Exogenous delivery of chaperonin subunit fragment ApiCCT1 modulates mutant Huntingtin cellular phenotypes.

Aggregation of misfolded proteins is characteristic of a number of neurodegenerative diseases, including Huntington disease (HD). The CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring) chaperonin complex can inhibit aggregation and cellular toxicity induced by expanded repeat Huntingtin (mHtt) fragments. The substrate-binding apical domain of CCT/TRiC subunit CCT1, ApiCCT1, is sufficient to inhibit aggregation of expanded repeat mHtt fragments in vitro, providing therapeutic promise for HD. However, a key hurdle in considering ApiCCT1 as a potential treatment is in delivery. Because ApiCCT1 has a region of similarity to the HIV Tat protein cell-transduction domain, we tested whether recombinant ApiCCT1 (ApiCCT1(r)) protein could enter cells following exogenous delivery and modulate an established panel of mHtt-mediated cell-based phenotypes. Cell fractionation studies demonstrate that exogenous ApiCCT1(r) can penetrate cell membranes and can localize to the nucleus, consistent with a strategy that can target both cytosolic and nuclear pathogenic events in HD. ApiCCT1(r) application does indeed modulate HD cellular phenotypes by decreasing formation of visible inclusions, fibrillar oligomers, and insoluble mHtt derived from expression of a truncated mHtt exon 1 fragment. ApiCCT1(r) also delays the onset of inclusion body formation as visualized via live imaging. ApiCCT1(r) reduces mHtt-mediated toxicity in immortalized striatal cells derived from full-length knock-in HD mice, suggesting that therapeutic benefit may extend beyond effects on aggregation. These studies provide the basis for a potentially robust and unique therapeutic strategy to target mHtt-mediated protein pathogenesis.

Nasal immunization with heat shock protein 65 attenuates atherosclerosis and reduces serum lipids in cholesterol-fed wild-type rabbits probably through different mechanisms.

In recent years atherosclerosis has proved to be associated with microbial infection, inflammation and autoimmunity. Conservative heat shock protein (HSP) 65/60 is a major autoantigen of atherosclerosis. In the current study, experiments were specifically designed to investigate whether a nasal immunization with HSP65 could attenuate atherosclerosis in a cholesterol-fed wild-type animal model and explore its influence on serum lipids. Wild-type rabbits were nasally treated with HSP65 10 times on alternate days. At the end of the experiment, the rabbits showed remarkably lightened lesions in aortas. The suppression of T cell proliferation, increase of IL-10 production and absence of related antibodies implied that a tolerance to HSP65 was successfully established. Simultaneously, the serum lipid levels were down-regulated significantly in this group. Further results of another group immunized with conjugated protein HSP65+CTB-P277 showed that the lipid reduction could also be achieved by an immunization without inducing tolerance. But this simple reduction of lipids could not eventually alleviate atherosclerosis. In conclusion, nasal administration of HSP65 can effectively attenuate atherosclerosis in cholesterol-fed wild-type rabbits primarily by inducing an unresponsive state of tolerance. The accompanying reduction of lipids, which probably results from a different immune mechanism other than tolerance, cannot ultimately prevent the development of atherosclerotic lesions alone.

Protection against tuberculosis by a single intranasal administration of DNA-hsp65 vaccine complexed with cationic liposomes.

