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The Journal of Immunology, 2006, 176: 7471-7481.
Copyright © 2006 by The American Association of Immunologists

The HIV-Neutralizing Monoclonal Antibody 4E10 Recognizes N-Terminal Sequences on the Native Antigen1

Christine Hager-Braun2,*, Hermann Katinger{dagger} and Kenneth B. Tomer*

* National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709; and {dagger} University for Natural Resources and Applied Life Sciences, Institute of Applied Microbiology, Vienna, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Characterization of the epitope recognized by the broadly neutralizing anti-HIV Ab 4E10 has, heretofore, focused on a linear sequence from the gp41 pretransmembrane region (PTMR). Attempts to generate neutralizing Abs based on this linear epitope sequence have been unsuccessful. We have characterized the antigenic determinants on recombinant glycosylated full-length Ags, and nonglycosylated and truncated Ags recognized by 4E10 using epitope extraction and excision assays in conjunction with MALDI mass spectrometry. The mAb recognized the peptides 34LWVTVYYGVPVWK46 and 512AVGIGAVFLGFLGAAGSTMGAASMTLTVQAR542 located at the N-terminal region of gp120 and gp41, respectively. Immunoassays verified AV(L/M)FLGFLGAA as the gp41 epitope core. Recognition of the peptide from the gp41 PTMR was detected only in constructs in which the N termini of the mature envelope proteins were missing. In this region, the epitope core is located in the sequence 672WFDITNWLWY681. We hypothesize that the hydrophobic surface of the paratope functions as a "trap" for the viral sequences, which are responsible for insertion into the host cell membrane. As the N-terminal region of gp120, the fusogenic peptide of gp41, and the PTMR of gp41 show high sequence homology among various HIV strains, this model is consistent with the broadly neutralizing capabilities of 4E10.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Infection of host cells with HIV occurs through attachment of the virus, a sequence of conformational changes of the proteins involved in virus cell interaction, and, finally, merger of the viral membrane with the host cell membrane. The major HIV proteins involved in viral fusion are the envelope proteins (Env), which are expressed as the precursor protein gp160 and subsequently mature through cleavage into the envelope protein gp120 and the transmembrane protein gp41 (see Fig. 1A). The majority of identified anti-HIV envelope human Abs recognize highly immunogenic epitopes, but have only strain-specific neutralizing capabilities. However, a few Abs have been isolated that are protective against HIV strains from various clades (reviewed in Refs. 1 and 2). The Ab 4E10 is highly protective against primary isolates as well as laboratory-adapted HIV strains (3, 4, 5, 6). The epitope for 4E10 determined by ELISA-based peptide screening and phage display has been identified as the hexamer NWFN/DIT (7, 8). Cocrystallization of 4E10 with the synthetic peptide KG671WNWFDITNW679GK revealed a helical conformation of the peptide with the hydrophobic residues located on one side of the helix. This allows binding to the largely hydrophobic paratope of 4E10 (9). The Ag interacts only with the base and central residues of the CDR H3 loop, whereas a large surface of CDR H3 is not involved in Ag binding. The tip of CDR H3 contains several nonpolar residues and bends away from the epitope binding site, suggesting interaction with the membrane of the virus or other residues from gp41 or even gp120 in the native protein Ag (9).


Figure 1
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  		FIGURE 1. Schematic representation of the envelope proteins (Env), which mature into gp120 and gp41 A, Sequence alignment revealed that gp120 consists of the conserved regions C1-C5 and the variable regions V1–V5. V1–V4 form loops through disulfide bridges between cysteine residues at their stems. Analyses of gp41 resulted in the identification of the fusogenic peptide (FP), the N-terminal heptad repeat (NHR), a helix-turning loop, the C-terminal heptad repeat (CHR), the pre-TM, the transmembrane domain (TMD) and the endodomain. The numbers reflect the start and end of each domain in the aa sequence of the reference strain HIVHXB2 (37 48 ). B, Alignment of the amino acid sequences of the proteins HIVMN gp160, HIVJR-FL SOSgp140, HIVSF2 gp120, HIVSF162 gp120, HIVMN gp4126–167 + 194–258, HIVHXBc2 gp41546–682, and HIVHXBc2 gp41541–682+His-tag, and of the synthetic peptides N15, N31, and C22 used in this study in comparison with the Env sequence of the reference HIV-strain HXBc2. Italicized letters correspond to amino acids that are part of the expression system and not part of the Env sequence. Amino acids in bold show sequences identified as antigenic determinants (see text for details). For sequence alignment the software MultAlin (<http://prodes.toulouse.inra.fr/multalin/multalin.html>) was used (49 ).

 
The linear epitope recognized by 4E10 would be a good candidate to serve as mimotope for active immunization but a trimeric construct of the transmembrane region and the extracellular C-terminal region of gp41 embedded in liposomes, though recognized by 4E10, did not induce a neutralizing immune response (10). Because some HIV strains cannot be neutralized by 4E10 despite the presence of NWFN/DIT and in other strains sequence variability in positions one, four, and six of the hexamer epitope does not interfere with neutralization (6, 8). Also, because the synthetic epitope-containing peptide left a significant portion of the binding site on 4E10 vacant in the crystal structure (9), the epitope might be more complex than previously considered. We have previously applied a mass spectrometry-based epitope extraction and excision approach to characterize the epitope recognized by the Ab 2F5 (11) and here we apply the same approach to the determination of the epitope recognized by 4E10. We show that 4E10 recognizes the N-terminal sequences of gp120 and gp41 on the native protein. In constructs missing these sequences, binding to 4E10 occurs through the gp41 pretransmembrane (pre-TM)3 region (PTMR). We conclude that the neutralizing Ab 4E10 is polyspecific and interacts with a discontinuous/conformational epitope. Although providing an alternative explanation, our findings are consistent with previously reported observations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Preparation of affinity microcolumns for indirect immunoadsorption

