1 Unidade de Inmunología Viral, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra Pozuelo km 2, E-28220 Majadahonda (Madrid), Spain
2 Unidade de Biología Viral, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra Pozuelo km 2, E-28220 Majadahonda (Madrid), Spain
Correspondence
Margarita Del Val
mdval{at}isciii.es
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ABSTRACT |
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INTRODUCTION |
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Immune mechanisms involved in RSV disease and protection are not well understood. Protection is mediated mainly by neutralizing antibodies directed to the two virion surface glycoproteins, while clearance of virus-infected cells requires CD8+ T lymphocytes. Studies in the mouse model of human RSV have shown that CD8+ T lymphocytes play a role both in lung pathology and viral clearance (Cannon et al., 1988; Graham et al., 1991
). CD8+ T lymphocytes are thought to regulate differentiation and activation of Th2 CD4+ T lymphocytes, which mediate lung pathology by recruiting eosinophils into the lungs during RSV infection (Srikiatkhachorn & Braciale, 1997
). On the other hand, CD8+ T lymphocytes specific for the matrix 2 (M2) M28292 epitope have been found to be the sole mediators of resistance to RSV infection in BALB/c mice infected with a recombinant vaccinia virus (rVV) expressing the M2 protein of RSV (Kulkarni et al., 1995
). Further studies found that RSV infection altered CD8+ effector activity and memory T lymphocytes selectively in the lungs (Chang & Braciale, 2002
; Chang et al., 2001
). This RSV-induced immune dysregulation of virus-specific CD8+ T lymphocytes has been suggested as a possible mechanism for the absence of durable long-lived immunity to RSV infection (Chang & Braciale, 2002
).
The fusion (F) protein of RSV, one of the two major surface glycoproteins in the virion, has long been recognized as a major vaccine candidate as it is an important target antigen for virus-specific cytotoxic T lymphocytes (CTL) (Pemberton et al., 1987) and neutralizing antibodies (Olmsted et al., 1986
). Monoclonal antibodies to the F glycoprotein, which is highly conserved among the two antigenic groups of human RSV, passively protect against human RSV challenge in the mouse (Taylor et al., 1984
) and reduce the severity of disease in premature and newborn babies (The IMpact-RSV Study Group, 1998
). Furthermore, immunization of mice with rVV (Olmsted et al., 1986
; Stott et al., 1987
) or plasmid DNA encoding the F protein (Bembridge et al., 2000
; Li et al., 1998
), which induce F-specific antibodies and CTL, confers protection against challenge with RSV of either antigenic group.
Human RSV F glycoprotein is synthesized as a 574 aa F0 precursor that is processed at two cleavage residues (109 and 136) by furin-like proteases (González-Reyes et al., 2001; Zimmer et al., 2001
). Cleavage yields two chains that remain linked by a disulfide bond. The larger carboxy-terminal F1 chain hosts the fusion peptide, the transmembrane region and several neutralizing antibody antigenic sites (López et al., 1998
). The amino-terminal signal sequence is followed by the F2 chain, which has recently been found to be responsible for RSV host-cell specificity suggesting it is the binding partner for specific RSV entry receptors (Schlender et al., 2003
). Furthermore, two murine H-2Kd-restricted epitopes recognized by CTL, F8593 and F92106, have been identified in the F2 chain of the A2 strain of human RSV (Chang et al., 2001
; Jiang et al., 2002
).
In this study, we used a novel approach to generate RSV-specific CTL lines by stimulating primed mouse splenocytes with the BCH4 fibroblast cell line that is persistently infected with the Long strain of RSV (Fernie et al., 1981). We found that the F92106 epitope is not present in RSV Long whereas the F8593 epitope is conserved between strains. We searched for CTL epitopes in the F1 chain by using CTL lines, epitope prediction programs and an rVV encoding a mutant F protein, in which the F2 chain was deleted. A novel H-2Kd-restricted epitope, F249258, was identified. No hierarchy in CD8+ T-lymphocyte responses to F8593 and F249258 epitopes was found in vivo during a primary response to an rVV expressing the full-length F glycoprotein. In contrast, F249258 was found subdominant with respect to F8593 in in vivo memory and secondary responses, and in CTL lines generated in vitro.
