DNAhsp65 vaccination induces protection in mice against Paracoccidioides brasiliensis infection.

Heat-shock proteins are molecules with extensive data showing their potential as immunomodulators of different types of diseases. The gene of HSP65 from Mycobacterium leprae has shown prophylactic and immunotherapeutic effects against a broad arrays of experimental models including tuberculosis, leishmaniasis, arthritis and diabetes. With this in mind, we tested the DNAhsp65 vaccine using an experimental model of Paraccocidiodomycosis, an important endemic mycosis in Latin America. The intramuscular immunization with DNAhsp65 induced, in BALB/c mice, an increase of Th1-levels cytokines and a reduction of fungal burdens resulted in a marked reduction of collagen and lung remodeling. DNAhsp65 may be an attractive candidate for prevention, therapy and as an adjuvant for mycosis treatment.

Protective efficacy of different strategies employing Mycobacterium leprae heat-shock protein 65 against tuberculosis.

BACKGROUND: Tuberculosis is a major threat to human health. The high disease burden remains unaffected and the appearance of extremely drug-resistant strains in different parts of the world argues in favor of the urgent need for a new effective vaccine. One of the promising candidates is heat-shock protein 65 when used as a genetic vaccine (DNAhsp65). Nonetheless, there are substantial data indicating that BCG, the only available anti-TB vaccine for clinical use, provides other important beneficial effects in immunized infants. METHODS: We compared the protective efficacy of BCG and Hsp65 antigens in mice using different strategies: i) BCG, single dose subcutaneously; ii) naked DNAhsp65, four doses, intramuscularly; iii) liposomes containing DNAhsp65, single dose, intranasally; iv) microspheres containing DNAhsp65 or rHsp65, single dose, intramuscularly; and v) prime-boost with subcutaneous BCG and intramuscular DNAhsp65. RESULTS: All the immunization protocols were able to protect mice against infection, with special benefits provided by DNAhsp65 in liposomes and prime-boost strategies. CONCLUSION: Among the immunization protocols tested, liposomes containing DNAhsp65 represent the most promising strategy for the development of a new anti-TB vaccine.

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.

Immune modulation induced by tuberculosis DNA vaccine protects non-obese diabetic mice from diabetes progression.

We have described previously the prophylactic and therapeutic effect of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in experimental murine tuberculosis. However, the high homology of this protein to the corresponding mammalian 60 kDa heat shock protein (Hsp60), together with the CpG motifs in the plasmid vector, could trigger or exacerbate the development of autoimmune diseases. The non-obese diabetic (NOD) mouse develops insulin-dependent diabetes mellitus (IDDM) spontaneously as a consequence of an autoimmune process that leads to destruction of the insulin-producing beta cells of the pancreas. IDDM is characterized by increased T helper 1 (Th1) cell responses toward several autoantigens, including Hsp60, glutamic acid decarboxylase and insulin. In the present study, we evaluated the potential of DNA-HSP65 injection to modulate diabetes in NOD mice. Our results show that DNA-HSP65 or DNA empty vector had no diabetogenic effect and actually protected NOD mice against the development of severe diabetes. However, this effect was more pronounced in DNA-HSP65-injected mice. The protective effect of DNA-HSP65 injection was associated with a clear shift in the cellular infiltration pattern in the pancreas. This change included reduction of CD4(+) and CD8(+) T cells infiltration, appearance of CD25(+) cells influx and an increased staining for interleukin (IL)-10 in the islets. These results show that DNA-HSP65 can protect NOD mice against diabetes and can therefore be considered in the development of new immunotherapeutic strategies.

Immunogenicity of Mycobacterium ulcerans Hsp65 and protective efficacy of a Mycobacterium leprae Hsp65-based DNA vaccine against Buruli ulcer.

Buruli ulcer, a disease caused by Mycobacterium ulcerans, is emerging as an increasingly important cause of morbidity throughout the world, for which surgery is the only efficient treatment to date. The aim of this work was to identify potential vaccine candidates in an experimental model of mouse infection. In BALB/c mice infected with M. ulcerans subcutaneously, Hsp65 appeared to be an immunodominant antigen eliciting both humoral and cellular responses. However, vaccination of mice with a DNA vector encoding Mycobacterium leprae Hsp65 only poorly limited the progression of M. ulcerans infection. In contrast, a substantial degree of protection was conferred by subcutaneous vaccination with BCG, suggesting that BCG antigens that are conserved in M. ulcerans, such as TB10.4, the 19 kDa antigen, PstS3 and Hsp70, may be interesting to consider as subunit vaccines in future prospects.

