WO2010006607A1

WO2010006607A1 - Vaccines comprising tb10.4

Info

Publication number: WO2010006607A1
Application number: PCT/DK2009/000171
Authority: WO
Inventors: Jes Dietrich; Claus Aagaard; Peter Andersen
Original assignee: Satens Serum Institut
Priority date: 2008-07-15
Filing date: 2009-07-14
Publication date: 2010-01-21
Also published as: DK2712625T3; CA2734714A1; US20120014980A1; CA2734714C; USRE46583E1; CN102149404A; US20100015171A1; US8557258B2; EP2320944A1; CN107050441A; EP2712625B1; EP2712625A1; EP2712625A3

Abstract

Vaccination with the combination of Ag85B-TB10.4 and IC31 generated a high amount of polyfunctional CD4⁺T cells expressing high levels of IFN-γ, TNF-α, and IL-2. This in turn led to significant protection against infection with M. tuberculosis in the mouse aerosol challenge model of tuberculosis. Importantly, our results also showed that both the irnmuno- genicity of the vaccine and its ability to protect against TB infection was highly dependent on the antigen dose. Thus, whereas the standard antigen dose of 5.0μg, as well as 15.0μg, did not induce significant protection against M. tuberculosis, reducing the dose to 0.5 μg increased both the immunogenicity of the vaccine as well as its protective efficacy to a level comparable to that observed in BCG vaccinated mice. Thus, the adjuvant IC31®, with the optimal antigen dose, can induce a strong protective Th1 response against M. tuberculosis.

Description

Vaccines comprising TBl 0.4

Field of the invention

The invention discloses a vaccine for human use with a low dose of an antigen comprising TB 10.4 fused to a polypeptide of the antigen 85-complex e.g. Ag85B in an adjuvant and methods of immunization and treatment of MΛuberculosis.

Background.

The global effort to develop a more effective Mycobacterium tuberculosis (M. tuberculosis) vaccine than the currently used Bacillus of Calmette and Guerin (BCG) vaccine involves different strategies such as live attenuated vaccines (9), virally vectored M. tuberculosis vaccines (11), and subunit vaccines (12, 14). The subunit approach holds a number of advantages, such as increased safety and stability as well as the demonstrated ability to boost prior BCG vaccination (4, 7). In addition, as subunit vaccines appear not to be influenced by environmental mycobacteria this type of vaccine may be of particular use in the developing world (3). However, progress in this field has been delayed by the lack of adjuvants that induce a strong cell-mediated immune (CMI) response. Therefore, there still remains a need for an immunogenic composition which can generate polyfunctional immune cells thereby providing greater protection against M. tuberculosis.

Summary

The present work examined the combination of Ag85B-TB10.4 (Hyvac 4) and the IC31® adjuvant as a new vaccine against infection with M. tuberculosis. The results show that Ag85B-TB 10.4 and the IC31® adjuvant induces high amounts of polyfunctional CD4⁺ T cells and provides significant protection against M. tuberculosis. Surprisingly, the combination of the Ag85B-TB10.4 antigen and the IC31® adjuvant was sensitive to the antigen dose. Thus, whereas a standard dose in mice of 5.0μg of Ag85B-TB10.4 in IC31® did not lead to protection against M. tuberculosis, 0.5 μg Ag85B-TB10.4 in IC31® induced protec- tion comparable to that of BCG. Detailed description

The present invention discloses an immunogenic composition for human use comprising a TB 10.4 protein and an Ag85-complex protein which optionally can be fused together or provided as separate proteins wherein the total amount of protein is less than about 25.0μg preferably less than lO.Oμg or equal to about 0.5μg per antigen dose.

The disclosed immunogenic composition can additionally comprise an adjuvant. The preferred adjuvant of the invention having at least one polycationic peptide and at least one oligonucleotide preferably the oligonucleotide is a TLR9 agonist.

The preferred adjuvant of the invention is IC31® and the preferred protein from the Ag85- complex is an Ag85B protein.

The invention discloses a vaccine for human use comprising the above mentioned immunogenic composition.

The invention further discloses a method of inducing protection against M. tuberculosis in a human, the method comprising introducing into the human an immunogenic composition as described above.

