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J Virol. 2010 March; 84(6): 2955–2962.
Published online 2009 December 30. doi:  10.1128/JVI.02257-09
PMCID: PMC2826063

Relationship between Antibody 2F5 Neutralization of HIV-1 and Hydrophobicity of Its Heavy Chain Third Complementarity-Determining Region [down-pointing small open triangle]


The membrane-proximal external region (MPER) of the HIV-1 gp41 transmembrane glycoprotein is the target of the broadly neutralizing antibody 2F5. Prior studies have suggested a two-component mechanism for 2F5-mediated neutralization involving both structure-specific recognition of a gp41 protein epitope and nonspecific interaction with the viral lipid membrane. Here, we mutationally alter a hydrophobic patch on the third complementarity-determining region of the heavy chain (CDR H3) of the 2F5 antibody and assess the abilities of altered 2F5 variants to bind gp41 and to neutralize diverse strains of HIV-1. CDR H3 alterations had little effect on the affinity of 2F5 variants for a peptide corresponding to its gp41 epitope. In contrast, strong effects and a high degree of correlation (P < 0.0001) were found between virus neutralization and CDR H3 hydrophobicity, as defined by predicted free energies of transfer from water to a lipid bilayer interface or to octanol. The effect of CDR H3 hydrophobicity on neutralization was independent of isolate sensitivity to 2F5, and CDR H3 variants with tryptophan substitutions were able to neutralize HIV-1 ~10-fold more potently than unmodified 2F5. A threshold was observed for increased hydrophobicity of the 2F5 CDR H3 loop beyond which effects on 2F5-mediated neutralization leveled off. Together, the results provide a more complete understanding of the 2F5 mechanism of HIV-1 neutralization and indicate ways to enhance the potency of MPER-directed antibodies.

The membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane glycoprotein is the target of three broadly neutralizing anti-HIV-1 antibodies, 2F5, Z13e, and 4E10, and is thus a potential site of HIV-1 vulnerability to the humoral immune response (21, 24, 27, 48). The MPER encompasses ~25 residues at the carboxyl-terminal end of the predicted gp41 ectodomain, just before the transmembrane region, and is rich in aromatic residues, typical of bilayer-interfacial regions of membrane proteins (26, 36, 40). Mutation of selected MPER tryptophans abrogates gp41-mediated fusion of the viral and target cell membranes, indicating that this region is crucial for HIV-1 infectivity (23, 28). Structural studies of unbound forms of the gp41 MPER both in solution and in lipid contexts have demonstrated that it adopts a number of conformations, many of which are α-helical, and electron-paramagnetic resonance measurements have indicated lipid bilayer immersion depths for MPER residues that range from acyl to phospholipid headgroup regions (4, 7, 8, 19, 32, 37). The binding of neutralizing antibodies, such as 2F5, to the MPER must therefore account for the membrane milieu in which the epitope is found.

The 2F5 antibody has been shown to exhibit ~100-fold-enhanced binding to its epitope on uncleaved gp140s when presented in the context of lipid proteoliposomes (11, 25), and other studies have shown that 2F5 can contact phospholipids directly in the absence of gp41 (1, 3, 12, 22, 29, 30). The latter finding has led to the suggestion that 2F5 might be autoreactive (12), although passive transfusion of 2F5 does not appear to have deleterious effects (38) and 2F5 failed to react in some clinically based assays for autoreactive lipid antibodies (31, 39). The crystal structures of the 2F5 antibody in complex with its gp41 MPER epitope revealed that, despite the 22-residue length of the 2F5 heavy chain third complementarity-determining region (CDR H3) loop, contacts with the gp41 MPER peptide are made predominantly at the loop base. In some crystal structures, the tip of the loop protrudes away from gp41, while in others, it is disordered (9, 14, 25). A unique feature of the tip of the CDR H3 loop is that it contains a patch of hydrophobic residues, including residues L100A, F100B, V100D, and I100F (Kabat numbering), which, with the exception of I100F, do not contact gp41 (9, 10, 14, 25) (Fig. (Fig.1).1). While a prior study revealed the importance of residue F100B of the CDR H3 loop in 2F5-neutralizing activity, nonconservative residue substitutions at this position also appeared to diminish 2F5 binding to the immobilized MPER peptide and gp41 in enzyme-linked immunosorbent assay (ELISA) formats (47). Conversely, a more recent study has shown that alanine mutations in the 2F5 CDR H3 loop can affect neutralization without affecting gp41 binding (2).

