scholarly journals Genetically Engineered DENV Produces Antigenically Distinct Mature Particles

2021 ◽  
Author(s):  
Longping V. Tse ◽  
Rita M. Meganck ◽  
Stephanie Dong ◽  
Lily E. Adams ◽  
Laura J. White ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Reverse Genetics ◽  
High Titer ◽  
Evolutionary Relationship ◽  
Clinical Importance ◽  
Genetically Engineered ◽  
Dominant Form ◽  
Live Virus ◽  
Viral Yield

Maturation of Dengue viruses (DENV) alters the structure, immunity and infectivity of the virion and highly mature particles represent the dominant form in vivo. The production of highly mature virions principally relies on the structure and function of the viral premature protein (prM) and its cleavage by the host protease furin. We developed a reliable clonal cell line which produces single-round mature DENVs without the need for DENV reverse genetics. More importantly, using protein engineering coupled with natural and directed evolution of the prM cleavage site, we engineered genetically stable mature DENVs without comprising viral yield and independent of cell, host, or passage. Using these complementary strategies to regulate maturation, we demonstrate that the resulting mature DENVs are antigenically distinct from their isogenic immature forms. Given the clinical importance of mature DENVs in immunity, our strategy provides a reliable strategy for the production of stable, high-titer mature candidate DENV live virus vaccines, genetically stabilized viruses for DENV maturation and immunity studies, and models for maturation-regulated experimental evolution in mammalian and invertebrate cells. Our data from directed-evolution across host species reveals distinct maturation-dependent selective pressures between mammalian and insect cells, which sheds light on the divergent evolutionary relationship of DENVs between its host and vector.

scholarly journals Influenza B Virus NS1-Truncated Mutants: Live-Attenuated Vaccine Approach

2008 ◽  
Vol 82 (21) ◽  
pp. 10580-10590 ◽  
Author(s):  
Rong Hai ◽  
Luis Martínez-Sobrido ◽  
Kathryn A. Fraser ◽  
Juan Ayllon ◽  
Adolfo García-Sastre ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Influenza Viruses ◽  
Influenza B Virus ◽  
Influenza B ◽  
Type I ◽  
Live Attenuated Vaccine ◽  
Live Virus ◽  
B Virus

ABSTRACT Type B influenza viruses can cause substantial morbidity and mortality in the population, and vaccination remains by far the best means of protection against infections with these viruses. Here, we report the construction of mutant influenza B viruses for potential use as improved live-virus vaccine candidates. Employing reverse genetics, we altered the NS1 gene, which encodes a type I interferon (IFN) antagonist. The resulting NS1 mutant viruses induced IFN and, as a consequence, were found to be attenuated in vitro and in vivo. The absence of pathogenicity of the NS1 mutants in both BALB/c and C57BL/6 PKR−/− mice was confirmed. We also provide evidence that influenza B virus NS1 mutants induce a self-adjuvanted immune response and confer effective protection against challenge with both homologous and heterologous B virus strains in mice.

Generation of Dendritic Cells after One-Hit Lentiviral Transduction of Hematopoietic Precursor Cells: Proof of Concept for the Human and Mouse Systems.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3448-3448
Author(s):  
Richard C. Koya ◽  
Nori Kasahara ◽  
Takahiro Kimura ◽  
Antoni Ribas ◽  
Renata Stripecke
Keyword(s):  
Tumor Antigens ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
High Titer ◽  
Lentiviral Vectors ◽  
Genetically Engineered ◽  
Interleukin 4 ◽  
Lentiviral Transduction ◽  
Gm Csf

