Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/MIV-prime-boost influenza vaccine trial, we sorted hemagglutinin-cross reactive memory-B cells and identified three antibody classes, each capable of neutralizing diverse subtypes of group 1 and 2-influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences from at least one multidonor class, and -in half the subjects- multidonor-class sequences were recovered from >40% of cross reactive-B cells. By contrast, these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency of B cells encoding broad influenza A-neutralizing antibodies; we propose the sequence signature-quantified prevalence of these B cells as a metric to guide universal influenza A-immunization strategies.

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.

While the HIV-1-glycan shield is known to shelter Env from the humoral immune response, its quantitative impact on antibody elicitation has been unclear. Here we use targeted deglycosylation to measure the impact of the glycan shield on elicitation of antibodies against the CD4 supersite. We engineered diverse Env trimers with select glycans removed proximal to the CD4 supersite, characterized their structures and glycosylation, and immunized guinea pigs and rhesus macaques. Immunizations yielded little neutralization against wild-type viruses, but potent CD4-supersite neutralization (titers 1:>1,000,000 against 4-glycan-deleted autologous viruses with over 90% breadth against 4-glycan-deleted heterologous strains exhibiting tier-2 neutralization character). To a first approximation, the immunogenicity of the glycan-shielded protein surface was negligible, with Env-elicited neutralization (ID50) proportional to the exponential of the protein-surface area accessible to antibody. Based on these high titers and exponential relationship, we propose site-selective deglycosylated trimers as priming immunogens to increase the frequency of site-targeting antibodies.

The escape mutant of HIV-1 protease (PR) containing 20 mutations (PR20) undergoes efficient polyprotein processing even in the presence of clinical protease inhibitors (PIs). PR20 shows >3 orders of magnitude decreased affinity for PIs darunavir (DRV) and saquinavir (SQV) relative to PR. Crystal structures of PR20 crystallized with yttrium, substrate analogue p2-NC, DRV, and SQV reveal three distinct conformations of the flexible flaps and diminished interactions with inhibitors through the combination of multiple mutations. PR20 with yttrium at the active site exhibits widely separated flaps lacking the usual intersubunit contacts seen in other inhibitor-free dimers. Mutations of residues 35–37 in the hinge loop eliminate interactions and perturb the flap conformation. Crystals of PR20/p2-NC contain one uninhibited dimer with one very open flap and one closed flap and a second inhibitor-bound dimer in the closed form showing six fewer hydrogen bonds with the substrate analogue relative to wild-type PR. PR20 complexes with PIs exhibit expanded S2/S2′ pockets and fewer PI interactions arising from coordinated effects of mutations throughout the structure, in agreement with the strikingly reduced affinity. In particular, insertion of the large aromatic side chains of L10F and L33F alters intersubunit interactions and widens the PI binding site through a network of hydrophobic contacts. The two very open conformations of PR20 as well as the expanded binding site of the inhibitor-bound closed form suggest possible approaches for modifying inhibitors to target extreme drug-resistant HIV.

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.

The structural and kinetic effects of amprenavir (APV), a clinical HIV protease (PR) inhibitor, were analyzed with wild‐type enzyme and mutants with single substitutions of V32I, I50V, I54V, I54M, I84V and L90M that are common in drug resistance. Crystal structures of the APV complexes at resolutions of 1.02–1.85 Å reveal the structural changes due to the mutations. Substitution of the larger side chains in PRV32I, PRI54M and PRL90M resulted in the formation of new hydrophobic contacts with flap residues, residues 79 and 80, and Asp25, respectively. Mutation to smaller side chains eliminated hydrophobic interactions in the PRI50V and PRI54V structures. The PRI84V–APV complex had lost hydrophobic contacts with APV, the PRV32I–APV complex showed increased hydrophobic contacts within the hydrophobic cluster and the PRI50V complex had weaker polar and hydrophobic interactions with APV. The observed structural changes in PRI84V–APV, PRV32I–APV and PRI50V–APV were related to their reduced inhibition by APV of six‐, 10‐ and 30‐fold, respectively, relative to wild‐type PR. The APV complexes were compared with the corresponding saquinavir complexes. The PR dimers had distinct rearrangements of the flaps and 80′s loops that adapt to the different P1′ groups of the inhibitors, while maintaining contacts within the hydrophobic cluster. These small changes in the loops and weak internal interactions produce the different patterns of resistant mutations for the two drugs. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7966480: HIV‐1 PR (uniprotkb:http://www.uniprot.org/uniprot/P03366) and HIV‐1 PR (uniprotkb:http://www.uniprot.org/uniprot/P03366) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by x‐ray crystallography (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114)

Highlights d Multiple MPER-directed bNAb lineages developed in a single individual d The broadest lineage belongs to the same antibody class as the 4E10 antibody d Low levels of somatic hypermutation of the RV217-VRC42 lineage can impart breadth d A multimeric immunogen activates VRC42 precursor B cells

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...