We have investigated if the administration of plasmid vectors engineered for gene delivery into mammalian muscle induced the production of anti-double stranded (ds) DNA and anti-nuclear autoantibodies in normal and autoimmunity-prone mouse models. In normal mice, repeated injection of plasmid DNA did not trigger an anti-DNA response. The presence of eukaryotic transcription factor binding sites in plasmid vectors did not increase autoantibody formation in these animals. In contrast, repeated injection of such plasmids in autoimmunity-prone MRL/MpJ mice caused a significant increase in both anti-dsDNA and anti-nuclear antibody levels. Thus the repeated administration of bacterial plasmids containing eukaryotic promoter elements may induce immune responses with generation of antibodies cross-reacting not only with the mammalian DNA, but also with nuclear antigens. The potential for iatrogenic autoimmunity in susceptible individuals should be considered.
Nucleic acid vaccines and gene therapy using intramuscular injection of plasmid DNA has entered clinical trials and may ultimately find a widespread clinical use.1,2 It is, therefore, important to identify and eliminate safety concerns associated with plasmid DNA injection, such as the potential for chromosomal integration, germ line transfer, induction of immune tolerance and plasmid immunogenicity.3,4
Plasmid DNA has an adjuvant property, which partially accounts for why genetic vaccines generate such effective immunity. This immunostimulatory property of plasmid DNA is contained within short unmethylated immunostimulatory sequences (ISS), consisting of a CpG dinucleotide flanked by two 5′ purines and two 3′ pyrimidines.5 ISS stimulate B cell division in vivo, activate macrophages to produce nuclear factor kappa B (NF-κB) and tumor necrosis factor alpha (TNF-α) mRNA, which are produced during inflammatory responses.6 Furthermore, ISS induce the production of cytokines and promote a helper T cell type 1 (TH1) response.7 This unique ability of plasmid-based DNA vaccines to stimulate cellular and humoral immune responses has led to novel vaccine preparations that are as effective as traditional products.8
However, an undesirable consequence of using plasmid DNA for vaccination or gene delivery may be the stimulation of potentially dangerous inflammatory and immune responses. The eukaryotic promoters within plasmid expression cassette contain transcription factor binding sites, and this combination – of ISS, plasmid DNA and bound transcription complex – could form a strong antigen-hapten combination. Potentially, injection of plasmids containing ISS may, under specific circumstances, initiate or increase anti-DNA and antinuclear autoimmune responses. Such antibodies are diagnostic markers for autoimmune diseases and the possibility of DNA injection triggering development of such autoimmunity should be considered.
The work described here examines the ability of plasmid DNA to induce systemic anti-DNA and anti-nuclear responses in normal (BALB/c) and autoimmunity-prone MRL/MpJ mice. This strain is characterised by low-level autoantibody production to double-stranded DNA9 and is bred as a control for MRL/MpJ-Faslpr mice. The latter strain is a model for systemic lupus erythematosus-like autoimmune syndromes.
Different types of plasmids, all containing ISS but with various combinations of prokaryotic and eukaryotic sequences were used to establish if the sequences contained within these vectors could influence the production of autoantibodies in BALB/c mice. One type (pBS) had no mammalian DNA sequences (bacterial plasmid) and the second type (pXAV and pcDNA3) had combinations of mammalian promoter/enhancer elements typical for plasmids used in nucleic acid vaccination and gene therapy trials. It has been shown that multiple injection of plasmid vaccine is required to generate protective immunity against pathogens in different species8,10 and similarly, repeated administration of plasmids may be needed in many gene therapy applications. Therefore, to mimic these gene delivery protocols, 25 μl of plasmid (2 mg/ml) were administered via single or multiple injections in the tibilais anterior muscle. Intraperitoneal injection was also used as a delivery route.
Following injections the serum titers of anti-double stranded DNA (dsDNA) antibodies were measured using a calf thymus dsDNA ELISA. None of the three plasmids (pBS, pXAV or pcDNA3) injected four times at 2-week intervals caused an increase in anti-dsDNA antibody levels compared with saline-injected controls. Moreover, injection of the pcDNA3 by an i.p route also had no effect on anti-dsDNA antibody production in this model (Figure 1a).
