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

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

A Broadly Neutralizing Antibody Targets the Dynamic HIV Envelope Trimer Apex via a Long, Rigidified, and Anionic ß-Hairpin Structure.

Broadly neutralizing antibodies (bnAbs) to HIV delineate vaccine targets and are prophylactic and therapeutic agents. Some of the most potent bnAbs target a quaternary epitope at the apex of the surface HIV envelope (Env) trimer. Using cryo-electron microscopy, we solved the atomic structure of an apex bnAb, PGT145, in complex with Env. We showed that the long anionic HCDR3 of PGT145 penetrated between glycans at the trimer 3-fold axis, to contact peptide residues from all three Env protomers, and thus explains its highly trimer-specific nature. Somatic hypermutation in the other CDRs of PGT145 were crucially involved in stabilizing the structure of the HCDR3, similar to bovine antibodies, to aid in recognition of a cluster of conserved basic residues hypothesized to facilitate trimer disassembly during viral entry. Overall, the findings exemplify the creative solutions that the human immune system can evolve to recognize a conserved motif buried under a canopy of glycans.

Glycan heterogeneity as a cause of the persistent fraction in HIV-1 neutralization.

Neutralizing antibodies (NAbs) to multiple epitopes on the HIV-1-envelope glycoprotein (Env) have been isolated from infected persons. The potency of NAbs is measured more often than the size of the persistent fraction of infectivity at maximum neutralization, which may also influence preventive efficacy of active or passive immunization and the therapeutic outcome of the latter. Many NAbs neutralize HIV-1 CZA97.012, a clone of a Clade-C isolate, to ~100%. But here NAb PGT151, directed to a fusion-peptide epitope, left a persistent fraction of 15%. NAb PGT145, ligating the Env-trimer apex, left no detectable persistent fraction. The divergence in persistent fractions was further analyzed by depletion of pseudoviral populations of the most PGT151- and PGT145-reactive virions. Thereby, neutralization by the non-depleting NAb increased, whereas neutralization by the depleting NAb decreased. Furthermore, depletion by PGT151 increased sensitivity to autologous neutralization by sera from rabbits immunized with soluble native-like CZA97.012 trimer: substantial persistent fractions were reduced. NAbs in these sera target epitopes comprising residue D411 at the V4-ß19 transition in a defect of the glycan shield on CZA97.012 Env. NAb binding to affinity-fractionated soluble native-like CZA97.012 trimer differed commensurately with neutralization in analyses by ELISA and surface plasmon resonance. Glycan differences between PGT151- and PGT145-purified trimer fractions were then demonstrated by mass spectrometry, providing one explanation for the differential antigenicity. These differences were interpreted in relation to a new structure at 3.4-Å resolution of the soluble CZA97.012 trimer determined by cryo-electron microscopy. The trimer adopted a closed conformation, refuting apex opening as the cause of reduced PGT145 binding to the PGT151-purified form. The evidence suggests that differences in binding and neutralization after trimer purification or pseudovirus depletion with PGT145 or PGT151 are caused by variation in glycosylation, and that some glycan variants affect antigenicity through direct effects on antibody contacts, whereas others act allosterically.

Conformational antigenic heterogeneity as a cause of the persistent fraction in HIV-1 neutralization.

