Trimeric Glycoprotein Research Articles

The Impact of United Kingdom SARS-CoV-2 Spike Mutations on Spike-ACE2 Interactions Investigated Through Molecular Dynamic Simulation

Background: A newly emergent betacoronavirus, SARS-CoV-2, poses a tremendous global threat and causes severe acute respiratory syndrome coronavirus-2, a major pandemic that began worldwide in 2019. The trimeric spike glycoprotein (S) is located on the envelope of the virus and facilitates the fusion of viral and host cell membranes by interacting with a cellular receptor called angiotensin-converting enzyme 2 (ACE2). In the United Kingdom (UK), a variant of SARS-CoV-2 has been detected using sequencing technology. There has been a significant surge in the number of COVID-19 cases in South East England. These lineages are significantly more transmissible than previously circulating variants. Objectives: The aim of this study is to investigate the effects of UK SARS-CoV-2 spike mutations on its interactions with ACE2. Methods: In this study, the structure of the UK spike protein with multiple mutations, including deletion 69 - 70, deletion 144, N501Y, A570D, T716I, D614G, P681H, S982A, and D1118H, was investigated. The research focuses on studying the impact of these mutations on spike function and its interactions with ACE2 through molecular dynamic simulation. Results: The results indicated that hydrophobic interactions, hydrogen bonds, and double/triple bonds are more prevalent in the mutated spike-ACE2 complex. Additionally, the UK spike-ACE2 complex maintained a consistent conformation throughout the simulation, experiencing minimal changes in structure. The mutant spike protein structure is less stable compared to the wild-type spike structure. Conclusions: Multiple mutations, including deletion 69 - 70, deletion 144, N501Y, A570D, T716I, D614G, P681H, S982A, and D1118H in the spike protein of UK SARS-CoV-2, can affect its interaction with the ACE2 receptor and the transmissibility of this variant of SARS-CoV-2.

Functional comparison of Fc-engineering strategies to improve anti-HIV-1 antibody effector functions

Substantial reduction of the intact proviral reservoir is essential towards HIV-1 cure. In vivo administration of broadly neutralizing antibodies (bNAbs) targeting the HIV-1 envelope glycoprotein (Env) trimer can decrease the viral reservoir, through Fc-mediated killing of infected cells. In this study, we compared three commonly used antibody engineering strategies to enhance Fc-mediated effector functions: (i) glyco-engineering, (ii) protein engineering, and (iii) subclass/hinge modifications in a panel of anti-HIV-1 antibodies. We found that antibody-dependent cellular phagocytosis (ADCP) was improved by elongating the hinge domain and switching to an IgG3 constant domain. In addition, potent NK cell activation and ADCC activity was observed for afucosylated antibodies and antibodies bearing the GASDALIE mutations. The combination of these engineering strategies further increased NK cell activation and induced antibody dependent cytotoxicity (ADCC) of infected cells at low antibody concentrations. The bNAb N6 was most effective at killing HIV-1 infected cells, likely due to its high affinity and optimal angle of approach. Overall, the findings of this study are applicable to other antibody formats, and can aid the development of effective immunotherapies and antibody-based treatments for HIV-1 cure strategies.

Conformations of membrane human immunodeficiency virus (HIV-1) envelope glycoproteins solubilized in Amphipol A18 lipid-nanodiscs.

