Structural and functional studies of HIV envelope glycoprotein (Env) as a transmembrane protein have long been complicated by challenges associated with inherent flexibility of the molecule and the membrane-embedded hydrophobic regions. Here, we present approaches for incorporating full-length, wild-type HIV-1 Env, as well as C-terminally truncated and stabilized versions, into lipid assemblies, providing a modular platform for Env structural studies by single particle electron microscopy. We reconstitute a full-length Env clone into a nanodisc, complex it with a membrane-proximal external region (MPER) targeting antibody 10E8, and structurally define the full quaternary epitope of 10E8 consisting of lipid, MPER, and ectodomain contacts. By aligning this and other Env-MPER antibody complex reconstructions with the lipid bilayer, we observe evidence of Env tilting as part of the neutralization mechanism for MPER-targeting antibodies. We also adapt the platform toward vaccine design purposes by introducing stabilizing mutations that allow purification of unliganded Env with a peptidisc scaffold.

Recent efforts toward HIV vaccine development include the design of immunogens that can engage B cell receptors with the potential to affinity mature into broadly neutralizing antibodies (bnAbs). V2-apex bnAbs, which bind a protein-glycan region on HIV envelope glycoprotein (Env) trimer, are among the most broad and potent described. We show here that a rare “glycan hole” at the V2 apex is enriched in HIV isolates neutralized by inferred precursors of prototype V2-apex bnAbs. To investigate whether this feature could focus neutralizing responses onto the apex bnAb region, we immunized wild-type rabbits with soluble trimers adapted from these Envs. Potent autologous tier 2 neutralizing responses targeting basic residues in strand C of the V2 region, which forms the core epitope for V2-apex bnAbs, were observed. Neutralizing monoclonal antibodies (mAbs) derived from these animals display features promising for subsequent broadening of the response.

A summary report from work presented at the 2017 Keystone Symposium on HIV Vaccines.

The HIV-1 envelope (Env) is a glycoprotein consisting of a trimer of heterodimers containing gp120 and gp41 subunits that mediates virus entry and is a major target of broadly neutralizing antibodies (bnAbs) developed during infection in some individuals. The engagement of the HIV-1 gp120 glycoprotein to the host CD4 protein triggers conformational changes in gp120 that allow its binding to co-receptors and is necessary for virus entry to establish infection. Native-like HIV-1 Env immunogens representing distinct clades have been proposed to improve immunogenicity. In the present study, we examined the basis of resistance of an HIV-1 B/C recombinant Env (LT5.J4b12C) to non-neutralizing antibodies targeting CD4-induced Env epitopes in the presence of soluble CD4 (sCD4). Using native polyacrylamide gel shift assay and negative-stain EM, we found that the prefusion conformational state of LT5.J4b12C trimeric Env was largely unaffected in the presence of excess sCD4 with most Env trimers appearing to be in a ligand-free state. This resistance to CD4-induced conformational changes was associated with a lower affinity for CD4. Moreover, the LT5.J4b12C trimeric Env preferentially bound to the neutralizing antibodies compared with non-neutralizing antibodies. Taken together, we report on an HIV-1 B/C recombinant, native-like trimeric Env protein that is highly resistant to CD4-induced conformational changes but displays epitopes recognized by a diverse array of bnAbs. Such features make this B/C recombinant trimeric Env a useful addition to the pool of other recently identified native-like HIV-1 Env trimers suitable for use as antigenic bait for bnAb isolation, structural studies, and use as potential immunogens.

