Biology subjects

Gale, M.

Publications and source records attributed to Gale, M..

31 records · Page 2Linked to original sources

bioRxiv2021

SARS-CoV2 variant-specific replicating RNA vaccines protect from disease and pathology and reduce viral shedding following challenge with heterologous SARS-CoV2 variants of concern

Despite mass public health efforts, the SARS-CoV2 pandemic continues as of late-2021 with resurgent case numbers in many parts of the world. The emergence of SARS-CoV2 variants of concern (VoC) and evidence that existing vaccines that were designed to protect from the original strains of SARS-CoV-2 may have reduced potency for protection from infection against these VoC is driving continued development of second generation vaccines that can protect against multiple VoC. In this report, we evaluated an alphavirus-based replicating RNA vaccine expressing Spike proteins from the original SARS-CoV-2 Alpha strain and recent VoCs delivered in vivo via a lipid inorganic nanoparticle. Vaccination of both mice and Syrian Golden hamsters showed that vaccination induced potent neutralizing titers against each homologous VoC but reduced neutralization against heterologous challenges. Vaccinated hamsters challenged with homologous SARS-CoV2 variants exhibited complete protection from infection. In addition, vaccinated hamsters challenged with heterologous SARS-CoV-2 variants exhibited significantly reduced shedding of infectious virus. Our data demonstrate that this vaccine platform elicits significant protective immunity against SARS-CoV2 variants and supports continued development of this platform.

microbiology↗

bioRxiv2021

Single cell based high-throughput Ig and TCR repertoire sequencing analysis in rhesus macaques

Recent advancements in microfluidics and high-throughput sequencing technologies have enabled recovery of paired heavy- and light-chains of immunoglobulins (Ig) and VDJ- and VJ-chains of T cell receptors (TCR) from thousands of single cells simultaneously in humans and mice. Despite rhesus macaques being one of the most well-studied model organisms for the human adaptive immune response, high-throughput single cell immune repertoire sequencing assays are not yet available due to the complexity of these polyclonal receptors. Here we employed custom primers that capture all known rhesus macaque Ig and TCR isotypes and chains that are fully compatible with a commercial solution for single cell immune repertoire profiling. Using these rhesus specific assays, we sequenced Ig and TCR repertoires in over 60,000 cells from cryopreserved rhesus PBMC, splenocytes, and FACS-sorted B and T cells. We were able to recover every Ig isotype and TCR chain, measure clonal expansion in proliferating T cells, and pair Ig and TCR repertoires with gene expression profiles of the same single cells. Our results establish the ability to perform high-throughput immune repertoire analysis in rhesus macaques at the single cell level.

immunology↗

bioRxiv2021

Multivalent designed proteins protect against SARS-CoV-2 variants of concern

Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness. One-Sentence SummaryWe designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.

synthetic biology↗

bioRxiv2021

Endomembrane targeting of human OAS1 p46 augments antiviral activity

Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from host cell innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense viral RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flavivirus, picornavirus, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform strongly associates with COVID-19 severity. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests early control of SARS-CoV-2 replication through OAS1-p46 is an important determinant of COVID-19 severity.

immunology↗

bioRxiv2021

Collaborative Cross Founder Expression Analysis (CCFEA)

To understand the role of host genetic variation in infection by SARS-CoV and influenza A virus we developed the Collaborative Cross Founder Expression Analysis (CCFEA), a shiny visualization tool using public RNAseq data from the collaborative cross (CC) founder strains (A/J, C57BL/6J, 129s1/SvImJ, NOD/ShILtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) that underwent infection by either virus, linked with genetic analyses to define loci linked to infection, immunity, and disease phenotypes. Individual gene expression data is displayed across founders, viral infections and days post infection. Resource Table O_TBL View this table: org.highwire.dtl.DTLVardef@4a284borg.highwire.dtl.DTLVardef@9b6168org.highwire.dtl.DTLVardef@11831baorg.highwire.dtl.DTLVardef@168e351org.highwire.dtl.DTLVardef@123e9ec_HPS_FORMAT_FIGEXP M_TBL C_TBL

