The viral entry process, mediated by the envelope (Env) gp41–gp120 complex [1–3], represents a major target for HIV-1 inhibition. Due to their conserved structure and specific functions, envelope proteins seem to display promising targets for a potential vaccine development against HIV-1 . So far, few broad neutralizing monoclonal antibodies (mAbs) have been isolated from HIV-1-infected patients [4,5]. 2F5 and 4E10 are two of these and recognize epitopes localized on the gp41 membrane-proximal external region (MPER) [4,5]. MPER is a tryptophan (Trp)-rich conserved region that is located near the viral membrane and seems to have important functions during the membrane fusion process [6,7]. Nevertheless, the efforts to obtain neutralizing antibodies using only MPER epitopes as antigens were proven to be difficult [8,9]. Due to the proximity of MPER to the viral membrane, lipid bilayers may be required for the development of antibodies against this region. In the native gp41, MPER corresponds to the extracellular region directly adjacent to the viral membrane  (see Fig. 1a). Peptides corresponding to this region are mainly unstructured in aqueous solution ; however, acquire defined α-helical secondary structure within lipid bilayers [12,13]. Recent studies have provided additional insights concerning MPER's structure, demonstrating that this region possesses two helices linked by a kink, with the N-terminal region more exposed to the aqueous medium and the C-terminal region inserted in a shallow position on lipid membranes . Additionally, it was demonstrated that MPER-derived peptides interact both with fluid (liquid disordered – ld) and cholesterol-enriched (liquid ordered – lo) membranes [15,16]. All of this demonstrates the importance of lipid membranes for the orientation and presentation of the MPER epitopes to neutralizing antibodies.
2F5 and 4E10 [7,17,18] have been studied as HIV-targeted antiretrovirals [19,20]. These mAbs possess structural adaptations to bind their epitopes in the lipid membrane interface [21–23]. For instance, they possess longer and more hydrophobic third complementarity-determining regions on the heavy chain (CDR H3) [23–25]. Furthermore, it has been reported that these Abs, mostly 4E10, interact with zwitterionic and anionic lipid membranes prior or during epitope recognition [26–39]. Recent studies reported that most of the residues in the CDR H3 region are not participating in epitope recognition, but are essential for membrane binding [11,40–44]. Modifications in some critical residues in the hydrophobic CDR H3 region can decrease lipid binding without affecting significantly the epitope recognition.
Despite the recent developments regarding epitope and membrane affinities, there is still a lot of information to be unraveled regarding 2F5 and 4E10 mode of action at the molecular level. Therefore, the aim of this work is to elucidate the neutralizing mAbs 2F5 and 4E10 molecular interaction with their epitopes at the membrane level. The membrane-binding properties of 2F5 and 4E10 were studied by the use of supported lipid bilayers (SLBs) as biomembrane models, with and without phase separation. Atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM) were used to evaluate the mAbs–membrane interactions, in the presence and absence of a MPER-derived peptide containing both mAbs epitopes. 2F5 is able to dock the MPER peptide on the membrane, whereas 4E10 is more intrusive and extracts the MPER from the lipid bilayer. The results obtained in this work contribute to the understanding of the molecular determinants underneath the differential efficacy of 2F5 and 4E10 targeting the HIV-1 MPER and distinguish their modes of action.
Materials and methods
MPER (NEQELLELDKWASLWNWFNITNWLWYIK-amide; >95% purity) and Fam-MPER (5,6-carboxyfluorescein-NEQELLELDKWASLWNWFNITNWLWYIK-amide; >92% purity) peptides were obtained from Bachem AG (Bubendorf, Switzerland). mAbs 2F5 and 4E10 were purchased from Polymun Scientific (Vienna, Austria). 1-Palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleyl-sn-glycro-3-phosphocholine (DOPC), chicken egg yolk sphingomyelin and cholesterol (Chol) were from Avanti Polar Lipids (Alabaster, Alabama, USA), whereas cholesteryl-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoate (FL-Bodipy-Chol) and 4,4-difluoro-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoate (TMR-Bodipy-Chol) were obtained from Molecular Probes (Eugene, Oregon, USA). Ten millimolar HEPES buffer pH 7.4, 150 mmol/l NaCl and 3 mmol/l NaN3 was used throughout the studies.
