scholarly journals Enhancing the Protective Immune Response to Administration of a LIVP-GFP Live Attenuated Vaccinia Virus to Mice

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 377
Author(s):  
Sergei N. Shchelkunov ◽  
Stanislav N. Yakubitskiy ◽  
Kseniya A. Titova ◽  
Stepan A. Pyankov ◽  
Alexander A. Sergeev
Keyword(s):  
Genetic Engineering ◽  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Protein Product ◽  
Smallpox Vaccination ◽  
Green Fluorescent Protein Gene ◽  
Kinase Gene ◽  
Foreign Genes ◽  
Green Fluorescent

Following the WHO announcement of smallpox eradication, discontinuation of smallpox vaccination with vaccinia virus (VACV) was recommended. However, interest in VACV was soon renewed due to the opportunity of genetic engineering of the viral genome by directed insertion of foreign genes or introduction of mutations or deletions into selected viral genes. This genomic technology enabled production of stable attenuated VACV strains producing antigens of various infectious agents. Due to an increasing threat of human orthopoxvirus re-emergence, the development of safe highly immunogenic live orthopoxvirus vaccines using genetic engineering methods has been the challenge in recent years. In this study, we investigated an attenuated VACV LIVP-GFP (TK-) strain having an insertion of the green fluorescent protein gene into the viral thymidine kinase gene, which was generated on the basis of the LIVP (Lister-Institute for Viral Preparations) strain used in Russia as the first generation smallpox vaccine. We studied the effect of A34R gene modification and A35R gene deletion on the immunogenic and protective properties of the LIVP-GFP strain. The obtained data demonstrate that intradermal inoculation of the studied viruses induces higher production of VACV-specific antibodies compared to their levels after intranasal administration. Introduction of two point mutations into the A34R gene, which increase the yield of extracellular enveloped virions, and deletion of the A35R gene, the protein product of which inhibits presentation of antigens by MHC II, enhances protective potency of the created LIVP-TK--A34R*-dA35R virus against secondary lethal orthopoxvirus infection of BALB/c mice even at an intradermal dose as low as 103 plaque forming units (PFU)/mouse. This virus may be considered not only as a candidate attenuated live vaccine against smallpox and other human orthopoxvirus infections but also as a vector platform for development of safe multivalent live vaccines against other infectious diseases using genetic engineering methods.

scholarly journals Mutations in the A34R gene increase the immunogenicity of vaccinia virus

2021 ◽  
Vol 25 (2) ◽  
pp. 139-146
Author(s):  
S. N. Shchelkunov ◽  
T. V. Bauer ◽  
S. N. Yakubitskiy ◽  
A. A. Sergeev ◽  
A. S. Kabanov ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Point Mutations ◽  
World Health ◽  
Smallpox Vaccination ◽  
Virus Infections ◽  
Pathogenic Viruses ◽  
Foreign Genes ◽  
Health Organization ◽  
Human Infections

Vaccination is the most simple and reliable approach of protection to virus infections. The most effective agents are live vaccines, usually low-virulence organisms for humans and closely related to pathogenic viruses or attenuated as a result of mutations/deletions in the genome of pathogenic virus. Smallpox vaccination with live vaccinia virus (VACV) closely related to smallpox virus played a key role in the success of the global smallpox eradication program carried out under the World Health Organization auspices. As a result of the WHO decision as of 1980 to stop smallpox vaccination, humankind has lost immunity not only to smallpox, but also to other zoonotic, orthopoxviruscaused human infections. This new situation allows orthopoxviruses to circulate in the human population and, as a consequence, to alter several established concepts of the ecology and range of sensitive hosts for various orthopoxvirus species. Classic VACV-based live vaccine for vaccination against orthopoxvirus infections is out of the question, because it can cause severe side effects. Therefore, the development of new safe vaccines against orthopoxviral infections of humans and animals is an important problem. VACV attenuation by modern approaches carried out by targeted inactivation of certain virus genes and usually leads to a decrease in the effectiveness of VACV in vivo propagation. As a result, it can cause a diminishing of the immune response after administration of attenuated virus to patients at standard doses. The gene for thymidine kinase is frequently used for insertion/inactivation of foreign genes and it causes virus attenuation. In this research, the effect of the introduction of two point mutations into the A34R gene of attenuated strain LIVP-GFP (ТК–), which increase the yield of extracellular enveloped virions (EEV), on the pathogenicity and immunogenicity of VACV LIVP-GFP-A34R administered intranasally to laboratory mice were studied. It was shown that increase in EEV production by recombinant strain VACV LIVP-GFP-A34R does not change the attenuated phenotype characteristic of the parental strain LIVP-GFP, but causes a significantly larger production of VACV-specific antibodies.

