NEW VACCINAL STRATEGY TO PREVENT OR TREAT RHUMATOID ARTHRITIS

Abstract

The present invention relates to field of treatment of rheumatoid arthritis. The inventors propose that PAD4, one of the enzymes which convert arginine into citrulline, is a target antigen for T cells that help the production of ACPA. They recently demonstrated that PAD immunization triggers anti-citrullinated fibrinogen antibody production in normal mice. Here, they demonstrate that the risk (OR) to develop RA associated with each of 12 HLA-DRB1 genotype correlates with the likelihood for the two HLA-DR molecules encoded by each genotype to bind at least one random peptide from PAD4, but not from citrullinated or native fibrinogen. PBLs from patients with RA, PsA and controls proliferate to PAD4 and they identify, notably, a peptide from PAD4, p8 (SEQ ID NO: 6), that stimulates T cells from RA patients and a few patients with PsA. Proliferative responses to p8 are associated with RA, shared epitope positive HLA-DR alleles and antibodies to PAD4. Thus the present invention relates to a peptide derived from the PAD4 protein and its use in the treatment and prevention of rheumatoid arthritis.

Claims

1. A peptide derived from the PAD4 protein of SEQ ID NO°: 2.

2. The peptide according to claim 1 wherein said peptide is described in table 1.

3. The peptide according to claim 1 wherein said peptide binds to a HLA-DRB1*0101, HLA-DRB1*0401, HLA-DRB1*0404, HLA-DRB1*0402 or HLA-DRB1*0701 molecule.

4. The peptide according to claim 1 wherein said peptide comprises or consists of the amino acid sequence of amino acids 61 to 100 of SEQ ID NO°: 2.

5. The peptide according to claim 4 wherein said peptide comprises or consists of the amino acid sequence SEQ ID NO: 3: or SEQ ID NO: 4.

6. The peptide according to claim 1 wherein said peptide comprises or consists of the amino acid sequence SEQ ID NO: 5: DPGVEVTLTMK-Xaa12-ASGSTGDQ wherein Xaa12 is an alanine (Ala or A), or a valine (Val or V) or a function-conservative variant thereof.

7. The peptide according to claim 6 wherein said peptide has the amino acid sequence SEQ ID NO: 6 or SEQ ID NO: 7 or a function-conservative variant thereof.

8. The peptide according to claim 1 wherein said peptide has the amino acid sequence SEQ ID NO: 8 or a function-conservative variant thereof.

9. A nucleic acid sequence encoding a peptide derived from PAD4 protein according to claim 1.

10. (canceled)

11. (canceled)

12. A method for treating or preventing rheumatoid arthritis or psoriatic arthritis comprising, administrating to a subject in need thereof a therapeutically effective amount of a peptide according to claim 1 or a nucleic acid sequence encoding the peptide.

13. A vaccine composition comprising a peptide derived from PAD4 protein according to claim 1 or a nucleic acid sequence encoding the peptide.

14. The method of claim 12, wherein the peptide is administered in a vaccine composition.

15. A T lymphocyte that recognizes specifically a peptide according to claim 1.

Description

FIGURES and TABLES

[0097] FIG. 1: Proliferative response to human PAD4 in 41 patients with RA, 25 patients with psoriatic arthritis and 11 healthy controls. 41 patients with RA, 25 patients with Psoriatic arthritis and 11 normal controls were tested for proliferative response to PAD4, native and citrullinated fibrinogen. For each protein, BRDU incorporation was tested in 4 replicates. Positivity was defined by a ratio between the OD for a well with cells and protein and the OD for a well with cells but without protein higher than 2 (OD ratio higher than 2). Mean OD ratios are indicated in red.

[0098] FIG. 2: Proliferative response to peptides from PAD4 in 22 patients with RA, 16 patients with psoriatic arthritis and 11 healthy controls. 22 patients with RA, 16 patients with Psoriatic arthritis and 11 healthy controls were tested for proliferative response to PAD4 peptides. For each peptide, BRDU incorporation was tested in 4 replicates. Positivity was defined by a ratio between the OD for a well with cells and peptide and the OD for a well with cells but without peptide higher than 2 (OD ratio higher than 2). Mean OD ratios are indicated in red.

