261. Technology Transfer of a High Yield Fermentation Process and cGMP Production of Antibiotic-Free Plasmid DNA Vaccines

The purpose of this work was to assess the ability of plasmid DNA encoding hepatitis B virus (HBV) HBsAg encapsulated in poly(dl-lactide-co-glycolic acid) (PLGA) microparticles to induce local and systemic HBsAg-specific immunity following a single dose of oral immunization. RT-PCR analysis demonstrated prolonged transcription of plasmid DNA, consistent with the sustained expression and presentation of target antigen observed by confocal laser scanning microscopy, in gut-associated lymphocyte tissue (GALT) from mice immunized orally with plasmid DNA encapsulated into PLGA microparticles. Oral administration of PLGA-DNA microparticles induced a long-lasting and stable antigen-specific antibody response, both serum total antibody and intestinal IgA, in BALB/c mice. Mice immunized orally exhibited antigen-specific gamma interferon production and cytotoxic T lymphocyte responses in spleen and GALT after restimulation in vitro with HBsAg or tumour cells stably expressing HBsAg. In contrast, naked DNA vaccines given by intramuscular injection induced only systemic cellular and humoral responses to HBsAg, which were much lower than the responses elicited by oral DNA encapsulated in PLGA microparticles at equivalent doses. The results are encouraging with regard to obtaining good compliance and vaccination coverage with candidate plasmid DNA vaccines, especially in developing countries.

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Improved downstream process for the production of plasmid DNA for gene therapy.

Acta Biochimica Polonica ◽

10.18388/abp.2005_3434 ◽

2005 ◽

Vol 52 (3) ◽

pp. 703-711 ◽

Cited By ~ 47

Author(s):

Jochen Urthaler ◽

Wolfgang Buchinger ◽

Roman Necina

Keyword(s):

Gene Therapy ◽

Plasmid Dna ◽

Viral Vectors ◽

Continuous System ◽

Size Exclusion ◽

High Yield ◽

Downstream Process ◽

Naked Dna ◽

Supercoiled Form ◽

Exclusion Chromatography

Gene therapy and genetic vaccines promise to revolutionize the treatment of inherited and acquired diseases. Since viral vectors are generally associated with numerous disadvantages when applied to humans, the administration of naked DNA, or DNA packed into lipo- or polyplexes emerge as viable alternatives. To satisfy the increasing demand for pharmaceutical grade plasmids we developed a novel economic downstream process which overcomes the bottlenecks of common lab-scale techniques and meets all regulatory requirements. After cell lysis by an in-house developed gentle, automated continuous system the sequence of hydrophobic interaction, anion exchange and size exclusion chromatography guarantees the separation of impurities as well as undesired plasmid isoforms. After the consecutive chromatography steps, adjustment of concentration and final filtration are carried out. The final process was proven to be generally applicable and can be used from early clinical phases to market-supply. It is scaleable and free of animal-derived substances, detergents (except lysis) and organic solvents. The process delivers high-purity plasmid DNA of homogeneities up to 98% supercoiled form at a high yield in any desired final buffer.

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532. An Inducible Fed-Batch Fermentation Process for Scale-Up and Production of DNA Vaccines and Gene Medicines

Molecular Therapy ◽

10.1016/j.ymthe.2006.08.603 ◽

2006 ◽

Vol 13 ◽

pp. S204

Author(s):

Aaron E. Carnes ◽

James A. Williams ◽

Clague P. Hodgson

Keyword(s):

Batch Fermentation ◽

Fermentation Process ◽

Dna Vaccines ◽

Scale Up ◽

Fed Batch ◽

Fed Batch Fermentation

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A Comparison of Plasmid DNA and mRNA as Vaccine Technologies

Vaccines ◽

10.3390/vaccines7020037 ◽

2019 ◽

Vol 7 (2) ◽

pp. 37 ◽

Cited By ~ 46

Author(s):

Liu

Keyword(s):

Clinical Trials ◽

Dna Vaccine ◽

Plasmid Dna ◽

Dna Vaccines ◽

Delivery Systems ◽

Potential Toxicity ◽

Vaccine Candidates ◽

The Status ◽

Experimental Findings ◽

Theoretical Issues

This review provides a comparison of the theoretical issues and experimental findings for plasmid DNA and mRNA vaccine technologies. While both have been under development since the 1990s, in recent years, significant excitement has turned to mRNA despite the licensure of several veterinary DNA vaccines. Both have required efforts to increase their potency either via manipulating the plasmid DNA and the mRNA directly or through the addition of adjuvants or immunomodulators as well as delivery systems and formulations. The greater inherent inflammatory nature of the mRNA vaccines is discussed for both its potential immunological utility for vaccines and for the potential toxicity. The status of the clinical trials of mRNA vaccines is described along with a comparison to DNA vaccines, specifically the immunogenicity of both licensed veterinary DNA vaccines and select DNA vaccine candidates in human clinical trials.

