TECHNICAL FIELD

This invention is in the field of in vitro assays for assessing the potency of proteincontaining 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 and protein-containing vaccines for protecting against Neisseria meningitidis (meningococcus) 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 SBA, 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 ELISA potency assay disclosed in WO2013132040. 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.

However, the assay disclosed in WO2013132040 requires a distinct analysis of each single immunogen in the vaccine and does not allow the simultaneous analysis of a plurality of immunogens. No multiplex assays are available for assessing the potency of protein-containing vaccines for protecting against Neisseria meningitidis (meningococcus).

There remains a need for further and improved in vitro assays for assessing the suitability of meningococcal vaccines for batch release. Such in vitro assays could be used to confirm that a particular vaccine will have an expected in vivo activity in human recipients.

DISCLOSURE OF THE INVENTION

The invention provides multiplexed binding assays, such as a Luminex assay, 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 standard or reference vaccine of known potency, it is possible to determine the relative potency of the test vaccine with an in vitro relative potency assay (IVRP).

Quality by Design together with the 3R principles are the main drivers for introducing IVRP assays in replacement of in vivo investigation when vaccines enter late development stages and commercialization. Through the use and characterization of functional monoclonal antibodies that recognize important target epitopes, IVRP assays guarantee product consistency and effectiveness.

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 that has known potency/efficacy in humans or a batch that 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 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 preferably at least 0.50 with respect to the potency of the reference 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 an in vitro analysis of a meningococcal vaccine sample including more than one meningococcal immunogen, comprising the steps of:

(i) allowing the interaction of a plurality of meningococcal immunogens within the sample with a plurality of anti-meningococcal immunogen-specific monoclonal antibodies which are bactericidal for meningococcus and/or are capable of binding to a conformational epitope of a meningococcal protein immunogen in said meningococcal vaccine;

(ii) measuring the interaction between each of said meningococcal immunogens and its corresponding anti-immunogen-specific monoclonal antibody with an immunoassay wherein each unbound anti- meningococcal immunogen-specific monoclonal antibody is allowed to interact with a plurality of colour-coded beads conjugated with an antigen selectively bound by said anti-immunogen-specific monoclonal antibody, said beads having a different colour-code for each different antigen.

The invention also provides method for in vitro relative potency analysis of a test meningococcal vaccine sample, comprising steps of:

(iii) performing steps (i) and (ii) of the binding assay herein disclosed and claimed on the test meningococcal vaccine sample and, optionally, on at least one dilution of the test meningococcal vaccine sample;

(iv) performing steps (i) and (ii) of the binding assay herein disclosed and claimed on a standard meningococcal vaccine sample of known in vivo potency and, optionally, on at least one dilution of the standard meningococcal vaccine sample; and

(v) comparing the results from steps (i) and (ii) obtained in (iii) and (iv) to determine the potency of immunogen(s) in the test meningococcal vaccine sample relative to the potency of immunogen(s) in the standard meningococcal vaccine sample.

The invention also provides a method for analysing a batch of vaccine, comprising steps of:

(1) assaying the relative potency of immunogen(s) in at least one meningococcal vaccine sample from the batch by the method for in vitro analysis of a meningococcal test vaccine sample comprising steps of:

(iii) performing steps (i) and (ii) of the binding assay herein disclosed and claimed on the test sample and, optionally, on at least one dilution of the test sample;

(iv) performing steps (i) and (ii) of the binding assay herein disclosed and claimed on a standard vaccine sample of known in vivo potency and, optionally, on at least one dilution of the standard vaccine sample; and

(v) comparing the results from steps (i) and (ii) obtained in (iii) and (iv) to determine the potency of immunogen(s) in the test vaccine relative to the potency of immunogen(s) in the standard vaccine, and, if the results of step (1) indicate an acceptable relative potency,

(2) releasing further vaccines from said batch for in vivo use.

The invention also provides a method for analysing a bulk vaccine, comprising steps of:

(1) assaying the relative potency of immunogen(s) in the bulk by the method for in vitro analysis of a meningococcal test vaccine sample comprising steps of:

(iii) performing steps (i) and (ii) of the binding assay herein disclosed and claimed on the test sample and, optionally, on at least one dilution of the test sample;

(iv) performing steps (i) and (ii) of the binding assay herein disclosed and claimed on a standard vaccine sample of known in vivo potency and, optionally, on at least one dilution of the standard vaccine sample; and

(v) comparing the results from steps (i) and (ii) obtained in (iii) and (iv) to determine the potency of immunogen(s) in the test vaccine relative to the potency of immunogen(s) in the standard vaccine; and, if the results of step (1) indicate an acceptable relative potency,

(2) preparing unit doses of vaccine from the bulk.

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

The invention also provides a kit for performing the assay of the invention, i.e. a kit for in vitro multiplex assay of a meningococcal vaccine sample.

The kit may comprise (i) a plurality of solution-phase anti -immunogen specific monoclonal antibodies each of which is capable of binding to a conformational epitope of one of a meningococcal immunogen in the vaccine of interest (ii) a plurality of colour-coded beads, each conjugated to an antigen selectively bound by one of said anti-immunogen-specific monoclonal antibodies, said beads having a different colour-code for each different antigen, and (iii) a labelled antibody which binds to said anti-immunogen specific-monoclonal antibodies, wherein at least two of said meningococcal immunogens are selected from NHBA, NadA and/or fHbp immunogens.

The invention also provides a binding assay for in vitro analysis of a meningococcal protein-immunogens-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.

Since that ability of purified protein immunogens to elicit a functional immune response is associated with the maintenance of native structure, binding of selected mAbs, to these conformational epitopes, acts as a readout of structural integrity and consequently vaccine immunogenicity. In other words, IVRP rely on the ability of mAbs to recognize reproducibly epitopes with structural and immunological relevance on the target vaccine antigens. Suitable mAbs for carrying out the assays of the invention are hence mAbs that are able to discriminate the degraded antigen from the native form.

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

The invention also provides said antibodies. The invention also provides monoclonal antibodies suitable for carrying out the assays of the invention such as antibodies that are capable of binding, with a selective (specific binding) 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 bind with higher affinity to the native immunogen vs. its denatured form, and that preferably bind to the native immunogen but do not bind to its denatured form.

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):

-CDRL1 having SEQ ID NO:35,

-CDRL2 having SEQ ID NO: 103,

-CDRL3 having SEQ ID NO: 36,

-CDRH1 having SEQ ID NO:39,

-CDRH2 having SEQ ID NO:40,

-CDRH3 having SEQ ID NO:41.

A monoclonal antibody which is capable of binding (selectively binding) to an epitope of meningococcal antigen NHBA , in particular of a meningococcal NHBA immunogen, comprising or consisting in the amino acid sequence of SEQ ID NO: 42, preferably of SEQ ID NO 43, even more preferably of SEQ ID 44.

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 SEQ ID NO: 38 (VH) and SEQ ID NO: 34 (VL) respectively.

A monoclonal antibody which is capable of binding (selectively binding) to meningococcal NHBA antigen, in particular to a meningococcal NHBA immunogen, whose VL region has the amino acid sequence of SEQ ID NO: 34 and whose VH region has the amino acid sequence of SEQ ID NO: 38

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

-CDRL1 having SEQ ID NO:47, -CDRL2 having SEQ ID NO: 104,

-CDRL3 having SEQ ID NO:48,

-CDRH1 having SEQ ID NO:51,

-CDRH2 having SEQ ID NO: 52,

-CDRH3 having SEQ ID NO:53

A monoclonal antibody which is capable of binding (selectively binding) an epitope, preferably a conformational epitope, in the region of meningococcal antigen NadA, in particular of a meningococcal NadA immunogen, corresponding to the amino acid sequence of residues 206-249 of SEQ ID NO:9.

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 identity with the amino acid sequence SEQ ID NO: 50 (VH) and SEQ ID NO: 46 (VL).

A monoclonal antibody which 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: 46 and whose VH region has the amino acid sequence of SEQ ID NO: 50.

A monoclonal antibody which is capable of binding (selectively binding) meningococcal fHbp v3, in particular fHbpv (3.2) 3.28 antigen, in particular an immunogen, of (SEQ ID 64 or SEQ ID NO: 10 or SEQ ID NO: 6), whose VH and VL comprise the following complementarity-determining regions (CDRs):

-CDRL1 having SEQ ID NO:56,

-CDRL2 having SEQ ID NO:57

-CDRL3 having SEQ ID NO: 58,

-CDRH1 having SEQ ID NO:61,

-CDRH2 having SEQ ID NO: 62,

-CDRH3 having SEQ ID NO: 63.

A monoclonal antibody which is capable of binding (selectively binding) an epitope in the region of meningococcal fHbp v3, in particular fHbpv (3.2) 3.28 antigen, in particular an immunogen, said epitope being absent from variants 1 and 2, the epitope comprising NRH amino acid residues in positions 169, 171 and 173 of fHbpv (3.2) 3.28 of SEQ ID NO 64 or SEQ ID NO: 10 or the corresponding amino acids in SEQ ID NO: 6.

A monoclonal antibody able to differentiate between native and denatured form of antigen fHbp v3, in particular fHbpv (3.2) 3.28, 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: 60 (VH) and SEQ ID NO: 55 (VL).

A monoclonal antibody which is capable of binding (selectively binding) meningococcal fHbp v3, in particular fHbpv (3.2) 3.28 antigen, in particular an immunogen, whose VL region has the amino acid sequence of SEQ ID NO: 55 and whose VH region has the amino acid sequence of SEQ ID NO: 60.

A monoclonal antibody which is capable of binding (selectively binding) meningococcal fHbp vl.13 antigen, in particular an immunogen, whose VH and VL comprise the following complementarity-determining regions (CDRs):

- CDRL1 having SEQ ID NO: 67,

-CDRL2 having SEQ ID NO: 68

-CDRL3 having SEQ ID NO: 69,

-CDRH1 having SEQ ID NO:71,

-CDRH2 having SEQ ID NO: 72,

-CDRH3 having SEQ ID NO: 73.

A monoclonal antibody which is capable of binding to the surface of MenB strain bacteria carrying the variant 1.13 of fHbp and kill them in a serum bactericidal assay, and/or recognizes conformational, functional and clinically relevant epitopes of the fHbp 1.13NB antigen within a 2-3-1.13 NB fusion protein comprising or consisting of SEQ ID NO 7 or 23.

A monoclonal antibody which is capable of binding to an epitope in the region of meningococcal fHbp variant 1.13 antigen, the epitope comprising at least EH amino acid residues in position 234 and 240 respectively of fHbp variant 3.28 of SEQ ID NO 4 or SEQ ID NO: 102, preferably EAH amino acid residues in positions position 234, 236 and 240 respectively of fHbp variant 3.28 of SEQ ID NO 4 or SEQ ID NO: 102.

A monoclonal antibody able to differentiate between native and denatured form of fHbp vl.13 antigen 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: 70 (VH) and SEQ ID NO: 66 (VL).

A monoclonal antibody which is capable of binding (selectively binding) meningococcal fHbp vl.13 antigen, in particular an immunogen, whose VL region has the amino acid sequence of SEQ ID NO: 66 and whose VH region has the amino acid sequence of SEQ ID NO: 70.

A monoclonal antibody which is capable of binding (selectively binding) meningococcal fHbp vl.13 antigen, in particular an immunogen, whose VH and VL comprise the following complementarity-determining regions (CDRs):

- CDRL1 having SEQ ID NO:75,

-CDRL2 having SEQ ID NO: 76

-CDRL3 having SEQ ID NO:77.

-CDRH1 having SEQ ID NO:79,

-CDRH2 having SEQ ID NO: 80,

-CDRH3 having SEQ ID NO:81,

A monoclonal antibody which is capable of binding (selectively binding) to the surface of MenB strain bacteria carrying the variant 1.13 of fHbp and kill them in a serum bactericidal assay, and recognize conformational, functional and clinically relevant epitopes of the fHbp 1.13NB antigen within a 2-3-1.13 NB fusion protein comprising or consisting of SEQ ID NO 7 or 23.

A monoclonal antibody able to differentiate between native and denatured form of fHbp vl.13 antigen 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: 78 (VH) and SEQ ID NO: 74 (VL).

A monoclonal antibody which is capable of binding (selectively binding) meningococcal fHbp vl.13 antigen, in particular an immunogen, whose VL region has the amino acid sequence of SEQ ID NO: 74 and whose VH region has the amino acid sequence of SEQ ID NO: 78.

A monoclonal antibody which is capable of binding (selectively binding) meningococcal fHbp vl.13 antigen, in particular an immunogen, whose VH and VL comprise the following complementarity-determining regions (CDRs): - CDRL1 having SEQ ID NO: 83,

-CDRL2 having SEQ ID NO: 84

-CDRL3 having SEQ ID NO:85.

-CDRH1 having SEQ ID NO:87,

-CDRH2 having SEQ ID NO:88,

-CDRH3 having SEQ ID NO: 89,

A monoclonal antibody which is capable of binding (selectively binding) the surface of MenB strain bacteria carrying the variant 1.13 of fHbp and kill them in a serum bactericidal assay, and recognize conformational, functional and clinically relevant epitopes of the fHbp 1.13NB antigen within a 2-3-1.13 NB fusion protein comprising or consisting of SEQ ID NO 7 or 23.

A monoclonal antibody able to differentiate between native and denatured form of fHbp vl.13 antigen 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: 86 (VH) and SEQ ID NO: 82 (VL).

A monoclonal antibody which is capable of binding (selectively binding) meningococcal fHbp vl.13 antigen, in particular an immunogen, whose VL region has the amino acid sequence of SEQ ID NO: 82 and whose VH region has the amino acid sequence of SEQ ID NO: 86.

Binding assays

The invention provides a multiplex binding immunoassay.

The term multiplex when referred to an immunoassay is intended as an immunoassay that allows the simultaneous analysis of more than one analyte in a single experiment.

The binding assay of the invention allows the in vitro analysis of a meningococcal vaccine sample including more than one meningococcal immunogen, comprising the steps of:

(i) allowing the interaction of a plurality of meningococcal immunogens within the sample with a plurality of anti-meningococcal immunogen-specific monoclonal antibodies which are bactericidal for meningococcus and/or are capable of binding to a conformational epitope of a meningococcal protein immunogen in said meningococcal vaccine;

(ii) measuring the interaction between each of said meningococcal immunogens and its corresponding anti-immunogen-specific monoclonal antibody with an immunoassay wherein each unbound anti- meningococcal immunogen-specific monoclonal antibody is allowed to interact with a plurality of colour-coded beads conjugated with an antigen selectively bound by said anti-immunogen-specific monoclonal antibody, said beads having a different colour-code for each different antigen.

Advantageously, the assay of the invention allows the simultaneous analysis of a plurality of meningococcal immunogens within the vaccine sample. The sample to be analysed will typically comprise at least two different meningococcal immunogens that are allowed to interact with at least two anti-immunogen-specific monoclonal antibodies, each mAb being specific for (i.e. capable of selectively binding) a different meningococcal immunogen in the sample, said antibodies being bactericidal for meningococcus and/or being capable of binding a conformational epitope of an meningococcal immunogen in said vaccine sample.

Therefore, when a given number of meningococcal immunogens within the vaccine sample is analysed at least the same number of anti- meningococcal immunogenspecific monoclonal antibodies, each of said antibodies selectively binding a different meningococcal immunogen will be used in order to allow the interaction in step (i).

In an embodiment of the invention a plurality (more than one, at least two) anti- meningococcal immunogen-specific monoclonal antibodies can be used so that one or more, or two or more, meningococcal immunogen can be selectively bound by a different monoclonal antibody.

Hence, step (i) of the assay of the invention involves permitting a plurality of meningococcal immunogens within the sample to interact, each, with an anti- meningococcal immunogen specific monoclonal antibody. The interaction between the monoclonal antibody and the immunogen to which it specifically binds is then measured in step (ii).

According to one embodiment, the measuring in (ii) provides the amount of each anti -meningococcal immunogen-specific monoclonal antibody’s target epitope within said meningococcal vaccine sample.

Therefore, according to the invention, the interaction can be measured qualitatively such that step (ii) provides a result which indicates the amount of each monoclonal antibody’s target epitope within the vaccine sample. By using monoclonal antibodies which selectively bind to a bactericidal or conformational epitope of one of the immunogens, the result in step (ii) indicates the amount of the corresponding functional epitope in the vaccine sample, and can allow the distinction 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 amounts obtained with a standard vaccine of known potency, results from step (ii) can be used to calculate relative potency of a test vaccine.

The interaction can be carried out for a suitable period of time at a suitable temperature allowing the formation of a complex meningococcal immunogen+anti- meningococcal 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 to allow the binding of immunogens with the respective anti-immunogen specific monoclonal antibodies.

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

In an embodiment of the invention, 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 2% 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 Al(0H)3 and provide a limited desorbption of the antigen from Al(0H)3 (e.g. maximum 20%) are preferred.