BACKGROUND: Neutralizing antibodies (NAbs) protect against HIV-1 acquisition in animal models and show promise in treatment of infection. They act by binding to the viral envelope glycoprotein (Env), thereby blocking its receptor interactions and fusogenic function. The potency of neutralization is largely determined by affinity. Less well explained is the persistent fraction, the plateau of remaining infectivity at the highest antibody concentrations. RESULTS: We observed different persistent fractions for neutralization of pseudovirus derived from two Tier-2 isolates of HIV-1, BG505 (Clade A) and B41 (Clade B): it was pronounced for B41 but not BG505 neutralization by NAb PGT151, directed to the interface between the outer and transmembrane subunits of Env, and negligible for either virus by NAb PGT145 to an apical epitope. Autologous neutralization by poly- and monoclonal NAbs from rabbits immunized with soluble native-like B41 trimer also left substantial persistent fractions. These NAbs largely target a cluster of epitopes lining a hole in the dense glycan shield of Env around residue 289. We partially depleted B41-virion populations by incubating them with PGT145- or PGT151-conjugated beads. Each depletion reduced the sensitivity to the depleting NAb and enhanced it to the other. Autologous neutralization by the rabbit NAbs was decreased for PGT145-depleted and enhanced for PGT151-depleted B41 pseudovirus. Those changes in sensitivity encompassed both potency and the persistent fraction. We then compared soluble native-like BG505 and B41 Env trimers affinity-purified by each of three NAbs: 2G12, PGT145, or PGT151. Surface plasmon resonance showed differences among the fractions in antigenicity, including kinetics and stoichiometry, congruently with the differential neutralization. The large persistent fraction after PGT151 neutralization of B41 was attributable to low stoichiometry, which we explained structurally by clashes that the conformational plasticity of B41 Env causes. CONCLUSION: Distinct antigenic forms even of clonal HIV-1 Env, detectable among soluble native-like trimer molecules, are distributed over virions and may profoundly mold neutralization of certain isolates by certain NAbs. Affinity purifications with some antibodies may yield immunogens that preferentially expose epitopes for broadly active NAbs, shielding less cross-reactive ones. NAbs reactive with multiple conformers will together reduce the persistent fraction after passive and active immunization.

Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens.

Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Computational design of protein nanoparticles with differing sizes and geometries enables combination with antigens of choice to test novel multimerization concepts in immunization strategies where the goal is to improve the induction and maturation of neutralizing antibody lineages. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to an underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. When the immunogenicity of ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle or as soluble proteins were compared in rabbits, the two immunogens elicited similar serum antibody binding titers against the trimer component. Neutralizing antibody titers were slightly elevated in the animals given the nanoparticle immunogen and were initially more focused to the trimer apex. Altogether, our findings indicate that tetrahedral nanoparticles can be successfully applied for presentation of HIV Env trimer immunogens; however, the optimal implementation to different immunization strategies remains to be determined.

Neutralizing Antibody Responses Induced by HIV-1 Envelope Glycoprotein SOSIP Trimers Derived from Elite Neutralizers.

The induction of broadly neutralizing antibodies (bNAbs) is a major goal in vaccine research. HIV-1-infected individuals that develop exceptionally strong bNAb responses, termed elite neutralizers, can inform vaccine design by providing blueprints for the induction of similar bNAb responses. We describe a new recombinant native-like envelope glycoprotein (Env) SOSIP trimer, termed AMC009, based on the viral founder sequences of an elite neutralizer. The subtype B AMC009 SOSIP protein formed stable native-like trimers that displayed multiple bNAb epitopes. Overall, its structure at 4.3-Å resolution was similar to that of BG505 SOSIP.664. The AMC009 trimer resembled one from a second elite neutralizer, AMC011, in having a dense and complete glycan shield. When tested as immunogens in rabbits, the AMC009 trimers did not induce autologous neutralizing antibody (NAb) responses efficiently while the AMC011 trimers did so very weakly, outcomes that may reflect the completeness of their glycan shields. The AMC011 trimer induced antibodies that occasionally cross-neutralized heterologous tier 2 viruses, sometimes at high titer. Cross-neutralizing antibodies were more frequently elicited by a trivalent combination of AMC008, AMC009, and AMC011 trimers, all derived from subtype B viruses. Each of these three individual trimers could deplete the NAb activity from the rabbit sera. Mapping the polyclonal sera by electron microscopy revealed that antibodies of multiple specificities could bind to sites on both autologous and heterologous trimers. These results advance our understanding of how to use Env trimers in multivalent vaccination regimens and the immunogenicity of trimers derived from elite neutralizers.IMPORTANCE Elite neutralizers, i.e., individuals who developed unusually broad and potent neutralizing antibody responses, might serve as blueprints for HIV-1 vaccine design. Here, we studied the immunogenicity of native-like recombinant envelope glycoprotein (Env) trimers based on viral sequences from elite neutralizers. While immunization with single trimers from elite neutralization did not recapitulate the breadth and potency of neutralization observed in these infected individuals, a combination of three subtype B Env trimers from elite neutralizers resulted in some neutralization breadth within subtype B viruses. These results should guide future efforts to design vaccines to induce broadly neutralizing antibodies.

SOS and IP Modifications Predominantly Affect the Yield but Not Other Properties of SOSIP.664 HIV-1 Env Glycoprotein Trimers.

