HIV-1 clade C envelope immunogens that elicit both neutralizing and non-neutralizing V1V2-scaffold-specific antibodies (protective correlates from RV144 human trial) are urgently needed due to the prevalence of this clade in the most impacted regions worldwide. To achieve this, we introduce structure-guided changes followed by consensus-C-sequence-guided optimizations at the V2 region to generate UFO-v2-RQH173 trimer. This improves the abundance of well-formed trimers. Following the immunization of rabbits, the wild-type protein fails to elicit any autologous neutralizing antibodies, but UFO-v2-RQH173 elicits both autologous neutralizing and broad V1V2-scaffold antibodies. The variant with a 173Y modification in the V2 region, most prevalent among HIV-1 sequences, shows decreased ability in displaying a native-like V1V2 epitope with time in vitro and elicited antibodies with lower neutralizing and higher V1V2-scaffold activities. Our results identify a stabilized clade C trimer capable of eliciting improved neutralizing and V1V2-scaffold antibodies and reveal the importance of the V2 region in tuning this.

Mapping polyclonal serum responses is critical to rational vaccine design. However, most high-resolution mapping approaches involve isolating and characterizing individual antibodies, which incompletely defines the polyclonal response. Here we use two complementary approaches to directly map the specificities of the neutralizing and binding antibodies of polyclonal anti-HIV-1 sera from rabbits immunized with BG505 Env SOSIP trimers. We used mutational antigenic profiling to determine how all mutations in Env affected viral neutralization and electron microscopy polyclonal epitope mapping (EMPEM) to directly visualize serum Fabs bound to Env trimers. The dominant neutralizing specificities were generally only a subset of the more diverse binding specificities. Additional differences between binding and neutralization reflected antigenicity differences between virus and soluble Env trimer. Furthermore, we refined residue-level epitope specificity directly from sera, revealing subtle differences across sera. Together, mutational antigenic profiling and EMPEM yield a holistic view of the binding and neutralizing specificity of polyclonal sera.

Flycatcher1 (FLYC1), a MscS homolog, has recently been identified as a candidate mechanosensitive (MS) ion channel involved in Venus flytrap prey recognition. FLYC1 is a larger protein and its sequence diverges from previously studied MscS homologs, suggesting it has unique structural features that contribute to its function. Here, we characterize FLYC1 by cryo-electron microscopy, molecular dynamics simulations, and electrophysiology. Akin to bacterial MscS and plant MSL1 channels, we find that FLYC1 central core includes side portals in the cytoplasmic cage that regulate ion preference and conduction, by identifying critical residues that modulate channel conductance. Topologically unique cytoplasmic flanking regions can adopt ‘up’ or ‘down’ conformations, making the channel asymmetric. Disruption of an up conformation-specific interaction severely delays channel deactivation by 40-fold likely due to stabilization of the channel open state. Our results illustrate novel structural features and likely conformational transitions that regulate mechano-gating of FLYC1. Flycatcher1 (FLYC1) is a candidate mechanosensitive channel involved in Venus flytrap touch-induced prey capture. Here, the authors report structural and functional details of FLYC1, with insights into gating conformational transitions.

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.

Monoclonal antibodies (mAbs) have proven effective for the treatment of ebolavirus infection in humans, with two mAb-based drugs Inmazeb™ and Ebanga™ receiving FDA approval in 2020. While these drugs represent a major advance in the field of filoviral therapeutics, they are composed of antibodies with single-species specificity for Zaire ebolavirus. The Ebolavirus genus includes five additional species, two of which, Bundibugyo ebolavirus and Sudan ebolavirus, have caused severe disease and significant outbreaks in the past. There are several recently identified broadly neutralizing ebolavirus antibodies, including some in the clinical development pipeline, that have demonstrated broad protection in preclinical studies. In this review, we describe how structural biology has illuminated the molecular basis of broad ebolavirus neutralization, including details of common antigenic sites of vulnerability on the glycoprotein surface. We begin with a discussion outlining the history of monoclonal antibody therapeutics for ebolaviruses, with an emphasis on how structural biology has contributed to these efforts. Next, we highlight key structural studies that have advanced our understanding of ebolavirus glycoprotein structures and mechanisms of antibody-mediated neutralization. Finally, we offer examples of how structural biology has contributed to advances in anti-viral medicines and discuss what opportunities the future holds, including rationally designed next-generation therapeutics with increased potency, breadth, and specificity against ebolaviruses.