The present work examined the combination of an Ag85B-TB10.4 (Hyvac 4) fusion protein and IC31® as a new vaccine against infection with M. tuberculosis. The IC31® adjuvant comprises cationic peptides and a TLR9 receptor agonist.

In an effort to generate an efficient vaccine against infection with M. tuberculosis, the combination of the Ag85B-TB10.4 fusion protein and IC31® is attractive for the following rea- sons:

(1) IC31® can promote a strong ThI response; (2) IC31® has a promising profile in clinical trials; it has successfully been tested in clinical phase 1 trial in combination with a trivalent influenza vaccine as well as in combination with a tuberculosis vaccine (50 ug Ag85B-ESAT-6 in IC31®; clinical data not published yet). Animal studies with Ag85B-ESAT6 in IC31® is described in (1); (3) Ag85B-TB 10.4 fusion protein has the advantage that it does not include any of the proteins that are useful for diagnostic purposes such as e.c. ESAT-6. ESAT-6 is an extremely valuable diagnostic reagent and the basis of a number of commercial diagnostic tests (5, 10, 13). Leaving ESAT-6 out of a future vaccine will allow the diagnostic tests and the vaccine to be used in parallel since the Ag85B-TB10.4 fusion protein -does not compromise any of the specific diagnostic tests;

(4) Since both Ag85B and TB10.4 are expressed by BCG, the vaccine of the current invention may also be employed as a BCG booster vaccine.

The results show that the Ag85B-TB10.4 and IC31® combination induces a high amount of polyfunctional CD4⁺T cells and provides significant protection against M. tuberculosis. Surprisingly, the combination of the Ag85B-TB 10.4 fusion protein and the IC31 ® adjuvant is extremely sensitive to the antigen dose. Thus, whereas a dose of 5.0μg of Ag85B-TB10.4 in IC31® does not lead to significant protection against M. tuberculosis, 0.5μg Ag85B-TB10.4 in IC31® induces a strong protection comparable to that of BCG (Ex.3 & figure 4). In con- trast, previous work with other antigens, such as Ag85B-ESAT6 in MPL-DDA showed that the optimal antigen dose for this fusion protein was approximately lO.Oμg (16) and that a dose of 5μg of Ag85B-TB10.4 in MPL-DDA provided significant protection comparable to BCG (8) indicating that Ag85B-TB10.4 is an extraordinary immunogenic molecule.

This is the first study to show that the Ag85B-TB 10.4 fusion protein in the IC31 ® adjuvant constitutes an effective vaccine against infection with M. tuberculosis.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations thereof such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. Antigens

The antigen of the present invention may comprise a protein of the Ag85 -complex fused to TB 10.4 protein, including, for example, proteins Ag85A, Ag85B or Ag85C of the Ag85 complex.

Ag85B-TB10.4 is an extraordinary immunogenic molecule which must be given in low doses to initiate the maximum amount of polyfunctional immune cells inducing more inter- feron-γ expression and increased protection against M .tuberculosis. Throughout this specification Ag85B-TB10.4 fusion protein is interchangeable with the terms H4 and HyVac4.

In another embodiment the antigen may comprise a protein of the Ag85 complex and TB 10.4 protein wherein the Ag85 complex protein is not fused to the TB 10.4 protein. The preferred protein of the Ag85 complex is Ag85B.

