HK1179855B - Tuberculosis vaccines comprising antigens expressed during the latent infection phase - Google Patents

Description

Tuberculosis vaccine comprising antigens expressed during latent infection phase

The application is a divisional application of Chinese patent application with the application date of 2006, 6 and 20, the application number of 200680022323.4, and the name of tuberculosis vaccine containing antigen expressed during latent infection stage.

Technical Field

The present invention discloses starvation induced antigens or novel fusion polypeptides based on immunogenic polypeptides of polypeptides derived from mycobacterium tuberculosis induced during starvation, the use of one or more fusion polypeptides or starvation induced antigens of the invention for the preparation of an immunogenic composition, vaccine or pharmaceutical composition for administration to a human/animal, and such immunogenic composition, vaccine or pharmaceutical composition.

Background

Human tuberculosis caused by mycobacterium tuberculosis (m.tuberculosis) is a serious global health problem, which, according to WHO data, causes about 3 million deaths per year. During the 60 and 70 decades of the twentieth century, new cases of Tuberculosis (TB) development have declined worldwide, but in recent years this trend has changed significantly due in part to the advent of AIDS and the emergence of multidrug-resistant (multidrug) strains of mycobacterium tuberculosis.

The only vaccine currently available for clinical use is BCG (bacille calmette-guerin), the efficacy of which remains somewhat controversial. BCG generally induces high levels of acquired resistance in animal models of TB and confers protection against transmissible forms of tuberculosis such as meningitis (meningitis) and miliary tuberculosis in the human population. BCG, when given to young children, protects against tuberculosis within a few years, but changes in efficacy thereafter. Comparison of different control experiments (controlled trials) revealed that the protective efficacy of BCG in adults varied significantly within the range of efficacy from ineffective to 80% protection. This makes the development of new and improved anti-mycobacterium tuberculosis vaccines a very urgent matter, which has been given very high priority by the World Health Organization (WHO).

There have been many attempts to elucidate protective mycobacterial agents and various researchers have reported increased resistance following experimental vaccination. Mycobacterium tuberculosis possesses and secretes several proteins that are potentially suitable for use in the generation of novel mycobacterium tuberculosis vaccines. The search for candidate molecules has mainly concentrated on proteins released from differentiated bacteria (differentiating bacteria). Although a large number of these proteins have been characterized, only a few of them have been demonstrated to induce protective immune responses as subunit vaccines in animal models, the most significant of which are ESAT-6 and Ag85B (Brandt et al, 2000). However, no examples have been obtained of specific long-term protective immune responses with BCG potential or to enhance the ability of BCG to vaccinate humans (demonstration). BCG augmentation with BCG at best has no effect [ Colditz, 1994 ]. Although there was no significant improvement over BCG alone, some protection was elicited by BCG boost with Ag85a in inbred mouse strains (Brooks et al IAI 2001; WO 0204018). Since BCG requires the differentiation and secretion of proteins to induce a protective immune response, the lack of booster immune effect is mainly due to the sensitivity of environmental mycobacteria or the residual immune response from the initial BCG vaccination. Both cause an acute immune response against BCG and thus a rapid inhibition of growth, as well as clearance of BCG.

The process of mycobacterium tuberculosis infection is mainly through three stages. In the acute phase, the bacteria proliferate in the organ until the immune response is enhanced. The specific sensitized CD4T lymphocytes mediate the regulation of infection, the most important mediating molecule appears to be interferon gamma (IFN- γ). Bacterial load (bacterial load) begins to decrease, creating a latent period when bacterial load is steadily maintained at a low level. During this period, M.tuberculosis goes dormant from active proliferation, essentially turning to a non-replicating state and remaining within granulomas. In some cases, infection tends to be in a reactivation phase, where dormant bacteria resume replication. It has been revealed that the transition of M.tuberculosis from primary infection to latent phase is accompanied by changes in gene expression (Honner zu Bentrup, 2001). Changes in the antigen-specificity of the immune response are also possible, as the bacteria regulate gene expression during the transition from active replication to dormancy. All the properties controlling the immune response to latent infection and the factors causing reactivation are essentially unknown. However, there is some evidence to correlate with a shift in dominant cell (dominant cell) types. Although CD4T cells are necessary and sufficient for acute phase infection control, studies have revealed that CD8T cell responses are more important in the latent phase. In 1998, Cole et al disclosed the complete genomic sequence of M.tuberculosis and predicted the presence of about 4000 open reading frames therein, disclosing nucleotide sequences and putative protein sequences (Cole et al, 1998). Importantly, however, this sequence information cannot be used to predict whether the DNA is translated and expressed as a protein in vivo. It is well known that some genes of mycobacterium tuberculosis are up-regulated under conditions that mimic latency. However, these are a limited subset of the total gene expression during latent infection. In addition, one skilled in the art will readily appreciate that the expression of a gene is not sufficient to make it a good vaccine candidate. The only way to determine whether a protein is recognized by the immune system during latent infection with Mycobacterium tuberculosis is to prepare the given protein and test it according to the appropriate assay described herein. Some proteins are particularly important and have the potential to become late antigens (antigens recognized during latent infection) because after infection they are expressed for a large part for a relatively long time, when the immune system has already started the initial adaptive defense and the environment becomes more hostile to mycobacteria. Culture conditions that mimic hypoxia in vitro, which have previously been shown to be relevant in this regard, have now been used to analyze changes in gene expression. It has been found that under these conditions some antigens, such as the 16kDa antigen α -crystallin (α -crystalin) (Sherman, 2001), Rv2660c and Rv2659c (Betts, 2002) can be induced or significantly up-regulated (our own application). Another environmental stimulus that may be of particular interest is starvation, which is designed to reflect that nutrients are localized within granulomas (sites of latent infection) and that gene expression products are up-regulated under starvation and thus likely to be antigenic targets in the latent phase of infection of particular interest.

Of more than 20000 antigens known to be expressed during the primary infection phase and tested as vaccines, less than half a dozen showed significant potential. Only one antigen has been demonstrated to date with all the potential as a therapeutic vaccine (Lowrie, 1999). However this vaccine only worked when administered as a DNA vaccine and proved controversial, as there were other groups claiming that vaccination using this protocol induces nonspecific protection and even worsens the disease (Turner, 2000). In contrast, as shown in the examples provided, the fusion polypeptides described herein can be incorporated into vaccines using optimally recognized vaccination techniques.

Further, since TB vaccines do not elicit bactericidal immunity but control infection at subclinical (subclinical) levels (thereby leading to the subsequent establishment of latent infection), the present invention describes a multi-phase vaccine combining components with prophylactic and therapeutic activity. Following conventional vaccination, the escape of the primary immune response (evasion) and the subsequent development of latent disease is probably due at least in part to changes in the antigen profile of the invading bacteria. Therefore, vaccination with an antigen associated with latent TB should prevent or reduce the development of latent infection and thus improve long-term immunity when a vaccine incorporating the antigen expressed by the bacteria during the first logarithmic growth phase and the latent pathogenesis is used as a prophylactic vaccine. Since this multi-phase vaccine is also clearly an effective therapeutic vaccine, it can solve the problem that most people in the third world who will receive future TB vaccines may have been latently infected.

Summary of The Invention

The present invention relates to immunogenic compositions, vaccines or pharmaceutical compositions (including booster vaccines and multi-phase vaccines) for the prevention or/and treatment of infections caused by species of the mycobacterium tuberculosis complex (complex) (mycobacterium tuberculosis, mycobacterium bovis, mycobacterium africanum, etc.) comprising a starvation induced antigen or a fusion polypeptide comprising one or more starvation induced mycobacterium tuberculosis antigens, wherein the unit of the fusion polypeptide is a mycobacterium tuberculosis antigen. The invention also relates to fusion polypeptides as such and to nucleic acid sequences encoding the fusion polypeptides. Still further, the present invention relates to the use of short overlapping peptide(s) or long overlapping peptide(s) or non-overlapping peptide(s) prepared synthetically or recombinantly. Still further, the present invention relates to the use of a starvation induced antigen or a fusion polypeptide sequence or a nucleic acid sequence of the present invention for the preparation of said immunogenic, vaccine or pharmaceutical composition, and a vaccine or pharmaceutical composition prepared by the method. Further, the present invention relates to the use of a vaccine comprising a starvation induced antigen or fusion polypeptide sequence or nucleic acid sequence of the present invention for the preparation of said immunogenic composition, vaccine or pharmaceutical composition, which vaccine is administered simultaneously with BCG, which vaccine may also be administered in admixture with BCG or separately at a different location or by a different route. Still further, the present invention relates to the use of a vaccine comprising a starvation induced antigen or fusion polypeptide sequence or nucleic acid sequence as a BCG booster. Further, by including antigens expressed both early and late during the natural infection, the vaccine will elicit a two-step immune response to defend the immune system against the pathogen, regardless of which of the most effective epitopes is at a point in time, including the latency period.

