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.

SUMMARY The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.

An important class of HIV-1 broadly neutralizing antibodies, termed the VRC01 class, targets the conserved CD4-binding site (CD4bs) of the envelope glycoprotein (Env). An engineered Env outer domain (OD) eOD-GT8 60-mer nanoparticle has been developed as a priming immunogen for eliciting VRC01-class precursors and is planned for clinical trials. However, a substantial portion of eOD-GT8-elicited antibodies target non-CD4bs epitopes, potentially limiting its efficacy. We introduced N-linked glycans into non-CD4bs surfaces of eOD-GT8 to mask irrelevant epitopes and evaluated these mutants in a mouse model that expressed diverse immunoglobulin heavy chains containing human IGHV1-202, the germline VRC01 V segment. Compared to the parental eOD-GT8, a mutant with five added glycans stimulated significantly higher proportions of CD4bs-specific serum responses and CD4bs-specific immunoglobulin G B cells including VRC01-class precursors. These results demonstrate that glycan masking can limit elicitation of off-target antibodies and focus immune responses to the CD4bs, a major target of HIV-1 vaccine design.

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 cryo-electron microscopy structures of these antibodies revealed fusion peptide-conformational diversity as a molecular explanation for the crossclade 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. 3 Since crossing from chimpanzees ~100 years ago 1 , HIV-1 has evolved to be one of the more diverse viruses to infect humans 2 . While antibodies capable of neutralizing ~50% of circulating HIV-1 strains arise in half of those infected after several years 3 , the vaccine elicitation of antibodies capable of neutralizing divergent strains of HIV-1 remains an unsolved problem: antibodies elicited by current candidate vaccines fail to neutralize more than a small fraction of the diverse primary isolates that typify transmitted strains of 5 ).We and others have isolated broadly neutralizing antibodies from HIV-1-infected donors and coupled antibody identification with structural characterization to delineate sites of vulnerability to neutralizing antibodies 6,7 . These antibodies target the viral entry machinery, the envelope (Env) trimer, composed of three gp120 and three gp41 subunits. Dozens of structurally defined epitopes have been determined that can be categorized into a handful of Env regions.The majority of identified neutralizing antibodies have characteristics that may make them difficult to elict by vaccination, including those to the CD4-binding site 8,9 , where extensive somatic hypermutation (SHM) appears to be required 10-12 , those to a quaternary site at the trimer apex [13][14][15][16] , where unusual recombination appears to be required 13,14,17,18 , those to a glycan-V3 supersite 19-21 , where recognition of N-linked glycan appears to be required [20][21][22] , and those to the membrane-proximal external region 23-26 , where co-recognition of membrane 27-29 and immune tolerance appear to be required 30 .Recently, we identified an antibody, N123-VRC34.01 (ref. 31 ), named for donor (N123), lineage (VRC34) and clone number (01), and hereafter referenced without donor prefix. VRC34.01 targets primarily the conserved N-terminal region of the HIV-1 fusion peptide (FP), a critical component of viral entry machinery 32 . FP is compo...