Abstract Conventional, ex vivo culture of monocytes with recombinant proteins for their differentiation into DCs involves considerable manipulation under “Good Manufacturing Practices” conditions, and is not only more labor intensive but importantly, after ex vivo produced DCs are administered, they lack the stimulatory signals to keep them alive and functional and therefore are short lived. Because of these problems, we have evaluated an one-hit lentiviral transduction approach for genetically modifying monocytes in order to promote autocrine and paracrine production of factors required for their differentiation into immature DCs. High-titer third generation self-inactivating lentiviral vectors expressing granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) efficiently achieved simultaneous and persistent co-delivery of the transgenes into purified human CD14+ monocytes. Co-expression of GM-CSF and IL-4 in monocytes was sufficient to induce their differentiation into lentivirus-modified DCs (“DC/LVs”), as evidenced by their morphology, immunophenotype and immune-function*. Mixed lymphocyte reactions showed that the T-cell stimulating activity of DC/LVs was superior to that of DCs grown by conventional methods. DC/LVs displayed efficient antigen-specific, MHC Class-I restricted stimulation of autologous CD8+ T-cells, as shown by IFN-G production and CTL assays. Importantly, DC/LVs could be maintained metabolically active and viable in culture for 2–3 weeks in the absence of exogenously added growth factors, unlike conventional DCs *. We are now evaluating whether DC/LVs can be re-infused immediately after gene transfer to achieve stable and long-lasting differentiation in vivo. Additionally, the genetic engineering of monocytes is anticipated to generate DCs after one hit of lentiviral transduction, instead of the three consecutive steps for development of DCs (differentiation, maturation, gene delivery of tumor antigens). We have thus established a mouse model for testing DC/LVs in vivo for the treatment of melanoma. Bone marrow cells from C57BL/6 mice transduced with lentiviral vectors expressing GM-CSF and IL-4 recapitulated the same DC/LV morphology and immunophenotype obtained in the human system. Mouse DC/LVs were also more viable in vitro and outperformed conventional mouse DCs in pilot immunization assays as followed by CTL assays and IFN-G ELISPOT. We are currently evaluating the immunotherapeutic efficacy of DC/LVs injected into mice developing B16 melanoma tumors. Co-delivery of a gene for DC maturation (CD40L) and of gene encoding a tumor-associated antigens (MART-1) is being performed. Our goal is to evaluate the implications of simultaneous co-expression of GM-CSF/ IL-4/ CD40L/ MART-1 in DC/LV differentiation and migration to lymph nodes in vivo, immunopotency and safety. Once these pre-clinical considerations are addressed, we foresee a broad clinical application of genetically engineered DCs for vaccination purposes against cancer and chronic infectious diseases.

scholarly journals Reverse Genetics for Type I Feline Coronavirus Field Isolate To Study the Molecular Pathogenesis of Feline Infectious Peritonitis

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Rosina Ehmann ◽  
Claudia Kristen-Burmann ◽  
Barbara Bank-Wolf ◽  
Matthias König ◽  
Christiane Herden ◽  
...  
Keyword(s):  
Cell Culture ◽  
Reverse Genetics ◽  
Rna Viruses ◽  
High Titer ◽  
Field Isolate ◽  
Molecular Pathogenesis ◽  
Feline Infectious Peritonitis ◽  
Persistent Infections

ABSTRACTFeline infectious peritonitis (FIP), one of the most important lethal infections of cats, is caused by feline infectious peritonitis virus (FIPV), the high-virulence biotype of feline coronaviruses (FCoVs). FIPVs are suggested to emerge from feline enteric coronaviruses (FECVs) by acquiring mutations in specific genes in the course of persistent infections. Although numerous studies identified mutations predicted to be responsible for the FECV-FIPV biotype switch, the presumed roles of specific genetic changes in FIP pathogenesis have not been confirmed experimentally. Reverse genetics systems established previously for serotype I and the less common serotype II FCoVs were based on cell culture-adapted FIPV strains which, however, were shown to be unsuitable for FIP pathogenesis studiesin vivo. To date, systems to produce and manipulate recombinant serotype I field viruses have not been developed, mainly because these viruses cannot be grownin vitro. Here, we report the first reverse genetics system based on a serotype I FECV field isolate that is suitable to produce high-titer stocks of recombinant FECVs. We demonstrate that these recombinant viruses cause productive persistent infections in cats that are similar to what is observed in natural infections. The system provides an excellent tool for studying FCoVs that do not grow in standard cell culture systems and will greatly facilitate studies into the molecular pathogenesis of FIP. Importantly, the system could also be adapted for studies of other RNA viruses with large genomes whose production and characterizationin vivoare currently hampered by the lack ofin vitropropagation systems.IMPORTANCEThe availability of recombinant serotype I FCoV field isolates that are amenable to genetic manipulation is key to studying the molecular pathogenesis of FIP, especially since previous studies using cell culture-adapted FIPVs had proven unsuccessful. To our knowledge, we report the first serotype I FECV field isolate-based reverse genetics system that allows the production of high-titer recombinant virus stocks that can be used for subsequentin vivostudies in cats. The system represents a milestone in FCoV research. It provides an essential tool for studying the molecular pathogenesis of FIP and, more specifically, the functions of specific gene products in causing a fundamentally different progression of disease following acquisition of specific mutations. The system developed in this study will also be useful for studying other coronaviruses or more distantly related RNA viruses with large genomes for which suitablein vitroculture systems are not available.

