Immunoassay involving antibodies binding vaccine adjuvants

Abstract

Provided herein are assays and methods of assessing a vaccine comprising a liposome, and antibodies and kits used in such assays and methods.

Description

[0001] IMMUNOASSAY INVOLVING ANTIBODIES BINDING VACCINE ADJUVANTS

[0002] Field of the invention

[0003] The invention relates to methods of assessing liposomal vaccines.

[0004] Background

[0005] Liposomal vaccines have received an increased interest over recent years. Liposomes are artificial vesicles, mostly made of (phospho)lipids, that may be used inter alia as vehicles for the presentation of antigens. Methods of preparing liposomes that display an antigen on their surface have been disclosed, for example, in WO2012 / 055933 or WO2012 / 020124.

[0006] Antigens presented via liposomes can induce humoral as well as cellular immune responses. By way of example, liposomal vaccines are disclosed in W02007 / 068411 , which pertains to a liposomal vaccine composition comprising (i) an amyloid-beta (Abeta)-derived peptide antigen displayed on the surface of the liposome, and (ii) an adjuvant, and in WO2019 / 197414, which pertains to a liposomal vaccine composition comprising (i) an amyloid-beta (Abeta)-derived peptide antigen displayed on the surface of the liposome, (ii) a peptide comprising a T-cell epitope, and (iii) an adjuvant. The vaccine compositions are disclosed for use in the treating, preventing, or alleviating the symptoms associated with an Abeta associated disease.

[0007] The properties of a liposomal vaccine, such as its immunogenicity or potency, is determined by numerous factors. For example, the immunogenicity of a vaccine comprising liposomes that display an antigenic peptide on the surface may depend not only on the amino acid sequence of the antigenic peptide, but also inter alia on the manner in which it is displayed on the liposomal surface.

[0008] The immunogenicity of a liposomal vaccine may be assessed via animal studies, for example by administering the vaccine to an animal and measuring the titers of the antigen-specific antibodies generated in response to such administration. However, animal studies have drawbacks, particularly in terms of time and costs. Predicting immune responses before vaccination remains challenging because of the complexity of the governing parameters. Accordingly, there remains a need for effective methods for assessing and / or predicting the antibody response or other properties of a liposomal vaccine.

[0009] Description

[0010] The inventors have now provided in vitro methods of assessing liposomal vaccines that have significant utility in vaccine development and production, inter alia in assessing and / or predicting the antibody response to a liposomal vaccine; predicting the in vivo immunogenicity of a liposomal vaccine; assessing the antigen presentation by a liposomal vaccine; assessing the heterogeneity of a liposomal vaccine; and the like. Thus, in one aspect, provided is a method of assessing a vaccine comprising a liposome that comprises both a vaccine adjuvant and an antigen displayed on the surface of the liposome, the method comprising the steps of

[0011] (a) contacting the vaccine with:

[0012] (i) a capture agent immobilised on a solid support, and

[0013] (ii) a detectable analyte-binding agent; wherein either the capture agent is specific for the vaccine adjuvant and the detectable analyte-binding agent is specific for the antigen, or the capture agent is specific for the antigen and the detectable analyte-binding agent is specific for the vaccine adjuvant;

[0014] (b) incubation under conditions allowing the formation of a complex between the capture agent, the liposome and the detectable analyte-binding agent; and

[0015] (c) detecting the complex by detecting the detectable analyte-binding agent.

[0016] Detecting the detectable analyte-binding agent may involve the mere determination whether the detectable analyte-binding agent (and hence a complex comprising the same) is present or not. However, in preferred embodiments of all aspects provided herein, detection includes measuring the intensity of a signal emitted or generated by a label linked to the detectable analyte-binding agent. As explained elsewhere herein, the label may be linked directly or indirectly to the detectable analyte-binding agent, and this linking may in some embodiments take place after step (b). Thus, in some embodiments of any of the methods provided herein, step (c) comprises measuring the intensity of a signal emitted or generated by a label linked to the detectable analyte-binding agent.

[0017] The signal strength / intensity may be compared to a reference and / or a control, or may be used to calculate the half maximal effective concentration (EC50) of the liposomal vaccine.

[0018] Thus, in some embodiments of any of the methods provided herein, step (c) comprises measuring the intensity of a signal emitted or generated by a label linked to the detectable analyte-binding agent. In such embodiments, the method may further comprise a step (d) of comparing the measured signal intensity to a reference and / or a control. The method may further comprise a step (e) of determining one or more properties of the liposomal vaccine on the basis of the result of step (c) and / or (d).

[0019] The signal intensity of a suitable control or reference may be known, or may have been predetermined, for example it may be a predetermined standard curve or a predetermined threshold. Alternatively, the method may comprise assessing at least two liposomal populations, such that one liposomal population may serve as a reference or control for the other.

[0020] The skilled person will be able to identify a suitable reference and / or a suitable control, but exemplary, non-exhaustive guidance is provided below, in which the liposomal vaccine that is assessed via the provided methods (i.e. the liposomal vaccine of interest) is referred to as the “test vaccine”.

[0021] It will be understood that by comparison to “a reference and / or a control” is meant that the signal intensity of the test vaccine may be compared to the signal intensity of a negative control, the signal intensity of a positive control, and / or the signal intensity of a reference.

[0022] Thus, the control may be a negative control or a positive control.

[0023] A negative control should be a liposomal population that is not capable of forming a complex with the capture agent and the detectable analyte-binding agent, and consequently not capable of generating a significant signal intensity. Any signal intensity generated by such a negative control may be considered to be background signal intensity. A negative control should typically lack the antigen and / or the vaccine adjuvant (i.e. lack the antigen and / or the vaccine adjuvant displayed on the surface of the liposome of the test vaccine). The negative control may thus, for example, be a liposome that

[0024] (a) does not display on its surface the vaccine adjuvant; and / or

[0025] (b) does not display on its surface the antigen.

[0026] For example, the liposome of the negative control may display on its surface the vaccine adjuvant, but lack the antigen. In another example, it may display on its surface the antigen, but lack the vaccine adjuvant. In another example, it may be a liposome lacking both the antigen and the vaccine adjuvant.

[0027] By a positive control is meant a liposomal vaccine that is known to form a complex with the capture agent and the detectable analyte-binding agent, and consequently to generate a significant signal intensity. Thus, the liposome of the positive control displays on its surface both the antigen and the vaccine adjuvant comprised by the test vaccine. A positive control may, for example, be a first batch of a vaccine that has been determined to generate a significant signal, and this may be used to assess other batches of the vaccine.

[0028] The term “reference” is used to mean any liposomal vaccine that may be used as a comparator and thus a “positive control” is a specific example of a “reference”, but the term “reference” also encompasses other embodiments, as explained below.

[0029] The reference should be a liposomal population that is capable of forming a complex with a suitable capture agent and a suitable detectable analyte-binding agent, and consequently be capable of generating a significant signal intensity. In many embodiments, for example where the reference comprises the same or a variant antigen compared to the test vaccine, the reference must be capable of forming a complex with the same capture agent and detectable analyte-binding agent as the test vaccine. In other embodiments, the reference may comprise a different antigen compared to the test vaccine and a suitable capture agent and a suitable detectable analyte-binding agent must be selected accordingly to allow complex formation of the reference liposome with these agents.

[0030] The reference should be a liposomal vaccine having known properties. In some embodiments, the reference may differ from the test vaccine by a feature of interest, but otherwise be similar or identical to the test vaccine. The feature of interest may, for example, be the antigen, for example the sequence or orientation thereof.

[0031] For example, the reference may display on its surface (instead of an antigen that is identical to the antigen of the test vaccine) a different antigen. By “different antigen” is meant that the antigen is significantly different to the antigen of the test vaccine, for example if the antigen is an antigenic peptide, the antigenic peptide of the reference may have an amino acid sequence that shares no more than 20, or 10% sequence identity, or may share no sequence identity, with the amino acid sequence of the antigenic peptide of the test vaccine.

[0032] A “different” antigen would typically not exhibit any significant binding to an antibody that is specific for the surface-displayed antigen of the test vaccine. In particular, the antigenspecific agent (which may be the capture agent or the detectable analyte-binding agent) may exhibit substantially no binding to a “different” antigen. By way of an illustrative example, if the antigen of the test vaccine is an Abeta- derived antigenic peptide, the “different” antigen may be an antigenic peptide derived from a protein that is unrelated to Abeta.

[0033] Alternatively, the reference may display on its surface (instead of an antigen that is identical to the antigen of the test vaccine) a variant antigen. By “variant antigen” is meant that the antigen is similar, but not identical, to the antigen of the test vaccine. A “variant” antigen would typically exhibit at least some binding to an antibody that is specific for the antigen of the test vaccine. Accordingly, the antigen-specific agent (which may be the capture agent or the detectable analyte-binding agent) may exhibit at least some binding to a “variant” antigen.

[0034] Thus, a variant antigen may, for example, comprise a variation in the antigenic moiety compared to the antigen of the test vaccine. In the case of an antigen that is an antigenic peptide, a variant may, for example, comprise up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid changes compared to the test vaccine. A variant antigen that differs in the antigenic moiety may be referred to as an “antigenic moiety variant”.

[0035] Alternatively, or in addition, a variant antigen may comprise a variation in a moiety that anchors it to the liposome. For example, it may differ in the type, location and / or number of hydrophobic moieties. A variant antigen that differs in the moiety that anchors it to the liposome may be referred to as an “anchoring moiety variant”. An anchoring moiety variant may impact on the presentation of the antigen on the liposomal surface. For example, it may impact on the orientation of the antigen, and / or its accessibility to an agent that is specific for the antigen.

[0036] In some embodiments, the variant antigen is an antigenic moiety variant only, i.e. it does not vary in the anchoring moiety. In some embodiments, the variant antigen is an anchoring moiety variant only, i.e. it does not vary in the antigenic moiety. In some embodiments, the variant antigen is an antigenic moiety variant and anchoring moiety variant.

[0037] In some embodiments, the reference or (positive) control is a liposomal vaccine having a known vaccine property, such as a known antibody response, a known in vivo immunogenicity and / or a known potency.

[0038] In some embodiments, the reference or (positive) control is a liposomal vaccine that is not known to differ from the test vaccine. For example, the reference or (positive) control may have been prepared using the same components as the test vaccine, for example be a different batch of the same vaccine. Thus, in some embodiments the method may be used to compare different batches of the same vaccine.

[0039] The method is typically carried out using a liposomal vaccine composition at a selected concentration (which is typically determined and expressed as the concentration of a surface-displayed antigenic peptide in the composition, using the molecule weight of the full antigen, which may for example be the molecule weight of the antigenic peptide plus its anchoring moiety). The reference or control should typically be used at an equivalent concentration or equivalent dilution.

[0040] In some embodiments, the method is carried out using a serial dilution of the liposomal vaccine. Thus, the method may be carried out using two or more different concentrations of the liposomal vaccine. The signal measured from each different concentration may be compared and may, for example, be used to calculate the EC50.

[0041] The method of assessing a vaccine advantageously allows an assessment of the surface presentation of an antigen. Thus, any of the methods provided herein may comprise a step of assessing the surface presentation of an antigen. For example, step (e) may comprise assessing or determining the surface presentation of an antigen.

[0042] Thus, also provided is a method of assessing antigen presentation of a vaccine comprising a liposome that comprises both a vaccine adjuvant and an antigen displayed on the surface of the liposome, the method comprising the steps of

[0043] (a) contacting the vaccine with:

[0044] (i) a capture agent immobilised on a solid support, and

[0045] (ii) a detectable analyte-binding agent; wherein either the capture agent is specific for the vaccine adjuvant and the detectable analyte-binding agent is specific for the antigen, or the capture agent is specific for the antigen and the detectable analyte-binding agent is specific for the vaccine adjuvant;

[0046] (b) incubation under conditions allowing the liposome to form a complex with the capture agent and the detectable analyte-binding agent; and

[0047] (c) detecting the complex by detecting the detectable analyte-binding agent; wherein step (c) preferably comprises measuring the intensity of a signal emitted or generated by a label linked (directly or indirectly) to the detectable analyte-binding agent.

[0048] Preferably, the method further comprises

[0049] (d) comparing the measured signal intensity to a reference and / or a control; and / or

[0050] (e) assessing or determining the surface presentation of the antigen of the vaccine on the basis of the result of step (c) and / or (d).

[0051] In one embodiment of such a method of assessing antigen presentation, the reference is preferably a liposomal vaccine comprising an antigen that is an anchoring moiety variant compared to the test vaccine.

[0052] For example, as illustrated in Example 1, the signal intensity obtained with a liposomal vaccine displaying an antigen may be compared to the signal intensity obtained with liposomal vaccines displaying an anchoring moiety variant of the antigen. As illustrated in that Example, the anchoring moiety variants may have the same antigenic moiety (peptide sequence) but differ in the anchoring moiety. The method may thus be used inter alia to obtain information regarding the orientation of an antigen on the surface of a liposome, and this information may, for example, be used to optimise the anchoring moiety and the like.

[0053] Alternatively, the method of assessing a vaccine may be used to compare antigenic moiety variants, to assess the impact of altering the antigenic moiety, to optimise the antigenic moiety and the like. In such a method, the signal intensity obtained with an antigen may be compared to the signal intensity obtained with one or more antigenic moiety variants. This information may, for example, be used to optimise the antigenic moiety.

[0054] The method of assessing a vaccine advantageously allows the antibody response to a liposomal vaccine to be assessed and / or predicted. Accordingly, any of the methods provided herein may comprise a step of assessing and / or predicting antibody response to a liposomal vaccine. For example, step (e) may comprise assessing and / or predicting antibody response to the liposomal vaccine. Thus, the method may, for example, be a method of assessing and / or predicting antibody response to a liposomal vaccine. Thus, provided is a method of assessing and / or predicting antibody response to a vaccine, wherein the vaccine comprises a liposome that comprises both a vaccine adjuvant and an antigen displayed on the surface of the liposome, the method comprising the steps of

[0055] (a) contacting the vaccine with:

[0056] (i) a capture agent immobilised on a solid support, and

[0057] (ii) a detectable analyte-binding agent; wherein either the capture agent is specific for the vaccine adjuvant and the detectable analyte-binding agent is specific for the antigen, or the capture agent is specific for the antigen and the detectable analyte-binding agent is specific for the vaccine adjuvant;

[0058] (b) incubation under conditions allowing the formation of a complex between the capture agent, the liposome and the detectable analyte-binding agent; and

[0059] (c) detecting the complex by detecting the detectable analyte-binding agent; wherein step (c) preferably comprises measuring the intensity of a signal emitted or generated by a label linked (directly or indirectly) to the detectable analyte-binding agent.

[0060] Preferably, the method further comprises

[0061] (d) comparing the measured signal intensity to a reference and / or a control; and / or

[0062] (e) assessing and / or predicting antibody response to the vaccine on the basis of the result of step (c) and / or (d),

[0063] In such a method of assessing and / or predicting antibody response, the reference or control may, for example, be a vaccine having a known antibody response.

[0064] The method of assessing a vaccine advantageously allows the prediction of the in vivo immunogenicity of a liposomal vaccine. Thus, any of the methods provided herein may comprise a step of predicting the in vivo immunogenicity of a vaccine. For example, step (e) may comprise prediction of the in vivo immunogenicity of the vaccine.

[0065] Thus, provided is a method of predicting the in vivo immunogenicity of a vaccine, wherein the vaccine comprises a liposome that comprises both a vaccine adjuvant and an antigen displayed on the surface of the liposome, the method comprising the steps of

[0066] (a) contacting the vaccine with:

[0067] (i) a capture agent immobilised on a solid support, and

[0068] (ii) a detectable analyte-binding agent; wherein either the capture agent is specific for the vaccine adjuvant and the detectable analyte-binding agent is specific for the antigen, or the capture agent is specific for the antigen and the detectable analyte-binding agent is specific for the vaccine adjuvant;

[0069] (b) incubation under conditions allowing the formation of a complex between the capture agent, the liposome and the detectable analyte-binding agent; and (c) detecting the complex by detecting the detectable analyte-binding agent; wherein step (c) preferably comprises measuring the intensity of a signal emitted or generated by a label linked (directly or indirectly) to the detectable analyte-binding agent.