Protein amino acid sequence

Ag85A MQLVDRVRGA VTGMSRRLW GAVGAALVSG LVGAVGGTAT

AGAFSRPGLP VEYLQVPSPS MGRDIKVQFQ SGGANSPALY

SEQID LLDGLRAQDD FSGWDINTPA FEWYDQSGLS WMPVGGQSS

NO.1 FYSDWYQPAC GKAGCQTYKW ETFLTSELPG WLQANRHVKP

TGSAWGLSM AASSALTLAI YHPQQFVYAG AMSGLLDPSQ

AMGPTLIGLA MGDAGGYKAS DMWGPKEDPA WQRNDPLLNV

GKLIANNTRV WVYCGNGKPS DLGGNNLPAK FLEGFVRTSN

IKFQDAYNAG GGHNGVFDFP DSGTHSWEYW GAQLNAMKPD

LQRALGATPN TGPAPQGA

Ag85B MTDVSRKIRA WGRRLMIGTA AAWLPGLVG LAGGAATAGA

FSRPGLPVEY LQVPSPSMGR DIKVQFQSGG NNSPAVYLLD

SEQID GLRAQDDYNG WDINTPAFEW YYQSGLSIVM PVGGQSSFYS

NO.2 DWYSPACGKA GCQTYKWETF LTSELPQWLS ANRAVKPTGS

AAIGLSMAGS SAMILAAYHP QQFIYAGSLS ALLDPSQGMG

PSLIGLAMGD AGGYKAADMW GPSSDPAWER NDPTQQIPKL

VANNTRLWVY CGNGTPNELG GANIPAEFLE NFVRSSNLKF QDAYNAAGGH NAVFNFPPNG THSWEYWGAQ LNAMKGDLQS

SLGAG

Ag85C MTFFEQVRRL RSAATTLPRR LAIAAMGAVL VYGLVGTFGG

PATAGAFSRP GLPVEYLQVP SASMGRDIKV QFQGGGPHAV

SEQID NO.3 YLLDGLRAQD DYNGWDINTP AFEEYYQSGL SVIMPVGGQS

SFYTDWYQPS QSNGQNYTYK WETFLTREMP AWLQANKGVS

PTGNAAVGLS MSGGSALILA AYYPQQFPYA ASLSGFLNPS

EGWWPTLIGL AMNDSGGYNA NSMWGPSSDP AWKRNDPMVQ

IPRLVANNTR IWVYCGNGTP SDLGGDNIPA KFLEGLTLRT

NQTFRDTYAA DGGRNGVFNF PPNGTHSWPY WNEQLVAMKA

DIQHVLNGAT PPAAPAAPAA

TB10.4 MSQIMYNYPA MLGHAGDMAG YAGTLQSLGA EIAVEQAALQ

SEQID SAWQGDTGIT YQAWQAQWNQ AMEDLVRAYH AMSSTHEANT

NO.4 MAMMARDTAE AAKWGG

Each protein may be modified by glycosylation, or lipidation (Mowat et al. 1991 ;Lustig et al. 1976). Each protein may be modified by the addition of prosthetic groups, a purification moiety, or a signal peptide. Each protein may be modified one or more times or not undergo any modification. Each protein may be modified singly or in combination. Each protein will be characterised by specific amino acids and be encoded by specific nucleic acid sequences. Within the scope of the present invention are such sequence and analogues and variants produced by recombinant or synthetic methods wherein such amino acid sequences have been modified by substitution, insertion, addition or deletion of one or more amino acid residues in the recombinant protein while retaining immunogenicity as confirmed by any one or all of the biological assays described herein.

Substitutions are preferably "conservative". These are defined according to the following table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other. The amino acids in the third column are indicated in one-letter code.

Adjuvants

In one embodiment of the present invention the antigen may comprise an Ag85 complex protein fused to TB 10.4 protein in an adjuvant.

In another embodiment the antigen may comprise an Ag85 complex protein and a TB 10.4 protein (i.e. non-fused) in an adjuvant.

Ag85B-TB 10.4 is an extraordinary immunogenic molecule which must be given in low doses to initiate the maximum amount of polyfunctional immune cells inducing more inter- feron-γ expression and increased protection against M .tuberculosis. It is exemplified with an adjuvant comprising a polycationic peptide [e.g. polylysin (KLK peptide) or polyarginin] and oligodeoxynucleic molecules but is not limited to this adjuvant.

The preferred adjuvant of the present invention is a mixture of a polycationic peptide [e.g. polylysin (KLK peptide) or polyarginin] and oligodeoxynucleic molecules [1-ODNs]. An example of a suitable I-ODN for use with the current invention is oligo dIC e.g. 5^λ- ICI CIC ICI CIC ICI CIC ICI CIC IC-3\ A thorough review I-ODN' s appropriate for use with the present invention is provided in Patent Cooperation Treaty International Application Publication No. WO0193905 and WO0193903 which are hereby incorporated by reference. An example of KLK peptides suitable for use with the current invention are NH2KLKLLLLLKLK-COOH or KLKLLLLLKLK-NH2. Additional examples of appropriate polycationic peptides are RLRLLLLLRLR-NH2, RLKLLLLLKLR-NH2, KFKFFFFFKFK-NH2, KWKWWWWWKWK-NH2 or KVKVVVVVKVK-NH2. A thorough review of the polycationic peptides suitable for use with the current invention is pro- vided in Patent Cooperation Treaty International Application Publication No. WO0232451 which is hereby incorporated by reference.