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

The amount of blocking buffer in order to saturate Al(0H)3 can be adjusted by the skilled person. In a preferred embodiment said blocking buffer, in particular a caseinbased blocking buffer as described above, is at a final concentration of 3-1% in the assay buffer, more preferably, said blocking buffer is at a final concentration of about 1,5%.

In a preferred embodiment, the assay buffer can comprise or consist of IX PBS 1,5% blocking buffer 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 nondenatured form or will preferably bind the immunogen in non-denatured form.

The interaction step is carried out with known amounts of vaccine and with known amounts of each anti-immunogen monoclonal antibody used.

Once the interaction in step (i) has taken place, said interaction can be measured by detecting and quantifying all the unbound anti-immunogen monoclonal antibody (i.e. the antibody that did not form the complex with the immunogen) thereby allowing the quantification of the amount of antibody that created an immunogen-antibody complex. The multiple detection of unbound antibody for each monoclonal antibody used can be carried out by adding to the assay mixture of step (i) a plurality of colour coded beads conjugated with an antigen of said anti-meningococcal immunogen monoclonal antibody, the beads having a different colour code for each antigen used. The antigen in this case does not need necessarily to be immunogenic, provided that it shares the epitope specifically bound by the anti-meningococcal immunogen monoclonal antibody, and the conformation thereof, and therefore provided that it can be selectively bound by said antibody.

Therefore, when two different meningococcal immunogens A and B in the vaccine sample are analysed, immunogen A will be allowed to interact with a selective anti- A monoclonal antibody -step (i)- in conditions suitable for the immunogen-antibody binding. The mixture resulting from step (i), or the mixture deprived of the formed immunogen-antibody complex, will be allowed to interact with an antigen selectively bound by antibody A, said antigen being conjugated with a colour coded bead having a spectral signature A; immunogen B will be allowed to interact with a selective anti-B monoclonal antibody -step (i)- in conditions suitable for the immunogen-antibody binding. The mixture resulting from step (i), or the mixture deprived of the formed immunogenantibody complex, will be allowed to interact with an antigen selectively bound by antibody B, said antigen being conjugated with a colour coded bead having a spectral signature B distinct from spectral signature A.

When a third different immunogen C in the vaccine sample is also analysed, immunogen C will be allowed to interact with a selective anti-C monoclonal antibody -step (i)- in conditions suitable for the immunogen-antibody binding. The mixture resulting from step (i), or the mixture deprived of the formed immunogenantibody complex, will be allowed to interact with an antigen selectively bound by antibody C, said antigen being conjugated with a colour coded bead having a spectral signature C distinct from spectral signature C and so on for each additional different immunogen analysed in the vaccine sample.

An anti-meningococcal immunogen-specific monoclonal antibody according to the present description is a monoclonal antibody (the term antibody is intended as defined below) that specifically and selectively binds a single meningococcal immunogen in the vaccine sample and that does not bind to other (different) meningococcal immunogens that are present in the vaccine sample used in the assay. As used herein, ’’colour-coded beads” are beads, such as magnetic beads, that are differentiated from each other by intrinsic fluorescence internal dyes, i.e. the beads being internally dyed with different proportions of red and infrared fluorophores, each bead corresponding to a distinct spectral signature. Suitable colour coded beads can be Luminex® beads or MagPlex® beads. See Figure 20 depicting the schematic representation of beads, the graphic shows the increasing amount of red dye in each bead in the abscissa and the increasing amount of infrared dye in each bead in the ordinate and the resulting distinct beads each one having a different spectral signature, and possible beads selection. Each antigen selected for each antiimmunogen-antibody in step (ii) is conjugated with a distinct pre-selected colour coded bead.

In one embodiment of the invention, starting from a menincococcal vaccine of known composition in terms of meningococcal immunogens, the measuring in step (ii) provides the amount of monoclonal antibody’s target epitopes within said vaccine sample, i.e. it provides the amount of each immunogen of interest. Advantageously, the use of antibodies that have higher affinity for, or selectively binds only to a nondenatured immunogen vs. its denatured form (i.e. differentiates between the native and the denatured form of the immunogen for which said antibody is specific) provides the amount of non-denatured immunogens in the sample.

The measuring in (ii) can be carried out on the reaction mixture of (i) or it can comprise the step of separating the unbound-anti immunogen specific monoclonal antibodies from the reaction mixture of (i), e.g. by centrifugation in order to precipitate the immunogen-antibody complexes and retaining the unbound antibodies in the surnatant and therefore can be carried out on the reaction mixture of (i) deprived of the immunogen-antibody complex(es) formed.

In an embodiment of the invention said measuring in (ii) comprises the steps of

(a) separating the unbound anti-immunogen-specific monoclonal antibodies from the immunogen bound anti-immunogen-specific monoclonal antibodies;

(b) contacting said unbound anti-immunogen-specific monoclonal antibodies with said plurality of colour-coded beads conjugated with the corresponding antigen of said anti-immunogen-specific monoclonal antibody wherein said beads have a different colour-code for each different antigen, and incubating the resulting mixture at a suitable temperature for a period of time allowing the binding of said plurality of colour-coded beads conjugated antigens with the respective anti-immunogen-specific monoclonal antibody to form bead-antigen-monoclonal antibody complexes;

(c) incubating the resulting bead-antigen-monoclonal antibody complexes with a labelled secondary antibody thereby forming complexes comprising also the secondary antibody

(d) carrying out a dual detection on the complexes obtained in step (c) thereby quantifying each bead-antigen-monoclonal antibody complex formed.

When step a. is carried out by centrifugation, the supernatant comprising the unbound monoclonal antibodies is collected and used in step b. Said supernatant is also defined in the present description as the mixture of (i) deprived of the immunogen-antibody complex formed, more precisely it can also be defined as the mixture of (i) deprived of the meningococcal immunogen+anti-meningococcal immunogen monoclonal specific monoclonal antibody complex formed in (i). Either definition can be used in any part of the description and of the claims as aliases.

Suitable centrifugation conditions are at about 1000g at room temperature ± 5°C, for 15-25 minutes.

Step b comprises contacting the unbound anti-immunogen-specific monoclonal antibodies resulting from (i) with a plurality of colour-coded beads conjugated with an antigen that is specifically (selectively) bound by said anti-meningococcal immunogen specific monoclonal antibody wherein said beads have a different colour-code for each different antigen, and incubating the resulting mixture at a suitable temperature for a period of time allowing the binding of said plurality of colour-coded beads conjugated antigens with the respective anti-meningococcal immunogen-specific monoclonal antibody.

The colour-coded beads-antigen conjugate can be prepared by following the manufacturer’s instructions. As each colour-coded bead has a different nonoverlapping spectral signature, each colour-coded bead-antigen conjugate will also have a different non-overlapping spectral signature corresponding to the bead signature.

The incubation in b. can be carried out by transferring each unbound anti- meningococcal immunogen specific-antibody (the number will vary depending on the number of immunogens tested) in a detection plate and they can be captured with a suitable amount of antigen-conjugated beads mix each previously coupled with the relative specific antigen for each antibody of interest. Suitable incubating conditions in b. allowing the formation of beads-antigen-antibody complexes are at about room temperature for a period of time of about one hour.

The resulting bead-antigen-monoclonal antibody complexes are then incubated in c. with a secondary antibody labelled with a fluorescent dye thereby forming complexes comprising also the secondary antibody.

The selection of the suitable secondary antibody depends on the primary antibody used as the skilled person knows in order to carry out the detection. In preferred embodiments, a single secondary antibody will be sufficient. Suitable conditions and buffers are known to the skilled person depending on the labelled secondary antibody used. Suitable labelling can be, e.g. with R-phycoerythrin. Suitable incubation conditions in c. can be at about RT for a period of time of 20-40 minutes, such as about 30 minutes. Dual detection d. can be carried out by reading the complexes obtained in c. on a dual-laser flow-based detection instrument emitting suitable laser beans, such as a dual detection flow cytometer or dual-laser flow cytometer. A suitable instrument is the Luminex 200™ or FlexMap® analyser or a bio-plex array reader. One laser classifies the bead and determines the analyte that is being detected. The second laser determines the magnitude of the PE-derived signal, which is in direct proportion to the amount of analyte bound.

In addition to flow-based analysers, magnetic beads can be read using the Luminex MAGPIX® Analyzer. A magnet in the MAGPIX analyzer captures and holds the magnetic beads in a monolayer, while two spectrally distinct light-emitting diodes (LEDs) illuminate the beads. One LED identifies the analyte that is being detected and, the second LED determines the magnitude of the PE-derived signal. Each well is imaged with a CCD camera.

The assay of the invention can take place in microwell plates.

By way of example the assay can take place in 24, 48, 96, 384 or 1536 microwell plates.

Advantages of the assay of the invention are that the interaction phase between the immunogen-specific mAb and the vaccine immunogens (i) is not performed individually, i.e. with a single antibody at the time as in an ELISA assay, but simultaneously, through a mix of all anti-immunogen-specific mAbs in which each one binds a different immunogen present in the vaccine of interest.

Therefore, the assay allows the binding of a plurality of different meningococcal immunogens with its anti-meningococcal immunogen specific mAb; and is therefore time and cost saving when compared to multiple singleplex assays.

The capture and quantification of the anti-immunogen specific free mAb (unbound after (i) in the detection phase (ii) occurs through the use of a mix of colour-coded magnetic beads previously coupled with an antigen selectively bound by each antiimmunogen specific mAb, each magnetic bead-antigen complex having a different spectral signature, and not by antigen specific ELISA plates.

The multiplex approach provided herein can drastically reduce the timing for reagents and sample preparation and consequently the entire assay execution. Furthermore, multiplex is more informative because it allows the introduction of multiple anti-immunogen specific mAbs directed against the same antigen with no impact on the entire assay workload.

Tipically, the assay of the invention will use the Luminex’ s xMAP Technology, which is a bead-based multiplexed immunoassay system in a microplate format. The system can simultaneously detect many targets in a single sample, depending on system design. The Luminex system uses microspheres (or beads) of 6.5 micron that are composed of polystyrene, divinylbenzene and methacrylic acid. Each bead has different spectral colour codes (also called regions). These unique spectral addresses are created by internally labelling beads with different ratios of two fluorophores, one in a red wavelength and the other infrared. Proteins including antibodies, ligands, immunogens, antigens, and nucleic acids specific to the desired targets can be coupled to the beads thanks to the functional carboxyl groups present on their outer surface, that are activated by using a N-hydroxysplfosuccinimide (sulfo-NHS) enhanced carbodiimide-mediated conjugation chemistry.

In the present invention, the desired targets are antigenic peptides that are selectively bound by a mAb used in step (i) of the assay and the specific moiety bound to the beads are antigens selectively bound by the antibody used in (i).

In the Luminex assay of the invention, each set of antigen-coupled beads is incubated with the specific target analyte i.e. the corresponding unbound anti-immunogen specific monoclonal antibody resulting from step (i).

By using a monoclonal antibody which binds to a bactericidal or conformational epitope, the result in step (ii) provides the amount of the corresponding functional epitope in the vaccine sample, and can allow to 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 vaccine sample

Assays of the invention are used to analyse meningococcal 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 :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°, E2 ¹ , 1 :2 ² , ..., 1 :2 ⁹ , and l :2 ¹⁰ -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 two meningococcal immunogens, in particular protein immunogens, i.e. a protein which, when administered to human beings, elicits an 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 [2, 3], Further protein immunogens which can be analysed are specific fHbp variants or a fusion protein comprising different fHbp variants as disclosed in W02020/030782. W02020/030782 provides mutated fHbp variant 1.13 or variant 1.15 (vl.13 or vl.15) polypeptides that are immunogenic and can be combined with existing meningococcal vaccines. A vaccine may include one or more of these various immunogens e.g. it can comprise each of NHBA, fHbp single variants, fusion protein comprising more than 1 fHbp variants and NadA. Therefore, the vaccine can also comprise, as immunogens, 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 [4] or one fHpb protein and a fusion protein comprising two or even three different fHbp variants or mutant forms of fHbp variants), using different monoclonal anti-fHbp antibodies to recognise each different variant separately (either in the form of a single peptide or within a fusion protein comprising more than 1 variant as described herein).

Preferably, the vaccine will comprise NHBA, NadA, a single fHbp variant, and a fusion protein comprising or consisting of three fHbp different variants or mutants thereof. In one embodiment, the fusion protein comprises or consists of. variant 2, variant 3 and variant 1.15 as disclosed in W02020/030782. In another embodiment, the fusion protein comprises or consists of. variant 2, variant 3 and variant 1.13 as disclosed in W02020/030782.

The vaccine can further comprise 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 5 to 20) 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 comprise vesicles, the vesicle component can be analysed in a different assay.

Hence, according to the invention, said meningococcal vaccine sample comprises at least two meningococcal immunogens, selected from NHBA, NadA and/or fHbp immunogens.

Preferably, said meningococcal vaccine sample comprises one or more fHbp immunogens selected from fHbp variant 1 (non-limiting example fHbp variant 1.1 and/or fHbp variant 1.13), fHbp variant 2 (non-limiting example fHbp variant 2.16) and/or fHbp variant 3 (non-limiting example fHbp variant 3.28) immunogens.

More preferably, said meningococcal vaccine sample comprises two or more different fHbp immunogens selected from fHbp variant 1 (non-limiting example fHbp variant 1.1 and/or fHbp variant 1.13), fHbp variant 2 (non-limiting example fHbp variant 2.16) and/or fHbp variant 3 (non-limiting example fHbp variant 3.28) immunogens.

In a further preferred embodiment, said meningococcal vaccine sample comprises a fHbp antigen immunogen and an fHbp fusion protein, wherein the fHbp fusion protein comprises or consists of two (or even more preferably three) different variants of the fHbp antigen or including mutant forms thereof.

Preferably, said fHbp fusion protein comprises or consists of a vl.13 fHbp mutated variant immunogen consisting of amino acid sequence SEQ ID NO: 4, a fHbp variant v2 mutated variant immunogen (e.g. consisting of amino acid sequence SEQ ID NO: 5) and, a fHbp variant v3 mutated sequence immunogen (e.g. consisting of amino acid sequence SEQ ID NO: 6).

Preferably, said fHbp fusion protein comprises or consists of the amino acid sequence SEQ ID NO: 7 or SEQ ID 23. In an embodiment the fusion protein is (also named herein fHbp2-3-1.13_NB) is a final tripartite fusion protein of SEQ ID NO: 7 composed of fused fH non-binding fHbp2.16, fHbp3.28 and fHbp 1.13 peptides.

In a most preferred embodiment wherein the meningococcal vaccine sample comprises a NHBA immunognen, a NadA immunogen and at least two fHbp immunogens (e.g. an fHbp variant 1 immunogen and an fHbp fusion protein comprising or consisting of an fHbp variant 2 immunogen, an fHbp variant 3 immunogen and an fHbp variant 1 immunogen).

Preferably, said meningococcal vaccine sample comprises the immunogens meningococcal immunogens of SEQ ID NOs 1-3 and 7.

Additionally, said meningococcal vaccine sample may comprise an aluminium hydroxide adjuvant.

The assay of the invention can be carried out on each of the meningococcal vaccines (vaccine samples) indicated above.

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 20. Other strains can also be tested for vaccine efficacy [21] but a response against MC58 is easily tested.

A vaccine which can be analysed according to the invention is BEXSERO [2] or a vaccine comprising at least all the BEXSERO immunogens. The BEXSERO vaccine includes three different recombinant proteins, consisting of amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. It also contains NZ98/254 outer membrane vesicles.

A further vaccine which can be analysed according to the invention is a vaccine comprising the proteins included in the BEXSERO vaccine, and a fusion protein comprising all three of vl, v2 and v3 meningococcal fHbp polypeptides. Preferably in the fusion protein comprised in the vaccine the variant fHbp sequences are in the order v2-v3-vl from N to C terminus. The fusion protein can have a formula NHz — A-[-X-L ]3-B — COOH, wherein each X is a different variant fHbp sequence and L is an optional linker amino acid sequence as disclosed in W02020/030782.

The fusion protein may be in any form disclosed in W02020/030782. A preferred vaccine comprises all the proteins included in the BEXSERO vaccine indicated above (SEQ ID NOs 1-3), and a fusion protein comprising or consisting of a vl.13 fHbp mutated variant consisting of amino acid sequence SEQ ID NO: 4, a v2 mutated variant consisting of amino acid sequence SEQ ID NO: 5 and a v3 mutated sequence consisting of amino acid sequence SEQ ID NO: 6.

A further preferred vaccine comprises all the proteins included in the BEXSERO vaccine indicated above (SEQ ID NOs 1-3), and a fusion protein comprising or consisting of the amino acid sequence SEQ ID NO: 7 or SEQ ID 23.