FIG. 1.
2F5 CDR H3 loop mutagenesis. (A) Structure of 2F5 Fab (blue and gray) in complex with a gp41 peptide (red). The 2F5 CDR H3 (purple) contacts gp41 only at its base, while the tip extends away from the peptide. (B) Close-up view of the 2F5 CDR H3 loop, ...

In this study, we sought to examine the role of the chemical nature of residues at the tip of the 2F5 CDR H3 loop in neutralization of HIV-1. Mutations were introduced into the 2F5 CDR H3 loop that altered its hydrophobicity, and the resulting 2F5 mutants were tested both for binding to a gp41 epitope peptide and for neutralization of HIV-1. The results showed that the tip of the 2F5 CDR H3 loop, and specifically its hydrophobic nature, is required for 2F5-mediated neutralization of HIV-1 by means that appear to be independent both of gp41 affinity and of isolate-specific sensitivity to neutralization by 2F5.



The heavy and light chains of the 2F5 antibody were codon optimized for mammalian expression, synthesized, and transferred separately into the pVRC8400 (CMV/R) mammalian expression vector (5). Mutations within the 2F5 CDR H3 loop were analyzed for structural compatibility with neighboring residues and were then introduced into the 2F5 heavy-chain plasmid using standard site-directed mutagenesis techniques, implemented by ACGT, Inc., Chicago, IL. The wild-type and mutant 2F5 heavy- and light-chain plasmids were transiently transfected into 293 Freestyle cells using 293fectin (Invitrogen), and supernatants containing secreted IgGs were harvested 72 to 96 h posttransfection. The IgGs were purified by flowing the supernatants over a protein A-agarose column (Pierce), followed by elution with IgG elution buffer (Pierce).


A wild-type gp41 MPER peptide corresponding to residues 657 to 669 of gp41 (HxB2 numbering) linked to a C-terminal C9 tag was used and was comprised of the sequence EQELLELDKWASLGGTETSQVAPA (American Peptide).

Surface plasmon resonance.

Biacore 3000 (GE Healthcare) was used in all experiments, as previously described (46). 2F5 wild-type and mutant IgGs were coupled directly to Biacore CM5 chips at final densities of ~4,000 to 5,000 response units (RU). The gp41 MPER peptide was used as the analyte and flowed over at 2-fold serial dilutions ranging from 500 to 0.49 nM at a flow rate of 30 μl/min for 3 min, followed by injection of standard Biacore HEPES buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.01% P-20) for 3 min. Binding profiles were analyzed using either Biaevaluation software (GE Healthcare) or Scrubber version 2 (Biologic).

Virus neutralization assays.

A single-cycle infectivity assay using Env-pseudotyped virus and Tzm-bl target cells (NIH AIDS Research and Reference Reagent Program) was used to assess the neutralization capacities of the 2F5 variants, as previously described (18, 44). Env from the HIV-1 strains MN, HxB2, JR-FL, SC422661.8, RHPA4259.7, and TRO.11 were used, as were Env from the HIV-2 strain 7312a and the HIV-2-HIV-1 chimera 7312a-C1 (which were gifts from George M. Shaw) (6). Murine leukemia virus (MuLV) Env was used as a negative control.

Hydrophobicity analysis.

Analysis of the free energies of partitioning the 2F5 CDR H3 loop tip into octanol or a lipid bilayer interface was performed using MPex 3.1 software ( and reference 35). The analysis was performed on residues 100A to 100F of the 2F5 CDR H3 loop using the Totalizer function, with no end groups added and the ΔCONH value set at 0.

Statistical analysis.

Statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software Inc.) and Origin 7 (Originlab Corporation).


