Cryo-ET of Env on intact HIV virions reveals structural variation and positioning on the Gag lattice.

HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here, we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1-Å sub-tomogram-averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers, and a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.

How a broadly neutralizing antibody grapples with antigenic and conformational diversity in dengue virus.

In this issue of Cell, two studies apply powerful structural approaches to probe the modes of interaction between a broadly neutralizing antibody and a conserved epitope found on four dengue virus serotypes and Zika virus. These findings offer new insights into how a broadly neutralizing antibody surmounts antigenic and conformational variation.

Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2.

A safe, effective, and scalable vaccine is needed to halt the ongoing SARS-CoV-2 pandemic. We describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 SARS-CoV-2 spike receptor-binding domains (RBDs) in a highly immunogenic array and induce neutralizing antibody titers 10-fold higher than the prefusion-stabilized spike despite a 5-fold lower dose. Antibodies elicited by the RBD nanoparticles target multiple distinct epitopes, suggesting they may not be easily susceptible to escape mutations, and exhibit a lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the assembled nanoparticles suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms and have launched cGMP manufacturing efforts to advance the SARS-CoV-2-RBD nanoparticle vaccine into the clinic.

Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a worldwide public health concern for which no vaccine is available. Elucidation of the prefusion structure of the RSV F glycoprotein and its identification as the main target of neutralizing antibodies have provided new opportunities for development of an effective vaccine. Here, we describe the structure-based design of a self-assembling protein nanoparticle presenting a prefusion-stabilized variant of the F glycoprotein trimer (DS-Cav1) in a repetitive array on the nanoparticle exterior. The two-component nature of the nanoparticle scaffold enabled the production of highly ordered, monodisperse immunogens that display DS-Cav1 at controllable density. In mice and nonhuman primates, the full-valency nanoparticle immunogen displaying 20 DS-Cav1 trimers induced neutralizing antibody responses ∼10-fold higher than trimeric DS-Cav1. These results motivate continued development of this promising nanoparticle RSV vaccine candidate and establish computationally designed two-component nanoparticles as a robust and customizable platform for structure-based vaccine design.

Single-Molecule Analysis of a Viral Fusion Protein Illuminates a Fusion-Active Intermediate State.

The influenza virus hemagglutinin (HA) fusion glycoprotein mediates viral entry into host cells through its receptor binding and membrane fusion activities. In this issue of Cell, Das et al. use single-molecule Förster resonance energy transfer (smFRET) to monitor HA conformational dynamics. Their study reveals this prototypical class I fusion protein to be a highly dynamic molecule capable of reversibly sampling multiple states, including on-pathway fusion intermediates between pre-fusion and post-fusion endpoints. These findings challenge long-held ideas for how HA functions and move the field closer to obtaining a mechanistic understanding of how class I fusion proteins mediate membrane fusion.

Vaccination with a structure-based stabilized version of malarial antigen Pfs48/45 elicits ultra-potent transmission-blocking antibody responses.

Malaria transmission-blocking vaccines (TBVs) aim to elicit human antibodies that inhibit sporogonic development of Plasmodium falciparum in mosquitoes, thereby preventing onward transmission. Pfs48/45 is a leading clinical TBV candidate antigen and is recognized by the most potent transmission-blocking monoclonal antibody (mAb) yet described; still, clinical development of Pfs48/45 antigens has been hindered, largely by its poor biochemical characteristics. Here, we used structure-based computational approaches to design Pfs48/45 antigens stabilized in the conformation recognized by the most potently inhibitory mAb, achieving >25°C higher thermostability compared with the wild-type protein. Antibodies elicited in mice immunized with these engineered antigens displayed on liposome-based or protein nanoparticle-based vaccine platforms exhibited 1-2 orders of magnitude superior transmission-reducing activity, compared with immunogens bearing the wild-type antigen, driven by improved antibody quality. Our data provide the founding principles for using molecular stabilization solely from antibody structure-function information to drive improved immune responses against a parasitic vaccine target.

HIV-1 Neutralizing Antibodies with Limited Hypermutation from an Infant.

