key: cord-0008033-r0f257jv
authors: Trirawatanapong, Thaweesak; Chandran, Bala; Putnak, Robert; Padmanabhan, R.
title: Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody
date: 1992-07-15
journal: Gene
DOI: 10.1016/0378-1119(92)90509-n
sha: e2e24b371002ae84f05ed7ec76ad923a2a90f573
doc_id: 8033
cord_uid: r0f257jv

Envelope glycoprotein E of flaviviruses is exposed at the surface of the virion, and is responsible for eliciting a neutralizing antibody (Ab) response, as well as protective immunity in the host. In this report, we describe a method for the fine mapping of a linear sequence of the E protein of dengue virus type-2 (DEN-2), recognized by a type-specific and neutralizing monoclonal Ab (mAb), 3H5. First, an Escherichia coli expression vector containing a heat-inducible λ pl promoter was used to synthesize several truncated, and near-full length E polypeptides. Reactivities of these polypeptides with polyclonal mouse hyperimmune sera, as well as the 3H5 mAb revealed the location of the 3H5-binding site to be within a region of 166 amino acids (aa) between aa 255 and 422. For fine mapping, a series of targeted deletions were made inframe within this region using the polymerase chain reaction (PCR). The hydrophilicity pattern of this region was used as a guide to systematically delete the regions encoding the various groups of surface aa residues within the context of a near-full-length E polypeptide by using PCR. The 3H5-binding site was thus precisely mapped to a region encoding 12 aa (between aa 386 and 397). A synthetic peptide containing this sequence was able to bind to the 3H5 mAb specifically, as shown by enzyme-linked immunosorbent assay. In addition, we show that rabbit Abs raised against the synthetic peptide of 12 aa were able to bind to the authentic E protein, and to neutralize DEN-2 virus in a plaque reduction assay.

Dengue is a human disease caused by dengue virus (DEN), a member of the family flaviviridae (Westaway et al., 1985) . The viral genome is a single-stranded RNA of 10723 nt (New Guinea C strain) in length, having a type-1 cap at the 5' end, but lacking a poly(A) tract at the 3' end (for a review, see Chambers 1990) . The genomic RNA is of positive-strand polarity having a single ORF that encodes a polyprotein of 3391 aa in length, which is then processed into three structural and at least seven nonstrucrural proteins so far identified. The four distinct serotypes of DEN are transmitted to humans principally by Aedes aegypti mosquitos.

The three structural proteins are the C, prM and E in mature flavivirus virion (Stollar, 1969) , and the E protein has been shown to exhibit multiple biological activities. It is the major target for the induction of neutralizing antibodies, and has the property of inducing a protective immune response in the host (Buckley and Gould, 1985; Della-Porta and Westaway, 1977; Kitano etal., 1974; Heinz et al., 1981; Monath et al., 1984) . It can also passively protect mice in vivo against lethal intracerebral DEN-2 challenge (Kaufman et al., 1987) . The E protein is 4 (irie et al., 1989) was digested with B, BamHI; E, E¢oRI; N, Ncol (partial); P, Pvull; S, Seal; Sp, Sphl; or 1", Tth 1111, and the fragments were made blunt by treatment with Pollk in each case. These fragments were cloned into the pOTS vector (pOTSV) plasmid [conraining ; . phage left operator and promoter (OtPL), and a Cll ribosome-binding site (¢!! RBS)], which was digested with BmnHl and treated with Pulik prior to cloning using standard techniques (Maniatis et al., 1982) . The expression plasmids of general structure pOTS-D2E were obtained in which the size ofthe DEN-2 E coding sequence varied as follows: p'i'F 10 (Pvull-EcoRi), pTTI5 (Tth I 1 ll-EcoRl), pTT20 ($cal-EcoRl), pTT25 (Sphl-EcoRl), pTT30 (Nc'oi-EcoRI) , and p'I-r35 (deletion of nt 1697-2203 between the two BamHl sites), in all cases, the reading frame for the expression of the corresponding E polypeptide was kept in-frame and was verified by the m sequence analysis. For transformation, and initial screening of the proper orientation of the eDNA insert, E. coli MM 294(2cl ÷ ) was used, and for expression N515 l(ta:lts857) was used.

