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Department of Biological and Physical Sciences, University of Southern Queensland, Toowoomba 4350, Queensland, Australia
Correspondence
Trilochan K. S. Mukkur
tk_mukkur{at}hotmail.com
Received 23 July 2007
Accepted 13 September 2007
) and interleukin (IL)-2 detected in the S1-stimulated splenocyte supernatants and no serum IgG. Despite the lack of an antibody response, the lungs of DNA-immunized mice were cleared of B. pertussis at a significantly faster rate compared with mock-immunized mice following an aerosol challenge. To gauge the true potential of this S1 DNA vaccine, the immune response and protective efficacy of the commercial diphtheria–tetanus–acellular pertussis (DTaP) vaccine were included as the gold standard. Immunization with DTaP elicited a typically strong T-helper (Th)2-polarized immune response with significantly higher titres of serum IgG than in the DNA vaccine group, but a relatively weak Th1 response with low levels of IFN- and IL-2 detected in the supernatants of antigen-stimulated splenocytes. DTaP-immunized mice cleared the aerosol challenge more efficiently than DNA-immunized mice, with no detectable pathogen after day 7 post-challenge.
Abbreviations: aP, acellular pertussis; CHO, Chinese hamster ovary; CMI, cell-mediated immunity; ConA, concanavalin A; DTaP, diphtheria–tetanus–acellular pertussis; HRP, horseradish peroxidase; i.d., intradermal; IFN-, gamma interferon; IL, interleukin; i.m., intramuscular; IMAC, immobilized metal affinity chromatography; i.n., intranasal; i.p., intraperitoneal; PT, pertussis toxin; s.c., subcutaneous; Th, T-helper; wP, whole-cell pertussis.
Present address: Panbio Limited, Brisbane, Queensland, Australia. 
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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DNA vaccines may represent an alternative to conventional vaccines for immunization against infectious diseases such as pertussis, with the potential to induce both humoral and cell-mediated immunity (CMI). Ulmer et al. (1993) were among the first to report that a protective antigen-specific antibody and cytotoxic T-cell response could be generated in mice following immunization with DNA. Since this early finding, DNA vaccines have now been shown to be effective against a number of viral, parasitic and bacterial pathogens (Vanderzanden et al., 1998; Wang et al., 1998; Delogu et al., 2000).
Pertussis toxin (PT) is an obvious candidate for DNA vaccination against pertussis, as it is the major virulence factor of B. pertussis and, as a toxoid, is a component of all modern aP vaccines. It is a hexamer consisting of five subunits (S1, S2, S3, S4 and S5) arranged in a 1 : 1 : 1 : 2 : 1 stoichiometry (Tamura et al., 1982), with the S1 subunit possessing ADP-ribosylating activity. Of the five subunits, S1 is the immunodominant domain (De Magistris et al., 1988). Passive immunization with S1-specific antibodies has been reported to neutralize the toxin in vitro and protect mice against aerosol or intracerebral challenge (Sato et al., 1984, 1991; Halperin et al., 1991). Systemic and mucosal humoral responses have been reported following both oral (Walker et al., 1992) and intranasal (i.n.) (Lee et al., 2003) immunization of mice with recombinant S1 antigens. Other studies have also had success using S1-derived vaccines (Boucher et al., 1994; Barry et al., 1996; Lee et al., 1999; Nascimento et al., 2000). More recently, DNA vaccines comprising the full S1 subunit (Kamachi et al., 2003) or an N-terminal fragment of S1 (Kamachi & Arakawa, 2004, 2007) were reported to induce antibody responses when delivered by the intradermal (i.d.) route using a gene gun, and protected immunized mice against a lethal challenge.
It has been demonstrated that endogenous expression of S1 has a cytotoxic effect on Chinese hamster ovary (CHO) cells (Castro et al., 2001). Therefore, an important aspect in the development of our DNA vaccine was the inactivation of the S1 gene. Pizza et al. (1989) showed that substitution of Glu-129 with Gly-129 in combination with either Arg-9 to Lys-9, Arg-13 to Leu-13 or Trp-26 to Ile-26 abolished the ADP-ribosylase activity but did not adversely affect immunogenicity. Nencioni et al. (1990) later characterized the 9K/129G toxoid and showed that it had the same physical and immunological properties as wild-type PT. For this study, we chose the 13L/129G mutation as it is reported to have marginally less leukocytosis-promoting activity and greater T-cell recognition, and mice had a higher survival rate following lethal challenge compared with the 9K/129G toxoid (Pizza et al., 1989).
