Biology subjects

Shin, J.

Publications and source records attributed to Shin, J..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

Long-term Immunity of a Microneedle Array Patch of SARS-CoV-2 S1 Protein Subunit Vaccine Irradiated by Gamma Rays in Mice

COVID-19 vaccines effectively prevent symptomatic infection and severe disease, including hospitalization and death. However, unequal vaccine distribution during the pandemic, especially in low- and middle-income countries, has led to the emergence of vaccine-resistant strains. This underscores the need for alternative, safe, and thermostable vaccine platforms, such as dissolved microneedle array patches (MAP) delivering a subunit vaccine, which eliminate the need for cold chain and trained healthcare personnel. This study demonstrates that the SARS-CoV-2 S1 monomer with RS09, a TLR-4 agonist peptide, serves as an optimal protein subunit immunogen. This combination stimulates a stronger S1-specific immune response, resulting in binding to the membrane-bound spike on the cell surface and ACE2-binding inhibition, compared to the monomer S1 alone or trimer S1, regardless of RS09. MAP delivery of the rS1RS09 subunit vaccine elicited higher and longer-lasting immunity compared to conventional intramuscular injection. S1-specific IgG levels remained significantly elevated for up to 70 weeks post-administration. Additionally, different doses of 5, 15, and 45 g of MAP vaccines induced robust and sustained Th2-prevalent immune responses, suggesting a dose-sparing effect and inducing significantly high neutralizing antibodies against the Wuhan, Delta, and Omicron variants at 15 and 45 g dose. Moreover, gamma irradiation as a terminal sterilization method did not significantly affect immunogenicity, with irradiated vaccines maintaining comparable efficacy to non-irradiated ones. The stability of MAP vaccines was evaluated after long-term storage at room temperature and refrigeration for 19 months, showing minimal protein degradation. Further, after an additional one-month of storage at elevated temperature (42{degrees}C), rS1RS09 in both non-irradiated and irradiated MAP degraded less than 3%, while the liquid subunit vaccine degraded over 23%. Overall, these results indicate that gamma irradiation sterilized MAP-rS1RS09 vaccines maintain stability during extended storage without refrigeration, supporting their potential for mass production and widespread use in global vaccination efforts.

microbiology↗

bioRxiv2024

Tau condensation on DNA and localization on centromeres: A potential link to cell division

Tau protein, traditionally recognized for stabilizing microtubules and forming pathological aggregates, has recently been observed to form condensates in various contexts. While its condensation with RNA has been well studied, the interaction between tau and DNA, along with its biological significance, remains less explored. Here, using single-molecule experiments, we found that tau binds stably to naked DNA at nanomolar concentrations, leading to the local co- condensation of tau and DNA. These tau condensates on DNA can also interface with microtubules, leveraging taus known role in promoting microtubule growth and organization. The dynamic nature of these condensates facilitates the remodeling of the DNA-microtubule assembly. Interestingly, two phosphomimetic tau mutants, T231D/S235D and S262D, retained their affinity for DNA but differed in their ability to link microtubules to DNA. Furthermore, imaging of HEK-293 and SH-SY5Y cells in early mitosis revealed that tau localizes on centromeres, poised to capture nascent mitotic spindles. Building on these observations, we speculate that tau may play a novel role in mitosis, where tau clusters facilitate the early registration of mitotic spindles to chromosomes before kinetochore-mediated attachment. We also discuss the potential implications of this model in conditions where abnormal cell cycle re-entry and tau activity may disrupt cell division.

biophysics↗

bioRxiv2024

AKT-mediated phosphorylation of TSC2 controls stimulus and tissue-specific mTORC1 signaling and organ growth

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates diverse intracellular and extracellular growth signals to regulate cell and tissue growth. How the molecular mechanisms regulating mTORC1 signaling established through biochemical and cell biological studies function under physiological states in specific mammalian tissues are unknown. Here, we characterize a genetic mouse model lacking the 5 phosphorylation sites on the tuberous sclerosis complex 2 (TSC2) protein through which the growth factor-stimulated protein kinase AKT can active mTORC1 signaling in cell culture models. These phospho-mutant mice (TSC2-5A) are developmentally normal but exhibit reduced body weight and the weight of specific organs, such as brain and skeletal muscle, associated with cell intrinsic decreases in growth factor-stimulated mTORC1 signaling. The TSC2-5A mouse model demonstrates that TSC2 phosphorylation is a primary mechanism of mTORC1 activation in some, but not all, tissues and provides a genetic tool to facilitate studies on the physiological regulation of mTORC1.

