Mycobacterial antigen composition

Abstract

There is provided an antigenic composition comprising (a) a first mycobacterial antigenic polypeptide or a first mycobacterial polynucleotide; and (b) a second mycobacterial antigenic polypeptide or a second mycobacterial polynucleotide; wherein: (i) said first mycobacterial antigenic polypeptide comprises a polypeptide sequence having at least 70% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or 7, or a fragment thereof having at least 7 consecutive amino acids thereof; (ii) said first mycobacterial polynucleotide comprises a polynucleotide sequence encoding said first mycobacterial antigenic polypeptide; (iii) said second mycobacterial antigenic polypeptide comprises a polypeptide sequence having at least 70% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 5, or a fragment thereof having at least 7 consecutive amino acids thereof; and (iv) said second mycobacterial polynucleotide comprises a polynucleotide sequence encoding said second mycobacterial polypeptide.

Claims

1. A composition comprising: a) a first antibody, wherein said first antibody specifically binds a first mycobacterial antigenic polypeptide within the amino acid sequence of SEQ ID NO: 1; and (b) a second antibody, wherein said second antibody specifically binds a second mycobacterial antigenic polypeptide within the amino acid sequence of SEQ ID NO: 5; the composition further comprising an adjuvant, an antimicrobial compound, an immunoregulatory agent, or a combination thereof.

2. The composition according to claim 1, wherein the antibodies are tagged with a detectable label or a functional label.

3. The composition according to claim 1, further comprising at least one additional antibody, which binds a mycobacterial antigenic polypeptide that is different from said first and second mycobacterial antigenic polypeptides.

4. The composition according to claim 3, wherein the at least one additional antibody specifically binds within a polypeptide having an amino acid sequence selected from any of SEQ ID NOs: 3, 7, 9-20, 34-44 or 56.

5. The composition according to claim 4, wherein the at least one additional antibody is tagged with a detectable label or a functional label.

6. The composition according to claim 1, wherein the composition comprises the adjuvant, and wherein the adjuvant is selected from the group consisting of: complete Freunds adjuvant (CFA), Incomplete Freunds adjuvant (IVA), Saponin, a purified extract fraction of Saporin such as Quil A, a derivative of Saporin such as QS-21, lipid particles based on Saponin such as ISCOM/ISCOMATIX, E. coli heat labile toxin (LT) mutants such as LTK63 and/or LTK72, aluminium hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1-2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryl oxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, and combinations thereof.

7. A method for producing a therapeutic or prophylactic formulation, the method comprising mixing a pharmaceutically acceptable carrier and at least one component that is an adjuvant, an antimicrobial compound, or an immunoregulatory agent, with: (a) a first antibody, wherein said first antibody specifically binds a first mycobacterial antigenic polypeptide within the amino acid sequence of SEQ ID NO: 1; and (b) a second antibody, wherein said second antibody specifically binds a second mycobacterial antigenic polypeptide within the amino acid sequence of SEQ ID NO: 5.

8. The method of claim 7, wherein the mixing comprises mixing at least two of the components with the pharmaceutically acceptable carrier and the first and the second antibody.

9. A method for producing a therapeutic or prophylactic formulation, the method comprising: (a) mixing a pharmaceutically acceptable carrier and at least one component that is an adjuvant, an antimicrobial compound, or an immunoregulatory agent, with a first antibody to form a first mixture, wherein the first antibody specifically binds a first mycobacterial antigenic polypeptide within the amino acid sequence of SEQ ID NO: 1; and (b) mixing a pharmaceutically acceptable carrier and at least one component that is an adjuvant, an antimicrobial compound, or an immunoregulatory agent, with a second antibody to form a second mixture, wherein the second antibody specifically binds a second mycobacterial antigenic polypeptide within the amino acid sequence of SEQ ID NO: 5; and (c) combining said first mixture and said second mixture to form the therapeutic or prophylactic formulation.

Description

EXAMPLES

Example 1Sub-Unit Vaccines Containing Polypeptides of the Invention

(1) To prepare sub-unit vaccines comprising polypeptides it is first of all necessary to obtain a supply of polypeptide to prepare the vaccine. This can be achieved by purifying proteins of interest from TB culture, or by cloning the gene of interest and producing a recombinant protein.

(2) The coding sequences for the genes of interest are amplified by PCR with restriction sites inserted at the N terminus and C terminus to permit cloning in-frame into a protein expression vector such as pET-15b. The genes are inserted behind an inducible promoter such as lacZ. The vector is then transformed into E. coli which is grown in culture. The recombinant protein is over-expressed and is purified.

