scholarly journals Reciprocal Inhibition of Immunogenic Performance in Mice of Two Potent DNA Immunogens Targeting HCV Related Liver Cancer

2021 ◽  
Author(s):  
Juris Jansons ◽  
Dace Skrastina ◽  
Alisa Kurlanda ◽  
Stefan Petkov ◽  
Darya Avdoshina ◽  
...  
Keyword(s):  
Immune Response ◽  
Liver Cancer ◽  
Reverse Transcriptase ◽  
Core Protein ◽  
Protein Translation ◽  
Reciprocal Inhibition ◽  
Full Length ◽  
Dna Immunization ◽  
Malignant Cells

Chronic HCV infection and associated liver cancer impose a heavy burden on the healthcare system. Direct acting antivirals eliminate HCV, unless it is drug resistant, and partially reverse liver disease, but they cannot cure HCV-related cancer. Possible remedy could be a multi-component immunotherapeutic vaccine targeting both HCV-infected and malignant cells, also those not infected with HCV. To meet this need we developed a two-component DNA vaccine based on the highly conserved core protein of HCV to target HCV-infected cells, and a renowned tumor associated antigen telomerase reverse transcriptase (TERT) based on the rat TERT, to target malignant cells. Their synthetic genes were expression-optimized, and HCV core was truncated after aa 152 (Core152opt) to delete the domain interfering with immunogenicity. Core152opt and TERT DNA were highly immunogenic in BALB/c mice, inducing IFN-γ/IL-2/TNF-α response of CD4+ and CD8+ T cells. Also, DNA-immunization with TERT enhanced cellular immune response against luciferase encoded by a co-delivered plasmid (Luc DNA). However, DNA-immunization with Core152opt and TERT mix resulted in abrogation of immune response against both components. A loss of bioluminescent signal after co-delivery of TERT and Luc DNA into mice indicated that TERT affects the in vivo expression of luciferase directed by the immediate early cytomegalovirus and interferon-β promoters. Panel of mutant TERT variants was created and tested for their expression effects. TERT with deleted N-terminal nucleoli localization signal and mutations abrogating telomerase activity still suppressed the IFN-β driven Luc expression, while the inactivated reverse transcriptase domain of TERT and its analogue, enzymatically active HIV-1 reverse transcriptase, exerted only weak suppressive effects, implying that suppression relied on the presence of the full-length/nearly full-length TERT, but not its enzymatic activity. The effect(s) could be due to interference of the ectopically expressed xenogeneic rat TERT with biogenesis of mRNA, ribosomes and protein translation in murine cells, affecting the expression of immunogens. HCV core can aggravate this effect, leading to early apoptosis of co-expressing cells, preventing the induction of immune response.

scholarly journals Reciprocal Inhibition of Immunogenic Performance in Mice of Two Potent DNA Immunogens Targeting HCV-Related Liver Cancer

2021 ◽  
Vol 9 (5) ◽  
pp. 1073
Author(s):  
Juris Jansons ◽  
Dace Skrastina ◽  
Alisa Kurlanda ◽  
Stefan Petkov ◽  
Darya Avdoshina ◽  
...  
Keyword(s):  
Immune Response ◽  
Liver Cancer ◽  
Reverse Transcriptase ◽  
Core Protein ◽  
Protein Translation ◽  
Reciprocal Inhibition ◽  
Full Length ◽  
Dna Immunization ◽  
Malignant Cells

