Helicobacter caga peptides and their use in diagnosis

Abstract

The invention relates to use of peptides derived from the CagA protein of Helicobacter pylori for improved prevention and prognosis of a vascular or epithelial pathology such as pre-eclampsia and / or other inflammatory conditions, and assessment of such pathologies.

Description

HELICOBACTER CAGA PEPTIDES AND THEIR USE IN DIAGNOSISTechnical Field

[0001] This invention relates to peptides from the CagA protein of Helicobacter pylori. The peptides can be used for improved prevention and prognosis of a vascular or epithelial pathology such as pre-eclampsia, and assessment of such pathologies.Background Art

[0002] The following discussion of the background art is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.

[0003] Helicobacter pylori is a bacterium usually found in the stomach. Certain H. pylori strains carry the cagA (cytotoxicity-associated antigen A) gene which encodes a virulence factor. The cagA gene encodes the 1140 to 1180-amino acid protein CagA which is a bacterial oncoprotein that is translocated into stomach epithelial cells at the site of infection. Upon translocation, it affects intracellular signalling pathways of the epithelial cell. Generally, H. pylori- nfected individuals will raise antibodies against H. pylori proteins, including CagA. Thus, the presence of CagA-specific antibodies indicates H. pylori infection.

[0004] Pre-eclampsia is a major cause of morbidity and mortality among pregnant women. Pre-eclampsia involves high blood pressure caused by pregnancy and affects around 5% of pregnant women. Around 76,000 women die from the condition each year, as do up to 500,000 babies. Several published studies state a correlation between H. pylori infection in the pregnant mother and risk for pre-eclampsia. For example, a recent meta-analysis which reviewed 31 different research studies, involving 22,800 patients in total, showed a 2.5-fold increased risk of pre-eclampsia in mothers infected with H. pylori compared to uninfected mothers (Zhan et al 2019). In addition, sensitivity analysis on meta-analyses showed a significant association between H. pylori infection and low birthweight (Zhan et al 2019).

[0005] A general hypothesis emanating from these studies is that H. pylori infection and its associated chronic inflammation of the stomach somehow causes increased inflammation in the placental blood vessels, which may lead to - or may increase the risk of - pre-eclampsiain a subject. Importantly, there is great variability in the DNA- and protein sequences among different H. pylori isolates. Certain CagA-variants appear to be more strongly associated with pre-eclampsia risk, but no underlying mechanism has been elucidated of which the Applicant is aware.

[0006] Unhelpfully, certain detection methods have, in the past, yielded wildly differing results. Depending on the method of assessment, the risk of pre-eclampsia due to H. pylori infection have varied from minor to significant between studies. Specifically, Shiadeh et al (2017) conducted a meta-analysis which indicated that in subgroup analysis, CagA seropositivity was a substantial risk factor for pre-eclampsia when immunoblotting methods (Odds-ratio [OR], 11.12; 95% Cl, 5.34-23.16; 2= 6.42; I2= 53.3%, 95% Cl 0-85) were used, whereas it was not a potential risk factor for pre-eclampsia when ELISA was used as a detecting method (OR, 1.11 ; 95% Cl, 0.6-2.06; x2= 1.83; I2= 0%, 95% Cl 0-90). Currently, Western blot appears to be the most effective method for CagA-antibody analysis, and when it was used the risk of pre-eclampsia in women positive for such antibodies was 11 times higher than in women without the antibodies (OR 11.1).

[0007] Therefore, it would be useful to be able to identify one or more risk factors, such as specific H. pylori CagA markers or strain types that are associated with increased risk of pre- eclampsia in subjects and that could provide a more accurate determination of a patient’s risk of developing a vascular or epithelial pathology, such as pre-eclampsia. Moreover, the ability to provide such a determination agnostic of the immune assay utilised would be of great help in producing more accurate diagnosis and potential treatment of subjects that are predisposed to developing a vascular or epithelial pathology, such as pre-eclampsia.

[0008] It is therefore an object of this invention to address some of the shortcomings of prior detection systems for identifying subjects with an increased risk of a vascular or epithelial pathology, such as pre-eclampsia.Summary of Invention

[0009] Broadly, the invention relates to peptides comprising linear epitopes from CagA+ H. pylori that find use in diagnostic applications related to H. py / or / -associated diseases including, specifically, identification of subjects at an increased risk of developing pathologies of the vascular or epithelial system, such as pre-eclampsia.

[0010] The term “linear epitope” or a “sequential epitope” as used herein is an epitope that is recognised by antibodies by its linear sequence of amino acids, or primary structure. In contrast, most antibodies recognise a conformational epitope that has a specific three- dimensional shape and its protein structure. This has implications for increased sensitivity and specificity when constructing immunological tests or assays, by making use of the peptides of the present invention to identify subjects with a predisposition to these pathologies.

[0011] According to the invention, there is provided use of at least one peptide sequence derived from a linear epitope of the CagA protein of H. pylori, for the identification of a subject at increased risk of developing pre-eclampsia. The invention further provides use of at least one peptide sequence derived from a linear epitope of the CagA protein of H. pylori as a marker for increased risk of developing pre-eclampsia in a subject.

[0012] The invention further provides a method of using at least one peptide sequence derived from a linear epitope of the CagA protein of H. pylori, for the identification of a subject at increased risk of developing pre-eclampsia. Also provided is a method of using at least one peptide sequence derived from a linear epitope of the CagA protein of H. pylori as a marker for increased risk of developing pre-eclampsia in a subject.

[0013] According to a first aspect of the invention, the at least one peptide sequence may be derived from any one or more peptides derived from linear epitopes of the CagA protein selected from the group consisting of:CagA_01 to CagA_18 (we refer to the description in Example 1 below), including a mixture of such peptides.

[0014] The at least one peptide sequence may be derived from one or more linear epitopes of H. pylori CagA_09.

[0015] As such, the at least one peptide sequence may comprise the amino acid sequence DX3IX1X4KX2X5L, wherein:Xi may be a positively charged amino acid (R, H or K)X2 may be a negatively charged amino acid (D or E),X3 may be absent or present and, when present, may be a hydrophobic amino acid (A, I, L, M, F, V, P, G, or L)X4 may be absent or present and, when present, may be an uncharged polar amino acid (S, T, N, Q, or N)X5: may be absent or present and, if present, may be an uncharged polar amino acid (S, T, N, Q, or N); and wherein the order of X2 and X5 may be X2X5 or X5X2.

[0016] As such, the at least one peptide sequence may comprise the sequence DI(X1)K(X2)L, wherein: one hydrophobic amino acid (A, I, L, M, F, V, P, G, or L) may be inserted at any position of the sequence; and an uncharged polar amino acid (S, T, N, Q, or N) may be inserted in up to two places, at any position of the sequence.

[0017] In one embodiment, the at least one peptide sequence may comprise the sequence DIX1KX2L, wherein:Xi may be any positively charged amino acid (R, H or K)X2 may be any negatively charged amino acid (D or E),

[0018] More specifically, the at least one peptide sequence may comprise any one or more of the following sequences:DIKKEL (SEQ ID NO 1)DIRKDL (SEQ ID NO 2)DIRKEL (SEQ ID NO 3)DLINKDNL (SEQ ID NO 4)DLINKNDL (SEQ ID NO 5)

[0019] In an embodiment of the invention, there is provided use of a peptide comprising at least one peptide sequence selected from the group consisting of SEQ ID NO 6 to SEQ ID NO 38, for determining the risk of a subject for developing a vascular or epithelial pathology, such as pre-eclampsia. SEQ ID NO 39 is derived from beta-actin and may also be useful for detecting CagA-specific antibodies or may serve as a control peptide in immunological assays. As such, the invention extends to the use of SEQ ID NO 39 for the detection of a vascular or epithelial pathology, such as pre-eclampsia.

[0020] The minimal binding regions of SEQ ID NOs 1 to 38 are especially useful for detecting CagA-specific antibodies that cross-react with beta-actin, and beta-actin-like, sequences in the vascular or epithelial system. As mentioned hereinbefore, SEQ ID NO 39 is derived from beta-actin and may also be useful for detecting CagA-specific antibodies or serve as control in immunological assays.

[0021] According to a second aspect of the invention, the at least one peptide sequence preferably comprises the sequence AKIDQLN (SEQ ID NO 257). In other embodiments, the at least one peptide sequence may comprise any one or more of the following sequences:TQVAKKVNAKIDQLN (SEQ ID NO 236)TQVAKKVKAKIDQLN (SEQ ID NO 239)AQVAKKVSAKIDQLN (SEQ ID NO 240)AQVAKKVNAKIDQLN (SEQ ID NO 241 )VAKKVSAKIDQLNEA (SEQ ID NO 244)KKVSAKIDQLNQAAS (SEQ ID NO 246)KKVSAKIDQLNEATS (SEQ ID NO 248)KKVNAKIDQLNQIAS (SEQ ID NO 250)KKVNAKIDQLNQAAS (SEQ ID NO 253)VSAKIDQLNEATSAI (SEQ ID NO 254)

[0022] The at least one peptide sequence preferably comprises the sequence KI(X1 )QLN, wherein:X1 is a negatively charged amino acid (D or E).

[0023] In another embodiment, the at least one peptide sequence further comprises the sequence KIEQLN (SEQ ID NO 396).

[0024] According to an embodiment of the invention, the at least one peptide sequence comprises any one or more of the following sequences:RSVSPEPIYATIDDL (SEQ ID NO 284)EPIYATI (SEQ ID NO 574)EPIYAAI (SEQ ID NO 575) EPAYATI (SEQ ID NO 576) EPXYAXI (SEQ ID NO 756)

[0025] According to an embodiment of the invention, the at least one peptide sequence comprises any one or more of the following sequences:ENSTEPIYAKVNKKK (SEQ ID NO 223)EPIYAQV (SEQ ID NO 479)EPIYAAV (SEQ ID NO 480) EPIYAKV (SEQ ID NO 487) EPIYAXV (SEQ ID NO 703)

[0026] A peptide sequence according to the invention preferably comprises at most 35 amino acids, more preferably 30 amino acids, more preferably 27 amino acids, more preferably 15 amino acids, more preferably 8 amino acids, and even more preferably no more than 7 amino acids.