cell biology↗

bioRxiv2024

Progressive overfilling of readily releasable pool underlies short-term facilitation at recurrent excitatory synapses in layer 2/3 of the rat prefrontal cortex

Short-term facilitation of recurrent excitatory synapses within the cortical network has been proposed to support persistent activity during working memory tasks, yet the underlying mechanisms remain poorly understood. We characterized short-term plasticity at the local excitatory synapses in layer 2/3 of the rat medial prefrontal cortex and studied its presynaptic mechanisms. Low-frequency stimulation induced slowly developing facilitation, whereas high-frequency stimulation initially induced strong depression followed by rapid facilitation. This non-monotonic delayed facilitation after a brief depression resulted from a high vesicular fusion probability and slow activation of Ca2+-dependent vesicle replenishment, which led to the overfilling of release sites beyond their basal occupancy. Pharmacological and gene knockdown (KD) experiments revealed that the facilitation was mediated by phospholipase C/diacylglycerol signaling and synaptotagmin 7 (Syt7). Notably, Syt7 KD abolished facilitation and slowed the refilling rate of vesicles with high fusion probability. Furthermore, Syt7 deficiency in layer 2/3 pyramidal neurons impaired the acquisition of trace fear memory and reduced c-Fos activity. In conclusion, Ca2+- and Syt7-dependent overfilling of release sites mediates synaptic facilitation at layer 2/3 recurrent excitatory synapses and contributes to temporal associative learning.

neuroscience↗

bioRxiv2024

Machine Learning Driven Optimization for High Precision Cellular Droplet Bioprinting

Controlled volume microliter cell-laden droplet bioprinting is important for precise biologics deposition, reliably replicating 3D microtissue environments for building cell aggregates or organoids. To achieve this, we propose an innovative machine-learning approach to predict cell-laden droplet volumes according to input parameters. We developed a novel bioprinting platform capable of collecting high-throughput droplet images and generating an extensive dataset for training machine learning and deep learning algorithms. Our research compared the performance of three machine learning and two deep learning algorithms that predict droplet volume based on numerous bioprinting parameters. By adjusting bioink viscosity, nozzle size, printing time, printing pressure, and cell concentration as input parameters, we precisely could control droplet sizes, ranging from 0.1 {micro}L to 50 {micro}L in volume. We utilized a hydrogel precursor composed of 5% gelatin methacrylate and a mixture of 0.5% and 1% alginate, respectively. Additionally, we optimized the cell bioprinting process using green fluorescent protein-tagged 3T3 fibroblast cells. These models demonstrated superior predictive accuracy and revealed the interrelationships among parameters while taking minimal time for training and testing. This method promises to advance the mass production of organoids and microtissues with precise volume control for various biomedical applications.

bioengineering↗

bioRxiv2024

Proteome-wide neuropeptide identification using NeuroPeptide-HMMer (NP-HMMer)

Neuropeptides are essential neuronal signaling molecules that orchestrate animal behavior and physiology via actions within the nervous system and on peripheral tissues. Due to the small size of biologically active mature peptides, their identification on a proteome-wide scale poses a significant challenge using existing bioinformatics tools like BLAST. To address this, we have developed NeuroPeptide-HMMer (NP-HMMer), a hidden Markov model (HMM)-based tool to facilitate neuropeptide discovery, especially in underexplored invertebrates. NP-HMMer utilizes manually curated HMMs for 46 neuropeptide families, enabling rapid and accurate identification of neuropeptides. Validation of NP-HMMer on Drosophila melanogaster, Daphnia pulex, Tribolium castaneum and Tenebrio molitor demonstrated its effectiveness in identifying known neuropeptides across diverse arthropods. Additionally, we showcase the utility of NP-HMMer by discovering novel neuropeptides in Priapulida and Rotifera, identifying 22 and 19 new peptides, respectively. This tool represents a significant advancement in neuropeptide research, offering a robust method for annotating neuropeptides across diverse proteomes and providing insights into the evolutionary conservation of neuropeptide signaling pathways.

