Surface display of antigens on Gram-negative outer membrane vesicles

Abstract

The present invention relates to vaccine compositions based on Gram-negative outer membrane vesicles displaying antigens of pathogens expressed as part of a fusion protein comprising N-terminal parts of surface expressed lipoproteins of Gram-negative bacteria, and use of such compositions in vaccination. The invention further relates to the fusion lipoproteins comprising N-terminal parts of surface expressed lipoproteins of Gram-negative bacteria and antigens of pathogens fused thereto, DNA constructs and bacterial host cells for expressing these fusion lipoproteins and to methods for producing outer membrane vesicles displaying the fusion lipoproteins.

Claims

1. An OMV comprising a fusion lipoprotein, wherein the fusion lipoprotein comprises an N-terminal and a C-terminal fusion partner, wherein: (a) the N-terminal fusion partner comprises in N- to C-terminal order: i) a lipidated N-terminal cysteine; ii) a tether of a surface exposed lipoprotein of a Gram-negative bacterium; and, optionally, iii) a stretch of at least 5 contiguous amino acids that are located C-terminally of the tether of a surface exposed lipoprotein of a Gram-negative bacterium; wherein the N-terminal fusion partner causes presentation of the fusion lipoprotein on the extracellular outer membrane surface of a Gram-negative bacterium upon expression therein; and, (b) the C-terminal fusion partner comprises at least one epitope of an antigen associated with an infectious disease and/or a tumour, wherein the C-terminal fusion partner does not comprise an amino acid sequence of at least 10 contiguous amino acids from the surface exposed lipoprotein from which the sequence of the N-terminal fusion partner originates; and wherein the amino acid sequence of the fusion lipoprotein does not occur in nature.

2. The OMV according to claim 1, wherein the tether of the fusion lipoprotein is located adjacent to the lipidated N-terminal cysteine.

3. The OMV according to claim 1, wherein the N-terminal fusion partner of the fusion lipoprotein comprises an N-terminal fragment from a surface exposed lipoprotein of a Gram-negative bacterium and wherein the fragment causes surface expression of the fusion lipoprotein when expressed in the Gram-negative bacterium.

4. The OMV according to claim 1, wherein the tether of the fusion protein is of a Gram-negative bacterium of the genus Neisseria.

5. The OMV according to claim 3, wherein the surface exposed lipoprotein is selected from the group consisting of fHbp, LpbB, TbpB, NHBA and Ag473.

6. The OMV according to claim 1, wherein the N-terminal fusion partner of the fusion lipoprotein comprises at least one of: a) the amino acid sequence in positions 20-38 of SEQ ID NO: 1; b) the amino acid sequence in positions 21-61 or positions 21-63 of SEQ ID NO: 2; and, c) the amino acid sequence in positions 23-51 of SEQ ID NO: 3.

7. The OMV according to claim 6, wherein the N-terminal fusion partner of the fusion lipoprotein comprises at least one of: a) the amino acid sequence in positions 20-50 of SEQ ID NO: 1; b) the amino acid sequence in positions 21-73 or positions 21-75 of SEQ ID NO: 2; or, c) the amino acid sequence in positions 23-63 of SEQ ID NO: 3.

8. The OMV according to claim 1, wherein the C-terminal fusion partner of the fusion lipoprotein does not comprise a contiguous amino acid sequence of at least 5 amino acids from the surface exposed lipoprotein from which the sequence of the N-terminal fusion partner originates.

9. The OMV according to claim 8, wherein the C-terminal fusion partner of the fusion lipoprotein comprises surface exposed epitopes from a proteinaceous antigen of an infectious agent or tumour.

10. The OMV according to claim 9, wherein the C-terminal fusion partner of the fusion lipoprotein comprises a surface exposed domain of a surface exposed bacterial protein or lipoprotein.

11. The OMV according to claim 10, wherein the bacterial protein comprises a Borrelia surface lipoprotein selected from the group consisting of OspA, OspB, OspC, OspF, VlsE, BbCRASP1, Vsp1, P35 (BBK32), P37 (BBK50), P39, P66, DpbA and BB017.

12. The OMV according to claim 11, wherein the Borrelia surface lipoprotein comprises amino acids 29-273 of SEQ ID NO: 4, amino acids 29-273 of SEQ ID NO: 58 or amino acids 136-210 of SEQ ID NO: 59.

13. The OMV according to claim 1, wherein the OMV is not a detergent-extracted OMV.

14. The OMV according to claim 1, wherein the OMV is a supernatant OMV or a native OMV.

15. The OMV according to claim 1, wherein the OMV is obtainable from a Gram-negative bacterium that has one or more genetic modifications selected from the group consisting of: a) a genetic modification causing the bacterium to produce an LPS with reduced toxicity; b) genetic modification that increases vesicle formation; and, c) genetic modification that prevent proteolytic release of cell surface-exposed lipoprotein.

16. The OMV according to claim 15, wherein the Gram-negative bacterium belongs to a genus selected from the group consisting of Neisseria, Bordetella, Escherichia and Salmonella.

Description

DESCRIPTION OF THE FIGURES

(1) FIGS. 1A, 1B, and 1C. Schematic overview of fHbp (hatched), OspA (white) and fusion constructs. Amino acid numbering for fHbp and OspA is shown in boxed areas. S.p.=signal peptide, α1=N-terminal alpha-helix of fHbp including the subsequent loop, β1-4=first four N-terminal beta-sheets of fHbp. Numbered arrows refer to primers listed in Table 1, forward primers are shown above and reverse primers are shown below the schematic (fusion) genes, positioning of the arrow heads reflects the approximate positioning of the 3′end. The artificial linker (6) that was incorporated in some constructs is shown in white. Note that construct fA1 was used as template to create constructs fA3b and fA5c.

(2) FIG. 2. Western blot analysis of OspA expression in N. meningitidis cells (A) or nOMVs (B) carrying OspA or fusion constructs. Cells and nOMVs were normalized based on OD.sub.600 and protein content, respectively. Expected molecular mass of the different constructs is shown in kDa (in parentheses). Arrows indicate the full-length proteins. Wt: N. meningitidis cells (or nOMVs harvested from these cells) carrying ‘empty’ plasmid pEN11-Imp (see Materials and Methods).

