WO2013030799A1 - B cell assay - Google Patents

B cell assay Download PDF

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WO2013030799A1
WO2013030799A1 PCT/IB2012/054498 IB2012054498W WO2013030799A1 WO 2013030799 A1 WO2013030799 A1 WO 2013030799A1 IB 2012054498 W IB2012054498 W IB 2012054498W WO 2013030799 A1 WO2013030799 A1 WO 2013030799A1
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antigen
cells
influenza
ha
method
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PCT/IB2012/054498
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French (fr)
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Monia BARDELLI
Grazia Galli
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Novartis Ag.
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Priority to US61/573,314 priority
Priority to GB201212550A priority patent/GB201212550D0/en
Priority to GB1212550.6 priority
Application filed by Novartis Ag. filed Critical Novartis Ag.
Publication of WO2013030799A1 publication Critical patent/WO2013030799A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966Animal cells
    • G01N33/56972White blood cells
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses, e.g. hepatitis E virus
    • C07K16/1018Orthomyxoviridae, e.g. influenza virus
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/20Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Abstract

Improved assays for directly enumerating, sorting and cloning antigen-specific plasmablasts and MBCs by reducing non-specific binding of labeled antigen to cell-surface glycans, such as sialic acid, when labeling for antigen-specific plasmablasts and MBCs. Two approaches for reducing non-specific background binding include: (1) saturation or disruption of non-specific binding sites; (2) reduction of the ability of the labeled antigen to bind sialic acid sites. By saturating and/or disrupting antigen-binding sialic acid residues and other non-specific antigen-binding proteins on cell surfaces in the test sample, prior to the labeling of antigen-specific B cells with the antigen of interest, background labeling and aggregation of cells are reduced.

Description

B CELL ASSAY

TECHNICAL FIELD

This invention relates to assays for analysing B cell responses, and in particular assays that directly label and detect antigen-specific cells using labeled antigen. BACKGROUND ART

Induction of a strong B cell response following vaccination is crucial for immune protection from many viruses, such as the influenza virus. It has been shown that the frequency of antibody secreting cells (ASC) and memory B cells (MBC) following vaccination varies according to vaccinees' immune experience and time from antigenic challenge (Galli G. et al. Proc Natl Acad Sci USA. 12;106(19):7962-7. 2009; Wrammert J. et al. Nature 29;453(7195):667-71 2008). Analyses of such responses have relied heavily on indirect measures of B cell activation, such as the measurement of antibody titers in serum or other bodily fluids. The major limit of the assays used for these analyses is that they can be used only to assess changes in frequency of ASCs or MBCs providing only indirect information on their functionality. Owing to low frequency of antigen(Ag)-specific B cells in peripheral blood, direct and detailed analysis of their functionality and clonal repertoire requires long and laborious in vitro culturing procedures and properly designed screening strategies. Polyclonal analysis of MBCs is limited in a number of ways, for example, it cannot provide: ex vivo enumeration of Ag-specific MBCs; functional characterisation of MBC populations; information on the impact of age and immune experience on changes in the repertoire of plasma cells and MBCs induced by vaccination; or analysis of genes differentially expressed by circulating effector and MBC subsets. The ability to directly analyse the MBCs themselves following pathogen encounter would be highly valuable for vaccine-related studies and for assessment of vaccine efficacy in different vaccinee populations. However, direct assessment of Ag-specific effectors and MBC are rarely performed in the context of influenza clinical trials. MBCs express highly antigen-specific cell surface receptors (or B cell receptors [BcRs]) that bind to native non-processed antigen. A labeling method to detect antigen-specific MBCs would provide a potent tool to directly analyse frequencies of MBCs induced by vaccination directly on ex vivo peripheral blood mononuclear cell (PBMC) samples without biases due to culture conditions. Difficulties in the labeling are caused by the intrinsic binding of glycan-binding antigen to sialic acid residues, or other non-specific binding compounds, present on the surface of the cells. This makes the non-specific background labeling on B cells extremely high.

Binding of tagged antigen to B cells has been used previously as a strategy to identify Ag-binding B cells directly ex vivo (Slifka MK. Et al. J Immunol Methods. 2006), but attempts to apply this protocol to glycan-binding viruses, such as influenza virus, have been unsuccessful. For example, direct ex vivo identification of hemagglutinin(HA)-specific MBCs has proven to be very difficult. An early attempt was to try using biotinylated whole virus to label HA-specific B cells taken from immunized mice (Doucett et al. Journal of Immunological Methods 2005, 303:40-52). These experiments were unsuccessful because the virus adhered to a large number of B cells, most of which were not virus-specific. The same group had more success using purified biotinylated PR8 (A/Puerto Rico/8/34) HA to label HA-specific cells. They demonstrated about 1% HA-specific labeling in virus-immunized but not in control mice. These results suggest that labeling with purified HA more clearly identifies influenza HA-specific B cells ex vivo. However, this method is still limited by the very high labeling background caused by HA binding to sialic residues that are naturally present on the surface of all B cells. In addition, non-specific binding can lead to aggregation of cells, making it difficult to sort and detect cells of interest.

Some approaches for reducing background labeling in MBC analysis have been previously suggested but they have had limited success and come with several drawbacks. For example, Townsend et al. (J. Immunol. Method 249, 137, 2001) used mixtures of two antigen-conjugates in which the same antigen can be conjugated to two fluorochromes. Each conjugate was then used for labeling at subsaturating concentrations and only cells that showed dual fluorescence were identified as being specific. However, this method was limited by a relatively high frequency of "false negatives" resulting in a high risk of underestimating frequencies of specific MBCs and a risk that only the highest binding avidity MBCs would be selectively isolated. Another suggestion in Doucett was to use multiple labels (such as propidium iodide that binds to dead cells, T cell and macrophage markers etc) to eliminate non-specific B cells.

There is still a need for improved assays that can directly quantify and characterize antigen-specific B cells. There is also a need for methods of identifying and isolating and producing antibodies that are broadly neutralizing to viral antigens. DISCLOSURE OF THE INVENTION

The invention provides improved assays for directly enumerating, sorting and cloning antigen- specific plasmablasts and MBCs. In particular the invention can reduce non-specific binding of labeled antigen to cell-surface glycans, such as sialic acid, when labeling for antigen-specific plasmablasts and MBCs. Two possible approaches for reducing non-specific background binding include: (1) saturation or disruption of non-specific binding sites; (2) reduction of the ability of the labeled antigen to bind sialic acid sites. This invention focuses on methods to saturate or remove non-specific binding sites.

The inventors have developed an improved technique for labeling antigen-specific plasmablasts and MBCs. They have discovered that by saturating and/or disrupting antigen-binding sialic acid residues and other non-specific antigen-binding proteins on cell surfaces in the test sample, prior to the labeling of antigen-specific B cells with the antigen of interest, they can reduce background labeling and aggregation of cells. They have demonstrated that saturation and/or disruption of non-specific binding can be achieved by including an additional pre-incubation step in the method.

Therefore, the invention provides a method for detecting in a sample plasmablasts and/or MBCs which express an antibody specific for a glycan-binding antigen of interest, wherein the method comprises the steps of:

(a) pre-incubation of the sample with a saturator and/or disrupter of non-specific glycan binding sites to give a pre-incubated sample;

(b) labeling of the pre-incubated sample with labeled bait to give a labeled sample,

wherein the labeled bait binds B cell receptors (BcRs) on the plasmablasts/MBCs which bind to the glycan-binding antigen of interest; and

(c) detection of the plasmablasts/MBCs in the labeled sample.

Compared to traditional assays such as ELISPOT and limiting dilution assay, this labeling method is useful to detect antigen-specific MBCs directly ex vivo, without bias due to culture conditions or proliferation or antibody secretion, providing a more accurate representation of in vivo clonal composition of MBCs.

Compared to Doucett et al., 2005, this method provides reduced aggregation of cells and lower background labeling. This method also provides opportunities for double labeling approaches, identification of cross-reactive MBCs, and direct ex vivo analysis of plasmablasts as well as MBCs. Methods of the present invention have been shown to work using samples from humans, whereas the Doucett method was only demonstrated using samples from immunized mice. This makes the present invention more applicable to human patients that require treatment against viral infection, and also more applicable for testing immune responses to vaccination in human vaccinees. The labeled bait used in the Doucett method was a virion-derived HA protein. Isolation of HA from the surface of purified influenza virus particles is laborious and requires relatively large amounts of starting material. The current inventors have shown that recombinant proteins, such as recombinant HA, can be used as the bait in the present invention. Recombinant HA is commercially available and so makes the method simpler and cheaper than the method described by Doucett.

Therefore, the invention also provides a method for detecting plasmablasts which have antibodies that are cross-reactive to more than one antigen of interest, wherein the method comprises labeling plasmablasts and/or MBCs with two labeled baits, wherein each bait has a different label. The invention also provides a method for detecting plasmablasts which have antibodies that are cross-reactive with variants of an antigen of interest, such as influenza HA antigen, wherein the method comprises labeling plasmablasts with labeled bait, and wherein the labeled bait binds BcRs that are cross-reactive to the antigen of interest. The invention also provides a method for detecting human plasmablasts and/or MBCs which express antibody specific to an antigen of interest, wherein the method comprises labeling plasmablasts with labeled bait, wherein the labeled bait binds BcRs that are cross-reactive to the antigen of interest.

The invention also provides a method for directly detecting plasmablasts and/or MBCs which express antibody specific to an antigen of interest, wherein the method comprises labeling plasmablasts with labeled recombinant protein bait, wherein the labeled bait binds BcRs that are cross-reactive to the antigen of interest.

Sample

Typically, the sample is taken from a patient or a vaccinee after exposure to a glycan-binding antigen of interest. The patient or vaccinee may be animal or human. The sample may be blood, or blood that has been treated or purified by methods well known in the art e.g. to provide serum, plasma, or specific populations of blood cells. In a preferred embodiment, the sample is PBMCs isolated from blood. PBMCs can be isolated from patient blood samples by methods well known in the art.

Plasmablasts and memory B cells

Upon stimulation by a T cell, which usually occurs in germinal centers, activated B cells begin to differentiate into more specialized cells. Some will differentiate into MBCs, while most will become plasmablasts that secrete large volumes of antibodies and divide rapidly. Plasmablasts may stay in this state for several days and then either die or irrevocably differentiate into mature, fully differentiated plasma cells. Both plasmablasts and MBCs have specific cell-surface receptors (BcRs) which are raised in response to exposure to an antigen and are therefore antigen-specific. Analysis of antigen-specific plasmablasts is useful for detecting responses soon after antigen exposure (e.g. by vaccination), whereas the detection of antigen-specific MBCs is useful for analyzing responses a longer time after antigen exposure. Therefore, in some embodiments of the invention, the patient sample is taken up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, or 10 days after exposure to an antigen of interest. For example, for the detection of antigen-specific plasmablasts, the patient sample may be taken approximately 7 days after exposure to an antigen of interest. In other embodiments of the invention, the sample is taken from a patient or vaccinee more than 10 days after exposure to an antigen of interest. For example, the sample is taken more than 10 days, more than 15 days, more than 20 days, more than 25 days, more than 30 days, more than 35 days, more than 40 days after exposure to an antigen of interest. In a further embodiment, the sample is taken after a much longer period of time after exposure to the antigen of interest, such as more than 1 month, more than 6 months, more than 1 year, more than 5 years, more than 20 years, or any time up until the death of the patient or vaccinee. In a further embodiment, the sample is taken from a dead patient or vaccinee.

Subpopulations of PBMCs enriched for plasmablasts or MBCs may also be used as the sample. For example, to detect plasmablasts or MBCs directly from peripheral blood, this population could be selected by fluorescent activated cell sorting (FACS) using labeling of appropriate surface markers. Cell surface markers for MBCs include but are not limited to any one or more or all of the markers selected from the group consisting of: CD19, CD20, CD27, and CD38. Cell surface markers for MBCs include but are not limited to any one or more or all of the markers selected from the group consisting of: CD 19, CD20, and CD27. These markers can be labeled using labeled anti-Ig-mAbs (monoclonal antibodies).

Circulating plasmablasts can be detected in a patient sample by FACS gating strategies that select a specific CD3"CD19+/"CD20low/"CD27++CD38+ population. This is a commonly used gating strategy for circulating plasmablasts (see Jacobi AM et al. Arthritis Rheum. 2003 May;48(5): 1332-42).

CD138 could also be added to the gating strategy to identify the CD 138" and CD138+ subsets, which are thought to correspond to diverse differentiation stages.

Circulating MBCs can be detected in a patient sample by FACS gating strategies that select specific CD20+CD27+ or CD20+CD27dim populations. This is a commonly used gating strategy for detecting circulating MBCs (see Klein U et al., J Exp Med. 1998 Nov 2; 188(9): 1679-89). Exposure to antigen

Typically, the patient or vaccinee will be exposed to a glycan-binding antigen of interest by natural exposure to a viral pathogen or by vaccination. In some embodiments the sample is from a subject ho survived natural infection by a lethal pathogen.

A glycan-binding antigen is typically an antigen that binds carbohydrate moieties, for example on the host cell surface. Many viral proteins interact with glycans, such as sialic acid residues, on the host cell surface.

Influenza HA and neuraminidase (NA) are examples of glycan-binding antigens. Examples of influenza vaccines include but are not limited to MF59-adjuvanted 2010 seasonal influenza vaccine (Fluad), Focetria (HlNlCalifornia+MF59) and unadjuvanted TIV (Agrippal). Influenza virus strains used in vaccines change from season to season. In the current inter-pandemic period, trivalent vaccines include two influenza A strains (H1N1 and H3N2) and one influenza B strain. It will be understood that the invention is not limited to use with the vaccines described herein, but is designed to be suitable for analysis of antigen-specific responses following vaccination with any past, current or future vaccines against a glycan-binding virus, such as influenza virus, and any past, current or future vaccine doses.