evolutionary biology↗

bioRxiv2024

Loss of asgr1a leads to the secretion of excess dietary cholesterol in zebrafish

One of the major pathways to clear glycoproteins from circulation is via the liver-specific asialoglycoprotein receptor (ASGPR). Loss of asialoglycoprotein receptor 1 (ASGR1), the major subunit of ASGPR, was recently found to correlate with lower levels of plasma apolipoprotein B- containing lipoproteins (B-lps) and a profoundly reduced risk of cardiovascular disease in humans. We set out to identify the zebrafish ortholog of ASGR1 (asgr1a) and generated two independent mutations in asgr1a using CRISPR/Cas9. Neither asgr1a mutation displayed changes in larval, juvenile, and adult B-lp numbers or sizes. However, when challenged with a Western diet, asgr1a mutant zebrafish exhibit less hepatic steatosis and lower hepatic triglyceride levels compared to control animals. Instead, the excess dietary cholesterol was excreted. While these results do not explain the cardioprotective nature of ASGR1 in humans, they indicate the importance of ASGR1 in modulating whole animal cholesterol flux.

pathology↗

bioRxiv2024

ERK3/MAPK6 promotes triple-negative breast cancer progression through collective migration and EMT plasticity

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and is associated with high cell plasticity, recurrence, and metastatic rate. During epithelial-to-mesenchymal transition (EMT), cancer cells display EMT plasticity, or partial-EMT features, which are required for breast cancer metastasis, such as collective migration. ERK3 has been implicated in promoting migration and invasion of breast cancer, but the mechanisms remain elusive. Here, we investigated ERK3 expression across patient-derived datasets of breast cancer and established its association with aggressive breast cancer phenotypes and poor clinical outcomes. Leveraging the hypothesis that ERK3 contributes to TNBC progression by supporting a partial-EMT state, we showed that ERK3 is essential in different steps of the metastatic process, especially by enabling collective migration but also by modulating cell-extracellular matrix adhesion, anchorage-independent growth, extravasation and colonization. In conclusion, our results demonstrate that ERK3 contributes to TNBC progression and potentially metastasis by promoting EMT plasticity and collective migration.

cancer biology↗

bioRxiv2024

Transcriptomics and proteomics of projection neurons in a circuit linking hippocampus with dorsolateral prefrontal cortex in human brain

Transcriptome and proteome sequencing of brain tissue homogenate has helped unravel processes underlying schizophrenia (SCZ). However, most studies have lacked granularity at the cell type level and have focused on individual brain regions, rather than examining expression dynamics across multiple regions or illness-relevant circuitries. We used laser capture microdissection to collect excitatory neuron-enriched samples from hippocampal subregions CA1 and presubiculum (SUB), and from dorsolateral prefrontal cortex (DLPFC), a circuit prominently implicated in schizophrenia. Using RNA sequencing and quantitative proteomics, we show significantly superior discrimination of brain regional identity in the transcriptomic (>90% accuracy) and proteomic data (>97% accuracy) compared with gene-level expression data (<70% in bulk). Patients with SCZ show hippocampal-specific differential protein phosphorylation. SCZ risk co-expression gene-sets that replicate across transcript and protein networks are enriched for transmembrane transporters in the DLPFC and CA1 and postsynaptic processes in the SUB. We demonstrate a strong directional connectivity effect of SCZ risk in that excitatory synaptic genes in CA1 unidirectionally predict gene expression in SUB. Finally, parallel CA1 snRNA-seq results suggest that in SCZ excitatory efferents in CA1 are affected by interactions with glia and by downregulation of inhibitory neuropeptide inputs. Our study proposes molecular mechanisms by which hippocampal communication, previously associated with SCZ at the macroscopic level, may be altered at the inter-field and interregional circuit level.

neuroscience↗

bioRxiv2024

TRPV4-Expressing Tissue-Resident Macrophages Regulate the Function of Collecting Lymphatic Vessels via Thromboxane A2 Receptors in Lymphatic Muscle Cells

