MODULATION OF ANTIGEN IMMUNOGENICITY BY DELETING EPITOPES RECOGNIZED BY NKT CELLS

Abstract

The invention describes a method and compounds for the prevention of immune responses towards allofactors, towards viral vectors used for gene therapy and gene vaccination, towards proteins to which subjects are naturally exposed, towards genetically-modified organisms and towards undesirable effects related to vaccine administration for allergic or infectious diseases.

Claims

1.-14: (canceled)

15. An engineered peptide derived from a peptide selected from the group consisting of an allofactor, a viral vector, a genetically-modified organism and a vaccine for an allergen, the peptide naturally comprising an epitope comprising hydrophobic amino acid residues in position P1 and/or P7 and the peptide being capable of binding to CD1d molecule and/or activating NKT cells, the engineered peptide having been modified to eliminate said epitope by substituting hydrophobic amino acid residues in position P1 and/or P7 with a non-hydrophobic residue, so that the binding to CD1d molecule is reduced in the engineered peptide as compared with the peptide.

16. The engineered peptide of claim 15, wherein the peptide comprises one or more motif consisting of [FWTHY]-X2X3-[ILMV]-X5X6-[FWTHY] and wherein the peptide has been modified to eliminate the [FWTHY] amino acids at positions P1 and/or P7 of the motif comprised in the peptide by substitution of the [FWTHY] amino acid by an amino acid different from [FWTHY].

17. The engineered peptide of claim 15, further comprising a substitution of the amino acid [ILMV] at position P4.

18. The engineered peptide of claim 15, wherein the engineered peptide is derived from human adenovirus 5.

19. The engineered peptide of claim 18, comprising a variant of one or more or all of the sequences: FIGLMYY, FDSICLY, FKKVAIT, FTRLKTK, WFLVQM, FMSMGAL and FDVVRVH.

20. The engineered peptide of claim 19, wherein the engineered peptide is derived from Mal d 1.

21. The engineered peptide of claim 19, comprising a variant of the sequence FKLIESY.

22. The engineered peptide of claim 15, wherein the engineered peptide is derived from alpha-gliadin.

23. The engineered peptide of claim 22, comprising a variant of the sequence YLQLQPF and/or the sequence FEEIRNL.

24. The engineered peptide of claim 15, wherein the engineered peptide is derived from Derlp.

25. The engineered peptide of claim 24, comprising a variant of one or more or all of the sequences: FSGVAAT, HSAIAVI and YPYVVIL.

26. The engineered peptide of claim 15, wherein the engineered peptide is derived from an IgG.

27. The engineered peptide of claim 26, comprising a variant of one or more or all of the sequences: YRVVSVL, FRVVSVL and HEALHNH.

Description

DETAILED DESCRIPTION OF THE DRAWINGS

[0125] FIG. 1

[0126] NKT cells recognize factor VIII epitopes presented by CD1d

[0127] Hemophilia A mice were immunized with 2 IU Factor VIII on 4 occasions separated by one week. The spleen was then removed and CD4+ T cells were prepared by magnetic bead sorting. A fluorochrome-labeled tetramer of CD1d was obtained from a commercial supplier and loaded with 15 aminoacid long FVIII peptides, which included peptides containing SEQ ID2 and ID3. Loading was carried out at room temperature overnight in the dark. Tetramers were then incubated for 30 minutes at 4° C. with the CD4+ T cell population and the cell suspension was analyzed by Facs.

[0128] The figure shows that CD1d tetramers loaded with peptide of SEQ ID1 (44pept in the figure) are recognized by NKT cells. CD1d ctl(−) shows the % of NKT cells recognizing unloaded tetramers. CD1d ctl(+) shows the % of NKT cells recognizing tetramers loaded with alpha-gal ceramide, which recruits all NKT cells. Up to 45% of NKT cells recognize 44pept, which is compatible with the absence of polymorphism at CD1d level and very limited polymorphism at the level of the NKT T cell receptor.

