Laboratoire National de Santé, PO Box 1102, L-1011 Luxembourg, Luxembourg1
Medizinische Fakultät2 and Biologische Fakultät3, Universität Tübingen, D-72076 Tübingen, Germany
Author for correspondence: Claude Muller (at Laboratoire National de Santé). Fax +352 490686. e-mail claude.muller{at}santel.lu
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Abstract |
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Introduction |
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Antibodies have been shown to be sufficient for protection against MV infection even in the absence of a T cell response (Albrecht et al., 1977 ; Giraudon & Wild, 1985
). The major target of neutralizing and protective antibodies in humans and in animal models is the MV haemagglutinin (MV-H) protein (Norrby & Hammarskjold, 1972
; Varsanyi et al., 1984
; Fournier et al., 1997
). Such antibodies are mostly directed against conformational epitopes (Benjamin et al., 1984
; Fournier et al., 1997
) which are difficult to mimic with synthetic peptides. Therefore, the design of peptide-based vaccines is essentially limited to sequential B cell epitopes (BCEs).
Using a panel of neutralizing and protective MV-H-specific MAbs, we have identified two sequential BCEs: (i) H386400 (HNE, haemagglutinin noose epitope; Ziegler et al., 1996 ); and (ii) H236256 (NE, neutralizing epitope; Fournier et al., 1997
). We have recently shown that synthetic peptides based on the NE sequence induce protective and neutralizing antibodies in an animal model (El Kasmi et al., 1998
, 1999
). However, this epitope is only poorly conserved among different wild-type isolates (El Kasmi et al., 1999
). The HNE sequence represents a cystine loop domain of the MV-H. Its three cysteines (Cys-381, Cys-386 and Cys-394) are conserved among all MV isolates. In addition, this epitope is not recognized by maternal antibodies (Ziegler et al., 1996
).
Peptide vaccines against MV have mostly been evaluated in experiments using vaccine-related clade A viruses. Although in these studies anti-peptide sera neutralized MV in vitro and protected in vivo, neutralization of wild-type viruses was never demonstrated (Obeid et al., 1995 ; Partidos et al., 1997
; El Kasmi et al., 1999
).
Here, we describe the optimization of synthetic peptides, combining the HNE domain with a promiscuous human TCE, that mimic the HNE loop and induce neutralizing serum against early passage wild-type strains, even in the presence of passively acquired antibodies.
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Methods |
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Active and passive immunizations.
Groups of 35 male and female specific-pathogen-free BALB/c mice (H2d) (815 weeks old) were primed intraperitoneally with 100 µg (TB, BT, B and BB) or 150 µg (TTB and BTT) of peptide dissolved in water and emulsified (1:1) in complete Freunds adjuvant (Sigma). Mice were boosted on day 14 and 45 using incomplete Freunds adjuvant (Sigma). Serum was prepared 710 days after the second boost. Protective titres of anti-MV mouse serum (800 µl) were injected intraperitoneally on the day of peptide immunization and served as a model for passively acquired maternal antibodies.
ELISA and flow cytometry.
ELISAs were performed in microtitre plates coated with the biotinylated peptide H386400 as previously described (Fournier et al., 1997 ). This peptide served as a reporter antigen to test reactivity with this BCE. To determine antibody titres of BH6 or of antisera against the homologous peptide or the TCE, microtitre plates were coated overnight at 4 °C with free peptides (0·7 µg per well in 50 µl water). Antibody titres were defined as the highest serum dilution that yielded a signal corresponding to four times the background which was obtained with a naive control serum at a dilution of 1:500 (range of optical density background values 0·150·2). This serum was obtained from pre-immunization bleedings of individual mice or pooled from the pre-immunization sera. These mice were naive for MV by MV-ELISA (Enzygnost) and by FACS on MV-infected cells and control cells. The reactivity of immune sera (1:100) with virus was tested by flow cytometry using an MV-superinfected EBV-transformed human B cell line (WMPT; gift of B. Chain, London, UK) as described previously (Muller et al., 1995b
). The flow cytometry data correspond to a serum dilution of 1:100, since this concentration reflected most appropriately differences between sera. FITC-conjugate (Sigma) alone, naive serum on MV-positive and MV-negative cells or test serum on MV-negative cells served as negative controls. Data are expressed as arbitrary fluorescence units (AFU). In the rare samples which had a background above 1 AFU, the serum was discarded from the analysis. MAb BH6 (diluted 1:1000; Ziegler et al., 1996
) was used to standardize flow cytometry. A neutralizing anti-MV-H serum and an anti-peptide serum served as positive controls. Data are expressed as AFU. Most sera were tested individually and pooled sera (three animals per group) accurately matched the results obtained with single sera.
