Mechanically activated ion channels underlie touch, hearing, shear-stress sensing, and response to turgor pressure. OSCA/TMEM63s are a newly-identified family of eukaryotic mechanically activated ion channels opened by membrane tension. The structural underpinnings of OSCA/TMEM63 function are not explored. Here, we elucidate high resolution cryo-electron microscopy structures of OSCA1.2, revealing a dimeric architecture containing eleven transmembrane helices per subunit and surprising topological similarities to TMEM16 proteins. We locate the ion permeation pathway within each subunit by demonstrating that a conserved acidic residue is a determinant of channel conductance. Molecular dynamics simulations reveal membrane interactions, suggesting the role of lipids in OSCA1.2 gating. These results lay a foundation to decipher how the structural organization of OSCA/TMEM63 is suited for their roles as MA ion channels.

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 as a highly transmissible pathogenic human betacoronavirus. The viral spike glycoprotein (S) utilizes angiotensin-converting enzyme 2 (ACE2) as a host protein receptor and mediates fusion of the viral and host membranes, making S essential to viral entry into host cells and host species tropism. As SARS-CoV enters host cells, the viral S is believed to undergo a number of conformational transitions as it is cleaved by host proteases and binds to host receptors. We recently developed stabilizing mutations for coronavirus spikes that prevent the transition from the pre-fusion to post-fusion states. Here, we present cryo-EM analyses of a stabilized trimeric SARS-CoV S, as well as the trypsin-cleaved, stabilized S, and its interactions with ACE2. Neither binding to ACE2 nor cleavage by trypsin at the S1/S2 cleavage site impart large conformational changes within stabilized SARS-CoV S or expose the secondary cleavage site, S2′.

The circumsporozoite protein (CSP) on the surface of Plasmodium falciparum sporozoites is important for parasite development, motility, and host hepatocyte invasion. However, intrinsic disorder of the NANP repeat sequence in the central region of CSP has hindered its structural and functional characterization. Here, the cryo–electron microscopy structure at ~3.4-Å resolution of a recombinant shortened CSP construct with the variable domains (Fabs) of a highly protective monoclonal antibody reveals an extended spiral conformation of the central NANP repeat region surrounded by antibodies. This unusual structure appears to be stabilized and/or induced by interaction with an antibody where contacts between adjacent Fabs are somatically mutated and enhance the interaction. This maturation in non-antigen contact residues may be an effective mechanism for antibodies to target tandem repeat sequences and provide novel insights into malaria vaccine design.

The HIV-1-envelope glycoprotein (Env) is the main target of antigen design for antibody-based prophylactic vaccines. The generation of broadly neutralizing antibodies (bNAb) likely requires the appropriate presentation of stabilized trimers preventing exposure of non-neutralizing antibody (nNAb) epitopes. We designed a series of membrane-bound Envs with increased trimer stability through the introduction of key stabilization mutations. We derived a stabilized HIV-1 trimer, ConSOSL.UFO.750, which displays a dramatic reduction in nNAb binding while maintaining high quaternary and MPER-specific bNAb binding. Its soluble counterpart, ConSOSL.UFO.664, displays similar antigenicity, and its native-like Env structure is confirmed by negative stain-EM and glycosylation profiling of the soluble ConSOSL.UFO.664 trimer. A rabbit immunization study demonstrated that the ConSOSL.UFO.664 can induce autologous tier 2 neutralization. We have successfully designed a stabilized native-like Env trimer amenable to nucleic acid or viral vector-based vaccination strategies.

As the sole target of broadly neutralizing antibodies (bnAbs) to HIV, the envelope glycoprotein (Env) trimer is the focus of vaccination strategies designed to elicit protective bnAbs in humans. Because HIV Env is densely glycosylated with 75–90 N-glycans per trimer, most bnAbs use or accommodate them in their binding epitope, making the glycosylation of recombinant Env a key aspect of HIV vaccine design. Upon analysis of three HIV strains, we here find that site-specific glycosylation of Env from infectious virus closely matches Envs from corresponding recombinant membrane-bound trimers. However, viral Envs differ significantly from recombinant soluble, cleaved (SOSIP) Env trimers, strongly impacting antigenicity. These results provide a benchmark for virus Env glycosylation needed for the design of soluble Env trimers as part of an overall HIV vaccine strategy.

Monoclonal antibodies (mAbs) with pan-ebolavirus cross-reactivity are highly desirable, but development of such mAbs is limited by a lack of a molecular understanding of cross-reactive epitopes. The antibody ADI-15878 was previously identified from a human survivor of Ebola virus Makona variant (EBOV/Mak) infection. This mAb demonstrated potent neutralizing activity against all known ebolaviruses and provided protection in rodent and ferret models against three ebolavirus species. Here, we describe the unliganded crystal structure of ADI-15878 as well as the cryo-EM structures of ADI-15878 in complex with the EBOV/Mak and Bundibugyo virus (BDBV) glycoproteins (GPs). ADI-15878 binds through an induced-fit mechanism by targeting highly conserved residues in the internal fusion loop (IFL), bridging across GP protomers via the heptad repeat 1 (HR1) region. Our structures provide a more complete description of the ebolavirus immunogenic landscape, as well as a molecular basis for how rare but potent antibodies target conserved filoviral fusion machinery.