Influenza strains, subtypes and eludes

Influenza A virus currently displays sixteen HA serotypes: HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15 and H16. Any of these serotypes can be used in the context of the invention. The virus may have any of NA serotypes: Nl, N2, N3, N4, N5, N6, N7, N8 or N9. H1N1 and H3N2 are the two virus subtypes currently circulating in humans.

The invention can be used to analyse patient responses to pandemic influenza A virus strains.

Characteristics of a pandemic strain are: (a) it contains a new HA compared to the HA in currently- circulating human strains, i.e. one that has not been evident in the human population for over a decade (e.g. H2), or has not previously been seen at all in the human population (e.g. H6 or H9, that have generally been found only in bird populations), such that the vaccine recipient and the general human population are immunologically naive to the strain's HA; (b) it is capable of being transmitted horizontally in the human population; and (c) it is pathogenic to humans. The antigenic properties of HA allow the classification of influenza A viruses into two major groups 1 and 2, which are further classified into five clades and 16 subtypes. Group 1 comprises HI, H2, H5, H6, H8, H9, HI 1, H12, H13, and H16. Group 2 comprises H3, H4, H7, H10, H14 and H15. Therefore, HI and H3 are in different groups and H5 is more closely related to HI than to H3.

Influenza B virus currently does not display different HA subtypes, but influenza B virus strains do fall into two distinct lineages. These lineages emerged in the late 1980s and have HAs which can be antigenically and/or genetically distinguished from each other and are more closely related to either B/Victoria/2/87 or B/Yamagata/16/88 (Rota et al. (1992) J Gen Virol 73:2737-42).

Pre-incubation step

The aim of the pre-incubation step is to saturate and/or disrupt non-specific binding sites, so that the labeled bait binds primarily to antigen-specific BcRs and not to non-specific binding sites. In this way, background labeling is reduced and labeled cells represent those which express antigen-specific BcRs. The term "saturator" refers to any binding component that is able to bind and saturate non-specific binding sites. The term "non-specific" in the context of the present invention, means a binding site that the glycan-binding antigen of interest or the bait binds to, but that is not a BcR. The non-specific binding site is typically present on the surface of many cells, not only on MBCs or plasmablasts. The non-specific binding sites are typically formed by glycans, such as sialic acid. The aim of the saturator is to saturate the non-specific binding sites so that the glycan-binding antigen of interest or the bait cannot bind to the non-specific binding sites, and instead binds to BcRs that are expressed on the cell surface of plasmablasts and MBCs. In the context of the present invention, the term

"saturate" means that the percentage of the available binding sites that are filled is sufficient to reduce the non-specific background labeling by the bait. In a preferred embodiment, the preincubation step should result in the non-specific binding sites in the sample being more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%), more than 90% or more than 99% saturated. The saturator may be a protein, peptide, small-molecule, nucleic acid, antibody, or any other compound that binds to non-specific binding sites. The term "disrupter" refers to any component that can disrupt non-specific binding sites, for example, by disrupting intramolecular or intermolecular bonding within the binding site or near the binding site so that the conformation or local structure of the binding site is changed or destroyed. This may be by cleavage of molecular bonding or by binding of the disrupter to glycans in such a way that the conformation of the binding site is disrupted. The aim of the disrupter is to disrupt the non-specific binding sites so that the glycan-binding antigen of interest or the bait cannot bind to the non-specific binding sites, and instead binds to BcRs that are expressed on the cell surface of plasmablasts and MBCs. In a preferred embodiment, more than 20%, more than 30%, more than 40%), more than 50%o, more than 60%o, more than 70%o, more than 80%o, more than 90%o or more than 99%o of the available non-specific binding sites in the sample are disrupted. The disrupter may be a protein, such as an enzyme, a peptide, small-molecule, nucleic acid, antibody, or any other compound that disrupts non-specific binding sites.

In a preferred embodiment, the saturator and/or disrupter results in a reduction of non-specific binding of the bait of more than 20%, more than 30%o, more than 40%o, more than 50%o, more than 60%, more than 70%, more than 80%, more than 90% or more than 99%. The inventors found that pre-incubation of cells with soluble lectins, fetuin or alpha-2,6- sialopentasaccharides, which bind sialic acid residues, was not sufficient to saturate all influenza HA non-specific binding sites. These compounds did not significantly reduce background labeling and sometimes led to aggregation of cells, making discrimination of MBCs difficult. However, the inventors have shown surprisingly that pre-incubation with HA and NA antigens from heterologous serotypes (compared to the glycan-binding antigen of interest) is a successful method for saturating non-specific binding of an HA antigen. In addition, pre-incubation with HA and NA (HANA) or with only NA subunit antigens from heterologous serotypes reduced cell aggregation (compared to methods without pre-incubation and methods comprising pre-incubation with soluble lectins and alpha-2,6-sialopentasaccharides) without inducing changes in cell morphology.

Therefore, in some embodiments of the invention, the saturator and/or disrupter is HANA or NA subunit antigens from a heterologous serotype to the viral HA-antigen of interest.

For example, if the antigen of interest is an influenza A HI antigen, then the saturator and/or disrupter may comprise any one or more of the HA antigens selected from the group consisting of: H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, and H16. For example, the saturator may comprise HANA derived from H5N1 A/Vietnam/1203/2004, H3N2

A/Panama/2007/99, H3N2 A/Wisconsin/67/2005, H3N2 A/California/7/2004, H3N2

A/Brisbane/ 10/2007 or H3N2 A/Fujian/411/2002. As a further example, if the antigen of interest is an influenza A H3 antigen, then the saturator and/or disrupter may comprise any one or more of the HA antigens selected from the group consisting of: HI, H2, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, and H16. For example, the saturator and/or disrupter may comprise HANA derived from H5N1 A/Vietnam/1203/2004, H1N1 A/California/7/2009, H1N1 A/Solomon Islands/3/2006, H1N1 A/New Caledonia/20/99 or H1N1 A/Brisbane/59/2007.

As a further example, if the antigen of interest is an influenza A H5 antigen, then the saturator and/or disrupter may comprise any one or more of the HA antigens selected from the group consisting of: HI, H2, H3, H4, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, and H16. For example, the saturator and/or disrupter may comprise HANA derived from H1N1 A/California/7/2009, H1N1 A/Solomon Islands/3/2006, H1N1 A/New Caledonia/20/99, H1N1 A/Brisbane/59/2007, H3N2 A/Panama/2007/99, H3N2 A/Wisconsin/67/2005, H3N2 A/California/7/2004, H3N2

A/Brisbane/ 10/2007 or H3N2 A/Fujian/411/2002.

Neuraminidase enzymes are "disruptors" that cleave the glycosidic linkages of sialic acid residues on cell surfaces. Pre-incubation with NA strips alpha-2,6-sialic acid from the B-cell surface, thus disrupting non-specific binding of HA to sialic acid residues. Therefore, the saturator and/or disrupter may be NA, such as NA from Influenza or Clostridium difficile. Examples of NA that may be used in the pre-incubation step include but are not limited to N2876, N5631 and N5521. A preferred NA is N5631. HA acts as a "saturator" because it binds to glycan residues on the cell surface.

Pre-incubation with HANA or with only NA successfully prevents B cell aggregation and reduces non-specific binding. However, the inventors found that they could reduce background binding to a greater extent with HANA. Therefore, when the antigen of interest is an influenza antigen, the saturator and/or disruptor preferably comprises influenza HA.

In one embodiment, the glycan-binding antigen of interest is an influenza A HA, and the saturator is one or more influenza B antigen, particularly HANA. Examples of influenza B type strains include but are not limited to: B/Ann Arbor/1/94, B/Yamanashi/166/98, B/Johannesburg/5/99, B/Victoria/504/2000, B/Hong Kong/330/2001, B/Brisbane/32/2002, and B/Jilin/20/2003. Influenza A and influenza B viruses share many features. However, reassortment between influenza A and B virus RNA segments does not occur (Ghenkina & Ghendon, Acta Virol 23, 91-106, 1979; Gotlieb & Hirst, J Exp Med 99, 307-320, 1954). Thus, influenza B viruses do not have pandemic potential. Moreover, influenza B viruses harbour some interesting genetic differences (reviewed by Palese & Shaw, In Fields Virology, 5th edn, pp. 1647-1689. Edited by Knipe, D. M. & Howley, P. M. Philadelphia: Lippincott Williams & Wilkins, 2007), including additional encoded proteins like NB, lack of other proteins like PB 1-F2, difference in protein length and also length of non-coding regions of the genome, which suggest both convergent and divergent evolution. They also utilize unusual alternative coding strategies that allow expansion of the genetic repertoire achieved from a relatively small RNA virus genome. This means that HANA proteins from type B strains have fewer common epitopes than different HANA subtypes from type A strains. It is therefore highly surprising that the inventors have demonstrated that pre-incubation with type B HANA can successfully saturate non-specific binding sites to allow detection of MBCs specific for influenza A antigens, such as group 1 and group 2 HA.

An advantage of using influenza B antigens as the saturators in the pre-incubation step is that double labeling approaches can then be used to identify cells that have antigen-specific BcRs that are broadly cross-reactive against multiple HA-antigens from influenza A group 1, group 2 or against groups 1 and 2 (see sections below on double labeling and cross-reactivity). For detecting BcRs that are cross-reactive across multiple strains, a preferred saturator molecule will saturate the maximum number of non-specific glycan binding sites but will have the minimum affinity for epitopes that are conserved across group 1, or group 1 and 2. Therefore, by alternating the use of H3N2 and B antigens this method allows, for the first time, labeling for the three seasonal strains (HI , H3 and B) to be performed in the same PBMC sample. For example, one strategy is to split the same PBMC sample into different tubes, pre-incubating with B to stain for HI and H3, or pre-incubating with H3N2 to stain for B and HI, or with H1N1 to stain for B and H3. The saturator and/or disruptor may be labeled or unlabeled. However, in a preferred embodiment, the first binding compound is unlabeled because it is mostly used for saturation purposes and labeling may interfere with binding potential.

In some embodiments, the pre-incubation step involves adding a saturator and/or disruptor to the sample at a concentration between approximately 0.05 and 20 μg/107 PBMC, between approximately 0.1 and 15 μg/107 PBMC, between approximately 0.2 and 5 μg/107 PBMC, between approximately 1 and 2 μg/107 PBMC, between approximately 0.1 and 1 μg/107 PBMC or approximately 1 μg/107 PBMC.

The saturator and/or disruptor should be incubated with the sample for sufficient time to allow all non-specific binding sites to be saturated and/or disrupted. This may be at least approximately 5 minutes, at least approximately 10 minutes, at least approximately 15 minutes, or more than 15 minutes. The sample may be held at room temperature (RT) or another temperature to optimize the pre-incubation step, but is typically at below room temperature, such as 4°C, during pre-incubation.

In some embodiments the pre-incubated sample may be washed, for example in buffer, by methods well known in the art, prior to adding labeled bait. There may also be other intermediary steps to prepare the pre-incubated sample before the labeled bait is added. However, in some embodiments, the labeled bait is added directly to the pre-incubated sample without any washing of the cells.

Labeled Bait

The intention of the labeled bait is that it should specifically bind to specific cell-surface receptors (BcPvs) on plasmablasts and MBCs that have been generated in response to the antigen exposure. Any labeled cells can then be detected, sorted and enumerated using label detection methods, such as FACS, as described below.

The labeled bait is typically closely related to the glycan-binding antigen of interest that the patient or sample has already been exposed to. Typically the labeled bait will have very similar or the same binding epitopes as the glycan-binding antigen of interest. The labeled bait may be a recombinant version of an antigen. It may be a soluble version, for example a protein (such as influenza A HA) with deletion of the transmembrane domain. Similarly, the labeled bait might be a truncated version of the glycan-binding antigen of interest. The labeled bait may or may not be glycosylated. The labeled bait may have similar, different or the same glycosylation patterns as the glycan-binding antigen of interest.

For example, when the glycan-binding antigen of interest is an influenza HA antigen, the labeled bait will typically be a recombinant HA protein derived from influenza strains and generated using standard cloning and expression techniques. Recombinant HA is sometimes denoted herein as rHA (although the term HA can also refer to recombinant HA) and is readily available (e.g. from Protein Science). Typically, rHA proteins are produced in baculovirus, although other expression systems are commonly available.

Obtaining good levels of separation between labeled and non-labeled B cells is crucial for the accurate enumeration of HA-positive events by flow cytometry. The choice of label used for labeling strongly affects the levels of separation obtained (Baumgarth and Roederer, J. Immunol. Methods 243, 77, 2000). The choice of label used is also heavily dependent on the detection apparatus used. Methods for choosing appropriate labels and methods for labeling proteins, such as HA antigen, are well known in the art.

Typically the label will be a fluorescent label (also known as fluorophores, fluors, or fluorochromes), such as fluorescein-based dyes, Alexa-488, Alexa-647 or other fluorophores from the Alexa Fluor™ group (Invitrogen). Other commonly used dye groups which are suitable for use in the context of the invention include CF dye (Biotium), BODIPY (Invitrogen), DyLight Fluor (Thermo Scientific, Pierce), Atto and Tracy (Sigma Aldrich) FluoProbes (Interchim) and MegaStokes Dyes (Dyomics).

Quantum dots may also be used as antigen labels. Quantum dots have narrow emission peaks which facilitate the use of more channels when using FACS detection. This makes quantum dots particularly suitable for double or multiple labeling protocols (see section on Double Labeling Approaches).

Another important consideration when labeling the antigen, is to ensure that B cell epitopes are not masked by the label. One way to minimise interference is to carefully select the method by which the label is linked to the antigen.

The antigen, such as influenza HA, may be recombinantly fused to a fluorescent protein or covalently attached to biotin, which may then be attached to a label via streptavidin. Techniques for producing fusion proteins and for biotinylation are well known in the art.

Other labels can be directly chemically linked to chemical groups that may be naturally present on the antigen of interest, such as amino groups (Active ester, Carboxylate, Isothiocyanate, hydrazine), carboxyl groups (carbodiimide), thiols (maleimide, acetyl bromide), or azides (via click chemistry or non-specifically (glutaraldehyde).