As the induction of CMI is considered to be essential for immunity against pertussis (Mills et al., 1993; Mills, 2001), the aim of this study was to evaluate the potential of a parenterally delivered S1 DNA vaccine to induce both antibody and CMI, and to protect against an aerosol challenge, with a view to determining whether this mode of immunization may represent a viable alternative to wP and aP vaccines.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Construction of pcDNA3.1D/ptS1.13L.129G and pTrcHis2/ptS1.13L.129G plasmids. The gene sequence encoding the S1 domain of PT, referred to as ptS1, was amplified from B. pertussis genomic DNA by PCR using Platinum Pfx DNA polymerase (Invitrogen) to generate a blunt-ended product or Platinum Taq High Fidelity DNA polymerase (Invitrogen) for A-tailed products. Using the published sequence of the ptx gene (GenBank accession no. M13223), the primers 5'-CACCATGCGTTGCACTCGGGCAATTCGC-3' and 5'-GAACGAATACGCGATGCTTTCGTAGTACAC-3' (GeneWorks) were designed to amplify from nt 507 to 1313. Genomic DNA was extracted from B. pertussis Tohama I using an AquaPure DNA isolation kit (Bio-Rad) according to the manufacturer's instructions. PCR products were cloned into the eukaryotic expression vector pcDNA3.1D/V5-His-TOPO (Invitrogen) to evaluate expression in mammalian cells in vitro and for DNA vaccination in vivo. The ptS1 gene was also cloned into the prokaryotic expression vector pTrcHis2-TOPO (Invitrogen) for purification of recombinant S1. To generate this construct, the ptS1 gene was amplified by PCR with the sense primer 5'-ATGCGTTGCACTCGGGCAATTCGC-3' and the antisense primer 5'-GAACGAATACGCGATGCTTTCGTAGTA-3', and ligated into the pTrcHis2-TOPO vector.
The ADP-ribosyltransferase activity of the recombinant S1 (rPTS1) encoded by pTrcHis2/pts1 and pcDNA3.1/ptS1 was inactivated by two point mutations in which Arg-13 was changed to Leu-13 and Glu-129 was changed to Gly-129 (Pizza et al., 1989). Site-directed mutagenesis was carried out using a QuikChange XL mutagenesis kit (Stratagene) according to the manufacturer's instructions. Primers used for introduction of the R13L mutation were 5'-TACCGCTATGACTCCCTGCCGCCGGAGGAC-3' and 5'-GAAAACGTCCTCCGGCGGCAGGGAGTCATAGCGGTA-3', and for the E129G mutation were 5'-GCCACCTACCAGAGCGGCTATCTGGCACACCGG-3' and 5'-CCGGTGTGCCAGATAGCCGCTCTGGTAGGTGGC-3'.
Expression of recombinant S1 antigens in COS-7 cells. COS-7 cells were transfected with pcDNA3.1/ptS1, pcDNA3.1/ptS1.13L.129G or pcDNA3.1 vector (negative control) using Lipofectamine 2000 (Invitrogen) as described previously (Chen et al., 2006). Expression of rPTS1 and rPTS1.13L.129G was detected by Western blotting with an alkaline phosphatase-labelled anti-V5 antibody (Invitrogen) specific for the V5 epitope, which was fused to the C terminus of the recombinant S1.
CHO cell assay. To verify inactivation of rPTS1.13L.129G, a CHO cell assay was performed according to the method of Castro et al. (2001) with some modifications. CHO-K1 cells were transfected with pcDNA3.1/ptS1, pcDNA3.1/ptS1.13L or pcDNA3.1/ptS1.13L.129G using Lipofectamine 2000 as described for the transfection of COS-7 cells. As a positive control, CHO-K1 cells were treated with 800 ng commercially purified PT (List Biologicals) suspended in 0.5 ml Opti-MEM (Invitrogen). As negative controls, CHO-K1 cells were incubated with pcDNA3.1D/ptS1, Lipofectamine 2000 or Opti-MEM only. The morphology of cells in the test and control wells was recorded after 20 h at 37 °C with 5 % CO2, with clustering used as an indicator of intoxication.
Expression of rPTS1.13L.129G in E. coli and purification by immobilized metal affinity chromatography (IMAC). His-tagged rPTS1.13L.129G was purified by IMAC using Ni-NTA resin (Qiagen) as described previously (Chen et al., 2006). A 100 ml culture of XL10-Gold E. coli harbouring the pTrcHis2/ptS1.13L.129G plasmid was grown in LB broth to OD600 0.7 and then induced with 1 mM IPTG for 4 h at 37 °C with shaking at 200 r.p.m. As a positive control, XL10-Gold E. coli transformed with the pTrcHis2/lacZ plasmid (Invitrogen), encoding β-galactosidase, was co-induced under identical conditions. Expression of rPTS1.13L.129G and β-galactosidase was detected by Western blotting with a horseradish peroxidase (HRP)-labelled anti-His-tag antibody (Invitrogen).