[0099] FIG. 3: Antibody responses to human PAD4 in 41 patients with RA, 25 patients with psoriatic arthritis and 11 normal controls. 41 patients with ACPA positive RA, 25 patients with Psoriatic arthritis and 11 healthy controls were tested for anti PAD4 IgM and IgG by ELISA. Each antibody assay was performed in duplicate. Positivity was defined by a ratio between the OD for a well with serum and PAD4 protein and the OD for a well with serum but without PAD4 protein higher than 2 (OD ratio higher than 2) for IgG and higher than 3 for IgM. Mean OD ratios are indicated in red.

[0100] FIG. 4: antibody and proliferative responses to PAD4 in 41 patients with RA, 25 patients with Psoriatic arthritis and 11 healthy controls. 41 patients with ACPA positive RA, 25 patients with Psoriatic arthritis and 11 healthy controls were tested for T cell proliferation and antibody response to PAD4 and classified into 4 groups according to the results. SE: Shared epitope.

TABLE-US-00003 TABLE 2 Likelihood to bind PALM or fibrinogen peptides for each of 12 different HLA-DRB1 genotypes. Likelihood to bind at least one peptide from Risk to Citrul- HLA-DRB1* develop RA Native linated Genotypes (Odd Ratio) PAD4 fibrinogen fibrinogen DRB1*07/07 0.4 0.090 0.270 0.340 DRB1*01:01/07 0.9 0.076 0.324 0.382 DRB1*04:04/07 1.5 0.134 0.350 0.382 DRB1*01:01/01:01 1.6 0.061 0.373 0.422 DRB1*04:01/07 2.4 0.178 0.253 0.281 DRB1*01:01/04:04 2.7 0.120 0.398 0.422 DRB1*04:01/04:01 4.1 0.258 0.234 0.216 DRB1*04:02/07 4.2 0.090 0.306 0.365 DRB1*01:01/04:01 5 0.165 0.307 0.327 DRB1*04:04/04:02 5.1 0.134 0.382 0.406 DRB1*04:04/04:04 10.3 0.176 0.422 0.422 DRB1*04:01/04:04 13.4 0.218 0.335 0.327 Pearson rho 0.594 0.303 −0.023 p 0.042 0.339 0.945

TABLE-US-00004 TABLE 3 Subjects tested for Ab and T cell proliferation to PAD4. RA PsA Healthy Number 41 25  11  Sex 30F 18F 8F Disease duration (years) 5 +/− 7 7 +/− 7 ACPA+ (3N)  41/41 (100%) 0 0 RF+ 24/33 (72%) 1/15 (7%)  0 SE+ 26/41 (63%) 6/25 (24%) 4/11 (36%) Anti PAD4 Ig 21/41 (51%) 4/25 (16%) 0 Anti PAD4 LT 19/41 (46%) 8/25 (32%) 4/11 (36%) Treatment 5 none 4 none 14 MTX 1 MTX 20 biotherapies 16 biotherapies RA: rheumatoid arthritis, PsA: psoriatic arthritis, F: female. ACPA: autoantibodies to citrullinated proteins, RF: rheumatoid factor. PALM: peptidylarginine deiminase, SE: shared epitope. MTX: methotrexate

TABLE-US-00005 TABLE 4 Subjects tested for immune responses to PAD4 and PAD4 peptides. RA PsA Healthy Number 22 16  11  Sex 19F 12F 8F Disease duration (years) 7 +/− 7 8 +/− 7 ACPA+ (3N)  22/22 (100%) 0 0 RF+ 14/16 (87%) 1/8 (12%) 0 SE+ 14/22 (63%)   6/16 (37.5%) 4/11 (36%) Anti PAD4 Ig 10/22 (45%) 3/16 (19%) 0 Anti PAD4 LT 11/22 (50%) 5/16 (31%) 4/11 (36%) Anti Peptide 8 LT  9/22 (41%) 3/16 (19%) 0 Treatment 3 none 2 none 5 MTX 1 MTX 11 biotherapies 10 biotherapies RA: rheumatoid arthritis, PsA: psoriatic arthritis, F: female. ACPA: autoantibodies to citrullinated proteins, RF: rheumatoid factor. PALM: peptidylarginyl deiminase, SE: shared epitope. MTX: methotrexate.

