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1.
Biomater Sci ; 10(11): 2940-2952, 2022 May 31.
Article in English | MEDLINE | ID: covidwho-1815640

ABSTRACT

Ionizable cationic lipids play a critical role in developing new gene therapies for various biomedical applications, including COVID-19 vaccines. However, it remains unclear whether the formulation of lipid nanoparticles (LNPs) using DLin-MC3-DMA, an optimized ionizable lipid clinically used for small interfering RNA (siRNA) therapy, also facilitates high liver-selective transfection of other gene therapies such as plasmid DNA (pDNA). Here we report the first investigation into pDNA transfection efficiency in different mouse organs after intramuscular and intravenous administration of lipid nanoparticles (LNPs) where DLin-MC3-DMA, DLin-KC2-DMA or DODAP are used as the ionizable cationic lipid component of the LNP. We discovered that these three benchmark lipids previously developed for siRNA delivery followed an unexpected characteristic rank order in gene expression efficiency when utilized for pDNA. In particular, DLin-KC2-DMA facilitated higher in vivo pDNA transfection than DLin-MC3-DMA and DODAP, possibly due to its head group pKa and lipid tail structure. Interestingly, LNPs formulated with either DLin-KC2-DMA or DLin-MC3-DMA exhibited significantly higher in vivo protein production in the spleen than in the liver. This work sheds light on the importance of the choice of ionizable cationic lipid and nucleic acid cargo for organ-selective gene expression. The study also provides a new design principle towards the formulation of more effective LNPs for biomedical applications of pDNA, such as gene editing, vaccines and immunotherapies.


Subject(s)
COVID-19 , Nanoparticles , Animals , COVID-19 Vaccines , Cations/chemistry , DNA/genetics , Gene Expression , Humans , Lipids/chemistry , Liposomes , Mice , Nanoparticles/chemistry , Plasmids/genetics , RNA, Small Interfering/chemistry
2.
Int J Mol Sci ; 23(6)2022 Mar 11.
Article in English | MEDLINE | ID: covidwho-1742489

ABSTRACT

The pandemic emergency determined by the spreading worldwide of the SARS-CoV-2 virus has focused the scientific and economic efforts of the pharmaceutical industry and governments on the possibility to fight the virus by genetic immunization. The genetic material must be delivered inside the cells by means of vectors. Due to the risk of adverse or immunogenic reaction or replication connected with the more efficient viral vectors, non-viral vectors are in many cases considered as a preferred strategy for gene delivery into eukaryotic cells. This paper is devoted to the evaluation of the gene delivery ability of new synthesized gemini bis-pyridinium surfactants with six methylene spacers, both hydrogenated and fluorinated, in comparison with compounds with spacers of different lengths, previously studied. Results from MTT proliferation assay, electrophoresis mobility shift assay (EMSA), transient transfection assay tests and atomic force microscopy (AFM) imaging confirm that pyridinium gemini surfactants could be a valuable tool for gene delivery purposes, but their performance is highly dependent on the spacer length and strictly related to their structure in solution. All the fluorinated compounds are unable to transfect RD-4 cells, if used alone, but they are all able to deliver a plasmid carrying an enhanced green fluorescent protein (EGFP) expression cassette, when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 1:2 ratio. The fluorinated compounds with spacers formed by six (FGP6) and eight carbon atoms (FGP8) give rise to a very interesting gene delivery activity, greater to that of the commercial reagent, when formulated with DOPE. The hydrogenated compound GP16_6 is unable to sufficiently compact the DNA, as shown by AFM images.