HIV-1 broadly neutralizing antibodies (bnAbs) develop in a subset of infected adults and exhibit high levels of somatic hypermutation (SHM) due to years of affinity maturation. There is no precedent for eliciting highly mutated antibodies by vaccination, nor is it practical to wait years for a desired response. Infants develop broad responses early, which may suggest a more direct path to generating bnAbs. Here, we isolated ten neutralizing antibodies (nAbs) contributing to plasma breadth of an infant at ∼1 year post-infection, including one with cross-clade breadth. The nAbs bind to envelope trimer from the transmitted virus, suggesting that this interaction may have initiated development of the infant nAbs. The infant cross-clade bnAb targets the N332 supersite on envelope but, unlike adult bnAbs targeting this site, lacks indels and has low SHM. The identification of this infant bnAb illustrates that HIV-1-specific neutralization breadth can develop without prolonged affinity maturation and extensive SHM.

Immunogenicity of Stabilized HIV-1 Envelope Trimers with Reduced Exposure of Non-neutralizing Epitopes.

The envelope glycoprotein trimer mediates HIV-1 entry into cells. The trimer is flexible, fluctuating between closed and more open conformations and sometimes sampling the fully open, CD4-bound form. We hypothesized that conformational flexibility and transient exposure of non-neutralizing, immunodominant epitopes could hinder the induction of broadly neutralizing antibodies (bNAbs). We therefore modified soluble Env trimers to stabilize their closed, ground states. The trimer variants were indeed stabilized in the closed conformation, with a reduced ability to undergo receptor-induced conformational changes and a decreased exposure of non-neutralizing V3-directed antibody epitopes. In rabbits, the stabilized trimers induced similar autologous Tier-1B or Tier-2 NAb titers to those elicited by the corresponding wild-type trimers but lower levels of V3-directed Tier-1A NAbs. Stabilized, closed trimers might therefore be useful components of vaccines aimed at inducing bNAbs.

Quadrivalent influenza nanoparticle vaccines induce broad protection.

Influenza vaccines that confer broad and durable protection against diverse viral strains would have a major effect on global health, as they would lessen the need for annual vaccine reformulation and immunization1. Here we show that computationally designed, two-component nanoparticle immunogens2 induce potently neutralizing and broadly protective antibody responses against a wide variety of influenza viruses. The nanoparticle immunogens contain 20 haemagglutinin glycoprotein trimers in an ordered array, and their assembly in vitro enables the precisely controlled co-display of multiple distinct haemagglutinin proteins in defined ratios. Nanoparticle immunogens that co-display the four haemagglutinins of licensed quadrivalent influenza vaccines elicited antibody responses in several animal models against vaccine-matched strains that were equivalent to or better than commercial quadrivalent influenza vaccines, and simultaneously induced broadly protective antibody responses to heterologous viruses by targeting the subdominant yet conserved haemagglutinin stem. The combination of potent receptor-blocking and cross-reactive stem-directed antibodies induced by the nanoparticle immunogens makes them attractive candidates for a supraseasonal influenza vaccine candidate with the potential to replace conventional seasonal vaccines3.

Identification of broad, potent antibodies to functionally constrained regions of SARS-CoV-2 spike following a breakthrough infection.

The antiviral benefit of antibodies can be compromised by viral escape especially for rapidly evolving viruses. Therefore, durable, effective antibodies must be both broad and potent to counter newly emerging, diverse strains. Discovery of such antibodies is critically important for SARS-CoV-2 as the global emergence of new variants of concern (VOC) has compromised the efficacy of therapeutic antibodies and vaccines. We describe a collection of broad and potent neutralizing monoclonal antibodies (mAbs) isolated from an individual who experienced a breakthrough infection with the Delta VOC. Four mAbs potently neutralize the Wuhan-Hu-1 vaccine strain, the Delta VOC, and also retain potency against the Omicron VOCs through BA.4/BA.5 in both pseudovirus-based and authentic virus assays. Three mAbs also retain potency to recently circulating VOCs XBB.1.5 and BQ.1.1 and one also potently neutralizes SARS-CoV-1. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs that had been approved for therapeutic applications. The mAbs target distinct epitopes on the spike glycoprotein, three in the receptor-binding domain (RBD) and one in an invariant region downstream of the RBD in subdomain 1 (SD1). The escape pathways we defined at single amino acid resolution with deep mutational scanning show they target conserved, functionally constrained regions of the glycoprotein, suggesting escape could incur a fitness cost. Overall, these mAbs are unique in their breadth across VOCs, their epitope specificity, and include a highly potent mAb targeting a rare epitope outside of the RBD in SD1.