[0027] A peptide according to the invention may be a non-naturally occurring peptide.

[0028] The peptides of the invention have the advantage that they can be used for identification, diagnosis, prediction, or prognosis of CagA+ H. py / or / -associated diseases of the vascular or epithelial system, such as, pre-eclampsia. The Applicant believes that diagnosis of, especially, subjects predisposed to pre-eclampsia using the peptides of the invention results in fewer false positives than existing diagnostic systems of which the Applicant is aware.

[0029] Given that the peptides of the present invention are short, the Applicant is of the opinion that there is decreased background binding of antibodies from subjects not predisposed to pre-eclampsia when compared to conventional peptide-based diagnostic systems of which the Applicant is aware. Furthermore, the peptides of the invention are short and can therefore be manufactured at low cost.

[0030] The invention further provides one or more peptides of the invention as herein described for use in a diagnostic test for determining whether a subject may develop, or may be predisposed to developing, vascular or endothelial dysfunctions or pathologies, such as pre-eclampsia.

[0031] The invention further provides a kit comprising a peptide according to an embodiment of the invention or a mixture of peptides according to the invention. The kit is preferably a kit for diagnosis, more specifically diagnosis of a predisposition to pre-eclampsia. The kit may also be for diagnosis, forming a prognosis, or monitoring of a subject having a predisposition to pre-eclampsia or another vascular or epithelial pathology. The kit may be used for assessing the binding of the at least one peptide comprising a peptide sequence according to the invention as described herein, to antibodies in a sample from the subject.

[0032] In an embodiment of the invention, there is provided a method of determining the risk of a vascular or epithelial pathology, such as pre-eclampsia, in a subject, the method comprising the steps of:(i) isolating or providing a sample from a subject;(ii) contacting said sample with a peptide as described herein or a mixture of one or more peptides as described herein; and(iii) detecting specific binding of antibodies in the sample to the peptide.

[0033] In another embodiment of the invention, there is provided a method for preventing a vascular or epithelial pathology, such as pre-eclampsia, in a subject, the method comprising the steps of:(i) carrying out diagnosis as described herein; and(ii) treating the H. pylori CagA+ infection in the subject.

[0034] The method may comprise the steps of using the diagnosis method described herein to determine whether the subject has a particular Helicobacter pylori infection that may indicate a predisposition to, or increased risk of, a vascular or epithelial pathology such as pre-eclampsia, specifically a CagA+ H. pylori infection, and then, if so, treating said infection.

[0035] The treatment may involve administering one or more antibiotics selected from a class of antibiotics. The class of antibiotics may be selected from the group consisting of: macrolides, beta-lactams, nitroimidazoles, tetracyclines, fluoroquinolones, and functional equivalents thereof.

[0036] The treatment may involve administering at least two antibiotics from said classes, where the at least two antibiotics are from different classes. The treatment may also involve administering a proton pump inhibitor to the subject, preferably in combination with antibiotics, either contemporaneously or in short succession of one another.

[0037] Accordingly, there is provided a method of detecting a predisposition in a subject to developing a vascular or epithelial pathology, such as pre-eclampsia, in a sample obtained from said subject, the method comprising contacting a biological sample with one or more peptides according to one embodiment of the invention and detecting binding of antibodies in the sample to the one or more peptides.

[0038] The sample may be a liquid biopsy sample such as a blood, serum, saliva, or plasma sample which contains antibodies, or a tissue sample, for example a gastric tissue sample.

[0039] The invention extends to a method for the identification, diagnosis, prediction, or prognosis of the onset of pregnancy-induced hypertension and / or pre-eclampsia or for the assessment of the risk of a pregnant subject developing hypertension and / or pre-eclampsia, the method comprising the steps of:(i) providing a sample comprising one or more of a biopsy, blood, serum, plasma, saliva and urine, containing antibodies of the subject;(ii) bringing the sample into contact with any one or more of the peptides of the invention; and(iii) detecting the binding of the antibodies with the any one or more peptides of the invention.

[0040] In an embodiment of the invention there is provided a mixture of at least two peptides of the invention. Such a mixture has the advantage that it can be used for detecting two or more different CagA-positive strains of H. pylori in an efficient manner which are related to a subject being predisposed to, or at higher risk of, developing a vascular or epithelial pathology, such as pre-eclampsia. The mixtures can be used in the same manner as the peptides herein.

[0041] In an embodiment of the invention, there is provided an antibody specific to a peptide comprising a peptide sequence according to the invention as described herein. The antibody can preferably cross-react with the human LLRC19 receptor. The cross-reacting with the human LLRC19 receptor preferably triggers inflammation that contributes to development of preeclampsia and / or other inflammatory conditions.

[0042] In an embodiment of the invention, there is provided a peptide comprising a peptide sequence according to the invention as described herein, for use in a diagnostic test for determining whether a subject may develop, or is predisposed to developing, vascular orendothelial dysfunctions or pathologies. The vascular or endothelial dysfunction or pathology is preferably pre-eclampsia.

[0043] In an embodiment of the invention, there is provided the use of a peptide comprising a peptide sequence according to the invention as described herein as a biomarker of potential for inflammation and preeclampsia development in a subject. The use of the peptide is preferably for diagnosis or as a prognostic indicator of pre-eclampsia or another vascular or epithelial pathology in a subject.

[0044] In an embodiment of the invention, there is provided an assay for diagnosing, monitoring, and / or forming a prognosis of a potential for inflammation and preeclampsia development in a subject, the assay comprising detecting specific binding of antibodies from a sample from the subject to the one or more peptide comprising a peptide sequence according to the invention as described herein.

[0045] In an embodiment of the invention, there is provided a screening method to assess the potential for inflammation and preeclampsia development in a subject using methods as described herein.

[0046] In an embodiment of the invention, there is provided a method of determining the risk of a subject for developing a vascular or epithelial pathology, such as pre-eclampsia, the method comprising the steps of comparing the subject’s antibody-binding to different CagA sequences with their profilin-beta-actin sequences and determining a score factor, wherein increased homology between the sequences of CagA the subject’s antibodies reacts to and the same subject’s profilin-beta-actin sequences indicates an increased score factor, indicative of an increased risk of developing pre-eclampsia.

[0047] As such, the invention extends to a method of assessing the risk profile of a subject for developing pre-eclampsia, the method comprising comparing a beta-actin sequence obtained from said subject with a CagA reference sequence and calculating the percentage identity with any one or more of the CagA sequences SEQ ID NOs 1 to 39, wherein a higher homology between the sequences indicates a higher risk of developing pre-eclampsia.

[0048] In an embodiment of the invention, there is provided a method of determining the risk of a vascular or epithelial pathology, such as pre-eclampsia, in a subject, the method comprising the steps of:(i) isolating or providing a sample from a subject;(ii) contacting said sample with one or more peptide comprising a peptide sequence according to the invention as described herein; and(iii) detecting specific binding of antibodies in the sample to the one or more peptide.

[0049] In an embodiment of the invention, there is provided a method for prognosing development of preeclampsia and / or other inflammatory conditions in a subject, the method comprising the step of identifying the presence of an antibody as described herein in a sample from the subject.

[0050] In an embodiment of the invention, there is provided a method of treating a subject at risk of developing preeclampsia and / or other inflammatory conditions, or for preventing a vascular or epithelial pathology, such as pre-eclampsia, the method comprising the steps:(i) identifying the presence of an antibody as described herein in a sample from the subject; and(ii) treating a H. pylori CagA+ infection in the subject.

[0051] The method preferably comprises the additional step of quantifying the concentration of the antibody in the sample from the subject. Treating the H. pylori CagA+ infection in the subject preferably comprises administering to the subject at least one antibiotic effective for treating a H. pylori infection. The antibiotic preferably comprises one or more selected from the group consisting of: macrolides, beta-lactams, nitroimidazoles, tetracyclines, fluoroquinolones, and functional equivalents thereof. The antibiotic preferably comprises at least one of clarithromycin, metronidazole, amoxicillin, or tetracycline.

[0052] In an embodiment, the subject is treated with an additional agent in combination with the at least one antibiotic, the additional agent comprising one or more selected from the group comprising: proton pump inhibitor or bismuth subsalicylate. The proton pump inhibitor preferably comprises esomeprazole, lansoprazole, omeprazole, pantoprazole, or rabeprazole.

[0053] In an embodiment of the invention, there is provided a method comprising the additional step of administering a therapeutic agent to the subject to reduce the risk of developing preeclampsia and / or other inflammatory conditions. The therapeutic agent preferably prevents the binding of the antibody to the LLRC19 receptors in cells of the subject. The therapeutic agent preferably prevents the binding of antibodies to the LLRC19 receptorsin cells of the subject. The cells of the subject are preferably located in the gastrointestinal tract.

[0054] In an embodiment of the invention, there is provided a method of treating a subject to reduce their risk of inflammation and preeclampsia development, the method comprising depleting B-cells in the subject that produce antibodies which cross-react with the human LLRC19 receptor. A method of treating a subject as described herein, wherein the antibodies are bispecific antibodies.Brief Description of Drawings

[0055] In order to provide a better understanding, embodiments of the present invention will be described, by way of example only, with reference to the accompanying drawings, in which:Figure 1 . Association between IgG or IgA antibodies to CagA peptides and preclampsia development. Antibodies to 1089 different 15-mer peptides of H. pylori CagA were measured in serum samples from women developing preeclampsia and pregnant women not developing preeclampsia. The association was calculated as the odds- ratio, which is plotted on the y-axis. The location of each peptide along the CagA protein is indicated on the x-axis. Each peptide is shown as one dot, and the odds- ratio of 2.8 is indicated with a horizontal dashed line.Description of Embodiments

[0056] The following embodiments, given by way of non-limiting example only, are described in order to provide a more precise understanding of the subject matter of a preferred embodiment or embodiments. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above.

[0057] The Applicant has identified a new use of Helicobacter pylori peptides as markers for identifying subjects at an increased risk of developing a vascular or epithelial pathology, including those associated with LLRC19 and / or beta-actin including, specifically, preeclampsia.