To ensure that a strong signal is obtained from the labeled antigens, more than one label may be attached to a single antigen protein. However, tagging with too many labels may interfere with the binding epitopes. Therefore, in some embodiments, a single labeled antigen of interest has 1 , 2, 3, 4 or 5 flurophores. The most preferred ratio is 2 to 3 fluorophores per single labeled antigen. This ratio can be achieved, for example, by incubation of antigens, such as rHA, with fluorophores at approximately 5-15 fold, approximately 8-12 fold or approximately 10 fold molar excess of fluorochrome to protein.

Typical examples of labeled antigens that can be used in the context of this invention include, but are not limited to rHl, rH3 and rH5 conjugated to Alexa-488 or Alexa-647. rHl may, for example, be derived from any one of the group consisting of: H1N1 A/California/7/2009, H1N1 A/Solomon Islands/3/2006, H1N1 A/New Caledonia/20/99 and H1N1 A/Brisbane/59/2007. rH3 may, for example, be derived from any one of the group consisting of: H3N2 A/Panama/2007/99, H3N2 A/Wisconsin/67/2005, H3N2 A/California/7/2004, H3N2 A/Brisbane/ 10/2007 or H3N2 A/Fujian/411/2002. rH5 may, for example, be derived from H5N1 A Vietnam/ 1203/2004. Negative control labels may be used in conjunction with the method of the invention. For example human serum albumin (HSA) can be used as a negative control label for fluorescence background.

The labeled bait may be added to the pre-incubated sample at a concentration between approximately 0.05 and 20 μg/107 PBMC, between approximately 0.1 and 15 μg/107 PBMC, between approximately 0.2 and 5 μg/107 PBMC, between approximately 1 and 2 μg/107 PBMC, between approximately 0.1 and 1 μg/107 PBMC or approximately 1 μg/107 PBMC. Typically, the labeled bait is added at the same or similar concentration as the saturator and/or disruptor was added to the same sample.

After or at the same time as addition of the labeled bait to the pre-incubated sample, a mixture of antibodies may also be added to the sample. The sample to which the labelled bait and optionally the mixture of antibodies has been added will then be incubated for sufficient time for the labelled bait to bind to the specific BcRs on the B cells in the sample. This may be at least approximately 10 minutes, at least approximately 15 minutes, at least approximately 30 minutes, at least approximately 45 minutes, at least approximately 60 minutes or more than 60 minutes. The sample may be held at room temperature or another temperature to optimize the labeling step, but is typically at below room temperature, such as 4°C, during labeling.

The labeled sample may be treated further prior to the detection step. For example, the labeled sample may be washed e.g. with staining buffer followed by resuspension of the cells in EDTA.

Double labeling approach

In some embodiments, a double labeling approach can be used in which two labeled baits are used in step (b) of the method of the invention. In some embodiments the two baits are labeled with different fluorochromes. In some embodiments, the two labeled baits are used for simultaneous labeling. In other embodiments, the two labeled baits are used for sequential labeling. In a preferred embodiment, two baits labeled with two different fluorochromes are used to simultaneously label MBCs and/or plasmablasts that specifically bind to both labeled antigens of interest. For example, the same samples may be simultaneously labeled with Hl-Alexa488 and H5-Alexa647 antigens.

The inventors have shown that this double labeling approach allows both common and serotype- specific epitopes to be detected. The presence of MBCs with such cross-reactive capacity in seasonal influenza has been repeatedly inferred from the relatively high frequency of B-cell clones reactive to both HI and H5 sub-serotypes among early plasmablasst induced by influenza infection or vaccination (Wrammert Nature 453, 667-671, 2008; Wrammert JEM 208, 181-193, 2011). It was predicted that shared epitopes are very rare. However, a direct estimate of their frequency ex vivo had never previously been obtained. Now the present inventors have shown that MBCs with restricted or cross-reactive specificity for HI and H5 influenza receptors can be identified by FACS analysis of PBMC labeled simultaneously with HA baits from the two serotypes, and pre-incubated with H3 or with influenza B HA. The close similarity of the results with conventional ELISPOT assays shows that obtaining reliable estimates of HA-specific IgG MBC frequency directly in ex vivo PBMC samples is possible.

Therefore, this double labeling approach makes it possible to better define origin and maturation stages of Ag-specific plasmablasts and MBCs and to analyse clonal size and composition of the Ag- specific antibody repertoire induced by vaccination.

In a further embodiment, a multiple labeling approach can be used in which more than two antigens (e.g. 3, 4, 5, 6, 7, 8, 9, 10 or more antigens) labeled with different fluorochromes can be used for simultaneous or sequential labeling of the pre-incubated patient sample.

Cross-reactivity

The double labeling approach means that the invention provides methods for detecting plasmablasts and/or MBCs that are cross-reactive against distant viral serotypes. The term "cross-reactive" means that the cell or BcR specifically binds to more than one antigen. For example, the invention provides methods for detecting plasmablasts and/or MBCs that are cross-reactive against two or more distant influenza A group 1 HA serotypes. The invention also provides methods for detecting plasmablasts and/or MBCs that are cross-reactive against two or more influenza A group 1 and group 2 HA serotypes. The inventors have shown that an MBC population selected based on binding to HI after pre- saturation with H3N2, does not contain cells cross-reactive to H3, but comprises a consistent number of cells that bind to epitopes conserved across distant group 1 HA serotypes. The inventors have also shown that it is possible to detect low frequencies of circulating MBCs reactive to H5, even in peripheral blood of people that have never experienced H5N1 virus or vaccine before.

The inventors have also shown that preincubation with type B strain allows detection of H1+ cells that also cross-react to H3. This is confirmation that pre-incubating with type B makes it possible to sort MBCs that are specific for epitopes shared by group 1 and 2.

Table 1: Examples of combinations of influenza saturators and labeled baits that are suitable for use in the method of the invention

Figure imgf000016_0001
B/Yamanashi/166/98 H3 H4, H7,H10,H14orH15

B/Johannesburg/5/99 H3 H4, H7,H10,H14orH15

B/Victoria/504/2000 H3 H4, H7,H10,H14orH15

B/Hong Kong/330/2001 H3 H4, H7,H10,H14orH15

B/Jilin/20/2003 H3 H4, H7,H10,H14orH15

Influenza B Influenza A Group 1 + Influenza A Group 2

B/Brisbane/32/2002 H2, H5, H6, H8, H9, Hll, H12, H13, or H16 + H3 H4, H7,

H10,H14orH15

B/Ann Arbor/ 1/94 H2, H5, H6, H8, H9, Hll, H12, H13, or H16 + H3 H4, H7,

H10,H14orH15

B/Yamanashi/166/98 H2, H5, H6, H8, H9, Hll, H12, H13, or H16 + H3 H4, H7,

H10,H14orH15

B/Johannesburg/5/99 H2, H5, H6, H8, H9, Hll, H12, H13, or H16 + H3 H4, H7,

H10,H14orH15

B/Victoria/504/2000 H2, H5, H6, H8, H9, Hll, H12, H13, or H16 + H3 H4, H7,

H10,H14orH15

B/Hong Kong/330/2001 H2, H5, H6, H8, H9, Hll, H12, H13, or H16 + H3 H4, H7,

H10,H14orH15

B/Jilin/20/2003 H2, H5, H6, H8, H9, Hll, H12, H13, or H16 + H3 H4, H7,

H10,H14orH15

Influenza A Influenza B

HINlCal B/Ann Arbor/1/94, B/Yamanashi/166/98, B/Johannesburg/5/99,

B/Victoria/504/2000, B/Hong Kong/330/2001,

B/Brisbane/32/2002, and B/Jilin/20/2003.

H3N2Brisbane B/Ann Arbor/1/94, B/Yamanashi/166/98, B/Johannesburg/5/99,

B/Victoria/504/2000, B/Hong Kong/330/2001,

B/Brisbane/32/2002, and B/Jilin/20/2003.

HINlSol. B/Ann Arbor/1/94, B/Yamanashi/166/98, B/Johannesburg/5/99,

B/Victoria/504/2000, B/Hong Kong/330/2001,

B/Brisbane/32/2002, and B/Jilin/20/2003.

H5NlViet. B/Ann Arbor/1/94, B/Yamanashi/166/98, B/Johannesburg/5/99,

B/Victoria/504/2000, B/Hong Kong/330/2001,

B/Brisbane/32/2002, and B/Jilin/20/2003.

H3N2Panama B/Ann Arbor/1/94, B/Yamanashi/166/98, B/Johannesburg/5/99,

B/Victoria/504/2000, B/Hong Kong/330/2001,

B/Brisbane/32/2002, and B/Jilin/20/2003.

Detection

Detection and sorting of the labeled cells will typically be carried out by fluorescent activated cell sorting (FACS). Methods for performing FACS on labeled cells are well known in the art (see for example Herzenberg and Herzenberg, Clin Lab Med.27(3) 453 (2007)). FACS allows single-cell analysis and sorting of each individual cell in a population. Cells labeled with the labeled antigen of interest are detected and sorted into separate channels. Sorted cells can be counted and isolated for further characterisation and analysis.

An important principle of flow cytometry data analysis is to selectively visualize the cells of interest while eliminating results from unwanted particles e.g. dead cells and debris. This procedure is called gating. Cells have traditionally been gated according to physical characteristics. For instance, subcellular debris and clumps can be distinguished from single cells by size, estimated by forward scatter. Also, dead cells have lower forward scatter and higher side scatter than living cells. Typically, to identify MBC and plasmablast subsets, a gating strategy based upon one or more or all of the following parameters may be used: SSC-A, SSC-W, FSC-A, CD20, CD27, CD19, CD38 and CD3.

Propidium iodide labeling is often used to sort dead cells from live cells.

In some embodiments, anti-hlgG is added to the labeling protocol. This allows calculation of the percentage of HA-specific B cells among the total number of B cells expressing IgG, for example using an ELISPOT assay (see below).

Isotope labeling

An alternative approach to FACS for detecting cells might be to use mass spectrometry in combination with lanthanide isotope labeling of the antigen of interest. This method could theoretically allow the use of 40 to 60 distinguishable labels and has been demonstrated for 30 labels (Ornatsky, O. et al., (2010). Journal of Immunological Methods 361 1, 1-20). Cells are introduced into a plasma, ionizing them and allowing time-of- flight mass spectrometry to identify the associated isotopes. Although this method permits the use of a large number of labels, it currently has lower throughput capacity than traditional flow cytometry. It also destroys the analysed cells, precluding their recovery by sorting. Analysis of sorted population

The sorted cell population can be cloned by methods well known in the art. The sorted and/or cloned population can be analyzed in various ways.

For example, the population of sorted B cells can be analyzed for expression or activation of pro- and anti-apoptotic genes and cytokine-related transcription factors as well as expression levels of other proteins for analysing phenotype or assessing the level of activities of various important signalling pathways. Gene expression analysis of the sorted B cells can help to expand the current understanding of their biology, for example through the further characterisation of their differentiation stage.

The specificity of the labeling can be independently analysed and verified using a separate assay, such as a limited dilution assay or an enzyme-linked immunosorbent spot (ELISPOT) assay. Differentiated MBCs are obtained for an ELISPOT assay, typically by 5 days of in vitro activation in the presence of B cell polyclonal activators, such as CpG and IL-2. The differentiated MBCs are plated on the wells and antibodies specific for the antigens of interested are detected by enzyme- conjugated antibodies that can activate a chromogen added at the end of the wells. A coloured spot develops if the antibody interaction takes place. The specificity of the sorted population can be estimated by counting antigen-specific Ig ASC as a percentage of total IgG ASC in the same sample.

In some embodiments of the invention, at least 10%, at least 20%, at least 30%), at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more of the cell population sorted by the method of the invention will be antigen specific, as determined by the ELISPOT assay. The term "specific" means that the cell has substantially greater affinity for the antigen of interest than its affinity for other antigens. By "substantially greater affinity" it is meant that there is a measurable increase in the affinity for the antigen of interest as compared with the affinity for other known antigens.

In some embodiments of the invention, the sorted population are enriched by at least 10 fold, at least 50 fold, at least 70 fold, at least 100 fold, at least 130 fold, at least 210 fold, at least 250 fold or more for antigen-specific B cells, compared to the pre-sorted population, as determined by the ELISPOT assay.

Uses of the invention

The method of the invention can be used for detecting the frequency of antigen-specific plasmablasts and MBCs circulating after vaccination or exposure to pathogen. This is useful, for example, for ex vivo clinical trial analysis, for assessing individual patient response to vaccination or for assessing immune response after exposure to pathogen.

The method of the invention can be used to enumerate different subsets of antigen-specific MBCs, for example, the invention can be used to analyse the distribution of HA+ MBCs across different B cell subsets. In some embodiments, the method of the invention can be used to analyse B-cell responses to vaccination. The method of the invention can also be used to document the differences in memory repertoires elicited by adjuvanted (e.g. MF59 adjuvanted) and un-adjuvanted vaccines, thus helping the development of acceptable adjuvants. Population of sorted B cells

The invention provides a population of sorted cells obtained by methods of the invention.

The invention also provides a population of cells that specifically bind a glycan-binding antigen of interest. The cells in the sorted population express antigen-specific BcRs on the cell surface. In some embodiments, the BcRs are cross-reactive against heterologous serotypes of influenza A HA antigens.

For example, the invention provides populations of sorted cells expressing antigen-specific BcRs that are cross-reactive against any two or more group 1 influenza A HA antigens. For example, the invention provides populations of sorted cells expressing antigen-specific BcRs that bind specifically to HI and to H5.

