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bioRxiv2024

Protection against N. gonorrhoeae induced by OMV-based Meningococcal Vaccines are associated with cross-species directed humoral and cellular immune responses

Limited protective immunologic responses to natural N. gonorrhoeae infection and a lack of knowledge about mechanisms of protection have hampered development of an effective vaccine. Recent studies in humans and mice have found meningococcal outer membrane vesicle-containing vaccines (OMV) induce cross species immune responses against gonococci and are associated with protection. The exact mechanisms or how humoral and cellular immunity are related to protection, remain unclear. To study this, we immunized mice with two meningococcal OMV-containing vaccines known to accelerate clearance of N. gonorrhoeae, 4CMenB and OMV from an engineered N. meningitidis strain lacking major surface antigens PorA, PorB, and Rmp (MC58 {Delta}ABR). We assessed serologic and cellular immune signatures associated with these immunizations and assessed bacterial clearance in the mice using a vaginal/cervical gonococcal infection model. Mice immunized with 4CMenB or MC58 {Delta}ABR demonstrated shortened courses of recovery of vaginal N. gonorrhoeae compared to control mice immunized with alum alone. Vaccination with 4CMenB or MC58{Delta}ABR OMV elicited serum and vaginal cross-reactive anti-Ng-OMV antibody responses that were augmented after vaginal challenge with N. gonorrhoeae. Further, splenocytes in 4CMenB and MC58 {Delta}ABR immunized mice exhibited elevated cytokine production after restimulation with heterologous N. gonorrhoeae OMV when compared to splenocytes from Alum immunized mice. We further tested for correlations between bacterial burden and the measured anti-gonococcal immune responses within each vaccination group and found different immunologic parameters associated with reduced bacterial burden for each vaccine. Our findings suggest the cross-protection against gonococcal infection induced by different meningococcal OMV vaccines is likely multifactorial and mediated by different humoral and cellular immune responses induced by these two vaccines.

microbiology↗

bioRxiv2024

Statins inhibit onco-dimerization of the 4Ig isoform of B7-H3

B7-H3 (CD276), a member of the B7-family of immune checkpoint proteins, has been shown to have immunological and non-immunological effects promoting tumorigenesis [1, 2] and expression correlates with poor prognosis for many solid tumors, including cervical, ovarian and breast cancers [3-6]. We recently identified a tumor-cell autochthonous tumorigenic role for dimerization of the 4Ig isoform of B7-H3 (4Ig-B7-H3) [7], where 4Ig-B7-H3 dimerization in cis activated tumor-intrinsic cellular proliferation and tumorigenesis pathways, providing a novel opportunity for therapeutic intervention. Herein, a live cell split-luciferase complementation strategy was used to visualize 4Ig-B7-H3 homodimerization in a high-throughput small molecule screen (HTS) to identify modulators of this protein-protein interaction (PPI). Notably, the HTS identified several compounds that converged on lipid metabolism (including HMG-CoA reductase inhibitors, also known as statins) as significant inhibitors of 4Ig-B7-H3 dimerization (p < 0.01). In vitro and in vivo murine studies provided evidence that statin-mediated disruption of 4Ig-B7-H3 dimerization was associated with anti-tumor effects. Statin-mediated anti-cancer efficacy was selective for B7-H3-expressing tumors and retrospective analysis of clinical tumor specimens supported the hypothesis that concurrent statin use enhanced clinical outcomes for patients in a B7-H3 restricted manner. Thus, disruption of 4Ig-B7-H3 dimerization provides an unanticipated molecular mechanism linking statin use in cancer therapy and prevention with immune checkpoint.

cancer biology↗

bioRxiv2024

Anti-Cancer Immune Priming with Beta-Radioligand Therapy and Isoform-Selective Targeting of 4Ig-B7-H3

