WO2005061534A2

WO2005061534A2 - Improved tuberculosis vaccines

Info

Publication number: WO2005061534A2
Application number: PCT/DK2004/000907
Authority: WO
Inventors: Dietrich Jes; Peter Andersen; Claus Aagaard
Original assignee: Statens Serum Institut
Priority date: 2003-12-23
Filing date: 2004-12-22
Publication date: 2005-07-07
Also published as: WO2005061534A3

Abstract

The invention is related to an immunogenic composition, vaccine or pharmaceutical composition for preventing boosting or treating infection caused by a species of the tuberculosis complex (M. tuberculosis, M. bovis, M. africanum, M. microti). The immunogenic composition, vaccine or pharmaceutical composition comprise a fusion polypeptide, the units of the fusion polypeptide being M. tuberculosis antigens. Further, the invention is relates to the use of a vaccine comprising a fusion polypeptide sequence or nucleic acid sequence of the invention given at the same time as BCG, either mixed with BCG or administered separately at different sites or routes for preparing said immunogenic composition, vaccine, or pharmaceutical composition. Further, the invention is related to the use of a vaccine comprising a fusion polypeptide sequence or nucleic acid sequence given as BCG booster vaccine.

Description

IMPROVED TUBERCULOSIS VACCINES

FIELD OF INVENTION

The present invention discloses new fusion polypeptides of immunogenic polypeptides based on polypeptides derived from M. tuberculosis, the use of one or more of the fusion polypeptides of the invention for the preparation of an immunogenic composition, vaccine or pharmaceutical composition to be used for administration to a person which has previously been vaccinated with BCG and the immunogenic compositions, vaccines or pharmaceutical compositions as such..

GENERAL BACKGROUND

Human tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) is a severe global health problem, responsible for approx. 3 million deaths annually, according to the WHO. The worldwide incidence of new tuberculosis (TB) cases had been falling during the 1960s and 1970s but during recent years this trend has markedly changed in part due to the advent of AIDS and the appearance of multidrug resistant strains of M. tuberculosis.

The only vaccine presently available for clinical use is BCG, a vaccine whose efficacy remains a matter of controversy. BCG generally induces a high level of acquired resistance in animal models of TB, and in humans it is protective against disseminated forms of tuberculosis such as meningitis and miliary tuberculosis. When given to young children it is protective against tuberculosis for years but then the efficacy vanes. Comparison of various controlled trials revealed that the protective efficacy of BCG in adults varied dramatically with an efficacy range from ineffective to 80% protection. At best, boost of BCG with BCG has no effect [Colditz, 1994]. Since BCG needs to divide and secrete proteins in order to induce a protective immune response, the lack of booster effect is primarily due to either sensitisation with environmental mycobacteria or a residual immune response from the primary BCG vaccination. Both events lead to a rapid immune response against BCG and therefore quick inhibition of growth and elimination of BCG.

Boosting of BCG has been done with Ag85a (Brooks et al IAI 2001; WO0204018) in an inbred mice strain leading to some protection, although compared to BCG alone it was not significantly better.

This makes the development of a new and improved vaccine against TB an urgent matter, which has been given a very high priority by the WHO. Many attempts to define protective mycobacterial substances have been made, and different investigators have reported increased resistance after experimental vaccination. However, the demonstration of a specific long-term protective immune response with the potency of BCG or the capability of boosting in a BCG vaccinating person has not yet been achieved.

Immunity to M. tuberculosis is characterized by some basic features; specifically sensitized T lymphocytes mediates protection, and the most important mediator molecule seems to be interferon gamma (IFN-gamma).

M. tuberculosis holds, as well as secretes, several proteins of potential relevance for the generation of a new TB vaccine. For a number of years, a major effort has been put into the identification of new protective antigens for the development of a novel vaccine against TB. The search for candidate molecules has primarily focused on proteins released from dividing bacteria. Despite the characterization of a large number of such proteins only a few of these have been demonstrated to induce a protective immune response as subunit vaccines in animal models, most notably ESAT-6 and Ag85B (Brandt et al 2000). Since only one of the partners in the Ag85b-ESAT6 fusion is found in M. bovis BCG it is not the optimum combination to boost a vaining BCG response; two or more antigens from BCG will have a higher capacity of boosting the BCG response.

In 1998 Cole et al published the complete genome sequence of M. tuberculosis and predicted the presence of approximately 4000 open reading frames (Cole et al 1998) disclosing nucleotide sequences and putative protein sequences. However importantly, this sequence information cannot be used to predict if the DNA is translated and expressed as proteins in vivo. More importantly, it is not possible on the basis of the sequences to predict whether a given sequence will encode an immunogenic or an inactive protein. The only way to determine if a protein is recognized by the immune system during or after an infection with M. tuberculosis is to produce the given protein and test it in an appropriate assay as described herein.

Diagnosing M. tuberculosis infection in its earliest stage is important for effective treatment of the disease. Current diagnostic assays to determine M. tuberculosis infection are expensive and labour-intensive. In the industrialised part of the world the majority of patients exposed to M. tuberculosis receive chest x-rays and attempts are made to culture the bacterium in vitro from sputum samples. X-rays are insensitive as a diagnostic assay and can only identify infections in a very progressed stage. Culturing of M. tuberculosis is also not ideal as a diagnostic tool, since the bacteria grows poorly and slowly outside the body, which can produce false negative test results and take weeks before results are obtained. The standard tuberculin skin test is an inexpensive assay, used in third world countries, however it is far from ideal in detecting infection because it cannot distinguish M. tuberculosis-^'mfected individuals from M. bovis BCG-vaccinated individuals and therefore cannot be used in areas of the world where patients receive or have received childhood vaccination with bacterial strains related to M. tuberculosis, e.g. a BCG vaccination.

Animal tuberculosis is caused by Mycobacterium bovis, which is closely related to M. tuberculosis and within the tuberculosis complex. M. bovis is an important pathogen that can infect a range of hosts, including cattle and humans. Tuberculosis in cattle is a major cause of economic loss and represents a significant cause of zoonotic infection. A number of strategies have been employed against bovine TB, but the approach has generally been based on government-organised programmes by which animals deemed positive to defined screening test are slaughtered. The most common test used in cattle is Delayed-type hypersensitivity with PPD as antigen, but alternative in vitro assays are also developed. However, investigations have shown that both the in vivo and the in vitro tests have a relative low specificity, and the detection of false-positive is a significant economic problem (Pollock et al 2000). There is therefore a great need for a more specific diagnostic reagent, which can be used either in vivo or in vitro to detect M. bovis infections in animals.

SUMMARY OF THE INVENTION

The invention is related to an immunogenic composition, vaccine or pharmaceutical composition for preventing (including booster vaccination) or treating infection caused by a species of the tuberculosis complex (M. tuberculosis, M. bovis, M. af canum), the immunogenic composition, the vaccine or pharmaceutical composition comprising a fusion polypeptide, the units of the fusion polypeptide being M. tuberculosis antigens. Also, the invention relates to the fusion polypeptides as such and to a nucleic acid sequence encoding a such fusion polypeptide. Further, the invention relates to the use of a fusion polypeptide sequence or nucleic acid sequence of the invention for preparing said immunogenic composition, vaccine, or pharmaceutical composition. Further, the invention relates to the use of a vaccine comprising a fusion polypeptide sequence or nucleic acid sequence of the invention given at the same time as BCG, either mixed with BCG or administered separately at different sites or routes for preparing said immunogenic composition, vaccine, or pharmaceutical composition. Further the invention relates to the use of a vaccine comprising a fusion polypeptide sequence or nucleic acid sequence given as a BCG booster.