The affinity columns were prepared as previously reported (11, 12). Briefly, the Fc-specific anti-human IgG (Sigma-Aldrich) was bound to cyanogen-activated Sepharose beads (Amersham Biosciences) at a concentration of 5–9 µg of Ab/1-µl initial volume of bead slurry. The mAb 4E10 was affinity-bound overnight (ON) at a concentration of 1.0–2.7 µg/µl and a total amount of 2–5 µg of 4E10/1-µl initial volume of bead slurry. The Abs were cross-linked with a 10 mM solution of bis(sulfosuccinimidyl)suberate (Pierce). Finally, the beads were split into aliquots of 8- to 10-µl initial volume of bead slurry for subsequent experiments.

Epitope mapping by epitope extraction (13)

For epitope extraction, 10 µg of HIVJR-FL SOSgp140 (0.9 µg/µl in 0.1 M NaP/0.15 M NaCl (pH 7.2); expressed in CHO cells (14)) was mixed with 0.7 µl of acetonitrile, and 2 µl of trypsin solution (0.1 µg/µl, enzyme:substrate 1:50; Roche Molecular Biologicals) and incubated ON at 35°C. To determine trypsin autodigest products, a sample was prepared similar to the SOSgp140 sample but lacking the Ag. The digested samples were loaded on the affinity columns containing beads corresponding to a 10-µl aliquot of the initial beads slurry. The microcolumns with the SOSgp140JR-FL digest and the trypsin autodigest, respectively, were incubated for 19 h at 25°C slowly rotating. After the incubation, the beads were drained, washed with 0.4 ml of 20 mM NaP (pH 7.2), and resuspended in 25 µl of 20 mM NaP (pH 7.2).

Epitope mapping by epitope excision (12)

In independent experiments, various proteins were tested for binding to 4E10. The proteins included HIVMN gp160, expressed in baculovirus (Protein Sciences); HIVJR-FL SOSgp140, expressed in CHO cells (14); HIVSF2 gp120, expressed in CHO cells (Austral Biologicals); HIVMN gp4126–167 + 194–258, expressed in Escherichia coli (ImmunoDiagnostics, obtained through National Institute for Biological Standards and Control (NIBSC)); HIVHXBc2 gp41546–682, expressed in Pichia pastoris, (U.K. Medical Research Council, obtained through NIBSC); and HIVHXBc2 gp41541–682+His-tag, expressed in P. pastoris (Viral Therapeutics). A sequence alignment of the proteins is shown in Fig. 1B. Some proteins required purification before their use in the epitope-mapping studies. For removal of urea and NaCl, 25 µl of HIVHXBc2 gp41541–682+His-tag was dialyzed against excess 20 mM NaP (pH 7.2) at 4°C ON. For the purification of proteins from buffer components which were incompatible with mass spectrometry (MS) such as imidazole, Triton X-100, or SDS, the samples were loaded on an HPLC reverse-phase C4 column (4.6 x 250 mm; Vydac), washed with 5% acetonitrile/0.1% trifluoroacetic acid for 5 min and eluted with a linear gradient from 5% acetonitrile/0.1% trifluoroacetic acid to 95% acetonitrile/0.1% trifluoroacetic acid within 50 min. The elution of protein was monitored based on the absorption at 214 and 260 nm. Individual fractions (1 ml each) were lyophilized, resuspended in 5–20 µl of 50% acetonitrile, and a 0.3 µl-aliquot was analyzed by MALDI/MS. Fractions containing the protein of interest were combined. For Ab binding studies, the sample was typically diluted 1/10 with 20 mM NaP (pH 7.2), 0.1% n-dodecyl beta-D-maltoside (DDM; Sigma-Aldrich) to reduce the concentration of acetonitrile to 5%. Typically, a microcolumn with immobilized 4E10 (corresponding to 8–10 µl of the initial bead slurry) was incubated with either 10 µg of SOSgp140JR-FL, 20 µg of gp160MN, 9 µg of gp120SF2, 10 µg of gp41MN (HPLC purified), 4.5 µg of gp41(546–682)HXBc2 (HPLC purified), or gp41(541–682+His-tag,)HXBc2 (27.5 µg of dialyzed Ag or HPLC-purified fraction 33) in 20 mM NaP (pH 7.2), 0.1% DDM ON at 37°C. The beads were then drained, washed twice with 0.4 ml of 20 mM NaP (pH 7.2), 0.1% DDM (except of gp120-containing beads, which were washed with 0.4 ml of 20 mM NaP (pH 7.2)), and resuspended in 20–25 µl of buffer. For digestion with endoproteinases, the immobilized mAb 4E10 with affinity-bound Ag was resuspended in an appropriate buffer (see below). Immobilized 4E10 without Ag was used as a control and treated in parallel to the sample. For trypsin digestions, the sample was incubated with 1 µg of proteinase in 50 µl of NH4HCO3 (20–50 mM), 0.1% DDM, ON at 37°C or in case of gp120 with 0.1 µg of trypsin in 20 µl of 20 mM NaP (pH 7.2), 4 h at 37°C. The GluC-digest was performed with 1 µg of GluC (Roche Molecular Biologicals) in 50 µl of 25 mM NH4HCO3, 0.1% DDM, ON at 25°C, and the chymotrypsin digest was performed with 1 µg of chymotrypsin (Roche Molecular Biologicals) in 50 µl of 0.1 M Tris-HCl (pH 8.0), 10 mM CaCl2, 0.1% DDM, ON at 25°C. Deglycosylation was achieved with 1 µl of PNGaseF (5 U; Sigma-Aldrich) in 50 µl of 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, ON at 37°C. For salt-washing steps, beads were washed first with 0.4 ml of 20 mM NaP (pH 7.2), 150 mM NaCl, 0.1% DDM and then with 0.4 ml of 20 mM NaP (pH 7.2), 0.1%DDM.

