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Short Communication |
1 Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK
2 Grupo de Investigaciones en Enfermedades Tropicales, Departamento de Ciencias Basicas Medicas, Universidad del Norte, Km 5 Antigua via Puerto Colombia, Barranquilla, Colombia
Correspondence
Andrew K. I. Falconar
afalconar{at}uninorte.edu.co
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ABSTRACT |
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). Because there are marked cDNA sequence differences in the main envelope (E) glycoprotein, particularly in dengue type-2 virus (DENV-2) strains amongst the four DENV serotypes (Holmes, 2006), it is important to identify conserved DENV-neutralizing epitopes. Epitopes on this protein, defined by neutralizing monoclonal antibodies (mAbs), have been located by escape mutation analyses or by the reduction/abrogation of mAb reactions against recombinant DENV-2 E glycoproteins that contained specific amino acid substitutions. Few studies have, however, shown that neutralizing epitopes on these glycoproteins can be represented faithfully using short synthetic peptides, despite their potential importance for diagnostic, pathogenesis and active protection studies. In an early study, the flavivirus subgroup-reactive neutralizing mAb, 1B7, most strongly identified three peptide sequences (50-AKQPATLR-57, 127-GKVVLPEN-134 and 349-GRLITVNP-356) amongst a set of peptides spanning the DENV-2 E glycoprotein sequence (Aaskov et al., 1989). The flavivirus group-reactive neutralizing mAbs, 2C5.1 and 4G2, however, reacted only weakly with the 106-GLFGKGSLVT-115 peptide overlapping the highly conserved 98–111 fusion sequence (Falconar, 1999), which contained the critical 106-G or 107-L residues for mAb 4G2 reaction against the native E glycoproteins of DENV-2 (Crill & Chang, 2004) and other flaviviruses (Stiasny et al., 2006; Crill et al., 2007; Trainor et al., 2007). The 98-DRGWGNGSGLFGKGG-112 peptide, containing 106-G, 107-L, and a 105-C (cysteine) by S (serine) substitution (underlined), reduced the binding of a recombinant Fab fragment to this target sequence on DENV-2 particles by less than 65 % at very high (1 mM) concentrations (Goncalvez et al., 2004). Further studies are, therefore, required to represent more faithfully the epitopes defined by mAbs 2C5.1 and 4G2. High-resolution X-ray crystallographic structural determinations of the native, dimeric form of the DENV-2 E glycoprotein (Modis et al., 2003; http://www.ncbi.nlm.nih.gov NCBI structural database MMDB ID: 23080) allows surface-exposed sequences to be identified and represented as synthetic peptides. For these epitope-mapping studies, it is essential that each surface-exposed amino acid side chain is available for mAb reaction and, therefore, their preparation with spacer molecules as described previously (Aaskov et al., 1989; Falconar et al., 1994; Falconar, 1999) offers an ideal methodology. The relatively high background signals obtained using this method, particularly using mAbs generated against the DENV E glycoproteins (Aaskov et al., 1989; Falconar, 1999), however, necessitates using an absorbance threshold and an appropriate negative-control mAb, to identify the DENV E glycoprotein peptide-specific mAb reactions.
In this study, a DENV E glycoprotein complex-reactive mAb, 3A8.1, a flavivirus subcomplex-reactive mAb, 2F2.1, two flavivirus group-reactive mAbs, 2C5.1 and 4G2, and a control mAb, 3D1.4, which defined the LX1 epitope on the DENV non-structural 1 (NS1) glycoproteins (Falconar, 2007), were used. mAbs 3A8.1, 2F2.1 and 2C5.1 and mAb 3D1.4 were generated against live DENV-2 (PR159 strain) and the purified DENV-2 (PR159 strain) NS1 glycoprotein (Falconar & Young, 1991; Falconar, 1999). The preparation and characterization of mAb 4G2 has been described previously (Henchal et al., 1985) and it was obtained from the ATCC. Each of these mAbs was affinity purified and their reciprocal log10 50 % end-point ELISA titres against DENV-2 particles (New Guinea-C strain) or the purified DENV-2 (PR159 strain) NS1 glycoprotein (mAb 3D1.4) was determined as described previously (Falconar, 1999, 2007). Synthetic peptides were prepared using activated Fmoc-amino acids (Novabiochem) on 60–64 nM ‘gears’ (Chiron Mimetopes) and these peptide-coated ‘gears’ were processed in 96-well ELISA format as described previously (Falconar, 1997, 1999, 2007). After determining the absorbance at 492 nm (MRX; Dynex), specific mAb reactions were identified as those above the 50 % average maximum absorbance. These peptide-coated ‘gears’ were then recycled as described previously (Falconar et al., 1994).
