scholarly journals Unraveling the Genome-Wide Impact of Recombinant Baculovirus Infection in Mammalian Cells for Gene Delivery

Genes ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 1306
Author(s):  
Ha Youn Shin ◽  
Hanul Choi ◽  
Nahyun Kim ◽  
Nayoung Park ◽  
Heesun Kim ◽  
...  
Keyword(s):  
Immune Response ◽  
Gene Delivery ◽  
Mammalian Cells ◽  
Vaccine Development ◽  
Recombinant Baculovirus ◽  
Cytokine Signaling ◽  
Rna Seq ◽  
Immune Response Genes ◽  
Genome Wide ◽  
Baculovirus Infection

Baculovirus expression systems have been widely used to produce recombinant mammalian proteins owing to the lack of viral replication in vertebrates. Although several lines of evidence have demonstrated impacts of baculovirus infection in mammalian hosts, genome-wide effects have not been fully elucidated. Here, we provide comparative transcriptome profiles of baculovirus and host-immune response genes in recombinant baculovirus-infected mammalian and insect cells. Specifically, to decipher the impacts of baculovirus infection in mammalian cells, we conducted total RNA-seq on human 293TT cells and insect Sf9 cells infected with recombinant baculovirus. We found that baculovirus genes were rarely expressed under the control of baculoviral promoters in 293TT cells. Although some baculovirus early genes, such as PE38 and IE-01, showed limited expression in 293TT cells, baculoviral late genes were mostly silent. We also found modest induction of a small number of mammalian immune response genes associated with Toll-like receptors, cytokine signaling, and complement in baculovirus-infected 293TT cells. These comprehensive transcriptome data will contribute to improving recombinant baculovirus as tools for gene delivery, gene therapy, and vaccine development.

scholarly journals Baculovirus as a Tool for Gene Delivery and Gene Therapy

Viruses ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 510 ◽  
Author(s):  
Chikako Ono ◽  
Toru Okamoto ◽  
Takayuki Abe ◽  
Yoshiharu Matsuura
Keyword(s):  
Gene Delivery ◽  
Mammalian Cells ◽  
Recombinant Baculovirus ◽  
Foreign Gene ◽  
Type I ◽  
Adjuvant Activity ◽  
Baculovirus Infection ◽  
Immunodeficiency Virus ◽  
Host Innate Immune Response ◽  
Infected Cells

Based on its ability to express high levels of protein, baculovirus has been widely used for recombinant protein production in insect cells for more than thirty years with continued technical improvements. In addition, baculovirus has been successfully applied for foreign gene delivery into mammalian cells without any viral replication. However, several CpG motifs are present throughout baculoviral DNA and induce an antiviral response in mammalian cells, resulting in the production of pro-inflammatory cytokines and type I interferon through a Toll-like receptor (TLR)-dependent or -independent signaling pathway, and ultimately limiting the efficiency of transgene expression. On the other hand, by taking advantage of this strong adjuvant activity, recombinant baculoviruses encoding neutralization epitopes can elicit protective immunity in mice. Moreover, immunodeficient cells, such as hepatitis C virus (HCV)- or human immunodeficiency virus (HIV)-infected cells, are more susceptible to baculovirus infection than normal cells and are selectively eliminated by the apoptosis-inducible recombinant baculovirus. Here, we summarize the application of baculovirus as a gene expression vector and the mechanism of the host innate immune response induced by baculovirus in mammalian cells. We also discuss the future prospects of baculovirus vectors.

scholarly journals Baculovirus-Mediated Gene Delivery into Mammalian Cells Does Not Alter Their Transcriptional and Differentiating Potential but Is Accompanied by Early Viral Gene Expression

2006 ◽  
Vol 80 (8) ◽  
pp. 4135-4146 ◽  
Author(s):  
Christos Kenoutis ◽  
Rodica C. Efrose ◽  
Luc Swevers ◽  
Alexandros A. Lavdas ◽  
Maria Gaitanou ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Schwann Cells ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Primary Cultures ◽  
Recombinant Baculovirus ◽  
Hek293 Cells ◽  
Viral Gene ◽  
Transcriptional Responses ◽  
Baculovirus Infection