scholarly journals Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System

2019 ◽  
Author(s):  
Asha A. Philip ◽  
Jacob L. Perry ◽  
Heather E. Eaton ◽  
Maya Shmulevitz ◽  
Joseph M. Hyser ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Rna Virus ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Genetically Engineered ◽  
Genome Segment ◽  
Fusion Product ◽  
Infected Cells

AbstractRotavirus is a segmented double-stranded (ds)RNA virus that causes severe gastroenteritis in young children. We have established an efficient simplified rotavirus reverse genetics (RG) system that uses eleven T7 plasmids, each expressing a unique simian SA11 (+)RNA, and a CMV support plasmid for the African swine fever virus NP868R capping enzyme. With the NP868R-based system, we generated recombinant rotavirus (rSA11/NSP3-FL-UnaG) with a genetically modified 1.5-kB segment 7 dsRNA that encodes full-length NSP3 fused to UnaG, a 139-aa green fluorescent protein (FP). Analysis of rSA11/NSP3-FL-UnaG showed that the virus replicated efficiently and was genetically stable over 10 rounds of serial passage. The NSP3-UnaG fusion product was well expressed in rSA11/NSP3-FL-UnaG-infected cells, reaching levels similar to NSP3 in wildtype rSA11-infected cells. Moreover, the NSP3-UnaG protein, like functional wildtype NSP3, formed dimersin vivo. Notably, NSP3-UnaG protein was readily detected in infected cells via live cell imaging, with intensity levels ~3-fold greater than that of the NSP1-UnaG fusion product of rSA11/NSP1-FL-UnaG. Our results indicate that FP-expressing recombinant rotaviruses can be made through manipulation of the segment 7 dsRNA without deleting or interrupting any of the twelve open reading frames of the virus. Because NSP3 is expressed at levels higher than NSP1 in infected cells, rotaviruses expressing NSP3-based FPs may be a more sensitive tool for studying rotavirus biology than rotaviruses expressing NSP1-based FPs. This is the first report of a recombinant rotavirus containing a genetically engineered segment 7 dsRNA.ImportancePrevious studies have generated recombinant rotaviruses that express fluorescent proteins (FPs) by inserting reporter genes into the NSP1 open reading frame (ORF) of genome segment 5. Unfortunately, NSP1 is expressed at low levels in infected cells, making viruses expressing FP-fused NSP1 less than ideal probes of rotavirus biology. Moreover, FPs were inserted into segment 5 in such a way as to compromise NSP1, an interferon antagonist affecting viral growth and pathogenesis. We have identified an alternative approach for generating rotaviruses expressing FPs, one relying on fusing the reporter gene to the NSP3 ORF of genome segment 7. This was accomplished without interrupting any of the viral ORFs, yielding recombinant viruses likely expressing the complete set of functional viral proteins. Given that NSP3 is made at moderate levels in infected cells, rotavirus encoding NSP3-based FPs should be more sensitive probes of viral infection than rotaviruses encoding NSP1-based FPs.

scholarly journals Deletion of the SH gene from avian metapneumovirus has a greater impact on virus production and immunogenicity in turkeys than deletion of the G gene or M2-2 open reading frame

2008 ◽  
Vol 89 (2) ◽  
pp. 525-533 ◽  
Author(s):  
Roger Ling ◽  
Sabrina Sinkovic ◽  
Didier Toquin ◽  
Olivier Guionie ◽  
Nicolas Eterradossi ◽  
...  
Keyword(s):  
Cell Culture ◽  
Antibody Response ◽  
Antibody Production ◽  
Reverse Genetics ◽  
Open Reading Frame ◽  
Virus Growth ◽  
Reading Frame ◽  
Viral Yield

Subgroup A avian metapneumoviruses lacking either the SH or G gene or the M2-2 open reading frame were generated by using a reverse-genetics approach. The growth properties of these viruses were studied in vitro and in vivo in their natural host. Deletion of the SH gene alone resulted in the generation of a syncytial-plaque phenotype and this was reversed by the introduction of the SH gene from a subgroup B, but not a subgroup C, virus. Infected turkeys were assessed for antibody production and the presence of viral genomic RNA in tracheal swabs. The virus with a deleted SH gene also showed the greatest impairment of replication both in cell culture and in infected turkeys. This contrasts with the situation with other pneumoviruses in culture and in model animals, where deletion of the SH gene results in little effect upon viral yield and a good antibody response. Replication of the G- and M2-2-deleted viruses was impaired more severely in turkeys than in cell culture, with only some animals showing evidence of virus growth and antibody production. There was no correlation between virus replication and antibody response, suggesting that replication sites other than the trachea may be important for induction of antibody responses.

