WO2023178390A1

WO2023178390A1 - Methods of diagnosing vitt and at-risk individuals

Info

Publication number: WO2023178390A1
Application number: PCT/AU2023/050219
Authority: WO
Inventors: Thomas Paul Gordon; Jing Jing Wang
Original assignee: The Flinders University Of South Australia
Priority date: 2022-03-25
Filing date: 2023-03-24
Publication date: 2023-09-28

Abstract

Methods for diagnosing individuals with vaccine-induced immune thrombotic thrombocytopenia (VITT) or for identifying individuals at risk of VITT are disclosed. The methods involve genotyping and/or the detection of peptide "barcode" sequences characteristic of anti-platelet factor 4 (PF4) antibody clonotypes which mediate the VITT syndrome. Methods of treatment of individuals with VITT and for monitoring treatment responses are also disclosed.

Description

METHODS OF DIAGNOSING VITT AND AT-RISK INDIVIDUALS

TECHNICAL FIELD

[0001] The present disclosure relates to methods (assays) for diagnosing individuals with vaccine- induced immune thrombotic thrombocytopenia (VITT) or for identifying individuals at risk of VITT by genotyping and/or the detection of peptide "barcode" sequences characteristic of anti-platelet factor 4 (PF4) antibody clonotypes which mediate the VITT syndrome.

PRIORITY DOCUMENT

[0002] The present application claims priority from Australian Provisional Patent Application No. 2022900751 titled "METHODS OF TREATMENT WITH VITT ANTIBODY MOTIFS AND USES THEREOF" and filed on 25 March 2022, the content of which is hereby incorporated by reference in its entirety.

BACKGROUND

[0003] The syndrome of vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare thromboembolic complication of the adenoviral-vectored SARS-CoV2 vaccines known as ChAdOxl nCoV-19 (AstraZeneca) and Ad26.COV2.S (Janssen/Johnson & Johnson) mediated by antibodies directed against platelet factor 4 (PF4)^{1 5}. The mechanisms by which the adenoviral DNA vectors break immune tolerance to PF4 and trigger B-cell clonal expansion and secretion of anti-PF4 IgGs are under intense investigation but most likely involve the formation of immunogenic complexes of PF4 with vaccine components in a pro-inflammatory setting⁶. Pathogenic anti-PF4 immunoglobulin G molecules (IgGs) may then subsequently form circulating immune complexes (ICs) with PF4 tetramers which are thought to drive thrombotic events by Fc gamma receptor Ila (FcyRIIa) -dependent platelet activation which, in turn, may activate granulocytes to release procoagulant neutrophil extracellular traps (NETS)^{6 8}. Further, it has been observed that serum anti-PF4 antibodies are mostly transient and appear in serum within days of vaccination, which suggests a recall immune response on memory B-cells⁹.

[0004] Given their causal role in VITT, the identification of the molecular composition of the anti-PF4 antibodies and their antigenic target(s) is crucial for developing better diagnostics and treatments, and also for precise tracking of PF4-specific B-cell clones and secreted clonotypes. In a key advance, Huynh et al. mapped the antibody-binding site to a single conformational epitope on the PF4 molecule which is located within the heparin-binding site and distinct from epitopes bound by serum from patients with heparin-induced thrombocytopenia (HIT)^{10, 25}. Moreover, a recent intact mass spectrometric analysis of anti-PF4 IgGs in patients with VITT and HIT revealed the expression of monoclonal and oligoclonal light chains in the former as distinct from a polyclonal light -chain pattern in the latter. However, while intact light-chain mass measurements were performed to inform clonality, direct amino acid (aa) sequencing of light- or heavy-chains was not investigated^{11, 12}.

[0005] The present inventors have developed a novel proteomic workflow based upon high-resolution de novo mass spectrometric sequencing of immunopurified serum antibodies to profile the anti-PF4 antibodies in VITT patients, and unexpectedly revealed stereotypic (also termed "public") LCDR3 and HCDR3 amino acid (aa) sequences with near perfect light chain stereotypy. This points to highly convergent pathways of anti-PF4 antibody production and the potential use of shared peptide "barcodes" provided by these sequences as novel molecular biomarkers for these highly pathogenic anti-PF4 antibody clonotypes.

SUMMARY

[0006] Accordingly, the present disclosure relates to methods (assays) for diagnosing individuals with vaccine-induced immune thrombotic thrombocytopenia (VITT) or for identifying individuals at risk of VITT by genotyping and/or the detection of peptide "barcode" sequences characteristic of anti-platelet factor 4 (PF4) antibody clonotypes which mediate the VITT syndrome.

[0007] In a first aspect, the present disclosure provides a method of diagnosing vaccine-induced immune thrombotic thrombocytopenia (VITT) syndrome in a subject, comprising the step of detecting the presence of antibodies directed against platelet factor 4 (PF4) in said subject, wherein the anti-PF4 antibodies are characterised by: (i) a first amino acid sequence motif DDX'D (SEQ ID NO: 1) present in a light chain complementarity determining region 2 (LCDR2), where X¹ is any proteinogenic amino acid; (ii) a second amino acid sequence motif QX²WD (SEQ ID NO: 2) present in a light chain complementarity determining region 3 (LCDR3), where X² is any proteinogenic amino acid; and (iii) a third amino acid sequence motif X³LED (SEQ ID NO: 3) present in a heavy chain complementarity determining region 3 (HCDR3), where X³ is any proteinogenic amino acid.

[0008] The method of the first aspect provides an opportunity to diagnose a vaccinated individual for VITT or the risk of developing VITT soon after the vaccination and thereby enable early or otherwise prompt treatment with appropriate treatments.

[0009] In a second aspect, the present disclosure provides a method of treating a subject with vaccine- induced immune thrombotic thrombocytopenia (VITT) syndrome or at risk of developing VITT, the method comprising the steps of: 1. Testing for the presence of antibodies directed against platelet factor 4 (PF4) in said subject, wherein the anti-PF4 antibodies are characterised by: (i) a first amino acid sequence motif DDX ¹ D (SEQ ID NO: 1) present in a light chain complementarity determining region 2 (LCDR2), where X¹ is any proteinogenic amino acid; (ii) a second amino acid sequence motif QX²WD (SEQ ID NO: 2) present in a light chain complementarity determining region 3 (LCDR3), where X² is any proteinogenic amino acid; and (iii) a third amino acid sequence motif X³LED (SEQ ID NO: 3) present in a heavy chain complementarity determining region 3 (HCDR3), where X³ is any proteinogenic amino acid; and

2. Where the presence of said anti-PF4 antibodies is detected, treating the subject so as to treat VITT and/or prevent the development or progression of VITT.

