# Patent

## Title
Strain of Salmonella enterica s. typhimurium, its use and a method to obtain a therapeutic vaccine vector

## Abstract

The present invention relates to a new bacterial strain of Samonella enterica serovar Typhimurium VNP20009 deposited in the Polish Collection of Microorganisms under access no. B/00024 and its us in the production of a vaccine, especially an anti-cancer vaccine. The present invention also relates to a method of obtaining a therapeutic vaccine vector, characterized in that a genetic modification is introduced into the vector strain specific to cancer cells, resulting in the delayed over expression of a gene encoding a protein responsible for the invasive ability of this strain.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Phase Application under 35 U.S.C. §371 of International Application PCT/PL2010/050005, filed on Feb. 23, 2010, which claims the benefit of Polish Application No. PL387319, filed on Feb. 23, 2009, the entire contents of which are hereby incorporated by reference in their entireties.
The present invention relates to the field of pharmacy, particularly the preparation of vaccines, especially of anti-cancer bacterial therapeutic vaccines. Bacteria belonging to the genus Salmonella are Gram-negative bacteria from the Enterobacteriaceae family which are disease-causing in humans and animals, and represent a serious medical and veterinary problem.
Vaccination with live, attenuated strains of Salmonella is an effective way to prevent the infection. Attenuated strains of Salmonella were obtained by introducing specific irreversible mutations into certain genes in the chromosome of Salmonella, such as: aroA (1), aroC (2), surA (3), htrA (4), rpoS (5), and galE (6).
Current methods of Salmonella attenuation which are used to obtain vaccine material are based on the introduction of genetically stable deletions that reduce or eliminate the virulence of bacteria, while they do not change or intensify their immunogenicity.
Through attenuation of wild strain Salmonella typhi, a new strain Ty21a was created and used for vaccination against typhoid fever. Attenuation of this strain has been achieved through a number of mutations induced by chemical mutagens. This has resulted in a strain that is sensitive to galactose (a mutation in galE gene), auxotrophic for isoleucine and valine (mutations in ilvD genes), has reduced resistance to stress (a mutation in rpoS), and is also unable to produce the Vi polysaccharide. The plurality of mutations makes the strain genetically stable and safe. Revertant mutations within the virulence genes were not observed either in vitro or in vivo (7, 8).
The strain CVD908 has clearly defined mutations in the genes aroC and aroD, but causes fever and other adverse reactions in volunteers vaccinated with high doses. Further attenuation of this strain by the deletion of htrA gene, which encodes a serine protease essential for bacterial survival in macrophages, led to strain CVD908-htrA, which is well tolerated even at high doses and shows high immunogenicity (9).
Mutants of Salmonella enterica serovar Typhimurium, lacking the transcription regulator RfaH effectively prevent salmonellosis when used to vaccinate mice. Lack of RfaH affects the expression of genes involved in the synthesis of lipopolysaccharide core and O-antigen. Such mutants do not differ in their ability to proliferate, but show increased susceptibility to antibacterial peptides (10).
Due to the specificity of preventive vaccines, aiming to elicit immune memory after administration, their activity may be based on the ability of attenuated bacteria to stimulate a humoral immune response (production of neutralising antibodies). The generation of an effective humoral response does not require the ability to invade host cells by Samonella sp. vaccines. This means that even relatively large deletions in the chromosome of Salmonella sp. may be tolerated in vaccinting material prepared for use as a preventive vaccine. However, the relative ease of attenuation does not always coincide with the retention of the original (effective) immunogenicity.
A therapeutic vaccine vector should be highly attenuated and possibly poorly immunogenic to provide effectiveness of repeated administration into a patient. In the case of conjugated heterogeneous (allo- or xenogenic) antigens (epitopes), the vector should demonstrate adjuvant properties (while maintaining its low immunogenicity). A therapeutic vector should be capable of stimulating a specific type of immune response, that is to induce a humoral (Th2) or cellular (Th1) response as needed.
The VNP20009 strain was developed as a vaccine vector whose task was to deliver non-toxic cytostatic drug precursors (prodrugs) to tumour tissue (9, 10). Deletions within the genes purl and msbB result in auxotrophism of the strain with a reduced capacity to stimulate TNF in the infected organism. Preliminary studies have shown the ability of preferential accumulation of VNP20009 within tumours in mice (11). VNP20009 was also found to have an inhibitory effect on tumour development. Phase I clinical studies have not confirmed the effects observed in small mammals. So far, there are no studies on the immunogenicity of VNP20009 in mice nor humans (12).
Surface expression of the variable fragment of a CEA-specific antibody (T84.66) improved the ability of selective accumulation of bacteria in malignant tumours (13). However, the stress caused by overexpression of the fusion protein OmpA-scFv (CEA-specific) substantially (100-fold) reduced the invasiveness of transformed VNP20009 (designated VNP/scFv). Despite the reduced invasiveness, an increase in therapeutic effects was observed for VNP/scFv versus VNP20009 in murine models of transplantable tumours stably transfected with the human CEA gene (MC38CEA and CT26CEA adenocarcinomas).
Another reason for the reduced effectiveness of VNP/scFv is probably genetic instability of the strain involving the loss of the plasmid (and hence the loss of ability to express OmpA-scFv) in the absence of antibiotic selection pressure. Reversion to wild type VNP20009 in the patient would not promote the targeting of the bacteria towards CEA-rich sites (tumour tissue).
In the case of a Salmonella sp., vector it is important to retain the invasive features of the bacteria, which are related to the quality of the immune mechanisms stimulated by those bacteria.
There is still a great need to develop expertise in the field of therapeutic vaccines. A particularly important issue is the impact of attenuation-related effects on the strength and type of immune response dependent on the type of microorganism.
The purpose of this invention is to provide an effective vaccine vector, particularly effective for the treatment of cancer.
The aim of the present invention is therefore to obtain the appropriate strain of Salmonella sp., pursuant to demand in the field of therapeutic vaccines as a vector for delivery of therapeutic material to target cells, particularly cancer cells, cells infected with a virus, etc., with immunogenicity resulting from the physiological intracellular location of bacteria. The subject of the present invention is a strain of Salmonella enlerica serovar Typhimurium VNP/sipB deposited in the Polish Collection of Microorganisms under access no. B/00024. Another subject of the present invention is the use of the strain of Salmonella enterica serovar Typhimurium VNP/sipB deposited in the Polish Collection of Microorganisms under access no. B/00024 to produce a vaccine, especially anti-cancer vaccine.
Another subject of the present invention is a method of obtaining a therapeutic vaccine vector, characterized in that a genetic modification is introduced into the vector strain specific to cancer cells, resulting in a delayed overexpression of a gene encoding a protein responsible for the invasive features of this strain.
Preferably, this method of obtaining a therapeutic vaccine vector according to the present invention is characterized in that an expression cassette is obtained, containing a gene encoding a protein under the control of a promoter that controls its delayed overexpression, and then the cassette is integrated into the bacterial chromosome.
Preferably, the therapeutic vaccine vector according to the present invention is a bacterial strain of Salmonella genus, especially Salmonella enterica species, preferably a strain of Salmonella enterica serovar Typhimurium VNP20009.
Preferably, the gene encoding the protein is the sipB gene.
Preferably, the promoter controlling the delayed gene overexpression is the PsifB promoter.
Preferably, the expression cassette is integrated into the bacterial chromosome is the cassette PsifB-sipB according to SEQ ID 1 or the cassette ushA-5′-PsifB-sipB-ushA-3′ according to SEQ ID 2.
In a preferred embodiment of the present invention, the method includes an additional final step of removing a gene for antibiotic resistance from the bacterial genome.



