Immunity against Yersinia enterocolitica by vaccination with Yersinia HSP60 immunostimulating complexes or Yersinia HSP60 plus interleukin-12.

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+ alphabeta 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.     

Immune responses to Yersinia enterocolitica in susceptible BALB/c and resistant C57BL/6 mice: an essential role for gamma interferon.

Susceptibility of mice to infection with Yersinia enterocolitica has been shown to be related to neither the Ity locus encoding for resistance to Salmonella typhimurium and other pathogens nor the H-2 locus. Recent studies in our laboratory have demonstrated that T-cell-mediated immune responses are required for overcoming primary Yersinia infection. In the present study, we investigated the course of infection with Y. enterocolitica and the resulting immune responses in Yersinia-susceptible BALB/c and Yersinia-resistant C57BL/6 mice. In the early phase of infection, the clearance of the pathogen was comparable in both strains of mice, suggesting similar mechanisms of innate resistance. Splenic T cells from Yersinia-infected C57BL/6 mice exhibited marked proliferative responses and produced gamma interferon (IFN-gamma) upon exposure to heat-killed yersiniae. By contrast, the Yersinia-specific T-cell response in BALB/c mice was weak, and IFN-gamma production could not be detected before day 21 postinfection. T cells isolated from C57BL/6 mice 7 days after infection mediated immunity to Y. enterocolitica but those from BALB/c mice did not, while at 21 days postinfection T cells from both strains mediated protection. Neutralization of IFN-gamma abrogated resistance to yersiniae in C57BL/6 mice but to a far smaller extent in BALB/c mice. Administration of recombinant IFN-gamma or anti-interleukin-4 antibodies rendered BALB/c mice resistant to yersiniae, whereas this treatment did not significantly affect the course of the infection in C57BL/6 mice. These results indicate that the cellular immune response, in particular the production of IFN-gamma by Yersinia-specific T cells, is associated with resistance of mice to Y. enterocolitica.     

T lymphocytes mediate protection against Yersinia enterocolitica in mice: characterization of murine T-cell clones specific for Y. enterocolitica.

Yersinia enterocolitica is enteropathogenic for humans and rodents, causing intestinal and extraintestinal diseases. The cellular immune response of the infected host has not yet been analyzed in detail. Therefore, we used a parenteral mouse infection model to determine the role of T lymphocytes in immunity against Y. enterocolitica. We report the generation and characterization of Y. enterocolitica-specific T-cell clones isolated from spleens of intravenously infected C57BL/6 mice. The T-cell clones obtained showed the phenotype of helper T cells (L3T4) or cytotoxic T cells (Lyt2). All T-cell clones were positive for the interleukin-2 (IL-2) receptor (Tac antigen, p55 subunit) and were negative for the gamma delta T-cell receptor. L3T4+ clones produced small quantities of IL-2 (less than 1 U/ml) when stimulated with heat-killed Y. enterocolitica, whereas Lyt2+ clones produced no or extremely low levels of IL-2. In contrast to IL-2 production, both L3T4+ and Lyt2+ T-cell clones produced considerable quantities of gamma interferon (500 U/ml). When transferred into nonimmune mice, some of the L3T4+, as well as the Lyt2+, T-cell clones could mediate at least partial protection against a challenge of a lethal dose of Y. enterocolitica. These data demonstrate for the first time the generation and characterization of Y. enterocolitica-specific T-cell clones and provide evidence that T cells may be involved in protection against enteropathogenic Y. enterocolitica.     

Isolation of Yersinia enterocolitica serovar O8 from free-living small rodents in Japan.

Yersinia species were isolated from 65 of 223 free-living small mammals trapped in 10 regions on Honshu Island in Japan. Of the 65 strains isolated, 1 was Yersinia enterocolitica serovar O3, 8 were Y. enterocolitica O5, 6 were Y. enterocolitica O8, 3 were Y. enterocolitica O9, and 1 was Yersinia pseudotuberculosis 4b. Of the six Y. enterocolitica O8 strains, five were positive for autoagglutination, Ca2+ dependence, and the 45-MDa virulence plasmid and showed high pathogenicity for mice.     