[0010] In a third aspect, the present disclosure provides a method for monitoring treatment responses in a subject with vaccine-induced immune thrombotic thrombocytopenia (VITT) syndrome, said method comprising quantifying, at two or more timepoints following and/or during the course of the subject's treatment, the presence of antibodies directed against platelet factor 4 (PF4) in said subject, wherein the anti-PF4 antibodies are characterised by: (i) a first amino acid sequence motif DDX¹ D (SEQ ID NO: 1) present in a light chain complementarity determining region 2 (LCDR2), where X¹ is any proteinogenic amino acid; (ii) a second amino acid sequence motif QX²WD (SEQ ID NO: 2) present in a light chain complementarity determining region 3 (LCDR3), where X² is any proteinogenic amino acid; and (iii) a third amino acid sequence motif X³LED (SEQ ID NO: 3) present in a heavy chain complementarity determining region 3 (HCDR3), where X³ is any proteinogenic amino acid.

[0011] In a fourth aspect, the present disclosure provides a method for identifying a subject at risk of vaccine-induced immune thrombotic thrombocytopenia (VITT) syndrome following administration with an adenoviral-vectored vaccine (preferably an adenoviral-vectored SARS-CoV2 vaccine), said method comprising the step of assaying a suitable sample from said subject for the presence or absence of a IGLV3-21*02 gene variant characterised by a nucleotide sequence encoding a first amino acid sequence motif DDX ¹ D (SEQ ID NO: 1) present in a light chain complementarity determining region 2 (LCDR2), where X¹ is any proteinogenic amino acid.

[0012] Where the step of assaying reveals the presence of the IGLV3-21*02 gene variant, the subject is considered to be at risk of vaccine-induced immune thrombotic thrombocytopenia (VITT) syndrome should they be administered with an adenoviral-vectored vaccine.

BRIEF DESCRIPTION OF FIGURES

[0013] Figure 1 provides the mass spectrometry-based characterisation of PF4-specific clonotypic antibodies and the approach taken in the work described hereinafter: (A) Schematically depicts the proteomics workflow to identify molecular signatures (peptide "barcodes") of anti-PF4 antibodies purified from serum of VITT patients. The Ig variable (V) region peptide sequences were analysed by combined de novo sequencing and IMGT database matching; (B) Illustrates the specificity of the purified anti-PF4 antibodies. Magnetic bead-purified anti-PF4 IgGs from serum of five VITT patients (patient code: VITT 1, VITT 2, VITT 3, VITT 4 and VITT 5) were tested with PF4, SARS-CoV-2 spike SI and S2 ELISAs (SS, starting serum; eluted anti-PF4, eluted anti-PF4 antibody fraction; unbound, anti-PF4 unbound fraction). Data are shown as mean ± SD (n=2); and

[0014] Figure 2 provides representative annotated MS/MS spectra (A-J) obtained from the proteomics workflow of Figure 1 A using anti-PF4 antibodies purified from the serum of one of the five VITT patients investigated (namely, patient VITT 1). The HCDR3 and LCDR3 -containing peptides were identified by de novo sequencing and the HCDR3 and LCDR3 regions are highlighted by boxing. Underlining of amino acids in the peptide sequences indicates a post-translational modification: that is, M (oxidised methionine), W (oxidised tryptophan), and C (carbamidomethylated cysteine). The sequences and m/z and z values of each of the individual peptides are shown on the top of the respective annotated MS/MS spectra. Matched b ions are indicated as dashed peak lines and y ions are shown as dotted peak lines. m=mass, z=charge.

DETAILED DESCRIPTION

[0015] The present inventors have identified stereotypic ("public") LCDR2, LCDR3 and HCDR3 amino acid (aa) sequences providing shared CDR peptide "barcodes" which are useful as molecular biomarkers for anti-platelet factor 4 (PF4) antibody clonotypes which mediate the VITT syndrome.

[0016] Thus, in a first aspect, the present disclosure provides a method of diagnosing vaccine-induced immune thrombotic thrombocytopenia (VITT) syndrome in a subject, comprising the step of detecting the presence of antibodies directed against platelet factor 4 (PF4) in said subject, wherein the anti-PF4 antibodies are characterised by: (i) a first amino acid sequence motif DDX'D (SEQ ID NO: 1) present in a light chain complementarity determining region 2 (LCDR2), where X¹ is any proteinogenic amino acid; (ii) a second amino acid sequence motif QX²WD (SEQ ID NO: 2) present in a light chain complementarity determining region 3 (LCDR3), where X² is any proteinogenic amino acid; and (iii) a third amino acid sequence motif X³LED (SEQ ID NO: 3) present in a heavy chain complementarity determining region 3 (HCDR3), where X³ is any proteinogenic amino acid.

[0017] To the best of the present inventors' knowledge, this combination of amino acid sequence motifs (peptide barcodes) in the LCDR2, LCDR3 and HCDR3 has not been observed before in the amino acid sequences of any antibodies of human origin or otherwise. [0018] The method of the first aspect is applicable to a subject who has been administered with an adenoviral-vectored vaccine, preferably an adenoviral-vectored SARS-CoV2 vaccine such as the vaccine known as ChAdOxl nCoV-19 (AstraZeneca) or the vaccine known as Ad26.COV2.S (Janssen/Johnson & Johnson). While VITT may occur as early as 5 days post vaccination, a pre-VITT syndrome (usually clinically manifested as severe headache) is more likely within this early period (eg 4-18 days post vaccination) with VITT following (but not always) later such as after 18-20 days post vaccination²⁶. In some cases, VITT may first manifest clinically up to 6 weeks or more post vaccination²⁰. However, since the anti-PF4 antibodies which mediate the VITT syndrome appear in serum within days (eg 5 days) of vaccination^{1 3}, the method of the first aspect provides an opportunity to diagnose a vaccinated individual for VITT or the risk of developing VITT soon after the vaccination and thereby enable early or otherwise prompt treatment with appropriate treatments such as therapeutic dose anticoagulants, intravenous (iv) high dose immunoglobulins (IVIG) and/or corticosteroids²². Early recognition and treatment of VITT is expected to lower morbidity and mortality^{20, 21}.

[0019] The presence in the subject of anti-PF4 antibodies characterised by the sequence motifs mentioned above will typically involve detecting the antibodies in a suitable sample obtained from the subject, particularly a whole blood, plasma or serum sample. Optionally, the sample may be treated so to be enriched for immunoglobulin such as immunoglobulin G (eg by any standard IgG purification process well known to those skilled in the art including Melon™ gel IgG purification). The sample may be taken within days of the vaccination (eg within 1-20 days, or 1-18 days, or 1-10 days, 1-5 days or 5-10 days post vaccination) or may be taken much later when significant VITT symptoms (eg persistent and severe headache, focal neurological symptoms, blurred vision, shortness of breath, abdominal pain, unusual bleeding or bruising, swelling and redness in a limb etc), first appear. In other embodiments, the sample may be taken from subject when symptoms of pre-VITT syndrome appear (eg typically severe headache).

