Modified bcg vaccine

Abstract

The present invention relates to a recombinant Mycobacterium bovis BCG strain comprising a plasmid having a short guide RNA (sgRNA) target sequence for knockdown of the Mb3739 gene. Also provided are vaccine compositions for eliciting an immune response against Mycobacterium tuberculosis comprising the recombinant Mycobacterium bovis BCG strain engineered to activate the NOD-1 pathway. The recombinant Mycobacterium bovis BCG strain or the vaccine compositions may be useful in methods of eliciting an immune response against Mycobacterium tuberculosis. The invention also relates to methods of obtaining the recombinant Mycobacterium bovis BCG strain.

Description

[0001] MODIFIED BCG VACCINE

[0002] BACKGROUND OF THE INVENTION

[0003] The present invention relates to a recombinant Mycobacterium bovis BCG strain and to vaccine compositions for eliciting an immune response against Mycobacterium tuberculosis. The recombinant Mycobacterium bovis BCG strain or the vaccine compositions may be useful in methods of eliciting an immune response against Mycobacterium tuberculosis and for use as a tuberculosis (TB) vaccine. The invention also relates to methods of producing the recombinant Mycobacterium bovis BCG strain.

[0004] Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains the leading cause of death from an infectious disease worldwide. Approximately 2 billion people worldwide are latently infected with Mtb and represent a reservoir of future active disease, despite the availability of a TB vaccine - the Bacille Calmette Guerin (BCG) vaccine. The BCG vaccine is the only licensed TB vaccine and has been in use since the 1920s, with close to 100 million infants worldwide vaccinated to date. The BCG vaccine protects against TB meningitis and milliary TB in children; however, this vaccine is not entirely protective against TB in adults, showing a range of protective efficacy between 0-80%. In fact, BCG efficacy is thought to wane away completely by adulthood. The conventional BCG vaccine strain was made by repeatedly growing a pathogenic Mycobacterium bovis strain in the lab until the strain lost genetic material and became too weak to cause disease but is still sufficient enough to induce immune responses to act as a vaccine.

[0005] Several TB vaccine candidates have been developed and some have successfully entered clinical trials. However, despite decades of research, a TB vaccine candidate that induces sterilizing immunity remains elusive. This is partly because of limited knowledge of correlates of protection required for elimination of Mtb infection. Most pre-clinical TB vaccine candidates reduce Mtb CFU by only 1 -1.5 logs in mice challenge experiments when administered intradermally or subcutaneously and this is usually considered sufficiently protective for further development. Intravenous vaccination of mice and non-human primates with the conventional BCG vaccine has demonstrated sterilizing immunity against Mtb infection. Some studies have highlighted a role of BCG vaccine induced trained innate immunity, a de facto innate immune memory dependent on the NOD-2 pathway for development of anti-tuberculous immunity, in addition to the role of T helper-1 cell-mediated immune responses and antibody responses. These findings renewed interest in targeting innate immunity with recombinant BCG strains modified to produce various immune- modulatory molecules including pro-inflammatory cytokines, bacterial toxins as well as other non-protein immunomodulatory molecules. Other strategies to improve BCG efficacy involve the deletion of immuno-evasion / suppressive genes from BCG to enable induction of enhanced anti-tuberculous immune responses.

[0006] NOD-1 is a ubiquitously expressed intracellular innate pathogen recognition receptor (PRR) that senses iE-DAP, a component of bacterial peptidoglycan (PG), during infection. NOD-1 appears to be important for rapid detection of bacterial infection, specifically for detection of gram-negative bacterial infection. Activation of NOD-1 triggers the production of pro-inflammatory cytokines through nuclear factor KB (NF-KB) and mitogen-activated protein kinase (MAPK). Mycobacteria evade NOD-1 activation through modification of the NOD-1 ligand iE-DAP into iQ-DAP, adding to immunoevasion / suppressive strategies. The amidation of the peptidoglycan serves as an immune evasion strategy as amidated peptidoglycan cannot be recognised by the NOD-1 system in humans. As a result, BCG does not produce a long-lasting immune response and whilst this vaccine is given to infants, the protective immunity it confers wanes by adolescence. Protection in adults is highly variable. Thus, the present invention seeks to address at least some of the limitations of the current BCG vaccine.

[0007] SUMMARY OF THE INVENTION

[0008] The invention relates to a Mycobacterium bovis BCG strain comprising disrupted Mb3739 gene expression, particularly a recombinant Mycobacterium bovis BCG strain comprising a plasmid having a short guide RNA (sgRNA) target sequence for knockdown of the Mb3739 gene. Also provided is a method of producing a Mycobacterium bovis BCG strain comprising a disrupted Mb3739 gene. The invention further relates to vaccine compositions comprising the recombinant Mycobacterium bovis BCG strain. The recombinant Mycobacterium bovis BCG strain or the vaccine compositions may be useful in methods of eliciting an immune response against Mycobacterium tuberculosis in a subject.

[0009] According to a first aspect of the present invention there is provided for a Mycobacterium bovis BCG strain comprising disrupted expression of a Mb3739 gene. It will be appreciated by those of skill in the art that several methods of disruption of gene expression are known, including using recombinant DNA technology. Preferably, the Mycobacterium bovis BCG strain presented herein is a recombinant Mycobacterium bovis BCG strain.

[0010] In a first embodiment of the Mycobacterium bovis BCG strain of the invention, the recombinant Mycobacterium bovis BCG strain may comprise a plasmid having a short guide RNA (sgRNA) target sequence for knockdown of expression of the Mb3739 gene.

[0011] According to a second embodiment of the Mycobacterium bovis BCG strain of the invention, the sgRNA may target the 5’ region of the murT-gatD operon. In a third embodiment of the Mycobacterium bovis BCG strain of the invention, the sgRNA may comprise a nucleotide sequence of SEQ ID NO:3.

[0012] In a fourth embodiment of the Mycobacterium bovis BCG strain of the invention, the plasmid may be a CRISPRi-dCas9 plasmid.

[0013] According to a fifth embodiment of the Mycobacterium bovis BCG strain of the invention, the disruption or knockdown of the expression of the Mb3739 gene may result in modification of a peptidoglycan in the Mycobacterium bovis BCG strain.

[0014] In another embodiment of the Mycobacterium bovis BCG strain of the invention, the modification reduces amidation of the peptidoglycan to activate the NOD-1 pathway.

[0015] In yet a further embodiment of the Mycobacterium bovis BCG strain of the invention, the recombinant Mycobacterium bovis BCG strain comprising the modification is capable of eliciting an immune response against Mycobacterium tuberculosis.

[0016] According to a second aspect of the present invention, there is provided for a method of producing a recombinant Mycobacterium bovis BCG strain comprising disrupted expression of a Mb3739 gene, the method comprising the steps of: a) cloning a short guide RNA (sgRNA) target sequence for knockdown of expression of the Mb3739 gene into a CRISPRi-dCas9 plasmid to obtain a cloned plasmid; and b) introducing the cloned plasmid from step a) into Mycobacterium bovis BCG, wherein the cloned plasmid causes knockdown of the expression of the Mb3739 gene in the recombinant Mycobacterium bovis BCG strain.

[0017] In a first embodiment of the method of producing a recombinant Mycobacterium bovis BCG strain of the invention, the sgRNA may comprise a nucleotide sequence of SEQ ID NO:3.

[0018] According to a second embodiment of the method of producing a recombinant Mycobacterium bovis BCG strain, the knockdown of the expression of the Mb3739 gene may result in modification of a peptidoglycan in the Mycobacterium bovis BCG strain.

[0019] In a third embodiment of the method of producing a recombinant Mycobacterium bovis BCG strain of the invention, the modification may reduce amidation of the peptidoglycan to activate the NOD-1 pathway.

[0020] According to a fourth embodiment of the method of producing a recombinant Mycobacterium bovis BCG strain of the invention, the cloned plasmid may be introduced into the Mycobacterium bovis BCG strain by electroporation.

[0021] In a third aspect of the present invention there is provided for a recombinant Mycobacterium bovis BCG strain produced by the method of producing a recombinant Mycobacterium bovis BCG strain of the invention. According to a fourth aspect of the present invention there is provided for a vaccine composition comprising a Mycobacterium bovis BCG strain of the invention, as described herein.

[0022] In one embodiment of the vaccine composition of the invention, the composition may further comprise a pharmaceutically acceptable carrier or adjuvant.

