The rockefeller university

Broad immune responses are needed to mitigate viral evolution and escape. To induce antibodies against conserved receptor-binding domain (RBD) regions of SARS-like betacoronavirus (sarbecovirus) spike proteins that recognize SARS-CoV-2 variants of concern and zoonotic sarbecoviruses, we developed mosaic-8b RBD nanoparticles presenting eight sarbecovirus RBDs arranged randomly on a 60-mer nanoparticle. Mosaic-8b immunizations protected animals from challenges from viruses whose RBDs were matched or mismatched to those on nanoparticles. Here, we describe neutralizing mAbs isolated from mosaic-8b-immunized rabbits, some on par with Pemgarda, the only currently FDA-approved therapeutic mAb. Deep mutational scanning, in vitro selection of spike resistance mutations, and single-particle cryo-electron microscopy structures of spike-antibody complexes demonstrated targeting of conserved RBD epitopes. Rabbit mAbs included critical D-gene segment RBD-recognizing features in common with human anti-RBD mAbs, despite rabbit genomes lacking an equivalent human D-gene segment, thus demonstrating that the immune systems of humans and other mammals can utilize different antibody gene segments to arrive at similar modes of antigen recognition. These results suggest that animal models can be used to elicit anti-RBD mAbs with similar properties to those raised in humans, which can then be humanized for therapeutic use, and that mosaic RBD nanoparticle immunization coupled with multiplexed screening represents an efficient way to generate and select broadly cross-reactive therapeutic pan-sarbecovirus and pan-SARS-CoV-2 variant mAbs.

The most dynamic and repetitive regions of great ape genomes have traditionally been excluded from comparative studies(1-3). Consequently, our understanding of the evolution of our species is incomplete. Here we present haplotype-resolved reference genomes and comparative analyses of six ape species: chimpanzee, bonobo, gorilla, Bornean orangutan, Sumatran orangutan and siamang. We achieve chromosome-level contiguity with substantial sequence accuracy (<1 error in 2.7 megabases) and completely sequence 215 gapless chromosomes telomere-to-telomere. We resolve challenging regions, such as the major histocompatibility complex and immunoglobulin loci, to provide in-depth evolutionary insights. Comparative analyses enabled investigations of the evolution and diversity of regions previously uncharacterized or incompletely studied without bias from mapping to the human reference genome. Such regions include newly minted gene families in lineage-specific segmental duplications, centromeric DNA, acrocentric chromosomes and subterminal heterochromatin. This resource serves as a comprehensive baseline for future evolutionary studies of humans and our closest living ape relatives.

Realistic paths to gene therapy for the X-linked bleeding disorder hemophilia started to materialize in the mid 1990s, resulting in disease correction in small and large animal models. Out of a diversity of approaches, in vivo adeno-associated viral (AAV) gene transfer to hepatocytes emerged as the most promising strategy, eventually forming the basis for multiple advanced clinical trials and regulatory approval of two products for the treatment of hemophilia B (coagulation factor IX deficiency) and one for hemophilia A (factor VIII deficiency). Ideally, gene therapy is effective with a single administration, thus providing therapeutic factor levels over a period of years, without the need for frequent injections. Overcoming multiple obstacles, some not predicted by preclinical studies, sustained partial to complete correction of coagulation for several years to an entire decade has now been documented in patients, with observation ongoing. A hyperactive form of FIX improved efficacy in hemophilia B, and superior engineered variants of FVIII are emerging. Nonetheless, challenges remain, including pre-existing immunity to AAV capsids, toxicities, inter-patient variability in response to treatment, and difficulty in obtaining durable therapeutic expression of FVIII. In alternative approaches, in vivo gene editing and ex vivo gene therapies targeting hemopoietic cells are in development.

Spermatogenesis is a key developmental process underlying the origination of newly evolved genes. However, rapid cell type-specific transcriptomic divergence of the Drosophila germline has posed a significant technical barrier for comparative single- cell RNA- sequencing studies. By quantifying a surprisingly strong correlation between species- and cell type-specific divergence in three closely related Drosophila species, we apply a statistical procedure to identify a core set of 198 genes that are highly predictive of cell type identity while remaining robust to species- specific differences that span over 25 to 30 My of evolution. We then utilize cell type classifications based on the 198- gene set to show how transcriptional divergence in cell type increases throughout spermatogenic developmental time. After validating these cross- species cell type classifications using RNA fluorescence in situ hybridization and imaging, we then investigate the influence of genome organization on the molecular evolution of spermatogenesis vis- a- vis transcriptional bursting. We first show altering transcriptional burst size contributes to premeiotic transcription and altering bursting frequency contributes to postmeiotic expression. We then report global differences in autosomal vs. X chromosomal transcription may arise in a developmental stage preceding full testis organogenesis by showing evolutionarily conserved decreases in X- linked transcription bursting kinetics in all examined somatic and germline cell types. Finally, we provide evidence supporting the cultivator model of de novo gene origination by demonstrating how the appearance of newly evolved testis- specific transcripts potentially provides short- range regulation of neighboring genes' transcriptional bursting properties during key stages of spermatogenesis.

The primary motor cortex (M1) is a central hub for motor learning and execution. M1 is composed of heterogeneous cell types with varying relationships to movement. Here, we tagged active neurons at different stages of motor task performance in mice and characterized cell type composition. We identified corticothalamic neurons (M1CT) as consistently enriched with training progression. Using two-photon calcium imaging, we found that M1CT activity is largely suppressed during movement, and this negative correlation augments with training. Increasing M1CT activity through closed-loop optogenetic manipulations during forelimb movement significantly hinders execution, an effect that became stronger with training. Similar manipulations, however, had little effect on locomotion. In contrast, M1 corticospinal neurons positively correlate with movement, with an increase during training. We uncovered that M1CT neurons suppress corticospinal activity via feedforward inhibition, also scaling with training. These results identify a permissive role of corticothalamic neurons in movement execution through disinhibition of corticospinal neurons.

