Biology subjects

Sharma, N.

Publications and source records attributed to Sharma, N..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

Prediction and scanning of IL-5 inducing peptides using alignment-free and alignment-based method

Interleukin-5 (IL-5) is the key cytokine produced by T-helper, eosinophils, mast and basophils cells. It can act as an enticing therapeutic target due to its pivotal role in several eosinophil-mediated diseases. Though numerous methods have been developed to predict HLA binders and cytokines-inducing peptides, no method was developed for predicting IL-5 inducing peptides. All models in this study have been trained, tested and validated on experimentally validated 1907 IL-5 inducing and 7759 non-IL-5 inducing peptides obtained from IEDB. First, alignment-based methods have been developed using similarity and motif search. These alignment-based methods provide high precision but poor coverage. In order to overcome this limitation, we developed machine learning-based models for predicting IL-5 inducing peptides using a wide range of peptide features. Our random-forest model developed using selected 250 dipeptides achieved the highest performance among alignment-free methods with AUC 0.75 and MCC 0.29 on validation dataset. In order to improve the performance, we developed an ensemble or hybrid method that combined alignment-based and alignment-free methods. Our hybrid method achieved AUC 0.94 with MCC 0.60 on validation/ independent dataset. The best model developed in this study has been incorporated in the web server IL5pred (https://webs.iiitd.edu.in/raghava/il5pred/). Key PointsO_LIIL-5 is a regulatory cytokine that plays a vital role in eosinophil-mediated diseases C_LIO_LIBLAST-based similarity search against IL-5 inducing peptides was employed C_LIO_LIA hybrid approach combines alignment-based and alignment-free methods C_LIO_LIAlignment-free models are based on machine learning techniques C_LIO_LIA web server IL5pred and its standalone software have been developed C_LI Authors BiographyO_LIDr. Naorem Leimarembi Devi is currently working as a DBT-Research Associate in Department of Computational Biology, Indraprastha Institute of Information Technology, New Delhi, India. C_LIO_LINeelam Sharma is pursuing her Ph.D. in Computational Biology from the Department of Computational Biology, Indraprastha Institute of Information Technology, New Delhi, India. C_LIO_LIProf. G.P.S. Raghava is currently working as Professor and Head of Department of Computational Biology, Indraprastha Institute of Information Technology, New Delhi, India. C_LI

bioinformatics↗

bioRxiv2022

Categorising update mechanisms for graph-structured metapopulations

The structure of a population strongly influences its evolutionary dynamics. In various settings ranging from biology to social systems, individuals tend to interact more often with those present in their proximity and rarely with those far away. A common approach to model the structure of a population is Evolutionary Graph Theory. In this framework, each graph node is occupied by a reproducing individual. The links connect these individuals to their neighbours. The offspring can be placed on neighbouring nodes, replacing the neighbours - or the progeny of its neighbours can replace a node during the course of ongoing evolutionary dynamics. Extending this theory by replacing single individuals with subpopulations at nodes yields a graph-structured metapopulation. The dynamics between the different local subpopulations is set by an update mechanism. There are many such update mechanisms. Here, we classify update mechanisms for structured metapopulations, which allows to find commonalities between past work and illustrate directions for further research and current gaps of investigation.

evolutionary biology↗

bioRxiv2022

Ancestral SARS-CoV-2 driven antibody repertoire diversity in an unvaccinated individual correlates with expanded neutralization breadth

