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.

Summary 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 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.

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. IMPORTANCEThe 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. Immunogens capable of inducing protective titers of broadly neutralizing antibodies (bNAbs) are being widely sought for use in vaccine design strategies for human immunodeficiency virus type 1 (HIV-1) (1). The basis of this approach is that bNAbs can prevent globally diverse HIV-1 strains from infecting target cells. They do so via binding to the envelope glycoprotein (Env) complex on the virion surface, an event that is both necessary and sufficient to neutralize HIV-1 infectivity (2, 3). One of the more common strategies to induce bNAbs involves the design of soluble, recombinant protein mimics of the native Env complex, a meta-stable structure comprising three gp120 and three gp41 subunits. The production of soluble Env trimers involves introducing a stop codon to truncate the gp41 ectodomain (gp41 ECTO ) subunit prior to the transmembrane region to yield soluble gp140 proteins (4-6). The fragility of the Env complex is, however, a substantial problem from a protein engineering perspective, as the natural noncovalent interactions between the six subunits are not robust enough to allow so...

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 (gp41 ECTO ) 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 gp41 ECTO . Antigenicity studies with neutralizing and nonneutralizing antibodies are consistent with the EM images; cleaved, SOSIP-stabilized trimers express quaternary structuredependent epitopes, whereas uncleaved trimers expose nonneutralizing gp120 and gp41 ECTO 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.T rimeric envelope glycoprotein (Env gp) spikes on the HIV type 1 (HIV-1) surface mediate entry of the viral genome into the target cell (1, 2). When spikes interact with their cellsurface receptors, a series of conformational changes within the Env culminates in virus-cell membrane fusion. Neutralizing antibodies (NAbs) against various Env epitopes antagonize these events (2, 3). Hence, Env glycoproteins are a focus of vaccine design programs intended to induce NAbs and thereby prevent HIV-1 transmission (3, 4). Env trimers are composed of three gp120 surface glycoprotein subunits and three gp41 transmembrane glycoproteins, the six subunits all associated via noncovalent interactions (5, 6). A critical event in trimer assembly is proteolytic cleavage of the gp160 precursor into its gp120 and gp41 components, a process essential for HIV-1 entry not least because it liberates the fusion peptide (FP) at the gp41 N terminus (5, 6).Trimer-based vaccine strategies involve expressing soluble, recombinant versions of the virion-associated (i.e., native) spikes. To facilitate production and purification, the membrane-spanning and cytoplasmic domains that anchor spikes to the virion, but that are not NAb targets, are eliminated (7-12). However, the resulting proteins, known as gp140s, are highly unstable and disintegrate into their gp120 and gp41-ectodomain (gp41 ECTO ) components, making them useless as immunogens. Two fundamentally different protein-engineering strategies have been used to create gp140s t...

SummaryBroadly 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.