Biology subjects

Kundu, R.

Publications and source records attributed to Kundu, R..

At least 19 recordsLinked to original sources

bioRxiv2026

The Genome In A Bottle HG002 assembly-based variant benchmark set enables comprehensive benchmarking of small and structural variants.

High-accuracy, high-resolution benchmark data are fundamental to improving the accuracy and generalizability of sequencing technologies and associated algorithms. Most existing benchmarks for DNA sequencing rely on mapping to a single linear reference, omitting inaccessible and highly repetitive complex genomic regions. Here, we present the Genome in a Bottle (GIAB) Consortium's HG002 v5.0q benchmark set, a more comprehensive variant benchmark derived from a fully phased, highly curated, and accurate diploid assembly of HG002. Our approach leverages recent advances in long-read sequencing and assembly to provide confident small- and structural-variant calls across previously inaccessible regions, including segmental duplications, homopolymers, tandem repeats, and complex immune loci. The v5.0 benchmark significantly expands the callable genome relative to v4.2.1, adding x200 Mbp of sequence and increasing variant detection by 18 % for small variants and 3-fold for structural variants compared to previous benchmarks. The benchmark regions include more variants in repetitive regions, including 1.2 x segmental duplications, 1.8 x tandem repeats, and 2.0 x homopolymers, compared to v4.2.1. Separate and combined small- and structural-variant benchmarks are available for three versions of the human reference genome (GRCh37, GRCh38, and CHM13). We validate the reliability of this benchmark set by comparing multiple sequencing technologies and variant calling pipelines and curating differences. The v5.0q benchmark provides a critical resource for the development and validation of next-generation sequencing technologies and analysis methods.

genomics↗

bioRxiv2026

Bistability to Quad-stability: Emergence of Hybrid Phenotypes & Enhanced Spatio-temporal Plasticity in Presence of Host-Circuit Coupling

In the context of multistability-driven diseases, like cancer, spatio-temporal plasticity plays a significant role to achieve a spectrum of phenotypic variations. The interplay between gene regulatory networks and environmental factors, such as resource competition and spatial diffusion, plays a crucial role in determining cellular behaviour and phenotypic heterogeneity. Though reaction-diffusion frameworks have been widely applied in developmental biology, less attention has been paid to the simultaneous effects of resource competition and growth feedback on spatial organization. In this paper, we observed that a bistable genetic circuit under high resource competition due to growth feedback gives rise to multiple emergent phenotypes, as observed in cancer systems. Furthermore, we observed how spatial diffusion coupled with intrinsic nonlinearity can drive the emergence of distinct spatial dynamics over time. The observed spatiotemporal plasticity can also be driven by the comparative stability of the fixed points, diffusivity, and asymmetry of diffusion. Our findings highlight that growth-induced resource competition combined with diffusion can provide deeper insights into metastasis and cancer progression.

cancer biology↗

bioRxiv2026

Spatial pattern formation as a consequence of Protease Competition

Exploring the emergence of spatio-temporal patterns due to nonlinearities in gene expression is a relatively new development. In this work, we explore the effect of resource constraint on gene regulatory motif from both equilibrium and spatio-temporal standpoint, taking into consideration the degradation class of resource, protease. We have demonstrated that protease-tagged degradation can cause an emergent bistability to form in the system in a steady-state scenario. Instead of a graded linear response in protein synthesis, two Saddle-node bifurcations caused by protease competition provide a switch-like response with hysteresis, where two drastically differing protein concentrations can coexist. We next turn our attention to spatio-temporal analysis: we extend our study for a two-dimensional sheet of cells with diffusible protein molecules and report the stationary patterns.To investigate the reasons behind these non-homogeneous stationary patterns, we investigate the traveling wave solution and observe that a stationary pattern is formed by the traveling wave solution. Considering that proteases play a major role in the regulation and expression of genes in a variety of diseased scenarios, the repercussions of this spatial patterning caused by protease competition can be extensive in gene regulatory systems.

synthetic biology↗

bioRxiv2025

Chronic Electronic Cigarette Exposure Promotes Atherosclerosis and Chondrogenic Modulation of Smooth Muscle Cells