Upon binding to the host cell receptor, CD4, the pretriggered (State-1) conformation of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer undergoes transitions to downstream conformations important for virus entry. State 1 is targeted by most broadly neutralizing antibodies (bNAbs), whereas downstream conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. Extraction of Env from the membranes of viruses or Env-expressing cells disrupts the metastable State-1 Env conformation, even when detergent-free approaches like styrene-maleic acid lipid nanoparticles (SMALPs) are used. Here, we combine three strategies to solubilize and purify mature membrane Envs that are antigenically native (i.e., recognized by bNAbs and not pNAbs): (1) solubilization of Env with a novel amphipathic copolymer, Amphipol A18; (2) use of stabilized pretriggered Env mutants; and (3) addition of the State-1-stabilizing entry inhibitor, BMS-806. Amphipol A18 was superior to the other amphipathic copolymers tested (SMA and AASTY 11-50) for preserving a native Env conformation. A native antigenic profile of A18 Env-lipid-nanodiscs was maintained for at least 7 days at 4°C and 2 days at 37°C in the presence of BMS-806 and was also maintained for at least 1 h at 37°C in a variety of adjuvants. The damaging effects of a single cycle of freeze-thawing on the antigenic profile of the A18 Env-lipid-nanodiscs could be prevented by the addition of 10% sucrose or 10% glycerol. These results underscore the importance of the membrane environment to the maintenance of a pretriggered (State-1) Env conformation and provide strategies for the preparation of lipid-nanodiscs containing native membrane Envs.IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoproteins (Envs) mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins like Env rely on purification procedures that maintain their natural conformation. In this study, we show that an amphipathic copolymer A18 can directly extract HIV-1 Env from a membrane without the use of detergents. A18 promotes the formation of nanodiscs that contain Env and membrane lipids. Env in A18-lipid nanodiscs largely preserves features recognized by broadly neutralizing antibodies (bNAbs) and conceals features potentially recognized by poorly neutralizing antibodies (pNAbs). Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful for future studies of HIV-1 Env structure, interaction with receptors and antibodies, and immunogenicity.

Impact of glycan depletion, glycan debranching and increased glycan charge on HIV-1 neutralization sensitivity and immunogenicity.

Broadly neutralizing antibodies (bNAbs) isolated from HIV-1 infected donors are vaccine paradigms. These bNAbs recognize envelope glycoprotein trimers that carry 75-90 oligomannose and complex-type glycans. Although bNAbs and their precursors must navigate past glycans, they usually also make some glycan contacts. Glycan-modified vaccines may therefore be useful to initiate and guide bNAb development. Here, we describe two ways to modify Env glycans for possible vaccine use: 1) using a cocktail of glycosidases (termed "NGAF3" (Neuraminidase, β-Galactosidase, N-Acetylglucosaminidase, endoglycosidase F3 (endo F3)) to deplete complex glycans to try to minimize bNAb-glycan clashes and 2) co-expressing β-1,4-galactosyltransferase 1 (B4G) and β-galactoside α-2,6 sialyltransferase 1 (ST6) during Env biosynthesis, creating bNAb-preferred glycan structures. Mass spectrometry revealed that NGAF3 removed glycan heads at 3/7 sites occupied by complex glycans. B4G overexpression resulted in hybrid glycan development whenever complex glycans were closely spaced. The glycan at position 611 in of Env's gp41 transmembrane subunit was uniquely isolated from the effects of both endo F3 and B4G. B4G and ST6 co-expression increased hybrid and sialylated glycan abundance, reducing glycan complexity. In rabbit vaccinations, B4G+ST6 virus-like particles (VLPs) induced less frequent, weaker titer NAbs, implying that ST6-mediated increased Env charge dampens vaccine antibodies. In some cases, vaccine sera preferentially neutralized B4G+ST6-modified pseudovirus. HIV-1+ donor plasma NAbs were generally more effective against B4G+ST6 modified pseudovirus, suggesting a preference for less complex and/or α-2,6 sialylated Env trimers. Collectively, our data suggest that B4G and ST6 Env modifications are best suited for intermediate or late vaccine shots.

Assembly of respiratory syncytial virus matrix protein lattice and its coordination with fusion glycoprotein trimers

Respiratory syncytial virus (RSV) is an enveloped, filamentous, negative-strand RNA virus that causes significant respiratory illness worldwide. RSV vaccines are available, however there is still significant need for research to support the development of vaccines and therapeutics against RSV and related Mononegavirales viruses. Individual virions vary in size, with an average diameter of ~130 nm and ranging from ~500 nm to over 10 µm in length. Though the general arrangement of structural proteins in virions is known, we use cryo-electron tomography and sub-tomogram averaging to determine the molecular organization of RSV structural proteins. We show that the peripheral membrane-associated RSV matrix (M) protein is arranged in a packed helical-like lattice of M-dimers. We report that RSV F glycoprotein is frequently observed as pairs of trimers oriented in an anti-parallel conformation to support potential interactions between trimers. Our sub-tomogram averages indicate the positioning of F-trimer pairs is correlated with the underlying M lattice. These results provide insight into RSV virion organization and may aid in the development of RSV vaccines and anti-viral targets.

