1 Institut für Virologie und Immunbiologie, Versbacher Str. 7, 97078 Würzburg, Germany
2 Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, München, Germany
Correspondence
Stefan Niewiesk
niewiesk{at}vim.uni-wuerzburg.de
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ABSTRACT |
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MAIN TEXT |
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Yersinia expresses a number of virulence-associated proteins, namely the Yersinia outer proteins (Yops). Central to the export of Yops is the function of a specialized protein secretion apparatus termed type III. The close contact between Y. enterocolitica and the surface of the eukaryotic cell triggers secretion and subsequent translocation of Yops into the cytoplasm of host cells. YopE is a 25 kDa protein containing an N-terminal secretion domain of 15 amino acids and a translocation domain of 50 amino acids. Plasmid pHR43 (Russmann et al., 2001) carries the genetic information for YopE with a deletion of aa 1973 (YopE
1973). This truncated version of YopE lacks part of its translocation domain and is therefore secreted but not translocated into the cytoplasm of eukaryotic cells. The full-length gene of MV N was amplified and inserted into pHR43. The N protein was expressed in large amounts in bacterial cells but no secretion could be detected (data not shown). This was probably due to the fact that N proteins form a large nucleocapsid in the bacterial cell, which subsequently cannot be secreted. As secretion of protein is crucial to induce a T cell response, a gene fragment encoding aa 79161 from MV N was instead amplified from plasmid pSC-N (forward primer BamHI 5'-ATAGGATCCTTATTTGTGGAGTCTCC-3', reverse primer BamHI 5'-GTTGGATCCCTCAGGGTCTTGCAC-3') (Tober et al., 1998
) and inserted into the BamHI site of pHR43 to create pHR356 (Fig. 1
a).
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The Y. enterocolitica WA-314 sodA mutant strain is tolerated by different inbred mouse strains to various degrees after oral inoculation (C57BL/6 with 107108 c.f.u. (Igwe et al., 1999); BALB/c mice with 104105 c.f.u.; H. Rüssmann, unpublished data). To investigate the susceptibility of C3H mice, animals were fed 3x103, 3x105 or 3x107 c.f.u. of Y. enterocolitica WA-314 sodA (pHR356) in a 30 µl volume of PBS using a microlitre pipette. After inoculation, no clinical signs were observed.
Ten days after oral immunization with 3x107 c.f.u. Y. enterocolitica WA-314 sodA, or 3x103, 3x105 or 3x107 c.f.u. Y. enterocolitica WA-314 sodA (pHR356), mice were challenged intracerebrally with 104 TCID50 CAM/RB MV and 4 days later the Yersinia- and MV-specific proliferative T cell response and cytokine secretion were tested. Spleen cells (5x105) were dispensed in a 200 µl volume of RPMI containing 2 % mouse serum with or without 10 µg gradient-purified UV-inactivated MV antigen ml-1 or 10 µg heat-inactivated Yersinia lysate ml-1. After 48 h, 0·5 µCi [3H]thymidine was added and cells were harvested 18 h later on to filters. Thymidine incorporation was measured using a betaplate scintillation counter (Weidinger et al., 2000). Immunization with either 3x107 c.f.u. Y. enterocolitica WA-314 sodA or 3x105 or 3x107 c.f.u. Y. enterocolitica WA-314 sodA (pHR356) resulted in a strong Yersinia-specific T cell proliferation (stimulation index of 3040), in agreement with published results (Igwe et al., 1999
). At an inoculation dose of 3x103 c.f.u. Y. enterocolitica WA-314 sodA (pHR356), Yersinia-specific T cell proliferation was not above background levels seen with spleen cells from naive animals (Fig. 2
).
