extent of supercoiling in plasmid dna vaccines

Am J Drug Deliv 2006; 4 (4): 195-199CURRENT OPINION 1175-9038/06/0004-0195/$39.95/0

© 2006 Adis Data Information BV. All rights reserved.

Extent of Supercoiling in Plasmid DNA VaccinesShould Restrictions be Relaxed?

Manmohan Singh, Mildred Ugozzoli, Elawati Soenawan, Amarjit Pannu, Elena Pushnova, Judith Allen andDerek T. O’Hagan

Novartis Vaccines, Emeryville, California, USA

Plasmid DNA is currently being considered for immunization as an alternative approach to protein-basedAbstractvaccines. The prevailing opinion is that the linear and open-circular forms of plasmid DNA are less effectivethan the supercoiled isoform in inducing immune responses. However, it can be argued that the plasmid is likelyto be nicked and relaxed during its transport into the nucleus, regardless of the level of supercoiling present. Thisarticle investigates the validity of the recommendations regarding supercoiling requirements for optimalefficiency of DNA vaccines.

Some studies appear to be consistent with the notion that cell transfection with plasmids is better with thesupercoiled than the relaxed open-circular isoform. Other studies have shown that there is a minimum limit ofsupercoiled plasmid that needs to be present for potent in vivo responses to DNA vaccines, with the limit beingreported to be >70% in one study. However, other data indicate that gene transfer is not significantly affected bythe level of supercoiling of the gene, and cationic lipids and microparticles can be used to deliver both isoformswithout significant loss of efficiency in vivo.

To further address this issue, we evaluated the in vitro and in vivo potency of DNA plasmids encoding theHIV proteins, gp140 env and p55 gag. The in vitro results showed no relationship between the degree ofsupercoiling and cellular transfection efficiency. Similarly, the antibody titers measured in mice immunized withfully relaxed open-circle plasmids were not significantly different from responses elicited with supercoiledplasmid. However, linearized DNA was ineffective.

Based on the findings of ourselves and others that the relaxed open-circular form of a plasmid DNA is no lessefficient in transfecting cells or in eliciting antibody responses than its supercoiled form, we propose that it ispossible to lower the requirement for a high percentage of supercoiled plasmid in a DNA vaccine formulation.

DNA vaccines have been generating excitement since 1993, cines composed of inactivated pathogens, isolated subcellularfractions, or recombinant proteins.when the first publications reported that the direct injection of

naked DNA was able to induce immune responses in mice.[1] Since Clinical trials have shown that DNA vaccines appear to be safethen, DNA vaccines have proven to be effective in inducing and well tolerated,[3] but naked DNA vaccines need to be madeprotective immunity against a wide variety of pathogens in various more potent to be effective in humans. Various approaches, suchanimal models.[2] DNA vaccines differ from traditional vaccines in as vector modification,[4,5] adjuvants,[6] and facilitation of DNAthat DNA encoding for a component of a disease-causing organ- delivery, have been used to successfully increase DNA vaccineism is injected into the body and the production of the protein takes potency.[7,8] Ultimately, the potential of DNA vaccines to induceplace in situ in the vaccinated host, eliminating any risk of infec- an effective immune response is directly dependent on the level oftion associated with live attenuated viruses. The safety profile of expression of the encoded protein in host cells. As yet, there is noplasmids in conjunction with the ability to induce both humoral definitive consensus of opinion as to whether the effectiveness ofand cellular immune responses has made plasmid DNA a highly cell transfection and expression is negatively affected by theattractive approach for vaccination. In addition, from the commer- detailed topological structure of the plasmid DNA.[9] However, thecial point of view, the manufacturing process is easier than vac- current recommendation is that the plasmid vectors should be

196 Singh et al.

mostly present in the supercoiled form, which is thought to be 2. What Happens When Supercoiling is Relaxed?more effective at inducing gene expression than open-circular,

