Abstract
The invention relates to a modified Neisseria lactamica, wherein the modified Neisseria lactamica is transformed with recombinant DNA encoding a heterologous protein; a method of prophylactic treatment for pathogenic infection of a subject comprising nasopharyngeal inoculation of a modified Neisseria lactamica; a method of reducing or preventing meningococcal colonisation of a subject; a method of modifying the microbiome of a subject; a wild-type Neisseria lactamica for use for the prophylactic treatment of meningococcal infection of a subject or reducing colonisation of a subject, wherein the prophylactic treatment comprises nasopharyngeal inoculation of the wild-type Neisseria lactamica; associated nucleic acid for mutagenesis of Neisseria lactamica; methods of mutagenesis; outer membrane vesicle (OMV) vaccines; and associated compositions and methods thereof.
Claims
1. A modified Neisseria lactamica, wherein the modified Neisseria lactamica is transformed with recombinant DNA encoding a heterologous protein, and wherein the modified Neisseria lactamica is modified by wild-type lacZ gene knockout or removal of wild-type lacZ gene sequence.
2. The modified Neisseria lactamica according to claim 1, wherein the heterologous protein is a Neisseria meningitidis protein.
3. The modified Neisseria lactamica according to claim 1, wherein the heterologous protein is a eukaryote protein, viral protein, or non-Neisseria prokaryote protein.
4. The modified Neisseria lactamica according to claim 1, wherein: the heterologous protein comprises meningococcal adhesin NadA, or a functional homologue thereof having at least 70% sequence identity to meningococcal adhesin NadA; and/or the heterologous protein comprises N. meningitidis PorA, or a functional homologue thereof having at least 70% sequence identity to N. meningitidis PorA.
5. The modified Neisseria lactamica according to claim 1, wherein the recombinant DNA is: inserted into the chromosome of Neisseria lactamica to provide the modified Neisseria lactamica; and/or inserted into an intergenic chromosomal sequence; and/or inserted into NHCIS1 or NHCIS2, or an equivalent thereof in other Neisseria lactamica strains.
6. The modified Neisseria lactamica according to claim 5, wherein the intergenic chromosomal sequence is: between genes NLY_27080 and NLY_27100 of Neisseria lactamica strain Y92-1009, or an equivalent loci thereof in other Neisseria lactamica strains; or between genes NLY_36160 and NLY_36180 of Neisseria lactamica strain Y92-1009, or an equivalent loci thereof in other Neisseria lactamica strains.
7. The modified Neisseria lactamica according to claim 1, wherein the recombinant DNA encoding the heterologous protein is at least 600 bp in length.
8. The modified Neisseria lactamica according to claim 1, wherein: the modified Neisseria lactamica is a -galactosidase (lacZ) deficient mutant; and/or the modified Neisseria lactamica does not comprise a functional or non-functional wild-type lacZ gene.
9. The modified Neisseria lactamica according to claim 1, wherein the recombinant DNA encodes a promoter to promote expression of the heterologous protein.
10. The modified Neisseria lactamica according to claim 9, wherein: the recombinant DNA further encodes a selection marker and encodes a further promoter that promotes expression of the selection marker.
11. The modified Neisseria lactamica according to claim 1, wherein the modified Neisseria lactamica does not comprise wild type lacZ gene sequence.
12. A method of prophylactic treatment for pathogenic infection of a subject comprising nasopharyngeal inoculation of the modified Neisseria lactamica, according to claim 1, wherein the heterologous protein is an antigen from the pathogen.
13. The method of prophylactic treatment according to claim 12, wherein the pathogenic infection is meningococcal infection and the heterologous protein is a meningococcal antigen.
14. The method according to claim 12, wherein the modified Neisseria lactamica is provided in a suspension comprising between 210.sup.2 per ml and 210.sup.8 per ml of the modified Neisseria lactamica.
15. The method according to claim 14, wherein the suspension of modified Neisseria lactamica comprises a pharmaceutically acceptable carrier.
Description
(1) Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.
(2) FIG. 1: Plasmid map of pUC19nlaIII::CLOVER-aphA3.
(3) FIG. 2: Nucleotide sequence (SEQ ID NO: 3) of tandemly-expressed, N. lactamica-codon-optimised CLOVER and aphA3 genes. The sequence of the CLOVER gene is shown as white text against a black background and the aphA3 sequence is shown as boxed black text. The NotI and NdeI restriction sites are shown as black text against a grey background. The DUS and RBS are shown in lower case letters, with the DUS sequence underlined.
(4) FIGS. 3A and 3B: Confocal microscopy of wild type (FIG. 3A) and CLOVER-expressing strains (FIG. 3B) of Neisseria lactamica Y91-1009.
(5) FIG. 4: Plasmid map of pUC19nlaIII::aphA3.
(6) FIG. 5: Transformation efficiency of wild type Neisseria lactamica using (hypermethylated) PCR products amplified from pUC19nlaIII::aphA3.
(7) FIG. 6: Transformation efficiency of wild type Neisseria lactamica using (hypermethylated) PCR products amplified from pUC19nlaIII::CLOVER-aphA3 and derivatives thereof, wherein site-directed mutagenesis has been used to remove CATG sequences from the CLOVER coding sequence. Hm=hypermethylated; 0=PCR product contains no sequences of CATG; 1a and 1b=duplicates for PCR products containing one sequence of CATG; 2=PCR product contains two sequences of CATG.
(8) FIG. 7: Hypermethylation of PCR product blocks restriction activity of NlaIII. Hm=hypermethylated. N=normal, non-hypermethylated. CATG free=no CATG sequence present; CATG=1a and CATG-1b=duplicates for PCR products containing one sequence of CATG; CATG-2=PCR product contains two sequences of CATG.
(9) FIG. 8: Effect of the length of the flanking region and amount of DNA used to transform wild type Neisseria lactamica using PROTOCOL A.
(10) FIG. 9: Effect of the neisserial DNA Uptake Sequence (DUS) on the transformation efficiency of Neisseria lactamica using PROTOCOL A. S-DUS=scrambled DUS.
(11) FIG. 10: Chromosomal schematic and nucleotide sequence of NHCIS1 (N. lactamica Y92-1009) (SEQ ID NOs: 4 and 5).
(12) FIG. 11: Chromosomal schematic and nucleotide sequence of NHCIS2 (N. lactamica Y92-1009) (SEQ ID NOs: 6 and 7).
(13) FIG. 12: Annotated nucleotide sequence of HAEC1 (SEQ ID NO: 8).
(14) FIG. 13: Annotated nucleotide sequence of HAEC2 (SEQ ID NO: 9). AT-DUS=AT-variant of DUS.
(15) FIG. 14: Plasmid map of pUC19NHCIS1::HAEC1:(Z)-lacZ.
(16) FIG. 15: Plasmid map of pUC19NHCIS1::HAEC2:(Z)-lacZ.
(17) FIG. 16: Plasmid map of pUC19NHCIS2::HAEC1:(Z)-lacZ.
(18) FIG. 17: Bar graph showing Specific Activity of lacZ, expressed from different NHCIS loci.
(19) FIG. 18: Plasmid map of pUC19NHCIS1::HAEC2:porAplusprom-lacZ.
(20) FIGS. 19A and 19B: Expression of PorA in recombinant N. lactamica has no appreciable effect on growth rate in TSB.
(21) FIG. 20: PorA is surface-expressed in recombinant N. lactamica strain 2Pp7.A.
(22) FIG. 21: Promoter constructs for investigating transcriptional enhancement by sequence associated with the N. meningitidis porA gene (SEQ ID NOs: 10-16).
(23) FIG. 22: Transcriptional enhancement of the lst promoter by sequence cloned from upstream of the porA gene from Neisseria meningitidis strain MC58.
(24) FIG. 23: Promoter construct designed to investigate the mechanism of porA transcriptional enhancement (SEQ ID NO: 17).
(25) FIG. 24: DNA bending is partially responsible for the transcriptional enhancement activity of the porA enhancer sequence.
(26) FIG. 25: Annotated nucleotide sequence of HAEC3 (SEQ ID NO: 18).
(27) FIG. 26: Annotated nucleotide sequence of HAEC4 (SEQ ID NO: 19).
(28) FIG. 27: Plasmid map of pUC19NHCIS1::HAEC4:nadA-lacZ
(29) FIG. 28: Plasmid map of pUC19NHCIS1::HAEC4:(Z)-lacZ
(30) FIG. 29: NadA-expressing N. lactamica have increased adherence to HEP-2 cells.
(31) FIG. 30: Plasmid map of pSC101NHCIS1::HAEC4:opc-lacZ
(32) FIG. 31: Plasmid map of pUC19nhba::HAEC3:(Z)-lacZ
(33) FIG. 32: Plasmid map of pUC19nhba
(34) FIG. 33: Repeated transformation of N. lactamica does NOT select for a more transformable phenotype.
(35) FIG. 34: Effect of the amount of donor material used to transform wild type Neisseria lactamica: supplemental.
(36) FIG. 35: Plasmid map of pSC101::lacZ:opcA
(37) FIG. 36: Plasmid map of pSC101::lacZ-synth.lacZ-3ENDNHCIS1
(38) FIG. 37 (SEQ ID NO: 20): Coding sequence of synth.lacZ
(39) FIG. 38: Expression of NadA and Opc outer membrane proteins on the surface of recombinant strains of N. lactamica
(40) FIG. 39: Expression of NadA, Opc or a combination of both antigens in recombinant N. lactamica has no appreciable effect on growth rates in TSB
(41) FIGS. 40A and 40B: NadA-expressing strains of N. lactamica have increased adherence to HEP-2 cells (FIG. 40A), whilst Opc-expressing strains of N. lactamica are internalized by HEP-2 cells in significantly larger numbers (FIG. 40B).
(42) FIGS. 41A and 41B: The NadA-expressing strain of N. lactamica, 4NB1, is recovered in significantly higher numbers from within human nasopharyngeal tissue explants than is the wild type
(43) FIG. 42: Recombinant strains of N. lactamica expressing meningococcal adhesin proteins have similar sensitivity profiles to front-line antibiotics as the wild type
(44) FIG. 43: Recombinant strains of N. lactamica expressing meningococcal adhesin proteins are competent for uptake of exogenous DNA and can be transformed with efficiencies similar to the wild type
(45) FIG. 44: Wild type and recombinant strains of N. lactamica are completely refractory to transformation by chromosomal DNA derived from N. meningitidis
(46) FIG. 45: Plasmid map of pSC101NHCIS1::HAEC4:porA(P1.7,16)-lacZ
(47) FIG. 46: Plasmid map of pSC101NHCIS1::PVporA(P1.7,16)-lacZ
(48) FIGS. 47A-47C: Recombinant Nlac strains express Porin A (P1.7, 16) at the cell surface and generate PorA-containing OMV
(49) FIGS. 48A and 48B: Immunisation with OMV from recombinant, PorA-expressing Nlac generates anti-meningococcal SBA
(50) FIG. 49: Immunisation with OMV from recombinant, NadA-expressing Nlac generates anti-meningococcal SBA
(51) Trials of experimental human nasopharyngeal inoculation with wild type Neisseria lactamica strain Y92-1009 have shown that successful colonisation of volunteers by the bacterium can lead to persistent carriage of the organism for up to six months, and that inoculation with the live bacterium also broadens the range of humoral immune responses directed against a narrow panel of Neisseria meningitidis isolates. Colonisation of the nasopharynx by Neisseria lactamica is completely benign. Therefore, the ability to modify the outer membrane components of Neisseria lactamica to include proteins not present in the wild type organism would be extremely beneficial, as it would allow the use of these modified strains as either a means of generating novel OMV that contain antigens from biotic sources other than Neisseria lactamica, or as a safe means of delivering antigen to the nasopharyngeal mucosa for prolonged periods as an alternative means of vaccination.
(52) The most likely cause of the lack of methods to manipulate the genetics of Neisseria lactamica is the battery of restriction enzymes encoded in the genome of Neisseria lactamica, most notably the 4-cutter restriction enzyme, NlaIII, which cuts dsDNA at the short palindromic sequence CATG. By random chance and in the absence of negative selection pressure, this sequence motif occurs with high frequency in a given stretch of nucleotides, meaning that uptake of DNA containing one or more of these sequences results in the intracellular degradation of the material before homologous recombination can take place and integrate the exogenous DNA into the Neisseria lactamica chromosome. This invention describes a process to circumvent the barriers to transformation of Neisseria lactamica and allow targeted genetic modification of this organism to be performed for the first time. The invention allows stable integration of DNA constructs into loci of the Neisseria lactamica chromosome, with utility for both deletion of existing genes (i.e. targeted mutagenesis) or insertion of genes from other biotic sources. This procedure differs from normal methods used to transform Neisseria in its use of hypermethylated PCR products as the donor material, whereby all the deoxycytosine residues of the nucleotide product have been replaced with methyl-deoxycytosine. Methylation of restriction sites can inhibit the DNA cleavage action of restriction endonucleases, which prevents degradation of the product following uptake and therefore provides sufficient time for homologous recombination to take place and the construct to become integrated into the chromosomal locus of choice. The restriction enzyme NlaIII is inhibited in this way (see FIG. 7), and the use of hypermethylated PCR products to transform Neisseria lactamica is demonstrably more efficient than using non-methylated, normal PCR products (see FIG. 6).
(53) A number of cloning vectors have been developed in accordance with the invention. These cloning vectors are based upon the standard cloning vector pUC19, but come in two varieties dependent upon the application. For most routine manipulations and cloning strategies, plasmids containing the pMB1 origin of replication (ori), which maintains a relatively high number of plasmids per cell, can be used. pMB1 is the original ori for pUC19, and all plasmids described in accordance with this invention that contain pMB1 are described as pUC19 derivatives (e.g. pUC19nlaIII::CLOVER-aphA3). In instances where a gene produces a product toxic to E. coli (the organism used for the maintenance and amplification of the plasmids), or where high-level expression of a gene is deleterious, a variant plasmid backbone has been developed in which the pMB1 ori has been replaced with the minimally required replication region of pSC101 (repA/ori). Whilst the rest of the plasmid backbone, including the selection marker, remains unchanged from pUC19, these plasmids are maintained at a much lower copy number per cell (i.e. five). The reduction in copy number may therefore prove permissive for cloning of deleterious sequences with high fidelity. All plasmids that contain the repA/ori region of pSC101 are described as pSC101 derivatives (i.e. pSC101NHCIS1::HAEC4:opcA-lacZ).
