Glycoconjugate vaccines
10039814 ยท 2018-08-07
Assignee
Inventors
Cpc classification
A61K45/06
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K47/646
HUMAN NECESSITIES
A61K2039/6037
HUMAN NECESSITIES
A61K2039/57
HUMAN NECESSITIES
A61K2039/55572
HUMAN NECESSITIES
International classification
A61K39/00
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K47/64
HUMAN NECESSITIES
Abstract
The disclosure relates to a glycoconjugate vaccine conferring protection against Francisella tularensis infections and a method to manufacture a glycoconjugate antigen.
Claims
1. A process for manufacturing a glycoconjugate polypeptide, comprising: i) providing a bacterial cell in cell culture medium, wherein said bacterial cell is genetically modified to include: a) a nucleic acid molecule comprising the nucleotide sequence of the Francisella O-antigen biosynthetic polysaccharide locus set forth in SEQ ID NO: 7; b) a nucleic acid molecule comprising the oligosaccharyltransferase nucleotide sequence set forth in SEQ ID NO: 8 or a functional variant thereof, wherein said functional variant comprises a nucleic acid molecule the complementary strand of which shares at least 90% identity to the sequence set forth in SEQ ID NO: 8 and wherein said nucleic acid molecule encodes an oligosaccharyltransferase; and c) a nucleic acid molecule comprising a nucleotide sequence of a carrier polypeptide, wherein the carrier polypeptide comprises one or more T-cell dependent epitopes and one or more amino acid sequences having the amino acid motif D/E-X-N-X-S/T, wherein X is any amino acid except proline; wherein the bacterial cell is adapted for expression of each nucleic acid molecule set forth in a), b), and c); ii) growing the bacterial cell in the cell culture medium, wherein the oligosaccharyltransferase of b) glycosylates the carrier polypeptide of c) to form a glycoconjugate polypeptide comprising a Francisella O-antigen; and iii) isolating the glycoconjugate polypeptide comprising a Francisella O-antigen from the bacterial cell or the cell culture medium, thereby manufacturing a glycoconjugate polypeptide.
2. The process according to claim 1, wherein at least the oligosaccharyltransferase is integrated into the bacterial genome of said bacterial cell.
3. The process according to claim 1, wherein one or more nucleic acid molecules encoding the carrier polypeptide is integrated into the bacterial genome of said bacterial cell.
4. The process according to claim 1, wherein said Francisella O-antigen comprises 4)-?-D-GalNAcAN-(1-4)-?-D-GalNAcAN-(1-3)-?-D-QuiN Ac-(1-2)-?-D-Qui4NFm-(1-), wherein GalNAcAN is -2-acetamido-2-deoxy-O-D-galact-uronamide, 4NFm is 4,6-dideoxy-4-formamido-D-glucose and the reducing end group QuiNAc is 2-acetamido-2,6-dideoxy-O-D-glucose.
5. The process according to claim 4, wherein said Francisella O-antigen is a tetrasaccharide.
6. A glycoconjugate polypeptide obtained by the process according to claim 1.
7. A vaccine or immunogenic composition comprising the glycoconjugate polypeptide according to claim 6 and an adjuvant and/or carrier.
8. A method for treating a Francisella infection, comprising administering an effective amount of the vaccine or immunogenic composition according to claim 7.
