Distinct allosteric remodeling of HIV-1 Env dynamics on virions by gp41-directed antibodies reveals two modes of neutralization
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eLife Assessment
This manuscript reports an important study in which the authors apply smFRET imaging to probe HIV-1 Env conformational dynamics in the presence of antibodies. Previous implementations of smFRET imaging of HIV-1 Env, which focus on gp120 conformation, have yielded limited information on antibodies that target gp41. Through the cutting-edge application of smFRET imaging, the study provides convincing insights into the mechanisms of action of relevant antibodies.
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Abstract
HIV-1 envelope glycoprotein (Env), a gp120–gp41 trimer, undergoes coordinated conformational changes that drive membrane fusion and allow immune evasion by transiently concealing neutralization-sensitive epitopes. Most broadly neutralizing antibodies (bNAbs) target gp120, whereas a distinct subset recognizes conserved gp41 regions, such as the fusion peptide and the membrane-proximal external region; however, their impact on Env dynamics and associated neutralization mechanisms remains unclear. Using bioorthogonal tagging for single-molecule FRET, we monitored real-time bNAb-induced conformational sampling of Env on intact virions. Most gp41-directed bNAbs allosterically stabilized the prefusion-closed (PC) state, whereas the bivalent 10E8.4/iMab favored both PC and CD4-bound open (predominant) states. Antibodies redistributed the conformational populations of Env with modest kinetic effects, preserving the sequential transition pathway. These findings reveal two modes of neutralization for gp41-directed antibodies, fixing the prefusion-closed conformation and opening it up – in both cases, with neutralization occurring via long-range allosteric control of Env dynamics.
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eLife Assessment
This manuscript reports an important study in which the authors apply smFRET imaging to probe HIV-1 Env conformational dynamics in the presence of antibodies. Previous implementations of smFRET imaging of HIV-1 Env, which focus on gp120 conformation, have yielded limited information on antibodies that target gp41. Through the cutting-edge application of smFRET imaging, the study provides convincing insights into the mechanisms of action of relevant antibodies.
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Reviewer #1 (Public review):
The authors have considered a panel of antibodies that target epitopes at the gp120/gp41 interface (8ANC195 and PGT151), the fusion peptide in the gp41 domain (VRC34), and the MPER region of gp41 (DH511.2_K3 and VRC42). They also investigate 10E8.4/iMab, which is an engineered bispecific antibody that targets the MPER and the CD4 receptor. On a technical note, they have applied a double amber codon-readthrough strategy to incorporate the non-natural TCO*A amino acid, which gets labeled through click chemistry. This approach should result in less disruption of the native Env structure as compared to the peptide insertion previously used for smFRET imaging of Env. Furthermore, previous implementations of smFRET imaging of HIV-1 Env, which focus on gp120 conformation, have yielded limited information on …
Reviewer #1 (Public review):
The authors have considered a panel of antibodies that target epitopes at the gp120/gp41 interface (8ANC195 and PGT151), the fusion peptide in the gp41 domain (VRC34), and the MPER region of gp41 (DH511.2_K3 and VRC42). They also investigate 10E8.4/iMab, which is an engineered bispecific antibody that targets the MPER and the CD4 receptor. On a technical note, they have applied a double amber codon-readthrough strategy to incorporate the non-natural TCO*A amino acid, which gets labeled through click chemistry. This approach should result in less disruption of the native Env structure as compared to the peptide insertion previously used for smFRET imaging of Env. Furthermore, previous implementations of smFRET imaging of HIV-1 Env, which focus on gp120 conformation, have yielded limited information on antibodies that target gp41. Altogether, through the cutting-edge application of smFRET imaging, the study provides novel insights into the mechanisms of action of interesting and clinically relevant antibodies.
