MUTANT HSP70i TO PREVENT AUTOIMMUNE DISEASE

Abstract

A vaccine and method of treatment suitable for treating autoimmune diseases, such as Vitiligo, by using variant peptides representing a sequence of amino acids found in heat shock protein 70. The vaccine includes a peptide derived from inducible heat shock protein 70 and a plasmid containing a full inducible heat shock protein 70 DNA sequence encoding the peptide.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 61/529,050, filed Aug. 30, 2011, the contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention generally relates to treatments for autoimmune diseases. More particularly, this invention relates to the use of variant peptides representing a sequence of amino acids found in heat shock protein 70 (HSP70) to treat autoimmune diseases such as Vitiligo.

[0003] Vitiligo is an autoimmune disorder of the skin presenting with depigmenting lesions, affecting approximately 0.5% of the world's population. Vitiligo lesions are milky-white patches that can increase in shape and size over time and the distribution of these lesions defines disease classification. In particular among dark skinned individuals, emotional effects can range from mild embarrassment to severe social anxiety reinforcing the impact of Vitiligo on a patient's quality of life. Meanwhile, treatment opportunities are very limited and largely unsuccessful.

[0004] Support for Vitiligo as an autoimmune disease is provided by infiltrates of T cells and macrophages described for skin of actively depigmenting patients. Circulating autoantibodies towards melanocyte antigens have also been detected. Mutations in genes associated with autoimmunity have been identified by family studies, such as a human leukocyte antigen (HLA) system which defines antigen presentation, and Vitiligo melanocytes often display anomalies in gene expression of proteins essential in antigen processing and protein folding. In addition, Vitiligo patients often develop other autoimmune disorders such as thyroid disease, pernicious anemia, addison's disease, and lupus. These and several other autoimmune disorders have been linked with HLA molecules, particularly HLA class II (HLA-DR). Patients with HLA molecules susceptible to binding autoantigens have higher frequencies of autoreactive T cells in their circulation. As often observed in autoimmune-affected tissue, perilesional skin from Vitiligo patients displays elevated expression of HLA-DR.

[0005] Immunohistochemical staining of Vitiligo skin with a panel of antibodies reactive with different melanocyte-specific features has revealed in previous studies that depigmentation is due to a loss of melanocytes rather than a disruption of melanin production. The role of such infiltrates in mediating an immune response specifically to melanocytes is supported by increased frequencies of T cells reactive with melanocyte antigens in both the skin and circulation of Vitiligo patients. Increased infiltration of T cells in Vitiligo skin involves primarily CD8+, that is, cytotoxic T cells pointing to a Th1 mediated immune response.

[0006] Patients frequently indicate that their Vitiligo lesions were augmented or initiated under the influence of stress, including triggering factors such as UV overexposure, mechanical trauma or contact with bleaching phenols. Psychological stress may also contribute to Vitiligo development. Such precipitating factors then translate into an autoimmune response specifically targeting melanocytes. To further understand this, stress can directly affect the immune system, as evidenced by changes in CD4+/CD8+ T cell ratios following stress mediated activation of R-adrenergic receptors of the sympathetic nervous system. Upon activation of the sympathetic nervous system, heat shock proteins (HSP) released into the extracellular milieu can drive the immune response. Thus HSPs can mediate the translation between causative stress factors and the autoimmune response to follow.

[0007] HSP70 is a family of proteins including constitutive HSP70 and inducible HSP70 (HSP70i). HSP70i is among cytoplasmic chaperones upregulated in response to temperature changes and other forms of stress. HSP70i is involved in maintaining molecular and cellular integrity, preventing protein misfolding and subsequent apoptosis. Extracellular HSP70i in turn serves as an alarm signal to the immune system by supporting uptake, processing and presentation of chaperoned antigens in the context of MHC class I and II, and invoking both the innate and adaptive responses. Dendritic cell (DC) activation allows for subsequent recruitment of CD8+ T cells from draining lymph nodes reactive with chaperoned proteins and peptides.

[0008] The unique role of HSP70i in precipitating autoimmunity may be better understood considering that HSP70i is actively secreted by living cells. Melanocytes established from non-lesional Vitiligo skin treated with skin bleaching 4-tertiary butyl phenol (4-TBP) has been shown in previous studies to increase secretion of HSP70i. The same stress also confers sensitivity to DC-mediated cytotoxicity through tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). HSP70i is released into the circulation via necrosis, or secreted by active transport through exosomal release. Similar to the proposed mechanism for inducing autoimmunity in Vitiligo, in vivo studies have shown that HSP70i secreted from tumors confer anti-tumor responses via the peptides they chaperone.

[0009] The role HSP70 has on the immune response can in part be explained by the activation of DCs). This is supported by evidence that HSPs chaperoning antigenic peptides can induce DC-mediated activation of cytotoxic T lymphocytes (CTLs). In addition, HSPs enhanced the presentation of tyrosinase peptide to specific CTLs. HSP70 also activates DCs directly, as indicated by upregulation of the maturation markers CD40, CD83, and CD86. A 20-mer peptide sequence within microbial HSP70 has been assigned an immune regulatory function. HSP70 is believed to bind multiple receptor molecules on the DC membrane including TLR4, TLR2, CD14, CD91, and CD40. Antibodies that bind these receptors are generally capable of activating DCs as well. Thus, inhibiting DC activation by blocking receptors from binding HSP70 is not expected to meet with success as a measure to prevent autoimmune responses.

[0010] In view of the above, it can be appreciated that improved methods of treating patients with autoimmune diseases, such as Vitiligo, are needed.

BRIEF DESCRIPTION OF THE INVENTION

[0011] The present invention provides a vaccine and method of treatment suitable for treating autoimmune diseases, such as Vitiligo, by using variant peptides representing a sequence of amino acids found in heat shock protein 70 (HSP70).

[0012] According to a first aspect of the invention, a vaccine for treating an autoimmune disease includes a peptide derived from inducible heat shock protein 70 and a plasmid containing a full inducible heat shock protein 70 DNA sequence encoding the peptide.

[0013] According to a second aspect of the invention, a method of treating an autoimmune disease includes deriving a peptide from an inducible heat shock protein 70, forming a vaccine including the peptide derived from inducible heat shock protein 70 and a plasmid containing a full inducible heat shock protein 70 DNA sequence encoding the peptide, and then vaccinating a human patient with the vaccine.

[0014] A technical effect of the invention is the ability to treat autoimmune diseases, such as vitiligo by reducing activation of dendritic cells. In particular, it is believed that, treating a patient with a vaccine comprising a variant peptide representing a sequence of amino acids found in heat shock protein 70 (HSP70) will reduce activation of dendritic cells and thereby reduce the symptoms of autoimmune diseases.

[0015] Other aspects and advantages of this invention will be better appreciated from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 is a scanned image showing C57BL/6 wild-type (wild-type) mice and inducible heat shock protein 70 knockout (HSP70-1 KO) mice that were gene gun vaccinated with either antigenic Tyrp1ee (TRP-1) or TRP-1 plus inducible heat shock protein 70 (HSP70i) and imaged 4 weeks after the final vaccination.

[0017] FIG. 2 is a graph representing image analysis of depigmentation in the wild-type mice and the HSP70-1 KO mice of FIG. 1.

