Impact of plasmid stability on oral DNA delivery by Salmonella enterica serovar Typhimurium

Vaccine 25 (2007) 1476–1483

Impact of plasmid stability on oral DNA delivery by Salmonella enterica serovar Typhimurium Michelle E. Gahan a,b,c,∗ , Diane E. Webster a,d , Steven L. Wesselingh a,c , Richard A. Strugnell b a

Children’s Vaccines Unit, Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Victoria 3001, Australia b Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3052, Australia c Department of Medicine, Monash University, Alfred Hospital, Prahran, Victoria 3181, Australia d Department of Immunology & School of Biological Sciences, Monash University, Clayton, Victoria 3800, Australia Received 14 July 2006; received in revised form 13 October 2006; accepted 18 October 2006 Available online 7 November 2006

Abstract Live attenuated Salmonellae may overcome limitations with conventional methods of DNA immunisation. This study examined the impact of plasmid stability on oral DNA delivery by the attenuated Salmonella enterica serovar Typhimurium vaccine strain BRD509. A DNA vaccine cassette comprising the C fragment of tetanus toxin under control of the cytomegalovirus (CMV) promoter was ligated into plasmid pcDNA3, pUC18, pBBR122, pACYC184, pRSF1010/CAT, pBR322 and pAT153. In vitro and in vivo stability studies revealed that, with the exception of pcDNA3 and pUC18, the plasmids were retained by BRD509. However, pAT153 was the only plasmid to induce a tetanus toxoid-specific antibody response following oral delivery. Plasmid copy number was found to impact on plasmid stability and the induction of antigen-specific humoral responses. © 2006 Elsevier Ltd. All rights reserved. Keywords: Salmonella; DNA; Oral

1. Introduction Conventional methods of DNA immunisation have induced immune responses in experimental animals, however, they have not proven as successful in human clinical studies (reviewed in Refs. [1,2]). It has been argued that their poor immunogenicity may be due, at least in part, to an inability to reproduce the DNA delivery conditions used in mice (i.e. volume, amount and concentration). To circumvent this problem, there is a need to develop a more efficient DNA delivery system. Live attenuated Salmonella enterica serovar Typhi (S. typhi) vaccines, such as S. typhi Ty21a, have provided pro∗ Corresponding author at: Children’s Vaccines Group, Macfarlane Burnet Institute for Medical Research and Public Health, GPO Box 2284, Melbourne, Victoria 3001, Australia. Tel.: +61 3 9282 2279; fax: +61 3 9282 2100. E-mail address: [email protected] (M.E. Gahan).

0264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2006.10.042

tection in humans against typhoid fever [3–5]. Salmonella vaccines for veterinary applications have also been developed [6–8]. The use of various attenuated strains of Salmonella enterica serovar Typhimurium (S. typhimurium) and S. typhi as vaccine vectors to orally deliver heterologous antigens to the immune system of the vaccine recipient is a logical extension of the success of these strains to act safely and efficaciously as Salmonella vaccines. Recombinant Salmonellae have been used as vaccine vectors to deliver both DNA and protein vaccines from a wide variety of bacterial, viral and parasitic sources (for a review see Refs. [9–11]). Benefits associated with Salmonella delivery include oral administration, induction of effective mucosal, humoral and cellular immune responses to the vector and heterologous antigen, availability of a wide choice of candidate attenuated Salmonella strains and extended antigen presentation due to the use of a live delivery system. Specifically, oral delivery overcomes limitations associated with other routes of administration including risks associated