Radioligand therapy (RLT), a re-emerging oncologic strategy using molecularly-targeted therapeutic radioisotopes, clinically reduces tumor burden and enhances survival for select patients with otherwise unresponsive advanced prostate cancer and neuroendocrine tumors. Developing new approaches to next generation targets and a better understanding of systemic immune effects could broaden the impact of RLT. Aside from contributions to immune checkpoint, B7-H3 (CD276) is an attractive oncologic target because of its widespread and high differential expression across a variety of solid tumors compared to normal tissues. However, B7-H3 has two isoforms: a 4Ig-B7-H3 isoform, the dominant transmembrane protein expressed on tumors and tumor immune microenvironments (TIME), and a 2Ig-B7-H3 isoform, a soluble ectodomain protein, representing a circulating, and in the context of RLT, significant shed (decoy) antigen. To enhance tumor-specific binding and circumvent confounding soluble 2Ig-B7-H3, a novel IgG2a monoclonal antibody (MIL33B) was generated with high affinity for 4Ig-B7-H3 (72 picomolar) and 8- to 18-fold selectivity over soluble 2Ig-B7-H3. Live cell fluorescence microscopy using AF594-labeled MIL33B demonstrated strong membranous localization and target specificity. PET-CT imaging with 89Zr-labeled MIL33B confirmed robust tumor-selective target binding in vivo in murine xenograft (HeLa cervical) and syngeneic tumor models (4T1 breast, B16F10 melanoma, and CT26 colorectal) expressing human 4Ig-B7-H3. As a single dose beta-emitting systemic RLT therapeutic, 90Y-labeled MIL33B (100 Ci) produced 53% long-term survival in a 4Ig-B7-H3-dependent manner in an otherwise fatal established CT26 colorectal tumor model. Immunologic analysis showed that 90Y-MIL33B RLT functioned as an immune priming event, engaging downstream CD8+ T-cell activation and inducing immunological memory in vivo, thus illustrating the potential of systemic beta-RLT to target both primary and metastatic sites. Thus, MIL33B showcases a strategy to selectively target 4Ig-B7-H3 for beta-RLT, warranting further investigation as an immune priming tactic alone or in combination for cancer therapy. Statement of significanceModest antigen expression levels, even if target tissue-selective, combined with ectodomain shedding (soluble decoy antigens) can generally hinder targeted diagnostic and therapeutic strategies, but are especially challenging for radioligand therapy, PET imaging, and in vivo diagnostics wherein high specific activity radioisotopes necessitate use of low masses of biocarrier. Binding, absorption and non-specific tissue deposition of radiolabeled biocarriers by decoy antigens can significantly misdirect systemic radiation, reducing therapeutic efficacy. An antibody development process with a focus aimed at on-target affinity for folded proteins on live cells resulted in a novel picomolar affinity antibody selectively targeting membranous 4Ig-B7-H3 over soluble decoy 2Ig-B7-H3. This antibody shows promise as a transformative systemic beta-radioligand therapy platform for immune priming applications in oncology, and potentially in cardiology, rheumatology, and autoimmunity.

cancer biology↗

bioRxiv2024

Conditional fusogenic lipid nanocarriers for cytosolic delivery of macromolecular therapeutics

Macromolecular therapeutics designed for intracellular targets must overcome systemic delivery barriers, target cell membrane impermeability, and inefficient endosomal escape. Here, we engineer a class of conditional fusogenic liposomes (C-FLIPs) that harness the catalytic activity of extracellular proteases present in the pathological microenvironment. This context-specific activation enables on-target membrane fusion with cells in diseased tissue, resulting in cytosolic delivery of therapeutic payloads. We describe the cytoplasmic delivery of three prototypic macromolecular therapeutic classes: peptide degraders, cytotoxic proteins, and ribonucleoprotein particles (RNPs). We further develop C-FLIP to deliver granzyme B (GzmB) to the cytoplasm of cancer cells in vivo and induce pyroptosis in immunologically-inert tumors. Treatment with C-FLIP/GzmB reprograms the immunosuppressive tumor microenvironment and synergizes with checkpoint blockade to result in the regression of established tumors and induce immunological memory. This modular, non-viral, cytosolic delivery platform represents a promising approach to leverage pathological protease activity for targeted delivery of biologics.