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METHODS |
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Construction of recombinant vvF containing the F gene of RSV Long (VRBF) has been described (Bembridge et al., 1998). An rVV expressing an F protein with aa 34128 (both included) deleted, vvF-
F2, was obtained as follows: plasmid LF1, containing a cDNA copy of the entire F protein gene of the Long strain (Cristina et al., 1990
), was mutagenized to introduce a restriction site recognized by BbsI (nt 106, Long sequence; López et al., 1988
) and AflII (nt 397), using the Quick-Change site-directed mutagenesis kit (Stratagene) and appropriate oligonucleotides. The resulting plasmid was digested with BbsI and AflII, treated with Klenow polymerase to make it blunt-ended, and ligated before being used to transform DH5 bacterial cells. Transformants were tested by PCR for the presence of plasmid with the deleted sequence that removed aa 34128 (both inclusive) from the F2 chain of the F protein. In addition, residue 31 was changed from glutamic to aspartic acid as a consequence of the cloning strategy. The entire F segment of one of the plasmids (LF1-
F2) was sequenced and
F2 insert was subcloned into plasmid pRB21 (Blasco & Moss, 1995
) after digestion with BamHI and EcoRI. This plasmid was used to select an rVV of the vRB12 strain by the method of Blasco and Moss (1995)
. The rVV was plaque-purified three times and expression of F protein was tested by immunofluorescence with specific monoclonal antibody (mAb) 47F (García-Barreno et al., 1989
). rVV stocks were grown in CV-1 monolayers and consisted of clarified sonicated cell extracts purified by ultracentrifugation through a 36 % sucrose cushion.
Cell lines.
P13.1 cell line, a derivative from P815 mastocytoma cells (H-2d) by transfection with lacZ gene encoding -galactosidase (Carbone & Bevan, 1990
), was provided by H.-G. Rammensee (Tübingen University, Tübingen, Germany) and was cultured in Iscove's modified Dulbecco's medium supplemented with 10 % fetal bovine serum and 5x105 M 2-mercaptoethanol. Human lymphoblastoid T2 cells deficient in transporters associated with antigen processing and transfected with Kd (Zhou et al., 1994
) were provided by G. Hämmerling (German Cancer Research Centre, Heidelberg, Germany) and were cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum and 5x105 M 2-mercaptoethanol. BALB/c fibroblast cells and human RSV Long persistently infected BCH4 fibroblast cells (Fernie et al., 1981
) were obtained from B. Fernie (Georgetown University School of Medicine and Dentistry, Manassas, VA) through G. Taylor (Institute for Animal Health, Compton, UK), and were cultured in Dulbecco's modified Eagle medium supplemented with 10 % fetal bovine serum. All cell lines were cultured at 37 °C in a 5 % CO2 atmosphere.
Synthetic peptides.
Peptides were synthesized in an Applied Biosystems peptide synthesizer (model 433A) and purified. Identity was confirmed by electrospray mass spectrometry by D. López (Centro Nacional de Microbiología, Madrid, Spain). All peptides that tested positive in cytotoxicity assays were found to be homogeneous by reversed-phase high-performance liquid chromatography. Peptides are named indicating the amino and carboxyl termini in the amino acid sequence of the F protein of RSV Long. Sequences are F3140, EFYQSTCSAV; F3240, FYQSTCSAV; F8593, KYKNAVTEL; F92106, ELQLLMQSTPAANNR; F249257, TYMLTNSEL; F249258, TYMLTNSELL; F365373, VFCDTMNSL and F467475, LYVKGEPII.
Polyclonal CTL lines and cytotoxicity assays.