Understanding the quality of immune repertoire triggered during natural infection can provide vital clues that form the basis for development of humoral immune response in some individuals capable of broadly neutralizing pan SARS-CoV-2 variants. We assessed the diversity of neutralizing antibody responses developed in an unvaccinated individual infected with ancestral SARS-CoV-2 by examining the ability of the distinct B cell germline-derived monoclonal antibodies (mAbs) in neutralizing known and currently circulating Omicron variants by pseudovirus and authentic virus neutralization assays. The ability of the antibodies developed post vaccination in neutralizing Omicron variants was compared to that obtained at baseline of the same individual and to those obtained from Omicron breakthrough infected individuals by pseudovirus neutralization assay. Broadly SARS-CoV-2 neutralizing mAbs representing unique B cell lineages with non-overlapping epitope specificities isolated from a single donor varied in their ability to neutralize Omicron variants. Plasma antibodies developed post vaccination from this individual demonstrated neutralization of Omicron BA.1, BA.2 and BA.4 with increased magnitude and found to be comparable with those obtained from other vaccinated individuals who were infected with ancestral SARS-CoV-2. Development of B cell repertoire capable of producing antibodies with distinct affinity and specificities for the antigen immediately after infection capable of eliciting broadly neutralizing antibodies offers highest probability in protecting against evolving SARS-CoV-2 variants. ImportanceDevelopment of robust neutralizing antibodies in SARS-CoV-2 convalescent individuals is known, however varies at population level. We isolated monoclonal antibodies from an individual infected with ancestral SARS-CoV-2 in early 2020 that not only varied in their B cell lineage origin but also varied in their capability and potency to neutralize all the known VOC and currently circulating Omicron variants. This indicated establishment of unique lineages that contributed in forming B cell repertoire in this particular individual immediately following infection giving rise to diverse antibody responses that could compensate each other in providing broadly neutralizing polyclonal antibody response. Individuals who were able to produce such potent polyclonal antibody responses after infection have a higher chance of being protected from evolving SARS-CoV-2 variants.

immunology↗

bioRxiv2022

Fgf8 promotes survival of nephron progenitors by regulating BAX/BAK-mediated apoptosis

Fibroblast growth factors (Fgfs) have long been implicated in processes critical to embryonic development, such as cell survival, migration, and differentiation. Several mouse models of organ development ascribe a prosurvival requirement specifically to FGF8. Here, we explore the potential role of prosurvival FGF8 signaling in kidney development. We have previously demonstrated that conditional deletion of Fgf8 in the mesodermal progenitors that give rise to the kidney leads to renal aplasia in the mutant neonate. Deleterious consequences caused by loss of FGF8 begin to manifest by E14.5 when massive aberrant cell death occurs in the cortical nephrogenic zone in the rudimentary kidney as well as in the renal vesicles that give rise to the nephrons. To rescue cell death in the Fgf8 mutant kidney, we inactivate the genes encoding the pro-apoptotic factors BAK and BAX. In a wild-type background, the loss of Bak and Bax abrogates normal cell death and has minimal effect on renal development. However, in Fgf8 mutants, the combined loss of Bak and Bax rescues aberrant cell death in the kidneys and restores some measure of kidney development: 1) the nephron progenitor population is greatly increased; 2) some glomeruli form, which are rarely observed in Fgf8 mutants; and 3) kidney size is rescued by about 50% at E18.5. The development of functional nephrons, however, is not rescued. Thus, FGF8 signaling is required for nephron progenitor survival by regulating BAK / BAX and for subsequent steps involving, as yet, undefined roles in kidney development.

developmental biology↗

bioRxiv2022

Genomewide basis for nitrogen use efficiency in contrasting genotypes of rice

Rice is an ideal crop with huge germplasm diversity and post-genomic resources for improvement of nitrogen (N) use efficiency (NUE). There is a paucity of comparative studies on rice genotypes contrasting for NUE, especially with urea, the predominant fertilizer in rice growing countries. In this study, low urea-responsive transcriptomes of contrasting rice genotypes namely Nidhi (low NUE) and Panvel1 (high NUE) were compared. They were based on whole plants grown for 21 days in pots containing nutrient-depleted soil fertilized with normal (15 mM) and low urea (1.5 mM) media. There were 1497 and 2819 differentially expressed genes (DEGs) in Nidhi and Panvel1, respectively, of which 271 were common. Though 1226 DEGs were genotype-specific in Nidhi and 2548 in Panvel1, there was far higher commonality in underlying processes. High NUE is associated with the urea-responsive regulation of other nutrient transporters, miRNAs, transcription factors and better photosynthesis, water use efficiency and post translational modifications. Many of their genes co-localized to NUE QTLs on chromosomes 1, 3 and 9. Field evaluation of the contrasting genotypes under different doses of urea revealed better performance of Panvel1 in different agronomic parameters including grain yield, transport/uptake efficiencies and NUE. Comparison of our urea-based transcriptomes with our previous nitrate-based transcriptomes from the same contrasting rice genotypes revealed many common processes despite large differences in their expression profiles. Our model proposes that differential involvement of transporters and transcription factors among others contributes to better urea uptake, translocation, utilization, flower development and yield for high NUE. SummaryRice genotypes with contrasting urea use efficiency differ in the role of transporters, transcription factors, miRNAs, post-translational modifications, photosynthesis and water use efficiency

plant biology↗

bioRxiv2022

Ifit2 regulates murine-coronavirus spread to the spinal cord white matter and its associated myelin pathology