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with needle reuse and disposal, such as the transmission of blood-borne viruses [12–14] and negates the need for highly trained medical personnel to administer the vaccines. Furthermore it is anticipated that the ease of administration will lead to enhanced availability, vaccine coverage and compliance. DNA vaccine delivery by Salmonella is a promising and versatile oral delivery system, applicable to the large-scale vaccination of a range of antigens. However, the advancement of this vaccination strategy has been hampered by issues pertaining to the stability of the plasmid encoded antigen. The ability of bacteria to stably retain plasmids during replication is influenced by a variety of factors attributable to the plasmid vectors, including the origin of replication, copy number, size and complexity. In the laboratory plasmids can be stably maintained in bacteria through the use of antibiotic selection genes encoded on the plasmid. However, in animal and particularly human models, concomitant antibiotic administration is neither feasible nor appropriate in an era of increasing antibiotic resistance. Plasmid stability in S. typhimurium has been addressed by approaches including integration of the foreign gene into the chromosome of the bacteria [15–17], codon optimisation of the heterologous antigen [18], in vivo inducible promoters [19,20] and post-segregational killing systems [21–23]. However, to enable the delivery of multiple copies of a plasmid encoded DNA vaccine, it is essential to address plasmid stability by analysing plasmid retention rates in the absence of antibiotic selection. Inherently stable plasmids should be identified for oral DNA delivery by Salmonella. The aims of this study were to analyse the in vitro and in vivo stability of selected DNA vaccine plasmids encoding a 451 amino acid region of the non-toxic binding portion of tetanus toxin (C fragment) in attenuated S. typhimurium vaccine strain BRD509. The in vitro and in vivo stability of the C fragment DNA plasmids were determined, as was the ability of each to induce S. typhimurium lipopolysaccharide (LPS)- and tetanus toxoid (TT)-specific antibody responses following oral delivery. These studies were undertaken to elucidate the factors affecting plasmid stability and determine the optimal plasmid for oral DNA delivery by S. typhimurium.

2. Materials and methods 2.1. Bacterial strains and plasmids S. typhimurium strain BRD509 is an aroA/aroD mutant of SL1344 and was the gift of Prof. G. Dougan, Imperial College, London, UK. All DNA manipulations were carried out in Escherichia coli laboratory strain JM109 or DH5␣. Bacterial strains were routinely cultured in Luria Bertani (LB) or LB agar and when required, were supplemented with antibiotics at the following concentrations: 100 ␮g ml−1 ampicillin, 30 ␮g ml−1 chloramphenicol,

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30 ␮g ml−1 kanamycin, 25 ␮g ml−1 streptomycin and 12.5 ␮g ml−1 tetracycline. The plasmids pCR® 2.1-TOPO (Invitrogen, Mount Waverley, Australia), pBR322 [24], pACYC184 [25], pBBR122 (MoBiTec, Germany), pUC18 [26,27], pRSF1010/CAT [28], pcDNA3 (Invitrogen, Mount Waverley, Australia) and pAT153 [29] were used in this study. 2.2. Construction of C fragment DNA plasmids The plasmid pcDNA3/Cfrag was the gift of L. Sait, Department of Microbiology and Immunology, University of Melbourne, Melbourne, Australia. Plasmid pcDNA3/Cfrag has previously been shown to be immunogenic and expressed in eukaryotic cells following intramuscular immunisation (Gahan et al. unpublished data). The C fragment DNA vaccine cassette (comprising of the cytomegalovirus promoter, C fragment gene and the bovine growth hormone polyadenylation signal and transcription termination sequence) was amplified by PCR from pcDNA3/Cfrag using primers C8 (5 ACATTCTAGACTGCTTCGCGATGTACG-3 ) and C9 (5 GACTTCTAGACACCGCATCCCCAGCATG-3 ) and ligated into pCR® 2.1-TOPO. Following sequence confirmation the plasmid was digested with BstX I and T4 DNA polymerase (New England Biolabs, Beverly, MA, USA) was used to generate cohesive ends. The blunted C fragment DNA vaccine cassette was ligated into Ssp I digested pBR322 to generate pBR322/Cfrag, Sma I digested pUC18 to generate pUC18/Cfrag, EcoR V digested pACYC184 to generate pACYC184/Cfrag, Sma I digested pRSF1010CAT to generate pRSF1010/Cfrag, Dra I digested pBBR122 to generate pBBR122/Cfrag and Sma I digested pAT153 to generate pAT153/Cfrag. Following confirmation by restriction enzyme digest and PCR screening the C fragment DNA plasmids were transformed into S. typhimurium BRD509 by electroporation. 2.3. In vitro plasmid stability To determine in vitro plasmid stability, cultures of S. typhimurium BRD509 transformed with the C fragment DNA plasmids were passaged daily for 5 days (approximately 100 generations) in the absence of antibiotic selection. The percentage stability of the plasmid was determined by dividing the number of bacteria containing the plasmid (viable counts on LB agar containing antibiotics) by the total number of bacteria (viable count on LB agar). 2.4. Oral immunisation with S. typhimurium Female 6–8 week old BALB/c mice were obtained from The University of Melbourne, Department of Microbiology and Immunology animal facility (Parkville, Victoria, Australia). Experiments were conducted under the guidance of the University of Melbourne animal ethics committee. Mice were orally immunised under anaesthesia (Penthrane) via