EXAMPLE

[0101] Material & Methods

[0102] Synthetic peptides from human PAD4 and human fibrinogen. Peptides were synthesized using the solid-phase system and purified (>70%)(Neosystem, Strasbourg, France). We synthesized 65 20-mers, encompassing residues 1-663 of wild-type PAD4 (residues S55, A82, and A112, on locus NM 012387) and overlapping on 10 amino acids. Residues S55, A82, and A112, which can be polymorphic, were detected under their native, unmutated form on peptides 5-6, 8-9, and 11-12, respectively. We synthesized 167 15-mers (71 native peptides, 71 citrullinated peptides and 25 peptides containing neither arginin nor citrulline) from the A and B chains of human fibrinogen (locus NP 000499, locus NP 005132). Whenever we thought the position of the R residue on the peptide might influence interaction with the P4 pocket, we synthesized extra, overlapping peptides (11).

[0103] Purification of HLA-DRB1 molecules from lymphoblastoid cell lines. The HLA homozygous lymphoblastoid cell lines JESTHOM (HLA-DRB1*01:01), SAVC (HLA-DRB1*04:01), YAR (HLA-DRB1*04:02), PEYSSON (HLA-DRB1*04:04), MOU (HLA-DRB1*07:01) were cultured in RPMI 1640 with 10% fetal calf serum. 2×109 cells were lysed in 10 mM Tris pH8, 10 mM NaCl, 10 mM MgCl2, 1% Triton X100, 0.05 mg/ml Dnase and protease inhibitors. These homozygous cell lines were chosen because they express 3 shared epitope positive alleles, HLA-DRB1*01:01, *04:01, *04:04 and two shared epitope negative alleles, not associated with RA, DRB1*04:02 and DRB1*07:01. Total protein extracts were immunoprecipitated by anti-HLA-DR LB3.1 antibody covalently coupled on cyanogen bromide-activated sepharose 4B (Sigma Aldrich, St. Quentin-Fallavier, France). After washing, HLA-DR molecules were eluted in PBS pH2 with 0.5% n-octylglucoside, neutralized in 1M Tris and quantified (11).

[0104] HLA-DR Peptide-binding assay. ELISA plates were coated with 10 μg of PAD4 or fibrinogen peptide/well and blocked in 1% bovine serum albumin (BSA). One microgram of purified HLA-DR molecule was added to plates. After washing, bound HLA-DR was detected by biotinylated anti HLA-DR antibody B8122 (Immunotech, Marseille, France), followed by peroxidase-conjugated avidin. Peroxidase-conjugated anti-mouse IgG and peroxidase-conjugated avidin were supplied by Sigma Aldrich (St. Quentin-Fallavier, France). Optical density was read at 405 nm. The binding of each of the purified HLA-DR alleles was assayed on ELISA plates coated with peptides from PAD4 or Fibrinogen (each in duplicate wells) and, as controls, 2 wells coated with a classical positive binder, influenza hemagglutinin (HA) peptide (PKYVKQNTLKLAT). Positive binding was defined as an OD value equal to the OD for the HA peptide.

[0105] Calculation of the likelihood to bind at least one peptide from a given protein for a particular HLA-DRB1 genotype. Briefly, we considered that the likelihood for a particular HLA-DRB1 allele to bind a given (unknown) peptide from a given protein was the ratio between number of bound peptides and total number of peptides from this protein. This ratio can be evaluated by studying the binding of a set of peptides covering the protein. Then, the likelihood for a given allele not to bind a peptide from the same protein is 1 minus the likelihood to bind a peptide. The likelihood for the two HLA-DRB1 alleles encoded by a genotype not to bind any peptide from the same protein is the product of the two likelihoods: (likelihood not to bind allele 1) (likelihood not to bind allele 2). Finally, the likelihood for the products of a given HLA-DRB1 genotype to bind at least one peptide from a protein is: 1 minus ((likelihood not to bind allele 1)×(likelihood not to bind allele 2)) (Table 2).

