A morphological transformation in respiratory syncytial virus leads to enhanced complement deposition
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Evaluation Summary:
This paper is of broad interest to virologists, immunologists, and structural biologists as it provides new insights into how the biophysical properties of viruses shape the development of immune responses. Overall, the data presented support key claims of the paper, and the strengths and limitations of the approach are properly acknowledged. However, the working model and its implications could be clarified and extended.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
The complement system is a critical host defense against infection, playing a protective role that can also enhance disease if dysregulated. Although many consequences of complement activation during viral infection are well established, mechanisms that determine the extent to which viruses activate complement remain elusive. Here, we investigate complement activation by human respiratory syncytial virus (RSV), a filamentous respiratory pathogen that causes significant morbidity and mortality. By engineering a strain of RSV harboring tags on the surface glycoproteins F and G, we are able to monitor opsonization of single RSV particles using fluorescence microscopy. These experiments reveal an antigenic hierarchy, where antibodies that bind toward the apex of F in either the pre- or postfusion conformation activate the classical pathway whereas other antibodies do not. Additionally, we identify an important role for virus morphology in complement activation: as viral filaments age, they undergo a morphological transformation which lowers the threshold for complement deposition through changes in surface curvature. Collectively, these results identify antigenic and biophysical characteristics of virus particles that contribute to the formation of viral immune complexes, and suggest models for how these factors may shape disease severity and adaptive immune responses to RSV.
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Author Response:
Reviewer #1 (Public Review):
In this study, Kuppan, Mitrovich, and Vahey investigated the impact of antibody specificity and virus morphology on complement activation by human respiratory syncytial virus (RSV). By quantifying the deposition of components of the complement system on RSV particles using high-resolution fluorescence microscopy, they found that antibodies that bind towards the apex of the RSV F protein in either the pre- or post-fusion conformation activated complement most efficiently. Additionally, complement deposition was biased towards globular RSV particles, which were frequently enriched in F in the post-fusion conformation compared to filamentous particles on which F exists predominantly in the pre-fusion conformation.
Strengths:
- While many previous studies have examined the properties of …
Author Response:
Reviewer #1 (Public Review):
In this study, Kuppan, Mitrovich, and Vahey investigated the impact of antibody specificity and virus morphology on complement activation by human respiratory syncytial virus (RSV). By quantifying the deposition of components of the complement system on RSV particles using high-resolution fluorescence microscopy, they found that antibodies that bind towards the apex of the RSV F protein in either the pre- or post-fusion conformation activated complement most efficiently. Additionally, complement deposition was biased towards globular RSV particles, which were frequently enriched in F in the post-fusion conformation compared to filamentous particles on which F exists predominantly in the pre-fusion conformation.
Strengths:
- While many previous studies have examined the properties of antibodies that impact Fc-mediated effector functions, this study offers a conceptual advance in its demonstration that heterogeneity in virus particle morphology impacts complement activation. This novel finding will motivate further research on this topic both in the context of RSV and other viral infections.
- The use of site-specific labeling of viral proteins and high-resolution fluorescence microscopy represents a technical advance in monitoring interactions among different components of antiviral immune responses at the level of single virus particles.
- The paper is well written, data are clearly presented and support key claims of the paper with caveats appropriately acknowledged.
We appreciate the reviewer’s supportive comments. In our revised manuscript, we have focused on improving clarity regarding the minor weaknesses noted below.
Minor weaknesses:
Working models and their implications could be clarified and extended. Specifically:
- The finding that globular particles enriched in F proteins in the post-fusion conformation (Fig 3F) are dominant targets of complement activation as measured by C3 deposition by not only post-F- but also pre-F-specific antibodies (Fig 4B, left) is interesting. This is despite the fact that, as expected, pre-F antibodies bind less efficiently to globular particles (Fig 4B, right). How do the authors reconcile these observations, given that C3 deposition seems to be IgG-concentration-dependent (Fig 2E)?