9. The method according to claim 8, wherein said Francisellainfection is caused by Francisella tularensis.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) An embodiment of the invention will now be described by example only and with reference to the following figures:
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DETAILED DESCRIPTION
(25) TABLE-US-00001 TABLE 1 Strains and plasmids used Strain/plasmid Description Source E. coli DH5? F-?80lacZ?M15 Invitrogen ?(lacZYA-argF) U169 deoRrecA1 endA1 hsdR17 (rk?, mk+), gal- phoAsupE44? - thi-1 gyrA96 relA1 E. coli XL-1 endA1 gyrA96(nalr)thi-1 Stratagene relA1 lac gln V44 F[::Tn10 proAB+ laclq ? (lacZ)M15] hsdR17(r.sub.k.sup.?m.sub.k.sup.+) E. coli CLM24 rph-l IN(rrnD-rrnE) 1, 5 ?waaL F. tularensis subs. Type A strain DSTL, Porton tularensis strain SchuS4 Down laboratories F. tularensis subs. Type B strain, isolated in Green, M., et al., holarctica strain HN63 Norway from an infected Efficacy of the live attenuated Hare Francisella tularensis vaccine (LVS) in a murine model of disease. Vaccine, 2005. 23(20): p. 2680-6 pGEM-T Easy TA cloning vector, amp.sup.r Promega pLAFR1 Low copy expression Vanbleu E, Marchal K, vector, tet.sup.r Vanderleyden J. Genetic and physical map of the pLAFR1 vector. DNA Seq. 2004 June; 15(3): 225-7. pGAB1 F. tularensis O antigen This study coding region inserted into MCS of pGEM-T easy pGAB2 F. tularensis subs. This study tularensis strain SchuS4 O antigen coding region inserted into Ecorl site of pLAFR. pGVXN114 Expression plasmid for GlycoVaxyn CjPglB regulated from the Lac promoter in pEXT21. IPTG inducible, HA tag, Spec.sup.r. pGVXN115 Expression plasmid for GlycoVaxyn C. jejuninon functionalPglB due to a mutation at .sub.457WWDYGY.sub.462 to .sub.457WAAYGY.sub.462, regulated from the Lac promoter in pEXT21. IPTG inducible, HA tag, Spec.sup.r. pGVXN150.sub.260DNQNS.sub.264 Expression plasmid for This study Pseudomonas aeruginosa PA103(DSM111/) Exotoxin A with the signal peptide of the E.coliDsbA protein, two inserted bacterial N-glycosylation sites, AA at position 262 altered from N to Q and a hexahis tag at the C-terminus. Induction under control of an arabinose inducible promoter, Amp.sup.r pGVXN150.sub.402DQQRT.sub.406 Expression plasmid for This study Pseudomonas aeruginosa PA103(DSM111/) Exotoxin A with the signal peptide of the E. coliDsbA protein, two inserted bacterial N-glycosylation sites, AA at position 404 altered from N to Q and a hexahis tag at the C-terminus. Induction under control of an arabinose inducible promoter, Amp.sup.r pGVXN150.sub.260DNQNS.sub.264/ Expression plasmid for This study .sub.402DQQRT.sub.406 Pseudomonas aeruginosa PA103(DSM111/) Exotoxin A with the signal peptide of the E. coliDsbA protein, two inserted bacterial N-glycosylation sites, AA at position 262 and 404 altered from N to Q and a hexahis tag at the C-terminus. Induction under control of an arabinose inducible promoter, Amp.sup.r pACYCpgl pACYC184 carrying the 5 CjPglB locus, Cm.sup.r E. coli CedAPglB E. coli strain CLM24 with a This study chromosomally inserted IPTG inducible copy of PglB
Materials and Methods
Bacterial Strains and Plasmids
(26) Escherichia coli strains were grown in LB at 37? C., 180 r.p.m. Antibiotics were used at the following concentrations; tetracycline 20 ?g/ml, ampicillin 100 ?g/ml, spectinomycin 80 ?g/ml, chloramphenicol 30 ?g/ml. The host strain for initial cloning experiments was E. coli XL-1, subsequent strains used for glycoconjugate production were E. coli DH5? and CLM24 ( Table 1). For efficacy studies, mice were challenged with F. tularensis subsp. holarctica strain HN63. The bacterium was cultured on blood cysteine glucose agar plates (supplemented with 10 ml of 10% (wt/vol) histidine per litre) at 37? C. for 18 hours.
(27) Cloning, Sequencing and Expression of the F. tularensis O Antigen Coding Region
(28) DNA was prepared from the F. tularensis subsp. tularensis strain SchuS4 by phenol extraction as described by Karlsson et al. Microb Comp Genomics. 5(1):25-39, (2000). The O-antigen coding region was amplified using the primers FTfragment2rev (5-GGATCATTAATAGCTAAATGTAGTGCTG-3; SEQ ID 10) and Oantlftfwd (5-TTTTGAATTCTACAGGCTGTCAATGGAGAATG-3; SEQ ID 11) using the following cycling conditions: 94? C., 15 sec, 55? C., 15 sec, 68? C., 20 min; 35 cycles using Accuprime TaqHifi (Invitrogen U.K.). This was cloned into the TA cloning vector pGEM-T Easy to generate the vector pGAB1. The plasmid pGAB1 was digested with EcoRI and the insert was subcloned into the vector pLAFR to generate the construct pGAB2.