Subject(s)
DNA/genetics , Gene Transfer Techniques , Methane/chemistry , Pyridinium Compounds/chemistry , Surface-Active Agents/chemistry , Transfection/methods , A549 Cells , Cell Survival , DNA/chemistry , DNA/metabolism , Genetic Therapy/methods , Halogenation , Humans , Hydrogenation , Methane/metabolism , Microscopy, Atomic Force , Molecular Structure , Plasmids/chemistry , Plasmids/genetics , Plasmids/metabolism , Pyridinium Compounds/metabolism , Reproducibility of Results , Surface-Active Agents/metabolism
3.
Front Immunol ; 12: 824728, 2021.
Article in English | MEDLINE | ID: covidwho-1686477

ABSTRACT

We generated an optimized COVID-19 vaccine candidate based on the modified vaccinia virus Ankara (MVA) vector expressing a full-length prefusion-stabilized SARS-CoV-2 spike (S) protein, termed MVA-CoV2-S(3P). The S(3P) protein was expressed at higher levels (2-fold) than the non-stabilized S in cells infected with the corresponding recombinant MVA viruses. One single dose of MVA-CoV2-S(3P) induced higher IgG and neutralizing antibody titers against parental SARS-CoV-2 and variants of concern than MVA-CoV2-S in wild-type C57BL/6 and in transgenic K18-hACE2 mice. In immunized C57BL/6 mice, two doses of MVA-CoV2-S or MVA-CoV2-S(3P) induced similar levels of SARS-CoV-2-specific B- and T-cell immune responses. Remarkably, a single administration of MVA-CoV2-S(3P) protected all K18-hACE2 mice from morbidity and mortality caused by SARS-CoV-2 infection, reducing SARS-CoV-2 viral loads, histopathological lesions, and levels of pro-inflammatory cytokines in the lungs. These results demonstrated that expression of a novel full-length prefusion-stabilized SARS-CoV-2 S protein by the MVA poxvirus vector enhanced immunogenicity and efficacy against SARS-CoV-2 in animal models, further supporting MVA-CoV2-S(3P) as an optimized vaccine candidate for clinical trials.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccines, DNA/immunology , Viral Vaccines/immunology , Aged , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/mortality , COVID-19 Vaccines/genetics , Cell Line, Tumor , Chick Embryo , Chlorocebus aethiops , Cytokines/analysis , Female , HeLa Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Plasmids/genetics , Spike Glycoprotein, Coronavirus/genetics , Vaccines, DNA/genetics , Vaccinia virus/immunology , Vero Cells , Viral Vaccines/genetics
4.
Microbiol Spectr ; 10(1): e0201521, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1622005

ABSTRACT

Emergency department areas were repurposed as intensive care units (ICUs) for patients with acute respiratory distress syndrome during the initial months of the coronavirus disease 2019 (COVID-19) pandemic. We describe an outbreak of New Delhi metallo-ß-lactamase 1 (NDM-1)-producing Escherichia coli infections in critically ill COVID-19 patients admitted to one of the repurposed units. Seven patients developed infections (6 ventilator-associated pneumonia [VAP] and 1 urinary tract infection [UTI]) due to carbapenem-resistant E. coli, and only two survived. Five of the affected patients and four additional patients had rectal carriage of carbapenem-resistant E. coli. The E. coli strain from the affected patients corresponded to a single sequence type. Rectal screening identified isolates of two other sequence types bearing blaNDM-1. Isolates of all three sequence types harbored an IncFII plasmid. The plasmid was confirmed to carry blaNDM-1 through conjugation. An outbreak of clonal NDM-1-producing E. coli isolates and subsequent dissemination of NDM-1 through mobile elements to other E. coli strains occurred after hospital conversion during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. This emphasizes the need for infection control practices in surge scenarios. IMPORTANCE The SARS-CoV-2 pandemic has resulted in a surge of critically ill patients. Hospitals have had to adapt to the demand by repurposing areas as intensive care units. This has resulted in high workload and disruption of usual hospital workflows. Surge capacity guidelines and pandemic response plans do not contemplate how to limit collateral damage from issues like hospital-acquired infections. It is vital to ensure quality of care in surge scenarios.


