Biology subjects

Ma, W.

Publications and source records attributed to Ma, W..

At least 19 recordsLinked to original sources

bioRxiv2026

GLOM TGITO: multiscale protein modelling and multiview landscape-guided sequence discovery

Motivation: Offline protein design must seek high-activity variants while retaining support for their predicted performance. The challenge is to explore multiple promising regions without confining the search to average-performing sequences. We test whether multiscale representations and local evidence can guide this search. Results: GLOM-TGITO links a global-local fluorescence predictor to multiview landscape-guided optimization. Across 31,434 measured ppluGFP2 sequences, GLOM achieved random/depth-holdout RMSEs of 0.170/0.247 versus 0.247/0.324 for the strongest SaProt-650M controls. Averaged across three training seeds, 96.3%/97.3% of prediction-ranked Top 100 test variants were measured bright (log10 fluorescence >=4.2). Search from 900 peak regions yielded 2,594 distinct candidate sequences. The selected Top 100 showed 2.9-8.2% lower mean representation deviation than four comparator pipelines; 38 versus 11-16 met joint novelty-reliability filters. Deviation identified measured test errors >0.1 (AUROC 0.773). The first molecular-dynamics run of the selected Top 30 identified smaller surface hydrophobic patches in 20 candidates relative to their starts, with representative variants showing more persistent chromophore contacts or narrower torsional fluctuations.

bioinformatics↗

bioRxiv2026

How Much Can AI Virtual Cells Gain from Unified Perturbational Modeling?

Predicting how individual cells respond to perturbations is a central task in artificial intelligence virtual cell (AIVC) modeling. However, integrating perturbation datasets is challenging because they differ in measured gene sets, perturbation types and cellular contexts. Existing AIVC predictors are typically developed for specific settings, limiting their applicability across heterogeneous datasets. Here, we investigate the extent to which AIVC predictors can benefit from unified perturbational modeling (UPM), a novel paradigm designed to integrate heterogeneous Perturb-seq datasets into a single predictive model. To this end, we introduce three unification modules into a variational autoencoder (VAE)-based AIVC predictor to address these sources of heterogeneity: gene-channel alignment, shared perturbation encoding, and joint embedding of gene expression and identity. We systematically evaluate and validate the individual contributions of these modules. Building on UPM, we assemble a dataset of 110 million cells spanning over 22,000 genes, four perturbation types and 76 cellular contexts, and use 89.7 million cells from 71 contexts to train a unified AIVC predictor with more than one billion parameters. Across genetic perturbation benchmarks, UPM achieves the highest PES and gene-wise response correlation among the evaluated models. It also improves all four evaluation metrics over State on the five held-out Tahoe-100M cell lines and over Stack on the cytokine benchmark. UPM also improves combinatorial perturbation prediction relative to specialized predictors. In the HSC application, our model achieves a 40.0% improvement in accuracy on a downstream task involving the recapitulation of effects induced by the knockdown of hepatic stellate cell (HSC)-related targets. Our implementation is openly available at https://github.com/youlab2025/UPM.

bioinformatics↗

bioRxiv2026

Niche-dependent epistasis within the cancer genome shapes metastatic fitness

Epistasis, the genetic interaction between loci, directs non-additive genotype-to-phenotype outcomes. Within a multicellular organism, how the tissue niche (E) shapes gene-by-gene epistasis (GxG) and its phenotypic consequences remains poorly studied. Using tumor lung metastasis as an experimental model, we mapped GxGxE through combinatorial CRISPR perturbation coupled with depletion of defined host myeloid populations in mice. The metastatic niche first selects disseminated tumor cells on GxG epistatic fitness, then drives transcriptome convergence regardless of genotype. Removing anatomically distinct myeloid populations reshaped the metastatic epistasis landscape despite identical tumor genotypes. Gene-by-gene-by-environment epistasis therefore operates at the organismal level, with interaction magnitude and sign set by niche cells. Beyond metastasis, our data suggest that the tissue niche conditions how genetic interactions translate into divergent phenotypes.