[0070] Preferably, the method further comprises

[0071] (d) comparing the measured signal intensity to a reference and / or a control; and / or

[0072] (e) predicting the in vivo immunogenicity of the vaccine on the basis of the result of step (c) and / or (d),

[0073] In such a method of predicting the in vivo immunogenicity, the reference or control may, for example, be a liposomal vaccine having a known in vivo immunogenicity.

[0074] Any of the methods provided herein may comprise a step of predicting the potency of a liposomal vaccine. For example, step (e) may comprise predicting the potency of the liposomal vaccine. Thus, the method may, for example, be a method of predicting the potency of a liposomal vaccine.

[0075] In such a method of predicting the potency, the reference or control may, for example, be a liposomal vaccine having a known potency.

[0076] Any of the methods provided herein may comprise assessing two or more different liposomal vaccines and ranking them according to the measured signal intensity. For example, the vaccines may be ranked according to their EC50 or according to their predicted in vivo immunogenicity.

[0077] In some embodiments, any of the provided methods may allow the identification of a defective batch of liposomal vaccines.

[0078] The method of assessing a liposomal vaccine allows an assessment of the heterogeneity of a liposomal vaccine. Thus, any of the methods provided herein may comprise a step of assessing the degree of heterogeneity of a liposomal vaccine. For example, step (e) may comprise assessing or determining the degree of heterogeneity of the liposomal vaccine.

[0079] In such a method of assessing the heterogeneity, the reference or control may, for example, be a liposomal vaccine having a known degree of heterogeneity, or a liposomal vaccine that is considered to have a desired homogeneity, for example on the basis of its known immunogenicity.

[0080] It is contemplated that any of the provided methods may be used in any areas of vaccine development or testing. Any of the provided methods may further comprise a step (f) of making a modification to an antigen on the basis of the determination made in step (e). In some embodiments, the signal intensity measured in step (c) may be inversely proportional to the level of heterogeneity. For example, a low signal intensity may be indicative of a high degree of heterogeneity; whereas a high signal intensity may be indicative of a low degree of heterogeneity, such as substantial homogeneity.

[0081] In some embodiments, the signal intensity measured in step (c) may be proportional to, for example, one or more of the following:

[0082] (i) the antibody response to the liposomal vaccine;

[0083] (ii) the (predicted) in vivo immunogenicity of the liposomal vaccine;

[0084] (iii) the (predicted) potency of the liposomal vaccine.

[0085] It may typically be desired for the signal intensity measured in step (c) to be equivalent to, or higher than, the signal intensity of a positive control or of a reference that has a known favourable antibody response and / or in vivo immunogenicity. Thus, as a general guide, a signal intensity that is substantially equivalent to, or higher than, the signal intensity of such a reference or positive control, may be indicative that the vaccine composition has advantageous properties.

[0086] Any of the methods provided herein are in vitro methods.

[0087] As will be apparent from the present disclosure, in certain preferred embodiments of any of the methods provided herein, the antigen is an antigenic peptide; the capture agent is an antibody specific for the antigenic peptide; the vaccine adjuvant is MPLA, and the detectable analyte-binding agent is an anti-MPLA antibody, wherein the MPLA is preferably 3D-(6-Acyl) PHAD®.

[0088] In particular, the detectable analyte-binding agent may be an anti-MPLA antibody wherein the antibody comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5 and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0089] The antigenic peptide may, for example, comprise, consist essentially of, or consist of SEQ ID NO: 9 or SEQ ID NO: 10 (which may optionally be linked to an anchoring moiety).

[0090] In certain especially preferred embodiments of any of the methods provided herein, the antigen is an antigenic peptide; the detectable analyte-binding agent is an antibody specific for the antigenic peptide; the vaccine adjuvant is MPLA, and the capture agent is an anti- MPLA antibody, wherein the MPLA is preferably 3D-(6-Acyl) PHAD®. In particular, the capture agent may be an anti-MPLA antibody, wherein the antibody comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1 , a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5 and a VL- CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0091] The antigenic peptide may, for example, comprise, consist essentially of, or consist of SEQ ID NO: 9 or SEQ ID NO: 10 (which may optionally be linked to an anchoring moiety).

[0092] Antigens

[0093] The vaccine comprises a liposome comprising at least one antigen displayed on the surface of the liposome (as well as at least one vaccine adjuvant displayed on the surface of the liposome) and the methods employ an agent specific for that antigen. In some embodiments, the agent specific for the antigen is the capture agent. In other embodiments, the agent specific for the antigen is the detectable analyte-binding agent.

[0094] As the antigen is displayed on the surface of the liposome, it may be referred to as a “surface-displayed antigen”.

[0095] The term “antigen” as used herein refers to any molecule comprising an antigenic moiety that is capable upon administration to a suitable subject of inducing an immune response in said subject. The subject may be a mammal, such as a human. The antigen-induced immune response may be humoral or cell-mediated, or both. Typically, the antigen is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody), a B-cell receptor (BCR), and / or T cell antigen receptor (TCR).

[0096] In some embodiments, the antigen is a B-cell antigen, i.e. an antigen that is capable of inducing a B-cell response in the subject. Preferably, it is capable of specifically interacting with a BCR.

[0097] In some embodiments, the antigen is a T-cell antigen, i.e. an antigen that is capable of inducing a T-cell response in the subject. Preferably, it is capable of specifically interacting with a TCR.

[0098] The antigen may, for example, be selected from a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid or any combination of any of the foregoing. Preferably, it comprises, consists essentially of, or consists of an (antigenic) peptide (which may optionally be linked to an anchoring moiety). In embodiments where the antigen comprises an antigenic peptide, the peptide will typically have a length of at least 8 amino acids and less than 100 amino acids. Preferably, the peptide comprises about 10-60 amino acids, for example about 10-50, 10-40, 10-30 or 10-20 amino acids.

[0099] An antigen may comprise an antigenic moiety and a further moiety, such as an anchoring moiety. It will be understood that in the context of an antigen displayed on the surface of a liposome, the term “peptide” or “antigenic peptide” is used to denote the antigenic moiety portion of the antigen. This will typically correspond to the moiety that is displayed on the surface of the liposome and that is the target for the capture agent or detectable analyte binding agent. The surface-displayed antigen may comprise a further moiety, such as a hydrophobic moiety, that inserts into the lipid layer. Such a moiety is also referred to herein as an anchoring moiety. Thus, in the context of a surface-displayed antigen, the expression that the antigen “is a peptide” or “is an antigenic peptide” or that the antigen “consists of” a particular amino acid sequence refers to the antigenic moiety of the antigen, and the antigen may optionally further comprise an additional moiety, such as an anchoring moiety. Thus, in some embodiments the antigen comprises, consists essentially of, or consists of an antigenic peptide linked to one or more anchoring moieties. Details of suitable anchoring moieties are discussed elsewhere herein.

[0100] In some embodiments, the antigenic peptide may be derived from a foreign (external) antigen, or may be derived from a self-antigen.

[0101] In some embodiments, the antigenic peptide may be a protein characteristic of a diseased state, a fragment of such a protein, or a variant thereof. In some embodiments, the protein characteristic of a diseased state is a protein expressed by cancer cells, a protein expressed by a pathogen, or a protein involved in a proteinopathy.

[0102] The term “self-antigen”, as used herein, refers to any antigen (e.g. antigenic peptide) derived from an antigen naturally produced by an individual. In general, the immune system of the individual is tolerant to self-antigens and therefore no immune reaction occurs. In some cases, the immune system is not tolerant to the self-antigens and auto-immune diseases may occur. The use of a self-antigen allows the targeting of a molecule to which the immune system is typically tolerant and therefore may help to induce an immune response that otherwise would not occur.

[0103] In some embodiments, the self-antigen may be selected from cytokines such as, for example, IL-17, IL-27; or from a protein involved in a proteinopathy, such as Abeta, Tau, a- synuclein, huntingtin, prion, or an amylin protein.

[0104] In embodiments where the self-antigen is derived from a protein involved in a proteinopathy, the proteinopathy may, for example be selected from Alzheimer's Disease (AD), mild cognitive impairment (MCI), Down syndrome (DS), including Down syndrome-related Alzheimer's disease, cardiac amyloidosis, cerebral amyloid angiopathy (CAA), multiple sclerosis, amyotrophic lateral sclerosis (ALS), Adult Onset Diabetes, inclusion body myositis (IBM), ocular amyloidosis, glaucoma, macular degeneration, lattice dystrophy, optic neuritis, Creutzfeldt-Jacob disease, Dementia pugilistica, Gerstmann Straussler-Scheinker disease, prion protein cerebral amyloid angiopathy, traumatic brain injury, Non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangles with calcification, frontotemporal dementia (preferably frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP- 17)), frontotemporal lobar dementia, Hallevorden-Spatz disease, Niemann-Pick disease type C, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, Subacute sclerosing panencephalitis, Tangle only dementia, Postencephalitic Parkinsonism, Myotonic dystrophy, chronic traumatic encephalopathy (CTE), Primary age-related tauopathy (PART), or Lewy Body Disorders (LBDs), wherein the LBD may, for example, be selected from Parkinson's Disease (PD), Parkinson's Disease with Dementia (PDD), Dementia with Lewy Bodies (DLB), Multiple System Atrophy (MSA), or Neurodegeneration with Brain Iron Accumulation type I (NBIA Type I).

[0105] In some embodiments, the antigen may be an amyloid-beta (Ap) peptide or a fragment thereof. For example, it may comprise, consist essentially of, or consist of, amino acids 1-15 of amyloid-beta (SEQ ID NO: 9) (which may optionally be linked to an anchoring moiety).

[0106] In some embodiments, the antigen may be an a-synuclein derived peptide. Non-limiting examples of suitable antigenic peptides derived from a-synuclein include those described in W02009 / 103105, WO2022 / 029181 and WO2023 / 152260. For example, it may comprise, consist essentially of or consist of an antigenic peptide of the sequence GGKESMPVDPDNEA (SEQ ID NO: 10) (which may optionally be linked to an anchoring moiety).

[0107] In some embodiments, the self-antigen is a cancer antigen or a tumour antigen.

[0108] In some embodiments, the antigen may be a foreign antigen, for example an antigen derived from a pathogen or an allergen. The term “pathogen”, as used herein, refers to any organism that can cause disease. The pathogen may for example be selected from viruses, fungi, parasites, yeast, bacteria, and protozoa. Examples of viral antigens include, but are not limited to, antigens from rhinoviruses, coronaviruses, enteroviruses, adenoviruses, parainfluenza viruses and respiratory syncytial viruses. The antigen may be a viral fusion protein in some embodiments.

[0109] In some embodiments, the antigen may comprise or be a T-cell antigen, which may, for example, comprise, consist essentially or consist of, a peptide (which may optionally be linked to an anchoring moiety). The T-cell antigen may, for example, comprise, consist essentially of, or consist of, one or more universal T-cell epitopes (which may optionally be linked to an anchoring moiety).

[0110] By “universal T-cell epitope” is meant an epitope that is specific to T-cells that are present in the majority of the human population. They commonly originate from antigens to which humans are normally exposed during their lifetime. Examples include antigens incorporated in routinely administered vaccines. Specific examples are T-cell epitopes included in tetanus, influenza and diphtheria, pan DR-binding epitope peptide (PADRE; WO2010 / 086294), and also Keyhole limpet hemocyanin (KLH) and Epstein Barr virus (EBV). The “universal” ability of a T-cell epitope to activate T cells is the result of at least two complementary properties: i) affinity of binding to the HLA groove, meaning the strength of the binding, as well as ii) its capacity to bind different HLA haplotypes in a promiscuous manner, meaning the ability to cover very diverse human populations, with regards to the differences in the expression of HLA molecules. The universal T-cell epitopes may bind to a majority of MHC class II alleles present in the human population. The universal T-cell epitopes which may be employed in the methods provided herein may thus be capable of stimulating a CD4 T-cell response. The universal T-cell epitopes may thus be capable of stimulating a helper T-cell response that enhances antibody production by B-cells.

[0111] The minimum length of a T-cell epitope peptide to ensure a sufficient immunogenicity is typically around 10 amino acids. Thus, the minimum length of the peptide is typically around 10 amino acids to ensure a sufficiently immunogenic T-cell epitope is generated. In some embodiments, the antigen may comprise two or more different T-cell epitopes.

[0112] An antigenic moiety of the antigen, for example an antigenic peptide, may in some embodiments be modified through at least one lipophilic or hydrophobic moiety to facilitate display on the surface of the lipid-based nanostructure. Such a moiety may be referred to as an anchoring moiety. Optionally the antigenic moiety (e.g. peptide) may be modified through multiple lipophilic or hydrophobic moieties. For example, the antigenic moiety may comprise two, three or four lipophilic or hydrophobic moieties. The lipophilic or hydrophobic moieties may connect the antigenic moiety to the lipid-based nanostructure. The one or more lipophilic or hydrophobic moieties may insert at least partly into the outer surface of the lipid-based nanostructure, i.e. into the lipid bilayer of the lipid-based nanostructure. The one or more lipophilic or hydrophobic moieties are preferably hydrophobic moieties for ease of insertion into the lipid bilayer. In some embodiments, the one or more moieties may be one or more of: a fatty acid, a triglyceride, diglyceride, steroid, sphingolipid, glycolipid, or a phospholipid.

[0113] The at least one lipophilic or hydrophobic moiety may facilitate connecting or anchoring of the antigenic moiety to / into the lipid bilayer of the liposome. The nature and / or location of the anchoring moiety may impact on the location and orientation of the antigenic moiety of the antigen. Preferably, the at least one lipophilic or hydrophobic moiety is a fatty acid. The fatty acid may comprise a carbon backbone of at least 3 carbon atoms. Optionally, the fatty acid may comprise a carbon backbone having at least or about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 carbon atoms.

[0114] Hydrophobic moieties may include, but are not limited to: palmitic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid, and linolenic acid, cholesterol or 1 ,2-distearoyl- sn- glycero-3-phosphatidylethanolamine (DSPE).

[0115] In a preferred embodiment the anchoring moiety or moieties may comprise a palmitoyl residue. Thus, the antigen may for example be an antigenic peptide that is palmitoylated, i.e. mono-palmitoylated or multi-palmitoylated. For example, the antigenic peptide may be modified by at least two palmitoyl residues, i.e. dipalmitoylated. Alternatively, the antigenic peptide may be modified by at least four palmitoyl residues, i.e. tetrapalmitoylated. The antigenic peptide can include additional residues, such as lysine residues to facilitate palmitoylation. Those residues are typically found at the N and / or C terminus of the peptide. In some embodiments, there may be 1-4 lysine residues added to the N and / or C terminus. In one embodiment, a preferred construction comprises the antigenic peptide attached to two palmitoyl residues in the N and / or C terminal regions of the peptide. Thus, the antigenic peptide is dipalmitoylated or tetrapalmitoylated. This may be facilitated by incorporating two lysine residues in the N and / or C terminal regions of the peptide antigen, whereby the lysine residues are palmitoylated.

[0116] However, in some embodiments, the antigen may not comprise an anchoring moiety. For example, the antigen may be able to stick to the surface of a liposome without being anchored into the lipid layer.

[0117] It will be understood that the antigen and vaccine adjuvant that are displayed on the surface of the liposome are different from one another. In particular, the antigen and vaccine adjuvant differ such that an agent specific for the antigen is not specific for the vaccine adjuvant, and vice versa.

[0118] Adjuvants

[0119] The vaccine comprises a liposome comprising at least one vaccine adjuvant displayed on the surface of the liposome (as well as at least one antigen displayed on the surface of the liposome) and the methods employ an agent specific for the vaccine adjuvant. In some embodiments, the agent specific for the vaccine adjuvant is the capture agent. In other embodiments, the agent specific for the vaccine adjuvant is the detectable analyte-binding agent. As discussed elsewhere herein, optionally, the liposomal vaccines may comprise multiple adjuvants.

