Multicomponent meningococcal vaccine

Abstract

A composition is provided comprising N. meningitidis outer membrane vesicles, wherein said outer membrane vesicles are enriched with at least one antigenic component. The composition is suitable for use in vaccines and for treatment of infection, particularly meningococcal infection, demonstrating a broad spectrum of protection. A number of preferred antigenic components are described and include antigenic proteins and proteoglycans derived from N. meningitidis.

Claims

1. A composition comprising: (i) isolated N. meningitidis outer membrane vesicles; (ii) at least one isolated N. meningitidis antigenic protein, wherein said at least one isolated N. meningitidis antigenic protein (a) has been extracted from an outer membrane of an N. meningitidis bacterium; or (b) is a recombinant N. meningitidis antigenic protein; and (iii) alum in an amount effective as an adjuvant; wherein said isolated N. meningitidis outer membrane vesicles are from a first strain of N. meningitidis and said at least one isolated N. meningitidis antigenic protein is from a second strain of N. meningitidis different from the first, said at least one isolated N. meningitidis antigenic protein is not transferrin binding protein A (TbpA) nor transferrin binding protein B (TbpB), and the N. meningitidis outer membrane vesicles are isolated by desoxycholate treatment.

2. The composition of claim 1, wherein said composition comprises a plurality of isolated N. meningitidis antigenic proteins from different strains of N. meningitidis.

3. The composition of claim 1, wherein said at least one isolated N. meningitidis antigenic protein is an isolated N. meningitidis antigenic proteoglycan.

4. The composition of claim 1, wherein said at least one isolated N. meningitidis antigenic protein is an isolated N. meningitidis protein selected from the group consisting of a surface antigen, a periplasmic protein, a superoxide dismutase, and a glycoprotein.

5. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.

6. The composition of claim 1, wherein the composition is obtained by a process comprising: (i) obtaining the isolated N. meningitidis outer membrane vesicles from a first strain of N. meningitidis; (ii) obtaining the at least one isolated N. meningitidis antigenic protein from a second strain of N. meningitidis different from the first; and (iii) combining the at least one isolated N. meningitidis antigenic protein with the isolated N. meningitidis outer membrane vesicles.

7. A vaccine composition comprising: (i) outer membrane vesicles from a first strain of N. meningitidis; (ii) an isolated antigenic protein from a second strain of N. meningitidis different from the first, wherein said isolated antigenic protein (a) has been extracted from an outer membrane of an N. meningitidis bacterium; or (b) is a recombinant N. meningitidis antigenic protein; and (iii) a pharmaceutically acceptable carrier comprising alum in an amount effective as an adjuvant, wherein said isolated antigenic protein is not transferrin binding protein A (TbpA) nor transferrin binding protein B (TbpB), and the N. meningitidis outer membrane vesicles are isolated by desoxycholate treatment.

8. The vaccine composition of claim 7, wherein said isolated antigenic protein is an N. meningitidis protein selected from the group consisting of a surface antigen, a periplasmic protein, a superoxide dismutase, and a glycoprotein.

9. The composition of claim 1, wherein said at least one isolated N. meningitidis antigenic protein comprises a surface antigen.

10. The composition of claim 2, wherein said at least one isolated N. meningitidis antigenic protein comprises a surface antigen.

11. The composition of claim 5, wherein said at least one isolated N. meningitidis antigenic protein comprises a surface antigen.

12. The composition of claim 6, wherein said at least one isolated N. meningitidis antigenic protein comprises a surface antigen.

13. The composition of claim 7, wherein said isolated N. meningitidis antigenic protein comprises a surface antigen.

14. The vaccine composition of claim 7, wherein said vaccine composition comprises a plurality of isolated N. meningitidis antigenic proteins from different strains of N. meningitidis.

15. A vaccine composition comprising: (i) outer membrane vesicles from a first strain of N. meningitidis; (ii) an isolated antigenic protein from a second strain of N. meningitidis different from the first, wherein said isolated antigenic protein (a) has been extracted from an outer membrane of an N. meningitidis bacterium; or (b) is a recombinant N. meningitidis antigenic protein; and (iii) a pharmaceutically acceptable carrier comprising an adjuvant, wherein the adjuvant is alum adjuvant, wherein said isolated antigenic protein is not transferrin binding protein A (TbpA) nor transferrin binding protein B (TbpB), and the N. meningitidis outer membrane vesicles are isolated by desoxycholate treatment.

