Protection of immunosuppressed mice against translocation of Pseudomonas aeruginosa from the gut by oral immunization with recombinant Pseudomonas aeruginosa outer membrane protein I expressing Salmonella dublin

Protection of immunosuppressed mice against translocation of Pseudomonas aeruoinosa from the gut by oral immunization with recombinant Pseudomonas aeruoinosa outer membrane protein I expressing Salmonella dublin A n d o r T o t h * , F l o r i a n S c h 6 d e l *~, M i c h a e l D u c h e n e ,it, K y r o s M a s s a r r a t * ' , , B a r b a r a B l u m ~, A n j a Schmitt*, H o r s t D o m d e y * a n d B e r n d - U l r i c h v o n S p e c h t *.~

AroA Salmonella dublin was used as recipient for a plasmid coding for the outer membrane protein I (OprI) of P s e u d o m o n a s aeruginosa. Oral immunization of Balb/c mice with recombinant S. dublin induced serum lgG and IgA antibodies against P. aeruginosa. In spleen and Peyer's patches anti-P, aeruginosa IgG- and IgA-secreting cells could be measured by the E L I S P O T technique. In an oral challenge of immunosuppressed mice with P. aeruginosa the orally immunized animals had a 58-fold higher LDso than control animals'. Keywords: Pseudomonasaeruginosa;outer membrane protein; Salmonelladublin

Translocation of Pseudomonas aeruginosa from the gut is thought to be a major cause of systemic infections in neutropenic patients with haematological malignancies 1 s. In humans and other mammals, the predominant immunoglobulin in external secretions is secretory IgA, which is a first line of defence of mucosal surfaces against microbial pathogens 6'~. However, parenteral vaccination against P. aeruginosa leads predominantly to the induction of IgG and IgM antibodies 8. In the past few years, several methods have been tested for the induction of specific secretory IgA antibodies in mucosal secretions 6,7'9 12. Among other methods tested, *Chirurgische Forschung, Chirurgische Universit~.tskiinik, Hugstetterstrasse 55, D-79106 Freiburg im Breisgau, Germany. tMax-Planck-lnstitut for Biochemie, 82152 Martinsried, Germany. tLaboratorium for Molekulare Biologie, Genzentrum der LMU MLinchen, Am Klopferspitz, 82152 Martinsried, Germany. ~°Present address: Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA. ttPresent address: Institut fLir allgemeine und experimentelle Pathologie, UniversitAt Wien, A-1090 Wien, Austria. '~Present address: Klinik Steglitz der FU Berlin, Klinik for Anaesthesiologie und operative Intensivmedizin, Hindenburgdamm 30, 12203 Berlin 30, Germany..~To whom correspondence should be addressed. (Received 2 November 1993; revised 1 February 1994; accepted 7 February 1994) 0264-410X/94/1311215-07 ,:(~ 1994 Butterworth-Heinemann Ltd

antigen delivery by recombinant avirulent Salmonella strains expressing foreign antigens has been shown to induce intestinal and serum IgG and IgA antibodies against the delivered antigen ~3,14 We have recently published the cloning and the amino acid sequences of the two major outer membrane proteins (Oprs) of P. aeruginosa, outer membrane protein F (OprF) is and outer membrane protein I (OprI) 16. Immunization of Balb/c mice with recombinant OprI synthesized in Escherichia coli was shown to be highly protective against P. aeruginosa infection in an immunosuppressed mouse model ~T. The present study examines the capability of an attenuated strain of Salmonella dublin to serve as a vehicle for delivery of the cloned antigen OprI of P. aeruginosa to the murine gut-associated lymphoid tissue, and to compare the importance of either an oral or parenteral induced immune response against P. aeruginosa for the protection against translocation of this Gram-negative pathogen. MATERIALS AND METHODS Expression of P. aeruginosa OprI in S. dublin The 625 bp fragment of plasmid pITaq116 containing the complete OprI gene was cloned into the replicative form of M13mpl9 ~8. After transformation into E. coli strain CJ236, single-stranded phage DNA was isolated.