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a 4 cm gastric lavage needle with approximately 1010 bacteria in phosphate buffered saline (PBS) (200 ␮l/mouse) as determined by retrospective viable count. Bacteria for immunisation were grown static in LB containing antibiotics at 37 ◦ C for 24 h. Thirty minutes prior to immunisation mice were orally administered 100 ␮l of 10% (w/v) sodium bicarbonate to neutralise stomach acidity. 2.5. In vivo plasmid stability Ten and 20 days post-oral administration of S. typhimurium mice were killed by carbon dioxide asphyxiation and their spleens and mesenteric lymph nodes (MLN) removed aseptically. Organs were homogenised separately in sterile plastic bags containing 5 ml of PBS using a Stomacher 80 homogeniser (Seward Medical, London, UK). The total number of S. typhimurium in each organ and the number containing the plasmid was enumerated by viable count on LB agar plates, and LB agar plates containing antibiotic selection, respectively. 2.6. Measurement of antibody response by enzyme-linked immunosorbent assay (ELISA)

Fig. 1. In vitro stability of C fragment DNA plasmids 100. S. typhimurium BRD509 containing the plasmids pAT153/Cfrag, pRSF1010/Cfrag, pBR322/Cfrag, pBBR122/Cfrag, pAYCY184/Cfrag, pUC18/Cfrag and pcDNA3/Cfrag were passaged for approximately 100 generations in the absence of antibiotic selection. The percentage of bacteria containing the plasmid was determined by viable count on media with and without the appropriate antibiotic selection.

3. Results 3.1. In vitro plasmid stability in S. typhimurium BRD509

On days 14, 27, 56, 84 and 168 mice were bled from the tail vein and the titre of antibody present in the mouse sera determined by end-point ELISA. Briefly, 96-well immunoplates (Nunc A/S, Kamstrupp, Denmark) were coated overnight at 4 ◦ C with antigen diluted in PBS: either 1Lf/ml (flocculating units) tetanus toxoid (CSL Ltd., Parkville, VIC, Australia) or 0.01 mg/ml Salmonella LPS (Sigma, Sydney, NSW, Australia). Following blocking with 4% skim milk powder in PBS for 1 h at 37 ◦ C, serial dilutions of mouse sera, in PBST (0.05% Tween20 in PBS) containing 0.4% skim milk powder, were added to the wells and the plates incubated for 2 h at 37 ◦ C. Bound antibody was detected by the addition of an anti-mouse Ig Horseradish peroxidase conjugate (Chemicon, Temecula, CA, USA) diluted 1/1000 in PBST containing 0.4% skim milk powder and incubated for 2 h at 37 ◦ C. Reactions were developed using immunopure ophenylenediamine (Pierce, Rockford, IL, USA) with H2 O2 as the substrate. Absorbance was read at 492 nm in a Multiskan® Multisoft plate reader (Labsystems, Helsinki, Finland). The serum antibody titre was designated as the reciprocal of the dilution of specific antibody that gave an OD492 value of five times the background value. 2.7. Statistical analysis The non-parametric Mann–Whitney test was used to compare data from groups of animals. A probability (P) value of less than 0.05 indicated the groups were significantly different. To perform statistical analysis on data groups which contained values that were below the point of detection, an arbitrary value one below the limit of the assay was assigned.

To investigate the affect of an antibiotic free environment, similar to an immunised host, on plasmid segregation the in vitro stability of the C fragment DNA plasmids in S. typhimurium BRD509 was determined. BRD509 containing the plasmids pAT153/Cfrag, pRSF1010/Cfrag, pBR322/Cfrag, pBBR122/Cfrag, pACYC184/Cfrag, pUC18/Cfrag and pcDNA3/Cfrag were grown in the absence of antibiotic selection for 100 generations and the percentage of bacteria retaining the plasmid determined (Fig. 1). Following growth in antibiotic free media, pAT153/Cfrag was stably retained by 100% of the bacteria. Plasmid pRSF1010/Cfrag was retained by 59% of the bacteria, pBR322/Cfrag by 48% and pBBR122/Cfrag by 42%. Plasmid pACYC184/Cfrag could only be recovered from 0.002% of the S. typhimurium after 100 generations. No bacteria containing pUC18/Cfrag or pcDNA3/Cfrag could be recovered after 87 and 37 generations, respectively. 3.2. In vivo plasmid stability in S. typhimurium BRD509 Based on the in vitro plasmid stability results five of the plasmids, encompassing the four most stable plasmids and the least stable yet frequently used plasmid pcDNA3 were further characterised in vivo. Groups of five BALB/c mice were orally immunised with BRD509 containing the plasmids pAT153/Cfrag, pBBR122/Cfrag, pRSF1010/Cfrag, pBR322/Cfrag or pcDNA3/Cfrag. The plasmid stability and persistence of the bacteria in vivo was investigated at 10 and 20 days post-immunisation in the spleen and MLN (Fig. 2). Carriage of the plasmids did not affect the ability of the bac-