(3) FIG. 3. Western blot analysis of mouse sera. Lanes were loaded with E. coli carrying pEN11-OspA (‘OspA’) or pEN11-Imp (‘Imp’). (A) From left to right; (1) control blot with anti-OspA showing the expected band of ˜28 kDa and blots with pooled sera of groups of five mice immunized with (2) PBS, (3) high dose empty nOMVS, (4) high dose OspA nOMVs, (5) high dose fA1 nOMVs, (6) low and (7) high dose fA4b nOMVs, and (8) low and (9) high dose of fA6 nOMVS. (B) Blots with sera from individual mice immunized with a high dose of fA6 nOMVs (pooled sera of this group are at the far right in FIG. 4A). Arrows point at bands with a molecular weight similar to that of OspA (˜28 kDa).

(4) FIG. 4. ELISA data for sera from individual mice immunized with 20 μg/ml nOMVs carrying construct fA4b, fA4c, fA6, or fA7. Pooled sera of mice immunized with 20 μg/ml nOMVs carrying empty vector pEN11-Imp were used as negative control and subtracted from data before plotting. Note that sera of mice immunized with fA6 tested here are the same as the ones in the Western blot shown in FIG. 4b, which is reflected in the lower responsiveness of individual #2.

(5) FIGS. 5A and 5B Schematic overview of TbpB (hatched) and OspA (white) fusion constructs. Amino acid numbering for TbpB and OspA is shown in boxed areas. S.p.=signal peptide. Numbered arrows refer to primers listed in Table 1, forward primers are shown above and reverse primers are shown below the schematic (fusion) genes, positioning of the arrow heads reflects the approximate positioning of the 3′ end.

(6) FIG. 6. Western blot analysis of OspA expression in N. meningitidis cells carrying constructs fTA1-4 using OspA polyclonal (Rockland Immunochemicals). Expected molecular mass of the different constructs is shown in kDa (in parentheses). Arrows indicate full-length proteins.

(7) FIG. 7. Schematic overview and western blot analysis of fHbp-OspA and TbpB-OspA fusion constructs. A) Schematic overview of the constructs. fHbp fragments are hatched, OspA fragments are grey and TbpB fragments are white. Amino acid numbering for fHbp, TbpB and OspA is shown in boxed areas. S.p.=signal peptide. Numbered arrows refer to primers listed in Table 1, forward primers are shown above and reverse primers are shown below the schematic (fusion) genes, positioning of the arrow heads reflects the approximate positioning of the 3′end. B) Western blot analysis of OspA expression in N. meningitidis cells carrying constructs fA10 and fTA5 using OspA monoclonal antibody (Santa Cruz Biotechnologies). Expected molecular mass of the different constructs is shown in kDa (in parentheses). Arrows indicate full-length proteins.

(8) FIG. 8. Schematic overview and western blot analysis of fHbp-OspC fusion constructs. A) Schematic overview of the constructs. fHbp fragments are hatched and OspC fragments are white. Amino acid numbering for fHbp and OspC is shown in boxed areas. S.p.=signal peptide. Numbered arrows refer to primers listed in Table 1, forward primers are shown above and reverse primers are shown below the schematic (fusion) genes, positioning of the arrow heads reflects the approximate positioning of the 3′ end. B) Western blot analysis of OspC expression in N. meningitidis cells with constructs fC9 and OspC. No clear expression for OspC was observed. Expected molecular mass of the different constructs is shown in kDa (in parentheses). The construct fC9 does not form homodimers.

(9) FIG. 9. Schematic overview and western blot analysis of the fHbp-RmpM and TbpB-RmpM fusion constructs. A) Schematic overview of the constructs. fHbp fragments are hatched, RmpM fragments are grey and TbpB fragments are white. Amino acid numbering for fHbp, TbpB and RmpM is shown in boxed areas. S.p.=signal peptide. Numbered arrows refer to primers listed in Table 1, forward primers are shown above and reverse primers are shown below the schematic (fusion) genes, positioning of the arrow heads reflects the approximate positioning of the 3′end. B) Western blot analysis of RmpM expression in N. meningitidis ΔRmpM cells with constructs fR1 and fTR1. Expected molecular mass of the different constructs is shown in kDa (in parentheses). Arrows indicate full-length proteins.

EXAMPLES

1. Materials and Methods

1.1 Antibiotics

(10) Ampicillin (Amp) and chloramphenicol (Cam) were purchased from Sigma. Stock solution were prepared in Milli-Q (MQ) water, filter-sterilized using a 0.22 μm Steriflip (Milipore), and stored at 4° C.

1.2 Bacterial Strains and Growth Conditions

(11) Escherichia coli strains JM109 (Promega) and TOP1OF′ (Invitrogen) were used for cloning steps involving vectors pGEM-T Easy and pEN11, respectively. Both strains were grown at 37° C. on Luria Bertani medium (MP Biomedicals) supplemented with 15 gram agar/liter and appropriate antibiotics (50 μg/ml Amp for pGEM-T Easy and 25 μg/ml Cam for pEN11). For blue/white screening of JM109, plates were supplemented with 50 μg/ml 5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside (X-gal, Fermentas) and 0.1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG, Thermo Scientific). Liquid cultures were grown at 37° C. and 200 RPM.

(12) Neisseria meningitidis strain HB-1 carrying the lpxL1 deletion (52) was grown on Difco GC medium base supplemented with IsoVitaleX (both Becton Dickinson) at 37° C. in a humid atmosphere containing 5% CO.sub.2. Plates were supplemented with 3 μg/ml Cam in case of transformation with pEN11. Liquid cultures were grown in Tryptic Soy Broth (TSB, Becton Dickinson) with 3 μg/ml Cam and 1 mM IPTG at 37° C. and 150 RPM.

(13) Borrelia burgdorferi strain B31 was kindly supplied by the lab of J. Hovius (Amsterdam Medical Centre). Genomic DNA of B. burgdorferi was extracted using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's instructions.

