WO2015084986A1 - Methods of evaluating immunodominant epitopes - Google Patents

Abstract

Provided herein are methods for determining whether a prospective epitope is an immunodominant epitope. Also provided are methods useful for designing immunodominant epitopes or epitopes that are less immunogenic.

Description

METHODS OF E VAL UA TING IMMUNODOMINANT EPITOPES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/911,215, filed on December 3, 2013, the disclosure of which is incorporated herein by reference in its entirety.

GOVERNMENT INTEREST

This invention was made with Government support under National Institutes of Health Grants AI063764 and GM053549. The Government has certain rights in the invention.

BACKGROUND

Immunodominance is a well-established phenomenon whereby a few specific peptides are selected as representative epitopes of a given protein antigen to the immune system. A restricted response to a given antigen might be necessary for keeping the sheer number of memory T cells raised against the antigen within numbers that can be accommodated by the lymph nodes. Over the last decades, there have been many studies aimed toward understanding the mechanisms of epitope selection and immunodominance. A wide range of hypotheses related to the properties of the specific T cells, the processing and presentation of antigens, or both, have been proposed and argued. Among these are intrinsic structural features of protein antigens, sensitivity to proteases, epitope affinity for MHC class II, T cell precursor frequency, and T cell receptor affinity for peptide/MHC II. These studies suggest that the mechanisms evolved for the selection of the dominant epitopes could be rather complex. Part of the difficulty is due to the multiplicity of steps involved in antigen processing and the participation of many chaperones and accessory proteins. In order to process protein antigens for presentation to CD4⁺ T cells, antigens are taken up by the cells from exogenous sources and move through a series of endosomal compartments. These compartments contain suitable denaturing environment, accessory chaperones, and cathepsins that process protein antigens and allow binding of some peptide fragments to the groove of MHC class II molecules. Newly synthesized MHC class II molecules associate with the invariant chain (Ii), which protects the MHC groove from binding to peptides present in the ER. The invariant chain is sequentially proteolysed until only a fragment, known as the class II-associated invariant chain peptide (CLIP) remains bound in the MHC II peptide-binding site. An important function of CLIP is to maintain the groove in a peptide-receptive conformation. Different cathepsins, each having their own signature pattern for protein cleavage, and the proper microenvironment to support their function, are provided in the MIIC. Efficient displacement of CLIP from the MHC groove requires the accessory molecule HLA-DM (DM).

The mechanism of DM interaction with MHC class II, which has been studied in great detail, indicates that DM crucially influences the selection of peptides that bind to MHC molecules. DM operates by generating a peptide-receptive MHC class II, which it accomplishes by exerting conformational changes in class Il/peptide complexes through preventing the formation of H-bonds between MHC II and the peptide main chain. A peptide-receptive MHC II can quickly sample a large pool of peptides derived from exogenously acquired proteins, and DM helps in shaping epitope selection. The molecular details of the interaction between DM and MHC class II hints at a critical role for DM in selection of immunodominant epitopes. However, this role has not been fully evaluated. Importantly, there is no consensus within the literature on whether epitope selection for immunodominance takes place during the binding of a whole protein antigen to MHC II (or its large fragments), or after smaller peptides are already generated by lysosomal proteases. It is not clear at what stage during this process DM exerts its function.

Moreover, there is no report on the direct roles that cathepsins might play during the selection of the immunodominant epitopes. Multiple studies using protease inhibitors and protease-deficient mice revealed that several enzymes, including (S, L, F) might contribute to antigen processing and removal of CLIP. Asparaginyl endopeptidase (AEP) present in the late endosomal compartment is necessary for cleavage and activation of cathepsins, although a role for generation, as well as the destruction of antigenic epitopes by AEP have been reported. Cathepsin S is a key player in invariant chain processing in DCs and B cells.

In light of the uncertainties surrounding antigen processing and presentation, the identification of immunodominant epitopes for autoimmune diseases remains a challenge. The development of autoimmune therapeutics would be improved if it were possible to determine whether a prospective immunodominant epitope was a true immunodominant autoimmune epitope. Thus, there is a great need for methods of determining whether a prospective immunodominant autoimmune epitopes is an authentic autoimmune epitope. SUMMARY

In one aspect, provided herein is a method of determining whether a prospective autoimmune epitope is an authentic autoimmune epitope. In some embodiments, the method includes the steps of incubating the prospective epitope with in a reaction solution that includes one or more cathepsins. In some embodiments, the reaction solution includes cathepsin B, cathepsin H and/or cathepsin S. In some embodiments, the reaction solution does not include HLA-DR1 and/or HLA-DM. In some embodiments, the method includes the step of assessing the digestion of the prospective autoimmune epitope by the reaction solution. In some embodiments, the digestion of the prospective autoimmune epitope is assessed by mass spectrometry. In some embodiments, the prospective autoimmune epitope is an authentic autoimmune epitope if it is resistant to digestion by the reaction solution.

In another aspect, the disclosure features a method for identifying an

immunodominant epitope of interest. The method comprises: incubating a prospective epitope within a reaction solution comprising cathepsin B, cathepsin H and cathepsin S; and assessing the digestion of the prospective epitope by the reaction solution, wherein the prospective epitope is identified as an immunodominant epitope if it is resistant to digestion by the reaction solution.

In some embodiments, any of the methods described herein can further comprise administering the prospective epitope or the identified immunodominant epitope to a mammal. In some embodiments, any of the methods described herein can further comprise detecting the presence or absence of an immune response to the prospective epitope or the identified immunodominant epitope. The immune response can be, e.g., a cellular immune response or a humoral immune response.

In another aspect, the disclosure features a method for generating an

immunodominant epitope. The method comprises incubating a prospective epitope within a reaction solution comprising one or more of cathepsin B, cathepsin H, and cathepsin S; assessing the digestion of the prospective epitope by the reaction solution; and if the prospective epitope is not resistant to digestion by the reaction solution, designing a variant version of the prospective epitope that is resistant to digestion, or is more resistant to digestion than the prospective epitope, by the reaction solution, to thereby generate an immunodominant epitope. In another aspect, the disclosure features a method for generating an

immunodominant epitope, which method comprises designing a variant version of a prospective epitope, wherein the prospective epitope is sensitive to digestion by a reaction solution comprising one or more of cathepsin B, cathepsin H, and cathepsin S, and wherein the variant version of the prospective epitope is resistant to digestion, or is more resistant to digestion than the prospective epitope, by the reaction solution, to thereby generate an immunodominant epitope.

In some embodiments of any of the methods described herein, the designing comprises engineering one or more mutations into a nucleotide sequence encoding the prospective epitope. In some embodiments, the one or more mutations remove from the prospective epitope at least one cleavage site of cathepsin B, cathepsin H, or cathepsin S.

In some embodiments, any of the methods described herein can comprise identifying in a prospective epitope a cleavage site for cathepsin B, cathepsin H, or cathepsin S.

In some embodiments of any of the methods described herein, a prospective epitope contains a cleavage site for cathepsin B, cathepsin H, or cathepsin S. In some embodiments of any of the methods described herein, a prospective epitope contains a cleavage site for cathepsin B, cathepsin H, and cathepsin S. In some embodiments of any of the methods described herein, a prospective epitope contains a cleavage site for at least two of cathepsin B, cathepsin H, and cathepsin S.

In some embodiments, the prospective epitope is from a microbial polypeptide. For example, the microbial polypeptide can be a viral polypeptide, a bacterial polypeptide, or a protozoan polypeptide.

In yet another aspect, the disclosure features a method for reducing the

immunogenicity of an epitope. The method comprises: incubating a prospective epitope within a reaction solution comprising one or more of cathepsin B, cathepsin H, and cathepsin S; assessing the digestion of the prospective epitope by the reaction solution; and if the prospective epitope is resistant to digestion by the reaction solution, designing a variant version of the prospective epitope that is sensitive to digestion by the reaction solution, to thereby reduce the immunogenicity of the prospective epitope.

In another aspect, the disclosure features a method for reducing the immunogenicity of an epitope, the method comprises: designing a variant version of a prospective epitope, wherein the prospective epitope is resistant to digestion by a reaction solution comprising one or more of cathepsin B, cathepsin H, and cathepsin S, and wherein the variant version of the prospective epitope is sensitive to digestion by the reaction solution, to thereby reduce the immunogenicity of the prospective epitope.

In some embodiments of any of the methods described herein, the designing comprises engineering one or more mutations into a nucleotide sequence encoding the prospective epitope. The one or more mutations can, e.g., introduce into the prospective epitope at least one cleavage site for cathepsin B, cathepsin H, or cathepsin S.

In some embodiments of any of the methods described herein, the prospective epitope is part of a therapeutic polypeptide, e.g., an antibody, such as a humanized or fully human antibody.

In some embodiments of any of the methods described herein, the designing is performed in silico.

In some embodiments, any of the methods described herein further comprise incubating the variant version of the prospective epitope within a reaction solution comprising one or more of cathepsin B, cathepsin H, and cathepsin S; and assessing the digestion of the variant version of the prospective epitope by the reaction solution.

In some embodiments, any of the methods described herein further comprise incubating the prospective epitope within a reaction solution comprising one or more of cathepsin B, cathepsin H, and cathepsin S; and assessing the digestion of the prospective epitope by the reaction solution.

In some embodiments of any of the methods described herein, the reaction solution comprises cathepsin B, cathepsin H, and cathepsin S.

In some embodiments of any of the methods described herein, the reaction solution comprises one or more of a cathepsin B activity, a cathepsin H activity, and a cathepsin S activity. For example, it is understood that the reaction mixtures can contain wild-type, active cathepsin enzymes or variant (e.g., amino acid sequence variants, e.g., at least 80, 85, 90, or 95% sequence identity) cathepsin enzymes possessing the relevant activity.