Triple tandem trimer immunogens for HIV-1 and influenza nucleic acid-based vaccines

Recombinant native-like HIV-1 envelope glycoprotein (Env) trimers are used in candidate vaccines aimed at inducing broadly neutralizing antibodies. While state-of-the-art SOSIP or single-chain Env designs can be expressed as native-like trimers, undesired monomers, dimers and malformed trimers that elicit non-neutralizing antibodies are also formed, implying that these designs could benefit from further modifications for gene-based vaccination approaches. Here, we describe the triple tandem trimer (TTT) design, in which three Env protomers are genetically linked in a single open reading frame and express as native-like trimers. Viral vectored Env TTT induced similar neutralization titers but with a higher proportion of trimer-specific responses. The TTT design was also applied to generate influenza hemagglutinin (HA) trimers without the need for trimerization domains. Additionally, we used TTT to generate well-folded chimeric Env and HA trimers that harbor protomers from three different strains. In summary, the TTT design is a useful platform for the design of HIV-1 Env and influenza HA immunogens for a multitude of vaccination strategies.

Humoral anti-SARS-CoV-2 immune response for different strains after Sinovac-CoronaVac and Oxford/AstraZeneca (ChAdOx1-S) full vaccination on a healthcare population in Brazil

COVID-19, caused by the SARS-CoV-2 virus, is a global respiratory syndrome with high mortality rates. Vaccination is currently the only proven method to prevent the disease, although the role of lab data in assessing efficacy remains uncertain. This study aimed to assess spike-binding and neutralizing antibody levels following full vaccination with Oxford/AstraZeneca (ChAdOx1 nCoV-19) or CoronaVac in healthcare workers in southeastern Brazil. ChAdOx1 nCoV-19 and CoronaVac induced IgG antibodies against trimeric spike glycoproteins in 99.5% and 80.9% of individuals, respectively. Neutralizing antibodies were produced against two viral strains groups: variants group 1 (Wuhan-Hu-1, Alpha) and variants group 2 (Beta, Gamma) with neutralization rates of 88.3% and 78.2% for ChAdOx1 nCoV-19, and 68.1% and 48.9% for CoronaVac. No associations were found between neutralizing levels and comorbidities, age, or side effects. A positive correlation was observed between IgG antibody concentrations against trimeric spike glycoproteins and neutralizing levels for both vaccines and variants. These findings indicate that both vaccines induced reasonable levels of neutralizing antibodies against variants group 1, but only ChAdOx1 nCoV-19 maintained acceptable levels against a variant strain. The study suggests that evaluating vaccine responses to different pathogen strains can aid in managing healthcare workforce concerns and improve vaccine selection, thereby enhancing overall vaccination strategies.

Broad Epitope Coverage of Therapeutic Multi-Antibody Combinations Targeting SARS-CoV-2 Boosts In Vivo Protection and Neutralization Potency to Corner an Immune-Evading Virus.

Therapeutic antibodies (Abs) which act on a broader range of epitopes may provide more durable protection against the genetic drift of a target, typical of viruses or tumors. When these Abs exist concurrently on the targeted antigen, several mechanisms of action (MoAs) can be engaged, boosting therapeutic potency. This study selected combinations of four and five Abs with non- or partially overlapping epitopes to the SARS-CoV-2 spike glycoprotein, on or outside the crucial receptor binding domain (RBD), to offer resilience to emerging variants and trigger multiple MoAs. The combinations were derived from a pool of unique-sequence scFv Ab fragments retrieved from two SARS-CoV-2-naïve human phage display libraries. Following recombinant expression to full-length human IgG1 candidates, a biolayer interferometric analysis mapped epitopes to bins and confirmed that up to four Abs from across the bins can exist simultaneously on the spike glycoprotein trimer. Not all the bins of Abs interfered with the spike protein binding to angiotensin converting enzyme 2 (ACE2) in competitive binding assays, nor neutralized the pseudovirus or authentic virus in vitro, but when combined in vivo, their inclusion resulted in a much stronger viral clearance in the lungs of intranasally challenged hamsters, compared to that of those treated with mono ACE2 blockers. In addition, the Ab mixtures activated in vitro reporter cells expressing Fc-gamma receptors (FcγRs) involved in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). The best four-Ab combination neutralized seventeen variants of concern from Wuhan-Hu1 to Omicron BA.4/BA.5 in vitro.

