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The immune response to a polyoma virus replicon-based DNA vaccine
Vaccine 19 (2001) 68±74
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The immune response to a polyoma virus replicon-based DNA vaccine A. Mena a, B.E.H. Coupar b, M.E. Andrew b,* a
The Flinders University of South Australia, Australia CSIRO Animal Health, Geelong, Victoria, Australia
b
Received 19 November 1999; received in revised form 2 December 1999; accepted 4 April 2000
Abstract DNA vaccination has proven to be eective against a number of tumours and microbial diseases. As DNA vaccines are unable to replicate, plasmid copy number per cell is dependent on in vivo transfection eciency, which is usually quite low. Consequently, immune responses generated are likely to be sub-optimal due to low antigen expression levels in transfected cells. During this study, replicating DNA vaccines delivered intra-epidermally by gene gun, were assessed for their ability to more eciently generate immune responses in mice. The data demonstrate that, using a polyoma virus-based system of replication, 10fold less DNA expressing the haemagglutinin gene of in¯uenza virus, was required to stimulate a humoral immune response, compared to an equivalent non-replicating vaccine. This observation suggests that the use of replicating DNA vaccines in some delivery systems may enhance the eectiveness of immune responses. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: DNA vaccine; Polyoma replicon; Antibody
1. Introduction Naked DNA plasmids can potentially provide safe and eective vaccines for human and veterinary use and are likely to be particularly useful for those organisms where there is no safe, attenuated strain, and/or where killed organisms or denatured proteins are ineffective. However, the immune response to a naked DNA vaccine is often lower than that to a whole organism and considerable eort is being put into increasing their immunogenicity. Plasmid DNA is most commonly delivered by intramuscular inoculation or via the gene gun, which propels DNA-coated gold particles into the skin. Cellular uptake of DNA is relatively inecient, particularly following intramuscular inoculation, and therefore expression of the encoded antigen is low [1,2]. Strong * Corresponding author. Tel: +61-3-5227-5745; fax: 61-3-52275555. E-mail address: [email protected] (M.E. Andrew).
promoters have been shown to increase expression levels [3±5] and the immediate/early promoter from cytomegalovirus (CMV) is currently the most eective and frequently used. For gene gun inoculation, Eisenbraun et al. [6] showed that antigen expression and antibody response are directly correlated to the plasmid copy number per gold particle. Several agents have been used in attempts to facilitate the entry of DNA into cells [3,5,7±9]. Muscledamaging agents, such as bupivicaine, have increased gene expression slightly, but the need to inoculate the bupivicaine several days before DNA inoculation reduces its suitability for widespread use in either human or veterinary vaccines. Increasing gene expression through plasmid replication would be a more practical strategy for increasing antigen expression. Bacterial plasmids do not replicate in eukaryotic cells. Plasmids with the ability to replicate in eukaryotic cells have been constructed [10±13] and have been shown to increase gene expression in vitro [14,15]. The polyoma virus genome replicates as an unintegrated mini chromosome in the nucleus of permissive
0264-410X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 0 ) 0 0 1 4 7 - X
A. Mena et al. / Vaccine 19 (2001) 68±74
cells [16]. The three components essential for replication are host derived replication factors, virally-derived large T antigen and a viral origin of replication [17]. Soon after infection, polyoma virus expresses three related proteins translated from dierent mRNAs generated by alternative splicing of a primary transcript [13]. Large T antigen is involved in viral DNA replication [16]; middle T antigen is associated with viral transforming activity [18,19]; and the small T antigen is suspected of playing a subsidiary role in DNA replication or transformation [20]. Replication begins with the large T antigen binding to the viral origin of replication and the local unwinding of the origin. Bi-directional replication, facilitated by host cell replication factors then follows. Deletion of the splice site used in the formation of the middle and small T antigens creates a gene that can only express large T antigen. Gassmann et al. [13] have constructed a plasmid capable of replicating in eukaryotic cells by including the mutated T antigen gene and the viral origin of replication, thus preventing the plasmid from transforming cells, but maintaining its ability to replicate in eukaryotic cells. In this report, the immunogenicity of a replicating DNA vaccine, constructed using the polyoma origin of replication and mutated T antigen, has been assessed. To avoid dierences in immunostimulatory DNA sequences [21,22], and transfection eciencies, polyoma virus based replicating and non-replicating plasmid with near identical sequences, expressing the luciferase reporter gene or the haemagglutinin (HA) gene of in¯uenza virus, were used to demonstrate increased antigen expression in vitro and improved antibody responses in mice. 2. Materials and methods 2.1. Plasmid construction The polyoma virus based replicating plasmid, pMGD20neo [13], was digested with AvaI, 730 bp into the polyoma virus large T antigen. Restriction ends were ®lled in with DNA polymerase I Klenow fragment and DNA religated to form pMGDko. The expected 4 bp insertion was con®rmed by sequencing using forward (5 '-TCCTCCTCCTCCTCCTCCAG-3 ' (5688±5707)) and reverse (5 '-GCTACCAGTCGCCGCCTAAG-3 ' (5929±5910)) primers. Primer positions are relative to pMGD20neo. A BamHI/BglII fragment encoding in¯uenza A/PR/8/34 HA gene under the control of the human cytomegalovirus immediate-early enhancer promoter (CMV IE) and SV40 polyadenylation signal (SV40pA), was cloned into the BamHI site of pMGD20neo and pMGDko to form pMGDHA and pMGDkoHA respectively. A blunt end fragment