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After immunization with 3x107 c.f.u. Y. enterocolitica WA-314 sodA (pHR356), supernatants from spleen cells stimulated with Yersinia or MV antigen were tested by ELISA for interferon- and IL4 secretion (Weidinger et al., 2001
). Monoclonal antibody pairs for a sandwich ELISA for the detection of IFN-
(clone R4-6A2 and XMG1.2) and IL4 (clone 11B11 and BVD6-24G2) as well as the respective recombinant cytokines as standards were obtained from Pharmingen. Cytokine ELISA was performed according to the manufacturer's recommendations. The strong Yersinia-specific T cell proliferation (Fig. 2
) correlated with the secretion of IFN-
(22 µg per 107 spleen cells) whereas no IL4 was found (data not shown). Stimulation with MV led to secretion of IFN-
(6 µg per 107 spleen cells) but not of IL4, clearly indicating a TH1 response. It is of interest to note that high-dose inoculation with 107 p.f.u. MV induces a TH2 response in C3H mice (Finke et al., 1995
; Weidinger et al., 2001
), whereas high-dose inoculation of 3x107 c.f.u. of the recombinant Y. enterocolitica WA-314 sodA (pHR356) strain resulted in a TH1 response. Obviously, induction of the Yersinia-specific TH1 response also directed the MV-specific TH response. Because the MV N fragment secreted by Y. enterocolitica WA-314 sodA (pHR356) contains the aa 8188 epitope for Kk-restricted cytotoxic T lymphocytes (CTLs), the induction of CTLs was also tested. Spleen cells from immunized animals were stimulated in vitro with peptide aa 8188 and 5 days later tested for cytotoxicity in a chromium-release assay using L929 cells infected with a vaccinia virus recombinant expressing the MV N protein and peptide-pulsed cells as described (Neumeister & Niewiesk, 1998
). However, no cytotoxicity was observed (data not shown), indicating that the secreted N fragment induced a CD4 T cell response only. This is in keeping with the fact that YopEMV-N (aa 79161) is only secreted but not translocated into the cytoplasm of eukaryotic cells.
In order to test whether the T cell response induced by oral inoculation of mutant Y. enterocolitica WA-314 sodA (pHR356) was protective against MVE, C3H mice were orally immunized with Yersinia and challenged by intracerebral inoculation with 104 TCID50 of the neurotropic rodent-adapted MV CAM/RB strain. Mice were weighed and their reflexes tested every day, as previously described (Weidinger et al., 2000), and moribund animals were killed. Seven days after immunization with either 2x107 c.f.u. Y. enterocolitica WA-314 sodA (pHR356) or Y. enterocolitica WA-314 sodA, animals were not protected against infection with CAM/RB (data not shown). In contrast, 10 days after immunization with 2x107 c.f.u. Y. enterocolitica WA-314 sodA (pHR356), animals were found to be protected against intracerebral challenge (Fig. 3
). The level of protection against encephalitis was comparable with peptide immunization or adoptive transfer of MV-specific CD4 T cells (Weidinger et al., 2000
, 2001
). An immunization dose of 2x105 c.f.u. Y. enterocolitica WA-314 sodA (pHR356) resulted in partial protection, whereas immunization with 2x103 c.f.u., which did not induce a MV CD4 T cell response (see Fig. 2
), did not protect against challenge (Fig. 3
).
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One concern raised in respect of subunit vaccines against MV is the occurrence of atypical measles where immunization with a killed vaccine has led to severe measles after contact with wild-type virus (Hilleman, 2001). Although the reasons for this condition are not understood, there is some evidence that the induction of a TH2 response and immune complex depositions are crucial in this context (Polack et al., 1999
, 2002
). As the vector system used in this study induces a strong TH1 response, it might be beneficial in directing T cell responses towards a protective rather than atypical response.
In summary, our data demonstrate that a single oral immunization via the mucosa with attenuated Y. enterocolitica is able to induce a T cell response protective in a time- and dose-dependent manner against viral infection of the central nervous system. Because Y. enterocolitica induces a mucosal and systemic immune response and due to its good protective capacity in the mouse model, it would be worth while testing this vector system in the presence of maternal antibody.
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Received 17 September 2002;
accepted 28 November 2002.