The IC31® adjuvant comprises a mixture of the peptide NH₂-KLKLLLLLKLK-COOH (ob- tained from Multiple Peptide Systems, San Diego, CA, USA) and the oligonucleotide 5^λ- ICI CIC ICI CIC ICI CIC ICI CIC IC-3' (purchased from Proligo, Boulder, USA).

Previous studies have shown that the Ag85B-TB10.4 fusion protein or Ag85B-ESAT-6 fu- sion protein in cationic liposomes induces efficient protection against M. tuberculosis and that Ag85B-ESAT-6 in IC31® is as protective as BCG in a mouse M. tuberculosis animal model (1, 8, 12). In each of these studies a standard antigen dose of 5.0μg was employed.

In the current study, a dose of 0.5 μg Ag85B-TB10.4 fusion protein in IC31 [100 nmol pep- tide (NH_2-KLKLLLLLKLK-COOH) and 5 nmol oligonucleotide (5^λ- ICI CIC ICI CIC ICI CIC ICI CIC IC-3')] induced a strong INF -γ response. This strong INF-γ response was in approximately the same range as 5.0μg Ag85B-TB10.4 fusion protein, without IC31. Moreover, 0.5 μg Ag85B-TB10.4 fusion protein in IC31 [100 nmol peptide (NH₂- KLKLLLLLKLK-COOH) and 5 nmol oligonucleotide (5'- ICI CIC ICI CIC ICI CIC ICI CIC IC-3")] provided a strong protection in approximately the same range as BCG. This is surprisingly better than 5.0μg or 15.0μg of Ag85B-TB10.4 fusion protein alone (i.e. no IC31), revealing that the optimal dose of Ag85B-TB10.4 fusion protein alone is less than about 5.0μgper dose.

Moreover, the lowest dose of Ag85B-TB10.4 fusion protein (0.5μg) in IC31 gave the highest IFN-γ response after stimulation with either of the vaccine components (see Figures 1 and 2). Finally, vaccination with Ag85B-TB10.4 fusion protein in IC31 induced two major CD4⁺T cell populations, one expressing IFN-γ, IL-2, and TNF-α, and another expressing IL-2 and TNF-α. Both of these T cell populations belong to central memory T cells, and are necessary for long term memory (17). Importantly, as seen with the IFN-γ expression measured by ELISA, using a dose of 0.5μg HyVac4 fusion protein in IC31 induced the highest cell num- bers within the polyfunctional population that expressed IFN-γ, IL-2, and TNF-α, (see Figure 3).

The present invention demonstrates the following: (1) the adjuvants IC31 and cationic liposomes exhibit different sensitivities towards the antigen dose; and

(2) the optimal antigen dose in IC31 is antigen-dependent.

For example, 0.5μg Ag85B-TB10.4 fusion protein in IC31 induced significant protection whereas 5.0μg Ag85B-TB10.4 fusion protein in IC31 does not (figure 4). Taken together, these results highlight the importance of analyzing the antigen dose when testing a new adjuvant/antigen formulations and teach that the optimal antigen dose in a vaccine depends both on the antigen and on the adjuvant.

Moreover, in the present invention, the observed correlation between the amount of poly- functional T cells and the strength of protection induced by the Ag85B-TB 10.4 vaccine in IC31 is in agreement with a recent study in which protective immunity against infection with Leishmania major was directly correlated with long lived memory response and the number of polyfunctional T cells generated (6).

Figure Legends.

Figure 1. Immune recognition of vaccination antigens. PBMCs isolated from groups of mice vaccinated with different doses of H4 in IC31 and a saline control group were stimulated with either 1.0 or 5.0μg/ml of Ag85B, TB10.4 or CFPlO. After 72 hours the concentra- tion of cell released IFN-γ was determined by ELISA. PBMCs were isolated 1 week after third vaccination and were pooled from five mice per group. Values represent the means of triplicate and SEM' s are indicated by bars.

