Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin.

Broadly neutralizing antibodies against highly variable pathogens have stimulated the design of vaccines and therapeutics. We report the use of diverse camelid single-domain antibodies to influenza virus hemagglutinin to generate multidomain antibodies with impressive breadth and potency. Multidomain antibody MD3606 protects mice against influenza A and B infection when administered intravenously or expressed locally from a recombinant adeno-associated virus vector. Crystal and single-particle electron microscopy structures of these antibodies with hemagglutinins from influenza A and B viruses reveal binding to highly conserved epitopes. Collectively, our findings demonstrate that multidomain antibodies targeting multiple epitopes exhibit enhanced virus cross-reactivity and potency. In combination with adeno-associated virus-mediated gene delivery, they may provide an effective strategy to prevent infection with influenza virus and other highly variable pathogens.

A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen.

The identification of human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem revitalized hopes of developing a universal influenza vaccine. Using a rational design and library approach, we engineered stable HA stem antigens ("mini-HAs") based on an H1 subtype sequence. Our most advanced candidate exhibits structural and bnAb binding properties comparable to those of full-length HA, completely protects mice in lethal heterologous and heterosubtypic challenge models, and reduces fever after sublethal challenge in cynomolgus monkeys. Antibodies elicited by this mini-HA in mice and nonhuman primates bound a wide range of HAs, competed with human bnAbs for HA stem binding, neutralized H5N1 viruses, and mediated antibody-dependent effector activity. These results represent a proof of concept for the design of HA stem mimics that elicit bnAbs against influenza A group 1 viruses.

Characterization of a broadly neutralizing monoclonal antibody that targets the fusion domain of group 2 influenza A virus hemagglutinin.

UNLABELLED: Due to continuous changes to its antigenic regions, influenza viruses can evade immune detection and cause a significant amount of morbidity and mortality around the world. Influenza vaccinations can protect against disease but must be annually reformulated to match the current circulating strains. In the development of a broad-spectrum influenza vaccine, the elucidation of conserved epitopes is paramount. To this end, we designed an immunization strategy in mice to boost the humoral response against conserved regions of the hemagglutinin (HA) glycoprotein. Of note, generation and identification of broadly neutralizing antibodies that target group 2 HAs are rare and thus far have yielded only a few monoclonal antibodies (MAbs). Here, we demonstrate that mouse MAb 9H10 has broad and potent in vitro neutralizing activity against H3 and H10 group 2 influenza A subtypes. In the mouse model, MAb 9H10 protects mice against two divergent mouse-adapted H3N2 strains, in both pre- and postexposure administration regimens. In vitro and cell-free assays suggest that MAb 9H10 inhibits viral replication by blocking HA-dependent fusion of the viral and endosomal membranes early in the replication cycle and by disrupting viral particle egress in the late stage of infection. Interestingly, electron microscopy reconstructions of MAb 9H10 bound to the HA reveal that it binds a similar binding footprint to MAbs CR8020 and CR8043. IMPORTANCE: The influenza hemagglutinin is the major antigenic target of the humoral immune response. However, due to continuous antigenic changes that occur on the surface of this glycoprotein, influenza viruses can escape the immune system and cause significant disease to the host. Toward the development of broad-spectrum therapeutics and vaccines against influenza virus, elucidation of conserved regions of influenza viruses is crucial. Thus, defining these types of epitopes through the generation and characterization of broadly neutralizing monoclonal antibodies (MAbs) can greatly assist others in highlighting conserved regions of hemagglutinin. Here, we demonstrate that MAb 9H10 that targets the hemagglutinin stalk has broadly neutralizing activity against group 2 influenza A viruses in vitro and in vivo.

A common solution to group 2 influenza virus neutralization.

The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.

Antibody recognition of the pandemic H1N1 Influenza virus hemagglutinin receptor binding site.

Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in antigenicity but fortunately was not nearly as deadly. 5J8 is a human antibody that potently neutralizes a broad spectrum of H1N1 viruses, including the 1918 and 2009 pandemic viruses. Here, we present the crystal structure of 5J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the A/California/07/2009 (H1N1) pandemic virus. 5J8 recognizes a conserved epitope in and around the receptor binding site (RBS), and its HCDR3 closely mimics interactions of the sialic acid receptor. Electron microscopy (EM) reconstructions of 5J8 Fab in complex with an HA trimer from a 1986 H1 strain and with an engineered stabilized HA trimer from the 2009 H1 pandemic virus showed a similar mode of binding. As for other characterized RBS-targeted antibodies, 5J8 uses avidity to extend its breadth and affinity against divergent H1 strains. 5J8 selectively interacts with HA insertion residue 133a, which is conserved in pandemic H1 strains and has precluded binding of other RBS-targeted antibodies. Thus, the RBS of divergent HAs is targeted by 5J8 and adds to the growing arsenal of common recognition motifs for design of therapeutics and vaccines. Moreover, consistent with previous studies, the bacterially expressed H1 HA properly refolds, retaining its antigenic structure, and presents a low-cost and rapid alternative for engineering and manufacturing candidate flu vaccines.