Subject(s)
Cross Infection/microbiology , Escherichia coli Infections/microbiology , Escherichia coli/enzymology , Escherichia coli/isolation & purification , beta-Lactamases/metabolism , Adult , Aged , COVID-19/epidemiology , COVID-19/virology , Conjugation, Genetic , Cross Infection/epidemiology , Disease Outbreaks , Escherichia coli/classification , Escherichia coli/genetics , Escherichia coli Infections/epidemiology , Escherichia coli Infections/mortality , Female , Humans , Intensive Care Units/statistics & numerical data , Male , Mexico/epidemiology , Middle Aged , Plasmids/genetics , SARS-CoV-2/physiology , Tertiary Care Centers/statistics & numerical data , beta-Lactamases/genetics
5.
Biologicals ; 75: 12-15, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1616379

ABSTRACT

BACKGROUND: The successful development of messenger RNA vaccines for SARS-CoV-2 opened up venues for clinical nucleotide-based vaccinations. For development of DNA vaccines, we tested whether the EGF domain peptide of Developmentally regulated endothelial locus1 (E3 peptide) enhances uptake of extracellularly applied plasmid DNA. METHODS: DNA plasmid encoding lacZ or GFP was applied with a conditioned culture medium containing E3 peptide to cell lines in vitro or mouse soleus muscles in vivo, respectively. After 48 h incubation, gene expression was examined by ß-galactosidase (ß-gal) assay and fluorescent microscope, respectively. RESULTS: Application of E3 peptide-containing medium to cultured cell lines induced intense ß-gal activity in a dose-dependent manner. Intra-gastrocnemius injection of E3 peptide-containing medium to mouse soleus muscle succeeded in the induction of GFP fluorescence in many cells around the injection site. CONCLUSIONS: The administration of E3 peptide facilitates transmembrane uptake of extracellular DNA plasmid which induces sufficient extrinsic gene expression.


Subject(s)
DNA/genetics , Epidermal Growth Factor/chemistry , Gene Expression , Peptides , Plasmids/genetics , Plasmids/metabolism , Protein Domains , Animals , COVID-19 Vaccines , Cell Membrane/metabolism , DNA/metabolism , Genes, Reporter , Green Fluorescent Proteins/genetics , Mice , Muscle, Skeletal , Vaccines, DNA/genetics , Vaccines, DNA/metabolism
6.
J Virol ; 96(3): e0156121, 2022 02 09.
Article in English | MEDLINE | ID: covidwho-1529876

ABSTRACT

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae. While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae. Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.


Subject(s)
DNA, Complementary , Genome, Viral , Reverse Genetics , Torovirus/genetics , Animals , Cattle , Cattle Diseases/virology , Cell Line , Cells, Cultured , Chromosomes, Artificial, Bacterial , Cloning, Molecular , Genes, Reporter , Hemagglutinins, Viral/genetics , Hemagglutinins, Viral/metabolism , Mutation , Plasmids/genetics , Torovirus/isolation & purification , Torovirus Infections , Transfection
7.
J Antimicrob Chemother ; 76(10): 2538-2545, 2021 09 15.
Article in English | MEDLINE | ID: covidwho-1447597

ABSTRACT

OBJECTIVES: To assess the spread of New Delhi metallo-ß-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae ST147 organisms in Poland since an introduction from Tunisia in March 2015, including their phylogenetic position in the global population of the high-risk clone. METHODS: Out of 8925 unique NDM-positive K. pneumoniae isolates identified in Poland from April 2015 till December 2019, 126 isolates, including the Tunisian imports, were related by PFGE and blaNDM gene-carrying Tn125 transposon derivatives. Forty-seven representative isolates were sequenced by Illumina MiSeq. The phylogeny, resistome, virulome and plasmid replicons were analysed and compared with the international ST147 strains. Plasmids of six isolates were studied by the MinION sequencing. RESULTS: A high homogeneity of the 47 isolates was observed, with minor variations in their resistomes and plasmid replicon profiles. However, the detailed SNP comparison discerned a strict outbreak cluster of 40 isolates. All of the organisms were grouped within the ST147 phylogenetic international lineage, and four NDM-1 producers from Tunisia, Egypt and France were the closest relatives of the Polish isolates. Yersiniabactin genes (YbST280 type) were located within the ICEKpn12-like element in most of the outbreak isolates, characterized by O2v1 and KL64 antigen loci. The blaNDM-1 genes were located in double-replicon IncFIIK2+IncFIBK plasmids. CONCLUSIONS: The continuous spread of K. pneumoniae ST147 NDM-1 in Poland since 2015, largely in the Warsaw area, is demonstrated by this genomic analysis. The isolates showed a high degree of homogeneity, and close relatedness to organisms spreading in the Mediterranean region.


