The trimeric HIV-1 envelope glycoprotein (Env) is critical for host immune recognition and neutralization. Despite advances in trimer design, the roots of Env trimer metastability remain elusive. Here we investigate the contribution of two Env regions to metastability. First, we computationally redesign a largely disordered bend in heptad region 1 (HR1) of SOSIP trimers that connects the long, central HR1 helix to the fusion peptide, substantially improving the yield of soluble, well-folded trimers. Structural and antigenic analyses of two distinct HR1 redesigns confirm that redesigned Env closely mimics the native, prefusion trimer with a more stable gp41. Next, we replace the cleavage site between gp120 and gp41 with various linkers in the context of an HR1 redesign. Electron microscopy reveals a potential fusion intermediate state for uncleaved trimers containing short but not long linkers. Together, these results outline a general approach for stabilization of Env trimers from diverse HIV-1 strains.

Structures of BG505 SOSIP.664 trimer in complex with broadly neutralizing antibodies (bNAbs) have revealed the critical role of trimeric context for immune recognition of HIV-1. Presentation of trimeric HIV-1 antigens on nanoparticles may thus provide promising vaccine candidates. Here we report the rational design, structural analysis and antigenic evaluation of HIV-1 trimer-presenting nanoparticles. We first demonstrate that both V1V2 and gp120 can be presented in native-like trimeric conformations on nanoparticles. We then design nanoparticles presenting various forms of stabilized gp140 trimer based on ferritin and a large, 60-meric E2p that displays 20 spikes mimicking virus-like particles (VLPs). Particle assembly is confirmed by electron microscopy (EM), while antigenic profiles are generated using representative bNAbs and non-NAbs. Lastly, we demonstrate high-yield gp140 nanoparticle production and robust stimulation of B cells carrying cognate VRC01 receptors by gp120 and gp140 nanoparticles. Together, our study provides an arsenal of multivalent immunogens for HIV-1 vaccine development.

Previous efforts to identify cross-neutralizing antibodies to the receptor-binding site (RBS) of ebolavirus glycoproteins have been unsuccessful, largely because the RBS is occluded on the viral surface. We report a monoclonal antibody (FVM04) that targets a uniquely exposed epitope within the RBS; cross-neutralizes Ebola (EBOV), Sudan (SUDV), and, to a lesser extent, Bundibugyo viruses; and shows protection against EBOV and SUDV in mice and guinea pigs. The antibody cocktail ZMapp™ is remarkably effective against EBOV (Zaire) but does not cross-neutralize other ebolaviruses. By replacing one of the ZMapp™ components with FVM04, we retained the anti-EBOV efficacy while extending the breadth of protection to SUDV, thereby generating a cross-protective antibody cocktail. In addition, we report several mutations at the base of the ebolavirus glycoprotein that enhance the binding of FVM04 and other cross-reactive antibodies. These findings have important implications for pan-ebolavirus vaccine development and defining broadly protective antibody cocktails.

Targeting germline (gl-) precursors of broadly neutralizing antibodies (bNAbs) is acknowledged as an important strategy for HIV-1 vaccines. The VRC01-class of bNAbs is attractive because of its distinct genetic signature. However, VRC01-class bNAbs often require extensive somatic hypermutation, including rare insertions and deletions. We describe a BG505 SOSIP trimer, termed GT1.2, to optimize binding to gl-CH31, the unmutated common precursor of the CH30-34 bNAb lineage that acquired a large CDRH1 insertion. The GT1.2 trimer activates gl-CH31 naive B cells in knock-in mice, and B cell responses could be matured by selected boosting immunogens to generate cross-reactive Ab responses. Next-generation B cell sequencing reveals selection for VRC01-class mutations, including insertions in CDRH1 and FWR3 at positions identical to VRC01-class bNAbs, as well as CDRL1 deletions and/or glycine substitutions to accommodate the N276 glycan. These results provide proof of concept for vaccine-induced affinity maturation of B cell lineages that require rare insertions and deletions.