The BEXSERO vaccine is described in reference 2, and it comprises 50 pg of each of NadA (subvariant 3.2; SEQ ID NO: 3), fHbp subvariant 5) (as a GNA2091-fHbp fusion protein; SEQ ID NO: 2), and NHBA subvariant 1.2 (as a NHBA-GNA1030 fusion protein; SEQ ID NO: 1), adsorbed onto 1.5 mg aluminium hydroxide, and with 25pg OMVs from N.meningitidis strain NZ98/254.

An example of a preferred vaccine according to the invention is an immunogenic composition which comprises the A, C, W, and Y capsular polysaccharides conjugated with CRM197, recombinant Neisseria meningitidis serogroup B NHsaBA fused to NUbp, NadA protein, fHbp Var 1.1 fused to GNA2091, Outer membrane vesicles (OMV) from Neisseria meningitidis group B strain NZ98/254 and the fHbp Var 1.13, Var 2.16 and Var 3.28 as unique fusion polypeptide (fHbp 2-3-1.13 NB). The vaccine is adjuvanted with alum hydroxide. Except for the fHbp Var 1.13, Var 2.16 and Var 3.28 as unique fusion polypeptide (fHbp 2-3-1.13 NB) the other immunogens can be the same immunogens described in reference 2 for the BEXSERO vaccine.

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 22.

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. An analysed vaccine comprises an immunologically effective amount of immunogenic antigen(s) also defined herein as immunogens, 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 individuals 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 immunogen 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 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. 23), 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 0.05pg) 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.

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 vaccine, 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 a reference batch of said vaccine 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 simultaneously. OMVs when present can be tested separately. These results allow the assay user to simultaneously identify the specific cause of any loss of overall potency.

The antibody

Assays of the invention use monoclonal antibodies which recognise protein immunogens which are present within the analysed vaccines. The invention can use antibodies which are bactericidal for meningococcus and/or which recognise conformational epitopes in the protein immunogens. In both cases the antibodies can thus differentiate 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 SB A [24-27], Reference 28 reports good interlaboratory 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.

In order to determine whether an antibody recognises a conformational epitope the skilled person may apply various 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.

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 for each immunogen tested: a single monoclonal antibody which recognises a single immunogen; a plurality of different monoclonal antibodies which recognise a single immunogen (typically different epitopes on the immunogen). The invention provides a single assay to recognise multiple immunogens simultaneously. The results on each immunogen allows an overall analysis of the vaccine sample. The assay enables to simultaneously differentiate each immunogen within a multi-immunogen vaccine and therefore 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. 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 R ² >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 the assay of the invention.

The monoclonal antibodies can have any heavy chain type e.g. it can have a, 5, a, 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.

Therefore, in any embodiment herein disclosed of the assay of the invention, at least one of said anti-immunogen-specific monoclonal antibodies can be a murine monoclonal IgG antibody, in particular IgGl or IgG2.

The monoclonal antibodies can have any light chain type e.g. it can have either a kappa (K) or a lambda (A) 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 a multiplex immunoassay such as a Luminex assay. Luminex assay protocols are readily available to the skilled person. 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 “single-chain 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 recognize 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 immune 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 NSO 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.

Tests to determinate whether an antibody is able to differentiate between native and denatured forms of a given immunogen include testing the antibody against a panel of linear peptide fragments from the target antigen (e.g. using the Pepscan technique) and comparing the binding can 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 thermal denaturation as disclosed in the present application.

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. The invention does not require the use of particular known antibodies, but a number of antibodies useful for analysis of the immunogens in the vaccine according to the description are herein disclosed, are described below:

- for assaying fHbp antigen as found in the BEXSERO product include, but are not limited to the 12C1/D7 antibody (disclosed in W02013/132040) and/or the 11F10/G6 antibody (disclosed in WO2013/132040);

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

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

- for assaying, in a preferred vaccine according to the specification, a fHbp antigen in the form of a fusion protein comprising two or even three different fHbp variants or mutant forms of fHbp variants, such as a fusion protein as disclosed in W02020/03078, such as the fusion protein as herein defined, preferably a fusion protein comprising or consisting of a vl.13 fHbp mutated variant consisting of amino acid sequence SEQ ID NO: 4, a v2 mutated variant consisting of amino acid sequence SEQ ID NO: 5 and a v3 mutated sequence consisting of amino acid sequence SEQ ID NO: 6, even more preferably a fusion protein comprising or consisting of the amino acid sequence SEQ ID NO: 7 or SEQ ID 23, include, but are not limited to one or more of the 191, mAb 6, mAb 36, mAb 44 (see below).

The sentence “ able to differentiate (or able to distinguish) between native and denatured form of antigen” means that the antibody is capable of binding to the native form of antigen with a higher affinity than the denatured form, or more preferably means that the antibody is not capable of binding to the denatured form of the antigen. In the present description, the sentence “the antibody is capable to bind” has the same meaning of “the antibody recognises”.

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 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 29-35, 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 [36, 37] or similar methods), or they can be predicted (e.g. using the Jameson-Wolf antigenic index [38, matrix-based approaches [39], MAPITOPE [40], TEPITOPE [41, 42], neural networks [43], OptiMer & EpiMer [44, 45], ADEPT [46], Tsites [47], hydrophilicity [48], antigenic index [49] or the methods disclosed in references 50 to 54, 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 55. 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. 56.

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 [57] was included in the published genome sequence for meningococcal serogroup B strain MC58 [20] as gene NMB2132 (GenBank accession number GE7227388; SEQ ID NO: 8 herein). 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 ref. 58, and in Example 13 and Figure 21 of reference 58 (SEQ IDs 3179 to 3184 therein). Various immunogenic fragments of NHBA have also been reported.

Preferred NHBA immunogens 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: 8; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 8, 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: 8.

The most useful NHBA immunogens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 8. Advantageous NHBA immunogens 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

The NadA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [20] as gene NMB1994 (GenBank accession number GE7227256; SEQ ID NO: 9 herein). 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 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; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 9, 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.

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: 9. Advantageous NadA immunogens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject. SEQ ID NO: 3 is one such fragment.

HmbR

The full-length HmbR sequence was included in the published genome sequence for meningococcal serogroup B strain MC58 [20] as gene NMB1668 (SEQ ID NO: 11). Reference 60 reports a HmbR sequence from a different strain (SEQ ID NO: 12 herein), and reference 61 reports a further sequence (SEQ ID NO: 13herein). SEQ ID NOs: 11 and 12 differ in length by 1 amino acid and have 94.2% identity. SEQ ID NO: 13 is one amino acid shorter than SEQ ID NO: 11 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: 11, 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: 11, where the value of j 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: 11 and/or (ii) comprising a fragment of at least j consecutive amino acids from SEQ ID NO: 11.

Preferred fragments of j amino acids comprise an epitope from SEQ ID NO: 11. 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 62. 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: 14 and 15. If soluble HmbR is used, however, sequences omitting the transmembrane sequence, but typically retaining epitope(s) from the extracellular portion, can be used.

The most useful HmbR antigens can elicit antibodies which, after administration to a subject, can bind to a meningococcal polypeptide consisting of amino acid sequence SEQ ID NO: 11. Advantageous HmbR antigens for use with the invention can elicit bactericidal anti -meningococcal antibodies after administration to a subject. 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. 59], ‘NMB1870’, ‘GNA1870’ 63-65], ‘P2086’, ‘LP2086’ or ‘ORF2086’ [66-68], 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 [69], 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.

As different fHbp classification schemes have been proposed, a dedicated database is available with sub-variants: a unified fHbp nomenclature for the assignment of new (http)://neisseria.org/nm/typing/fhbp (also as (https)://pubmlst.org/neisseria/fHbp/). fHbp has been classified into three (main) variants 1, 2 and 3, which were further divided into sub-variants fHbp-l.x, fHbp-2.x and fHbp-3.x, where x denotes the specific peptide sub-variant. In contrast to v2 and v3, fHbp vl is highly heterogeneous and contains several subvariants. In a different nomenclature scheme, the sub/variants are grouped into subfamily A (corresponding to variants 2 and 3) and subfamily B (corresponding to variant 1) based on sequence diversity.

Bactericidal activity is variant specific; antibodies raised against one variant are not necessarily cross-protective against other variants, although some cross-reactivity has been described between fHbp v2 and v3 (63). Antibodies raised against sub-variant fHbpvl .l, included in the BEXSERO vaccine, are highly cross-reactive with the most frequently occurring fHbp vl sub-variants but are less cross-reactive with vl sub-variants that are most distantly related to vl.l. Furthermore, antibodies raised against sub-variant fHbpvl .1 included in the BEXSERO vaccine are poorly cross- reactive with fHbp v2 and v3 (70). As indicated above, the vaccine of the invention may comprise also immunogens that are effective against meningococcal strains carrying fHbp v2, v3, or strains carrying some vl sub-variants.

The fHbp antigen falls into three distinct variants [71] 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. A preferred vaccine will also include a fusion protein comprising different fHbp variants or mutants thereof as disclosed in W02020/030782. Thus it may use a combination of two or more different fHbps, selected from: (a) a first protein, comprising an amino acid sequence having at least a% sequence identity to SEQ ID NO: 5 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 5; (b) a second protein, comprising an amino acid sequence having at least b% sequence identity to SEQ ID NO: 6 and/or comprising an amino acid sequence consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 6; and/or (c) a third protein, comprising an amino acid sequence having at least c% sequence identity to SEQ ID NO: 4 and/or comprising an amino acid sequence consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 4 and/or a fusion protein comprising more than one different fHbp variant or mutants thereof as disclosed in W02020/030782, preferably a fusion protein comprising all three of vl, v2 and v3 meningococcal fHbp polypeptides or mutants thereof. Advantageously, mutated variants that reduce binding to hfH can be used. Preferably in the fusion protein comprised in the vaccine the variant fHbp sequences are in the order v2-v3-vl from N to C terminus. The fusion protein can have a formula bffib — A-[-X-L ]a-B — COOH, wherein each X is a different variant fHbp sequence and L is an optional linker amino acid sequence as disclosed in W02020/030782.

When the fusion protein comprises a vl.13 mutated variant, the fusion protein will comprise aa polypeptide comprising an amino acid sequence having at least k% sequence identity to SEQ ID NO: 7, with the proviso that the amino acid sequence of said mutant vl.13 meningococcal fHbp polypeptide includes a substitution mutation at one or more of residues E211, S216 or E232 of SEQ ID NO: 7. The value of K may be selected from 80, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100. It is preferably 80 (i.e. the mutant fHbp vl.13 amino acid sequence has at least 80% identity to SEQ ID NO: 7) and is more preferably 85, more preferably 90 and more preferably 95. Most preferably, the mutant fHbp vl.13 amino acid sequence has at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 7.

Preferably, the amino acid sequence comprises at least one or more of the substitutions E211A, S216R or E232A. More preferably, the amino acid sequence comprises substitutions at multiple residues selected from the following (i) E211A and E232A, or (11) E211A and S216R. More preferably, the amino acid sequence comprises substitutions at residues E211 A and S216R, relative to SEQ ID NO. 7.

In preferred embodiments, the mutant vl.13 polypeptide in the fusion protein has the amino acid sequence of SEQ ID NO: 20 (vl.13 AO E211A/E232A) or SEQ ID NO: 4 (vl l3 AO (E211A/S216R). More preferably, mutant vl.13 polypeptide of the invention has the amino acid sequence of SEQ ID NO:4.

The v2 and v3 fHbp polypeptide components of the fusion protein are preferably mutant v2 and v3 polypeptides having enhanced stability and reduced ability to bind to hfH, compared to the wild-type v2 and v3 polypeptides.

In a preferred embodiment, the fusion protein comprises a mutant v2 fHbp polypeptide comprising an amino acid sequence having at least % sequence identity to SEQ ID NO: 21, with the proviso that the v2 fHbp amino acid sequence includes a substitution mutation at residues S32 and L123 of SEQ ID NO: 21. Preferably the substitutions are S32V and L123R.

The value of K may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100. It is preferably 80 (i.e. the mutant fHbp v2 amino acid sequence has at least 80% identity to SEQ ID NO: 21) and is more preferably 85, more preferably 90 and more preferably 95.

In some embodiments, the fHbp v2 polypeptide included in the fusion protein of the invention is truncated relative to SEQ ID NO: 21.

In a preferred embodiment, the v2 fHbp polypeptide included in the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 5.

In a preferred embodiment, the fusion polypeptide of the invention comprises a mutant v3 fHbp polypeptide comprising an amino acid sequence having at least k% sequence identity to SEQ ID NO: 22, with the proviso that the v3 fHbp amino acid sequence includes substitution mutations at residues S32 and L126 of SEQ ID NO: 22. Preferably the substitutions are S32V and L126R.

The value of K may be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100. It is preferably 80 (i.e. the mutant fHbp v2 amino acid sequence has at least 80% identity to SEQ ID NO: 15) and is more preferably 85, more preferably 90 and more preferably 95.

In some embodiments, the fHbp v3 polypeptide included in the fusion protein of the invention is truncated relative to SEQ ID NO: 22.

In a preferred embodiment, the v3 fHbp polypeptide included in the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 6. In a preferred embodiment, the fusion protein comprises or consists of a vl.13 fHbp mutated variant consisting of amino acid sequence SEQ ID NO: 4, a v2 mutated variant consisting of amino acid sequence SEQ ID NO: 5 and a v3 mutated sequence consisting of amino acid sequence SEQ ID NO: 6, such as a fusion protein comprising or consisting of the amino acid sequence SEQ ID NO: 7 or a fusion protein comprising or consisting of the amino acid sequence SEQ ID NO 23.

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 of z 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 of x, 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: 5 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 5; or (b) an amino acid sequence having at least b% sequence identity to SEQ ID NO: 6 and/or comprising an amino acid sequence consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 6; or (c) an amino acid sequence having at least c% sequence identity to SEQ ID NO: 4 and/or comprising an amino acid sequence consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 4.

Where the invention uses a fHbp from two or more 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: 5 and/or comprising an amino acid sequence consisting of a fragment of at least x contiguous amino acids from SEQ ID NO: 5; (b) a second polypeptide, comprising an amino acid sequence having at least b% sequence identity to SEQ ID NO: 6 and/or comprising an amino acid sequence consisting of a fragment of at least y contiguous amino acids from SEQ ID NO: 6; and/or (c) a third polypeptide, comprising an amino acid sequence having at least c% sequence identity to SEQ ID NO: 4 and/or comprising an amino acid sequence consisting of a fragment of at least z contiguous amino acids from SEQ ID NO: 4, and/or a fusion protein comprising or consisting of a v2 mutated variant consisting of amino acid sequence SEQ ID NO: 5 and a v3 mutated sequence consisting of amino acid sequence SEQ ID NO: 6, a vl.13 fHbp mutated variant consisting of amino acid sequence SEQ ID NO: 4, such as a fusion protein comprising or consisting of the amino acid sequence SEQ ID NO: 7 or a fusion protein comprising or consisting of the amino acid sequence SEQ ID NO 23. The first, second and third polypeptides have different amino acid sequences. fHbp protein(s) in a OMV will usually be lipidated e.g. at a N-terminus cysteine. In other embodiments, they will not be lipidated.

One vaccine which can be analysed by the methods of the invention includes one or more, or two of more, different variant(s) of fHbp in the form of a single polypeptide and/or of a fusion protein comprising more than one, preferably three, different variants and/or mutants thereof as defined above. The first variant can have amino acid sequence SEQ ID NO: 24, and the second can have amino acid sequence SEQ ID NO: 25, the fusion protein can comprise or consist of amino acid sequence SEQ ID 7 or SEQ ID 23. 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 (Neisseria! surface protein A)

The NspA antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [20] as gene NMB0663 (GenBank accession number GE7225888; SEQ ID NO: 26 herein). The antigen was previously known from references 72 and 73. 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 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: 26; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 26, wherein 'ri 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: 26. 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: 26. 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 [20] as gene NMB0992 (GenBank accession number GE7226232; SEQ ID NO: 27 herein). The sequences of NhhA antigen from many strains have been published since e.g. refs. 58 & 74 and various immunogenic fragments of NhhA have been reported. It is also known as Hsf.

Preferred NhhA immunogens 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: 27; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 27, wherein 'ri 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: 27.

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: 27. Advantageous NhhA immunogens 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 [20] as gene NMB1985 (GenBank accession number GE7227246; SEQ ID NO: 28 herein). The sequences of App antigen from many strains have been published since then. It has also been known as ‘ORFE and ‘Hap’. Various immunogenic fragments of App have also been reported.

Preferred App immunogens 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: 28; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 13, wherein 'ri 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: 28. 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: 28. Advantageous App immunogens 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 [20] as gene NMB0182 (GenBank accession number GE7225401; SEQ ID NO: 29 herein). The sequences of Omp85 antigen from many strains have been published since then. Further information on Omp85 can be found in references 75 and 75. Various immunogenic fragments of Omp85 have also been reported.

