The present invention relates to vaccines, in particular, to an attenuated gram-negative cell comprising the SPI2 gene locus, wherein at least one gene of the SPI2 locus is inactivated, wherein said inactivation results in an attenuation/reduction of virulence compared to the wild type of said cell, and to a carrier for the presentation of an antigen to a host, which carrier is said attenuated gram-negative cell, wherein said cell comprises at least one heterologous nucleic acid molecule comprising a nucleic acid sequence coding for said antigen, wherein said cell is capable of expressing said nucleic acid molecule or capable of causing the expression of said nucleic acid molecule in a target cell.

Description of the Invention

BACKGROUND OF THE INVENTION

In 1996, over 17 million people world-wide, mainly in developing countries, were killed by various infections. The appearance and spread of antibiotic resistances coupled with the increase in world-wide travel has led to an increasing risk for the outbreak of pandemic infections. This possibility must be taken very seriously since, for some pathogenic bacteria, the therapeutic alternatives available have been reduced to a single option. Intriguingly, pathogenic bacteria have also been discovered to be a relevant factor in many chronic diseases. Stomach cancer, for example, is the second most common cancer world-wide and is directly linked with chronic Helicobacter pylori infections. Chlamydia pneumoniae has been detected in arteriosclerotic plaques and recently this bacterium has been found in the diseased regions of the brain of people suffering from Alzheimer's disease. Many autoimmune diseases, such as rheumatoid arthritis, seem to have bacterial origin. Borrelia burgdorferi is, in addition to many other bacteria, a prominent example of an organism causing disease affecting increasing numbers of people. Finally, Nanobacteria have been identified in the chronically diseased kidneys of patients with crystalline deposits. Other serious chronic diseases are caused by viral pathogens, the most clinically relevant are Hepatitis B and C viruses (liver cancer) and the human papilloma virus (cervical cancer).

The increasing clinical importance of bacterial pathogens has provoked increased discussion regarding the paradigm of medicinal treatment or prevention as the means to handle chronic diseases, Consistently, some chronic diseases have been successfully cured by antibiotic treatment. However, as indicated above, all micro-organisms are genetically capable of rapidly generating progenies with adequate antibiotic resistances, thus impeding efficient routine treatment. Conclusively, vaccines represent an excellent alternative to pharmacological drugs, and, considering the financial aspect that disease prevention is less cost-intensive than therapy, the option of vaccination is even more attractive. Therefore, the therapeutic vaccination approach has become particularly relevant, especially with respect to the treatment of cancer and chronic bacterial or viral diseases.

The most frequently practised approach uses oral delivery of either inactivated pathogens (dead vaccine) or parenteral injections of a defined mixture of purified components (subunit vaccines). Most of the dead vaccines are efficacious, however, the risk that the inactivation procedure was incomplete and that the vaccinee may become infected remains a problem. Furthermore, dead vaccines very often do not cover all genetic variants that appear in nature. The subunit vaccines abolish most of the disadvantages of the traditional dead vaccines. However, they require technologically advanced antigen and adjuvant preparations, which makes such vaccines relatively expensive. Furthermore, the subunit vaccines are preferentially inoculated by the parenteral route, which is not the optimal route for eliciting a broad immune response. In particular, the mucosal branch of the immune system, which is the primary line of protection against many pathogens, is strongly neglected by parenteral immunisations.

Another generation of vaccines is represented by live attenuated vaccines, which are based on pathogenic bacteria or viruses that have been mutated to apathogenic variants. These variants multiply in vivo for a limited period of time before they are completely cleared by the host. Their limited prevalence in the host tissue is sufficient to adequately provoke the host immune system, which is then able to establish a protective immune response. From the safety aspect, live attenuated bacterial vaccines are more favoured than live attenuated viral vaccines. Should a live bacterial vaccine becomes threating for a vaccinee, the attenuated bacteria can generally be controlled by antibiotic treatment. In contrast, live viral vaccines, which use the replication apparatus of the host cell, are almost impossible to control. Live bacterial vaccines are typically administered orally and serve as excellent stimulators of the mucosal immune system. Moreover, live bacterial vaccines are also good stimulators of the systemically active immune system, namely the humoral and cellular branches. Due to these excellent immuno-stimulatory characteristics, live bacterial vaccine strains, such as Salmonella, are ideal carriers for expressing antigens from a heterologous pathogen. Such bivalent (or multivalent) vaccines mediate protection against two pathogens: the pathogen homologous to the carrier as well as the pathogen whose protective antigen(s) are expressed by the carrier. Although no bivalent bacterial vaccine expressing heterologous antigens is currently in use, potential carriers currently under investigation include Bacille Calmette-Guerin (BCG), Salmonella species, Vibrio cholerae and Escherichia coli.

In the attenuation process, mutations are preferentially targeted to genes that support the survival of the pathogen in the host. Initially, chemical mutation regimes were applied to the Salmonella typhi strain Ty2, resulting in what were thought to be perfectly attenuated pathogens capable of mediating protective immunity, in contrast to the dead homologue. However, subsequent large-scale clinical trails revealed that such strains were still not sufficiently efficacious in the prevention of typhoid fever. It appears that such strains were mutated in several genes, resulting in an over-attenuation, which adversely affects the immunogenic potential of the strain. Novel typhoid vaccine strains have been developed by the introduction of genetically defined mutations. Most of these mutations have been established in S. typhimurium. Infection with S. typhimurium causes typhoid fever-like symptoms in mice and murine salmonellosis is a well accepted model for human typhoid. Such vaccine strains contain mutations in proteins causing deficiencies in the biosynthesis of aromatic amino acids (e.g. aroA, aroC and aroD) or purines (e.g. purA and pure), in the adenylate cyclase gene (cya) or the cAMP receptor protein (crp), or possess mutations affecting the regulation of several virulence factors (phoP and phoQ). However, although a number of attenuated mutants have been generated and characterised in the mouse model with regard to their role in virulence, relatively few of them have been evaluated as vaccine carriers in humans. The reason for this is that the mutants used are either still too virulent, causing severe side effects in the host, or are not sufficiently immunogenic, due to inadequate presentation to the immune system, which requires a critical level of persistence of the vaccine strain in the host for activation.