Chronic HCV infection and associated liver cancer impose a heavy burden on the healthcare system. Direct acting antivirals eliminate HCV, unless it is drug resistant, and partially reverse liver disease, but they cannot cure HCV-related cancer. A possible remedy could be a multi-component immunotherapeutic vaccine targeting both HCV-infected and malignant cells, but also those not infected with HCV. To meet this need we developed a two-component DNA vaccine based on the highly conserved core protein of HCV to target HCV-infected cells, and a renowned tumor-associated antigen telomerase reverse transcriptase (TERT) based on the rat TERT, to target malignant cells. Their synthetic genes were expression-optimized, and HCV core was truncated after aa 152 (Core152opt) to delete the domain interfering with immunogenicity. Core152opt and TERT DNA were highly immunogenic in BALB/c mice, inducing IFN-γ/IL-2/TNF-α response of CD4+ and CD8+ T cells. Additionally, DNA-immunization with TERT enhanced cellular immune response against luciferase encoded by a co-delivered plasmid (Luc DNA). However, DNA-immunization with Core152opt and TERT mix resulted in abrogation of immune response against both components. A loss of bioluminescence signal after co-delivery of TERT and Luc DNA into mice indicated that TERT affects the in vivo expression of luciferase directed by the immediate early cytomegalovirus and interferon-β promoters. Panel of mutant TERT variants was created and tested for their expression effects. TERT with deleted N-terminal nucleoli localization signal and mutations abrogating telomerase activity still suppressed the IFN-β driven Luc expression, while the inactivated reverse transcriptase domain of TERT and its analogue, enzymatically active HIV-1 reverse transcriptase, exerted only weak suppressive effects, implying that suppression relied on the presence of the full-length/nearly full-length TERT, but not its enzymatic activity. The effect(s) could be due to interference of the ectopically expressed xenogeneic rat TERT with biogenesis of mRNA, ribosomes and protein translation in murine cells, affecting the expression of immunogens. HCV core can aggravate this effect, leading to early apoptosis of co-expressing cells, preventing the induction of immune response.

scholarly journals Inhibition of the HCV Core Protein on the Immune Response to HBV Surface Antigen and on HBV Gene Expression and Replication In Vivo

PLoS ONE ◽  
2012 ◽  
Vol 7 (9) ◽  
pp. e45146 ◽  
Author(s):  
Wenbo Zhu ◽  
Chunchen Wu ◽  
Wanyu Deng ◽  
Rongjun Pei ◽  
Yun Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
Surface Antigen ◽  
Core Protein ◽  
Hcv Core Protein

scholarly journals Tumour secreted grp170 chaperones full-length protein substrates and induces an adaptive anti-tumour immune response in vivo

2010 ◽  
Vol 26 (4) ◽  
pp. 366-375 ◽  
Author(s):  
Hilal Arnouk ◽  
Evan R. Zynda ◽  
Xiang-Yang Wang ◽  
Bonnie L. Hylander ◽  
Masoud H. Manjili ◽  
...  
Keyword(s):  
Immune Response ◽  
Full Length ◽  
Protein Substrates

scholarly journals Development of a flexible split prime editor using truncated reverse transcriptase

2021 ◽  
Author(s):  
Wen Xue ◽  
Chunwei Zheng ◽  
SHUNQING LIANG ◽  
Pengpeng Liu ◽  
Bin Liu ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Mammalian Cells ◽  
Stop Codon ◽  
Protein Biosynthesis ◽  
Potential Effect ◽  
Rnase H ◽  
Full Length ◽  
Large Size ◽  
Efficient Delivery

Prime Editor (PE) has tremendous promise for gene therapy. However, it remains a challenge to deliver PE (>6.3 kb) in vivo. Although PE can be split into two fragments and delivered using dual adeno-associated viruses (AAVs), choice of split sites within Cas9, which affects editing efficiency, is limited due to the large size of PE. Furthermore, the potential effect of overexpressing RT in mammalian cells is largely unknown. Here, we developed a compact PE with complete deletion of the RNase H domain of reverse transcriptase (RT), which showed comparable editing to full-length PE. Using compact PE, we tested the effect of 4 different Cas9 split sites and found that the Glu 573 split site supports robust editing (up to 93% of full-length PE). The compact PE, but not PE2, abolished its binding to eRF1 and showed minimal effect on stop codon readthrough, which therefore might reduce the effects on protein biosynthesis. This study identifies a safe and efficient compact PE2 that enables flexible split-PE design to facilitate efficient delivery in vivo and advance the utility of prime editing.

scholarly journals Released Tryptophanyl-tRNA Synthetase Stimulates Innate Immune Responses against Viral Infection