For purification under denaturing conditions, cells were pelleted at 12 500 g for 5 min, resuspended in 6 M guanidine hydrochloride, 100 mM NaH2PO4, 10 mM Tris/HCl (pH 8) and then incubated at room temperature for 1 h with shaking at 60 r.p.m. The lysate was sonicated for five cycles of 30 s (60 % duty and maximum output) at 30 s intervals using a Branson 250 Sonifier followed by centrifugation at 10 000 g for 25 min. Ni-NTA agarose (1 ml) was added to the clarified lysate and incubated for 40 min at room temperature. The slurry was loaded onto a disposable column and the matrix washed with eight bed volumes 8 M urea, 100 mM NaH2PO4, 10 mM Tris/HCl (pH 6.3) and then eluted with two bed volumes 8 M urea, 100 mM NaH2PO4, 10 mM Tris/HCl (pH 5.9), followed by two bed volumes 8 M urea, 100 mM NaH2PO4, 10 mM Tris/HCl (pH 4.5). The purity of rPTS1.13L.129G was checked by SDS-PAGE according to the method of Laemmli (1970) and the yield determined using a Coomassie Plus protein assay reagent (Pierce) according to the manufacturer's instructions.
Vaccination of mice. Six-week-old female BALB/c mice were purchased from the Animal Resources Centre (Canning Vale, Western Australia). Mice were housed in a charcoal- and HEPA-filtered isolation cabinet (Techno-Plas) and fed pellet food and water ad libitum. Four groups [pcDNA3.1/ptS1.13L.129G, pcDNA3.1 vector, diphtheria–tetanus–acellular pertussis (DTaP) and a placebo] of 17 mice were given three doses at 3 week intervals. Two weeks after the final booster, five mice within each treatment group were sacrificed to determine the immunogenicity of the vaccines and controls at the time of challenge. The remaining 12 mice per group were challenged with virulent B. pertussis. DNA vaccine test doses consisted of 100 µg plasmid DNA in 100 µl PBS. Self-ligated pcDNA3.1 vector and PBS were used as negative controls and 0.2 standard human dose of the commercial DTaP vaccine (Infanrix) was included as a positive control. Each dose was administered via the intramuscular (i.m.) route with a 29-gauge needle (50 µg injected into each quadricep) with the exception of the DTaP vaccine, which was administered via the subcutaneous (s.c.) route. Prior to i.m. or s.c. injection, mice were anaesthetised with an intraperitoneal (i.p.) dose of 80 mg ketamine kg–1 and 16 mg xylazine kg–1. The experiment was performed in compliance with the Animal Care and Protection Act 2001 and was approved by the Animal Ethics Committee of the University of Southern Queensland.
Analysis of serum antibody responses. The titres of anti-PT and anti-rPTS1.13L.129G IgG antibodies in mouse sera were measured by ELISA as described previously (Chen et al., 2006), with the following modifications. Nunc MaxiSorp plates were coated with 5 µg PT ml–1 (Seikagaku) or 5 µg rPTS1.13L.129G ml–1. End-point titres were determined as the reciprocal of the highest dilution at a cut-off point 3 SD above the mean absorbance of sera from placebo-immunized mice and presented as mean titres±SEM. In the event that the highest dilution of test serum exceeded the cut-off value, regression was used to estimate the end-point titre.
Estimation of cytokine levels.
Splenocytes from each group were pooled and resuspended in Dulbecco's modified Eagle's medium (Gibco) containing 100 µg penicillin ml–1, 100 µg streptomycin ml–1, 10 % fetal bovine serum and 50 µM 2-mercaptoethanol, and seeded at 5x106 cells per well. Cells were stimulated in vitro with 5 µg rPTS1.13L.129G ml–1, 5 µg heat-killed B. pertussis ml–1 or 2.5 µg concanavalin A (ConA) ml–1. Supernatants were removed after 24 h incubation at 37 °C with 5 % CO2 to determine interleukin (IL)-2 production and after 72 h for gamma interferon (IFN-) and IL-4 measurements. The levels of the respective cytokines were determined using a mouse sandwich ELISA (Pierce) according to the manufacturer's instructions.