The subject of the present invention in an embodiment is made clear in the figure, in which:
FIG. 1A shows the electrophoretic separation of a PCR product in 1% agarose gel in the presence of ethidium bromide for the sipB gene sequence (1782 bp) obtained from the genomic DNA of a reference strain, S. typhimurium SL5319;
FIG. 1B shows the results obtained using Western blotting confirming the functionality of the cloned sipB gene;
FIG. 2 shows the results obtained as a result of the isolation of the sifB gene promoter and the results of functionality assays of the PsifB-GFP construct:
FIG. 2A shows the image of the electrophoretic separation of the PCR product in 1.2% agarose gel in the presence of ethidium bromide;
FIG. 2B shows a microscopic image of the RAW264.7 macrophage cell line infected with VNP20009 transformed with the pBR322-PsifB-GFP plasmid;
FIG. 2C shows the results of the cytofluorimetric analysis of bacterial cultures. VNP20009/PsifB-sipB-GFP: induction of the expression of the SipB-GFP fusion protein (right panel), GFP-negative bacteria (left panel);
FIG. 3 shows a diagram of the integration of the plasmid into the genome and DNA fragment exchange, stimulated by the cleavage of both DNA strands;
FIG. 4 shows the relative position of the sipB gene sequence, PsifB promoter and the insertion site of the PsifB-sipB cassette (ushA gene) in the genome of Salmonella typhimurium LT2;
FIG. 5 shows a diagram of the pSG76C plasmid including cloned cassette ushA-5′-PsifB-sipB-ushA-3′;
FIG. 6 shows a diagram of the genomic sequence generated through the recombination of the ushA gene with a homologous region 5′-ushA of the plasmid. Primer sequences used for the analysis of chloramphenicol-resistant clones were indicated on the figure;
FIG. 7 shows the electrophoretic separation of RT-PCR products in 1% agarose gel in the presence of ethidium bromide: DNA of the mutated clone was amplified with the following primers: 1—A and B (2350 bp) 2—A and G (3755 bp) 3—A and F (4560 bp) 4—A and D, 5 —C and D (2411 bp); M—mass standard;
FIG. 8 shows the images obtained by electrophoretic separation of RT-PCR products in 1% agarose gel in the presence of ethidium bromide:
in FIG. 8A the image of the product of cDNA amplification with primers specific for PsifB-sipB (1222 bp): 1—VNP20009, 2—modified VNP20009, M—mass standard;
in FIG. 8B the image of the product of cDNA amplification with primers specific for sipB (389 bp; on the upper panel): 1—VNP20009, 2—modified VNP20009; product of cDNA amplification of 16S rRNA (350 bp; on the bottom panel); M—mass standard;
FIG. 9 shows the image of electrophoretic separation of the PCR product with primer sequences complementary to sequences flanking the ushA gene performed on the genomic DNA of a clone obtained through the procedure of PsifB-sipB integration into the VNP20009 chromosome; 1—amplification product with primers E and D (1526 bp), 2—product amplification with primer A and D (4709 bp), M—mass standard;
FIG. 10 shows a graph representing the results of the invasion of RAW264 cells by VNP20009 and VNP/sipB;
FIG. 11 shows the results of an experiment performed on lung metastasis model CT26CEA in Balb/c mice. The upper row shows the lungs of the control mice isolated on day 15 after the intravenous administration of 5×105 CT26CEA cells. White spots visible on the dark background are small tumour foci. The bottom row shows lungs explanted from mice, which were administered Salmonella typhimurium VNP/sipB bacteria intranasally in the amount of 2×107 CFU/mouse 48 hours after the injection of cancer cells;
FIG. 12 presents the result of an experiment performed on lung metastasis model CT26CEA in Balb/c mice. The upper row shows lungs of the control mice isolated on day 15 after intravenous administration of 5×105 CT26CEA cells. White spots visible on the dark background are small tumour foci. The bottom row shows lungs taken from mice, which were administered various modifications of Salmonella typhimurium intranasally in the amount of 2×107 CFU/mouse 96 hours after the injection of cancer cells; the mean numbers of tumour foci in the lungs are shown on the graph in the lower part of the figure.
FIG. 13 shows the results of an experiment performed on lung metastasis model B16F10 in C57Bl/6 mice. The upper row shows lungs of the control mice isolated on day 28 after intravenous administration of 5×105 B16F10 cells. Tumour sites are visible as dark spots. The middle and lower rows shows lungs isolated from mice, which were administered VNP (middle row) or VNP/sipB (bottom row) bacteria intranasally in the amount of 2×107 CFU/mouse 96 hours after the injection of cancer cells;
FIG. 14 shows the effect of antibody isotype skewing caused by selective stimulation of a Th1-type immune response by VNP/sipB. The results were produced by vaccination of Swiss mice (outbred) with VNP20009 strains: mice vaccinated with “wild type” VNP20009 (upper panel), mice vaccinated with VNP/sipB (lower panel);
FIG. 15 shows the result of DNA sequencing performed on a recombinant genomic DNA fragment of the modified strain of Salmonella enterica s. Typhimurium VNP20009:
FIG. 15A shows a DNA sequence, which includes sequences flanking ushA gene and complete sequence of the integrated PsifB-sipB construct that in VNP/sipB bacteria is located within the ushA gene.
FIG. 15B-15D present the results which confirm the identity of the sequence from VNP/sipB with template DNA from a model strain of Salmonella typhimurium LT2. Lower strand: template (lower strand of DNA), the location of the each element of the construct is indicated (sifB promoter, sipB ORF, ushA integration site) in accordance with the numbering of LT2 strain deposited in GenBank (NCBI) (SEQ ID NOS: 23, 25, 27, and 29). Upper strand: marked as “Query”, shows the sequence obtained from the VNP/sipB (SEQ ID NOS: 22, 24, 26, and 28).
FIG. 16 shows the sequences of primers used in the sequencing of genomic DNA (top to bottom; SEQ ID NOS: 30, 31, 32, 5, 9, 33, and 16). A schematic location of the primers is indicated in FIG. 15 within the flanking sequences and the integrated construct.
FIG. 17 shows the Apoptosis of VNP20009- or VNP/sipB-infected MC38CEA cells, measured as a percentage of annexin V binding cells. Bacteria used for infection were expressing RFP. Annexin V-APC fluorescence was analyzed in RFP-positive population of MC38CEA cells.
In the present invention the VNP/sipB vector was obtained with the full retention of invasive ability (FIG. 10).