Structural dynamics reveal subtype-specific activation and inhibition of influenza virus hemagglutinin.

Influenza hemagglutinin (HA) is a prototypical class 1 viral entry glycoprotein, responsible for mediating receptor binding and membrane fusion. Structures of its prefusion and postfusion forms, embodying the beginning and endpoints of the fusion pathway, have been extensively characterized. Studies probing HA dynamics during fusion have begun to identify intermediate states along the pathway, enhancing our understanding of how HA becomes activated and traverses its conformational pathway to complete fusion. HA is also the most variable, rapidly evolving part of influenza virus, and it is not known whether mechanisms of its activation and fusion are conserved across divergent viral subtypes. Here, we apply hydrogen-deuterium exchange mass spectrometry to compare fusion activation in two subtypes of HA, H1 and H3. Our data reveal subtype-specific behavior in the regions of HA that undergo structural rearrangement during fusion, including the fusion peptide and HA1/HA2 interface. In the presence of an antibody that inhibits the conformational change (FI6v3), we observe that acid-induced dynamic changes near the epitope are dampened, but the degree of protection at the fusion peptide is different for the two subtypes investigated. These results thus provide new insights into variation in the mechanisms of influenza HA's dynamic activation and its inhibition.

Cytoplasmic Tail Truncation Stabilizes S1-S2 Association and Enhances S Protein Incorporation into SARS-CoV-2 Pseudovirions.

Truncations of the cytoplasmic tail (CT) of entry proteins of enveloped viruses dramatically increase the infectivity of pseudoviruses (PVs) bearing these proteins. Several mechanisms have been proposed to explain this enhanced entry, including an increase in cell surface expression. However, alternative explanations have also been forwarded, and the underlying mechanisms for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein remain undetermined. Here, we show that the partial or complete deletion of the CT (residues 19 to 35) does not modify SARS-CoV-2 S protein expression on the cell surface when the S2 subunit is measured, whereas it is significantly increased when the S1 subunit is measured. We also show that the higher level of S1 in these CT-truncated S proteins reflects the decreased dissociation of the S1 subunit from the S2 subunit. In addition, we demonstrate that CT truncation further promotes S protein incorporation into PV particles, as indicated by biochemical analyses and cryo-electron microscopy. Thus, our data show that two distinct mechanisms contribute to the markedly increased infectivity of PVs carrying CT-truncated SARS-CoV-2 S proteins and help clarify the interpretation of the results of studies employing such PVs. IMPORTANCE Various forms of PVs have been used as tools to evaluate vaccine efficacy and study virus entry steps. When PV infectivity is inherently low, such as that of SARS-CoV-2, a CT-truncated version of the viral entry glycoprotein is widely used to enhance PV infectivity, but the mechanism underlying this enhanced PV infectivity has been unclear. Here, our study identified two mechanisms by which the CT truncation of the SARS-CoV-2 S protein dramatically increases PV infectivity: a reduction of S1 shedding and an increase in S protein incorporation into PV particles. An understanding of these mechanisms can clarify the mechanistic bases for the differences observed among various assays employing such PVs.

Structurally related but genetically unrelated antibody lineages converge on an immunodominant HIV-1 Env neutralizing determinant following trimer immunization.

Understanding the molecular mechanisms by which antibodies target and neutralize the HIV-1 envelope glycoprotein (Env) is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies (NAbs). Here, we analyzed monoclonal antibodies (mAbs) isolated from non-human primates (NHPs) immunized with variants of a native flexibly linked (NFL) HIV-1 Env stabilized trimer derived from the tier 2 clade C 16055 strain. The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from 0.005 to 3.68 µg/ml (IC50). Structural characterization using negative-stain EM and X-ray crystallography identified the variable region 2 (V2) of the 16055 NFL trimer to be the common epitope for these antibodies. The crystal structures revealed that the V2 segment adopts a ß-hairpin motif identical to that observed in the 16055 NFL crystal structure. These results depict how vaccine-induced antibodies derived from different clonal lineages penetrate through the glycan shield to recognize a hypervariable region within V2 (residues 184-186) that is unique to the 16055 strain. They also provide potential explanations for the potent autologous neutralization of these antibodies, confirming the immunodominance of this site and revealing that multiple angles of approach are permissible for affinity/avidity that results in potent neutralizing capacity. The structural analysis reveals that the most negatively charged paratope correlated with the potency of the mAbs. The atomic level information is of interest to both define the means of autologous neutralization elicited by different tier 2-based immunogens and facilitate trimer redesign to better target more conserved regions of V2 to potentially elicit cross-neutralizing HIV-1 antibodies.