BACKGROUND: The greatest challenges in vaccine development include optimization of DNA vaccines for use in humans, creation of effective single-dose vaccines, development of delivery systems that do not involve live viruses, and the identification of effective new adjuvants. Herein, we describe a novel, simple technique for efficiently vaccinating mice against tuberculosis (TB). Our technique consists of a single-dose, genetic vaccine formulation of DNA-hsp65 complexed with cationic liposomes and administered intranasally. RESULTS: We developed a novel and non-toxic formulation of cationic liposomes, in which the DNA-hsp65 vaccine was entrapped (ENTR-hsp65) or complexed (COMP-hsp65), and used to immunize mice by intramuscular or intranasal routes. Although both liposome formulations induced a typical Th1 pattern of immune response, the intramuscular route of delivery did not reduce the number of bacilli. However, a single intranasal immunization with COMP-hsp65, carrying as few as 25 microg of plasmid DNA, leads to a remarkable reduction of the amount of bacilli in lungs. These effects were accompanied by increasing levels of IFN-gamma and lung parenchyma preservation, results similar to those found in mice vaccinated intramuscularly four times with naked DNA-hsp65 (total of 400 microg). CONCLUSION: Our objective was to overcome the significant obstacles currently facing DNA vaccine development. Our results in the mouse TB model showed that a single intranasal dose of COMP-hsp65 elicited a cellular immune response that was as strong as that induced by four intramuscular doses of naked-DNA. This formulation allowed a 16-fold reduction in the amount of DNA administered. Moreover, we demonstrated that this vaccine is safe, biocompatible, stable, and easily manufactured at a low cost. We believe that this strategy can be applied to human vaccines to TB in a single dose or in prime-boost protocols, leading to a tremendous impact on the control of this infectious disease.

The dynamics of articular leukocyte trafficking and the immune response to self heat-shock protein 65 influence arthritis susceptibility.

INTRODUCTION: Adjuvant arthritis (AA) shares several features with human rheumatoid arthritis, and it can be induced in the Lewis (LEW) rat but not the Wistar Kyoto (WKY) rat (both RT.1(l)) by immunization with heat-killed Mycobacterium tuberculosis (Mtb). We set out to unravel the mechanisms underlying the differential susceptibility to AA of these MHC-compatible rat strains. MATERIALS AND METHODS: We compared the levels of T-cell proliferative and cytokine response to the immunoregulatory self (rat) hsp65 (Rhsp65) after an arthritogenic (Mtb) challenge and the kinetics of migration of adoptively transferred, (111)Indium-labeled, Mtb-primed leukocytes into the hind paw joints of recipient rats. RESULTS AND DISCUSSION: The WKY rats raised a significantly higher level of T-cell proliferative response coupled with a temporally opposite cytokine profile against the disease-regulating Rhsp65 compared to that of LEW rats. Moreover, the arthritogenic leukocytes accumulated into the joints of WKY rats at significantly lower numbers than that in LEW rats. CONCLUSIONS: These results offer novel insights into the immune events influencing the pathogenesis of autoimmune arthritis.

Th1 polarized response induced by intramuscular DNA-HSP65 immunization is preserved in experimental atherosclerosis

We previously reported that a DNA vaccine constructed with the heat shock protein (HSP65) gene from Mycobacterium leprae (DNA-HSP65) was protective and also therapeutic in experimental tuberculosis. By the intramuscular route, this vaccine elicited a predominant Th1 response that was consistent with its protective efficacy against tuberculosis. It has been suggested that the immune response to Hsp60/65 may be the link between exposure to microorganisms and increased cardiovascular risk. Additionally, the high cholesterol levels found in atherosclerosis could modulate host immunity. In this context, we evaluated if an atherogenic diet could modulate the immune response induced by the DNA-HSP65 vaccine. C57BL/6 mice (4-6 animals per group) were initially submitted to a protocol of atherosclerosis induction and then immunized by the intramuscular or intradermal route with 4 doses of 100 mug DNA-HSP65. On day 150 (15 days after the last immunization), the animals were sacrificed and antibodies and cytokines were determined. Vaccination by the intramuscular route induced high levels of anti-Hsp65 IgG2a antibodies, but not anti-Hsp65 IgG1 antibodies and a significant production of IL-6, IFN-g and IL-10, but not IL-5, indicating a Th1 profile. Immunization by the intradermal route triggered a mixed pattern (Th1/Th2) characterized by synthesis of anti-Hsp65 IgG2a and IgG1 antibodies and production of high levels of IL-5, IL-6, IL-10, and IFN-g. These results indicate that experimentally induced atherosclerosis did not affect the ability of DNA-HSP65 to induce a predominant Th1 response that is potentially protective against tuberculosis.

Th1 polarized response induced by intramuscular DNA-HSP65 immunization is preserved in experimental atherosclerosis.