1.3 Recombinant DNA Technology

(14) All primers used in this study are shown in Table 1. Hybrids were made of fHbp (N. meningitidis), TbpB (N. meningitidis), OspA (B. burgdorferi or B. afzelii), OspC (B. burgdorferi) and RmpM (N. meningitidis). All hybrids were constructed using Overlap Extension PCR (53). All PCRs were carried out using the Accuprime Taq DNA Polymerase System (Invitrogen) to ensure both high fidelity amplification and the addition of 3′ A-overhangs. See FIGS. 1 and 5 for a schematic overview of the different constructs.

(15) TABLE-US-00001 TABLE 1  Oligonucleotide primers used in this study SEQ Primer ID Primer number NO name Sequence (5′->3′) 1 9 fHbp-F CATATGCGCCGTTCGGACGACATTTGATTTTTGC 2 10 fHbp-R GACGTCACGGTAAATTATCGTGTTCG 3 11 OspA-F CATATGAAGGAGAATATATTATGAAAAAATATTTATTGG 4 12 OspA-R GACGTCTAAAGCTAACGCTAAAGCAAATCC 5 13 M13-F GTAAAACGACGGCCAG 6 14 M13-R CAGGAAACAGCTATGAC 7 15 pEN11-F AAACCGCATTCCGCACCACAAG 8 16 pEN11-R GGGCGACACGGAAATGTTGAATAC 9 17 fA1-F GTGTCGCCGCCGACATCGGTGCGAGCGTTTCAGTAGATTTGC 10 18 fA1-R GCAAATCTACTGAAACGCTCGCACCGATGTCGGCGGCGACAC 11 19 fA2b-F GGCGACCCGGGTGGCTCAGGTGCTAGCGTTTCAGTAGATTTGC 12 20 fA2b-R AAACGCTAGCACCTGAGCCACCCGGGTCGCCGATTCTGAACTG 13 21 fA3b-F TTAAAACCGGGTGGCTCAGGTGCTAGGGCGACATATCGCGGGACG 14 22 fA3b-R CGCCCTAGCACCTGAGCCACCCGGTTTTAAAGCGTTTTTAATTTC 15 23 fA4b-F AAGCAACCGGGTGGCTCAGGTGCTAGCGTTTCAGTAGATTTGC 16 24 fA4b-R AAACGCTAGCACCTGAGCCACCCGGTTGCTTGGCGGCAAG 17 25 fA4c-F CCTTGCCGCCAAGCAATGTAAGCAAAATGTTAGC 18 26 fA4c-R GCTAACATTTTGCTTACATTGCTTGGCGGCAAGG 19 27 fA5c-F GAAATTAAAAACGCTTTAAAATGCAGCAGCGGAGGGGGTGGTG 20 28 fA5c-R CACCACCCCCTCCGCTGCTGCATTTTAAAGCGTTTTTAATTTC 21 29 fA6-F CCATAAAGACAAAGGTTTGAGCGTTTCAGTAGATTTGC 22 30 fA6-R GCAAATCTACTGAAACGCTCAAACCTTTGTCTTTATGG 23 31 fA7-F CAATACGGGCAAATTGAAGAGCGTTTCAGTAGATTTGC 24 32 fA7-R GCAAATCTACTGAAACGCTCTTCAATTTGCCCGTATTG 25 33 pfHbpTbpB-F CGTATGACTAGGAGTAAACCTATGAACAATCCATTGGT GAATCAGG 26 34 pfHbpTbpB-R CCAATGGATTGTTCATAGGTTTACTCCTAGTCATACG 27 35 TbpB-R GACGTCCGTCTGAAGCCTTATTCTCG 28 36 OspA afz-R GACGTCTACTTTTTGGCTCAGTACC long 29 37 OspC-F CATATGAATAAAAAGGAGGCACAAATTAATG 30 38 OspC-R GACGTCTTAATTAAGGTTTTTTTGGACTTTCTG 31 39 RmpM-R GACGTCGCATCGGCAAGATATTGC 32 40 fA10-F CCATAAAGACAAAGGTTTGAGCGCTTCAGTAGATTTGC 33 41 fA10-R GCAAATCTACTGAAGCGCTCAAACCTTTGTCTTTATGG 34 42 fTA1-F CCTGTGTTTTTGTTGAGTGCTTGTAAGCAAAATGTTAG CAGCCTTG 35 43 fTA1-R CAAGGCTGCTAACATTTTGCTTACAAGCACTCAACAAA AACACAGG 36 44 fTA2-F GCAAGCCCAAAAAGACCAAAGCGTTTCAGTAGATTTGC 37 45 fTA2-R GCAAATCTACTGAAACGCTTTGGTCTTTTTGGGCTTGC 38 46 fTA3-F CAAGCGGCGGAATTGGTATCAGAGGAGCGTTTCAGTAG ATTTGC 39 47 fTA3-R GCAAATCTACTGAAACGCTCCTCTGATACCAATTCCGC CGCTTG 40 48 fTA4-F GGATGATGGTGATATCAAAAGCGTTTCAGTAGATTTGC CTGGTG 41 49 fTA4-R CACCAGGCAAATCTACTGAAACGCTTTTGATATCACCA TCATCC 42 50 fTA5-F GCAAGCCCAAAAAGACCAAAGCGCTTCAGTAGATTTGC 43 51 fTA5-R GCAAATCTACTGAAGCGCTTTGGTCTTTTTGGGCTTGC 44 52 fC9-F GACCATAAAGACAAAGGTTTGAATAAATTAAAAGAAAA ACACACAG 45 53 fC9-R CTGTGTGTTTTTCTTTTAATTTATTCAAACCTTTGTCT TTATGGTC 46 54 fR1-F CCATAAAGACAAAGGTTTGCCGCAATATGTTGATGAAACC 47 55 fR1-R GGTTTCATCAACATATTGCGGCAAACCTTTGTCTTTATGG 48 56 fTR1-F GCAAGCCCAAAAAGACCAACCGCAATATGTTGATGAAACC 49 57 fTR1-R GGTTTCATCAACATATTGCGGTTGGTCTTTTTGGGCTTGC