BackgroundElectronic cigarette (E-cig) use has reached epidemic proportions worldwide, yet its cardiovascular consequences remain poorly defined. While several lines of evidences in human epidemiological and animal studies suggest chronic aerosol exposure accelerates atherosclerosis; the cellular and molecular mechanisms underlying this pathological remain unknown. MethodsWe exposed hyperlipidemic mice to chronic e-cigarette aerosol inhalation and characterized the plaque cellular landscape by coupling SMC lineage tracing with single-cell transcriptomic/epigenomic profiling and histologic phenotyping. We subsequently leveraged human coronary artery smooth muscle cells (HCASMCs) to validate in vivo discovery and identified E-cig specific pathological signaling pathways relevant to human vascular disease risk. ResultsChronic E-cig aerosol exposure accelerated atherosclerosis, increasing both SMC phenotypic modulation and plaque macrophage burden in a lipid-independent manner. Transcriptomically, SMCs are particularly more sensitive to E-cig than other vascular cell types. E-cig exposure reprogrammed SMCs toward a pro-calcifying, chondrogenic phenotype, thereby enhancing vascular ossification in vivo and in HCASMCs in vitro. Mechanistically, E-cig mediated SMC fate alteration occurs through activation of a glutamatergic/NMDAR signaling program, that increased NMDAR-dependent Ca2+ influx in a GRIN2A dependent manner. Notably, inhibition of GRIN2A mediated signaling reversed E-cig-induced pathological shifts in SMC phenotype. ConclusionsSMC chondrogenic reprogramming and subsequent vascular calcification are central to the detrimental cardiovascular consequences of E-cig exposure. Our findings implicate a GRIN2A-dependent glutamatergic/NMDAR signaling axis in SMC as a primary driver of this calcifying remodeling program. These findings define a SMC-specific vulnerability to E-cig aerosols and establish the GRIN2A/NMDAR pathway as potential therapeutic targets for mitigating E-cig-associated cardiovascular disease.

genomics↗

bioRxiv2025

BLASE: Bulk Linkage Analysis for Single Cell Experiments - Teasing Out the Secrets of Bulk Transcriptomics with Trajectory Analysis

1MotivationscRNA-seq experiments can capture cell process trajectories. Bulk RNA-seq is more practical, however does not have the granularity to elucidate cell-type specific trajectories. Deconvolution methods can estimate cell-types in RNA-seq data, but there is a need for methods characterising their pseudotime. ResultsWe show that our method, BLASE, can identify the progress of an RNA-seq sample through a trajectory in a scRNA-seq reference. ConclusionBLASE can be used to a) annotate scRNA-seq data from existing RNA-seq, b) identify progress of RNA-seq data through a process based on scRNA-seq data, and c) be used to correct developmental differences in RNA-seq differential expression analysis.

bioinformatics↗

bioRxiv2025

Vascular smooth muscle cell atherosclerosis trajectories characterized at single cell resolution identify causal transcriptomic and epigenomic mechanisms of disease risk

Vascular smooth muscle cells (SMC) contribute to heritable coronary artery disease (CAD) risk and undergo complex cell state transitions to multiple disease related phenotypes. To investigate the genetic basis of SMC state trajectories that underlie the SMC component of CAD causality we have developed a dense timecourse single cell transcriptomic and epigenetic map of atherosclerosis in a murine disease animal model. Cellular trajectories were derived from the temporal data and probabilistic fate modeling with Waddington-Optimal Transport (WOT). We created transcription factor (TF) centered regulons mapped across the developmental timeline and through network-based prioritization with WOT predicted TFs and in silico TF perturbation, identified key drivers of cell state changes associated with EMT, vascular development, and circadian clock functions. Integration of mouse disease data with human CAD genetic findings identified transition SMC phenotypes that mediate disease risk and point to causal disease mechanisms. Parallel studies using knockout of the validated CAD gene Tcf21 revealed its impact on SMC transition cellular phenotypes and disease risk genes, due in part to a role regulating the transition of SMC precursor cells in the secondary heart field. Together, these studies characterize atherosclerosis trajectories at single cell resolution and identify genetic causal transcriptomic and epigenomic mechanisms of CAD risk.

genomics↗

bioRxiv2024

Super Resolved Structural Imaging of Mitochondrial Network using Orange Emissive Carbon Nanodot as Fluorescent Probe

