Immunology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK1
Laboratory of Molecular Medicine, Childrens Hospital, Boston MA 02115, USA2
Author for correspondence: Michael W. Steward. Fax: +44 171 9272378. e-mail michael.steward{at}lshtm.ac.uk
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Abstract |
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Introduction |
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Studies in the mouse model of RSV have demonstrated that both CD4+ and CD8+ T cells are crucial for the termination of the primary infection but that these cells may also be pathogenic (Graham et al., 1991 ). The vaccine-enhanced illness is thought to have resulted from an inappropriate immune response leading to an excess of TH2-type cytokines, particularly IL-4 and IL-5, and an influx of eosinophils (Connors et al., 1994
; Waris et al., 1997
; Graham, 1996
). The attachment glycoprotein (G) of RSV has been implicated (Openshaw et al., 1992
; Hsu et al., 1999
). Furthermore, although CTL are likely to be important in both recovery and protection from RSV, the G protein does not induce detectable CTL responses (Bangham et al., 1986
; Srikiatkhachorn et al., 1997
). In contrast, the fusion protein (F) of RSV induces a TH1-type cytokine response and the development of CD8+ CTL (Bangham et al., 1986
; Alwan et al., 1993
) while immunization of CD8-deficient mice or mice depleted of CD8+ T cells with the F protein led to a TH2 response and eosinophilia (Srikiatkhachorn & Braciale, 1997
). Thus, to avoid immunopathology, it is necessary to design vaccines that promote CD8+ T cell responses: the F protein, a major target of CTL isolated during primary RSV infection in mice (Alwan et al., 1993
; Kulkarni et al., 1993
) and humans (Cherrie et al., 1992
), is a likely candidate.
In this study we describe a peptide from the F protein of RSV that behaves as an allele-specific CTL epitope in BALB/c mice (H-2d). Despite being motif-negative, this peptide was not only recognized by RSV-specific CTL but, following immunization, generated virus-specific CTL responses which significantly reduced the viral load in the lungs following challenge with RSV.
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Methods |
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H-2Kd:
2-microglobulin:peptide P8:F92106 complex assembly.
Constructs encoding amino acids 1280 of the heavy chain of H-2Kd and amino acids 199 of 2-microglobulin were cloned and expressed in the T7 promoter-based vector pGMT7 in E. coli (Gao et al., 2000
) and inclusion bodies of both Kd and
2-microglobulin were prepared. Complex refolding with or without peptide P8:F92106 was carried out by dilution in a redox buffer in a standard MHC class I refolding assay (Garboczi et al., 1992
). The MHCpeptide complex formed was separated by gel filtration on a Superdex 200 column (Amersham Pharmacia) and the eluted peaks analysed by SDSPAGE.
RSV and cell lines.
The A2 strain of RSV was grown and titrated on Hep-2C cells as previously described (Hsu et al., 1999 ). BALB/c fibroblasts (H-2d) and BCH4 cells, persistently infected with RSV (R. Gaddum, AFRC, Compton, UK) were used as target cells in the CTL assays. L929 cells (H-2k) transfected with H-2Ld molecules (T1.1.1) or with H-2Dd/Ld molecules (T37.2.1) were kindly provided by Carol S. Reiss, New York University, USA. L929 cells (H-2k) transfected with H-2Kd molecules (Lkd) were kindly provided by Jonathan Yewdell of NIH, USA.
Mice and immunization.
Mice, BALB/c (H-2d), C57BL/6 (H-2b) and CBA (H-2k) mice (NIMR, Mill Hill, UK) and BALB/c CB-17 scid mice (LSHTM, London) were either infected intranasally (i.n.) with RSV (106 p.f.u./100 µl/mouse) or immunized subcutaneously (s.c.) with 100 µg peptide in complete Freunds adjuvant (Difco) followed 3 weeks later by a booster of peptide in incomplete Freunds adjuvant (Difco). For lymphocyte proliferation assays, mice were immunized s.c. with P8:F92106 or with SH4560 twice at weekly intervals, and the spleens removed 1 week after the second immunization.
Pulmonary and splenic CTL assays.