16. The vaccine composition of claim 15, wherein said isolated antigenic protein is an N. meningitidis protein selected from the group consisting of a surface antigen, a periplasmic protein, a superoxide dismutase, and a glycoprotein.

17. The composition of claim 15, wherein said isolated N. meningitidis antigenic protein comprises a surface antigen.

18. The vaccine composition of claim 15, wherein said vaccine composition comprises a plurality of isolated N. meningitidis antigenic proteins from different strains of N. meningitidis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The embodiments of the invention are discussed in more detail by means of the Example described below. The results referred to in the Example are illustrated by the accompanying drawings, in which:

(2) FIG. 1 shows immunisation of mice with TbpA+B and outer membrane vesicles;

(3) FIG. 2 shows immunisation of mice with TbpA+B;

(4) FIG. 3 shows protection of mice against IP infection after immunisation with TbpA+B, isolated TbpA or isolated TbpB;

(5) FIGS. 4A and 4B show protection of mice against meningococcal infection following immunisation with either neisserial TbpA+B, TbpB or TbpA (nTbps) or recombinant Tbps (rTbps);

(6) FIGS. 5A and 5B show protection of mice against challenges of 10.sup.6 and 10.sup.7 organisms/mouse of N. meningitidis strain K454 respectively, following immunisation with recombinant TbpA+B, TbpB or TbpA;

(7) FIG. 6 shows protection against challenge with heterologous serogroup;

(8) FIG. 7 shows protection against challenge with B16B6;

(9) FIG. 8 shows insertion of a promoter construct into the N. meningitidis genome for up regulation of endogenous TbpB in N. meningitidis by homologous recombination;

(10) FIG. 9. shows insertion of an expression cassette into the N. meningitidis genome by homologous recombination;

(11) FIG. 10 illustrates IgG surface labelling of N. meningitidis strains NZ98/254 and M01-240149. Total IgG binding to both strains NZ98/254 and M01-240149 was increased when mice were vaccinated with OMV+PorA, as compared to vaccination with OMVs alone. The symbol ** indicates a greater than 95% certainty of a significant difference between these values, determined using a z-test; and

(12) FIG. 11 illustrates the OPA response against N. meningitidis strains NZ98/24 and M01-240149. Mice immunised with OMV+PorA elicited a greater OPA response against strains NZ98/254 and M01-240149, as compared to the OPA response to immunisation with OMVs alone. The symbol ** indicates a greater than 95% certainty of a significant difference between these values, determined using a z-test.

DETAILED DESCRIPTION

(13) The invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1

(14) Mouse Protective Data

(15) Mice (CAMR-NIH) were immunised with Tbps and/or outer membrane vesicles and challenged with either the homologous or a heterologous meningococcal strain. Survivors per group of mice immunised with Tbps from strain K454 and the outer membrane vesicle vaccine following challenge by strain K454 are shown in FIG. 1. Compared to Tbps, the outer membrane vesicle vaccine gave reduced protection but this may be because it is produced from a different group B strain (H44/76). Animals immunised with TbpA+B isolated from strain K454 were also protected against challenge with other serogroup B organisms (FIG. 1), with greater protection seen with the homologous strain and strains expressing a TbpB with a similar molecular weight. Little or no protection was observed against challenge with meningococci possessing TbpB with a very different molecular weight (strain B16B6). With the heterologous challenge strains, there is a slightly greater number survivors in the groups vaccinated with the combination of Tbps+outer membrane vesicles. However, the numbers involved are small and no definite conclusions can be reached. It is interesting to observe that mice immunised with TbpA+B from strain K454 were also protected against infection with a serogroup C but not a serogroup A strain (FIG. 2).

(16) Protection against challenge with strain K454 with mice immunised with co-purified TbpA+B and isolated TbpA and TbpB is shown in FIG. 3. It can be seen that TbpB is the predominant antigen responsible for protection, with little protection afforded by TbpA alone.

(17) Recombinant TbpA and TbpB

(18) TbpA and TbpB from N. meningitidis strain K454 were cloned and overexpressed in E. coli. The proteins were purified using affinity chromatography and used to determine their protective potency in a mouse model of meningococcal disease. Recombinant Tbps showed equivalent protection to that provided by Tbps isolated from iron-stressed N. meningitidis (FIGS. 4A and 4B). These recombinant Tbps were also utilised in two further larger IP challenge experiments (FIGS. 5A-B).

(19) The strong and consistent protective potency of Tbps against mouse I.P. infection with N. meningitidis is probably the most compelling evidence for their vaccine potential.