genetics↗

bioRxiv2026

Bacteria hyperpolarize in response to oxygen drop

Aerobic microbes use oxygen as a terminal electron acceptor to establish an electrochemical gradient across their cell membranes. While this membrane potential ({triangleup}{Psi}) is essential for multiple cellular processes, it is unknown if and how it is maintained in bacteria during shifts in oxygen availability. We find that bacteria hyperpolarize in response to drops in oxygen. Focusing on Bacillus subtilis and guided by modeling, we report that hyperpolarization magnitude scales with the degree of electron acceptor deprivation. We find that potassium efflux contributes to the response. Surveying mutants, we uncover genes involved in potassium homeostasis and oxygen stress that contribute to hyperpolarization. We show that hyperpolarization scales with faster growth rates resulting from nutrient type and colony spatial heterogeneity. Finally, we demonstrate that oxygen shift increases susceptibility to antibiotics. Our results demonstrate a conserved link between {triangleup}{Psi} and oxygen stress, suggesting a common mechanism for responding to shifting oxygen availability.

microbiology↗

bioRxiv2026

Neurons in the lateral spinal nucleus give rise to the spinotectal component of the anterolateral system in the mouse

The anterolateral system (ALS) originates from neurons in various regions of the spinal cord and conveys somatosensory information related to pain, skin temperature and itch to several brain targets. One component of the ALS is the spinotectal tract, which projects to the superior colliculus (SC) and is thought to underlie directed movements of the head towards a painful stimulus. Using anterograde and retrograde tracing, we demonstrate that the input from the spinal enlargements to the SC originates predominantly from neurons in the lateral spinal nucleus (LSN), many of which express calbindin. This input, which mainly targets the caudal and lateral parts of the SC, is somatotopically organised, and is associated with the clusters of Pitx2-expressing neurons that have been identified in previous studies. Since activation of Pitx2 neurons drives directed head movements, input from LSN neurons to the Pitx2 cells is likely to be a critical component of orientation and attention towards a noxious stimuli. We also provide evidence that spinotectal LSN cells receive excitatory synaptic input from neurons in superficial dorsal horn that express substance P or the gastrin-releasing peptide receptor (GRPR). Since GRPR neurons are strongly implicated in itch, our findings raise the possibility that the SC has a role in coordinating movements in response to pruritic stimuli.

neuroscience↗

bioRxiv2026

Deciphering Mechanistic Signatures in Drug-Drug Interactions with Dual Topology Graphs

Drug-drug interactions (DDIs) represent a critical challenge in drug development and clinical practice, as they can lead to severe adverse effects, including toxicity and reduced therapeutic efficacy. Deep learning methods have shown promise in large-scale, rapid DDI prediction; however, current approaches suffer from significant limitations in providing mechanistic insights into these interactions. Here, we propose DualTopoDDI, a dual-topology-enhanced interpretable deep learning model for DDI prediction. We applied DualTopoDDI to predict 9.2 billion potential interactions among approved drugs, achieving 97.99% high-confidence predictions. The model demonstrates molecular-level interpretability, identifying key substructures responsible for drug actions in terms of both atom-centric and bond-centric views. DualTopoDDI also excels in elucidating particular DDI toxicity mechanisms, which is validated by its successful explanation of the controversial cardiac toxicity in two COVID-19 drug combination regimens at the time. Evaluations across 11 benchmark datasets demonstrates that DualTopoDDI not only achieves state-of-the-art performance but also showcases robust generalizability and good interpretability. Overall, DualTopoDDI offers a powerful, interpretable tool for understanding and predicting drug-drug interactions, providing critical insights for drug safety and design.