We previously reported that a DNA vaccine constructed with the heat shock protein (HSP65) gene from Mycobacterium leprae (DNA-HSP65) was protective and also therapeutic in experimental tuberculosis. By the intramuscular route, this vaccine elicited a predominant Th1 response that was consistent with its protective efficacy against tuberculosis. It has been suggested that the immune response to Hsp60/65 may be the link between exposure to microorganisms and increased cardiovascular risk. Additionally, the high cholesterol levels found in atherosclerosis could modulate host immunity. In this context, we evaluated if an atherogenic diet could modulate the immune response induced by the DNA-HSP65 vaccine. C57BL/6 mice (4-6 animals per group) were initially submitted to a protocol of atherosclerosis induction and then immunized by the intramuscular or intradermal route with 4 doses of 100 microg DNA-HSP65. On day 150 (15 days after the last immunization), the animals were sacrificed and antibodies and cytokines were determined. Vaccination by the intramuscular route induced high levels of anti-Hsp65 IgG2a antibodies, but not anti-Hsp65 IgG1 antibodies and a significant production of IL-6, IFN-g and IL-10, but not IL-5, indicating a Th1 profile. Immunization by the intradermal route triggered a mixed pattern (Th1/Th2) characterized by synthesis of anti-Hsp65 IgG2a and IgG1 antibodies and production of high levels of IL-5, IL-6, IL-10, and IFN-g. These results indicate that experimentally induced atherosclerosis did not affect the ability of DNA-HSP65 to induce a predominant Th1 response that is potentially protective against tuberculosis.

Induction of CD4-independent E7-specific CD8+ memory response by heat shock fusion protein.

Infection with human papillomavirus type 16 (HPV16) is strongly associated with a number of disease states, of which cervical and anal cancers represent the most drastic endpoints. Induction of T-cell-mediated immunity, particularly cytotoxic T lymphocytes (CTL), is important in eradication of HPV-induced lesions. Studies have shown that heat shock protein fusion proteins are capable of inducing potent antigen-specific CTL activity in experimental animal models. In addition, E7-expressing tumors in C57BL/6 mice can be eradicated by treatment with HspE7, an Hsp fusion protein composed of Mycobacterium bovis BCG Hsp65 linked to E7 protein of HPV16. More importantly, HspE7 has also displayed significant clinical benefit in phase II clinical trials for the immunotherapy of HPV-related diseases. To delineate the mechanisms underlying the therapeutic effects of HspE7, we investigated the capability of HspE7 to induce antigen-specific protective immunity. Here, we demonstrate that HspE7 primes potent E7-specific CD8(+) T cells with cytolytic and cytokine secretion activities. These CD8(+) T cells can differentiate into memory T cells with effector functions in the absence of CD4(+) T-cell help. The HspE7-induced memory CD8(+) T cells persist for at least 17 weeks and confer protection against E7-positive murine tumor cell challenge. These results indicate that HspE7 is a promising immunotherapeutic agent for treating HPV-related disease. Moreover, the ability of HspE7 to induce memory CD8(+) T cells in the absence of CD4(+) help indicates that HspE7 fusion protein may have activity in individuals with compromised CD4(+) functions, such as those with invasive cancer and/or human immunodeficiency virus infection.

Antibody responses to mycobacterial and self heat shock protein 65 in autoimmune arthritis: epitope specificity and implication in pathogenesis.

Many autoimmune diseases are believed to involve primarily T cell-mediated effector mechanisms. There is increasing realization, however, that Abs may also play a vital role in the propagation of T cell-driven disorders. In this study, on the rat adjuvant-induced arthritis (AA) model of human rheumatoid arthritis, we examined the characteristics of serum Ab response to mycobacterial heat shock protein (hsp) 65 (Bhsp65), self (rat) hsp65 (Rhsp65), and linear peptides spanning these two molecules. The AA-resistant WKY (RT.1(l)) rat responded to the heat-killed Mycobacterium tuberculosis immunization with a rapid burst of Abs to both Bhsp65 and Rhsp65. These Abs reacted with numerous peptide epitopes; however, this response was reduced to a few epitopes with time. On the contrary, the susceptible Lewis (RT.1(l)) rat developed a relatively lower Ab response to Bhsp65, and Abs to Rhsp65 did not appear until the recovery from the disease. The Ab response in Lewis rats diversified with progression of AA, and there was an intriguing overlap between the repertoire of Bhsp65-reactive B and T cells during the recovery phase of AA. Nonetheless, subsets of the repertoire of the late Abs in both rat strains became focused on the same epitope regions of Bhsp65 and Rhsp65. The functional relevance of these Abs was evident from the results showing that sera from recovery phase Lewis or WKY rats, but not that of naive rats, afforded protection against subsequent AA. These results are of significance in further understanding of the role of humoral immunity in the pathogenesis of autoimmune arthritis.

