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1.
J Immunol Methods ; 500: 113182, 2022 01.
Article in English | MEDLINE | ID: covidwho-1768318

ABSTRACT

Serology tests for SARS-CoV-2 have proven to be important tools to fight against the COVID-19 pandemic. These serological tests can be used in low-income and remote areas for patient contact tracing, epidemiologic studies and vaccine efficacy evaluations. In this study, we used a semi-stable mammalian episomal expression system to produce high quantities of the receptor-binding domain-RBD of SARS-CoV-2 in a simple and very economical way. The recombinant antigen was tested in an in-house IgG ELISA for COVID-19 with a panel of human sera. A performance comparison of this serology test with a commercial test based on the full-length spike protein showed 100% of concordance between tests. Thus, this serological test can be an attractive and inexpensive option in scenarios of limited resources to face the COVID-19 pandemic.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/economics , COVID-19 Serological Testing/economics , Costs and Cost Analysis , Enzyme-Linked Immunosorbent Assay , Genetic Engineering , Humans , Immunoglobulin G/genetics , Immunoglobulin G/metabolism , Protein Binding , Protein Interaction Domains and Motifs/genetics , Spike Glycoprotein, Coronavirus/genetics
2.
Int J Mol Sci ; 23(6)2022 Mar 13.
Article in English | MEDLINE | ID: covidwho-1765731

ABSTRACT

Crop breeding has mainly been focused on increasing productivity, either directly or by decreasing the losses caused by biotic and abiotic stresses (that is, incorporating resistance to diseases and enhancing tolerance to adverse conditions, respectively). Quite the opposite, little attention has been paid to improve the nutritional value of crops. It has not been until recently that crop biofortification has become an objective within breeding programs, through either conventional methods or genetic engineering. There are many steps along this long path, from the initial evaluation of germplasm for the content of nutrients and health-promoting compounds to the development of biofortified varieties, with the available and future genomic tools assisting scientists and breeders in reaching their objectives as well as speeding up the process. This review offers a compendium of the genomic technologies used to explore and create biodiversity, to associate the traits of interest to the genome, and to transfer the genomic regions responsible for the desirable characteristics into potential new varieties. Finally, a glimpse of future perspectives and challenges in this emerging area is offered by taking the present scenario and the slow progress of the regulatory framework as the starting point.


Subject(s)
Biofortification , Plant Breeding , Biofortification/methods , Crops, Agricultural/genetics , Genetic Engineering/methods , Plant Breeding/methods , Plants, Genetically Modified/genetics
3.
Int J Mol Sci ; 22(20)2021 Oct 09.
Article in English | MEDLINE | ID: covidwho-1736951

ABSTRACT

Throughout history, nature has been acknowledged for being a primordial source of various bioactive molecules in which human macular carotenoids are gaining significant attention. Among 750 natural carotenoids, lutein, zeaxanthin and their oxidative metabolites are selectively accumulated in the macular region of living beings. Due to their vast applications in food, feed, pharmaceutical and nutraceuticals industries, the global market of lutein and zeaxanthin is continuously expanding but chemical synthesis, extraction and purification of these compounds from their natural repertoire e.g., plants, is somewhat costly and technically challenging. In this regard microbial as well as microalgal carotenoids are considered as an attractive alternative to aforementioned challenges. Through the techniques of genetic engineering and gene-editing tools like CRISPR/Cas9, the overproduction of lutein and zeaxanthin in microorganisms can be achieved but the commercial scale applications of such procedures needs to be done. Moreover, these carotenoids are highly unstable and susceptible to thermal and oxidative degradation. Therefore, esterification of these xanthophylls and microencapsulation with appropriate wall materials can increase their shelf-life and enhance their application in food industry. With their potent antioxidant activities, these carotenoids are emerging as molecules of vital importance in chronic degenerative, malignancies and antiviral diseases. Therefore, more research needs to be done to further expand the applications of lutein and zeaxanthin.


