Wirganowicz, A. | bioRxiv Science

bioRxiv2025

Repurposed endogenous virus-like vesicles mediate dendritic cell long-range antigen presentation and T cell activation for enhanced cancer vaccination

Dendritic cells (DCs) release extracellular vesicles (DEVs) that amplify antigen presentation while incorporating patient-derived, rapidly evolving antigens. By enriching peptide-MHC and co-stimulatory ligands orders-of-magnitude above donor-cell levels, DEVs emerge as potent vesicle-vaccines, although efficacy remains limited by unclear mechanisms. We show that DCs repurpose viral components to generate endogenous virus-like vesicles (VLVs) that preferentially carry peptide-MHC and high-density co-stimulatory ligands, intensifying and extending antigen presentation. Upon antigen exposure, Arc partners with endogenous envelope proteins to assemble VLVs that directly engage T cells and trigger intrinsic adjuvanticity via viral mimicry. Arc-/- DEVs failed to prime antigen-specific T cell responses, whereas Arc overexpression with its 5'-UTR stem-loop shifted DEVs toward VLVs that trafficked to lymphoid organs, drove rapid CD4-assisted priming and durable CD8-biased memory, suppressed melanoma, and prolonged survival. These reveal a viral-mimicry mechanism enabling long-range immune activation and support Arc+ VLVs as an antigen-agnostic vaccine for cancer immunotherapy. HighlightsO_LIArc+ VLVs intensify and extend DC antigen presentation in vivo C_LIO_LIArc+ VLVs directly engage T cells and trigger viral-mimic adjuvanticity C_LIO_LIArc-/- DEVs fail to prime antigen-specific T cell responses C_LIO_LIEngineered Arc+ VLVs drive durable CD8-biased memory and tumor control C_LIO_LIArc+ VLV vaccination provides long-range and cross-tumor protection in vivo C_LI

bioengineering↗

bioRxiv2025

END nucleases: Antiphage defense systems targeting multiple hypermodified phage genomes

Prokaryotes carry clusters of phage defense systems in "defense islands" that have been extensively exploited bioinformatically and experimentally for discovery of immune functions. However, little effort has been dedicated to determining which specific system(s) within defense islands limit lytic phage reproduction in clinical bacterial strains. Here, we employed the CRISPR-based Cascade-Cas3 system to delete defense islands in a Pseudomonas aeruginosa clinical isolate to identify mechanisms of lytic phage antagonism. Deletion of one island in a cystic fibrosis-derived clinical isolate sensitized the strain to phages from the Pbunavirus family, which are commonly used as therapeutics. The causal defense system is a Type IIS restriction endonuclease-like protein (ENDPaCF1), common in Pseudomonads, however it lacks an associated methyltransferase typical Type IIS R-M systems. ENDPaCF1 protects bacteria against phages with hypermodified DNA and is surprisingly agnostic to the specific structure of the modification, which is unlike typical type IV restriction endonucleases. In ENDPaCF1, the endonuclease domain is fused to a catalytically inactive Endonuclease III (iEndoIII), a domain that recognizes non-canonical bases to repair DNA in prokaryotes and eukaryotes. We therefore propose that nucleases containing an iEndoIII domain (END nucleases) can sense diverse DNA hypermodifications. Our findings reveal modularity of the sensing and cleavage domains, as expected of a modification-dependent endonucleases. We further show that some hypermodified phages, including Pbunavirus family members and Wrowclawvirus family (Pa5oct-like) of jumbo phages, encode END nuclease inhibitors that directly bind to the nuclease, likely via the iEndoIII domain. These inhibitors are necessary for Pbunavirus to plaque on clinical isolates and sufficient to enable other hypermodified phages to plaque in the presence of this defense system.

microbiology↗