involved in binding of the virus to the host cell, and is also the antigen responsible for hemagglutination of erythrocytes at acid pH (Sweet and Sabin, 1954) . Similar to the membrane glycoproteins of other enveloped viruses, the flavivirus E protein is quite variable at least in certain antigenically active domains (Heinz, 1986) . The variable portion of the E protein contains epitopes which determine the type specificity and complex specificity. These antigenic specificities form the basis for serological classification of flaviviruses as determined by HI and neutralization tests. Identification of the neutralizing epitopes of the E protein is important to understand the mechanism of virus-host cell interaction, and to develop an effective vaccine to control the dengue virus infections. Various methods are available to determine the precise location of antigenic sites on a protein molecule (Benjamin et al., 1984; Berzofsky, 1985) . One approach involves the use ofmAbs as specific reagents for defining single epitopes on the complex antigenic structure of protein molecules. Using methods involving screening of purified proteolytic fragments of a protein molecule and/or screening a collection of overlapping synthetic peptides, the antigenic determinants of a number of viral glycoproteins have been studied (Emini et al., 1982; Lubeck and Gerhard, 1982; Massey and Schochetman, 1981; Mehra et al., 1986; Roehrig et al., 1982; Volk et al., 1982) . Using RNA recombination as a genetic tool, the antigenic determinants involved in neutralization, as well as virus neuropathogenicity of the peplomer protein encoded by gene C of murine coronavirus were localized at the C-terminal one third of the peplomer (Makino et al., 1987) . Lobigs et al. (1987) defined the antigenic determinant involved in the neutralization of YF on   TABLE I Map positions of E polypeptides encoded by expression clones the N-terminal region of the E protein, which is hydrophilic, rich in Cys, and not conserved among flavivirus subgroups. The approach used was to select neutralization escape variants against two neutralizing mAbs, and sequence the variants in the region of the genomc encoding the E and M proteins. It was found that each of the variants resulted from a single base change in the E protein coding sequence leading to a nonconservative substitution at aa ~ or aa 72. An alternative method involves the construction of expression libraries using the ).gtl 1 system and fragments ofgene encoding the antigen, and sequence all the cDNA expression clones that reacted with a specific mAb. The DNA sequence encoding the epitope is attributed to sequences that are shared by multiple antibody-positive recombinant clones (Mehra et al., 1986) .

The aim of the present study was to map the antigenic determinant of the DEN-2 E protein recognized by the 3H5, a type-specific mAb, which was shown to have a significantly high neutralization titer Henchal et al., 1995) . The expression system used by us for mapping the 3HS-binding site on the E protein significantly differs from the previously reported method (Mehra et al., 1986) in that in our system the polypeptides produced in E. coli cells are not fused to any bacterial protein.

In order to map the neutralizing determinant of DEN-2 E recognized by the 3H5 mAb, we have made use of an E. coil expression vector pOTS (Fig. 1 BamHl site for cloning foreign genes downstream from the PL promoter, the ribosome-binding site, and the translation-initiation site (Rosenberg et al., 1983) . For expression, the cDNA fragment encoding the E polypeptide between PvulI and EcoRI sites of the pKT2.4 plasmid (Irie et al., 1989) was cloned into the pOTS vector (pTT10 in Table I ) was used. The polypeptide encoded by pTT10 contains the E sequence from aa 1 to aa 470, as well as seven hydrophobic aa upstream from the N terminus, but lacks 25 aa at the C terminus of the E protein.

Next, several deletion constructs were made in the Nterminal, C-terminal, or internal regions such that the translational reading frame was maintained (Fig. 1) . The progressively shortened N-terminal deletions of the E protein were made by cleaving the PvuII-EcoRI fragment with restriction enzymes Tth l l lI, Scal, Sphl and Ncol (see Table I ). These DNA fragments were cloned at the BamHI site of pOTS vector to generate the clones, pTT 15, pTT20, pTT25, and pTT30, respectively. These clones had the correct orientation for expression of the E polypeptides with progressively longer N-terminal deletions (see Table I ). The internal deletion ofthe E protein was made by removing the region encoding 166 aa flanked by the two BamHI restriction sites from the plasmid pTT15 ( Fig. 1 and Table I ). This plasmid encodes the polypeptide lacking aa 255 to 422 in the vicinity of the C terminus. The parent and the truncated expression constructs of E polypeptide were analyzed for their levels of expression in E. coil N5151 strain, and tested for their reactivities with the polyclonal HMAF (Fig. 2 , panel A), as well as the 3H5 mAb (Fig. 2, panel B) , by Western blotting. The expression plasmid pTTI0 gave rise to an E polypeptide which was lower in size than the expected 51 kDa ( Fig. 2A and 2B, lane 1), most likely due to its instability in E. coll. However, the polypeptide expressed from pTTIS was of expected size (see Fig. 2A and 2B, lane 2), suggesting that removal of the N-terminal 29 aa, including the seven hydrophobic residues, stabilized this protein considerably. The level of this polypeptide was high enough to be visualized directly by Coomassie blue staining of the polyacrylamide gel (data not shown). The E polypeptides containing progressively shorter N-terminal sequences expressed from the plasmids pTT20, pTT25 and pTT30, respectively, were loaded in lanes 3, 4 and 5, and the authentic E protein present in the DEN-2-infected mosquito (C6/ 36) cell lysates was loaded in lane C of Fig. 2 . The levels of expression, and their immunoreactivities with HMAF and the 3H5 mAb of these E polypeptides varied. However, the size of the largest polypeptide expressed from a deletion construct, which was reactive with HMAF and/or the 3H5 mAb correlated well with its predicted value. The 3H5 mAb reacted with all the E polypeptides with Nterminal deletions, except with the one lacking aa 255 to 422 (Fig. 2B, lane 6) . These data indicated that the anti- Reactivitics of E polypeptides with the polyclonal HMAF and DEN-2 type-specific 3H5 mAb. E. cog N5151 cells were transformed by the expression plasmids and tested for the synthesis of E polypeptides as described for pKC 30 and its derivatives (Rosenberg et al., 1983) . Briefly, the bacterial cells were grown at 32°C to A65 o = 1.0 in LB medium containing 100/~g Ap/ml, and then the temperature was shifted to 42°C by adding equal volume of LB medium prewarmed to 65 °C. The culture was incubated at 42 °C for an additional 60 rain. The cells were harvested by centrifugation and washed once with TES buffe) (10 mM Tris.HCl, pH 7.5/! mM EDTA/0.1 M NaCI). Total bacterial pellets resuspended in 300 gl of Laemmli (1970) sample buffer were heated to 100°C for 5 rain and were analysed by 0.1% SDS/10• PAGE (Laemmli, 1970) . Proteins were then transferred to nitrocellulose (Towbin and Gordon, 1984) in a buffer containing 25raM Tris'HCI/150mM glycine, pH 8.3/20% methanol/l% SDS at 200 mA for 4 h. Blots were blocked for 30 rain in 22~ nonfat dry milk/0.9% NaCI/10 mM Tris.HCI, pH 7.5. After washing with 50 mM Tris.HCI, pH 7.5/200 mM NaC! (TBS), the blots were then incubated sequentially with 1:500 dilution of HMAF (panel A), or 1:500 dilution of 3H5 mAb (panel B), goat anti-mouse IgG conjugated with alkaline phosphatase (Hyclone, Logan, UT), and a mixture of 5bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. Blots were washed 3 times between incubations with TBS for 10 min each. When color had developed sufficiently, the reaction was stopped by adding stop buffer (20 mM Tris. HCI/5 mM EDTA). Panels A and B, lanes 1-6: bacterial extracts containing E polypeptides expressed from pTTI0, pTT15, p'l"r20, pTT25, pTr30 and pTT35, respectively. The E coding sequences contained in these plasmids are described under Fig. 1 and Table I . In lane C, the cell lysate from C6/36 (mosquito) cells infected with DEN-2 was loaded as a positive control. The preparation and characterization of 3H5 mAb and the polyclonal HMAF antibodies against DEN-2 viral antigens have previously been reported (Brandt et al., 1967; Henchal et al., 1982) .

