A tale of two fusion proteins: understanding the metastability of human respiratory syncytial virus and metapneumovirus and implications for rational design of uncleaved prefusion-closed trimers.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause human respiratory diseases and are major targets for vaccine development. In this study, we designed uncleaved prefusion-closed (UFC) trimers for the fusion (F) proteins of both viruses by examining mutations critical to F metastability. For RSV, we assessed four previous prefusion F designs, including the first and second generations of DS-Cav1, SC-TM, and 847A. We then identified key mutations that can maintain prefusion F in a native-like, closed trimeric form (up to 76%) without introducing any interprotomer disulfide bond. For hMPV, we developed a stable UFC trimer with a truncated F2-F1 linkage and an interprotomer disulfide bond. Tens of UFC constructs were characterized by negative-stain electron microscopy (nsEM), x-ray crystallography (11 RSV-F and one hMPV-F structures), and antigenic profiling. Using an optimized RSV-F UFC trimer as bait, we identified three potent RSV neutralizing antibodies (NAbs) from a phage-displayed human antibody library, with a public NAb lineage targeting sites Ø and V and two cross-pneumovirus NAbs recognizing site III. In mouse immunization, rationally designed RSV-F and hMPV-F UFC trimers induced robust antibody responses with high neutralizing titers. Our study provides a foundation for future prefusion F-based RSV and hMPV vaccine development.

Mispacking of the F87 sidechain drives aggregation-promoting conformational fluctuations in the subunit interfaces of the transthyretin tetramer.

Aberrant formation and deposition of human transthyretin (TTR) aggregates causes transthyretin amyloidosis. To initialize aggregation, transthyretin tetramers must first dissociate into monomers that partially unfold to promote entry into the aggregation pathway. The native TTR tetramer (T) is stabilized by docking of the F87 sidechain into an interfacial cavity enclosed by several hydrophobic residues including A120. We have previously shown that an alternative tetramer (T*) with mispacked F87 sidechains is more prone to dissociation and aggregation than the native T state. However, the molecular basis for the reduced stability in T* remains unclear. Here we report characterization of the A120L mutant, where steric hindrance is introduced into the F87 binding site. The X-ray structure of A120L shows that the F87 sidechain is displaced from its docking site across the subunit interface. In A120S, a naturally occurring pathogenic mutant that is less aggregation-prone than A120L, the F87 sidechain is correctly docked, as in the native TTR tetramer. Nevertheless, 19F-NMR aggregation assays show an elevated population of a monomeric aggregation intermediate in A120S relative to a control containing the native A120, due to accelerated tetramer dissociation and slowed monomer tetramerization. The mispacking of the F87 sidechain is associated with enhanced exchange dynamics for interfacial residues. At 298 K, the T* populations of various naturally occurring mutants fall between 4-7% (ΔG ~ 1.5-1.9 kcal/mol), consistent with the free energy change expected for undocking and solvent exposure of one of the four F87 sidechains in the tetramer (ΔG ~ 1.6 kcal/mol). Our data provide a molecular-level picture of the likely universal F87 sidechain mispacking in tetrameric TTR that promotes interfacial conformational dynamics and increases aggregation propensity.

Immunization of cows with HIV envelope trimers generates broadly neutralizing antibodies to the V2-apex from the ultralong CDRH3 repertoire.