Predictive measures of hydrophobicity, as determined by scales of free energy for partitioning whole residues from water to a lipid bilayer interface, ΔGwif, or to octanol, ΔGoct (40, 43), were used to design mutations that would either disrupt or augment the predicted partitioning of the 2F5 CDR H3 tip (residues L100A to I100F) (Fig. (Fig.1).1). The amino acid serine, which is found midway on the ΔGwif and ΔGoct hydrophobicity scales, was chosen as a single or double substitution in order to achieve a gradual decrease in hydrophobicity (Fig. (Fig.1C1C and Table Table1).1). Conversely, tryptophan, which is the residue most favored to partition into the bilayer interface or octanol (40, 43), was used to augment the hydrophobicity of the loop (Fig. (Fig.1C1C and Table Table11).

2F5 CDR H3 partition free energies, binding constants to gp41 peptide, and neutralization IC50se

To ascertain the effect of the CDR H3 tip mutations on binding of 2F5 to the gp41 MPER, the affinities of the expressed 2F5 variants for a gp41 MPER peptide were determined using surface plasmon resonance. The 2F5 wild-type and mutant IgGs were coupled directly to the biosensor surface, and a gp41 MPER peptide comprised of residues 657 to 669 of gp41 (isolate HxB2 numbering) linked to a C-terminal C9 tag was used as analyte, at 2-fold serial dilutions ranging from 500 to 0.49 nM. As shown in Table Table11 (see Fig. S1 in the supplemental material), all of the mutants maintained nanomolar affinity to the gp41 peptide. In some cases, such as for mutants of I100F, there was a moderate reduction in affinity, likely due to minor contacts made by this residue with gp41.

The 2F5 mutants were then tested for neutralization of the laboratory-adapted strain HxB2, which is highly sensitive to wild-type 2F5. A single-cycle infectivity assay using Env-pseudotyped virus and Tzm-bl target cells was employed for this purpose, as previously described (18, 44). As shown in Fig. Fig.2,2, top, even single replacements of hydrophobic residues of the CDR H3 loop with serine were able to reduce the neutralization capacity of 2F5 against HxB2 by several orders of magnitude. The most noticeable single-mutation effect was observed for the F100BS mutation, which led to a more than 500-fold increase in the 50% inhibitory concentration (IC50) relative to wild-type 2F5 (Fig. (Fig.2,2, top, and Table Table1).1). The single mutations L100AS and I100FS likewise led to 100-fold increases in the IC50s, while the V100DS mutation increased the IC50 by about 15-fold. Introduction of double mutations to serine at the same residue locations, furthermore, led to complete abrogation of 2F5-mediated neutralization of HxB2, with neutralization profiles indistinguishable from those of the negative control mouse 1D4 anti-rhodopsin antibody (20) (Fig. (Fig.2,2, middle, and Table Table1).1). Thus, modification of residues at the tip of the CDR H3 loop can reduce and completely disrupt 2F5 neutralization, despite maintaining nanomolar affinity for gp41.

FIG. 2.
Neutralization of HxB2 by CDR H3 mutants of antibody 2F5. (Top) Neutralization profiles of 2F5 variants with single mutations to serine. Single serine substitutions resulted in a 15- to 500-fold reduction in neutralization potency. (Middle) Neutralization ...

Since these findings could be the result either of a disruption of specific protein contacts made by these residues or of a disruption of nonspecific interactions mediated by their chemical nature, mutation of the same CDR H3 residues to tryptophan was undertaken. Substitutions to tryptophan served two purposes: first, they augmented the hydrophobicity and predicted favorability of free energies of transfer from water to a bilayer interface or to octanol (ΔGwif and ΔGoct), and second, the bulky nature of the tryptophan side chain had the potential to disrupt protein-protein contacts, should they exist. When tested for neutralization of HxB2, the tryptophan mutants were either commensurate with 2F5 wild-type neutralization, such as L100AW and F100BW, or were even more potent, such as V100DW and the double mutant L100AW V100DW, both of which showed an ~10-fold decrease in the neutralization IC50 relative to wild-type 2F5 (Fig. (Fig.2,2, bottom).