In validating the functionality of the S401TAG/R542TAG Env, the authors performed infectivity assays and observed 20% infectivity as compared to wild-type (Figure S2A). However, the text equates this with "20% dual-amber suppression efficiency". This would benefit from some explanation. Why do the authors interpret infectivity as reporting on amber suppression efficiency, and not the functional cost of modifying Env, which is probably unavoidable? Or a combination of both? Is there data to suggest that 100% amber suppression would leave Env 100% functional? If so, this would be valuable to show. If not, the text should be clarified.
The authors state that the contour plots in Figure 2E reveal "dynamic sampling" of the observed FRET states. Strictly speaking, as presented, the contour plots (and FRET histograms) provide no information on dynamics per se. They indicate only the relative thermodynamic stabilities of the FRET states; transitions between states are a matter of interpretation. The TDPs, shown later in Figure 5A, nicely display the dynamics. More importantly, interpretation of the contour plots is challenging, as some seem to suggest an evolution toward lower FRET states. This is especially evident in Figures 2F and 3D, which suggest that the system evolves into a stable 0.1-FRET state (CO) after about 3 sec. Unless the authors want to conclude something from this, I would suggest that they consider removing the contour plots, since their interpretations are fully supported by the FRET histograms alone.
The data indicating that Env conformation is manipulated by 10E8.4/iMab is interesting. If I understand correctly, 10E8.4/iMab is an engineered antibody with one Fab targeting MPER and the second Fab targeting CD4. In the absence of CD4, could the difference between 10E8.4/iMab and the other MPER antibodies be due to 10E8.4/iMab being monovalent with respect to MPER binding?
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Reviewer #2 (Public review):
Summary:
In this paper, Xu and co-workers unveil two distinct modes of neutralisation by gp41-targeted broadly neutralizing antibodies on HIV-1 Env. So far, it was unclear as to how the mechanism of neutralisation occurred for this subset of neutralising antibodies (that can target the fusion peptide or the membrane proximal external region of the gp41 subunit). Thanks to single-molecule FRET, the authors show that the majority of broadly neutralizing antibodies stabilize the closed Env conformation (named State 1 since the original work by Munro and colleagues PMID: 25298114). Interestingly, the bivalent 10E8.4/iMab stabilized in turn a CD4-bound open state of Env. The two modes of neutralization described for these antibodies show previously unknown allosteric mechanisms that stabilize closed and open Env …
Reviewer #2 (Public review):
Summary:
In this paper, Xu and co-workers unveil two distinct modes of neutralisation by gp41-targeted broadly neutralizing antibodies on HIV-1 Env. So far, it was unclear as to how the mechanism of neutralisation occurred for this subset of neutralising antibodies (that can target the fusion peptide or the membrane proximal external region of the gp41 subunit). Thanks to single-molecule FRET, the authors show that the majority of broadly neutralizing antibodies stabilize the closed Env conformation (named State 1 since the original work by Munro and colleagues PMID: 25298114). Interestingly, the bivalent 10E8.4/iMab stabilized in turn a CD4-bound open state of Env. The two modes of neutralization described for these antibodies show previously unknown allosteric mechanisms that stabilize closed and open Env conformation, stressing the importance of Env conformational dynamics and its efficiency during the process of fusion.
Strengths:
The article is well-written, and the figures fully depict the data in a convincing way. The authors have used smFRET, which is now established in the field as a good tool to assess Env dynamics.
Weaknesses:
(1) The limited controls on how click chemistry affects Env (as labelled Env HIV virions were not evaluated).
(2) Photobleaching of donor and acceptor molecules occurs right after 10sec exposure.
(3) Other limitations are well described in the corresponding section.
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Author response:
eLife Assessment
This manuscript reports an important study in which the authors apply smFRET imaging to probe HIV-1 Env conformational dynamics in the presence of antibodies. Previous implementations of smFRET imaging of HIV-1 Env, which focus on gp120 conformation, have yielded limited information on antibodies that target gp41. Through the cutting-edge application of smFRET imaging, the study provides convincing insights into the mechanisms of action of relevant antibodies.
We appreciate this positive assessment and thank the reviewers for their time and constructive comments. We will make the following changes in the revised manuscript.