In a further example, the invention provides populations of sorted cells expressing antigen-specific BcRs that are cross-reactive against at least one group 1 and at least one group 2 influenza A HA antigen. For example, the invention provides populations of sorted cells expressing antigen-specific BcRs that bind specifically to HI and to H3. Uses of sorted B cells

A population of B cells sorted by the methods of the invention can be used to analyse the repertoire of antibodies developed after vaccination, for example by gene expression profiling and deep sequencing and/or fingerprinting of immunoglobulin variable regions. cDNAs of VL and VH can be retrotranscribed and amplified from single cells for sequence analysis, somatic hypermutation analysis and clonal families clustering. Sequencing data can be used to elucidate how genes involved in differentiation and affinity maturation processes are expressed in single plasma cells and MBCs. cDNAs can also be cloned in expression vectors to transfect eukaryotic cells. Antibodies can then be cloned from culture supernatants.

Alternatively, sorted cells can be cultured with stimuli to induce differentiation to Ig secreting MBCs. These cells can then be cloned and immortalised (e.g. by Epstein Barr virus transformation) to obtain monoclonal cell lines that produce specific antibodies.

The clones generated by either of these methods are stable and secrete antibodies that can be selected for affinity to antigen, avidity, specificity and microneutralization effect. The most desirable antibodies can be amplified and purified for research, commercial or therapeutic uses, for example vaccine preparation.

The invention provides a method for producing anti-HA antibodies, wherein the method comprises: (a) isolation of nucleic acid encoding an anti-HA antibody from a population of cells sorted according to the invention;

(b) cloning into expression vectors; and

(c) expressing the anti-HA antibodies in an expression system. The invention also provides a method for producing anti-HA antibodies, wherein the method comprises:

(a) obtaining a population of cells sorted according to methods of the invention;

(b) culturing the cells with stimuli to induce differentiation to Ig secreting MBCs;

(c) cloning and immortalising cells. Isolated antibodies

The invention provides antibodies that have been identified and/or produced by methods of the invention.

The invention also provides antibodies that are cross-reactive against heterologous serotypes of influenza A HA antigens. For example, the invention provides antibodies that are cross-reactive against any two or more group 1 influenza A HA antigens. For example, the invention provides antibodies that bind specifically to HI and to H5.

In a further example, the invention provides antibodies that are cross-reactive against at least one group 1 and at least one group 2 influenza A HA antigen. For example, the invention provides antibodies that bind specifically to HI and to H3.

Current flu vaccines provide only limited coverage against seasonal strains of influenza viruses. However, predicting which strains will dominate annually is difficult and mismatches between the vaccine and circulating viruses can lead to little or no protective effect. The identification of antibodies that broadly neutralize heterologous serotypes of influenza A and B antigens would enable development of universal influenza vaccines and broad antibody therapies. Recently, antibodies with broader neutralizing activity have described. For example, VHl-69 antibodies broadly neutralize almost all influenza A group 1 viruses (Ekiert et al., Science 324, 246, (2009); Throsby et al., PLoS ONE 3, e3942 (2008)), and even more recently a highly conserved neutralizing epitope on group 2 influenza A viruses was reported along with the human monoclonal antibody CR8020 which binds to this epitope and can neutralise most group 2 viruses (Corti et al., Science, 333, 850 (2011)). There is still a need for broadly neutralising antibodies and methods for identifying such antibodies. Antibodies of the invention can take various forms. For instance, they may be native antibodies, as naturally found in mammals. Native antibodies are made up of heavy chains and light chains. The heavy and light chains are both divided into variable domains and constant domains. The ability of different antibodies to recognize different antigens arises from differences in their variable domains, in both the light and heavy chains. Light chains of native antibodies in vertebrate species are either kappa (κ) or lambda (λ), based on the amino acid sequences of their constant domains. The constant domain of a native antibody's heavy chains will be α, δ, ε, γ or μ, giving rise respectively to antibodies of IgA, IgD, IgE, IgG, or IgM class. Classes may be further divided into subclasses or isotypes e.g. IgGl, IgG2, IgG3, IgG4, IgA, IgA2, etc. Antibodies may also be classified by allotype e.g. a γ heavy chain may have Glm allotype a, f, x or z, G2m allotype n, or G3m allotype bO, bl, b3, b4, b5, c3, c5, gl, g5, s, t, u, or v; a κ light chain may have a Km(l), Km(2) or Km(3) allotype. A native IgG antibody has two identical light chains (one constant domain CL and one variable domain VL) and two identical heavy chains (three constant domains CHI CH2 & CH3 and one variable domain VH), held together by disulfide bridges. The domain and three-dimensional structures of the different classes of native antibodies are well known.

Where an antibody of the invention has a light chain with a constant domain, it may be a κ or λ light chain. Where an antibody of the invention has a heavy chain with a constant domain, it may be a α, δ, ε, γ or μ heavy chain. Heavy chains in the γ class (i.e. IgG antibodies) are preferred. The IgGl subclass is preferred (although, in some embodiments, antibodies do not have a IgGl heavy chain). Antibodies of the invention may have any suitable allotype (see above).

Antibodies of the invention may be fragments of native antibodies that retain antigen binding activity. For instance, papain digestion of native antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment without antigen-binding activity. Pepsin treatment yields a "F(ab')2" fragment that has two antigen-binding sites. "Fv" is the minimum fragment of a native antibody that contains a complete antigen-binding site, consisting of a dimer of one heavy chain and one light chain variable domain. Thus an antibody of the invention may be Fab, Fab', F(ab')2, Fv, or any other type, of fragment of a native antibody.

An antibody of the invention may be a "single-chain Fv" ("scFv" or "sFv"), comprising a VH and VL domain as a single polypeptide chain (Worn & Pluckthun (2001) J Mol Biol. 305(5):989-1010; W093/16185; Adams & Schier (1999) J Immunol Methods. 231(l-2):249-60). Typically the VH and VL domains are joined by a short polypeptide linker (e.g. >12 amino acids) between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. Multiple scFvs can be linked in a single polypeptide chain (Gruber et al. (1994) J Immunol 152(11):5368-74). An antibody of the invention may be a "diabody" or "triabody" etc. (US5591828; WO 93/11161 ; Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Hudson & Kortt (1999) J Immunol Methods 231 : 177-89), comprising multiple linked Fv (scFv) fragments. By using a linker between the VH and VL domains that is too short to allow them to pair with each other {e.g. <12 amino acids), they are forced instead to pair with the complementary domains of another Fv fragment and thus create two antigen-binding sites. An antibody of the invention may be a single variable domain or VHH antibody. Antibodies naturally found in camelids {e.g. camels and llamas) and in sharks contain a heavy chain but no light chain. Thus antigen recognition is determined by a single variable domain, unlike a mammalian native antibody (Muyldermans (2001) J Biotechnol 74(4):277-302; Dumoulin et al. (2002) Protein Sci. 1 1(3):500- 15; Sidhu et al. (2004) J Mol Biol. 338(2):299-310). The constant domain of such antibodies can be omitted while retaining antigen-binding activity.

An antibody of the invention may be a "domain antibody" (dAb). Such dAbs are based on the variable domains of either a heavy or light chain of a human antibody and have a molecular weight of approximately 13 kDa (less than one-tenth the size of a full antibody). By pairing heavy and light chain dAbs that recognize different targets, antibodies with dual specificity can be made, and a dAbs of the invention will include at least one domain that can bind to the antigen of interest. dAbs are cleared from the body quickly, but can be sulabeled in circulation by fusion to a second dAb that binds to a blood protein {e.g. to serum albumin), by conjugation to polymers {e.g. to a polyethylene glycol), or by other techniques.

Thus the term "antibody" as used herein encompasses a range of proteins having diverse structural features (usually including at least one immunoglobulin domain having an all-β protein fold with a 2- layer sandwich of anti-parallel β-strands arranged in two β-sheets), but all of the proteins possess the ability to bind to an antigen of interest

Antibodies of the invention may include a single antigen-binding site {e.g. as in a Fab fragment or a scFv) or multiple antigen-binding sites {e.g. as in a F(ab')2 fragment or a diabody or a native antibody). Where an antibody has more than one antigen-binding site then advantageously it can result in cross-linking of antigens.

Where an antibody has more than one antigen-binding site, the antibody may be mono-specific {i.e. all antigen-binding sites recognize the same antigen) or it may be multi-specific {i.e. the antigen- binding sites recognise more than one antigen). Thus, in a multi-specific antibody, at least one antigen-binding site will recognise an antigen of interest and at least one antigen-binding site will recognise a different antigen.

An antibody of the invention may include a non-protein substance e.g. via covalent conjugation. For example, an antibody may include a radio-isotope e.g. the Zevalin™ and Bexxar™ products include 90Y and 13 T isotopes, respectively. As a further example, an antibody may include a cytotoxic molecule e.g. Mylotarg™ is linked to N-acetyl-y-calicheamicin, a bacterial toxin. As a further example, an antibody may include a covalently-attached polymer e.g. attachment of polyoxyethylated polyols or polyethylene glycol (PEG) has been reported to increase the circulating half- life of antibodies.

In some embodiments of the invention, an antibody can include one or more constant domains (e.g. including CH or CL domains). As mentioned above, the constant domains may form a κ or λ light chain or an α, δ, ε, γ or μ heavy chain. Where an antibody of the invention includes a constant domain, it may be a native constant domain or a modified constant domain. A heavy chain may include either three (as in α, γ, δ classes) or four (as in μ, ε classes) constant domains. Constant domains are not involved directly in the binding interaction between an antibody and an antigen, but they can provide various effector functions, including but not limited to: participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC); Clq binding; complement dependent cytotoxicity; Fc receptor binding; phagocytosis; and down-regulation of cell surface receptors.

The constant domains can form a "Fc region", which is the C-terminal region of a native antibody's heavy chain. Where an antibody of the invention includes a Fc region, it may be a native Fc region or a modified Fc region. A Fc region is important for some antibodies' functions e.g. the activity of Herceptin™ is Fc-dependent. Although the boundaries of the Fc region of a native antibody may vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226 or Pro230 to the heavy chain's C-terminus. The Fc region will typically be able to bind one or more Fc receptors, such as a FcyRI (CD64), a FcyRII (e.g. FcyRIIA, FcyRIIB l , FcyRIIB2, FcyRIIC), a FcyRIII (e.g. FcyRIIIA, FcyRIIIB), a FcRn, FcaR (CD89), Fc5R, FcμR, a FceRI (e.g. FceRIa y2 or FceRIo^), FceRII (e.g. FceRIIA or FceRIIB), etc. The Fc region may also or alternatively be able to bind to a complement protein, such as Clq. Modifications to an antibody's Fc region can be used to change its effector function(s) e.g. to increase or decrease receptor binding affinity. For instance, US patent 5,624,821 reports that effector functions may be modified by mutating Fc region residues 234, 235, 236, 237, 297, 318, 320 and/or 322. Similarly, Shields et al. (2001) J Biol Chem 276:6591-604 reports that effector functions of a human IgGl can be improved by mutating Fc region residues (EU Index Kabat numbering) 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 294, 295, 296, 298, 301 , 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331 , 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 and/or 439. Modification of Fc residues 322, 329 and/or 331 is reported in US patent 6,538,124 for modifying Clq affinity of human IgG antibodies, and residues 270, 322, 326, 327, 329, 331 , 333 and/or 334 are selected for modification in US patent 6,528,624. Mapping of residues important for human IgG binding to FcRI, FcRII, FcRIII, and FcRn receptors is reported in Shields et al. (2001) J Biol Chem 276:6591-604, together with the design of variants with improved FcR-binding properties. Whole CH domains can be substituted between isotypes e.g. WO2006/033386 discloses antibodies in which the CH3 domain (and optionally the CH2 domain) of human IgG4 is substituted by the CH3 domain of human IgGl to provide suppressed aggregate formation. It is also reported that mutation of arginine at position 409 (EU index Kabat) of human IgG4 to e.g. lysine shows suppressed aggregate formation. Mutation of the Fc region of available monoclonal antibodies to vary their effector functions is known e.g. Idusogie et al. (2000) J Immunol 164(8):4178-84 reports mutation studies for RITUXAN™ to change Clq-binding, and Dall'acqua et al. (2006) J Biol Chem 281(33):23514-24 reports mutation studies for NUMAX™ to change FcR-binding, with mutation of residues 252, 254 and 256 giving a 10-fold increase in FcRn-binding without affecting antigen-binding.

Antibodies of the invention will typically be glycosylated. N-linked glycans attached to the CH2 domain of a heavy chain, for instance, can influence Clq and FcR binding, with aglycosylated antibodies having lower affinity for these receptors. The glycan structure can also affect activity e.g. differences in complement-mediated cell death may be seen depending on the number of galactose sugars (0, 1 or 2) at the terminus of a glycan's biantennary chain. An antibody's glycans preferably do not lead to a human immunogenic response after administration.

Antibodies of the invention can be prepared in a form free from products with which they would naturally be associated. Contaminant components of an antibody's natural environment include materials such as enzymes, hormones, or other host cell proteins.

Antibodies of the invention can be used directly {e.g. as the active ingredient for pharmaceuticals or diagnostic reagents), or they can be used as the basis for further development work. For instance, an antibody may be subjected to sequence alterations or chemical modifications in order to improve a desired characteristic e.g. binding affinity or avidity, pharmacokinetic properties (such as in vivo half-life), etc. Techniques for modifying antibodies in this way are known in the art. For instance, an antibody may be subjected to "affinity maturation", in which one or more residues (usually in a CDR) is mutated to improve its affinity for a target antigen. Random or directed mutagenesis can be used, but Marks et al. (1992) Bio/Technology 10:779-83. describes affinity maturation by VH and VL domain shuffling as an alternative to random point mutation. Wu et al. (2005) J Mol Biol 350(1): 126- 44 reports how NUMAX™ was derived by a process of in vitro affinity maturation of the CDRs of the heavy and light chains of SYNAGIS™, giving an antibody with enhanced potency and 70-fold greater binding affinity for RSV F protein.