Use of recombinant antigens expressed in Escherichia coli K-12 to map B-cell and T-cell epitopes on the immunodominant 65-kilodalton protein of Mycobacterium bovis BCG

In gene libraries of Mycobacterium bovis BCG, Mycobacterium tuberculosis, and Mycobacterium leprae, recombinants were frequently encountered that expressed an immunodominant 65-kilodalton (kDa) protein antigen that was shown to react with a high proportion of mycobacterium-reactive human and murine T cells and murine monoclonal antibodies. In this study, recombinant antigens were used to map T-cell and B-cell epitopes on the M. bovis BCG 65-kDa protein that was previously designated MbaA. Four different T-cell-epitope-containing regions (amino acid residues 1 through 16, 17 through 61, 85 through 108, and 235 through 279) were defined that were recognized by seven T-cell clones from patients with tuberculoid leprosy. These regions are distinct from two previously described T-cell epitopes recognized by T cells from a tuberculosis patient. As T-cell clones restricted by different class II determinants were shown to be specific for different regions on the 65-kDa protein, the presented data suggested that the products of different human leukocyte antigen class II loci and alleles present different parts of MbaA to the immune system. B-cell epitopes recognized by 20 monoclonal antibodies were assigned to eight different regions of MbaA. Using 15 of these antibodies, we previously showed that MbaA was antigenically related to a common antigen present in many bacterial species. The dispersed localization of the involved epitopes defined here shows that various different parts of MbaA are indeed conserved. These results show that well-defined recombinant antigens are useful tools for the localization of both B- and T-cell-epitope-containing regions of a protein. Peptides synthesized from the sequences of such regions may then exactly define the epitopes relevant for the development of specific diagnostic tests or of vaccines against mycobacteria.     

Cross-resistance to fecal excretion of Yersinia enterocolitica in mice by oral vaccination of killed cells.

Mice vaccinated orally with heat-killed cells of serovar O3 of Yersinia enterocolitica were protected only against fecal excretion of the homologous serovar, whereas Formalin-killed cells provided cross-protection against serovar O9 and vice versa. Serovar IVA of Yersinia pseudotuberculosis, when Formalin-killed, also provided cross-protection against Y. enterocolitica. On the other hand, Serratia liquefaciens and serovar O6 strains did not furnish cross-protection.     

Arginine-143 of Yersinia enterocolitica YopP Crucially Determines Isotype-Related NF-κB Suppression and Apoptosis Induction in Macrophages

Pathogenic Yersinia spp. counteract host defense mechanisms by modulating the cellular signal relay in response to infection. Subversion of the antiapoptotic NF-kappaB signaling pathway by the Yersinia enterocolitica virulence protein YopP crucially determines the induction of apoptosis in Yersinia-infected macrophages. Here, we analyzed a panel of pathogenic, phylogenetically distinct Y. enterocolitica serotypes for their abilities to trigger macrophage apoptosis. Y. enterocolitica from the highly pathogenic serogroup O8 was substantially more effective in apoptosis induction than Yersinia from the serogroups O3 and O9. Complementation of yopP-knockout mutants revealed that this effect was specifically conferred by the serogroup O8 YopP. The amino acid sequences of YopPO8 and YopPO9 share 94% identity, and both YopP isotypes were found to interact with the NF-kappaB-activating kinase IKKbeta in macrophages. However, selectively, YopPO8 mediated efficient inhibition of IKKbeta activities, which led to substantial suppression of NF-kappaB activation. To localize the YopPO8-related effector domain, we interchanged stretches of amino acids and single amino acid residues between YopPO8 and YopPO9. Functional characterization of the resulting mutants revealed a major role of the arginine-143 residue in determining the inhibitory impact of YopP on IKKbeta activity and survival of macrophages.     

Immortalized B-lymphocytes from rheumatoid synovial tissue show specificity for bacterial HSP 60

