Biology subjects

Alabi, C. A.

Publications and source records attributed to Alabi, C. A..

8 recordsLinked to original sources

bioRxiv2025

Stabilized Full-Length Measles Fusion Protein Elicits Potent Immunity and Protection In Vivo

Measles virus (MeV) is a highly contagious pathogen that causes significant morbidity and mortality in populations with low vaccination coverage. Infection typically leads to immune amnesia and, in rare cases, fatal neurological disease. While current live-attenuated vaccines are highly effective, they primarily elicit neutralizing antibodies against the hemagglutinin (H) glycoprotein, with a less robust response to the fusion (F) protein, a key protein for viral entry. To improve the immunogenicity of the F protein, we designed and characterized stabilized, prefusion MeV F protein antigens. We engineered both soluble ectodomains (FECTO) and full-length, membrane-embedded proteins (FFL) with mutations that confer thermal stability. Cryo-electron microscopy confirmed that these engineered antigens faithfully maintain the native prefusion conformation. When evaluated in a cotton rat model, immunization with either FECTO or FFL constructs induced neutralizing antibodies and elicited protection against viral challenge. The most stable full-length construct (FFL 3M) elicited a more potent neutralizing antibody response than its ectodomain counterpart. Importantly, no evidence of vaccine-enhanced respiratory disease was observed. These findings establish that a thermostable, full-length F protein is a superior immunogen to its soluble ectodomain. This work presents a promising candidate for next-generation, non-replicating measles vaccines intended to complement current vaccination strategies and provide a safe option for immunocompromised individuals and others who cannot receive live-virus vaccines. One-Sentence SummaryA prefusion-stabilized, full-length measles Fusion glycoprotein immunogen induces strong neutralizing responses and offers protection against challenge with wild-type virus.

immunology↗

bioRxiv2025

Intracellular delivery of full-length antibodies via organ targeted lipid nanoparticles

Antibodies are proteins prized for their ability to bind to extracellular antigens with exceptionally high affinities and specificities. These features have motivated researchers to utilize antibody-antigen binding to inhibit intracellular disease targets in the proteome, yet delivery of antibodies into the cytosol of cells has long been a considerable challenge. Here, we outline the development of a novel lipid nanoparticle (LNP) platform for delivering antibodies into cells to selectively inhibit disease-relevant cytosolic targets. This approach efficiently delivers various therapeutic antibodies into multiple cancer cell lines, inhibiting key transcription factors in inflammatory and cancer signaling pathways. We further demonstrate systemic delivery of therapeutic antibodies in disease models, including alpha-synuclein-specific antibodies for Parkinsons disease and RelA-specific IgGs for acute lung injury using targeted LNP formulations. This work establishes a promising method for using LNPs for the delivery of antibody and antibody-derived therapeutics intracellularly to treat numerous proteome targets.

bioengineering↗

bioRxiv2024

Programmable protein degraders enable selective knockdown of pathogenic β-catenin subpopulations in vitro and in vivo

Aberrant activation of Wnt signaling results in unregulated accumulation of cytosolic {beta}-catenin, which subsequently enters the nucleus and promotes transcription of genes that contribute to cellular proliferation and malignancy. Here, we sought to eliminate pathogenic {beta}-catenin from the cytosol using designer ubiquibodies (uAbs), chimeric proteins composed of an E3 ubiquitin ligase and a target-binding domain that redirect intracellular proteins to the proteasome for degradation. To accelerate uAb development, we leveraged a protein language model (pLM)-driven algorithm called SaLT&PepPr to computationally design "guide" peptides with affinity for {beta}-catenin, which were subsequently fused to the catalytic domain of a human E3 called C-terminus of Hsp70-interacting protein (CHIP). Expression of the resulting peptide-guided uAbs in colorectal cancer cells led to the identification of several designs that significantly reduced the abnormally stable pool of free {beta}-catenin in the cytosol and nucleus while preserving the normal membrane-associated subpopulation. This selective knockdown of pathogenic {beta}-catenin suppressed Wnt/{beta}-catenin signaling and impaired tumor cell survival and proliferation. Furthermore, one of the best degraders selectively decreased cytosolic but not membrane-associated {beta}-catenin levels in livers of BALB/c mice following delivery as a lipid nanoparticle (LNP)-encapsulated mRNA. Collectively, these findings reveal the unique ability of uAbs to selectively eradicate abnormal proteins in vitro and in vivo and open the door to peptide-programmable biologic modulators of other disease-causing proteins.

bioengineering↗

bioRxiv2024

Discovery of a single-subunit oligosaccharyltransferase that enables glycosylation of full-length IgG antibodies in Escherichia coli