Subject(s)
Antioxidants/chemistry , Lutein/chemistry , Zeaxanthins/chemistry , Biological Factors/chemistry , Drug Compounding , Drug Stability , Esterification , Gene Editing , Genetic Engineering , Humans , Macula Lutea/chemistry
4.
J Healthc Eng ; 2022: 9311052, 2022.
Article in English | MEDLINE | ID: covidwho-1723969

ABSTRACT

Big data platforms can effectively analyze the data and maximize the value of the data by mining the text, digital, video, and image data in various industries. The combination of big data and various industries has brought great changes to the development of the industry. Providing data according to demand can save more time and promote the development of the industry. SARS-CoV-2 (COVID-19) is sweeping across the world, and it has spread to several countries and regions. Human infections have been reported all around the world. Due to the unique characteristics of COVID-19, no specific medicine is available yet to cure patients before the successful research and development of vaccines. Hence, it is of important significance to research and develop vaccines. Guided by the biological characteristics of COVID-19 and the philosophy of synthetic biology, this study reviews the developed genetic engineering vaccines.


Subject(s)
COVID-19 , Vaccines , Big Data , COVID-19/prevention & control , COVID-19 Vaccines , Genetic Engineering , Humans , SARS-CoV-2
5.
Plant Biotechnol J ; 20(2): 360-373, 2022 02.
Article in English | MEDLINE | ID: covidwho-1621953

ABSTRACT

In the age of synthetic biology, plastid engineering requires a nimble platform to introduce novel synthetic circuits in plants. While effective for integrating relatively small constructs into the plastome, plastid engineering via homologous recombination of transgenes is over 30 years old. Here we show the design-build-test of a novel synthetic genome structure that does not disturb the native plastome: the 'mini-synplastome'. The mini-synplastome was inspired by dinoflagellate plastome organization, which is comprised of numerous minicircles residing in the plastid instead of a single organellar genome molecule. The first mini-synplastome in plants was developed in vitro to meet the following criteria: (i) episomal replication in plastids; (ii) facile cloning; (iii) predictable transgene expression in plastids; (iv) non-integration of vector sequences into the endogenous plastome; and (v) autonomous persistence in the plant over generations in the absence of exogenous selection pressure. Mini-synplastomes are anticipated to revolutionize chloroplast biotechnology, enable facile marker-free plastid engineering, and provide an unparalleled platform for one-step metabolic engineering in plants.


Subject(s)
Genetic Engineering , Plastids , Metabolic Engineering , Plants/genetics , Plastids/genetics , Synthetic Biology , Transgenes
6.
Front Immunol ; 12: 795741, 2021.
Article in English | MEDLINE | ID: covidwho-1581316

ABSTRACT

Glycan-masking the vaccine antigen by mutating the undesired antigenic sites with an additional N-linked glycosylation motif can refocus B-cell responses to desired epitopes, without affecting the antigen's overall-folded structure. This study examined the impact of glycan-masking mutants of the N-terminal domain (NTD) and receptor-binding domain (RBD) of SARS-CoV-2, and found that the antigenic design of the S protein increases the neutralizing antibody titers against the Wuhan-Hu-1 ancestral strain and the recently emerged SARS-CoV-2 variants Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2). Our results demonstrated that the use of glycan-masking Ad-S-R158N/Y160T in the NTD elicited a 2.8-fold, 6.5-fold, and 4.6-fold increase in the IC-50 NT titer against the Alpha (B.1.1.7), Beta (B.1.351) and Delta (B.1.617.2) variants, respectively. Glycan-masking of Ad-S-D428N in the RBD resulted in a 3.0-fold and 2.0-fold increase in the IC-50 neutralization titer against the Alpha (B.1.1.7) and Beta (B.1.351) variants, respectively. The use of glycan-masking in Ad-S-R158N/Y160T and Ad-S-D428N antigen design may help develop universal COVID-19 vaccines against current and future emerging SARS-CoV-2 variants.


Subject(s)
Antigens, Viral/immunology , COVID-19/immunology , Epitopes/immunology , Protein Interaction Domains and Motifs/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adenoviridae/genetics , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody Formation/immunology , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , Disease Models, Animal , Dose-Response Relationship, Immunologic , Female , Genetic Engineering , Genetic Vectors/genetics , Humans , Immunization , Mice , Neutralization Tests , Polysaccharides , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity Relationship
7.
J Virol ; 96(3): e0183721, 2022 02 09.
Article in English | MEDLINE | ID: covidwho-1546443