To measure primary CTL responses in the lung, mice were infected i.n. with 106 p.f.u. RSV in 100 µl. Lungs were removed after 7 days and homogenized in cold DMEM-5 (Life Technologies). Lymphocytes were isolated by gradient centrifugation on Histopaque-1083 (Sigma) and cultured in RPMI 1640 (Life Technologies) containing 10% FCS, 10 mM HEPES (Life Technologies), 50 µM 2-mercaptoethanol (Sigma) and antibiotics at the required concentration as effectors. For the splenic CTL assays, splenic mononuclear cells were isolated from immunized mice and restimulated in vitro with 0·5 M peptide for 67 days.
Target cells for the CTL assays were BALB/c fibroblasts and BCH4 cells. These were labelled with 51Cr (200 µCi/2x107 cells; Amersham Pharmacia). Targets were pulsed with 10 µM peptides for 1 h at 37 °C. Titration experiments confirmed this to be the optimal concentration of peptide for these experiments. 51Cr-labelled non-peptide-pulsed BALB/c fibroblasts were used as a control. In the experiments with C57BL/6 mice, the target cells were C57BL/6 fibroblasts either pulsed with peptides or infected with RSV 12 h before the experiment. In the experiments with CBA mice, L929 cells were used as targets. Effectors and targets were co-cultured for 6 h in a standard chromium release assay (Hsu et al., 1999 ). In all experiments shown, the spontaneous release of 51Cr from target cells incubated alone was less than 20% of the total release from target cells lysed with Triton X-100. All the assays were repeated at least twice and the data presented are representative of repeated assays.
In some experiments, effector cells were incubated with 10 µg/ml of either anti-CD8 or anti-CD4 monoclonal antibodies prior to the assay. For experiments using enzyme inhibitors, 51Cr-labelled target cells (106) were incubated for 1 h with either 10 µM lactacystin (ICN Pharmaceuticals) or with 20 or 30 µl of a protease inhibitor cocktail containing a mixture of inhibitors for the inhibition of serine, cysteine and aspartic proteases (Sigma; cat. no. P8340). The cytotoxicity assay was performed as described above. Effectors were splenocytes from P8:F92106-immunized BALB/c mice.
Lymphocyte purification and flow cytometric analysis.
Adherent cells were removed by plastic adherence. B cells and contaminating macrophages were depleted using anti-MHC class II antibody (clone T1B 120) followed by addition of rabbit anti-rat immunoglobulin-coated Dynabeads (Dynal). The T cells were further depleted of either CD4+ or CD8+ subsets using anti-CD4 (clone YTS 191.1; a gift from X. Xu, Institute of Molecular Medicine, Oxford, UK) or anti-CD8 (clone 53-6.7, BD Biosciences, Cowley, Oxford, UK), respectively followed by rabbit anti-rat Ig-coated Dynabeads as before. The enriched CD4+ and CD8+ subsets were analysed for purity by flow cytometry.
Lymphocyte proliferation assay.
Splenic lymphocytes were isolated and cultured at 2x106/ml in RPMI 1640 plus 10% FCS plus antibiotics in the presence of peptide (12·50 µg/ml) for 5 days. Six hours before harvesting, cells were pulsed-labelled with 1 µCi per well of [3H]thymidine (Amersham Pharmacia). Results are expressed as c.p.m. (c.p.m. of peptide stimulated-c.p.m. of unstimulated cells)±SD.
ELISA.
These assays were performed as previously described (Hsu et al., 1999 ). Briefly, 96-well microplates were coated with 50 µl peptide P8:F92106 or P18:F192206 at 5 µg/ml or with 50 µl RSV at 5 µg/ml. Sera from mice immunized with P8:F92106 or control peptide P18:F192206 or with RSV were added at 1:200. Peroxidase-conjugated anti-mouse Ig (H and L chains, Nordic Immunological Laboratories) was added and 0·04% O-phenylenediamine/0·004% hydrogen peroxidase (Sigma) used as the substrate and the absorbance at 490 nm measured. RSV neutralization titres were assessed as previously described (Hsu et al., 1999
). The neutralizing antibody titre was determined as the reciprocal of the highest dilution of antiserum which reduced the number of plaques by 50% of the mean value observed in control wells containing serum from unimmunized mice.
Challenge experiments.