bioengineering↗

bioRxiv2025

Cancer cells suppress NK activity by actin-driven polarisation of inhibitory ligands at the synapse

Natural killer (NK) cells engage target cells via the immunological synapse, where inhibitory and activating signals determine whether NK cell cytotoxicity is suppressed or activated. We report that cancer cells can rapidly remodel their actin cytoskeleton upon NK cell engagement, leading to F-actin accumulation at the synapse. This process inhibits NK cell activation as indicated by impaired MTOC and lytic granule polarization. Exploring the underlying mechanism, we found that actin remodelling drives the recruitment of inhibitory ligands, such as HLA-A, -B, and -C, to the synapse. Disrupting HLA interaction with their cognate inhibitory receptors KIRs restored NK cell activation. Using NK cells expressing inhibitory KIR receptors, matched or unmatched to HLA molecules on cancer cells, we show that synaptic F-actin accumulation and matching KIR-HLA interactions jointly suppress NK cell cytotoxicity. Our findings reveal a novel immune evasion strategy in which cancer cells impair NK cell activation by altering synaptic signalling through actin cytoskeleton-driven recruitment of inhibitory signals to the immunological synapse.

cancer biology↗

bioRxiv2025

Single-cell transcriptomics identifies a dampened neutrophil function and accentuated T-cell cytotoxicity in tobacco flavored e-cigarette exposed mouse lungs

E-cigarettes (e-cigs) are a public health concern for young adults due to their rising popularity despite evidence of harmful effects. Yet an extensive study defining the cell-specific immune changes upon exposure to flavored e-cigs remains elusive. To determine the immunological lung landscape upon acute nose-only exposure of C57BL/6J to flavored e-cig aerosols, we performed single-cell RNA sequencing (scRNA seq). Analyses of the levels of metals in the e-cig aerosol generated daily during exposure revealed a flavor-dependent variation in the day-to-day leaching of the levels of metals like Ni, Cu, K and Zn, among others. scRNA profiles of 71,725 cells generated from control and treatment groups (n=2/sex/group) found maximum dysregulation of (a) myeloid cell function in menthol (324 differentially enriched genes (DEG)) and tobacco (553 DEGs) -flavor exposed and (b) lymphoid cell function in fruit (112 DEGs)-flavored e-cig aerosol exposed mouse lungs as compared to air. Flow cytometry analyses identified marked increase in the neutrophil percentage and a decrease in the eosinophil count in menthol and tobacco-flavored e-cig aerosol exposed mouse lungs which corroborated with our scRNA seq data. We further found an: (a) increase in CD8+ T cell percentages, (b) upregulation of inflammatory genes like Stat4, Il1bos, Il1b, Il1ra and Cxcl3 and (c) enrichment of terms like T-helper cell 1cytokine function and NK cell degranulation in the lungs of e-cig aerosol exposed mouse when compared to control. Interestingly, the increase in the level of immature neutrophils characterized by Ly6G deficiency and reduction in the S100A8 (marker for neutrophil activation) using immunofluorescence in tobacco-flavored e-cig aerosol exposed mouse lung sections point towards a possible shift in the neutrophil dynamics upon exposure to e-cigs. Overall, this study identifies flavor dependent changes in myeloid and lymphoid-cell mediated responses in the mouse lungs exposed to acute nose-only e-cig aerosols and provides a resource to influence future research in select /specific cell types to understand the immunological implications of long-term use of e-cigs.

pharmacology and toxicology↗

bioRxiv2025

Unveiling Interaction Signatures Across Viral Pathogens through VASCO: Viral Antigen-Antibody Structural COmplex dataset

