Mice immunized with DNA encoding a modified Pseudomonas aeruginosa exotoxin A develop protective immunity against exotoxin intoxication

Vaccine 19 (2001) 1106 – 1112 www.elsevier.com/locate/vaccine

Mice immunized with DNA encoding a modified Pseudomonas aeruginosa exotoxin A develop protective immunity against exotoxin intoxication Jen-Wen Shiau a,b, Tswen-Kei Tang b, Yung-Luen Shih c, Chein Tai d, Yung-Yi Sung e, Jau-Lan Huang f, Huey-Lang Yang b,d,* a

Department of Animal Physiology, Taiwan Li6estock Research Institute, Hsinhau, Tainan, Taiwan b Institute of BioAgricultural Science, Academia Sinica, Taipei, Taiwan c Department of Medical Technology, China Medical College, Taichang, Taiwan d Institute of Biotechnology, National Cheng-Kung Uni6ersity, Tainan, Taiwan e Department of Animal Production, National Taiwan Uni6ersity, Taipei, Taiwan f Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Received 8 May 2000; received in revised form 2 August 2000; accepted 29 August 2000

Abstract A recombinant plasmid, which contains the Pseudomonas aeruginosa exotoxin A (PE) gene with a C-terminal deletion, was inserted into expression vector pSecTag Xpress. The expression of this bacterial exotoxin in an animal cell was first demonstrated in 3T3 cell by transient transfection and western blot assay. Recombinant plasmid DNA was then injected intramuscularly to BALB/c mice, anti-PE specific antibodies were found in all animals vaccinated with plasmid containing the PE gene and with ‘detoxicated’ recombinant PE protein. Mice vaccinated with DNA were protected from the intoxication of lethal dosage of P. aeruginosa exotoxin A. Our results indicated that mice vaccinated with DNA encoding the PE gene could express PE protein in vivo, induced specific immune response, and provided sufficient protective immunity that safeguarded mice from the injection of lethal dosage of PE toxin. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: DNA vaccine; Pseudomonas aeruginosa exotoxin A (PE); Toxin intoxication

1. Introduction Pseudomonas aeruginosa is an opportunistic pathogen, which often causes infection in immunocompromised patients, burn and wound patients or animals that have their skin barrier breached. The toxicity of P. aeruginosa is caused primarily by the exotoxin A (PE), an ADP-ribosyl transferase [1]. Recent developments on vaccine research have demonstrated the feasibility of inoculation of DNA for vaccination [2]. This invention was built on the observations that nucleic acid, after intramuscular injection in muscle cells, could induce immune response [3]. This * Corresponding author. Present address: Institute of BioAgricultural Science, Academia Sinica, Nankang, Taipei 11529, Taiwan. Tel.: +886-2-2785-5696, Ext. 8020; fax: +886-2-2651-5120. E-mail address: [email protected] (H.-L. Yang).

novel vaccination route has then commenced a unique vaccine delivery system [4,5]. Subsequently, numerous DNA vaccine researchers employing intramuscular injection have reported success in various animal models ranging from fish [6], rodent [7], rabbit [8], cattle [9], chicken [10], and primate [11]. Similar to the development of subunit vaccine, the efficacy of DNA vaccine still needs improvement. One approach undertaken is the coupling of specific interleukin genes that has been proven effective in stimulating an immune response [12]. Alternatively, several bacterial toxins, such as tetanus and cholera toxins that have been demonstrated to be effective as ‘biological adjuvant’, have been employed in the subunit vaccine research to enhance the immune response [13–15]. The ability of bacterial toxin to induce immune response has been demonstrated with Clostridium tetanus toxin and