DETAILED DISCLOSURE OF THE INVENTION

In order to obtain significantly better BCG booster vaccines, fusion polypeptides or a mix of several polypeptides will be needed. These will also have a better chance of presentation when tested in more heterogeneous populations with different MHC haplotypes. In a first aspect, the invention discloses a fusion polypeptide which comprises an amino acid sequence selected from the amino acid sequences encoding the fusion polypeptides Ag85B-TB10.4 Ag85B-TB10.4-Ag85A Ag85B-TB10.4-ORF2c Ag85B-TB10.4-ORF2c-Ag85A Ag85B-TB10.4-Rvl036 Ag85A-TB10.4 Ag85B-TB10.4-Ag85A-TB10.4 TB10.4-Rv0285-Ag85A TB10.4-Rvl036-Ag85A TB10.4-ORF2c-Ag85A Ag85A-Rv0287 Rv0287-TB10.4

The amino acid and nucleic acid sequences of these fusion polypeptides appear from the sequence listing as follows:

In the present context the individual immunogenic polypeptide based on polypeptides derived from M. tuberculosis is termed a "unit" of the fusion polypeptide. The fusion may comprise 2, 3, 4, 5, 6, 7, 8, 9 or event 10 different units. The order of the units of the fusion polypeptide can be any combination. In order terms, fusion polypeptides of all of the above antigens in any combination are within the scope of the present invention. The fusion polypeptides of the invention are useful for the preparation of an immunogenic composition, vaccine or pharmaceutical composition, in particular a BCG booster vaccine, as will be described in detail in the following.

The polypeptides making up units of the fusion polypeptides have the following Sanger identity number and amino acid sequences:

The amino acid sequences of the units of fusion polypeptides appear from the following:

Within the scope of the present invention is an analogue of a fusion polypeptide which has an amino acid sequence with a sequence identity of at least 80% to any one of the fusion polypeptides of the invention and which is immunogenic, and a nucleic acid sequence which encodes a such polypeptide. Such analogues are comprised within the term "polypeptide of the invention" or "fusion polypeptide of the invention" which terms are used interchangeably throughout the specification and claims. By the term "nucleic acid sequence of the invention" is meant a nucleic acid sequence encoding a such polypeptide.

A presently preferred embodiment of the invention is a vaccine to boost immunity from prior BCG vaccination, i.e. the vaccine is administered to individuals previously vaccinated with BCG.

This first aspect of the invention comprises a variant of the above mentioned fusion polypeptide which is lipidated so as to allow a self-adjuvating effect of the polypeptide.

The immunogenic composition, vaccine or pharmaceutical composition of the invention can be administered by mucosal delivery, e.g. orally, nasally, buccally, or traditionally intramuscularly, intradermally, by subcutaneous injection or transdermally or any other suitable route, e.g rectally.

In another embodiment, the invention discloses the use of a fusion polypeptide as defined above for the preparation of an immunogenic composition, vaccine or pharmaceutical composition which can be used for a prophylactic vaccination together with BCG, a booster vaccine or therapeutical vaccination against an infection caused by a virulent mycobacterium, e.g. by e.g. by Mycobacterium tuberculosis, Mycobacterium af canum, Mycobacterium bovis, Mycobacterium lepra or Mycobacterium ulcerans

In a second aspect, the invention discloses an immunogenic composition, vaccine or pharmaceutical composition which comprises a nucleotide sequence which encodes a fusion polypeptide as defined above, or comprises a nucleic acid sequence complementary thereto which is capable of hybridizing to the nucleic acid sequence of the invention under stringent conditions.

The nucleic acid fragment is preferably a DNA fragment. The fragment can be used as a pharmaceutical as discussed in the following.

In one embodiment, the invention discloses a an immunogenic composition, vaccine or pharmaceutical composition comprising a nucleic acid fragment according to the invention, optionally inserted in a vector, the vaccine effecting in vivo expression of antigen by an animal, including a human being, to whom the vaccine has been administered, the amount of expressed antigen being effective to confer substantially increased resistance to tuberculosis caused by virulent mycobacteria, e.g. by e.g. by Mycobacterium tuberculosis, Mycobacterium af^ήcanum, Mycobacterium bovis,

Mycobacterium lepra or Mycobacterium ulcerans,, in an animal, including a human being.

In a further embodiment, the invention discloses the use of an immunogenic composition, vaccine or pharmaceutical composition comprising a nucleic acid fragment according to the invention for therapeutic vaccination against tuberculosis caused by a virulent mycobacterium.

In a still further embodiment, the invention discloses an immunogenic composition, vaccine or pharmaceutical composition which can be used for a prophylactic vaccination together with BCG or as a booster vaccine to a person previously vaccinated with BCG for immunizing an animal, including a human being, against tuberculosis caused by a virulent mycobacterium, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium lepra or Mycobacterium ulcerans, comprising as the effective component a non-pathogenic microorganism, such as vaccinia, adenovirus or Mycobacterium bovis BCG, wherein at least one copy of a DNA fragment comprising a DNA sequence encoding a fusion polypeptide as defined above has been incorporated into the microorganism (e.g. placed on a plasmid or in the genome) in a manner allowing the microorganism to express and optionally secrete the fusion polypeptide.

In another embodiment, the invention discloses an infectious expression vector, such as vaccinia, adenovirus or Mycobacterium bovis BCG which comprises a nucleic acid fragment according to the invention, and a transformed cell harbouring at least one such vector.

In a third aspect, the invention discloses a method for immunising and boosting the immunity of an animal, including a human being, against tuberculosis caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium lepra or Mycobacterium ulcerans, the method comprising administering to the animal the fusion polypeptide as defined above, the immunogenic composition according to the invention, or the vaccine according to the invention.

In a fourth aspect, the invention discloses a method for treating an animal, including a human being, having tuberculosis, active or latent, caused by virulent mycobacteria, e.g. by Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium lepra or Mycobacterium ulcerans, the method comprising administering to the animal the immunogenic composition, vaccine or pharmaceutical composition as defined above.

In a fifth aspect, the invention discloses the use of a fusion polypeptide or nucleic acid fragment as defined above for the preparation of an immunogenic composition, vaccine or pharmaceutical composition in combination with M. bovis BCG, e.g. for a prophylactic (including boosting) or therapeutical vaccination against an infection caused by a virulent mycobacterium, e.g. by e.g. by Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium lepra or Mycobacterium ulcerans.

The vaccine, immunogenic composition, vaccine and pharmaceutical composition according to the invention can be used prophylactically in a subject not infected with a virulent mycobacterium or in an individual previously vaccinated with M. tuberculosis BCG or therapeutically in a subject infected with a virulent mycobacterium.

It is to be understood that the embodiments of the first aspect of the invention, such as the immunogenic polypeptides described also apply to all other aspects of the invention; and vice versa.

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.

DEFINITIONS

Combination with M. bovis BCG

By the term "combination with M. bovis BCG" is understood co-administration with any M. bovis BCG strain including, Pasteur, Phipps, Frappier, Connaught, Tice, Denmark, Glaxo, Prague, Birkhaug, Sweden, Japan, Moreau and Russia in quantities that lead either to a significant increased specific immune response or to a significant protection in an animal model or a human either together with one or more of the fusion polypeptides defined above or with one or more of the nucleic acid fragments encoding these, or administered at the same time but at separate sites or routes.

