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1.
Front Cell Infect Microbiol ; 12: 802147, 2022.
Article in English | MEDLINE | ID: covidwho-1753359

ABSTRACT

Owing to the outbreak of the novel coronavirus (SARS-CoV-2) worldwide at the end of 2019, the development of a SARS-CoV-2 vaccine became an urgent need. In this study, we developed a type 9 adeno-associated virus vectored vaccine candidate expressing a dimeric receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) and evaluated its immunogenicity in a murine model. The vaccine candidate, named AAV9-RBD virus, was constructed by inserting a signal peptide to the N-terminus of two copies of RBD, spaced by a linker, into the genome of a type 9 adeno-associated virus. In vitro assays showed that HeLa cells infected by the recombinant AAV virus expressed high levels of the recombinant RBD protein, mostly found in the cell culture supernatant. The recombinant AAV9-RBD virus was cultured and purified. The genome titer of the purified recombinant AAV9-RBD virus was determined to be 2.4 × 1013 genome copies/mL (GC/mL) by Q-PCR. Balb/c mice were immunized with the virus by intramuscular injection or nasal drip administration. Eight weeks after immunization, neutralizing antibodies against the new coronavirus pseudovirus were detected in the sera of all mice; the mean neutralizing antibody EC50 values were 517.7 ± 292.1 (n=10) and 682.8 ± 454.0 (n=10) in the intramuscular injection group and nasal drip group, respectively. The results of this study showed that the recombinant AAV9-RBD virus may be used for the development of a SARS-CoV-2 vaccine.


Subject(s)
COVID-19 Vaccines , COVID-19 , Animals , COVID-19/prevention & control , Dependovirus/genetics , HeLa Cells , Humans , Mice , Mice, Inbred BALB C , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus
2.
Viruses ; 14(2)2022 01 27.
Article in English | MEDLINE | ID: covidwho-1667342

ABSTRACT

The hepatitis E virus (HEV) is a major global health problem, leading to large outbreaks in the developing world and chronic infections in the developed world. HEV is a non-enveloped virus, which circulates in the blood in a quasi-enveloped form. The quasi-envelope protects HEV particles from neutralising anti-capsid antibodies in the serum; however, most vaccine approaches are designed to induce an immune response against the HEV capsid. In this study, we explored systemic in vivo administration of a novel synthetic and myotropic Adeno-associated virus vector (AAVMYO3) to express the small HEV phosphoprotein ORF3 (found on quasi-enveloped HEV) in the musculature of mice, resulting in the robust and dose-dependent formation of anti-ORF3 antibodies. Neutralisation assays using the serum of ORF3 AAV-transduced mice showed a modest inhibitory effect on the infection of quasi-enveloped HEV in vivo, comparable to previously characterised anti-ORF3 antibodies used as a control. The novel AAVMYO3 capsid used in this study can serve as a versatile platform for the continued development of vector-based vaccines against HEV and other infectious agents, which could complement traditional vaccines akin to the current positive experience with SARS-CoV-2.


Subject(s)
Dependovirus/genetics , Genetic Vectors , Hepatitis Antibodies/blood , Hepatitis E virus/immunology , Muscles/virology , Viral Proteins/immunology , Absorption, Physiological , Animals , Dependovirus/immunology , Female , Hepatitis Antibodies/immunology , Hepatitis E virus/genetics , Mice , Mice, Inbred BALB C , Viral Proteins/administration & dosage , Viral Proteins/genetics
3.
Mol Ther ; 30(5): 1994-2004, 2022 May 04.
Article in English | MEDLINE | ID: covidwho-1612107

ABSTRACT

Adeno-associated virus (AAV)-mediated gene delivery holds great promise for gene therapy. However, the non-invasive delivery of AAV for lung tissues has not been adequately established. Here, we revealed that the intratracheal administration of an appropriate amount of AAV2/8 predominantly targets lung tissue. AAV-mediated gene delivery that we used in this study induced the expression of the desired protein in lung parenchymal cells, including alveolar type II cells. We harnessed the technique to develop severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-susceptible mice. Three kinds of immune function-relevant gene knockout (KO) mice were transduced with AAV encoding human angiotensin-converting enzyme 2 (hACE2) and then injected with SARS-CoV-2. Among these mice, type I interferon receptor (IFNAR) KO mice showed increased viral titer in the lungs compared to that in the other KO mice. Moreover, nucleocapsid protein of SARS-CoV-2 and multiple lesions in the trachea and lung were observed in AAV-hACE2-transduced, SARS-CoV-2-infected IFNAR KO mice, indicating the involvement of type I interferon signaling in the protection of SARS-CoV-2. In this study, we demonstrate the ease and rapidness of the intratracheal administration of AAV for targeting lung tissue in mice, and this can be used to study diverse pulmonary diseases.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , COVID-19/therapy , Dependovirus/genetics , Disease Models, Animal , Disease Susceptibility , Lung/pathology , Mice , Mice, Transgenic , SARS-CoV-2/genetics
4.
Nucleic Acid Ther ; 31(5): 321-323, 2021 10.
Article in English | MEDLINE | ID: covidwho-1467290