[0058] From a large array of CagA peptides that may be present in H. pylori infected individuals, the Applicant previously (in International Patent Application No. WO2018153991 ,incorporated herein by reference, including the sequences disclosed therein) defined which subset of CagA peptides are immunogenic and elicit an antibody-response with low levels of false positives and may be indicative to an increased predisposition to developing certain types of cancers, such as stomach cancer. Generally, with these types of immunological tests a significant number of peptides tend to cross-react with serum from non-infected patients, unless due care is taken to identify specific peptides which are suitably sensitive and specific.

[0059] Within the subset of immunogenic peptides, the Applicant has identified a more specific subset of peptides that has improved diagnostic capacity for a vascular or epithelial pathology, such as pre-eclampsia; and finally, within this subset of diagnostic peptides, the Applicant has identified crucial amino acid sequence(s) common to the peptides having the highest diagnostic capacity. In other words, the diagnostic capacity does not stem from only the presence / absence of peptides in the infected individual, but crucially also from only a small subset of the immunogenic peptides consistently eliciting an antibody-response that is absent in non-infected individuals and that may be indicative of a predisposition to developing a vascular or epithelial pathology, such as pre-eclampsia.

[0060] By utilising high-precision serology, with resolution at the peptide level, specifically at the level of linear epitopes (instead of at the protein level), the Applicant has now identified peptides containing highly specific linear epitopes to which there is a strong antibodyresponse only in individuals carrying a subset of very particular CagA+ H. pylori sequences. These sequences are predicted to be indicative of a vascular or epithelial pathology, such as pre-eclampsia, which involve binding of H. pylori CagA antibodies to components of the cell walls involved in a vascular or epithelial pathology, such as pre-eclampsia, while excluding peptides that cause false positives due to a cross-reactive antibody-response in individuals lacking said specific CagA+ H. pylori infection. Previous studies have shown significant variance in detecting subjects with a predisposition to a vascular or epithelial pathology, such as pre-eclampsia, with wildly varying results depending on the types of immunological tests used. Specifically, a need exists for a test which can lessen the variability of detection of potential risk, irrespective of the immunological detection method used.

[0061] Therefore, the diagnostic peptides containing linear epitopes that the Applicant has identified are predicted to have both high sensitivity and specificity as determined by ROC AUG values and are useful for diagnostic applications and address the shortcomings of the currents tests of which the Applicant is aware.

[0062] Reference is made herein to an interval of sequences. This refers to all the sequences in the interval, thus for example “SEQ ID NO 2 to SEQ ID NO 5” or “SEQ ID NOs 2 to 5” refers, inclusively, to SEQ ID NO 2, 3, 4, and 5. Sequences are written using the standard one-letter annotation for amino acid residues. The amino acid residues are preferably connected with peptide bonds but may, in certain instances, be connected with alternative bonds known to those skilled in the field of the invention.

[0063] Some peptides herein may have sequence variability. Thus, certain sequences may specify a position in the sequence that can be any amino acid. This may be indicated with an X or, in the sequence listing, Xaa. The X or Xaa can be replaced with any amino acid, preferably any L-amino acid, including amino acids resulting from post translational modification, such as citrulline. The amino acid does not have to be a naturally occurring amino acid. Preferably the amino acid does not have a bulky side chain, as a bulky side chain could prevent antibody binding. A suitable molecular weight of the amino acid may be from 85 D to 300 D, more preferably from 89 D to 220 D.

[0064] In a first aspect of the invention, in general, the peptide may comprise or consist of an amino acid or peptide sequence selected from the group consisting of SEQ ID NO 1 to SEQ ID NO 39 (Table 1 , wherein “n_strains” refers to the number of H. pylori strains assessed that possessed that linear epitope sequence). SEQ ID NO 1 and SEQ ID NOs 3 to 38 are H. pylori linear epitopes, while SEQ ID NOs 2 and 39 are short stretches derived from human betaactin protein that are predicted to cross-react with antibodies to CagA+ H. pylori epitopes disclosed herein.

[0065] Table 1. High-specificity diagnostic peptide sequences of CagA linear epitopes.

[0066] The peptides of the invention may comprise parts or functional fragments of the sequences of SEQ ID NO 1 to SEQ ID NO 38 to which antibodies can be generated that can be used for the positive identification of subjects with a predisposition to developing a vascular or epithelial pathology such as pre-eclampsia. These fragments will typically contain the following sequence: DX3IX1X4KX2X5L, wherein:Xi may be a positively charged amino acid (R, H or K)X2 may be a negatively charged amino acid (D or E),X3 may be absent or present and, when present, may be a hydrophobic amino acid (A, I, L, M, F, V, P, G, or L)X4 may be absent or present and, when present, may be an uncharged polar amino acid (S, T, N, Q, or N)X5: may be absent or present and, if present, may be an uncharged polar amino acid (S, T, N, Q, or N); and wherein the order of X2 and X5 may be X2X5 or X5X2.

[0067] When the peptide comprises or consists of 6 or more of the amino acids of SEQ ID NOs 6 to 39 comprising the DX3IX1X4KX2X5L motif, the remaining amino acid positions can be replaced with any amino acid as described above for X and Xaa, while the remaining amino acids have the positions as in SEQ ID NOs 1 to 39. In certain embodiments, the amino acid may be replaced in a conserved manner, wherein, for example, a hydrophobic amino acid is replaced with a different hydrophobic amino acid, or where a polar amino acid is replaced with a different polar amino acid.

[0068] In a preferred embodiment a peptide comprising or consisting of one of SEQ ID NO 1 to SEQ ID NO 5 (Table 1) is used. These sequences comprise the minimal binding regions of certain antibodies that find use in the present invention. These peptides have the advantage that the diagnostic accuracy is higher than conventional tests of which the Applicant is aware, since they are predicted to elicit a strong, highly selective antibodyresponse in a high percentage of individuals carrying a CagA+ H. pylori infection and that are pre-disposed to pre-eclampsia. Certain of these peptides (SEQ ID NO 1 to SEQ ID NO 3) all relate to the same H. pylori linear epitopes (Cag_09).

[0069] As mentioned hereinbefore, the peptides have common structural features in that they all comprise amino acid sequence DX3IX1X4KX2X5L, wherein:Xi is a positively charged amino acid (R, H or K)X2 is a negatively charged amino acid (D or E),X3 is absent or present and, when present, may be a hydrophobic amino acid (A, I, L, M, F, V, P, G, or L)X4 is absent or present and, when present, may be an uncharged polar amino acid (S, T, N, Q, or N)X5: is absent or present and, if present, may be an uncharged polar amino acid (S, T, N, Q, or N); andwherein the order of X2 and X5 is X2X5 or X5X2, respectively.

[0070] As such, the at least one peptide sequence comprises, in certain embodiments, the sequence DI(X1 )K(X2)L, where:

[0071] one hydrophobic amino acid (A, I, L, M, F, V, P, G, or L) is inserted at any position of the sequence; and

[0072] an uncharged polar amino acid (S, T, N, Q, or N) is inserted in up to two places, at any position of the sequence.

[0073] In one embodiment, the at least one peptide sequence comprises the sequence DIX1KX2L, wherein:Xi is any positively charged amino acid (R, H or K)X2 is any negatively charged amino acid (D or E),

[0074] Examples of useful peptides that comprise SEQ ID NO 1 to SEQ ID NO 5 and functional analogues from CagA strains include, but are not limited to, sequences SEQ ID NO 6 to SEQ ID NO 38.

[0075] The invention further extends to any protein product of the cagA gene which includes a peptide of SEQ ID NOs 1 to 39, or containing the DX3IX1X4KX2X5L motif described hereinbefore.

[0076] Not wishing to be bound by theory, the Applicant believes that pre-eclampsia is caused by cross-reacting antibodies that bind to linear epitopes of H. pylori CagA protein and at the same time also bind to beta-actin (typically, present in cell walls or epithelial or other cells). Therefore, the Applicant investigated which linear epitopes of CagA possessed similarities with any part of beta-actin. Peptide sequences, initially investigated in CagA_09, identified a sequence that was similar, but not identical, to a part of beta-actin. This part of beta-actin has also been shown to be exposed on the surface of the protein, according to published data from analysis of crystal structures of beta-actin.

[0077] The most prominent sequence identified was DIRKDL (starting at amino acid position 288 in beta-actin) and DIKKEL in CagA_09 (4 of 6 identical amino acids, the two remaining chemically similar).

[0078] Out of 180 different CagA sequences investigated by the Applicant (i.e. CagA sequences from 180 different H. pylori isolates), 141 had the DIKKEL sequence. A further 28 had the sequence DIRKEL (16% of the available sequences). The R (Arg 290) of beta-actin, which is present in the more similar CagA-sequences, is exposed on the surface of the betaactin helix.

[0079] The Applicant has found that antibodies to CagA linear epitopes such as those found in CagA_09 can, surprisingly, bind to at least parts of beta-actin, particularly those exposed on the cell surface. The Applicant believes that the higher the degree of similarity between at least part of the CagA linear epitope sequence to the DIRKDL-sequence of beta-actin, the higher the risk of pre-eclampsia or other vascular pathologies developing in a subject, including during pregnancy. Therefore, antibodies to H. pylori epitope CagA_09 specifically, and to H. pylori sequences which contain the DIKKEL, DIRKEL and DIRKDL, DLINKDNL, and DLINKNDL sequences generally, can be used to diagnose the risk for developing preeclampsia.

[0080] H. pylori displays some genetic diversity in the CagA sequence and it may be desirable to use a peptide or a group of peptides that identifies several strains. SEQ ID NO 1 and SEQ ID NOs 3 to 5 represents such a group of peptides, since 96% of all CagA+ H. pylori isolates carry at least one of these sequence variants. Thus, it may be useful to provide a mixture (a “cocktail”) of two or more peptides described herein (for example, SEQ ID NOs 1 to 38 or including SEQ ID NO 39 if it is to be included as a control). In one embodiment such a mixture comprises at least two, preferably three, more preferably four, more preferably five, more preferably six and more preferably seven peptides selected from peptides that comprise or consist of SEQ ID NO 1 to SEQ ID NO 39. In one embodiment the sequences are selected from SEQ ID NO 1 to SEQ ID NO 5. Preferred mixtures include SEQ ID NOs 1 , 2, 3, 4, and 5. In another embodiment the peptides are selected from the peptides of SEQ ID NO 6 to SEQ ID NO 39.