The reviewer raises an excellent point: globular particles, which accumulate as the virus ages, contain more post-F and less pre-F than particles that have recently been shed from infected cells. These ‘aged’ particles nonetheless accumulate more C3 when incubated with pre-F mAbs than ‘younger’ particles, where the proportion of pre-F is higher. We attribute this to the lower surface curvature of globular particles: they accumulate more C3 in the presence of pre-F mAbs in spite of the reduced availability of pre-F epitopes. Figure 1C and 1F help to support this point. This data shows C3 deposition driven by different antibodies bound to particles enriched in either pre-F (Figure 1C) or post-F (Figure 1F). Importantly, for this experiment the conversion to post-F was driven in such a way that virion morphology is preserved (Figure 1E). In this case, we see a clear reduction in C3 deposition by pre-F mAbs on post-F particles (e.g. for CR9501, the percentage of C3-positive particles drops from 24% on pre-F virus to 6% on post-F-enriched virus). This demonstrates that, in the absence of other changes, conversion of pre-F to post-F reduces complement deposition by pre-F specific mAbs.
Similarly, the reviewer correctly points out that reduced levels of antibody binding lead to lower levels of C3 deposition (Figure 2E); however, as in Figure 1, this data is collected from particles with the same morphologies. Thus, in the absence of additional factors, reduction in mAbs bound to pre-F leads to a reduction in C3 deposition driven by these mAbs. The fact that we observe the opposite trend when changes in particle morphology accompany changes in post-F abundance points to an important role for particle shape in activation of the classical pathway.
- Based on data in Figure 5-figure supplement 2, the authors argue that "large viruses are poised to evade complement activation when they emerge from cells as highly-curved filaments, but become substantially more susceptible as they age or their morphology is physically disrupted." Could the increase in C3 deposition be alternatively explained by a higher density of F proteins on larger particles instead of / in addition to a larger potential decrease in membrane curvature?
We agree that the density of F on a virus – the number of F trimers per unit surface area - likely contributes to the efficiency of C3 deposition. In Figure 6 – figure supplement 2 (Figure 5 – figure supplement 2 in the original submission), we control for this potential effect by comparing viruses that have the same amount of F (as measured by fluorescence intensities of SrtA-labeled F) that are either in filamentous form or globular form (induced through osmotic swelling). The total amount of F per virus is preserved during swelling, and the membrane surface area will remain constant due to the limited ability of lipid bilayers to stretch7. As a result, the input material for these comparisons is the same in terms of F trimers per unit area, yet the C3:F ratio differs substantially. This leads us to conclude that the differences must be attributable to factors other than the density of F. Importantly, this does not mean that the amount of F per unit surface area does not matter for C3 deposition – only that this is not the effect we are observing here. We have added text (Line 299) to help clarify this point: “This effect is unlikely to arise due to changes in the abundance or density of F in the viral membrane, both of which will remain constant following swelling. Similarly, it does not appear to be purely related to size, as larger viral filaments show similar C3:F ratios as smaller viral filaments.”
- In the discussion, the authors acknowledge that the implications based on the findings are speculative. However, more clarity on the basis of these speculative models would be useful. For example, it is not clear how the findings directly inform the presented model of immunodominance hierarchies in infants.
We agree that this was unclear in the original manuscript. We have rewritten paragraph 4 of the Discussion to clarify how our results may contribute to the changes in immunodominance that have been observed in RSV between infants and adults.
Reviewer #2 (Public Review):
This is an intriguing study that investigates the role of virus particle morphology on the ability of the first few components in the complement pathway to bind and opsonize RSV virions. The authors use primarily fluorescence microscopy with fluorescently tagged F proteins and fluorescently labeled antibodies and complement proteins (C3 and C4). They observed that antibodies against different epitopes exhibited different abilities to induce C3 binding, with a trend reflecting positioning of IgG Fc more distal to the viral membrane resulting in better complement "activation". They also compared the ability of C3 to deposit on virus produced from cells +/- CD55, which inhibits opsonization, and showed knockout led to greater C3 binding, indicating a role for this complement "defense protein" in RSV opsonization. They also examined kinetics of complement protein deposition (probed by C4 binding) to globular vs filamentous particles, observing that deposition occurred more rapidly to non-filaments.