[0020] Anti-PF4 antibodies characterised by the sequence motifs mentioned above can be detected by amino acid sequencing of antibodies (eg as present in a sample such as a serum sample). Methods for sequencing proteins such as antibodies are well known to those skilled in the art. Preferably, the sequencing of the antibodies will be conducted by a method suited for direct sequencing and/or high throughput sequencing such as high-resolution de novo mass spectrometric (MS) sequencing. Such methods may be performed in a multiplexed manner. Examples of such methods are described elsewhere. Typically, the sequencing will be conducted using suitable purified antibodies, preferably anti-PF4 antibodies that have been immunopurified from a whole blood, plasma or serum sample by binding the antibodies to PF4 or a suitable fragment thereof (eg by using PF4 protein-coupled capture beads such as suitable magnetic beads well known to those skilled in the art). As required by the method of the first aspect, the sequencing will need to minimally include the portion(s) of the sequences of the antibodies including, potentially, the sequence motifs mentioned above. Thus, if the sequencing reveals one or more anti-PF4 antibody comprising a sequence with the first amino acid sequence motif (of LCDR2), the second amino acid sequence motif (of LCDR3), and the third amino acid sequence motif (of HCDR3), then the method of the first aspect identifies a vaccinated subject with VITT or at risk of developing VITT.

[0021] The anti-PF4 antibodies to be detected are characterised by a first amino acid sequence motif DDX'D (SEQ ID NO: 1) present in LCDR2, where X¹ is any proteinogenic amino acid; however, in some embodiments, the anti-PF4 antibodies to be detected are characterised by a first amino acid sequence motif DDX'D (SEQ ID NO: 1) present in LCDR2, where X¹ is selected from serine (Ser, S), glycine (Gly, G), alanine (Ala, A), threonine (Thr, T), cysteine (Cys, C), asparagine (Asn, N), glutamine (Gin, Q) and tyrosine (Tyr, Y). Preferably, X¹ is Ser (S). Thus, in some particularly preferred embodiments, the anti-PF4 antibodies to be detected are characterised by a DDSD (SEQ ID NO: 4) motif in LCDR2.

[0022] The anti-PF4 antibodies to be detected are characterised by a second amino acid sequence motif QX²WD (SEQ ID NO: 2) present in LCDR3, where X² is any proteinogenic amino acid; however, in some embodiments, the anti-PF4 antibodies to be detected are characterised by a second amino acid sequence motif QX²WD (SEQ ID NO: 2) present in LCDR3, where X² is selected from valine (Vai, V), alanine (Ala, A), leucine (Leu, L), isoleucine (He, I), threonine (Thr, T), methionine (Met, M), phenylalanine (Phe, F), Tryptophan (Trp, W) and proline (Pro, P). Preferably, X² is Vai (V), Met (M) or Pro (P). Thus, in some particularly preferred embodiments, the anti-PF4 antibodies to be detected are characterised by a QVWD (SEQ ID NO: 5), QMVD (SEQ ID NO: 6) or QPWD (SEQ ID NO: 7) motif in LCDR3.

[0023] Further, the anti-PF4 antibodies to be detected are characterised by a third amino acid sequence motif X³LED present in HCDR3, where X³ is any proteinogenic amino acid; however, in some embodiments, the anti-PF4 antibodies to be detected are characterised by a third amino acid sequence motif X³LED (SEQ ID NO: 3) present in HCDR3, where X³ is selected from asparagine (Asn, N), glutamine (Gin, Q), tyrosine (Tyr, Y), serine (Ser, S), glycine (Gly, G), threonine (Thr, T), cysteine (Cys, C) and alanine (Ala, A). Preferably, X³ is Asn (N) or Gly (G). Thus, in some particularly preferred embodiments, the anti-PF4 antibodies to be detected are characterised by a GLED (SEQ ID NO: 8) or NEED (SEQ ID NO: 9) motif.

[0024] In some particularly preferred embodiments of the method of the first aspect, the anti-PF4 antibodies to be detected are characterised by: (i) a first amino acid sequence motif DDSD (SEQ ID NO: 4) present in a light chain complementarity determining region 2 (LCDR2); (ii) a second amino acid sequence motif selected from QVWD (SEQ ID NO: 5), QMVD (SEQ ID NO: 6) and QPWD (SEQ ID NO: 7) present in a light chain complementarity determining region 3 (LCDR3); and (iii) a third amino acid sequence motif selected from GLED (SEQ ID NO: 8) and NEED (SEQ ID NO: 9) present in a heavy chain complementarity determining region 3 (HCDR3).

[0025] In some embodiments, the anti-PF4 antibodies to be detected are further characterised as IgGl, IgG2 or IgG3 antibodies.

[0026] In a second aspect, the present disclosure provides a method of treating a subject with vaccine- induced immune thrombotic thrombocytopenia (VITT) syndrome or at risk of developing VITT, the method comprising the steps of:

1. Testing for the presence of antibodies directed against platelet factor 4 (PF4) in said subject, wherein the anti-PF4 antibodies are characterised by: (i) a first amino acid sequence motif DDX ¹ D (SEQ ID NO: 1) present in a light chain complementarity determining region 2 (LCDR2), where X¹ is any proteinogenic amino acid; (ii) a second amino acid sequence motif QX²WD (SEQ ID NO: 2) present in a light chain complementarity determining region 3 (LCDR3), where X² is any proteinogenic amino acid; and (iii) a third amino acid sequence motif X³LED (SEQ ID NO: 3) present in a heavy chain complementarity determining region 3 (HCDR3), where X³ is any proteinogenic amino acid; and

[0027] The step of testing for (detecting) the anti-PF4 antibodies (ie step 1.) may be conducted as described above in connection to the method of the first aspect. As such, in preferred embodiments, anti- PF4 antibodies will be detected using a suitable sample such as immunopurified antibodies from a serum sample of the subject. The antibodies will then, preferably, be subjected to a suitable method of direct sequencing to determine whether anti-PF4 antibodies are present which are characterised by the sequence motifs mentioned above (ie peptide barcodes). As will be appreciated, in some particularly preferred embodiments of the method of the second aspect, it will be determined whether anti-PF4 antibodies present are characterised by: (i) a first amino acid sequence motif DDSD (SEQ ID NO: 4) present in a light chain complementarity determining region 2 (LCDR2); (ii) a second amino acid sequence motif selected from QVWD (SEQ ID NO: 5), QMVD (SEQ ID NO: 6) and QPWD (SEQ ID NO: 7) present in a light chain complementarity determining region 3 (LCDR3); and (iii) a third amino acid sequence motif selected from GLED (SEQ ID NO: 8) and NEED (SEQ ID NO: 9) present in a heavy chain complementarity determining region 3 (HCDR3). The subject will be a vaccinated subject (ie a subject vaccinated with an adenoviral-vectored vaccine, preferably an adenoviral-vectored SARS-CoV2 vaccine) who may be experiencing one or more symptoms of pre-VITT (eg typically severe headache) or VITT (eg persistent and severe headache, focal neurological symptoms, blurred vision, shortness of breath, abdominal pain, unusual bleeding or bruising, swelling and redness in a limb etc). [0028] The step of treating the subject (ie step 2.) may, for example, involve any one or more of the treatments for VITT and/or VITT symptoms well known to those skilled in the art. Such treatments for VITT include the administration of anticoagulants, intravenous (iv) immunoglobulins (IVIG) and/or corticosteroids.

