Biology subjects

Ellis, J.

Publications and source records attributed to Ellis, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Disrupted Brain Organoid Circuitry, Structural Organization, and Spine Morphology in 7q11.23 Copy Number Variant Syndromes

The 7q11.23 chromosomal region represents a model of gene dosage-dependence, where a hemizygous deletion causes Williams Syndrome (WS) and a duplication leads to 7q11.23 Duplication Syndrome (Dup7). It is not understood how these copy number variations (CNVs) disrupt development and functional cortical circuit assembly. Utilizing iPSC-derived cerebral organoids and longitudinal imaging from post-differentiation day 30 to 150, we characterized the aberrant neural rosette morphogenesis in WS and Dup7 during early stages, establishing an early structural divergence from control lines. We observed accelerated early cortical rosette morphogenesis in WS, characterized by a premature increase in both rosette number and layer thickness compared to controls. In contrast, Dup7 organoids consistently exhibit a significantly lower number and reduced thickness of rosettes from early stages onward. This early structural disruption progressively impacted synaptic-level architecture as the organoids matured. Dendritic spine characterization at later stages revealed Dup7 organoids exhibited a significantly higher dendritic spine density compared to WS. Pharmacological antagonism of CCR5 (C-C chemokine receptor type 5) with Maraviroc significantly enhanced dendritic spine density in control and WS organoids; however, this effect was absent in Dup7. To determine how these structural anomalies translate into circuit-level behavior, we performed longitudinal calcium imaging using GCaMP. Control organoids sustained synchronized activity and high spike correlations at all time points. This synchronization was delayed and highly transient in WS organoids, and completely abolished in Dup7 organoids, which exhibit significantly low spike correlations at all stages. Developmentally, GABA changes from acting as an excitatory signal in the immature brain to an inhibitory signal as the brain matures. As control lines matured gabazine-induced desynchronization progressively diminished, but persisted in WS organoids. Dup7 organoids failed to establish synchronization at any developmental time point, but displayed a negligible increased synchrony following gabazine treatment. These functional aberrations were paralleled by genotype-specific defects in structural organization, specifically in rosette morphogenesis and dendritic spine density. Collectively, our findings demonstrate that 7q11.23 CNVs trigger pathogenic neurodevelopmental defects by derailing the trajectories of structural organization, circuit assembly, and functional synchronization during cortical maturation.

neuroscience↗

bioRxiv2025

A Wildlife Health Outbreak Response Table-top Exercise for Pandemic Preparedness Planning

Zoonotic diseases have received significantly more attention over the last few decades, emerging with increasing frequency and causing the majority of notable disease outbreaks in this century, including the COVID-19 pandemic. As human activities and shifting climate patterns induce changes in the environment that alter habitat and range of reservoir species, the potential for human and animal interactions will increase and enhance the opportunity for spillover. Thus, any emergency response preparedness planning must take into account the function and coordination of agencies across the sectors of human, animal and environmental health. Within the Commonwealth of Pennsylvania a table-top exercise was performed to evaluate a multi-agency response during a hypothetical zoonotic disease investigation. The exercise was evaluated by the participants to gain feedback on the overall process and lessons learned. Here, we describe the tabletop exercise scenario and the insights gained. We found that differences in operational structure create challenges for interdepartmental communication and in the ability to resource a coordinated response, highlighting opportunities to develop infrastructure that will facilitate future actions. A set of recommendations are outlined that may enhance cross-agency activities and promote more effective and efficient emergency response.

scientific communication and education↗

bioRxiv2024

Disease-linked mutations dysregulate neuronal condensate physical properties, composition, and RNA translation

Local RNA translation is essential for development. In neurons, deficient local translation linked with mutations in scaffold proteins results in dysregulated dendrite and dendritic spine growth. However, mechanisms by which these proteins control translation and how disease-linked mutations induce aberrant translation were unclear. We use biochemical reconstitution and neuronal assays to show that mutations to the neuronal condensate scaffold shank2 cause physical hardening and altered composition of condensates; a key RNA translation-modulator FMRP is excluded from mutant condensates. Functionally, shank2 condensates repress translation while condensates composed of shank2 with intrinsically disordered region-localized missense mutations promote translation. These results demonstrate that disease-linked dysregulation of condensate physical properties and composition is an underlying mechanism of aberrant RNA translation often observed in disease. One Sentence SummaryDisease-linked missense mutations in the postsynaptic density scaffold protein shank2 dysregulate phase separated biomolecular condensate physical properties and composition resulting in aberrant RNA translation.