Super-resolution microscopy (SRM), coupled with appropriate bright and photostable fluorescent probes, has revolutionized the ability to study organelle dynamics with unprecedented spatial and temporal resolution. An increasing trend of designing nanomaterial probes that has unprecedented advantages over the organic molecular probes has become the frontier in SRM based imaging of subcellular organelles. Herein, we report the development of orange-emissive fluorescent carbon nanodots (CNDs) via a one-pot synthesis that has excellent capabilities to target mitochondria. Spectroscopic analysis confirms the presence of guanidine on the surface of CNDs, thus facilitating its ability to selectively target mitochondria. The CNDs were highly capable for the super resolution radial fluctuation (SRRF) imaging of mitochondrial network and the morphology. The synthesized CNDs exhibited excellent photoluminescent properties along with high biocompatibility and non-toxicity, which could be used for their application in mitochondria-based imaging modalities.

biochemistry↗

bioRxiv2024

Activation of IL1A/IRAK1 axis and downstream proinflammatory signaling in healthy adult and neonatal African American skin

Differences in prevalence of inflammatory skin diseases including atopic dermatitis and psoriasis in African American (AA) versus White Non-Hispanic (WNH) population are well recognized. However, the underlying mechanisms are largely unknown. We previously observed significant differences in healthy AA skin transcriptome with differentially expressed genes (DEG) enriched for inflammation and cornification processes. Here we analyzed proteome in skin biopsies from healthy AA and WNH volunteers using Olink(R) Explore Inflammation 384 biomarker panel. Among proteins with higher expression in AA skin were IRAK1, IL1A, IL4, IL22RA1. IL1A binding to IL1R1 receptor is known to result in recruitment of adapter molecules such as IRAK1, and activation of downstream NF-{kappa}B and MAPK signaling. We confirmed NF-{kappa}B and ERK1/2 activation in AA skin by Western blot analysis of their phosphorylation at specific activating sites. Importantly, we observed similar differences between AA and WNH neonatal foreskin and between AA and WNH 3D skin organoids. Further analysis of DEG promoters by Gene Transcription Regulation Database (GTRD) pointed to NF-{kappa}B and AP1 as key transcription factors involved in AA DEG regulation. Overall, proinflammatory signaling in healthy AA skin starting early in childhood may contribute to the increased risk of certain inflammatory skin diseases within the AA population.

immunology↗

bioRxiv2024

Carbon nanodots as a red emissive fluorescent probe for the super-resolution microscopy of DNA dynamics during paclitaxel treatment

Paclitaxel is a commonly used frontline chemotherapeutic drug for cancer treatment. It is known to be functional by arresting the microtubule disassembly during mitosis. Recently, a non-mitotic pathway has been evolving and thus contemplating the mitotic mechanism. Here, using super-resolution microscopy (SRM), we directly visualized the nuclear dynamics and unveiled the mechanism of paclitaxel treatment. A new class of non-toxic, biocompatible and highly fluorescent carbon nanodots (CNDs) were used as a fluorescent probe that were highly capable to directly stain the nuclear DNA, capture the SRM imaging of chromosome and the chromatin structures. Apart from SRM imaging of chromosomes during all stages of normal mitotic cell division, CNDs successfully visualized the formation of lagging, mis-segregated and bridging chromosomes which leads to the multi-micronucleus formation upon paclitaxel treatment. A detailed chromatin remodelling analysis suggested that heterochromatin played an important role in the formation of condensed multi-micronucleus, ultimately leading to cell death. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/620846v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1d5bb12org.highwire.dtl.DTLVardef@ecd313org.highwire.dtl.DTLVardef@15bb3corg.highwire.dtl.DTLVardef@b79273_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Aryl-hydrocarbon receptor in smooth muscle cells protect against dioxin induced adverse remodeling of atherosclerosis

