Author Correction: Mosaic nanoparticle display of diverse influenza virus hemagglutinins elicits broad B cell responses.

In the version of this article initially published, the labels (50 Å) above the scale bars in Fig. 1b were incorrect. The correct size is 50 nm. The error has been corrected in the HTML and PDF versions of the article.

Mosaic nanoparticle display of diverse influenza virus hemagglutinins elicits broad B cell responses.

The present vaccine against influenza virus has the inevitable risk of antigenic discordance between the vaccine and the circulating strains, which diminishes vaccine efficacy. This necessitates new approaches that provide broader protection against influenza. Here we designed a vaccine using the hypervariable receptor-binding domain (RBD) of viral hemagglutinin displayed on a nanoparticle (np) able to elicit antibody responses that neutralize H1N1 influenza viruses spanning over 90 years. Co-display of RBDs from multiple strains across time, so that the adjacent RBDs are heterotypic, provides an avidity advantage to cross-reactive B cells. Immunization with the mosaic RBD-np elicited broader antibody responses than those induced by an admixture of nanoparticles encompassing the same set of RBDs as separate homotypic arrays. Furthermore, we identified a broadly neutralizing monoclonal antibody in a mouse immunized with mosaic RBD-np. The mosaic antigen array signifies a unique approach that subverts monotypic immunodominance and allows otherwise subdominant cross-reactive B cell responses to emerge.

Trimeric HIV-1-Env Structures Define Glycan Shields from Clades A, B, and G.

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ∼90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B, and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, that encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed, and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans among known broadly neutralizing antibodies that target the glycan-shielded trimer.

Vaccine-Induced Antibodies that Neutralize Group 1 and Group 2 Influenza A Viruses.

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 group 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.

Rational Design of an Epstein-Barr Virus Vaccine Targeting the Receptor-Binding Site.

Epstein-Barr virus (EBV) represents a major global health problem. Though it is associated with infectious mononucleosis and ∼200,000 cancers annually worldwide, a vaccine is not available. The major target of immunity is EBV glycoprotein 350/220 (gp350) that mediates attachment to B cells through complement receptor 2 (CR2/CD21). Here, we created self-assembling nanoparticles that displayed different domains of gp350 in a symmetric array. By focusing presentation of the CR2-binding domain on nanoparticles, potent neutralizing antibodies were elicited in mice and non-human primates. The structurally designed nanoparticle vaccine increased neutralization 10- to 100-fold compared to soluble gp350 by targeting a functionally conserved site of vulnerability, improving vaccine-induced protection in a mouse model. This rational approach to EBV vaccine design elicited potent neutralizing antibody responses by arrayed presentation of a conserved viral entry domain, a strategy that can be applied to other viruses.

A Neutralizing Antibody Recognizing Primarily N-Linked Glycan Targets the Silent Face of the HIV Envelope.

Virtually the entire surface of the HIV-1-envelope trimer is recognized by neutralizing antibodies, except for a highly glycosylated region at the center of the "silent face" on the gp120 subunit. From an HIV-1-infected donor, #74, we identified antibody VRC-PG05, which neutralized 27% of HIV-1 strains. The crystal structure of the antigen-binding fragment of VRC-PG05 in complex with gp120 revealed an epitope comprised primarily of N-linked glycans from N262, N295, and N448 at the silent face center. Somatic hypermutation occurred preferentially at antibody residues that interacted with these glycans, suggesting somatic development of glycan recognition. Resistance to VRC-PG05 in donor #74 involved shifting of glycan-N448 to N446 or mutation of glycan-proximal residue E293. HIV-1 neutralization can thus be achieved at the silent face center by glycan-recognizing antibody; along with other known epitopes, the VRC-PG05 epitope completes coverage by neutralizing antibody of all major exposed regions of the prefusion closed trimer.

Structure-based design of a quadrivalent fusion glycoprotein vaccine for human parainfluenza virus types 1-4.