Boost of M. bovis BCG By the term" boost of M. bovis BCG" is understood administration of one or more fusion polypeptides as defined above or one or more nucleic acid fragments encoding these at any period after vaccination with any M. bovis BCG strain including, Pasteur, Phipps, Frappier, Connaught, Tice, Denmark, Glaxo, Prague, Birkhaug, Sweden, Japan, Moreau and Russia in quantities that lead either to a significant increased specific immune response or a significant increased protection in an animal model or a human.

Polypeptide

A preferred polypeptide to be used as a unit of the fusion polypeptides of the present invention is an immunogenic polypeptide from M. tuberculosis. Such polypeptide can for example be based on a polypeptide derived from the M. tuberculosis cell and/or M. tuberculosis culture filtrate. The polypeptide will normally be a recombinant or synthetic polypeptide and may consist of the immunogenic polypeptide, an immunogenic portion thereof or may contain additional sequences. The additional sequences may be derived from the native M. tuberculosis antigen or be heterologous and such sequences may, but need not, be immunogenic.

By the term "fusion polypeptide" is understood a random order of two or more immunogenic polypeptides from M. tuberculosis or analogues thereof fused together with or without an amino acid spacer(s) of arbitrary length and sequence.

The word "polypeptide" in the present invention should have its usual meaning. That is an amino acid chain of any length, including a full-length protein, oligopeptide, short peptide and fragment thereof and fusion polypeptide, wherein the amino acid residues are linked by covalent peptide bonds.

The polypeptide may be chemically modified by being glycosylated, by being lipidated (e.g. by chemical lipidation with palmitoyloxy succinimide as described by Mowat et al. 1991 or with dodecanoyl chloride as described by Lustig et al. 1976), by comprising prosthetic groups, or by containing additional amino acids such as e.g. a his-tag or a signal peptide.

Each immunogenic polypeptide 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 polypeptide sequences have been modified by substitution, insertion, addition or deletion of one or more amino acid residues in the recombinant polypeptide while still being immunogenic in any 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.

Each polypeptide is encoded by a specific nucleic acid sequence. Within the scope of the present invention are analogues and such nucleic acid sequences which have been modified by substitution, insertion, addition or deletion of one or more nucleic acids. Substitutions are preferably silent substitutions in the codon usage which will not lead to any change in the amino acid sequence, but may be introduced to enhance the expression of the protein.

Nucleic acid fragment By the terms "nucleic acid fragment" and "nucleic acid sequence" are understood any nucleic acid molecule including DNA, RNA , LNA (locked nucleic acids), PNA, RNA, dsRNA and RNA-DNA-hybrids. Also included are nucleic acid molecules comprising non-naturally occurring nucleosides. The term includes nucleic acid molecules of any length e.g. from 10 to 10000 nucleotides, depending on the use. When the nucleic acid molecule is for use as a pharmaceutical, e.g. in DNA therapy, or for use in a method for producing a polypeptide according to the invention, a molecule encoding at least one epitope is preferably used, having a length from about 18 to about 1000 nucleotides, the molecule being optionally inserted into a vector. When the nucleic acid molecule is used as a probe, as a primer or in antisense therapy, a molecule having a length of 10-100 is preferably used. According to the invention, other molecule lengths can be used, for instance a molecule having at least 12, 15, 21, 24, 27, 30, 33, 36, 39, 42, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or 1000 nucleotides (or nucleotide derivatives), or a molecule having at most 10000, 5000, 4000, 3000, 2000, 1000, 700, 500, 400, 300, 200, 100, 50, 40, 30 or 20 nucleotides (or nucleotide derivatives). The term "stringent" when used in conjunction with hybridization conditions is as defined in the art, i.e. the hybridization is performed at a temperature not more than 15-20°C under the melting point Tm, cf. Sambrook et al, 1989, pages 11.45-11.49. Preferably, the conditions are "highly stringent", i.e. 5-10°C under the melting point Tm. Sequence identity

The term "sequence identity" indicates a quantitative measure of the degree of homology between two amino acid sequences of substantially equal length or between two nucleic acid sequences of substantially equal length. The two sequences to be compared must be aligned to best possible fit possible with the insertion of gaps or alternatively, truncation at the ends of the protein sequences. The sequence identity can be calculated as ^r^ — , wherein N_d|_f is the total number of non-identical residues in the two sequences when aligned and wherein N_ref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (N_dlf=2 and N_ref=8). A gap is counted as non-identity of the specific residue(s), i.e. the DNA sequence AGTGTC will have a sequence identity of 75% with the DNA sequence AGTCAGTC (N_dif=2 and N_ref=8). Sequence identity can alternatively be calculated by the BLAST program e.g. the BLASTP program (Pearson W.R and DJ. Lipman (1988))(www. ncbi.nlm.nih.gov/cgi-bin/BLAST). In one embodiment of the invention, alignment is performed with the sequence alignment method ClustalW with default parameters as described by Thompson J., et al 1994, available at http://www2.ebi.ac.uk/clustalw/.

A preferred minimum percentage of sequence identity is at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%. Preferably, the numbers of substitutions, insertions, additions or deletions of one or more amino acid residues in the fusion polypeptide is limited, i.e. no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 substitutions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 insertions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 additions, and no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 deletions compared to the immunogenic polypeptide units based on polypeptides derived from M. tuberculosis.

Immunogenic portion

The polypeptide of the invention comprises an immunogenic portione, such as an epitope for a B-cell or T-cell. The immunogenic portion of an immunogenic polypeptide is the part of the polypeptide, which elicits an immune response in an animal or a human being, and/or in a biological sample determined by any of the biological assays described herein. The immunogenic portion of a polypeptide may be a T-cell epitope or a B-cell epitope. Immunogenic portions can be related to one or a few relatively small parts of the polypeptide, they can be scattered throughout the polypeptide sequence or be situated in specific parts of the polypeptide. For a few polypeptides epitopes have even been demonstrated to be scattered throughout the polypeptide covering the full sequence (Ravn et al 1999).

In order to identify relevant T-cell epitopes which are recognised during an immune response, it is possible to use a "brute force" method: Since T-cell epitopes are linear, deletion mutants of the polypeptide will, if constructed systematically, reveal what regions of the polypeptide are essential in immune recognition, e.g. by subjecting these deletion mutants e.g. to the IFN-γ assay described herein. Another method utilises overlapping oligopeptides for the detection of MHC class II epitopes, preferably synthetic, having a length of e.g. 20 amino acid residues derived from the polypeptide. These peptides can be tested in biological assays (e.g. the IFN-γ assay as described herein) and some of these will give a positive response (and thereby be immunogenic) as evidence for the presence of a T cell epitope in the peptide. For the detection of MHC class I epitopes it is possible to predict peptides that will bind (Stryhn et al. 1996) and hereafter produce these peptides synthetically and test them in relevant biological assays e.g. the IFN-γ assay as described herein. The peptides preferably having a length of e.g. 8 to 11 amino acid residues derived from the polypeptide. B-cell epitopes can be determined by analysing the B cell recognition to overlapping peptides covering the polypeptide of interest as e.g. described in Harboe et al 1998.

Immunogenic portions of polypeptides may be recognised by a broad part (high frequency) or by a minor part (low frequency) of the genetically heterogenic human population. In addition some immunogenic portions induce high immunological responses (dominant), whereas others induce lower, but still significant, responses (subdominant). High frequency > < low frequency can be related to the immunogenic portion binding to widely distributed MHC molecules (HLA type) or even by multiple MHC molecules (Kilgus et al. 1991, Sinigaglia et al 1988 ).