At odds with the proposed essential role of supercoiling oflinear, multimeric, or partially denatured DNA.[3] However, argu-plasmids in DNA vaccines (as described in the previous section), itments have been made against this specification for high levels ofhas been reported that gene transfer in vitro and in vivo by cationicsupercoiled plasmid DNA, given that the plasmid is likely to belipids is not significantly affected by the level of supercoiling of anicked and relaxed during its transport into the nucleus, regardlessreported gene.[19] Furthermore, once formulated the two isoformsof the level of supercoiling present.[10]

(open circular and supercoiled) can be delivered in the sameThe aim of this article is to investigate the validity of themanner into the cells of choice for maximum gene expression.recommendations regarding supercoiling and consider whether orCationic lipids and microparticles can be used to deliver bothnot high levels of supercoiling are in fact necessary for optimalisoforms without significant loss of efficiency in vivo.[7]

efficiency of DNA vaccines.Why do these results differ from those reported in the previous

section? The discrepancies between these results are probably1. Why is Supercoiling Thought to be so Important?based on several factors, such as the type of cell line used and thetransfection agents employed, in addition to other variables, such

DNA vaccines rely on the plasmid entering the nucleus of theas salt concentration, pH, and electrostatic effect.[14,15]

target cells in the host, where the gene would be transcribed. SomeBecause there is still a question as to whether the efficiency ofstudies appear to be consistent with the notion that supercoiled

cell transfection is affected by the detailed topological structure ofplasmid is better for cell transfection.[11,12] These studies claim thatDNA, we evaluated both the in vitro efficiency and in vivoa supercoiled plasmid has greater in vitro transfection ability thanimmunogenicity of different isoforms of HIV plasmid DNA vac-relaxed or linear forms of plasmids. While that may be true forcines (unpublished data). The methods are briefly outlined in thecertain plasmids, other parameters – such as vector modification,Appendix.size of the plasmid, the protein being expressed, intracellular

We found that both fully relaxed (open-circular) and super-versus extracellular secretion, etc. – would equally affect transfec-coiled isoforms of a HIV plasmid DNA vaccine were able totion.transfect cells in vitro. Transfection of the cells with supercoiled orOther studies have shown that it is essential for a minimumopen-circular plasmid DNA (based on the p24 gag concentrationpercentage of supercoiled plasmid to be present for potent in vivosecreted into the media or present in the cell lysates) is shown inresponses to DNA vaccines, with this minimum limit being report-figure 1. For both types of DNA (supercoiled and open-circular),ed to be relatively high for maximal potency.[1] For instance,

Cupillard et al.[13] concluded that their DNA vaccine against rabiesneeded at least 70% of the plasmids to be in the supercoiledisoform because they found that the open-circular form was lessefficient than the supercoiled form. However, such results may bevery antigen specific, making it difficult to compare results be-tween different systems. Furthermore, very limited data exist thatdirectly compare the two forms of plasmid in vaccine applications.

It is usually during the extraction, purification, and preparationsteps that many plasmids undergo significant stress that results inconversion to the relaxed form of the plasmid. The generation of asmall fraction of relaxed plasmid during storage may not necessa-rily compromise the potency of the formulation. Thus, some of therequirements for a ‘supercoiled’-only DNA vaccine formulationmay not really need to be this stringent during the course of itsshelf-life.[14-18] It would be very beneficial to have the minimumacceptable level of supercoiled plasmid in DNA vaccines set in the50% range. Therefore, if during the storage period or shelf-life ofthe dosage form the percentage of supercoiled plasmid fell from avery high range of >90% to around >50%, the product could stillbe considered to be active.

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Fig. 1. Transfection of cells with supercoiled (SC) or open-circular (OC)plasmid DNA. Human 293 T cells were transfected with 1.4, 0.7, or 0.35μgper well of OC or SC DNA of pCMVKm2.GagMod.SF2 (HIV Gag) plasmidDNA previously mixed with Mirus Trans IT-LT1 polyamine transfectionreagent. The bars represent the mean ± standard error of p24 gag proteinconcentration in the media or present in the cell lysates 48 hours aftertransfection for four independent determinations.