[0120] Adjuvants serve to enhance the immune response when used in pharmaceutical compositions, e.g. vaccines. Adjuvants typically stimulate the immune system so as to induce a stronger and / or longer lasting immune response to an antigen. The term “vaccine adjuvant” refers to any substance that acts to accelerate, prolong, or enhance an immune response induced by an antigen in the context of vaccination. A vaccine adjuvant typically does not specifically interact with a B-cell receptor (BCR) or T cell antigen receptor (TCR); instead, it typically interacts with a Toll-like receptor.

[0121] Optionally, the vaccine adjuvant(s) of the liposomal vaccine may be selected from one or more of: monophosphoryl lipid A (MPLA); diphosphoryl lipid A (DPLA); Alum; Pam2CSK4; Pam3CSK4; Pam3CAG; saponins; CpG; lipidated CpG, such as CpG-Cholesterol; cationic lipids; phosphorothioated PS-CpG-ODNs; CpG oligodeoxynucleotides (CpG-ODN); CpG-A; CpG-B or CpG-C.

[0122] Further non-limiting examples of vaccine adjuvants that may be used include QuilA, QS-21 , trehalose dimycolate (TDM), lipoteichoic acid (purified from Staphylococcus aureus), DDAB (dimethyldioctadecylammonium (bromide salt)), L18-MDP & B30-MDP (hydrophobic muramyl-dipeptide derivatives), C12-iE-DAP (diamino-pimelic acid).

[0123] The at least one vaccine adjuvant(s) may be associated with the liposome via any appropriate manner provided that the vaccine adjuvant is displayed, at least in part, on the surface of the liposome. For example, the vaccine adjuvant may form part of the liposome, for example, the vaccine adjuvant(s) may from part of the lipid bilayer of the liposome, or may be covalently linked to the lipid bilayer of the liposome through a covalent linkage Optionally the adjuvant(s) may be at least in part in part integrated in the lipid bilayer of the liposome.

[0124] In a preferred embodiment the at least one vaccine adjuvant is a lipid-based vaccine adjuvant. Optionally the lipid-based vaccine adjuvant may be selected from, monophosphoryl lipid A (MPLA); diphosphoryl lipid A (DPLA); Pam2CSK4; Pam3CSK4; Pam3CAG; saponins; CpG; lipidated CpG, such as CpG-Cholesterol; cationic lipids; phosphorothioated PS-CpG-ODNs; CpG oligodeoxynucleotides (CpG-ODN); CpG-A; CpG-B or CpG-C.

[0125] The lipid-based vaccine adjuvant may form part of the lipid bilayer of the liposome. Thus, the lipid-based vaccine adjuvant may be at least in part integrated within the lipid bilayer of the liposome, provided that it is at least in part displayed on the surface of the liposome.

[0126] Conveniently, the lipid-based vaccine adjuvant may be incorporated into a liposome during liposomal synthesis, for example by mixing the lipid-based vaccine adjuvant with the other components that form the lipid bilayer. For example, in preferred embodiments the liposome may comprise dimyristoylphosphatidyl-choline (DM PC), dimyristoylphosphatidyl-glycerol (DMPG), cholesterol and the lipid-based vaccine adjuvant. The molar ratios of these four components may be 9:1:7:0.05 in some embodiments.

[0127] In some embodiments the vaccine adjuvant may be a Toll-like receptor (TLR) agonist, in particular a TLR4 agonist or a TLR9 agonist.

[0128] As used herein, the term "toll-like receptor 4 agonist" refers to any compound that acts as an agonist of TLR4. Examples of TLR4 agonist useful for the invention include, but not limited to, monophosphoryl lipid A (MPLA). MPLA useful for the invention can be obtained using methods known in the art, or from a commercial source, such as 3D-(6-acyl) PHAD®, PHAD®, PHAD®-504, or 3D-PHAD® from Avanti Polar Lipids (Alabaster, Alabama, USA), or MPL™ from various commercial sources.

[0129] The vaccine adjuvant may for example, be derived from, or based on, a glycolipid and may thus be referred to as a “glycolipid-based” vaccine adjuvant.

[0130] In a preferred embodiment, the at least one vaccine adjuvant is a glycolipid-based vaccine adjuvant. According to particular embodiments, the at least one vaccine adjuvant is the glycolipid-based vaccine adjuvant MPLA. In particular embodiments the MPLA may be selected from Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (Synthetic) (3D-(6-acyl) PHAD®), PHAD® (Phosphorylated HexaAcyl Disaccharide), PHAD®-504, 3D-PHAD® from Avanti Polar Lipids (Alabaster, Alabama, USA)) or MPL. In a preferred embodiment, the vaccine adjuvant may be 3D-(6-acyl) PHAD®.

[0131] The glycolipid-based vaccine adjuvant may for example, be derived from, or based on, the glycolipid Lipid A, or the glycolipid monophosphoryl lipid A (MPLA).

[0132] According to particular embodiments, the at least one vaccine adjuvant is the glycolipid- based vaccine adjuvant MPLA.

[0133] “MPLA” refers to a modified form of lipid A, which is the biologically active part of Gramnegative bacterial lipopolysaccharide (LPS) endotoxin. MPLA provides immunostimulatory activity, but is less toxic than LPS. The term “MPLA” also encompasses MPLA-derivatives such as Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (Synthetic), PHAD® (Phosphorylated HexaAcyl Disaccharide), PHAD®-504, 3D-PHAD® from Avanti Polar Lipids (Alabaster, Alabama, USA)) or MPL. Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (Synthetic) is also known as (3D-(6-acyl) PHAD®), so these terms are used interchangeably herein.

[0134] MPLA is an agonist of Toll-like receptor 4 (TLR4). Accordingly, the glycolipid-based vaccine adjuvant is preferably a TLR4 agonist. The glycolipid-based vaccine adjuvant may thus, for example, be the TLR4 agonist MPLA or an analog thereof that is a TLR4 agonist.

[0135] Accordingly, the glycolipid-based vaccine adjuvant may, for example, comprise two glucosamine units, at least 1 acyl chain and one or more phosphate groups.

[0136] Preferably, the glucosamine units are in a P(1 — >6) linkage.

[0137] Preferably, the (glycolipid-based) vaccine adjuvant comprises only a single phosphate group.

[0138] Typically, it may comprise 1-6 acyl chains, preferably 2-6, more preferably 4-6, e.g. 6. Each acyl chain may be about C10 to C16 (i.e. 10-16 carbons in length), preferably C12-C14. Six C14 acyl chains are especially preferred.

[0139] Especially preferred is Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (Synthetic) which is also referred to herein as 3D-(6-acyl) PHAD®.

[0140] As used herein, the term "toll-like receptor 9 agonist" (TLR9 agonist) refers to any compound that acts as an agonist of TLR9. Examples of suitable TLR9 agonist include, but not limited to, CpG oligonucleotides. As used herein, the term "CpG oligonucleotide", "CpG oligodeoxynucleotide" or "CpG ODN" refers to an oligonucleotide comprising at least one CpG motif. As used herein, "oligonucleotide," "oligodeoxynucleotide" or "ODN" refers to a polynucleotide formed from a plurality of linked nucleotide units. Such oligonucleotides can be obtained from existing nucleic acid sources or can be produced by synthetic methods. As used herein, the term "CpG motif' refers to a nucleotide sequence which contains unmethylated cytosine-phosphateguanine (CpG) dinucleotides (i.e., a cytosine (C) followed by a guanine (G)) linked by a phosphate bond or a phosphodiester backbone or other internucleotide linkages. Examples of synthetic CpG oligonucleotides include, but are not limited to, CpG 2006 (also known as CpG 7909), CpG 1018, CpG 2395, CpG 2216 or CpG 2336.

[0141] In one embodiment, the vaccine adjuvant(s) of the liposomal construct may comprise CpG, for example it may be a lipid-based vaccine adjuvant comprising CpG. The CpG oligonucleotide may, for example, be covalently linked to the lipid bilayer of the liposome through a covalent linkage.

[0142] The terms “lipid-based adjuvant” and “lipid-based vaccine adjuvant” are used interchangeably herein.

[0143] Agents The methods provided herein employ an agent specific for the antigen and an agent specific for the vaccine adjuvant respectively. One of these agents is the capture agent and the other is the detectable binding agent. Thus, the antigen and vaccine adjuvant are targets of the capture agent and the detectable binding agent respectively.

[0144] The terms “capture agent” and “detectable analyte-binding agent” are terms of art. The capture agent binds to its target and captures it to a solid support (to which the capture agent is attached), thereby capturing the liposome comprising the target onto the solid support. The detectable analyte-binding agent binds to its target. As explained elsewhere herein, a label may be linked directly or indirectly to the detectable analyte-binding agent, and this linking may in some embodiments take place after step (b). The label emits or generates the signal that may be detected or measured in step (c).

[0145] It will be understood that the antigen and the vaccine adjuvant are different. In particular, the antigen and vaccine adjuvant differ such that an agent specific for the antigen is not specific for the vaccine adjuvant, and vice versa.

[0146] In some embodiments, the capture agent is specific for the antigen and the detectable analyte-binding agent is specific for the vaccine adjuvant.

[0147] In preferred embodiments, the capture agent is specific for the vaccine adjuvant and the detectable analyte-binding agent is specific for the antigen.

[0148] The term “agent” used herein encompasses inter alia “capture agent” and “detectable analyte-binding agent”. By the terms “specific for”, “specific binding” or “selective binding” is meant that the agent preferentially binds to its specified target. Typically, it does not significantly bind, or does not bind, to any other unrelated species, particularly any other unrelated species present on the surface of the liposome or otherwise present in the assay.

[0149] In particular, an agent specific for the vaccine adjuvant preferentially binds to the vaccine adjuvant (i.e. its target) and does not significantly bind to the antigen, nor any other molecules at the surface of the liposome. An agent specific for the antigen preferentially binds to the antigen (i.e. its target) and does not significantly bind to the vaccine adjuvant, nor any other molecules at the surface of the liposome.

[0150] Any of the agents disclosed herein may, for example, be an antibody.

[0151] In general, the term "antibody" is used herein in the broadest sense and encompasses any antibody structures that exhibit the desired binding activity, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific, biparatopic antibodies), fully-human antibodies and antigen-binding fragments. Preferably, the antibody is not polyclonal. Thus, monoclonal antibodies or antigen-binding fragments thereof are preferred. Antibodies which may be used according to the methods of present invention may be chimeric antibodies, recombinant antibodies, or humanized antibodies, or an antigen-binding fragment of any thereof.

[0152] An "antigen-binding fragment" of an antibody refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Accordingly, examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab' -SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

[0153] Monoclonal antibodies or antigen-binding fragments thereof are preferred. The term “monoclonal antibody” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as being amongst a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies may be made by the hybridoma method described in KOHLER, G., MILSTEIN, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-497 (1975).

[0154] The capture agent is preferably an antibody.

[0155] The detectable analyte-binding agent is preferably an antibody.

[0156] In embodiments where the capture agent is specific for the antigen, preferred capture agents may be selected from those that are specific for one of the preferred antigens described herein. Similarly, in embodiments where the detectable analyte-binding agent is specific the antigen, preferred detectable analyte-binding agents may be selected from those that are specific for one of the preferred antigens described herein.

[0157] In embodiments where the capture agent is specific for the vaccine adjuvant, preferred capture agents may be selected from those that are specific for one of the preferred vaccine adjuvants described herein. Similarly, in embodiments where the detectable analyte-binding agent is specific the vaccine adjuvant, preferred detectable analyte-binding agent may be selected from those that are specific for one of the preferred vaccine adjuvants described herein.

[0158] The vaccine adjuvant is preferably MPLA. Accordingly, the agent specific for the vaccine adjuvant (which may be the capture agent or the detectable analyte-binding agent), is preferably specific for MPLA. The MPLA may, for example, be 3D-(6-Acyl) PHAD®. Most preferably, the agent specific for the vaccine adjuvant is an anti-MPLA antibody, for example an anti-MPLA antibody as disclosed herein by reference to any one of SEQ ID Nos 1-8.

[0159] In some embodiments the antigen is an antigenic peptide. Accordingly, the agent specific for the antigen (which may be the capture agent or the detectable analyte-binding agent) is preferably specific for such an antigenic peptide. By way of example, the agent may be an antibody specific for an antigenic peptide. Suitable antibodies are well known.

[0160] In some embodiments, the antigenic peptide is an amyloid-beta (Ap) peptide or a fragment thereof, and the agent specific for the antigen (which may be the capture agent or the detectable analyte-binding agent) is an A binding antibody that can specifically bind that peptide or to an epitope in that peptide, for example an epitope within SEQ ID NO: 9. In some embodiments, the agent specific for the antigen is a commercially available Ap binding antibody. In some embodiments, the antibody is selected from: NAB228 (Invitrogen), 6E10 (Covance), 2C8 (Invitrogen), 4G8 (Chemicon), WO-2 (Merck) and DE2 (Chemicon). In preferred embodiments, the antibody is 6E10.

[0161] In some embodiments, the antigenic peptide is a Tau peptide or fragment thereof, and the agent specific for the antigen (which may be the capture agent or the detectable analytebinding agent) is a Tau binding antibody that can specifically bind that peptide or to an epitope in that peptide. In some embodiments, the agent specific for the antigen is a commercially available Tau binding antibody. In some embodiments, the antibody is selected from: Tau12 (Covance), Tau13 (Covance), 43D (Covance), 3H6.H7 (Covance), 77E9 (Covance), Tau1 (Chemicon), BT-2 (Pierce), Tau46 (Abeam), Tau2 (Covance), AT270 (ThermoFisher), HT7 (ThermoFisher), Tau5 (ThermoFisher), 77G7 (Covance), AT100 (ThermoFisher), AT180 (ThermoFisher), PHF13 (ThermoFisher) and AT8 (ThermoFisher).

[0162] In some embodiments, the antigenic peptide is an alpha-synuclein peptide or fragment thereof, and the agent specific for the antigen (which may be the capture agent or the detectable analyte-binding agent) is an alpha-synuclein binding antibody that can specifically bind that peptide or to an epitope in that peptide, for example an epitope within SEQ ID NO: 10. In some embodiments, the agent specific for the antigen is a commercially available alpha-synuclein binding antibody. In some embodiments, the antibody is selected from MJFR1 (Abeam), Syn211 (ThermoFisher) and LB-509 (BioLegend). In preferred embodiments, the antibody is MJFR1.

[0163] The capture agent is immobilized on a solid support. Methods for immobilising agents on a solid support, such as coupling antibodies to solid support, are well known to those of ordinary skill in the art.

[0164] Suitable solid supports are well known in the field of radioimmunoassay and enzyme immunoassay. Exemplary solid support substances include, but are not limited to, microtiter plates, multiwell plates, test tubes, beads and slides. The solid support may be made of a suitable material such as plastic or glass; in the case of e.g. beads, it may alternatively be made of polystyrene or be magnetic. In particular, the solid support may comprise a porous material such as nylon, nitrocellulose, cellulose acetate, glass fibers and other porous polymers.

[0165] In some embodiments, the capture agent may be immobilized on the surface of a well of the solid support, such as a multiwell plate.

[0166] Detection of the detectable analyte-binding agent involves detection of a signal emitted or generated by a label linked to the detectable analyte-binding agent. The label may be linked directly or indirectly to the detectable analyte-binding agent and indirect linking may in some embodiments take place after step (b).

[0167] Thus, in some embodiments, the detectable analyte-binding agent comprises a label that is directly linked to the detectable analyte-binding agent, for example through a covalent bond.

[0168] In other embodiments, a further agent is used to (indirectly) label the detectable analytebinding agent, for example a labelled secondary antibody may be used to bind to, and thereby label, the detectable analyte-binding agent.

[0169] As such, in some embodiments, the detectable analyte-binding agent may comprise a label. In alternative embodiments, the methods of the invention may further comprise a step of (indirectly) linking a label to the detectable analyte-binding agent.

[0170] Suitable labels are known in the art and include enzymes; radioisotopes; and fluorescent, luminescent or chromogenic substances. The label may emit a signal, for example a fluorescent signal. Alternatively, the label may generate a signal, for example enzymes generate a signal by converting a substrate into a detectable product. Thus, for example, the label may be selected from horseradish peroxidase, alkaline phosphatase, a coloured particle or a fluorescent moiety, preferably horseradish peroxidase or alkaline phosphatase.