Observing Dynamic Conformational Changes within the Coiled-Coil Domain of Different Laminin Isoforms Using High-Speed Atomic Force Microscopy.

Laminins are trimeric glycoproteins with important roles in cell-matrix adhesion and tissue organization. The laminin α, ß, and γ-chains have short N-terminal arms, while their C-termini are connected via a triple coiled-coil domain, giving the laminin molecule a well-characterized cross-shaped morphology as a result. The C-terminus of laminin alpha chains contains additional globular laminin G-like (LG) domains with important roles in mediating cell adhesion. Dynamic conformational changes of different laminin domains have been implicated in regulating laminin function, but so far have not been analyzed at the single-molecule level. High-speed atomic force microscopy (HS-AFM) is a unique tool for visualizing such dynamic conformational changes under physiological conditions at sub-second temporal resolution. After optimizing surface immobilization and imaging conditions, we characterized the ultrastructure of laminin-111 and laminin-332 using HS-AFM timelapse imaging. While laminin-111 features a stable S-shaped coiled-coil domain displaying little conformational rearrangement, laminin-332 coiled-coil domains undergo rapid switching between straight and bent conformations around a defined central molecular hinge. Complementing the experimental AFM data with AlphaFold-based coiled-coil structure prediction enabled us to pinpoint the position of the hinge region, as well as to identify potential molecular rearrangement processes permitting hinge flexibility. Coarse-grained molecular dynamics simulations provide further support for a spatially defined kinking mechanism in the laminin-332 coiled-coil domain. Finally, we observed the dynamic rearrangement of the C-terminal LG domains of laminin-111 and laminin-332, switching them between compact and open conformations. Thus, HS-AFM can directly visualize molecular rearrangement processes within different laminin isoforms and provide dynamic structural insight not available from other microscopy techniques.

HIV-1 entry stoichiometry differs across strains, distribution of surface envelope (Env) glycoprotein trimer, and inter-protomer cooperativity in Env trimer

HIV-1 entry stoichiometry differs across strains, distribution of surface envelope (Env) glycoprotein trimer, and inter-protomer cooperativity in Env trimer

Structural and biochemical rationale for Beta variant protein booster vaccine broad cross-neutralization of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, uses a surface expressed trimeric spike glycoprotein for cell entry. This trimer is the primary target for neutralizing antibodies making it a key candidate for vaccine development. During the global pandemic circulating variants of concern (VOC) caused several waves of infection, severe disease, and death. The reduced efficacy of the ancestral trimer-based vaccines against emerging VOC led to the need for booster vaccines. Here we present a detailed characterization of the Sanofi Beta trimer, utilizing cryo-EM for structural elucidation. We investigate the conformational dynamics and stabilizing features using orthogonal SPR, SEC, nanoDSF, and HDX-MS techniques to better understand how this antigen elicits superior broad neutralizing antibodies as a variant booster vaccine. This structural analysis confirms the Beta trimer preference for canonical quaternary structure with two RBD in the up position and the reversible equilibrium between the canonical spike and open trimer conformations. Moreover, this report provides a better understanding of structural differences between spike antigens contributing to differential vaccine efficacy.

Cleavage-intermediate Lassa virus trimer elicits neutralizing responses, identifies neutralizing nanobodies, and reveals an apex-situated site-of-vulnerability

Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into LASV neutralization, here we develop a prefusion-stabilized LASV glycoprotein trimer (GPC), pan it against phage libraries comprising single-domain antibodies (nanobodies) from shark and camel, and identify one, D5, which neutralizes LASV. Cryo-EM analyses reveal D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. The recognized site appears specific to GPC intermediates, with protomers lacking full cleavage between GP1 and GP2 subunits. Guinea pig immunizations with the prefusion-stabilized cleavage-intermediate LASV GPC, first as trimer and then as a nanoparticle, induce neutralizing responses, targeting multiple epitopes including that of D5; we identify a neutralizing antibody (GP23) from the immunized guinea pigs. Collectively, our findings define a prefusion-stabilized GPC trimer, reveal an apex-situated site-of-vulnerability, and demonstrate elicitation of LASV-neutralizing responses by a cleavage-intermediate LASV trimer.