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encoding the ®re¯y luciferase gene (Luc) under the control of CMV IE promoter and SV40pA was cloned into a Klenow repaired SalI site in pMGD20neo and pMGDko to form pMGDLuc and pMGDkoLuc respectively. A KpnI/NotI fragment encoding the Luc gene was cloned between the KpnI and NotI site of pCI (Promega) to form pCILuc. A neomycin resistance cassette from pMGD20neo was cloned as an EcoRIrepaired/BamHI fragment into pCI between ClaIrepaired and BamHI sites to form pCIneo. Plasmids were propagated in E.coli strain DH5a and DNA was isolated and puri®ed using Qiagen Plasmid Giga Kits. 2.2. Cell culture and transfections Mouse L929 ®broblasts were cultured in Earle's minimal essential medium supplemented with 5% foetal bovine serum at 378C in a 5% CO2 atmosphere. All transfections were conducted using LipofectAMINE PLUS reagent (GibcoBRL) according to the manufacturer's instructions. 2.3. Luciferase assay L929 cells were transfected with plasmid and cells harvested for assay using Promega luciferase cell culture lysis reagent with bovine serum albumin. Luciferase activity was determined with a luminometer (Berthold Lumate LB9501) using Promega's luciferase assay system. 2.4. Back transformation assay Plasmid DNA was extracted using a modi®ed version of the protocol described by Hirt [23], which separates low molecular weight plasmids from high molecular weight chromosomal DNA. Two days posttransfection, L929 cells were rinsed twice with cold phosphate-buered saline (PBS), then overlaid with 1% Nonidet P-40 in PBS and incubated at room temperature for 1 min [24]. Following aspiration of the PBS, the cells were treated according to the standard Hirt protocol, which involved disruption with SDS and sodium chloride, phenol/choroform extraction and ethanol precipitation in the presence of glycogen. Extracted DNA was digested overnight with DpnI to digest DH5a derived plasmid [25] and then transformed into competent DH5a cells by electroporation. Cells were plated onto LB agar plates containing ampicillin, incubated at 378C overnight and the next day, colony numbers recorded. 2.5. Vaccination of mice and serum collection Female CBA/H mice (Animal Resource Centre, Perth), 6±8 weeks old, were used and maintained in ac-
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A. Mena et al. / Vaccine 19 (2001) 68±74
cordance with institutional animal ethics committee approved protocols. Mice were sedated using alcohol, chloroform and ether vapour (1:2:3) and DNA was delivered to shaved abdominal skin using the Helios Gene Gun (BioRad). DNA plasmid preparations were quantitated by scanning gel bands. Both plasmids were then diluted to 1 mg/ml and the concentration rechecked on gels. A helium discharge pressure of 250 psi was used to deliver 0.4 mg of 1 mm gold beads (BioRad) coated with the speci®ed amounts of DNA. Cartridges containing coated gold beads were visually inspected to ensure even distribution of DNA-coated gold. Previous elution of DNA from multiple cartridges had shown that DNA batches were consistently loaded on the gold beads and that DNA was evenly distributed between the cartridges.
oma origin of replication and T antigen could replicate in eukaryotic cells, DNA was recovered from transfected cells and digested with DpnI, as detailed in Section 2. The methylation of bacterial DNA renders it senstive to digestion with DpnI, whereas unmethylated plasmid DNA that has been produced in the transfected cells would be insensitive to digestion by DpnI. Assays were performed in triplicate and mean colony numbers calculated for each transfection plasmid. Transfection with pMGDLuc or pMGDHA resulted in more than a thousand colonies, while transfection with pMGDkoLuc or pMGDkoHA, which encode the mutated large T antigen, or with pCILuc, resulted in very low numbers of colonies (Fig. 1).
2.6. Haemagglutination inhibition assay
To determine whether plasmid replication resulted in increased antigen production, dose response and time course analyses were conducted. Cells were transfected with 1000, 100, 10 or 1 ng of plasmid in triplicate and harvested 2 days post-transfection. Three fold higher expression was observed in cells transfected with 1000 ng of pMGDkoLuc, compared to the same dose of pMGDLuc (Fig. 2). At 100 ng DNA, luciferase activity was slightly higher in cells transfected with pMGDLuc, but at 10 and 1 ng DNA, luciferase activity was approximately 3-fold higher in cells transfected with pMGDLuc compared to those transfected with pMGDkoLuc. These results were con®rmed in other experiments (data not shown) and demonstrate that, at low doses, gene expression from replicating plasmids is higher than that generated from non-replicating plasmids. To determine the time course of luciferase expression, cells were transfected with 10 ng of pMGDLuc or PMGDkoLuc in triplicate. Flasks were harvested 6, 24 and 48 h post-transfection. Luciferase activity was barely detectable 6 h post-transfection (Fig. 3). At 24 h, cells transfected with pMGDLuc expressed 3.9-fold higher amounts of luciferase than
Serum (20 ml) was mixed with 80 ml of neuraminidase (100 U/ml in PBS) (BioWhittaker) and incubated at 378C for 16 h. Sixty microlitres of 2.5% sodium citrate were then added before incubation at 568C for 30 min, followed by the addition of 40 ml of PBS. Two-fold dilutions of neuraminidase-treated serum, starting at 1:10, were made in PBS in 96 well U-bottomed microtitre plates. In¯uenza virus (A/PR/8/34) at a concentration of 4 haemagglutination units/25 ml was added to all wells and the plates incubated at room temperature for 30 min. Packed, washed chicken red blood cells, from speci®c pathogen free birds, were diluted to 0.5% in PBS and 50 ml added to each well. After incubation at room temperature for 1 h, the plates were scored for haemagglutination inhibition. 3. Results 3.1. Back transformation analysis of plasmids To con®rm that the plasmids containing the poly-
3.2. In vitro luciferase analysis
Fig. 1. Back transformation analysis. Assays were performed in triplicate and mean colony numbers 2standard error are shown.