Soluble recombinant native-like (NL) envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design, designated BG505 SOSIP.664, incorporates an intersubunit disulfide bond (SOS) to covalently link the gp120 and gp41 ectodomain (gp41ECTO) subunits and a point substitution, I559P (IP), to further stabilize the gp41ECTO components. Without the SOS and IP changes, proteolytically cleaved trimers tend to disintegrate into their constituent gp120 and gp41ECTO subunits. We show, however, that NL trimers lacking the SOS and/or IP change can be affinity purified in amounts sufficient for analyses of their antigenicity and thermal stability. In general, these trimer variants have properties highly comparable to those of the fully stabilized SOSIP.664 version. We conclude that the major effect of the SOS and IP changes is to substantially increase trimer stability during and after the expression process, thereby allowing useful amounts to be produced. However, once the trimers have been purified, the SOS and IP changes have only subtle impacts on thermostability and the antigenicity of bNAb and other epitopes.IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. One vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. A commonly used design is designated SOSIP.664, a term reflecting the sequence changes that are used to stabilize the trimers and allow their production in practically useful amounts. Here, we show that these stabilizing changes act to increase trimer yield during the biosynthesis process within the producer cell but have little impact on the properties of purified trimers.

High-Throughput Protein Engineering Improves the Antigenicity and Stability of Soluble HIV-1 Envelope Glycoprotein SOSIP Trimers.

Soluble envelope glycoprotein (Env) trimers (SOSIP.664 gp140) are attractive HIV-1 vaccine candidates, with structures that mimic the native membrane-bound Env spike (gp160). Since engineering trimers can be limited by the difficulty of rationally predicting beneficial mutations, here we used a more comprehensive mutagenesis approach with the goal of identifying trimer variants with improved antigenic and stability properties. We created 341 cysteine pairs at predicted points of stabilization throughout gp140, 149 proline residue substitutions at every residue of the gp41 ectodomain, and 362 space-filling residue substitutions at every hydrophobic and aromatic residue in gp140. The parental protein target, the clade B strain B41 SOSIP.664 gp140, does not bind the broadly neutralizing antibody PGT151 and so was used here to identify improved variants that also provide insight into the structural basis for Env antigenicity. Each of the 852 mutants was expressed in human cells and screened for antigenicity using four different monoclonal antibodies (MAbs), including PGT151. We identified 29 trimer variants with antigenic improvements derived from each of the three mutagenesis strategies. We selected four variants (Q203F, T538F, I548F, and M629P) for more comprehensive biochemical, structural, and antigenicity analyses. The T538F substitution had the most beneficial effect overall, including restoration of the PGT151 epitope. The improved B41 SOSIP.664 trimer variants identified here may be useful for vaccine and structural studies.IMPORTANCE Soluble Env trimers have become attractive HIV-1 vaccine candidates, but the prototype designs are capable of further improvement through protein engineering. Using a high-throughput screening technology (shotgun mutagenesis) to create and evaluate 852 variants, we were able to identify sequence changes that were beneficial to the antigenicity and stability of soluble trimers based on the clade B B41 env gene. The strategies described here may be useful for identifying a wider range of antigenically and structurally improved soluble trimers based on multiple genotypes for use in programs intended to create a broadly protective HIV-1 vaccine.

Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes.

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.

Reducing V3 Antigenicity and Immunogenicity on Soluble, Native-Like HIV-1 Env SOSIP Trimers.

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.

cGMP production and analysis of BG505 SOSIP.664, an extensively glycosylated, trimeric HIV-1 envelope glycoprotein vaccine candidate.

We describe the properties of BG505 SOSIP.664 HIV-1 envelope glycoprotein trimers produced under current Good Manufacturing Practice (cGMP) conditions. These proteins are the first of a new generation of native-like trimers that are the basis for many structure-guided immunogen development programs aimed at devising how to induce broadly neutralizing antibodies (bNAbs) to HIV-1 by vaccination. The successful translation of this prototype demonstrates the feasibility of producing similar immunogens on an appropriate scale and of an acceptable quality for Phase I experimental medicine clinical trials. BG505 SOSIP.664 trimers are extensively glycosylated, contain numerous disulfide bonds and require proteolytic cleavage, all properties that pose a substantial challenge to cGMP production. Our strategy involved creating a stable CHO cell line that was adapted to serum-free culture conditions to produce envelope glycoproteins. The trimers were then purified by chromatographic methods using a 2G12 bNAb affinity column and size-exclusion chromatography. The chosen procedures allowed any adventitious viruses to be cleared from the final product to the required extent of >12 log10 . The final cGMP production run yielded 3.52 g (peptidic mass) of fully purified trimers (Drug Substance) from a 200 L bioreactor, a notable yield for such a complex glycoprotein. The purified trimers were fully native-like as judged by negative-stain electron microscopy, and were stable over a multi-month period at room temperature or below and for at least 1 week at 50°C. Their antigenicity, disulfide bond patterns, and glycan composition were consistent with trimers produced on a research laboratory scale. The methods reported here should pave the way for the cGMP production of other native-like Env glycoprotein trimers of various designs and genotypes.