Transgenic oil palm: production and projection

2000 ◽  
Vol 28 (6) ◽  
pp. 969-972 ◽  
Author(s):  
Ghulam Kadir Ahmad Parveez ◽  
Masnita Mohd Masri ◽  
Alizah Zainal ◽  
Na'imatulapidah Abdul Majid ◽  
Abdul Masani Mat Yunus ◽  
...  
Keyword(s):  
Genetic Engineering ◽  
Palm Oil ◽  
Oil Palm ◽  
Fluorescent Protein ◽  
Marker Gene ◽  
Value Added ◽  
Green Fluorescent Protein Gene ◽  
Regeneration System ◽  
System A ◽  
Green Fluorescent

Oil palm is an important economic crop for Malaysia. Genetic engineering could be applied to produce transgenic oil palms with high value-added fatty acids and novel products to ensure the sustainability of the palm oil industry. Establishment of a reliable transformation and regeneration system is essential for genetic engineering. Biolistic was initially chosen as the method for oil palm transformation as it has been the most successful method for monocotyledons to date. Optimization of physical and biological parameters, including testing of promoters and selective agents, was carried out as a prerequisite for stable transformation. This has resulted in the successful transfer of reporter genes into oil palm and the regeneration of transgenic oil palm, thus making it possible to improve the oil palm through genetic engineering. Besides application of the Biolistics method, studies on transformation mediated by Agrobacterium and utilization of the green fluorescent protein gene as a selectable marker gene have been initiated. Upon the development of a reliable transformation system, a number of useful targets are being projected for oil palm improvement. Among these targets are high-oleate and high-stearate oils, and the production of industrial feedstock such as biodegradable plastics. The efforts in oil palm genetic engineering are thus not targeted as commodity palm oil. Due to the long life cycle of the palm and the time taken to regenerate plants in tissue culture, it is envisaged that commercial planting of transgenic palms will not occur any earlier than the year 2020.

scholarly journals Yaba-Like Disease Virus: an Alternative Replicating Poxvirus Vector for Cancer Gene Therapy

2001 ◽  
Vol 75 (21) ◽  
pp. 10300-10308 ◽  
Author(s):  
Yun Hu ◽  
John Lee ◽  
J. Andrea McCart ◽  
Hui Xu ◽  
Bernard Moss ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
High Titer ◽  
Human Tumor ◽  
Cancer Gene Therapy ◽  
Smallpox Vaccination ◽  
Cancer Gene ◽  
Virus Rna ◽  
Green Fluorescent

ABSTRACT Vaccinia virus is being investigated as a replicating vector for tumor-directed gene therapy. However, the majority of cancer patients have preformed immunologic reactivity against vaccinia virus, as a result of smallpox vaccination, which may limit its use as a vector. The Yaba-like disease (YLD) virus was investigated here as an alternative, replicating poxvirus for cancer gene therapy. We have demonstrated that the YLD virus does not cross-react with vaccinia virus antibodies, and it replicates efficiently in human tumor cells. YLD virus can be expanded and purified to high titer in CV-1 cells under conditions utilized for vaccinia virus. The YLD virus RNA polymerase was able to express genes regulated by a synthetic promoter designed for use in orthopoxviruses. We sequenced the YLD virus TK gene and created a shuttle plasmid, which allowed the recombination of the green fluorescent protein (GFP) gene into the YLD virus. In a murine model of ovarian cancer, up to 38% of cells in the tumor expressed the GFP transgene 12 days after intraperitoneal virus delivery. YLD virus has favorable characteristics as a vector for cancer gene therapy, and this potential should be explored further.

scholarly journals Essential and Dispensable Virus-Encoded Replication Elements Revealed by Efforts To Develop Hypoviruses as Gene Expression Vectors

2000 ◽  
Vol 74 (16) ◽  
pp. 7568-7577 ◽  
Author(s):  
Nobuhiro Suzuki ◽  
Lynn M. Geletka ◽  
Donald L. Nuss
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Cryphonectria Parasitica ◽  
Expression Vectors ◽  
Green Fluorescent Protein Gene ◽  
Reading Frame ◽  
Chestnut Blight Fungus ◽  
Virulence Attenuation ◽  
Foreign Genes ◽  
Green Fluorescent