[0129] FIG. 2

[0130] Immunization of hemophilia A mice with a CD1 d-restricted peptides elicits anti-factor VIII antibodies

[0131] Hemophilia A mice were immunized 3 times subcutaneously with 50 μg of an equimolar mixture of peptides of SEQ IDI (44pept) and peptide of SEQ ID2 (256pept) adsorbed on aluminum hydroxide. A control group received physiological serum instead of peptides. Plasma was taken 10 days after the last immunization and assessed for the presence of anti-Factor VIII antibodies, using a direct binding assay. Briefly, Factor VIII (10 IU/ml) was insolubilized on polystyrene plates, which were washed and incubated with a 1/10 dilution of plasma. After a further washing, a HRP-labeled goat anti-mouse antiserum was added, followed by an enzyme substrate. Colour development was read as OD.

[0132] The figure shows that hemophilia A mice immunized with peptides of SEQ ID1 and of SEQ ID2 develop antibodies to Factor VIII.

[0133] FIG. 3

[0134] Hemophilia A mice reconstituted with bone marrow from CD1d KO mice do not produce antibodies to Factor VIII

[0135] Hemophilia A mice were lethally irradiated and reconstituted with the bone marrow of CD1d KO mice (5×10.sup.6/mouse), which lack NKT cells. Six weeks after bone marrow reconstitution the mice received 4 IV injections of 2 IU/ml separated by one week. Mice were bled 10 days after the last immunization and the plasma was assayed for the presence of anti-Factor VIII antibodies using a direct binding assay as described in the legend of FIG. 2. A control group of irradiated hemophilia A mice was reconstituted with a normal bone marrow.

[0136] The figure indicates that, although control mice produce high concentrations of anti-FVIII antibodies after the fourth injection of Factor VIII (left panel), mice reconstituted with the bone marrow of NKT cell deficient mice did not (right panel).

Example 2: Adenovirus 5 Viral Vectors

[0137] Viral vectors are commonly used for gene therapy and gene vaccination. One of the most common of these viral vectors is derived from adenovirus, serotype 5. Adenoviruses (Ad) are non-enveloped viruses possessing a linear, double-stranded DNA genome of about 35 kb. Human Ad5 has a capsid consisting of 3 major structural proteins: hexon, penton, and fiber. Neutralizing antibodies are raised towards hexon proteins. Such antibodies are very common in humans as a consequence of viral infection. The presence of such antibodies blocks the entry of the viral vector and, consequently, prevents expression of the transgene protein carried by the vector. Anti-Ad5 antibodies are generated in the course of an adaptive response, which depends on activation of CD4+ T cells specific for epitopes presented in the context of MHC class II molecules.

[0138] It is known that Ad5 activates the innate immune system, though the precise mechanism by which it occurs and the location where it takes place remain unclear. Yet, activation of the innate immune system could be a required step for neutralizing antibodies to be formed.

[0139] Using algorithms, we identified 7 aminoacid sequences matching with the general motif [FWTHY]-X.sub.2X.sub.3-[ILMV]-X.sub.5X.sub.6-[FWTHY] of a CD1d binding sequence (SEQ ID7, with motifs underlined) in hexon 6.

[0140] Mice were injected intravenously with 10.sup.9 PFU Ad5 vector on 3 occasions at 10-day intervals. CD4+ T cells were then prepared from the spleen by magnetic bead sorting. CD4+ T cells were incubated with CD1d tetramers loaded with peptides corresponding to each of the 7 sequences identified. It showed that a significant proportion (±10%) of CD4+ NKT cells were labeled by tetramers, indicating that Ad5 vector injections activated NKT cells specific for the peptide of SEQ ID7. In addition, such mice produced specific antibodies of the IgG2a isotype, characteristic of neutralizing antibodies in the mouse.

[0141] A viral vector was prepared which contained a substitution of [FW] by serine S for each of the 7 aminoacid sequences identified. This mutated viral vector (SEQ ID8, with underlined motifs) was used to immunize animals according to the same protocol as described above for the natural sequence. The proportion of NKT cells as assessed using tetramers loaded with the peptide in natural sequence (SEQ ID7) was <1% and the concentration of Ad5 virus specific antibodies was significantly reduced (up to 10-fold).

