WO2025224251A1

WO2025224251A1 - Modified microorganisms

Info

Publication number: WO2025224251A1
Application number: PCT/EP2025/061241
Authority: WO
Inventors: Marc Biarnes CARRERA
Original assignee: Prokarium Ltd
Current assignee: Prokarium Ltd
Priority date: 2024-04-24
Filing date: 2025-04-24
Publication date: 2025-10-30
Anticipated expiration: 2026-10-24

Abstract

The present invention relates to a live attenuated Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is split into a first segment and a second segment, the first segment being operably linked to a first independently controlled promoter, wherein the first independently controlled promoter is a strong constitutive promoter or a strong vacuole-induced promoter, and the second segment being operably linked to a second independently controlled promoter, wherein the second independently controlled promoter is a strong vacuole-induced promoter, and wherein the first segment comprises a heterologous polynucleotide encoding a lysin upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding one or more cargo molecules replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

Description

MODIFIED MICROORGANISMS

FIELD OF THE INVENTION

The present invention relates to live attenuated Gram-negative bacteria modified to enable delivery of a biological molecule.

BACKGROUND

Recombinant protein secretion from the bacterial chassis has been a strategy explored to deliver relevant cargo from within the cellular envelope [Freudl et al., 2018], This can facilitate downstream purification and processing of biotechnologically relevant proteins [Freudl, R., et al., 2018] or deliver actively pharmacological molecules in appropriate tissues (e.g., tumours in immunooncology) and cellular compartments (e.g., cytosol or membrane-bound) in vivo [Chabloz, A., et al., 2020; Carrier, M.J., et al., 1992; Yang, E.Y. and Shah, K., 2020; Ruano-Gallego, D., et al., 2019],

Bacteria such as Salmonella enterica Typhi can naturally home in and colonise the tumour microenvironment (TME) [Hoffman, R.M., 2011], This can allow targeted delivery of relevant proteins and peptides in vivo to boost the initial immune response elicited by the exposure to pathogen-associated molecular patterns from Salmonella [Chen, J., et al., 2021], Salmonella can invade mammalian cells after secreting proteins using specific pathways that rely on needle-like structures to inject protein effectors into mammalian cells [Lhocine, N., et al., 2015; Park, D., et al., 2018], After assembly of the needle (SPI-1) and piercing of the mammalian host, these effectors, which contain specific signal peptides, are translocated to the host’s cytosol, where they can, for example, induce the engulfment of the bacterium [Park, D., et al., 2018] into a Salmonellacontaining vacuole (SCV). During the natural life cycle of Salmonella, the injected SPI-1 effectors ensure that the SCV does not fuse with the lysosome but instead is translocated into a juxtanuclear position, triggering the expression of a second needle-like secretion system (SPI-2) that drives vacuole maturation and bacterial replication. Attenuated Salmonella strains can be engineered to prevent functional assembly or expression of SPI-2. Attenuated strains have been used in therapeutic payload delivery. However, these bacterial strains typically reside within the SCV, and do not have access to the host’s cytosol, which is critical for the delivery of certain therapeutic payloads (e.g., nucleic acids or proteins). Currently, only one viable strategy has been reported to enforce bacterial release from the SCV (vacuole escape, VE) relies on the deletion of the gene sifA, involved in vacuole maturation, and requires a functional SPI-2. Therefore, there is a need to develop alternative strategies that warrant bacterial access into the cytosol and that are compatible with SPI-2 -deficient mutants (attenuated strains).

One strategy relies on the use of the Type 1 Secretion System from the uropathogenic strains of Escherichia coli (E. coli) that can export a pore-forming toxin (hemolysin), involved in the lysis of erythrocytes, into the interstitial space using a specialised secretion pathway, named Type 1 Secretion System (T1SS) [Thomas, S., et al., 2014], The pathway relies on two transport proteins, HlyB and HlyD, which are bound to the bacterium’s inner membrane and periplasm, respectively [Gentschev, I., et al., 2002]; and a constitutively expressed outer membrane protein, TolC. HlyB and HlyD are involved in secretion. Upon expression of the toxin, HlyA, and recognition of a signal peptide (HlyAs) on its C- terminus end, the H ly B 1 D2 complex interacts with TolC, opening a pore that allows translocation of HlyA from the cytoplasm whilst the protein is still not folded. The last protein in the pathway, HlyC, activates the toxicity of HlyA by transferring an acyl group into two internal lysins (Lys564 and Lys690) whilst this is still in the bacterial cytosol (/.e., before export).

In the chromosome, the hly genomic island is composed of four genes, hlyCABD, and one regulatory activator, hlyR [Gentschev, I., et al., 2002; Nagamatsu, K., et al., 2015; Nieto, J.M., et al., 2000; Pourhassan, N.Z., et al., 2022; Khosa, S., et al., 2018; Madrid, C., et al., 2002], Beyond the control exerted by the product of hlyR, the expression of the toxin is controlled by multiple genetic elements, such as an operator polarization sequence (ops) and RfaH binding sequence [Gentschev, I., et al., 2002; Nagamatsu, K., et al., 2015; Wang, B., M. et al., 2022] downstream of the poorly characterized promoter (P/?/y), which are involved in the in-trans suppression of a Rho-independent terminator between hlyA and hlyB that splits the operon in two [Gentschev, I., et al., 2002;], a stress-related sensor CpxR, physicochemical sensors (e.g., pH or osmolarity) HhA or H-NS [Gentschev, I., et al., 2002; Nieto, J.M., et al., 2000; Madrid, C., et al., 2002], and enhancer elements such as the AU-rich region between hlyC and hlyA that can recruit RpsA (also known as S1) [Pourhassan, N.Z., et al., 2022; Khosa, S., et al., 2018],

The use of T1SS to enforce vacuole escape has been reported (Gentschev, I., et al., 1996) by fusing Listeriolysin O (LLO) to HlyA_s. LLO is a hemolysin produced by Listeria monocytogenes that permits escape of bacteria from the vacuole into the cytosol by inducing the formation of pores within the vacuolar membrane. However, when designing synthetic organisms for the delivery of relevant cargo molecules directly into eukaryotic cells, such as within the Salmonella-containing vacuole (SCV), the complex regulation of the hly operon (for example hlyR, ops, RfaH, pHlyR, CpxR, HhA, H-NS, RpsA as shown in Figure 1) can interfere with endogenous and synthetic circuitry and limit the yields and robustness of recombinant protein delivery. Therefore, there is a need to provide a streamlined operon which can enable the efficient delivery of therapeutic cargo via bacteria.

SUMMARY OF INVENTION

The inventors of the present invention have surprisingly found that the hlyCABD operon (the unmodified operon is shown in Figure 1) can be modified in such a way using an optimised set of promoters so as to enable the delivery of cargo molecules outside of the bacterial cell and into the Salmonella-containing vacuole (SCV) (a modified operon of the invention is shown Figure 2). When the cargo is LLO, this allows enforced vacuole escape and grants bacterial access to the cytosol, where relevant therapeutic payloads (e.g., nucleic acid or protein) can be further delivered. In a modified hlyCABD operon split into two segments (a ‘cargo segment’ and a ‘secretion segment’), the inventors have presently identified the optimal promoters and promoter strength necessary to drive the expression of each segment to increase the efficiency of LLO delivery, and consequently, vacuole escape. In particular, the inventors have surprisingly found that the optimal conditions to maximise the export of cargo molecules, such as LLO, are primarily those that maximise the expression of the secretion machinery, followed by expression of the cargo molecule itself.

Therefore, in a first aspect, the invention provides a live attenuated Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is split into a first segment and a second segment, the first segment being operably linked to a first independently controlled promoter, wherein the first independently controlled promoter is a strong constitutive promoter or a strong vacuole-induced promoter, and the second segment being operably linked to a second independently controlled promoter, wherein the second independently controlled promoter is a strong vacuole-induced promoter, and wherein the first segment comprises a heterologous polynucleotide encoding a lysin upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding one or more cargo molecules replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

In a second aspect, the present invention provides a live attenuated Gramnegative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is split into a first segment and a second segment, the first segment being operably linked to a first independently controlled promoter, wherein the first independently controlled promoter is any of J23119 or sse t, and the second segment being operably linked to a second independently controlled promoter, wherein the second independently controlled promoter is a strong vacuole-induced promoter, and wherein the first segment comprises a heterologous polynucleotide encoding one or more cargo molecules upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding one or more cargo molecules replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

In a third aspect, the present invention provides a live attenuated Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is split into a first segment and a second segment, the first segment being operably linked to a first independently controlled promoter, wherein the first independently controlled promoter is not pipB2, ssaG or proC, and the second segment being operably linked to a second independently controlled promoter, wherein the second independently controlled promoter is a strong vacuole- induced promoter, and wherein the first segment comprises a heterologous polynucleotide encoding one or more cargo molecules upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding one or more cargo molecules replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

In a fourth aspect, the present invention provides a live attenuated Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is split into a first segment and a second segment, the first segment being operably linked to an independently controlled promoter, wherein the first independently controlled promoter is any of J23119 or sseA, and the second segment being operably linked to a second independently controlled promoter, wherein the second independently controlled promoter is any of ssaG, sseJ or sseA, and wherein the first segment comprises a heterologous polynucleotide encoding one or more cargo molecules upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding one or more cargo molecules replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

In a fifth aspect, the present invention provides a vaccine composition comprising a live attenuated Gram-negative bacterium according to any of the first, second, third and fourth aspects.

In a sixth aspect, the present invention provides a method of treating, preventing, inhibiting, preventing recurrence or controlling a disease in a subject, wherein the method comprises administering to a subject a live attenuated Gram-negative bacterium according to any of the first, second, third and fourth aspects.

In a seventh aspect, the present invention provides a use of a live attenuated Gram-negative bacterium according to any of the first, second, third and fourth aspects. BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows a schematic representation of the native hly operon which encodes the Type 1 Secretion System (T1SS). The hly operon is subject to stringent regulation by multiple proteins and signals.

Figure 2 shows a schematic representation of the refactored T1SS derived from E. coli (HlyR removed). The cargo region and the structural region are shown to be separated into two transcriptional units/segments independently controlled by promoter P1 and promoter P2. Additionally, two variants of cargo region were built, one variant in which the hlyC gene was upstream of the secretable cargo, and one variant lacking the hlyC gene in order to assess the effect of the hlyC gene on cargo secretion yield.

Figure 3 shows the secretion of reporter protein mScarlet under different configurations of the T1SS. Figure 3A demonstrates the impact on the export of the ratio of transcription of cargo versus structural proteins which was assessed by manipulating the strength of promoter P1 and promoter P2. Analysis of protein content in the supernatant shows that increased strength of cargo promoter results in enhanced protein detected in the supernatant up to 10 times. Increasing strengths in the promoter controlling the expression of the structural genes boosted the protein yields up to 64 times. Figure 3B demonstrates that when the same circuits are utilised, plus the addition of hlyC upstream of the cargo, the overall pattern previously observed was conserved, albeit at lower levels of proteins export (4 times lower). This could be due to the binding of H-NS of Hha downstream of hlyC or cell burden due to the addition of hlyC expression.

Figure 4 shows the secretion of recombinant protein LLO (also known as listerolysin O) as a cargo molecule under different configurations of the T1SS circuit. Figure 4A shows that the reporter gene mScarlet was exchanged by hly from Listeria monocytogenes and its export into the culture supernatant was assessed under all configurations. Overall protein levels exported were lower than with reporter protein mScarlet, although the observed pattern of increased secretion with increased promoter P2 strength was conserved. Figure 4B demonstrates that contrary to the previous observation as shown in Figure 3, the addition of hlyC does not result in decreased export, and the pattern observed, where enhanced expression of hlyBD correlates with higher secretion, is also conserved. This suggests that the decrease previously observed in Figure 3 may be due to the protein expression burden on the cell.

Figure 5 shows a two-plasmid system used to allow modulation of cargo export yields. The split of two circuits into independent plasmids supports modulation of cargo export yields by the combination of promoter strength and copy number. This allows screening for optimal experimental conditions that allow optimizing yields without over-burdening the bacterial carrier. The experiment evaluated the export of mScarlet in a T1 SS dual-plasmid system, where the mscarlet-hlyA fusion was controlled by increasingly strong promoters (2 (SEQ ID NO: 22), 4 (SEQ ID NO: 23), 6 (SEQ ID NO:24)) and hlyBD was under the control of the same increasing promoters. It was found that, while export yield was highest in the strongest promoter combination (cargo-hlyA 6, hlyBD: 6) it inflicted cell burden (see Figure 5B). Export was evaluated as relative luminescence units (RLU) from NanoLuc that emitted proportionally to cargo in supernatant due to a Hi Bit tag in the N-terminus of the cargo; growth was recorded as absorbance at 600 nm (see Figure 5C).

Figure 6 shows that the optimal conditions within the modified operon to maximise the export of cargo (LLO) are those that maximise expression of the export/secretion machinery. As expression of hlyBD increases, levels of LLO in culture supernatant increases.

Figure 7 shows the results of a vacuole escape CCF4 assay, in which HeLa cells infected with Salmonella were stained with the CCF4 reagent. HeLa cells were invaded with S. Typhi (STy) ZH9 or S. Typhimurium (STyM) CD12 with and without a plasmid bearing the beta-lactamase gene and treated with CCF4 at 24h postinvasion.

Figure 8 shows a schematic representation of the present circuits and the different promoters evaluated therein. Each circuit comprises a cargo segment, with either LLO or mScarlet and hlyAs under the control of various promoters (C3, C6, vac8, or vac12, corresponding to proB, J23119, pipB and sseA), and a T1SS secretion segment with hlyB and hlyD under the control of various promoters (C6, vac1 , vac2, or vac12, corresponding to J23119, ssaG, sseJ and sseA). Each plasmid backbone had a p15a origin of replication (oh) and the bla gene that processes the CCF4 dye in the event of vacuole escape.

Figure 9 shows the effect of different combinations of promoters on cargo secretion and vacuole escape. Blue cell counts at 24 hours post-invasion of HeLa cells with ZH9 strains bearing the vacuole escape circuits show that strong expression of LLO (either under constitutive C6 or vacuole-inducible vac12 promoters) results in forced vacuole escape when combined with strong expression of hlyBD operon. Strong constitutive expression (C6) of both the LLO and hlyBD segments abolished the escape phenotype.

DETAILED DESCRIPTION

In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

As used herein, the term “non-natural bacterium or bacteria” refers to bacterial (prokaryotic) cells that have been genetically modified or “engineered” such that it is altered with respect to the naturally occurring cell. Such genetic modification may for example be the incorporation of additional genetic information into the cell, modification of existing genetic information or indeed deletion of existing genetic information. This may be achieved, for example, by way of transfection of a recombinant plasmid into the cell or modifications made directly to the bacterial genome. Additionally, a bacterial cell may be genetically modified by way of chemical mutagenesis, for example, to achieve attenuation, the methods of which will be well known to those skilled in the art. As such, the term “non-natural bacterium or bacteria” may refer to both recombinantly modified and non- recombinantly modified strains of bacteria. As used herein “heterologous polynucleotide” refers to a polynucleotide that has been introduced into the live attenuated Gram-negative bacterium, i.e., the introduction of a polynucleotide that was not previously present. Accordingly, the live attenuated Gram-negative bacteria herein disclosed is a recombinant strain of bacteria. The heterologous polynucleotide in the context of the present invention may be a DNA molecule or RNA molecule intended for delivery to a eukaryotic cell. The heterologous polynucleotide in the context of the present invention may encode for a protein or peptide intended for delivery to a eukaryotic cell. The heterologous polynucleotide in the context of the present invention may encode for an RNA molecule intended for delivery to a eukaryotic cell. In a particularly preferred embodiment, the DNA or RNA molecule to be encoded is a mammalian DNA or RNA molecule.