The term "monoclonal" as originally used in relation to antibodies referred to antibodies produced by a single clonal line of immune cells, as opposed to "polyclonal" antibodies that, while all recognizing the same target protein, were produced by different B cells and would be directed to different epitopes on that protein. As used herein, the word "monoclonal" does not imply any particular cellular origin, but refers to any population of antibodies that all have the same amino acid sequence and recognize the same epitope in the same target protein. Thus a monoclonal antibody may be produced using any suitable protein synthesis system, including immune cells, non-immune cells, acellular systems, etc. This usage is usual in the field e.g. the product datasheets for the CDR-grafted humanised antibody Synagis™ expressed in a murine myeloma NSO cell line, the humanised antibody Herceptin™ expressed in a CHO cell line, and the phage-displayed antibody Humira™ expressed in a CHO cell line all refer the products as monoclonal antibodies.

Preferred antibodies of the invention are specific for glycan-binding antigens, such as influenza HA- antigen. Thus the antibody will have a tighter binding affinity for that antigen than for an arbitrary control antigen e.g. than for a human protein. Preferred antibodies have nanomolar or picomolar affinity constants for target antigens e.g. 10"9 M, 10"10 M, 10"11 M, 10"12 M, 10"13 M or tighter). Such affinities can be determined using conventional analytical techniques e.g. using surface plasmon resonance techniques as embodied in BIAcore™ instrumentation and operated according to the manufacturer's instructions. Radio-immunoassay using radiolabeled target antigen (HA) is another method by which binding affinity may be measured.

The invention allows fully human monoclonal antibodies to be obtained. Neutralizing activity

Antibodies of the invention can be used to neutralize the HA of an influenza A virus that can infect human beings. Thus they can neutralize the ability of the virus to initiate and/or perpetuate an infection in a human host. Various assays can be used to determine neutralizing activity, such as the microneutralization assay described herein. Preferred antibodies can neutralize the infectivity of 100 TCID50 (50% Tissue Culture Infective Dose) of a virus for MDCK cells. As an alternative to using viruses for influenza neutralisation assays, in some embodiments of the invention retroviral pseudotypes bearing influenza HA can be used instead (Temperton et al. (2007) Influenza and other Respiratory Viruses. DOI: 10.1111/j. l750-2659.2007.00016.x).

Nucleic acids and recombinant antibody expression

The invention also encompasses nucleic acid sequences encoding antibodies of the invention. Where an antibody of the invention has more than one chain {e.g. a heavy chain and a light chain), the invention encompasses nucleic acids encoding each chain. The invention also encompasses nucleic acid sequences encoding the amino acid sequences of CDRs of antibodies of the invention.

Nucleic acids encoding the antibodies can be prepared from cells, viruses or phages that express an antibody of interest. For instance, nucleic acid {e.g. mRNA transcripts, or DNA) can be prepared from plasmablasts/MBCs of interest, and the gene(s) encoding the antibody of interest can then be cloned and used for subsequent recombinant expression. Expression from recombinant sources is more common for pharmaceutical purposes than expression from B cells or hybridomas e.g. for reasons of stability, reproducibility, culture ease, etc. Methods for obtaining and sequencing immunoglobulin genes from B cells are well known in the art e.g. see Chapter 4 of Kuby Immunology (4th edition, 2000; ASIN: 0716733315. Thus various steps of culturing, sub-culturing, cloning, sub-cloning, sequencing, nucleic acid preparation, etc. can be performed in order to perpetuate the antibody expressed by a cell or phage of interest. The invention encompasses all cells, nucleic acids, vectors, sequences, antibodies etc. used and prepared during such steps.

The invention provides a method for preparing one or more nucleic acid molecules {e.g. heavy and light chain genes) that encodes an antibody of interest, comprising the steps of: (i) providing plasmablasts/MBCs expressing an antibody of interest; (ii) obtaining from the plasmablasts/MBCs nucleic acid that encodes the antibody of interest. The nucleic acid obtained in step (ii) may be inserted into a different cell type, or it may be sequenced.

The invention also provides a method for preparing a recombinant cell, comprising the steps of: (i) obtaining one or more nucleic acids {e.g. heavy and/or light chain genes) from plasmablasts/MBCs that encodes an antibody of interest; and (ii) inserting the nucleic acid into an expression host in order to permit expression of the antibody of interest in that host.

Similarly, the invention provides a method for preparing a recombinant cell, comprising the steps of: (i) sequencing nucleic acid(s) from plasmablasts/MBCs that encodes the antibody of interest; and (ii) using the sequence information from step (i) to prepare nucleic acid(s) for inserting into an expression host in order to permit expression of the antibody of interest in that host.

Recombinant cells produced in these ways can then be used for expression and culture purposes. They are particularly useful for expression of antibodies for large-scale pharmaceutical production.

The invention provides a method for preparing an antibody of the invention, comprising a step of culturing a cell such that it produces the antibody. The methods may further comprise a step of recovering the antibody that has been produced, to provide a purified antibody. A cell used in these methods may, as described elsewhere herein, be a recombinant cell, an immortalised B cell, or any other suitable cell. Purified antibody from these methods can then be used in pharmaceutical and/or diagnostic compositions, etc.

Cells for recombinant expression include bacteria, yeast and animal cells, particularly mammalian cells {e.g. CHO cells, human cells, etc.), as well as plant cells. Preferred expression hosts can glycosylate the antibody of the invention, particularly with carbohydrate structures that are not themselves immunogenic in humans (see above). Expression hosts that can grow in serum- free media are preferred. Expression hosts that can grow in culture without the presence of animal-derived products are preferred. The expression host may be cultured to give a cell line.

Nucleic acids used with the invention may be manipulated to insert, delete or amend certain nucleic acid sequences. Changes from such manipulation include, but are not limited to, changes to introduce restriction sites, to amend codon usage, to add or optimise transcription and/or translation regulatory sequences, etc. It is also possible to change the nucleic acid to alter the encoded amino acids. For example, it may be useful to introduce one or more (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, one or more (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid deletions, and/or one or more (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, efc.) amino acid insertions into the antibody's amino acid sequence. Such point mutations can modify effector functions, antigen-binding affinity, post- translational modifications, immunogenicity, etc. , can introduce amino acids for the attachment of covalent groups (e.g. labels) or can introduce tags (e.g. for purification purposes). Mutations can be introduced in specific sites or can be introduced randomly, followed by selection (e.g. molecular evolution).

Nucleic acids of the invention may be present in a vector (such as a plasmid) e.g. in a cloning vector or in an expression vector. Thus a sequence encoding an amino acid sequence of interest may be downstream of a promoter such that its transcription is suitable controlled. The invention provides such vectors, and also provides cells containing them.

Uses of Antibodies

Antibodies of the invention can be used for treatment or prevention of infection by influenza viruses. Antibodies of the invention can be used to help develop a universal influenza vaccine that can neutralise multiple strains or all strains of influenza virus.

Antibodies of the invention, whether polyclonal or monoclonal, may have additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA) or protein arrays. In these applications, the antibodies can be labeled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme.

General

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

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

The term "about" in relation to a numerical value x means, for example, x±10%.

Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc. BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 B cells specific for H1N1 A/California/04/09 can be sorted by labeling with rHlCalifornia- Alexa 488. A CD19+ cells labeling with rHICal-Alexa 488 compared to HSA-Alexa 488 as negative control. Populations sorted as Ag-specific shown in red, Ag- in blue and flow through in green have been cultured for 5 days with polyclonal stimuli (CpG 2.5 mg/ml and IL-2 1000 U/ml). B The specificity of the sorted populations has been analyzed by ELISPOT on HANA proteins of different strains.

Fig. 2 HA-Alexa 488 labeling is useful to follow immune response to vaccination. PBMC from a donor that received concomitant vaccination with Focetria (Hl lCalifornia+MF59) and unadjuvanted TIV (Agrippal). The same donor received H5N1 Vietnam + MF59 3 years before. A. MBC CD20+CD27+ labeled for HI California show increase in frequency of positive populations 32 day post vaccination with Focetria. B. Consistent increase in HINlCalifornia MBC is also demonstrated by sLDA. C. Plasmablasts can be identified as a CD3-CD19+CD20dimCD27+CD38+ population around day7 post vaccination. The specificity of ACS is analyzed by intracellular labeling with rHl California-Alexa 488. The induced population is also reactive to rH5 common epitopes, as confirmed by ELISPOT with total unstimulated PBMC (upper panel a and b).

Fig. 3 A. Gating strategy to detect HICal-specific MBC as CD20+CD27+H1+ and CD20+CD27dimHl+. Neither lectin pre-treatment of cell surface (data not shown) nor saturation of HA with sialopentasaccarides mantain cell morphology. Pre-incubation with unlabeled H3N2 is suitable for specific HA labeling. B. and C. ELISPOT analysis of sorted HlCal+ population shows that Neuraminidase pre-treatment compared to pre-incubation with unlabeled H3N2 detects higher amount of HlCal+ MBC, but only at least 20% are HICal-specific compared to 100% obtained with the other protocol. Alexa488-labeled HSA is used as negative control. Donor 1 vaccinated with Focetria different sample. Fig. 4 A. H5Vietnam+ and B. HlNlSolomon+ MBC detected with rHA are sorted and compared to pre-sorting PBMC by ELISPOT to assess the enrichment of Ag-specific IgG among total IgG.

Fig. 5 PBMC of a healthy donor (# 1) are labeled ex vivo to detect MBC as CD20+CD27+/dimIgG+ population. Frequencies of Ag-specific MBC are assessed as percentage of HA+ events among IgG+ population or in single labeling step or in double labeling one. In the double labeling approach the frequencies of both common and serotype-specific epitopes could be detected.

Fig. 6 A. PBMC from a vaccinee 10 months post vaccination with H1N1 pandemic vaccine and MF59-adjuvanted H5N1 vaccine were stained as CD20+ B cell. HlCal+ cells are distributed among CD20+CD27+/dim memory B cells, as compared with HSA used as negative control. B cell populations were sorted into CD20+ HlCal+ (right gate) and CD20+ HlCal- (left gate). The sorted populations were cultured for 5 days in presence of stimuli (CpG and IL2) and the quality of secreting antibodies analyzed by ELISPOT. B. Results of ELISPOT analysis are represented as spot/million cells for HANA and total IgG.

Fig. 7 A double positive (H5+/H1N1+) B cell population were gated and sorted with H5+ or H1N1+ according to the brightness of their positivity to give an H5++/H1N1+ and an H5+/H1/N1++ population.

Fig. 8 Frequencies of HA-specific IgG (expressed as % among total IgG) detected by staining. PBMC of a healthy donor (# 3) were stained ex vivo to detect MBC as CD20+CD27+/dimIgG+ population. Frequencies of Ag-specific MBC are assessed as percentage of HA+ events among IgG+ population or in single staining step (A) or in double staining one (B). In the double staining approach the frequencies of both common and serotype-specific epitopes could be detected. Alexa- labeled HSA is used as negative control.

Fig. 9 Pre-incubation with B vaccine subunit antigens.

Fig.10 Both influenza A and B vaccine subunits antigens prevent rHA binding to all B cells and allow the detection of BCR-specific binding. A) B cells binding to HSA or rHl (top panels), to HSA or rH3 (middle panels), and to HSA or B/HA (bottom panels) were identified gating in CD20+ B cells, following pre-incubation of PBMC with influenza B/Brisbane/60/08 and A/Panama/07/99, respectively. B) Number of antibody secreting plasma cells detected by ELISPOT in unsorted PBMCs and in HA+ sorted B cells. Results are expressed as number of PCs inl million cultured cells. Fig.ll Alternate use of mismatched influenza subunits for simultaneous identification of B cells specific for two different HA subtypes. B cells binding to rHl and / or to rH3 (top panels), or to rHl or to B/rHA (bottom panels) were identified in the CD20 gate, following pre-incubation of sample tubes containing PBMC from the same donor with influenza B/Brisbane/60/08 or A/Panama/07/99 vaccine subunits, respectively. HSA conjugated with Alexa 647 and Alexa 488 were used as negative control.

Fig. 12 Identification of HI Sol+ B cell in human PBMC sample. A FACS analysis of HI Sol+ B cells in human PBMC gated on the CD20+ cells. HSA was used as negative control sample. B ELISPOT analysis of total and H1N1 -specific PCs recovered from cultures of sorted H1+ B cells and of unfractionated PBMC. Results are expressed as number of PCs in 1 million of cultured cells. Fig.13 A significant linear correlation was found between the frequencies of HA+ MBCs measured by FACS or by ELISPOT assay.

Fig.14 FACS analysis of Hl-specific B cells before and after seasonal vaccination. PBMC samples, collected from volunteers enrolled in 2008/2009 vaccination studies, were analyzed by FACS in order to determine the frequency of HI Sol+ B lymphocytes at baseline (day 0) and at 3 and 6 weeks after vaccination (day 21 and 43, respectively). Dot plots depicts the distribution of H1+ B cells in CD20+ gated PBMC across the CD27+ and CD27- B cell subsets, in one representative subject out of the 3 analyzed.

Fig.15 Phenotypic characterization of H1+ and HI- B cells. Dot plots show the expression of CD27 and IgG in PBMC gated on the H1+CD20+ (upper panels) and H1-CD20+ regions at day 0, and 21 days and 43 days post-vaccination.

Fig. 16 The distribution of HA+ MBC across different B cell subsets (e.g. IgG and IgA MBC populations) was analysed by FACs.

Fig. 17 The sequence of paired heavy and light chain coding genes expressed by single sorted HA+ B cells has also been determined.