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Fig. 2. Dose response analysis of luciferase gene expression in vitro. Assays were performed in triplicate and results show mean relative light units (RLU) 2standard error.
did cells transfected with pMGDkoLuc, and the dierence increased to 5.3-fold at 48 h. For pMGDLuc, there was approximately 5-fold more activity at 48 h, compared to 24 h. 3.3. Antibody responses The plasmid pMGDHA, expressing the HA of in¯uenza virus was used to assess whether the ability of the plasmid to replicate would result in higher antibody responses. As the ability of the plasmid to replicate was dependent upon host cell replication factors, it was important to ensure that the DNA-coated gold particles reached the skin layer containing replicating cells. Gold particles were propelled into shaved mouse skin using the gene gun at helium pressures of 100, 200, 250 and 300 psi. Biopsies were taken immediately and examined histologically. Using 100 psi, the gold particles were deposited in the super®cial skin layers (data not shown). With increasing pressure, the particles penetrated deeper into the skin. A helium pressure of 250 psi propelled the gold into the stratum germinativum layer of skin where the replicating cells reside
(Fig. 4) and subsequent experiments used 250 psi helium. pMGDHA and pMGDkoHA were delivered intraepidermally to groups of 8 or 9 mice, using the gene gun and doses of 1000, 100, 10 or 5 ng. The control plasmid, pCIneo, which does not express HA, was used at 1000 ng in four mice. The mice were vaccinated at 0 and 4 weeks. When 1000 ng pMGDHA or pMGDkoHA were delivered (Fig. 5a), the replicating plasmid, pMGDHA, stimulated slightly higher primary antibody at weeks 3 and 4 ( p = 0.056 and p = 0.043, respectively, using 2tailed t-test), but the secondary responses were the same. Similarly, using 100 ng DNA, the pMGDHA stimulated higher antibody at weeks 3 and 4, ( p = 0.032 and p = 0.040) but not 5 and 7 weeks (Fig. 5b). At the 10 ng dose (Fig. 5c), pMGDHA stimulated higher antibody at all timepoints ( p = 0.050 at 3 weeks and <0.001 for the other timepoints), and at 7 weeks post-primary vaccination, the mean antibody titre in the group receiving pMGDHA was 10-fold higher than that in the group receiving pMGDkoHA. Sera collected at all timepoints from mice vaccinated
Fig. 3. Time course analysis of luciferase gene expression in vitro. Cells were transfected with 10 ng of DNA and harvested at the indicated times. Luciferase assays were performed in triplicate and results are expressed as mean RLU 2standard error.
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sequences present on bacterially derived DNA can have a profound eect on the immunogenicity of DNA vaccines [21,22]. Consequently the use of pMGDkoLuc and pMGDkoHA with near identical
Fig. 4. Penetration of gold particles into skin. Gold particles were ®red into skin using 250 psi and biopsies taken immediately and processed for light microscopy (400 magni®cation). The gold particles are seen as many small dots.
with pCIneo showed no signs of haemagglutination inhibition (results not shown). Comparison of mean antibody titres from groups receiving 10 ng pMGDHA or 100 ng pMGDkoHA demonstrated that, with the exception of the 5 week timepoint, mean antibody titres generated from both groups were statistically indistinguishable. Few mice vaccinated with 5 ng of plasmid seroconverted before the secondary inoculation. Three weeks after the second inoculation, 6 of 8 mice receiving pMGDHA had seroconverted as opposed to 1 of 9 mice in the group receiving pMGDkoHA (Fig. 5d). The results obtained in this experiment were reproduced in two further experiments, except that at the higher doses of 1000 and 100 ng, the replicating plasmid did not always stimulate a higher response at early timepoints. However, in all experiments, low doses of pMGDHA stimulated higher antibody responses. To measure in vivo plasmid activity, gold particles with 10, 100 or 1000 ng pMGDLuc or pMGDkoLuc were ®red into the abdomenal skin of groups of eight mice and skin and draining lymph nodes removed 24 or 48 h later. Consistent dierences in luciferase activity were not observed in either of two experiments performed (data not shown). The plasmids were also inoculated intramuscularly and muscle and draining lymph nodes removed 48 h later. Again, no consistent dierences were observed.
4. Discussion The results shown here demonstrate that plasmids containing the polyoma origin of replication and large T antigen can replicate in eukaryotic cells and therefore require less input DNA to stimulate an antibody response. The presence of immunostimulatory DNA
Fig. 5. HAI antibody responses in mice. Nine mice/group were vaccinated by gene gun at times 0 and 4 weeks. Mean HAI titres 2standard error are shown are shown for 1000 (a), 100 (b) and 10 ng (c) doses. Individual values at 7 weeks after immunization are shown for 5 ng DNA (d).