The prepared vaccine vector utilised the natural ability of Salmonella sp. to infect host cells. The invasiveness of Salmonella sp. is a multifactorial process, which involves components of both the bacterial cells and the host cells (14). Adhesion of bacteria to a host cell causes the activation of Type III Secretion Systems genes (TTSS) grouped into the “Salmonella Pathogenicity Island-1” (SPI-1). Products of those genes, including the SipB protein, allow bacteria to penetrate the cell (15). Expression of another set of genes is activated inside the host cell. Those genes are located within the SPI-2 and their products facilitate the proliferation of the bacteria within the cytoplasm. The expression of SPI-2 genes is regulated by interaction of endoplasmatic factors with the SPI-2 promoters. The promoter of the sifB gene is one of those promoters (16).
Proliferation of the bacteria in the cytoplasm leads to apoptosis of the host cell, which in turn allows the bacteria to escape and subsequently infect other cells. One of the factors that induce apoptosis is the already mentioned SipB protein (14).
It was assumed that delayed (by using the promoter of sifB gene) overexpression of SipB (PsifB-sipB) will result in intracellular elimination of infective bacteria with simultaneous apoptosis/necrosis of the infected cells. Interim infection will form a strong signal for leukocyte migration to the infection site, but will not result in the infection of the population of newly-migrating immune cells.
The result produced by such a vaccine vector will be the eradication of tumours through the concerted action of the bacteria and the immune system.
The present invention is illustrated by the following embodiments:
EXAMPLE 1
Cloning of the SipB Gene
The sequence of the SipB gene (1782 bp) (FIG. 1A) was obtained from the genomic DNA of a reference strain S. typhimurium SL5319 using a PCR technique with the following primers:









(SEQ ID NO: 3)



5′ AACTGCAGAACCAATGCATTGGTTTCTCCCTTTATTTTGGCA



 



(SEQ ID NO: 4)



3′ CGGGATCCCGAAGTAGCATTAGCCGTAGCG,






which contain restriction site PstII BamHI.
cDNA for sipB was cloned into the expression cassette of plasmid pQE30 to obtain a sequence encoding the fusion protein RGS-6His-SipB. The correctness of the sipB sequence was confirmed by sequencing of both strands of sipB cDNA.
The functionality of the cloned sipB gene was confirmed using a standard Western blot technique (FIG. 1B) performed on lysates of E. coli M15 incubated in the presence of IPTG (0.5 mM), using anti-His-tag antibodies.
The results of Western blot analysis showed that the expression of SipB with an inducible promoter was toxic to bacteria, as induced in the E. coli M15 strain (containing the plasmid pREP-4 with the lacI gene encoding the repressor protein). Protein expression was induced with 0.5 mM IPTG in E. coli M15 grown in TB medium at 25° C. up to an optical density of OD600≈1.0. Bacterial lysate was prepared from 120 ml of culture after a 2.5-hour induction and was purified on an IMAC chromatography resin, TALON BD. The collected fractions were separated with SDS-PAGE on 10% gel and the blots were probed with α-RGS-6His antibodies (Qiagen).
Molecular weight of the obtained product corresponded to the mass of SipB protein (62 kDa).
EXAMPLE 2
Isolation of SifB Gene Promoter And Construction of Sipb Gene Controlled by PsifB
The coding sequence of the promoter region of sifB gene was obtained through PCR on genomic DNA of Salmonella typhimurium VNP20009, using the following primers: “forward” CCCAAGCTTGGGCCTTAGCCATTCTGACTG (SEQ ID NO: 5) with a HindIII restriction site and “reverse” GAAGATCTTCACTTCATTACTGGAATAGGTGGT (SEQ ID NO: 6) with a BglII restriction site. Concurrently, a sequence encoding GFP was obtained from the pGFPuv plasmid (Clontech) using PCR and the following primers: “forward” GAAGATCTTCTCACACAGGAAACAGCTATGAC (SEQ ID NO: 7) with a BglII restriction site and “reverse” GAAGATCTTCGCGCTCAGTTGGAATTCA (SEQ ID NO: 8), also with a BglII restriction site.
PCR products were cloned into the plasmid pGEM-TEasy (Promega) and propagated in Escherichia coli DH5α. Upon confirmation of sequence identity with a sequence obtained from GenBank database, the gfp gene was cloned into the BglII restriction site of the pGEM-TEasy-PsifB plasmid, which had been previously obtained from plasmid pGEM-TEasy-gfp. Intracellular induction of the PsifB promoter was confirmed in vitro by infection of the RAW264.7 macrophage cell line with VNP20009 bacteria containing the pGEM-TEasy-PsifB-gfp plasmid, with the gfp sequence cloned in forward or reverse orientation to the promoter. The sipB coding sequence with the RBS and 6His sequences was obtained from the pQE-sipB plasmid using PCR and the following primers:









(SEQ ID NO: 9)



“forward” GGAAGATCTTCCAGAGGAGAAATTAACTATGAGA,



 



(SEQ ID NO: 10)



“reverse” GAAGATCTTCGGAGTCCAAGCTCAGCTA






(both primers with a BglII restriction site).
The PCR product was cloned into the pGEM-TEasy-PsifB plasmid into the BglII site. PsifB-sipB cassette was excised from pGEM-TEasy-PsifB-sipB plasmid with the restrictase NotI and after filling the sticky ends, it was cloned into a low-copy pBR322 plasmid (in EcoRV-NruI cloning site) and into pMoPac2-lpp-ompA-scFv plasmid.
FIG. 2 presents the results of sifB gene promoter isolation and functional tests of PsifB-GFP construct.
FIG. 2A shows an electrophoretic image of PCR product separation in 1.2% agarose gel in the presence of ethidium bromide. The coding sequence of the promoter region of sifB gene (603 bp) was obtained using PCR from the genomic DNA of Salmonella typhimurium. The identity of the sequence was confirmed with the sequence obtained from GenBank (Salmonella typhimurium LT2, 1 691 572-1 692 152 bp). To confirm the functionality of the obtained promoter sequence, a transcriptional fusion protein PsifB-GFP was produced (gfp sequence amplified from the pGFPuv plasmid (Clontech) using PCR). Concurrently, the RBS-RGS-6His-sipB sequence was obtained from plasmid pQE30-sipB via PCR.
FIG. 2B shows a microscopic image obtained of the RAW264.7 macrophage cell line infected with VNP20009 transformed with the pBR322-PsifB-gfp plasmid. Induction of the PsifB promoter in intracellular bacteria was evaluated microscopically. VNP20009 transformed with low-copy plasmid (pBR322) or high-copy plasmid (pGEM-TEasy), both including the PsifB-gfp cassette, did not demonstrate any microscopically detectable GFP expression when grown in TB medium.
FIG. 2C presents the results of cytofluorimetric analysis performed on the bacterial culture. VNP20009/PsifB-sipB-gfp (but not E. coli DH5α/PsifB-sipB-gfp), cultured for 12 hours at 30° C. with shaking (180 RPM) in eukaryotic cell culture medium OPTI-MEM® (reduced serum medium) (Invitrogen), showed the induced expression of the fusion protein SipB-GFP (right panel). The same bacteria cultured in TB were GFP-negative (left panel).
The use of plasmid constructs is limited in vivo due to the frequent loss of plasmids from bacteria in the absence of selection pressure (antibiotics). Yet genetic stability is one of the fundamental features of bacteria that allows their use as vaccine material. A method of obtaining genetically stable bacterial strains is the integration of functional cassettes (promoter-gene) into the bacterial chromosome.
EXAMPLE 3
Integration of the PsifB-SipB Expression Cassette into the VNP20009 Chromosome
In order to obtain a genetically stable, attenuated strain of Salmonella typhimurium with the overexpression of endogenous SipB protein induced inside an infected cell, the PsifB-sipB expression cassette was integrated into VNP20009 genome. Integration was performed by the homologous recombination method, based on the natural recombination and repair system of the bacteria (Rec-A protein activity) and using a conditionally replicating plasmid as a vector for delivering a mutant allele into genomic DNA (17). At a temperature not permitting multiplication of the plasmid, clones which integrated the plasmid (as a result of recombination with wild and mutant copies of the gene) and acquired chromosomal resistance are selected in the presence of an antibiotic. At this stage, homologous regions of the modified gene are duplicated in the genome. In the next stage, an exchange occurs between the duplicated sequences, stimulated by cleavage of DNA sequences within the plasmid sequence present in the genome. The result is a return to the wild allele form or replacement into the mutated form, with simultaneous excision of the antibiotic resistance gene. A diagram of gene exchange by recombination of homologous segments stimulated by DNA cleavage is shown in FIG. 3.
The region of the ushA gene (STM0494) was chosen as the target site for integration, as it has a high homology with the ushA gene of Escherichia coli, but very low expression and low activity of the encoded enzyme in Salmonella typhimurium (UDP-glucose hydrolase with a point mutation, “silent gene”). The ushA gene has been inactivated in strains of S. typhimurium and its active functional homologue is ushB. The DNA sequences of these genes do not show a significant homology (18, 19). Considerable distance between ushA and sequences PsifB and sipB in S. typhimurium genome (FIG. 4) should reduce the genomic instability of the modified strain, associated with the introduction of additional copies of those sequences, which may be a substrate for homologous recombination.
A conditionally replicating plasmid, pSG76-C, was used for the integration. The PsifB-sipB sequence was cloned into the plasmid, flanked with regions of homology, i.e. segments of sequence identical to the target sequence of integration within the bacterial chromosome (5′-ushA and 3′-ushA); chloramphenicol resistance gene for selection of clones, in which crossing-over occurred; ori R6Kγ and an extremely rare restriction site for endonuclease I-SceI (FIG. 4).
Replication of the pSG76-C plasmid requires protein II, supplied with an auxiliary plasmid pPIR-A (with thermosensitive ori pSC101). Plasmid pSG76C-ushA-PsifB-sipB can be built into the genome by a single crossing-over involving a region of homology and the corresponding chromosomal region. At a temperature preventing the replication of the plasmid pPIR-A (37-42° C.), and thus also plasmid pSG76C, in the presence of the antibiotic, there is a selection of clones with the plasmid sequence integrated into the genome. At this stage, flanking regions of homology are duplicated in the genome. Then, expression of I-SceI meganuclease is induced from the pSTKST auxiliary plasmid. The cleavage of both DNA strands within the integrated plasmid sequence stimulates the Rec-A-dependent intramolecular recombination (double strand break-stimulated gene replacement)—a repair of the DNA break occurs, with the use of adjacent flanking regions of homology. Single crossing-over could happen with the participation of the regions of 5′-ushA or 3′-ushA. In the first case the region of integration returns to the wild form (restoration of the ushA gene sequence integrity); in the second case, a productive re-arrangement occurs, with simultaneous removal of the antibiotic resistance gene. A modified bacterial strain is obtained as a result, without any selection marker in the form of antibiotic resistance gene or any other exogenous sequence.
EXAMPLE 4
Cloning the ushA-5′-PsifB-sipB-ushA-3′ Cassette into the pSG76-C Plasmid
Complementary DNA for ushA was obtained from the genomic DNA of VNP20009 using PCR and the following primers:









(SEQ ID NO: 11)



forward FushA GGGGTACCCCGCGATGTTGGAGATAGTAGG,



 



(SEQ ID NO: 12)



reverse RushA GGGGTACCCCTACAGCCAGCTCACCTCA,






both containing a restriction site for the enzyme KpnI. The PCR product (1825 bp) was cloned into the pGEM-TEasy plasmid (Promega) and propagated in Escherichia coli DH5α. Upon confirmation of sequence identity with a sequence obtained from the GenBank database, the PsifB-sipB sequence (obtained from pGEM-TEasy-PsifB-sipB) was cloned into the restriction site HpaI, located within ushA. The orientation of the PsifB-sipB sequence was the reverse of the orientation of the ushA gene.
In the thusly obtained construct, the PsifB-sipB sequence is flanked with a 1091 by fragment of ushA at 5′-end and a 732 by fragment at 3′-end. Then, the ushA-5′ -PsifBsipB-ushA-3′ cassette was cut out with KpnI enzyme and cloned into pSG76-C in KpnI site, yielding pSG76C-USS (FIG. 5).
EXAMPLE 5
Integration of the pSG76C-USS Plasmid into VNP20009 Chromosome
The plasmid was amplified in Escherichia coli DH5α.pir (with the genomic copy of pir gene, encoding II protein) and transformed into the VNP20009 strain (by electroporation), which had been previously transformed with the pPIR-A plasmid. Then, the VNP20009 bacteria were cultured on solid medium as follows: 40 hours at 30° C. with ampicillin and chloramphenicol, 5 hours at 30° C. with chloramphenicol, 17 hours at 42° C. with chloramphenicol and 7 hours at 37° C. with chloramphenicol, for the selection of clones that have acquired the chromosomal antibiotic resistance. Among the transformants resistant to chloramphenicol, 5 large colonies of bacteria were selected, transferred to solid medium with chloramphenicol and cultured for additional 20 hours at 37° C. Then the selected clones were tested for integration of the plasmid into chromosome, that is, whether the plasmid was inserted into the ushA gene. PCR was carried out with a pair of primers complimentary to sequences flanking the insertion site and to the plasmid sequence and PsifB-sipB (FIG. 5, Table 1), with the following conditions: 94° C. 90 sec, 94° C. 45 s, 58° C. 30 s, 72° C. for 2 min 30 sec, 28 cycles.
Accordingly, a diagram of genomic sequence generated by recombination between ushA gene and homology region 5′-ushA of the plasmid in shown FIG. 6. The position of the primers used for the analysis of chloramphenicol-resistant clones is also indicated.
The primers used in the experiment are listed in Table 1:







TABLE 1







Primer sequences used for analysis of recombinant clones and mass 


of PCR products confirming the integration of the plasmid into the 


genome within the ushA gene sequence.












PRODUCT-
PRODUCT AFTER


PRIMER
SEQUENCE (5′-3′)
WILD TYPE
INTEGRATION





FushA
GGGGTACCCCGCGATGTTGGAGATAGTAGG
1825 bp
8366 bp



(SEQ ID NO: 13)




 


RushA
GGGGTACCCCTACAGCCAGCTCACCTCA

4366 bp (after removal



(SEQ ID NO: 14)

of resistance gene)


 


A
GCGACTGGATCATATCGT
—
2350 bp



(SEQ ID NO: 15)




 


B
CGCCTCACTATGCTCATG





(SEQ ID NO: 16)




 


C
CTGAACGGTCTGGTTATAGG
—
2411 bp



(SEQ ID NO: 17)




 


D
CTGGATATTGAACTGGCG

- (after removal of



(SEQ ID NO: 18)

resistance gene)


 


A
GCGACTGGATCATATCGT
2168 bp
8709 bp



(SEQ ID NO: 15)




 


D
CTGGATATTGAACTGGCG

4709 bp (after removal



(SEQ ID NO: 18)

of resistance gene)


 


E
CCCAAGCTTGGGCCTTAGCCATTCTGACTG
—
5140 bp



(SEQ ID NO: 19)




 


D
CTGGATATTGAACTGGCG

1526 bp (after removal



(SEQ ID NO: 18)

of resistance gene)


 


A
GCGACTGGATCATATCGT
—
4560 bp



(SEQ ID NO: 15)




 


F
GCAGGTCGACTCTAGAGGAT

- (after removal of



(SEQ ID NO: 20)

resistance gene)


 