© 2006 Adis Data Information BV. All rights reserved. Am J Drug Deliv 2006; 4 (4)

Extent of Supercoiling in Plasmid DNA Vaccines 197

the transfection activity was shown to be dose dependent and 3. What are the Advantages of Relaxingalthough the highest concentration of DNA (1.4μg) showed lower Supercoiling Requirements?levels of activity when fully relaxed DNA was used comparedwith supercoiled DNA the overall results showed no major differ- The manufacturing of plasmid DNA demands significant pro-ences. Therefore, these data suggest that the ability of HIV Gag cess development to produce clinical-grade material. There areDNA to transfect cells is not exclusive to the supercoiled form of

numerous challenges that must be faced including, but not limitedthe plasmid. Whether the plasmid is supercoiled or fully relaxed,

to, fermentation, purification, and characterization, followed bythe topological state of the plasmid has minimal effect in formingscaling up and formulation.[16] The determination of the percent-active lipid-DNA complexes.age of supercoiled form is a key indicator of plasmid conservation

However, one could argue that the reagents used for the in vitroduring the manufacturing process and for product stability. Sometransfection are facilitating the DNA transfection. Therefore, be-changes in the process may provide a higher yield of thecause our main purpose for DNA application was vaccination, wesupercoiled form and a slightly lower percentage of open-circularfurther explored the potency of the DNA using an in vivo mouseform of plasmid in the mixture. Relaxing the upper limit onmodel by directly injecting different isoforms of plasmid DNA

into the muscle in the absence of the transfection agents. In this relaxed open-circular form allowed in a preparation can havemodel, it was observed that the open-circular versions of the two dramatic effect on recovery, yield, and cost. Also, extending theDNA vaccine components (pCMVKm2.GagMod.SF2 and pSINCP shelf-life of the plasmid preparation to 2 years or beyond could begp140 dV2 SF162), which are currently in clinical trials, were

very favorable from a product distribution and storage standpoint.active in expressing the HIV p55 gag and HIV gp140 proteins, asdemonstrated by measuring the antibody responses after immuni-zation (figure 2). Based on the antibody geometric mean titers, theimmunogenicities of the supercoiled and relaxed open-circularplasmid forms were not significantly different.

Curiously, mice immunized with the linear plasmid generatedby a double-strand break in the plasmid backbone with no inter-ruption of the coding sequence (‘linear-backbone’ plasmid)showed no significant decrease in antibody response comparedwith the plasmid open-circular conformation (untreated DNA) forHIV p55 gag. In contrast, the linear form generated by digestingthe plasmid in the p55Gag gene sequence (‘linear-coding’ plas-mid) elicited significantly lower antibody titers compared with theuntreated plasmid (figure 2a). Both linear forms of the plasmid forHIV gp140 elicited a significantly lower antibody response com-pared with untreated DNA (figure 2b). The immunogenicity of thelinear form in which the double-strand was digested in the gp140coding region (‘linear-coding’ plasmid) was particularly low.

Thus, our results suggest that there is no relationship betweenthe degree of supercoiling and the efficiency of cellular transfec-tion in vitro, and that in vivo immunological responses to DNAvaccine components expressing the HIV p55 gag and HIV gp140proteins are comparable with fully relaxed open-circular plasmidisoforms and supercoiled plasmids. However, linearized DNAdoes not appear to produce sufficient immunological responses. Inparticular, we saw significantly lower antibody responses whenthe plasmid was digested within the protein-coding region. Thiscould be interpreted to suggest that the truncated protein is lessimmunogenic than the fully formed protein.