Broadly neutralizing antibodies that target epitopes of haemagglutinin on the influenza virus have the potential to provide near universal protection against influenza virus infection1. However, viral mutants that escape broadly neutralizing antibodies have been reported2,3. The identification of broadly neutralizing antibody classes that can neutralize viral escape mutants is critical for universal influenza virus vaccine design. Here we report a distinct class of broadly neutralizing antibodies that target a discrete membrane-proximal anchor epitope of the haemagglutinin stalk domain. Anchor epitope-targeting antibodies are broadly neutralizing across H1 viruses and can cross-react with H2 and H5 viruses that are a pandemic threat. Antibodies that target this anchor epitope utilize a highly restricted repertoire, which encodes two public binding motifs that make extensive contacts with conserved residues in the fusion peptide. Moreover, anchor epitope-targeting B cells are common in the human memory B cell repertoire and were recalled in humans by an oil-in-water adjuvanted chimeric haemagglutinin vaccine4,5, which is a potential universal influenza virus vaccine. To maximize protection against seasonal and pandemic influenza viruses, vaccines should aim to boost this previously untapped source of broadly neutralizing antibodies that are widespread in the human memory B cell pool. A distinct class of broadly neutralizing antibodies to the influenza virus target a membrane-proximal anchor epitope of the haemagglutinin stalk domain.

Recognition of N-linked glycan at residue N276 (glycan276) at the periphery of the CD4-binding site (CD4bs) on the HIV-envelope trimer is a formidable challenge for many CD4bs-directed antibodies. To understand how this glycan can be recognized, here we isolate two lineages of glycan276-dependent CD4bs antibodies. Antibody CH540-VRC40.01 (named for donor-lineage.clone) neutralizes 81% of a panel of 208 diverse strains, while antibody CH314-VRC33.01 neutralizes 45%. Cryo-electron microscopy (cryo-EM) structures of these two antibodies and 179NC75, a previously identified glycan276-dependent CD4bs antibody, in complex with HIV-envelope trimer reveal substantially different modes of glycan276 recognition. Despite these differences, binding of glycan276-dependent antibodies maintains a glycan276 conformation similar to that observed in the absence of glycan276-binding antibodies. By contrast, glycan276-independent CD4bs antibodies, such as VRC01, displace glycan276 upon binding. These results provide a foundation for understanding antibody recognition of glycan276 and suggest its presence may be crucial for priming immunogens seeking to initiate broad CD4bs recognition.

Ebolaviruses cause a severe and often fatal illness with the potential for global spread. Monoclonal antibody-based treatments that have become available recently have a narrow therapeutic spectrum and are ineffective against ebolaviruses other than Ebola virus (EBOV), including medically important Bundibugyo (BDBV) and Sudan (SUDV) viruses. Here, we report the development of a therapeutic cocktail comprising two broadly neutralizing human antibodies, rEBOV-515 and rEBOV-442, that recognize non-overlapping sites on the ebolavirus glycoprotein (GP). Antibodies in the cocktail exhibited synergistic neutralizing activity, resisted viral escape, and possessed differing requirements for their Fc-regions for optimal in vivo activities. The cocktail protected non-human primates from ebolavirus disease caused by EBOV, BDBV, or SUDV with high therapeutic effectiveness. High-resolution structures of the cocktail antibodies in complex with GP revealed the molecular determinants for neutralization breadth and potency. This study provides advanced preclinical data to support clinical development of this cocktail for pan-ebolavirus therapy.