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Protection against Lymphocytic Choriomeningitis Virus Infection Induced by a Reduced Peptide Bond Analogue of the H-2Db-restricted CD8+ T Cell Epitope GP33

1999; Elsevier BV; Volume: 274; Issue: 9 Linguagem: Inglês

10.1074/jbc.274.9.5550

1083-351X

Christine Stemmer, Anne Quesnel-Barbet, Armelle Prévost‐Blondel, Christine Zimmermann, Sylviane Muller, Jean‐Paul Briand, Hanspeter Pircher,

Immune Cell Function and Interaction

Recent investigations have suggested that pseudopeptides containing modified peptide bonds might advantageously replace natural peptides in therapeutic strategies. We have generated eight reduced peptide bond Ψ(CH2–NH) analogues corresponding to the H-2Db-restricted CD8+ T cell epitope (called GP33) of the glycoprotein of the lymphocytic choriomeningitis virus. One of these pseudopeptides, containing a reduced peptide bond between residues 6 and 7 (Ψ(6–7)), displayed very similar properties of binding to major histocompatibility complex (MHC) and recognition by T cell receptor transgenic T cells specific for GP33 when compared with the parent peptide. We assessed in vitro and in vivo the proteolytic resistance of GP33 and Ψ(6–7) and analyzed its contribution to the priming properties of these peptides. The Ψ(6–7) analogue exhibited a dramatically increased proteolytic resistance when compared with GP33, and we show for the first time that MHC-peptide complexes formed in vivo with a pseudopeptide display a sustained half-life compared with the complexes formed with the natural peptide. Furthermore, in contrast to immunizations with GP33, three injections of Ψ(6–7) in saline induced significant antiviral protection in mice. The enhanced ability of Ψ(6–7) to induce antiviral protection may result from the higher stability of the analogue and/or of the MHC-analogue complexes. Recent investigations have suggested that pseudopeptides containing modified peptide bonds might advantageously replace natural peptides in therapeutic strategies. We have generated eight reduced peptide bond Ψ(CH2–NH) analogues corresponding to the H-2Db-restricted CD8+ T cell epitope (called GP33) of the glycoprotein of the lymphocytic choriomeningitis virus. One of these pseudopeptides, containing a reduced peptide bond between residues 6 and 7 (Ψ(6–7)), displayed very similar properties of binding to major histocompatibility complex (MHC) and recognition by T cell receptor transgenic T cells specific for GP33 when compared with the parent peptide. We assessed in vitro and in vivo the proteolytic resistance of GP33 and Ψ(6–7) and analyzed its contribution to the priming properties of these peptides. The Ψ(6–7) analogue exhibited a dramatically increased proteolytic resistance when compared with GP33, and we show for the first time that MHC-peptide complexes formed in vivo with a pseudopeptide display a sustained half-life compared with the complexes formed with the natural peptide. Furthermore, in contrast to immunizations with GP33, three injections of Ψ(6–7) in saline induced significant antiviral protection in mice. The enhanced ability of Ψ(6–7) to induce antiviral protection may result from the higher stability of the analogue and/or of the MHC-analogue complexes. major histocompatibility complex antigen-presenting cell cytotoxic T lymphocyte high performance liquid chromatography incomplete Freund's adjuvant lymphocytic choriomeningitis virus phosphate-buffered saline T cell receptor transgenic T cells recognize antigenic peptides in association with MHC1 molecules and play a key role in protection against harmful pathogens and in tumor elimination. Over the past years many attempts have been made to use synthetic peptides as potential vaccines and immunoregulatory agents (1Liu M.A. Nat. Med. 1998; 4 Suppl. 5: 503Google Scholar, 2Stemmer C. Guichard G. Exp. Opin. Ther. Patents. 1998; 8: 819-830Crossref Scopus (1) Google Scholar). Recent studies have provided evidence that pseudopeptides, in which one or several of the natural amide bonds (CO–NH) are replaced by CO–NH isosters (3Gante J. Angew. Chem. Int. Ed. Engl. 1994; 33: 1699-1720Crossref Scopus (883) Google Scholar), can have enhanced antigenic and immunogenic properties (4Guichard G. Benkirane N. Graff R. Muller S. Briand J.-P. Pept. Res. 1994; 7: 308-321PubMed Google Scholar, 5Benkirane N. Guichard G. Briand J.-P. Muller S. J. Biol. Chem. 1996; 271: 33218-33224Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 6Briand J.-P. Benkirane N. Guichard G. Newman J.F.E. Van Regenmortel M.H.V. Brown F. Muller S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12545-12550Crossref PubMed Scopus (70) Google Scholar). Most interestingly, it has been shown that such peptide analogues can bind to class I and II MHC molecules (7Guichard G. Calbo S. Muller S. Kourilsky P. Briand J.-P. Abastado J.-P. J. Biol. Chem. 1995; 270: 26057-26059Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 8Guichard G. Connan F. Graff R. Ostankovitch M. Muller S. Guillet J.-G. Choppin J. Briand J.-P. J. Med. Chem. 1996; 39: 2030-2039Crossref PubMed Scopus (46) Google Scholar, 9Hill C.M. Liu A. Marshall K.W. Mayer J. Jorgensen B. Yuan B. Cubbon R.M. Nichols E.A. Wicker L.S. Rothbard J.B. J. Immunol. 1994; 152: 2890-2898PubMed Google Scholar, 10Ettouati L. Salvi J.-P. Trescol-Biemont M.C. Walchshofer N. Gerlier D. Rabourdin-Combe C. Paris J. Pept. Res. 1996; 9: 248-253PubMed Google Scholar, 11Mézière C. Viguier M. Dumortier H. Lo-Man M.R. Leclerc C. Guillet J.-G. Briand J.-P. Muller S. J. Immunol. 1997; 159: 3230-3237PubMed Google Scholar, 12Cotton J. Hervé M. Pouvelle S. Maillère B. Ménez A. Int. Immunol. 