Several studies indicate a pathogenetic role of T-lymphocytes with specificity for heat shock proteins (HSP) in rheumatoid arthritis (RA). Surprisingly, there are no experimental data for B-lymphocytes with specificity for HSP. To investigate whether B-lymphocytes from rheumatoid synovial tissue show a specificity for HSP 60 we immortalized synovial tissue B-lymphocytes by the electrofusion technique and tested the specificity of the B-cell clones for HSP 60 by ELISA. Tissue samples from four patients with classic, active RA were used in this study. The isolated cells were electrofused in strongly hypo-osmolar medium with cells either of the mouse strain X63-Ag8-653 (Ag8) or the heteromyeloma strain HAB-1. Clones positive for IgG, the IgG fraction of the supernatant of the isolated synovial cells and the IgG of the serum of the patients were tested in an ELISA for reactivity to the recombinant HSP 60 of Yersinia enterocolitica, which shows great homology with mycobacterial HSP 65 and human HSP 60. The expression of this HSP 60 was studied in normal and rheumatoid synovial tissue using a polyclonal rabbit serum against HSP 60 from Y. enterocolitica (Ye HSP 60). In this way we investigate differences in the expression of HSP 60 and compared the pattern of this HSP60 with the pattern of mycobacterial HSP65 and human HSP 60 described by others. In three of four patients 10 IgG secreting B-cell clones showing a specificity for HSP 60 were detected. IgG specific for HSP 60 was also detected in the supernatant of the isolated synovial cells before fusion and in the serum of these patients. HSP 60 was demonstrated immunohistochemically within the rheumatoid synovial tissue and showed stronger expression with a different distribution when compared with the expression in normal synovial tissue. B-cell clones from rheumatoid synovial tissue thus exhibit a specificity for bacterial HSP 60, and a monospecific rabbit serum against this HSP shows strong reactivity within the rheumatoid synovial tissue. It may be postulated that a humoral HSP 60 response, initially directed against an infectious agent, could react with cross-reactive epitopes of rheumatoid synovial tissue or with self-HSP perpetuating the local inflammatory process.     

Genes for immunodominant protein antigens are highly homologous in Mycobacterium tuberculosis, Mycobacterium africanum, and the vaccine strain Mycobacterium bovis BCG.

The relatedness of immunodominant protein antigens in Mycobacterium tuberculosis, M. africanum, and M. bovis BCG was investigated by comparing the genes that encode major protein antigens in M. tuberculosis with their counterparts in the other two mycobacteria. Genes encoding homologs of M. tuberculosis major protein antigens were isolated from M. africanum and M. bovis BCG by constructing lambda gt11 recombinant DNA expression libraries and screening them with murine monoclonal antibodies and DNA probes. The antibodies were directed against four major protein antigens of M. tuberculosis with molecular masses of 71, 65, 19, and 14 kilodaltons. The isolated M. africanum and M. bovis BCG DNA clones were mapped with restriction endonucleases, and the maps of the mycobacterial genes were confirmed by Southern analysis of mycobacterial genomic DNA. The restriction maps of DNA containing the four genes in M. tuberculosis, M. africanum, and M. bovis BCG are identical, indicating that the immunodominant proteins that they encode are highly homologous in the three mycobacteria. Thus, the immunity against tuberculosis engendered by M. bovis BCG vaccination could be provided, at least in part, by the immune response to these homologous antigens.     

Role of YadA-mediated collagen binding in arthritogenicity of Yersinia enterocolitica serotype O:8: experimental studies with rats.

Outer membrane protein YadA, Yersinia adhesin, is one of the plasmid-encoded virulence factors of yersiniae. YadA protects bacteria against host defense through several different mechanisms. One important role of YadA is to mediate binding to several collagen types. Our recent study revealed that a yadA null mutant of Yersinia enterocolitica serotype O:8 has a drastically reduced arthritogenic capacity when injected intravenously into Lewis rats. To further characterize the arthritogenic role of YadA, we repeated the rat experiments with strain Y. enterocolitica O:8/pYV082; this strain expresses a YadA deletion derivative lacking 22 amino acids from the amino-terminal hydrophobic region and does not bind to collagen. Y. enterocolitica O:8/pYV082 induced arthritis in 5 to 14% of rats inoculated with arthritogenic doses, whereas the arthritis incidence with the wild-type parent strain was 65%. The parent strain was slightly more virulent than Y. enterocolitica O:8/pYV082, as determined by rat mortality. It also frequently induced skin abscesses, whereas Y. enterocolitica O:8/pYV082 did not. Infection kinetics in spleen and mesenteric lymph nodes were about the same with both of the bacterial strains used, and the same was true of the Yersinia-specific antibody response. Altogether, these results suggest that YadA-mediated collagen binding contributes to the arthritogenicity of Y. enterocolitica O:8.     