IntroductionEnvironmental exposure to dioxin has been linked to increased myocardial infarction. Smooth muscle cells (SMC) in the coronary vasculature play a critical role in atherosclerotic plaque remodeling due to their phenotypic plasticity, however, the detailed mechanism linking dioxin exposure to adverse SMC modulation is not well understood. MethodsSingle-cell RNA and ATAC sequencing and histological analyses were performed on the aorta from mouse models of atherosclerosis exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) or control. Primary human coronary artery SMC (HCASMC) treated in culture with TCDD were used to perform RNA-Seq, ATAC-Seq, and functional phenotypic assays. ChIP-Seq was performed with antibodies against Aryl-hydrocarbon receptor (AHR) and TCF21, two of known SMC modulating transcription factors. ResultsModulated SMC were the most transcriptionally responsive cell type to dioxin in the atherosclerotic aorta. Dioxin accelerated disease phenotype by promoting a modulated SMC phenotype early, resulting in increased lesion size, migration of SMC, and macrophage recruitment to the lesion. We found C3 expressing modulated SMCs to be likely contributing to the increased macrophage recruitment and inflammation. Analysis of the RNA-Seq data from HCASMC treated with TCDD showed differential enrichment of biological pathways related to cell migration, localization, and inflammation. Furthermore, ATAC-Seq data showed a significant activation for pathways regulating vascular development, cell migration, inflammation, and apoptosis. With TCDD treatment, there was also enrichment of AHR ChIP-Seq peaks, while the TCF21 enrichment decreased significantly. The SMC-specific Ahr knockout resulted in increased oxidative stress in SMC, increased lesion size and macrophage content, and loss of SMC lineage cells in the lesion cap when exposed to TCDD, consistent with a more vulnerable plaque phenotype. ConclusionDioxin adversely remodels atherosclerotic plaque by accelerating the SMC- phenotypic modulation, and increasing inflammation and oxidative stress resulting in increased macrophage recruitment and lesion size. Dioxin may adversely affect the SMC phenotype and disease state by affecting the TCF21 occupancy in the open chromatin regions. Furthermore, we observed that SMC-specific deletion of Ahr in mice resulted in worsening of dioxin mediated SMC modulation and atherosclerosis, suggesting that Ahr in SMC confers protection against dioxin by promoting a stable plaque phenotype and reducing dioxin induced oxidative stress. SummaryO_LIExposure to dioxin, an environmental pollutant present in tobacco smoke and air pollution, accelerates smooth muscle cell modulation, and atherosclerosis. C_LIO_LIDioxin exposure leads to inflammatory smooth muscle cell phenotype characterized by complement pathway activation and increased macrophage recruitment to plaque C_LIO_LIAryl-hydrocarbon receptor in SMC protects against oxidative stress, and promotes a stable plaque phenotype C_LI

molecular biology↗

bioRxiv2024

Direct measurement of PIP2 densities in biological membranes using a peptide-based sensor

Organization and composition of the plasma membrane are important modulators of many cellular programs. Phosphatidylinositol phosphate (PIP) lipids are low abundance membrane constituents with different arrangements of phosphate groups around an inositol head group that regulate a large number of signaling pathways. Many strategies have been developed to detect and track PIP species to monitor their clustering, mobility, and interaction with binding partners. We implement a peptide-based ratiometric sensor for the detection of PI(4,5)P2 lipids in reconstituted membrane systems that permit absolute quantification of PI(4,5)P2 densities down to physiological levels. The sensor is membrane permeable and easily applicable to measurements in living cells. Application of calibrated sensors to cells expressing common mutations in the small GTPase, Ras, showed a reshaping of surface PI(4,5)P2 levels and distributions in a mutation-specific manner. The rapid implementation of this quantitative sensing strategy to cellular studies of cellular signaling, membrane organization and dynamics should be broadly applicable.

biochemistry↗

bioRxiv2024

A cell and transcriptome atlas of the human arterial vasculature

Contiguous arterial segments show different propensities for different vascular pathologies, yet mechanisms explaining these fundamental differences remain unknown. We sought to build a transcriptomic, cellular, and spatial atlas of human arterial cells across multiple different arterial segments to understand these underlying differences. Analysis of multiple isogenic arterial segments from healthy donors reveals a significant stereotyped pattern of cell type-specific segmental heterogeneity in healthy arteries. Combining single cell analysis with spatial transcriptomic data reveals cellular heterogeneity not captured by commonly used cell-type marker genes. Determinants of arterial transcriptomic identities are predominantly encoded in fibroblasts and smooth muscle cells (SMC), and their differentially expressed genes are particularly enriched for different vascular disease-associated genetic risk- loci and risk-genes. Adventitial fibroblast-specific heterogeneity in gene expression coincides with a disproportionally large number of vascular disease genetic signals, suggesting a previously unrecognized role for this cell type in disease risk. Adult arterial cells from different segments cluster not by anatomical proximity, but by embryonic origin. Global regulon analysis of disease related segment-specific gene expression program in fibroblast and SMC enriches for binding sites of transcription factors that are developmental master regulators whose expression persists into adulthood, suggesting an important functional role of the same developmental master regulators in adult gene expression and disease. Lastly, non-coding transcriptomes across arterial cells contain extensive variation in lncRNAs expressed in cell type- and segment-specific patterns, rivaling heterogeneity in protein coding transcriptomes. Differentially expressed LncRNA demonstrate enrichment for non-coding genetic signals for vascular diseases, suggesting a potential global role of segmental specific LncRNAs in regulating inherited human vascular disease risk.