Viral antigen-antibody (Ag-Ab) interactions shape immune responses, drive pathogen neutralization, and inform vaccine strategies. Understanding their structural basis is crucial for predicting immune recognition, optimizing immunogen design to induce broadly neutralizing antibodies (bnAbs), and developing antiviral therapeutics. However, curated structural benchmarks for viral Ag-Ab interactions remain scarce. To address this, we present VASCO (Viral Antibody-antigen Structural COmplex dataset), a high-resolution, non-redundant collection of [~]1225 viral Ag-Ab complexes sourced from the Protein Data Bank (PDB) and refined via energy minimization. Spanning Coronaviruses, Influenza, Ebola, HIV, and others, VASCO provides a comprehensive structural reference for viral immune recognition. By comparing VASCO against general protein-protein interactions (GPPI), we identify distinct sequence and structural features that define viral Ag-Ab binding. While conventional descriptors show broad similarities across datasets, deeper analyses reveal key sequence-space interactions, secondary structure preferences, and manifold-derived latent features that distinguish viral complexes. These insights highlight the limitations of GPPI-trained predictive models and the need for specialized computational frameworks. VASCO serves as a critical resource for advancing viral immunology, improving predictive modeling, and guiding immunogen design to elicit protective antibody responses. By bridging sequence and structural immunological datasets, VASCO should enable better docking, affinity prediction, and antiviral therapeutic development--key to pandemic preparedness and emerging pathogen response.

biophysics↗

bioRxiv2025

Early-peaking caspase-7 activity at the plasma membrane drives apoptotic phosphatidylserine exposure

Apoptosis is an immunologically silent form of regulated cell death executed by caspase1. Caspase cleaves hundreds of substrates throughout the cell to regulate apoptotic processes2, including phosphatidylserine (PS) externalization3-5. However, spatio-temporal regulation of caspase activity in dying cells remains unclear. Here, we show that caspase activity peaks earlier at the plasma membrane (PM) during apoptosis by establishing a Dual Forster resonance energy transfer (Dual FRET) imaging system that combines subcellularly targeted FRET-based caspase biosensors with a cytosolic reference counterpart6,7. Genetic analysis identified caspase-7, an executioner caspase considered an inefficient backup for caspase-3, the major executioner caspase8,9, as a caspase responsible for the Early-Peaking Caspase Activity at the PM (EP-CAP). Mechanistically, EP-CAP is mediated via electrostatic interactions between PS in the inner leaflet of the PM and polybasic residues in the N-terminal intrinsically disordered region (IDR) of caspase-7, which are liberated by the caspase-mediated removal of polyacidic residues. Physiologically, EP-CAP facilitates the efficient cleavage of phospholipid scramblases for the rapid externalization of PS and subsequent efferocytosis. Accordingly, we propose that caspase-7, but not caspase-3, is a bona fide immunologically silent death caspase reinforcing the non-inflammatory nature of apoptosis via EP-CAP.

cell biology↗

bioRxiv2025

Insects fight low-dose infections with terminal investment, not innate immunity

Though the innate immune system is considered to be the primary defence promoting survival against pathogenic microbes, non-immunological strategies may provide cost-effective responses against ubiquitous low-dose infections. However, research using model systems has yet to develop a framework for systematically varying topical exposure doses, much less for examining how hosts mitigate the fitness costs associated with immune deployment. Here, we demonstrate that insects respond to low-dose infections with terminal investment. Female fruit flies, Drosophila melanogaster, have higher egg-to adult viability and lower survival when exposed to low-dose or sexually transmitted infections of an endemic fungus, Aspergillus austwickii. We identify Turandot C (TotC), a humoral stress response gene in insects, as the first non-immunological regulator of fecundity compensation. Strikingly, TotC-mediated fecundity compensation imposes negligible lifetime fitness costs, whereas expression of the canonical immune gene Dorsal-related immunity factor (Dif) triggers reproductively costly antagonistic pleiotropy. Contrary to foundational ecological theory, we show that terminal investment arises from immediate survival-reproduction trade-offs, not truncated reproductive potential. Our findings reveal that adaptive evolution of innate immunity is constrained by classical fitness trade-offs in response to ubiquitous low-dose infections, which permits the evolution of mechanisms in which the host strategically surrenders to the pathogen. Our study refines how we conceptualise host-pathogen evolutionary conflicts and underscores a need to understand how immunosuppression evolves in hosts under ecologically prevalent low-dose infections.