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Vibrio cholera toxin [16,17]. Consequently, these bacterial toxins could also be used as ‘biological adjuvant’ to stimulate immune response in DNA delivery vaccine system, providing that the bacterial toxin gene would be expressed in eukaryotic cells. Pseudomonas exotoxin A (PE) is a very unique bacterial toxin; it has several features that could be useful as a biological adjuvant for DNA vaccines. The Pseudomonas exotoxin A consists of a three-domain structure. Domain I, the amino-terminal of PE toxin, has a specific cell binding ability to eukaryotic cells [18]. Domain II accounts for the ability to translocate toxin across cell membranes [19,20]. The carboxyl-terminal domain III catalyzes the ADP-ribosylation activity that has the major cytotoxic function [21]. Both domains I and II are also essential for the toxicity and the cytotoxic effect is active broadly in almost every eukaryotic cell [21–23] indicating that domain I and II could also function in various eukaryotic cells. Our laboratory and others have indicated that PE protein can stimulate immune response with a heterologous antigen fused onto it. The immuno-stimulator effect of Pseudomonas exotoxin A has been demonstrated in subunit vaccines of HIV and found it also can stimulate mucosal immunity [15,24]. Our laboratory has observed that PE and gonadotropin release hormone conjugate could induce sufficient amount specific antibody and inhibited reproductive functions in animals. To utilize the ‘adjuvant’ effects of Pseudomonas exotoxin A in DNA vaccines, one has to assure that this bacterial protein can be expressed in animal cell. In term of deveoping protein vaccine for P. aeruginosa, recombinant PE protein has been used to vaccinate mice, and it has shown some degree of protection from P. aeruginosa infection [25,26]. Here we reported the success of vaccination with a recombinant plasmid that contains a truncated non-cytotoxic PE gene. The plasmid was inoculated directly into BALB/c mice muscle cells, the encoded PE gene expressed in mice, and induced sufficient amount of specific immune response that effectively protected mice from the intoxication caused by Pseudomonas exotoxin A.

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peptide, a C-terminal histidine sequence and a myctagged DNA sequence fused in frame proximal to the PE gene (Fig. 1). Therefore, in this construct, the PE transcript was fused downstream and was put under the control of human CMV immediate-early promoter/enhancer. The recombinant plasmid was transformed into Escherichia coli strain DH5a. Plasmid DNA of the transformants was then isolated and confirmed by restriction mapping and nucleotide sequence analysis. The correct transformant was incubated in LuriaBertani (LB) broth with shaking in an orbital shaker at 37°C for overnight. Bacterial cells were harvested when the cell density reached O.D.600 of 0.6. The plasmid DNA was purified following the method provided by the manufacturer of Plasmid Giga kit (QIAGEN). The concentration of plasmid DNA was determined by measuring the O.D.260 and the quality of DNA was analyzed by comparing the ratio of O.D.260/O.D.280.

2.2. Vaccination of plasmid DNA and subunit protein

2. Materials and methods

Six- to eight-week-old female BALB/c mice were purchased from the Animal Center of the National Taiwan University Hospital. These mice were divided randomly into four experimental groups, with five to six mice in each group. The first group (group I) was a negative control in which the mice were injected subcutaneously with 100 ml of phosphate-buffered saline (PBS). The second group (group II) was a positive control in which the mice were injected subcutaneously with 100 ml of vaccine containing 25 mg of ‘detoxicated’ PE protein synthesized and isolated from recombinant E. coli. The recombinant protein was emulsified in Complete Freund’s Adjuvant (CFA, Pierce) before injection. Mice in group III were injected with 100 mg of pSecTagC-PE DNA in 100 ml PBS. Mice in group IV were vaccinated the same way as those mice in group III except 50 ml of 25% sucrose was injected at the same site 15 min prior to the injection of plasmid DNA. DNA used for vaccination was prepared with 100 mg of plasmid DNA dissolved in 100 ml of PBS, and was injected intramuscularly with a No. 31-gauge needle into the right hind thigh (quadriceps muscles). Within the vaccinated groups, all animals were vaccinated five times at 2 weeks intervals. Sera were collected from the retro-orbital plexus 7 days after inoculation, pooled and were used to measure anti-PE specific antibody.