Subject(s)
Klebsiella Infections , Klebsiella pneumoniae , Anti-Bacterial Agents , Humans , Klebsiella Infections/epidemiology , Klebsiella pneumoniae/genetics , Microbial Sensitivity Tests , Phylogeny , Plasmids/genetics , Poland/epidemiology , beta-Lactamases/genetics
8.
Int J Mol Sci ; 22(19)2021 Sep 22.
Article in English | MEDLINE | ID: covidwho-1438627

ABSTRACT

The ongoing pandemic coronavirus (CoV) disease 2019 (COVID-19) by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has already caused substantial morbidity, mortality, and economic devastation. Reverse genetic approaches to generate recombinant viruses are a powerful tool to characterize and understand newly emerging viruses. To contribute to the global efforts for countermeasures to control the spread of SARS-CoV-2, we developed a passage-free SARS-CoV-2 clone based on a bacterial artificial chromosome (BAC). Moreover, using a Lambda-based Red recombination, we successfully generated different reporter and marker viruses, which replicated similar to a clinical isolate in a cell culture. Moreover, we designed a full-length reporter virus encoding an additional artificial open reading frame with wild-type-like replication features. The virus-encoded reporters were successfully applied to ease antiviral testing in cell culture models. Furthermore, we designed a new marker virus encoding 3xFLAG-tagged nucleocapsid that allows the detection of incoming viral particles and, in combination with bio-orthogonal labeling for the visualization of viral RNA synthesis via click chemistry, the spatiotemporal tracking of viral replication on the single-cell level. In summary, by applying BAC-based Red recombination, we developed a powerful, reliable, and convenient platform that will facilitate studies answering numerous questions concerning the biology of SARS-CoV-2.


Subject(s)
COVID-19/virology , Cloning, Molecular/methods , Genome, Viral , SARS-CoV-2/genetics , Animals , Chlorocebus aethiops , HEK293 Cells , Humans , Mutagenesis , Plasmids/genetics , Recombination, Genetic , Vero Cells
9.
Curr Opin Allergy Clin Immunol ; 21(6): 569-575, 2021 12 01.
Article in English | MEDLINE | ID: covidwho-1356717

ABSTRACT

PURPOSE OF REVIEW: Molecular forms of allergen-specific immunotherapy (AIT) are continuously emerging to improve the efficacy of the treatment, to shorten the duration of protocols and to prevent any side effects. The present review covers the recent progress in the development of AIT based on nucleic acid encoding allergens or CpG oligodeoxynucleotides (CpG-ODN). RECENT FINDINGS: Therapeutic vaccinations with plasmid deoxyribonucleic acid (DNA) encoding major shrimp Met e 1 or insect For t 2 allergen were effective for the treatment of food or insect bite allergy in respective animal models. DNA expressing hypoallergenic shrimp tropomyosin activated Foxp3+ T regulatory (Treg) cells whereas DNA encoding For t 2 down-regulated the expression of pruritus-inducing IL-31. Co-administrations of major cat allergen Fel d 1 with high doses of CpG-ODN reduced Th2 airway inflammation through tolerance induction mediated by GATA3+ Foxp3hi Treg cells as well as early anti-inflammatory TNF/TNFR2 signaling cascade. Non-canonical CpG-ODN derived from Cryptococcus neoformans as well as methylated CpG sites present in the genomic DNA from Bifidobacterium infantis mediated Th1 or Treg cell differentiation respectively. SUMMARY: Recent studies on plasmid DNA encoding allergens evidenced their therapeutic potential for the treatment of food allergy and atopic dermatitis. Unmethylated or methylated CpG-ODNs were shown to activate dose-dependent Treg/Th1 responses. Large clinical trials need to be conducted to confirm these promising preclinical data. Moreover, tremendous success of messenger ribonucleic acid (mRNA) vaccines against severe acute respiratory syndrome coronavirus 2 must encourage as well the re-exploration of mRNA vaccine platform for innovative AIT.