Direct imaging of liquid domains in membranes by cryo-electron tomography.

Images of micrometer-scale domains in lipid bilayers have provided the gold standard of model-free evidence to understand the domains' shapes, sizes, and distributions. Corresponding techniques to directly and quantitatively assess smaller (nanoscale and submicron) liquid domains have been limited. Researchers commonly seek to correlate activities of membrane proteins with attributes of the domains in which they reside; doing so hinges on identification and characterization of membrane domains. Although some features of membrane domains can be probed by indirect methods, these methods are often constrained by the limitation that data must be analyzed in the context of models that require multiple assumptions or parameters. Here, we address this challenge by developing and testing two methods of identifying submicron domains in biomimetic membranes. Both methods leverage cryo-electron tomograms of ternary membranes under vitrified, hydrated conditions. The first method is optimized for probe-free applications: Domains are directly distinguished from the surrounding membrane by their thickness. This technique quantitatively and accurately measures area fractions of domains, in excellent agreement with known phase diagrams. The second method is optimized for applications in which a single label is deployed for imaging membranes by both high-resolution cryo-electron tomography and diffraction-limited optical microscopy. For this method, we test a panel of probes, find that a trimeric mCherry label performs best, and specify criteria for developing future high-performance, dual-use probes. These developments have led to direct and quantitative imaging of submicron membrane domains in vitrified, hydrated vesicles.

Prebiotic amino acids bind to and stabilize prebiotic fatty acid membranes.

The membranes of the first protocells on the early Earth were likely self-assembled from fatty acids. A major challenge in understanding how protocells could have arisen and withstood changes in their environment is that fatty acid membranes are unstable in solutions containing high concentrations of salt (such as would have been prevalent in early oceans) or divalent cations (which would have been required for RNA catalysis). To test whether the inclusion of amino acids addresses this problem, we coupled direct techniques of cryoelectron microscopy and fluorescence microscopy with techniques of NMR spectroscopy, centrifuge filtration assays, and turbidity measurements. We find that a set of unmodified, prebiotic amino acids binds to prebiotic fatty acid membranes and that a subset stabilizes membranes in the presence of salt and Mg2+ Furthermore, we find that final concentrations of the amino acids need not be high to cause these effects; membrane stabilization persists after dilution as would have occurred during the rehydration of dried or partially dried pools. In addition to providing a means to stabilize protocell membranes, our results address the challenge of explaining how proteins could have become colocalized with membranes. Amino acids are the building blocks of proteins, and our results are consistent with a positive feedback loop in which amino acids bound to self-assembled fatty acid membranes, resulting in membrane stabilization and leading to more binding in turn. High local concentrations of molecular building blocks at the surface of fatty acid membranes may have aided the eventual formation of proteins.

Deep Mutational Scan of the Highly Conserved Influenza A Virus M1 Matrix Protein Reveals Substantial Intrinsic Mutational Tolerance.