Figure 2. Recognition of Ag85B in different organs. PBMCs (A) and slenocytes (B) iso- Iated from groups of mice immunized with 3 with different doses of H4 formulated in IC31 or a saline control group were stimulated with Ag85B or TB 10.4 for 72 hours where after IFN-γ cytokine secretion was measured by ELISA. The bars represent means of 3 individual mice. SEMs are indicated. In both (A) and (B) a vaccination dose of 0.5 μg H4 gave significantly (p < 0.05, one-way ANOVA and Tukey's post test) higher antigen responses, compared to vaccination doses of 5.0μg and 15.0μg.

Figure 3. Ag85B and TB 10.4 specific T cells are poly-functional. (A) Production of IFN-γ, TNF-α and IL-2 was assessed following antigenic stimulation of PBMCs and spleenocytes 2 weeks post vaccination by flow cytometry. The pie charts are grouped after vaccination dose and colour coded according to the cytokine production profile and summarizes the fractions of the CD4⁺ T cell response that are positive for a given cytokine production profile. (B) Every possible combination of cytokines is shown on the x-axis of the bar chart and the percentage of Hyvac4 (H4) specific CD4⁺ T cells expressing any combination of cytokines is given for each immunization group. No responses were seen in the CD8⁺ T cell subset. (C) Dot plots from the FACS analysis in (A) showing IL-2 and IFN-γ expression by CD4⁺ spleen cells from mice vaccinated with 0.5μg or 5.0μg HyVac4. Percentages in each quad- rant are indicated.

Figure 4. Protective efficacy of H4. In two independent experiments (A and B) groups of mice were vaccinated with three different doses of H4 formulated in IC31 and compared to saline and BCG-vaccinated controls. All groups were challenged by the aerosol route with virulent M. tuberculosis ten weeks after the first vaccination. Six weeks post-challenge, all mice were killed and the bacterial burden (CFU) was measured in the lung by bacterial count. In both experiments data are presented as mean values from six animals per group and standard errors of the means are indicated by bars. Statistical comparison among the vaccination groups were done by one-way ANOVA and Tukey's post test. Significant dif- ferences are only shown for selected groups. ***: p < 0.001, *: p < 0.05.

Materials and Methods

Animals,

Studies were performed with 8 to 12 week-old C57BL/6xBalb/c Fl female mice, purchased from Taconic, Ej by, Denmark. Infected animals were housed in cages contained within laminar flow safety enclosures in a BSL-3 facility. The use of mice was done in accordance with the regulations set forward by the Danish Ministry of Justice and Animal Protection Committees and in compliance with EC Directive 86/609 and the US ALAC recommendations for the care and use of Laboratory animals.

Bacteria. M. tuberculosis Erdman were grown at 37°C on Lowenstein- Jensen medium or in suspension in Sauton medium enriched with 0.5% sodium pyruvate and 0.5% glucose.

Immunization.

Mice were immunized three times at 2-week intervals subcutaneously on the back with ex- perimental vaccines containing 0.5, 5.0 or 15.0μg of Ag85B-TB10.4 fusion protein

(H4)/dose, emulsified in IC31 in a total volume of 0.2 ml/dose. Doses were 100 nmol peptide and 5 nmol oligonucleotide. All vaccines were formulated using 10 mM Tris-HCL/270 mM sorbitol buffer (pH 7.9) as previously described [12] to obtain a final volume of 0.2 ml/mouse. At the time of the first subunit vaccination, one group of mice received a single dose of BCG Danish 1331 (2.5 x 10⁵ CFU) injected subcutaneously at the base of the tail and one group received a saline injection. AU groups of mice were challenged 10 weeks after the first vaccination.

Experimental infections When challenged by the aerosol route, the animals were infected with approximately 50

CFU of M. tuberculosis Erdman/mouse. These mice were sacrificed 6 weeks after challenge. Numbers of bacteria in the spleen or lung were determined by serial threefold dilutions of individual whole-organ homogenates in duplicate on 7Hl 1 medium. Organs from the BCG- vaccinated animals were grown on medium supplemented with 2.0μg of 2-thiophene- carboxylic acid hydrazide (TCH)/ml to selectively inhibit the growth of the residual BCG bacteria in the test organs. Colonies were counted after 2 to 3 weeks of incubation at 37°C. Bacterial burden in the lungs was expressed as log 10 of the bacterial counts based on vaccination groups of six animals.

