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 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 envelope [Freudl, R., et al., 2018] or deliver actively pharmacological molecules in appropriate tissues (e.g., tumours in Immuno Oncology) in vivo [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 establish within 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 typically secrete 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 and piercing of the mammalian host, a series of proteins (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], However, the fusion of recombinant proteins to recognized signal peptides would only allow the secretion of protein inside cells and not the interstitial space, which may be more therapeutically relevant.

The uropathogenic strains of Escherichia coli (E. coli) 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], Upon expression of the toxin, HlyA, and recognition of an approximately 60 base pair-long signal peptide (HlyAs) on its C- terminus end, the TolB1 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 then-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],

When designing synthetic organisms for the delivery of relevant proteins in the interstitial space between eukaryotic cells, such as that within a tumour microenvironment (TME), the complex regulation of the hly operon can limit the yields and robustness of recombinant protein production.

Therefore, there exists a need for new delivery systems of cargo molecules to the interstitial space between eukaryotic cells. SUMMARY OF INVENTION

The inventors of the present invention have surprisingly found that the hlyCABD operon (shown in Figure 1) can be modified in such a way so as to enable the delivery of cargo molecules to the interstitial space between eukaryotic cells (Figure 2).

In 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, each segment being operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding a cargo molecule replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

In a second aspect, the present invention provides a vaccine composition comprising 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, each segment being operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding a cargo molecule replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

In a third 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, said 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, each segment being operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding a cargo molecule replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

In a fourth aspect, the present invention provides a method of delivering a therapeutic molecule to the interstitial space in between eukaryotic cells of the tumour microenvironment in a subject suffering from a tumour, said method comprising the steps of: i) modifying a live attenuated Gram-negative bacterium, said live attenuated Gram-negative bacteria comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is split into a first segment and a second segment, each segment being operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding a cargo molecule replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion and ii) administering said modified Gram-negative bacterium to the subject in need thereof.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows a schematic representation of the 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. The cargo region and the structural region are shown to be separated into two transcriptional units 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 in the export of the ratio of transcription of cargo versus structural proteins 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. Interestingly, 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) under different configurations of T1SS. 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 the 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 the T1SS dual-plasmid system, where 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).

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. The resulting RNA molecule or protein is also referred to herein as “cargo” or a “cargo molecule”. 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 (shown in Figure 1) can be modified in such a way so as to enable the delivery of cargo molecules to the interstitial space between eukaryotic cells (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. As a starting point, the hly operon was separated into two segments: a first segment (“cargo region”) and a second segment (“structural region”). The process of separating the four genes of the hlyCABD operon into two segments allows the genes involved in cargo production and/or activation (hlyC, hlyA) to be transcriptionally separated from those involved in secretion (hlyB, hlyD). The toxin sequence of hlyA can be replaced by a reporter gene (eg mScarlet), or indeed a heterologous nucleotide encoding any other cargo molecule, while maintaining the translocation peptide (HlyAs). 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. Accordingly, the present invention relates to a live attenuated Gram-negative bacterium comprising a modified hly operon.

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. 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.

Accordingly, in 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, each segment being operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding a cargo molecule replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

It is therefore 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 extracellular surroundings, including the interstitial space between eukaryotic cells. 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 term “cargo” will be well known to those in the art, and refers to a specific molecule of interest which is intended to be translocated, delivered, transported, or exported from one place to another. In particular, a cargo molecule may be translocated from the bacterial cytoplasm to the extracellular environment surrounding eukaryotic cells. In a preferred embodiment, the cargo molecule is a protein and/or peptide. Cargo molecules may be heterologous proteins which do not occur naturally to the carrier bacterial cell. The cargo peptide and/or protein may be a therapeutic peptide and/or a therapeutic protein. 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. 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), micro RNA (miRNA), transfer RNA (tRNA), self-amplifying 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 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 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 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 (protein) molecules, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 different cargo (protein) molecules.

It is also 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. 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. 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 trans-periplasm 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.

In one embodiment, the heterologous polynucleotide encoding the cargo molecule is positioned upstream of the HlyAs sequence. In another embodiment, the heterologous polynucleotide encoding the cargo molecule is positioned downstream of the independently controlled promoter. In yet another embodiment, the heterologous polynucleotide encoding the cargo molecule 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, 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 T 1 SS 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.

The inventors of the present invention surprisingly found that splitting the operon into two segments resulted in functional translocation, which was increased upon increasing the transcription levels of both segments. The modified hlyCABD operon of the present invention is therefore split into two segments: a first segment and a second segment, each segment being operably linked to its own independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding a cargo molecule 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 Gram-negative 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. 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 including invasion-associated SPI-1 , SPI-4, orflagella-related promoters. Suitable promoters may include, but are not limited to, invF, hilA, hilD, sicA, siiE, flhDC, and fliC.