Aerosol challenge with B. pertussis. Mice were exposed to a sublethal infectious dose of virulent B. pertussis by aerosol challenge based on the method of Xing et al. (1999). B. pertussis Tohama I phase I were grown on BG agar for 4 days at 37 °C. Bacteria were then cultured in SSH broth at 37 °C and 150 r.p.m. until the OD625 reached 0.5. Cells were pelleted at 2500 g for 10 min and, based on a predetermined growth curve, were resuspended to a concentration of 1x1010 c.f.u. ml–1 in 1 % (w/v) casein (Difco). Mice were exposed to aerosols from a suspension of B. pertussis Tohama I for 10 min, which was previously determined to deliver a sublethal infectious dose of 5.7x105 (±0.1) c.f.u. per lung. Four mice per group were sacrificed at days 4, 7 and 14 post-challenge to determine the number of B. pertussis in the lungs. Lungs were removed aseptically and homogenized in 1 ml 1 % casein on ice. Serial dilutions of each homogenate were spotted in quadruplicate onto BG agar and incubated at 37 °C for 4 days. Bacterial counts were expressed as mean c.f.u. per lung±SEM.
Statistical analysis of data.
Significant differences in antibody titres and clearance data were identified using a paired t-test or analysis of variance for multiple comparisons. Differences between groups were considered significant if P
0.05.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). A dominant band of 30 kDa was observed and corresponded well to the calculated molecular mass of rPTS1.13L.129G, as determined from the amino acid sequence (VectorNTI; Invitrogen). When constitutively expressed in COS-7 cells following transfection with pcDNA3.1D/pts1.13L.129G, rPTS1.13L.129G had a molecular mass that was equivalent to rPTS1.13L.129G expressed and purified from E. coli (Fig. 1b). Following expression in the mammalian cell line COS-7, rPTS1.13L.129G appeared as a doublet, indicating that there were two isoforms of the recombinant protein, which are likely to have occurred through atypical post-translational modification. The level of rPTS1.13L.129G expression in XL10-Gold E. coli was found to be sufficient for purification by IMAC (Fig. 1c).
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), CHO-K1 cells transfected with pcDNA3.1D/ptS1 (endogenous expression of the non-mutated rPTS1 protein) developed a clustered appearance within 12 h (Fig. 2a). This clustered morphology was also observed for the cells treated with wild-type PT (Fig. 2d). Overall, the clustering appeared to be slightly more pronounced and compact in the PT treatment group compared with cells transfected with pcDNA3.1D/ptS1. Endogenous expression of rPTS1.13L.129G following transfection of CHO cells with pcDNA3.1D/ptS1.13L.129G had little or no effect on the cell morphology (Fig. 2c) and the cells were similar in appearance to untreated cells. The cumulative effect of two point mutations versus a single amino acid substitution was tested by including a group of CHO cells transfected with pcDNA3.1D/ptS1.13L for the endogenous expression of rPTS1.13L (Fig. 2b). Whilst there appeared to be a low level of clustering that was not present in the pcDNA3.1D/ptS1.13L.129G treatment group, the clustering was less obvious than with cells transfected with pcDNA3.1D/ptS1 (endogenous expression of rPTS1) or treated with native PT. Castro et al. (2001) also observed clustering in CHO cells that expressed the wild-type S1 and a normal CHO cell morphology after endogenous expression of a 9K/129G mutant. The results of our modified CHO cell assay were consistent with those of Castro et al. (2001) as rPTS1.13L.129G had no apparent ADP-ribosylase activity but cells transfected with the non-mutated S1 construct (pcDNA3.1D/pts1) showed a rounded and clustered morphology that was comparable with exposure of cells to native PT.
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when cultured in the presence of either rPTS1.13L.129G or ConA, with 5253 and 6000 pg ml–1, respectively (Fig. 3). When stimulated with a heat-killed B. pertussis lysate, these cells produced less IFN- (1629 pg ml–1). Splenocytes from vector- and DTaP-immunized mice produced a low level of IFN- when stimulated with rPTS1.13L.129G or the B. pertussis lysate (Fig. 3). Splenocytes from mice immunized with pcDNA3.1D/ptS1.13L.129G, vector and the DTaP vaccine all produced low levels of IL-2 when stimulated with rPTS1.13L.129G (Fig. 4). Moreover, there was virtually no IL-2 detected when the splenocytes from mice immunized with pcDNA3.1D/ptS1.13L.129G were stimulated with a heat-killed B. pertussis lysate. In contrast, a large amount of IL-2 (12.3 ng ml–1) was produced when these same cells were stimulated with ConA (Fig. 4). In mice vaccinated with pcDNA3.1D/ptS1.13L.129G, there was little IL-4 produced when stimulated with rPTS1.13L.129G (15.3 pg ml–1); in contrast, there was a relatively large amount of IL-4 produced by these splenocytes following stimulation with ConA (468.2 pg ml–1) (data not shown). Mice vaccinated with pcDNA3.1D vector produced equivalent levels of IL-4 to mice given the DNA vaccine (12.8 pg ml–1).