Characterizing polyclonal antibody responses via currently available methods is inherently complex and difficult. Mapping epitopes in an immune response is typically incomplete, which creates a barrier to fully understanding the humoral response to antigens and hinders rational vaccine design efforts. Here, we describe a method of characterizing polyclonal responses by using electron microscopy, and we applied this method to the immunization of rabbits with an HIV-1 envelope glycoprotein vaccine candidate, BG505 SOSIP.664. We detected known epitopes within the polyclonal sera and revealed how antibody responses evolved during the prime-boosting strategy to ultimately result in a neutralizing antibody response. We uncovered previously unidentified epitopes, including an epitope proximal to one recognized by human broadly neutralizing antibodies as well as potentially distracting non-neutralizing epitopes. Our method provides an efficient and semiquantitative map of epitopes that are targeted in a polyclonal antibody response and should be of widespread utility in vaccine and infection studies.

Ebolaviruses cause severe disease in humans, and identification of monoclonal antibodies (mAbs) that are effective against multiple ebolaviruses are important for therapeutics development. Here we describe a distinct class of broadly neutralizing human mAbs with protective capacity against three ebolaviruses infectious for humans: Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) viruses. We isolated mAbs from human survivors of ebolavirus disease and identified a potent mAb, EBOV-520, which bound to an epitope in the glycoprotein (GP) base region. EBOV-520 efficiently neutralized EBOV, BDBV, and SUDV and also showed protective capacity in relevant animal models of these infections. EBOV-520 mediated protection principally by direct virus neutralization and exhibited multifunctional properties. This study identified a potent naturally occurring mAb and defined key features of the human antibody response that may contribute to broad protection. This multifunctional mAb and related clones are promising candidates for development as broadly protective pan-ebolavirus therapeutic molecules.

SWELL1 (LRRC8A) is the only essential subunit of the Volume Regulated Anion Channel (VRAC), which regulates cellular volume homeostasis and is activated by hypotonic solutions. SWELL1, together with four other LRRC8 family members, potentially forms a vastly heterogeneous cohort of VRAC channels with different properties; however, SWELL1 alone is also functional. Here, we report a high-resolution cryo-electron microscopy structure of full-length human homo-hexameric SWELL1. The structure reveals a trimer of dimers assembly with symmetry mismatch between the pore-forming domain and the cytosolic leucine-rich repeat (LRR) domains. Importantly, mutational analysis demonstrates that a charged residue at the narrowest constriction of the homomeric channel is an important pore determinant of heteromeric VRAC. Additionally, a mutation in the flexible N-terminal portion of SWELL1 affects pore properties, suggesting a putative link between intracellular structures and channel regulation. This structure provides a scaffold for further dissecting the heterogeneity and mechanism of activation of VRAC.

Antibodies are promising post-exposure therapies against emerging viruses, but which antibody features and in vitro assays best forecast protection are unclear. Our international consortium systematically evaluated antibodies against Ebola virus (EBOV) using multidisciplinary assays. For each antibody, we evaluated epitopes recognized on the viral surface glycoprotein (GP) and secreted glycoprotein (sGP), readouts of multiple neutralization assays, fraction of virions left un-neutralized, glycan structures, phagocytic and natural killer cell functions elicited, and in vivo protection in a mouse challenge model. Neutralization and induction of multiple immune effector functions (IEFs) correlated most strongly with protection. Neutralization predominantly occurred via epitopes maintained on endosomally cleaved GP, whereas maximal IEF mapped to epitopes farthest from the viral membrane. Unexpectedly, sGP cross-reactivity did not significantly influence in vivo protection. This comprehensive dataset provides a rubric to evaluate novel antibodies and vaccine responses and a roadmap for therapeutic development for EBOV and related viruses.

The high rate of antigenic drift in seasonal influenza viruses necessitates frequent changes in vaccine composition. Recent seasonal H3 vaccines do not protect against swine-origin H3N2 variant (H3N2v) strains that recently have caused severe human infections. Here, we report a human VH1-69 gene-encoded monoclonal antibody (mAb) designated H3v-47 that exhibits potent cross-reactive neutralization activity against human and swine H3N2 viruses that circulated since 1989. The crystal structure and electron microscopy reconstruction of H3v-47 Fab with the H3N2v hemagglutinin (HA) identify a unique epitope spanning the vestigial esterase and receptor-binding subdomains that is distinct from that of any known neutralizing antibody for influenza A H3 viruses. MAb H3v-47 functions largely by blocking viral egress from infected cells. Interestingly, H3v-47 also engages Fcγ receptor and mediates antibody dependent cellular cytotoxicity (ADCC). This newly identified conserved epitope can be used in design of novel immunogens for development of broadly protective H3 vaccines.