VRC01-class antibodies neutralize diverse HIV-1 strains by targeting the conserved CD4-binding site. Despite extensive investigations, crucial events in the early stage of VRC01 development remain elusive. We demonstrated how VRC01-class antibodies emerged in a Chinese donor by antigen-specific single B cell sorting, structural and functional studies, and longitudinal antibody and virus repertoire analyses. A monoclonal antibody DRVIA7 with modest neutralizing breadth was isolated that displayed a subset of VRC01 signatures. X-ray and EM structures revealed a VRC01-like angle of approach, but less favorable interactions between the DRVIA7 light-chain CDR1 and the N terminus with N276 and V5 glycans of gp120. Although the DRVIA7 lineage was unable to acquire broad neutralization, longitudinal analysis revealed a repertoire-encoded VRC01 light-chain CDR3 signature and VRC01-like neutralizing heavy-chain precursors that rapidly matured within 2 years. Thus, light chain accommodation of the glycan shield should be taken into account in vaccine design targeting this conserved site of vulnerability.

Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.

The HIV-1 envelope glycoprotein trimer is covered by an array of N-linked glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints limiting the actions of glycan-processing enzymes, so that multiple under-processed structures remain on specific areas. These oligomannose glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, paradoxically, target it. Our site-specific glycosylation analysis of a soluble, recombinant trimer (BG505 SOSIP.664) maps the extremes of simplicity and diversity of glycan processing at individual sites and reveals a mosaic of dense clusters of oligomannose glycans on the outer domain. Although individual sites usually minimally affect the global integrity of the glycan shield, we identify examples of how deleting some glycans can subtly influence neutralization by bNAbs that bind at distant sites. The network of bNAb-targeted glycans should be preserved on vaccine antigens.

The envelope glycoprotein trimer (Env) on the surface of HIV-1 recognizes CD4+ T cells and mediates viral entry. During this process, Env undergoes substantial conformational rearrangements, making it difficult to study in its native state. Soluble stabilized trimers have provided valuable insights into the Env structure, but they lack the hydrophobic membrane proximal external region (MPER, an important target of broadly neutralizing antibodies), the transmembrane domain, and the cytoplasmic tail. Here we present (i) a cryogenic electron microscopy (cryo-EM) structure of a clade B virus Env, which lacks only the cytoplasmic tail and is stabilized by the broadly neutralizing antibody PGT151, at a resolution of 4.2 angstroms and (ii) a reconstruction of this form of Env in complex with PGT151 and MPER-targeting antibody 10E8 at a resolution of 8.8 angstroms. These structures provide new insights into the wild-type Env structure.

Antibodies targeting the Ebola virus surface glycoprotein (EBOV GP) are implicated in protection against lethal disease, but the characteristics of the human antibody response to EBOV GP remain poorly understood. We isolated and characterized 349 GP-specific monoclonal antibodies (mAbs) from the peripheral B cells of a convalescent donor who survived the 2014 EBOV Zaire outbreak. Remarkably, 77% of the mAbs neutralize live EBOV, and several mAbs exhibit unprecedented potency. Structures of selected mAbs in complex with GP reveal a site of vulnerability located in the GP stalk region proximal to the viral membrane. Neutralizing antibodies targeting this site show potent therapeutic efficacy against lethal EBOV challenge in mice. The results provide a framework for the design of new EBOV vaccine candidates and immunotherapies.