In the context of providing candidate molecules for a new vaccine against tuberculosis, the subdominant epitopes are however as relevant as are the dominant epitopes since it has been show (Olsen et al 2000) that such epitopes can induce protection even though subdominant. Analogues

A common feature of the fusion polypeptides of the invention is their capability to induce an immunological response as illustrated in the examples. It is understood that within the scope of the present invention are analogues of a fusion polypeptide of the invention produced by substitution, insertion, addition or deletion is also immunogenic determined by any of the assays described herein.

Substantially pure In the present context the term "substantially pure polypeptide" means a polypeptide preparation which contains at most 5% by weight of other polypeptide material with which it is associated natively or during recombinant or synthetic production (lower percentages of other polypeptide material are preferred, e.g. at most 4%, at most 3%, at most 2%, at most 1%, and at most ¹/2%). It is preferred that the substantially pure polypeptide is at least 96% pure, i.e. that the polypeptide constitutes at least 96% by weight of total polypeptide material present in the preparation, and higher percentages are preferred, such as at least 97%, at least 98%, at least 99%, at least 99,25%, at least 99,5%, and at least 99,75%. It is especially preferred that the polypeptide is in "essentially pure form", i.e. that the polypeptide is essentially free of any other antigen with which it is natively associated, i.e. free of any other antigen from bacteria belonging to the tuberculosis complex or a virulent mycobacterium. This can be accomplished by preparing the polypeptide by means of recombinant methods in a non-mycobacterial host cell as will be described in detail below, or by synthesizing the polypeptide by the well-known methods of solid or liquid phase peptide synthesis, e.g. by the method described by Merrifield or vari- ations thereof, and by using appropriate purification procedures well known to the person of ordinary skill in the art.

Virulent mycobacterium, individual currently infected and immune individual

By the term "virulent mycobacterium" is understood a bacterium capable of causing the tuberculosis disease in an animal or in a human being. Examples of virulent mycobacteria are Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium lepra or Mycobacterium ulcerans. Examples of relevant animals are cattle, possums, badgers and kangaroos.

By "an animal or human currently infected with a virulent mycobacterium" is understood an individual with culture or microscopically proven infection with virulent mycobacteria, and/or an individual clinically diagnosed with TB and who is responsive to anti-TB chemotherapy. Culture, microscopy and clinical diagnosis of TB are well known by any person skilled in the art.

An immune individual is defined as a person or an animal, which has cleared or controlled an infection with a virulent mycobacterium or has received a vaccination with M. bovis BCG.

Immunogenic An immunogenic polypeptide is defined as a polypeptide that induces an immune response. The immune response may be monitored by one of the following methods:

An in vitro cellular response is determined by release of a relevant cytokine such as IFN-γ, from lymphocytes withdrawn from an animal or human currently or previously infected with virulent mycobacteria, or by detection of proliferation of these T cells. The induction is performed by addition of the polypeptide or the immunogenic portion to a suspension comprising from lxlO⁵ cells to 3xl0⁵ cells per well. The cells are isolated from either blood, the spleen, the liver or the lung and the addition of the polypeptide or the immunogenic portion of the polypeptide result in a concentration of not more than 20 μg per ml suspension and the stimulation is performed from two to five days. For monitoring cell proliferation the cells are pulsed with radioactive labeled Thymidine and after 16-22 hours of incubation the proliferation is detected by liquid scintillation counting. A positive response is a response more than background plus two standard deviations. The release of IFN-γ can be determined by the ELISA method, which is well known to a person skilled in the art. A positive response is a response more than background plus two standard deviations. Other cytokines than IFN-γ could be relevant when monitoring an immunological response to the polypeptide, such as IL-12, TNF-α, IL-4, IL-5, IL-10, IL-6, TGF-β. Another and more sensitive method for determining the presence of a cytokine (e.g. IFN-γ) is the ELISPOT method where the cells isolated from either the blood, the spleen, the liver or the lung are diluted to a concentration of preferable of 1 to 4 x 10⁶ cells /ml and incubated for 18-22 hrs in the presence of the polypeptide or the immunogenic portion of the polypeptide resulting in a concentration of not more than 20 μg per ml. The cell suspensions are hereafter diluted to 1 to 2 x 10⁶/ ml and transferred to Maxisorp plates coated with anti-IFN-γ and incubated for preferably 4 to 16 hours. The IFN-γ producing cells are determined by the use of labelled secondary anti-IFN-γ antibody and a relevant substrate giving rise to spots, which can be enumerated using a dissection microscope. It is also a possibility to determine the presence of mRNA coding for the relevant cytokine by the use of the PCR technique. Usually one or more cytokines will be measured utilizing for example the PCR, ELISPOT or ELISA. It will be appreciated by a person skilled in the art that a significant increase or decrease in the amount of any of these cytokines induced by a specific polypeptide can be used in evaluation of the immunological activity of the polypeptide.

An in vitro cellular response may also be determined by the use of T cell lines derived from an immune individual or an M. tuberculosis infected person where the T cell lines have been driven with either live mycobacteria, extracts from the bacterial cell or culture filtrate for 10 to 20 days with the addition of IL-2. The induction is performed by addition of not more than 20 μg polypeptide per ml suspension to the T cell lines containing from lxlO⁵ cells to 3xl0⁵ cells per well and incubation is performed from two to six days. The induction of IFN-γ or release of another relevant cytokine is detected by ELISA. The stimulation of T cells can also be monitored by detecting cell proliferation using radioactively labeled Thymidine as described above. For both assays a positive response is a response more than background plus two standard deviations.

An in vivo cellular response may be determined as a positive DTH response after intradermal injection or local application patch of at most 100 μg of the polypeptide or the immunogenic portion to an individual who is clinically or subclinically infected with a virulent Mycobacterium, a positive response having a diameter of at least 5 mm 72-96 hours after the injection or application.

An in vitro humoral response is determined by a specific antibody response in an immune or infected individual. The presence of antibodies may be determined by an ELISA technique or a Western blot where the polypeptide or the immunogenic portion is absorbed to either a nitrocellulose membrane or a polystyrene surface. The serum is preferably diluted in PBS from 1: 10 to 1: 100 and added to the absorbed polypeptide and the incubation being performed from 1 to 12 hours. By the use of labeled secondary antibodies the presence of specific antibodies can be determined by measuring the presence or absence of a specific label e.g. by ELISA where a positive response is a response of more than background plus two standard deviations or alternatively a visual response in a Western blot.

Another relevant parameter is measurement of the protection in animal models induced after vaccination with the polypeptide in an adjuvant or after DNA vaccination. Suitable animal models include primates, guinea pigs or mice, which are challenged with an infection of a virulent Mycobacterium. Readout for induced protection could be decrease of the bacterial load in target organs compared to non-vaccinated animals, prolonged survival times compared to non-vaccinated animals and diminished weight loss or pathology compared to non-vaccinated animals.

Preparation methods In general the fusion polypeptides of the invention, and DNA sequences encoding such fusion polypeptides, may be prepared by use of any one of a variety of procedures.