A recent study revealed that the inactivation of individual Salmonella genes causing attenuation of virulence directly influences the quality of an immune response against the vaccine carrier strain. From this finding, one can conclude that it might be possible to generate a variety of differently attenuated Salmonella vaccine strains, each with a unique profile and individual capabilities for eliciting an immune response. With this repertoire, it might be possible to tailor a vaccine strain according to specific immunological demands. As a logical consequence, one should also be able to develop attenuated Salmonella vaccine strains for either prophylactic or therapeutic purposes. However, the means by which such a representative repertoire of Salmonella vaccine strains is obtained and further developed into an efficacious vaccine must be determined.

In cases in which a Salmonella vaccine strain is used as a carrier for heterologous antigens, additional parameters must be considered. Traditionally, heterologous antigens have been expressed in the Salmonella cytosol. In the mouse typhoid model, it was demonstrated that, when heterologous antigens are expressed at high levels in the Salmonella cytosol, inclusion bodies are often formed, which negatively influence the immunogenicity of the recombinant live vaccine strain in the vaccinated host. It was concluded that the formation of inclusion bodies might be fatal for the bacterium, further decreasing vitality and increasing attenuation, and thus lowering the immunogenicity. Indeed, specific expression systems that circumvent this secondary attenuation principle, e.g. the 2-phase regulated expression system, can improve the efficacy of the presentation of heterologous antigens to the host immune system.

It has been demonstrated that secretion of antigens by live attenuated Salmonella can be superior to intracellular expression of the same antigens both in eliciting protective T-cell responses (Hess et al., 1996; Hess et al., 1997b) and in eliciting elevated levels of antigen-specific antibody (Gentschev et al., 1998). Efficiencies of HlyA-directed secretion systems, however, are usually low (30% or less of total synthesized antigen) (Hess et al. 1997a; Hess et al., 1996), and the system seems to be problematical in S. typhi for export of heterologous antigens (Orr et al., 1999).

A similar immunological profile is induced by the two type III secretion systems, which are encoded by the Salmonella Pathogenicity Islands 1 and 2. These complex secretion machineries naturally deliver "effector proteins" into the cytosol of the infected host cell, supporting the survival of the pathogen within the host cell. By means of gene technology, the "effector proteins" can be converted into carrier vehicles for epitopes from heterologous antigens. Such chimeric "effector proteins" lose their virulent character but retain their secretory character. Consequently, the chimeric "effector protein" is delivered into the lumen of the host cell, where it is appropriately processed and subsequently stimulates the cytotoxic branch of the host immune system.

The most abundant protein secreted by Salmonella is flagellin (see, for example (Hueck et al., 1995)). In S. typhimurium, flagellin occurs in two allelic variants, FliC or FljB, while S. typhi carries only the FliC gene. Flagellin is secreted via the flagellum-specific export (FEX) pathway (Macnab, 1996, Minamino and Macnab, 1999), which is homologous to the type III secretion pathway (Hueck, 1998). It also has been shown recently that the FEX pathway functions in secretion of non-flagella proteins in Yersinia enterocolitica (Young et al., 1999). Like in type III secretion, the amino terminus of FliC directs secretion. Thus, a truncated version of 183 amino terminal amino acids of FliC (full length is 495 aa) is constitutively secreted in large amounts (Kuwajima et al., 1989). In analogy to type III secretion, the effective secretion signal in FliC may be as short as 10 to 20 amino acids. The FliC or FliB secretion signals can potentially be used to secrete large quantities of a heterologous protein which can serve as an antigen in heterologous vaccination. It is likely that the amount of secreted antigen can be even further increased in regulatory mutants affecting the expression of flagella biosynthesis genes (Macnab, 1996; Schmitt et al., 1996) or by using recombinant promoters to drive expression of the flagellin gene.

Secretion via the FEX pathway can allow the delivery of large amounts of antigen into the Salmonella-containing phagosome for early and efficient antigen processing and antigen presentation to the host immune system. Especially the MHC class II dependent branch of the host immune system is strongly supported by the FEX pathway mediated antigen delivery.

The other known export machineries and surface display systems of Gram-negative bacteria can be also applied to bacterial vaccine carriers such as Salmonella. In general, a good immune response is achieved when the antigen is presented on the Salmonella surface. However, as little is known about the immunological consequence of such antigen presentation systems, further experimental work is needed.

Additional immuno-modulatory effects can be achieved when environmentally regulated Salmonella promoters are used for the expression of heterologous antigens. For instance, the expression of a heterologous gene in a Salmonella carrier strain under control of the in vivo regulated stress response htrA gene promoter resulted in a stronger immune response than was obtained when under control of the anaerobically inducible promoter of the nirB gene.

According to a first aspect, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence comprising at least 50 nucleotides a) of the nucleic acid sequence of one of FIGS. 21A, B, b (see Original Patent)) of an allele of the nucleic acid sequence of one of FIGS. 21A, B or c) of a nucleic acid sequence which under stringent conditions hybridizes with the nucleic acid sequence of one of FIGS. 21A, B (see Original Patent).

Stringent hybridization conditions in the sense of the present invention are defined as those described by Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), 1.101-1.104. According to this, hybridization under stringent conditions means that a positive hybridization signal is still observed after washing for 1 hour with 1.times.SSC buffer and 0.1% SDS at 55.degree. C., preferably at 62.degree. C. and most preferably at 68.degree. C., in particular, for 1 hour in 0.2.times.SSC buffer and 0.1% SDS at 55.degree. C., preferably at 62.degree. C. and most preferably at 68.degree. C.