Ifit2, an interferon-induced protein with tetratricopeptide repeats 2, plays a critical role in restricting neurotropic murine {beta}-coronavirus RSA59 infection. RSA59 intracranial injection of Ifit2 deficient (-/-) compared to wild type (WT) mice results in impaired acute microglial activation, associated with reduced CX3CR1 expression, which consecutively limits migration of peripheral lymphocytes into the brain, leading to impaired virus control followed by severe morbidity and mortality. While the protective role of Ifit2 is established for acute viral encephalitis, less is known about its influence on demyelination during the chronic phase of RSA59 infection. Our current study demonstrates that Ifit2 deficiency causes extensive RSA59 viral spread throughout both the spinal cord grey and white matter and is associated with impaired CD4+ T cell infiltration. Cervical lymph nodes of RSA59 infected Ifit2-/- mice showed reduced activation of CD4+ T cells and impaired IFN{gamma} expression during acute encephalomyelitis. Furthermore, blood-brain-barrier integrity was preserved in the absence of Ifit2 as evidenced by integral, tight junction protein ZO-1 expression surrounding the meninges and blood vessels and decreased Texas red dye uptake. In contrast to WT mice exhibiting only sparse myelin loss, the chronic disease phase in Ifit2-/- mice was associated with severe demyelination and persistent viral load, even at low infection doses. Overall, our study highlights that Ifit2 provides antiviral functions by promoting acute neuroinflammation and thereby aiding virus control and limiting severe demyelination. Author SummaryThe role of interferons in providing protective immunity against viral spread and pathogenesis is well known. Interferons execute their function by inducing certain genes collectively called as interferon stimulated genes (ISGs) among which Interferon-induced protein with tetratricopeptide repeats 2, Ifit2, is known for restricting neurotropic viral replication and spread in the brain. So far, not much has been investigated about its role in viral spread to the spinal cord and its associated myelin pathology. Towards this our study using neurotropic murine-{beta}-coronavirus and Ifit2 deficient mice demonstrate that Ifit2 deficiency causes extensive viral spread throughout grey and white matter of spinal cord accompanied by impaired microglial activation and CD4+ T cell infiltration. Furthermore, infected Ifit2 deficient mice showed impaired activation of T cells in cervical lymph node and Blood-Brain-Barrier was relatively intact. Ifit2 deficient mice developed viral induced severe chronic neuroinflammatory demyelination accompanied by the presence of ameboid shaped phagocytotic microglia/macrophages.

neuroscience↗

bioRxiv2022

Anticodon Table of the Chloroplast Genome and Identification of Putative Quadruplet Anticodons in Chloroplast tRNAs

The chloroplast genome of 5959 species was analyzed to construct the anticodon table of the chloroplast genome. Analysis of the chloroplast transfer ribonucleic acid (tRNA) revealed the presence of a putative quadruplet anticodon containing tRNAs in the chloroplast genome. The tRNAs with putative quadruplet anticodons were UAUG, UGGG, AUAA, GCUA, and GUUA, where the GUUA anticodon putatively encoded tRNAAsn. The study also revealed the complete absence of tRNA genes containing ACU, CUG, GCG, CUC, CCC, and CGG anticodons in the chloroplast genome from the species studied so far. The chloroplast genome was also found to encode tRNAs encoding N-formylmethionine (fMet), Ile2, selenocysteine, and pyrrolysine. The chloroplast genomes of mycoparasitic and heterotrophic plants have had heavy losses of tRNA genes. Furthermore, the chloroplast genome was also found to encode putative spacer tRNA, tRNA fragments (tRFs), tRNA-derived, stress-induced RNA (tiRNAs), and group I introns. An evolutionary analysis revealed that chloroplast tRNAs had evolved via multiple common ancestors and the GC% had more influence toward encoding the tRNA number in the chloroplast genome compared to the genome size.

genomics↗

bioRxiv2022

Do amplifiers of selection maximise average fitness?

