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1.
Sci Rep ; 12(1): 5424, 2022 Mar 31.
Article in English | MEDLINE | ID: covidwho-1768856

ABSTRACT

The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene "landing pad" composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free integration of donor constructs at efficiencies > 40% with up to ~ 43 kb transgenes. Coupled with a nanopore-based Cas9-targeted sequencing (nCATS), complete verification of precise insertion sequence was achieved. As a proof-of-concept we describe the development of C57BL/6J and NSG Krt18-ACE2 models for SARS-CoV2 research with verified heterozygous N1 animals within ~ 4 months. Additionally, we created a series of mice with diverse backgrounds carrying a single att site including FVB/NJ, PWK/PhJ, NOD/ShiLtJ, CAST/EiJ and DBA/2J allowing for rapid transgene insertion. Combined, this system enables predictable, rapid development with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds.


Subject(s)
Bacteriophages , COVID-19 , Animals , Bacteriophages/genetics , DNA , Gene Transfer Techniques , Genetic Background , Integrases/genetics , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Inbred NOD , RNA, Viral , SARS-CoV-2
2.
Viruses ; 14(2)2022 02 14.
Article in English | MEDLINE | ID: covidwho-1687052

ABSTRACT

The evolution of the SARS-CoV-2 virus during the COVID-19 pandemic was accompanied by the emergence of new heavily mutated viral variants with increased infectivity and/or resistance to detection by the human immune system. To respond to the urgent need for advanced methods and materials to empower a better understanding of the mechanisms of virus's adaptation to human host cells and to the immuno-resistant human population, we suggested using recombinant filamentous bacteriophages, displaying on their surface foreign peptides termed "mimotopes", which mimic the structure of viral receptor-binding sites on the viral spike protein and can serve as molecular probes in the evaluation of molecular mechanisms of virus infectivity. In opposition to spike-binding antibodies that are commonly used in studying the interaction of the ACE2 receptor with SARS-CoV-2 variants in vitro, phage spike mimotopes targeted to other cellular receptors would allow discovery of their role in viral infection in vivo using cell culture, tissue, organs, or the whole organism. Phage mimotopes of the SARS-CoV-2 Spike S1 protein have been developed using a combination of phage display and molecular mimicry concepts, termed here "phage mimicry", supported by bioinformatics methods. The key elements of the phage mimicry concept include: (1) preparation of a collection of p8-type (landscape) phages, which interact with authentic active receptors of live human cells, presumably mimicking the binding interactions of human coronaviruses such as SARS-CoV-2 and its variants; (2) discovery of closely related amino acid clusters with similar 3D structural motifs on the surface of natural ligands (FGF1 and NRP1), of the model receptor of interest FGFR and the S1 spike protein; and (3) an ELISA analysis of the interaction between candidate phage mimotopes with FGFR3 (a potential alternative receptor) in comparison with ACE2 (the authentic receptor).


Subject(s)
Bacteriophages/genetics , Cell Surface Display Techniques/methods , Molecular Mimicry , Receptors, Cell Surface/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Bacteriophages/metabolism , Binding Sites , Humans , Protein Binding , Receptors, Cell Surface/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Virus Attachment
3.
ACS Synth Biol ; 11(2): 528-537, 2022 02 18.
Article in English | MEDLINE | ID: covidwho-1655460

ABSTRACT

Over the past decades, there have been numerous outbreaks, including parasitic, fungal, bacterial, and viral infections, worldwide. The rate at which infectious diseases are emerging is disproportionate to the rate of development for new strategies that could combat them. Therefore, there is an increasing demand to develop novel, specific, sensitive, and effective methods for infectious disease diagnosis and treatment. Designed synthetic systems and devices are becoming powerful tools to treat human diseases. The advancement in synthetic biology offers efficient, accurate, and cost-effective platforms for detecting and preventing infectious diseases. Herein we focus on the latest state of living theranostics and its implications.


Subject(s)
Communicable Disease Control/methods , Synthetic Biology , Bacterial Physiological Phenomena , Bacteriophages/genetics , COVID-19/therapy , COVID-19/virology , Humans , Pandemics , Precision Medicine , SARS-CoV-2/isolation & purification , SARS-CoV-2/pathogenicity
4.
Nat Biotechnol ; 40(3): 374-381, 2022 03.
Article in English | MEDLINE | ID: covidwho-1483138

ABSTRACT

Multimodal measurements of single-cell profiles are proving increasingly useful for characterizing cell states and regulatory mechanisms. In the present study, we developed PHAGE-ATAC (Assay for Transposase-Accessible Chromatin), a massively parallel droplet-based method that uses phage displaying, engineered, camelid single-domain antibodies ('nanobodies') for simultaneous single-cell measurements of protein levels and chromatin accessibility profiles, and mitochondrial DNA-based clonal tracing. We use PHAGE-ATAC for multimodal analysis in primary human immune cells, sample multiplexing, intracellular protein analysis and the detection of SARS-CoV-2 spike protein in human cell populations. Finally, we construct a synthetic high-complexity phage library for selection of antigen-specific nanobodies that bind cells of particular molecular profiles, opening an avenue for protein detection, cell characterization and screening with single-cell genomics.


Subject(s)
Bacteriophages , COVID-19 , Bacteriophages/genetics , Chromatin/genetics , Humans , SARS-CoV-2 , Single-Cell Analysis/methods , Spike Glycoprotein, Coronavirus
5.
Proc Natl Acad Sci U S A ; 118(30)2021 07 27.
Article in English | MEDLINE | ID: covidwho-1301236

ABSTRACT

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.


Subject(s)
Bacteriophages/genetics , COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Immunization Programs , Administration, Inhalation , Animals , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/immunology , Dependovirus/genetics , Drug Storage , Female , Immunization Programs/methods , Immunogenicity, Vaccine , Mice , Mice, Inbred BALB C , Proof of Concept Study , Temperature
6.
Curr Opin Biotechnol ; 68: 160-165, 2021 04.
Article in English | MEDLINE | ID: covidwho-970472

ABSTRACT

Bacteriophages have attracted extensive interest in vaccine design. This includes the use of phage display technology to select antigens, the use of engineered phages displaying target antigens in vaccine formulations, and phage DNA vaccines. However, the development of these approaches is limited in part by uncertainty regarding the underlying mechanisms by which phages elicit immunity. This has stymied the clinical development of this technology. Here we review the immunology of phage vaccines and highlight the gaps in our knowledge regarding the underlying mechanisms. First, we review the basic biology of phages and their use in vaccines. Next we discuss what is known about the mechanisms of immunity against engineered phages and phage DNA. Finally, we highlight the gaps in our understanding regarding the immunogenicity of these preparations. We argue that mechanistic insight into the immunology of phage vaccines is essential for the further development and clinical utility of these technologies.


Subject(s)
Bacteriophages , Vaccines , Bacteriophages/genetics , Cell Surface Display Techniques
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