The development of novel vaccines to eradicate herpes simplex virus (HSV) is a global public health priority. HerpV is intended as a therapeutic vaccine, designed to treat, not prevent, herpes infection. Plasmid DNA encoding each glycoprotein was purified on a cesium chloride gradient. VP22 has been reported as being able to exit the cell in which it is synthesised via an uncharacterised, golgi-independent secretory pathway and subsequently enter surrounding cells by a non-endocytic mechanism. Only one survived in the pVAX and pVAX–Hsp70 groups (Fan and Yang, 2010). Mice were followed for 20 days after infection and scored for morbidity and mortality (Haynes et al., 2006). Journal of leukocyte biology.
Efficacy: When mice were immunized with gD plasmid DNA and then challenged with 4 LD50, 60% survival of gD plasmid-vaccinated animals was noted. For PBLs, the most significant stimulation of thymidine incorporation was registered at a gD1/313 concentration of 5 microg/100 microl, while the splenocytes from DNA vaccine-immunized mice responded already at a concentration of 1 microg/100 microl. The genome of the human papillomavirus type 16 (HPV-16), the most cancer-prone HPV type, is found in at least 50% of the detected HPV-associated malignancies (2). Williams JA, Luke J, Langtry S, Anderson S, Hodgson CP, Carnes AE. Generic plasmid DNA production platform incorporating low metabolic burden seed-stock and fed-batch fermentation processes. In addition, a single vaccine dose consisting of the co-administration of pgD-E7E6E5 and the vector encoding interleukin-12 or granulocyte-macrophage colony-stimulating factor further enhanced the therapeutic anti-tumor effects and conferred protection to 60 and 50% of the vaccinated mice, respectively. In addition, some component of gram-negative bacteria such as lipopolysaccharide (LPS) has been shown to act as a potent adjuvant which can enhance immune responses .
Following fermentation, plasmid DNA is typically isolated using an alkaline lysis-based process. The challenges of scaling up alkaline lysis are well known, most notably achieving sufficient mixing without damaging the plasmid DNA and creating difficult to remove host cell DNA fragments.66. Hoare M, Levy MS, Bracewell DG, Doig SD, Kong S, Titchener-Hooker N, et al. Bioprocess engineering issues that would be faced in producing a DNA vaccine at up to 100 m3 fermentation scale for an influenza pandemic. Biotechnol Prog 2005; 21:1577 – 92; http://dx.doi.org/10.1021/bp050190n; PMID: 16321039 [CrossRef], [PubMed], [Web of Science ®] View all references-88. Hebel H, Attra H, Khan A, Draghia-Akli R. Successful parallel development and integration of a plasmid-based biologic, container/closure system and electrokinetic delivery device.
At maximum cytopathic effect, the virus was harvested by thrice freezing and thawing. Devices and Methods for Biomaterial Production, 2007, US Patent 7 238 522). Many approaches to HSV vaccines have been evaluated, including killed whole virus, subunit, live attenuated, vectored, and DNA vaccines (reviewed in [5, 6, 23]). For example, Atanasiu et al. 2A). Furthermore, use of chemokine gene-delivered adjuvants, in particular IL-8 and RANTES, could be important in crafting more efficacious vaccines as immune therapies or contributors to immune therapies for HSV. HyperGRO™ process: (A) process illustration; (B-D): Growth and plasmid yield profiles for HyperGRO™ fermentations with plasmid NTC8485-O2-gD2; the arrow marks the time of the temperature shift from 30 to 42 ºC: (B) 10L NTC process development fermentation; (C) 10L VGXI process development fermentation; (D) 320L VGXI cGMP fermentation.
where F(t) is the feed rate (L/h), μ is the desired specific growth rate during fed-batch phase (h−1), XB is the biomass concentration at the end of the batch phase (g dry cell weight/L), VB is the initial liquid volume of culture (L), Sf is the limiting substrate concentration in nutrient feed medium (g/L), YX/S is the yield coefficient of biomass from substrate (g/g) and t is the time since beginning of fed-batch phase (h). The cowpox viral DNA is extracted and cleaved 3, and the herpes gene is then combined with the cowpox DNA 4. At both the 10 L process development scale and the 320 L cGMP scale, feeding was started 15 h following bioreactor inoculation. Because we observed that there existed the inhibition due to the gDNA matrix, which affected quantification using qPCR (data not shown). If they are transfected themselves, which has been shown to happen after DNA immunization, they present the endogenously synthesized antigen via MHC class I (7, 53). Carnes AE, Luke JM, Vincent JM, Anderson S, Schukar A, Hodgson CP, et al. Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields.