ABSTRACT

The utilization of the mRNA-based Pfizer-BioNTech and Moderna coronavirus disease 2019 (COVID-19) vaccines represents the culmination of many years of nonviral nucleic acid delivery, but more importantly, they signify a massive clinical scientific success. Scientists working in the area of nucleic acid delivery using lipid nanoparticles will undoubtedly be energized by the success of these vaccines and begin to collect much needed data in the realm of nonviral-based RNA and DNA delivery, specifically, the use of lipid nanoparticles, the immune response, safety, and efficacy. It is easily conceivable that in the future we can utilize these data to help streamline our approach for the delivery of DNA for gene therapy and regulatory RNAs for therapeutic and regenerative medicine (ie, wound repair) applications.


Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , DNA/pharmacokinetics , Gene Transfer Techniques , RNA, Messenger/pharmacokinetics , Biotechnology/trends , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/biosynthesis , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/genetics , DNA/chemistry , Data Mining , Dependovirus/genetics , Dependovirus/immunology , Humans , Liposomes/chemistry , Liposomes/pharmacokinetics , Nanoparticles/administration & dosage , Nanoparticles/chemistry , RNA, Messenger/chemistry , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity
5.
Stem Cell Res Ther ; 11(1): 448, 2020 10 23.
Article in English | MEDLINE | ID: covidwho-1388825

ABSTRACT

Gene therapy is being investigated for a range of serious lung diseases, such as cystic fibrosis and emphysema. Recombinant adeno-associated virus (rAAV) is a well-established, safe, viral vector for gene delivery with multiple naturally occurring and artificial serotypes available displaying alternate cell, tissue, and species-specific tropisms. Efficient AAV serotypes for the transduction of the conducting airways have been identified for several species; however, efficient serotypes for human lung parenchyma have not yet been identified. Here, we screened the ability of multiple AAV serotypes to transduce lung bud organoids (LBOs)-a model of human lung parenchyma generated from human embryonic stem cells. Microinjection of LBOs allowed us to model transduction from the luminal surface, similar to dosing via vector inhalation. We identified the naturally occurring rAAV2 and rAAV6 serotypes, along with synthetic rAAV6 variants, as having tropism for the human lung parenchyma. Positive staining of LBOs for surfactant proteins B and C confirmed distal lung identity and suggested the suitability of these vectors for the transduction of alveolar type II cells. Our findings establish LBOs as a new model for pulmonary gene therapy and stress the relevance of LBOs as a viral infection model of the lung parenchyma as relevant in SARS-CoV-2 research.


Subject(s)
Dependovirus/genetics , Genetic Therapy/methods , Human Embryonic Stem Cells/cytology , Lung Diseases/therapy , Organoids/cytology , Cell Line , Dependovirus/immunology , Gene Transfer Techniques , Genetic Vectors/genetics , Humans , Lung/metabolism , Models, Biological , Parenchymal Tissue/cytology
6.
PLoS Pathog ; 17(8): e1009758, 2021 08.
Article in English | MEDLINE | ID: covidwho-1352713

ABSTRACT

Since the pandemic of COVID-19 has intensely struck human society, small animal model for this infectious disease is in urgent need for basic and pharmaceutical research. Although several COVID-19 animal models have been identified, many of them show either minimal or inadequate pathophysiology after SARS-CoV-2 challenge. Here, we describe a new and versatile strategy to rapidly establish a mouse model for emerging infectious diseases in one month by multi-route, multi-serotype transduction with recombinant adeno-associated virus (AAV) vectors expressing viral receptor. In this study, the proposed approach enables profound and enduring systemic expression of SARS-CoV-2-receptor hACE2 in wild-type mice and renders them vulnerable to SARS-CoV-2 infection. Upon virus challenge, generated AAV/hACE2 mice showed pathophysiology closely mimicking the patients with severe COVID-19. The efficacy of a novel therapeutic antibody cocktail RBD-chAbs for COVID-19 was tested and confirmed by using this AAV/hACE2 mouse model, further demonstrating its successful application in drug development.


Subject(s)
COVID-19 , Communicable Diseases, Emerging , Disease Models, Animal , 3T3 Cells , Angiotensin-Converting Enzyme 2/genetics , Animals , Antibodies, Viral/immunology , Antibodies, Viral/therapeutic use , COVID-19/immunology , COVID-19/pathology , COVID-19/physiopathology , Chlorocebus aethiops , Dependovirus/genetics , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Transduction, Genetic , Vero Cells
7.
Cell Host Microbe ; 29(9): 1437-1453.e8, 2021 09 08.
Article in English | MEDLINE | ID: covidwho-1347535