Evolutionary dynamics on graphs has remarkable features: For example, it has been shown that amplifiers of selection exist that - compared to an unstructured population - increase the fixation probability of advantageous mutations, while they decrease the fixation probability of disadvantageous mutations. So far, the theoretical literature has focused on the case of a single mutant entering a graph structured population, asking how the graph affects the probability that a mutant takes over a population and the time until this typically happens. For continuously evolving systems, the more relevant case is when mutants constantly arise in an evolving population. Typically, such mutations occur with a small probability during reproduction events. We thus focus on the low mutation rate limit. The probability distribution for the fitness in this process converges to a steady-state at long times. Intuitively, amplifiers of selection are expected to increase the populations mean fitness in the steady-state. Similarly, suppressors of selection are expected to decrease the populations mean fitness in the steady-state. However, we show that another category of graphs, called suppressor of fixation, can attain the highest population mean fitness. The key reason behind this is their ability to efficiently reject deleterious mutants. This illustrates the importance of the deleterious mutant regime for the long-term evolutionary dynamics, something that seems to have been overlooked in the literature so far.

evolutionary biology↗

bioRxiv2022

The aggregation potential of Zika virus proteome

The ability of human encoded soluble proteins to convert into amyloid fibrils is now recognized as a generic phenomenon in several human illnesses. Typically, such disease causal proteins/peptides consist of aggregation-prone regions (APR) that make them susceptible to misfolding and assemble into highly ordered {beta}-sheet rich fibrils, distinct from their native soluble state. Here, we show that the zika virus (ZIKV) consists of several such aggregation prone hotspots spread across its entire proteome. Using a combination of high-accuracy prediction tools, we identified APRs in both structural and non-structural proteins of ZIKV. Furthermore, we have experimentally validated the bioinformatic results by subjecting the ZIKV proteins and peptides to artificial aggregation inducing environment. Using a combination of dye-based assays (ThT and ANS) and microscopy techniques (HR-TEM and AFM), we further characterized the morphological features of amyloid-like fibrils. We found that Envelope domain III (EDIII) protein, NS1 {beta}-roll peptide, membrane-embedded signal peptide 2K, and cytosolic region of NS4B protein to be highly aggregating in the experimental setup. Our findings also pave the way for an extensive and detailed functional analysis of these predicted APRs in the future to enhance our understanding of the role played by amyloids in the pathogenesis of flavivirus. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/485915v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1bedf4dorg.highwire.dtl.DTLVardef@364676org.highwire.dtl.DTLVardef@14bf54aorg.highwire.dtl.DTLVardef@1d4fc19_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Transcriptome-wide Analyses of Adipose Tissue in Outbred Rats Reveal Genetic Regulatory Mechanisms Relevant for Human Obesity

Transcriptomic analysis in metabolically active tissues allows a systems genetics approach to identify causal genes and networks involved in metabolic disease. Outbred heterogeneous stock (HS) rats are used for genetic mapping of complex traits, but to-date, a systems genetics analysis of metabolic tissues has not been done. We investigated whether adiposity-associated genes and gene co-expression networks in outbred heterogeneous stock (HS) rats overlap those found in humans. We analyzed RNAseq data from adipose tissue of 415 male HS rats, correlated these transcripts with body weight (BW) and compared transcriptome signatures to two human cohorts: the African American Genetics of Metabolism and Expression and Metabolic Syndrome in Men. We used weighted gene co-expression network analysis to identify adiposity-associated gene networks and mediation analysis to identify genes under genetic control whose expression drives adiposity. We identified 554 orthologous "consensus genes" whose expression correlates with BW in the rat and with body mass index (BMI) in both human cohorts. Consensus genes fell within eight co-expressed networks and were enriched for genes involved in immune system function, cell growth, extracellular matrix organization and lipid metabolic processes. We identified 19 consensus genes for which genetic variation may influence BW via their expression, including those involved in lipolysis (e.g., Hcar1), inflammation (e.g., Rgs1), adipogenesis (e.g., Tmem120b) or no previously known role in obesity (e.g., St14, Msa4a6). Strong concordance between HS rat and human BW/BMI associated transcripts demonstrates translational utility of the rat model, while identification of novel genes expands our knowledge of the genetics underlying obesity.

genomics↗

bioRxiv2022

Tissue-specific and repeat length-dependent somatic instability of the X-linked dystonia parkinsonism-associated CCCTCT repeat