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Fig. 2. Results from in vivo (a) serum IgG p55 gag antibody titers in mice (n= 10) immunized with 10μg of different isoforms of HIV Gag DNA(pCMVKm2.GagMod.SF2). Antibody responses are shown as geometricmean titers (GMT) ± standard error (SE) at day 28; and (b) serum IgGgp140 antibody titers in mice (n = 10) immunized with 20μg of differentisoforms of HIV Env DNA (pSINCP gp140 dV2 SF162). Antibody re-sponses are shown as GMT ± SE at day 35. For both plasmids, HIV Gagand HIV Env, no significant differences were found when mice were immu-nized with supercoiled or open-circular plasmid DNA. ‘Linear-backbone’refers to the linear plasmid generated by a double-strand break in theplasmid backbone with no interruption of the coding sequence. ‘Linear-coding’ refers to the linear isoform generated by digesting the plasmid inthe target protein coding sequence.


198 Singh et al.

4. Summary and Opinion backbone’ plasmid). The SgrAI enzyme was used to cut inside theenv coding sequence (‘linear-coding’ plasmid). Similarly, 250μg

Our opinion from our studies and other data is that the immu-of HIV Gag plasmid was digested with 215 units of topoisomerase

nogenicity of the relaxed form of a plasmid DNA vaccine may beI at 37°C for 2 hours, and the linear isoforms of the plasmid were

equally as efficient as the supercoiled form in terms of elicitingobtained by cutting it inside the p55 gag sequence with 50 units of

protective humoral and cellular responses in vivo. We believe thatrestriction enzyme StuI (‘linear-coding’ plasmid), or digesting

raising the restriction concerning the amount of relaxed open-within the nonfunctional sequence with the SfuI enzyme (‘linear-

circular DNA acceptable in a vaccine product would not onlybackbone’ plasmid).

facilitate DNA manufacturing, but could also prolong the shelf-lifeThe efficiency of plasmid relaxation and linearization reactionsof the product without compromising its quality.

was confirmed by electrophoresis. Images of the agarose gels wereHowever, this observation needs to be reinforced with furthersubjected to peak integration analysis to determine the percentagestudies testing other plasmids encoding for various complex anti-of different isoforms present in each sample.gens in other animal models and detailed serological and cellular

The supercoiled and open-circular isoforms of the HIV Gagdata generated to establish the limits of each DNA vaccine.plasmid were compared in the in vitro study. The supercoiled,

Acknowledgments open-circular and both linearized isoforms of the HIV Gag andHIV Env plasmids were compared in the in vivo study.We would like to thank Nelle Cronen for her help in preparing the

manuscript. Manmohan Singh, Mildred Ugozzoli, and Derek T. O’Hagan areemployees of Novartis Vaccines; Elawati Soenawan, Amarjit Pannu, Elena

1.2 In Vitro Study: Transfection and Expression ofPushnova, and Judith Allen have previously worked for Novartis Vaccines.

gag Protein

AppendixSamples of HIV Gag plasmid untreated or digested with to-

poisomerase I restriction enzymes were used for the assay. Human1. Methodology of Studies293 T cells were seeded 1 day prior to the transfection at a densityof 5 × 105 cells per 35mm diameter well and grown at 37°C, 5%

1.1 DNA Preparation and Quantification CO2 in a final volume of 2mL Dulbecco’s Modified Eagle Medi-um (DMEM) from Aldrich (St Louis, MO, USA). The next day,The codon-optimized CMVKm2.GagMod.SF2 (HIV Gag) plas-the cells (60–70% confluent) were transfected with 0.35–1.4μg ofmid encoding the HIV-1 p55 gag protein and SINCP gp140 dV2plasmid previously mixed with Mirus TransIT-LT1 polyamineSF162 (HIV Env) plasmid encoding the HIV gp140 protein (previ-transfection reagent (Sigma, St Louis, MO, USA) in serum-freeously described by zur Megede et al.[5]) were used for these studiesmedia. Therefore, submaximal concentrations of DNA were used(Plasmid Purification Group, Chiron Corp., Emeryville, CA,to perform the comparison between the two isoforms of theUSA).plasmid (open circular and supercoiled). The cells were trans-To identify and quantify the different isoforms of both plas-fected in 2mL per well of transfection media for 5 hours; then themids, purified GMP-grade plasmid materials were subjected tomedia was replaced with complete media (Dulbecco’s medium,agarose gel electrophoresis and densitometry analysis (Biorad,10% fetal calf serum [Sigma, St Louis, MO, USA]).Hercules, CA, USA). The DNA samples were run in 0.8% agarose