In some embodiments of any of the methods described herein, the reaction solution does not comprise HLA-DR1 and/or HLA-DM. In some embodiments of any of the methods described herein, the digestion of the prospective epitope is assessed by mass spectrometry. "Polypeptide," "peptide," and "protein" are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the presently disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Other features and advantages of the present disclosure, e.g., methods for identifying an immunodominant epitope, will be apparent from the following description, the examples, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows HLA-DM and cathepsins together select the immunodominant epitope of type II collagen. (A-E). DRl was incubated with the following components: with or without MMP9-fragmented bCII (MMP9-bCII), with or without DM, and with or without Cathepsins B and H. Mass spectra of peptides eluted from DRl under these conditions. A-D shows the reactions containing MMP9-fragmented bCII. E shows the negative control reactions carried out without MMP9-bCII. DRl used in all experiments shown here was pre-incubated with HA_Y308A (2-3 days at 37°C) prior to use except in sample D. The 1258.4 Da peak seen in E is a background peak that was present in some preparations of DRl but not others. The peaks in the shaded area represent post- translational modification variants of a dominant peptide composed of residues 273-305 of bCII (bCII₂7₃_₃05). Mass species in red represent CH-derived peptides containing the immunodominant CII₂g₂-₂89 epitope. Non-dominant peptides are shown in green.

Background peptide species are labeled in grey. (F) Proliferation of T cells isolated from DRl -transgenic mice immunized with native CII protein in CFA in response to stimulation with, CII₂8o-₂94, CII954968, or CLIP₈₉ 105 in vitro. Cellular proliferation was measured by [³H] thymidine incorporation (Left panel). IL-2 ELISA was performed from supernatant collected from in vitro culture of three individual mice immunized with CII protein/CFA. Cell culture supernatants were removed after 48 h culture. (Right panel).

(G-H). Dissociation assay of fluorescently labeled non-dominant epitope, CII954-968 and immunodominant epitope CII280-294 from DRl . Fluorescently labeled CII954-968//DRI complexes (G), or fluorescently labeled CII280-294/DRI complexes (H) were dissociated in the presence of 100 times molar excess of unlabeled HA₃₀6-3i8 at 37° C for the indicated times in the absence (square), or presence of DM (circle). The fluorescence of the labeled complex before dissociation is arbitrarily assigned a value of 1.0, and fluorescence after dissociation is expressed as a fraction of fluorescence before dissociation. Figure 2 shows type II collagen-derived immunodominant peptides are resistant to cathepsin digestion. (A-E) Mass spectra of peptides eluted from DRl are shown between m/z 1400-3900 Da. MMP9-bCII was incubated with DRl and DM for 3hrs (A). MMP9- bCII was first exposed to cathepsins B and H for 3h (B), lh (C), 15min (D), and then incubated with DRl and DM for additional 3hrs. Mass spectrum of peptides eluted from DRl (E), when all components of the cell-free assay were mixed simultaneously and incubated together for 3 hrs. As in Figure 1, the peaks in the shaded area represent post- translational modification variants of a dominant peptide composed of residues 273-305 of bCII (bCII₂73_305). (F-G) Mass spectra of synthetic peptides, CII280-294 (F) and CIl95₄_9₆8 (G), that were directly exposed to the cathepsins, B, H, and S for lh at 37°C. Top spectrum shows synthetic peptides alone as a control. Middle spectrum shows the samples incubated with the cathepsins. Peptide at m/z 1338.8 Da is the background peak. Bottom is the background samples containing only cathepsins.

Figure 3 shows degradation of HA₃₀6-3i8 by the cathepsins outcompetes its capture by HLA-DRl . (A-D) The mass spectrum of peptides eluted from DRl (enlarged spectrum between m/z 1950-2550 Da), when rHAl is first exposed to cathepsins B and H for 3h (A), lh (B), 15min (C), and then incubated with DRl and DM for 2 hrs at 37°C. Mass spectrum of peptides eluted from DRl, when all components of the cell-free system are mixed simultaneously and incubated together for 3 hrs (D). (E) Mass spectra of synthetic HA₃₀6-3i8 peptide exposed to the cathepsins. Sensitivity of synthetic HA₃₀6-3i8 to the cathepsins tested by directly exposing it to the cathepsins B, H, and S. Top spectrum shows HA₃₀6-3i8 peptide alone and middle spectrum is the sample reaction containing HA306-318 incubated with the cathepsins for lh at 37°C. Background sample contains the cathepsin mix without the synthetic peptide shown in the bottom spectrum.

Figure 4 shows immunodominant epitope of H5Nl-rHAl is sensitive to the cathepsins. (A-C) The mass spectrum of peptides eluted from DRl (enlarged spectrum between m/z 1100-2900 Da), when denatured H5Nl-rHAl (A/Vietnam/ 1203/2004 H5N1 strain, Genebank No. AY651334) was first incubated with DRl and DM and then exposed to cathepsins B, H, and S (A), or when denatured H5Nl-rHAl is first exposed to the cathepsins for 3hrs (B) and then incubated with DRl and DM. The background spectrum is shown in C. Mass species that are boxed represent H5Nl-rHAl fragments containing the DRl restricted immunodominant HA₂59_274 epitope. (D-E) Similar to the previous experiment done in Figure 3E, the mass spectrum of synthetic HA₂₅9-₂74 alone (top), HA259- 274 digested with the cathepsins (middle), and cathepsins alone as a background control (bottom) (D). The mass spectrum of peptide eluted from DRl, when HA₂₅9-₂74 peptide alone, HA₂59_₂74 digested with the cathepsins, or the cathepsins alone and then incubated with receptive DRl for additional 3 hrs (E). Baculovirus-derived peptide CLi₃_₂3 are present in every spectra of samples containing DRl .

Figure 5 shows intact protein antigens form complexes with HLA-DR.

(A-B) SDS-stable complexes formation with intact proteins and DRl . Various

combinations of DRl, DM, rHAl, and synthetic HA306-318 peptide were incubated in citrate- phosphate buffer pH 5 for 2h (A) or overnight (B) at 37°C. Different combinations of proteins and peptides (shown in different lanes) were analyzed by "gentle" SDS-PAGE in which samples are not boiled prior to loading and Silver-stained (A) and western blotted (B) either with anti-His antibody or polyclonal anti-DRl serum (CHAMP2). The burned films associated with lanes 6, 13, 15-17 occurred because of the extremely high signal intensities despite only for 1 sec exposure of membranes to the film. Arrows point to position of rHAl protein/DRl complexes (96 kD), rHAl (45 kD), and DRl/peptide (51kD). B, boiled, NB, non-boiled. rHAl/DRl complexes were estimated to migrate at 96 kD molecular mass in both A and B. (C) Two DR alleles binding to H5Nl-rHAl protein detected by BIAcore. Biotinylated DR4-HA_Y308A complexes were incubated with denatured H5Nl-rHAl protein for 20 min at 37°C in the presence of DM. Immediately after, DR4- H5Nl-rHAl protein complexes were injected on Sensor chip SA (streptavidin).

DR4/H5Nl-rHAl protein complexes immobilization on the chip was about 3500 RU (data not shown) Then, receptive DR1 (pre-incubated with HA_Y308A) or HA₃₀₆_₃i8 bound DR1 were injected over H5Nl-rHAl bound DR4 immobilized surface in the presence of DM.

Figure 6 shows HLA-DR3 binding core region of self-antigen derived

immunodominant epitope show resistant to the cathepsins and HLA-DM. (A-B) The mass spectrum of retinal arrestin derived dominant epitope, hSA(291-306), and thyroglobulin,- derived dominant epitope, Tg(2098-2112), digestion with the cathepsins. Synthetic peptides alone, hSA(291-306) (A) or Tg(2098-2112) (B) are shown in top spectrum. Peptides incubated with cathepsin B, H, and S for lh at 37°C are shown in middle spectrum. The background control is shown in bottom spectrum that included cathepsins alone. (C-D) Dissociation assay of fluorescently labeled hSA(291-306) or Tg(2098-2112) from DR3. Fluorescently labeled hSA(291-306)/DR3 (C) or Tg(2098-2112)/DR3 (D) complexes were dissociated in the presence of 100 times molar excess of unlabeled peptides at 37° C for indicated time in the presence (circle) or absence of DM (square). The fluorescence of the labeled complex before dissociation is arbitrarily assigned a value of 1.0, and fluorescence after dissociation is expressed as a fraction of fluorescence before dissociation.

Figure 7 is a pair of bar graphs depicting the results of experiments evidencing that Cathepsin B is a critical endosomal protease for the generation of CII(280-294) and H5N1- HA (259-274) immunodominant epitopes. ELISPOT assay measuring IFN-γ production of T cells isolated from DR1 -transgenic mice immunized with 01(280-294) (a), or H5N1- HA(259-274) (b) in CFA. Cells were stimulated with peptides or proteins in vitro for 24 hours (a) or 48 hours (b) in the presence or absence of cell-permeable cathepsin B inhibitor, CA-074ME. Data shown here are representative of three independent experiments. Error bars are defined as SD.

Figure 8 is a series of photographs depicting the results of experiments evidencing that the cathepsin B inhibitor, CA-074ME, blocks the presentation of type II collagen and H5N1 HA protein derived immunodominant epitopes. (A-B) show IFN-γ production detected by the ELISPOT assay. HLA-DR1 mice were immunized with either CII(280- 294) (A) and HA(259-274) peptide (B). At day 8, LN cells were harvested and stimulated with peptides, or proteins in the absence of presence of CA-074ME. The data shown in this figure are representative of three independent experiments.