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Oral immunization against Pseudomonas aeruginosa: A. Toth et al.

By site-directed mutagenesis 19, an EcoRI site was introduced either immediately upstream of the original methionine initiation codon, or upstream of the first codon of the mature protein, a cystine which was replaced simultaneously by a methionine residue. The oligonucleotides 5'-TTGTTCATCGAATTCCCCTTAA-3' and 5'-GTGGCTGCTCATGAATTCGGCCAGAAC-3' were used, respectively, for the mutagenesis. The generated sequences were confirmed by DNA-sequence analysis according to Chen and Seeburg 2°. The EcoRI inserts from the mutagenized constructs were excised and cloned into the EcoRI site of the expression vector pKK223-321 to give the plasmids pEXIP (expression with OprI signal peptide) and pEXI (expression without OprI signal peptide). AroA S. dublin (SL 1438)22 (a kind gift from Bruce Stocker, Stanford University) was transformed with these two plasmids by electroporation as described 23. Plasmid DNA was extracted and analysed by restriction enzyme digestion and agarose gel electrophoresis using standard techniques 24. Expression of recombinant Oprl in Salmonella was verified by SDS PAGE and immunoblotting of bacterial whole-cell lysates from overnight cultures with the monoclonal antibody 2AI 25 Recombinant SL1438 were stored in broth containing 100 #g ml -~ ampicillin and 20% glycerol at -70°C.

Preparation and purification of recombinant P. aeruoinosa OprI from E. coli The P. aeruginosa OprI gene sequence contained in plasmid pITaq116 was amplified by PCR with the two primers 5'-GCGGATCCATCGAGGGTAGAATGTGCAGCAGCCACTCCAAAGAAACCG-3' and 5'-CCAAGCTTATTACTTGCGGCTGGCTTTTTC-3'. The amplified DNA was purified, digested with HindIII and BamHI, and ligated into the corresponding sites of the expression vector pQE8 (Diagen, Diisseldorf, Germany) downstream of a sequence encoding an oligo-histidin peptide of six residues. The resulting construct pQE8-I was transformed into competent M15/pREP4 cells (Diagen). Expression of the recombinant Oprl-(His)6 fusion protein was induced by 4 h incubation in 1 litre TB-Medium 24 containing 1 mM IPTG. Cells (around 6 g wet weight) were lysed for I h at room temperature in 30 ml lysis buffer (6 Mguanidinium HC1, 0.1 MNaH2PO4, 0.1 MTris, pH 8.0). After 20 min centrifugation at 10 0009, the supernatant was loaded onto an equilibrated Ni NTA column (9 ml bed volume, Diagen). The column was washed first with 50ml of lysis buffer, and subsequently with 50 ml of 8 M urea, 0.1 M NaH2PO4, 0.1 M Tris, pH 8.0 and 50ml 50ram sodium phosphate, pH8.0, 100mM KC1, 0.1% Tween-20, 10mM /L mercaptoethanol, 10ram EDTA, 10mM EGTA. The recombinant protein was eluted with 250 mM imidazole in the same buffer. The yield of recombinant protein was generally around 150 mg 1-1 of IPTG-induced E. coli culture with an estimated purity of more than 95%.

SDS-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of Oprl Sodium dodecyl sulfate (SDS)-polyacrylamide (12%) slab gel electrophoresis and Western blotting were performed as previously described 17. After blotting, nitrocellulose sheets were incubated with diluted mouse serum or 1:90 diluted 2A1 hybridoma supernatant. Binding was visualized with alkaline phosphatase-linked

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goat anti-mouse IgG diluted 1:3000 (BioRad, Hercules, USA) and 5-bromo-4-chloro-3-indolyl-phosphate, as substrate.

Monoclonal antibody The induction, preparation, and purification of the OprI specific monoclonal antibody (mAb) designated 2A1 immunoglobulin IgG 2b has been described 25.