Female BALB/c mice (H-2d haplotype) bred in our colony were infected by intraperitoneal injection of 107 p.f.u. of vvF or vvF-F2. Three weeks post-infection an identical booster was given, and three or more weeks later mice were sacrificed and spleen cells obtained. CTL lines were named indicating the virus used for priming in vivo and the agent used for restimulation in vitro (Table 1
). Thus CTL F/RSV derive from vvF-primed mice and were restimulated in vitro with RSV-infected splenocytes, mimicking a described method (Gaddum et al., 1996
). Splenocytes from vvF-primed mice were stimulated with naïve splenocytes infected for 90 min with RSV Long at a m.o.i. of 0·4 p.f.u. per cell. Recombinant human interleukin 2 (IL2), generously provided by Hoffman-La Roche, was added 5 days later at 25 U ml1. Long-term cultures were restimulated weekly with IL2 and RSV-infected splenocytes at an effector : stimulator ratio of 3 : 1. The CTL F/BCH4 and CTL
F2/BCH4 lines were generated by stimulating 5x106 splenocytes ml1 with 2x105 BCH4 cells ml1 treated with 90 µg mitomycin C ml1 (Sigma). IL2 (25 U ml1) was added after 5 days. Long-term cultures were restimulated weekly with IL2 and mitomycin C-treated splenocytes and BCH4 cells at an effector : stimulator ratio of 3 : 1 and 5 : 1, respectively. The CTL F/F8593 and CTL F/F249257 lines were generated by stimulating 107 splenocytes ml1 with 1010 M of the relevant peptide. Five days after selection, IL2 (25 U ml1) was added. Long-term cultures were maintained by weekly restimulation with mitomycin C-treated 106 M peptide-pulsed splenocytes (effector : stimulator ratio of 3 : 1) and cultured in medium with IL2 and 1010 M peptide. All CTL lines were cultured in minimal essential medium
supplemented with 10 % inactivated fetal bovine serum and 5x105 M 2-mercaptoethanol at 37 °C in a 5 % CO2 atmosphere. Standard 34 h 51Cr release cytotoxicity assays (Del Val et al., 1988
) were performed incubating CTL and target cells that had been previously labelled for 6090 min with Na51CrO4 at 37 °C in the presence or absence of 106 M peptide and washed. Down to 100-fold lower peptide concentrations gave similar results. Alternatively, P13.1 cells were infected with rVV at a m.o.i. of 310 p.f.u. per cell as described (Eisenlohr et al., 1992
), virus inoculum was thoroughly washed after 1 h adsorption, and infection was allowed to proceed for 3 h prior to cytotoxicity assay.
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RESULTS AND DISCUSSION |
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To identify epitopes presented by Kd to multispecific CTL F/RSV and CTL F/BCH4, we first assessed in cytotoxicity assays whether protein epitopes known in the A2 strain were recognized. Peptides F8593 and F92106 corresponding to these epitopes were synthesized and used to sensitize cells. We found that peptide F92106 is not recognized by either CTL line (Fig. 1c) and also fails to restimulate F-primed CTL (data not shown). Despite 98 % aa identity between the F glycoproteins of RSV strains A2 and Long (López et al., 1988
), residues 11 and 12 of this 15-mer F92106 epitope are not conserved (PT in A2 and AA in Long) and may be critical for the unpredictable non-canonical binding of the epitope to Kd. It cannot be excluded either that the F92106 peptide is not a good reagent to mimic this potential epitope in the Long strain. Therefore, in RSV Long we have no evidence of the presence of this epitope.
On the other hand, both multispecific CTL F/RSV and CTL F/BCH4 lines were able to recognize F8593 sensitized cells (Fig. 1c). Furthermore, a long-term polyclonal monospecific CTL F/F8593 line was generated by stimulating splenocytes from vvF-primed mice with F8593 peptide. Titration assays were performed by pulsing targets with increasing concentrations of peptide, and both multispecific CTL F/BCH4 and monospecific CTL F/F8593 were found to recognize down to 1011 M F8593 (Fig. 1d
). Therefore, as expected from the conserved sequence within the epitope and flanking regions, F8593 is also a CTL epitope in the Long strain.