[0023] According to a fifth aspect of the present invention there is provided for a method of eliciting an immune response against Mycobacterium tuberculosis in a subject, preferably a human subject, the method comprising administering an immunogenically effective amount of the recombinant Mycobacterium bovis BCG strain of the invention as described herein, or the vaccine composition of the invention as described herein, to the subject.

[0024] In a further aspect of the present invention there is provided for a Mycobacterium bovis BCG strain of the invention as described herein, or a vaccine composition of the invention as described herein, for use in a method of eliciting an immune response against Mycobacterium tuberculosis in a subject, preferably a human subject, the method comprising administering an immunogenically effective amount of the recombinant Mycobacterium bovis BCG strain or vaccine composition to the subject.

[0025] BRIEF DESCRIPTION OF THE FIGURES

[0026] Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

[0027] Figure 1 : Knockdown of murT-gatD gene expression using CRISPRi-dCas9. A short-guide RNA that targets knockdown of the murT-gatD two-gene operon was introduced into the BCG strain.

[0028] Figure 2: Schematic of the CRISPRi mechanism in bacteria. Schematic of CRISPRi-mediated transcriptional repression. Anhydrotetracycline (ATc)-inducible (PTet) dcas9 is directed to specific DNA targets by ATc-inducible sgRNA, which then prevents transcription initiation or elongation. RNAP: RNA polymerase; NT: non-template; T: template.

[0029] Figure 3: MurT-GatD operon showing the MurT (Mb3739)-GatD (Mb3740) sequences. The PAM sequence is shown in the block and sgRNA sequence is underlined. This sequence is provided as SEQ ID NO:1 .

[0030] Figure 4: Schematic diagram depicting the models of WT BCG and rBCG induced training of monocytes. CRISPRi mediated depletion of MurT-GatD in rBCG::iE-DAP is hypothesized to reduce PG amidation leading to both NOD-1 and NOD-2 activation. Figure 5: qPCR data for CRISPRi depletion of MurT-GatD in rBCG::CRISPRi-

[0031] MurT-GatD. CRISPRi activation with ATc (200 ng / ml) resulted in inhibition of murT expression.

[0032] Figure 6: NOD-1 and NOD-2 gene expression measured by quantitative PCR in non-activated (A & B) and INFy activated (C & D) THP-1 macrophages stimulated with E. coli, WT BCG and rBCG::iE-DAP.

[0033] Figure ?: Schematic representation of MurT-GatD mediated PG precursor amidation.

[0034] Figure 8: Phenotypic characterization of rBCG::CRISPRi-MurT-GatD: Scanning electron micrographs of WT BCG and rBCG-CRISPRi-MurT-GatD grown in media supplemented with 200 ng / ml ATc. Depletion of MurT-GatD in rBCG-CRISPRi-MurT-GatD causes outer cell wall defects (wrinkled and dents in the cell wall).

[0035] Figure 9: Phenotypic characterization of rBCG::CRISPRi-MurT-GatD: Frequency of cells with cell wall defects.

[0036] Figure 10: Phenotypic characterization of rBCG::CRISPRi-MurT-GatD: Minimum inhibitory concentration of antibiotics targeting cell wall biosynthesis against Wildtype BCG and rBCG-CRISPRi-MurT-GatD grown in media supplemented with 200 ng / ml Ate. AMX: amoxicillin, CLV: clavulanate, MPM: meropenem, VANC: vancomycin and ETM: ethionamide. Depletion of MurT-GatD in rBCG-CRISPRi-MurT-GatD causes increased sensitivity to cell wall targeting antibiotics.

[0037] Figure 11 : Phenotypic characterization of rBCG::CRISPRi-MurT-GatD: MurT- GatD depleted cells labelled with fluorescent BODIPY-FL vancomycin reveals side-wall labelling.

[0038] Figure 12: Phenotypic characterization of rBCG::CRISPRi-MurT-GatD: Transmission electron micrographs of WT BCG and rBCG-CRISPRi-MurT-GatD grown in media supplemented with 200 ng / ml ATc. Depletion of MurT-GatD causes cell wall defects.

[0039] Figure 13: Phenotypic characterization of rBCG::CRISPRi-MurT-GatD: Frequency of cells with cell wall defects.

[0040] Figure 14: Phenotypic characterization of rBCG::CRISPRi-MurT-GatD: Flow cytometry analysis of WT BCG and rBCG-CRISPRi-MurT-GatD cells labelled with a PG amidation reporter probe (TAMRA-Ala-D-glutamine-L-Lys-D-Ala [TetraFI]). Depletion of MurT-GatD causes increased labelling with the PG amidation reporter indicative of reduced PG amidation in the MurT-GatD depleted cells.

[0041] Figure 15: Depletion of MurT and GatD causes reduced PG amidation: (A) Flow chart representation of the protocol used for assessing PG amidation in MurT-GatD depleted cells by Alexa Fluor 488 NHS Ester labelling of PG in comparison with control cells (ATc- and WT BCG). The Alexa Fluor 488 NHS Ester labels primary amines (R-NH2) also found in PG as a result of amidation. (B). Quantification of Alexa Fluor 488 NHS Ester labelled PG from MurT-GatD depleted cells in comparison to the no ATc control cells. MurT- GatD depletion causes decreased PG amidation which results in decreased labelling with Alexa Fluor 488 NHS Ester.

[0042] Figure 16: rBCG::CRISPRi-MurT-GatD enhances the Mtb H37Rv killing ability of macrophages derived from trained U937 monocytes: Macrophages derived from heat-killed rBCG-trained monocytes were able to control Mtb H37Rv growth significantly different from macrophages derived from BCG, LPS, MDP trained / stimulated monocytes and nonstimulated macrophages (RPMI control). Statistical analysis was conducted using Student t- test *: p-value: <0.01 .

[0043] Figure 17: Survival of rBCG-CRISPRi-MurT-GatD in IFNy activated bone marrow derived macrophages (BMDMs) and analysis of pro-inflammatory cytokine release: IFNy activated BMDMs (1 x 106cells) were infected at MOI: 1 with BCG and rBCG. ATc was added to culture media for induction of the CRISPRi system in rBCG at concentrations ranging from 100 ng / ml - 500 ng / ml and growth of the strains was assessed after 3 and 5 days. Supplementation of culture media with 400-500 ng / ml ATc resulted in significantly reduced growth of rBCG on day 5 in comparison to WT BCG.

[0044] Figure 18: Analysis of pro-inflammatory cytokine release: Secreted TNFa and IL- 6 levels from non-activated and IFNy-activated BMDMs infected with WT BCG and rBCG- CRISPRi-MurT-GatD at MOI 1 :20. No difference in IL-6 secretion from WT BCG or rBCG- CRISPRi-MurT-GatD infected BMDMs was observed. Increased TNFa secretion was observed from rBCG-CRISPRi-MurT-GatD infected IFNy-activated BMDMs cultured in media supplemented with 500 ng / ml ATc. LPS was used as a control.

[0045] Figure 19: In vitro growth rates of WT BCG by OD600(Panel A) and CFU counting (Panel B) and of rBCG-CRISPRi-MurT-GatD by OD600(Panel C) and CFU counting (Panel D) in complete 7H9 medium at varying concentrations of ATc (A & C) and Dox (B & D).

[0046] Figure 20: Determination of the Dox concentration for activation of rBCG- CRISPRi-MurT-GatD in vivo. Mice were aerosol infected with ~2.5 log °H1n0 CFU of rBCG and

[0047] Dox (at doses of 0.125-1 mg / kg / day) - was administered by oral gavage for 10 days. CFU counts from the experiment are shown. A dose of 1 mg / kg / day Dox resulted in reduced growth of rBCG-CRISPRi-MurT-GatD indicative of sufficient activation of the CRISPRi system. Lung homogenates were plated on both 7H11 with and without kanamycin (25 pg / ml) to assess the loss of the CRISPRi plasmid during in vivo growth. There was no difference in the growth of recovered bacteria on media containing kanamycin, indicating the presence of the CRISPRi plasmid in recovered bacteria. Student t-test was used for statistical analysis. * :p-value <0.05.

[0048] Figure 21 : Efficacy of 1 mg / kg / day dose of Doxycycline for CRISPRi activation: Schematic representation of mice aerosol infection with WT BCG and rBCG::CRISPRi- MurT-GatD and analysis of the efficacy of 1 mg / kg / day dose of doxycycline for CRISPRi- MurT-GatD activation in vivo.