Figure 9 is a pair of bar graphs showing that hSA(291-306) and Tg(2098-2112) binding to soluble HLA-DR3 is enhanced by HLA-DM. Binding of fluorescently labeled hSA(291-306) (left) and Tg(2098-2122) (right) to DR3 in the presence or absence of DM for indicated times at 37°C. Experiments were repeated twice.

Figure 10 is a series of mass spectra detecting Myosin(334-352) peptide after digestion with CatB, CatH, and CatS. The spectra show that Myosin(334-352) survives cathepsin digestion. Undigested synthetic peptide, Myosin(334-352), is shown in top spectrum. Myosin(334-352) digested with the cathepsins for 1 hour is shown in the middle spectrum. The bottom spectrum represents sample containing cathepsins only. Samples were run on MALDI.

Figure 11 is a series of mass spectra detecting MBP(89-101) peptide after digestion with CatB, CatH, and CatS . The spectra show that MBP(89- 101) survives cathepsin digestion. Undigested synthetic peptide, MBP(89-101), is shown in top spectrum. MBP(89- 101) digested with the cathepsins for lh, is shown in the middle spectrum. The bottom spectrum represents sample containing cathepsins only. Samples were run on MALDI. Anchor residues that bind to HLA-DR2b are underlined. Figure 12 is a series of mass spectra detecting synthetic insulin B7-23, after digestion with CatB, CatH, and CatS for 1 hour at 37°C. The spectra show that insulin B7- 23 survives cathepsin digestion. Untreated synthetic peptide, insulin B7-23, is shown in top spectrum. Synthetic insulin B7-23 digested with the cathepsins for lh, is shown in the middle spectrum. Background control that include cathepsins are shown in the bottom spectrum. Samples were run on MALDI. The experiments were done three times.

Figure 13 is a series of photographs depicting the results of experiments evidencing that cathepsin D/E inhibitors do not inhibit processing and generation of immunodominant epitopes from three protein antigens. (A-C) show IFN-g production as detected by the ELISPOT assay. HLA-DR1 mice were immunized with peptides HA(306-318) (A), CII(280-294) (B), or H5Nl-HA(259-275) (C) in CFA. On day 8, cells from the draining LNs were cultured and stimulated with peptides, or proteins in the presence, or absence of CatD/E inhibitors, Pepstatin A (PepA) and Pepstatin A-Penetratin (PepA-P), or CatB inhibitor CA-074ME. Results show raw data of IFN-γ ELISPOT plates. Peptide presentation data are on the left and protein processing and presentation are to the right of the central axes.

Figure 14 is a series of mass spectra evidencing that the repertoire of H5Nl-rHAl derived peptides appears the same with or without DM. Samples containing equal starting materials (same amount of DR1 and rHAl protein) were prepared in the presence, or absence of DM and the eluted peptides were analyzed by MALDI. (a-d) Native form of H5Nl-rHAl protein, DR1, and the cathepsins B, and H, and S were incubated in the (a) presence or (b) absence of DM. (c) and (d) are background spectra for samples a and b, respectively, from reactions not containing H5Nl-rHAl . Immunodominant epitopes, HA(259-274) (SNGNFIAPEYAYKIVK), and HA(259-278)

(SNGNFIAPEYAYKIVK GDS), are shown at m/z 1814.7Da and 2200.8Da. The experiments were repeated three times.

Figure 15 is a series of mass spectra showing that DM increases the abundance of dominant epitope. Quantitation of HA(259-274) epitopes obtained in samples with or without DM by liquid chromatography (LC) combined with Linear Ion Trap Quadruple tandem mass spectrometry (LC-LTQ MS/MS) is shown. Samples containing equal starting materials (same amount of DR1 and rHAl protein) were prepared in the presence, or absence of DM and the eluted peptides were analyzed. For relative quantification of this peptide, the samples were rerun on LTQ mass spectrometry and HA(259-274) was detected as doubly charged ions at m/z 907 Da. (15A) The MS/MS profile of the doubly charged ions produced two major daughter ions at m z 1110 and 1181Da. (15B) The base peak area values from extraction ion chromatograms of 1110 Da or (15C) 1181 Da daughter ions in samples with or without DM are shown and were estimated as 3356 and 3064 in the presence of DM, versus 626 and 597 without DM accordingly. Neither of those daughter ions were detected in the background samples. (BP -base peak; AA - automated area calculated using Genesis peak detection algorithm). The experiments were repeated twice and each experiment was quantified two times.

Figure 16 is a series of mass spectra (A) and a bar graph (B) showing that vaccinia I6L(338-352) is both sensitive to cathepsin digestion and HLA-DM. (A) Mass spectra detecting I6L(338-352) peptide, after digestion with CatB, CatH, and CatS for 1 hour at 37°C. Top spectrum shows untreated synthetic I6L(338-352). I6L(338-352) peptide digested with the cathepsins for 1 hour at 37 °C is shown in the middle spectrum, and background is shown in the bottom spectrum. The samples were analyzed by MALDI. The experiments were repeated three times. When exposed to the cathepsins I6L(338-352), the peptide was completely lost and the cleavage product, SSFPVPT, was detected at m/z 734Da was much too short to bind DR1 stably. (B) Dissociation of fluorescently labeled I6L(338-352) from DR1 in the presence or absence of DM for indicated times at 37°C. Dissociation experiment was repeated three times.

Figure 17 is a series of mass spectra showing that HLA-DR1 selectively captures the immunodominant epitope of type II collagen. (A-H) The mass spectra of peptides eluted from DR1 were analyzed under these following conditions. DR1 is incubated with the following components: with or without MMP9-fragmented bCII (bCII+/-), with or without DM (DM+/-), and with or without Cathepsins B and H (CatB+CatH +/-). All eight permutations are tested. (A-D) show the reactions containing MMP9-fragmented bCII. (E- H) show the negative control reactions carried out without MMP9-fragmented bCII. With the exception of (D), DR1 used in all experiments shown here was pre-incubated with HA(Y308A) (2-3 days at 37°C) prior to use. CL(13-23) is residues 13-23 of the

Autographica Californica nucleopolyhedrovirus conotoxin-like peptide (NCBI accession number NP 054032), and is present as a background peptide in nearly all of our DR1 preparations. (bCIl₉₅₄_9₆8)20H is residues 954-968 of bovine type II collagen (bCII) with two hydroxylated residues. (bCIl₂₇₃-305)4OH is residues 273-305 of bCII with four hydroxylated residues, which contains CII282-289, the core DR1 -restricted immunodominant epitope of CII.

Figure 18 is a series of mass spectra (A) and a bar graph (B) showing that HLA- DR1 restricted immunodominant epitope HA(306-318) is sensitive to cathepsin digestion. (A) Mass spectra of detecting HA(306-318) peptide, after digestion with CatB, CatH, and CatS. DR1 used in all experiments shown here was pre-incubated with HA(Y308A) (2-3 days at 37°C) prior to use. Synthetic peptide, HA(306-318) incubated with

HA(Y308A)/DR1 for 3h is shown in top spectrum. Synthetic peptide, HA(306-318), digested first with the cathepsins for lh, and then incubated with HA(Y308A)/DR1 for 3h at 37 °C is shown in the middle spectrum. Incubation of cathepsins and HA(Y308A)/DR1 in the absence of HA(306-318) is shown in bottom spectrum. The peptides were eluted from DR1 and they were run on MALDI. The experiments were repeated three times. (B) The HA(306-318) peptide is incubated with cathepsins B and H in citrate phosphate buffer pH 4.0 at 37°C. A control reaction without cathepsins is assembled in parallel. After the incubation, the pH is adjusted to pH 7.4 and the cathepsins are heat inactivated by incubating at 80°C for 3 hrs. Both samples are then added to DR1+ EBV transformed B cells to yield a final HA(306-318) concentration of ΙμΜ. After a 4 hours incubation, the B cells are irradiated and then incubated with Clone 1 cells, a cultured human CD4⁺ T cell population specific for HA(306-318)/DR1. Proliferation of Clone 1 cells is measured by H³ -Thymidine incorporation.

DETAILED DESCRIPTION

The disclosure provides, among other things, methods for determining whether a prospective autoimmune epitope is an authentic autoimmune epitope. Such methods are useful, for example, in the development of novel autoimmune therapeutics.

As described herein, dominant epitopes from self-antigens are resistant to cathepsin digestion. The experimental results provided herein are in sharp contrast to the 'binding first, trim later' model of antigen processing. As reported herein, all self-derived autoimmune epitopes tested resisted digestion by the cathepsins. Thus, it can be determined whether a prospective autoimmune epitope is an authentic autoimmune epitope by determining whether the prospective epitope is susceptible to cathepsin digestion.

In certain aspects, provided herein is a method of determining whether a prospective autoimmune epitope is an authentic autoimmune epitope. In some embodiments, the prospective autoimmune epitope can be any epitope of a self-protein associated with an autoimmune response. In some embodiments, the prospective epitope has been identified as a potential immunodominant epitope, for example, using a method described in U.S. Pat. Pub. Nos. 2012/0076811 and 2011/0091497, each of which is incorporated by reference in its entirety.

In some embodiments, the method includes the steps of incubating the prospective epitope with in a reaction solution that includes one or more cathepsins. In some embodiments, the reaction solution includes cathepsin B, cathepsin H and/or cathepsin S. In some embodiments, the reaction solution does not include HLA-DRl and/or HLA-DM. In some embodiments, the prospective epitope is incubated for a period of time and under conditions such that an immunodominant epitope from a pathogen would be digested. In some embodiments, the prospective epitope is incubated for a period of time and under conditions such that the A/Texas/ 1/77-derived HA₃₀₆-3i8 (PKYVKQNTLKLAT) epitope would be digested. In some embodiments, the prospective epitope is incubated for at least 15, 30, 45, 60, 90, 120, 150 or 180 minutes.