bioinformatics↗

bioRxiv2026

Model-based assessment of race, sex, and electrode montage in ECT

Objective: This computational modeling study quantified the influence of sex and race-related cranial anatomy on predicted brain-wide current flow during electroconvulsive therapy (ECT) across conventional (bifrontal (BF), bitemporal/bilateral (BL), right unilateral (RUL)) and experimental (focal electrically administered seizure therapy (FEAST) and frontomedial (FM)) electrode montages. The objective was to determine whether race-associated variability meaningfully contributes to differences in ECT stimulation metrics across montages. Methods: Finite element head models of Chinese, Black, and Caucasian subjects were developed using high-resolution magnetic resonance imaging and analyzed using the Realistic vOlumetric- Approach-based Stimulator for Transcranial electric stimulation (ROAST) pipeline (N = 150 total; n = 50 per cohort, comprising 25 M and 25 F, age range: 20-30 years). Five ECT montages were simulated under a constant-current condition (900mA). Stimulation strength (Ebrain/Eth) was quantified as 90th percentile of brain-wide E-field magnitude (Ebrain) relative to neuronal activation threshold (Eth = 0.25 V/cm) quantified stimulation strength. Overall focality was evaluated as a percentage of brain volume stimulated above the neural activation threshold (Ebrain [≥] Eth), while laterality was quantified as the median right-to-left hemispheric E-field magnitude ratio. The effects of race, sex, and montage on stimulation strength, focality, and hemispheric laterality were statistically analyzed. Results: Substantial race- and sex-related differences observed in cranial anatomy resulted in systematic variation in predicted ECT-induced E-field intensity. Brain-wide E-field magnitude varied by both race and montage, with the largest fields generally observed in Caucasian head models and during BL stimulation. Montage exerted the strongest effect on stimulation strength (Ebrain/Eth) with BL and FEAST producing the highest stimulation strengths, followed by RUL and FM, while BF produced the lowest. Caucasian subjects generally predicted higher stimulation strengths than Black and Chinese subjects, whereas females predicted modestly higher stimulation strengths than males. Laterality was primarily determined by montage, with FEAST producing the greatest hemispheric asymmetry, followed by RUL. Chinese subjects demonstrated higher laterality ratios than both Black and Caucasian subjects. BL, RUL, and FEAST stimulated substantially larger brain volumes above neural activation threshold (less focal stimulation) than BF. Lower focality was observed in Caucasian subjects relative to Black and Chinese subjects, and in females relative to males. Conclusions: Electrode montage was the primary determinant of predicted ECT stimulation strength, focality, and laterality. Race-related anatomical differences and, to a lesser extent, sex-related differences systematically altered stimulation patterns, supporting consideration of individualized anatomy in ECT dosing and treatment optimization.

neuroscience↗

bioRxiv2026

Predicting Protein-RNA Binding Affinity Changes via Spatial Coupling-Aware State Space Modeling

Accurately predicting the effects of mutations on protein-RNA binding is crucial for elucidating disease mechanisms. Yet, exhaustively exploring the space of all possible variants is prohibitively expensive, motivating computational methods that can quantify mutation-induced changes in binding affinity (aka {Delta}{Delta}G) accurately and efficiently. We present iSCALE, an interpretable and generalizable deep learning method that adopts an implicit Spatial Coupling-Aware Ligand Encoding strategy to predict mutation-induced binding affinity changes. By injecting this implicit multiscale encoding scheme into a bidirectional state space modeling architecture, iSCALE learns a generalizable multiscale coupling pattern that achieves superior performances on not only the protein-RNA binding {Delta}{Delta}G, but also the protein stability {Delta}{Delta}G and protein-protein binding {Delta}{Delta}G predictions. Detailed analyses demonstrate that the model attention scores align well with structural characteristics. In addition, iSCALE shows good discriminative ability when predicting close samples such as complexes of same mutation but with different ligands or the same complex but with different mutation sites. In summary, iSCALE serves as an effective in silico tool for large-scale protein-RNA binding {Delta}{Delta}G prediction, which pushes the border of understanding in mutation-induced pathological outcomes.

