The need to diversify genomic studies: Insights from Andean highlanders and Amazonians.

More globally diverse perspectives are needed in genomic studies and precision medicine practices on non-Europeans. Here, we illustrate this by discussing the distribution of clinically actionable genetic variants involved in drug response in Andean highlanders and Amazonians, considering their environment, history, genetic structure, and historical biases in the perception of biological diversity of Native Americans.

A budding new approach strengthens an important message.

In vaccinology, both mRNA-based delivery of genes encoding antigens as well as nanoparticle-based vaccines have shown great promise in tackling challenging pathogens. In this issue of Cell, Hoffmann et al. combine these two approaches, harnessing the same cellular pathway hijacked by many viruses to boost immune responses to SARS-CoV-2 vaccination.

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.

Neutralizing and protective human monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein.

Most human monoclonal antibodies (mAbs) neutralizing SARS-CoV-2 recognize the spike (S) protein receptor-binding domain and block virus interactions with the cellular receptor angiotensin-converting enzyme 2. We describe a panel of human mAbs binding to diverse epitopes on the N-terminal domain (NTD) of S protein from SARS-CoV-2 convalescent donors and found a minority of these possessed neutralizing activity. Two mAbs (COV2-2676 and COV2-2489) inhibited infection of authentic SARS-CoV-2 and recombinant VSV/SARS-CoV-2 viruses. We mapped their binding epitopes by alanine-scanning mutagenesis and selection of functional SARS-CoV-2 S neutralization escape variants. Mechanistic studies showed that these antibodies neutralize in part by inhibiting a post-attachment step in the infection cycle. COV2-2676 and COV2-2489 offered protection either as prophylaxis or therapy, and Fc effector functions were required for optimal protection. Thus, natural infection induces a subset of potent NTD-specific mAbs that leverage neutralizing and Fc-mediated activities to protect against SARS-CoV-2 infection using multiple functional attributes.

Therapeutic alphavirus cross-reactive E1 human antibodies inhibit viral egress.

Alphaviruses cause severe arthritogenic or encephalitic disease. The E1 structural glycoprotein is highly conserved in these viruses and mediates viral fusion with host cells. However, the role of antibody responses to the E1 protein in immunity is poorly understood. We isolated E1-specific human monoclonal antibodies (mAbs) with diverse patterns of recognition for alphaviruses (ranging from Eastern equine encephalitis virus [EEEV]-specific to alphavirus cross-reactive) from survivors of natural EEEV infection. Antibody binding patterns and epitope mapping experiments identified differences in E1 reactivity based on exposure of epitopes on the glycoprotein through pH-dependent mechanisms or presentation on the cell surface prior to virus egress. Therapeutic efficacy in vivo of these mAbs corresponded with potency of virus egress inhibition in vitro and did not require Fc-mediated effector functions for treatment against subcutaneous EEEV challenge. These studies reveal the molecular basis for broad and protective antibody responses to alphavirus E1 proteins.

Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions for optimal therapeutic protection.

SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes and CD8+ T cells for optimal clinical and virological benefit. Thus, potently neutralizing mAbs utilize Fc effector functions during therapy to mitigate lung infection and disease.

Imaging of DNA and RNA in Living Eukaryotic Cells to Reveal Spatiotemporal Dynamics of Gene Expression.

This review focuses on imaging DNA and single RNA molecules in living cells to define eukaryotic functional organization and dynamic processes. The latest advances in technologies to visualize individual DNA loci and RNAs in real time are discussed. Single-molecule fluorescence microscopy provides the spatial and temporal resolution to reveal mechanisms regulating fundamental cell functions. Novel insights into the regulation of nuclear architecture, transcription, posttranscriptional RNA processing, and RNA localization provided by multicolor fluorescence microscopy are reviewed. A perspective on the future use of live imaging technologies and overcoming their current limitations is provided.

Human Antibodies Protect against Aerosolized Eastern Equine Encephalitis Virus Infection.

Eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has recently shown an increase in human cases. Here, we characterize human monoclonal antibodies (mAbs) isolated from a survivor of natural EEEV infection with potent (<20 pM) inhibitory activity of EEEV. Cryo-electron microscopy reconstructions of two highly neutralizing mAbs, EEEV-33 and EEEV-143, were solved in complex with chimeric Sindbis/EEEV virions to 7.2 Å and 8.3 Å, respectively. The mAbs recognize two distinct antigenic sites that are critical for inhibiting viral entry into cells. EEEV-33 and EEEV-143 protect against disease following stringent lethal aerosol challenge of mice with highly pathogenic EEEV. These studies provide insight into the molecular basis for the neutralizing human antibody response against EEEV and can facilitate development of vaccines and candidate antibody therapeutics.

Mortality Risk Profiling of Staphylococcus aureus Bacteremia by Multi-omic Serum Analysis Reveals Early Predictive and Pathogenic Signatures.

Staphylococcus aureus bacteremia (SaB) causes significant disease in humans, carrying mortality rates of ∼25%. The ability to rapidly predict SaB patient responses and guide personalized treatment regimens could reduce mortality. Here, we present a resource of SaB prognostic biomarkers. Integrating proteomic and metabolomic techniques enabled the identification of >10,000 features from >200 serum samples collected upon clinical presentation. We interrogated the complexity of serum using multiple computational strategies, which provided a comprehensive view of the early host response to infection. Our biomarkers exceed the predictive capabilities of those previously reported, particularly when used in combination. Last, we validated the biological contribution of mortality-associated pathways using a murine model of SaB. Our findings represent a starting point for the development of a prognostic test for identifying high-risk patients at a time early enough to trigger intensive monitoring and interventions.

Intracellular mRNA transport and localized translation.

Fine-tuning cellular physiology in response to intracellular and environmental cues requires precise temporal and spatial control of gene expression. High-resolution imaging technologies to detect mRNAs and their translation state have revealed that all living organisms localize mRNAs in subcellular compartments and create translation hotspots, enabling cells to tune gene expression locally. Therefore, mRNA localization is a conserved and integral part of gene expression regulation from prokaryotic to eukaryotic cells. In this Review, we discuss the mechanisms of mRNA transport and local mRNA translation across the kingdoms of life and at organellar, subcellular and multicellular resolution. We also discuss the properties of messenger ribonucleoprotein and higher order RNA granules and how they may influence mRNA transport and local protein synthesis. Finally, we summarize the technological developments that allow us to study mRNA localization and local translation through the simultaneous detection of mRNAs and proteins in single cells, mRNA and nascent protein single-molecule imaging, and bulk RNA and protein detection methods.

Extrafollicular B cell responses correlate with neutralizing antibodies and morbidity in COVID-19.

A wide spectrum of clinical manifestations has become a hallmark of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) COVID-19 pandemic, although the immunological underpinnings of diverse disease outcomes remain to be defined. We performed detailed characterization of B cell responses through high-dimensional flow cytometry to reveal substantial heterogeneity in both effector and immature populations. More notably, critically ill patients displayed hallmarks of extrafollicular B cell activation and shared B cell repertoire features previously described in autoimmune settings. Extrafollicular activation correlated strongly with large antibody-secreting cell expansion and early production of high concentrations of SARS-CoV-2-specific neutralizing antibodies. Yet, these patients had severe disease with elevated inflammatory biomarkers, multiorgan failure and death. Overall, these findings strongly suggest a pathogenic role for immune activation in subsets of patients with COVID-19. Our study provides further evidence that targeted immunomodulatory therapy may be beneficial in specific patient subpopulations and can be informed by careful immune profiling.

Oncolytic Virotherapy Promotes Intratumoral T Cell Infiltration and Improves Anti-PD-1 Immunotherapy.

Here we report a phase 1b clinical trial testing the impact of oncolytic virotherapy with talimogene laherparepvec on cytotoxic T cell infiltration and therapeutic efficacy of the anti-PD-1 antibody pembrolizumab. Twenty-one patients with advanced melanoma were treated with talimogene laherparepvec followed by combination therapy with pembrolizumab. Therapy was generally well tolerated, with fatigue, fevers, and chills as the most common adverse events. No dose-limiting toxicities occurred. Confirmed objective response rate was 62%, with a complete response rate of 33% per immune-related response criteria. Patients who responded to combination therapy had increased CD8+ T cells, elevated PD-L1 protein expression, as well as IFN-γ gene expression on several cell subsets in tumors after talimogene laherparepvec treatment. Response to combination therapy did not appear to be associated with baseline CD8+ T cell infiltration or baseline IFN-γ signature. These findings suggest that oncolytic virotherapy may improve the efficacy of anti-PD-1 therapy by changing the tumor microenvironment. VIDEO ABSTRACT.