MODES FOR CARRYING OUT THE INVENTION

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

Example 1

Induction of a strong B cell response following vaccination is crucial for immune protection from influenza virus. Results available from our and other groups show that frequency of antibody secreting cells (ASC) and memory B cells (MBC) following influenza vaccination varies according to vaccine formulation and vaccinees' immune experience (Galli G. et al. Proc Natl Acad Sci U S A. 2009 Mar 10;106(10):3877-82; Galli G. et al. Proc Natl Acad Sci U S A. 2009 May 12;106(19):7962-7; Wrammert J. Et al. Nature. 2008 May 29;453(7195):667-71). A major limit of these analyses is that they rely on standardized polyclonal assays that can be used only to assess changes in frequency of ASC or their MBC precursors providing only indirect information on functionality. Direct assessment of Ag (antigen)-specific effectors and MBC are rarely performed in the context of influenza clinical trials. Because of low frequency of Ag-specific B cells in peripheral blood, direct and detailed analysis of their functionality and clonal repertoire requires long and laborious cloning procedures and properly designed screening strategies. Binding of tagged antigen to B cells has been used previously (Slifka MK. Et al. J Immunol Methods. 2006. (17): 175-85) as strategy to identify Ag-binding B cells directly ex vivo, but attempts to apply this protocol to influenza hemagglutinin (HA) so far have been unsuccessful.

Here we show the labeling method obtained using Alexa 488 labeled rHA from A/Vietnam/ 1203/2004 H5N1 and A/California/04/09 H1N1. Results obtained by multicolor flow cytometry demonstrate that it is possible to enumerate and sort HA-specific memory B cells elicited by vaccination and to identify and enumerate HA specific ASC among the bulk of plasmablasts circulating in the blood during the first week following the vaccination. Methods

Protein labeling. HSA (Sigma- Aldrich), rH5 and rHl (Protein Science) antigens were conjugated with Alexa 488 fluorochrome (Invitrogen).

Labeling for FACS analysis and sorting. For MBC 8x106 PBMC were pre-incubated with 0.3 mg of unlabeled H3N2 A Panama/2007/99. After 15 min in ice, 0.3 mg of labeled Ag was added to the sample with a-CD19, a-CD20 and a-CD27 mAbs (BD) for 1 hour in ice. Samples have been acquired on Canto II, while sorting has been performed by FACS-Aria (BD). For plasmablasts labeling, 10x106 PBMC were first labeled with a-CD3, a-CD19, a-CD20, a-CD27 and a-CD38. Cells were then fixed and permeabilized (CitoFix/CitoPerm BD) before being incubated with unlabeled H3N2 and Alexa 488- Ags, as described above.

ELISPOT. ELISPOT plates (MultiScreen HTSTM HA, Millipore) were coated with BSA, rHAs, H1N1 A/California/04/09, H5N1 A Vietnam/ 1203/2004, H1N1 A/Solomon Island/3/2006 or H3N2 A/Panama/2007/99 at 10 mg/ml O/N. Cells, stimulated or not with CpG 2.5 mg/ml and IL-2 lOOOU/ml, were plated into wells O/N. IgG secretion was revealed with biotinilated a-hlgG (Southern Biotech), followed by extravidine-HPR (TEMA ricerca) and developed with AEC chromogen (Sigma). Spots were counted by ELISPOT Analyzer (C.T.L.)

Results

Labeling with Alexa 488 is specific and allows sorting of highly enriched population of HA-specific Memory B cells (Fig.l). Table 2: Frequencies of Ag-specific IgG of sorted populations compared with pre-sorting PBMC.

Figure imgf000032_0001

Labeling of HA-specific B cells can be useful to follow the development of Ag-specific memory B cells (Fig. 2 A) long time after the vaccination (day 32) and plasmablasts response immediately after (7 days) the vaccination with Focetria (Fig.2 C).

Conclusions

We propose a rational strategy for enumerating, sorting and cloning of plasmablasts and memory B cells, suitable for more in depth analysis of phenotypic and functional profiles of the HA-specific B cells repertoires elicited by vaccination. We are now testing Ags labeled with different fluorochromes to detect simultaneously populations that recognize common epitopes and strain specific epitopes between HAs in order to sort and analyze them separately.

Because of the stickiness of HA to the surface of the cells, it would be useful to work with recombinant antigens lacking the transmembrane domain and defective for sialic acid binding.

Example 2

As already mentioned, induction of a potent, durable and broadly cross-reactive B cell response to influenza vaccination is crucial to prevent infection from influenza virus. The assays currently available to estimate the frequency of memory B cells (MBC) developed following influenza vaccination require PBMC activation and in vitro culturing, thus providing only indirect information on numbers and functionality of antigen-specific B cells circulating in vivo (Galli G. et al. Proc Natl Acad Sci U S A. 2009 Mar 10;106(10):3877-82; Galli G. et al. Proc Natl Acad Sci U S A. 2009 May 12;106(19):7962-7; Wrammert J. Et al. Nature. 2008 May 29;453(7195):667-71). Binding of tagged antigens to B cells has been used previously (Slifka MK. Et al. J Immunol Methods. 2006. (17): 175-85) to identify circulating MBC with defined antigen specificity by FACS, but attempts to apply this protocol to identify MBC specific for influenza hemagglutinin (HA) have so far been unsuccessful. We developed a labeling method to identify human MBC specific for HA from different influenza strains that is specific and allows sorting of a highly enriched population of HA-specific memory B cells. Results obtained by multicolor flow cytometry demonstrate that it is possible to enumerate HA-specific MBC in ex vivo PBMC and assess changes in their frequency following vaccination. In addition, by labeling PBMC with HA from two different serotypes, it is possible to identify and estimate the presence of MBC recognizing shared or serotype-specific epitopes.

Methods

HSA (Sigma-Aldrich), rHA (Protein Science) antigens were conjugated with Alexa 488 and Alexa 647 fluorochromes (Invitrogen). For FACS analysis and sorting 10x106 PBMC were pre-incubated with 0.3 mg of egg-derived unlabeled H3N2, 0.3 mg of labeled Ag was added to the sample with a-CD19, a- CD20, a-CD27 and a-IgG mAbs (BD) for 1 hour in ice and analyzed on Canto II or sorted by FACS- Aria (BD). ELISPOT: cells stimulated with CpG/IL-2 were plated into ELISPOT plates (MultiScreen HTSTM HA, Millipore) coated with BSA, HANA or a-hlgG. Secreted IgG was revealed with FITC a- hlgG (Southern Biotech) and developed with AEC chromogen (Sigma). Spots were counted by ELISPOT Analyzer (C.T.L.)

Results

MBC specific for HI California can be detected and enumerated among CD20+CD27+ and CD20+CD27dim B cells by labeling with labeled rHICalifornia (Fig.3A). ELISPOT analysis of the H1+ sorted population shows that only pre-incubation with unlabeled egg- derived HANA confers specificity to the labeling, compared to neuraminidase treatment of cell surface (Fig.3B).

H1 N1 Solomon H5N1 Vietnam

ELISPOT SINGLE STAINING DOUBLE STAINING ELISPOT SINGLE STAINING DOUBLE STAINING

# 1 0.8-1.1 0.7-1.1 0.8-0.4 D.7-0.8 0.6-0.4 0.3-0.4

# 2 0.6-0.6 0.6-0.9 0.6-0.7 D.3-0.2 0.3-0.3 0.1 -0.2

# 3 0.3-0.2 0.5-0.6 0.4-0.5 D.04-0.1 0.2-0.2 0.1 -0.2

# 4 0.2-0.03 0.4-0.6 0.3-0.5 D.1-0.1 0.1-0.3 0.1 -0.6 Table 3: Frequencies of HA-IgG MBC among total IgG estimated by ELISPOT and frequencies of HA+ events among IgG+ MBC estimated by FACS labeling in 4 different healthy donors. Frequencies detected in two independent experiments.

Frequencies of HA-specific IgG (expressed as % among total IgG) detected by labeling (Fig.5) are consistent with frequencies detected by ELISPOT (Table 3). Labeling PBMC with HA of 2 different serotypes it is possible to identify and estimate the presence of MBC recognizing shared or serotype-specific epitopes (Fig.5) (Table 3).

Conclusions

We propose a new approach for ex vivo enumerating, sorting and cloning of memory B cells, suitable for more in depth analysis of phenotypic and functional profiles of the HA-specific B cells repertoires elicited by vaccination.

Labeling with two different HAs labeled with different fluorochromes it is possible to identify and estimate the frequencies of MBC recognizing shared or serotype-specific epitopes in the same sample. Alternatively pre-incubating with one serotype HANA and labeling with two different labeled-HA serotypes it is possible to follow the frequencies of HA-specific MBC developed following seasonal or pandemic vaccination.

Example 3:

Effective saturation of a(2,6) sialic acid residues on the B cell surface allows detection of BcR specific binding to rHA baits

In order to identify memory B cells with BcR specific binding capacity to defined rHA influenza antigens, we did some preliminary experiments incubating PBMC samples directly with a mixture of pre-titrated amounts of monoclonal antibodies against CD20 and CD27 and increasing amount of Alexa-488 conjugated rHl molecules used as bait (from the pandemic H1N1 A/California/07/2009 strain, 0.1 to 9 μg /107 PBMC). Alexa-488 conjugated human serum albumin (HSA) was included as negative antigen bait. The results from these experiments (figure 3A) showed that, differently from HSA, rHl molecules bound to all naive and memory B cells causing their aggregation, thus making it impossible to discriminate B lymphocytes specifically binding to rHl through their BcR. HA is known to mediate influenza virus adhesion to human epithelial cells lining the respiratory tract by binding to a(2,6) sialic acid residues. a(2,6) sialic acid residues are also present on surface glycoproteins abundantly expressed by B lymphocytes (e.g. CD22). In order to prevent not specific HA binding to all B cells we tried different approaches. First we pre-incubated PBMC with increasing amounts of different compounds known to bind to a(2,6) sialic acid residues (e.g. a- fetuin, the Sambucus nigra lectin) prior adding a mixture of fluorochrome-tagged rHl and the CD20 and CD27 mAbs. Then we tried to saturate sialic acid binding sites on HA molecules by pre- incubating rHA with soluble o2,6 sialopentasaccharides before using it to stain PBMC. Unfortunately, none of these compounds blocked not specific binding of HA to B cells, or B cell aggregation (figure 3 A). Then we tried to prevent HI non-specific binding either by pre-incubating PBMC with the egg- derived vaccine subunits from a mismatched serotype (i.e. H3N2 from A/Panama/2007/99), or by stripping a (2,6) sialic acid from the B cell surface by pre-treatment with neuraminidase from C, difficile. Interestingly, both procedures prevented B cell aggregation and minimized rHl not specific binding. Following both treatments B cells putatively binding rHl baits through BcR- specific interactions become detectable across the CD27+/dun memory B cell subset, but not among CD27- naive B cells (figure 3B). The subset of H1+ memory B cell detected in PBMC treated with neuraminidase was larger than that detected in PBMC pre-incubated with H3N2 ((2.18% and 0.48%) of total B cells, respectively). H1+ B cells were then sorted and kept in culture in the presence of CpG and IL-2 for 5 days to induce their differentiation into antibody secreting cells (ASCs) and test their specificity in terms of HINl-IgG and total IgG by ELISPOT assay. A fraction of unsorted PBMC was also cultured and analyzed in the same ELISPOT assays to measure the frequency of Hl- specific IgG in the original sample. The results from this experiment (figure 3C) were surprising. In fact, compared to cultures of unsorted PBMC where Hl-IgG ASCs were 1.4% of the total IgG ASCs, cultures from both populations of H1+ sorted B cells contained an enriched proportion of Hl- specific IgG ASCs. However, in cultures of B cells stained and sorted from PBMC pre-incubated with H3N2 vaccine subunits (H3N2 H1+) ASCs secreting Hl-specific IgG accounted for 94.4% of the total IgG ASCs. Instead, in cultures of B cell sorted from neuraminidase-treated PBMC ASCs secreting Hl-specific IgG were accounted only for 18.5%> of total IgG ASCs (figure 3C). Comparable results were obtained with PBMC from 2 different donors and using neuraminidase concentrations up to 5 M (data not shown). In conclusion, these results demonstrate that pre-incubation of PBMC with commonly available a(2,6) sialic acid ligands is not sufficient for preventing unspecific binding of rHl baits to all B cells. This phenomenon is instead greatly reduced when sialic acid residues expressed on the surface of PBMC are stripped by neuraminidase pre -treatment, or saturated by pre-incubation with mismatched H3N2 vaccine subunit antigens prior to perform the staining with fluorochrome-tagged HI baits. However, while either approach allows detecting H1+ specific memory B cells by FACS, the specificity of the staining performed after H3N2-presaturation is superior.

The population of HA-specific memory B cells identified by FACS includes all B cells inducible to secrete specific IgG-switched antibodies in vitro.

Next we assessed whether the H3N2-pretreatment approach was suitable to detect all circulating IgG memory B cells binding to pandemic rHl through BcR specific interactions. To address this question we first stained PBMC from a donor who in the previous 10 years had been regularly vaccinated against seasonal influenza and also had received 3 doses of an MF59-adjuvanted vaccines against avian H5N1 influenza and 1 dose of an MF59-adjuvanted vaccine against 2009 pandemic A/H1N1 influenza (3 years and ten months before blood drawn, respectively). As in previous experiments, H1+ B cells were detected only across the CD27+ and the CD27dim memory B cell subsets (figure 6 A). The populations of H1+ and HI - memory B cells identified by FACS were then sorted, placed in culture with CpG and IL-2 for 5 days, and analyzed by ELISPOT for their capacity to generate cell secreting IgG antibodies binding to pandemic H1N1, or to seasonal influenza A and avian H5N1 vaccine antigens. A fraction of unsorted PBMC was also cultured and analyzed in the same ELISPOT assay to determine the frequency of IgG memory B cells specific for each antigen in the original sample. Figure 6 B depicts the results from this experiment. ASCs specific for pandemic or H1N1, seasonal H1N1 , H3N2, or for avian H5N1 antigens were detected at comparable frequencies in cultures of unsorted PBMC (-2% of total IgG ASCs). After sorting, ASCs secreting IgG antibodies binding to pandemic H1N1 antigens were found only within the population of H1+ B cells and accounted for 35% of the total IgG ASCs. Remarkably, almost all ASCs secreting IgG binding to seasonal H1N1 or avian H5N1 antigens also segregated in the H1+ B cell subset. Conversely, the population of HI- B cells generated approximately the same number of IgG ASCs as the H1+ B cell subset, but no IgG specific for the H1N1 pandemic vaccine subunit and only few IgG specific for seasonal H1N1 or avian H5N1 antigens (0.7% and 1.9% respectively). It is of note that, while H3N2- specific ASCs were detected in cultures of unsorted PBMCs, none of them were found in cultures from either H1+ or HI- sorted B cells, suggesting that H3N2 molecules used to saturate not specific binding sites in PBMC were carried over by all sorted B cells precluding the detection of H3-specific IgG in the ELISPOT assay. Comparable results were obtained in a second experiment with PBMC donated from a different donor after vaccination against 2009 pandemic influenza (data not shown). The results from these experiments show that the staining protocol we developed is suitable to detect all memory B cells carrying an IgG-switched BcR specific for the pandemic rHl antigen used as bait.

