HIV-1 Envelope Trimer Research Articles

Enhancing and shaping the immunogenicity of native-like HIV-1 envelope trimers with a two-component protein nanoparticle

The development of native-like HIV-1 envelope (Env) trimer antigens has enabled the induction of neutralizing antibody (NAb) responses against neutralization-resistant HIV-1 strains in animal models. However, NAb responses are relatively weak and narrow in specificity. Displaying antigens in a multivalent fashion on nanoparticles (NPs) is an established strategy to increase their immunogenicity. Here we present the design and characterization of two-component protein NPs displaying 20 stabilized SOSIP trimers from various HIV-1 strains. The two-component nature permits the incorporation of exclusively well-folded, native-like Env trimers into NPs that self-assemble in vitro with high efficiency. Immunization studies show that the NPs are particularly efficacious as priming immunogens, improve the quality of the Ab response over a conventional one-component nanoparticle system, and are most effective when SOSIP trimers with an apex-proximate neutralizing epitope are displayed. Their ability to enhance and shape the immunogenicity of SOSIP trimers make these NPs a promising immunogen platform.

Neutralization-guided design of HIV-1 envelope trimers with high affinity for the unmutated common ancestor of CH235 lineage CD4bs broadly neutralizing antibodies.

The CD4 binding site (CD4bs) of the HIV-1 envelope glycoprotein is susceptible to multiple lineages of broadly neutralizing antibodies (bnAbs) that are attractive to elicit with vaccines. The CH235 lineage (VH1-46) of CD4bs bnAbs is particularly attractive because the most mature members neutralize 90% of circulating strains, do not possess long HCDR3 regions, and do not contain insertions and deletions that may be difficult to induce. We used virus neutralization to measure the interaction of CH235 unmutated common ancestor (CH235 UCA) with functional Env trimers on infectious virions to guide immunogen design for this bnAb lineage. Two Env mutations were identified, one in loop D (N279K) and another in V5 (G458Y), that acted synergistically to render autologous CH505 transmitted/founder virus susceptible to neutralization by CH235 UCA. Man5-enriched N-glycans provided additional synergy for neutralization. CH235 UCA bound with nanomolar affinity to corresponding soluble native-like Env trimers as candidate immunogens. A cryo-EM structure of CH235 UCA bound to Man5-enriched CH505.N279K.G458Y.SOSIP.664 revealed interactions of the antibody light chain complementarity determining region 3 (CDR L3) with the engineered Env loops D and V5. These results demonstrate that virus neutralization can directly inform vaccine design and suggest a germline targeting and reverse engineering strategy to initiate and mature the CH235 bnAb lineage.

Structure of Antibody CAP256-VRC26.25 in Complex with HIV-1 Envelope Reveals a Combined Mode of Trimer-Apex Recognition

Antibodies targeting the V1V2-apex of the HIV-1 envelope (Env) trimer comprise one of the most commonly elicited categories of broadly neutralizing antibodies. Structures of these antibodies indicate diverse modes of Env recognition typified by antibodies of the PG9 class and the PGT145 class. The mode of recognition, however, has been unclear for the most potent of the V1V2-apex-targeting antibodies, CAP256-VRC26.25 (named for donor-lineage.clone). Here we determine the cryo-electron microscopy structure at 3.8-A resolution of the antigen-binding fragment of CAP256-VRC26.25 in complex with the Env trimer thought to have initiated the lineage. Remarkably, the 36-residue protruding loop of CAP256-VRC26.25 displayed recognition incorporating both strand-C interactions similar to the PG9 class and V1V2-apex insertion similar to the PGT145 class. Structural elements of separate antibody classes can thus intermingle to form a “combined” class, which in this case yields an antibody of extraordinary potency.