The generation of broadly neutralizing antibodies (bnAbs) to specific HIV epitopes of the HIV Envelope (Env) is one of the cornerstones of HIV vaccine research. The current animal models we use have been unable to reliable produce a broadly neutralizing antibody response, with the exception of cows. Cows have rapidly and reliably produced a CD4 binding site response by homologous prime and boosting with a native-like Env trimer. In small animal models other engineered immunogens previously have been able to focus antibody responses to the bnAb V2-apex region of Env. Here, we immunized two groups of cows (n=4) with two regiments of V2-apex focusing immunogens to investigate whether antibody responses could be directed to the V2-apex on Env. Group 1 were immunized with chimpanzee simian immunodeficiency virus (SIV)-Env trimer that shares its V2-apex with HIV, followed by immunization with C108, a V2-apex focusing immunogen, and finally boosted with a cross-clade native-like trimer cocktail. Group 2 were immunized with HIV C108 Env trimer followed by the same HIV trimer cocktail as Group 1. Longitudinal serum analysis showed that one cow in each group developed serum neutralizing antibody responses to the V2-apex. Eight and 11 bnAbs were isolated from Group 1 and Group 2 cows respectively. The best bnAbs had both medium breadth and potency. Potent and broad responses developed later than previous CD4bs cow bnAbs and required several different immunogens. All isolated bnAbs were derived from the ultralong CDRH3 repertoire. The finding that cow antibodies can target multiple broadly neutralizing epitopes on the HIV surface reveals important insight into the generation of immunogens and testing in the cow animal model. The exclusive isolation of ultralong CDRH3 bnAbs, despite only comprising a small percent of the cow repertoire, suggests these antibodies outcompete the long and short CDRH3 antibodies during the bnAb response.

Synthetic development of a broadly neutralizing antibody against snake venom long-chain α-neurotoxins.

Snakebite envenoming is a major global public health concern for which improved therapies are urgently needed. The antigenic diversity present in snake venom toxins from various species presents a considerable challenge to the development of a universal antivenom. Here, we used a synthetic human antibody library to find and develop an antibody that neutralizes long-chain three-finger α-neurotoxins produced by numerous medically relevant snakes. Our antibody bound diverse toxin variants with high affinity, blocked toxin binding to the nicotinic acetylcholine receptor in vitro, and protected mice from lethal venom challenge. Structural analysis of the antibody-toxin complex revealed a binding mode that mimics the receptor-toxin interaction. The overall workflow presented is generalizable for the development of antibodies that target conserved epitopes among antigenically diverse targets, and it offers a promising framework for the creation of a monoclonal antibody-based universal antivenom to treat snakebite envenoming.

Probing the Dissociation Pathway of a Kinetically Labile Transthyretin Mutant.

Aggregation of transthyretin (TTR) is associated with devastating amyloid diseases. Amyloidosis begins with the dissociation of the native homotetramer (a dimer of dimers) to form a monomeric intermediate that assembles into pathogenic aggregates. This process is accelerated in vitro at low pH, but the process by which TTR dissociates and reassembles at neutral pH remains poorly characterized due to the low population of intermediates. Here, we use 19F-nuclear magnetic resonance (NMR) and a highly sensitive trifluoromethyl probe to determine the relative populations of the species formed by the dissociation of a destabilized variant, A25T. The A25T mutation perturbs both the strong dimer and weak dimer-dimer interfaces. A tetramer ⇌ dimer ⇌ monomer (TDM) equilibrium model is proposed to account for concentration- and temperature-dependent population changes. Thermodynamic and kinetic parameters and activation energetics for dissociation of the native A25T tetramer, as well as a destabilized alternative tetramer (T*) with a mispacked F87 side chain, were extracted by van't Hoff and 19F-NMR line shape analysis, saturation transfer, and transition state theory. Chemical shifts for the dimer and T* species are degenerate for 19F and methyl probes close to the strong dimer interface, implicating interfacial perturbation as a common structural feature of these destabilized species. All-atom molecular dynamics simulations further suggest more frequent F87 ring flipping on the nanosecond time scale in the A25T dimer than in the native A25T tetramer. Our integrated approach offers quantitative insights into the energy landscape of the dissociation pathway of TTR at neutral pH.

The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2.

Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This "knob" domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.

Divergent Total Synthesis and Characterization of Maxamycins.