cell biology↗

bioRxiv2024

Smooth muscle expression of RNA editing enzyme ADAR1 controls vascular integrity and progression of atherosclerosis

Mapping the genomic architecture of complex disease has been predicated on the understanding that genetic variants influence disease risk through modifying gene expression. However, recent discoveries have revealed that a significant burden of disease heritability in common autoinflammatory disorders and coronary artery disease (CAD) is mediated through genetic variation modifying post-transcriptional modification of RNA through adenosine-to-inosine (A-to-I) RNA editing. This common RNA modification is catalyzed by ADAR enzymes, where ADAR1 edits specific immunogenic double stranded RNA (dsRNA) to prevent activation of the double strand RNA (dsRNA) sensor MDA5 (IFIH1) and stimulation of an interferon stimulated gene (ISG) response. Multiple lines of human genetic data indicate impaired RNA editing and increased dsRNA sensing by MDA5 to be an important mechanism of CAD risk. Here, we provide a crucial link between observations in human genetics and mechanistic cell biology leading to progression of CAD. Through analysis of human atherosclerotic plaque and culture of human coronary artery vascular smooth muscle cells (SMCs) we implicate the SMC to have a distinct requirement for RNA editing, and that MDA5 activation regulates SMC phenotypic modulation. Through generation of a conditional SMC specific Adar1 deletion mouse model on a pro-atherosclerosis background with additional constitutive deletion of MDA5 (Ifih1), and with incorporation of single cell RNA sequencing cellular profiling, we further show that Adar1 controls SMC phenotypic state by regulating Mda5 activation, is required to maintain vascular integrity, and controls progression of atherosclerosis and vascular calcification. Finally, we further corroborate our findings in a large human carotid endarterectomy dataset (Athero-Express) where we show that ISG activation is strongly associated with decreased plaque stability, increased SMC phenotypic modulation, and increased plaque calcification. Through this work, we describe a fundamental mechanism of CAD, where cell type and context specific RNA editing and sensing of dsRNA mediates disease progression, bridging our understanding of human genetics and disease causality. One Sentence SummarySmooth muscle expression of RNA editing enzyme ADAR1 regulates activation of double strand RNA sensor MDA5 in novel mechanism of atherosclerosis.

genomics↗

bioRxiv2024

A Cell-Permeable Fluorescent Probe Reveals Temporally Diverse PI(4,5)P2 Dynamics Evoked by Distinct GPCR Agonists in Neurons

Lipids, key constituents of cell-membranes, are the first responders to cell signals. At the crux of spatiotemporal dynamics of lipid-signaling response are phosphoinositides. Indeed, phosphoinositides like phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2), present in the inner-leaflet of eukaryotic cell-membranes, form the link between signal reception and downstream signal-transmission. In this backdrop, reversible fluorescent probes that can track live PI(4,5)P2 dynamics on a seconds time-scale will afford key insights into lipid-mediated signaling. However, realizing cell-permeable PI(4,5)P2-selective sensors for imaging dynamics remains a challenge due to the presence of structurally similar lipids and low levels of PI(4,5)P2. We report a computationally-designed, rapid-response, reversible, photo-stable, fluorescent sensor that permeates living cells, neurons, and a multicellular organism within few min of direct incubation and distinctly visualizes PI(4,5)P2 pools. We used the sensor to interrogate the role of PI(4,5)P2 in driving heterogeneity of signaling responses and contrasting behavioral effects that ensue upon binding of distinct ligands to the same G protein-coupled receptor. Specifically, we asked whether probing PI(4,5)P2 dynamics using our novel sensor could uncover the earliest of signaling differences evoked by hallucinogenic versus non-hallucinogenic ligands at the serotonin2A (5-HT2A) receptor. Our results reveal that a hallucinogenic ligand at the 5-HT2A receptor leads to a slower rate of PI(4,5)P2-depletion when compared to a non-hallucinogenic ligand, within the initial seconds of ligand addition, but has a sustained effect. The ability of our designer chemical probe in timing early seconds-minute timescale lipid-dynamics in living cells opens avenues for tracking early time-point molecular events in neuronal response to chemical and physical stimuli.