[0081] The peptides of the invention may comprise parts or functional fragments of the sequences of SEQ ID NOs 236, 241 , 250, 246, 253, 248, 254, 257 (or 396) to which antibodies can be generated that can be used for the positive identification of subjects with a predisposition to developing a vascular or epithelial pathology such as pre-eclampsia.

[0082] The invention also extends to combinations of such peptides for use in identification, diagnosis, or prognosis or pre-eclampsia risk.

[0083] The herein described sequences comprise the minimal binding regions of certain antibodies that find use in the present invention. These peptides have the advantage that the diagnostic accuracy is higher than conventional tests of which the Applicant is aware, since they are predicted to elicit a strong, highly selective antibody-response in a high percentage of individuals carrying a CagA+ H. pylori infection and that are pre-disposed to pre-eclampsia.

[0084] Preferably, said peptide sequence comprises at most 35 amino acids, more preferably 30 amino acids, even more preferably, at most 27 amino acids. A shorter peptide may be desirable because it results in less unspecific binding (by an antibody) and therefore less background. However, a longer peptide may in some cases be desirable to allow for exposing the linear epitope to allow antibody binding without steric hindrance. Thus, more preferably the peptide is 20 amino acid residues, more preferably 15 amino acid residues, even more preferably 12, 11 , 10, 9, and even more preferably 8 or 7 amino acids (SEQ ID Nos 404 to 780), or 6 amino acid residues (7 also applies to SEQ ID NOs 4, 5 and 257 only, and 6 applies to SEQ ID NOs 1 to 3 and 396 only).

[0085] Preferably the peptide binds specifically (in the immunological sense) and with high affinity to an antibody, preferably an antibody from a subject sample that also binds to linear epitopes the H. pylori CagA protein. An antibody-peptide interaction is said to exhibit “specific binding” or “preferential binding” in the immunological sense if it reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody “specifically binds” or “preferentially binds” to a peptide if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. Binding can be determined with any suitable method. Binding can be determined by methods known in the art, for example ELISA, surface plasmon resonance, Western blot or the other methods described herein (see below). Such methods can be used by those skilled in the art to determine suitable lengths or amino acid sequences of the peptide.

[0086] Preferably the use of the peptide has both a high diagnostic specificity and a high diagnostic sensitivity. In any diagnostic test, these two properties are dependent on what level is used as the cut-off for a positive test. To assess diagnostic accuracy independently of a set cut-off, a receiver operator characteristic curve (ROC curve) can be used. In an ROC curve, true positive rate (sensitivity) is plotted against false positive rate (1 -specificity) as the cut-off is varied from 0 to infinity. The area under the ROC curve (ROC AUG) is then used to estimate the overall diagnostic accuracy. Preferably the use of the peptide has an ROC AUGof at least 0.55, for example an ROC AUG of at least, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, 0.99 or an ROC AUG of 1.00. Preferably, the use of the peptide has ROC AUG of at least 0.85, and most preferably an ROC AUG of 1 .

[0087] Under some circumstances, for example when only a specific subset of individuals are identified by a diagnostic test, it may be beneficial to use a diagnostic accuracy measure other than the ROC AUG. In such cases, a Diagnostic odds ratio (DOR) - also called Odds ratio (OR) - can be used to estimate the discriminative power of a diagnostic test. When referring to diagnostic tests, the OR is the ratio of the odds of test positivity in subjects with disease relative to the odds in subjects without disease, and is calculated by the formula (TP / FN) / (FP / TN), where TP, FN, FP and TN means the number of subjects that are True Positive, False Negative, False Positive and True Negative for the test in question, respectively.

[0088] In an embodiment of the invention, a herein described peptide preferably has an OR of at least 2, but more preferably an OR of equal to or more than 5, 10, or 20.

[0089] As used herein, the term “peptide” is used to mean peptides, proteins, fragments of proteins and the like, including peptidomimetic compounds. The term “peptidomimetic”, means a peptide-like molecule that has the activity of the peptide upon which it is structurally based, the activity being specific and high affinity binding to antibodies that bind to linear epitopes of the H. pylori CagA protein. Such peptidomimetics include chemically modified peptides, peptide-like molecules containing non-naturally occurring amino acids (see, for example, Goodman and Ro, Peptidomimetics for Drug Design, in “Burger's Medicinal Chemistry and Drug Discovery” Vol. 1 (ed. M. E. Wolff; John Wiley & Sons 1995), pages 803- 861 ). A variety of peptidomimetics are known in the art including, for example, peptide-like molecules which contain a constrained amino acid. In certain embodiments circular peptides may be used. The term “functional fragment” as used herein refers to truncated forms of SEQ ID NO 1 to 39, 236, 241 , 250, 246, 253, 248, and 254 which consist of contiguous amino acid sequences identical to contiguous amino acid sequences of such sequences and which are capable of being used in the methods of the invention to identify subjects with a predisposition to a vascular or epithelial pathology such as, for example, pre-eclampsia.

[0090] As mentioned hereinbefore, the term “linear epitope” or a “sequential epitope” as used herein is an epitope that is recognised by antibodies by its linear sequence of amino acids, or primary structure. In contrast, most antibodies recognise a conformational epitope that has a specific three-dimensional shape and its protein structure.

[0091] The peptide may be an isolated peptide meaning a peptide in a form other than it occurs in nature, e.g. in a buffer, in a dry form awaiting reconstitution, as part of a kit, etc.

[0092] The peptide may be substantially purified or isolated, meaning a peptide that is substantially free of other proteins, lipids, carbohydrates, nucleic acids and other biological materials with which it is naturally associated. For example, a substantially pure peptide can be at least about 60% of dry weight, preferably at least about 70%, 80%, 90%, 95%, or 99% of dry weight.

[0093] A peptide of the present invention can be in the form of a salt. Suitable acids and bases that are capable of forming salts with the peptides are well known to those of skill in the art, and include inorganic and organic acids and bases, including potassium, calcium, magnesium, or sodium salts. The peptide can be provided in a solution, for example an aqueous solution. Such a solution may comprise suitable buffers, salts, protease inhibitors, or other suitable components as is known in the art.

[0094] The peptide may be associated with (e.g. coupled, fused or linked to, directly or indirectly) one or more additional moieties as is known in the art. Non-limiting examples of such moieties include peptide or non-peptide molecules such as biotin, a poly his tag, GST, a FLAG-tag, or a linker or a spacer. The association may be a covalent or non-covalent bond. The association may be, for example, via a terminal cysteine residue or a chemically reactive linking agent, the biotin-avidin system or a poly-his tag. For example, the peptide may be linked with a peptide bond to a single biotin-conjugated lysine residue, in which the lysine is biotinylated via the epsilon amino groups on its side chain, such as the peptide example H- XXXXXXXXXXXXXXX(K(Biotin))-NH2, where X indicates the amino acids of the peptide.

[0095] The associated moiety may be used to attach or link the peptide, to improve purification, to enhance expression of the peptide in a host cell, to aid in detection, to stabilise the peptide, etc. In the case of a short peptide attached to a substrate, for example a solid phase, it may be desirable to use a linker or a spacer to ensure exposure of the peptide to antibodies so that the antibodies can bind.

[0096] The peptide may be associated with a substrate that immobilises the peptide. The substrate may be, for example, a solid or semi-solid carrier, a solid phase, support or surface. The peptide may be immobilised on a solid support. Examples includes beads or wells in plates, such as microtitre plates, such as 96-well plates, and also include surfaces of lab-on- a-chip diagnostic or similar devices. The association can be covalent or non-covalent andcan be facilitated by a moiety associated with the peptide that enables covalent or non- covalent binding, such as a moiety that has a high affinity to a component attached to the carrier, solid phase, support or surface. For example, the biotin-avidin system can be used.

[0097] The peptides of the present invention find application in detecting H. pylori CagA- specific linear epitope antibodies in a sample from a subject, the method comprising contacting a biological sample with a peptide as described herein and detecting binding of antibodies in the sample to the peptide to infer the risk of the subject to developing a vascular or epithelial pathology such as pre-eclampsia. The peptide may be associated with a substrate that immobilises the peptide, as described herein, for example attached to a solid support. The method may include incubation to allow binding, washing, and detection of antibodies as described herein. Methods for detecting binding of antibodies are described below and include, for example, immunoblotting, ELISA, or Western blot.

[0098] The peptides can be used for diagnosis and / or prognosis, in particular for identifying subjects predisposed to developing pre-eclampsia. It is known that CagA H. pylori infection, generally, correlates with an increased risk for pre-eclampsia. Thus, the novel peptides of the invention present novel diagnostic targets that can be used for assessing the risk of a subject developing pre-eclampsia and address the shortcomings of conventional CagA- immunological detection methods such as those disclosed in Franceschi et al (2012).

[0099] The risk of developing a vascular or epithelial pathology such as pre-eclampsia may include the risk of proceeding from not being at risk of developing pre-eclampsia (e.g. during pregnancy) to having pre-eclampsia of any stage of pregnancy. For sake of clarity, the risk may include the risk of developing pre-eclampsia in the future if the H. pylori infection is left untreated. In a preferred embodiment the peptide is used for assessing the risk of a subject for developing pre-eclampsia in the future.

[0100] The peptides can also be used for identification, diagnosis, prediction, or prognosis of other diseases that are associated with vascular or epithelial development or maintenance such as foetal growth retardation, eclampsia, hypertension, and cardiovascular diseases such as atherosclerosis, coronary heart disease, ischaemic stroke, unstable angina, and cardiac X syndrome.

[0101] The present invention further relates to the use of the described methods and kits for the diagnosis, prognosis and risk assessment of gestational hypertension and / or pre- eclampsia in pregnant women.

[0102] The term “gestational hypertension” or gestational hypertension is defined as the onset of new arterial hypertension (systolic and diastolic blood pressure = 140 and 90 mmHg, respectively) in pregnant women after 20 weeks of gestation.

[0103] The term “pre-eclampsia” includes hypertensive multi-organ diseases of pregnant women characterised by hypertension and proteinuria. The most common symptoms of pre-eclampsia are hypertension, increased protein in the urine, and hand and face swelling or oedema. In certain embodiments of the invention, pre-eclampsia is associated with hypertension (systolic and diastolic blood pressure >140 and 90 mm Hg, respectively) and proteinuria (>300 mg protein excretion in a 24-hour urine collection).