X-linked dystonia-parkinsonism (XDP) is a progressive adult-onset neurodegenerative disorder caused by insertion of a SINE-VNTR-Alu (SVA) retrotransposon in the TAF1 gene. The SVA retrotransposon contains a CCCTCT hexameric repeat tract of variable length, whose length is inversely correlated with age at onset. This places XDP in a broader class of repeat expansion diseases, characterized by the instability of their causative repeat mutations. Here, we observe similar inverse correlations between CCCTCT repeat length with age at onset and age at death and no obvious correlation with disease duration. To gain insight into repeat instability in XDP we performed comprehensive quantitative analyses of somatic instability of the XDP CCCTCT repeat in blood and in seventeen brain regions from affected males. Our findings reveal repeat length-dependent and expansion-based instability of the XDP CCCTCT repeat, with greater levels of expansion in brain than in blood. The brain exhibits regional-specific patterns of instability that are broadly similar across individuals, with cerebellum exhibiting low instability and cortical regions exhibiting relatively high instability. The spectrum of somatic instability in the brain includes a high proportion of moderate repeat length changes of up to 5 repeats, as well as expansions of [~]20->100 repeats and contractions of [~]20-40 repeats at lower frequencies. Comparison with HTT CAG repeat instability in postmortem Huntingtons disease brains reveals similar brain region-specific profiles, indicating common trans-acting factors that contribute to the instability of both repeats. Analyses in XDP brains of expansion of a different SVA-associated CCCTCT located in the LIPG gene, and not known to be disease-associated, reveals repeat length-dependent expansion at overall lower levels relative to the XDP CCCTCT repeat, suggesting that expansion propensity may be modified by local chromatin structure. Together, the data support a role for repeat length-dependent somatic expansion in the process(es) driving the onset of XDP and prompt further investigation into repeat dynamics and the relationship to disease.

genetics↗

bioRxiv2022

The CRISPR-Cas System differentially regulates surface-attached and pellicle-biofilm in Salmonella enterica serovar Typhimurium

CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) system has been studied for its role in biofilm regulation and expression of outer membrane proteins in Salmonella. We investigated the CRISPR-Cas mediated biofilm regulation in Salmonella enterica serovar Typhimurium by deleting CRISPR-Cas components,{Delta} crisprI,{Delta} crisprII, {Delta}{Delta}crisprI crisprII, and{Delta} cas op. We determined that the system positively regulates surface-biofilm while inhibiting pellicle-biofilm. In knockout strains, flagellar (fliC, flgK) and curli (csgA) genes were repressed, causing reduced surface-biofilm. Conversely, they displayed altered pellicle-biofilm architecture possessing bacterial multilayers and a denser ECM with enhanced cellulose and lesser Curli, ergo weaker pellicle. The intracellular cellulose concentration was less in the knockout strains due to upregulation of bcsC, necessary for cellulose export. We hypothesized that exported cellulose integrates into the pellicle. We determined that crp is upregulated in the knockout strains, thereby inhibiting the expression of csgD, hence csgA and bcsA. The conflicting upregulation of bcsC, the last gene of bcsABZC operon, could be independently regulated by the CRISPR-Cas system owing to a partial match between the CRISPR-spacers and bcsC gene. The CRP-mediated regulation of the flagellar genes in the knockout strains was probably circumvented through the regulation of Yddx governing the availability of {sigma}28 factor that further regulates class3 flagellar genes (fliC, flgK). Additionally, the variations in the LPS profile and expression of LPS-related genes (rfaC, rfbG, rfbI) in the knockout strains could also contribute to the altered pellicle architecture. Collectively, we establish that the CRISPR-Cas system differentially regulates the formation of surface-attached and pellicle-biofilm.

microbiology↗

bioRxiv2022

Polyfunctional IL-17A+ MAIT cells are expanded in the peripheral blood of patients with HLA-B27+ axial spondyloarthritis