genic determinant of the protein recognized by the 3H5 mAb is located within a region of 166 aa (between aa 255 and 422) (Fig. 3 ).

In order to map the region recognized by the 3H5 mAb more precisely, we developed a strategy to construct a series of targeted deletions within this region of 166 aa using PCR (Saiki et al., 1988) . It is a generally accepted notion that most, if not all, of the antigenic determinants reactive with antibodies are exposed to the surface of the antigen molecule (Todd et al., 1982; Sutcliffe et al., 1983; Benjamin et al., 1984; Berzofsky, 1985) , and hence are hydrophilic. Based on this notion, algorithms for hydrophilicity (Hopp 

and Woods, 1981; Kyte and Doolittle, 1982; Parker et al., 1986) have been used to predict antigenicity. The hydropathy plot ofthe region of 166 aa containing the 3H5 mAbbinding site is shown in Fig. 3B . The predicted surface residues containing stretches of hydrophilic aa are indicated with negative values (Kyte and Doolittle, 1982) . The hydropathy profile predicts three major hydrophilic regions (ten or more aa in length) at aa 285-298, aa 358-374 and aa 386-397, and three minor regions (five or more aa in length) at aa 325-333, aa 341-347 and aa 404-410. A set of oligos, 1-6, were synthesized corresponding to the regions shown in Fig. 3A , and used for PCR. The amplification reactions were carried out using the oligo 1, and any (Kyte and Doolittle, 1982) of the region shown in A. The numbered horizontal b;~'s representing the PCR primers (1-6) correspond to the hydrophobic aa residues flanking the various hydrophilic (surface) regions, and their sequences are underlined in panel A. The filled-in hydrophilic region represents the region required for binding by 3H5 mAb, which was identified by fine mapping (see Fig. 4 ).