bioinformatics↗

bioRxiv2026

Ribosome-tethered in situ sequencing for single-cell translatome analysis

Translational regulation plays a critical role in shaping cellular states and functions, yet approaches for spatially resolved translatome profiling at single-cell resolution remain limited. Here, we develop Ribosome-tethered In Situ Sequencing (Ribo-ISS), an imaging-based spatial translatomics technology that enables high-throughput mapping of ribosome-associated mRNAs in intact tissues. Ribo-ISS integrates ribosome-dependent molecular anchoring with multiplexed in situ sequencing, allowing specific detection of translation-associated transcripts without genetic manipulation or exogenous ribosome labeling. We demonstrate that Ribo-ISS achieves high specificity and enables single-cell spatial translatome profiling in mouse brain, accurately recapitulating major cell types and their anatomical organization. Applied to a sleep deprivation model, Ribo-ISS simultaneously resolved transcriptional and translational changes, revealing extensive transcription-translation uncoupling and distinct cell-type-specific translational responses. Ribo-ISS provides a scalable framework for investigating spatially organized translational programs and expands the capability of spatial omics toward understanding gene regulation beyond transcription. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/740970v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@40da8dorg.highwire.dtl.DTLVardef@1cc76b4org.highwire.dtl.DTLVardef@8535c8org.highwire.dtl.DTLVardef@18e009d_HPS_FORMAT_FIGEXP M_FIG C_FIG Ribo-ISS integrates ribosome-dependent molecular anchoring with multiplexed in situ sequencing, enabling specific detection of ribosome-bound transcripts in intact tissues without genetic manipulation or exogenous labeling. As an imaging-based spatial translatomics technology, it resolves translatome profiling at single-cell resolution. This scalable approach reveals spatial translational programs, extending spatial omics beyond transcription.

molecular biology↗

bioRxiv2026

Transcriptomic analysis of cecal mucosal immunity in SPF White Leghorn chicks infected with precocious and parent strains of Eimeria tenella

Eimeria tenella (E. tenella) preferentially invades the cecum of young chickens and causes enormous economic losses to the global poultry industry. In this study, chick infection models of virulent parent strain and precocious attenuated line were established with schizogony (2 dpi) and gametogony (6 dpi) as two critical sampling time points. Combined with pathogenicity detection, transcriptome sequencing, mucosal immune index measurement and homologous challenge protection assays, we systematically deciphered the differential molecular mechanisms underlying pathogenicity and immune regulation between the two strains. Pathogenicity results showed that increased infection dosages suppressed weight gain and aggravated bloody diarrhea and oocyst shedding in both strains. In particular, infection with 1 x 105 sporulated oocysts of the parent strain caused massive chick mortality, while the precocious line exhibited markedly lower virulence. Transcriptomic data revealed that gametogony (6 dpi) represented the peak of host immune response. The parent strain persistently overactivated the NF- {kappa}B - mediated positive feedback cascade of coagulation and complement as well as ECM remodeling, triggering steroid metabolic disorder and antioxidant exhaustion, which ultimately induced severe hemorrhagic necrosis of the cecum. By contrast, the precocious line specifically activated the PPAR - {gamma} signaling pathway to negatively restrain excessive inflammation, accompanied by enriched TLR signaling and leukocyte transendothelial migration pathways, thereby forming an immune cascade of "lipid anti - inflammation - pathogen elimination - mucosal repair". Immunoprotection trials verified that priming with 1 x 104 sporulated oocysts of the precocious line significantly alleviated cecal lesions and reduced oocyst output upon secondary challenge, conferring stable mucosal immune protection. The expression trends of seven hub genes (PPARG, PLIN1, CYP1A1, THBS1, FMO4, CYP2C18, CYP14) detected via qRT - PCR were consistent with RNA - seq data. This study identified a dual regulatory paradigm consisting of NF - {kappa}B - mediated tissue injury and PPAR - {gamma} - dependent anti - inflammatory responses, refined the mucosal immune theoretical framework for commercially available precocious attenuated strains, and provided candidate molecular targets for targeted anti - coccidial intervention in chickens.