In particular, the present invention relates to such a nucleic acid molecule which comprises the complete coding regions or parts thereof of the genes ssaD, ssaE, sseA, sseB, sscA, sseC, sseD, sseE, sscB, sseF, sseG, ssaG, ssaH, ssae, ssaJ, ssrA and ssrB. The invention pertains also to such nucleic acids, wherein at least one coding region of said genes is functionally deleted.

In one embodiment, the nucleic acid molecule comprises an insertion cassette to facilitate the insertion of a heterologous nucleic acid molecule by transposon or phage mediated mechanism.

Furthermore, said nucleic acid molecules can comprise at least one heterologous nucleic acid molecule. In this case the heterologous nucleic acid molecule may be fused 5' or 3', inserted or deletion-inserted to the inventive nucleic acid molecule. By the term "deletion-inserted" it is understood that the insertion of the heterologous nucleic acid molecule is associated with a concurrent deletion of parts of the inventive nucleic acid molecule. Preferably, the nucleic acid molecule is inserted or deletion-inserted and in one preferred embodiment the heterologous nucleic acid molecule is flanked 5' and 3' by sequences of the nucleic acid molecule according to the invention, wherein each of said sequences has a length of at least 50 nucleotides, preferably 200-250 nucleotides.

Preferred, the heterologous nucleic acid molecule codes for a polypeptide or peptide, more preferred it codes for a bacterial or viral antigen or a homologue thereof or for a tumor antigen.

It is preferred that the nucleic acid molecule also comprises at least one gene expression cassette to allow for efficient expression of the heterologous nucleic acid molecule. Such gene expression cassette usually comprises elements such as promoters and/or enhancers which improve the expression of the heterologous nucleic molecule acids. Usually, such gene expression cassette comprises elements for the termination of transcription. The presence of transcription terminators, however, may be not preferred in cases where the heterologous nucleic acid molecule is to be transcribed together with other genes into a cistronic mRNA.

The nucleic acid molecule, one or more selective marker cassettes and one or more transactivator cassettes and optionally invertase cassettes for allowing the expression of the heterologous nucleic acid molecules in a one-phase system or a two-phase system. Furthermore, sequences may be present which code for a polypeptide or peptide-targeting domain and, thus, allow for the targeting of the expression product of the heterologous nucleic acid molecule to a predetermined cell compartment such as cytosol, periplasma or outer membrane, or the secretion of said expression product, or which code for an immunostimulatory domain.

According to another aspect, the invention relates to a recombinant vector which comprises the nucleic acid molecule described above. Another aspect of the invention pertains to a cell comprising a modified inventive nucleic acid molecule as described above by insertion of a heterologous sequence or the recombinant vector. The cell may be a prokaryotic cell such as a gram-negative cell, e.g. a Salmonella cell, or it can be a eukaryotic cell such as a mammalian cell, e.g. a human cell, and, in particular, a macrophage.

According to a still further aspect, the present invention relates to a peptide or polypeptide comprising a peptide sequence comprising at least 20 amino acids a) of the sequence of one of FIGS. 23A-Q, or b (see Original Patent)) of a sequence which is 60%, preferred 65% and more preferred 70% homologous to the sequence of one of FIGS. 23A-Q. In particular, the invention relates to a polypeptide comprising the sequence a) of one of FIGS. 23A-Q, or b) which is 60%, preferred 65% and more preferred 70% homologous to the sequence of one of FIGS. 23A-Q.

Percent (%) homology are determined according to the following equation -- see Original Patent.

Another aspect of the present invention relates to an antibody which is directed against an epitope which is comprised of the aforementioned peptide or polypeptide. The antibody may be polyclonal or monoclonal. Methods for producing such an antibody are known to the person skilled in the art.

A further aspect of the present invention relates to a fusion protein comprising the polypeptide according to any one of the claims 17 and 18 having inserted or deletion-inserted or being fused C- or NH.sub.2-terminally with at least one heterologous polypeptide. The heterologous polypeptide preferred is an antigen, more preferred a bacterial or viral antigen or a tumor antigen.

The present invention furthermore provides instructions for the development of a variety of potential live Salmonella vaccine strains with different attenuation levels, which subsequently serve as platforms for the development of recombinant live Salmonella vaccine carrier strains that express antigens from heterologous pathogens, thus serving as multivalent vaccines. Such recombinant live Salmonella vaccine carriers are equipped with modules comprising variable gene cassettes that regulate the expression of heterologous antigens in Salmonella and determine presentation of the heterologous antigens to the host immune system. By combinations of both systems, differently attenuated live Salmonella vaccine strains and variable gene cassettes, a variety of recombinant live vaccine carrier strains can be generated that have, due to their variable immunogenic characteristics, a broad application spectrum for both prophylactic and therapeutic use. The basic attenuation principle originates from novel mutations in the Salmonella Pathogenicity island 2 (SPI2) gene locus. Additional mutations, which can be used either alone or in combination with mutations in sse or SPI-2 genes or in combination with the aroA mutation for optimal attenuation of live vaccine carrier strains, have been reported recently (Heithoff et al., 1999; Valentine et al., 1998). By combination of the individual mutations in the SPI-2 gene locus with each other and with other known attenuating gene mutations, such as aroA, etc., a broad repertoire of attenuation and immunogenicity can be achieved. Different expression cassettes can be introduced on these platforms, allowing further modulation of the immune response directed against the heterologous antigens. Finally, a library of individual recombinant live Salmonella vaccine carrier strains is generated, covering a broad spectrum of immuno-stimulatory potential, from which a genuine live vaccine strain can be tailored for the optimal protection or treatment of humans and/or animals against specific pathogens or disease.

Thus, in a further aspect, the present invention is an attenuated gram-negative cell comprising the SPI2 gene locus, wherein at least one gene of the SPI2 locus is inactivated, wherein said inactivation results in an attenuation/reduction of virulence compared to the wild type of said cell.