kDa 84- 4 . lmmunoreactivity of truncated E proteins. Total lysates from E. coli transformed by the various expression plasmids were electrophoresed in 0.1% SDS/10% PAGE system (Laemmli, 1970) and transferred to nitrocellulose paper. The filter was either reacted with the polyclonal HMAF (Fig. 4A) , or with the 3H5 mAb (Fig. 4B) one of the oligos 2-6 as primers, and the 506-bp region between the two BamHl sites as the template. When the primer pairs of I and 2, 1 and 3, 1 and 4, 1 and 5, and 1 and 6 were used in PCR, DNA fragments of sizes 148 bp, 245 bp, 388 bp, 439 bp and 500 bp, respectively, were generated. Each amplified DNA fragment was ligated to the blunt-ended BamHl sites of the expression plasmid pTT15 (see Table I ). The plasmid clones with the correct orientations were selected by restriction enzyme digestions, and the accuracy of PCR-amplifications was verified by DNA sequence analysis. The plasmids selected for expression of E polypeptides with their aa coordinates are shown in Table  II , and the E. coli N5151 strain transformed by these plasraids was analyzed. The lysates oftransformants obtained from deletion constructs were tested by Western blot for the reactivities of the truncated E polypeptides with polyclonal HMAF (Fig. 4A) , as well as the 3H5 mAb (Fig. 4B ). The sizes of the expressed polypeptides agreed well with the predicted values. The plasmid containing E-coding sequence between Tth 11 ll-EcoRl (pTTI5) expressed a polypeptide of about 48 kDa in size ( Fig. 4A and 4B, lane 6) . The plasmid pTT35 with the deletion of coding sequence between the two BamHl sites expressed a polypeptide of about 36 kDa ( Fig. 2A, lane 6) . All the other clones obtained from inframe insertion of PCR-amplified DNA fragments at the BamHl site of the plasmid pTT35 expressed polypeptides in the size range of 36-48 kDa (Fig. 4A , lanes 1-6; see also Table II ). When these E polypeptides were tested for their reactivities with the 3H5 mAb, only the 47-and 48-kDa polypeptides were immunoreactive (see Fig. 4B , lanes 4 and 5, and Table II) . The E polypeptide having a deletion between aa 385-423 did not react with the 3H5 (Fig. 4B,  lane 3) , but the one having the deletion between aa 401-423 did react (Fig. 4B, lane 4) . Therefore, the 3H5 binding site is located between aa 385-401. In Fig. 4B , lane 5, the E polypeptide expressed from the plasmid pTT35.6, which was the equivalent of pTT15 in lane 6, was loaded. This polypeptide also reacted with the 3H5 mAb as expected. These results suggested that the hydrophilic region containing residues QLKLNWFKKGSS (aa 386-397) between the two PCR primers No. 4 and No. 5 (see Fig. 3B , and lane 4 in Fig. 4B ), were required for the immunoreactivity with the 3H5 mAb. " The sequences of PCR primers No. 1-6 art~ given in Fig. 3A (underlined). The BamHl fragment (nt 1697-2203) was used as template (100 ng) in a reaction mixture (100 ld) that contained dNTP (125/aM), one/AM each of the two primers, and 4 units of Taq polymerase. PCR was carried out for 25 cycles.

b Sizes of the amplified DNA from each of the PCR between the primer pair 1:2, 1:3, 1:4, 1:5 and 1:6, respectively. They were ligated to the expression plasmid, pTT15 (Table l) , which was previously digested with BamHl and blunt-ended by treatment with Pollk enzyme. Screening of the transformants for the proper orientation of the insert was carried out by restriction analysis, and the PCR-amplified regions (shown as thick lines in column 5) in each of the clones were verified by nt sequence analysis. 

It has been reported that many of the mAbs to 17D strain of YF were able to distinguish between the wt and the vaccine strains of the virus by neutralization and/or HI assays. There were also differences between individual 17D vaccine strains (Buckley and Gould, 1985; Schlesinger et al., 1983) . However, the 3H5 mAb reacted with several DEN-2 strains from different geographical areas, indicating that this antibody is not DEN-2 strain-specific . It is interesting to note that the aa sequences among the three DEN-2 strains: NGS-C (Irie et al., 1989) , Jamaica strain (Deubel et al., 1986) , and PR-159/S1 (Hahn et al., 1988) in the region required for the 3H5-binding are identical except for the substitution of the D residue in PR-159/SI for N (aa 390) in the other two strains. It was found that the 3H5 was just as effective in neutralizing either of the two DEN-2 strains, NGS-C, and PR-159/SI (R. Putnak, unpublished results), suggesting that N/D substitution in PR-159/S1 did not contribute to ~ny significant change in the immunoreactivity of the 3H5 mAbo However, earlier studies have shown that the 3H5 was not effective in neutralizing DEN-1 (Henchal et al., 1985) . When the sequence of the 3H5-binding region of DEN-2 was compared with the corresponding regions of DEN-1 (Mason et al., 1987b) , DEN-3 (Osatomi and Sumiyoshi, 1990) , and DEN-4 (Zhao et al., 1986) , the variations in the sequence are the greatest in DEN-4 and DEN-3 with 4 aa changes, whereas in DEN-I, there are only two changes (see Table III ). Since the DEN-2 E sequence corresponding to this 3H5-binding site differs from that of the DEN-1 E sequence only in the N-terminal half of the region, this portion of the sequence may be critical for the 3H5 recognition. Roehrig et al. (1990) reported that the peptide from aa 388-400 neither bound to the 3H5, nor elicited neutralizing antibody in mice. It should be noted that the peptide 388 differs from the 3H5 peptide studied here in lacking two N-terminal aa, but having three addi-tional C-terminal residues. It is possible that the peptide 388 may assume a different conformation from that of the 3H5 peptide (aa 386-397) (see also section e). Another important difference is that Roehrig et al. (1990) used mice for immunization with the synthetic peptide, whereas rabbits were used in this study.