In some embodiments, the method includes the step of assessing the digestion of the prospective autoimmune epitope by the reaction solution. In some embodiments, any method can be used to assess the digestion of the prospective autoimmune epitope. In some embodiments, the digestion of the prospective autoimmune epitope is assessed by mass spectrometry.

In some embodiments, the prospective autoimmune epitope is identified as an authentic autoimmune epitope if it is resistant to digestion by the reaction solution. In some embodiments, the prospective autoimmune epitope is identified as an authentic autoimmune epitope if it is more resistant to digestion by the reaction solution than the A/Texas/1/77- derived HA₃₀6-3 i8 (PKYVKQNTLKLAT) epitope. In some embodiments, the prospective autoimmune epitope is identified as an authentic autoimmune epitope if it is no more resistant to digestion by the reaction solution than the immunodominant epitope of retinal arrestin, hSA(291-306) (NRERRGIALDGKIKHE). In some embodiments, the prospective autoimmune epitope is identified as an authentic autoimmune epitope if it is no more resistant to digestion by the reaction solution than thyroglobulin peptide, Tg(2098-2112), (LSSVVVDPSIRHFDV). In some embodiments, the methods of disclosed herein may be used to evaluate prospective epitopes for the development of autoimmune therapeutics. Examples of autoimmune diseases for which epitopes could be evaluated include but are not limited to, glomerular nephritis, arthritis, dilated cardiomyopathy-like disease, ulceous colitis, Sjogren syndrome, Crohn disease, systemic erythematodes, chronic rheumatoid arthritis, multiple sclerosis, psoriasis, allergic contact dermatitis, polymyosiis, pachyderma, periarteritis nodosa, rheumatic fever, vitiligo vulgaris, insulin dependent diabetes mellitus, Behcet disease, Hashimoto disease, Addison disease, dermatomyositis, myasthenia gravis, Reiter syndrome, Graves' disease, anaemia perniciosa, Goodpasture syndrome, sterility disease, chronic active hepatitis, pemphigus, autoimmune thrombopenic purpura, and autoimmune hemolytic anemia, active chronic hepatitis, Addison's disease, anti-phospholipid syndrome, atopic allergy, autoimmune atrophic gastritis, achlorhydra autoimmune, celiac disease, Cushing's syndrome, dermatomyositis, discoid lupus, erythematosis, Goodpasture's syndrome, Hashimoto's thyroiditis, idiopathic adrenal atrophy, idiopathic

thrombocytopenia, insulin-dependent diabetes, Lambert-Eaton syndrome, lupoid hepatitis, some cases of lymphopenia, mixed connective tissue disease, pemphigoid, pemphigus vulgaris, pernicious anema, phacogenic uveitis, polyarteritis nodosa, polyglandular autosyndromes, primary biliary cirrhosis, primary sclerosing cholangitis, Raynaud's syndrome, relapsing polychondritis, Schmidt's syndrome, limited scleroderma (or crest syndrome), sympathetic ophthalmia, systemic lupus erythematosis, Takayasu's arteritis, temporal arteritis, thyrotoxicosis, type b insulin resistance, ulcerative colitis and Wegener's granulomatosis.

As described above, the disclosure also features methods for generating

immunodominant epitopes from prospective epitopes or reducing the potential

immunogenicity in a mammal of prospective epitopes that may have an immunodominant character. In some embodiments, these methods can include designing variant versions of the epitopes that contain fewer cleavage sites for cathepsin B, cathepsin H and/or cathepsin S. The methods can include, in some embodiments, designing variant versions of the epitopes that contain a greater number cleavage sites for cathepsin B, cathepsin H and/or cathepsin S. Methods for identifying potential cleavage sites for each of these enzymes are well known in the art and described in, e.g., Biniossek et al. (2011) J Proteosome Res

10(12 :5363-5373; Choe et al. (2006) J Biol Chem 281(18}: 12824- 12832; and Turk et al. (2012) Biochem et Biophys Acta 1824(1 V68-89. The designing can be performed in silico, e.g., computer-assisted methods to study or review the amino acid sequence of, or the nucleotide sequence encoding, an epitope and design variant versions with altered sequence (e.g., with more or fewer cathepsin cleavage sites). In some embodiments, mutations can be introduced into the nucleotide sequence encoding an epitope. Such methods for introducing mutations are well known in the art of molecular biology and protein chemistry. {See, e.g., Current Protocols in Molecular

Biology, Wiley & Sons, and Molecular Cloning—A Laboratory Manual—3rd Ed., Cold Spring Harbor Laboratory Press, New York (2001)). In some embodiments, a variant version of an epitope can be chemically synthesized.

In some embodiments, the epitope is all or part of a polypeptide antigen from a microorganism., or a protein (or antigenic fragment thereof) derived from a microorganism. While in no way limiting, exemplary antigens can be from any one of the following: viruses (e.g., HIV. rotavirus, influenza, parainfluenza, herpes (e.g., VZV, HSV-1, HAV-6, HSV-II, CMV, and Epstein Barr virus) Chicken pox, small pox, rabies, polio, Hepatitis A, Hepatitis B, Hepatitis C, measles, Dengue, mumps, Coxsackie virus, ftavivirases, adenoviruses, distemper, reovirus, respirator sync tial vims, ebola, hanta virus, papillomavirus, and parvovirus), bacteria (e.g., Bordetella pertussis, Brucella abortis, Escherichia coli,

Salmonella species, Streptococci, Cholera, Shigella, Pseudomonas, Tuberculosis, Pertussis, pneumonococci, meningococci, Klebsiella proteus, legionella, anthrax, leptospirosis), parasites (e.g., Plasmodium, falciparum, P. vivax, P. malariae, Entamoeba histolytica, Baiantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondi, an Nippostrongylus brasiliensis), or Candida (e.g., albicans, krusei, glabrata, or tropicalis), Cryptococcus neoformans, Aspergillus (Q.g. umigatus or niger), Mucorales (e.g., mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, or Histoplasma capsulatum). Antigens also include Sporozoan antigens, Plasmodium antigens, such as all or a portion of Circumsporozoite protein, a

Sporozoite surface protein, a liver stage antigen, an apical membrane associated protein, or a Meroz.oi.te surface protein.

In some embodiments, the antigen is a tumor antigen, including: alpha-actinin-4, Bcr-Abl, Casp-8, beta-catenin, cdc27, cdk4, edkii2a, coa-1, dek-can, EF2, ETV6-AML1, LDLR-fucosyitransferaseAS, HLA.-A2, HLA-A11, hsp70-2, KIAA02G5, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARa, PTPR , K-ras, N-ras, Triosephosphate isomerase, Bage-1, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, Mage-A l ,2,3,4,6,10,.12, Mage-C2, NA-88, NY-Eso-l/Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART -I), g lOO (Pniel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5(58), CEA, RAGE, NY-ESO (LAGE), SCP-1 , Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Ban- virus antigens, EBNA, human papillomavirus (HFV ) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG- 72- 4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenm, CDK.4, Mu.m-1, p i . TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, a-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29YBCAA), CA 195, CA 242, CA-50, CAM43,

( 1⁾68 I . CO-029, FGF-5, G250, Ga733 (EpCAM ), HTgp-175, M344, MA-50, MG7- Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein/cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS. In some embodiments, the method includes the steps of incubating the prospective epitope with in a reaction solution that includes one or more cathepsins. In some embodiments, the reaction solution includes cathepsin B, cathepsin H and/or cathepsin S. In some embodiments, the reaction solution does not include HLA-DR1 and/or HLA-DM. In some embodiments, the prospective epitope is incubated for a period of time and under conditions such that an immunodominant epitope from a pathogen would be digested. In some embodiments, the prospective epitope is incubated for a period of time and under conditions such that the A/Texas/ 1/77-derived HA₃₀6-3i8

(PKYVKQNTLKLAT) epitope would be digested. In some embodiments, the prospective epitope is incubated for at least 15, 30, 45, 60, 90, 120, 150 or 180 minutes.

In some embodiments, the method includes the step of assessing the digestion of the prospective epitope by the reaction solution. In some embodiments, any method can be used to assess the digestion of the prospective epitope. In some embodiments, the digestion of the prospective epitope is assessed by mass spectrometry.

In some embodiments, the prospective epitope is identified as an immunodominant epitope if it is resistant to digestion by the reaction solution. In some embodiments, the prospective epitope is identified as an immunodominant epitope if it lacks (or has fewer to no) cleavage sites for one or more of the proteases described herein or if it is no less resistant to cathepsin digestion than any of the other immunodominant epitopes described herein. In some embodiments, identifying the epitope as being immunodominant further identifies the epitope as likely to induce an immune response in a mammal, e.g., a human or being immunogenic in a mammal.

The following examples are meant to exemplify, not to limit, the disclosure. EXEMPLIFICATION

Experimental Methods

Production of Recombinant Proteins

Soluble HLA-DR 1 *0101 was produced in baculovirus-transduced insect cells. Soluble HLA-DM was expressed in the same manner and affinity-purified with M2 mAb sepharose (Sigma) at pH 6.0 through the FLAG tag placed on the a chain C-terminus.

Recombinant influenza hemagglutinin (rHAl) was produced as described previously in E. coli ⁴³. H5Nl-rHAl from strain A/Vietnam/ 1203/2005 was purified from 293 cells

(eEnzyme). Biotinylated HLA-DR4/CLIP and HLA-DR3/CLIP were received from NIH tetramer core facility (Atlanta, GA).

