To investigate the role of heat shock proteins (HSP) of Yersinia enterocolitica for the host immune response against this pathogen, we cloned and expressed a 60-kDa HSP of Y. enterocolitica serotype 08. A fragment of Y. enterocolitica 08 HSP60 encoded by amino acids 90 to 286 was sequenced and showed more than 90%Yo homology with HSP60 of Y. enterocolitica 03 and GroEL of Escherichia coli and 59%o homology with HSP65 of Mycobacterium bovis. The arthritogenic T-cell epitope of mycobacterial HSP65 (amino acid residues 180 to 188) was not found on Yersinia HSP60. To determine whether Yersinia HSP60 is an immunodominant antigen, the immune responses of Yersinia-infected C57BL/6 mice were analyzed. Yersinia-infected mice evolved a significant serum antibody and splenic T-cell response against Yersinia HSP60. CD4+ at, T-cell clones which were generated from splenic T cells isolated from either Yersinia-infected or Yersinia HSP60-immunized mice, recognized both heat-killed Yersinia serotypes 03 and 08 as well as recombinant Yersinia HSP60 but not
Microbial heat shock proteins (HSP) are dominant antigens for the host immune response. Because of the high sequence homology between mammalian and microbial HSP, their value as component of a subunit vaccine has been the subject of controversy. Previous work from this laboratory, however, demonstrated for the first time that the adoptive transfer of HSP60-reactive CD4 ؉ ␣ T-cell clones confers protection against bacterial infection in mice but does not induce autoimmunity. In the present study, we have therefore evaluated the potential role of Yersinia HSP60 (Y-HSP60) as a vaccine in the Yersinia enterocolitica mouse infection model. For this purpose, immunostimulating complexes (ISCOM) which included Y-HSP60 were constructed. Parenteral administration of this vaccine induced high Y-HSP60-specific serum antibody responses as well as T-cell responses. This reaction was parallelled by immunity against a lethal challenge with Y. enterocolitica. In contrast, mucosal application of Y-HSP60-ISCOM failed to induce systemic Y-HSP60-specific T-cell responses and thus failed to induce immunity against yersiniae. Likewise, vaccination with purified recombinant Y-HSP60 induced antibody responses but only weak T-cell responses. Therefore, this vaccination protocol was not protective. However, when interleukin-12 was used as an adjuvant, purified Y-HSP60 induced significant Y-HSP60-specific T-cell responses and thus induced protection against subsequent challenge with yersiniae. These studies suggest that (i) microbial HSP might be promising candidates for the design of subunit vaccines and (ii) interleukin-12 is an efficient alternative adjuvant to ISCOM particles for induction of protective CD4 Th1-cell-dependent immune responses against bacterial pathogens.
SUMMARYHeat shock proteins (hsp) are immunodominant antigens in microbial infections. Previous work from this laboratory demonstrated that Yersinia-hsp60 (Y-hsp60)-reactive CD4 + ® ¯T cells play an important role for resolution of Y. enterocolitica infections in mice. In the present study we identified two epitopes of Y-hsp60 recognized by CD4 Th1 cell clones. The epitopes comprise 12 (214-225) and 13 (74-86) amino acid (aa) residues of Y-hsp60, and are the first described for MHC class II (I-A b ) molecules. Both epitopes are also recognized by T cells isolated from mesenteric lymph nodes from mice orogastrically infected with yersiniae. Stimulation of T cells with peptides of 12 and 13 aa residues of Y-hsp60 caused highly efficient proliferation compared with longer peptides, full-length recombinant Y-hsp60, or heatkilled Yersinia (HKY). Incubation of antigen-presenting cells with chloroquine blocked both peptide and HKY-triggered T cell proliferation, whereas cytochalasin B only blocked HKY-induced proliferation and to a lesser extent peptide-induced proliferation. The identified epitopes reside in a region of Yhsp60 that is conserved between Enterobacteriaceae but highly variable when compared with murine or human hsp60. Although both epitopes are identical to the related sequence of hsp60 (GroEL) of Escherichia coli, only weak T cell responses were observed upon stimulation with GroEL of E. coli, suggesting that other factors, e.g. flanking amino acid residues, might be important for antigen processing and T cell stimulation in a class II-restricted manner. Furthermore, these observations might be of significance for the rational design of subunit vaccines.
Naked plasmid DNA (pRc/Y-hsp60) with a cytomegalovirus promoter and a sequence encoding Yersinia enterocolitica 60-kDa heat shock protein (Y-HSP60) was used for vaccination. After intramuscular injection of pRc/Y-hsp60, Y-hsp60 mRNA could be detected by reverse transcription-PCR in muscle, liver and spleen. A single immunization with pRc/Y-hsp60 induced significant Y-HSP60-specific T cell responses after 1 week. IFN-gamma production by spleen cells upon stimulation with Y-HSP60 was strictly dependent on the presence of CD4+ T cells, indicating the generation of a Th1 response upon DNA immunization. DNA immunization in addition induced strong Y-HSP60-specific IgG2a, weak IgG1, but not IgA antibodies. Immunization of BALB/c and C57BL/6 mice with pRc/Y-hsp60 conferred protection against disseminated Y. enterocolitica infection in spleen, but not at the site of mucosal entry, the Peyer's patches. Furthermore, pRc/Y-hsp60 vaccination did not induce cross-protection against related pathogens. Vaccination of beta2-microglobulin- and H2-I-Abeta-deficient mice was not protective, suggesting that both CD4+ and CD8+ T cells are required for protective immunity induced by DNA vaccination.
Endogenous interleukin-12 (IL-12) mediates protection againstYersinia enterocolitica in C57BL/6 mice by triggering gamma interferon (IFN-γ) production in NK and CD4+ T cells. Administration of exogenous IL-12 confers protection against yersiniae in Yersinia-susceptible BALB/c mice but exacerbates yersiniosis in resistant C57BL/6 mice. Therefore, we wanted to dissect the different mechanisms exerted by IL-12 during Yersiniainfections by using different models of Yersinia-resistant and -susceptible mice, including resistant C57BL/6 mice, susceptible BALB/c mice, intermediate-susceptible wild-type 129/Sv mice, 129/Sv IFN-γ-receptor-deficient (IFN-γR−/−) mice and C57BL/6 tumor necrosis factor (TNF) receptor p55 chain-deficient (TNFR p55−/−) mice. IFN-γR−/− mice turned out to be highly susceptible to infection by Y. enterocoliticacompared with IFN-γR+/+ mice. Administration of IL-12 was protective in IFN-γR+/+ mice but not in IFN-γR−/− mice, suggesting that IFN-γR-induced mechanisms are essential for IL-12-induced resistance against yersiniae. BALB/c mice could be rendered Yersinia resistant by administration of anti-CD4 antibodies or by administration of IL-12. In contrast, C57BL/6 mice could be rendered more resistant by administration of transforming growth factor β (TGF-β). Furthermore, IL-12-triggered toxic effects in C57BL/6 mice were abrogated by coadministration of TGF-β. While administration of IL-12 alone increased TNF-α levels, administration of TGF-β or TGF-β plus IL-12 decreased both TNF-α and IFN-γ levels inYersinia-infected C57BL/6 mice. Moreover, IL-12 did not induce toxicity in Yersinia-infected TNFR p55−/− mice, suggesting that TNF-α accounts for IL-12-induced toxicity. Taken together, IL-12 may induce different effector mechanisms in BALB/c and C57BL/6 mice resulting either in protection or exacerbation. These results are important for understanding the critical balance of proinflammatory and regulatory cytokines in bacterial infections which is decisive for beneficial effects of cytokine therapy.
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