J Gene Med 2010; 12:818 – 31; http://dx.doi.org/10.1002/jgm.1499; PMID: 20806425 [CrossRef], [PubMed], [Web of Science ®] View all references High plasmid yields achieved at the 10 L process development scale were maintained at the 320 L cGMP scale ( and Fig. Presumably, it is to the host’s benefit to recognize such early proteins, because it gives the host a chance to destroy infected cells before the virus can produce infectious progeny. However, before these cells can activate cytotoxic T cells, the killer T cells must be primed, and this requires the interaction of ‘professional’ APC. Alkaline lysis using the proprietary AIRMIXTM technology followed by lysate clarification and subsequent binding to an anion exchange (AEX) membrane showed minimal loss through clarification steps and no detrimental effect on the plasmid product. In-process analysis showed that the bind and elute conditions allowed the flow through of impurities contained in the clarified lysate without compromising plasmid binding affinity. The mutations in the d106 viral genome were confirmed by sequencing of another d106-derived recombinant, d106-27lacZ . Consequentially, approximately 50% of the available plasmid DNA in the clarified lysate was bound to and recovered from the AEX membrane.
More significantly, this DNA prime-protein boost formation also elicited high frequency of responders with HIV-1 antigen specific and polyfunctional T cell immune responses [16, 17]. Because of the high prevalence of existing HPV infection and years required for the development of cervical cancer, preventive HPV vaccines would need to be in widespread use for many years to reduce the number of HPV-associated cervical cancer. For other test vaccines, investigator-specific ELISA antigens will be required. The elute conditions resulted in the selective elution of supercoiled pDNA as evidenced by in process analysis and release testing of the plasmid product (). In both cases the dynamic binding capacity (2.5–2.8 mg/mL) was reached after approximately 50–60% of the conditioned load was processed, the remaining conditioned load was not passed through the column. 1090 mg (NTC8485-O2-gD2) and 1220 mg of (NTC8485-O2-UgD2tr) of pDNA were recovered in the HIC eluate. In order to maximize plasmid recovery from the UF/DF process a final nucleic acid concentration was targeted to over 150% of the final required concentration.
Nucleic acid concentrations of 4.7 mg/mL (NTC8485-O2-gD2) and 6.2 mg/mL (NTC8485-O2-UgD2tr) were achieved with over 90% plasmid recovery. Following dilution of the UF/DF product to the required concentration (2.8 mg/ml, ) and aseptic processing, samples were analyzed by Quality Control. The plasmid product met all established limits for quality and purity assays; with up to 99% supercoiled plasmid and host cell impurities near the assay detection limit (). The active bulk constituent from the purification processes, excluding filtered plasmid product for release testing, totaled 828 mg of NTC8485-O2-gD2 and 969 mg of NTC8485-O2-UgD2tr. Using the plasmid mass and gel analysis from each step in purification an approximate yield of > 2.4 g of purified pDNA/ kg WCW can be inferred. If purification processes were scaled to suit the expected biomass and specific plasmid yield, a single 320 L HyperGRO fermentation followed by the VGXI purification process should yield a minimum of 175 g of each active bulk constituent for the HSV-2 DNA vaccine, which translates to approximately 40 000 filled units of HSV-2 vaccine. Fermentation The HSV-2 DNA vaccine plasmids were transformed into E.
The vaginal washings were then centrifuged to remove particulate matter and the supernatants were stored at −20°C. Fermentations were performed in semi-defined media according to the HyperGRO™ fed-batch fermentation process as described previously.44. Carnes AE, Hodgson CP, Williams JA. Inducible Escherichia coli fermentation for increased plasmid DNA production. Biotechnol Appl Biochem 2006; 45:155 – 66; http://dx.doi.org/10.1042/BA20050223; PMID: 16819941 [CrossRef], [PubMed], [Web of Science ®] View all references,55. Williams JA, Luke J, Langtry S, Anderson S, Hodgson CP, Carnes AE. Generic plasmid DNA production platform incorporating low metabolic burden seed-stock and fed-batch fermentation processes.