[0142] It was therefore concluded that substitution of F to S in each P1 location of CD1d binding motifs was sufficient as to reduce NKT cell activation and thereby reduce the production of anti-Ad5 antibodies.

Example 3: Genetically-Modified Proteins

[0143] Proteins to which subjects are exposed by way of inhalation or ingestion are frequently eliciting unwanted reactions in predisposed subjects. Allergic asthma affects millions of people across the world. Food allergy on the other hand has an overall prevalence of ±2.5% in the general population. Allergens either airborne, ingested or penetrating the skin could share properties by which they activate NKT cells.

[0144] One of the most common food allergen is apple (Malus domesticus), and allergenicity is almost exclusively borne by the Mal d 1 protein, a 159 aminoacid long protein, which protects the plant against infectious agents. A sequence motif was identified using computer algorithms, which corresponds to the general motif [FWTHY]-X.sub.2X.sub.3-[ILMV]-X.sub.5X.sub.6-[FWTHY] of a CD1d binding sequence.

[0145] FKLIESY corresponding to aminoacids 144-150 of Mal d 1 (SEQ ID9)

[0146] A recombinant form of Mal d 1, in which F144 and Y150 were mutated in S was produced by genetic engineering. The recombinant form of Mal d 1 therefore encompasses peptide of sequence:

[0147] SKLIESS (SEQ ID10)

[0148] Synthetic peptides corresponding to SEQ ID9 and SEQ ID10 were produced. Their capacity to activate NKT cells was determined in vitro using human dendritic cells derived from peripheral blood monocytes of an individual sensitized to Mal d 1. Dendritic cells loaded with each one of the two peptides were incubated in the presence of NKT cells obtained from the same individual by sorting peripheral lymphocytes using specific markers such as CD4 and NKG2D. It was observed that NKT cells incubated with peptide of SEQ ID9 activated a significant proportion of NKT cells, while the mutated peptide of SEQ ID10 did not. Additionally, human CD1d tetramers loaded with peptides of SEQ ID9 were recognized by a significant proportion of NKT cells, but tetramers loaded with the mutated peptide of SEQ ID were recognized by less than 1% of NKT cells.

[0149] The two F144S and Y1505 mutations are introduced directly in clonal cells by site-directed mutagenesis. The full organism is then produced by conventional growth strategies. Apples produced by this GMO do not elicit allergic reactions.

[0150] One specific application of the peptides or polypeptides of the present invention is celiac disease (gluten intolerance). This disease is among the most commons in human beings and is related to T cell activation to gliadin epitopes which are presented in the context of MHC class II determinants. A genetic susceptibility has been described, with human beings carrying the HLA-DQ2 or DQ8 class II determinant being predisposed to disease. These class II determinants present peptides which have been submitted to deamidation by transglutaminase. However, these events are the results of intestinal inflammatory reaction, likely related to the innate immune system.

[0151] Gliadins are monomers of 250-300 aminoacid residues. A search for the general motif [FW]-XX-[ILM]-XX-[FWTHY] of a CD1d binding sequence using computer algorithms identified such sequence (SEQ ID11, see listing of sequences) in alpha-gliadin. A mutated form of alpha-gliadin was then produced in which the F residue of the motif was substituted by a S residue (SEQ ID12, see addendum).

[0152] The same procedure as for Mal d 1 was followed to show that, although polypeptide of SEQ ID11 activated a significant proportion of NKT cells when presented by antigen-presenting dendritic cells, the mutated form of the polypeptide (SEQ ID12) failed to do so. As for Mal d 1, human CD1d tetramers loaded with a synthetic peptide representing the motif identified in the polypeptide of

[0153] SEQ ID11 were recognized by NKT cells, while tetramers loaded with the mutated form of the motif as shown in SEQ ID12 were not.