Enzyme-linked immunosorbent assay IgG and IgA antibodies against P. aeruginosa were measured by an enzyme-linked immunosorbent assay as previously described 26. Specific peroxidase-linked antibodies against mouse IgG (7 chain specific, Zymed, diluted 1:4000) and biotinylated rabbit anti-mouse IgA (diluted 1:4000)followed by peroxidase-coupled streptavidin (diluted 1:4000) (Zymed) were used as secondary antibodies.

Bacterial strains and growth conditions P. aeruginosa International Antigenic Typing Scheme serogroup 1 (ATCC 33348) was obtained from K.D. Hungerer, Behringwerke Marburg, Germany. Bacteria were grown and adjusted to the required concentration as previously described 17 Recombinant S. dublin (SL 1438) (pEXIP) were grown in tryptone-yeast medium. Tryptone (10g) (Merck, Darmstadt), 5 g yeast extract (Merck), 100 mg ampicillin (Bayer, Leverkusen) and 1 nag 2,3-dihydroxybenzoic acid (Fluka) were dissolved in 1 litre of distilled water and sterilized by filtration. S. dublin without expression plasmid was grown identically without ampicillin. Log-phase cultures were adjusted densitometrically to the desired bacterial concentration, which was confirmed by plating serial tenfold dilutions on endoagar (Merckoplate, Merck, Darmstadt, Germany).

ELISPOT assay The Elispot assay was modified from the method of Sedgwick and Holt 2~'2~ for use with P. aeruginosa. Multiscreen plates (96 well) (Millipore, Eschborn, Germany) were coated with 100/tl (0.7/~g) per well of sonicated P. aeruginosa overnight at 4°C. Plates were washed three times with phosphate-buffered saline (PBS) and blocked by incubation with 1% bovine serum albumin in PBS for 1 h at 37°C. After washing with PBS 1.25 x 104 spleen cells or Peyer's patch cells in 50 #1 PBS were added to each of 80 wells of the plate. To eight of the remaining wells of the plate 500 hybridoma cells secreting the P. aeruginosa OprI-specific mAb 2AI (IgG2b) were added as IgG-positive control. To the remaining eight wells normal spleen ceils were added as negative control. The plates were incubated for 4 h at 37°C in a humidified CO2 incubator (5% CO2), washed four times with PBS followed by the addition of the secondary antibody as described below for 1 h at 37°C. For the IgA spots, 50/~1 of 1:1000 diluted biotinylated rabbit anti-mouse IgA (alpha-chain specific, Zymed) were added, followed by 1 h incubation with 1:1000 diluted peroxidase-linked streptavidin (Zymed). For the IgG spots, 50 #1 per well of 1:1000 diluted peroxidase-linked rabbit anti-mouse IgG (Zymed) were added, After washing, spots were visualized with 3-amino-9-ethylcarbazole (AEC) (Sigma, Taufkirchen, Germany) at room

Oral immunization against Pseudomonas aeruginosa: A. Toth et al.

temperature. Spots were counted under the microscope at a 16-25-fold magnification.

Animals Female Balb/c mice, 8 weeks old, were obtained from the Zentralinstitut flit Versuchstierkunde, Hannover, Germany.

Immunization The 252 mice were divided into three groups, A, B and C. The animals of groups A and B received eight doses of 200/A each containing either 2 × 109 recombinant OprI expressing S. dublin or the corresponding OprI-negative bacteria, suspended in 0.1 MNaHCO 3 intragastrically (by garage) every second week. Mice of group C received two oral doses of recombinant S. dublin followed by six intraperitoneal (i.p.) injections of 100#g (1 mgm1-1) purified recombinant OprI suspended with an equal volume of AI(OH) 3 (Behringwerke Marburg, Germany). Blood was taken from the tail vein for serum antibody determination 1 week after each immunization. One week after the fifth immunization six mice of each group were killed for the determination of P. aeruginosa specific spot-forming cells (SFC). One week after the seventh immunization cycle six animals of each group were selected for the determination of antibody titres and SFC against P. aeruginosa during immunosuppression. The animals received three cycles of cyclophosphamide, as described below, at days 0, 2 and 4. At days 5, 6, 11, 12, 24, 25 one animal of each group was killed for the ELISPOT assay. The remaining animals were allowed to rest during this period (1 month). Then an eighth immunization was performed followed by determination of IgG and IgA antibody titres against P. aeruginosa 1 week later and subsequent immunosuppression and challenge with P. aeruginosa.