A
GCGACTGGATCATATCGT
—
3755 bp



(SEQ ID NO: 15)




 


G
CCCAAGCTTGGGCCTTAGCCATTCTGACTG





(SEQ ID NO: 21)









In the case of plasmid integration into ushA gene in PCR with primers A and B, a product of 2350 by was obtained and with primers C and D—a 2411 by product, while the standard PCR with primers A and D did not yield any product (FIG. 7).
FIG. 7 shows an electrophoretic image of the separation of PCR in a 1% agarose gel in the presence of ethidium bromide. DNA of the mutated clone was amplified using the following primers: 1—A and B (2350 bp) 2—A and G (3755 bp) 3—A and F (4560 bp) 4—A and D, 5—C and D (2411 bp).
Total RNA was isolated from wild-type VNP20009 bacteria and a clone positive for the integration, cultured in conditions that induced the PsifB promoter activity. In a RT-PCR with primer specific for the sipB copy newly introduced into the genome (complementary to the sequence including the synthetic RBS sequence) performed on a template derived from the mutant clone, a product was obtained with molecular mass corresponding to 1222 by (FIG. 8).
FIG. 8 shows the images obtained by electrophoretic separation of RT-PCR products in 1% agarose gel in the presence of ethidium bromide. RNA was isolated from bacteria cultured under PsifB promoter-inducing conditions. (A) Product of cDNA amplification with primers specific for PsifB-sipB (1222 bp): 1—VNP20009, 2—modified VNP20009; (B) product of cDNA amplification with primers specific for sipB (389 bp): 1—VNP20009, 2—modified VNP20009 and product of cDNA amplification for 16S rRNA (350 bp).
EXAMPLE 6
Removal of Antibiotic Resistance Gene from the VNP20009 Genome
The above-mentioned genetic manipulations required the presence of an antibiotic resistance gene, which is undesirable in the final vaccine material. Therefore, this gene was deleted in the following stage. The cleavage of genomic DNA at the restriction site for I-SceI enzyme, which had been introduced into the genome together with the plasmid sequence, stimulated recombination with the participation of neighbouring regions of homology and selection of the clones, in which the DNA break was repaired. Clones with confirmed integration of the pSG76C-USS plasmid were subsequently transformed with plasmid pSTKST (with thermosensitive ori sequence pSC101) containing a gene for I-SceI meganuclease under the control of the tetracycline promoter. The clones were cultured on solid medium with kanamycin at 30° C. Then, individual colonies were transferred into liquid LB medium with kanamycin (20 μg/ml) and autoclaved chlortetracycline (cTc, 30 μg/ml, induces the expression of I-SceI by inactivating the tetracycline repressor) and incubated for 24 hours at 30° C. The culture was diluted 1:106, transferred to solid medium with kanamycin and cTc and cultured at 30° C. for 20 hours. Then, the clones were tested for the deletion of the resistance gene and for planned recombination with a PCR using pair of primers E and D—in this case the obtained product had 1526 by (FIG. 9, lane 1). PCR was performed using the primers A and D and a programme as follows: 94° C. 2 min, 94° C. 30 s, 58° C. 30 s, 70° C. 4 min, 10 cycles; 94° C. 30 s, 58° C. 30 s, 70° C. 4 min +10 sec/cycle, 20 cycles. The resulting product was longer than 4500 by and corresponded to the length of the ushA sequence with the integrated PsifB-sipB cassette (FIG. 9, path 2).
FIG. 9 shows the image of electrophoretic separation of the PCR product with primer sequences complementary to sequences flanking the ushA gene performed on the genomic DNA of a clone obtained with the procedure of PsifB-sipB integration into the VNP20009 chromosome. Amplification products were obtained with primers E and D (1526 bp) and with primers A and D (4709 bp).
Integration of the PsifB-sipB functional cassette into the VNP20009 chromosome is such a significant genetic modification of the bacteria, that it results in obtaining of a new bacterial strain.
EXAMPLE 7
Testing of the VNP/sipB (INT) Functionality
EXAMPLE 7A
Comparison of the Invasive Ability of VNP20009 And VNP/sipB Bacteria
The invasiveness VNP20009 and VNP/sipB strains was tested on RAW264.7 cells. No statistically significant differences in the invasiveness were found between “wild” and recombinant strains of VNP. RAW264.7 macrophage cell line was cultured on 48-well plates at the density of 2.5 ×104 in DMEM medium with 10% serum for 24 hours. The cells were infected with VNP20009 and VNP/sipB in 100 μl of OPTI-MEM® (reduced serum medium) (Invitrogen) without serum at MOI 5, determined by measuring optical density at 600 nm. After 1-hour of co-incubation of the cells with bacteria (37° C., 5% CO2), another 100 μl of OPTI-MEM® (reduced serum medium) (Invitrogen) supplemented serum and gentamicin (2% serum, 100μg/ml gentamicin) was added and incubated for 3 hours (37° C., 5% CO2) to eliminate extracellular bacteria. Then, the cells were harvested and transferred to LB/agar plates. The number of live intracellular bacteria was assessed based on the number of bacterial colonies present after 24-hour incubation and referred to the number of cells in the specific well.
Concurrently, the number of CFU per well of the start of infection was determined, based on the amount of bacterial colonies after 24-hour culturing on LB/agar plates, obtained from an appropriately diluted bacterial suspension used for the infection. Invasiveness of the bacteria was defined as the fraction of intracellular bacteria from the number of bacteria used for the infection (FIG. 10).
EXAMPLE 7B
Therapeutic Effects of VNP/sipB In A Mouse Tumour Model
The experiment was performed on the CT26CEA lung metastasis model in Balb/c mice. Lungs were stained with Indian ink. FIG. 11 (upper row) shows lungs of the control mice isolated on day 15 after the intravenous administration of 5×105 CT26CEA cells. White spots visible on the dark background are small tumour foci and their number (>300 per lung) and randomly assessed volume illustrate the effectiveness of metastasis. The bottom row shows lungs taken from mice, which were administered Salmonella typhimurium VNP/sipB bacteria intranasally in the amount of 2×107 CFU/mouse 48 hours after the injection of cancer cells. In those lungs, an average of <20 tumours/lung were found. In the group of mice that were vaccinated with VNP/sipB, no detectable tumours were found in 50% of the examined lungs.