gel, 1X TAE buffer at 4.4 V/cm, and the plasmid isoforms were Cells were harvested 48 hours post-transfection, the media wasseparated and identified in supercoiled, open-circular, and linear then collected and frozen until assayed. The cells were washedforms after staining with ethidium bromide. twice in phosphate-buffered saline (PBS), and then lysed on ice

To completely convert the plasmid into its open-circular con- with 40μL of buffer containing 1% NP-40 and 0.1 mol/L Tris-formation, 500μg of DNA was digested with 475 units of to- HCL, pH 7.5 (Sigma, St Louis, MO, USA). To remove cell debris,poisomerase I (Sigma, St Louis, MO, USA) for 2 hours at 37°C. cell lysates were subsequently clarified by centrifugation in anThe linear HIV Env plasmid derivatives were obtained by di- Eppendorf microcentrifuge (Eppendorf Centrifuge, Westbury,gesting the plasmid with either SgrAI or PsiI restriction enzymes NY, USA) for 10 minutes. Secreted and intracellular p24 gag(Sigma, St Louis, MO, USA). To linearize the plasmid, but con- protein concentrations were measured using the HIV p24 gagserve intact the gp140 sequence, 500μg of the plasmid was di- Antigen ELISA Test System (Coulter Corporation, Fullerton, CA,gested within the nonfunctional sequence at the backbone site with USA). The two isoforms of the plasmid HIV Gag, the completely100 units of the PsiI enzyme for 20 hours at 37°C (‘linear- relaxed (open-circular) form or the predominantly supercoiled,


Extent of Supercoiling in Plasmid DNA Vaccines 199

were used at subsaturation concentrations (0.35, 0.7, and 1.4μg) to determined using ANOVA of log-transformed data by Fisher’spaired least-squares difference test for pairwise comparisons.transfect 293 T cells for 5 hours.

1.3 In Vivo Study: Immunogenicity of Supercoiled,References

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by injection of DNA encoding a viral protein. Science 1993; 259: 1745-9

2. Donnelly J, Ulmer JB, Liu MA. DNA vaccines. Life Sci 1997; 60: 163-721.3.1 Immunization Studies

3. Food and Drug Administration, Center for Biologics Evaluation and ResearchOffice of Vaccine Research and Review. Points to consider on plasmid DNAGroups of ten female 6- to 8-week-old BalB/C mice werevaccines for preventive infectious disease indications. Issued 1996 Dec [on-immunized with 10μg of HIV Gag DNA or 20μg of HIV Env DNAline]. Available from URL: http://www.fda.gov/cber/gdlns/plasmid.pdf [Ac-

in 100μL of formulation buffer (5 mmol/L citrate, 1 mmol/L cessed 2006 Aug 2]

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either the gag or the env plasmid in the supercoiled, open-circular, 2000; 74: 2628-35linear-backbone or linear-coding isoform, plus a control group that 6. Ulmer JB, DeWitt CM, Chastain M, et al. Enhancement of DNA vaccine potency

using conventional aluminum adjuvants. Vaccine 1999; 18: 18-28received DNA formulation buffer. The DNA formulation bufferwas used as a negative control. Mice were injected with HIV Gag 7. O’Hagan D, Singh M, Ugozzoli M, et al. Induction of potent immune responses by

cationic microparticles with adsorbed HIV DNA vaccines. J Virol 2001; 75:DNA on days 0 and 14, and sera were collected on day 28 by9037-43

cardiac puncture. For HIV Env DNA, mice were dosed on days 08. Widera G, Austin M, Rabussay D, et al. Increased DNA vaccine delivery and

and 28 and blood collected on day 35. immunogenicity by electroporation in vivo. J Immunol 2000; 164: 4635-40