Memory B cells with restricted HI or H5 specificity, or cross-reactive across the two sub-serotypes can be identified in the same PBMC sample obtaining reliable estimates of their frequency ex vivo The remarkable enrichment in H5N1 -specific IgG ASC observed in cultures of rHl + memory B cells sorted from a donor vaccinated against avian H5N1 influenza (Fig.6) suggested that a fraction of these cells could be capable of cross-reactive binding to H5 avian influenza antigens. Interestingly, the presence of memory B cells with such cross-reactive capacity in seasonal influenza experienced adults has been repeatedly inferred from the relatively high frequency of B cell clones reactive to both HI and H5 sub-serotypes among early plasmablasts induced by influenza infection or vaccination (Wrammert J. et al. Nature 29;453(7195):667-71 2008; and Wrammert JEM 208, 181- 193, 2011). However, a direct estimate of their frequency ex vivo has never been obtained.

To address this question we first assessed the feasibility of a double staining approach with fluorochrome-tagged rH5 and seasonal HI antigens used as baits. PBMC from the donor vaccinated against avian H5N1 influenza were pre-incubated with H3N2 antigens then simultaneously stained with Alexa 488- conjugated rH5 (from the A/Vietnam/1194/04 strain) and Alexa 647-conjugated HI subunit antigens (from the 2007 seasonal strain A/Solomon Islands/3/2006) used in equal amount as baits. The results obtained from FACS analysis of this sample showed, once again, a restricted distribution of HA-binding cells across the CD27+/dim memory B cell subsets (data not shown). In addition, the dot plot distribution of B cells binding to the HA baits was consistent with that expected from a mixed population of memory B cells binding to H5 only (H5+/H1N1-: 0.57% of total B cells), to HlNl only (H5-/H1N1+: >0.09% of total B cells), or to both antigens (H5+/H1N1+: >0.23% of total B cells) (figure 7 A). Due to the limited number of PBMC in this sample, gates were set aiming at sorting the H5+ and H1+ memory B cell subsets in numbers sufficient to verify by ELISPOT the specificity of their binding to each of the two baits (figure 7A). For this reason the double positive population (H5+/H1N1+ B cells) were gated and sorted with H5+ or H1N1+ according to the brightness of their positivity, so that H5++/H1N1+ were sorted together with H5+B cells in one tube (named H5+/H1N1+), while few H5+/H1N1++ B cells were sorted together with H1N1+ B cells in a second tube (named H1N1+). Following activation in vitro, the population sorted as H5+/H1N1+ B cells generated a majority (62%) of IgG ASCs specific for H5 and a consistent amount (19%) of Hi- specific IgG ASCs (Figure 7B). Due to the low number of cells recovered in the population sorted as H1N1+, we could confirm their capacity to secrete HINl-specific antibodies, but not assess whether the same sorted population also contained ASC capable to secrete H5-specific IgG (figure 7B). It is worth noting, however, that the ratio between numbers of HI specific and H5-specific ASCs generated from the mix of H5+/H1N1+ memory B cells was strikingly close to the ratio between frequencies of H5+/H1N1+ memory B cells ad total H5+ memory B cells detected by FACS (0.32 and 0.31, respectively).

Since these results supported the possibility of using two different baits to identify memory B cells specifically binding to seasonal HI and / or avian H5 influenza antigens, we asked whether the same staining could be applied to obtain reliable estimates of their frequency in PBMC ex vivo.

To this aim, frozen PBMC from 4 healthy blood donors not vaccinated against avian H5N1 influenza were thawed and distributed in equal amounts in different tubes, saturated with H3N2, then stained with a mix of anti-CD20, anti-CD27 and anti-hlgG mAbs, containing the Alexa 488-rHl , or the Alexa647 rH5 bait (from A/Solomon Islands/3/2006 and A/Vietnam/ 1 194/04, respectively), or the two combined. PBMC stained with the same mix of mAbs and Alexa 488- or Alexa647- HSA, alone or in combination, were used as negative controls. In order to assess whether the frequency of H5+ and H1+ memory B cells estimated by FACS were comparable to those obtained by conventional ELISPOT assays, an unstained fraction from each PBMC sample was placed in culture with CpG and IL-2 for 5 days and then assayed in H1N1 , H5N1 or total IgG ELISPOT assays. In addition, to compare results obtained from FACS analysis and by ELISPOT for their reproducibility this experiment was repeated 2-3 times using different frozen aliquots from each of the 4 donors. The results from these experiments are summarized in figure 8. The analysis done by FACS on PBMC samples stained with both rH5 and rHl revealed distinct populations of H5+/H1- and H5-/H1+ memory B cells and a small, but detectable subset of H5+/H1+ IgG memory B cells (0.01% of total IgG memory B cells for donor 1 in the example reported in fig.8 A). It is worth nothing that in the example of donor 1 the frequency of H5+/H1- and H5-/H1+ IgG memory B cells detected after double staining (0.18% and 0.45% of total IgG memory B cells, respectively) was only slightly lower than the frequency of H5+ and H1+ IgG memory B cells detected in PBMC samples stained with only rH5 or rHl (0.20% and 0.50% of total IgG memory B cells, figure 8B). In addition, and most importantly, frequencies of H5+ and H1+ IgG memory B cells detected by FACS were very close to those derived analyzing the same PBMC sample by a conventional ELISPOT assay (see Table 3). Remarkably, when applied in parallel to the analysis of multiple aliquots of the same PBMC samples, the two methods also demonstrated comparable reproducibility of results.

Taken as a whole, these results demonstrate that memory B cells with restricted, or cross-reactive specificity for HI and H5 influenza receptors can be identified by FACS analysis of PBMC stained simultaneously with HA baits from the two sub-serotypes. In addition, the close similarity of the results obtained estimating the frequency of H5- and Hl-specific IgG memory B cells by FACS analysis and conventional ELISPOT assays shows that obtaining reliable estimates of HA-specific IgG memory B cell frequency directly in ex vivo PBMC samples is possible. Alternative use of serotype A and B vaccine subunits as saturation agents allows identifying memory B cells specific for HI, H3 and B influenza antigens

In order to extend the analysis to memory B cells specific for HA receptor molecules from the three influenza strains included in human seasonal influenza vaccines (i.e. A/H1N1, A/H3N2 and B) we explored the possibility to stain PBMC with HA baits from two vaccine strains, while using the third to prevent not specific binding to a (2,6) sialic acid residues.

First we assessed the specificity of the staining with a fluorochrome-tagged rH3 bait (from A/Brisbane/dd/07) done on PBMC pre-saturated with B vaccine subunit antigens (Figure 9A). The results obtained from the FACS analysis of this samples showed a restricted distribution of H3+ B cells across the CD27+/dim memory B cell subset (figure 9B). In addition, the population of ASCs generated by H3+ memory B cells after activation in vitro was greatly enriched in cells secreting H3- specific IgG (figure 9B).

Example 4:

Alternate use of mismatched influenza A and B vaccine antigens as saturating agents allows to analyze memory B cells with restricted and cross-reactive specificity to two HA serotypes

Since it is generally recognized that cross-reactive immune responses to influenza A and B strains is minimal if not at all absent, we decided to assess whether alternate use of mismatched influenza A and B vaccine antigens could allow to combine HA baits from 2 different (sub)types to stain the same PBMC samples. To this aim, we first checked the specificity of HA+ B cells sorted from PBMC pre-incubated with vaccine subunit antigens from the B/Brisbane/60/2008 strain and then stained with rA/HA baits (either from the H1N1 A/California/07/2009 or from the H3N2 A/Brisbane/ 10/2007 strains, respectively), or from PBMC pre-incubated with influenza A subunit antigens (from the A/Panama/2007/99 strain) and then stained with the rB/HA subunits from B/Brisbane/60/2008. As negative control for BCR independent binding the same PBMC samples was stained with fluorochrome-conjugated HSA.

As shown in figure 10 A, both influenza A and B vaccine subunits antigens efficiently prevented rHA binding to all B cells. To verify the specificity of the staining HA+ cells were sorted and tested by ELISPOT for their capacity of generating HA-specific IgG, as in previous experiments. Of note, to obtain sufficient numbers of cells for this analysis, we fixed 'generous' gates for sorting accepting the risk of including HA- B cells (figure 10 A). Figure 10 B shows that, the populations of PCs generated from the A/H1+, the A/H3+ or the B/HA+ sorted B cells were all highly enriched in PCs producing antigen-specific IgG as compared to unsorted PBMC samples, even if at variable extent (44, 210 and 180 folds, respectively). Comparable results were obtained in three different experiments with PBMC from multiple donors.

Next we verified whether it was possible to stain the same PBMC samples with 2 HA baits, either from type A and type B influenza strains, or from 2 influenza strains from different influenza A subtypes. PBMC from the same donor were then split in 4 tubes. PBMC in 2 tubes were pre- incubated with 0.6 μg of H3N2 vaccine subunits from the B/Brisbane/60/2008A strain and stained either with A488 rHl and A647 rH3, or with A488 HSA and A647 HSA (0.3 μg/each); PBMC in the other 2 tubes were pre-incubated with 0.6 μg of vaccine subunits from the A/Panama/2007/99 strain and stained either with A488 rHl and A647 B/rHA, or with A488 HSA and A647 HSA (0.3 μg/each).

After gating PBMC samples on CD20+ B cells, B cells bound to either rHA baits become detectable (figure 11). Consistently with the poor antigenic homology between influenza A and B strains, B cells binding simultaneously to both rHl and B/rHA baits were not detectable and HA+ B cells observed in PBMC stained simultaneously with these two baites displayed a restricted pattern of specificity (figure 11 bottom panels). A similar restricted distribution was also observed in PBMC stained simultaneously with rHl and rH3; strikingly, however, rare 'bona fide' H1+H3+ B cells were observed in some donors (figure 11 upper panels). Of note, comparable frequencies of H1+ B cells were detected in tubes from the same PBMC samples, independently from the vaccine subunit used as saturating agent, as well as from the nature of the paired rHA bait. Validation of the staining protocol for the analysis of B cell responses to vaccination

To verify the possibility of monitoring by FACS analysis the dynamic changes in HA-specific B cells induced by vaccination we took advantage of the availability of PBMC samples collected in a clinical trial performed between 2008 and 2009. These samples were collected during the influenza season and cases of infections were mostly driven by the H3N2 strain; for this reason we decided to restrict our analysis to MBC response to the HI A/Solomon Island/3/2006 vaccine strain, which should have been less affected by concomitant natural exposure to influenza antigens.

Before proceeding to this analysis, we first assessed the specificity of the staining with the rHl bait. As in previous experiments, this assessment was done by sorting H1+ B cells from healthy donors' PBMC and by comparing, in conventional ELISPOT assays, the frequency of HINl-IgG PCs they generated after activation with CpG and IL-2 in vitro, to that observed in parallel cultures of unsorted PBMC. As shown in figure 12, 63% of IgG PCs detected in cultures of sorted H1+ B cells secreted IgG specific for H1N1 , while only 0.3% of PCs recovered from cultures of not fractioned PBMC displayed the same specificity. These results confirmed that population of B cells identified based on their binding to rHl A/Solomon Island/03/06 bait was enriched (143-fold) in HINl-specific memory B cells. Next we assessed the reproducibility of H1+ MBC enumeration by FACS analysis. For this purpose PBMCs from 4 blood bank donations were frozen in multiple vials to be thawed over three different days. On each day, an aliquot of PBMC from each donor was thawed and split in 2 parts: one was processed for FACS analysis of H1+ MBCs, the other to assess the frequency of HlNl-IgG specific MBC by conventional IgG ELISPOT assay. In order to make possible comparing the results from the two analytical procedures, a monoclonal antibody against human IgG was included in the staining solution and the frequencies of H1+ IgG+ MBCs determined by enumerating H1+ events observed in the CD20+ IgG+ gate.

