Biology subjects

Pinsky, B. A.

Publications and source records attributed to Pinsky, B. A..

15 recordsLinked to original sources

bioRxiv2024

XBB.1.5 monovalent booster improves antibody binding and neutralization against emerging SARS-CoV-2 Omicron variants

The rapid emergence of divergent SARS-CoV-2 variants has led to an update of the COVID-19 booster vaccine to a monovalent version containing the XBB.1.5 spike. To determine the neutralization breadth following booster immunization, we collected blood samples from 24 individuals pre- and post-XBB.1.5 mRNA booster vaccination ([~]1 month). The XBB.1.5 booster improved both neutralizing activity against the ancestral SARS-CoV-2 strain (WA1) and the circulating Omicron variants, including EG.5.1, HK.3, HV.1, XBB.1.5 and JN.1. Relative to the pre-boost titers, the XBB.1.5 monovalent booster induced greater total IgG and IgG subclass binding, particular IgG4, to the XBB.1.5 spike as compared to the WA1 spike. We evaluated antigen-specific memory B cells (MBCs) using either spike or receptor binding domain (RBD) probes and found that the monovalent booster largely increases non-RBD cross-reactive MBCs. These data suggest that the XBB.1.5 monovalent booster induces cross-reactive antibodies that neutralize XBB.1.5 and related Omicron variants.

immunology↗

bioRxiv2023

Convergent Evolution of A-Lineage (Clade 19B) SARS-CoV-2 Spike Sequences with B-Lineage Variants of Concern Affects Virus Replication in a Temperature-Dependent Manner on Human Nasal Epithelial Cell Cultures

The first three months of the COVID-19 pandemic was dominated by two SARS-CoV-2 lineages: A-lineages (Clade 19B) and B-lineages (Clade 19A). However, with the emergence of the Spike D614G substitution in B.1 lineages (Clade 20A), both early lineages were outcompeted and remained near-extinction from mid-2020 onwards. In early-2021, there was a re-emergence and persistence of novel A-lineage variants with substitutions in the Spike gene resembling those found in Variants of Concern (VOCs). An early A.3 variant (MD-HP00076/2020) and three A.2.5 variants (MD-HP02153/2021, MD-HP05922/2021 and CA-VRLC091/2021) were isolated and characterized for their genomic sequences, antibody neutralization, and in vitro replication. All A.2.5 isolates had five Spike mutations relative to the A.3 variant sequence: D614G, L452R, {Delta}141-143, D215A, and ins215AGY. Plaque reduction neutralization assays demonstrated that A.2.5 isolates had a 2.5 to 5-fold reduction in neutralization using contemporaneous COVID-19 convalescent plasma when compared to A.3. In vitro viral characterization in VeroE6 cell lines revealed that the A.3 isolate grew faster and spread more than A.2.5. On VeroE6-TMPRSS2 cells, significant syncytia formation was also observed with the A.2.5 isolates, however Spike cleavage efficiency did not explain these differences. In human nasal epithelial cell (hNEC) cultures, the A.2.5 isolates grew significantly faster and to higher total infectious virus titers than A.3. All A.2.5 lineage isolates grew significantly faster at 37{degrees}C than at 33{degrees}C irrespective of cell type, and to higher peak titers except compared to A.3. This suggests A.2.5s adapted to improve replication using similar mutations found in the B-lineage SARS-CoV-2 variants. ImportanceWhile both A- and B-lineage SARS-CoV-2 variants emerged and circulated together during the early months of the pandemic, the B-lineages that acquired Spike D614G eventually outcompeted all other variants. We show that the A-lineage variants eventually evolved mutations including Spike D614G and Spike L452R that improved their in vitro replication in human nasal epithelial cells in a temperature dependent manner, suggesting there are some highly selectable mutation landscapes that SARS-CoV-2 can acquire to adapt to replication and transmission in humans.

microbiology↗

bioRxiv2022

Global and cell type-specific immunological hallmarks of severe dengue progression