(54) The majority of cloning vectors developed in accordance with this invention contain large regions of homology to the Neisseria lactamica chromosome, with added restriction sites for ease of cloning. The regions of homology are designated the Neisseria Heterologous Construct Insertion Sites (NHCIS) and are located in areas of relative gene paucity in the chromosome of the vaccine strain of Neisseria lactamica, Y92-1009. The purpose of these plasmids is to streamline the process of inserting DNA coding for heterologous antigen(s) into the Y92-1009 chromosome, specifically into loci where there will be minimal disruption of other genes that are perhaps essential or beneficial to the biological fitness of the bacterium. Using these vectors we have also developed derivatives that contain a novel means of screening for successful transformation of Neisseria lactamica, whereby the regions of homology flank Heterologous Antigen Expression Constructs (HAEC), each of which consists of two Neisseria lactamica promoter sequences surrounded by a number of unique restriction sites for streamlined manipulation and directional cloning of desired sequences for downstream introduction into the Y92-1009 genome. At the current time there are 4 such constructs, designated HAEC1 through HAEC4, named in the order in which they were created. HAEC1 contains the lst promoter followed (3) by the porB promoter, and this is reversed in HAEC2. HAEC3 contains a hybrid porA/porB promoter, wherein the homopolymeric tract of G nucleotides that renders the wild type porA gene phase variable has been replaced with sequence derived from the wild type, non-phase variable porB promoter of N. lactamica. This promoter is preceded by 250 bp of transcriptional enhancer sequence, derived from the wild type porA gene of N. meningitidis. The porA/porB hybrid promoter is followed (3) by a second hybrid promoter, wherein the 17 bp that separate the 10 and 35 boxes of the RNA Polymerase binding site of the wild type porB gene have been replaced with 17 bp of sequence derived from the lst promoter. The rest of the promoter is identical to that of the wild type, N. lactamica porB gene (FIG. 25). HAEC4 contains an identical porA/porB hybrid promoter to HAEC3, but which is preceded by only 200 bp of porA-derived enhancer sequence. This length of enhancer sequence has been demonstrated to be more potent than the 250 bp version (see FIG. 22). In HAEC4, the lst promoter follows (3) the highly transcriptionally active hybrid promoter (FIG. 26). In each plasmid designed to transform N. lactamica in a way suitable for use in human challenge, the second promoter sequence of each HAEC drives a copy of the Neisseria lactamica -galactosidase (lacZ) gene, which we have used as a means of screening for successful transformation of a lacZ mutant derivative of Y92-1009. To construct the lacZ mutant, the procedure described in Protocol A was utilised to completely remove the coding sequence of the lacZ genethus minimising the potential for off-target recombination of NHCIS-targeted constructs. Taken together, the lacZ Y92-1009 derivative and the NHCIS(X)::HAEC(Y):(Z)-lacZ vector system constitute a useful tool for generating the recombinant strains of Neisseria lactamica described above in a way free from the use of antibiotic resistance cassettes, which would otherwise preclude their use in human challenge studies.
(55) In some examples a lacZ mutant derivative of N. lactamica Y92-1009 is further genetically manipulated. This strain was created using the transformation protocol detailed in Protocol A. The strain has had all of the coding sequence for the wild type lacZ gene removed from the chromosome to avoid non-specific recombination events with the Nlac lacZ-containing Cassettes and concomitant mis-targeting of the Cassette to loci other than that intended. This strain provides the background for insertion of genes coding for heterologous antigens without need for antibiotic resistance markers, a preferred state for potential human challenge with recombinant Neisseria lactamica.
(56) When referring to the use of restriction sites for restriction/ligation cloning of sequences into various constructs, the same restriction site must be absent from the oligonucleotide sequence being cloned.
(57) PROTOCOL A: Transformation Protocol for Neisseria lactamica:
(58) 1. Inoculate TSB with multiple colonies of the recipient strain of Neisseria lactamica (hereafter, the Culture). This could be a recognised laboratory strain such as Y92-1009, a mutant derivative thereof, or wild type or mutant strains of N. lactamica isolated from volunteers. 2. Incubate the Culture at 37 C., 5% CO.sub.2 with shaking until an OD.sub.600nm is reached of 0.3. 3. Dilute the Culture 100 in fresh TSB. 4. Spot 10 l aliquots of the 100 dilution of the Culture onto Tryptone Soy Broth +0.2% yeast extract agar plates (hereafter, TSB agar) and allow to dry in a Microbiological Safety Cabinet. 5. Replace the lids and transfer the plate(s) (right side up) to a CO.sub.2 incubator set to 30 C., 5% CO.sub.2. Incubate for 6 h. 6. Onto the surface of the now-visible N. lactamica colonies, pipette 10 l of Tris HCl buffer containing an appropriate amount of Donor material and allow to dry in a Microbiological Safety Cabinet. 7. Transfer the plate(s) to the incubator at 30 C., 5% CO.sub.2 and incubate for 9-10 h.
PROTOCOL A.1: Alternative transformation protocol for Neisseria lactamica: 1. Grow the recipient strain of N. lactamica on suitable (i.e. growth sustaining) agar plates overnight at 37 C., 5% CO.sub.2. This could be a recognised laboratory strain such as Y92-1009, a mutant derivative thereof, or wild type or mutant strains of N. lactamica isolated from volunteers. 2. Using a sterile microbiological loop, transfer a multitude of colonies to fresh, sterile TSB medium supplemented with 10 mM Mg.sup.2+ ions (solutions of either MgSO.sub.4 or MgCl.sub.2 are suitable). Continue this process until the OD.sub.600m of the suspension is greater than or equal to 0.3. 3. Transfer aliquots of the concentrated bacterial suspension to fresh, sterile microcentrifuge tubes and adjust the OD.sub.600m to 0.3 using fresh, sterile TSB medium supplemented with 10 mM Mg.sup.2+ 4. Prepare the Donor material in sterile PBS. The Donor material could be genomic DNA, a PCR or hypermethylated PCR product, or an artificially synthesized nucleic acid. 5. Add the Donor material to the microcentrifuge tubes, mix by vortexing or pipetting and incubate the tubes at 37 C., 5% CO.sub.2 for 3 h.
(59) Continue with Protocol B for constructs using 3-galactosidase activity as the means of identifying transformed bacteria; alternatively, plate the suspensions on an appropriately selective agar medium prior to overnight incubation.
(60) PROTOCOL B: Screening for transformed Neisseria lactamica containing (3-galactosidase as part of the genetic construct:
(61) 8. Harvest each patch of putatively-transformed N. lactamica into 1 ml of fresh, sterile TSB. 9. Perform a 10 serial dilution of the bacterial suspension in TSB: i.e. 10, 10.sup.2 and 10.sup.3. 10. Further dilute the 10.sup.3 suspension 5 (i.e. 100 l of 10.sup.3 bacterial suspension in to 400 l TSB). 11. On 5 separate TSB agar plates supplemented with 20 ng/ml 5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside (hereafter X-GAL), spread 100 l of the 510.sup.3 bacterial suspension and allow to dry in a Microbiological Safety Cabinet. 12. Incubate overnight at 37 C., 5% CO.sub.2. 13. Isolate each BLUE colony into 1 ml TSB and culture at 37 C., 5% CO.sub.2 for production of stocks and isolation of DNA.
Plasmid Map of pUC19nlaIII::CLOVER-aphA3.
(62) With reference to FIG. 1, the pUC19nlaIII::CLOVER-aphA3 Construct is a pUC19-derived plasmid designed to introduce codon-optimised CLOVER and aphA3, under the control of the promoter from the -2,3-Sialyltransterase gene (lst), into the ORF of the restriction enzyme, NlaIII (nlaIII, NLY_28620). The Cassette, which has been cloned into the HincII restriction site of pUC19 via Gibson Assembly (NEB), consists of sequence amplified from the region of the N. lactamica chromosome containing the nlaIII gene, including most of the nlaIII coding sequence, into which has been cloned versions of the CLOVER and aphA3 genes that are codon-optimised for expression in Neisseria lactamica. Expression of these genes is controlled by a modified lst promoter, into the 5 untranslated region (hereafter, 5 UTR) of which was introduced an XhoI restriction site to simplify downstream manipulation of the Cassette. The CLOVER and aphA3 genes are tandemly expressed from this promoter, and are separated by (in 5 to 3 order), a NotI restriction site (to facilitate substitution of the CLOVER gene for any other nucleotide sequence), a DUS (to enhance uptake of the Cassette by N. lactamica as part of PROTOCOL A), and a modified Ribosome Binding Site (hereafter, RBS) (to ensure translation of mRNA coding aphA3) (see FIG. 2). Immediately 3 of aphA3, the chromosomal sequence has been modified to introduce an XbaI restriction site. This site enables XbaI-digestion of the Construct to release the Cassette, for use as Donor material in PROTOCOL A. The sequence derived from the N. lactamica nlaIII-containing chromosomal region also contains an inv. AT-DUS, 5 of the start codon of the nlaIII gene. The plasmid map is presented showing all relevant features, including the insertionally-inactivated ORF for nlaIII, and detailing the location of unique restriction sites with recognition sequences 6 nucleotides or greater.
(63) Nucleotide Sequence of Tandemly-Expressed, N. lactamica-Codon-Optimised CLOVER and aphA3 Genes.
(64) With reference to FIG. 2, the sequences of these genes have been codon-optimised for expression in N. lactamica. The activity of NlaIII is a significant barrier to successful transformation of N. lactamica (see FIG. 6). This piece of the Cassette could be controlled from any N. lactamica-compatible promoter sequence and be flanked by a contiguous nucleotide sequence from theoretically any locus in the N. lactamica chromosome.
(65) Confocal Microscopy of Wild Type and CLOVER-Expressing Strains of Neisseria lactamica Y91-1009.
(66) With reference to FIG. 3, wild type N. lactamica Y92-1009 was transformed with donor material derived by hypermethylated PCR from pUC19nlaII::CLOVER-aphA3 (FIG. 1), as described in PROTOCOL A. Transformants, which were selected for on the basis of resistance to kanamycin (50 g/ml in TSB agar plates), were isolated and cultured. Chromosomal integration of the donor material into the nlaIII locus was determined by PCR (data not shown). Stationary phase (overnight) colonies of both wild type Y92-1009 (A) and a transformed, putatively CLOVER-expressing derivative thereof (B), were dispersed into sterile PBS, spread across the surface of a microscope slide and allowed to air dry in a Class II microbiological safety cabinet. Bacteria were visualised under the confocal microscope using light at a wavelength of 988 nm and the images digitally captured. It is important to note that whilst the overall level of fluorescence is low, there is green fluorescence of the CLOVER-transformed bacteria, which is not evident in the wild type, parental strain. This is the first demonstration of significant eukaryotic gene expression in N. lactamica. Furthermore, this is the first demonstration of heterologous protein expression in this species of bacterium.
(67) Plasmid Map of pUC19 nlaIII::aphA3.
(68) With reference to FIG. 4, the pUC19nlaIII::aphA3 Construct is a derivative of pUC19nlaIII::CLOVER-aphA3, wherein the aphA3 gene has been placed under direct transcriptional control of the lst promoter, and that which remains of the nlaIII coding sequence has been further truncated. The sequence derived from the N. lactamica nlaIII-containing chromosomal region also contains an inv. AT-DUS, 5 of the start codon of the nlaIII gene. The plasmid map is presented showing all relevant features, including the insertionally-inactivated ORF for nlaIII, and detailing the location of unique restriction sites with recognition sequences 6 nucleotides or greater.
(69) Transformation Efficiency of Wild Type Neisseria lactamica Using (Hypermethylated) PCR Products Amplified from pUC19nlaIII:aphA3.
(70) With reference to FIG. 5, XbaI-digested pUC19nlaIII::aphA3 was used as a template to amplify the nlaIII::aphA3 Cassette using both traditional (Non-methylated) and Hypermethylated PCR. Traditional PCR uses an equimolar mixture of the four, unmodified deoxyribonucleotides; whereas hypermethylated PCR uses a nucleotide mixture that substitutes 5-methyl-dCTP instead of unmodified dCTP and results in a PCR product in which every C residue is methylated. The primers used in the reaction produced products of identical sequence with 1200 bp of flanking DNA on either side of the aphA3 gene. The transformation was carried out according to Protocol A, using a total of 1000 ng of each PCR product (=0.46 pmol). The number of transformants was considered to be equal to the number of kanamycin-resistant colonies that grew overnight on selective agar plates (TSB+0.2% yeast extract supplemented with 50 g/ml kanamycin). Indeed, PCR analysis of the nlaIII chromosomal locus from 50 individual, kanamycin resistant colonies derived from transformation with either construct showed that the aphA3 gene had been accurately targeted. The graph shows that transformation into the nlaIII locus is significantly higher through the use of hypermethylated PCR product as compared to an identical, unmodified PCR product. However, it is important to note that the nlaII::aphA3 Cassette is free of CATG sites, the recognition and cleavage site of the NlaIII restriction enzyme.
(71) Transformation efficiency of wild type Neisseria lactamica using (hypermethylated) PCR products amplified from pUC19A nlaIII::CLOVER-aphA3 and derivatives thereof, wherein site-directed mutagenesis has been used to remove CATG sequences from the CLOVER coding sequence.
(72) With reference to FIG. 6, the coding sequence of the Neisseria lactamica-codon optimised CLOVER gene contains two CATG sequences, which is the recognition and cleavage site for the NlaIII restriction enzyme. As a four-cutter restriction enzyme, the frequency of this sequence in exogenous genetic material is likely to be high, and we hypothesise that the restriction activity of NlaIII is one of the main components of the observed resistance of Neisseria lactamica to genetic manipulation. To investigate this, a series of plasmids were generated in which one or both of the two CATG sequences had been removed from the CLOVER gene, using the Q5 Site-directed mutagenesis kit (NEB). The remainder of the construct contained no CATG sequences. These plasmids were used as templates for traditional (i.e. Non-methylated) or hypermethylated PCR. Identical primers were used for both types of PCR, producing PCR products with 600 bp of sequence flanking either side of the CLOVER-aphA3 cassette. Transformation of wild type Neisseria lactamica was carried out as described in Protocol A, using 634 ng (=0.46 pmol) of each PCR or hmPCR product. The number of transformants was considered to be equal to the number of kanamycin-resistant colonies that grew overnight on selective agar plates (TSB+0.2% yeast extract supplemented with 50 g/ml kanamycin), adjusted for dilution and plating volume. Where the transformation efficiency was below the limits of detection, the appropriate data points were calculated as if the transformation derived a single transformant. The graph shows that the transformation efficiency of wild type Neisseria lactamica is significantly effected by the presence of CATG sequences, wherein the introduction of a single CATG sequence into an otherwise CATG-free construct reduces the transformation efficiency by an average of 33 fold. The graph also shows that the use of hypermethylated PCR products enhances the transformation efficiency of wild type Neisseria lactamica, in keeping with the data presented in FIG. 4. Where two CATG sequences are present in the transformation construct, use of hypermethylated PCR product is approximately 4000 times more efficient at transforming wild type Neisseria lactamica than using an equivalent, Non-methylated PCR product. Importantly, there are no differences in the transformation efficiencies measured using any of the hypermethylated PCR products, which implies complete blocking of all restriction endonuclease recognition and cleavage sites. Hypermethylation is also likely to block the restriction function of other endonuclease enzymes in Neisseria lactamica, as transforming bacteria with a hypermethylated PCR product derived from the CATG-free template was on average 22 times more efficient than using a Non-methylated PCR product.