Tumor Interferon Signaling Regulates a Multigenic Resistance Program to Immune Checkpoint Blockade.

Therapeutic blocking of the PD1 pathway results in significant tumor responses, but resistance is common. We demonstrate that prolonged interferon signaling orchestrates PDL1-dependent and PDL1-independent resistance to immune checkpoint blockade (ICB) and to combinations such as radiation plus anti-CTLA4. Persistent type II interferon signaling allows tumors to acquire STAT1-related epigenomic changes and augments expression of interferon-stimulated genes and ligands for multiple T cell inhibitory receptors. Both type I and II interferons maintain this resistance program. Crippling the program genetically or pharmacologically interferes with multiple inhibitory pathways and expands distinct T cell populations with improved function despite expressing markers of severe exhaustion. Consequently, tumors resistant to multi-agent ICB are rendered responsive to ICB monotherapy. Finally, we observe that biomarkers for interferon-driven resistance associate with clinical progression after anti-PD1 therapy. Thus, the duration of tumor interferon signaling augments adaptive resistance and inhibition of the interferon response bypasses requirements for combinatorial ICB therapies.

Unique pattern of neutrophil migration and function during tumor progression.

Although neutrophils have been linked to the formation of the pre-metastatic niche, the mechanism of their migration to distant, uninvolved tissues has remained elusive. We report that bone marrow neutrophils from mice with early-stage cancer exhibited much more spontaneous migration than that of control neutrophils from tumor-free mice. These cells lacked immunosuppressive activity but had elevated rates of oxidative phosphorylation and glycolysis, and increased production of ATP, relative to that of control neutrophils. Their enhanced spontaneous migration was mediated by autocrine ATP signaling through purinergic receptors. In ectopic tumor models and late stages of cancer, bone marrow neutrophils demonstrated potent immunosuppressive activity. However, these cells had metabolic and migratory activity indistinguishable from that of control neutrophils. A similar pattern of migration was observed for neutrophils and polymorphonuclear myeloid-derived suppressor cells from patients with cancer. These results elucidate the dynamic changes that neutrophils undergo in cancer and demonstrate the mechanism of neutrophils' contribution to early tumor dissemination.

Real-time single-molecule imaging of transcriptional regulatory networks in living cells.

Gene regulatory networks drive the specific transcriptional programmes responsible for the diversification of cell types during the development of multicellular organisms. Although our knowledge of the genes involved in these dynamic networks has expanded rapidly, our understanding of how transcription is spatiotemporally regulated at the molecular level over a wide range of timescales in the small volume of the nucleus remains limited. Over the past few decades, advances in the field of single-molecule fluorescence imaging have enabled real-time behaviours of individual transcriptional components to be measured in living cells and organisms. These efforts are now shedding light on the dynamic mechanisms of transcription, revealing not only the temporal rules but also the spatial coordination of underlying molecular interactions during various biological events.

Quantifying Protein-mRNA Interactions in Single Live Cells.

Specific binding proteins are crucial for the correct spatiotemporal expression of mRNA. To understand this process, a method is required to characterize RNA-protein interactions in single living cells with subcellular resolution. We combined endogenous single RNA and protein detection with two-photon fluorescence fluctuation analysis to measure the average number of proteins bound to mRNA at specific locations within live cells. We applied this to quantify the known binding of zipcode binding protein 1 (ZBP1) and ribosomes to ß-actin mRNA within subcellular compartments of primary fibroblasts and neurons. ZBP1-mRNA binding did not occur in nuclei, contrary to previous conclusions. ZBP1 interaction with ß-actin mRNA was enhanced perinuclearly in neurons compared to fibroblasts. Cytoplasmic ZBP1 and ribosome binding to the mRNA were anti-correlated depending on their location in the cell. These measurements support a mechanism whereby ZBP1 inhibits translation of localizing mRNA until its release from the mRNA peripherally, allowing ribosome binding.

Computationally restoring the potency of a clinical antibody against Omicron.