Identification of CTL epitope F249258 in the F glycoprotein of the Long strain of RSV
To identify other potential CTL epitopes in the F glycoprotein of the Long strain we constructed an rVV, vvF-F2, encoding a mutant F protein in which aa 34128 have been deleted, and which therefore does not contain the F8593 epitope. Although the mutant was constructed in such a way as to allow maturation of the full F1 chain, reduced levels of expression compared with the wild-type protein were detected (data not shown). In spite of the lower expression of the mutant protein, cells infected with vvF-
F2 were efficiently lysed by CTL F/BCH4, thus demonstrating presentation of one or more undefined epitopes from vvF-
F2-infected cells to multispecific CTL F/BCH4 (Fig. 2
a). It can be argued that a F8593 mimotope may be recognized by F8593-specific CTL clones present in the multispecific CTL line, or that an artificial epitope is being generated from the mutant protein. To address this issue the same targets were assayed with monospecific CTL F/F8593 and no lysis was observed (Fig. 2a
). Furthermore, a multispecific CTL
F2/BCH4 line that did not include F8593-specific clones was generated from mice that had been primed with vvF-
F2 (see Table 1
). Cytotoxicity assays were performed and as expected CTL
F2/BCH4 were unable to lyse targets exogenously loaded with F8593, but were, however, able to recognize vvF-infected targets (Fig. 2b
). We therefore demonstrate the existence of one or more novel uncharacterized RSV Long F glycoprotein epitopes in the F1 chain or in the first 33 aa of the protein.
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In contrast, neither recognition of peptide-pulsed target cells nor generation of monospecific CTL lines were observed with other predicted epitope peptides (F365373 and F467475, data not shown). The possibility cannot be excluded that other epitopes may exist within the deleted region, but a further predicted epitope located in the region deleted in vvF-F2 represented by synthetic peptides F3140 and F3240 was also tested and found negative (data not shown). The results are summarized in Fig. 5
.
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Shifting immunodominance pattern of CD8+ T-lymphocyte responses to F8593 and F249258 after in vivo vvF infection and in CTL lines
A potential competition between the two epitopes presented by Kd and located in the F protein of RSV Long (F8593 and F249257/8) led us to examine the hierarchy of CD8+ T-lymphocyte responses. Factors that potentially contribute to immunodominance include efficiency of antigen processing, binding affinity of peptides to class I molecules, presence of T lymphocytes capable of responding to a given peptide/MHC complex and inhibition of responses to subdominant epitopes by CD8+ T lymphocytes specific for dominant epitopes (Chen et al., 2000; Yewdell & Bennink, 1999
). The fact that recognition of peptide F249257 by monospecific CTL F/F249257 was better than by CTL F/BCH4 (Figs 4a and 3b
, respectively), suggested that F249257 might be a subdominant determinant in the latter multispecific CTL line. Furthermore, when T lymphocyte clones specific for the immunodominant F8593 epitope were not present in the CTL line, as in CTL
F2/BCH4 or in CTL F/F249257 (see Table 1
), targets pulsed with down to 1011 M F249257 peptide were efficiently lysed (Fig. 6
a). Therefore, the limit in F249257 peptide recognition by CTL F/BCH4 is imposed by F8593-specific T lymphocyte clones, not by BCH4 cells, as these are able to select more sensitive CTL when stimulating splenocytes from vvF-
F2-primed mice.
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To assess whether responses to the immunodominant determinant F8593 were preventing the emergence of responses to the subdominant determinant F249257/8, we quantified CD8+ T lymphocytes specific for each epitope in an intracellular cytokine staining assay using peptide-pulsed cells to stimulate CTL F/BCH4 and CTL F2/BCH4. Fig. 7
(a) (upper panels) shows that when targets were pulsed with peptide F8593, 77 % of the CD8+ lymphocytes in the CTL F/BCH4 line were activated to produce IFN-
, whereas only 9·8 and 9·4 % of the CD8+ lymphocytes were activated by peptides F249257 and F249258, respectively. Therefore CTL lines obtained from vvF-primed memory mice show immunodominance, as CTL responses are strongly skewed to F8593 at the expense of responses to F249257/8. Interestingly, when the immunodominant epitope F8593 was absent in the in vivo priming event that led to the generation of the CTL
F2/BCH4 line, 98 and 52 % activation was achieved by stimulation with peptides F249258 and F249257, respectively, showing that in the absence of the F8593 epitope, responses to the F249257/8 epitope are very efficiently generated. Thus, there is no limitation in the in vivo availability of CD8+ T lymphocytes capable of responding to F249257/8.