(73) Hypermethylation of PCR Product Blocks Restriction Activity of NlaIII.
(74) With reference to FIG. 7, the series of pUC19nlaII::CLOVER-aphA3 plasmids, each with a different number of CATG motifs within the CLOVER coding sequence, were used as templates in both normal (i.e. Non-methylated) and hypermethylated PCR. Amplification of the nlaIII::CLOVER-aphA3 Cassettes was followed by in vitro digestion of 1 g of each with recombinant NlaIII (NEB) in Cutsmart buffer. Two hundred nanograms of each digested product was then loaded onto a 0.7% agarose gel and electrophoresis was carried out to separate any products. An inverted gel image was captured from the UV transilluminator using a digital camera. Whilst each hypermethylated PCR product (Hm) ran at a slightly increased molecular weight cf. its non-methylated equivalent (N), the former was completely protected from NlaIII enzymatic digest.
(75) Effect of the Length of the Flanking Region and Amount of DNA Used to Transform Wild Type Neisseria lactamica Using PROTOCOL A.
(76) With reference to FIG. 8, to investigate the effect of the size of the regions of homology to the N. lactamica chromosome on transformation efficiency (A), a series of hypermethylated PCR products of different lengths were derived from XbaI-digested pUC19nlaIII::aphA3 plasmid. In each product the aphA3 gene and its promoter were identical, whilst the length of the nlaIII chromosomal regions surrounding this sequence was varied. The flanking lengths used in this experiment (i.e. the length of the DNA sequence on each side of the aphA3 gene) were: 75 bp, 150 bp, 300 bp, 600 bp and 1200 bp. As shown in FIGS. 6 and 7, the fact that the PCR products were hypermethylated ruled out the presence of CATG sequences in shorter products as a confounding factor, as it is likely that all restriction endonuclease recognition and cleavage sites were blocked by the inclusion of mdCTP. Transformation of wild type Neisseria lactamica was carried out according to Protocol A, using 0.46 pmol of each hypermethylated PCR product. The number of transformants was considered to be equal to the number of kanamycin-resistant colonies that grew overnight on selective agar plates (TSB+0.2% yeast extract supplemented with 50 g/ml kanamycin), adjusted for dilution and plating volume. Graph A shows that with increasing length of the Cassette flanking sequence, there is an increase in the transformation efficiency of Neisseria lactamica into the nlaIII locus. However, the presence of longer flanking sequences results in increased inter-experimental variation in transformation efficiency.
(77) To investigate the effect of the amount of donor DNA on the transformation efficiency of Neisseria lactamica (B), increasing amounts of hypermethylated PCR product with 600 bp of flanking sequence were used as described in Protocol A. This PCR product was used because there is inherently less inter-experimental variability then when using a product that contains 1200 bp of flanking sequence, implying a better signal-to-noise ratio in the event the effect was a subtle one. Wild type Neisseria lactamica were transformed using 0.06, 0.26, 0.46, 0.66, 0.86 and 1.06 pmol of hypermethylated PCR product. The number of transformants was considered to be equal to the number of kanamycin-resistant colonies that grew overnight on selective agar plates (TSB+0.2% yeast extract supplemented with 50 g/ml kanamycin), adjusted for dilution and plating volume. Graph B shows that there is a potentially bi-phasic peak in transformation efficiency, although the mechanism for this remains even theoretically unclear. The only significant difference in transformation efficiency is between the transformations carried out using 0.26 and 0.46 pmol of DNA, where the lower amount of DNA yields the most transformants. However, this is most probably a statistical anomaly that we predict will disappear with repetition of the experiment.
(78) Effect of the neisserial DNA Uptake Sequence (DUS) on the transformation efficiency of Neisseria lactamica using PROTOCOL A.
(79) With reference to FIG. 9, the non-palindromic repeat sequence 5-GCCGTCTGAA-3 (SEQ ID NO: 1), or close derivatives/relatives thereof, occurs with high frequency in the chromosomes of many Neisseria species. Previous studies have demonstrated that bacterial uptake of DNA molecules containing these sequences is enhanced, hypothetically through a charge interaction with a positively charged region of ComP, a pilus-associated protein. The effect of the standard DNA Uptake Sequence (hereafter, DUS) on transformation efficiency is further enhanced by the presence of AT nucleotides at the 5 end. The AT-variant of the DUS (hereafter, AT-DUS) is the dialect of DUS repeated at the highest frequency in the wild type Neisseria lactamica chromosome. The nlaIII chromosomal region included in pUC19nlaII::aphA3 contains an inverted AT-DUS sequence in its 5 end. As a result, the primer pairs used to generate the PCR products containing 300 bp, 150 bp and 75 bp of flanking nlaIII chromosomal sequence (see FIG. 6) each contain only one copy of the DUS (that which was deliberately included after the aphA3 coding sequence), as compared to the PCR products containing 600 bp and 1200 bp of flanking nlaIII sequence, which contain two. To investigate the role of an additional AT-DUS on the transformation efficiency of wild type Neisseria lactamica, two versions of each PCR product were amplified from XbaI-digested pUC19nlaII:aphA3, using alternative 5 primers: one primer in each set contained a canonical, inverted AT-DUS at the 5 terminus (DUS, black circles), whilst the alternate version contained a scrambled DUS (S-DUS, red squares). The S-DUS contains the same proportions of the same nucleotides as the DUS, but in a configuration designed to ensure no similarity to the various dialects of DUS. Transformation of wild type Neisseria lactamica was carried out as described in Protocol A, using 0.46 pmol of each PCR or hmPCR product. The number of transformants was considered to be equal to the number of kanamycin-resistant colonies that grew overnight on selective agar plates (TSB+0.2% yeast extract supplemented with 50 g/ml kanamycin), adjusted for dilution and plating volume. The graph shows that inclusion of a second DUS in the PCR products used to transform Neisseria lactamica increases the transformation efficiency, and that this effect is more enhanced (to the point of becoming statistically significant) with increasing flanking sequence length.
(80) Chromosomal Schematic and Nucleotide Sequence of NHCIS1 (N. lactamica Y92-1009).
(81) With reference to FIG. 10, the schematic shows the chromosomal locus of NHCIS1 and its surrounding genes, which are shaded (greyscale) on the basis of GC nucleotide pair content (modified and reproduced from: http://www.xbase.ac.uk/genome/neisseria-lactamica-y92-1009/CACL01000001/NLY_27080;/viewer). The nucleotide sequences of the NHCIS1 regions are as detailed on the plasmid map for pUC19NHCIS1::HAEC1:(Z)-lacZ (FIG. 14) (i.e. 5ENDNHCIS1 and 3ENDNHCIS1).
(82) Chromosomal Schematic and Nucleotide Sequence of NHCIS2 (N. lactamica Y92-1009).
(83) With reference to FIG. 11, the schematic shows the chromosomal locus of NHCIS2 and its surrounding genes, which are shaded (greyscale) on the basis of GC nucleotide pair content (modified and reproduced from: http://www.xbase.ac.uk/genome/neisseria-lactamica-y92-1009/CACL01000018/NLY_36160;/viewer). The sequences of the NHCIS2 regions as detailed on the plasmid map for pUC19NHCIS2::HAEC1:(Z)-lacZ (FIG. 16) (i.e. 5ENDNHCIS2 and 3ENDNHCIS2). The italicised text represent nucleotides comprising part of the coding sequence for NLY_36160. The 5 NHCIS2 sequence contains an AT-DUS and an inv-AT-DUS, which are highlighted in bold text.
(84) Annotated Nucleotide Sequence of HAEC1.
(85) With reference to FIG. 12, the sequence consists of the N. lactamica lst promoter, preceded by a DUS and a SalI restriction site (to facilitate cloning and replacement of the lst promoter sequence), separated from the N. lactamica porB promoter sequence by an XhoI restriction site, an interchangeable LINKER sequence and a NotI restriction site. It is important to note that either LINKER sequence can be any given nucleotide sequence (represented as poly-N). By including an XhoI restriction site immediately 5 of the START codon and a NotI restriction site immediately 3 of the STOP codon of any given coding sequence (e.g. through the use of extended primer sequences that incorporate these sites), that sequence can be directionally cloned, IN-FRAME into the Construct for expression from the lst promoter. The featured NdeI restriction site can be used in conjunction with the HindIII restriction site present immediately 5 of the porB terminator sequence to clone any given coding sequence into the Construct. By including an NdeI restriction site immediately 5 of the START codon and a HindIII restriction site immediately 3 of the STOP codon of any given coding sequence (e.g. through the use of extended primer sequences that incorporate these sites), that sequence can be directionally cloned, IN-FRAME into the Construct for expression from the porB promoter. Immediately after the HindIII restriction site, there is a transcriptional terminator sequence, derived from downstream of the N. lactamica porB gene. This is immediately followed by an AT-DUS. In the context of heterologous antigen expression in recombinant Neisseria lactamica, HAEC1 is flanked on either side by sequences homologous to the N. lactamica chromosome (e.g. NHCIS1).
(86) Annotated Nucleotide Sequence of HAEC2.
(87) With reference to FIG. 13, the sequence consists of the N. lactamica porB promoter, preceded by a DUS and a SalI restriction site (to facilitate cloning and replacement of the lst promoter sequence), separated from the N. lactamica lst promoter sequence by an XhoI restriction site, an interchangeable LINKER sequence and a NotI restriction site. It is important to note that either LINKER sequence can be any given nucleotide sequence (represented as poly-N). By including an XhoI restriction site immediately 5 of the START codon and a NotI restriction site immediately 3 of the STOP codon of any given coding sequence (e.g. through the use of extended primer sequences that incorporate these sites), that sequence can be directionally cloned, IN-FRAME into the Construct for expression from the porB promoter. The featured NdeI restriction site can be used in conjunction with the HindIII restriction site present immediately 5 of the porB terminator sequence to clone any given coding sequence into the Construct. By including an NdeI restriction site immediately 5 of the START codon and a HindIII restriction site immediately 3 of the STOP codon of any given coding sequence (e.g. through the use of extended primer sequences that incorporate these sites), that sequence can be directionally cloned, IN-FRAME into the Construct for expression from the lst promoter. Immediately after the HindIII restriction site, there is a transcriptional terminator sequence, derived from downstream of the N. lactamica porB gene. This is immediately followed by an AT-DUS. In the context of heterologous antigen expression in recombinant Neisseria lactamica, HAEC2 is flanked on either side by sequences homologous to the N. lactamica chromosome (e.g. NHCIS1).
(88) Plasmid Map of pUC19NHCIS1::HAEC1:(Z)-lacZ.
(89) With reference to FIG. 14, the pUC19NHCIS1::HAEC1:(Z)-lacZ plasmid consists of a Cassette, comprised of the intergenic chromosomal sequence between NLY_27080 and NLY_27100 of N. lactamica (i.e. NHCIS1, see FIG. 10), disrupted by HAEC1:(Z)-lacZ (where Z represents any given coding sequence) (FIG. 12); cloned into pUC19 between two XbaI restriction sites. In one aspect of the invention, the N. lactamica lacZ gene is included as a screening marker, to enable identification of successfully transformed N. lactamica on the basis of BLUE/WHITE colony formation on plates containing 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal). This is deemed essential to the production of strains of N. lactamica for potential inoculation into human volunteers. Immediately 3 of the lacZ gene the Construct includes the terminator sequence from the N. lactamica porB gene, to ensure there is no translation of downstream, cistronic transcripts. Immediately 5 to the porB terminator sequence there is a unique HindIII restriction site, to facilitate removal of the lacZ gene from the Construct and its substitution with potentially any other oligonucleotide sequence. In other aspects of the invention, the Construct can be manipulated to introduce either one or two of potentially any given oligonucleotide sequence into the chromosome of N. lactamica at the NHCIS1 locus, although it is anticipated that one of the two sequences encodes a marker gene to enable screening or selecting for successfully transformed N. lactamica. The marker gene could plausibly encode proteins conferring antibiotic resistance, but it is preferable to avoid the use of such markers for producing strains intended for human challenge. The plasmid map is presented showing all relevant features and detailing the location of unique restriction sites with recognition sequences 6 nucleotides or greater.
(90) Plasmid Map of pUC19NHCIS1::HAEC2:(Z)-lacZ.
(91) With reference to FIG. 15, the pUC19NHCIS1::HAEC2:(Z)-lacZ Construct contains many elements identical to pUC19NHCIS1::HAEC1:(Z)-lacZ; except that HAEC1 (FIG. 12) has been replaced by HAEC2 (FIG. 13). The functional difference of this change is that the expression of any gene of interest cloned correctly into this plasmid will be driven by the N. lactamica porB promoter, whilst the expression of lacZ is driven by the N. lactamica lst promoter. The plasmid map is presented showing all relevant features and detailing the location of unique restriction sites with recognition sequences 6 nucleotides or greater.
(92) Plasmid Map of pNHCIS2::HAEC1:(Z)-lacZ.
(93) With reference to FIG. 16, the pNHCIS2::HAEC1:(Z)-lacZ plasmid consists of a Cassette, comprised of the intergenic chromosomal sequence between NLY_36160 and NLY_36180 of N. lactamica (i.e. NHCIS2, see FIG. 11), disrupted by HAEC1:(Z)-lacZ (where Z represents any given coding sequence) (FIG. 12); cloned into a truncated version of pUC19 between two XbaI restriction sites. The plasmid map is presented showing all relevant features and detailing the location of unique restriction sites with recognition sequences 6 nucleotides or greater.
(94) Specific Activity of lacZ, Expressed from Different NHCIS Loci.