In a preferred embodiment, the independently controlled promoters are strong promoters, such that when the 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, and 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. Whilst any strong promoter that fulfils the function herein disclosed is suitable, particular examples of suitable strong promoters include, but are not limited to, a ptrc promoter, a ptet promoter, a pcon5 promoter, a pBAD promoter, a placllV5 promoter, invasion-associated promoters (e.g., SPI-1 , SPI-4, or flagella), intracellular promoters (SPI-2), host-cytosolic promoters or a pTac promoter. Other suitable examples include, but are not limited to, uphT (Glucose- 6-phosphate), frubKA (fructose). SPI-1 are invasion-associated promoters that are active outside of the host cell and when the bacterium is attempting to invade the host cell. Additional promoters for use in the present invention may include, but are not limited to, a trc promoter, a tac promoter, a trp promoter, a lac operon promoter, a lac/tac promoter, a tac/trc promoter, a trp/lac promoter, a bad/ara promoter, an ssaG promoter, a pagC promoter, a nirB promoter, a dps promoter or an spv promoter. The term “cytosolic promoter”, as used herein, refers to intracellular promoters in bacteria. Host-cytosolic promoters for use in the present invention may include, but are not limited to, uhpT, mntH, entC, fhuE, iroN, fepB, fepA, fhuA, sitA, stn3250, sufA, yjjZ, soxS, sfbA. 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, ssaO, ssaP, ssaQ, ssaR, ssaS, ssaT, ssaD, ssaE, ssaG, ssa/, ssaC and ssaH. Other vacuolar promoters include PipB2, zinT, mtgC. In a preferred embodiment, PipB2 and ssaG may be used for intracellular delivery. PipB2 is a strong SPI-2-dependent promoter, relative to other SPI-2 promoters. 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 1 details the promoters used in screening.

Table 1 : 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.

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 2 details the sequences used to build expression plasmids.

Table 2: 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.

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 molecules can be delivered to the extracellular space, including the interstitial space, between eukaryotic cells. 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 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, IFNct, IFN|3, IL-1 , or any combination thereof. In one embodiment, the live attenuated Gram-negative bacteria comprising a modified hlyCABD operon encoding a therapeutic cargo molecule which is intended to be secreted into the extracellular environment between eukaryotic cells. As used herein, the terms “extracellular environment”, “extracellular space”, “extracellular compartment”, or “extracellular surroundings” are used interchangeably and refer to the region within a multicellular organism which is outside the cells, i.e. , beyond the plasma membrane, and is occupied by extracellular matrix. The extracellular environment comprises three compartments: the interstitial, intravascular and transcellular compartments.

In one embodiment, the cargo molecule is intended to be secreted into the interstitial space. As used herein, the terms “interstitial compartment”, “interstitial environment”, “interstitial space”, “tissue space” or “interstitial surroundings” are used interchangeably and refer to the space surrounding tissue cells (i.e., the space outside blood and lymph vessels and parenchymal cells), also known as the tissue microenvironment. The interstitial space consists of two major phases: interstitial fluid which provides the immediate microenvironment between eukaryotic cells, and the structural molecules comprising the extracellular matrix. 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 bacteria is administered intratumourally, peritoumorally, intravenously, intraperitoneally, subcutaneously, intradermally, or orally administered. In a more preferred embodiment, the live attenuated Gram-negative bacteria 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 to the interstitial space 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 10 ⁵ and 10 ¹² CFU, where CFU is a colony-forming unit. For example, suitable doses may be between 10 ⁵ and 10 ⁶ CFU, 10 ⁵ and 10 ⁷ CFU, 10 ⁵ and 10 ⁸ CFU, 10 ⁵ and 10 ⁹ CFU, 10 ⁵ and 10 ¹ ° CFU, 10 ⁵ and 10 ¹¹ CFU, 10 ⁶ and 10 ⁷ CFU, 10 ⁶ and 10 ⁸ CFU, 10 ⁶ and 10 ⁹ CFU, 10 ⁶ , and 10 ¹ ° CFU, 10 ⁶ and 10 ¹¹ CFU, 10 ⁶ and 10 ¹² CFU, 10 ⁷ and 10 ⁸ CFU, 10 ⁷ and 10 ⁹ CFU, 10 ⁷ and 10 ¹ ° CFU, 10 ⁷ and 10 ¹¹ CFU, 10 ⁷ and 10 ¹² CFU, 10 ⁸ and 10 ⁹ CFU, 10 ⁸ and 10 ¹ ° CFU, 10 ⁸ and 10 ¹¹ CFU, 10 ⁸ and 10 ¹² CFU, 10 ⁹ and 10 ¹ ° CFU, 10 ⁹ and 10 ¹¹ CFU, 10 ⁹ and 10 ¹² CFU, 10 ¹ ° and 10 ¹¹ CFU, 10 ¹ ° and 10 ¹² CFU, or 10 ¹¹ and 10 ¹² 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 interstitial space in between 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 Gram-negative 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 Gram-negative 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 a second aspect, the present invention provides a vaccine composition comprising 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, each segment being operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding a cargo molecule replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

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 third 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, said 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, each segment being operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding a cargo molecule replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion.