Human immunoglobulin G (IgG) antibodies are one of the most important classes of biotherapeutic agents and undergo glycosylation at the conserved N297 site in the CH2 domain, which is critical for IgG Fc effector functions and anti-inflammatory activity. Hence, technologies for producing authentically glycosylated IgGs are in high demand. While attempts to engineer Escherichia coli for this purpose have been described, they have met limited success due in part to the lack of available oligosaccharyltransferase (OST) enzymes that can install N-linked glycans within the QYNST sequon of the IgG CH2 domain. Here, we identified a previously uncharacterized single-subunit OST (ssOST) from the bacterium Desulfovibrio marinus that exhibited greatly relaxed substrate specificity and, as a result, was able to catalyze glycosylation of native CH2 domains in the context of both a hinge-Fc fragment and a full-length IgG. Although the attached glycans were bacterial in origin, conversion to a homogeneous, asialo complex-type G2 N-glycan at the QYNST sequon of the E. coli-derived hinge-Fc was achieved via chemoenzymatic glycan remodeling. Importantly, the resulting G2-hinge-Fc exhibited strong binding to human Fc{gamma}RIIIa (CD16a), one of the most potent receptors for eliciting antibody-dependent cellular cytotoxicity (ADCC). Taken together, the discovery of a unique ssOST from D. marinus provides previously unavailable biocatalytic capabilities to the bacterial glycoprotein engineering toolbox and opens the door to using E. coli for the production and glycoengineering of human IgGs and fragments derived thereof.

synthetic biology↗

bioRxiv2024

Nanoparticle-mediated delivery of peptide-based degraders enables targeted protein degradation

The development of small molecule-based degraders against intracellular protein targets is a rapidly growing field that is hindered by the limited availability of high-quality small molecule ligands that bind to the target of interest. Despite the feasibility of designing peptide ligands against any protein target, peptide-based degraders still face significant obstacles such as, limited serum stability and poor cellular internalization. To overcome these obstacles, we repurposed lipid nanoparticle (LNP) formulations to facilitate the delivery of Peptide-based proteolysis TArgeting Chimeras (PepTACs). Our investigations reveal robust intracellular transport of PepTAC-LNPs across various clinically relevant human cell lines. Our studies also underscore the critical nature of the linker and hydrophobic E3 binding ligand for efficient LNP packaging and transport. We demonstrate the clinical utility of this strategy by engineering PepTACs targeting two critical transcription factors, {beta}-catenin and CREPT (cell-cycle-related and expression-elevated protein in tumor), involved in the Wnt-signalling pathway. The PepTACs induced target-specific protein degradation and led to a significant reduction in Wnt-driven gene expression and cancer cell proliferation. Mouse biodistribution studies revealed robust accumulation of PepTAC-LNPs in the spleen and liver, among other organs, and PepTACs designed against {beta}-catenin and formulated in LNPs showed a reduction in {beta}-catenin levels in the liver. Our findings demonstrate that LNPs can be formulated to encapsulate PepTACs, thus enabling robust delivery and potent intracellular protein degradation.

bioengineering↗

bioRxiv2024

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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/578333v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@68fb6aorg.highwire.dtl.DTLVardef@52166aorg.highwire.dtl.DTLVardef@13dbfaeorg.highwire.dtl.DTLVardef@f352ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

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 {beta}-catenin signaling. Further, we show that LNPs encapsulating cloaked fluorescent proteins distribute to major organs in mice following systemic administration. Overall, our results point towards a generalizable platform that can be employed for intracellular delivery of a wide range of protein cargos.

bioengineering↗

bioRxiv2020

Intranasal fusion inhibitory lipopeptide prevents direct contact SARS-CoV-2 transmission in ferrets

Containment of the COVID-19 pandemic requires reducing viral transmission. SARS-CoV-2 infection is initiated by membrane fusion between the viral and host cell membranes, mediated by the viral spike protein. We have designed a dimeric lipopeptide fusion inhibitor that blocks this critical first step of infection for emerging coronaviruses and document that it completely prevents SARS-CoV-2 infection in ferrets. Daily intranasal administration to ferrets completely prevented SARS-CoV-2 direct-contact transmission during 24-hour co-housing with infected animals, under stringent conditions that resulted in infection of 100% of untreated animals. These lipopeptides are highly stable and non-toxic and thus readily translate into a safe and effective intranasal prophylactic approach to reduce transmission of SARS-CoV-2. One-sentence summaryA dimeric form of a SARS-CoV-2-derived lipopeptide is a potent inhibitor of fusion and infection in vitro and transmission in vivo.

microbiology↗