Analyses by MALDI/MS and MALDI/MS/MS

For MS-analyses at various stages of the experiments, 0.3–0.4 µl of the beads were spotted on a stainless-steel MALDI target together with equal volume of saturated {alpha}-hydroxycinnamic acid solution (in acetonitrile/water/formic acid 60/30/10 v/v/v) and analyzed by MALDI/MS on a Voyager Super DE-STR (Applied Biosystems). Typically, 100 spectra were summed, and the ions were calibrated externally and/or internally. For acquisition of an MS/MS, beads were spotted on a target in a 1:1 ratio with {alpha}-hydroxycinnamic acid solution and analyzed by MALDI/MS/MS on a 4700 Proteomics Analyzer (Applied Biosystems).

ELISA

Immulon 1B 96-well plates (Thermo Electron) were coated with Ags in 0.1 M NaHCO3 at room temperature (RT) ON (for a complete list of the peptides analyzed, see Table I). The wells were washed with TBS (10 mM Tris-HCl (pH 7.4), 150 mM NaCl) containing 0.1% DDM, blocked with 0.5% BSA, and 10% low IgG FCS (Invitrogen Life Technologies) in TBS for 1 h at 35°C and washed again with TBS/0.1% DDM. Subsequently, the wells were incubated with 0.5 µg/ml 4E10 in TBS for 2 h at 35°C, washed with TBS, incubated with the alkaline phosphatase-conjugated IgG (Sigma-Aldrich) diluted 1/5000 with TBS/20% low IgG FCS/2% skim milk for 1 h at 35°C and washed again with TBS. Phosphatase substrate (Sigma-Aldrich) was added (100 µl, 1 mg/ml) and the reaction was stopped after 1 h at RT with 25 µl of 3 N NaOH. The absorption was measured at 405 nm. Alternatively, for amplification of the alkaline phosphatase reaction, the AMPAK system was used based on the instructions of the manufacturer (DakoCytomation). The absorption was measured at 492 nm. To determine whether detergents would influence the binding of N15, N31, C22, and no. 6373 to 4E10, the ELISA was performed with DDM substituted with 0.1% Tween 20 or without any detergent in the washing buffer (n = 2). For the initial screening of peptides recognized by 4E10, a concentration range of 0.1–10 µM was tested (n = 3, DDM in accordance to mass spectrometric experiments). Reactive peptides as well as the peptides with the adjacent amino acid sequence upstream and downstream of the reactive peptide were subjected to additional testing (two independent experiments with n = 2 each). Peptides N15, N31, and C22 were analyzed in five separate experiments with a total of n = 11. Gramicidin S (Sigma-Aldrich) and peptide nos. 8916 and 8917 were used to evaluate nonspecific binding of hydrophobic peptides to 4E10. For the determination of the apparent affinity of antigenic peptides, microtiter plates were coated with various concentrations of peptides N15, N31, and C22 (one experiment, n = 3) and peptide nos. 6338 and 6376 (two independent experiments, n = 3 each). Samples were washed with 20 mM NaP (pH 7.2)/0.1% DDM before incubation with 0.05 µg/ml 4E10 in 20 mM NaP (pH 7.2). For amplification of the alkaline phosphatase signal, the AMPAK system was used, however, with a 1:0.5 ratio of substrate:amplifier (DakoCytomation). Initial screening of the proteins gp160MN, gp120SF, gp120SF162 (expressed in CHO cells, obtained from the National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases), gp41MN, gp41SF2 (expressed in yeast cells, Austral Biologicals), gp41HXBc2(541–682+His-tag) and the His-tagged sporulation initiation phosphotransferase F (Spo0F-His6, obtained as a gift from J. Cavanagh, North Carolina State University, Raleigh, NC) was performed at a concentration range of 0.1–10 µM. Additional experiments were done with gp160MN, gp120SF162, gp41MN, and gp41SF2 only.


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  		Table I. Synthetic peptides used for epitope mapping

 
Nonquantitative Bradford assay

After incubation of the microtiter plates ON, 2 20-µl aliquots of the "supernatant" were carefully removed and transferred into microtiter plates for protein analyses. A Bradford assay was performed by adding 30 µl of Bradford solution (Bio-Rad, diluted 1/3 with H20). After 5 min at RT, 50 µl of H2O was added to achieve a homogeneous mixture for absorption measurements at 595 nm. The assay was performed in a nonquantitative manner, i.e., without standard proteins. According to the manufacturer, the linear range for detection is 8–80 µg/ml.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Mass spectrometric epitope mapping using protein Ags

To map the epitope recognized by the broadly neutralizing Ab 4E10, we tested binding of various glycosylated full-length Ags, nonglycosylated and truncated proteins, as well as synthetic peptides. The alignment of the sequences of all Ags used in this study with the Env sequence of the reference strain HXBc2 is presented in Fig. 1B. For detection of affinity-bound Ags, Sepharose beads with immobilized 4E10 and noncovalently attached Ag are spotted on a stainless steel target and analyzed directly by MALDI/MS (15).