[0018] FIG. 3A is a pair of graphs represent fluorescence-activated cell sorting (FACS) used to determine peptide-specific cytotoxic T lymphocyte (CTL) activity for in vivo cytotoxicity assays.

[0019] FIG. 3B is a graph representing cytotoxicity in the mice of FIG. 3A.

[0020] FIGS. 4A and 4B are scanned images showing skin near the dermo-epidermal junction from wild-type mice and HSP70-1 mice one week after a final booster gene gun vaccination and their respective graphs representing quantification of T cell infiltration. CD3+ T cells are represented by solid arrows, hair follicles are represented by asterisks (*) and gold particles are represented by open arrows.

[0021] FIG. 5A is a scanned image of wild-type mice and constitutive HSP70 knockout mice (HSP70-2) that were gene-gun vaccinated with either TRP-2 or control DNA (plasmid containing no gene insert).

[0022] FIGS. 5B and 5C are graphs representing the depigmentation and cytotoxicity, respectively, observed in the mice of FIG. 5A.

[0023] FIG. 6A is a Western Blot representing HSP70i protein expression and antibody recognition.

[0024] FIG. 6B is a graph representing image analysis of depigmentation in mice vaccinated with mutant versions of HSP70i and TRP-2 as compared to wild-type HSP70i.

[0025] FIG. 7 is a scanned image of wild-type and Vitiligo-prone, 78B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J gp100 T cell receptor transgenic (pmel-1) mice that were vaccinated with either wild-type (WT), HSP70i(Q435A) (MT), or control DNA (vector) three months after the final gene gun vaccination.

[0026] FIG. 8 is a pair of graphs representing quantification of depigmentation of the mice of FIG. 7 relative to control DNA.

[0027] FIGS. 9A and 9B are graphs representing quantification of splenocytes obtained from pmel-1 mice nine months after gene gun vaccination with wild-type (WT) or mutant HSP70iQ435A (MT), and control DNA (vector) and were stained to discriminate leukocyte subpopulations, and staining was quantified by flow cytometry.

[0028] FIGS. 10 and 11 are scanned images representing wild-type mice and CD8 knockout mice that were vaccinated with either control DNA and TRP-1, HSP70i(Q435A) and TRP-1, or wild-type HSP70i and TRP-1.

[0029] FIGS. 12 and 13 are graphs representing observed tumor growth in the mice of FIGS. 10 and 11.

[0030] FIGS. 14 and 15 are graphs representing quantification of monocytes harvested and magnetically sorted from peripheral blood of the mice of FIGS. 10 and 11 and stained for CD11C, CD80, CD86, CD83, HLA-DR, HLA-DR, all directly with conjugated fluorochromes.

[0031] FIG. 16 is a scanned image of a wild-type mouse and a pmel-1 mouse and their respective skin samples prior to testing.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention will be described in terms of methods for treatment of autoimmune diseases. In one aspect of the invention, a mutant inducible heat shock protein 70 is used to treat depigmintation resulting from the autoimmune disease Vitiligo.

[0033] Vaccines including melanocyte-specific antigens in combination with HSP70 have been shown in previous studies to elicit immune responses towards tumorigenic and healthy melanocytes. Whereas intracellular heat shock proteins serve a cytoprotective function, upon secretion, an inducible isoform of HSP70 (HSP70i) functions as a chaperokine, eliciting innate and adaptive immune responses to surrounding tissue. Under stressful conditions, secreted, HSP70i is believed to serve as an adjuvant in Vitiligo, binding melanocytic antigens to be processed by antigen presenting cells (APC) and eliciting autoimmune responses to melanocytes in active disease. HSP70i may also activate dendritic cell (DC) directly through specific receptor binding, leading to production of various cytokines such as IL-12, and IFN-γ and the subsequent activation and recruitment of effector cells from draining lymph nodes. Thus, the identification of a peptide within HSP70 crucial to eliciting autoimmunity would open doors to preventing the causative chain of events leading to depigmentation in Vitiligo.

[0034] As previously discussed, heat shock protein 70 (HSP70) as well as various other heat shock proteins, have been implicated in DC activation. It is believed that inhibiting DC activation by blocking receptors from binding HSP70 is not expected to meet with success as a measure to prevent autoimmune responses. Therefore, moieties within HSP70 capable of binding and activating DCs were investigated in order to modulate the development of autoimmune Vitiligo. Specifically, HSP70i was investigated to determine whether its role in Vitiligo is unique or redundant in order to justify targeting a single stress protein as a treatment measure for autoimmune disease. HSP70i was chosen for its advantages over other heat shock proteins. For example, unlike other heat shock proteins, HSP70i is secreted by living cells, thus its actions occur naturally in vivo. In addition, as compared to other heat shock proteins, HSP70i is more effective at eliciting immune responses.

[0035] The following trials were conducted using C57BL/6 wild-type (wild-type) mice, HSP70i knockout (HSP70-1 KO) mice, constitutive HSP70 knockout (HSP70-2 KO) mice, and Vitiligo-prone, 78B6.Cg-Thy1a/Cy Tg(TcraTcrb)8Rest/J gp100 T cell receptor transgenic (pmel-1) mice. All mice were six to eight weeks of age when included in experiments, excluding the HSP70-2 KO mice which were eighteen to twenty-four months of age, and all mice were gender matched to wild-type mice. The mice were maintained in facilities approved by institutional IACUC regulations.

[0036] A first trial was performed to assess whether there is a sole requirement for HSP70i in mediating depigmentation. Wild-type mice and HSP70-1 KO mice were gene gun vaccinated with antigenic Tyrp1ee (TRP-1) encoding plasmid. The mice were gene gun vaccinated five times every six days with DNA encoding either six μg of optimized TRP-1 or three μg each of optimized TRP-1 plus HSP70i and imaged four weeks after the final vaccination. As represented in FIG. 1, depigmentation was virtually absent in the HSP70-1 KO mice. Image analysis confirmed significant depigmentation in the wild-type mice after vaccination with optimized TRP-1, indicating that expression of HSP70i is required for efficient induction of autoimmunity, as represented in the chart of FIG. 2. These results demonstrated that including the human HSP70i encoding plasmid in the vaccine is insufficient to restore the depigmentation process in the HSP70-1 KO mice. Therefore, it is believed that depigmentation is the result of HSP70i-enhanced DC function, accompanied by activation of T cells specifically reactive with melanocytes.

