WO2023067002A1

WO2023067002A1 - In vitro potency assay for protein-based meningococcal vaccines using monoclonal antibodies

Info

Publication number: WO2023067002A1
Application number: PCT/EP2022/079103
Authority: WO
Inventors: Carmine MALZONE; Werner Pansegrau; Laura SANTINI
Original assignee: Glaxosmithkline Biologicals Sa
Priority date: 2021-10-21
Filing date: 2022-10-19
Publication date: 2023-04-27
Also published as: GB202115072D0

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.

Description

IN VITRO POTENCY ASSAY FOR PROTEIN-BASED MENINGOCOCCAL VACCINES USING MONOCLONAL ANTIBODIES

TECHNICAL FIELD

This invention is in the field of in vitro assays for assessing the potency of protein-containing vaccines for protecting against Neisseria meningitidis (meningococcus).

BACKGROUND ART

Unlike live vaccines that are quantified by in vitro titration, the potency of inactivated or subunit vaccines normally requires an in vivo test for each batch prior to its release for public use [1], although a number of exceptions exist e.g. the SRID (single radial immunodiffusion) potency test for the influenza vaccine and the use of ELISA for hepatitis B vaccines.

Typical in vivo tests involve an immunisation-challenge test using small rodents (mice or rats) as the experimental model. Depending on the type of vaccine, different endpoints are used, such as death/survival ratios (whole cell pertussis, diphtheria toxoid and tetanus toxoid, rabies vaccine), clinical signs (diphtheria, tetanus) or colonisation (whole cell and acellular pertussis). By establishing a dose-response curve in parallel to a standard preparation with known potency, the potency of the vaccine can be expressed relative to that preparation e.g. in standard units.

A challenge model is not always available. In those cases potency testing is usually limited to serological responses, with antibody responses being measured after immunisation of test animals. At least part of the functionality of these antibodies can be determined by their ability to neutralise the pathogen in vitro or to their ability to kill bacteria in the presence of complement (such as the serum bactericidal antibody assay, or SB A, for meningococcus).

The SBA assay is useful but cumbersome, and involves the sacrifice of many mice. As explained in reference 1 it is thus desirable to provide in vitro alternatives for assessing vaccine potency.

One in vitro assay for analysing MenB vaccines is the “MATS” ELISA test disclosed in references 2 and 3. The relative potency measured by MATS was shown to correlate with the ability of MenB strains to be killed in SBA.

The MATS test is used to evaluate the strain coverage of a MenB vaccine, rather than to analyse the vaccine’s immunogenicity. There remains a need for further and improved in vitro assays for assessing the immunogenicity of meningococcal vaccines. Such in vitro assays could be used to confirm that a particular vaccine will have an expected in vivo activity in human recipients.

The International Patent Application WO2013132040 discloses binding assays, such as ELISA, for analysing a meningococcal vaccine. DISCLOSURE OF THE INVENTION

The invention uses binding assays, such as ELISA, 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 is capable of binding 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 standard or 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. Assays of the invention are particularly useful for analysing vaccines which contain multiple different antigens and/or which contain adsorbed antigen(s).

Thus the invention provides a binding assay for in vitro analysis of a meningococcal vaccine sample, comprising steps of: (i) allowing a meningococcal immunogen within the sample to interact with a monoclonal antibody which is bactericidal for meningococcus and/or capable of binding a conformational epitope of an immunogen in said meningococcal vaccine then (ii) measuring the interaction between the immunogen and antibody from step (i).

The invention also provides a binding assay for in vitro analysis of a meningococcal vaccine sample, comprising the following steps of: (i) incubating the sample with the monoclonal antibody so that complexes can form between the antibody and meningococcal immunogen in the sample; (ii) separating the unbound monoclonal antibody from immunogen-bound monoclonal antibody; (iii) adding the unbound monoclonal antibody to a container in which antigens of said monoclonal antibody are immobilised, wherein the immobilised antigens can form a complex with said unbound monoclonal antibody; (iv) determining the amount of the complex formed in step (iii) and (v) measuring the interaction between the immunogen and antibody from step (i).

The invention also provides a binding assay for in vitro analysis of a meningococcal vaccine sample, comprising steps of: (i) allowing a meningococcal immunogen within the sample to interact with a monoclonal antibody which is bactericidal for meningococcus and/or capable of binding a conformational epitope of a protein immunogen in said meningococcal vaccine then (ii) measuring the interaction between the immunogen and antibody from step (i), wherein the vaccine includes an adsorbed meningococcal immunogen and the assay comprises a desorption step in order to separate adsorbed meningococcal immunogens.

The invention also provides an assay for in vitro analysis of a meningococcal test vaccine sample, comprising steps of: (i) performing the above binding assay on the test sample and, optionally, on at least one dilution of the test sample; (ii) performing the above binding assay on a standard vaccine sample and, optionally, on at least one dilution of the standard vaccine sample; and (iii) comparing the results from steps (i) and (ii) to determine the potency of immunogen(s) in the test vaccine relative to the potency of immunogen(s) in the standard vaccine.

The invention also provides a binding assay for in vitro analysis of a meningococcal protein-containing vaccine sample from a batch of final vaccine in the form in which it would be released to the public, comprising the steps of any one of the assays or methods herein disclosed.

The invention also provides method for in vitro relative potency analysis of a meningococcal test vaccine sample, comprising steps of: (i) performing the in vitro assay according to any embodiments herein discloses on a test sample and, optionally, on at least one dilution of the test sample; (ii) performing the in vitro assay performing the assay according to any embodiments herein discloses on a standard vaccine sample of known in vivo potency and, optionally, on at least one dilution of the standard vaccine sample; and (iii) comparing the results from steps (i) and (ii) to determine the potency of immunogen(s) in the test vaccine relative to the potency of immunogen(s) in the standard vaccine.

The invention also provides a method for detecting or measuring a change in conformation of a meningococcal immunogen in a vaccine sample, comprising steps of: (i) performing the in vitro assay according to any embodiments herein discloses on a test sample and, optionally, on at least one dilution of the test sample; (ii) performing the in vitro assay performing the assay according to any embodiments herein discloses on a standard vaccine sample of known native antigenic form and, optionally, on at least one dilution of the standard vaccine sample; and (iii) comparing the results from steps (i) and (ii) to determine the amount of immunogen(s) in the native form of the test vaccine relative to the amount of immunogen(s) in the native form in the standard vaccine.

The invention also provides a method for analysing a bulk vaccine, comprising steps of: (i) assaying the relative potency of immunogen(s) in the bulk as described above; and, if the results of step (i) indicate an acceptable relative potency, (ii) preparing unit doses of vaccine from the bulk.

The invention also provides a method for analysing a batch of vaccine, comprising steps of: (i) assaying the relative potency of immunogen(s) in at least one vaccine from the batch as described above; and, if the results of step (i) indicate an acceptable relative potency, (ii) releasing further vaccines from the batch for in vivo use.

The invention also provides a vaccine, which has been released following use of an assay as described herein.

The invention also provides a kit for performing the assay of the invention comprising (i) a solutionphase anti-vaccine monoclonal antibody (ii) an immobilised antigen which is capable of binding by the anti-vaccine antibody, and (iii) a labelled antibody which binds to the anti-vaccine antibody, wherein the anti -vaccine antibody (a) is bactericidal for meningococcus and/or (b) capable of binding a conformational epitope of the meningococcal NHBA or NadA antigen.

The invention also provides monoclonal antibodies, which is capable of binding (selectively binding) meningococcal antigens, in particular wherein said monoclonal antibodies are bactericidal for meningococcus and is capable of binding a conformational epitope of said meningococcal antigens. These antibodies can be used with the assays of the invention, or can be used more generally.

The invention also provides monoclonal antibodies suitable for carrying out the assays of the invention such as antibodies that selectively bind one of the immunogens in the vaccine. In particular the invention also provides monoclonal antibodies that are able to differentiate between native and denatured immunogens in the vaccine, i.e. monoclonal antibodies that do not bind to denatured immunogens. Such antibodies are for example:

A monoclonal antibody which is capable of binding (selectively binding) to the meningococcal NHBA antigen, in particular to a meningococcal NHBA immunogen, whose VH and VL comprise the following complementarity-determining regions (CDRs):

-CDR1 of VH having SED ID NO: 7,

-CDR2 of VH having SED ID NO: 8,

-CDR3 of VH having SED ID NO: 9,

-CDR1 of VL having SED ID NO: 3,

-CDR2 of VL having the sequence RMS and

-CDR3 of VL having SED ID NO:4.

A monoclonal antibody that is capable of binding (selectively binding) to an epitope, preferably a conformational epitope, of the meningococcal antigen NHBA comprising or consisting in the amino acid sequence of SEQ ID NO:20, preferably in SEQ ID NO:20, more preferably in SEQ ID NO:21.

A monoclonal antibody able to differentiate between native and denatured form of antigen NHBA and whose VH and VL shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more of identities with the amino acid sequence of SEQ ID NO: 6 (VH) and SEQ ID NO: 2(VL) respectively.

A monoclonal antibody that is capable of binding (selectively binding) to meningococcal NadA antigen, in particular to a meningococcal NadA immunogen, whose VL region has the amino acid sequence of SEQ ID NO: 11 and whose VH region has the amino acid sequence of SEQ ID NO: 15.

A monoclonal antibody that is capable of binding (selectively binding) meningococcal NadA antigen, in particular to a meningococcal NadA immunogen, whose VH and VL comprise the following complementarity-determining regions (CDRs): -CDR1 of VH having SED ID NO: 16,

-CDR2 of VH having SED ID NO: 17,

-CDR3 of VH having SED ID NO: 18,

-CDR1 of VL having SED ID NO: 12,

-CDR2 of VL having the sequence ATS and

-CDR3 of VL having SED ID NO: 13.

A monoclonal antibody that is capable of binding (selectively binding) an epitope, preferably a conformational epitope, in the region of meningococcal antigen NadA corresponding to the amino acid sequence of residues 206-249 of SEQ ID NO:22.

A monoclonal antibody able to differentiate between native and denatured form of antigen NadA and whose VH and VL region shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more of identities with the amino acid sequence SEQ ID NO: 15 (VH) and SEQ ID NO: 11 (VL).

Binding assays and ELISA formats

The invention uses a binding immunoassay. Typically, this will be an enzyme-linked immunosorbent assay (ELISA) as is well known in the art. The invention can use any ELISA format, including those conventionally known as direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA.

Step (i) of the ELISA assay of the invention involves permitting a meningococcal immunogen within the sample to interact with a monoclonal antibody. The characteristics of this interaction (e.g. homogeneous or heterogeneous) will vary according to the chosen ELISA format. The interaction between the monoclonal antibody and the immunogen is then detected in step (ii). As typical for ELISA, the interaction can be measured quantitatively, such that step (ii) provides the amount of the monoclonal antibody’s target epitope within the vaccine sample. By using a monoclonal antibody which binds to a bactericidal and/or a conformational epitope, the result in step (ii) indicates the concentration of the corresponding functional epitope in the vaccine sample, and can differentiate between immunogens which retain the relevant epitope (and function) and those which have lost the epitope (e.g. due to denaturation, aggregation or breakdown during storage or by mishandling). By comparison with values obtained with a standard vaccine of known potency, results from step (ii) can be used to calculate relative potency of a test vaccine.

The preferred ELISA format for use with the invention is the competitive ELISA. In this format the vaccine sample is incubated with the monoclonal antibody (primary antibody) so that complexes can form between the antibody and immunogens in the sample. These complexes are then added to a container in which competitor antigens are immobilised. Antibody which is not complexed with immunogens from the vaccine sample is able to bind to these immobilised competitor antigens; if the sample contains a lot of target for the antibody then there will be less uncomplexed antibody to bind to the immobilised competitor antigens, whereas less target in the sample (whether due to lower amounts of immunogen, for example after dilution, or to loss of the antibody’s epitope, for example after denaturation of immunogens) leads to more uncomplexed antibody. The antibody which is bound to the immobilised competitor antigens (after usual washing steps, etc.) can then be detected by adding a labelled secondary antibody which binds to the monoclonal anti -vaccine (i.e. primary) antibody. The label is used to quantify the amount of immobilised primary antibody in the normal ways. The use of competitive ELISA avoids the need to have two different anti-immunogen antibodies which recognise different epitopes on the same immunogen (i.e. sandwich ELISA), and also can give better results in vaccines which include multiple different immunogen components. It also permits the test vaccine to be analysed directly, without requiring any manipulation prior to testing (although such manipulations can be performed if desired).

In one preferred embodiment after the incubation of the vaccine sample with the monoclonal antibody (primary antibody), the antibody which is not complexed with immunogen from the vaccine sample (defined also as unbound antibody or uncomplexed antibody) is separated from the immunogen-bound monoclonal antibody. This separation step is carried out preferably by a centrifugation step, for example centrifuging between 500 and 1500 g for at least 5, 6, 7, 8, 9, 10 minutes, preferably at 1000 g for 20 minutes at room temperature.

Suitable competitor antigens for immobilisation include the meningococcal proteins which are present in the vaccine, or proteins comprising these vaccine proteins (e.g. fusion proteins), or proteins comprising fragments of the vaccine proteins (e.g. truncated forms). The immobilised competitor antigen must retain the epitope recognised by the relevant monoclonal antibody, so that it can compete with the vaccine’s immunogens for binding to the antibody. Typically this can be achieved by immobilising antigen from fresh batches of bulk vaccine or, preferably, from fresh batches of bulk purified immunogen prior to preparation of bulk vaccine.

Labelling of antibodies in an ELISA can take various forms. In the preferred competitive format the secondary antibody is labelled. In an ELISA the antibody is labelled with an enzyme, which is then used to catalyse a reaction whose product is readily detectable. The linked enzyme can cause a detectable change in an enzyme substrate which is added to the labelled antibody after it becomes immobilised e.g. to modify a substrate in a manner which causes a colour change. For example the enzyme may be a peroxidase (e.g. horseradish peroxidase, HRP), or a phosphatase (e.g. alkaline phosphatase, AP). Other enzymes can also be used e.g. laccase, P-galactosidase, etc.

The choice of substrate will depend on the choice of linked enzyme. Moreover, substrates differ in terms of cost, ease-of-use, sensitivity (i.e. lower limit of detection) and compatibility with available imaging equipment. These parameters are familiar to those skilled in ELISA. Preferred substrates undergo a colorimetric change, a chemiluminescent change, or a chemifluorescent change when contacted with the linked enzyme. Colorimetric substrates (and their enzymatic partners) include, but are not limited to: PNPP or p-Nitrophenyl Phosphate (AP); ABTS or 2,2'-Azinobis [3- ethylbenzothiazoline-6-sulfonic acid] (HRP); OPD or o-phenylenediamine dihydrochloride (HRP); and TMB or 3,3',5,5'-tetramethylbenzidine (HRP). Chemiluminescent substrates include luminol or 5- amino-2,3-dihydro-l,4-phthalazinedione (HRP), particularly in the presence of modified phenols such as p-iodophenol. Chemifluorescent substrates include p-hydroxyhydrocinnamic acid. Various proprietary substrates are also available and these can be used with the invention if desired e.g. QuantaBlu, QuantaRed, SuperSignal, Turbo TMB, etc.