scholarly journals Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System

2019 ◽  
Vol 93 (24) ◽  
Author(s):  
Asha A. Philip ◽  
Jacob L. Perry ◽  
Heather E. Eaton ◽  
Maya Shmulevitz ◽  
Joseph M. Hyser ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
African Swine Fever Virus ◽  
Genetically Engineered ◽  
Genome Segment ◽  
Fusion Product ◽  
Wild Type ◽  
Infected Cells

ABSTRACT Rotavirus is a segmented double-stranded RNA (dsRNA) virus that causes severe gastroenteritis in young children. We have established an efficient simplified rotavirus reverse genetics (RG) system that uses 11 T7 plasmids, each expressing a unique simian SA11 (+)RNA, and a cytomegalovirus support plasmid for the African swine fever virus NP868R capping enzyme. With the NP868R-based system, we generated recombinant rotavirus (rSA11/NSP3-FL-UnaG) with a genetically modified 1.5-kb segment 7 dsRNA encoding full-length nonstructural protein 3 (NSP3) fused to UnaG, a 139-amino-acid green fluorescent protein (FP). Analysis of rSA11/NSP3-FL-UnaG showed that the virus replicated efficiently and was genetically stable over 10 rounds of serial passaging. The NSP3-UnaG fusion product was well expressed in rSA11/NSP3-FL-UnaG-infected cells, reaching levels similar to NSP3 levels in wild-type recombinant SA11-infected cells. Moreover, the NSP3-UnaG protein, like functional wild-type NSP3, formed dimers in vivo. Notably, the NSP3-UnaG protein was readily detected in infected cells via live-cell imaging, with intensity levels ∼3-fold greater than those of the NSP1-UnaG fusion product of rSA11/NSP1-FL-UnaG. Our results indicate that FP-expressing recombinant rotaviruses can be made through manipulation of the segment 7 dsRNA without deletion or interruption of any of the 12 open reading frames (ORFs) of the virus. Because NSP3 is expressed at higher levels than NSP1 in infected cells, rotaviruses expressing NSP3-based FPs may be more sensitive tools for studying rotavirus biology than rotaviruses expressing NSP1-based FPs. This is the first report of a recombinant rotavirus containing a genetically engineered segment 7 dsRNA. IMPORTANCE Previous studies generated recombinant rotaviruses that express FPs by inserting reporter genes into the NSP1 ORF of genome segment 5. Unfortunately, NSP1 is expressed at low levels in infected cells, making viruses expressing FP-fused NSP1 less than ideal probes of rotavirus biology. Moreover, FPs were inserted into segment 5 in such a way as to compromise NSP1, an interferon antagonist affecting viral growth and pathogenesis. We have identified an alternative approach for generating rotaviruses expressing FPs, one relying on fusing the reporter gene to the NSP3 ORF of genome segment 7. This was accomplished without interrupting any of the viral ORFs, yielding recombinant viruses that likely express the complete set of functional viral proteins. Given that NSP3 is made at moderate levels in infected cells, rotaviruses encoding NSP3-based FPs should be more sensitive probes of viral infection than rotaviruses encoding NSP1-based FPs.

scholarly journals Ultra-strong bio-glue from genetically engineered polypeptides

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chao Ma ◽  
Jing Sun ◽  
Bo Li ◽  
Yang Feng ◽  
Yao Sun ◽  
...  
Keyword(s):  
Soft Tissues ◽  
Covalent Bond ◽  
Genetically Engineered ◽  
Supramolecular Interactions ◽  
Molar Ratio ◽  
High Adhesion ◽  
Strong Adhesion ◽  
Order Of Magnitude

AbstractThe development of biomedical glues is an important, yet challenging task as seemingly mutually exclusive properties need to be combined in one material, i.e. strong adhesion and adaption to remodeling processes in healing tissue. Here, we report a biocompatible and biodegradable protein-based adhesive with high adhesion strengths. The maximum strength reaches 16.5 ± 2.2 MPa on hard substrates, which is comparable to that of commercial cyanoacrylate superglue and higher than other protein-based adhesives by at least one order of magnitude. Moreover, the strong adhesion on soft tissues qualifies the adhesive as biomedical glue outperforming some commercial products. Robust mechanical properties are realized without covalent bond formation during the adhesion process. A complex consisting of cationic supercharged polypeptides and anionic aromatic surfactants with lysine to surfactant molar ratio of 1:0.9 is driven by multiple supramolecular interactions enabling such strong adhesion. We demonstrate the glue’s robust performance in vitro and in vivo for cosmetic and hemostasis applications and accelerated wound healing by comparison to surgical wound closures.

