Department of Immunology, Imperial College School of Medicine (St Marys Campus), Norfolk Place, London W2 1PG, UK1
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK2
Author for correspondence: Keith Gould. Fax +44 20 7402 0653. e-mail k.gould{at}ic.ac.uk
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Abstract |
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Introduction |
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Vaccinia virus (VV), the prototypic orthopoxvirus, has a double-stranded DNA genome of 191 kb and replicates in the cell cytoplasm. The virus has been used extensively as an expression vector to study antigen presentation, but it interferes with the presentation of some CTL epitopes. This effect was first described with a recombinant VV expressing influenza virus haemagglutinin (HA) in which the presentation of HA epitopes to CTLs was inhibited during the late phase of infection (Coupar et al., 1986 ). Subsequently, we showed that the presentation of certain other influenza virus epitopes to CTLs is also inhibited by VV infection in murine cells and that the blockage is present during both the early and the late phases of infection, although the blockage is more extensive at late times (Townsend et al., 1988
; Gould et al., 1991
; Cossins et al., 1993
). The inhibitory effect of VV is limited neither to a particular epitope nor to presentation by a particular MHC class I molecule.
Because the presentation of only certain epitopes is affected, the question arises as to whether or not this actually represents a mechanism of virus evasion of the immune response. It was proposed previously that the mechanism of interference with antigen presentation by VV is at the level of proteolytic processing of antigen (Townsend et al., 1988 ). This hypothesis is supported by the observation that the inhibitory effect can be overcome by expression of rapidly degraded forms of the antigen which are processed differently, such as a ubiquitinnucleoprotein (NP) fusion protein (Townsend et al., 1988
), or by redirecting HA into the cytosol by deletion of the amino-terminal signal sequence (Townsend et al., 1988
; Gould et al., 1991
). The subsequent identification of VV proteins with amino acid similarity to serine protease inhibitors (serpins) led to the suggestion that they may be involved in the inhibition of antigen proteolysis (Smith et al., 1989
). However, no evidence for this hypothesis could be found when it was tested experimentally (Blake et al., 1995
) and the mechanism of interference with antigen presentation by VV remains ill-defined.
In this study, we have investigated the presentation of the well-characterized Db-restricted epitope in influenza virus NP using CTLs that are cross-reactive for two strains of influenza virus. Surprisingly, VV infection had very different effects on the presentation of this epitope from the two different strains of influenza virus NP. This was shown to be due to intrinsic differences in the efficiency of processing and presentation of the two Db-restricted NP epitopes, even in the absence of VV infection. Therefore, the differential effects of VV infection are most likely to be due to a general decrease in antigen presentation, which has a more significant effect on epitopes that are presented less efficiently.
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Methods |
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Synthetic peptides.
The synthetic peptides ASNENMETM and ASNENMDAM, corresponding to the Db-restricted NP epitope of influenza A/PR/8/34 and A/NT/60/68, respectively, were purchased from GENOSYS Biotechnologies at a grade of greater than 95% purity. The peptides were dissolved in RPMI-1640 medium for use in assays.
Recombinant VV.
The Western Reserve (WR) strain recombinant viruses Ub-Arg-NP-VAC (Townsend et al., 1988 ), NT60NP-VAC (Townsend et al., 1988
), PR8NP-VAC (Smith et al., 1987
) and a recombinant of the modified virus Ankara (MVA) strain MVA.NP, which expresses the A/NT/60/68 NP (Hanke et al., 1998
; Schneider et al., 1998
), have all been described previously. All four recombinant viruses use the 7·5 KDa VV promoter for expression. MVA.pSC11, a negative control virus, was generated using the shuttle plasmid pSC11 (Chakrabarti et al., 1985
) without a cloned insert by standard methods (Hanke et al., 1998
).
Generation of cross-reactive NP-specific CTL cell lines and clones.
Female C57BL/6 (H-2b) and CBA/Ca mice (H-2k), 812 weeks old, were obtained from the specific-pathogen-free mouse-breeding unit (Sir William Dunn School of Pathology, University of Oxford, UK) or purchased from Harlan UK. Mice were immunized by intravenous injection of 107 p.f.u. of recombinant VV Ub-Arg-NP-VAC (Townsend et al., 1988 ) for C57BL/6 mice and PR8NP-VAC (Smith et al., 1987
) for CBA/Ca mice. Two weeks later, spleens were removed. Prepared spleen cells were then restimulated in vitro by using influenza A/PR/8/34 virus-infected syngeneic feeder spleen cells, as described previously (Gould et al., 1991
). The effector cells were then restimulated with antigen at weekly intervals in the same way, and after 3 weeks of culture in vitro, human recombinant interleukin 2 (rIL-2) (Cetus) was added to a final concentration of 10 U/ml. CTL clones were established by limiting dilution to 0·5 cells per well in 96-well dishes. Actively growing clones were expanded into 24-well dishes and later into flasks, with weekly restimulation as described above. Effector cells were used in chromium-release assays on day 4 or 5 after restimulation with antigen.