The term “prophylactic treatment”, as used herein, refers to a medical procedure whose purpose is to prevent, rather than treat or cure, an infection or disease. In the present invention, this applies particularly to the vaccine composition. The term “prevent” as used herein is not intended to be absolute and may also include the partial prevention of the infection or disease and/or one or more symptoms of said infection or disease. In contrast, the term “therapeutic treatment” refers to a medical procedure with the purpose of treating or curing an infection or disease or the associated symptoms thereof, as would be appreciated within the art.

The terms "tumour," "cancer", “malignancy” and "neoplasia" are used interchangeably and refer to a cell or population of cells whose growth, proliferation or survival is greater than growth, proliferation or survival of a normal counterpart cell, e.g., a cell proliferative or differentiative disorder. Typically, the growth is uncontrolled. The term "malignancy" refers to invasion of nearby tissue. The term "metastasis" refers to spread or dissemination of a tumour, cancer or neoplasia to other sites, locations, or regions within the subject, in which the sites, locations or regions are distinct from the primary tumour or cancer. In one embodiment, the cancer is malignant. In an alternative embodiment, the cancer is non-malignant. The terms "effective amount" or "pharmaceutically effective amount" refer to a sufficient amount of an agent to provide the desired biological or therapeutic result. That result can be reduction, amelioration, palliation, lessening, delaying, and/or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In reference to cancer, an effective amount may comprise an amount sufficient to cause a tumour to shrink and/or to decrease the growth rate of the tumour (such as to suppress tumour growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development or prolong survival or induce stabilisation of the cancer or tumour.

In some embodiments, a therapeutically effective amount is an amount sufficient to prevent or delay recurrence. A therapeutically effective amount can be administered in one or more administrations. The therapeutically effective amount of the agent or combination may result in one or more of the following: (i) reduce the number of cancer cells; (ii) reduce tumour size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumour metastasis; (v) inhibit tumour growth; (vi) prevent or delay occurrence and/or recurrence of tumour; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.

For example, for the treatment of tumours, a "therapeutically effective dosage" may induce tumour shrinkage by at least about 5% relative to baseline measurement, such as at least about 10%, or about 20%, or about 60% or more. The baseline measurement may be derived from untreated subjects.

A therapeutically effective amount of a therapeutic compound can decrease tumour size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. The term "treatment" or "therapy" refers to administering an active agent with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a condition (e.g., a disease), the symptoms of the condition, or to prevent or delay the onset of the symptoms, complications, biochemical indicia of a disease, or otherwise arrest or inhibit further development of the disease, condition, or disorder in a statistically significant manner.

As used herein, the term "subject" is intended to include human and non-human animals. Preferred subjects include human patients in need of enhancement of an immune response. The methods are particularly suitable for treating human patients having a disorder that can be treated by augmenting the immune response. In a particular embodiment, the methods are particularly suitable for treatment of neoplastic disease or infectious disease in vivo.

The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any recited or enumerated component.

As used herein, "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" can mean a range of up to 20%. When particular values are provided in the application and claims, unless otherwise stated, the meaning of "about" should be assumed to be within an acceptable error range for that particular value.

The inventors of the present invention have surprisingly found that the hlyCABD operon (Figure 1) can be modified in such a way so as to enable the delivery of cargo molecules to the Sa/mone//a-containing vacuole of eukaryotic cells infected with Salmonella (Figure 2). To provide a more rational control and standardised application of the system, the inventors of the present invention carried out a rational refactorisation of the hly genomic island. The modified operon disclosed herein need not rely on the stringent regulation by multiple proteins and signals, such as those in Figure 1 (hlyR, ops, RfaH, pHlyR, CpxR, HhA, H-NS, RpsA , such that it is not hindered by complex native regulation. Therefore, the modified hlyCABD operon may not include the following regulatory elements: hlyR, ops, RfaH, pHlyR, CpxR, HhA, H-NS, or RpsA. The removal of these elements in the refactorization of the operon as in Figure 2 overcomes the problems associated with complex native regulation.

As a starting point, the hly operon was separated into two segments: a first segment (“cargo region”) and a second segment (“structural region”), each segment being operably linked to its own independently controlled promoter. The process of separating the four genes of the hly operon into two segments allows the genes involved in cargo production and/or activation (hlyA and optionally hlyC) to be transcriptionally separated from those involved in secretion (hlyB, hlyD). The toxin sequence (hlyA) can be replaced by a reporter gene (e.g., mScarlet), or indeed a heterologous nucleotide encoding any other cargo molecule, such as LLO, while maintaining the translocation peptide (HlyAs). As used herein, “maintaining” HlyAs refers to the HlyAs sequence being intact or undisrupted by any other molecule. For instance, an unmaintained HlyAs sequence may have another molecule, for example a cargo molecule, inserted within the HlyAs sequence such that HlyAs flanks the cargo molecule. “Maintaining” HlyAs may also refer to the entire, full length HlyAs sequence being present without disruption.

The first segment may comprise or consist essentially of a heterologous polynucleotide encoding a lysin upstream of a hlyAs translocation sequence. The second segment may comprise or consist essentially of the hly genes involved in secretion.

The optimal levels of transcription of the cargo and the secretion modules were screened by manipulating the strength of the promoters upstream of each segment. The results described herein show that splitting the operon into two segments results in functional translocation, which is increased upon increasing the transcription levels of both segments. Further, the inventors have surprisingly identified the optimal combinations of promoters to control each of the two segments of the modified /?/y operon Accordingly, the present invention relates to a live attenuated Gram-negative bacterium comprising a modified hly operon.

The inventors of the present invention surprisingly found that splitting the hly operon into two segments resulted in functional translocation, which was increased upon increasing the transcription levels of both segments. The present inventors have further identified the optimal combination of promoters to be operably linked to each segment in order to maximise cargo export in vitro and in vivo. The inventors have demonstrated that cargo secretion increases with increases in cargo expression and hlyBD expression. While increasing the expression of cargo contributed to increased yield of export, the major parameter responsible for high titres was found to be strong expression of the hlyBD segment. For example, Figure 6 demonstrates that LLO secretion increases with increased strength of promoter controlling LLO expression (16C1 , 16C3 and 16C6 promoters), but that the major driver of increased LLO secretion was due to hlyBD expression (C1-C6 promoters increasing in strength, with C1 being the weakest promoter and C6 being the strongest promoter).

The modified hlyCABD operon of the present invention is therefore split into two segments: a first segment and a second segment, and in accordance with a first aspect, the present invention provides a live attenuated Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is split into a first segment and a second segment, the first segment being operably linked to a first independently controlled promoter, wherein the first independently controlled promoter is a strong constitutive promoter or a strong vacuole-induced promoter, and the second segment being operably linked to a second independently controlled promoter, wherein the second independently controlled promoter is a strong vacuole-induced promoter, and wherein the first segment comprises a heterologous polynucleotide encoding a lysin upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding one or more cargo molecules replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

In order to allow for transcription and translation of the heterologous polynucleotide and production of the cargo molecule in the live attenuated Gramnegative bacterium, the heterologous polynucleotide is operably linked to an independently controlled promoter. Further, in order to allow for transcription and translation of the hly genes involved in secretion, the hly genes involved in secretion is operably linked to an independently controlled promoter.

As used herein, the term “independently controlled promoter” refers to a promoter which is controlled by regulatory elements which are distinct to that of another promoter in the system. The utilisation of a first and second independently controlled promoter allows for the sequences to which they are operably linked to be transcriptionally separated. In particular, the promoter controlling the expression of the first segment may be different, or have distinct regulatory mechanisms, to that of the promoter controlling the expression of the second segment. Temporal control of expression can be achieved by using different promoters.

The first and second independently controlled promoters described herein are strong promoters. When a strong promoter is used, a high rate of transcription is initiated. As used herein, the term “strong promoter” or “active promoter” may be used interchangeably. The skilled person will be familiar with the term “strong promoter” as a widely used term in the art. For the avoidance of doubt, these terms refer to a promoter which yields a high rate of transcription of the genes under its regulation. Genes regulated by strong promoters more frequently recruit RNA polymerases and therefore yield more mRNA and therefore more product protein than genes regulated by weak promoters. Any strong promoter that fulfils the function herein disclosed may be suitable. However, the present inventors have identified a number of promoters to control each segment of the modified operon such that cargo secretion is maximised. Figure 8 shows a schematic representation of the different promoters that may be selected to control expression of the two segments of the modified operon. The first independently controlled promoter may be a strong constitutive or strong vacuole induced promoter. As used herein, the term “constitutive promoter” has its usual meaning in the art. For the avoidance of doubt, constitutive promoters are active in the cell and allow for continual transcription of its associated gene. Constitutive promoters differ in strength, and may be weak, medium or strong constitutive promoters. For example, J23119 is a strong constitutive promoter, whereas proB is a medium to weak constitutive promoter. As used herein, the term “vacuole-induced promoter” has its usual meaning in the art. For the avoidance of doubt, vacuole-induced promoters are promoters which initiate the transcription of particular genes when the bacterium is present in the Sa/mone//a-containing vacuole or any other eukaryotic vacuole. Vacuole-induced promoters vary in strength. Examples of strong vacuole-induced promoters include ssaG, sseJ, pipB and sseA.

In a second aspect of the invention, there is provided a live attenuated Gramnegative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is split into a first segment and a second segment, the first segment being operably linked to a first independently controlled promoter, wherein the first independently controlled promoter is any of J23119 or sseA, and the second segment being operably linked to a second independently controlled promoter, wherein the second independently controlled promoter is a strong vacuole-induced promoter, and wherein the first segment comprises a heterologous polynucleotide encoding one or more cargo molecules upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding one or more cargo molecules replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

Therefore, in accordance with the second aspect, the first independently controlled promoter (P1) may be a strong constitutive promoter or a strong vacuole induced promoter, such as J23119 or sseA. In this embodiment, the second independently controlled promoter (P2) is a strong vacuole-induced promoter. The second independently controlled promoter may be any suitable vacuole- induced promoter. In some embodiments, the second independently controlled promoter may be ssaG. In some embodiments, the second independently controlled promoter may be sseJ. In some embodiments, the second independently controlled promoter may be sseA. In some embodiments, the second independently controlled promoter may be ssrA. In some embodiments, the second independently controlled promoter may be pipB. In some embodiments, the second independently controlled promoter may be pipB2. In some embodiments, the second independently controlled promoter may be ssaB. In some embodiments, the second independently controlled promoter may be sifA. In some embodiments, the second independently controlled promoter may be sifB. In some embodiments, the second independently controlled promoter may be zinT. In some embodiments, the second independently controlled promoter may be mgtC.

In a preferred embodiment, the first independently controlled promoter is J23119 or sseA, and the second independently controlled promoter is any of ssaG, sseJ, pipB and sseA, even more preferably ssaG, sseJ and sseA. In a most preferred embodiment, the first independently controlled promoter is J23119 and the second independently controlled promoter is ssaG.

In an alternative embodiment, the second independently controlled promoter may be a constitutive promoter, for example, a strong, medium or weak constitutive promoter.

In accordance with a third aspect, the present invention provides a live attenuated Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is split into a first segment and a second segment, the first segment being operably linked to a first independently controlled promoter, wherein the first independently controlled promoter is not pipB2, ssaG or proC, and the second segment being operably linked to a second independently controlled promoter, wherein the second independently controlled promoter is a strong vacuole-induced promoter, and wherein the first segment comprises a heterologous polynucleotide encoding one or more cargo molecules upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding one or more cargo molecules replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

The first independently controlled promoter may be any suitable promoter, in particular any suitable strong constitutive promoter or strong vacuole induced promoter that is not pipB2, ssaG or proC. In one embodiment, the first independently controlled promoter is not pipB2. In another embodiment, the first independently controlled promoter is not ssaG. In another embodiment, the first independently controlled promoter is not proC. In another embodiment, the first promoter is none of pipB2, ssaG or proC. In this embodiment, the second independently controlled promoter (P2) is a strong vacuole induced promoter and the first independently controlled promoter is any suitable promoter that is not pipB2, ssaG or proC. In a preferred embodiment, the first independently controlled promoter is a strong promoter, more preferably a strong constitutive or strong vacuole induced promoter. In a most preferred embodiment, the first independently controlled promoter is J23119 or sseA. In some embodiments, the second independently controlled promoter is any of ssaG, sseJ, pipB2 and sse/t, even more preferably ssaG, sseJ and sseA. In a most preferred embodiment, the first independently controlled promoter is J23119 and the second independently controlled promoter is ssaG.

In a fourth aspect, the present invention provides a live attenuated Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is split into a first segment and a second segment, the first segment being operably linked to an independently controlled promoter, wherein the first independently controlled promoter is any of J23119 or sse and the second segment being operably linked to a second independently controlled promoter, wherein the second independently controlled promoter is any of ssaG, sseJ or sseA and wherein the first segment comprises a heterologous polynucleotide encoding one or more cargo molecules upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding one or more cargo molecules replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion. The first segment of the modified operon (the cargo region) is operably linked to a first independently controlled promoter, which may be a strong constitutive promoter or a strong vacuole-inducible promoter. In one embodiment, when the first independently controlled promoter is a strong constitutive promoter, the strong constitutive promoter may be any of proB or J23119. In another embodiment, when the first independently controlled promoter is a strong vacuole-inducible promoter, the strong vacuole-inducible promoter may be any of pipB or sseA.

In a preferred embodiment, the first independently controlled promoter is any of J23119 orsse t (denoted as C6 and vac12 in Figures 8 and 9). In a most preferred embodiment, the first independently controlled promoter is J23119.

The second segment of the modified operon (the secretion region) is operably linked to a second independently controlled promoter, which may be a may be a strong constitutive promoter or a strong vacuole-inducible promoter. In one embodiment, where the second independently controlled promoter is a strong constitutive promoter, the strong constitutive promoter may be J23119. In another embodiment, when the second independently controlled promoter is a strong vacuole-inducible promoter, the strong vacuole-inducible promoter may be any of ssaG, sseJ or sseA.

In a preferred embodiment, the second independently controlled promoter is any of ssaG, sseJ or sse t (denoted as vac1 , vac2 and vac12 in Figures 8 and 9). In a most preferred embodiment, the second independently controlled promoter is ssaG.

In the modified operon, in a preferred embodiment, the combination of promoters for the first independently controlled promoter (P1) and second independently controlled promoter (P2) is as follows:

Table 1. Preferred combinations of promoters within modified operon.

Further combinations of promoters may include any of the following sequences, as shown in Figure 8. For example, in one embodiment, the first independently controlled promoter (P1) may be proB, and the second independently controlled promoter (P2) may be ssaG. In another embodiment, the first independently controlled promoter (P1) may be proB, and the second independently controlled promoter (P2) may be sseJ. In another embodiment, the first independently controlled promoter (P1) may be proB, and the second independently controlled promoter (P2) may be sseA. In another embodiment, the first independently controlled promoter (P1) may be p/pB, and the second independently controlled promoter (P2) may be ssaG. In another embodiment, the first independently controlled promoter (P1) may be p/pB, and the second independently controlled promoter (P2) may be sseJ. In another embodiment, the first independently controlled promoter (P1) may be p/pB, and the second independently controlled promoter (P2) may be sseA.

As is disclosed in Table 1 , in a most preferred embodiment, the first independently controlled promoter is J23119 and the second independently controlled promoter is ssaG.

In another preferred embodiment, the first independently controlled promoter is J23119 and the second independently controlled promoter is sseA. In another preferred embodiment, the first independently controlled promoter is J23119 and the second independently controlled promoter is sseJ.

In another most preferred embodiment, the first independently controlled promoter is sse t and the second independently controlled promoter is ssaG.

In another preferred embodiment, the first independently controlled promoter is sse t and the second independently controlled promoter is sseA.