[0049] Figure 22: Efficacy of 1 mg / kg / day dose of Doxycycline for CRISPRi activation: Day 1 implantation of WT BCG and rBCG::CRISPRi-MurT-GatD in the lungs of aerosol infected mice.

[0050] Figure 23: Efficacy of 1 mg / kg / day dose of Doxycycline for CRISPRi activation: Panel A = Day 28 Lung bacterial loads of WT BCG and rBCG::CRISPRi-MurT-GatD aerosol infected mice; Panel B = Graph of Day 28 Lung bacterial load CPUs; Panel C = Day 56 Lung bacterial loads of WT BCG and rBCG::CRISPRi-MurT-GatD aerosol infected mice; Panel D = Graph of Day 56 Lung bacterial load CFUs.

[0051] Figure 24: Efficacy of 1 mg / kg / day dose of Doxycycline for CRISPRi activation: PCR amplification of dCas9 in recovered Big or Small rBCG::CRISPRi-MurT-GatD colonies as shown in Figure 23.

[0052] Figure 25: Efficacy of 1 mg / kg / day dose of Doxycycline for CRISPRi activation: Growth kinetics of Big and Small colonies of rBCG::CRISPRi-MurT-GatD (shown in Figure 21 ) in liquid broth supplemented with Doxycycline. Statistical analysis was conducted using Student t-test. *: p-value: <0.01 .

[0053] Figure 26: Analysis of rBCG::CRISPRi-MurT-GatD strain attenuation: Schematic representation of SCID mice aerosol infection with WT BCG and rBCG::iE-DAP for analysis of strain attenuation. rBCG::iE-DAP activation in vivo was achieved by administration of Dox at 1 mg / kg / day. SCID mice (n = 5 per group) were aerosol infected with -100 colonyforming units (CFUs) / mouse of WT BCG or rBCG::iE-DAP, a WT BCG + Dox group was included as a control.

[0054] Figure 27: Analysis of rBCG::CRISPRi-MurT-GatD strain attenuation: Percent survival of SCID mice following low-dose challenge with WT BCG and rBCG compared to WT BCG+Dox or rBCG+Dox groups.

[0055] Figure 28: Efficacy of rBCG::CRISPRi-MurT-GatD in comparison to standard WT BCG for protection against Mtb H37Rv infection in mice: Schematic representation of the mouse immunization and Mtb H37Rv challenge protocol.

[0056] Figure 29: Efficacy of rBCG::CRISPRi-MurT-GatD in comparison to standard WT BCG for protection against Mtb H37Rv infection in mice: Percentage weight change immediately prior to Mtb challenge. Figure 30: Efficacy of rBCG::CRISPRi-MurT-GatD in comparison to standard WT BCG for protection against Mtb H37Rv infection in mice: Lung (Panel A) and Spleen (Panel B) weights at Week 4 and Week 8 post-challenge with Mtb. Lung (Panel C) and Spleen (Panel D) bacterial burdens at Week 4 and Week 8 post-challenge with Mtb. rBCG::CRISPRi-MurT-GatD+Dox was superior to WT BCG or WT BCG+Dox in controlling Mtb H37Rv growth in the lung at week 4 and week 8 post challenge with Mtb. Statistical analysis was conducted using Student t-test. * :p-value <0.05.

[0057] Figure 31 : Histopathological analysis of lung samples: Panels A and B = Histological haematoxylin and eosin (H&E) staining of lung samples at week 4 post Mtb challenge. Analysis of percentage of inflamed area from each mouse lung per immunized group (n=5 per group), shows that rBCG::CRISPRi-MurT-GatD+Dox immunized mice present with early lung inflammation compared to WT BCG+Dox. Panels C and D = Histological haematoxylin and eosin (H&E) staining of lung samples at week 8 post Mtb H37Rv infection. Analysis of percentage of inflamed area from each mouse lung (n=5 per group), shows that rBCG::CRISPRi-MurT-GatD+Dox immunized mice present with sustained lung inflammation compared to WT BCG and WT BCG+Dox. The percentage inflamed area was evaluated using Imaged software (NIH) and plotted as whisker boxplots (whiskers represent minimum and maximum values). Student t-test was used for statistical analysis. * :p-value <0.05.

[0058] SEQUENCE LISTING

[0059] The nucleic acid and amino acid sequences listed herein or in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and the standard one or three letter abbreviations for amino acids. It will be understood by those of skill in the art that only one strand of each nucleic acid sequence is shown, but that the complementary strand is included by any reference to the displayed strand.

[0060] SEQ ID NO:1 - nucleotide sequence of MurT-GatD operon

[0061] SEQ ID NO:2 - nucleotide sequence of PAM located on template strand

[0062] SEQ ID NO:3 - nucleotide sequence of sgRNA located on template strand

[0063] SEQ ID NO:4 - nucleotide sequence of CRISPRi forward primer SEQ ID NO:5 - nucleotide sequence of CRISPRi reverse primer SEQ ID NO:6 - nucleotide sequence of the MurT (Mb3739) gene SEQ ID NO:7 - nucleotide sequence of the GatD (Mb3740) gene SEQ ID NO:8 - nucleotide sequence of PAM binding region SEQ ID NO:9 - nucleotide sequence of sgRNA binding region DETAILED DESCRIPTION OF THE INVENTION

[0064] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

[0065] The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0066] As used throughout this specification and in the claims which follow, the singular forms “a”, “an” and “the” include the plural form, unless the context clearly indicates otherwise.

[0067] The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms “comprising”, “containing”, “having” and “including” and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items. It is, however, contemplated as a specific embodiment of the present disclosure that the term “comprising” encompasses the possibility of no further members being present, i.e., in some embodiments “comprising” is to be understood as having the meaning of “consisting of”.

[0068] The inventors of the present invention have developed a modified Mycobacterium bovis BCG vaccine. In this study the inventors have characterized a BCG vaccine reengineered through CRISPRi to activate the NOD-1 pathway by targeted depletion of peptidoglycan (PG) amidation enzymes (MurT and GatD). In the present invention, the inventors provide a recombinant BCG bacterial strain carrying a CRISPRi-dCas9 plasmid with a short guide RNA for targeting the promoter region of the gene Mb3739 (MurT) which encodes a glutaminase which forms an enzyme complex with an amidotransferase (GatD) that modifies the stem peptide structure of peptidoglycan in Mycobacterium bovis BCG vaccine, that enables transcriptional repression of the target gene Mb3739. Induction of the CRISPRi system in this bacterial strain results in changes in the bacterial cell wall, particularly the amidation of peptidoglycan subunits which are believed to have the ability to activate enhanced innate immune responses. The inventors further show activation of the NOD-1 pathway by the recombinant BCG vaccine invented herein by quantitative PCR analysis of NOD-1 transcription in in vitro grown THP-1 macrophages.

[0069] Mycobacteria amidate the stem peptides of their peptidoglycan, this modification occurs on the peptidoglycan monomers when they are still being synthesised inside the cell. When the monomers are exported outside of the cell to build the cell wall, these modified peptidoglycan monomers are crosslinked and exposed on the cell surface. Thus, the inventors of the present invention targeted the amidotransferase complex - MurT-GatD - essential for PG amidation (i.e., modifying iE-DAP to iQ-DAP) to develop a recombinant BCG vaccine with reduced PG amidation. Depletion of MurT-GatD through CRISPRi reduced the growth of the bacteria, caused perturbations in cell wall morphology observed through scanning and transmission electron microscopy and sensitized the bacteria to cell wall targeting antibiotics. Labeling of the MurT-GatD depleted cells with a fluorescent PG- amidation reporter probe indicated increased labeling of the cells with this probe suggestive of reduced PG-amidation upon MurT-GatD depletion. This was confirmed by labelling of PG derived from the recombinant BCG strain with an amide reactive dye in comparison to PG derived from wildtype (WT) BCG.