NK cells modulate the cytotoxic activity generated by Mycobacterium leprae-hsp65 in leprosy patients: role of IL-18 and IL-13.

Protection against intracellular pathogens such as Mycobacterium leprae is critically dependent on the function of NK cells at early stages of the immune response and on Th1 cells at later stages. In the present report we evaluated the role of IL-18 and IL-13, two cytokines that can influence NK cell activity, in the generation of M. leprae-derived hsp65-cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) of leprosy patients. We demonstrated that IL-18 modulates hsp65-induced CTL generation and collaborates with IL-12 for this effect. In paucibacillary (PB) patients and normal controls (N) depletion of NK cells reduces the cytolytic activity. Under these conditions, IL-12 cannot up-regulate this CTL generation, while, in contrast, IL-18 increases the cytotoxic activity both in the presence or absence of NK cells. IL-13 down-regulates the hsp65-induced CTL generation and counteracts the positive effect of IL-18. The negative effect of IL-13 is observed in the early stages of the response, suggesting that this cytokine affects IFNgamma production by NK cells. mRNA coding for IFNgamma is induced by IL-18 and reduced in the presence of IL-13, when PBMC from N or PB patients are stimulated with hsp65. Neutralization of IL-13 in PBMC from multibacillary (MB) leprosy patients induces the production of IFNgamma protein by lymphocytes. A modulatory role on the generation of hsp65 induced CTL is demonstrated for IL-18 and IL-13 and this effect takes place through the production of IFNgamma.

Anti-mycobacterial immunity induced by a single injection of M. leprae Hsp65-encoding plasmid DNA in biodegradable microparticles.

A single sub-cutaneous injection of a plasmid DNA encoding a mycobacterial heat shock protein 65 (Hsp65) entrapped in biodegradable poly(lactic-co-glycolic acid) microspheres produced high titers of antibodies, measured 5 months after the injection in BALB/c mice. Splenocytes secreted IFN-gamma and exerted an anti-bacterial effect on macrophages infected in vitro with Mycobacterium tuberculosis. The results are encouraging with regard to obtaining good compliance and vaccination coverage with candidate plasmid DNA vaccines, especially in developing countries.

Effect of hsp65 DNA vaccination carrying immunostimulatory DNA sequences (CpG motifs) against Mycobacterium leprae multiplication in mice.

A DNA expressing hsp65 of Mycobacterium leprae (pACB/hsp65) was constructed by using a vector containing immunostimulatory DNA sequences (pACB). At 12 weeks post-immunization, spleen cells from BALB/cA mice immunized with pACB/hsp65, produced a significantly higher amount of IFN-gamma than mice immunized with pACB in the absence of any in vitro stimulation, and further enhanced its production upon secondary in vitro stimulation with M. leprae lysate and hsp65. On the other hand, while production of IL-12 was observed in mice immunized with pACB/hsp65 12 weeks before, the cytokine production was inhibited by in vitro secondary stimulation with M. leprae or hsp65. At 18 weeks post-immunization, the production of both IFN-gamma and IL-12 was apparently down-regulated, but that of IL-10 was up-regulated. IL-10 seemed to suppress the IFN-gamma and IL-12 productions, because their production was recovered by neutralization of IL-10 with anti-IL-10 mAb. Furthermore, when the efficiency of pACB/hsp65 as a vaccine against M. leprae was evaluated in vivo, the mice immunized with pACB/hsp65 suppressed the multiplication of subsequently challenged M. leprae. These results suggest that a DNA containing M. leprae-derived hsp65 and immunostimulatory sequences might be a potent vaccine candidate against M. leprae infection.

Identification of an I-Ad restricted peptide on the 65-kilodalton heat shock protein of Mycobacterium avium.