Subject(s)
Desensitization, Immunologic/methods , Hypersensitivity, Immediate/therapy , Oligodeoxyribonucleotides/administration & dosage , Vaccines, DNA/administration & dosage , Vaccines, Synthetic/administration & dosage , Allergens/administration & dosage , Allergens/genetics , Allergens/immunology , Animals , Clinical Trials as Topic , Desensitization, Immunologic/trends , Disease Models, Animal , Drug Evaluation, Preclinical , Humans , Hypersensitivity, Immediate/immunology , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/immunology , Plasmids/administration & dosage , Plasmids/genetics , Plasmids/immunology , Treatment Outcome , Vaccines, DNA/genetics , Vaccines, DNA/immunology , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology
10.
Methods Enzymol ; 660: 321-339, 2021.
Article in English | MEDLINE | ID: covidwho-1309118

ABSTRACT

Described here is the use of piggyBac transposase generated HEK293 stable cell pools for doxycycline-inducible protein production. The key benefits of the system are that low amounts of plasmid DNA are needed for transfection, high levels of protein expression can be achieved also for toxic proteins at robust scalability and reproducibility and the recombinant cell line can be stored as frozen cell bank. Transfection, selection, expression and purification of enhanced green fluorescence protein (eGFP) and SARS-CoV-2 Spike protein are described in this chapter.


Subject(s)
COVID-19 , Transposases , DNA Transposable Elements/genetics , Genetic Vectors/genetics , HEK293 Cells , Humans , Plasmids/genetics , Reproducibility of Results , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Transfection , Transposases/genetics , Transposases/metabolism
11.
PLoS One ; 16(3): e0248007, 2021.
Article in English | MEDLINE | ID: covidwho-1145483

ABSTRACT

More than 65 million people have been confirmed infection with SARS-CoV-2 and more than 1 million have died from COVID-19 and this pandemic remains critical worldwide. Effective vaccines are one of the most important strategies to limit the pandemic. Here, we report a construction strategy of DNA vaccine candidates expressing full length wild type SARS-CoV-2 spike (S) protein, S1 or S2 region and their immunogenicity in mice. All DNA vaccine constructs of pCMVkan-S, -S1 and -S2 induced high levels of specific binding IgG that showed a balance of IgG1/IgG2a response. However, only the sera from mice vaccinated with pCMKkan-S or -S1 DNA vaccines could inhibit viral RBD and ACE2 interaction. The highest neutralizing antibody (NAb) titer was found in pCMVkan-S group, followed by -S1, while -S2 showed the lowest PRNT50 titers. The geometric mean titers (GMTs) were 2,551, 1,005 and 291 for pCMVkan-S, -S1 and -S2, respectively. pCMVkan-S construct vaccine also induced the highest magnitude and breadth of T cells response. Analysis of IFN-γ positive cells after stimulation with SARS-CoV-2 spike peptide pools were 2,991, 1,376 and 1,885 SFC/106 splenocytes for pCMVkan-S, -S1 and -S2, respectively. Our findings highlighted that full-length S antigen is more potent than the truncated spike (S1 or S2) in inducing of neutralizing antibody and robust T cell responses.


Subject(s)
Immunity, Humoral , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Th1 Cells/immunology , Vaccines, DNA/immunology , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/blood , COVID-19/prevention & control , COVID-19/virology , Cytokines/metabolism , Female , Immunoglobulin G/blood , Interferon-gamma/metabolism , Mice , Mice, Inbred ICR , Plasmids/genetics , Plasmids/metabolism , Protein Binding , Th1 Cells/cytology , Th1 Cells/metabolism , Vaccines, DNA/genetics
12.
PLoS Biol ; 19(2): e3001091, 2021 02.
Article in English | MEDLINE | ID: covidwho-1102372

ABSTRACT

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.