1998; 10: 159-166Crossref PubMed Scopus (19) Google Scholar, 13Bianco A. Brock C. Zabel C. Walk T. Walden P. Jung G. J. Biol. Chem. 1998; 273: 28759-28765Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), and some of them also induce differential effects on T cell responsiveness (14Ostankovitch M. Guichard G. Connan F. Muller S. Chaboissier A. Hoebeke J. Choppin J. Briand J.-P. Guillet J.-G. J. Immunol. 1998; 161: 200-208PubMed Google Scholar) similar to those described with altered peptide ligands, which contain single amino acid replacements (15Sloan-Lancaster L.J. Allen P.M. Curr. Opin. Immunol. 1995; 7: 103-109Crossref PubMed Scopus (71) Google Scholar). These several recent studies thus demonstrated the potential interest of pseudopeptides as possible therapeutic strategies in T cell-mediated disorders.The high susceptibility of synthetic peptides to proteases is considered to be a major drawback for their use as vaccines or immunoregulatory molecules (16Muller S. Briand J.-P. Res. Immunol. 1998; 149: 55-57Crossref Scopus (2) Google Scholar). We have previously shown that several pseudopeptides with enhanced antigenic activity are more resistant to proteolytic degradation in vitro (5Benkirane N. Guichard G. Briand J.-P. Muller S. J. Biol. Chem. 1996; 271: 33218-33224Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 6Briand J.-P. Benkirane N. Guichard G. Newman J.F.E. Van Regenmortel M.H.V. Brown F. Muller S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12545-12550Crossref PubMed Scopus (70) Google Scholar, 14Ostankovitch M. Guichard G. Connan F. Muller S. Chaboissier A. Hoebeke J. Choppin J. Briand J.-P. Guillet J.-G. J. Immunol. 1998; 161: 200-208PubMed Google Scholar, 17Guichard G. Benkirane N. Zeder-Lutz G. Van Regenmortel M.H.V. Briand J.-P. Muller S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9765-9769Crossref PubMed Scopus (154) Google Scholar). However, no information on in vivo stability of such peptide analogues and the possible direct contribution of this resistance in their ability to modulate the immune response is available. Increased biological activity of protease-resistant pseudopeptide analogues has already been widely shown in other fields of medical chemistry (for review, see Ref. 18Fauchère J.-L. Thurieau C. Adv. Drug. Res. 1992; 23: 127-159Google Scholar, 19Buss J.E. Marsters J.C.J. Chem. Biol. 1995; 2: 787-791Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 20Teal P.E. Nachman R.J. Regul. Pept. 1997; 72: 161-167Crossref PubMed Scopus (22) Google Scholar, 21Azay J. Nagain C. Llinares M. Devin C. Fehrentz J.A. Bernad N. Roze C. Martinez J. Peptides. 1998; 19: 57-63Crossref PubMed Scopus (16) Google Scholar, 22Verdini A.S. Silvestri S. Becherucci C. Longobardi M.G. Parente L. Peppoloni S. Perretti M. Pileri P. Pinori M. Viscomi G.C. Nencioni L. J. Med. Chem. 1991; 34: 3372-3379Crossref PubMed Scopus (42) Google Scholar). The retro-inverso analogue of the immunostimulatory molecule tuftsin is an outstanding example of a bioactive tetrapeptide for which biological efficiency in vivo has been remarkably increased by introducing one modified peptide bond (22Verdini A.S. Silvestri S. Becherucci C. Longobardi M.G. Parente L. Peppoloni S. Perretti M. Pileri P. Pinori M. Viscomi G.C. Nencioni L. J. Med. Chem. 1991; 34: 3372-3379Crossref PubMed Scopus (42) Google Scholar). To examine the possible influence of proteolytic resistance of MHC class I binding peptides on their biological activity, we designed a series of eight reduced peptide bond pseudopeptides of the immunodominant CD8+ T cell epitope of LCMV in C57BL/6 (B6, H-2b) mice. This CTL epitope (called GP33) is located in residues 33–41 of the LCMV glycoprotein (23Pircher H. Moskophidis D. Rohrer U. Burki K. Hengartner H. Zinkernagel R.M. Nature. 1990; 346: 629-633Crossref PubMed Scopus (473) Google Scholar). We tested the capacity of these eight analogues, each containing one reduced peptide bond Ψ(CH2–NH) at successive positions, to bind to H-2Db MHC molecules. The peptides that bound significantly to H-2Db were further analyzed with respect to their recognition by GP33-specific T cells from a Tg mouse that expresses a H-2Db-restricted T cell receptor specific for this epitope and for their ability to induce antiviral protection.DISCUSSIONThe use of peptides corresponding to MHC class I epitopes to induce a protective CTL response against viruses or tumors is of particular interest in the development of peptide-based vaccines. Recent investigations have suggested that antigenic pseudopeptides containing one or several peptide bond isosters might advantageously replace natural peptides in therapeutic strategies because they can bind to MHC class I molecules and generate an efficient T cell response (7Guichard G. Calbo S. Muller S. Kourilsky P. Briand J.-P. Abastado J.-P. J. Biol. Chem. 1995; 270: 26057-26059Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 13Bianco A. Brock C. Zabel C. Walk T. Walden P. Jung G. J. Biol. Chem. 1998; 273: 28759-28765Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 14Ostankovitch M. Guichard G. Connan F. Muller S. Chaboissier A. Hoebeke J. Choppin J. Briand J.-P. Guillet J.-G. J. Immunol. 1998; 161: 200-208PubMed Google Scholar). In this study, we have examined the antigenic and immunogenic properties of pseudopeptide analogues derived from the LCMV glycoprotein peptide 33–41 (GP33) in which one CO–NH amide bond at a time was replaced by a reduced peptide bond Ψ(CH2–NH) in the native sequence. GP33 is presented by H-2Db MHC molecules and recognized in this context by specific CD8+ T cells. This model was particularly interesting for at least three reasons. First, although infection with cytopathic viruses (e.g. vaccina, vesicular stomatis, Semliki Forest, or influenza virus) is controlled by soluble mediators such as antibodies and cytokines, T cell-mediated cytotoxicity is crucial for the resolution of infections with noncytopathic viruses such as LCMV (31Kägi D. Hengartner H. Curr. Opin. Immunol. 