[0029] In a third aspect, the present disclosure provides a method for monitoring treatment responses in a subject with vaccine-induced immune thrombotic thrombocytopenia (VITT) syndrome, said method comprising quantifying, at two or more timepoints following and/or during the course of the subject's treatment, the presence of antibodies directed against platelet factor 4 (PF4) in said subject, wherein the anti-PF4 antibodies are characterised by: (i) a first amino acid sequence motif DDX ’ D (SEQ ID NO: 1) present in a light chain complementarity determining region 2 (LCDR2), where X¹ is any proteinogenic amino acid; (ii) a second amino acid sequence motif QX²WD (SEQ ID NO: 2) present in a light chain complementarity determining region 3 (LCDR3), where X² is any proteinogenic amino acid; and (iii) a third amino acid sequence motif X³LED (SEQ ID NO: 3) present in a heavy chain complementarity determining region 3 (HCDR3), where X³ is any proteinogenic amino acid.

[0030] As mentioned above, the serum anti-PF4 antibodies of VITT patients are mostly transient, however studies have found that these antibodies may continue to be present for 3-4 months or longer^{9, 23}. It is anticipated that effective treatment of VITT will result in a reduction or disappearance of the anti- PF4 antibodies in the patient within a shorter time period to that typically seen (eg less than 3-4 months). By tracking the presence of the anti-PF4 antibodies following and/or during treatment, the method of the third aspect provides a means for monitoring for the effectiveness or otherwise of the VITT treatment.

[0031] In some embodiments of the method of the third aspect, the presence of the anti-PF4 antibodies will be quantified at a first time point, being the time of the treatment or commencement of the treatment wherein the treatment is ongoing, and at least one further time point thereafter. For example, the anti-PF4 antibodies may be quantified at least at a second time point that is one, two, three, four, five or seven days after the first time point. Thereafter, the anti-PF4 antibodies may optionally be quantified at least at a third time point which may be one, two, three, four, five or seven days after the first time point. In some embodiments, the anti-PF4 antibodies may be quantified at a second time point which is one day after the first time point, a third time point which is one day after the second time point, a fourth time point that is one day after the third time point, and if a reduction of the anti-PF4 antibodies has been observed at this time, a fifth time point which is one week after the fourth time point. In practice, the period between each time point will be dependent upon, for example, the seriousness of the VITT and/or VITT symptoms being experienced by the subject and the determination of any reduction in the anti-PF4 antibodies, and the degree of any such reduction, as well as the implementation of any variation(s) to the treatment. In some embodiments, the anti-PF4 antibodies will also be quantified prior to treatment. [0032] The step of quantifying the anti-PF4 antibodies in the method of third aspect (ie at each of the two or more timepoints) may be conducted by MS-based quantitative proteomic methodologies such as those well known to those skilled in the art. As such, in preferred embodiments, anti-PF4 antibodies will be quantified using a suitable sample such as immunopurified antibodies from a serum sample of the subject. The antibodies will then, preferably, be subjected to a suitable method of direct sequencing to determine whether anti-PF4 antibodies are present which are characterised by the sequence motifs mentioned above (ie peptide barcodes). As will be appreciated, in some particularly preferred embodiments of the method of the third aspect, the anti-PF4 antibodies to be quantified are characterised by: (i) a first amino acid sequence motif DDSD (SEQ ID NO: 4) present in a light chain complementarity determining region 2 (LCDR2); (ii) a second amino acid sequence motif selected from QVWD (SEQ ID NO: 5), QMVD (SEQ ID NO: 6) and QPWD (SEQ ID NO: 7) present in a light chain complementarity determining region 3 (LCDR3); and (iii) a third amino acid sequence motif selected from GLED (SEQ ID NO: 8) and NEED (SEQ ID NO: 9) present in a heavy chain complementarity determining region 3 (HCDR3). The subject will be a vaccinated subject (ie a subject vaccinated with an adenoviral-vectored vaccine, preferably an adenoviral-vectored SARS-CoV2 vaccine) who may be experiencing one or more symptoms of VITT (eg persistent and severe headache, focal neurological symptoms, blurred vision, shortness of breath, abdominal pain, unusual bleeding or bruising, swelling and redness in a limb etc).

[0033] In the work described hereinafter, the present inventors have also identified that the anti-platelet factor 4 (PF4) antibody clonotypes (from five unrelated VITT patients) which mediate the VITT syndrome, comprise a single IgG2 heavy (H)-chain species paired with a single lambda (I) light (L)-chain species, and that, remarkably, all of the L-chains are encoded by the identical IGLV3-21*02 gene subfamily and showed identical LCDR3 region lengths (ie 17 amino acids) consistent with a high degree of L-chain stereotypy. This shared IGLV3-21*02 allele expresses the LCDR2 amino acid sequence motif DDX'D (SEQ ID NO: 1) and enables the opportunity to identify individuals at risk of VITT before vaccination, by genotyping (genetic screening) for the IGLV3-21*02 light chain gene variant. Consequently, where an individual tests "positive" for this IGLV3-21*02 light chain gene variant, an informed selection of a SARS-CoV2 vaccine for administration may be made (ie an mRNA vaccine may be selected instead of an adenoviral-vectored vaccine).

[0034] Thus, in a fourth aspect, the present disclosure provides a method for identifying a subject at risk of vaccine-induced immune thrombotic thrombocytopenia (VITT) syndrome following administration with an adenoviral-vectored vaccine (preferably an adenoviral-vectored SARS-CoV2 vaccine), said method comprising the step of assaying a suitable sample from said subject for the presence or absence of a IGLV3-21*02 gene variant characterised by a nucleotide sequence encoding a first amino acid sequence motif DDX ¹ D (SEQ ID NO: 1) present in a light chain complementarity determining region 2 (LCDR2), where X¹ is any proteinogenic amino acid.

[0035] Where the step of assaying reveals the presence of the IGLV3-21*02 gene variant, the subject is considered to be at risk of vaccine-induced immune thrombotic thrombocytopenia (VITT) syndrome should they be administered with an adenoviral-vectored vaccine such as an adenoviral-vectored SARS- CoV2 vaccine (eg the vaccine known as ChAdOxl nCoV-19 (AstraZeneca) or the vaccine known as Ad26.COV2.S (Janssen/Johnson & Johnson)).