[0104] In certain embodiments of the invention, gestational hypertension and I or pre- eclampsia is asymptomatic and / or does not appear at the time of measurement. Thus, asymptomatic and / or not appearing means less than 140 and 90 mm Hg systolic and relaxant blood pressure and / or less than 300 mg protein excretion in a 24-hour urine collection.

[0105] “Prognosis” relates to predicting the outcome or inherent risk of a subject suffering from a particular disease or clinical condition. This may include an estimate of the subject's chances of recovery or adverse outcome.

[0106] The term “sample” as used herein refers to a bodily fluid sample obtained for the purpose of diagnosis, prognosis or evaluation of a subject in question, e.g, a patient. Preferred test samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva and pleural effusion. In addition, those skilled in the art will appreciate that some test samples are easily analysed according to fractionation or purification means, such as separation of whole blood into serum or plasma components. In one embodiment, the sample is preferably a blood sample.

[0107] Thus, in a preferred embodiment of the invention, the sample is selected from the group consisting of a blood sample, a serum sample, a plasma sample, a cerebrospinal fluid sample, a saliva sample, and a urine sample or any extract of said sample. Preferably, the sample is a blood sample, most preferably a serum sample or a plasma sample. The sample may also be a tissue sample or may be derived from a harvesting procedure, such as during an endoscopy.

[0108] In the present specification, the subject may be assessed, tested, or prognosed prior to pregnancy having occurred, which will allow for treatment of the H. pylori infectionprior to pregnancy occurring with any one or more treatments commonly used in the field, such as the antibiotics listed herein. However, the subject may already be pregnant at the time of diagnosis or at the time of performing the method of diagnosis, prognosis or treatment in accordance with the invention.

[0109] Identification, diagnosis, or prognosis can be carried out using any suitable method. In a preferred method, antibodies in a sample from a subject are allowed to bind to one or more peptides of the invention, and binding is detected using detection methods known in the art. The subject can be a human or an animal, preferably a human. Binding in vitro of antibodies from the subject to one or more peptides of the invention indicates that the immune system of the subject has generated antibodies against that particular peptide and thus that said at least one peptide and hence that linear epitopes of CagA H. pylori of the present invention are associated with increased risk of pre-eclampsia is present in the subject.[001 10] The method, in one embodiment, thus comprises the steps of (1 ) isolating, from a subject, a sample of body fluid or tissue likely to contain antibodies or providing, in vitro, such a sample; (2) contacting the sample with a peptide, under conditions effective for the formation of a specific peptide-antibody complex (for specific binding of the peptide to the antibody), e.g., reacting or incubating the sample and a peptide; and (3) assaying the contacted (reacted) sample for the presence of an antibody-peptide reaction (for example determining the amount of an antibody-peptide complex). The method may involve one or more washing steps, as is known in the art. Steps 2 and 3 are preferably carried out in vitro, that is, using the sample after the sample has been isolated from the subject, in a sample previously isolated from a subject.[001 1 1 ] Antibody-response to the peptides can be detected by different immunological / serological methods. Suitable formats of detecting presence of the antibody using the peptides includes peptide micro arrays, ELISA, chromatography, Western blot, lab- on-a chip formats, microbead-based single- or multiplex immunoassays, lateral-flow assays etc.[001 12] Often these methods involve proving the peptide bound to stationary phase (such as the well of an ELISA plate or the surface of a microbead) and adding the sample to be analysed in the liquid phase, allowing antibodies to bind and then washing away unbound antibodies.[001 13] Antibody binding can be detected in vitro by using a labelled secondary antibody that binds to a specific type of human antibody for example IgG, IgA, IgG 1 , lgG2 or lgG3, lgG4. In ELISA, the secondary antibody is labelled with an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). The secondary antibody is suitably from another species than human, for example from rabbit or goat. Alternatively, a fluorescence label or radioactive label can be used.[001 14] A protocol for using the peptides in an ELISA can be easily optimised by a person skilled in the art with regard to which secondary antibody to use, its dilution, buffers, blocking solution, wash etc. An outline of an example of an ELISA protocol using plates can be as follows: Polystyrene microtiter plates are coated with optimal concentrations, as determined by checkerboard titrations, of the peptides of interest dissolved in PBS at room temperature overnight. After two washes with PBS, wells are blocked with 0.1% (wt / vol) bovine serum albumin-PBS at 37°C for 30 min. Subsequent incubations are performed at room temperature, and plates are washed three times with PBS containing 0.05% Tween (PBS-Tween) between incubations. Samples of serum or other bodily fluids are added in duplicates or triplicates in initial dilutions of for example 1 / 10 and diluted for example in a three-fold dilution series. Control samples previously tested and found to have antibodies to the peptides are used as positive controls. Samples with known concentrations of antibodies may be used for creating a standard curve. Wells to which only PBS-Tween® are added are used as negative controls for determination of background values. After incubation at room temperature for 90 min, HRP-labeled rabbit anti-human IgA or IgG antibodies are added and incubated for 60 min. Plates are thereafter read in a spectrophotometer 20 min after addition of H2O2 and orf o-phenylene-diamine dihydrochloride in 0.1 M sodium citrate buffer, pH 4.5. The end point titers of each sample are determined as the reciprocal interpolated dilution giving an absorbance of for example 0.4 above background at 450 nm. Alternatively, as the final read-out value, the absorbance value can be used. The skilled person recognises that this ELISA protocol is an example only and many different variants and alterations of this protocol are possible.[001 15] Alternatively, in one embodiment, B-cells are isolated from the subject, and it is analysed if the cells are able to produce antibodies that bind to the peptide. This can be done by using the ELISPOT method, ALS (antibodies in lymphocyte secretions), or similar methods.[001 16] Diagnosis can also be carried out by detecting the presence of linear epitopes of CagA protein of the present invention in a tissue sample from a patient using antibodies specific for a peptide selected from peptides comprising or consisting of those according to the invention as described herein.[001 17] Antibodies with the desired binding specificity can be generated by a person skilled in the art. The antibody can be a polyclonal or a monoclonal antibody, with monoclonal antibodies being preferred. The antibody can be used in any useful format to detect the proteins or peptides, for example Western blot, ELISA, immunohistochemistry, etc. The antibody can be used for the diagnostic methods herein.[001 18] The method may be such that it can result in two possible outcomes: CagA+ H. pylori infection present or CagA+ H. pylori infection absent. CagA+ H. pylori infection can for example be determined on the basis of a signal cut off in the assay. There may also be an intermediate result: outcome uncertain that warrants further investigation or re-sampling or reanalysis of samples.[001 19] Once it has been established that a CagA+ H. pylori infection that places a subject at risk or pre-eclampsia is present, it may be useful to treat the H. pylori infection, for example in order to decrease the risk of the subject developing pre-eclampsia. Treatment can be done by methods known in the art, for example with the use of antibiotics. For different reasons, some being low availability of active antibiotics in the stomach as well as problems with antibiotic resistance, there are many different antibiotic treatment regimens for H. pylori infection, and the efficacy of these generally differ in different parts of the world. In general, the treatment regimens include at least two different antibiotics selected from the groups of macrolides, beta-lactams, nitroimidazoles, tetracyclines and fluoroquinolones, with or without the addition of bismuth subcitrate potassium, where one antibiotic is preferably selected from each group. One or more antibiotics may be administered in combination with a proton pump inhibitor. One treatment includes administration of the proton pump inhibitor omeprazole or esomeprazole, and the antibiotics amoxicillin and clarithromycin for 7 to 14 days.

[0120] Thus, there is also provided a method for preventing pre-eclampsia comprising the steps of 1 ) carrying out diagnosis as described herein on a subject and 2) treating the CagA+ H. pylori infection in the subject. Preferably treatment is made so that the subject is free of CagA+ H. pylori infection.

[0121] Once it has been established that a CagA+ H. pylori infection is present it may also be useful to perform further investigations to assess the presence of other a vascular or epithelial pathology (including atherosclerosis), or hypertensive disorders. This may be relevant for all subjects, not just pregnant subjects.

[0122] The peptides can be synthesised by methods known in the art. The peptides can be obtained substantially pure and in large quantities by means of organic synthesis, such as solid phase synthesis. Methods for peptide synthesis are well known in the art, for example using a peptide synthesis machine. Of course, the peptides may be ordered from a peptide synthesis company.

[0123] The peptides can also be of animal, plant, bacterial or virus origin. The peptide may then be purified from the organism, as is known in the art. The peptide can be produced using recombinant technology, for example using eukaryotic cells, bacterial cells, or virus expression systems. It is referred to Current Protocols in Molecular Biology, (Ausubel et al, Eds.,) John Wiley & Sons, NY (current edition) for details.

[0124] One or more peptides may be included in a kit. The kit may be used for diagnosis as described herein. A kit may comprise one or more peptides or mixtures thereof, binding buffer, and detection agents such as a secondary antibody. The kit can include a substrate that immobilises the peptide, such as a solid support, such as microtiter plates, such as ELISA plates to which the peptide(s) of the invention have been pre-adsorbed, various diluents and buffers, labelled conjugates or other agents for the detection of specifically bound antigens or antibodies, such as secondary antibodies, and other signalgenerating reagents, such as enzyme substrates, cofactors and chromogens. Other suitable components of a kit can easily be determined by one of skill in the art.EXAMPLES

[0125] Example 1 - Identification of epitopes of CagA indicative of increased risk of

[0126] In WO2018153991 , the Applicant of the present invention disclosed an exhaustive map of peptide sequences that described which CagA-peptides induced antibodyresponses in individuals infected with CagA-carrying H. pylori (i.e. so-called linear epitopes of CagA) Specifically, Table 1 in that patent application discloses 18 different peptide sequences (“CagA_01” to “CagA_18”, each of 15 to 31 amino acid length) that include all linear epitopes of CagA.