A better understanding of complement activation in response to viruses can lead to a more comprehensive understanding of the immune response to antigen both beneficial and detrimental, when dysfunctional, during infection as well as mechanisms of combating the viral infection. The study provides new mechanistic information for understanding the properties of an enveloped virus that can influence complement activation, at least in an in vitro setting. It remains to be determined whether these effects manifest in the considerably more complex setting of natural infection or even in the presence of a polyclonal antibody mixture.
The studies are elegantly designed and carefully executed with reasonable checks for reproducibility and controls, which is important especially in a relatively complex and heterogeneous experimental system.
We thank the reviewer for the insightful comments. We have revised the manuscript to help to clarify points of confusion and to address some of the technical points raised here.
Specific points:
- "Complement activation" involves much more than C3 or C4 binding. Better to use more specific terminology relating to the observable (i.e. fluorescently labeled complement component binding)
We agree with the reviewer. We have revised the manuscript throughout to make our language more accurate and precise.
- What is the rationalization for concentrations of antibodies used? What range was tested, and how dependent on antibody concentration were the observed complement deposition trends? How do they relate to physiological concentrations, and how would the presence of a more complex polyclonal response that is typically present (e.g. as the authors noted, the serum prior to antibody depletion already mediates complement activation) affect the complement activation trends? The neat, uniform display of Fc for monoclonals that were tested is likely to be quite garbled in more natural antibody response situations. This should be discussed.
We have added discussion of antibody concentrations and possible differences between monoclonal and polyclonal responses to the revised manuscript. Below, we address the specific questions raised here by the reviewer.
We chose to use antibody concentrations that are comparable to the concentrations of dominant clonotypes in post-vaccination serum1. Our goal in selecting relatively high antibody concentrations for our experiments was to focus on understanding the capacity of an antibody to drive complement deposition when it has reached maximum densities on RSV particles. This is discussed starting on Line 125 of Results, and in paragraph 2 of Discussion. Experiments testing a range of antibody concentrations would be valuable, but are likely to strongly reflect differences in the binding affinities of these antibodies, which have been characterized previously.
Although we have not performed titrations for each of the antibodies tested due to the large number of conditions needed and the limited throughput of our experimental approach, the manuscript does present a dilution series for CR9501, the IgG1 mAb with the greatest potency in driving C3 deposition among those tested here. This data (shown in Figure 3E & F in the revised manuscript) shows that as the amount of antibody added in solution decreases over a 16-fold range, C3 deposition decreases as well. The decrease in C3 deposition is roughly commensurate with the reduction in antibody binding, reaching levels that are just above background at an antibody concentration of ~0.6μg/ml (1:800 dilution). We think it is likely that other activating antibodies would show similar trends, while antibodies that do not activate the classical pathway at saturating concentrations would be unlikely to do so across a range of lower concentrations.
We agree with the reviewer that complement deposition driven by polyclonal antibodies is more complex than the monoclonal responses studied here. As discussed in paragraph 2 of our revised Discussion, one effect that polyclonal serum might have is to increase the density of Fcs on the virus by providing antibody mixtures that bind to multiple non-overlapping antigenic sites. We speculate that this would generally increase complement deposition, provided that sufficient antibodies are present that bind to productive antigenic sites (e.g. sites 0/ , II, and V).
Finally, we note that we observe a similar phenomenon where globular particles are preferentially opsonized with C3 in our experiments with polyclonal serum where IgG and IgM have not been depleted (Figure R1). The major limitation of this data – which is resolved by using monoclonal antibodies – is the difficulty of determining to what extent this bias arises due to the epitopes targeted by the polyclonal serum versus the intrinsic sensitivity of the virus particles.
Figure R1: RSV opsonized with polyclonal human serum. A similar bias towards globular particles (white dashed circles) is observed as in experiments with monoclonal antibodies.
- Are there artifacts or caveats resulting from immobilization of virus particles on the coverslips?