Parainfluenza virus types 1-4 (PIV1-4) are highly infectious human pathogens, of which PIV3 is most commonly responsible for severe respiratory illness in newborns, elderly, and immunocompromised individuals. To obtain a vaccine effective against all four PIV types, we engineered mutations in each of the four PIV fusion (F) glycoproteins to stabilize their metastable prefusion states, as such stabilization had previously enabled the elicitation of high-titer neutralizing antibodies against the related respiratory syncytial virus. A cryoelectron microscopy structure of an engineered PIV3 F prefusion-stabilized trimer, bound to the prefusion-specific antibody PIA174, revealed atomic-level details for how introduced mutations improved stability as well as how a single PIA174 antibody recognized the trimeric apex of prefusion PIV3 F. Nine combinations of six newly identified disulfides and two cavity-filling mutations stabilized the prefusion PIV3 F immunogens and induced 200- to 500-fold higher neutralizing titers in mice than were elicited by PIV3 F in the postfusion conformation. For PIV1, PIV2, and PIV4, we also obtained stabilized prefusion Fs, for which prefusion versus postfusion titers were 2- to 20-fold higher. Elicited murine responses were PIV type-specific, with little cross-neutralization of other PIVs. In nonhuman primates (NHPs), quadrivalent immunization with prefusion-stabilized Fs from PIV1-4 consistently induced potent neutralizing responses against all four PIVs. For PIV3, the average elicited NHP titer from the quadrivalent immunization was more than fivefold higher than any titer observed in a cohort of over 100 human adults, highlighting the ability of a prefusion-stabilized immunogen to elicit especially potent neutralization.

Self-Competitive Inhibition of the Bacteriophage P22 Tailspike Endorhamnosidase by O-Antigen Oligosaccharides.

The P22 tailspike endorhamnosidase confers the high specificity of bacteriophage P22 for some serogroups of Salmonella differing only slightly in their O-antigen polysaccharide. We used several biophysical methods to study the binding and hydrolysis of O-antigen fragments of different lengths by P22 tailspike protein. O-Antigen saccharides of defined length labeled with fluorophors could be purified with higher resolution than previously possible. Small amounts of naturally occurring variations of O-antigen fragments missing the nonreducing terminal galactose could be used to determine the contribution of this part to the free energy of binding to be ∼7 kJ/mol. We were able to show via several independent lines of evidence that an unproductive binding mode is highly favored in binding over all other possible binding modes leading to hydrolysis. This is true even under circumstances under which the O-antigen fragment is long enough to be cleaved efficiently by the enzyme. The high-affinity unproductive binding mode results in a strong self-competitive inhibition in addition to product inhibition observed for this system. Self-competitive inhibition is observed for all substrates that have a free reducing end rhamnose. Naturally occurring O-antigen, while still attached to the bacterial outer membrane, does not have a free reducing end and therefore does not perform self-competitive inhibition.

A Frizzled-Like Cysteine-Rich Domain in Glypican-3 Mediates Wnt Binding and Regulates Hepatocellular Carcinoma Tumor Growth in Mice.

Wnt signaling is one of the key regulators of hepatocellular carcinoma (HCC) tumor progression. In addition to the classical receptor frizzled (FZD), various coreceptors including heparan sulfate proteoglycans (HSPGs) are involved in Wnt activation. Glypican-3 (GPC3) is an HSPG that is overexpressed in HCC and functions as a Wnt coreceptor that modulates HCC cell proliferation. These features make GPC3 an attractive target for liver cancer therapy. However, the precise interaction of GPC3 and Wnt and how GPC3, Wnt, and FZD cooperate with each other are poorly understood. In this study, we established a structural model of GPC3 containing a putative FZD-like cysteine-rich domain at its N-terminal lobe. We found that F41 and its surrounding residues in GPC3 formed a Wnt-binding groove that interacted with the middle region located between the lipid thumb domain and the index finger domain of Wnt3a. Mutating residues in this groove significantly inhibited Wnt3a binding, ß-catenin activation, and the transcriptional activation of Wnt-dependent genes. In contrast with the heparan sulfate chains, the Wnt-binding groove that we identified in the protein core of GPC3 seemed to promote Wnt signaling in conditions when FZD was not abundant. Specifically, blocking this domain using an antibody inhibited Wnt activation. In HCC cells, mutating residue F41 on GPC3 inhibited activation of ß-catenin in vitro and reduced xenograft tumor growth in nude mice compared with cells expressing wild-type GPC3. Conclusion: Our investigation demonstrates a detailed interaction of GPC3 and Wnt3a, reveals the precise mechanism of GPC3 acting as a Wnt coreceptor, and provides a potential target site on GPC3 for Wnt blocking and HCC therapy.

The crystal structure of human GlnRS provides basis for the development of neurological disorders.