[0070] The inventors hypothesized that depletion of MurT-GatD in BCG will result in increased expression of the NOD-1 ligand (iE-DAP) and increase the immunogenicity of the recombinant strain. Thus, they further tested the activation of the NOD-1 pathway in THP-1 macrophages and indeed observed activation of NOD-1 as assessed by quantitative PCR showing increased NOD-1 transcription in THP-1 macrophages infected with the recombinant BCG strain. The survival and immunogenicity of the recombinant BCG strain in mouse bone marrow derived macrophages (BMDMs) was also tested. Firstly, the inventors analysed the survival of the strain in IFNy activated BMDMs upon CRISPRi activation and secondly analysed cytokine expression by ELISA. CRISPRi activation during infection of BMDMs with rBCG::CRISPRi MurT-GatD revealed reduced survival of the bacteria in vivo and increased production of the pro-inflammatory cytokine TNFa but not IL-6. Mouse aerosol infection experiments with rBCG::CRISPRi-MurT-GatD in comparison to wildtype (WT) BCG revealed activation of rBCG::CRISPRi MurT-GatD after administration of doxycyline (a tetracycline anolog enabling CRISPRi activation) as evidenced by the recovery of bacteria forming small colonies as compared to WT BCG which formed typical mycobacterial colonies on solid agar media, although colony forming units were similar among the groups tested.

[0071] Vaccination of mice with rBCG::CRISPRi MurT-GatD and subsequent activation of the CRISPRi system by administration of doxycycline to induce MurT-GatD depletion prior to Mtb infection, significantly reduced Mtb load in the lungs of rBCG::CRISPRi MurT-GatD vaccinated mice in comparison to unvaccinated and WT BCG vaccinated mice or WT BCG vaccinated mice given doxycycline as a control. This study demonstrates that targeting essential mycobacterial genes producing immunonomodulatory molecules enhances the immunogenicity of BCG and improves the efficacy of BCG against Mtb infection in mice.

[0072] A “protein,” “peptide” or “polypeptide” is any chain of two or more amino acids, including naturally occurring or non-naturally occurring amino acids or amino acid analogues, irrespective of post-translational modification (e.g., glycosylation or phosphorylation). An “antigen” is a compound, composition, or substance that can stimulate the production of antibodies and / or a CD4+ or CD8+ T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term “antigen” includes all related antigenic epitopes. An “epitope” refers to a site on an antigen, including chemical groups or peptide sequences on a molecule that are antigenic, i.e., that elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide.

[0073] The terms “nucleic acid”, “nucleic acid molecule” and “polynucleotide” are used herein interchangeably and encompass both ribonucleotides (RNA) and deoxyribonucleotides (DNA), including cDNA, genomic DNA, and synthetic DNA. The nucleic acid may be double-stranded or single-stranded. Where the nucleic acid is singlestranded, the nucleic acid may be the sense strand or the antisense strand. A nucleic acid molecule may be any chain of two or more covalently bonded nucleotides, including naturally occurring or non-naturally occurring nucleotides, or nucleotide analogs or derivatives. By “RNA” is meant a sequence of two or more covalently bonded, naturally occurring or modified ribonucleotides. The term “DNA” refers to a sequence of two or more covalently bonded, naturally occurring or modified deoxyribonucleotides. By “cDNA” is meant a complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase). Accordingly, a “cDNA clone” refers to a duplex DNA sequence which is complementary to an RNA molecule of interest, and which is carried in a cloning vector.

[0074] The term “complementary” refers to two nucleic acids molecules, e.g., DNA or RNA, which are capable of forming Watson-Crick base pairs to produce a region of double- strandedness between the two nucleic acid molecules. It will be appreciated by those of skill in the art that each nucleotide in a nucleic acid molecule need not form a matched Watson- Crick base pair with a nucleotide in an opposing complementary strand to form a duplex. One nucleic acid molecule is thus “complementary” to a second nucleic acid molecule if it hybridizes, under conditions of high stringency, with the second nucleic acid molecule. A nucleic acid molecule according to the invention includes both complementary molecules.

[0075] In some embodiments of the invention nucleic acid molecules are described for use in a modified BCG vaccine. The nucleic acid molecules may be substantially identical to the nucleotide sequences described herein.

[0076] As used herein a “substantially identical” sequence is an amino acid or nucleotide sequence that differs from a reference sequence only by one or more conservative substitutions, or by one or more non-conservative substitutions, deletions, or insertions located at positions of the sequence that do not destroy or substantially reduce the antigenicity of one or more of the expressed polypeptides or of the polypeptides encoded by the nucleic acid molecules. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the knowledge of those with skill in the art. These include using, for instance, computer software such as ALIGN, Megalign (DNASTAR), CLUSTALW or BLAST software. Those skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In one embodiment of the invention there is provided for a polypeptide or polynucleotide sequence that has at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the sequences described herein.

[0077] Alternatively, or additionally, two nucleic acid sequences may be “substantially identical” if they hybridize under high stringency conditions. The “stringency" of a hybridisation reaction is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation which depends upon probe length, washing temperature, and salt concentration. In general, longer probes required higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridisation generally depends on the ability of denatured DNA to re-anneal when complementary strands are present in an environment below their melting temperature. A typical example of such “stringent” hybridisation conditions would be hybridisation carried out for 18 hours at 65°C with gentle shaking, a first wash for 12 min at 65°C in Wash Buffer A (0.5% SDS; 2XSSC), and a second wash for 10 min at 65°C in Wash Buffer B (0.1% SDS; 0.5% SSC).

[0078] Those skilled in the art will appreciate that polypeptides, peptides or peptide analogues can be synthesised using standard chemical techniques, for instance, by automated synthesis using solution or solid phase synthesis methodology. Automated peptide synthesisers are commercially available and use techniques known in the art. Polypeptides, peptides and peptide analogues can also be prepared from their corresponding nucleic acid molecules using recombinant DNA technology.

[0079] As used herein, the term “gene” refers to a nucleic acid that encodes a functional product, for instance an RNA, polypeptide or protein. A gene may include regulatory sequences upstream or downstream of the sequence encoding the functional product.

[0080] As used herein, the term “coding sequence” refers to a nucleic acid sequence that encodes a specific amino acid sequence. On the other hand, a “regulatory sequence” refers to a nucleotide sequence located either upstream, downstream or within a coding sequence. Generally regulatory sequences influence the transcription, RNA processing or stability, or translation of an associated coding sequence. Regulatory sequences include but are not limited to: effector binding sites, enhancers, introns, polyadenylation recognition sequences, promoters, RNA processing sites, stem-loop structures and translation leader sequences.

[0081] In some embodiments, the genes used in the method of the invention may be operably linked to other sequences. By “operably linked” is meant that the nucleic acid molecules encoding the recombinant polypeptides of the invention and regulatory sequences are connected in such a way as to permit expression of the proteins when the appropriate molecules are bound to the regulatory sequences. Such operably linked sequences may be contained in vectors or expression constructs which can be transformed or transfected into host cells for expression. It will be appreciated that any vector or vectors can be used for the purposes of expressing the recombinant antigenic polypeptides of the invention.

[0082] The term “promoter” refers to a DNA sequence that is capable of controlling the expression of a nucleic acid coding sequence or functional RNA. A promoter may be based entirely on a native gene, or it may be comprised of different elements from different promoters found in nature. Different promoters are capable of directing the expression of a gene in different cell types, or at different stages of development, or in response to different environmental or physiological conditions. A “constitutive promoter” is a promoter that direct the expression of a gene of interest in most host cell types most of the time.

[0083] The term “recombinant” means that something has been recombined. When used with reference to a nucleic acid construct the term refers to a molecule that comprises nucleic acid sequences that are joined together or produced by means of molecular biological techniques. The term “recombinant” when used in reference to a protein or a polypeptide refers to a protein or polypeptide molecule which is expressed from a recombinant nucleic acid construct created by means of molecular biological techniques. Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Accordingly, a recombinant nucleic acid construct indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e., by human intervention. Recombinant nucleic acid constructs may be introduced into a host cell by transformation. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species.

[0084] The term “vector” refers to a means by which polynucleotides or gene sequences can be introduced into a cell. There are various types of vectors known in the art including plasmids, viruses, bacteriophages and cosmids. Generally, polynucleotides or gene sequences are introduced into a vector by means of a cassette. The term “cassette” refers to a gene sequence or gene sequences inserted into a vector, which in some embodiments, provides regulatory sequences for expressing the polynucleotide or gene sequences. In other embodiments, the vector provides the regulatory sequences for the expression of the polypeptides of the invention. In further embodiments, the vector provides some regulatory sequences, and the nucleotide or gene sequence provides other regulatory sequences. “Regulatory sequences” include but are not limited to promoters, transcription termination sequences, enhancers, splice acceptors, donor sequences, introns, ribosome binding sequences, poly(A) addition sequences, and / or origins of replication.