Structural analysis of the E protein of WN (Novak and Wengier, 1987) , and TBE (Heinz et al., 1983a,b; Mandl et al., 1989) revealed three distinct antigenic domains, A, B and C. In TBE, it has been shown that the domain A essentially contains flavivirus-cross reactive epitopes, as well as some subtype-specific epitopes, which are also present in domain C. Epitopes of domain B, on the other hand, are specific for the tick-borne complex of flaviviruses (Heinz, 1986) , which were lost upon reduction of disulfide bonds, suggesting that the three-dimensional structure of the E protein is important for the integrity of these determinants. In order to map the antigenic determinant(s) involved in neutralization, Mandl et al. (1989) isolated and sequenced the regions encoding E and M proteins of the escape variants of TBE virus, which were selected in the presence of neutralizing mAbs. Two of the variants resulted from single aa substitutions in the B domain of the E protein (Ser3Sg-.Arg and Tyr3S4-,His). Interestingly, these neutralization escape mutations map within, or in the vicinity of the regior, identified by us as the neutralizing 3H5 mAbbinding site.

The domain B of DEN-2 E protein (aa 301-395) contains or, e disulfide bridge formed between aa 302 and aa 333. This region has been shown to fold independent of other disulfide bridges in the protein ofTBE (Winkler et al., 1o87) . The domain B is also resistant to acid pH, guanidine-HCl, and SDS treatment. However, it is destroyed by reduction and carboxymethylation. The antige- (Deubel et al., 1986) , and DEN-2 (NGS-C) (Irie et al., 1989) . The synthetic peptide, aa 388-400 was used by Roehrig et al. (1990) (see section c). The other sequences are derived from: DEN-2 (PR159-SI; Hahn et al., 1988) ; DEN-I (Mason et al., 1987b) ; DEN-4 (Zhao et al., 1986) ; DEN-3 (Osatomi and Sumiyoshi, 1990) . The dashes denote identical aa in that position. nicity of the domain B could be restored by the removal of the reducing agent (Heinz, 1986) , suggesting that this domain has the potential to renature very rapidly. This property of the domain B is consistent with our observation that the 3H5 mAb reacted with the E polypeptides subsequent to boiling in the presence of 2-mercaptoethanol. However, the reactivity of the 3H5 mAb reduced upon carboxymethylation (data not shown). This result suggests that the conformation ofthe B domain may play a role in presenting this linear sequence to the surface of the protein molecule for efficient binding by the 3H5 mAb.

Since the 3H5 is a DEN-2 type-specific anti-E mAb having a high neutralizing titer (Henchal et al., 1985) , we were interested to examine whether the linear sequence of the E protein recognized by the 3H5 mAb can elicit neutralizing immune response. We synthesized a peptide of 13 aa residues (including the N-terminal Cys added for coupling to KLH) corresponding to the 3HS-binding site, and used it to immunize rabbits. The reactivity of the rabbit antisera raised against the peptide was monitored by . Synthetic peptide was purified by HPLC. The coupling reagent MBS (Pierce Chemical Co., Rockford, IL) was used to attach the peptide, via the N-terminal Cys residue, to the KLH (Sigma Chemical Co., St. Louis, MO) as described (Liu et ai., 1979) . Antiserum to synthetic peptide-KLH conjugate was obtained from 10-week-old New Zealand white male rabbit. The animal was first inoculated intramuscularly with peptide-KLH conjugate (200/~g) emulsified in Freund's complete adjuvanl fDifco Laboratories, Detroit, MI). On day 14, 100/~g peptide-KLH conjugate in 0.5 ml PBS (10 mM Na.phosphatc, pH 7.2/150 mM NaCI), mixeci ~i~!, 0.5 ml of Freund incomplete adjuvant was injected subcutaneously at multiple sites in the back and was subsequently boosted four times at ,, e, cl.ly intervals with the same amount used in the second inoculation, The rabbit was bled from the ear vein 10 days after last injection. Reactivity of antisera against the synthetic peptide was measured by a modification of the ELISA (Engvall, 1980) . Microtiter plates (Coming, NY) were coated with 50 ~l/well of peptide (20 pg/ml) in 50 mM carbonate-bicarbonate buffer (pH 9.6) overnight at 37°C. The antigen-coated wells were then washed 3 times with 0.85% NaCI/0.05% Tween 20. After washing, the plates were blocked with 5% BSA in PBSfrween 20 (100/li/well for 1 h at 37°C. Plates were then incubated sequentially with 50 pl/weU of diluted antiserum in PBS/Tween 20/1% BSA, goat anti-rabbit Ab conjugated to horseradish peroxidase (Hyclone, Logan, UT), and the substrate orthophenylenediamine (Sigma Chemical Co., St. Louis, MO). Plates were washed between incubations with 0.05% Tween 20/0.85% NaCI. The final enzyme reaction was stopped with 2N H2SO 4, and the color change was measured at 492 nm on a Titertek Multiskan plate reader (Flow Laboratories, McLean, VA). In ELISA, A at 492 nm is plotted against the log serum dilution.