The COVID-19 pandemic underscored the promise of monoclonal antibody-based prophylactic and therapeutic drugs1-3 and revealed how quickly viral escape can curtail effective options4,5. When the SARS-CoV-2 Omicron variant emerged in 2021, many antibody drug products lost potency, including Evusheld and its constituent, cilgavimab4-6. Cilgavimab, like its progenitor COV2-2130, is a class 3 antibody that is compatible with other antibodies in combination4 and is challenging to replace with existing approaches. Rapidly modifying such high-value antibodies to restore efficacy against emerging variants is a compelling mitigation strategy. We sought to redesign and renew the efficacy of COV2-2130 against Omicron BA.1 and BA.1.1 strains while maintaining efficacy against the dominant Delta variant. Here we show that our computationally redesigned antibody, 2130-1-0114-112, achieves this objective, simultaneously increases neutralization potency against Delta and subsequent variants of concern, and provides protection in vivo against the strains tested: WA1/2020, BA.1.1 and BA.5. Deep mutational scanning of tens of thousands of pseudovirus variants reveals that 2130-1-0114-112 improves broad potency without increasing escape liabilities. Our results suggest that computational approaches can optimize an antibody to target multiple escape variants, while simultaneously enriching potency. Our computational approach does not require experimental iterations or pre-existing binding data, thus enabling rapid response strategies to address escape variants or lessen escape vulnerabilities.

Targeted RNA editing: novel tools to study post-transcriptional regulation.

RNA binding proteins (RBPs) regulate nearly all post-transcriptional processes within cells. To fully understand RBP function, it is essential to identify their in vivo targets. Standard techniques for profiling RBP targets, such as crosslinking immunoprecipitation (CLIP) and its variants, are limited or suboptimal in some situations, e.g. when compatible antibodies are not available and when dealing with small cell populations such as neuronal subtypes and primary stem cells. This review summarizes and compares several genetic approaches recently designed to identify RBP targets in such circumstances. TRIBE (targets of RNA binding proteins identified by editing), RNA tagging, and STAMP (surveying targets by APOBEC-mediated profiling) are new genetic tools useful for the study of post-transcriptional regulation and RBP identification. We describe the underlying RNA base editing technology, recent applications, and therapeutic implications.

Molecular Mechanisms for Iron Uptake and Homeostasis in Marine Eukaryotic Phytoplankton.

The micronutrient iron is essential for phytoplankton growth due to its central role in a wide variety of key metabolic processes including photosynthesis and nitrate assimilation. As a result of scarce bioavailable iron in seawater, marine primary productivity is often iron-limited with future iron supplies remaining uncertain. Although evolutionary constraints resulted in high cellular iron requirements, phytoplankton evolved diverse mechanisms that enable uptake of multiple forms of iron, storage of iron over short and long timescales, and modulation of their iron requirement under stress. Genomics continues to increase our understanding of iron-related proteins that are homologous to those characterized in other model organisms, while recently, molecular and cell biology is revealing unique genes and processes with connections to iron acquisition or use. Moreover, there are an increasing number of examples showing the interplay between iron uptake and extracellular processes such as boundary layer chemistry and microbial interactions.

Epigenetic priming of memory updating during reconsolidation to attenuate remote fear memories.

Traumatic events generate some of the most enduring forms of memories. Despite the elevated lifetime prevalence of anxiety disorders, effective strategies to attenuate long-term traumatic memories are scarce. The most efficacious treatments to diminish recent (i.e., day-old) traumata capitalize on memory updating mechanisms during reconsolidation that are initiated upon memory recall. Here, we show that, in mice, successful reconsolidation-updating paradigms for recent memories fail to attenuate remote (i.e., month-old) ones. We find that, whereas recent memory recall induces a limited period of hippocampal neuroplasticity mediated, in part, by S-nitrosylation of HDAC2 and histone acetylation, such plasticity is absent for remote memories. However, by using an HDAC2-targeting inhibitor (HDACi) during reconsolidation, even remote memories can be persistently attenuated. This intervention epigenetically primes the expression of neuroplasticity-related genes, which is accompanied by higher metabolic, synaptic, and structural plasticity. Thus, applying HDACis during memory reconsolidation might constitute a treatment option for remote traumata.