[0085] The vaccine of the present invention comprises a recombinant M. bovis BCG strain with an altered cell wall following the removal of the amidation modification. This strain was used to immunize U937 monocyte derived macrophage cells and subsequently showed improved protection than the currently used BCG vaccine. The vaccine of the present invention can be provided either alone or in combination with other compounds (for example, nucleic acid molecules, small molecules, peptides, or peptide analogues), in the presence of a liposome, an adjuvant, or any carrier, such as a pharmaceutically acceptable carrier and in a form suitable for administration to mammals, for example, humans, cattle, sheep, etc.

[0086] As used herein a “pharmaceutically acceptable carrier” or “excipient” includes any and all antibacterial and antifungal agents, coatings, dispersion media, solvents, isotonic and absorption delaying agents, and the like that are physiologically compatible. A “pharmaceutically acceptable carrier” may include a solid or liquid filler, diluent or encapsulating substance which may be safely used for the administration of the recombinant antigen or vaccine composition to a subject. The pharmaceutically acceptable carrier can be suitable for intramuscular, intradermal, intravenous, intraperitoneal, subcutaneous, oral or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions, dispersions and sterile powders for the preparation of sterile solutions. The use of media and agents for the preparation of pharmaceutically active substances is well known in the art. Where any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is not contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0087] Suitable formulations or compositions to administer the vaccine construct comprising the recombinant M. bovis BCG strain to subjects infected with Mycobacterium tuberculosis. Any appropriate route of administration may be employed, such as, parenteral, intravenous, intradermal, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracistemal, intraperitoneal, intranasal, aerosol, topical, or oral administration. Vaccine formulations and compositions that are useful in the present invention include a construct comprising the recombinant M. bovis BCG strain that primes and / or boosts an immune response to M. tuberculosis infection.

[0088] Typically, an effective amount of the vaccine or a formulation or composition comprising the vaccine will be administered to a subject. As used herein the term “subject” includes all mammals, and in particular a human subject.

[0089] The invention also relates in part to a method of eliciting a protective immune response against Mycobacterium tuberculosis or treating a TB infection in a subject comprising administering to a subject in need thereof a therapeutically effective amount, an immunogenically effective amount, or a prophylactically effective amount of the vaccine, or compositions or formulations thereof of the present invention, in order to prevent or treat TB in the subject.

[0090] An “effective amount” of the recombinant M. bovis BCG strain or vaccine compositions comprising the strain includes a therapeutically effective amount, immunologically effective amount, or a prophylactically effective amount. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as treatment of an infection or a condition associated with such infection. The outcome of the treatment may for example be measured by a decrease in bacteraemia, delay in development of a pathology associated with TB infection, stimulation of the immune system, or any other method of determining a therapeutic benefit. A therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.

[0091] Preferably, the recombinant M. bovis BCG strain or vaccine compositions of the present invention are for administration to babies, young children or healthy adults. In some embodiments, the recombinant M. bovis BCG strain or vaccine compositions of the present invention may be suitable for administration to subjects with TB infection. The dosage of the recombinant M. bovis BCG strain or vaccine compositions of the present invention will vary depending on the symptoms, age and body weight of the subject, the nature and severity of the disorder to be treated or prevented, the route of administration, and the form of the composition. Any of the compositions of the invention may be administered in a single dose or in multiple doses. The dosages of the compositions of the invention may be readily determined by techniques known to those of skill in the art or as taught herein.

[0092] By “immunogenically effective amount” is meant an amount effective, at dosages and for periods of time necessary, to achieve a desired immune response. The desired immune response may include stimulation or elicitation of an immune response, for instance a T-cell response.

[0093] A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result, such as prevention of onset of a condition associated with a TB infection. Typically, a prophylactic dose is used in a subject prior to or at an earlier stage of infection, so that a prophylactically effective amount may be less than a therapeutically effective amount.

[0094] Dosage values may vary and be adjusted over time according to the individual need. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected. The amount of modified BCG strain(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single dose may be administered, or multiple doses may be administered over time. It may be advantageous to formulate the compositions in dosage unit forms for ease of administration and uniformity of dosage.

[0095] The term “preventing”, when used in relation to an infectious disease, such as TB, is well understood in the art, and includes administration of a composition which reduces the frequency of or delays the onset of symptoms of a condition in a subject relative to a subject which does not receive the composition. Prevention of a disease includes, for example, reducing the number of diagnoses of the infection in a treated population versus an untreated control population, and / or delaying the onset of symptoms of the infection in a treated population versus an untreated control population.

[0096] The term “prophylactic or therapeutic” treatment is well known to those of skill in the art and includes administration to a subject of one or more of the compositions of the invention. If the composition is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the subject) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

[0097] The vaccination protocol for eliciting an immune response against a TB infection in a subject as defined herein typically comprises a series of single doses of the recombinant BCG or compositions described herein. A single dose or dosage, as used herein, refers to the priming dose (i.e., initial first or second dose with the same antigens), and any subsequent dose, respectively, which are preferably administered in order to "boost" the immune reaction. In this context, each single dosage comprises the administration of the recombinant BCG strain or compositions according to the invention, wherein the interval between the administration of two single dosages can vary from at least one week, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 weeks apart. It will be appreciated that the intervals between single dosages may be constant or vary over the course of the immunization protocol, e.g., the intervals may be shorter in the beginning and longer towards the end of the protocol. Additionally, depending on the total number of single dosages and the interval between single dosages, the immunization protocol may extend over a period of time. Each single dosage encompasses the administration of one of the constructs comprising the M. bovis BCG strain described herein.

[0098] Toxicity and therapeutic efficacy of compositions of the invention may be determined by standard pharmaceutical procedures in cell culture or using experimental animals, such as by determining the LD5o and the ED5o. Data obtained from the cell cultures and / or animal studies may be used to formulate a dosage range for use in a subject. The dosage of any composition of the invention lies preferably within a range of circulating concentrations that include the ED5o, but which has little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilised. For compositions of the present invention, the therapeutically effective dose may be estimated initially from cell culture assays.

[0099] In some embodiments, the modified BCG vaccine strains or compositions according to the invention may be provided in a kit, optionally with a carrier and / or an adjuvant, together with instructions for use.

[0100] The following examples are offered by way of illustration and not by way of limitation.

[0101] EXAMPLE 1

[0102] Construction and cloning of a recombinant BCG::CRISPRi MurT-GatD strain

[0103] Growth conditions for E. coli DH5a and derivative strains

[0104] E. coli DH5a and derivative strains were grown in Luria-Bertani broth (LB) or on Luria-Bertani agar (LA) at 37 °C with supplementation of the media with appropriate antibiotics. The antibiotic concentrations used were as follows: Kanamycin (Kan): 50 pg / ml. Liquid cultures were grown at 37 °C with shaking at a 100 rpm.

[0105] Growth conditions for Mycobacterial and derivative strains

[0106] M. bovis, M tuberculosis H37Rv and the recombinant BCG::CRISPRi MurT-GatD strain were grown at 37 °C in Middlebrook 7H9 broth supplemented with OADC enrichment, 0.5% glycerol, 0.05% Tween 80 and appropriate antibiotics (hereafter referred to as Middlebrook 7H9 broth) or on Middlebrook 7H1 1 agar supplemented with OADC enrichment and 0.5% glycerol and appropriate antibiotics. Antibiotic concentration used Kan: 50 pg / ml. Construction of a recombinant BCG::CRISPRi MurT-GatD strain

[0107] The programmable mycobacterial CRISPRi system for repression of gene transcription was used as previously described by Rock et al. (2017), which is incorporated in its entirety herein by reference., to generate the recombinant BCG::CRISPRi MurT-GatD strain. Briefly, a short-guide RNA (sgRNA) sequence that targets knockdown of the murT- gatD two-gene operon was introduced into the BCG strain (Figure 1). The CRISPRi system utilizes a catalytically inactivated anhydrotetracycline / doxycycline (ATc / Dox)-inducible CRISPRi dcas9 from Streptococcus thermophiles which is directed by a (ATc)-inducible sgRNA to specific target genes to prevent transcription initiation or elongation (Figure 2 and Figure 3). A sgRNA (near the PAM sequence of 5’-NNAGCAA-3’ (SEQ ID NO:2)), with sequence 5’-CCAGCTGGTCTCGGGAGAGGT-3’ (SEQ ID NO:3), targeting the 5’ region of the murT-gatD operon (SEQ ID NO:1) was synthesized by annealing the sgRNA primer sets (SEQ ID NO:4 and SEQ ID NO:5) and cloned into BsmBI-digested CRISPRi vector PLJR965, which express the dCas9 endonuclease. The sgRNA sequence binds to the complementary sequences on the MurT-GatD operon depicted by SEQ ID NO:8 and SEQ ID NO:9, respectively.