Enhancing humoral immunity via sustained-release implantable microneedle patch vaccination

Sustained exposure of lymphoid tissues to vaccine antigens promotes humoral immunity, but traditional bolus immunizations lead to rapid antigen clearance. We describe a technology to tailor vaccine kinetics in a needle-free platform translatable to human immunization. Solid pyramidal microneedle (MN) arrays were fabricated with silk fibroin protein tips encapsulating a stabilized HIV envelope trimer immunogen and adjuvant, supported on a dissolving polymer base. Upon brief skin application, vaccine-loaded silk tips are implanted in the epidermis/upper dermis where they release vaccine over a time period determined by the crystallinity of the silk matrix. Following MN immunization in mice, Env trimer was released over 2 wk in the skin, correlating with increased germinal center (GC) B cell responses, a ∼1,300-fold increase in serum IgG titers and a 16-fold increase in bone marrow (BM) plasma cells compared with bolus immunization. Thus, implantable MNs provide a practical means to substantially enhance humoral immunity to subunit vaccines.

Neutralizing antibodies for HIV-1 prevention.

In the absence of a protective vaccine against HIV-1, passive immunization using novel broadly neutralizing antibodies (bNAbs) is an attractive concept for HIV-1 prevention. Here, we summarize the results of preclinical and clinical studies of bNAbs, discuss strategies for optimizing bNAb efficacy and lay out current pathways for the development of bNAbs as prophylaxis. Passive transfer of second-generation bNAbs results inpotent protection against infection in preclinical animal models. Furthermore, multiple bNAbs targeting different epitopes on the HIV-1 envelope trimer are in clinical evaluation and have demonstrated favorable safety profiles and robust antiviral activity in chronically infected individuals. The confirmation that passive immunization with bNAb(s) will prevent HIV-1 acquisition in humans is pending and the focus of ongoing investigations. Given the global diversity of HIV-1, bNAb combinations or multispecific antibodies will most likely be required to produce the necessary breadth for effective protection. Encouraging results from preclinical and clinical studies support the development of bNAbs for prevention and a number of antibodies with exceptional breadth and potency are available for clinical evaluation. Further optimization of viral coverage and antibody half-life will accelerate the clinical implementation of bNAbs as a critical tool for HIV-1 prevention strategies.

Structure and immunogenicity of a stabilized HIV-1 envelope trimer based on a group-M consensus sequence

Stabilized HIV-1 envelope glycoproteins (Env) that resemble the native Env are utilized in vaccination strategies aimed at inducing broadly neutralizing antibodies (bNAbs). To limit the exposure of rare isolate-specific antigenic residues/determinants we generated a SOSIP trimer based on a consensus sequence of all HIV-1 group M isolates (ConM). The ConM trimer displays the epitopes of most known bNAbs and several germline bNAb precursors. The crystal structure of the ConM trimer at 3.9 Å resolution resembles that of the native Env trimer and its antigenic surface displays few rare residues. The ConM trimer elicits strong NAb responses against the autologous virus in rabbits and macaques that are significantly enhanced when it is presented on ferritin nanoparticles. The dominant NAb specificity is directed against an epitope at or close to the trimer apex. Immunogens based on consensus sequences might have utility in engineering vaccines against HIV-1 and other viruses.

A Single Substitution in gp41 Modulates the Neutralization Profile of SHIV during In Vivo Adaptation

The HIV-1 envelope glycoprotein (Env) maintains a delicate balance between mediating viral entry and escaping antibody neutralization. Adaptation during transmission of neutralization-sensitive Envs with an "open" conformation remains poorly understood. By passaging a replication-competent simian-human immunodeficiency virus carrying a highly neutralization-sensitive Env (SHIVCNE40) in rhesus macaques, we show that SHIVCNE40 develops enhanced replication kinetics associated with neutralization resistance against antibodies and autologous serum. Agp41 substitution, E658K, functions as the majordeterminant for these properties. Structural modeling and functional verification indicate that the substitution disrupts an intermolecular salt bridge with the neighboring protomer, thereby promoting fusion and facilitating immune evasion. This effect is applicable across diverse HIV-1 subtypes. Our results highlight the critical role of gp41 in shaping the neutralization profile and the overall conformation of Env during viral adaptation. The unique intermolecular salt bridge could potentially be utilized for rational vaccine design involving more stable HIV-1 envelope trimers.

