Pan-ebolavirus protective therapy by two multifunctional human antibodies.

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.

Systematic Analysis of Monoclonal Antibodies against Ebola Virus GP Defines Features that Contribute to Protection.

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.

Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection against Ebolaviruses.

Experimental monoclonal antibody (mAb) therapies have shown promise for treatment of lethal Ebola virus (EBOV) infections, but their species-specific recognition of the viral glycoprotein (GP) has limited their use against other divergent ebolaviruses associated with human disease. Here, we mined the human immune response to natural EBOV infection and identified mAbs with exceptionally potent pan-ebolavirus neutralizing activity and protective efficacy against three virulent ebolaviruses. These mAbs recognize an inter-protomer epitope in the GP fusion loop, a critical and conserved element of the viral membrane fusion machinery, and neutralize viral entry by targeting a proteolytically primed, fusion-competent GP intermediate (GPCL) generated in host cell endosomes. Only a few somatic hypermutations are required for broad antiviral activity, and germline-approximating variants display enhanced GPCL recognition, suggesting that such antibodies could be elicited more efficiently with suitably optimized GP immunogens. Our findings inform the development of both broadly effective immunotherapeutics and vaccines against filoviruses.

Cross-Reactive and Potent Neutralizing Antibody Responses in Human Survivors of Natural Ebolavirus Infection.

Recent studies have suggested that antibody-mediated protection against the Ebolaviruses may be achievable, but little is known about whether or not antibodies can confer cross-reactive protection against viruses belonging to diverse Ebolavirus species, such as Ebola virus (EBOV), Sudan virus (SUDV), and Bundibugyo virus (BDBV). We isolated a large panel of human monoclonal antibodies (mAbs) against BDBV glycoprotein (GP) using peripheral blood B cells from survivors of the 2007 BDBV outbreak in Uganda. We determined that a large proportion of mAbs with potent neutralizing activity against BDBV bind to the glycan cap and recognize diverse epitopes within this major antigenic site. We identified several glycan cap-specific mAbs that neutralized multiple ebolaviruses, including SUDV, and a cross-reactive mAb that completely protected guinea pigs from the lethal challenge with heterologous EBOV. Our results provide a roadmap to develop a single antibody-based treatment effective against multiple Ebolavirus infections.

Integrated single-cell profiling dissects cell-state-specific enhancer landscapes of human tumor-infiltrating CD8+ T cells.

Despite extensive studies on the chromatin landscape of exhausted T cells, the transcriptional wiring underlying the heterogeneous functional and dysfunctional states of human tumor-infiltrating lymphocytes (TILs) is incompletely understood. Here, we identify gene-regulatory landscapes in a wide breadth of functional and dysfunctional CD8+ TIL states covering four cancer entities using single-cell chromatin profiling. We map enhancer-promoter interactions in human TILs by integrating single-cell chromatin accessibility with single-cell RNA-seq data from tumor-entity-matching samples and prioritize cell-state-specific genes by super-enhancer analysis. Besides revealing entity-specific chromatin remodeling in exhausted TILs, our analyses identify a common chromatin trajectory to TIL dysfunction and determine key enhancers, transcriptional regulators, and deregulated genes involved in this process. Finally, we validate enhancer regulation at immunotherapeutically relevant loci by targeting non-coding regulatory elements with potent CRISPR activators and repressors. In summary, our study provides a framework for understanding and manipulating cell-state-specific gene-regulatory cues from human tumor-infiltrating lymphocytes.

Single-cell chromatin accessibility landscape identifies tissue repair program in human regulatory T cells.

Murine regulatory T (Treg) cells in tissues promote tissue homeostasis and regeneration. We sought to identify features that characterize human Treg cells with these functions in healthy tissues. Single-cell chromatin accessibility profiles of murine and human tissue Treg cells defined a conserved, microbiota-independent tissue-repair Treg signature with a prevailing footprint of the transcription factor BATF. This signature, combined with gene expression profiling and TCR fate mapping, identified a population of tissue-like Treg cells in human peripheral blood that expressed BATF, chemokine receptor CCR8 and HLA-DR. Human BATF+CCR8+ Treg cells from normal skin and adipose tissue shared features with nonlymphoid T follicular helper-like (Tfh-like) cells, and induction of a Tfh-like differentiation program in naive human Treg cells partially recapitulated tissue Treg regenerative characteristics, including wound healing potential. Human BATF+CCR8+ Treg cells from healthy tissue share features with tumor-resident Treg cells, highlighting the importance of understanding the context-specific functions of these cells.