[0154] The mutation was introduced directly in clonal cells by site-directed mutagenesis. The full organism was then produced by conventional growth strategies. Cereals containing the mutated form of gliadin do not elicit reactions of intolerance.

[0155] It should be obvious for those skilled in the art that the present invention can also be applied to proteins which are added to, for instance, genetically-modified organisms to increase their resistance to insecticides, pesticides or any other modifications judged to be beneficial. Such modifications carry the risk of creating new CD1d binding motifs.

[0156] Additional examples of genetically-modified proteins with reduced allergenicity/immunogenicity are: [0157] food allergens such as soybean, peanut and fruits of the Rosaceous family [0158] milk proteins [0159] airborne allergens such as latex (Hevea brasiliensis), pollens of grasses such as Rye grass (Lolium perenne), Timothy (Phleum pratense) or Kentucky blue grass (Poa pratensis) [0160] fish parvalbumin [0161] honey bee phospholipase A2

[0162] It should also be clear for the one skilled in the art that the invention extends to methods by which peptides or polypeptides of the invention are produced, including the production of transgenic plants and animals.

Example 4: Allergen Der p 1

[0163] Der p 1 is a cysteine protease which is the main allergen of the so-called house dust mite (HDM), D. pteronyssinus. Sensitization to HDM is by far the commonest trigger of allergic asthma and rhinitis worldwide. Der p 1 contains 3 motifs matching the general CD1d binding motif [FWTHY]-X.sub.2X.sub.3-[ILMV]-X.sub.5X.sub.6-[FWTHY], as identified using computer algorithms and which are:

[0164] SEQ ID13: FSGVAAT aminoacids 38-44 of Der p 1

[0165] SEQ ID14: HSAIAAVI aminoacids 135-141 of Der p 1

[0166] SEQ ID15: YPYVVIL aminoacids 216-222 of Der p 1

[0167] Peptides of SEQ ID13, SEQ ID14 and SEQ ID15 were synthesized and used to load CD1d tetramers.

[0168] BALB/c mice were submitted to intranasal administration of Der p 1, using 50 μl of saline containing 100 μs of Der p 1. This challenge procedure was repeated twice on three consecutive days at one-week interval. The mice were sacrificed 5 days after the last nasal instillation and the spleen was removed. CD4+ T cells were purified by magnetic bead sorting and incubated in the presence of the CD1d tetramers loaded with peptides of SEQ ID13, SEQ ID or SEQ ID15. By fluorescence-activated cell sorter (facs) determination, it was observed that a significant percentage of cells (±10%) were stained with the tetramers, identifying them as CD4+ NKT cells. It was therefore concluded that peptides of SEQ ID13, SEQ ID14 and SEQ ID15 were functional in binding to CD1d and in being recognized by NKT cells.

[0169] CD4+ T cells obtained from the above experiments were incubated in culture medium in the presence of an antigen-presenting cell which expresses the CD1d molecule. Such cells are commercially available, as for instance the JAWS2 cells, which do not express MHC class II determinants. JAWS2 cells were loaded with Der p 1 and presentation of Der p 1-derived epitopes by CD1d was evaluated by measuring the production of cytokines such as IFN-gamma and IL-4 as markers of NKT activation. It could be observed that a significant production of cytokines was present, confirming that Der p 1 contained epitopes presented by CD1d molecules.

[0170] Next, a mutated form of Der p 1 was prepared by genetic engineering, in which the 3 aminoacid residues predicted to be in position P1 for CD1d binding of peptides of SEQ ID13, SEQ ID14 and SEQ ID15 were substituted by serine. The mutated Der p 1 (SEQ ID16) was used for nasal instillation as described above with Der p 1 in natural sequence (SEQ ID17). In such a case, no significant binding of CD4+ T cell splenocytes was observed when incubated with the tetramers loaded with peptide of SEQ ID13, peptide of SEQ ID14 or peptide of SEQ ID15, indicating that the mutated Der p 1 had lost its capacity to activate NKT cells specific for these peptides.