ObjectivesStudies in axial spondyloarthritis (axSpA) have yielded conflicting results regarding the identity of the major IL-17A-producing lymphocyte populations. The goal of this study was to comprehensively assess the production of IL-17A and related cytokines by peripheral blood lymphocytes in axSpA. MethodsPeripheral blood mononuclear cells were isolated from patients with axSpA and healthy controls matched for age, sex and HLA-B27 status. Unstimulated cells and cells activated with PMA/Ionomycin were analyzed by 25-parameter fluorescent flow cytometry. Data were analyzed by hierarchical gating, UMAP and SPICE. ResultsExcept for a reduced frequency of mucosal-associated invariant T (MAIT) cells and natural killer (NK) cells, there were no other significant differences in abundance of major lymphocyte populations in axSpA patients compared with controls. Increased IL-17A production in axSpA was observed in total non-B lymphocytes and in MAIT cells. The fraction of MAIT cells expressing the tissue residency markers CD69 and CD103 was increased in axSpA. CD103 positive MAIT cells were enriched for IL-17A producers. axSpA patients demonstrated an expansion of MAIT cell subsets producing IL-17A, IL-17F, GM-CSF and TNF. This expansion was only observed in HLA-B27+ patients. ConclusionsWe document an expansion of polyfunctional IL-17A+ MAIT cells in the peripheral blood of HLA-B27+ patients with axSpA. These results are consistent with the implied role of intestinal dysbiosis or inflammation in axSpA pathogenesis. Key messagesWhat is already known about this subject? O_LIVarious IL-17A-producing lymphocyte populations have been implicated in the pathogenesis of axSpA. C_LI What does this study add? O_LIPolyfunctional MAIT cells capable of producing IL-17A, IL-17F, GM-CSF and TNF are expanded in the peripheral blood of HLA-B27+ patients with axSpA. C_LI How might this impact on clinical practice or future developments? O_LIOverproduction of IL-17A by MAIT cells is the most consistent finding of peripheral blood lymphocyte studies in axSpA. C_LIO_LIOur data support the pathogenetic link between intestinal and axial inflammation in axSpA. C_LI

immunology↗

bioRxiv2021

Finger stability in precision grips

Stable precision grips using the fingertips are a cornerstone of human hand dexterity. Occasionally, however, our fingers become unstable and snap into a hyper-extended posture. This is because multi-link mechanisms, like our fingers, can buckle under tip forces. Suppressing this instability is crucial for hand dexterity, but how the neuromuscular system does so is unknown. Here we show that finger stability is due to the stiffness from muscle contraction and likely not feedback control. We recorded maximal force application with the index finger and found that most buckling events lasted less than 50ms, too fast for sensorimotor feedback to act. However, a biomechanical model of the finger predicted that muscle-induced stiffness is also insufficient for stability at maximal force unless we add springs to stiffen the joints. We tested this prediction in 39 volunteers. Upon adding stiffness, maximal force increased by 34{+/-}3%, and muscle electromyography readings were 21{+/-}3% higher for the finger flexors (mean{+/-}standard error). Hence, people refrain from applying truly maximal force unless an external stabilizing stiffness allows their muscles to apply higher force without losing stability. Muscle recordings and mathematical modeling show that the splint offloads the demand for muscle co-contraction and this reduced co-contraction with the splint underlies the increase in force. But more stiffness is not always better. Stiff fingers would interfere the ability to passively adapt to complex object geometries and precisely regulate force. Thus, our results show how hand function arises from neurally tuned muscle stiffness that balances finger stability with compliance.

neuroscience↗

bioRxiv2021

Peptide-antibody Fusions Engineered by Phage Display Exhibit Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants

The COVID-19 pandemic has been exacerbated by the emergence of variants of concern (VoCs). Many VoC mutations are found in the viral spike protein (S-protein), and are thus implicated in host infection and response to therapeutics. Bivalent neutralizing antibodies (nAbs) targeting the S-protein receptor-binding domain (RBD) are promising therapeutics for COVID-19, but are limited due to low potency and vulnerability to RBD mutations found in VoCs. To address these issues, we used naive phage-displayed peptide libraries to isolate and optimize 16-residue peptides that bind to the RBD or the N-terminal domain (NTD) of the S-protein. We fused these peptides to the N-terminus of a moderate affinity nAb to generate tetravalent peptide-IgG fusions, and showed that both classes of peptides were able to improve affinities for the S-protein trimer by >100-fold (apparent KD < 1 pM). Critically, cell-based infection assays with a panel of six SARS-CoV-2 variants demonstrate that an RBD-binding peptide was able to enhance the neutralization potency of a high-affinity nAb >100-fold. Moreover, this peptide-IgG was able to neutralize variants that were resistant to the same nAb in the bivalent IgG format. To show that this approach is general, we fused the same peptide to a clinically approved nAb drug, and showed that it rescued neutralization against a resistant variant. Taken together, these results establish minimal peptide fusions as a modular means to greatly enhance affinities, potencies, and breadth of coverage of nAbs as therapeutics for SARS-CoV-2.