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Fig. 2. In vivo stability of C fragment DNA plasmids in S. typhimurium. Groups of BALB/c mice were orally immunised with attenuated S. typhimurium BRD509 containing the plasmids pBR322/Cfrag, pAT153/Cfrag, pcDNA3/Cfrag, pBBR122/Cfrag or pRSF1010/Cfrag. Ten or 20 days later mice were killed and the number of bacteria present in the spleen and mesenteric lymph nodes (MLN) was determined by viable count. The total number of bacteria isolated on LB agar and LB agar containing antibiotic selection is represented by the open and closed shapes, respectively. Each point denotes the count for an individual mouse. The broken line indicates the limit of the assay. The symbol (##) denotes that the total number of bacteria isolated is significantly higher than the number of bacteria containing the plasmid (P < 0.05).

teria to colonise as S. typhimurium was recovered from the organs of all immunised mice. On average, the number of bacteria isolated from the MLN was 10 fold higher than the number of bacteria isolated from the spleen. In both organs the bacterial load reduced over time, resulting in bacteria being unable to be recovered from the spleen of some of the

individual mice at day 20. All C fragment plasmids were stably retained by BRD509 in vivo at days 10 and 20 with the exception of pcDNA3/Cfrag. Plasmid pcDNA3/Cfrag was significantly unstable at days 10 and 20 with no bacteria containing pcDNA3/Cfrag isolated from either the spleen or MLN.

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Fig. 3. S. typhimurium LPS and tetanus toxoid-specific serum Ig antibody titres. Groups of BALB/c mice were immunised on days 0 and 28 with attenuated S. typhimurium BRD509 containing the plasmids pBR322/Cfrag, pAT153/Cfrag, pcDNA3/Cfrag, pBBR122/Cfrag or pRSF1010/Cfrag or with S. typhimurium BRD509 alone. Serum samples were collected on days 14, 27, 56, 84 and 168. The amount of (a) S. typhimurium LPS-specific and (b) tetanus toxoid-specific total serum Ig was determined by endpoint ELISA. Each symbol represents a titre from an individual mouse. Serum antibody titres were designated as the reciprocal of the dilution of specific antibody that gave an OD492 value five times the background of the assay. The broken line indicates the limit of the assay and the closed symbols indicate values obtained are below the limit of the assay (1/50). The symbol (##) denotes the antibody titre is significantly higher than all the other groups (P < 0.05).

3.3. Humoral immune responses induced following oral delivery by S. typhimurium BRD509 Groups of five BALB/c mice were orally immunised on days 0 and 28 with S. typhimurium BRD509

containing the plasmids pAT153/Cfrag, pBBR122/Cfrag, pRSF1010/Cfrag, pBR322/Cfrag or pcDNA3/Cfrag, or with BRD509 alone. Serum antibody response (total Ig) specific for S. typhimurium LPS and tetanus toxoid (TT) was determined by ELISA at days 14, 27, 56, 84 and 168. Anti-LPS

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total Ig was detected in the sera of all immunised mice (Fig. 3a). The pattern and titre of antibody response was similar in all groups of mice, including the group immunised with BRD509 alone, suggesting LPS antibody response is not affected by plasmid carriage. Anti-LPS responses on average peaked 27–56 days post-immunisation, following which they levelled out but were still persistent 168 days post-immunisation. Of the six groups of mice, an antiTT antibody response was only detected in the sera of mice immunised with BRD509 containing pAT153/Cfrag (Fig. 3b). The response peaked on average at day 56, where it was significantly higher than all other groups (P < 0.05), then levelled out and remained detectable at day 168. There was no significant difference between the groups immunised with pBBR122/Cfrag, pRSF1010/Cfrag, pBR322/Cfrag and pcDNA3/Cfrag and mice immunised with S. typhimurium BRD509 alone. An anti-TT antibody response was detected in a limited number of mice immunised with pBBR122/Cfrag, pRSF1010/Cfrag, pBR322/Cfrag and BRD509 alone, however, the response was not detected over multiple time points.