A. Mena et al. / Vaccine 19 (2001) 68±74
sequences to pMGDLuc and pMGDHA, respectively, was important. The dierences observed in vitro and in vivo can be directly attributed to either the plasmids ability or inability to replicate, but not to immunostimulatory sequences, or to transfection eciencies due to dierences in plasmid sequence or structure. Camenisch et al. [14] have demonstrated 6- to 10fold greater gene expression in cells transfected with replicating plasmids. The lower gene expression observed in L929 cells transfected with 1000 ng of pMGDLuc may be due to redirection of limiting host cell replication, transcriptional and translational factors within the cell from their original role in the host cell, thus killing or damaging some cells. However, at lower doses, there were clearly higher levels of gene expression from the replicating plasmids. Time-course analysis of luciferase activity in cells transfected with 10 ng of pMGDLuc or pMGDkoLuc showed negligible luciferase activity at 6 h, but high levels at 24 h and a further ®ve-fold increase at 48 h. The dierence between the two plasmids was higher at 48 h than at 24 h, suggesting that plasmid replication occurs within the ®rst 24 h post-transfection and continues after this period. Other studies have shown that DNA can move rapidly from the site of inoculation [26] (A. Mena, unpublished, 1997), which may account for the failure to ®nd higher luciferase activity in muscle or draining lymph nodes. pMGDLuc is likely to replicate only in replicating host cells that have sucient induction of replicative enzymes and factors, further reducing likelihood of demonstrating in vivo replication. Only in mice receiving 10 or 5 ng of DNA was pMGDHA consistently more ecient at generating antibody titres than pMGDkoHA, which may be due to the average number of plasmids present per cell nuclei after vaccination. Eisenbraun et al. [6] demonstrated that a single gold particle coated with approximately 300 copies of plasmid, (coding for an antigen under the control of the CMV promoter), was sucient to give maximum gene expression and antibody titre. Increasing plasmid copy number above this level did not greatly increase gene expression or antibody titre. However, decreasing plasmid copy number per particle did reduce gene expression and antibody titres. Eisenbraun et al. [6] and Pertmer et al. [27] showed a plateau eect which may be due to limited transcriptional and translational factors within the cell. Gold particles coated to give doses of 1000 or 100 ng DNA, delivered approximately 2770 or 277 plasmid copies/ particle respectively. The failure to increase antibody responses with 100 ng or more of replicating plasmid, therefore is consistent with the ®ndings of Eisenbraun et al. [6]. At 10 ng, however, the mean antibody titre stimulated by pMGDkoHA was approximately 10-fold lower, while that stimulated by pMGDHA was equiv-
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alent to levels stimulated by 1000 or 100 ng doses. This suggests that plasmid copy number/cell in groups receiving pMGDHA at 10 ng remained above the threshold required for maximal gene expression while that in groups receiving pMGDkoHA at the same dose did not. The inability of DNA vaccines to replicate in vivo has been cited as a safety factor [1,28]. Data obtained during this study suggested that replicating plasmids did not persist in the skin of mice for longer periods than the non-replicating plasmids (results not shown), probably due to the process of keratinization and shedding of skin cells. However, it is possible that replicating plasmids could move to other cells, or other sites, and persist there. Although there may be fewer concerns about persistence of veterinary vaccines, than of human vaccines, further research is required to address safety issues of replicating DNA vaccines, such as transfer of genes to humans via processing or consumption of meat, transfer of antibiotic resistance genes or increased cancer rates. These data have demonstrated that a DNA vaccine that is able to replicate reduces the dose of DNA required to stimulate an antibody response and may pave the way towards increasing the eectiveness of DNA vaccines.
Acknowledgements We gratefully acknowledge Dr Greg Donoho from Lexicon Genetics Inc., 4000 Research Forest Drive, The Woodlands, TX 77381, for his gift of plasmid pMGD20neo. We would also like to thank Dr Cor Lenghaus for his technical support and advice.
References [1] Wol JA, Malone RW, William P, et al. Direct gene transfer into mouse muscle in vivo. Science 1990;247:1465±8. [2] Acsadi G, Dickson G, Love DR, et al. Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs. Nature 1991;352:815±8. [3] Davis HL, Whalen RG, Demeneix BA. Direct gene transfer into skeletal muscle in vivo: factors aecting eciency of transfer and stability of expression. Human Gene Therapy 1993;4:151±9. [4] Manthorpe M, Jensen FC, Hartikka J, et al. Gene therapy by intramuscular injection of plasmid DNA: studies on ®re¯y luciferase gene expression in mice. Human Gene Therapy 1993;4:419±31. [5] Levy MN, Barron LG, Meyer KB, Szoka FC. Characterization of plasmid DNA transfer into mouse skeletal muscle: evaluation of uptake mechanism, expression and secretion of gene products in blood. Gene Therapy 1995;3:201±11. [6] Eisenbraun MD, Fuller DH, Haynes JR. Examination of parameters aecting the elicitation of humoral immune responses by particle bombardment-mediated genetic immunization. DNA and Cell Biology 1993;2:791±7.
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[7] Danko I, Fritz JD, Jiao S, Hogan K, Latendresse JS, Wol JA. Pharmacological enhancement of in vivo foreign gene expression in muscle. Gene Therapy 1994;1:114±21. [8] Li L, Homan RM. The feasibility of targeted selective gene therapy of the hair follicle. Nature Medicine 1995;1:705±6. [9] Winegar RA, Monforte JA, Suing KD, O'Loughlin KG, Rudd CJ, Macgregor JT. Determination of tissue distribution of an intramuscular plasmid vaccine using PCR and in situ DNA hybridisation. Human Gene Therapy 1996;7:2185±94. [10] O'Hare K. Replication of polyoma plasmid recombinants in mouse cells. J Molec Biol 1981;151:203±10. [11] Mungal S, Steinberg BM, Taichman LB. Replication of plasmid-derived human papillomavirus type 11 DNA in cultured keratinocytes. J Virol 1992;66:3220±4. [12] Iguchi-Ariga SMM, Ogawa N, Ariga H. Identi®cation of the initiation in the murine immunoglobulin heavy chain gene and possible function of the octamer motif as a putative DNA replication origin in mammalian cells. Biochim Biophys Acta 1993;1172:73±81. [13] Gassmann M, Donoho G, Berg P. Maintenance of an extrachromosomal plasmid vector in mouse embryonic stem cells. Proc Natl Acad Sci USA 1995;92:1292±6. [14] Camenisch G, Gruber M, Donoho G, Sloun PV, Wenger RH, Gassmann M. A polyoma-based episomal vector eciently expresses exogenous genes in mouse embryonic stem cells. Nucl Acid Res 1996;24:3707±13. [15] Mucke S, Polack A, Pawlita M et al. Suitability of Epstein± Barr virus based episomal vectors for expression of cytokine genes in human lymphoma cells. 1997;4:82±92. [16] Fried M, Prives C. The biology of simian virus 40 and polyomavirus. Cancer Cells 1986;4:1±16. [17] Weinberg DH, Collins KL, Simancek P, et al. Reconstitution of simian virus 40 DNA replication with puri®ed proteins. Proc Natl Acad Sci USA 1990;87:8692±6.