Destabilization of YopE by the ubiquitin-proteasome pathway fine-tunes Yop delivery into host cells and facilitates systemic spread of Yersinia enterocolitica in host lymphoid tissue

Pathogenic Yersinia species inject a panel of Yop virulence proteins by type III protein secretion into host cells to modulate cellular defense responses. This enables the survival and dissemination of the bacteria in the host lymphoid tissue. We have previously shown that YopE of the Y. enterocolitica serogroup O8 is degraded in the host cell through the ubiquitin-proteasome pathway. YopE normally manipulates rearrangements of the actin cytoskeleton and triggers phagocytosis resistance. To shed light into the physiological role of YopE inactivation, we mutagenized the lysine polyubiquitin acceptor sites of YopE in the Y. enterocolitica serogroup O8 virulence plasmid. The resulting mutant strain escaped polyubiquitination and degradation of YopE and displayed increased intracellular YopE levels, which was accompanied by a pronounced cytotoxic effect on infected cells. Despite its intensified activity on cultured cells, the Yersinia mutant with stabilized YopE showed reduced dissemination into liver and spleen following enteral infection of mice. Furthermore, the accumulation of degradation-resistant YopE was accompanied by the diminished delivery of YopP and YopH into cultured, Yersinia-infected cells. A role of YopE in the regulation of Yop translocation has already been described. Our results imply that the inactivation of YopE by the proteasome could be a tool to ensure intermediate intracellular YopE levels, which may effectuate optimized Yop injection into host cells. In this regard, Y. enterocolitica O8 appears to exploit the host ubiquitin proteasome system to destabilize YopE and to fine-tune the activities of the Yop virulence arsenal on the infected host organism.     

Yersinia enterocolitica induces apoptosis and inhibits surface molecule expression and cytokine production in murine dendritic cells

Yersinia enterocolitica evades innate immunity by expression of a variety of pathogenicity factors. Therefore, adaptive immunity including CD4(+) T cells plays an important role in defense against Y. enterocolitica. We investigated whether Y. enterocolitica might target dendritic cells (DC) involved in adaptive T-cell responses. For this purpose, murine DC were infected with Y. enterocolitica wild-type and mutant strains prior to incubation with ovalbumin (OVA) as antigen and 5-(6)-carboxyfluorescein diacetate N-succinimidyl ester-labeled OVA-specific T cells from DO11.10 mice. While T-cell proliferation was partially affected by infection of DC with plasmid-cured and YopP-deficient Yersinia mutant strains, no T-cell proliferation occurred after infection of DC with wild-type Y. enterocolitica. Infection of DC with Y. enterocolitica wild type resulted in decreased up-regulation of major histocompatibility complex class II, CD54 (intercellular adhesion molecule 1), CD 80, and CD86 expression. Experiments with plasmid-cured Y. enterocolitica or a YopP-deficient mutant strain revealed that YopP accounts for inhibition of surface molecule expression. Wild-type Y. enterocolitica suppressed the release of KC, tumor necrosis factor alpha, interleukin-10 (IL-10), and IL-12 by DC, while infection of DC with plasmid-cured Y. enterocolitica or with the YopP-deficient mutant resulted in the production of these cytokines. Moreover, infection with wild-type Y. enterocolitica induced apoptosis in DC mediated by YopP. Apoptosis occurred despite translocation of NF-kappaB to the nucleus, as demonstrated by electromobility shift assays. Together, these data demonstrate that Y. enterocolitica targets functions of murine DC that are required for T-cell activation. This might contribute to evasion of adaptive immune responses by Y. enterocolitica.     

Human gamma delta T-cell recognition of Yersinia enterocolitica.

We have studied the human gamma delta T-cell response to Yersinia enterocolitica, a facultative intracellular bacterium which causes gastroenteritis and, particularly in human leucocyte antigen (HLA)-B27+ individuals, reactive arthritis (ReA). A marked proliferation of that cytotoxic gamma delta T cells is seen when Yersinia-infected lymphoblastoid cell lines or fixed intact Yersinia are added to cultures of mononuclear cells derived from the synovial fluid of ReA patients or from the peripheral blood of healthy donors. In contrast, heat-inactivated Yersinia fail to stimulate the gamma delta T-cell response. The gamma delta T-cell lines generated killed both autologous and allogeneic infected cell lines. Interestingly, a T-cell line generated from synovial fluid mononuclear cells (SFMC) killed infected autologous cell lines and a cell line matched for HLA-B27 less well than infected allogeneic target cells. gamma delta T-cell clones isolated from this line were found to express V gamma 9V delta 2 T-cell receptor (TCR) and also killed infected mismatched cells more efficiently than autologous targets. Moreover, from experiments using major histocompatability complex (MHC)-deficient cell lines, it was apparent that target cell recognition was MHC independent. Our results suggest that gamma delta T cells can be involved in immunity to Yersinia enterocolitica and should be taken into account when considering immunopathological mechanisms leading to reactive arthritis.     