ABSTRACT

Research activities with infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are currently permitted only under biosafety level 3 (BSL3) containment. Here, we report the development of a single-cycle infectious SARS-CoV-2 virus replicon particle (VRP) system with a luciferase and green fluorescent protein (GFP) dual reporter that can be safely handled in BSL2 laboratories to study SARS-CoV-2 biology. The spike (S) gene of SARS-CoV-2 encodes the envelope glycoprotein, which is essential for mediating infection of new host cells. Through deletion and replacement of this essential S gene with a luciferase and GFP dual reporter, we have generated a conditional SARS-CoV-2 mutant (ΔS-VRP) that produces infectious particles only in cells expressing a viral envelope glycoprotein of choice. Interestingly, we observed more efficient production of infectious particles in cells expressing vesicular stomatitis virus (VSV) glycoprotein G [ΔS-VRP(G)] than in cells expressing other viral glycoproteins, including S. We confirmed that infection from ΔS-VRP(G) is limited to a single round and can be neutralized by anti-VSV serum. In our studies with ΔS-VRP(G), we observed robust expression of both luciferase and GFP reporters in various human and murine cell types, demonstrating that a broad variety of cells can support intracellular replication of SARS-CoV-2. In addition, treatment of ΔS-VRP(G)-infected cells with either of the anti-CoV drugs remdesivir (nucleoside analog) and GC376 (CoV 3CL protease inhibitor) resulted in a robust decrease in both luciferase and GFP expression in a drug dose- and cell-type-dependent manner. Taken together, our findings show that we have developed a single-cycle infectious SARS-CoV-2 VRP system that serves as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high-throughput screening of antiviral drugs under BSL2 containment. IMPORTANCE Due to the highly contagious nature of SARS-CoV-2 and the lack of immunity in the human population, research on SARS-CoV-2 has been restricted to biosafety level 3 laboratories. This has greatly limited participation of the broader scientific community in SARS-CoV-2 research and thus has hindered the development of vaccines and antiviral drugs. By deleting the essential spike gene in the viral genome, we have developed a conditional mutant of SARS-CoV-2 with luciferase and fluorescent reporters, which can be safely used under biosafety level 2 conditions. Our single-cycle infectious SARS-CoV-2 virus replicon system can serve as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high-throughput screening of antiviral drugs under BSL2 containment.


Subject(s)
Genetic Engineering , Recombination, Genetic , Replicon , SARS-CoV-2/genetics , COVID-19/virology , Cell Culture Techniques , Cell Line , Containment of Biohazards/standards , Genes, Reporter , Humans , Laboratories/standards , Viral Proteins/genetics , Virus Replication
8.
J Immunol Methods ; 500: 113195, 2022 01.
Article in English | MEDLINE | ID: covidwho-1536656

ABSTRACT

COVID-19 pandemic poses a serious threat to human health; it has completely disrupted global stability, making vaccine development an important goal to achieve. Monoclonal antibodies play an important role in subunit vaccines strategies. In this work, nine murine MAbs against the RBD of the SARS-CoV-2 spike protein were obtained by hybridoma technology. Characterization of purified antibodies demonstrated that five of them have affinities in the order of 108 L/mol. Six MAbs showed specific recognition of different recombinant RBD-S antigens in solution. Studies of the additivity index of anti-RBD antibodies, by using a novel procedure to determine the additivity cut point, showed recognition of at least five different epitopes. The MAbs CBSSRBD-S.11 and CBSSRBD-S.8 revealed significant neutralizing capacity against SARS-CoV-2 in an ACE2-RBD binding inhibition assay (IC50 = 85.5pM and IC50 = 122.7pM, respectively) and in a virus neutralizing test with intact SARS-CoV-2 (VN50 = 0.552 nM and VN50 = 4.854 nM, respectively) when D614G strain was used to infect Vero cells. Also CBSSRBD-S.11 neutralized the SARS-CoV-2 strains Alpha and Beta: VN50 = 0.707 nM and VN50 = 0.132 nM, respectively. The high affinity CBSSRBD-S.8 and CBSSRBD-S.7 recognized different epitopes, so they are suitable for the development of a sandwich ELISA to quantitate RBD-S recombinant antigens in biomanufacturing processes, as well as in pharmacokinetic studies in clinical and preclinical trials.