evolutionary biology↗

bioRxiv2025

Epigenetic memory astrocytes are likely an artifact of immune cell contamination

Innate immune memory, in which prior immune stimuli can "train" certain immune cells to respond more aggressively to subsequent challenges, is crucial for immune system plasticity in disease. Lee et al. [1] recently described a similar kind of immune memory state in astrocytes which they termed "epigenetic memory astrocytes". The discovery of astrocytes with immune memory could have tremendous importance in understanding and treating neurological disease. However, the RNA-seq data and in vitro experiments presented by Lee et al. to claim astrocytes possess pro-inflammatory immune memory show signs of immune cell contamination. Further, astrocyte-specific knockout of Ep300, the purported epigenetic regulator of this memory, did not reduce expression of any memory astrocyte signature genes. The FIND-seq signature used to verify the presence of epigenetic memory astrocytes in experimental autoimmune encephalomyelitis (EAE) also shows signs of immune cell contamination, and the cells identified as memory astrocytes in previously published EAE single-cell RNA-seq data are misannotated macrophages. Lastly, we find the purported epigenetic memory astrocytes identified in single-nucleus RNA-seq data of multiple sclerosis (MS) tissue are an artifact of ambient RNA, low quality nuclei, and non-astrocyte contamination. We conclude that the epigenetic memory astrocyte signature is likely driven by immune cell contamination and the existence of astrocyte immunological memory is insufficiently evidenced. We caution that astrocyte transcriptomic, epigenomic, and functional assays must take care to exclude contamination by immune cells, especially when evaluating the potential of astrocytes to perform immunological functions.

neuroscience↗

bioRxiv2025

Gut bacteria gatekeep host-specific colonization of a commensal fungus that boosts type 2 immunity

Naturalized, wilded, wildling, and dirty/pet store mouse models represent a spectrum of approaches designed to make laboratory mice more immunologically and physiologically similar to wild or human contexts by increasing their exposure to naturally occurring microbes and pathogens. In this study, we screened the gut mycobiome of pet store mice, and identified Kazachstania pintolopesii as a dominant fungus in pet store mice across various geographical locations. K. pintolopesii strains isolated from mice in geographically distinct pet stores stably colonize the gastrointestinal tract of laboratory mice, independent of gut bacterial composition, maintaining high fungal burdens for extended periods. K. pintolopesii rapidly became the dominant fungus in the mouse gut in conventional, antibiotic, and germ-free settings, outcompeting other non-murine fungal strains. Pet store-derived K. pintolopesii exhibited unique immunological properties distinct from typical anti-fungal responses. Unlike C. albicans colonization, K. pintolopesii did not induce circulating neutrophil expansion or Th17 cell populations in the gut mucosa. When administered systemically, K. pintolopesii-infected mice showed 100% survival with minimal fungal burden in kidneys, contrasting sharply with lethal C. albicans infections. Adaptive immune deficiency (Rag1 knockout mice) did not affect K. pintolopesii colonization or host response, indicating that B and T cell-mediated immunity does not restrain this fungus. K. pintolopesii colonization provided no cross-protection against systemic candidiasis further establishing lack of immune activation. These findings demonstrate that K. pintolopesii establishes a benign host-fungal relationship through neutrophil-independent mechanisms, avoiding classical anti-fungal immune activation while maintaining stable gut colonization. Instead, it selectively induces strong type 2 mucosal immune responses, increasing tuft and goblet cell counts and stimulating Th2 and group 2 innate lymphoid cell (ILC2) populations. This immune profile confers notable protection against intestinal nematode infection, demonstrated by reduced Heligmosomoides polygyrus egg counts. Altogether, K. pintolopesii serves as an exemplary model for commensal mycobiota, revealing distinct mechanisms for host tolerance and immune modulation.