[0037] To determine if HSP70i mediates the immune activation of cytotoxic T cells (CTL), wild-type mice and HSP70-1 KO mice vaccinated with a combination of optimized TRP-1 and HSP70i encoding plasmids were assessed for in vivo cytotoxicity towards the encoded antigen as well as reactivity towards tyrosinase-related protein 2 (TRP-2). For in vivo cytotoxicity assays, the mice were challenged with splenocytes pulsed with either TRP-1, TRP-2, or irrelevant control peptides plus differing concentrations of carboxyfluorescein succinimidyl ester (CFSE). The mice received two booster gene gun vaccinations three days apart. Spleens were harvested 18 hours after challenge, and fluorescence-activated cell sorting (FACS) was used to determine peptide-specific CTL activity. Data from individual wild-type mice and HSP70-1 KO mice are represented in FIGS. 3A and 3B. The wild-type mice displayed significantly increased killing of splenocytes pulsed with a peptide derived from TRP-1 as compared to the HSP70-1 KO mice following gene gun vaccination, that is, the wild-type mice depigmented significantly more as compared to the HSP70-1 KO mice after either vaccination. The data also reveal cytotoxicity towards the TRP-2 peptide-pulsed splenocytes in all groups, indicating that epitope spreading had occurred. A correlation was observed between increased depigmentation and cytotoxicity towards splenocytes, both of which are augmented by HSP70i. In all, data confirm a solid requirement for HSP70i in inducing CTL mediated autoimmune Vitiligo and showed that the HSP70-1 mice are resistant to cytotoxic depigmentation after TRP-1 vaccinations.

[0038] To further support the belief that HSP70-induced skin immune reactivity is responsible for the Vitiligo phenotype, local differences in immune reactivity in response to HSP70i were investigated. Skin from wild-type mice and HSP70-1 KO mice were probed one week following booster gene gun vaccinations using antibodies against the pan T lymphocyte marker CD3 and the density and location of the stained cells was evaluated. FIG. 4A is a scanned image showing skin samples near the dermo-epidermal junction from the wild-type mice containing an increased number of CD3+ T cells (indicated by solid arrows) as compared to the HSP70-1 KO mice one week after a final booster gene gun vaccination. CD3+T cells are more abundant near hair follicles (indicated by *) in the wild-type mice. Gold particles can also be observed (indicated by open arrow). FIG. 4A further includes a graph representing quantification of T cell infiltration within the skin samples. The vaccinated skin of the wild-type mice contained significantly more T cells than the HSP70-1 KO mice. Consistent with depigmentation data, immunodetection of melanocyte antigen TRP-1 indicated less melanocyte-containing hair follicles were maintained within the wild-type mouse skin as compared to skin from the HSP70-1 KO mice. FIG. 4B is a scanned image showing skin samples with more melanocyte-containing hair follicles in the HSP70-1 KO mice as compared to the wild-type mice after vaccination. TRP-1 expressing melanocytes (indicated by solid arrows) are shown within hair follicles (indicated by *) in the HSP70-1 KO mouse. FIG. 4B further includes a graph representing quantification of melanocyte containing follicles. Depigmentation is shown to correlate with peptide-specific CTL activity, supporting the causative involvement of CD8+ T cells. Taken together, this immunohistology data firmly establish HSP70i induced Vitiligo developing in the skin of depigmenting mice indicating that HSP70i is necessary for loss of melanocytes by T cells.

[0039] In an additional trial, HSP70i was confirmed to be a sole isoform responsible for accelerating autoimmune depigmentation by gene gun vaccinating HSP70-2 KO mice with TRP-2 encoding plasmid in concentrations sufficient to induce depigmentation in wild-type mice. The wild-type and the HSP70-2 KO mice were gene-gun vaccinated five times every six days with six μg of either TRP-2 or control DNA (plasmid containing no gene insert). FIG. 5A is an image representing depigmentation in the wild-type mice and the HSP70-2 KO mice. Unlike the above trials engaging the HSP70-1 KO mice, no difference in depigmentation was observed between the wild-type and the HSP70-2 KO mice. Conversly, similar amounts of depigmentation were observed in both the wild-type and the HSP70-2 mice vaccinated with TRP-2 as compared to control DNA vaccinations. Visual observations were supported by statistical analysis of quantified data, as represented in the graph of FIG. 5B.

[0040] In addition, a graph of FIG. 5C represents that there were no differences in cytotoxicity towards peptide pulsed splenocytes from the wild-type mice and the HSP70-2 KO mice following gene gun vaccination with the TRP-2 encoding plasmid. This is unlike the HSP70-1 KO mice which displayed less killing towards the vaccinated antigen (TRP-1). Together, these data show that constitutive HSP70 is irrelevant for inducing an autoimmune response and further confirm the unique requirement for HSP70i expression in precipitating autoimmune Vitiligo.

[0041] Due to the results of the above trials, mammalian HSP70i was investigated to determine if it contains a peptide moiety responsible for binding and activation of DC that may be targeted to modulate autoimmune depigmentation. A database search identified the peptide QPGVLIQVYEGER in human HSP70i as maximally homologous with a DC binding region of microbial HSP70. To assess whether this region is indeed crucial for the autoimmune activation, mutations were inserted via site directed mutagenesis, results of which are represented in a Western Blot of FIG. 6A.

[0042] For site directed mutagenesis human and microbial inducible HSP70 were aligned in a BLAST search to identify a 13 amino acid stretch that was maximally homologous to the microbial DC peptide as the putative DC binding domain within human HSP70. Vectors were created with single or double mutations in the putative DC binding region of human HSP70i (HSP70i435-447) using appropriate primers to induce mutations in a sequence QPGVLIQVYEGER. As a template for the site directed mutagenesis, an expression vector encoding HSP70i was used as described. In vitro mutagenesis of HSP70 residues was performed on double-stranded template using polymerase chain reaction as previously described. Mutant sequences were verified by DNA sequencing. To verify protein expression, COS7 (CV-1 (simian) in origin, and carrying SV40 genetic material) cells were transfected with individual plasmids encoding human HSP70i, or the mutant HSP70i plasmids using lipofectamine and verified protein expression by Western blotting. Blots were probed with anti-HSP70 Ab (rabbit polyclonal Ab SPA-811 or mouse mAb SPA-810) and alkaline phosphatase-conjugated secondary Abs (goat anti-rabbit, or goat anti-mouse).

[0043] A mutant plasmid HSP70i(Q435A) is recognized equally efficiently as wild-type HSP70i whereas monoclonal antibody SPA-810 shows 20-30% reduced reactivity towards the mutant HSP70i plasmids HSP70i(V438K,I440A), HSP70i(V442A,Y443V), and HSP70i(E446V,R447A), as calculated by comparing band intensities for those observed using antibody SPA-811. Thus PGVLIQVYEGER is part of the SPA-810 binding epitope. The resulting plasmids were introduced into COS7 cells to confirm functional protein expression. To identify HSP70i expression, monoclonal antibody SPA-810 generated against a 67-mer peptide partially overlapping with the peptide of interest, and polyclonal antibody SPA-811 which recognizes a C-terminal peptide within HSP70i downstream of a peptide of interest was used. Some expression of HSP70i was observed in COS7 cells as a consequence of the transfection process as indicated by SPA-810 and SPA-811 staining in lanes representing cells transfected with control DNA (vector), as represented in FIG. 6A. Of interest, antibody SPA-810 demonstrated reduced reactivity towards mutants HSP70i(V438K,I440A), HSP70i(V442A,Y443V), and HSP70i(E446V), (R447A), with resulting staining similar to that of endogenous HSP70i. However, there were no differences in reactivity to mutant HSP70i(Q435A). Together, these data demonstrate that the HSP70i sequence PGVLIQVYEGER is part of an epitope recognized by SPA-810.