The 65 kilodalton heat shock protein (Hsp65) from mycobacterial species elicits immune responses and in some cases protective immunity. Here we have used a DNA sublibrary approach to identify antigenic fragments of Mycobacterium avium Hsp65 and a synthetic peptide approach to delineate CD4+ T cell determinants. A panel of Hsp65 reactive CD4+ T cell clones was established from lymph node cells obtained from BALB/c mice immunized with recombinant Hsp65. The clones were tested for proliferative reactivity against the products of the DNA sublibrary of the hsp65 gene. A T cell epitope, restricted by the I-Ad molecule, was identified within the C-terminal region of Hsp65 and the minimal epitope (amino acid residues 489-503) delineated using overlapping peptides spanning the C-terminal fragment. Additionally, the CD4+ T cell clone recognizing this epitope also responded to native Hsp65 present in M. avium lysates by both proliferation and cytokine production, indicating that the epitope was present and processed similarly both in the native and the recombinant forms of Hsp65. This sequence identified in BALB/c mice (Hsp65 489-503) is identical in other mycobacteria, notably M. tuberculosis, M. bovis and M. leprae, suggesting the epitope may have wider application in murine models of other mycobacterial infections.

The detection of Mycobacterium leprae protein and carbohydrate antigens in skin and nerve from leprosy patients with type 1 (reversal) reactions.

Type 1 (reversal) reactions are the most common immunological complications of leprosy. These episodes of delayed hypersensitivity produce severe local immunopathology and ultimately nerve damage. To date, the Mycobacterium leprae antigens associated with type 1 reactions have not been identified. Using monoclonal antibodies to defined protein and carbohydrate M. leprae epitopes (65, 35 and 28 kd and lipoarabinomannan [LAM]) in a two-step immunoperoxidase staining technique, M. leprae antigens were demonstrated in skin and nerve biopsies from patients in reversal reaction. Antigen presence and staining patterns were similar in skin and nerve lesions, implying that the pathological processes are similar in the two sites. Antigens were present both in macrophages and Schwann cells but also as a diffuse extracellular infiltrate associated with the inflammatory infiltrate. The 28-kd antigen was present most strongly and may be a potential candidate antigen for initiating type 1 reactions. LAM also stained strongly and persisted after treatment. The possible roles of LAM and 65 kd in the cellular events of type 1 reactions are discussed.

Cellular immune response to Klebsiella pneumoniae antigens in patients with HLA-B27+ ankylosing spondylitis.

OBJECTIVE: To study the reactivity of peripheral blood mononuclear cells (PBMC) of patients with ankylosing spondylitis (AS) and rheumatoid arthritis (RA) and healthy controls to Klebsiella pneumoniae antigens and to the GroEL-like proteins from K. pneumoniae (HSP60Kp) and Mycobacterium leprae recombinant heat shock protein 65 (rHSP65Ml). METHODS: PBMC of 13 patients with AS and 9 with RA and 10 controls were stimulated in vitro by heat shock induced K. pneumoniae antigens in a cell blot assay, by insolubilized HSP60Kp, by cytosolic proteins (CP) from K. pneumoniae cultivated at 37 degrees C or 45 degrees C, by soluble HSP60Kp, or by rHSP65Ml. RESULTS: In the cell blot assay 7/13 AS and 3/9 RA patients responded to fraction 4, which contains mainly HSP60Kp, and no controls responded (AS vs. controls: p = 0.007). The response to the insolubilized HSP60Kp was positive in 6/13 AS patients but negative in RA patients and controls (p = 0.004). The response to CP45 degrees C was positive in 7/13 AS, in 2/9 RA, and no controls (AS vs controls: p<0.015). Response to the soluble HSP60Kp was found in 7/13 AS and 5/9 RA patients, but no controls (AS vs. controls: p = 0.0075). Response to rHSP65Ml was positive in 3/13 AS, 7/9 RA patients, and 1/10 controls (AS vs RA: p = 0.027; RA vs. controls: p = 0.005; AS vs. controls: nonsignificant). CONCLUSION: In PBMC of the majority of patients with AS and in some with RA, but not in healthy controls, there are cells that proliferate in the presence of HSP60 of K. pneumoniae.

Interleukin-12 amplifies the M. leprae hsp65-cytotoxic response in the presence of tumour necrosis factor-alpha and interferon-gamma generating CD56+ effector cells: interleukin-4 downregulates this effect.