The fusion polypeptide may be produced recombinantly using a DNA sequence encoding the polypeptide, which has been inserted into an expression vector and expressed in an appropriate host. Examples of host cells are E. coli. The fusion polypeptides can also be produced synthetically having fewer than about 100 amino acids, and generally fewer than 50 amino acids and may be generated using techniques well known to those ordinarily skilled in the art, such as commercially available solid-phase techniques where amino acids are sequentially added to a growing amino acid chain.

The fusion polypeptides may also be produced with an additional fusion partner, by which methods superior characteristics of the polypeptide of the invention can be achieved. For instance, fusion partners that facilitate export of the polypeptide when produced recombinantly, fusion partners that facilitate purification of the polypeptide, and fusion partners which enhance the immunogenicity of the polypeptide of the invention are all interesting. The invention in particular pertains to a fusion polypeptide comprising fusions of two or more immunogenic polypeptides based on polypeptides derived from M. tuberculosis.

Other fusion partners, which could enhance the immunogenicity of the product, are lymphokines such as IFN-γ, IL-2 and IL-12. In order to facilitate expression and/or purification, the fusion partner can e.g. be a bacterial fimbrial protein, e.g. the pilus components pilin and papA; protein A; the ZZ-peptide (ZZ-fusions are marketed by Pharmacia in Sweden); the maltose binding protein; gluthatione S-transferase; β-galac- tosidase; or poly-histidine. Fusion proteins can be produced recombinantly in a host cell, which could be E. coli, and it is a possibility to induce a linker region between the different fusion partners. The linker region between e.g. the individual immunogenic polypeptide units may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

Interesting fusion polypeptides are polypeptides of the invention, which are lipidated so that the immunogenic polypeptide is presented in a suitable manner to the immune system. This effect is e.g. known from vaccines based on the Borrelia burgdorferi OspA polypeptide as described in e.g. WO 96/40718 A or vaccines based on the Pseudomonas aeruginosa Oprl lipoprotein (Cote-Sierra J 1998). Another possibility is N-terminal fusion of a known signal sequence and an N-terminal cystein to the immunogenic polypeptide. Such a fusion results in lipidation of the immunogenic fusion polypeptide at the N-terminal cystein, when produced in a suitable production host.

Vaccine

An important aspect of the invention pertains to a vaccine composition comprising a fusion polypeptide according to the invention. In order to ensure optimum performance of such a vaccine composition it is preferred that it comprises an immunologically and pharmaceutically acceptable carrier, vehicle or adjuvant.

An effective vaccine, wherein a fusion polypeptide of the invention is recognized by the animal, will in an animal model be able to decrease bacterial load in target organs, prolong survival times and/or diminish weight loss or pathology after challenge with a virulent Mycobacterium, compared to non-vaccinated animals

Suitable carriers are selected from the group consisting of a polymer to which the polypeptide(s) is/are bound by hydrophobic non-covalent interaction, such as a plastic, e.g. polystyrene, or a polymer to which the polypeptide(s) is/are covalently bound, such as a polysaccharide, or a polypeptide, e.g. bovine serum albumin, ovalbumin or keyhole limpet haemocyanin. Suitable vehicles are selected from the group consisting of a diluent and a suspending agent. The adjuvant is preferably selected from the group consisting of dimethyldioctadecylammonium bromide (DDA), dimethyldioctadecenylammonium bromide (DODAC), Quil A, poly I:C, aluminium hydroxide, Freund's incomplete adjuvant, IFN-γ, IL- 2, IL-12, monophosphoryl lipid A (MPL), Treholose Dimycolate (TDM), Trehalose Dibehenate and muramyl dipeptide (MDP) or mycobacterial lipid extract, in particular apolar lipid extracts as disclosed in DK 2003 01403.

Preparation of vaccines which contain polypeptides as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251; 4,601,903; 4,599,231 and 4,599,230, all incorporated herein by reference.

Other methods of achieving adjuvant effect for the vaccine include use of agents such as aluminum hydroxide or phosphate (alum), synthetic polymers of sugars (Carbopol), aggregation of the protein in the vaccine by heat treatment, aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed. Other possibilities involve the use of immune modulating substances such as cytokines or synthetic IFN-γ inducers such as poly I:C in combination with the above-mentioned adjuvants.

Another interesting possibility for achieving adjuvant effect is to employ the technique described in Gosselin et al., 1992 (which is hereby incorporated by reference herein). In brief, a relevant antigen such as an antigen of the present invention can be conjugated to an antibody (or antigen binding antibody fragment) against the Fcγ receptors on monocytes/macrophages.

To improve the BCG vaccine, one or more relevant antigen(s) such as one or more fusion polypeptides of the present invention can be mixed with a BCG vaccine before administration and injected together with the BCG vaccine thereby obtaining a synergistic effect leading to a better protection. Another interesting possibility for achieving a synergistic effect is to keep the BCG vaccine and the fusion polypeptide(s) of the present invention separate but use them at the same time and administer them at different sites or through different routes.

To boost the currently used BCG vaccines a relevant antigen such as one or more of the fusion polypeptides of the present invention can be administrated at the time where the BCG vaccines typically start waning or even before, such as 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65 or 70 years after BCG vaccination. It could thereafter be given at regular intervals, such as 1, 2, 3, 4, 5 or 10 years, for up to 5 times.

The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactic or therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of protection desired. Suitable dosage ranges are of the order of several hundred micro- grams of the fusion polypeptide of the invention per vaccination with a preferred range from about 0.1 μg to 1000 μg, such as in the range from about 1 μg to 300 μg, and especially in the range from about 10 μg to 50 μg. Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These include oral, nasal or mucosal application in either a solid form containing the active ingredients (such as a pill, suppository or capsule) or in a physiologically acceptable dispersion, such as a spray, powder or liquid, or parenterally, by injection, for example, subcutaneously, intradermally or intramuscularly or transdermally applied. The dosage of the vaccine will depend on the route of administration and will vary according to the age of the person to be vaccinated and, to a lesser degree, the size of the person to be vaccinated. Currently, most vaccines are administered intramuscularly by needle injection and this is likely to continue as the standard route. However, vaccine formulations which induce mucosal immunity have been developed, typically by oral or nasal delivery. One of the most widely studies delivery systems for induction of mucosal immunity contain cholera toxin (CT) or its B subnit. This protein enhances mucosal immune responses and induce IgA production when administered in vaccine formulations. An advantage is the ease of delivery of oral or nasal vaccines. Modified toxins from other microbial species, which have reduced toxicity but retained immunostimulatory capacity, such as modified heat-labile toxin from Gram- negative bacteria or staphylococcal enterotoxins may also be used to generate a similar effect. These molecules are particularly suited to mucosal administration.

The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and advantageously contain 10-95% of active ingredient, preferably 25-70%.

In many instances, it will be necessary to have multiple administrations of the vaccine. Especially, vaccines can be administered to prevent an infection with virulent mycobacteria and/or to treat established mycobacterial infection or to boost a previous BCG vaccinated person. When administered to prevent an infection, the vaccine is given prophylactically, before definitive clinical signs or symptoms of an infection are present.

Due to genetic variation, different individuals may react with immune responses of varying strength to the same polypeptide. Therefore, the vaccine according to the invention may comprise several different fusion polypeptides and/or polypeptides in order to increase the immune response. The vaccine may comprise two or more fusion polypeptides or polypeptides or immunogenic portions hereof, where all of the fusion polypeptides are as defined above, or some but not all of the polypeptides may be derived from virulent mycobacteria. In the latter example, the polypeptides not necessarily fulfilling the criteria set forth above for fusion polypeptides may either act due to their own immunogenicity or merely act as adjuvants.