(3) One of the common purification methods is to produce a recombinant protein with an N-terminal tag for purificationeg. a His-tag. The protein can then be purified on the basis of the affinity of the His-tag for metal ions on a Ni-NTA column after which the His-tag is cleaved. The purified protein is then administered to animals in a suitable adjuvant.

(4) Where at least 2 mycobacterial antigens are used in combination, the 1st and 2nd mycobacterial antigens may be expressed as separate polypeptides and used in combination by mixing with adjuvant and inoculating at a single site. Alternatively, the 1st and 2nd mycobacterial antigens may be expressed as a fusion protein, mixed with adjuvant and used to inoculate at a single site.

Example 2Use of BCG as a Microbial Carrier

(5) The polynucleotide sequence of interest is amplified by PCR. The amplified product is purified and cloned into a plasmid (pMV306) that integrates site specifically into the mycobacterial genome at the attachment site (attB) for mycobacteriophage L5.

(6) BCG is transformed with the plasmid by electroporation, which involves damaging the cell envelope with high voltage electrical pulses, resulting in uptake of the DNA. The plasmid integrates into the BCG chromosome at the attB site, generating stable recombinants. Recombinants are selected and are checked by PCR or Southern blotting to ensure that the gene has been integrated. The recombinant strain is then used for protection studies.

(7) The polynucleotide sequence of interest may comprise a single mycobacterial antigen, 1st and 2nd mycobacterial antigens, or fragments thereof as defined herein.

Example 3Viral Vectors (Eg. Attenuated Vaccinia Virus) Expressing Mycobacterial Genes

(8) Left Flanking RegionPromotorTarget gene(s)Right Flanking Region

(9) One of the best examples of this type of approach is the use of Modified Vaccinia virus Ankara (MVA). Methodologies permitting recombination of foreign or target genes into the genome of MVA are well known in the art [1,2].

(10) Insertion of the target gene(s) is mediated by transfer DNA with features similar to those shown above. The transfer DNA may be in the form of a plasmid that can be propagated in a bacterial strain optimized for routine cloning procedures. The target gene(s) is introduced to the cassette downstream of a promoter such as mH5, p7.5 or another. The target gene(s) may comprise one or more of the polynucleotides of the invention and/or fragments thereof. The target gene(s) may also comprise adjuvanting cofactors such as B7-1 or IL-12, as is well described in the art [3]. The target gene(s) are positioned downstream and in frame with an optimized Kozak sequenceeg. GCCACCATGG (SEQ ID NO:58). The target gene(s) may also be positioned downstream and in frame with a leader sequenceeg. tPA. The target gene(s) may be positioned upstream of an in-frame tageg. V5, HIS or another. Transfer of the cassette into the genome of MVA is mediated by homologous flanking regions well known in the arteg. Del I-VI. 1. Earl P L et al. Current Protocols in Protein Science, (2001) Generation of recombinant vaccinia viruses. 2. Earl P L et al. Current Protocols in Protein Science, (2001) Preparation of cell cultures and vaccine virus stocks. 3. Carroll M W et al. Journal of the National Cancer Institute, (1998) Construction and characterization of a triple-recombinant vaccine virus encoding B7-1, Interleukin 12 and a model tumor antigen.

Example 4Plasmid DNA Vaccines Carrying Mycobacterial Polynucleotides

(11) A polynucleotide sequence of interest is amplified by PCR, purified and inserted into specialized vectors developed for vaccine development, such as pVAX1. These vectors contain promoter sequences (eg. CMV or SV40 promoters), which direct strong expression of the introduced polynucleotide (encoding the candidate antigen) in eukaryotic cells; and polyadenylation signals (eg. SV40 or bovine growth hormone) to stabilize the mRNA transcript.

(12) The vector is transformed into E. coli and transformants are selected using a marker, such as kanamycin resistance, encoded by the plasmid. The plasmid is then recovered from transformed colonies and is sequenced to check that the polynucleotide of interest is present and encoded properly without PCR generated mutations.

(13) Large quantities of the plasmid are then produced in E. coli and the plasmid is recovered and purified using commercially available kits (e.g. Qiagen Endofree-plasmid preparation). The vaccine is then administered to animals (eg. by intramuscular injection) in the presence or absence of an adjuvant.