Where an ELISA reagent is immobilised on a solid surface, this surface take various forms. Usually the reagent is immobilised on a plastic surface, such as a surface made from polystyrene, polypropylene, polycarbonate, or cyclo-olefin. The plastic will usually be transparent and colourless, particularly when using chromogenic enzyme substrates. White or black plastics may be preferred used when using luminescent or fluorescent substrates, as known in the art. The plastic will generally be used in the form of a microwell plate (microtitre plate) as known in the art for ELISA (a flat plate having multiple individual and reaction wells). Such plates include those with 6, 24, 96, 384 or 1536 sample wells, usually arranged in a 2:3 rectangular matrix. Microwell plates facilitate the preparation of dilution series and also the transfer of materials from one plate to another while maintaining spatial relationships e.g. in the step of transferring a mixture of antibody and vaccine into a different micro well plate for measuring the interaction between the antibody and vaccine.

Antigens are coated on the plate using for example PBS, TRIS-HC1 or carbonate buffer.

During an ELISA it may be desirable to add a blocking reagent and/or detergent e.g. to reduce nonspecific binding interactions which might distort the assay’s results. Blocking procedures are familiar to people working in the ELISA field.

In addition to the ELISA formats discussed above, the invention can use any suitable variants of ELISA, such as M&P ELISA or ELISA Reverse [4], the rapid ELISA of reference 5, etc., and can also be extended to use alternatives to ELISA, such as flow injection immunoaffinity analysis (FIIAA), AlphaLISA or AlphaScreen [6], dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA), ELAST, the BIO-PLEX Suspension Array System, MSD, etc. Any of these binding assays can be used.

As an alternative to using a conjugated enzyme as the label, other labelling is possible. For instance, other indirect labels (i.e. alternative to enzymes) can be used, but it is also possible to label the antibody by conjugation to a direct label such as a coloured particle, an electrochemically active reagent, a redox reagent, a radioactive isotope, a fluorescent label or a luminescent label. As a further alternative, the primary antibody can be conjugated to a high affinity tag such as biotin, avidin or streptavidin. An enzyme conjugated to a ligand for the tag, such as avidin, streptavidin or biotin can then be used to detect immobilised primary antibody.

Any of these variations can be used within the scope and spirit of the overall invention.

In some ELISA formats, rather than labelling a secondary antibody, the anti-vaccine monoclonal antibody (whether a bactericidal antibody or one which is capable of binding a conformational epitope) will be labelled. Thus the invention provides a monoclonal antibody which immuno-specifically binds to a meningococcal protein (such as NHBA, etc., as disclosed herein) and which is conjugated to an enzyme (such as AP or HRP). Immunospecific binding can be contrasted with non-specific binding, and antibodies of the invention will thus have a higher affinity (e.g. at least 100-fold higher affinity) for the meningococcal target protein than for an irrelevant control protein, such as bovine serum albumin.

The interaction can be carried out for a suitable period of time at a suitable temperature allowing the formation of a complex immunogen+anti-immunogen specific monoclonal antibody. Suitable conditions for the binding between the immunogens and their respective the anti-immunogen specific monoclonal antibodies are known to the skilled person.

In an embodiment the interaction leading to said binding in (i) can be carried out at a temperature of 37°C ± 2°C for a period of time of 30±5 minutes. The assay can be carried out in any buffer known to the skilled person suitable for allowing the binding of immunogens with the respective antiimmunogen specific monoclonal antibodies.

A suitable buffer can be, without limiting the invention to it, IX PBS 0.05% Tween 20.

The vaccine sample

Assays of the invention are used to analyse vaccines. The assay is performed on at least one sample of the vaccine, and this analysis reveals information about the sampled vaccine. The assay can be performed on a sample(s) taken from a bulk vaccine, in which case the assay’s results can be used to determine the fate of that bulk e.g. whether it is suitable for further manufacturing use (e.g. for preparing packaged doses of the vaccine), or whether it should instead be modified or discarded. The assay can also be performed on a sample(s) taken from a batch of vaccines, in which case the assay’s results can be used to determine the fate of that batch e.g. whether the batch is suitable for release for use by healthcare professionals. Usually, enough samples will be taken from bulks/batches to ensure compliance with statistical practices which are normal for vaccine release assays. Testing of batches of final vaccine (formulated and packaged) in the form in which they would be released to the public is most useful. The vaccine sample can be analysed at full strength i.e. in the form in which it is taken from the bulk or batch. In some cases, however, it is useful to analyse the vaccine at a fraction of full strength e.g. after dilution. The most useful assays analyse a series of strengths, the strongest of which may be a full strength sample or may be at fractional strength. Dilutions will typically be achieved using buffer rather than with plain water. Such buffers can sometimes include surfactants such as polysorbate 20 or polysorbate 80.

It is useful to analyse a series of dilutions of the vaccine. For instance, serial 1:1,5, 1:2, 1:5 or 1: 10 (by volume) dilutions can be used. The dilution series will include at least 2 members, but usually will include more e.g. 5, 10, or more members. For instance, 9 serial dilutions at 1:2 gives 10 samples at 1:2°, E2¹, 1:2², ... , 1:2⁹, and l:2¹⁰-fold strengths relative to the strongest sample. The dilution series can be tested using the assays of the invention to provide a series of measurements which can be plotted (literally or notionally) against dilution. This series of measurements can be used to assess the vaccine’s relative potency, as described below. The vaccine includes at least one meningococcal protein immunogen i.e. a protein which, when administered to human beings, elicits a bactericidal immune response. Various such proteins are known in the art, including but not limited to NHBA, fHbp and NadA as found in the BEXSERO product [7,8]. Further protein immunogens which can be analysed are HmbR, NspA, NhhA, App, Omp85, TbpA, TbpB, and Cu,Zn-superoxide dismutase. A vaccine may include one or more of these various antigens e.g. it can include each of NHBA, fHbp and NadA. It can also include variant forms of a single antigen e.g. it can include more than one variant of meningococcal fHbp (i.e. two fHbp proteins with different sequences [9]), using different monoclonal anti-fHbp antibodies to bind each different variant separately.

The vaccine can include meningococcal vesicles i.e. any proteoliposomic vesicle obtained by disruption of or blebbing from a meningococcal outer membrane to form vesicles therefrom that retain antigens from the outer membrane. Thus, this term includes, for instance, OMVs (sometimes referred to as ‘blebs’), microvesicles (MVs) and ‘native OMVs’ (‘NOMVs’). Various such vesicles are known in the art (e.g. see references 10 to 24) and any of these can be included within a vaccine to be analysed by the invention. In some embodiments, however, the vaccine is vesicle-free. Where a vaccine does include vesicles, it is preferred to use a competitive ELISA format, as this tends to give better results in samples, which contain multiple components.

An analysed vaccine can preferably elicit an immune response in human beings, which is protective against serogroup B meningococcus. For instance, the vaccine may elicit an immune response which is protective at least against a prototype serogroup B strain such as MC58, which is widely available (e.g. ATCC BAA-335) and was the strain sequenced in reference 25. Other strains can also be tested for vaccine efficacy [2] but a response against MC58 is easily tested. A preferred vaccine which can be analysed according to the invention is BEXSERO [7], This vaccine includes three different recombinant proteins, consisting of amino acid sequences disclosed in WO2013132040 as SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 and herein incorporated by reference. It also contains NZ98/254 outer membrane vesicles.

In addition to meningococcal protein immunogens, a vaccine can include other immunogens. These can be non-protein immunogens from meningococcus and/or immunogens from other bacteria and/or immunogens from non-bacterial pathogens, such as viruses. Thus, for instance, an analysed vaccine might include: (a) one or more capsular saccharides from meningococci e.g. from serogroups A, C, W135 and/or Y, as in the MENVEO, MENACTRA, and NIMENRIX products which all include conjugated capsular saccharides; (b) an antigen from Streptococcus pneumoniae, such as a saccharide (typically conjugated), as in the PREVNAR and SYNFLORIX products; (c) an antigen from hepatitis B virus, such as the surface antigen HBsAg; (d) an antigen from Bordetella pertussis, such as pertussis holotoxin (PT) and filamentous haemagglutinin (FHA) from B.pertussis, optionally also in combination with pertactin and/or agglutinogens 2 and 3; (e) a diphtheria antigen, such as a diphtheria toxoid; (f) a tetanus antigen, such as a tetanus toxoid; (g) a saccharide antigen from Haemophilus influenzae B (Hib), typically conjugated; and/or (h) inactivated poliovirus antigens.

The vaccine is a pharmaceutical composition and so, in addition to its immunogens, typically includes a pharmaceutically acceptable carrier, and a thorough discussion of such carriers is available in reference 26.

The pH of an analysed vaccine is usually between 6 and 8, and more preferably between 6.5 and 7.5 (e.g. about 7). Stable pH in an analysed vaccine may be maintained by the use of a buffer e.g. a Tris buffer, a citrate buffer, phosphate buffer, or a histidine buffer. Thus an analysed vaccine will generally include a buffer.

An analysed vaccine may be sterile and/or pyrogen-free. Compositions of the invention may be isotonic with respect to humans.

An analysed vaccine comprises an immunologically effective amount of antigen(s), as well as any other components, as needed. By ‘immunologically effective amount’, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. The antigen content of compositions of the invention will generally be expressed in terms of the mass of protein per dose. A dose of 10-500pg (e.g. 50pg) per immunogen can be useful.

Analysed vaccines may include an immunological adjuvant. Thus, for example, they may include an aluminium salt adjuvant or an oil-in-water emulsion (e.g. a squalene-in-water emulsion). Suitable aluminium salts include hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), (e.g. see chapters 8 & 9 of ref. 27), or mixtures thereof. The salts can take any suitable form (e.g. gel, crystalline, amorphous, etc.), with adsorption of antigen to the salt being preferred. The concentration of Al⁺⁺⁺ in a composition for administration to a patient is preferably less than 5mg/ml e.g. <4 mg/ml, <3 mg/ml, <2 mg/ml, <1 mg/ml, etc. A preferred range is between 0.3 and Img/ml. A maximum of 0.85mg/dose is preferred. Aluminium hydroxide adjuvants are particularly suitable for use with meningococcal vaccines. The invention has been shown to give useful results despite the adsorption of protein immunogens within the vaccine, and analysis is possible without requiring a desorption step (i.e. analysis can be performed without a desorption pre-treatment of the vaccine).

Analysed vaccines may include an antimicrobial, particularly when packaged in multiple dose format. Antimicrobials such as thiomersal and 2-phenoxyethanol are commonly found in vaccines, but it is preferred to use either a mercury-free preservative or no preservative at all.

Analysed vaccines may comprise detergent e.g. a Tween (polysorbate), such as Tween 80. Detergents are generally present at low levels e.g. <0.01%. Analysed vaccines may include residual detergent (e.g. deoxycholate) from OMV preparation. The amount of residual detergent is preferably less than 0.4pg (more preferably less than 0.2pg) for every pg of MenB protein.

If an analysed vaccine includes LOS, the amount of LOS is preferably less than 0.12pg (more preferably less than 0.05 pg) for every pg of protein.

Analysed vaccines may include sodium salts (e.g. sodium chloride) to give tonicity. A concentration of 10+2 mg/ml NaCl is typical e.g. about 9 mg/ml.

Adsorbed immunogen

In one preferred embodiment of the invention, e.g. when the vaccine sample includes an adsorbed immunogen, e.g. when the vaccine sample comprises aluminium hydroxide, the aluminium hydroxide can be saturated by adding a suitable saturating agent to the assay buffer in (i) in order to separate the immunogens from A1(OH)₃, alternatively, the immunogens can be separated by A1(OH)₃ by previous desorption with commonly used methods such as sodium citrate treatment. In a preferred embodiment A1(OH)₃ is saturated without previous desorption without altering the concentrations of each immunogen in the vaccine composition.

A1(OH)₃ can be successfully saturated by adding in the assay buffer blocking agents such as commercially available blocking solutions for ELISA plates or the like e.g. comprising casein, modified casein, BSA and the like. Suitable commercially available blocking buffers/solutions can be buffers or solutions based on chemically modified and fragmented purified casein such as The Blocking Solution provided by Candor; peptides-based blocking solutions BSA free such as SmartBlock provided by Candor, BSA based blocking solutions such as BSA-Block provided by Candor, animal-free and protein-free blocking buffers such as PlateBlock provided by Candor. Other suitable ELISA blocking buffers can be purchased by ThermoFisher. Suitable blocking solution/buffers can also be prepared according to standard protocols known to the skilled person, e.g. as described in ELISA technical guide and protocols by Thermo scientific and the like.

In a preferred embodiment casein and/or fragmented casein and/or modified casein-based blocking buffers are preferred. Several ready to use casein based blocking buffers are available in the market. In general, said buffers comprise an amount of casein or casein derivatives (such as fragments) of about 0.5 to 4% and they can be used according to the manufacturer’s instructions. Also dry milk powder can be used for the preparation of a suitable blocking buffer according to commonly used standard protocols for ELISA and the like.

Suitable casein based blocking buffers can be commercial buffers such as ThermoFisher Blocker™ Casein in PBS or in TBS, Candor The Blocking Solution by Bioscience GmbH, abeam Protein Block ab64226.

In particular, blocking buffers that saturate free A1(OH)3 and provide a limited desorbption of the antigen from A1(OH)3 (e.g. maximum 20%) are preferred, the inventors found that such buffers are for example casein and/or fragmented casein and/or modified casein-based blocking buffers.

The skilled person can adjust the amount of saturation buffer depending on the buffer used.

The amount of blocking buffer in order to saturate A1(OH)3 can be adjusted by the skilled person. In a preferred embodiment said blocking buffer, in particular a casein-based blocking buffer as described above, is at a final concentration of 0.5-4% in the assay buffer, more preferably, said blocking buffer is at a final concentration of about 0.5, 1, 1.5, 2, 3 or 4%.

In a preferred embodiment, the assay buffer can comprise or consist of IX PBS, 0.5% blocking buffer and 0.05% Tween 20. The interaction in step (i) will allow the binding of each anti-immunogen specific monoclonal antibody to the respective immunogen if present.

Advantageously the antibody will selectively bind only the immunogen in non-denatured form.