(29) Immunofluorescence Imaging of E. coli Cells Carrying F. tularensis O Antigen Coding Region
(30) Immunofluorescence was carried out as previously described [17] with the modification that the IgG2a mouse monoclonal antibody FB11 was used to detect F. tularensis O antigen (1 ?l/ml in 5% (v/v) FCS/PBS).
(31) Production and Purification of Glycoconjugate Vaccine
(32) E. coli CLM24 carrying the vectors pGAB2, pGVXN114 and pGVXN150 was grown for 16 h in 200 mL LB broth at 37? C., 180 r.p.m. This was used to inoculate 1.8 L of LB broth and further incubated at 110 r.p.m. 37? C. to an OD.600 nm reading of 0.4 to 0.6. L-arabinose was then added to a final concentration of 0.2% and IPTG to a final concentration of 1 mM to induce expression of exoA and CjpglB respectively; following 5 hours of incubation, 0.2% L-arabinose was added again and the culture left to incubate o/n.
(33) Cells were harvested by centrifugation at 6,000 r.p.m. for 30 m, and pelleted cells were incubated at room temperature for 30 m in a lysis solution composed of 10? BugBuster protein extraction reagent (Novagen) diluted to 1? in 50 mM NaH2PO.sub.4, 300 mM NaCl, 10 mM imidazole, pH 8.0 supplemented with 0.1% Tween, 1 mg/ml lysozyme and 1 ?l/ml Benzonase nuclease (Novagen). Cell debris was removed by centrifugation at 10,000 r.p.m. for 30 m, the supernatant was collected and 1 ml Ni-NTA agarose (QIAgen) was added to the supernatant. The slurry-lysate was incubated for 1 h at 4? C. with shaking then loaded into 10 ml polypropylene columns (Thermo scientific). His tagged ExoA was purified according to manufacturer's instructions (QIA expressionist, QIAGEN) with the addition of 20% glycerol and 5% glucose to the elution buffer. Protein yields were estimated using a bicinchonic acid assay kit according to manufacturer's instructions (Pierce? Biotechnology BCA protein Assay Kit, U.S.A.).
(34) For large-scale protein purification, material was isolated using GE Healthcare His Trap columns and an AKTA purifier with an imidazole gradient of 30-500 mM. The collected fraction containing ExoA glycosylated with F. tularensis O-antigen was further purified using a resource Q anionic exchange column (GE Healthcare) with a NaCl gradient from 0 to 500 mM in 20 mM TrisHCl pH 8.0. This generated a typical yield of 2-3 mg/ml of glycoconjugate per 2 L of E. coli culture.
(35) The same techniques were used for the generation of the sham C. jejuni heptasaccharide ExoA glycoconjugate encoded by pACYCpgl [18].
(36) Using the E. coli chromosomally inserted strain CLM 24 CedAPglB: Escherichia coli strain CLM24 with a chromosomally inserted copy of pgIB were grown in Luria-Bertani (LB) broth at 37? C., with shaking. Antibiotics were used at the following concentrations: tetracycline 20 ?g ml.sup.?1 and ampicillin 100 ?g ml.sup.?1. Tetracycline was used to maintain the plasmid pGAB2 coding for Francisella tularensis O antigen and ampicillin was used to maintain the plasmid coding for the acceptor carrier protein.
(37) Escherichia coli cells were grown for 16 h in 200 ml LB broth at 37? C., with shaking. This was used to inoculate 1.8 l of LB broth and further incubated with shaking at 37? C. until an OD600 reading of 0.4-0.6 was reached. At this point L-arabinose was added to a final concentration of 0.2 per cent and IPTG to a final concentration of 1 mM to induce expression of the acceptor protein and pgIB, respectively; after another 5 h of incubation, 0.2% L-arabinose was added again and the culture left to incubate overnight.