ELISA. The anti-3H5 peptide antibody reacted specifically with the 3H5 peptide at the dilution of 1:10000, and no reactivity was seen with preimmune sera (Fig. 5A, profiles  1 and 4) . As a control, we tested the reactivities of two other peptides from the glycoprotein sequences of HHV-6 and HSV-2gG with the anti-3H5 peptide antibody (Fig. 5A , profiles 2 and 3, respectively). The reactivities of these three peptides with the preimmune sera are shown by profiles 4-6 in Fig. 5A . The reactivities of the anti-HSV-2gG peptide antiserum, as well as the anti-HHV-6 peptide antiserum were lower, compared to that of the anti-3H5 peptide serum tested with the respective homologous peptides (Fig. 5B , profiles 1 and 2 vs. profile 1 in Fig. 5A ). The reactivities of the heterologous peptides with the peptide antisera (e.g., the anti-3H5 peptide antibody with the HHV-6 peptide, and the anti-HHV-6 antibody with the 3H5 peptide etc.), were all much lower and grouped together (see 1:1000 dilution, profiles 2 and 3 in Fig. 5A and profiles 3 to 6 in Fig. 5B ). Next, the reactivities of the 3H5 peptide with the various antibodies were compared. As shown in Fig. 5A , the reactivity between the 3H5 peptide and the anti-3H5 peptide serum is the greatest, whereas its reactivity with the HMAF and the 3H5 antibody were threefold and fivefold less, respectively (Fig. 5C , profiles 1 and 2). However, when the ELISA values at 1:330 were compared for the reactivities of (i) the 3H5 peptide vs. the 3H5 mAb (profile 2), (ii) the 3H5 peptide vs. another DEN-2 E-specific 6B6 mAb (profile 3), or (iii)the 3H5 peptide vs. the nonspecific mAb (13~C5) raised against the HSV-2gG protein (profile 4), the reactivity of (i) was about fivefold higher than Nos. 2 and 3 (see Fig. 5C ). The reactivities of an unrelated HHV-6 peptide with the HMAF, the 3H5 mAb, the DEN-2 specific 6B6 mAb or the HSV-2gGspecific 130~C5 mAb, were all very low and similar to those of the 3H5 peptide vs. the DEN-2-6B6 mAb and the 130cC5 mAb (Fig. 5D , profiles 1-4 vs. Fig. 5C , profiles 3 and 4).

In addition, the specificity of this immunoreactivity of the 3HS-peptide was also tested by using other unrelated mAbs as controls: a mAb against DEN-2 NS-1 protein (3E9) and the AI6 mAb against HSV-2 glycoprotein D (Balachandran and Hurt-Fletcher, 1985) and the data indicated that the reactivity between the 3H5 peptide and the 3H5 mAb is very specific (data not shown). These data clearly supported that the 12-aa region (aa 386-397) of the DEN-2 E protein constituted at least a portion of the antigenic determinant recognized by the 3H5 mAb.

(f) Anti-3H5 peptide antibody reacts with the authentic DEN-2 E protein Next, we examined whether the rabbit anti-3H5 peptide antiserum could recognize the authentic E glycoprotein from the DEN-2-infected cells. Monkey kidney (CV-1) cells were infected with DEN-2, and the cells were metabolically methionine (10pCi/ml) was added in methionine-deficient medium for 3 h. The cells were collected by scraping and low speed cantrifugation. Cells were solubilized in RIPA buffer (50 mM Tris.HC! pH 7.5/150 mM NaCl/1% DOC/0.5% NP40/ 0.1 mM PMSF), and sonicated for I min. The lysates were centrifuged at 100000 x g for I h. Supernatants were mixed with appropriate antibodies and protein A-agarose beads. The precipitates were washed, dissociated by boiling in sample buffer, and analysed by 0.1 ~ SDS/10% PAGE (Laemmli, 1970) . Gels were fixed, stained and processed for fluorography. (Panel A) Soluble cell lysates from uninfected CV-I cells (lanes I, 3 and 5), and DEN-2-infected cells (lanes 2, 4, 6 and 7) were reacted with the following antibodies: HMAF (lanes 1 and 2); 3H5 mAb (lanes 3 and 4); rabbit antiserum to the synthetic peptides (lanes 5 and 6); rabbit preimmune serum (lane 7). The mobility of molecular weight markers is indicated. (Panel B) Labeled cell lysates as in panel A, but using additional E-specific mAbs were analyzed by immunoprecipitation. C and V denote the control uninfected and DEN-2-infected CV-I cell lysates, respectively. Lanes: I and 2, mouse preimmune serum; 3 and 4, HMAF; 5 and 6, 3H5 mAb; 7 and 8, E-specific mAb, DEN-2-6B6; 9 and 10, E-specific mAb DEN-I-13B7; 11 and 12, E-specific mAb, DEN-2-2H3; 13 and 14, Especific mAb DEN-2-4ES; 15 and 16, E-specific mAb DEN-I-gDI2 Henchal et ai., 1982) . The arrowheads in panels A and B denote the E protein (gp60).