(14) Plasmid DNA encoding the 1st mycobacterial antigens or the 2nd mycobacterial antigens separately may be mixed and inoculated at a single site of administration. A single plasmid may be constructed that expresses both the 1st and the 2nd mycobacterial antigens (and optionally the third mycobacterial antigen).

Example 5Plasmid DNA Vaccines Carrying Multiple Mycobacterial Polynucleotides

(15) Further plasmid DNA encoding a 3rd and/or further (eg. 4.sup.th and 5.sup.th) mycobacterial antigens separately may be prepared as described in Example 4. The separate plasmids encoding the 3.sup.rd and/or further mycobacterial antigens may be inoculated at a single site of administration simultaneously or sequentially with plasmid DNA encoding the 1.sup.st and 2.sup.nd mycobacterial antigens (eg. as prepared in Example 4).

(16) Alternatively, a single plasmid may be constructed as described in Example 4 that expresses the 3.sup.rd and one or more further (eg. 4.sup.th and 5.sup.th) mycobacterial antigens. This single plasmid may be inoculated at a single site of administration simultaneously or sequentially with plasmid DNA encoding the 1.sup.st and 2.sup.nd mycobacterial antigens separately or from a single plasmid. Alternatively, a single plasmid may be constructed as described in Example 4 that expresses the 1.sup.st, 2.sup.nd and 3.sup.rd (and optionally one or more furthereg. 4.sup.th and 5.sup.th) mycobacterial antigens.

Example 6Preparation of DNA Expression Vectors

(17) DNA vaccines consist of a nucleic acid sequence of interest cloned into a bacterial plasmid. The plasmid vector pVAX1 is commonly used in the preparation of DNA vaccines. The vector is designed to facilitate high copy number replication in E. coli and high level transient expression of the peptide of interest in most mammalian cells (for details see manufacturers protocol for pVAX1 (catalog No. V260-20 www.invitrogen.com).

(18) The vector contains the following elements: Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a variety of mammalian cells T7 promoter/priming site to allow in vitro transcription in the sense orientation and sequencing through the insert Bovine growth hormone (BGH) polyadenylation signal for efficient transcription termination and polyadenylation of mRNA Kanamycin resistance gene for selection in E. coli A multiple cloning site pUC origin for high-copy number replication and growth in E. coli BGH reverse priming site to permit sequencing through the insert

(19) Vectors may be prepared by means of standard recombinant techniques that are known in the art, for example Sambrook et al. (1989). Key stages in preparing the vaccine are as follows: The polynucleotide of interest is ligated into pVAX1 via one of the multiple cloning sites The ligation mixture is then transformed into a competent E. coli strain (e.g. TOP10) and LB plates containing 50 pg/ml kanamycin are used to select transformants. Clones are selected and may be sequenced to confirm the presence and orientation of the gene of interest. Once the presence of the gene has been verified, the vector can be used to transfect a mammalian cell line to check for protein expression. Methods for transfection are known in the art and include, for example, electroporation, calcium phosphate, and lipofection. Once polypeptide expression has been confirmed, large quantities of the vector can be produced and purified from the appropriate cell host, eg. E. coli.

(20) pVAX1 does not integrate into the host chromosome. All non-essential sequences have been removed to minimise the possibility of integration. When constructing a specific vector, a leader sequence may be included to direct secretion of the encoded protein when expressed inside the eukaryotic cell.

(21) Other examples of vectors that can be used include V1Jns.tPA and pCMV4.

(22) Expression vectors may be used that integrate into the genome of the host, however, it is more common and more preferable to use a vector that does not integrate. Integration would lead to the generation of a genetically modified host which raises other issues.

Example 7Preparation of DNA Expression Vectors Containing Multiple Antigens

(23) DNA vaccines consist of a nucleic acid sequence of interest cloned into a bacterial plasmid. The plasmid vector pVAX1 is commonly used in the preparation of DNA vaccines. The vector is designed to facilitate high copy number replication in E. coli and high level transient expression of the peptide of interest in most mammalian cells (for details see manufacturers protocol for pVAX1 (catalog No. V260-20 www.invitrogen.com).