biophysics↗

bioRxiv2024

Expression-Driven Genetic Dependency Reveals Targets for Precision Medicine

Cancer cells are heterogeneous, each harboring distinct molecular aberrations and are dependent on different genes for their survival and proliferation. While successful targeted therapies have been developed based on driver DNA mutations, many patient tumors lack druggable mutations and have limited treatment options. Here, we hypothesize that new precision oncology targets may be identified through "expression-driven dependency", whereby cancer cells with high expression of a targeted gene are more vulnerable to the knockout of that gene. We introduce a Bayesian approach, BEACON, to identify such targets by jointly analyzing global transcriptomic and proteomic profiles with genetic dependency data of cancer cell lines across 17 tissue lineages. BEACON identifies known druggable genes, e.g., BCL2, ERBB2, EGFR, ESR1, MYC, while revealing new targets confirmed by both mRNA- and protein-expression driven dependency. Notably, the identified genes show an overall 3.8-fold enrichment for approved drug targets and enrich for druggable oncology targets by 7 to 10-fold. We experimentally validate that the depletion of GRHL2, TP63, and PAX5 effectively reduce tumor cell growth and survival in their dependent cells. Overall, we present the catalog of express-driven dependency targets as a resource for identifying novel therapeutic targets in precision oncology.

genomics↗

bioRxiv2024

Hypersynchronous iPSC-derived SHANK2 neuronal networks are rescued by mGluR5 agonism

Variants in the gene encoding the postsynaptic scaffolding protein SHANK2 are associated with several neurodevelopmental disorders, including autism spectrum disorder. Here, we used in vitro multielectrode arrays and pharmacological manipulations to characterize how functional connectivity and network-level firing properties were altered in cultures of human iPSC-derived SHANK2 neurons. Using two isogenic pairs of SHANK2 cell lines, we showed that the SHANK2 hyperconnectivity phenotype was recapitulated at the network level. SHANK2 networks displayed significantly increased frequency and reduced duration of network burst events relative to controls. SHANK2 network activity was hypersynchronous, with increased functional correlation strength between recording channels. Analysis of intra-network burst firing dynamics revealed that spikes within SHANK2 network bursts were organized into high-frequency trains, producing a distinctive network burst shape. Calcium-dependent events called reverberating super bursts (RSBs) were observed in control networks but rarely occurred in SHANK2 networks. SHANK2 network hypersynchrony and numbers of strong correlations were fully rescued by the group 1 mGluR agonist DHPG, that also restored detection of RSBs and significantly improved network burst frequency and duration metrics. Our results demonstrate that SHANK2 variants produce a functional hyperconnectivity phenotype that deviates from the developmental trajectory of isogenic control networks. Furthermore, the hypersynchronous phenotype was rescued by pharmacologically regulating glutamatergic neurotransmission.

neuroscience↗

bioRxiv2024

Mutation of two intronic nucleotide alters RNA structure and dynamics, inhibiting MBNL1 and RBFOX1 regulated splicing of the Insulin Receptor

Alternative splicing (AS) of Exon 11 of the Insulin Receptor (INSR) is highly regulated and disrupted in several human disorders. To better understand INSR exon 11 AS regulation, splicing activity of an INSR exon 11 minigene reporter was measured across a gradient of the AS regulator muscleblind-like 1 protein (MBNL1). The RNA-binding protein Fox-1 (RBFOX1) was added to determine its impact on MBNL1-regulated splicing. The role of the RBFOX1 UGCAUG binding site within intron 11 was assessed across the MBNL1 gradient. Mutating the UGCAUG motif inhibited RBFOX1 regulation of exon 11 and had the unexpected effect of reducing MBNL1 regulation of this exon. Molecular dynamics simulations showed that exon 11 and the adjacent RNA adopts a dynamically stable conformation. Mutation of the RBFOX1 binding site altered RNA structure and dynamics, while a mutation that created an optimal MBNL1 binding site at the RBFOX1 site shifted the RNA back to wild type. An antisense oligonucleotide (ASO) was used to confirm the structure in this region of the pre-mRNA. This example of intronic mutations shifting pre-mRNA structure and dynamics to modulate splicing suggests RNA structure and dynamics should be taken into consideration for AS regulation and therapeutic interventions targeting pre-mRNA. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/574689v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@17e410org.highwire.dtl.DTLVardef@7deb00org.highwire.dtl.DTLVardef@cf69f9org.highwire.dtl.DTLVardef@13684e8_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract figure:C_FLOATNO Model for INSR exon 11 splicing regulation through the UGCAUG motif. We propose that the UGCAUG motif, MBNL1, and RBFOX1 cooperatively regulate INSR exon 11 splicing. Mutating this UGCAUG motif is sufficient to alter RNA structural dynamics to disrupt this regulation. C_FIG