Preferred Omp85 immunogens 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: 29; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 29, wherein 'ri 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: 29.

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: 29. Advantageous Omp85 immunogens 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 [20] as gene NMB0461 (GenBank accession number GE7225687; SEQ ID NO: 30 herein). The sequences of TbpA from many strains have been published since then. Various immunogenic fragments of TbpA have also been reported.

Preferred TbpA immunogens 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: 30; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 30, wherein 'ri 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: 30.

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: 30. Advantageous TbpA immunogens 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 [20] as gene NMB1398 (GenBank accession number GE7225686; SEQ ID NO: 31 herein). The sequences of TbpB from many strains have been published since then. Various immunogenic fragments of TbpB have also been reported.

Preferred TbpB immunogens 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: 31; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 31, wherein 'ri 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: 31.

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: 31. Advantageous TbpB immunogens for use with the invention can elicit bactericidal anti-meningococcal antibodies after administration to a subject.

Cu.Zn-super oxide dismutase

The Cu,Zn-superoxide dismutase antigen was included in the published genome sequence for meningococcal serogroup B strain MC58 [20] as gene NMB1398 (GenBank accession number GE7226637; SEQ ID NO: 32 herein). 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: 32; and/or (b) comprising a fragment of at least 'n' consecutive amino acids of SEQ ID NO: 32, 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: 32.

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: 32. 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 immunogen in a meningococcal vaccine sample, comprising steps of: (i) performing the binding assay according of the invention on a test meningococcal vaccine sample and, optionally, on at least one dilution of the test meningococcal vaccine sample; (ii) performing the binding assay of the invention on a standard meningococcal vaccine sample of known native antigenic form and, optionally, on at least one dilution of the standard meningococcal 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 meningococcal vaccine sample relative to the amount of immunogen(s) in the native form in the standard meningococcal vaccine sample.

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 test meningococcal vaccine sample, comprising steps of:

(iii) performing steps (i) and (ii) of the binding assay of the invention on the test meningococcal vaccine sample and, optionally, on at least one dilution of the test meningococcal vaccine sample;

(iv) performing steps (i) and (ii) of the binding assay of the invention on a standard meningococcal vaccine sample of known in vivo potency and, optionally, on at least one dilution of the standard meningococcal vaccine sample; and

(v) comparing the results from steps (i) and (ii) obtained in (iii) and (iv) to determine the potency of immunogen(s) in the test meningococcal vaccine sample relative to the potency of immunogen(s) in the standard meningococcal vaccine sample Monoclonal antibodies

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

One antibody of the invention is a monoclonal antibody (10E8) capable of binding to meningococcal NHBA antigen, whose light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 34 and whose heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 38

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

-CDRL1 having SEQ ID NO:35,

-CDRL2 having SEQ ID: 103,

-CDRL3 having SEQ ID NO: 36,

-CDRH1 having SEQ ID NO:39,

-CDRH2 having SEQ ID NO:40,

-CDRH3 having SEQ ID NO:41,

In one embodiment the antibody of the invention is a monoclonal antibody which recognises an epitope of meningococcal antigen NHBA comprising or consisting in the amino acid sequence of SEQ ID NO: 42, preferably of SEQ ID NO 43, even more preferably of SEQ ID 44.

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: 38) and VL (SEQ ID NO: 34) region of the antibody 10E8.

The invention provides also a monoclonal antibody which binds to antigen NHBA and competes or cross-competes 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) capable of binding to meningococcal NadA antigen, whose VL region has the amino acid sequence of SEQ ID NO: 46 and whose VH region has the amino acid sequence of SEQ ID NO: 50.

One antibody of the invention is a monoclonal antibody (6E3) capable of binding to meningococcal NadA antigen whose VH and VL comprise the following complementarity-determining regions (CDRs): -CDRL1 having SEQ ID NO:47, -CDRL2 having SEQ ID: 104, -CDRL3 having SEQ ID NO:48, -CDRH1 having SEQ ID NO:51, -CDRH2 having SEQ ID NO: 52, -CDRH3 having SEQ ID NO:53.

In one embodiment, the antibody of the invention is a monoclonal antibody which recognises an epitope in the region of meningococcal antigen NadA corresponding to the amino acid sequence of residues 206-249 of SEQ ID NOV.

In one embodiment, the antibody of the invention is a monoclonal antibody able to differentiate between native and denatured form of antigen (immunogen) 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 of VH (SEQ ID NO: 50) and VL (SEQ ID NO: 46) region of the antibody 6E3.

The invention provides also a monoclonal antibody which binds to antigen (immunogen) NadA and competes or cross-competes with and/or binds the same epitope as the antibody 6E3. If two antibodies reciprocally compete with each other for binding to antigen (immunogen) NadA, they are said to cross-compete.

One antibody of the invention is a monoclonal antibody (191 herein referred to also as V3/19I or Var3/19I) capable of binding to meningococcal fHbp v3, in particular fHbpv (3.2) 3.28 antigen (immunogen) (SEQ ID 64 or SEQ ID NO: 10 or SEQ ID NO: 6), and capable of binding to meningococcal fHbp v3 (3.28) when present within an fhbp fusion protein as described herein, whose VL region has the amino acid sequence of SEQ ID NO: 55 and whose VH region has the amino acid sequence of SEQ ID NO: 60.

One antibody of the invention is a monoclonal antibody (191) capable of binding to meningococcal fHbp v3, in particular fHbpv (3.2) 3.28 antigen whose VH and VL comprise the following complementarity-determining regions (CDRs): -CDRL1 having SEQ ID NO:56, -CDRL2 having SEQ ID NO:57 -CDRL3 having SEQ ID NO: 58. -CDRH1 having SEQ ID NO:61,

-CDRH2 having SEQ ID NO: 62,

-CDRH3 having SEQ ID NO:63,

In one embodiment the antibody of the invention is a monoclonal antibody (191) capable of binding to an epitope in the region of meningococcal fHbp v3, in particular fHbpv (3.2) 3.28 antigen, said epitope being absent from variants 1 and 2, the epitope comprising NRH amino acid residues in positions 169, 171 and 173 of fHbpv (3.2) 3.28 of SEQ ID NO 64 or SEQ ID NO: 10 or the corresponding amino acids in SEQ ID NO: 6.

In one embodiment, the antibody of the invention is a monoclonal antibody able to differentiate between native and denatured form of antigen fHbp v3, in particular fHbpv (3.2) 3.28 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: 60) and VL (SEQ ID NO: 55) region of the antibody 191.

The invention provides also a monoclonal antibody which binds to antigen fHbp v3, in particular fHbpv (3.2) 3.28 and competes or cross-competes with and/or binds the same epitope as the antibody 191. If two antibodies reciprocally compete with each other for binding to antigen fHbp v3, in particular fHbpv (3.2) 3.28, they are said to cross-compete.

One antibody of the invention is a monoclonal antibody (191) capable of binding to meningococcal fHbp v3, in particular fHbpv (3.2) 3.28 antigen (immunogen) (SEQ ID NO: 6), and whose VL region has the amino acid sequence of SEQ ID NO: 55 and whose VH region has the amino acid sequence of SEQ ID NO: 60. One antibody of the invention is a monoclonal antibody (191) capable of binding to meningococcal fHbp fusion protein as described herein (e.g. SEQ ID NO: 7) and whose VL region has the amino acid sequence of SEQ ID NO: 55 and whose VH region has the amino acid sequence of SEQ ID NO: 60.

One antibody of the invention is a monoclonal antibody (mAb 6) capable of binding to meningococcal fHbp vl.13 antigen (SEQ ID NO: 4 or SEQ ID NO: 102), whose VL region has the amino acid sequence of SEQ ID NO: 65 and whose VH region has the amino acid sequence of SEQ ID NO: 70.

One antibody of the invention is a monoclonal antibody (mAb 6) capable of binding to meningococcal fHbp vl.13 antigen whose VH and VL comprise the following complementarity-determining regions (CDRs): -CDRL1 having SEQ ID NO: 67, -CDRL2 having SEQ ID NO: 68 -CDRL3 having SEQ ID NO:69.

-CDRH1 having SEQ ID NO:71,

-CDRH2 having SEQ ID NO: 72,

-CDRH3 having SEQ ID NO:73,

In one embodiment the antibody of the invention is a monoclonal antibody (mAb 6) is able to bind the surface of MenB strain carrying the variant 1.13 (e.g. SEQ ID NO: 4 or SEQ ID NO: 102) of fHbp and kill them in a serum bactericidal assay, and recognize conformational, functional and clinically relevant epitopes of the fHbp 1.13NB antigen within the 2-3-1.13 NB fusion protein comprising or consisting of SEQ ID NO 7 or 23.

In one embodiment, the antibody of the invention is a monoclonal antibody able to differentiate between native and denatured form of antigen fHbp vl.13 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: 70) and VL (SEQ ID NO: 66) region of the antibody mAb 6.

The invention provides also a monoclonal antibody which binds to antigen fHbp vl.13 and competes or cross-competes with and/or binds the same epitope as the antibody mAb 6. If two antibodies reciprocally compete with each other for binding to antigen fHbp vl.13, they are said to cross-compete.

One antibody of the invention is a monoclonal antibody (mAb 6) capable of binding to meningococcal fHbp vl.13 antigen (SEQ ID NO: 4), whose VL region has the amino acid sequence of SEQ ID NO: 65 and whose VH region has the amino acid sequence of SEQ ID NO: 70. One antibody of the invention is a monoclonal antibody (mAb 6) capable of binding to meningococcal fHbp fusion protein as described herein (SEQ ID NO: 7), whose VL region has the amino acid sequence of SEQ ID NO: 65 and whose VH region has the amino acid sequence of SEQ ID NO: 70.

One antibody of the invention is a monoclonal antibody (mAb 36) capable of binding to meningococcal fHbp vl.13 antigen (e.g. SEQ ID NO: 4 or SEQ ID NO: 102), whose VL region has the amino acid sequence of SEQ ID NO: 74 and whose VH region has the amino acid sequence of SEQ ID NO: 78.

One antibody of the invention is a monoclonal antibody (mAb 36) capable of binding to meningococcal fHbp vl.13 antigen whose VH and VL comprise the following complementarity-determining regions (CDRs):

-CDRL1 having SEQ ID NO:75, -CDRL2 having SEQ ID NO: 76 -CDRL3 having SEQ ID NO:77. -CDRH1 having SEQ ID NO:79,

-CDRH2 having SEQ ID NO: 80,

-CDRH3 having SEQ ID NO: 81,

In one embodiment, the antibody of the invention is a monoclonal antibody (mAb 36) is able to bind the surface of MenB strain carrying the variant 1.13 of fHbp and kill them in a serum bactericidal assay, and recognize conformational, functional and clinically relevant epitopes of the fHbp 1.13NB antigen within the 2-3-1.13 NB fusion protein comprising or consisting of SEQ ID NO 7 or 23.

In one embodiment, the antibody of the invention is a monoclonal antibody able to differentiate between native and denatured form of antigen fHbp vl.13 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: 78) and VL (SEQ ID NO: 74) region of the antibody mAb 36.

The invention provides also a monoclonal antibody which binds to antigen fHbp vl.13 and competes or cross-competes with and/or binds the same epitope as the antibody mAb 36. If two antibodies reciprocally compete with each other for binding to antigen fHbp vl.13, they are said to cross-compete.

One antibody of the invention is a monoclonal antibody (mAb 36) capable of binding to meningococcal fHbp vl.13 antigen (e.g. SEQ ID NO: 4), whose VL region has the amino acid sequence of SEQ ID NO: 74 and whose VH region has the amino acid sequence of SEQ ID NO: 78. One antibody of the invention is a monoclonal antibody (mAb 36) capable of binding to meningococcal fHbp fusion protein as described herein (e.g. SEQ ID NO: 7), whose VL region has the amino acid sequence of SEQ ID NO: 74 and whose VH region has the amino acid sequence of SEQ ID NO: 78.

One antibody of the invention is a monoclonal antibody (mAb 44) capable of binding to meningococcal fHbp vl.13 antigen, whose VL region has the amino acid sequence of SEQ ID NO: 82 and whose VH region has the amino acid sequence of SEQ ID NO: 86.

One antibody of the invention is a monoclonal antibody (mAb 44) capable of binding to meningococcal fHbp vl.13 antigen whose VH and VL comprise the following complementarity-determining regions (CDRs):

-CDRL1 having SEQ ID NO: 83,

-CDRL2 having SEQ ID NO: 84 -CDRL3 having SEQ ID NO:85. -CDRH1 having SEQ ID NO:87, -CDRH2 having SEQ ID NO:88, -CDRH3 having SEQ ID NO: 89,

In one embodiment, the antibody of the invention is a monoclonal antibody (mAb 44) is able to bind the surface of MenB strain carrying the variant 1.13 of fHbp and kill them in a serum bactericidal assay, and recognize conformational, functional and clinically relevant epitopes of the fHbp 1.13NB antigen within the 2-3-1.13 NB fusion protein comprising or consisting of SEQ ID NO 7 or 23.

In one embodiment, the antibody of the invention is a monoclonal antibody able to differentiate between native and denatured form of antigen fHbp vl.13 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: 86) and VL (SEQ ID NO: 82) region of the antibody mAb 44.

The invention provides also a monoclonal antibody which binds to antigen fHbp vl.13 and competes or cross-competes with and/or binds the same epitope as the antibody mAb 44. If two antibodies reciprocally compete with each other for binding to antigen fHbp vl.13, they are said to cross-compete.

One antibody of the invention is a monoclonal antibody (mAb 44) capable of binding to meningococcal fHbp vl.13 antigen (e.g. SEQ ID NO: 4), whose VL region has the amino acid sequence of SEQ ID NO: 82 and whose VH region has the amino acid sequence of SEQ ID NO: 86. One antibody of the invention is a monoclonal antibody (mAb 44) capable of binding to meningococcal fHbp fusion protein as described herein (e.g. SEQ ID NO: 7), whose VL region has the amino acid sequence of SEQ ID NO: 82 and whose VH region has the amino acid sequence of SEQ ID NO: 86.

• mAb 191 - selectively binds fHbp v3 as defined above.

• mAbs 6, 36 and 44 - selectively bind fHbp vl.13.

The use of one of these mAbs alone is able to differentiate one part of the fHbp 231.13 fusion, therefore any of mAb 191, mAb 6, mAb 36 or mAb 44 is sufficient for the assays of the invention.

Each of the antibodies disclosed herein has the following features:

-specificity for the target

-binds a function 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)

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 antigen-binding 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 33, SEQ ID NO 37, SEQ ID NO 45, SEQ ID 49, SEQ ID 54, SEQ ID 59 or SEQ ID NO:65.

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 (dashed line) and the calculated curve (solid line) 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 mAb 6E3 indicated. 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 or bound to the mAh 6E3. Incorporation substantially overlaps except in the two boxed graphs wherein the free form is in the upper line and the form bound to mAb 63E is in the lower line where significant difference of deuterium uptake is observed.

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

Figure 6. SCK results showing the experimental curve (dashed line) and the calculated curve (solid line) 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 or bound to the mAb 10E8. Lines are substantially overlapping, in the boxed graph upper line is free form and bound to mAb 10E8 is the bottom line where significant difference of deuterium uptake is observed.

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 reports the rSBA titers obtained against the mutant 5/99 AAC3.28 (in bold in Table 1). The curves represent the percentage of bacterial survival at each sample dilution.

Figure 12. FACS analysis of mAb 191 IgG2a and mAb 121 with M1239 (top) and 5/99 AAC3.28 (bottom) strains. Monoclonal antibodies were tested at 2pg/ml.

Figure 13. Overlapping of FACS analysis results with M1239 (solid line) and 5/99 AAC3.28 (dashed) for mAb 191 IgG2a (on the left) and mAb 121 (on the right). Monoclonal antibodies were tested at 2pg/ml.

Figure 14 represents gating strategy to sort only varl,13NB-specific MBC excluding all the cross-reactive or less specific B cells.

Figure 15 shows the results of SPR analysis for specificity and affinity of mAbs 6, 36 and 44. For each sensorgram, a blank subtraction was performed, based on the corresponding captured mAb but with injections of buffer instead of antigen dilutions.

Figure 16 mAbs binding specificity to fHbp variant 1.13 and not to the close variant 1. Selected indicator stains for fHbp var 1.13 (DEI 1301) and fHbp var 1 (MC58) were used for this scope and results are reported. Panel a mAb 6, panel b mAb 36 panel c mAb 44.

Figure 17. Sensorgrams of mAbs against not stressed and thermal stressed fHbp2- 31.13_NB antigen. Sensorgrams legend: -70°C in 1, +4°C in 2, +70°C in 3 and +80°C in 4. Panel a mAb 6, panel b mAb 36 panel c mAb 44.