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

In a fourth aspect, the present invention provides a method of delivering a therapeutic molecule to the interstitial space in between eukaryotic cells of the tumour microenvironment in a subject suffering from a tumour, said method comprising the steps of: i) modifying a live attenuated Gram-negative bacterium, said live attenuated Gram-negative bacteria comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is split into a first segment and a second segment, each segment being operably linked to an independently controlled promoter, wherein the first segment comprises a heterologous polynucleotide encoding a cargo molecule upstream of a hlyAs translocation sequence, wherein the heterologous polynucleotide encoding a cargo molecule replaces a hlyA gene, and wherein the second segment comprises hly genes involved in secretion and ii) administering said modified Gram-negative bacterium to the subject in need thereof.

In one embodiment of the fourth aspect of the present invention, 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 method of delivering a therapeutic molecule to the interstitial space in between eukaryotic cells of the tumour microenvironment in a subject suffering from a tumour of the fourth aspect may comprise one or more of the aforementioned embodiments in respect to any preceding aspect.

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.

EXAMPLES

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

In order to navigate the optimal expression ratio between secretable elements (e.g. , mScarlet fused with T 1 SS 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), T = 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).

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 OD ₆ oo ~ 0.5 and then an aliquot was taken and processed as: (1) an aliquot was extracted from the deepwell plate (100 pL) and used to measure the cell density (OD ₆ oo), (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 OD ₆ oo. 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.

Exchange of mScarlet cargo for LLO results in reproducible secretion outcomes

The plasmid library with LLO (also known as ListeroLysin O) 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. 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 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 OD ₆ oo). 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.

Dual-plasmid expression system

To provide a higher level of control over the expression levels of the two systems, a dual-plasmid expression system was designed and built. In this, a cargocontaining 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 J23199 (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 dual-plasmid expression system, which allows for optimizing yields without over-burdening the bacterial carrier.

SEQUENCES FORMING PART OF THE DESCRIPTION

SEQ ID NO:1- /7/y(LLO) (DNA sequence)

GCTAGGAGTCGTCTGGTGGAAGACTAGCGATGGCAAAGGATGCATCTGCATTC

AATAAAGAAAATTCAATTTCATCCATGGCACCACCAGCATCTCCGCCTGCAAGTC

CTAAGACGCCAATCGAAAAGAAACACGCGGATGAAATCGATAAGTATATACAAGG ATTGGATTACAATAAAAACAATGTATTAGTATACCACGGAGATGCAGTGACAAATG TGCCGCCAAGAAAAGGTTACAAAGATGGAAATGAATATATTGTTGTGGAGAAAAA