(16) Amplicons were ligated blunt-end in the pGEM-T Easy vector (Promega) and subsequently heat-shock transformed into E. coli JM109 cells (Promega) according to the manufacturer's instructions. Transformants were screened for insert of the correct length using primers M13-F and M13-R (see Table 1). Plasmids were isolated from overnight cultures of positive JM109 transformants using the Wizard Plus SV Plasmid Miniprep System (Promega). Isolated plasmids were digested using restriction enzymes AatII and NdeI (Fermentas). The resulting fragments were then separated by gel-electrophoresis, and subsequently gel-purified using the Wizard SV Gel and PCR Cleanup System (Promega). Universal plasmid pEN11 (54) was used as vector after digestion with AatII and NdeI in the presence of Shrimp Alkaline Phosphatase (Roche). Inserts and vector were ligated using T4 DNA ligase (Promega) according to the manufacturer's instruction and the resulting plasmids were then heat-shock transformed into E. coli One Shot TOP10F′ competent cells (Invitrogen). Transformants were screened for insert of the correct length using primer pEN11-F and a reverse primer of the respective construct. Approximately 1 μg of isolated pEN11 plasmid (isolation procedure as described before) carrying OspA or fHbp-OspA fusions was added to a 1 ml suspension of N. meningitidis cells (OD.sub.600≈0.2) and grown in TSB supplemented with 10 mM MgCl.sub.2 at 37° C. for 6 hours (no shaking). Bacteria were then plated on GC plates containing 3 μg/ml Cam. Colonies were isolated after 24-48 hours and screened for the presence of pEN11 with correct insert as before.

1.4 Expression of Constructs in N. meningitidis Cells and nOMVs

(17) N. meningitidis cells were streaked out on GC II plates (Becton Dickinson) and grown overnight as described. The following day, colonies were harvested with a sterile cotton swab were suspended in TSB containing 1 mM IPTG and 3 μg/ml Cam and further diluted to an OD.sub.600 of 0.2 in 5 ml of the same broth with supplements. Cells were grown for 4 hours at 37° C. and 170 RPM after which OD.sub.600 was again determined and aliquots corresponding to 4.0×10.sup.8 CFU were centrifuged for 5 min at 13,000 RPM and resuspended in phosphate buffered saline (PBS). Cells were then centrifuged as before, after which the resulting pellet was dissolved in 40 μl MQ, combined with 10 μl 5× sample buffer (50% glycerol, 0.25% Tris pH 6.8, 10% Sodium Dodecyl Sulphate, 10% dithiothreitol and 0.05% Bromophenol blue), and boiled for 10 minutes. Samples were then stored at −20° C. for further analysis.

(18) N. meningitidis native OMVs (nOMVs) were diluted to 20 μg total protein per ml in PBS, after which 40 μl nOMV suspension was boiled with 10 μl 5× sample buffer as before.

(19) Protein samples of cells of nOMVs were separated by SDS-PAGE on 12% Precise Protein Gels (Thermo Scientific). Separated proteins were then transferred to 0.45 μm nitrocellulose membranes (BioRad). Membranes were incubated for 1 hour on a rolling table in a 1:1000 dilution of anti-OspA (Rockland) in buffer containing 0.1 M Tris, 1.54 M NaCl, and 5% Tween-80. The membrane was then transferred to a 1:2000 dilution of goat-anti-rabbit IgG AP (Southern BioTech) in the same buffer supplemented with 0.5% Protifar (Nutricia). Blots were developed using the AP Conjugate Substrate Kit (BioRad).

1.5 Immunostaining

(20) N. meningitidis cells carrying pEN11 with the various constructs were immobilized on coverslips coated with poly-1-lysine (Sigma). Cells were fixated with 2% formaldehyde in PBS for 10 minutes. After blocking in PBS containing 3% Bovine Serum Albumin (BSA, Sigma), the coverslips were first incubated in a 1:300 dilution of a mix of anti-OspA (Rockland) and anti-fHbp (variant 1, NIBSC) in PBS with 0.5% BSA. After washing, they were incubated in a 1:300 dilution of a mix of Alexa Fluor 488 goat-anti-rabbit IgG and Alexa Fluor 594 goat-anti-mouse IgG (Life Technologies). Slides were post-fixed in 2% formaldehyde in PBS and viewed under an Olympus CKX41 fluorescence microscope at 40× magnification using appropriate filters.

1.6 Purification of nOMV Vaccines

(21) Glycerol-stocks of clones selected for the immunization experiment were streaked out on GC II plates and grown overnight under conditions described above. The following day, colonies were harvested and used to start a 200 ml culture of OD.sub.600=0.05 in TSB with 1 M IPTG and 3 μg/ml Cam. These cultures were grown at 37° C. and 130 RPM and OD.sub.600 was measured at regular intervals. When cultures reached an OD.sub.600 of 1.5 (after ˜6 hours) they were placed on ice and subsequently centrifuged at 3,500 RPM and 4° C. for 30 minutes. Pellets were resuspended in a Tris-EDTA buffer (100 mM Tris, 10 mM EDTA, pH=8.6) and incubated on a horizontal shaking table for 30 minutes. Since this buffer contains a chelating agent (EDTA) that destabilizes the OM, the release of OMVs is stimulated. The suspensions were centrifuged at 13,000 RPM for 30 minutes and the supernatant was sterilized using a Steriflip 0.22 μm filter (Millipore). The sterile supernatant was then centrifuged at 40,000 RPM for 65 minutes, after which the resulting OMV pellet was allowed to dry before being resuspended in 1 ml sucrose buffer. Suspensions were again filtered as before and stored at 4° C.

(22) The nOMV isolation procedure described above was developed in order to harvest as many nOMVs as possible without the hitchhiking of other cellular proteins due to lysis. As we noticed that the expression of OspA, fA1, fA2b, fA3b, and fA5c in nOMVs harvested using this procedure could be increased by allowing the respective cultures to grow for 12 hours, without significantly increasing the hitchhiking of other bacterial proteins (data not shown). We therefore decided to use the alternative isolation procedure for these constructs, in order to equalize the expression of constructs as much as possible.