Antigenic properties and processing requirements of 65-kilodalton mannoprotein, a major antigen target of anti-Candida human T-cell response, as disclosed by specific human T-cell clones

T-cell-mediated immunity is known to play a central role in the host response to Candida albicans. T-cell clones are useful tools for the exact identification of fungal T-cell epitopes and the processing requirements of C. albicans antigens. We isolated human T-cell clones from an HLA-DRB1*1101 healthy donor by using an antigenic extract (MP-F2) of the fungus. Specific clones were T-cell receptor alpha/beta and CD4(+)/CD8(-) and showed a T-helper type 1 cytokine profile (production of gamma interferon and not interleukin-4). The large majority of these clones recognized both the natural (highly glycosylated) and the recombinant (nonglycosylated) 65-kDa mannoprotein (MP65), an MP-F2 minor constituent that was confirmed to be an immunodominant antigen of the human T-cell response. Surprisingly, most of the clones recognized two synthetic peptides of different MP65 regions. However, the peptides shared the amino acid motif IXSXIXXL, which may be envisaged as a motif sequence representing the minimal epitope recognized by these clones. Three clones recognized natural and pronase-treated MP65 but did not detect nonglycosylated, recombinant MP65 or the peptides, suggesting a possible role for polysaccharides in T-cell recognition of C. albicans. Finally, lymphoblastoid B-cell lines were efficient antigen-presenting cells (APC) for recombinant MP65 and peptides but failed to present natural, glycosylated antigens, suggesting that nonprofessional APC might be defective in processing highly glycosylated yeast proteins. In conclusion, this study provides the first characterization of C. albicans-specific human T-cell clones and provides new clues for the definition of the cellular immune response against C. albicans.     

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.     

High frequency of CD4+ T cells specific for the TB10.4 protein correlates with protection against Mycobacterium tuberculosis infection

TB10.4 is a newly identified antigen of Mycobacterium tuberculosis recognized by human and murine T cells upon mycobacterial infection. Here, we show that immunization with Mycobacterium bovis BCG induces a strong, genetically controlled, Th1 immune response against TB10.4 in mice. BALB/c and C57BL/6 strains behave as high and low responders to TB10.4 protein, respectively. The TB10.4:74-88 peptide was identified as an immunodominant CD4+ T-cell epitope for H-2d mice. Since recent results, as well as the present study, have raised interest in TB10.4 as a subunit vaccine, we analyzed immune responses induced by this antigen delivered by a new vector, the adenylate cyclase (CyaA) of Bordetella pertussis. CyaA is able to target dendritic cells and to deliver CD4+ or CD8+ T-cell epitopes to the major histocompatibility complex class II/I molecule presentation pathways, triggering specific Th1 or cytotoxic T-lymphocyte (CTL) responses. Several CyaA harboring either the entire TB10.4 protein or various subfragments containing the TB10.4:20-28 CTL epitope were shown to induce TB10.4-specific Th1 CD4+ and CD8+ T-cell responses. However, none of the recombinant CyaA, injected in the absence of adjuvant, was able to induce protection against M. tuberculosis infection. In contrast, TB10.4 protein administered with a cocktail of strong adjuvants that triggered a strong Th1 CD4+ T-cell response induced significant protection against M. tuberculosis challenge. These results confirm the potential value of the TB10.4 protein as a candidate vaccine and show that the presence of high frequencies of CD4+ T cells specific to this strong immunogen correlates with protection against M. tuberculosis infection.     

Evaluation of enzyme immunoassay for the detection of pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis strains.

To determine the virulence plasmid-harboring strains of Yersinia enterocolitica, we prepared antiserum against plasmid-encoded proteins of Y. enterocolitica serotype O3 and carried out an enzyme immunoassay (EIA) against temperature-inducible released proteins. This serum reacted with proteins released from not only a Y. enterocolitica serotype O3 strain but also Y. enterocolitica serotype O5:27, O8, and O9 and Y. pseudotuberculosis serotype 1b, 2a, 2b, 2c, 3, 4a, 4b, 5a, 5b, 6, 7, and 8 strains, which all harbored plasmids. Plasmid-cured Y. enterocolitica and Y. pseudotuberculosis strains did not react in the EIA, nor did nonpathogenic Y. enterocolitica strains or Y. frederiksenii, Y. intermedia, and Y. kristensenii strains. These observations demonstrated that this EIA was useful for determining whether the isolated Yersinia strains were pathogenic or not.     