labeled with [3SS]methionine. Infected and uninfected cell lysates were used for immunoprecipitation using the polyclonal HMAF, 3H5 mAb, the rabbit anti-3H5 peptide antiserum, and other E-specific mAbs. As shown in Fig. 6A and B, the HMAF reacted with a number of proteins specific for DEN-2 infected cell proteins, and the E glycoprotein gp60 was very prominent among them (Fig. 6A, lane  2, and 6B, lane 4) . The rabbit anti-3H5 peptide antibody also immunoprecipitated the prominent gp60 (Fig. 6A , lm~e 6). The gp60 band immunoprecipitated by the 3H5 mAb (IgG! subclass) was much weaker (Fig. 6A , lane 4) than that by HMAF (Fig. 6A, lane 2) , or by the anti-3H5 peptide antiserum (Fig. 6A, lane 6) . In order to show that the putative gp60 band was the authentic E protein, the [3SS]methionine-labeled DEN-2-infected cell lysates were immunoprecipitated by a number of other anti-E mAbs including 3H5 (Fig. 6B) . "['he gp60 band immunoprecipirated by some of the other E-specific mAbs (DEN-2-6B6, DEN-2-2H3 and DEN-2-4E5 in Fig. 6B , lanes 7 and 8, 11 and 12, and 13 and 14, respectively) was much stronger than that obtained with the 3H5 mAb, the DEN-I-13B7 mAb and the DEN-I-9DI2 (Fig. 6B , lanes 5 and 6, 9 and 10, 15 and 16, respectively). These results indicated that the gp60 band was the authentic E protein. The weaker gp60 band seen with the 3H5 and other E-specific mAbs might be due to the weak reactivity of the protein A-agarose beads to a different subclass of IgG. These data clearly demonstrated the specificity of the anti-3H5 peptide antibody in binding to the DEN-2 E protein.

Rabbit anti-serum against the 3H5 peptide was next tested for its ability to neutralize DEN.2 in a plaque reduction neutralization test (PRNT) in a complementindependent reaction as described by Russell and Nisaluk (1967) . The anti-peptide antibodies showed neutralization of the infectivity of DEN-2 virus (Table IV) with the 70% neutralizing antibody titer (PRNTTo) of 1:80 reproducibly. With the sera from a different rabbit, a PRNT7o neutralization titer of only 1:23 was obtained in duplicate experiments (data not shown). These results strongly supported that the 3H5-binding site on the E protein represents a portion of the antigenic determinant of the E protein involved in neutralization.

Previous studies using synthetic peptides to elicit antisera reactive with the hepatitis B surface antigen have shown that the determinant carried within a short peptide is sufficient to elicit reactivity against a much larger protein molecule with a complex tertiary and quaternary structure (Sutcliffe et al., 1983) . Mason et al. (1987a; expressed the eDNA encoding E protein of JE in E. coli using ggtl 1 system to localize the antigenic determinants recognized by mAbs. Using BAL31 for deletion mutagenesis from the 5' and 3' ends of the cDNA encoding the E protein, a 95-aa fragment between Met3°3-Lys 39a was found to be the minimal structure capable of binding to any of ten mAbs, and the immunoreactivity of this fragment was dependent on the formation of disulfide bonds. However, a recombinant fusion protein containing this 95-aa sequence reacted with the neutralizing and protective mAb in direct binding assays, but it failed to induce a neutralizing immune response when injected into mice (Mason et al., 1989) . Interestingly, this antigenic domain mapped in JE-E protein falls within the corresponding domain of DEN-2 E reactive with the 3H5 mAb. However, the ability of our synthetic peptide to induce neutralizing antibody in rabbits might be due to conformational differences assumed by the short synthetic peptide, compared to the antigenic domain of 95-aa expressed in E. coli in the form of a fusion protein (Mason et al., 1989) . Similar differences have been observed by others. For example, Eisenberg et al. (1985) identified an antigenic site in the aa 268-287 sequence of glycoprotein Rabbit anti-peptide serum obtained from rabbit immunized with 3H5-reactive peptide. b Serum dilution made in Hanks" balanced salt solution containing 2% fetal bovine serum.

[ _ plaques with immune serum -] Mean percent plaque reduction calculated from the average of duplicate titration as ,_ 1 pl~it~n~m]

x 100 (i.e., expressed as percent). D of HSVol, by showing reactivity of this peptide with the mAb against glycoprotein D. This peptide was conjugated to KLH via C-terminal Cys, but failed to elicit antibody in rabbits that could recognize either the glycoprotein D or the intact virions. In contrast, Strynadka et al. (1988) found that antibodies to the KLH, conjugated via the N-terminus of the peptide encoding aa 267-276, recognized both glycoprotein D and HSV-I virions. They also neutralized the infectivity of H SV-1. To explain the differences between these results obtained with two closely related peptides from the same region, Strynadka et al. (1988) speculated that the longer peptide (267-287 aa) might assume a conformation distinct from that of the native epitope.

The method described in this report is generally applicable to define precisely any short linear sequence of a protein molecule, which is required for the presentation of a larger antigenic determinant recognized by a mAb. The notable unique feature of this method is that the truncated polypeptides ate not fused to the E. coli//-galactosidase, and therefore the antigenic determinants of interest are not subjected to any major conformational changes during folding of the proteins. An alternative method for mapping the neutralizing determinants involves the isolation of a large number of neutralization escape variants, their propagation, cDNA cloning and sequence analysis of these variants in order to be able to define the antibody binding site. In some cases, selective synthetic peptides based on such data have failed to elicit a neutralizing antibody response in an appropriate host due to the possibility that in the immune-selected variant, a conformational change might alter a neutralization epitope at a distant site (Wilson et al., 1990) .