In MRL/MpJ mice, the titer of anti-dsDNA and anti-nuclear immunoglobulins in the sera was measured following single and multiple injections of plasmid using the same protocol as for the experiment with BALB/c mice. In this model, the mean serum level of anti-dsDNA antibodies in multiple plasmid-injected MRL/MpJ mice was significantly higher than in the saline-injected group (0.68 ± 0.11 versus 0.42 ± 0.06, P < 0.05) (Figure 1b). This indicated that i.m. injection of bacterial plasmid DNA could increase the production of antibodies with affinity for mammalian dsDNA.
The sera from MRL/MpJ animals were then analysed for antibodies, which cross-reacted with epitopes in cell nuclei, using a qualitative fluorescence assay. The positive control serum from a MRL/MpJ.Faslpr mouse gave a strong pattern of immunoreactivity against the cell nucleus (Figure 2a). Six of the seven MRL/MpJ mice injected with plasmid had antibodies, which reacted with the cell nuclei of HEp-2 cells. Moreover, the patterns of immunoreactivity revealed great variability of antinuclear antibodies that were generated in response to plasmid injection. Three sera gave a fine speckled granular pattern (Figure 2c), a fourth serum generated a nucleolar pattern of immunofluorescence (Figure 2d) and two sera gave a weaker fine-speckled pattern of staining.
This immunoreactivity was in a marked contrast to the negative staining in control sera from a saline-injected MRL/MpJ mice (shown in Figure 2b). Of the seven sera from MRL/MpJ mice injected with saline, only two generated a weak pattern of immunoreactivity on HEp-2 cells (data not shown).
In contrast to the multiple injections, the single injection of 25 μl of plasmid (2 mg/ml) in the tibilais anterior muscle of MRL/MpJ mice did not result in any significant changes in autoantibody levels as compared with controls (data not shown).
We have shown that in normal mice, repeated injection of plasmid DNA typical of a plasmid immunisation regime does not trigger an autoimmune response. This is in agreement with results by others showing no or only very low-level anti-DNA responses induced in normal animals in similar paradigms.11,12,13 Furthermore, the inclusion of eukaryotic TF binding sites did not increase the risk of autoantibody formation in immunologically naive mice. However, multiple plasmid injections in the autoimmunity-prone MRL/MpJ mice caused a significant increase of anti-DNA antibodies, which was accompanied by an increase in the number of sera containing anti-nuclear antibodies. Thus, repeated administration of bacterial plasmids containing eukaryotic promoter elements may stimulate immune responses with antibodies reacting not only with the mammalian DNA, but also with endogenous cell nuclear antigens.
Sera from autoimmune systemic lupus erythematosus (SLE) patients14,15 and a small proportion of the normal population16,17,18,19 contain anti-DNA antibodies recognising epitopes present on mammalian DNA and we have shown here that in predisposed mice, this mechanism can be augmented in response to repeated overload with bacterial plasmid DNA.
The structure of the variable regions of SLE autoantibodies against double-stranded DNA indicates that they are produced in response to an antigen-selective stimulation.20 Furthermore, the combination of ISS, plasmid DNA and bound transcription complex may form a strong antigen-hapten combination and such complexes may be released from intracellular compartments in response to muscle damage and inflammation caused by the DNA injection procedure. There is mounting evidence that anti-DNA antibodies are also produced in response to DNA–protein complexes rather than naked DNA, as combinations of DNA with various DNA- binding proteins including nucleosomal peptides21 and non-self antigenic peptides22 have been shown to trigger anti-DNA responses.
The recent animal model data indicated that anti-DNA antibodies promote pathological mechanisms in chronic inflammatory disorders23 and bacterial DNA can exacerbate autoimmune responses.24 However, it must be stressed that the clinical implications of the plasmid-induced autoimmune phenomenon described here are not clear. In addition to the potential safety considerations, binding of antibodies to plasmid DNA may result in rapid elimination or sequestration of DNA and the development of autoantibodies could also decrease the efficacy of subsequent applications of nucleic acids as vaccines or gene therapy vectors.
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MacColl, G., Bunn, C., Goldspink, G. et al. Intramuscular plasmid DNA injection can accelerate autoimmune responses. Gene Ther 8, 1354–1356 (2001). https://doi.org/10.1038/sj.gt.3301537
- plasmid vector
- anti-DNA antibodies
- anti-nuclear antibodies
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