[0171] In embodiments where linkage of the detectable analyte-binding agent to the label is indirect, any suitable method may be used to (indirectly) label the detectable analyte-binding agent by contacting the detectable analyte-binding agent with a secondary agent that comprises a label, i.e. a labelled secondary agent. The labelled secondary agent is specific for a moiety comprised by the detectable analyte-binding agent.

[0172] For example, in embodiments where the detectable analyte-binding agent comprises an Fc domain, the labelled secondary agent may be specific for the Fc domain, for example it may comprise protein A or G, or a secondary antibody. Alternatively, in embodiments where the detectable analyte-binding agent comprises biotin, the labelled secondary agent may comprise avidin or streptavidin; or in embodiments where the detectable analyte-binding agent comprises a hapten, the labelled secondary agent may comprise an anti-hapten binding peptide.

[0173] Preferably, the detectable analyte-binding agent is a primary antibody and the secondary agent is a labelled secondary antibody, most preferably labelled with horseradish peroxidase or alkaline phosphatase.

[0174] Conjugation methods are well known in the art and several technologies are commercially available for conjugating antibodies to a label or other moiety. Conjugation is typically through amino acid residues contained within the binding agent (such as lysine, histidine or cysteine). They may rely upon methods such as the NHS (Succinimidyl) ester method, isothiocyanate method, carbodiimide method and periodate method. Conjugation may be achieved through creation of fusion proteins for example. This is appropriate where the detectable analyte-binding agent is conjugated with another protein molecule. Thus, suitable genetic constructs may be formed that permit the expression of a fusion of the detectable analyte-binding agent of the invention with the label or other molecule. Conjugation may be via a suitable linker moiety to ensure suitable spatial separation of the antibody and conjugated molecule, such as detectable label. However, a linker may not be required in all instances.

[0175] Those of ordinary skill in the art will know of these and other suitable labels which may be employed in accordance with the present invention. The binding of these labels to the detectable analyte-binding agent or to the secondary agent can be accomplished using standard techniques commonly known to those of ordinary skill in the art. Typical techniques are described by Kennedy, J. H., et al., 1976 (Clin. Chim. Acta 70:1-31), and Schurs, A. H. W. M., et al. 1977 (Clin. Chim Acta 57:1-40). Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, and others.

[0176] As mentioned above, in some embodiments, the label is capable of emitting a signal, for example it may be radioactive or fluorescent.

[0177] In other embodiments, the label is capable of generating a signal. This typically requires a suitable substrate to be present. Accordingly, in some embodiments, the method further comprises adding a suitable substrate for signal generation by the label. For example, the label may be an enzyme requiring a suitable substrate to generate a signal. The enzyme may interact with a substrate to induce a quantifiable colour change or fluorescence. In an example embodiment, the detectable analyte-binding agent may be labelled with horseradish peroxidase (HRP). Upon incubation with a HRP substrate, HRP catalyses a reaction causing a detectable colour change. Common suitable HRP substrates include O- Phenylenediamine (OPD), tetramethyl benzidine (TMB) or ABTS (2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid). Following addition of a suitable substrate, signal intensity may be measured by measuring the optical density (OD) of the reaction product, for example via spectrophotometry.

[0178] Complex

[0179] The methods of the invention comprise a step of allowing the formation of a complex between the capture agent, the liposome and the detectable analyte-binding agent. The complex is formed through specific binding of the agents to their targets such that the resulting complex comprises the capture agent bound to its target on the surface of the liposome and the detectable analyte-binding agent bound to its target on the surface of the liposome. Thus, by “complex” is meant herein a complex that comprises the capture agent bound to its target on the surface of the liposome and the detectable analyte-binding agent bound to its target on the surface of the liposome. Alternatively viewed, the “complex” is a liposome having specifically bound to it (more specifically to its antigen and its vaccine adjuvant) a capture agent and a detectable analyte-binding agent respectively. Accordingly, a liposome having bound thereto no capture agent and / or no detectable analyte-binding agent is not a complex as defined herein.

[0180] It will be understood that complex formation relies inter alia on the antigen being accessible to take part in the complex formation. Accordingly, liposomes that lack the antigen will substantially not take part in the complex formation. Liposomes that display the antigen on their surface may take part in complex formation, but their ability to take part in the complex formation may be affected by factors such as the affinity of the antigen-specific agent (capture agent or detectable analyte-binding agent, as the case may be) for the antigen; and how accessible the antigen is to the antigen-specific agent.

[0181] In some embodiments, the capture agent is specific for the antigen of the liposome and the detectable analyte-binding agent is specific for the vaccine adjuvant of the liposome. As such, the capture agent binds the antigen of the liposome and the detectable analyte-binding agent binds the vaccine adjuvant so as to form a complex.

[0182] In some embodiments, the capture agent is specific for the vaccine adjuvant of the liposome and the detectable analyte-binding agent is specific for the antigen of the liposome. As such, the capture agent binds the vaccine adjuvant of the liposome and the detectable analytebinding agent binds the antigen so as to form a complex.

[0183] Contacting the liposome with the capture agent and the detectable analyte-binding agent may be simultaneous or sequential. Thus, in some embodiments, the liposome, the capture agent and the detectable analyte-binding agent are contacted simultaneously, for example by simultaneously, or substantially simultaneously adding each of these components to an assay compartment, or by adding two of these components to an assay compartment already containing the remaining component. In other embodiments, the liposome, the capture agent and the detectable analyte-binding agent are contacted sequentially. For example, the liposome may in a first step be contacted with the capture agent. During this first step, the capture agent may be allowed to bind to its target on the liposome (if its target is present). In a second step, the liposome (and any capture agent bound thereto) may be contacted with the detectable analyte-binding agent. During this second step, the detectable analyte-binding agent may be allowed to bind to its target on the liposome (if its target is present).

[0184] Alternatively, the liposome may in a first step be contacted with the detectable analytebinding agent. During this first step, the detectable analyte-binding agent may be allowed to bind to its target on the liposome (if its target is present). In a second step, the liposome (and any detectable analyte-binding agent bound thereto) may be contacted with the capture agent. During this second step, the capture agent may be allowed to bind to its target on the liposome (if its target is present).

[0185] The method may comprise one or more steps of removing free assay components. By “free” is meant that the assay component is not bound to its target. The “assay component” may be the capture agent, the liposome, the detectable analyte-binding agent, or any other component used in the method, such as any component used to detect the detectable analyte-binding agent. A convenient example of such a step is a wash step, in which a free component is removed using a suitable wash solution. A step of removing free assay components, such as a wash step, may, e.g., be carried out after the step of contacting the liposome with the capture agent and / or after the step of contacting the liposome with the detectable analyte-binding agent. In particular, the method may comprise a wash step after complex formation has been allowed to take place. Such a wash step may remove any liposomes that are not bound to a capture agent (including liposome bound solely to a detectable analyte-binding agent), and any detectable analyte-binding agent that is not bound to a liposome. Preferably, an appropriate wash step is carried out to remove any free detectable analyte-binding agent, such that detection of the detectable analyte-binding agent detects substantially only detectable analyte-binding agent that forms part of a complex as defined herein.

[0186] Exemplary suitable conditions for incubation to allow the formation of a complex are provided in the Examples. Based on this information, together with their common general knowledge, the skilled person will be able to select suitable incubation conditions.

[0187] A liposomal vaccine comprises a population of liposomes, so any reference herein to a “liposomal vaccine” should be understood to encompass a reference to a population of liposomes. By “liposomal vaccine” is meant a vaccine comprising a liposome that comprises both a vaccine adjuvant and an antigen displayed on its surface. For the avoidance of any doubt, by this is meant that the vaccine adjuvant is displayed on the liposome’s surface and the antigen is displayed on the liposome’s surface. Typically, at least a proportion of the liposomes comprise the antigen displayed on the surface. In some embodiments, all, substantially all, or at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the liposomes in the liposomal vaccine comprise the antigen displayed on the surface of the liposome. In some embodiments, the method is carried out on a liposomal vaccine comprising a significant proportion of liposomes that do not comprise the antigen displayed on the surface, for example at least 20%, at least 30%, at least 40% or at least 50% of liposomes may not comprise the antigen displayed on the surface of the liposome.

[0188] Typically, at least a proportion of the liposomes comprise the vaccine adjuvant displayed on the surface of the liposome. In some embodiments, all, substantially all, or at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the liposomes in the liposomal vaccine comprise the vaccine adjuvant displayed on the surface of the liposome.

[0189] Thus, by “a vaccine comprising a liposome that comprises both a vaccine adjuvant and an antigen displayed on the surface of the liposome” is meant that at least a proportion of the liposomes of the vaccine (liposomal population) comprise both a vaccine adjuvant and an antigen displayed on the surface of the liposome. However, in some embodiments, at least a proportion of the liposomes of the liposomal vaccine comprise a vaccine adjuvant displayed on the surface of the liposome but do not comprise an antigen displayed on the surface of the liposome. Thus, the liposomal population may be considered heterogeneous with respect to the presence or absence of the antigen.

[0190] The provided methods may be used to assess the degree of heterogeneity of the liposomal vaccine with regard to the presence or absence of a target. The target is typically the antigen, but may alternatively be the vaccine adjuvant or a difference surface molecule. Thus, “heterogeneity” or “homogeneity” is used herein with respect to the presence or absence of a target. The term “heterogeneity” as used here does not refer to the inherent polydispersity of liposomal populations. A population of liposomes in which all or substantially all of the liposomes comprise the target may be considered to be “homogeneous” (with regard to the presence of that target, meaning that there are substantially no liposomes in the population that lack the target). Thus, the term “homogeneous” as used herein does not necessarily mean that there are no differences between the liposomes in the population.

[0191] Thus, the “degree of heterogeneity” as used herein refers to the proportion of liposomes in the population that lack the target. By a “low degree of heterogeneity” is meant that at least 80%, at least 85%, at least 90%, or at least 95% of the liposomes comprise the target. By a “high degree of heterogeneity” is meant that at least at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of liposomes may not comprise the target. As mentioned elsewhere, the methods provided herein have significant utility in vaccine characterization, development and production, inter alia in assessing and / or predicting the antibody response to a liposomal vaccine; predicting the in vivo immunogenicity of a liposomal vaccine; or predicting the potency of a liposomal vaccine.

[0192] "Antibody response” as used herein refers to the production of circulating antibodies by B- cells and their progeny in response to an antigen.

[0193] “In vivo immunogenicity” as used herein refers to a measure of the ability of the liposomal vaccine to elicit an immune response (humoral and cellular) when administered to a recipient.

[0194] “Potency” refers to the biological activity of a vaccine required in the treatment or prevention of diseases, disorders, or conditions.

[0195] Liposomes

[0196] Liposomes are widely used as model systems for cell membrane and as drug carriers in drug delivery systems. Liposomes that can be used in the methods of the present invention include those known to one skilled in the art. Any of the standard lipids useful for making liposomes may be used. Any method of making liposomes known to one skilled in the art may be used, for example, the method disclosed in Alving et al., Infect. Immun. 60:2438- 2444, 1992.

[0197] Liposomes are vesicles composed of (phospho)lipid molecules comprising a hydrophilic head group and a hydrophobic tail. The (phospho)lipid molecules assemble in an aqueous solution such that the hydrophobic parts get oriented toward each other to avoid contact with the aqueous phase, whereas the hydrophilic head groups are oriented such that they make maximal contact with the aqueous surrounding. This leads to spontaneous self-assembly into spherical structures that contain an inner aqueous compartment surrounded by a lipid bilayer.

[0198] A liposome can be used as a carrier for presenting an antigen on its outer surface; as well as comprising an adjuvant to increase or stimulate the immune response against the antigen within the target animal or human.

[0199] By “surface” of a liposome is meant the external surface, i.e. the surface which is oriented towards the aqueous solution surrounding the liposome. The terms “surface molecule”, “surface antigen”, “surface-displayed antigen”, “displayed on the surface of the liposome” and the like mean that the molecule (e.g. the antigen or the vaccine adjuvant respectively) is presented, at least partially, on the external surface of the (intact) liposome, as would be understood by one skilled in the art (see e.g. Muhs, 2007, Pihlgren, 2013). This is typically by insertion of the molecule (e.g. the antigen or the vaccine adjuvant respectively) into, or otherwise anchoring of the molecule to, the outer surface of the liposome, for example via a hydrophobic moiety.

[0200] In embodiments where the vaccine adjuvant is lipid-based, it intrinsically comprises a suitable hydrophobic moiety.

[0201] In other embodiments, the vaccine adjuvant may for example comprise a vaccine adjuvant moiety that has been covalently linked to one or more anchoring moieties.

[0202] In some embodiments, the antigen may for example comprise an antigenic moiety that has been covalently linked to one or more anchoring moieties.

[0203] Suitable anchoring moieties are discussed elsewhere herein.

[0204] The liposomes may also comprise additional components that are not targets for any of the agents used in the methods provided herein. In particular, such additional components, if present, are neither a target for the capture agent nor a target for the detectable analytebinding agent. Thus, neither the capture agent nor the detectable analyte-binding agent are specific for such additional components. Such an additional component may, for example be a further adjuvant, provided that the further adjuvant is sufficiently different from the vaccine adjuvant that is the target of the capture agent or the detectable binding agent respectively so as to not interfere with the method. A suitable example is CpG, but other adjuvants are well known such as alum (e.g. aluminium phosphate or aluminium hydroxide), calcium phosphate, cytokines (e.g. interleukin-1), muramyl peptides (e.g., N-acetylmuramyl-L- threonyl-D-isoglutamine (thrMDP) or N-acetylglucosaminyl-N-acetylinuramyl-L-Ala-D-isoGlu- L-Ala-dipalmitoxy propylamide (DTP-DPP) Theramide™), oil (e.g. MF59 or squalene), Freund's incomplete adjuvant, Freund's complete adjuvant, polydispersed |3-(1 ,4) linked acetylated mannan ("Acemannan"), TITERMAX® (polyoxyethylene-polyoxypropylene copolymer adjuvants from CytRx Corporation), modified lipid adjuvants from Chiron Corporation, saponin derivative adjuvants from Cambridge Biotech, killed Bordetella pertussis or large polymeric anions such as dextran sulfate. Alternatively or in addition, an additional component may, for example be a further antigen, provided that the further antigen is sufficiently different from the antigen that is the target of the capture agent or the detectable binding agent respectively so as to not interfere with the method.

[0205] Such additional components are not required for the purpose of the methods provided herein. Their presence may serve to provide additional functionality to liposome. For example, the liposome may comprise one or more additional antigenic peptides and / or adjuvants. Any such additional component may be encapsulated by the liposome or be partially or fully displayed on its surface. For example, the liposome may comprise a vaccine adjuvant and an antigen displayed on its surface, and may further comprises one or more additional components, optionally selected from one or more encapsulated universal T-cell epitopes; one or more different adjuvants; and / or one or more different surface-displayed antigens.

[0206] In some embodiments, the vaccine comprises at least a B-cell antigen displayed on the surface of the liposome and at least one T-cell antigen displayed, at least partially, on the surface of the liposome.

[0207] Anti-MPLA binding molecule

[0208] As indicated in Example 6, the inventors have generated an anti-MPLA antibody. To our knowledge, this is the first report of an anti-MPLA antibody. In particular, the antibody is capable of binding to 3D-(6-acyl) PHAD® and may thus also be referred to as an anti-3D-(6- acyl) PHAD® antibody.

[0209] Accordingly, in a further aspect, provided is an antigen binding molecule capable of binding MPLA, i.e. is an anti-MPLA binding molecule. Preferably, the anti-MPLA binding molecule is an anti-MPLA antibody. It may be referred to as an anti-3D-(6-acyl) PHAD® antibody.

[0210] Preferably, the anti-MPLA binding molecule comprises a Heavy Chain Variable Region comprising:

[0211] (i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto;

[0212] (ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 2, or a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto; and

[0213] (iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto; and / or a Light Chain Variable Region comprising:

[0214] (iv) a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID

[0215] NO: 4, or a sequence having at least 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto;

[0216] (v) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 5, or a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto; and

[0217] (vi) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 6, or a sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto.