microbiology↗

bioRxiv2025

TCRanalyzer: A user-friendly tool for comprehensive analysis of T-cell diversity, dynamics and potential antigen targets

T cells are critical for immune responses, recognizing antigens via their unique T-cell receptors (TCRs). Analyzing the diverse TCR repertoires, especially the hypervariable CDR3 region, is essential for understanding immune function in health and disease. Current TCR analysis tools often require specialized expertise, computational resources, or sacrifice biological information for efficiency. To address these limitations, we developed TCRanalyzer, a fast and comprehensive TCR analysis pipeline within a user-friendly graphical interface. TCRanalyzer covers all steps from data loading, aggregation and optional sequence clustering, to the analysis of TCR diversity metrics, clonal expansion and antigen specificity. Applied to datasets from patients with either benign or malignant tumors, TCRanalyzer identified changes in TCR clonality, clonal expansion and shifts in antigen specificity across different cohorts or following immunotherapy, thereby demonstrating its potential to dissect critical immunological processes. TCRanalyzer provides a robust and user-friendly tool for TCR sequence analysis, enhancing research in immunology and related fields. AvailabilityTCRanalyzer is available at https://hub.docker.com/r/tcranalyzer/application. Contactnicole.seifert@bioinf.med.uni-goettingen.de

bioinformatics↗

bioRxiv2025

Single-Cell Analysis of Meningiomas Reveals Mutation-Associated Tumor and Immune Cell Gene Expression Programs

Genomic and epigenetic profiling, particularly DNA methylation analysis, have refined the molecular classification of meningiomas and revealed marked intratumoral heterogeneity. To further characterize heterogeneity within and between patients, we analyzed meningiomas using single-cell RNA sequencing (n=11), whole-exome sequencing (n=9), and spatial transcriptomics (n=3). Single-cell analysis revealed six transcriptionally distinct tumor cell states that corresponded to unique biological processes. Integration of the single-cell data with published exome and bulk RNA sequencing data from a large cohort revealed significant associations among somatic variants, tumor cell states, and immunological signatures. Notably, NF2-altered tumors were enriched for an epithelial-to-mesenchymal transition (EMT) cell state and immune cells, whereas NF2-intact tumors were enriched for a sterol metabolism cell state. Spatial transcriptomic analysis confirmed co-localization of immune cells and EMT tumor cells. Comparisons with immune cells from other brain tumors and peripheral tissues highlighted immunological cell states specific to meningioma. Collectively, these findings refine our genetic and molecular understanding of meningioma heterogeneity and underscore the link between genotype and molecular phenotype.

cancer biology↗

bioRxiv2025

Total immunoglobulin G variation through the phenological cycle of migrant and resident Long-nosed bats (Leptonycteris yerbabuenae) in the drylands of Mexico and its relationship with bacterial-killing ability of plasma.

Immunological variations of bats throughout their life cycle represent an underexplored and controversial topic that has great potential for understanding their immunology. This gap is particularly large regarding the state of immunity during early life and migration, and how different immune components interact with each other. In this study, the partial migratory long-nosed bat (Leptonycteris yerbabuenae) was used as a model to assess the state of acquired humoral immunity (total IgG concentration, tIgG) during early life, during the reproductive cycle and at the extremes of the migratory range of females. We also determined the relationship of tIgG with plasma bactericidal activity against Escherichia coli. The concentration of tIgG was stable throughout the reproductive cycle of adults, and no significant changes were detected at the extremes of the migratory range of females. However, tIgG concentration was reduced during early life (2 to 3 months of age) and in non-reproductive males in March-May 2019 relative to non-reproductive males in March 2020. The tIgG concentration had a positive relationship with plasma bactericidal ability in non-reproductive females, but no relationship was observed in non-reproductive males and reproductive females. These results suggests that tIgG concentration is resilient to physiological and ecological changes experienced by adults throughout their reproductive and migratory cycle. The reduction of tIgG during early life suggests that young individuals have not developed an IgG pool comparable to that of adults. Finally, the positive relationship observed between tIgG and BKA in non-reproductive females suggests that E. coli clearance is enhanced by the presence of antibodies that facilitate the elimination of this bacteria.