[0044] Depigmenting effects of mutant HSP70i were next investigated in vivo. Wild-type mice were gene gun vaccinated with wild-type or mutant HSP70i and/or TRP-2 encoding plasmids, and assessed for depigmentation. Results of these trials are represented in a graph of FIG. 6B. Image analysis indicated depigmentation is halted in mice receiving vaccinations with mutant versions of HSP70i and TRP-2 as compared to wild-type HSP70i. As represented in FIG. 6B, depigmentation was significantly decreased in the presence of variant sequences HSP70i(V438K,I440A), HSP70i(V442A,Y443V), and HSP70i(Q435A) as compared to unadulterated HSP70i or HSP70i(E446V,R447A). This indicates that a single amino acid difference in the target peptide region is sufficient to inactivate the depigmentation accelerating effects of HSP70i and supports the crucial involvement of the QPGVLIQVYEG moiety in inducing autoimmune depigmentation.

[0045] Next, a trial was performed to determine whether HSP70i alone is sufficient to establish depigmentation in animals predisposed to developing autoimmune depigmentation, akin to human Vitiligo. Pmel-1 mice and wild-type mice were gene gun vaccinated with wild-type HSP70i and mutant HSP70i(Q435A) plasmids, as well as control DNA. The wild-type and the pmel-1 mice were vaccinated three times every seven days with four μg of either wild-type (WT), HSP70i(Q435A) (MT), or control DNA (vector). Three months after the final vaccination, the pmel-1 mice vaccinated with wild-type HSP70i displayed significantly increased ventral and dorsal depigmentation compared to mutant HSP70i or control DNA, as represented in FIG. 7. Graphs of FIG. 8 represent that upon quantification, the pmel-1 mice receiving wild-type HSP70i-encoding DNA displayed significantly more depigmentation as compared to control DNA vaccinations and wild-type HSP70i, confirming that focal overexpression of HSP70i is sufficient to induce Vitiligo in disease-prone mice. As expected, no depigmentation was observed in the wild-type mice gene gun vaccinated with any plasmid. Importantly, ventral and dorsal depigmentation was significantly decreased in the pmel-1 mice gene gun vaccinated with mutant HSP70i(Q435A) plasmid as compared to the pmel-1 mice vaccinated with control DNA. FIG. 16 is a scanned image of a wild-type mouse and a pmel-1 mouse and their respective skin samples prior to testing. As shown in FIG. 16, the pmel-1 mouse has already lost its differentiated melanocytes prior to testing. This indicates that a single missense nucleotide variation in HSP70i derivative peptide region is sufficient not only to abrogate the Vitiligo-inducing effect of natural HSP70i, but also to actively interfere with immune activation and subsequent depigmentation.

[0046] As the QPGVLIQVYEG moiety proved crucial to the depigmentation process, its role in DC activation was further analyzed by FACS analysis. DC profiles were obtained from splenocytes of mice euthanized nine months after vaccination. The mouse splenocytes were stained for lymphocyte markers CD3, CD8, and IgM-Thy1.2, non-lymphocyte markers CD11 b, CD11 c, and F4/80, as well as DC maturation markers CD80, CD86, and MHC II. Stained cells were then followed by flow cytometry to delineate cell populations and their state of activation. For trials including pmel-1 knockout mice, splenocytes were recovered twenty-five weeks after the final vaccination, and immediately stained. Initial gating was performed on live non-debris singlets, with subsequent gating towards CD11c versus CD11b cells using FACS canto equipment. Thy1.2- IgM, and CD3 positive cells were excluded from the final gating. Notably, marked differences were observed in the abundance of monocyte-derived subpopulations discriminated based on CD11 b and CD11c expression levels, shown as R1 through R3 in graphs represented in FIG. 9A. Three distinct populations of cells were observed after gating for CD11 b and CD11 c cells in the non-lymphocyte population, with high (hi), low (lo) or intermediate (int) levels of expression. Macrophages are observed in the R1 population as CD11bint/hiCD11clo, while DCs are amongst the R2 and R3 populations as CD11 bintCD11 cint and CD11bloCD11chi respectively. The R3 conventional DC population remained unchanged, yet monocyte derivative populations were markedly biased towards CD11b expression within mice vaccinated with DNA encoding mutant HSP70i when compared to wild-type HSP70i encoding or control DNA. Graphs in FIG. 9B represent quantification of CD11b and CD11c expressing cells determined differential expression of these markers among leukocytes after vaccination with wild-type or mutant HSP70i(Q435A) plasmids. Intriguingly, the population R1 was increased by the mutant HSP70i(Q435A) vaccination, whereas the same population is suppressed after vaccination with wild-type HSP70i. The DC population R2 (CD11bintCD11cint) representative of pro-inflammatory DCs shows an opposite trend. These data demonstrate that HSP70i can drive the relative abundance of immune cell subpopulations associated with autoimmune depigmentation. The QPGVLIQVYEG moiety is involved in depigmentation by defining the prevalence of dendritic cell subsets important for CTL activation, and mutations in this region alter the macrophage and DC ratios.

[0047] After determining that mutant HSP70i can halt disease in Vitiligo-prone mice, trials were performed to investigate potential side effects. Specifically, when inhibiting autoimmunity, there is also a concern of inhibiting immunity in general and patients may not be able to mount an anti-tumor response. Therefore, wild-type and CDS knockout mice were gene gun vaccinated first with autoimmune treatments as described above and then gene gun vaccinated with tumors.

[0048] Wild-type and CD8 KO mice were vaccinated with control DNA and TRP-1, Wild-type HSP70i and TRP-1, or mutant HSP70i(Q435A) and TRP-1. The wild-type and CD8 KO mice were given a dosage of three μg of each plasmid for a total of six μg each and were vaccinated five times, six days apart. FIGS. B1 and B2 are images showing the wild-type and CD8 KO mice six weeks after final vaccination when they were assessed for depigmentation. Thereafter, the wild-type and CD8 KO mice were injected with two booster vaccinations of 100,000 B16F2 cells, five days apart. FIGS. 10 and 11 are graphs representing the observed tumor growth in the wild-type and CD8 KO mice.

[0049] To determine DC stimulation within the wild-type and CD8 KO mice, monocytes were harvested and magnetically sorted from peripheral blood. 250,000 cells were added to five milliliters of media (RPMI, 10% FBS, Pen/strep) in teflon containers. GM-CSF and IL-4 were added to drive DC differentiation and half of the media was changed every other day. On day 6, one ug/ul of either His tag sorted wild-type or mutant HSP70i(Q435A), commercially available HSP70i, LPS, or nothing was added. On day 7, the cells were stained for CD11C, CD80, CD86, CD83, HLA-DR, and/or HLA-DR all directly with conjugated fluorochromes. FACS analysis gating on DC population was performed (measured by forward and side scatter, and CD11C positivity). The results of the trial are represented in graphs of FIGS. 12, 13, 14, and 15. These results reveal that animals treated with mutant HSP70i are still protected from tumors compared to animals treated with wild-type HSP70i.

[0050] More recently, trials have been performed to understand the differential effects of wildtype HSP70i and mutant HSP70i on DC activation by studying enhanced expression of activation markers in response to either isoform. It is believed that the natural variant activates DC in vitro while the mutant HSP70i does not.