Lymphocyte cultures

Lymphocytes from spleens were obtained as described previously (2). Blood lymphocytes (PBMCs) were purified on a density gradient. Cells pooled from five mice in each experi- ment were cultured in microtiter wells (96-well plates; Nunc, Roskilde, Denmark) containing 2 x 10⁵ cells in a volume of 200μl of RPMI 1640 supplemented with 5 x 10^"5 M 2- mercaptoethanol, 1% penicillin-streptomycin, 1 mM glutamine, and 5% (vol/vol) fetal calf serum. Based on previous dose-response investigations, the mycobacterial antigens were all used at 15μg/ml or 5μg/ml, while concanavalin A was used at a concentration of lμg/ml as a positive control for cell viability. All preparations were tested in cell cultures and found to be nontoxic at the concentrations used in the present study. Supernatants were harvested from cultures after 72h of incubation for the investigation of IFN-γ.

IFN-γ enzyme-linked immunosorbent assay (ELISA).

Microtiter plates (96 wells; Maxisorb; Nunc) were coated with monoclonal hamster anti- murine IFN-γ (Genzyme, Cambridge, Mass.) in PBS at 4°C. Free binding sites, were blocked with 1% (wt/vol) bovine serum albumin-0.05% Tween 20. Culture supernatants were tested in triplicate, and IFN-γ was detected with a biotin-labelled rat anti-murine monoclonal anti- body (clone XMGl .2; Pharmingen, San Diego, CA). Recombinant IFN-γ (Pharmingen, San Diego, CA) was used as a standard.

FACS analysis of lymphocytes.

Intracellular cytokine staining procedure: Cells from blood, spleen or lungs of mice were stimulated for 1-2 h with 2μg/ml Ag and subsequently incubated for 6 h with lOμg/ml bre- feldin A (Sigma-Aldrich, USA) at 37⁰C. Thereafter, cells were stored overnight at 4°C. The following day, Fc receptors were blocked with 0.5μg/ml anti-CD 16/CD32 mAb (BD Pharmingen, USA) for 10 minutes, where after the cells were washed in FACS buffer (PBS containing 0.1% sodium azide and 1% FCS), and stained for surface markers as indicated using 0.2μg/ml anti-CD4 (clone: RM4-5), anti-CD8 (clone: 53-6,7) mAb's, Cells were then washed in FACS buffer, permeabilized using the Cytofix/Cytoperm kit (BD Pharmingen, Denmark) according to the manufacturers instructions, and stained intracellularly with 0.2 μg/ml anti-IFN- r (clone: XMGl .2), anti-TNF-α (clone: MP6-XT22), or anti-IL-2 (clone:

JES6-5H4) mAb's. After washing, cells were re-suspended in formaldehyde solution 4% (w/v) pH 7.0 (Bie & Berntsen, Denmark) and analysed by flow cytometry on a six-colour BD FACSCanto flow cytometer (BD Biosciences, USA). Statistical methods

The data obtained were tested by analysis of variance. Differences between means were assessed for statistical significance by Tukey's test. A P value of < 0.05 was considered sig- nificant.

Example 1

Immune response induced after immunization with Ag85 B-TB 10.4 fusion protein inIC31 We first analyzed the immunogenicity of Ag85B-TB10.4 fusion protein delivered in IC31 and whether both components of the fusion protein were recognized by the immune system after immunization. Groups of mice were immunized with Ag85B-TB10.4 fusion protein in IC31. As negative control, a group of mice received the adjuvant alone (data not shown). To examine the optimal antigen dose in IC31, we used 15.0, 5.0 and 0.5μg of Ag85B-TB10.4 fusion protein. One week after the last injection, mice were bled, and the IFN-γ release was evaluated after in vitro stimulation of purified PBMCs with different concentrations of Ag85B and TB 10.4 proteins (5μg/ml and lμg/ml) (Fig. IA). Immunization with Ag85B- TB 10.4 fusion protein in IC31 induced a strong IFN-γ response specific for Ag85B and TB 10.4 proteins. Surprisingly, this response was sensitive to the antigen immunization dose. Thus, the lowest dose of Ag85B-TB10.4 fusion protein in IC31 gave the highest IFN-γ re- sponse after stimulation with either Ag85B (9401 +/- 3668 pg/ml IFN-γ) or TB10.4 (4694 +/- 3992 pg/ml IFN-γ) (Fig. IA). Using a dose of 5.0μg or 15.0μg Ag85B-TB10.4 fusion protein significantly reduced the IFN-γ response against both Ag85B and TB 10.4 proteins relative to mice vaccinated with 0.5μg Ag85B-TB10.4 fusion protein (pO.OOl). This was particularly apparent for the high immunization dose - 15μg Ag85B-TB10.4 fusion protein per immunization dose - which gave IFN-γ responses that did not differ from the observed responses in non- vaccinated mice (or in Ag85B-TB10.4 fusion protein vaccinated mice stimulated in vitro with control antigen CFPlO). The same dose dependency was subsequently repeated in an independent experiment where the immune responses were analyzed in both blood and spleen (Fig 2). These results show that the lowest dose of 0.5μg Ag85B- TB 10.4 fusion protein in IC31 induced the strongest systemic response of the antigen doses tested. Example 2