Severe dengue (SD) is a major cause of morbidity and mortality impacting approximately 5 million of the 400 million people infected with dengue virus (DENV) annually. To define DENV target cells and immunological hallmarks of SD progression in childrens blood, we integrated virus-inclusive single cell RNA-Seq 2 (viscRNA-Seq 2) with functional assays. Beyond myeloid cells, in natural infection, B cells harbor replicating DENV capable of infecting permissive cells. Alterations in cell type abundance, gene and protein expression and secretion, and cell-cell communications point towards increased migration and inflammation in SD progressors (SDp). Concurrently, antigen presenting cells from SDp demonstrate intact uptake, yet impaired interferon responses and antigen presentation, in part DENV-modulated. Increased activation, regulation, and exhaustion of effector responses and expansion of HLA-DR-expressing, possibly compensatory, adaptive-like NK cells also characterize SDp. These findings reveal DENV target cells in the human blood and provide insight into SD pathogenesis beyond antibody-mediated enhancement.

immunology↗

bioRxiv2022

mRNA bivalent booster enhances neutralization against BA.2.75.2 and BQ.1.1

The emergence of the highly divergent SARS-CoV-2 Omicron variant has jeopardized the efficacy of vaccines based on the ancestral spike. The bivalent COVID-19 mRNA booster vaccine within the United States is comprised of the ancestral and the Omicron BA.5 spike. Since its approval and distribution, additional Omicron subvariants have been identified with key mutations within the spike protein receptor binding domain that are predicted to escape vaccine sera. Of particular concern is the R346T mutation which has arisen in multiple subvariants, including BA.2.75.2 and BQ.1.1. Using a live virus neutralization assay, we evaluated serum samples from individuals who had received either one or two monovalent boosters or the bivalent booster to determine neutralizing activity against wild-type (WA1/2020) virus and Omicron subvariants BA.1, BA.5, BA.2.75.2, and BQ.1.1. In the one monovalent booster cohort, relative to WA1/2020, we observed a reduction in neutralization titers of 9-15-fold against BA.1 and BA.5 and 28-39-fold against BA.2.75.2 and BQ.1.1. In the BA.5-containing bivalent booster cohort, the neutralizing activity improved against all the Omicron subvariants. Relative to WA1/2020, we observed a reduction in neutralization titers of 3.7- and 4-fold against BA.1 and BA.5, respectively, and 11.5- and 21-fold against BA.2.75.2 and BQ.1.1, respectively. These data suggest that the bivalent mRNA booster vaccine broadens humoral immunity against the Omicron subvariants.

immunology↗

bioRxiv2022

Magnitude and kinetics of the human immune cell response associated with severe dengue progression by single-cell proteomics

Approximately five million dengue virus-infected patients, particularly children, progress to a potentially life-threatening severe dengue (SD) infection annually. To identify the immune features and temporal dynamics underlying SD progression, we performed deep immune profiling by mass cytometry of PBMCs collected longitudinally from SD progressors (SDp) and uncomplicated dengue (D) patients. While D is characterized by early activation of innate immune responses, in SDp there is rapid expansion and activation of IgG-secreting plasma cells and memory and regulatory T cells. Concurrently, SDp, particularly children, demonstrate increased proinflammatory NK cells, inadequate expansion of CD16+ monocytes, and high expression of the Fc{gamma}R, CD64 on myeloid cells, yet diminished antigen presentation. Syndrome-specific determinants include suppressed dendritic cell abundance in shock/hemorrhage vs. enriched plasma cell expansion in organ impairment. This study reveals uncoordinated immune responses in SDp and provides insights into SD pathogenesis in humans with potential implications for prediction and treatment.

immunology↗

bioRxiv2022

Disease diagnostics using machine learning of immune receptors

Clinical diagnosis typically incorporates physical examination, patient history, and various laboratory tests and imaging studies, but makes limited use of the human systems own record of antigen exposures encoded by receptors on B cells and T cells. We analyzed immune receptor datasets from 593 individuals to develop MAchine Learning for Immunological Diagnosis (Mal-ID), an interpretive framework to screen for multiple illnesses simultaneously or precisely test for one condition. This approach detects specific infections, autoimmune disorders, vaccine responses, and disease severity differences. Human-interpretable features of the model recapitulate known immune responses to SARS-CoV-2, Influenza, and HIV, highlight antigen-specific receptors, and reveal distinct characteristics of Systemic Lupus Erythematosus and Type-1 Diabetes autoreactivity. This analysis framework has broad potential for scientific and clinical interpretation of human immune responses.