(24) The vector contains the following elements: Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a variety of mammalian cells T7 promoter/priming site to allow in vitro transcription in the sense orientation and sequencing through the insert Bovine growth hormone (BGH) polyadenylation signal for efficient transcription termination and polyadenylation of mRNA Kanamycin resistance gene for selection in E. coli A multiple cloning site pUC origin for high-copy number replication and growth in E. coli BGH reverse priming site to permit sequencing through the insert

(25) Vectors may be prepared by means of standard recombinant techniques that are known in the art, for example Sambrook et al. (1989), Gateway cloning (Invitrogen, UK). Key stages in preparing the vaccine are as follows: The polynucleotides of interest are ligated into pVAX1 via one of the multiple cloning sites or introduced via Gateway cloning. Polynucleotides for more than one antigen can be expressed as a recombinant fusion. A competent E. coli strain (e.g. TOP10) is transformed and LB plates containing 50 g/ml kanamycin are used to select transformants. Clones are selected and may be sequenced to confirm the presence and orientation of the genes of interest. Once the presence of the genes has been verified, the vector can be used to transfect a mammalian cell line to check for protein expression. Methods for transfection are known in the art and include, for example, electroporation, calcium phosphate, and lipofection. Once polypeptide expression has been confirmed, large quantities of the vector can be produced and purified from the appropriate cell host, eg. E. coli.

(26) pVAX1 does not integrate into the host chromosome. All non-essential sequences have been removed to minimise the possibility of integration. When constructing a specific vector, a leader sequence may be included to direct secretion of the encoded protein when expressed inside the eukaryotic cell.

(27) Other examples of vectors that can be used include V1Jns.tPA and pCMV4.

(28) Expression vectors may be used that integrate into the genome of the host; however, it is more common and more preferable to use a vector that does not integrate. Integration would lead to the generation of a genetically modified host which raises other issues.

(29) A single plasmid may be thus constructed that expresses multiple mycobacterial antigens. For example, the single plasmid may encode both the 1.sup.st and 2.sup.nd mycobacterial antigens. The single plasmid may additionally encode one or more further mycobacterial antigens, such as a 3.sup.rd mycobacterial antigen (and optionally one or more furthereg. 4.sup.th and 5.sup.th) mycobacterial antigens.

Example 8RNA Vaccine

(30) RNA can be introduced directly into the host. Thus, a vector construct may be used to generate RNA in vitro and the purified RNA is then injected into the host. The RNA then serves as a template for translation in the host cell. In this embodiment, integration would not normally occur.

(31) An alternative option is to use an infectious agent such as the retroviral genome carrying RNA corresponding to the gene of interest. In this embodiment, integration into the host genome will occur.

(32) Another option is the use of RNA replicon vaccines which can be derived from virus vectors such as Sindbis virus or Semliki Forest virus. These vaccines are self-replicating and self-limiting and may be administered as either RNA or DNA which is then transcribed into RNA replicons in vivo. The vector eventually causes lysis of the transfected cells thereby reducing concerns about integration into the host genome.

Example 9Diagnostic Assays Based on Assessing Immune Cell Responses

(33) For a diagnostic assay based on assessing immune cell responses (eg. T cell responses) it would be sufficient to obtain a sample of blood from the patient. Mononuclear cells (monocytes, T and B lymphocytes) can be separated from the blood using density gradients such as Ficoll gradients.

(34) Both monocytes and B-lymphocytes are both able to present antigen, although less efficiently than professional antigen presenting cells (APCs) such as dendritic cells. The latter are more localized in lymphoid tissue.

(35) The simplest approach would be to add antigen to the separated mononuclear cells and incubate for a week and then assess the amount of proliferation. If the individual had been exposed to the antigen previously through infection, then immune cell clones (eg. T-cell clones) specific to the antigen should be more prevalent in the sample and should respond.

(36) It is also possible to separate the different cellular populations should it be desired to control the ratio of T cells to APCs.

(37) Another variation of this type of assay is to measure cytokine production by the responding lymphocytes as a measure of response. The ELISPOT assay is a suitable example of this assay.

Example 10Detection of Latent Mycobacteria

(38) The presence of latent mycobacteria-associated antigen may be detected either by detecting antigen-specific antibody, or by detecting immune cells such as T-cells in blood samples.

(39) A 96 well plate is coated with cytokine (e.g. interferon-, IL-2)-specific antibody. Peripheral blood monocytes are then isolated from patient whole blood and are applied to the wells.

(40) Antigen is added to stimulate specific immune cells (eg. T cells) that may be present and the plates are incubated for 24 h. The antigen stimulates the immune cells (eg. T-cells) to produce cytokines, which bind a specific antibody.