(23) The total protein concentration of the isolated nOMVs was measured using the BCA Protein Assay Kit (Pierce) and nOMVs were then further diluted in PBS to 5 or 20 μg total protein per ml on the day of vaccination.

1.7 Mice and Immunization

(24) Groups of five female, six- to eight-week-old BALB/cOlaHsd mice (Harlan) were immunized subcutaneously with 200 μl of nOMVs, at either low concentration (5 μg/ml) or high concentration (20 μg/ml). Next to the groups that received nOMVs ‘loaded’ with OspA (two groups) or fHbp-OspA fusions (sixteen groups), two control groups received ‘empty’ nOMVS harvested form cells carrying the pEN11 plasmid with the imp gene replacing the ospA-constructs (54). An additional control group was immunized with PBS, resulting in a total of 21 groups. Mice were immunized at days 0 and 28 and sacrificed 14 days after the last immunization. Blood was collected in Vacuette Z Serum Clot Activator tubes (Greiner Bio-One) and centrifuged at 2000 RPM for 15 minutes. Subsequently, sera were collected and stored at −20° C. for further analysis.

1.8 Analysis of Sera

(25) Sera were first pooled by group (five mice) and analyzed for the presence of antibodies by Western blot. Membranes were loaded with proteins from E. coli TOP10F′ cells carrying either pEN11-Imp or pEN11-OspA. Membranes were incubated for one hour on a rolling table with pooled sera diluted 1:1000 in Tris buffer (described previously), followed by incubation in a 1:2000 dilution of secondary antibody (goat-anti-mouse IgG AP, Southern BioTech) in the same buffer and blot development as described previously. The ten individual sera of all mice from the two most strongly reacting groups (fA4b, 20 μg/ml and fA6, 20 μg/ml) were analyzed in the same manner.

(26) For ELISAs, 100 μl of 0.5 μg/ml OspA control protein (Rockland) diluted in PBS was coated on the surface of wells in Microlon 96-well plates (GreinerBio) and incubated overnight at room temperature (RT). The following day, plates were blocked by adding 200 μl 0.5% Protifar in PBS followed by incubation for 30 minutes at RT. Plates were then washed three times in wash buffer (water with 0.05% tween-80). Sera of individual mice were suitably diluted in PBS with 0.1% tween-80 and 100 μl was added per well, followed by incubated for 1 hour at RT. Plates were then again washed three times in wash buffer, after which 100 μl of goat-anti-mouse IgG HRP (SouthernBiotech) was added (diluted 1:4000 in PBS with 0.1% tween-80). After incubation for 1 hour, the plates were washed as before and 100 μl of TMB was added. Plates were then incubated for 10 minutes after which coloring was stopped with 100 μl 2 M H.sub.2SO.sub.4. OD.sub.450 was subsequently measured on a SynergyMx plate-reader (Biotek).

2. Results

2.1 Construction of fHbp-OspA Fusion-Genes

(27) Adjacent to their lipidated N-terminal cysteine, most characterized lipoproteins contain a stretch of amino acids with a low propensity for the formation of secondary structure. This so-called ‘tether’ is thought to act as a flexible linker between the lipid ‘anchor’ (the lipidated cysteine) and the structurally confined part of the protein (55). Tethers also play a role in the transport of lipoproteins over the outer membrane, since deletions in this region can result in mislocalization of surface-exposed lipoproteins to the inside of the outer membrane (55).

(28) Since we found no evidence for the surface exposure of OspA expressed in Neisseria, we hypothesized that the switch of bacterial host resulted in mislocalization of the protein to the inside of the outer membrane. We then set out to test whether the addition of parts of fHbp, a surface-exposed Neisserial lipoprotein, might correct this mislocalization. Since the sorting rules for transport over the outer membrane in Neisseria have not yet been elucidated, we designed hybrid genes that combined various parts of fHbp with the globular domain of OspA.

(29) A schematic overview of fHbp and OspA, as well as the fusion genes created from these two genes by overlap extension PCR, is given in FIG. 1. Information regarding the structure of both lipoproteins was obtained from published crystal structures (48, 56). Both fHbp and OspA contain a signal peptide (that is cleaved after transport over the inner membrane) and a tether region. The globular domain of fHbp consists of an N-terminal and a C-terminal domain that are separated by a 15 amino acid linker.

(30) Genomic DNA of N. meningitidis strain 44/76 was used as template in all PCR reactions involving the amplification of parts of fHbp. The fHbp-F primer anneals upstream of the fHbp promoter (57), and therefore all fusion-genes contain the fHbp promoter. OspA was amplified from genomic DNA of B. burgdorferi strain B31 using primers OspA-F and OspA-R (Table 1), and was successfully expressed in E. coli TOP10F′ from the pEN11 plasmid, which contains an IPTG-inducible tac-lacUV5 promoter (54).

(31) The six amino acid linker peptide that was introduced in constructs fA2b, fA3b, and fA4b was previously used to successfully link OspA to calmodulin (58).

(32) We created eight different fusion constructs between fHbp and OspA. From here on we refer to these constructs as ‘fA’. Primers used for the construction of fusion genes are shown above (forward primers) or below (reverse primers) the schematic genes in FIG. 1 (primer numbers refer to Table 1). All fusion genes were created by overlap extension PCR, a two-step PCR protocol (53). In short, the gene parts to be fused were first amplified separately using partially overlapping primers, after which both parts (that can anneal to each other) were used as template in a second reaction yielding the fusion gene. As an example, the first step PCRs for construct fA1 used primer pairs 1-10 (to amplify the fHbp promoter, signal peptide, and tether from the N. meningitidis genome) and 4-9 (to amplify the globular domain of OspA from the B. burgdorferi genome). The resulting PCR products were then mixed and used as template in a second PCR reaction. In this second reaction, both PCR products anneal to each other and at the same time serve as template for primer pair 1-4 resulting in the full-length PCR product fA1. All other fusion genes were created using the same method. Note that construct fA1 served as template for the construction of fA3b and fA5c.