immunology↗

bioRxiv2022

Numb-associated kinases are required for SARS-CoV-2 infection and are cellular targets for therapy

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose serious threats to global health. We previously reported that AAK1, BIKE and GAK, members of the Numb-associated kinase family, control intracellular trafficking of multiple RNA viruses during viral entry and assembly/egress. Here, using both genetic and pharmacological approaches, we probe the functional relevance of NAKs for SARS-CoV-2 infection. siRNA-mediated depletion of AAK1, BIKE, GAK, and STK16, the fourth member of the NAK family, suppressed SARS-CoV-2 infection in human lung epithelial cells. Both known and novel small molecules with potent AAK1/BIKE, GAK or STK16 activity suppressed SARS-CoV-2 infection. Moreover, combination treatment with the approved anti-cancer drugs, sunitinib and erlotinib, with potent anti-AAK1/BIKE and GAK activity, respectively, demonstrated synergistic effect against SARS-CoV-2 infection in vitro. Time-of-addition experiments revealed that pharmacological inhibition of AAK1 and BIKE suppressed viral entry as well as late stages of the SARS-CoV-2 life cycle. Lastly, suppression of NAKs expression by siRNAs inhibited entry of both wild type and SARS-CoV-2 pseudovirus. These findings provide insight into the roles of NAKs in SARS-CoV-2 infection and establish a proof-of-principle that pharmacological inhibition of NAKs can be potentially used as a host-targeted approach to treat SARS-CoV-2 with potential implications to other coronaviruses.

microbiology↗

bioRxiv2021

An intranasal ASO therapeutic targeting SARS-CoV-2

The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity1. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic2. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identified an LNA ASO binding to the 5 leader sequence of SARS-CoV-2 ORF1a/b that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the K18-hACE2 humanized COVID-19 mouse model potently (98-99%) suppressed viral replication in the lungs of infected mice, revealing strong prophylactic and treatment effects. We found that the LNA ASO also represses viral infection in golden Syrian hamsters, and is highly efficacious in countering all SARS-CoV-2 "variants of concern" tested in vitro and in vivo, including B.1.427, B.1.1.7, and B.1.351 variants3. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce transmission of variants partially resistant to vaccines and monoclonal antibodies, and could be deployed intranasally for prophylaxis or via lung delivery by nebulizer to decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences4, and they may have particular impact in areas where vaccine distribution is a challenge, and could be stockpiled for future coronavirus pandemics.

microbiology↗

bioRxiv2021

Pan-ErbB inhibition protects from SARS-CoV-2 replication, inflammation, and injury

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, 2 and 4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, pro-inflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production and disruption of the blood-brain barrier integrity in microfluidic-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof-of-principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

microbiology↗

bioRxiv2021

Infection and vaccine-induced neutralizing antibody responses to the SARS-CoV-2 B.1.617.1 variant

SARS-CoV-2 has caused a devastating global pandemic. The recent emergence of SARS-CoV-2 variants that are less sensitive to neutralization by convalescent sera or vaccine-induced neutralizing antibody responses has raised concerns. A second wave of SARS-CoV-2 infections in India is leading to the expansion of SARS-CoV-2 variants. The B.1.617.1 variant has rapidly spread throughout India and to several countries throughout the world. In this study, using a live virus assay, we describe the neutralizing antibody response to the B.1.617.1 variant in serum from infected and vaccinated individuals. We found that the B.1.617.1 variant is 6.8-fold more resistant to neutralization by sera from COVID-19 convalescent and Moderna and Pfizer vaccinated individuals. Despite this, a majority of the sera from convalescent individuals and all sera from vaccinated individuals were still able to neutralize the B.1.617.1 variant. This suggests that protective immunity by the mRNA vaccines tested here are likely retained against the B.1.617.1 variant. As the B.1.617.1 variant continues to evolve, it will be important to monitor how additional mutations within the spike impact antibody resistance, viral transmission and vaccine efficacy.

immunology↗

bioRxiv2021

Long-read sequencing of SARS-CoV-2 reveals novel transcripts and a diverse complex transcriptome landscape.

Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2 (COVID-19), is a positive single-stranded RNA virus with a 30 kb genome that is responsible for the current pandemic. To date, the genomes of global COVID-19 variants have been primarily characterized via short-read sequencing methods. Here, we devised a long-read RNA (IsoSeq) sequencing approach to characterize the COVID-19 transcript landscape and expression of its [~]27 coding regions. Our analysis identified novel COVID-19 transcripts including a) a short [~]65-70 nt 5-UTR fused to various downstream ORFs encoding accessory proteins such as the envelope, ORF 8, and ORF 9 (nucleocapsid) proteins, that are relatively highly expressed, b) novel SNVs that are differentially expressed, whereby a subset are suggestive of partial RNA editing events, and c) SNVs at functional sites, whereby at least one is associated with a differentially expressed spike protein isoform. These previously uncharacterized COVID-19 isoforms, expressed genes, and gene variants were corroborated using ddPCR. Understanding this transcriptional complexity may help provide insight into the biology and pathogenicity of SARS-CoV-2 compared to other coronaviruses.

genetics↗

bioRxiv2021

Integrated plasma proteomic and single-cell immune signaling network signatures demarcate mild, moderate, and severe COVID-19

The biological determinants of the wide spectrum of COVID-19 clinical manifestations are not fully understood. Here, over 1400 plasma proteins and 2600 single-cell immune features comprising cell phenotype, basal signaling activity, and signaling responses to inflammatory ligands were assessed in peripheral blood from patients with mild, moderate, and severe COVID-19, at the time of diagnosis. Using an integrated computational approach to analyze the combined plasma and single-cell proteomic data, we identified and independently validated a multivariate model classifying COVID-19 severity (multi-class AUCtraining = 0.799, p-value = 4.2e-6; multi-class AUCvalidation = 0.773, p-value = 7.7e-6). Features of this high-dimensional model recapitulated recent COVID-19 related observations of immune perturbations, and revealed novel biological signatures of severity, including the mobilization of elements of the renin-angiotensin system and primary hemostasis, as well as dysregulation of JAK/STAT, MAPK/mTOR, and NF-{kappa}B immune signaling networks. These results provide a set of early determinants of COVID-19 severity that may point to therapeutic targets for the prevention of COVID-19 progression. SummaryFeyaerts et al. demonstrate that an integrated analysis of plasma and single-cell proteomics differentiates COVID-19 severity and reveals severity-specific biological signatures associated with the dysregulation of the JAK/STAT, MAPK/mTOR, and NF-{kappa}B immune signaling networks and the mobilization of the renin-angiotensin and hemostasis systems.

immunology↗

bioRxiv2020

Electric-field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2

The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In a basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex is activated when it highly specifically binds to target DNA, and the activated complex non-specifically cleaves single-stranded DNA reporter probes labeled with a fluorophore-quencher pair. We recently discovered that electric field gradients can be used to control and accelerate this CRISPR assay by co-focusing Cas12-gRNA, reporters, and target. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab sample. We here combine this ITP purification with loop-mediated isothermal amplification, and the ITP-enhanced CRISPR assay to achieve detection of SARS-CoV-2 RNA (from raw sample to result) in 30 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables a new modality for a suite of microfluidic CRISPR-based diagnostic assays. Significance statementRapid, early-stage screening is especially crucial during pandemics for early identification of infected patients and control of disease spread. CRISPR biology offers new methods for rapid and accurate pathogen detection. Despite their versatility and specificity, existing CRISPR-diagnostic methods suffer from the requirements of up-front nucleic acid extraction, large reagent volumes, and several manual steps--factors which prolong the process and impede use in low resource settings. We here combine on-chip electric-field control in combination with CRIPSR biology to directly address these limitations of current CRISPR-diagnostic methods. We apply our method to the rapid detection of SARS-CoV-2 RNA in clinical samples. Our method takes 30 min from raw sample to result, a significant improvement over existing diagnostic methods for COVID-19.