Design and structure of two HIV-1 clade C SOSIP.664 trimers that increase the arsenal of native-like Env immunogens.

A key challenge in the quest toward an HIV-1 vaccine is design of immunogens that can generate a broadly neutralizing antibody (bnAb) response against the enormous sequence diversity of the HIV-1 envelope glycoprotein (Env). We previously demonstrated that a recombinant, soluble, fully cleaved SOSIP.664 trimer based on the clade A BG505 sequence is a faithful antigenic and structural mimic of the native trimer in its prefusion conformation. Here, we sought clade C native-like trimers with comparable properties. We identified DU422 and ZM197M SOSIP.664 trimers as being appropriately thermostable (Tm of 63.4 °C and 62.7 °C, respectively) and predominantly native-like, as determined by negative-stain electron microscopy (EM). Size exclusion chromatography, ELISA, and surface plasmon resonance further showed that these trimers properly display epitopes for all of the major bnAb classes, including quaternary-dependent, trimer-apex (e.g., PGT145) and gp120/gp41 interface (e.g., PGT151) epitopes. A cryo-EM reconstruction of the ZM197M SOSIP.664 trimer complexed with VRC01 Fab against the CD4 binding site at subnanometer resolution revealed a striking overall similarity to its BG505 counterpart with expected local conformational differences in the gp120 V1, V2, and V4 loops. These stable clade C trimers contribute additional diversity to the pool of native-like Env immunogens as key components of strategies to induce bnAbs to HIV-1.

Comprehensive antigenic map of a cleaved soluble HIV-1 envelope trimer.

The trimeric envelope (Env) spike is the focus of vaccine design efforts aimed at generating broadly neutralizing antibodies (bNAbs) to protect against HIV-1 infection. Three recent developments have facilitated a thorough investigation of the antigenic structure of the Env trimer: 1) the isolation of many bNAbs against multiple different epitopes; 2) the generation of a soluble trimer mimic, BG505 SOSIP.664 gp140, that expresses most bNAb epitopes; 3) facile binding assays involving the oriented immobilization of tagged trimers. Using these tools, we generated an antigenic map of the trimer by antibody cross-competition. Our analysis delineates three well-defined epitope clusters (CD4 binding site, quaternary V1V2 and Asn332-centered oligomannose patch) and new epitopes at the gp120-gp41 interface. It also identifies the relationships among these clusters. In addition to epitope overlap, we defined three more ways in which antibodies can cross-compete: steric competition from binding to proximal but non-overlapping epitopes (e.g., PGT151 inhibition of 8ANC195 binding); allosteric inhibition (e.g., PGT145 inhibition of 1NC9, 8ANC195, PGT151 and CD4 binding); and competition by reorientation of glycans (e.g., PGT135 inhibition of CD4bs bNAbs, and CD4bs bNAb inhibition of 8ANC195). We further demonstrate that bNAb binding can be complex, often affecting several other areas of the trimer surface beyond the epitope. This extensive analysis of the antigenic structure and the epitope interrelationships of the Env trimer should aid in design of both bNAb-based therapies and vaccines intended to induce bNAbs.

Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers.