1996; 8: 472-477Crossref PubMed Scopus (162) Google Scholar). LCMV is thus an excellent model to study in vivo the efficacy of the CTL response induced by modified peptides. Second, it is known that although the parent peptide GP33 is particularly efficient to induce protection against a viral challenge when it is injected in the presence of IFA, this peptide is unable to generate protection when used in saline solution. Third, a transgenic model containing within the CD8+ T cell population 40–60% transgenic TCR+ (Vα2/Vβ8) T cells specific for peptide GP33 presented in the H-2Db MHC context is available (27Battegay M. Cooper S. Althage A. Baenziger J. Hengartner H. Zinkernagel R. Pircher H. J. Virol. Methods. 1991; 33: 191-198Crossref PubMed Scopus (375) Google Scholar), thus allowing in vivo study of the recognition of the peptide (or pseudopeptide)-MHC complexes by this TCR.Two of eight analogues studied, namely Ψ(5–6) and Ψ(6–7), were able to bind to H-2Db molecules with an apparent affinity similar to that of the parent GP33 peptide. Guichard et al.(7Guichard G. Calbo S. Muller S. Kourilsky P. Briand J.-P. Abastado J.-P. J. Biol. Chem. 1995; 270: 26057-26059Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) previously found that five of eight reduced peptide bond analogues derived from a Plasmodium berghei MHC class I epitope could bind to soluble recombinant H-2Kd molecules. However, the relative affinity of these pseudopeptides to MHC molecules was 5–10-fold lower than that of the parent peptide. The present finding that most of the reduced peptide bond analogues of GP33 exhibited a decreased or no MHC binding capacity correlates with crystallographic data indicating that the peptide backbone plays an important role for binding of peptide to MHC class I molecules (32Young A.C. Zhang W. Sacchettini J.C. Nathenson S.G. Cell. 1994; 76: 39-50Abstract Full Text PDF PubMed Scopus (239) Google Scholar). It also suggests that in this case, the carbonyl oxygens of the residues in positions 5 and 6 are not essential for peptide-MHC binding.CD8+ T cells from LCMV TCR+ mice recognized equally well the parent GP33 peptide and the Ψ(6–7) analogue. In contrast, they did not recognize the Ψ(5–6) analogue presented in the H-2Db context. This result suggests either that the carbonyl oxygen of the residue in position 5 is directly involved in the interaction with the TCR or that this oxygen atom influences the orientation of the Phe6 side chain that has been shown to be crucial for TCR recognition (23Pircher H. Moskophidis D. Rohrer U. Burki K. Hengartner H. Zinkernagel R.M. Nature. 1990; 346: 629-633Crossref PubMed Scopus (473) Google Scholar, 33Sebzda E. Kundig T.M. Thomson C.T. Aoki K. Mak S.Y. Mayer J.P. Zamborelli T. Nathenson S.G. Ohashi P.S. J. Exp. Med. 1996; 183: 1093-1104Crossref PubMed Scopus (144) Google Scholar, 34Hudrisier D. Oldstone M.B. Gairin J.-E. Virology. 1997; 234: 62-73Crossref PubMed Scopus (73) Google Scholar).Because the parent and Ψ(6–7) peptides share similar antigenic properties, the role of their respective susceptibility to proteases in relation to their biological activity could be investigated. We found that the level of GP33 resistance to mouse proteases drastically increased when a single peptide bond located between residues 6 and 7 was replaced by a reduced peptide bond in analogue Ψ(6–7). This result fits well with the observation that a highly protease-sensitive cleavage site is located between positions 6 and 7 in GP33. The detailed molecular mechanisms of GP33 degradation have not yet been elucidated. Cleavage by an endopeptidase remains the most likely possibility, although intervention of carboxypeptidases cannot be excluded. When examined in vivo, the stability of the Ψ(6–7) analogue was also significantly increased. Its half-life in the serum of injected mice was increased by >10 times compared with GP33. The rapid disappearance of GP33 from serum (by renal clearance or most probably as a result of proteolytic degradation), however, may be balanced by the very fast loading of GP33 to MHC molecules from APC. Ten minutes after peptide injection, GP33 (as well as the Ψ(6–7) peptide) was present on splenocytes. This rapid loading of exogenously provided peptides suggests a direct binding to MHC molecules without internalization.In good agreement with previous results (35Romero P. Corradin G. Luescher I.F. Maryanski J.L. J. Exp. Med. 1991; 174: 603-612Crossref PubMed Scopus (140) Google Scholar, 36Eberl G. Widmann C. Corradin G. Eur. J. Immunol. 1996; 26: 1993-1999Crossref PubMed Scopus (21) Google Scholar), the estimated functional half-life of complexes formed in vivo by H-2Db and GP33 was ∼10–15 h. In the same test, the half-life of the Ψ(6–7)-MHC complexes on APC was increased by a factor of two. Several possibilities may account for this increased stability of Ψ(6–7)-MHC complexes. As shown in a stabilization assay with RMA-S cells, the binding of Ψ(6–7) and GP33 peptides to MHC molecules was similar. Nevertheless, we cannot rule out the possibility that the affinity equilibrium constant of the parent peptide and the Ψ(6–7) analogue to Db molecules are slightly different. A second possibility is that reloading on the cell surface after dissociation from the MHC groove is increased in the case of the Ψ(6–7) analogue because this analogue probably also exhibits an enhanced resistance to proteases present in the extracellular matrix. Finally, it is possible that peptide-MHC complexes are internalized after a few hours, and that because of increased proteolytic resistance