biochemistry↗

bioRxiv2023

Cutaneous T cell lymphoma atlas reveals malignant Th2 cells supported by a B cell-rich microenvironment

Cutaneous T-cell lymphoma (CTCL) is a potentially fatal clonal malignancy of T cells primarily affecting the skin. The most common form of CTCL, mycosis fungoides (MF), can be difficult to diagnose resulting in treatment delay. The pathogenesis of CTCL is not fully understood due to limited data from patient studies. We performed single-cell RNA sequencing and spatial transcriptomics profiling of skin from patients with MF-type CTCL, and an integrated comparative analysis with human skin cell atlas datasets from healthy skin, atopic dermatitis and psoriasis. We reveal the co-optation of Th2-immune gene programmes by malignant CTCL cells and modelling of the tumour microenvironment to support their survival. We identify MHC-II+ fibroblast subsets reminiscent of lymph node T-zone reticular cells and monocyte-derived dendritic cells that can maintain Th2-like tumour cells. CTCL Th2-like tumour cells are spatially associated with B cells, forming aggregates reminiscent of tertiary lymphoid structures which are more prominent with progressive disease. Finally, we validated the enrichment of B cells in CTCL skin infiltrates and its association with disease progression across three independent patient cohorts. Our findings provide diagnostic aids, potential biomarkers for disease staging and therapeutic strategies for CTCL.

genomics↗

bioRxiv2023

Hyperexcitability in human MECP2 null neuronal networks manifests as calcium-dependent reverberating super bursts

Rett syndrome (RTT) patients show abnormal developmental trajectories including loss of language and repetitive hand movements but also have signs of cortical hyperexcitability such as seizures. RTT is predominantly caused by mutations in MECP2 and can be modelled in vitro using human stem cell-derived neurons. MECP2 null excitatory neurons are smaller in soma size and have reduced synaptic connectivity but are also hyperexcitable, due to higher input resistance, which increases the chance to evoke action potentials with a given depolarized current. Few studies examine how single neuron activity integrates into neuronal networks during human development. Paradoxically, networks of MECP2 null neurons show a decrease in the frequency of bursting patterns consistent with synaptic hypoconnectivity, but no hyperexcitable network events have been reported. Here, we show that MECP2 null neurons have an increase in the frequency of a network event described as reverberating super bursts (RSBs) relative to isogenic controls. RSBs can be mistakenly called as a single long duration burst by standard burst detection algorithms. However, close examination revealed an initial large amplitude network burst followed by high frequency repetitive low amplitude mini-bursts. Using a custom burst detection algorithm, we unfolded the multi-burst structure of RSBs revealing that MECP2 null networks increased the total number of bursts relative to isogenic controls. Application of the Ca2+ chelator EGTA-AM selectively eliminated RSBs and rescued the network burst phenotype relative to the isogenic controls. Our results indicate that during early development, MECP2 null neurons are hyperexcitable and produce hyperexcitable networks. This may predispose them to the emergence of hyper-synchronic states that potentially translate into seizures. Network hyperexcitability is dependent on asynchronous neurotransmitter release driven by pre-synaptic Ca2+ and can be rescued by EGTA-AM to restore typical network dynamics. HIGHLIGHTSO_LIReverberating super-bursts (RSBs) follow a stereotypic form of a large initial network burst followed by several smaller amplitude high-frequency mini-bursts. C_LIO_LIRSBs occur more often in MECP2 null excitatory networks. C_LIO_LIMECP2 null excitatory networks with increased RSBs show a hyperexcitable network burst phenotype relative to isogenic controls. C_LIO_LIThe calcium chelator, EGTA-AM, decreases RSBs and rescues the dynamics of MECP2 null hyperexcitable networks. C_LI

neuroscience↗

bioRxiv2023

Pan-tissue mitochondrial phenotyping reveals lower OXPHOS expression and function across tumor types