Production and Purification of Filovirus Glycoproteins.

Ebola (EBOV) and Marburg (MARV) viruses cause hemorrhagic fever disease in humans and non-human primates (NHPs) with case-fatality rates as high as 90%. The 2013-2016 Ebola virus disease (EVD) outbreak led to over 28,000 cases and 11,000 deaths and took an enormous toll on the economy of West African nations, in the absence of any vaccine or therapeutic options. Like EVD, there have been at least 6 outbreaks of MVD with ~88% case-fatality and the most recent cases emerging in Equatorial Guinea in February 2023. These outbreaks have spurred an unprecedented global effort to develop vaccines and therapeutics for EVD and MVD and led to an approved vaccine (ERVEBO™) and two monoclonal antibody (mAb) therapeutics for EBOV. In contrast to EVD, therapeutic options against Marburg and another Ebola-relative Sudan virus (SUDV) are lacking. The filovirus glycoprotein (GP), which mediates host cell entry and fusion, is the primary target of neutralizing antibodies. In addition to its pre- and post-fusion trimeric states, the protein is highly glycosylated making production of pure and homogeneous trimers on a large scale, a requirement for subunit vaccine development, a challenge. In efforts to address this roadblock, we have developed a unique combination of structure-based design, selection of expression system, and purification methods to produce uniform and stable EBOV and MARV GP trimers at scales appropriate for vaccine production.

Human PBMCs Form Lipid Droplets in Response to Spike Proteins.

Intracellular lipid droplets (LDs) can accumulate in response to inflammation, metabolic stresses, and other physiological/pathological processes. Herein, we investigated whether spike proteins of SARS-CoV-2 induce LDs in human peripheral blood mononuclear cells (PBMCs) and in pulmonary microvascular endothelial cells (HPMECs). PBMCs or HPMECs were incubated alone or with endotoxin-free recombinant variants of trimeric spike glycoproteins (Alpha, Beta, Delta, and Omicron, 12 µg/mL). Afterward, cells were stained with Oil Red O for LDs, cytokine release was determined through ELISA, and the gene expression was analyzed through real-time PCR using TaqMan assays. Our data show that spikes induce LDs in PBMCs but not in HPMECs. In line with this, in PBMCs, spike proteins lower the expression of genes involving lipid metabolism and LD formation, such as SREBF1, HMGCS1, LDLR, and CD36. On the other hand, PBMCs exposed to spikes for 6 or 18 h did not increase in IL-1β, IL-6, IL-8, MCP-1, and TNFα release or expression as compared to non-treated controls. Thus, spike-induced LD formation in PBMCs seems to not be related to cell inflammatory activation. Further detailed studies are warranted to investigate in which specific immune cells spikes induce LDs, and what are the pathophysiological mechanisms and consequences of this induction in vivo.

Omicron variants of SARS CoV-2 indicate how small molecules can interfere with spike glycoprotein trimerization

In our search for a possible achilles’ heel of SARS-CoV-2, we explored the variability of 1,382,462 complete sequences of the viral spike glycoprotein, all the ones that we could retrieve from the NCBI SARS-CoV-2 databank as of 6 March 2023. Then, by using the Shannon entropy algorithm, we quantified the sequence variability of SARS-CoV-2 spike glycoprotein. With PDBePISA, we have performed a detailed analysis of protomer-protomer interfaces of the spike glycoprotein for two representative structures of different viral variants. The largest protomer-protomer contact patch that is present in the stem region of both structures is highly conserved. It is remarkable that the Asp796Tyr mutation, centered in this patch, is always present in all the Omicron variants. The structure of the SARS-CoV-2 Omicron spike glycoprotein trimer indicates that Tyr796 and Phe898 of the same protomer form a network of aromatic sidechains with Tyr707 of another protomer, yielding a strong constraint that stabilizes the spike glycoprotein quaternary assembly. We believe that the resulting structural stability of the viral trimer is among the key features for the successful proliferation of Omicron variants. This finding also supports the fact that disrupting this network of aromatic moieties with suitable small molecules would represent a powerful antiviral strategy.