to cytoplasmic proteases, the Ψ(6–7) analogue can be reloaded on MHC molecules and represented at the cell surface.Because of the fact that GP33 and Ψ(6–7) are loaded to MHC molecules with a similar initial efficacy, when we assessed the immunogenic activity of the Ψ(6–7) analogue in antiviral protection experiments (peptides injected in IFA), we did not find an improved T cell response to the more stable peptide Ψ(6–7). The pseudopeptide effectively showed antiviral protection properties, which is a novel observation, but these were not significantly different from those observed with GP33. This result suggests that possibly because of the presence of oil in the adjuvant, the advantage of increased proteolytic resistance and prolonged in vivo persistence did not improve the apparent biological activity of the analogue. Furthermore, once peptides are bound to MHC molecules they are apparently protected from digestion (37Mouritsen S. Meldal M. Werdelin O. Hansen A.S. Buus S. J. Immunol. 1992; 149: 1987-1993PubMed Google Scholar). It is known that injection of GP33 inoculated without adjuvant is not able to protect mice from LCMV after challenge infection (Ref. 38Aichele P. Brduscha R.K. Zinkernagel R.M. Hengartner H. Pircher H. J. Exp. Med. 1995; 182: 261-266Crossref PubMed Scopus (205) Google Scholarand Fig. 5 B). An important observation shown in this study is that three subcutaneous injections of the free analogue Ψ(6–7) in saline were able to reduce the virus titer in the spleen of immunized mice. This result suggests that Ψ(6–7) but not GP33 is able to prime T cells in the absence of IFA. It remains to be analyzed whether this result is attributable to the higher stability of the Ψ(6–7) analogue in the circulation or to the prolonged half-life of the Ψ(6–7) peptide-MHC complexes. However, the reduction of virus titers obtained with Ψ(6–7) in PBS was not as impressive as observed with peptides in IFA, suggesting the need of an inflammatory process to reach complete antiviral protection. Because our conclusions are of immediate relevance to vaccination, it would be interesting to test the presence and reactivity of memory CD8+ T cells generated after pseudopeptide vaccination. Finally, to better understand the possible mechanisms involved in the clearance of virus by peptide-activated T cells, it will be important to examine the immunological properties of modified peptide ligands containing different types of amide bond isosteric replacements in different viral systems. T cells recognize antigenic peptides in association with MHC1 molecules and play a key role in protection against harmful pathogens and in tumor elimination. Over the past years many attempts have been made to use synthetic peptides as potential vaccines and immunoregulatory agents (1Liu M.A. Nat. Med. 1998; 4 Suppl. 5: 503Google Scholar, 2Stemmer C. Guichard G. Exp. Opin. Ther. Patents. 1998; 8: 819-830Crossref Scopus (1) Google Scholar). Recent studies have provided evidence that pseudopeptides, in which one or several of the natural amide bonds (CO–NH) are replaced by CO–NH isosters (3Gante J. Angew. Chem. Int. Ed. Engl. 1994; 33: 1699-1720Crossref Scopus (883) Google Scholar), can have enhanced antigenic and immunogenic properties (4Guichard G. Benkirane N. Graff R. Muller S. Briand J.-P. Pept. Res. 1994; 7: 308-321PubMed Google Scholar, 5Benkirane N. Guichard G. Briand J.-P. Muller S. J. Biol. Chem. 1996; 271: 33218-33224Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 6Briand J.-P. Benkirane N. Guichard G. Newman J.F.E. Van Regenmortel M.H.V. Brown F. Muller S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12545-12550Crossref PubMed Scopus (70) Google Scholar). Most interestingly, it has been shown that such peptide analogues can bind to class I and II MHC molecules (7Guichard G. Calbo S. Muller S. Kourilsky P. Briand J.-P. Abastado J.-P. J. Biol. Chem. 1995; 270: 26057-26059Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 8Guichard G. Connan F. Graff R. Ostankovitch M. Muller S. Guillet J.-G. Choppin J. Briand J.-P. J. Med. Chem. 1996; 39: 2030-2039Crossref PubMed Scopus (46) Google Scholar, 9Hill C.M. Liu A. Marshall K.W. Mayer J. Jorgensen B. Yuan B. Cubbon R.M. Nichols E.A. Wicker L.S. Rothbard J.B. J. Immunol. 1994; 152: 2890-2898PubMed Google Scholar, 10Ettouati L. Salvi J.-P. Trescol-Biemont M.C. Walchshofer N. Gerlier D. Rabourdin-Combe C. Paris J. Pept. Res. 1996; 9: 248-253PubMed Google Scholar, 11Mézière C. Viguier M. Dumortier H. Lo-Man M.R. Leclerc C. Guillet J.-G. Briand J.-P. Muller S. J. Immunol. 1997; 159: 3230-3237PubMed Google Scholar, 12Cotton J. Hervé M. Pouvelle S. Maillère B. Ménez A. Int. Immunol. 1998; 10: 159-166Crossref PubMed Scopus (19) Google Scholar, 13Bianco A. Brock C. Zabel C. Walk T. Walden P. Jung G. J. Biol. Chem. 1998; 273: 28759-28765Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), and some of them also induce differential effects on T cell responsiveness (14Ostankovitch M. Guichard G. Connan F. Muller S. Chaboissier A. Hoebeke J. Choppin J. Briand J.-P. Guillet J.-G. J. Immunol. 1998; 161: 200-208PubMed Google Scholar) similar to those described with altered peptide ligands, which contain single amino acid replacements (15Sloan-Lancaster L.J. Allen P.M. Curr. Opin. Immunol. 1995; 7: 103-109Crossref PubMed Scopus (71) Google Scholar). These several recent studies thus demonstrated the potential interest of pseudopeptides as possible therapeutic strategies in T cell-mediated disorders. The high susceptibility of synthetic peptides to proteases is considered to be a major drawback for their use as vaccines or immunoregulatory molecules (16Muller S. Briand J.-P. Res. Immunol. 