The results of these experiments are summarized in figure 13, where frequencies of H1+ IgG+ B cells detected by FACS analysis are plotted on the y-axis against frequency of HlNl-IgG PCs detected by ELISPOT in the same PBMC samples, which are plotted on the x-assay. Of note, values of antigen specific IgG MBC detected by the two assays were in significant linear correlations (R2= 0.63; p = 0.0013; y= 0.49 + 0.72 x). Eventually, the frequency of H1+ MBC detected by FACS tend to be slightly higher, as indicated the positive value of the curve's intercept on the y-axis. Finally, we applied the staining protocol to evaluate quantitative and qualitative changes in the pool of HI -specific memory B cells induced by vaccination. Accordingly, PBMC from 3 healthy volunteers were pre-incubated with B mismatched influenza antigens serotype are then stained with HSA or rHl baits. Pre-titrated amounts of monoclonal antibodies against human CD20, CD27 and IgG were added to the staining solution in order to analyze the distribution of H1+ B cells across the mature (CD27+IgG+) and recently generated (CD27-IgG+) IgG-switched memory B subsets (Magdalena A. Berkowska et al. Blood 118, 2011). Figures 14 and 15 summarize the results from the analysis of a representative vaccinee. A clear increase in the frequency of H1+ CD20+ CD27+ MBCs over baseline values was detected in PBMC collected 21 days after vaccination, followed by a slight and not significant contraction at day 43 (Figure 14). In order to best characterize the distribution of H1+ B cells across different memory B cell subsets before and after vaccination samples were gated on CD20+ H1+ or CD20+ HI- B cells and further characterized for CD27 and IgG expression (figure 15). At all the analyzed time points the majority of H1+ B cells expressed the CD27 mature memory marker, and about 50% of them were IgG- switched; strikingly however, at baseline a consistent proportion (36%) of H1+ B cells were CD27- and only one third of them expressed an IgG-switched BCR. Following vaccination instead, we found that >90% of H1+ B cells were found to express the CD27 marker and a slight increased proportion of them were also co-expressing IgG-switched BCR (55-60%) in example depicted in figure 14). Conversely, constant frequencies of CD27+ and/or IgG+ cells were detected across HI - B cells. Overall, these results demonstrate that the staining protocol we have developed is sufficiently robust to monitor quantitative and qualitative changes induced by antigenic challenge in the pool of HA- specific memory B cells. Of note, the results from our preliminary analysis of the distribution of HA- specific MBC across different B cell subsets might provide a most comprehensive understanding of B cell response to vaccination as compared to natural infection, as well as to vaccines differing in antigenic composition or for type of adjuvants included in their formulation. Discussion

The aim of this study was to develop a staining protocol suitable to analyze and sort by flow cytometry human memory B lymphocytes binding to influenza soluble HA baits through BCR- antigen specific interactions. Similar approaches have been successfully designed to detect B lymphocytes specific for various protein antigens (Amanna et al. Journal of Immunology Methods 317 (2006) 175-185). Their application for direct analysis of influenza HA-specific human memory B cells in ex vivo PBMCs has been so far limited by the high unspecific binding of HA baits to human cells (Doucett et al. Journal of Immunological Methods 2005). We demonstrate here that by alternate use of mismatched mono bulk vaccine subunits from influenza A or B serotypes it is possible to saturate unspecific binding sites and to reveal B cells carrying BCR specific for the HA subtypes of interest. We also provide strong evidences that this approach is sufficiently robust to measure by FACS analysis changes in the frequency of HA-specific B cells in human PBMC collected before and after vaccination, as well as for monitoring variations in their distribution across the different memory B cell subsets . Finally, we show that, by staining the same PBMC sample with two HA baits tagged with different fluoro chromes, it is possible to discriminate memory B cells with restricted specificity for type A or type B influenza HA, for HI or H3 influenza A subtypes, and also to identify rare 'bona fide' H1/H3 cross-reactive memory B cells.

In our preliminary experiments we confirmed previous findings (Doucett et al. Journal of Immunological Methods 2005) that, when added directly to PBMC suspensions, HA binds to the surface of almost all leucocytes. We found that this stickiness is not restricted to recombinant HA molecules, nor restricted to a peculiar influenza (sub)type. Multiple evidences indicate that this type of binding is largely due to the capacity of influenza HA trimers to establish multimeric interactions with sialic acid residues, which are abundantly present on blood cells surface. Compounds that bind with high avidity to HA receptor binding sites, such as sialopentasaccarides, efficiently prevent HA binding to all blood cells other than B lymphocytes. A clear reduction of HA unspecific binding to B cells was instead observed when PBMC were stained after being pre-treated with neuraminidase, which removes sialic acid residues from cell surfaces' glycoproteins and glycolipids. Once verified by ELISPOT, however, the specificity of HA+ B cells sorted after neuraminidase treatment was consistently lower than that of HA+ B cells sorted from PBMC pre-incubated with mismatched influenza subunit antigens. The expression of CD27 has been established as a reliable marker to identify human MBCs (Agematsu et al. 1997, Eur. J.Immunol.27(8)). A minority of CD27 negative IgG+ and IgA+ memory B cells has also been observed in human blood by several groups (Wirths et al. Eur. J. Immunol. 2005. 35: 3433-3441; Magdalena A. Berkowska et al. Blood 118, 2011). Multiple evidences indicate that circulating CD27 negative Ig-switched B cells might correspond to recently generated MBC, which eventually acquire CD27 expression upon antigen re-encounter, or to memory cells primed in mucosal-associated lymphoid tissues. Our results demonstrate that in resting condition about 60% of HA+ B cells express the CD27 memory marker and up to 50% of these are IgG-switched. However, 30% of IgG-switched HA+ B lymphocytes do not express CD27, while 24 % of HA+ B cells do not express CD27 or IgG. About 15% of these CD27- IgG- HA+ B cells express IgA while the remaining might express IgD and or IgM BCR, suggesting that they might be naive B cells binding with low avidity to influenza HA. It is remarkable, however, that after vaccination we found that the frequency of HA+ B lymphocytes expressing CD27 increased to >90% while the frequency of HA+ B cells expressing CD27+ and IgG-switched BCR rose to >50%. Comparable changes were not observed among CD27+ and CD27- B cells not binding to HA. All together, these observations support the conclusion that by our staining protocol we can identify MBC capable of BCR-specific interaction with HA antigens in vivo.

The assays currently in use for the analysis of memory B cell response to vaccination, i.e. ELISPOT and limiting dilution assays, can provide reliable information on quantitative changes in the frequency of antigen specific MBC induced by vaccination or infection. In their most sophisticated format, e.g. dual color or fluorescent ELISPOT, they also allow to discriminate MBC expressing BCR of different isotypes. However, it is important to note that all these assays require an obligatory step where PBMC are cultured with B cell polyclonal stimuli for several days. This step unavoidably introduces biases that are difficult if not impossible to be controlled.

In recent years we started to gain a best knowledge of the repertoires of HA specific B cells in influenza patients and vaccinees. This has been possible thanks to the development of high throughput systems for B cell immortalization, cloning and expression of Ig VH and VL genes expressed by single B cells and, most importantly, to the availability of specific markers to identify and sort by flow cytometry antibody secreting plasmablasts that circulate in the blood early after influenza vaccination or infection. We still do not know however, if the clonal repertoire of HA specific plasmablasts accurately reflects the clonal composition of all memory B cells reacting to antigenic challenge. According to the results obtained in several mouse vaccination models, plasmablasts early appearing in the blood originate from the most avid portion of pre-existing antigen-specific MBC, which preferentially take the extra-follicular pathway and differentiate into short lived antibody secreting cells (Paus D et al J Exp Med 2006; 203: 1081-1091). MBC with low and intermediate affinity for antigen binding seem to be more prone to enter into germinal centers, where, after new rounds of somatic hypermutations and competition for antigen binding, they acquire affinity matured BCR before generating new memory B cells and long lived plasma cells (Paus D et al J Exp Med 2006; 203: 1081-1091). Evidences that human memory B cells might behave similarly have been reported (Magdalena A. Berkowska et al. Blood 118, 2011). The observation that human HA-specific plasmablast have a remarkably oligoclonal composition and express a high number of mutation in their Ig V genes are also consistent with the hypothesis that they might derive from a small fraction of MBC that have already acquired high binding avidity for HA through multiple germinal center reactions triggered by previous influenza infections or vaccinations (Wrammert et al. Nature Vol 453, 2008 and Wrammert et al. Journal of Experimental Medicine Vol.208 Nol, January 2011; M. Anthony Moody et al. PLoS ONE 2011 Vol.6 No.10).

Direct investigations aimed at understanding how the clonal repertoire of HA-specific memory B cells evolves after repeated infections, or following the administration of different vaccine formulations, has been so far limited by the lack of reliable protocols for their direct identification ex vivo. Laborious screenings of large libraries of EBV-transformed human IgG memory B cells or of phages displaying antibodies from unswitched memory B cells have been so far successfully applied only in the quest for human monoclonal antibodies broadly neutralizing multiple HA subserotypes (Corti D et al The Journal of Clinical Investigation 2010 Vol.120 No.5 1663-1673); Throsby M et al. PLoS ONE 2008; 3(12):e3942).

The staining protocol we have developed to identify HA-specific MBCs in ex vivo PBMC samples can greatly simplify the analysis of human repertoires of memory B cells specific for influenza HA. In particular, by pairing the use HA baits from different (sub)serotypes with that of monoclonal antibodies against diverse MBC subsets, our approach might result in an extremely powerful tool to analyze large number of single HA-specific MBC in comparison to that of plasmablasts isolated from in the same subjects, as well as to deep sequence changes in repertoire of HA memory B cells elicited by influenza infection or vaccination in cohorts of subjects differing by age or underlying health conditions.

EXAMPLE 5

Characterising the repertoire of HA binding B cells in human PBMC

The distribution of HA+ MBC across different B cell subsets (e.g. IgG and IgA MBC populations) was analysed by FACs (see figure 16).

The sequence of paired heavy and light chain coding genes expressed by single sorted HA+ B cells has also been determined (see figure 17).

Claims

I) A method for detecting in a sample plasmablasts and/or MBCs which express an antibody specific for a glycan-binding antigen of interest, wherein the method comprises the steps of:
(a) pre-incubation of the sample with a saturator and/or disruptor of non-specific glycan binding sites to give a pre-incubated sample;
(b) labeling of the pre-incubated sample with labeled bait to give a labeled
sample, wherein the labeled bait binds B cell receptors (BcRs) on the plasmablasts/MBCs which bind to the glycan-binding antigen of interest; and
(c) detection of the plasmablasts and/or MBCs in the labeled sample. 2) The method of claim 1 , wherein the antigen of interest is an influenza virus antigen.
3) The method of claim 2, wherein the influenza antigen is an influenza A antigen.
4) The method of claim 2, wherein the influenza antigen is an influenza B antigen.
5) The method of claim 3, wherein the influenza A antigen is a HA, NA or HANA antigen.
6) The method of claim 5, wherein the HA or HANA antigen has any serotype from the group consisting of: HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15,
H16, H17.
7) The method of claim 5, wherein the HA or HANA antigen has any serotype from the group consisting of: HI, H2, H5, H6, H8, H9, Hl l, H12, H13, and H16.
8) The method of claim 5, wherein the HA or HANA antigen has any serotype from the group consisting of: H3, H4, H7, H10, H14 and H15.
9) The method of any of the preceding claims, wherein the saturator and/or disruptor consists of or comprises an influenza A antigen from a different serotype to the antigen of interest.
10) The method of claim 3, wherein the influenza A antigen is a group 1 HA, selected from the group consisting of: HI, H2, H5, H6, H8, H9, Hl l, H12, H13, and H16.
I I) The method of claim 3, wherein the influenza A antigen is a group 2 HA, selected from the group consisting of: H3, H4, H7, H10, H14 and H15.
12) The method of any of the preceding claims, wherein the saturator and/or disruptor consists of or comprises Neuraminidase. 13) The method of any of the preceding claims, wherein the saturator and/or disruptor consists of or comprises an influenza B antigen, such as influenza B HANA.
14) The method of any of the preceding claims, wherein the at least one or more labeled bait is related to or the same as the antigen of interest. 15) The method of any of the preceding claims, wherein the at least one or more labeled bait is a recombinant HA protein.
16) The method of any of the preceding claims, wherein the method comprises 1 , 2, 3, 4, or 5 labeled baits.
17) The method of any of the preceding claims, wherein the method comprises two labeled baits.
18) The method of claim 15, wherein the two labeled baits of interest are two HA proteins from two distinct serotypes and which are labeled with two distinct labels.
19) The method of any of the preceding claims, wherein the at least one labeled antigen of interest is labeled with a fluorescent label. 20) The method of claim 1 , wherein the patient sample is a blood sample or PBMCs extracted from a blood sample.
21) The method of claim 1 , wherein the patient sample is taken at least 5, at least 6, at least 7, at least 8, at least, at least 9, at least 10 days after exposure to the at least one or more antigen of interest or more than 15, more than 20, more than 25, more than 30, more than 35, more than 40 days, more than 1 month, more than 6 months, more than 1 year, more than 5 years, or more than 20 years after exposure to the at least one or more antigen of interest.
22) A population of cells, obtained by the method of any of the preceding claims, wherein the cell population comprises at least 10%, at least 20%, at least 30%), at least 40%o, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% antigen-specific B cells, as determined by the ELISPOT assay.
23) A population of cells, obtained by the method of any of the preceding claims, wherein the cell population is enriched by at least 10 fold, at least 50 fold, at least 70 fold, at least 100 fold, at least 130 fold, at least 210 fold, at least 250 fold or more for antigen-specific B cells, compared to the pre-sorted population, as determined by the ELISPOT assay.
24) A method for producing anti-HA antibodies, wherein the method comprises: (a) isolation of nucleic acid encoding the anti-HA antibody from a population of cells sorted according to any preceding claim;
(b) cloning into expression vectors; and
(c) expressing the anti-HA antibodies in an expression system. 25) A method for producing anti-HA antibodies, wherein the method comprises:
(a) obtaining a population of cells sorted according to any preceding claim;
(b) culturing the cells with stimuli to induce differentiation to Ig secreting MBCs;
(c) cloning and immortalising cells.
26) An antibody produced by methods of claim 24 or 25.
27) An antibody that is cross-reactive against two or more influenza group 1 antigens, such as HI and H5.
28) An antibody that is cross-reactive against two or more influenza group 2 antigens, such as H3 and H7.
29) An antibody that is cross-reactive against two or more influenza antigens in group 1 and in group 2, such as HI and H3.
30) An antibody that is cross-reactive against two or more influenza antigens, wherein at least one antigen is influenza A and at least one antigen is influenza B.
31) The antibody of any of claims 26 to 30, wherein the antibody is an IgG antibody.
32) The antibody of any of claims 26 to 31 , wherein the antibody is a humanised antibody. 33) A nucleic acid encoding the antibody of any of claims 26 to 33.
34) The method of claim 1 , wherein the saturator is influenza B and the one or more labeled bait comprises influenza HI and H3.
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WO2014096389A1 (en) * 2012-12-21 2014-06-26 Ucb Pharma S.A. Method for identifying antibody producing cells

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