4. Discussion This study investigated the comparative in vitro and in vivo stability in S. typhimurium BRD509 of multicopy number plasmids encoding a C fragment DNA vaccine. The ability of the plasmids to induce a TT- and Salmonella LPS-specific total Ig antibody response in BALB/c mice was examined. Of the plasmids examined only pAT153/Cfrag was able to induce a TT antibody response following oral immunisation. Plasmid pAT153/Cfrag was stable in vitro and in vivo. Although pBBR122/Cfrag, pBR322/Cfrag and pRSF1010/Cfrag were also stable in vivo the TT antibody response was not significantly different to mice immunised with BRD509 alone. The high copy number plasmid pcDNA3/Cfrag was highly unstable and no plasmid containing bacteria could be isolated from the spleen or MLN. The results of this study suggest plasmid copy number impacts on both plasmid stability in S. typhimurium BRD509 and the induction of an antigen-specific immune response. This study highlights the importance of addressing the issue of plasmid stability early in the development of a vaccine strategy involving S. typhimurium delivery of a plasmid DNA vaccine. Plasmids pUC18/Cfrag and pcDNA3/Cfrag were highly unstable in S. typhimurium in vitro with no bacteria containing the plasmid isolated after 87 and 37 generations, respectively. Furthermore no bacteria containing pcDNA3/Cfrag could be isolated from the spleen and MLN. Plasmid pcDNA3 is derived from pUC and, due to a mutation in the site controlling replication, these plasmids have the highest copy number of the plasmids examined in this study (∼500–700 copies/cell) [30]. In comparison, the other plasmids have copy numbers ranging from 30 copies/cell (pAT153) to 10 copies/cell (pBBR122), suggesting a rela-

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tionship between high plasmid copy number and instability. In the absence of antibiotic selection the increased copy number may place too great a metabolic burden on the bacteria to retain the plasmid whilst undergoing replication, resulting in rapid plasmid loss. The results of this study are in agreement with similar studies identifying a relationship between the copy number of DNA vaccine plasmids and stability [31,32]. Two days after intragastric inoculation with S. typhimurium aroA SL7207 containing a high copy number plasmid (∼500–700 copies/cell) encoding for ␤-galactosidase, no plasmid containing bacteria was recovered from the Peyer’s patches (PP), MLN, liver and spleen. In contrast, lower copy number plasmids (15–20, 10–12 and 5 copies/cell) were stably maintained out to 21 days in these organs [31]. Similarly, Garmory et al. [32] reported a ␤-galactosidase DNA plasmid with a copy number of ∼5 copies/cell was 99% stable in vivo 3 days post-oral immunisation with S. typhimurium aroA SL7207 [32]. A relationship between plasmid instability and copy number has also been reported following oral delivery by S. typhimurium expressing plasmid encoded antigens [28,33,34]. In addition to identifying a relationship between plasmid copy number and stability in S. typhimurium, the results of this study suggest plasmid copy number impacts on the induction of an antigen-specific immune response. Oral delivery of the stable plasmid pAT153/Cfrag resulted in the induction of an anti-TT antibody response in contrast to pcDNA3/Cfrag for which no response was detected. Oral delivery by S. typhimurium of a stable plasmid encoding ␤-galactosidase has previously been reported to result in superior induction of antigen-specific cellular and antibody responses than delivery of an unstable plasmid [31]. Despite the importance of a stable plasmid in the generation of antigen-specific responses, the results of this study also suggest in vivo stability does not solely correlate with antibody responses. Plasmids pBBR122/Cfrag, pRSF1010/Cfrag and pBR322/Cfrag failed to induce a TT-specific response despite being stable in vivo and all immunised mice developing an anti-LPS response to the vaccine vector. These three plasmids have copy numbers less than that of pAT153/Cfrag, suggesting a low copy number may be detrimental. This finding is supported by published reports utilising stable low copy number plasmids (5 copies/cell) which report the absence of antigen-specific antibody responses [32] and weaker T cell response compared with plasmids with copies numbers of 10–12 and 15–20 copies/cell [31]. A low copy number plasmid may result in an insufficient dose of plasmid being delivered by S. typhimurium than is required to stimulate a response. To induce a specific immune response following oral delivery of DNA vaccines by S. typhimurium, it appears a balance is required between plasmid stability and ensuring the delivery of a sufficient dose of the DNA vaccine. Plasmid stability has been reported to affect bacterial colonisation of mouse organs. Coulson et al. found that intravenous injection of BALB/c mice with S. typhimurium aroA SL3261 carrying the high copy number plasmids pUC19