As pointed out by the reviewer, a few possible artifacts or caveats could arise due to the immobilization of viruses on coverslips. These include (1) spurious binding of C1 or other complement components to the immobilizing antibody (3D3); (2) reduced access to viral antigens as a result of immobilization; and (3) inhibition of antibody-induced viral aggregation. We are able to rule out issues associated with (1), because we do not see attachment of C1 or C3 to the coverslip (i.e. outside regions occupied by virus particles). This is consistent with the fact that the antibodies are immobilized on the surface via a C-terminal biotin attached to the heavy chain, which would limit access for C1 binding and prevent the formation of Fc hexamers.
Immobilization on coverslips could reduce the accessibility of a portion of the virus for binding by antibodies and complement proteins. This could effectively shield a portion of the viral surface from assembly of an activating complex, which we estimate requires ~35nm of clearance above the targeted epitope on F8. Importantly, the fraction of the viral surface area that would be shielded would vary for filaments and spheres; to determine if this could influence our results, we calculated the expected magnitude of this effect (Figure R2). To do this, we modeled the virus as being tethered to the surface via a 25nm linkage. This accounts for the length of the biotinylated PEG (~5-15nm for PEG2K, depending on the degree of extension), streptavidin (~5nm), and the anti-G antibody (~10-15nm including the biotinylated C-terminal linker). Although limited structural information is available for RSV G, the ~100 residue, heavily glycosylated region between the viral membrane and the 3D3 epitope likely extends above the height of F (~12nm). Our model assumes that a shell of thickness d surrounding the virus is necessary for antibody-C1 complexes to fit without clashing with the surface (this shell is shaded in gray in the schematic from Figure R2). Tracing the angles at which this shell clashes with the coverslip allows us to calculate the fraction of total surface area that is inaccessible for activation of the classical pathway. The results are plotted on the right side of Figure R2. The relative surface area accessible to a 35nm activating antibody-C1 complex differs between a filament and a sphere of equivalent surface area by about 15%. We conclude that this difference is modest compared to the ~5-fold difference in deposition kinetics we observe between viral filaments and spheres (Figure 4), or the 3- to 10-fold difference in relative C3 deposition we observe on larger filamentous particles after conversion to spheres (Figure 6 – figure supplement 2C).
Finally, by performing experiments on immobilized viruses, we eliminate the possibility for antibody-dependent particle aggregation. While this was necessary for us to get interpretable results, the formation of viral aggregates could affect the dynamics and extent of complement deposition. For example, activation of the classical pathway on one particle in an aggregate could spread to non-activating particles through a “bystander effect”, as has been reported in other contexts9. We are interested in this question and have begun preliminary experiments in this direction; however, we believe that a definitive answer is outside the scope of this current work. To alert readers to this consideration, we have added this to paragraph 2 of the revised Discussion (Line 359).
Figure R2: Estimating the surface accessibility of RSV particles bound to coverslips. Definition of variables: af: radius of cylindrical RSV filament; as: radius of spherical RSV particle of equivalent surface area (see Figure 6 – figure supplement 2A); d: distance needed above the viral surface to accommodate IgG-C1 activating complexes; h: height of viral surface above the coverslip; L: length of the viral filament.
- How is the "density of antigen" quantitated? What fraction of F or G is labeled? For fluorescence intensity measurements in general, how did the authors ensure their detection was in a linear sensitivity range for the detectors for the various fluorescent channels? Since quantitation of fluorescence intensities is important in this study, some discussion in methods would be valuable.
We have performed this important additional characterization of our fluorescence system and our overall labeling and quantification strategy to address these concerns. The results of this characterization are now included in two new figure supplements in the revised manuscript (Figure 1 – figure supplements 2 & 3).
- The authors also show that the particle morphology, whether globular or filamentous, as well as relative size and resulting apparent curvature, correlate with ability of C3 to bind. Some link to the abundance of post-fusion F (post-F) is examined and discussed, but I found the back and forth discussion between morphology, C3 binding, and post-F abundance to be confusing and in need of clarification and streamlining. Is there a mechanistic link between morphology changes and post-F level increases? Are the two linked or coincidental (for example does pre-F interaction with matrix help stabilize that conformation, and if lost lead to spontaneous conversion to post-F?). Please clarify.