[0171] Further, mutated Der p 1 (SEQ ID16) was used to load JAWS2 cells and tested for its capacity to activate NKT cells. For this experiment, NKT cells were used as obtained from mice immunized with Der p 1 in either natural or mutated configuration. The production of IFN-gamma and IL-4 was taken as an indication of NKT activation. It was observed that NKT cells obtained from mice immunized with natural sequence Der p 1 failed to be activated when incubated in the presence of JAWS2 cells loaded with mutated Der p 1.

[0172] It was therefore concluded that Der p 1 in natural sequence contained functional CD1d restricted T cell epitopes activating NKT cells. Further, elimination of such functional CD1d-restricted epitopes by mutation was sufficient to eliminate NKT cell activation.

Example 5: Antibodies

[0173] Antibodies are used as therapeutic agents in a large number of indications, from chronic inflammatory diseases such as rheumatoid arthritis (e.g., anti-TNF-alpha antibodies) or allergic asthma (e.g. anti-IgE antibodies), to tumors (e.g., anti-CD20 antibodies). More than 120 therapeutic antibodies are presently used for clinical applications at various stages from preclinical to phase III trials and accepted for routine clinical practice.

[0174] Therapeutic antibodies are either chimeric or fully humanized, which contains sequence of foreign origin only in the complementarity determining regions of the variable parts. A minority of such antibodies are derived from the human repertoire and, as such, considered as poorly immunogenic. However, antibodies towards the therapeutic antibody, even when directly derived from the human repertoire, are produced by a majority of the patients under treatment, with, in a significant proportion of the cases, the production of antibodies neutralizing the activity of the therapeutic agent.

[0175] A search for epitopes matching the CD1d binding motif in human IgG antibody sequence was carried out using computer algorithms. One of such motif was identified in the CH2 region (second domain of the heavy chain constant part) of each of the 4 IgG subclass (IgG1, IgG2, IgG3 and IgG4) and a second motif was identified in the CH3 loop of IgG1, IgG2 and IgG4:

[0176] SEQ ID18: YRVVSVL (CH2 of IgG1 and IgG4)

[0177] SEQ ID19: FRVVSVL (CH2 of IgG2 and IgG3)

[0178] SEQ ID20: HEALHNH (CH3 loop of IgG1, IgG2 and IgG4)

[0179] Synthetic peptides corresponding to SEQ ID18, SEQ ID19 and SEQ ID20 were produced and used to load human CD1d tetramers as for the examples above (see for instance example 4 for allergen Der p 1). Peripheral blood cells were obtained by venous puncture of patients who had received an injection of a therapeutic antibody during the previous 5 days. CD4+ T cells were purified by magnetic bead sorting. The cells were then incubated with tetramers loaded with peptides of SEQ ID18, SEQ ID19 or SEQ ID20. Analysis by facs identifies a significant proportion of NKT cells (±10%) labeled by tetramers.

[0180] Monoclonal human antibodies of the IgG4 isotype were derived from the peripheral blood B lymphocytes by transformation with the Epstein-Barr virus. The genomic sequence of such antibodies was obtained from transformed B cells. A viral vector containing the corresponding cDNA sequence was constructed and used for transfection of CHO cells. All these methods are known in the art (see for instance, Jacquemin et al Blood 92: 496-506, 1998).

[0181] The hydrophobic aminoacid residues located in position 1 in the peptides of SEQ ID18 and SEQ ID20 were mutated to a serine and the mutated antibody produced by transfected CHO cells.

[0182] Peripheral blood CD4+ T cells obtained as above were exposed in culture medium to human dendritic cells (derived from human peripheral blood monocytes by methods known in the art) and loaded with either the antibody in natural configuration (SEQ ID21) or its mutated counterpart (SEQ ID22). After culturing the cells with CD4+ T cells for 5 to 7 days, the population of CD4+ T cells activated by either natural or mutated antibody was evaluated. CD4+ NKT cells were separated from CD4+ T cells using an antibody to NKG2D, a surface marker associated with NK or NKT cells only.