Biotechnol Bioeng 2009; 103:1129 – 43; http://dx.doi.org/10.1002/bit.22347; PMID: 19408315 [CrossRef], [PubMed], [Web of Science ®] View all references,1111. Carnes AE, Luke JM, Vincent JM, Schukar A, Anderson S, Hodgson CP, et al. Plasmid DNA fermentation strain and process-specific effects on vector yield, quality, and transgene expression. Biotechnol Bioeng 2011; 108:354 – 63; http://dx.doi.org/10.1002/bit.22936; PMID: 20830679 [CrossRef], [PubMed], [Web of Science ®] View all references Inocula were prepared from RSBs or MCBs and grown at 30 °C in seed medium containing 6% (w/v) sucrose. Batch phase fermentation media contained 5 g/L sucrose for plasmid selection. During fermentation, pH was controlled at 7.0 ± 0.1 by the automatic addition of 30% ammonium hydroxide or 10% phosphoric acid. The cultures were aerated at 0.5 to 1 VVM (volume of gas/volume of medium per min) and dissolved oxygen was maintained at 30% air saturation by proportional-integral control of agitation.
Oxygen gas supplementation was initiated automatically as needed during fermentations to maintain dissolved oxygen at 30% air saturation. A semi-defined nutrient feed containing glycerol and yeast extract substrate was added according to a predetermined exponential feeding strategy to target a specific growth rate of µ = 0.12 h−1. Under the same conditions, no anti-tumor protection was observed in mice immunized with a single dose of pgD-E7E6E5 (Figure 3). Culture samples were taken at key points and at regular intervals during all fermentations. Samples were analyzed immediately for biomass (OD600) and for plasmid yield. Plasmid yield was determined by quantification of plasmid obtained from Qiagen Spin Miniprep Kit preparations as described.44. Carnes AE, Hodgson CP, Williams JA.
Inducible Escherichia coli fermentation for increased plasmid DNA production. Biotechnol Appl Biochem 2006; 45:155 – 66; http://dx.doi.org/10.1042/BA20050223; PMID: 16819941 [CrossRef], [PubMed], [Web of Science ®] View all references 10L scale fermentations were performed at VGXI and NTC with a starting culture volume of 8L, in either New Brunswick Scientific BioFlo 3000 or BioFlo 110 bioreactors with a maximum working volume of 10.6 L. 320 L scale GMP fermentations were performed at VGXI with a starting culture volume of 220 L in a New Brunswick Scientific BioFlo Pro bioreactor with a 400 L maximum working volume. Downstream purification process Downstream processing of the HSV-2 DNA vaccine plasmids was performed by VGXI and consisted of cell lysis, solid/liquid separation, anion exchange membrane chromatography, hydrophobic interaction chromatography (HIC), ultrafiltration/diafiltration (UF/DF), and aseptic filtration. The cell lysis step was conducted using a proprietary lysis skid and AIRMIXTM technology (Hebel, et al. Devices and Methods for Biomaterial Production, 2007, US Patent 7 238 522) as previously described which was developed on the principle of alkaline lysis.88. Hebel H, Attra H, Khan A, Draghia-Akli R.
It has been reported that local immunization provides more local protection than systemic immunization. Vaccine 2006; 24:4607 – 14; http://dx.doi.org/10.1016/j.vaccine.2005.08.049; PMID: 16150516 [CrossRef], [PubMed], [Web of Science ®] View all references,1212. DNA encoding cytosolic gD2 provided no protection from disease symptoms (excluding viability; mean total lesion score, 15.7 ± 2.3). Consequently, both cellular (50, 51) and hybrid (52) promoters are currently being tested as possible alternatives to viral promoters. Vaccine 2010; 28:2046 – 52; http://dx.doi.org/10.1016/j.vaccine.2009.10.057; PMID: 19896448 [CrossRef], [PubMed], [Web of Science ®] View all references The neutralized lysate was clarified with a decreasing pore size nylon monofilament mesh bag filter (Filter Specialist Inc.) followed by a 0.2 µm depth filter (Millipore) before being subjected to chromatography purifications. The anion exchange membrane chromatography employing Mustang Q capsules (Pall) captured the majority of plasmid DNA from crude lysate while allowing the majority of impurities to pass through. Plasmid product was eluted with a solution containing a higher ion concentration than the membrane equilibration.