Cytotoxic assay.
A standard 5 h 51Cr-release assay was used with modifications for the use of adherent target cell lines, as described previously (Townsend et al., 1984 ). The target cell lines used were L-Db (KkDkDb) (Townsend et al., 1985
) and EL-4 (KbDb). Virus infections of target cells, the use of synthetic peptides and the calculation of the percentage of specific lysis were all described previously (Gould et al., 1991
). Briefly, target cells (4x106) were either left uninfected or infected with 10 p.f.u. per cell of either VV or influenza virus and labelled with 51Cr for 90 min. After two washes, target cells were left at 37 °C for 2 h to allow antigen expression, washed twice more and plated out in the assay. Each point was measured in duplicate against quadruplicate controls and average values are shown in all of the figures. All experiments were repeated at least twice, with similar results. Spontaneous 51Cr-release was less than 23% in all experiments.
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Results |
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Discussion |
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Further experiments with additional cross-reactive CTL cell lines that gave lower levels of specific lysis revealed a similar difference in efficiency of presentation of the two Db-restricted NP epitopes in influenza virus-infected cells (Fig. 6), showing that the difference was independent of VV infection. Therefore, there is an intrinsic difference between the efficiency of presentation of the Db-restricted NP epitope in influenza virus A/NT/60/68 and A/PR/8/34, but this only becomes apparent when the CTLs are recognizing either target cells inefficiently or VV-infected cells. It is well-established that different MHC class I-associated peptides may be produced from gene products with very different efficiency (Anton et al., 1997
) and we propose that this is the case for the Db epitope in the A/NT/60/68 NP as compared with the equivalent epitope in the A/PR/8/34 NP. Previous work using the proteasome inhibitor lactacystin has shown that generation of the Db epitope from the A/NT/60/68 NP is extremely sensitive to the inhibitor, whereas generation of the A/PR/8/34 NP epitope is less so (Cerundolo et al., 1997
). This is consistent with a difference in processing of the two equivalent epitopes. The reason why there should be such a difference in the processing of the two epitopes in the different strains of virus is unclear, but must be related to the 31 out of the 498 amino acid differences between the two NP molecules. An interesting possibility is that the A/PR/8/34 sequence leads to the formation of more NP-derived defective ribosomal products, which have been proposed to be a major source of peptide epitopes for presentation to CTLs (Schubert et al., 2000
).
VV has been extensively used as an expression vector for the investigation of CTL responses, but at the same time has been shown to interfere with the presentation of certain CTL epitopes (Bennink & Yewdell, 1990 ). Because VV is such a widely used vector and because of its potential use in vaccines, it is important to understand the mechanism of VV interference with antigen presentation and to establish whether or not a specific virus gene is involved. Our results suggest that rather than specifically interfering with the presentation of certain CTL epitopes, VV infection causes a general decrease in MHC class I-restricted antigen presentation, probably because of the induced shutdown of host cell protein synthesis (Moss, 1968
). The differences in presentation that are observed with the recombinant NP-VACs may be explained by the intrinsic differences in efficiency of presentation of the two equivalent epitopes. This difference becomes significant because of the general reduction in antigen presentation that is caused by VV infection which caused the efficiency of presentation of the A/NT/60/68 NP epitope to be reduced below the threshold that is required for CTL recognition. VV infection would be expected to have a more significant effect on epitopes that are processed and presented less efficiently, and susceptibility to inhibition by VV infection may be a useful indicator of the efficiency of processing of a particular CTL epitope.
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Acknowledgments |
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Footnotes |
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c Present address: MRC Laboratories, Fajara, PO Box 273 Banjul, The Gambia, West Africa.
d Present address: The WrightFleming Institute, Imperial College School of Medicine (St Marys Campus), Norfolk Place, London W2 1PG, UK.
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References |
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Received 10 October 2000;
accepted 5 January 2001.