ABSTRACT Gene delivery to neural cells is central to the development of transplantation therapies for neurological diseases. In this study, we used a baculovirus derived from the domesticated silk moth, Bombyx mori, as vector for transducing a human cell line (HEK293) and primary cultures of rat Schwann cells. Under optimal conditions of infection with a recombinant baculovirus containing the reporter green fluorescent protein gene under mammalian promoter control, the infected cells express the transgene with high efficiency. Toxicity assays and transcriptome analyses suggest that baculovirus infection is not cytotoxic and does not induce differential transcriptional responses in HEK293 cells. Infected Schwann cells retain their characteristic morphological and molecular phenotype as determined by immunocytochemistry for the marker proteins S-100, glial fibrillary acidic protein, and p75 nerve growth factor receptor. Moreover, baculovirus-infected Schwann cells are capable of differentiating in vitro and express the P0 myelination marker. However, transcripts for several immediate-early viral genes also accumulate in readily detectable levels in the transduced cells. This transcriptional activity raises concerns regarding the long-term safety of baculovirus vectors for gene therapy applications. Potential approaches for overcoming the identified problem are discussed.

scholarly journals VELCRO-IP RNA-seq explores ribosome expansion segment function in translation genome-wide

2020 ◽  
Author(s):  
Kathrin Leppek ◽  
Gun Woo Byeon ◽  
Kotaro Fujii ◽  
Maria Barna
Keyword(s):  
Ribosomal Rna ◽  
Mammalian Cells ◽  
Genetically Modify ◽  
Rna Seq ◽  
Eukaryotic Evolution ◽  
Novel Technology ◽  
Genome Wide ◽  
Chromosomal Loci ◽  
Independent Translation ◽  
Specific Mrna

SUMMARYRoles for ribosomal RNA (rRNA) in gene regulation remain largely unexplored. With hundreds of rDNA units scattered across multiple chromosomal loci, it is not possible to genetically modify rRNA in mammalian cells, hindering understanding of ribosome function. Emerging evidence suggests that expansion segments (ESs), tentacle-like rRNA extensions that vary in sequence and size across eukaryotic evolution, may provide platforms for the binding of proteins and mRNAs. Here, we develop VELCRO-IP RNA-seq: a versatile methodology to generate species-adapted ESs and map specific mRNA regions across the transcriptome that preferentially associate with ESs. By applying VELCRO-IP RNA-seq to a mammalian ES, ES9S, we identified a large array of mRNAs that are selectively recruited to ribosomes via an ES. We further characterize a set of specific 5’ UTRs that facilitate cap-independent translation through ES9S-mediated ribosome recruitment. These data provide a novel technology for studying the enigmatic ESs of the ribosome in gene-specific translation.

scholarly journals Potential Functional Variants in Innate Immune Response Genes Associated with Feed Efficiency in Beef Cattle

2020 ◽  
Author(s):  
Gabriela Ribeiro ◽  
Aline Silva Mello Cesar ◽  
Pâmela Almeida Alexandre ◽  
José Bento Sterman Ferraz ◽  
Heidge Fukumasu
Keyword(s):  
Immune Response ◽  
Beef Cattle ◽  
Innate Immune Response ◽  
Feed Efficiency ◽  
Factor H ◽  
Innate Immune ◽  
Functional Enrichment ◽  
Rna Seq ◽  
Immune Response Genes ◽  
Functional Variants

Abstract Background: Identifying and selecting animals for feed efficiency (FE) is extremely important for the beef production chain. Currently, the most common parameter to access the FE animals is residual feed intake (RFI), which is the residual of the linear regression that estimates DMI based on average daily gain and mid-test metabolic body weight. However, it relies on costly and time-consuming data collection, creating a growing demand for alternative approaches to identify genetically superior animals for FE. This study aimed to detect potential liver-specific functional variants from RNA-seq data of 16 Nellore bulls divergently selected for FE. Results: The variant call analysis detected 247 missense SNPs and nine insertion-deletions (INDELs) that alter the protein functions. These variants were found within 190 genes differentially found (P < 0.05) in liver tissue between high FE (HFE) and low FE (LFE) animals. To better understand the role of these variants in biological pathways, we performed a functional enrichment analysis, which highlighted six genes involved in complement cascade and cascade complement regulation pathways, and 20 genes involved in the regulation of the innate immune system. They had four different significant variants in the complement factor H (CFH) family genes, and all were homozygous in HFE animals rather than some degree of heterozygous in LFE animals. Conclusion: We developed a pipeline to detect potential liver-specific functional variants from RNA-seq data from animals divergently selected for feed efficiency. With this approach, we found potential functional variants in innate immune response genes associated with feed efficiency in beef cattle.

scholarly journals Baculovirus Transduction in Mammalian Cells Is Affected by the Production of Type I and III Interferons, Which Is Mediated Mainly by the cGAS-STING Pathway