2018 ◽  
Vol 93 (2) ◽  
Author(s):  
Hyun-Cheol Lee ◽  
Eun-Seo Lee ◽  
Md Bashir Uddin ◽  
Tae-Hwan Kim ◽  
Jae-Hoon Kim ◽  
...  
Keyword(s):  
Immune Response ◽  
Immune Responses ◽  
Viral Infection ◽  
Innate Immune Response ◽  
Trna Synthetase ◽  
Innate Immune ◽  
Full Length ◽  
Type I ◽  
Innate Immune Responses

ABSTRACT Tryptophanyl-tRNA synthetase (WRS) is one of the aminoacyl-tRNA synthetases (ARSs) that possesses noncanonical functions. Full-length WRS is released during bacterial infection and primes the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex to elicit innate immune responses. However, the role of WRS in viral infection remains unknown. Here, we show that full-length WRS is secreted by immune cells in the early phase of viral infection and functions as an antiviral cytokine. Treatment of cells with recombinant WRS protein promotes the production of inflammatory cytokines and type I interferons (IFNs) and curtails virus replication in THP-1 and Raw264.7 cells but not in TLR4−/− or MD2−/− bone marrow-derived macrophages (BMDMs). Intravenous and intranasal administration of recombinant WRS protein induces an innate immune response and blocks viral replication in vivo. These findings suggest that secreted full-length WRS has a noncanonical role in inducing innate immune responses to viral infection as well as to bacterial infection. IMPORTANCE ARSs are essential enzymes in translation that link specific amino acids to their cognate tRNAs. In higher eukaryotes, some ARSs possess additional, noncanonical functions in the regulation of cell metabolism. Here, we report a novel noncanonical function of WRS in antiviral defense. WRS is rapidly secreted in response to viral infection and primes the innate immune response by inducing the secretion of proinflammatory cytokines and type I IFNs, resulting in the inhibition of virus replication both in vitro and in vivo. Thus, we consider WRS to be a member of the antiviral innate immune response. The results of this study enhance our understanding of host defense systems and provide additional information on the noncanonical functions of ARSs.

scholarly journals Mutagenesis of the NS2B-NS3-Mediated Cleavage Site in the Flavivirus Capsid Protein Demonstrates a Requirement for Coordinated Processing

1999 ◽  
Vol 73 (10) ◽  
pp. 8083-8094 ◽  
Author(s):  
Sean M. Amberg ◽  
Charles M. Rice
Keyword(s):  
Cleavage Site ◽  
Yellow Fever Virus ◽  
Core Protein ◽  
Biological Significance ◽  
Full Length ◽  
Signal Peptidase ◽  
Amino Terminus ◽  
Efficient Production

ABSTRACT Analysis of flavivirus polyprotein processing has revealed the presence of a substrate for the virus-encoded NS2B-NS3 protease at the carboxy-terminal end of the C (capsid or core) protein. Cleavage at this site has been implicated in the efficient generation of the amino terminus of prM via signal peptidase cleavage. Yellow fever virus has four basic residues (Arg-Lys-Arg-Arg) in the P1 through P4 positions of this cleavage site. Multiple alanine substitutions were made for these residues in order to investigate the substrate specificity and biological significance of this cleavage. Mutants were analyzed by several methods: (i) a cell-free trans processing assay for direct analysis of NS2B-NS3-mediated cleavage; (ii) a transprocessing assay in BHK-21 cells, using a C-prM polyprotein, for analysis of prM production; (iii) an infectivity assay of full-length transcripts to determine plaque-forming ability; and (iv) analysis of proteins expressed from full-length transcripts to assess processing in the context of the complete genome. Mutants that exhibited severe defects in processing in vitro and in vivo were incapable of forming plaques. Mutants that contained two adjacent basic residues within the P1 through P4 region were processed more efficiently in vitro and in vivo, and transcripts bearing these mutations were fully infectious. Furthermore, two naturally occurring plaque-forming revertants were analyzed and shown to have restored protein processing phenotypes in vivo. Finally, the efficient production of prM was shown to be dependent on the proteolytic activity of NS3. These data support a model of two coordinated cleavages, one that generates the carboxy terminus of C and another that generates the amino terminus of prM. A block in the viral protease-mediated cleavage inhibits the production of prM by the signal peptidase, inhibits particle release, and eliminates plaque formation.

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