The vaccine may comprise 1-20, such as 2-20, or even 3-20 different polypeptides or fusion polypeptides, such as 3-10 different polypeptides or fusion polypeptides.

The invention also pertains to a method for immunising an animal, including a human being, against TB caused by virulent mycobacteria, comprising administering to the animal the fusion polypeptide of the invention, or a vaccine composition of the invention as described above, or a live vaccine described above. In a presently preferred embodiment, the animal or human is an immune individual as defined above.

The invention also pertains to a method for producing an immunogenic composition accor- ding to the invention, the method comprising preparing, synthesising or isolating a fusion polypeptide according to the invention, and solubilizing or dispersing the fusion polypeptide in a medium for a vaccine, and optionally adding other M. tuberculosis antigens and/or a carrier, vehicle and/or adjuvant substance.

The nucleic acid fragments of the invention may be used for effecting in vivo expression of immunogenic polypeptides, i.e. the nucleic acid fragments may be used in so-called DNA vaccines as reviewed in Ulmer et al 1993, which is included by reference.

In the construction and preparation of plasmid DNA encoding a fusion polypeptide to be used defined for DNA vaccination a host strain such as E. coli can be used. Plasmid DNA can then be prepared from overnight cultures of the host strain carrying the plasmid of interest, and purified using e.g. the Qiagen Giga -Plasmid column kit (Qiagen, Santa Clarita, CA, USA) including an endotoxin removal step. It is essential that plasmid DNA used for DNA vaccination is endotoxin free.

Hence, the invention also relates to a vaccine comprising a nucleic acid fragment according to the invention, the vaccine effecting in vivo expression of the immunogenic polypeptide by an animal, including a human being, to whom the vaccine has been administered, the amount of expressed polypeptide being effective to confer substantially increased resis- tance to infections caused by virulent mycobacteria in an animal, including a human being.

The efficacy of such a DNA vaccine can possibly be enhanced by administering the gene encoding the expression product together with a DNA fragment encoding a polypeptide which has the capability of modulating an immune response. One possibility for effectively activating a cellular immune response can be achieved by expressing the relevant immunogenic polypeptide in a non-pathogenic microorganism or virus. Well-known examples of such microorganisms are Mycobacterium bovis BCG, Salmonella and Pseudomona and examples of viruses are Vaccinia Virus and Adenovirus.

Therefore, another important aspect of the present invention is an improvement of the live BCG vaccine presently available, wherein one or more copies of a DNA sequence encoding one or more fusion polypeptides as defined above has been incorporated into the genome of the micro-organism in a manner allowing the micro-organism to express and secrete the fusion polypeptide. The incorporation of more than one copy of a nucleic acid sequence of the invention is contemplated to enhance the immune response.

Another possibility is to integrate the DNA encoding the fusion polypeptide according to the invention in an attenuated virus such as the vaccinia virus or Adenovirus (Rolph et al

1997). The recombinant vaccinia virus is able to enter within the cytoplasma or nucleus of the infected host cell and the fusion polypeptide of interest can therefore induce an immune response, which is envisioned to induce protection against TB.

The invention also relates to the use of a fusion polypeptide or nucleic acid of the invention for use as therapeutic vaccines as have been described in the literature exemplified by D. Lowry (Lowry et al 1999). Antigens with therapeutic properties may be identified based on their ability to diminish the severity of M. tuberculosis infection in experimental animals or prevent reactivation of previous infection, when administered as a vaccine. The composition used for therapeutic vaccines can be prepared as described above for vaccines.

FIGURE LEGENDS

Figure 1. Systemic immune responses as assessed by IFN-gamma ELISA

Groups of Balb/c-C57BL/6 FI mice were subcutaneously vaccinated three times at two- week intervals with TB10.4 in DDA/MPL. One week after the final vaccination, blood was drawn and the immune responses, measured by INF-gamma secretion following stimulation with 0.25, 1.25, or 5 microg/ml TB10.4, were assessed by ELISA.

Figure 2. Immunogenicity of Hyvacc4

Groups of Balb/c-C57BL/6 FI mice were subcutaneously vaccinated three times at two- week intervals with Ag85B-TB10.4 (Hyvacc4) in DDA/MPL. Tree weeks after the final vaccination, spleen cells were analyzed by ELISA for INF-gamma secretion following stimulation with 0.25, 1.25, or 5 microgram/ml Hyvacc4, Ag85B, or TB10.4 (as noted in the figure).

Figure 3. Hyvacc4 induce protection against infection with M. Tuberculosis Groups of Balb/c-C57BL/6 mice were subcutaneously vaccinated three times at two-week intervals with Ag85B-TB10.4 (Hyvac4), Ag85B, TB10.4, or Ag85B+TB10.4 and protective efficacy was assessed by reduction in CFU compared to unimmunized mice from lungs and spleens (data not shown) of mice 6 weeks after aerosol infection. Results are expressed as logio colony forming units (CFU) in the lung and are mean results from 5 mice per experimental group. As a positive control, a single dose of BCG Danish 1331 (5xl0⁴ bacilli/mouse) was injected s.c. at the base of the tail at the same time as the first subunit vaccination; no booster injections were administered.

Figure 4. HyVac4 boosts BCG generated immunity Groups of Balb/c-C57BL/6 FI mice were subcutaneously vaccinated with BCG Danish 1331 (5x104 bacilli/mouse), rested for 8 month, and vaccinated two times, at two-week intervals, with HyVac4 in DDA/MPL ("BCG+HyVac4"). Thereafter, in vitro IFN-gamma responses to Ag85B and TB10.4 (5, 1, 0.2 ug/ml as noted in the figure) of PBMC taken one week after final vaccination was examined and compared to PBMCs taken from mice boosted only with DDA/MPL ("BCG"), or mice boosted with BCG once ("BCG+BCG"). Naive untreated mice served as a negative control group. Values are means and SEMs of groups of 5 mice.

Figure 5. HyVac4 can boost BCG generated immunity. Groups of Balb/c-C57BL/6 FI mice were subcutaneously vaccinated with BCG Danish 1331 (5x104 bacilli/mouse) and rested for 8 month. Thereafter, the mice were vaccinated two times at two-week intervals with HyVac4 in DDA/MPL ("BCG+HyVac4"). Ten weeks after the first vaccination, the mice were challenged by the aerosol route with virulent M. tuberculosis. Six weeks post challenge, the mice were killed and protective efficacy of the HyVac4 booster vaccinations was assessed by reduction in colony forming units (CFU) in lungs or spleens (data not shown) compared with mice mice boosted only with DDA/MPL ("BCG"), or mice boosted with BCG once ("BCG+BCG"). Naive untreated mice served as a negative control group. Results are expressed as loglO CFU in the lung and are mean results from 5 mice per experimental group.

EXAMPLES Materials and methods

Animals

Female specific-pathogen-free C57BL/6xBalb/C FI mice, 8 to 16 weeks of age, obtained from Bomholtegaard, Denmark were used for analysis of immune responses and studies of protection as assessed by CFU analysis. Infection studies were performed in the BSL3 facilities of the Statens Serum Institute. Animals were housed in isolator cages and fed water and sterile food ad libitum. All animals were allowed a 1-week rest period after delivery before the initiation of experiments.