Detailed description of the invention

Disclosed are immunogenic compositions, vaccines or pharmaceutical compositions comprising starvation-induced antigens or fusion polypeptides containing one or more starvation-induced antigens.

The amino acid and nucleic acid sequences of these starvation-induced (more than 6.5-fold up-regulated during starvation or genetically linked to starvation-induced genes) antigens appear in the sequence listing as shown below:

starvation-induced antigens	DNA SEQ ID NO	aa SEQ ID NO
			Rv2655	1	2
Rv2656	3	4
			Rv2657	5	6
Rv2658	7	8
			Rv2659c	9	10
Rv2660c	11	12

Rv2661	13	14
			Rv2662	15	16
Rv2663	17	18
			Rv0188	19	20
Rv3290c	21	22
			Rv3289c	23	24
Rv2034	25	26
			Rv2169c	27	28
Rv0116c	29	30
			Rv2558	31	32
Rv1152	33	34
			Rv3291c	35	36
Rv1284	37	38
			Rv1954c	39	40
Rv3810	41	42
			Rv2517c	43	44
Rv3288c	45	46
			Rv0789c	47	48
Rv1955	49	50
			Rv3735	51	52
Rv3675	53	54
			Rv2270	55	56
Rv2050	57	58
			Rv3287c	59	60
Rv2557	61	62
			Rv0122	63	64
Rv2497c	65	66
			Rv1250	67	68
Rv1552	69	70
			Rv2526	71	72
Rv1809	73	74
			Rv0918	75	76
Rv0516c	77	78
			Rv2745c	79	80
Rv1472	81	82
			Rv1660	83	84
Rv2302	85	86

In the present context, each immunogenic polypeptide based on polypeptides derived from mycobacterium tuberculosis is defined as a "unit" of the fusion polypeptide. The fusion may comprise 2, 3, 4,5, 6, 7, 8, 9 or even 10 different units.

The order of the units in the fusion polypeptide may be in any combination. In sequential terms, fusion polypeptides of all of the above antigens in any combination are within the scope of the invention. The fusion polypeptides of the invention are useful in the preparation of immunogenic compositions, vaccines or pharmaceutical compositions, particularly BCG booster vaccines, described in detail below.

Preferred polypeptides which together with the hunger polypeptide constitute a fusion polypeptide unit have the following Sanger identity number and amino acid sequence:

plain (trivisual) names	Sanger ID
		ESAT6	Rv3875
TB10.4	Rv0288
		Ag85A	Rv3804c
Ag85B	Rv1886c
		ORF2c	Rv3871 (C terminal)
TB13.0	Rv1036
		TB9.56	Rv0285
TB9.8	Rv0287

Preferred fusion polypeptide compounds comprise the following polypeptides having one or more starvation-induced antigens (X) combined in different unit orders: ESAT6-Ag85A-X, ESAT6-Ag85B-X, Ag8A-X, Ag85B-X, TB10-Ag85A-X, TB10-Ag85B-X, wherein X is any one of the starvation induced antigens, and the order of the antigenic units can be any combination, e.g., wherein the order is reversed or X is placed in the middle, etc.

Fusion polypeptides may be constructed based on any other combination of one or more starvation-induced antigens and one or more mycobacterium tuberculosis antigens.

Analogs of the fusion polypeptides having an amino acid sequence with at least 80% sequence identity to any portion of any of the fusion polypeptides of the invention and having immunogenicity, and nucleic acid sequences encoding such polypeptides, are within the scope of the invention. Such analogs are encompassed by the term "polypeptide of the invention" or "fusion polypeptide of the invention", which terms are used interchangeably throughout the specification and claims. According to the term "nucleic acid sequence of the invention", it is intended to mean a nucleic acid sequence encoding such a polypeptide. Still further, short overlapping peptide(s) or long overlapping peptide(s) or non-overlapping peptide(s) having an amino acid sequence with 80% sequence identity to any of the fusion polypeptides of the invention and being immunogenic are also included within the scope of the invention.

A presently preferred embodiment of the present invention is a vaccine that boosts the immunity of a prior BCG vaccination, i.e. the vaccine is administered to previously vaccinated BCG individuals.

A first aspect of the invention comprises a variant of the above-described starvation-induced antigen or fusion polypeptide, which is lipidated (lipidated) so as to provide a self-adjuvanting (self-adjuvating) effect of the polypeptide.

The immunogenic composition, vaccine or pharmaceutical composition of the invention may be administered, for example, via the mucosa, orally, nasally, buccally or by conventional intramuscular, intradermal, by subcutaneous injection or transdermally, or by any other suitable route, for example rectally.

In another embodiment, the invention discloses the use of a starvation induced antigen or fusion polypeptide as defined above in the preparation of an immunogenic composition, a vaccine or a pharmaceutical composition which can be used together with a BCG vaccine for vaccination, booster vaccination or for therapeutic vaccination against infection caused by a virulent mycobacterium such as mycobacterium tuberculosis, mycobacterium africanum, mycobacterium bovis, mycobacterium leprae or mycobacterium ulcerous.

In a second aspect, the invention discloses an immunogenic composition, a vaccine or a pharmaceutical composition comprising a nucleotide sequence encoding a starvation induced antigen or fusion polypeptide as defined above, or comprising a complementary nucleic acid sequence capable of hybridising under stringent conditions to a nucleic acid sequence of the invention.

The nucleic acid fragment is preferably a DNA fragment. The fragments can be used as medicaments as discussed below.

In one embodiment, the invention discloses immunogenic compositions, vaccines or pharmaceutical compositions comprising a nucleic acid fragment of the invention optionally inserted into a vector. Upon administration of a vaccine causing expression of an antigen in an animal including a human to the animal including the human, the amount of the antigen expressed is effective to impart substantially enhanced resistance to tuberculosis caused by a virulent mycobacterium such as mycobacterium tuberculosis, mycobacterium africanum, mycobacterium bovis, mycobacterium leprae or mycobacterium ulcerosa to the animal including the human.

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

In another further embodiment, the present invention discloses immunogenic compositions, vaccines or pharmaceutical compositions which can be used for vaccination with BCG, or as a booster vaccine to a human previously vaccinated with BCG to immunize an animal including a human against tuberculosis caused by a virulent Mycobacterium such as Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium leprae or Mycobacterium ulcerosa, the immunogenic composition, vaccine or pharmaceutical composition comprises a non-pathogenic microorganism such as vaccinia, adenovirus or Mycobacterium bovis BCG as an active ingredient, wherein a DNA fragment comprising at least one copy of a DNA sequence encoding the above-described fusion polypeptide is introduced into the microorganism (e.g., placed in a plasmid or genome) in a manner that allows the microorganism to express and selectively secrete the fusion polypeptide.

In another embodiment, the invention discloses infectious expression vectors, such as vaccinia, adenovirus or Mycobacterium bovis BCG, comprising a nucleic acid fragment of the invention, as well as transformed cells comprising at least one of the vectors.

In a third aspect, the present invention discloses a method of immunizing animals, including humans, or boosting their immunity to tuberculosis caused by virulent mycobacteria, such as mycobacterium tuberculosis, mycobacterium africanum, mycobacterium bovis, mycobacterium leprae or mycobacterium ulcerosa, comprising administering to the animal a fusion polypeptide as defined above, an immunogenic composition of the invention or a vaccine of the invention.

In a fourth aspect, the invention discloses a method for the treatment of an animal (including a human) suffering from active or latent tuberculosis caused by a virulent mycobacterium such as mycobacterium tuberculosis, mycobacterium africanum, mycobacterium bovis, mycobacterium leprae or mycobacterium ulcerosa, which method comprises administering to the animal an immunogenic composition, vaccine or pharmaceutical composition as defined above.

In a fifth aspect, the present invention discloses the use of a starvation induced antigen or fusion polypeptide or nucleic acid fragment as defined above in the preparation of an immunogenic composition, a vaccine or a pharmaceutical composition for prophylactic vaccination (including booster vaccination) or therapeutic vaccination against infection caused by a virulent mycobacterium such as mycobacterium tuberculosis, mycobacterium africanum, mycobacterium bovis, mycobacterium leprae or mycobacterium ulcerosa in combination with mycobacterium bovis BCG.

The vaccines, immunogenic compositions, vaccines and pharmaceutical compositions of the present invention can be used prophylactically in subjects not infected with virulent mycobacteria, or in individuals previously vaccinated with mycobacterium tuberculosis BCG, or therapeutically in subjects infected with virulent mycobacteria.