[18] Treisman R, Novak U, Favaloro J, Kaman R. Transformantion of rat cells by an altered polyoma virus genome expressing only the middle-T protein. Nature 1981;292:595±600. [19] Bouchard L, Gelinas C, Asselin C, Bastin M. Tumorigenic activity of polyoma virus and SV40 DNAs in newborn rodents. Virology 1984;135:53±64. [20] Berger H, Wintersberger E. Polyomavirus small T antigen enhances replication of viral genomes in 3T6 mouse ®broblasts. J Virol 1986;60:768±70. [21] Sato Y, Roman M, Tighe H, et al. Immunostimuatory DNA sequences necessary for eective intradermal gene immunization. Science 1986;273:352±4. [22] Yi A, Tuetken R, Redford T, Waldschmidt M, Kirsch J, Krieg AM. CpG motifs in bacterial DNA activate leukocytes through the pH-dependent generation of reactive oxygen species. J Immunol 1998;160:4755±61. [23] Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Molec Biol 1967;26:365±9. [24] Cahill KB, Roome AJ, Carmichael GG. Replication-dependent transactivation of the polyomavirus late promoter. J Virol 1990;64:992±1001. [25] Lacks S, Greenberg B. Complementary speci®city of restriction endonucleases of Diplococcus pnemoniae with respect to DNA methylation. J Molec Biol 1977;114:153±68. [26] Torres AT, Iwasaki A, Barber BH, Robinson HL. Dierential dependence on target site tissue for gene gun and intramuscular DNA immunizations. J Immunol 1997;158:4529±32. [27] Pertmer TM, Eisenbraun MD, McCade D, Prayaga SK, Fuller DH, Haynes JR. Gene gun-based nucleic acid immunization: elicitation of humoral and cytotoxic T lymphocyte responses following epidermal delivery of nanogram quantities of DNA. Vaccine 1995;13:1427±30. [28] Kucerova L. DNA/genetic vaccination. Viral Immunol 1998;11:55±63.
www.elsevier.com/locate/vaccine
The immune response to a polyoma virus replicon-based DNA vaccine A. Mena a, B.E.H. Coupar b, M.E. Andrew b,* a
The Flinders University of South Australia, Australia CSIRO Animal Health, Geelong, Victoria, Australia
b
Received 19 November 1999; received in revised form 2 December 1999; accepted 4 April 2000
Abstract DNA vaccination has proven to be eective against a number of tumours and microbial diseases. As DNA vaccines are unable to replicate, plasmid copy number per cell is dependent on in vivo transfection eciency, which is usually quite low. Consequently, immune responses generated are likely to be sub-optimal due to low antigen expression levels in transfected cells. During this study, replicating DNA vaccines delivered intra-epidermally by gene gun, were assessed for their ability to more eciently generate immune responses in mice. The data demonstrate that, using a polyoma virus-based system of replication, 10fold less DNA expressing the haemagglutinin gene of in¯uenza virus, was required to stimulate a humoral immune response, compared to an equivalent non-replicating vaccine. This observation suggests that the use of replicating DNA vaccines in some delivery systems may enhance the eectiveness of immune responses. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: DNA vaccine; Polyoma replicon; Antibody
1. Introduction Naked DNA plasmids can potentially provide safe and eective vaccines for human and veterinary use and are likely to be particularly useful for those organisms where there is no safe, attenuated strain, and/or where killed organisms or denatured proteins are ineffective. However, the immune response to a naked DNA vaccine is often lower than that to a whole organism and considerable eort is being put into increasing their immunogenicity. Plasmid DNA is most commonly delivered by intramuscular inoculation or via the gene gun, which propels DNA-coated gold particles into the skin. Cellular uptake of DNA is relatively inecient, particularly following intramuscular inoculation, and therefore expression of the encoded antigen is low [1,2]. Strong * Corresponding author. Tel: +61-3-5227-5745; fax: 61-3-52275555. E-mail address: [email protected] (M.E. Andrew).