1998; 149: 55-57Crossref Scopus (2) Google Scholar). We have previously shown that several pseudopeptides with enhanced antigenic activity are more resistant to proteolytic degradation in vitro (5Benkirane N. Guichard G. Briand J.-P. Muller S. J. Biol. Chem. 1996; 271: 33218-33224Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 6Briand J.-P. Benkirane N. Guichard G. Newman J.F.E. Van Regenmortel M.H.V. Brown F. Muller S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12545-12550Crossref PubMed Scopus (70) Google Scholar, 14Ostankovitch M. Guichard G. Connan F. Muller S. Chaboissier A. Hoebeke J. Choppin J. Briand J.-P. Guillet J.-G. J. Immunol. 1998; 161: 200-208PubMed Google Scholar, 17Guichard G. Benkirane N. Zeder-Lutz G. Van Regenmortel M.H.V. Briand J.-P. Muller S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9765-9769Crossref PubMed Scopus (154) Google Scholar). However, no information on in vivo stability of such peptide analogues and the possible direct contribution of this resistance in their ability to modulate the immune response is available. Increased biological activity of protease-resistant pseudopeptide analogues has already been widely shown in other fields of medical chemistry (for review, see Ref. 18Fauchère J.-L. Thurieau C. Adv. Drug. Res. 1992; 23: 127-159Google Scholar, 19Buss J.E. Marsters J.C.J. Chem. Biol. 1995; 2: 787-791Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 20Teal P.E. Nachman R.J. Regul. Pept. 1997; 72: 161-167Crossref PubMed Scopus (22) Google Scholar, 21Azay J. Nagain C. Llinares M. Devin C. Fehrentz J.A. Bernad N. Roze C. Martinez J. Peptides. 1998; 19: 57-63Crossref PubMed Scopus (16) Google Scholar, 22Verdini A.S. Silvestri S. Becherucci C. Longobardi M.G. Parente L. Peppoloni S. Perretti M. Pileri P. Pinori M. Viscomi G.C. Nencioni L. J. Med. Chem. 1991; 34: 3372-3379Crossref PubMed Scopus (42) Google Scholar). The retro-inverso analogue of the immunostimulatory molecule tuftsin is an outstanding example of a bioactive tetrapeptide for which biological efficiency in vivo has been remarkably increased by introducing one modified peptide bond (22Verdini A.S. Silvestri S. Becherucci C. Longobardi M.G. Parente L. Peppoloni S. Perretti M. Pileri P. Pinori M. Viscomi G.C. Nencioni L. J. Med. Chem. 1991; 34: 3372-3379Crossref PubMed Scopus (42) Google Scholar). To examine the possible influence of proteolytic resistance of MHC class I binding peptides on their biological activity, we designed a series of eight reduced peptide bond pseudopeptides of the immunodominant CD8+ T cell epitope of LCMV in C57BL/6 (B6, H-2b) mice. This CTL epitope (called GP33) is located in residues 33–41 of the LCMV glycoprotein (23Pircher H. Moskophidis D. Rohrer U. Burki K. Hengartner H. Zinkernagel R.M. Nature. 1990; 346: 629-633Crossref PubMed Scopus (473) Google Scholar). We tested the capacity of these eight analogues, each containing one reduced peptide bond Ψ(CH2–NH) at successive positions, to bind to H-2Db MHC molecules. The peptides that bound significantly to H-2Db were further analyzed with respect to their recognition by GP33-specific T cells from a Tg mouse that expresses a H-2Db-restricted T cell receptor specific for this epitope and for their ability to induce antiviral protection. DISCUSSIONThe use of peptides corresponding to MHC class I epitopes to induce a protective CTL response against viruses or tumors is of particular interest in the development of peptide-based vaccines. Recent investigations have suggested that antigenic pseudopeptides containing one or several peptide bond isosters might advantageously replace natural peptides in therapeutic strategies because they can bind to MHC class I molecules and generate an efficient T cell response (7Guichard G. Calbo S. Muller S. Kourilsky P. Briand J.-P. Abastado J.-P. J. Biol. Chem. 1995; 270: 26057-26059Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 13Bianco A. Brock C. Zabel C. Walk T. Walden P. Jung G. J. Biol. Chem. 1998; 273: 28759-28765Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 14Ostankovitch M. Guichard G. Connan F. Muller S. Chaboissier A. Hoebeke J. Choppin J. Briand J.-P. Guillet J.-G. J. Immunol. 1998; 161: 200-208PubMed Google Scholar). In this study, we have examined the antigenic and immunogenic properties of pseudopeptide analogues derived from the LCMV glycoprotein peptide 33–41 (GP33) in which one CO–NH amide bond at a time was replaced by a reduced peptide bond Ψ(CH2–NH) in the native sequence. GP33 is presented by H-2Db MHC molecules and recognized in this context by specific CD8+ T cells. This model was particularly interesting for at least three reasons. First, although infection with cytopathic viruses (e.g. vaccina, vesicular stomatis, Semliki Forest, or influenza virus) is controlled by soluble mediators such as antibodies and cytokines, T cell-mediated cytotoxicity is crucial for the resolution of infections with noncytopathic viruses such as LCMV (31Kägi D. Hengartner H. Curr. Opin. Immunol. 1996; 8: 472-477Crossref PubMed Scopus (162) Google Scholar). LCMV is thus an excellent model to study in vivo the efficacy of the CTL response induced by modified peptides. Second, it is known that although the parent peptide GP33 is particularly efficient to induce protection against a viral challenge when it is injected in the presence of IFA, this peptide is unable to generate protection when used in saline solution. Third, a transgenic model containing within the CD8+ T cell population 40–60% transgenic TCR+ (Vα2/Vβ8) T cells specific for peptide GP33 presented in the H-2Db MHC context is available (27Battegay M. Cooper S. Althage A. Baenziger J. Hengartner H. Zinkernagel R. Pircher H. J. Virol. Methods. 