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Hierarchy in primary, memory and secondary CD8+ T-lymphocyte responses to F8593, F249257 and F249258 was studied in vivo after vvF infection (Fig. 7b). CD8+ lymphocytes activated with each peptide in an intracellular cytokine staining assay are observed with a frequency similar to that reported in other systems (Chen et al., 2000
). Staining with tetramers Kd/F8593 and Kd/F249258 essentially gave the same results (data not shown). Low frequencies are probably due to competition with vaccinia virus determinants, which for example have previously been suggested to account for difficulty in detection of the NP147155 epitope following infection with an rVV encoding the complete influenza virus nucleoprotein, NP (Chen et al., 2000
; Restifo et al., 1995
). This has been discussed in terms of the limitations of viral vectors that elicit massive CTL responses to their own antigens.
In the primary immune response to vvF (Fig. 7b, white bars), there is no limitation for the generation of a similarly effective CD8+ T-lymphocyte response to either epitope, as frequencies are comparable after stimulation with either peptide, thus concluding that no significant hierarchy exists in the in vivo primary response in vvF-infected mice. However, frequencies of epitope-specific populations in the memory immune response (Fig. 7b
, grey bars) increase for F8593 and decrease for F249257/8 in comparison with the primary immune response (Fig. 7b
, white bars). Therefore, in the initial experimental setting for in vitro selection of CTL lines by restimulation with BCH4 cells, the F8593-specific CD8+ T lymphocyte population is already in advantage. This difference is probably amplified by restimulation with BCH4 cells to that observed in the CTL F/BCH4 line (Fig. 7a
).
Furthermore, in a secondary immune response (Fig. 7b, black bars) the mean percentage of activated CD8+ lymphocytes after stimulation with peptide F8593 is almost 10-fold higher than after stimulation with peptides F249257 or F249258. This difference in the number of F8593 and F249257/8 epitope-specific CD8+ T lymphocytes is similar in proportion to that observed in the established in vitro generated CTL F/BCH4 line (Fig. 7a
). Therefore, the CTL F/BCH4 line is representative of an in vivo secondary response to vvF infection.
Different patterns of T cell immunodominance in primary and secondary responses have been described previously (Belz et al., 2000; Crowe et al., 2003
). In influenza virus primary infection, similar frequencies of T cells specific for epitopes DbPA224 and DbNP366 are observed, whereas the secondary response is dominated by T cells specific for DbNP366 (Belz et al., 2000
). The mechanism underlying this changing pattern of immunodominance has been investigated in a recent study that proposes a model in which differential antigen presentation by dendritic and non-dendritic cells together with the capacity of T cells to perceive the antigens presented by these cells, regulate the T cell specificity pattern (Crowe et al., 2003
). Along with this hypothesis, we suggest that in our experimental system professional antigen presenting cells with a similar proportion of epitopes F8593 and F249258 would activate naïve T cells during the primary immune response, whereas during the secondary immune response memory T cells would also be activated by cross-priming or by non-professional antigen presenting cells with a higher proportion of epitope F8593. This would lead to the gradual establishment of a CD8+ T-lymphocyte response strongly skewed to epitope F8593.
The different immunodominance pattern for epitopes F8593 and F249258 observed in primary and secondary vvF infection must be taken into account when considering single epitope vaccines. However, the finding that CD8+ T lymphocytes specific for either epitope are generated in vivo holds promise for the potential to generate a multispecific CD8+ response in natural infection or after vaccination. In conjunction with neutralizing antibodies targeting the F protein, a multispecific CD8+ response would better contribute to clearing RSV infection, as it would hinder the emergence of viral CTL escape mutants lacking single determinants. Definition of novel CTL epitopes thus contributes to addressing these issues in the mouse model.
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ACKNOWLEDGEMENTS |
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Received 23 April 2004;
accepted 21 July 2004.
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