GAAGAAATCCATCAATCAAAATAATGCAGACATTCAAGTTGTGAATGCAATTTCG

AGCCTAACCTATCCAGGTGCTCTCGTAAAAGCGAATTCGGAATTAGTAGAAAATC

AACCAGATGTTCTCCCTGTAAAACGTGATTCATTAACACTCAGCATTGATTTGCC

AGGTATGACTAATCAAGACAATAAAATCGTTGTAAAAAATGCCACTAAATCAAACG

TTAACAACGCAGTAAATACATTAGTGGAAAGATGGAATGAAAAATATGCTCAAGC

TTATCCAAATGTAAGTGCAAAAATTGATTATGATGACGAAATGGCTTACAGTGAAT

CACAATTAATTGCGAAATTTGGTACAGCATTTAAAGCTGTAAATAATAGCTTGAAT

GTAAACTTCGGCGCAATCAGTGAAGGGAAAATGCAAGAAGAAGTCATTAGTTTT

AAACAAATTTACTATAACGTGAATGTTAATGAACCTACAAGACCTTCCAGATTTTT

CGGCAAAGCTGTTACTAAAGAGCAGTTGCAAGCGCTTGGAGTGAATGCAGAAA

ATCCTCCTGCATATATCTCAAGTGTGGCGTATGGCCGTCAAGTTTATTTGAAATTA

TCAACTAATTCCCATAGTACTAAAGTAAAAGCTGCTTTTGATGCTGCCGTAAGCG GAAAATCTGTCTCAGGTGATGTAGAACTAACAAATATCATCAAAAATTCTTCCTTC

AAAGCCGTAATTTACGGAGGTTCCGCAAAAGATGAAGTTCAAATCATCGACGGC

AACCTCGGAGACTTACGCGATATTTTGAAAAAAGGCGCTACTTTTAATCGAGAAA

CACCAGGAGTTCCCATTGCTTATACAACAAACTTCCTAAAAGACAATGAATTAGC

TGTTATTAAAAACAACTCAGAATATATTGAAACAACTTCAAAAGCTTATACAGATG

GAAAAATTAACATCGATCACTCTGGAGGATACGTTGCTCAATTCAACATTTCTTG

GGATGAAGTAAATTATGATCCTGAAGGTAACGAAATTGTTCAACATAAAAACTGG

AGCGAAAACAATAAAAGCAAGCTAGCTCATTTCACATCGTCCATCTATTTGCCAG

GTAACGCGAGAAATATTAATGTTTACGCTAAAGAATGCACTGGTTTAGCTTGGGA

ATGGTGGAGAACGGTAATTGATGACCGGAACTTACCACTTGTGAAAAATAGAAAT ATCTCCATCTGGGGCACCACGCTTTATCCGAAATATAGTAATAAAGTAGATAATCC AATCGAATATGCAGTGAGCTGGTCTTCCTATCGGGTGGTTGCGAAG

SEQ ID NO: 2- LLO (amino acid sequence)

MAKDASAFNKENSISSMAPPASPPASPKTPIEKKHADEIDKYIQGLDYNKNNVLVYH

GDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSE LVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEK YAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVI

SFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKL STNSHSTKVKAAFDAAVSGKSVSGDVELTN 11 KNSSFKAVIYGGSAKDEVQI I DGN LG DLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDH SGGYVAQFNISWDEVNYDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNINV

YAKECTGLAWEWWRTVIDDRNLPLVKNRNISIWGTTLYPKYSNKVDNPIE

SEQ ID NO: 3- Pro1_1 (P1)