The Standard vaccine

The assay of the invention can provide information about the amount of functional epitopes in a vaccine. If this amount is compared to the amount in a vaccine of known potency then it is possible to calculate the relative potency of a test vaccine. Thus in some embodiments the analysed vaccine is a standard vaccine which has known potency in an in vivo assay e.g. it has a known SBA titre. In other embodiments the analysed vaccine is a test vaccine which does not have a known potency in an in vivo assay. In further embodiments the assay is used to analyse both a standard vaccine and a test vaccine, and the results of the analysis of the test vaccine are compared to the analysis of the standard vaccine, and this comparison is used to express the test vaccine’s potency relative to the known potency of the standard vaccine.

For instance, after manufacture of a new bulk preparation of BEXSERO, or after storage of a batch or bulk of manufactured vaccine, a test sample from the batch/bulk can be tested using the assay of the invention, and the results can be compared to those obtained with BEXSERO having known in vivo potency. This comparison will reveal whether the new/stored batch/bulk (the test sample) is as potent as it should be. If so, the batch/bulk can be released for further use; if not, it can be investigated and/or discarded. For instance, unit doses can be prepared from the bulk, or the batch can be released for public distribution and use.

For assessing relative potency, it is useful to analyse the test vaccine and the standard vaccine at a variety of strengths. As discussed above, a series of dilutions of the vaccines can be analysed. The dilution series can be tested using the assays of the invention to provide a curve (literally or notionally) of binding assay results against dilution. This curve can be compared to a standard curve (i.e. the same curve, but obtained with the standard vaccine) to determine relative potency. For instance, by plotting the logarithm of the binding titer against the logarithm of dilution for the test and reference vaccines, the horizontal distance between the two parallel regression lines indicates relative potency (no horizontal separation indicating a relative potency of 100% or 1.0).

To simplify comparisons, the dilutions used for the test vaccine should be the same as those used for the reference vaccine (e.g. a series of 1:2, 1:5, or 1:10 dilutions for both vaccines).

A test for relative potency can be carried out multiple times in order to determine variance of the assay e.g. multiple times (duplicate, triplicate, etc. ) on a single sample, and/or performed on multiple samples from the same bulk/batch. The invention can involve determining the variation in such multiple assays (e.g. the coefficient of variation) as a useful parameter, and in some embodiments the results of the assay are considered as useful only where variation falls within acceptable limits e.g. <15%. Sometimes a wider variation is permitted e.g. <20%, depending whether tests are performed within (intra-assay) or in different (inter-assay) experimental sessions.

Where a vaccine includes multiple different immunogen, the potency of each of these is ideally tested separately. These results can then be combined for an analysis of the vaccine sample as a whole, but it is useful to identify the specific cause of any loss of overall potency.

According to USP 1032 “Relative potency is a unitless measure obtained from a comparison of the dose-response relationships of Test and Standard drug preparations. For the purpose of the relative comparison of Test to Standard, the potency of the Standard is usually assigned a value of 1”.

Therefore, the Standard vaccine, in an IVRP assay, is a reference vaccine with a known potency (e.g. a batch which has known potency/efficacy in humans or a batch which has been proven to be immunogenic in an animal model), preferably said potency being assigned as 1 in the assay. The range of acceptable relative potency results is thus preferably defined between 0.50 to 2.00, and includes the specification range established for the product, this means that the potency of vaccine tested with respect to the reference vaccine is acceptable when the potency of the tested vaccine is at least 0.50 with respect to the potency of the reference/standard vaccine.

The antibody

Assays of the invention use monoclonal antibodies which is capable of binding (selectively binding) protein immunogens which are present within the analysed vaccines. The invention can use antibodies which are bactericidal for meningococcus and/or which is capable of binding conformational epitopes in the protein immunogens. In both cases the antibodies can thus distinguish between functional immunogen and denatured or non-functional immunogen. The use of bactericidal antibodies is preferred.

Determining whether an antibody is bactericidal against meningococcus is routine in the art, and can be assessed by SBA [28-31], Reference 32 reports good inter-laboratory reproducibility of this assay when using harmonised procedures. SBA can be run against strain H44/76 (reference strain 237 from the PubMLST database; strain designation B: Pl.7, 16: F3-3: ST-32 (cc32); also known as 44/76-3 or Z3842). For present purposes, however, an antibody can be regarded as bactericidal if it kills strain MC58 using human complement.

Determining whether an antibody is capable of binding a conformational epitope is also possible using known techniques. For instance, the antibody can be tested against a panel of linear peptide fragments from the target antigen (e.g. using the Pepscan technique) and the binding can be compared to the antibody’s binding against the complete antigen. As an alternative, binding can be compared before and after denaturation of the target antigen.

Assays of the invention can use a single monoclonal antibody or a mixture of monoclonal antibodies. Typically, a vaccine will include multiple different immunogens and each of these will require a different monoclonal antibody for its analysis. Thus an assay can use: a single monoclonal antibody which is capable of binding (selectively binding) a single immunogen; a plurality of different monoclonal antibodies which is capable of binding (selectively binding) a single immunogen (typically different epitopes on the immunogen); a plurality of different monoclonal antibodies which is capable of binding (selectively binding) a plurality of different immunogens, in which there is one or more antibody/s per immunogen (typically recognising different epitopes if they target the same immunogen). Rather than perform a single assay to recognise multiple immunogens simultaneously, it is preferred to perform multiple assays with a single monoclonal antibody per assay. These results can then be combined for an overall analysis of the vaccine sample. By using multiple assays, each immunogen within a multi-immunogen vaccine can be assessed separately e.g. to isolate the cause of any loss of potency relative to a standard vaccine.

An antibody can be tested to ensure that it does not cross-react with other antigens which might be present in a vaccine. This test is straightforward, and such cross-reacting antibodies can either be used with caution and proper controls, or can be rejected in favour of antibodies which do not have the cross-reacting activity.

To facilitate determination of relative potency, the monoclonal antibody should show a linear binding response when a target antigen diluted i.e. dilution of the target antigen should bring about a corresponding reduction in binding by the antibody. Linearity can be assessed by linear regression e.g. to have R²>0.95.

The monoclonal antibodies can be obtained from any suitable species e.g. murine, rabbit, sheep, goat, or human monoclonal antibodies. Advantageously, the chosen species can be selected such that secondary antibodies are readily available e.g. labelled goat anti-mouse secondary antibodies are easy to obtain, so mouse monoclonal antibodies are easily usable in ELISA.

The monoclonal antibodies can have any heavy chain type e.g. it can have a, 8, s, y or p heavy chain, giving rise respectively to antibodies of IgA, IgD, IgE, IgG, or IgM class. Classes may be further divided into subclasses or isotypes e.g. IgGl, IgG2, IgG3, IgG4, IgA, IgA2, etc. Antibodies may also be classified by allotype e.g. a y heavy chain may have Glm allotype a, f, x or z, G2m allotype n, or G3m allotype bO, bl, b3, b4, b5, c3, c5, gl, g5, s, t, u, or v; a K light chain may have a Km(l), Km(2) or Km(3) allotype. IgG monoclonal antibodies are preferred. A native IgG antibody has two identical light chains (one constant domain CL and one variable domain VL) and two identical heavy chains (three constant domains CHI CH2 & CH3 and one variable domain VH), held together by disulfide bridges.

The monoclonal antibodies can have any light chain type e.g. it can have either a kappa (K) or a lambda (X) light chain.

The term “antibody” is not limited to native antibodies, as naturally found in mammals. The term encompasses any similar molecule which can perform the same role in an immunoassay such as ELISA. Thus the antibody may be, for example, a fragment of a native antibody which retains antigen binding activity (e.g. a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment), a “singlechain Fv” comprising a VH and VL domain as a single polypeptide chain, a “diabody”, a “triabody”, a single variable domain or VHH antibody, a “domain antibody” (dAb), a chimeric antibody having constant domains from one organism but variable domains from a different organism, a CDR-grafted antibody, etc. The antibody may include a single antigen binding site (e.g. as in a Fab fragment or a scFv) or multiple antigen binding sites (e.g. as in a F(ab’)2 fragment or a diabody or a native antibody). Where an antibody has more than one antigen-binding site, however, it is preferably a mono-specific antibody i.e. all antigen-binding sites is capable of binding the same antigen. The antibody may have a constant domain (e.g. including CH or CL domains), but this is not always required. Thus the term “antibody” as used herein encompasses a range of proteins having diverse structural features (usually including at least one immunoglobulin domain having an all-P protein fold with a 2-layer sandwich of anti-parallel P-strands arranged in two P-sheets), but all of the proteins possess the ability to bind to the target protein immunogens.

The term “monoclonal” as originally used in relation to antibodies referred to antibodies produced by a single clonal line of B cells, as opposed to “polyclonal” antibodies that, while all recognizing the same target protein, were produced by different B cells and would be directed to different epitopes on that protein. As used herein, the word “monoclonal” does not imply any particular cellular origin, but refers to any population of antibodies that all have the same amino acid sequence and recognize the same epitope(s) in the same target protein(s). Thus a monoclonal antibody may be produced using any suitable protein synthesis system, including immune cells, non-immune cells, acellular systems, etc. This usage is usual in the field e.g. the product datasheets for the CDR grafted humanised antibody Synagis™ expressed in a murine myeloma NS0 cell line, the humanised antibody Herceptin™ expressed in a CHO cell line, and the phage-displayed antibody Humira™ expressed in a CHO cell line all refer the products as monoclonal antibodies. The term “monoclonal antibody” thus is not limited regarding the species or source of the antibody, nor by the manner in which it is made.

In the present description the sentence “able to differentiate between native and denatured form of antigen” or “able to distinguish between native and denatured form of antigen” means that the antibody is capable to bind the native form of the antigen or immunogen with a higher affinity than the denatured form, preferably means that is not capable to bind the denatured form of the antigen or immunogen, hence such antibodies can distinguish between functional immunogen and denatured or non-functional immunogen. Determining whether an antibody is able to differentiate between native and denatured form of antigen is known from the skilled person in the art. For instance, the antibody can be tested against a panel of linear peptide fragments from the target antigen (e.g. using the Pepscan technique) and the binding can be compared to the antibody’s binding against the complete antigen. As an alternative, binding can be compared before and after denaturation of the target antigen. For example using the termal denaturation as disclosed in the present application.

In the present description the sentence “the antibody is capable to bind” has the same meaning of “the antibody recognises”.

In the present description the term “the antibody is capable to bind” has the same meaning of “the antibody recognises”.

In the present description the term “antigen” means a molecule or molecular structure, such as may be present on the outside of a pathogen that can be bound by an antigen-specific antibody.

In the present description the term “immunogen” means an antigen that is capable of inducing humoral and/or cell-mediated immune response.

In the present description the term “immunogen” means also a protein immunogen, either definition can be used in any part of the description and of the claims as aliases. Known monoclonal antibodies can be used with the invention, or new monoclonal antibodies can be generated using known techniques (e.g. injection of a reference vaccine’s immunogen into mice with Freund’s complete adjuvant), followed by screening for those with suitable properties e.g. for bactericidal activity, etc. Preferred antibodies for analysis of the immunogens in BEXSERO are herein disclosed (see below) for example:

- for assaying fHbp antigen as found in the BEXSERO product include, but are not limited tothe 12C1/D7 antibody (disclosed in W02013/132040) and the 11F10/G6 antibody (disclosed in W02013/132040);

- for assaying NHBA antigen as found in the BEXSERO product include, but are not limited to the 10E8 antibody (see below);

- for assaying NadA antigen as found in the BEXSERO product include, but are not limited to the 6E3 antibody (see below).

Assaying a vesicle component in a vaccine can use any antigen in the vesicle, but it is convenient to use anti-PorA antibodies as these are readily available for serosubtype analysis (e.g. from NIBSC). Thus for assaying the OMV component as found in the BEXSERO product a suitable monoclonal antibody recognises serosubtype PorA1.4. A secondary antibody used with the invention (e.g. in the assay’s competitive format) can recognise the primary antibody when the primary antibody has become immobilised. The secondary antibody is typically polyclonal. For instance, if the primary antibody is murine then the secondary antibody can be an anti-murine antibody e.g. goat anti-mouse IgG. Suitable criteria for choosing secondary antibodies are well known in the ELISA field.

General

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., references 33-39, etc.

The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.

The term “about” in relation to a numerical value x is optional and means, for example, x+10%.

Where the invention concerns an “epitope”, this epitope may be a B-cell epitope and/or a T-cell epitope, but will usually be a B-cell epitope. Such epitopes can be identified empirically (e.g. using PEPSCAN [40,41] or similar methods), or they can be predicted (e.g. using the Jameson-Wolf antigenic index [42], matrix-based approaches [43], MAPITOPE [44], TEPITOPE [45,46], neural networks [47], OptiMer & EpiMer [48, 49], ADEPT [50], Tsites [51], hydrophilicity [52], antigenic index [53] or the methods disclosed in references 54-58, etc.). Epitopes are the parts of an antigen that are recognised by and bind to the antigen binding sites of antibodies or T-cell receptors, and they may also be referred to as “antigenic determinants”.

References to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences. This alignment and % homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. 59. A preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is disclosed in ref. 60.

The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention. Meningococcal protein immunogens

NHBA (Neisserial Heparin Binding Antigen)

NHBA [63] was included in the published genome sequence for meningococcal serogroup B strain MC58 [25] as gene NMB2132 (GenBank accession number GI:7227388; SEQ ID NO: 9 disclosed in W02013/132040). Sequences of NHBA from many strains have been published since then. For example, allelic forms of NHBA (referred to as protein ‘287’) can be seen in Figures 5 and 15 of reference 61, and in example 13 and figure 21 of reference 62. Various immunogenic fragments of NHBA have also been reported.

Preferred NHBA antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 9 disclosed in W02013/132040; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 9 SEQ ID NO: 9 disclosed in W02013/132040, wherein 'n' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 9 disclosed in W02013/132040.

The most useful NHBA antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 9 disclosed in W02013/132040. Advantageous NHBA antigens for use with the invention can elicit bactericidal anti- meningococcal antibodies after administration to a subject.

Over-expression of NHBA has previously been achieved in various ways e.g. introduction of a NHBA gene under the control of an IPTG-inducible promoter [63],

NadA (Neisserial adhesin A)

The NadA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [25] as gene NMB1994 (GenBank accession number GE7227256; SEQ ID NOTO disclosed in W02013/132040). The sequences of NadA antigen from many strains have been published since then, and the protein’s activity as a Neisserial adhesin has been well documented. Various immunogenic fragments of NadA have also been reported.

Preferred NadA immunogens or antigen for use with the invention comprise an amino acid sequence:

(a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 10 disclosed in W02013/132040; and/or

(b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 10 disclosed in W02013/132040, wherein 'n' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NOTO disclosed in W02013/132040. The most useful NadA immunogens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 10 disclosed in W02013/132040.

