On-Demand Vaccine Production via Dock-and-Display of Biotinylated Antigens on Bacterial Extracellular Vesicles.

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising vaccine technology for developing immunity against diverse pathogens. However, antigen display on OMVs can be challenging to control and highly variable due to bottlenecks in protein expression and localization to the bacterial host cell's outer membrane, especially for bulky and complex antigens. Here, we describe methods related to a universal vaccine technology called AvidVax (avidin-based vaccine antigen crosslinking) for rapid and simplified assembly of antigens on the exterior of OMVs during vaccine development. The AvidVax platform involves remodeling the OMV surface with multiple copies of a synthetic antigen-binding protein (SNAP), which is an engineered fusion protein comprised of an outer membrane scaffold protein linked to a biotin-binding protein. The resulting SNAPs enable efficient decoration of OMVs with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, nucleic acids, and short peptides. We detail the key steps in the AvidVax vaccine production pipeline including preparation and isolation of SNAP-OMVs, biotinylation and enrichment of vaccine antigens, and formulation and characterization of antigen-loaded SNAP-OMVs.

De Novo Design of Peptide Binders to Conformationally Diverse Targets with Contrastive Language Modeling.

Designing binders to target undruggable proteins presents a formidable challenge in drug discovery, requiring innovative approaches to overcome the lack of putative binding sites. Recently, generative models have been trained to design binding proteins via three-dimensional structures of target proteins, but as a result, struggle to design binders to disordered or conformationally unstable targets. In this work, we provide a generalizable algorithmic framework to design short, target-binding linear peptides, requiring only the amino acid sequence of the target protein. To do this, we propose a process to generate naturalistic peptide candidates through Gaussian perturbation of the peptidic latent space of the ESM-2 protein language model, and subsequently screen these novel linear sequences for target-selective interaction activity via a CLIP-based contrastive learning architecture. By integrating these generative and discriminative steps, we create a Peptide Prioritization via CLIP (PepPrCLIP) pipeline and validate highly-ranked, target-specific peptides experimentally, both as inhibitory peptides and as fusions to E3 ubiquitin ligase domains, demonstrating functionally potent binding and degradation of conformationally diverse protein targets in vitro. Overall, our design strategy provides a modular toolkit for designing short binding linear peptides to any target protein without the reliance on stable and ordered tertiary structure, enabling generation of programmable modulators to undruggable and disordered proteins such as transcription factors and fusion oncoproteins.

Programmable Protein Stabilization with Language Model-Derived Peptide Guides.

Dysregulated protein degradation via the ubiquitin-proteasomal pathway can induce numerous disease phenotypes, including cancer, neurodegeneration, and diabetes. Stabilizing improperly ubiquitinated proteins via target-specific deubiquitination is thus a critical therapeutic goal. Building off the major advances in targeted protein degradation (TPD) using bifunctional small-molecule degraders, targeted protein stabilization (TPS) modalities have been described recently. However, these rely on a limited set of chemical linkers and warheads, which are difficult to generate de novo for new targets and do not exist for classically "undruggable" targets. To address the limited reach of small molecule-based degraders, we previously engineered ubiquibodies (uAbs) by fusing computationally-designed "guide" peptides to E3 ubiquitin ligase domains for modular, CRISPR-analogous TPD. Here, we expand the TPS target space by engineering "deubiquibodies" (duAbs) via fusion of computationally-designed guides to the catalytic domain of the potent OTUB1 deubiquitinase. In human cells, duAbs effectively stabilize exogenous and endogenous proteins in a DUB-dependent manner. To demonstrate duAb modularity, we swap in new target-binding peptides designed via our generative language models to stabilize diverse target proteins, including key tumor suppressor proteins such as p53 and WEE1, as well as heavily-disordered fusion oncoproteins, such as PAX3::FOXO1. In total, our duAb system represents a simple, programmable, CRISPR-analogous strategy for TPS.

Intranasal Vaccination with Recombinant TLR2-Active Outer Membrane Vesicles Containing Sequential M2e Epitopes Protects against Lethal Influenza a Challenge.