In another preferred embodiment, the first independently controlled promoter is sse t and the second independently controlled promoter is sseJ.

In another preferred embodiment, the first independently controlled promoter is sse t and the second independently controlled promoter is J23119.

In another embodiment, the first independently controlled promoter is proB and the second independently controlled promoter is ssaG.

In another embodiment, the first independently controlled promoter is proB and the second independently controlled promoter is sseA.

In another embodiment, the first independently controlled promoter is proB and the second independently controlled promoter is sseJ.

It is envisaged that, in one embodiment, if one promoter is a strong constitutive promoter, the other promoter is not a strong constitutive promoter. Figure 9 demonstrates that strong constitutive expression of both segments abolished the vacuole escape phenotype.

Other intracellular promoters (SPI-2) may be suitable. In one embodiment, the SPI-2 gene is an ssa gene. Suitable promoters may include, but are not limited to, ssa ssaJ, ssaU, ssaK, ssaL, ssaM, ssaG, ssaP, ssaQ, ssaR, ssaS, ssaT, ssaD, ssaE, ssaG, ssa/, ssaC and ssaH. SPI-2 promoters can also be used to activate the expression of Type 1 Secretion Systems (T1SS) which facilitate the export of hemolysins from Listeria. The strength of the promoters selected for each segment may be manipulated to enhance the efficiency of secretion of the cargo molecule (Figures 3 and 4). Table 2 details other promoters used in screening.

Table 2. Promoters used in the screening and their corresponding sequences. Shown doubly underlined, promoter sequence with the -10 consensus region (variable) highlighted in bold. Shown dashed underlined, a riboJ or vtmoJ ribozyme sequence. On sequences from promoter 2, BBa_B1006 terminator is shown singly underlined. As would be appreciated by the skilled person, sequences sharing a certain percent identity with any of the sequences disclosed herein may also be utilised in accordance with the invention. As used herein, the term “sequence identity” and “sequence homology” are interchangeable and refers to the number of identical residues over a defined length into a given alignment. To calculate % sequence identity of any of the sequences herein disclosed, sequence comparison software may be used, for example, using the default settings on the BLAST software package (V2.10.1). For instance, there may be a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to any of the sequences disclosed herein.

The bacterium of the present invention is a live attenuated Gram-negative bacterium. Examples of live attenuated Gram-negative bacteria for use in the present invention include, but are not limited to, Salmonella, Escherichia coli, Shigella, Pseudomonas, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, Legionella, Chlamydia and Yersinia. However, in one embodiment, the live attenuated Gram-negative bacterium is Salmonella. The Salmonella may be Salmonella Typhi or Salmonella Typhimurium.

In another embodiment of the present invention, the live attenuated Gramnegative bacterium is a genetically engineered non-natural bacterium.

Accordingly, the present invention discloses live attenuated Gram-negative bacteria that have been genetically altered to produce bacterial strains that can effectively deliver various cargo molecules. As would be understood by a person of skill in the art, genes may be mutated by a number of well-known methods in the art, such as homologous recombination with recombinant plasmids targeted to the gene of interest, in which case an engineered gene with homology to the target gene is incorporated into an appropriate nucleic acid vector (such as a plasmid or a bacteriophage), which is transfected into the target cell. The homologous engineered gene is then recombined with the natural gene to either replace or mutate it to achieve the desired inactivating mutation. Such modification may be in the coding part of the gene or any regulatory portions, such as the promoter region. As would be understood by a person of skill in the art, any appropriate genetic modification technique may be used to mutate the genes of interest, such as the CRISPR/Cas system, e.g., CRISPR/Cas 9, to produce the bacterial strains herein disclosed. Table 3 details the sequences used to build expression plasmids.

Table 3. Sequences used to build expression plasmids, including conserved regions for amplification of DNA blocks and Bsal-dependent cleavage sites for Golden Gate Assembly. Thus, numerous methods and techniques for genetically engineering bacterial strains will be well known to the person skilled in the art. These techniques include those required for introducing heterologous genes into the bacteria either via chromosomal integration or via the introduction of a stable autosomal selfreplicating genetic element. Exemplary methods for genetically modifying (also referred to as "transforming" or “engineering”) bacterial cells include bacteriophage infection, transduction, conjugation, lipofection or electroporation. A general discussion on these and other methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); which are hereby incorporated by reference.

Several Gram-negative bacteria use a Type 1 Secretion System (T1SS) to translocate proteins across their inner and outer membranes into the extracellular environment. Of these T1SS, the Escherichia coli a-hemolysin (HlyA) secretion system is the most thoroughly characterised. Exploitation of the T1SS enables proteins and other cargo molecules to be actively presented to eukaryotic cells of a host immune system through export from the bacterial cytoplasm, rather than merely becoming accessible to the eukaryotic cells once the bacterium has been engulfed and disintegrated. HlyA is a bacterial toxin and virulence factor, and is hence replaced in the presently modified operon. The secretion and activation of HlyA is determined by the hlyCABD operon. Briefly, once HlyA has been transcribed and translated, there are three components that modulate the export of HlyA: HlyB, HlyD and TolC. HlyB and HlyD are inner membrane proteins (which may be found in a HlyB-HlyD complex anchored to the inner membrane of the Gram-negative bacterial cell), whereas TolC is located on the outer membrane of the Gram-negative bacterial cell. HlyA carries a translocation signal sequence, known as HlyAs, on its C-terminus. Recognition of HlyAs by the HlyB-HlyD complex induces contact with TolC which forms a trans-periplasmic export channel between the inner and outer membrane. HlyC plays a role in the activation of HlyA. Therefore, the replacement of the HlyA toxin with a heterologous nucleotide encoding a protein, or other cargo molecule, enables the export of specific heterologous proteins or other molecules of interest, via the C-terminal HlyAs sequence, from a carrier bacterium to the extracellular surroundings.

It is envisaged that the live attenuated Gram-negative bacterium of the present invention can act as an efficient and reliable method of delivering or exporting cargo molecules from the bacterial cytoplasm into the Sa/mone//a-containing vacuole. Accordingly, the bacterial strains herein disclosed are recombinant strains comprising a modified hlyCABD operon which comprises a heterologous polynucleotide encoding a cargo molecule. The heterologous polynucleotide therefore has a nucleotide-encoding structure which allows for its transcription, and, in the case where the cargo molecule is a protein, its subsequent translation into the encoded cargo molecule.

It is envisaged that the modified hlyCABD operon is split into a first segment and a second segment, each segment being operably linked to an independently controlled promoter.

The first segment (the ‘cargo region’) is envisaged to comprise the heterologous polynucleotide which encodes a cargo molecule, upstream of a hlyAs translocation sequence.

As used herein, the terms “cargo” or “cargo molecule” may be used interchangeably and refer to a specific molecule of interest which is intended to be translocated, delivered, transported, or exported from one place to another. The skilled person will be highly familiar with these terms. In particular, a cargo molecule may be translocated from the bacterial cytoplasm to the extracellular environment, for example into the SCV. The cargo molecules encoded by the modified operon are to be secreted by the secretion machinery of the modified operon. The skilled person will understand the various types of cargo which can be encoded in the modified operon. In the context of the modified operon, the cargo molecule may be encoded by a polynucleotide sequence, for example, where the cargo molecule is a protein, said protein is encoded by a polynucleotide sequence, such as DNA or mRNA. Where the cargo is a lysin, such as LLO, the lysin protein would be expressed and subsequently secreted outside of the bacteria via hlyBD and into the vacuole to allow VE. Whereas the “cargo” is to be encoded by the modified hly operon, the bacteria comprising the modified operon may further comprise a “payload”. The payload may be a therapeutic payload as described herein. The one or more cargo may also comprise a therapeutic molecule. The payload within the bacteria may be released once the bacteria achieve VE and reach the host cytosol. Therefore, the term “payload” is used where the modified operon is utilised to export LLO and thus grant access to the cytosol. The payload need not be encoded on the modified operon, and could be encoded by or comprised within the bacterial genome or on a plasmid.

The heterologous polynucleotide encodes one or more cargo molecules, for example one, two, three, four, five, six, seven, eight, nine, ten, or more than ten cargo molecules. Within the bacteria there may be any number of payloads encoded, for example one, two, three, four, five, six, seven, eight, nine, ten, or more than ten payload molecules. In a preferred embodiment, the heterologous polynucleotide encodes one cargo molecule.

In a preferred embodiment, the cargo molecule is a protein and/or peptide. Cargo molecules may or may not be heterologous proteins which do not occur naturally to the carrier bacterial cell. In a preferred embodiment, the cargo is a lysin. As used herein, the term “lysin” has its conventional meaning in the art, and refers to a protein (for example a hydrolytic enzyme) that digests, destroys or causes pore formation in membranes and/or cell walls. Types of lysins include, but are not limited to, hemolysins. Listeriolysin O (LLO) is a hemolysin, therefore in a more preferred embodiment, the cargo molecule is LLO. As the skilled person will appreciate, any suitable lysin may be utilised, including derivatives, homologous and other related or similar enzymes to LLO. The amino acid sequence of LLO is given as SEQ ID NO: 2. Therefore, any sequence encoding LLO of SEQ ID NO: 2 may be utilised including a sequence having at least 70% identity thereto, or a sequence having at least 75%, at least 80%, at least 90%, at least 95% or at least 100% identity to SEQ ID NO: 2.

The cargo molecule may consist of LLO, or may comprise LLO and one or more other cargo molecules.

In accordance with the invention, the heterologous polynucleotide encoding one or more cargo molecules replaces the hlyA gene (whereby hlyAs is retained as the signal peptide for secretion via hlyBD). The cargo may be LLO. In this embodiment, LLO is fused upstream of hlyAs (i.e. , llo.hlyAs). In one embodiment, the llo gene may comprise the one or more cargo molecules and thereby itself entirely or partly replace the hlyA gene (whereby hlyAs is retained as the signal peptide for secretion via hlyBD). In another embodiment, the llo gene as fused to the hlyAs signal peptide may be present anywhere else within the modified operon or even outside of the modified operon. The llo gene as fused to the hlyAs signal peptide may be present anywhere in bacterial genome or as a plasmid, provided that it is within the same bacteria.

Where the modified operon comprises llo, in one embodiment, the first independently controlled promoter (P1 ) that is operably linked to the heterologous polynucleotide encoding a lysin may be a strong promoter, in particular a strong constitutive or a strong vacuole-inducible promoter. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be proB. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be J23119. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be pipB. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be sseA. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be ssaG. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be sseJ. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be ssrA. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be pipB2. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be pipB2 extended. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be ssaB. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be sifA. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be sifB. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be zinT. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be mgtC. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be pro1. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be proA. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be proC. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be proD. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be rpsM. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be dnaK. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be sicAp. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be sicDp. In another embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin may be sopEp.

In a most preferred embodiment, the first independently controlled promoter that is operably linked to the heterologous polynucleotide encoding a lysin is J23119 or sseA.

In preferred embodiments, the lysin is LLO.

A modified operon encoding a lysin such as LLO may have any of the promoters described herein at P2, i.e., any of J23119, ssaG, sseJ or sseA. Where the promoter at P1 is a strong constitutive promoter, the promoter at P2 is not a strong constitutive promoter (i.e., J23119 at P1 and J23119 at P2 is not a preferred combination of promoters).

While it is envisaged that the cargo molecule of the present invention may be a protein or peptide, other cargo types include DNA and RNA molecules. Therefore, in another embodiment, the cargo molecule is an RNA molecule. Similarly, the payload may be a protein, peptide, or nucleic acid. As used herein, the terms “RNA” and “ribonucleic acid” are used interchangeably, and refer to nucleic acids composed of uracil, adenine, guanine, and cytosine ribonucleic acid bases. These terms and concepts will be well known to those in the art. Types of RNA molecules include, for example, messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), transfer RNA (tRNA), selfamplifying RNA (saRNA) and ribosomal RNA (rRNA). Accordingly, the RNA cargo molecule may be an mRNA molecule. As used herein, the terms “mRNA” and “messenger RNA” are used interchangeably and refer to a single-stranded RNA molecule involved in protein synthesis. Eukaryotic mRNA molecules are transcribed from DNA in the nucleus of a eukaryotic cell, and subsequently exported from the nucleus into the cytoplasm of the eukaryotic cell, where translation of the mRNA molecule into proteins takes place. Bacterial mRNA molecules are transcribed from DNA that is non-compartmentalised and translated in the cytosol coupled to transcription. These terms and concepts will be well known to those in the art. RNA molecules are transcribed and translated within the bacterium itself. For example, the live attenuated Gram-negative bacterium may encode up to 10 different heterologous mRNA molecules, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 different heterologous mRNA molecules. A cargo or payload mRNA molecule itself may encode a peptide and/or protein, whereby the peptide and/or protein may be a therapeutic peptide and/or therapeutic protein.

In one embodiment, where the cargo or payload is a therapeutic peptide and/or therapeutic protein, the therapeutic peptide and/or therapeutic protein may be a cytokine, a chemokine, an antibody or a functional fragment thereof, a cytotoxic agent, a cancer agent or any combination thereof. The invention herein disclosed provides a live attenuated Gram-negative bacterium in which the cargo or payload molecule is expressed within the bacterium itself prior to export. For example, the live attenuated Gram-negative bacterium may encode up to 10 different cargo or payload (protein) molecules, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 different cargo or payload (protein) molecules.

It is envisaged that the heterologous polynucleotide encoding the cargo molecule is fused to a hlyAs sequence encoding the translocation peptide, HlyAs. In one embodiment, where the cargo molecule is a protein or peptide, the HlyAs translocation peptide is positioned on the C-terminus of the cargo molecule, since the cargo molecule replaces HlyA, thus retaining HlyAs. The HlyAs protein may be approximately 50 to 220 amino acids in length. In some embodiments, the HlyAs protein is 218 amino acids in length. The optimal, minimal, recognition site of HlyAs may be 60 amino acids in length (Hess, J., et al, 1990; Jarchau, T., et al., 1994). Several structural and sequence motifs within HlyAs have been identified as being important for its signal function through site-directed mutagenesis, CD and NMR spectroscopy studies [Holland, I.B., et al., 1990; Koronakis, V., 1989; Jarchau, T., et al., 1994], As used herein, the terms “translocation sequence”, “signal sequence”, and “target sequence” may be used interchangeably, and refer to a gene encoding a translocation peptide or protein which is recognised by cellular export machinery and targeted for export or secretion from the cell. Translocation peptides are typically found on the N- or C-terminus of a peptide or protein which is intended to be translocated from one location to another. Translocation sequences generally encode peptides with a specific amino acid sequence or motif which can be recognised by cellular export machinery. In the case of the hlyCABD operon, HlyAs translocation peptide is recognised by the HlyB and HlyD structural proteins which engage with TolC to create a transperiplasm channel, thus enabling the translocation of HlyAs (and any cargo to which it may be fused) through the inner and outer membrane of the live attenuated Gram-negative bacterium.

Preferably, the full length HlyAs is maintained at the C-terminus of the cargo molecule. The full translocation sequence includes three glycine- and aspartic- rich repeats known as repeats in toxins (RTX). RTX repeats play an important role in facilitating translocation and secretion by providing binding sites for calcium ions, which help to improve stabilisation of the protein to be translocated as it passes through the secretion machinery. Advantageously, use of the full length HlyAs may therefore allow for increasingly efficient secretion as compared to other operons in the art which insert cargo within the translocation sequence or utilise truncated translocation sequences. Thus, using a longer, and in some embodiments the full length, HlyAs sequence, enables improved secretion efficiency and results in the system being payload independent (Holland, LB., et al., 2016).

In one embodiment, the heterologous polynucleotide encoding one or more cargo molecules is positioned upstream of the HlyAs sequence. In another embodiment, the heterologous polynucleotide encoding one or more cargo molecules is positioned downstream of the independently controlled promoter. In yet another embodiment, the heterologous polynucleotide encoding one or more cargo molecules is positioned upstream of the HlyAs sequence and downstream of the independently controlled promoter.