(38) Cells were harvested by centrifugation at 5300 g for 30 min, and pelleted cells were incubated at room temperature for 30 min in a lysis solution composed of 10? BugBuster protein extraction reagent (Novagen) diluted to 1? in 50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 10 mM imidazole, pH 8.0 supplemented with 0.1 per cent Tween, 1 mg ml.sup.?1 lysozyme and 1 ?l ml.sup.?1 Benzonase nuclease (Novagen). Cell debris was removed by centrifugation at 7840 g for 30 min, the supernatant was collected and 1 ml Ni-NTA agarose (QIAGEN) was added to the supernatant. The slurry-lysate was incubated for 1 h at 4? C. with shaking then loaded into 10 ml polypropylene columns (Thermo Scientific). His-tagged ExoA was purified by the addition of an elution buffer according to manufacturer's instructions (QIA expressionist, QIAGEN) containing 250 mM imidazole with the addition of 20 per cent glycerol and 5 per cent glucose.
(39) Alternatively cells were grown in LB agar plates containing tetracycline, ampicillin, IPTG to a final concentration of 50 ?M and L-arabinose to a final concentration of 0.2% for 16 h at 37 ? C. Cells were subsequently harvested by scraping and protein purified as indicated above.
(40) Immunoblot Analysis
(41) To verify transfer and presence of the F. tularensis O antigen, samples were analysed by western blotting. E. coli cells were grown o/n in 10 ml LB broth and diluted to an O.D.600 nm of 1.0. Cells were centrifuged at 13,000 r.p.m. for 10 min, supernatant was removed and cells were resuspended in 100 ?l Laemmli buffer and lysed by boiling for 10 min before analysis by western blotting or silver staining. Mouse anti F. tularensis O-antigen monoclonal antibody FB011 (AbCam U.K.) was used at a dilution of 1:1,000, rabbit anti HIS monoclonal antibody was used to detect ExoA at a dilution of 1:10,000 (AbCam U.K.). Secondary antibodies used were goat anti mouse IRDye680 and IRDye800 conjugates used at 1:5000 dilutions. Signal detection was undertaken using the Odyssey? LI-COR detection system (LI_COR Biosciences GmbH).
(42) Cytokine Response Analysis
(43) Spleen supernatants were assessed using mouse inflammatory cytometric bead array kit (CBA-BD biosciences) for IL-10, IL-12p70, IFN-?, IL-6, TNF-?, and MCP-1. Samples were incubated with the combined capture bead cocktail, and incubated for 1 h at room temperature. Following incubation, PE detection antibodies were added and incubated for a further 1 h. Samples were then washed and resuspended in FACS buffer. Cytokine concentrations were measured via quantification of PE fluorescence of samples in reference to a standard curve.
(44) BALB/c Mouse Challenge Studies
(45) Female Balb/C mice were obtained from Charles River Laboratories (Kent, U.K.) at 6-8 weeks of age. The pilot study was done in groups of 10 mice immunised with either 0.5 ?g F. tularensis LPS, 0.5 ?g F. tularensis glycoconjugate, 0.5 ?g F. tularensis glycoconjugate +SAS, 0.5 ?g sham glycoconjugate +SAS, 0.5 ?g sham glycoconjugate or SAS only. One group of mice were left untreated as challenge efficacy controls. Immunisations occurred on days 0, 14 and 28 via intra-peritoneal (IP) route. Mice were challenged 35 days post-immunisation with 100 CFU of F. tularensis strain HN63 by the IP route, delivered in 0.1 ml. Subsequent experiments used the same schedule with 15 mice per group and doses of 10 ?g of material per immunisation. Four weeks following final vaccination 5 mice from each group were tail bled to obtain sera for antibody analysis and culled at day 3 post-infection with spleens harvested to analyse bacterial load and cytokine response. For the enumeration of bacteria, spleen samples were homogenized in 2 ml of PBS through 40 ?m cell sieves (BD Biosciences) and 100 ?l aliquots were plated onto BCGA plates. F. tularensis LPS-specific IgM and total IgG levels were determined by ELISA as previously described [19]. All work was performed under the regulations of the Home Office Scientific Procedures Act (1986).