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Based on both human and animal studies of immunity against B. pertussis, PT-neutralizing IgG1 antibodies are believed to be an important protective mechanism (Sato et al., 1984; Halperin et al., 1991; Schneerson et al., 1996), albeit in the early phases of infection. Kamachi et al. (2003) reported that i.d. immunization of mice with an S1 DNA vaccine generated significant IgG1 titres that protected against both the toxic effects of PT and a lethal i.n. challenge with virulent B. pertussis. In our investigation involving DNA vaccination by the i.m. route, no IgG could be detected. It was thus apparent that unless strategies to induce a Th2-mediated humoral immune response, for example by simultaneous immunization with the DNA vaccine by both the i.m. and i.d. routes or the co-administration of purified antigen, are investigated, the lack of toxin-neutralizing IgG in the serum would be a limitation of this vaccine.
Protective efficacy of pcDNA3.1/ptS1.13L.129G injected via the i.m. route
As a measure of protective efficacy, bacterial numbers from the lungs of challenged mice were recorded at days 4, 7 and 14 post-challenge. The results of the challenge experiment showed that mice immunized with pcDNA3.1D/ptS1.13L.129G cleared the B. pertussis infection more effectively than mice given the placebo or vector only, but not when compared with the DTaP vaccine, which cleared the infection at a significantly faster rate than the DNA vaccine (Fig. 5). At day 4 post-challenge, mice vaccinated with either pcDNA3.1D/ptS1.13L.129G, the placebo or vector only showed a twofold to fourfold increase in bacterial numbers compared with day 0 counts. By day 7, the DNA-vaccinated mice showed a 10-fold reduction in numbers followed by a more than 6000-fold reduction after 2 weeks, whereas the negative-control mice failed to show any reduction in the c.f.u. counts. The clearance data showed that bacterial counts in mice immunized with pcDNA3.1D/ptS1.13L.129G were reduced to 1.46 % of the placebo controls at day 7 post-challenge and then to 0.001 % at day 14 post-challenge. Three of the four mice immunized with the DNA vaccine failed to clear the B. pertussis infection completely within 14 days of challenge. In contrast, mice immunized with the DTaP vaccine demonstrated a more rapid and complete clearance with no bacteria detected in the lungs at day 7 post-challenge, with a significantly improved rate of clearance compared with mice given the DNA vaccine, vector only or placebo (P<0.05) (Fig. 5).
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produced from splenocytes restimulated with rPTS1.13L.129G. In fact, levels of this cytokine approached that following stimulation with ConA, a natural and potent T-cell mitogen. No IgG to rPTS1.13L.129G or native PT could be detected in the sera of mice immunized with our parenteral S1 DNA vaccine. Although there has been relatively little insight into the mechanisms of DNA uptake and processing of endogenously expressed antigen, it has become apparent that the mode of delivery of DNA vaccines is a major influence on whether a humoral or cellular response is generated. Most DNA vaccines administered via the i.m. route have induced a Th1-biased response, whereas DNA delivered via the i.d. route by gene gun typically directs a Th2-type response (Donnelly et al., 1997; Klinman et al., 1997; Gurunathan et al., 2000). Interestingly, Redhead et al. (1993) found that although high serum antibody levels resulted in an earlier decline in the numbers of B. pertussis recovered from the lungs of challenged mice, complete clearance was dependent on CMI. Also, the dichotomous albeit slightly Th1-biased response to wP vaccines has been shown to produce a greater reduction in bacteria compared with the purely Th2 response induced by acellular vaccines (Mills et al., 1998). From the results of this study, we believe that a composite vaccine, in which purified toxoid is either co-administered or used as a booster for DNA vaccination, may be more efficacious against B. pertussis than immunization with purified antigen or DNA alone. It will be interesting to determine whether immunization with a combination DNA–aP vaccine will generate both antibody and CMI responses and protect mice against challenge with B. pertussis.
In conclusion, we have shown that DNA vaccination of mice with the S1 subunit of PT can enhance the rate of clearance of a B. pertussis infection through the induction of a Th1 immune response. Although the S1 DNA vaccine was not as efficacious in clearing the aerosol challenge as the DTaP vaccine, the result was nevertheless encouraging considering it involved the coverage of only one of the virulence antigens of B. pertussis and the absence of any adjuvant.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, pcDNA3.1D/ptS1.13L.129G; 
, pcDNA3.1D; 
, placebo; 
, DTaP.