The protein SOSgp140JR-FL was initially used to characterize the epitope. This construct consists of gp120 and the gp41 ectodomain and is heavily glycosylated. The precursor matures by cleavage at the native cleavage site, however, the mature proteins remain covalently connected through an engineered disulfide bond between gp120 and gp41 (14). SOSgp140 bound to the immobilized 4E10. To determine the epitope recognized on this Ag, we first used an epitope extraction assay. For this, the protein in solution was digested with trypsin and the peptide mixture was subsequently added to the immobilized Ab. Fig. 2A compares the MALDI spectrum of the SOSgp140-derived peptide mixture in solution (top panel) with the MALDI spectrum of the beads with the immunoextracted peptides (bottom panel). Assignment of the peptide ions from the solution digest to the aa sequence of SOSgp140 showed a sequence coverage of 61% (395–646 aa). Ions of m/z 1609.60, 2925.06, and 4271.58 were selectively affinity bound to 4E10. These ions correspond to aa 7–19 (theoretical monoisotopic M + H+ 1609.88), aa 477–507 (M + H+mono. 2925.53), and aa 508–544 (M + H+mono. 4271.43). The sequences of the ions 1609.6 and 2925.1 were confirmed by MALDI/MS/MS. For the ion of m/z 1609.6, the immonium ions of all amino acids except glycine, the b ions b2-b9, and the y ions y2-y10 were observed, thereby confirming the sequence LWVTVYYGVPVWK (Fig. 2B). For the ion of m/z 2925.1, the sequence AVGIGAVFLGFLGAAGSTMGAASMTLTVQAR was verified with the b ions b1-b7, the y ions y1–8 and y12–13, and the presence of the immonium ions of V, L/I, F, T, and R (Fig. 2C). Although an ion of m/z 2778.46 corresponding to the tryptic peptide 621NEQELLELDKWASLWNWFDITK642 with a theoretical monoisotopic M + H+ of 2778.38 was detected with low abundance in the in-solution digest (Fig. 2A), this ion was not observed bound to the Ab. Other ions whose sequence would correspond to the C-terminal region of the gp41 ectodomain containing the previously determined epitope NWFDIT were not detected.


Figure 2
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  		FIGURE 2. MALDI/MS spectra of the HIVJR-FL SOSgp140 tryptic digest used for the epitope extraction assay (A, top panel) and of the beads with the affinity-extracted peptides (B, bottom panel). Ions marked with asterisks correspond to sodium adducts. Signal-to-noise ratios for the ions of interest are given in parenthesis. MALDI/MS/MS spectra of the affinity-bound singly charged peptide at m/z 1609.6 (B) and the affinity-bound singly charged peptide at m/z 2925.1 (C) confirmed the amino acid sequences.

 
In addition to the epitope extraction assay, an epitope excision assay was performed, in which the Ag is affinity bound to the immobilized Ab and subsequently digested with proteinases. The peptides that remain bound to the Ab contain the epitope. After a trypsin digest of SOSgp140 bound to the immobilized 4E10, MALDI/MS analysis yielded ions of m/z 1610.7, 2928.0, 3083.2, 4620, and 7184 (linear mode, data not shown). The ions were assigned to the peptides 7LWVTVYYGVPVWK19 (theoretical average M + H+: 1610.9), and 477AVGIGAVFLGFLGAAGSTMGAASMTLTVQAR507 (M + H+avg: 2927.5). Ions of higher m/z were assigned to peptide 476–507 (M + H+avg: 3083.7), peptide 477–522 (M + H+avg: 4618.5), and peptide 477–544 (M + H+avg: 7183). These peptides are located at the N-terminal region of gp41 and contain up to four missed cleavage sites. All peptides remained bound to 4E10 when the beads were washed with 150 mM NaCl in sodium-phosphate buffer.

Binding of the uncleaved precursor protein HIVMN gp160 to 4E10 was analyzed to determine whether the free N-terminal end of gp41 is required for the interaction with the Ab. The glycosylated full-length protein (32 potential N-glycosylation sites) bound to 4E10, and the antigenic determinants were characterized. The detergent DDM was included in the buffer to reduce nonspecific binding of hydrophobic peptides to the hydrophobic binding site of 4E10. DDM is a mild detergent, which does not affect the conformation of hydrophobic proteins (16). In addition, this detergent is compatible with MALDI/MS. The epitope excision assay with consecutive digests using trypsin, endoproteinase GluC, deglycosylation with PNGase F as well as salt-washing steps resulted in observation of ions of m/z 1609.78 (4LWVTVYYGVPVWK16, M + H+mono. 1609.88) and 2822.37 (485AAIGALFLGFLGAAGSTMGAASVTLTVQAR 514, M + H+mono. 2822.52) (Fig. 3A). To determine whether the N terminus of gp120 could be affinity captured in the absence of the N terminus of gp41, the immobilized Ab was incubated with HIVSF2 gp120. The glycosylated full-length envelope protein bound to 4E10 and a subsequent epitope excision assay with trypsin showed that a peptide of m/z 1866.97 (1EKLWVTVYYGVPVWK15 M + H+mono. 1867.02) remained bound to the Ab (data not shown).


Figure 3
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  		FIGURE 3. MALDI/MS spectra of affinity-bound peptides obtained by epitope excision assays. A, Ions detected after the digest of gp160MN with trypsin followed by endoproteinase GluC, salt-washing steps, and deglycosylation with PNGaseF were assigned to peptide 4LWVTVYYGVPVWK16 (M + H+mono. 1609.88) and 485AAIGALFLGFLGAAGSTMGAASVTLTVQAR514 (M + H+mono. 2822.53). B, Trypsin digest of gp41541–682+His-tag followed by chymotrypsin digest of affinity-bound peptides resulted in ions that were assigned to 136NWFNITNWLWYIHHHHHH153 (M + H+mono 2492.17), 132ASLWNWFNITNWLWYIHHHHHH153 (M + H+mono. 2949.40), or 121NEQELLELDKWASLWNWFNITNW143 (M + H+mono. 2949.42), and 127ELDKWASLWNWFNITNWLWYIHHHHHH153 (M + H+mono. 3620.73). Asterisks mark sodium adducts and triangles indicate background ions.