Peptides synthesis and peptide labeling

Human short CLIP₈₉ 105 peptide (KMRMATPLLMQALPM), HA_Y308A

(PKA VKQNTLKL AT) , CH259-273 (CAGF GEQGPKGEP (CI I,,, , ,,,, .ΌΗ (FTGLQGLPO_HGPPO_HGPSGC) were synthesized by Global Peptide (currently Pi

Proteomics, Huntsviile, AL, USA). HA₃₀₆-3i8 peptide (PKYVKQNTLKLAT), H5N1- HA259 -274 (S G FIAPEYAYK IV ), MBP₈₄ 102 (CVHFFK IVTPRTP), hSA(291-306) (NRERRGIALDGKIKHE), and hSA(291-306) (LSSVVVDPSIRHFDV) were synthesized by Elim Biopharmaceuticals. hSA(291 -306), hSA(291 -306) are fluorescently labeled at N- termini of the peptides.

Peptide binding and dissociating assays

Prior to set up binding assay, HLA-DR3/CLIP complexes were thrombin (Novagen, EMD Millipore chemicals, Billerica, MA, USA) cleaved for lh at room temperature. After the thrombin cleavage reaction, phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA) was added to inactivate enzymes (500 μΜ). For binding assay, DR3 (1 μΜ) was incubated for various times at 37 °C in the presence or absence of 1 μΜ DM with 50 μΜ fluorescence-labeled peptides in citrate phosphate buffer, pH 5. After removal of free peptides by a Sephadex G-50 spin column equilibrated with PBS/0.1% filtered non- fat milk, pH 7.4, fluorescence signal was measured at 25 °C and 514-516 nm with excitation at 492 nm on a Fluoromax3 spectrofluorometer (Horiba Jobin-Yvon, Kyoto, Japan) with a slit width of 2 nm. For dissociation assays, DR1 or thrombin cleaved DR3 was incubated at 37 °C with fluorescent peptides in PBS, pH 7.4, for 3 days to yield maximal loading. Samples were then spun through a Sephadex G-50 spin column equilibrated with PBS/0.1% filtered non-fat milk, pH7.4, for removal of excess unbound peptides. Samples were then incubated for the required length of time at 37 °C with excess unlabeled competitor peptide (100-molar excess) in the presence or absence of DM. After one more spin through a Sephadex G-50 spin column (block the G-50 column with 0.1% milk) for removal of dissociated fluorescent peptide, fluorescence was measured as described above.

Detection of SDS-stable HLA-DRl-antigen complexes by SDS-PAGE

DR1 (1 μΜ ) was incubated with various combinations of the following: rHAl (2.7 μΜ), and HA₃₀6-₃ i8 (300 μΜ) and HLA-DM (1 μΜ). The incubations were in citrate phosphate buffer pH 5.0 + 0.05%> sodium azide for 3 hrs at 37°C. Following incubation, pH was adjusted to 7-7.5 with either 0.2M sodium phosphate dibasic or 1M Tris-HCl pH 8.0 and the samples were mixed with a modified Laemmli buffer with 0.1% SDS final concentration, and no reducing agents. The samples were resolved as is, without boiling, on 12%) polyacrylamide gels. They were silver stained. Various combination of DR1(1 μΜ), HPLC purified DM (0.5 μΜ), rHAl (2 μΜ), and HA306-318 (300 μΜ) were incubated in citrate-phosphate buffer pH 5 overnight at 37°C. After the pH was readjusted to 7.5 and the samples were analyzed on 10% SDS-PAGE (BioRad, Hercules, CA, USA) in which samples are not boiled or boiled prior to loading and western blotted with anti-His or CHAMP2 (Rabbit polyclonal antiDRl serum, 1 :4000 dilution was used). Sample buffer contained 0.1 %> SDS and 2.5%> reducing agent.

Surface Plasmon Resonance Experiments

Biotinylated DR4/CLIP was thrombin cleaved for lh at room temperature and incubated with excess 100 times molar excess of HA_Y308A- After removing unbound

HA_Y3o8A by filtering through Sephadex G-50 column (in PBS/0.05% NaN₃), DR4-HA_Y308A (5 μΜ) complexes were incubated with denatured H5Nl-rHAl protein (eEnzyme) (2 μΜ) for 20 min at 37°C in the presence of DM (1 μΜ). Immediately after, DR4-H5Nl-rHAl protein complexes were injected on Sensor chip SA (streptavidin) (GE Healthcare

Biosciences, Waukesha, WI, USA). Receptive DR1 or HA₃₀6-₃i8 bound DR1 (2 μΜ) were injected over H5Nl-rHAl bound DR4 immobilized surface in the presence of DM (1 μΜ). All protein solutions were diluted in the running buffer composed of citrate-phosphate ρϊ I 5.5 with 150 mM NaCi, 0.005% Tween-20 and 0.05% Na ₃. Measurements were done at 37°C with flow rates ranging between 1-3 μΐ,/min. Binding sensogram was col lected on a BIAcore 1000 instrument (GE Healthcare Biosciences, Waukesha, WI, USA).

Mass Spectrometry

HLA-DRl, antigen and HLA-DM were incubated in citrate phosphate buffer (pH 5.0-5.2) at 37°C for 3hrs, after which cathepsin B (bovine spleen, Sigma) and cathepsin H (human liver, Calbiochem) were added with 6 mM L-Cysteine and 4 mM EDTA for an additional 2-3hrs. For type II collagen and rHAl protein sample preparation, cathepsin B and H were added. For H5Nl-rHAl, cathepsins B, H, and S (Calbiochem) were included. After this, the pH was adjusted to 7.5, 10 mM iodoacetamide was added, and HLA-DRl was immunoprecipitated with Sepharose conjugated with HLA-DR specific mAb (L243). Bound peptide was eluted with TFA ¹⁰² , filtered through a 10 kDa MWCO Microcon (Millipore) and lyophilized. vMALDI-LTQ XL analysis. Lyophilized samples were analyzed without further clean up and were re-suspended in 5-10ul 50%> ethanol/ 50%> water/0.2%oTFA. 0.5ul of re-suspended sample was spotted, dried, and covered with 0.5uL matrix (either 2,5-Dihydroxybenzoic acid, 40-50mg/ml in 50% ethanol/ 50% water/0.15 %TF A or a-Cyano-4-hydroxycinnamic acid, 2.5mg/ml in 50% ethanol/ 50% water/0.15%TF A). First Full MS (1100-4000Da mass range) was acquired on a vMALDI- LTQ XL (Thermo Fisher Scientific), and then MS2 and MS3 CID fragmentation spectra were acquired on peptides of interest. Spectra were manually acquired using CPS plate motion, 2 microscans, 0 sweep scans, automatic gain control (AGC) feature "on", automatic scan filtering (ASF) "on" and set on 3000 for Full MS and 200 for MSⁿ, and accumulating 10- 15 scans for Full MS and 15-25 scans for MSⁿ.

Data analysis included (1) visual analysis of Full MS spectra to find peptides of interest; (2) MSn data collected for the peptides of interest, were search with Bioworks 3.3.1 SPl (Thermo Fisher) against custom-built database, containing all protein and other components present in the sample, with no enzyme, monoisotopic precursor and fragment ions, 2 missed cleavage sites, peptide tolerance l-2Da, fragment ion tolerance IDa, and variable modifications: cysteine carboamidomethylated, methionine oxidized, methionine with 48Da loss for MS3.

Synthetic peptide digestion sample preparation: Synthetic peptides were incubated in citrate phosphate buffer (pH 5.0) at 37°C for lh with the cathepsin mix together with 6 mM L-Cysteine and 4 mM EDTA. After the digestion, cathepsins were inactivated with 10 μΜ iodoacetamide. Then, samples were filtered through a 10 kDa MWCO Microcon (Millipore) and washed with 10% MeOH/0.1% TFA and lyophilized. Lyophilized filtrate was resuspended in 10 μΐ 0.1% TFA and zip tipped (Millipore).

Example 1: HLA-DM alone is not sufficient for the selection of Immunodominant epitope of type II collagen

The determinants of immunodominance were addressed using a simplified antigen processing system. A reductionist system for MHC class II molecules that incorporates steps of antigen processing and selection, and accurately predicts immunodominant epitopes was used. It included soluble purified HLA-DR1 (DR1), HLA-DM (DM), and cathepsins B (cat B), H (cat H), and S (cat S). Mass spectrometry was used for identifying and sequencing the unique peaks derived from each protein.

Whether HLA-DM is the critical component of the antigen processing machinery contributing to the selection of the immunodominant epitopes was evaluated by analyzing the processing of type II collagen (CII) in the simplified antigen processing system. It has been documented that upon inflammation in joints and leukocyte activation, proteases are released that include a member of matrix metalloproteinases so called gelatinase B or MMP-9. MMP-9 degrades CII into fragments, which includes a longer variant of the known immunodominant CII epitope. MMP-9 pre-cut bCII was used in the system for identification of the immunodominant epitope to confirm epitope selection as well as contribution of each component to immunodominance. MMP-9 fragmented bCII was incubated with DRl and DM for three hours before adding cathepsins. DRl was immunoprecipitated after another 2-3h incubation and the bound peptides were eluted and subjected to mass spectrometry and sequencing. Figure 1 depicts a comparison of mass spectra from five samples incorporating all, or partial mixtures of the components in the system in different combinations. Samples A-D contained MMP9 digested bCII peptide mixtures whereas sample E served as the background missing the antigen (unabridged spectra are shown in Figure 17). The majority of bCII derived peptides appeared on spectra between the m/z 3000-3500 Da range (Figure 1). The known immunodominant collagen peptide was identified as a cluster of variants with different posttranslational modifications (PTM). The most prominent peak of this cluster was peptide 273-305 of bCII (OTGEPGIAGFKGEQGPKGEPGPAGVOGAPGPAG with four hydroxylated residues ((bCII₂73-305)₄O_H). This fragment contained the core DRl -restricted

immunodominant bCII₂₈₂-₂₈₉ epitope (underlined). This cluster was present in all samples that contained bCII (Figure 1 A-D) and was absent in all control experiments that were missing collagen fragments (Figure IE). Another fragment was present in all experiments containing cathepsins (m/z -2850 Da) is a variant of the bCII₂₇₃-₃₀₅ fragment.