(20) Interestingly, the results shown in FIGS. 4B and 5A-B, although arising from experiments of similar design, show the surprising variability associated with vaccine compositions that depend solely upon either TbpA or TbpB alone. In FIG. 4B the recombinant TbpA composition displayed low levels of protection compared to TbpB or TbpA+B. However, in FIGS. 5A and 5B the recombinant TbpA vaccine composition that showed high levels of protection comparable to the TbpA+B complex and the TbpB alone composition demonstrated poor protection to infection. This latter result is completely contrary to the teaching of the prior art.

(21) Human Immune Response to TbpA and TbpB in Convalescent Sera

(22) We have undertaken a number of studies looking at the antibody response in humans to TbpA and TbpB following meningococcal disease. The general conclusions are that both TbpA and TbpB are expressed during meningococcal disease and that an immune response is raised against them. The response is functional (opsonic) and is more cross-reactive between different meningococcal strains than is the response induced by immunisation of animals with Tbps. The immune response to TbpA appears to be stronger and more cross-reactive than that to TbpB, confirming the importance of the vaccine potential of TbpA.

(23) TbpA and TbpB Form a Transferrin Receptor

(24) Our structural studies indicate that the transferrin receptor on the meningococcal surface consists of two TbpA molecules and one TbpB molecule that act together.

(25) Effect of a Vaccine Containing A+B Tbps

(26) We carried out further tests of the efficacy of recombinant TbpB versus recombinant TbpA+B containing vaccine formulations against challenge from N. meningitidis strains L91 705 and B16B6, the results of which are illustrated in FIGS. 6 and 7 respectively. In both cases some improved protection was conferred by A+B compared to B alone.

Example 2

Up Regulation of Genes in Meningococcal Species

(27) These methods can be used to enhance expression of TbpB but can equally be applied to other meningococcal outer membrane proteins with known sequence such as NspA (Martin et al., 1997), OMP85 (Manning et al., 1998) or FrpB (Pettersson et al., 1995). Any of the antigenic component sequences disclosed herein (SEQ ID NOS: 1; 3; 5; 7; 9; 11; 13; 15; 17; 19; 21; 23; 25; 27; 29; 31; 33; 35; 37; 39; 41; 43; 45; 47; 49; 51; 53; 55; 57; 59; 61; 63; 65; 67; 69; and 71) would also be suitable for over expression in this system as would any immunogenic meningococcal sequence.

(28) In the following methods primers are designed using the primer select program from the >DNA star=software package (www.dnastar.com). Primers to flanking sequences are designed following sequencing from the known coding region.

(29) Up Regulation of TbpB in N. meningitidis by Promoter Delivery

(30) Using homologous recombination (Frosch et al., 1990, van der Ley et al., 1995) a strong promoter such as the porA promoter is inserted into the N. meningitidis genome upstream of the tbpB coding region (Legrain et al., 1993). A selectable marker, encoding resistance to kanamycin, is also included in the delivery cassette upstream from the promoter. The integration locus is selected by sequencing upstream from the tbpB coding region.

(31) PCR primers are designed to the regions flanking the porA promoter and incorporate restriction endonuclease sites for ligation into this amplified sequence. This is then inserted into the multiple cloning site of a vector containing the kanamycin resistance gene. Also inserted through PCR produced products are integration loci that complement a region directly before the coding region for tbpB in the N. meningitidis genome. Through homologous recombination the construct shown in FIG. 8 will be produced.

(32) From the published sequence of porA (McGuinnes et al. 1990) (SEQ ID NO: 1) a primer is constructed at the N-terminal region in order to sequence up steam of the coding region. This sequence data yields the promoter region of the highly expressed porA gene. Primers are designed to this sequence so as to amplify any conserved hexamers centred around −35 and −10 the consensus sequences of TTGACA and TATAAT with a 16-18 bp gap between them and up to 67 bp upstream of the start of transcription (ATG). The designed primers have restriction endonuclease sites incorporated in order to insert the amplified region into a suitable vector.

(33) In order to identify a suitable integration locus in front of the tbpB coding region a primer is designed to the start of the tbpB coding region, using the published sequence (Legrain et al. 1993), running up stream into the tbpB promoter region. The promoter region itself as well as some sequence upstream is suitable as the integration locus. This section is amplified in two halves using 4 primers and restriction endonucleases are incorporated in order to ligate the sections into a suitable vector.

(34) The vector used already contains an antibiotic resistance marker (such as kanamycin) and this is suitably positioned between the integration loci but upstream from the incorporated porA promoter, as illustrated in FIG. 8. This means that the vector used has cloning sites either side of the selectable marker.