EXAMPLE 7C
Comparison of Therapeutic Effects of Various VNP20009 Modifications In A Mouse Tumour Model
The experiment was performed on the CT26CEA lung metastasis model in Balb/c mice. Lungs were stained with Indian ink. FIG. 12 (upper row) shows lungs of the control mice isolated on day 15 after intravenous administration of 5×105 CT26CEA cells. White spots visible on the dark background are small tumour foci and their number (>300 per lung) and randomly assessed volume illustrate the effectiveness of metastasis. The bottom row shows lungs taken from mice, which were administered various modifications of Salmonella typhimurium bacteria intranasally in the amount of 2×107 CFU/mouse 96 hours after the injection of cancer cells. In the lungs VNP/sipB vaccinated animals an average of 54 tumours/lung was found and 50% of the examined lungs did not have any detectable tumours; the mean numbers of tumour foci in the lungs are shown on the graph in the lower part of the figure.
EXAMPLE 7D
Comparison of Therapeutic Effects of VNP20009 And VNP/sipB In Mouse Tumour Model of B16F10 Melanoma
The experiment was performed on the B16F10 lung metastasis model in C57B1/6 mice (FIG. 13). Lungs were stained with picric acid. FIG. 13 (upper row) shows lungs of the control mice isolated on day 28 after intravenous administration of 5×105 B16F10 cells. Tumours are shown as dark dots and their number and randomly assessed size illustrate the effectiveness of metastasis. The middle and lower rows shows lungs isolated from mice, which were administered VNP (middle row) or VNP/sipB (bottom row) bacteria intranasally in the amount of 2×107 CFU/mouse 96 hours after the injection of cancer cells. In these lungs, less than 2 tumours/lung were found on average. In the group of mice that were vaccinated with VNP/sipB, no detectable tumours were found in 75% of the examined lungs.
EXAMPLE 7E
Selective Stimulation of Th1-Type Response By VNP/sipB
As an effect of the vaccination of Swiss mice (outbred) with the modified VNP20009 strains, immune response skewing towards a Th1-type response is found in half of the vaccinated animals (FIG. 14). While administration of the “wild type” VNP20009 (top panel) elicited all analysed antibody isotypes (IgG1, IgG2a and IgM), only high levels of IgG2a and the almost complete absence of IgG1 were found in the serum of mice vaccinated with VNP/sipB (bottom panel). It is very likely that the observed isotype skewing forms an important link in the chain of immune responses stimulated by VNP/sipB, which result, inter alia, in growth inhibition (or elimination) of tumours in the vaccinated mice.
EXAMPLE 7F
Apoptosis of VNP/sipB Infected MC38CEA Cells
Apoptosis induction in MC38CEA adenocarcinoma cells infected with VNP20009 or VNP/sipB was analyzed by flow cytometry of annexin V-stained cells. Bacteria transformed with the pDsRed2 plasmid encoding RFP (Red Fluorescent Protein) were used for the infection.
Cells were seeded on 24-well plate at 7.5·104 per well in DMEM containing 10% FBS and cultured at 37° C. in 5% CO2. Infection was carried out with bacteria suspended in 200 μl Opti-MEM at the multiplicity of infection equal to 20 bacteria per cell. Bacteria were allowed to invade cells for 45 min at 37° C. in 5% CO2. Then, 200 μl of gentamicin-containing Opti-MEM was added to kill extracellular bacteria (final concentration of gentamicin was 100 μg/ml). Incubation was continued for 1.5 h (37° C., 5% CO2). The media were then replaced with 0.5 ml Opti-MEM with 20 μg/ml gentamicin. After 20 h of incubation, cells were collected, stained with APC-labeled annexin V for 15 min at room temperature and analyzed by flow cytometry. FIG. 17 shows the percentage of annexin V binding cells gated on an RFP-positive, bacteria-infected population. There was around 10% more annexin V positive MC38CEA cells following infection with VNP/sipB than VNP20009.
EXAMPLE 8
Sequencing of the Recombinant Fragment of Genomic DNA of the Modified Salmonella enterica s. Typhimurium VNP20009 Strain
The following DNA sequences were analysed: the ushA gene with its flanking sequences and the entire sequence of the integrated PsifB-sipB construct that in VNP/sipB bacteria is located within the ushA gene (FIG. 15A). The identity of the sequence from VNP/sipB with template DNA from a model strain of Salmonella typhimurium, LT2, was confirmed using BLAST software (NCBI). On the template (lower strand of DNA) in FIGS. 15B-15D, the location of the each element of the construct is indicated (sifB promoter, sipB ORF, ushA integration site) in accordance with the numbering of the LT2 strain deposited in GenBank (NCBI). Upper strand, marked as “Query”, shows the sequence obtained from the VNP/sipB. The PsifB sequence in the genome of Salmonella typhimurium LT2 is located in the position 1 691 578-1 692 147 bp. The sipB sequence in the genome of Salmonella typhimurium LT2 is located in the position of 3 029 114-3 030 895 bp. The ushA sequence in the genome of Salmonella typhimurium LT2 is located in the position 553 634-555 286 bp.
Oligonucleotides used for sequencing of genomic DNA are listed in FIG. 16. The schematic location of the primers is indicated in FIG. 15 within the flanking sequences and the integrated construct.
As a result of the production of a properly modified strain of Salmonella enterica s. Typhimurium, a favourable therapeutic vaccine vector was produced, which is particularly suitable for use as an anti-cancer bacterial vaccine vector.
Deposited Materials
The stain of Salmonella enterica serovar Typhimurium VNP/sipB was deposited with the Polish Collection of Microorttanisms, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114, Wroclaw, Poland, on Nov. 26, 2008, and has been assigned the indicated accession no. B/00024. The strain of Salmonellal enterica serovar Typhimurium VNP/sipB is a strain of Salmonella enterica serovar Typhimurium VNP20009 with a PsifB-sipB functional cassette integrated into its chromosome.
Literature