[0051] In the above trials, the exclusive, causative role of HSP70i in inducing autoimmune depigmentation in mice was examined. Using constitutive and inducible HSP70 knockout mouse models, it was determined that the inducible, but not constitutive isoform of HSP70 is solely responsible for inducing and perpetuating an autoimmune response.

[0052] Previous vaccination studies using HSP70 in combination with melanocyte antigens at suboptimal levels have shown that immune responses can be elicited towards cells bearing these target antigens. This specificity is best attributed to the chaperone function of HSP70 in binding these antigens. In addition, these trials showed that depigmentation preferentially occurred after inclusion of HSP70i in the vaccine, demonstrating the importance of this protein in perpetuating and precipitating autoimmune responses.

[0053] Importantly, these trials show that injecting mice with a combination of HSP70 and melanocyte antigen encoding plasmids is sufficient for inducing Vitiligo, and this response is mediated by CD8+ T cells. These trials demonstrate the shared relationship between Vitiligo and melanoma in the targeting of specific cells mediated by HSPs. Here, it was determined that depigmentation in wild-type mice occurred within and distal to the site of the gene gun vaccination, as represented in FIGS. 1, 2, 3A, 3B, 7, and 8. Cytotoxicity towards melanocytes is now believed to be due to an antigen-specific and systemic immune response. Here, the HSP70i KO mice displayed reduced killing of melanocyte antigen pulsed splenocytes, indicating a role for HSP70 in the immunogenicity towards these cells.

[0054] In the case of anti-tumor immunity, HSP70i is believed to efficiently chaperone tumor antigens, drive DC activation, or directly activate the adaptive immune system. In the latter, APCs will be activated leading to the recruitment of effector cells, and the eventual killing of target cells. Interestingly, in the above trials both in the wild-type and HSP70i KO mice that were vaccinated with TRP-1 plasmid, killing of TRP-2 pulsed splenocytes was observed indicating that epitope spreading had occurred.

[0055] Previous studies have observed that perilesional skin of expanding lesions in Vitiligo patients contains greater numbers of T cell infiltrates as compared to control skin. T cell mediated autoimmune responses to melanocytes are accompanied by the presence of melanocyte specific CTLs in Vitiligo patient skin. The histological staining of the above trials show that the skin from the wild-type mice had greater numbers of T cells as compared to the HSP70-1 KO mice after vaccinating each with TRP-1, and there was an even greater increase with the addition of HSP70i plasmid (not shown). These observations were inversely related to the loss of melanocytes within the wild-type mice, which can be attributed to increases in self-reactive CTL. Generally, vaccinating with TRP-1 (Tyrp1ee) elicits highly effective T cell responses; however, it was showed that in the absence of endogenous HSP70i, T cell tolerance towards melanocytes is maintained. On the other hand, removing constitutive HSP70 still resulted in melanocyte death. This confirms that only HSP70i is required to elicit an autoimmune response.

[0056] Extracellular HSP70 has been shown in previous studies to activate DC directly, and via the peptides they chaperone. These studies have shown that the C-terminal portion of HSP70 (residues 359-610) stimulates DC, and supports Th1 polarization, driving cytokine production of TNF-a, IL-12, and release of NO release. Therefore, a peptide sequence QPGVLIQVYEG (HSP70434-444) was identified as a potential target for blocking entities to prevent DC activation. From the trials above, the peptide sequence QPGVLIQVYEG was implicated as a novel target in preventing an autoimmune response, and a potential therapy in autoimmune disorders such as Vitiligo.

[0057] The stand-alone function of HSP70i was evaluated in a spontaneous mouse model of Vitiligo to demonstrate its immune enhancing effects. The pmel-1 mice were engineered to have self reactive T cells expressing a T cell receptor reactive to the melanocyte antigen gp100. These pmel-1 mice faithfully mimic human Vitiligo in which melanocyte reactive T cells escape clonal deletion, and stress is shown to augment the T cell response. The p-mell mice were vaccinated not only with wild-type HSP70i to look for accelerated establishment of autoimmune disease, but also with a representative mutant sequence to look for abrogation of the same response. Comparison of FIGS. 7 and 16 show that the mutant HSP70i may be used to reduce the progression of Vitiligo but to regain pigmentation of the white skin.

[0058] Importantly, it had already been shown that as little as a single residue change in this region is sufficient to fully prevent the depigmentation inducing effects in mice when vaccinated in combination with TRP-2. Vaccination with mutant HSP70i(Q435A) fully abrogated the gradual depigmentation observed in aging mice or animals vaccinated with the plasmid control DNA. The trials showed that depigmentation is accelerated after vaccination with wild-type HSP70i, again demonstrating the importance of this protein in precipitating disease.

[0059] Here, FACS analysis of the pmel-1 mice splenocytes revealed phenotypes of CD11c and CD11b cells as reported in the wild-type mice. Previous studies reported that Treg homing to skin is severely reduced in Vitiligo patients, which may in part be associated with a decline of the skin homing chemokine CCL22. Importantly, the above trials demonstrate that the same subset of Treg inducing macrophages is down or upregulated by wild-type and mutant HSP70i(Q435A) respectively. Thus, wild-type HSP70i may contribute to Vitiligo through the inhibition of Treg via reduced macrophage activity, and through supporting Th17 mediated autoimmunity by inflammatory DC. It is believed then that HSP70i may be acting on two fronts, by activating CTLs via DC activation, and simultaneously downregulating Treg activity. By contrast, HSP70i(Q435A) drives an opposite, anti-Vitiligo phenotype. The differential immune effects of wild-type versus mutant HSP70i are believed to be related to mutations in the DC activating peptide QPGVLIQVYEG.

[0060] HSP70 has been implicated in previous studies in various autoimmune disorders including diabetes and rheumatoid arthritis. In addition, the release of HSP70i by necrotic cells may augment inflammation such as that seen with acute myocardial infarction (AMI). This renders therapeutic measures that interfere with immune functions of HSP70 of primary importance. Here, the trials showed that HSP70i plays a crucial role in another autoimmune disorder, Vitiligo. The data indicate that HSP70i has an important role in inducing depigmentation by activating DC and subsequent breaking of T cell tolerance to self antigens. This implicates HSP70i as a crucial molecule defining a bottleneck in the etiopathology of Vitiligo.

[0061] Preventing secreted HSP70i from inducing an autoimmune response will prove as an important strategy for treating autoimmune disorders. Currently, treatments for Vitiligo typically involve local immune suppression through topical hydrocortisone application, and psoralen plus UVA (PUVA) therapy; however, the success rate is quite limited and involves long lag periods. Several groups have implicated the local production of reactive oxygen species (ROS) in Vitiligo as well as other autoimmune disorders. Therefore, it has been proposed in the art to increase catalase activity to protect melanocytes; however, this will similarly support T cell longevity. Thus there is a dire need for treatment measures that can inhibit and preferentially invert depigmentation. The trials above show that the peptide region QPGVLIQVYEG is crucial for this response. The trials not only implicate HSP70i in Vitiligo, but provide a mechanism by which the autoimmune response is activated and maintained. In knockout animals, depigmentation could not be established with or without wild-type, or mutant HSP70i. Therefore, it is now believed that depigmentation is fully dependent on CDS cytotoxic T cells. Using HSP70i expressing variants to the DC binding peptide QPGVLIQVYEG in vaccinations, therapeutic measures can be established to reduce or prevent autoimmune diseases including Vitiligo, for which effective measures do not exist to date.