Extensive shielding by N-glycans on the surface of the HIV envelope glycoproteins (Env) restricts B cell recognition of conserved neutralizing determinants. Elicitation of broadly neutralizing antibodies (bNAbs) in selected HIV-infected individuals reveals that Abs capable of penetrating the glycan shield can be generated by the B cell repertoire. Accordingly, we sought to determine if targeted N-glycan deletion might alter antibody responses to Env. We focused on the conserved CD4 binding site (CD4bs) since this is a known neutralizing determinant that is devoid of glycosylation to allow CD4 receptor engagement, but is ringed by surrounding N-glycans. We selectively deleted potential N-glycan sites (PNGS) proximal to the CD4bs on well-ordered clade C 16055 native flexibly linked (NFL) trimers to potentially increase recognition by naïve B cells in vivo. We generated glycan-deleted trimer variants that maintained native-like conformation and stability. Using a panel of CD4bs-directed bNAbs, we demonstrated improved accessibility of the CD4bs on the N-glycan-deleted trimer variants. We showed that pseudoviruses lacking these Env PNGSs were more sensitive to neutralization by CD4bs-specific bNAbs but remained resistant to non-neutralizing mAbs. We performed rabbit immunogenicity experiments using two approaches comparing glycan-deleted to fully glycosylated NFL trimers. The first was to delete 4 PNGS sites and then boost with fully glycosylated Env; the second was to delete 4 sites and gradually re-introduce these N-glycans in subsequent boosts. We demonstrated that the 16055 PNGS-deleted trimers more rapidly elicited serum antibodies that more potently neutralized the CD4bs-proximal-PNGS-deleted viruses in a statistically significant manner and strongly trended towards increased neutralization of fully glycosylated autologous virus. This approach elicited serum IgG capable of cross-neutralizing selected tier 2 viruses lacking N-glycans at residue N276 (natural or engineered), indicating that PNGS deletion of well-ordered trimers is a promising strategy to prime B cell responses to this conserved neutralizing determinant.

Induction of broadly neutralizing antibodies (bNAbs) by HIV-1 envelope glycoprotein immunogens would be a major advance toward an effective vaccine. A critical step in this process is the activation of naive B cells expressing germline (gl) antibody precursors that have the potential to evolve into bNAbs. Here, we reengineered the BG505 SOSIP.664 glycoprotein to engage gl precursors of bNAbs that target either the trimer apex or the CD4-binding site. The resulting BG505 SOSIP.v4.1-GT1 trimer binds multiple bNAb gl precursors in vitro. Immunization experiments in knock-in mice expressing gl-VRC01 or gl-PGT121 show that this trimer activates B cells in vivo, resulting in the secretion of specific antibodies into the sera. A crystal structure of the gl-targeting trimer at 3.2-Å resolution in complex with neutralizing antibodies 35O22 and 9H+109L reveals a native-like conformation and the successful incorporation of design features associated with binding of multiple gl-bNAb precursors.

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.

Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed nanoparticles from eukaryotic cells would be advantageous, but in practice, they often secrete poorly. Here we show that designed hydrophobic interfaces that drive nanoparticle assembly are often predicted to form cryptic transmembrane domains, suggesting that interaction with the membrane insertion machinery could limit efficient secretion. We develop a general computational protocol, the Degreaser, to design away cryptic transmembrane domains without sacrificing protein stability. The retroactive application of the Degreaser to previously designed nanoparticle components and nanoparticles considerably improves secretion, and modular integration of the Degreaser into design pipelines results in new nanoparticles that secrete as robustly as naturally occurring protein assemblies. Both the Degreaser protocol and the nanoparticles we describe may be broadly useful in biotechnological applications.

Broadly neutralizing antibodies (bnAbs) against HIV-1 protect from infection and reduce viral load upon therapeutic applications. However no vaccine was able so far to induce bnAbs demanding their expensive biotechnological production. For clinical applications, nanobodies (VHH) derived from heavy chain only antibodies from Camelidae, may be better suited due to their small size, high solubility/stability and extensive homology to human VH3 genes. Here we selected broadly neutralizing nanobodies by phage display after immunization of dromedaries with different soluble trimeric envelope proteins derived from HIV-1 subtype C. We identified 25 distinct VHH families binding trimeric Env, of which 6 neutralized heterologous primary isolates of various HIV-1 subtypes in a standardized in vitro neutralization assay. The complementary neutralization pattern of two selected VHHs in combination covers 19 out of 21 HIV-1 strains from a standardized panel of epidemiologically relevant HIV-1 subtypes. The CD4 binding site was preferentially targeted by the broadly neutralizing VHHs as determined by competition ELISAs and 3D models of VHH-Env complexes derived from negative stain electron microscopy. The nanobodies identified here are excellent candidates for further preclinical/clinical development for prophylactic and therapeutic applications due to their potency and their complementary neutralization patterns covering the majority of epidemiologically relevant HIV-1 subtypes.