Genes present in the Salmonella pathogenicity island 2 that encode for a variety of proteins involved in type III secretion and those that are required for systemic spread and survival within phagocytic cells are ideal candidates for attenuation of pathogenic Salmonella ssp.

Several gram-negative bacterial pathogens secrete certain virulence proteins via specialised type III secretion systems. Virulence factors enable pathogenic bacteria to colonise a niche in the host despite specific attacks of the immune system. The type III secretion systems comprise a large number of proteins required to transfer specific effector proteins into eukaryotic host cells in a contact-dependent manner, thus they have also been called contact-dependent secretory systems. Although several components of the secretion system apparatus show evolutionary and functional conservation across bacterial species, the effector proteins are less well conserved and have different functions. The Yersinia effectors YpkA and YopH have threonine/serine kinase and tyrosine phosphatase activities, respectively. The actions of these and other Yops inhibit bacterial phagocytosis by host cells, which is thought to enable extracellular bacterial proliferation. The Shigella Ipa proteins, secreted by the mxi/spa type III secretion system, promote entry of this bacterium into epithelial cells. EspA, EspB and EspD, encoded by the locus of enterocyte effacement (LEE) of enteropathogenic Escherichia coli (EPEC) are required for translocation of proteins that cause cytoskeletal rearrangements and the formation of pedestal-like structures on the host cell surface.

For the purposes of the present invention an "gram-negative cell comprising the SPI2 gene locus" is a cell having a gene locus that harbors genes required for the systemic spread and survival within phagocytic cells and, thus, is a homologue or functional equivalent of the SPI2 locus from Salmonella. Preferred, the inventive attenuated gram-negative cell is an Enterobactericae cell, more preferred, a Salmonella cell, a Shigella cell or a Vibrio cell. In general, cells having a broad host range are preferred. Typical hosts are mammals, e.g. man, and birds, e.g. chicken. Salmonella cells are more preferred, and particularly preferred is Salmonella serotype typhimurium Definitive Type 104 (DT 104).

Salmonella typhimurium is unusual in that it contains two type III secretion systems for virulence determinants. The first controls bacterial invasion of epithelial cells, and is encoded by genes within a 40 kb pathogenicity island (SPI1). The other is encoded by genes within a second 40 kb pathogenicity island (SPI2) and is required for systemic growth of this pathogen within its host. The genes located on pathogenicity island SPI1 are mainly responsible for early steps of the infection process, the invasion of non-phagocytic host cells by the bacterium. For most of the SPI1 genes, mutations result in a reduced invasiveness in vitro. However, mutants that are defective in invasion are not necessarily avirulent; studies in mice demonstrated that, while these mutations in SPI1 genes significantly reduced virulence upon delivery by the oral route, they had no influence on virulence following an intraperitoneal route of infection. Taken together, these results indicate that mutations in genes within the pathogenicity island SPI1 do not abolish systemic infection and are therefore not very useful for the development of a safe, attenuated Salmonella carrier strain. In comparison, virulence studies of SPI2 mutants have shown them to be attenuated by at least five orders of magnitude compared with the wild-type strain after both oral and intraperitoneal inoculation of mice.

Many of the genes encoding components of the SPI2 secretion system are located in a 25 kb segment of SPI2. SPI2 contains genes for a type III secretion apparatus (ssa) and a two component regulatory system (ssr), as well as candidate genes for a set of secreted effectors (sse) and their specific chaperones (ssc). On the basis of similarities with genes present in other bacterial pathogens, the first 13 genes within the ssaK/U operon and ssaJ encode components of the secretion system apparatus. A number of additional genes, including ssaC (orf 11 in Shea et al., 1996; spiA in Ochman et al., 1996) and ssrA (orf 12 in Shea et al., 1996; spiR in Ochman et al., 1996), which encode a secretion system apparatus protein and a two component regulatory protein, respectively, are found in a region approximately 8 kb from ssaJ.

Preferably, the inventive attenuated gram-negative cell has inactivated at least one gene selected from effector (sse) gene secretion apparatus (ssa) genes, chaperon (ssc) genes and regulation (ssr) genes. More preferably, the at least one inactivated gene is an sse, ssc and/or ssr gene, even more preferred is an sse and/or ssc gene.

As far as the sse genes are affected by the inactivation, the inactivated gene is preferably sseC, sseD, sseE or a combination thereof. As far as the ssr genes are affected by the inactivation, preferably at least ssrB is inactivated. As far as the ssc genes are affected by the inactivation, preferably at least sscB is inactivated.

The inactivation of said gene of the SPI2 locus or functional homologue thereof in cells other than Salmonella) is effected by a mutation which may comprise deletion. Preferred are deletions of at least six nucleotides, and more preferred is a deletion of the partial and, in particular, the complete coding sequence for said gene. The mutation may also comprise the insertion of a heterologous nucleic acid molecule into said gene to be inactivated or a combination of deletion and insertion.

Pathogenic Salmonella ssp. serve a basis for the construction of a panel of different live Salmonella vaccine prototypes generated by gradual attenuations accomplished through the introduction of defined SPI2 gene locus mutations. Each resulting individual live Salmonella vaccine prototype is further transformed into a multivalent recombinant vaccine by the introduction of exchangeable DNA modules carrying (1) genetically engineered genes from heterologous pathogens and (2) adequate expression systems executing efficacious antigen presentation to the host immune system. In concert, these features elicit a specific immune response that either protects vaccinated hosts against subsequently invading Salmonella and/or other pathogens (prophylactic vaccination) or eliminates persistent pathogens, such as Helicobacter pylori (therapeutic vaccination).