Interleukin-12 (IL-12) is a major immunomodulatory cytokine that represents a functional bridge between the early resistance and the subsequent antigen specific adaptive immunity. TNF-alpha and IFN-gamma have an important role in the generation of hsp65 specific cytotoxic T lymphocytes (CTL) that lyse hsp65-pulsed autologous macrophages (hsp65 CTL). Since a positive feedback mechanism between TNF-alpha, IFN-gamma and IL-12 has been described, we undertook to evaluate the role of IL-12 on the hsp65 CTL generation in leprosy patients. Our results show that the presence of IL-12 during the first 24 h of the in vitro antigen stimulation amplifies the hsp65 cytotoxic response whenever both IFN-gamma and TNF-alpha are present. The addition of these three cytokines (CKs) was able to abrogate the inhibitory effect of IL-10 on hsp65 CTL in cells from paucibacillary patients (PB) but not that of IL-4 in PB and normal controls (N). Both IL-12 or anti IL-4 enhanced the cytotoxic activity in cells from multibacillary patients (MB). Anti IL-4 upregulated the binding of IFN-gamma and did not modify that of TNF-alpha so the low CTL activity could be as a result of IL-4 by a decrease of the IFN-gamma binding on MB cells. Cells from those MB patients taking thalidomide (MB-T) did neither bind IFN-gamma nor TNF-alpha even when antigen or anti-IL-4 were added, demonstrating that thalidomide inhibits either the in vitro binding or receptor expression of both TNF-alpha and IFN-gamma. Development of CD56 effector cells during the hsp65 stimulation was observed in PB and N by the addition of IL-12 plus TNF-alpha and IFN-gamma, while in MB and MB-T anti IL-4 was also required. So, the inhibitory effect of IL-4 on either production of IFN-gamma, TNF-alpha and/or IL-12 or their receptors could be the mechanism underlying the lack of the hsp65 CTL generation in cells from MB.

Identification of promiscuous epitopes from the Mycobacterial 65-kilodalton heat shock protein recognized by human CD4(+) T cells of the Mycobacterium leprae memory repertoire.

By using a synthetic peptide approach, we mapped epitopes from the mycobacterial 65-kDa heat shock protein (HSP65) recognized by human T cells belonging to the Mycobacterium leprae memory repertoire. A panel of HSP65 reactive CD4(+) T-cell lines and clones were established from healthy donors 8 years after immunization with heat-killed M. leprae and then tested for proliferative reactivity against overlapping peptides comprising both the M. leprae and Mycobacterium tuberculosis HSP65 sequences. The results showed that the antigen-specific T-cell lines and clones established responded to 12 mycobacterial HSP65 peptides, of which 9 peptides represented epitopes crossreactive between the M. tuberculosis and M. leprae HSP65 (amino acids [aa] 61 to 75, 141 to 155, 151 to 165, 331 to 345, 371 to 385, 411 to 425, 431 to 445, 441 to 455, and 501 to 515) and 3 peptides (aa 343 to 355, 417 to 429, and 522 to 534) represented M. leprae HSP65-specific epitopes. Major histocompatibility complex restriction analysis showed that presentation of 9 of the 12 peptides to T cells were restricted by one of the 2 HLA-DR molecules expressed from self HLA-DRB1 genes, whereas 3 peptides with sequences completely identical between the M. leprae and M. tuberculosis HSP65 were presented to T cells by multiple HLA-DR molecules: peptide (aa 61 to 75) was presented by HLA-DR1, -DR2, and -DR7, peptide (aa 141 to 155) was presented by HLA-DR2, -DR7, and -DR53, whereas both HLA-DR2 and -DR4 (Dw4 and Dw14) were able to present peptide (aa 501 to 515) to T cells. In addition, the T-cell lines responding to these peptides in proliferation assays showed cytotoxic activity against autologous monocytes/macrophages pulsed with the same HSP65 peptides. In conclusion, we demonstrated that promiscuous peptide epitopes from the mycobacterial HSP65 antigen can serve as targets for cytotoxic CD4(+) T cells which belong to the human memory T-cell repertoire against M. leprae. The results suggest that such epitopes might be used in the peptide-based design of subunit vaccines against mycobacterial diseases.

Immunohistological analysis of in situ expression of mycobacterial antigens in skin lesions of leprosy patients across the histopathological spectrum. Association of Mycobacterial lipoarabinomannan (LAM) and Mycobacterium leprae phenolic glycolipid-I (PGL-I) with leprosy reactions.