promoters have been shown to increase expression levels [3±5] and the immediate/early promoter from cytomegalovirus (CMV) is currently the most eective and frequently used. For gene gun inoculation, Eisenbraun et al. [6] showed that antigen expression and antibody response are directly correlated to the plasmid copy number per gold particle. Several agents have been used in attempts to facilitate the entry of DNA into cells [3,5,7±9]. Muscledamaging agents, such as bupivicaine, have increased gene expression slightly, but the need to inoculate the bupivicaine several days before DNA inoculation reduces its suitability for widespread use in either human or veterinary vaccines. Increasing gene expression through plasmid replication would be a more practical strategy for increasing antigen expression. Bacterial plasmids do not replicate in eukaryotic cells. Plasmids with the ability to replicate in eukaryotic cells have been constructed [10±13] and have been shown to increase gene expression in vitro [14,15]. The polyoma virus genome replicates as an unintegrated mini chromosome in the nucleus of permissive
0264-410X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 0 ) 0 0 1 4 7 - X
A. Mena et al. / Vaccine 19 (2001) 68±74
cells [16]. The three components essential for replication are host derived replication factors, virally-derived large T antigen and a viral origin of replication [17]. Soon after infection, polyoma virus expresses three related proteins translated from dierent mRNAs generated by alternative splicing of a primary transcript [13]. Large T antigen is involved in viral DNA replication [16]; middle T antigen is associated with viral transforming activity [18,19]; and the small T antigen is suspected of playing a subsidiary role in DNA replication or transformation [20]. Replication begins with the large T antigen binding to the viral origin of replication and the local unwinding of the origin. Bi-directional replication, facilitated by host cell replication factors then follows. Deletion of the splice site used in the formation of the middle and small T antigens creates a gene that can only express large T antigen. Gassmann et al. [13] have constructed a plasmid capable of replicating in eukaryotic cells by including the mutated T antigen gene and the viral origin of replication, thus preventing the plasmid from transforming cells, but maintaining its ability to replicate in eukaryotic cells. In this report, the immunogenicity of a replicating DNA vaccine, constructed using the polyoma origin of replication and mutated T antigen, has been assessed. To avoid dierences in immunostimulatory DNA sequences [21,22], and transfection eciencies, polyoma virus based replicating and non-replicating plasmid with near identical sequences, expressing the luciferase reporter gene or the haemagglutinin (HA) gene of in¯uenza virus, were used to demonstrate increased antigen expression in vitro and improved antibody responses in mice. 2. Materials and methods 2.1. Plasmid construction The polyoma virus based replicating plasmid, pMGD20neo [13], was digested with AvaI, 730 bp into the polyoma virus large T antigen. Restriction ends were ®lled in with DNA polymerase I Klenow fragment and DNA religated to form pMGDko. The expected 4 bp insertion was con®rmed by sequencing using forward (5 '-TCCTCCTCCTCCTCCTCCAG-3 ' (5688±5707)) and reverse (5 '-GCTACCAGTCGCCGCCTAAG-3 ' (5929±5910)) primers. Primer positions are relative to pMGD20neo. A BamHI/BglII fragment encoding in¯uenza A/PR/8/34 HA gene under the control of the human cytomegalovirus immediate-early enhancer promoter (CMV IE) and SV40 polyadenylation signal (SV40pA), was cloned into the BamHI site of pMGD20neo and pMGDko to form pMGDHA and pMGDkoHA respectively. A blunt end fragment
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encoding the ®re¯y luciferase gene (Luc) under the control of CMV IE promoter and SV40pA was cloned into a Klenow repaired SalI site in pMGD20neo and pMGDko to form pMGDLuc and pMGDkoLuc respectively. A KpnI/NotI fragment encoding the Luc gene was cloned between the KpnI and NotI site of pCI (Promega) to form pCILuc. A neomycin resistance cassette from pMGD20neo was cloned as an EcoRIrepaired/BamHI fragment into pCI between ClaIrepaired and BamHI sites to form pCIneo. Plasmids were propagated in E.coli strain DH5a and DNA was isolated and puri®ed using Qiagen Plasmid Giga Kits. 2.2. Cell culture and transfections Mouse L929 ®broblasts were cultured in Earle's minimal essential medium supplemented with 5% foetal bovine serum at 378C in a 5% CO2 atmosphere. All transfections were conducted using LipofectAMINE PLUS reagent (GibcoBRL) according to the manufacturer's instructions. 2.3. Luciferase assay L929 cells were transfected with plasmid and cells harvested for assay using Promega luciferase cell culture lysis reagent with bovine serum albumin. Luciferase activity was determined with a luminometer (Berthold Lumate LB9501) using Promega's luciferase assay system. 2.4. Back transformation assay Plasmid DNA was extracted using a modi®ed version of the protocol described by Hirt [23], which separates low molecular weight plasmids from high molecular weight chromosomal DNA. Two days posttransfection, L929 cells were rinsed twice with cold phosphate-buered saline (PBS), then overlaid with 1% Nonidet P-40 in PBS and incubated at room temperature for 1 min [24]. Following aspiration of the PBS, the cells were treated according to the standard Hirt protocol, which involved disruption with SDS and sodium chloride, phenol/choroform extraction and ethanol precipitation in the presence of glycogen. Extracted DNA was digested overnight with DpnI to digest DH5a derived plasmid [25] and then transformed into competent DH5a cells by electroporation. Cells were plated onto LB agar plates containing ampicillin, incubated at 378C overnight and the next day, colony numbers recorded. 2.5. Vaccination of mice and serum collection Female CBA/H mice (Animal Resource Centre, Perth), 6±8 weeks old, were used and maintained in ac-
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cordance with institutional animal ethics committee approved protocols. Mice were sedated using alcohol, chloroform and ether vapour (1:2:3) and DNA was delivered to shaved abdominal skin using the Helios Gene Gun (BioRad). DNA plasmid preparations were quantitated by scanning gel bands. Both plasmids were then diluted to 1 mg/ml and the concentration rechecked on gels. A helium discharge pressure of 250 psi was used to deliver 0.4 mg of 1 mm gold beads (BioRad) coated with the speci®ed amounts of DNA. Cartridges containing coated gold beads were visually inspected to ensure even distribution of DNA-coated gold. Previous elution of DNA from multiple cartridges had shown that DNA batches were consistently loaded on the gold beads and that DNA was evenly distributed between the cartridges.
oma origin of replication and T antigen could replicate in eukaryotic cells, DNA was recovered from transfected cells and digested with DpnI, as detailed in Section 2. The methylation of bacterial DNA renders it senstive to digestion with DpnI, whereas unmethylated plasmid DNA that has been produced in the transfected cells would be insensitive to digestion by DpnI. Assays were performed in triplicate and mean colony numbers calculated for each transfection plasmid. Transfection with pMGDLuc or pMGDHA resulted in more than a thousand colonies, while transfection with pMGDkoLuc or pMGDkoHA, which encode the mutated large T antigen, or with pCILuc, resulted in very low numbers of colonies (Fig. 1).