[0036] Preferably, the immunoglobulin lambda variable 3-21 gene variant (IGLV3-21*02) is characterised by a nucleotide sequence encoding a first amino acid sequence motif DDX ¹ D (SEQ ID NO: 1) present in LCDR2, where X¹ is selected from serine (Ser, S), glycine (Gly, G), alanine (Ala, A), threonine (Thr, T), cysteine (Cys, C), asparagine (Asn, N), glutamine (Gin, Q) and tyrosine (Tyr, Y). More preferably, X¹ is Ser (S).

[0037] Further, in some embodiments, the method of the fourth aspect may further comprise determining whether the IGLV3-21*02 gene variant is characterised by a nucleotide sequence encoding a light chain complementarity determining region 3 (LCDR3) that is 17 amino acids in length and, preferably, comprises an amino acid sequence as follows:

QX²WDX^aX^bX^cDX^dX^eX^fFGGGTK (SEQ ID NO: 10); wherein

X² is as described above, X^a is Ser (S) or Gly (G); X^b is Ser (S) or Arg (R); X^c is Ser (S), Asn (N) or Arg (R); X^d is His (H) or Glu (Q);

X^e is Pro (P) or Vai (V); and X^f is Vai (V) or Leu (L).

[0038] The suitable sample utilised in the method of the fourth aspect may be any suitable sample taken from the subject that contains genomic DNA (eg a blood sample, hair sample, serum sample, saliva sample, cheek cell sample, semen sample, etc). The step of assaying may be conducted directly on the sample or, otherwise, the assaying methodology may comprise one or more treatment(s) to isolate or, at least, partially purify genomic DNA from the sample in accordance with standard techniques well known to those skilled in the art. The step of assaying may employ any of the techniques for assaying nucleotide sequences that are well known to those skilled in the art. For example, the step of assaying may comprise amplifying a suitable target region of the subject's genome including the locus of the IGLV3-21*02 gene using any of the typical nucleotide sequence amplification methodologies (eg polymerase chain reaction (PCR)-based protocols) followed by nucleotide sequencing of the amplification products (amplicons). In some embodiments, the target region may include only the region that may include a nucleotide sequence encoding a first amino acid sequence motif DDX ¹ D (SEQ ID NO: 1) present in LCDR2, while in other embodiments, the target region may additionally include the region comprising the nucleotide sequence encoding LCDR3. Further, in some preferred embodiments, the target region may include the region comprising the nucleotide sequence encoding LCDR2 and LCDR3. Suitable primer sequences for amplification of such target regions may be readily designed and prepared by routine methodologies well known to those skilled in the art such as, for example, those described in Sambrook J and DW Russell, Molecular Cloning: a laboratory manual, Third Edition (Cold Spring Harbor Laboratory Press; 2001), see in particular Chapters 8 and 10.

[0039] In this specification, a number of terms and phrases are used which are well known to those skilled in the art. Nevertheless, for the purposes of clarity, a number of these terms and phrases are hereinafter defined.

[0040] The term "proteogenic amino acid" refers to an amino acid that is genetically encoded; of which there are 20 in the standard genetic code and an additional two (namely, selenocysteine and pyrrolysine) that can be incorporated by special translation mechanisms.

[0041] The term "adenoviral-vectored vaccine" refers to any vaccine based upon a replication-defective vector molecule derived from an adenovirus (eg from a simian adenovirus).

[0042] The term "treating", as used herein, includes prophylaxis as well as the alleviation of established symptoms of a disease or condition. As such, the act of "treating" a disease or condition therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the disease or condition developing in a subject afflicted with or predisposed to the disease or condition; (2) inhibiting the disease or condition (ie arresting, reducing or delaying the development of the disease or condition or a relapse thereof (in case of a maintenance treatment)) or at least one clinical or subclinical symptom thereof; and (3) relieving or attenuating the disease or condition (ie causing regression of the disease or condition or at least one of its clinical or subclinical symptoms).

[0043] The methods and uses of the disclosure are hereinafter further described with reference to the following, non-limiting example(s) and accompanying figure(s). EXAMPLES

Example 1

[0044] A novel proteomics workflow based on high-resolution de novo mass spectrometric sequencing of anti-PF4 serum antibodies from VITT patients was used to identify immunoglobulin variable (IgV) subfamily expression profiles; particularly to determine whether shared peptide "barcodes" may exist in the complementarity determining regions (CDRs) of these highly pathogenic anti-PF4 antibody clonotypes.

Materials and Methods

[0045] Human subjects

Diagnostic serum specimens were obtained from five patients with VITT following vaccination with the ChAdOxl nCoV-19 vaccine. The demographic, clinical and serological findings for these five patients are summarised in Table 1. All five cases satisfied the case definition criteria for "Definite VITT" ²⁴, and four of the five cases were characterised by thrombosis in critical/unusual sites, mainly cerebral venous sinus thrombosis (CVST) and splanchnic vein thrombosis (SVT). Commercially available assay kits were used for diagnostic detection of anti-PF4 antibodies (ie the Asserachrom HPIA IgG assay kit (Stago S.A.S, France) and Lifecodes PF4 IgG assay kit (Immucor, Peachtree Corners, GA, United States of America).

0046] Table 1: Demographic, laboratory and clinical features of AstraZeneca-associated VITT patients

VST, cerebral venous sinus thrombosis; DVT, deep vein thrombosis of legs; ICH, intracerebral haemorrhage; PE, pulmonary embolism; SVT, splanchnic vein thrombosis. Reference range: 150-450 xlO⁹/L Reference range: 1.5-4.0 g/L Cutoff value: <0.5 mg/L ELISA cutoff value: OD<0.21 *ELISA cutoff value: OD<0.4

[0047] Anti-PF4 antibody purification

Antibodies against PF4 were affinity-purified from the serum of the VITT patients using PF4 protein- coupled MyOne Carboxylic Acid Dynabeads (ThermoFisher, Waltham, United States of America). Briefly, the beads were washed twice in 1ml of 15mM MES buffer (pH6) followed by activation with lOOpL of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (lOmg/ml) and incubation on a rotator for 30 min at room temperature. Following activation, the beads were washed with 15mM MES buffer and coated with human PF4 protein (ChromaTec, Greifswald, Germany) in 15mM MES buffer. Samples were then incubated overnight on a rotator at room temperature. After antigen coating, the beads were washed twice in 0.1% Tween 20 PBS and blocked with PBS 0.1% BSA for 10 min on a rotator. Diluted serum was then added to the beads and mixed on a rotator for 2 h at room temperature. After the mixing, unbound sera were removed, and the beads were washed 4 times with PBS. To elute the antibodies, lOOmM glycine elution buffer (pH 11) was added to each sample, vortexed and incubated for 5 min. Eluted anti-PF4 were then transferred to lOkd spin columns (Amicon® Ultra; Merck Millipore, Burlington, MA, United States of America) with Milli Q water for buffer exchange and stored at -80°C until required. To validate the antibody purification method, serum samples from two healthy donors were used as negative controls and no IgG was eluted from these samples, indicating no non-specific binding to the antigen. Moreover, rabbit polyclonal anti -human PF4 antibody (ThermoFisher) and a serum sample from one patient with HIT were used as positive controls. The eluted anti-PF4 IgGs from rabbit anti-human PF4 antibodies and the serum sample from patient with HIT-expressed polyclonal kappa and light chains, IGKV1S66, IGKV1S15 and IGLV3S9 for the former, IGKV3-20, IGKV2D-29, IGKV3-11, IGLV3-21 and IGLV6-57 for the latter.