Pathogenic Salmonella ssp. are gradually attenuated by mutations in individual virulence genes that are part of the SPI2 gene locus, e.g. an sse gene coding for an effector protein, such as sseC, sseD or sseE, or an ssc gene, such as sscB, coding for a chaperone, or an ssr gene, such as ssrB, coding for a regulator. Individual mutation of each of these genes leads to a unique individual grade of attenuation, which, in turn, effects a characteristic immune response at the mucosal, humoral and cellular levels. The individual grade of attenuation can be moderately increased by combinations of at least two gene mutations within the SPI2 gene locus or by combination with a mutation in another Salmonella gene known to attenuate virulence, e.g. an aro gene, such as aroA. A stronger grade of attenuation is achieved by mutation of a virulence gene that is part of a polycistronic gene cluster encoding several virulence factors, such as the transcriptional unit comprising the sseC, sseD, sseE and sscB genes, such that the mutation exerts a polar effect, disrupting expression of the following genes. The grade of attenuation may directly depend on the number of virulence genes that are affected by the polar mutation as well as their individual characteristics. Finally, the strongest attenuation is achieved when regulatory genes, such as ssrB, are mutated. Again, each mode of attenuation of a Salmonella ssp. leads to the generation of a live Salmonella vaccine strain that evokes an immune response at the mucosal, humoral and cellular levels that is characteristic for the type and/or combination of attenuating mutations present in that strain. The panel of differently attenuated live Salmonella vaccine strains that is generated represents a pool of potential carrier strains from which that carrier can be selected that provokes the most efficacious immune response for either the prevention or eradiction of disease in conjunction with the heterologous antigens that are expressed.

Mutations leading to attenuation of the indicated Salmonella virulence genes are preferentially introduced by recombinant DNA technology as defined deletions that either completely delete the selected virulence gene or result in a truncated gene encoding an inactive virulence factor. In both cases, the mutation involves a single gene and does not affect expression of neighbouring genes (non-polar mutation). An insertional mutation in one of the indicated virulence genes is preferred when the selected gene is part of a polycistronic virulence gene cluster and all of the following virulence genes are included in the attenuation process (polar mutation). Insertional mutations with non-polar effects are in general restricted to genes that are either singly transcribed or are localised at the end of a polycistronic cluster, such as ssrB. However, other attenuating mutations can arise spontaneously, by chemical, energy or other forms of physical mutagenesis or as a result of mating or other forms of genetic exchange.

Thus, the mutation which results in the preparation of the inventive attenuated gram-negative cell may be a polar or non-polar mutation. Furthermore, the grade of attenuation may be modified by inactivating an additional gene outside of the SPI2 locus, for example, another virulence gene or a gene that is involved in the biosynthesis of a metabolite or a precursor thereof such as the aro genes, in particular, aroA, or any other suitable gene such as superoxide dismutase (SOD).

The attenuated cell according to the invention may furthermore comprise elements which facilitate the detection of said cell and/or the expression of an inserted heterologous nucleic acid molecule. An example of an element which facilitates the detection of the attenuated cell is a selective marker cassette, in particular, a selective marker cassette which is capable of conferring antibiotic resistance to the cell. In one embodiment, the selective marker cassette confers an antibiotic resistance for an antibiotic which is not used for therapy in a mammal. Examples of elements which facilitate the expression of a heterologous nucleic acid molecule are a gene expression cassette which may comprise one or more promoter, enhancer, optionally transcription terminator or a combination thereof, a transactivator cassette, an invertase cassette for 1-phase or 2-phase expression of a heterologous nucleic acid. An example of an element which facilitates the insertion of a heterologous nucleic acid molecule is an insertion cassette.

In another aspect, the invention provides a carrier for the presentation of an antigen to a host, which carrier is an attenuated gram-negative cell, wherein said cell comprises at least one heterologous nucleic acid molecule comprising a nucleic acid sequence coding for said antigen, wherein said cell is capable of expressing said nucleid acid molecule or capable of causing the expression of said nucleic acid molecule in a target cell.

Preferably, said nucleic acid molecules comprises a nucleic acid sequence coding for a bacterial or viral antigen or for a tumor antigen. Examples of bacterial antigens are antigens from Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi and Nanobacteria. Examples of viral antigens are antigens from Hepatitis virus, erg. Hepatitis B and C, human papilloma virus and Herpes virus. The heterologous nucleic acid molecule may comprise a nucleic acid sequence which codes for at least one polypeptide or peptide-targeting domain and/or immunostimulatory domain. Thus, the expression product of said heterologous nucleic acid molecule may be targeted specifically to predetermined compartments such as periplasma, outer membrane, etc. The heterologous nucleic acid molecule may code for a fusion protein.

According to one embodiment the heterologous nucleic acid molecule is inserted into the SPI2 locus, preferred, into an sse gene and, more preferred, into sseC, sseD and/or sseE, in particular, sseC.

The insertion may be a polar insertion or an unpolar insertion. Generally, the introduction of an unpolar insertion is preferred, since it allows for the expression of the remaining genes of a polycistronic gene cluster, which can be used for the generation of carriers having different grades of attenuation.

Attenuated live Salmonella vaccines are used as carriers for specific antigens from heterologous pathogens, e.g. Helicobacter, etc., thus acting as a multivalent vaccine. The heterologous antigens are provided by a gene expression cassette (GEC) that is inserted by genetic engineering into the genome of an attenuated Salmonella strain. Preferentially, insertion of the gene expression cassette is targeted to one of the indicated virulence genes, thereby causing an insertional mutation as described in previous paragraph. In another application form, expression of the heterologous genes in the gene expression cassette is regulated by trans-acting factors encoded by a trans-activator cassette (TC) or an invertase cassette performing a 2-phase variable expression mode. Preferentially, the insertion of the trans-activator cassette is targeted to a second chosen virulence gene, which is then inactivated. Alternatively, the gene expression cassette or the trans-activator cassette or the invertase cassette can be introduced into the Salmonella genome by transposon-mediated insertion, which has no attenuation effect.