Subject(s)
COVID-19 Vaccines , COVID-19/diagnosis , COVID-19/virology , Reverse Genetics , SARS-CoV-2/genetics , A549 Cells , Angiotensin-Converting Enzyme 2/metabolism , Animals , Chlorocebus aethiops , Codon , Humans , Hydrazones/pharmacology , Mice , Morpholines/pharmacology , Open Reading Frames , Plasmids/genetics , Pyrimidines/pharmacology , Serine Endopeptidases/metabolism , Vero Cells , Viral Proteins/metabolism
13.
Emerg Microbes Infect ; 10(1): 342-355, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1069193

ABSTRACT

The current study aims to develop a safe and highly immunogenic COVID-19 vaccine. The novel combination of a DNA vaccine encoding the full-length Spike (S) protein of SARS-CoV-2 and a recombinant S1 protein vaccine induced high level neutralizing antibody and T cell immune responses in both small and large animal models. More significantly, the co-delivery of DNA and protein components at the same time elicited full protection against intratracheal challenge of SARS-CoV-2 viruses in immunized rhesus macaques. As both DNA and protein vaccines have been proven safe in previous human studies, and DNA vaccines are capable of eliciting germinal center B cell development, which is critical for high-affinity memory B cell responses, the DNA and protein co-delivery vaccine approach has great potential to serve as a safe and effective approach to develop COVID-19 vaccines that provide long-term protection.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Vaccines, DNA/immunology , Vaccines, Subunit/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Cell Line , DNA/immunology , HEK293 Cells , Humans , Lymphocyte Count , Macaca mulatta , Mice , Mice, Inbred C57BL , Plasmids/genetics , Rabbits , Recombinant Proteins/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , T-Lymphocytes/immunology
14.
J Virol ; 94(24)2020 11 23.
Article in English | MEDLINE | ID: covidwho-985727

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness and has a high mortality of ∼34%. However, since its discovery in 2012, an effective vaccine has not been developed for it. To develop a vaccine against multiple strains of MERS-CoV, we targeted spike glycoprotein (S) using prime-boost vaccination with DNA and insect cell-expressed recombinant proteins for the receptor-binding domain (RBD), S1, S2, SΔTM, or SΔER. Our S subunits were generated using an S sequence derived from the MERS-CoV EMC/2012 strain. We examined humoral and cellular immune responses of various combinations with DNA plasmids and recombinant proteins in mice. Mouse sera immunized with SΔER DNA priming/SΔTM protein boosting showed cross-neutralization against 15 variants of S-pseudovirions and the wild-type KOR/KNIH/002 strain. In addition, these immunizations provided full protection against the KOR/KNIH/002 strain challenge in human DPP4 knock-in mice. These findings suggest that vaccination with the S subunits derived from one viral strain can provide cross-protection against variant MERS-CoV strains with mutations in S. DNA priming/protein boosting increased gamma interferon production, while protein-alone immunization did not. The RBD subunit alone was insufficient to induce neutralizing antibodies, suggesting the importance of structural conformation. In conclusion, heterologous DNA priming with protein boosting is an effective way to induce both neutralizing antibodies and cell-mediated immune responses for MERS-CoV vaccine development. This study suggests a strategy for selecting a suitable platform for developing vaccines against MERS-CoV or other emerging coronaviruses.IMPORTANCE Coronavirus is an RNA virus with a higher mutation rate than DNA viruses. Therefore, a mutation in S-protein, which mediates viral infection by binding to a human cellular receptor, is expected to cause difficulties in vaccine development. Given that DNA-protein vaccines promote stronger cell-mediated immune responses than protein-only vaccination, we immunized mice with various combinations of DNA priming and protein boosting using the S-subunit sequences of the MERS-CoV EMC/2012 strain. We demonstrated a cross-protective effect against wild-type KOR/KNIH/002, a strain with two mutations in the S amino acids, including one in its RBD. The vaccine also provided cross-neutralization against 15 different S-pseudotyped viruses. These suggested that a vaccine targeting one variant of S can provide cross-protection against multiple viral strains with mutations in S. The regimen of DNA priming/Protein boosting can be applied to the development of other coronavirus vaccines.