[0183] It was observed that CD4+ T cells and NKT cells were activated when the antibody in natural sequence was used (SEQ ID21), while the mutated form of the antibody (SEQ ID22) only activate class II restricted CD4+ T cells and not NKT cells.

[0184] It was concluded that human IgG antibodies contained epitopes corresponding to the [FWTHY]-X.sub.2X.sub.3-[ILMV]-X.sub.5X.sub.6-[FWTHY] motif, having the capacity to be recognized by and to activate NKT cells. Further, mutation of key hydrophobic aminoacid residues within such motif was sufficient to prevent activation of NKT cells.

[0185] It should be understood that the examples provided here are not exhaustive and that combinations of proteins or peptides containing various numbers of aminoacid substitutions or deletions can be envisioned. For instance, in example 1, various combinations of substitution of hydrophobic aminoacids can be delineated.

TABLE-US-00001 Sequence listings Factor VIII aminoacids 309-315 (human) SEQ ID1 FCHISSH Factor VIII aminoacids 1816-1822 (human) SEQ ID2 FWKVQHH Factor VIII aminoacids 1918-1924 (human) SEQ ID3 FHAINGY Factor A1 domain (mutations F309S and H315S underlined) (human) SEQ ID4    1 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPF   41 NTSVVYKKTLFVEFTVHLFNIAKPRPPWMGLLGPTIQAEV   81 YDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQ  121 REKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLS  161 HVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFA  201 VFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNR  241 SLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRN  281 HRQASLEISPITFLTAQTLLMDLGQFLLSCHISSSQHDGM  321 EAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRF  361 DDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVL  401 APDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTRE  441 AIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGI  481 TDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDG  521 PTKSDPRCLTRYYSSFVNMERDLASG Factor VIII A3 domain (mutations F1816S and H18225 underlined) (human) SEQ ID5 1637 SQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKED 1677 FDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPH 1717 VLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHL 1757 GLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQR 1798 QGAEPRKNFVKPNETKTYSWKVQHSMAPTKDEFDCKAW 1836 AYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQE 1876 FALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKEN 1916 YRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHS 1956 IHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGI 1996 WRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDF 2036 QITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDL 2076 LAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTY 2116 RGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTH 2156 YSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSY 2196 FTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQ 2236 KTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFF 2276 QNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVH 2316 QIALRMEVLGCEAQDLY* Factor VIII (mutations F309S, H315S, F1816S and F1918S underlined) (human) SEQ ID6 ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTVHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHA VGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASD PLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFA VFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL MDLGQFLLSCHISSSQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDL TDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVL APDDRSYKSQYLNNGPQRIGRKYKKVREMAYTDETEKTREAIQUESGILG PLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKD FPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSEVNMERDLASGLIGP LLICYKESVDQRGNQIMSDKRNVILFSVEDENRSWYLTENIQRFLPNPAG VQLEDPEFQASNIMHSINGYVEDSLQLSVCLHEVAYWYILSIGAQTDFLS VFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNR GMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPS TRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTP HGLSLSDLQEAKYETESDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFT PESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDN TSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLES GLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKT NKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRM LMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKML FLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKV VVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEK KETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYEGAYAPVLQD FRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPN TSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPS TLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIR PIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTL EMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHI YQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVA TESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILS LNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREI TRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFI AAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRG ELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGA EPRKNFVKPNETKTYSWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSG LIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCR APCNIQMEDPTFKENYRSHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSN ENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVEC LIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKL ARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQ FIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIR LHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMF ATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKS LLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPP LLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY*GWPLQHLPLPSPLPPQL QGSVPPWLAFYLCAKS*QTLP*SLL Hexon, Human adenovirus 5, (CD1d binding motifs underlined): (virus) SEQ ID7 MATPSMMPQWSYMHISGQDASEYLSPGLVQFARATETYFSLNNKFRNPTVAPTHDV TTDRSQRLTLRFIPVDREDTA YSYKARFTLAVGDNRVLDMASTSFDIRGVLDRGPTFKPYSGTAYNALAPKGAPNPCE WDEAATALEINLEEEDDDNE DEVDEQAEQQKTHVFGQAPYSGINITKEGIQIGVEGQTPKYADKTFQPEPQIGESQWY ETEINHAAGRVLKKTTPMK PCYGSYAKPTNENGGQGILVKQQNGKLESQVEMQFFSTTEAAAGNGDNLTPKVVLY SEDVDIETPDTHISYMPTIKE GNSRELMGQQSMPNRPNYIAFRDNFIGLMYYNSTGNMGVLAGQASQLNAVVDLQD RNTELSYQLLLDSIGDRTRYFS MWKQAVDSYDPDVRIIENHGTEDELPNYCFPLGGVINTETLTKVKPKTGQENGWEK DATEFSDKNEIRVGNNFAMEI NLNANLWRNFLYSNIALYLPDKLKYSPSNVKISDNPNTYDYMNKRVVAPGLVDCYI NLGARWSLDYMDNVNPFNHHR NAGLRYRSMLLGNGRYVPFHIQVPQKFFAIKNLLLLPGSYTYEWNFRKDVNMVLQSS LGNDLRVDGASIKFDSICLY ATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNWA AFRGWAFTRLKTKETPSLGS GYDPYYTYSGSIPYLDGTFYLNHTFKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDG EGYNVAQCNMTKDWFLVQM LANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDYQQVGILHQHNNS GFVGYLAPTMREGQAYPANFP YPLIGKTAVDSITQKKELCDRTLWRIPFSSNFMSMGALTDLGQNLLYANSAHALDMT FEVDPMDEPTLLYVLFEVFD VVRVHRPHRGVIETVYLRTPFSAGNATT Hexon, Human adenovirus 5 (mutations of P1 anchoring residue underlined): (virus) SEQ ID8 MATPSMNIPQWSYMHISGQDASEYLSPGLVQFARATETSFSLNNKFRNPTVAPTHDV TTDRSQRLTLRFIPVDREDTA