Vaccination with Ag85B-TB10.4 fusion protein in IC31 induce polyfunctioned CD4⁺T cells. We next analyzed the phenotype of the T cells induced by immunizing with Ag85B-TB10.4 fusion protein in IC31. In particular, we were interested in the ability of this vaccine to in- duce polyfunctional (IFN-γ⁺IL-2⁺TNF-α⁺) CD4⁺ T cells as these have been shown to correlate with protective immunity against infections such as Leishmania major and to form the basis for a long lived memory response (6, 15). PBMC 's from Ag85B-TB10.4 fusion protein in IC31 vaccinated mice were stimulated in vitro with Ag85B or TB 10.4 fusion protein and analyzed by flow cytometry for expression of CD4, CD8, IFN-γ, TNF- Oi , and IL-2. The results showed that immunizing with Ag85B-TB10.4 fusion protein in IC31 induced two major polyfunctional T cell populations; CD4⁺IFN-γ⁺IL-2⁺TNF-α⁺ and CD4⁺IL-2⁺TNF-α⁺ T cells (Fig. 3). This was seen for Ag85B and TB 10.4 specific T cells. Interestingly, as observed in figure 1, there is a higher response in the group immunized with 0.5μg HyVac4 in IC31 compared to the group immunized with 5.0μg HyVac4 fusion protein in IC31 (see Figure 3), and that the major difference was that the group being vaccinated with only 0.5μg showed significantly more polyfunctional T cells. Taken together, immunizing with Ag85B- TB 10.4 fusion protein in IC31 induced poly-functional CD4⁺ T cells and confirmed that lowering the amount of Ag85B-TB10.4 fusion protein increased the immunogenicity of the vaccine in terms of not only IFN-γ expression but also the number of polyfunctional T cells.

Example 3

Protective efficacy of Ag85B-TB 10.4 fusion protein andIC31 in a mouse M. tuberculosis infection model

We finally examined the protective efficacy of Ag85B-TB10.4 fusion protein in IC31, and whether the dose dependency regarding the immunogenicity of the vaccine was also reflected in the protective efficacy of the vaccine. Mice were vaccinated three times at two weeks interval with Ag85B-TB10.4 fusion protein in IC31. As a positive control for protection, a group of mice were immunized once with BCG. Ten weeks after the first vaccination, the mice were challenged by the aerosol route with virulent M. tuberculosis Erdman. Six weeks post challenge, the mice were killed and the bacterial numbers were determined in the lungs. As observed with the immunogenicity of the vaccines, the lowest Ag85B-TB10.4 fu- sion protein immunization dose induced the highest protection. Thus, mice vaccinated with 0.5μg Ag85B-TB10.4 fusion protein in IC31 contained a bacterial number of 5.0 +/- 0.2 Logio CFU in the lungs. This was equal to the numbers observed in BCG vaccinated mice (4.90 +/- 0.35 Log₁₀ CFU), but significantly lower (pO.OOl) compared to the bacterial numbers in non-vaccinated mice (5.83 +/- 0.12 Log₁₀ CFU) (Fig. 4A). In contrast, the bacterial numbers in mice vaccinated with 5.0μg or 15.0μg of Ag85B-TB10.4 fusion protein in IC31, were not significantly different from the levels found in the lungs of non- vaccinated mice (Fig. 4A). Repeating the experiment led to the same conclusion although the overall bacterial numbers were slightly lower in all the groups (Fig. 4B).Thus the ability of the vac- cine, Ag85B-TB10.4 fusion protein in IC31, to induce protection against M. tuberculosis correlated with the immunogenicity of the vaccine, in terms of INF -γ production and the number of polyfunctional T cells, and was highest when the lowest antigen dose was used.