Subject(s)
Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Cross Protection , Middle East Respiratory Syndrome Coronavirus/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccines, DNA/immunology , Viral Vaccines/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Coronavirus Infections/mortality , Coronavirus Infections/virology , Disease Models, Animal , Female , Humans , Immunity, Cellular , Immunization, Secondary , Immunogenicity, Vaccine , Mice , Plasmids/administration & dosage , Plasmids/genetics , Plasmids/immunology , Spike Glycoprotein, Coronavirus/genetics , Vaccination , Vaccines, DNA/administration & dosage , Viral Vaccines/administration & dosage
16.
J Virol ; 94(17)2020 08 17.
Article in English | MEDLINE | ID: covidwho-831425

ABSTRACT

Accessory genes occurring between the S and E genes of coronaviruses have been studied quite intensively during the last decades. In porcine epidemic diarrhea virus (PEDV), the only gene at this location, ORF3, encodes a 224-residue membrane protein shown to exhibit ion channel activity and to enhance virus production. However, little is known about its intracellular trafficking or about its function during PEDV infection. In this study, two recombinant PEDVs were rescued by targeted RNA recombination, one carrying the full-length ORF3 gene and one from which the gene had been deleted entirely. These viruses as well as a PEDV encoding a naturally truncated ORF3 protein were employed to study the ORF3 protein's subcellular trafficking. In addition, ORF3 expression vectors were constructed to study the protein's independent transport. Our results show that the ORF3 protein uses the exocytic pathway to move to and accumulate in the Golgi area of the cell similarly in infected and transfected cells. Like the S protein, but unlike the other structural proteins M and N, the ORF3 protein was additionally observed at the surface of PEDV-infected cells. In addition, the C-terminally truncated ORF3 protein entered the exocytic pathway but it was unable to leave the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartment (ERGIC). Consistently, a YxxØ motif essential for ER exit was identified in the C-terminal domain. Finally, despite the use of sensitive antibodies and assays no ORF3 protein could be detected in highly purified PEDV particles, indicating that the protein is not a structural virion component.IMPORTANCE Coronaviruses typically express several accessory proteins. They vary in number and nature, and only one is conserved among most of the coronaviruses, pointing at an important biological function for this protein. PEDV is peculiar in that it expresses just this one accessory protein, termed the ORF3 protein. While its analogs in other coronaviruses have been studied to different extents, and these studies have indicated that they share an ion channel property, little is still known about the features and functions of the PEDV ORF3 protein except for its association with virulence. In this investigation, we studied the intracellular trafficking of the ORF3 protein both in infected cells and when expressed independently. In addition, we analyzed the effects of mutations in five sorting motifs in its C-terminal domain and investigated whether the protein, found to follow the same exocytic route by which the viral structural membrane proteins travel, is also incorporated into virions.


Subject(s)
Coronavirus Infections/veterinary , Exocytosis , Host-Pathogen Interactions , Open Reading Frames , Porcine epidemic diarrhea virus/genetics , Swine Diseases/metabolism , Swine Diseases/virology , Viral Proteins/metabolism , Amino Acid Sequence , Animals , Genetic Engineering , Metabolic Networks and Pathways , Plasmids/genetics , Protein Transport , Proteomics , Swine , Viral Proteins/chemistry , Viral Proteins/genetics
17.
Viruses ; 12(9)2020 09 10.
Article in English | MEDLINE | ID: covidwho-769396