2020 ◽  
Vol 94 (21) ◽  
Author(s):  
Sabrina Amalfi ◽  
Guido Nicolás Molina ◽  
Romina Jimena Bevacqua ◽  
María Gabriela López ◽  
Oscar Taboga ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Gene Delivery ◽  
Epithelial Cells ◽  
Mammalian Cells ◽  
Viral Vectors ◽  
Type I ◽  
Dependent Manner ◽  
Human Epithelial Cells ◽  
Baculovirus Infection ◽  
Sting Pathway

ABSTRACT The baculovirus Autographa californica multiple nucleopolyhedrovirus is an insect virus with a circular double-stranded DNA genome, which, among other multiple biotechnological applications, is used as an expression vector for gene delivery in mammalian cells. Nevertheless, the nonspecific immune response triggered by viral vectors often suppresses transgene expression. To understand the mechanisms involved in that response, in the present study, we studied the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway by using two approaches: the genetic edition through CRISPR/Cas9 technology of genes encoding STING or cGAS in NIH/3T3 murine fibroblasts and the infection of HEK293 and HEK293 T human epithelial cells, deficient in cGAS and in cGAS and STING expression, respectively. Overall, our results suggest the existence of two different pathways involved in the establishment of the antiviral response, both dependent on STING expression. Particularly, the cGAS-STING pathway resulted in the more relevant production of beta interferon (IFN-β) and IFN-λ1 in response to baculovirus infection. In human epithelial cells, IFN-λ1 production was also induced in a cGAS-independent and DNA-protein kinase (DNA-PK)-dependent manner. Finally, we demonstrated that these cellular responses toward baculovirus infection affect the efficiency of transduction of baculovirus vectors. IMPORTANCE Baculoviruses are nonpathogenic viruses that infect mammals, which, among other applications, are used as vehicles for gene delivery. Here, we demonstrated that the cytosolic DNA sensor cGAS recognizes baculoviral DNA and that the cGAS-STING axis is primarily responsible for the attenuation of transduction in human and mouse cell lines through type I and type III IFNs. Furthermore, we identified DNA-dependent protein kinase (DNA-PK) as a cGAS-independent and alternative DNA cytosolic sensor that contributes less to the antiviral state in baculovirus infection in human epithelial cells than cGAS. Knowledge of the pathways involved in the response of mammalian cells to baculovirus infection will improve the use of this vector as a tool for gene therapy.

HIV-1 Accessory Proteins: Which one is Potentially Effective in Diagnosis and Vaccine Development?

2020 ◽  
Vol 28 ◽  
Author(s):  
Alireza Milani ◽  
Kazem Baesi ◽  
Elnaz Agi ◽  
Ghazal Marouf ◽  
Maryam Ahmadi ◽  
...  
Keyword(s):  
Immune Response ◽  
Vaccine Development ◽  
Treated Group ◽  
Vaccine Antigen ◽  
Accessory Proteins ◽  
Serological Method ◽  
Th2 Immune Response ◽  
Untreated Individuals ◽  
Immunogenic Properties ◽  
Hiv 1

Background:: The combination antiretroviral therapy (cART) could increase the number of circulating naive CD4 T lymphocytes, but was not able to eradicate human immunodeficiency virus-1 (HIV-1) infection. Objective:: Thus, induction of strong immune responses is important for control of HIV-1 infection. Furthermore, a simple and perfect serological method is required to detect virus in untreated-, treated- and drug resistant- HIV-1 infected individuals. Methods:: This study was conducted to assess and compare immunogenic properties of Nef, Vif, Vpr and Vpu accessory proteins as an antigen candidate in mice and their diagnostic importance in human as a biomarker. Results:: Our data showed that in mice, all heterologous prime/ boost regimens were more potent than homologous prime/ boost regimens in eliciting Th1 response and Granzyme B secretion as CTL activity. Moreover, the Nef, Vpu and Vif proteins could significantly increase Th1 immune response. In contrast, the Vpr protein could considerably induce Th2 immune response. On the other hand, among four accessory proteins, HIV-1 Vpu could significantly detect treated group from untreated group as a possible biomarker in human. Conclusion:: Generally, among accessory proteins, Nef, Vpu and Vif antigens were potentially more suitable vaccine antigen candidates than Vpr antigen. Human antibodies against all these proteins were higher in HIV-1 different groups than healthy group. Among them, Vpu was known as a potent antigen in diagnosis of treated from untreated individuals. The potency of accessory proteins as an antigen candidate in an animal model and a human cohort study are underway.