To rule out the possibility that these results were specific to the HxB2 isolate, the 2F5 mutants were tested for neutralization of a panel of tier 1 and tier 2 HIV-1 isolates, ranging from highly sensitive strains, such as MN, to more resistant ones, such as JR-FL (17), and also to an HIV-2-HIV-1 2F5 epitope chimera, 7312a-C1 (6). As shown in Table Table1,1, single mutations that decreased the hydrophobicity of the 2F5 CDR H3 loop led to decreases in neutralization potency, and double mutations that decreased hydrophobicity completely abrogated neutralization. Likewise, single and double mutations that increased the hydrophobicity of the loop led to increased neutralization potencies (Table (Table1).1). Meanwhile, neutralization of the Tro.11 isolate, which has a K665S point mutation in the core of the epitope, was virtually undetectable for all 2F5 mutants, similar to what was observed for viruses pseudotyped with MuLV and parental HIV-2 7312a Env, which were used as negative controls.

To determine if there was a statistical relationship between the hydrophobicity of the 2F5 CDR H3 loop and the neutralization capacity of the antibody, neutralization IC50s of the 2F5 mutants were plotted against the estimated free energies of transfer, ΔGwif, of the mutant CDR H3s for each of the strains tested. As shown in Fig. Fig.3A3A and Table Table2,2, with the exception of RHPA-4259, statistically significant linear relationships were observed between neutralization IC50s and CDR H3 ΔGwif, with P values ranging from 0.0012 for HxB2 to 0.018 for JR-FL. Linear relationships with even more stringent P values were observed between neutralization IC50s and the predicted free energy of transfer to octanol, ΔGoct (see Fig. S2A and Table S1 in the supplemental material). Associations between the neutralization IC50s and the affinities of the various 2F5 mutants for gp41 MPER peptide were also examined. As shown in Fig. Fig.3B3B and Table Table2,2, despite the fact that the fits appear to be largely driven by the KD (dissociation constant) of the I100FS 2F5 mutant for gp41 (which makes minor contacts with the peptide), no statistically significant relationships were observed between the neutralization IC50s of the 2F5 CDR H3 variants and their affinities for the gp41 MPER peptide.

FIG. 3.
Relationship between 2F5-mediated HIV-1 neutralization and the hydrophobicity of its CDR H3 loop. (A) 2F5 variant neutralization IC50s plotted against the calculated ΔGwif of the 2F5 CDR H3 loop for each virus strain tested. Linear regressions ...
Linear correlation statistics of neutralization IC50 and ΔΔGN versus ΔGwif and KD gp41 peptidea

Though the overall levels of the IC50s varied per strain tested, likely a reflection of the strain sensitivity to 2F5 itself, the mutations appeared to exhibit similar effects on neutralization regardless of the strain used. Specifically, no significant differences were observed in the slopes of the linear fits of the neutralization IC50s versus ΔGwif or versus ΔGoct, unlike their y intercepts, which did display significant differences (Fig. (Fig.3A3A and Table Table2;2; see Fig. S2A and Table S1 in the supplemental material). The similarities in the slopes of the regressions across all strains suggested that the neutralization effects mediated by 2F5 CDR H3 loop hydrophobicity were largely independent of strain sensitivity to 2F5.

The free energy of antibody neutralization (ΔGN) can be viewed as a sum of free energies that contribute to its functional interactions with a specific strain of virus (13). It can also be viewed as a binding association of an antibody “n” with the virion: ΔGN = −RT ln K(n), where K(n) is defined as IC50/f(n) and f(n) is a function that accounts for variables such as strain sensitivity and assay used. Although an absolute value for this free energy requires the definition of f(n), if f(n1) can be approximated to equal f(n2) for two variants n1 and n2 of the same antibody neutralizing the same strain of virus, a relative free energy, ΔΔGN, can be obtained (13). In the case of 2F5, we sought to verify how the free energy of partitioning the CDR H3 loop from water to a lipid bilayer interface, ΔGwif, or to octanol, ΔGoct, correlated with the relative free energy of neutralization (ΔΔGN = ΔG2F5wtN − ΔG2F5mutN). Linear models were used to fit curves of ΔΔGN versus ΔGwif (Fig. (Fig.3C)3C) or ΔGoct (see Fig. S2C in the supplemental material) for each individual strain and for all strains together. The shared correlations obtained were statistically significant, with P < 0.0001, although end points at high hydrophobicity suggested linear fits might not be ideal (see below). Nonetheless, the results confirmed that the effects of ΔGwif and ΔGoct of the 2F5 CDR H3 loop on virus neutralization were largely independent of the virus strain, with the normalization relative to 2F5 wild type in the ΔΔGN calculation making the fits for all the strains virtually superimposable (Fig. (Fig.3C;3C; see Fig. S2C in the supplemental material). The shared slopes from these correlations were 1.3 and 1.2 for ΔGwif and ΔGoct, respectively. Thus, the calculated change in the free energy of partitioning the 2F5 CDR H3 loop translates almost directly into changes in neutralization. The 30% enhancement in neutralization, ΔΔGN, over the calculated partition free energy may reflect the planar positioning of these residues in the CDR H3 structure and/or their positioning induced by recognition of the gp41 protein component (42).