RationaleTRPV4 channels are critical regulators of blood vascular function and have been shown to be dysregulated in many disease conditions in association with inflammation and tissue fibrosis. These are key features in the pathophysiology of lymphatic system diseases, including lymphedema and lipedema; however, the role of TRPV4 channels in the lymphatic system remains largely unexplored. TRPV4 channels are calcium permeable, non-selective cation channels that are activated by diverse stimuli, including shear stress, stretch, temperature, and cell metabolites, which may regulate lymphatic contractile function. ObjectiveTo characterize the expression of TRPV4 channels in collecting lymphatic vessels and to determine the extent to which these channels regulate the contractile function of lymphatics. Methods and ResultsPressure myography on intact, isolated, and cannulated lymphatic vessels showed that pharmacological activation of TRPV4 channels with GSK1016790A (GSK101) led to contractile dysregulation. The response to GSK101 was multiphasic and included, 1) initial robust constriction that was sustained for [≥]1 minute and in some instances remained for [≥]4 minutes; and 2) subsequent vasodilation and partial or complete inhibition of lymphatic contractions associated with release of nitric oxide. The functional response to activation of TRPV4 channels displayed differences across lymphatics from four anatomical regions, but these differences were consistent across different species (mouse, rat, and non-human primate). Importantly, similar responses were observed following activation of TRPV4 channels in arterioles. The initial and sustained constriction was prevented with the COX inhibitor, indomethacin. We generated a controlled and spatially defined single-cell RNA sequencing (scRNAseq) dataset from intact and microdissected collecting lymphatic vessels. Our data uncovered a subset of macrophages displaying the highest expression of Trpv4 compared to other cell types within and surrounding the lymphatic vessel wall. These macrophages displayed a transcriptomic profile consistent with that of tissue-resident macrophages (TRMs), including differential expression of Lyve1, Cd163, Folr2, Mrc1, Ccl8, Apoe, Cd209f, Cd209d, and Cd209g; and at least half of these macrophages also expressed Timd4. This subset of macrophages also highly expressed Txa2s, which encodes the thromboxane A2 (TXA2) synthase. Inhibition of TXA2 receptors (TXA2Rs) prevented TRPV4-mediated contractile dysregulation. TXA2R activation on LMCs caused an increase in mobilization of calcium from intracellular stores through Ip3 receptors which promoted store operated calcium entry and vasoconstriction. ConclusionsClinical studies have linked cancer-related lymphedema with an increased infiltration of macrophages. While these macrophages have known anti-inflammatory and pro-lymphangiogenic roles, as well as promote tissue repair, our results point to detrimental effects to the pumping capacity of collecting lymphatic vessels mediated by activation of TRPV4 channels in macrophages. Pharmacological targeting of TRPV4 channels in LYVE1-expressing macrophages or pharmacological targeting of TXA2Rs may offer novel therapeutic strategies to improve lymphatic pumping function and lymph transport in lymphedema. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/595189v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1ce67fforg.highwire.dtl.DTLVardef@dd1beorg.highwire.dtl.DTLVardef@1454c78org.highwire.dtl.DTLVardef@9f76a1_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

Chaperonin-mediated Winter Cold Response via Circadian Clock Components in Arabidopsis

Vernalization and cold acclimation are plant strategies that evolved to enhance their fitness during the winter season. The molecular mechanisms behind these processes in Arabidopsis have been intensively studied. However, how plants measure the duration of long-term cold exposure has not been fully understood. Our research shows that cytosolic chaperonin is crucial for appropriate long-term cold responses by regulating plant circadian clocks. Furthermore, one of the clock components, REVEILLE4 (RVE4) and RVE8 directly activate VERNALIZATION INSENSITIVE 3 (VIN3) and the C-REPEAT BINDING FACTOR (CBF)/DEHYDRATION-RESPONSIVE ELEMENT BINDING 1 (DREB1) family genes, which are key regulators of vernalization and cold acclimation, respectively. The activation of the VIN3 and CBF/DREB1 genes was specific to the time of day, indicating that cold exposure during the day is critical for long-term cold responses. Our research delves deeper into understanding the regulatory mechanism governing these two distinct long-term cold responses.

plant biology↗

bioRxiv2024

Limosilactobacillus fermentum ATG-V5 stimulates the immune response by enhancing gut microbiome and metabolite in a cyclophosphamide-induced immunosuppression mouse model