bioinformatics↗

bioRxiv2021

Sustained IL-15 response signature predicts RhCMV/SIV vaccine efficacy

Simian immunodeficiency virus (SIV) challenge of rhesus macaques (RMs) vaccinated with Rhesus Cytomegalovirus (RhCMV) vectors expressing SIV proteins (RhCMV/SIV) results in a binary outcome: stringent control and subsequent clearance of highly pathogenic SIV in ~55% of vaccinated RMs with no protection in the remaining 45%. Although previous work suggests that unconventionally restricted, SIV-specific, effector-memory (EM)-biased CD8+ T cell responses are necessary for efficacy, the magnitude of these responses does not predict efficacy, and the basis of protection vs. non-protection in RhCMV/SIV vector-vaccinated RMs has not been elucidated. Here, we report that RhCMV/SIV vector administration strikingly alters the whole blood transcriptome of vaccinated RMs, with the sustained induction of specific immune-related pathways, including non-canonical T cell receptor (TCR), toll-lie receptor (TLR), inflammasome/cell death, and interleukin-15 (IL-15) signaling, significantly predicting protection. The IL-15 gene expression signature was further evaluated in an independent RM IL-15 treatment cohort, revealing that in whole blood the response to IL-15 is inclusive of innate and adaptive immune gene expression networks that link with RhCMV/SIV vaccine efficacy. We also show that this IL-15 response signature similarly tracks with vaccine protection in an independent RhCMV/SIV vaccination/SIV challenge RM validation cohort. Thus, the RhCMV/SIV vaccine imparts a coordinated and persistent induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ T cell function, that enable vaccine-elicited CD8+ T cells to mediate protection against highly pathogenic SIV challenge. Author SummarySIV insert-expressing vaccine vectors based on strain 68-1 RhCMV elicit robust, highly effector-memory-biased T cell responses that are associated with an unprecedented level of SIV control after challenge (replication arrest leading to clearance) in slightly over half of vaccinated monkeys. Since efficacy is not predicted by standard measures of immunogenicity, we used functional genomics analysis of RhCMV/SIV vaccinated monkeys with known challenge outcomes to identify immune correlates of protection. We found that arrest of viral replication after challenge significantly correlates with a vaccine-induced response to IL-15 that includes modulation of T cell, inflammation, TLR signaling, and cell death programming. These data suggest that RhCMV/SIV efficacy is not based on chance, but rather, results from a coordinated and sustained vaccine-mediated induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ effector-memory T cell function, that enable vaccine-elicited CD8+ T cells to mediate efficacy.

genomics↗

bioRxiv2020

SARS-CoV-2 infects human pluripotent stem cell-derived cardiomyocytes, impairing electrical and mechanical function

Global health has been threatened by the COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2)1. Although considered primarily a respiratory infection, many COVID-19 patients also suffer severe cardiovascular disease2-4. Improving patient care critically relies on understanding if cardiovascular pathology is caused directly by viral infection of cardiac cells or indirectly via systemic inflammation and/or coagulation abnormalities3,5-9. Here we examine the cardiac tropism of SARS-CoV-2 using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and three-dimensional engineered heart tissues (3D-EHTs). We observe that hPSC-CMs express the viral receptor ACE2 and other viral processing factors, and that SARS-CoV-2 readily infects and replicates within hPSC-CMs, resulting in rapid cell death. Moreover, infected hPSC-CMs show a progressive impairment in both electrophysiological and contractile properties. Thus, COVID-19-related cardiac symptoms likely result from a direct cardiotoxic effect of SARS-CoV-2. Long-term cardiac complications might be possible sequelae in patients who recover from this illness.