[0218] Thus, the anti-MPLA binding molecule may, for example, comprise a Heavy Chain Variable Region comprising:

[0219] (i) a VH CDR1 that has the amino acid sequence of SEQ ID NO: 1;

[0220] (ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 2; and (iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 3; and / or a Light Chain Variable Region comprising:

[0221] (iv) a VL CDR1 that has the amino acid sequence of SEQ IDNO: 4;

[0222] (v) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 5; and

[0223] (vi) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 6.

[0224] Preferably, it comprises a Heavy Chain Variable Region comprising:

[0225] (i) a VH CDR1 that has the amino acid sequence of SEQ ID NO: 1;

[0226] (ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 2; and

[0227] (iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 3; and a Light Chain Variable Region comprising:

[0228] (iv) a VL CDR1 that has the amino acid sequence of SEQ ID NO: 4;

[0229] (v) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 5; and

[0230] (vi) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 6.

[0231] The anti-MPLA binding molecule binding molecule may, for example, comprise a Heavy Chain Variable Region comprising the amino acid sequence of SEQ ID NO: 7 or a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto; and / or a Light Chain Variable Region comprising the amino acid sequence of SEQ ID NO: 8 or a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto. Thus, it may comprise a Heavy Chain Variable Region comprising the amino acid sequence of SEQ ID NO: 7 or a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto, and a Light Chain Variable Region comprising the amino acid sequence of SEQ ID NO: 8 or a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto. Preferably, it may comprise a Heavy Chain Variable Region comprising the amino acid sequence of SEQ ID NO: 7 and a Light Chain Variable Region comprising the amino acid sequence of SEQ ID NO: 8

[0232] An “antigen binding molecule,” as used herein, is any molecule that can specifically or selectively bind to an antigen. A binding molecule may include or be an antibody or an antigen-binding fragment thereof. An anti-MPLA binding molecule is a molecule that binds to a region of MPLA that is exposed on the outer surface of a liposome that comprises MPLA.

[0233] The term “CDR” as employed herein relates to “complementary determining region”, which is well known in the art. The CDRs are parts of immunoglobulins that determine the specificity of said molecules and make contact with a specific ligand. The CDRs are the most variable part of the molecule and contribute to the diversity of these molecules. There are three CDR regions CDR1 , CDR2 and CDR3 in each V domain. CDR-H depicts a CDR region of a variable heavy chain and CDR-L relates to a CDR region of a variable light chain. VH means the variable heavy chain and VL means the variable light chain. The CDR regions of an Ig- derived region may be determined as described in Kabat “Sequences of Proteins of Immunological Interest”, 5th edit. NIH Publication no. 91-3242 U.S. Department of Health and Human Services (1991). CDR sequences provided herein are defined according to Kabat.

[0234] However, it will be understood by the skilled person that the invention is intended to encompass anti-MPLA binding molecules in which the CDR sequences are defined according to any useful identification / numbering scheme. For example, Chothia (Canonical structures for the hypervariable regions of immunoglobulins. Chothia C, LeskAM. J Mol Biol. 1987 Aug 20; 196(4):901-17), IMGT (IMGT, the international ImMunoGeneTics database. Giudicelli V, Chaume D, Bodmer J, Muller W, Busin C, Marsh S, Bontrop R, Marc L, Malik A, Lefranc MP. Nucleic Acids Res. 1997 Jan 1; 25(l):206-l I and Unique database numbering system for immunogenetic analysis. Lefranc MP. Immunol Today. 1997 Nov; 18(11):509), MacCallum (MacCallum RM, Martin AC, Thornton JM, J Mol Biol. 1996 Oct 11 ; 262(5):732- 45) and Martin (Abhinandan KR, Martin ACR. Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Mol Immunol. (2008) 45:3832-9. 10.1016 / j.molimm.2008.05.022) numbering schemes may be adopted in order to define the CDRs.

[0235] In a further aspect, provided is a kit comprising: a) a capture agent; and b) detectable analyte-binding agent; wherein the capture agent or detectable analyte-binding agent specifically binds to a vaccine adjuvant.

[0236] Preferably, the vaccine adjuvant is a glycolipid-based vaccine adjuvant, more preferably MPLA, for example 3D-(6-acyl) PHAD®. For example, the capture agent or detectable analyte-binding agent may be an anti- MPLA binding agent, such as an anti-MPLA binding agent as described herein by reference to one or more of SEQ ID Nos 1-8.

[0237] The kit may further comprise a solid support. For example, it may comprise a solid support having the capture agent immobilised thereon.

[0238] The definitions and preferred embodiments of any relevant components, particularly capture agents, detectable analyte-binding agents and solid supports, set out herein in connection with the provided methods or anti-MPLA binding agent apply mutatis mutandis to the corresponding components of the kit.

[0239] The kit may in some embodiments comprise all necessary components for performing the herein provided methods, such as, for example, buffers, detectable labels, substrates, reaction containers, and the like. The kit may optionally be provided with suitable instructions for use. The instructions for use may further explain the storage conditions for the compositions therein. These kits may be applied to all relevant methods of the invention as disclosed herein.

[0240] Certain further or specific aspects and embodiments of the methods provided herein are set out below.

[0241] Further embodiments

[0242] Throughout the disclosure reference is made to “liposomal vaccine”. This term may be used interchangeably with “liposomal population” or “vaccine comprising a liposome”. In the context of this disclosure, the term “vaccine” should be understood to refer to a vaccine comprising a liposome unless explicitly stated otherwise.

[0243] Liposomes are a type of lipid-based nanovesicles, and the provided methods are equally applicable to other types of lipid-based nanovesicles or lipid-based nanoparticles, collectively referred to herein as lipid-based nanostructures. Thus, any reference herein to a “liposome” may, where appropriate, be used interchangeably with “lipid-based nanostructure” and thus any reference to a “liposomal vaccine” may, where appropriate, be used interchangeably with “population of lipid-based nanostructures”.

[0244] By “lipid-based nanostructure” is meant a sphere-shaped structure comprising a lipid outer layer that encapsulates an inner compartment. The lipid outer layer may be a monolayer or may be a bilayer. The structure may have a single monolayer, a single bilayer, or more than one mono- and / or bi-layer. The inner compartment may be aqueous or non-aqueous. For example, the nanostructure may be a vesicle comprising an aqueous inner compartment. In particular, the nanostructure may be a self-assembled spherical structure that contains an inner aqueous compartment surrounded by a lipid bilayer typically composed of phospholipids and sterols, such as a liposome. Standard bilayer and multi-layer lipid-based nanostructures may be used in the methods of the present invention.

[0245] It will also be apparent that an antigen is an example of a surface molecule; and a vaccine adjuvant is another example of a surface molecule.

[0246] The terms “lipid adjuvant” and “lipid-based adjuvant” are used interchangeably herein.

[0247] Thus, also provided are aspects and embodiments in accordance with following numbered clauses. 1. A method of assessing a population of lipid-based nanostructures, wherein the population of lipid-based nanostructures preferably comprises a lipid-based nanostructure comprising a first surface molecule and a second surface molecule, the method comprising

[0248] (a) contacting the population of lipid-based nanostructures with:

[0249] (i) a capture agent, and

[0250] (ii) a detectable analyte-binding agent;

[0251] (b) incubation under conditions allowing the formation of a complex between the capture agent, the lipid-based nanostructure and the detectable analyte-binding agent; wherein the capture agent is specific for the first surface molecule of the nanostructure and the detectable analyte-binding agent is specific for the second surface molecule of the nanostructure; wherein the capture agent is immobilised on a solid support, or the method comprises a step of immobilising the capture agent on a solid support; and

[0252] (c) detecting the complex by detecting the detectable analyte-binding agent, which may preferably comprise measuring the signal intensity of a signal emitted or generated by a label linked directly or indirectly to the detectable analyte-binding agent.

[0253] 2. The method according to clause 1, wherein

[0254] (i) the first surface molecule is a vaccine adjuvant and the capture agent is specific for the vaccine adjuvant; or

[0255] (ii) the second surface molecule is a vaccine adjuvant and the detectable analyte-binding agent is specific for the vaccine adjuvant.

[0256] 3. The method according to clause 2, wherein the vaccine adjuvant is a lipid-based vaccine adjuvant.

[0257] 4. The method according to clause 2 or 3, wherein the vaccine adjuvant is a glycolipid-based vaccine adjuvant.

[0258] 5. The method according to any one of clauses 2-4, wherein the vaccine adjuvant is an agonist of Toll-like receptor 4 (TLR4).

[0259] 6. The method according to any one of any one of clauses 2-5, wherein the vaccine adjuvant is monophosphoryl lipid A (MPLA).

[0260] 7. The method according to any one of clauses 2-6, wherein the vaccine adjuvant comprises, consists essentially of, or consists of, Monophosphoryl Hexa-acyl Lipid A, 3- Deacyl (3D-(6-acyl) PHAD®). 8. The method according to any one of the preceding clauses, wherein the first surface molecule or the second surface molecule is an antigen.

[0261] 9. The method according to clause 8, wherein the antigen is an antigenic peptide, wherein the antigenic peptide is optionally linked to an anchoring moiety.

[0262] 10. The method according to clause 9, wherein the antigenic peptide is derived from a protein derived from a pathogen or a protein characteristic of a diseased state, for example a protein derived from a viral pathogen, a protein expressed by cancer cells, or a protein involved in a proteinopathy.

[0263] 11. The method according to any one of clauses 8-10, wherein the antigen is derived from a self-antigen.

[0264] 12. The method according to clause 11, wherein the self-antigen is selected from p-amyloid (Ap), Tau, a-synuclein, huntingtin, prion, an amylin protein, IL-17 or IL-27.

[0265] 13. The method according to any one of clauses 8 to 12, wherein the antigen is an antigenic peptide derived from p-amyloid (Ap), preferably wherein the antigenic peptide comprises, consists essentially of, or consists of, amino acids 1-15 of p-amyloid (Ap) (DAEFRHDSGYEVHHQ, SEQ ID NO: 9).

[0266] 14. The method according to any one of clauses 8 to 12, wherein the antigen is an antigenic peptide derived from a-synuclein, preferably wherein the antigenic peptide comprises, consists essentially of, or consists of, SEQ ID NO: 10 (GGKESMPVDPDNEA).

[0267] 15. The method according to any one of the preceding clauses, wherein the first surface molecule is a lipid-based vaccine adjuvant and the capture agent is specific for the lipid- based vaccine adjuvant, and wherein the second surface molecule is an antigen and the detectable analyte-binding agent is specific for the antigen, wherein the lipid-based adjuvant is preferably a glycolipid-based vaccine adjuvant.

[0268] 16. The method according to any one of clauses 1-14, wherein the first surface molecule is an antigen and the capture agent is specific for the antigen, and wherein the second surface molecule is a lipid-based vaccine adjuvant and the detectable analyte-binding agent is specific for the lipid-based vaccine adjuvant.

[0269] 17. The method according to clause 15 or 16, wherein the lipid-based adjuvant is a glycolipid-based vaccine adjuvant and wherein the antigen is an antigenic peptide. 18. The method according to any preceding clause, wherein one of the surface molecules is a glycolipid-based vaccine adjuvant that is an agonist of Toll-like receptor 4 (TLR4), wherein the TLR4 agonist is preferably MPLA or an analog thereof, most preferably Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (3D-(6-acyl) PHAD®).

[0270] 19. The method according to any preceding clause, wherein the capture agent and / or the analyte-binding agent is an antibody, preferably a monoclonal antibody.

[0271] 20. The method according to any preceding clause, wherein (a) the capture agent is an anti- MPLA antibody; or (b) the analyte-binding agent is an anti-MPLA antibody.

[0272] 21. The method according to clause 20, wherein the antibody comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1 , a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5 and / or a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein the antibody preferably comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH- CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5 and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0273] 22. The method according to clause 20 or 21 , wherein the antibody comprises a VH chain comprising the amino acid sequence of SEQ ID NO: 7 or a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto and / or VL chain comprising the amino acid sequence of SEQ ID NO: 8 or a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto.

[0274] 23. The method according to any one of clauses 20 to 22, wherein the antibody comprises a VH chain comprising the amino acid sequence of SEQ ID NO: 7 and / or VL chain comprising the amino acid sequence of SEQ ID NO: 8.

[0275] 24. The method according to any one of clauses 1-13 or 15 to 23, wherein one agent out of the detectable analyte-binding agent and the capture agent is an antibody that is specific for a surface molecule that is an antigenic peptide and wherein the antibody that is specific for the antigenic peptide is preferably an antibody that is specific for an antigenic peptide derived from p-amyloid (Ap), such as an antibody specific for an antigenic peptide comprising, consisting essentially of, or consisting of, amino acids 1-15 of p-amyloid (AP) (DAEFRHDSGYEVHHQ, SEQ ID NO: 9).

[0276] 25. The method according to clause 24, wherein the antibody is selected from antibodies 6E10, NAB228, 2C8, 4G8, WO-2 and DE2, preferably 6E10. 26. The method according to any one of clauses 1-12 or 14 to 23, wherein one agent out of the detectable analyte-binding agent and the capture agent is an antibody that is specific for a surface molecule that is an antigenic peptide and wherein the antibody that is specific for the antigenic peptide is preferably an antibody that is specific for an antigenic peptide antigenic peptide derived from a-synuclein, preferably wherein the antigenic peptide comprises, consists essentially of, or consists of, SEQ ID NO: 10 (GGKESMPVDPDNEA).

[0277] 27. The method according to clause 26, wherein the antibody is selected from antibodies MJFR1 , Syn211 and LB-509, preferably MJFR1.

[0278] 28. The method according to any preceding clause, wherein the lipid-based nanostructure is a liposome, preferably wherein the population of lipid-based nanostructures is a liposomal vaccine.

[0279] 29. The method according to clause 8, wherein the antigen is a T-cell antigen, wherein the antigen preferably comprises, consists essentially of, or consists of, one or more universal T- cell epitopes.

[0280] 30. The method according to clause 1, wherein the first surface molecule is a glycolipid- based vaccine adjuvant that is an agonist of Toll-like receptor 4 (TLR4), wherein the TLR4 agonist is preferably MPLA and most preferably 3D-(6-acyl) PHAD® and wherein the capture agent is specific for said glycolipid-based vaccine adjuvant and is preferably an anti-MPLA antibody, such as an anti-MPLA antibody as defined in any one of clauses 21-23, and wherein the second surface molecule is an antigenic peptide and the detectable analyte-binding agent is specific for that peptide, wherein the antigenic-peptide is preferably derived from a self-antigen.

[0281] 31. The method according to clause 1, wherein the first surface molecule is an antigenic peptide and the capture agent is specific for said antigenic peptide, wherein the antigenic peptide is preferably derived from a self-antigen, and wherein the second surface molecule is a glycolipid-based vaccine adjuvant that is an agonist of Toll-like receptor 4 (TLR4), wherein the TLR4 agonist is preferably MPLA and most preferably 3D-(6-acyl) PHAD® and the detectable analyte-binding agent is specific for that glycolipid-based vaccine adjuvant and is preferably an anti-MPLA antibody, such as an anti-MPLA antibody as defined in any one of clauses 21-23. 32. A method of assessing a population of lipid-based nanostructures, which may be a liposomal vaccine, the method comprising

[0282] (a) contacting the nanostructures with:

[0283] (i) a capture agent that is an anti-MPLA antibody, such as an anti-MPLA antibody as defined in any one of clauses 21-23

[0284] (ii) a detectable analyte-binding agent that is specific for an antigenic peptide;

[0285] (b) incubation under conditions allowing the formation of a complex between the capture agent, the lipid-based nanostructure and the detectable analyte-binding agent; wherein the capture agent is immobilised on a solid support; and

[0286] (c) detecting the complex by detecting the detectable analyte-binding agent, which may preferably comprise measuring the signal intensity of a signal emitted or generated by a label linked directly or indirectly to the detectable analyte-binding agent, wherein preferably

[0287] (i) the antigenic peptide is derived from p-amyloid (Ap), preferably wherein the antigenic peptide comprises, consists essentially of, or consists of, amino acids 1-15 of p-amyloid (Ap) (DAEFRHDSGYEVHHQ, SEQ ID NO: 9) (which may optionally be linked to an anchoring moiety) and the detectable analyte-binding agent is an antibody that binds thereto, such as antibody 6E10; or

[0288] (ii) the antigenic peptide is derived from a-synuclein, preferably wherein the antigenic peptide comprises, consists essentially of, or consists of, SEQ ID NO: 10 (GGKESMPVDPDNEA) (which may optionally be linked to an anchoring moiety) and the detectable analyte-binding agent is an antibody that binds thereto, such as antibody MJFR1.