9. Reschel T, Konak C, Oupicky D, et al. Physical properties and in vitro transfectionefficiency of gene delivery vectors based on complexes of DNA with synthetic

1.3.2 ELISA Assay polycations. J Control Release 2002; 81: 201-17

10. Prazeres DM, Monteiro GA, Ferreira GN, et al. Purification of plasmids for geneThe presence of IgG antibody was determined for each mousetherapy and DNA vaccination. Biotechnol Annu Rev 2001; 7: 1-30by testing eight serial dilutions of serum starting at 1/10 on plates

11. Cherng JY, Schuurmans-Nieuwenbroek NME, Jiskoot W, et al. Effect of DNAthat were coated with HIV p55 gag or gp140 antigen, which weretopology on the transfection efficiency of poly((2-dimethylamino)ethyl

both obtained from Chiron’s manufacturing division (Chiron methacrylate)-plasmid complexes. J Control Release 1999; 60: 343-53

Corp., Emeryville, CA, USA). A positive control and positive 12. Adami RC, Collard WT, Gupta SA, et al. Rice Stability of peptide-condensedplasmid DNA formulations. J Pharm Sci 1998; 87: 678-83mouse serum reference were tested on each plate for as control.

13. Cupillard L, Juliard V, Latour S, et al. Impact of supercoiling on the efficacy of aThe presence of serum antibodies was detected with a second rabies DNA vaccines to protect cats. Vaccine 2005; 23: 1910-6

antibody conjugated to horseradish peroxidase in combination 14. Eastman SJ. Biophysical characterization of cationic lipid: DNA complexes.Biochim Biophys Acta 1997; 1325: 41-62with a colorimetric substrate, which adsorbs at 450nm. The dilu-

tion at which the absorbance was equal to the absorbance of the 15. Zuidam NJ, Barenholz Y. Electrostatic and structural properties of complexesinvolving plasmid DNA and cationic liposomes with a cationic cholesterolpositive mouse serum reference, to produce an absorbance of 0.50derivative. Biochim Biophys Acta 1998; 1368: 115-28

versus 40 ng/mL mouse IgG was defined as the titer. Titers were16. Middaugh CR, Evans RK, Montgomery DL, et al. Analysis of plasmid DNA from a

obtained by interpolation from a 4-parameter curve fit of ab- pharmaceutical perspective. J Pharm Sci 1998; 87: 131-46

sorbance versus dilution. The geometric mean titers were calculat- 17. Robertson RM, Laib S, Smith DE. Diffusion of isolated DNA molecules: depen-dance on length and topology. Proc Natl Acad Sci U S A 2006; 103: 7310-4ed and the mice having a titer ≥20 (2-fold higher than background)

18. Campeau P, Chapdelaine P, Tremblay JP. Transfection of large plasmids inwere reported as responders.primary human myoblasts. Gene Ther 2001; 8: 1387-94

All samples were run in duplicate on separate plates, and the19. Bergan D, Galbraith T, Sloane DL. Gene transfer in vitro and in vivo by cationic

titers were calculated as the average of the two. lipids is not significantly affected by levels of supercoiling of a reporterplasmid. Pharm Res 2000; 17: 967-73

1.3.3 StatisticsCorrespondence and offprints: Dr Manmohan Singh, Novartis Vaccines, M/

Values for each group were reported as the geometric mean and S 4.3, 4560 Horton Street, Emeryville, CA 94608, USA.standard error for ten animals. Differences among the groups were E-mail: [email protected]


extent of supercoiling in plasmid dna vaccines

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