ABSTRACT

The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on "pseudotyping" can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and MN results. The overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Lentivirus/genetics , Neutralization Tests/methods , Pandemics , Pneumonia, Viral/virology , Animals , Antibodies, Neutralizing , Antibodies, Viral/immunology , Betacoronavirus/immunology , COVID-19 , Cell Line , Coronavirus Infections/epidemiology , Humans , Immune Sera/immunology , Italy/epidemiology , Plasmids/genetics , Pneumonia, Viral/epidemiology , SARS-CoV-2 , Seroepidemiologic Studies , Spike Glycoprotein, Coronavirus/biosynthesis , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/physiology , Transfection , Vesiculovirus/genetics , Viral Load
18.
Chemistry ; 26(66): 15259-15269, 2020 Nov 26.
Article in English | MEDLINE | ID: covidwho-671205

ABSTRACT

Original molecular vectors that ensure broad flexibility to tune the shape and surface properties of plasmid DNA (pDNA) condensates are reported herein. The prototypic design involves a cyclodextrin (CD) platform bearing a polycationic cluster at the primary face and a doubly linked aromatic module bridging two consecutive monosaccharide units at the secondary face that behaves as a topology-encoding element. Subtle differences at the molecular level then translate into disparate morphologies at the nanoscale, including rods, worms, toroids, globules, ellipsoids, and spheroids. In vitro evaluation of the transfection capabilities revealed marked selectivity differences as a function of nanocomplex morphology. Remarkably high transfection efficiencies were associated with ellipsoidal or spherical shapes with a lamellar internal arrangement of pDNA chains and CD bilayers. Computational studies support that the stability of such supramolecular edifices is directly related to the tendency of the molecular vector to form noncovalent dimers upon DNA templating. Because the stability of the dimers depends on the protonation state of the polycationic clusters, the coaggregates display pH responsiveness, which facilitates endosomal escape and timely DNA release, a key step in successful transfection. The results provide a versatile strategy for the construction of fully synthetic and perfectly monodisperse nonviral gene delivery systems uniquely suited for optimization schemes.


Subject(s)
Cyclodextrins , DNA/chemistry , Gene Transfer Techniques , Plasmids/genetics , Transfection
19.
G3 (Bethesda) ; 10(9): 3399-3402, 2020 09 02.
Article in English | MEDLINE | ID: covidwho-695997

ABSTRACT

The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.


Subject(s)
Betacoronavirus/genetics , Open Reading Frames/genetics , Betacoronavirus/isolation & purification , COVID-19 , Cloning, Molecular , Coronavirus Infections/pathology , Coronavirus Infections/virology , Escherichia coli/metabolism , Humans , Pandemics , Plasmids/genetics , Plasmids/metabolism , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Potyvirus/genetics , SARS-CoV-2
20.
Med Hypotheses ; 143: 110084, 2020 Oct.
Article in English | MEDLINE | ID: covidwho-643765

ABSTRACT

COVID-19 is the pandemic outbreak that is caused by SARS-CoV-2 virus from December, 2019. Human race do not know the curative measure of this devastating disease. In today's era of nanotechnology, it may use its knowledge to develop molecular vaccine to combat this disease. In this article we are intended to propose a hypothesis on the development of a vaccine that is molecular in nature to work against COVID-19. The nanoconjugate may comprise with the inorganic nanoparticle layered double hydroxide intercalated with shRNA-plasmid that have a sequence targeting towards the viral genome or viral mRNA. This nanoconjugate may be used as a nasal spray to deliver the shRNA-plasmid to the target site. The nanoconjugate will have several advantages such as they are biocompatible, they forms as stable knockdown to the target cells and they are stable in the nasal mucosa.


Subject(s)
Betacoronavirus , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Viral Vaccines/genetics , Betacoronavirus/genetics , Betacoronavirus/immunology , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/virology , Humans , Models, Biological , Nanoconjugates/chemistry , Plasmids/genetics , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , RNA, Small Interfering/genetics , RNA, Viral/genetics , SARS-CoV-2 , Vaccines, Synthetic/chemistry , Vaccines, Synthetic/genetics , Viral Vaccines/chemical synthesis
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