Immunomodulation and protection induced by DNA-hsp65 vaccination in an animal model of arthritis.

We described a prophylactic and therapeutic effect of a DNA vaccine encoding the Mycobacterium leprae 65-kDa heat shock protein (DNA-hsp65) in experimental murine tuberculosis. However, high homology of the vaccine to the corresponding mammalian hsp60, together with the CpG motifs in the plasmidial vector, could trigger or exacerbate an autoimmune disease. In the present study, we evaluate the potential of DNA-hsp65 vaccination to induce or modulate arthritis in mice genetically selected for acute inflammatory reaction (AIR), either maximal (AIRmax) or minimal (AIRmin). Mice immunized with DNA-hsp65 or injected with the corresponding DNA vector (DNAv) developed no arthritis, whereas pristane injection resulted in arthritis in 62% of AIRmax mice and 7.3% of AIRmin mice. Administered after pristane, DNA-hsp65 downregulated arthritis induction in AIRmax animals. Levels of interleukin (IL)-12 were significantly lower in mice receiving pristane plus DNA-hsp65 or DNAv than in mice receiving pristane alone. However, when mice previously injected with pristane were inoculated with DNA-hsp65 or DNAv, the protective effect was significantly correlated with lower IL-6 and IL-12 levels and higher IL-10 levels. Our results strongly suggest that DNA-hsp65 has no arthritogenic potential and is actually protective against experimentally induced arthritis in mice.

Prime-boost vaccination based on DNA and protein-loaded microspheres for tuberculosis prevention.

We evaluated the use of a vaccine formulation based on a mixture of two different PLGA microspheres, composed by faster and slower release profiles, containing DNA encoding hsp65 and the recombinant hsp65 protein, respectively, aiming to DNA priming and protein boost after a single dose vaccination. The combination of PLGA50:50 microspheres containing DNA-hsp65 and trehalose dimycolate (TDM) with PLGA75:25 microspheres containing recombinant hsp65 (prime-boost Me) was able to induce high levels of anti-hsp65 specific antibodies. The serum levels of these specific antibodies remained high during 90 days after vaccination, whereas the DNA Me formulation based only in DNA-hsp65 plus TDM-loaded microspheres was not able to sustain the high antibody levels during the same period. Production of IFN-gamma was significant in animals vaccinated with both formulations, while the prime-boost Me vaccinated mice sustained higher levels of this cytokine during all the evaluation period. Thus, prime-boost strategy by using biodegradable microspheres seems to be a promising strategy to stimulate long-lasting immune response.

Long-lasting specific antibodies against CETP induced by subcutaneous and mucosal administration of a 26-amino acid CETP epitope carried by heat shock protein 65 kDa in the absence of adjuvants.

The heat shock protein 65 kDa (Hsp65) of Mycobacterium tuberculosis var. bovis was fused with the linear polypeptide epitope of cholesteryl ester transfer protein C-terminal fragment (CETPC) and expressed as soluble protein in Escherichia coli. The fusion protein Hsp65-CETPC was purified by anion exchange column and eluted at 100-130 mM NaCl in 10mM phosphate buffer (pH 8.0), and then used to immunize mice via subcutaneous injection or intranasal delivery in the absence of adjuvants. Antibodies against CETPC were detected in immunized mice sera by enzyme-linked immunosorbent assay (ELISA) and verified by Western blot analysis. Specific antibodies were successfully induced and lasted for more than 12 weeks in animals immunized with the fusion protein via both subcutaneous and intranasal routes even in the absence of adjuvants. Results showed that Hsp65 could be used as a convenient carrier molecule for presenting foreign polypeptide epitopes, such as CETPC, to the immune system in vivo. Antibodies induced by Hsp65-CETPC could partially inhibit the excessive activity of CETP to normal level. Therefore, Hsp65-CETPC might be further developed to a vaccine against atherosclerosis.