ecology↗

bioRxiv2025

Metatranscriptomic Insights into Host-Microbiome Interactions Underlying Asymptomatic COVID-19 Cases

In recent years, the research on Coronavirus disease 2019 (COVID-19) has surged rapidly due to its infectious nature and epidemiological importance as a pandemic. Association of other pathogenic microbes or different human microbiomes with COVID-19 severity is well established. While much is known about COVID-19, studies exploring how host-pathogen interactions at the transcriptomic level influence disease severity are still limited. This research focuses on metatranscriptomic perspective of COVID-19 patients from different Bangladeshi cohorts. Metatranscriptomic sequencing was performed using the extracted RNA of forty different nasopharyngeal samples. After preprocessing and assembly of the genome sequence data, taxonomic identification and diversity along with antibiotic resistance pattern was analyzed using different bioinformatic pipelines. COVID-19 positive and asymptomatic positive patients had a higher abundance of pathogenic and multidrug resistant bacteria whereas the Healthy or recovered patients had higher fungal population. Differential gene expression analysis was also performed to identify the upregulated and downregulated genes responsible for different biological and immunological pathways of humans. Here, immunological response related genes were mostly upregulated in positive cases which was also evident in the proinflammatory cytokines upregulation. Moreover, asymptomatic positive cases showed low TLR-4 expression, which is key to recognizing pathogen-associated molecular patterns (PAMPs) and triggering pro-inflammatory immune responses. This study can clarify the gene expression and signaling pattern of COVID-19 patients with different severity. This may also suggest that some populations exhibit reduced basal expression of TLR-4, which may suppress innate immune activation following infection and contribute to asymptomatic clinical outcomes; however, this hypothesis requires further investigation. Future studies should aim to validate these associations using larger, ethnically diverse cohorts with comprehensive clinical and demographic data.

microbiology↗

bioRxiv2025

5'' cap sequestration is required for sensing of unspliced HIV-1 RNA by MDA5

Heterogenous transcription start site (TSS) usage dictates the structure and function of unspliced HIV-1 RNAs (usRNA). We and others have previously reported that expression and Rev/CRM1-mediated nuclear export of HIV-1 usRNA in macrophages activates MDA5, MAVS, and innate immune signaling cascades. In this study, we reveal that MDA5 sensing of viral usRNA is strictly determined by TSS, 5 leader structure, and RNA function. We show that cap-sequestered HIV-1 usRNAs (cap1G) destined for viral genome packaging are specifically targeted by MDA5, while translation-destined (cap3G) usRNAs are remarkably immunologically silent. Using mutant viruses which express usRNA with altered 5 cap-exposed leader structure, or inclusion of a retroviral constitutive transport element which drives mRNA-like NXF1-dependent nuclear export of viral usRNA, we show that cap exposure and nuclear export pathway choice are major determinants of both lentiviral RNA immunogenicity and function. In total, we identify innate immune system evasion as a possible rationale for the universal conservation of heterogenous TSS usage among ancestral and extant HIV-1 isolates and shed light on how MDA5 fundamentally discriminates between self and non-self RNAs. SIGNIFICANCEInnate immune activation is critical to both the process of initial infection establishment and ongoing chronic inflammation in HIV-1 infection. While MDA5 has been identified as the sensor which detects unspliced HIV-1 RNA produced in infected cells, it remains unclear how HIV-1 unspliced RNAs, which are generated by cellular transcriptional processes, are recognized as non-self. Here, we reveal that HIV-1 RNA function determines MDA5-driven immunogenicity. We show that only unspliced RNAs which traffic to membrane-associated viral assembly sites are immunogenic, while unspliced RNAs which are ribosomally translated to produce viral proteins are immunologically silent. These findings not only advance our knowledge of how the human innate immune system recognizes HIV-1 unspliced RNAs as foreign but also provide a rationale for the selective advantage to generate two pools of unspliced RNAs during HIV-1 replication.