 
Additionally, the binding of gp41 in the absence of gp120 was tested. The gp41 constructs HIVHXBc2 gp41546–682, HIVMN gp4126–167 + 194–258, and HIVHXBc2 gp41541–682+His-tag contain the gp41-ectodomain (residues 512–683 in env-HXBc2, see Fig. 1B). HIVMN gp4126–167 + 194–258 contains also 65 residues of the endodomain without the transmembrane segment. All three commercially available gp41 constructs lack the N-terminal fusogenic peptide of gp41 but contain the previously reported NWFN/DIT antigenic determinant (8). Again, DDM was added to prevent precipitation of the hydrophobic protein and to minimize nonspecific binding to 4E10. Binding of HPLC-purified glycosylated HIVHXBc2 gp41546–682 to the mAb was observed with low abundance (S/N 4:1) and did not yield any results after protease digestion (data not shown). Epitope excision of the HPLC-purified nonglycosylated HIVMN gp4126–167 + 194–258 yielded no ions that could be assigned to the C-terminal region of the gp41 ectodomain containing NWFDIT (data not shown). After a trypsin digest of affinity-bound glycosylated HIVHXBc2 gp41541–682+His-tag (either HPLC purified or dialyzed), an ion of m/z 4349.72 was detected (data not shown) and assigned to peptide 121NEQELLELDKWASLWNWFNITNWLWYIHHHHHH153 (M + H+avg. 4349.82). Subsequently, the affinity bound peptide was further digested with chymotrypsin. Ions of m/z 2492.02, 2949.21, and 3620.70 were observed (Fig. 3B). These protonated ions were assigned to peptide 136–153, (M + H+mono. 2492.17), peptide 132–153 (M + H+mono. 2949.40) or peptide 121–143 (M + H+mono. 2949.42), and peptide 127–153 (M + H+mono. 3620.73), and all peptides remained bound after a salt wash.

Epitope analysis using synthetic peptides

To determine whether the N-terminal sequences of gp120 and gp41 as well as the pre-TM sequence of gp41 could bind to 4E10 in the absence of their native protein environment, the synthetic peptides N15 (amino acid sequence EKLWVTVYYGVPVWK), N31 (AVGIGAVFLGFLGAAGSTMGAASMTLTVQAR), and C22 (NEQELLELDKWASLWNWFNITNWLWYI) were added to immobilized 4E10 either individually or in a molar ratio of 1:1:1. MALDI/MS analyses showed that all three peptides bound to the Ab separately as well as in combination (Fig. 4A). However, after washing with 150 mM NaCl in sodium-phosphate buffer, only N15 and N31 remained bound while C22 was lost (Fig. 4B).


Figure 4
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  		FIGURE 4. Epitope analysis using synthetic peptides: MALDI/MS spectra of synthetic peptides bound in combination (N15/N31/C22) or individually (N31 and C22) to the immobilized Ab 4E10 before (A) and after (B) washing steps with 0.15 M NaCl in sodium-phosphate buffer. The ion of m/z 1866 is not shown due to the high sensitivity of the singly charged peptide N15. For the combined peptides, N15, N31, and C22 were preincubated at an equimolar ratio and 100 µl were added to the 4E10 microcolumn after dilution to 3.38 µM/peptide. For the individual peptides, the concentration was 5 µM N15/N31/C22. Microcolumns were extensively washed with sodium-phosphate buffer before MS analysis. C, ELISA-based epitope mapping using peptides from the N-terminal region of gp120 and gp41, respectively, and the gp41 PTMR. The absorption values at 492 nm for microtiter plates coated with 100 µl of 10 µM peptide are shown as an example (n = 2). D, ELISA of various concentrations of the peptide nos. 6338 (dashed line with stars) and 6376 (dotted line with squares) with data points fitted (solid line). The data points reflect the mean absorption of three samples per concentration.

 
To further characterize the antigenic determinants, binding of synthetic peptides to 4E10 was tested by ELISA. Due to the hydrophobicity of the peptides, the immunoassays were performed using microtiter plates with medium binding affinity for hydrophobic biomolecules. As the conformation of hydrophobic peptides and proteins strongly depends on their lipophilic environment, binding of 4E10 to various peptides was tested under different conditions (Table II). Binding of peptides N31 and C22 to 4E10 was observed for washing buffer containing 0.1% DDM. As this result is consistent with the mass spectrometric data, the subsequent immunoassays were performed with DDM. A total of 34 synthetic peptides corresponding to the N-terminal sequences and the PTMR of the native Ag were analyzed. The results of the binding assays with peptides from the N-terminal region of gp41 are summarized in Table III. Based on these data, the epitope core is contained within the sequence AXFLGFLGAA with X being valine (HXBc2), leucine (MN), and a methionine in the consensus sequence of HIV subtype B. The results for gp41 PTMR peptides are presented in Table IV. These data indicate that the epitope core of this region is contained within the sequence WFDITNWLWY. Peptide N31 and the peptides with the sequence RAAIGALFLGFLGAA (no. 6337) and GALFLGFLGAAGSTM (no. 6338) from the N terminus of gp41 were recognized by 4E10 with an absorption similar to peptide C22 and peptides SLWNWFDITNWLWYI (no. 6376) and WFDITNWLWYIKIFI (no. 6377) (Fig. 4C). The gp120 N-terminal sequence EKLWVTVYYGVPVWK showed only low absorption. The hydrophobic peptides gramicidin S and peptide nos. 8916 and 8917 did not bind to 4E10, thereby suggesting that nonspecific binding of hydrophobic peptides to the Ab does not occur. To determine the apparent affinity of the antigenic peptides GALFLGFLGAAGSTM (no. 6338) from the N terminus of gp41 and SLWNWFDITNWLWYI (no. 6376) from the PTMR to 4E10, various concentrations of Ag were bound to the mAb and analyzed by ELISA (Fig. 4D). The mean apparent affinity was calculated as 0.157 ± 0.008 µM for peptide no. 6338 and 0.055 ± 0.002 µM for peptide no. 6376. Similar values were determined for N31 with ~0.152 µM and C22 with ~0.07 µM. For the peptide N15 resembling the gp120 N-terminal region, an apparent affinity of ~0.27 µM was calculated.