Though linear regressions provided a reasonable first approximation of the relationship between neutralization IC50s and the free energy of hydrophobic transfer of the tip of the 2F5 CDR H3 loop, we observed that beyond a certain threshold of loop hydrophobicity, the effects on neutralization appeared to level off. To account for this observation, a quadratic term was added to the linear regressions. This yielded better fits of the data, as judged by an extra sums-of-squares F test (Fig. (Fig.4A;4A; see Fig. S2B and Table S2 in the supplemental material) (15). Based on these quadratic models, IC50 minima were interpolated for each of the strains tested (see Table S3 in the supplemental material). On average, the interpolated minimum IC50s were approximately 0.96 log units or 9.2-fold lower than the corresponding experimental IC50s of wild-type 2F5 (see Table S3 in the supplemental material). Compared to the interpolated IC50s corresponding to a ΔGwif of 0, in which no transfer is predicted to occur, the mean minimum IC50s were approximately 5.1 log units or 13,000-fold lower than those predicted for a 2F5 variant with no capacity for hydrophobic transfer (see Table S3 in the supplemental material).

FIG. 4.
Threshold for effects of increased 2F5 CDR H3 loop hydrophobicity. To account for the observed leveling off of the effects of increased 2F5 CDR H3 loop hydrophobicity, quadratic fits were applied to the data shown in Fig. Fig.3.3. (A) 2F5 variant ...

A quadratic term was also added to the fits of the plots of the relative free energies of neutralization, ΔΔGN, versus the predicted free energies of transfer of the 2F5 CDR H3 loop to a bilayer interface, ΔGwif, or octanol, ΔGoct (Fig. (Fig.4B;4B; see Fig. S2D in the supplemental material). Shared quadratic fits of ΔΔGN versus ΔGwif and ΔGoct were also performed, and ΔΔGN minima were observed at 2F5 CDR H3 ΔGwif and ΔGoct values of −4.08 and −5.69 kcal/mol, with corresponding ΔΔGN values of −1.28 and −1.18 kcal/mol, respectively (Fig. (Fig.4C;4C; see Fig. S2D in the supplemental material).


The results presented in this study suggest that in addition to gp41 MPER binding, interactions mediated by the tip of the 2F5 CDR H3 loop are also required for 2F5-mediated neutralization of HIV-1. For the elements of 2F5-mediated neutralization described here, the free energy of 2F5-mediated virus neutralization, ΔGN, can thus be viewed as the sum of the free energy of 2F5 structure-specific recognition of gp41 combined with the free energy of transfer of its CDR H3 loop into a hydrophobic milieu (Fig. (Fig.5).5). While a number of possibilities exist to explain how the tip of the 2F5 CDR H3 mediates neutralization of HIV-1, the finding that mutations to tryptophan were tolerated at three separate locations within the CDR H3 loop, in some cases even augmenting 2F5-mediated neutralization, is consistent with the loop mediating nonspecific hydrophobic interactions. We surmise that such interactions are more likely to occur at a lipid bilayer interface than within a protein-protein interface, though 2F5 has been shown to tolerate a great deal of sequence variation at its interface with gp41 (9). The finding that correlations of hydrophobicity of the loop and neutralization capacity are largely independent of HIV-1 isolate sensitivity to 2F5, furthermore, suggests that the contacts mediated by the 2F5 CDR H3 loop are distinct from gp41 binding, at least in terms of elements of gp41 not conserved across all strains. The fact that this is true not only for HIV-1 isolates, but also for divergent simian immunodeficiency virus (SIV)-HIV-1 and HIV-2-HIV-1 chimeras, provides additional evidence that 2F5 CDR H3 interactions are not specific for HIV-1 envelope (6, 45).

