Glioblastoma persists as a uniformly deadly diagnosis for patients and effective therapeutic options are gravely needed. Recently, targeted gene therapy approaches are re-emerging as attractive experimental clinical agents. Our ligand-directed hybrid virus of adeno-associated virus and phage (AAVP) is a targeted gene delivery vector that has been used in several formulations displaying targeting ligand peptides to deliver clinically applicable transgenes. Here we compared different constructs side-by-side in a tumor model, an orthotopic model of xenograft human glioblastoma cells stereotactically-implanted in immunodeficient mice. We have used divergent therapeutic strategies for two AAVP constructs, both displaying a double-cyclic RGD4C motif ligand specific for alpha V integrins expressed in tumor vascular endothelium but carrying different genes of interest for the treatment of intracranial xenografted tumors. One construct delivered tumor necrosis factor (TNF), a purely cytotoxic gene for anti-tumor activity (RGD4C-AAVP-TNF); in the other construct, we delivered Herpes simplex virus thymidine kinase (HSVtk) for in tandem molecular-genetic imaging and targeted therapy (RGD4C-AAVP- HSVtk) utilizing ganciclovir (GCV) for suicide gene therapy. Both AAVP constructs demonstrated anti-tumor activity, with damage to the tumor-associated neovasculature and induction of cell death evident after treatment. In addition, the ability to monitor transgene expression with a radiolabeled HSVtk substrate pre and post GCV treatment demonstrated the theranostic potential of RGD4C-AAVP-HSVtk. We conclude that targeted AAVP constructs delivering either cytotoxic TNF or theranostic HSVtk followed by suicide gene therapy with GCV have comparable preclinical efficacy, at least in this standard experimental model. The results presented here provide a blueprint for future studies of targeted gene delivery against human glioblastomas and other brain tumors.
Development of effective vaccines against coronavirus disease 2019 (COVID-19) is a global imperative. Rapid immunization of the entire human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and different vaccine approaches are being pursued. Engineered filamentous bacteriophage (phage) particles have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the development and initial evaluation of two targeted phage-based vaccination approaches against SARS-CoV-2: dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. For peptide-targeted phage, we performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein. One of these epitopes displayed on the major capsid protein pVIII of phage induced a specific and sustained humoral response when injected in mice. These phage were further engineered to simultaneously display the peptide CAKSMGDIVC on the minor capsid protein pIII to enable their transport from the lung epithelium into the systemic circulation. Aerosolization of these “dual-display” phage into the lungs of mice generated a systemic and specific antibody response. In the second approach, targeted AAVP particles were engineered to deliver the entire S protein gene under the control of a constitutive CMV promoter. This induced tissue-specific transgene expression, stimulating a systemic S protein-specific antibody response in mice. With these proof-of-concept preclinical experiments, we show that both targeted phage- and AAVP-based particles serve as robust yet versatile platforms that can promptly yield COVID-19 vaccine prototypes for translational development.
Summary Large-scale mapping of antigens and epitopes is pivotal for developing immunotherapies but challenging, especially for eukaryotic pathogens, owing to their large genomes. Here, we developed an integrated platform for genome phage display (gPhage) to show that unbiased libraries of the eukaryotic parasite Trypanosoma cruzi enable the identification of thousands of antigens recognized by serum samples from patients with Chagas disease. Because most of these antigens are hypothetical proteins, gPhage provides evidence of their expression during infection. We built and validated a comprehensive map of Chagas disease antibody response to show how linear and putative conformation epitopes, many rich in repetitive elements, allow the parasite to evade a buildup of neutralizing antibodies directed against protein domains that mediate infection pathogenesis. Thus, the gPhage platform is a reproducible and effective tool for rapid simultaneous identification of epitopes and antigens, not only in Chagas disease but perhaps also in globally emerging/reemerging acute pathogens.
Development of effective vaccines against Coronavirus Disease 2019 (COVID-19) is a global imperative. Rapid immunization of the world human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and many different vaccine approaches are being pursued to meet this task. Engineered filamentous bacteriophage (phage) have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the design, development, and initial evaluation of targeted phage-based vaccination approaches against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) by using dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. Towards a unique phage- and AAVP-based dual-display candidate approach, we first performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein for display on the recombinant major capsid coat protein pVIII. Targeted phage particles carrying one of these epitopes induced a strong and specific humoral response. In an initial experimental approach, when these targeted phage particles were further genetically engineered to simultaneously display a ligand peptide (CAKSMGDIVC) on the minor capsid protein pIII, which enables receptor-mediated transport of phage particles from the lung epithelium into the systemic circulation (termed dual-display), they enhanced a systemic and specific spike (S) protein-specific antibody response upon aerosolization into the lungs of mice. In a second line of investigation, we engineered targeted AAVP particles to deliver the entire S protein gene under the control of a constitutive cytomegalovirus (CMV) promoter, which induced tissue-specific transgene expression stimulating a systemic S protein-specific antibody response. As proof-of-concept preclinical experiments, we show that targeted phage- and AAVP-based particles serve as robust yet versatile enabling platforms for ligand-directed immunization and promptly yield COVID-19 vaccine prototypes for further translational development.
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