(95) With reference to FIG. 17, a lacZ mutant derivative of N. lactamica Y92-1009 was transformed with Cassettes derived from either pUC19NHCIS1::HAEC1:(Z)-lacZ or pNHCIS2::HAEC1:(Z)-lacZ according to the procedure detailed in PROTOCOL A. Chromosomal integration of HAEC1:(Z)-lacZ into either NHCIS1 (Strain NHCIS1) or NHCIS2 (Strain NHCIS2) was confirmed by PCR of the respective locus (data not shown) and functionality of the lacZ gene was confirmed by growth of the strains as BLUE colonies on Tryptone Soy Broth+0.2% yeast extract agar plates (hereafter, TSB agar), supplemented with X-Gal (20 ng ml.sup.1). Wild type Y92-1009, the lacZ mutant derivative thereof and Strains NHCIS1/2 were cultured in Tryptone Soy Broth, supplemented with 0.2% yeast extract (hereafter, TSB) at 37 C., 5% CO.sub.2 and 320 rpm, until reaching mid-log phase (OD.sub.600nm=0.3). Bacteria were pelleted by centrifugation, washed once in sterile PBS then resuspended into 200 l of Bacterial Lysis Buffer (BLB) before being lysed through sonication. The supernatant was diluted 5 with BLB, and 5, 2 and 1 l aliquots were assayed for B-galactosidase activity using the chromogenic substrate ONPG. The activities in each of these aliquots were averaged to provide a value for each sample in each experiment. The protein concentration of each supernatant was measured using the DC Protein Assay Reagent (Bio Rad) and these values were used to normalise the measured -galactosidase activities (yielding Specific Activity). Values shown are the mean of three independent experiments. Error bars represent the standard error of the mean. Where no error bars are visible, they fall within the line at the top of the column.
(96) These data show that gene expression, driven by identical promoter sequences, is different when the same gene construct is chromosomally integrated into different loci. Assuming that -galactosidase specific activity is proportional to the amount of -galactosidase enzyme present, which is itself proportional to the level of transcriptional activity from the porB promoter sequence; the graph shows a significantly higher -galactosidase activity, indicative of increased levels of gene transcription, were measured in Strain NHCIS2 than in Strain NHCIS1. In both instances expression of the lacZ gene is being controlled by the porB promoter. In the wild type and lacZ strains, -galactosidase expression is being controlled by its native, inducible lac promoter, except that the coding sequence for -galactosidase is missing in the lacZ strain, eliminating almost all of -galactosidase activity. This graph shows that the choice of locus for chromosomal integration of genes coding for heterologous antigen is important. Based on these data; if a high level of protein expression is desired, then the gene coding for the heterologous antigen could be targeted to NHCIS2; conversely, genes coding for either potentially toxic products, or proteins that have a detrimental, concentration-dependent effect on the host organism should be targeted to NHCIS1, where the overall level of gene activity is lower.
(97) Plasmid Map of pUC19NHCIS1::HAEC2:porAplusprom-lacZ
(98) With reference to FIG. 18, the pUC19NHCIS1::HAEC2:porAplusprom-lacZ plasmid is a derivative of pUC19NHCIS1::HAEC2:(Z)-lacZ (FIG. 15), whereby the porA gene sequence, derived from Neisseria meningitidis strain H44/76, has been cloned, together with the native porA promoter and 107 bp of upstream chromosomal sequence, in between the SalI and NotI restriction sites of pUC19NHCIS1::HAEC2:(Z)-lacZ. Note that the plasmid map is based upon the ideal sequence for this plasmid, wherein the homopolymeric G tract, which separates the 10 and 35 boxes of the native porA promoter contains 11 contiguous guanosine nucleotide residues. The plasmid map is presented showing all relevant features and detailing the location of unique restriction sites with recognition sequences 6 nucleotides or greater.
(99) Expression of PorA in Recombinant N. lactamica has No Appreciable Effect on Growth Rate in TSB.
(100) With reference to FIG. 19, the lacZ mutant derivative of N. lactamica Y92-1009 was transformed with donor material derived by hypermethylated PCR from pUC19NHCIS1::HAEC2:porAplusprom-lacZ (Clone #7), as described in PROTOCOL A. Individual transformants, screened for on the basis of BLUE/WHITE colony formation on X-gal-containing TSB agar plates, were isolated. Chromosomal integration of the HAEC2:porAplusprom-lacZ cassette into the NHCIS1 locus was demonstrated through PCR (data not shown). Following DNA sequencing of the porA coding sequence in this locus, Strain A (hereafter: 2Pp7.A) was determined to contain genetic material with 100% identity to the porA gene.
(101) Overnight cultures of wild type N. meningitidis strain, H44/76 (grown in Mueller Hinton Broth: MHB), the lacZ mutant derivative of N. lactamica strain, Y92-1009 and the recombinant N. lactamica strain, 2Pp7.A (both grown in TSB+0.2% yeast extract), were washed once in PBS then resuspended into Bacterial Lysis Buffer (hereafter; BLB) supplemented with 100 g/ml lysozyme, protease inhibitor cocktail and 1 mM PMSF. Suspensions were incubated at 30 C. for 30 minutes before the bacteria were lysed via sonication on ice (315 second bursts). Sonicated lysates were supplemented with 2 l of a 5 dilution of rDNase and adjusted to a final [Mg.sup.2+] of 2 mM before a further 30 minute incubation at 30 C. Bacterial membrane fractions were collected by centrifugation at 17,000 g for 30 minutes at 4 C., then resolubilised for 10 minutes at 95 C. into 1LDS reducing sample buffer (Life Technologies). Insoluble material was removed by centrifugation at 17,000 g for 10 minutes at 4 C., following which the protein concentration of the solubilised membrane fraction was measured using the RC DC Protein Assay kit from Bio Rad. A total of 50 g of protein was loaded into each well of a 4-12% polyacrylamide gel and proteins were separated by electrophoresis. The proteins were then transferred to a methanol-activated PVDF membrane and the membrane was blocked using 5% milk-TBS for 1 h. The membrane was interrogated with a 1:1000 dilution of SM300 (anti-PorA P 1,7.16 monoclonal antibody) in 5% milk-TBS and an anti-mouse IgG-HRP conjugate (1:10,000 dilution in 5% milk-TBS). Washed membranes were exposed to ECL reagents, and bands were visualised on photographic film.
(102) The Western blot (left) of Neisseria membrane fractions shows that the recombinant strain of N. lactamica, 2Pp7.A produces a membrane-associated protein that is recognised by the SM300 MAb, similar to but in much less abundance than the meningococcal strain known to express the cognate PorA protein, H44/76. The absence of a similar band in the membrane fraction derived from the lacZ mutant derivative of N. lactamica Y92-1009 suggests that this band is the PorA protein. This is, to the best of our knowledge, the first example of meningococcal gene expression in N. lactamica. The comparative growth curve (right) of different N. lactamica strains in TSB medium supplemented with 0.2% yeast extract shows that the expression of PorA by 2Pp7.A is not detrimental to the aerobic growth of the bacterium.
(103) It is important to note that the relatively low levels of PorA expression in strain 2Pp7.A (as compared to wild type N. menigitidis strain H44/76), are attributable to the phase-variable nature of the endogenous porA promoter. Sequencing of the porA promoter region from the chromosome of 2Pp7.A revealed that the homopolymeric G tract that separates the 10 and 35 boxes (RNA Polymerase binding sites) in this promoter was only 10 nucleotides long. Previous work has shown that when replicating this promoter, slipped strand mispairing mutagenesis can lead to variation in the number of G residues in this tract, resulting in a heterologous bacterial population. Whilst a tract length of 10 nucleotides is permissive for gene expression in N. meningitidis, the overall levels of gene expression are lower than from porA promoters that contain a tract length of 11 nucleotides (the optimum length). Future iterations of recombinant N. lactamica strains expressing PorA will need to either contain non phase variable promoters or have the optimum number of G residues in the homopolymeric tract.
(104) PorA is Surface-Expressed in Recombinant N. lactamica Strain 2Pp7.a
(105) With reference to FIG. 20, both wild type N. lactamica strain Y92-1009 and its recombinant derivative, the PorA (P1.7,16) expressing strain, 2Pp7.A, were cultured to mid-log phase (OD.sub.600nm=0.4) in TSB supplemented with 0.2% yeast extract. Aliquots of each culture containing 110.sup.7 cfu were transferred to fresh microcentrifuge tubes and washed twice in PBS containing 5% foetal calf serum (hereafter, Wash Buffer). Washed bacteria were resuspended into 100 l of Wash Buffer, supplemented with a 1:100 dilution of SM300 MAb and incubated at 4 C. for 30 minutes. The primary Mab was removed by washing twice with 1 ml of Wash Buffer, and the bacteria were resuspended into 100 l of Wash Buffer containing a 1:100 dilution of anti-mouse IgG-AlexaFluor488. The secondary antibody was allowed to bind over the course of 30 minutes at 4 C., before the bacteria were again washed twice in Wash Buffer. After labelling, the bacteria were resuspended into 100 l formalin and were fixed for 10 minutes at room temperature. Once formalin had been removed and the labelled, fixed bacteria washed twice more in Wash Buffer, they were resuspended into 200 l of Wash Buffer and transferred to FACS tubes for quantitative analysis of AlexaFluor488 fluorescence by Flow Cytometry.
(106) The graph shows that the Mean fluorescence intensity of the sample composed of the recombinant strain of N. lactamica (2Pp7.A; BLUE plot), is greater than the Mean fluorescence intensity of the sample composed of wild type N. lactamica (BLACK plot). This indicates surface expression of the target for SM300, which is most plausibly the (P1.7, 16) PorA protein. Together with the data from FIG. 19, these data show that the gene for the meningococcal-derived antigen porA has been successfully introduced into the chromosome of N. lactamica strain Y92-1009, that the PorA protein is associated with the membrane of the bacterium, and that the PorA protein is expressed on the surface of recombinant strain 2Pp7.A.
(107) Promoter Constructs for Investigating Transcriptional Enhancement by Sequence Associated with the N. meningitidis porA Gene
(108) With reference to FIG. 21, in previous work conducted on the expression of the porA gene in N. meningitidis, it was remarked upon that the sequence immediately 5 of the 35 box of the porA promoter bore resemblance to transcriptional enhancer elements, originally characterised in E. coli. These sequences are characterised by multiple poly-A and/or poly-T nucleotide tracts, which confers on those molecules an ability to physically bend. It has been shown that such DNA bending brings distal DNA sequence elements into close proximity, such that proteins bound to these sequence elements are brought together to interact and potentially to form complexes. To investigate the role of a putative porA enhancer sequence on gene expression, a series of plasmids were developed in which the N. lactamica lacZ gene was placed under the transcriptional control of the lst promoter and targeted to NHCIS1 (FIG. 10). Other plasmids in this series were otherwise identical, but also included incrementally larger sequences derived from immediately upstream of the 35 box from the porA promoter in N. meningitidis strain MC58. The length of the putative enhancer sequence preceding and abutted to the lst promoter ranged from 50 bp to 400 bp (GREY text). In the diagram, each promoter and enhancer sequence shows restriction sites as black text against a boxed, grey background, the 10 and 35 RNA Polymerase binding sites as white text against a black background and the lst 5 UTR as lower case letters, the 17 bp separating the 10 and 35 boxes of the lst promoter are underlined. The half site shown, CAT, is from the NdeI restriction site immediately preceding the lacZ coding sequence. The second half of this site is the ATG START codon of the lacZ gene.
(109) With the naked (i.e. unenhanced) lst promoter providing a basal level of lacZ gene activity in the NHCIS1 locus, it was hypothesised that any enhancement to gene expression mediated through the upstream, putative enhancer sequences would lead to increased lacZ gene expression, higher amounts of -galactosidase per bacterium and therefore increased levels of -galactosidase Specific Activity.
(110) Transcriptional Enhancement of the Lst Promoter by Sequence Cloned from Upstream of the porA Gene from Neisseria meningitidis Strain MC58.
(111) With reference to FIG. 22, the series of plasmids containing the N. lactamica lacZ gene under transcriptional control of the lst promoter and incrementally larger, putative enhancer sequences derived from chromosomal sequence immediately 5 to the 35 box of the porA gene of N. meningitidis strain MC58, were used as PCR templates for the generation of donor genetic material with which to transform the lacZ mutant derivative of N. lactamica. Transformation was carried out using hypermethylated PCR product as described in PROTOCOL A. Individual transformants, screened for on the basis of BLUE/WHITE colony formation on X-gal-containing TSB agar plates, were isolated. Chromosomal integration of the lst(X)::lacZ cassettes into the NHCIS1 locus was demonstrated through PCR (data not shown). Following DNA sequencing of the lacZ promoter sequences in this locus, individual clones containing constructs with identical sequences were pooled.
(112) Recombinant N. lactamica strains containing the lst promoter-driven lacZ gene, were cultured in TSB at 37 C., 5% CO.sub.2 and 320 rpm, until reaching mid-log phase (OD.sub.600nm=0.3). Bacteria were pelleted by centrifugation, washed once in sterile PBS then resuspended into 200 l of BLB supplemented with protease inhibitor cocktail, before being lysed through sonication (315 seconds pulses) on ice. Immediately after the last round of sonication, lysates were supplemented to a final concentration of 1 mM PMSF. Cell debris was removed by centrifugation at 17,000 g for 10 minutes ands the supernatant transferred to a fresh microcentrifuge tube. The supernatant was diluted 5 with BLB, and 5, 2 and 1 l aliquots were assayed for (-galactosidase activity using the chromogenic substrate ONPG. The activities in each of these aliquots were averaged to provide a value for each sample in each experiment. The protein concentration of each supernatant was measured using the DC Protein Assay Reagent (Bio Rad) and these values were used to normalise the measured -galactosidase activities (yielding Specific Activity). Values shown are the mean of three independent experiments. Error bars represent the standard error of the mean. Where no error bars are visible, they fall within the line at the top of the column.
(113) These data show that the sequence immediately upstream of the RNA Polymerase binding site of the meningococcal porA gene acts as a transcriptional enhancer. The native lst promoter provides a baseline level of -galactosidase activity when lacZ is expressed from this promoter at the NHCIS1 locus, but the Specific Activity of 3-galactosidase is significantly increased when the upstream sequence of nucleotides is at least 150 bp long and optimal at 200 bp. Further increases to the length of the enhancer sequence disrupt its function in the NHCIS1 context and leads to a reduced Specific Activity in these samples (i.e. at 250 and 400 bp).