[0048] Specificity analysis of anti-PF4 antibodies

The activity and specificity of purified anti-PF4 IgGs was determined by testing starting sera (diluted 1:100), eluted anti-PF4 fraction and unbound fractions (normalised to each starting serum) for reactivity against PF4, SARS-CoV-2 spike SI and S2 proteins (the Native Antigen Company, Kidlington, England) by ELISA. Briefly, Maxisorp nunc immune plates (ThermoFisher) were coated with 100 pl of individual PF4, SI and S2 protein at 4 pg/ml in PBS buffer overnight at 4°C. Plates were blocked with PBS 1% BSA (Sigma- Aldrich, A3059) and subsequently incubated with patient serum (diluted to 1:100) for 2 h at 37 °C. After washing plates four times with PBS 0.05% Tween 20, anti-human IgG (Sigma-Aldrich A3187), secondary antibodies were added and incubated for 1 h at 37 °C. Plates were then washed six times with PBS 0.05% Tween 20 and phosphatase substrate (Sigma, S0942) added. Optical density (OD) at 405nm was measured by a plate reader (Spectramax Id5) at 30 minutes of incubation. Blank measurements were subtracted from each sample measurement. [0049] Mass Spectrometry (MS) sequencing

Purified anti-PF4 IgG heavy (H) and light (L)-chains were isolated by reduced SDS-PAGE (criterion stain-free TGX gels; Bio-Rad, Hercules, CA, United States of America). The gel bands were excised and digested with Pierce trypsin protease (ThermoFisher) and chymotrypsin (Promega, Madison, WI, United States of America), separately.

[0050] Peptides were analysed with a Dionex Ultimate 3000 UPLC coupled to a Thermo Fusion Lumos tandem mass spectrometer (ThermoFisher). Peptides were applied to a PepMap™ 100 trap cartridge (0.3 x 5 mm, 5 pm Cl 8, ThermoFisher) and separated on an in-house 40 cm pulled column created from 75 pm inner diameter fused silica capillary packed with 1.9 pm ReproSil-Pur C18 beads (Dr. Maisch, Ammerbuch, Germany). Solvent A was 0.1% formic acid in water and solvent B was 0.1% formic acid in 80% acetonitrile. For each injection, approximately 1 pg peptides were loaded and separated using a 60- min gradient from 3 to 31.2% B, followed by a 25 min washing and re-equilibration step.

[0051] The mass spectrometer was operated in positive ion mode with a MSI resolution of 60,000, normalised AGC target of 8e5, scan range of 350-1200m/z and with the maximum injection time set to auto for all precursor scans. Data dependent mode was set to a cycle time of 3 seconds with MS/MS performed on the most intense precursors exceeding an intensity threshold of 5e5. Additional MS/MS settings were a 1.4 m/z quadrupole isolation width, normalised HCD (higher-energy collisional dissociation) energy of 32% and analysis of fragment ions in the orbitrap with a resolution of 15,000, dynamic exclusion was set to 30 seconds, monoisotopic precursor selection (MIPS) was set to Peptide, maximum injection time was set to Dynamic mode, AGC target set to Standard, charge states unknown, +1 or >+7 were excluded, advanced peak determination was toggled on and the EASY-IC internal mass calibration was employed.

[0052] Purification of anti-PF4 IgGs from individual patient sera was carried out on at least two independent occasions, and the purified antibodies were run for MS at two technical replicates. The peptides were only included if they were found in at least three out of four technical replicates.

[0053] Protein sequence data analysis

Peptide sequence analysis was performed by de novo sequencing and International ImMunoGeneTics (IMGT) database matching using Peaks studio XPro software (Bioinformatics Solution Inc., Waterloo, ON, Canada). Briefly, a maximum of two missed cleavages, precursor tolerance of < 15 parts per million, product ion tolerance of 0.02 Da, precursor charge state of +2 to +4, fixed modification carbamidomethylation, variable modifications oxidation and deamidation, a maximum of three modifications allowed and non-specific cleavage at one end. High-quality de novo peptides were selected based on sequences having an average local confidence score threshold greater than or equal to 80% and inspected manually to ensure correct assignments. A false discovery rate (FDR) threshold of 1.0% was applied at the peptide level to each data set. The Ig variable region subfamily was assigned from the presence of a unique peptide corresponding to the subfamily.

Results and Discussion

[0054] A proteomics discovery approach (schematically depicted in Figure 1A) was used to profile serum anti-PF4 antibodies in VITT patients and unexpectedly revealed stereotypic ("public") amino acid (aa) sequences in the heavy (H) and light (L)-chains of the third complementarity determining regions (ie LCDR3 and HCDR3) with near perfect light chain stereotypy.

[0055] Prior to MS sequencing, the monospecificity of the anti-PF4 IgGs was verified by testing the starting serum (diluted 1/100), bead-purified anti-PF4 antibody fraction (eluted with lOOmM glycine, 0.1% sodium deoxycholate, pHl l) and unbound fractions, using ELISAs coated with individual PF4, SARS-CoV-2 spike (SI) and S2 proteins at 4 pg/ml. There was no cross-reactivity between eluted, purified anti-PF4 IgGs and SI and S2 proteins in the VITT serum samples (see Figure IB). As denoted in the proteomics discovery approach (Figure 1A), purified anti-PF4 IgGs were then separated by SDS- PAGE; H and L-chain bands excised for in-gel digestion with trypsin and chymotrypsin, respectively; and analysis of peptides performed in a Thermo Orbitrap Fusion Lumos Tribrid mass spectrometer with peptide de novo sequencing performed by Peaks Studio XPro and IMGT databases as detailed previously¹³. Representative (from the patient, "VITT 1") annotated de novo peptide spectra from HCDR3 and LCDR3 clonotypic peptide barcodes are shown in Figure 2A-J.

[0056] The sequencing of the anti-PF4 IgGs revealed a single IgG2 H-chain species paired with a single lambda L-chain species in all five of the unrelated VITT patients. Remarkably, all L-chains were encoded by the identical IGLV3-21*02 gene subfamily and showed identical LCDR3 peptide lengths consistent with a high degree of L-chain stereotypy (Table 2). Notably, the shared IGLV3-21*02 allele expresses an acidic (ie negatively charged) DDxD (SEQ ID NO: 1) motif in the CDR2 region which is likely to be of importance in antibody binding to the positively charged PF4 epitope¹⁰. Another shared amino acid motif of interest, namely QxWD (SEQ ID NO: 2), is located in the LCDR3 region (see Table 2). Interestingly, a stereotypic IGLV3-21 B-cell receptor defines a subgroup of chronic lymphocytic leukemia with a poor prognosis¹⁴. 0057] Table 2: Clonotypic H and L-chain third complementarity-determining region (CDR3) molecular signatures in the anti-PF4 antibodies

old amino acids in the HCDR3 and LCDR3 sequences denote shared motifs across unrelated patients, and underlined amino acids denote amino acid replacement mutations found in thendividual patient HCDR3 and LCDR3 regions.