(1) Clarify the distinction between suppression efficiency and functional cost.
(2) Add controls: smFRET experiments in the presence of monovalent 10E8.4 and iMab individually and …
Author response:
eLife Assessment
This manuscript reports an important study in which the authors apply smFRET imaging to probe HIV-1 Env conformational dynamics in the presence of antibodies. Previous implementations of smFRET imaging of HIV-1 Env, which focus on gp120 conformation, have yielded limited information on antibodies that target gp41. Through the cutting-edge application of smFRET imaging, the study provides convincing insights into the mechanisms of action of relevant antibodies.
We appreciate this positive assessment and thank the reviewers for their time and constructive comments. We will make the following changes in the revised manuscript.
(1) Clarify the distinction between suppression efficiency and functional cost.
(2) Add controls: smFRET experiments in the presence of monovalent 10E8.4 and iMab individually and compare results with the bivalent 10E8.4/iMab that we currently have.
(3) Increase the number of repeats in neutralization experiments to reduce variability and, where feasible, perform infectivity and neutralization assays after click chemistry labeling.
(4) Add discussion on conformational populations probed by smFRET versus structural analyses, Env conformational heterogeneity, ligand effects, and how these approaches complement each other.
(5) Further clarify the assignments of multiple conformational states by smFRET, the heterogeneity of Env spikes and virion morphology by cryoET, and the focus of the current smFRET-focused storyline.
Please find below our provisional responses to the public reviews. We will provide detailed point-by-point responses upon submission of the revised manuscript.
Public Reviews:
Reviewer #1 (Public review):
The authors have considered a panel of antibodies that target epitopes at the gp120/gp41 interface (8ANC195 and PGT151), the fusion peptide in the gp41 domain (VRC34), and the MPER region of gp41 (DH511.2_K3 and VRC42). They also investigate 10E8.4/iMab, which is an engineered bispecific antibody that targets the MPER and the CD4 receptor. On a technical note, they have applied a double amber codon-readthrough strategy to incorporate the non-natural TCO*A amino acid, which gets labeled through click chemistry. This approach should result in less disruption of the native Env structure as compared to the peptide insertion previously used for smFRET imaging of Env. Furthermore, previous implementations of smFRET imaging of HIV-1 Env, which focus on gp120 conformation, have yielded limited information on antibodies that target gp41. Altogether, through the cutting-edge application of smFRET imaging, the study provides novel insights into the mechanisms of action of interesting and clinically relevant antibodies.
Thank you for the positive comments!
In validating the functionality of the S401TAG/R542TAG Env, the authors performed infectivity assays and observed 20% infectivity as compared to wild-type (Figure S2A). However, the text equates this with "20% dual-amber suppression efficiency". This would benefit from some explanation. Why do the authors interpret infectivity as reporting on amber suppression efficiency, and not the functional cost of modifying Env, which is probably unavoidable? Or a combination of both? Is there data to suggest that 100% amber suppression would leave Env 100% functional? If so, this would be valuable to show. If not, the text should be clarified.
We acknowledge this concern and will clarify the distinction between suppression efficiency and functional cost in the revision. The observed reduction in infectivity does not translate into the functional loss; instead, it more reflects the efficiency of suppression (one of the critical limitations of applying genetic code expansion in mammalian cells), as evidenced by reduced Env expression and incorporation on virions (Fig. 1B). In support of the preservation of Env functionality, tag-free and dual-ncAA-incorporated Env virions exhibited similar dose-dependent neutralization sensitivity against trimer-specific neutralizing antibodies (Fig.1D). We have previously discussed several limitations of amber suppression in mammalian cells combined with smFRET viral systems (PMID: 38232732; PMID: 40716060). In brief, orthogonal tRNA/aaRS pair–mediated amber suppression (reassigning/repurposing amber stop codons to non-canonical amino acids) of the introduced ambers in the target protein (Env in our case) must compete with the cellular translation system, particularly release factors that recognize amber codons and terminate translation. Readthrough of endogenous amber codons in virus-producing cells (in our case, HEK293T) can disrupt normal protein expression and virus production. Similarly, readthrough of preexisting amber codons in HIV-1 ORFs other than the targeted ambers in Env can disrupt virus assembly, which we addressed by generating an amber-free provirus (PMID: 38232732). Introducing two amber codons into Env further reduces efficiency, as dual suppression requires two sequential successful suppression events within the same Env molecule.