Specifically, we have separated the discussion of pre-F versus post-F abundance and particle morphology into two different sections in Results, and we have rearranged Figures 4 and 5 (Figures 3 and 4 in the original submission) to improve clarity.
Regarding the question of whether changes in morphology and the pre-F to post-F conversion are coincidental or mechanistically linked: the answer is not entirely clear, although we have collected new data that suggests a connection. We first want to note that the two effects are at least partly separable: brief treatment with a low osmolarity solution causes particle shape to change while preserving pre-F (Figure 6A & B), whereas treating with an osmotically balanced solution with low ionic strength converts pre-F to post-F without affecting virus shape (Figure 1E). However, we were motivated by the reviewer’s questions to look into this further. To determine if the change in viral shape may serve to destabilize the pre-F conformation over time, we compared the relative amounts of pre-F and post-F present in particles that were osmotically swollen to those that were not at 0h and at 24h. In these experiments, particles were swollen using a brief (~1 minute) exposure to low osmolarity conditions before returning them to PBS (Figure R3, left). As expected, we observe no immediate change in pre-F abundance following the brief osmotic shock (Figure R3, right: 0h time point), consistent with Figure 6B. After incubating the particles an additional 24h at 37oC, the post-F-to-pre-F ratio is ~3.5-fold higher in osmotically-swollen particles than in those where filamentous morphology was initially preserved (Figure R3, right: 24h time point). This supports the reviewer’s suggestion that interactions with the matrix may help to stabilize F in the prefusion conformation, since the conversion to post-F is faster when this interaction is disrupted. Whether or not this has any relevance for RSV entry into cells remains to be determined; however, it is worth noting that we observed no clear loss or gain of infectivity in RSV particles following osmotic swelling (Figure 6 – figure supplement 1A). Since this result may be of interest to readers, we have included this new data in Figure 6 – figure supplement 1B, and it is discussed briefly in Results (Line 250).
Figure R3: Determining stability of pre-F following matrix detachment. Left: Experimental design. Right: Comparison of pre-F stability on untreated particles (gray) and particles subjected to brief osmotic swelling (magenta). Distributions show the ratio of post-F (ADI-14353) to pre-F (5C4) intensities per particle, combined for four biological replicates, sampled at 0h (immediately after swelling) and after an additional incubation at 37oC for 24h. Black points show median values for each individual replicate. P-values are determined from a two-sample T test.
- Since their conclusion is that curvature of the virus surface is a major influence on the ability of complement proteins to bind, I feel that some effort at modeling this effect based upon known structures is warranted. One might also anticipate then that there would be some epitope-dependent effect as a result of changes in curvature that may lead to an exaggeration of the epitope-specific effects for more highly curved particles perhaps than those with lower curvature? Is this true?
The reviewer raises two excellent points: that it may be possible to gain insight into the mechanisms through which curvature dictates C1 binding and other aspects of complement activation through structural modeling, and that such a model may help to identify specific epitope effects that could contribute to curvature dependence.
We developed simulations based on the geometry of RSV, F, and hexameric IgG to try to better understand how curvature may influence initiation of the classical pathway. This model is described in the Methods section (Modeling IgG hexamers on curved surfaces), and the results are discussed in the final two paragraphs of the Results section. In addition, we have included a new figure (Figure 7) to summarize the model’s predictions. This model corroborates the curvature sensitivity of IgG hexamer formation and suggests a possible intuitive explanation for our findings: high curvature effectively increases the distance between epitopes that sit high above the viral membrane, decreasing the likelihood of hexamer formation (Figure 7D). Regarding epitope specific effects, this model suggests that the further the epitope is above the viral membrane, the greater the effect that decreasing curvature will have. However, we find that epitopes closer to the membrane (e.g. those bound by 101F or ADI-19425) are overall very inefficient at activating the classical pathway, potentially due to steric obstruction of the formation of IgG hexamers. Thus, there may be an inherent tradeoff between overcoming steric obstruction (by binding to epitopes distal to the membrane) and sensitivity to surface curvature.