It will be appreciated that embodiments of the first aspect of the invention such as the immunogenic polypeptides described may also be used in all other aspects of the invention; and vice versa.

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

Definition of

Starvation of

By the term "starvation" it is understood that an organism is deprived of carbon, nitrogen or energy sources, any combination thereof or even all thereof.

Starvation induced proteins

By the term "starvation-induced protein" it is understood any protein that is induced (increased) at least 6.5-fold at the transcriptional or protein level after starvation stress of the mycobacteria. Combination with Mycobacterium bovis BCG

According to the term "combination with mycobacterium bovis BCG", it is understood that any mycobacterium bovis BCG strain comprising Pasteur (Pasteur), phips, frappeer, connaut, tie, danish, glaucon (Glaxo), bragg, Birkhaug, sweden, japan, Moreau and russia strains, administered simultaneously but in different locations or by different routes, in an amount that can elicit a significantly improved specific immune response or effective protection in animal models or humans, is administered in combination with one or more fusion polypeptides as defined above or one or more nucleic acid fragments encoding these fusion polypeptides.

Reinforcement of Mycobacterium bovis BCG

According to the term "boosting of mycobacterium bovis BCG" it is understood that at any time after inoculation with any mycobacterium bovis BCG strain, including Pasteur, Phipps, frapper, Connaught, tie, denmark, Glaxo, bragg, birkhaus, sweden, japan, Moreau and russia, one or more fusion polypeptides as defined above or one or more nucleic acid fragments encoding them are administered in an amount that causes a significantly improved specific immune response or effective protection in animal models or humans.

Polypeptides

The polypeptide used as a unit of the fusion polypeptide of the invention is preferably an immunogenic polypeptide from mycobacterium tuberculosis. Such polypeptides may for example be based on polypeptides derived from mycobacterium tuberculosis cells and/or mycobacterium tuberculosis culture filtrate. The polypeptide is typically a recombinant or synthetic polypeptide, and may consist of an immunogenic polypeptide, an immunogenic portion thereof, or may comprise additional sequences. The additional sequences may be derived from a natural mycobacterium tuberculosis antigen or be heterologous, and such sequences may, but need not, be immunogenic.

By the term "fusion polypeptide" it is understood two or more randomly arranged immunogenic polypeptides from mycobacterium tuberculosis or analogues thereof, with or without one or more amino acid spacers (spacers) of any length and sequence fused thereto.

The word "polypeptide" has its usual meaning in the present invention, i.e. amino acid chains of any length, including full length proteins, oligopeptides, short peptides and fragments thereof and fusion polypeptides, wherein the amino acid residues are linked via covalent peptide bonds.

The polypeptide may be chemically modified, including glycosylation, lipidation (e.g., with palmitoyl oxysuccinimide as described by Mowat et al 1991 or with lauroyl chloride as described by Lustig et al 1976 (ligation)), inclusion of a prosthetic group, or inclusion of an additional amino acid such as a histidine tag or signal peptide.

Each immunogenic polypeptide is characterized by a specific amino acid and is encoded by a specific nucleic acid sequence. Such sequences and analogs and mutants, prepared by recombinant or synthetic means in which such polypeptide sequences have been substituted, inserted, added or deleted for one or more amino acid residues, but are still immunogenic in any of the biological assays described herein, are also within the scope of the present invention.

Substitutions are preferably "conservative", and these conservative substitutions are shown in the table below. Amino acids in the same group in column two, preferably in the same line in column three, may be substituted for each other. The amino acids in the third column are indicated by the single letter codes.

Each polypeptide is encoded by a specific nucleic acid sequence. Analogs and such nucleic acid sequences modified by substitution, insertion, addition or deletion of one or more nucleic acids are within the scope of the invention. In codon usage, the substitutions are preferably silent substitutions, so as not to cause any change in the amino acid sequence, but can be introduced to enhance protein expression.

Nucleic acid fragments

By the terms "nucleic acid fragment" and "nucleic acid sequence" are understood any nucleic acid molecule, including DNA, RNA, LNA (locked nucleic acids), Pentose Nucleic Acids (PNA), RNA, dsRNA and RNA-DNA hybrid strands. 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 used as a pharmaceutical product, for example in DNA therapy, or in a method for preparing a polypeptide according to the invention, it is preferred to use a molecule encoding at least one epitope, which is from about 18 to about 1000 nucleotides in length, which can optionally be inserted into a vector. When the nucleic acid molecule is used as a probe, primer or antisense therapy, it is preferred to use a molecule of 10 to 100 nucleotides in length. Other molecular lengths can also be used according to the invention, for example molecules with 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 molecules with 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 connection with hybridization conditions is as defined in the literature, i.e.hybridization is carried out at a temperature of at most 15-20 ℃ below the melting temperature Tm as described in Sambrook et al, 1989, pages 11.45-11.49. Preferably, the conditions are "high stringency", i.e.5-10 ℃ below the Tm of the melting temperature.

Sequence identity

The term "sequence identity" refers to a quantitative measure of the degree of identity between two substantially equal length amino acid sequences or two substantially equal length nucleic acid sequences. The two sequences compared must be adjusted to have the best potential for an overlap with the inserted gap or truncation at the end of the protein sequence. Sequence identity can be determined by the formula (N)_ref-N_dif)100/N_refIs calculated, whereinN_difIs the total number of non-identical residues in the two sequences after alignment, where N_refIs the number of residues in one of the two sequences. Thus, the DNA sequence AGTCAGTC has 75% sequence identity (N) to the sequence AATCAATC_dif2 and N_ref8). Gaps are counted as a mismatch of specific residue(s), i.e.the DNA sequence AGTGTC will have 75% sequence identity with the DNA sequence AGTCAGTC (N)_dif2 and N_ref8). Sequence identity can additionally be calculated by BLAST programs, such as the BLASTP program (Pearson W.R and D.J. Lipman (1988)) (www.ncbi.nlm.nih.gov/cgi-bin/BLAST). In one embodiment of the invention, the calibration is performed with the sequence alignment method ClustalW, using default parameters as described by Thompson J.et al 1994, which can be obtained via the website http:// www2.ebi. ac. uk/ClustalW/.

Preferably, the 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 number of substitutions, insertions, additions or deletions of one or more amino acid residues in the fusion polypeptide of the invention is limited, i.e. at most 1,2, 3, 4,5, 6, 7, 8, 9, 10 substitutions, at most 1,2, 3, 4,5, 6, 7, 8, 9, 10 insertions, at most 1,2, 3, 4,5, 6, 7, 8, 9, 10 additions and at most 1,2, 3, 4,5, 6, 7, 8, 9, 10 deletions relative to the immunogenic polypeptide unit based on a polypeptide derived from mycobacterium tuberculosis.

Immunogenic moieties

The polypeptides of the invention comprise immunogenic portions such as B cell or T cell epitopes.

The immunogenic portion of an immunogenic polypeptide is the portion of the polypeptide that elicits an immune response in an animal or human and/or a biological sample determined by any of the biological assays described herein. The immunogenic portion of the polypeptide may be a T cell epitope or a B cell epitope. An immunogenic portion may refer to a portion of one or a few relatively small polypeptides, which may be dispersed throughout the polypeptide sequence or located in a specific portion of the polypeptide. For some polypeptide epitopes, it has even been demonstrated to be dispersed throughout the entire sequence of the polypeptide (Ravn et al, 1999).

In order to identify relevant T cell epitopes that are recognized during an immune response, it is possible to use a "brute force" method: because T cell epitopes are linear, if deletion mutants of a polypeptide are constructed systematically, these mutants will show which regions of the polypeptide are critical for immune recognition, e.g., as the subject of IFN detection as described herein. Another approach is to use overlapping oligopeptides for the detection of MHC class II epitopes, preferably synthetic epitopes having a length of e.g.20 amino acid residues from the polypeptide. These peptides can be assayed in biological assays (e.g., the IFN assay described herein), and some of them will give rise to a positive response (and thus immunogenic) that is evidence of the presence of T cell epitopes in the peptide. To find MHC class I epitopes, those peptides that will bind are predicted (Stryhn et al, 1996), and then these peptides are prepared synthetically and tested in relevant biological assays such as the IFN assay described herein. The peptide preferably has a length of, for example, 8 to 11 amino acid residues derived from the polypeptide. B cell epitopes can be determined by analyzing B cell recognition of overlapping peptides covering the polypeptide of interest as described by Harboe et al, 1998.

The immunogenic portion of the polypeptide can be recognized by a large (high frequency) or small (low frequency) portion of the genetic heterotypic hybrid population. In addition, some immunogenic parts induce a high immune response (predominant), while others induce a lower but still effective response (second-most predominant). High frequency > < Low frequency can be associated with widely distributed MHC molecules (HLA type) or even immunogenic portions bound by multiple MHC molecules (Kilgus et al, 1991; Sinigaglia et al, 1998).