pathology↗

bioRxiv2020

De novo design of ACE2 protein decoys to neutralize SARS-CoV-2

There is an urgent need for the ability to rapidly develop effective countermeasures for emerging biological threats, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a generalized computational design strategy to rapidly engineer de novo proteins that precisely recapitulate the protein surface targeted by biological agents, like viruses, to gain entry into cells. The designed proteins act as decoys that block cellular entry and aim to be resilient to viral mutational escape. Using our novel platform, in less than ten weeks, we engineered, validated, and optimized de novo protein decoys of human angiotensin-converting enzyme 2 (hACE2), the membrane-associated protein that SARS-CoV-2 exploits to infect cells. Our optimized designs are hyperstable de novo proteins ([~]18-37 kDa), have high affinity for the SARS-CoV-2 receptor binding domain (RBD) and can potently inhibit the virus infection and replication in vitro. Future refinements to our strategy can enable the rapid development of other therapeutic de novo protein decoys, not limited to neutralizing viruses, but to combat any agent that explicitly interacts with cell surface proteins to cause disease.

biochemistry↗

bioRxiv2020

Single-dose replicating RNA vaccine induces neutralizing antibodies against SARS-CoV-2 in nonhuman primates

The ongoing COVID-19 pandemic, caused by infection with SARS-CoV-2, is having a dramatic and deleterious impact on health services and the global economy. Grim public health statistics highlight the need for vaccines that can rapidly confer protection after a single dose and be manufactured using components suitable for scale-up and efficient distribution. In response, we have rapidly developed repRNA-CoV2S, a stable and highly immunogenic vaccine candidate comprised of an RNA replicon formulated with a novel Lipid InOrganic Nanoparticle (LION) designed to enhance vaccine stability, delivery and immunogenicity. We show that intramuscular injection of LION/repRNA-CoV2S elicits robust anti-SARS-CoV-2 spike protein IgG antibody isotypes indicative of a Type 1 T helper response as well as potent T cell responses in mice. Importantly, a single-dose administration in nonhuman primates elicited antibody responses that potently neutralized SARS-CoV-2. These data support further development of LION/repRNA-CoV2S as a vaccine candidate for prophylactic protection from SARS-CoV-2 infection.

immunology↗

bioRxiv2019

The early local and systemic Type I interferon responses to ultraviolet B light exposure are cGAS dependent.

Most systemic lupus erythematosus (SLE) patients are photosensitive and ultraviolet B light (UVB) exposure worsens cutaneous disease and precipitates systemic flares of disease. The pathogenic link between skin disease and systemic exacerbations in SLE remains elusive. In an acute model of UVB-triggered inflammation, we observed that a single UV exposure triggered a striking IFN-I signature not only in the skin, but also in the blood and kidneys. The early IFN-I signature was significantly higher in female compared to male mice. The early IFN-I response in the skin was almost entirely, and in the blood partly, dependent on the presence of cGAS, as was skin inflammatory cell infiltration. Inhibition of cGAMP hydrolysis augmented the UVB-triggered IFN-I response. UVB skin exposure leads to cGAS-activation and both local and systemic IFN-I signature and could contribute to acute flares of disease in susceptible subjects such as patients with SLE.

immunology↗

bioRxiv2019

Systematic profiling of full-length immunoglobulin and T-cell receptor repertoire diversity in rhesus macaque through long read transcriptome sequencing