The non-dominant epitopes were detected in experimental conditions utilized in panels A-C in Figure 1, where either DRl, or DRl and DM, or DRl and cathepsins were present, but not all together. All those epitopes became undetectable in condition D where all the components of the reductionist system were included (Figure ID). The two most prominent bCII-derived peptides at m/z 1413 and 1435 Da, which were reproducibly eliminated by the addition of DM and cathepsins, were sodiated and non-sodiated forms of the same peptide sequence,

(FTGLQGLP_OHGPP_0HGPSG) (Figure 1 A-C). To assess whether these peptides are non-dominant in mice, DRl (DR Bl *0101) transgenic mice were immunized with CII protein in complete Freund's adjuvant (CFA) and cells from draining lymph nodes were cultured in the presence of either the dominant, or the non- dominant (CII95₄- 68)₂O_H, peptide. As shown in Figure IF, cells responded to the dominant epitope as measured by IL-2 production and T cell proliferation, but did not response to (CIl₉₅₄_968)20H peptide. These observations highly suggest that the reductionist system can reproducibly identify dominant and non-dominant epitopes from CII.

To confirm the sensitivity of (CIl954_968)20H and immunodominant epitope to DM mediated dissociation, the dissociation kinetics of fluorescein-labeled (CIl954- 6s)20H and CII280-294 (AGFKGEQGPKGEPGP) in complex with DR1 in the presence or the absence of DM were measured. In the absence of DM, dissociation of (Οΐ₉₅₄_9₆8)20Η peptide from DR1 consists of a fast phase (t ₂ ~30min) and a slow phase (ti/₂ ~13h). When DM was included, (CIl95₄- 68)20H peptide dissociated much more rapidly (Ti/₂~15 min) (Figure 1G). The CII dominant epitope, which dissociated much slower (ti/₂~ 90h), also showed sensitivity to DM-mediated dissociation (t ₂~ lh) (Figure 1H). These finding suggest that DM-sensitive non-dominant epitopes, are dissociated by DM and digested away by the cathepsins. The dominant epitope, CII₂₈₀-294, despite its sensitivity to DM, survives the enzymatic digestion and hence emerges as dominant.

To verify if this explanation is true, MMP9 digested CII fragments were either incubated with only DR1 and DM in the absence of cathepsins (Figure 2A), or exposed to cathepsins (3h, lh, or 15min) prior to the incubation with DRland DM (Figure 2 B-D). Lastly, all the components were included altogether (Figure 2E). Once all the components were included, cathepsin-sensitive epitopes at m/z -3357 Da, and m/z -1413, and m/z -2778 Da were digested away within an hour (Figure 2C), or instantly (Figure 2E), It was intriguing that the clusters of peptides containing the core dominant epitope (m/z 3000- 3500 Da range) were detected regardless of the length of exposure to the cathepsins.

To even further examine the hypothesis, synthetic dominant (CII₂₈₀-294) (Figure 2F) and non-dominant (CIIc>_54-968)20H, peptides were directly exposed to CatB, H, and S for one hour and analyzed by mass spectrometry (Figure 2G). As such, the non-dominant (CIl₉₅₄_ 96δ)20Η peptide was no longer detectable (Figure 2G, middle) whereas, the dominant CILso- ₂₉₄, was prominently detected at m/z 1471.8 Da (Figure 2F, middle). There was a new peptide peak shown at m z 1338.8 Da, which when sequenced, turned out unrelated to (CIl954-968)20H- This means that non-dominant (CIl954-96s)20H was completely digested away by the enzymes. The samples including only cathepsins B, H, and S were used as the background (Figure 2F-G, bottom).

These results confirm that complexes of DR1/ CII immunodominant epitope is sensitive to DM-mediated dissociation, but is insensitive to the cathepsin degradation. Example 2: Immunodominant epitopes of HA1 Antigens are sensitive to cathepsins digestion

To find out if the results obtained with CII were applicable to other antigens, rHAl of influenza strain A/PR/8/34, which contains the immunodominant epitope from

A/Texas/ 1/77-derived HA₃₀₆-₃i8 (PKYVKQNTLKLAT) epitope attached at its C-terminus was used. HA protein was exposed to cathepsins prior to interacting with DR1 and DM, similar to CII. However, the results were in contrast to the CII data; 3 h pre-digestion with cathepsins resulted in nearly all rHAl -derived peptides being completely digested except for a trace amount of peptide at m/z 2339.09 Da (Figure 3A). To find out how long exposure to the mixture of cathepsins could be tolerated, two additional samples were run in parallel each having exposed for either lh, or only 15 min prior to the addition of DR1 and DM respectively. HA₃₀6-₃ i8 epitope was undetectable in either case while other rHAl- derived peptides were still detectable (Figure 3B-C). All the components were then mixed together, i.e., protein Ag, DR1, DM and the cathepsins, to find out that the

immunodominant epitopes could now be detected (Figure 3D). These data suggest different temporal relationship between epitope capture and processing for HA versus CII.

The order of reaction was changed and full-length rHAl protein was incubated with DR1 and DM for three hours before adding the cathepsin mix. The immunodominant epitope was successfully captured at m/z 2217.3, 2281.3 and 2524.6 Da. Other rHAl- derived peptides were observed at m z 1955.2, 2102.2, 2265.1, and 2339.1 Da as well.

To validate cathepsin sensitivity of the dominant epitope of rHAl, synthetic

HA306-318 rather than rHAl protein was exposed to the cathepsin mix for one hour and analyzed the samples by mass spectrometry (Figure 3E). Compared to the sample containing only HA306-318, which was detected at 1504.1 Da (Figure 3E, top), cathepsin exposed HA306-318 became shorter and appeared as a new peptide peak at m/z 1218.8 Da. The sequence was identified as PKYVKQNTLK, which is missing LAT at its C-terminus including Leu at P9 (Figure 3E, middle). The binding ability of cathepsin exposed synthetic HA306-318 to DR1 was next tested. When HA306-318 was exposed to cathepsins for one hour prior to incubation with peptide -receptive DR1 and the bound peptides were assessed by mass spec, only the baculovirus-derived background peptide was detected. In that sample only the bacculovirus-derived background peptide CL(13-23) that co-purifies with DR1 was detected (Figure 18).

To ensure sensitivity of the HA306-318 peptide to degradation by the cathepsins, Clone 1 T cells, a cultured human CD4 T cell population specific for HA₃₀6-3i8 HLA-DRl, were used. As before, HA₃₀6-₃i8 peptide was incubated with cathepsin mix, followed by heat-inactivation of the cathepsins and then used for pulsing HLA-DR1⁺ EBV-transformed B cells. After a 4 h incubation, B cells were irradiated and then incubated with clone 1 T cells and proliferation were measured. The peptide subjected to digestion did not activate the specific T cells, whereas the control group, which contained HA₃₀6_₃i8 without prior cathepsin digestion, did.

To examine if epitope protection by DR1 was not unique to HA₃₀6-₃i8 epitope of rHAl, an immunodominant epitopes from rHAl of H5N1 strain of influenza (H5N1- rHAl,HA₂59_274) was analyzed using the reductionist system. This epitope from H5N1 was verified in DR1 transgenic mice as immunodominant. Samples were assembled as before with recombinant proteins from H5Nl-rHAl either preincubated with DR1 and DM for three hours before adding the cathepsin mix, or first incubated with the proteases and then with DR1 and DM (Figure 4). Peptide repertoires in two different sample reactions were compared. As before, when H5Nl-rHAl was incubated with DR1 and DM first, followed by cathepsin digestion, peptide species at m/z 1726.8, 1813.9, and 2202.1 Da (Figure 4A) were detected. They shared the same core dominant epitope, ΗΑ₂₆ο-₂74

(NGNFI APE Y AYKI VK) . Three hours predigestion of H5N1 -rHAl with cathepsins peptide led to a significantly lower peak intensity at m/z 1814.9 Da. Other peptide species at m/z 1726.8 and 2202.1 Da were no longer detected (Figure 4B) indicating that this epitope was sensitive to cathepsin digestion. None of H5Nl-rHAl derived peptides were detected in the background sample (Figure 4C).

To further determine sensitivity of H5Nl-rHAl dominant epitope to the cathepsins, synthetic HA₂₅9-₂74 (SNGNFIAPEY AYKIVK) was digested with three cathepsins for one hour. Figure 4D, top panel shows undigested HA₂₅9-₂74 detected at m/z 1814.9 Da on mass spectrometry. A shorter peptide detected at m/z 1111.5 Da upon HA₂59_₂74 exposure to the cathepsins (Figure 4D, middle). It was sequenced as SNGNFIAPEY, which is short of four a.a. from C-terminus. In a parallel set of experiments, when HA259-274 and DR1 were pre -incubated for three hours and then exposed to cathepsins, (Figure 4E, top), HA₂59_274 was detected uncut. When synthetic HA₂59_₂74 was digested with the cathepsins for one hour prior to incubation with DR1, no trace of the HA259-274 was detected, only the baculovirus-derived background peptide. The peptide detected at m z 1111.5 Da (Figure 4D, middle) was not captured by DRl (Figure 4E, middle). This again demonstrates that H5N1-HA derived HA259-274 epitope is sensitive to degradation by cathepsins.