    1. Hoiseth, S. K., and B. A. Stocker. 1981. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291:238-239.
    2. Dougan, G., S. Chatfield, D. Pickard, J. Bester, D. O'Callaghan, and D. Maskell. 1988. Construction and characterization of vaccine strains of Salmonella harboring mutations in two different aro genes. J Infect Dis 158:1329-1335.
    3. Sydenham, M., G. Douce, F. Bowe, S. Ahmed, S. Chatfield, and G. Dougan. 2000. Salmonella enterica serovar typhimurium surA mutants are attenuated and effective live oral vaccines. Infect Immun 68:1109-1115.
    4. Chatfield, S. N., K. Strahan, D. Pickard, I. G. Charles, C. E. Hormaeche, and G. Dougan. 1992. Evaluation of Salmonella typhimurium strains harbouring defined mutations in htrA and aroA in the murine salmonellosis model. Microb Pathog 12:145-151.
    5. Coynault, C., V. Robbe-Saule, and F. Norel. 1996. Virulence and vaccine potential of Salmonella typhimurium mutants deficient in the expression of the RpoS (sigma S) regulon. Mol Microbiol 22:149-160.
    6. Dougan, G., C. E. Hormaeche, and D. J. Maskell. 1987. Live oral Salmonella vaccines: potential use of attenuated strains as carriers of heterologous antigens to the immune system. Parasite Immunol 9:151-160.
    7. Gentschev, I., S. Spreng, H. Sieber, J. Ures, F. Mollet, A. Collioud, J. Pearman, M. E. Griot-Wenk, J. Fensterle, U. R. Rapp, W. Goebel, S. A. Rothen, and G. Dietrich. 2007. Vivotif—a ‘magic shield’ for protection against typhoid fever and delivery of heterologous antigens. Chemotherapy 53:177-180.
    8. Cheminay, C., and M. Hensel. 2008. Rational design of Salmonella recombinant vaccines. Int J Med Microbiol 298:87-98.
    9. Bermudes, D., L. M. Zheng, and I. C. King. 2002. Live bacteria as anticancer agents and tumor-selective protein delivery vectors. Curr Opin Drug Discov Devel 5:194-199.
    10. Pawelek, J. M., K. B. Low, and D. Bermudes. 1997. Tumor-targeted Salmonella as a novel anticancer vector. Cancer Res 57:4537-4544.
    11. Clairmont, C., K. C. Lee, J. Pike, M. Ittensohn, K. B. Low, J. Pawelek, D. Bermudes, S. M. Brecher, D. Margitich, J. Turnier, Z. Li, X. Luo, I. King, and L. M. Zheng. 2000. Biodistribution and genetic stability of the novel antitumor agent VNP20009, a genetically modified strain of Salmonella typhimurium. J Infect Dis 181:1996-2002.
    12. Toso, J. F., V. J. Gill, P. Hwu, F. M. Marincola, N. P. Restifo, D. J. Schwartzentruber, R. M. Sherry, S. L. Topalian, J. C. Yang, F. Stock, L. J. Freezer, K. E. Morton, C. Seipp, L. Haworth, S. Mavroukakis, D. White, S. MacDonald, J. Mao, M. Sznol, and S. A. Rosenberg. 2002. Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol 20:142-152.
    13. Bereta, M., A. Hayhurst, M. Gajda, P. Chorobik, M. Targosz, J. Marcinkiewicz, and H. L. Kaufman. 2007. Improving tumor targeting and therapeutic potential of Salmonella VNP20009 by displaying cell surface CEA-specific antibodies. Vaccine 25:4183-4192.
    14. Cossart, P., and P. J. Sansonetti. 2004. Bacterial invasion: the paradigms of enteroinvasive pathogens. Science 304:242-248.
    15. Hersh, D., D. M. Monack, M. R. Smith, N. Ghori, S. Falkow, and A. Zychlinsky. 1999. The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1. Proc Natl Acad Sci USA 96:2396-2401.
    16. Freeman, J. A., M. E. Ohl, and S. I. Miller. 2003. The Salmonella enterica serovar typhimurium translocated effectors SseJ and SifB are targeted to the Salmonella-containing vacuole. Infect Immun 71:418-427.
    17. Posfai, G., V. Kolisnychenko, Z. Bereczki, and F. R. Blattner. 1999. Markerless gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome. Nucleic Acids Res 27:4409-4415.
    18. Burns, D. M., and I. R. Beacham. 1986. Identification and sequence analysis of a silent gene (ushAO) in Salmonella typhimurium. J Mol Biol 192:163-175.
    19. Innes, D., I. R. Beacham, C. A. Beven, M. Douglas, M. W. Laird, J. C. Joly, and D. M. Burns. 2001. The cryptic ushA gene (ushA(c)) in natural isolates of Salmonella enterica (serotype Typhimurium) has been inactivated by a single missense mutation. Microbiology 147:1887-1896.

## Claims

1. A method of producing a vector, comprising introducing a nucleic acid containing a gene encoding sipB under the control of the PsifB promoter into Salmonella enterica serovar Typhimurium VNP20009.

2. The method of claim 1, wherein the nucleic acid is an expression cassette, and the expression cassette is integrated into the bacterial chromosome.

3. The method of claim 2, wherein the expression cassette is a PsifB-sipB cassette comprising SEQ ID NO 1 or a ushA-5′-PsifB-sipB-ushA-3′ cassette comprising SEQ ID NO: 2.

4. The method of claim 2, the method further comprising removing it gene for antibiotic. resistance from Salmonella enterica serovar Typhimurium VNP20009.

5. The method of claim 1, wherein the vector is the strain of Salmonella enterica serovar Typhimurium VNP/sipB deposited at the Polish Collection of Microorganisms under the accession number B/00024.