Analogues

The fusion polypeptides of the invention have the common feature that they are capable of inducing an immune response, as exemplified in the examples. Of course, analogs of the fusion polypeptides of the invention prepared by substitution, insertion, addition or deletion that are also immunogenic as determined by any of the assays described herein are also within the scope of the invention.

Substantially purified

In the present context, the term "substantially purified polypeptide" refers to a polypeptide preparation comprising up to 5% by weight of other polypeptide material associated with the polypeptide either naturally or in recombinant or synthetic production (lower percentages of other polypeptide material are preferred, e.g. up to 4%, up to 3%, up to 2%, up to 1% and up to 0.5%). Preferably, the substantially purified polypeptide is at least 96% purified, i.e., the polypeptide comprises 96% by weight of the total polypeptide material present in the preparation, and preferably higher percentages, e.g., at least 97%, at least 98%, at least 99%, at least 99.25%, at least 99.5% and at least 99.75%. It is particularly preferred that the polypeptide is in "substantially pure form", i.e. that the polypeptide is substantially free of any other antigen with which it is naturally associated, i.e. free of any other antigen from a bacterium belonging to the tuberculosis complex or virulent mycobacterium. This can be achieved by producing the polypeptide by recombinant means in a non-mycobacterial host cell, as will be described in detail below, or by synthesizing the polypeptide by well-known solid-phase or liquid-phase peptide synthesis methods, such as the method described by Merrifield or derived therefrom, and by using appropriate purification steps well known to those skilled in the art.

Virulent mycobacteria, currently infected individuals and immunized individuals

By the term "virulent mycobacteria" it is understood bacteria capable of causing tuberculosis disease in animals or humans. Examples of toxic mycobacteria are e.g. mycobacterium tuberculosis, mycobacterium africanum, mycobacterium bovis, mycobacterium leprae or mycobacterium ulcerosa. Examples of related animals are cattle, opossum, badgers, buffalo, lions, spanish mackerel (kurus) and kangaroo.

By "animal or human currently infected with virulent mycobacteria" it is understood an individual who has been confirmed by culture or microscopy to be infected with virulent mycobacteria, and/or an individual who has been clinically diagnosed with TB and who has responded to anti-TB chemotherapy. Culture, microscopy and clinical diagnosis of TB are well known to any person skilled in the art.

An immunized individual is defined as a human or animal that has cleared or controlled infection by virulent mycobacteria or has received a mycobacterium bovis BCG vaccination.

Immunogenicity

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

in vitro cellular responses are determined by release of relevant cytokines such as IFN from lymphocytes isolated from animals or humans currently or previously infected with virulent Mycobacteria, or by detection of proliferation of these T cells induction is by addition of polypeptides or immunogenic moieties to cells containing 1x10⁵Cell to 3X10⁵In suspension per well of each cell. Cells are isolated from blood, spleen, liver or lung, and the polypeptide or immunogenic portion of the polypeptide is added to a concentration of no more than 20(g/ml suspension, stimulated for two to five days, for monitoring cell proliferation, the cells are pulsed with radiolabeled thymidine and after incubation for 16-22 hours, proliferation is detected by liquid scintillation counting, a positive response is a response above background plus two standard deviations, the release of IFN- (is determined by ELISA methods well known to those skilled in the art. a positive response is a response above background plus two standard deviations, in monitoring immune responses to the polypeptide, in addition to IFN- (, other cytokines are also suitable, such as IL-12, TNF- (. IL-4, IL-5, IL-10, IL-6, TGF- (. TGF) -, another and more sensitive method for determining the presence of a cytokine (such as IFN- () is the ELISPOT method, wherein cells isolated from blood, spleen, liver or lung are diluted to preferably 1X10⁶Cells/ml to 4X 10⁶Cell/ml concentration, in the presence of a concentration of no more than 20(g/ml of polypeptide or immunogenic portion of polypeptide), incubation for 18-22 hours after cell suspension is diluted to 1X10⁶From/ml to 2X10⁶The IFN- (producing cells) are determined by using spots produced with labeled secondary anti-IFN- (antibodies and related substrates, which spots can be counted using a dissecting microscope.

The in vitro cellular response can also be determined by using T cell lines derived from immunized individuals or M.tuberculosis infected individuals, which have been initiated by live Mycobacteria plus IL-2 extracted from bacterial cells or culture filtrate for 10 to 20 days. By adding no more than 20(g/ml suspension of polypeptide to a suspension containing 1X10⁵Cell to 3X10⁵Induction was achieved with T cell lines per well of cells and incubation was performed for two to six days. Stimulation of T cells can also be monitored by detecting cell proliferation using radiolabeled thymidine as described above.

After intradermal injection or topical application of patches (patch) up to 100 g of polypeptide or immunogenic portion to a clinically or sub-clinically infected individual with virulent mycobacteria, the in vivo cellular response can be determined as a positive DTH response if a positive response of at least 5mm diameter is produced 72-96 hours after injection or application.

The in vitro humoral response can be determined by specific antibody responses in the immunized or infected individual. The presence of antibodies can be determined by ELISA techniques or western blotting, where the polypeptide or immunogenic portion is adsorbed to the surface of nitrocellulose membrane or polystyrene. The serum is preferably diluted with PBS in a ratio of 1: 10 to 1: 100 and added to the absorbed polypeptide and incubated for 1 to 12 hours. The presence or absence of a specific antibody can be determined by using a labeled secondary antibody, e.g., by ELISA, by determining the presence or absence of the specific label, wherein a positive reaction is a reaction greater than background plus two standard deviations, or alternatively, by a visual reaction in a western blot.

Another relevant parameter after vaccination with a polypeptide or DNA vaccine present in an adjuvant is the determination of the protection induced in animal models. Suitable animal models include primates, guinea pigs or mice challenged with an infection with a virulent mycobacterium. The readout for induced protection can be reduced bacterial inoculum size in the target organ relative to the unvaccinated animal, prolonged survival time relative to the unvaccinated animal and reduced weight loss or pathology relative to the unvaccinated animal.

Preparation method

In general, the fusion polypeptides of the invention, and the DNA sequences encoding such fusion polypeptides, can be prepared using any of a variety of methods.

Fusion polypeptides may be recombinantly produced using a DNA sequence encoding the polypeptide, which is inserted into an expression vector and expressed in a suitable host. An example of a host cell is E.coli. Fusion polypeptides having less than about 100 amino acids and typically less than 50 amino acids can also be prepared synthetically by techniques well known to those skilled in the art, such as commercially available solid phase techniques, which add amino acids sequentially to a growing amino acid chain.

Fusion polypeptides can also be prepared by adding fusion partners, by means of which the advantageous properties of the polypeptides of the invention can be achieved. For example, fusion partners that facilitate export (export) of the polypeptide during recombinant production, that facilitate purification of the polypeptide, and that increase the immunogenicity of the polypeptide of the invention are of interest. The invention specifically includes fusion polypeptides comprising two or more immunogenic polypeptides based on polypeptides derived from mycobacterium tuberculosis.

Other fusion partners that increase the immunogenicity of the product are cytokines, such as IFN-. gamma.IL-2 and IL-12. To facilitate expression and/or purification, the fusion partner may, for example, be a bacterial pilin (bacterial protein), such as the pilus components pilin (pilin) and papA; protein A; ZZ-peptide (ZZ-fusion peptide is sold by Pharmacia, Framex, Sweden); a maltose binding protein; glutathione (glutathione) S-transferase; the (-galactosidase or poly-histidine. fusion protein may be produced recombinantly in a host cell, such as E.coli, and it is also possible to form a linker region between the different fusion partners the linker region between, for example, the individual immunogenic polypeptide units may comprise 1,2, 3, 4,5, 6, 7, 8, 9 or 10 amino acids.

The fusion polypeptide of interest is a polypeptide of the invention that is lipidated such that the immunogenic polypeptide is present in the immune system in a suitable manner. This effect is known from vaccines based on the polypeptides of Borrelia burgdorferi OspA or on the lipoproteins of Pseudomonas aeruginosa (Pseudomonas aeruginosa) as described in WO96/40718A (Cote-Sierra J, 1998). Another possibility is to fuse a known signal sequence and an N-terminal cysteine to the N-terminus of the immunogenic polypeptide. When prepared in a suitable production host, such fusion results in lipidation of the immunogenic fusion polypeptide at the N-terminal cysteine.

Vaccine

An important aspect of the present invention relates to a vaccine composition comprising the fusion polypeptide of the present invention. In order to ensure optimal performance of such a vaccine composition, it preferably comprises an immunologically and pharmaceutically acceptable carrier, vehicle or adjuvant.