[0106] Patients. We tested 41 patients with rheumatoid arthritis (RA), 25 patients with psoriatic arthritis (PsA) from the rheumatology unit at Sainte Marguerite Hospital in Marseille, France and 11 healthy controls from the staff of the laboratory and the rheumatology ward. RA patients fulfilled the 2010 ACR/EULAR criteria and had ACPA titers higher than 3 times the upper limit of normal (4). Psoriatic arthritis patients fulfilled the the CASPAR criteria (26). HLA-DRB1 typing was performed by PCR/sequence specific oligonucleotide analysis in every patient and control (3). Anti-cyclic citrullinated peptide IgG antibodies were detected by a second-generation ELISA (Immunoscan RA Mark 2, Eurodiagnostica, Malmö, Sweden). Rheumatoid factors were detected by ELISA using the Orgentec Kit (Mainz, Germany). Baseline characteristics of patients are presented in Table 3 and Table 4.

[0107] Proteins. Human PAD4 protein was produced in baculovirus expression system and purified (Proteogenix, Schiltigheim, France). Activity and autocitrullination status were tested before T cell proliferation assay. Human fibrinogen (Merck Millipore, Darmstadt, Germany) was incubated in 1 M Tris HCl (pH7.4), 100 mM CaCl2, 50 mM dithiothreitol buffer at a concentration of 1 mg/ml with rabbit PAD2 protein (Sigma Aldrich, St. Quentin-Fallavier, France). Citrullination was performed for 2 hours at 37° C. Non citrullinated fibrinogen was treated identically, except that water was added instead of PAD.

[0108] T cell proliferation assay. Mononuclear cells from patients were isolated from 20 ml of heparinized blood by centrifugation through Ficoll-Histopaque (Sigma Aldrich, St. Quentin-Fallavier, France). Cells were cultured at a density of 106 cells/ml in RPMI 1640 with 10% self-serum in the presence of 1 μg/ml of human PAD4 or human fibrinogen or PHA (phytohemagglutinin) or 5 μg/ml of PAD4 peptide. After 6 days of culture at 37° C., proliferative response to proteins was evaluated using the colorimetric bromodeoxyuridine kit (Roche Diagnostics, Meylan, France). Positive T cell responses were defined by optical densities (OD) higher than twice the ODs for cells cultured without protein or peptide.

[0109] Detection of anti-PAD4 antibodies. Plates were coated with 0.5 μg human PAD4 and blocked with 2% BSA (bovine serum albumin). Sera diluted to 1:100 were incubated on plates. After washing, peroxydase conjugated anti-human IgG or IgM was added. Optical density (OD) was read at 405 nm. Background OD was obtained by adding each serum to a well without protein. Positive sera were defined by OD values higher than twice background OD for IgG and three times background OD for IgM.

[0110] Statistics. Correlation between the HLA-DRB1 genotypic Odds Ratio to develop RA and the likelihood to bind at least one PAD4 or Fibrinogen peptide for a given genotype was evaluated by Pearson's correlation test. Comparisons between groups for T cell and antibody assays were performed using the Fisher's test. GraphPad Prism 5.02 (GraphPad Software) was used for all statistical analyses.

[0111] Results

[0112] Binding of PAD4 and Fibrinogen Peptides to 5 Different HLA-DR Molecules

[0113] Binding of 65 peptides from PAD4, 96 citrullinated or arginine free peptides from fibrinogen and their 96 non citrullinated counterparts to 5 purified HLA-DR molecules was tested (Table 1 and data not shown). Each purified HLA-DRB1 molecule bound 2 to 9 of 65 PAD4 peptides, 12 to 23 of 96 fibrinogen peptides, 11 to 23 of 96 citrullinated fibrinogen peptides.

[0114] Likelihood to Bind PAD4 Peptide for Each of 12 Different HLA-DRB1 Genotypes

[0115] The likelihood to bind at least one PAD4 peptide, one native Fibrinogen peptide and one citrullinated Fibrinogen peptide was calculated for 12 genotypes for which we had calculated the risk to develop RA (Table 2).