(33) In fA1, the signal peptide and tether of fHbp were fused to the globular domain of OspA. In constructs fA2b and fA3b, the C-terminal domain (fA2b) or N-terminal domain (fA3b) of fHbp was replaced by the globular domain of OspA and connected via an artificial linker (PGGSGA). In constructs fA4b and fA4c, the complete fHbp gene was linked to the globular domain of OspA using the artificial linker (fA4b) or the OspA tether (fA4c). Construct fA5c is fA1 linked to the globular domain of fHbp via the fHbp tether. Constructs fA6 is similar to fA1, but in addition to signal peptide and tether also contains the first alpha-helix and subsequent loop of the N-terminal domain of fHbp. Construct fA7 is similar to fA6, but additionally contains the first four beta-sheets of the N-terminal domain of fHbp. All constructs were successfully expressed from pEN11 in E. coli TOP10F′ before transformation in Neisseria (data not shown).

2.2 Construction of TbpB-OspA Fusion Genes

(34) TbpB of N. meningitidis H44/76 contains a signal peptide (residues 1-20), a 38 amino acid flexible linker or ‘tether’ (residues 21-58), and a large globular domain (residues 59-691). In order to test whether fusion of N-terminal parts of TbpB to OspA could rescue surface expression of OspA in N. meningitidis, four TbpB-OspA fusion constructs were designed. Because TbpB has an iron-regulated promoter, the TbpB promoter was first exchanged for the constitutive fHbp promoter using overlap extension PCR with primers 1, 25-27 (see Table 1). The resulting construct (‘TbpB with fHbp promoter’, see FIG. 5) was then used as template for fusion PCRs between TbpB and OspA.

(35) An overview of constructs fTA1-4 can be found in FIG. 5, while primers for the overlap-extension PCRs can be found in Table 1. Genomic DNA from Neisseria meningitidis H44/76 was used as template for amplification of parts of TbpB and genomic DNA of Borrelia burgdorferi strain B31 was used as template for the OspA part.

(36) Construct fTA1 contained residues 1-20 (the signal peptide) of TbpB fused to residues 17-273 (tether and globular domain) of OspA. Construct fTA2 contained residues 1-58 (signal peptide and tether) of TbpB fused to residues 29-273 (globular domain) of OspA. Construct fTA3 contained residues 1-75 (signal peptide, tether and first seventeen N-terminal amino acids of the globular domain) of TbpB fused to residues 29-273 (globular domain) of OspA. Construct fTA4 contained residues 1-99 (signal peptide, tether and first 41 N-terminal amino acids of the globular domain) of TbpB fused to residues 29-273 (globular domain) of OspA. TbpB fusion points for fTA3 and fTA4 were chosen in loops between secondary structure elements, based on the TbpB crystal structure (63).

(37) To test whether OspA serotypes other than serotype 1 (B. burgdorferi B31) could be transported to the meningococcal cell surface with the help of N-terminal parts of fHbp or TbpB, fusion constructs similar to fA6 and fTA2 were made, the only difference being that they contained the globular domain of OspA serotype 2 from Borrelia afzelii PKo (residues 29-273). All constructs were made by overlap extension PCR, see FIG. 7 and Table 1 for details.

2.3 Construction of fHbp—OspC Fusion Genes

(38) OspC is a surface-exposed borrelial lipoprotein. It is generally considered to be the most interesting vaccinogen after OspA (64). However, the expression of full-length OspC including its signal sequence has proven difficult in E. coli (65), possibly due to its tendency to aggregate; in the Borrelia outer membrane, it is present in the form of large multimeric complexes. Expression of full-length OspC in both E. coli and N. meningitidis previously led to similar problems in our hands, resulting in poor expression on Western blots (data not shown).

(39) In Borrelia, OspC forms homo-dimers on the cell-surface, mostly by interactions between the N-terminal al-helices (66, 67). Since most murine and human OspC epitopes are located on the C-terminal side of the protein (68, 69), fusion construct fC9 (FIG. 8) was generated, that combined the previously described N-terminal part of fHbp (as used in fA6) and C-terminal residues 136-210 of OspC (see FIG. 8). To this end, genomic DNA of N. meningitidis strain H44/76 was used as template for the amplification of fHbp and genomic DNA of B. burgdorferi strain B31 was used for amplification of OspC

2.4 Construction of fHbp-RmpM and TbpB-RmpM Fusion Genes

(40) RmpM is an outer membrane protein of N. meningitidis that is thought to associate non-covalently with the peptidoglycan layer (70). It consists of a signal peptide, a flexible N-terminal domain (which binds to integral outer membrane proteins), and an OmpA-like C-terminal domain (which is thought to associate with peptidoglycan). Since RmpM is generally considered to be mostly periplasmic, an attempt was made to express RmpM at the cell surface of N. meningitidis. It was decided to leave out the first 89 residues that consist of signal peptide and the unstructured N-terminal domain. The remaining C-terminal domain (residues 90-242) was fused to N-terminal parts of fHbp (residues 1-50) and TbpB (residues 1-58) that had been used previously for the successful surface localization of OspA. Genomic DNA of N. meningitidis strain H44/76 was used as template for the amplification of fHbp, TbpB, and RmpM. The constructs were named fR1 and fTR1 (see FIG. 9).

2.5 OspA and fHbp-OspA Fusions are Expressed in N. meningitidis Cells and nOMVs

(41) The expression of the different constructs in N. meningitidis is shown in FIG. 2A and expression in nOMVs harvested from these cells is shown in FIG. 2B. Expression levels in N. meningitidis cells clearly vary between the different constructs and similar variation is observed in the nOMVs, with high expression levels for constructs fA4b, fA4c, fA6, and fA7. Several constructs are apparently prone to degradation. For some constructs (fA3b, fA5c), degradation seems to be amplified in nOMVs compared to cells.