In an alternative embodiment, the hlyCABD operon may further comprise a hlyC gene. HlyC is relevant for the activation of HlyA. However, according to the literature, a region on the 3’ end of HlyC can influence secretion yields, and therefore in another embodiment, the hlyCABD operon may further comprise a functional fragment or portion of the hlyC gene. In one embodiment, the hlyC gene, or functional fragment thereof, is positioned upstream of the heterologous polynucleotide which encodes a cargo molecule upstream of a hlyAs translocation sequence. In another embodiment, the hlyC gene, or functional fragment thereof, is positioned downstream of the independently controlled promoter. In yet another embodiment, the hlyC gene, or functional fragment thereof, is positioned upstream of the heterologous polynucleotide which encodes a cargo molecule upstream of a hlyAs translocation sequence and downstream of the independently controlled promoter.

In an alternative embodiment, after secretion, the cargo molecule retains the HlyAs translocation peptide. In an alternative embodiment, the HlyAs translocation peptide is removed or cleaved from the cargo molecule after secretion. It is envisaged that the secretion of any given cargo molecule may be optimised. In one embodiment, where the cargo molecule is a peptide or a protein, the folding rate of cargo may be modified, as it is suggested in the literature that cargo molecules exhibiting a lower folding rate experience a higher rate of secretion. In another embodiment, the translation efficiency of the cargo molecule may be modified through the adaptation of ribosome binding sequences. In yet another embodiment, the coding sequence may be modified such that one or more codons within the heterologous polynucleotide encoding the cargo molecule may be changed without altering the encoded amino acid (synonymous codon change) due to the redundancy of the genetic code. One of skill in the art will recognise that the folding rate, translational efficiency, and coding sequence can be optimised for each peptide, protein, or gene involved, and will depend on the nature of the cargo molecule (gene, peptide, or protein cargo molecule) and its envisaged use.

The second segment (the ‘structural region’) is envisaged to comprise the hly genes involved in secretion. In the context of the hlyCABD operon, as would be readily appreciated by the skilled person, the hly genes involved in secretion are hlyB and hlyD. Accordingly, in one embodiment, the second segment comprises a hlyB gene and a hlyD gene. The hlyB and hlyD genes are also referred to as the T1SS structural genes. In one embodiment, the hlyB gene is upstream of the hlyD gene. In another embodiment, the hlyB gene is downstream of the independently controlled promoter. In yet another embodiment, the hlyB gene is upstream of the hlyD gene and downstream of the independently controlled promoter.

In a preferred embodiment, the live attenuated Gram-negative bacterium is Salmonella. The live attenuated Gram-negative bacterium may be selected from the group comprising Ty21a, CVD 908-htrA, CVD 909, Ty800, M01ZH09 (also known as ZH9), x9633, x639, x9640, x8444, DTY88, ZH9PA, MD58, WT05, ZH26, SL7838, SL7207, VNP20009, A1-R, or any combinations thereof. In a more preferred embodiment, the live attenuated Gram-negative bacterium is M01ZH09 (also known as ZH9). These live attenuated strains are readily available and would be easily identifiable and commonly used by those in the art. For example, EP 2 801 364 A1 discloses Ty21a, CVD 908-htrA, CVD 909, Ty800, M01ZH09, x9633, X9640, and x8444. In addition, EP 3 917 565 A1 discloses in detail ZH9 strains and derivatives thereof, including ZH9PA. Further references to these strains can be found in the literature, in particular in Petrovska 2004, Hindle 2002, Lehouritis 2017, and Kimura 2010. Also intended to be included are any derivatives or variants of the strains, including genetically engineered or genetically modified strains.

The genetically engineered non-natural bacterium may further comprise one or more gene cassettes. Such gene cassettes may be used to deliver additional prokaryotic molecules to support the function of the genetically engineered nonnatural bacterium to condition the immune system, or to support the activity of the cargo molecule.

The present invention provides a way in which cargo and payload molecules can be delivered to the Sa/mone//a-containing vacuole of eukaryotic cells after bacterial invasion. Accordingly, the present invention provides a bacterial delivery system with broad usability across numerous disease areas. As the skilled person will appreciate, such a system has a significant and wide-reaching therapeutic benefit. The heterologous polypeptide may encode a cargo or payload molecule which may be a therapeutic peptide, therapeutic protein and/or a heterologous antigen (dependent on the indication to be treated). In a preferred embodiment, the therapeutic peptide or protein is a cytokine, a chemokine, an antibody, or a functional fragment thereof, a cytotoxic agent, a cancer agent, or any combination thereof. Even more preferably, the resulting therapeutic protein may be IL-15, IL- 21 , CXCL9, CXCL10, IL-18, IL-27, IFNy, IFNa, IFNp, IL-1 , or any combination thereof. In one embodiment, the live attenuated Gram-negative bacteria comprising a modified hlyCABD operon encoding a therapeutic cargo or payload molecule which is intended to be secreted into the SCV or cytosol of eukaryotic cells. The eukaryotic cells may be mammalian cells. In a preferred embodiment, the eukaryotic cells are human cells. Where the eukaryotic cell is a human cell, the target cell may be a cancerous human cell or a non-cancerous human cell.

The tissue microenvironment is relevant to solid and haematological cancers. As used herein, the terms “tumour microenvironment” or “TME” are used interchangeably and refer to the local environment surrounding a tumour, tumour interstitial space and interstitial fluid. The TME is created by the tumour and is dominated by tumour-induced interactions but may also comprise immune effector cells which have been recruited to the tumour, fibroblasts, signalling molecules, and blood vessels. Therefore, it is envisaged that the live attenuated Gramnegative bacteria can be modified to deliver therapeutically relevant proteins in the interstitial space of the TME in a subject suffering from a tumour.

In one embodiment of the present invention, the live attenuated Gram-negative bacterium is administered intratumourally, peritoumorally, intravenously, intraperitoneally, subcutaneously, intradermally, or orally administered. In a more preferred embodiment, the live attenuated Gram-negative bacterium is administered intratumourally. However, it is also contemplated that other methods of administration may be used in some cases. Therefore, in certain instances the live attenuated Gram-negative bacterium of the present invention may be administered by injection, infusion, continuous infusion, intradermally, intraarterially, intralesionally, intravaginally, intrarectally, intramuscularly, subcutaneously, subconjunctival, mucosally, intrapericardially, intraumbilically, intraocularally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, via a catheter, via a lavage, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990).

The amount of the live attenuated Gram-negative bacterium administered to the subject is sufficient to deliver the cargo molecule in high enough concentrations for it to have the desired effect. The skilled person will readily understand that the precise amount to be administered will be dependent on a number of factors, for example, the disease to be treated and the medical history of the subject to be treated.

The live attenuated Gram-negative bacterium may be administered at a dose of between 10⁵ and 10¹² CFU, where CFU is a colony-forming unit For example, suitable doses may be between 10⁵ and 10⁶ CFU, 10⁵ and 10⁷ CFU, 10⁵ and 10⁸ CFU, 10⁵ and 10⁹ CFU, 10⁵ and 10¹° CFU, 10⁵ and 10¹¹ CFU, 10⁶ and 10⁷ CFU, 10⁶ and 10⁸ CFU, 10⁶ and 10⁹ CFU, 10⁶, and 10¹° CFU, 10⁶ and 10¹¹ CFU, 10⁶ and 10¹² CFU, 10⁷ and 10⁸ CFU, 10⁷ and 10⁹ CFU, 10⁷ and 10¹° CFU, 10⁷ and 10¹¹ CFU, 10⁷ and 10¹² CFU, 10⁸ and 10⁹ CFU, 10⁸ and 10¹° CFU, 10⁸ and 10¹¹ CFU, 10⁸ and 10¹² CFU, 10⁹ and 10¹° CFU, 10⁹ and 10¹¹ CFU, 10⁹ and 10¹² CFU, 10¹° and 10¹¹ CFU, 10¹° and 10¹² CFU, or 10¹¹ and 10¹² CFU. The live attenuated Gram-negative bacterium may be administered in a single dose or in multiple doses. The specific number of doses to be administered are understood to be dependent on the specific cargo molecule to be delivered, as well as the specific indication to be treated.

In one embodiment, the live attenuated Gram-negative bacterium of the present invention is envisaged to allow for the delivery of a therapeutically relevant cargo molecule to the cytosol of eukaryotic cells of the TME in a subject suffering from a tumour. Accordingly, the live attenuated Gram-negative bacterium herein disclosed may be for therapeutic use. For example, the live attenuated Gramnegative bacteria may be used in the treatment, reduction, inhibition, prevention, prevention of recurrence, or control of a disease. In a preferred embodiment the disease is a human disease. In a more preferred embodiment, the disease may be a neoplastic disease, an infectious disease, a cardiovascular disease, a neurodegenerative disease, a gastrointestinal disease, a respiratory disease, a renal disease, a liver disease, an autoimmune disease, an inflammatory disease or a genetic disorder. In a preferred embodiment, the live attenuated Gramnegative bacterium is for use in the treatment, reduction, inhibition, prevention, prevention of recurrence, or control of a neoplastic disease or an infectious disease. Where the disease to be treated is a neoplastic disease, the neoplastic disease may be associated with a solid tumour or haematological tumour. In particular aspects, the neoplastic disease is associated with a cancer selected from prostate cancer, oesophageal cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, endometrial cancer, vulvar cancer, vaginal cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma.

Neoplasia, tumours, and cancers include benign, malignant, metastatic and non- metastatic types, and include any stage (I, II, III, IV or V) or grade (G1 , G2, G3, etc.) of neoplasia, tumour, or cancer, or a neoplasia, tumour, cancer or metastasis that is progressing, worsening, stabilized or in remission. Cancers that may be treated according to the invention include but are not limited to cells or neoplasms of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestines, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to the following: neoplasm, malignant; carcinoma; undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumour, malignant; bronchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumour, malignant; thecoma, malignant; granulosa cell tumour, malignant; androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumour, malignant; lipid cell tumour, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumour; Mullerian mixed tumour; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; Brenner tumour, malignant; phyllodes tumour, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumour of bone; Ewing's sarcoma; odontogenic tumour, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumour; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumour, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. Preferably, the neoplastic disease may be tumours associated with a cancer selected from prostate cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, pancreatic cancer, brain cancer, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, endometrial cancer, vulvar cancer, vaginal cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, head and neck cancer, skin cancer and soft tissue sarcoma and/or other forms of carcinoma. The tumour may be metastatic or a malignant tumour.

In a preferred embodiment, the neoplastic disease is associated with a cancer selected from bladder cancer, prostate cancer, lung cancer, mesothelioma, hepatocellular cancer, melanoma, oesophageal cancer, gastric cancer, endometrial cancer, vulvar cancer, vaginal cancer, cervical cancer, ovarian cancer, colorectal cancer, head and neck cancer or breast cancer.

In an embodiment, the vaccine composition of the present invention may be for therapeutic use. For example, the live attenuated Gram-negative bacterium may be for use in the treatment, reduction, inhibition, prevention, prevention of recurrence, or control of a disease.

It is particularly envisaged that the vaccine composition herein disclosed may be used in the treatment, reduction, inhibition, prevention of recurrence or control of an infectious disease, for example, a disease caused by a bacteria, a virus, a parasite or a fungi. In such instances, the heterologous polynucleotide of the present invention may encode for an antigen of the causative agent of the specific infectious disease in order to produce an immune response in the host. Alternatively, it is envisaged that the vaccine composition herein disclosed may be used as a cancer vaccine. In such instances the vaccine composition comprises Gram-negative bacteria comprising a heterologous polynucleotide encoding a cancer antigen that is capable of producing an immune response in the host. It is therefore appreciated that a wide-range of cancers and infectious diseases can be prevented/treated using the bacterium and methods herein disclosed. In other instances, the heterologous polynucleotide may encode an siRNA or shRNA molecule, which is designed to enhance immune anti-infectious function or tissue anti-infectious defences.

The vaccine composition of the present invention may further comprise an adjuvant, a pharmaceutically acceptable carrier or excipient.

As used herein, "pharmaceutically acceptable camer/adjuvant/diluent/excipient" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavouring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329). Examples include, but are not limited to disodium hydrogen phosphate, soya peptone, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, magnesium sulphate, calcium chloride, sucrose, borate buffer, sterile saline solution (0.9 % NaCI) and sterile water.

Suitable aqueous and non-aqueous carriers that may be employed in the vaccine compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

The vaccine compositions herein disclosed may further contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of unwanted microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminium monostearate and gelatin. The vaccine composition may also optionally include additional therapeutic agents, known to be efficacious in, for example, infectious disease or neoplastic disease. Accordingly, the vaccine composition herein disclosed may also comprise antiretroviral drugs, antibiotics, antifungals, antiparasitics and anticancer agents.

The vaccine composition may also comprise additional components intended for enhancing an immune response in a subject following administration. Examples of such additional components include but are not limited to; aluminium salts such as aluminium hydroxide, aluminium oxide and aluminium phosphate, oil-based adjuvants such as Freund's Complete Adjuvant and Freund's Incomplete Adjuvant, mycolate-based adjuvants (e.g., trehalose dimycolate), bacterial lipopolysaccharide (LPS), peptidoglycans (e.g., mureins, mucopeptides, or glycoproteins such as N-Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans (e.g., extracted from Klebsiella pneumoniae), streptococcal preparations (e.g., OK432), muramyldipeptides, Immune Stimulating Complexes (the "Iscoms" as disclosed in EP 109942, EP 180564 and EP 231 039), saponins, DEAE-dextran, neutral oils (such as miglyol), vegetable oils (such as arachis oil), liposomes, polyols, the Ribi adjuvant system (see, for instance, GB-A-2 189 141 ), vitamin E, Carbopol, interferons (e.g., IFN-alpha, IFN-gamma, or IFN-beta) or interleukins, particularly those that stimulate cell mediated immunity (e.g., IL-2, IL- 3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1 , IL-12, IL-13, IL-14, IL-15, IL-16 and IL-17).

The live attenuated Gram-negative bacterium of the vaccine composition herein disclosed may include any one of, or any combination of the features of the live attenuated Gram-negative bacterium herein disclosed. In a sixth aspect, the present invention provides a method of treating, preventing, inhibiting, preventing recurrence or controlling a disease in a subject, wherein the method comprises administering to a subject a live attenuated Gram-negative bacterium according to any of the first, second, third and fourth aspects.

The method of treating, preventing, inhibiting, preventing recurrence or controlling a disease in a subject of the fifth aspect may comprise one or more of the aforementioned embodiments in respect to any preceding aspect.

In one embodiment, the method of treatment may involve the delivery of a therapeutic molecule. The therapeutic molecule may be a protein or peptide. In another embodiment of the present invention, the therapeutic molecule may be a RNA molecule that is subsequently translated into a protein or peptide. The term “therapeutic molecule” refers to any molecule that may result in the reduction, amelioration, palliation, lessening, delaying, and/or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, in the context of cancer, a therapeutic molecule may be one which results in the size of a tumour shrinking.

The live attenuated Gram-negative bacteria comprising the modified hylCABD operon of the present invention may also have applications in allowing for the safe, efficient, and reliable delivery of RNA molecules into target eukaryotic cells. Accordingly, in one embodiment, a method for delivering an RNA molecule into a eukaryotic cell using the modified live attenuated Gram-negative bacterium herein disclosed is provided. As used herein, the term “bactofection” refers to the process of transduction of genetic material from a bacterium (e.g., Salmonella) into a mammalian cell. Specifically, the term “bactofection” in the context of the present invention refers to the use of live attenuated Gram-negative bacteria to deliver RNA molecules to the cytosol of a eukaryotic cell following delivery of the live attenuated Gram-negative bacteria to the target eukaryotic cell. SPI-2 promoters can be used to activate the expression of Type 1 Secretion Systems (T1SS) which facilitate the export of hemolysins from Listeria and enable, for example, the release of bacterial cells from a vacuole into the cytoplasm of a eukaryotic cell, thus granting bacteria access to the cytoplasm of a eukaryotic cell.