An HIV-1 broadly neutralizing antibody overcomes structural and dynamic variation through highly focused epitope targeting

The existence of broadly cross-reactive antibodies that can neutralize diverse HIV-1 isolates (bnAbs) has been appreciated for more than a decade. Many high-resolution structures of bnAbs, typically with one or two well-characterized HIV-1 Env glycoprotein trimers, have been reported. However, an understanding of how such antibodies grapple with variability in their antigenic targets across diverse viral isolates has remained elusive. To achieve such an understanding requires first characterizing the extent of structural and antigenic variation embodied in Env, and then identifying how a bnAb overcomes that variation at a structural level. Here, using hydrogen/deuterium-exchange mass spectrometry (HDX-MS) and quantitative measurements of antibody binding kinetics, we show that variation in structural ordering in the V1/V2 apex of Env across a globally representative panel of HIV-1 isolates has a marked effect on antibody association rates and affinities. We also report cryo-EM reconstructions of the apex-targeting PGT145 bnAb bound to two divergent Env that exhibit different degrees of structural dynamics throughout the trimer structures. Parallel HDX-MS experiments demonstrate that PGT145 bnAb has an exquisitely focused footprint at the trimer apex where binding did not yield allosteric changes throughout the rest of the structure. These results demonstrate that structural dynamics are a cryptic determinant of antigenicity, and mature antibodies that have achieved breadth and potency in some cases are able to achieve their broad cross-reactivity by “threading the needle” and binding in a highly focused fashion, thus evading and overcoming the variable properties found in Env from divergent isolates.

Modulation of immune responses to liposomal vaccines by intrastructural help

The encapsulation of HIV-unrelated T helper peptides into liposomal vaccines presenting trimers of the HIV-1 envelope glycoprotein (Env) on the surface (T helper liposomes) may recruit heterologous T cells to provide help for Env-specific B cells. This mechanism called intrastructural help can modulate the HIV-specific humoral immune response. In this study, we used cationic T helper liposomes to induce intrastructural help effects in a small animal model. The liposomes were functionalized with Env trimers by a tag-free approach designed to enable a simplified GMP production. The pre-fusion conformation of the conjugated Env trimers was verified by immunogold electron microscopy (EM) imaging and flow cytometry. The liposomes induced strong activation of Env-specific B cells in vitro. In comparison to previously established anionic liposomes, cationic T helper liposomes were superior in CD4+ T cell activation after uptake by dendritic cells. Moreover, the T helper liposomes were able to target Env-specific B cells in secondary lymphoid organs after intramuscular injection. We also observed efficient T helper cell activation and proliferation in co-cultures with Env-specific B cells in the presence of cationic T helper liposomes. Mouse immunization experiments with cationic T helper liposomes further revealed a modulation of the Env-specific IgG subtype distribution and enhancement of the longevity of antibody responses by ovalbumin- and Hepatitis B (HBV)-specific T cell help. Thus, clinical evaluation of the concept of intrastructural help seems warranted.

Structural basis of the American mink ACE2 binding by Y453F trimeric spike glycoproteins of SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) enters the host cell by binding to angiotensin-converting enzyme 2 (ACE2). While evolutionarily conserved, ACE2 receptors differ across various species and differential interactions with Spike (S) glycoproteins of SARS-CoV-2 viruses impact species specificity. Reverse zoonoses led to SARS-CoV-2 outbreaks on multiple American mink (Mustela vison) farms during the pandemic and gave rise to mink-associated S substitutions known for transmissibility between mink and zoonotic transmission to humans. In this study, we used bio-layer interferometry (BLI) to discern the differences in binding affinity between multiple human and mink-derived S glycoproteins of SARS-CoV-2 and their respective ACE2 receptors. Further, we conducted a structural analysis of a mink variant S glycoprotein and American mink ACE2 (mvACE2) using cryo-electron microscopy (cryo-EM), revealing four distinct conformations. We discovered a novel intermediary conformation where the mvACE2 receptor is bound to the receptor-binding domain (RBD) of the S glycoprotein in a "down" position, approximately 34° lower than previously reported "up" RBD. Finally, we compared residue interactions in the S-ACE2 complex interface of S glycoprotein conformations with varying RBD orientations. These findings provide valuable insights into the molecular mechanisms of SARS-CoV-2 entry.