2.6 at Least Four fHbp-OspA Hybrids are Surface Exposed

(42) We tested whether or not OspA and the eight fHbp-OspA fusions were surface-localized in N. meningitidis using immunostaining. Briefly, cells containing plasmid pEN11 with the various constructs were incubated with a mix of anti-OspA and anti-fHbp (positive control), followed by incubation with fluorescent secondary antibodies (green for OspA and red for fHbp). Cells containing constructs fA4b, fA4c, fA6, and fA7 showed clear green fluorescence, indicating surface exposure of these constructs (data not shown). No green fluorescence was observed for OspA or any of the other constructs (data not shown), indicating that they were not surface exposed, although we cannot rule out the possibility that this is due to their lower expression level (see FIG. 2A).

2.7 Expression and Surface-Exposure TbpB-OspA Hybrids

(43) Successful expression of all four constructs in E. coli and N. meningitidis was confirmed by Western blot using a polyclonal antiserum against OspA (Rockland Immunochemicals—see FIG. 6 for Neisseria Western blots). Immunostaining (as described above) showed no signal for construct fTA1, but a strong signal for constructs fTA2, fTA3, and fTA4 (data not shown). This proves that N-terminal parts of TbpB can be used for the surface localization of OspA and that at least signal peptide and tether are required for this.

2.8 Expression and Surface-Exposure of Alternative C-Protein Fusion Partners

2.8.1 Expression and Surface Exposure of an Alternative OspA Serotype

(44) Successful expression of both constructs in E. coli and N. meningitidis was confirmed by Western blot using a polyclonal antiserum against OspA (Rockland Immunochemicals—see FIG. 7 for Neisseria Western blots). Immunostaining with the OspA polyclonal (Rockland Immunochemicals) proved difficult, most probably because the antiserum (which was raised against serotype 1 OspA from B. burgdorferi) showed considerably weaker binding to the B. afzelii OspA. Therefore, an OspA monoclonal antibody (Santa Cruz Biotechnology) was used for immunostaining. N. meningitidis cells expressing constructs fA10 and fTA5 showed a strong signal when this monoclonal was used for immunostaining (data not shown), indicating that the serotype 2 OspA was indeed successfully expressed at the cell surface.

2.8.2 Expression and Surface Exposure of OspC

(45) Despite poor expression in N. meningitidis (FIG. 8), cells carrying the fC9 construct surprisingly showed a signal with immunostaining (data not shown). This indicates that the N-terminal part of fHbp (as previously described in fA6) can be used for the surface localization of at least C-terminal parts of OspC.

2.8.3 Expression and Surface Exposure of the Non-Borrelial Non-Lipoprotein RmpM

(46) Both constructs (named fR1 and fTR1, see FIG. 11), were successfully expressed in E. coli and N. meningitidis.

(47) Although immunostaining yielded bright signals for both constructs, the negative control (N. meningitidis cells without construct) showed bright fluorescence as well. This indicated either a-specific binding of the MN2D6D monoclonal antibody to Neisseria cells, or unanticipated surface localization of (parts of) RmpM. Therefore, a RmpM knockout was created using plasmid pCF13 (71). Plasmids carrying constructs fR1 and fTR1 were transformed in this ΔRmpM strain as before and successful expression of both constructs was determined by Western blot (see FIG. 12).

(48) Immunostaining of N. meningitidis cells and the RmpM knockout showed that there was a-specific binding of the RmpM antibody at high antibody concentrations (strong staining of the knockout at 1:300 dilution and weak staining of the knockout at 1:2000 dilution, data not shown), while at low antibody concentrations (1:10,000) staining of the knockout completely vanished (data not shown). However, some normal Neisseria cells still showed staining at this low concentration (data not shown), indicating that at least for part of the cells RmpM might be (partially) surface exposed.

(49) When placed in the RmpM knockout background, both fR1 and fTR1 showed a fluorescent signal at the lowest antibody concentration (1:10,000), while staining was completely absent for the RmpM knockout without construct at this concentration (data not shown). This indicates that the N-terminal parts of both fHbp and TbpB that were previously used for the surface localization of OspA can also be used for the surface localization of an otherwise (predominantly) periplasmic non-lipoprotein of Neisseria meningitidis.

2.9 Immunogenicity of nOMVs Carrying OspA and fHbp-OspA Hybrids

(50) Next, we investigated the sera of mice immunized with nOMVs carrying heterologous (fusion) proteins using Western blot. Pooled sera of all 21 groups were blotted on membranes loaded with total protein content of E. coli with either pEN11-OspA or pEN11-Imp (see FIG. 3A for some of these groups). Pooled sera of the groups immunized with nOMVs carrying both low and high doses of constructs fA4b, fA4c, fA6, and fA7 all showed a strong band with the molecular mass of OspA (˜28 kDa), indicating a strong antibody response. Additionally, the pooled sera of the group immunized with the 20 μg/ml dose of OspA-carrying nOMVS showed a very weak band at ˜28 kDa (data not shown). All other groups showed no detectable response.

(51) To see whether all individual mice raised OspA-specific antibodies, we blotted the sera of all ten individual mice from two highly responsive groups (20 μg/ml fA4b and 20 μg/ml fA6). In all cases a ˜28 kDa band was detected (see FIG. 3B for the five mice immunized with 20 μg/ml fA6, data for fA4b not shown). This shows that the observed signals in the pooled sera were not due to single hyper-responders in the groups. Note that the ˜36 kDa background signal in the pooled sera of the high dose fA6 group (FIG. 3A) is clearly caused by a single individual (mouse #4 in FIG. 4B).

(52) In order quantify the immune response, sera of all individual mice immunized with 20 μg/ml nOMVs carrying constructs fA4b, fA4c, fA6, or fA7 were tested for their binding capacity to purified OspA protein. Results for constructs fA4b and fA6 are shown in FIG. 4. The signal detected for fA4b was ˜1.5 times higher than that observed for fA6 and fA7 and ˜2.5 times higher than that observed for fA4c (data not shown).

3. Discussion

(53) OMVs are gaining attention as a robust and engineerable vaccine platform against many bacterial diseases. With the recognition of their vaccine potential, interest in heterologous expression in OMVs has flourished. One of the unresolved issues regarding heterologous expression in OMVs is whether the location of the heterologous antigen (lumen, inside of OM, outside of OM) affects the immune response that it evokes. For whole cells, some studies indicate that heterologous antigens are more immunogenic when they are located on the cell surface than when they reside beneath it (59, 60), and it has been suggested that the same may be true for OMVs (21). This makes the display of heterologous proteins on the OMV surface of special interest.