It is important to note that this model is reductionist and does not include detailed structural information. Additional factors may be important for considering epitope-specific effects. For example, antibodies that bind equatorially on F (e.g. ADI-19425, which binds to antigenic site III), show minimal complement deposition in our experiments. However, particles whose curvature approaches the diameter of hexameric IgG or IgM (~20nm) may display these epitopes in a manner that is more accessible. If the curvature necessary to observe such an effect falls outside of the biologically accessible range, it would not be observable in our experiments. Nonetheless, it is possible that a different set of antibodies may drive complement deposition on highly-curved nanoparticle vaccines that are in development10. We have added this important point to the second paragraph of the Discussion.
- Line 265: it would be useful to confirm the increase C1 binding as a function of morphology as was done for antibody-angle of binding experiments.
We believe that this data is shown in Figure 6B (Figure 5B in the original manuscript).
Reviewer #3 (Public Review):
Overall the manuscript is clearly written and the data are displayed well, with helpful diagrams in the figures to illustrate assays and RSV F epitopes. The engineering of the RSV strain to include a fluorescent reporter and tags on F and G that serve as substrates for fluorophore attachment is impressive and is a strength. The RSV literature is well cited and the interpretation of the results is consistent with structure/function data on RSV F and its interaction with antibodies. This reviewer is not an expert on the experiments performed in this manuscript, but they appear to be rigorously performed with appropriate controls. As such, the conclusions are justified by the data. One weakness is the extent to which the results regarding virion morphology are biologically relevant. Non-filamentous forms of the virion are generally obtained only in vitro as a result of virion purification or biochemical treatment. However, these results may be relevant for certain vaccine candidates, including the failed formalin-inactivated RSV vaccine that was evaluated in the late 1960s and caused vaccine-enhanced disease upon natural RSV infection.
Thank you for these suggestions, which have helped us to better place our results regarding RSV morphology in the context of prior work. We agree with the reviewer that non-filamentous RSV particles are commonly obtained in vitro, and that this morphology does not reflect the structure of the virus as it is budding from infected cells. Our work has characterized the transition from filament to globular / amorphous form, with the finding that it can occur rapidly upon physical or chemical perturbations, as well as more gradually during natural aging: i.e. in the absence of handling or purification. We are also able to detect globular particles accumulating in cultured A549 cells, where no handling has occurred prior to observation (Figure 5 – figure supplement 1). While we do not currently know how well this reflects the tendency of RSV to undergo conversion from filament to sphere in vivo, we propose that it is plausible that such a transformation could occur. To distinguish between what we demonstrate and what we speculate, we write (Line 401): “Although more work is needed to understand the prevalence of globular particles during in vivo infection, our observations that these particles accumulate over time through the conversion of viral filaments – even under normal cell culture conditions - suggest that their presence in vivo is feasible, where the physical and chemical environment would be considerably harsher and more complex.”
We agree with the reviewer that our results may have relevance towards understanding the failed formalin-inactivated vaccine trial. We have added this to paragraph 5 of the Discussion section.
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Evaluation Summary:
This paper is of broad interest to virologists, immunologists, and structural biologists as it provides new insights into how the biophysical properties of viruses shape the development of immune responses. Overall, the data presented support key claims of the paper, and the strengths and limitations of the approach are properly acknowledged. However, the working model and its implications could be clarified and extended.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
In this study, Kuppan, Mitrovich, and Vahey investigated the impact of antibody specificity and virus morphology on complement activation by human respiratory syncytial virus (RSV). By quantifying the deposition of components of the complement system on RSV particles using high-resolution fluorescence microscopy, they found that antibodies that bind towards the apex of the RSV F protein in either the pre- or post-fusion conformation activated complement most efficiently. Additionally, complement deposition was biased towards globular RSV particles, which were frequently enriched in F in the post-fusion conformation compared to filamentous particles on which F exists predominantly in the pre-fusion conformation.