[0289] 33. A method of assessing a population of lipid-based nanostructures, which may be a liposomal vaccine, the method comprising:

[0290] (a) contacting the nanostructures with:

[0291] (i) a capture agent that is specific for an antigenic peptide, and

[0292] (ii) a detectable analyte-binding agent that is an anti-MPLA antibody, such as an anti-MPLA antibody as defined in any one of clauses 21-23;

[0293] (b) incubation under conditions allowing the formation of a complex between the capture agent, the lipid-based nanostructure and the detectable analyte-binding agent; wherein the capture agent is immobilised on a solid support; and

[0294] (c) detecting the complex by detecting the detectable analyte-binding agent, which may preferably comprise measuring the signal intensity of a signal emitted or generated by a label linked directly or indirectly to the detectable analyte-binding agent, wherein preferably

[0295] (i) the antigenic peptide is derived from p-amyloid (A ), preferably wherein the antigenic peptide comprises, consists essentially of, or consists of, amino acids 1-15 of p-amyloid (Ap) (DAEFRHDSGYEVHHQ, SEQ ID NO: 9) (which may optionally be linked to an anchoring moiety) and the detectable analyte-binding agent is an antibody that binds thereto, such as antibody 6E10; or (ii) the antigenic peptide is derived from a-synuclein, preferably wherein the antigenic peptide comprises, consists essentially of, or consists of, SEQ ID NO: 10 (GGKESMPVDPDNEA) (which may optionally be linked to an anchoring moiety) and the detectable analyte-binding agent is an antibody that binds thereto, such as antibody MJFR1.

[0296] 34. The method according to any one of the preceding clauses, wherein in step (a) the nanostructure is contacted with the capture agent prior to, simultaneously with, or after it is contacted with the detectable analyte-binding agent.

[0297] 35. The method according to any one of the preceding clauses, wherein the method further comprises one or more wash or separation steps to remove any detectable analyte-binding agent that is not part of the complex prior to step (c).

[0298] 36. The method according to any one of the preceding clauses, wherein the detectable analyte-binding agent comprises a label, or wherein prior to step (c), the method further comprises a step of contacting the detectable analyte-binding agent with a labelled secondary agent, thereby linking the label indirectly to the detectable analyte-binding agent.

[0299] 37. The method according to clause 36, wherein the detectable analyte-binding agent is a primary antibody and the labelled secondary agent is a labelled secondary antibody.

[0300] 38. The method according to any one of the preceding clauses, wherein the label is selected from horseradish peroxidase, alkaline phosphatase, a coloured particle or a fluorescent moiety, preferably horseradish peroxidase or alkaline phosphatase.

[0301] 39. The method according to any one of the preceding clauses , wherein the method comprises adding a suitable substrate for the label and detecting or measuring the resulting signal.

[0302] 40. The method according to any preceding clause, wherein the method is a method of assessing a vaccine comprising a liposome that comprises both a vaccine adjuvant and an antigen displayed on the surface of the liposome, the method comprising the steps of

[0303] (a) contacting the vaccine with:

[0304] (i) a capture agent immobilised on a solid support, and

[0305] (ii) a detectable analyte-binding agent; wherein either the capture agent is specific for the vaccine adjuvant and the detectable analyte-binding agent is specific for the antigen, or the capture agent is specific for the antigen and the detectable analyte-binding agent is specific for the vaccine adjuvant;

[0306] (b) incubation under conditions allowing the formation of a complex between the capture agent, the liposome and the detectable analyte-binding agent; and (c) detecting the complex by detecting the detectable analyte-binding agent.

[0307] 41. The method according to clause 40, wherein the vaccine is a test vaccine and wherein step (c) comprises measuring the intensity of a signal emitted or generated by a label linked directly or indirectly to the detectable analyte-binding agent, and the method further comprises

[0308] (d) comparing the measured signal intensity of the test vaccine to a reference and / or a control; and / or

[0309] (e) determining one or more properties of the test vaccine on the basis of the result of step (c) and / or (d).

[0310] 42. The method according to clause 41 , wherein the reference is the signal intensity of a reference vaccine comprising a liposome that

[0311] (i) comprises the same antigen displayed on the surface of the liposome;

[0312] (ii) comprises a variant antigen displayed on the surface of the liposome;

[0313] (iii) comprises a different antigen displayed on the surface of the liposome; and / or

[0314] (iv) has a known vaccine property, such as a known ability to induce an antibody response, a known in vivo immunogenicity, or a known potency.

[0315] 43. The method according to any one of clauses 41 or 42, wherein the control is the signal intensity of a control liposomal population that is not capable of forming a complex with the capture agent and the detectable analyte-binding agent because the liposome of the control population

[0316] (a) does not display on its surface the vaccine adjuvant; and / or

[0317] (b) does not display on its surface the antigen.

[0318] 44. The method according to any one of the preceding clauses, wherein the method comprises assessing antigen presentation of the liposomal vaccine.

[0319] 45. The method according to any one of the preceding clauses, wherein the method comprises assessing the heterogeneity of the liposomal vaccine.

[0320] 46. The method according to any one of clauses 41 to 45, wherein step (e) comprises assessing the antibody response to the liposomal vaccine; predicting the in vivo immunogenicity of the liposomal vaccine; and / or predicting the potency of the liposomal vaccine.

[0321] 47. The method according to any one of clauses 41 to 46, wherein the method comprises assessing at least two liposomal vaccines and further comprises ranking the liposomal vaccines according to the signal intensity measured in step (c).

[0322] 48. The method according to clause 47, wherein the method comprises comparing multiple batches of the liposomal vaccine comprising the same antigenic peptide. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications and patents specifically mentioned herein are incorporated by reference in their entirety for all purposes in connection with the invention.

[0323] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended clauses. Moreover, all aspects and embodiments of the invention described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, including those taken from other aspects of the invention (including in isolation) as appropriate.

[0324] Brief description of the drawings

[0325] Non-limiting embodiments of the invention are now described by way of example only, with reference to the accompanying drawings, in which:

[0326] Fig. 1 Diagram of sandwich ELISA in an exemplary setup wherein A is an antigen and capture of the complex onto a solid support is via B, a vaccine adjuvant. This setup may be referred to as “adjuvant-based capture”. The detectable analyte-binding agent is shown lacking a covalently linked label; and use of a labelled secondary agent is shown to indirectly link a label to the detectable analyte-binding agent, but alternatively the detectable analytebinding agent may comprise a covalently linked label.

[0327] Fig. 2 Diagram of sandwich ELISA in an exemplary setup wherein A is a vaccine adjuvant and B is an antigen and capture of the complex onto a solid support is via this antigen. This setup may be referred to as “antigen-based capture”. The detectable analyte-binding agent is shown lacking a covalently linked label; and use of a labelled secondary agent is shown to indirectly link a label to the detectable analyte-binding agent, but alternatively the detectable analyte-binding agent may comprise a covalently linked label.

[0328] Fig. 3 Analysis of the immunogenicity of a-synuclein liposomal vaccines in vitro.

[0329] The graph shows resulting OD values for sandwich ELISA analysis. Samples were tested in duplicates. Optical density at 405 nm (average ± standard deviation) is plotted against peptide concentration. ACI-IL.062 is a negative control having the same composition as ACI- 7110.062, ACI-7111.062 and ACI-7108.062 but which does not comprise the alpha-synuclein antigenic peptide.

[0330] Fig. 4 Graph showing IgG titers against an a-synuclein antigenic peptide in mice immunized with ACI-7108.062, ACI-7110.062 or ACI-7111.062 liposomal preparations (vaccines). The Y axis shows anti-a-synuclein antigenic peptide IgG titers in All / mL and the X axis shows days post immunization. ACI-7108.062 liposomal vaccine treated group (left), ACI-7110.62 treated group (middle) and ACI-7111.062 treated group (right); at theoretical a-synuclein antigenic peptide dose of 0.2 pg dose (squares), 2 pg (circles) or 80 pg (triangles). Geometric mean ±95% confidence interval (Cl) is also indicated (with n=10 for groups 2, 3, 5, 6, 8 and 9; and n=5 for groups 1, 4 and 7).

[0331] Fig. 5. Sandwich ELISA results for liposomal formulations “ACI-24.060200 pg / mL” and “ACI-24.060400 pg / mL: ACI-24E.060 (1:1)”. Optical density at 405 nm (average ± standard deviation) is plotted against liposome dilution factor. Samples were tested in duplicates. The ELISA set up was as shown in Figure 1.

[0332] Fig. 6 Sandwich ELISA results for liposomal formulations “ACI-24.060200 pg / mL” and “ACI- 24.060400 pg / mL: ACI-24E.060 (1:1)”. Optical density at 405 nm (average ± standard deviation) is plotted against liposome dilution factor. Samples were tested in duplicates. The ELISA set up was as shown in Figure 2.

[0333] Fig.7 Graph showing results of standard competitive ELISA assay of Example 4.

[0334] Fig. 8 Sandwich ELISA results for liposomal formulations “ACI-24.060” (control sample, stored at 2-8 °C) and “ACI-24.0606M 25C” (stressed sample, stored 6 months at 23-27 °C). Optical density at 405 nm (average ± standard deviation) is plotted against liposome dilution factor. Samples were tested in duplicates. The ELISA set up was as shown in Figure 1.

[0335] Fig. 9 Sandwich ELISA results for liposomal formulations “ACI-24.060” (positive control sample, stored at 2-8 °C) and “ACI-24.0606M 25C” (stressed sample, stored 6 months at 23-27 °C). Optical density at 405 nm (average ± standard deviation) is plotted against liposome dilution factor. Samples were tested in duplicates. The ELISA set up was as shown in Figure 2.

[0336] Fig. 10 Graph showing IgG titers against Abetal -42 in mice immunized with “ACI-24.060” (positive control sample, stored at 2-8 °C) and “ACI-24.0606M 25C” (stressed sample, stored 6 months at 23-27 °C). The Y axis shows anti-Abeta1-42 IgG titers (AU / mL) and the X axis shows time measured by days post immunization. “ACI-24.060” (control sample, stored at 2-8 °C) vaccine treated group (black circles), “ACI-24.0606M 25C” (stressed sample, stored 6 months at 23-27 °C treated group (grey squares). Geometric mean ±95% confidence interval (Cl) is also indicated (with n=10 animals / group).

[0337] Fig. 11A and 11B Graphical representation of ACI-8039.044-918.14C2-Ab1 and 6E10 titration on ELISA plates coated with 3D-(6-acyl) PHAD® or biotinylated liposomes captured onto Neutravidin-coated ELISA plates. 6E10 was used as a control for liposomes displaying Abeta1-15 antigenic peptide. The following sequences are disclosed herein:

[0338] SEQ ID Nos 1-8 are sequences of the anti-MPLA antibody as shown in Example 6.

[0339] SEQ ID NO: 9 is amino acids 1-15 of -amyloid (A ) (DAEFRHDSGYEVHHQ).

[0340] SEQ ID NO: 10 is GGKESMPVDPDNEA.

[0341] Non-limiting embodiments of the invention are now described by way of example only, with reference to the accompanying drawings.

[0342] Examples

[0343] Example 1

[0344] A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to assess antibody binding to an antigen displayed on the surface of a liposome. A schematic of the ELISA set up is shown in Figure 1.

[0345] Three liposomal formulations were prepared, each displaying a surface molecule that is a peptide antigen derived from alpha-synuclein. The three peptide antigens shared the same peptide sequence, GGKESMPVDPDNEA (SEQ ID NO: 10), and all comprised a hydrophobic moiety, comprising palmitic acid chains, but the location at which the palmitic acid chains were attached to the peptide differed between the three liposomal formulations.

[0346] In ACI-7110.062, the peptide was tetrapalmitoylated via its N and C terminus; in ACI-7111.062, the peptide was dipalmitoylated via its N and C terminus; and in ACI-7108.062, the peptide was dipalmitoylated via its N terminus

[0347] The three formulations contained comparable peptide concentrations (464 to 491 pg / mL) and comparable lipid concentrations (16.2 to 16.8 mg / mL).

[0348] Any indication provided herein of the weight of a peptide should be understood to refer to the weight of the peptide including any covalently attached moieties, such as lipid chains. Thus, the reference to 464 to 491 pg / mL of the peptide should be understood to refer to the weight of the palmitoylated peptide (peptide sequence + palmitic chains). The Mw of the three palmitoylated peptides are in the same range (2236.71 to 2969.87).

[0349] The liposomes all comprised the vaccine adjuvant MPLA, specifically 3D-(6-acyl) PHAD®. The anti-MPLA antibody provided in Example 6 was used as a capture agent to capture the liposomes onto a solid support.

[0350] Using an anti-alpha-synuclein antibody that is specific for the antigen (antigenic peptide SEQ ID NQ:10) comprised in ACI-7110.062, ACI-7111.062 and ACI-7108.062, an ELISA was carried out to assess the capacity of the antibody to bind the antigens when displayed on the surface of a liposome. Any anti-alpha-synuclein antibody having an epitope within the antigenic region of the antigen may suitably be used. Suitable antibodies include any antialpha synuclein antibody having an epitope binding the antigenic peptide SEQ ID NO: 10 . Thus, here the commercially available anti-alpha-synuclein [MJFR1] antibody was used as a detectable analyte-binding agent. ACI-IL.062 was used as a negative control (ACI-IL.062 has the same composition as ACI-7110.062, ACI-7111.062 and ACI-7108.062 but does not comprise the alpha-synuclein antigenic peptide).

[0351] Methods

[0352] ELISA plates (Nunc MicroWell 96-Well, Non-Treated, Flat-Bottom Microplate, ThermoFisher Scientific) were coated with 5 pg / mL of ACI-8039.044-918.14C2-Ab1 (anti-MPLA murine lgG2a recombinant antibody) in PBS, overnight at 4 °C. Plates were then washed with 0.0025% Tween-20 / PBS and blocked with 1% bovine serum albumin (BSA) in 0.0025% Tween-20 / PBS for 1 hour at 37 °C. The blocking buffer was removed by flipping the plate and patting on a paper napkin.

[0353] Serial dilutions of liposomal formulations were made using PBS buffer comprising 1% BSA and 0.0025% Tween-20. The concentration of the liposomal formulation is expressed as the peptide concentration.

[0354] Serial dilutions of liposomal formulations were then added to the plates. Samples were tested in duplicates. Liposomal samples were added in a 2-fold serial dilution starting at 1 pg / mL peptide concentration (ACI-7110.062, ACI-7108.062) or 50 pg / mL peptide concentration (ACI-7111.062). Plates were incubated for 1 hour at 37 °C, and then washed as described above. Anti-alpha-synuclein [MJFR1] antibody (a rabbit monoclonal antibody available from Abeam, catalogue ref. ab209420) was then added to the wells at 0.5 pg / mL (ACI-7110.062, ACI-7108.062) or 5 pg / mL (ACI-7111.062) and incubated for 1 hour at 37 °C, after which the plates were washed.

[0355] Anti-rabbit IgG-AP (an antibody that binds to the rabbit Fc of Anti-alpha-synuclein [MJFR1] antibody and is labelled with alkaline phosphatase; available from Abeam, catalogue ref. ab6722) was subsequently added to the wells at 1 :1000 dilution as a secondary agent to (indirectly) label the detectable analyte binding agent. Plates were incubated for 1 hour at 37 °C, and then washed. p-Nitrophenyl Phosphate (pNPP) substrate was added and plates incubated at RT for 20 min (ACI-7110.062, ACI-7108.062) or 30 min (ACI-7111.062). The plate was read at 405 nm using an ELISA plate reader (Tecan, Switzerland).