A new streamlined and scaled divergent total synthesis of pocket-modified vancomycin analogs is detailed that provides a common late-stage intermediate [Ψ[C(═S)NH]Tpg4]vancomycin (LLS = 18 steps, 12% overall yield, >5 g prepared) to access both existing and future pocket modifications. Highlights of the approach include an atroposelective synthesis of [Ψ[C(═S)NH]Tpg4]vancomycin aglycon (11), a one-pot enzymatic glycosylation for direct conversion to [Ψ[C(═S)NH]Tpg4]vancomycin (12), and new powerful methods for the late-stage conversion of the embedded thioamide to amidine/aminomethylene pocket modifications. Incorporation of two peripheral modifications provides a scalable total synthesis of the maxamycins, all prepared from aglycon 11 without use of protecting groups. Thus, both existing and presently unexplored pocket-modified analogues paired with a range of peripheral modifications are accessible from this common thioamide intermediate. In addition to providing an improved synthesis of the initial member of the maxamycins, this is illustrated herein with the first synthesis and examination of maxamycins that contain the most effective of the pocket modifications (amidine) described to date combined with two additional peripheral modifications. These new amidine-based maxamycins proved to be potent, durable, and efficacious antimicrobial agents that display equipotent activity against vancomycin-sensitive and vancomycin-resistant Gram-positive organisms and act by three independent synergistic mechanisms of action. In the first such study conducted to date, one new maxamycin (21, MX-4) exhibited efficacious in vivo activity against a feared and especially challenging multidrug-resistant (MRSA) and vancomycin-resistant (VRSA) S. aureus bacterial strain (VanA VRS-2) for which vancomycin is inactive.

Fully synthetic platform to rapidly generate tetravalent bispecific nanobody-based immunoglobulins.

Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human VH3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the VH and VL domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.

Complementary antibody lineages achieve neutralization breadth in an HIV-1 infected elite neutralizer.

Broadly neutralizing antibodies (bNAbs) have remarkable breadth and potency against most HIV-1 subtypes and are able to prevent HIV-1 infection in animal models. However, bNAbs are extremely difficult to induce by vaccination. Defining the developmental pathways towards neutralization breadth can assist in the design of strategies to elicit protective bNAb responses by vaccination. Here, HIV-1 envelope glycoproteins (Env)-specific IgG+ B cells were isolated at various time points post infection from an HIV-1 infected elite neutralizer to obtain monoclonal antibodies (mAbs). Multiple antibody lineages were isolated targeting distinct epitopes on Env, including the gp120-gp41 interface, CD4-binding site, silent face and V3 region. The mAbs each neutralized a diverse set of HIV-1 strains from different clades indicating that the patient's remarkable serum breadth and potency might have been the result of a polyclonal mixture rather than a single bNAb lineage. High-resolution cryo-electron microscopy structures of the neutralizing mAbs (NAbs) in complex with an Env trimer generated from the same individual revealed that the NAbs used multiple strategies to neutralize the virus; blocking the receptor binding site, binding to HIV-1 Env N-linked glycans, and disassembly of the trimer. These results show that diverse NAbs can complement each other to achieve a broad and potent neutralizing serum response in HIV-1 infected individuals. Hence, the induction of combinations of moderately broad NAbs might be a viable vaccine strategy to protect against a wide range of circulating HIV-1 viruses.

Identification of IOMA-class neutralizing antibodies targeting the CD4-binding site on the HIV-1 envelope glycoprotein.

A major goal of current HIV-1 vaccine design efforts is to induce broadly neutralizing antibodies (bNAbs). The VH1-2-derived bNAb IOMA directed to the CD4-binding site of the HIV-1 envelope glycoprotein is of interest because, unlike the better-known VH1-2-derived VRC01-class bNAbs, it does not require a rare short light chain complementarity-determining region 3 (CDRL3). Here, we describe three IOMA-class NAbs, ACS101-103, with up to 37% breadth, that share many characteristics with IOMA, including an average-length CDRL3. Cryo-electron microscopy revealed that ACS101 shares interactions with those observed with other VH1-2 and VH1-46-class bNAbs, but exhibits a unique binding mode to residues in loop D. Analysis of longitudinal sequences from the patient suggests that a transmitter/founder-virus lacking the N276 glycan might have initiated the development of these NAbs. Together these data strengthen the rationale for germline-targeting vaccination strategies to induce IOMA-class bNAbs and provide a wealth of sequence and structural information to support such strategies.