Stabilization of the V2 loop improves the presentation of V2 loop–associated broadly neutralizing antibody epitopes on HIV-1 envelope trimers

A successful HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs) that target the envelope glycoprotein (Env) spike on the virus. Native-like recombinant Env trimers of the SOSIP design now serve as a platform for achieving this challenging goal. However, SOSIP trimers usually do not bind efficiently to the inferred germline precursors of bNAbs (gl-bNAbs). We hypothesized that the inherent flexibilities of the V1 and V2 variable loops in the Env trimer contribute to the poor recognition of gl-bNAb epitopes at the trimer apex that extensively involve V2 residues. To reduce local V2 flexibility and improve the binding of V2-dependent bNAbs and gl-bNAbs, we designed BG505 SOSIP.664 trimer variants containing newly created disulfide bonds intended to stabilize the V2 loop in an optimally antigenic configuration. The first variant, I184C/E190C, contained a new disulfide bond within the V2 loop, whereas the second variant, E153C/R178C, had a new disulfide bond that cross-linked V2 and V1. The resulting engineered native-like trimer variants were both more reactive with and were neutralized by V2 bNAbs and gl-bNAbs, a finding that may be valuable in the design of germline targeting and boosting trimer immunogens to create an antigenic conformation optimal for HIV vaccine development.

I-101 Quaternary contact improves the potency of broadly neutralizing antibodies to the CD4-binding site of HIV-1

Broadly neutralizing antibodies (bNAb) directly cloned from HIV-infected individuals are attracting increasing consideration for the prevention and treatment of HIV-1 infection. We recently reported the identification of a second, quaternary CD4-binding site (CD4-BS) in the native HIV-1 envelope trimer, showing that selected anti-CD4BS NAbs also establish quaternary interactions with a neighboring gp120 protomer through the extended framework 3 (FR3) loop of their heavy chain. In contrast, some potent bNAbs like VRC01 and N6 contain a shorter FR3 loop and appear to interact with a single gp120 protomer. Here, we used structure-based engineering to engraft the extended FR3 loop of VRC03 onto 4 potent anti-CD4 supersite antibodies, namely, VRC01, VRC07, N6 and 3BNC117, with the aim of enabling them to contact an adjacent gp120 promoter. With the exception of 3BNC117, all the chimeric antibodies showed an enhanced neutralizing capacity against the majority of a large HIV-1 envelope panel (208 strains), which correlated with an increased binding affinity for the pre-fusion envelope trimer. The crystal structure of one engineered antibody, N6 FR3-03, was solved in complex with a soluble stabilized trimer, BG505 SOSIP.664, which allowed to visualize the quaternary interaction of the engrafted FR3 loop with residues at the base of the V3 loop of a neighboring gp120 protomer. Thus, engraftment of the VRC03 FR3 loop enabled potent anti-CD4BS antibodies to establish quaternary contact with a neighboring protomer of the HIV-1 Env timer, leading to increased neutralization potency. FR3 loop-chimeric bNAbs may be considered for applications in HIV-1 prevention and treatment.

The HIV-1 Envelope Glycoprotein C3/V4 Region Defines a Prevalent Neutralization Epitope following Immunization

SummaryDespite recent progress in engineering native trimeric HIV-1 envelope glycoprotein (Env) mimics as vaccine candidates, Env trimers often induce vaccine-matched neutralizing antibody (NAb) responses. Understanding the specificities of autologous NAb responses and the underlying molecular mechanisms restricting the neutralization breadth is therefore informative to improve vaccine efficacy. Here, we delineate the response specificity by single B cell sorting and serum analysis of guinea pigs immunized with BG505 SOSIP.664 Env trimers. Our results reveal a prominent immune target containing both conserved and strain-specific residues in the C3/V4 region of Env in trimer-vaccinated animals. The defined NAb response shares a high degree of similarity with the early NAb response developed by a naturally infected infant from whom the HIV virus strain BG505 was isolated and later developed a broadly NAb response. Our study describes strain-specific responses and their possible evolution pathways, thereby highlighting the potential to broaden NAb responses by immunogen re-design.