The principles of genetic engineering are required to generate either deletion or insertional mutations in Salmonella virulence genes. Generally, a suicide plasmid carrying a mutated virulence gene cassette containing a selective marker cassette (SMC) either alone or in combination with a gene expression cassette or a trans-activator cassette or the invertase cassette is introduced into the receptor Salmonella strain by conjugation. The original virulence gene is replaced with the mutated virulence gene cassette via homologous recombination, and the suicide plasmid, unable to replicate in the Salmonella receptor strain, becomes rapidly depleted. Successfully recombined Salmonella can be selected based on properties (such as, but not limited to, antibiotic resistance) conferred by the product of the gene(s) within the selective marker cassette. The mutated virulence gene cassette comprises DNA sequences that are homologous to the genome of the receptor Salmonella strain where the original virulence gene is localised. In the case where the original virulence gene is to be completely deleted, only those genomic DNA sequences that border the original virulence gene (indicated as flanking regions) are included in the mutated virulence gene cassette. The general architecture of a mutated virulence gene cassette includes at each end a DNA sequence of at least 50 nucleotides, ideally 200-250 nucleotides, that is homologous to the genome segment where the original virulence gene is localised. These DNA sequences flank a selective marker cassette and the other cassettes, such as the gene expression cassette (GEC) or the trans-activator cassette (TC) or the invertase cassette. As indicated above, these cassettes are used to generate insertional mutations which disrupt original gene expression. For in-frame deletions, a selective marker cassette is preferentially used.

The selective marker cassette (SMC) principally consists of a gene mediating resistance to an antibioticum which is able to inactivate the receptor Salmonella strain but which is actually not used in the treatment of Salmonellosis. Alternatively, another selectable marker can be used. The selective marker cassette is inserted in-frame in the targeted virulence gene and, consequently, the expression of the marker gene is under the control of the virulence gene promoter. Alternatively, the cassette is inserted within a polycistronic transcriptional unit, in which case the marker gene is under control of the promoter for this unit. In another application, the selective marker gene is under control of its own promoter; in this case a transcriptional terminator is included downstream of the gene. The selective marker is needed to indicate the successful insertion of the mutated virulence gene cassette into the genome of the receptor Salmonella strain. Furthermore, the antibiotic resistance marker is needed to facilitate the pre-clinical immunological assessment of the various attenuated Salmonella strains. In another application form, the selective marker is flanked by direct repeats, which, in the absence of selective pressure, lead to the recombinatorial excision of the selective marker cassette from the genome, leaving the short sequence of the direct repeat. Alternatively, the selective marker cassette can be completely removed by recombinant DNA technology. Firstly, the selective marker cassette is removed by adequate restriction endonuclease from the original mutated virulence gene cassette on the suicide plasmid leaving the flanking region sequences which are homologous to the Salmonella genome The suicide plasmid is then transferred into the attenuated receptor Salmonella strain by conjugation where the SMC-depleted mutated virulence gene cassette replaces the SMC-carrying mutated virulence gene cassette by recombination. After removal of the selective marker, the attenuated Salmonella strain is free for the application in humans. Transcriptional terminator sequences are generally included in the cassettes when polar mutations are established.

The gene expression cassette (GEC) comprises elements that allow, facilitate or improve the expression of a gene. In a functional mode the gene expression cassette additionally comprises one or more gene expression units derived from either complete genes from a heterologous source or fragments thereof, with a minimal size of an epitope. Multiple gene expression units are preferentially organised as a concatemeric structure. The genes or gene fragments are further genetically engineered, such that the resulting proteins or fusion proteins are expressed in the cytosol, in the periplasm, surface displayed or secreted. Furthermore the genes or gene fragments can be fused with DNA sequences encoding immunologically reactive protein portions, e.g. cytokines or attenuated bacterial toxins. The genes or gene fragments are either controlled in a one-phase mode from a promoter within the gene expression cassette or in a 2-phase mode or indirectly by a trans-activator cassette (TC). In the one-phase mode the promoter is preferentially a Salmonella promoter that is activated, i.e. induced, by environmental signals but also constitutive promoters of different strength can be used. In the 2-phase mode, the expression of the gene cassette is controlled by an invertase that derived from an invertase cassette. The invertase catalyses the inversion of a DNA segment comprising the gene cassette. The DNA segment is flanked on each end by an inverted repeat which is the specific substrate for the invertase finally causing two orientation of the gene cassette with respect to the gene expression cassette promoter. In the ON-orientation the gene cassette is correctly placed allowing transcription of the gene cassette. In OFF, the orientation of the gene cassette is incorrect and no transcription occurs. The invertase cassette comprises of an invertase that is controlled by a constitutive promoter or a Salmonella promoter induced or derepressed by environmental signals.

Heterologous antigens encoded within the gene expression cassette can be expressed under the control of a promoter, e.g. a tissue-specific promoter, which may be constitutive or inducible. The expression can be activated in a target cell, whereby a signal is transmitted from the target cell to the interior of the Salmonella cell, which signal induces the expression. The target cell, for example, can be a macrophage. The expression product may comprise a targeting domain or immunostimulatory domain, e.g. in the form of a fusion protein. The heterologous protein itself also may be a fusion protein. The heterologous antigens can be optionally expressed as cytosolic, periplasmic, surface displayed or secretory proteins or fusion proteins in order to achieve an efficacious immune response. The antigen encoding sequences may be fused to accessory sequences that direct the proteins to the periplasm or outer membrane of the Salmonella cell or into the extracellular milieu. If the heterologous polypeptides are secreted, secretion can occur using a type III secretion system. Secretion by the SPI2 type III secretion system is suitable. Proteins that are destined for the cytosolic compartment of the Salmonella do not need accessory sequences, in this case, naturally occurring accessory sequences must be removed from the genes encoding such antigens.

The accessory sequences for the periplasmatic compartment of Salmonella comprise a DNA sequence deduced from the amino-terminally localised signal peptide of a heterologous protein naturally translocated via the general secretion pathway, e.g. CtxA, etc.

