regimens, in Exp-II, a modified protocol was employed allowing the comparison of different regimens with a constant time interval for all the groups. The guinea pigs in this experiment were challenged at 12 weeks post primary immunization and were euthanized at 16 weeks post-infection prolonging the time of euthanasia helped in evaluating the long -term effect of various vaccination regimens.
Example 12: Measurement of protective efficacy
Animals were monitored regularly for change in body weight and general body condition as an indicator of disease progression and were euthanized at specified time points. In addition to the measurement of bacillary load in lung and spleen, gross and histopathological changes in various organs and extent of pulmonary fibrosis were evaluated. A significant reduction in these parameters in vaccinated animals was considered as a protective effect of the vaccine.
Example 13: Necropsy procedure and gross pathological evaluation
Guinea pigs were euthanized by i.p. injection of Thiopentone sodium (100 mg/kg body weight) (Neon Laboratories Ltd., India). After aseptically dissecting the animals, lung, liver and spleen were examined for gross pathological changes and scored using the Mitchison scoring system (Mitchison, D. A., Wallace, J. G., Bhatia, A. L., Selkon, J. B., Subbaiah, T. V. and Lancaster, M. C. (1960). A comparison of the virulence in guinea pigs of South Indian and British tubercle bacilli. Tubercle 41, 1-22) with minor modifications (Table III), wherein equal emphasis was given to each organ. For histopathological evaluation, three lung lobes (right caudal, middle and cranial) and a

portion of left dorsal lobe of liver were removed and fixed in 10% neutral buffered formalin. Left caudal lung lobe and cranial portion of spleen were aseptically removed for the measurement of bacillary load.
Table III: Post-mortem gross pathological scoring system
(Table Removed)

Example 14: Bacterial enumeration
Specific portions of lungs and spleen were weighed and homogenized separately in 5 ml saline in a Teflon glass homogenizer. Appropriate dilutions of the homogenates were inoculated on to MB7H11 agar plates in duplicates and incubated at 37°C in a CO2 incubator for three to four weeks. The number of colonies were counted and

expressed as logio CFU/g of tissue. The detection limit in case of both lung and spleen CFUwas l.0 log10CFU/g.
Example 15: Histopathological evaluation
Sections of 5 µm thickness from formalin fixed and paraffin embedded tissues were cut on to glass slides and stained with haematoxylin and eosin for histo-pathological examination. The percent granuloma in lung and liver, type and extent of necrosis, organization of granuloma along with the type of infiltrating cells were assessed as described earlier ( Shakila, H., Jayasankar, K. and Ramanathan, V. D. (1999). The clearance of tubercle bacilli & mycobacterial antigen vis a vis the granuloma in different organs of guinea pigs. Indian J Med Res 110, 4-10). In order to determine the extent of collagen deposition and fibrosis, the lung sections were also stained with Van Gieson stain.
Example 16: Statistical analysis
Mean differences for Logio CFU was analyzed by one-way analysis of variance (ANOVA). Least square difference and Duncan's post hoc-tests were also carried out to determine the significance of differences between various groups. The differences between scores allotted for gross pathological lesions and granuloma percent across different groups were analysed by non-parametric methods. The non-parametric Kruskal-Wallis test was employed for comparison of multiple groups, followed by the Mann-Whitney U test for comparison between two groups. The differences were considered statistically significant when the p values were less than 0.05. These statistical tests were run on SPSS software (Version. 10.0, SPSS Inc., Illinois, USA).
Example 17: Immunization and challenge of guinea pigs with M. tuberculosis by subcutaneous route
For protective efficacy studies, pathogen free 200-300 g female guinea pigs (outbred Dunkin Hartley strain) were employed. Groups of 6 guinea pigs were immunized with

one of the following: (i) 5X106 CFU of either BCG (Danish strain) or rBCG strains in 100 µ1 of saline by i.d. route and (ii) 100 µ1 of saline by i.d. route as a negative control. For protective efficacy studies, guinea pigs were challenged at 8 weeks after the immunization with 7.5X105 bacilli of virulent Mtb H37Rv via the sub-cutaneous route in and were euthanized at 8 weeks post-infection.
SEQ ID NO: 1- Ag85C nucleotide sequence (1023 nt)
(Sequence Removed)
SEQ ID NO: 2 - Ag85C amino acid sequence (340 aa)
(Sequence Removed)

l/We Claim:
1. A recombinant BCG over-expressing antigen 85C from Mycobacterium for use in generating an immunogenic response in a subject against Mycobacterium, wherein said recombinant BCG having recombinant vector comprising nucleotide sequence coding for antigen 85C.
2. The recombinant BCG over-expressing antigen 85C as claimed in claim 1, wherein said antigen 85C is from Mycobacterium tuberculosis.
3. The recombinant BCG over-expressing antigen 85C as claimed in claim 1, wherein said antigen 85C is from Mycobacterium bovis.
4. The recombinant BCG as claimed in claim 1, wherein said nucleotide sequence coding for antigen 85C is as set forth in SEQ ID NO: 1.
5. The recombinant BCG as claimed in claim 1, wherein amino acid sequence of said antigen 85C is as set forth in SEQ ID NO: 2.
6. The recombinant BCG as claimed in claim 1, wherein said BCG is selected from a group consisting of Danish strain, Glaxo strain, Pasteur strain, Tice strain and Connaught strain; preferably Danish strain.
7. A vaccine formulation for generating an immunogenic response in a subject against Mycobacterium, said composition comprising an immunologically effective amount of the recombinant BCG as claimed in claim 1.
8. The vaccine formulation as claimed in claim 7 comprises an adjuvant and a pharmaceutically effective carrier.
9. A process for producing a recombinant BCG over-expressing antigen 85C from Mycobacterium as claimed in claim 1, said process comprising, introducing a nucleotide sequence coding for antigen 85C from Mycobacterium into a suitable expression vector, to obtain a recombinant vector and transforming BCG with the recombinant vector to obtain a recombinant BCG over-expressing antigen 85C.

10. The process as claimed in claim 9, wherein said Mycobacterium is Mycobacterium tuberculosis or Mycobacterium bovis.
11. The process as claimed in claim 9, wherein said nucleotide sequence coding for antigen 85C is as set forth in SEQ ID NO: 1.
12. The process as claimed in claim 9, wherein amino acid sequence of said antigen 85C is as set forth in SEQ ID NO: 2.