Mechanism of human antibody-mediated neutralization of Marburg virus.

The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition.

Mechanical activation opens a lipid-lined pore in OSCA ion channels.

OSCA/TMEM63 channels are the largest known family of mechanosensitive channels1-3, playing critical roles in plant4-7 and mammalian8,9 mechanotransduction. Here we determined 44 cryogenic electron microscopy structures of OSCA/TMEM63 channels in different environments to investigate the molecular basis of OSCA/TMEM63 channel mechanosensitivity. In nanodiscs, we mimicked increased membrane tension and observed a dilated pore with membrane access in one of the OSCA1.2 subunits. In liposomes, we captured the fully open structure of OSCA1.2 in the inside-in orientation, in which the pore shows a large lateral opening to the membrane. Unusually for ion channels, structural, functional and computational evidence supports the existence of a 'proteo-lipidic pore' in which lipids act as a wall of the ion permeation pathway. In the less tension-sensitive homologue OSCA3.1, we identified an 'interlocking' lipid tightly bound in the central cleft, keeping the channel closed. Mutation of the lipid-coordinating residues induced OSCA3.1 activation, revealing a conserved open conformation of OSCA channels. Our structures provide a global picture of the OSCA channel gating cycle, uncover the importance of bound lipids and show that each subunit can open independently. This expands both our understanding of channel-mediated mechanotransduction and channel pore formation, with important mechanistic implications for the TMEM16 and TMC protein families.

A lanthanide-rich kilonova in the aftermath of a long gamma-ray burst.

Observationally, kilonovae are astrophysical transients powered by the radioactive decay of nuclei heavier than iron, thought to be synthesized in the merger of two compact objects1-4. Over the first few days, the kilonova evolution is dominated by a large number of radioactive isotopes contributing to the heating rate2,5. On timescales of weeks to months, its behaviour is predicted to differ depending on the ejecta composition and the merger remnant6-8. Previous work has shown that the kilonova associated with gamma-ray burst 230307A is similar to kilonova AT2017gfo (ref. 9), and mid-infrared spectra revealed an emission line at 2.15 micrometres that was attributed to tellurium. Here we report a multi-wavelength analysis, including publicly available James Webb Space Telescope data9 and our own Hubble Space Telescope data, for the same gamma-ray burst. We model its evolution up to two months after the burst and show that, at these late times, the recession of the photospheric radius and the rapidly decaying bolometric luminosity (Lbol ∝ t-2.7±0.4, where t is time) support the recombination of lanthanide-rich ejecta as they cool.

Genome-wide DNA-methylation landscape defines specialization of regulatory T cells in tissues.

Regulatory T cells (Treg cells) perform two distinct functions: they maintain self-tolerance, and they support organ homeostasis by differentiating into specialized tissue Treg cells. We found that epigenetic modifications defined the molecular characteristics of tissue Treg cells. Tagmentation-based whole-genome bisulfite sequencing revealed more than 11,000 regions that were methylated differentially in pairwise comparisons of tissue Treg cell populations and lymphoid T cells. Similarities in the epigenetic landscape led to the identification of a common tissue Treg cell population that was present in many organs and was characterized by gain and loss of DNA methylation that included many gene sites associated with the TH2 subset of helper T cells, such as the gene encoding cytokine IL-33 receptor ST2, as well as the production of tissue-regenerative factors. Furthermore, the ST2-expressing population was dependent on the transcriptional regulator BATF and could be expanded by IL-33. Thus, tissue Treg cells integrate multiple waves of epigenetic reprogramming that define their tissue-restricted specialization.

Corrigendum: Genome-wide DNA-methylation landscape defines specialization of regulatory T cells in tissues.

This corrects the article DOI: 10.1038/ni.3799.

Precursors for Nonlymphoid-Tissue Treg Cells Reside in Secondary Lymphoid Organs and Are Programmed by the Transcription Factor BATF.