Limosilactobacillus fermentum is known to exert immunomodulatory effects. In this study, we elucidated the immunoenhancing effects and mechanisms of a novel strain of Limosilactobacillus fermentum ATG-V5. Our findings demonstrate that ATG-V5 regulates the activation and proliferation of natural killer (NK) and T cells. It induced activation effects such as increased nitric oxide (NO) production, phagocytic capacity, and cytokine generation in Raw264.7 cells, which were confirmed to occur through the ERK and pp65 pathways. Furthermore, using a cyclophosphamide (CPP) mouse model, we demonstrated the immunomodulatory efficacy of ATG-V5, including enhanced activation of NK cells, increased levels of antibodies and cytokine secretion, in mice with immunosuppression induced by CPP. Additionally, by analyzing the gut bacterial community in the mouse model, we confirmed the ability of ATG-V5 consumption to regulate the gut microbiota, thereby substantiating the potential of this strain as a promising immunoenhancing agent.

immunology↗

bioRxiv2024

FOXA1 is required for ErbB2 expression and luminal differentiation in HER2-positive breast cancer

The function of the pioneer factor forkhead box A1 (FOXA1) in estrogen receptor (ER)-negative, human epidermal growth factor receptor 2 (HER2, encoded by the ERBB2 gene)-positive breast cancer represents a critical gap in our understanding of lineage identity in tumorigenesis. Here, we solve this enigma by addressing this question through the identification of an indispensable, co-dependent regulatory circuit formed between FOXA1 and the SAM pointed domain containing ETS-family transcription factor (SPDEF). Using integrative multi-omics analyses of patient tumors and experimental models, we demonstrate that this circuit functions as the master guardian of the HER2-positive luminal state. It executes this role via two distinct, essential mechanisms: it directly binds regulatory elements to drive high-level expression of the ERBB2 oncogene, and it simultaneously preserves epithelial identity by suppressing a latent program of epithelial-to-mesenchymal transition (EMT). Mechanistically, the FOXA1/SPDEF circuit promotes the expression of the transcriptional repressor TRPS1, which in turn antagonizes the pro-mesenchymal activity of the TEAD/YAP complex. Our findings define a central regulatory node that couples lineage fidelity to oncogenic output in HER2-positive cancer, revealing the FOXA1/SPDEF-TRPS1 axis as a critical determinant of tumor plasticity and a potential therapeutic target for overcoming resistance in HER2-positive breast cancer.

cancer biology↗

bioRxiv2024

Dysfunctional β-cell longevity in diabetes relies on high energy conservation and positive epistasis

Long-lived PFKFB3 expressing {beta}-cells are dysfunctional cells because of prevailing glycolysis that compromises metabolic coupling of insulin secretion. Their accumulation in type-2 diabetes (T2D) appears to be related to the loss of apoptotic competency of cell fitness competition (CFC) that maintains tissue function by favoring constant selection of healthy "winner" cells. To investigate how PFKFB3 can disguise the competitive traits of dysfunctional "loser" {beta}-cells, we analyzed the overlap between human {beta}-cells with bona-fide "loser signature" across diabetes pathologies utilizing the HPAP scRNA-seq and spatial transcriptomics of PFKFB3 positive {beta}-cells from nPOD T2D pancreata. The overlapping transcriptional profile of "loser" {beta}-cells was represented by downregulated ribosomal biogenesis- and genes encoding for mitochondrial respiration. PFKFB3 positive "loser" {beta}-cells had reduced expression of HLA Class I and II genes. Gene-gene interaction analysis revealed that PFKFB3 rs1983890 can interact with anti-apoptotic gene MAIP1 implicating positive epistasis as a mechanism for prolonged survival of "loser" {beta}-cells in T2D. Inhibition of PFKFB3 resulted in the clearance of dysfunctional "loser" {beta}-cells leading to restored glucose tolerance in mouse model of T2D.

cell biology↗

bioRxiv2023

VPS13B is localized at the cis-trans Golgi complex interface and is a functional partner of FAM177A1

Mutations in VPS13B, a member of a protein family implicated in bulk lipid transport between adjacent membranes, cause Cohen syndrome. VPS13B is known to be concentrated in the Golgi complex, but its precise location within this organelle and thus the site(s) where it achieves lipid transport remains unclear. Here we show that VPS13B is localized at the interface between cis and trans Golgi sub-compartments and that Golgi complex re-formation after Brefeldin A (BFA) induced disruption is delayed in VPS13B KO cells. This delay is phenocopied by loss of FAM177A1, a Golgi complex protein of unknown function reported to be a VPS13B interactor and whose mutations also result in a developmental disorder. In zebrafish, the vps13b orthologue, not previously annotated in this organism, genetically interacts with fam177a1. Collectively, these findings raise the possibility that bulk lipid transport by VPS13B may play a role in expanding Golgi membranes and that VPS13B may be assisted in this function by FAM177A1.