ABSTRACT

The SARS-CoV-2 pandemic has affected more than 185 million people worldwide resulting in over 4 million deaths. To contain the pandemic, there is a continued need for safe vaccines that provide durable protection at low and scalable doses and can be deployed easily. Here, AAVCOVID-1, an adeno-associated viral (AAV), spike-gene-based vaccine candidate demonstrates potent immunogenicity in mouse and non-human primates following a single injection and confers complete protection from SARS-CoV-2 challenge in macaques. Peak neutralizing antibody titers are sustained at 1 year and complemented by functional memory T cell responses. The AAVCOVID vector has no relevant pre-existing immunity in humans and does not elicit cross-reactivity to common AAVs used in gene therapy. Vector genome persistence and expression wanes following injection. The single low-dose requirement, high-yield manufacturability, and 1-month stability for storage at room temperature may make this technology well suited to support effective immunization campaigns for emerging pathogens on a global scale.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Vaccines/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/genetics , Dependovirus/genetics , Dependovirus/metabolism , Female , Humans , Immunogenicity, Vaccine/immunology , Immunologic Memory/immunology , Macaca fascicularis , Macaca mulatta , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , T-Lymphocytes/immunology , Transgenes/genetics , Vaccination/methods , Viral Load/immunology
8.
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
9.
Nature ; 593(7859): 424-428, 2021 05.
Article in English | MEDLINE | ID: covidwho-1152859

ABSTRACT

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.


Subject(s)
Antibodies, Bispecific/immunology , Antibodies, Neutralizing/immunology , COVID-19/immunology , COVID-19/virology , Immunoglobulin G/immunology , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Bispecific/therapeutic use , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/therapeutic use , Body Weight , COVID-19/drug therapy , COVID-19/prevention & control , Dependovirus/genetics , Disease Models, Animal , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Female , Humans , Immune Evasion/genetics , Mice , Mice, Inbred C57BL , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
10.
Int J Mol Sci ; 21(14)2020 Jul 14.
Article in English | MEDLINE | ID: covidwho-649852

ABSTRACT

Effective treatment of retinal diseases with adeno-associated virus (AAV)-mediated gene therapy is highly dependent on the proportion of successfully transduced cells. However, due to inflammatory reactions at high vector doses, adjunctive treatment may be necessary to enhance the therapeutic outcome. Hydroxychloroquine and chloroquine are anti-malarial drugs that have been successfully used in the treatment of autoimmune diseases. Evidence suggests that at high concentrations, hydroxychloroquine and chloroquine can impact viral infection and replication by increasing endosomal and lysosomal pH. This effect has led to investigations into the potential benefits of these drugs in the treatment of viral infections, including human immunodeficiency virus and severe acute respiratory syndrome coronavirus-2. However, at lower concentrations, hydroxychloroquine and chloroquine appear to exert immunomodulatory effects by inhibiting nucleic acid sensors, including toll-like receptor 9 and cyclic GMP-AMP synthase. This dose-dependent effect on their mechanism of action supports observations of increased viral infections associated with lower drug doses. In this review, we explore the immunomodulatory activity of hydroxychloroquine and chloroquine, their impact on viral infections, and their potential to improve the efficacy and safety of retinal gene therapy by reducing AAV-induced immune responses. The safety and practicalities of delivering hydroxychloroquine into the retina will also be discussed.


Subject(s)
Chloroquine/therapeutic use , Genetic Therapy , Hydroxychloroquine/therapeutic use , Retinal Diseases/therapy , Virus Diseases/drug therapy , Animals , Betacoronavirus/drug effects , Chloroquine/pharmacology , Dependovirus/genetics , Humans , Hydroxychloroquine/pharmacology , Immunomodulation/drug effects , Retinal Diseases/pathology , SARS-CoV-2
11.
J Exp Med ; 217(12)2020 12 07.
Article in English | MEDLINE | ID: covidwho-709757

ABSTRACT

Severe acute respiratory syndrome-coronavirus 2 (SARS-Cov-2) has caused over 13,000,000 cases of coronavirus disease (COVID-19) with a significant fatality rate. Laboratory mice have been the stalwart of therapeutic and vaccine development; however, they do not support infection by SARS-CoV-2 due to the virus's inability to use the mouse orthologue of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis, these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno-associated virus (AAV)-mediated expression of hACE2. These mice support viral replication and exhibit pathological findings found in COVID-19 patients. Moreover, we show that type I interferons do not control SARS-CoV-2 replication in vivo but are significant drivers of pathological responses. Thus, the AAV-hACE2 mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds.


Subject(s)
Betacoronavirus/metabolism , Coronavirus Infections/metabolism , Disease Models, Animal , Interferon Type I/metabolism , Mice/genetics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/metabolism , Angiotensin-Converting Enzyme 2 , Animals , COVID-19 , Cell Line, Tumor , Coronavirus Infections/pathology , Coronavirus Infections/virology , Dependovirus/genetics , Female , Humans , Inflammation/metabolism , Lung/pathology , Lung/virology , Male , Mice, Inbred C57BL , Mice, Transgenic , Pandemics , Parvoviridae Infections/metabolism , Parvoviridae Infections/virology , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , SARS-CoV-2 , Signal Transduction/genetics , Virus Replication/genetics
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