Influenza is a highly contagious respiratory disease, resulting in an estimated 3 to 5 million cases of severe illness annually. While most influenza vaccines are administered parenterally via injection, one shortcoming is that they do not generate a strong immune response at the site of infection, which can become important in a pandemic. Intranasal vaccines can generate both local and systemic protective immune responses, can reduce costs, and enhance ease of administration. Previous studies showed that parenterally administered outer membrane vesicles (OMVs) that carry sequences of the M2e protein (OMV-M2e) protect against influenza A/PR8 challenge in mice and ferrets. In the current study, we measured the effectiveness of the intranasal route of the OMV-M2e vaccine against the influenza A/PR8 strain in mice. We observed high anti-M2e IgG and IgA titers post-challenge in mice vaccinated intranasally with OMV-M2e. In addition, we observed a Th1/Tc1 bias in the vaccinated mice, and an increased Th17/Tc17 response, both of which correlated with survival to A/PR8 challenge and significantly lower lung viral titers. We conclude that the intranasal-route administration of the OMV-M2e vaccine is a promising approach toward generating protection against influenza A as it leads to an increased proinflammatory immune response correlating with survival to viral challenge.

Bacterial glycoengineering: Cell-based and cell-free routes for producing biopharmaceuticals with customized glycosylation.

Glycosylation plays a pivotal role in tuning the folding and function of proteins. Because most human therapeutic proteins are glycosylated, understanding and controlling glycosylation is important for the design, optimization, and manufacture of biopharmaceuticals. Unfortunately, natural eukaryotic glycosylation pathways are complex and often produce heterogeneous glycan patterns, making the production of glycoproteins with chemically precise and homogeneous glycan structures difficult. To overcome these limitations, bacterial glycoengineering has emerged as a simple, cost-effective, and scalable approach to produce designer glycoprotein therapeutics and vaccines in which the glycan structures are engineered to reduce heterogeneity and improve biological and biophysical attributes of the protein. Here, we discuss recent advances in bacterial cell-based and cell-free glycoengineering that have enabled the production of biopharmaceutical glycoproteins with customized glycan structures.

Novel human recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in a glyco-engineered Escherichia coli strain.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in the accumulation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). Previously, it was reported the production of an active human recombinant GALNS (rGALNS) in E. coli BL21(DE3). However, this recombinant enzyme was not taken up by HEK293 cells or MPS IVA skin fibroblasts. Here, we leveraged a glyco-engineered E. coli strain to produce a recombinant human GALNS bearing the eukaryotic trimannosyl core N-glycan, Man3GlcNAc2 (rGALNSoptGly). The N-glycosylated GALNS was produced at 100 mL and 1.65 L scales, purified and characterized with respect to pH stability, enzyme kinetic parameters, cell uptake, and KS clearance. The results showed that the addition of trimannosyl core N-glycans enhanced both protein stability and substrate affinity. rGALNSoptGly was capture through a mannose receptor-mediated process. This enzyme was delivered to the lysosome, where it reduced KS storage in human MPS IVA fibroblasts. This study demonstrates the potential of a glyco-engineered E. coli for producing a fully functional GALNS enzyme. It may offer an economic approach for the biosynthesis of a therapeutic glycoprotein that could prove useful for MPS IVA treatment. This strategy could be extended to other lysosomal enzymes that rely on the presence of mannose N-glycans for cell uptake.

Bioreversible Anionic Cloaking Enables Intracellular Protein Delivery with Ionizable Lipid Nanoparticles.

Protein-based therapeutics comprise a rapidly growing subset of pharmaceuticals, but enabling their delivery into cells for intracellular applications has been a longstanding challenge. To overcome the delivery barrier, we explored a reversible, bioconjugation-based approach to modify the surface charge of protein cargos with an anionic "cloak" to facilitate electrostatic complexation and delivery with lipid nanoparticle (LNP) formulations. We demonstrate that the conjugation of lysine-reactive sulfonated compounds can allow for the delivery of various protein cargos using FDA-approved LNP formulations of the ionizable cationic lipid DLin-MC3-DMA (MC3). We apply this strategy to functionally deliver RNase A for cancer cell killing as well as a full-length antibody to inhibit oncogenic ß-catenin signaling. Further, we show that LNPs encapsulating cloaked fluorescent proteins distribute to major organs in mice following systemic administration. Overall, our results point toward a generalizable platform that can be employed for intracellular delivery of a wide range of protein cargos.

Establishing a Cell-Free Glycoprotein Synthesis System for Enzymatic N-GlcNAcylation.