(114) Promoter Construct Designed to Investigate the Mechanism of porA Transcriptional Enhancement
(115) With reference to FIG. 23, to investigate whether DNA bending is responsible for the enhancement of gene expression observed in FIG. 22, site-directed mutagenesis using the Q5 site-directed mutagenesis kit (NEB) was employed to introduce 5 additional nucleotides into the junction between the enhancer sequence (GREY text) and the 35 box of the lst(200):lacZ promoter. The additional five residues, along with the ultimate A residue of the enhancer region (shown as white text), constitute a novel restriction site for NsiI in this construct (shown as black text in grey box: ATGCAT). The resulting construct was termed NHCIS1::lst(200+5):lacZ. The presence of an additional 5 nucleotides at the junction of the enhancer and promoter sequences has the effect of turning the enhancer region through half a helix relative to the promoter, given there are 10-11 nucleotide residues per turn of the DNA helix. This hypothetically reverses the directionality of the DNA bending, such that the DNA still bends, but in the opposite direction to the original construct. The consequence of this is that any distal sequence elements and their associated proteins, which would normally be brought into close association with RNA Polymerase and act to enhance open complex formation, would instead be brought into close association with the non-coding strand of the DNA, on the opposite face of the DNA to RNA Polymerase. It was hypothesised that, if the porA enhancer sequence functions through a DNA bending phenomenon, then there will be a significantly lower level of -galatosidase Specific Activity in N. lactamica transformed with the lst(200+5):lacZ construct, as compared to N. lactamica transformed with lst(200):lacZ. In the diagram, restriction sites are shown as black text against a boxed, grey background, the 10 and 35 RNA Polymerase binding sites are shown as white text against a black background and the lst 5 UTR as lower case letters, with the 17 bp separating the 10 and 35 boxes of the lst promoter underlined. The half site shown, CAT, is from the NdeI restriction site immediately preceding the lacZ coding sequence. The second half of this site is the ATG START codon of the lacZ gene.
(116) DNA Bending is at Least Partially Responsible for the Transcriptional Enhancement Activity of the Sequence Upstream of the N. meningitidis porA Gene.
(117) With reference to FIG. 24, the lacZ mutant derivative of N. lactamica strain Y92-1009 and the recombinant N. lactamica strains lst:lacZ, lst(200):lacZ and lst(200+5):lacZ, were cultured in TSB at 37 C., 5% CO.sub.2 and 320 rpm, until reaching mid-log phase (OD.sub.600nm=0.3). Bacteria were pelleted by centrifugation, washed once in sterile PBS then resuspended into 200 l of BLB supplemented with protease inhibitor cocktail, before being lysed through sonication (315 seconds pulses) on ice. Immediately after the last round of sonication, lysates were supplemented to a final concentration of 1 mM PMSF. Cell debris was removed by centrifugation at 17,000 g for 10 minutes and the supernatant transferred to a fresh microcentrifuge tube. The supernatant was diluted 5 with BLB, and 5, 2 and 1 l aliquots were assayed for 3-galactosidase activity using the chromogenic substrate ONPG. The activities in each of these aliquots were averaged to provide a value for each sample in each experiment. The protein concentration of each supernatant was measured using the DC Protein Assay Reagent (Bio Rad) and these values were used to normalise the measured 3-galactosidase activities (yielding Specific Activity). Values shown are the mean of three independent experiments. Error bars represent the standard error of the mean. Where no error bars are visible, they fall within the line at the top of the column.
(118) These data show that the addition of 5 extra nucleotides at the junction of the 200 nucleotide-long porA transcriptional enhancer and the 35 box of the lst promoter reduces by approximately 50% the Specific Activity of -galactosidase measured in bacterial lysates. The graph shows that the addition of the enhancer sequence to the 5 end of the lst promoter results in a large increase in -galactosidase Specific Activity, as compared to bacteria expressing lacZ from the naked (i.e. unenhanced) lst promoter. Despite being otherwise identical to the lst(200):lacZ construct, the presence of 5 extra nucleotides in the lst(200+5):lacZ construct significantly reduces the Specific Activity of -galactosidase, indicating less transcriptional activity from this promoter construct.
(119) Annotated Nucleotide Sequence of HAEC3.
(120) With reference to FIG. 25, this Cassette has been designed to enable non-phase variable, high level gene expression of heterologous antigen. The sequence consists of a modified N. meningitidis porA promoter, wherein the phase variable, 17 bp tract that separates the 10 and 35 boxes in the wild type porA promoter has been replaced with 17 bp of sequence identical to that which separates the 10 and 35 boxes of the N. lactamica porB promoter. The 35 box of the modified porA promoter is preceded by 250 bp of the porA-derived transcriptional enhancer sequence (FIG. 22), and most distally a SalI restriction site, to facilitate cloning and replacement of the promoter sequence (when used in conjunction with the XhoI restriction site). The modified, optimally-enhanced porA promoter is separated from the 3, modified N. lactamica porB promoter sequence by an XhoI restriction site, an interchangeable LINKER sequence and a NotI restriction site. It is important to note that either LINKER sequence can be any given nucleotide sequence (represented as poly-N). It is also important to note that the 17 bp sequence separating the 10 and 35 boxes of the RNA Polymerase binding site in this porB promoter have been replaced by 17 bp of sequence derived from the lst promoter. This is to avoid recombination between identical sequences within the Cassette that might otherwise lead to loss of the gene coding for heterologous antigen. By including an XhoI restriction site immediately 5 of the START codon and a NotI restriction site immediately 3 of the STOP codon of any given coding sequence (e.g. through the use of extended primer sequences that incorporate these sites), that sequence can be directionally cloned, IN-FRAME into the Construct for expression from the modified, optimally-enhanced porA promoter. The featured NdeI restriction site can be used in conjunction with the HindIII restriction site present immediately 5 of the porB terminator sequence to clone any given coding sequence into the Construct. By including an NdeI restriction site immediately 5 of the START codon and a HindIII restriction site immediately 3 of the STOP codon of any given coding sequence (e.g. through the use of extended primer sequences that incorporate these sites), that sequence can be directionally cloned, IN-FRAME into the Construct for expression from the lst promoter. Immediately after the HindIII restriction site, there is a transcriptional terminator sequence, derived from downstream of the N. lactamica porB gene. This is immediately followed by an AT-DUS. In the context of heterologous antigen expression in recombinant Neisseria lactamica, HAEC3 is flanked on either side by sequences homologous to the N. lactamica chromosome (e.g. NHCIS1).
(121) Annotated Nucleotide Sequence of HAEC4.
(122) With reference to FIG. 26, this Cassette has been designed to enable non-phase variable, high level gene expression of heterologous antigen. The sequence consists of a modified N. meningitidis porA promoter, wherein the phase variable, 17 bp tract that separates the 10 and 35 boxes in the wild type porA promoter has been replaced with 17 bp of sequence identical to that which separates the 10 and 35 boxes of the N. lactamica porB promoter. The 35 box of the modified porA promoter is preceded by 200 bp of the porA-derived transcriptional enhancer sequence (FIG. 22), and most distally a SalI restriction site, to facilitate cloning and replacement of the promoter sequence (when used in conjunction with the XhoI restriction site). The modified, optimally-enhanced porA promoter is separated from the 3 N. lactamica lst promoter sequence by an XhoI restriction site, an interchangeable LINKER sequence and a NotI restriction site. It is important to note that either LINKER sequence can be any given nucleotide sequence (represented as poly-N). By including an XhoI restriction site immediately 5 of the START codon and a NotI restriction site immediately 3 of the STOP codon of any given coding sequence (e.g. through the use of extended primer sequences that incorporate these sites), that sequence can be directionally cloned, IN-FRAME into the Construct for expression from the modified, optimally-enhanced porA promoter. The featured NdeI restriction site can be used in conjunction with the HindIII restriction site present immediately 5 of the porB terminator sequence to clone any given coding sequence into the Construct. By including an NdeI restriction site immediately 5 of the START codon and a HindIII restriction site immediately 3 of the STOP codon of any given coding sequence (e.g. through the use of extended primer sequences that incorporate these sites), that sequence can be directionally cloned, IN-FRAME into the Construct for expression from the lst promoter. Immediately after the HindIII restriction site, there is a transcriptional terminator sequence, derived from downstream of the N. lactamica porB gene. This is immediately followed by an AT-DUS. In the context of heterologous antigen expression in recombinant Neisseria lactamica, HAEC4 is flanked on either side by sequences homologous to the N. lactamica chromosome (e.g. NHCIS1).
(123) Plasmid Map of pUC19NHCIS1::HAEC4:nadA-lacZ
(124) With reference to FIG. 27, the pUC19NHCIS1::HAEC4:nadA-lacZ plasmid is a derivative of pUC19NHCIS1::HAEC2:porAplusprom-lacZ (Clone #7) (FIG. 18), wherein the majority of the plasmid architecture is identical to pUC19NHCIS1::HAEC2:porAplusprom-lacZ; but where the wild type, non-optimally enhanced porA promoter and the porA coding sequence have been replaced by the optimally enhanced, modified porA/porB hybrid promoter and a N. lactamica codon-optimised version of the nadA gene. The nadA gene codes for the membrane associated adhesin, Neisseria Adhesin A, one of the four immunogenic components of the Bexsero anti-meningococcal serogroup B vaccine. The hybrid promoter and the nadA coding sequence were synthesised as a gBLOCK gene fragment (Integrated DNA Technologies) and cloned into the SalI-NotI double-digested plasmid vector using Gibson Assembly (NEB). The plasmid map is presented showing all relevant features and detailing the location of unique restriction sites with recognition sequences 6 nucleotides or greater.
(125) Plasmid Map of pUC19NHCIS1::HAEC4:(Z)-lacZ
(126) With reference to FIG. 28, the pUC19NHCIS1::HAEC4:(Z)-lacZ plasmid is a derivative of pUC19NHCIS1::HAEC4:nadA-lacZ, wherein the nadA coding sequence has been replaced by a 14 bp LINKER sequence. To create this plasmid, the nadA coding sequence was excised from pUC19NHCIS1::HAEC4:nadA-acZ using PCR and the plasmid re-circularised using the KLD enzyme mix from the Q5 site-directed mutagenesis kit (NEB). The plasmid map is presented showing all relevant features and detailing the location of unique restriction sites with recognition sequences 6 nucleotides or greater.
(127) NadA-Expressing N. lactamica have Increased Adherence to HEP-2 Cells.
(128) With reference to FIG. 29, the pUC19NHCIS1::HAEC4:nadA-lacZ and pUC19NHCIS1::HAEC4:(Z)-lacZ plasmids were used as templates for hypermethylated PCR. Hypermethylated donor genetic material was used to transform the lacZ mutant derivative of N. lactamica strain Y92-1009 as described in PROTOCOL A. Individual transformants, screened for on the basis of BLUE/WHITE colony formation on X-gal-containing TSB agar plates, were isolated. Chromosomal integration of either the HAEC4:(Z)-lacZ or HAEC4:nadA-lacZ cassettes into the NHCIS1 locus was demonstrated through PCR (data not shown). Following DNA sequencing of this locus, NadA-expressing N. lactamica strain B1 (hereafter: 4NB1) was determined to contain genetic material with 100% identity to the nadA gene, the modified porA/porB hybrid promoter and porA enhancer sequence. NB: Transformation with the HAEC4:(Z)-lacZ cassette provides a procedural control and a transformed derivative (hereafter: 4YB2) that contains identical elements to 4NB1, but without the nadA coding sequence.
(129) Two days prior to infection, HEP-2 cells were seeded into 24 well plates at a density of 210.sup.5 cells/well. HEP-2 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% Foetal Calf Serum (FCS) at 37 C., 5% CO.sub.2. On the morning of infection, duplicate wells of cells were treated with trypsin/EDTA and the average number of cells per well was measured by counting using a haemocytometer. Wild type N. lactamica strain Y92-1009, the NadA-expressing strain 4NB1 and the control strain 4YB2 were cultured in TSB at 37 C., 5% CO.sub.2 and 320 rpm, until reaching mid-log phase (OD.sub.600nm=0.3). Aliquots of each culture were isolated, pelleted by centrifugation and then resuspended in fresh DMEM+FCS. The final concentration of bacteria was adjusted so that 500 l of DMEM contained sufficient bacteria to infect cells at MOI=100. At t=ZERO, HEP-2 cells were washed twice in sterile PBS, then infected at MOI=100 with the relevant bacterial strain. Plates were transferred to the incubator and cultured at 37 C., 5% CO.sub.2 until the appropriate time point. At t=2 h, 4 h and 6 h the plates were removed from the incubator, supernatants were carefully aspirated and each well washed 5 with excess, sterile PBS. The final two washes were accompanied by complete aspiration of liquid from the well. A 250 l aliquot of a 2% saponin solution in PBS was added to each well and the plates were again incubated for 15 minutes at 37 C., 5% CO.sub.2. To fully disrupt the HEP-2 monolayers and disperse adherent/internalised bacteria, 750 l of sterile PBS was added to each well and the cells were mechanically agitated through pipetting. The diluted lysate was serially diluted 10-fold in PBS, and the number of viable bacteria determined by plating onto Columbia Blood Agar (CBA) plates. The viability of each lysate was normalised to the estimated number of HEP-2 cells per well.
(130) The graph shows the number of HEP-2 adherent bacteria of each different strain recovered from infected wells at 2 h, 4 h and 6 h. At all time points there is a trend for the transformed strain, that which putatively expresses the NadA adhesin (4NB1), to bind more readily to HEP-2 cells than either the wild type strain or the transformation control strain (4YB2). Over time, this difference becomes more pronounced, but is similarly more variable. Whether this simple difference in adherence will reflect a higher propensity of this strain to colonise the human nasopharynx has yet to be determined.
(131) Plasmid Map of pSC101NHCIS1::HAEC4:opcA-lacZ
(132) With reference to FIG. 30, each of the pUC-derived plasmids used in this series has a large copy number (50-300 copies per bacterial cell). Whilst this simplifies the harvest of these plasmids, the high copy number may also result in toxicityespecially if the plasmid codes for a genetic system designed to express high levels of a particular product. The metabolic burden to E. coli transformed with these plasmids perhaps renders them non-viable, meaning that the only plasmids recovered during transformation are those with mutations in either the coding sequence or the promoter region. One potential solution to the problem of gene dose-mediated toxicity is to change the plasmid's origin of replication. By substituting the pUC origin of replication (modified pMB1) for the minimally-required region for propagation of plasmid pSC101 (repA/ori), the derivative plasmids will have a copy number of only 5 in daughter cells. It is hypothesised that the propensity for mutated plasmids to be selected for will be lower in the pSC101-based plasmids than the pUC19-based ones, increasing the frequency with which high fidelity plasmids containing the correct sequence for the gene of interest are recovered.
(133) The pSC101NHCIS1::HAEC4:opcA-lacZ plasmid contains a N. lactamica codon-optimised version of the opcA gene under the control of the optimally-enhanced, modified porA/porB hybrid promoter. The plasmid is designed for use as a template in hypermethylated PCR, so as to generate donor genetic material suitable for the transformation of N. lactamica according to PROTOCOL A. The opcA and lacZ genes are targeted to NHCIS1. The plasmid map is presented showing all relevant features and detailing the location of unique restriction sites with recognition sequences 6 nucleotides or greater.