Myeloid differentiation factor 88 is required for cross-priming in vivo.

We describe a role for myeloid differentiation factor 88 (MyD88) in the induction of functional CTLs in vivo, in response to exogenously administered Ag, using a heat shock fusion protein, hsp65-P1, as a model Ag. CD8 T cells transferred into MyD88-deficient animals produce normal numbers of CD8 effector cells that have normal activation marker profiles after immunization with hsp65-P1. However, these CD8 T cells produced significantly less IFN-gamma and showed reduced killing activity. This reduction in activation of functional CTLs appears to be unrelated to Toll-like receptor 4 function, because in vitro hsp65-P1-experienced Toll-like receptor 4-deficient dendritic cells (DCs), but not MyD88-deficient DCs, activated CD8 T cells to a similar extent to wild-type DCs. We identify a cross-presentation defect in MyD88-deficient DCs that, when treated with hsp65-P1 fusion protein, results in surface display of fewer SIYRYYGL/class I MHC complexes. Thus, MyD88 plays a role in the developmental maturation of DCs that allows them to prime CD8 T cells through cross-presentation.

The T cells specific for the carboxyl-terminal determinants of self (rat) heat-shock protein 65 escape tolerance induction and are involved in regulation of autoimmune arthritis.

Immunization of Lewis rats with heat-killed Mycobacterium tuberculosis H37Ra leads to development of polyarthritis (adjuvant-induced arthritis; AA) that shares several features with human rheumatoid arthritis (RA). Immune response to the 65-kDa mycobacterial heat-shock protein (Bhsp65) is believed to be involved in induction of AA as well as in experimental modulation of this disease. However, the understanding of several critical aspects of the pathogenesis of AA in the Lewis rat has severely been hampered by the lack of information both regarding the level as well as epitope specificity of tolerance to the mammalian self (rat) homologue of Bhsp65, 65-kDa rat heat-shock protein (Rhsp65), and about the functional attributes of the T cell repertoire specific for this self protein. In this study, we established that tolerance to Rhsp65 in the Lewis rat is incomplete, and that the residual T cells primed upon challenge with this self hsp65 are disease regulating in nature. We also have defined the T cell epitopes in the C-terminal region within Rhsp65 that contribute predominantly to the immune reactivity as well as the AA-protective effect of this self protein. Furthermore, the T cells primed by peptides comprising these C-terminal determinants can be efficiently restimulated by the naturally generated epitopes from endogenous Rhsp65, suggesting that self hsp65 might also be involved in natural remission from acute AA. These novel first experimental insights into the self hsp65-directed regulatory T cell repertoire in AA would help develop better immunotherapeutic approaches for autoimmune arthritis.

A role for Toll-like receptor 4 in dendritic cell activation and cytolytic CD8+ T cell differentiation in response to a recombinant heat shock fusion protein.

Recombinant heat shock fusion proteins (Hsfp) injected into mice without added adjuvants can stimulate production of CD8 cytolytic T cells. Because initiation of productive immune responses generally requires dendritic cell (DC) activation, the question arises as to whether the Hsfp can activate DC independently of contaminating LPS. Using microarray analyses of DC from LPS-insensitive mice having a point mutation in Toll-like receptor 4 (Tlr4) (C3H/HeJ), or lacking Tlr4 (B10/ScNCr), we show here that unlike a LPS standard, Hsfp activated DC from HeJ mice almost as well as DC from wild-type mice. Consistent with the microarray analysis, the Hsfp's ability to activate DC was not eliminated by polymyxin B but was destroyed by proteinase K. The Hsfp did not, however, stimulate DC from mice lacking Tlr4. In vivo the CD8 T cell response to the Hsfp in mice lacking Tlr4 was impaired: the responding CD8 cells initially proliferated vigorously but their development into cytolytic effector cells was diminished. Overall, the results indicate that this Hsfp can activate DC independently of LPS but still requires Tlr4 for an optimal CD8 T cell response.