[0108] The clone was amplified in E. coli to generate the plasmid PLJR965-CRISPRi MurT- GatD. This plasmid was introduced into M. bovis BCG by electroporation using the following cloning strategy:

[0109] 1 . Amplify the CRISPRi vector PLJR965 in E. coli (kanamycin selection), digest them with BsmBI (NEB), and gel purify prior to cloning sgRNAs.

[0110] 2. Anneal primers (Table 1 ) and ligate them into agarose gel purified BsmBI-digested CRISPRi vector.

[0111] 3. Transform ligation reaction into E. coli DH5a competent cells and plate onto Kanamycin 50 pg / ml containing Luria-Bertani agar (LA) and incubate at 37 °C. Surviving clones will contain the clones.

[0112] 4. Amplify CRISPRi construct in E. coli DH5a, extract the plasmid, purify and electroporate into M. bovis BCG and plate transformants on 7H11 Middlebrook agar supplemented with OADC enrichment, 0.5% and Kanamycin. Incubate at 37 °C for 3 weeks. Pick colonies and assess CRISPRi mediated MurT-GatD knockdown.

[0113] Table 1 . Primers used for CRISPRi amplification in E. coli DH5a. EXAMPLE 2

[0114] In vitro testing of recombinant BCG::CRISPRi MurT-GatD strain rBCG::CRISPRi-MurT-GatD activates the NOD-1 pathway and training of U937 monocytes and Mtb H37Rv killing ability

[0115] The standard BCG vaccine in use possesses immune evasion proteins, lipids or molecules that limit its efficacy during vaccination. Transposon mutagenesis screens have previously been used successfully to identify non-essential immunomodulatory proteins that limit the efficacy of the BCG vaccine. However, essential mycobacterial immunomodulatory proteins remain to be studied to evaluate their role in limiting BCG vaccine efficacy. The inventors analyzed a list of essential genes of M. bovis BCG Danish and M. bovis BCG Pasteur strains published from a transposon mutagenesis screen (in Mtb) to identify essential immunomodulatory proteins or enzymes involved in the generation of immunomodulatory molecules (Mendum et al, 2019). Essential enzymes associated with cell wall biosynthesis were the focus of the study, as mycobacteria are known to produce a cell envelope with associated modifications implicated in immune modulation during infection. The inventors targeted genes involved in biosynthesis of different layers or components of the cell wall and identified essential genes: treS - involved in the production of cell wall glycolipids, IpqW - involved in the production of lipoarabinomannan, mmaA4 - which encodes a methyl transferase required for introducing the distal oxygen-containing modifications of mycolic acids, embC - involved in arabinogalactan biosynthesis and murT- gatD genes encoding an amidotransferase enzyme complex known to modify peptidoglycans as potential essential cell wall associated enzymes producing immunomodulatory cell wall components. To study the effect of these genes on limiting BCG vaccine efficacy, the inventors first used an in vitro macrophage training assay with CRISPRi engineered M. bovis BCG strains individually depleted of the essential enzymes encoded by the genes listed above to assess the Mtb H37Rv bactericidal activity of the trained macrophages.

[0116] For cell-based in vitro infection assays the human monocyte U937 cell line and BMDMs extracted from the bone marrow (BM) of 6-8 week old female wildtype Balb / c mice were cultivated in RPMI-Glutamax (Cat. 61870-036, Fischer Scientific) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Cat. 10082147, Fischer Scientific) at 37 °C with 5% CO2. BMDMs were generated as previously described by Toda et aL, (23), which is incorporated herein by reference. Briefly, for differentiation of BM cells into macrophages, BM cells were seeded in BMDM differentiation media (RPMI-Glutamax supplemented with 10% FBS and 10% L929-conditioned media) and differentiated for 6 days. Non-adherent cells were washed out with warm BMDM differentiation media and adherent macrophages were used for in vitro infection assays. In vitro training of monocytes was performed according to the model by Bekkering et al, 2016 and Pan et al, 2020, both of which are incorporated herein by reference. Briefly, LI937 monocytes (1 x lO6 / mL) were transferred into a 24-well plate and cells were incubated with either culture medium only as a negative control or MDP (Muramyl dipeptide), LPS (lipopolysaccharide), heat killed WT BCG or heat killed rBCG::CRISPRi strains at 37 °C, and 5% CO2 for 24 h. Cells were washed twice with 1 mL of warm PBS and then incubated for 2 days in RPMI with 10% FBS and penicillin streptomycin in the presence of 25 nM phorbol 12-myristate 13-acetate (PMA) (which can induce the differentiation of monocytes to macrophages). After washing twice with 1 mL of warm PBS, the differentiated macrophages were infected with Mtb H37Rv at MOI:1 and incubated for 24h. After 24 h, cells were lysed, and bacterial load was enumerated by plating for CPU counts. CRISPRi mediated depletion of MurT-GatD in rBCG::CRISPRi-MurT-GatD is hypothesized to reduce PG amidation leading to both NOD1 and NOD2 activation (Figure 4). Activation of CRISPRi with ATc led to a 1000 fold reduction in murT (Mb3739) expression (Figure 5). As shown in Figure 6, NOD-1 and NOD-2 expression was present at basal levels in non-activated and uninfected cells as previously shown; WT BCG infection did not induce significant changes in NOD expression (Figure 6). Infection with rBCG::CRISPRi-MurT-GatD led to a ~15-fold increase in NOD-2 expression in IFNy activated macrophages, a ~55 fold and -200 fold increase in NOD-1 expression in both non-activated and IFNy activated THP-1 macrophages, respectively (Figure 6).

[0117] Effect of modification on cell wall morphology

[0118] The generated modified M. bovis BCG strains have an altered cell wall as the amidation modification is removed (Figure 7-9). Depletion of MurT-GatD through CRISPRi reduced the in vitro growth of the bacteria; caused perturbations in bacterial cell wall morphology observed through scanning and transmission electron microscopy and sensitized the bacteria to cell wall targeting antibiotics (Figures 8-13). The MurT-GatD enzymes function as an amidotransferase complex required for amidation of D-isoglutamate to D-isoglutamine during peptidoglycan biosynthesis, a modification essential for peptidoglycan cross-linking and evasion of activation of the pathogen recognition receptor NOD-1 during infection (Figure 7). Labeling of the MurT-GatD depleted cells with a fluorescent PG-amidation reporter chemical-probe showed increased labeling of the cells with this probe indicative of reduced PG-amidation upon MurT-GatD depletion (Figure 14). This was confirmed by labelling of PG derived from the recombinant BCG strain with an amide reactive dye in comparison to PG derived from wildtype (WT) BCG (Figure 15). Further microscopic imaging of the MurT-GatD depleted cells labelled with a fluorescent peptidoglycan dye BODIPY-Vancomycin which specifically labels uncross-linked peptidoglycan showed complete labeling of the cells with this dye in contrast to cell pole labeled wildtype cells (Figure 11), a phenotype suggestive of reduced peptidoglycan crosslinking in rBCG::CRISPRi-MurT-GatD resulting in the cell wall defects observed through electron microscopy and the increased sensitivity to cell wall targeting antibiotics. rBCG::CRISPRi-MurT-GatD trained macrophages displayed increased control of Mtb H37Rv compared to wildtype M. bovis BCG trained macrophages (Figure 16). The inventors therefore further characterized the rBCG::CRISPRi-MurT-GatD strain to evaluate the potential role of the MurT-GatD complex in limiting BCG vaccine efficacy.

