Genetic design of an optimized packaging cell line for gene vectors transducing human B cells

Hettich, E; Janz, Annette; Zeidler, Reinhard; Pich, Dagmar; Hellebrand, E; Weissflog, B; Moosmann, Andreas; Hammerschmidt, Wolfgang

doi:10.1038/sj.gt.3302714

Cited by 19 publications

(17 citation statements)

References 59 publications

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“…The construction of the first-generation packaging cell line, TR-2/293, and its successor, 293-VIIϩ, and the BZLF1 expression plasmid p509 have been described elsewhere (9,16,19).…”

Section: Methodsmentioning

confidence: 99%

“…However, it remained unclear whether particles from TR-2/293 are entirely free of viral DNA due to illegitimate recombination and thus retained transformation capacity (13). In order to meet these major safety issues, we generated a next-generation packaging cell line, termed 293-VIIϩ, in that we additionally deleted or functionally inactivated six viral genes (EBNA2, LMP1, EBNA3A, -B, and -C, BZLF1) which contribute to the transformation of human B cell or virus synthesis (19). We show here that these packaging cell lines are also appropriate producer cells for virus-like particles (VLPs) which maintain key characteristics of EBV, such as B cell tropism and antigenicity.…”

mentioning

confidence: 99%

“…Transient cotransfection of these cell lines with an expression plasmid encoding BZLF1, the viral transactivator of the lytic cycle (16), and appropriate vector plasmid DNA results in the induction of EBV's lytic phase and concomitant release of recombinant viral vectors with potential to efficiently deliver genes of interest to human B lymphocytes (17,19). The first-generation packaging cell line, termed TR-2/293, contained a helper genome that lacked the packaging element TR essential for encapsidation and thus did not release detectable amounts of infectious helper EBV (9).…”

mentioning

confidence: 99%

“…In a phase I clinical study in children awaiting kidney transplantation, adequate gp350 antibody responses were obtained, but insufficient B cell memory was achieved, probably due to the weak adjuvant used (38). Thus, it is currently not known whether this vaccine also prevents the development of posttransplant lymphoproliferative disorder.Previously, we described HEK293 cell-based EBV packaging cell lines that harbor helper EBV genomes for the generation of EBV-based viral gene vectors (9,19). Transient cotransfection of these cell lines with an expression plasmid encoding BZLF1, the viral transactivator of the lytic cycle (16), and appropriate vector plasmid DNA results in the induction of EBV's lytic phase and concomitant release of recombinant viral vectors with potential to efficiently deliver genes of interest to human B lymphocytes (17, 19).…”

mentioning

confidence: 99%

“…Previously, we described HEK293 cell-based EBV packaging cell lines that harbor helper EBV genomes for the generation of EBV-based viral gene vectors (9,19). Transient cotransfection of these cell lines with an expression plasmid encoding BZLF1, the viral transactivator of the lytic cycle (16), and appropriate vector plasmid DNA results in the induction of EBV's lytic phase and concomitant release of recombinant viral vectors with potential to efficiently deliver genes of interest to human B lymphocytes (17, 19).…”

mentioning

confidence: 99%

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A Virus-Like Particle-Based Epstein-Barr Virus Vaccine

Ruiss

Jochum

Wanner

et al. 2011

J Virol

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Epstein-Barr Virus (EBV) is an ubiquitous human herpesvirus which can lead to infectious mononucleosis and different cancers. In immunocompromised individuals, this virus is a major cause for morbidity and mortality. Transplant patients who did not encounter EBV prior to immunosuppression frequently develop EBV-associated malignancies, but a prophylactic EBV vaccination might reduce this risk considerably. Viruslike particles (VLPs) mimic the structure of the parental virus but lack the viral genome. Therefore, VLPs are considered safe and efficient vaccine candidates. We engineered a dedicated producer cell line for EBV-derived VLPs. This cell line contains a genetically modified EBV genome which is devoid of all potential viral oncogenes but provides viral proteins essential for the assembly and release of VLPs via the endosomal sorting complex required for transport (ESCRT). Human B cells readily take up EBV-based VLPs and present viral epitopes in association with HLA molecules to T cells. Consequently, EBV-based VLPs are highly immunogenic and elicit humoral and strong CD8؉ and CD4 ؉ T cell responses in vitro and in a preclinical murine model in vivo. Our findings suggest that VLP formulations might be attractive candidates to develop a safe and effective polyvalent vaccine against EBV.Epstein-Barr Virus (EBV) infects more than 90% of the human population worldwide and persists for life. Primary infection normally occurs during early childhood and is usually asymptomatic, but EBV causes a lymphoproliferative syndrome termed infectious mononucleosis (IM) in about 50% of adolescents and adults on their first contact with EBV (40). Although the disease is normally self-limiting, prolonged forms of IM (52) and chronic active EBV infection with a fatal outcome have been reported (30). IM also significantly increases the risk of developing Hodgkin's disease and other types of lymphoma (14) and is an independent risk factor for multiple sclerosis (51). In addition, EBV is causally associated with a heterogeneous group of malignancies, including undifferentiated nasopharyngeal carcinoma, gastric carcinoma, and various types of lymphoma (29). As a consequence, the International Agency for Research on Cancer (IARC) classifies the virus as a class I carcinogen (1).Opportunistic infections represent severe complications in patients with HIV/AIDS and patients treated with immunosuppressive drugs. Transplant recipients who are immunologically naïve for EBV at the onset of iatrogenic immunosuppression are at a particularly high risk of developing life-threatening EBV-positive posttransplant lymphoproliferative disease (PTLD). The exposure to immunosuppressive drugs impairs T cell immunity, and the patients are unable to effectively prime EBV-specific T cells, which control and prevent proliferation of EBV-infected B cells in vivo. Patients who are EBV seropositive at transplant have a much lower risk for PTLD (see reference 12 for a review), and the adoptive transfer of EBV-specific effector T cells can sometimes cure thi...