The accessory sequences for the outer membrane compartment of Salmonella preferentially comprise DNA sequences deduced from the functionally relevant portions of a type IV secretory (autotransporter) protein, e.g. AIDA or IgA protease. The appropriate fusion protein contains an amino-terminally localised signal peptide and, at the carboxy-terminus, a .beta.-barrel shaped trans-membrane domain to which the foreign passenger protein is coupled via a spacer that anchors the passenger protein to the bacterial surface.

The accessory sequences for secretion into the extracellular milieu comprise DNA sequences deduced from proteins naturally secreted by the type III secretion system. In a generally functional fusion protein, the heterologous antigen is fused in the centre of a protein naturally secreted by the type III pathway or at the carboxy-terminal end of the respective protein.

The transactivator cassettes (TC) provide activators which generally improve expression of the heterologous antigens encoded by the various gene expression cassettes. Such activators either directly (RNA polymerase) or indirectly (transcriptional activator) act on the transcription level in a highly specific order. Preferentially, the expression of such activators are controlled by Salmonella promoters which are induced in vivo by environmental signals. In another application form the synthesis of the activator within the transactivator cassette is regulated in a 2-phase mode. The invertase expressed by the invertase cassette places the activator encoding DNA fragment in two orientations with respect to the transcriptional promoter. In the ON-orientation the activator gene is in the correct transcriptional order. In the OFF-modus the activator is incorrectly orientated and no expression occurs.

In the simple system, the gene product of the transactivator cassette exerts its effect directly on the promoter present in the gene expression cassette, directly activating or de-repressing expression of the heterologous gene. In the complex system, activation of the promoter in the heterologous gene expression cassette is dependent upon two or more interacting factors, at least one of which (encoded in the transactivator cassette) may be regulated by external signals. Further complexity is found in cascade systems, in which the external signal does not directly exert its effect on the transactivator cassette, but rather through a multi-step process, or in which the gene product of the transactivator cassette does not directly exert its effect on the heterologous gene expression cassette, but rather through a multi-step process.

According to still another aspect, the present invention is an attenuated gram-negative cell comprising the SPI2 gene locus, characterized by a lack of at least one SPI2 polypeptide, wherein said lack results in an attenuation/reduction of virulence compared to the wild type of said cell. Preferably, said missing SPI2 polypeptide is one or more effector polypeptide, secretion apparatus polypeptide, chaperon polypeptide or regulatory polypeptide. Furthermore, said attenuated cell may be a carrier which then is characterized by the presence of at least one heterologous peptide or polypeptide having immunogenic properties.

A further aspect of the present invention is a pharmaceutical composition which comprises as an active agent an immunologically protective living vaccine which is an attenuated gram-negative cell or carrier according to the invention. The pharmaceutical composition will comprise additives such as pharmaceutically acceptable diluents, carriers and/or adjuvants. These additives are known to the person skilled in the art. Usually, the composition will administered to a patient via a mucosa surface or via or via the parenteral route.

Further aspects of the present invention include a method for the preparation of a living vaccine, which comprises providing a living gram-negative cell comprising the SPI2 locus and inactivating at least one gene of the SPI2 locus to obtain an attenuated gram-negative cell of the invention, and optionally inserting at least one heterologous nucleic acid molecule coding for an antigen to obtain a carrier according to the invention. A further aspect pertains to a method for the preparation of a living vaccine composition comprising formulating an attenuated cell or a carrier according to the invention in a pharmaceutically effective amount together with pharmaceutically acceptable diluents, carriers and/or adjuvants. A further aspect of the invention relates to a method for the detection of an attenuated cell or a carrier according to the invention, comprising providing a sample containing said cell and detecting a specific property not present in a wild type cell. Methods for detecting a specific property of the attenuated cell or carrier, which is not present in wild type, are known to the person skilled in the art. For example, if this specific property of the attenuated cell comprises a deletion of one or more parts of the SPI2 locus, then the presence of said cell can be detected by providing a pair of specific primers which are complementary to sequences flanking this deletion and amplifying a fragment of specific length using amplification methods such as PCR. Methods for detecting the presence of an inventive carrier comprise PCR amplification of an inserted fragment or a fragment spanning the insertion boundary, hybridization methods or the detection of the heterologous expression product or of a selective marker.

A further aspect of the invention is a method for establishing a library of attenuated gram-negative cells or carriers, respectively, according to the invention. The method comprises the preparation of attenuated recombinant vaccine strains, each having a different mutation in the SPI2 locus which results in a different degree of attenuation. The pathogenicity or virulence potential of said strains can then be determined using known methods such as determination of the LD50, and the strains are rated according to the different pathogenicities, i.e. a different grade of attenuation. Preferably, the method comprises also the determination of other parameters of interest such as the immunogenicity or the immuno-stimulatory response raised in a host. Methods for determining the immuno-stimulatory potential are known to the person skilled in the art and some of them are described in Example 6. Preferably, the immuno-stimulatory potential of the inventive attenuated cells or carriers is determined at humoral, cellular and/or mucosal level. In this way it is possible to establish a library of attenuated cells or carriers having a predetermined attenuation degree and predetermined immuno-stimulatory properties. Thus, for each application, the strain having the desired properties can be selected specifically. For example, it wilt be usually preferred to select a strong attenuated strain for administration to patients which receive immunosuppressive drugs.

In a similar way, the invention allows for the establishment of libraries of attenuated carriers having defined pathogenicities and optionally immunogenicities. The establishment of a carrier library additionally will comprise the determination of the antigen presentation of said carrier strains to a host, whereby a panel of different carriers strains will be obtained having defined properties with respect to pathogenicity, immuno-stimulatory potential of carrier antigens and immuno-stimulatory potential of the heterologous antigen.

Another aspect of the invention is the use of the attenuated cell or carrier according to the invention for the preparation of a drug for the preventive or therapeutic treatment of an acute or chronic disease caused essentially by a bacterium or virus. For example, for the prevention or treatment of a Salmonella infection one will administer an attenuated Salmonella cell to raise the immune response of an affected patient. Similarly, a carrier according to the invention may be used for the preparation of a drug for the preventive or therapeutic treatment of a tumor.