Recombinant Antigen Preparations

Recombinant Ag85B (Rvl886c) was produced as previously described (Olsen, van Pinxteren et al. 2001). Briefly, the His-tagged protein was expressed in Esche chia coli XL- 1 Blue and purified on a Talon column followed by protein anion-exchange chromatography using a HiTrap Q column (Pharmacia, Uppsala, Sweden). The sample was dialyzed against 25 mM HEPES buffer (pH 8.0)-0.15 M NaCI-10% glycerol-0.01% Tween 20 before dilution and storage.

Recombinant TB10.4 (Rv0288) was produced as described previously (Skjot, Oettinger et al. 2000). Briefly, the full-length TB10.4 gene was PCR-amplified from cloned M. tuberculosis genomic DNA and subcloned into the plasmid pMCT6. The recombinant protein was produced in Escherichia coli XL1 blue and purified by metal ion affinity chromatography on a Ni+ column esentially as described previously (Theisen, Vuust et al. 1995) but with phosphate buffers containing 8 M urea, which was removed after the purification.

The Ag85B-Tbl0.4 fusion was cloned in the expression vector pQE60 (Qiagen) using the Ncol and Hindlll sites. The N-terminus (between the Ncol and EcoRV) part of the Ag85b was optimized to give an expression in E. coli. The Ag85B and Tbl0.4 parts are joined directly using a Narl site in the C-terminal part of Ag85B.

The DNA sequence encoding Ag85B-TB10.4 is disclosed in the sequence listing as SEQ ID NO: l.The encoded protein sequence is disclosed in the sequence listing as SEQ ID NO: 2.

The protein was expressed in E.coli strain NF1830 (N Fiil) after induction by IPTG. The recombinant Ag85B-Tbl0.4 protein was collected in inclusion bodies after disruption of the cells by glass beads in a bead beather. The purified inclusion bodies were dissolved in 20mM NaOAc + 8 M urea at pH 4.9 and passed over an Q sepharose column to capture endotoxin. The collected run-through was diluted in Bis-tris buffer + 8 M urea pH 6.5 and the pH was adjusted to pH 6.5. The protein was then passed over a CM sepharose to capture impurities and then captured on a Q sepharose column. The column was washed with bis-tris buffer pH 6.5 + 3 M urea. The Ag85b-Tbl0.4 protein was eluted with NaCI. The protein was then buffer exchanged on af Sephadex column to 25 mM tris-HCI pH 8 + 10 % glycerol.

Vaccine preparation and immunization procedure Mice were immunized with 5 microg recombinant vaccine (either Ag85B, TB10.4 or a fusion protein composed of Ag85B and TB10.4 (designated Hyvac4)) which was delivered subcutaneously at the base of the tail in 25 μg monophosphoryl lipid A (MPL, Corixa, WA, USA) emulsified in dioctadecylammonium bromide (DDA, 250μg/dose, Eastman Kodak, Inc., Rochester, N.Y.) in a total volume of 200 μl, as recently described (Olsen, van Pinxteren et al. 2001). The vaccines (0.2 ml/mice) were injected three times subcutaneously (s.c.) on the back with 2-week interval. A single dose of BCG Danish 1331 (5xl0⁴ bacilli/mouse) was injected s.c. at the base of the tail at the same time as the first subunit vaccination; no booster injections were administered. The prechallenge immunity was evaluated with blood lymphocytes 5 weeks after the first vaccination

Experimental infections and bacterial enumeration in organs

To evaluate the level of protection, mice were challenged 10 weeks after the first immunization either by the aerosol route in a Glas-Col inhalation exposure system, calibrated to deliver approximately 100 CFU of M. tuberculosis Erdman per lung. Mice were sacrificed 6 weeks (aerosol route) later, and lungs and spleens were removed for bacterial enumeration. The organs were homogenized separately in sterile saline, and serial dilutions were plated onto Middlebrook 7H11 agar supplemented with 2 mg of 2-thiophene- carboxylic acid hydrazide per ml to selectively inhibit the growth of residual BCG in the test organs. Colonies were counted after 2 to 3 weeks of incubation at 37°C.

Lymphocyte Cultures

Organs were homogenized by maceration through a fine mesh stainless steel sieve into complete RPMI (GIBCO, Grand Island, NY, including 2 mM glutamine, 100 U/ml each of penicillin 6-potassium and streptomycin sulphate, 10% FCS and 50 mM 2-ME).

Blood lymphocytes were purified on a density gradient lympholyte (Cedarlane, Hornby, Ontario, Canada). Cells were pooled from five mice in each group and cultured in triplicate in round-bottomed microtiter wells (96 well; Nunc, Roskilde, Denmark) containing 2xl0⁵ cells in a volume of 200 microl of RPMI 1640 medium supplemented with 5xl0^"5 M 2- mercaptoethanol, 1 mM glutamine, penicillin-streptomycin 5% (vol/vol) fetal calf serum. The mycobacterial antigens (Hyvacc4, TB10.4, or Ag85) were used in concentrations ranging from 5 to 0.2 mg/ml. Cultures were incubated at 37°C in 10% C02 for 3 days, before the removal of lOOμl of supernatant for gamma interferon (IFN-gamma determination by enzyme-linked immunosorbent assay (ELISA) as described below.

Enzyme-Linked Immunosorbent Assay (ELISA) for IFN-gamma

A double sandwich ELISA method was used to quantify the levels of IFN-gamma in duplicate titrations of culture supernatants, using a commercial kit for IFN- gamma assay, in accordance with the manufacturer's instructions (Mabtech, AB. Sweden). Concentrations of IFN- gamma in the samples were calculated using a standard curve generated from recombinant IFN- gamma (Life Technologies) and results are expressed in pg/ml. The difference between the duplicate wells was consistently less than 10% of the mean.

Example 1

Immunogenicity of the fusion polypeptide (or the components of the fusion polypeptide)

Groups of mice are subcutaneously vaccinated three times at two-week intervals with the fusion polypeptide (or with the single components in the fusion polypeptide, in separate experiments) in adjuvans (e.g. DDA/MPL). One week after the final vaccination, blood cells are analyzed by ELISA for INF-gamma secretion following stimulation with 0.25, 1.25, or 5 microgram/ml fusion protein or single components in the fusion protein. Three weeks after the final vaccination, spleen cells are analyzed by ELISA for INF-gamma secretion following stimulation with 0.25, 1.25, or 5 microgram/ml of the fusion protein or single components in the fusion Protein (Fig. l and 2).

The ability of the fusion polypeptide (or the components in the fusion polypeptide) to induce protection against infection with M. tuberculosis

Groups of mice are subcutaneously vaccinated three times at two-week intervals with the fusion polypeptide (or the single components in the fusion polypeptide) in adjuvans (e.g. DDA/MPL) and protective efficacy are assessed by reduction in colony forming units (CFU) from lungs and spleens when compared to naϊve (non-vaccinated) mice 6 weeks after aerosol infection. As a positive control for protection, a single dose of BCG Danish 1331 (5x104 bacilli/mouse) is injected s.c. at the base of the tail at the same time as the first subunit (or single component) vaccination (Fig. 3).

Conclusion

In this study, the potential of a tuberculosis subunit vaccine based on fusion proteins of the antigens TB10.4 (Rv0288) and antigen 85B (Rvl886) was investigated. When the fusion proteins were administered to mice in the adjuvant combination dimethyl dioctadecylammonium bromide-monophosphoryl lipid A, a strong dose-dependent immune response was induced to both single components but the response was stronger for the fusion proteins. The immune response induced was accompanied by high levels of protective immunity and reached the level of Mycobacterium bovis BCG-induced protection. The fusion polypeptide Ag85B-TB10.4 is strongly immunologically recognized in BCG vaccinated individuals (data not shown), even better than the hitherto best vaccine candidate ESAT6-Ag85B, and is therefore a candidate for a BCG booster vaccine.