[0119] EXAMPLE 3

[0120] BMDM immunogenicity of rBCG::CRISPRi-MurT-GatD and activation in vitro and in mice-aerosol infections with Doxycycline

[0121] Immunogenicity in BMDMs

[0122] To test the hypothesis that inhibition of MurT-GatD expression in rBCG::CRISPRi- MurT-GatD improves the immunogenicity of the recombinant strain through increased expression of the NOD-1 ligand iE-DAP, the inventors infected IFNy activated bone marrow derived macrophages (BMDMs) with rBCG::CRISPRi-MurT-GatD and supplemented the growth media with increasing concentrations of anhydrotetracycline (ATc) to assess activation of the CRISPRi system ex vivo and recorded this by plating for rBCG::CRISPRi- MurT-GatD colony forming unit (CFU) counts at day 3 and day 5 post-infection.

[0123] At day 3 bacterial killing was observed with all ATc concentrations tested for activating CRISPRi, contributing to growth inhibition by activated BMDMs and a dose dependent inhibition of growth of rBCG::CRISPRi-MurT-GatD was observed at day 5, with 500 ng / ml ATc (the maximum concentration used) resulting in a ~3-fold difference in growth inhibition of rBCG::CRISPRi-MurT-GatD in comparison to WT BCG and rBCG::CRISPRi- MurT-GatD without ATc supplementation (Figure 17).

[0124] Secondly, sandwiched ELISA was performed for cytokine (TNF-a and IL-6) measurement in culture supernatants. The inventors assessed the expression of the pro- inflammatory cytokines TNFa and IL-6 as rBCG::CRISPRi-MurT-GatD is designed to express the NOD-1 ligand iE-DAP and potentially induce increased NF-KB activation leading to potentially increased pro-inflammatory cytokine expression. Culture supernatants were used immediately after harvest for ELISA. Sandwiched ELISAs (R&D systems) were performed as per manufacturer’s recommendations.

[0125] Activation of rBCG::CRISPRi-MurT-GatD by supplementation of growth media with ATc (to reduce murT-gatD expression) resulted in a dose dependent increase in TNFa expression but not IL-6 expression in comparison to WT BCG (Figure 18). These results demonstrate that rBCG::CRISPRi-MurT-GatD is responsive to activation ex vivo and signals for increased pro-inflammatory cytokine expression.

[0126] In vitro doxycycline activation of rBCG::CRISPRi-MurT-GatD and aerosol infection of BALB / c mice with doxycycline

[0127] Doxycycline (Dox), a tetracycline analog, is used in TB in vivo models for temporal regulation of mycobacterial gene expression. The CRISPRi platform used in rBCG::CRISPRi-MurT-GatD is also based on a Dox-responsive TetR-tetO unit (Rock et al, 2017). rBCG::CRISPRi-MurT-GatD was grown in an increasing range of Dox concentrations to assess the activation of CRISPRi in vitro. In comparison to the WT BCG control which displayed minimal sensitivity to Dox during growth in liquid broth, activation of CRISPRi in rBCG::CRISPRi-MurT-GatD with Dox resulted in a dose dependent inhibition of growth of rBCG::CRISPRi-MurT-GatD as the MurT-GatD enzymes are essential for growth (Figure 19).

[0128] To determine the lung bacillary burden of wild-type and rBCG::CRISPRi-MurT-GatD strains, 6-8 week-old female BALB / c mice were infected using the aerosol route in a Glasscol inhalation exposure system (Glasscol). The inoculum implanted in the lungs at day 1 (n = 3 mice per group) in female BALB / c mice was determined by plating the whole-lung homogenate on 7H11 -selective plates containing carbenicillin (50 mg / ml), Trimethoprim (20 mg / ml), Polymyxin B (25 mg / ml) and Cycloheximide (10 mg / ml). Doxycycline was administered at determined doses for CRISPRi activation by daily oral gavage in vivo and following infection, mice lungs were harvested (n = 5 animals / group), homogenized in sterile PBS and plated on 7H11 -selective plates at different dilutions. The 7H11 -selective plates were incubated at 37 °C and single colonies were enumerated at day 10, week 4 and week 8. Single colonies were expressed at log CFU per organ.

[0129] To test the activation of rBCG::CRISPRi-MurT-GatD in vivo and the minimum effective Dox dose, the inventors aerosol infected Balb / C mice as set out above with -100 CFU of rBCG::CRISPRi-MurT-GatD and administered Dox for 10 days at doses ranging from 0.125 - 1 mg / kg / day. They found that 1 mg / kg / day resulted in a significant reduction in growth of rBCG::CRISPRi-MurT-GatD in the lungs (Figure 20). To assess retention of the CRISPRi plasmid (PLRJ965, which has the kanamycin [Kan] resistance cassette) by rBCG::CRISPRi-MurT-GatD in vivo, lung homogenates were plated on media with or without Kan and it was found that rBCG::CRISPRi-MurT-GatD bacilli recovered from the lungs of aerosol infected mice formed similar CFU counts on both media containing Kan and media without Kan (Figure 20). These results demonstrate long term retention of the CRISPRi plasmid by rBCG::CRISPRi-MurT-GatD during in vivo conditions. The inventors further evaluated the long-term efficacy of 1 mg / kg / day Dox for rBCG::CRISPRi-MurT-GatD activation at 4 weeks and 8 weeks post aerosol infection of mice (Figure 21-22) and found that recovered rBCG::CRISPRi-MurT-GatD bacilli formed small colonies on solid agar in comparison to WT BCG recovered from infected mice also exposed to 1 mg / kg / day Dox dose, indicative of the long-term efficacy of Dox in vivo for CRISPRi activation (Figure 23). Genomic DNA was extracted from small colonies (SC) and big colonies (BC) recovered and a PCR was performed for verification of the presence of the CRISPRi plasmid. PCR amplification of the dCas9 ORF (encoding the degenerate endonuclease dCas9 used in this CRISPRi platform) demonstrated the presence of the dCas9 in all colonies analyzed (Figure 24). The rBCG::CRISPRi-MurT-GatD SCs and BCs were then regrown in the presence of Dox and all displayed reduced growth rate in liquid broth supplemented with Dox, while the WT strain displayed minimal sensitivity to Dox (Figure 25). These results showed that 1 mg / kg / day Dox is effective for rBCG::CRISPRi- MurT-GatD activation in vivo and enabled determination of the minimum Dox dose for CRISPRi activation in vivo as administration of Dox at high doses is known to have immuno-modulatory effects.

[0130] EXAMPLE 4

[0131] Analysis of rBCG::CRISPRi-MurT-GatD attenuation in SCID mice rBCG::CRISPRi-MurT-GatD is not more attenuated than WT BCG and does not cause more disease in SCID mice

[0132] To further explore the attenuation of rBCG::CRISPRi-MurT-GatD, we aerosol infected female SCID (severe combined immunodeficiency) mice with a low dose (-100 CFU) of WT BCG and rBCG::CRISPRi-MurT-GatD, and included Dox receiving groups (i.e. WT BCG+Dox and rBCG::CRISPRi-MurT-GatD+Dox) (Figure 26). WT BCG infected mice displayed early decreased survival as expected, followed by the WT BCG+Dox group of mice (Figure 27). Although rBCG::CRISPRi-MurT-GatD infected mice, either receiving Dox or not, displayed slight increased survival, this data was not signicantly different from WT BCG infected mice (Figure 27). This suggested that rBCG::CRISPRi-MurT-GatD upon CRISPRi activation is not more attenuated than WT BCG and does not cause more disease in SCID mice compared to WT BCG.

[0133] EXAMPLE 5

[0134] In vivo testing of recombinant BCG::CRISPRi MurT-GatD strain

[0135] Mouse immunization and determination of protective efficacy of rBCG::CRISPRi- MurT-GatD and lung histopathology analysis To test the efficacy of rBCG::CRISPRi-MurT-GatD, BalB / c mice (n = 10 per group) were immunized intradermally with 105 CFU / 100 pL of WT BCG or rBCG::CRISPRi-MurT- GatD strains (Figure 28). Mice were sham immunized with saline and Dox was administered by daily oral gavage to the Saline+Dox (n=5), WT BCG+Dox (n=5) and the rBCG::CRISPRi- MurT-GatD+Dox (n=5) groups for 6 weeks. Mice were weighed per week to monitor the effect of Dox administration on the health of the mice. The inventors assessed the percentage weight change of mice receiving Dox in comparison to no-Dox groups and found that the percentage weight change of WT BCG immunized mice receiving Dox was significantly different from WT BCG immunized mice without Dox treatment (Figure 29). However, the rBCG::CRISPRi-MurT-GatD immunized group receiving Dox did not display significant weight change compared to all groups (Figure 29). Importantly, the Saline+Dox, WT BCG+Dox and rBCG::CRISPRi-MurT-GatD+Dox groups were similar in weight at 6 weeks pre-challenge with Mtb H37Rv.