microbiology↗

bioRxiv2025

Deep generative modeling reveals maturation-linked pairing signatures in human antibodies

Understanding how antibody heavy and light chains pair is critical for decoding immune repertoire architecture and designing therapeutic antibodies. However, most antibody sequence databases lack paired chain information. To address this gap, we developed a two-stage deep learning framework. First, we pre-trained separate transformer-based language models on large corpora of unpaired heavy and light chain sequences to capture patterns of gene usage and somatic hypermutation. These models were then integrated via lightweight adapters into a sequence-to-sequence model trained in a machine translation setting, enabling light chain generation conditioned on heavy chain input. Although native light chain recovery was moderate, the model consistently captured functionally meaningful constraints: generated sequences exhibited high germline identity, improved structural quality of predicted folds, and broader coverage of framework and CDR regions. Immunologically, heavy chains from memory B cells preferentially generated light chains with more restricted V gene usage, reflecting maturation-dependent selection. Additionally, generated{kappa} light chains displayed a trimodal similarity distribution, suggesting distinct functional pairing modes ranging from promiscuous to highly specific. This work shows that sequence-to-sequence modeling can uncover inter-chain dependencies and generate structurally and immunologically plausible antibody pairs, providing a foundation for computational repertoire analysis and therapeutic design. HighlightsO_LIA deep generative modeling framework enables conditional generation of light chains from heavy chains, leveraging unpaired data. C_LIO_LIConditioning enhances the structural quality and germline coherence of predicted antibodies. C_LIO_LIMemory B cell-derived heavy chains preferentially generate light chains with restricted V gene usage, consistent with maturation-dependent selection. C_LIO_LIGenerated{kappa} light chains show a trimodal similarity distribution, suggesting discrete pairing modes ranging from promiscuous to highly specific. C_LI

bioinformatics↗

bioRxiv2025

USP18 Inhibition Enhances Type I Interferon Signalling and Immune Activation in the Tumour Microenvironment of Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is one of the most aggressive and treatment-resistant breast cancers. Although immunotherapy has emerged as a promising treatment option, clinical benefit is limited, with only around half of patients responding, even when combined with standard chemotherapeutic agents. This limited efficacy is often attributed to immunologically "cold" tumour microenvironments (TME), which are resistant to current immunotherapies. Addressing this challenge requires approaches that can reprogram "cold" TMEs into "hot" immune-responsive states. USP18, a negative regulator of type I interferon (IFN) signalling, suppresses immune activation by removing ISG15 from target proteins and disrupting IFNAR-STAT2 interactions. Here, we show that both genetic ablation and catalytic inactivation of USP18 enhance type I IFN signalling in TNBC cells, leading to sustained STAT1/STAT2 phosphorylation. This increased IFN responsiveness promotes antigen presentation via MHC-I upregulation and increases expression of pro-apoptotic ligands such as FAS. Proteomic profiling and immunophenotyping revealed that USP18 inhibition in vivo reduces tumour growth and increases immunogenicity, accompanied by cancer-immune infiltration modulation including CD8 T cells, Th1 cells, NK cells, cDC1, and pro-inflammatory M1-like macrophages. These changes reflect a shift in the TME from an immunosuppressive to an immunostimulatory state, driven by heightened and prolonged type I IFN signalling. Our findings highlight the therapeutic potential of USP18 inhibition to convert immunologically "cold" tumours into "hot" tumours, by enhancing IFN-driven immune activation and improving the efficacy of immunotherapy in TNBCs.

cancer biology↗