An effective vaccine comprising a fusion polypeptide of the invention recognized by an animal will result in a reduction of bacterial load in target organs, a prolonged survival time and/or a reduction of weight loss or pathological conditions in animal models challenged with virulent mycobacteria relative to non-vaccinated animals.

Suitable carriers are selected from the group consisting of polymers to which the polypeptide(s) are bound via hydrophobic non-covalent interactions, such as plastics, e.g. polystyrene, or covalently bound to which the polypeptide(s) are bound, such as polysaccharides or polypeptides, e.g. bovine serum albumin, ovalbumin or keyhole limpet hemocyanin. Suitable excipients are selected from the group consisting of diluents and suspending agents. The adjuvant is preferably selected from the group consisting of: dimethyl octacosyl ammonium bromide (DDA), dimethyl dioctadecyl ammonium bromide (DODAC), Quil A, poly I: C, aluminum hydroxide, Freund's incomplete adjuvant, IFN- (, IL-2, IL-12, monophosphoryl lipid A (MPL), Trehalose Dimycolate (TDM), trehalose dibehenate (dibehenate), and Muramyl Dipeptide (MDP), or a mycobacterial lipid extract, especially a non-polar lipid extract as disclosed in PCT/DK 2004/000488.

The preparation of vaccines comprising polypeptides as active ingredients is generally known in the art, as exemplified in US4,608,251, 4,601,903, 4,599,231 and 4,599,230, which are all incorporated herein by reference.

Other methods for obtaining vaccine adjuvant effect include using agents such as aluminium hydroxide or phosphate (alum), synthetic polymers of sugars (Carbopol) by heat treatment to coagulate proteins in vaccines, by coagulation of albumin reactivated by pepsin treated (Fab) antibodies, mixed with bacterial cells such as cryptosporidium parvum (c.parvum) or endotoxins or lipopolysaccharide components of gram negative bacteria, emulsified in physiologically acceptable oil excipients such as mannide monooleate (aracole) or emulsified with 20% perfluorocarbon solution as blocking substitute (block subcostinate) (perfluorodecalin and perfluorotripropylamine mixed emulsion (Fluosol-DA)). Other possibilities include the use of immunomodulatory substances such as cytokines or synthetic IFN- γ inducers such as poly I: C in combination with the adjuvants described above.

Another possibility of interest in achieving adjuvant effect is the use of the technique described in Gosselin et al, 1992 (which is incorporated herein by reference). Briefly, a relevant antigen such as an antigen of the present invention may be conjugated to an antibody (or antigen-binding antibody fragment) against an Fc-receptor on monocytes/macrophages.

To improve the BCG vaccine, one or more relevant antigens such as one or more fusion polypeptides of the invention can be mixed with BCG prior to administration, while simultaneously injecting with BCG to obtain a synergistic effect leading to better protection. Another interesting possibility for achieving a synergistic effect is to keep BCG and the fusion polypeptide(s) of the invention used independently but simultaneously, administering them at different locations or by different routes.

To boost the currently used BCG vaccine, relevant antigens such as one or more fusion polypeptides of the invention may be administered at a time when BCG typically begins to be attenuated or even before it begins to be attenuated, e.g. 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65 or 70 years after BCG vaccination. Thereafter, administration may be at regular intervals, such as 1,2, 3, 4,5 or 10 years, for up to 5 times.

The vaccine is administered in a manner consistent with the dosage formulation, in an amount that will be prophylactically or therapeutically effective and immunogenic, for example. The amount administered will depend on the capacity of the subject being treated, e.g., the individual's immune system to elicit an immune response, and the degree of protection desired. Suitable dosage ranges are of the order of several hundred micrograms of fusion polypeptide of the invention per vaccination, preferably in the range from about 0.1. mu.g to 1000. mu.g, for example in the range from about 1. mu.g to 300. mu.g, in particular in the range from about 10. mu.g to 100. mu.g. The appropriate regimen for initial administration and booster injections may also vary, but is typically followed by vaccination or other administration.

The mode of administration may vary widely. Any conventional method for vaccine administration is suitable. These include oral, nasal or mucosal administration, in solid form containing the active ingredient (e.g. pills, suppositories or capsules), or in the form of a dispersion in a physiologically acceptable medium, e.g. a spray, powder or liquid, or by parenteral injection, e.g. subcutaneous, intradermal or intramuscular or transdermal administration. The dose of the vaccine depends on the route of administration and varies according to the age and, to a lesser extent, the size of the person to be vaccinated. Currently, most vaccines are administered by needle injection by intramuscular injection, which is likely to continue as the standard route of administration. However, vaccine formulations have been developed which induce mucosal immunity, typically by oral or nasal delivery. The most widely studied delivery system for inducing mucosal immunity at present comprises Cholera Toxin (CT) or its B subunit. When this protein is present in a vaccine formulation for administration, it enhances the mucosal immune response and induces IgA production. Oral and nasal vaccines have the advantage of convenient delivery. Modified toxins from other microbial species have reduced toxicity but retain the ability to be immunostimulatory, e.g., modified heat-labile enterotoxins from gram-negative bacteria or staphylococcal-produced toxins may be used to produce similar effects. These molecules are particularly suitable for mucosal administration.

Vaccines are usually administered parenterally by injection, for example by subcutaneous or intramuscular injection. Other formulations suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, conventional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably in the range of 1-2%. Oral formulations include such excipients as commonly used, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin (sodium saccharate), cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release preparations or powders, advantageously containing 10 to 95% of active ingredient, preferably 25 to 70%.

In many cases, multiple administrations of the vaccine are necessary. In particular, these vaccines can be administered to prevent virulent mycobacterial infections and/or to treat established mycobacterial infections or to boost the condition of persons previously vaccinated with BCG. When the vaccine is administered for the prevention of infection, it is administered prophylactically prior to the occurrence of the clinical signs or symptoms of a defined infection.

Due to genetic variation, different individuals may mount immune responses of different strengths to the same polypeptide. Thus, the vaccine of the invention may comprise several different fusion polypeptides and/or polypeptides to enhance the immune response. The vaccine may comprise two or more fusion polypeptides or starvation-induced polypeptides or immunogenic portions thereof, wherein all starvation-induced antigens or fusion polypeptides are as defined above, or some (but not all) of the polypeptides may be derived from a virulent mycobacterium. In the latter example, polypeptides that do not necessarily meet the above criteria for fusion polypeptides may function due to their own immunity 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 relates to a method of immunizing an animal, including a human, against TB caused by virulent mycobacteria, said method comprising administering to the animal a fusion polypeptide of the invention, or a vaccine composition of the invention as described above, or a live vaccine as described above. In a presently preferred embodiment, the animal or human is an immunized individual as defined above.

The invention also relates to a process for the preparation of the immunogenic composition of the invention, said process comprising the preparation, synthesis or isolation of the fusion polypeptide of the invention and the dissolution or dispersion of the fusion polypeptide in a medium for a vaccine, optionally with the addition of other mycobacterium tuberculosis antigens and/or carrier, excipient and/or adjuvant substances.

The nucleic acid fragments of the invention can be used to achieve expression of the immunogenic polypeptide in vivo, i.e.the nucleic acid fragments can be used in so-called DNA vaccines, as reviewed in Ulmer et al, 1993, which is incorporated herein by reference.

In constructing and preparing plasmid DNA encoding fusion polypeptides defined for use in DNA vaccination, a host strain such as E.coli may be used. The host strain carrying the Plasmid of interest is then cultured overnight and purified to produce Plasmid DNA using, for example, the Qiagen Giga-Plasmid column kit (Qiagen, Santa Clarita, Calif., USA) including an endotoxin removal step. Plasmid DNA for DNA vaccination must be endotoxin free.

Thus, the invention also relates to a vaccine comprising a nucleic acid fragment of the invention, which vaccine causes expression of immunogenic polypeptides in animals including humans, in amounts effective to confer substantially enhanced resistance to tuberculosis caused by virulent mycobacteria, to animals including humans, after administration of the vaccine.

The efficacy of such DNA vaccines can be enhanced by the combined use of genes encoding expression products and DNA fragments encoding polypeptides having the ability to modulate immune responses.

One possibility for achieving an immune response for efficient activation of cells may be by expression of the relevant immunogenic polypeptide in a non-pathogenic microorganism or virus. The most notable examples of such microorganisms are mycobacterium bovis BCG, salmonella and pseudomonas, and examples of viruses are vaccinia virus and adenovirus. Thus, another important aspect of the present invention is the boosting of the currently available live BCG vaccines, wherein one or more copies of a DNA sequence encoding one or more fusion polypeptides as defined above are introduced into the genome of the microorganism in a manner that allows the microorganism to express and secrete the fusion polypeptides. The introduction of more than one copy of the nucleic acid sequence of the invention is expected to enhance the immune response.