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“…The construction of the first-generation packaging cell line, TR-2/293, and its successor, 293-VIIϩ, and the BZLF1 expression plasmid p509 have been described elsewhere (9,16,19).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

A Virus-Like Particle-Based Epstein-Barr Virus Vaccine

Ruiss

Jochum

Wanner

et al. 2011

J Virol

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Developing Extrachromosomal Gene Expression Vector Technologies: An Overview

Wade‐Martins

2011

Methods in Molecular Biology

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Extrachromosomal, or episomal, vectors offer a number of advantages for therapeutic and scientific applications compared to integrating vectors. Extrachromosomal vectors persist in the nucleus without the requirement to integrate into the host genome, hence avoiding the recent concerns surrounding the genotoxic effects of vector integration. By avoiding integration, episomal vectors avoid vector rearrangement, which can occur at integration, and also avoid any effect of surrounding DNA activity on transgene expression ("position effect"). Extrachromosomal vectors offer a very high transgene capacity, allowing either the incorporation of large promoter and regulatory elements into an expression cassette, or the use of complete genomic loci of up to 100 kb or larger as transgenes. Whole genomic loci transgenes offer an elegant means to express genes under physiological and developmental-stage regulation, to express multiple transcript variants from a single locus, and to express multiple genes from a single tract of genomic DNA. The combined advantages of episomal vectors of prolonged transgene persistence in the absence of vector integration, avoiding silencing by flanking heterochromatin, and high capacity, facilitating delivery and expression of genomic DNA transgenes, will be reviewed here and potential therapeutic and scientific uses outlined.

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Vaccine Development for Epstein-Barr Virus

Cohen

2018

Advances in Experimental Medicine and Biology

108

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Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and is associated with several malignancies, including nasopharyngeal carcinoma, gastric carcinoma, Hodgkin lymphoma, Burkitt lymphoma, and lymphomas in immunocompromised persons, as well as multiple sclerosis. A vaccine is currently unavailable. While monomeric EBV gp350 was shown in a phase 2 trial to reduce the incidence of infectious mononucleosis, but not the rate of EBV infection, newer formulations of gp350 including multimeric forms, viruslike particles, and nanoparticles may be more effective. A vaccine that also includes additional viral glycoproteins, lytic proteins, or latency proteins might improve the effectiveness of an EBV gp350 vaccine. Clinical trials to determine if an EBV vaccine can reduce the rate of infectious mononucleosis or posttransplant lymphoproliferative disease should be performed. The former is important since infectious mononucleosis can be associated with debilitating fatigue as well as other complications, and EBV infectious mononucleosis is associated with increased rates of Hodgkin lymphoma and multiple sclerosis. A vaccine to reduce EBV posttransplant lymphoproliferative disease would be an important proof of principle to prevent an EBV-associated malignancy. Trials of an EBV vaccine to reduce the incidence of Hodgkin lymphoma, multiple sclerosis, or Burkitt lymphoma would be difficult but feasible.

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Genetic design of an optimized packaging cell line for gene vectors transducing human B cells

Cited by 19 publications

References 59 publications

A Virus-Like Particle-Based Epstein-Barr Virus Vaccine

A Virus-Like Particle-Based Epstein-Barr Virus Vaccine

Developing Extrachromosomal Gene Expression Vector Technologies: An Overview

Vaccine Development for Epstein-Barr Virus

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