[0062] In view of the above, a natural, human HSP70i derived peptide that is believed to be pivotal to depigmentation has the sequence: QPGVLIQVYEG. The mutant for which autoimmune-inhibiting properties were investigated is APGVLIQVYEG. Both depigmentation-enhancing and depigmentation-inhibiting properties are seen with the encoding sequence incorporated into a plasmid containing the full HSP70i eDNA sequence.

[0063] Although the invention has been described in terms of the peptide comprising the sequence APGVLIQVYEG, other peptides derived from the inducible heat shock protein 70 may be used for treating autoimmune diseases and are therefore within the scope of this invention. These autoimmune-inhibiting peptides are characterized by their ability to reduce or prevent activation of Dcs due to HSP70i. Such autoimmune-inhibiting peptides are preferably adapted to a vaccine comprising the peptide derived from HSP70i and a plasmid containing the full HSP70i DNA sequence encoding the peptide. The resulting vaccine may be combined with a vaccine administration device to form a vaccination kit. An example of the administration device is, but should not be limited to, a gene gun.

[0064] According to a preferred aspect of the invention, human patients susceptible to or suffering from autoimmune diseases can be treated using vaccines formed from the autoimmune-inhibiting peptides. In the case of Vitiligo, it is believed that the patients will experience reduced depigmentation in their skin once vaccinated with the vaccine as described above.

{The figures in Attachment A labeled Pmel and B6 Vitiligo figures Ai, Aii, B, and C do not have descriptions. If this application would benefit from their inclusion, please supply a short description of the figures so they may be added.}

[0065] While the invention has been described in terms of specific embodiments, it is apparent that other forms could be adopted by one skilled in the art. For example, the specific peptide derived from the HSP70i could differ from that shown, and materials and processes other than those noted could be used. Therefore, the scope of the invention is to be limited only by the following claims.