2.6. Haemagglutination inhibition assay
To determine whether plasmid replication resulted in increased antigen production, dose response and time course analyses were conducted. Cells were transfected with 1000, 100, 10 or 1 ng of plasmid in triplicate and harvested 2 days post-transfection. Three fold higher expression was observed in cells transfected with 1000 ng of pMGDkoLuc, compared to the same dose of pMGDLuc (Fig. 2). At 100 ng DNA, luciferase activity was slightly higher in cells transfected with pMGDLuc, but at 10 and 1 ng DNA, luciferase activity was approximately 3-fold higher in cells transfected with pMGDLuc compared to those transfected with pMGDkoLuc. These results were con®rmed in other experiments (data not shown) and demonstrate that, at low doses, gene expression from replicating plasmids is higher than that generated from non-replicating plasmids. To determine the time course of luciferase expression, cells were transfected with 10 ng of pMGDLuc or PMGDkoLuc in triplicate. Flasks were harvested 6, 24 and 48 h post-transfection. Luciferase activity was barely detectable 6 h post-transfection (Fig. 3). At 24 h, cells transfected with pMGDLuc expressed 3.9-fold higher amounts of luciferase than
Serum (20 ml) was mixed with 80 ml of neuraminidase (100 U/ml in PBS) (BioWhittaker) and incubated at 378C for 16 h. Sixty microlitres of 2.5% sodium citrate were then added before incubation at 568C for 30 min, followed by the addition of 40 ml of PBS. Two-fold dilutions of neuraminidase-treated serum, starting at 1:10, were made in PBS in 96 well U-bottomed microtitre plates. In¯uenza virus (A/PR/8/34) at a concentration of 4 haemagglutination units/25 ml was added to all wells and the plates incubated at room temperature for 30 min. Packed, washed chicken red blood cells, from speci®c pathogen free birds, were diluted to 0.5% in PBS and 50 ml added to each well. After incubation at room temperature for 1 h, the plates were scored for haemagglutination inhibition. 3. Results 3.1. Back transformation analysis of plasmids To con®rm that the plasmids containing the poly-
3.2. In vitro luciferase analysis
Fig. 1. Back transformation analysis. Assays were performed in triplicate and mean colony numbers 2standard error are shown.
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Fig. 2. Dose response analysis of luciferase gene expression in vitro. Assays were performed in triplicate and results show mean relative light units (RLU) 2standard error.
did cells transfected with pMGDkoLuc, and the dierence increased to 5.3-fold at 48 h. For pMGDLuc, there was approximately 5-fold more activity at 48 h, compared to 24 h. 3.3. Antibody responses The plasmid pMGDHA, expressing the HA of in¯uenza virus was used to assess whether the ability of the plasmid to replicate would result in higher antibody responses. As the ability of the plasmid to replicate was dependent upon host cell replication factors, it was important to ensure that the DNA-coated gold particles reached the skin layer containing replicating cells. Gold particles were propelled into shaved mouse skin using the gene gun at helium pressures of 100, 200, 250 and 300 psi. Biopsies were taken immediately and examined histologically. Using 100 psi, the gold particles were deposited in the super®cial skin layers (data not shown). With increasing pressure, the particles penetrated deeper into the skin. A helium pressure of 250 psi propelled the gold into the stratum germinativum layer of skin where the replicating cells reside
(Fig. 4) and subsequent experiments used 250 psi helium. pMGDHA and pMGDkoHA were delivered intraepidermally to groups of 8 or 9 mice, using the gene gun and doses of 1000, 100, 10 or 5 ng. The control plasmid, pCIneo, which does not express HA, was used at 1000 ng in four mice. The mice were vaccinated at 0 and 4 weeks. When 1000 ng pMGDHA or pMGDkoHA were delivered (Fig. 5a), the replicating plasmid, pMGDHA, stimulated slightly higher primary antibody at weeks 3 and 4 ( p = 0.056 and p = 0.043, respectively, using 2tailed t-test), but the secondary responses were the same. Similarly, using 100 ng DNA, the pMGDHA stimulated higher antibody at weeks 3 and 4, ( p = 0.032 and p = 0.040) but not 5 and 7 weeks (Fig. 5b). At the 10 ng dose (Fig. 5c), pMGDHA stimulated higher antibody at all timepoints ( p = 0.050 at 3 weeks and <0.001 for the other timepoints), and at 7 weeks post-primary vaccination, the mean antibody titre in the group receiving pMGDHA was 10-fold higher than that in the group receiving pMGDkoHA. Sera collected at all timepoints from mice vaccinated
Fig. 3. Time course analysis of luciferase gene expression in vitro. Cells were transfected with 10 ng of DNA and harvested at the indicated times. Luciferase assays were performed in triplicate and results are expressed as mean RLU 2standard error.