cell biology↗

bioRxiv2023

The biochemical impact of extracting an embedded adenylate kinase domain using circular permutation

Adenylate kinases (AKs) are phosphotransferases that are frequently employed as models to investigate protein structure-function relationships. Prior studies have shown that AK homologs of different stabilities retain cellular activity in cells following circular permutation that split the AMP binding domain into fragments coded at different ends of the primary structure, such that this domain was no longer embedded as a continuous polypeptide within the core domain. Herein, we show mesophilic and thermophilic AKs having this topological restructuring retain activity and substrate-binding characteristics of the parental AK. While permutation decreased the activity of both AK homologs at physiological temperatures, the catalytic activity of the thermophilic AK increased upon permutation when assayed >30{degrees}C below the melting temperature of the native AK. The thermostabilities of the permuted AKs were uniformly lower than native AKs, and they exhibited multi-phasic unfolding transitions, unlike the native AKs, which presented cooperative thermal unfolding. In addition, proteolytic digestion revealed that permutation destabilized each AK, and mass spectrometry suggested that the new termini within the AMP binding domain were responsible for the increased proteolysis sensitivity. These findings illustrate how changes in contact order can be used to tune enzyme activity and alter folding dynamics in multidomain enzymes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/564053v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1ee9ecdorg.highwire.dtl.DTLVardef@fbb415org.highwire.dtl.DTLVardef@ebdd90org.highwire.dtl.DTLVardef@11f4271_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Open-top Bessel beam two-photon light sheet microscopy for three-dimensional pathology

Nondestructive pathology based on three-dimensional (3D) optical microscopy holds promise as a complement to traditional destructive hematoxylin and eosin (H&E) stained slide-based pathology by providing cellular information in high throughput manner. However, conventional techniques provided superficial information only due to shallow imaging depths. Herein, we developed open-top two-photon light sheet microscopy (OT-TP-LSM) for intraoperative 3D pathology. An extended depth of field two-photon excitation light sheet was generated by scanning a nondiffractive Bessel beam, and selective planar imaging was conducted with cameras at 400 frames/s max during the lateral translation of tissue specimens. Intrinsic second harmonic generation was collected for additional extracellular matrix (ECM) visualization. OT-TP-LSM was tested in various human cancer specimens including skin, pancreas, and prostate. High imaging depths were achieved owing to long excitation wavelengths and long wavelength fluorophores. 3D visualization of both cells and ECM enhanced the ability of cancer detection. Furthermore, an unsupervised deep learning network was employed for the style transfer of OT-TP-LSM images to virtual H&E images. The virtual H&E images exhibited comparable histological characteristics to real ones. OT-TP-LSM may have the potential for histopathological examination in surgical and biopsy applications by rapidly providing 3D information.

pathology↗

bioRxiv2023

Fourth dose of Microneedle Array Patch of SARS-CoV-2 S1 Protein Subunit Vaccine Elicits Robust Long-lasting Humoral Responses in mice

The COVID-19 pandemic has underscored the pressing need for safe and effective booster vaccines, particularly in considering the emergence of new SARS-CoV-2 variants and addressing vaccine distribution inequalities. Dissolving microneedle array patches (MAP) offer a promising delivery method, enhancing immunogenicity and improving accessibility through the skins immune potential. In this study, we evaluated a microneedle array patch-based S1 subunit protein COVID-19 vaccine candidate, which comprised a bivalent formulation targeting the Wuhan and Beta variant alongside a monovalent Delta variant spike proteins in a murine model. Notably, the second boost of homologous bivalent MAP-S1(WU+Beta) induced a 15.7-fold increase in IgG endpoint titer, while the third boost of heterologous MAP-S1RS09Delta yielded a more modest 1.6-fold increase. Importantly, this study demonstrated that the administration of four doses of the MAP vaccine induced robust and long-lasting immune responses, persisting for at least 80 weeks. These immune responses encompassed various IgG isotypes and remained statistically significant for one year. Furthermore, neutralizing antibodies against multiple SARS-CoV-2 variants were generated, with comparable responses observed against the Omicron variant. Overall, these findings emphasize the potential of MAP-based vaccines as a promising strategy to combat the evolving landscape of COVID-19 and to deliver a safe and effective booster vaccine worldwide.

immunology↗