YSYKARFTLAVGDNRVLDMASTSFDIRGVLDRGPTEKPYSGTASNALAPKGAPNPCE WDEAATALEINLEEEDDDNE DEVDEQAEQQKTHVFGQAPSSGINITKEGIQIGVEGQTPKYADKTFQPEPQIGESQWY ETEINHAAGRVLKKTTPMK PCYGSYAKPTNENGGQGILVKQQNGKLESQVEMQFFSTTEAAAGNGDNLTPKVVLY SEDVDIETPDTHISYMPTIKE GNSRELMGQQSMPNRPNYIAFRDNSIGLMYYNSTGNMGVLAGQASQLNAVVDLQD RNTELSYQLLLDSIGDRTRYFS MWKQAVDSYDPDVRIIENHGTEDELPNYCFPLGGVINTETLTKVKPKTGQENGWEK DATEFSDKNEIRVGNNFAMEI NLNANLWRNFLSSNIALYLPDKLKYSPSNVKISDNPNTYDYMNKRVVAPGLVDCYIN LGARWSLDYMDNVNPFNHHR NAGLRSRSMLLGNGRYVPFSIQVPQKSFAIKNLLLLPGSYTYEWNFRKDVNMVLQSS LGNDLRVDGASIKSDSICLY ATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNVPISIPSRNWA AFRGWASTRLKTKETPSLGS GYDPYYTYSGSIPYLDGTFYLNHTSKKVAITFDSSVSWPGNDRLLTPNEFEIKRSVDG EGYNVAQCNMTKDSFLVQM LANYNIGYQGFYIPESYKDRMYSFFRNFQPMSRQVVDDTKYKDSQQVGILHQHNNS GFVGYLAPTMREGQAYPANFP SPLIGKTAVDSITQKKFLCDRTLWRIPFSSNSMSMGALTDLGQNLLYANSAHALDMT FEVDPMDEPTLLYVLFEVSD VVRVHRPSRGVIETVYLRTPFSAGNATT Mal d 1, malus domesticus, aminoacids 144-150 SEQ ID9 FKLIESY Mal d 1, malus domesticus, F144S and Y150S mutations underlined (vegetal) SEQ ID10 SKLIESS Alpha-Gliadin (CD1d binding motif underlined) SEQ ID11 MVRVPVPQLQPQNPSQQQPQEQVPLVQQQQFPGQQQPFPPQQPYPQPQPFPSQQPYL QLQPFPQPQLPYPQPQLPY PQPQLPYPQPQPFRPQQPYPQSQPQYSQPQQPISQQQQQQQQQQQQKQQQQQQQQIL QQILQQQLIPCRDVVLQQH SIAYGSSQVLQQSTYQLVQQLCCQQLWQIPEQSRCQAIHNVVHAIILHQQQQQQQQQ QQQPLSQVSFQQPQQQYPS GQGSFQPSQQNPQAQGSVQPQQLPQFEEIRNLALETLPAMCNVYIPPYCTIAPVGIFGT NYR Alpha-Gliadin (mutation underlined) SEQ ID12 MVRVPVPQLQPQNPSQQQPQEQVPLVQQQQFPGQQQPFPPQQPYPQPQPFPSQQPSL QLQPFPQPQLPYPQPQLPY PQPQLPYPQPQPFRPQQPYPQSQPQYSQPQQPISQQQQQQQQQQQQKQQQQQQQQIL QQILQQQLIPCRDVVLQQH SIAYGSSQVLQQSTSQLVQQLCCQQLWQIPEQSRCQAISNVVHAIILHQQQQQQQQQ QQQPLSQVSFQQPQQQYPS GQGSFQPSQQNPQAQGSVQPQQLPQSEEIRNLALETLPAMCNVYIPPSCTIAPVGIFGT NYR D. pteronyssinus Der p 1, aminoacids 38-44 (pyroglyphidae, Dermatophagoides pteronyssinus, European house dust mite) SEQ ID13 FSGVAAT D. pteronyssinus Der p 1, aminoacids 135-141 (pyroglyphidae, Dermatophagoides pteronyssinus, European house dust mite) SEQ ID14 HSAIAAVI D. pteronyssinus Der p 1, aminoacids 216-222 (pyroglyphidae, Dermatophagoides pteronyssinus, European house dust mite) SEQ ID15 YPYVVIL Mature Der p 1 (mutations of P1 anchoring residues F38S, H135S and Y216S underlined) (pyroglyphidae, Dermatophagoides pteronyssinus, European house dust mite) SEQ ID16 ETNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWASSGVAATESAYLAYRNQSLD LAEQELVDCASQHGCHGDTI PRGIEYIQHNGVVQESYYRYVAREQSCRRPNAQRFGISNYCQTYPPNVNKIREALAQT SSAIAVIIGIKDLDAFRHY DGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAANI DLMMIEESPYVVIL Mature Der p 1 (CD1d epitopes underlined): (pyroglyphidae, Dermatophagoides pteronyssinus, European house dust mite) SEQ ID17 ETNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLD LAEQELVDCASQHGCHGDTI PRGIEYIQHNGVVQESYYRYVAREQSCRRPNAQRFGISNYCQTYPPNVNKIREALAQT HSAIAVIIGIKDLDAFRHY DGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAANI DLMMIEEYPYVVIL IgG antibody, CH2 domain of IgG1 and IgG4 (human) SEQ ID18 YRVVSVL IgG antibody, CH2 domain of IgG2 and IgG3 (human) SEQ ID19 FRVVSVL IgG antibody, CH3 domain of IgGl, IgG2 and IgG4 (human) SEQ ID20 HEALHNH Human IgG4 FC fragment (CD1d epitopes underlined) (human) SEQ ID21 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSL GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSLGK Human IgG4 FC fragment (mutated aminoacids underlined) (human) SEQ ID22 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSL GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTSRVVSVLTVLHQDWLNGKEYKCKVSNKG LPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFS CSVMSEALHNHYTQKSLSLSLGK