[0356] Results

[0357] Results are shown in Fig. 3. The half maximal effective concentration (EC50) was calculated by plotting the optical density at 405 nm against the logarithms of concentrations of peptide using a four-parameter logistics (4-PL) fit, using the GraphPad Prism (version 10.0.2) application. The EC50 values are summarized in Table 1.

[0358] Table 1

[0359] These results confirm that the ELISA is able to differentiate between different presentations of an antigen on the surface of liposomal vaccine preparations.

[0360] Example 2 - Immunogenicity study of alpha-synuclein liposomal vaccines in mice

[0361] An in vivo study was carried out using the liposomal preparations (liposomal vaccines) of Example 1 to evaluate the immunogenicity of these three liposomal preparations (liposomal vaccines) in C57BL / 6J female mice at three different doses. a) Study design

[0362] A total of 75 C57BL / 6J female mice, approximately 10 weeks old at 1stimmunization, were allocated to nine groups (with 10 or 5 animals / group) as indicated in Table 2.

[0363] The nine groups were immunized three times by subcutaneous (s.c) injection into the subcutis of the dorsum on Days 1, 15 and 29 with either ACI-7108.062 (Groups 1 to 3), ACI- 7110.062 (Groups 4 to 6) or ACI-7111.062 (Groups 7 to 9) at 80, 2 or 0.2 pg of alpha- synuclein antigenic peptide / injection.

[0364] Table 2 s.c: subcutaneous

[0365] A fixed volume of 0.2 mL / animal was administered for each subcutaneous injection. For the groups 1, 4 and 7 (immunized with 0.2 pg dose), or 2, 5 and 8 (immunized with 2 pg dose), the corresponding vaccine was diluted 400 or 40 times respectively in formulation buffer (10 mM histidine, 250 mM sucrose) before injection. b) Immunogenicity results in plasma:

[0366] Immunogenicity against an a-synuclein-derived antigenic peptide (SEQ ID NO: 10) was assessed in plasma samples collected at pre-dose and one week after each immunization (on Days 8, 22 and 36).

[0367] The anti-a-synuclein-derived antigenic peptide IgG titers were analyzed at each timepoint by an enzyme-linked immune sorbent assay (ELISA). Briefly, BSA conjugated to SEQ ID NO: 10 peptide was immobilized on 96-well micro titers plates overnight. After washing and blocking, plates were incubated with the plasma samples for two hours at 37°C, allowing the antibodies present in plasma to bind the peptide. After incubation, the plates were washed to remove non-reactive plasma components.

[0368] The antibody / antigen complex was detected via a secondary anti-mouse IgG antibody conjugated to alkaline phosphatase. pNPP (p-Nitrophenyl Phosphate) substrate was added to the wells and optical density was read at 405nM in an ELISA plate reader. The anti-a- synuclein-derived antigenic peptide IgG titers were back-calculated against a calibration curve in eight two-fold serial dilution, using an unweighted four-parameter logistic regression model using the Gen5 software (BioTek, Switzerland). Results are expressed as AU / mL.

[0369] Data are expressed as individual values. Geometric mean ±95% confidence interval (Cl) is also indicated. Figures 4 shows that animals immunized with ACI-7108.062 and ACI-7110.062 vaccines developed a robust antibody response against the peptide of SEQ ID NO: 10 after one immunization, at all doses tested and in a dose dependent manner, with higher IgG titers observed in animals immunized at highest dose (80 pg dose), whereas animals immunized with ACI-7111.062 showed only low IgG titers at the highest dose tested (80 pg dose), with no IgG titers induced in 0.2 or 2 pg dose immunized animals.

[0370] The results confirm the potential of liposomal vaccines ACI-7108.062 and ACI-7110.062 to induce a strong immune response in vivo.

[0371] The results are in agreement with previous findings that the manner in which an antigen is anchored to a liposome and displayed on its surface impacts the antibody response to the liposomal formulation.

[0372] Example 3

[0373] An analysis of the results of Examples 1 and 2 shows that the results of the in vitro assay of Example 1 are in good agreement with the results of the in vivo assay of Example 2. The EC50 determined in vitro serves as a good indicator of antibody titres against the target antigen induced by the liposomal vaccine in vivo.

[0374] Thus, an assay as exemplified in Example 1 provides an effective method for predicting antibody response to a liposomal vaccine or predicting the in vivo immunogenicity of a liposomal vaccine.

[0375] Example 4

[0376] The Sandwich ELISA was also shown to be effective for determining the distribution of surface molecules, such as antigens, in a liposomal population.

[0377] Two liposomal formulations were prepared containing the same concentration of the same surface-displayed peptide antigen (Abeta1-15 (SEQ ID NO: 9)) but the peptide antigen was differently distributed in the liposomal population.

[0378] Concentrations are expressed as the concentration of the tetrapalm itoylated Abeta1-15 peptide antigen.

[0379] In one formulation (denoted “ACI-24.060200 pg / mL”), the peptide antigen was added to all liposomes in the liposome population to reach a final concentration of 200 pg / ml.

[0380] In the other formulation (denoted “ACI-24.060400 pg / mL+ACI-24E.O6O”), the peptide antigen was in a first step added to all liposomes in the liposome population to reach a final concentration of 400 pg / mL. The resulting (intermediate) liposome population was subsequently mixed 1:1 with a liposome population lacking the peptide antigen to yield a heterogeneous liposomal vaccine preparation (i.e. a liposomal vaccine preparation comprising a substantial proportion of liposomes lacking the peptide antigen) denoted ACI- 24.060400 pg / mL+ACI-24E.O6O.

[0381] In both formulations, all (or substantially all) of the liposomes comprised 3D-(6-acyl) PHAD®, which served as a vaccine adjuvant targeted by a capture or analyte-binding agent.

[0382] Using a standard competitive ELISA set up, no difference in antigen distribution could be detected (see Fig.7). Briefly, ELISA plates were coated with an antigen comprising an antigenic peptide derived from Ap. The liposomal formulation comprising the antigenic peptide derived from Ap as a surface-displayed antigen were used to compete with the ELISA plates for binding to an anti-Abeta antibody (antibody 6E10).

[0383] By contrast, the difference in antigen distribution could be detected by sandwich ELISA using an “adjuvant-based capture” set-up (see Figure 1 for illustration), with results shown in Fig. 5. The difference could also be detected using an “antigen-based capture” set up (see Figure 2 for illustration), with results shown in Fig. 6.

[0384] Methods

[0385] Adjuvant-based capture

[0386] ELISA plates (Nunc MicroWell 96-Well, Non-Treated, Flat-Bottom Microplate, ThermoFisher Scientific) were coated with 5 pg / mL of ACI-8039.044-918.14C2-Ab1 (anti-MPLA human I gG 1 recombinant antibody) in PBS, overnight at 4 °C. Plates were then washed with 0.0025% Tween-20 / PBS and blocked with 1% bovine serum albumin (BSA) in 0.0025% Tween-20 / PBS for 1 hour at 37 °C. The blocking buffer was removed by flipping the plate and patting on paper napkin.

[0387] Serial dilutions of liposomal formulations were then added to the plates. Samples were tested in duplicates. Liposomal samples were added in a 2-fold serial dilution starting at 1:50 dilution factor. Plates were incubated for 1 hour at 37 °C, and then washed. The 6E10 antibody (BioLegend Inc., ref. 803002) which is specific for the surface-displayed antigen, was then added to the wells at 0.5 pg / mL and incubated for 1 hour at 37 °C, after which the plates were washed. Anti-mouse IgG-AP, a secondary antibody labelled with alkaline phosphatase, (Jackson ImmunoResearch Europe Ltd. ref. 115-055-164) was subsequently added to the wells at 1 : 1000 dilution. Plates were incubated for 1 hour at 37 °C, and then washed. p-Nitrophenyl Phosphate (pNPP) substrate was added and plates incubated at RT for 2 hours.

[0388] The plate was read at 405 nm using an ELISA plate reader (Tecan, Switzerland).

[0389] The maximum effect (Emax) was calculated by plotting the optical density (OD) at 405 nm against the logarithms of concentrations of peptide using a four-parameter logistics (4-PL) with shared bottom and hill slope, using the GraphPad Prism (version 10.0.2) application. Results are summarized in Table 3.

[0390] Antigen-based capture

[0391] ELISA plates (Nunc MicroWell 96-Well, Non-Treated, Flat-Bottom Microplate, ThermoFisher Scientific) were coated with 8 pg / mL of 6E10 (BioLegend Inc., ref. 803002) in PBS, overnight at 4 °C. Plates were then washed with 0.0025% Tween-20 / PBS and blocked with 1% bovine serum albumin (BSA) in 0.0025% Tween-20 / PBS for 1 hour at 37 °C. The blocking buffer was removed by flipping the plate and patting on a paper napkin.

[0392] Serial dilutions of liposomal formulations were then added to the plates. Samples were tested in duplicates. Liposomal samples were added in a 2-fold serial dilution starting at 1 :10 dilution factor. Plates were incubated for 1 hour at 37 °C, and then washed. ACI-8039.044- 918.14C2-Ab1 (anti-MPLA human lgG1 recombinant antibody) was then added to the wells at 0.1 pg / mL and incubated for 1 hour at 37 °C, after which the plates were washed. Antihuman IgG-HRP (Thermo Fisher Scientific, ref. 31420) was subsequently added to the wells at 1 : 1000 dilution. Plates were incubated for 1 hour at 37°C, and then washed. 2,2' -Azino- bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS, Biotium ref. BI010050) was added and plates incubated at RT for 45 min. The plate was read at 405 nm using an ELISA plate reader (Tecan, Switzerland).

[0393] The half maximal effective concentration (EC50) was calculated by plotting the optical density at 405 nm against the logarithms of dilution factor using a four-parameter logistics (4- PL) with shared bottom, top and hill slope, using the GraphPad Prism (version 10.0.2) application. Results are summarized in Table 3.

[0394] Table 3

[0395] Figure 5 and Table 3 (second column) show that a heterogeneous liposomal vaccine preparation (i.e. a liposomal vaccine preparation comprising a substantial proportion of liposomes lacking the peptide antigen) presents a decreased Emax OD compared to a homogeneous liposomal vaccine preparation (i.e. liposomal vaccine preparation wherein the peptide antigen was added to all liposomes in the liposome population) in the adjuvant-capture set-up. Figure 6 and Table 3 (third column) show that a heterogeneous liposomal vaccine preparation presents a decreased EC50 compared to homogeneous liposomal vaccine preparations in the antigen-capture set-up.

[0396] These results confirm that the method of the invention is able to detect differences in antigen distribution in both antigen-capture and adjuvant-capture set-ups and assess the heterogeneity of liposomal vaccine preparations.

[0397] Example 5 - In vivo concordance for ACI-24.060

[0398] Two liposomal vaccine preparations were produced containing comparable concentration of the tetrapalmitoylated Abeta1-15 antigenic peptide, which comprises SEQ ID NO: 9 (400- 460 pg / mL range), this concentration serving as a proxy for the liposome concentration. The reference liposomal vaccine preparation was stored at long-term storage temperature (2- 8°C) and the stressed liposomal vaccine preparation was stored at 23-27 °C for 6 months. No change in peptide concentration was observed.

[0399] Method descriptions: sandwich ELISA adjuvant capture

[0400] The capacity of antibodies to bind the vaccine adjuvant 3D-(6-acyl) PHAD® and the antigen Abeta1-15 antigenic peptide (SEQ ID NO:9), which is derived from Ap, displayed on the surface of the liposomes was determined using a sandwich enzyme-linked immunosorbent assay (ELISA) using an adjuvant-capture set up.

[0401] ELISA plates (Nunc MicroWell 96-Well, Non-Treated, Flat-Bottom Microplate, ThermoFisher Scientific) were coated with 5 pg / mL of ACI-8039.044-918.14C2-Ab1 (anti-MPLA human I gG 1 recombinant antibody) in PBS, overnight at 4 °C. Plates were then washed with 0.0025% Tween-20 / PBS and blocked with 1% bovine serum albumin (BSA) in 0.0025% Tween-20 / PBS for 1 hour at 37 °C. The blocking buffer was removed by flipping the plate and patting on a paper napkin.

[0402] Serial dilutions of liposomal formulations were then added to the plates. Samples were tested in duplicates. Liposomal samples were added in a 2-fold serial dilution starting at 1:50 dilution factor. Plates were incubated for 1 hour at 37 °C, and then washed. 6E10 antibody (BioLegend Inc., ref. 803002) was then added to the wells at 0.5 pg / mL and incubated for 1 hour at 37 °C, after which the plates were washed. Anti-mouse IgG-AP (Jackson ImmunoResearch Europe Ltd., ref. 115-055-164) was subsequently added to the wells at 1 :1000 dilution. Plates were incubated for 1 hour at 37 °C, and then washed. p-Nitrophenyl Phosphate (pNPP) substrate was added and plates incubated at RT for 75 min. The plate was read at 405 nm using an ELISA plate reader (Tecan, Switzerland).

[0403] The maximum effect (Emax) was calculated by plotting the optical density at 405 nm against the logarithms of concentrations of peptide using a four-parameter logistics (4-PL) with shared bottom and hill slope, using the GraphPad Prism (version 10.0.2) application. Results are shown in Figure 8 and summarized in Table 4.

[0404] Method descriptions: sandwich ELISA antigen capture

[0405] The capacity of antibodies to bind 3D-(6-acyl) PHAD® vaccine adjuvant and the antigen Abeta1-15 antigenic peptide (SEQ ID NO:9), which is derived from Ap, displayed on the surface of the liposomes was also determined using a sandwich enzyme-linked immunosorbent assay (ELISA) using an antigen capture set up.

[0406] ELISA plates (Nunc MicroWell 96-Well, Non-Treated, Flat-Bottom Microplate, ThermoFisher Scientific) were coated with 4 pg / mL of 6E10 (BioLegend Inc., ref. 803002) in PBS, overnight at 4 °C. Plates were then washed with 0.0025% Tween-20 / PBS and blocked with 1% bovine serum albumin (BSA) in 0.0025% Tween-20 / PBS for 1 hour at 37 °C. The blocking buffer was removed by flipping the plate and patting on a paper napkin.

[0407] Serial dilutions of liposomal formulations were then added to the plates. Samples were tested in duplicates. Liposomal samples were added in a 2-fold serial dilution starting at 1:10 dilution factor. Plates were incubated for 1 hour at 37 °C, and then washed. ACI-8039.044- 918.14C2-Ab1 (anti-MPLA human lgG1 recombinant antibody) was then added to the wells at 0.1 pg / mL and incubated for 1 hour at 37 °C, after which the plates were washed. Antihuman IgG-HRP (ThermoFisher Scientific, ref. 31420) was subsequently added to the wells at 1:1000 dilution. Plates were incubated for 1 hour at 37°C, and then washed. 2,2'-Azino- bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS, Roche ref. 10-102-946-001) was added and plates incubated at RT for 75 min. The plate was read at 405 nm using an ELISA plate reader (Tecan, Switzerland).

[0408] The maximum effect (Emax) was calculated by plotting the optical density at 405 nm against the logarithms of concentrations of peptide using a four-parameter logistics (4-PL) with shared bottom, top and hill slope, using the GraphPad Prism (version 10.0.2) application. Results are shown in Figure 9 and summarized in Table 4.

[0409] Table 4 Both the adjuvant-capture set-up (Figure 8 and Table 4 (second column)) and the antigencapture set-up (Figure 9 and Table 4 (third column)) showed a decrease in Emax OD for the stressed liposomal vaccine preparation.

[0410] Immunogenicity testing

[0411] Study design

[0412] A total of 20 C57BL / 6J female mice, approximately 10 weeks old at 1stimmunization, were allocated to two groups (with 10 animals / group) as indicated in Table 5. The two groups were immunized three time by intramuscular immunization in the lateral surface of left quadriceps muscle on Days 1 , 15 and 29 with “ACI-24.060” (reference sample, stored at 2-8 °C) or “ACI-24.0606M 25C” (stressed sample, stored 6 months at 23-27 °C) at 0.02 pg of Pal1-15 / injection.