MPER and Fam-MPER stock solutions were prepared in dimethyl sulfoxide (DMSO) and diluted to desired concentrations with HEPES buffer. In order not to perturb the lipid organization , DMSO concentration was maintained below 0.14% (v/v) in the peptide samples throughout the experiments. 2F5 and 4E10 stock solutions (11 mg/ml) were stored in 2 mmol/l acetate buffer with 10% maltose and diluted to the desired concentrations using the HEPES buffer.
SLBs were prepared by deposition on freshly cleaved mica, in the presence of 1 mmol/l CaCl2, of POPC or DOPC: sphingomyelin: Chol (3: 3: 2) small unilamellar vesicles obtained by sonication, as described in . FL-Bodipy-Chol and TMR-Bodipy-Chol molar percentages in the lipid membranes were kept below 0.005 and 0.01% for POPC and DOPC: sphingomyelin: Chol (3: 3: 2), respectively.
AFM measurements were performed on a JPK Instruments Nanowizard BioAFM (Berlin, Germany) mounted on a Carl Zeiss laser scanning microscope (LSM) Meta 510 system (Jena, Germany) . Intermittent contact imaging was performed using uncoated silicon cantilevers CSC38 from MikroMasch (Tallinn, Estonia) with typical spring constants of 0.01–0.2 N/m. The scan rate was set to less than 1 Hz and the cantilever oscillation frequency between 10 and 20 kHz. The force applied on the sample was minimized by continuously adjusting the set point and gain during the imaging.
CLSM and line-scan fluorescence correlation spectroscopy (FCS) measurements were performed on the LSM Meta 510 system as described elsewhere . Highly sensitive fluorescence confocal microscopy measurements were conducted using a commercial Carl Zeiss ConfoCor3 system (Jena, Germany) with avalanche photodiode (APD) detectors. The 488-nm line of the argon-ion laser (to excite Fam-MPER and FL-Bodipy-Chol), the 543 nm helium–neon laser (to excite TMR-Bodipy-Chol) and a 40 × NA 1.2 UV–VIS–IR C Apochromat water-immersion objective were used in both setups. CLSM images were acquired with a 512 pixel × 512 pixel resolution and typical 6.4 μs per pixel scanning rate.
For all AFM and CLSM studies, the interactions of 2F5 or 4E10 mAbs (0.3–300 nmol/l) and MPER/Fam-MPER peptides (0.2–500 nmol/l) with POPC or DOPC: sphingomyelin: Chol (3: 3: 2) SLB (containing Bodipy-Chol) were followed upon successive additions of small volumes of stock solutions to the desired concentrations. The samples were allowed to incubate for 15 min at room temperature after each addition. Controls, with buffer instead of mAbs or MPER, were performed. When two components (mAbs and peptides) were being evaluated, the experiments were carried out as follows (see also Fig. 1b–d). Whenever a component B (mAbs or MPER, respectively) was added to membranes in which a component A (MPER or mAbs, respectively) was already present, the excess of free component A in solution was always rinsed with buffer to ensure the interaction of only component B with the membrane-bound component A. Images were obtained at least for three to five regions of the lipid bilayers. Statistical analysis was performed using repeated or one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test (interval of confidence 95%).
Confocal and atomic force microscopy studies
Interaction of the antibodies with lipid bilayers in the absence of the peptide epitopes
The interaction of both 2F5 and 4E10 (0.3–300 nmol/l) with SLB composed by POPC or the ternary mixture DOPC: sphingomyelin: Chol (3: 3: 2) deposited on freshly cleaved mica were analyzed by AFM and CLSM. POPC was used to mimic the ld bulk of an eukaryotic plasma membrane, whereas the DOPC: sphingomyelin: Chol mixture was used to mimic the coexistence of a ld phase with lo domains, enriched in Chol and sphingomyelin, usually termed lipid rafts [49,50]. The HIV-1 membrane that constitutes the enveloped is highly ordered and rich in cholesterol . Using AFM and CLSM, we can easily distinguish both ld and lo domains on SLB, as seen in Fig. 2a and d (see legend for more details).
In the presence of either 2F5 or 4E10, no extensive modifications in POPC bilayer thickness (data not shown – DNS) nor perturbations on the phase-separated domains of DOPC: sphingomyelin: Chol bilayers occur, as revealed by the AFM results (Fig. 2a–c). Absence of significant changes in domain morphology was also corroborated by CLSM (Fig. 2d–f). Moreover, by APD imaging, no significant modifications of the lipid fluorescence intensities were obtained (DNS). Additional FCS measurements to assess the dynamics of POPC phospholipids in the presence and absence of 2F5 or 4E10 did not demonstrate significant interference of either mAbs on the lipid diffusion coefficient (∼3 μm2/s, in agreement with ).