Groups of four BALB/c mice were immunized s.c. with peptides on day 0 and day 21 and were challenged i.n. with 106 p.f.u./100 µl RSV 3 weeks after the second immunization. Four days after challenge, the lungs were removed, weighed and homogenized. Supernatants were assayed immediately for RSV, expressed as log10 p.f.u./g lung. For passive transfer experiments splenocytes from P8:F92106-primed BALB/c mice were restimulated with P8:F92106 in vitro for 7 days, separated into three populations CD8+ enriched, CD4+ enriched and CD8+ plus CD4+ enriched and transfused 106/mouse) into three groups of five BALB/c scid mice. A fourth group was reconstituted with CD4+ and CD8+ T cells from naïve animals. All four groups of mice and untreated mice were challenged i.n. with RSV 2 days after passive transfer of the lymphocytes. Four days later, all mice were killed and lungs removed for assessment of virus load. Means were compared using Students t-test.
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Results |
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Kd heavy chain:2-microglobulin:peptide complex formation
In vitro refolding experiments with Kd heavy chain and 2-microglobulin showed that in the presence of P8:F92106, a complex of approximately 43 kDa was formed as assessed by Superdex 200 chromatography (Fig. 5a
). This complex was shown by SDSPAGE analysis to contain both Kd heavy chain and
2-microglobulin (Fig. 5b
). In contrast, there was no specific complex peak observed without peptide (data not shown). This indicates that the peptide is specific for Kd.
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Immunization of mice with peptide P8:F92106 was also able to prime lymphocytes for a dose-dependent proliferative response upon subsequent restimulation in vitro (data not shown). The levels of proliferation seen were similar to those induced by the positive control, peptide SH-45, an H-2d T helper cell epitope from the SH protein of RSV (Kulkarni et al., 1993 ). In ELISA, immune serum from P8:F92106-immunized mice was able to recognize P8:F92106 but not RSV (A490 at 1/200 dilution of sera: P8:F92106 2·00 0·11, RSV 0·16 0·03, P18:F192206 0·20 0·01). These anti-P8:F92106 antibodies were, however, unable to neutralize RSV in vitro (neutralizing titre, log2 anti-RSV 6·5 2·0, anti-P8:F92106 0·7 0·05, anti-P18:F192206 0·6 0·04). Thus, although immunization with P8:F92106 induced peptide-specific antibodies, it is very unlikely that this peptide represents an important protective B cell epitope.
Effect of P8:F92106 immunization on virus recovery following challenge with RSV
To investigate if immunization with P8:F92106 could reduce viral load following infection with RSV, groups of mice were immunized with either P8:F92106, F/M29, P18:F192206 or with adjuvant only and then challenged with RSV. Titres from mice immunized with P8:F92106 and the positive control F/M29 were 630 and 160 p.f.u./g respectively, near the limit of detection of the assay (102 p.f.u./g tissue). These were both significantly lower than those recovered from control, adjuvant only immunized mice (12500 p.f.u./g) or P18:F192206-immunized mice (4000 p.f.u./g; P<0·001; Fig. 7a). Thus, immunization with P8:F92106 can significantly reduce virus titres in the lung following RSV challenge.
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Discussion |
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The lymphocytes used to screen for peptide-specific CTL responses were isolated from the lungs of mice during primary RSV infection. This is a useful approach because it allows the investigation of effector cells that have differentiated in vivo in response to the virus and migrated back into the tissue. The recognition of P8:F92106 by these CTL indicates that this peptide must be processed and presented naturally during RSV infection resulting in CTL proliferation and differentiation. The high levels of killing suggest it is likely to be an important epitope in these animals. Although P8:F92106-specific CD4+ and CD8+ T cells were isolated from the lungs, the blocking studies suggest that the main CTL activity was contained within the CD8+ subset. The decreased CTL activity seen following blocking with anti-CD4 antibody may have been a non-specific effect although further studies would be needed to confirm this. CTL that are CD4+ and MHC class II restricted have been described in a number of viral infections but these are usually only detected after culture in vitro, and are not detected if cells are assayed directly during acute infections (Enssle & Fleischer, 1990 ). In RSV infection, spleen cells from mice infected with live RSV and restimulated for only one cycle in vitro have CD8+ CTL but repeated re-stimulation in vitro leads to the generation and outgrowth of CD4+ CTL (Nicholas et al., 1990
).