[0413] Table 5: i.m: intramuscular

[0414] A fixed volume of 0.05 mL / animal was administered for each injection, diluting the vaccine 1000-times in formulation buffer (10 mM histidine, 250 mM sucrose, pH 6.5) before injection.

[0415] Immunogenicity results in plasma:

[0416] Immunogenicity against Abetal -42 was assessed in plasma samples collected at pre-dose and one week after each immunization (on Days 8, 22 and 36).

[0417] The anti-Abeta1-42 IgG titers were analyzed at each timepoint by an enzyme-linked immune sorbent assay (ELISA). Briefly, Abeta1-42 peptide film was immobilized on 96-well micro titers plates overnight. After washing and blocking, plates were incubated with the plasma samples for two hours at 37°C, allowing the antibodies present in plasma to bind the Abetal- 42. After incubation, the plates were washed to remove non-reactive plasma components.

[0418] The antibody / antigen complex was detected via a secondary anti-mouse IgG antibody conjugated to alkaline phosphatase. pNPP (p-Nitrophenyl Phosphate) substrate was added to the wells and optical density was read at 405nM in an ELISA plate reader. The anti- Abeta1-42 IgG titers were back-calculated against a calibration curve in eight two-fold serial dilution, using a four-parameter logistic regression model using the SoftMax Pro. Results are expressed as AU / mL. Results are shown in Figure 10 and Table 6. Data are expressed as individual values. Geometric mean ±95% confidence interval (Cl) is also indicated. Table 6

[0419] Results summary

[0420] As seen from (Figure 10 and Table 6) the stressed liposomal vaccine preparation induced a lower antibody response in-vivo than the reference liposomal vaccine.

[0421] The in-vitro results described above for both the adjuvant capture and antigen capture set-ups (Table 4, Figures 8 and 9) were consistent with the lower antibody response induced in-vivo by the stressed liposomal vaccine preparation.

[0422] These results demonstrate that stability-related differences can be detected by the method of the invention, using either the antigen-capture or adjuvant-capture set-ups, and that they correlate with in vivo immunogenicity.

[0423] Example 6 - Anti-MPLA antibody generation and characterization

[0424] The liposome-based vaccines were prepared according to the protocols published in W02012 / 055933. Liposomal vaccine preparations comprising the Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (Synthetic) (3D-(6-acyl) PHAD®) adjuvant, hereinafter 3D-(6-acyl) PHAD®, were used for antibody generation.

[0425] Female C57BL / 6JOIaHsd (C57BL / 6) and BALB / c OlaHsd (BALB / c) wild-type mice (Harlan, USA) were received at 9 weeks of age. Vaccinations started at 10 weeks. Mice were vaccinated with the liposomal vaccine preparations by subcutaneous injection (s.c.) on days 0, 7, 28, 49. Mice were bled and heparinized plasma prepared 2 days before immunization (pre-immune plasma) and on days 14, 35 and 56 after first immunization. The presence of antibodies binding to 3D-(6-acyl) PHAD® was evaluated in mice plasma collected at day 56. Briefly, biotinylated control liposomal vaccine comprising only the 3D-(6-acyl) PHAD® adjuvant was captured on pre-coated Neutravidin plates to evaluate the specific response to the adjuvant by ELISA. The dose response of mice presenting an anti-3D-(6-acyl) PHAD® titer was determined and the two mice showing the highest response were selected for the generation of hybridomas.

[0426] Mice were euthanized and fusion with myeloma cells was performed using cells from lymph nodes and spleen. Screening for antibodies from the successfully fused hybridoma cell lines was performed by ELISA. The evaluation of the antibody response in hybridoma supernatant was carried out at multiple timepoints of sequential cell cloning steps leading to the final isolation of single cells defining a monoclonal hybridoma and the identification of ACI- 8039.044-918.14C2-Ab1 antibody.

[0427] Antibody specificity

[0428] ACI-8039.044-918.14C2-Ab1 antibody was further characterized by ELISA against various liposomal vaccine preparations comprising either MPLA, 3D-(6-acyl) PHAD® and / or Abetal- 15 antigenic peptide, as well as liposome controls without adjuvant to assess the specificity (Figure 11 A). For the ELISA, the different liposomal vaccine preparations were biotinylated and captured on pre-coated Neutravidin plates, or plates directly coated with 3D-(6-acyl) PHAD®. The 6E10 antibody was used as a positive control for liposome displaying Abeta 1- 15 antigenic peptide (Figure 11 B).

[0429] As seen in Figure 11 A, ACI-8039.044-918.14C2-Ab1 antibody binds to both liposome- displayed MPLA and 3D-(6-acyl) PHAD® as well as isolated 3D-(6-acyl) PHAD® and does not exhibit any cross-reactivity to Abeta1-15 antigenic peptide or any other components of the liposome. These results confirm the selectivity of the ACI-8039.044-918.14C2-Ab1 antibody for MPLA, including 3D-(6-acyl) PHAD®.

[0430] Antibody sequencing

[0431] Sequencing of the ACI-8039.044-918.14C2-Ab1 antibody was performed, and the sequence of the antibody heavy and light chain variable region are shown in Table 7.

[0432] Table 7

[0433] VH (SEQ ID NO: 7)

[0434] QVQLQQSGAELARPGALVRLSCKASGYTFTSYAISWVKQRTGQGLEWIGDIYPRRGNAYYN

[0435] ENFKDKATLTADKSSSTAYMELRSLTSEDSAVYFCARSGVYYSSYYAMDYWGQGTSVTVSS

[0436] VL (SEQ ID NO: 8)

[0437] QAVVTQESALTTSPGGTVILTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTSNRAPGVPV

[0438] RFSGSLIGDKAALTITGAQTEDDAMYFCALWYSTHYVFGGGTKVTVL.

Claims

CLAIMS1. A method of assessing a vaccine comprising a liposome that comprises both a vaccine adjuvant and an antigen displayed on the surface of the liposome, the method comprising the steps of(a) contacting the vaccine with:(i) a capture agent immobilised on a solid support, and(ii) a detectable analyte-binding agent; wherein either the capture agent is specific for the vaccine adjuvant and the detectable analyte-binding agent is specific for the antigen, or the capture agent is specific for the antigen and the detectable analyte-binding agent is specific for the vaccine adjuvant;(b) incubation under conditions allowing the formation of a complex between the capture agent, the liposome and the detectable analyte-binding agent; and(c) detecting the complex by detecting the detectable analyte-binding agent.

2. The method according to claim 1, wherein the capture agent is specific for the vaccine adjuvant and the detectable analyte-binding agent is specific for the antigen.

3. The method according to claim 1, wherein the capture agent is specific for the antigen and the detectable analyte-binding agent is specific for the vaccine adjuvant.

4. The method according to any one of the preceding claims, wherein the vaccine adjuvant is a lipid-based vaccine adjuvant.

5. The method according to claim 4, wherein the lipid-based vaccine adjuvant is a glycolipid- based vaccine adjuvant.

6. The method according to any one of the preceding claims, wherein the vaccine adjuvant is an agonist of Toll-like receptor 4 (TLR4).

7. The method according to any one of the preceding claims, wherein the vaccine adjuvant is monophosphoryl lipid A (MPLA).

8. The method according to any one of the preceding claims, wherein the vaccine adjuvant comprises, consists essentially of, or consists of, Monophosphoryl Hexa-acyl Lipid A, 3- Deacyl (3D-(6-acyl) PHAD®).

9. The method according to any one of the preceding claims, wherein the antigen is an antigenic peptide, wherein the antigenic peptide is optionally linked to an anchoring moiety.

10. The method according to any one of the preceding claims, wherein the antigen is derived from a self-antigen.

11. The method according to claim 10, wherein the self-antigen is selected from p- amyloid (A ), Tau, a-synuclein, huntingtin, prion, an amylin protein, IL-17 or IL-27.

12. The method according to any one of claims 9-11 , wherein the antigen is an antigenic peptide derived from p-amyloid (Ap).

13. The method according to claim 12, wherein the antigenic peptide comprises, consists essentially of, or consists of, amino acids 1-15 of p-amyloid (AP) (DAEFRHDSGYEVHHQ, SEQ ID NO: 9).

14. The method according to any one of claims 9-11 , wherein the antigenic peptide is derived from a-synuclein.

15. The method according to claim 14, wherein the antigenic peptide comprises, consists essentially of, or consists of, SEQ ID NO: 10 (GGKESMPVDPDNEA).

16. The method according to any one of the preceding claims, wherein the capture agent is an antibody, preferably a monoclonal antibody.

17. The method according to any one of the preceding claims, wherein the detectable analyte-binding agent is an antibody, preferably a monoclonal antibody.

18. The method according to claim 16 or claim 17, wherein the antibody is an anti-MPLA antibody.

19. The method according to claim 18, wherein the antibody comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1 , a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5 and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

20. The method according to claim 18 or 19, wherein the antibody comprises a VH chain comprising the amino acid sequence of SEQ ID NO: 7 or a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto and / or VL chain comprising the amino acid sequence of SEQ ID NO: 8 or a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto.

21. The method according to claim 16 or claim 17, wherein the antibody is specific for an antigenic peptide derived from p-amyloid (Ap).

22. The method according to claim 21, wherein the antibody is selected from antibodies 6E10, NAB228, 2C8, 4G8, WO-2 and DE2.

23. The method according to claim 16 or claim 17, wherein the antibody is specific for an antigenic peptide derived from alpha-synuclein.

24. The method according to claim 23, wherein the antibody is selected from antibodies MJFR1, Syn211 and LB-509.

25. The method according to any one of claims 1 to 9 or 16 to 20, wherein the antigen is derived from a pathogen.

26. The method according to any one of claims 1 to 9, 16 to 20 or 25, wherein the antigen is a T-cell antigen.

27. The method according to claim 26, wherein the antigen comprises, consists essentially of, or consists of, a universal T-cell epitope.

28. The method according to any one of claims 1 to 2 or 4 to 24, wherein the vaccine adjuvant is Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (3D-(6-acyl) PHAD®); the capture agent is an anti-MPLA antibody that comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL- CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5 and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; the antigen is an antigenic peptide derived from a self-antigen; and the detectable analyte-binding agent is an antibody specific for said antigenic peptide.

29. The method according to any one of claims 1 to 2, 4 to 13, or 16 to 22, wherein the vaccine adjuvant is Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (3D-(6-acyl) PHAD®); the capture agent is an anti-MPLA antibody that comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence ofSEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL- CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5 and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; the antigen is an antigenic peptide that comprises, consists essentially of, or consists of, amino acids 1-15 of -amyloid (Ap) (DAEFRHDSGYEVHHQ, SEQ ID NO: 9); and the detectable analyte-binding agent is an antibody specific for said antigenic peptide, preferably antibody 6E10.

30. The method according to any one of claims 1 to 2, 4 to 11, 14 to 20 or 23 to 24, wherein the vaccine adjuvant is Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (3D-(6-acyl) PHAD®); the capture agent is an anti-MPLA antibody that comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL- CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5 and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; the antigen is an antigenic peptide that comprises, consists essentially of, or consists of, SEQ ID NO: 10 (GGKESMPVDPDNEA); and the detectable analyte-binding agent is an antibody specific for said antigenic peptide, preferably antibody MJFR1.

31. The method according to any one of claims 1, 3 to 13, or 16 to 22, wherein the vaccine adjuvant is Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (3D-(6-acyl) PHAD®); the detectable analyte-binding agent is an anti-MPLA antibody that comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1 , a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5 and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; the antigen is an antigenic peptide that comprises, consists essentially of, or consists of, amino acids 1-15 of p-amyloid (Ap) (DAEFRHDSGYEVHHQ, SEQ ID NO: 9); and the capture agent is an antibody specific for said antigenic peptide, preferably antibody 6E10.

32. The method according to any one of claims 1, 3 to 11, 14 to 20, or 23 to 24 wherein the vaccine adjuvant is Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (3D-(6-acyl) PHAD®);the detectable analyte-binding agent is an anti-MPLA antibody, wherein the antibody comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1 , a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5 and a VL- CDR3 comprising the amino acid sequence of SEQ ID NO: 6; the antigen is an antigenic peptide that comprises, consists essentially of, or consists of, SEQ ID NO: 10 (GGKESMPVDPDNEA); and the capture agent is an antibody specific for said antigenic peptide, preferably antibody MJFR1.

33. The method according to any one of the preceding claims, wherein step (c) comprises measuring the intensity of a signal emitted or generated by a label linked directly or indirectly to the detectable analyte-binding agent, and the method further comprises(d) comparing the measured signal intensity to a reference or control; and / or(e) determining one or more properties of the vaccine on the basis of the result of step (c) and / or (d).

34. The method according to claim 33, wherein prior to step (c), the method further comprises a step of contacting the detectable analyte-binding agent with a labelled secondary agent, thereby linking the label indirectly to the detectable analyte-binding agent.

35. The method according to claim 34, wherein labelled secondary agent is a secondary antibody labelled with horseradish peroxidase or alkaline phosphatase.

36. The method according to any one of claims 33-35, wherein the reference is the signal intensity of a reference vaccine comprising a liposome that(i) comprises the (same) antigen displayed on the surface of the liposome;(ii) comprises a variant antigen displayed on the surface of the liposome;(iii) comprises a different antigen displayed on the surface of the liposome; and / or(iv) has a known vaccine property, such as a known ability to induce an antibody response, a known in vivo immunogenicity, or a known potency.

37. The method according to any one of claims 33-36, wherein the control is the signal intensity of a control liposomal population that is not capable of forming a complex with the capture agent and the detectable analyte-binding agent because the liposome of the control population(a) does not display on its surface the vaccine adjuvant; and / or(b) does not display on its surface the antigen.

38. The method according to any one of claims 33 to 37, wherein the method comprises assessing antigen presentation of the liposomal vaccine.

39. The method according to any one of claims 33 to 37, wherein the method comprises assessing the heterogeneity of the liposomal vaccine.

40. The method according to any one of claims 33 to 39, wherein step (e) comprises assessing the antibody response to the liposomal vaccine; predicting the in vivo immunogenicity of the liposomal vaccine; and / or predicting the potency of the liposomal vaccine.

41. The method according to any one of claims 33 to 40, wherein the method comprises assessing at least two liposomal vaccines and further comprises ranking the liposomal vaccines according to the signal intensity measured in step (c).

42. The method according to claim 41, wherein the method comprises comparing multiple batches of the liposomal vaccine comprising the same antigenic peptide.

43. An anti-MPLA antibody that comprises a Heavy Chain Variable Region comprising:(i) a variable heavy (VH) CDR1 that has the amino acid sequence of SEQ ID NO: 1;(ii) a VH CDR2 that has the amino acid sequence of SEQ ID NO: 2; and(iii) a VH CDR3 that has the amino acid sequence of SEQ ID NO: 3; and / or wherein the antibody comprises a Light Chain Variable Region comprising:(iv) a variable light (VL) CDR1 that has the amino acid sequence of SEQ ID NO: 4;(v) a VL CDR2 that has the amino acid sequence of SEQ ID NO: 5; and(vi) a VL CDR3 that has the amino acid sequence of SEQ ID NO: 6.

44. The antibody according to claim 43, wherein the antibody comprises a Heavy Chain Variable Region comprising the amino acid sequence of SEQ ID NO: 7 or a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto and / or a Light Chain Variable Region comprising the amino acid sequence of SEQ ID NO: 8 or a sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity thereto.

45. The antibody according to any one of claims 43 or 44, wherein the antibody comprises a Heavy Chain Variable Region comprising the amino acid sequence of SEQ ID NO: 7 and / or a Light Chain Variable Region comprising the amino acid sequence of SEQ ID NO: 8.

46. Use of an antibody according to any one of claims 43 to 45 as an in-vitro tool or an analytical reference.

47. A kit comprising: a) a capture agent; and b) detectable analyte-binding agent; wherein the capture agent or detectable analyte-binding agent is an anti-MPLA antibody.

48. A kit according to claim 47, wherein the capture agent or detectable analyte-binding agent is an antibody according to any one of claims 43 to 45.

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