[0116] Correlation Between OR to Develop RA and Likelihood to Bind at Least One PAD4 or Fibrinogen Peptide for 12 HLA-DRB1 Genotypes

[0117] There is a strong correlation between the risk to develop RA carried by each HLA-DRB1 genotype and the likelihood for its two encoded HLA-DRB1 molecules to bind at least one PAD4 peptide (p=0.042 Pearson's) (data not shown). Conversely, there is no correlation between the risk to develop RA carried by each HLA-DRB1 genotype and the likelihood for its two encoded HLA-DRB1 molecules to bind at least one peptide from native or citrullinated Fibrinogen (data not shown).

[0118] T Cell Proliferative Response to Human PAD4 is Common in Patients with RA, Psoriatic Arthritis and Normal Controls

[0119] PBLs from 41 patients with ACPA positive RA, 25 patients with Psoriatic arthritis and 11 healthy controls were tested for T cell proliferative response to human PAD4, human native and citrullinated fibrinogen, by BRDU incorporation (Table 3, FIG. 1).

[0120] 19/41 (46%) RA patients, 8/25 (32%) PsA patients, 4/11 (36%) controls had T cells proliferating to PAD4 (RA versus others, p=0.35 Fisher's test. N.S.).

[0121] One patient with RA (2.4%) and none of the patients with psoriatic arthritis or healthy controls had proliferative response to citrullinated fibrinogen. To confirm that the proliferative response observed in PBLs cultured with PAD4 was due to T cells, we ran flow cytometry analysis in an extra 10 RA patients and 7 controls. In this analysis, CD4 T cells showed increased expression of CD154, a T cell early activation marker and increased production of TNF alpha, in whole blood samples stimulated with PAD4 (data not shown).

[0122] T Cell Proliferative Responses to Peptides from PAD4 in 22 Patients with RA, 16 Patients with Psoriatic Arthritis and 11 Healthy Controls

[0123] To identify which epitope(s) on PAD4 were recognized by T cells, we studied proliferative responses to 11 20 mers from PAD4 which we had found to be good binders of HLA-DRB1*04:01, *04:04, *01:01, *04:02 or *07:01, in 22 RA patients, 16 PsA patients and 11 healthy controls (Table 4, FIG. 2).

[0124] Proliferative response to peptide 22 (p22, sequence: VRVFQATRGKLSSKCSVVLG, SEQ ID NO: 8) a peptide found to bind HLA-DRB1*04:01 only), was observed in 2/22 (9%) RA patients, 2/16 (12.5%) patients with PsA and none of 11 controls (RA versus others, Fisher's test, N.S.).

[0125] Proliferative response to peptide 8 (p8, sequence: DPGVEVTLTMKAASGSTGDQ, SEQ ID NO: 6, a peptide that bound all five tested HLA-DRB1 alleles) was associated with RA: indeed 9/22 (41%) RA patients, 3/16 (19%) patients with PsA and 0/11 controls proliferated to p8 (RA versus others p=0.02, Fisher's test).

[0126] Proliferative response to peptide 8 was associated with the shared epitope: 75% (9/12) in responders against 40% (15 of 37) in non responders (responders against non responders p=0.05, Fisher's test).

[0127] Proliferative response to peptide 8 was associated with anti PAD4 antibodies (all subjects with proliferation to peptide 8 against all subjects with anti PAD4 antibodies p=0.05, Fisher's).

[0128] Antibody Responses to Human PAD4 in 41 Patients with RA, 25 with Psoriatic Arthritis and 11 Normal Controls

[0129] IgG anti PAD4 antibodies were detected in 11/41 (27%) patients with RA, 1/25 (4%) patients with PsA and 0/11 healthy controls (RA versus others, Fisher's test, p=0.004) (FIG. 3).

[0130] IgM antibodies to human PAD4 were detected in 14/41 (34%) patients with RA against 3 of 25 (12%) patients with psoriatic arthritis and none of 11 healthy controls (RA versus others, Fisher's test, p=0.01) (FIG. 3).

[0131] Positive anti PAD4 IgM responses in RA patients are most likely caused by recognition of IgM rheumatoid factors bound to IgG anti PAD4 antibodies by the peroxydase labelled anti IgM antibody used in the ELISA. Indeed, 11 of the 14 patients with RA who tested positive for anti PAD4 IgM also tested positive for IgM rheumatoid factors. Therefore the IgM anti PAD4 antibodies we detected in patients with RA most likely indicate the presence of IgG anti PAD4 antibodies in these patients. Thus, we pooled IgM and IgG anti PAD4 antibodies and called them “anti PAD4 antibodies”.