(35) Up Regulation of TbpB in N. meningitidis by Gene Delivery

(36) A construct is produced containing a selectable marker, such as kanamycin resistance, up stream from a strong promoter (porA) which is in turn upstream from the coding region for tbpB. Using homologous recombination (Frosch et al., 1990, van der Ley et al., 1995), the cassette is inserted into the N. meningitidis genome. The integration locus is selected by sequencing upstream from a known sequenced area that is also known to actively expressed.

(37) PCR is used to amplify the tbpB coding region and porA promoter region with suitable restriction endonucleases incorporated so they can be inserted in series, into a vector containing kanamycin resistance. Also inserted into the construct are two flanking integration loci complementing a region of the N. meningitidis genome (see FIG. 9).

(38) From the published sequence of porA (McGuinness et al. 1990) (SEQ ID NO: 1) a primer can be constructed to complement the N-terminal region in order to sequence up steam of the coding region. This sequence data yields the promoter region of the highly expressed porA gene. Primers are designed to this sequence so as to amplify any conserved hexamers centred around −35 and −10 the consensus sequences of TTGACA and TATAAT with a 16-18 bp gap between them and up to 67 bp upstream of the start of transcription (ATG). The reverse primer is designed to be as close as possible to the start of the coding region, but does not actually include any of it. This ensures that when it is positioned in series with any inserted DNA it acts as an efficient promoter. The designed primers have restriction endonuclease sites incorporated in order to insert the amplified region into a suitable vector.

(39) From the published sequence of tbpB (Legrain et al. 1993) primers are constructed to complement the C-terminal and N-terminal regions in order to sequence the flanking regions of the gene. From this sequence primers are constructed which amplify the entire gene. The forward primer incorporates the first coding triplet (ATG) of the gene and does not include the full native promoter region. The reverse is as close to the end of the coding region as possible while still being a good partner to the forward primer. It is accepted that this means the reverse primer may be some distance downstream of the gene of interest.

Example 3

(40) A combination of outer membrane vesicles (OMV)+PorA extends the cross-reactivity of the antibody response.

(41) We have demonstrated that the addition of recombinant PorA from strain NZ98/254 (PorA serosubtype P1.7-2, 4) to OMVs isolated from strain 44/76 (PorA serosubtype P1.7, 16) broadens the protective response elicited by OMVs from strain 44/76.

(42) Serum bactericidal antibody (SBA) is a correlate of protection (Frasch et al., Vaccine, 2009; 27 Suppl 2:B112-6).

(43) We have shown that the SBA titre against strain NZ98/254 in serum from mice vaccinated with 44/76 OMVs alone was 16 (Table 1). However, the SBA titre obtained against this strain with OMV+PorA was 256.

(44) TABLE-US-00001 TABLE 1 SBA titres: SBA titre against Vaccine strain NZ98/254 OMVs from 44/76 16 PorA from NZ98/254 4 OMVs from 44/76 + 256 PorA from NZ98/254

(45) Binding of IgG antibody to the surface of intact N. meningitidis can be determined by flow cytometry. Antibody binding to the organism is essential for bactericidal activity and also opsonophagocytic activity. FIG. 10 shows that the total IgG binding to both strains NZ98/254 and M01-240149 was increased when mice were vaccinated with OMV+PorA, as compared to vaccination with OMVs alone.

(46) Opsonophagocytosis (OPA) is also important for protection against meningococcal disease (Granoff, Vaccine, 2009; 27 Suppl 2:B117-25). FIG. 11 shows that mice immunised with OMV+PorA elicited a greater OPA response against strains NZ98/254 and M01-240149, as compared to the OPA response to immunisation with OMVs alone.

(47) In summary, the SBA, SLA and OPA assays all confirm that the OMV+PorA vaccine elicits greater cross-strain protection than OMV alone.

(48) Methods:

(49) Animal Vaccination

(50) NIH mice (six to eight weeks old, Harlan, UK) were immunized on days 0, 21, and 28 by subcutaneous injection (0.2 ml), with:

(51) MPL adjuvant only;

(52) MPL and 10 μg Neisseria meningitidis 44/76-SL OMVs;

(53) MPL and 10 μg PorA (P1.7-2,4); or

(54) MPL with 10 μg Neisseria meningitidis 44/76-SL and 10 μg PorA (P1.7-2,4)

(55) On Day 35 terminal sera were collected from mice.