Neutralization assay (NT).
Triplicates of two-fold serial dilutions of immune and pre-immune serum (75 µl per well; starting concentration 1/48) were pre-incubated for 3 h at 4 °C with 100 TCID50 (75 µl per well) of different viruses (Table 2) in 96-well microtitre plates (Nunc) as described previously (Huiss et al., 1997
). Wild-type viruses were passaged not more than eight times on B95a cells. After adding B95a cells (3·5x104 cells in 75 µl per well), the plates were incubated under standard tissue culture conditions. On day 5, cytopathic effects were evaluated. The serum dilution that prevented cytopathic effects in at least two out of three wells corresponded to the neutralization titre (NT). All peptides were tested in two to four independent immunizations and neutralization experiments. Unless otherwise stated, neutralization was tested against the rodent-adapted MV (Table 2
). The sequence of the BCE H379400 of all viruses was confirmed by sequencing and corresponds to that of the Edmonston strain (Alkhatib & Briedis, 1986
).
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Results |
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Tandem repeats induce neutralizing serum
In previous studies, we and others have observed that tandem TCEs may be more efficient in inducing functional antibodies (El Kasmi et al., 1999 ; Obeid et al., 1995
). Therefore, BCCC was coupled in two different orientations to a tandem TCE. Since the results shown in Fig. 1(b)
indicated that the BCCC contained a TCE, this sequence was also tested as a free tandem repeat (BCCCBCCC). All of these peptides cross-reacted with BH6 (1:103·56) and induced peptide cross-reactive (e.g. anti-BCE, 1:105·56·8) and MV cross-reactive antisera (mean AFU of 535) (Fig. 1c
). The best reactivity against MV was obtained with anti-BCCCBCCC serum. More importantly, however, antibodies generated with TaTaBCCC and with BCCCBCCC neutralized the neuro-adapted MV in vitro (Fig. 1 c
). The consecutive replacement of cysteines by alanines in TaTaBCCC peptides confirmed the results obtained with the alanine-substituted T/B peptides (Table 1
), inasmuch as all three cysteines were required to induce neutralizing serum (Table 2
).
Anti-peptide serum neutralizes wild-type strains
As part of a subunit vaccine, peptides must induce antibodies that are able to neutralize not only laboratory strains but also wild-type viruses. For this purpose, a pool of anti-BCCCBCCC serum was produced and tested against representative viruses of clades A, B, C and D. These viruses represent early passage isolates from different geographic origins (Europe, America and Africa). Table 2 shows that the antisera neutralized all viruses tested. It can be concluded that the neutralizing antibodies are solely directed against the HNE domain, since no additional TCE was used to generate these antisera. Anti-TaTaBCCC serum also neutralized wild-type viruses with similar titres (data not shown).
HNE peptides are not recognized by passively acquired antibodies
To demonstrate immunogenicity of the BCE in the presence of maternal antibodies, the MV Edmonston strain was used to generate a large pool of neutralizing (titre 1:1024) and protective serum. Adult mice were injected intra-peritoneally (i.p.) with protective levels of this serum (800 µl, giving at least 80% protection). Within 1 h after the injection, mice were actively immunized i.p. with the TaTaBCCC peptide. Serum from each mouse was tested by ELISA against the reporter BCE and against the peptide used for immunization (Fig. 2a). Neither the pre-immune sera nor the anti-MV serum pool reacted with the reporter peptide H386400 (Fig. 2a
). The peptide induced BCE-reactive antibody titres in all infused mice that were similar to those observed in the absence of passive MV serum (P value not significant). These results demonstrate that such a peptide can induce virus-specific antibodies in the presence of pre-existing passive protective antibodies.