A 4.0 Å resolution cryo-electron microscopy structure of the pre-fusion form of the trimeric spike from the human coronavirus HKU1 provides insight into how the spike protein mediates host-cell attachment and membrane fusion. Coronaviruses are responsible for respiratory infections worldwide, many of them mild, but also including severe pneumonia and the recent SARS and MERS outbreaks. The entry of coronaviruses into cells is mediated by the virus glycoprotein spike trimer, which contains the receptor-binding domain, as well as membrane fusion domains. Two papers published in this issue of Nature provide high-resolution (4Å) cryo-electron microscopy structures of pre-fusion coronavirus spike trimers. David Veesler and colleagues studied the trimer from murine hepatitis virus; Andrew Ward and colleagues used the human betacoronavirus HKU1, a cause of mild respiratory disease. The structures reveal mechanistic insights into the viral fusion process and architectural similarities to paramyxovirus F proteins, suggesting that these fusion proteins may have evolved from a distant common ancestor. HKU1 is a human betacoronavirus that causes mild yet prevalent respiratory disease1, and is related to the zoonotic SARS2 and MERS3 betacoronaviruses, which have high fatality rates and pandemic potential. Cell tropism and host range is determined in part by the coronavirus spike (S) protein4, which binds cellular receptors and mediates membrane fusion. As the largest known class I fusion protein, its size and extensive glycosylation have hindered structural studies of the full ectodomain, thus preventing a molecular understanding of its function and limiting development of effective interventions. Here we present the 4.0 Å resolution structure of the trimeric HKU1 S protein determined using single-particle cryo-electron microscopy. In the pre-fusion conformation, the receptor-binding subunits, S1, rest above the fusion-mediating subunits, S2, preventing their conformational rearrangement. Surprisingly, the S1 C-terminal domains are interdigitated and form extensive quaternary interactions that occlude surfaces known in other coronaviruses to bind protein receptors. These features, along with the location of the two protease sites known to be important for coronavirus entry, provide a structural basis to support a model of membrane fusion mediated by progressive S protein destabilization through receptor binding and proteolytic cleavage. These studies should also serve as a foundation for the structure-based design of betacoronavirus vaccine immunogens.

Broadly neutralizing antibodies targeting a highly conserved region in the hemagglutinin (HA) stem protect against influenza infection. Here, we investigate the protective efficacy of a protein (HB36.6) computationally designed to bind with high affinity to the same region in the HA stem. We show that intranasal delivery of HB36.6 affords protection in mice lethally challenged with diverse strains of influenza independent of Fc-mediated effector functions or a host antiviral immune response. This designed protein prevents infection when given as a single dose of 6.0 mg/kg up to 48 hours before viral challenge and significantly reduces disease when administered as a daily therapeutic after challenge. A single dose of 10.0 mg/kg HB36.6 administered 1-day post-challenge resulted in substantially better protection than 10 doses of oseltamivir administered twice daily for 5 days. Thus, binding of HB36.6 to the influenza HA stem region alone, independent of a host response, is sufficient to reduce viral infection and replication in vivo. These studies demonstrate the potential of computationally designed binding proteins as a new class of antivirals for influenza.

Recent studies have suggested that antibody-mediated protection against the Ebolaviruses may be achievable, but little is known about whether or not antibodies can confer cross-reactive protection against viruses belonging to diverse Ebolavirus species, such as Ebola virus (EBOV), Sudan virus (SUDV), and Bundibugyo virus (BDBV). We isolated a large panel of human monoclonal antibodies (mAbs) against BDBV glycoprotein (GP) using peripheral blood B cells from survivors of the 2007 BDBV outbreak in Uganda. We determined that a large proportion of mAbs with potent neutralizing activity against BDBV bind to the glycan cap and recognize diverse epitopes within this major antigenic site. We identified several glycan cap-specific mAbs that neutralized multiple ebolaviruses, including SUDV, and a cross-reactive mAb that completely protected guinea pigs from the lethal challenge with heterologous EBOV. Our results provide a roadmap to develop a single antibody-based treatment effective against multiple Ebolavirus infections.