Strengths:
While many previous studies have examined the properties of antibodies that impact …
Reviewer #1 (Public Review):
In this study, Kuppan, Mitrovich, and Vahey investigated the impact of antibody specificity and virus morphology on complement activation by human respiratory syncytial virus (RSV). By quantifying the deposition of components of the complement system on RSV particles using high-resolution fluorescence microscopy, they found that antibodies that bind towards the apex of the RSV F protein in either the pre- or post-fusion conformation activated complement most efficiently. Additionally, complement deposition was biased towards globular RSV particles, which were frequently enriched in F in the post-fusion conformation compared to filamentous particles on which F exists predominantly in the pre-fusion conformation.
Strengths:
While many previous studies have examined the properties of antibodies that impact Fc-mediated effector functions, this study offers a conceptual advance in its demonstration that heterogeneity in virus particle morphology impacts complement activation. This novel finding will motivate further research on this topic both in the context of RSV and other viral infections.
The use of site-specific labeling of viral proteins and high-resolution fluorescence microscopy represents a technical advance in monitoring interactions among different components of antiviral immune responses at the level of single virus particles.
The paper is well written, data are clearly presented and support key claims of the paper with caveats appropriately acknowledged.
Minor weaknesses:
Working models and their implications could be clarified and extended. Specifically:The finding that globular particles enriched in F proteins in the post-fusion conformation (Fig 3F) are dominant targets of complement activation as measured by C3 deposition by not only post-F- but also pre-F-specific antibodies (Fig 4B, left) is interesting. This is despite the fact that, as expected, pre-F antibodies bind less efficiently to globular particles (Fig 4B, right). How do the authors reconcile these observations, given that C3 deposition seems to be IgG-concentration-dependent (Fig 2E)?
Based on data in Figure 5-figure supplement 2, the authors argue that "large viruses are poised to evade complement activation when they emerge from cells as highly-curved filaments, but become substantially more susceptible as they age or their morphology is physically disrupted." Could the increase in C3 deposition be alternatively explained by a higher density of F proteins on larger particles instead of / in addition to a larger potential decrease in membrane curvature?
In the discussion, the authors acknowledge that the implications based on the findings are speculative. However, more clarity on the basis of these speculative models would be useful. For example, it is not clear how the findings directly inform the presented model of immunodominance hierarchies in infants.
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Reviewer #2 (Public Review):
This is an intriguing study that investigates the role of virus particle morphology on the ability of the first few components in the complement pathway to bind and opsonize RSV virions. The authors use primarily fluorescence microscopy with fluorescently tagged F proteins and fluorescently labeled antibodies and complement proteins (C3 and C4). They observed that antibodies against different epitopes exhibited different abilities to induce C3 binding, with a trend reflecting positioning of IgG Fc more distal to the viral membrane resulting in better complement "activation". They also compared the ability of C3 to deposit on virus produced from cells +/- CD55, which inhibits opsonization, and showed knockout led to greater C3 binding, indicating a role for this complement "defense protein" in RSV …
Reviewer #2 (Public Review):
This is an intriguing study that investigates the role of virus particle morphology on the ability of the first few components in the complement pathway to bind and opsonize RSV virions. The authors use primarily fluorescence microscopy with fluorescently tagged F proteins and fluorescently labeled antibodies and complement proteins (C3 and C4). They observed that antibodies against different epitopes exhibited different abilities to induce C3 binding, with a trend reflecting positioning of IgG Fc more distal to the viral membrane resulting in better complement "activation". They also compared the ability of C3 to deposit on virus produced from cells +/- CD55, which inhibits opsonization, and showed knockout led to greater C3 binding, indicating a role for this complement "defense protein" in RSV opsonization. They also examined kinetics of complement protein deposition (probed by C4 binding) to globular vs filamentous particles, observing that deposition occurred more rapidly to non-filaments.