[0058] Analysis of the clonal HCDR3 peptide sequences of Table 2 readily revealed striking stereotypic features characterised by identical HCDR3 lengths and homologous sequences, together with a shared binding motif G/NLED (SEQ D NO: 8/9) located in immunoglobulin heavy chain diversity (IGHD) regions known to confer antigen binding specificity¹⁵. Notwithstanding these convergent HCDR3 regions, individual patient anti-PF4 Ig proteomes were encoded by distinct immunoglobulin heavy chain variable (IGHV) region subfamilies including 3-7, 7-4, 2-5, 3-48 and 3-53 (Table 2), emphasising the critical role of HCDR3s in PF4 epitope binding as opposed to the divergent IGHV regions.

[0059] Amino acid replacement mutations were also found in individual patient HCDR3 and LCDR3 regions (Table 2), consistent with a model of PF4 antigen-driven intraclonal diversification as observed for systemic autoantibodies in lupus and Sjogren’s syndrome^{16 18}. In addition, glutamic acid (E) and aspartic acid (D) replacement mutations of potential binding significance were identified in the IgV regions of H- and L-chains, indicating recall immune responses on PF4-specific memory B cells. The full length amino acid sequences for the variable (V) and constant (C) regions from multiple different anti- PF4 antibodies from each of the five individual VITT patients are provided in Table 3.

Conclusion

[0060] The finding of a stereotyped clonotypic anti-PF4 antibody in this example represents a significant advance in elucidating the molecular pathways of pathologic antibody production in VITT and offers a rare example in human disease of a dangerous small B-cell clone that undergoes rapid clonal expansion and secretion of a harmful monoclonal antibody¹⁹. Moreover, the identification herein of molecular signatures (peptide "barcodes") of anti-PF4 antibodies from VITT patients enables individuals at risk of VITT to be identified before vaccination by genotyping for individuals carrying the IGLV3-21*02 light chain gene variant (thereby enabling an informed selection of a more appropriate SARS-CoV2 vaccine) as well as rapid diagnosis of vaccinated individuals shortly after, or even prior to, early VITT symptoms (for example, symptoms of pre- VITT syndrome such as severe headache occurring 4-18 days after vaccination) to enable prompt and suitable medical treatment to prevent or treat serious life-threatening symptoms or complications of VITT, particularly cerebral venous sinus thrombosis (CVST) and other thromboses. [0061] Throughout the specification and the claims that follow, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprising" and "including" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0062] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge.

[0063] It will be appreciated by those skilled in the art that the present disclosure is not restricted in its use to the particular application described. Neither is the present disclosure restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be also appreciated that the disclosure is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the disclosure as set forth and defined by the following claims.

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Claims

1. A method of diagnosing vaccine-induced immune thrombotic thrombocytopenia (VITT) syndrome in a subject, comprising the step of detecting the presence of antibodies directed against platelet factor 4 (PF4) in said subject, wherein the anti-PF4 antibodies are characterised by: (i) a first amino acid sequence motif DDX ¹ D (SEQ ID NO: 1) present in a light chain complementarity determining region 2 (LCDR2), where X¹ is any proteinogenic amino acid; (ii) a second amino acid sequence motif QX²WD (SEQ ID NO: 2) present in a light chain complementarity determining region 3 (LCDR3), where X² is any proteinogenic amino acid; and (iii) a third amino acid sequence motif X³LED (SEQ ID NO: 3) present in a heavy chain complementarity determining region 3 (HCDR3), where X³ is any proteinogenic amino acid.

2. The method of claim 1, wherein the presence of the anti-PF4 antibodies comprises detecting the antibodies in a serum sample.

3. The method of claim 2, wherein the sample is taken from the subject within 1-20 days or 5-10 days post vaccination.

4. The method of claim 2, wherein the sample is taken from the subject when significant VITT symptoms first appear.

5. The method of any one of claims 1 to 4, wherein the anti-PF4 antibodies to be detected are characterised by a first amino acid sequence motif DDX ¹ D (SEQ ID NO: 1) present in LCDR2, where X¹ is selected from serine (Ser, S), glycine (Gly, G), alanine (Ala, A), threonine (Thr, T), cysteine (Cys, C), asparagine (Asn, N), glutamine (Gin, Q) and tyrosine (Tyr, Y).

6. The method of claim 5, wherein the anti-PF4 antibodies to be detected are characterised by a DDSD (SEQ ID NO: 4) motif in LCDR2.

7. The method of any one of claims 1 to 6, wherein the anti-PF4 antibodies to be detected are characterised by a second amino acid sequence motif QX²WD (SEQ ID NO: 2) present in LCDR3, where X² is selected from valine (Vai, V), alanine (Ala, A), leucine (Leu, L), isoleucine (He, I), threonine (Thr, T), methionine (Met, M), phenylalanine (Phe, F), Tryptophan (Trp, W) and proline (Pro, P).

8. The method of claim 7, wherein the anti-PF4 antibodies to be detected are characterised by a QVWD (SEQ ID NO: 5), QMVD (SEQ ID NO: 6) or QPWD (SEQ ID NO: 7) motif in LCDR3.

9. The method of any one of claims 1 to 8, wherein the anti-PF4 antibodies to be detected are characterised by a third amino acid sequence motif X³LED (SEQ ID NO: 3) present in HCDR3, where X³ is selected from asparagine (Asn, N), glutamine (Gin, Q), tyrosine (Tyr, Y), serine (Ser, S), glycine (Gly, G), threonine (Thr, T), cysteine (Cys, C) and alanine (Ala, A).

10. The method of claim 9, wherein the anti-PF4 antibodies to be detected are characterised by a GLED (SEQ ID NO: 8) or NEED (SEQ ID NO: 9) motif.

11. The method of any one of claims 1 to 10, wherein the anti-PF4 antibodies to be detected are characterised by: (i) a first amino acid sequence motif DDSD (SEQ ID NO: 4) present in a light chain complementarity determining region 2 (LCDR2); (ii) a second amino acid sequence motif selected from QVWD (SEQ ID NO: 5), QMVD (SEQ ID NO: 6) and QPWD (SEQ ID NO: 7) present in a light chain complementarity determining region 3 (LCDR3); and (iii) a third amino acid sequence motif selected from GLED (SEQ ID NO: 8) and NEED (SEQ ID NO: 9) present in a heavy chain complementarity determining region 3 (HCDR3).

