As the sole viral antigen on the HIV-1-virion surface, trimeric Env is a focus of vaccine efforts. Here we present the structure of the ligand-free HIV-1-Env trimer, fix its conformation, and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies, but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C-433C (DS) variant, specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer bound by a single CD4 without the typical antigenic hallmarks of CD4 induction. Antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like particle and soluble formats providing a new generation of vaccine antigens.

The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic 1 – 8 . Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3) 8 – 10 . It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers) 5 , 11 – 18 . Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1 JR-FL strain in complex with the antibody PGT151 19 . Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pretriggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies—and thus of interest for the design of immunogens—remains unknown.

A central goal of HIV-1 vaccine research is the elicitation of antibodies capable of neutralizing diverse primary isolates of HIV-1. Here we show that focusing the immune response to exposed N-terminal residues of the fusion peptide, a critical component of the viral entry machinery and the epitope of antibodies elicited by HIV-1 infection, through immunization with fusion peptide-coupled carriers and prefusion stabilized envelope trimers, induces cross-clade neutralizing responses. In mice, these immunogens elicited monoclonal antibodies capable of neutralizing up to 31% of a cross-clade panel of 208 HIV-1 strains. Crystal and cryoelectron microscopy structures of these antibodies revealed fusion peptide conformational diversity as a molecular explanation for the cross-clade neutralization. Immunization of guinea pigs and rhesus macaques induced similarly broad fusion peptide-directed neutralizing responses, suggesting translatability. The N terminus of the HIV-1 fusion peptide is thus a promising target of vaccine efforts aimed at eliciting broadly neutralizing antibodies.