Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.10.24.761556

A Synthetic Virology Platform for Rapid Prototyping of Influenza A Virus mutants

Murti, J. R.·Schön, K.·Angeletti, D.
View original source ↗Export metadata ↓

Abstract

Influenza A virus (IAV) remains a significant public health threat, with the need for effective vaccines and antiviral therapies remaining a high priority. In vitro testing is paramount in studying novel IAV viruses and their immunogenicity. To address this need, we developed a synthetic virology approach to rapidly prototype and test IAV mutants with altered antigenic sites. By strategically inserting restriction sites within an ambisense plasmid, we generated a tool where we can seamlessly replace either 1) the full hemagglutinin (HA), 2) the HA globular head domain to generate head/stalk chimeras or 3) the five canonical antigenic sites. The cloning inserts can be computationally designed and artificially synthesized, enabling rescue of synthetic Influenza A viruses. Using this platform we rescued IAV H1N1, H3N2 & H5N1, and went on to prototype their respective antigenic site mutants & chimeric IAVs using viral genome sequence alone. This platform could be useful for generating vaccine seed strains or to study anti-viral immunity.

Explore related subjects

synthetic biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Murti, J. R., Schön, K., Angeletti, D.. 2026-10-29. A Synthetic Virology Platform for Rapid Prototyping of Influenza A Virus mutants. https://doi.org/10.64898/2026.10.24.761556

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

E. coli-produced nanobody fusions as a pan-serotype platform for AAV purification, quantification, and detection

Research, production and application of the recombinant adeno-associated virus (rAAV) require versatile tools for rapid and cost-effective advances. We developed fusion constructs with the pan-serotype, AAV-binding VHH-antibody-fragment named nanobody AAVX and devised procedures for AAV purification, quantification, and detection. Starting from an amino acid sequence, we cytosolically expressed in E. coli nanobody AAVX carrying various tags and fusion partners. Very efficient soluble production followed by a one-step immobilized metal-ion affinity chromatography (IMAC) yielded mostly >90% pure protein. Crosslinking AAVX with an agarose matrix enabled efficient purification of intact AAV2 and AAV5 capsids from cell lysates, as assessed by qPCR and ELISA. AAVX also supported effective AAV quantification by ELISA, as capture reagent and, when fused to luciferase, as detection reagent. Using tagged AAVX and streptavidin biosensors, we developed a rapid and label-free quantification assay of AAV capsids by biolayer interferometry (BLI). Finally, immunostaining with an AAVX-eGFP fusion visualized AAV capsids in fixed HEK-293 cells by fluorescence microscopy. Collectively, these results establish nanobody AAVX fusion proteins as versatile and readily customizable tools for AAV research and bioprocessing.

synthetic biology↗
bioRxiv2026

Building synthetic cell-cell synapses using engineered scaffold protein condensates

Synaptic assemblies that form at cell-cell junctions are often organized by multivalent scaffold proteins with condensate forming properties. An emerging hypothesis is that these scaffolds provide a thresholding mechanism whereby small increases in the density of cell-cell recognition receptors can trigger all-or-none assembly selectively at the correct partner cell interface. We test this hypothesis by engineering synthetic scaffolded synapses. Coarse-grained simulations suggest that properly-tuned scaffold interactions can yield a sharp condensation threshold. Guided by these simulations, we designed modular scaffold proteins consisting of PDZ domains and intrinsically disordered regions (IDRs) of different molecular grammars. Within a specific region of parameter space, controlled by valency, receptor binding affinity and IDR-driven condensation, the synthetic scaffold proteins exhibit switch-like condensation behaviors, where co-condensates of receptor and scaffold selectively form only at interfaces with the proper partner cell. Notably, orthogonal receptor-scaffold pairs enable a single receiver cell to form multiple distinct interfaces, each associated with a different partner cell. We demonstrate that these synthetic synapse assemblies can recruit different client proteins to enhance intercellular signaling, orient cell division, or sculpt complex synaptic morphologies. Our results illustrate how the ultrasensitive condensation properties of modular scaffold proteins can be harnessed to create novel and highly specific cell-cell interfaces.

synthetic biology↗
bioRxiv2026

Expanding the therapeutic window of bacteria for cancer therapy

Engineered bacteria enable self-amplifying therapeutic delivery and immune activation, yet these same features drive systemic toxicity and off-target growth. Here, we create a "tumorobe" that overcomes these limitations by integrating reduced immunogenicity, tumour-restricted growth, and enhanced payload release. Specifically, we identify lipopolysaccharide modifications that reduce immunogenicity while preserving fitness, restrict growth to tumours using a hypoxia-lactate "AND" gate, and enhance extracellular payload release via lipoprotein deletion. This strain expands the maximum tolerated dose in a route-dependent manner by up to 1,000-fold. Across multiple murine models, including colorectal cancer and glioblastoma, the strain drives robust antitumour efficacy as both a standalone therapeutic and an immunotherapeutic delivery platform, achieving durable complete responses in up to 75% of colorectal tumours when armed with a cytokine payload. Together, these results establish a broadly applicable platform for safe and effective bacterial cancer therapy.

synthetic biology↗