The presence of mycobacterial antigens in leprosy skin lesions was studied by immunohistological methods using monoclonal antibodies (MAbs) to Mycobacterium leprae-specific phenolic glycolipid I (PGL-I) and to cross-reactive mycobacterial antigens of 36 kd, 65 kd, and lipoarabinomannan (LAM). The staining patterns with MAb to 36 kd and 65 kd were heterogeneous and were also seen in the lesions of other skin diseases. The in situ staining of PGL-I and LAM was seen only in leprosy. Both antigens were abundantly present in infiltrating macrophages in the lesions of untreated multibacillary (MB) patients, whereas only PGL-I was occasionally seen in scattered macrophages in untreated paucibacillary lesions. During treatment, clearance of PGL-I from granulomas in MB lesions occurred before that of LAM, although the former persisted in scattered macrophages in some treated patients. This persistence of PGL-I in the lesions paralleled high serum anti-PGL-I antibody titers but was not indicative for the presence of viable bacilli in the lesions. Interestingly, we also observed a differential expression pattern of PGL-I and LAM in the lesions of MB patients with reactions during the course of the disease as compared with those without reactions. In conclusion, the in situ expression pattern of PGL-I and LAM in MB patients may assist in early diagnosis of reactions versus relapse.

Interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) are necessary in the early stages of induction of CD4 and CD8 cytotoxic T cells by Mycobacterium leprae heat shock protein (hsp) 65 kD.

Cytotoxic T cells (CTL) may play an important role in host defence against mycobacterial infections. CD4 CTL are preferentially induced by mycobacteria, but both CD4 and CD8 CTL may be necessary components of a protective immune response. The 65-kD mycobacterium heat shock protein (hsp65) is a poor inducer of CTL in multibacillary leprosy (MB) patients. In this study we evaluate the possible role of cytokines in modulating the cytotoxic activity of CTL from leprosy patients and normal individuals (N) against autologous macrophages presenting Mycobacterium leprae hsp65. Our results show that hsp65-specific CTL were generated from both CD4 and CD8 lymphocytes. In N, individual cytokines as well as the combination of them were able to modify the hsp65-induced cytotoxic activity. The effect of cytokines on leprosy patients' lymphocytes was different in MB and paucibacillary (PB) patients. Thus, IL-6, IL-2, IFN-gamma or TNF-alpha did not modify the generation of hsp65-CTL from either MB (with or without an erythema nodosum episode (ENL)) or PB. In all the patients the simultaneous addition of two cytokines was required in order to increase CTL generation. In MB, IL-6 plus IFN-gamma or IL-2 increased both CD4 and CD8 CTL, while TNF-alpha plus IFN-gamma up-regulated only CD4 CTL. In PB, CD8 CTL were prominent with IL-6 plus IFN-gamma, while the increase was significant in CD4 CTL with IL-6 plus IL-2. Down-regulation of CTL was observed by addition of IL-4, IL-10, anti-IFN-gamma or anti-TNF-alpha in N controls. Our data demonstrate that IFN-gamma and TNF-alpha must be present for at least the first 60 h of the induction stage in order to generate full hsp65 CTL. Hence, IFN-gamma and TNF-alpha would be key factors in the generation of hsp65 CTL.

Antibody response to Klebsiella pneumoniae 60 kDa protein in familial and sporadic ankylosing spondylitis: role of HLA-B27 and characterization as a GroEL-like protein.

OBJECTIVE: To study the antibody response of HLA-B27+ patients with ankylosing spondylitis (AS) and their first degree relatives to the 60 kDa protein of Klebsiella pneumoniae and to characterize this protein. METHODS: Sera from 84 individuals were analyzed by ELISA to determine the titer of antibodies against the 60 kDa protein of K. pneumoniae. Subjects were divided into 3 categories: Group 1: 44 HLA-B27+ AS related individuals (35 patients, 9 healthy controls); Group 2: 28 healthy B27- AS related individuals; and Group 3: 12 healthy B27- non-AS related subjects. The 60 kDa protein of K. pneumoniae was induced at 45 degrees C and purified by electroelution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was characterized as a GroEL-like heat shock protein (HSP). The recognition of GroEL-like protein was confirmed by immunoblot of 2 dimension electrophoresis. The response to GroEL-like protein from other bacteria and the response to lipopolysaccharide (LPS) was also analyzed by immunoblot. RESULTS: HLA-B27+ individuals (Group 1), independent of their disease status, showed a significant higher response to the 60 kDa protein of K. pneumoniae than HLA-B27- subjects from Groups 2 and 3 (p < 0.0001). This protein was characterized as a HSP of the GroEL family and designated HSP60Kp. The GroEL of other enterobacteria as well as that of Mycobacterium leprae were recognized by HLA-B27+ individuals by immunoblot, whereas HLA-B27- individuals did not. LPS was not recognized by HLA-B27 positive or negative subjects. CONCLUSION: These findings suggest a relationship between HLA-B27 and the response to a GroEL-like protein that could have implications in AS.