[0132] Anti PAD4 antibodies are present in 21/41 (51%) patients with RA, 4/25 (16%) patients with PsA and 0/11 healthy controls (RA versus others p=0.0002, Fisher's test).

[0133] 13 of 14 Patients with Anti PAD4 Antibodies and T Cell Proliferation to PAD4 have RA

[0134] Among the 77 subjects that were tested, T cell proliferation and antibody response to PAD4 allowed us define 4 subgroups (FIG. 4):

[0135] 35/77 subjects were negative for both antibody and proliferation to PAD4. They included 14 (34%) of 41 patients with RA, 14 (56%) of 25 patients with PsA and 7 (64%) of 11 healthy controls. RA patients were underrepresented in this group (RA versus others, p=0.04, Fisher's test.).

[0136] 17/77 subjects were negative for antibodies and positive for T cell proliferation to PAD4. They included 6 (15%) of 41 patients with RA, 7 (28%) of 25 patients with PsA and 4 (36%) of 11 healthy controls (RA versus others, p=0.1, N. S.).

[0137] 11/77 subjects were positive for antibodies and negative for T cell proliferation to PAD4. They included 8 (19.5%) of 41 patients with RA, 3 (12%) of 25 patients with PsA and none of 11 healthy controls (RA versus others, p=0.2, NS).

[0138] 14/77 subjects were positive for both antibodies and T cell proliferation to PAD4. They included 13 (32%) of 41 patients with RA, 1 (4%) of 25 patients with PsA and none of 11 healthy controls (RA versus others, p=0.0009, Fisher's test) (FIG. 4).

[0139] In this “double positive” group, 71% (10 of 14) patients express the shared epitope against 41% in other groups (Fisher's, p=0.07).

[0140] Moreover, 25 had antibodies to PAD4, of whom 16 expressed SE positive HLA-DR alleles and 52 were negative for anti PAD4, of whom 20 expressed shared epitope positive HLA-DR alleles (Fisher's test, p=0.051) (FIG. 4).

[0141] 31 subjects had proliferative response to PAD4, of whom 15 expressed SE positive HLADR alleles and 46 had no proliferative response to PAD4, of whom 21 expressed SE positive HLA-DR alleles (p=0.8, Fisher's test) (FIG. 4).

[0142] Thus, the HLA-DR SE is associated with anti PAD4 antibodies, but not with anti PAD4 proliferative response.

Conclusion

[0143] The inventors have developed an alternative to the “Shared Epitope binds Citrullinated peptides” hypothesis. Indeed, the development of RA is preceded by the emergence of IgG antibodies to PAD4 (Peptidyl Arginyl Deiminase, the citrullinating enzyme) (17-21). This suggests the existence of helper T cells specific for PAD4 in patients with RA. Since PAD4 binds and citrullinates multiple proteins, any protein bound and being citrullinated by PAD4 may benefit from the help by a classical hapten carrier mechanism. Indeed, B cells specific for citrullinated residues on proteins bound to PAD4 could internalize and process the PAD4/citrullinated protein complex and present PAD4 peptides to helper T cells. This would lead to the production of IgG antibodies to multiple citrullinated proteins. To prove this point, the inventors have recently immunized normal non autoimmune mice with human and/or murine PADs and found that 20% of the mice developed anti citrullinated fibrinogen antibodies, in the absence of any T cell response to citrullinated fibrinogen (22).