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

TACAAATAATTTTGTTTAACACA

SEQ ID NO: 8- PROA_2

GTAAGTCCCCAGGCATTACTAGAGTCACACTAAAAAAAAACCCCGCCCCTG

ACAGGGCGGGGTTTTTTTTTTTATAGCACAGCTAACACCACGTCGTCCCTA

TCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCA

TAAGGCTCGTAGGCTATATTCAGGCACAGCACAACGGTTTCCTTTTAGTCC

GTAGTGGATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTC

TACAAATAATTTTGTTTAACACA

SEQ ID NO: 9- PROB_2

GTAAGTCCCCAGGCATTACTAGAGTCACACTAAAAAAAAACCCCGCCCCTG

ACAGGGCGGGGTTTTTTTTTTTATAGCACAGCTAACACCACGTCGTCCCTA

TCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCA

TAAGGCTCGTAATATATATTCAGGCACAGCACAACGGTTTCCTTTTAGTCC

GTAGTGGATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTC

TACAAATAATTTTGTTTAACACA

SEQ ID NO: 10- PROC_2

GTAAGTCCCCAGGCATTACTAGAGTCACACTAAAAAAAAACCCCGCCCCTG

ACAGGGCGGGGTTTTTTTTTTTATAGCACAGCTAACACCACGTCGTCCCTA TCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTTACGGGCATGCA

TAAGGCTCGTATGATATATTCAGGCACAGCACAACGGTTTCCTTTTAGTCC

GTAGTGGATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGACCTC

TACAAATAATTTTGTTTAACACA

SEQ ID NO: 11- hlyA _s

GCTAGGAGTCGTCTGGTGGGTCTCTAAAGACAACGGTTTCCCTCTAGAAATAAT

TTTGTTTAACTTTAAGAAGGAGATATATTCATGGCTGTGAGCGGCTGGCGTCTG

TTCAAGAAAATTAGCGCGACCGTCTTCGGCGGTGGTGGCAGCGTTAGCAAAGG

CGAGGCGGTTATCAAGGAGTTTATGCGTTTTAAGGTTCACATGGAGGGTAGCA

TGAATGGTCACGAGTTCGAGATCGAGGGTGAAGGCGAGGGTCGTCCGTACGA

AGGCACCCAGACCGCGAAGCTGAAAGTGACCAAGGGTGGCCCGCTGCCGTTC

AGCTGGGACATCCTGAGCCCGCAGTTCATGTATGGCAGCCGTGCGTTTACCAA

ACACCCGGCGGACATTCCGGATTACTATAAGCAAAGCTTCCCGGAAGGTTTTA

AATGGGAGCGTGTTATGAACTTCGAAGATGGTGGCGCGGTGACCGTTACCCAG

GACACCAGCCTGGAGGATGGCACCCTGATTTACAAGGTGAAACTGCGTGGCA

CCAACTTTCCGCCGGATGGTCCGGTTATGCAGAAGAAAACGATGGGTTGGGAA

GCGAGCACCGAGCGTCTGTATCCGGAAGATGGCGTGCTGAAGGGTGATATCA

AAATGGCGCTGCGTCTGAAGGACGGTGGCCGTTACCTGGCGGATTTTAAGACC

ACCTATAAAGCGAAGAAACCGGTGCAAATGCCGGGTGCGTACAACGTTGACCG

TAAACTGGATATTACCAGCCACAACGAGGATTATACCGTGGTTGAGCAATATGA

GCGTAGCGAGGGTCGCCACAGCACCGGCGGCATGGACGAACTGTATAAGGGA

TCCGAAGACGCGAGCACGCCCGGGGGTGCGCCGGTGCCGTATCCGGATCCG

CTGGAACCGGCCGGGGAAAATTCTCTTGCTAAAAATGTATTATCCGGTGGAAA

AGGTAATGACAAGTTGTACGGCAGTGAGGGAGCAGACCTGCTTGATGGCGGA

GAAGGGAATGATCTTCTGAAAGGTGGATATGGTAATGATATTTATCGTTATCTTT

CAGGATATGGCCATCATATTATTGACGATGAAGGGGGGAAAGACGATAAACTC

AGTTTAGCTGATATAGATTTCCGGGACGTTGCCTTTAAGCGAGAAGGGAATGA

CCTCATTATGTATAAAGCTGAAGGTAATGTTCTTTCTATTGGCCACAAAAATGGT

ATTACATTTAAAAACTGGTTTGAAAAAGAGTCAGATGATCTCTCTAATCATCAGA

TAGAGCAGATTTTTGATAAAGACGGCAGGGTAATCACACCAGATTCTCTTAAAA

AAGCATTTGAATATCAGCAGAGTAATAACAAGGTAAGTTATGTGTATGGACATG

ATGCATCAACTTATGGGAGCCAGGACAATCTTAATCCATTAATTAATGAAATCA

GCAAAATCATTTCAGCTGCAGGTAACTTCGATGTTAAGGAGGAAAGATCTGCC

GCTTCTTTATTGCAGTTGTCCGGTAATGCCAGTGATTTTTCATATGGACGGAAC

TCAATAACTTTGACAGCATCAGCATAATATATTAGTAATGAGACCTATCGGGTG

GTTGCGAAG

SEQ ID NO: 12- hlyCA _s

GCTAGGAGTCGTCTGGTGGGTCTCTAAAGATATTTTTGCCACAATATTTAATCA

TATAATTTAAGTTGTAGTGAGTTTATTATGAATATAAACAAACCATTAGAGATTCT

TGGGCATGTATCCTGGCTATGGGCCAGTTCTCCACTACACAGAAACTGGCCAG TATCTTTGTTTGCAATAAATGTATTACCCGCAATACAGGCTAACCAATATGTTTT

ATTAACCCGGGATGATTACCCTGTCGCGTATTGTAGTTGGGCTAATTTAAGTTT

AGAAAATGAAATTAAATATCTTAATGATGTTACCTCATTAGTTGCAGAGGACTGG

ACTTCAGGTGATCGTAAATGGTTCATTGACTGGATTGCTCCTTTCGGGGATAAC