2.1. Construction and preparation of plasmid DNA

2.3. Preparation of ‘detoxicated’ PE toxin

A 1.66 Kb HindIII-Xhol DNA fragment encoding the 2–552 amino acid residues of the PE gene [22] was cloned into the eukaryotic expression vector, pSecTag Xpress (Invitrogene, Netherlands). The resulting recombinant plasmid (pSecTagC-PE) contained an N-terminal immunoglobulin k (kappa) light chain signal

Detoxicated PE toxin was purified from E. coli recombinant cell harboring plasmid containing a truncated PE gene that has the C-terminal nucleotide sequence coding for 533 a.a to 613 a.a deleted [22]. The detoxicated PE protein was expressed and purified as inclusion body from E. coli, and dissolved with the

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Protein Refolding Kit (Novagen). The dissolved PE proteins were dialyzed and diluted in 20 mM Tris (pH 8.5) before used for injection. The safety of the detoxicated PE protein was evaluated by intramuscular injection of 50 mg recombinant protein per mouse (50 times LD50). It was found that the test mice did not have any noticeable morphological, histological abnormality and no mortality was observed.

2.4. Assay of the expression of detoxicated PE gene by transient trasfection BALB/c mice 3T3 cells (embryo fibroblast, ATCC CCL-163) were grown in 12-well plates at 37°C with Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS). When the cells reached approximately 50% confluent, they were transfected with different doses of purified pSecTagC-PE DNA using Lipofectamine 2000 (Gibco BRL), and

were incubated at 37°C in a CO2 incubator. We found that the optimal dosage of Lipofectamine 2000 for transfection was around 3 ml in our experimental condition. Increasing the amount of Lipofectamine 2000 to 6 ml did not help the efficiency of transfection. The culture medium was removed and the transfected cells were detached by adding 1 ml of trypsin-EDTA solution after incubation for 2 days, cells were collected in 1.5 ml microcentrifuge tubes and were then centrifuged at 3000× g for 10 min. The cell pellet were resuspended in 100 ml of lysis buffer (20 mM Tris–HCl at pH 7.0, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF and 1 mM sodium orthovanadate) and kept on ice for 10 min. The cell lysates were collected and then sonicated on ice by Sonicator (model W-380, Ultrasonics) three times with a 10 s sonication at 50% power output, followed by centrifugation at 15 000 × g for 20 min at 4°C to remove cell

Fig. 1. The map of ‘detoxicated’ PE protein (A) and the construct of plasmid pSecTagC-PE plasmid (B). Panel A shows the 1.66 Kb HindIII-Xhol DNA fragment encoding 2–532 amino acid residues of PE. Panel B is a map of pSecTagC-PE constructed by inserting the 1.66 Kb fragment into eukaryotic expression vector, pSecTagC. The resulting plasmid (pSecTagC-PE) has this portion of PE gene fused downstream to and under the control of human CMV immediate-early promoter and enhancer sequences.

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debris. The protein concentration of the supernatant was determined by using the BCA protein assay kit obtained from Pierce, Rockford, IL. Two micrograms of cell extracts were analyzed on a 10% SDS-PAGE followed by western blot analysis. The rabbit anti-PE antibody was prepared and conjugated with alkaline phosphatase in our laboratory, and was used in all immuno-reaction.

2.5. Measurement of Anti-PE antibody by the enzyme-linked immunosorbant assay (ELISA) Microtiter plates (Nunc, 467466) were coated with recombinant ‘detoxicated’ PE protein (10 mg/ml in 0.05 M carbonate/bicarbonate coating buffer; pH 9.6, 100 ml per well) and incubated overnight at 4°C. After washing with PBS/T (0.05% Tween-20 in PBS, pH 7.4), the plates were blocked with 0.5% BSA in PBS and incubated overnight at 4°C, and stored at 4°C until use. Three days after the injection, serum samples were drawn from each animal. Serum samples of the same group were pooled, and diluted 1:1000 with 0.5% BSA in PBS before assay. Test was performed by adding the diluted serum to the PE protein coated micro-wells and incubated at room temperature for 2 h. These plates were washed four times with PBS/T and incubated with alkaline phosphate-conjugated goat anti-mouse IgG (Sigma, diluted 1:10 000 with 0.5% BSA in PBS) for 2 h at room temperature. Plates were then washed four times with PBS/T, followed by the addition of chromogen (10 mg p-nitrophenyl phosphate dissolved in 10 ml diethanolamine buffer with 0.5 mM MgCl2, pH at 9.1). Thirty minutes after the addition of the substrates, the reaction was stopped with 100 ml of 2.5 N NaOH and result was measured at the absorbency of 405 nm using a Multiscan MCC ELISA reader (Flow Laboratories, VA).