Figure 18 is a schematic representation of the multiplex assay of the invention, exemplified for 2 distinct immunogens, 1 and 2, each allowed to interact with it’s respective anti-immunogen specific mAb (depicted as mAb 1 and mAb 2). Bead 1 conjugated to a mAb 1 antigen and Bead 2 conjugated with mAb 2 antigen are also represented. Just two immunogens are represented in order to simplify the scheme.

Figure 19. Dose-response curve obtained for vaccine components. For mAb 191, 12C1D7 and mAb 6 open and closed circle traces overlapped.

Figure 20. Schematic representation of beads selection.

Figure 21 results of the potency test disclosed in the specification “The IVRP assay” The clear rectangles represent the accuracy range of [-20%; +25%] (i.e., ±0.22 in In scale) around the expected RP value and the black dots are the observed relative potency (RP) result. Despite a single analytical session was performed, the RP results are well within the accuracy range and the regression line log-measured potency vs log-expected potency has a slope close to 1 for all the antigens.

Figure 22 Plate layout used for the mAb binding step of IVRP assay.

Summarising, the invention relates to:

A binding assay for an in vitro analysis of a meningococcal vaccine sample including more than one meningococcal immunogen, comprising the steps of:

(i) allowing the interaction of a plurality of meningococcal immunogens within the sample with a plurality of anti-meningococcal immunogen-specific monoclonal antibodies which are bactericidal for meningococcus and/or are capable of binding to a conformational epitope of a meningococcal protein immunogen in said meningococcal vaccine;

(ii) measuring the interaction between each of said meningococcal immunogens and its corresponding anti-immunogen-specific monoclonal antibody with an immunoassay wherein each unbound anti- meningococcal immunogen-specific monoclonal antibody is allowed to interact with a plurality of colour-coded beads conjugated with an antigen selectively bound by said anti-immunogen-specific monoclonal antibody, said beads having a different colour-code for each different antigen.

The binding assay as defined above wherein said measuring in (ii) provides the amount of each anti -meningococcal immunogen-specific monoclonal antibody’s target epitope within said meningococcal vaccine sample.

The binding assay according to any one of the two definitions above wherein said each of said colour-coded beads is internally dyed with different proportions of red and infrared fluorophores that correspond to a distinct spectral signature.

The binding assay of any one of the three definitions above wherein said measuring in (ii) comprises the steps of

(a) separating the unbound anti-immunogen-specific monoclonal antibodies from the immunogen bound anti-immunogen-specific monoclonal antibodies;

(b) contacting said unbound anti-immunogen-specific monoclonal antibodies with said plurality of colour-coded beads conjugated with the corresponding antigen of said anti-immunogen-specific monoclonal antibody wherein said beads have a different colour-code for each different antigen, and incubating the resulting mixture at a suitable temperature for a period of time to allow the binding of said plurality of colour-coded beads conjugated antigens with the respective anti-immunogen-specific monoclonal antibody to form bead-antigen-monoclonal antibody complexes;

(c) incubating the resulting bead-antigen-monoclonal antibody complexes with a labelled secondary antibody thereby forming complexes comprising also the secondary antibody

(d) carrying out a dual detection on the complexes obtained in step (c) thereby quantifying each bead-antigen-monoclonal antibody complex formed.

The binding assay according to the previous definition, wherein said separation in step (a) is carried out by centrifugation.

The binding assay of any of the five definitions above, wherein said interaction (i) and/or said measuring (ii) takes place in microwell plates.

The binding assay of any one of the six definitions above wherein said meningococcal vaccine sample comprises at least two meningococcal immunogens, selected from NHBA, NadA and/or fHbp immunogens

The binding assay according to the previous definition, wherein said meningococcal vaccine sample comprises one or more fHbp immunogens selected from fHbp variant 1 (e.g. fHbp variant 1.1 and/or fHbp variant 1.13), fHbp variant 2 (e.g. fHbp variant 2.16) and/or fHbp variant 3 (e.g. fHbp variant 3.28) immunogens.

The binding assay according to the previous definition, wherein said meningococcal vaccine sample comprises two or more different fHbp immunogens selected from fHbp variant 1 (e.g. fHbp variant 1.1 and/or fHbp variant 1.13), fHbp variant 2 (e.g. fHbp variant 2.16) and/or fHbp variant 3 (e.g. fHbp variant 3.28) immunogens.

The binding assay of any one the three previous definitions, wherein said meningococcal vaccine sample comprises an fHbp immunogen and an fHbp fusion protein, wherein the fHbp fusion protein comprises or consists of two (or preferably three) different variants of the fHbp antigen including mutant forms thereof.

The binding assay according to the previous definition, wherein said fHbp fusion protein comprises or consists of a fHbp variant 2 immunogen (e.g. consisting of amino acid sequence SEQ ID NO: 5), a fHbp variant 3 immunogen (e.g. consisting of amino acid sequence SEQ ID NO: 6), and a fHbp mutated variant 1.13 immunogens (e.g. consisting of amino acid sequence SEQ ID NO: 4),

The binding assay of any one the two previous definitions, wherein said fHbp fusion protein comprises or consists of the amino acid sequence SEQ ID NO: 7 or SEQ ID 23.

The binding assay of any one the six previous definitions, wherein the meningococcal vaccine sample comprises an NHBA immunogen, a NadA immunogen and at least two fHbp immunogens (e.g. an fHbp variant 1 immunogen and an fHbp fusion protein comprising or consisting of an fHbp variant 2 immunogen, an fHbp variant 3immunogen and an fHbp variant limmunogen).

The binding assay of any one of the definitions above wherein the meningococcal vaccine sample comprises the meningococcal immunogens of SEQ ID NOs 1-3 and 7.

The binding assay of any one of the definitions above wherein said meningococcal vaccine sample comprises an aluminium hydroxide adjuvant.

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

The binding assay according to the previous definition, wherein said blocking buffer comprises casein or derivatives or fragments thereof.

The binding assay according to the previous definition, wherein said blocking buffer is at a final concentration of about 1,5%.

The binding assay of any one of the definitions above wherein at least one of said anti-immunogen-specific monoclonal antibodies is a murine monoclonal IgG antibody, in particular IgGl or IgG2.

The binding assay of any one of the definitions above, wherein at least one anti- immunogen-specific monoclonal antibodies is selected from a monoclonal antibody as defined below.

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

A method for in vitro relative potency analysis of a test meningococcal vaccine sample, comprising steps of:

(iii) performing steps (i) and (ii) of the binding assay of any one of the definitions above on the test meningococcal vaccine sample and, optionally, on at least one dilution of the test meningococcal vaccine sample;

(iv) performing steps (i) and (ii) of the binding assay of any one of the definitions above on a standard meningococcal vaccine sample of known in vivo potency and, optionally, on at least one dilution of the standard meningococcal vaccine sample; and

(v) comparing the results from steps (i) and (ii) obtained in (iii) and (iv) to determine the potency of immunogen(s) in the test meningococcal vaccine sample relative to the potency of immunogen(s) in the standard meningococcal vaccine sample.

A method for analysing a batch of vaccine, comprising steps of:

(1) assaying the relative potency of immunogen(s) in at least one meningococcal vaccine sample from the batch by the method according to the previous definition and, if the results of step (1) indicate an acceptable relative potency, (2) releasing vaccine from said batch for in vivo use.

A kit for in vitro multiplex assay of a meningococcal vaccine sample comprising (i) a plurality of solution-phase anti-immunogen specific monoclonal antibodies each of which is capable of binding to a conformational epitope of one of a meningococcal immunogen in the vaccine of interest (ii) a plurality of colour-coded beads, each conjugated to an antigen selectively bound by one of said anti-immunogen-specific monoclonal antibodies, said beads having a different colour-code for each different antigen, and (iii) a labelled antibody which binds to said anti-immunogen specific- monoclonal antibodies, wherein at least two of said meningococcal immunogens are selected from NHBA, NadA and/or fHbp immunogens.

The kit as defined above comprising two or more anti-immunogen specific monoclonal antibodies capable of binding to different fHbp variants selected from fHbp variant 1 (e.g. fHbp variant 1.1 and/or fHbp variant 1.13), fHbp variant 2 (e.g. fHbp variant 2.6) and/or fHbp variant 3 (e.g. fHbp variant 3.28).

The kit of any of the definitions above wherein at least one of said anti-immunogen specific monoclonal antibodies is capable of binding to a conformational epitope of an fHbp immunogen comprised in a fusion protein, wherein said fusion protein comprises or consists of a vl.13 fHbp mutated variant consisting of amino acid sequence SEQ ID NO: 4, a v2 mutated variant consisting of amino acid sequence SEQ ID NO: 5 and a v3 mutated sequence consisting of amino acid sequence SEQ ID NO: 6.

The kit according to the previous definition wherein said fusion protein comprises or consists of the amino acid sequence SEQ ID NO: 7 or SEQ ID 23.

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

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

-CDRL1 having SEQ ID NO:35,

-CDRL2 having SEQ ID: 103,

-CDRL3 having SEQID NO: 36,

-CDRH1 having SEQ ID NO:39, -CDRH2 having SEQ ID NO:40, -CDRH3 having SEQ ID NO:41.

A monoclonal antibody which is capable of binding to the meningococcal antigen NHBA, whose light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 34 and whose heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 38. A monoclonal antibody which is capable of binding to an epitope of meningococcal antigen NHBA comprising or consisting in the amino acid sequence of SEQ ID NO: 42, preferably of SEQ ID NO 43, even more preferably of SEQ ID 44.

A monoclonal antibody capable of differentiating between native and denatured forms of meningococcal 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 SEQ ID NO: 38 (VH) and SEQ ID NO: 34 (VL) respectively.

A monoclonal antibody which is capable of binding to meningococcal antigen NHBA and competes or cross-competes with and/or binds the same epitope of the monoclonal antibody according to any one of the four definitions above.

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

-CDRL1 having SEQ ID NO:47,

-CDRL2 having SEQ ID NO: 104,

-CDRL3 having SEQ ID NO:48,

-CDRH1 having SEQ ID NO:51,

-CDRH2 having SEQ ID NO: 52,

-CDRH3 having SEQ ID NO:53

A monoclonal antibody which is capable of binding the meningococcal antigen NadA, whose light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 46 and whose heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 50.

A monoclonal antibody which is capable of binding to 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:9.

A monoclonal antibody capable of differentiating between the native and denatured forms of meningococcal 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: 50 (VH) and SEQ ID NO: 46 (VL).

A monoclonal antibody which binds to meningococcal antigen NadA and competes or cross-competes with and/or binds the same epitope of the monoclonal antibody according to according to any one of the four definitions above. A monoclonal antibody which is capable of binding to meningococcal antigen fHbp variant 3, in particular fHbpv 3.28 variant (SEQ ID 64 or SEQ ID NO: 10 or SEQ ID NO: 6), whose VH and VL comprise the following complementarity-determining regions (CDRs):

-CDRL1 having SEQ ID NO:56,

-CDRL2 having SEQ ID NO:57

-CDRL3 having SEQ ID NO: 58,

-CDRH1 having SEQ ID NO:61,

-CDRH2 having SEQ ID NO: 62,

-CDRH3 having SEQ ID NO: 63.

A monoclonal antibody which is capable of binding to the meningococcal fHbp variant 3, in particular fHbp variant 3.28 variant whose light chain variable domain (VL) has the amino acid sequence of SEQ IDNO: 55 and whose heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 60.

A monoclonal antibody which is capable of binding to an epitope in the region of meningococcal fHbp variant 3, (in particular fHbp variant 3.28) antigen, the epitope comprising NRH amino acid residues in positions 169, 171 and 173 of fHbp variant 3.28 of SEQ ID NO 64 or SEQ ID NO: 10 or the corresponding amino acids in SEQ ID NO: 6.

A monoclonal antibody capable of differentiating between the native and denatured forms of meningococcal antigen fHbp variant 3 (in particular fHbp variant 3.28), 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: 60 (VH) and SEQ ID NO: 55 (VL).

A monoclonal antibody which is capable of binding to meningococcal antigen fHbp variant 3 (in particular fHbp variant 3.28) and competes or cross-competes with and/or binds the same epitope of the monoclonal antibody according to according to any one of the four definitions above.

A monoclonal antibody which is capable of binding to meningococcal antigen fHbp variant 1.13, whose VH and VL comprise the following complementaritydetermining regions (CDRs):

- CDRL1 having SEQ ID NO: 67,

-CDRL2 having SEQ ID NO: 68,

-CDRL3 having SEQ ID NO: 69, -CDRH1 having SEQ ID NO:71,

-CDRH2 having SEQ ID NO: 72,

-CDRH3 having SEQ ID NO: 73.

A monoclonal antibody which is capable of binding to the meningococcal antigen fHbp variant 1.13, whose light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 66 and whose heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 70.

A monoclonal antibody which is capable of binding to the surface of MenB strain bacteria carrying the meningococcal antigen fHbp variant 1.13 and killing them in a serum bactericidal assay, and capable of binding to an epitope of the meningococcal antigen fHbp 1.13 within a 2-3-1.13 NB fusion protein comprising or consisting of SEQ ID NO 7 or 23.

A monoclonal antibody which is capable of binding to an epitope in the region of meningococcal fHbp variant 1.13 antigen, the epitope comprising at least EH amino acid residues in position 234 and 240 respectively of fHbp variant 3.28 of SEQ ID NO 4 or SEQ ID NO: 102, preferably EAH amino acid residues in positions position 234, 236 and 240 respectively of fHbp variant 3.28 of SEQ ID NO 4 or SEQ ID NO: 102.

A monoclonal antibody capable of differentiating between the native and denatured forms of the meningococcal antigen fHbp variant 1.13 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: 70 (VH) and SEQ ID NO: 66 (VL).

A monoclonal antibody which is capable of binding to meningococcal antigen fHbp variant 1.13 and competes or cross-competes with and/or binds the same epitope of the monoclonal antibody according to according to any one of the five definitions above.

A monoclonal antibody which is capable of binding to meningococcal antigen fHbp variant 1.13, whose VH and VL comprise the following complementaritydetermining regions (CDRs):

- CDRL1 having SEQ ID NO:75,

-CDRL2 having SEQ ID NO: 76

-CDRL3 having SEQ ID NO:77.

-CDRH1 having SEQ ID NO:79, -CDRH2 having SEQ ID NO: 80,

-CDRH3 having SEQ ID NO:81.

A monoclonal antibody which is capable of binding to meningococcal antigen fHbp variant 1.13, whose light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 74 and whose heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 78.

A monoclonal antibody which is capable of binding to the surface of MenB strain bacteria carrying the meningococcal antigen fHbp variant 1.13 and killing them in a serum bactericidal assay, and capable of binding to an epitope of the meningococcal antigen fHbp variant 1.13 within a 2-3-1.13 NB fusion protein comprising or consisting of SEQ ID NO 7 or 23.

A monoclonal antibody capable to differentiate between the native and the denatured form of the meningococcal antigen fHbp variant 1.13 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: 78 (VH) and SEQ ID NO: 74 (VL).

A monoclonal antibody which is capable of binding to a meningococcal antigen fHbp variant 1.13 and competes or cross-competes with and/or binds the same epitope of the monoclonal antibody according to according to any one of the four definitions above.

A monoclonal antibody which is capable of binding to meningococcal antigen fHbp variant 1.13, whose VH and VL comprise the following complementaritydetermining regions (CDRs):

- CDRL1 having SEQ ID NO: 83,

-CDRL2 having SEQ ID NO: 84

-CDRL3 having SEQ ID NO:85,

-CDRH1 having SEQ ID NO:87,

-CDRH2 having SEQ ID NO:88,

-CDRH3 having SEQ ID NO:89.

A monoclonal antibody which is capable of binding to the meningococcal antigen fHbp vl.13, whose light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 82 and whose heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 86. A monoclonal antibody which is capable of binding to the surface of a MenB strain bacteria carrying the of the meningococcal antigen fHbp variant 1.13 and killing them in a serum bactericidal assay, and capable of binding to an epitope of the meningococcal antigen fHbp variant 1.13 within a 2-3-1.13 NB fusion protein comprising or consisting of SEQ ID NO 7 or 23.

. A monoclonal antibody capable of differentiating between the native and denatured forms of meningococcal antigen fHbp variant 1.13 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: 86 (VH) and SEQ ID NO: 82 (VL).

. A monoclonal antibody which is capable of binding to meningococcal antigen fHbp variant 1.13 and competes or cross-competes with and/or binds the same epitope of the monoclonal antibody according to according to any one of the four definitions above.

Embodiments of the invention are further described in the subsequent numbered paragraphs:

1. A binding assay for an in vitro analysis of a meningococcal vaccine sample including more than one meningococcal immunogen, comprising the steps of:

(i) allowing the interaction of a plurality of meningococcal immunogens within the sample with a plurality of anti-meningococcal immunogen-specific monoclonal antibodies which are bactericidal for meningococcus and/or are capable of binding to a conformational epitope of a meningococcal protein immunogen in said meningococcal vaccine;

(ii) measuring the interaction between each of said meningococcal immunogens and its corresponding anti-immunogen-specific monoclonal antibody with an immunoassay wherein each unbound anti- meningococcal immunogen-specific monoclonal antibody is allowed to interact with a plurality of colour-coded beads conjugated with an antigen selectively bound by said anti-immunogen-specific monoclonal antibody, said beads having a different colour-code for each different antigen.