In a seventh aspect, the present invention provides a use of a live attenuated Gram-negative bacterium according to any of the first, second, third and fourth aspects, in the manufacture of a medicament.

EXAMPLES

Example 1 - Construction of a combinatorial library with different promoters controlling T1SS cargo and structural genes expression

In order to initially navigate the optimal expression ratio between secretable elements (e.g., mScarlet fused with T1SS signal peptide hlyAs, fused at the C- terminus end) and structural elements (i.e., HlyB and HlyD) these two moieties were separated by a bidirectional terminator (BBa_B1006) and controlled independently by promoters of different strengths (pro1, proA, proB, proC). Additionally, the gene hlyC, encoded upstream of hlyA in the native genomic island, has been reported to contain encoded regions that affect the secretion efficiency of hlyA or any other cargo. Thus, two variants of the secretion moiety were generated: one with hlyC and one without hlyC. All of this considered, a total of 32 plasmids were designed, each with a unique P1/P2 and hlyC composition.

Fragments were as dsDNA blocks through integrated DNA technologies (IDT). The dsDNA blocks were cloned into pJET plasmid and validated by sequencing and further used as reference material. Fragments for Golden Gate Assembly (GGA) were generated from validated plasmids via PCR. The plasmid library was assembled via Bsal-dependent GGA and using the ECHO 525 Liquid Handling Platform to perform the reaction mixtures, on a 96-well PCR plate (reaction volume, 5 pL) with mixture DNA volume brought to 3 pL and topped up with 2 pL of NEB Bridge + Bsal (1.667 pL NEB Bridge and 0.333 pL of Bsal) per reaction. To ensure a successful reaction, after the mixture samples were mixed properly, centrifuged, and cycled at 37°C, 4 min and 16°C, 2 min for 30 cycles (~4h run). The resulting assemblies were transformed into DH5a (2 pL only).

Plasmid assembly was initially confirmed visually (no red background colonies) and then 2x each were grown overnight. The plasmid was miniprepped and validated via PCR using primers T1SSVal_PF/R01 (CGACTGAGCCTTTCGTTTTATTTGATGCC (SEQ ID NO: 15),

GGTCATTACTGGATCTATCAACAGGAGTCCAAG (SEQ ID NO: 16), TA = 58°C, text = 35 sec, amplicon = 6379/5866 bp) with CloneAmp. Successfully amplified plasmids were sent for sequencing (one clone per plasmid) with primers T1SSVal_PF01 and SQ_mscarlet_for (gcatggacgaactgtataagggatcc (SEQ ID NO: 17).

Example 2 - Evaluation of the expression landscape of T1SS

Plasmids from the library were transformed into DH5a cells via Heat Shock (standard protocol), plated in vLBA and grown at 37°C overnight. A total of 3x single colonies per construct were picked and inoculated in 1 mL of vLBA on a 96- deep well plate supplemented with chloramphenicol 12.5 pg mL'¹ and grown overnight at 200rpm and 37°C. Samples were then diluted down 1 :100 in 500 uL of vLBA supplemented with Chloramphenicol. A volume of 100 uL was transferred to a 96-well plate and used to monitor growth over time on a ClarioStar by shaking at 700rpm and at 37°C. The remaining volume was grown on the deep-well plate under the same conditions.

Hi Bit assay was performed on supernatant from bacterial cultures grown at 37°C in the deep-well plate. Samples were allowed to reach OD₆oo ~ 0.5 and then an aliquot was taken and processed as: (1) an aliquot was extracted from the deep- well plate (100 pL) and used to measure the cell density (OD₆oo), (2) cells were spun down from all plates at 4000xg for 10 min at room temperature, (3) the supernatant was carefully transferred to a fresh plate, (4) 10 pL of the supernatant were mixed with HiBit MasterMix (10 pL) previously prepared and used to determine the amount of HiBit tag in the supernatant by measuring luminescence.

There were no significant differences in OD₆oo. When analysing with HiBit Assay, the results showed that increasing strength of P1 (cargo promoter) resulted in higher cargo presence in the supernatant, independent of the expression of structural genes from P2. This can be explained by the self-secreting ability of fluorescent proteins and protein released from cell turnover. Therefore, the results were normalised to the P1 strength to assess the effect of increased P2 strength over secretion. In this, secretion is enhanced at higher levels of structural genes. The experiment was repeated using further 3x independent clones, different than those already tested. The results obtained are also consistent with the previously obtained data.

Example 3 - Exchange of mScarlet cargo for LLO results in reproducible secretion outcomes

The plasmid library with LLO instead of mScarlet was obtained by performing Bbsl-dependent Golden Gate Assembly on a 96-well PCR plate (reaction volume, 2uL) of the previously generated library and a block encoding hly from Listeria monocytogenes surrounded by appropriate Bbsl sites. The conditions used the were standard ones (0.67 pL of NEB Bridge, 0.264 pL of 10 pM hly insert, 0.5 pL of 2.5 pM vector, 0.132 pL of Bbsl, 0.434 pL of water). To ensure a successful reaction, after the mixture samples were mixed properly, centrifuged, and cycled at 37°C for 4 min and 16°C for 2 min for 30 cycles (~4h run). The resulting assemblies were transformed into DH5a. Plasmid assembly was confirmed by sequencing.

Validated plasmids from the library were transformed into DH5alpha cells via Heat Shock (standard protocol), plated in vLBA, and grown at 37°C overnight. A total of 3x single colonies per construct were picked and inoculated in 1 mL of vLBA on a 96-deep well plate supplemented with chloramphenicol 12.5 ug ml_’¹ and grown overnight at 200 rpm and 37°C. Samples were then diluted down 1 :100 in 500 uL of vLBA supplemented with Chloramphenicol. Avolume of 100 uL was transferred to a 96-well plate and used to monitor growth over time on a ClarioStar by shaking at 700rpm and 37°C. The remaining volume was grown on the deep well plate under the same conditions.

Cells were processed as previously described (see previous section) and the secretion in all samples measured using the HiBit-induced luminescence. Overall, all samples grew similarly (same range of endpoint OD₆oo). As previously observed, higher strength of p2 (T1SS genes) results in higher export rates. Interestingly, higher p1 also typically results in higher export, contradicting the hypothesis of "less is more" when expressing cargo cytosolically.

Finally, as it has been observed elsewhere, the substitution of fluorescent protein (mScarlet) for a complex cargo (hly) results in circa 10-fold decrease of exported cargo - this is possibly due to better expression rates of fluorescent protein as well as an inherent ability to cross membranes.

Example 4 - Dual-plasmid expression system

To provide a higher level of control over the expression levels of the two segments, a dual-plasmid expression system was designed and built. It should be noted however that the two segments are not required to be separated into two different plasmids, and a single plasmid may be used provided there is a form of separation between the segments (e.g., a terminator sequence or any other suitable means). Nonetheless, in the dual plasmid expression system, a cargo-containing plasmid is integrated into a p15A ori plasmid (circa 10 copies I cell) that allows for in-frame cloning (Bbsl-dependent) of any relevant cargo with the hlyAs sequence. Additional control over the expression levels can be achieved by Bsal-dependent cloning of a promoter of interest. Export of such cargo is then achieved by introduction of a second plasmid with a compatible origin of replication (e.g., pSC101 , ~5 copies I cell) expressing the hlyBD operon under the control of a relevant promoter, which has been introduced via Bsal-dependent Golden Gate, as previously done with cargo-hlyAs fusion. Both plasmids were built using the same eBlocks previously employed for the one-plasmid system.

To test the system, three cargo plasmids with increasing relative expression strength were built by cloning pro1 (hlyAs-1), proC (hlyAs-4), and J23119 (hlyAs- 6) promoters upstream of an mscarlet-hlyAs fusion. In parallel, three plasmids with hlyBD (hlyBD-1 , hlyBD-4, hlyBD-6) were built under the control of the same promoters. These were co-transformed into Salmonella enterica ZH9 bacteria by electroporation, using standard protocol and 100 ng of each vector, and selected in LB Agar media supplemented with 25 pg mL'¹ of each antibiotic (Carbenicil lin and Kanamycin) for 16 h at 37 °C. Single plasmid (only cargo) controls were also transformed and selected in Carbenicillin.

Single colonies were then picked (3 per construct) and inoculated into LB media supplemented with appropriate antibiotics at the same concentration as in solid media. Samples were grown in 96-deep well plates for 16 h and diluted 1 :500 in 1 mL of fresh media. Growth was then monitored from a 100 pL aliquot grown at 700 rpm and 37 °C for 16 h and the remaining culture was grown in the same conditions in a shaking incubator. After growth, samples were spun down at 4000xg for 15 min and the supernatant transferred into a fresh 96-well plate. A 10 pL aliquot of each supernatant was then subject to HiBit analysis as specified previously.

The results herein disclosed therefore support the use of a two-segmented operon, for example in a dual-plasmid expression system, which allows for optimizing yields without over-burdening the bacterial carrier.

Example 5 - Identification of hlyBD expression level as main driver of cargo secretion

In order to test whether the expression of the secretion or cargo segment boosted cargo secretion, levels of LLO in culture supernatant from cultures of ZH9 carrying dual plasmids with Ho on a p15A oh plasmid under the control of C1 , C3, or C6, and a pSC101 ori plasmid containing hlyB and hlyD under the control of C1 , C3, C4, C5 or C6 (hlyBD1-5) were tested in a HiBit assay (Promega) that detects the HiBit tag on the N-terminus of LLO (Figure 6). The results of this assay demonstrated that whilst increasing the expression of llo contributed to yield of export, the major parameter responsible for high titres is a strong expression of the hlyBD segment.

Example 6 - CCF4 vacuole escape assay

A cell-based assay based on the addition of the CCF4 reagent was developed to validate the vacuole escape nature of Salmonella ZH9 strains bearing the in vivo- inducible circuits described above. CCF4 is a Forster Resonance Energy Transfer (FRET) substrate composed of a cephalosporin core linking 7-hydroxycummarine to fluorescein. After administration, the substrate accumulates within the cytosol of mammalian cells. Cleavage of the beta-lactam ring through a Beta-lactamase enzyme (provided by the bacterial strain) causes the split and release of fluorescein, shifting the emission wavelength from 520 nm to 447 nm after excitation with a 409 nm wavelength.

Bacterial invasion stocks were prepared by subculturing 1 :100 of a pre-culture of the desired Salmonella bacterial strain in 10 mL of Lysogeny Broth (Miller) supplemented with 100 pg/mL of Carbenicillin/Ampicillin, followed by growth at 37 °C and 250 rpm for approximately 3 h (OD₆oo~0.8). Bacterial cells were spun down and resuspended in 1 mL of 10 % ice-cold glycerol. Stocks were aliquoted and flash-frozen, and stored at -80 °C until required.

In the CCF4 cell-based assay, 15,000 HeLa cells were seeded overnight in a black-walled 96 well plates. On day of invasion, bacterial invasion stocks were thawed and diluted in cell culture media to appropriate concentrations to give an MOI of 200. Bacterial dilutions were added to HeLa cells and incubated for 1 h at 37 °C. Then, the HeLa cells were washed with PBS three times, and incubated overnight in cell culture media containing 50pg/mL Gentamicin. At 24 h post invasion, cell culture media was aspirated and replaced with 100pL fresh media. LiveBLAzer™ FRET-B/G Loading Kit with CCF4-AM (Thermofisher; K1095) was prepared with Solution D (Thermofisher; K1156) according to the manufacturer’s instructions. 20 pL of CCF4 was then added to each well and incubated for 45 min at room temperature in the dark. The media was replaced with fresh media and imaged on a Leica DMi8 inverted microscope. Fluorescence images were captured using two filter sets: Blue (excitation -405 nm; emission -450 nm) and Green (excitation -405 nm; emission -535). Image analysis was performed in Cell Profiler (www.cellprofiler.org). The number of blue cells and number of green cells was counted per well and the ratio of blue to green cells calculated to give a percentage of vacuole escape.

Invasiveness of the tested strains was evaluated in parallel with the CCF4 assay by performing replica invasion experiments on 15,000 HeLa cells previously seeded overnight, and as described above. At 24 h post-invasion, cell culture media was aspirated from each well and the cells washed once with 200 pL/well of sterile DPBS. Cells were detached by removing the DPBS wash and replacing with 200 pL/well Trypsin EDTA, followed by incubation at 37 °C and 5% CO2 for 2 to 3 min. Medium was neutralised by adding 70 pL/well of 2%FBS/medium. Detached cells were transferred into a U-shaped 96-well plate and spun down at 300 xg for 5 min. Supernatant was removed and cells permeabilised and dyed for 30 min with CSA-I-FITC antibody. After wash, cells were resuspended in ice-cold FACS buffer and analysed by flow cytometry to detect FITC⁺ cell harbouring Salmonella. Non-invasive samples were discarded from further analysis by CCF4 (Figure 7).

Example 7 - Construction of in vivo-inducible LLO export circuits

As shown in Figure 8, a number of modified operons were designed and built in which each of the two segments was operably linked to a strong promoter, in particular a strong constitutive or strong vacuole-inducible promoter as described herein.

Type 1 Secretion System (T1SS) investigated is based on the expression of two genes, hlyB and hlyD, encoding the secretion machinery, and the fusion of the hlyA signal sequence (hlyAs) to a recombinant cargo of interest. Previous work where the T1SS was split into two segments (cargo and secretion) controlled by independent promoters suggested that ensuring strong expression from hlyBD was a critical parameter to optimise the yield of cargo secretion. Here, these findings were confirmed by evaluating LLO secretion at 24 h post-inoculation of Salmonella ZH9 strains bearing the dual-plasmid circuit with lysterolysin O- encoding gene (//o) fused to hlyAs in a p15A oh plasmid (Carbenicillin-resistant, bla) under the control of C1 , C3, or C6; and a pSC101 oh plasmid (Kanamycin- resistant, aac3(IV)) with hlyBD under the control of C1 C6. Results showed that whilst increasing the expression of llo contributed to yield of export, the major parameter responsible for high titres is a strong expression of the hlyBD segment.

Whilst these circuits bearing constitutive promoters are useful in determining the optimal circuit parameters to optimise cargo secretion, they tend to introduce burden to the bacterial cells, derived from the both the dual-plasmids and constitutive expression, which make them less able to invade host cells (e.g., cancer cells) and thus ineffective for intracellular delivery of therapeutics (protein or nucleic acids) against cancer via bacterial carriers. To overcome this limitation, promoters that are induced in the Sa/mone//a-containing vacuole (SCV) offer a strategy to induce the secretion systems in vivo. A set of single-plasmid systems were constructed that contained the cargo segment (llo or mscarlet) under control of constitutive (C3 or C6) or vacuole-inducible (pipB or sseA) promoters, and the secretion machinery segment (hlyBD) under the control of strong constitutive (C6) or vacuole-inducible (ssaG, sseJ, or sseA) promoters. Plasmid assembly was done via Gibson of PCR-amplified fragments onto a p15A oh plasmid (Carbenicillin resistant, bla).

Example 8 - reproducible vacuole escape with T1SS on optimised modified operons

Figure 9 shows the results of blue cell counts at 24 h post-invasion of HeLa cells with ZH9 strains bearing the vacuole escape operons designed as described in Example 7. The results showed that strong expression of llo (either under constitutive C6 or vacuole-inducible vac12) results in forced vacuole escape when combined with strong expression of hlyBD operon. Of note is that strong constitutive expression (C6) of both the llo and hlyBD segments abolished the escape phenotype.