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  		Table II. Binding of synthetic peptides to 4E10 as determined by ELISA

 

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  		Table III. Epitope mapping by ELISA using synthetic peptides corresponding to the N-terminal sequence of gp41

 

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  		Table IV. Epitope mapping by ELISA using synthetic peptides corresponding to the PTMR of gp41

 
ELISA-based epitope mapping using protein Ags

The initial screening of protein Ags in the concentration range of 0.1–10 µM showed binding of 4E10 to gp160MN, gp41MN, gp41HXBc2(541–682+His-tag), and gp41SF2 but not to gp120SF2 or gp120SF162 when immobilized on Immulon 1B plates. A Bradford assay of the protein solution of gp120SF162 after coating the microtiter plates was positive for concentration >0.1 µM, which is slightly above the detection limit for the linear range of protein concentrations (8–80 µg/ml). The lack of signal in ELISA was, therefore, caused by insufficient binding of gp120 to the plates and not by a lack of recognition of the protein by the Ab. The proteins gp160MN, gp41MN, gp41HXBc2(541–682+His-tag), and gp41SF2 on Immulon 1B plates were recognized by 4E10 with saturation at a protein concentration of 0.1 µM for all four Ags. To determine the sensitivity of 4E10, the Ags gp160MN, gp41MN, and gp41SF2 were further analyzed. As depicted in Fig. 5A, gp160 was recognized with greater sensitivity than the gp41 proteins. Half-maximal binding occurred at ~0.003 µM gp160MN and at concentrations of 0.008–0.01 µM for gp41MN and gp41SF2. To explore the possibility of a conformational or discontinuous epitope, immunoassays were performed with gp160MN and gp41MN with and without thermal denaturation. For gp160MN, binding to 4E10 was reduced by ~50% when incubated at 95°C for 5 min compared with native protein (Fig. 5B, left panel), whereas for gp41MN binding to 4E10 was slightly enhanced after denaturation (Fig. 5B, right panel).


Figure 5
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  		FIGURE 5. A, Binding of the glycosylated Ags gp160MN and gp41SF2 and the nonglycosylated Ag gp41MN to 4E10 as determined by ELISA. The signal for each Ag (n = 2) was normalized to its maximal absorption at 492 nm. B, Effect of thermal denaturation of the Ags gp160MN and gp41MN on the binding to 4E10 analyzed by ELISA. The relative absorption of the denatured Ag (n = 2) was calculated in respect to the normalized value of the maximal absorption of the native protein in the corresponding experiment. For native gp160 MN, data of n = 7 in four separate experiments and for gp41MN of n = 6 in three separate experiments were combined.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
4E10 has been described as a broadly neutralizing mAb (6) and, therefore, its characterization might provide information relevant to vaccine development. Determination of the epitope recognized by 4E10 has been attempted previously: Buchacher et al. (17) identified the sequence AEGTDRV located in the gp41 endodomain (Env-HXBc2 aa 823–828). Stiegler et al. (7) found that 4E10 bound a peptide (termed peptide 2031) with the sequence LLELDKWASLWNWFDITNWL (Env-HXBc2 aa 660–679). The synthetic peptides resembling sequences of gp41 that were used for the ELISAbased screening were mostly comprised of 20 aa with 10-aa overlap between sequential peptides (7). However, the peptides corresponding to the N-terminal region of gp41 in the study by Stiegler et al. (7) had the sequences TKAKRRVVQREKRAAIGALF, LGFLGAAGST, GAAGSTMGAAS, and SMTLTVQARQL. As none of these peptides bound to 4E10 and overlapping peptides were not tested, the antigenic determinants 34LWVTVYYGVPVWK46 and 512AVGIGAVFLGFLGAAGSTMGAASMTLTVQAR542 identified in the present study would not have been detected. Screening of an HXBc2 gp160 fragment expression library revealed NWFNIT as the epitope (8). The HXBc2 env gene used for the generation of the fragment library, however, missed the first 12 residues of gp120 (8), which includes the first 10 residues of the N-terminal sequence 34LWVTVYYGVPVWK46 we found to be recognized by the mAb. Moreover, the hydrophobic fusogenic peptide and its consecutive residues might not have been presented for efficient recognition by 4E10 either due to the lack of correct conformation within the surrounding M13-spike protein or due to interaction with the detergent Tween 20 in the washing buffer for panning. It would be interesting to determine whether repeating the clone selection in the presence of DDM would result in the identification of the antigenic determinants presented here. In an epitope-mapping serology assay, various biotinylated gp41 peptides were tested for their binding to 4E10 (18). Despite the presence of the sequence NWFNIT (underlined), a relatively low fluorescence intensity was detected for the modified peptide biotin-KWASLWNWFNITNWLWYIK bound to 4E10, whereas the peptide QELLELDKWASLWNWFNITNWLWYIKKKK with a biotinylated zinc-finger scaffold at the N terminus was bound with high affinity, which emphasizes the need for a correct presentation of the epitope. 4E10 binding of the peptide AVGLGAHFLGHLG with a biotinylated zinc-finger scaffold at the N terminus was not reported (18). The substitution of amino acids, which we located within the epitope core AXGIGAVFLGFLG with X being V, L, or M, might have abolished the binding properties of the peptide.