(134) Plasmid Map of pUC19Nhba::HAEC1:(Z)-lacZ
(135) With reference to FIG. 31, the pUC19nhba::HAEC1:(Z)-lacZ Construct comprises many features identical to those of pUC19NHCIS1::HAEC1:(Z)-lacZ (FIG. 14), but where the sequences homologous to the NHCIS1 locus are replaced by sequences homologous to the N. lactamica nhba gene (alternatively, gna2132 OR NLY_32180). The genome of N. lactamica strain 020-06 contains the gene: NLA_20270, which codes for the putative lipoprotein GNA2132, otherwise known as the Neisseria Heparin Binding Antigen (NHBA). NHBA is one of the four immunogenic components of the 4CMenB (Bexsero) vaccine. The genome of N. lactamica strain Y92-1009 contains the NLY_32180 open reading frame, which has an 87.5% similarity with NLA_20270 at the nucleotide sequence level, and codes for a protein with the characteristic, arginine-rich region of NHBA. Therefore NLY_32180 likely codes for the Y92-1009 homologue of NHBA.
(136) The pUC19nhba::HAEC1:(Z)-lacZ Construct was designed as the first step in of a two-step strategy to truncate the coding sequence (and therefore effectively delete) the NHBA homologue from the lacZ derivative of Y92-1009, without the need for a screening/selection marker being present in the final strain. Initially, lacZ N. lactamica were transformed with the nhba::HAEC1:(Z)-lacZ Cassette according to PROTOCOL A, which disrupted the nhba gene with a functional copy of the N. lactamica lacZ gene. Successful transformants were screened for on medium containing X-Gal in accordance with PROTOCOL B, and grew as blue colonies. Successful transformants were verified by PCR of the nhba locus and had the genotype: lacZ nhba::HAEC1:(Z)-lacZ. One of these strains provided the background for the second transformation event, which used the nhba Cassette, amplified from pUC19nhba (FIG. 32), to remove the lacZ coding sequence and replace the nhba gene with a truncated version of itself (nhba). The plasmid map is presented showing all relevant features and detailing the location of unique restriction sites with recognition sequences 6 nucleotides or greater.
(137) Plasmid Map of pUC19Nhba
(138) With reference to FIG. 32, the pUC19nhba Construct comprises a truncated copy of the N. lactamica nhba gene cloned into a modified, truncated version of pUC19. The pUC19nhba Construct was designed as the second step in a two-step strategy to truncate the coding sequence (and therefore effectively delete) the NHBA homologue from the lacZ derivative of Y92-1009, without the need for a screening/selection marker being present in the final strain. The nhba Cassette was amplified from this plasmid and transformed into lacZ nhba::HAEC1:(Z)-lacZ N. lactamica according to PROTOCOL A. Successful transformants were screened for on medium containing X-Gal in accordance with PROTOCOL B, and grew as white colonies. Successful transformants were verified by PCR of the nhba locus and had the genotype: lacZ nhba. These strains will provide the background for subsequent transformation events, wherein components of the 4CMenB (Bexsero) vaccine will be expressed in N. lactamica in a way suitable for future use of those strains in human challenge. The plasmid map is presented showing all relevant features and detailing the location of unique restriction sites with recognition sequences 6 nucleotides or greater.
(139) Repeated Transformation of N. lactamica does NOT Select for a More Transformable Phenotype.
(140) With reference to FIG. 33, a justified cause for concern in using genetically modified N. lactamica in a human challenge model is whether recombinant derivatives are more readily transformable than their parent cells. A higher propensity to become transformed by genetic material from the nasopharyngeal milieu means an increased risk of recombinant N. lactamica taking up and integrating into its genome the means to synthesise extracellular capsule, which could transition the organism from harmless commensal to potential pathogen. If the process of transforming N. lactamica also unintentionally selects for an enhanced inherent transformability, then the serial transformation of strains is likely to result in increased transformation efficiency under experimental conditions.
(141) In creating a nhba mutant in N. lactamica, bacteria were serially transformed a total of three times. The first transformation removed the coding sequence for -galactosidase (lacZ) from the wild type organism (to produce lacZ). The lacZ strain provided the background for the knock-out of the nhba gene, which was achieved using a two-step approach. The first step was the introduction of the HAEC1:(Z)-lacZ cassette) into the nhba chromosomal locus (to produce lacZ nhba::HAEC1:(Z)-lacZ). The HAEC1 cassette was subsequently removed from sequence-verified transformants, along with a large section of the nhba coding sequence (to produce lacZ nhba). Each of the above steps was dependent upon BLUE/WHITE colony formation on X-gal-supplemented TSB agar plates.
(142) Each of the above strains was experimentally transformed with 0.46 pmol of hypermethylated PCR product, amplified from pUC19nlaII:aphA3 (FIG. 4). The number of transformants was considered to be equal to the number of kanamycin-resistant colonies that grew overnight on selective agar plates (TSB+0.2% yeast extract supplemented with 50 g/ml kanamycin), adjusted for dilution and plating volume. The graph shows that there are no significant INCREASES in the transformation efficiencies of any of the serially-transformed strains, which suggests that we are not inadvertently selecting for an inherently more transformable phenotype.
(143) OMV Production from Neisseria meningitidis and Neisseria lactamica
(144) Note: Neisseria meningitidis is a BSL-2 organism that is spread via creation of aerosols and droplets. Infection rate among laboratory workers is very low, estimated to be 13/100,000, but is approximately 10-fold higher than the general population. Mortality among infected workers is approximately 50%. Historical cases of laboratory-acquired infection are traceable to manipulation of the organism on the open bench by clinical microbiologists. Therefore, all manipulations of the live organism, including sub-culturing of colonies from plates, must only be performed within a class II containment hood.
(145) Effect of the Amount of Donor Material Used to Transform Wild Type Neisseria lactamica: Supplemental.
(146) With reference to FIG. 34, wild type N. lactamica strain Y92-1009 was transformed with a broad range of dilutions of a hypermethylated PCR construct amplified from pUC19nlaII:aphA3, (nlaII:aphA3(600)) according to Protocol A. The number of transformants was considered to be equal to the number of kanamycin resistant colonies evident after overnight growth on selective media (TSB agar supplemented with 50 g/ml kanamycin), corrected for dilution factor and plating volume. The total number of viable bacteria was determined by tenfold serial dilution of each suspension of putatively transformed N. lactamica. This graph shows a strong relationship between the amount of donor material and the frequency of transformation of wild type N. lactamica. The relationship appears to be linear over the dilution range (0.05 pmol-0.000005 pmol), but appears to approach saturation when transforming using 0.5 pmol of donor material. This is consistent with FIG. 8 (B), wherein small changes in the amount of available donor material when provided in relatively large quantities (0.06 pmol-1 pmol) do not appear to appreciably impact the frequency of Nlac transformation. Points represent the MeanSEM of six biological replicates.
(147) Plasmid Map of pSC101::lacZ:opcA
(148) With reference to FIG. 35, the vector component of this plasmid, comprising the origin of replication (repA/ori) from pSC101 and the ampicillin resistance gene and M13 primer binding sites of pUC19, is identical to that of pSC101NHCIS1::HAEC4:opc-lacZ (FIG. 30). The pSC101::lacZ:opcA plasmid contains an N. lactamica codon-optimized version of the opcA gene (eg. NMB1053) under the control of the optimally enhanced, modified porA/porB hybrid promoter and flanked on either side by sequences derived from the 5 and 3 ends of the Nlac lacZ gene. The purpose of the lacZ:opcA construct is to introduce the opcA gene into the coding sequence of the lacZ gene, which was itself introduced into the NHCIS1 locus along with the nadA gene (using the NHCIS1::HAEC4:nadA-lacZ constructFIG. 27). Transformation of N. lactamica strain 4NB1 with this construct generated the strain lacZ NHCIS1::HAEC4:nadA-lacZ:opcA, which contained chromosomal sequences with 100% identity to both the porA(P1.7,16) and opcA genes and grew as WHITE colonies on X-gal-containing TSB agar plates. The plasmid map is presented showing all relevant features and unique restriction sites.
(149) Plasmid Map of pSC101::lacZ-Synth.lacZ-3ENDNHCIS1
(150) With reference to FIG. 36, the vector component of this plasmid, comprising the origin of replication (repA/ori) from pSC101 and the ampicillin resistance gene and M13 primer binding sites of pUC19, is identical to that of pSC101NHCIS1::HAEC4:opc-lacZ (FIG. 30). The pSC101::lacZ-synth.lacZ-3ENDNHCIS1 plasmid contains a synthetic version of the lacZ gene (synth.lacZ), in which the coding sequence has been adjusted to maximize its diversity from the endogenous Nlac version of the lacZ gene at the nucleotide sequence level, whilst maintaining the fidelity of the amino acid sequence of -galactosidase (see FIG. 37). The synth.lacZ gene is flanked at its 5 end by nucleotide sequence identical to the 3 end of the endogenous Nlac version of the lacZ gene, and at its 3 end by nucleotide sequence identical to the 3 end of NHCIS1. The purpose of the lacZ-synth.lacZ-3ENDNHCIS1 construct is to introduce a functional copy of the lacZ gene back into the chromosome of Nlac strain lacZ NHCIS1::HAEC4:nadA-lacZ:opcA, so that all strains in the NadA/Opc mutant panel retain -galactosidase activity. Transformation of N. lactamica strain lacZ NHCIS1::HAEC4:nadA-lacZ:opcA with this construct generated the strain, lacZ NHCIS1::HAEC4:nadA-lacZ:opcA-lacZ (hereafter, 4NOA1), which contained chromosomal sequences with 100% identity to both the porA(P1.7,16) and opcA coding sequences, and grew as BLUE colonies on X-gal-containing TSB agar plates. The plasmid map is presented showing all relevant features and unique restriction sites.
(151) Coding Sequence of Synth.lacZ
(152) With reference to FIG. 37, this coding sequence has been designed to maximize diversity from the nucleotide sequence of the endogenous Nlac version of lacZ, whilst maintaining the fidelity of the amino acid sequence of -galactosidase (CAI: 0.687). These adjustments were necessary in order to minimize the likelihood of the synthetic lacZ gene undergoing homologous recombination with the remaining lacZ fragments left in the NHCIS1 locus of Nlac strain lacZ NHCIS1::HAEC4:nadA-lacZ:opcA, which would have most probably resulted in the excision of the opcA gene from the chromosome. Where nucleotides have been manually substituted in order to increase nucleotide sequence diversity, they are in lower case.
(153) Expression of NadA and Opc Outer Membrane Proteins on the Surface of Recombinant Strains of N. lactamica
(154) With reference to FIG. 38, wild type N. lactamica strain Y92-1009 and its recombinant derivatives, the putatively NadA-expressing strain, 4NB1 and the putatively Opc-expressing strain, 4OA2, were cultured to mid-log phase (OD.sub.600nm=0.4) in TSB supplemented with 0.2% yeast extract. Aliquots of each culture containing 210.sup.7 CFU were transferred to fresh microcentrifuge tubes and washed twice in Wash Buffer. Washed bacteria were resuspended into 100 l of Wash Buffer, supplemented with either a 1:200 dilution of anti-NadA monoclonal antibody, 6E3 (WT and 4NB1) or a 1:50 dilution of anti-Opc monoclonal antibody, 279/5c (WT and 4OA2) and incubated at 4 C. for 30 minutes. The primary mAbs were removed by washing twice with 1 ml of Wash Buffer, and the bacteria were resuspended into 100 l of Wash Buffer containing a 1:100 dilution of anti-mouse IgG-AlexaFluor488 (1.5 mg/ml). The secondary antibody was allowed to bind over the course of 30 minutes at 4 C., before the bacteria were again washed twice in Wash Buffer. After labelling, the bacteria were resuspended into 100 l formalin and were fixed for 10 minutes at room temperature. Once formalin had been removed and the labelled, fixed bacteria washed twice more in Wash Buffer, they were resuspended into 200 l of Wash Buffer and transferred to FACS tubes for quantitative analysis of AlexaFluor488 fluorescence by Flow Cytometry.
(155) The graphs in this figure show that, in both instances, the Mean fluorescence intensity of the samples composed of the recombinant strains of N. lactamica (either 4NB1, GREEN plot or 4OA2, BLUE plot), are greater than the Mean fluorescence intensity of the samples composed of wild type N. lactamica (RED plots). This indicates surface expression of the targets for 6E3 and 279/5c mAbs, which are most plausibly the NadA and Opc outer membrane proteins, respectively. Combined with the binding and internalisation data collected from analysis of the interaction of these recombinant Nlac strains with HEP-2 cells (FIG. 40) and (in the case of 4NB1) human nasopharyngeal tissue explants (FIG. 41), these graphs suggest the NadA and Opc outer membrane proteins are functionally expressed on the surface of recombinant Nlac strains 4NB1 and 4OA2, respectively.
(156) Expression of NadA, Opc or a Combination of Both Antigens in Recombinant N. lactamica has No Appreciable Effect on Growth Rates in TSB
(157) With reference to FIG. 39, it is plausible that expression of one or more outer membrane protein(s) in additional to the usual complement of Nlac proteins, might constitute a metabolic burden for recombinant strains of N. lactamica. A significant metabolic burden might manifest as impairment in the growth characteristics of the strains as compared to wild type. To investigate this, wild type N. lactamica strain Y92-1009 and the mutant derivatives thereof, 4NB1, 4OA2, 4NOA1 and 4YB2 were cultured in TSB and the OD.sub.600nm and viability of each culture was measured hourly. Area Under Curve analyses of these data show that there are no significant differences between the growth rates or viability of these strains, suggesting that expression of additional outer membrane protein(s) do(es) not constitute a significant metabolic burden in rich medium. Points represent MeanSD of four biological replicates, where no bars are visible, they fall within the points.