Targeting mitochondrial oxidative phosphorylation (OXPHOS) to combat cancer is increasingly being investigated using a variety of small molecule inhibitors. Clinical success for these inhibitors has been hampered due to serious side-effects potentially arising from the inability to discriminate between non-cancerous and cancerous mitochondria. Although mitochondrial oxidative metabolism is essential for malignant growth, mitochondria OXPHOS is also essential to the physiology of all organs, including high-energy-demand organs like the heart. In comparing tumor OXPHOS reliance to these preeminent oxidative organs it is unclear if a therapeutic window for targeting mitochondrial OXPHOS in cancer exists. To address this gap in knowledge, mitochondrial OXPHOS was comprehensively evaluated across various murine tumors and compared to both matched normal tissues and other organs. When compared to both matched normal tissues, as well as high OXPHOS reliant organs like heart, intrinsic expression of the OXPHOS complexes, as well as OXPHOS flux were consistently lower across distinct tumor types. Operating on the assumption that intrinsic OXPHOS expression/function predicts OXPHOS reliance in vivo, these data suggest that pharmacologic blockade of mitochondrial OXPHOS likely compromises bioenergetic homeostasis in healthy oxidative organs prior to impacting tumor mitochondrial flux in a clinically meaningful way. Although these data caution against the use of indiscriminate mitochondrial inhibitors for cancer treatment, considerable heterogeneity was observed across tumor types with respect to both mitochondrial proteome composition and substrate-specific flux, highlighting the possibility for targeting discrete mitochondrial proteins or pathways unique to a given tumor type.

biochemistry↗

bioRxiv2023

iPSC-derived healthy human astrocytes selectively load miRNAs targeting neuronal genes into extracellular vesicles

Astrocytes are in constant communication with neurons during the establishment and maturation of functional networks in the developing brain. Astrocytes release extracellular vesicles (EVs) containing microRNA (miRNA) cargo that regulates transcript stability in recipient cells. Astrocyte released factors are thought to be involved in neurodevelopmental disorders. Healthy astrocytes partially rescue Rett Syndrome (RTT) neuron function. EVs isolated from stem cell progeny also correct aspects of RTT. EVs cross the blood-brain barrier (BBB) and their cargo is found in peripheral blood which may allow non-invasive detection of EV cargo as biomarkers produced by healthy astrocytes. Here we characterize miRNA cargo and sequence motifs in healthy human astrocyte derived EVs (ADEVs). First, human induced Pluripotent Stem Cells (iPSC) were differentiated into Neural Progenitor Cells (NPCs) and subsequently into astrocytes using a rapid differentiation protocol. iPSC derived astrocytes expressed specific markers, displayed intracellular calcium transients and secreted EVs. miRNAs were identified by RNA-Seq on astrocytes and ADEVs and target gene pathway analysis detected brain related terms. The miRNA profile was consistent with astrocyte identity, and included approximately 80 miRNAs found in astrocytes that were relatively depleted in ADEVs suggestive of passive loading. About 120 miRNAs were relatively enriched in ADEVs and motif analysis discovered binding sites for RNA binding proteins FUS, SRSF7 and CELF5. miRNA-483-5p was the most significantly enriched in ADEVs. This miRNA regulates MECP2 expression in neurons and has been found differentially expressed in blood samples from RTT patients. Our results identify potential miRNA biomarkers selectively sorted into ADEVs and implicate RNA binding protein sequence dependent mechanisms for miRNA cargo loading.

molecular biology↗

bioRxiv2023

Myosin inhibitor reverses hypertrophic cardiomyopathy in pediatric iPSC-cardiomyocytes to mirror variant correction

Hypertrophic cardiomyopathy (HCM) is mainly caused by sarcomere gene variants in MYH7 and MYBPC3. Targeted drugs like myosin ATPase inhibitors have shown efficacy in adult HCM but have not been evaluated in children. We generated iPSC-cardiomyocytes (CMs) from four children with HCM harboring variants in MYH7 (V606M; R453C) or MYBPC3 (G148R; P955fs and TNNI3_A157P), variant-corrected controls, and a healthy individual. All CMs showed hypertrophy and sarcomere disorganization. All 3 single variant CMs showed higher contractility, slower relaxation, higher calcium transients and higher ATPase activity. Only MYH7 variant CMs showed stronger myosin-actinin binding. Targeted myosin ATPase inhibitor showed complete rescue of the phenotype in affected CMs and in cardiac Biowires to mirror isogenic controls. The response was stronger compared to verapamil or metoprolol, highlighting the need for clinical trials of myosin targeted therapy in pediatric HCM patients. The phenotype and response to drug therapy are influenced by the underlying genotype.