Influenza A virus matrix protein M1 is involved in multiple stages of the viral infectious cycle. Despite its functional importance, our present understanding of this essential viral protein is limited. The roles of a small subset of specific amino acids have been reported, but a more comprehensive understanding of the relationship between M1 sequence, structure, and virus fitness remains elusive. In this study, we used deep mutational scanning to measure the effect of every amino acid substitution in M1 on viral replication in cell culture. The map of amino acid mutational tolerance we have generated allows us to identify sites that are functionally constrained in cell culture as well as sites that are less constrained. Several sites that exhibit low tolerance to mutation have been found to be critical for M1 function and production of viable virions. Surprisingly, significant portions of the M1 sequence, especially in the C-terminal domain, whose structure is undetermined, were found to be highly tolerant of amino acid variation, despite having extremely low levels of sequence diversity among natural influenza virus strains. This unexpected discrepancy indicates that not all sites in M1 that exhibit high sequence conservation in nature are under strong constraint during selection for viral replication in cell culture.IMPORTANCE The M1 matrix protein is critical for many stages of the influenza virus infection cycle. Currently, we have an incomplete understanding of this highly conserved protein's function and structure. Key regions of M1, particularly in the C terminus of the protein, remain poorly characterized. In this study, we used deep mutational scanning to determine the extent of M1's tolerance to mutation. Surprisingly, nearly two-thirds of the M1 sequence exhibits a high tolerance for substitutions, contrary to the extremely low sequence diversity observed across naturally occurring M1 isolates. Sites with low mutational tolerance were also identified, suggesting that they likely play critical functional roles and are under selective pressure. These results reveal the intrinsic mutational tolerance throughout M1 and shape future inquiries probing the functions of this essential influenza A virus protein.

Dissection of Epitope-Specific Mechanisms of Neutralization of Influenza Virus by Intact IgG and Fab Fragments.

The neutralizing antibody (nAb) response against the influenza virus hemagglutinin (HA) fusion glycoprotein is important for preventing viral infection, but we lack a comprehensive understanding of the mechanisms by which these antibodies act. Here we investigated the effect of nAb binding and the role of IgG bivalency in the inhibition of HA function for nAbs targeting distinct HA epitopes. HC19 targets the receptor binding pocket at the distal end of HA, while FI6v3 binds primarily to the HA2 fusion subunit toward the base of the stalk. Surprisingly, HC19 inhibited the ability of HA to induce lipid mixing by preventing the structural rearrangement of HA under fusion-activating conditions. These results suggest that nAbs such as HC19 not only act by blocking receptor binding but also inhibit key late-stage HA conformational changes required for fusion. Intact HC19 IgG was also shown to cross-link separate virus particles, burying large proportions of HA within aggregates where they are blocked from interacting with target membranes; Fabs yielded no such aggregation and displayed weaker neutralization than IgG, emphasizing the impact of bivalency on the ability to neutralize virus. In contrast, the stem-targeting nAb FI6v3 did not aggregate particles. The Fab fragment was significantly less effective than IgG in preventing both membrane disruption and fusion. We infer that interspike cross-linking within a given particle by FI6v3 IgG may be critical to its potent neutralization, as no significant neutralization occurred with Fabs. These results demonstrate that IgG bivalency enhances HA inhibition through functionally important modes not evident in pared-down Fab-soluble HA structures.IMPORTANCE The influenza virus hemagglutinin (HA) fusion glycoprotein mediates entry into target cells and is the primary antigenic target of neutralizing antibodies (nAbs). Our current structural understanding of mechanisms of antibody (Ab)-mediated neutralization largely relies on the high-resolution characterization of antigen binding (Fab) fragments in complex with soluble, isolated antigen constructs by cryo-electron microscopy (EM) single-particle reconstruction or X-ray crystallography. Interactions between full-length IgG and whole virions have not been well characterized, and a gap remains in our understanding of how intact Abs neutralize virus and prevent infection. Using structural and biophysical approaches, we observed that Ab-mediated inhibition of HA function and neutralization of virus infectivity occur by multiple coexisting mechanisms, are largely dependent on the specific epitope that is targeted, and are highly dependent on the bivalent nature of IgG molecules.

Extracellular Matrix Proteins Mediate HIV-1 gp120 Interactions with α4ß7.