Another possibility is to incorporate the DNA encoding the fusion polypeptide according to the invention into attenuated viruses such as vaccinia virus or adenovirus (Rolph et al, 1997). Recombinant vaccinia virus can enter the cytoplasm or nucleus of an infected host cell, whereby the fusion polypeptide of interest is expected to induce a protective immune response against TB.

The invention also relates to the use of the fusion polypeptides or nucleic acids of the invention as therapeutic vaccines, as has been exemplified in the literature by d.lowry (Lowry et al, 1999). Antigens with therapeutic properties, when used as vaccines, can be identified by their ability to reduce the severity of a mycobacterium tuberculosis infection in an experimental animal or to prevent reactivation of a previous infection. The composition to be used as a therapeutic vaccine may be prepared for use in a vaccine by the method described above.

Drawings

FIG. 1: antibody responses to Rv2660c from ubida-negative (TB +/HIV-) and HIV-positive (TB +/HIV +) TB patients and from danish healthy controls (control). Cut-off lines (cut-off) were based on ROC curve analysis at a 97% specificity level. The observed sensitivity is shown above the graphical representation of the data;

FIG. 2: immunogenicity of Rv2659c

Groups of Fl (Balb/cxC 57 BL/6) mice were inoculated subcutaneously three times with DDA/MPL containing Rv2659c at two week intervals. One week after the last inoculation, INF- γ secretion from PBMCs stimulated with 5 μ gg/ml Rv2659c was analyzed by ELISA;

FIG. 3: rv2659c induces protection against mycobacterium tuberculosis infection

Groups of Balb/C-C57BL/6 mice were vaccinated subcutaneously three times at two week intervals with Rv2659C, and the protective efficacy was assessed 12 weeks after vaccination by comparing the reduction in CFU counts in the lungs of non-immunized and BCG-immunized mice. Results are expressed as log10 Colony Forming Units (CFU) in the lungs and the average of 6 mice per experimental group;

FIG. 4: immunogenicity of Rv2660c

Fl (Balb/cxC 57 BL/6) mice were inoculated subcutaneously three times with DDA/MPL containing recombinant Rv2660c protein, at two week intervals. (A) One week after the last vaccination, PBMCs were analyzed for IFN- γ secretion after stimulation with Rv2660c at 0.2, 1 or 5 μ gg/ml by ELISA. Three weeks after the final inoculation, splenocytes (B) stimulated with 0.2, 1 or 5 μ gg/ml recombinant Rv2660C were analyzed for INF- γ secretion by ELISA and Peripheral Blood Mononuclear Cells (PBMCs) (C) stimulated with 0.2, 1 or 5 μ gg/ml recombinant Rv2660C were used for proliferative response studies;

FIG. 5: protection against mycobacterium tuberculosis infection induced by Rv2660c

Groups of Balb/C-C57BL/6 mice were vaccinated subcutaneously three times at two week intervals with Rv2660C and protective efficacy was assessed 6 weeks after aerosol infection by CFU counts in the lungs and comparison with non-immunized and BCG-immunized mice. The results are expressed as log in lung₁₀Colony Forming Units (CFU) and average of 6 mice per experimental group. Simultaneously, a single dose of BCG Danish1331 (5X 10)⁴Bacillus/mouse) as the first subunit vaccine was injected subcutaneously (s.c.) into the tail root (the base of the tail) as a positive control, no booster injection was given;

FIG. 6: immunogenicity of Hybrid56, HyVac21 and HyVac28

Groups of Fl (Balb/cxC 57 BL/6) mice were inoculated subcutaneously three times every two weeks with DDA/TDB (Lipovac) containing 5 micrograms of Ag85b-ESAT6-Rv2660c (H56), Ag85a-TB10.4-Rv2660c (H21), or Ag85b-TB10.4-Rv2660c (H28). One week after the last vaccination, PBMCs were analyzed for IFN-. gamma.release by ELISA after stimulation with 1. mu.g/ml of fusion protein Ag85b, TB10.4 or Rv2660C for immunization (FIGS. 6A-C).

Three weeks after the final inoculation with Ag85b-ESAT6-Rv2660c splenocytes (D) were analyzed by ELISA for INF- γ secretion after stimulation with 0.2, 1 or 5 μ gg/ml recombinant Ag85B, ESAT6 or Rv2660c PBMCs (E) were analyzed for proliferative responses against 1 μ g/ml of the same antigen;

FIG. 7: strong protection against Mycobacterium tuberculosis infection after immunization with Hybrid56

(A) Groups of Balb/C-C57BL/6 mice were inoculated subcutaneously three times with Ag85B-ESAT6-Rv2660C (Hybrid56 (Hybrid 56)) at two week intervals and the protective efficacy was assessed by comparing CFU counts in the lungs of non-immunized and BCG-immunized mice after 2, 6, 12 and 24 weeks of aerosol infection. (B) Groups of B6 mice were vaccinated subcutaneously three times with Ag85B-ESAT6 (Hybrid) or Ag85B-ESAT6-Rv2031c (Hybrid 32) at two week intervals, and protective efficacy was assessed by comparing CFU counts in the lungs of non-immunized and BCG-immunized mice at 7, 13, 24, 35 and 44 weeks post aerosol infection. The results are expressed as log in lung₁₀Colony Forming Units (CFU) and average of 6 mice per experimental group. Simultaneously, a single dose of BCG Danish1331 (5X 10)⁴Bacillus/mouse) was injected subcutaneously (s.c.) as a first subunit vaccine into the tail root as a positive control, no booster injection was given;

FIG. 8: Kaplan-Meier survival curve (n = 7) immunization of guinea pigs with Ag85b-ESAT6-Rv2660c fusion protein after aerosol challenge with low doses of mycobacterium tuberculosis extended their survival time to levels of BCG immunized animals;

FIG. 9: immunity and protection induced by Hybrid56 (Ag 85b-ESAT6-Rv2660 c)

Fl (Balb/cxC 57 BL/6) mice were inoculated subcutaneously three times with DDA/MPL containing recombinant Ag85b-ESAT6-Rv2660c (hybrid56) with an interval of two weeks. Ten weeks after the final inoculation, for analysis by ELISA0.2, 1 or 5 μ g/ml of Ag85b, ESAT6 or Rv2660c stimulated IFN- γ secretion by splenocytes (as shown in FIG. 9A). Ten weeks after vaccination, protective efficacy was assessed by a reduction in CFU counts in the lungs of immunized mice relative to adjuvant control. Results are expressed as log of lung in 12 mice per experimental group₁₀Colony Forming Units (CFU) (fig. 9B).

Examples

Materials and methods

Animal(s) production

Female C57BL/6xBalb/C F1 or C57BL/6 mice, 8 to 16 weeks old, from bom holtegaard, denmark, were used for analysis of immune response and study of protection, with protection assessed by CFU analysis. Infection studies were performed in the BSL3 facility of the national Serum Institute (Statens Serum Institute). Animals were housed in isolated cages, fed water and optionally sterile food. All animals were given a 1-week rest period (rest period) before starting the experiment. Recombinant antigen preparation recombinant Ag85B-ESAT6 (Hybrid 1) was prepared according to the previously described method (Olsen, van Pinxteren et al, 2001). Briefly, the His-tagged protein was expressed in E.coli XL-1Blue and subjected to protein anion exchange chromatography using HiTrap Q column (Pharmacia, Uppsala, Sweden) after purification on Talon column. Prior to dilution and storage, the samples were dialyzed against 25mM HEPES buffer (pH 8.0) -0.15M NaCl-10% glycerol-0.01% Tween 20.

Recombinant Rv2660c was prepared by the same method previously described for other small mycobacterial proteins (Skjot, Oettinger et al, 2000). Briefly, the full-length Rv2660c gene was PCR amplified from mycobacterium tuberculosis genomic DNA and the full-length Rv2660c gene was subcloned into the expression plasmid pDestl 7. The recombinant protein was produced in E.coli Bl21blue, purified by metal ion affinity chromatography on a Ni + column essentially as described previously (Theisen, Vuust et al, 1995) but using phosphate buffer containing 8M urea, and removed after purification.

Hybrid56 (Ag 85B-ESAT6-Rv2660 c), Hybrid32 (Ag 85b-ESAT6-Rv2031 c), HyVac21 (Ag 85a-TB10.4-Rv2660 c) and HyVac28 (Ag 85b-TB10.4-Rv2660 c) fusion proteins were cloned into the expression vector pDestl7 (Invitrogen) by site-specific recombination according to the instructions.

