IN-VITRO POTENCY ASSAY FOR PROTEIN-BASED MENINGOCOCCAL VACCINES

Abstract

The invention uses ELISA or similar assays for analysing a meningococcal vaccine. The assay uses antibodies which bind to meningococcal proteins within the vaccine, and in particular monoclonal antibodies which are bactericidal for meningococcus and/or which recognise conformational epitopes within the meningococcal proteins. By performing the assay on a series of dilutions of a test vaccine, and by comparing the results with those obtained using a reference vaccine of known potency, it is possible to determine the relative potency of the test vaccine. This value can be used as a parameter for determining whether a manufactured batch of a vaccine is suitable for release to the public, or whether it has experienced a production failure and so should not be used.

Claims

1-18. (canceled)

19. A kit comprising: (i) an anti-vaccine monoclonal antibody; (ii) an immobilized antigen which is recognized by the anti-vaccine monoclonal antibody, wherein the anti-vaccine monoclonal antibody (a) is bactericidal for meningococcus or (b) recognizes a conformational epitope in the meningococcal antigen.

20. The kit of claim 19, further comprising a labeled antibody which binds to the anti-vaccine monoclonal antibody.

21. The kit of claim 19, wherein the immobilized antigen is from a meningococcal protein, a fusion protein comprising a meningococcal protein, or a truncated form of a meningococcal protein.

22. The kit of claim 21, wherein the immobilized antigen is meningococcal Neisserial Heparin Binding Antigen (NHBA), meningococcal factor H binding protein (fHbp), or meningococcal Neisserial adhesin A (NadA).

23. The kit of claim 20, wherein the labeled antibody is labelled with an enzyme.

24. The kit of claim 23, wherein the enzyme is a peroxidase, a phosphatase, a laccase or a beta-galactosidase.

25. The kit of claim 19, wherein the anti-vaccine monoclonal antibody comprises a monoclonal antibody with a variable light (VL) region comprising the amino acid sequence of SEQ ID NO:21.

26. The kit of claim 19, wherein the anti-vaccine monoclonal antibody comprises a monoclonal antibody with a variable heavy (VH) region comprising the amino acid sequence of SEQ ID NO:22.

27. The kit of claim 19, wherein the anti-vaccine monoclonal antibody comprises a monoclonal antibody with variable light (VL) and variable heavy (VH) regions comprising the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:22.

28. The kit of claim 19, wherein the anti-vaccine monoclonal antibody comprises CDRs from the variable light (VL) and variable heavy (VH) regions of SEQ ID NO:21 and SEQ ID NO:22.

29. The kit of claim 19, wherein the anti-vaccine monoclonal antibody is a murine monoclonal IgG antibody.

30. The kit of claim 29, wherein the murine monoclonal IgG antibody is a murine monoclonal IgG2b antibody.

31. The kit of claim 30, wherein the immobilized meningococcal antigen is fHbp.

32. The kit of claim 19, wherein the anti-vaccine monoclonal antibody comprises a high-affinity tag.

33. The kit of claim 32, wherein the high-affinity tag is biotin, avidin or streptavidin.

34. The kit of claim 33, further comprising an enzyme conjugated to a ligand of the high affinity tag.

35. The kit of claim 19, wherein the immobilized meningococcal antigen is immobilized on a surface.

36. The kit of claim 19, further comprising a buffer and a microwell plate, wherein the immobilized meningococcal antigen is immobilized on the microwell plate.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0145] FIGS. 1A-1F shows relative potency plots for NHBA, fHbp, NadA and OMV immunogens in BEXSERO™ using monoclonal antibodies (A) 42A4A2 (B) MAb502 (C) 12C1/D7 (D) 11F10/G6 (E) 9F11/19 (F) Anti-PorA. Each plot shows log(OD.sub.405-620nm) against log(dilution). Circles show data for the standard vaccine; triangles for the test vaccine.

[0146] FIGS. 2A-2B shows relative potency plots for two further batches of OMV in BEXSERO™.

[0147] FIG. 3 shows RP values for vaccines heated overnight. The four groups of four bars are, from left to right: fHbp; NHBA; NadA; and OMVs. Within each group, the four bars are: 37° C.; 50 C; 60° C.; and 80° C.

[0148] FIGS. 4A-4D shows RP plots for standard vaccine (circles) and for adjuvant (triangles) using monoclonal antibodies (A) MAb502 (B) 42A4A2 (C) 9F11/19 and (D) Anti-PorA.