A better understanding of complement activation in response to viruses can lead to a more comprehensive understanding of the immune response to antigen both beneficial and detrimental, when dysfunctional, during infection as well as mechanisms of combating the viral infection. The study provides new mechanistic information for understanding the properties of an enveloped virus that can influence complement activation, at least in an in vitro setting. It remains to be determined whether these effects manifest in the considerably more complex setting of natural infection or even in the presence of a polyclonal antibody mixture.
The studies are elegantly designed and carefully executed with reasonable checks for reproducibility and controls, which is important especially in a relatively complex and heterogeneous experimental system.
Specific points:
"Complement activation" involves much more than C3 or C4 binding. Better to use more specific terminology relating to the observable (i.e. fluorescently labeled complement component binding)
What is the rationalization for concentrations of antibodies used? What range was tested, and how dependent on antibody concentration were the observed complement deposition trends? How do they relate to physiological concentrations, and how would the presence of a more complex polyclonal response that is typically present (e.g. as the authors noted, the serum prior to antibody depletion already mediates complement activation) affect the complement activation trends? The neat, uniform display of Fc for monoclonals that were tested is likely to be quite garbled in more natural antibody response situations. This should be discussed.
Are there artifacts or caveats resulting from immobilization of virus particles on the coverslips?
How is the "density of antigen" quantitated? What fraction of F or G is labeled? For fluorescence intensity measurements in general, how did the authors ensure their detection was in a linear sensitivity range for the detectors for the various fluorescent channels? Since quantitation of fluorescence intensities is important in this study, some discussion in methods would be valuable.
The authors also show that the particle morphology, whether globular or filamentous, as well as relative size and resulting apparent curvature, correlate with ability of C3 to bind. Some link to the abundance of post-fusion F (post-F) is examined and discussed, but I found the back and forth discussion between morphology, C3 binding, and post-F abundance to be confusing and in need of clarification and streamlining. Is there a mechanistic link between morphology changes and post-F level increases? Are the two linked or coincidental (for example does pre-F interaction with matrix help stabilize that conformation, and if lost lead to spontaneous conversion to post-F?). Please clarify.
Since their conclusion is that curvature of the virus surface is a major influence on the ability of complement proteins to bind, I feel that some effort at modeling this effect based upon known structures is warranted. One might also anticipate then that there would be some epitope-dependent effect as a result of changes in curvature that may lead to an exaggeration of the epitope-specific effects for more highly curved particles perhaps than those with lower curvature? Is this true?
Line 265: it would be useful to confirm the increase C1 binding as a function of morphology as was done for antibody-angle of binding experiments.
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Reviewer #3 (Public Review):
Overall the manuscript is clearly written and the data are displayed well, with helpful diagrams in the figures to illustrate assays and RSV F epitopes. The engineering of the RSV strain to include a fluorescent reporter and tags on F and G that serve as substrates for fluorophore attachment is impressive and is a strength. The RSV literature is well cited and the interpretation of the results is consistent with structure/function data on RSV F and its interaction with antibodies. This reviewer is not an expert on the experiments performed in this manuscript, but they appear to be rigorously performed with appropriate controls. As such, the conclusions are justified by the data. One weakness is the extent to which the results regarding virion morphology are biologically relevant. Non-filamentous forms of the …
Reviewer #3 (Public Review):
Overall the manuscript is clearly written and the data are displayed well, with helpful diagrams in the figures to illustrate assays and RSV F epitopes. The engineering of the RSV strain to include a fluorescent reporter and tags on F and G that serve as substrates for fluorophore attachment is impressive and is a strength. The RSV literature is well cited and the interpretation of the results is consistent with structure/function data on RSV F and its interaction with antibodies. This reviewer is not an expert on the experiments performed in this manuscript, but they appear to be rigorously performed with appropriate controls. As such, the conclusions are justified by the data. One weakness is the extent to which the results regarding virion morphology are biologically relevant. Non-filamentous forms of the virion are generally obtained only in vitro as a result of virion purification or biochemical treatment. However, these results may be relevant for certain vaccine candidates, including the failed formalin-inactivated RSV vaccine that was evaluated in the late 1960s and caused vaccine-enhanced disease upon natural RSV infection.
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