Subject(s)
Antibodies, Monoclonal/metabolism , COVID-19 Vaccines/immunology , COVID-19/diagnosis , SARS-CoV-2/physiology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Monoclonal/genetics , COVID-19/immunology , COVID-19 Vaccines/genetics , Clinical Trials as Topic , Female , Genetic Engineering , Humans , Mice , Mice, Inbred BALB C , Protein Interaction Domains and Motifs/genetics , Vaccines, Subunit/genetics
9.
Viruses ; 13(10)2021 10 04.
Article in English | MEDLINE | ID: covidwho-1481009

ABSTRACT

The livestock industry is constantly threatened by viral disease outbreaks, including infections with zoonotic potential. While preventive vaccination is frequently applied, disease control and eradication also depend on strict biosecurity measures. Clustered regularly interspaced palindromic repeats (CRISPR) and associated proteins (Cas) have been repurposed as genome editors to induce targeted double-strand breaks at almost any location in the genome. Thus, CRISPR/Cas genome editors can also be utilized to generate disease-resistant or resilient livestock, develop vaccines, and further understand virus-host interactions. Genes of interest in animals and viruses can be targeted to understand their functions during infection. Furthermore, transgenic animals expressing CRISPR/Cas can be generated to target the viral genome upon infection. Genetically modified livestock can thereby reduce disease outbreaks and decrease zoonotic threats.


Subject(s)
Gene Editing/methods , Livestock/virology , Viruses/genetics , Animal Husbandry/methods , Animals , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Genetic Engineering , Host Microbial Interactions/genetics , Virus Diseases/prevention & control , Viruses/pathogenicity
10.
Hum Mol Genet ; 30(R2): R274-R284, 2021 10 01.
Article in English | MEDLINE | ID: covidwho-1455301

ABSTRACT

The mouse is the pre-eminent model organism for studies of mammalian gene function and has provided an extraordinarily rich range of insights into basic genetic mechanisms and biological systems. Over several decades, the characterization of mouse mutants has illuminated the relationship between gene and phenotype, providing transformational insights into the genetic bases of disease. However, if we are to deliver the promise of genomic and precision medicine, we must develop a comprehensive catalogue of mammalian gene function that uncovers the dark genome and elucidates pleiotropy. Advances in large-scale mouse mutagenesis programmes allied to high-throughput mouse phenomics are now addressing this challenge and systematically revealing novel gene function and multi-morbidities. Alongside the development of these pan-genomic mutational resources, mouse genetics is employing a range of diversity resources to delineate gene-gene and gene-environment interactions and to explore genetic context. Critically, mouse genetics is a powerful tool for assessing the functional impact of human genetic variation and determining the causal relationship between variant and disease. Together these approaches provide unique opportunities to dissect in vivo mechanisms and systems to understand pathophysiology and disease. Moreover, the provision and utility of mouse models of disease has flourished and engages cumulatively at numerous points across the translational spectrum from basic mechanistic studies to pre-clinical studies, target discovery and therapeutic development.


Subject(s)
Genetic Association Studies , Genetic Predisposition to Disease , Genome , Genomics , Alleles , Animals , Disease Models, Animal , Drug Discovery , Gene Expression Regulation , Genetic Association Studies/methods , Genetic Engineering , Genome-Wide Association Study , Genomics/methods , High-Throughput Screening Assays , Humans , Mice , Mutagenesis , Mutation , Phenomics/methods , Phenotype , Precision Medicine , Signal Transduction
12.
Proc Natl Acad Sci U S A ; 118(36)2021 09 07.
Article in English | MEDLINE | ID: covidwho-1370748

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 4 million humans globally, but there is no bona fide Food and Drug Administration-approved drug-like molecule to impede the COVID-19 pandemic. The sluggish pace of traditional therapeutic discovery is poorly suited to producing targeted treatments against rapidly evolving viruses. Here, we used an affinity-based screen of 4 billion DNA-encoded molecules en masse to identify a potent class of virus-specific inhibitors of the SARS-CoV-2 main protease (Mpro) without extensive and time-consuming medicinal chemistry. CDD-1714, the initial three-building-block screening hit (molecular weight [MW] = 542.5 g/mol), was a potent inhibitor (inhibition constant [K i] = 20 nM). CDD-1713, a smaller two-building-block analog (MW = 353.3 g/mol) of CDD-1714, is a reversible covalent inhibitor of Mpro (K i = 45 nM) that binds in the protease pocket, has specificity over human proteases, and shows in vitro efficacy in a SARS-CoV-2 infectivity model. Subsequently, key regions of CDD-1713 that were necessary for inhibitory activity were identified and a potent (K i = 37 nM), smaller (MW = 323.4 g/mol), and metabolically more stable analog (CDD-1976) was generated. Thus, screening of DNA-encoded chemical libraries can accelerate the discovery of efficacious drug-like inhibitors of emerging viral disease targets.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/genetics , Drug Discovery/methods , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Animals , COVID-19/drug therapy , COVID-19/virology , Cells, Cultured , Coronavirus 3C Proteases/metabolism , Dose-Response Relationship, Drug , Enzyme Activation , Genetic Engineering , Humans , Models, Molecular , Molecular Conformation , Molecular Structure , SARS-CoV-2/metabolism , Structure-Activity Relationship , Virus Replication
13.
Int J Mol Sci ; 22(7)2021 Mar 24.
Article in English | MEDLINE | ID: covidwho-1369767