HmbR

The full-length HmbR sequence was included in the published genome sequence for meningococcal serogroup B strain MC58 [25] as gene NMB1668 (SEQ ID NO: 7 disclosed in W02013/132040). Reference 64 reports a HmbR sequence from a different strain (SEQ ID NO: 8 disclosed in W02013/132040), and reference 65 reports a further sequence (SEQ ID NO: 19 disclosed in W02013/132040). SEQ ID NOs: 7 and 8 differ in length by 1 amino acid and have 94.2% identity. SEQ ID NO: 19 disclosed in W02013/132040 is one amino acid shorter than SEQ ID NO: 7 disclosed in W02013/132040 and they have 99% identity (one insertion, seven differences) by CLUSTALW. The invention can use any such HmbR polypeptide.

The invention can use a polypeptide that comprises a full-length HmbR sequence, but it will often use a polypeptide that comprises a partial HmbR sequence. Thus in some embodiments a HmbR sequence used according to the invention may comprise an amino acid sequence having at least i% sequence identity to SEQ ID NO: 7 disclosed in W02013/132040, where the value of i is 50, 60, 70, 80, 90, 95, 99 or more. In other embodiments a HmbR sequence used according to the invention may comprise a fragment of at least j consecutive amino acids from SEQ ID NO: 7 disclosed in W02013/132040, where the value of is 7, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more. In other embodiments a HmbR sequence used according to the invention may comprise an amino acid sequence (i) having at least i% sequence identity to SEQ ID NO: 7 disclosed in W02013/132040 and/or (ii) comprising a fragment of at least j consecutive amino acids from SEQ ID NO: 7 disclosed in W02013/132040.

Preferred fragments of j amino acids comprise an epitope from SEQ ID NO: 7 disclosed in W02013/132040. Such epitopes will usually comprise amino acids that are located on the surface of HmbR. Useful epitopes include those with amino acids involved in HmbR’s binding to haemoglobin, as antibodies that bind to these epitopes can block the ability of a bacterium to bind to host haemoglobin. The topology of HmbR, and its critical functional residues, were investigated in reference 66. Fragments that retain a transmembrane sequence are useful, because they can be displayed on the bacterial surface e.g. in vesicles. Examples of long fragments of HmbR correspond to SEQ ID NOs: 15 and 16 disclosed in W02013/132040. If soluble HmbR is used, however, sequences omitting the transmembrane sequence, but typically retaining epitope(s) from the extracellular portion, can be used. fHbp (factor H binding protein)

The fHbp antigen has been characterised in detail. It has also been known as protein ‘741’ [SEQ IDs 2535 & 2536 in ref. 62], ‘NMB1870’, ‘GNA1870’ [67-69], ‘P2086’, ‘LP2086’ or ‘ORF2086’ [70-72], It is naturally a lipoprotein and is expressed across all meningococcal serogroups. The structure of fHbp’s C-terminal immunodominant domain (‘fHbpC’) has been determined by NMR [73], This part of the protein forms an eight-stranded P-barrel, whose strands are connected by loops of variable lengths. The barrel is preceded by a short a-helix and by a flexible N-terminal tail.

The fHbp antigen falls into three distinct variants [74] and it has been found that serum raised against a given family is bactericidal within the same family, but is not active against strains which express one of the other two families i.e. there is intra-family cross-protection, but not inter-family cross-protection. The invention can use a single fHbp variant, but a vaccine will usefully include a fHbp from two or three of the variants. Thus it may use a combination of two or three different fHbps, selected from: (a) a first protein, comprising an amino acid sequence having at least a% sequence identity to SEQ ID NO: 1 disclosed in W02013/132040 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1 disclosed in W02013/132040; (b) a second protein, comprising an amino acid sequence having at least b%> sequence identity to SEQ ID NO: 2 disclosed in W02013/132040 and/or comprising an amino acid sequence consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 2 disclosed in W02013/132040; and/or (c) a third protein, comprising an amino acid sequence having at least c%> sequence identity to SEQ ID NO: 3 disclosed in W02013/132040 and/or comprising an amino acid sequence consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 3 disclosed in W02013/132040.

The value of a is at least 85 e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or more.

The value of b is at least 85 e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or more.

The value of c is at least 85 e.g. 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or more.

The values of a, b and c are not intrinsically related to each other.

The value of x is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value of y is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value ofz is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The values ofx. y and z are not intrinsically related to each other.

Where the invention uses a single fHbp variant, a vaccine composition may include a polypeptide comprising (a) an amino acid sequence having at least a% sequence identity to SEQ ID NO: 1 disclosed in W02013/132040 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1 disclosed in W02013/132040; or (b) an amino acid sequence having at least b%> sequence identity to SEQ ID NO: 2 disclosed in W02013/132040 and/or comprising an amino acid sequence consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 2 disclosed in W02013/132040; or (c) an amino acid sequence having at least c% sequence identity to SEQ ID NO: 3 disclosed in W02013/132040 and/or comprising an amino acid sequence consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 3 disclosed in W02013/132040.

Where the invention uses a fHbp from two or three of the variants, a vaccine composition may include a combination of two or three different fHbps selected from: (a) a first polypeptide, comprising an amino acid sequence having at least a%> sequence identity to SEQ ID NO: 1 disclosed in W02013/132040 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1 disclosed in W02013/132040; (b) a second polypeptide, comprising an amino acid sequence having at least b%> sequence identity to SEQ ID NO: 2 disclosed in W02013/132040 and/or comprising an amino acid sequence consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 2 disclosed in W02013/132040; and/or (c) a third polypeptide, comprising an amino acid sequence having at least c%> sequence identity to SEQ ID NO: 3 disclosed in W02013/132040 and/or comprising an amino acid sequence consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 3 disclosed in W02013/132040. The first, second and third polypeptides have different amino acid sequences.

Where the invention uses a fHbp from two of the variants, a composition can include both: (a) a first polypeptide, comprising an amino acid sequence having at least a%> sequence identity to SEQ ID NO:

1 disclosed in W02013/132040 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1 disclosed in W02013/132040; and (b) a second polypeptide, comprising an amino acid sequence having at least b%> sequence identity to SEQ ID NO:

2 disclosed in W02013/132040 and/or comprising an amino acid sequence consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 2 disclosed in W02013/132040. The first and second polypeptides have different amino acid sequences.

Where the invention uses a fHbp from two of the variants, a composition can include both: (a) a first polypeptide, comprising an amino acid sequence having at least a%> sequence identity to SEQ ID NO: 1 disclosed in W02013/132040 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 1 disclosed in W02013/132040; (b) a second polypeptide, comprising an amino acid sequence having at least c%> sequence identity to SEQ ID NO:

3 disclosed in W02013/132040 and/or comprising an amino acid sequence consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 3 disclosed in W02013/132040. The first and second polypeptides have different amino acid sequences.

Another useful fHbp which can be used according to the invention is one of the modified forms disclosed, for example, in reference 75 e.g. comprising SEQ ID NO: 20 or 23 therefrom. These modified forms can elicit antibody responses which are broadly bactericidal against meningococci. SEQ ID NO: 77 in reference 75 is another useful fHbp sequence which can be used. fHbp protein(s) in a OMV will usually be lipidated e.g. at aN-terminus cysteine. In other embodiments they will not be lipidated.

One vaccine which can be analysed by the methods of the invention includes two different variants of fHbp. The first variant can have amino acid sequence SEQ ID NO: 29 disclosed in W02013/132040, and the second can have amino acid sequence SEQ ID NO: 30 disclosed in W02013/132040. These are preferably lipidated at their N-terminus cysteines. This vaccine can include an aluminium phosphate adjuvant, and can also include a histidine buffer and polysorbate 80. Ideally it includes equal masses of the two different fHbp polypeptides.

NspA (Neisserial surface protein A)

The NspA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [25] as gene NMB0663 (GenBank accession number GE7225888; SEQ ID NO: 11 disclosed in W02013/132040). The antigen was previously known from references 76 & 77. The sequences of NspA antigen from many strains have been published since then. Various immunogenic fragments of NspA have also been reported.

Preferred NspA immunogens or antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 11; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 11 disclosed in W02013/132040, wherein 'n' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 11 disclosed in W02013/132040.

The most useful NspA immunogens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 11 disclosed in W02013/132040. Advantageous NspA antigens for use with the invention can elicit bactericidal anti- meningococcal antibodies after administration to a subject.

NhhA (Neisseria hia homologue)

The NhhA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [25] as gene NMB0992 (GenBank accession number GE7226232; SEQ ID NO: 12 disclosed in W02013/132040). The sequences of NhhA antigen from many strains have been published since e.g. refs 61 & 78, and various immunogenic fragments of NhhA have been reported. It is also known as Hsf

Preferred NhhA immunogens or antigens for use with the invention comprise an amino acid sequence:

(a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 12 disclosed in W02013/132040; and/or

(b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 12 disclosed in W02013/132040, wherein 'n' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 12 disclosed in W02013/132040.

The most useful NhhA immunogens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 12 disclosed in W02013/132040. Advantageous NhhA antigens for use with the invention can elicit bactericidal anti- meningococcal antibodies after administration to a subject.

App (Adhesion and penetration protein)

The App antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [25] as gene NMB1985 (GenBank accession number GE7227246; SEQ ID NO: 13 disclosed in W02013/132040). The sequences of App antigen from many strains have been published since then. It has also been known as ‘ORFF and ‘Hap’. Various immunogenic fragments of App have also been reported.

Preferred App immunogens or antigens for use with the invention comprise an amino acid sequence:

(a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 13 disclosed in W02013/132040; and/or

(b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 13 disclosed in W02013/132040, wherein 'n' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 13 disclosed in W02013/132040.

The most useful App immunogens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 13 disclosed in W02013/132040. Advantageous App antigens for use with the invention can elicit bactericidal anti- meningococcal antibodies after administration to a subject.

Omp85 (85kDa outer membrane protein)

The Omp85 antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [25] as gene NMB0182 (GenBank accession number GE7225401; SEQ ID NO: 14 disclosed in W02013/132040). The sequences of Omp85 antigen from many strains have been published since then. Further information on Omp85 can be found in references 79 and 80. Various immunogenic fragments of Omp85 have also been reported.

Preferred Omp85 immunogens or antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 14; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 14, wherein 'n' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 14 disclosed in W02013/132040.

The most useful Omp85 immunogens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 14 disclosed in W02013/132040. Advantageous Omp85 antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.

TbpA

The TbpA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [25] as gene NMB0461 (GenBank accession number GE7225687; SEQ ID NO: 17 disclosed in W02013/132040). The sequences of TbpA from many strains have been published since then. Various immunogenic fragments of TbpA have also been reported.

Preferred TbpA immunogens or antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 17; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 17, wherein 'n' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 17 disclosed in W02013/132040.

The most useful TbpA immunogens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 17 disclosed in W02013/132040. Advantageous TbpA antigens for use with the invention can elicit bactericidal anti- meningococcal antibodies after administration to a subject.

TbpB

The TbpB antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [25] as gene NMB1398 (GenBank accession number GE7225686; SEQ ID NO: 18 disclosed in W02013/132040). The sequences of TbpB from many strains have been published since then. Various immunogenic fragments of TbpB have also been reported. Preferred TbpB immunogens or antigens for use with the invention comprise an amino acid sequence:

(a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 18 disclosed in W02013/132040; and/or

(b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 18 disclosed in W02013/132040, wherein 'n' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 18 disclosed in W02013/132040.

The most useful TbpB immunogens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 18 disclosed in W02013/132040. Advantageous TbpB antigens for use with the invention can elicit bactericidal anti- meningococcal antibodies after administration to a subject.

Cu.Zn-superoxide dismutase

The Cu,Zn-superoxide dismutase antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [25] as gene NMB1398 (GenBank accession number GI: 7226637; SEQ ID NO: 20 disclosed in W02013/132040). The sequences of Cu,Zn-superoxide dismutase from many strains have been published since then. Various immunogenic fragments of Cu,Zn-superoxide dismutase have also been reported.

Preferred Cu,Zn-superoxide dismutase antigens for use with the invention comprise an amino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 20 disclosed in W02013/132040; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 20 disclosed in W02013/132040, wherein 'n' is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise an epitope from SEQ ID NO: 20 disclosed in W02013/132040.

The most useful Cu,Zn-superoxide dismutase antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 20 disclosed in W02013/132040. Advantageous Cu,Zn-superoxide dismutase antigens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.

Method for detecting or measuring a change in conformation of a meningococcal protein immunogen

In one embodiment the invention provides a method for detecting or measuring a change in conformation of a meningococcal protein immunogen in a vaccine sample, said method comprising steps of: (i) performing the in vitro assay herein disclosed on a test sample and; (ii) performing the in vitro assay herein disclosed on a standard vaccine sample of known native antigenic form and; and (iii) comparing the results from steps (i) and (ii) to determine the amount of immunogen(s) in the native form of the test vaccine relative to the amount of immunogen(s) in the native form in the standard vaccine.

Method for in vitro relative potency analysis of a meningococcal test vaccine sample

In one embodiment the invention provides a method for in vitro relative potency analysis of a meningococcal test vaccine sample, comprising steps of: (i) performing the assay herein disclosed on the test sample and, optionally; (ii) performing the assay herein disclosed on a standard vaccine sample of known in vivo potency and, optionally, on at least one dilution of the standard vaccine sample; and (iii) comparing the results from steps (i) and (ii) to determine the potency of immunogen(s) in the test vaccine relative to the potency of immunogen(s) in the standard vaccine.

Monoclonal antibodies

The invention also provides monoclonal antibodies, which are capable of binding to meningococcal antigens, in particular wherein said monoclonal antibodies are bactericidal for meningococcus, in particular against homologous reference strains, and are capable of binding a conformational epitope of said meningococcal antigens. These antibodies can be used with the assays of the invention, or can be used more generally.

Each of the antibodies disclosed herein has the following features:

-specificity for the target;

-binds to a functional epitope on the antigen (e.g., competes for binding with neutralizing antibodies);

-is sensitive to degradation of the target (i.e., binds an epitope which is lost during degradation) or in general to be stability indicating.

One antibody of the invention is a monoclonal antibody (10E8) that is capable of binding (selectively binding) meningococcal NHB A antigen, whose light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 2 and whose heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 6

One antibody of the invention is a monoclonal antibody (10E8) that is capable of binding (selectively binding) meningococcal NHBA antigen whose VH and VL comprise the following complementarity-determining regions (CDRs):

-CDR1 of VH having SED ID NO: 7,

-CDR2 of VH having SED ID NO: 8,

-CDR3 of VH having SED ID NO: 9,

-CDR1 of VL having SED ID NO: 3, -CDR2 of VL having the sequence RMS and -CDR3 of VL having SED ID NO:4.

In one embodiment the antibody of the invention is a monoclonal antibody that is capable of binding (selectively binding) an epitope of meningococcal antigen NHBA comprising or consisting in the amino acid sequence of SEQ ID NO: 19, preferably in SEQ ID NO:20, more preferably in SEQ ID NO:21.