After induction with IPTG, the fusion protein was expressed in e.coli strain BL 21. Inclusion bodies of all four fusion proteins were collected after lysis with mild detergent (B-PER, Sigma (Sigma)) and sonication. The washed inclusion bodies were dissolved in 20mM NaOAc +8M urea, pH4.9 and passed through a Q sepharose (sepharose) column to capture endotoxin. The collected flow was diluted in Bis-tris buffer +8M urea pH6.5 and the pH was adjusted to 6.5. The protein is then passed through CM sepharose to capture impurities, after which the protein is collected on a Q sepharose column. The column was washed with bis-tris buffer (pH 6.5) +3M urea. Bound protein was eluted with NaCl. Then passed through a Sephadex column and the buffer was changed to 25mM tris-HCl pH8 and 10% glycerol.

Human cognition-serology

Before use in ELISA, all sera were depleted by cross-reactive antibodies by adding 20. mu.l of E.coli extract (S3761, Promega, Madison, Wis.) to 200. mu.l of serum samples and then incubating with mixing at room temperature for 4 hours. After centrifugation (10.000 Xg, 10 minutes), 0.05% sodium azide was added to the supernatant. The ELISA was performed as follows, 96-well Maxisorp (Nunc, Roskilde, Denmark) microtiter plates were coated with 1.0. mu.g/ml (100. mu.l per well) of antigen-containing carbonate-bicarbonate buffer (pH 9.6) overnight at 4 ℃. The plates were then washed 3 times with PBS containing 0.05% Tween20 (PBS-T). Mixing the raw materials in a ratio of 1: 100 serum samples were diluted with PBS (dilution buffer) containing 0.2% Tween20 and 1.0% (wt/vol) bovine serum albumin, 0.1ml of diluted serum was added to the paired wells in duplicate and incubated at room temperature for one hour. Wash with PBS-TAfter 3x, 100 μ l of a 1: 8000 peroxidase-conjugated rabbit anti-human Ig (P212, DAKO, Glostrup, Denmark) diluted in dilution buffer, incubated for 1 hour. The plates were washed 3 times with PBS-T, incubated with N-tetramethylbiphenyl substrate (TMB plus, Kem-En-Tec, Denmark) for 30 min, by adding 1M H₂SO₄The reaction was terminated. The Optical Density (OD) at 405nm was then determined₄₀₅). Vaccine preparation and immunization procedure

Mice were immunized with 5 micrograms of recombinant vaccine (Rv 2659c, Rv2660c, Hybrid56, HyVac21, HyVac28 or Hybrid 32) delivered as 25 μ g monophosphoryl lipid a (MPL, Corixa, WA, usa) emulsified in dioctadecylammonium bromide (DDA, 250 μ g/dose, Eastman Kodak, inc., Rochester, n.y.) to a total volume of 200 μ l as recently described (Olsen, van Pinxteren et al, 2001). The vaccine (0.2 ml/mouse) was injected subcutaneously (s.c.) into the back three times every two weeks. Simultaneously, a single dose of BCG vaccine Danish1331 (5X 10)⁴Bacillus/mouse) was injected subcutaneously (s.c.) into the tail root as a first subunit vaccine, and no booster injections were given. The pre-challenge immunity was assessed by blood lymphocytes 5 and 7 weeks after the first vaccination and splenocytes 7 weeks after the first vaccination. Experimental infection and bacterial count in organs

To assess the level of protection, mice were challenged 10 weeks after the first immunization or approximately 100CFU of mycobacterium tuberculosis Erdman was delivered to each lung by aerosol means using a calibrated Glas-Col inhalation contact system. Mice were sacrificed after 2, 6, 12 or 24 weeks (Hybrid56) or after 7, 13, 24, 35 or 44 weeks (Hybrid 32) and lungs and spleen were removed for bacterial enumeration. The organs were evenly dispersed in sterile saline and serial dilutions were plated on Middlebrook7H11 agar supplemented with 2mg of 2-thiophene-carboxylic acid hydrazide per ml to selectively inhibit the growth of residual BCG in the tested organs. Colonies were counted after 2 to 3 weeks incubation at 37 ℃.

Lymphocyte culture

Organs were homogenized by segregation through a fine-meshed stainless steel sieve into complete RPMI (GIBCO, Grand Island, NY, containing 2mM glutamine, 100U/ml penicillin 6-potassium and 100U/ml streptomycin sulfate, 10% FCS and 50mM 2-ME). Blood lymphocytes were purified on a density gradient of lympholyte (Cedarlane, Hornby, Ontario, canada). Cells from each group of five mice were pooled and cultured in triplicate in round bottom microtiter wells (96 wells, Nunc, Roskilde, Denmark), each well containing a volume of 200. mu.l of RPMI1640 medium containing 2X10⁵Cells, supplemented with 5x10^-5M2-mercaptoethanol, 1mM glutamine, penicillin-streptomycin 5% (v/v) fetal bovine serum. The concentration of the mycobacterial antigen used ranges from 5 to 0.2 mg/ml. Culture at 37 deg.C with 10% CO₂For 3 days, then 100. mu.l of the supernatant was removed for determination of interferon gamma (IFN-. gamma.by enzyme-linked immunosorbent assay (ELISA) described below).

Enzyme-linked immunosorbent assay (ELISA) for IFN-gamma

A double sandwich ELISA method was used to quantify IFN- γ levels in duplicate titrations of culture supernatants using a commercial kit for IFN- γ determination according to the manufacturer's instructions (Mabtech, ab. The concentration of IFN-. gamma.in the sample was calculated using a standard curve generated from recombinant IFN-. gamma. (Life technologies) and the results are expressed in pg/ml. The difference between duplicate wells was consistently less than 10% of the mean.

Experimental infection and vaccine efficacy assessment in guinea pig model

Inbred female Hartley guinea pigs purchased from Charles River laboratories (North Wilmington, Mass.) or given 10 intradermally³CFU doses of BCG were administered once, or three times with 20 μ g of Ag85b-ESAT6 or Ag85b-ESAT6-Rv2660c emulsified in DDA/MPL, with a 3-week interval between immunizations. Six weeks after the third immunization, a calibrated device (Glas) was usedCol, Terre Haute, Ind.) deliver approximately 20 bacilli into the lungs of each guinea pig given an aerosol MTB challenge. The survival time of infected guinea pigs was determined by observing changes in food consumption, evidence of dyspnea and behavioral changes on a daily basis in the animals. In addition, animals were weighed on a weekly basis until a sustained loss of body weight indicating disease was observed for more than a few days.

Example 1

Human recognition of starvation-induced antigens

Human identification of Rv2660c was assessed in a panel of lung TB patients from udata (Uganda) provided by the WHO Tuberculosis Specimen Bank. Among these are patients with negative and positive HIV infection characteristics (N =94 and N =73, respectively). The control group consisted of one hundred healthy donors living in denmark, with estimated BCG coverage greater than 90%.

Microtiter plates were coated with 1.0. mu.g/ml (100. mu.l per well) of Rv2660c protein and incubated with 100 Xdilution of serum samples, and developed using peroxidase-conjugated rabbit anti-human Ig and tetramethylbiphenyl as substrates (results are shown in FIG. 1).

Conclusion

In this study, the recognition of starvation-induced proteins was tested. Based on the cut-off line (cutoff) determined from the control group with a sensitivity of 97%, it was possible to confirm TB infection in 45% of HIV-cases and 61% of HIV + cases. Experiments clearly show that during MTB infection, RV2660c protein is expressed and recognized by the immune system.

Example 2

Prevention of immunogenicity and reactivation by post-contact (post-exposure) administration of starvation induced antigen (Rv 2659 c)

Mice were infected with mycobacterium tuberculosis and treated with antibiotics to reduce bacterial load (burden) and entered a latent infection stage with bacterial load close to the detection level. Mice were vaccinated three times every two weeks with adjuvant (e.g. DDA/MPL) containing Rv2659c during the latent phase of infection. One week after the last inoculation, blood cells were analyzed by ELISA for INF- γ secretion after stimulation with Rv2659c (fig. 2). The ability of the starvation-induced protein Rv2659c to induce protection against reactivation of mycobacterium tuberculosis

Groups of mice with latent mycobacterium tuberculosis were inoculated subcutaneously three times every two weeks with Rv2659c formulated in adjuvant (e.g., DDA/MPL) and protective efficacy was assessed as a reduction in Colony Forming Units (CFU) in lung and spleen relative to non-inoculated (latently infected) mice. Protection against reactivation was assessed three months after vaccination. Rv2659c induced a 3 to 90 fold reduction in pulmonary bacterial levels relative to reactivated non-immunized latently infected mice (figure 3). To evaluate the effect of Rv2659c vaccination on the pathology likely to occur in latently infected mice, lung tissue was removed from latently infected vaccinated mice for histopathological examination. No significant cheesy necrosis, fibrosis or mineralization was detected at the lesion, nor increased infiltration of inflammatory cells was found.