12. The method of any one of claims 1 to 11, wherein the anti-PF4 antibodies to be detected are further characterised as IgGl, IgG2 or IgG3 antibodies.

13. A method of treating a subject with vaccine-induced immune thrombotic thrombocytopenia (VITT) syndrome or at risk of developing VITT, the method comprising the steps of:

14. The method of claim 13, wherein the step of testing for the anti-PF4 antibodies (step 1.) comprises detecting the antibodies in a serum sample.

15. The method of claim 14, wherein the sample is taken from the subject within 1-20 days or 5- 10 days post vaccination.

16. The method of any one of claims 13 to 15, wherein the anti-PF4 antibodies are characterised by a first amino acid sequence motif DDX ¹ D (SEQ ID NO: 1) present in LCDR2, where X¹ is selected from serine (Ser, S), glycine (Gly, G), alanine (Ala, A), threonine (Thr, T), cysteine (Cys, C), asparagine (Asn, N), glutamine (Gin, Q) and tyrosine (Tyr, Y).

17. The method of any one of claims 13 to 16, wherein the anti-PF4 antibodies are characterised by a second amino acid sequence motif QX²WD (SEQ ID NO: 2) present in LCDR3, where X² is selected from valine (Vai, V), alanine (Ala, A), leucine (Leu, L), isoleucine (He, I), threonine (Thr, T), methionine (Met, M), phenylalanine (Phe, F), Tryptophan (Trp, W) and proline (Pro, P).

18. The method of any one of claims 13 to 17, wherein the anti-PF4 antibodies are characterised by a third amino acid sequence motif X³LED (SEQ ID NO: 3) present in HCDR3, where X³ is selected from asparagine (Asn, N), glutamine (Gin, Q), tyrosine (Tyr, Y), serine (Ser, S), glycine (Gly, G), threonine (Thr, T), cysteine (Cys, C) and alanine (Ala, A).

19. The method of any one of claims 13 to 18, wherein the anti-PF4 antibodies are characterised by: (i) a first amino acid sequence motif DDSD (SEQ ID NO: 4) present in a light chain complementarity determining region 2 (LCDR2); (ii) a second amino acid sequence motif selected from QVWD (SEQ ID NO: 5), QMVD (SEQ ID NO: 6) and QPWD (SEQ ID NO: 7) present in a light chain complementarity determining region 3 (LCDR3); and (iii) a third amino acid sequence motif selected from GLED (SEQ ID NO: 8) and NEED (SEQ ID NO: 9) present in a heavy chain complementarity determining region 3 (HCDR3).

20. A method for monitoring treatment responses in a subject with vaccine-induced immune thrombotic thrombocytopenia (VITT) syndrome, said method comprising quantifying, at two or more timepoints following and/or during the course of the subject's treatment, the presence of antibodies directed against platelet factor 4 (PF4) in said subject, wherein the anti-PF4 antibodies are characterised by: (i) a first amino acid sequence motif DDX ¹ D (SEQ ID NO: 1) present in a light chain complementarity determining region 2 (LCDR2), where X¹ is any proteinogenic amino acid; (ii) a second amino acid sequence motif QX²WD (SEQ ID NO: 2) present in a light chain complementarity determining region 3 (LCDR3), where X² is any proteinogenic amino acid; and (iii) a third amino acid sequence motif X³LED (SEQ ID NO: 3) present in a heavy chain complementarity determining region 3 (HCDR3), where X³ is any proteinogenic amino acid.

21. The method of claim 20, wherein the presence of the anti-PF4 antibodies (step 1.) is quantified by detecting the antibodies in a serum sample.

22. The method of claim 20 or 21, wherein the anti-PF4 antibodies are characterised by a first amino acid sequence motif DDX'D (SEQ ID NO: 1) present in LCDR2, where X¹ is selected from serine (Ser, S), glycine (Gly, G), alanine (Ala, A), threonine (Thr, T), cysteine (Cys, C), asparagine (Asn, N), glutamine (Gin, Q) and tyrosine (Tyr, Y).

23. The method of any one of claims 20 to 22, wherein the anti-PF4 antibodies are characterised by a second amino acid sequence motif QX²WD (SEQ ID NO: 2) present in LCDR3, where X² is selected from valine (Vai, V), alanine (Ala, A), leucine (Leu, L), isoleucine (He, I), threonine (Thr, T), methionine (Met, M), phenylalanine (Phe, F), Tryptophan (Trp, W) and proline (Pro, P).

24. The method of any one of claims 20 to 23, wherein the anti-PF4 antibodies are characterised by a third amino acid sequence motif X³LED (SEQ ID NO: 3) present in HCDR3, where X³ is selected from asparagine (Asn, N), glutamine (Gin, Q), tyrosine (Tyr, Y), serine (Ser, S), glycine (Gly, G), threonine (Thr, T), cysteine (Cys, C) and alanine (Ala, A).

25. The method of any one of claims 20 to 24, wherein the anti-PF4 antibodies are characterised by: (i) a first amino acid sequence motif DDSD (SEQ ID NO: 4) present in a light chain complementarity determining region 2 (LCDR2); (ii) a second amino acid sequence motif selected from QVWD (SEQ ID NO: 5), QMVD (SEQ ID NO: 6) and QPWD (SEQ ID NO: 7) present in a light chain complementarity determining region 3 (LCDR3); and (iii) a third amino acid sequence motif selected from GLED (SEQ ID NO: 8) and NEED (SEQ ID NO: 9) present in a heavy chain complementarity determining region 3 (HCDR3).

26. A method for identifying a subject at risk of vaccine-induced immune thrombotic thrombocytopenia (VITT) syndrome following administration with an adenoviral-vectored vaccine (preferably an adenoviral-vectored SARS-CoV2 vaccine), said method comprising the step of assaying a suitable sample from said subject for the presence or absence of a IGLV3-21*02 gene variant characterised by a nucleotide sequence encoding a first amino acid sequence motif DDX ¹ D (SEQ ID NO: 1) present in a light chain complementarity determining region 2 (LCDR2), where X¹ is any proteinogenic amino acid.

27. The method of claim 26, wherein the IGLV3-21*02 gene variant is characterised by a nucleotide sequence encoding a first amino acid sequence motif DDX ¹ D (SEQ ID NO: 1) present in LCDR2, where X¹ is selected from serine (Ser, S), glycine (Gly, G), alanine (Ala, A), threonine (Thr, T), cysteine (Cys, C), asparagine (Asn, N), glutamine (Gin, Q) and tyrosine (Tyr, Y). More preferably, X¹ is Ser (S).

28. The method of claim 26 or 27, wherein the IGLV3-21*02 gene variant is characterised by a DDSD (SEQ ID NO: 4) motif in LCDR2.

29. The method of any one of claims 26 to 28, wherein the IGLV3-21*02 gene variant is characterised by a nucleotide sequence encoding a light chain complementarity determining region 3 (LCDR3) that is 17 amino acids in length