Example 3: Protein antigen binds to MHC class II molecules

If binding of protein antigen to DRl is a requirement for epitope protection, DR should form a complex with the undigested protein. To test this, a gentle SDS-PAGE assay in which samples were not boiled was used to detect binding of intact proteins to DRl . Figure 5A shows a silver stained gel, and Figure 5B depicts a parallel Western blot showing binding of full-length rHAl protein to DRl . The binding of the rHAl protein to DRl was readily detected by the appearance of a slower migrating molecular species in the presence of DRl , rHAl and DM (Figure 5A and B). This interaction occurred specifically through the peptide-binding groove of HLA-DR1 as the inclusion of a molar excess of HA₃₀6-3i8 prevented the formation of the rHAl/HLA-DRl complex (Figure 5A, Lane 8, Figure 5B, Lane 8 and 17). Importantly, the molecular mass of the slower migrating complex was estimated ~ 96 kD that is the sum of rHAl (~45kD) and DRl (~51 kD). The new band that appeared in the presence of excess HA306-318 exactly matches the migration pattern of HA306-318 DRI (Figure 5 A, Lane 9). Consistent with the role for DM in generating the peptide-receptive conformation of DR molecules, inclusion of DM together with DRl and the rHAl proteins resulted in more intensely stained bands corresponding to the

protein/DRl complexes as compared to the samples missing DM (Figure 5A, Lane 6 and 7, and in 5B Lane 4 and 6). Similarly, DRl bound to H5Nl-rHAl protein through its peptide-binding groove as detected by a gentle SDS-PAGE.

Example 4: Dominant epitopes from self-antigens are less susceptible to cathepsin digestion

The immunodominant epitopes described above have one of these two

characteristics: they are either DM-sensitive but cathepsin-insensitive (e.g. CII antigen), or DM-resistant but cathepsin-sensitive (e.g. influenza HA). Additional immunodominant epitopes were examined. Two known epitopes from self-antigens were analyzed; Uveitis- associated epitopes from retinal antigens, and autoimmune thyroiditis associated epitope from thyroglobulin. Retinal arrestin (soluble- Ag or "S-Ag") is the antigen that is thought to be involved in Uveitis in human and also in murine experimental autoimmune uveitis (EAU). Immunodominant epitope of retinal arrestin was identified as hSA(291-306) (NRERRGIALDGKIKHE), which induces EAU in HLA-DR3 (HLA-DRB 1 *03:01) transgenic mice. Thyroglobulin peptide Tg(2098-2112), LSSVVVDPSIRHFDV, has been identified as an immunodominant epitope that induces murine experimental autoimmune thyroiditis (EAT) in DR3 bearing transgenic mice.

To test cathepsin sensitivity of synthetic hSA(291-306) and Tg(2098-2112), peptides were directly exposed to the cathepsin mixture for lh, and the reaction products were analyzed by mass spectrometry. hSA(291-306) and Tg(2098-2112) lacked

susceptibility to cathepsin digestion while being resistant to DM-mediated dissociation. Figure 6A, top panel shows undigested hSA(291-306) at m/z 1892.0 Da. Upon digestion with cathepsins, the full-length peptide peak at m/z 1892.0 Da was minimized and a new peptide peak was detected at m/z 1627.0 Da (Figure 6A, middle), was sequenced as NRERRGIALDGKIK that is missing two amino acids off of its C-terminus. Other peptides detected at m/z 1177.7, 1275.7, 1346.8, 1429.8, and 1487.8 Da were detected in the background sample as well which contains all three cathepsins without the hSA(291-306) peptide. Importantly, loss of the two C-terminal residues does not abrogate the ability of this peptide to induce EAU in mice.

Thyroglobulin (2098-2112) peptide was next examined for its cathepsin sensitivity in the same way. Compared to the sample containing thyroglobulin peptide Tg(2098-2112) alone detected at m/z 1669.9 Da (Figure 6B, top), cathepsin exposed Tg(2098-2112) produced different peptide peaks at m/z 1171.8 (LSSVVVDPSIR), 1255.8

(SVVVDPSIRHF), 1456.93 (LSSVVVDPSIRHF), and 1469.9 (SVVVDPSIRHFDV) Da, along with reduced intensity of intact peptide at m/z 1670.0 Da (Figure 6B, middle). All those peptide species were different truncated versions of the immunodominant epitope of Tg and were identified among the nested set of peptides eluted from DR3 in our cell free minimalist system (data not shown). The 10 residue SVVVDPSIRH was identified as the smallest peptide eluted from DR3 in our cell-free system containing DR3, cathepsins, and DM. These observations indicate that similar to CII, the core of the dominant epitopes of arrestin and Tg, hSA(291-306) and Tg(2098-2112), remain resistant to cathepsin digestion.

To test the sensitivity of hSA(291-306) and Tg(2098-2112) to DM mediated dissociation, the dissociation kinetics of fluorescein- hSA(291-306) and fluorescein-

Tg(2098-2112) in complex with DR3 in the presence or the absence of DM was measured. To ensure DM functionality for dissociation assay, binding abilities of fluorescein-labeled peptides to DR3 were examined with or without DM with satisfactory results (Figure 9). DM did not facilitate the dissociation of either hSA(291-306), or Tg(2098-2112) from DR3 (Figure 6C-D). These results suggest that these autoantigen derived-epitopes are selected as immunodominant by having two advantages; resistance to cathepsin digestions, and resistance to DM-mediated dissociation. Sensitivity of Myosin(334-352) to cathepsin digestion was examined as with hSA and thyroglobulin peptides. While the majority of Myosin(334-352) remained intact after cathepsin digestion, minor fractions of shortened epitopes were also detected (Figure 10).

Next, the following proteins were examined: Myelin basic protein, MBP(84-102) that has been considered as a potential immunodominant epitope in relation to multiple sclerosis (MS) restricted to HLA-DR2b (DRB1 * 15:01) (Ota et al. (1990) Nature 346: 183- 187, and insulin B7-23 epitope, a candidate antigen for the induction of diabetes in NOD mice (Alleva et al. (2001) J Clin Invest 106: 173-180). When MBP(89-101) peptide was directly exposed to the cathepsin mixture, although some trimming occurred, the major epitope, VHFFKNIVTPR at m/z 1357Da maintained its corresponding anchor residues for binding to DR2b. Interestingly, two shorter peptide fragments, FFKNIVTPR at m/z 1121Da, and HFFKNIVTPR at m/z 1258Da, maintained at least some of their anchors including the major P4 fitting residues (Figure 11). (Krogsgaard et al. (2000) J Exp Med 191 : 1395-1412 and Smith et al. (1998) J Exp Med 188:1511-1520). MBP(89-101) peptide has been reported to have two registers for binding to DR2a and DR2b. One of the shorter peptides shown above lost PI anchor for binding to DR2b, and the other lost P9 anchor for binding to DR2a. MBP(89-101) epitope is sensitive to DM-mediated dissociation

(Nicholson et al. (2006) J Immunol 176:4208-4220), hence lack of sensitivity to cathepsins allows its immunodominance. Similar to MBP peptide, digestion of insulin peptide at m/z 1774Da by the cathepsin mixture did not destroy the peptide (Figure 12). The only other fragment detected post digestion of insulin B7-23 was a previously described epitope register for binding to I-A^g7. ^{3 '39} In all, the trend seems to hold that immunodominant autoantigens are less susceptible to cathepsin digestion as compared to dominant epitopes from external pathogens (Table 1). Table 1. Summary of Cathepsin and HLA-DM Sensitivity for Epitopes Tested

A destructive potential of CatD in MHC II antigen processing has been reported Moss et al. (2005) Eur J Immunol 35 :3442-3451. In light of resistance to the cathepsin mix, it was of interest to evaluate whether CatD or CatE might destroy the dominant epitopes from autoantigens during antigen processing and epitope selection. This potential was tested in a cellular IFN-γ ELISPOT assay in combination with pharmacological inhibitors of CatD/E, Pepstatin A and a cell penetrating variant of it, Pepstatin A-Penetratin (PepA-P). Zaidi et al. (2007) Biochem Biophys Res Commun 364:243-249. The results depicted in Figure 13 indicate that inclusion of PepA or PepA-P did not significantly reduce generation of immunodominant epitopes of three antigens tested; recombinant influenza HA1, influenza H5N1, and collagen II. This was in sharp contrast to the complete inhibition of the dominant epitope generation in the presence of CatB inhibitor, CA-074ME in agreement with data mentioned above. These results suggest that CatD/E may not have a critical role in generation of immunodominant epitopes during MHC class II antigen processing.

Another self-derived epitope considered as immunodominant is Multiple sclerosis (MS) associated epitopes from myelin basic protein (MBP), MBP(84-102). This epitope has been extensively studied and has been assigned as a possible immunodominant epitope for binding to HLA-DR2b (DRB1 * 1501). When MBP(89-101) peptide was directly exposed to the cathepsin mixture for lh, and then analyzed by mass spectrometry, three different peptide fragments were generated; FFK IVTPR at m/z 1121.8 Da,

HFFKNIVTPPv at m z 1258.8 Da, and VHFFK IVTPR at m/z 1357.9 Da. Two of the peptides, at 1258.8 Da and 1121.8 Da, had their PI anchor residue truncated whereas peptide at m/z 1357.9 Da contains all of its anchoring residues, although P4 pocket for DR2 is the strongest anchor. MBP(89-101) peptide has been reported to have two registers for binding to DR2, hence losing its PI anchor likely will not make it ineffective in the induction of MS. MBP(89-101) epitope has also been reported as sensitive to DM- mediated dissociation. Because of sensitivity to DM-mediated dissociation as well as partial sensitivity to cathepsins in our system, MBP(89-101) may not be the most effective epitope associated with inducing MS.