Native-like, soluble, recombinant SOSIP trimers of various designs and based on several env genes of human immunodeficiency virus type 1 (HIV-1) are being tested as immunogens in different animal models. These experiments almost always involve coformulating the trimers with an adjuvant to boost the magnitude of the immune responses. One factor relevant to the choice of an adjuvant is that it should not physically damage the immunogen or impede its ability to present relevant epitopes. As examples, an adjuvant formulation that includes harsh detergents could disrupt the structural integrity of a trimer, and any charged compounds in the formulation could bind to countercharged regions of the trimer and physically occlude nearby epitopes. While a few adjuvants have been tested for their potential effects on SOSIP trimers in vitro, there has been no systematic study. Here, we have assessed how nine different adjuvants of various compositions affect SOSIP trimers of the BG505 and B41 genotypes. We used negative-stain electron microscopy, thermal denaturation, and gel electrophoresis to evaluate effects on trimer integrity and immunoassays to measure effects on the presentation of various epitopes. We conclude that most of the tested adjuvants are benign from these perspectives, but some raise grounds for concern. An acidified alum formulation is highly disruptive to trimer integrity, and a DNA-based polyanionic CpG oligodeoxynucleotide adjuvant binds to trimers and occludes the trimer apex epitope for the PGT145 neutralizing antibody. The methods described here should be generalizable to protein subunit vaccines targeting various pathogens. IMPORTANCE Adjuvant formulations increase the magnitude of immune responses to vaccine antigens. They are critically important for formulation of HIV-1 envelope glycoprotein (Env) vaccines intended to induce antibody production, as Env proteins are otherwise only very weakly immunogenic. The HIV-1 vaccine field now uses the well-defined structures of trimeric Env glycoproteins, like SOSIPs, to present multiple known epitopes for broad and potent neutralizing human antibodies in a native-like conformation. Successful adjuvant formulations must not disrupt how the trimers are folded, as that could adversely affect their performance as immunogens. We studied whether the various adjuvants most commonly used in animal experiments affect the integrity of two different SOSIP trimers in vitro. Most adjuvant classes are not problematic, but an aluminum sulfate formulation is highly damaging, as it exposes trimers to acidic pH and a nucleic acid-based adjuvant can bind to the trimer and block access to a key neutralizing epitope.

Broadly neutralizing antibodies (bnAbs) targeting the HIV envelope glycoprotein (Env) typically take years to develop. Longitudinal analyses of both neutralizing antibody lineages and viruses at serial time points during infection provide a basis for understanding the co-evolutionary contest between HIV and the humoral immune system. Here, we describe the structural characterization of an apex-targeting antibody lineage and autologous clade A viral Env from a donor in the Protocol C cohort. Comparison of Ab-Env complexes at early and late time points reveals that, within the antibody lineage, the CDRH3 loop rigidifies, the bnAb angle of approach steepens, and surface charges are mutated to accommodate glycan changes. Additionally, we observed differences in site-specific glycosylation between soluble and full-length Env constructs, which may be important for tuning optimal immunogenicity in soluble Env trimers. These studies therefore provide important guideposts for design of immunogens that prime and mature nAb responses to the Env V2-apex.

Ebola virus (EBOV) in humans causes a severe illness with high mortality rates. Several strategies have been developed in the past to treat EBOV infection, including the antibody cocktail ZMappTM that has been shown to be effective in nonhuman primate models of infection1 and has been used under compassionate-treatment protocols in humans2. ZMappTM is a mixture of three chimerized murine monoclonal antibodies (mAbs)3–6 that target EBOV-specific epitopes on the surface glycoprotein (GP)7,8. However, ZMappTM mAbs do not neutralize other species from the Ebolavirus genus, such as Bundibugyo virus (BDBV), Reston virus (RESTV) or Sudan virus (SUDV). Here we describe three naturally-occurring human cross-neutralizing mAbs, from BDBV survivors, that target an antigenic site in the canonical heptad repeat 2 (HR2) region near the membrane proximal external region (MPER) of GP. The identification of a conserved neutralizing antigenic site in the GP suggests that these mAbs could be used to design universal antibody therapeutics against diverse ebolavirus species. Furthermore, we found that immunization with a peptide comprising the HR2/MPER antigenic site elicits neutralizing antibodies in rabbits. Structural features determined by conserved residues in the antigenic site described here could inform an epitope-based vaccine design against infection caused by diverse ebolavirus species.