2.6. E6aluation of the effect of 6accination by intoxication with nati6e PE toxin To determine the efficacy of PE-DNA vaccination, mice were injected intraperitoneally with 1 mg of native Pseudomonas exotoxin A (Sigma, isolated from P. aeruginosa, supplemented with 1.0 mg/ml bovine serum albumin and dissolved in sterile saline) 8 weeks after the last vaccination. The physiological conditions of the mice in each group after exotoxin intoxication were carefully observed and recorded every day. The injection of native Pseudomonas exotoxin A usually killed mice within 4 days, therefore, individual mice surviving 7 days after toxin injection was considered was protected by vaccination.

Fig. 2. The expression of PE gene in 3T3 cell. Lane M is the molecular weight marker. Lane 1 was a control lane loaded with cell extracts from 3T3 cells transfected only with Lipofectamine 2000 in concentration of 3 ml. Lane 2 was loaded with cell extracts of 3T3 cells transfected with 3 mg of pSecTagC-PE plasmid DNA together with 3 ml of Lipofectamine. Lane 3 was loaded with cell extracts of 3T3 cells transfected with 5 mg of pSecTagC-PE plasmid DNA together with 3 ml of Lipofectamine.

3. Results

3.1. Plasmid construction and expression in 6i6o The PE gene coding for P. aeruginoas exotoxin A amino acid residues 2–532, was demonstrated to be ‘detoxicated’ and biologically safe, was inserted on the eukaryotic expression plasmid pSecTagC-PE (Fig. 1). After purification, the supercoiled form of plasmid DNA was diluted with PBS and injected directly into mouse muscles.

3.2. Transient expression of PE gene in eukaryotic cell Before injecting PE DNA into mice, the ability of the PE gene to express in eukaryotic cells was analyzed by transient transfection using a mouse 3T3 cell line as host cells. As shown in Fig. 2, the expression of PE was confirmed by western blot analysis using cell lysates prepared from the pSecTagC-PE DNA transfected 3T3 cells (Fig. 2).

3.3. Humoral immune response to DNA 6accine The sera antibody specific to PE was assayed by ELISA to assess the ability of direct PE-DNA inoculation in eliciting immune response. As shown in Fig. 3, vaccination with PE-DNA did produce significant amount of anti-PE antibody. No PE-specific antibodies were detected in mice in the negative control group that were injected with PBS alone. We also found that injection of E. coli synthesized ‘detoxicated’ PE protein did induce higher antibody

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Fig. 3. Humoral immune response after vaccination with pSecTagC-PE plasmid DNA. Control group was injected with 100 ml PBS (-"-) or with 25 mg of ‘detoxicated’ PE protein (- -, mixed with CFA). 100 mg of DNA was injected into the quadriceps muscles in the right hind thigh whether pre-treatment with (- ×-) and without (--) 100 ml of 25% hypertonic sucrose 15 min prior to injection. (¡) indicated the time of vaccination. Sera were drawn from every mouse of each group 7 days after each vaccination; sera were pooled and diluted 1000 fold with PBS, and assayed with the ELISA method.

titers than those from the DNA vaccinated mice. Nevertheless, both the protein and the DNA antigens have produced sufficient amount of protective antibody to safeguard mice from exotoxin intoxication. In spite of several reports on the effect of pretreated the muscle with hypertonic sucrose injection to enhance the effect of DNA vaccination [11,27,28], we did not observe a similar enhancement effect in our experiments (Fig. 3).