(158) NadA-Expressing Strains of N. lactamica have Increased Adherence to HEP-2 Cells, Whilst Opc-Expressing Strains of N. lactamica are Internalized by HEP-2 Cells in Significantly Larger Numbers
(159) With reference to FIG. 40, wild type N. lactamica strain Y92-1009 and the recombinant, (meningococcal adhesin-expressing) derivatives thereof: 4NB1, 4OA2, 4NOA1 and 4YB2 were grown to mid-log phase, washed, resuspended into Dulbecco's modified Eagles medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and used to infect duplicate confluent monolayers of HEP-2 epithelial cells at a multiplicity of infection (MOI) of 100 (i.e. 100 bacteria per HEP-2 cell). Infected cells were incubated at 37 C., 5% CO2 and samples were processed every 2 h for a total of 6 h. Thirty minutes prior to each sampling, the infected supernatant was removed from one of each pair of duplicates and the HEP-2 cells were gently washed with sterile PBS. Pre-warmed (37 C.) DMEM+10% FBS, supplemented with 100 g/ml gentamicin and g/ml penicillin G was then added to each aspirated well and the plates returned to the incubator, with the intention of killing all bacteria adherent to the surface of the cells. At the appropriate time points, all media was aspirated from the HEP-2 cells, which were subsequently washed 5 times with an excess of sterile PBS. Following aspiration of the final wash, 250 l of a 2% saponin solution in PBS was added to each well and, after 15 minutes incubation at 37 C. and repeated pipetting to break up the HEP-2 monolayers, was supplemented with 750 l of sterile PBS (yielding all HEP-2 associated bacteria, or all HEP-2 internalised bacteria from a given well in a total volume of 1 ml). These suspensions were serially diluted in sterile PBS and the number of viable CFU enumerated on CBA agar. Viable counts were normalized to the number of HEP-2 cells present in each well. These data show that: (A). By 4 h of infection (light gray bars), Nlac strains expressing the meningococcal adhesin NadA on their surface (4NB1 and 4NOA1) associate in significantly higher numbers with HEP-2 cells than do any of the other strains examined. The wild type parental strain (WT), the Opc-expressing strain (4OA2) and the transformation procedure-control strain (4YB2) all associate in similar numbers with HEP-2 cells at every time point studied, but in consistently lower numbers when compared to both 4NB1 and 4NOA1. For all strains, these data suggest that the binding capacity of the HEP-2 cells becomes saturated at or before 4 h of infection, but that a significantly larger population of the NadA-expressing strains can associate with this cell line. This likely indicates the existence of an as-yet unidentified receptor for NadA binding on the HEP-2 cell surface, which is not bound by the surface armamentarium of wild type Nlac. *p0.05, RM 2-way ANOVA with Tukey's Multiple Comparisons test, n=4. (B). By 6 h of infection (dark gray bars, also shown in isolation in blow-out below main graph), the Nlac strain expressing the meningococcal adhesin Opc on its surface (4OA2) becomes internalized by HEP-2 cells in significantly higher numbers than every other strain examined except for 4NOA1. Although 4NOA1 also expresses Opc along with NadA, the internalization of this strain by HEP-2 cells is not significantly different from that of any other strain examined. Whether the presence of NadA in addition to Opc somehow interferes with the internalization process is not yet clear, although the relative expression levels of each adhesin on the surface of these bacteria, as compared to strains expressing one or the other protein, have not yet been determined. Bars represent MeanSD; *p0.05, ***p0.001 and ****p0.0001, RM 2-way ANOVA with Tukey's Multiple Comparisons test, n=4.
(160) The NadA-Expressing Strain of N. lactamica, 4NB1, is Recovered in Significantly Higher Numbers from within Human Nasopharyngeal Tissue Explants than is the Wild Type
(161) With reference to FIG. 41, wild type N. lactamica strain Y92-1009 and the recombinant, NadA-expressing derivative thereof, 4NB1 were grown to mid-log phase, washed, resuspended in sterile PBS and used to infect the epithelium of agarose-mounted, transwell-suspended nasopharyngeal tissue explants derived from human turbinets from consenting donors. Each strain was used to infect four replicate explants. Explants were incubated at 37 C., 5% CO2 for 24 h, after which they were carefully removed from the agarose, weighed and processed. Duplicate explants were washed either three times in sterile PBS by vortexing (30 seconds per wash) or were vortexed for 30 seconds in a 0.2% solution of sodium taurocholate (bile salts) in PBS, followed by an additional 2 washes in sterile PBS. The wash in bile salts effectively sterilizes the surface of the explant, isolating the population of viable bacteria that have penetrated the epithelium (i.e. sub-epithelial bacteria). Each explant was subsequently disrupted into 1 ml of a sterile 2% saponin solution in PBS, using a high-pressure, one-shot tissue disruptor. The resultant cell suspension was incubated for 15 minutes at 37 C., 5% CO2 to release any intracellular bacteria. An aliquot of each saponised lysate was serially diluted and plated on CBA agar to enumerate viable bacteria (CFU/ml). Viable counts were normalized to the weight of each explant (g). Points on each graph represent the average of values derived from duplicate explants. Paired Explants (derived from the same donor but infected with different bacterial strains) are joined by dotted lines. Although there is no significant difference between the total number of wild type or 4NB1 bacteria associated with the explants (i.e. surface-bound and those within the body of the explant), a significantly larger number of CFU were recovered from inside explants infected with strain 4NB1 than with wild type Nlac (*p=0.0313, Wilcoxon matched pairs signed rank test, n=5). These data suggest that although surface expression of meningococcal NadA by N. lactamica does not significantly enhance the ability of the strain to bind to the epithelial surface of nasopharyngeal explants compared to wild type, a significantly larger number of 4NB1 bacteria are able to penetrate the epithelium. Whether these sub-epithelial bacteria are intracellular or not remains to be elucidated; however, given the existence of sub-epithelial Neisseria microcolonies in tonsillar crypts (Sim et al, 2000 Research Letters| Volume 356, Issue 9242, P1653-1654), we hypothesize that an increased propensity to cross the nasopharyngeal epithelium ex vivo may impact upon the colonisation dynamics of this recombinant strain in human challenge. Penetration of the mucosal epithelium could plausibly mean the bacteria are protected from elements of the innate immune system operating at the mucosal service, effectively sheltering the strain from killing and promoting its persistence in a given human host. However, we must be mindful that the sub-epithelial location of these bacteria might preclude their recovery by swabbing of the nasopharynx, leading to false negative results in terms of an individual's colonization status.
(162) Recombinant Strains of N. lactamica Expressing Meningococcal Adhesin Proteins have Similar Sensitivity Profiles to Front-Line Antibiotics as the Wild Type
(163) With reference to FIG. 42, and in the context of potentially using these strains in experimental human challenge, it is important to determine whether the process of creating recombinant strains of N. lactamica has decreased their sensitivity to clinically relevant antibiotics. Although the expectation is that these strains pose no threat to human health, the ability to kill these bacteria using front-line antibiotics should it become necessary needs to be assessed. Wild type N. lactamica strain Y92-1009 and recombinant Nlac strains 4NB1, 4OA2, 4NOA1 and 4YB2 were cultured in TSB to mid log phase. Sterile cotton swabs were saturated with these suspensions, which were then used to inoculate the surface of TSB agar plates supplemented with 5% horse blood in such a way as to generate a confluent lawn of bacteria after overnight growth. Onto the surface of each inoculated plate, an E-test strip was placed containing one of rifampicin, ciprofloxacin or ceftriaxone across a range of concentrations. The minimum inhibitory concentration (MIC) of each antibiotic was determined to be the concentration at which the bacterial lawn failed to develop following overnight growth, characterized by an area of clearance surrounding the E-test strip (Table 1). With reference to Table 2, which details the MIC and antibiotic sensitivity levels of the pathogenic species of Neisseria to each of these antimicrobials, it is evident that each strain remains sensitive to these antibiotics. These data demonstrate that the process of generating our recombinant strains of N. lactamica has not significantly altered the susceptibility profile of these bacteria to the antimicrobial agents used to treat infections of the pathogenic Neisseria species.
(164) Recombinant Strains of N. lactamica Expressing Meningococcal Adhesin Proteins are Competent for Uptake of Exogenous DNA and can be Transformed with Efficiencies Similar to the Wild Type
(165) In reference to FIG. 43, wild type N. lactamica strain Y92-1009 and recombinant Nlac strains 4NB1, 4OA2, 4NOA1 and 4YB2 were transformed with 0.5 pmol of hypermethylated PCR construct: nlaII:aphA3(600), as described in Protocol A. The number of transformants was considered to be equal to the number of kanamycin-resistant colonies that grew overnight on selective agar plates (TSB+0.2% yeast extract supplemented with 50 g/ml kanamycin), adjusted for dilution factor and plating volume. This experiment was performed to determine whether serial transformation of N. lactamica had inadvertently selected for a more transformable phenotype compared to the WT strain. This is important, as our argument that recombinant Nlac is safe for use in human challenge experiments is predicated on the fact that they are no more likely to assimilate genetic information from the environment than the parental strain. An increased propensity to take up exogenous DNA could plausibly translate into an increased likelihood to assimilate capsule synthesis genes from the nasopharyngeal milieu, with the potential to transform commensal strains into facultative pathogens. Strains 4NB1, 4OA2 and 4YB2 have each undergone two transformations from the wild type background, whereas strain 4NOA1 has been transformed a total of four times. These data show no significant differences in the transformation efficiency between any of the strains investigated and is consistent with the data presented in FIG. 33. This implies that isolation of serially-transformed bacteria does not also select for an inherently more transformable phenotype.
(166) Wild Type and Recombinant Strains of N. lactamica are Completely Refractory to Transformation by Chromosomal DNA Derived from N. meningitidis
(167) In reference to FIG. 44, the wild type meningococcal strain MC58, along with the wild type N. lactamica strain Y92-1009 and the recombinant derivatives thereof, 4NB1, 4OA2 and 4NOA1, were exposed to 110.sup.4 pmol of chromosomal DNA isolated from four mutant strains of Neisseria, as described in Protocol A. 1. Chromosomal DNA was isolated from the Nlac mutant derivative of Y92-1009 containing an insertionally-inactivated copy of the nlaIII gene (nlaIII) and mutant derivatives of MC58 containing insertionally-inactivated copies of the opc, nadA and siaD genes, (opc, nadA and siaD, respectively). All insertionally-inactivated meningococcal genes contained the full coding sequence of the appropriate gene, disrupted by identical copies of the kanamycin resistance gene, aphA3, transcriptionally controlled by the Nlac lst gene promoter. The same antibiotic resistance-conferring marker was used to disrupt the nlaIII coding sequence. The number of transformants was considered to be equal to the number of kanamycin-resistant colonies that grew overnight on selective agar plates (Columbia agar supplemented with horse blood and saturated then allowed to dry with 3 ml of a 2 g/ml kanamycin solution), adjusted for dilution factor and plating volume.
(168) This experiment was designed to determine the propensity of (recombinant) Nlac strains to take up and become transformed with chromosomal DNA from a meningococcal donor, which is the most plausible source of capsule synthesis genes these strains might encounter in the nasopharyngeal milieu. As such, all the strains were exposed to chromosomal DNA derived from MC58 siaD. Because Nlac does not express capsule, and instead contains the capsule null locus (cnl) in a position homologous to the capsule synthesis locus in Nmen, the incorporation of the insertionally-inactivated siaD gene into the Nlac chromosome would represent a de novo, untargeted recombination event and could theoretically occur at any locus. This is also true for all the recombinant Nlac strains. However, each of the recombinant strains contains at least one additional region of similarity to the meningococcal chromosome not present in the WT strain, i.e. the opc and nadA genes incorporated into NHCIS1. Both coding sequences for the Nlac versions of opc and nadA are approximately 80% similar to the appropriate MC58 homologue, which could plausibly facilitate homologous recombination of meningococcal DNA into the Nlac genome. As such, strains 4NB1 and 4OA2 were exposed to DNA derived from MC58 nadA and MC58 opc, respectively, whilst strain 4NOA1 was exposed to DNA derived from both of these sources. Because we hypothesize that exogenous, non-Nlac derived chromosomal DNA taken up into N. lactamica will be degraded by potent restriction endonuclease activities (specifically, NlaIII), it was important to demonstrate that each of the Nlac strains in this experiment were actually competent for DNA uptake and homologous recombination. As such, each Nlac strain was also exposed to chromosomal DNA isolated from Y92-1009 nlaIII, which reproducibly transformed wild type Nlac with high efficiency in preliminary experiments. As a demonstration that the isolated, meningococcal chromosomal DNA was capable of transforming a competent and compatible recipient, wild type MC58 bacteria were exposed to chromosomal DNA from all mutant sources. These data demonstrate that each strain of Nlac is highly competent for DNA uptake and homologous recombination when transformed with chromosomal DNA from a compatible source (nlaIII, black bars). Interestingly, nlaIII chromosomal DNA was also capable of transforming wild type MC58 with low efficiency, in what is presumably a de novo, untargeted recombination event due to the absence of an nlaIII coding sequence in the MC58 genome. This observation is in keeping with data that demonstrates horizontal gene transfer from Nlac into the pathogenic Neisseria in vivo. Each pool of chromosomal DNA derived from the MC58 mutant strains was capable of transforming wild type MC58 with low efficiency, as compared to the analogous back-cross into Nlac. However, each strain of Nlac was completely refractory to transformation with meningococcal DNA isolated from any mutant source (opc, checked bars; nadA, striped bars or siaD, dotted bars), with absolutely no recovery of kanamycin-resistant colonies across biological replicates (n=6). Bars denote MeanSD.
(169) Plasmid Map of pSC101NHCIS1::HAEC4:porA(P1.7,16)-lacZ
(170) With reference to FIG. 45, the pSC101NHCIS1::HAEC4:porA(P1.7,16)-lacZ plasmid is a derivative of pSC101NHCIS1::HAEC4:opcA-lacZ (FIG. 30), wherein the majority of the plasmid architecture is identical to pSC101NHCIS1::HAEC4:opcA-lacZ; but where the opcA coding sequence has been replaced by an Nlac codon-optimized version of the gene coding for Porin A (P1.7,16) (porA(P1.7,16)). Important to note is the fact that the nucleotide sequence of porA (P1.7,16) has been adjusted to remove the homopolyadenosine tract present at the 5 end of the wild type porA gene, so as to reduce the likelihood of porA(P1.7,16) expression being downregulated through phase variation. Transformation of strain lacZ with hypermethylated NHCIS1::HAEC4:porA(p1.7,16)-lacZ construct gave rise to strain lacZ NHCIS1::HAEC4:porA(p1.7,16)-lacZ (hereafter, 4PA1), which contained chromosomal sequences with 100% identity to the porA(P1.7,16) gene and grew as BLUE colonies on TSB agar plates supplemented with X-gal. The plasmid map is presented showing all relevant features and detailing the location of unique restriction sites.