N-linked glycosylation plays a key role in the efficacy of many therapeutic proteins. One limitation to the bacterial glycoengineering of human N-linked glycans is the difficulty of installing a single N-acetylglucosamine (GlcNAc), the reducing end sugar of many human-type glycans, onto asparagine in a single step (N-GlcNAcylation). Here, we develop an in vitro method for N-GlcNAcylating proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. We use cell-free protein synthesis (CFPS) to test promiscuous PglB variants previously reported in the literature for the ability to produce N-GlcNAc and successfully determine that PglB with an N311V mutation (PglBN311V) exhibits increased GlcNAc transferase activity relative to the wild-type enzyme. We then improve the transfer efficiency by producing CFPS extracts enriched with PglBN311V and further optimize the reaction conditions, achieving a 98.6 ± 0.5% glycosylation efficiency. We anticipate this method will expand the glycoengineering toolbox for therapeutic research and biomanufacturing.

Cell-Free Systems for the Production of Glycoproteins.

Cell-free protein synthesis (CFPS), whereby cell lysates are used to produce proteins from a genetic template, has matured as an attractive alternative to standard biomanufacturing modalities due to its high volumetric productivity contained within a distributable platform. Initially, cell-free lysates produced from Escherichia coli, which are both simple to produce and cost-effective for the production of a wide variety of proteins, were unable to produce glycosylated proteins as E. coli lacks native glycosylation machinery. With many important therapeutic proteins possessing asparagine-linked glycans that are critical for structure and function, this gap in CFPS production capabilities was addressed with the development of cell-free expression of glycoproteins (glycoCFE), which uses the supplementation of extracted lipid-linked oligosaccharides and purified oligosaccharyltransferases to enable glycoprotein production in the CFPS reaction environment. In this chapter, we highlight the basic methods for the preparation of reagents for glycoCFE and the protocol for expression and glycosylation of a model protein using a more productive, yet simplified, glycoCFE setup. Beyond this initial protocol, we also highlight how this protocol can be extended to a wide range of alternative glycan structures, oligosaccharyltransferases, and acceptor proteins as well as to a one-pot cell-free glycoprotein synthesis reaction.

Immunoengineering can overcome the glycocalyx armour of cancer cells.

Cancer cell glycocalyx is a major line of defence against immune surveillance. However, how specific physical properties of the glycocalyx are regulated on a molecular level, contribute to immune evasion and may be overcome through immunoengineering must be resolved. Here we report how cancer-associated mucins and their glycosylation contribute to the nanoscale material thickness of the glycocalyx and consequently modulate the functional interactions with cytotoxic immune cells. Natural-killer-cell-mediated cytotoxicity is inversely correlated with the glycocalyx thickness of the target cells. Changes in glycocalyx thickness of approximately 10 nm can alter the susceptibility to immune cell attack. Enhanced stimulation of natural killer and T cells through equipment with chimeric antigen receptors can improve the cytotoxicity against mucin-bearing target cells. Alternatively, cytotoxicity can be enhanced through engineering effector cells to display glycocalyx-editing enzymes, including mucinases and sialidases. Together, our results motivate the development of immunoengineering strategies that overcome the glycocalyx armour of cancer cells.

Bio-orthogonal Glycan Imaging of Culture Cells and Whole Animal C. elegans with Expansion Microscopy.

Complex carbohydrates called glycans play crucial roles in the regulation of cell and tissue physiology, but how glycans map to nanoscale anatomical features must still be resolved. Here, we present the first nanoscale map of mucin-type O -glycans throughout the entirety of the Caenorhabditis elegans model organism. We construct a library of multifunctional linkers to probe and anchor metabolically labelled glycans in expansion microscopy (ExM), an imaging modality that overcomes the diffraction limit of conventional optical microscopes through the physical expansion of samples embedded in a polyelectrolyte gel matrix. A flexible strategy is demonstrated for the chemical synthesis of linkers with a broad inventory of bio-orthogonal functional groups, fluorophores, anchorage chemistries, and linker arms. Employing C. elegans as a test bed, we resolve metabolically labelled O -glycans on the gut microvilli and other nanoscale anatomical features using our ExM reagents and optimized protocols. We use transmission electron microscopy images of C. elegans nano-anatomy as ground truth data to validate the fidelity and isotropy of gel expansion. We construct whole organism maps of C. elegans O -glycosylation in the first larval stage and identify O -glycan "hotspots" in unexpected anatomical locations, including the body wall furrows. Beyond C. elegans , we provide validated ExM protocols for nanoscale imaging of metabolically labelled glycans on cultured mammalian cells. Together, our results suggest the broad applicability of the multifunctional reagents for imaging glycans and other metabolically labelled biomolecules at enhanced resolutions with ExM.

Proteomic profiling of membrane vesicles from Mycobacterium avium subsp. paratuberculosis: Navigating towards an insilico design of a multi-epitope vaccine targeting membrane vesicle proteins.