genetics↗

bioRxiv2023

Pathology of natural infection with highly pathogenic avian influenza virus (H5N1) clade 2.3.4.4b in wild terrestrial mammals in the United States in 2022

This article describes the first detections of disease due to natural infection with highly pathogenic avian influenza virus (HPAIv) H5N1 of the Eurasian lineage goose/Guangdong clade 2.3.4.4b in wild terrestrial mammals throughout the United States during 2021-2022. Affected mammalian species include 50 red foxes (Vulpes vulpes), 6 striped skunks (Mephitis mephitis), 4 raccoons (Procyon lotor), 2 bobcats (Lynx rufus), 2 Virginia opossums (Didelphis virginiana), 1 coyote (Canis latrans), 1 fisher (Pekania pennanti), and 1 gray fox (Urocyon cinereoargenteus). Infected mammals primarily exhibited neurological signs. Necrotizing meningoencephalitis, interstitial pneumonia, and myocardial necrosis were the most common lesions; however, species variations in lesion distribution were observed. Genotype analysis of sequences from 48 animals indicates that these cases represent spillover infections from wild birds.

pathology↗

bioRxiv2023

Transcriptional buffering and 3'UTR lengthening are shaped during human neurodevelopment by shifts in mRNA stability and microRNA load

The contribution of mRNA half-life is commonly overlooked when examining changes in mRNA abundance during development. mRNA levels are regulated by transcription rate or mRNA half-life, but combinations of both could boost or buffer the final steady-state. We measured transcription rate and mRNA half-life changes during human induced pluripotent stem cell (iPSC)-derived neuronal development using RATE-seq. The most prevalent mode of gene regulation during transitions to neuronal progenitors and neurons was transcriptional buffering. Less prevalent modes consisted of transcriptional boosting events, transcription rate only shifts, or mRNA half-life only shifts. Buffered and novel boosted gene sets were enriched in RNA-binding protein (RBP) motifs and microRNA sites. Global mRNA half-life decreased two-fold in neurons. microRNA copy number per cell increased during neurodevelopment coincident with destabilization of a subset of short isoforms that contributed to increased average 3'UTR length. Our findings identify mRNA instability mechanisms that regulate transcript levels and 3'UTR isoform abundance, and provide a precedent for transcriptional boosting or buffering during development of other human tissues.

molecular biology↗

bioRxiv2023

Directed differentiation of human hindbrain neuroepithelial stem cells recapitulates cerebellar granule neurogenesis

Cerebellar granule neurons (CGNs) are the most abundant neurons in the human brain and modulate cerebellar output to the motor cortex. Dysregulation of CGN development underlies movement disorders and medulloblastomas. It is suspected that these disorders arise in progenitor states of the CGN lineage, for which human models are lacking. Here, we have differentiated human hindbrain neuroepithelial stem (hbNES) cells to CGNs in vitro using soluble growth factors, recapitulating key progenitor states in the lineage. We show that hbNES cells are not lineage committed and retain rhombomere 1 (r1) regional identity. Upon differentiation, hbNES cells first transit through a rhombic lip (RL) progenitor state at day 7, demonstrating human specific sub-ventricular cell identities. This RL state is followed by an ATOH1+ CGN progenitor state at day 14. By the end of a 56-day differentiation procedure, we obtain mature neurons expressing CGN markers GABAAa6 and vGLUT2. These neurons generate spontaneous and evoked action potentials. A small fraction of endpoint neurons were unipolar brush cells (UBC). We noted maintenance of a RL population throughout differentiation, as is consistent with human development. We show that sonic hedgehog (SHH) promotes {gamma}-aminobutyric acid (GABA)-ergic lineage specification and is a positive regulator of CGN progenitor proliferation. Interestingly, we observed that functional neuronal maturation is impaired by either elevated or absent SHH signaling. Impaired maturation under high SHH levels represents the potential of our system to model cerebellar tumorigenesis. Further, our data suggest a potential pro-differentiation role of SHH within a certain concentration range. Our work is, to our knowledge, the first detailed temporal characterization of the complete human CGN lineage in vitro. Our system recapitulates developmentally relevant progenitor states and is a new tool to model this specific cerebellar lineage, and how it may be disrupted to cause human disease.