Specialized regulatory T (Treg) cells accumulate and perform homeostatic and regenerative functions in nonlymphoid tissues. Whether common precursors for nonlymphoid-tissue Treg cells exist and how they differentiate remain elusive. Using transcription factor nuclear factor, interleukin 3 regulated (Nfil3) reporter mice and single-cell RNA-sequencing (scRNA-seq), we identified two precursor stages of interleukin 33 (IL-33) receptor ST2-expressing nonlymphoid tissue Treg cells, which resided in the spleen and lymph nodes. Global chromatin profiling of nonlymphoid tissue Treg cells and the two precursor stages revealed a stepwise acquisition of chromatin accessibility and reprogramming toward the nonlymphoid-tissue Treg cell phenotype. Mechanistically, we identified and validated the transcription factor Batf as the driver of the molecular tissue program in the precursors. Understanding this tissue development program will help to harness regenerative properties of tissue Treg cells for therapy.

Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization.

Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus. We systematically analyzed the antibody repertoire in human survivors and identified a pair of potently neutralizing mAbs that cooperatively bound to the ebolavirus glycoprotein (GP). High-resolution structures revealed that in a two-antibody cocktail, molecular mimicry was a major feature of mAb-GP interactions. Broadly neutralizing mAb rEBOV-520 targeted a conserved epitope on the GP base region. mAb rEBOV-548 bound to a glycan cap epitope, possessed neutralizing and Fc-mediated effector function activities, and potentiated neutralization by rEBOV-520. Remodeling of the glycan cap structures by the cocktail enabled enhanced GP binding and virus neutralization. The cocktail demonstrated resistance to virus escape and protected non-human primates (NHPs) against Ebola virus disease. These data illuminate structural principles of antibody cooperativity with implications for development of antiviral immunotherapeutics.

Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal.

Decades of previous efforts to develop renal-sparing polyene antifungals were misguided by the classic membrane permeabilization model1. Recently, the clinically vital but also highly renal-toxic small-molecule natural product amphotericin B was instead found to kill fungi primarily by forming extramembraneous sponge-like aggregates that extract ergosterol from lipid bilayers2-6. Here we show that rapid and selective extraction of fungal ergosterol can yield potent and renal-sparing polyene antifungals. Cholesterol extraction was found to drive the toxicity of amphotericin B to human renal cells. Our examination of high-resolution structures of amphotericin B sponges in sterol-free and sterol-bound states guided us to a promising structural derivative that does not bind cholesterol and is thus renal sparing. This derivative was also less potent because it extracts ergosterol more slowly. Selective acceleration of ergosterol extraction with a second structural modification yielded a new polyene, AM-2-19, that is renal sparing in mice and primary human renal cells, potent against hundreds of pathogenic fungal strains, resistance evasive following serial passage in vitro and highly efficacious in animal models of invasive fungal infections. Thus, rational tuning of the dynamics of interactions between small molecules may lead to better treatments for fungal infections that still kill millions of people annually7,8 and potentially other resistance-evasive antimicrobials, including those that have recently been shown to operate through supramolecular structures that target specific lipids9.

Re-evaluating TRP channel mechanosensitivity.

Transient receptor potential (TRP) channels are implicated in a wide array of mechanotransduction processes. However, a question remains whether TRP channels directly sense mechanical force, thus acting as primary mechanotransducers. We use several recent examples to demonstrate the difficulty in definitively ascribing mechanosensitivity to TRP channel subfamilies. Ultimately, despite being implicated in an ever-growing list of mechanosignalling events in most cases limited robust or reproducible evidence supports the contention that TRP channels act as primary transducers of mechanical forces. They either (i) possess unique and as yet unspecified structural or local requirements for mechanosensitivity; or (ii) act as mechanoamplifiers responding downstream of the activation of a primary mechanotransducer that could include Ca2+-permeable mechanosensitive (MS) channels or other potentially unidentified mechanosensors.

Affinity for self antigen selects Treg cells with distinct functional properties.

The manner in which regulatory T cells (Treg cells) control lymphocyte homeostasis is not fully understood. We identified two Treg cell populations with differing degrees of self-reactivity and distinct regulatory functions. We found that GITR(hi)PD-1(hi)CD25(hi) (Triple(hi)) Treg cells were highly self-reactive and controlled lympho-proliferation in peripheral lymph nodes. GITR(lo)PD-1(lo)CD25(lo) (Triple(lo)) Treg cells were less self-reactive and limited the development of colitis by promoting the conversion of CD4(+) Tconv cells into induced Treg cells (iTreg cells). Although Foxp3-deficient (Scurfy) mice lacked Treg cells, they contained Triple(hi)-like and Triple(lo)-like CD4(+) T cells zsuper> T cells infiltrated the skin, whereas Scurfy Triple(lo)CD4(+) T cells induced colitis and wasting disease. These findings indicate that the affinity of the T cell antigen receptor for self antigen drives the differentiation of Treg cells into distinct subsets with non-overlapping regulatory activities.