FIG. 5.
The two-component mechanism of 2F5-mediated neutralization of HIV-1. The free energy of 2F5-mediated neutralization of HIV-1, ΔGN (diagonal), can be expressed as the sum of the free energy of its structure-specific recognition of its gp41 MPER ...

Although we did not experimentally measure nonspecific direct binding of 2F5 to lipids in our study, this type of experiment is relevant and has been performed elsewhere (1-3, 12). Notably, a recent study published while this article was under review shows that mutation of residues L100A and F100B of the 2F5 CDR H3 loop to alanine leads to a reduction in binding to lipid vesicles (2). These same mutations lead to what appear to be more pronounced reductions in 2F5-mediated neutralization. Because the hydrophobic tip of the CDR H3 represents only a small portion of the surface of antibody 2F5, it seems likely that the reduction in direct binding to lipid vesicles represents an averaging of the alteration in CDR H3 tip hydrophobicity relative to the entire 2F5 antibody. It thus seems reasonable to expect that the full effects of the mutations described herein for neutralization can likely be recapitulated in in vitro lipid binding assays only if the 2F5 CDR H3 loop is properly oriented relative to gp41 and the viral membrane, as it is in the virion/neutralization context, or when the gp41 MPER is presented in a proteoliposome context (11, 38). The difference between an oriented CDR H3 effect (large and significant) and an overall effect on direct biding to lipid vesicles (weak and less significant) may provide an explanation for the lack of 2F5 autoreactivity in in vivo studies (38); in the former case, the effect is amplified by the precise orientation of the CDR H3 to the viral membrane through binding to the protein component of the MPER epitope, while in the latter case, the effect is minimized by entropic effects and by averaging over the entire surface of the 2F5 antibody.

Finally, we note that a number of proteins have interfacial binding properties similar to those proposed for 2F5. Soluble phospholipases A2, for instance, show dramatic interfacial activation of catalytic activity and require attachment to membranes to appropriately position a substrate for catalysis (33, 34, 41). Such systems may allow additional insight into the neutralization mechanism of 2F5. The sizes and hydrophobicities of likely membrane attachment surfaces, for example, are decreased in neurotoxic phospholipases A2, which need to avoid nonspecific membrane interactions during diffusion to the neuronal synapse (16). Indeed, quadratic fits of our neutralization versus CDR H3 hydrophobicity data resulted in improved correlations and appeared to reveal a threshold beyond which additional hydrophobicity did not enhance neutralization. Overall, our findings have numerous implications for optimization of 2F5 potency and for recreating 2F5-like antibodies in vaccine settings. Variants of 2F5 with tryptophan substitutions are already ~10-fold more potent than the wild type in terms of neutralization, and future designs of 2F5-based vaccine immunogens may thus need to account not only for structure-specific recognition of gp41 but also for the hydrophobic interactions mediated by the tip of the 2F5 CDR H3 loop.

Supplementary Material

[Supplemental material]


We thank the members of the Structural Biology Section, VRC, and S. H. White for discussions and comments on the manuscript, W. R. Schief for comments on the CDR H3 mutants, G. M. Shaw for providing HIV-2 strains 7312a and 7312aC1, and J. Stuckey for assistance with figures and tables.

G.O., F.J.G., R.W., M.B.Z., J.R.M., and P.D.K. designed research; G.O., K.M., Y.Y., and Z.-Y.Y. performed research; Z.-Y.Y. and G.J.N. contributed new reagents; G.O., K.M., J.S., J.R.M., and P.D.K. analyzed data; G.O. and P.D.K. wrote the paper.

Support for this work was provided by the Intramural Research Program of the NIH, by a grant from the Bill and Melinda Gates Foundation Grand Challenges in Global Health Initiative, and by the International AIDS Vaccine Initiative (IAVI).


[down-pointing small open triangle]Published ahead of print on 30 December 2009.

Supplemental material for this article may be found at

The authors have paid a fee to allow immediate free access to this article.


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