1991; 33: 191-198Crossref PubMed Scopus (375) Google Scholar), thus allowing in vivo study of the recognition of the peptide (or pseudopeptide)-MHC complexes by this TCR.Two of eight analogues studied, namely Ψ(5–6) and Ψ(6–7), were able to bind to H-2Db molecules with an apparent affinity similar to that of the parent GP33 peptide. Guichard et al.(7Guichard G. Calbo S. Muller S. Kourilsky P. Briand J.-P. Abastado J.-P. J. Biol. Chem. 1995; 270: 26057-26059Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) previously found that five of eight reduced peptide bond analogues derived from a Plasmodium berghei MHC class I epitope could bind to soluble recombinant H-2Kd molecules. However, the relative affinity of these pseudopeptides to MHC molecules was 5–10-fold lower than that of the parent peptide. The present finding that most of the reduced peptide bond analogues of GP33 exhibited a decreased or no MHC binding capacity correlates with crystallographic data indicating that the peptide backbone plays an important role for binding of peptide to MHC class I molecules (32Young A.C. Zhang W. Sacchettini J.C. Nathenson S.G. Cell. 1994; 76: 39-50Abstract Full Text PDF PubMed Scopus (239) Google Scholar). It also suggests that in this case, the carbonyl oxygens of the residues in positions 5 and 6 are not essential for peptide-MHC binding.CD8+ T cells from LCMV TCR+ mice recognized equally well the parent GP33 peptide and the Ψ(6–7) analogue. In contrast, they did not recognize the Ψ(5–6) analogue presented in the H-2Db context. This result suggests either that the carbonyl oxygen of the residue in position 5 is directly involved in the interaction with the TCR or that this oxygen atom influences the orientation of the Phe6 side chain that has been shown to be crucial for TCR recognition (23Pircher H. Moskophidis D. Rohrer U. Burki K. Hengartner H. Zinkernagel R.M. Nature. 1990; 346: 629-633Crossref PubMed Scopus (473) Google Scholar, 33Sebzda E. Kundig T.M. Thomson C.T. Aoki K. Mak S.Y. Mayer J.P. Zamborelli T. Nathenson S.G. Ohashi P.S. J. Exp. Med. 1996; 183: 1093-1104Crossref PubMed Scopus (144) Google Scholar, 34Hudrisier D. Oldstone M.B. Gairin J.-E. Virology. 1997; 234: 62-73Crossref PubMed Scopus (73) Google Scholar).Because the parent and Ψ(6–7) peptides share similar antigenic properties, the role of their respective susceptibility to proteases in relation to their biological activity could be investigated. We found that the level of GP33 resistance to mouse proteases drastically increased when a single peptide bond located between residues 6 and 7 was replaced by a reduced peptide bond in analogue Ψ(6–7). This result fits well with the observation that a highly protease-sensitive cleavage site is located between positions 6 and 7 in GP33. The detailed molecular mechanisms of GP33 degradation have not yet been elucidated. Cleavage by an endopeptidase remains the most likely possibility, although intervention of carboxypeptidases cannot be excluded. When examined in vivo, the stability of the Ψ(6–7) analogue was also significantly increased. Its half-life in the serum of injected mice was increased by >10 times compared with GP33. The rapid disappearance of GP33 from serum (by renal clearance or most probably as a result of proteolytic degradation), however, may be balanced by the very fast loading of GP33 to MHC molecules from APC. Ten minutes after peptide injection, GP33 (as well as the Ψ(6–7) peptide) was present on splenocytes. This rapid loading of exogenously provided peptides suggests a direct binding to MHC molecules without internalization.In good agreement with previous results (35Romero P. Corradin G. Luescher I.F. Maryanski J.L. J. Exp. Med. 1991; 174: 603-612Crossref PubMed Scopus (140) Google Scholar, 36Eberl G. Widmann C. Corradin G. Eur. J. Immunol. 1996; 26: 1993-1999Crossref PubMed Scopus (21) Google Scholar), the estimated functional half-life of complexes formed in vivo by H-2Db and GP33 was ∼10–15 h. In the same test, the half-life of the Ψ(6–7)-MHC complexes on APC was increased by a factor of two. Several possibilities may account for this increased stability of Ψ(6–7)-MHC complexes. As shown in a stabilization assay with RMA-S cells, the binding of Ψ(6–7) and GP33 peptides to MHC molecules was similar. Nevertheless, we cannot rule out the possibility that the affinity equilibrium constant of the parent peptide and the Ψ(6–7) analogue to Db molecules are slightly different. A second possibility is that reloading on the cell surface after dissociation from the MHC groove is increased in the case of the Ψ(6–7) analogue because this analogue probably also exhibits an enhanced resistance to proteases present in the extracellular matrix. Finally, it is possible that peptide-MHC complexes are internalized after a few hours, and that because of increased proteolytic resistance to cytoplasmic proteases, the Ψ(6–7) analogue can be reloaded on MHC molecules and represented at the cell surface.Because of the fact that GP33 and Ψ(6–7) are loaded to MHC molecules with a similar initial efficacy, when we assessed the immunogenic activity of the Ψ(6–7) analogue in antiviral protection experiments (peptides injected in IFA), we did not find an improved T cell response to the more stable peptide Ψ(6–7). The pseudopeptide effectively showed antiviral protection properties, which is a novel observation, but these were not significantly different from those observed with GP33. This result suggests that possibly because of the presence of oil in the adjuvant, the advantage of increased proteolytic resistance and prolonged in vivo persistence did not improve the apparent biological activity of the analogue. Furthermore, once peptides are bound to MHC molecules they are apparently protected from digestion (37Mouritsen S. Meldal M. Werdelin O. Hansen A.S. Buus S. J. Immunol. 