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Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. However, antigen display on OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. Here, we describe a universal approach for avidin-based vaccine antigen crosslinking (AvidVax) whereby biotinylated antigens are linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen-binding protein (SNAP) comprised of an outer membrane scaffold protein fused to a biotin-binding protein. We show that SNAP-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations are injected in mice, strong antigen-specific antibody responses are observed that depend on the physical coupling between the antigen and SNAP-OMV delivery vehicle. Overall, these results demonstrate AvidVax as a modular platform that enables rapid and simplified assembly of antigen-studded OMVs for application as vaccines against pathogenic threats.

 

The practice of serial X-ray crystallography (SX) depends on efficient, continuous delivery of hydrated protein crystals while minimizing background scattering. Of the two major types of sample delivery devices, fixed-target devices offer several advantages over widely adopted jet injectors, including: lower sample consumption, clog-free delivery, and the ability to control on-chip crystal density to improve hit rates. Here we present our development of versatile, inexpensive, and robust polymer microfluidic chips for routine and reliable room temperature serial measurements at both synchrotrons and X-ray free electron lasers (XFELs). Our design includes highly X-ray-transparent enclosing thin film layers tuned to minimize scatter background, adaptable sample flow layers tuned to match crystal size, and a large sample area compatible with both raster scanning and rotation based serial data collection. The optically transparent chips can be used both for in situ protein crystallization (to eliminate crystal handling) or crystal slurry loading, with prepared samples stable for weeks in a humidified environment and for several hours in ambient conditions. Serial oscillation crystallography, using a multi-crystal rotational data collection approach, at a microfocus synchrotron beamline (SSRL, beamline 12-1) was used to benchmark the performance of the chips. High-resolution structures (1.3–2.7 Å) were collected from five different proteins – hen egg white lysozyme, thaumatin, bovine liver catalase, concanavalin-A (type VI), and SARS-CoV-2 nonstructural protein NSP5. Overall, our modular fabrication approach enables precise control over the cross-section of materials in the X-ray beam path and facilitates chip adaption to different sample and beamline requirements for user-friendly, straightforward diffraction measurements at room temperature. 

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native‐like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent‐solubilized protein‐lipid mixture. Recently, an alternative method has been developed using direct cell‐free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell‐free expression method are structurally indistinguishable from those produced using detergent removal methodologies. This further supports the utility of single step cell‐free methods for the production of lipid binding proteins. In addition, detailed structural information describing these NLPs can be obtained by fitting a capped core‐shell cylinder type model to all SANS/SAXS data simultaneously.