Protective role of interleukin-6 during Yersinia enterocolitica infection is mediated through the modulation of inflammatory cytokines

Yersinia enterocolitica is a gram-negative enteric pathogen responsible for a number of gastrointestinal disorders. A striking feature of the pathology of a Y. enterocolitica infection is inflammation. Recently, we demonstrated a role for interleukin-1alpha (IL-1alpha) in the establishment of intestinal inflammation in response to a Y. enterocolitica infection. A cytokine directly affected by IL-1 levels is IL-6. A previous report suggested that IL-6 plays an anti-inflammatory role during Y. enterocolitica infection, and in other systems IL-6 has been shown to be proinflammatory. Therefore, a closer examination of the roles of IL-6 and inflammatory cytokines in the control of Y. enterocolitica infection in IL-6(-/-) mice was undertaken. Y. enterocolitica organisms were more virulent in the IL-6(-/-) mice (60-fold decreased 50% lethal dose) and colonized systemic tissues more rapidly and to a higher level than in the wild-type mice. One role of IL-6 during a Y. enterocolitica infection may be the downmodulation of the inflammatory response. The IL-6(-/-) mice have a more robust T(H)1 T-cell response, as well as hyperinflammatory pathologies. These phenotypes appear to be due to the misregulation of tumor necrosis factor alpha, monocyte chemotactic protein 1, IL-10, transforming growth factor beta1, and gamma interferon in the IL-6(-/-) mouse. These data provide further insight into the intricate cytokine signaling pathways involved in the regulation of inflammatory responses and the control of bacterial infections.     

Analysis of the Yersinia enterocolitica 0:8 V antigen for cross protectivity

The plasmid encoded V antigen (Vag) of pathogenic Yersinia spp. is a major virulence factor as well as a protective immunogen. Recently, two main types of Vag, represented by either Yersinia enterocolitica 0:8 or Yersinia pseudotuberculosis, have been identified and it has been suggested, that antibodies generated against one type are unable to protect against Yersinia spp. carrying the other type. By using a recombinant Vag (rVagHis) of the Y. enterocolitica 0:8 type we show here, that actively immunized mice were completely protected against challenge with both, Y. enterocolitica 0:8 and Y. pseudotuberculosis serotype III. In addition, passive protection was possible with polyclonal rabbit anti-rVagHisIgG. However, while a single antibody dose (200 microgramg) was sufficient to protect against challenge with Y. enterocolitica 0:8, repetitive injections at intervals of 2 to 3 days were needed to protect against challenge with Y. pseudotuberculosis III. The apparent difference in protection correlated with a rapid disappearance of anti-rVagHisIgG from the circulation by days 3 to 4. The data therefore indicate, that expression of distinct types of Vag by Yersinia spp. does not necessarily exclude immunoprotection in mice immunized with the other type of Vag. It rather appears, that differences in immunoprotection between Yersinia species relate to the amount of cross-protective antibody. Finally, as revealed by the lack of complement-mediated killing and the lack of immunostaining of Yersiniae with anti-rVagHisantibodies, evidence is provided to indicate that immunoprotection does not occur via opsonisation or complement lysis.     

Yersinia pestis V protein epitopes recognized by CD4 T cells

Pneumonic plague, an often-fatal disease for which no vaccine is presently available, results from pulmonary infection by the bacterium Yersinia pestis. The Y. pestis V protein is a promising vaccine candidate, as V protein immunizations confer to mice significant protection against aerosolized Y. pestis. CD4 T cells play central roles during vaccine-primed immune responses, but their functional contributions to Y. pestis vaccines have yet to be evaluated and optimized. Toward that end, we report here the identification of three distinct epitopes within the Y. pestis V protein that activate CD4 T cells in C57BL/6 mice. To our knowledge, these are the first identified CD4 T-cell epitopes in any Y. pestis protein. The epitopes are restricted by the I-A(b) class II major histocompatibility complex molecule and are fully conserved between Y. pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica. Immunizing mice with a V protein-containing vaccine or with short peptides containing the identified epitopes primes antigen-specific production of interleukin 2 and gamma interferon by CD4 T cells upon their restimulation in vitro. Consistent with prior studies documenting protective roles for CD4 T cells during Y. enterocolitica infection, vaccinating mice with a 16-amino-acid peptide encoding one of the epitopes suffices to protect against an otherwise lethal Y. enterocolitica challenge. The identification of these epitopes will permit quantitative assessments of V-specific CD4 T cells, thereby enabling researchers to evaluate and optimize the contribution of these cells to vaccine-primed protection against pneumonic plague.