[0149] FIG. 5 illustrates an ELISA of the invention in competitive format. At the top, monoclonal antibody (step A) for one of the vaccine immunogens is mixed with the vaccine sample (step B) in ten wells having increasingly-diluted vaccine in each well. In step c this mixture is transferred into the wells of a second plate, the wells of which are coated with immobilised vaccine immunogen. After incubation the plates are washed (step D), then enzyme-conjugated anti-mAb serum is added in step E, after which the enzyme is used to catalyse a detectable reaction for ELISA output.

MODES FOR CARRYING OUT THE INVENTION

[0150] The BEXSERO™ product is described in reference 7, and it includes 50 μg of each of NadA (subvariant 3.1; SEQ ID NO: 6), fHbp subvariant 1.1 (as a GNA2091-fHbp fusion protein; SEQ ID NO: 5), and NHBA subvariant 1.2 (as a NHBA-GNA1030 fusion protein; SEQ ID NO: 4), adsorbed onto 1.5 mg aluminium hydroxide, and with 25 μg OMVs from N. meningitidis strain NZ98/254.

[0151] The following monoclonal antibodies are available: [0152] (A) 42A4A2 (murine IgG1 against NHBA) [0153] (B) MAb502 (murine IgG2a against fHbp) [0154] (C) 12C1/D7 (murine IgG2b against fHbp) [0155] (D) 11F10/G6 (murine IgG2b against fHbp) [0156] (E) 9F11/19 antibody (murine IgG2b against NadA) [0157] (F) Anti-PorA(P1.4), available from NIBSC.

[0158] These antibodies are bactericidal, except for 42A4A2 (which is non-bactericidal but seems to recognise a conformational epitope).

[0159] The BEXSERO™ product is serially diluted 9 times, either 1:2 or 1:5 each time. Six of these dilution series are present in rows (A) to (F) of a first microtitre plate (plate 1), from columns 1 (strongest) to 10 (most dilute). Each row receives one of the six monoclonal antibodies (A) to (F) described above, each used at the same strength in each column. After incubation the contents of these 60 wells are transferred into 60 wells in a second plate (plate 2). The wells in rows (A) to (F) in plate 2 are coated with the individual recombinant proteins (A) NHBA (B-D) fHbp (E) NadA and (F) PorA. In other embodiments, all wells in a single ELISA plate are coated using the same antigen, and each antigen is tested separately by using a different ELISA microtiter plate.

[0160] The mixture is incubated for 2 hours at 37° C. (for fHbp) or at room temperature (for NHBA, NadA and PorA), then washed. Monoclonal antibodies which were unbound to the vaccine antigens are retained on the plates. Anti-mouse IgG, conjugated to alkaline phosphatase, is then added to all 60 wells with pNPP and the amount of retained monoclonal antibody is assessed by OD.sub.405-620nm. Thus the vaccine immunogen (serially diluted) inhibits the binding of the monoclonal antibodies to the immobilised antigens in plate 2. Higher levels of epitope in the vaccine sample will lead to more inhibition of this binding, and thus to less detectable signal after adding the pNPP.

[0161] FIGS. 1A to 1F show the results from the six rows. The graphs also include data measured with a reference vaccine, and comparison of the two parallel lines reveals the following relative potencies:

TABLE-US-00001 A B C D E F R.P. 0.915 2.344 0.859 0.895 1.037 1.033

[0162] The aberrant value in FIG. 1B (i.e. using MAb502) arose because the curves were not linear and were not parallel to each other. In all other cases the curves were linear with good R.sup.2 values. Thus the assay is suitable for assessing relative potency.

[0163] To check for inter-assay consistency the anti-PorA measurement was checked for two further BEXSERO™ batches (FIGS. 2A and 2B). The results in FIGS. 1F, 2A and 2B show no big differences, and RP was 1.033, 0.917 and 0.893 in the three different vaccine batches.

[0164] The ability of this assay to identify damaged vaccine was tested by artificially exposing a BEXSERO™ product to thermal stress. Relative potency values for each of the four immunogen components after 2 hours at 80° C. were as follows:

TABLE-US-00002 NHBA fHbp NadA OMV R.P. 0.25 0.08 0.01 0.55

[0165] FIG. 3 shows relative potency values for each of the four immunogen components after overnight incubation at 37° C., 50° C., 60° C. and 80° C. Thus the assay can detect losses in potency caused by thermal mistreatment.

[0166] To confirm that the aluminium hydroxide adjuvant did not interfere with the assay, antibodies (A), (B), (E) and (F) were tested with standard vaccine or with adjuvant. As shown in FIGS. 4A-4D the adjuvant always showed its inability to compete and/or interfere with the binding of each monoclonal antibody to the respective immunogen(s).