Presentation of HIV-1 envelope glycoprotein trimers on diverse nanoparticle platforms.

Purpose of reviewWe will discuss recent advances in the development of nanoparticle vaccines presenting HIV-1 envelope trimer vaccines and the immunological mechanisms by which they act.Recent findingsThe multivalent presentation of Env trimers on nanoparticles is a promising strategy to increase Env immunogenicity. Recent studies have shed light on how Env nanoparticles increase lymph node trafficking and germinal center formation by using the lectin-mediated complement pathway and enhancing the interaction with naïve B cells. Meanwhile, research on different nanoparticle platforms has resulted in improved designs, such as liposomes with improved stability, and the emergence of novel platforms such as protein nanoparticles that self-assemble in vitro. Immmunogenicity studies with these nanoparticles delineate the advantages and expose the limitations of the different nanoparticle platforms.SummaryIt is becoming increasingly clear that HIV-1 vaccine research might benefit greatly from using nanoparticles presenting Env trimers, particularly during the priming stage of immunization. Among the different nanoparticles that are being pursued, in vitro-assembling nanoparticles allow for greater control of Env quality making them a promising nanoparticle platform.

Characterization of native-like HIV-1 gp140 glycoprotein expressed in insect cells

The trimeric HIV-1 envelope glycoprotein (Env) is critical for vaccine development aimed at achieving broadly-neutralizing antibody responses. The use of various recombinant expression systems and construct designs are associated with the resultant nature of produced proteins, especially in terms of glycosylation, antigenicity, and immunogenicity of the glycoprotein. Here, we explored an otherwise baculovirus cassette than classical one designed to express HIV-1 Env protein, including SOSIP mutation and Foldon moiety involvement. This improved design increased the ratio of the Env trimer fraction from ∼40% to ∼60% with respect to that of prototypical design, as indicated by high-performance size-exclusion chromatography and sedimentation velocity analysis. In addition, the design prolonged cell viability and enhanced the final yield (approximately 13–15 mg/L) after affinity purification. gp140 produced from insect cells mimicked the native-like trimer and mainly adopted glycosylation pattern of oligomannose glycans. The native-like Env proteins conferred cross-clade neutralizing antibody production in BALB/c mice. In summary, the expression of Env in insect cells by optimizing the baculovirus vector provides an alternative strategy for HIV-1 immunogen production and may benefit future Env-based HIV vaccine design.

Lattice engineering enables definition of molecular features allowing for potent small-molecule inhibition of HIV-1 entry

Diverse entry inhibitors targeting the gp120 subunit of the HIV-1 envelope (Env) trimer have been developed including BMS-626529, also called temsavir, a prodrug version of which is currently in phase III clinical trials. Here we report the characterization of a panel of small-molecule inhibitors including BMS-818251, which we show to be >10-fold more potent than temsavir on a cross-clade panel of 208-HIV-1 strains, as well as the engineering of a crystal lattice to enable structure determination of the interaction between these inhibitors and the HIV-1 Env trimer at higher resolution. By altering crystallization lattice chaperones, we identify a lattice with both improved diffraction and robust co-crystallization of HIV-1 Env trimers from different clades complexed to entry inhibitors with a range of binding affinities. The improved diffraction reveals BMS-818251 to utilize functional groups that interact with gp120 residues from the conserved β20-β21 hairpin to improve potency.