scholarly journals The Role of Melatonin on NLRP3 Inflammasome Activation in Diseases

Antioxidants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1020
Author(s):  
Burak Ibrahim Arioz ◽  
Emre Tarakcioglu ◽  
Melis Olcum ◽  
Sermin Genc
Keyword(s):  
Nlrp3 Inflammasome ◽  
Intracellular Signaling ◽  
Metabolic Diseases ◽  
Metabolic Stress ◽  
Clinical Importance ◽  
Rapid Identification ◽  
In Vivo Studies ◽  
Inflammasome Activation

NLRP3 inflammasome is a part of the innate immune system and responsible for the rapid identification and eradication of pathogenic microbes, metabolic stress products, reactive oxygen species, and other exogenous agents. NLRP3 inflammasome is overactivated in several neurodegenerative, cardiac, pulmonary, and metabolic diseases. Therefore, suppression of inflammasome activation is of utmost clinical importance. Melatonin is a ubiquitous hormone mainly produced in the pineal gland with circadian rhythm regulatory, antioxidant, and immunomodulatory functions. Melatonin is a natural product and safer than most chemicals to use for medicinal purposes. Many in vitro and in vivo studies have proved that melatonin alleviates NLRP3 inflammasome activity via various intracellular signaling pathways. In this review, the effect of melatonin on the NLRP3 inflammasome in the context of diseases will be discussed.

scholarly journals Designing P. aeruginosa synthetic phages with reduced genomes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Diana P. Pires ◽  
Rodrigo Monteiro ◽  
Dalila Mil-Homens ◽  
Arsénio Fialho ◽  
Timothy K. Lu ◽  
...  
Keyword(s):  
Galleria Mellonella ◽  
Antibacterial Properties ◽  
Genetically Engineered ◽  
In Vivo Studies ◽  
Infection Model ◽  
Promising Therapeutic Approach ◽  
Genes Encoding ◽  
First Time

AbstractIn the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem. Genetically engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage. To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48% of the genes encoding hypothetical proteins from the genome of the newly isolated Pseudomonas aeruginosa phage vB_PaeP_PE3. The antibacterial efficacy of the wild-type and the synthetic phages was assessed in vitro as well as in vivo using a Galleria mellonella infection model. Overall, both in vitro and in vivo studies revealed that the knock-outs made in phage genome do not impair the antibacterial properties of the synthetic phages, indicating that this could be a good strategy to clear space from phage genomes in order to enable the introduction of other genes of interest that can potentiate the future treatment of P. aeruginosa infections.

scholarly journals Characterization and visualization of murine coagulation factor VIII-producing cells in vivo

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Morisada Hayakawa ◽  
Asuka Sakata ◽  
Hiroko Hayakawa ◽  
Hikari Matsumoto ◽  
Takafumi Hiramoto ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Fluorescent Protein ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Genetically Engineered ◽  
Coagulation Factor Viii ◽  
Liver Sinusoidal Endothelial Cells ◽  
Genetically Engineered Mice

AbstractCoagulation factors are produced from hepatocytes, whereas production of coagulation factor VIII (FVIII) from primary tissues and cell species is still controversial. Here, we tried to characterize primary FVIII-producing organ and cell species using genetically engineered mice, in which enhanced green fluorescent protein (EGFP) was expressed instead of the F8 gene. EGFP-positive FVIII-producing cells existed only in thin sinusoidal layer of the liver and characterized as CD31high, CD146high, and lymphatic vascular endothelial hyaluronan receptor 1 (Lyve1)+. EGFP-positive cells can be clearly distinguished from lymphatic endothelial cells in the expression profile of the podoplanin− and C-type lectin-like receptor-2 (CLEC-2)+. In embryogenesis, EGFP-positive cells began to emerge at E14.5 and subsequently increased according to liver maturation. Furthermore, plasma FVIII could be abolished by crossing F8 conditional deficient mice with Lyve1-Cre mice. In conclusion, in mice, FVIII is only produced from endothelial cells exhibiting CD31high, CD146high, Lyve1+, CLEC-2+, and podoplanin− in liver sinusoidal endothelial cells.

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