The individual immuno-protective potential of each of the established recombinant Salmonella vaccine strains is determined in a mouse model using a pathogenic Salmonella typhimurium as the challenge strain. Determination of the virulence potential of the recombinant Salmonella vaccine strain: (1) Competitive index or LD50; (2) Systemic prevalence in blood, liver and spleen strictly excluded. Determination of the immuno-stimulatory potential of the carrier strain with a cytosolically expressed heterologous test antigen: (1) Single oral immunisation and subsequent evaluation of the short- and long-term immune response: (a) analysis of the humoral immune response profile, (b) analysis of the mucosal immune response profile, (c) analysis of the cellular immune response profile; (2) Multiple oral immunisations and subsequent evaluation of the short- and long-term immune response: (a) analysis of the humoral immune response profile, (b) analysis of the mucosal immune response profile, (c) analysis of the cellular immune response profile. Determination of the immuno-stimulatory potential of the carrier strain for the delivery of heterologous DNA (DNA vaccination).

Preferentially, the Salmonella acceptor strain has a broad host range, exhibiting significant pathogenicity in both animals and humans. Ideally, this is a Salmonella strain that is strongly pathogenic for mice, such as S. typhimurium. After successful development of the recombinant Salmonella vaccine strain, the strain is directly applicable for use in both animals and humans. If such an ideal Salmonella acceptor strain is not satisfactory for the respective host, other host-specific Salmonella must be selected, such as S. typhi for humans.

Other aspects of the invention relate to the use of a nucleic acid molecule as shown in FIG. 21A or B or one of the FIGS. 22A-Q, optionally modified as described hereinabove or of a vector as described hereinabove for the preparation of an attenuated cell, a living vaccine or a carrier for the presentation of an antigen to a host and to the use of the Salmonella SPI2 locus for the preparation of an attenuated cell, a living vaccine or preferably a carrier for the presentation of an antigen to a host. In this context the term "Salmonella SPI2 locus" refers to any nucleic acid sequence, coding or not coding, and to the expression product of coding sequences.

A still further aspect of the present invention is the use of a virulence gene locus of a gram-negative cell for the preparation of a carrier for the presentation of an antigen to a host.

Another aspect of the invention relates to a method of therapeutically or prophylactically vaccinating an animal, e.g. a mammal, e.g. a human, against a chronic disease caused primarily by a infectious organism including preparation and administering a vaccine of the invention.

Still another aspect of the present invention is an isolated nucleic acid molecule comprising a nucleic acid of at least 100 nucleotides a) of the nucleic acid sequence of one of FIGS. 24A, B, b (see Original Patent)) of a nucleic acid sequence which under stringent conditions hybridizes with the nucleic acid sequence of one of FIGS. 24A, B.

In particular, said aspect relates to said nucleic acid molecule which is capable of inducing the expression of a nucleic acid sequence conding for a peptide or polypeptide operatively linked to said nucleic acid molecule.

The in vivo inducible promoter Pivi comprises a DNA fragment which carries sequences for an operator and a transcriptional promoter. Such in vivo inducible promoter can be identified by applying an adequate reporter gene approach. Two of such in vivo inducible promoters have been identified within the SPI2 locus which initiate expression of the ssaBCDE operon (promoter A2) and the sseABsscAsseCDEsscBsseFG operon (promoter B), respectively. These promoters are induced by a regulative system comprising the ssrA and ssrB gene products. This regulative system is part of the SPI2 locus responsible for the activation of additional SPI2 locus genes. The regulative system is activated in macrophages by environmental signal(s) via sensor protein SsrA. The SsrB protein finally binds at a defined DNA sequence which initiates transcription through the RNA polymerase.

In an application form the DNA fragment comprising operator/promoter sequences is inserted in front of an invertase gene or an activator gene or a gene expression cassette, thereby executing an in vivo inducible expression in bacteria carrying at least the ssrA and ssrB genes or the complete SPI2 locus.

Thus, in a further aspect, the invention relates to an expression system for the in vivo inducible expression of a heterologous nucleic acid in a target cell, comprising a carrier cell for said heterologous nucleic acid, wherein said carrier cell comprises (a) a polypeptide having the amino acid sequence shown in FIG. 23P (ssrA) or a functional homologue thereof, (b) a polypeptide having the amino acid sequence shown in FIG. 23Q (ssrB) or a functional homologue thereof, and (c) the nucleic acid molecule of one of FIGS. 24A, B or a functional homologue thereof, as described above.

The target cell may be any suitable cell but preferably it is a macrophage. The carrier cell preferably is a Salmonella cell. The target cell may also comprise one or more of the elements described above such as selective marker cassettes, gene expression cassettes, transactivator cassettes, invertase cassettes and/or insertion cassettes. Furthermore, it may comprise a heterologous nucleic acid, in particular, the heterologous nucleic acids may be inserted into a gene expression cassette, thus rendering the GEC functional.

A still further aspect of the invention relates to the use of a nucleic acid molecule comprising at least 100 nucleotides of the nucleic acid sequence shown in one of FIGS. 24A, B or hybridizing therewith and having promoter activity, for the in vivo inducible expression of a heterologous nucleic acid molecule.

A further aspect of the present invention is the use of said nucleic acid molecule for the detection of in vivo inducible promoters.
 

Claim 1 of 18 Claims

1. An isolated attenuated Salmonella cell comprising the SPI2 gene locus, wherein at least one secretion apparatus (ssa) gene selected from the group consisting of ssaB, ssaC, ssaD, ssaE, ssaG, ssaH, ssaI, ssaJ, ssaP, ssaT, and ssaV of the SPI2 locus is inactivated, wherein said inactivation results in an attenuation/reduction of virulence compared to the wild type of said cell and wherein at least one additional gene located outside the SPI2 locus is inactivated, wherein the inactivation results in further attenuation/reduction of virulence compared to the wild type.
 

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