DNA vaccination with heat shock protein 60 inhibits cyclophosphamide-accelerated diabetes.

Nonobese diabetic (NOD) mice spontaneously develop diabetes as a consequence of an autoimmune process that can be inhibited by immunotherapy with the 60-kDa heat shock protein (hsp60), with its mycobacterial counterpart 65-kDa (hsp65), or with other Ags such as insulin and glutamic acid decarboxylase (GAD). Microbial infection and innate signaling via LPS or CpG motifs can also inhibit the spontaneous diabetogenic process. In addition to the spontaneous disease, however, NOD mice can develop a more robust cyclophosphamide-accelerated diabetes (CAD). In this work, we studied the effect on CAD of DNA vaccination with constructs encoding the Ags human hsp60 (phsp60) or mycobacterial hsp65 (phsp65). Vaccination with phsp60 protected NOD mice from CAD. In contrast, vaccination with phsp65, with an empty vector, or with a CpG-positive oligonucleotide was not effective, suggesting that the efficacy of the phsp60 construct might be based on regulatory hsp60 epitopes not shared with its mycobacterial counterpart, hsp65. Vaccination with phsp60 modulated the T cell responses to hsp60 and also to the GAD and insulin autoantigens; T cell proliferative responses were significantly reduced, and the pattern of cytokine secretion to hsp60, GAD, and insulin showed an increase in IL-10 and IL-5 secretion and a decrease in IFN-gamma secretion, compatible with a shift from a Th1-like toward a Th2-like autoimmune response. Our results extend the role of specific hsp60 immunomodulation in the control of beta cell autoimmunity and demonstrate that immunoregulatory networks activated by specific phsp60 vaccination can spread to other Ags targeted during the progression of diabetes, like insulin and GAD.

The beta 2-adrenergic agonist salbutamol potentiates oral induction of tolerance, suppressing adjuvant arthritis and antigen-specific immunity.

Therapeutic protocols for treating autoimmune diseases by feeding autoantigens during the disease process have not been very successful to date. In vitro it has been shown that beta-adrenergic agonists inhibit pro-inflammatory cytokine production and up-regulate anti-inflammatory cytokine production. We hypothesized that the protective effect of oral administration of Ag would be enhanced by oral coadministration of the beta(2)-adrenergic agonist salbutamol. Here we demonstrate that oral administration of salbutamol in combination with the Ag mycobacterial 65-kDa heat shock protein increased the efficacy of disease-suppressive tolerance induction in rat adjuvant arthritis. To study the mechanism of salbutamol in more detail, we also tested oral administration of salbutamol in an OVA tolerance model in BALB/c mice. Oral coadministration of OVA/salbutamol after immunization with OVA efficiently suppressed both cellular and humoral responses to OVA. Coadministration of salbutamol was associated with an immediate increase in IL-10, TGF-beta, and IL-1R antagonist in the intestine. The tolerizing effect of salbutamol/OVA was maintained for at least 12 wk. At this time point IFN-gamma production in Ag-stimulated splenocytes was increased in the OVA/salbutamol-treated animals. In conclusion, salbutamol can be of great clinical benefit for the treatment of autoimmune diseases by promoting oral tolerance induction.

Mucosal administration of heat shock protein-65 decreases atherosclerosis and inflammation in aortic arch of low-density lipoprotein receptor-deficient mice.

BACKGROUND: Increasing evidence supports the involvement of inflammation and immunity in atherogenesis as well as the role of autoimmunity to heat shock proteins (HSPs) in the progression of atherosclerosis. Mucosal administration of autoantigens decreases organ-specific inflammation and disease in several models of autoimmunity (diabetes, arthritis, and encephalomyelitis) and is also being tested in human clinical trials. METHODS AND RESULTS: We examined the effect of nasal or oral administration of mycobacterial HSP-65 on atherosclerotic lesion formation in mice lacking the receptor for LDL that were maintained on a high-cholesterol diet. Animals were nasally or orally treated for 1 week with HSP-65, and a high-cholesterol diet was started after the last treatment. The mice were mucosally treated once a week for 8 or 12 weeks, at which time pathological analysis was performed. We found a significant decrease in the size of atherosclerotic plaques, a reduction in macrophage-positive area in the aortic arch, increased interleukin-10 expression, and a reduced number of T cells in nasally treated animals compared with control animals. A similar trend was observed in orally treated mice, but it was not significant. CONCLUSIONS: Our results demonstrate that nasal vaccination with HSP reduces the inflammatory process associated with atherosclerosis and provides a new immunologic approach for the treatment of atherosclerosis.