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Plasmid DNA encoding influenza virus haemagglutinin induces Th1 cells and protection against respiratory infection despite its limited ability to generate antibody responses

Microbiology ◽

10.1099/0022-1317-81-7-1737 ◽

2000 ◽

Vol 81 (7) ◽

pp. 1737-1745 ◽

Cited By ~ 45

Author(s):

Patricia A. Johnson ◽

Margaret A. Conway ◽

Janet Daly ◽

Carolyn Nicolson ◽

James Robertson ◽

...

Keyword(s):

Influenza Virus ◽

T Cell ◽

Immune Responses ◽

Plasmid Dna ◽

Dna Vaccines ◽

Dose Range ◽

Th1 Cells ◽

Dna Encoding ◽

Live Virus ◽

Limited Ability

Direct intramuscular injection of plasmid DNA can generate immune responses against encoded antigens. However, the relative ability of DNA vaccines to induce cellular and humoral immunity after a single or booster immunization and the persistence of this response have not been fully elucidated. In this study, induction and maintenance of antibody and T cell subtypes with different doses of naked DNA encoding the haemagglutinin (HA) gene of influenza virus were examined and compared to the immune responses and protection induced by respiratory tract infection and immunization with a killed virus vaccine. Like natural infection, immunization with HA DNA induced potent Th1 responses. Spleen cells from mice immunized once with HA DNA in the dose range 10 ng to 100 μg secreted significant levels of IFN-γ, but low or undetectable IL-5, in response to influenza virus in vitro. Furthermore, CD4+ HA-specific Th1 clones were generated from spleens of immunized mice. Although T cell responses waned 12 weeks after a single immunization, antigen-specific Th1 cells persisted in the spleen for at least 6 months after two booster immunizations. In contrast, influenza virus-specific ELISA IgG titres were low after a single immunization and required two booster immunizations to reach significant levels. Furthermore, haemagglutination inhibition (HI) antibodies were weak or undetectable after two immunizations. Nevertheless, two doses of HA DNA conferred almost complete protection against respiratory challenge with live virus. Thus, despite the limited ability to induce antibodies, DNA vaccines confer protective immunity against influenza virus infection, which appears to be mediated by Th1 cells.

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Application of Plasmid Engineering to Enhance Yield and Quality of Plasmid for Vaccine and Gene Therapy

Bioengineering ◽

10.3390/bioengineering6020054 ◽

2019 ◽

Vol 6 (2) ◽

pp. 54

Author(s):

Folarin ◽

Nesbeth ◽

Ward ◽

Keshavarz-Moore

Keyword(s):

Plasmid Dna ◽

Downstream Processing ◽

Point Of View ◽

High Yield ◽

Bacteriophage Mu ◽

E Coli ◽

Yield And Quality ◽

Superhelical Density ◽

High Level ◽

Binding Sequence

There is an increased interest in plasmid DNA as therapeutics. This is evident in the number of ongoing clinical trials involving the use of plasmid DNA. In order to be an effective therapeutic, high yield and high level of supercoiling are required. From the bioprocessing point of view, the supercoiling level potentially has an impact on the ease of downstream processing. We approached meeting these requirements through plasmid engineering. A 7.2 kb plasmid was developed by the insertion of a bacteriophage Mu strong gyrase-binding sequence (Mu-SGS) to a 6.8 kb pSVβ-Gal and it was used to transform four different E. coli strains, and cultured in order to investigate the Mu-SGS effect and dependence on strain. There was an increase of over 20% in the total plasmid yield with pSVβ-Gal398 in two of the strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The extent of supercoiling was examined using superhelical density (σ) quantification with pSVβ-Gal398 maintaining a superhelical density of −0.022, and pSVβ-Gal −0.019, in both strains. This study has shown that plasmid modification with the Mu-phage SGS sequence has a beneficial effect on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing.

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Engineered Nanodelivery Systems to Improve DNA Vaccine Technologies

Pharmaceutics ◽

10.3390/pharmaceutics12010030 ◽

2020 ◽

Vol 12 (1) ◽

pp. 30 ◽

Cited By ~ 12

Author(s):

Michael Lim ◽

Abu Zayed Md Badruddoza ◽

Jannatul Firdous ◽

Mohammad Azad ◽

Adnan Mannan ◽

...

Keyword(s):

Immune Responses ◽

Clinical Application ◽

Dna Vaccine ◽

Immune Cells ◽

Plasmid Dna ◽

Dna Vaccines ◽

Immunological Response ◽

Main Challenge ◽

Human Immunodeficiency Viruses ◽

Stimulation Of

DNA vaccines offer a flexible and versatile platform to treat innumerable diseases due to the ease of manipulating vaccine targets simply by altering the gene sequences encoded in the plasmid DNA delivered. The DNA vaccines elicit potent humoral and cell-mediated responses and provide a promising method for treating rapidly mutating and evasive diseases such as cancer and human immunodeficiency viruses. Although this vaccine technology has been available for decades, there is no DNA vaccine that has been used in bed-side application to date. The main challenge that hinders the progress of DNA vaccines and limits their clinical application is the delivery hurdles to targeted immune cells, which obstructs the stimulation of robust antigen-specific immune responses in humans. In this updated review, we discuss various nanodelivery systems that improve DNA vaccine technologies to enhance the immunological response against target diseases. We also provide possible perspectives on how we can bring this exciting vaccine technology to bedside applications.

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