By the use of epitope extraction and epitope excision of glycosylated and unglycosylated, full-length or truncated, precursor and cleaved mature proteins, we characterized the N termini of gp120 and gp41, respectively, as antigenic determinants, thereby identifying two peptides with 15 and 31 residues, respectively, from the 827-residue long precursor protein gp160. Even if an antigenic peptide might not be observed in the mass spectrum of the digest due to ion suppression effects, it would be observed after enrichment by the mAb.

HIVSF2 gp120 bound to the immobilized 4E10 and allowed the identification of the N-terminal region as being recognized by the Ab in an epitope excision assay. In our immunoassays, however, binding of gp120 to 4E10 could not be observed. Zwick et al. (8) also reported a low absorption for gp120–4E10 binding in an ELISA. We could show in our case that the lack of 4E10 binding is caused by insufficient coating of the plates with Ag. For either of the antigenic determinants derived from the N terminus of gp41 and the PTMR of gp41, respectively, the apparent affinity to 4E10 was calculated to be in the submicromolar range. Nevertheless, a comparison of 4E10 binding in immunoassays showed increased binding in the sequence synthetic peptides < gp41MN < gp160MN despite increasing conformational complexity of the Ags, again indicating different epitopes and/or different conformations. It has been postulated that the residues NWFN/DIT form a cryptic epitope, which would only be accessible in a virus-cell fusion intermediate stage (7). Denaturation of gp41 (for example, by high temperature) would expose the cryptic epitope and, therefore, should increase binding to 4E10. This was demonstrated for gp41MN, which lacked the N-terminal region of the mature gp41. In contrast, Zwick et al. (8) found reduced binding after denaturation of gp41MN. Decreased binding to 4E10 was also observed for denatured gp160MN (8), which we could confirm in this study. These data clearly indicate that, in the native envelope protein, the epitope is conformational and/or discontinuous.

Analyses of the three-dimensional structure of synthetic peptides corresponding to the sequence of the C-terminal region in the gp41 ectodomain by infrared spectroscopy (19) and nuclear magnetic resonance spectroscopy (20) showed that the PTMR forms an {alpha}-helix. The crystal structure of this peptide bound to 4E10 verifies a helical conformation, in which the hydrophobic residues are located on one face of the helix (9). The fusogenic peptide, however, could adopt an {alpha}-helical shape or a beta-sheet conformation depending on the peptide concentration and environmental factors such as type of lipids or the presence of cations (21, 22, 23, 24, 25, 26). The PTMR as well as the N-terminal regions of gp120 and gp41 contain several hydrophobic residues. As depicted in Fig. 6A, when arranged in a helical conformation the gp41 N-terminal residues form a large hydrophobic face similar to the PTMR (Fig. 6B; Ref. 9). On the opposing side, the small residues (Ala and Gly) would provide flexibility for the helix as well as sufficient space for the interaction with other hydrophobic residues with large side chains. The hydrophobic residues of the gp120-derived sequence form one side of the helix with tightly packed hydrophobic residues, whereas on the opposite side the hydrophobic residues are more widely separated (Fig. 6C). This conformation might allow for a mediator function of this peptide between two hydrophobic surfaces such as another hydrophobic peptide (such as the gp41 N-terminal fusogenic peptide) or a membrane. The N-terminal region of gp120 matches the sequence L/VX1–5YX1–5R/K (Fig. 6D), which is considered to be the binding motif for cholesterol (27). The membranes of HIV and the host cells are rich in cholesterol (28) and interaction of gp160 with cholesterol was demonstrated (29). Recombinant gp120LAI did not bind to the cholesteryl hemisuccinate-derivatized resin and, therefore, further analysis focused on another segment, which contained the binding motif. The sequence LWYIK is located within the PTMR and binds to cholesterol (29, 30). However, a 19-residue long peptide, which contains also the sequence NWFNIT, was less able to sequester cholesterol into domains than the shorter N-acetyl-LWYIK-amide (30), indicating that further analysis of potential cholesterol binding sites is necessary. The functional relevance, however, of the gp41 N-terminal fusogenic peptide for the fusion process has been established (21, 31, 32, 33, 34, 35, 36, 37, 38, 39). A synthetic 33-mer peptide corresponding to the gp41 N-terminal region could induce self-association in phospholipid membranes (21, 31), but a peptide with a single amino acid substitution (V2E) abolished liposome destabilization or vesicle fusion (31). This mutation as well as others in the N-terminal region of gp41 interfered with syncytium formation and infectivity (32, 33, 34, 35, 36, 37). As the fusogenic peptide induced membrane fusion and permeabilization and in equimolar mixtures with the pre-TM peptide acted in a cooperative way, Suarez et al. (40) speculated that in the hairpin structure of gp41 both peptides would be in close proximity at the same end of the molecule.


Figure 6
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  		FIGURE 6. Representation of the helical wheel for the peptides identified as antigenic determinants of 4E10: (A) the first 16 residues of the gp41 N terminus. The inner circle reflects the sequence AVGIGAVFLGFLGAAG from HIVJR-LF, whereas the residues in the outer circle correspond to the sequence in HIVHXBc2. B, LWVTVYYGVPVWK located at the N-terminal region of gp120. The structure has not been experimentally characterized and a helical conformation is hypothetical. C, Helical wheel of NWFDITNWGK from the PTMR as presented in Ref. 9 . For A and B, the direction of the helical wheel N->C is going into the page. Hydrophobic residues are shown with shaded circles. D, Analysis of the gp120 N-terminal sequence and the pre-TM peptide for correlation with the motif for cholesterol binding (27 ). The number of residues X between L/V in the first position and Y40 of the gp120 N-terminal sequence is given in parentheses.