SEQUENCES FORMING PART OF THE DESCRIPTION

SEQ ID NO: 1 - hly(LLO) (DNA sequence)

GCTAGGAGTCGTCTGGTGGAAGACTAGCGATGGCAAAGGATGCATCTGCATTCAATAAA GAAAAT T C AAT T T CAT C CAT G G CAC C AC C AGC AT CTCCGCCT GC AAGT C C T AAGAC G C C AAT C GAAAAGAAAC AC G C GGAT GAAAT C GAT AAG T AT AT AC AAG GAT T G GAT T ACAAT A AAAACAATGTATTAGTATACCACGGAGATGCAGTGACAAATGTGCCGCCAAGAAAAGGT TACAAAGAT GGAAATGAATATATT GT TGT GGAGAAAAAGAAGAAAT CCAT CAAT CAAAA TAATGCAGACATTCAAGTTGTGAATGCAATTTCGAGCCTAACCTATCCAGGTGCTCTCG TAAAAGCGAATTCGGAATTAGTAGAAAATCAACCAGATGTTCTCCCTGTAAAACGTGAT TCATTAACACTCAGCATTGATTTGCCAGGTATGACTAATCAAGACAATAAAATCGTTGT AAAAAATGCCACTAAAT CAAACGT TAACAACGCAGTAAATACAT TAGT GGAAAGAT GGA ATGAAAAATATGCTCAAGCTTATCCAAATGTAAGTGCAAAAATTGATTATGATGACGAA ATGGCTTACAGTGAATCACAATTAATTGCGAAATTTGGTACAGCATTTAAAGCTGTAAA TAATAGCT T GAAT GTAAACT T CGGCGCAATCAGT GAAGGGAAAATGCAAGAAGAAGT CA TTAGTTTTAAACAAATTTACTATAACGTGAATGTTAATGAACCTACAAGACCTTCCAGA TTTTTCGGCAAAGCTGTTACTAAAGAGCAGTTGCAAGCGCTTGGAGTGAATGCAGAAAA TCCTCCTGCATATATCTCAAGTGTGGCGTATGGCCGTCAAGTTTATTTGAAATTATCAA CTAATTCCCATAGTACTAAAGTAAAAGCTGCTTTTGATGCTGCCGTAAGCGGAAAATCT GTCTCAGGTGATGTAGAACTAACAAATATCATCAAAAATTCTTCCTTCAAAGCCGTAAT TTACGGAGGTTCCGCAAAAGATGAAGTTCAAATCATCGACGGCAACCTCGGAGACTTAC GCGATATTTTGAAAAAAGGCGCTACTTTTAATCGAGAAACACCAGGAGTTCCCATTGCT TATACAACAAACT T CCTAAAAGACAATGAATTAGCT GT TAT TAAAAACAACT CAGAATA T AT T GAAAC AAC T T CAAAAG C T T AT ACAGAT G GAAAAAT T AACAT C GAT CAC T C T G GAG GATACGTTGCTCAATTCAACATTTCTTGGGATGAAGTAAATTATGATCCTGAAGGTAAC GAAATTGTTCAACATAAAAACTGGAGCGAAAACAATAAAAGCAAGCTAGCTCATTTCAC ATCGTCCATCTATTTGCCAGGTAACGCGAGAAATATTAATGTTTACGCTAAAGAATGCA CTGGTTTAGCTTGGGAATGGTGGAGAACGGTAATTGATGACCGGAACTTACCACTTGTG AAAAATAGAAATATCTCCATCTGGGGCACCACGCTTTATCCGAAATATAGTAATAAAGT AGATAATCCAATCGAATATGCAGTGAGCTGGTCTTCCTATCGGGTGGTTGCGAAG

SEQ ID NO: 2 - LLO (amino acid sequence) MAKDASAFNKENS I S SMAPPAS PPAS PKT P IEKKHADE I DKYI QGLDYNKNNVLVYHGD AVTNVPPRKGYKDGNEYIWEKKKKS INQNNADIQWNAI SSLTYPGALVKANSELVEN QPDVLPVKRDSLTLSIDLPGMTNQDNKIWKNATKSNVNNAVNTLVERWNEKYAQAYPN VSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAI SEGKMQEEVI SFKQIYYNV NVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYI SSVAYGRQVYLKLSTNSHSTKVKA AFDAAVSGKSVS GDVELTNI I KNS S FKAVI YGGSAKDEVQI I DGNLGDLRDI LKKGAT F NRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNI SW DEVNYDPEGNEIVQHKNWSENNKSKLAHFTSS IYLPGNARNINVYAKECTGLAWEWWRT

VIDDRNLPLVKNRNIS IWGTTLYPKYSNKVDNPIE

SEQ ID NO: 3 - Pro1_1

CTCGGTCCCCAGGCATTACTAGAGTCACACTGGCTCACCTTCGGGTGGGC

CTTTCTGCGTTTATAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCT

AGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCGGT

ATCTATATTCAGGCACAGCACAACGGTTTCCTTTTAGCTGTCACCGGATGT GCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATT TTGTTTAAAAAG

SEQ ID NO: 4 - PROA_1

CTCGGTCCCCAGGCATTACTAGAGTCACACTGGCTCACCTTCGGGTGGGC

CTTTCTGCGTTTATAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCT

AGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCGTA

GGCTATATTCAGGCACAGCACAACGGTTTCCTTTTAGCTGTCACCGGATGT

GCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATT TTGTTTAAAAAG

SEQ ID NO: 5 - PROB_1

CTCGGTCCCCAGGCATTACTAGAGTCACACTGGCTCACCTTCGGGTGGGC

CTTTCTGCGTTTATAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCT

AGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCGTA ATATATATTCAGGCACAGCACAACGGTTTCCTTTTAGCTGTCACCGGATGT

GCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATT TTGTTTAAAAAG

SEQ ID NO: 6 - PROC_1

CTCGGTCCCCAGGCATTACTAGAGTCACACTGGCTCACCTTCGGGTGGGC

CTTTCTGCGTTTATAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCT

AGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAGGCTCGTA

TGATATATTCAGGCACAGCACAACGGTTTCCTTTTAGCTGTCACCGGATGT

GCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATT TTGTTTAAAAAG

SEQ ID NO: 7 - PRO1_2

GTAAGTCCCCAGGCATTACTAGAGTCACACTAAAAAAAAACCCCGCCCCTG

ACAGGGCGGGGTTTTTTTTTTTATAGCACAGCTAACACCACGTCGTCCCTA

TCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCA

TAAGGCTCGGTATCTATATTCAGGCACAGCACAACGGTTTCCTTTTAGTCC

GTAGTGGATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTC

T ACAAAT AAT T T T G T T T AACAC A

SEQ ID NO: 8 PROA_2

GTAAGTCCCCAGGCATTACTAGAGTCACACTAAAAAAAAACCCCGCCCCTG

ACAGGGCGGGGTTTTTTTTTTTATAGCACAGCTAACACCACGTCGTCCCTA

TCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCA

TAAGGCTCGTAGGCTATATTCAGGCACAGCACAACGGTTTCCTTTTAGTCC

GTAGTGGATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTC

T ACAAAT AAT T T T G T T T AACAC A

SEQ ID NO: 9 - PR0B_2 GTAAGTCCCCAGGCATTACTAGAGTCACACTAAAAAAAAACCCCGCCCCTG

ACAGGGCGGGGTTTTTTTTTTTATAGCACAGCTAACACCACGTCGTCCCTA

TCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCA

TAAGGCTCGTAATATATATTCAGGCACAGCACAACGGTTTCCTTTTAGTCC

GTAGTGGATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTC

T ACAAAT AAT T T T G T T T AACAC A

SEQ ID NO: 10 - PROC_2

GTAAGTCCCCAGGCATTACTAGAGTCACACTAAAAAAAAACCCCGCCCCTG

ACAGGGCGGGGTTTTTTTTTTTATAGCACAGCTAACACCACGTCGTCCCTA

TCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCA

TAAGGCTCGTATGATATATTCAGGCACAGCACAACGGTTTCCTTTTAGTCC

GTAGTGGATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTC

T ACAAAT AAT T T T G T T T AACAC A

SEQ ID NO: 11 - hlyA_s

GCTAGGAGTCGTCTGGTGGGTCTCTAAAGACAACGGTTTCCCTCTAGAAATAATTTTGT

TTAACTTTAAGAAGGAGATATATTCATGGCTGTGAGCGGCTGGCGTCTGTTCAAGAAAA

TTAGCGCGACCGTCTTCGGCGGTGGTGGCAGCGTTAGCAAAGGCGAGGCGGTTATCAAG

GAGTTTATGCGTTTTAAGGTTCACATGGAGGGTAGCATGAATGGTCACGAGTTCGAGAT

CGAGGGTGAAGGCGAGGGTCGTCCGTACGAAGGCACCCAGACCGCGAAGCTGAAAGTGA

CCAAGGGTGGCCCGCTGCCGTTCAGCTGGGACATCCTGAGCCCGCAGTTCATGTATGGC

AGCCGTGCGTTTACCAAACACCCGGCGGACATTCCGGATTACTATAAGCAAAGCTTCCC

GGAAGGTTTTAAATGGGAGCGTGTTATGAACTTCGAAGATGGTGGCGCGGTGACCGTTA

CCCAGGACACCAGCCTGGAGGATGGCACCCTGATTTACAAGGTGAAACTGCGTGGCACC

AACTTTCCGCCGGATGGTCCGGTTATGCAGAAGAAAACGATGGGTTGGGAAGCGAGCAC

CGAGCGTCTGTATCCGGAAGATGGCGTGCTGAAGGGTGATATCAAAATGGCGCTGCGTC

TGAAGGACGGTGGCCGTTACCTGGCGGATTTTAAGACCACCTATAAAGCGAAGAAACCG

GTGCAAATGCCGGGTGCGTACAACGTTGACCGTAAACTGGATATTACCAGCCACAACGA

GGATTATACCGTGGTTGAGCAATATGAGCGTAGCGAGGGTCGCCACAGCACCGGCGGCA

TGGACGAACTGTATAAGGGATCCGAAGACGCGAGCACGCCCGGGGGTGCGCCGGTGCCG TATCCGGATCCGCTGGAACCGGCCGGGGAAAATTCTCTTGCTAAAAATGTATTATCCGG TGGAAAAGGTAATGACAAGTTGTACGGCAGTGAGGGAGCAGACCTGCTTGATGGCGGAG AAGGGAATGATCTTCTGAAAGGTGGATATGGTAATGATATTTATCGTTATCTTTCAGGA TATGGCCATCATATTATTGACGATGAAGGGGGGAAAGACGATAAACTCAGTTTAGCTGA TATAGATTTCCGGGACGTTGCCTTTAAGCGAGAAGGGAATGACCTCATTATGTATAAAG CTGAAGGTAATGTTCTTTCTATTGGCCACAAAAATGGTATTACATTTAAAAACTGGTTT GAAAAAGAG T CAGAT GAT C T C T C T AAT CAT CAGAT AGAGC AGAT T T T T GAT AAAGAC G G CAGGGTAATCACACCAGATTCTCTTAAAAAAGCATTTGAATATCAGCAGAGTAATAACA AGGTAAGTTATGTGTATGGACATGATGCATCAACTTATGGGAGCCAGGACAATCTTAAT CCATTAATTAATGAAATCAGCAAAATCATTTCAGCTGCAGGTAACTTCGATGTTAAGGA GGAAAGATCTGCCGCTTCTTTATTGCAGTTGTCCGGTAATGCCAGTGATTTTTCATATG GACGGAACTCAATAACTTTGACAGCATCAGCATAATATATTAGTAATGAGACCTATCGG GTGGTTGCGAAG

SEQ ID NO: 2 - hlyCAs

GCTAGGAGTCGTCTGGTGGGTCTCTAAAGATATTTTTGCCACAATATTTAATCATATAA T T TAAGTT GTAGT GAGT T TAT TAT GAATATAAACAAACCAT TAGAGAT T CTT GGGCAT G TATCCTGGCTATGGGCCAGTTCTCCACTACACAGAAACTGGCCAGTATCTTTGTTTGCA ATAAATGTATTACCCGCAATACAGGCTAACCAATATGTTTTATTAACCCGGGATGATTA CCCTGTCGCGTATTGTAGTTGGGCTAATTTAAGTTTAGAAAATGAAATTAAATATCTTA ATGATGTTACCTCATTAGTTGCAGAGGACTGGACTTCAGGTGATCGTAAATGGTTCATT GACTGGATTGCTCCTTTCGGGGATAACGGTGCCCTGTACAAATATATGCGAAAAAAATT CCCTGATGAACTATTCAGAGCCATCAGGGTGGATCCCAAAACTCATGTTGGTAAAGTAT CAGAAT TT CAT GGAGGTAAAATT GATAAACAGTTAGCGAATAAAAT T TT TAAACAATAT CACCACGAGTTAATAACTGAAGTAAAAAGAAAGTCAGATTTTAATTTTTCATTAACTGG TTAAACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATATTC ATGGCTGTGAGCGGCTGGCGTCTGTTCAAGAAAATTAGCGCGACCGTCTTCGGCGGTGG TGGCAGCGTTAGCAAAGGCGAGGCGGTTATCAAGGAGTTTATGCGTTTTAAGGTTCACA TGGAGGGTAGCATGAATGGTCACGAGTTCGAGATCGAGGGTGAAGGCGAGGGTCGTCCG TACGAAGGCACCCAGACCGCGAAGCTGAAAGTGACCAAGGGTGGCCCGCTGCCGTTCAG CTGGGACATCCTGAGCCCGCAGTTCATGTATGGCAGCCGTGCGTTTACCAAACACCCGG CGGACATTCCGGATTACTATAAGCAAAGCTTCCCGGAAGGTTTTAAATGGGAGCGTGTT ATGAACTTCGAAGATGGTGGCGCGGTGACCGTTACCCAGGACACCAGCCTGGAGGATGG CACCCTGATTTACAAGGTGAAACTGCGTGGCACCAACTTTCCGCCGGATGGTCCGGTTA TGCAGAAGAAAACGATGGGTTGGGAAGCGAGCACCGAGCGTCTGTATCCGGAAGATGGC GTGCTGAAGGGTGATATCAAAATGGCGCTGCGTCTGAAGGACGGTGGCCGTTACCTGGC GGATTTTAAGACCACCTATAAAGCGAAGAAACCGGTGCAAATGCCGGGTGCGTACAACG TTGACCGTAAACTGGATATTACCAGCCACAACGAGGATTATACCGTGGTTGAGCAATAT GAGCGTAGCGAGGGTCGCCACAGCACCGGCGGCATGGACGAACTGTATAAGGGATCCGA AGACGCGAGCACGCCCGGGGGTGCGCCGGTGCCGTATCCGGATCCGCTGGAACCGGCCG GGGAAAATTCTCTTGCTAAAAATGTATTATCCGGTGGAAAAGGTAATGACAAGTTGTAC GGCAGTGAGGGAGCAGACCTGCTTGATGGCGGAGAAGGGAATGATCTTCTGAAAGGTGG ATATGGTAATGATATTTATCGTTATCTTTCAGGATATGGCCATCATATTATTGACGATG AAGGGGGGAAAGACGATAAACTCAGTTTAGCTGATATAGATTTCCGGGACGTTGCCTTT AAGCGAGAAGGGAATGACCTCATTATGTATAAAGCTGAAGGTAATGTTCTTTCTATTGG C C AC AAAAAT GG T AT T AC AT T T AAAAAC T G GT T T GAAAAAGAGT C AGAT GAT C T C T C T A ATCATCAGATAGAGCAGATTTTTGATAAAGACGGCAGGGTAATCACACCAGATTCTCTT AAAAAAGCAT TT GAATAT CAGCAGAGTAATAACAAGGTAAGT TAT GTGTATGGACAT GA T GCAT CAACT TAT GGGAGCCAGGACAAT CT TAAT CCAT TAAT TAAT GAAATCAGCAAAA TCATTTCAGCTGCAGGTAACTTCGATGTTAAGGAGGAAAGATCTGCCGCTTCTTTATTG CAGTTGTCCGGTAATGCCAGTGATTTTTCATATGGACGGAACTCAATAACTTTGACAGC

