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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.

The elicitation of autologous neutralizing responses by immunization with HIV-1 envelope (Env) trimers conformationally stabilized in a prefusion closed state has generated considerable interest in the HIV-1 vaccine field. However, soluble prefusion closed Env trimers have been produced from only a handful of HIV-1 strains, limiting their utility as vaccine antigens and B cell probes. Here, we report the engineering from 81 HIV-1 strains of soluble, fully cleaved, prefusion Env trimers with appropriate antigenicity. We used a 96-well expression-screening format to assess the ability of artificial disulfides and Ile559Pro substitution (DS-SOSIP) to produce soluble cleaved-Env trimers; from 180 Env strains, 20 yielded prefusion closed trimers. We also created chimeras, by utilizing structure-based design to incorporate select regions from the well-behaved BG505 strain; from 180 Env strains, 78 DS-SOSIP-stabilized chimeras, including 61 additional strains, yielded prefusion closed trimers. Structure-based design thus enables the production of prefusion closed HIV-1-Env trimers from dozens of diverse strains.

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 four-glycan-deleted autologous viruses with over 90% breadth against four-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.

Structure-based design of vaccines, particularly the iterative optimization used so successfully in the structure-based design of drugs, has been a long-sought goal. We previously developed a first-generation vaccine antigen called DS-Cav1, comprising a prefusion-stabilized form of the fusion (F) glycoprotein, which elicits high-titer protective responses against respiratory syncytial virus (RSV) in mice and macaques. Here we report the improvement of DS-Cav1 through iterative cycles of structure-based design that significantly increased the titer of RSV-protective responses. The resultant second-generation 'DS2'-stabilized immunogens have their F subunits genetically linked, their fusion peptides deleted and their interprotomer movements stabilized by an additional disulfide bond. These DS2 immunogens are promising vaccine candidates with superior attributes, such as their lack of a requirement for furin cleavage and their increased antigenic stability against heat inactivation. The iterative structure-based improvement described here may have utility in the optimization of other vaccine antigens.

Mitomycin C (MC) is a potent antitumor antibiotic which alkylates DNA through covalent linkage of its C-1″ position with the exocyclic N2amino group of guanine to yield the [MC]dG adduct at the duplex level. We report on the solution structure of the monoalkylated MC-DNA 9-mer complex where the [MC]dG5 adduct is positioned opposite dLC14 in the d(A3-C4-[MC]G5-T6).d(A13-C14-G15-T16) sequence context. The solution structure was solved based on a combined NMR-molecular dynamics study including NOE intensity based refinement. The formation of the [MC]dG adduct occurs with retention of the Watson-Crick alignment at the [MC]dG5.dC14 base-pair and flanking pairs in the complex. The MC ring is positioned in the minor groove with its indoloquinone aromatic ring system at a ∼45° angle relative to the helix axis and directed towards the 3′-direction on the unmodified strand. The MC indoloquinone chromphore is asymmetrically positioned in a slightly widened minor groove so that its plane is parallel to and stacked over the d(C14-G15-T16) segment on the unmodified strand with its other face exposed to solvent. The MC five-membered ring adopts an envelope pucker with its C-2″ atom displaced from the main plane and directed away from the unmodified strand. We observe conformational perturbations in the DNA 9-mer duplex on formation of the monoalkylated MC complex. Specifically, the base-pairs are displaced by ∼−3.0 Å towards the mjor groove on positioning the MC in the minor groove. This perturbation is accompanied by base stacking patterns similar to those observed inA-DNA while the majority of the sugars adopt puckers characteristic ofB-DNA. Conformational perturbations as monitored by helix twist, sugar pucker pseudorotation and glycosidic torsion angles are also observed for th 6(T6-C7-I8).d(C11-G12-A13) segment that is adjacent to but does not overlap the MC binding on the 9-mer duplex. We note that the O-10″ atom on the carbamate side-chain of MC forms an intermolecular hydrogen bond with the exocyclic amino group of dG15 in two of the three refined structures of the complex. The solution structure of the complex containing this intramolecular hydrogen bond readily explains both the previously observed d(C-G).d(C-G) sequence requirement for cross-linking and the observed, somewhat less stringent, requirement of the same sequence for the initial monoalkylation step. The structure indicates that the former is determined by the specific alignment of the MC monoadduct in the minor groove, while the specificity of the monoalkylation step likely reflects a specific hydrogen-bond between MC and DNA at the d(C-G).d(C-G) step in the precovalent complex.

Mithramycin (MTH) is a DNA-binding antitumor agent containing A-B disaccharide and C-D-E trisaccharide segments projecting from opposite ends of an aglycone chromophore. We have previously reported on the solution structure of the MTH-DNA 6-mer complex based on a combined NMR and molecular dynamics study. This study established that the Mg²⁺-coordinated mithramycin dimer bound to a widened minor groove centered about the sequence-specific (G-C)·(G-C) site and that the C-D-E trisaccharide segments from individual monomers were directed towards opposite ends of the helix spanning a six base-pair segment. This research is now extended to the binding of mithramycin dimers to partially overlapping sites on the self-complementary d(T-A-G-C-T-A-G-C-T-A) 10-mer duplex. The six base-pair mithramycin dimer footprint centered about (G-C)·(G-C) steps should result in a potential steric clash in the center of the helix involving the inwardly pointing E-sugars of the pair of mithramycin dimers bound to the DNA 10-mer duplex. The MTH-d(T-A-G-C-T-A-G-C-T-A) complex (two MTH dimers per duplex) yields narrow and well-resolved NMR spectra, which have been assigned to identify intramolecular and intermolecular nuclear Overhauser enhancement (NOE) connectivities in the complex. The solution structure of the MTH-DNA 10-mer complex based on distance-restrained molecular dynamics calculations has defined the conformation of the drug and the DNA necessary for accommodation of the pair of mithramycin dimers on the DNA10-mer helix. Specifically, the inwardly pointing E-sugars retain their face-down alignment towards the floor of the minor groove and occupy adjacent binding sites in the center of the duplex. This is achieved, in part, through torsion angle differences in the glycosidic linkage bonds along the length of the inwardly pointing aglycone-C-D-E trisaccharide segment relative to its outwardly pointing aglycone-C-D-E trisaccharide counterpart in the complex. In addition, a pronounced kink at the central (T-A)·(T-A) step opens the minor groove and generates additional space to accommodate the inwardly pointing E-sugars at adjacent sites in the MTH-DNA 10-mer complex. These studies establish conformational plasticity in the C-D-E trisaccharide segment of the mithramycin dimer and deformability of the DNA helix allowing mithramycin dimers to bind to partially overlapping minor groove sites on the DNA helix.

Antibodies to the prefusion conformation of the RSV F glycoprotein neutralize natural infection.