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Discussion |
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This problem, therefore, cannot necessarily be circumvented by designing peptides that contain mutations of the wild-type viruses or by targeting a highly conserved BCE. We have evidence that antibodies binding to the same sequence of a vaccine-like virus and wild-type viruses, only neutralize the former (unpublished data). Neutralization of wild-type viruses after immunization with peptide-based immunogens is therefore critical and has not been demonstrated in previous studies with MV peptides (El Kasmi et al., 1999 ; Obeid et al., 1995
). Protection against MV wild-type strains can only be inferred from in vitro neutralization assays since small animal models using wild-type virus are only just being developed (Niewiesk, 1999
). In this study, we induced antibodies to a sequential epitope that mediates neutralization of both vaccine-like viruses and wild-type virus. After iterative optimization, peptides based on the sequence of the HNE domain neutralized early passage isolates from different geographic origins and clades.
Several other features of these peptides make them attractive candidates as a component of a subunit vaccine. (i) In infants, current live-attenuated vaccines are neutralized by transplacentally acquired maternal antibodies. HNE-based peptides are not recognized by passively transferred anti-MV immunoglobulins in the mouse. This explains why even protective levels of anti-whole virus antibodies do not suppress active seroconversion induced by the peptide antigen. We have previously shown that humans, particularly women of a child-bearing age, do not produce antibodies against the HNE domain (Ziegler et al., 1996 ). Our results therefore suggest that vaccine formulations based on such peptides could be administered to infants, irrespective of the level of persisting maternal antibodies.
(ii) Another problem that may limit the use of peptide-vaccines is MHC restriction. This is further complicated by the fact that the choice of the TCE influences the conformation of the BCE, and its ability to induce functional antibodies (El Kasmi et al., 1999 ). HNE peptides combined with promiscuous human TCEs induced neutralizing antibodies. Such peptides could also be immunogenic in large parts of the human population. The TCE derived from the MV-F protein may induce memory T cells that could become activated when challenged with MV (Partidos & Steward, 1990
).
(iii) Finally, an increasing number of immune-suppressed children, who might develop complications with a live vaccine, may benefit from a subunit vaccine.
The loop between Cys-381 and Cys-394 is thought to account for the principal tertiary structure of the HNE domain (Hu & Norrby, 1994 ). The iterative optimization of the peptide demonstrated that the induction of MV cross-reactive serum required one of the loops formed by Cys-386 or Cys-381 with Cys-394. However, neutralizing antibodies were solely induced with peptides containing all three cysteines (BCCC). Either one of the two loops is also present in the virus, since MAb BH6, specific for the sequences H381394 and H386400, does not recognize the virus in its reduced form. This MAb also reacts only with oxidized peptides that contain either the Cys-386/Cys-394 or the Cys-381/Cys-394 loop (Ziegler et al., 1996
). In keeping with previous studies, we could also attribute an important role to Cys-394 in the binding of neutralizing antibodies (Ziegler et al., 1996
).
Whereas Hu & Norrby (1994) suggest that Cys-386 may not participate into the loop formation, our data are ambiguous as to the role of Cys-381 and Cys-386. The present results, however, do not allow any conclusion as to which loop is actually required to induce neutralizing antibodies. Consistent with previous observations (El Kasmi et al., 1999
; Obeid et al., 1995
) regarding other sequential epitopes, tandem repeats of TCEs considerably improved the functional activity of the antibodies. Since the BCE used here also contained a TCE, the BCCC dimer may have benefited from the same enhanced T cell help; moreover, the BCCC dimer may enhance B cell activation via surface Ig cross-linking (Rehe et al., 1990
; Wortis et al., 1995
). The TCEs as flanking sequences have profound influences on the structural presentation of the BCE but all TCEs used here appeared to preserve the loop conformation of the BCE. Conversely, the orientation of the BCEs as flanking sequences can influence the immunogenicity of the TCE due to modulation of processing and presentation (Levely et al., 1990
; Vacchio et al., 1989
; Martineau et al., 1996
; Partidos & Steward, 1992
). However, all TCEs in TB orientation supported the induction of neutralizing serum.