Alterations in gp120 glycans or the gp41 fusion peptide-proximal region modulate the stability of the human immunodeficiency virus (HIV-1) envelope glycoprotein pretriggered conformation.

The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer mediates entry into host cells by binding receptors, CD4 and CCR5/CXCR4, and fusing the viral and cell membranes. In infected cells, cleavage of the gp160 Env precursor yields the mature Env trimer, with gp120 exterior and gp41 transmembrane Env subunits. Env cleavage stabilizes the State-1 conformation, which is the major target for broadly neutralizing antibodies, and decreases the spontaneous sampling of more open Env conformations that expose epitopes for poorly neutralizing antibodies. During HIV-1 entry into cells, CD4 binding drives the metastable Env from a pretriggered (State-1) conformation into more "open," lower-energy states. Here, we report that changes in two dissimilar elements of the HIV-1 Env trimer, namely particular gp120 glycans and the gp41 fusion peptide-proximal region (FPPR), can independently modulate the stability of State 1. Individual deletion of several gp120 glycans destabilized State 1, whereas removal of a V1 glycan resulted in phenotypes indicative of a more stable pretriggered Env conformation. Likewise, some alterations of the gp41 FPPR decreased the level of spontaneous shedding of gp120 from the Env trimer and stabilized the pretriggered State-1 Env conformation. State-1-stabilizing changes were additive and could suppress the phenotypes associated with State-1-destabilizing alterations in Env. Our results support a model in which multiple protein and carbohydrate elements of the HIV-1 Env trimer additively contribute to the stability of the pretriggered (State-1) conformation. The Env modifications identified in this study will assist efforts to characterize the structure and immunogenicity of the metastable State-1 conformation. IMPORTANCE The elicitation of antibodies that neutralize multiple strains of HIV-1 is an elusive goal that has frustrated the development of an effective vaccine. The pretriggered shape of the HIV-1 envelope glycoprotein (Env) spike on the virus surface is the major target for such broadly neutralizing antibodies. The "closed" pretriggered Env shape resists the binding of most antibodies but is unstable and often assumes "open" shapes that elicit ineffective antibodies. We identified particular changes in both the protein and the sugar components of the Env trimer that stabilize the pretriggered shape. Combinations of these changes were even more effective at stabilizing the pretriggered Env than the individual changes. Stabilizing changes in Env could counteract the effect of Env changes that destabilize the pretriggered shape. Locking Env in its pretriggered shape will assist efforts to understand the Env spike on the virus and to incorporate this shape into vaccines.

Multivalent antigen display on nanoparticle immunogens increases B cell clonotype diversity and neutralization breadth to pneumoviruses

Nanoparticles for multivalent display and delivery of vaccine antigens have emerged as a promising avenue for enhancing B cell responses to protein subunit vaccines. Here, we evaluated B cell responses in rhesus macaques immunized with prefusion-stabilized respiratory syncytial virus (RSV) F glycoprotein trimer compared with nanoparticles displaying 10 or 20 copies of the same antigen. We show that multivalent display skews antibody specificities and drives epitope-focusing of responding B cells. Antibody cloning and repertoire sequencing revealed that focusing was driven by the expansion of clonally distinct B cells through recruitment of diverse precursors. We identified two antibody lineages that developed either ultrapotent neutralization or pneumovirus cross-neutralization from precursor B cells with low initial affinity for the RSV-F immunogen. This suggests that increased avidity by multivalent display facilitates the activation and recruitment of these cells. Diversification of the B cell response by multivalent nanoparticle immunogens has broad implications for vaccine design.