Cytosolic glutaminyl-tRNA synthetase (GlnRS) is the singular enzyme responsible for translation of glutamine codons. Compound heterozygous mutations in GlnRS cause severe brain disorders by a poorly understood mechanism. Herein, we present crystal structures of the wild type and two pathological mutants of human GlnRS, which reveal, for the first time, the domain organization of the intact enzyme and the structure of the functionally important N-terminal domain (NTD). Pathological mutations mapping in the NTD alter the domain structure, and decrease catalytic activity and stability of GlnRS, whereas missense mutations in the catalytic domain induce misfolding of the enzyme. Our results suggest that the reduced catalytic efficiency and a propensity of GlnRS mutants to misfold trigger the disease development. This report broadens the spectrum of brain pathologies elicited by protein misfolding and provides a paradigm for understanding the role of mutations in aminoacyl-tRNA synthetases in neurological diseases.

Design of a Multicompartment Hydrogel that Facilitates Time-Resolved Delivery of Combination Therapy and Synergized Killing of Glioblastoma.

There is significant current interest in identifying new combination therapies that synergize to treat disease, and it is becoming increasingly clear that the temporal resolution of their administration greatly impacts efficacy. To facilitate effective delivery, a multicompartment hydrogel material was developed that is composed of spherical vesicles interlaced within a self-assembled peptide-based network of physically crosslinked fibrils that allows time-resolved independent co-delivery of small molecules. This material architecture effectively delivers the EGFR kinase inhibitor Erlotinib (ERL) and Doxorubicin (DOX, DNA intercalator) in an ERL→DOX sequential manner to synergistically kill glioblastoma, the most aggressive form of brain cancer.

Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein.

Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens.

Single-Chain Soluble BG505.SOSIP gp140 Trimers as Structural and Antigenic Mimics of Mature Closed HIV-1 Env.

UNLABELLED: Similar to other type I fusion machines, the HIV-1 envelope glycoprotein (Env) requires proteolytic activation; specifically, cleavage of a gp160 precursor into gp120 and gp41 subunits creates an N-terminal gp41 fusion peptide and permits folding from an immature uncleaved state to a mature closed state. While the atomic-level consequences of cleavage for HIV-1 Env are still being determined, the uncleaved state is antigenically distinct from the mature closed state, and cleavage has been reported to be essential for mimicry of the mature viral spike by soluble versions of Env. Here we report the redesign of a current state-of-the-art soluble Env mimic, BG505.SOSIP, to make it cleavage independent. Specifically, we replaced the furin cleavage site between gp120 and gp41 with Gly-Ser linkers of various lengths. The resultant linked gp120-gp41 constructs, termed single-chain gp140 (sc-gp140), exhibited different levels of structural and antigenic mimicry of the parent cleaved BG505.SOSIP. When constructs were subjected to negative selection to remove subspecies recognized by poorly neutralizing antibodies, trimers of high antigenic mimicry of BG505.SOSIP could be obtained; negative-stain electron microscopy indicated these to resemble the mature closed state. Higher proportions of BG505.SOSIP-trimer mimicry were observed in sc-gp140s with linkers of 6 or more residues, with a linker length of 15 residues exhibiting especially promising traits. Overall, flexible linkages between gp120 and gp41 in BG505.SOSIP can thus substitute for cleavage, and sc-gp140s that closely mimicked the vaccine-preferred mature closed state of Env could be obtained. IMPORTANCE: The trimeric HIV-1 envelope glycoprotein (Env) is the sole target of virus-directed neutralizing antibody responses and a primary focus of vaccine design. Soluble mimics of Env have proven challenging to obtain and have been thought to require proteolytic cleavage into two-component subunits, gp120 and gp41, to achieve structural and antigenic mimicry of mature Env spikes on virions. Here we show that replacement of the cleavage site between gp120 and gp41 in a lead soluble gp140 construct, BG505.SOSIP, with flexible linkers can result in molecules that do not require cleavage to fold efficiently into the mature closed state. Our results provide insights into the impact of cleavage on HIV-1 Env folding. In some contexts such as genetic immunization, optimized cleavage-independent soluble gp140 constructs may have utility over the parental BG505.SOSIP, as they would not require furin cleavage to achieve mimicry of mature Env spikes on virions.

Conformational switching in PolyGln amyloid fibrils resulting from a single amino acid insertion.