GGTGCCCTGTACAAATATATGCGAAAAAAATTCCCTGATGAACTATTCAGAGCC

ATCAGGGTGGATCCCAAAACTCATGTTGGTAAAGTATCAGAATTTCATGGAGGT

AAAATTGATAAACAGTTAGCGAATAAAATTTTTAAACAATATCACCACGAGTTAA

TAACTGAAGTAAAAAGAAAGTCAGATTTTAATTTTTCATTAACTGGTTAAACAAC

GGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATATTCATGGC

TGTGAGCGGCTGGCGTCTGTTCAAGAAAATTAGCGCGACCGTCTTCGGCGGT

GGTGGCAGCGTTAGCAAAGGCGAGGCGGTTATCAAGGAGTTTATGCGTTTTAA

GGTTCACATGGAGGGTAGCATGAATGGTCACGAGTTCGAGATCGAGGGTGAA

GGCGAGGGTCGTCCGTACGAAGGCACCCAGACCGCGAAGCTGAAAGTGACCA

AGGGTGGCCCGCTGCCGTTCAGCTGGGACATCCTGAGCCCGCAGTTCATGTA

TGGCAGCCGTGCGTTTACCAAACACCCGGCGGACATTCCGGATTACTATAAGC

AAAGCTTCCCGGAAGGTTTTAAATGGGAGCGTGTTATGAACTTCGAAGATGGT

GGCGCGGTGACCGTTACCCAGGACACCAGCCTGGAGGATGGCACCCTGATTT

ACAAGGTGAAACTGCGTGGCACCAACTTTCCGCCGGATGGTCCGGTTATGCAG

AAGAAAACGATGGGTTGGGAAGCGAGCACCGAGCGTCTGTATCCGGAAGATG

GCGTGCTGAAGGGTGATATCAAAATGGCGCTGCGTCTGAAGGACGGTGGCCG

TTACCTGGCGGATTTTAAGACCACCTATAAAGCGAAGAAACCGGTGCAAATGC

CGGGTGCGTACAACGTTGACCGTAAACTGGATATTACCAGCCACAACGAGGAT

TATACCGTGGTTGAGCAATATGAGCGTAGCGAGGGTCGCCACAGCACCGGCG

GCATGGACGAACTGTATAAGGGATCCGAAGACGCGAGCACGCCCGGGGGTGC

GCCGGTGCCGTATCCGGATCCGCTGGAACCGGCCGGGGAAAATTCTCTTGCT

AAAAATGTATTATCCGGTGGAAAAGGTAATGACAAGTTGTACGGCAGTGAGGG

AGCAGACCTGCTTGATGGCGGAGAAGGGAATGATCTTCTGAAAGGTGGATATG

GTAATGATATTTATCGTTATCTTTCAGGATATGGCCATCATATTATTGACGATGA

AGGGGGGAAAGACGATAAACTCAGTTTAGCTGATATAGATTTCCGGGACGTTG

CCTTTAAGCGAGAAGGGAATGACCTCATTATGTATAAAGCTGAAGGTAATGTTC

TTTCTATTGGCCACAAAAATGGTATTACATTTAAAAACTGGTTTGAAAAAGAGTC

AGATGATCTCTCTAATCATCAGATAGAGCAGATTTTTGATAAAGACGGCAGGGT

AATCACACCAGATTCTCTTAAAAAAGCATTTGAATATCAGCAGAGTAATAACAAG

GTAAGTTATGTGTATGGACATGATGCATCAACTTATGGGAGCCAGGACAATCTT

AATCCATTAATTAATGAAATCAGCAAAATCATTTCAGCTGCAGGTAACTTCGATG

TTAAGGAGGAAAGATCTGCCGCTTCTTTATTGCAGTTGTCCGGTAATGCCAGTG

ATTTTTCATATGGACGGAACTCAATAACTTTGACAGCATCAGCATAATATATTAG

TAATGAGACCTATCGGGTGGTTGCGAAG

SEQ ID NO: 13- hlyB

GCTAGGAGTCGTCTGGTGGGTCTCTCACAGATATTTTTTTGGAGTCATAATGGA

TTCTTGTCATAAAATTGATTATGGGTTATACGCCCTGGAGATTTTAGCCCAATAC

CATAACGTGTCTGTTAACCCGGAAGAAATTAAACATAGATTTGACACAGACGGG

ACTGGTCTGGGATTAACGTCATGGTTGCTTGCTGCGAAATCTTTAGAACTAAAG GTAAAACAGGTAAAAAAAACAATTGACCGATTAAACTTTATTTCTCTGCCCGCAT

TAGTCTGGAGAGAGGATGGACGTCATTTTATTCTGACTAAAGTCAGTAAAGAAG

CAAACAGATATCTTATTTTTGATCTGGAGCAGCGAAATCCCCGTGTTCTCGAAC

AGTCTGAGTTTGAGGCGTTATATCAGGGGCATATTATTCTTATCGCTTCCCGTT

CTTCTGTGGCGGGCAAACTGGCGAAATTTGACTTTACCTGGTTTATTCCTGCCA

TTATAAAATACAGGAGAATATTTATTGAAACCCTTGTTGTGTCTGTTTTTTTACAA

TTATTTGCATTAATAACCCCCCTTTTTTTTCAGGTGGTTATGGACAAAGTATTAG

TGCACAGGGGATTTTCAACTCTTAATGTTATTACTGTCGCATTATCTGTTGTGGT

GGTGTTTGAGATTATACTCAGCGGTTTAAGAACTTACATTTTTGCACATAGTACA

AGTCGGATTGATGTTGAGTTGGGTGCCAAACTCTTCCGGCATTTACTGGCGCT

ACCGATCTCTTATTTTGAGAGTCGTCGTGTTGGTGATACTGTTGCCAGGGTAAG

AGAATTAGACCAGATCCGTAATTTTCTGACAGGACAGGCATTAACATCTGTTCT

GGACTTATTATTTTCATTCATATTTTTTGCGGTAATGTGGTATTACAGTCCAAAG

CTTACTCTGGTGATCTTATTTTCGCTGCCTTGTTATGCTGCATGGTCTGTTTTTA

TTAGCCCCATTTTGCGACGTCGCCTTGATGATAAGTTTTCACGGAATGCGGATA

ATCAATCTTTCCTGGTGGAATCAGTCACGGCGATTAACACTATAAAAGCTATGG

CAGTCTCACCTCAGATGACGAACATATGGGACAAACAATTGGCAGGATATGTT

GCTGCAGGCTTCAAAGTGACAGTATTAGCAACCATTGGTCAACAAGGAATACA

GTTAATACAAAAGACTGTTATGATCATCAACCTGTGGTTGGGAGCACACCTGGT

TATTTCCGGGGATTTAAGTATTGGTCAGTTAATTGCTTTTAATATGCTTGCTGGT