[0127] The entire CagA-sequence was screened by assessing serum antibodybinding to overlapping 15-mer peptides and pools of serum samples. Medium-density arrays of example 2 spotted with peptides covering the entirety of the CagA-sequence, with a sequential overlap of 10 amino acids (n = 234 peptides) were used. In follow-up experiments, high-density arrays of example 3 with 15-mer peptides covering the entirety of the CagA- sequence were used, but this time with a sequential overlap of 14 amino acids (n = 1172 peptides). In both cases, the H. pylori strain 26695 was used as the source of the CagA peptide sequences. Antibody-binding to each peptide was assessed individually on the array, and two serum pools - one consisting of pooled sera from 10 H. pylori- nfected (Hp+) individuals and the other consisting of sera from 10 uninfected (Hp-) individuals were used.

[0128] The antibody-binding of the Hp+ serum pool was compared to the binding of the Hp- pool. A linear B-cell epitope was defined as a stretch of at least four amino acids where the antibody-binding was at least 2x higher in the Hp+ group than in the Hp- group. In this way it was determined that H. pylori CagA contains 18 different linear B-cell epitopes, with an average length of 22 amino acids (Table 1 and Fig 1 in WO2018153991 ). These epitopes are all useful for diagnosis of a CagA+ H. py / or / -infection.

[0129] Example 2 - Association between pre-eclampsia and CaqA+ H. pylori.

[0130] Previous studies have shown a stronger association between pre-eclampsia and H. pylori carrying the protein CagA, than with H. py / or / -infection alone (Shiadeh et al 2017). This has been demonstrated in studies measuring antibodies to CagA and correlating the presence of such antibodies in serum with the risk of pre-eclampsia development. Depending on method of measurement, the risk of pre-eclampsia between studies have varied from minor to significant. Western blot is the most effective method for CagA-antibody analysis, and when it was used the risk of pre-eclampsia in women positive for such antibodies was 11 times higher than in women without the antibodies (Odds-ratio 11.1).

[0131] A link has been proposed between anti-CagA antibodies and diseases of the circulatory and vascular system, including atherosclerosis. A causal relationship has been proposed by microscopy studies showing that antibodies to CagA from H. pylori can bind to blood vessel cells (Franceschi et al 2002). More recently this was also studied in relation to pre-eclampsia (Franceschi et al 2012). In this study, antibodies to CagA were shown to bind to the surface of placental cells (trophoblasts). It was suggested that this happens since the CagA-antibodies also can bind the human protein beta-actin, which can be bound to the surface of these cells, but neither a mechanism for this, nor specific epitopes or sequencesthat find application in such differentiation of subjects, were elucidated in these disclosures. As such, the binding of CagA-antibodies (directed to the sequences of the present invention) to blood vessel cells or to placental cells would cause inflammation and contribute to disease formation.

[0132] Example 3: Sequence similarity between one CagA epitope and beta-actin

[0133] A BLAST analysis of the 18 linear epitope sequences of CagA mentioned hereinbefore revealed that one of the epitope sequences has a 6-amino-acid similarity with the amino acid sequence of beta-actin. In the stretch of these 6 amino acids, 4 are identical in the most common published CagA sequences. It has been found in crystal structure studies of beta-actin (Schutt et al 1993) that the 6-amino acid stretch is exposed on the surface of the protein which indicates that antibodies binding to it can access the protein in its native form.

[0134] As such, certain CagA sequences have higher homology and are thus more strongly associated with disease conditions.

[0135] In a database of 180 CagA peptide sequences, 78% exhibited identity with beta-actin in 4 out of 6 amino acids in the stretch of interest, viz. SEQ ID NOs 1 , 6, 7, 9-13, 16, 22, 24-25, 27, 31 -34, and 38.

[0136] Interestingly, 16% of the CagA-sequences exhibited a 5 out of 6 amino acid identity in the same stretch, viz. SEQ ID NOs 3, 8, 14, 17, 21 , and 23.

[0137] The Applicant is of the opinion that pre-eclampsia risk is thus not only related to the presence of antibodies to CagA, but also that the specific sequence of CagA sequences the individual carries is of importance. Under this model, a greater similarity between the sequence of CagA and that of beta-actin is likely to cause a higher risk for pre-eclampsia development.

[0138] Example 4 - Identification of epitopes for use in predicting risk for pre- eclampsia development in pregnant women

[0139] Serum samples were tested from a cohort of pregnant women for presence of antibodies to the proprietary H. pylori CagA epitopes / peptides of the Applicant, to identify if any of the epitopes can be used to predict risk for pre-eclampsia development in pregnant women.

[0140] Methods

[0141] Samples

[0142] Two sample collections were used.

[0143] Cohort 1 : Plasma samples from pregnant women, retrieved at 26-28 weeks gestation. Samples were provided from the GUSTO cohort, by scientists from the Singapore Institute for Clinical Sciences (SICS).

[0144] Two groups of samples were tested from cohort 1 : samples from women that later developed preeclampsia (n=22), and samples from healthy subjects (n=22).

[0145] Cohort 2: Serum samples from pregnant women, retreived during the first trimester of pregnancy, were provided by scientists in the Swedish region of Kronoberg.

[0146] Two groups of samples were tested from cohort 2: samples from women that later developed preeclamspia (n=15), and samples from healthy controls (n=16).

[0147] Antibody assays for linear epitope identification

[0148] IgG and IgA antibody-binding was investigated by peptide microarray; 1204 different 15 amino acid long peptides were printed on the surface of a glass slide (PEPperPRINT GmBH, Heidelberg, Germany). Plasma (Cohort 1 ) or serum (Cohort 2) samples were incubated on the array, followed by washing and incubation with fluorochrome- conjugated anti-human IgG and anti-human IgA antibodies. Binding to each individual peptide was quantified by image analysis using the Applicant’s proprietary software. A cutoff for presence or absence of anti-peptide antibodies was set to 3 SD above the average background signal.

[0149] Peptides screened in the array experiments included overlapping peptides from the Helicobacter pylori protein CagA (n=1089 peptides), peptides from a variety of other H. pylori proteins (n=31 peptides); and control peptides from an organism unrelated to H. pylori (Epstein-Barr virus, n=24 peptides).

[0150] Results

[0151] Association between CagA epitopes and preeclampsia in one CagA region

[0152] For each CagA peptide, the association between antibodies to the peptide and the occurrence of preeclampsia was estimated by calculating the odds-ratio for the association (Table 2). An association was deemed to be present with an odds-ratio above 1 ; to be useful in identifying risk of preeclampsia, an odds-ratio for a peptide should be above 1 , but preferably above 2 or even 5 or 10. Surprisingly, although there was a significant antibody-response to 292 CagA peptides (27% of tested peptides), there were strong and consistent associations to preeclampsia development in only 70 (24%) of those CagA peptides. One particular region of the CagA protein, just adjacent to the so-called EPIYA-B motif, had a very high association to preeclampsia. Seventeen of the peptides with an OR of above 5 (37% of all such peptides) for IgG were from this EPIYA-B-adjacent region, and 37 out of 79 peptides from this region had odds-ratios above 2 (Figure 1 ).

[0153] Table 2. High-specificity diagnostic peptide sequences of CagA linear epitopes and IgG and IgA ORs. Column * provides the corresponding SEQ ID NOs as they were labelled in priority document AU2023902106.

[0154] A preeclampsia-associated peptide motif with potential cross-reactivity to the human inflammation-triggering receptor LLRC19

[0155] A group of peptides with OR above 5 (SEQ ID NOs 236, 239, 240, 241 , 244, 253 and 254) all share the motif sequence AKIDQLN (SEQ ID NO 257).

[0156] Interestingly, this sequence SEQ ID NO 257 is very similar to a sequence in the human protein LLRC19; the LLRC19 sequence is KIEQLN (SEQ ID NO 396). The difference between SEQ ID NOs 257 and 396 is only one amino acid - a D vs an E in position 3; these amino acids are both negatively charged and have similar properties. Antibodies associated to preeclampsia, binding to SEQ ID NO 257, are therefore, based on these findings, highly likely to cross-react and bind to SEQ ID NO 396 of the LLRC19 protein. This protein is located in the cell membrane, and SEQ ID NO 396 is located in the extracellular portion of the protein. LLRC19 functions as an inflammatory receptor triggering NF-kB and cytokine production (Chai et al 2009).

[0157] IgG and IgA-antibody binding to 20 different 15-mer peptides from the LLRC19 protein was tested, and 16 of these peptides had an OR of above 2 (SEQ ID NOs 387 to 395, and 397 to 403 - see Table 3).

[0158] Applicant therefore proposes that some H. pylori-i nfected individuals produce antibodies to CagA, including SEQ ID NO 257; and these SEQ ID NO 257-specific antibodies cross-reacts with the human LLRC19 receptor and trigger inflammation that contributes to development of preeclampsia and possibly other inflammatory conditions.

[0159] In addition, IgG and IgA-antibody binding to 124 different 15-mer peptides from the Beta-actin protein was tested, and 52 of these peptides had an OR of above 2 (SEQ ID NOs 335 to 386 - see Table 3).

[0160] Table 3. High-specificity diagnostic peptide sequences of beta-actin linear epitopes and LLRC19-peptides and IgG and IgA ORs. Column * provides the corresponding SEQ ID NOs as they were labelled in priority document AU2023902106.

[0161] SEQ ID NO 257 and SEQ ID NO 396, as well as SEQ ID NOs 387 to 395, and 397 to 403 (i.e. the LLRC19-peptides of Table 3), can therefore be used in a diagnostic test that identifies such antibodies, and thereby diagnoses risk for preeclampsia development in a blood sample or in a saliva sample. Individuals positive for such a test could be closely monitored for development of preeclampsia, and / or treated with antibiotics to clear their H. pylori infection, which will over time lead to reduction in anti-CagA antibodies and a lowering of the risk of preeclampsia development.

[0162] Alternatively, individuals carrying such antibodies could be treated with therapies targeting antigen-specific B cells, for example bispecific antibodies, which would deplete B-cells producing the cross-reactive antibodies and thereby reduce the risk of inflammation and preeclampsia development.

[0163] Example 5 - Identification of peptide motifs for use in predicting risk for preeclamspia development in pregnant women

[0164] In order to identify the minimal useful peptide sequences for preeclampsia prediction, the Applicant tested serum samples from pregnant women later developing preeclampsia to investigate to what extent their antibodies bound specifically to sequence variants of peptides that were identified in Example 4 to be useful for prediction of preeclampsia (Tables 2 and 3).