(46) Statistical Analysis
(47) Statistical analyses were performed using the program PASW (SPSS release 18.0). Survival data was analysed by pair-wise Log Rank test stratified by experiment. Cytokine and bacterial load data were analysed using univariate general linear models, using Bonferroni's post tests to further clarify significant differences.
(48) Production and Purification of Glycoconjugate Vaccine
(49) E. coli CLM24 carrying the vectors pGAB2, pGVXN114 and pGVXN150 was grown for 16 h in 200 mL LB broth at 37? C., 180 r.p.m. This was used to inoculate 1.8 L of LB broth and further incubated at 110 r.p.m. 37? C. until an O.D600 reading of 0.4 to 0.6 was reached. At this point L-arabinose was added to a final concentration of 0.2% and IPTG to a final concentration of 1 mM to induce expression of exoA and CjpglB respectively; after another 5 hours of incubation, 0.2% L-arabinose was added again and the culture left to incubate overnight.
(50) Cells were harvested by centrifugation at 6,000 r.p.m. for 30 m, and pelleted cells were incubated at room temperature for 30 m in a lysis solution composed of 10? BugBuster protein extraction reagent (Novagen) diluted to 1? in 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0 supplemented with 0.1% Tween, 1 mg/ml lysozyme and 1 ?l/ml Benzonase nuclease (Novagen). Cell debris was removed by centrifugation at 10,000 r.p.m. for 30 m, the supernatant was collected and 1 ml Ni-NTA agarose (QIAgen) was added to the supernatant. The slurry-lysate was incubated for 1 h at 4? C. with shaking then loaded into 10 ml polypropylene columns (Thermo scientific). His tagged ExoA was purified by the addition of an elution buffer according to manufacturer's instructions (QIA expressionist, QIAGEN) containing 250 mM imidazole with the addition of 20% glycerol and 5% glucose. Protein yields were estimated using a bicinchonic acid assay kit according to manufacturer's instructions (Pierce? Biotechnology BCA protein Assay Kit, U.S.A.).
(51) For large-scale protein purification, material was isolated using GE Healthcare HIS trap columns and an AKTA purifier with an imidazole gradient of 30 mM to 500 mM. The collected fraction containing ExoA glycosylated with F. tularensis O-antigen was further purified using a resource Q anionic exchange column (GE Healthcare) with a NaCl gradient from 0 to 500 mM in 20 mM TrisHCl pH 8.0. This generated a typical yield of 2-3 mg/ml of glycoconjugate per 2 L of E. coli culture.
(52) The same techniques were used for the generation of the sham C. jejuni heptasaccharide ExoA glycoconjugate. The plasmid coding for this heptasaccharide was pACYCpgl carrying the entire Cjpgl cluster from C. jejuni 81116 [1].
(53) Protein Expression
(54) Escherichia coli strain CLM24 with a chromosomally inserted copy of pglB were grown in Luria-Bertani (LB) broth at 37? C., with shaking. Antibiotics were used at the following concentrations: tetracycline 20 ?g ml-1 and ampicillin 100 ?g ml-1. Tetracycline was used to maintain the plasmid pGAB2 coding for Francisella tularensis O antigen and ampicillin was used to maintain the plasmid coding for the acceptor carrier protein.
(55) Escherichia coli cells were grown for 16 h in 200 ml LB broth at 37? C., with shaking. This was used to inoculate 1.8 l of LB broth and further incubated with shaking at 37? C. until an OD600 reading of 0.4-0.6 was reached. At this point L-arabinose was added to a final concentration of 0.2 per cent and IPTG to a final concentration of 1 mM to induce expression of the acceptor protein and pglB, respectively; after another 5 h of incubation, 0.2 per cent L-arabinose was added again and the culture left to incubate overnight.