The surprising in vivo results from these well recognized M. tuberculosis animal models, supports the use of the immunogenic compositions of the current invention as a M. tuberculosis vaccine in humans.

Example 4 Protective efficacy of Ag85 B-TB 10.4 fusion protein and ICSl in a clinical trial

In human clinical trials subjects will be vaccinated with less than about 5.0μg to 25.0μg of Ag85B-TB10.4 in IC31.

This low dose of Ag85B-TB10.4 is in stark contrast with other subunit M. tuberculosis vaccines currently in clinical trials. For example, 40.0μg of MTB72F in AS02A per dose (19) and 50.0μg of Ag85B-ESAT-6 in IC31 per dose (clinical data not published yet).

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation. References.

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Claims

1. An immunogenic composition for human use comprising a TB 10.4 protein and an Ag85-compleχ protein, wherein the total amount of the TB 10.4 protein and the Ag85-complex protein is less than about 25.0μg.

2. An immunogenic composition according to claim 1, wherein the TB 10.4 protein and the Ag85-complex protein are present as a fusion protein.

3. An immunogenic composition of claim 1 or 2 further comprising an adjuvant

4. The immunogenic composition of claim 3 wherein the adjuvant comprises at least one polycationic peptide and at least one oligonucleotide.

5. The immunogenic composition according to any one of claims lto 4 wherein the Ag85-complex protein is an Ag85B protein.

6. The immunogenic composition according to any one of claims 1 to 5 wherein the fusion protein is present in amount of less than about lO.Oμg.

7. The immunogenic composition according to any one of claims 6 wherein the fusion protein is present in amount equal to about 0.5μg.

8. The immunogenic composition of claim 4 wherein the at least one oligonucleotide is a TLR9 agonist.

9. The immunogenic composition according to claim 8, wherein the adjuvant is a mixture of peptide NH₂-KLKLLLLLKLK-COOH and oligonucleotide 5'-ICI CIC ICI CIC ICI CIC ICI CIC IC-3¹.

10. The immunogenic composition of claim 8 wherein the adjuvant is IC31®.

11. A vaccine for human use comprising a TB 10.4 protein and an Ag85-complex protein an adjuvant comprising at least one polycationic peptide and at least one oligonucleotide, wherein the total amount of the TB 10.4 protein and the Ag85-complex protein is less than about 25. Oμg.

12. A vaccine according to claim 10, wherein the TB 10.4 protein and the Ag85- complex protein are present as a fusion protein.

13. A vaccine of claim 10 to 11 further comprising an adjuvant.

14. A vaccine of claim 12 wherein the adjuvant comprises at least one polycationic peptide and at least one oligonucleotide.

15. The vaccine according to any one of claims 10 to 13 wherein the Ag85-complex protein is an Ag85B protein.

16. The vaccine according to any one of claims 10 to 14 wherein the total amount of TB10.4 protein and the Ag85-complex protein less than about 10. Oμg.

17. The vaccine of claim 15 wherein the total amount of TB 10.4 protein and the Ag85- complex protein equal to about 0.5 μg.

18. The vaccine of claim 13 wherein the at least one oligonucleotide is a TLR9 agonist.

19. The vaccine according to claim 14, wherein the adjuvant is a mixture of peptide NH₂-KLKLLLLLKLK-COOH (SEQ ID NO: ) and oligonucleotide 5'-ICI CIC ICI CIC ICI CIC ICI CIC IC-3' (SEQ ID NO: ).

20. The vaccine of claim 13 wherein the adjuvant is IC31®.

21. A method of inducing protection against M. tuberculosis in a human, the method comprising introducing into the human an immunogenic composition according to any one of claims 1-9.

22. A method of inducing polyfunctional CD4⁺T cells in a human, the method comprising introducing into the human an immunogenic composition according to any one of claims 1-9.