(56) Serum bactericidal assay (SBA):

(57) SBA assays were performed using a standardized assay with a starting dilution of 1:2. Briefly, human serum, at 25%, was used as an exogenous source of human complement, with titers expressed as the reciprocal of the final dilution giving ≥50% SBA killing at 60 min compared with the level for the control (inactive complement and no test serum). Single determinations were made using each serum sample, the human complement used had previously been screened for lack of intrinsic bactericidal activity.

(58) Surface Labelling and Opsonophagocytosis Assays

(59) Bacteria: Bacteria used were killed using a protocol designed to minimise chemical alteration of surface epitopes. Briefly, live meningococci were incubated with 0.2% (w/v) sodium azide and 17 μg.Math.ml.sup.−1 phenylmethylsulphonyl fluoride for 48 hours at 37° C. Bacteria used in the opsonophagocytosis assay were first stained for 1 hour with 20 μM of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine-5,5′-disulfonic acid (DiIC.sub.18(5)-DS) (Invitrogen, UK).

(60) Complement: Human plasma was used as a complement source and was IgG depleted using a protein G Sepahrose column immediately before use. Briefly, the column was equilibrated with three column volumes of hanks buffered saline solution before one column volume of plasma was added. This was incubated for 5 minutes at 4° C. before one column volume of hanks buffered saline solution was used to displace the complement.

(61) Opsonophagocytosis assay (OPA): Opsonophagocytosis (OP) of meningococci by HL-60 cells (American Type Culture Collection, Rockville Md., USA) differentiated to granulocytes with 0.8% N,N dimethyleformamide (Sigma, UK) for 5 days, was measured using a flow cytometric assay performed in U-bottom 96-well microtitre plates (Sterilin, UK). 20 μl of each test serum diluted 1:10 in OP buffer (Hanks balanced salts solution (Sigma, UK) containing 2% skimmed milk powder (Marvel, Premier International Foods, UK), 1.2 mM CaCl.sub.2, and 1 mM MgSO.sub.4 (Sigma, UK)) was added to 10 μl of target bacteria at 6.25×10.sup.8 ml.sup.−1 in OP buffer, followed by 10 μl of IgG depleted Human plasma as a complement source, and then incubated for 7.5 min with shaking (900 rpm) at 37.0° C.

(62) Differentiated HL60 cells at 2.5×10.sup.7 ml.sup.−1 in OP buffer (50 μl) were added and incubation continued, with shaking, at 37.0° C. for 7.5 min. Opsonophagocytosis was stopped by addition of 80 μl of ice-cold Dulbecco phosphate-buffered saline (PBS; Sigma, UK) containing 0.02% EDTA (Sigma, UK).

(63) Horizontal gates in the DiIC.sub.18(5)-DS channel of a flow cytometer (Beckman Coulter FC500) were set against a complement-only, no-antibody control to include approximately 10% of the population. For each sample, 7,500 live HL60 cells were measured, and the percentage of cells showing DilC.sub.18(5)-DS fluorescence in the appropriate gate (% gated) was multiplied by the mean fluorescence of the gated population (X−mean) to calculate a fluorescence index (FI). The FI of each test was subtracted from the FI of the complement-only no antibody control to give FI minus complement control (FI−C′).

(64) Total antibody binding assay (SLA): IgG binding to the surface of meningococci was measured using a flow cytometric assay performed in U-bottom 96-well microtitre plates (Sterilin, UK).

(65) For the assay, 2 μl of each test serum was added to 198 μl of target bacteria at an O.D.sup.6000.1 in a blocking buffer of 1% bovine serum albumin (BSA) in PBS and the mixture incubated for 30 min with shaking (900 rpm) at 25.0° C. This was then centrifuged at 3050 g for 5 minutes and the supernatant removed and the pellet was then washed with 200 μl of blocking buffer. The process was repeated twice before the addition of 200 μl FITC-labelled goat anti-human IgG (Biodesign, UK) at 1:500 in blocking buffer, followed by incubation for 20 minutes at 4° C., before being washed twice more with blocking buffer.

(66) Horizontal gates in the FITC channel of a flow cytometer (Beckman Coulter FC500) were set against a conjugate-only, no-antibody control to include approximately 10% of the population. For each sample, 7,500 bacteria were measured, and the percentage of cells showing FITC fluorescence in the appropriate gate (% gated) was multiplied by the mean fluorescence of the gated population (X−mean) to calculate a fluorescence index (FI).; the FI of the conjugate only control was subtracted from the FI of each test sera to give FI minus complement control (FI−Conj).

(67) From the Foregoing, it will be appreciated that although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.