In one embodiment, the antibody of the invention is a monoclonal antibody able to differentiate between native and denatured form of antigen NHBA and whose VH and VL region shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity with the amino acid sequence of VH (SEQ ID NO: 6) and VL (SEQ ID NO: 2) region of the antibody 10E8.

The invention provides also a monoclonal antibody that binds to antigen NHBA and competes or crosscompetes with and/or binds the same epitope as the antibody 10E8. If two antibodies reciprocally compete with each other for binding to antigen NHBA, they are said to compete.

One antibody of the invention is a monoclonal antibody (6E3) that is capable of binding (selectively binding) meningococcal NadA antigen, whose VL region has the amino acid sequence of SEQ ID NO: 11 and whose VH region has the amino acid sequence of SEQ ID NO: 15.

One antibody of the invention is a monoclonal antibody (6E3) that is capable of binding (selectively binding) meningococcal NHBA antigen whose VH and VL comprise the following complementarity-determining regions (CDRs):

-CDR1 of VH having SED ID NO: 16,

-CDR2 of VH having SED ID NO: 17,

-CDR3 of VH having SED ID NO: 18,

-CDR1 of VL having SED ID NO: 12,

-CDR2 of VL having the sequence ATS and

-CDR3 of VL having SED ID NO: 13.

In one embodiment the antibody of the invention is a monoclonal antibody that is capable of binding (selectively binding) an epitope in the region of meningococcal antigen NadA corresponding to the amino acid sequence of residues 206-249 of SEQ ID NO:22.

In one embodiment, the antibody of the invention is a monoclonal antibody able to differentiate between native and denatured form of antigen NadA and whose VH and VL region shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more of identities with the amino acid sequence of VH (SEQ ID NO: 15) and VL (SEQ ID NO: 11) region of the antibody 6E3. The invention provides also a monoclonal antibody that binds to antigen NadA and competes or crosscompetes with and/or binds the same epitope as the antibody 6E3. If two antibodies reciprocally compete with each other for binding to antigen NadA, they are said to cross-compete.

One can determine whether an antibody binds to the same epitope or cross competes for binding with an anti-meningococcal-antigen-antibody by using methods known in the art. For example, allowing one of the antibody of the invention to bind to the target meningococcal antigen under saturating conditions and then measures the ability of the test antibody to bind to the target meningococcal antigen. If the test antibody is able to bind to the same meningococcal antigen at the same time as the antibody of the invention, then the test antibody binds to a different epitope as the antibody of the invention. However, if the test antibody is not able to bind to the same meningococcal antigen at the same time, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope that is in close proximity to the epitope bound by the antibody of the invention. This experiment can be performed using ELISA, RIA, BIACORE(TM), flow cytometry or other methods known in the art. One may use the competition method described above in two directions i.e. determining if the reference antibody blocks the test antibody and vice versa.

In certain aspects, the invention provides an isolated cell line that produces the antibody or antigenbinding portion thereof according to any one of the embodiments herein disclosed.

In certain aspects, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes the antibody or antigen-binding portion thereof according to any one of the embodiments herein disclosed, such isolated nucleic acid molecule have for example a sequence selected from SEQ ID NO: 1, SEQ ID NO:5 SEQ ID NOTO or SEQ ID NO: 14.

In certain aspects, the invention provides a vector comprising the nucleic acid molecule encoding the antibody or antigen-binding portion thereof embodiments according to any one of the embodiments herein disclosed, wherein the vector optionally comprises an expression control sequence operably linked to the nucleic acid molecule.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1: A) NadA binds with high affinity to mAb 6E3. Panel A shows both the experimental curve and the calculated curve based on fitting to a 1 : 1 binding model. B) A summary of the kinetic values for the interaction.

Figure 2. FACS analysis was performed on strain NMB using anti-mouse IgG-FITC conjugated as secondary antibody

Figure 3. Results of the Protein Chip Analysis for mAb6E3 on the NadA antigen. Figure 4. Panel A: in-silico model of NadA with the epitope recognized by mAh 6E3. The model was generated on the basis of sequence homology to NadA var5, whose xray structure was recently solved. Dashes show regions with low sequence homology or unknown secondary structure that were not included in the in-silico model. Panel B: time course of deuterium incorporation for the peptides covering the entire peptide map of NadA, as free form (solid line) or bound to the mAb 6E3 (dashed line). The peptides with a significant difference of deuterium uptake are highlighted.

Figure 5. SPR binding results obtained for NadA and NadA heat-treated to immobilized mAb6E3

Figure 6. SCK results showing the experimental curve and the calculated curve based on fitting to a 1 : 1 binding model for antigen NHBA binding to mAbl0E8.

Figure 7. Results of the Protein Chip analysis with mAbl0E8 on fragments of different length spanning the entire sequence of NHBA. Only fragments containing the common 84-115 amino acid sequence are recognized by mAbl0E8

Figure 8. Results of the PeptideScanning analysis with mAbl0E8 on the full length sequence of NHBA-953 fusion protein. The sequences of the different NHBA peptide variants (pl, p2, etc) are aligned

Figure 9. Time course of deuterium incorporation for the peptides covering the entire peptide map of NHBA, as free form (solid line) or bound to the mAb 10E8 (dashed line). The peptide with a significant difference of deuterium uptake is shown in the bold dash-line box.

Figure 10. SPR binding results obtained with 287-953 antigen kept treated at different temperatures to mAbl0E8. Binding is strongly reduced when the protein is treated at 40°C for 2 weeks or at 95°C for 15 hours.

Figure 11. Schematic representation of the four steps of the assay according to one embodiment of the invention. In the competition step Ref & Test Vaccine may in duplicate be three-fold diluted in 96 w LB-DW plate. In the centrifugation step only supernatant (free mAbs) is transferred into the ELISA plate (coated and blocked).

Figure 12: Results obtained for 287-953 as overall Optical Density response (no RP values have been calculated). Dashed and solid lines represent Reference and treated sample respectively.

Figure 13: Results for 287 antigen/10E8 mAb, for each session and operator, as overall Optical Density response in log natural scale.

Figure 14: Results for 741 antigen/12ClD7 mAb, for each session and operator, as overall Optical Density response in log natural scale. Figure 15: Results for 961 antigen/6E3 mAb, for each session and operator, as overall Optical Density response in log natural scale.

Figure 16: Results for OMV antigen/PorA1.4 mAb, for each session and operator, as overall Optical Density response in log natural scale.

Summarising, the invention relates to:

A binding assay for in vitro analysis of a meningococcal vaccine sample, comprising steps of: (i) allowing a meningococcal immunogen within the sample to interact with a monoclonal antibody which is bactericidal for meningococcus and/or capable of binding a conformational epitope of an immunogen in said meningococcal vaccine then (ii) measuring the interaction between the immunogen and antibody from step (i).

The assay as defined above, wherein the measurement in step (ii) provides the amount of the monoclonal antibody’s target epitope within said meningococcus vaccine sample.

The assay of any one of the definitions above, wherein the binding assay is an ELISA, in particular a competitive ELISA.

The assay of any one of the definitions above, comprising the following steps: (i) incubating the sample with the monoclonal antibody so that complexes can form between the antibody and meningococcal immunogen in the sample; (ii) separating the unbound monoclonal antibody from immunogen-bound monoclonal antibody; (iii) adding the unbound monoclonal antibody to a container in which antigens of said monoclonal antibody are immobilised, wherein the immobilised antigens can form a complex with said unbound monoclonal antibody; (iv) determining the amount of the complex formed in step (iii) and (v) measuring the interaction between the immunogen and antibody from step (i).

The assay of any one of the definitions above, wherein said separation of the unbound monoclonal antibody from immunogen-bound monoclonal antibody is carried out by centrifugation.

The assay of any one of the definitions above, wherein the measurement in step (ii) uses a secondary antibody labelled with an enzyme.

The assay of any one of the definitions above, wherein the vaccine includes meningococcal NHB A, fHbp and/or NadA immunogen, and wherein the monoclonal antibody used in step (i) capable of binding meningococcal NHB A, fHbp or NadA immunogen.

The assay of any one of the definitions above, wherein the vaccine includes more than one meningococcal immunogen.

The assay of any one of the definitions above, wherein the vaccine includes meningococcal outer membrane vesicles.

The assay of any one of the definitions above, which uses a single monoclonal antibody in step (i). The assay of any one of the definitions above, which uses a murine monoclonal IgG antibody in step (i), in particular IgGl or IgG2b.

The assay of any one of the definitions above, wherein the vaccine includes one or more adsorbed meningococcal immunogen.

The assay of any one of the definitions above, wherein said meningococcal immunogen is adsorbed with aluminium hydroxide adjuvant.

The assay of any one of the definitions above further comprising a desorption step in order to separate adsorbed meningococcal immunogens, preferably said desorption step is performed before the step (i).

The assay of any one of the definitions above, wherein in said step (i) said interaction in step (i) is carried out in a medium comprising a blocking buffer.

The assay as defined above, wherein said blocking buffer comprises casein or casein derivatives or fragments thereof.

The assay as defined above, wherein said adsorbed immunogen is NHBA, fHbp or NadA and the blocking buffer is at a concentration of about 0.5% or the adsorbed immunogen is OMV and the blocking buffer is at a concentration of about 4%.

The assay of any one of the definitions above, wherein said monoclonal antibody is one or more monoclonal antibody selected from the monoclonal antibody of any one of claims 24 to 32.

A method for detecting or measuring a change in conformation of a meningococcal immunogen in a vaccine sample, comprising steps of: (i) performing the in vitro assay according of any one of the definitions above on a test sample and, optionally, on at least one dilution of the test sample; (ii) performing the in vitro assay according to any one of the definitions above on a standard vaccine sample of known native antigenic form and, optionally, on at least one dilution of the standard vaccine sample; and (iii) comparing the results from steps (i) and (ii) to determine the amount of immunogen(s) in the native form of the test vaccine relative to the amount of immunogen(s) in the native form in the standard vaccine.

A method for in vitro relative potency analysis of a meningococcal test vaccine sample, comprising steps of: (i) performing the assay of any one of the definitions above on the test sample and, optionally, on at least one dilution of the test sample; (ii) performing the assay of any one of the definitions above on a standard vaccine sample of known in vivo potency and, optionally, on at least one dilution of the standard vaccine sample; and (iii) comparing the results from steps (i) and (ii) to determine the potency of immunogen(s) in the test vaccine relative to the potency of immunogen(s) in the standard vaccine.

A method for analysing a batch of vaccine, comprising steps of: (i) assaying the relative potency of immunogen(s) in at least one vaccine from the batch by the method as defined above, and, if the results of step (i) indicate an acceptable relative potency, (ii) releasing further vaccines from the batch for in vivo use.

A Kit for in vitro ELISA assay of a meningococcal vaccine sample comprising (i) a solution-phase anti-vaccine monoclonal antibody (ii) an immobilised antigen which is recognised by the anti-vaccine antibody, and (iii) a labelled antibody which binds to the anti-vaccine antibody, wherein the anti-vaccine antibody (a) is bactericidal for meningococcus and/or (b) capable of binding a conformational epitope of the meningococcal NHBA or NadA antigen.

A vaccine which has been released following the use of an assay of any one of the definitions above or a method according to any one of the definitions above.

A monoclonal antibody capable of binding the meningococcal NHBA antigen, whose VH and VL comprise the following complementarity-determining regions (CDRs):

-CDR1 of VH having SED ID NO: 7,

-CDR2 of VH having SED ID NO: 8,

-CDR3 of VH having SED ID NO: 9,

-CDR1 of VL having SED ID NO: 3,

-CDR2 of VL having the aminoacid sequence RMS and

-CDR3 of VL having SED ID NO:4.

A monoclonal antibody capable of binding the meningococcal NHBA antigen, whose light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 2 and whose heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 6.

A monoclonal antibody capable of binding an epitope of the meningococcal antigen NHBA comprising or consisting in the amino acid sequence of SEQ ID NO: 19, preferably of SEQ ID NO: 20, more preferably in SEQ ID NO:21.

A monoclonal antibody able to distinguish between native and denatured form of antigen NHBA and whose VH and VL shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity with the amino acid sequence of SEQ ID NO: 6 (VH) and SEQ ID NO: 2(VL) respectively.

A monoclonal antibody that binds to antigen NHBA and competes or cross-competes with and/or binds the same epitope of the monoclonal antibody according to any one of the definitions above.

A monoclonal antibody capable of binding meningococcal NadA antigen, whose VH and VL comprise the following complementarity-determining regions (CDRs):

-CDR1 of VH having SED ID NO: 16,

-CDR2 of VH having SED ID NO: 17,

-CDR3 of VH having SED ID NO: 18, -CDR1 of VL having SED ID NO: 12,

-CDR2 of VL having the amino acid sequence ATS and

-CDR3 of VL having SED ID NO: 13.

A monoclonal antibody capable of binding meningococcal NadA antigen, whose VL region has the amino acid sequence of SEQ ID NO: 11 and whose VH region has the amino acid sequence of SEQ ID NO: 15.

A monoclonal antibody capable of binding an epitope in the region of meningococcal antigen NadA corresponding to the amino acid sequence of residues 206-249 of SEQ ID NO:22.

A monoclonal antibody able to distinguish between native and denatured form of antigen NadA and whose VH and VL region shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity with the amino acid sequence SEQ ID NO: 15 (VH) and SEQ ID NO: 11 (VL).

A monoclonal antibody that binds to antigen NadA and competes or cross-competes with and/or binds the same epitope of the monoclonal antibody according to any one of the definitions above.

Embodiments of the invention are further described in the subsequent numbered paragraphs: A binding assay for in vitro analysis of a meningococcal vaccine sample, comprising steps of: (i) allowing a meningococcal immunogen within the sample to interact with a monoclonal antibody which is bactericidal for meningococcus and/or capable of binding a conformational epitope of an immunogen in said meningococcal vaccine then (ii) measuring the interaction between the immunogen and antibody from step (i). The assay of the preceding paragraph, wherein the measurement in step (ii) provides the amount of the monoclonal antibody’s target epitope within said meningococcus vaccine sample. The assay of any preceding paragraphs, wherein the binding assay is an ELISA, in particular a competitive ELISA. The assay of any preceding paragraphs, comprising the following steps: (i) incubating the sample with the monoclonal antibody so that complexes can form between the antibody and meningococcal immunogen in the sample; (ii) separating the unbound monoclonal antibody from immunogen-bound monoclonal antibody; (iii) adding the unbound monoclonal antibody to a container in which antigens of said monoclonal antibody are immobilised, wherein the immobilised antigens can form a complex with said unbound monoclonal antibody; (iv) determining the amount of the complex formed in step (iii) and (v) measuring the interaction between the immunogen and antibody from step (i). 5. The assay of the preceding paragraph, wherein said separation of the unbound monoclonal antibody from immunogen-bound monoclonal antibody is carried out by centrifugation.