bioengineering↗

bioRxiv2020

Comparison of the Accula SARS-CoV-2 Test with a Laboratory-Developed Assay for Detection of SARS-CoV-2 RNA in Clinical Nasopharyngeal Specimens

BackgroundSeveral point-of-care (POC) molecular tests have received emergency use authorization (EUA) from the Food and Drug Administration (FDA) for diagnosis of SARS-CoV-2. The test performance characteristics of the Accula (Mesa Biotech) SARS-CoV-2 POC test need to be evaluated to inform its optimal use. ObjectivesThe aim of this study was to assess test performance of the Accula SARS-CoV-2 test. Study designThe performance of the Accula test was assessed by comparing results of 100 nasopharyngeal swab samples previously characterized by the Stanford Health Care EUA laboratory-developed test (SHC-LDT) targeting the envelope (E) gene. Assay concordance was assessed by overall percent agreement, positive percent agreement (PPA), negative percent agreement (NPA), and Cohens kappa coefficient. ResultsOverall percent agreement between the assays was 84.0% (95% confidence interval [CI] 75.3 to 90.6%), PPA was 68.0% (95% CI 53.3 to 80.5%) and the kappa coefficient was 0.68 (95% CI 0.54 to 0.82). Sixteen specimens detected by the SHC-LDT were not detected by the Accula test, and showed low viral load burden with a median cycle threshold value of 37.7. NPA was 100% (95% CI 94.2 to 100%). ConclusionCompared to the SHC-LDT, the Accula SARS-CoV-2 test showed excellent negative agreement. However, positive agreement was low for samples with low viral load. The false negative rate of the Accula POC test calls for a more thorough evaluation of POC test performance characteristics in clinical settings, and for confirmatory testing in individuals with moderate to high pre-test probability of SARS-CoV-2 who test negative on Accula.

microbiology↗

bioRxiv2018

Virus-inclusive single cell RNA sequencing reveals molecular signature predictive of progression to severe dengue infection

Dengue virus (DENV) infection can result in severe complications. Yet, the understanding of the molecular correlates of severity is limited, partly due to difficulties in defining the peripheral blood mononuclear cells (PBMCs) that are associated with DENV in vivo. Additionally, there are currently no biomarkers predictive of progression to severe dengue (SD). Bulk transcriptomics data are difficult to interpret because blood consists of multiple cell types that may react differently to infection. Here we applied virus-inclusive single cell RNA-seq approach (viscRNA-Seq) to profile transcriptomes of thousands of single PBMCs derived early in the course of disease from six dengue patients and four healthy controls, and to characterize distinct DENV-associated leukocytes. Multiple genes, particularly interferon response genes, were upregulated in a cell-specific manner prior to progression to SD. Expression of MX2 in naive B cells and CD163 in CD14+ CD16+ monocytes was predictive of SD. The majority of DENV-associated cells in the blood of two patients who progressed to SD were naive IgM B cells expressing the CD69 and CXCR4 receptors and antiviral genes, followed by monocytes. Bystander uninfected B cells also demonstrated immune activation, and plasmablasts from two patients exhibited antibody lineages with convergently hypermutated heavy chain sequences. Lastly, assembly of the DENV genome revealed diversity at unexpected genomic sites. This study presents a multi-faceted molecular elucidation of natural dengue infection in humans and proposes biomarkers for prediction of SD, with implications for profiling any tissue and viral infection, and for the development of a dengue prognostic assay.\n\nSignificanceA fraction of the 400 million people infected with dengue annually progresses to severe dengue (SD). Yet, there are currently no biomarkers to effectively predict disease progression. We profiled the landscape of host transcripts and viral RNA in thousands of single blood cells from dengue patients prior to progressing to SD. We discovered cell-type specific immune activation and candidate predictive biomarkers. We also revealed preferential virus association with specific cell populations, particularly naive B cells and monocytes. We then explored immune activation of bystander cells, clonality and somatic evolution of adaptive immune repertoires, and viral genomics. This multi-faceted approach could advance understanding of pathogenesis of any viral infection, map an atlas of infected cells and promote the development of prognostics.

immunology↗