(41) The plates are washed leaving a footprint where antigen-specific immune cells (eg. T cells) were present. A second antibody coupled with a suitable detection system, e.g. enzyme, is then added and the number of spots is enumerated after the appropriate substrate has been added. The number of spots, each corresponding to a single antigen-specific immune cell (eg. T cell), is related to the total number of cells originally added.

(42) The above-described assay may also be used to distinguish TB-infected individuals from BCG-vaccinated individuals.

Example 11Antigenic Activity of Multiple Antigens

(43) Mice are immunized with at least a 1st and 2nd mycobacterial antigen. Delivery systems include (but are not restricted to) DNA vaccines, recombinant MVA, adjuvanted protein. Delivery routes include (but are not restricted to) sub-cutaneous, intra-dermal, intra-muscular administration. The immunization regimen may involve heterologous prime-boostingeg. priming with a DNA vaccine followed by boosting with an MVA vaccine. The immunization regimen may involve multiple doses.

(44) After vaccination (eg. about 2 weeks later), splenocytes are removed from the vaccinated animals and stimulated with a polypeptide(s) representative of the immunizing antigen or antigens. An immune response is measurable through antigen-specific induction of cytokine releaseeg. IFN-, and is evidence of immunization against the target antigen.

(45) Where an animal has been immunized with a vaccine comprising a 1st and 2nd mycobacterial antigen, an antigen recall response to the 1st and 2nd mycobacterial antigen in the same sample demonstrates immunogenicity of both antigens when co-administered. Immunogenicity is a pre-requisite for protective efficacy.

(46) Data generated according to this Example are illustrated in FIGS. 1-7.

(47) FIGS. 1A and B illustrate the splenocyte response to antigens Ag85A and Rv1807 alone and in combination (FIG. 1A=intra-muscular; FIG. 1B=intra-dermal).

(48) FIG. 2 illustrates the splenocyte response to antigens Ag85A and Rv0111 alone and in combination.

(49) FIG. 3 illustrates the splenocyte response to antigens Ag85A and Rv0198 alone and in combination.

(50) FIG. 4 illustrates the splenocyte response to antigens Ag85A and Rv1807 alone and in combination (repeat of the IM data in FIG. 1B).

(51) FIG. 5 illustrates the splenocyte response to antigens Ag85A and Rv1806 alone and in combination.

(52) FIG. 6 illustrates the splenocyte response to antigensRv1806, Rv1807, Rv0198 and Rv0111, alone and in combination.

(53) FIG. 7 illustrates the reduction in bacterial load in response to each of antigens Rv0111, Rv0198c, Rv3812, Rv1086 and Rv180 alone, and in response to a combination of antigens Rv0111 and Rv0198c.

Example 12Demonstrating Vaccine Efficacy in an Experimental Model

(54) The efficacy of vaccine candidates in guinea pigs may be assessed on the basis of reducing the bacterial burden of M. tuberculosis in the lungs and/or spleens at 4 weeks post-aerosol challenge.

(55) The 1st and 2nd mycobacterial antigens are delivered as sub-unit DNA vaccines or protein in a Th1-inducing adjuvant such as DDA/MPL, or by expression vectors such as recombinant viruses or BCG (see Examples 1-4). The 1st and 2nd mycobacterial antigens are delivered in a manner designed to prime the immune system, which includes all of the above. At least one boost to the initial prime is given through inoculation of either DNA, polypeptide or viral vector or (less commonly) recombinant BCG. Groups of six to eight guinea pigs are immunized two or three times with a 2 to 3 week rest between each immunization. Following the final inoculation, the guinea pigs are rested for 6 weeks prior to challenge.

(56) A group of positive control animals are inoculated subcutaneously with 510.sup.4 colony forming units (CFU) of BCG Danish (1331), and a group of negative control animals are given saline.

(57) Six weeks following the final vaccination, fine particle aerosols of M. tuberculosis (2 m mean diameter; generated in a Collison nebuliser), are delivered directly to the animal snout using a contained Henderson apparatus. A suspension of the challenge strain, M. tuberculosis H37Rv (NCTC 7416), cultured under defined conditions in a chemostat is diluted to 110.sup.6 CFU/ml in order to achieve an estimated retained, inhaled dose of approximately 10 CFU/lung.

(58) Four weeks after aerosol challenge, the animals are humanely killed, and the lungs removed for CFU determination.

(59) Homogenized samples are serially diluted and plated on Middlebrook 7H11 selective agar and the mean CFU for each treatment group is determined. Vaccine efficacy is assessed in terms of reduction in bacterial counts in lungs or spleens compared to the saline control group. The mean logo CFU of test vaccines is compared with the negative controls and differences between groups are analyzed statistically using an appropriate test such Mann-Whitney.