mAbs 4G2, 2C5.1 and 2F2.1 bound to a recombinant domain III fragment of the DENV-2 E glycoprotein (Megret et al., 1992), against which specific mAbs were the most efficient blockers of DENV-2 adsorption to Vero cells (Crill & Roehrig, 2001). Their reactions were, therefore, initially assessed against duplicate sets of 47 overlapping synthetic peptides, 9 aa in length, sequentially spanning domain III of DENV-2 (New Guinea-C strain) and the negative-control DENV NS1 glycoprotein epitope, LX1 (113-YSWKTWGKA-121) (Falconar, 1997, 2007), against which none of these DENV E glycoprotein-reactive mAbs should cross-react. In this study, these four DENV E glycoprotein-specific mAbs very weakly cross-reacted [absorbance (Abs) 0.02–0.06] with the LX1 epitope (negative-control peptide), while it was strongly identified by the control mAb, 3D1.4 (Abs 1.75) (data not shown). An average maximal absorbance of 1.59 was, however, obtained by mAbs 3A8.1, 2C5.1 and 4G2 (Abs 1.38, 1.60 and 1.78, respectively) against the duplicate set of DENV-2 E glycoprotein domain III peptides, from which the 0.80 absorbance threshold (Absmax1.59/2=0.80) was established (Fig. 1). Both flavivirus group-reactive mAbs, 2C5.1 and 4G2, reacted most strongly with peptide 26 (351-LITVNPIVT-359) (mAb 2C5.1: Abs 1.60; mAb 4G2: Abs 1.78), but mAb 2C5.1 also reacted strongly with peptide 25 (349-GRLITVNPI-357: Abs 1.39). These results were consistent with these mAbs, showing strong reactions against the recombinant 298–397 DENV-2 E glycoprotein fragment (Megret et al., 1992) and the ability of a DENV-2 E glycoprotein 352–368 peptide, containing the 351–357 sequence, to generate neutralizing antibodies (Roehrig et al., 1990). This 351–357 sequence was, however, inaccessible in the native, dimeric DENV-2 E glycoprotein structure (Modis et al., 2003), suggesting that it may mimic a surface-exposed neutralizing epitope located elsewhere on this glycoprotein. The DENV complex-reactive mAb 3A8.1 reacted strongly with peptide 2 (303-TGKFKVVKE-311: Abs 0.99) and 3 (305-KFKVVKEIA-313: Abs 1.03), but reacted even more strongly with peptide 47 (393-KKGSSIGQM-401: Abs 1.38). Both the 303–311 and 305–313 sequences were highly exposed on the top side face of the native DENV-2 E glycoprotein within the 302–333 cysteine bridge-stabilized loop (Modis et al., 2003). These peptides contained 305-K, 307-K and 310-K identified by some DENV subcomplex-reactive neutralizing mAbs (Sukupolvi-Petty et al., 2007), and a 307-K by E escape mutation was generated by a neutralizing mAb (Lin et al., 1994). The 393–401 sequence was, however, not included in the high resolution X-ray diffraction determination of the dimeric DENV-2 E glycoprotein (aa 1–395) (Modis et al., 2003), but was identified as a highly flexible [high B (norm)] exposed coil located immediately adjacent to the 303–313 sequence in the lower (9.5 Å, 0.95 nm) resolution cryo-electron microscopic structure determination of aa 1–495 (Zhang et al., 2003; NCBI structural database MMDB ID: 25032). The 303–313 and 393–401 sequences were, therefore, likely to be identified by mAb 3A8.1 either as a single-discontinuous epitope or possibly as two individual epitope sequences. In contrast, the flavivirus subgroup-reactive mAb, 2F2.1, as well as the control mAb, 3D1.4, reacted very weakly with this duplicate set of 47 peptides from domain III.