The Electron Microscopy Hole Punch (EMHP) is a streamlined suite of tools for quick assessment, sorting and hole masking of electron micrographs. With recent advances in single-particle electron cryo-microscopy (cryo-EM) data processing allowing for the rapid determination of protein structures using a smaller computational footprint, we saw the need for a fast and simple tool for data pre-processing that could run independent of existing high-performance computing (HPC) infrastructures. EMHP provides a data preprocessing platform in a small package that requires minimal python dependencies to function. https://www.bitbucket.org/chazbot/emhp Apache 2.0 License Supplementary data are available at Bioinformatics online.

No immunogen to date has reliably elicited broadly neutralizing antibodies (bnAbs) to HIV in humans or animal models. Advances in the design of immunogens (BG505 SOSIP) that antigenically mimic the HIV envelope glycoprotein (Env)1 have improved the elicitation of potent isolate-specific Ab responses in rabbits2 and macaques3, but so far failed to induce bnAbs. One possible contributor to this failure is that the relevant antibody repertoires are poorly suited to target somewhat occluded conserved epitope regions on Env relative to exposed variable epitopes. To test this hypothesis, we immunized four cows with BG505 SOSIP. The antibody repertoire of cows contains long third heavy chain complementary determining regions (HCDR3) with an ultralong subset that can reach over 70 amino acids in length4–9. Remarkably, BG505 SOSIP immunization resulted in rapid elicitation of broad and potent serum antibody responses in all four cows. Longitudinal serum analysis for one cow showed the development of neutralization breadth (20%, n = 117 cross-clade isolates) in 42 days and 96% breadth (n = 117) at 381 days. A monoclonal antibody (mAb) isolated from this cow harbored an ultralong HCDR3 of 60 amino acids and neutralized 72% of cross-clade isolates (n = 117) with a potent median IC50 of 0.028 μg/ml. We note that breadth was elicited with a single trimer immunogen and did not require additional envelope diversity. Immunization of cows may provide an avenue to rapidly generate antibody prophylactics and therapeutics to address disease agents that have evolved to avoid human antibody responses.

Native-like trimers of the SOSIP design are being developed as immunogens in human immunodeficiency virus type 1 (HIV-1) vaccine development programs. These trimers display the epitopes for multiple broadly neutralizing antibodies (bNAbs) but can also expose binding sites for some types of nonneutralizing antibodies (non-NAbs). Among the latter are epitopes in the gp120 V3 region that are highly immunogenic when SOSIP trimers are evaluated in animal models. It is presently uncertain whether antibodies against V3 can interfere with the induction of NAbs, but there are good arguments in favor of suppressing such “off-target” immune responses. Accordingly, we have assessed how to minimize the exposure of V3 non-NAb epitopes and thereby reduce their immunogenicity by introducing N-glycans within the V3 region of BG505 SOSIP trimers. We found that inserting glycans at positions 306 and 314 (termed M1 and M7) markedly reduced V3 antigenicity while improving the presentation of trimer apex bNAb epitopes. Both added glycans were shown to be predominantly of the Man6GlcNAc2 form. The additional introduction of the E64K ground-state stabilizing substitution markedly reduced or ablated soluble CD4 (sCD4) induction of non-NAb epitopes in V3 and/or associated with the coreceptor binding site. When a V3 glycan- and E64K-modified trimer variant, BG505 SOSIP.664-E64K.M1M7, was tested in rabbits, V3 immunogenicity was eliminated while the autologous NAb response was unchanged. IMPORTANCE Trimeric proteins are being developed for future HIV-1 vaccine trials in humans, with the goal of eliciting broadly active neutralizing antibodies (NAbs) that are active against a wide variety of circulating strains. In animal models, the present generation of native-like trimer immunogens, exemplified by the BG505 SOSIP.664 construct, induces narrow-specificity antibodies against the neutralization-resistant (tier-2), sequence-matched virus and more broadly active antibodies against sequence-divergent atypically neutralization-sensitive (tier-1) viruses. A concern in the trimer immunogen design field has been whether the latter off-target antibodies might interfere with the induction of the more desired responses to tier-2 epitopes. Here, we have inserted two glycans into the dominant site for tier-1 NAbs, the gp120 V3 region, to block the induction of off-target antibodies. We characterized the new trimers, tested them as immunogens in rabbits, and found that the blocking glycans eliminated the induction of tier-1 NAbs to V3-epitopes.