Conclusion

In this study, the potential of the starvation induced protein Rv2659c as a therapeutic vaccine was tested. When Rv2659c protein-containing adjuvant combination dimethyldioctadecylammonium bromide-monophosphoryl lipid a was administered to mice, a strong immune response was induced/boosted. Immunization resulted in a 0.5-1.0log reduction in bacterial load in the lungs. Thus, our studies indicate that post-contact vaccination reduces or delays reactivation of mycobacterium tuberculosis without eliciting immunopathological symptoms in the lung.

Example 3

Immunogenicity and protection against Aerosol Mycobacterium tuberculosis infection induced by starvation-induced antigen Rv2660c

Mice were vaccinated three times every two weeks with adjuvant (e.g. DDA/MPL) containing Rv2660 c. One week after the last inoculation, INF- γ secretion after stimulation of blood cells with Rv2660c was analyzed by ELISA (fig. 4A). Three weeks after the final inoculation, splenocytes were used to determine IFN- γ secretion after stimulation by Rv2660C (fig. 4B), and blood cells were used to determine antigen-specific proliferative responses (fig. 4C).

Groups of mice vaccinated three times subcutaneously every two weeks with Rv2659c formulated in adjuvant (e.g. DDA/MPL) were challenged with aerosol infection with mycobacterium tuberculosis, and their protective efficacy was assessed by the reduction in Colony Forming Units (CFU) isolated from the lungs relative to uninoculated mice. Protection was assessed 12 weeks after vaccination. Rv2660c induced an approximately 0.5log (10) reduction in pulmonary bacterial levels relative to non-immunized infected mice (figure 5).

Conclusion

In this study, the potential of the starvation induced protein Rv2660c as a vaccine antigen was tested. When an adjuvant combination of dimethyldioctadecylammonium bromide-monophosphoryl lipid a containing Rv2660c protein was administered to mice, it induced a strong immune response. Immunization resulted in an approximately 0.5log (10) reduction in bacterial load in the lungs.

Example 4

Starvation-induced antigen fusion to form a prophylactic vaccine (multi-phase vaccine)

Immune response following immunization with three fusion proteins

Groups of mice were inoculated subcutaneously twice every two weeks with adjuvant (e.g., DDA/MPL) containing the fusion polypeptide Hybrid56, HyVac21, or HyVac 28. One week after the final inoculation, blood cells were analyzed for IFN-. gamma.secretion following stimulation with 1. mu.g/ml of the fusion protein or single components of the fusion protein (FIGS. 6A-C). Three weeks after the last vaccination with Hybrid56, splenocytes were assayed for IFN- γ secretion by ELISA after stimulation with 0.2, 1 or 5 μ g/ml of the single component of the fusion protein (see fig. 6D). Antigen-specific proliferative responses in blood cells were measured three weeks after the last inoculation (fig. 6E).

Ability of three fusion polypeptides to induce anti-mycobacterium tuberculosis infection in mice

Groups of mice were inoculated subcutaneously three times every two weeks with Hybrid, Hybrid56 and Hybrid32 formulated in adjuvant (e.g., DDA/MPL), and protective efficacy was assessed by reduction of Colony Forming Units (CFU) in the lungs and spleen relative to naive (uninoculated) mice following aerosol infection. Simultaneously, a single dose of BCG Danish1331 (5X 10)⁴Bacillus/mouse) was injected subcutaneously (s.c.) into the tail root as a first subunit vaccine as a positive control for protection studies (fig. 7A and 7B). Protection of polypeptide Hybrid56 (Ag 85b-ESAT6-Rv2660 c) in guinea pig against Aerosol Mycobacterium tuberculosis

Groups of guinea pigs were inoculated subcutaneously three times every two weeks with an adjuvant (e.g., DDA/MPL) containing the fusion polypeptide, and each animal was weighed on a weekly basis to assess primary protective efficacy. Simultaneously, a single dose of BCG Danish1331 (5X 10)⁴Bacillus/mouse) was injected intradermally (i.d.) as a first subunit vaccine as a positive control for the protection study. The results are the survival curves shown in figure 8.

Conclusion

In this study, the immunological potential of three fusion proteins (Hybrid56, HyVac21 and HyVac 28) was studied. When the fusion protein-containing adjuvant combination dimethyl dioctadecyl ammonium bromide-monophosphoryl lipid a was administered to mice, all three single protein components induced a strong dose-dependent immune response, indicating their potential as a multi-phase vaccine. The immune response induced by the selected Hybrid56 was accompanied by a high level of protective immunity that increased over time to a level significantly above that achieved by vaccination with M.bovis BCG, the standard MTB vaccine. Furthermore, a similar three-unit (triple) fusion protein containing the standard MTB potential antigen Rv2031c (Ag 85b-ESAT6-Rv2031 c) instead of Rv2660c showed no improved protection over time. Finally, high levels of protection against Hybrid56 were enhanced in a more susceptible (succineptibel) guinea pig model.

Example 5

Activity of fused starvation-induced antigens and preventive vaccines (multi-phase vaccines) administered (therapeutically) after contact

Mice are infected with mycobacterium tuberculosis and treated with antibiotics to reduce bacterial load and enter a latent infection phase of low bacterial load. Mice are vaccinated three times every two weeks with an adjuvant (e.g., DDA/MPL) containing the fusion polypeptide during the latent phase of infection. Fifteen weeks after the final inoculation, IFN-. gamma.secretion was measured by ELISA on blood cells stimulated with 0.2, 1 or 5. mu.g/ml of the single component of the fusion protein (FIG. 9A). Protective capacity of fusion polypeptide to induce reactivation of mycobacterium tuberculosis

Groups of mice with latent Mycobacterium tuberculosis are inoculated subcutaneously three times every two weeks with the fusion polypeptide prepared in adjuvant (e.g., DDA/MPL) and protective efficacy is assessed by the reduction of Colony Forming Units (CFU) in the lungs relative to non-inoculated (latently infected) mice. Protection against reactivation was assessed three months after vaccination. The fusion polypeptide induced significantly reduced reactivation, resulting in a reduction in pulmonary bacterial levels relative to reactivated non-immunized latently infected mice (fig. 9B).

Conclusion

In this study, the potential of a tuberculosis subunit vaccine based on a fusion protein of the antigens Rv2660c, ESAT6 (Rv 3875) and antigen 85B (Rv 1886 c) as a therapeutic vaccine was investigated. When the adjuvant combination dimethyl dioctadecyl ammonium bromide-monophosphoryl lipid a containing the fusion protein was administered to mice, a strong immune response was induced/boosted. During the reactivated latent infection phase, the immune effect results in a reduction of the bacterial load in the lungs. Our studies thus show that vaccination with fused starvation-induced antigens following exposure reduces or delays reactivation of mycobacterium tuberculosis (multi-phase vaccines).

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Claims (9)

1. An immunogenic composition, vaccine or pharmaceutical composition comprising: a B-cell or T-cell epitope of polypeptide Rv1284 or polypeptide Rv 1284.

2. An immunogenic composition, a vaccine or a pharmaceutical composition according to claim 1 for prophylactic use, therapeutic use, a multi-stage vaccine, or for boosting immunity from prior bcg vaccination.

3. An immunogenic composition, a vaccine or a pharmaceutical composition according to any one of claims 1-2, which is administered intradermally, transdermally, subcutaneously, intramuscularly or mucosally.

4. Use of an immunogenic composition, a vaccine or a pharmaceutical composition according to any one of claims 1 to 3 in the manufacture of a medicament for prophylactic use, therapeutic use, or both, a multi-phase vaccine, or a medicament for boosting immunity from prior BCG vaccination.

5. Use of an immunogenic composition, a vaccine or a pharmaceutical composition according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment of active tuberculosis or latent tuberculosis caused by virulent mycobacteria or for the manufacture of a medicament for enhancing immunity from prior BCG vaccination.

6. Use of an immunogenic composition, a vaccine or a pharmaceutical composition according to any one of claims 1 to 3 in the manufacture of a medicament for the prevention of infection by a virulent mycobacterium.

7. Use according to claim 5 or 6, wherein the mycobacteria are selected from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium leprae or Mycobacterium ulcerosa.

8. Use of an immunogenic composition, a vaccine or a pharmaceutical composition according to any one of claims 1 to 3 in the preparation of a composition for prophylactic, booster, multi-phase or therapeutic vaccination against mycobacteria.

9. Use according to claim 8, wherein the mycobacterium is selected from mycobacterium tuberculosis, mycobacterium africanum, mycobacterium bovis, mycobacterium leprae or mycobacterium ulcerosa.