Nevertheless, the mAbs formed lipid-segregated aggregates within the SLB on mica. CLSM reveals that both mAbs form segregated regions with irregular forms and lacking fluorescence signal (Fig. 2e and f), preferentially on the more fluid membrane domains. Using AFM, irregular aggregates on the lipid bilayers were observed (Fig. 2b and c), which are clearly related to the darkest regions observed on the CLSM images. 4E10 aggregates tend to be higher than 2F5 aggregates. Typical heights of 3.2 ± 1.3 and 16.9 ± 6.1 nm were retrieved for 100 nmol/l 2F5 and 4E10, respectively (Fig. S1, supplementary data, http://links.lww.com/QAD/A106). As such extensive effects were not observed when buffer (control) or even MPER peptide (see following section) were added, the aggregates here observed can be interpreted as direct consequence of at least a transient interaction and destabilization of intact lipid membranes by 2F5 and 4E10.
Interaction of the antibodies with lipid bilayers with inserted peptide epitopes
The study of 2F5 and 4E10 effects on lipid bilayers with the prebound MPER was performed using an MPER-derived peptide (see Fig. 1a). This peptide (amino acids 656–683 by HXBc2 numbering) overlaps both 2F5 epitope core sequence (ELDKWA, amino acids 662–667) and 4E10 epitope core sequence (NWFNIT, amino acids 671–676) and is extended by 6 amino acid residues at both N and C terminals. This peptide mimics better the regions of gp41 presented to 2F5 and 4E10 compared with the epitope cores alone .
Initially, the membrane interaction properties of the nonlabeled MPER-derived peptide were evaluated. CLSM images of POPC (Fig. 3e) and DOPC: sphingomyelin: Chol (DNS) bilayers in the presence of MPER (0.2–200 nmol/l) did not present significant perturbations when compared with the controls (Fig. 3d). Furthermore, line scan FCS measurements confirmed that the peptide (200 nmol/l) did not induce major changes in the lipid dynamics and diffusion coefficients of POPC SLB (DNS). Additionally, AFM experiments did not reveal major interferences of the peptide on the membrane bilayers structure (DNS). Nevertheless, it is worth stressing that prolonged AFM imaging in the presence of MPER peptide could contribute to an increased lipid bilayer perturbation and the formation of holes (as seen in Fig. 3b), which are not observed in the controls (Fig. 3a).
The addition of 2F5 (DNS) and 4E10 (Fig. 3c and f) to SLB on mica with prebound MPER revealed results similar to those observed for the interaction of both mAbs with the membranes alone (Fig. 3b and e). No major perturbations on the bilayer thickness and phase-separated domains were detected by CLSM or AFM imaging, except the formation of the lipid-segregated aggregates of mAbs, already discussed.
Highly sensitive avalanche photodiode fluorescence microscopy studies
To unravel details on the interplay between mAbs, MPER-derived peptides and lipids, an APD-imaging microscopy approach was utilized, following the fluorescence emission of a 5,6-carboxyfluorescein labeled MPER-derived peptide (Fam-MPER) at the membrane level. It is known that the MPER C terminal is highly enriched in Trp residues, which are essential for the MPER membrane binding [13–15,52]. Therefore, to ensure minimal interference of the probe on the binding of the peptide to membranes, the peptide was labeled at the N terminus.
Fam-MPER demonstrated a preferential interaction toward the more fluid ld membrane domains. As shown in Fig. 4a, the peptide's fluorescence emission is more intense in POPC (ld) and less intense in the overall domains coexisting in DOPC: sphingomyelin: Chol (ld + lo) membranes. Nonetheless, if we only count for the fluorescence intensities on ld of phase-separated bilayers, the intensities are similar to those observed for the peptide interacting with POPC membranes. This indicates that the peptide presents a preferential and facilitated insertion in ld membranes compared with cholesterol-enriched lo domains. It is also to notice that no significant changes on the lipid TMR-Bodipy-Chol fluorescence intensity were reported as the peptide concentration was increased (DNS), indicating an absence of major lipid perturbation or significant lipid removal.