ABSTRACT Due to high viral diversity, an effective HIV-1 vaccine will likely require Envs derived from multiple subtypes to generate broadly neutralizing antibodies (bNAbs). Soluble Env mimics, like the native flexibly linked (NFL) and SOSIP trimers, derived from the subtype A BG505 Env, form homogeneous, stable native-like trimers. However, other Env sequences, such as JRFL and 16055 from subtypes B and C, do so to a lesser degree. The high-resolution BG505 SOSIP crystal structures permit the identification and redesign of Env elements involved in trimer stability. Here, we identified structure trimer-derived (TD) residues that increased the propensity of the subtype B JRFL and subtype C 16055 Env sequences to form well-ordered, homogenous, and highly stable soluble trimers. The generation of these spike mimics no longer required antibody-based selection, positive or negative. Using the redesigned subtype B and C trimer representatives as respective foundations, we further stabilized the NFL TD trimers by engineering an intraprotomer disulfide linkage in the prebridging sheet, I201C-A433C (CC), that locks the gp120 in the receptor nontriggered state. We demonstrated that this disulfide pair prevented CD4 induced-conformational rearrangements in NFL trimers derived from the prototypic subtype A, B, and C representatives. Coupling the TD-based design with the engineered disulfide linkage, CC, increased the propensity of Env to form soluble highly stable spike mimics that are resistant to CD4-induced changes. These advances will allow testing of the hypothesis that such stabilized immunogens will more efficiently elicit neutralizing antibodies in small-animal models and primates. IMPORTANCE HIV-1 displays unprecedented global diversity circulating in the human population. Since the envelope glycoprotein (Env) is the target of neutralizing antibodies, Env-based vaccine candidates that address such diversity are needed. Soluble well-ordered Env mimics, typified by NFL and SOSIP trimers, are attractive vaccine candidates. However, the current designs do not allow most Envs to form well-ordered trimers. Here, we made design modifications to increase the propensity of representatives from two of the major HIV subtypes to form highly stable trimers. This approach should be applicable to other viral Envs, permitting the generation of a repertoire of homogeneous, highly stable trimers. The availability of such an array will allow us to assess if sequential or cocktail immune strategies can overcome some of the vaccine challenges presented by HIV diversity.

We describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as immunogens. In the absence of nonionic detergents, gp140 of the KNH1144 genotype, terminating at residue 681 in gp41 (SOSIP.681), has a tendency to form higher-order complexes or aggregates, which is particularly undesirable for structure-based research. We found that this aggregation in the absence of detergent does not involve the V1, V2, or V3 variable regions of gp120. Moreover, we observed that detergent forms micelles around the membrane-proximal external region (MPER) of the SOSIP.681 gp140 trimers, whereas deletion of most of the MPER residues by terminating the gp140 at residue 664 (SOSIP.664) prevented the aggregation that otherwise occurs in SOSIP.681 in the absence of detergent. Although the MPER can contribute to trimer formation, truncation of most of it only modestly reduced trimerization and lacked global adverse effects on antigenicity. Thus, the MPER deletion minimally influenced the kinetics of the binding of soluble CD4 and a CD4-binding site antibody to immobilized trimers, as detected by surface plasmon resonance. Furthermore, the MPER deletion did not alter the overall three-dimensional structure of the trimers, as viewed by negative-stain electron microscopy. Homogeneous and aggregate-free MPER-truncated SOSIP Env trimers are therefore useful for immunogenicity and structural studies.