microbiology↗

bioRxiv2026

Designing Fidelity of CRISPR-Cas Endonucleases by Kinetic Insights

Finding high-fidelity CRISPR-Cas variants is critical for both the precision of in vitro DNA detection and the safety of in vivo gene-editing therapeutics. However, the large size of the Cas enzyme and the distinct selection criteria between its natural evolution and clinical practice lead to extensive experimental trials and limited success rate for de novo design, directed evolution, protein language model (PLM)-based filtering. Here, we present a PLM-assisted physics-driven approach that utilizes atomistic molecular dynamics simulations and automated path searching to efficiently obtain the complete kinetic insights, including the transition state structures, for the conformational changes of Cas before DNA cleavage. We show that these kinetic insights can pinpoint a few fidelity-diminishing protein residues during the early stage of target recognition, and have led to SpyCas9 and FnCas12a variants with ultra-high fidelity surpassing previously reported counterparts at minimal cost of a few wet-lab trials.

biophysics↗

bioRxiv2026

An engineered IdeS variant with enhanced activity and performance for IgG degradation

IgG-degrading enzymes have emerged as innovative therapeutic agents for treating conditions driven by pathogenic antibodies. Here, we used structure-guided rational design to engineer IdeSM33, a double mutant (K167R/D226E) of the IgG-specific bacterial protease IdeS from Streptococcus pyogenes, with improved catalytic efficiency. Biolayer interferometry revealed a fourfold increase in binding affinity relative to wild-type IdeS (IdeSWT). This enhancement is likely attributable to mutations that strengthen hydrogen bonding at the enzyme-IgG Fc interface. In vitro, IdeSM33 has higher performance than IdeSWT in cleaving serum IgG. In vivo studies in rabbits demonstrated that IdeSM33 effectively depleted circulating IgG and showed better performance at a dose of 0.005 mg/kg than the IdeSWT. Although doses greater than 0.2 mg/kg demonstrated higher plasma concentrations of IdeS and a larger AUClast, they did not show a significant enhancement in the pharmacodynamics of IgG degradation. Importantly, a single dose of IdeSM33 (0.2 mg/kg) potently degraded binding and neutralizing antibodies against AAV9 within 1-2 days and restored hepatic AAV9 transduction in pre-immunized animals. Together, these findings highlight IdeSM33 as a potent and safe engineered enzyme with therapeutic potential for autoimmune disorders, transplant rejection, and overcoming pre-existing humoral immunity in gene therapy.

bioengineering↗

bioRxiv2026

The Two-Component System CrdRS Mediates Sub-inhibitory Antibiotic-Induced Biofilm Formation in Helicobacter pylori via Bidirectional Regulation of Transporters PlpA and GlnP

Biofilm formation by Helicobacter pylori is a major driver of antibiotic tolerance and treatment failure, yet the signaling pathways that trigger biofilm development under sub-inhibitory antibiotic pressure remain poorly understood. Here, we show that sub-MIC levels of metronidazole, amoxicillin, and ciprofloxacin potently induce dense H. pylori biofilms. Through transcriptomic and genetic analyses, we identify the two-component system CrdRS as a central signaling hub that orchestrates this response via the bidirectional regulation of two ATP-binding cassette (ABC) transporters. Phosphorylated CrdR binds to AC-rich promoter motifs to directly activate plpA, which encodes a substrate-binding protein that drives exopolysaccharide secretion and matrix assembly. Concomitantly, CrdR represses glnP, which encodes an inner-membrane permease, thereby relieving transcriptional inhibition of the L-asparaginase gene ansB. This derepression triggers aberrant reactive oxygen species (ROS) accumulation, which promotes oxidative stress-dependent biofilm maturation. Through phenotypic analysis of crdRS deletion mutants, phosphorylation-defective point mutants (CrdRD53A and CrdSH173A), and exogenous hydrogen peroxide (H2O2) induction, we demonstrate that CrdRS specifically responds to antibiotic-induced stress signals in a manner genetically separable from ROS sensing. Collectively, our findings establish a dual-mechanism model in which CrdRS orchestrates antibiotic-induced biofilm formation by simultaneously controlling matrix production and intracellular ROS generation. Notably, glnP expression was significantly lower in clinical multidrug-resistant isolates than in drug-sensitive ones, whereas plpA showed the opposite trend. These findings provide a mechanistic foundation for developing CrdRS-targeted strategies to combat biofilm-associated H. pylori infections.