The development of stabilized recombinant HIV envelope trimers that mimic the virion surface molecule has increased enthusiasm for a neutralizing antibody (nAb)-based HIV vaccine. However, there is limited experience with recombinant trimers as immunogens in nonhuman primates, which are typically used as a model for humans. Here, we tested multiple immunogens and immunization strategies head-to-head to determine their impact on the quantity, quality, and kinetics of autologous tier 2 nAb development. A bilateral, adjuvanted, subcutaneous immunization protocol induced reproducible tier 2 nAb responses after only two immunizations 8 weeks apart, and these were further enhanced by a third immunization with BG505 SOSIP trimer. We identified immunogens that minimized non-neutralizing V3 responses and demonstrated that continuous immunogen delivery could enhance nAb responses. nAb responses were strongly associated with germinal center reactions, as assessed by lymph node fine needle aspiration. This study provides a framework for preclinical and clinical vaccine studies targeting nAb elicitation.

For many enveloped viruses, binding to a receptor(s) on a host cell acts as a first step in a series of events culminating in fusion with the host cell membrane and transfer of genetic material for replication [for review see1,2]. The envelope glycoprotein (Env) trimer on the surface of HIV is responsible for receptor binding and fusion. While Env can tolerate a high degree of mutation in five variable regions (V1-V5), and also at N-linked glycosylation sites that contribute roughly half the mass of Env, the functional sites for recognition of receptor CD4 and co-receptor CXCR4/CCR5 are conserved and essential for viral fitness. Soluble SOSIP Env trimers are structural and antigenic mimics of the pre-fusion native, surface-presented Env3,4, targets of broadly neutralizing antibodies (bnAbs). Thus, they are attractive immunogens for vaccine development [for review see5–8]. Here we present high-resolution cryo-electron microscopy (cryoEM) structures of subtype B B41 SOSIP Env trimers in complex with CD4 and antibody 17b, or with antibody b12, at resolutions of ~3.7 Å and ~3.6 Å, respectively, and compare them to cryoEM reconstructions of ligand-free B41 SOSIP Env trimers or in complex with either CD4 or CD4bs antibody PGV04, at ~5.6 Å, ~5.2 Å and ~7.4 Å, respectively. Consequently, we present the most complete description and understanding of the CD4/17b-induced intermediate and provide the molecular basis of the receptor-binding induced conformational change required for HIV-1 entry into host cells. Both CD4 and b12 induce large, previously uncharacterized conformational rearrangements in the gp41 subunits, and the fusion peptide becomes more buried in a newly formed pocket. These structures provide key details on the biological function of the type I viral fusion machine from HIV-1 as well as new templates for inhibitor design.