Although protection against MV encephalitis has been demonstrated after immunization with MV-derived peptides (El Kasmi et al., 1999 ; Obeid et al., 1995
; Partidos et al., 1997
), this is the first report showing that neutralization of wild-type MV strains after immunization with a peptide is possible.
A peptide-based vaccine with the above features could potentially be given at an early age irrespective of maternal antibody titres and it would also protect against infection with wild-type MV as demonstrated here. If such a vaccine could close the window of vulnerability that exists when maternal antibodies have waned until the child is protected by active immunization at the age of 915 months, considerable progress towards measles control in infants could be achieved
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Acknowledgments |
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References |
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Alkhatib, G. & Briedis, D. J. (1986). The predicted primary structure of the measles virus hemagglutinin. Virology 150, 479-490.[Medline]
Benjamin, D. C., Berzofsky, J. A., East, I. J., Gurd, F. R., Hannum, C., Leach, S. J., Margoliash, E., Michael, J. G., Miller, A. & Prager, E. M. (1984). The antigenic structure of proteins: a reappraisal. Annual Review of Immunology 2, 67-101.[Medline]
Demotz, S., Lanzavecchia, A., Eisel, U., Niemann, H., Widmann, C. & Corradin, G. (1989). Delineation of several DR-restricted tetanus toxin T cell epitopes. Journal of Immunology 142, 394-402.
El Kasmi, K. C., Theisen, D., Brons, N. H. & Muller, C. P. (1998). The molecular basis of virus crossreactivity and neutralisation after immunisation with optimised chimeric peptides mimicking a putative helical epitope of the measles virus hemagglutinin protein. Molecular Immunology 35, 905-918.[Medline]
El Kasmi, K. C., Theisen, D., Brons, N. H., Ammerlaan, W., Klingele, M., Truong, A. T. & Muller, C. P. (1999). A hemagglutinin-derived peptide-vaccine ignored by virus-neutralising passive antibodies, protects against murine measles encephalitis. Vaccine 17, 2436-2445.[Medline]
Fournier, P., Brons, N. H. C., Berbers, G. A. M., Wiesmüller, K.-H., Fleckenstein, B. T., Schneider, F., Jung, G. & Muller, C. P. (1997). Antibodies to a new linear site at the topographical or functional interface between the haemagglutinin and fusion proteins protect against measles encephalitis. Journal of General Virology 78, 1295-1302.[Abstract]
Giraudon, P. & Wild, T. F. (1985). Correlation between epitopes on hemagglutinin of measles virus and biological activities: passive protection by monoclonal antibodies is related to their hemagglutination inhibiting activity. Virology 144, 46-58.[Medline]
Hu, A. & Norrby, E. (1994). Role of individual cysteine residues in the processing and antigenicity of the measles virus haemagglutinin protein. Journal of General Virology 75, 2173-2181.[Abstract]
Huiss, S., Damien, B., Schneider, F. & Muller, C. P. (1997). Characteristics of asymptomatic secondary immune responses to measles virus in late convalescent donors. Clinical Experimental Immunology 109, 416-420.[Medline]
Levely, M. E., Mitchell, M. A. & Nicholas, J. A. (1990). Synthetic immunogens constructed from T-cell and B-cell stimulating peptides (T:B chimeras): preferential stimulation of unique T- and B-cell specificities is influenced by immunogen configuration. Cellular Immunology 125, 65-78.[Medline]
Martineau, P., Leclerc, C. & Hofnung, M. (1996). Modulating the immunological properties of a linear B-cell epitope by insertion into permissive sites of the MalE protein. Molecular Immunology 33, 1345-1358.[Medline]