microbiology↗

bioRxiv2026

Perturbation of genes linked to common schizophrenia risk variants identifies cilia programs

Schizophrenia (SCZ) is a common psychiatric disorder characterized by psychosis, emotional withdrawal, and cognitive deficits. Most SCZ risk variants reside in non-coding regions of the genome and are thought to influence disease risk by modulating gene regulation. However, the target genes, biological pathways, and cell types through which these variants exert their effects remain poorly understood. To address this gap, we employed in vivo CRISPR droplet sequencing (CROP-seq) in the postnatal mouse neocortex. We perturbed 12 SCZ risk genes previously linked to functionally validated risk variants, followed by single-cell RNA sequencing. We identified 3,031 differentially expressed genes (DEGs) that recapitulate transcriptional alterations observed in postmortem SCZ brains. Integrative analysis using DEG clustering, factor analysis, and gene regulatory network inference uncovered convergent gene programs with distinct biological functions and cell type specificity. Notably, ciliary transcriptional programs consistently emerged across analytical frameworks. The primary cilium is a neurocircuit modulating signaling organelle in neurons and glia that remains understudied in SCZ. Perturbation of key contributors to the ciliary transcriptional programs led to significant alterations in ciliary structure, suggesting that SCZ genetic risk factors may influence how brain cells sense and transduce extracellular signals through synapse-independent mechanisms. Together, this study provides the first in vivo characterization of the functional consequence of common variant architecture in SCZ and implicates ciliary dysfunction as a convergent downstream mechanism.

neuroscience↗

bioRxiv2026

Integrated optimization of experimental and computational workflows improves genome recovery in long-read gut metagenomics

Short-read metagenomic sequencing has been widely applied in microbial research due to its high quality and decreasing cost. However, short reads are inherently fragmented, which limits assembly contiguity and the recovery of complete microbial genomes. In contrast, long-read sequencing, with significantly longer read lengths, helps overcome these limitations. Achieving complete and accurate genome recovery is the core goal of metagenomics. To address this challenge, we systematically evaluated and optimized the long-read metagenomic workflow, from sample processing to computational assembly, using the CycloneSEQ platform.

bioinformatics↗

bioRxiv2026

Porcine Left Atrial and Ventricular Thick Filaments Exhibit Distinct Resting Structures and Calcium-dependent Responses

The heart maintains systemic perfusion through the coordinated function of its four chambers: the left and right atria and ventricles. Each chamber has distinct structural, functional, and molecular properties tailored to its role in circulation, which may result in chamber-specific differences in myofilament structure and regulation between atria and ventricles. To test this hypothesis, we employed muscle mechanics and X-ray diffraction to investigate functional and structural differences in porcine left atrial (LA) and left ventricular (LV) tissue. Here, we report the first X-ray diffraction study of atrial tissue, demonstrating that under resting conditions, myosin filaments in LA adopted a more ON-like, structurally distinct configuration compared with those in LV. Under contracting conditions, LV generated greater force and exhibited higher sinusoidal stiffness than LA across multiple calcium concentrations. LA showed faster kTR than in LV, with no calcium-dependence, in contrast to the calcium-dependence of kTR seen in LV. Structurally, the distinct myosin head configuration seen in the relaxed LA persisted during contraction. Furthermore, using the troponin inhibitor MYK-7660 to inhibit active contraction, we showed that, unlike LV, LA showed no direct calcium-dependent thick filament activation, reconciling discrepancies between fast rat and slow porcine ventricular myocardium regarding calciums role in thick filament regulation. Altogether, our study reveals that LA myosin filaments adopt a molecular architecture and regulatory mechanism distinct from their LV counterparts, suggesting that myosin filament structure and regulation have evolved differently to meet the unique functional demands of each cardiac chamber. Moreover, atrial disease is often associated with cardiomyopathy-related genetic variants, highlighting the atrial myocardium as an important therapeutic target and understanding atrial-specific regulatory mechanisms provides new insights into therapeutic strategies for atrial diseases.