1992; 149: 1987-1993PubMed Google Scholar). It is known that injection of GP33 inoculated without adjuvant is not able to protect mice from LCMV after challenge infection (Ref. 38Aichele P. Brduscha R.K. Zinkernagel R.M. Hengartner H. Pircher H. J. Exp. Med. 1995; 182: 261-266Crossref PubMed Scopus (205) Google Scholarand Fig. 5 B). An important observation shown in this study is that three subcutaneous injections of the free analogue Ψ(6–7) in saline were able to reduce the virus titer in the spleen of immunized mice. This result suggests that Ψ(6–7) but not GP33 is able to prime T cells in the absence of IFA. It remains to be analyzed whether this result is attributable to the higher stability of the Ψ(6–7) analogue in the circulation or to the prolonged half-life of the Ψ(6–7) peptide-MHC complexes. However, the reduction of virus titers obtained with Ψ(6–7) in PBS was not as impressive as observed with peptides in IFA, suggesting the need of an inflammatory process to reach complete antiviral protection. Because our conclusions are of immediate relevance to vaccination, it would be interesting to test the presence and reactivity of memory CD8+ T cells generated after pseudopeptide vaccination. Finally, to better understand the possible mechanisms involved in the clearance of virus by peptide-activated T cells, it will be important to examine the immunological properties of modified peptide ligands containing different types of amide bond isosteric replacements in different viral systems. The use of peptides corresponding to MHC class I epitopes to induce a protective CTL response against viruses or tumors is of particular interest in the development of peptide-based vaccines. Recent investigations have suggested that antigenic pseudopeptides containing one or several peptide bond isosters might advantageously replace natural peptides in therapeutic strategies because they can bind to MHC class I molecules and generate an efficient T cell response (7Guichard G. Calbo S. Muller S. Kourilsky P. Briand J.-P. Abastado J.-P. J. Biol. Chem. 1995; 270: 26057-26059Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 13Bianco A. Brock C. Zabel C. Walk T. Walden P. Jung G. J. Biol. Chem. 1998; 273: 28759-28765Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 14Ostankovitch M. Guichard G. Connan F. Muller S. Chaboissier A. Hoebeke J. Choppin J. Briand J.-P. Guillet J.-G. J. Immunol. 1998; 161: 200-208PubMed Google Scholar). In this study, we have examined the antigenic and immunogenic properties of pseudopeptide analogues derived from the LCMV glycoprotein peptide 33–41 (GP33) in which one CO–NH amide bond at a time was replaced by a reduced peptide bond Ψ(CH2–NH) in the native sequence. GP33 is presented by H-2Db MHC molecules and recognized in this context by specific CD8+ T cells. This model was particularly interesting for at least three reasons. First, although infection with cytopathic viruses (e.g. vaccina, vesicular stomatis, Semliki Forest, or influenza virus) is controlled by soluble mediators such as antibodies and cytokines, T cell-mediated cytotoxicity is crucial for the resolution of infections with noncytopathic viruses such as LCMV (31Kägi D. Hengartner H. Curr. Opin. Immunol. 1996; 8: 472-477Crossref PubMed Scopus (162) Google Scholar). LCMV is thus an excellent model to study in vivo the efficacy of the CTL response induced by modified peptides. Second, it is known that although the parent peptide GP33 is particularly efficient to induce protection against a viral challenge when it is injected in the presence of IFA, this peptide is unable to generate protection when used in saline solution. Third, a transgenic model containing within the CD8+ T cell population 40–60% transgenic TCR+ (Vα2/Vβ8) T cells specific for peptide GP33 presented in the H-2Db MHC context is available (27Battegay M. Cooper S. Althage A. Baenziger J. Hengartner H. Zinkernagel R. Pircher H. J. Virol. Methods. 1991; 33: 191-198Crossref PubMed Scopus (375) Google Scholar), thus allowing in vivo study of the recognition of the peptide (or pseudopeptide)-MHC complexes by this TCR. Two of eight analogues studied, namely Ψ(5–6) and Ψ(6–7), were able to bind to H-2Db molecules with an apparent affinity similar to that of the parent GP33 peptide. Guichard et al.(7Guichard G. Calbo S. Muller S. Kourilsky P. Briand J.-P. Abastado J.-P. J. Biol. Chem. 1995; 270: 26057-26059Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) previously found that five of eight reduced peptide bond analogues derived from a Plasmodium berghei MHC class I epitope could bind to soluble recombinant H-2Kd molecules. However, the relative affinity of these pseudopeptides to MHC molecules was 5–10-fold lower than that of the parent peptide. The present finding that most of the reduced peptide bond analogues of GP33 exhibited a decreased or no MHC binding capacity correlates with crystallographic data indicating that the peptide backbone plays an important role for binding of peptide to MHC class I molecules (32Young A.C. Zhang W. Sacchettini J.C. Nathenson S.G. Cell. 1994; 76: 39-50Abstract Full Text PDF PubMed Scopus (239) Google Scholar). It also suggests that in this case, the carbonyl oxygens of the residues in positions 5 and 6 are not essential for peptide-MHC binding. CD8+ T cells from LCMV TCR+ mice recognized equally well the parent GP33 peptide and the Ψ(6–7) analogue. In contrast, they did not recognize the Ψ(5–6) analogue presented in the H-2Db context. This result suggests either that the carbonyl oxygen of the residue in position 5 is directly involved in the interaction with the TCR or that this oxygen atom influences the orientation of the Phe6 side chain that has been shown to be crucial for TCR recognition (23Pircher H. Moskophidis D. Rohrer U. Burki K. Hengartner H. Zinkernagel R.M. Nature. 