Bacteria typically produce membrane vesicles (MVs) at varying levels depending on the surrounding environments. Gram-negative bacterial outer membrane vesicles (OMVs) have been extensively studied for over 30 years, but MVs from Gram-positive bacteria only recently have been a focus of research. In the present study, we isolated MVs from Mycobacterium avium subsp. paratuberculosis (MAP) and analyzed their protein composition using LC-MS/MS. A total of 316 overlapping proteins from two independent preparations were identified in our study, and topology prediction showed these cargo proteins have different subcellular localization patterns. When MVs were administered to bovine-derived macrophages, significant up-regulation of pro-inflammatory cytokines was observed via qRT-PCR. Proteome functional annotation revealed that many of these proteins are involved in the cellular protein metabolic process, tRNA aminoacylation, and ATP synthesis. Secretory proteins with high antigenicity and adhesion capability were mapped for B-cell and T-cell epitopes. Antigenic, Immunogenic and IFN-γ inducing B-cell, MHC-I, and MHC-II epitopes were stitched together through linkers to form multi-epitope vaccine (MEV) construct against MAP. Strong binding energy was observed during the docking of the 3D structure of the MEV with the bovine TLR2, suggesting that the putative MEV may be a promising vaccine candidate against MAP. However, in vitro and in vivo analysis is required to prove the immunogenic concept of the MEV which we will follow in our future studies. SIGNIFICANCE: Johne's disease is a chronic infection caused by Mycobacterium avium subsp. paratuberculosis that has a potential link to Crohn's disease in humans. The disease is characterized by persistent diarrhea and enteritis, resulting in significant economic losses due to reduced milk yield and premature culling of infected animals. The dairy industry in the United States alone experiences losses of approximately USD 250 million due to Johne's disease. The current vaccine against Johne's disease is limited by several factors, including variable efficacy, limited duration of protection, interference with diagnostic tests, inability to prevent infection, and logistical and cost-related challenges. Nevertheless, a multiepitope vaccine design approach targeting M. avium subsp. paratuberculosis has the potential to overcome these challenges and offer improved protection against Johne's disease.

SaLT&PepPr is an interface-predicting language model for designing peptide-guided protein degraders.

Protein-protein interactions (PPIs) are critical for biological processes and predicting the sites of these interactions is useful for both computational and experimental applications. We present a Structure-agnostic Language Transformer and Peptide Prioritization (SaLT&PepPr) pipeline to predict interaction interfaces from a protein sequence alone for the subsequent generation of peptidic binding motifs. Our model fine-tunes the ESM-2 protein language model (pLM) with a per-position prediction task to identify PPI sites using data from the PDB, and prioritizes motifs which are most likely to be involved within inter-chain binding. By only using amino acid sequence as input, our model is competitive with structural homology-based methods, but exhibits reduced performance compared with deep learning models that input both structural and sequence features. Inspired by our previous results using co-crystals to engineer target-binding "guide" peptides, we curate PPI databases to identify partners for subsequent peptide derivation. Fusing guide peptides to an E3 ubiquitin ligase domain, we demonstrate degradation of endogenous ß-catenin, 4E-BP2, and TRIM8, and highlight the nanomolar binding affinity, low off-targeting propensity, and function-altering capability of our best-performing degraders in cancer cells. In total, our study suggests that prioritizing binders from natural interactions via pLMs can enable programmable protein targeting and modulation.

Glycovaccinology: The design and engineering of carbohydrate-based vaccine components.

Vaccines remain one of the most important pillars in preventative medicine, providing protection against a wide array of diseases by inducing humoral and/or cellular immunity. Of the many possible candidate antigens for subunit vaccine development, carbohydrates are particularly appealing because of their ubiquitous presence on the surface of all living cells, viruses, and parasites as well as their known interactions with both innate and adaptive immune cells. Indeed, several licensed vaccines leverage bacterial cell-surface carbohydrates as antigens for inducing antigen-specific plasma cells secreting protective antibodies and the development of memory T and B cells. Carbohydrates have also garnered attention in other aspects of vaccine development, for example, as adjuvants that enhance the immune response by either activating innate immune responses or targeting specific immune cells. Additionally, carbohydrates can function as immunomodulators that dampen undesired humoral immune responses to entire protein antigens or specific, conserved regions on antigenic proteins. In this review, we highlight how the interplay between carbohydrates and the adaptive and innate arms of the immune response is guiding the development of glycans as vaccine components that act as antigens, adjuvants, and immunomodulators. We also discuss how advances in the field of synthetic glycobiology are enabling the design, engineering, and production of this new generation of carbohydrate-containing vaccine formulations with the potential to prevent infectious diseases, malignancies, and complex immune disorders.

Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli.

Here we describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered Escherichia coli cells. The method is based on the transport of a bifunctional substrate comprised of an antigen fused to chloramphenicol acetyltransferase, which allows positive selection of bacterial cells co-expressing cytoplasmic IgGs called cyclonals that specifically capture the chimeric antigen and sequester the antibiotic resistance marker in the cytoplasm. The utility of this approach is first demonstrated by isolating affinity-matured cyclonal variants that specifically bind their cognate antigen, the leucine zipper domain of a yeast transcriptional activator, with subnanomolar affinities, which represent a ~20-fold improvement over the parental IgG. We then use the genetic assay to discover antigen-specific cyclonals from a naïve human antibody repertoire, leading to the identification of lead IgG candidates with affinity and specificity for an influenza hemagglutinin-derived peptide antigen.

Rapid biosynthesis of glycoprotein therapeutics and vaccines from freeze-dried bacterial cell lysates.

The advent of distributed biomanufacturing platforms promises to increase agility in biologic production and expand access by reducing reliance on refrigerated supply chains. However, such platforms are not capable of robustly producing glycoproteins, which represent the majority of biologics approved or in development. To address this limitation, we developed cell-free technologies that enable rapid, modular production of glycoprotein therapeutics and vaccines from freeze-dried Escherichia coli cell lysates. Here, we describe a protocol for generation of cell-free lysates and freeze-dried reactions for on-demand synthesis of desired glycoproteins. The protocol includes construction and culture of the bacterial chassis strain, cell-free lysate production, assembly of freeze-dried reactions, cell-free glycoprotein synthesis, and glycoprotein characterization, all of which can be completed in one week or less. We anticipate that cell-free technologies, along with this comprehensive user manual, will help accelerate development and distribution of glycoprotein therapeutics and vaccines.

Profiling Germinal Center-like B Cell Responses to Conjugate Vaccines Using Synthetic Immune Organoids.

Glycoengineered bacteria have emerged as a cost-effective platform for rapid and controllable biosynthesis of designer conjugate vaccines. However, little is known about the engagement of such conjugates with naïve B cells to induce the formation of germinal centers (GC), a subanatomical microenvironment that converts naïve B cells into antibody-secreting plasma cells. Using a three-dimensional biomaterials-based B-cell follicular organoid system, we demonstrate that conjugates triggered robust expression of hallmark GC markers, B cell receptor clustering, intracellular signaling, and somatic hypermutation. These responses depended on the relative immunogenicity of the conjugate and correlated with the humoral response in vivo. The occurrence of these mechanisms was exploited for the discovery of high-affinity antibodies against components of the conjugate on a time scale that was significantly shorter than for typical animal immunization-based workflows. Collectively, these findings highlight the potential of synthetic organoids for rapidly predicting conjugate vaccine efficacy as well as expediting antigen-specific antibody discovery.

A low-cost recombinant glycoconjugate vaccine confers immunogenicity and protection against enterotoxigenic Escherichia coli infections in mice.

Enterotoxigenic Escherichia coli (ETEC) is the primary etiologic agent of traveler's diarrhea and a major cause of diarrheal disease and death worldwide, especially in infants and young children. Despite significant efforts over the past several decades, an affordable vaccine that appreciably decreases mortality and morbidity associated with ETEC infection among children under the age of 5 years remains an unmet aspirational goal. Here, we describe robust, cost-effective biosynthetic routes that leverage glycoengineered strains of non-pathogenic E. coli or their cell-free extracts for producing conjugate vaccine candidates against two of the most prevalent O serogroups of ETEC, O148 and O78. Specifically, we demonstrate site-specific installation of O-antigen polysaccharides (O-PS) corresponding to these serogroups onto licensed carrier proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. The resulting conjugates stimulate strong O-PS-specific humoral responses in mice and elicit IgG antibodies that possess bactericidal activity against the cognate pathogens. We also show that one of the prototype conjugates decorated with serogroup O148 O-PS reduces ETEC colonization in mice, providing evidence of vaccine-induced mucosal protection. We anticipate that our bacterial cell-based and cell-free platforms will enable creation of multivalent formulations with the potential for broad ETEC serogroup protection and increased access through low-cost biomanufacturing.

A modular vaccine platform enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens.

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.