ABSTRACT

According to Darwin's theory, endless evolution leads to a revolution. One such example is the Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas system, an adaptive immunity system in most archaea and many bacteria. Gene editing technology possesses a crucial potential to dramatically impact miscellaneous areas of life, and CRISPR-Cas represents the most suitable strategy. The system has ignited a revolution in the field of genetic engineering. The ease, precision, affordability of this system is akin to a Midas touch for researchers editing genomes. Undoubtedly, the applications of this system are endless. The CRISPR-Cas system is extensively employed in the treatment of infectious and genetic diseases, in metabolic disorders, in curing cancer, in developing sustainable methods for fuel production and chemicals, in improving the quality and quantity of food crops, and thus in catering to global food demands. Future applications of CRISPR-Cas will provide benefits for everyone and will save countless lives. The technology is evolving rapidly; therefore, an overview of continuous improvement is important. In this review, we aim to elucidate the current state of the CRISPR-Cas revolution in a tailor-made format from its discovery to exciting breakthroughs at the application level and further upcoming trends related to opportunities and challenges including ethical concerns.


Subject(s)
CRISPR-Cas Systems , Gene Editing/methods , Genetic Engineering/methods , Animals , Archaea/metabolism , Bacteria/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats , Crops, Agricultural/genetics , Genetic Engineering/history , Genome , History, 20th Century , History, 21st Century , Humans , Livestock
14.
Methods Mol Biol ; 2099: 137-159, 2020.
Article in English | MEDLINE | ID: covidwho-1292550

ABSTRACT

Since 2012, monthly cases of Middle East respiratory syndrome coronavirus (MERS-CoV) continue to cause severe respiratory disease that is fatal in ~35% of diagnosed individuals. The ongoing threat to global public health and the need for novel therapeutic countermeasures have driven the development of animal models that can reproducibly replicate the pathology associated with MERS-CoV in human infections. The inability of MERS-CoV to replicate in the respiratory tracts of mice, hamsters, and ferrets stymied initial attempts to generate small animal models. Identification of human dipeptidyl peptidase IV (hDPP4) as the receptor for MERS-CoV infection opened the door for genetic engineering of mice. Precise molecular engineering of mouse DPP4 (mDPP4) with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology maintained inherent expression profiles, and limited MERS-CoV susceptibility to tissues that naturally express mDPP4, notably the lower respiratory tract wherein MERS-CoV elicits severe pulmonary pathology. Here, we describe the generation of the 288-330+/+ MERS-CoV mouse model in which mice were made susceptible to MERS-CoV by modifying two amino acids on mDPP4 (A288 and T330), and the use of adaptive evolution to generate novel MERS-CoV isolates that cause fatal respiratory disease. The 288-330+/+ mice are currently being used to evaluate novel drug, antibody, and vaccine therapeutic countermeasures for MERS-CoV. The chapter starts with a historical perspective on the emergence of MERS-CoV and animal models evaluated for MERS-CoV pathogenesis, and then outlines the development of the 288-330+/+ mouse model, assays for assessing a MERS-CoV pulmonary infection in a mouse model, and describes some of the challenges associated with using genetically engineered mice.