Synthesis and processing of glycoprotein gG of herpes simplex virus type 2

The antigenic structure of proteins: a reappraisal

Intrinsic and extrinsic factors in protein antigenic structure

Production and characterization of arbovirus antibody in mouse ascitic fluid

Neutralization of yellow fever virus studied using monoclonal and polyclonal antibodies

Flavivirus genome organization expression, and replication

Immune response in rabbits to virion and non-virion antigens of the flavivirus Kunjin

Nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue type 2 virus, Jamaica genotype

Localization of epitopes of herpes simple~, virus type ! glycoprotein D

Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies

Enzyme immunoassay ELISA and EMIT

Identification of distinct antigenic determinants on dengue-2 virus using monoclonal antibodies

Nucleotide sequence of dengue 2 RNA and comparison of the encoded proteins with those of other flaviviruses

Epitope mapping of flavivirus glycoproteins

Antigenic and immunological properties of defined physical forms of tick-borne encephalitis virus structural proteins

A topological and functional model of epitopes on the structural glycoprotein of tickborne encephalitis virus defined by monoclonal antibodies

Location of immunodominant antigenic determinants on fragments of the tick-borne encephalitis virus glycoprotein: evidence for two different mechanisms by which antibodies mediate neutralization and haemagglutination inhibition

Dengue virus specific and flavivirus group determinants identified with monoclonal antibodies by indirect immunofluorescence

Epitopic analysis of antigenic determinants on the surface of dengue-2 virions using monoclonal antibodies

Prediction of protein antigenic determinants from amino acid sequences

Sequence analysis of cloned dengue virus type 2 genome

Monoclonal antibodies against dengue 2 virus E-glycoprotein protect mice against lethal dengue infection

Morphological, chemical, and biological characterization of Japanese encephalitis virus virion and its hemagglutinin

A simple method for displaying the hydropathic character of a protein. 3

Cleavage of structural proteins during the assembly of the head of bacteriophage "I"4

New procedures for preparation and isolation of conjugates of proteins and synthetic co-polymer of D-amino acids and immunochemical characterization of such conjugates

Location of a neutralization determinant in the E protein of yellow fever virus (17D vaccine strain)

Conformational changes at topologically distinct antigenic sites on the influenza A/PR/8/34 virus HA molecule are induced by the binding of monoclonal antibodies

RNA recombination of coronaviruses: localization of neutralizing epitopes and neuropathogenic determinants on the carboxyl terminus of peplomers

Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model

Expression of Japanese encephalitis virus antigens in Escherichia coli

Sequence of the dengue virus genome in the region encoding the three structural proteins and the major nonstructural protein NSI

Molecular characterization of a neutralizing domain of the Japanese encephalitis virus structural glycoprotein

Topographical analysis of viral epitopes using monoclonal antibodies: mechanisms of virus neutralization

Efficient mapping of protein antigenic determinants

Yellow fever monoclonal antibodies: type-specific and cross-reactive determinants identified by immunofluorescence

Analysis of disulfides present in the membrane proteins of the West Nile flavivirus

Complete nucleotide sequence of dengue virus type 3 virus genome RNA

A new hydrophilicity scale derived from HPLC peptide retention data: correlation of predicted surface residues with antigenicity and X-ray derived accessible sites

Antigenic analysis of the surface glycoproteins of a Venezuelan equine encephalomyelitis virus (TC-83) using monoclonal antibodies

Antibodies to dengue 2 virus E-glycoprotein synthetic peptides identify antigenic conformation

The use of pKC30 and its derivatives for controlled expression of genes

Dengue virus identification by the plaque reduction neutralization test

Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase

Monoclonal antibodies distinguish between wild and vaccine strains of yellow fever virus by neutralization, hemagglutination inhibition, and immune precipitation of the virus envelope protein

Studies on the nature of dengue viruses. IV. The structural proteins of type 2 dengue virus

Use of synthetic peptides to map the antigenic determinants of glycoprotein D of herpes simplex virus

Antibodies that react with predetermined sites on proteins

Properties and antigenic relationships of hemagglutinins associated with dengue viruses

The immunogenicity and antigenicity of proteins

Immunoblotting and dot immunobindingcurrent status and outlook

Monoclonal antibodies to the glycoprotein of vesicular stomatitis virus: comparative neutralizing activity

The site of an immune-selected point mutation in the transmembrane protein of human immunodeficiency virus type I does not constitute the neutralization epitope

Characterization of a disulfide bridge-stabilized antigenic domain of tick-borne encephalitis virus structural glycoprotein

Cloning full-length dengue type 4 viral DNA sequences: analysis of genes coding for structural proteins

We would like to thank Kevin Graham for expert technical assistance, Jon Hillyer for computer analysis and Rich Grabbe for peptide synthesis. We thank Dr. Mary K. Gentry for the generous gift of monoclonal antibody 3H5 used in this study. This work was supported by a research contract from the U.S. Army Medical Research and Development Command (DAMD 17-85-C-5273)to R.P., by a grant from the Department of Defense (DAMD17-86-G-6038) to Dr. Fred Samson, and by a Biomedical Research Support Grant/Shared Instrumentation Facility. The views, opinions, and/or findings in this report are those of the authors only and should not be construed as an official Department of the Army position, or decision unless so designated by other documentation.