3.4. Assay for the protection from exotoxin A intoxication To determine the efficacy of PE-DNA vaccination, we have injected vaccinated mice with native P. aeruginosa exotoxin A to evaluate the efficacy of DNA vaccination by toxin intoxication. Mice from control and vaccinated groups were injected 8 weeks after the last immunization with 1 mg native Pseudomonas toxin intraperitoneally that has been previously determined as

the minimal amount to cause a 100% mortality in mice. The result of survival rate to toxin intoxication test in each group of mice was shown in Table 1. Three mice in the control group (group 1) that were given PBS only were killed within 24 h of toxin intoxication. Mice Table 1 Survival rates of immunized mice after injection of native P. aeruginosa exotoxin A intoxicationa Treatment

Death after intoxication c animal death/c animal tested

1 2

PBS control PE protein injected

3/3 0/6

3 4

DNA injected PPE pPE (Sucrose pretreatment)

0/5 0/5

BALB/c mice were injected intraperitoneally with 1 mg/ml of native P. aeruginosa Exotoxin A (Sigma) contained in 1.0 ml sterile PBS with 10 mg/ml bovine serum albumin. a

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vaccinated with ‘detoxicated’ PE toxin (group 2, subunit vaccine vaccinated group) and with plasmid DNA encoding PE gene (group 3 and 4) were found that they were totally protected from Pseudomonas exotoxin A intoxication. We have also observed that the vaccinated group were protected even at 3 mg/mouse of Pseudomonas exotoxin A.

4. Discussion Gene expression in muscle cells following injection of plasmid DNA has been demonstrated successfully in experiments using some reporter genes [29], and proved to be an effective route of immunization [2 – 11,30]. In this study, we demonstrated that mice immunized with a recombinant DNA encoding Pseudomonas exotoxin could elicit sufficient immune response that could protect the animal from the lethal assault of native P. aeruginosa exotoxin A. In the comparison of the mouse group immunized with E. coli produced ‘‘detoxicated’’ PE (a subunit vaccine) and with PE-DNA vaccine, the titer of anti-PE antibody is always higher in the former one. Since we did not perform the titration experiment by using different amount of antigen in these vaccination routes, and both vaccination methods could produce sufficient protection from toxin induced death, we cannot elucidate the significance of these difference. Regarding to the number of DNA vaccination, we have vaccinated the mice with five consecutive DNA inoculations, but from the linear increasing of specific antibody as shown in Fig. 3, we anticipated that DNA vaccination twice could be sufficient to produce protective immunity. In addition, the optimization of the amounts of DNA and the selection of proper adjuvant could also decrease the number of vaccination. We also evaluated the enhancement effect of hypertonic sucrose pretreatment in DNA inoculation as reported [11,27,28]. In our study, it appears that sucrose pretreatment did not improve humoral immune response against Pseudomonas exotoxin A, as antibodies titer was not elevated (Fig. 3). It could be that PE protein itself has sufficiently enhanced the immune response. The potential enhancement mechanism of the Pseudomonas exotoxin A protein in DNA vaccine remained to be determined. We are currently investigating the different roles of each domain of Pseudomonas exotoxin A in its individual ability to increase the efficacy in DNA vaccine. Previous results using recombinant ‘‘detoxicated’’ PE toxin from E. coli to vaccinate mice has shown be effective but failed to totally protect the vaccinated animals from infection of P. aeruginosa [26]. It might due to the improper folding of PE protein in E. coil expression system. With the DNA delivery system, the

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PE antigen is expressed and synthesized in the animal cells, it might have a different outcome. We are currently testing the efficacy of PE-DNA vaccine against P. aeruginosa infection using the subcutaneously injection [25], intraperitoneal infection and in burn mouse model [31]. From the exotoxin A intoxication assay, we found that vaccinated mice survived from injection of lethal dosage of 1 mg/mouse. The maximum tolerant concentration of exotoxin A tried in our test in vaccinated groups was 3 mg/ml. Since P. aeruginosa infection is common in immuno-compromised patients and is a serious death-causative agent in domestic animals. This observation suggested that the exotoxin A DNA vaccine could be promising for clinical and veterinary application. In conclusion, we have demonstrated the expression of the Pseudomonas exotoxin A gene in mouse via DNA vaccination, and it could induce protective humoral immunity. In addition, the successful of expression of Pseudomonas exotoxin A gene in animal might open a new avenue for applying Pseudomonas exotoxin A as ‘biological adjuvant’ to enhance the immune response in nucleic acid vaccination.

Acknowledgements The authors like to thank Dr Mi-Hua Tao of the Institute of BioMedical Science, Academic Sinica for many useful suggestions, and Dr J. Palmy Rose Rajan for proof reading of this manuscript.

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