6. The assay of any preceding paragraphs, wherein the measurement in step (ii) uses a secondary antibody labelled with an enzyme.

7. The assay of any preceding paragraphs, wherein the vaccine includes meningococcal NHBA, fHbp and/or NadA immunogen, and wherein the monoclonal antibody used in step (i) capable of binding meningococcal NHBA, fHbp or NadA immunogen.

8. The assay of any preceding paragraphs, wherein the vaccine includes more than one meningococcal immunogen.

9. The assay of any preceding paragraphs, wherein the vaccine includes meningococcal outer membrane vesicles.

10. The assay of any preceding paragraphs, which uses a single monoclonal antibody in step (i).

11. The assay of any preceding paragraphs, which uses a murine monoclonal IgG antibody in step (i), in particular IgGl or IgG2b.

12. The assay of any preceding paragraphs, wherein the vaccine includes one or more adsorbed meningococcal immunogen.

13. The assay of the preceding paragraphs, wherein said meningococcal immunogen is adsorbed with aluminium hydroxide adjuvant.

14. The assay of any preceding paragraphs further comprising a desorption step in order to separate adsorbed meningococcal immunogens, preferably said desorption step is performed before the step (i).

15. The assay of any preceding paragraphs, wherein in said step (i) said interaction in step (i) is carried out in a medium comprising a blocking buffer.

16. The assay of the paragraph 15, wherein said blocking buffer comprises casein or casein derivatives or fragments thereof.

17. The assay of paragraph 16, wherein said adsorbed immunogen is NHBA, fHbp or NadA and the blocking buffer is at a concentration of about 0.5% or the adsorbed immunogen is OMV and the blocking buffer is at a concentration of about 4%.

18. The assay of any preceding paragraphs, wherein said monoclonal antibody is one or more monoclonal antibody selected from the monoclonal antibody of any one of paragraphs 24 to 32.

19. A method for detecting or measuring a change in conformation of a meningococcal immunogen in a vaccine sample, comprising steps of: (i) performing the in vitro assay according of any one of paragraphs 1 to 18 on a test sample and, optionally, on at least one dilution of the test sample; (ii) performing the in vitro assay according to any one of paragraphs 1 to 18 on a standard vaccine sample of known native antigenic form and, optionally, on at least one dilution of the standard vaccine sample; and (iii) comparing the results from steps (i) and (ii) to determine the amount of immunogen(s) in the native form of the test vaccine relative to the amount of immunogen(s) in the native form in the standard vaccine.

20. A method for in vitro relative potency analysis of a meningococcal test vaccine sample, comprising steps of: (i) performing the assay of any one of paragraphs 1 to 18 on the test sample and, optionally, on at least one dilution of the test sample; (ii) performing the assay of any one of paragraphs 1 to 18 on a standard vaccine sample of known in vivo potency and, optionally, on at least one dilution of the standard vaccine sample; and (iii) comparing the results from steps (i) and (ii) to determine the potency of immunogen(s) in the test vaccine relative to the potency of immunogen(s) in the standard vaccine.

21. A method for analysing a batch of vaccine, comprising steps of: (i) assaying the relative potency of immunogen(s) in at least one vaccine from the batch by the method of paragraph 19, and, if the results of step (i) indicate an acceptable relative potency, (ii) releasing further vaccines from the batch for in vivo use.

22. A Kit for in vitro ELISA assay of a meningococcal vaccine sample comprising (i) a solution-phase anti-vaccine monoclonal antibody (ii) an immobilised antigen which is recognised by the anti-vaccine antibody, and (iii) a labelled antibody which binds to the anti-vaccine antibody, wherein the anti-vaccine antibody (a) is bactericidal for meningococcus and/or (b) capable of binding a conformational epitope of the meningococcal NHBA or NadA antigen.

23. A vaccine which has been released following the use of an assay of any preceding paragraph 1 to 18 or a method according to anyone of paragraphs 19 to 21.

24. A monoclonal antibody capable of binding the meningococcal NHBA antigen, whose VH and VL comprise the following complementarity-determining regions (CDRs):

-CDR1 of VH having SED ID NO: 7,

-CDR2 of VH having SED ID NO: 8,

-CDR3 of VH having SED ID NO: 9,

-CDR1 of VL having SED ID NO: 3,

-CDR2 of VL having the aminoacid sequence RMS and

-CDR3 of VL having SED ID NO:4.

25. A monoclonal antibody capable of binding the meningococcal NHBA antigen according to paragraph 24, whose light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 2 and whose heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 6. A monoclonal antibody capable of binding an epitope of the meningococcal antigen NHBA comprising or consisting in the amino acid sequence of SEQ ID NO: 19, preferably of SEQ ID NO: 20, more preferably in SEQ ID NO:21. A monoclonal antibody able to distinguish between native and denatured form of antigen NHBA and whose VH and VL shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity with the amino acid sequence of SEQ ID NO: 6 (VH) and SEQ ID NO: 2(VL) respectively. A monoclonal antibody that binds to antigen NHBA and competes or cross-competes with and/or binds the same epitope of the monoclonal antibody according to any one of the paragraphs from 23 to 26. A monoclonal antibody capable of binding meningococcal NadA antigen, whose VH and VL comprise the following complementarity-determining regions (CDRs):

-CDR1 of VH having SED ID NO: 16,

-CDR2 of VH having SED ID NO: 17,

-CDR3 of VH having SED ID NO: 18,

-CDR1 of VL having SED ID NO: 12,

-CDR2 of VL having the amino acid sequence ATS and

-CDR3 of VL having SED ID NO: 13. A monoclonal antibody capable of binding meningococcal NadA antigen according to paragraph 28, whose VL region has the amino acid sequence of SEQ ID NO: 11 and whose VH region has the amino acid sequence of SEQ ID NO: 15. A monoclonal antibody capable of binding an epitope in the region of meningococcal antigen NadA corresponding to the amino acid sequence of residues 206-249 of SEQ ID NO:22. A monoclonal antibody able to distinguish between native and denatured form of antigen NadA and whose VH and VL region shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity with the amino acid sequence SEQ ID NO: 15 (VH) and SEQ ID NO: 11 (VL). A monoclonal antibody that binds to antigen NadA and competes or cross-competes with and/or binds the same epitope of the monoclonal antibody according to any one of the paragraphs from 28 to 31. EXAMPLES AND EXPERIMENTAL DATA

Anti- NadA-monoclonal antibody 6E3

The murine anti-NadA monoclonal antibody of IgGl subclass defined herein as 6E3 was obtained. RNA was isolated from the murine hybridoma cells using RNA prepared from dead cells using the Qiagen RNeasy mini kit (Cat No: 74104). RNA was eluted in 50pL water and checked by OD 260/280nm. The first strand cDNAs were amplified by PCR using oligo (dT) primers and VH and VK regions were amplified using several sets of degenerated primers. The amplified DNAs were gel- purified and cloned into a suitable vector. The VH and VK clones obtained were screened for inserts of the expected size. The DNA sequence of at least ten selected clones was determined in both directions by automated DNA sequencing. The locations of the complementarity determining regions (CDRs) in the sequences were determined with reference to other antibody sequences (Chothia & Lesk, 1987; Kabat EA et al., 1991; IMGT - Lefranc MP, 1997).

1. mAb 6E3 binds to recombinant NadA with high affinity

SPR analysis was performed to characterize the kinetics of binding of the NadA protein to mAb 6E3. The monoclonal antibody was captured by an immobilized anti-mouse antibody and NadA was injected at increasing concentrations. SCK was used with 10 mM potassium phosphate, 150 mM NaCl, 0.05% P20 surfactant, at pH 7.4 as running buffer (Figure 1 A and B).

2. mAb6E3 recognizes the native NadA protein on the surface of live meningococcal cells

By FACS analysis we confirmed that mAb6E3 is able to recognize NadA when natively expressed on the surface of meningococcal strain NMB, which expresses NadA variant 3 (Figure 2).

3. Epitope mapping of anti-NadA mAb 6E3

3. a. by Protein Chip Analysis

NadA fragments of different length were spotted on a microarray and used for hybridization with mAb 6E3. All the fragments containing the amino acid region between residues 219-255 were recognized by the mAb, suggesting that this region includes the epitope (Figure 3).

3.b. by HDX-MS approach

Epitope mapping by HDX-MS was performed in two parallel steps as previously described. Deuterium incorporation was performed on NadA alone (reference experiment) and on the antigen-antibody complex. Both samples were digested by pepsin and deuterium incorporation was monitored for 52 peptides covering 98% of NadA sequence and compared. In figure 4, the HDX-MS results have been simplified reporting only the extent of deuterium uptake for 18 sequential peptide fragments covering the entire peptide map. HDX-MS revealed that H-D exchange was reduced in presence of mAh 6E3 for 2 of the 18 NadA fragments, corresponding to the two overlapping peptides spanning 190-249 and 206-249 residues, respectively. The two protected peptides displayed the same difference in deuterium uptake suggesting that the epitope is included in the region 206-249.

4. mAb6E3 targets a conformational epitope on the stalk of NadA

In an attempt to define the mAh 6E3 epitope, an array of overlapping peptides each composed of 13 amino acid residues and spanning the entire NadA sequence was synthesized on a cellulose membrane and tested for binding to the monoclonal antibody. The Peptide Scanning analysis revealed that the mAb 6E3 did not recognize any synthetic peptide suggesting that the epitope is not linear and likely needs to adopt a conformational structure to be efficiently recognized. Moreover a monomeric form of the NadA variant 3 produced by heat treating the protein at 90°C for 120 seconds, was not able to bind captured murine mAb 6E3 in SPR assay (Figure 5).

Anti- NHBA-tnonoclonal antibody 10E8

The murine anti-NHBA monoclonal antibody of IgG2b subclass defined herein as 10E8 IG was obtained. RNA was isolated from the murine hybridoma cells using RNA prepared from dead cells using the Qiagen RNeasy mini kit (Cat No: 74104). RNA was eluted in 50pL water and checked by OD 260/280nm. The first strand cDNAs were amplified by PCR using oligo (dT) primers and VH and VK regions were amplified using several sets of degenerated primers. The amplified DNAs were gel- purified and cloned into a suitable vector. The VH and VK clones obtained were screened for inserts of the expected size. The DNA sequence of at least ten selected clones was determined in both directions by automated DNA sequencing. The locations of the complementarity determining regions (CDRs) in the sequences were determined with reference to other antibody sequences (Chothia & Lesk, 1987; Kabat EA et al., 1991; IMGT - Lefranc MP, 1997).

1. mAbl0E8 binds to purified recombinant NHBA with high affinity

SPR analysis was performed to characterize the kinetics of binding of the NHBA protein to mAb 10E8. The monoclonal antibody was captured by an immobilized anti-mouse antibody and NHBA was injected at increasing concentrations. SCK was used with 10 mM potassium phosphate, 150 mM NaCl, 0.05% P20 surfactant, at pH 7.4 as running buffer (Figure 6).

2. mAbl0E8 targets an epitope located on the N-term of NHBA

In a first attempt to map the epitope recognized by mAbl0E8, NHBA fragments of different length were spotted on a microarray and used for mAb hybridization. The results of the Protein Chip experiment indicate that mAb 10E8 targets epitopes on the N-terminus of 287 antigen, specifically located between amino acid 84-115 (Figure 7).

Pepti deScanning analysis was also performed on synthesized 11-mer NHBA peptides spanning the entire length of the protein. By this approach, we identified a single peptide recognized by the mAbl0E8.

This peptide nicely superimposes with the fragment identified by Protein Chip analysis.

To further confirm the exact position of this epitope, the HDX-MS technology was applied. By this technique we found that a single long fragment located on the N-terminal part of NHBA was impacted on its ability to exchange Deuterium upon binding to mAbl0E8 (Figure 9). This fragment partially overlaps with the segment previously identified by Protein Chip analysis.

4. The epitope targeted by mAbl0E8 on NHBA-953 protein is conformational (force degradation study)

A strong reduction of the binding to mAb 10E8 is observed for the heat treated proteins (Figure 10). For this experiment, protein NHBA-953 was treated as undiluted sample at three different temperatures and times

• 40°C 2 weeks (performed at TD)

• 95°C 15 hours (performed at CR)

Comparison to the protein kept at -20°C (benchmark), reduction of binding was 86% for the protein treated at 40°C for 2 weeks and 90% for the protein treated at 95°C for 15 hours.

According to this analysis, the recognition of the mAb 10E8 binding epitope on NHBA-953 is sensitive to heat treatment suggesting a conformational nature of the epitope.

MODES FOR CARRYING OUT THE INVENTION

1. INTRODUCTION AND SETTING OF THE ASSAY METHOD

The BEXSERO product is described in reference 7, and it includes 50 pg of each of NadA, fHbp subvariant 1.1, and NHBA subvariant 1.2, adsorbed onto 1.5 mg aluminium hydroxide, and with 25 g OMVs from N.meningitidis strain NZ98/254.

The following monoclonal antibodies are used:

(A) 10E8 (murine IgG2b against NHBA)

(B) 12C1/D7 (murine IgG2b against fHbp)

(C) 6E3 antibody (murine IgGl against NadA)

(D) Anti -PorA(P 1.4), available from NFBSC. Briefly, the principle of the method is an in vitro antibody binding inhibition assay, in which the monoclonal antibody binds to the specific epitope present in each vaccine component (741, 961c, 287, OMV). Then, the non -bound antibody is measured with an indirect ELISA assay and the inhibition curve obtained with an indirect ELISA assay and the inhibition curve obtained with a reference vaccine lot is compared to the one obtained with a vaccine lot under investigation by a relative potency calculation.

A schematic representation of Bexsero IVRP assay is shown in figure 11.

A Design of Experiment (DoE) was designed in order to optimize the binding phase with the following goals (details in addendum 2 of the present TR):

- Find the conditions that provide the best dose-response curves (well defined upper and lower asymptote similar for Ref and Test vaccines, good linear range with a high slope)

- Find the conditions that provide a Relative Potency close to 1

The following factors and levels were decided to be challenged in a dedicated Design of Experiments (DoE) in order to find the optimal conditions for the assay is shown in the following table 1 :

Table 1

In table 2 are reported batches, concentrations and buffers for each drug substance (DS) used for the ELISA coating. The assay diluent was IX PBS 0,5% Candor Blocking 0,05% Tween20.

Table 2

In the following table 3 are reported for each drug substance the monoclonal antibody used and its working dilution. The starting vaccine dilution and step dilution were selected in order to obtain a full curve with upper and lower asymptotes.

Table 3

In the following table 4 are reported the conditions for time, temperatures and buffers for each phase of the assay.

Table 4

2. RESULTS IVRP Evaluation of new monoclonal antibodies

The selected mAbs 6E3 and 10E8 gave the best results in the IVRP assay over a large number of tested monoclonal antibodies, in particular they showed the following excellent features:

-Goodness of the mAb curve after competition step;

- Potency of Freshly Formulated Vaccine (FFV) vs aged closed to 1;

-Trend over time;

-Conformational Epitope mapping;

-Conformational/ability to discriminate degraded material;

-Bactericidal;

-Clinical relevance of the epitope.