Subject(s)
Coronavirus Infections/virology , Dipeptidyl Peptidase 4/genetics , Disease Models, Animal , Mice/genetics , Middle East Respiratory Syndrome Coronavirus/physiology , Respiratory Distress Syndrome/virology , Animals , CRISPR-Cas Systems , Coronavirus Infections/pathology , Dipeptidyl Peptidase 4/metabolism , Disease Susceptibility , Female , Genetic Engineering , Humans , Lung/virology , Male , Mice, Inbred C57BL , Respiratory Distress Syndrome/pathology
15.
Methods Mol Biol ; 2099: 53-68, 2020.
Article in English | MEDLINE | ID: covidwho-1292546

ABSTRACT

Over the past two decades, several coronavirus (CoV) infectious clones have been engineered, allowing for the manipulation of their large viral genomes (~30 kb) using unique reverse genetic systems. These reverse genetic systems include targeted recombination, in vitro ligation, vaccinia virus vectors, and bacterial artificial chromosomes (BACs). Quickly after the identification of Middle East respiratory syndrome-CoV (MERS-CoV), both in vitro ligation and BAC-based reverse genetic technologies were engineered for MERS-CoV to study its basic biological properties, develop live-attenuated vaccines, and test antiviral drugs. Here, I will describe how lambda red recombination can be used with the MERS-CoV BAC to quickly and efficiently introduce virtually any type of genetic modification (point mutations, insertions, deletions) into the MERS-CoV genome and recover recombinant virus.


Subject(s)
Bacteriophage lambda/genetics , Chromosomes, Artificial, Bacterial/genetics , Coronavirus Infections/virology , Genome, Viral/genetics , Middle East Respiratory Syndrome Coronavirus/genetics , Viral Vaccines/genetics , Coronavirus Infections/prevention & control , Deoxyribonuclease I/genetics , Deoxyribonuclease I/metabolism , Genetic Engineering , Homologous Recombination , Humans , Middle East Respiratory Syndrome Coronavirus/immunology , Mutation , Vaccines, Attenuated/genetics , Vaccinia virus/genetics
17.
Eur J Immunol ; 51(8): 1992-2005, 2021 08.
Article in English | MEDLINE | ID: covidwho-1251932

ABSTRACT

The phenotype of infused cells is a major determinant of Adoptive T-cell therapy (ACT) efficacy. Yet, the difficulty in deciphering multiparametric cytometry data limited the fine characterization of cellular products. To allow the analysis of dynamic and complex flow cytometry samples, we developed cytoChain, a novel dataset mining tool and a new analytical workflow. CytoChain was challenged to compare state-of-the-art and innovative culture conditions to generate stem-like memory cells (TSCM ) suitable for ACT. Noticeably, the combination of IL-7/15 and superoxides scavenging sustained the emergence of a previously unidentified nonexhausted Fit-TSCM signature, overlooked by manual gating and endowed with superior expansion potential. CytoChain proficiently traced back this population in independent datasets, and in T-cell receptor engineered lymphocytes. CytoChain flexibility and function were then further validated on a published dataset from circulating T cells in COVID-19 patients. Collectively, our results support the use of cytoChain to identify novel, functionally critical immunophenotypes for ACT and patients immunomonitoring.


Subject(s)
Data Mining/methods , Flow Cytometry/methods , Receptors, Antigen, T-Cell/immunology , Receptors, Chimeric Antigen/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , COVID-19/blood , COVID-19/immunology , Cytokines/metabolism , Genetic Engineering , Humans , Immunologic Memory , Immunophenotyping , Immunotherapy, Adoptive , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Receptors, Chimeric Antigen/genetics , SARS-CoV-2/immunology
18.
Essays Biochem ; 65(3): 503-518, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1240779

ABSTRACT

Over the last few decades, the world has witnessed multiple viral pandemics, the current severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) pandemic being the worst and most devastating one, claiming millions of lives worldwide. Physicians, scientists, and engineers worldwide have joined hands in dealing with the current situation at an impressive speed and efficiency. One of the major reasons for the delay in response is our limited understanding of the mechanism of action and individual effects of the virus on different tissues and organs. Advances in 3D bioprinting have opened up a whole new area to explore and utilize the technology in fabricating models of these tissues and organs, recapitulating in vivo environment. These biomimetic models can not only be utilized in learning the infection pathways and drug toxicology studies but also minimize the need for animal models and shorten the time span for human clinical trials. The current review aims to integrate the existing developments in bioprinting techniques, and their implementation to develop tissue models, which has implications for SARS-CoV-2 infection. Future translation of these models has also been discussed with respect to the pandemic.