2. The binding assay according to paragraph 1 wherein said measuring in (ii) provides the amount of each anti-meningococcal immunogen-specific monoclonal antibody’s target epitope within said meningococcal vaccine sample.

3. The binding assay according to any one of paragraphs 1 or 2 wherein said each of said colour-coded beads is internally dyed with different proportions of red and infrared fluorophores that correspond to a distinct spectral signature. 4. The binding assay of any one of paragraphs 1 to 3 wherein said measuring in (ii) comprises the steps of

(a) separating the unbound anti-immunogen-specific monoclonal antibodies from the immunogen bound anti-immunogen-specific monoclonal antibodies;

(b) contacting said unbound anti-immunogen-specific monoclonal antibodies with said plurality of colour-coded beads conjugated with the corresponding antigen of said anti-immunogen-specific monoclonal antibody wherein said beads have a different colour-code for each different antigen, and incubating the resulting mixture at a suitable temperature for a period of time to allow the binding of said plurality of colour-coded beads conjugated antigens with the respective anti-immunogen-specific monoclonal antibody to form bead-antigen-monoclonal antibody complexes;

(c) incubating the resulting bead-antigen-monoclonal antibody complexes with a labelled secondary antibody thereby forming complexes comprising also the secondary antibody

(d) carrying out a dual detection on the complexes obtained in step (c) thereby quantifying each bead-antigen-monoclonal antibody complex formed.

5. The binding assay of the paragraph 4, wherein said separation in step (a) is carried out by centrifugation.

6. The binding assay of any one of paragraphs 1 to 5, wherein said interaction (i) and/or said measuring (ii) takes place in microwell plates.

7. The binding assay of any one of paragraphs 1 to 6 wherein said meningococcal vaccine sample comprises at least two meningococcal immunogens, selected from NHBA, NadA and/or fHbp immunogens

8. The binding assay of paragraph 7 wherein said meningococcal vaccine sample comprises one or more fHbp immunogens selected from fHbp variant 1 (e.g. fHbp variant 1.1 and/or fHbp variant 1.13), fHbp variant 2 (e.g. fHbp variant 2.16) and/or fHbp variant 3 (e.g. fHbp variant 3.28) immunogens.

9. The binding assay of paragraph 8 wherein said meningococcal vaccine sample comprises two or more different fHbp immunogens selected from fHbp variant 1 (e.g. fHbp variant 1.1 and/or fHbp variant 1.13), fHbp variant 2 (e.g. fHbp variant 2.16) and/or fHbp variant 3 (e.g. fHbp variant 3.28) immunogens.

10. The binding assay of any one of paragraphs 7 to 9 wherein said meningococcal vaccine sample comprises an fHbp immunogen and an fHbp fusion protein, wherein the fHbp fusion protein comprises or consists of two (or preferably three) different variants of the fHbp antigen including mutant forms thereof.

11. The binding assay of paragraph 10 wherein said fHbp fusion protein comprises or consists of a fHbp variant 2 immunogen (e.g. consisting of ammo acid sequence SEQ ID NO: 5), a fHbp variant 3 immunogen (e.g. consisting of amino acid sequence SEQ ID NO: 6), and a fHbp mutated variant 1.13 immunogen (e.g. consisting of amino acid sequence SEQ ID NO: 4),

11. The binding assay of any one of paragraphs 10 or 11 wherein said fHbp fusion protein comprises or consists of the amino acid sequence SEQ ID NO: 7 or SEQ ID 23.

12. The binding assay of any one of paragraphs 7 to 11 wherein the meningococcal vaccine sample comprises an NHBA immunogen, a NadA immunogen and at least two fHbp immunogens (e.g. an fHbp variant 1 immunogen and an fHbp fusion protein comprising or consisting of an fHbp variant 2 immunogen, an fHbp variant 3immunogen and an fHbp variant limmunogen).

13. The binding assay of any one of paragraphs 1 to 12 wherein the meningococcal vaccine sample comprises the meningococcal immunogens of SEQ ID NOs 1-3 and 7.

14. The binding assay of any one of paragraphs 1 to 13 wherein said meningococcal vaccine sample comprises an aluminium hydroxide adjuvant.

15. The binding assay of any one of paragraphs 1 to 14 wherein said interaction in step (i) is carried out in a medium comprising a blocking buffer.

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

17. The binding assay of paragraph 16 wherein said blocking buffer is at a final concentration of about 1,5%.

18. The binding assay of any one of paragraphs 1 to 17 wherein at least one of said anti-immunogen-specific monoclonal antibodies is a murine monoclonal IgG antibody, in particular IgGl or IgG2.

19. The binding assay of any one of paragraphs 1 to 18, wherein at least one anti- immunogen-specific monoclonal antibodies is selected from a monoclonal antibody as defined in any one of paragraphs 28 to 57.

20. A method for detecting or measuring a change in conformation of a meningococcal immunogen in a meningococcal vaccine sample, comprising steps of: (i) performing the binding assay according of any one of paragraphs 1 to 19 on a test meningococcal vaccine sample and, optionally, on at least one dilution of the test meningococcal vaccine sample; (ii) performing the binding assay according to any one of paragraphs 1 to 19 on a standard meningococcal vaccine sample of known native antigenic form and, optionally, on at least one dilution of the standard meningococcal vaccine sample; and (in) comparing the results from steps (i) and (11) to determine the amount of immunogen(s) in the native form of the test meningococcal vaccine sample relative to the amount of immunogen(s) in the native form in the standard meningococcal vaccine sample.

21. A method for in vitro relative potency analysis of a test meningococcal vaccine sample, comprising steps of:

(iii) performing steps (i) and (ii) of the binding assay of any one of paragraphs 1 to 19 on the test meningococcal vaccine sample and, optionally, on at least one dilution of the test meningococcal vaccine sample;

(iv) performing steps (i) and (ii) of the binding assay of any one of paragraphs 1 to 19 on a standard meningococcal vaccine sample of known in vivo potency and, optionally, on at least one dilution of the standard meningococcal vaccine sample; and

(v) comparing the results from steps (i) and (ii) obtained in (iii) and (iv) to determine the potency of immunogen(s) in the test meningococcal vaccine sample relative to the potency of immunogen(s) in the standard meningococcal vaccine sample.

22. A method for analysing a batch of vaccine, comprising steps of:

(1) assaying the relative potency of immunogen(s) in at least one meningococcal vaccine sample from the batch by the method of paragraph 21 and, if the results of step (1) indicate an acceptable relative potency, (2) releasing vaccine from said batch for in vivo use.

23. A kit for in vitro multiplex assay of a meningococcal vaccine sample comprising (i) a plurality of solution-phase anti-immunogen specific monoclonal antibodies each of which is capable of binding to a conformational epitope of one of a meningococcal immunogen in the vaccine of interest (ii) a plurality of colour- coded beads, each conjugated to an antigen selectively bound by one of said anti- immunogen-specific monoclonal antibodies, said beads having a different colourcode for each different antigen, and (iii) a labelled antibody which binds to said anti-immunogen specific-monoclonal antibodies, wherein at least two of said meningococcal immunogens are selected from NHBA, NadA and/or fHbp immunogens.

24. The kit of paragraph 21 comprising two or more anti -immunogen specific monoclonal antibodies capable of binding to different fHbp variants selected from fHbp variant 1 (e.g. fHbp variant 1.1 and/or fHbp variant 1.13), fHbp variant 2 (e.g. fHbp variant 2.6) and/or fHbp variant 3 (e.g. fHbp variant 3.28).

25. The kit of paragraph 23 or 24 wherein at least one of said anti -immunogen specific monoclonal antibodies is capable of binding to a conformational epitope of an fHbp immunogen comprised in a fusion protein, wherein said fusion protein comprises or consists of a vl.13 fHbp mutated variant consisting of amino acid sequence SEQ ID NO: 4, a v2 mutated variant consisting of amino acid sequence SEQ ID NO: 5 and a v3 mutated sequence consisting of amino acid sequence SEQ ID NO: 6.

26. The kit of paragraph 25 wherein said fusion protein comprises or consists of the amino acid sequence SEQ ID NO: 7 or SEQ ID 23.

27. A vaccine which has been released following the use of a binding assay of any one of paragraphs 1 to 19 or a method according to anyone of paragraphs 20 to 22.

28. A monoclonal antibody which is capable of binding to the meningococcal antigen NHBA, whose VH and VL comprise the following complementaritydetermining regions (CDRs):

-CDRL1 having SEQ ID NO:35,

-CDRL2 having SEQ ID: 103,

-CDRL3 having SEQID NO:36,

-CDRH1 having SEQ ID NO:39,

-CDRH2 having SEQ ID NO:40,

-CDRH3 having SEQ ID NO:41.

29. A monoclonal antibody which is capable of binding to the meningococcal antigen NHBA, whose light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 34 and whose heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 38.

30. A monoclonal antibody which is capable of binding to an epitope of meningococcal antigen NHBA comprising or consisting in the amino acid sequence of SEQ ID NO: 42, preferably of SEQ ID NO 43, even more preferably of SEQ ID 44.

31. A monoclonal antibody capable of differentiating between native and denatured forms of meningococcal 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 SEQ ID NO: 38 (VH) and SEQ ID NO: 34 (VL) respectively. 32. A monoclonal antibody which is capable of binding to meningococcal 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 28 to 31

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

-CDRL1 having SEQ ID NO:47,

-CDRL2 having SEQ ID NO: 104,

-CDRL3 having SEQ ID NO:48,

-CDRH1 having SEQ ID NO:51,

-CDRH2 having SEQ ID NO: 52,

-CDRH3 having SEQ ID NO:53.

34. A monoclonal antibody which is capable of binding the meningococcal antigen NadA, whose light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 46 and whose heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 50.

35. A monoclonal antibody which is capable of binding to 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: 9.

36. A monoclonal antibody capable of differentiating between the native and denatured forms of meningococcal 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: 50 (VH) and SEQ ID NO: 46 (VL).

37. A monoclonal antibody which binds to meningococcal 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 33 to 36

38. A monoclonal antibody which is capable of binding to meningococcal antigen fHbp variant 3, in particular fHbpv 3.28 variant (SEQ ID NO: 64 or SEQ ID NO: 10), whose VH and VL comprise the following complementarity-determining regions (CDRs):

-CDRL1 having SEQ ID NO:56,

-CDRL2 having SEQ ID NO:57 -CDRL3 having SEQ ID NO: 58,

-CDRH1 having SEQ ID NO:61,

-CDRH2 having SEQ ID NO: 62,

-CDRH3 having SEQ ID NO: 63.

39. A monoclonal antibody which is capable of binding to the meningococcal fHbp variant 3, in particular fHbp variant 3.28 variant whose light chain variable domain (VL) has the amino acid sequence of SEQ IDNO: 55 and whose heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 60.

40. A monoclonal antibody which is capable of binding to an epitope in the region of meningococcal fHbp variant 3, (in particular fHbp variant 3.28) antigen, the epitope comprising NRH amino acid residues in positions 169, 171 and 173 respectively, of fHbp variant 3.28 of SEQ ID NO 64 or SEQ ID NO: 10 or the corresponding amino acids of SEQ ID NO: 6.

41. A monoclonal antibody capable of differentiating between the native and denatured forms of meningococcal antigen fHbp variant 3 (in particular fHbp variant 3.28), 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: 60 (VH) and SEQ ID NO: 55 (VL).

42. A monoclonal antibody which is capable of binding to meningococcal antigen fHbp variant 3 (in particular fHbp variant 3.28) and competes or cross-competes with and/or binds the same epitope of the monoclonal antibody according to any one of the paragraphs from38 to 41

43. A monoclonal antibody which is capable of binding to meningococcal antigen fHbp variant 1.13, whose VH and VL comprise the following complementaritydetermining regions (CDRs):

- CDRL1 having SEQ ID NO: 67,

-CDRL2 having SEQ ID NO: 68,

-CDRL3 having SEQ ID NO: 69,

-CDRH1 having SEQ ID NO:71,

-CDRH2 having SEQ ID NO: 72,

-CDRH3 having SEQ ID NO: 73.

44. A monoclonal antibody which is capable of binding to the meningococcal antigen fHbp variant 1.13, whose light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 66 and whose heavy chain vanable domain (VH) has the amino acid sequence of SEQ ID NO: 70.

45. A monoclonal antibody which is capable of binding to the surface of MenB strain bacteria carrying the meningococcal antigen fHbp variant 1.13 and killing them in a serum bactericidal assay, and capable of binding to an epitope of the meningococcal antigen fHbp 1.13 within a 2-3-1.13 NB fusion protein comprising or consisting of SEQ ID NO 7 or 23.

46. A monoclonal antibody which is capable of binding to an epitope in the region of meningococcal fHbp variant 1.13 antigen, the epitope comprising at least EH amino acid residues in position 234 and 240 respectively of fHbp variant 3.28 of SEQ ID NO 4 or SEQ ID NO: 102, preferably EAH amino acid residues in positions position 234, 236 and 240 respectively of fHbp variant 3.28 of SEQ ID NO 4 or SEQ ID NO: 102.

47. A monoclonal antibody capable of differentiating between the native and denatured forms of the meningococcal antigen fHbp variant 1.13 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: 70 (VH) and SEQ ID NO: 66 (VL).

48. A monoclonal antibody which is capable of binding to meningococcal antigen fHbp variant 1.13 and competes or cross-competes with and/or binds the same epitope of the monoclonal antibody according to any one of the paragraphs from 43 to 47.

49. A monoclonal antibody which is capable of binding to meningococcal antigen fHbp variant 1.13, whose VH and VL comprise the following complementaritydetermining regions (CDRs):

- CDRL1 having SEQ ID NO:75,

-CDRL2 having SEQ ID NO: 76

-CDRL3 having SEQ ID NO:77.

-CDRH1 having SEQ ID NO:79,

-CDRH2 having SEQ ID NO: 80,

-CDRH3 having SEQ ID NO: 81.

50. A monoclonal antibody which is capable of binding to meningococcal antigen fHbp variant 1.13, whose light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 74 and whose heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 78.

51. A monoclonal antibody which is capable of binding to the surface of MenB strain bacteria carrying the meningococcal antigen fHbp variant 1.13 and killing them in a serum bactericidal assay, and capable of binding to an epitope of the meningococcal antigen fHbp variant 1.13 within a 2-3-1.13 NB fusion protein comprising or consisting of SEQ ID NO 7 or 23.

52. A monoclonal antibody capable to differentiate between the native and the denatured form of the meningococcal antigen fHbp variant 1.13 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: 78 (VH) and SEQ ID NO: 74 (VL).

53. A monoclonal antibody which is capable of binding to a meningococcal antigen fHbp variant 1.13 and competes or cross-competes with and/or binds the same epitope of the monoclonal antibody according to any one of the paragraphs from 49 to 52.

54. A monoclonal antibody which is capable of binding to meningococcal antigen fHbp variant 1.13, whose VH and VL comprise the following complementaritydetermining regions (CDRs):

- CDRL1 having SEQ ID NO: 83,

-CDRL2 having SEQ ID NO: 84

-CDRL3 having SEQ ID NO:85,

-CDRH1 having SEQ ID NO:87,

-CDRH2 having SEQ ID NO:88,

-CDRH3 having SEQ ID NO:89.

55. A monoclonal antibody which is capable of binding to the meningococcal antigen fHbp vl.13, whose light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 82 and whose heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 86.

56. A monoclonal antibody which is capable of binding to the surface of a MenB strain bacteria carrying the of the meningococcal antigen fHbp variant 1.13 and killing them in a serum bactericidal assay, and capable of binding to an epitope of the meningococcal antigen fHbp variant 1.13 within a 2-3-1.13 NB fusion protein comprising or consisting of SEQ ID NO 7 or 23. 57. A monoclonal antibody capable of differentiating between the native and denatured forms of meningococcal antigen fHbp variant 1.13 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: 86 (VH) and SEQ ID NO: 82 (VL).

58. A monoclonal antibody which is capable of binding to meningococcal antigen fHbp variant 1.13 and competes or cross-competes with and/or binds the same epitope of the monoclonal antibody according to any one of the paragraphs from 54 to 57.

EXAMPLES

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 antimouse 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 e 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 mAb 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 mAb 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-monoclonal 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 NHB A 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).

PeptideScanning 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 (in red in Figure 8).

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 mAh 10E8 binding epitope on NHBA-953 is sensitive to heat treatment suggesting a conformational nature of the epitope.