Forced Degradation Study

Considering its bactericidal activity, the 10E8 mAb has been included in a forced degradation study, designed for evaluating the ability of IVRP to discriminate between regular and sub-potent lots. Based on the knowledge gained working on Bexsero antigens, two conditions have been selected to stress the 112801 DP vaccine (3 years old):

• 15 hours at 95 °C - strong condition

• 15 days at 40°C - mild condition (linked to accelerate stability)

In Table 3 for each antigen are reported the mAb tested, the starting vaccine dilution and the sample’s dilution step used during this study. For each antigen a full curve has been obtained.

Each sample was tested in three independent analytical sessions by two different operators. In each plate, the reference vaccine lot 112801 was tested in parallel either with the sample kept for 2 weeks at 40°C (2w40°) or with the sample kept for 15 hours at 95°C (15h95°).

Aluminium hydroxide Saturation

A preliminary experiment was performed with the aim to verify the aluminium hydroxide saturation range at different vaccine and Candor percentages for OMV. Briefly, IVRP was executed in presence of vaccine or Placebo buffer alone (6.25 mg/mLNaCl, 2% Sucrose, 10 mMHystidinepH 6.5, 3 mg/mL aluminium hydroxide) with increasing Candor percentages (up to 4%). IVRP assay conditions are the same described in table 2 and 3, with the exception of Candor % in the binding phase. Results confirmed that, with 0.5% a not completed aluminium hydroxide saturation, resulting in a not specific mAb capture was observed for OMV at higher vaccine concentrations and therefore the saturation is dependent on Candor concentration.

An extended experimental plan was set up to fulfill both the need of investigating in depth detail the effect of Al(0H)3 and the need of identifying the linear range of the response.

Three independent analytical sessions were performed. In each analytica 1 session mAbs were incubated with the reference vaccine lot 112801 (as per standard procedure) or with the placebo alone (containing only aluminium hydroxide without antigens) were tested with different Candor percentages (0.5% up to 10%) for both OMV and recombinant proteins. During this experiment different Candor percentages were used only for the competition step while the remaining ELISA steps were performed in 0,5% Candor. Reference and placebo were analyzed in a range from undiluted up to 1024 by using a dilution step of 4. IVRP conditions are detailed in tables 3, 4 and 5. Three analytical sessions were performed, in each session five plates.

The plate layout was designed ad hoc for the experiment, considering the results from the plate effect study (see paragraph 3.5) that were already available at that time: the distribution of the different Candor % within the plate was done to minimize the plate effect observed for some antigen.

The results obtained as overall Optical Density response, represented in logarithmic scale for all antigens tested revealed a region of aspecific binding of mAb to Al(0H)3 (represented as gray area in the graphs “A”) which varied based on antigen tested and Candor % used for the saturation phase.For the three recombinant proteins, this region of aspecific binding is approximately up to 1/16 vaccine dilution for low Candor percentages (0.5%-l%), and decreases to 1/4 for higher Candor percentages (>2% for 741 and 961; >4% for 287). On the contrary, for OMV (figure 16A and 16B), the range of aspecific binding reached a vaccine dilution of 1/64 for the lowest Candor percentage (0,5%).

By looking at the linear part of the dose response curve it was evident that for the three recombinant proteins the linear range resulted in the range of fully saturation of Al(0H)3, even at the lowest 0,5% Candor, confirming the 0.5% a suitable Candor % for the IVRP assay. On the other hand, for OMV, the linear range of the response resulted outside the aspecific zone by using Candor percentages >4% (figure 16B), confirmed a possible interference of Al(0H)3 in the assay as observed in the forced degradation study. For this reason, a 4% Candor was selected for OMV for the further studies.

In conclusions, the selected Candor % for the binding phase was:

- 0.5% for recombinant proteins

- 4% for OMV By re-evaluating the forced degradation data, considering the outcome of the aluminium hydroxide saturation results, was observed that, for recombinant proteins, results generated within the vaccine dilution range 4-7 (linear part of the curve) are out of the impacted dilutions and can be considered valid. In particular, for 287 -953, the graphs are shown in figure 17, in which the linear ranges, as well as the interference zone are indicated. It can be noted that the two mAbs for 287 showed the same behaviors in term of IVRP performance and ability to discriminate degraded material. Among the two mAbs compared, due to its bactericidal activity, 10E8 was selected as mAb for 287.

In parallel to the activities performed for the selection of a new mAb against 287, the forced degradation study and the aluminium hydroxide saturation effect, a set of experiments was run with the aim to

- select the optimal mathematical model for IVRP;

- evaluate plate and edge effects

The studies were executed only with the mAb 31E10 for 287 since when the study started mAb 10E8 was not yet available. However, results and conclusions have been considered applicable also to the new selected 10E8 mAb for the 287 antigen.

As to the 4PL feasibility evaluation, performed analysis (not reported here) and graphical evaluations shown that the first four prerequisites can be considered as satisfied while deviations from 4PL shape resulted to be evident especially for some antigens.

More precisely, in order to better evaluate the 4PL shape, a transformation for curve linearization suggested by European Pharmacopoeia 7.0 chapter 5.3 was considered. When a dose-response curve with a perfect 4PL shape is transformed according to this linearization, a perfect linear shape is obtained. For this reason any deviation from linearity can be considered as deviation from 4PL shape. Deviation from linearity of the transformed curves was observed in the majority of the doseresponse curves.

For these reasons PLA resulted to be as the more promising model for IVRP assay and was further optimized with additional experiments reported in the following paragraphs.

PLATE EFFECT

In order to define the optimal plate layout, which will be designed based on the final model for calculation of RP, two experiments were designed with the aim to investigate the effect of position of samples within the 96 wells plate. In the first experiment, for each antigen a fixed vaccine dilution was selected and incubated with the specific mAb, and the non-bound mAb transferred to an ELISA plate, in order to obtain a value of optical density corresponding approximately to the ED50. The dose response curve. Vaccine and mAbs were incubated in a deep- well plate, in each well was dispensed 500 pL of a fixed vaccine concentration and 500 pL of a fixed mAb concentration in order to obtain a total volume of 1 mL/well. In particular in rows A and B were performed the binding reaction between vaccine and mAb anti -961 (clone 6E3), in rows C and D between vaccine and mAb anti -936-741 (clone 12C1D7), in rows E and F between vaccine and mAb anti-287-953 (clone 31E10) and in rows G and H between vaccine and mAb anti-OMV (clone PorA 1.4).

After the Binding Phase, the deep-well plate was centrifuged and the supernatant transferred in a plate coated with each specific antigen. For example from row A in the deep-well, 100 pL were transferred in row A, row C, row E and row G of an ELISA plate coated con DS 961c. Same operation was done for row B of deep-well plate but transferred in rows B, D, F and H of ELISA plate. At the end, the same sample was distributed in all the 96 wells of the ELISA plate, for each antigen.

Following USP <1032> suggestions a graphical evaluation of OD values was performed for each antigen. In the 3D plots reported in Figure 22, OD levels systematically higher at the edge are evident for the recombinant proteins. Moreover, for antigens 936 -741 and 961 the effect is evident also for internal wells. For OMV no systematic effect was observed.

Furthermore, in order to better investigate on the observed plate effect, OD levels were evaluated on the basis of wells position. More precisely a number level was assigned at each well moving from the external frame to the middle of the plate and a Wilcoxon test was performed in order to detect significant difference among OD values belonging to different levels (p - value of 0.01). Results confirm that, for recombinant proteins, position in the plate may significantly impact OD level following a systematic behavior. Based on the results, preferably the external edge was excluded and samples were plated from B2 to G11 position.

Vaccine Dilution Range Optimization

To confirm the agreement between technical feasibility and theoretical considerations extrapolated from data generated in aluminium hydroxide saturation study, and considering the conclusions from the evaluation of mathematical model to be applied for the calculation of the RP, ad hoc experiments were planned in order to:

- enlarge the linear dilution range by optimizing the vaccine dilution step;

- optimize the starting vaccine dilution for each antigen; with the final aim to have at least 4 points in the linear range for the application of the parallel line assay. In tables 5 and 6 are reported for each antigen vaccine starting dilutions, dilution steps and Candor percentage. Reference and samples were titrated in 6 following serial dilutions.

Table 5: Starting dilutions, dilution steps and candor percentage used for each antigen

Table 6: Starting dilutions, dilution steps and candor percentage used for each antigen

In figures 13-16 all the obtained curve profiles for each antigen and session are reported.

For 287-953 (10E8) a very high repeatability among sessions was obtained, For 936-741 (mAb 12C1D7) was obtained high repeatability among sessions but only 3/2 points in the linear range suggesting the need of a furthe r optimization of the linear range to avoid possible bias on RP estimation. In addition results showed a strong plate effect as indicated by an inflection point in the middle of the curves suggesting that a further optimization of plate layout is needed. For 961c (mAh 6E3) high repeatability was observed with 3 points in the linear range.

For OMV (mAb PorA 1.4) high repeatability and 4 points in the linear range were observed as well as random variability and no evident plate effect.

DISCUSSION & CONCLUSION

The IVRP is an ELISA-based assay performed using monoclonal antibodies specific for each vaccine component, aimed to substitute the current in-vivo potency assay for measurement of Bexsero vaccine. The inhibition curves obtained with a reference vaccine lot are compared to those obtained with a vaccine lot under testing by a relative potency calculation.

The assay has been fully developed. The following aspect has been deeply investigated and summarized in this document:

1. Selection of the optimal mAb for 287-953 antigen in which the results obtained on 10E8 revelead this mAb suitable for IVRP.

2. Deeply investigation of the aluminium hydroxide saturation effect in order to define the optimal Candor percentage in the assay diluent for each antigen component. The results revealed a region of aspecific binding of mAb to Al(0H)3 which varied based on antigen tested and Candor % used for the saturation phase. By looking at the linear part of the dose response curve it was evident that for the three recombinant proteins the linear range resulted in the range of fully saturation of Al(0H)3, even at the lowest 0.5% Candor, confirming the 0.5% a suitable Candor % for the IVRP assay. On the other hand, for OMV, the linear range of the response resulted outside the aspecific zone by using Candor percentages > 4%, confirmed a possible interference of Al(0H)3 in the assay as observed in the forced degradation study. For this reason, a 4% Candor was selected for OMV.

3. Vaccine starting dilution optimization as well as the identification of alternative buffers to use as coating to get the optimal linear range for the assay. Details reported in the table below.

Below summarized the optimized assay conditions for BEXSERO ELISA:

* Assay buffer is represented from PBS 0,5% Candor 0,05% Tween20

** Assay buffer is represented from PBS 4% Candor 0,05% Tween20

In addition, in the forced degradation study, results showed a drastic reduction of mAh binding when sample were treated at high temp, whereas at intermediate temp the binding was only partially reduced, confirming the suitability of the IVRP as stability indicating assay. In Stability Study of a FFV was monitored up to 7 months at two temperatures (2/8°C and 25°C) all the proteins did not show trend over time except for the 287-953.

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.

SEQUENCE LISTING IN THE DESCRIPTION

Antibody’s sequence following IMGT numbering

Sequences of monoclonal antibody 10E8

SEQ ID NO: 1 DNA coding for 10E8 light chain variable region

GATATTGTGATGACT CAGGCTGCACCCTCT GTATCTGTCACTCCT GGAGAGTCAGTATCC ATCTCCTGCAGGTCT AGTAAGAGTCTCCTG TATAGTAATGGCAAC ACTTACTTGTATTGG TTCCTGCAGAGGCCA GGCCAGTCTCCTCAG CTCCTGATATATCGG ATGTCCAACCTTGCC TCAGGAGTCCCAGAC AGGTTCAGTGGCAGT GGGTCAGGAACTGCT TTCACACTGAGAATC

AGTAGAGTGGAGGCT GAGGATGTGGGTGTT TATTACTGTATGCAA CATCTAGAATATCCG CTCACGTTCGGTGCT GGGACCAAGCTGGAG CTGAAA

SEQ ID NO:2 PROTEIN sequence 10E8 light chain variable region DIVMTQAAPSVSVTPGESVSISCRSSKSLLYSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPD

RFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPLTFGAGTKLELK

SEQ ID NO: 3 CDR-L1 10E8

KSLLYSNGNTY

NO SEQ ID Number Less than 4aa CDR-L2 10E8

RMS

SEQ ID NO: 4 CDR-L3 10E8

MQHLEYPLT

SEQ ID NO:5 DNA coding for 10E8 heavy chain variable region

CAGGTCCAACTGCAG CAGCCTGGGGCTGAG CTTGTGAAGCCTGGG GCTTCAGTGAAGATG TCCTGTAAGGCTTCT GGCTACACCTTCACC AGTCACTGGATAACC TGGGTGAAGCAGAGG CCTGGACAAGGCCTT GAGTGGATTGGAGAT ATTTATCCTGTTACT GGTCGTTTTTACTGC AATGAGAAGTTCAAG AACAAGACCACACTG ACTGTAGACACATCC TCCAGCACAGCCTAC ATGCAGCTCAGCAGC CTGACATCTGAGGAC TCTGCGGTCTATTAC TGTGCCGAGCGAGAC TACTGGGGCCAAGGC ACCACTCTCACAGTC TCCTCA

SEQ ID NO:6 PROTEIN sequence 10E8 heavy chain variable region

QVQLQQPGAELVKPGASVKMSCKASGYTFTSHWITWVKQRPGQGLEWIGDIYPVTGRFYCNEK FKNKTTLTVDTSSSTAYMQLSSLTSEDSAVYYCAERDYWGQGTTLTVSS

SEQ ID NO: 7 CDR-H1 10E8

GYTFTSHW

SEQ ID NO: 8 CDR-H2 10E8

IYPVTGRF

SEQ ID NO: 9 CDR-H3 10E8

AERDY

Sequences of monoclonal antibody 6E3

SEQ ID NO: 10 DNA coding for 6E3 light chain variable region

CAAATTGTTCTCTCC CAGTCTCCAGCAATC CTGTCTGCATCTCCA GGGGAGAAGGTCACA ATGACTTGCAGGGCC AGTTCAAGTGTAAAT TACATGTACTGGTAC CAGCAGAAGCCAGGA TCTTCCCCCAAAGTC TGGATTTATGCCACA TCCAACCTGGCTTCT GGAGTCCCTGCTCGC TTCAGTGGCAGTGGG TCTGGGACCTCTTAC TCTCTCACAATCAGC AGAGTGGAGGCTGAA GATGCTGCCACTTAT TACTGCCAGCAGTGG AGTAGTAATTCACGG ACGTTCGGTGGAGGC ACCAAGCTGGAAATC AAA