Enhancing Humoral Responses Against HIV Envelope Trimers via Nanoparticle Delivery with Stabilized Synthetic Liposomes

An HIV vaccine capable of eliciting durable neutralizing antibody responses continues to be an important unmet need. Multivalent nanoparticles displaying a high density of envelope trimers may be promising immunogen forms to elicit strong and durable humoral responses to HIV, but critical particle design criteria remain to be fully defined. To this end, we developed strategies to covalently anchor a stabilized gp140 trimer, BG505 MD39, on the surfaces of synthetic liposomes to study the effects of trimer density and vesicle stability on vaccine-elicited humoral responses in mice. CryoEM imaging revealed homogeneously distributed and oriented MD39 on the surface of liposomes irrespective of particle size, lipid composition, and conjugation strategy. Immunization with covalent MD39-coupled liposomes led to increased germinal center and antigen-specific T follicular helper cell responses and significantly higher avidity serum MD39-specific IgG responses compared to immunization with soluble MD39 trimers. A priming immunization with liposomal-MD39 was important for elicitation of high avidity antibody responses, regardless of whether booster immunizations were administered with either soluble or particulate trimers. The stability of trimer anchoring to liposomes was critical for these effects, as germinal center and output antibody responses were further increased by liposome compositions incorporating sphingomyelin that exhibited high in vitro stability in the presence of serum. Together these data highlight key liposome design features for optimizing humoral immunity to lipid nanoparticle immunogens.

HIV-1 envelope glycan modifications that permit neutralization by germline-reverted VRC01-class broadly neutralizing antibodies.

Broadly neutralizing antibody (bnAb) induction is a high priority for effective HIV-1 vaccination. VRC01-class bnAbs that target the CD4 binding site (CD4bs) of trimeric HIV-1 envelope (Env) glycoprotein spikes are particularly attractive to elicit because of their extraordinary breadth and potency of neutralization in vitro and their ability to protect against infection in animal models. Glycans bordering the CD4bs impede the binding of germline-reverted forms of VRC01-class bnAbs and therefore constitute a barrier to early events in initiating the correct antibody lineages. Deleting a subset of these glycans permits Env antigen binding but not virus neutralization, suggesting that additional barriers impede germline-reverted VRC01-class antibody binding to functional Env trimers. We investigated the requirements for functional Env trimer engagement of VRC01-class naïve B cell receptors by using virus neutralization and germline-reverted antibodies as surrogates for the interaction. Targeted deletion of a subset of N-glycans bordering the CD4bs, combined with Man5 enrichment of remaining N-linked glycans that are otherwise processed into larger complex-type glycans, rendered HIV-1 426c Env-pseudotyped virus (subtype C, transmitted/founder) highly susceptible to neutralization by near germline forms of VRC01-class bnAbs. Neither glycan modification alone rendered the virus susceptible to neutralization. The potency of neutralization in some cases rivaled the potency of mature VRC01 against wildtype viruses. Neutralization by the germline-reverted antibodies was abrogated by the known VRC01 resistance mutation, D279K. These findings improve our understanding of the restrictions imposed by glycans in eliciting VRC01-class bnAbs and enable a neutralization-based strategy to monitor vaccine-elicited early precursors of this class of bnAbs.

Structural Rearrangements Maintain the Glycan Shield of an HIV-1 Envelope Trimer After the Loss of a Glycan

The HIV-1 envelope (Env) glycoprotein is the primary target of the humoral immune response and a critical vaccine candidate. However, Env is densely glycosylated and thereby substantially protected from neutralisation. Importantly, glycan N301 shields V3 loop and CD4 binding site epitopes from neutralising antibodies. Here, we use molecular dynamics techniques to evaluate the structural rearrangements that maintain the protective qualities of the glycan shield after the loss of glycan N301. We examined a naturally occurring subtype C isolate and its N301A mutant; the mutant not only remained protected against neutralising antibodies targeting underlying epitopes, but also exhibited an increased resistance to the VRC01 class of broadly neutralising antibodies. Analysis of this mutant revealed several glycans that were responsible, independently or through synergy, for the neutralisation resistance of the mutant. These data provide detailed insight into the glycan shield’s ability to compensate for the loss of a glycan, as well as the cascade of glycan movements on a protomer, starting at the point mutation, that affects the integrity of an antibody epitope located at the edge of the diminishing effect. These results present key, previously overlooked, considerations for HIV-1 Env glycan research and related vaccine studies.