Multisubunit "DNA-dependent" RNA polymerases (RNAPs) have noncanonical RNA-directed RNA synthesis activity; this allows the synthesis of complementary RNA from RNA templates. Such noncanonical RNAP activities are biologically significant, serving RNA pathogens such as hepatitis delta virus (HDV) and contributing to cellular gene regulation. Despite the broad biological implications of these processes, our understanding of the underlying RNAP mechanisms remains incomplete. Using Escherichia coli RNAP, a multisubunit RNAP, as a model, we describe here the general RNA-templated RNA extension activity of that enzyme. Our data argue that the 3 ' end of an added RNA template can fold back and pair with upstream bases in the template, creating an intramolecular primer:template duplex as short as 1-2 base pairs. The RNAP then extends this intramolecular duplex, incorporating nucleotides complementary to the template. RNA-templated RNA extension occurred in minutes and did not appear to be suppressed by the presence of a promoter-containing DNA template. Excepting oligonucleotides implicitly designed to prevent any possibility of 3 ' end self-priming, every RNA template we tested could be extended by the enzyme, highlighting the general nature of this reaction. These data define a general activity of a cellular RNAP. Unrestricted, this activity could contribute to the emergence and replication of RNA-based agents such as HDV and viroids; if highly regulated, the activity could limit these same elements.

The impact of SARS-CoV-2 in the lung has been extensively studied, yet the molecular regulators of host-cell programs hijacked by the virus in distinct human airway epithelial cell populations remain poorly understood. Some of the reasons include overreliance on transcriptomic profiling and use of nonprimary cell systems. Here we report a network-based analysis of single-cell transcriptomic profiles able to identify master regulator (MR) proteins controlling SARS-CoV-2-mediated reprogramming in pathophysiologically relevant human ciliated, secretory, and basal cells. This underscored chromatin remodeling, endosomal sorting, ubiquitin pathways, as well as proviral factors identified by CRISPR assays as components of the viral-host response in these cells. Large-scale drug perturbation screens revealed 11 candidate drugs able to invert the entire MR signature activated by SARS-CoV-2. Leveraging MR analysis and perturbational profiles of human primary cells represents an innovative approach to investigate pathogen-host interactions in multiple airway conditions for drug prioritization.

CD4+ T cells are indispensable for optimal immunity to Mycobacterium tuberculosis (M.tb), a pathogen that triggers tuberculosis (TB) in humans. M.tb-specific human CD4+ T cells are known to polarize toward an interferon-gamma (IFN-gamma)-producing, CCR4-CCR6+CXCR3+T-bet+ROR gamma T+ T helper 1* cell (TH1*cell) memory phenotype. We report that autosomal recessive deficiency of the human lymphocytic surface receptor LY9 (SLAMF3 and CD229), which is found in less than 10-5 individuals in the general population, underlies TB in three unrelated patients due to selective impairment in IFN-gamma production by TH1* cells. TH1* cells express higher levels of LY9 than other CD4+ T cells. Mechanistically, LY9 polarizes na & iuml;ve CD4+ T cells toward memory TH1* cells by inducing T-bet via signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) and ROR gamma T (thymus-specific retinoid-related orphan receptor gamma) without SAP. LY9 costimulation enhances TCR-driven IFN-gamma production of memory TH1*, but not TH1, cells in a T cell-intrinsic manner via NFAT1 (nuclear factor of activated T cells 1) and ROR gamma T. LY9 is likely to govern an optimal TH1* cell- and IFN-gamma-dependent protective immunity to M.tb in humans.

We talk to authors Sukrit Silas and Heloise Carion about their paper "Activation of bacterial programmed cell death by phage inhibitors of host immunity" (in this issue of Molecular Cell) and the mutagenesis screen that it arose from.

Leukopoiesis is lethally arrested in mice lacking the master transcriptional regulator PU.1. Depending on the animal model, subtotal PU.1 loss either induces acute myeloid leukemia or arrests early B-cell and dendritic-cell development. Although humans with absolute PU.1 deficiency have not been reported, a small cadre of congenital agammaglobulinemia patients with sporadic, inborn PU.1 haploinsufficiency was recently described. To better estimate the penetrance, clinical complications, immunophenotypic features, and malignancy risks of PU.1-mutated agammaglobulinemia (PU.MA), a collection of 134 novel or rare PU.1 variants from publicly available databases, institutional cohorts, previously published reports, and unsolved agammaglobulinemia cases were functionally analyzed. In total, 25 loss-of-function (LOF) variants were identified in 33 heterozygous carriers from 21 kindreds across 13 nations. Of individuals harboring LOF PU.1 variants, 22 were agammaglobulinemic, 5 displayed antibody deficiencies, and 6 were unaffected, indicating an estimated disease penetrance of 81.8% with variable expressivity. In a cluster of patients, disease onset was delayed, sometimes into adulthood. All LOF variants conveyed effects via haploinsufficiency, either by destabilizing PU.1, impeding nuclear localization, or directly interfering with transcription. PU.MA patient immunophenotypes consistently demon strated B-cell, conventional dendritic-cell, and plasmacytoid dendritic-cell deficiencies. Associated infectious and noninfectious symptoms hewed closely to X-linked agammaglobulinemia and not monogenic dendritic-cell deficiencies. No carriers of LOF PU.1 variants experienced hematologic malignancies. Collectively, in vitro and clinical data indicate heterozygous LOF PU.1 variants undermine humoral immunity but do not convey strong leukemic risks.