Sequence CWU 1

1

111926PRTHomo sapiensMutation(1303)..(1304) 1Ala Thr Gly Gly Cys Cys Ala Ala Ala Gly Cys Cys Gly Cys Gly Gly 1 5 10 15 Cys Gly Ala Thr Cys Gly Gly Cys Ala Thr Cys Gly Ala Cys Cys Thr 20 25 30 Gly Gly Gly Cys Ala Cys Cys Ala Cys Cys Thr Ala Cys Thr Cys Cys 35 40 45 Thr Gly Cys Gly Thr Gly Gly Gly Gly Gly Thr Gly Thr Thr Cys Cys 50 55 60 Ala Ala Cys Ala Cys Gly Gly Cys Ala Ala Gly Gly Thr Gly Gly Ala 65 70 75 80 Gly Ala Thr Cys Ala Thr Cys Gly Cys Cys Ala Ala Cys Gly Ala Cys 85 90 95 Cys Ala Gly Gly Gly Cys Ala Ala Cys Cys Gly Cys Ala Cys Cys Ala 100 105 110 Cys Cys Cys Cys Cys Ala Gly Cys Thr Ala Cys Gly Thr Gly Gly Cys 115 120 125 Cys Thr Thr Cys Ala Cys Gly Gly Ala Cys Ala Cys Cys Gly Ala Gly 130 135 140 Cys Gly Gly Cys Thr Cys Ala Thr Cys Gly Gly Gly Gly Ala Thr Gly 145 150 155 160 Cys Gly Gly Cys Cys Ala Ala Gly Ala Ala Cys Cys Ala Gly Gly Thr 165 170 175 Gly Gly Cys Gly Cys Thr Gly Ala Ala Cys Cys Cys Gly Cys Ala Gly 180 185 190 Ala Ala Cys Ala Cys Cys Gly Thr Gly Thr Thr Thr Gly Ala Cys Gly 195 200 205 Cys Gly Ala Ala Gly Cys Gly Gly Cys Thr Gly Ala Thr Cys Gly Gly 210 215 220 Cys Cys Gly Cys Ala Ala Gly Thr Thr Cys Gly Gly Cys Gly Ala Cys 225 230 235 240 Cys Cys Gly Gly Thr Gly Gly Thr Gly Cys Ala Gly Thr Cys Gly Gly 245 250 255 Ala Cys Ala Thr Gly Ala Ala Gly Cys Ala Cys Thr Gly Gly Cys Cys 260 265 270 Thr Thr Thr Cys Cys Ala Gly Gly Thr Gly Ala Thr Cys Ala Ala Cys 275 280 285 Gly Ala Cys Gly Gly Ala Gly Ala Cys Ala Ala Gly Cys Cys Cys Ala 290 295 300 Ala Gly Gly Thr Gly Cys Ala Gly Gly Thr Gly Ala Gly Cys Thr Ala 305 310 315 320 Cys Ala Ala Gly Gly Gly Gly Gly Ala Cys Ala Cys Cys Ala Ala Gly 325 330 335 Gly Cys Ala Thr Thr Cys Thr Ala Cys Cys Cys Cys Gly Ala Gly Gly 340 345 350 Ala Gly Ala Thr Cys Thr Cys Gly Thr Cys Cys Ala Thr Gly Gly Thr 355 360 365 Gly Cys Thr Gly Ala Cys Cys Ala Ala Gly Ala Thr Gly Ala Ala Gly 370 375 380 Gly Ala Gly Ala Thr Cys Gly Cys Cys Gly Ala Gly Gly Cys Gly Thr 385 390 395 400 Ala Cys Cys Thr Gly Gly Gly Cys Thr Ala Cys Cys Cys Gly Gly Thr 405 410 415 Gly Ala Cys Cys Ala Ala Cys Gly Cys Gly Gly Thr Gly Gly Thr Cys 420 425 430 Ala Cys Cys Gly Thr Gly Cys Cys Gly Gly Cys Cys Thr Ala Cys Thr 435 440 445 Thr Cys Ala Ala Cys Gly Ala Cys Thr Cys Gly Cys Ala Gly Cys Gly 450 455 460 Cys Cys Ala Gly Gly Cys Cys Ala Cys Cys Ala Ala Gly Gly Ala Thr 465 470 475 480 Gly Cys Gly Gly Gly Thr Gly Thr Gly Ala Thr Cys Gly Cys Gly Gly 485 490 495 Gly Gly Cys Thr Cys Ala Ala Cys Gly Thr Gly Cys Thr Gly Cys Gly 500 505 510 Gly Ala Thr Cys Ala Thr Cys Ala Ala Cys Gly Ala Gly Cys Cys Cys 515 520 525 Ala Cys Gly Gly Cys Cys Gly Cys Cys Gly Cys Cys Ala Thr Cys Gly 530 535 540 Cys Cys Thr Ala Cys Gly Gly Cys Cys Thr Gly Gly Ala Cys Ala Gly 545 550 555 560 Ala Ala Cys Gly Gly Gly Cys Ala Ala Gly Gly Gly Gly Gly Ala Gly 565 570 575 Cys Gly Cys Ala Ala Cys Gly Thr Gly Cys Thr Cys Ala Thr Cys Thr 580 585 590 Thr Thr Gly Ala Cys Cys Thr Gly Gly Gly Cys Gly Gly Gly Gly Gly 595 600 605 Cys Ala Cys Cys Thr Thr Cys Gly Ala Cys Gly Thr Gly Thr Cys Cys 610 615 620 Ala Thr Cys Cys Thr Gly Ala Cys Gly Ala Thr Cys Gly Ala Cys Gly 625 630 635 640 Ala Cys Gly Gly Cys Ala Thr Cys Thr Thr Cys Gly Ala Gly Gly Thr 645 650 655 Gly Ala Ala Gly Gly Cys Cys Ala Cys Gly Gly Cys Cys Gly Gly Gly 660 665 670 Gly Ala Cys Ala Cys Cys Cys Ala Cys Cys Thr Gly Gly Gly Thr Gly 675 680 685 Gly Gly Gly Ala Gly Gly Ala Cys Thr Thr Thr Gly Ala Cys Ala Ala 690 695 700 Cys Ala Gly Gly Cys Thr Gly Gly Thr Gly Ala Ala Cys Cys Ala Cys 705 710 715 720 Thr Thr Cys Gly Thr Gly Gly Ala Gly Gly Ala Gly Thr Thr Cys Ala 725 730 735 Ala Gly Ala Gly Ala Ala Ala Ala Cys Ala Cys Ala Ala Gly Ala Ala 740 745 750 Gly Gly Ala Cys Ala Thr Cys Ala Gly Cys Cys Ala Gly Ala Ala Cys 755 760 765 Ala Ala Gly Cys Gly Ala Gly Cys Cys Gly Thr Gly Ala Gly Gly Cys 770 775 780 Gly Gly Cys Thr Gly Cys Gly Cys Ala Cys Cys Gly Cys Cys Thr Gly 785 790 795 800 Cys Gly Ala Gly Ala Gly Gly Gly Cys Cys Ala Ala Gly Ala Gly Gly 805 810 815 Ala Cys Cys Cys Thr Gly Thr Cys Gly Thr Cys Cys Ala Gly Cys Ala 820 825 830 Cys Cys Cys Ala Gly Gly Cys Cys Ala Gly Cys Cys Thr Gly Gly Ala 835 840 845 Gly Ala Thr Cys Gly Ala Cys Thr Cys Cys Cys Thr Gly Thr Thr Thr 850 855 860 Gly Ala Gly Gly Gly Cys Ala Thr Cys Gly Ala Cys Thr Thr Cys Thr 865 870 875 880 Ala Cys Ala Cys Gly Thr Cys Cys Ala Thr Cys Ala Cys Cys Ala Gly 885 890 895 Gly Gly Cys Gly Ala Gly Gly Thr Thr Cys Gly Ala Gly Gly Ala Gly 900 905 910 Cys Thr Gly Thr Gly Cys Thr Cys Cys Gly Ala Cys Cys Thr Gly Thr 915 920 925 Thr Cys Cys Gly Ala Ala Gly Cys Ala Cys Cys Cys Thr Gly Gly Ala 930 935 940 Gly Cys Cys Cys Gly Thr Gly Gly Ala Gly Ala Ala Gly Gly Cys Thr 945 950 955 960 Cys Thr Gly Cys Gly Cys Gly Ala Cys Gly Cys Cys Ala Ala Gly Cys 965 970 975 Thr Gly Gly Ala Cys Ala Ala Gly Gly Cys Cys Cys Ala Gly Ala Thr 980 985 990 Thr Cys Ala Cys Gly Ala Cys Cys Thr Gly Gly Thr Cys Cys Thr Gly 995 1000 1005 Gly Thr Cys Gly Gly Gly Gly Gly Cys Thr Cys Cys Ala Cys Cys 1010 1015 1020 Cys Gly Cys Ala Thr Cys Cys Cys Cys Ala Ala Gly Gly Thr Gly 1025 1030 1035 Cys Ala Gly Ala Ala Gly Cys Thr Gly Cys Thr Gly Cys Ala Gly 1040 1045 1050 Gly Ala Cys Thr Thr Cys Thr Thr Cys Ala Ala Cys Gly Gly Gly 1055 1060 1065 Cys Gly Cys Gly Ala Cys Cys Thr Gly Ala Ala Cys Ala Ala Gly 1070 1075 1080 Ala Gly Cys Ala Thr Cys Ala Ala Cys Cys Cys Cys Gly Ala Cys 1085 1090 1095 Gly Ala Gly Gly Cys Thr Gly Thr Gly Gly Cys Cys Thr Ala Cys 1100 1105 1110 Gly Gly Gly Gly Cys Gly Gly Cys Gly Gly Thr Gly Cys Ala Gly 1115 1120 1125 Gly Cys Gly Gly Cys Cys Ala Thr Cys Cys Thr Gly Ala Thr Gly 1130 1135 1140 Gly Gly Gly Gly Ala Cys Ala Ala Gly Thr Cys Cys Gly Ala Gly 1145 1150 1155 Ala Ala Cys Gly Thr Gly Cys Ala Gly Gly Ala Cys Cys Thr Gly 1160 1165 1170 Cys Thr Gly Cys Thr Gly Cys Thr Gly Gly Ala Cys Gly Thr Gly 1175 1180 1185 Gly Cys Thr Cys Cys Cys Cys Thr Gly Thr Cys Gly Cys Thr Gly 1190 1195 1200 Gly Gly Gly Cys Thr Gly Gly Ala Gly Ala Cys Gly Gly Cys Cys 1205 1210 1215 Gly Gly Ala Gly Gly Cys Gly Thr Gly Ala Thr Gly Ala Cys Thr 1220 1225 1230 Gly Cys Cys Cys Thr Gly Ala Thr Cys Ala Ala Gly Cys Gly Cys 1235 1240 1245 Ala Ala Cys Thr Cys Cys Ala Cys Cys Ala Thr Cys Cys Cys Cys 1250 1255 1260 Ala Cys Cys Ala Ala Gly Cys Ala Gly Ala Cys Gly Cys Ala Gly 1265 1270 1275 Ala Thr Cys Thr Thr Cys Ala Cys Cys Ala Cys Cys Thr Ala Cys 1280 1285 1290 Thr Cys Cys Gly Ala Cys Ala Ala Cys Gly Cys Ala Cys Cys Cys 1295 1300 1305 Gly Gly Gly Gly Thr Gly Cys Thr Gly Ala Thr Cys Cys Ala Gly 1310 1315 1320 Gly Thr Gly Thr Ala Cys Gly Ala Gly Gly Gly Cys Gly Ala Gly 1325 1330 1335 Ala Gly Gly Gly Cys Cys Ala Thr Gly Ala Cys Gly Ala Ala Ala 1340 1345 1350 Gly Ala Cys Ala Ala Cys Ala Ala Thr Cys Thr Gly Thr Thr Gly 1355 1360 1365 Gly Gly Gly Cys Gly Cys Thr Thr Cys Gly Ala Gly Cys Thr Gly 1370 1375 1380 Ala Gly Cys Gly Gly Cys Ala Thr Cys Cys Cys Thr Cys Cys Gly 1385 1390 1395 Gly Cys Cys Cys Cys Cys Ala Gly Gly Gly Gly Cys Gly Thr Gly 1400 1405 1410 Cys Cys Cys Cys Ala Gly Ala Thr Cys Gly Ala Gly Gly Thr Gly 1415 1420 1425 Ala Cys Cys Thr Thr Cys Gly Ala Cys Ala Thr Cys Gly Ala Thr 1430 1435 1440 Gly Cys Cys Ala Ala Cys Gly Gly Cys Ala Thr Cys Cys Thr Gly 1445 1450 1455 Ala Ala Cys Gly Thr Cys Ala Cys Gly Gly Cys Cys Ala Cys Gly 1460 1465 1470 Gly Ala Cys Ala Ala Gly Ala Gly Cys Ala Cys Cys Gly Gly Cys 1475 1480 1485 Ala Ala Gly Gly Cys Cys Ala Ala Cys Ala Ala Gly Ala Thr Cys 1490 1495 1500 Ala Cys Cys Ala Thr Cys Ala Cys Cys Ala Ala Cys Gly Ala Cys 1505 1510 1515 Ala Ala Gly Gly Gly Cys Cys Gly Cys Cys Thr Gly Ala Gly Cys 1520 1525 1530 Ala Ala Gly Gly Ala Gly Gly Ala Gly Ala Thr Cys Gly Ala Gly 1535 1540 1545 Cys Gly Cys Ala Thr Gly Gly Thr Gly Cys Ala Gly Gly Ala Gly 1550 1555 1560 Gly Cys Gly Gly Ala Gly Ala Ala Gly Thr Ala Cys Ala Ala Ala 1565 1570 1575 Gly Cys Gly Gly Ala Gly Gly Ala Cys Gly Ala Gly Gly Thr Gly 1580 1585 1590 Cys Ala Gly Cys Gly Cys Gly Ala Gly Ala Gly Gly Gly Thr Gly 1595 1600 1605 Thr Cys Ala Gly Cys Cys Ala Ala Gly Ala Ala Cys Gly Cys Cys 1610 1615 1620 Cys Thr Gly Gly Ala Gly Thr Cys Cys Thr Ala Cys Gly Cys Cys 1625 1630 1635 Thr Thr Cys Ala Ala Cys Ala Thr Gly Ala Ala Gly Ala Gly Cys 1640 1645 1650 Gly Cys Cys Gly Thr Gly Gly Ala Gly Gly Ala Thr Gly Ala Gly 1655 1660 1665 Gly Gly Gly Cys Thr Cys Ala Ala Gly Gly Gly Cys Ala Ala Gly 1670 1675 1680 Ala Thr Cys Ala Gly Cys Gly Ala Gly Gly Cys Cys Gly Ala Cys 1685 1690 1695 Ala Ala Gly Ala Ala Gly Ala Ala Gly Gly Thr Gly Cys Thr Gly 1700 1705 1710 Gly Ala Cys Ala Ala Gly Thr Gly Thr Cys Ala Ala Gly Ala Gly 1715 1720 1725 Gly Thr Cys Ala Thr Cys Thr Cys Gly Thr Gly Gly Cys Thr Gly 1730 1735 1740 Gly Ala Cys Gly Cys Cys Ala Ala Cys Ala Cys Cys Thr Thr Gly 1745 1750 1755 Gly Cys Cys Gly Ala Gly Ala Ala Gly Gly Ala Cys Gly Ala Gly 1760 1765 1770 Thr Thr Thr Gly Ala Gly Cys Ala Cys Ala Ala Gly Ala Gly Gly 1775 1780 1785 Ala Ala Gly Gly Ala Gly Cys Thr Gly Gly Ala Gly Cys Ala Gly 1790 1795 1800 Gly Thr Gly Thr Gly Thr Ala Ala Cys Cys Cys Cys Ala Thr Cys 1805 1810 1815 Ala Thr Cys Ala Gly Cys Gly Gly Ala Cys Thr Gly Thr Ala Cys 1820 1825 1830 Cys Ala Gly Gly Gly Thr Gly Cys Cys Gly Gly Thr Gly Gly Thr 1835 1840 1845 Cys Cys Cys Gly Gly Gly Cys Cys Thr Gly Gly Gly Gly Gly Cys 1850 1855 1860 Thr Thr Cys Gly Gly Gly Gly Cys Thr Cys Ala Gly Gly Gly Thr 1865 1870 1875 Cys Cys Cys Ala Ala Gly Gly Gly Ala Gly Gly Gly Thr Cys Thr 1880 1885 1890 Gly Gly Gly Thr Cys Ala Gly Gly Cys Cys Cys Cys Ala Cys Cys 1895 1900 1905 Ala Thr Thr Gly Ala Gly Gly Ala Gly Gly Thr Ala Gly Ala Thr 1910 1915 1920 Thr Ala Gly 1925