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sequences present on bacterially derived DNA can have a profound eect on the immunogenicity of DNA vaccines [21,22]. Consequently the use of pMGDkoLuc and pMGDkoHA with near identical
Fig. 4. Penetration of gold particles into skin. Gold particles were ®red into skin using 250 psi and biopsies taken immediately and processed for light microscopy (400 magni®cation). The gold particles are seen as many small dots.
with pCIneo showed no signs of haemagglutination inhibition (results not shown). Comparison of mean antibody titres from groups receiving 10 ng pMGDHA or 100 ng pMGDkoHA demonstrated that, with the exception of the 5 week timepoint, mean antibody titres generated from both groups were statistically indistinguishable. Few mice vaccinated with 5 ng of plasmid seroconverted before the secondary inoculation. Three weeks after the second inoculation, 6 of 8 mice receiving pMGDHA had seroconverted as opposed to 1 of 9 mice in the group receiving pMGDkoHA (Fig. 5d). The results obtained in this experiment were reproduced in two further experiments, except that at the higher doses of 1000 and 100 ng, the replicating plasmid did not always stimulate a higher response at early timepoints. However, in all experiments, low doses of pMGDHA stimulated higher antibody responses. To measure in vivo plasmid activity, gold particles with 10, 100 or 1000 ng pMGDLuc or pMGDkoLuc were ®red into the abdomenal skin of groups of eight mice and skin and draining lymph nodes removed 24 or 48 h later. Consistent dierences in luciferase activity were not observed in either of two experiments performed (data not shown). The plasmids were also inoculated intramuscularly and muscle and draining lymph nodes removed 48 h later. Again, no consistent dierences were observed.
4. Discussion The results shown here demonstrate that plasmids containing the polyoma origin of replication and large T antigen can replicate in eukaryotic cells and therefore require less input DNA to stimulate an antibody response. The presence of immunostimulatory DNA
Fig. 5. HAI antibody responses in mice. Nine mice/group were vaccinated by gene gun at times 0 and 4 weeks. Mean HAI titres 2standard error are shown are shown for 1000 (a), 100 (b) and 10 ng (c) doses. Individual values at 7 weeks after immunization are shown for 5 ng DNA (d).
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sequences to pMGDLuc and pMGDHA, respectively, was important. The dierences observed in vitro and in vivo can be directly attributed to either the plasmids ability or inability to replicate, but not to immunostimulatory sequences, or to transfection eciencies due to dierences in plasmid sequence or structure. Camenisch et al. [14] have demonstrated 6- to 10fold greater gene expression in cells transfected with replicating plasmids. The lower gene expression observed in L929 cells transfected with 1000 ng of pMGDLuc may be due to redirection of limiting host cell replication, transcriptional and translational factors within the cell from their original role in the host cell, thus killing or damaging some cells. However, at lower doses, there were clearly higher levels of gene expression from the replicating plasmids. Time-course analysis of luciferase activity in cells transfected with 10 ng of pMGDLuc or pMGDkoLuc showed negligible luciferase activity at 6 h, but high levels at 24 h and a further ®ve-fold increase at 48 h. The dierence between the two plasmids was higher at 48 h than at 24 h, suggesting that plasmid replication occurs within the ®rst 24 h post-transfection and continues after this period. Other studies have shown that DNA can move rapidly from the site of inoculation [26] (A. Mena, unpublished, 1997), which may account for the failure to ®nd higher luciferase activity in muscle or draining lymph nodes. pMGDLuc is likely to replicate only in replicating host cells that have sucient induction of replicative enzymes and factors, further reducing likelihood of demonstrating in vivo replication. Only in mice receiving 10 or 5 ng of DNA was pMGDHA consistently more ecient at generating antibody titres than pMGDkoHA, which may be due to the average number of plasmids present per cell nuclei after vaccination. Eisenbraun et al. [6] demonstrated that a single gold particle coated with approximately 300 copies of plasmid, (coding for an antigen under the control of the CMV promoter), was sucient to give maximum gene expression and antibody titre. Increasing plasmid copy number above this level did not greatly increase gene expression or antibody titre. However, decreasing plasmid copy number per particle did reduce gene expression and antibody titres. Eisenbraun et al. [6] and Pertmer et al. [27] showed a plateau eect which may be due to limited transcriptional and translational factors within the cell. Gold particles coated to give doses of 1000 or 100 ng DNA, delivered approximately 2770 or 277 plasmid copies/ particle respectively. The failure to increase antibody responses with 100 ng or more of replicating plasmid, therefore is consistent with the ®ndings of Eisenbraun et al. [6]. At 10 ng, however, the mean antibody titre stimulated by pMGDkoHA was approximately 10-fold lower, while that stimulated by pMGDHA was equiv-
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alent to levels stimulated by 1000 or 100 ng doses. This suggests that plasmid copy number/cell in groups receiving pMGDHA at 10 ng remained above the threshold required for maximal gene expression while that in groups receiving pMGDkoHA at the same dose did not. The inability of DNA vaccines to replicate in vivo has been cited as a safety factor [1,28]. Data obtained during this study suggested that replicating plasmids did not persist in the skin of mice for longer periods than the non-replicating plasmids (results not shown), probably due to the process of keratinization and shedding of skin cells. However, it is possible that replicating plasmids could move to other cells, or other sites, and persist there. Although there may be fewer concerns about persistence of veterinary vaccines, than of human vaccines, further research is required to address safety issues of replicating DNA vaccines, such as transfer of genes to humans via processing or consumption of meat, transfer of antibiotic resistance genes or increased cancer rates. These data have demonstrated that a DNA vaccine that is able to replicate reduces the dose of DNA required to stimulate an antibody response and may pave the way towards increasing the eectiveness of DNA vaccines.
Acknowledgements We gratefully acknowledge Dr Greg Donoho from Lexicon Genetics Inc., 4000 Research Forest Drive, The Woodlands, TX 77381, for his gift of plasmid pMGD20neo. We would also like to thank Dr Cor Lenghaus for his technical support and advice.
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