Discrete kinetic models from funneled energy landscape simulations.

A general method for facilitating the interpretation of computer simulations of protein folding with minimally frustrated energy landscapes is detailed and applied to a designed ankyrin repeat protein (4ANK). In the method, groups of residues are assigned to foldons and these foldons are used to map the conformational space of the protein onto a set of discrete macrobasins. The free energies of the individual macrobasins are then calculated, informing practical kinetic analysis. Two simple assumptions about the universality of the rate for downhill transitions between macrobasins and the natural local connectivity between macrobasins lead to a scheme for predicting overall folding and unfolding rates, generating chevron plots under varying thermodynamic conditions, and inferring dominant kinetic folding pathways. To illustrate the approach, free energies of macrobasins were calculated from biased simulations of a non-additive structure-based model using two structurally motivated foldon definitions at the full and half ankyrin repeat resolutions. The calculated chevrons have features consistent with those measured in stopped flow chemical denaturation experiments. The dominant inferred folding pathway has an "inside-out", nucleation-propagation like character.

Prediction of native-state hydrogen exchange from perfectly funneled energy landscapes.

Simulations based on perfectly funneled energy landscapes often capture many of the kinetic features of protein folding. We examined whether simulations based on funneled energy functions can also describe fluctuations in native-state protein ensembles. We quantitatively compared the site-specific local stability determined from structure-based folding simulations, with hydrogen exchange protection factors measured experimentally for ubiquitin, chymotrypsin inhibitor 2, and staphylococcal nuclease. Different structural definitions for the open and closed states based on the number of native contacts for each residue, as well as the hydrogen-bonding state, or a combination of both criteria were evaluated. The predicted exchange patterns agree with the experiments under native conditions, indicating that protein topology indeed has a dominant effect on the exchange kinetics. Insights into the simplest mechanistic interpretation of the amide exchange process were thus obtained.

Structure of the inhibitor W7 bound to the regulatory domain of cardiac troponin C.

The calmodulin antagonist W7 binds to troponin C in the presence of Ca(2+) and inhibits striated muscle contraction. This study integrates multiple data into the structure of the regulatory domain of human cardiac troponin C (cNTnC) bound to Ca(2+) and W7. The protein-W7 interface is defined through a three-dimensional {(1)H,(13)C}-edited-{(1)H,(12)C}-detected NOESY NMR experiment, and other aspects of the structure are modeled as perturbations to previously known coordinates and restraints. The structure determination protocol optimizes the protein-W7 contacts prior to the introduction of protein-W7 steric interactions or conformational changes in the protein. The structure determination protocol gives families of conformers that all have an optimal docking as assessed by satisfaction of the target function. The structure supports the previously proposed troponin I blocking mechanism for the activity of W7 in striated muscle and suggests a role for the flexible tail of W7 in stabilization of the bound state. This clarifies the structure-activity relationships of W7 and implicates an electrostatically mediated component of activity in common analogues of W7, including the antipsychotic trifluoroperazine and the cardiotonic levosimendan.

Isoform-specific variation in the intrinsic disorder of troponin I.

Various intrinsic disorder (ID) prediction algorithms were applied to the three tissue isoforms of troponin I (TnI). The results were interpreted in terms of the known structure and dynamics of troponin. In line with previous results, all isoforms of TnI were predicted to have large stretches of ID. The predictions show that the C-termini of all isoforms are extensively disordered as is the N-terminal extension of the cardiac isoform. Cardiac TnI likely belongs to the group of intrinsically disordered signalling hub proteins. For a given portion of the protein sequence, most ID prediction approaches indicate isoform-dependent variations in the probability of disorder. Comparison of machine learning and physically based approaches suggests the ID variations are only partially attributable to local variations in the ratio of charged to hydrophobic residues. The VSL2B algorithm predicts the largest variations in ID across the isoforms, with the cardiac isoform having the highest probability of structured regions, and the fast-skeletal isoform having no intrinsic structure. The region corresponding to residues 57-95 of the fast-skeletal isoform, known to form a coiled coil substructure with troponin T, was highly variable between isoforms. The isoform-specific ID variations may have mechanistic significance, modulating the extent to which conformational fluctuations in tropomyosin are communicated to the troponin complex. We discuss structural mechanisms for this communication. Overall, the results motivate the development of predictors designed to address relative levels of disorder between highly similar proteins.

An interplay between protein disorder and structure confers the Ca2+ regulation of striated muscle.