1990; 346: 629-633Crossref PubMed Scopus (473) Google Scholar, 33Sebzda E. Kundig T.M. Thomson C.T. Aoki K. Mak S.Y. Mayer J.P. Zamborelli T. Nathenson S.G. Ohashi P.S. J. Exp. Med. 1996; 183: 1093-1104Crossref PubMed Scopus (144) Google Scholar, 34Hudrisier D. Oldstone M.B. Gairin J.-E. Virology. 1997; 234: 62-73Crossref PubMed Scopus (73) Google Scholar). Because the parent and Ψ(6–7) peptides share similar antigenic properties, the role of their respective susceptibility to proteases in relation to their biological activity could be investigated. We found that the level of GP33 resistance to mouse proteases drastically increased when a single peptide bond located between residues 6 and 7 was replaced by a reduced peptide bond in analogue Ψ(6–7). This result fits well with the observation that a highly protease-sensitive cleavage site is located between positions 6 and 7 in GP33. The detailed molecular mechanisms of GP33 degradation have not yet been elucidated. Cleavage by an endopeptidase remains the most likely possibility, although intervention of carboxypeptidases cannot be excluded. When examined in vivo, the stability of the Ψ(6–7) analogue was also significantly increased. Its half-life in the serum of injected mice was increased by >10 times compared with GP33. The rapid disappearance of GP33 from serum (by renal clearance or most probably as a result of proteolytic degradation), however, may be balanced by the very fast loading of GP33 to MHC molecules from APC. Ten minutes after peptide injection, GP33 (as well as the Ψ(6–7) peptide) was present on splenocytes. This rapid loading of exogenously provided peptides suggests a direct binding to MHC molecules without internalization. In good agreement with previous results (35Romero P. Corradin G. Luescher I.F. Maryanski J.L. J. Exp. Med. 1991; 174: 603-612Crossref PubMed Scopus (140) Google Scholar, 36Eberl G. Widmann C. Corradin G. Eur. J. Immunol. 1996; 26: 1993-1999Crossref PubMed Scopus (21) Google Scholar), the estimated functional half-life of complexes formed in vivo by H-2Db and GP33 was ∼10–15 h. In the same test, the half-life of the Ψ(6–7)-MHC complexes on APC was increased by a factor of two. Several possibilities may account for this increased stability of Ψ(6–7)-MHC complexes. As shown in a stabilization assay with RMA-S cells, the binding of Ψ(6–7) and GP33 peptides to MHC molecules was similar. Nevertheless, we cannot rule out the possibility that the affinity equilibrium constant of the parent peptide and the Ψ(6–7) analogue to Db molecules are slightly different. A second possibility is that reloading on the cell surface after dissociation from the MHC groove is increased in the case of the Ψ(6–7) analogue because this analogue probably also exhibits an enhanced resistance to proteases present in the extracellular matrix. Finally, it is possible that peptide-MHC complexes are internalized after a few hours, and that because of increased proteolytic resistance to cytoplasmic proteases, the Ψ(6–7) analogue can be reloaded on MHC molecules and represented at the cell surface. Because of the fact that GP33 and Ψ(6–7) are loaded to MHC molecules with a similar initial efficacy, when we assessed the immunogenic activity of the Ψ(6–7) analogue in antiviral protection experiments (peptides injected in IFA), we did not find an improved T cell response to the more stable peptide Ψ(6–7). The pseudopeptide effectively showed antiviral protection properties, which is a novel observation, but these were not significantly different from those observed with GP33. This result suggests that possibly because of the presence of oil in the adjuvant, the advantage of increased proteolytic resistance and prolonged in vivo persistence did not improve the apparent biological activity of the analogue. Furthermore, once peptides are bound to MHC molecules they are apparently protected from digestion (37Mouritsen S. Meldal M. Werdelin O. Hansen A.S. Buus S. J. Immunol. 1992; 149: 1987-1993PubMed Google Scholar). It is known that injection of GP33 inoculated without adjuvant is not able to protect mice from LCMV after challenge infection (Ref. 38Aichele P. Brduscha R.K. Zinkernagel R.M. Hengartner H. Pircher H. J. Exp. Med. 1995; 182: 261-266Crossref PubMed Scopus (205) Google Scholarand Fig. 5 B). An important observation shown in this study is that three subcutaneous injections of the free analogue Ψ(6–7) in saline were able to reduce the virus titer in the spleen of immunized mice. This result suggests that Ψ(6–7) but not GP33 is able to prime T cells in the absence of IFA. It remains to be analyzed whether this result is attributable to the higher stability of the Ψ(6–7) analogue in the circulation or to the prolonged half-life of the Ψ(6–7) peptide-MHC complexes. However, the reduction of virus titers obtained with Ψ(6–7) in PBS was not as impressive as observed with peptides in IFA, suggesting the need of an inflammatory process to reach complete antiviral protection. Because our conclusions are of immediate relevance to vaccination, it would be interesting to test the presence and reactivity of memory CD8+ T cells generated after pseudopeptide vaccination. Finally, to better understand the possible mechanisms involved in the clearance of virus by peptide-activated T cells, it will be important to examine the immunological properties of modified peptide ligands containing different types of amide bond isosteric replacements in different viral systems. We thank S. Batsford and G. Guichard for comments on the manuscript, M. Rawiel for excellent technical assistance, and S. Denkler and T. Imhof for animal husbandry.