For many enveloped viruses, binding to a receptor(s) on a host cell acts as a first step in a series of events culminating in fusion with the host cell membrane and transfer of genetic material for replication [for review see1,2]. The envelope glycoprotein (Env) trimer on the surface of HIV is responsible for receptor binding and fusion. While Env can tolerate a high degree of mutation in five variable regions (V1-V5), and also at N-linked glycosylation sites that contribute roughly half the mass of Env, the functional sites for recognition of receptor CD4 and co-receptor CXCR4/CCR5 are conserved and essential for viral fitness. Soluble SOSIP Env trimers are structural and antigenic mimics of the pre-fusion native, surface-presented Env3,4, targets of broadly neutralizing antibodies (bnAbs). Thus, they are attractive immunogens for vaccine development [for review see5–8]. Here we present high-resolution cryo-electron microscopy (cryoEM) structures of subtype B B41 SOSIP Env trimers in complex with CD4 and antibody 17b, or with antibody b12, at resolutions of ~3.7 Å and ~3.6 Å, respectively, and compare them to cryoEM reconstructions of ligand-free B41 SOSIP Env trimers or in complex with either CD4 or CD4bs antibody PGV04, at ~5.6 Å, ~5.2 Å and ~7.4 Å, respectively. Consequently, we present the most complete description and understanding of the CD4/17b-induced intermediate and provide the molecular basis of the receptor-binding induced conformational change required for HIV-1 entry into host cells. Both CD4 and b12 induce large, previously uncharacterized conformational rearrangements in the gp41 subunits, and the fusion peptide becomes more buried in a newly formed pocket. These structures provide key details on the biological function of the type I viral fusion machine from HIV-1 as well as new templates for inhibitor design.

The production of native-like recombinant versions of the HIV-1 envelope glycoprotein (Env) trimer requires overcoming the natural flexibility and instability of the complex. The engineered BG505 SOSIP.664 trimer mimics the structure and antigenicity of native Env. Here, we describe how the introduction of new disulfide bonds between the glycoprotein (gp)120 and gp41 subunits of SOSIP trimers of the BG505 and other genotypes improves their stability and antigenicity, reduces their conformational flexibility, and helps maintain them in the unliganded conformation. The resulting next-generation SOSIP.v5 trimers induce strong autologous tier-2 neutralizing antibody (NAb) responses in rabbits. In addition, the BG505 SOSIP.v6 trimers induced weak heterologous NAb responses against a subset of tier-2 viruses that were not elicited by the prototype BG505 SOSIP.664. These stabilization methods can be applied to trimers from multiple genotypes as components of multivalent vaccines aimed at inducing broadly NAbs (bNAbs).

Many viral surface glycoproteins and cell surface receptors are homo-oligomers1-4, and hence can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5-8. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We used this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The best characterized of these designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.

Designing an effective HIV-1 envelope glycoprotein (Env) immunogen for elicitation of broadly neutralizing antibodies (bNAbs) is a challenging task because of the high sequence diversity, heavy glycosylation, and inherent meta-stability of Env. Based on the antigenic profile of recently isolated bNAbs, the rational approach to immunogen design is to make a stable version of the Env trimer, which mimics the native trimeric Env present on the viral surface. The SOSIP.664 form of a clade A Env, BG505, yields a homogeneous and well ordered prefusion trimeric form, which maintains structural integrity and desired antigenicity. Following the same approach, we attempted to stabilize a naturally occurring efficiently cleaved clade C Env, namely 4-2.J41, isolated from an Indian patient. Although the SOSIP form of 4-2.J41 failed to produce reasonably well ordered trimers, the 4-2.J41.SOSIP.664 Env could be stabilized in a native-like trimeric form by swapping a domain from BG505 Env to 4-2.J41 Env. Using various biochemical and biophysical means we confirmed that this engineered Env is cleaved, trimeric, and it retains its native-like quaternary conformation exposing mostly broadly neutralizing epitopes. Moreover, introduction of a disulfide bond in the bridging sheet region further stabilized the closed conformation of the Env. Thus, our 4-2.J41.SOSIP.664 Env adds to the increasing pool of potential immunogens for a HIV-1 vaccine, particularly for clade C, which is the most prevalent in India and many other countries. Besides, the approach used to stabilize the 4-2.J41 Env may be used successfully with Envs from other HIV-1 strains as well. Additionally, a soluble native trimeric form of an efficiently cleaved membrane-bound Env, 4-2.J41, may be beneficial for immunization studies using various prime-boost strategies.