scholarly journals Platforms for Production of Protein-Based Vaccines: From Classical to Next-Generation Strategies

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1072
Author(s):  
Raquel Cid ◽  
Jorge Bolívar
Keyword(s):  
Infectious Diseases ◽  
Mammalian Cells ◽  
Vaccine Development ◽  
Low Cost ◽  
Cost Effective ◽  
Insect Larvae ◽  
Subunit Vaccines ◽  
Effective Strategies ◽  
Alternative Systems ◽  
Inactivated Vaccines

To date, vaccination has become one of the most effective strategies to control and reduce infectious diseases, preventing millions of deaths worldwide. The earliest vaccines were developed as live-attenuated or inactivated pathogens, and, although they still represent the most extended human vaccine types, they also face some issues, such as the potential to revert to a pathogenic form of live-attenuated formulations or the weaker immune response associated with inactivated vaccines. Advances in genetic engineering have enabled improvements in vaccine design and strategies, such as recombinant subunit vaccines, have emerged, expanding the number of diseases that can be prevented. Moreover, antigen display systems such as VLPs or those designed by nanotechnology have improved the efficacy of subunit vaccines. Platforms for the production of recombinant vaccines have also evolved from the first hosts, Escherichia coli and Saccharomyces cerevisiae, to insect or mammalian cells. Traditional bacterial and yeast systems have been improved by engineering and new systems based on plants or insect larvae have emerged as alternative, low-cost platforms. Vaccine development is still time-consuming and costly, and alternative systems that can offer cost-effective and faster processes are demanding to address infectious diseases that still do not have a treatment and to face possible future pandemics.

scholarly journals Integration of tumor inflammation, cell proliferation, and traditional biomarkers improves prediction of immunotherapy resistance and response

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Sarabjot Pabla ◽  
R. J. Seager ◽  
Erik Van Roey ◽  
Shuang Gao ◽  
Carrie Hoefer ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Proliferation ◽  
Cell Activation ◽  
Response Rate ◽  
Checkpoint Inhibitors ◽  
Host Immune Response ◽  
Gene Signature ◽  
B Cell Activation ◽  
Rna Seq ◽  
Multiple Tumor

Abstract Background Contemporary to the rapidly evolving landscape of cancer immunotherapy is the equally changing understanding of immune tumor microenvironments (TMEs) which is crucial to the success of these therapies. Their reliance on a robust host immune response necessitates clinical grade measurements of immune TMEs at diagnosis. In this study, we describe a stable tumor immunogenic profile describing immune TMEs in multiple tumor types with ability to predict clinical benefit from immune checkpoint inhibitors (ICIs). Methods A tumor immunogenic signature (TIGS) was derived from targeted RNA-sequencing (RNA-seq) and gene expression analysis of 1323 clinical solid tumor cases spanning 35 histologies using unsupervised analysis. TIGS correlation with ICI response and survival was assessed in a retrospective cohort of NSCLC, melanoma and RCC tumor blocks, alone and combined with TMB, PD-L1 IHC and cell proliferation biomarkers. Results Unsupervised clustering of RNA-seq profiles uncovered a 161 gene signature where T cell and B cell activation, IFNg, chemokine, cytokine and interleukin pathways are over-represented. Mean expression of these genes produced three distinct TIGS score categories: strong (n = 384/1323; 29.02%), moderate (n = 354/1323; 26.76%), and weak (n = 585/1323; 44.22%). Strong TIGS tumors presented an improved ICI response rate of 37% (30/81); with highest response rate advantage occurring in NSCLC (ORR = 36.6%; 16/44; p = 0.051). Similarly, overall survival for strong TIGS tumors trended upward (median = 25 months; p = 0.19). Integrating the TIGS score categories with neoplastic influence quantified via cell proliferation showed highly proliferative and strong TIGS tumors correlate with significantly higher ICI ORR than poorly proliferative and weak TIGS tumors [14.28%; p = 0.0006]. Importantly, we noted that strong TIGS and highly [median = not achieved; p = 0.025] or moderately [median = 16.2 months; p = 0.025] proliferative tumors had significantly better survival compared to weak TIGS, highly proliferative tumors [median = 7.03 months]. Importantly, TIGS discriminates subpopulations of potential ICI responders that were considered negative for response by TMB and PD-L1. Conclusions TIGS is a comprehensive and informative measurement of immune TME that effectively characterizes host immune response to ICIs in multiple tumors. The results indicate that when combined with PD-L1, TMB and cell proliferation, TIGS provides greater context of both immune and neoplastic influences on the TME for implementation into clinical practice.

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