(54) Here, we demonstrate a novel approach for the surface display of heterologous antigens in OMVs. A detergent-free OMV extraction process was recently developed for Neisseria that allows surface-exposed lipoproteins to remain attached to the OMV. This new extraction protocol opens up the possibility to decorate the surface of Neisseria OMVs with heterologous lipoproteins. We therefore expressed OspA, a Borrelial surface lipoprotein, in N. meningitidis. Although OspA could be detected in Neisseria cells and OMVs, we found no evidence for its surface exposure. We then constructed fusions between OspA and fHbp, a meningococcal surface lipoprotein. Several of these constructs consisting of N-terminal parts of fHbp linked to OspA could be detected at the cell surface using immunostaining. Furthermore, we have demonstrated that technology is more broadly applicable as lipoprotein mediated surface expression could also be mediated by N-terminal parts of other lipoproteins, such as TbpB. In addition we demonstrated that a variety of antigenic proteins, including non-borrelial non-lipoproteins such as RmpM, could be surface expressed by fusion to N-terminal parts of lipoproteins. Hence, we have shown that the technology is broadly applicable to mediate the surface expression of specific antigens. The antigens may be associated with an infectious disease and/or a tumour. The fusion proteins of the invention cause surface expression of such antigens on e.g. OMVs, which triggers an immune response, resulting in the prevention or treatment of an infectious disease or tumour associated with the antigen.

(55) In this respect, OMVs carrying these constructs elicited a strong immune response in immunized mice, while OMVs carrying constructs that showed no evidence of surface exposure did not.

3.1 Outer Membrane Translocation

(56) Knowledge of factors that govern the translocation of lipoproteins over the OM is frugal and incomplete. In Borrelia, the lipoprotein tether (the unstructured region adjacent to the N-terminal cysteine) of several surface lipoproteins (OspA, OspC, and Vsp) seems to contain essential information for OM translocation, since deletion or mutation of amino acids in this region can lead to subsurface localization. Furthermore, fusion of the signal peptide and tether of these three lipoproteins to red fluorescent protein leads to surface localization of this otherwise periplasmic reporter-protein in Borrelia (47, 51, 55).

(57) Neisseria has only a few surface lipoproteins and what factors affect their translocation over the OM is unknown. Our data indicate that, at least for fHbp, these factors are different from those found in Borrelia.

(58) In our hands, expression of OspA in N. meningitidis leads to mislocalization in the periplasm or periplasmic side of the outer membrane. This is in line with previous findings that expression of a lipoprotein in an alternative host can lead to mislocalization, probably because host factors define the localization rules (51). We reasoned that hybridization with an autologous surface exposed lipoprotein from Neisseria like fHbp or TbpB might rescue this mislocalization and therefore constructed fusions between OspA and different parts of fHbp or TbpB. Replacement of the OspA signal peptide and tether with that of fHbp (construct fA1) did not restore OspA surface localization. However, when the fHbp part of this fusion gene was extended with the first 17 N-terminal amino acids of the globular domain of fHbp (consisting of the first alpha-helix and subsequent loop), the resulting construct (fA6) could be detected at the cell surface. This indicates that sorting rules may differ between Neisseria and Borrelia, since in the former the region affecting outer membrane translocation seems to extend beyond the tether region.

(59) Almost all constructs that consists of fusion of OspA to an N-terminal part of fHbp longer than that in fA6 were detected at the cell surface (fA4b, fA4c, fA7). The only exception is construct fA2b, which contains amino acids 1-152 of fHbp (the so-called N-terminal domain) coupled to OspA via an artificial linker. It is however important to stress that considering the extremely poor expression level of fA2b in cells (see FIG. 2A), it is not possible to discriminate whether the lack of signal after immunostaining resulted from subsurface localization or poor expression. We discuss this localization-expression problem further below. Constructs in which OspA was placed in between N-terminal and C-terminal parts of fHbp (fA3b, fA5c) were not detected at the cell surface.

3.2 Effect of Antigen Location on Immunogenicity

(60) We found evidence for surface exposure of constructs fA4b, fA4c, fA6, and fA7 in Neisseria cells. Coinciding with this, OMVs carrying these constructs were the only ones to elicit strong antibody responses in immunized mice. This suggests that only antigens that are located on the surface of the OMV can elicit antibody responses. However, it is important to realize that the expression level of the four ‘surface’ constructs is clearly higher than that of the other constructs, both in cells and nOMVs (see FIG. 2). Failure to detect a specific construct after immunostaining could therefore also result from low expression levels rather than subsurface localization. It is not possible to exclude this scenario for constructs that have a very low expression levels in cells, e.g. fA1 and fA2b. However, cells that express OspA, fA3b, or fA5c have considerably higher expression levels, and it seems unlikely that we would not observe them after immunostaining in case they were surface exposed.

(61) If we compare the Western blots of the groups of mice immunized with high dose OspA and low dose fA6 carrying OMVs (FIG. 2A, blots 4 and 5 from left), the difference in apparent immunogenicity is much higher than we would expect based on the observed expression levels of these constructs in OMVs (FIG. 2B). This indicates that surface display of a heterologous antigen in the OMV can indeed lead to enhanced immunogenicity.

3.3 Vaccine Potential

(62) Borrelia vaccines that are currently in development are subunit vaccines that are based on various recombinant lipidated OspA serotypes (38, 39). However, subunit vaccines suffer from poor immunogenicity and require the use of adjuvants. The presentation of OspA on the surface of Neisserial nOMVs may result in better immunogenicity because of the intrinsic adjuvant activity of the nOMV and the presentation of the antigen in its native conformation.

(63) Our OMV surface display method is generally applicable to other antigenic proteins from pathogens, including also non-lipoproteins, especially since it has been shown that lipidation of non-lipoproteins via fusion to lipoproteins can enhance immunogenicity (62). Furthermore it is possible to combine the expression and OMV surface display of multiple heterologous antigens in order to facilitate the production of multivalent heterologous OMVs.

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