The molecular basis of allostery in a facilitated dissociation process.

Facilitated dissociation provides a mechanism by which high-affinity complexes can be rapidly disassembled. The negative feedback regulator CITED2 efficiently downregulates the hypoxic response by displacing the hypoxia-inducible transcription factor HIF-1α from the TAZ1 domain of the transcriptional coactivators CREB-binding protein (CBP) and p300. Displacement occurs by a facilitated dissociation mechanism involving a transient ternary intermediate formed by binding of the intrinsically disordered CITED2 activation domain to the TAZ1:HIF-1α complex. The short lifetime of the intermediate precludes straightforward structural investigations. To obtain insights into the molecular determinants of facilitated dissociation, we model the ternary intermediate by generating a fusion peptide composed of the primary CITED2 and HIF-1α binding motifs. X-ray crystallographic and NMR studies of the fusion peptide complex reveal TAZ1-mediated negative cooperativity that results in nearly mutually exclusive binding of specific CITED2 and HIF-1α interaction motifs, providing molecular-level insights into the allosteric switch that terminates the hypoxic response.

Single-component multilayered self-assembling nanoparticles presenting rationally designed glycoprotein trimers as Ebola virus vaccines.

Ebola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design. Here, we first investigate the contribution of the stalk and heptad repeat 1-C (HR1C) regions to GP metastability. Specific stalk and HR1C modifications in a mucin-deleted form (GPΔmuc) increase trimer yield, whereas alterations of HR1C exert a more complex effect on thermostability. Crystal structures are determined to validate two rationally designed GPΔmuc trimers in their unliganded state. We then display a modified GPΔmuc trimer on reengineered protein nanoparticles that encapsulate a layer of locking domains (LD) and a cluster of helper T-cell epitopes. In mice and rabbits, GP trimers and nanoparticles elicit cross-ebolavirus NAbs, as well as non-NAbs that enhance pseudovirus infection. Repertoire sequencing reveals quantitative profiles of vaccine-induced B-cell responses. This study demonstrates a promising vaccine strategy for filoviruses, such as EBOV, based on GP stabilization and nanoparticle display.

50 Years of structural immunology.

Fifty years ago, the first landmark structures of antibodies heralded the dawn of structural immunology. Momentum then started to build toward understanding how antibodies could recognize the vast universe of potential antigens and how antibody-combining sites could be tailored to engage antigens with high specificity and affinity through recombination of germline genes (V, D, J) and somatic mutation. Equivalent groundbreaking structures in the cellular immune system appeared some 15 to 20 years later and illustrated how processed protein antigens in the form of peptides are presented by MHC molecules to T cell receptors. Structures of antigen receptors in the innate immune system then explained their inherent specificity for particular microbial antigens including lipids, carbohydrates, nucleic acids, small molecules, and specific proteins. These two sides of the immune system act immediately (innate) to particular microbial antigens or evolve (adaptive) to attain high specificity and affinity to a much wider range of antigens. We also include examples of other key receptors in the immune system (cytokine receptors) that regulate immunity and inflammation. Furthermore, these antigen receptors use a limited set of protein folds to accomplish their various immunological roles. The other main players are the antigens themselves. We focus on surface glycoproteins in enveloped viruses including SARS-CoV-2 that enable entry and egress into host cells and are targets for the antibody response. This review covers what we have learned over the past half century about the structural basis of the immune response to microbial pathogens and how that information can be utilized to design vaccines and therapeutics.