developmental biology↗

bioRxiv2022

Inferring transmission routes for foot-and-mouth disease virus within a cattle herd using approximate Bayesian computation

To control an outbreak of an infectious disease it is essential to understand the different routes of transmission and how they contribute to the overall spread of the pathogen. With this information, policy makers can choose the most efficient methods of detection and control during an outbreak. Here we demonstrate a method for assessing the contribution of different routes of transmission using approximate Bayesian computation with sequential Monte Carlo sampling (ABC-SMC). We apply this to infer parameters of an individual based model of within-herd transmission of foot-and-mouth disease virus (FMDV), incorporating transmission through direct contact and via environmental contamination. Additionally, we use ABC-SMC for model selection to assess the plausibility of either transmission route alone being responsible for all infections. We show that direct transmission likely contributes the majority of infections during an outbreak of FMD but there is an accumulation of environmental contamination that can cause infections within a farm and also have the potential to spread between farms via fomites.

zoology↗

bioRxiv2022

Advanced physiological maturation of iPSC-derived human cardiomyocytes using an algorithm-directed optimization of defined media components

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold tremendous promise for in vitro modeling to assess native myocardial function and disease mechanisms as well as testing drug safety and efficacy. However, current iPSC- CMs are functionally immature, resembling in vivo CMs of fetal or neonatal developmental states. The use of targeted culture media and organoid formats have been identified as potential high-yield contributors to improve CM maturation. This study presents a novel iPSC-CM maturation medium formulation, designed using a differential evolutionary approach targeting metabolic functionality for iterative optimization. Relative to gold-standard reference formulations, our medium significantly matured morphology, Ca2+ handling, electrophysiology, and metabolism, which was further validated by multiomic screening, for cells in either pure or co-cultured microtissue formats. Together, these findings not only provide a reliable workflow for highly functional iPSC-CMs for downstream use, but also demonstrate the power of high-dimensional optimization processes in evoking advanced biological function in vitro.

bioengineering↗

bioRxiv2022

Neuron-specific protein network mapping of autism risk genes identifies shared biological mechanisms and disease relevant pathologies

Manuscript summaryThere are hundreds of risk genes associated with autism spectrum disorder (ASD), but signaling networks at the protein level remain unexplored. We use neuron-specific proximity-labeling proteomics (BioID) to identify protein-protein interaction (PPI) networks for 41 ASD-risk genes. Neuron-specific PPI networks, including synaptic transmission proteins, are disrupted by de novo missense variants. The PPI network map reveals convergent pathways, including mitochondrial/metabolic processes, Wnt signaling, and MAPK signaling. CRISPR knockout reveal an association between mitochondrial activity and ASD-risk genes. The PPI network shows an enrichment of 112 additional ASD-risk genes and differentially expressed genes from post-mortem ASD patients. Clustering of risk genes based on PPI networks identifies gene groups corresponding to clinical behavior score severity. Our data reveal that cell type-specific PPI networks can identify individual and convergent ASD signaling networks, provide a method to assess patient variants, and reveal biological insight into disease mechanisms and sub-cohorts of ASD.

neuroscience↗

bioRxiv2021

Design, immunogenicity, and efficacy of a pan-sarbecovirus dendritic-cell targeting vaccine

The emergence of SARS-CoV-2 variants of concern (VOCs) that escape pre-existing antibody neutralizing responses increases the need for vaccines that target conserved epitopes and induce cross-reactive B- and T-cell responses. We used a computational approach and sequence alignment analysis to design a new-generation subunit vaccine targeting conserved sarbecovirus B- and T-cell epitopes from Spike (S) and Nucleocapsid (N) to antigen-presenting cells expressing CD40 (CD40.CoV2). We demonstrate the potency of CD40.CoV2 to elicit high levels of cross-neutralizing antibodies against SARS-CoV-2, VOCs, and SARS-CoV-1 in K18-hACE2 transgenic mice, associated with improved viral control and survival after challenge. In addition, we demonstrate the potency of CD40.CoV2 in vitro to recall human multi-epitope, functional, and cytotoxic SARS-CoV-2 S- and N-specific T-cell responses that are unaffected by VOC mutations and cross-reactive with SARS-CoV-1 and, to a lesser extent, MERS epitopes. Overall, these findings provide a framework for a pan-sarbecovirus vaccine.

immunology↗