The established correlation between neurodegenerative disorders and intracerebral deposition of polyglutamine aggregates motivates attempts to better understand their fibrillar structure. We designed polyglutamines with a few lysines inserted to overcome the hindrance of extreme insolubility and two D-lysines to limit the lengths of ß-strands. One is 33 amino acids long (PolyQKd-33) and the other has one fewer glutamine (PolyQKd-32). Both form well-dispersed fibrils suitable for analysis by electron microscopy. Electron diffraction confirmed cross-ß structures in both fibrils. Remarkably, the deletion of just one glutamine residue from the middle of the peptide leads to substantially different amyloid structures. PolyQKd-32 fibrils are consistently 10-20% wider than PolyQKd-33, as measured by negative staining, cryo-electron microscopy, and scanning transmission electron microscopy. Scanning transmission electron microscopy analysis revealed that the PolyQKd-32 fibrils have 50% higher mass-per-length than PolyQKd-33. This distinction can be explained by a superpleated ß-structure model for PolyQKd-33 and a model with two ß-solenoid protofibrils for PolyQKd-32. These data provide evidence for ß-arch-containing structures in polyglutamine fibrils and open future possibilities for structure-based drug design.

Structural dependence of HET-s amyloid fibril infectivity assessed by cryoelectron microscopy.

HET-s is a prion protein of the fungus Podospora anserina which, in the prion state, is active in a self/nonself recognition process called heterokaryon incompatibility. Its prionogenic properties reside in the C-terminal "prion domain." The HET-s prion domain polymerizes in vitro into amyloid fibrils whose properties depend on the pH of assembly; above pH 3, infectious singlet fibrils are produced, and below pH 3, noninfectious triplet fibrils. To investigate the correlation between structure and infectivity, we performed cryo-EM analyses. Singlet fibrils have a helical pitch of approximately 410 Å and a left-handed twist. Triplet fibrils have three protofibrils whose lateral dimensions (36 × 25 Å) and axial packing (one subunit per 9.4 Å) match those of singlets but differ in their supercoiling. At 8.5-Å resolution, the cross-section of the singlet fibril reconstruction is largely consistent with that of a ß-solenoid model previously determined by solid-state NMR. Reconstructions of the triplet fibrils show three protofibrils coiling around a common axis and packed less tightly at pH 3 than at pH 2, eventually peeling off. Taken together with the earlier observation that fragmentation of triplet fibrils by sonication does not increase infectivity, these observations suggest a novel mechanism for self-propagation, whereby daughter fibrils nucleate on the lateral surface of singlet fibrils. In triplets, this surface is occluded, blocking nucleation and thereby explaining their lack of infectivity.

Fibril structure of human islet amyloid polypeptide.

Misfolding and amyloid fibril formation by human islet amyloid polypeptide (hIAPP) are thought to be important in the pathogenesis of type 2 diabetes, but the structures of the misfolded forms remain poorly understood. Here we developed an approach that combines site-directed spin labeling with continuous wave and pulsed EPR to investigate local secondary structure and to determine the relative orientation of the secondary structure elements with respect to each other. These data indicated that individual hIAPP molecules take up a hairpin fold within the fibril. This fold contains two ß-strands that are much farther apart than expected from previous models. Atomistic structural models were obtained using computational refinement with EPR data as constraints. The resulting family of structures exhibited a left-handed helical twist, in agreement with the twisted morphology observed by electron microscopy. The fibril protofilaments contain stacked hIAPP monomers that form opposing ß-sheets that twist around each other. The two ß-strands of the monomer adopt out-of-plane positions and are staggered by about three peptide layers (∼15 Å). These results provide a mechanism for hIAPP fibril formation and could explain the remarkable stability of the fibrils. Thus, the structural model serves as a starting point for understanding and preventing hIAPP misfolding.

δ-Tocopherol reduces lipid accumulation in Niemann-Pick type C1 and Wolman cholesterol storage disorders.

Niemann-Pick disease type C (NPC) and Wolman disease are two members of a family of storage disorders caused by mutations of genes encoding lysosomal proteins. Deficiency in function of either the NPC1 or NPC2 protein in NPC disease or lysosomal acid lipase in Wolman disease results in defective cellular cholesterol trafficking. Lysosomal accumulation of cholesterol and enlarged lysosomes are shared phenotypic characteristics of both NPC and Wolman cells. Utilizing a phenotypic screen of an approved drug collection, we found that δ-tocopherol effectively reduced lysosomal cholesterol accumulation, decreased lysosomal volume, increased cholesterol efflux, and alleviated pathological phenotypes in both NPC1 and Wolman fibroblasts. Reduction of these abnormalities may be mediated by a δ-tocopherol-induced intracellular Ca(2+) response and subsequent enhancement of lysosomal exocytosis. Consistent with a general mechanism for reduction of lysosomal lipid accumulation, we also found that δ-tocopherol reduces pathological phenotypes in patient fibroblasts from other lysosomal storage diseases, including NPC2, Batten (ceroid lipofuscinosis, neuronal 2, CLN2), Fabry, Farber, Niemann-Pick disease type A, Sanfilippo type B (mucopolysaccharidosis type IIIB, MPSIIIB), and Tay-Sachs. Our data suggest that regulated exocytosis may represent a potential therapeutic target for reduction of lysosomal storage in this class of diseases.