ATCAGCATAATATATTAGTAATGAGACCTATCGGGTGGTTGCGAAG

SEQ ID NO: 13 - hlyB

GCTAGGAGTCGTCTGGTGGGTCTCTCACAGATATTTTTTTGGAGTCATAATGGATTCTT GTCATAAAATTGATTATGGGTTATACGCCCTGGAGATTTTAGCCCAATACCATAACGTG TCTGTTAACCCGGAAGAAATTAAACATAGATTTGACACAGACGGGACTGGTCTGGGATT AACGTCATGGTTGCTTGCTGCGAAATCTTTAGAACTAAAGGTAAAACAGGTAAAAAAAA CAATTGACCGATTAAACTTTATTTCTCTGCCCGCATTAGTCTGGAGAGAGGATGGACGT CAT T T T AT T C T GAC T AAAGT C AGT AAAGAAGC AAAC AGAT AT CTTATTTTT GAT C T G GA GCAGCGAAATCCCCGTGTTCTCGAACAGTCTGAGTTTGAGGCGTTATATCAGGGGCATA TTATTCTTATCGCTTCCCGTTCTTCTGTGGCGGGCAAACTGGCGAAATTTGACTTTACC TGGTTTATTCCTGCCATTATAAAATACAGGAGAATATTTATTGAAACCCTTGTTGTGTC TGTTTTTTTACAATTATTTGCATTAATAACCCCCCTTTTTTTTCAGGTGGTTATGGACA AAGTATTAGTGCACAGGGGATTTTCAACTCTTAATGTTATTACTGTCGCATTATCTGTT GTGGTGGTGTTTGAGATTATACTCAGCGGTTTAAGAACTTACATTTTTGCACATAGTAC AAGTCGGATTGATGTTGAGTTGGGTGCCAAACTCTTCCGGCATTTACTGGCGCTACCGA TCTCTTATTTTGAGAGTCGTCGTGTTGGTGATACTGTTGCCAGGGTAAGAGAATTAGAC CAGATCCGTAATTTTCTGACAGGACAGGCATTAACATCTGTTCTGGACTTATTATTTTC ATTCATATTTTTTGCGGTAATGTGGTATTACAGTCCAAAGCTTACTCTGGTGATCTTAT TTTCGCTGCCTTGTTATGCTGCATGGTCTGTTTTTATTAGCCCCATTTTGCGACGTCGC CTTGATGATAAGTTTTCACGGAATGCGGATAATCAATCTTTCCTGGTGGAATCAGTCAC GGCGAT TAACACTATAAAAGCTAT GGCAGT CT CACCT CAGAT GACGAACATAT GGGACA AACAATTGGCAGGATATGTTGCTGCAGGCTTCAAAGTGACAGTATTAGCAACCATTGGT CAACAAGGAATACAGT T AAT ACAAAAGAC T GT TAT GAT CAT CAAC C T GT GGT T GGGAGC ACACCTGGTTATTTCCGGGGATTTAAGTATTGGTCAGTTAATTGCTTTTAATATGCTTG CTGGTCAGATTGTTGCACCGGTTATTCGCCTTGCACAAATCTGGCAGGATTTCCAGCAG GTTGGTATATCAGTTACCCGCCTTGGTGATGTGCTTAACTCTCCAACTGAAAGTTATCA TGGGAAACTGGCATTACCGGAAATTAATGGTGATATCACTTTTCGTAATATCCGGTTTC GCTATAAGCCTGACTCTCCGGTTATTTTAGATAATATCAATCTCAGTATTAAGCAGGGG GAGGTTATTGGTATTGTCGGACGTTCTGGTTCAGGAAAAAGCACATTAACTAAATTAAT TCAACGTTTTTATATTCCTGAAAATGGCCAGGTCTTAATTGATGGACATGATCTTGCGT TGGCCGATCCTAACTGGTTACGTCGTCAGGTGGGGGTTGTGTTGCAGGACAATGTGCTG CTTAATCGCAGTATTATTGATAATATCTCACTGGCTAATCCTGGTATGTCCGTCGAAAA AGTTATTTATGCAGCGAAATTAGCAGGCGCTCATGATTTTATTTCTGAATTGCGTGAGG GGTATAACACCATTGTCGGGGAACAGGGGGCAGGATTATCCGGAGGTCAACGTCAACGC ATCGCAATTGCAAGGGCGCTGGTGAACAACCCTAAAATACTTATTTTTGATGAAGCAAC CAGTGCTCTGGATTATGAGTCGGAGCATATCATCATGCGCAATATGCACAAAATATGTA AGGGCAGAACGGTTATAATCATTGCTCATCGTCTGTCTACAGTAAAAAATGCAGACCGC AT TAT T GT CATGGAAAAAGGGAAAAT TGT T GAACAGGGTAAACATAAGGAACT GCT T T C T GAAC C GGAAAG T T T AT ACAG T T AC T TAT AT C AG T T AC AG T C AGAC T AAC AGAAAGAAC AGTGAGACCTATCGGGTGGTTGCGAAG

SEQ ID NO: 14 - hlyD

GCTAGGAGTCGTCTGGTGGGTCTCTACAGAAGAATATGAAAACATGGTTAATGGGGTTC AGCGAGTTCCTGTTGTGCTATAAACTTGTCTGGAGTGAAACATGGAAAATCCGGAAGCA ATTAGATACTCCGGTACGTGAAAAGGACGAAAATGAATTCTTACCCGCTCATCTGGAAT TAATTGAAACGCCGGTATCCCGCAGACCGCGTCTGGTTGCTTATTTTATTATGGGGTTT CTGGTTATTGCTGTCATTTTATCTGTTTTAGGTCAGGTGGAAATTGTTGCCACTGCAAA TGGGAAATTAACACTAAGTGGGCGTAGCAAAGAAATTAAACCTATTGAAAACTCAATAG T TAAAGAAAT TAT CGTAAAAGAAGGAGAGTCAGT CCGGAAAGGGGAT GT GT TAT TAAAG CTTACAGCACTGGGAGCTGAAGCTGATACGTTAAAAACACAGTCATCACTGTTACAGAC CAGGCTGGAACAAACTCGGTATCAAATTCTGAGCAGGTCAATTGAATTAAATAAACTAC C T GAAC T GAAGC T T C C T GAT GAGC C T T AT T T T C AGAAT GT AT C T GAAGAG GAAG T AC T G C GT T T AAC T T CT T T GAT AAAAGAACAGT T T T C CACAT GGCAAAAT CAGAAGT AT CAAAA AGAACTGAATCTGGATAAGAAAAGAGCAGAGCGATTAACAATACTTGCCCGTATAAACC GTTATGAAAATTTATCGAGAGTTGAAAAAAGCCGTCTGGATGATTTCAGGAGTTTATTG CATAAACAGGCAATTGCAAAACATGCTGTACTTGAGCAGGAGAATAAATATGTCGAGGC AGCAAATGAATTACGGGTTTATAAATCGCAACTGGAGCAAATTGAGAGTGAGATATTGT C T GC AAAAGAAGAAT AT C AG C T T G T C AC G C AG C T T T T T AAAAAT GAAAT T T T AGAC AAG CTAAGACAAACAACAGACAGCAT T GAGT TAT TAACT CT GGAGT TAGAGAAAAAT GAAGA GCGTCAACAGGCTTCAGTAATCAGGGCCCCTGTTTCGGGAAAAGTTCAGCAACTGAAGG TTCATACTGAAGGTGGGGTTGTTACAACAGCGGAAACACTGATGGTCATCGTTCCGGAA GATGACACGCTGGAGGTTACTGCTCTGGTACAAAATAAAGATATTGGTTTTATTAACGT CGGGCAGAATGCCATCATTAAAGTGGAGGCCTTTCCTTACACCCGATATGGTTATCTGG T GGGTAAGGT GAAAAATATAAAT T TAGAT GCAATAGAGGACCAGAAACT GGGACT CGT T T T TAAT GT CAT T GT T T C T GT T GAAGAGAAT GAT T T GT CAAC C GGGAAT AAGCACAT T C C ATTAAGCTCGGGTATGGCTGTCACTGCAGAAATAAAGACTGGAATGCGAAGCGTAATCA GCTATCTTCTTAGTCCTCTGGAAGAGTCTGTAACAGAAAGTTTACATGAGCGTTAAGTC TCAGAGCCGCGGTATCCGGCTCATATCTTCTCCTGTCGTCCTGAGACCTATCGGGTGGT TGCGAAG

SEQ ID NO: 15 - forward T1SS primer

CGACTGAGCCTTTCGTTTTATTTGATGCC

SEQ ID NO: 16 - reverse T1SS primer

GGTCATTACTGGATCTATCAACAGGAGTCCAAG SEQ ID NO: 17 -SQ_mscarlet primer gcatggacgaactgtataagggatcc

SEQ ID NO: 18 - riboJ

AGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTAC AAAT AAT T T T G T T T AA

SEQ ID NO: 19 - vtmoJ

AGTCCGTAGTGGATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTCTAC AAAT AAT T T T G T T TAA

SEQ ID NO: 20 - BBA_B1006 Terminator

AAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTTTTATAGCACAGCTAACACC ACGTCGTCCCTA

SEQ ID NO: 21 - PROMOTER 6 (J23119)

CAGAGTCCCCAGGCATTACTAGAGTCACACTTTTATAGCACAGCTAACACCACGTCGTC

CCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTGACAGCTAGCTCAGTC

CTAGGTATAATGCTAGCAGGCACAGCACAACGGTTTCCTTTTAGCTGTCACCGGATGTG

CTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAG GCA

SEQ ID NO: 22 - proB (C3)

CAGAGTCCccaggcattactagagtcacacttttataGCACAGCTAACACCACGTCGTC

CCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAG GCTCGTAATATATATTCAGGCACAGCACAACGGTTTCCTTTtAGCTGTCACCGGATGTG CTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAA

SEQ ID NO: 23 - J23119 (C6)

CAGAGTCCccaggcattactagagtcacacttttataGCACAGCTAACACCACGTCGTC CCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTGACAGCTAGCTCAGTC CTAGGTATAATGCTAGCAGGCACAGCACAACGGTTTCCTTTtAGCTGTCACCGGATGTG CTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAG GCA

SEQ ID NO: 24 - ssaG (vac1)

CAGAtattgccatcgcggatgtcgcctgtcttatctaccatcataaacatcatttgcct atggctcacgacagtataggcaatgccgttttttatattgctaattgtttcgccaatca acgcaaaagtatggcgattgctaaagccgtctccctgggcggtagattagccttaaccg cgacggtaatgactcattcatactggagtggtagtttgggactacagcctcatttatta gagcgtcttaatgatatt acct at ggactaatgagttt tact cgcttcggtatggatgg gatggcaatgaccggtatgcaggtcagcagcccattatatcgtttgctggctcaggtaa cgccagaacaacgtgcgccggagtaatcgttttcaggtatataccggatgttcattgct ttctaaattttgctatgttgccagtatccttacgatgtatttattttaaggaaaagcGG CA

SEQ ID NO: 25 - sseJ (vac2)

CAGATCACATAAAACACTAGCACTTTAGCAATAATAGTCGGATGATAAGTTTGTCTGTT TTTCCTGAGTATCAAGCCAGCTCATACTCACGCCAGCACACTAAAATCAGGAGTGGCTT _{CTTTTTTAGATCTTTGCCTTAGCCAGGCGCACACTCAATAATGATAGCAGTCAGATAAT} AT GT AC CAG G CAT T AAC C T C AC GT T G T T GAT GAT AT AT TTACTTCGTT GAAAAACAAT A AACATTGTATGTATTTTATTGGCGACGAAAAACTGTTAAAGAAGCGTAATTCCATATAC ACCATTTACCTGATTACTTTTCTTGCTAATATTTGCTAATTAATTATTTGCTAAAGCGT

GTTTAATAAAGTAAGGAGGAGGCA SEQ ID NO: 26 - pipB (vac8)

CAGAAAAAATATTGGTGCTTATTATTTTTTCTTTAAGTAAATTTTCGCTCAACAAACTT AATTGTTTATTCAATGATGATGAAGCGTAAGCTATGCTGGAAATGAAGGAAGTCAATAG CAAGGATAATCTTATTATTCACGGGTGATATTACTTCTGCTTCAGGCA

SEQ ID NO: 27 - sseA (vac12)

GAGAGGCGTATTCTTGATTTTCATCGGTGGAATGGgtgtcctgttaagtattagtggtc agcctgaaacggtaaatgactt acct ttgcgggttaagtttt tat tagacaaaagcaat attcattatgtgcgggcgcaatggaaagaagatggaagcctgcagttgtccggttattg ctcgtcaagcgaacagatgcaaaaggtgagagcgactctcgaatcatggggggtcatgt atcgggatggtgtaatctgtgatgacttattgatacgagaagtgcaggatgttttgata aaaatgggttacccgcatgctgaagtatccagcgaagggccggggagcgtgttaattca tgatgatatacaaatggatcagcaatggcgtaaggttcaaccattacttgcagatattc ccgggttattgcactggcagattagtcattctcatcagtctcagggggatgatattatt tctgcgataatagagaacggtttagtggggcttgtcaatgttacgccaatgcggcgctc ttttgttatcagtggtgtactggatgaatctcatcaacgcattttgcaagaaacgttag cagcattaaagaaaaaggaccccgctctttctttaatttatcaggatattgcgccttct catgatgaaagcaagtatctgcctgcgccagtggctggctttgtacagagtcgccatgg taattacttattactgacgaataaagagcgtttacgtgtaggggcattgttacccaatg ggggagaaattgtccatctgagtgccgatgtggtaacgattaaacataatgatactttg attaactatccattagattttaagtgagtggaaaatgacaactctgacccggttagaag atttgctgcttcattcgcgtgaagaggccaaaggcataattttacaattaagggctgcc cggaaacagttagaagagaacaacggtaggttacaggatccgcagcaatatcagcaaaa caccttattgcttgaagcgatcgagcaggccgaaaatatcatcaacattatttattatc gttaccataacagcgcacttgtagtgagtgagcaagagtaaagtaaaaatatcttagag cctatcccaccaggcgttaattggcgcagccagtttggacacggatagcgcgcaaaaac cggagcgtacacgtagtacgtgaggattttgagcactgcccaggttcaaaatggcaagt aaaatagccctaatgggacaggctcttagttagcacgttaattatctatcgtgtatatg gaggggaatgatgataaagaaaaaggctgcgtttagtgaatatcgtgatttagagcaaa gttacatgcagctaaatcactgtcttaaaaaatttcaccaaatccgggctaaggtgaGT CAACAGCTTGCTGAAAGGGCA SEQ ID NO: 28 - ssrA

CAGAGATGCTGTCTACATATACCTTGTCACAGGCGATTCTATCATTCGGATTTTCCGAT AAAT T C AC AAT T AC AT T T T C AG CAT T GAC AT AAAAAC T T AC AAT T T GAAAAAT T AT T T A T T AAAT AAAC T G T T AC GAT G T T T T T ACAT C GC C AT C T T AT T AAAAAGT AAT T G T AG T C A T C GAC T GG G T TAT AT AT GAAGAAAT T TAT C T T C C T AAT GAT AAC AC CAT C GAT T AAT C T