The troponin (Tn) complex regulates the thin filament of striated muscle by transducing [Ca2+] fluctuations into conformational changes. These changes propagate to tropomyosin (Tm), which then assumes a new disposition with respect to actin, reversibly exposing actin's binding sites for the thick filament motor-ATPase (myosin). To date, the structural biology of thin filament regulation has been studied in the context of two equilibrium states corresponding to high (contraction-activated) and low (contraction-inhibited) sarcomeric [Ca2+]. New electron micrographic reconstructions of the thin filament have resolved Tn, actin, and Tm in high and low [Ca2+] states, integrating high-resolution structures of the Tn core, actin, and Tm. The resultant picture of thin filament regulation does not resolve all of the functionally significant portions of troponin I (TnI) or troponin C (TnC). Those regions of Tn have been shown (using NMR relaxation spectroscopy) to undergo conformational fluctuations, rationalizing the absence of these regions from micrograph-based reconstructions. The disordered portions of Tn are, to date, being interpreted within a canonical structure-activity paradigm. Here we present a new mechanism for the regulation of Tn having explicit descriptions of the kinetic pathways of activation and inhibition. Our thesis is that the intrinsic disorder of TnI is mechanistically significant. As the coupling of folding to binding has been shown to confer an inherent kinetic advantage (known as flycasting activity), our thesis accounts for TnI's conformational heterogeneity and known structure-activity relationships in a parsimonious fashion. We integrate recent NMR structures of the C-terminus of TnI and NMR observations of the conformational dynamics of the Tn complex into high-resolution models of the thin filament. Ways of evaluating the mechanism are discussed. The novel conceptual framework presented here prompts new hypotheses regarding the mechanism of pH sensitivity and of pathogenic mutations in troponin.

Interaction of cardiac troponin C with calmodulin antagonist [corrected] W7 in the presence of three functional regions of cardiac troponin I.

W7 is a well-known calmodulin (CaM) antagonist and has been implicated as an inhibitor of the troponin C-mediated Ca(2+) activation of cardiac muscle contraction. In this study, we use NMR spectroscopy to study binding of W7 to cardiac troponin C (cTnC) free or in complex with cardiac troponin I (cTnI) peptides. Titration of cTnC.3Ca(2+) with W7 shows that residues throughout the sequence, including the N- and C-domains of cTnC and the central linker, are affected. Analysis of the binding stoichiometry and the trajectories of chemical shift changes indicate that W7 binding occurs at multiple sites. To address the issue of whether multiple-site binding is relevant within the troponin complex, W7 is titrated to a cTnC-cTnI complex (cTnC.3Ca(2+).cTnI(34)(-)(71).cTnI(128)(-)(163)). In the presence of the N-terminal (residues approximately 34-71), inhibitory (residues approximately 128-147), and switch (residues approximately 147-163) regions of cTnI, W7 induces chemical shift changes only in the N-domain and not in the C-domain or the central linker of cTnC. The results indicate that in the presence of cTnI, W7 no longer binds to multiple sites of cTnC but instead binds specifically to the N-domain, and the binding (K(D) = 0.5 +/- 0.1 mM) can occur together with the switch region of cTnI. Hence, W7 may play a role in directly modulating the Ca(2+) sensitivity of the regulatory domain of cTnC and the interaction of the switch region of cTnI and cTnC.

The binding of W7, an inhibitor of striated muscle contraction, to cardiac troponin C.

W7 is a well-characterized calmodulin antagonist. It decreases the maximal tension and rate of ATP hydrolysis in cardiac muscle fibers. Cardiac troponin C (cTnC) has been previously implicated as the mechanistically significant target for W7 in the myofilament. Two-dimensional NMR spectra ({1H,15N}- and {1H,13C}-HSQCs) were used to monitor the Ca2+-dependent binding of W7 to cTnC. Titration of cTnC x 3Ca2+ with W7 indicated binding to both domains of the protein. We examined the binding of W7 to the separated domains of cTnC to simplify the spectral analysis. In the titration of the C-terminal domain (cCTnC x 2Ca2+), the spectral peaks originating from a subset of residues changed nonuniformly, and could not be well-described as single-site binding. A global fit of the cCTnC x 2Ca2+ titration data to a two-site, sequential binding model (47 residues simultaneously fit) yielded a dissociation constant (Kd1) of 0.85-0.91 mM for the singly bound state, with the second dissociation constant fit to 3.40-3.65 mM (> or = 4 x Kd1). The titration data for the N-terminal domain (cNTnC x Ca2+) was globally fit to single-site binding model with a Kd of 0.15-0.30 mM (41 residues fit). The data are consistent with W7 binding to each domain's major hydrophobic pocket, coordinating side chains responsible for liganding cTnI. When in muscle fibers, W7 may compete with cTnI for target sites on cTnC.