Immunization with P8:F92106 alone resulted in CTL responses against both whole RSV and peptide-pulsed cells, confirming that P8:F92106 is processed and presented by infected cells. The CTL from immunized mice were again found to be CD8+ and these were able to reduce viral load in the lungs following RSV challenge. Immunization with a single CTL epitope has previously been shown to induce protection against viral infection or tumour growth (Feltkamp et al., 1993 ; Blaney et al., 1998
). In some instances, protective responses were only induced following immunization with the CTL epitope linked to a T helper epitope or other carrier (Partidos et al., 1996
). The differentiation of CTL from P8:F92106-immunized mice and the induction of P8:F92106-specific proliferative responses following in vitro restimulation strongly suggests that although the peptide does not contain a motif for binding to either MHC class I or class II, it does have epitopes for the generation of both CTL activity and T cell help. This dual function of the peptide makes it particularly interesting.
It is known that MHC class I molecules are capable of binding several different peptides. These peptides are generally 89 amino acids long and both N and C termini are bound by hydrogen bonds to conserved residues in the class I peptide binding cleft. In addition, two side-chains, one at the C terminus (usually position 9) and the other at the amino terminus (usually position 2), hold the peptide in allele-specific pockets (Falk et al., 1990 , 1991
). Using this information for a class I allelic product, it is possible to predict CTL epitopes from a protein sequence. A comparison of the sequence of peptide P8 (ELQLLMQSTPPTNNR) with the published H-2Kd anchor motif (1X345678X', where X=Y, F; X'=I, L) shows that this peptide is motif negative and reliance on motif prediction would have missed this epitope. There are several examples in the literature of other motif-negative CTL epitopes (Kast et al., 1993
, 1994
). Interestingly, peptide P7, with the sequence ELDKYKNAVTELQLL, has an appropriate H-2d anchor motif but did not prime targets for lysis by RSV-specific CTL.
Peptides presented by MHC class I are produced in the cytosol by the cleavage of partially digested protein antigens by the proteasome enzyme complex. In vivo experiments using purified 20S proteasome have shown that this complex preferentially cuts protein antigens into peptides of either 89 amino acids, the usual size of MHC class I epitopes, or 1415 amino acids, the length of P8:F92106 (Niedermann et al., 1996 ). An alternative explanation is that P8:F92106 was broken down into smaller fragments (>9-mers) by protease/s that can only cleave the 15-mer sequence. This is unlikely, given that lactacystin had no effect on CTL recognition of P8:F92106-pulsed targets. The observation that the use of a protease inhibitor cocktail (including lysosomal protease inhibitors) actually enhanced CTL recognition suggests that P8:F92106 is not cleaved and loaded onto MHC class I by an alternative lysosomal pathway as has been shown for some measles virus fusion protein epitopes. Minimal CTL epitopes of up to13 amino acids have been reported (Urban et al., 1994
). Since the MHC class I binding groove is closed at both ends and can only accommodate 810 amino acids in a linear form, it is considered that longer peptide epitopes are accommodated by the formation of a central loop or kink in the sequence (Fremont et al., 1992
; Gromme et al., 1999
) or even by zig-zagging (Madden et al., 1993
). It has also been suggested that longer peptides can be accommodated by a protrusion mechanism (Stryhn et al., 2000
). Other work has shown that the binding of longer peptides is restricted to peptides extended at their C terminus (Horig et al., 1999
). The demonstration that P8:F92106 can promote the in vitro folding of Kd heavy chain and
2-microglobulin suggests that the peptide is indeed specific for Kd.
Immunization with the 15-mer peptide P8:F92106 from the F protein of RSV results in the production of peptide- and virus-specific CTL and peptide-specific proliferative responses. Viral loads in the lungs of RSV-challenged, P8:F92106-immunized mice were reduced compared to controls. Viral loads in BALB/c scid mice receiving passively transferred purified CD8+ lymphocytes from P8:F92106-immunized mice were reduced compared to controls and these data suggest that P8:F92106 had induced protective CD8+ responses. It thus seems possible that appropriately designed epitope-specific peptide vaccines could be considered for use against RSV.
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Acknowledgments |
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Footnotes |
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References |
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Received 6 September 2001;
accepted 15 October 2001.