Anti-fHbp var3.28- monoclonal antibody 191

A mAh 191’ specific for fHbp var 3.28NB was as obtained by standard hybridoma method from the spleen cells of immunized mice with fHbp var 3.28 (SEQ ID 64 or SEQ ID NO: 10 or SEQ ID NO: 6) as IgGl subclass. The antibody was identified and characterized. mAb 191’ is an IgGl subclass antibody. From this antibody, a murine Anti-fHbp var3.28-monoclonal antibody of IgG2a subclass defined herein as 191 was obtained.

The VL and VH genes were sequenced from hybridoma cell line and gene fragments, optimized for mammalian expression, corresponding to VH and VL of mAb 191’ monoclonal antibody were synthetized by adding at the gene extremities the appropriate linker for single step cloning.

Synthetic DNA strings of VH and VL were cloned into PCR-amplified murine expression vectors (Invitrogen Thermo Fisher Scientific) additionally containing a human Ig gene signal peptide sequence and the murine IgG2a or IgK constant regions following manufacturer instructions.

Positive clones were selected based on resistance to ampicillin and clones sequence was further confirmed by Next-Generation Sequencing (NGS).

Transient production of recombinant antibodies in suspension Expi293F™ Cells (A14527 Thermo Fisher Scientific) was performed according to manufacturer protocol.

Protein G chromatography was used for antibody capture from cell culture supernatant and the integrity of the mAb was checked by analitycal gel filtration.

The capability of 191 IgG2a to induce complement mediated killing of N. meningitidis strains was assessed through the serum bactericidal activity assay in presence of baby rabbit serum (rSBA) or human serum (hSBA) as source of exogenous complement. The strains selected for the SBA analysis were the serogroup B M1239 carrying fHbp var 3.28 and the mutant 5/99 AAC3.28 (the nadA and nhba null mutant of 5/99 strain, EryR, KanR complemented with fHbp subvariant 3.28) (Brunelli B, et al, Vaccine. 2011 Jan 29;29(5): 1072-81). Moreover 191 IgG2a was tested with FACS analysis to verify the recognition and to evaluate the fHbp expression level on the bacterial surface of M1239 and 5/99 AAC3.28 strains. Functionality measured by SBA

Monoclonal antibody 191 IgG2a tested in SBA with baby rabbit serum as complement source against strain M1239 carrying fHbp var 3.28 showed negative titer. When 191 IgG2a was tested in rSBA against 5/99 AAC3.28, bactericidal activity (titers from 512 to 2048) was observed in three repeated experiments, whereas negative titer was obtained using human serum as source of complement. MAb 121 (Reference 2) was used as control and showed high rSBA titers (Table 4.3.2-1). rSBA titers obtained against the mutant 5/99 AAC3.28 (in bold in the Table 1) are reported in Figure 11 where the curves represent the percentage of bacterial survival at each sample dilution.

Table 1 : Bactericidal titers against M1239 and 5/99 AAC3.28. The concentration corresponding to the starting dilution 14 is 250pg/ml.

■tt : rabbit complement; HC ; human complement

FACS analysis

Monoclonal antibody 191 IgG2a tested in FACS analysis with M1239 and 5/99 AAC3.28 showed binding to the antigen exposed on the bacterial surface of both strains with a fluorescence intensity higher than mAb 121 used as control (Figure 12). The comparison of the signal on M1239 strain is comparable with the signal on mutant 5/99 AAC3.28 as the peaks overlap (Figure 13).

191 IgG2a is able to bind the surface of both strains carrying the variant 3.28 of fHbp and the expression level of the antigen is comparable between these two strains in FACS analysis. Nevertheless the functional activity of 191 IgG2a tested in SBA assay with rabbit complement on strain M1239 was negative, whereas the rSBA on strain 5/99 AAC3.28 showed positive bactericidal titer even if the percentage of bacterial survival remains about 25%-30% at the highest tested mAb concentration. Moreover 191 IgG2a doesn’t show killing on strain 5/99 AAC3.28 using human complement.

These results suggest a higher sensitivity of mutant 5/99 AAC3.28 compared to M1239 strain to the killing mediated by 191 IgG2a.

191 IgG2a is able to induce bactericidal killing in appropriate experimental conditions and that it recognizes an epitope with immunological relevance on the fHbp 3.28 vaccine antigen.

Identification of the epitope and specificity for fHbp Var3,28 The epitope recognized by mAb 191 (also fHbp Var3.28 herein) on fHbp Var3.28 was identified by X-ray crystallography of the fragment antigen binding (Fab) complex with fHbp Var3.28 mutant S43V-L137R. mAb 191 exclusively recognizes the fHbp 3.28 variant among the other variants present in the fusion protein of SEQ ID NO 7 or 23.

Murine mAb 191 recognizes, as target, a conformational epitope, which is shared with both fHbp Var3.28 domains (N-ter and C-ter). At least one of the amino acid residues asparagine, arginine or histidine in position 169, 171 and 173 respectively is key to define the specificity of the mAb for the fHbp Var3.28 (SEQ ID NO: 64 or SEQ ID NO: 10). SEQ ID NO: 10 comprises SEQ ID NO: 6. Amino acid 1 of SEQ ID NO: 6 corresponds to amino acid 12 of SEQ ID NO: 10.

The X-ray diffraction has proven to be a powerful technology to study the structural features of proteins and the details of interaction within protein and ligand, including mAb or Fab (Ref. 77-80).

The Fab/antigen complex was performed using the recombinant fHbp Var3.28 S43VL137R and the Fab V3/19I deriving from the mAb V3/19I.

The complex formed by fHbp Var3.28 S43V-L137R - Fab V3/19I at the concentration of 20 mg/mL in Tris-HCl 20 mM, NaCl 150 mM, pH 8.0, was mixed in a ratio 1 : 1 (v/v) with the buffers of 96 different solution conditions from commercially available crystallization kits which contain precipitants, additives used to reduce protein solubility and achieve supersaturation. Five different kits were used, corresponding to a total of 480 conditions of screening for the generation of the crystals. The X-ray diffraction experiments were held at 103 beamline of Diamond Light Source synchrotron (UK). 2. X-ray diffraction data were collected at 100K, at wavelength = 0.9763 A. 2000 frames were collected. Data were processed using Xia2 dials and were reduced using Scala within the CCP4 program suite.

The crystal structure of the fHbp-Var3/19I Fab complex reveals shape complementarity at the epitope-paratope interface. The X-ray crystal structure of the complex between fHbp and the Fab fragment of mAbVar3/19I was solved by molecular replacement at 2.9 A resolution and the resulting electron density maps allowed unambiguous model building.

Table 2 below reports the molecular interaction between key residues in the fHbp Var 3.28 of SEQ ID NO 64 or SEQ ID NO: 10 and Fab 191

VdW: Van tier Waals

The sequence alignment of fHbp Var3.28 (SEQ ID NO: 64 or SEQ ID NO: 10) with the Vari.13 and Var2.16 present in fusion fHbp 2-3-1.13 of SEQ ID NO 7 evidenced that among the 16 amino acid residues involved in the binding, only three of them are specific for the fHbp Var3.28. They are the asparagine, arginine and histidine residues in position 169, 171 and 173 respectively; they are all three involved in hydrogen bonds with the CDR H3 for the asparagine residue and LI for the arginine and histidine residues of SEQ ID NO 64 or SEQ ID NO: 10. At least one of these amino acid residues is therefore key to define the specificity of mAb 191 for the fHbp Var3.28.

Anti-fHbp varl.l3-monoclonal antibodies mAb 6, mAb 36 and mAb 44

The murine Anti-fHbp varl.13 (SEQ ID NO 4) monoclonal antibodies of IgG2a subclass defined herein as mAb 6, mAb 36 and mAb 44 were obtained.

A panel of murine mAbs derived from mice immunized with fHbp varl,13NB of SEQ ID NO 4 by isolating antigen specific memory B cells (MBC) was prepared.

The selected clones were retro-transcribed to recover paired VH and VL and expressed as recombinant murine IgG2a. Different qualitative features of the mAbs panel were investigated by several approaches, including biochemical characterization to determine specificity and affinity, flow cytometry to assess recognition of the antigen exposed on the surface of Neisseria meningitidis, bactericidal activity on a panel of selected meningococcal strains and their ability to discriminate degraded fHbp 2-3-1.13 NB fusion protein of SEQ ID 7 or 23 from integer form. Two mAbs with the desired characteristics were identified. These mAbs can discriminate between stressed and not stressed target resulting in promising candidate in terms of ability to detect structural changes in the epitope. Moreover, the selected mAbs are able to bind the surface of MenB strain carrying the variant 1.13 of fHbp and kill them in a serum bactericidal assay, this means that said antibodies should recognize conformational, functional and clinically relevant epitopes of the fHbp 1.13 NB antigen within the 2-3-1.13 NB fusion protein of SEQ ID 7 or 23. A third monoclonal is taken in exam for its ability to discriminate degraded fHbp protein from integer form even if its negative in rSBA assay. Said mAbs are suitable for the assays (including IVRP assay) described herein.

The selected mAbs are suitable for the assays of the invention also when the vaccine comprises a fusion protein as described herein, comprising fHbp Var3.28, Vari.13 and Var2.16 such as fusion protein fHbp 2-3-1.13 of SEQ ID NO 7 or 23.

The selected antibodies meet one or more of the following features:

Functionality: able to induce bactericidal activity.

Specificity: specific mAb a-fHbp var 1.13NB without cross interaction with fHbp var 1.1, 2.16NB or 3.28NB.

Stability indicator: able to discriminate degraded fHbp 2-3-1.13 NB fusion protein of SEQ ID NO 7 or 23from integer form.

Monoclonal antibodies specific for fHbp var 1.13NB were derived from mice immunized with fHbp varl, 13NB of SEQ ID NO 4, by isolating antigen specific memory B cells (MBC). Splenocytes were stained to identify variant 1.13NB- specific MBC, negatively selecting for cross-reactivity to variant 2.16, variant 3.28 and variant 1.1. The selected clones were retro-transcribed to recover paired VH and VL and expressed as recombinant murine IgG2a.

The ability of the mAbs to recognize the Ag expressed on the surface of the bacteria was tested by FACS and the bactericidal effect was evaluated by rSBA. Moreover, Surface Plasmon Resonance (SPR) was performed to confirm fHbpl, 13NB- specificity and evaluate the affinity for the single variant 1.13NB compared to the fusion protein.

Moreover, best candidate mAbs were additionally analysed in SPR assessing their capability to discriminate between not stressed and thermal stressed fHbp2-3- 1.13 NB (NB in the present description stands for Non Binding) fusion protein (- 70°C, +4°C 24 hours, +70°C 24 hours and +80°C 24 hours).

To identify fHbp 1.13 -specific mAbs, supernatant of each mono-clones was screened for specificity to variant 1.13NB by Gyros. The selected 20 clones were retro- transcribed to recover paired VH and VL and expressed as recombinant murine IgG2a for further investigations. Three antibodies were finally sub-selected: mAbs 6 and 44 specifically bind fHbp var 1.13NB of SEQ ID NO 4 (Gyros, SPR and

FACS analysis), show bactericidal activity and are able to discriminate between not stressed and thermal stressed fHbp2-3-l .13_NB fusion protein

Ab 36 specifically binds fHbp var 1.13NB of SEQ ID NO 4 (Gyros, SPR and FACS analysis) and can discriminate between not stressed and thermal stressed fHbp2-3- 1.13 NB fusion protein but the clone is negative in bactericidal assay.

Monoclonal antibodies specific for fHbp var 1.13NB were derived from mice immunized with fHbp var 1.13 mutated form (SEQ ID NO 4), by isolating antigen specific memory B cells.

Table 3 below summarize all steps followed to identified fHbp 1.13NB-specific mAbs.

Specificity and affinity analysis of recombinant mAbs by Biacore SPR analysis of supernatants containing recombinant mAbs to assess specificity and affinity for fHbpl.13 NB (SEP ID NO 4)

20 supernatants containing monoclonal antibodies (including mAbs 6, 36 and 44) against fHbpl.13 NB (SEQ ID NO 4) were analysed to test the specificity and affinity using fHbp single and fusion protein constructs.

Serum bactericidal activity of purified mAbs was measured on Neisseria meningitidis reference strains of fHbp var 1.1 (SEQ ID NO 5) or 1.13 (SEQ ID NO 4) by using rabbit sera complement as complement source (rSBA). fHbpl.13_NB-specific and purified mAbs were analysed to assess if mAbs were able to discriminate between not stressed and thermal stressed fHbp2-3-1.13_NB antigen by SPR analysis.

Samples were analysed by SDS-PAGE to confirm the integrity of the proteins and to check the presence of degraded forms.

To evaluate the ability of mAbs to recognize structural changes of fHbp2-3-1.13_NB antigen induced by thermal stress, SPR binding analysis was performed.

Each mAb (ligand) was captured at concentration of 20pg/ml on the surface of the CM5 sensor chip coated with polyclonal anti -mouse IgG Fc. Each sample was injected for 180 seconds on the surface of the CM5 sensor chip, at fixed concentration of 200 nM.

Dissociation was followed for 600 seconds. After each analysis cycle, the chip was regenerated injecting 10 mM glycine-HCl pH 1.7 (GE Healthcare, # BR-1008-38). In all experiments the running buffer used was HBS-EP+.

All experiments were performed using a Biacore T200 Instrument (GE Healthcare) and analysed with Biacore T200 Evaluation software 3.0 (GE Healthcare).

Based on SPR results obtained with fHbpl.13NB-specific and purified mAbs on thermal stressed fHbp2-3-1.13_NB antigen, SPR analysis of three mAbs (6, 36 and 44) was performed after purification to confirm specificity and affinity, using the five fHbp constructs reported in Table 4 below

Results:

Vari.13 -specific Memory B Cell sorting Varl. l3NB-specmc Memory B cells have been sorted based on positive signal on Alexa488 channel and negative signal on Alexa647 channel, respectively x and y axes, in order to excluded population of B cells crossreactive with the other variants because of specificity for epitopes shared between variants (Figure 14 is representative of the sorting strategy. For the details of each sorted sample refer to N65260-13).

A total of 1700 sorted B cells have been screened by Gyrolab to assess the presence of Ig in the supernatant: 104 wells presented IgG positive clones.

The 104 clones have been screened for their binding vs fHbp var 1.1; var 1.13NB, var 2.16 and var 3.28 to select the clones based on the specificity for fHbp varl,13NB and absence of cross reactivity to the other fHbp variants. In total 59 clones resulted var 1.13 positive. Of these one was also crossreactive to var 3.28 and 15 were also crossreactive to var 1.1. Overall, 43 clones out of 59 were highly var 1.13 -specific, with no crossreaction at all with the other fHbp variants. Screening results are summarized in the Figures.

PCR reaction was performed on all 43 clones for VH and VL paired sequence recovery by RT-PCR and sequencing.

20 clones (including mAbs 6. 36 and 44) were selected for specificity and affinity analysis for fHbpl, 13_NB (SEQ ID NO 4).

Results of SPR analyses are shown in Figure 15. For each sensorgram, a blank subtraction was performed, based on the corresponding captured mAb but with injections of buffer instead of antigen dilutions.

Other tested mAbs showed specificity for fHbpl,13_NB. mAb-fHbp variant 1.13 binding on live bacteria

Flow cytometry analysis was performed to assess recognition of the antigen exposed on the surface of Neisseria meningitidis strains with mAbs supernatant screened in the study. mAbs binding specifically to fHbp variant 1.13 and not to the close variant 1.1 were selected. Selected indicator stains for fHbp var 1.13 (DEI 1301) and fHbp var 1 (MC58) were used for this scope and results are reported in Figure 16.

Selectivity for non degraded form of the antigen.

SDS-PAGE analysis was performed to test four preparations of fHbp2-3-1.13_NB antigen received. Samples at -70°C and +4°C 24 hours gave a comparable pattern. At +70°C 24 hours thermal stress condition the protein degradation started, but there was even a good amount of intact protein. Instead, at +80°C 24 hours the protein was almost all degraded. Results of SPR analyses are shown below in Figure 17. For each sensorgram, a blank subtraction was performed, based on the corresponding captured mAb but with injections of buffer instead of sample preparations.

The sensorgrams are shown at individual scale for each mAb. Plot abscissae represent time in seconds [s], while ordinates represent resonance units [RU],

8 selected mAbs were tested, Table 5 below shows the Binding response measured for each mAb with not stressed and thermal stressed fHbp2-3-1.13_NB antigen

Regarding mAbs 6 and 36, samples at -70°C and +4°C 24 hours shown a comparable binding intensity. The binding response was reduced almost half at +70°C 24 hours and was almost abolished at +80°C 24 hours. Even if the binding response of mAb

33 decreased in line with increasing stress temperatures, sample stressed at +70°C shown a slower dissociation compared to not stressed samples that could be due to the protein unfolding and a more accessibility of the mAb binding site. With mAbs

34 and 54, stressed samples shown a higher or comparable binding intensity than samples at -70°C or +4°C 24 hours, while no difference of complex stability was observed. Finally, mAb 44 revealed a reduction of binding response by increasing stress temperature, even if the dissociation wasfast in all samples analysed.