Subject(s)
Bioprinting , COVID-19/physiopathology , COVID-19/therapy , Printing, Three-Dimensional , Angiotensin-Converting Enzyme 2/metabolism , Animals , Biomimetics , Biotechnology , Extracellular Matrix/pathology , Genetic Engineering , Humans , Immune System , Lung/physiopathology , Models, Biological , SARS-CoV-2
19.
mBio ; 12(3)2021 05 11.
Article in English | MEDLINE | ID: covidwho-1225697

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino-acid fragment of the 1,273-amino-acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here, we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD is expressed inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) is expressed markedly more efficiently and generates a more potent neutralizing responses as a DNA vaccine antigen than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers, such as a Helicobacter pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines.IMPORTANCE All available vaccines for coronavirus disease 2019 (COVID-19) express or deliver the full-length SARS-CoV-2 spike (S) protein. We show that this antigen is not optimal, consistent with observations that the vast majority of the neutralizing response to the virus is focused on the S-protein receptor-binding domain (RBD). However, this RBD is not expressed well as an independent domain, especially when expressed as a fusion protein with a multivalent scaffold. We therefore engineered a more highly expressed form of the SARS-CoV-2 RBD by introducing four glycosylation sites into a face of the RBD normally occluded in the full S protein. We show that this engineered protein, gRBD, is more immunogenic than the wild-type RBD or the full-length S protein in both genetic and protein-delivered vaccines.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19 Vaccines/immunology , Immunogenicity, Vaccine , Receptors, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/immunology , Animals , Binding Sites , COVID-19 Vaccines/chemistry , Female , Genetic Engineering , Glycosylation , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Models, Molecular , Protein Domains , Rats , Rats, Sprague-Dawley , Receptors, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Conjugate/genetics , Vaccines, Conjugate/immunology , Vaccines, Synthetic/chemistry , Vaccines, Synthetic/immunology
20.
mBio ; 12(2)2021 03 02.
Article in English | MEDLINE | ID: covidwho-1115089

ABSTRACT

There are no approved vaccines against the life-threatening Middle East respiratory syndrome coronavirus (MERS-CoV). Attenuated vaccines have proven their potential to induce strong and long-lasting immune responses. We have previously described that severe acute respiratory syndrome coronavirus (SARS-CoV) envelope (E) protein is a virulence factor. Based on this knowledge, a collection of mutants carrying partial deletions spanning the C-terminal domain of the E protein (rMERS-CoV-E*) has been generated using a reverse genetics system. One of these mutants, MERS-CoV-E*Δ2in, was attenuated and provided full protection in a challenge with virulent MERS-CoV after a single immunization dose. The MERS-CoV-E*Δ2in mutant was stable as it maintained its attenuation after 16 passages in cell cultures and has been selected as a promising vaccine candidate.IMPORTANCE The emergence of the new highly pathogenic human coronavirus SARS-CoV-2 that has already infected more than 80 million persons, killing nearly two million of them, clearly indicates the need to design efficient and safe vaccines protecting from these coronaviruses. Modern vaccines can be derived from virus-host interaction research directed to the identification of signaling pathways essential for virus replication and for virus-induced pathogenesis, in order to learn how to attenuate these viruses and design vaccines. Using a reverse genetics system developed in our laboratory, an infectious cDNA clone of MERS-CoV was engineered. Using this cDNA, we sequentially deleted several predicted and conserved motifs within the envelope (E) protein of MERS-CoV, previously associated with the presence of virulence factors. The in vitro and in vivo evaluation of these deletion mutants highlighted the relevance of predicted linear motifs in viral pathogenesis. Two of them, an Atg8 protein binding motif (Atg8-BM), and a forkhead-associated binding motif (FHA-BM), when deleted, rendered an attenuated virus that was evaluated as a vaccine candidate, leading to full protection against challenge with a lethal dose of MERS-CoV. This approach can be extended to the engineering of vaccines protecting against the new pandemic SARS-CoV-2.


Subject(s)
Middle East Respiratory Syndrome Coronavirus/pathogenicity , COVID-19/immunology , COVID-19/prevention & control , Genetic Engineering/methods , Humans , Middle East Respiratory Syndrome Coronavirus/immunology , Vaccines, Attenuated/therapeutic use , Viral Vaccines/therapeutic use
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