T CTGAT GAAACTATAT GTACT GCGATAGT GAT CAAGT GCCAAAGAT TT T GCAACAGGCA

SEQ ID NO: 29 - pipB2

CAGAtttatacgtgTTTGGCTGCTATTGTGTAAGCCAGACAGCAACGCGTCGTGATACG TTATTATGTAACCAGACGTAAAGGGGGTATTCACCTTATCTCTAAATGCAAATCTATAT GATAAATTTTATCATGCACTGTGTTGCTGTCTCTGGGAGAAAATATTAGCCGGTTTCCT TTtGGCA

SEQ ID NO: 30 - pipB2 extended

GCTAGGAGTCGTCTGGTGGGTCTCACAGAGTCTGTACAGACGATGTTGTTAAACAGTTT ATCAATGGCGGTAATAAAATGCCTGAACACGCTTTTTAATAGGTTTTCTGCTAATGAGT GGGATGAGTCCAGAACGTAGTATTAAAACGAATGCCGTGTTTATTATTTTAAATGATGT TATATACTCTAAATAATTCGAGTTGCAGGAAGGCGGCAAGCGAGCGAATCCCCAGGAGC

TTACTAAAGTAAGTGACTGGGGTGAGGGAACGCGGCCGCAGCACATGCAACTTGAAGTA TGACGAGTATAAGCCAATATATTTATTTGGCTGCTATTGTGTAAGCCAGACAGCAACGC GTCGTGATACGTTATTATGTAACCAGACGTAAAGGGGGTATTCACCTTATCTCTAAATG CAAATCTATATGATAAATTTTATCATGCACTGTGTTGCTGGCAGGAGACCTATCGGGTG

GTTGCGAAG

SEQ ID NO: 31 - ssaB

CAGAttatcggaaaatccgaatgatagaatcgcctgtgacaaggtatatgtagacagca tcctgatattgtacaagaagagtatagtcgaaataaatgtgaatcaggctttttacgga tgtggttgtgagcgaatttgatagaaactcccatttatgtctgGGCA SEQ ID NO: 32 - sifA

GCTAGGAGTCGTCTGGTGGGTCTCACAGAATAAGCGATTAATTGCGCAACGCTAACAAA TCCACACGCATCCAGGCATGAAGTTTATTCAAGGGTAAACTTCATGCCTTCGGCATAAA AAACGCATGAAAGAAGTTGCCGCCAGTATTGCAAATCTACAACATCATCCGCGGTAGTC CTTCTTTTATTTTTACCTGTAGCGACGCTATCACAGACAGTAATGCGTTTATACGCGAA GCTCTCAGGTTTTATACTGATTGCCAGTCTCTTTTAAAAATTATATTACATCCGATGCG CCCGCAGTTGAGATAAAAAGGGTCGATTTAATCAATTATGTAGTCATTTTTACTCCAGT ATAAGTGAGATTAAGGCAGGAGACCTATCGGGTGGTTGCGAAG

SEQ ID NO: 33 - sifB

CAGACTGCCCTACCGCTAAACATCTCATTGTTGTTAGCCTAATAATACTTTTAGTTTAA C T T C T T AT AAGAC AAT T T C T AC AC G G T T GAGC AAC TATTTACTTTCTC T AAAAAT AAT A T AGT GC GT AAT T AAT CAT TAC T CAT AGT ACAT GAT GAT GT GAGAAT TAAGAAAACC GT T TTACTTTCATTCGTTTTATCTGACATATTTCATGGCCAGGAGGCGTGGGCATGACTAAA GCTACGGGTCGATTTGAACAATTGAACAATAATGTTGACGGTTCAGGACAAAGCAAAAA TCAGGTGTTTCACCGATAGGCAAACCGATGGGCAACATGGGATAATATTTCGAATACCA CCTATTCCAGTAATGAAGTATCATATAATCACTTGTGGGGCA

SEQ ID NO: 34 - zinT

CAGAAAGCGAGTAGTCACAAAAATTATGCCgcctgtgcgcggatatctgcaaagcctgt gccgaagagtgtgcaaggcacgatcacgaccattgccagaattgcgcgcgggcatgcag ccaatgcgcagacgcctgccttaaaatggccgcgtaatttttcttccgccatt agetea accggatagagcatagagcttctacctctaaggttcggggttcaattcctcgatggcgg accagttgatatcaaaaaaggccacctgcgcggtggccgctgagtttctgttgaaataa atgcaatgttataatataacaatcatctttctaagaaagatgagggtaacgttttggtg attcatttaaaaaaactgacaatgcttctgGGAATGCTGTTGGTAAATAGTGGCA

SEQ ID NO: 35 - mgtC

CAGACGTTTAGCATCCCTTTTCTGGTGGAACCCATTTTTTCCTCGTCATGTTGTTTTAT TTTTTTACGTGCAGGCATCATAACAGAGCTATCGCCGGCATTAAGCAGGAATTTATTGT TTAATGATTTCAGACGAGCCTGTTATTGACATAATATTGTCATTTTTTTGTCACGGGAA AT AT C AAAC AAAC T T AAACAAAT C G T CAC TATCCCCGCCTTT GC AC T T T ACAGAAC AT A T T GACT GACTATAATAAGCGCAAAT T CAT GCAGGAGTAATAT GT T GGACAGT CACT T T T ACGTAAATCATCTGGCAAGTTAACGCACGCTATTCCTGCGCTGCTTGCCGAACCGGTGG GCAGCAATCTCCCCTTGTGACGATTGTCATCCCAATAATGTTACAACACGCGCATTGTC GCGAGGTAATCGTCATGTTCATGTTTAAACACGCTTTATTTCCTCCGCCGTTAACACGA CGCTAATTGCCTCAGGGCAGAAATTTGTCGTGTGCTAAATATAGCACGTACTTATTCTT CCAGAAAAAATGGAGGAACGTATGTTAATGTTTCCTTATATTTTAAATTTACTGGCCGC TATGCTGGCA

SEQ ID NO: 36 - proD

CAGAGTCCccaggcattactagagtcacacttttataGCACAGCTAACACCACGTCGTC CCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAG GCTCGTATAATATATTCAGGCACAGCACAACGGTTTCCTTTtAGCTGTCACCGGATGTG CTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAG GCA

SEQ ID NO: 37 - rpsM

CAGAGAAAGGCTACGGCCGTTAATtggtcgcctgagaagttacggagagtaaaaatgaa agttcgtgcttccgtcaagaaattatgccgtaactgcaaaatcgttaagcgtgatggtg tcatccgtgtgatttgcagtgccgagccgaagcataaacagcgccaaggctgatttttt cgcatatttttcttgcaaagttgggttgagctggctagattagccagccaatcttttgt atgtctgtacgtttccatttgagtatcctgaaaacgggcttttcagcatggtacgtaca tattaaatagtaggagtgccaGGCA

SEQ ID NO: 38 - dnaK

CAGAaaaagcacaaaaaatttttgcatctcccccttgatgacgtggtttacgaccccat ttagtagtcaaccgcagtgagtgagtctgcaaaaaaatgaaattgggcagttgaaacca gacgtttcgcccctattacagactcacaaccacatgatgaccgaatatatagtggaaac gtttagatgggGGCA SEQ ID NO: 39 - sicAp

CAGAGTCCccaggcattactagagtcacacttttataGCACAGCTAACACCACGTCGTC

CCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACaataggcgtatcacgagg ccctttcgtgttcacctcgagccacaagaaaacgaggtacggcattgagccgcgtaagg cagtagcgatgtattcattgggcgttttttgaatgttcactaaccaccgtcggggttta ataactgcatcagataaacgcagtcgttaagttctacaaagtcggtgacagatGGCA

SEQ ID NO: 40 - sicDp

CAGAGTCCccaggcattactagagtcacacttttataGCACAGCTAACACCACGTCGTC

CCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTATGAACATTTGATGTA

CCGATCTCCCCCATGATCGCCACTACGTATGGACGTCAGGATGCCTCCCCGCCTGATCA

GAAgcgtttcctcattaaaaaggacatttttttaaagttcctggtgcataaaagtcaca tccttttaaaGAGATTGTTAACCCTGTTGAATGTTCCCACTCCCCTATTCAGGCA

SEQ ID NO: 41 - sopEp

CAGAGTCCccaggcattactagagtcacacttttataGCACAGCTAACACCACGTCGTC

CCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACATCAGCTCACACTCCATT

TCAATGCCAGAACGGCAAGGCTCCTCCTGAGCGAAAAGGACTTTTTTTGAAAGTTTCTG

GAAAAT AAAAAT AG T AC T AT T T GT AG CAT T AAT T GAAT CAG C C GAAT T T T T C T AAT T C A TCAATCAGATGGGCA

SEQ ID NO: 42 - pro1

CAGAGTCCccaggcattactagagtcacacttttataGCACAGCTAACACCACGTCGTC

CCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAG

GCTCGGTACTATATTCAGGCACAGCACAACGGTTTCCTTTtAGCTGTCACCGGATGTGC

TTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGG

CA SEQ ID NO: 43 - proA

CAGAGTCCccaggcattactagagtcacacttttataGCACAGCTAACACCACGTCGTC

CCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAG

GCTCGTAGGCTATATTCAGGCACAGCACAACGGTTTCCTTTtAGCTGTCACCGGATGTG CTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAG

GCA

SEQ ID NO: 44 - proC

CAGAGTCCccaggcattactagagtcacacttttataGCACAGCTAACACCACGTCGTC

CCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCATAAG GCTCGTATGATATATTCAGGCACAGCACAACGGTTTCCTTTtAGCTGTCACCGGATGTG

CTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAG GCA

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Claims

1. A live attenuated Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is split into a first segment and a second segment, the first segment being operably linked to a first independently controlled promoter, wherein the first independently controlled promoter is a strong constitutive promoter or a strong vacuole-induced promoter, and the second segment being operably linked to a second independently controlled promoter, wherein the second independently controlled promoter is a strong vacuole-induced promoter, and wherein the first segment comprises a heterologous polynucleotide encoding a lysin upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding one or more cargo molecules replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

2. The live attenuated Gram-negative bacterium according to claim 1 , wherein the lysin is listeriolysin O.

3. A live attenuated Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is split into a first segment and a second segment, the first segment being operably linked to a first independently controlled promoter, wherein the first independently controlled promoter is any of J23119 or sseA, and the second segment being operably linked to a second independently controlled promoter, wherein the second independently controlled promoter is a strong vacuole-induced promoter, and wherein the first segment comprises a heterologous polynucleotide encoding one or more cargo molecules upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding one or more cargo molecules replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

4. A live attenuated Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is split into a first segment and a second segment, the first segment being operably linked to a first independently controlled promoter, wherein the first independently controlled promoter is not pipB2, ssaG or proC, and the second segment being operably linked to a second independently controlled promoter, wherein the second independently controlled promoter is a strong vacuole-induced promoter, and wherein the first segment comprises a heterologous polynucleotide encoding one or more cargo molecules upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding one or more cargo molecules replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

5. The live attenuated Gram-negative bacterium according to any of claims 1 to 4, wherein the second independently controlled promoter is any of ssaG, sseJ or sseA.

6. A live attenuated Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is split into a first segment and a second segment, the first segment being operably linked to a first independently controlled promoter, wherein the first independently controlled promoter is any of J23119 or sseA, and the second segment being operably linked to a second independently controlled promoter, wherein the second independently controlled promoter is any of ssaG, sseJ or sseA, and wherein the first segment comprises a heterologous polynucleotide encoding one or more cargo molecules upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding one or more cargo molecules replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

7. The live attenuated Gram-negative bacterium according to any preceding claim, wherein the first independently controlled promoter is J23119 and the second the independently controlled promoter is any of ssaG, sseJ or sseA.

8. The live attenuated Gram-negative bacterium according to any preceding claim, wherein the first independently controlled promoter is J23119 and the second the independently controlled promoter is ssaG.

9. The live attenuated Gram-negative bacterium according to any preceding claim, wherein the first independently controlled promoter is sse/t and the second the independently controlled promoter is any of: J23119, ssaG, sse/t or sseJ.

10. The live attenuated Gram-negative bacterium according to claim 9, wherein the first independently controlled promoter is sse/t and the second the independently controlled promoter is ssaG.

11 . The live attenuated Gram-negative bacterium according to any preceding claim, wherein the first independently controlled promoter is positioned upstream of the heterologous polynucleotide encoding the one or more cargo molecules.

12. The live attenuated Gram-negative bacterium according to any preceding claim, wherein the first segment further comprises a hlyC gene, or a fragment thereof, upstream of the heterologous polynucleotide encoding the one or more cargo molecules.

13. The live attenuated Gram-negative bacterium according to any preceding claim, wherein the first independently controlled promoter is positioned upstream of the hlyC gene, or a fragment thereof.

14. The live attenuated Gram-negative bacterium according to any preceding claim, wherein the hly genes involved in secretion are hlyB and hlyD.

15. The live attenuated Gram-negative bacterium according to any preceding claim, wherein the second independently controlled promoter is positioned upstream of the hlyB gene.

16. The live attenuated Gram-negative bacterium according to any preceding claim, wherein the one or more cargo molecules comprises a peptide and/or protein.

17. The live attenuated Gram-negative bacterium according to claim 16, wherein the one or more cargo molecules comprises a lysin.

18. The live attenuated Gram-negative bacterium according to claim 17, wherein the lysin is lysteriolysin O.

19. The live attenuated Gram-negative bacterium according to claim 18, wherein the peptide and/or protein is a therapeutic peptide and/or therapeutic protein.

20. The live attenuated Gram-negative bacterium according to claims 1 to 15, wherein the one or more cargo molecules comprises an RNA molecule.

21. The live attenuated Gram-negative bacterium according to claim 20, wherein the RNA molecule is an mRNA molecule.

22. The live attenuated Gram-negative bacterium according to claim 21 , wherein the mRNA molecule encodes a therapeutic protein and/or therapeutic peptide.

23. The live attenuated Gram-negative bacterium of any preceding claim for use in therapy.

24. The live attenuated Gram-negative bacterium for use of claim 23, wherein the therapy is treatment, reduction, inhibition, prevention, or control of a neoplastic disease, an infectious disease, a cardiovascular disease, a neurodegenerative disease, a gastrointestinal disease, a respiratory disease, a renal disease, a liver disease, an autoimmune disease, an inflammatory disease or a genetic disorder, preferably the live attenuated Gram-negative bacterium is for use in the treatment, reduction, inhibition, prevention of recurrence, or control of a neoplastic disease or an infectious disease.

25. The live attenuated Gram-negative bacterium for use of claim 24, wherein the neoplastic disease is a solid cancer and/or a haematological malignancy.

26. The live attenuated Gram-negative bacterium for use of claim 25, wherein the solid cancer and/or the haematological malignancy is a cancer selected from prostate cancer, oesophageal cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, mesothelioma, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, prostate cancer, endometrial cancer, endometrial cancer, vulvar/vaginal cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma, preferably wherein the neoplastic disease is associated with a cancer selected from bladder cancer, lung cancer, mesothelioma, hepatocellular cancer, melanoma, oesophageal cancer, gastric cancer, ovarian cancer, colorectal cancer, head and neck cancer, prostate cancer, endometrial cancer, cervical cancer or breast cancer

27. A vaccine composition comprising a live attenuated Gram-negative bacterium according to any of claims 1 to 22.

28. The vaccine composition of claim 27, wherein the vaccine composition further comprises a pharmaceutically acceptable adjuvant, carrier or excipient.

29. A method of treating, preventing, inhibiting, preventing recurrence or controlling a disease in a subject, wherein the method comprises administering to a subject a live attenuated Gram-negative bacterium according to any of claims 1 to 22, or the vaccine composition according to claim 27 to 28.

30. Use of a live attenuated Gram-negative bacterium according to any of claims 1 to 22, in the manufacture of a medicament.