After confirming the interaction of Fam-MPER with POPC and DOPC: sphingomyelin: Chol SLB (Fig. 4a), we assessed the effect of 2F5 and 4E10 on the binding of peptide to membranes, as illustrated in Fig. 1b–d. Fam-MPER fluorescence co-localizes within the lipid-segregated mAbs aggregates on the surface of lipid bilayers (Fig. 4b). This is an unequivocal demonstration of the binding of both 4E10 and 2F5 to their epitopes.
Interaction of the peptide epitopes with lipid bilayers preincubated with antibodies
As membrane binding of both 2F5 and 4E10 has been proposed [4,34], we analyzed how a mAbs preincubation at the bilayer level would affect MPER binding (Fig. 1b). Preincubation of 2F5 and 4E10 on the SLB indeed interfered with the MPER membrane binding. Both mAbs decreased the binding of the peptide to POPC (Fig. 4c) and DOPC: sphingomyelin: Chol (Fig. 4d) systems. This decrease may be related to the capture of the peptide by the mAbs still in solution and/or to a weaker interaction of the MPER–mAbs complex with membranes, when compared with MPER alone. It has been reported that 2F5 does not bind or binds less to membranes than 4E10 [4,27]. The higher decrease in the peptide fluorescence at the bilayer level caused by 2F5, in comparison with 4E10, can be a consequence of this differential binding extent. 4E10 would have more affinity to the membrane, promoting therefore more MPER binding compared with 2F5.
Interaction of the antibodies with lipid bilayers with inserted peptide epitopes
Our second approach consisted in determining the effects of mAbs on MPER already bound to membranes (Fig. 1c). This experimental design is a better mimetic of what happens in a biological setting, where the mAbs binds to the MPER in a lipid environment .
As the peptide is in equilibrium between the aqueous-soluble and membrane-bound forms, performing successive additions of mAbs would perturb this equilibrium. This would remove peptide from the lipid bilayer, consequently decreasing the fluorescence intensity comparative to controls. Therefore, we performed successive additions of fixed concentration of mAbs (100 nmol/l) to ensure that the effects of the mAbs binding on the membrane-bound MPER are not masked by this equilibrium shift.
As can be seen in Fig. 5a, for POPC, and in Fig. 5b for DOPC: sphingomyelin: Chol membranes, we observed that 4E10 promoted an extensive decrease in the peptide's fluorescence intensity compared with the control. In contrast, in the presence of 2F5, no reduction in the fluorescence intensity was observed. This indicates that 4E10 promotes the extraction of MPER from the SLB, whereas 2F5 increases the docking of the peptide to the SLB. Furthermore, the addition of either mAbs did not cause any perturbation on the lipid signal and promoted the formation of lipid-segregated complexes (Fig. 5c), as expected.
Interaction of antibody–epitope complexes with lipid bilayers
To evaluate the lipid-binding properties of mAbs–MPER complexes, 2F5 or 4E10 were preincubated with MPER (Fig. 1d). The mAbs binding to Fam-MPER was confirmed by FCS. An approximate three-fold increase in Fam-MPER diffusion time (τD) was observed (unbound: τD = 43 ± 4 ms; bound to mAb: τD = 118 ± 15 ms), confirming mAbs–epitope binding. The complexes were then allowed to interact with POPC membranes. For both mAbs–MPER preformed complexes (Fig. 5d), a reduction in the peptide's fluorescence intensity at the membrane level was observed, when compared with MPER alone. This result suggests that the mAbs hinder MPER-membrane binding. Furthermore, a more pronounced reduction was observed for 4E10–MPER complexes. This indicates that 4E10–MPER complex has less affinity for membranes, compared with the 2F5–MPER complex, which is in agreement with the previous results.
2F5 and 4E10 are mAbs directed against the HIV-1 gp41 MPER. It has been reported that 2F5 holds a stronger binding toward its ELDKW epitope core compared with 4E10 with its NWF(D/N)IT epitope core . Nevertheless, concerning the membrane binding properties, the interaction of 2F5 is still a matter of debate [4,26]. For 4E10, the interaction with membranes is generally well accepted (reviewed in ). Most of the studies agree, however, that both mAbs would bind to MPER in a lipid-bound state [4,13,27], confirming the importance of membranes in their mode of action.