[0144] Here, not knowing which peptide(s) from PAD4 or fibrinogen are critical to stimulate T cell responses, the inventors evaluated the likelihood for the 2 HLA-DR molecules encoded by each of 12 HLA-DRB1 genotypes to bind at least one of 65 peptides from the PAD4 protein and one of 96 native or 96 citrullinated peptides from human fibrinogen. To do so, the inventors studied the binding of overlapping peptides from PAD4 and native or citrullinated fibrinogen to 5 different HLA-DR alleles. The goal of this analysis was not to identify the actual peptide(s) from PAD4 or fibrinogen seen by helper T cells, but to quantify the likelihood for the two HLA-DR molecules encoded by a given genotype to bind an unknown relevant peptide from PAD4 or Fibrinogen. Regarding PAD4, a 663 amino acid protein, it can yield about 6500 relevant peptides (653 10mers, 652 11mers . . . 642 20 mers) and the inventors evaluated binding likelihoods using a sample of only 65 of them. Thus, this analysis can give us an indication of the ability for a pair of HLA-DR molecules to bind a peptide from a given protein, but not the certitude of having identified the relevant T cell epitope.

[0145] The inventors found a correlation between the likelihood that at least one the two HLA-DRB1 molecules encoded by each HLA-DRB1 genotype might bind at least one peptide from PAD4 and the OR to develop RA associated with this genotype. This was not observed with native or citrullinated peptides from fibrinogen.

[0146] The inventors then looked for T cell responses to PAD4 in the PBLs of patients with RA, psoriatic arthritis and healthy controls. They found that 19 of 41 patients with RA, 8 of 25 patients with PsA and 4 of 11 healthy controls had proliferative responses to PAD4. 13 of 41 RA patients had both proliferative response to PAD4 and anti PAD4 antibodies in their sera and this was different from all others (p=0.0009 Fisher's). This suggested that in these 13 patients, PAD4 specific T cells might provide help to PAD4 specific B cells. Thus, proliferative response to PAD4 is common, even in healthy subjects. However, when it is associated with anti PAD4 antibodies it is characteristic of ACPA positive RA. Among the 31 subjects (19 RA, 8 PsA and 4 controls) with proliferative responses to PAD4, SE HLA-DRB1 alleles were associated with anti PAD4 antibodies (Fisher's, p=0.03). This suggested that SE positive HLA-DRB1 alleles might allow PAD4 specific T cells to help anti PAD4 antibody production, maybe through binding of specific peptide(s).

[0147] To identity the peptides from PAD4 which are recognized by the T cells which proliferate to PAD4, the inventors performed proliferation studies with 11 peptides from PAD4 that were predicted to bind SE positive HLA-DRB1 alleles, in 22 patients with RA, 16 patients with PsA and 11 healthy controls. The inventors identified one particular peptide from PAD4, peptide 8 (p8), which was recognized by 9 of 22 RA patients, 3 of 16 patients with PsA and no healthy control. Proliferative response to p8 was associated with RA (p=0.02, Fisher's test), the HLA-DRB1 SE (p=0.05, Fisher's test) and antibodies to PAD4 (p=0.05, Fisher's test). The most straightforward explanation for these associations is that p8 may activate helper T cells. However, p8 is not a SE specific binder, it binds HLA-DRB1*04:01, 04:04, 01:01, 07:01, 04:02. Consistent with our binding data for p8, 12 of 13 patients with proliferative response to p8 express at least one of these 5 alleles. It is conceivable that presentation of p8 by SE positive HLA-DRB1 alleles might be more efficient than by shared epitope negative alleles, thus allowing activation of helper T cells. It is possible as well that p8 is better processed from PAD4 in subjects expressing SE positive than shared epitope negative HLA-DRB1 alleles, as already known of HLA-DRB1*04:01 (23).

[0148] Thus, the data of the inventors suggest a model for the development of ACPA in which T cells specific for PAD4 help, as expected, B cells specific for PAD4, leading to the production of IgG antibodies to PAD4, and at the same time, B cells specific for citrullinated antigens that have internalized and processed the PAD4/citrullinated antigen complex and present PAD4 peptide. These results place PAD4 at the center of the immunological conflict leading to RA. They remind us that the gene encoding PAD4 is associated with RA in Asians and in some European populations (24). Finally, if anti citrullinated protein immunity develops under the influence of helper T cells specific for peptide(s) from one protein, PAD4, as opposed to citrullinated peptide(s) from multiple proteins, it improves the prospects for prevention of RA by PAD4 vaccination in high risk individuals identified by their HLA-DRB1 genotypes.

[0149] In this respect, the identification of peptide 8 (and the others peptides of the invention) from PAD4 will be useful for peptide tolerization.

REFERENCES

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