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or pBluescript resulted in a 200 fold lower colonisation of the spleen at day seven compared to the lower copy number plasmid pBR322 [35]. Similarly, 3 days after intragastric inoculation with S. typhimurium aroA SL7207 containing a high copy number plasmid (∼500–700 copies/cell) no S. typhimurium could be isolated from the PP or spleen [32]. In contrast to these studies, no reduction was observed in our study at days 10 and 20 in the ability of S. typhimurium BRD509 (pcDNA3/Cfrag) to colonise the MLN and spleen compared with mice immunised with bacteria containing the stable plasmids (Fig. 2). This is consistent with a study reporting on oral delivery of S. typhimurium BRD509 expressing C fragment from plasmids of varying copy numbers [28]. The discrepancies regarding colonisation among these studies may be a consequence of the S. typhimurium strain employed or the oral dose delivered. In the reports where a reduction in the ability of bacteria carrying unstable plasmids to colonise was detected following oral delivery, doses of 1–5 × 109 [32] and 1–5 × 108 [31] were administered. These doses are lower than the oral dose of ∼1 × 1010 of BRD509 for which no effect of plasmid instability on colonisation was observed out to 20 days (present study and [28]). In comparison to the various doses administered in these studies the current Ty21a vaccine consists of 2–10 × 109 live bacteria. A reduction in the number of bacteria able to colonise following oral delivery will directly reduce the amount of vaccine being delivered, potentially impacting on the ability of the immune system to raise an adequate response. Plasmid carriage places a metabolic burden on the bacteria as a portion of its cellular resources must be diverted to plasmid replication, potentially resulting in a reduced growth rate of plasmid containing bacteria. Consequently, once a plasmid-free bacterium becomes present in the population it will be selected for resulting in a reduction in the number of plasmid containing bacteria [36]. Therefore, it is vital the plasmid is maintained by the entire bacterial population to enable the delivery of a sufficient quantity of the vaccine to induce an immune response. Limited strategies are available to ensure the stability of DNA vaccines. Woo et al. [37] found hepatitis B surface antibody-specific titres were significantly enhanced seven and 21 days after oral immunisation with S. typhi carrying a hepatitis B DNA vaccine when ampicillin was concomitantly administered [37]. However, concomitant administration of antibiotics is both impractical and highly undesirable in a clinical setting. Post-segregational killing systems such as the hok-sok stabilisation loci [23] and the asd+ vector/asd host lethal system [21,22] do not prevent plasmid segregation. Instead plasmid free bacteria lyse resulting in a reduction in the dose of plasmid being delivered. To address the issue of plasmid stability in the context of DNA vaccine delivery and deliver multiple copies of the plasmid it is necessary to focus on the plasmid vectors themselves and identify inherently stable plasmids for oral DNA delivery by S. typhimurium. This study highlights the importance of examining the in vitro and in vivo stability of plasmids when using S.

typhimurium to orally deliver DNA vaccines. Plasmids which are inherently stable in the absence of antibiotic selection should be identified early in the vaccine development process as plasmid stability appears to have a substantial impact on the induction of a humoral immune response to the heterologous antigen. Findings suggest plasmid copy number strongly impacts on plasmid stability and that the delivery and sustained presence of bacteria carrying moderate copies of the DNA plasmids is more efficient in inducing an immune response than the initial delivery of bacteria carrying high numbers of plasmids which then undergo rapid segregation. The use of a low copy number stable plasmid also appears to result in the delivery of an insufficient quantity of DNA vaccine to induce a specific immune response to the vaccine antigen. Of the plasmids examined in this study pAT153 appears to be an ideal candidate for the oral delivery of DNA vaccines by S. typhimurium. It is stably maintained in S. typhimurium in vitro and in vivo and has a copy number which is high enough to deliver a sufficient dose of the vaccine to induce a humoral immune response. The findings of this study, and those of others, highlight the difficulties associated with inducing a consistent and reproducible immune response to a DNA vaccine following oral delivery by S. typhimurium. Research into the processes by which Salmonella deliver DNA vaccines into eukaryotic cells will be critical in overcoming these difficulties. This, in conjunction with the determination of the most efficacious Salmonella strain, may result in further optimisation of DNA vaccine delivery by Salmonella and enhancement of the antigen-specific immune response.

Acknowledgment This work was carried out with the financial support of the National Health and Medical Research Council (NHMRC) of Australia.

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