biochemistry↗

bioRxiv2023

Molecular mechanisms of coronary artery disease risk at the PDGFD locus

Platelet derived growth factor (PDGF) signaling has been extensively studied in the context of vascular disease, but the genetics of this pathway remain to be established. Genome wide association studies (GWAS) for coronary artery disease (CAD) have identified a risk locus at 11q22.3, and we have verified with fine mapping approaches that the regulatory variant rs2019090 and PDGFD represent the functional variant and putative functional gene. Further, FOXC1/C2 transcription factor (TF) binding at rs2019090 was found to promote PDGFD transcription through the CAD promoting allele. Employing a constitutive Pdgfd knockout allele along with SMC lineage tracing in a male atherosclerosis mouse model we mapped single cell transcriptomic, cell state, and lesion anatomical changes associated with gene loss. These studies revealed that Pdgfd promotes expansion, migration, and transition of SMC lineage cells to the chondromyocyte phenotype and vascular calcification. This is in contrast to protective CAD genes TCF21, ZEB2, and SMAD3 which we have shown to promote the fibroblast-like cell transition or perturb the pattern or extent of transition to the chondromyocyte phenotype. Further, Pdgfd expressing fibroblasts and pericytes exhibited greater expression of chemokines and leukocyte adhesion molecules, consistent with observed increased macrophage recruitment to the plaque. Despite these changes there was no effect of Pdgfd deletion on SMC contribution to the fibrous cap or overall lesion burden. These findings suggest that PDGFD mediates CAD risk through promoting SMC expansion and migration, in conjunction with deleterious phenotypic changes, and through promoting an inflammatory response that is primarily focused in the adventitia where it contributes to leukocyte trafficking to the diseased vessel wall.

genetics↗

bioRxiv2022

Identification of potential natural compound inhibitors and drug like molecules against human METTL3 by docking and molecular dynamics simulation

Nucleotide level chemical modification in transcriptome is critical in regulating different cellular processes, including cancer. The most investigated epitranscriptomic modification is methylation at the N6-position of adenosine (m6A). This dynamic modification process is carried out by: writer, reader and eraser proteins. Writers are methyltransferases, METTL3 is the major writer that works in association with METTL14, an accessory protein. Extensive study revealed that cancer progression for acute myeloid leukaemia, gastric cancer, colorectal cancer, hepatocellular carcinoma, and lung cancer is directly contributed by irregular expression of METTL3. Targeting METTL3, has opened a new window in the development of new inhibitors/drugs. In this study, 80 commercially available compounds were found from an unbiased screening by molecular docking, showing better score when compared with the existing substrate/substrate-analogue and the inhibitor bound crystal structures in terms of docking score and binding energy calculation. Among this pool of compounds, the best seven small molecules, AMF, RAD, JNJ, MEH, ECP, MHN, SGI, have been selected and further validated by different computational tools like binding energy calculation, molecular dynamics simulation etc. The novel hits found in this study can function as lead compounds which can be developed into inhibitors as well as drugs, specific against METTL3.

bioinformatics↗

bioRxiv2021

Receptor-binding domain recombinant protein RBD219-N1C1 on alum-CpG induces broad protection against SARS-CoV-2 variants of concern

We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the Corbevax vaccine that recently received emergency use authorization by the Drugs Controller General of India. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum+CpG. We also evaluated mice immunized with RBD/alum+CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum+CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B. 1.351 (Beta), B. 1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum+CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2 including variants of concern.

molecular biology↗

bioRxiv2020

Process Development and Scale-up Optimization of the SARS-CoV-2 Receptor Binding Domain-Based Vaccine Candidate, RBD219-N1C1

A SARS-CoV-2 RBD219-N1C1 (RBD219-N1C1) recombinant protein antigen formulated on Alhydrogel(R) has recently been shown to elicit a robust neutralizing antibody response against SARS-CoV-2 pseudovirus in mice. The antigen has been produced under current good manufacturing practices (cGMP) and is now in clinical testing. Here, we report on process development and scale-up optimization for upstream fermentation and downstream purification of the antigen. This includes production at the 1 and 5 L scale in the yeast, Pichia pastoris, and the comparison of three different chromatographic purification methods. This culminated in the selection of a process to produce RBD219-N1C1 with a yield of >400 mg per liter of fermentation with >92% purity and >39% target product recovery after purification. In addition, we show the results from analytical studies, including SEC-HPLC, DLS, and an ACE2 receptor binding assay that were performed to characterize the purified proteins to select the best purification process. Finally, we propose an optimized upstream fermentation and downstream purification process that generates quality RBD219-N1C1 protein antigen and is fully scalable at a low cost.

immunology↗