Gut-homing α4ß7high CD4+ T lymphocytes have been shown to be preferentially targeted by human immunodeficiency virus type 1 (HIV-1) and are implicated in HIV-1 pathogenesis. Previous studies demonstrated that HIV-1 envelope protein gp120 binds and signals through α4ß7 and that this likely contributes to the infection of α4ß7high T cells and promotes cell-to-cell virus transmission. Structures within the second variable loop (V2) of gp120, including the tripeptide motif LDV/I, are thought to mediate gp120-α4ß7 binding. However, lack of α4ß7 binding has been reported in gp120 proteins containing LDV/I, and the precise determinants of gp120-α4ß7 binding are not fully defined. In this work, we report the novel finding that fibronectins mediate indirect gp120-α4ß7 interactions. We show that Chinese hamster ovary (CHO) cells used to express recombinant gp120 produced fibronectins and other extracellular matrix proteins that copurified with gp120. CHO cell fibronectins were able to mediate the binding of a diverse panel of gp120 proteins to α4ß7 in an in vitro cell binding assay. The V2 loop was not required for fibronectin-mediated binding of gp120 to α4ß7, nor did V2-specific antibodies block this interaction. Removal of fibronectin through anion-exchange chromatography abrogated V2-independent gp120-α4ß7 binding. Additionally, we showed a recombinant human fibronectin fragment mediated gp120-α4ß7 interactions similarly to CHO cell fibronectin. These findings provide an explanation for the apparently contradictory observations regarding the gp120-α4ß7 interaction and offer new insights into the potential role of fibronectin and other extracellular matrix proteins in HIV-1 biology.IMPORTANCE Immune tissues within the gut are severely damaged by HIV-1, and this plays an important role in the development of AIDS. Integrin α4ß7 plays a major role in the trafficking of lymphocytes, including CD4+ T cells, into gut lymphoid tissues. Previous reports indicate that some HIV-1 gp120 envelope proteins bind to and signal through α4ß7, which may help explain the preferential infection of gut CD4+ T cells. In this study, we demonstrate that extracellular matrix proteins can mediate interactions between gp120 and α4ß7 This suggests that the extracellular matrix may be an important mediator of HIV-1 interaction with α4ß7-expressing cells. These findings provide new insight into the nature of HIV-1-α4ß7 interactions and how these interactions may represent targets for therapeutic intervention.

Sequential and Simultaneous Immunization of Rabbits with HIV-1 Envelope Glycoprotein SOSIP.664 Trimers from Clades A, B and C.

We have investigated the immunogenicity in rabbits of native-like, soluble, recombinant SOSIP.664 trimers based on the env genes of four isolates of human immunodeficiency virus type 1 (HIV-1); specifically BG505 (clade A), B41 (clade B), CZA97 (clade C) and DU422 (clade C). The various trimers were delivered either simultaneously (as a mixture of clade A + B trimers) or sequentially over a 73-week period. Autologous, Tier-2 neutralizing antibody (NAb) responses were generated to the clade A and clade B trimers in the bivalent mixture. When delivered as boosting immunogens to rabbits immunized with the clade A and/or clade B trimers, the clade C trimers also generated autologous Tier-2 NAb responses, the CZA97 trimers doing so more strongly and consistently than the DU422 trimers. The clade C trimers also cross-boosted the pre-existing NAb responses to clade A and B trimers. We observed heterologous Tier-2 NAb responses albeit inconsistently, and with limited overall breath. However, cross-neutralization of the clade A BG505.T332N virus was consistently observed in rabbits immunized only with clade B trimers and then boosted with clade C trimers. The autologous NAbs induced by the BG505, B41 and CZA97 trimers predominantly recognized specific holes in the glycan shields of the cognate virus. The shared location of some of these holes may account for the observed cross-boosting effects and the heterologous neutralization of the BG505.T332N virus. These findings will guide the design of further experiments to determine whether and how multiple Env trimers can together induce more broadly neutralizing antibody responses.

Local and global structural drivers for the photoactivation of the orange carotenoid protein.

Photoprotective mechanisms are of fundamental importance for the survival of photosynthetic organisms. In cyanobacteria, the orange carotenoid protein (OCP), when activated by intense blue light, binds to the light-harvesting antenna and triggers the dissipation of excess captured light energy. Using a combination of small angle X-ray scattering (SAXS), X-ray hydroxyl radical footprinting, circular dichroism, and H/D exchange mass spectrometry, we identified both the local and global structural changes in the OCP upon photoactivation. SAXS and H/D exchange data showed that global tertiary structural changes, including complete domain dissociation, occur upon photoactivation, but with alteration of secondary structure confined to only the N terminus of the OCP. Microsecond radiolytic labeling identified rearrangement of the H-bonding network associated with conserved residues and structural water molecules. Collectively, these data provide experimental evidence for an ensemble of local and global structural changes, upon activation of the OCP, that are essential for photoprotection.