CAGATTGTTGCACCGGTTATTCGCCTTGCACAAATCTGGCAGGATTTCCAGCA

GGTTGGTATATCAGTTACCCGCCTTGGTGATGTGCTTAACTCTCCAACTGAAAG

TTATCATGGGAAACTGGCATTACCGGAAATTAATGGTGATATCACTTTTCGTAAT

ATCCGGTTTCGCTATAAGCCTGACTCTCCGGTTATTTTAGATAATATCAATCTCA

GTATTAAGCAGGGGGAGGTTATTGGTATTGTCGGACGTTCTGGTTCAGGAAAA

AGCACATTAACTAAATTAATTCAACGTTTTTATATTCCTGAAAATGGCCAGGTCT

TAATTGATGGACATGATCTTGCGTTGGCCGATCCTAACTGGTTACGTCGTCAGG

TGGGGGTTGTGTTGCAGGACAATGTGCTGCTTAATCGCAGTATTATTGATAATA

TCTCACTGGCTAATCCTGGTATGTCCGTCGAAAAAGTTATTTATGCAGCGAAAT

TAGCAGGCGCTCATGATTTTATTTCTGAATTGCGTGAGGGGTATAACACCATTG

TCGGGGAACAGGGGGCAGGATTATCCGGAGGTCAACGTCAACGCATCGCAAT

TGCAAGGGCGCTGGTGAACAACCCTAAAATACTTATTTTTGATGAAGCAACCAG

TGCTCTGGATTATGAGTCGGAGCATATCATCATGCGCAATATGCACAAAATATG

TAAGGGCAGAACGGTTATAATCATTGCTCATCGTCTGTCTACAGTAAAAAATGC

AGACCGCATTATTGTCATGGAAAAAGGGAAAATTGTTGAACAGGGTAAACATAA

GGAACTGCTTTCTGAACCGGAAAGTTTATACAGTTACTTATATCAGTTACAGTCA

GACTAACAGAAAGAACAGTGAGACCTATCGGGTGGTTGCGAAG

SEQ ID NO: 14- hlyD

GCTAGGAGTCGTCTGGTGGGTCTCTACAGAAGAATATGAAAACATGGTTAATG

GGGTTCAGCGAGTTCCTGTTGTGCTATAAACTTGTCTGGAGTGAAACATGGAAA

ATCCGGAAGCAATTAGATACTCCGGTACGTGAAAAGGACGAAAATGAATTCTTA

CCCGCTCATCTGGAATTAATTGAAACGCCGGTATCCCGCAGACCGCGTCTGGT

TGCTTATTTTATTATGGGGTTTCTGGTTATTGCTGTCATTTTATCTGTTTTAGGTC AGGTGGAAATTGTTGCCACTGCAAATGGGAAATTAACACTAAGTGGGCGTAGC

AAAGAAATTAAACCTATTGAAAACTCAATAGTTAAAGAAATTATCGTAAAAGAAG

GAGAGTCAGTCCGGAAAGGGGATGTGTTATTAAAGCTTACAGCACTGGGAGCT

GAAGCTGATACGTTAAAAACACAGTCATCACTGTTACAGACCAGGCTGGAACAA

ACTCGGTATCAAATTCTGAGCAGGTCAATTGAATTAAATAAACTACCTGAACTG

AAGCTTCCTGATGAGCCTTATTTTCAGAATGTATCTGAAGAGGAAGTACTGCGT

TTAACTTCTTTGATAAAAGAACAGTTTTCCACATGGCAAAATCAGAAGTATCAAA

AAGAACTGAATCTGGATAAGAAAAGAGCAGAGCGATTAACAATACTTGCCCGTA

TAAACCGTTATGAAAATTTATCGAGAGTTGAAAAAAGCCGTCTGGATGATTTCA

GGAGTTTATTGCATAAACAGGCAATTGCAAAACATGCTGTACTTGAGCAGGAGA

ATAAATATGTCGAGGCAGCAAATGAATTACGGGTTTATAAATCGCAACTGGAGC

AAATTGAGAGTGAGATATTGTCTGCAAAAGAAGAATATCAGCTTGTCACGCAGC

TTTTTAAAAATGAAATTTTAGACAAGCTAAGACAAACAACAGACAGCATTGAGTT

ATTAACTCTGGAGTTAGAGAAAAATGAAGAGCGTCAACAGGCTTCAGTAATCAG

GGCCCCTGTTTCGGGAAAAGTTCAGCAACTGAAGGTTCATACTGAAGGTGGGG

TTGTTACAACAGCGGAAACACTGATGGTCATCGTTCCGGAAGATGACACGCTG

GAGGTTACTGCTCTGGTACAAAATAAAGATATTGGTTTTATTAACGTCGGGCAG

AATGCCATCATTAAAGTGGAGGCCTTTCCTTACACCCGATATGGTTATCTGGTG

GGTAAGGTGAAAAATATAAATTTAGATGCAATAGAGGACCAGAAACTGGGACTC

GTTTTTAATGTCATTGTTTCTGTTGAAGAGAATGATTTGTCAACCGGGAATAAGC

ACATTCCATTAAGCTCGGGTATGGCTGTCACTGCAGAAATAAAGACTGGAATGC

GAAGCGTAATCAGCTATCTTCTTAGTCCTCTGGAAGAGTCTGTAACAGAAAGTT

TACATGAGCGTTAAGTCTCAGAGCCGCGGTATCCGGCTCATATCTTCTCCTGTC

GTCCTGAGACCTATCGGGTGGTTGCGAAG

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

AGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGC

CTCTACAAATAATTTTGTTTAA

SEQ ID NO: 19- vtmoJ

AGTCCGTAGTGGATGTGTATCCACTCTGATGAGTCCGAAAGGACGAAACGGAC

CTCTACAAATAATTTTGTTTAA SEQ ID NO: 20- BBA_B1006 Terminator

AAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTTTTATAGCACAGCTA

ACACCACGTCGTCCCTA SEQ ID NO: 21- PROMOTER 6 (J23199)

CAGAGTCCCCAGGCATTACTAGAGTCACACTTTTATAGCACAGCTAACACCACG

TCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTTGCTGGATAACTTGACAG

CTAGCTCAGTCCTAGGTATAATGCTAGCAGGCACAGCACAACGGTTTCCTTTTA

GCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCC TCTACAAATAATTTTGTTTAAGGCA

REFERENCES

Carrier, M.J., et al., Expression of human IL-1 beta in Salmonella typhimurium. A model system for the delivery of recombinant therapeutic proteins in vivo. J Immunol, 1992. 148(4): p. 1176-81.

Chen, J., et al., Salmonella flagella confer anti-tumor immunological effect via activating Flagellin/TLR5 signalling within tumor microenvironment. Acta Pharm Sin B, 2021. 11 (10): p. 3165-3177.

Freudl, R., Signal peptides for recombinant protein secretion in bacterial expression systems. Microb Cell Fact, 2018. 17(1): p. 52.

Gentschev, I., G. Dietrich, and W. Goebel, The E. coli alpha-hemolysin secretion system and its use in vaccine development. Trends Microbiol, 2002. 10(1): p. 39- 45.

Hoffman, R.M., Tumor-seeking Salmonella amino acid auxotrophs. Curr Opin Biotechnol, 2011. 22(6): p. 917-23.

Holland, I.B. et al. (1990) The mechanism of secretion of hemolysin and other polypeptides from Gram-negative bacteria. J. Bioenerg. Biomembr. 22, 473-491.

Jarchau, T. et al. (1994) Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HlyB/HlyDdependent secretion comprises the C-terminal 62 amino acids of HlyA. Mol. Gen. Genet. 245, 53-60.

Khosa, S., et al., AnA/U-Rich Enhancer Region Is Required for High-Level Protein Secretion through the HlyAType I Secretion System. Appl Environ Microbiol, 2018. 84(1).

Koronakis, V. (1989) Isolation and analysis of the C-terminal signal directing export of Escherichia coli hemolysin protein across both bacterial membranes. EMBO J. 8, 595-605. Lhocine, N., et al., Apical invasion of intestinal epithelial cells by Salmonella typhimurium requires villin to remodel the brush border actin cytoskeleton. Cell Host Microbe, 2015. 17(2): p. 164-77.

Madrid, C., et al., Temperature- and H-NS-dependent regulation of a plasmid- encoded virulence operon expressing Escherichia coli hemolysin. J Bacteriol, 2002. 184(18): p. 5058-66.

Nagamatsu, K., et al., Dysregulation of Escherichia coli alpha-hemolysin expression alters the course of acute and persistent urinary tract infection. Proc Natl Acad Sci U S A, 2015. 112(8): p. E871 -80.

Nieto, J.M., et al., Expression of the hemolysin operon in Escherichia coli is modulated by a nucleoid-protein complex that includes the proteins Hha and H- NS. Mol Gen Genet, 2000. 263(2): p. 349-58.

Park, D., et al., Visualization of the type III secretion mediated Sa/mone//a-host cell interface using cryo-electron tomography. Elife, 2018. 7.

Pourhassan, N.Z., et al., Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region. Chembiochem, 2022. 23(6): p. e202100702.

Ruano-Gallego, D., et al., Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system. Microbial Cell Factories, 2019. 18(1): p. 47.

Thomas, S., I.B. Holland, and L. Schmitt, The Type 1 secretion pathway - the hemolysin system and beyond. Biochim Biophys Acta, 2014. 1843(8): p. 1629-41.

Wang, B., M. Mittermeier, and I. Artsimovitch, RfaH May Oppose Silencing by H- NS and YmoA Proteins during Transcription Elongation. J Bacteriol, 2022. 204(4): p. e0059921. Yang, E.Y. and K. Shah, Nanobodies: Next Generation of Cancer Diagnostics and Therapeutics. Front Oncol, 2020. 10: p. 1182.