ABSTRACT Soluble, recombinant native-like envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed for structural studies and as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design is designated SOSIP.664, but many HIV-1 env genes do not yield fully native-like trimers efficiently. One such env gene is CZA97.012 from a neutralization-resistant (tier 2) clade C virus. As appropriately purified, native-like CZA97.012 SOSIP.664 trimers induce autologous neutralizing antibodies (NAbs) efficiently in immunized rabbits, we sought to improve the efficiency with which they can be produced and to better understand the limitations to the original design. By using structure- and antigenicity-guided mutagenesis strategies focused on the V2 and V3 regions and the gp120-gp41 interface, we developed the CZA97 SOSIP.v4.2-M6.IT construct. Fully native-like, stable trimers that display multiple bNAb epitopes could be expressed from this construct in a stable CHO cell line and purified at an acceptable yield using either a PGT145 or a 2G12 bNAb affinity column. We also show that similar mutagenesis strategies can be used to improve the yields and properties of SOSIP.664 trimers of the DU422, 426c, and 92UG037 genotypes. IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for future vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein (Env) structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. The vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. Because HIV-1 is extremely variable, a practical vaccine may need to incorporate Env trimers derived from multiple different virus sequences. Accordingly, we need to understand how to make recombinant trimers from many different env genes. Here, we show how to produce trimers from a clade C virus, CZA97.012, by using an array of protein engineering techniques to improve a prototypic construct. We also show that the methods may have wider utility for other env genes, thereby further guiding immunogen design.

The high-mannose patch on the HIV-1 envelope (Env) glycoprotein is the epicenter for binding of the potent broadly neutralizing PGT121 family of antibodies, but strategies for generating such antibodies by vaccination have not been defined. We generated structures of inferred antibody intermediates by X-ray crystallography and electron microscopy to elucidate the molecular events that occurred during evolution of this family. Binding analyses revealed that affinity maturation was primarily focused on avoiding, accommodating, or binding the N137 glycan. The overall antibody approach angle to Env was defined very early in the maturation process, yet some variation evolved in the PGT121 family branches that led to differences in glycan specificities in their respective epitopes. Furthermore, we determined a crystal structure of the recombinant BG505 SOSIP.664 HIV-1 trimer with a PGT121 family member at 3.0 Å that, in concert with these antibody intermediate structures, provides insights to advance design of HIV vaccine candidates.

Major neutralizing antibody immune evasion strategies of the HIV-1 envelope glycoprotein (Env) trimer include conformational and structural instability. Stabilized soluble trimers such as BG505 SOSIP.664 mimic the structure of virion-associated Env but nevertheless sample different conformational states. Here we demonstrate that treating BG505 SOSIP.664 trimers with glutaraldehyde or a heterobifunctional cross-linker introduces additional stability with relatively modest effects on antigenicity. Thus, most broadly neutralizing antibody (bNAb) epitopes were preserved after cross-linking, whereas the binding of most weakly or nonneutralizing antibodies (non-NAb) was reduced. Cross-linking stabilized all Env conformers present within a mixed population, and individual conformers could be isolated by bNAb affinity chromatography. Both positive selection of cross-linked conformers using the quaternary epitope-specific bNAbs PGT145, PGT151, and 3BC315 and negative selection with non-NAbs against the V3 region enriched for trimer populations with improved antigenicity for bNAbs. Similar results were obtained using the clade B B41 SOSIP.664 trimer. The cross-linking method may, therefore, be useful for countering the natural conformational heterogeneity of some HIV-1 Env proteins and, by extrapolation, also vaccine immunogens from other pathogens. IMPORTANCE The development of a vaccine to induce protective antibodies against HIV-1 is of primary public health importance. Recent advances in immunogen design have provided soluble recombinant envelope glycoprotein trimers with near-native morphology and antigenicity. However, these trimers are conformationally flexible, potentially reducing B-cell recognition of neutralizing antibody epitopes. Here we show that chemical cross-linking increases trimer stability, reducing binding of nonneutralizing antibodies while largely maintaining neutralizing antibody binding. Cross-linking followed by positive or negative antibody affinity selection of individual stable conformational variants further improved the antigenic and morphological characteristics of the trimers. This approach may be generally applicable to HIV-1 Env and also to other conformationally flexible pathogen antigens.