(60) Any combination of 1st and 2nd mycobacterial antigens giving a reduction in the number of viable M. tuberculosis that is statistically significantly (p=<0.05) lower than sham-vaccinated (saline) controls, demonstrates the protective efficacy of the antigens when co-administered.

(61) Protective efficacy in guinea pigs is indicative of the ability of the combination vaccine to protect humans and animals from pathogenic mycobacterial infection.

Example 13Demonstrating Vaccine Efficacy in an Experimental Model

(62) The efficacy of vaccine candidates in guinea pigs may be assessed on the basis of reducing the bacterial burden of M. tuberculosis in the spleens at 4 weeks post-aerosol challenge.

(63) The 1.sup.st and 2.sup.nd mycobacterial antigens are delivered as sub-unit DNA vaccines or protein in a Th1-inducing adjuvant such as DDA/MPL, or by expression vectors such as recombinant viruses or BCG (see Examples 1-4). The 1.sup.st and 2.sup.nd mycobacterial antigens are delivered in a manner designed to prime the immune system, which includes all of the above. At least one boost to the initial prime is given through inoculation of either DNA, polypeptide or viral vector or (less commonly) recombinant BCG. Groups of six to eight guinea pigs are immunized two or three times with a 2 to 3 week rest between each immunization. Following the final inoculation, the guinea pigs are rested for 6 weeks prior to challenge.

(64) A group of positive control animals are inoculated subcutaneously with 510.sup.4 colony forming units (CFU) of BCG Danish (1331), and a group of negative control animals are given saline or remain unvaccinated.

(65) Six weeks following the final vaccination, fine particle aerosols of M. tuberculosis (2 m mean diameter; generated in a Collison nebuliser), are delivered directly to the animal snout using a contained Henderson apparatus. A suspension of the challenge strain, M. tuberculosis H37Rv (NCTC 7416), cultured under defined conditions in a chemostat is diluted to 110.sup.6 CFU/ml in order to achieve an estimated retained, inhaled dose of approximately 10 CFU/lung.

(66) Four weeks after aerosol challenge, the animals are humanely killed, and the spleens removed for CFU determination.

(67) Homogenized samples are serially diluted and plated on Middlebrook 7H11 selective agar and the mean CFU for each treatment group is determined. Vaccine efficacy is assessed in terms of reduction in bacterial counts in spleens compared to the saline or unvaccinated control group. The mean log.sup.10 CFU of test vaccines is compared with the negative controls and differences between groups are analyzed statistically using an appropriate test such Mann-Whitney.

(68) Any combination of 1st and 2.sup.nd mycobacterial antigens giving a reduction in the number of viable M. tuberculosis that is statistically significantly (p=<0.05) lower than unvaccinated controls, demonstrates the protective efficacy of the antigens when co-administered.

(69) Protective efficacy in guinea pigs is indicative of the ability of the combination vaccine to protect humans and animals from pathogenic mycobacterial infection.

(70) Data generated according to this Example are illustrated in FIG. 7. FIG. 7 illustrates the reduction in bacterial load in response to each of antigens Rv0111, Rv0198c, Rv3812, Rv1806 and Rv180 alone, and in response to a combination of antigens Rv0111 and Rv0198c.

Example 14Antigenic Activity of Multiple Antigens

(71) Mice are immunized with at least a 1st and 2.sup.nd mycobacterial antigen. Delivery systems include (but are not restricted to) DNA vaccines, recombinant MVA or adjuvanted protein. Delivery routes include (but are not restricted to) subcutaneous, intra-dermal or intra-muscular administration. The immunization regimen may involve heterologous prime-boostingeg. priming with a DNA vaccine followed by boosting with an MVA vaccine, and/or may involve multiple doses.

(72) After vaccination (eg. about 2 weeks later), serum are removed from the vaccinated animals and screened for the presence of antibodies. An immune response is measurable through the detection of antibodies specific for the immunising antigeneg. as detected via ELISA.

(73) Where an animal has been immunized with a vaccine comprising a 1st and 2.sup.nd mycobacterial antigen, the presence in the same sample of antibodies to the 1.sup.st and 2.sup.nd mycobacterial antigen demonstrates immunogenicity of both antigens when co-administered. Immunogenicity is a pre-requisite for protective efficacy.