The eluate was conditioned with a high salt ammonium sulfate solution and loaded to an equilibrated XK 50/20 column (GE Healthcare) packed with Butyl 650M hydrophobic resin (Tosoh Biosep). Single step elution with a decreased concentration of ammonium sulfate was able to significantly reduce the open circular (OC) form of plasmid and selectively elute the SC plasmid, while separating residual contaminants. The recovered HIC product was further processed with an UF/DF step employing a disposable filter cassette (Novasep) of modified polyethersulfone membrane with a 50 kDa MWCO to displace the HIC elution buffer with 10 mM Tris (HCl) 1 mM EDTA pH 8 and increase the plasmid concentration to the target range. The UF product was filtered with 0.2 µm MiniKleenpak filters (Pall) and aliquoted for storage at -75 °C to -85 °C. Proliferation assays.Bovine blood was collected into citrate-dextran, and peripheral blood mononuclear cells (PBMC) were isolated on Ficoll-Paque PLUS (Pharmacia, Mississauga, Ontario, Canada). The concentration of total nucleic acid in the product sample was measured with a SpectraMAX Plus 384 UV/Vis spectrophotometer (Molecular Devices) at the wavelength of 260nm, and the purity of plasmid DNA was evaluated with A260/A280. The osmolality of final plasmid was determined with the Advanced Micro-Osmometer Model 3300 (Advanced Instruments, Inc.).
The host cell protein content was determined with a Micro BCA Protein Assay Reagent Kit (Pierce) using bovine serum albumin as the standard, and measuring the A562 with SpectraMAX PLUS 384 UV/Vis spectrophotometer (Molecular Devices). The phenomenon of apparent protein transfer, first observed in studies of minor histocompatibility responses,82 is termed “crosspriming”. When HIV infection first occurs the immune system suppresses the virus very well, but with time this ability is lost. The residual host cell DNA percentage was determined by Q-PCR using the Applied BioSystems Step One Plus Real Time PCR System). Plasmid isoform evaluation of final product was conducted by HPLC (Agilent 1100 Series), using a linear gradient with a TSKgel Butyl-NPR Column (Tosoh BioSep) to separate plasmid isoforms at A260. HSV recombinants delivered for clinical purposes need to be susceptible to standard anti-HSV drugs so that the drugs, in the unlikely event that any revertant or recombinant that might arise, could control this virus. VGXI’s downstream purification process with plasmid bearing cells produced using the NTC HyperGRO™ process yielded plasmid product with supercoiled pDNA percentages of up to 99% and a very low contaminant profile (), providing high quality plasmid DNA for Coridon’s HSV-2 DNA vaccine Phase I clinical trial.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Electroporation represents another way of increasing the number of HPV DNA–transfected cells and enhancing the magnitude of gene expression, while reducing inter subject variability and requiring less time to reach a maximal immune response compared to conventional intramuscular injection of the vaccine (for review, see ). Make the workstation as comfortable as possible, optimizing chair height, bench height, microscope eye piece angle, and distance from edge of bench to work area. (A) NTC8485-O2-gD2 vector map; (B) Left: In a plasmid-free cell, levansucrase is expressed from a chromosomally integrated SacB gene, leading to cell death in the presence of sucrose. Right: RNA-OUT from the plasmid represses translation of the SacB gene, achieving plasmid selection. Figure 2. HyperGRO™ process: (A) process illustration; (B-D): Growth and plasmid yield profiles for HyperGRO™ fermentations with plasmid NTC8485-O2-gD2; the arrow marks the time of the temperature shift from 30 to 42 ºC: (B) 10L NTC process development fermentation; (C) 10L VGXI process development fermentation; (D) 320L VGXI cGMP fermentation.