 
Although our immunoassay and MS data show binding of the synthetic peptide resembling the PTMR of gp41, we could not identify the PTMR peptide as part of the antigenic determinants in epitope excision or extraction assays using native protein Ag. Several explanations for this observation are possible: 1) 4E10 has been described as a polyspecific Ab, which recognizes cardiolipin, phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine (41) in addition to the pre-TM peptide and the N-terminal regions of gp120 and gp41. The antigenic determinants could bind independent of each other through induced fit of the Ab’s Fab-region, but with different binding affinities. 2) As shown by ELISA, the epitope core of the PTMR is contained within the sequence 672WFDITNWLWY681. Alanine-scanning mutagenesis of residues 660–680 identified W672, F673, and W680 as important for neutralization by 4E10 and the subtle changes W672Y and W672F significantly increased viral sensitivity for 4E10 (42). Nevertheless, the crystal structure of 4E10 with a synthetic peptide of the PTMR leaves a large area at the tip of the CDR H3 loop vacant (9). The peptides characterized in this study could bind simultaneously to different areas in the Ab-binding site. In this discontinuous conformational epitope, the pre-TM peptide does not provide sufficient binding energy to be retained to the Ab in the epitope excision assays. Analyses of the interaction sites between the envelope proteins showed that the conserved regions C1 (which contains the N15 sequence) and C5 of gp120 interact with gp41 (14, 43, 44, 45). Although the interaction site of gp120-C1 with gp41 has not been determined, our data indicate an association of gp120-C1 with the N-terminal region of gp41. 3) It is possible that ions from peptides of the PTMR are not observed in the mass spectrometric analysis due to ion suppression effects. However, as we could observe ions for the synthetic peptide C22 (Fig. 4A), we consider this explanation as unlikely.

Based on the results presented here and the above literature, we propose a model for the recognition of the epitope by 4E10 (Fig. 7): on the surface of the virions, gp41 exists in a native state with the N-terminal fusogenic peptides largely inaccessible (46, 47). After interaction of the trimeric complex (gp120/gp41)3 with the receptors on the host cell, the native gp41 changes into a transient prehairpin intermediate with the fusogenic peptide initially exposed. Based on the interaction of the hydrophobic N-terminal region of gp120, the fusogenic peptide binds to the host cell membrane, thereby anchoring the complex and directing the fusogenic peptide for membrane insertion. Subsequently, the heptad repeats align into the coiled-coil six-helix bundle structure termed hairpin, allowing the pre-TM peptide to interact with the host cell membrane and bringing the viral membrane and the target membrane into close proximity. Ultimately, the adjacent membranes of HIV and the human target cell fuse (46). However, in the presence of 4E10, the mAb binds to the fusogenic peptide of gp41 and the N-terminal region of gp120, which inhibits the insertion of the fusogenic peptide into the host cell membrane. The formation of the coiled-coil hairpin structure might be possible despite the presence of 4E10. Subsequent interaction of the Ab with the pre-TM peptide prevents the insertion of this hydrophobic sequence into the host cell membrane. Consequently, fusion of the viral membrane with the target cell membrane is inhibited. We propose that the Ab 4E10 functions as a "trap" for the viral sequences, which are responsible for insertion into the host cell membrane, through the hydrophobic surface in the paratope. As the N-terminal region of gp120, the fusogenic peptide of gp41, and the PTMR in gp41 show high sequence homology among various HIV strains, this model is consistent with the broadly neutralizing capabilities of 4E10.


Figure 7
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  		FIGURE 7. Model of the HIV entry and its inhibition by the broadly neutralizing Ab 4E10. After interaction of gp120 with the primary receptor CD4 and the secondary receptor, conformational changes in gp41 result in the formation of the prehairpin intermediate. In this transient conformation, the N-terminal fusogenic peptide (FP) of gp41 is exposed, and interacts with the N-terminal region of gp120 (C1). The fusogenic peptides insert into the membrane of the human cell, and the N- and C-terminal heptad repeats (NHR, CHR) collapse into the hairpin structure, thereby bringing the viral membrane and the target cell membrane into close proximity for fusion (46 ). In the presence of 4E10, the mAb binds to the hydrophobic N-terminal peptides of the mature proteins. After formation of the hairpin structure, the tryptophan-rich sequence in the PTMR adjacent to the transmembrane domain (TMD) can also interact with 4E10. By trapping the hydrophobic regions of gp120 and gp41, insertion into the host cell membrane is inhibited, thereby preventing infection of the human target cell by. The gp120 molecules were omitted from the representation of the hairpin structure for clarity and the endodomain of the gp41 molecule was omitted in all representations.

 

   Acknowledgments
 
We thank Norbert Schülke and John Moore for providing the Ag HIVJR-FL SOSgp140. Moreover, we gratefully acknowledge Norbert Schülke for his advice and discussion of this manuscript. We also acknowledge the reagents obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences. Back

2 Address correspondence and reprint requests to Dr. Christine Hager-Braun, National Institute of Environmental Health Sciences, 111 T. W. Alexander Drive, Research Triangle Park, NC 27709. E-mail address: christine_hager-braun{at}ncsu.edu Back

3 Abbreviations used in this paper: pre-TM, pretransmembrane; PTMR, pre-TM region; ON, overnight; MS, mass spectrometry; DDM, n-dodecyl beta-D-maltoside; RT, room temperature. Back

Received for publication October 5, 2005. Accepted for publication February 28, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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