(171) Plasmid Map of pSC101NHCIS1::PVporA(P1.7,16)-lacZ
(172) With reference to FIG. 46, the pSC101NHCIS1::PVporA(P1.7,16)-lacZ plasmid is a derivative of pSC101NHCIS1::HAEC4:opcA-lacZ (FIG. 30), wherein the majority of the plasmid architecture is identical to pSC101NHCIS1::HAEC4:opcA-lacZ; but where the porA/porB hybrid promoter and the opcA coding sequence have been replaced with the native porA promoter and porA(P1.7,16) coding sequence, amplified from the chromosome of wild type meningococcal strain H44/76. It is important to note that, unlike pUC19NHCIS1::HAEC2:porAplusprom-lacZ (FIG. 18), in which there was significant truncation of the porA transcriptional enhancer sequence (FIGS. 21 & 22), the pSC101NHCIS1::PVporA(P1.7,16)-lacZ plasmid contains the optimum length of 200 bp of enhancer sequence upstream of the porA promoter. Note that the plasmid map is based upon the ideal sequence for this plasmid, wherein the homopolymeric G tract, which separates the 10 and 35 boxes of the native porA promoter contains 11 contiguous guanosine nucleotide residues. The plasmid map is presented showing all relevant features and detailing the location of unique restriction sites.
(173) Recombinant Nlac Strains Express Porin a (P1.7, 16) at the Cell Surface and Generate PorA-Containing OMV
(174) In reference to FIG. 47, wild type N. lactamica strain Y92-1009 and the (putatively) PorA-expressing strains 2Pp7.A and 4PA1 were assessed for Porin A expression by western blot and flow cytometry (A and B, respectively). Outer membrane vesicles of the wild type and 4PA1 strains were analysed for PorA content by western blotting (C) prior to their use to immunize mice (see FIG. 48).
(175) (A) Wild type (WT) Nlac and strains 2Pp7.A and 4PA1 were grown to mid-log phase and lysed with sonication. Equal amounts (50 rig) of each crude membrane preparation were separated by SDS-PAGE and transferred to activated PVDF. Expression of PorA in the membrane was detected by interrogation with anti-P1.7, 16 mAb, SM300. PVDF was subsequently stripped and reprobed with antibody directed against the constitutively-expressed membrane protein LpdA.
(176) (B) WT Nlac and strains 2Pp7.A and 4PA1 were grown to mid-log phase and 210.sup.7 whole cells were labelled with anti-meningococcal serosubtype P1.7 mAb (NIBSC) and a goat-derived, anti-murine IgG-Alexafluor488 conjugate. After labelling, the bacteria were fixed in formalin prior to analysis on a FACSCalibur flow cytometer. Graph shows the Median Fluorescence Intensity of three independent bacterial cultures, lines denote Mean MFI. These data are consistent with that presented in FIG. 20, which showed a low level of PorA expression on the cell surface of strain 2Pp7.A. The addition of approximately 100 bp to the 5 end of the porA enhancer sequence (FIGS. 21 & 22) and the substitution of the native homopolymeric G tract present in the wild type porA promoter for 17 bp derived from the non-phase variable porB promoter, have resulted in an increased level of PorA expression in strain 4PA1.
(177) (C) WT Nlac and strain 4PA1 were grown overnight in modified Catlin medium (MC.7) to produce OMV, which was harvested with deoxycholate-extraction. Five micrograms (5 rig) of each OMV preparation were analysed for PorA by western blotting with anti-P1.7,16 mAb, SM300.
(178) Immunisation with OMV from Recombinant Nlac Generates Anti-Meningococcal SBA
(179) In reference to FIG. 48, mice were immunised i/p using a standard prime/boost strategy. Mice were injected with either Alum alone (AlOH) or Alum combined with deoxycholate-extracted OMV derived from one of: wild type Nlac (WT), the PorA-expressing recombinant Nlac strain 4PA1 (4PA1) or wild type serogroup B meningococcal strain H44/76 (H44/76). NB: H44/76 expresses the cognate PorA serosubtype, P1.7, 16. Sera from 5 immunised mice per group were pooled and doubling dilutions were assessed for SBA against (A) H44/76 and (B) MC58 (which expresses PorA serosubtype P1.7, 16-2). The maximum dilution of sera tested was 1:1024. OMV derived from strain 4PA1 elicited strong SBA against H44/76. Serum from these mice was more potent against strain MC58 than mice immunised with OMV from H44/76, suggesting Nlac OMV could provide broader anti-meningococcal adjuvant properties than Nmen-derived OMV.
(180) In reference to FIG. 49, mice were immunised i/p using a standard prime/boost strategy. Mice were injected with either Alum alone (AlOH) or Alum combined with deoxycholate-extracted OMV derived from the NadA-expressing recombinant Nlac strain 4NB1 (4NB1+AlOH). Sera from 5 immunised mice per group were pooled and doubling dilutions were assessed for SBA against Neisseria meningitidis strain 5/99 (a reference strain that expresses NadA to high levels). OMV derived from strain 4NB1 elicited strong SBA against 5/99 (SBA titre reciprocal: 170.6774). Serum from these mice was more potent against strain 5/99 than antisera from humans immunised with the 4CMenB (Bexsero) anti-meningococcal vaccine (SBA titre reciprocal: 640). Bars represent Mean SBA titre reciprocalSD. n2.
(181) Materials
(182) 50 ml bio-reaction tubes (50 ml tubes with 0.22 m filter cap) (GPE Scientific) 250 ml shaker flask, baffled with vented cap (Fisherbrand, pack of 12, Cat no 11735253): These flasks are fully autoclavable, including cap.
(183) Glass homogeniser and plunger (VWR: Vessel=5 ml capacity, cat no. 432-0201. Plunger=5 ml, cat no. 432-0207.
(184) *Ultracentrifuge bottles for rotor 55.2 Ti fixed-angle rotor: Polycarbonate bottle6 with cap assembly, 26.3 ml 2589 mm. Beckman Coulter, Cat no. 355618.
(185) Yeast extract powder (500 g, Fisher Scientific, Cat no. 10108202)
(186) Agar Plates
(187) Tryptone Soya Broth Media Powder (500 g, Fisher Scientific, Cat No. 10198002)
(188) Add 15 g of powder to 500 ml of water. Supplement with 0.2% (Ig) yeast extract. Sterilise by autoclaving at 121 C. for 15 minutes to make broth.
(189) To make agar, add 7.5 g of bacteriological agar to the above mix prior to autoclaving. Then swirl to mix agar and allow to cool until the bottle can comfortably be held in the palm of the bare hand.
(190) Agar Bacteriological (Agar No. 1) (500 g, Fisher Scientific, Cat No. 10351303)
(191) Pour into petri dishes within a class II containment hood. Leave half uncovered to cool before replacing lids to avoid build up of condensation.
(192) Media
(193) Modified Catlin Medium (MC.7)
(194) Dissolve the following components in distilled water for a final volume of 1000 ml and 500 ml respectively. pH will be approximately 7.
(195) TABLE-US-00001 1000 ml 500 ml NaCl 5.8 g 2.9 g K.sub.2HPO.sub.4 4 g 2 g NH.sub.4Cl 1 g 0.5 g K.sub.2SO.sub.4 1 g 0.5 g D-(+)-glucose 10 g 5 g MgCl.sub.26H.sub.2O 0.4 g 0.2 g CaCl.sub.22H.sub.2O 0.03 g 0.015 g Yeast extract 0.8 g 0.4 g HEPES 5.96 g 2.98 g EDDA 0.005 g 0.0025 g
(196) Dissolve the following reagents in distilled water and filter sterilise using a 0.2 m syringe filter.
(197) TABLE-US-00002 L-glutamic acid 2 1.95 g 1.95 g
(198) Weigh 1.95 g into 21 ml 1M HCl. Warm to 60 C. and add 6 M HCl dropwise until it dissolves. Bring pH back to 7 using 6 M NaOH. Top up to 50 ml with distilled water and filter sterilise. Make up 250 ml tube for 1000 ml of medium.
(199) TABLE-US-00003 L-CysteineHCl 0.1 g 0.05 g
(200) For 500 ml make up to 25 ml with water and filter sterilise. For 100 ml make up to 50 ml
(201) Add the above to the autoclaved medium within a class II containment hood to keep medium sterile.
(202) After autoclaving, the medium is yellow and may contain a large white precipitate that will not dissolve. In the event of precipitate formation, ensure thorough resuspension of the precipitate before use.
(203) Buffers
(204) Buffer 1
(205) 0.1M Tris-HCl pH 8.6
(206) 10 mM EDTA
(207) 0.5% (W/V) Deoxycholic acid sodium salt
(208) Buffer 2
(209) 50 mM Tris-HCl pH 8.6
(210) 2 mM EDTA
(211) 1.2% Deoxycholic acid sodium salt
(212) 20% sucrose (W/V)
(213) After addition of EDTA to above buffers, heat to 60 C. in a water batch to encourage it to dissolve. If it does not fully dissolve, add 1 M NaOH whilst stirring until a solution is obtained. Then add remaining reagents.
(214) Buffer 3
(215) 0.2M glycine buffer
(216) 3% sucrose
(217) Filter sterilise buffers.
(218) Protocol (Work within a Class II Containment Hood Until Step 20) for Use with Either MHB, MC.6 or MC.7 Media
(219) Note: All materials that come in direct contact with live Neisseria meningitidis must either be decontaminated overnight in 2% Virkon, or sealed prior to disposal via the clinical waste stream or by autoclaving. Keep a sealable plastic bag inside the hood for disposal of gloves. After use, seal the bag, place inside a clinical waste bag and dispose of the clinical waste at the end of the experiment by taping up the neck of the bag and placing inside a large yellow clinical waste bin. At the end of the experiment disinfect surfaces such as the centrifuge control panel, door handles and pipettes with 70% ethanol. 1. At the end of the day, thaw a stock of N. lactamica or N. meningitidis. Remove 200 l using a sterile pipette tip and plate onto the centre of a TSB+0.2% yeast extract agar plate. 2. Using a sterile cell spreader, place the spreader on the centre of the plate and rotate the centre of spreader so that the inoculum is spread evenly over the surface. 3. Replace the lid and place in a 37 C. 5% CO.sub.2 incubator for 20 minutes with the lid facing up to allow the inoculum to soak into the agar. Also prepare a streak plate to assess purity of culture. 4. Turn plate upside down so that the lid is facing down and culture overnight. 5. At the end of the next day there should be a bacterial lawn covering the plate. Assess streak plate to ensure there is a mono-culture of N. lactamica, with no obvious contaminants. 6. Add 3 ml of MC.7 medium to the edge of the bacterial lawn and use a sterile spreader to very gently scrape over the surface of the lawn to liberate the adhered bacteria into the medium. Caution: Aerosol formation. 7. Using a Pasteur pipette, transfer the bacterial suspension from the surface of the plate into a 50 ml tube containing a 0.2 m filter fitted into the cap (Bio-reaction tube). 8. Top the tube up to 30 ml with medium. 9. Incubate at 37 C. in a 5% CO.sub.2 shaking incubator at 350 RPM overnight. 10. Use 2 ml of the culture from step 9 to inoculate 150 ml of medium in a 250 ml baffled, vented flask. Use the remainder of the culture to prepare fresh glycerol stocks if required. Caution: Aerosol formation. 11. Incubate for 8 hours. Divide this culture into two other bottles, giving a total of 3 bottles containing 150 ml in each. Culture overnight until an OD.sub.600nm of at least 2.0 has been reached. 12. Pour the contents of the bottle into 50 ml tubes. Caution: Aerosol formation. Spray the outside of the tubes with 70% ethanol to decontaminate any spillages and spray your gloves to do the same. Dispose of gloves, wash hands and put on a fresh pair of gloves. 13. Place tubes in a water bath set to 56 C. and heat kill the bacteria by incubating for 45 minutes. 30 minutes is sufficient, but it will take 10 minutes for the suspension to reach temperature. 14. Centrifuge heat-killed bacteria at 4,500g for 1 hour at 20 C. to pellet. 15. Pour off supernatant. Pellet may be stored at 20 C. 16. Measure wet mass of cell pellet and Resuspend in buffer 1 using a buffer:biomass ratio of 5:1 (V/W). 17. Homogenise pellet using a glass homogeniser. Sterilise the glass homogeniser by filling with 70% ethanol. Leave for 5 minutes and then rinse with sterile water. 18. Centrifuge suspension at 20,000g for 30 minutes at 4 C. Sterilise the ultracentrifuge tubes by filling with 70% ethanol for 5 minutes. Spray caps with 70% ethanol. Then rinse tubes and caps with sterile water. Do not autoclave the ultracentrifuge tubes and do not expose to pure ethanol. 19. Retain supernatant and resuspend pellet in buffer 1 (a third of the volume used in step 5). 20. Repeat steps 17 and 18. 21. Retain supernatant and combine with that from step 19. 22. Ultracentrifuge suspension at 100,000g for 2 hours at 4 C. 23. Resuspend pellet in 2.5 ml of buffer 2 by flicking and vortexing. 24. Ultracentrifuge suspension at 100,000g for 2 hours at 4 C. 25. Resuspend pellet in 2 ml of buffer 3 and transfer to a sterile universal. 26. Homogenise dOMV suspension by adding 6 sterile glass beads to the suspension and vortexing for approximately 10 minutes. 27. Measure protein concentration of homogenised OMV. 28. Assess OMVs by SDS-PAGE followed by coomassie blue staining or silver staining. 29. Store OMV frozen at 20 C. or 80 C.
Plasmid Vector Sequences
(220) The following vectors are encompassed by the invention herein and their sequences are provided in the sequence listing as part of the description.
(221) pUC19nlaIII:CLOVER-aphA3 (SEQ ID NO: 21)
(222) pUC19nlaIII:aphA3 (SEQ ID NO: 22)
(223) pUC19NHCIS1::HAEC1:(Z)-lacZ (SEQ ID NO: 23)
(224) pUC19NHCIS1::HAEC2:(Z)-lacZ (SEQ ID NO: 24)
(225) pUC19NHCIS2::HAEC1:(Z)-lacZ (SEQ ID NO: 25)
(226) pUC19NHCIS1::HAEC2:porAplusprom-lacZ (SEQ ID NO: 26)
(227) pUC19NHCIS1::HAEC4:nadA-lacZ (SEQ ID NO: 27)
(228) pUC19NHCIS1::HAEC4:(Z)-lacZ (SEQ ID NO: 28)
(229) pSC101NHCIS1::HAEC4:opcA-lacZ (SEQ ID NO: 29)
(230) pUC19nhba::HAEC1:(Z)-lacZ (SEQ ID NO: 30)
(231) pUC19nhba (SEQ ID NO: 31)
(232) pSC101lacZ:opcA (SEQ ID NO: 32)
(233) pSC101lacZ-synth.lacZ-3NHCIS1 (SEQ ID NO: 33)
(234) pSC101NHCIS1::HAEC4:porA(P1.7,16)-lacZ (SEQ ID NO: 34)
(235) pSC101NHCIS1::PVporA(P1.7,16)-lacZ (SEQ ID NO: 35)