Muller, C. P., Bunder, R., Mayser, H., Ammon, S., Weinmann, M., Brons, N. H., Schneider, F., Jung, G. & Wiesmuller, K. H. (1995a). Intramolecular immunodominance and intermolecular selection of H2d-restricted peptides define the same immunodominant region of the measles virus fusion protein. Molecular Immunology 32, 37-47.[Medline]
Muller, C. P., Beauverger, P., Schneider, F., Jung, G. & Brons, N. H. C. (1995b). Cholera toxin B stimulates systemic neutralizing antibodies after intranasal co-immunization with measles virus. Journal of General Virology 76, 1371-1380.[Abstract]
Niewiesk, S. (1999). Cotton rats (Sigmodon hispidus): an animal model to study the pathogenesis of measles virus infection. Immunology Letters 65, 47-50.[Medline]
Norrby, E. & Hammarskjold, B. (1972). Structural components of measles virus. Microbios 5, 17-29.[Medline]
Obeid, O. E., Partidos, C. D., Howard, C. R. & Steward, M. W. (1995). Protection against morbillivirus-induced encephalitis by immunisation with a rationally designed synthetic peptide vaccine containing B- and T-cell epitopes from the fusion protein of measles virus. Journal of Virology 69, 1420-1428.[Abstract]
Partidos, C. D. & Steward, M. W. (1990). Prediction and identification of a T cell epitope in the fusion protein of measles virus immunodominant in mice and humans. Journal of General Virology 71, 2099-2105.[Abstract]
Partidos, C. D. & Steward, M. W. (1992). The effects of a flanking sequence on the immune response to a B and a T cell epitope from the fusion protein of measles virus. Journal of General Virology 73, 1987-1994.[Abstract]
Partidos, C. D., Ripley, J., Delmas, A., Obeid, O. E., Denbury, A. & Steward, M. W. (1997). Fine specificity of the antibody response to a synthetic peptide from the fusion protein and protection against measles virus-induced encephalitis in a mouse model. Journal of General Virology 78, 3227-3232.[Abstract]
Rehe, G. T., Katona, I. M., Brunswick, M., Wahl, L. M., June, C. H. & Mond, J. J. (1990). Activation of human B lymphocytes by nanogram concentrations of anti-IgM-dextran conjugates. European Journal of Immunology 20, 1837-1842.[Medline]
Sabin, A. B. (1992). My last will and testament on rapid elimination and ultimate global eradication of poliomyelitis and measles. Pediatrics 90, 162-169.[Medline]
Vacchio, M. S., Berzofsky, J. A., Krzych, U., Smith, J. A., Hodes, R. J. & Finnegan, A. (1989). Sequences outside a minimal immunodominant site exert negative effects on recognition by staphylococcal nuclease-specific T cell clones. Journal of Immunology 143, 2814-2819.
Varsanyi, T. M., Utter, G. & Norrby, E. (1984). Purification, morphology and antigenic characterization of measles virus envelope components. Journal of General Virology 65, 355-366.[Abstract]
Wiesmüller, K.-H., Spahn, G., Handtmann, D., Schneider, F., Jung, G. & Muller, C. P. (1992). Heterogeneity of linear B cell epitopes of the measles virus fusion protein reacting with late convalescent sera. Journal of General Virology 73, 2211-2216.[Abstract]
World Health Report (1996). Fighting Disease, Fostering Development. Geneva, Switzerland: WHO.
Wortis, H. H., Teutsch, M., Higer, M., Zheng, J. & Parker, D. C. (1995). B-cell activation by crosslinking of surface IgM or ligation of CD40 involves alternative signal pathways and results in different B-cell phenotypes. Proceedings of the National Academy of Sciences, USA 92, 3348-3352.[Abstract]
Ziegler, D., Fournier, P., Berbers, G. A. H., Steuer, H., Wiesmüller, K.-H., Fleckenstein, B., Schneider, F., Jung, G., King, C.-C. & Muller, C. P. (1996). Protection against measles virus encephalitis by monoclonal antibodies binding to a cystine loop domain of the H protein mimicked by peptides which are not recognized by maternal antibodies. Journal of General Virology 77, 2479-2489.[Abstract]
Received 12 October 1999;
accepted 16 November 1999.