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; NCBI sequence database). When the 0.80 absorbance threshold was applied, the flavivirus group-reactive mAbs, 2C5.1 and 4G2, only showed reactions above this value against the 101-WGNGCGLFG-109 peptide from the flavivirus-conserved fusion sequence (Table 1). mAb 2F2.1, however, showed a reaction just above the threshold value with peptide 48-TEAKQPATLR-57 amongst these peptides. This latter result was consistent with this peptide sequence also being identified by another flavivirus subgroup-reactive mAb, 1B7 (Aaskov et al., 1989), and a peptide containing the 35–55 sequence, in which only aa 35–38 and 48–56 were surface exposed in the DENV-2 E glycoprotein, generating neutralizing antibodies in mice (Roehrig et al., 1990). mAbs 3A8.1 and 2C5.1, but not mAb 4G2, also showed weak, but sub-absorbance threshold reactions (<Abs 0.80) against the 48–57 peptide, possibly due to the inclusion of 57-R, that was slightly recessed in the native, dimeric DENV-2 E glycoprotein. Interestingly, however, the 48–57 sequence on chain A was brought adjacent to the 101–109 fusion sequence of chain C in the transition from immature to mature DENV-2 virions (Zhang et al., 2003; NCBI MMDB ID: 25032; Zhang et al., 2004; NCBI MMDB IDs: 29434 and 29438) that could, therefore, also be part of a discontinuous epitope defined by mAb 2F2.1, and possibly mAb 2C5.1 (see below). The DENV complex-reactive mAb 3A8.1, however, reacted most strongly with synthetic peptides from domain III, as observed (Fig. 1
). This mAb showed the strongest reaction (Abs 1.24) against the corresponding 393-KKGSSIGKM-401 peptide sequence of one Asian 1 DENV-2 genotype strain, which contained a 400-Q by K substitution (underlined) (Lewis et al., 1993) (Table 1). This absorbance value was, therefore, similar to that obtained against the 393-KKGSSIGQM-401 peptide (Abs 1.38) present in other DENV-2 strains (Fig. 1). The 393-R/KKGSSIGQ/KM-401 sequence was conserved in the E glycoproteins of all four DENV serotypes and, therefore, the reaction of mAb 3A8.1 with this sequence as a single epitope, or possibly together with the adjacent 304–313 sequence (see below) as a discontinuous epitope, is likely to account for its DENV complex reactivity. mAb 3A8.1 also showed reactions against the 304-GKFKVVKEIA-313 and 304-GKFKIVKEIA-313 sequences present in nearly all DENV-2 strains, but only weakly with the 304-GKFKVVEEIA-313 sequence present in some DENV-2 strains of the Asian 1 genotype (Lewis et al., 1993) (Table 1). Thus, mAb 3A8.1 is likely to react only weakly against this sequence per se from these latter DENV-2 strains due to this single substitution of 310-K, which was also shown to be a critical amino acid defined by some DENV subcomplex neutralizing mAbs (Sukupolvi-Petty et al., 2007). In contrast, the control mAb 3D1.4 showed very weak cross-reactions with all of these peptides.
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, 2004; NCBI MMDB IDs: 23080 and 26046) and (ii) a synthetic peptide containing the 105-C by G substitution prepared in the reverse (negative) orientation to gauge mAb reaction-specificity as described previously (Falconar, 1999). In this study, only the flavivirus complex-reactive mAbs 2C5.1 and 4G2 showed reactions above the 0.80 absorbance threshold against the natural 101-WGNGCGLFG-109 sequence, but their reactions were greatly increased when it contained the 105-C by G substitution (101-WGNGGGLFG-109) (underlined) (Modis et al., 2003, 2004) (Table 2). The flavivirus subcomplex mAb, 2F2.1, also showed a reaction slightly above the 0.80 absorbance threshold (Abs 0.85) against the 101–109 peptide only when it contained the 105-C by G substitution. mAb 2F2.1, therefore, also appeared to define an epitope within the fusion sequence, but which was less well represented as a synthetic peptide. This mAb, and possibly mAb 2C5.1, may, however, also react more strongly with a discontinuous epitope, including the 48–57 sequence of chain A when it is brought adjacent to the 101–109 fusion sequence of chain C in mature DENV-2 virions (Zhang et al., 2003). As expected, the 101–109 sequence was weakly identified by mAbs 4G2, 2C5.1 and 2F2.1 when it was prepared in the reverse orientation containing the 105-C by G substitution, confirming their high specificity with this peptide sequence.
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; Stiasny et al., 2006; Crill et al., 2007; Trainor et al., 2007) as well as 104-G that was also required for optimal mAb 4G2 binding (Crill & Chang, 2004). This 101–109 peptide also contained 101-W that was essential for the binding of some neutralizing mAbs generated against the West Nile virus E glycoprotein (Oliphant et al., 2006). The 105-C by G substitution used to represent the rotated C side chain (-CH2-SH) bridged with 74-C (i.e. -CH2-S-S-CH2-), greatly increased the antigenicity of the 101–109 peptide in this study. These results may also account for the relatively weak ability of a 98–112 peptide sequence containing a 105-C by S substitution, which had a similar sized side chain (-CH2-OH), to inhibit only weakly a mAb binding to this target-epitope sequence (Goncalvez et al., 2007). This peptide sequence, possibly containing additional substitutions to represent more faithfully the native fusion sequence may, therefore, be useful for diagnostic assays. The flavivirus fusion sequence was, however, cryptic in the pre-fusion form (Allison et al., 2001; Stiasny et al., 2006). In contrast, the newly defined DENV conserved 393-R/KKGSSIGQ/KM-401 epitope was present as a highly flexible coil immediately flanked by the 385–393 sequence implicated in cell binding (Cologna & Rico-Hesse, 2003) and the 401–413 sequence implicated in E glycoprotein homo-trimer formation (Allison et al., 1999), which may make this sequence alone, or possibly as a discontinuous epitope with the adjacent 304–313 sequences, useful for diagnostic assays as well as suitable for generating active cross-protection against all DENV serotypes.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 6 December 2007;
accepted 1 March 2008.
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