[0167] Anti-fHbp Monoclonal Antibodies

[0168] Four bactericidal murine anti-fHbp IgG2b subclass monoclonal antibodies were obtained: 12C1/D7; 11F10/G6; 30G11/H3; and 14B3/D4. RNA was isolated from the murine hybridoma cells using an Oligotex Direct mRNA Mini Kit according to the manufacturer's instructions. cDNA was produced via reverse transcription using ˜200 ng of the poly(A)+RNA template, an oligo-(dT) primer, and SuperScript II RT. cDNA was amplified by PCR using immunoglobulin heavy (H)- and light (L)-chain degenerate primers as described in reference 86. The purified products were inserted into the pSTBlue-1 Perfectly Blunt vector for sequencing.

TABLE-US-00003 12C1/D7's V.sub.L region has amino acid sequence SEQ ID NO: 21: DIVLTQSPSSIYASLGERVTLTCKASQDIHNYLNW FQQKPGKSPKTLIYRANRLVDGVPSRFSGGGSGQD YSLTISSLEFEDIGIYYCLQYDEFPPTFGGGTRLE IKRADAAPTVS and its V.sub.H region has amino acid sequence SEQ ID NO: 22: QVQLQESGPELVKPGASVKISCKASGYSFSDYNMS WVKQSNGKSLEWIGIIDPKYGTINYNQKFKGKATL TVDQASSTAYMQLMSLTSEDSAVYYCVRDYYGSSY FDYWGQGTTLTVS 11F10/G6's V.sub.L region has amino acid sequence SEQ ID NO: 23: DIVLTQTPSSIYASLGERVTLTCKASQDIHNYLNW FQQKPGKSPKTLIYRANRLVDGVPSRFSGGGSGQD YSLTISSLEFEDIGIYYGLQYDEFPPTFGGGTRLE IKRADAAPTVS and its V.sub.H region has amino acid sequence SEQ ID NO: 24: EFQLQQSGPELVKPGASVKISCKASGYSFSDYNMS WVKQSNGK$LEWIGTIDPKYGTINYNQKFKGKATL TVDQASSTAYMQLNSLTSEDSAVYYCVRDYYGSSY FDYWGQGTTLTVS 30G11/H3's V.sub.L region has amino acid sequence SEQ ID NO: 25: DIVMTQSQKFMSTSVGDRVSITCKASQHVRTAVAW YQQKPGQSPKGLIYLASNRRTGVPDRFTASGSGTD FTLTITNVQSEDLADYFCLQHWNYPFTFGSGTKLE IKRADAAPTVS and its V.sub.H region has amino acid sequence SEQ ID NO: 26: EVQLEESGPELVKPGASVKISCKASGYSFSDYNMS WVKQSNGKSLEWIGIIDFKYGTINYNQKFKGKATL TVDQASSTAYMQLNSLTSEDSAVYYCVRDYYGSSY FDYWGQGTTLTVS 14B3/D4's V.sub.L region has amino acid sequence SEQ ID NO: 27: DIVLTQSPSSLTVTAGEKVTMSCRSSQSLLNSGNQ KNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTG SGSGTDFTLTISSVQAEDLAIYYCQNDYNYPLTFG AGTKLELKR and its V.sub.H region has amino acid sequence SEQ ID NO: 28: QVQLQQPGAELVKPGASVKLSCKASGYSFTTYYWM NWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKNKAT LTVDKSSSTAYIQLSSLTSEDSAVFYCAAHYNKYE GYFYAMDYWGQGTSVTVSS

[0169] In a FACS assay the 11F10/G6 and 30G11/H3 were able to bind to meningococcal strains having each of the three different fHbp variants: MC58 (variant 1); 961-5945 (variant 2); and M1239 (variant 3). Moreover, these two FACS-positive antibodies also showed bactericidal activity against strains having each of the three variants.

[0170] 14B3/D4 was FACS-positive and bactericidal against MC58 and 961-5945, but not against M1239. 12C1/D7 was FACS-positive and bactericidal against MC58, but not against 961-5945 or M1239. 12C1/D7 and 11F10/G6 competed with fH for binding to fHbp; the other two antibodies did not.

[0171] The epitope for 11F10/G6 seems to include residue Lys-268 in fHbp (var 1.1).

[0172] The epitope for 12C1/D7 seems to include residue Val-270 in fHbp (var 1.1).

[0173] The epitope for 14B3/D4 seems to include residues 60-90 in fHbp.

[0174] The epitope for 30H11/H3 seems to include residue Lys-257 in fHbp (var 1.1).

[0175] It will be understood that the invention is described above by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

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