Dynamics of mycobacterial HSP65-induced T-cell cytokine expression during oral tolerance induction in adjuvant arthritis.

OBJECTIVE: To investigate whether oral administration of mycobacterial heat-shock protein 65 (HSP65) during adjuvant arthritis (AA) induces regulatory cells and cytokines. METHODS: AA was induced in Lewis rats and from the time of disease onset HSP65 in the presence of soya bean trypsin inhibitor (STI) was administered orally every other day. The number of splenic CD4+CD25+ T cells and antigen-induced cytokine mRNA expression were determined. RESULTS: Oral treatment with HSP65/STI reduced AA symptoms. After one feeding of HSP65/STI, the number of CD4+CD25+ splenic T cells increased and HSP65-specific T cells expressed increased levels of interferon gamma and interleukin 10. After two feedings, the expression of interleukin-10 mRNA remained increased, whereas there was low expression of interferon gamma mRNA. The number of CD4+CD25+ splenic T cells remained increased. CONCLUSIONS: Oral treatment with HSP65/STI after AA onset reduces disease symptoms via dynamic changes in the number of CD4+CD25+ splenocytes and in antigen-induced cytokine production.

Resistance to adjuvant arthritis is due to protective antibodies against heat shock protein surface epitopes and the induction of IL-10 secretion.

Adjuvant arthritis (AA) is an experimental model of autoimmune arthritis that can be induced in susceptible strains of rats such as inbred Lewis upon immunization with CFA. AA cannot be induced in resistant strains like Brown-Norway or in Lewis rats after recovery from arthritis. We have previously shown that resistance to AA is due to the presence of natural as well as acquired anti-heat shock protein (HSP) Abs. In this work we have studied the fine specificity of the protective anti-HSP Abs by analysis of their interaction with a panel of overlapping peptides covering the whole HSP molecule. We found that arthritis-susceptible rats lack Abs to a small number of defined epitopes of the mycobacterial HSP65. These Abs are found naturally in resistant strains and are acquired by Lewis rats after recovery from the disease. Active vaccination of Lewis rats with the protective epitopes as well as passive vaccination with these Abs induced suppression of arthritis. Incubation of murine and human mononuclear cells with the protective Abs induced secretion of IL-10. Analysis of the primary and tertiary structure of the whole Mycobacterium tuberculosis HSP65 molecule indicated that the protective epitopes are B cell epitopes with nonconserved amino acid sequences found on the outer surface of the molecule. We conclude that HSP, the Ag that contains the pathogenic T cell epitopes in AA, also contains protective B cell epitopes exposed on its surface, and that natural and acquired resistance to AA is associated with the ability to respond to these epitopes.

Immunization with chaperone-peptide complex induces low-avidity cytotoxic T lymphocytes providing transient protection against herpes simplex virus infection.

Heat shock proteins loaded with viral peptides were shown to induce a CD8+ T cell response and confer protective immunity against challenge with herpes simplex virus (HSV). The delivery system consisted of recombinant human hsp70 coupled to the peptide SSIEFARL, which is the immunodominant peptide epitope, recognized by HSV specific T cells in C57BL/6 mice. Immunization resulted in CD8+ T-cell responses, measured by peptide-specific tetramers and peptide-induced intracellular gamma interferon expression and cytotoxicity, similar to responses resulting from immunization with a recombinant vaccinia virus that expressed SSIEFARL as a minigene (VvgB) and UV-inactivated HSV. However, the durability of the hsp70-SSIEFARL response was less than that resulting from VvgB and HSV immunization and in addition the CD8+ T-cell responses in the memory phase were functionally less effective. Mice challenged soon after immunization showed excellent immunity, but by 90 days postimmunization this had waned to be significantly less than the level of immunity in both VvgB- and HSV-immunized mice.