A ketogenic diet increases brown adipose tissue mitochondrial proteins and UCP1 levels in mice.

We evaluated the effects of feeding a ketogenic diet (KD) for a month on general physiology with emphasis on brown adipose tissue (BAT) in mice. KD did not reduce the caloric intake, or weight or lipid content of BAT. Relative epididymal fat pads were 40% greater in the mice fed the KD (P = 0.06) while leptin was lower (P < 0.05). Blood glucose levels were 30% lower while D-ß-hydroxybutyrate levels were about 3.5-fold higher in the KD group. Plasma insulin and leptin levels in the KD group were about half of that of the mice fed NIH-31 pellets (chow group). Median mitochondrial size in the interscapular BAT (IBAT) of the KD group was about 60% greater, whereas the median lipid droplet size was about half of that in the chow group. Mitochondrial oxidative phosphorylation proteins were increased (1.5-3-fold) and the uncoupling protein 1 levels were increased by threefold in mice fed the KD. The levels of PPARγ, PGC-1α, and Sirt1 in KD group were 1.5-3-fold while level of Sirt3 was about half of that in the chow-fed group. IBAT cyclic AMP levels were 60% higher in the KD group and cAMP response element binding protein was 2.5-fold higher, suggesting increased sympathetic system activity. These results demonstrate that a KD can also increase BAT mitochondrial size and protein levels.

Mitochondrial biogenesis and increased uncoupling protein 1 in brown adipose tissue of mice fed a ketone ester diet.

We measured the effects of a diet in which D-ß-hydroxybutyrate-(R)-1,3 butanediol monoester [ketone ester (KE)] replaced equicaloric amounts of carbohydrate on 8-wk-old male C57BL/6J mice. Diets contained equal amounts of fat, protein, and micronutrients. The KE group was fed ad libitum, whereas the control (Ctrl) mice were pair-fed to the KE group. Blood d-ß-hydroxybutyrate levels in the KE group were 3-5 times those reported with high-fat ketogenic diets. Voluntary food intake was reduced dose dependently with the KE diet. Feeding the KE diet for up to 1 mo increased the number of mitochondria and doubled the electron transport chain proteins, uncoupling protein 1, and mitochondrial biogenesis-regulating proteins in the interscapular brown adipose tissue (IBAT). [(18)F]-Fluorodeoxyglucose uptake in IBAT of the KE group was twice that in IBAT of the Ctrl group. Plasma leptin levels of the KE group were more than 2-fold those of the Ctrl group and were associated with increased sympathetic nervous system activity to IBAT. The KE group exhibited 14% greater resting energy expenditure, but the total energy expenditure measured over a 24-h period or body weights was not different. The quantitative insulin-sensitivity check index was 73% higher in the KE group. These results identify KE as a potential antiobesity supplement.

Degradation of fungal prion HET-s(218-289) induces formation of a generic amyloid fold.

The prion-forming domain of the fungal prion protein HET-s, HET-s(218-289), is known from solid-state NMR studies to have a ß-solenoidal structure; the ß-solenoid has the cross-ß structure characteristic of all amyloids, but is inherently more complex than the generic stacked ß-sheets found in studies of small synthetic peptides. At low pH HET-s(218-289) has also been reported to form an alternative structure, which has not been characterized. We have confirmed by x-ray fiber diffraction that HET-s(218-289) adopts a ß-solenoidal structure at neutral pH, and shown that at low pH, it forms either a ß-solenoid or a stacked ß-sheet structure, depending on the integrity of the protein and the conditions of fibrillization. The low pH stacked-sheet structure is usually formed only by proteolyzed HET-s(218-289), but intact HET-s(218-289) can form stacked sheets when seeded with proteolyzed stacked-sheet HET-s(218-289). The polymorphism of HET-s parallels the structural differences between the infectious brain-derived and the much less infectious recombinant mammalian prion protein PrP. Taken together, these observations suggest that the functional or pathological forms of amyloid proteins are more complex than the simple generic stacked-sheet amyloids commonly formed by short peptides.