Example 2. HyVac4 as a BCG booster vaccine

The aim was to examine whether vaccination with HyVac4 would increase BCG generated immunity and thereby also protection against infection with M. Tuberculosis

The ability of the fusion polypeptide to boost BCG generated immunity

To examine the ability of HyVac4 to boost BCG generated immunity, groups of Balb/c- C57BL/6 FI mice were subcutaneously vaccinated with BCG Danish 1331 (5x104 bacilli/mouse), rested for 8 month, and vaccinated two times, at two-week intervals, with HyVac4 in DDA/MPL. One week after the final vaccination, blod cells were analyzed by ELISA for IFN-gamma secretion following stimulation with 0.2, 1., or 5 microgram/ml Ag85B and TB10.4 and compared to PBMCs taken from mice boosted only with DDA/MPL, or mice boosted with BCG once. Naive untreated (non-vaccinated) mice served as a negative control group. Values are means and SEMs of groups of 5 mice (Fig. 4). The ability of the fusion polypeptide to boost BCG and thereby to increase protection against infection with M. tuberculosis.

Groups of Balb/c-C57BL/6 FI mice were subcutaneously vaccinated with BCG Danish 1331 (5x104 bacilli/mouse) and rested for 8 month. Thereafter, the mice were vaccinated two times at two-week intervals with HyVac4 in DDA/MPL. Ten weeks after the first vaccination, the mice were challenged by the aerosol route with virulent M. tuberculosis. Six weeks post challenge, the mice were killed and protective efficacy of the HyVac4 booster vaccinations was assessed by reduction in colony forming units (CFU) in lungs or spleens (data not shown) compared with mice mice boosted with Ag85B, TB10.4 (both in DDA/MPL) or only with DDA/MPL or mice boosted with BCG once. Naive untreated mice served as a negative control group. Results are expressed as loglO CFU in the lung and are mean results from 5 mice per experimental group (Fig. 5).

Conclusion The data clearly showed that HyVac4 is able to boost BCG generated immunity. Thus stimulation of PBMC's, from HyVac4 boosted mice, with Ag85B or TB10.4 induced a significant increase in secretion of IFN-gamma as compared to non-boosted mice (Fig. 4). This lead to a higher protection against infection with M. tuberculosis (Fig. 5). Importantly, the efficacy of HyVac4 as a BCG booster vaccine was higher than that observed with vaccines based only on either of the components, Ag85B or TB10.4. This demonstrate that that in order to obtain the maximum effect in terms of inducing protection in naϊve animals (please see above), or in BCG vaccinated animals (Fig. 5), Ag85B and TB10.4 must be fused together.

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Claims

1. An immunogenic composition, a vaccine or a pharmaceutical composition comprising a fusion polypeptide comprising antigens from BCG administered to a person together with BCG or to a person who has previously been vaccinated with BCG.

2. An immunogenic composition, a vaccine or a pharmaceutical composition comprising a fusion polypeptide according to claim 1, which comprises an amino acid sequence selected from the amino acid sequences encoding the fusion polypeptides Ag85B-TB10.4;

Ag85B-TB10.4-Ag85A;

Ag85B-TB10.4-ORF2c;

Ag85B-TB10.4-ORF2c-Ag85A;

Ag85B-TB10.4-Rvl036; Ag85A-TB10.4;

Ag85B-TB10.4-Ag85A-TB10.4;

TB10.4-Rv0285-Ag85A;

TB10.4-Rvl036-Ag85A; and

TB10.4-ORF2c-Ag85A, Ag85A-Rv0287

RV0287-TB10.4 or analogues thereof, wherein the immunogenic composition, vaccine or pharmaceutical composition is to be administered to a person together with BCG or to a person who has previously been vaccinated with BCG.

3. An immunogenic composition, a vaccine or a pharmaceutical composition as defined in any of claims 1-2 for prophylactic use, therapeutic use or to be used to boost immunity from prior BCG vaccination.

4. An immunogenic composition, a vaccine or a pharmaceutical composition as defined in any of claims 1-3 which is to be administered intradermally, transdermally, subcutaneously, intramuscularly or mucosally.

5. An immunogenic composition, a vaccine or a pharmaceutical composition comprising a fusion polypeptide which comprises two or more of the following immunogenic polypeptides or an analogue thereof: Ag85A; Ag85B; TB10.4;

ORF2c; Rv0285 Rv0287 and Rvl036c. 5 6. An immunogenic composition, a vaccine or a pharmaceutical composition as claimed in claim 5 where the fusion polypeptide comprises 2 different immunogenic polypeptides or analogues thereof.

10 7. An immunogenic composition, a vaccine or a pharmaceutical composition as claimed in claim 5 where the fusion polypeptide comprises 3 different immunogenic polypeptides or analogues thereof.

8. An immunogenic composition, a vaccine or a pharmaceutical composition as 15 claimed in claim 5 where the fusion polypeptide comprises 4 different immunogenic polypeptides or analogues thereof.

9. An immunogenic composition, a vaccine or a pharmaceutical composition comprising a fusion polypeptide which comprises an amino acid sequence selected from

20 the amino acid sequences encoding the fusion polypeptides Ag85B-TB10.4; Ag85B-TB10.4-Ag85A; Ag85B-TB10.4-ORF2c; Ag85B-TB10.4-ORF2c-Ag85A; 25 Ag85B-TB10.4-Rvl036; Ag85A-TB10.4; Ag85B-TB10.4-Ag85A-TB10.4; TB10.4-Rv0285-Ag85A; TB10.4-Rvl036-Ag85A; and 30 TB10.4-ORF2c-Ag85A, Ag85A-Rv0287 Rv0287-TB10.4 or analogues thereof.

35 10. An immunogenic composition, a vaccine or a pharmaceutical composition as defined in any of claims 4-9 for prophylactic use, therapeutic use or to be used to boost immunity from prior BCG vaccination.

11. An immunogenic composition, a vaccine or a pharmaceutical composition as defined in any of claims 4-10 which is to be administered intradermally, transdermally, subcutaneously, intramuscularly or mucosally.

12. Use of a fusion polypeptide as defined in any of claims 4-9 for the preparation of an immunogenic composition, vaccine or pharmaceutical composition for a prophylatic or therapeutical vaccination against an infection caused by a virulent mycobacterium.

13. Use according to claim 12 wherein the immunogenic composition is to be administered to a person which has previously been vaccinated with BCG.

14. A vaccine or pharmaceutical composition comprising a nucleic acid fragment , which comprises a nucleotide sequence encoding a fusion polypeptide according to any of claims 4-9.

15. A vaccine or pharmaceutical composition according to claim 14 for prophylactic use, therapeutic use or to be used to boost immunity from prior BCG vaccination.

16. A method for immunising an animal, which has previously been immunised against tuberculosis caused by a virulent mycobacterium with BCG, the method comprising administering to the animal the immunogenic composition, vaccine or pharmaceutical composition according to any of claims 1-9 or 14-15.

17. A method for treating an animal, which has previously been immunised with BCG, having active or latent tuberculosis caused by a virulent mycobacterium, the method comprising administering to the animal the immunogenic composition, vaccine or pharmaceutical composition according to any of claims 1-9 or 14-15.