Functional convergence of a germline-encoded neutralizing antibody response in rhesus macaques immunized with HCV envelope glycoproteins.

Human IGHV1-69-encoded broadly neutralizing antibodies (bnAbs) that target the hepatitis C virus (HCV) envelope glycoprotein (Env) E2 are important for protection against HCV infection. An IGHV1-69 ortholog gene, VH1.36, is preferentially used for bnAbs isolated from HCV Env-immunized rhesus macaques (RMs). Here, we studied the genetic, structural, and functional properties of VH1.36-encoded bnAbs generated by vaccination, in comparison to IGHV1-69-encoded bnAbs from HCV patients. Global B cell repertoire analysis confirmed the expansion of VH1.36-derived B cells in immunized animals. Most E2-specific, VH1.36-encoded antibodies cross-neutralized HCV. Crystal structures of two RM bnAbs with E2 revealed that the RM bnAbs engaged conserved E2 epitopes using similar molecular features as human bnAbs but with a different binding mode. Longitudinal analyses of the RM antibody repertoire responses during immunization indicated rapid lineage development of VH1.36-encoded bnAbs with limited somatic hypermutation. Our findings suggest functional convergence of a germline-encoded bnAb response to HCV Env with implications for vaccination in humans.

Structural basis for differential recognition of phosphohistidine-containing peptides by 1-pHis and 3-pHis monoclonal antibodies.

In 2015, monoclonal antibodies (mAbs) that selectively recognize the 1-pHis or 3-pHis isoforms of phosphohistidine were developed by immunizing rabbits with degenerate Ala/Gly peptides containing the nonhydrolyzable phosphohistidine (pHis) analog- phosphotriazolylalanine (pTza). Here, we report structures of five rabbit mAbs bound to cognate pTza peptides: SC1-1 and SC50-3 that recognize 1-pHis, and their 3-pHis-specific counterparts, SC39-4, SC44-8, and SC56-2. These cocrystal structures provide insights into the binding modes of the pTza phosphate group that are distinct for the 1- and 3-pHis mAbs with the selectivity arising from specific contacts with the phosphate group and triazolyl ring. The mode of phosphate recognition in the 3-pHis mAbs recapitulates the Walker A motif, as present in kinases. The complementarity-determining regions (CDRs) of four of the Fabs interact with the peptide backbone rather than peptide side chains, thus conferring sequence independence, whereas SC44-8 shows a proclivity for binding a GpHAGA motif mediated by a sterically complementary CDRL3 loop. Specific hydrogen bonding with the triazolyl ring precludes recognition of pTyr and other phosphoamino acids by these mAbs. Kinetic binding experiments reveal that the affinity of pHis mAbs for pHis and pTza peptides is submicromolar. Bound pHis mAbs also shield the pHis peptides from rapid dephosphorylation. The epitope-paratope interactions illustrate how these anti-pHis antibodies are useful for a wide range of research techniques and this structural information can be utilized to improve the specificity and affinity of these antibodies toward a variety of pHis substrates to understand the role of histidine phosphorylation in healthy and diseased states.

An alternate conformation of HCV E2 neutralizing face as an additional vaccine target.

To achieve global elimination of hepatitis C virus (HCV), an effective cross-genotype vaccine is needed. The HCV envelope glycoprotein E2 is the main target for neutralizing antibodies (nAbs), which aid in HCV clearance and protection. E2 is structurally flexible and functions in engaging host receptors. Many nAbs bind to the "neutralizing face" on E2, including several broadly nAbs encoded by the VH1-69 germline gene family that bind to a similar conformation (A) of this face. Here, a previously unknown conformation (B) of the neutralizing face is revealed in crystal structures of two of four additional E2-VH1-69 nAb complexes. In this conformation, the E2 front-layer region is displaced upon antibody binding, exposing residues in the back layer for direct antibody interaction. This E2 B structure may represent another conformational state in the viral entry process that is susceptible to antibody neutralization and thus provide a new target for rational vaccine development.