Molecular structure of ß-amyloid fibrils in Alzheimer's disease brain tissue.

In vitro, ß-amyloid (Aß) peptides form polymorphic fibrils, with molecular structures that depend on growth conditions, plus various oligomeric and protofibrillar aggregates. Here, we investigate structures of human brain-derived Aß fibrils, using seeded fibril growth from brain extract and data from solid-state nuclear magnetic resonance and electron microscopy. Experiments on tissue from two Alzheimer's disease (AD) patients with distinct clinical histories showed a single predominant 40 residue Aß (Aß40) fibril structure in each patient; however, the structures were different from one another. A molecular structural model developed for Aß40 fibrils from one patient reveals features that distinguish in-vivo- from in-vitro-produced fibrils. The data suggest that fibrils in the brain may spread from a single nucleation site, that structural variations may correlate with variations in AD, and that structure-specific amyloid imaging agents may be an important future goal.

An unusual topological structure of the HIV-1 Rev response element.

Nuclear export of unspliced and singly spliced viral mRNA is a critical step in the HIV life cycle. The structural basis by which the virus selects its own mRNA among more abundant host cellular RNAs for export has been a mystery for more than 25 years. Here, we describe an unusual topological structure that the virus uses to recognize its own mRNA. The viral Rev response element (RRE) adopts an "A"-like structure in which the two legs constitute two tracks of binding sites for the viral Rev protein and position the two primary known Rev-binding sites ~55 Å apart, matching the distance between the two RNA-binding motifs in the Rev dimer. Both the legs of the "A" and the separation between them are required for optimal RRE function. This structure accounts for the specificity of Rev for the RRE and thus the specific recognition of the viral RNA.

A kilonova following a long-duration gamma-ray burst at 350 Mpc.

Gamma-ray bursts (GRBs) are divided into two populations1,2; long GRBs that derive from the core collapse of massive stars (for example, ref. 3) and short GRBs that form in the merger of two compact objects4,5. Although it is common to divide the two populations at a gamma-ray duration of 2 s, classification based on duration does not always map to the progenitor. Notably, GRBs with short (≲2 s) spikes of prompt gamma-ray emission followed by prolonged, spectrally softer extended emission (EE-SGRBs) have been suggested to arise from compact object mergers6-8. Compact object mergers are of great astrophysical importance as the only confirmed site of rapid neutron capture (r-process) nucleosynthesis, observed in the form of so-called kilonovae9-14. Here we report the discovery of a possible kilonova associated with the nearby (350 Mpc), minute-duration GRB 211211A. The kilonova implies that the progenitor is a compact object merger, suggesting that GRBs with long, complex light curves can be spawned from merger events. The kilonova of GRB 211211A has a similar luminosity, duration and colour to that which accompanied the gravitational wave (GW)-detected binary neutron star (BNS) merger GW170817 (ref. 4). Further searches for GW signals coincident with long GRBs are a promising route for future multi-messenger astronomy.

A very luminous jet from the disruption of a star by a massive black hole.

Tidal disruption events (TDEs) are bursts of electromagnetic energy that are released when supermassive black holes at the centres of galaxies violently disrupt a star that passes too close1. TDEs provide a window through which to study accretion onto supermassive black holes; in some rare cases, this accretion leads to launching of a relativistic jet2-9, but the necessary conditions are not fully understood. The best-studied jetted TDE so far is Swift J1644+57, which was discovered in γ-rays, but was too obscured by dust to be seen at optical wavelengths. Here we report the optical detection of AT2022cmc, a rapidly fading source at cosmological distance (redshift z = 1.19325) the unique light curve of which transitioned into a luminous plateau within days. Observations of a bright counterpart at other wavelengths, including X-ray, submillimetre and radio, supports the interpretation of AT2022cmc as a jetted TDE containing a synchrotron 'afterglow', probably launched by a supermassive black hole with spin greater than approximately 0.3. Using four years of Zwicky Transient Facility10 survey data, we calculate a rate of [Formula: see text] per gigapascals cubed per year for on-axis jetted TDEs on the basis of the luminous, fast-fading red component, thus providing a measurement complementary to the rates derived from X-ray and radio observations11. Correcting for the beaming angle effects, this rate confirms that approximately 1 per cent of TDEs have relativistic jets. Optical surveys can use AT2022cmc as a prototype to unveil a population of jetted TDEs.