(56) Cells were harvested by centrifugation at 5300 g for 30 min, and pelleted cells were incubated at room temperature for 30 min in a lysis solution composed of 10? BugBuster protein extraction reagent (Novagen) diluted to 1? in 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0 supplemented with 0.1 per cent Tween, 1 mg ml-1 lysozyme and 1 ?l ml-1 Benzonase nuclease (Novagen). Cell debris was removed by centrifugation at 7840 g for 30 min, the supernatant was collected and 1 ml Ni-NTA agarose (QIAGEN) was added to the supernatant. The slurry-lysate was incubated for 1 h at 4? C. with shaking then loaded into 10 ml polypropylene columns (Thermo Scientific). His-tagged ExoA was purified by the addition of an elution buffer according to manufacturer's instructions (QIA expressionist, QIAGEN) containing 250 mM imidazole with the addition of 20 per cent glycerol and 5 per cent glucose.
EXAMPLES
Example 1
Expression of the F. tularensis SchuS4 O-antigen in E. coli DH5? Cells
(57) The 20 kb F. tularensis SchuS4 O-antigen coding region was PCR amplified and cloned into pGEM-T Easy to generate the plasmid pGAB1. All bacterial strains and vectors used in this study are summarized in table 1. To confirm O-antigen expression and transport to the outer cell surface of E. coli, pGAB1 was transformed into DH5? cells and probed by immunofluorescence using mAb FB11, specific to the F. tularensis O-antigen.
Example 2
CjPgIB can Transfer F. tularensis O-antigen to the Acceptor Protein Exotoxin A
(58) In order to generate a strong T-cell response and lasting immunity, a highly immunogenic protein is required as a carrier for the F. tularensis O-antigen. The selected carrier protein was an inactivated form of the P. aeruginosa Exotoxin A variant L552V, AE553 (ExoA) was selected [20]. The plasmid pGAB2 containing the F. tularensis O-antigen expressed in the low copy vector pLAFR1 [21] was transformed into E. coli CLM24 cells along with the plasmids pGVXN114 and pGVXN150 which contain CjPgIB and ExoA respectively. As negative glycosylation controls, CLM24 cells were transformed with either pGVXN150 alone or with the combination of pGAB2, pGVXN150 and pGVXN115, the latter coding for an inactive version of CjPgIB [18]. Following overnight induction of CjpglB and exoA expression with 1 mM IPTG and 0.2% L-arabinose (w/v) respectively, cells were lysed and HIS tagged ExoA purified using Nickel columns. Elution fractions from each sample were separated by SDS PAGE and tested by immunoblotting with mAbFB011 specific for F. tularensis LPS. A band matching the expected size of ExoA and an O-antigen ladder pattern could only be seen when a functional CjPgIB was present (
Example 3
Vaccination with the Glycoconjugate Provides Significant Protection against F. tularensis subsp. holarctica Infection in Mice
(59) In a pilot study we compared LPS alone against the glycoconjugate vaccine and monitored antibody levels and murine survival. The Sigma Adjuvant System? was selected for use in this study because it is based on monophosphoryl lipid A (MPL), a low toxicity derivative of LPS that has been demonstrated to be a safe and effective immunostimulant [22]. In order to demonstrate the specificity of the glycoconjugate we used controls including mice with SAS adjuvant alone, unvaccinated mice and mice vaccinated with a sham glycoconjugate control (C. jejuni heptasaccharide conjugated to ExoA). Only mice vaccinated with 0.5 ?g test glycoconjugate+SAS (P<0.05) or 0.5 ?g LPS (P<0.001) demonstrated increased survival compared to the appropriate controls as determined by log rank test (
Example 4
Mice Vaccinated with Test Glycoconjugate and Challenged with F. tularensis subsp. holarctica have Lower Bacterial Loads and Pro-Inflammatory Cytokines 3 Days Post Challenge
(60) Three days post challenge 5 mice per group were sacrificed and bacterial loads in the spleens and inflammatory responses were evaluated (
Example 5
Vaccination with the F. tularensis Glycoconjugate Induces a Greater IgG Immune Response
(61) The levels of LPS-specific IgG were assessed in mice 7 days prior to challenge for both experiments. Increased LPS-specific IgG was observed in the glycoconjugate+SAS vaccinated group when compared to animals vaccinated with LPS only (P<0.001). Although experiment 2 had higher levels of antibody (P<0.01), we observed no difference in pattern between experiments (P>0.05) (
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