[0165] Methods:

[0166] Samples

[0167] From Cohort 2 (see Example 4 above) the 9 serum samples with the strongest antibody-reactivity to the selected peptides were tested in peptide microarray experiments.

[0168] Antibody assays for linear epitope identification

[0169] IgG and IgA antibody-binding was investigated by peptide microarray; 1065 different 12 or 15 amino acid long peptides were printed on the surface of a glass slide (PEPperPRINT GmBH, Heidelberg, Germany). Serum samples were incubated on the array, followed by washing and incubation with fluorochrome-conjugated anti-human IgG and antihuman IgA antibodies. Binding to each individual peptide was quantified by image analysis using the Applicant’s proprietary software.

[0170] For this sequence variant testing, 49 peptides were selected from Tables 2 and 3 (these peptides are henceforth referred to as "Parent peptides"); see Table 4 for a list of all Parent peptides. Each Parent peptide was tested on the array. In addition, 7- and 8-mer peptide sequences were generated so that each 15-mer Parent peptide was represented on the array by 9 different 7-mer peptide variants and 4 different 8-mer variants covering the entirety of its 15-mer sequence. To allow sufficient space for antibodies to bind to these 7- and 8-mer peptides on the arrays, additional alanine amino acids were added to these sequences (henceforth referred to as "Alanine padding"): 2 alanines in the N-terminal end of both 7- and 8-mers, and 3 or 2 alanines in the C-terminal end of 7-mers and 8-mers respectively, to make each peptide on the array a 12-mer like so: AAXXXXXXXAAA where "XXXXXXX" represents the 7-mer sequence and AAYYYYYYYYAA where "YYYYYYYY" represents the 8-mer sequence. Inclusion of 7- and 8-mer variants of the Parent peptides ensured that the Applicant could identify which 7- and 8-mer sequences were the minimal useful peptide sequences of each of the Parent peptides.

[0171] To further identify which amino acid residues of the identified 7- and 8-mer sequences were redundant and therefore could be replaced with any other amino acid, the Applicant also tested alanine-substituted variants of each 7- and 8-mer peptide on the array. Thus, for each XXXXXXX 7-mer sequence, each position in the 7-mer was substituted for an alanine like so: AXXXXXX, XAXXXXX, XXAXXXX, XXXAXXX, etc. On the array, Alanine padding was used for these peptides as well, so the corresponding peptides tested on the array were AAAXXXXXXAAA, AAXAXXXXXAAA, AAXXAXXXXAAA, AAXXXAXXXAAA, etc. The peptides tested for each 8-mer sequence were likewise AAAYYYYYYYAA, AAYAYYYYYYAA, AAYYAYYYYYAA, etc.

[0172] Results

[0173] For each peptide tested, the Applicant calculated the mean array signal across the 9 different samples. For each Parent peptide and its associated sequence variants, the Applicant then compared each sequence variant and with its Parent peptide by calculatingthe fold-difference (mean array signal of the sequence variant divided by the mean signal of the Parent peptide), as well as the Cohen's d (mean signal of the sequence variant minus mean signal of the Parent peptide, divided by the pooled standard deviation of both). This revealed that out of the 1065 different peptide variants tested, only 245 7-mer or 8-mer peptide sequences (SEQ ID NOs 404-648) retained at least 25% of their antibody-binding across the preeclampsia cases as compared to their Parent peptides (Table 4).Table 4. Sequence variants that are highly diagnostic.Each 7- or 8-mer tested was compared to its Parent peptide (referenced in "Parent SEQ ID NOs"). "Fold diff" and "Cohen's d" provides the fold-difference and the Cohen's d metric, respectively, of antibody-binding to the tested peptide compared to its Parent peptide.

[0174] SEQ ID NOs 404 to 648 can therefore be used to predict preeclampsia development.

[0175] Upon further analysis, whereby the 7- and 8-mers and their alanine-substituted variants were analysed, it was found that in these short peptides, certain amino acids can be exchanged to other amino acids and the peptide in question still retaining its high binding by antibodies from preeclampsia cases. The Applicant identified 132 such common denominator peptides (SEQ ID NOs 649 to 780; Table 5).

[0176] Table 5. Common denominator peptides that are highly diagnostic for preeclampsia.

[0177] SEQ ID NOs 649 to 780 can therefore be used to predict preeclampsia development.

[0178] The Applicant is of the opinion that the present invention provides a new and useful diagnostic test, markers, and method for determining increased risk of pre-eclampsia in subjects based on the surprising discovery that the linear peptides of CagA+ H. pylorispecifically identified herein are useful for determining the risk of developing vascular pathologies, especially pre-eclampsia in a subject. There is also a need for CagA-peptides that bind specifically to antibodies, in particular antibodies that bind to the CagA protein and that can be used in determining subjects with an increased predisposition to developing vascular pathologies, including pre-eclampsia.

[0179] The Applicant is of the opinion that they have identified a need for a pre- eclampsia diagnostic test for CagA+ H. pylori with improved diagnostic properties, for example improved specificity and sensitivity.

[0180] Optional embodiments of the present invention may also be said to broadly consist in the parts, elements and features referred to or indicated herein, individually or collectively, in any or all combinations of two or more of the parts, elements or features, and wherein specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0181] It is to be appreciated that reference to "one example" or "an example" of the invention is not made in an exclusive sense. Accordingly, one example may exemplify certain aspects or embodiments of the invention, whilst other aspects or embodiments are exemplified in a different example. These examples are intended to assist the skilled person in performing the invention and are not intended to limit the overall scope of the invention in any way unless the context clearly indicates otherwise.

[0182] It is to be understood that the terminology employed above is for the purpose of description and should not be regarded as limiting. The described embodiment is intended to be illustrative of the invention, without limiting the scope thereof. The invention is capable of being practised with various modifications and additions as will readily occur to those skilled in the art.

[0183] Various substantially and specifically practical and useful exemplary embodiments of the claimed subject matter are described herein, textually and / or graphically, including the best mode, if any, known to the inventors for carrying out the claimed subject matter. Variations (e.g. modifications and / or enhancements) of one or more embodiments described herein might become apparent to those of ordinary skill in the art upon reading this application.

[0184] The inventor(s) expects skilled artisans to employ such variations as appropriate, and the inventor(s) intends for the claimed subject matter to be practiced other than as specifically described herein. Accordingly, as permitted by law, the claimed subject matter includes and covers all equivalents of the claimed subject matter and all improvements to the claimed subject matter. Moreover, every combination of the above-described elements, activities, and all possible variations thereof are encompassed by the claimed subject matter unless otherwise clearly indicated herein, clearly and specifically disclaimed, or otherwise clearly contradicted by context.

[0185] The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate one or more embodiments and does not pose a limitation on the scope of any claimed subject matter unless otherwise stated. No language in the specification should be construed as indicating any non-claimed subject matter as essential to the practice of the claimed subject matter.

[0186] The use of words that indicate orientation or direction of travel is not to be considered limiting. Thus, words such as "front", "back", "rear", "side", "up", down", "upper", "lower", "top", "bottom", "forwards", "backwards", "towards", "distal", "proximal", "in", "out" and synonyms, antonyms and derivatives thereof have been selected for convenience only, unless the context indicates otherwise. The inventor(s) envisage that various exemplary embodiments of the claimed subject matter can be supplied in any particular orientation and the claimed subject matter is intended to include such orientations.

[0187] The use of the terms "a", "an", "said", "the", and / or similar referents in the context of describing various embodiments (especially in the context of the claimed subject matter) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "including," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted.

[0188] Moreover, when any number or range is described herein, unless clearly stated otherwise, that number or range is approximate. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value and each separate sub-range defined by such separate values is incorporated into the specification as if it were individually recited herein. For example, if a range of 1 to 10 is described, that range includes all values there between, such as for example, 1.1 , 2.5, 3.335,5, 6.179, 8.9999, etc., and includes all sub-ranges there between, such as for example, 1 to 3.65, 2.8 to 8.14, 1.93 to 9, etc.

[0189] Accordingly, every portion (e.g., title, field, background, summary, description, abstract, drawing figure, etc.) of this application, other than the claims themselves, is to be regarded as illustrative in nature, and not as restrictive; and the scope of subject matter protected by any patent that issues based on this application is defined only by the claims of that patent.

[0190] Embodiments of the invention may also comprise any one or more or combinations of the claims.Citation List

[0191] Non-Patent Literature1 . Chai L, Dai L, Che Y, Xu J, Liu G, Zhang Z, et al. LRRC19, a novel member of the leucine- rich repeat protein family, activates NF-kappaB and induces expression of proinflammatory cytokines. Biochem Biophys Res Commun. 2009 Oct 23;388(3):543-8.2. Franceschi F, Sepulveda AR, Gasbarrini A, Pola P, Silveri NG, Gasbarrini G, Graham DY, Genta RM (2002). Cross-reactivity of anti-CagA antibodies with vascular wall antigens: possible pathogenic link between Helicobacter pylori infection and atherosclerosis. Circulation 106(4):430-4.3. Franceschi F, Di Simone N, D'Ippolito S, Castellani R, Di Nicuolo F, Gasbarrini G, Yamaoka Y, Todros T, Scambia G, Gasbarrini A (2012): Antibodies anti-CagA cross-react with trophoblast cells: a risk factor for pre-eclampsia? Helicobacter 17(6):426-34.4. Schutt CE, Myslik JC, Rozycki MD, Goonesekere NC, Lindberg U (1993): The structure of crystalline profilin-beta-actin. Nature 365(6449): 810-6.5. Shiadeh MN, Seyed MR, Adam I, Saber V, Moghadam ZB, Armon B, Spotin A, Kangavari HN & Rostami R (2017): Helicobacter pylori infection and risk of pre-eclampsia: a systematic review and meta-analysis. The Journal of Maternal-Fetal & Neonatal Medicine, DOI: 10.1080 / 14767058.2017.1378331 .6. Zhan Y, Si M, Li M, Jiang Y (2019): The risk of Helicobacter pylori infection for adverse pregnancy outcomes: A systematic review and meta-analysis. Helicobacter 24(2):e12562. doi: 10.1111 / hel.12562.

Claims

Claims1 . A method of using at least one peptide sequence derived from a linear epitope of the CagA protein of H. pylori, for the identification of a subject at increased risk of developing pre-eclampsia.