Generation of a stable, soluble mimic of the HIV-1 envelope glycoprotein (Env) trimer on the virion surface has been considered an important first step for developing a successful HIV-1 vaccine. Recently, a soluble native-like Env trimer (BG505 SOSIP.664) has been described. This protein has facilitated major advances in the HIV-1 vaccine field, since it was the first Env immunogen that induced consistent neutralizing antibodies against a neutralization-resistant (tier 2) virus. Moreover, BG505 SOSIP.664 enabled elucidation of the atomic resolution structure of the Env trimer and facilitated the isolation and characterization of new broadly neutralizing antibodies against HIV-1. Here, we designed and characterized the BG505 SOSIP.664 trimer fused to fluorescent superfolder GFP (sfGFP), a GFP variant that allows efficient folding (BG505 SOSIP.664-sfGFP). Despite the presence of the sfGFP, the Env protein largely retained its morphology, antigenicity, glycan composition, and thermostability. In addition, we show that BG505 SOSIP.664-sfGFP can be used for fluorescence-based assays, such as flow cytometry.

ABSTRACT Generating neutralizing antibodies (nAbs) is a major goal of many current HIV-1 vaccine efforts. To be of practical value, these nAbs must be both potent and cross-reactive in order to be capable of preventing the transmission of the highly diverse and generally neutralization resistant (Tier-2) HIV-1 strains that are in circulation. The HIV-1 envelope glycoprotein (Env) spike is the only target for nAbs. To explore whether Tier-2 nAbs can be induced by Env proteins, we immunized conventional mice with soluble BG505 SOSIP.664 trimers that mimic the native Env spike. Here, we report that it is extremely difficult for murine B cells to recognize the Env epitopes necessary for inducing Tier-2 nAbs. Thus, while trimer-immunized mice raised Env-binding IgG Abs and had high-quality T follicular helper (Tfh) cell and germinal center (GC) responses, they did not make BG505.T332N nAbs. Epitope mapping studies showed that Ab responses in mice were specific to areas near the base of the soluble trimer. These areas are not well shielded by glycans and likely are occluded on virions, which is consistent with the lack of BG505.T332N nAbs. These data inform immunogen design and suggest that it is useful to obscure nonneutralizing epitopes presented on the base of soluble Env trimers and that the glycan shield of well-formed HIV Env trimers is virtually impenetrable for murine B cell receptors (BCRs). IMPORTANCE Human HIV vaccine efficacy trials have not generated meaningful neutralizing antibodies to circulating HIV strains. One possible hindrance has been the lack of immunogens that properly mimic the native conformation of the HIV envelope trimer protein. Here, we tested the first generation of soluble, native-like envelope trimer immunogens in a conventional mouse model. We attempted to generate neutralizing antibodies to neutralization-resistant circulating HIV strains. Various vaccine strategies failed to induce neutralizing antibodies to a neutralization-resistant HIV strain. Further analysis revealed that mouse antibodies targeted areas near the bottom of the soluble envelope trimers. These areas are not easily accessible on the HIV virion due to occlusion by the viral membrane and may have resulted from an absence of glycan shielding. Our results suggest that obscuring the bottom of soluble envelope trimers is a useful strategy to reduce antibody responses to epitopes that are not useful for virus neutralization.

Secretory and membrane proteins from mammalian cells undergo post-translational modifications, including N-linked glycosylation, which can result in a large number of possible glycoforms. This sample heterogeneity can be problematic for structural studies, particularly X-ray crystallography. Thus, crystal structures of heavily glycosylated proteins such as the HIV-1 Env viral spike protein have been determined by removing the majority of glycans. This step is most frequently carried out using Endoglycosidase H (EndoH) and requires that all expressed glycans be in the high-mannose form, which is often not the native glycoform. With significantly improved technologies in single-particle cryoelectron microscopy, we demonstrate that it is now possible to refine and build natively glycosylated HIV-1 Env structures in solution to 4.36 Å resolution. At this resolution we can now analyze the complete epitope of a broadly neutralizing antibody (bnAb), PGT128, in the context of the trimer expressed with native glycans.