UNLABELLED: We have investigated factors that influence the production of native-like soluble, recombinant trimers based on the env genes of two isolates of human immunodeficiency virus type 1 (HIV-1), specifically 92UG037.8 (clade A) and CZA97.012 (clade C). When the recombinant trimers based on the env genes of isolates 92UG037.8 and CZA97.012 were made according to the SOSIP.664 design and purified by affinity chromatography using broadly neutralizing antibodies (bNAbs) against quaternary epitopes (PGT145 and PGT151, respectively), the resulting trimers are highly stable and they are fully native-like when visualized by negative-stain electron microscopy. They also have a native-like (i.e., abundant) oligomannose glycan composition and display multiple bNAb epitopes while occluding those for nonneutralizing antibodies. In contrast, uncleaved, histidine-tagged Foldon (Fd) domain-containing gp140 proteins (gp140UNC-Fd-His), based on the same env genes, very rarely form native-like trimers, a finding that is consistent with their antigenic and biophysical properties and glycan composition. The addition of a 20-residue flexible linker (FL20) between the gp120 and gp41 ectodomain (gp41ECTO) subunits to make the uncleaved 92UG037.8 gp140-FL20 construct is not sufficient to create a native-like trimer, but a small percentage of native-like trimers were produced when an I559P substitution in gp41ECTO was also present. The further addition of a disulfide bond (SOS) to link the gp120 and gp41 subunits in the uncleaved gp140-FL20-SOSIP protein increases native-like trimer formation to ∼20 to 30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140UNC-Fd-His proteins but very rare in native-like trimer populations. The design and stabilization method and the purification strategy are, therefore, all important influences on the quality of trimeric Env proteins and hence their suitability as vaccine components. IMPORTANCE: Soluble, recombinant multimeric proteins based on the HIV-1 env gene are current candidate immunogens for vaccine trials in humans. These proteins are generally designed to mimic the native trimeric envelope glycoprotein (Env) that is the target of virus-neutralizing antibodies on the surfaces of virions. The underlying hypothesis is that an Env-mimetic protein may be able to induce antibodies that can neutralize the virus broadly and potently enough for a vaccine to be protective. Multiple different designs for Env-mimetic trimers have been put forth. Here, we used the CZA97.012 and 92UG037.8 env genes to compare some of these designs and determine which ones best mimic virus-associated Env trimers. We conclude that the most widely used versions of CZA97.012 and 92UG037.8 oligomeric Env proteins do not resemble the trimeric Env glycoprotein on HIV-1 viruses, which has implications for the design and interpretation of ongoing or proposed clinical trials of these proteins.

A native-like SOSIP.664 trimer based on an HIV-1 subtype B env gene.

UNLABELLED: Recombinant trimeric mimics of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike should expose as many epitopes as possible for broadly neutralizing antibodies (bNAbs) but few, if any, for nonneutralizing antibodies (non-NAbs). Soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A strain BG505 approach this ideal and are therefore plausible vaccine candidates. Here, we report on the production and in vitro properties of a new SOSIP.664 trimer derived from a subtype B env gene, B41, including how to make this protein in low-serum media without proteolytic damage (clipping) to the V3 region. We also show that nonclipped trimers can be purified successfully via a positive-selection affinity column using the bNAb PGT145, which recognizes a quaternary structure-dependent epitope at the trimer apex. Negative-stain electron microscopy imaging shows that the purified, nonclipped, native-like B41 SOSIP.664 trimers contain two subpopulations, which we propose represent an equilibrium between the fully closed and a more open conformation. The latter is different from the fully open, CD4 receptor-bound conformation and may represent an intermediate state of the trimer. This new subtype B trimer adds to the repertoire of native-like Env proteins that are suitable for immunogenicity and structural studies. IMPORTANCE: The cleaved, trimeric envelope protein complex is the only neutralizing antibody target on the HIV-1 surface. Many vaccine strategies are based on inducing neutralizing antibodies. For HIV-1, one approach involves using recombinant, soluble protein mimics of the native trimer. At present, the only reliable way to make native-like, soluble trimers in practical amounts is via the introduction of specific sequence changes that confer stability on the cleaved form of Env. The resulting proteins are known as SOSIP.664 gp140 trimers, and the current paradigm is based on the BG505 subtype A env gene. Here, we describe the production and characterization of a SOSIP.664 protein derived from a subtype B gene (B41), together with a simple, one-step method to purify native-like trimers by affinity chromatography with a trimer-specific bNAb, PGT145. The resulting trimers will be useful for structural and immunogenicity experiments aimed at devising ways to make an effective HIV-1 vaccine.

Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation.