Structural basis of broad HIV neutralization by a vaccine-induced cow antibody.

Potent broadly neutralizing antibodies (bnAbs) to HIV have been very challenging to elicit by vaccination in wild-type animals. Here, by x-ray crystallography, cryo-electron microscopy, and site-directed mutagenesis, we structurally and functionally elucidate the mode of binding of a potent bnAb (NC-Cow1) elicited in cows by immunization with the HIV envelope (Env) trimer BG505 SOSIP.664. The exceptionally long (60 residues) third complementarity-determining region of the heavy chain (CDR H3) of NC-Cow1 forms a mini domain (knob) on an extended stalk that navigates through the dense glycan shield on Env to target a small footprint on the gp120 CD4 receptor binding site with no contact of the other CDRs to the rest of the Env trimer. These findings illustrate, in molecular detail, how an unusual vaccine-induced cow bnAb to HIV Env can neutralize with high potency and breadth.

A Conformational Switch in the Zinc Finger Protein Kaiso Mediates Differential Readout of Specific and Methylated DNA Sequences.

Recognition of the epigenetic mark 5-methylcytosine (mC) at CpG sites in DNA has emerged as a novel function of many eukaryotic transcription factors (TFs). It remains unclear why the sequence specificity of these TFs differs for CpG-methylated motifs and consensus motifs. Here, we dissect the structural and dynamic basis for this differential DNA binding specificity in the human zinc finger TF Kaiso, which exhibits high affinity for two consecutive mCpG sites in variable contexts and also for a longer, sequence-specific Kaiso binding site (KBS). By integrating structural analysis and DNA binding studies with targeted protein mutagenesis and nucleotide substitutions, we identify distinct mechanisms for readout of methylated and KBS motifs by Kaiso. We show that a key glutamate residue (E535), critical for mCpG site recognition, adopts different conformations in complexes with specific and methylated DNA. These conformational differences, together with intrinsic variations in DNA flexibility and/or solvation at TpG versus mCpG sites, contribute to the different DNA affinity and sequence specificity. With methylated DNA, multiple direct contacts between E535 and the 5' mCpG site dominate the binding affinity, allowing for tolerance of different flanking DNA sequences. With KBS, Kaiso employs E535 as part of an indirect screen of the 5' flanking sequence, relying on key tyrosine-DNA interactions to stabilize an optimal DNA conformation and select against noncognate sites. These findings demonstrate how TFs use conformational adaptation and exploit variations in DNA flexibility to achieve distinct DNA readout outcomes and target a greater variety of regulatory and epigenetic sites than previously appreciated.

The Impact of Sustained Immunization Regimens on the Antibody Response to Oligomannose Glycans.

The high mannose patch (HMP) of the HIV envelope protein (Env) is the structure most frequently targeted by broadly neutralizing antibodies; therefore, many researchers have attempted to use mimics of this region as a vaccine immunogen. In our previous efforts, vaccinating rabbits with evolved HMP mimic glycopeptides containing Man9 resulted in an overall antibody response targeting the glycan core and linker rather than the full glycan or Manα1→2Man tips of Man9 glycans. A possible reason could be processing of our immunogen by host serum mannosidases. We sought to test whether more prolonged dosing could increase the antibody response to intact glycans, possibly by increasing the availability of intact Man9 to germinal centers. Here, we describe a study investigating the impact of immunization regimen on antibody response by testing immunogen delivery through bolus, an exponential series of mini doses, or a continuously infusing mini-osmotic pump. Our results indicate that, with our glycopeptide immunogens, standard bolus immunization elicited the strongest HIV Env-binding antibody response, even though higher overall titers to the glycopeptide were elicited by the exponential and pump regimens. Antibody selectivity for intact glycan was, if anything, slightly better in the bolus-immunized animals.