physiology↗

bioRxiv2026

Colletotrichum higginsianum effector ChEC108 binds a plasmodesmal HMA protein and elicits plant defence

To establish infection, phytopathogens deploy effectors to compromise host defences and facilitate invasive growth. As part of this, the battle for control of symplastic connectivity via plasmodesmata is a key determinant of infection outcomes, yet little is known about how fungal effectors directly exploit these channels, and in turn, how hosts defend them. Here, we have identified ChEC108 as a plasmodesmal-targeting, cell-to-cell mobile effector from the anthracnose fungus, Colletotrichum higginsianum. ChEC108 binds the plasmodesmal protein HEAVY METAL-ASSOCIATED (HMA) ISOPRENYLATED PLANT PROTEIN 6 (HIPP6) from Arabidopsis via a tetrahedral metal ion coordination site with either of its HMA domains. Constitutive in planta expression of ChEC108 induces plasmodesmal closure and the upregulation of defence-associated genes in a manner dependent on its capacity to bind HIPP6. Further, HIPP6 binding impairs cell-to-cell mobility of ChEC108. Alongside the finding that loss of ChEC108 favoured C. higginsianum infection, this suggests ChEC108-HIPP6 interaction at plasmodesmata positively regulates defence.

plant biology↗

bioRxiv2026

Harnessing Human Immune Organoid model for Systemic and Comparative Analysis of Clinically Vaccine Adjuvants

Effective vaccine adjuvants are crucial for eliciting strong, specific, and long-lasting adaptive immune responses. However, traditional vaccine and adjuvant development is often hindered by prolonged animal testing, reliance on singular evaluation metrics, and batch variability, limiting the comparative analysis of immune responses induced by different adjuvants. To our knowledge, systematic adjuvant-focused analysis in human immune organoids remains limited, particularly across distinct antigen-conditioned immune baselines. Here, we established a matrix supported human tonsil immune organoid (M-hTIO) platform, derived from tonsil tissue for systemic adjuvant evaluation. Within 2 days, the platform replicated key germinal center (GC) features, with the potential for antigen-specific antibody production and plasmablast differentiation. Dynamic GC formation and T-B cell interactions were tracked using light-field imaging. Notably, IL-21 significantly accelerates GC-like remodeling and enhanced immune responses. Employing bulk cell transcriptomics we systematically analyzed early immune signatures and prolonged adaptive responses induced by five clinical-grade adjuvants (Alum, MnJ{beta}, AddaSO3, R848, ODN 1018) paired with influenza HA and SARS-CoV-2 RBD antigens across only adjuvant, weak and strong memory backgrounds. By integrating early and prolonged response phases, M-hTIO captured complementary dimensions of adjuvant activity, distinguishing rapid immune activation from sustained adaptive remodeling. Importantly, early activation signatures and prolonged adaptive remodeling provided complementary information, highlighting the value of temporally resolved adjuvant evaluation. AddaSO3 showed a consistent activity pattern across early and prolonged response phases, supporting both rapid immune activation and sustained adaptive remodeling. Moreover, our findings reveal shared/exclusive signaling pathways and gene networks. These results represent systematic and comparative analysis of clinically tested vaccine adjuvants in a human model, paving the way for high-throughput systems vaccinology research.

immunology↗