In this study, we evaluated the interaction of those mAbs with lipid membranes and with MPER-derived peptides containing their core epitopes. In terms of the membrane alterations caused by 2F5 and 4E10, we observed from the CLSM and AFM images that these mAbs do not cause significant perturbations on phase separation, lipid dynamics, bilayer thickness or lipid removal. Nevertheless, extensive aggregation of the mAbs, with lipid segregation, was imaged in the presence and absence of MPER. These results show the ability of the mAbs to be intrusive and induce confined local disorder in the membranes. Studies reporting the aggregation of mAbs on cholesterol-enriched membranes containing MPER  further illustrate the relevance of this observation, because they show the ability of the mAbs to perturb membranes locally exposing epitopes for interaction and to promote cooperative effects that may facilitate the interaction with gp41 because this protein is in trimeric state in the virus. It is worth stressing at this point that, relatively to the interaction of the MPER-derived peptide with SLB, no significant perturbations were detected. Moreover, using highly sensitive APD imaging, we observed that the peptide prefers ld domains in opposition to the cholesterol-enriched lo domains, which shows its tendency to co-localize in the mAbs-induced aggregates, favoring binding between both.
After evaluating the affinities of mAbs and MPER to lipid bilayers separately and more qualitatively, we were prompted to study more elaborated experimental designs to describe in more biologically accurate settings the mAbs–MPER combined membranes interactions. Using that approach, we could unravel distinct mode of actions for 2F5 and 4E10 on membrane-bound MPER. First of all, by preincubating the mAbs before the addition of the MPER-derived peptide, we retrieved less peptide binding toward membranes, as already described [36,37]. The differences obtained between 2F5 and 4E10 could be explained by the fact that 4E10 has a higher membrane affinity. Therefore, 4E10 would be more concentrated on the membrane, enabling more MPER to bind to membranes, in opposition to 2F5 [27,37]. Our second approach consisted in analyzing the effects of 2F5 and 4E10 on membrane-bound MPER, which is close to what actually happens on the viral surface. The mAbs presented the same effect either in POPC or DOPC: sphingomyelin: Chol membranes, demonstrating no clear membrane phase binding preferences for 2F5 and 4E10 in the presence of MPER peptide.
Nevertheless, significant differences relative to the mode of action of both mAbs were observed: it was clearly demonstrated that the binding of 2F5 causes peptide docking on the membrane, in opposition to 4E10 that promotes the peptide extraction (Fig. 5e). This may be due to the different in-depth location of both epitopes on the MPER. The ELDKW core (more N terminal) is more exposed to the aqueous medium than the NWF(D/N)IT core (more C terminal), which is more inserted and protected by the lipid bilayer [13,14]. The C-terminal region of MPER is highly hydrophobic and, therefore, crucial for lipid interactions. In contrast to 2F5, binding of 4E10 to its epitope hinders interaction of the C-terminal region of MPER to membranes. 2F5 can bind to its epitope at the surface of the membrane; at variance, 4E10 needs to interact more deeply with membranes, promoting the pulling and extraction of its epitope from an in-depth localization (Fig. 5e). In agreement, the 2F5–MPER complex has a higher membrane affinity than the 4E10–MPER complex (Fig. 5d). Other studies also reported different behaviors of 2F5 and 4E10 at the membrane level, supporting our observations [14,27,44,53,54]. For instance, in SPR experiments, higher dissociation rate constants were reported for 4E10 interacting with membranes in the presence of its epitope, in contrast to 2F5 where dissociation is almost irrelevant.
The results obtained in this work provide insights into the mode of action of 2F5 and 4E10 at the membrane level and are a valuable contribution to the understanding of their different biological efficacies. Although more efficient epitope binding for 2F5 was acknowledged  and lipid preferential reactivity was reported for 4E10 , the molecular reasons underlying these phenomena have so far remained uncertain. Those differences are related not only to the direct interaction with the lipids themselves but also with the specific microenvironments of the core epitopes in the lipid bilayer. While the core epitopes of both mAbs are very close in terms of gp41 amino-acid sequence, the short difference between them determines different degrees of exposure in the lipid environment, with 4E10 needing to be more intrusive in the perturbation of the interaction of its core epitope with the membrane lipids [14,53]. This may be the key to its improved neutralizing effect.
The authors are grateful for the support from the Max Planck Gesellschaft (Germany). Fundação para a Ciência e Tecnologia – Ministério da Ciência, Tecnologia e Ensino Superior (Portugal) is acknowledged for funding (SFRH/BD/39039/2007 grant to H.G.F. and projects PTDC/QUI-BIQ/104787/2008, PTDC/QUI/69937/2006 and REEQ/140/BIO/2005).
Experimental design: All.
Performing experiments: H.G.F. and S.C.
Data discussion: All.
Writing the paper: H.G.F. and M.C.
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