The authors state that the contour plots in Figure 2E reveal "dynamic sampling" of the observed FRET states. Strictly speaking, as presented, the contour plots (and FRET histograms) provide no information on dynamics per se. They indicate only the relative thermodynamic stabilities of the FRET states; transitions between states are a matter of interpretation. The TDPs, shown later in Figure 5A, nicely display the dynamics. More importantly, interpretation of the contour plots is challenging, as some seem to suggest an evolution toward lower FRET states. This is especially evident in Figures 2F and 3D, which suggest that the system evolves into a stable 0.1-FRET state (CO) after about 3 sec. Unless the authors want to conclude something from this, I would suggest that they consider removing the contour plots, since their interpretations are fully supported by the FRET histograms alone.
We agree and will remove the contour plots, as they do not add meaningful information beyond what the histograms show.
The data indicating that Env conformation is manipulated by 10E8.4/iMab is interesting. If I understand correctly, 10E8.4/iMab is an engineered antibody with one Fab targeting MPER and the second Fab targeting CD4. In the absence of CD4, could the difference between 10E8.4/iMab and the other MPER antibodies be due to 10E8.4/iMab being monovalent with respect to MPER binding?
We appreciate this question. To answer this, we will perform smFRET experiments in the presence of 10E8.4 and iMab individually and compare those with the bivalent 10E8.4/iMab.
Reviewer #2 (Public review):
Summary:
In this paper, Xu and co-workers unveil two distinct modes of neutralisation by gp41-targeted broadly neutralizing antibodies on HIV-1 Env. So far, it was unclear as to how the mechanism of neutralisation occurred for this subset of neutralising antibodies (that can target the fusion peptide or the membrane proximal external region of the gp41 subunit). Thanks to single-molecule FRET, the authors show that the majority of broadly neutralizing antibodies stabilize the closed Env conformation (named State 1 since the original work by Munro and colleagues PMID: 25298114). Interestingly, the bivalent 10E8.4/iMab stabilized in turn a CD4-bound open state of Env. The two modes of neutralization described for these antibodies show previously unknown allosteric mechanisms that stabilize closed and open Env conformation, stressing the importance of Env conformational dynamics and its efficiency during the process of fusion.
Strengths:
The article is well-written, and the figures fully depict the data in a convincing way. The authors have used smFRET, which is now established in the field as a good tool to assess Env dynamics.
We appreciate these positive comments!
Weaknesses:
(1) The limited controls on how click chemistry affects Env (as labelled Env HIV virions were not evaluated).
We agree. Our validation focused on ncAA-incorporated Env HIV-1 virions, but not the fluorescently labeled virions. To address this, we will increase the number of repeats in neutralization experiments to reduce variability and, where feasible, perform infectivity and neutralization assays after click chemistry labeling. We will attempt to do it. However, we expect that the additional handling time required for labeling and the centrifugation steps needed to remove free dyes, which can deform/disrupt viral membranes and degrade virions, together with the low dual-amber suppression efficiency, will make these experiments technically challenging as an additional layer of functional validation in live cells. On a related note, we have previously performed real-time tracking of single click-labeled Env virion internalization and trafficking in live cells (PMID: 38232732), supporting the retained functionality of click-chemistry-labeled Env.
(2) Photobleaching of donor and acceptor molecules occurs right after 10sec exposure.
We acknowledge this limitation and will include it in the corresponding section.
(3) Other limitations are well described in the corresponding section.
We appreciate this comment.
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