Based on this SPR analysis, mAb 6, 36 and 44 could recognize structural changes induced by thermal stress.

SPR analysis to confirm specificity and affinity of purified mAbs 6, 36 and 44.

For each of mAb 6, 36 and 44, kinetic parameters (ka. kd and KD) and Rmax values measured with fHbp antigens.

SPR analysis confirmed specificity of mAbs 6, 36 and 44 for fHbpl.13 protein, indeed no binding response was detected with fHbp2.16_NB, fHbp3.28_NB and 936-741 antigens. KD values measured for purified mAbs were comparable to KD values obtained with supernatants. The results are reported in the Table 6 below.

The X-ray diffraction has proven to be a powerful technology to study the structural features of proteins and the details of interaction within protein and ligand, including mAh or Fab.

The Fab/antigen complex was performed using the recombinant fHbp Vari.13 wt (SEQ ID NO: 102) and the Fab clone 6 deriving from the mAb clone 6.

The complex formed by fHbp Vari.13 wt - Fab clone 6 at the concentration of 25 mg/mL in Tris-HCl 20 mM, NaCl 150 mM, pH 8.0, was mixed in a ratio 1 : 1 (v/v) with the buffers of 96 different solution conditions from commercially available crystallization kits which contain precipitants, additives used to reduce protein solubility and achieve supersaturation. Five different kits were used, corresponding to a total of 480 conditions of screening for the generation of the crystals. The X-ray diffraction experiments were held at European Syncothron Radiation Facility ID30A1 beamline (France). X-ray diffraction data were collected at 100K, at wavelength = 0.965 A. 2420 frames were collected. Data were processed using autoPROC.

The crystal structure of the fHbp-Varl.13 wt/clone 6 Fab complex reveals shape complementarity at the epitope-paratope interface. The X-ray crystal structure of the complex between fHbp and the Fab fragment of mAb clone 6 was solved by molecular replacement at 1.6 A resolution and the resulting electron density maps allowed unambiguous model building. The epitope recognized by mAb clone 6 (also fHbp Vari.13 wt herein) on fHbp Vari.13 wt was identified by X-ray crystallography of the fragment antigen binding (Fab) complex with fHbp Vari.13 wt.

Clone 6 exclusively recognizes the fHbp 1.13 variant among the other variants present in the fusion protein of SEQ ID NO 7 or 23.

Murine mAb clone 6 recognizes, as target, a conformational epitope, which is shared with both fHbp Vari.13 domains (N-ter and C-ter). At least two of the amino acid residues glutammate and histidine, in position 234 and 240 respectively are key to define the specificity of the mAb for the fHbp Var 1.13 wt and DM form (SEQ ID NO: 102 or SEQ ID NO:4).

Table below reports the molecular interaction between key residues in the fHbp Var 1.13 wt of SEQ ID NO: 102 and Fab clone 6 VdW: Van der Waals

The sequence alignment of fHbp Vari.13 (SEQ ID NO: 4 or SEQ ID NO: 102) with the Varl. l of SEQ ID NO: 5 evidenced that among the 11 amino acid residues, crucial for binding, only three of them are specific for both fHbp Vari.13 (WT and DM). They are the glutamate, alanine and histidine residues in position 234, 236 and 240 respectively (SEQ ID NO: 4 or SEQ ID NO: 102); while glutamate and alanine are involved in hydrogen bonds and Van der Waals interactions with the CDR H2, histidine engages Van der Waals interactions with leucine 104 of CDR H3. At least glutamate 234 and histidine 240 of SEQ ID NO 4 or SEQ ID NO: 102 represent the key to define the specificity of mAb clone 6 for the fHbp Var 1.13.

The IVRP assay Best mode to carry out the invention

Analytical Method Procedure

The aim of the assay is to measure the level of recognition of each antigen (: PorA, 961c, 287-953, 936-741, and the fusion fHbp231.13NB) by functional and conformational mAbs (i.e. able to recognize conformational epitopes and/or to induce complement mediated killing*), in comparison to a reference batch (to provide a relative output), to confirm content and integrity of epitopes expected to be relevant for immunogenicity.

In the IVRP assay, in each plate one test vaccine is tested against a reference vaccine. The two samples are pre-diluted and then put on the plate making eight serial dilution steps.

In Table 7 a summary of vaccine composition for Reference and Test Vaccine is reported.

Table 7: Summary of vaccine composition for Reference and Test Vaccine

FHbpl. l SEQ ID NO: 5 Fusion protein fHbp231.13 SEQ ID NO: 7 or 23

NHBA (287-953) SEQ ID NO: 8

Nad A (961c) SEQ ID NO: 9

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 has been assigned a value of 1 to the Reference Vaccine used as Standard in the assay.

The range of acceptable relative potency results has been defined between 0.50 to 2.00, and includes the specification range established for the product.

Operative instructions for beads conjugation

The coupling is executed by reacting 2.5 x 10 ⁶ bead/region with 40 pg of specific Drug Substance DS, except for the 287-953 for which 80 pg have to be used.

The concentration of each (DS) used in the coupling is lot dependent and has to be re-assessed every time a new lot is introduced.

Lumninex beads were used.

Below are reported the association between antigen (DS) and bead region: MagPlex beads were used and coupling was carried out following the manufacturer’s instructions.

• Bead#57 (Luminex, cat. n° MC10057) used to conjugated antigen NadA

• Bead#44 (Luminex, cat. n° MCI 0044) used to conjugated antigen fHbpvarl. l

• Bead#27 (Luminex, cat. n° MC 10027) used to conjugated antigen fHbp23L13

• Bead#46 (Luminex, cat. n° MCI 0046) used to conjugated antigen NHBA

• Bead#72 (Luminex, cat. n° MCI 0072) used to conjugated antigen OMV

The coupling procedure has been carried out according to the manufacturer’s instructions.

Distribute each conjugated-bead in four aliquots (low binding eppendorf) of 1 ml/each (stock concentration 625.000beads/mL);

Store the beads at 2-8°C protected from the light. The conjugated-beads are stable 30 days from conjugation date. Protocol for IVRP assay applied to Meningococcal Vaccine Composition

Although the multiplex technology allows to combine more analytes together in the same wells, some times it is not possible to achieve this result due to different assay conditions to apply for each analyte of interest in order to obtain a larger linear range in the dose-response curve. mAb 6 for Var 1.13 of fHbp231.13, is tested in dualplex with OMV.

For recombinant proteins (936-741, fHbp231.13, 287-953 and 961c) dispense in black square (Ref) and red square (Test) shown in 23 of 96 deep-well plate a triplicates of Reference and Test vaccine pre-diluted one to four assay buffer (lx PBS 1% Candor 0,05% Tween20). Titrate the Reference and Test Vaccine in order to execute 8 two-fold dilution. Dispense in the wells indicated in the black square of Figure 1, 300 pl/w of mAb mix (clone 6E3/39 anti-NadA, clone 12C1D7 anti- fHbpvl, clone 191 anti-fHbp231.13 and clone 10E8/A5 anti-NHBA). The working dilution of each monoclonal antibody is not reported because is depending from the batch in use.

For OMV and mAb 6 dispense in black square (Ref) and red square (Test) shown in 23 of 96 deep-well plate a triplicates of Reference and Test vaccine pre-diluted one to two in assay buffer (lx PBS 3% Candor 0,05% Tween20). Titrate the Reference and Test Vaccine in order to execute eight 1,5-fold dilution. Dispense in the wells indicated in the black square of Figure 1, 300 pl/w of mAb (clone PorA1.4 anti- OMV) The working dilution is not reported because is depending from the batch in use.

Incubate the two deep-well plate2 at 37°C for 30 minutes.

Centrifuge the deep-well plate for 20 minutes at 1000g.

Transfer 100 pl/w of supernatants from the deep-well to the multiplex plates respecting the same plate layout shown in figure 1.

For plate dedicated to the recombinant proteins (936-741, fHbp231.13, 287-953 and 961c) dispense 10 pl/w of Ag-conjugated beads mix (antigen NadA-bead57, antigen fHbpVl-bead44, antigen fHbp231-bead27 and antigen NHBA-bead46) diluted 1 to 5 in PBS 0.05% Tween20. The stock concentration of each conjugated-bead is 625.000beads/mL.

For plate dedicated to OMV and fHbp231.13 when mAb 6 is used dispense 10 pl/w of Ag-conjugated beads mix (antigen OMV-bead72 and antigen fHbp231-bead27) diluted 1 to 5 in PBS 0.05% Tween20. The stock concentration of each conjugated- bead is 625.000beads/mL.

Incubate the multiplex plates for 60 minutes at room temperature and using shaker plate.

Wash 2X the multiplex plate with IxPBS.

Dispense 50 pl/w of of R-Phycoerythrin affine pure F (Ab) 2 fragment goat antimouse IgG-PE (Li StarFish cat n° 115-116-072) diluted 1 : 100 in PBS.

Incubate the multiplex plates for 30 minutes at room temperature.

Wash 2X the multiplex plate with IxPBS.

Dispense 100 pl/w of lx PBS.

Shake plate at 150-160 rpm/minute for 10 minutes at room temperature and acquire sample by Luminex Analyzer.

Raw Data Analysis

The primary data obtained for Reference and Test Vaccine by Luminex software are represented from median fluorescence intensity (MFI).

The inhibition curves obtained for test vaccines are compared to the one obtained for the reference vaccine by means of a parallel line assay model (PLA).

The PLA is conducted by Combistats software using the transformation of the data into a natural logarithm selecting, between the eight possible dilutions, at least 4 consecutive dilutions in the linear range of dose-response curve. Initially, it was established that RP calculation should be performed selecting the same range of dilutions for Reference Vaccine and Test Vaccine.

In Table 8 are reported the system suitability criteria qualified during the assay development.

Table 8: System Suitability Criteria Data for evaluation

The parameters assessed for qualification of IVRP Meningococcal Vaccine Composition were:

• Accuracy

• Intermediate precision

• Linearity

• Sample Dilutability

• Range

Two different experimental designs were planned. The first one to evaluate the accuracy, intermediate precision and linearity and the second one for sample dilutability.

For IVRP it was decided to use as reference standard a Meningococcal Vaccine Composition with fHbp231.13NB at 50 pg/dose (100 pg/ml).

Qualification Design for Accuracy, Intermediate Precision and Linearity

The accuracy of a relative potency assay is the relationship between measured relative potency and known relative potency. Samples with target potencies are obtained by dilution of the standard material or a Test sample with known potency (potency levels). This type of study is often referred to as a dilutional linearity study (USP < 1033> Biological assay validation).

In order to do that, the relative accuracy (as relation between the measured versus the expected RP) has been evaluated using mock samples with broader levels of target RP from 0.50 to 2.00.

As shown in Table 9 to evaluate the accuracy and the intermediate precision, a Meningococcal Vaccine Composition was formulated from TRD-DP at lab-scale with a concentration 2x for all MenB components respect to the target concentration.

Table 9. Meningococcal Vaccine Composition (Low Dose 2x) used for accuracy and intermediate precision evaluation

As Reference vaccine (100%), the Meningococcal Vaccine Composition Low Dose 2X diluted 1 :2 in formulation buffer was used, with an assigned RP=1.

Three different accuracy levels were executed and tested: • 200% represented from Meningococcal Vaccine Composition Low Dose 2X as it is,

• 100% represented from Meningococcal Vaccine Composition Low Dose 2X diluted 1 :2 in formulation buffer

• 50% represented from Meningococcal Vaccine Composition Low Dose 2X diluted 1 :4 in formulation buffer

Reference (100%) and test vaccines (200%, 100% and 50%) were prepared independently for each analytical session. o Accuracy Result

For the accuracy evaluation, for each antigen Relative accuracy has been assessed at individual levels following the Equation 1 :

Equation 1: Relative Bias (RB)% formula

SB = 100 where GM is the geometric mean of the RPs.

The Acceptance criteria was setted as one-sided 95%CI of RB% [-20%; +25%].

The Min and Max One-sided 95% CI of RB% observed for each antigen satisfied the acceptance criteria and results are reported below :

• 287-953: [-2.39%; 5.77%]

• 936-741 : [-6.94%; 13.85%]

• 961c: [-4.14%; 14.66%]

• fHbp231: [-7.19%; 9.01%]

• OMV: [-11.45%; 4.46%] o Assay Precision Result

The assay precision has been evaluated in terms of intermediate precision and using the same data of the accuracy experiments.

The Intermediate Precision of the In (RP) for each antigen have been assessed following USP <1033> suggestions using a variance decomposition approach. The variance component analysis was performed to decompose the total variability into two components: variability due to the operator and residual variability. The Intermediate Precision has been calculated as percentage of geometric coefficient of variation (%GCV) of RP as described in the Equation 2.

Equation 2. Intermediate precision formula

The acceptance was set as GCV<15%.

The Range of GCV% at the three RP levels (0.50, 1.00 and 2.00) for each antigen satisfied the acceptance criteria and are reported below :

• 287-953: 2.7%-3.2%

• 936-741 : 3.5%-5.4%

• 961c: 2.9%-6.1%

• fHbp231.13NB: 3.2%-7.9%

• OMV: 1.5%-7.2% o Linearity Result

As described in USP <1033>, relative accuracy in bioassay refers to a unit slope (slope = 1) between log measured relative potency and log known relative potency. Testing slope equal to 1 in the log-log system corresponds also to testing linearity of the response, as shown below:

InfAPj,,) = ct + & * l:n(RP _r ) exponentiating = e ^a * PP^ where RP _M is the Measured Relative Potency and RP _T is the Theoretical Relative Potency.

The same set of data used for accuracy and intermediate precision assessment, was used for the evaluation of the linearity of the response. A linear regression analysis was performed for each antigen on ln(Relative Potency) vs ln(Expected Potency).

Although the linearity of response has been calculated only ad info the literature reports that the 95% Confidence Interval of the slopes of the fitted regression line between ln(RP measured) and ln(RP theoretical) is included in the range 0.8 - 1.2, deemed acceptable for this type of assay.

The linearity of the response for each antigen satisfied the acceptance criteria and are reported below :

• Min and Max two-sided 95% CI slope of ln(measured potency) vs ln(expected potency): [0.95; 1.20] • R-squared>0.99

Qualification Design for Sample Dilutability

As shown in Table , to evaluate the dilutability a Meningococcal Vaccine Composition was formulated from TRD-DP at lab-scale with a concentration 2x for all MenB components (936-741, 287-953, 961c and OMV) and 4x for fHbp231.13NB respect to the target concentration, named Meningococcal Vaccine Composition High Dose 2X.

Table 10. Meningococcal Vaccine Composition (High Dose 2X) used for dilutability evaluation

As Reference vaccine was used the Meningococcal Vaccine Composition formal stability SAP batch EMN2A001 A (DP composition shown in Table ).

Table 11. Meningococcal Vaccine Composition formal stability lot composition, used as Reference Vaccine

As test vaccine, four dilutability levels were executed:

• Meningococcal Vaccine Composition High Dose 2X as it is

• Meningococcal Vaccine Composition High Dose 2X diluted 1 :2 in assay buffer

• Meningococcal Vaccine Composition High Dose 2X diluted 1 :4 in assay buffer

• Meningococcal Vaccine Composition High Dose 2X diluted 1 :8 in assay buffer

Test vaccines (Meningococcal Vaccine Composition High Dose 2X diluted 1 :2, 1 :4 and 1 :8) were prepared independently in each analytical session. o Sample Dilutability Result Relative potency was calculated against the Formal Stability lot, considered as Reference Standard. Then, the RPs were multiplied by the corresponding dilution factor and the GCV% was calculated for each antigen using a variance component analysis with Analyst as random factor.

The acceptance was set as GCV<15%.

For each antigen the GCV% (see below) is well below the ATP requirement of 15% and comparable to the Intermediate Precision results. This experiment confirms the possibility to change the starting dilution if the sample tested is outside the qualified range

• 287-953: 1.8%

• 936-741 : 3.6%

• 961c: 8.3%

• fHbp231.13NB: 8.4%

• OMV: 8.3% o Range Result

The range of theoretical RP where accuracy and precision is proved is 0.50-2.00.

The range is established based on accuracy experiments. It is defined as the measured levels where the assay is demonstrated accurate and precise as required. In this specific case, the limits for the range correspond to the minimum and maximum RP observed at the extreme of the range 0.50- 2.00, for each antigen. Based on the accuracy experiments, the qualified range is:

Table 12: Qualified Range

Figure 21 shows a graphical representation of the results obtained.

Performance results

For the validity of the results, system and sample suitability criteria defined during method development were applied to each qualification session. They are summarized in 13. Table 13: Summary of acceptance criteria

The RP calculation was performed using Combistats software version 6.1

Data were analysed with JMP14.

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

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