ABSTRACT We have investigated whether hypoviruses, viral agents responsible for virulence attenuation (hypovirulence) of the chestnut blight fungusCryphonectria parasitica, could serve as gene expression vectors. The infectious cDNA clone of the prototypic hypovirus CHV1-EP713 was modified to generate 20 different vector candidates. Although transient expression was achieved for a subset of vectors that contained the green fluorescent protein gene from Aequorea victoria, long-term expression (past day 8) was not observed for any vector construct. Analysis of viral RNAs recovered from transfected fungal colonies revealed that the foreign genes were readily deleted from the replicating virus, although small portions of foreign sequences were retained by some vectors after months of replication. However, the results of vector viability and progeny characterization provided unexpected new insights into essential and dispensable elements of hypovirus replication. The N-terminal portion (codons 1 to 24) of the 5′-proximal open reading frame (ORF), ORF A, was found to be required for virus replication, while the remaining 598 codons of this ORF were completely dispensable. Substantial alterations were tolerated in the pentanucleotide UAAUG that contains the ORF A termination codon and the overlapping putative initiation codon of the second of the two hypovirus ORFs, ORF B. Replication competence was maintained following either a frameshift mutation that caused a two-codon extension of ORF A or a modification that produced a single-ORF genomic organization. These results are discussed in terms of determinants of hypovirus replication, the potential utility of hypoviruses as gene expression vectors, and possible mechanisms by which hypoviruses recognize and delete foreign sequences.

scholarly journals Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

2021 ◽  
Vol 9 (5) ◽  
pp. 1005
Author(s):  
Olga Chervyakova ◽  
Elmira Tailakova ◽  
Nurlan Kozhabergenov ◽  
Sandugash Sadikaliyeva ◽  
Kulyaisan Sultankulova ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Viral Vector ◽  
Foot And Mouth Disease ◽  
Open Reading Frames ◽  
Foreign Gene ◽  
Mouth Disease Virus ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
Reading Frames

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

Efficient Knockout of Transplanted Green Fluorescent Protein Gene in Medaka Using TALENs

2014 ◽  
Vol 16 (6) ◽  
pp. 674-683 ◽  
Author(s):  
Chao Qiu ◽  
Bin Cheng ◽  
Yunsheng Zhang ◽  
Rong Huang ◽  
Lanjie Liao ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Gene ◽  
Green Fluorescent

scholarly journals Mechanism of Aurora-B Degradation and Its Dependency on Intact KEN and A-Boxes: Identification of an Aneuploidy-Promoting Property

2005 ◽  
Vol 25 (12) ◽  
pp. 4977-4992 ◽  
Author(s):  
Hao G. Nguyen ◽  
Dharmaraj Chinnappan ◽  
Takeshi Urano ◽  
Katya Ravid
Keyword(s):  
Chromosome Segregation ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Degradation Pathway ◽  
Aurora B ◽  
Stable Form ◽  
Wild Type ◽  
Green Fluorescent ◽  
Critical Regions

ABSTRACT The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.

scholarly journals Vaccinia Virus F13L Protein with a Conserved Phospholipase Catalytic Motif Induces Colocalization of the B5R Envelope Glycoprotein in Post-Golgi Vesicles

2001 ◽  
Vol 75 (16) ◽  
pp. 7528-7542 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Recombinant Vaccinia Virus ◽  
Open Reading Frames ◽  
Type I ◽  
Golgi Vesicles ◽  
Infected Cells ◽  
Catalytic Motif ◽  
Green Fluorescent

ABSTRACT The wrapping of intracellular mature vaccinia virions by modifiedtrans-Golgi or endosomal cisternae to form intracellular enveloped virions is dependent on at least two viral proteins encoded by the B5R and F13L open reading frames. B5R is a type I integral membrane glycoprotein, whereas F13L is an unglycosylated, palmitylated protein with a motif that is conserved in a superfamily of phospholipid-metabolizing enzymes. Microscopic visualization of the F13L protein was achieved by fusing it to the enhanced green fluorescent protein (GFP). F13L-GFP was functional when expressed by a recombinant vaccinia virus in which it replaced the wild-type F13L gene or by transfection of uninfected cells with a plasmid vector followed by infection with an F13L deletion mutant. In uninfected or infected cells, F13L-GFP was associated with Golgi cisternae and post-Golgi vesicles containing the LAMP 2 late endosomal-lysosomal marker. Association of F13L-GFP with vesicles was dependent on an intact phospholipase catalytic motif and sites of palmitylation. The B5R protein was also associated with LAMP2-containing vesicles when F13L-GFP was coexpressed, but was largely restricted to Golgi cisternae in the absence of F13L-GFP or when the F13L moiety was mutated. We suggest that the F13L protein, like its human phospholipase D homolog, regulates vesicle formation and that this process is involved in intracellular enveloped virion membrane formation.

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