Example 5: CA-074ME inhibits processing of type II collagen and H5N1-HA

As mentioned above, a cell-free antigen processing system was developed that includes cathepsins B, H and S (CatB, CatH, and CatS, respectively). To test their roles in dominant epitope selections in cells, a cell-permeable cathepsin B inhibitor, CA-074ME, was used during the processing and presentation of type II collagen and H5N1-HA proteins ex vivo. While CA-074 is a potent inhibitor of CatB, addition of methyl ester group to make it cell permeable has made it a partial inhibitor of CatS as well. However, upon entry into the cell methyl ester group is lost and the remarkable potency of CA-074 as CatB inhibitor is revealed. Buttle et al. (1992) Arch Biochem Biophys 299:377-380. Specific T cells were generated in DR1 (DRB1 *0101) transgenic mice by immunization with type II collagen (CII) derived-epitope CII(280-294), or H5N1-HA derived-epitope, H5Nl-HA(259-274), in CFA. Cells from draining lymph nodes were cultured in the presence of either peptides, or proteins in the presence or absence of the inhibitors. As shown in Figs. 7 and 8, addition of

CA-074ME in control groups did not affect the presentation of CII and H5N1-HA derived epitopes as measured by IFN-γ ELISPOT assay. In contrast, in the presence of CA-074ME, processing and presentations of both proteins were completely inhibited. These results confirm that cathepsin B is a crucial component of antigen processing for the generation of CII(280-294) or HA(259-274) dominant epitopes.

Example 6: DM increases the abundance of dominant epitope

To determine how DM influences peptide selection, two parallel samples assembled for processing of H5Nl-rHAl with or without DM were compared. The immunodominant peptide at m/z 1814Da was detected in both samples and that the repertoire of eluted peptides remained unchanged, although, the intensity of m/z 1814Da peak was higher in the presence of DM (Figure 14). To evaluate the relative quantities of the peak in two samples, Selected Reaction Monitoring (SRM) was used. Label-free mass spectrometry methods for relative quantitation of MS peaks have the advantage of experimental simplicity and applicability. In label free quantitation, protein profiling comparisons are based on the relative intensities of extracted ion chromatograms. This approach therefore, does not require any metabolic, chemical and enzymatic labeling.

Fresh samples containing equal starting materials (same amount of DR1 and rHAl protein) were prepared in the presence, or absence of DM and the eluted peptides were analyzed by liquid chromatography tandem mass spectrometry (LC MS/MS). The m/z 1814Da peak of HA(259-274) was detected by MALDI first (Figure 14). For relative quantification of this peptide, the samples were run on LTQ mass spectrometry and

HA(259-274) was detected as doubly charged ions at m/z 907Da. The MS2 profile of the doubly charged ions produced two major daughter ions at m z 1110 and 1181Da (Figure 15A). The peak area values from extraction ion chromatograms of 111 ODa or 118 IDa daughter ions in samples with or without DM are shown in Figures 15B and 15C and were estimated as 3356 and 3064 in the presence of DM, versus 626 and 597 without DM accordingly. Both daughter ions showed greater area and higher intensities in the presence of DM. None of those daughter ions were detected in the background samples. Thus, H5Nl-rHAl derived HA(259-274) was enriched in the presence of DM by about five fold. These data suggest that DM influences the amount of epitopes captured by DR1. Example 7: Exemplification Using a Vaccinia Epitope

The cell-free system was also used to test the vaccinia epitope I6L(338-352) that has previously been identified as an immunogenic epitope using peptide elution studies from DR1 expressing DC (Strug et al. (2008) J Proteome Res 7:2703-2711). Exposure to cathepsins destroyed this peptide leaving a cleavage product, SSFPVPT, which was too short to bind DR1 stably. Moreover, when examined for DM-mediated sensitivity, I6L(338-352)/DRl complex was DM sensitive (Figure 16). While the disclosure is not bound by any particular theory or mechanism of action, I6L(338-352) likely cannot be an immunodominant epitope as it is sensitive to both cathepsins and DM. Indeed, this epitope did not generate a primary response in CD4 T cells from DR1⁺ individuals; only after a boost, T cells responded to this peptide moderately (Strug et al. (2008)).

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. A method of determining whether a prospective autoimmune epitope is an authentic autoimmune epitope comprising the steps of:

incubating the prospective epitope with in a reaction solution comprising cathepsin B, cathepsin H and cathepsin S; and

assessing the digestion of the prospective autoimmune epitope by the reaction solution; wherein the prospective autoimmune epitope is an authentic autoimmune epitope if it is resistant to digestion by the reaction solution.

2. The method of claim 1 , wherein the reaction solution does not comprise HLA-DR1.

3. The method of claim 1, wherein the reaction solution does not comprise HLA-DM.

4. The method of claim 1, wherein the digestion of the prospective autoimmune epitope is assessed by mass spectrometry.

5. A method for identifying an immunodominant epitope of interest, the method comprising:

incubating a prospective epitope within a reaction solution comprising cathepsin B, cathepsin H and cathepsin S; and

assessing the digestion of the prospective epitope by the reaction solution, wherein the prospective epitope is identified as an immunodominant epitope if it is resistant to digestion by the reaction solution.

6. The method of claim 5, further comprising administering the prospective epitope or the identified immunodominant epitope to a mammal.

7. The method of claim 6, further comprising detecting the presence or absence of an immune response to the prospective epitope or the identified immunodominant epitope.

8. The method of claim 7, wherein the immune response is a cellular immune response or a humoral immune response.

9. A method for generating an immunodominant epitope, the method comprising: incubating a prospective epitope within a reaction solution comprising one or more of cathepsin B, cathepsin H, and cathepsin S;

assessing the digestion of the prospective epitope by the reaction solution; and if the prospective epitope is not resistant to digestion by the reaction solution, designing a variant version of the prospective epitope that is resistant to digestion, or is more resistant to digestion than the prospective epitope, by the reaction solution, to thereby generate an immunodominant epitope.

10. A method for generating an immunodominant epitope, the method comprising: designing a variant version of a prospective epitope, wherein the prospective epitope is sensitive to digestion by a reaction solution comprising one or more of cathepsin B, cathepsin H, and cathepsin S, and wherein the variant version of the prospective epitope is resistant to digestion, or is more resistant to digestion than the prospective epitope, by the reaction solution, to thereby generate an immunodominant epitope.

11. The method of claim 9 or 10, wherein the designing comprises engineering one or more mutations into a nucleotide sequence encoding the prospective epitope.

12. The method of claim 11 , wherein the one or more mutations remove from the prospective epitope at least one cleavage site of cathepsin B, cathepsin H, or cathepsin S.

13. The method of any one of claims 9 to 12, wherein the prospective epitope is from a microbial polypeptide.

14. The method of claim 13, wherein the microbial polypeptide is a viral polypeptide, a bacterial polypeptide, or a protozoan polypeptide.

15. A method for reducing the immunogenicity of an epitope, the method comprising: incubating a prospective epitope within a reaction solution comprising one or more of cathepsin B, cathepsin H, and cathepsin S;

assessing the digestion of the prospective epitope by the reaction solution; and if the prospective epitope is resistant to digestion by the reaction solution, designing a variant version of the prospective epitope that is sensitive to digestion by the reaction solution, to thereby reduce the immunogenicity of the prospective epitope.

16. A method for reducing the immunogenicity of an epitope, the method comprising: designing a variant version of a prospective epitope, wherein the prospective epitope is resistant to digestion by a reaction solution comprising one or more of cathepsin B, cathepsin H, and cathepsin S, and wherein the variant version of the prospective epitope is sensitive to digestion by the reaction solution, to thereby reduce the immunogenicity of the prospective epitope.

17. The method of claim 15 or 16, wherein the designing comprises engineering one or more mutations into a nucleotide sequence encoding the prospective epitope.

18. The method of claim 17, wherein the one or more mutations introduce into the prospective epitope at least one cleavage site of cathepsin B, cathepsin H, or cathepsin S.

19. The method of any one of claims 15 to 18, wherein the prospective epitope is part of a therapeutic polypeptide.

20. The method of claim 19, wherein the therapeutic polypeptide is an antibody.

21. The method of claim 20, wherein the antibody is a humanized or fully human antibody.

22. The method of any one of claims 9 to 21, wherein the designing is performed in silico.

23. The method of any one of claims 9 to 22, further comprising incubating the variant version of the prospective epitope within a reaction solution comprising one or more of cathepsin B, cathepsin H, and cathepsin S; and assessing the digestion of the variant version of the prospective epitope by the reaction solution.

24. The method of any one of claims 9 to 23, further comprising incubating the prospective epitope within a reaction solution comprising one or more of cathepsin B, cathepsin H, and cathepsin S; and assessing the digestion of the prospective epitope by the reaction solution.

25. The method of any one of claims 9 to 24, wherein the reaction solution comprises cathepsin B, cathepsin H, and cathepsin S.

26. The method of any one of claims 1 to 25, wherein the reaction solution does not comprise HLA-DR1.

27. The method of any one of claims 1 to 26, wherein the reaction solution does not comprise HLA-DM.

28. The method of any one of claims 9 to 27, wherein the digestion of the prospective epitope is assessed by mass spectrometry.