The diversity of immunoglobulin (Ig) and T-cell receptor (TCR) repertoires is a focal point of immunological studies. Rhesus macaques are key for modeling human immune responses, placing critical importance on the accurate annotation and quantification of their Ig and TCR repertoires. However, due to incomplete reference resources, the coverage and accuracy of the traditional targeted amplification strategies for profiling rhesus Ig and TCR repertoires are largely unknown. Here, using long read sequencing, we sequenced four Indian-origin rhesus macaque tissues and obtained high quality, full-length sequences for over 6,000 unique Ig and TCR transcripts, without the need for sequence assembly. We constructed the first complete reference set for the constant regions of all known isotypes and chain types of rhesus Ig and TCR repertoires. We show that sequence diversity exists across the entire variable regions of rhesus Ig and TCR transcripts. Consequently, existing strategies using targeted amplification of rearranged variable regions comprised of V(D)J gene segments miss a significant fraction (27% to 53% and 42% to 49%) of rhesus Ig/TCR diversity. To overcome these limitations, we designed new rhesus-specific assays that remove the need for primers conventionally targeting variable regions and allow single cell-level Ig and TCR repertoire analysis. Our improved approach will enable future studies to fully capture rhesus Ig and TCR repertoire diversity and is applicable for improving annotations in any model organism.

immunology↗

bioRxiv2019

Human DNA-PK activates a STING-independent DNA sensing pathway

Detection of intracellular DNA by the cGAS-STING pathway activates a type I interferon-mediated innate immune response that protects from virus infection and can be harnessed to promote anti-tumor immunity. Whether there are additional DNA sensing pathways, and how such pathways might function, remains controversial. We show here that humans - but not mice - have a second, potent, STING-independent DNA sensing pathway that is blocked by the E1A viral oncogene of human adenovirus 5. We identify human DNA-PK as the sensor of this pathway and demonstrate that DNA-PK kinase activity drives a robust and broad antiviral response. We discover that the heat shock protein HSPA8/HSC70 is a unique target of DNA-PK. Finally, we demonstrate that detection of foreign DNA and DNA damage trigger distinct modalities of DNA-PK activity. These findings reveal the existence, sensor, unique target, and viral antagonists of a STING-independent DNA sensing pathway (SIDSP) in human cells.

immunology↗

bioRxiv2018

Role of RIG-I-like receptors in innate immune sensing of Coxsackievirus B3 and encephalomyocarditis virus in murine macrophages and fibroblasts

Viral infections are sensed by pattern recognition receptors that trigger an innate immune response through the expression of interferons (IFNs) and other cytokines. Most RNA viruses are sensed by the RIG-I like receptors (RLR)s. The contributions of these receptors to sensing viruses of the Picornaviridae family were investigated. Encephalomyocarditis virus (EMCV) and Coxsackievirus B3 (CVB3), picornaviruses of the Cardiovirus and Enterovirus genera, are detected by both MDA5 and RIG-I in bone marrow derived macrophages. In macrophages from wild type mice, type I IFN is produced early after infection; IFN{beta} synthesis is reduced in the absence of each sensor, while IFN production is reduced in the absence of MDA5. EMCV and CVB3 do not replicate in murine macrophages, and their detection is different in murine embryonic fibroblasts (MEFs), in which the viruses replicate to high titers. In MEFs RIG-I was essential for the expression of type I IFNs but contributes to increased yields of CVB3, while MDA5 inhibited CVB3 replication but in an IFN independent manner. These observations demonstrate that innate sensing of similar viruses by RLRs depends upon the cell type.\n\nImportanceEnteroviruses such as Coxsackieviruses are the most common human respiratory pathogens. The hosts innate immune response, in particular that modulated by the production of type I and III interferons, is thought to restrict picornavirus infection. Two cytoplasmic proteins, MDA5 and RIG-I, are critical for initiating the early innate immune response against these viruses. Mutations within MDA5 encoding gene have been associated with the development of severe enterovirus associated respiratory illnesses in healthy children. To further understand how the innate immune response dependent upon MDA5 and Rig-I is initiated during picornavirus infection, macrophages from mice lacking MDA5 or RIG-I were infected with Coxsackievirus B3 (CVB3) and a related animal virus. RIG-I is essential for type I IFN production during CVB3 infection; when MDA5 is present, viral titers are reduced by an IFN-independent pathway. These observations demonstrate that innate sensing of viruses by MDA5 and RIG-I depends upon the cell type.

microbiology↗