Completeness of HIV-1 Envelope Glycan Shield at Transmission Determines Neutralization Breadth

SUMMARYDensely arranged N-linked glycans shield the HIV-1 envelope (Env) trimer from antibody recognition. Strain-specific breaches in this shield (glycan holes) can be targets of vaccine-induced neutralizing antibodies that lack breadth. To understand the interplay between glycan holes and neutralization breadth in HIV-1 infection, we developed a sequence-and structure-based approach to identify glycan holes for individual Env sequences that are shielded in most M-group viruses. Applying this approach to 12 longitudinally followed individuals, we found that transmitted viruses with more intact glycan shields correlated with development of greater neutralization breadth. Within 2 years, glycan acquisition filled most glycan holes present at transmission, indicating escape from hole-targeting neutralizing antibodies. Glycan hole filling generally preceded the time to first detectable breadth, although time intervals varied across hosts. Thus, completely glycan-shielded viruses were associated with accelerated neutralization breadth development, suggesting that Env immunogens with intact glycan shields may be preferred components of AIDS vaccines.

Interdomain Stabilization Impairs CD4 Binding and Improves Immunogenicity of the HIV-1 Envelope Trimer

The HIV-1 envelope (Env) spike is a trimer of gp120/gp41 heterodimers that mediates viral entry. Binding to CD4 on the host cell membrane is the first essential step for infection but disrupts the native antigenic state of Env, posing a key obstacle to vaccine development. We locked the HIV-1 Env trimer in a pre-fusion configuration, resulting in impaired CD4 binding and enhanced binding to broadly neutralizing antibodies. This design was achieved via structure-guided introduction of neo-disulfide bonds bridging the gp120 inner and outer domains and was successfully applied to soluble trimers and native gp160 from different HIV-1 clades. Crystallization illustrated the structural basis for CD4-binding impairment. Immunization of rabbits with locked trimers from two different clades elicited neutralizing antibodies against tier-2 viruses with a repaired glycan shield regardless of treatment with a functional CD4 mimic. Thus, interdomain stabilization provides a widely applicable template for the design of Env-based HIV-1 vaccines.

C-103 Germline-targeting vaccine design for HIV

Induction of broadly neutralizing antibodies (bnAbs) against HIV is a holy grail in the efforts to develop an HIV vaccine. The HIV envelope trimer, the target of both bnAbs and strain-specific neutralizing antibodies, is heavily glycosylated and has an antigenic surface that varies significantly among different HIV isolates. Therefore, vaccine elicitation of bnAbs using stable trimer immunogens that mimic native spikes presents major challenges. Potent HIV bnAbs are typically highly mutated and their germline-reverted forms typically show little or no reactivity to wild-type HIV antigens. Thus, key barriers to bnAb elicitation include activating bnAb precursor B cells and guiding bnAb maturation. With the ultimate aim of developing a vaccine that consistently induces potent bnAbs in humans, we are pursuing a strategy to develop germline-targeting priming immunogens to initiate bnAb development, following by structure-guided boost immunogens to shepherd B cell maturation to the development of bnAbs.

A Universal Approach to Optimize the Folding and Stability of Prefusion-Closed HIV-1 Envelope Trimers

The heavily glycosylated native-like envelope (Env) trimer of HIV-1 is expected to have low immunogenicity, whereas misfolded forms are often highly immunogenic. High-quality correctly folded Envs may therefore be critical for developing a vaccine that induces broadly neutralizing antibodies. Moreover, the high variability of Env may require immunizations with multiple Envs. Here, we report a universal strategy that provides for correctly folded Env trimers of high quality and yield through a repair-and-stabilize approach. In the repair stage, we utilized a consensus strategy that substituted rare strain-specific residues with more prevalentones. The stabilization stage involved structure-based design and experimental assessment confirmed by crystallographic feedback. Regions important for the refolding of Env were targeted for stabilization. Notably, the α9-helix and an intersubunit β sheet proved to be critical for trimer stability. Our approach provides a means to produce prefusion-closed Env trimers from diverse HIV-1 strains, a substantial advance for vaccine development.