Claims

1. A vaccine for treating an autoimmune disease comprising a peptide derived from inducible heat shock protein 70, the peptide encoded in a plasmid containing a full inducible heat shock protein 70 DNA sequence.

2. The vaccine of claim 1, wherein the peptide is characterized by its ability to reduce activation of dendritic cells.

3. The vaccine of claim 1, wherein the peptide is an inducible heat shock protein 70 expressing variant to the dendritic cell binding peptide with the sequence QPGVLIQVYEG.

4. The vaccine of claim 1, wherein the peptide has the sequence APGVLIQVYEG.

5. The vaccine of claim 1, wherein the peptide is derived from human inducible heat shock protein 70.

6. The vaccine of claim 1, wherein the autoimmune disease is Vitiligo and the vaccine is effective to reduce depigmentation in the skin of a human suffering from Vitiligo when administered to the human.

7. A vaccination kit comprising a vaccine administration device and the vaccine of claim 1.

8. The vaccination kit of claim 7, wherein the administration device is a gene gun.

9. A method of treating an autoimmune disease comprising administering to a human patient the vaccine of claim 1.

10. The method of to claim 9, wherein the patient is currently suffering from an autoimmune disease.

11. The method of to claim 9, wherein the patient is genetically prone to an autoimmune disease.

12. A method of treating an autoimmune disease, the method comprising: deriving a peptide from inducible heat shock protein 70; forming a vaccine comprising the peptide derived from inducible heat shock protein 70, the peptide encoded in a plasmid containing a full inducible heat shock protein 70 DNA sequence; and then vaccinating a human patient with the vaccine.

13. The method of claim 12, wherein the peptide is characterized by its ability to reduce activation of dendritic cells.

14. The method of claim 12, wherein the peptide is an inducible heat shock protein 70 expressing variant to the dendritic cell binding peptide QPGVLIQVYEG.

15. The method of claim 12, wherein the peptide is APGVLIQVYEG.

16. The method of claim 12, wherein the peptide is derived from human inducible heat shock protein 70.

17. The method of claim 12, wherein the autoimmune disease is Vitiligo and the vaccine is effective to reduce depigmentation in the skin of a human suffering from Vitiligo when administered to the human.

18. The method of claim 12, wherein the administration device is a gene gun.

19. The method of to claim 12, wherein the human patient is currently suffering from an autoimmune disease.

20. The method according to claim 12, wherein the human patient is genetically prone to an autoimmune disease.

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