We compare the antigenicity and conformation of soluble, cleaved vs. uncleaved envelope glycoprotein (Env gp)140 trimers from the subtype A HIV type 1 (HIV-1) strain BG505. The impact of gp120-gp41 cleavage on trimer structure, in the presence or absence of trimer-stabilizing modifications (i.e., a gp120-gp41 disulfide bond and an I559P gp41 change, together designated SOSIP), was assessed. Without SOSIP changes, cleaved trimers disintegrate into their gp120 and gp41-ectodomain (gp41ECTO) components; when only the disulfide bond is present, they dissociate into gp140 monomers. Uncleaved gp140s remain trimeric whether SOSIP substitutions are present or not. However, negative-stain electron microscopy reveals that only cleaved trimers form homogeneous structures resembling native Env spikes on virus particles. In contrast, uncleaved trimers are highly heterogeneous, adopting a variety of irregular shapes, many of which appear to be gp120 subunits dangling from a central core that is presumably a trimeric form of gp41ECTO. Antigenicity studies with neutralizing and nonneutralizing antibodies are consistent with the EM images; cleaved, SOSIP-stabilized trimers express quaternary structure-dependent epitopes, whereas uncleaved trimers expose nonneutralizing gp120 and gp41ECTO epitopes that are occluded on cleaved trimers. These findings have adverse implications for using soluble, uncleaved trimers for structural studies, and the rationale for testing uncleaved trimers as vaccine candidates also needs to be reevaluated.

A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies.

A desirable but as yet unachieved property of a human immunodeficiency virus type 1 (HIV-1) vaccine candidate is the ability to induce broadly neutralizing antibodies (bNAbs). One approach to the problem is to create trimeric mimics of the native envelope glycoprotein (Env) spike that expose as many bNAb epitopes as possible, while occluding those for non-neutralizing antibodies (non-NAbs). Here, we describe the design and properties of soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A transmitted/founder strain, BG505. These trimers are highly stable, more so even than the corresponding gp120 monomer, as judged by differential scanning calorimetry. They are also homogenous and closely resemble native virus spikes when visualized by negative stain electron microscopy (EM). We used several techniques, including ELISA and surface plasmon resonance (SPR), to determine the relationship between the ability of monoclonal antibodies (MAbs) to bind the soluble trimers and neutralize the corresponding virus. In general, the concordance was excellent, in that virtually all bNAbs against multiple neutralizing epitopes on HIV-1 Env were highly reactive with the BG505 SOSIP.664 gp140 trimers, including quaternary epitopes (CH01, PG9, PG16 and PGT145). Conversely, non-NAbs to the CD4-binding site, CD4-induced epitopes or gp41ECTO did not react with the trimers, even when their epitopes were present on simpler forms of Env (e.g. gp120 monomers or dissociated gp41 subunits). Three non-neutralizing MAbs to V3 epitopes did, however, react strongly with the trimers but only by ELISA, and not at all by SPR and to only a limited extent by EM. These new soluble trimers are useful for structural studies and are being assessed for their performance as immunogens.

Differential binding of neutralizing and non-neutralizing antibodies to native-like soluble HIV-1 Env trimers, uncleaved Env proteins, and monomeric subunits.

BACKGROUND: The trimeric envelope glycoproteins (Env) on the surface of HIV-1 virions are the targets for neutralizing antibodies (NAbs). No candidate HIV-1 immunogen has yet induced potent, broadly active NAbs (bNAbs). Part of the explanation may be that previously tested Env proteins inadequately mimic the functional, native Env complex. Trimerization and the proteolytic processing of Env precursors into gp120 and gp41 profoundly alter antigenicity, but soluble cleaved trimers are too unstable to serve as immunogens. By introducing stabilizing mutations (SOSIP), we constructed soluble, cleaved Env trimers derived from the HIV-1 subtype A isolate BG505 that resemble native Env spikes on virions both structurally and antigenically. RESULTS: We used surface plasmon resonance (SPR) to quantify antibody binding to different forms of BG505 Env: the proteolytically cleaved SOSIP.664 trimers, cleaved gp120-gp41ECTO protomers, and gp120 monomers. Non-NAbs to the CD4-binding site bound only marginally to the trimers but equally well to gp120-gp41ECTO protomers and gp120 monomers, whereas the bNAb VRC01, directed to the CD4bs, bound to all three forms. In contrast, bNAbs to V1V2 glycan-dependent epitopes bound preferentially (PG9 and PG16) or exclusively (PGT145) to trimers. We also explored the antigenic consequences of three different features of SOSIP.664 gp140 trimers: the engineered inter-subunit disulfide bond, the trimer-stabilizing I559P change in gp41ECTO, and proteolytic cleavage at the gp120-gp41ECTO junction. Each of these three features incrementally promoted native-like trimer antigenicity. We compared Fab and IgG versions of bNAbs and validated a bivalent model of IgG binding. The NAbs showed widely divergent binding kinetics and degrees of binding to native-like BG505 SOSIP.664. High off-rate constants and low stoichiometric estimates of NAb binding were associated with large amounts of residual infectivity after NAb neutralization of the corresponding BG505.T332N pseudovirus. CONCLUSIONS: The antigenicity and structural integrity of cleaved BG505 SOSIP.664 trimers render these proteins good mimics of functional Env spikes on virions. In contrast, uncleaved gp140s antigenically resemble individual gp120-gp41ECTO protomers and gp120 monomers, but not native trimers. Although NAb binding to functional trimers may thus be both necessary and sufficient for neutralization, the kinetics and stoichiometry of the interaction influence the neutralizing efficacy of individual NAbs.

Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies.

BACKGROUND: Recombinant soluble, cleaved HIV-1 envelope glycoprotein SOSIP.664 gp140 trimers based on the subtype A BG505 sequence are being studied structurally and tested as immunogens in animals. For these trimers to become a vaccine candidate for human trials, they would need to be made in appropriate amounts at an acceptable quality. Accomplishing such tasks by transient transfection is likely to be challenging. The traditional way to express recombinant proteins in large amounts is via a permanent cell line, usually of mammalian origin. Making cell lines that produce BG505 SOSIP.664 trimers requires the co-expression of the Furin protease to ensure that the cleavage site between the gp120 and gp41 subunits is fully utilized. RESULTS: We designed a vector capable of expressing Env and Furin, and used it to create Stable 293 T and CHO Flp-In™ cell lines through site-specific recombination. Both lines produce high quality, cleaved trimers at yields of up to 12-15 mg per 1 × 109 cells. Trimer expression at such levels was maintained for up to 30 days (10 passages) after initial seeding and was consistently superior to what could be achieved by transient transfection. Electron microscopy studies confirm that the purified trimers have the same native-like appearance as those derived by transient transfection and used to generate high-resolution structures. They also have appropriate antigenic properties, including the presentation of the quaternary epitope for the broadly neutralizing antibody PGT145. CONCLUSIONS: The BG505 SOSIP.664 trimer-expressing cell lines yield proteins of an appropriate quality for structural studies and animal immunogenicity experiments. The methodology is suitable for making similar lines under Good Manufacturing Practice conditions, to produce trimers for human clinical trials. Moreover, any env gene can be incorporated into this vector system, allowing the manufacture of SOSIP trimers from multiple genotypes, either by transient transfection or from stable cell lines.

Use of G-protein-coupled and -uncoupled CCR5 receptors by CCR5 inhibitor-resistant and -sensitive human immunodeficiency virus type 1 variants.

Small-molecule CCR5 inhibitors such as vicriviroc (VVC) and maraviroc (MVC) are allosteric modulators that impair HIV-1 entry by stabilizing a CCR5 conformation that the virus recognizes inefficiently. Viruses resistant to these compounds are able to bind the inhibitor-CCR5 complex while also interacting with the free coreceptor. CCR5 also interacts intracellularly with G proteins, as part of its signal transduction functions, and this process alters its conformation. Here we investigated whether the action of VVC against inhibitor-sensitive and -resistant viruses is affected by whether or not CCR5 is coupled to G proteins such as Gαi. Treating CD4(+) T cells with pertussis toxin to uncouple the Gαi subunit from CCR5 increased the potency of VVC against the sensitive viruses and revealed that VVC-resistant viruses use the inhibitor-bound form of Gαi-coupled CCR5 more efficiently than they use uncoupled CCR5. Supportive evidence was obtained by expressing a signaling-deficient CCR5 mutant with an impaired ability to bind to G proteins, as well as two constitutively active mutants that activate G proteins in the absence of external stimuli. The implication of these various studies is that the association of intracellular domains of CCR5 with the signaling machinery affects the conformation of the external and transmembrane domains and how they interact with small-molecule inhibitors of HIV-1 entry.