synthetic biology↗

bioRxiv2021

Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants by Modular, Tetravalent, Bi-paratopic Antibodies

Neutralizing antibodies (nAbs) that target the SARS-CoV-2 spike protein are approved for treatment of COVID-19. However, with the emergence of variants of concern, there is a need for new treatment options. We report a novel format that enables modular assembly of bi-paratopic, tetravalent nAbs with antigen-binding sites from two distinct nAbs. The tetravalent nAb was purified in high yield, and it exhibited biophysical characteristics that were comparable to those of approved therapeutic antibodies. The tetravalent nAb bound to the spike protein trimer at least 100-fold more tightly than bivalent IgGs (apparent KD < 1 pM), and it exhibited extremely high potencies against a broad array of pseudoviruses, chimeric viruses, and authentic virus variants. Together, these results establish the tetravalent diabody-Fc-Fab as a robust, modular platform for rapid production of drug-grade nAbs with potencies and breadth of coverage that greatly exceed those of conventional bivalent IgGs.

bioengineering↗

bioRxiv2021

Longitudinal characterization of circulating neutrophils uncovers distinct phenotypes associated with disease severity in hospitalized COVID-19 patients

Multiple studies have identified an association between neutrophils and COVID-19 disease severity; however, the mechanistic basis of this association remains incompletely understood. Here we collected 781 longitudinal blood samples from 306 hospitalized COVID-19+ patients, 78 COVID-19- acute respiratory distress syndrome patients, and 8 healthy controls, and performed bulk RNA-sequencing of enriched neutrophils, plasma proteomics, cfDNA measurements and high throughput antibody profiling assays to investigate the relationship between neutrophil states and disease severity or death. We identified dynamic switches between six distinct neutrophil subtypes using non-negative matrix factorization (NMF) clustering. At days 3 and 7 post-hospitalization, patients with severe disease had an enrichment of a granulocytic myeloid derived suppressor cell-like state gene expression signature, while non-severe patients with resolved disease were enriched for a progenitor-like immature neutrophil state signature. Severe disease was associated with gene sets related to neutrophil degranulation, neutrophil extracellular trap (NET) signatures, distinct metabolic signatures, and enhanced neutrophil activation and generation of reactive oxygen species (ROS). We found that the majority of patients had a transient interferon-stimulated gene signature upon presentation to the emergency department (ED) defined here as Day 0, regardless of disease severity, which persisted only in patients who subsequently died. Humoral responses were identified as potential drivers of neutrophil effector functions, as enhanced antibody-dependent neutrophil phagocytosis and reduced NETosis was associated with elevated SARS-CoV-2-specific IgG1-to-IgA1 ratios in plasma of severe patients who survived. In vitro experiments confirmed that while patient-derived IgG antibodies mostly drove neutrophil phagocytosis and ROS production in healthy donor neutrophils, patient-derived IgA antibodies induced a predominant NETosis response. Overall, our study demonstrates neutrophil dysregulation in severe COVID-19 and a potential role for IgA-dominant responses in driving neutrophil effector functions in severe disease and mortality.

immunology↗

bioRxiv2021

RAD51AP1 compensates for the loss of RAD54 in homology-directed DNA repair

Homologous recombination (HR) is a complex DNA damage repair pathway and an attractive target of inhibition in anti-cancer therapy. To help guide the development of efficient HR inhibitors, it is critical to identify compensatory sub-pathways. In this study, we describe a novel synthetic interaction between RAD51AP1 and RAD54, two structurally unrelated proteins that function downstream of the RAD51 recombinase in HR. We show that deletion of both RAD51AP1 and RAD54 synergistically sensitizes human cancer cell lines to treatment with a Poly(adenosine 5 s-diphosphate-ribose) polymerase inhibitor, to the DNA inter-strand crosslinking agent mitomycin C, and to hydroxyurea, which stalls the progression of DNA replication forks. We infer that HR-directed anti-cancer treatment modalities shall consider this intra-pathway functional overlap, and we hypothesize that in cancerous cells the simultaneous inactivation of both RAD54 and RAD51AP1 will accentuate tumor kill.

cell biology↗