SEQ ID NO: 11 PROTEIN sequence 6E3 light chain variable region

QIVLSQSPAILSASPGEKVTMTCRASSSVNYMYWYQQKPGSSPKVWIYATSNLASGVPARFSGSG SGTSYSLTISRVEAEDAATYYCQQWSSNSRTFGGGTKLEIK

SEQ ID NO: 12 CDR-L1 6E3

SSVNY

NO SEQ ID NO Less than 4aa CDR-L2 6E3

ATS

SEQ ID NO: 13 CDR-L3 6E3

QQWSSNSRT

SEQ ID NO: 14 DNA coding for 6E3 heavy chain variable region

CAGGTCCAGCTGCAG CAGCCTGGGAATGAA CTGGTGAAGCCTGGG GCTTCAGTGAAGCTG TCCTGCAAGGCTTCT GGCTACACGTTCACC AGCTACTGGATGCAC TGGGTGAAGCAGAGG CCTGGACAAGGCCTT GAGTGGATTGGAGAG ATTAACCCTATCGAC GGTCGTACTGACTAC AATGAGAACTTCAAG ACCAAGGCCACACTG ACTGTAGACAAATCC TCCAGCACAGCCTAC ATGCAACTCAGCAGC CTGACATCTGAGGAC TCTGCGGTCTATTAC TGTGCAAGAACGGCC TATGATGGTTACTAC GTTGCCTGGTTTGCT TACTGGGGCCAAGGG ACTCTGGTCACTGTC TCTGCA

SEQ ID NO: 15 PROTEIN sequence 6E3 heavy chain variable region

QVQLQQPGNELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPIDGRTDYNEN FKTKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARTAYDGYYVAWFAYWGQGTLVTVSA

SEQ ID NO: 16 CDR-H1 6E3

GYTFTSYW

SEQ ID NO: 17 CDR-H2 6E3

INPIDGRT

SEQ ID NO: 18 CDR-H3 6E3

ARTAGCAYDGYYVAWFAY SEQ ID NO: 19 sequence of a region of meningococcal antigen NHBA recognises by 10E8

VSEENTGNGGAAATDKPKNEDEGAQNDMPQNAADTDSLTPNHTPASNMPAGNMENQAPDAGE S

SEQ ID NO:20 sequence of the epitope recognised by 10E8

NAADTDSLTPNHTPASNMPAGNMENQAPDAGES

SEQ ID NO:21 sequence of the specific epitope recognised by 10E8

NAADTDSLTPN

SEQ ID NO:22 sequence of NadA

Met Ser Met Lys His Phe Pro Ser Lys Vai Leu Thr Thr Ala He Leu Ala Thr Phe Cys Ser Gly Ala Leu Ala Ala Thr Ser Asp Asp Asp Vai Lys Lys Ala Ala Thr Vai Ala He Vai Ala Ala Tyr Asn Asn Gly Gin Glu He Asn Gly Phe Lys Ala Gly Glu Thr He Tyr Asp He Gly Glu Asp Gly Thr He Thr Gin Lys Asp Ala Thr Ala Ala Asp Vai Glu Ala Asp Asp Phe Lys Gly Leu Gly Leu Lys Lys Vai Vai Thr Asn Leu Thr Lys Thr Vai Asn Glu Asn Lys Gin Asn Vai Asp Ala Lys Vai Lys Ala Ala Glu Ser Glu He Glu Lys Leu Thr Thr Lys Leu Ala Asp Thr Asp Ala Ala Leu Ala Asp Thr Asp Ala Ala Leu Asp Glu Thr Thr Asn Ala Leu Asn Lys Leu Gly Glu Asn He Thr Thr Phe Ala Glu Glu Thr Lys Thr Asn He Vai Lys He Asp Glu Lys Leu Glu Ala Vai Ala Asp Thr Vai Asp Lys His Ala Glu Ala Phe Asn Asp He Ala Asp Ser Leu Asp Glu Thr Asn Thr Lys Ala Asp Glu Ala Vai Lys Thr Ala Asn Glu Ala Lys Gin Thr Ala Glu Glu Thr Lys Gin Asn Vai Asp Ala Lys Vai Lys Ala Ala Glu Thr Ala Ala Gly Lys Ala Glu Ala Ala Ala Gly Thr Ala Asn Thr Ala Ala Asp Lys Ala Glu Ala Vai Ala Ala Lys Vai Thr Asp He Lys Ala Asp He Ala Thr Asn Lys Ala Asp He Ala Lys Asn Ser Ala Arg He Asp Ser Leu Asp Lys Asn Vai Ala Asn Leu Arg Lys Glu Thr Arg Gin Gly Leu Ala Glu Gin Ala Ala Leu Ser Gly Leu Phe Gin Pro Tyr Asn Vai Gly Arg Phe Asn Vai Thr Ala Ala Vai Gly Gly Tyr Lys Ser Glu Ser Ala Vai Ala He Gly Thr Gly Phe Arg Phe Thr Glu Asn Phe Ala Ala Lys Ala Gly Vai Ala Vai Gly Thr Ser Ser Gly Ser Ser Ala Ala Tyr His Vai Gly Vai Asn Tyr Glu Trp

SEQ ID NO:23 sequence of NHBA

Met Phe Lys Arg Ser Vai He Ala Met Ala Cys He Phe Ala Leu Ser Ala Cys Gly Gly Gly Gly Gly Gly Ser Pro Asp Vai Lys Ser Ala Asp Thr Leu Ser Lys Pro Ala Ala Pro Vai Vai Ser Glu Lys Glu Thr Glu Ala Lys Glu Asp Ala Pro Gin Ala Gly Ser Gin Gly Gin Gly Ala Pro Ser Ala Gin Gly Ser Gin Asp Met Ala Ala Vai Ser Glu Glu Asn Thr Gly Asn Gly Gly Ala Vai Thr Ala Asp Asn Pro Lys Asn Glu Asp Glu Vai Ala Gin Asn Asp Met Pro Gin Asn Ala Ala Gly Thr Asp Ser Ser Thr Pro Asn His Thr Pro Asp Pro Asn Met Leu Ala Gly Asn Met Glu Asn Gin Ala Thr Asp Ala Gly Glu Ser Ser Gin Pro Ala Asn Gin Pro Asp Met Ala Asn Ala Ala Asp Gly Met Gin Gly Asp Asp Pro Ser Ala Gly Gly Gin Asn Ala Gly Asn Thr Ala Ala Gin Gly Ala Asn Gin Ala Gly Asn Asn Gin Ala Ala Gly Ser Ser Asp Pro He Pro Ala Ser Asn Pro Ala Pro Ala Asn Gly Gly Ser Asn Phe Gly Arg Vai Asp Leu Ala Asn Gly Vai Leu He Asp Gly Pro Ser Gin Asn He Thr Leu Thr His Cys Lys Gly Asp Ser Cys Ser Gly Asn Asn Phe Leu Asp Glu Glu Vai Gin Leu Lys Ser Glu Phe Glu Lys Leu Ser Asp Ala Asp Lys He Ser Asn Tyr Lys Lys Asp Gly Lys Asn Asp Lys Phe Vai Gly Leu Vai Ala Asp Ser Vai Gin Met Lys Gly He Asn Gin Tyr He He Phe Tyr Lys Pro Lys Pro Thr Ser Phe Ala Arg Phe Arg Arg Ser Ala Arg Ser Arg Arg Ser Leu Pro Ala Glu Met Pro Leu He Pro Vai Asn Gin Ala Asp Thr Leu He Vai Asp Gly Glu Ala Vai Ser Leu Thr Gly His Ser Gly Asn He Phe Ala Pro Glu Gly Asn Tyr Arg Tyr Leu Thr Tyr Gly Ala Glu Lys Leu Pro Gly Gly Ser Tyr Ala Leu Arg Vai Gin Gly Glu Pro Ala Lys Gly Glu Met Leu Ala Gly Ala Ala Vai Tyr Asn Gly Glu Vai Leu His Phe His Thr Glu Asn Gly Arg Pro Tyr Pro Thr Arg Gly Arg Phe Ala Ala Lys Vai Asp Phe Gly Ser Lys Ser Vai Asp Gly He He Asp Ser Gly Asp Asp Leu His Met Gly Thr Gin Lys Phe Lys Ala Ala He Asp Gly Asn Gly Phe Lys Gly Thr Trp Thr Glu Asn Gly Ser Gly Asp Vai Ser Gly Lys Phe Tyr Gly Pro Ala Gly Glu Glu Vai Ala Gly Lys Tyr Ser Tyr Arg Pro Thr Asp Ala Glu Lys Gly Gly Phe Gly Vai Phe Ala Gly Lys Lys Glu Gin Asp.

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Claims

CLAIMS A binding assay for in vitro analysis of a meningococcal vaccine sample, comprising steps of: (i) allowing a meningococcal immunogen within the sample to interact with a monoclonal antibody which is bactericidal for meningococcus and/or capable of binding a conformational epitope of an immunogen in said meningococcal vaccine then (ii) measuring the interaction between the immunogen and antibody from step (i). The assay of the preceding claim, wherein the measurement in step (ii) provides the amount of the monoclonal antibody’s target epitope within said meningococcus vaccine sample. The assay of any preceding claims, wherein the binding assay is an ELISA, in particular a competitive ELISA. The assay of any preceding claims, comprising the following steps: (i) incubating the sample with the monoclonal antibody so that complexes can form between the antibody and meningococcal immunogen in the sample; (ii) separating the unbound monoclonal antibody from immunogenbound monoclonal antibody; (iii) adding the unbound monoclonal antibody to a container in which antigens of said monoclonal antibody are immobilised, wherein the immobilised antigens can form a complex with said unbound monoclonal antibody; (iv) determining the amount of the complex formed in step (iii) and (v) measuring the interaction between the immunogen and antibody from step (i). The assay of the preceding claim, wherein said separation of the unbound monoclonal antibody from immunogen-bound monoclonal antibody is carried out by centrifugation. The assay of any preceding claims, wherein the measurement in step (ii) uses a secondary antibody labelled with an enzyme. The assay of any preceding claims, wherein the vaccine includes one or more meningococcal immunogen selected from NHBA, fHbp and/or NadA immunogen, preferably the vaccine further includes meningococcal outer membrane vesicles and wherein the monoclonal antibody used in step (i) capable of binding meningococcal NHBA, fHbp or NadA immunogen. The assay of any preceding claims, which uses a single monoclonal antibody in step (i), preferably which uses a murine monoclonal IgG antibody in step (i), in particular IgGl or IgG2b. The assay of any preceding claims, wherein the vaccine includes one or more adsorbed meningococcal immunogen, in particular wherein said meningococcal immunogen is adsorbed with aluminium hydroxide adjuvant. The assay of any preceding claims, wherein in said step (i) said interaction in step (i) is carried out in a medium comprising a blocking buffer, in particular wherein said blocking buffer comprises casein or casein derivatives or fragments thereof.

56 The assay of any preceding claims, wherein said monoclonal antibody is one or more monoclonal antibody selected from the monoclonal antibody of any one of claims 16 to 25. A method for detecting or measuring a change in conformation of a meningococcal immunogen in a vaccine sample, comprising steps of: (i) performing the in vitro assay according of any one of claims 1 to 11 on a test sample and, optionally, on at least one dilution of the test sample; (ii) performing the in vitro assay according to any one of claims 1 to 11 on a standard vaccine sample of known native antigenic form and, optionally, on at least one dilution of the standard vaccine sample; and (iii) comparing the results from steps (i) and (ii) to determine the amount of immunogen(s) in the native form of the test vaccine relative to the amount of immunogen(s) in the native form in the standard vaccine. A method for in vitro relative potency analysis of a meningococcal test vaccine sample, comprising steps of: (i) performing the assay of any one of claims 1 to 11 on the test sample and, optionally, on at least one dilution of the test sample; (ii) performing the assay of any one of claims 1 to 11 on a standard vaccine sample of known in vivo potency and, optionally, on at least one dilution of the standard vaccine sample; and (iii) comparing the results from steps (i) and (ii) to determine the potency of immunogen(s) in the test vaccine relative to the potency of immunogen(s) in the standard vaccine. A method for analysing a batch of vaccine, comprising steps of: (i) assaying the relative potency of immunogen(s) in at least one vaccine from the batch by the method of claim 13, and, if the results of step (i) indicate an acceptable relative potency, (ii) releasing further vaccines from the batch for in vivo use. A vaccine which has been released following the use of an assay of any preceding claim 1 to 11 or a method according to anyone of claims 12 to 15. A monoclonal antibody capable of binding the meningococcal NHBA antigen, whose VH and VL comprise the following complementarity-determining regions (CDRs):

-CDR1 of VH having SED ID NO: 7,

-CDR2 of VH having SED ID NO: 8,

-CDR3 of VH having SED ID NO: 9,

-CDR1 of VL having SED ID NO: 3,

-CDR2 of VL having the aminoacid sequence RMS and

-CDR3 of VL having SED ID NO:4. A monoclonal antibody capable of binding the meningococcal NHBA antigen according to claim 16, whose light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 2 and whose heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 6.

57 A monoclonal antibody capable of binding an epitope of the meningococcal antigen NHBA comprising or consisting in the amino acid sequence of SEQ ID NO: 19, preferably of SEQ ID NO: 20, more preferably in SEQ ID NO:21. A monoclonal antibody able to distinguish between native and denatured form of antigen NHBA and whose VH and VL shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity with the amino acid sequence of SEQ ID NO: 6 (VH) and SEQ ID NO: 2(VL) respectively. A monoclonal antibody that binds to antigen NHBA and competes or cross-competes with and/or binds the same epitope of the monoclonal antibody according to any one of the claims from 16 to 19. A monoclonal antibody capable of binding meningococcal NadA antigen, whose VH and VL comprise the following complementarity-determining regions (CDRs):

-CDR1 of VH having SED ID NO: 16,

-CDR2 of VH having SED ID NO: 17,

-CDR3 of VH having SED ID NO: 18,

-CDR1 of VL having SED ID NO: 12,

-CDR2 of VL having the amino acid sequence ATS and

-CDR3 of VL having SED ID NO: 13. A monoclonal antibody capable of binding meningococcal NadA antigen according to claim 21, whose VL region has the amino acid sequence of SEQ ID NO: 11 and whose VH region has the amino acid sequence of SEQ ID NO: 15. A monoclonal antibody capable of binding an epitope in the region of meningococcal antigen NadA corresponding to the amino acid sequence of residues 206-249 of SEQ ID NO:22. A monoclonal antibody able to distinguish between native and denatured form of antigen NadA and whose VH and VL region shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity with the amino acid sequence SEQ ID NO: 15 (VH) and SEQ ID NO: 11 (VL). A monoclonal antibody that binds to antigen NadA and competes or cross-competes with and/or binds the same epitope of the monoclonal antibody according to any one of the claims from 21 to 24.

58