2. A method of using at least one peptide sequence derived from a linear epitope of the CagA protein of H. pylori as a marker for increased risk of developing preeclampsia in a subject.

3. A method according to claim 1 or claim 2, wherein the at least one peptide sequence comprises the sequence AKIDQLN (SEQ ID NO 257).

4. A method according to any one of the previous claims, wherein the at least one peptide sequence comprises any one or more of the following sequences:TQVAKKVNAKIDQLN (SEQ ID NO 236)TQVAKKVKAKIDQLN (SEQ ID NO 239)AQVAKKVSAKIDQLN (SEQ ID NO 240)AQVAKKVNAKIDQLN (SEQ ID NO 241 )VAKKVSAKIDQLNEA (SEQ ID NO 244)KKVSAKIDQLNQAAS (SEQ ID NO 246)KKVSAKIDQLNEATS (SEQ ID NO 248)KKVNAKIDQLNQIAS (SEQ ID NO 250)KKVNAKIDQLNQAAS (SEQ ID NO 253)VSAKIDQLNEATSAI (SEQ ID NO 254)5. A method according to claim 1 or claim 2, wherein the at least one peptide sequence comprises any one or more of the following sequences:RSVSPEPIYATIDDL (SEQ ID NO 284)EPIYATI (SEQ ID NO 574)EPIYAAI (SEQ ID NO 575)EPAYATI (SEQ ID NO 576)EPXYAXI (SEQ ID NO 756)6. A method according to claim 1 or claim 2, wherein the at least one peptide sequence comprises any one or more of the following sequences:ENSTEPIYAKVNKKK (SEQ ID NO 223)EPIYAQV (SEQ ID NO 479)EPIYAAV (SEQ ID NO 480)EPIYAKV (SEQ ID NO 487)EPIYAXV (SEQ ID NO 703)7. A method according to claim 1 or claim 2, wherein the at least one peptide sequence comprises the sequence KI(X1)QLN, wherein:X1 is a negatively charged amino acid (D or E).

8. A method according to claims 1 , 2, or 7, wherein the at least one peptide sequence comprises the sequence KIEQLN (SEQ ID NO 396).

9. A method according to any one of the previous claims, wherein the at least one peptide sequence comprises at most 35 amino acids.

10. A method according to any one of the previous claims, wherein the at least one peptide sequence comprises no more than 15 amino acids.

11. A method according to any one of the previous claims, wherein the at least one peptide sequence comprises no more than 8 amino acids.

12. A method according to any one of the previous claims, wherein the at least one peptide sequence comprises no more than 7 amino acids.

13. Use of at least one peptide sequence derived from a linear epitope of the CagA protein of H. pylori, for the identification of a subject at increased risk of developing pre-eclampsia.

14. Use of at least one peptide sequence derived from a linear epitope of the CagA protein of H. pylori as a marker for increased risk of developing pre-eclampsia in a subject.

15. Use according to claim 13 or claim 14, wherein the at least one peptide sequence comprises the sequence AKIDQLN (SEQ ID NO 257).

16. Use according to any one of claims 13 to 15, wherein the at least one peptide sequence comprises any one or more of the following sequences:TQVAKKVNAKIDQLN (SEQ ID NO 236)TQVAKKVKAKIDQLN (SEQ ID NO 239)AQVAKKVSAKIDQLN (SEQ ID NO 240)AQVAKKVNAKIDQLN (SEQ ID NO 241 )VAKKVSAKIDQLNEA (SEQ ID NO 244)KKVSAKIDQLNQAAS (SEQ ID NO 246)KKVSAKIDQLNEATS (SEQ ID NO 248)KKVNAKIDQLNQIAS (SEQ ID NO 250)KKVNAKIDQLNQAAS (SEQ ID NO 253)VSAKIDQLNEATSAI (SEQ ID NO 254)17. Use according to claim 13 or claim 14, wherein the at least one peptide sequence comprises any one or more of the following sequences:RSVSPEPIYATIDDL (SEQ ID NO 284)EPIYATI (SEQ ID NO 574)EPIYAAI (SEQ ID NO 575)EPAYATI (SEQ ID NO 576)EPXYAXI (SEQ ID NO 756)18. Use according to claim 13 or claim 14, wherein the at least one peptide sequence comprises any one or more of the following sequences:ENSTEPIYAKVNKKK (SEQ ID NO 223)EPIYAQV (SEQ ID NO 479)EPIYAAV (SEQ ID NO 480)EPIYAKV (SEQ ID NO 487)EPIYAXV (SEQ ID NO 703)19. Use according to claim 13 or claim 14, wherein the at least one peptide sequence comprises the sequence KI(Xi)QLN, wherein:Xi is a negatively charged amino acid (D or E).

20. Use according to claims 13, 14, or 19, wherein the at least one peptide sequence comprises the sequence KIEQLN (SEQ ID NO 396).

21. Use according to any one of claims 13 to 20, wherein the at least one peptide sequence comprises at most 35 amino acids.

22. Use according to any one of claims 13 to 20, wherein the at least one peptide sequence comprises no more than 15 amino acids.

23. Use according to any one of claims 13 to 20, wherein the at least one peptide sequence comprises no more than 8 amino acids.

24. Use according to any one of claims 13 to 20, wherein the at least one peptide sequence comprises no more than 7 amino acids.

25. An antibody specific to a peptide comprising a peptide sequence as used in any one of claims 1 to 24.

26. An antibody according to claim 25, wherein the antibody can cross-react with the human LLRC19 receptor.

27. An antibody according to claim 26, wherein the cross-reacting with the human LLRC19 receptor triggers inflammation that contributes to development of preeclampsia and / or other inflammatory conditions.

28. A method for prognosing development of preeclampsia and / or other inflammatory conditions in a subject, the method comprising the step of identifying the presence of an antibody according to any one of claims 25 to 27 in a sample from the subject.

29. A method of determining the risk of a vascular or epithelial pathology, such as preeclampsia, in a subject, the method comprising the steps of:(i) isolating or providing a sample from a subject;(ii) contacting said sample with one or more peptide comprising a peptide sequence as used in any one of claims 1 to 24; and(iii) detecting specific binding of antibodies in the sample to the one or more peptide.

30. A method for the identification, diagnosis, prediction, or prognosis of the onset of pregnancy-induced hypertension and / or pre-eclampsia or for the assessment of the risk of a pregnant subject developing hypertension and / or pre-eclampsia, the method comprising the steps of:(i) providing a biopsy sample containing antibodies of the subject;(ii) bringing the sample into contact with any one or more of the peptides comprising a peptide sequence as used in any one of claims 1 to 24; and(iii) detecting the binding of the antibodies with the any one or more peptides.

31. A method or use according to any one of claims 1 to 24, wherein the sample comprises a liquid biopsy sample such as a blood, serum, saliva, or plasma sample which contains antibodies, or a tissue sample, for example a gastric tissue sample.

32. A method of treating a subject at risk of developing preeclampsia and / or other inflammatory conditions, or for preventing a vascular or epithelial pathology, such as pre-eclampsia, the method comprising the steps:(i) identifying the presence of an antibody according to any one of claims 25 to 27 in a sample from the subject; and(ii) treating a H. pylori CagA+ infection in the subject.

33. A method according to claim 32, the method comprising the additional step of quantifying the concentration of the antibody in the sample from the subject.

34. A method according to claim 33, wherein treating the H. pylori CagA+ infection in the subject comprises administering to the subject at least one antibiotic effective for treating a H. pylori infection.

35. A method according to claim 34, wherein the antibiotic comprises one or more selected from the group consisting of: macrolides, beta-lactams, nitroimidazoles, tetracyclines, fluoroquinolones, and functional equivalents thereof.

36. A method according to claim 35, wherein the antibiotic comprises at least one of clarithromycin, metronidazole, amoxicillin, or tetracycline.

37. A method according to claim 35 or claim 36, wherein the subject is treated with an additional agent in combination with the at least one antibiotic, the additional agent comprising one or more selected from the group comprising: proton pump inhibitor or bismuth subsalicylate.

38. A method according to claim 37, wherein the proton pump inhibitor comprises esomeprazole, lansoprazole, omeprazole, pantoprazole, or rabeprazole.

39. A method according to any one of claims 32 to 38, the method comprising the additional step of administering a therapeutic agent to the subject to reduce the risk of developing preeclampsia and / or other inflammatory conditions.

40. A method according to claim 39, wherein the therapeutic agent prevents the binding of the antibody to the LLRC19 receptors in cells of the subject.

41. A method according to claim 40, wherein the therapeutic agent prevents the binding of antibodies to the LLRC19 receptors in cells of the subject.

42. A method according to claim 41 , wherein the cells of the subject are located in the gastrointestinal tract.

43. A method of treating a subject to reduce their risk of inflammation and preeclampsia development, the method comprising depleting B-cells in the subject that produce antibodies which cross-react with the human LLRC19 receptor.

44. A method of treating a subject according to claim 43, wherein the antibodies are bispecific antibodies.

45. A peptide comprising a peptide sequence as used in any one of claims 1 to 24, for use in a diagnostic test for determining whether a subject may develop, or is predisposed to developing, vascular or endothelial dysfunctions or pathologies.

46. The peptide of claim 45, wherein the vascular or endothelial dysfunction or pathology is pre-eclampsia.

47. Use of a peptide comprising a peptide sequence as used in any one of claims 1 to 24 as a biomarker of potential for inflammation and preeclampsia development in a subject.

48. Use of a peptide according to claim 47, for diagnosis or as a prognostic indicator of pre-eclampsia or another vascular or epithelial pathology in a subject.

49. A kit comprising at least one peptide comprising a peptide sequence as used in any one of claims 1 to 24, for diagnosis, forming a prognosis, or monitoring of a subject having a predisposition to pre-eclampsia or another vascular or epithelial pathology.

50. The kit according to claim 49 for assessing the binding of the at least one peptide to antibodies in a sample from the subject.51 . An assay for diagnosing, monitoring, and / or forming a prognosis of a potential for inflammation and preeclampsia development in a subject, the assay comprising detecting specific binding of antibodies from a sample from the subject to the one or more peptide comprising a peptide sequence as used in any one of claims 1 to 24.

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