[0136] Mice were challenged with -100 CFU of Mtb H37Rv strain by the aerosol route 6 weeks post immunization in a Glasscol inhalation exposure system (Glasscol). Lungs and spleens from infected animals were harvested at week 4 and week 8 post Mtb infection for analysis of lung bacterial burden by plating the whole-lung homogenate on 7H11 -selective plates containing carbenicillin (50 mg / ml), Trimethoprim (20 mg / ml), Polymyxin B (25 mg / ml) and Cycloheximide (10 mg / ml).

[0137] The WT BCG+Dox group displayed lower lung weights, the WT-BCG and rBCG::CRISPRi-MurT-GatD without Dox-treatment groups displayed similar lung weights while the Saline+Dox and rBCG::CRISPRi-MurT-GatD+Dox groups displayed increased lung weights indicative of increased lung inflammation (Figure 30A). The Saline+Dox group displayed increased spleen weights compared to the other groups (Figure 30B). Analysis of lung bacterial burden at week 4 revealed that rBCG::CRISPRi-MurT-GatD+Dox was superior to WT BCG and WT BCG+Dox in protecting against Mtb H37Rv challenge in the lungs and reduced bacterial dissemination to the spleen similar to WT BCG or WT BCG+Dox (Figure 30C and D). At week 8 post challenge, the rBCG::CRISPRi-MurT- GatD+Dox group displayed reduced lung weight indicative of control of bacterial burden and analysis of lung bacterial burden demonstrated again that rBCG::CRISPRi-MurT-GatD+Dox was superior to WT BCG or WT BCG+Dox in controlling Mtb H37Rv growth in the lung which displayed waning efficacy in this model (Figure 30A and C). In the spleen rBCG::CRISPRi-MurT-GatD+Dox displayed similar efficacy to WT BCG in control of infection compared to the Saline+Dox group (Figure 30D). Histopathological analysis of lung pathology after vaccination with rBCG::CRISPRi- MurT-GatD+Dox compared to WT BCG post Mtb Infection

[0138] For histopathology, vertical halves of the left lung were fixed in 10% buffered formalin and sections of 5 pm in thickness from formalin fixed and paraffin embedded tissues were cut onto glass slides and stained with H&E for histopathological examination.

[0139] Histopathological analysis of haematoxylin and eosin (H&E) stained lung samples from the vaccinated and Mtb challenged mice indicated that rBCG::CRISPRi MurT- GatD+Dox immunized mice presented with early increased lung inflammation compared to WT BCG+Dox vaccinated mice. At week 8 also, rBCG::CRISPRi MurT-GatD+Dox immunized mice presented with increased inflamed proportions of lung area compared to WT BCG+Dox immunized mice suggestive of sustained inflammation for control of infection (Figure 31). The increased early inflammation in rBCG::CRISPRi MurT-GatD+Dox immunized mice is reflective of early induction of anti-tuberculous immune responses which were able to control bacterial growth early before establishment of infection and the sustained inflammation at week 8 is suggestive of enhanced immune responses during chronic disease which enable control of disease progression.

[0140] REFERENCES

[0141] Bekkering, S., Blok, B.A., Joosten, L.A., Riksen, N.P., van Crevel, R. and Netea, M.G., 2016. In vitro experimental model of trained innate immunity in human primary monocytes. Clinical and Vaccine Immunology, 23(12), pp.926-933.

[0142] Mendum, T.A., Chandran, A., Williams, K., Vordermeier, H.M., Villarreal-Ramos, B., Wu, H., Singh, A., Smith, A. A., Butler, R.E., Prasad, A. and Bharti, N., 2019. Transposon libraries identify novel Mycobacterium bovis BCG genes involved in the dynamic interactions required for BCG to persist during in vivo passage in cattle. BMC genomics, 20(1 ), pp.1 -13.

[0143] Pan, W., Hao, S., Zheng, M., Lin, D., Jiang, P., Zhao, J., Shi, H., Yang, X., Li, X. and Yu, Y., 2020. Oat-derived p-glucans induced trained immunity through metabolic reprogramming. Inflammation, 43(4), pp.1323-1336.

[0144] Rock, J.M., Hopkins, F.F., Chavez, A., Diallo, M., Chase, M.R., Gerrick, E.R., Pritchard, J.R., Church, G.M., Rubin, E.J., Sassetti, C.M. and Schnappinger, D., 2017. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nature microbiology, 2(4), pp.1 -9.

[0145] Toda, G., Yamauchi, T., Kadowaki, T. and Ueki, K., 2021. Preparation and culture of bone marrow-derived macrophages from mice for functional analysis. STAR protocols, 2(1), p.100246.

Claims

CLAIMS1. A recombinant Mycobacterium bovis BCG strain comprising disrupted expression of a Mb3739 gene.

2. The recombinant Mycobacterium bovis BCG strain of claim 1 , wherein the recombinant Mycobacterium bovis BCG strain comprises a plasmid having a short guide RNA (sgRNA) target sequence for knockdown of expression of the Mb3739 gene.

3. The recombinant Mycobacterium bovis BCG strain of claim 2, wherein the sgRNA targets the 5’ region of the murT-gatD operon.

4. The recombinant Mycobacterium bovis BCG strain of claim 2 or 3, wherein the sgRNA comprises a nucleotide sequence of SEQ ID NO:3.

5. The recombinant Mycobacterium bovis BCG strain of any one of claims 2 to4, wherein the plasmid is a CRISPRi-dCas9 plasmid.

6. The recombinant Mycobacterium bovis BCG strain of any one of claims 1 to5, wherein the disruption or knockdown of the expression of the Mb3739 gene results in modification of a peptidoglycan in the recombinant Mycobacterium bovis BCG strain.

7. The recombinant Mycobacterium bovis BCG strain of claim 6, wherein the modification reduces amidation of the peptidoglycan and activates NOD-1 .

8. The recombinant Mycobacterium bovis BCG strain of claim 6 or 7, wherein the recombinant Mycobacterium bovis BCG strain comprising the modification is capable of eliciting an immune response against Mycobacterium tuberculosis.

9. A method of producing a recombinant Mycobacterium bovis BCG strain comprising disrupted expression of a Mb3739 gene, the method comprising: a) cloning a short guide RNA (sgRNA) target sequence for knockdown of expression of the Mb3739 gene into a CRISPRi-dCas9 plasmid to obtain a cloned plasmid; and b) introducing the cloned plasmid from a) into Mycobacterium bovis, wherein the cloned plasmid causes knockdown of expression of the Mb3739 gene in the recombinant Mycobacterium bovis BCG strain.

10. The method of claim 9, wherein the sgRNA comprises a nucleotide sequence of SEQ ID NO:3.11 . The method of claim 9 or 10, wherein the knockdown of the expression of the Mb3739 gene results in modification of a peptidoglycan in the Mycobacterium bovis BCG strain.

12. The method of claim 11 , wherein the modification reduces amidation of the peptidoglycan and activates NOD-1.

13. The method of any one of claims 9 to 12, wherein the cloned plasmid is introduced into the Mycobacterium bovis by electroporation.

14. A recombinant Mycobacterium bovis BCG strain produced by the method of any one of claims 9 to 13.

15. A vaccine composition comprising the recombinant Mycobacterium bovis BCG strain of any one of claims 1 to 8 and 14.

16. The vaccine composition of claim 15, further comprising a pharmaceutically acceptable carrier or adjuvant.

17. A method of eliciting an immune response against Mycobacterium tuberculosis in a subject, the method comprising administering an immunogenically effective amount of the recombinant Mycobacterium bovis BCG strain of any one of claims 1 to 8 and 14 or the vaccine composition of claim 15 or 16 to the subject.

18. The method of claim 17, wherein the subject is a human subject.

19. The recombinant Mycobacterium bovis BCG strain of any one of claims 1 to 8 and 14 or the vaccine composition of claim 15 or 16 for use in a method of eliciting an immune response against Mycobacterium tuberculosis in a subject, the method comprising administering an immunogenically effective amount of the recombinant Mycobacterium bovis BCG strain or vaccine composition to the subject.

20. The recombinant Mycobacterium bovis BCG strain or vaccine composition for use of claim 19, wherein the subject is a human subject.

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