Immunosuppression For immunosuppression mice received three i.p. injections of 150pgg -1 of cyclophosphamide (Serva, Heidelberg, Germany) dissolved in 0.2 ml PBS on days 0, 2 and 4.

immunization with 109 recombinant S. dublin. The organs were homogenized and added to 50ml of tryptone-yeast medium. The presence of OprI was verified by immunoblotting of overnight culture whole-cell lysates.

Isolation of spleen cells Mice were killed by CO2 inhalation. The spleen was removed aseptically and passed through a nylon mesh (cell strainer 70 pm, Falcon). After washing with Hank's balanced salt solution cells were centrifuged for 10 min at 200g at 4°C. The erythrocytes in the pellet were lysed with distilled water 29. Cells were washed with Dulbecco's modified Eagle's medium (DMEM), counted by trypan blue staining and finally resuspended in DMEM supplemented with 10% fetal calf serum (Gibco, Heidelberg, Germany) at 2.5 x 105 ml- 1.

Isolation of Peyer's patches After removal of the spleen the small intestine was placed under a microscope at tenfold magnification. Peyer's patches were localized and removed with a scalpel without opening the lumen of the gut. Lymphocytes were isolated as described for the spleen cells but omitting the step for the lysis of the erythrocytes.

Statistics Survival curves were compared by the logit regression model 3°. The LDso values were calculated by probit analysis3°. RESULTS

Stability of Oprl expression by S. dublin in vivo In the collected stool specimens, salmonellas could be isolated until day 3 postimmunization. OprI expression could be shown by Western blotting only at day 1 (Figure 1). From the spleens and Peyer's patches of orally immunized mice salmonella could be isolated during the observation period of 10 days. OprI expression could be shown by Western blotting of spleen-derived S. dublin at day 2 from S. dublin isolated from Peyer's patches at days 1, 2 and 5.

Challenge experiments

Serum antibodies against OprI

One week after the last (eighth) immunization the animals of groups A-C were divided into four subgroups, each containing 18 mice. The animals received three injections with cyclophosphamide on days 0, 2 and 4. One day later the animals were challenged orally via a tube with 4.7 x 104 subgroup 1, 4.7 x 105 subgroup 2, 4.7x 106 subgroup 3 and 4.7x 10~ subgroup 4 P. aeruginosa bacteria suspended in 100#1 NaHCO 3 solution. Survival of the animals was registered every 6 h for a 96 h period.

After oral immunization, serum IgG and IgA antibody titres against P. aeruginosa showed a slow rise following each boost (Figure2a). Maximum antibody titres of 1:2400 (IgG) and 1:100 (IgA) were observed. In the sera of animals that had received combined vaccination by the oral and i.p. routes, maximal mean serum IgG antibody titres of 1:250000 and 1:600 (IgA) could be measured (Figure2b). No antibody titres against P. aeruginosa could be measured in the control group that had received oral immunization with normal S. dublin. Western blot analysis of pooled sera of the different immunization groups showed the 8 kDa band of OprI only in the two groups that had received immunization with recombinant S. dublin or with recombinant OprI (data not shown). After immunosuppression, the serum IgG and IgA titres declined within 1 week of 50% of the peak values and had fallen to background values at day 15 (data not shown).

Stability of Oprl expression by S. dublin in vivo Three mice received 10 9 recombinant S. dublin bacteria orally. Stool specimens were collected daily over a 8-day period and sent to the Department of Microbiology. Salmonellas grown from the samples were checked by Western blotting for the presence of OprI. Peyer's patches and spleens of three mice each were removed aseptically at days 1, 2, 5 and 10 after oral

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In order to investigate whether the local immune response against OrpI is affected by the different immunization regimes, the numbers of lgA- and IgGsecreting B cells were determined in isolated spleen cells and Peyer's patch cells. The results of the ELISPOT are shown in Table 1. A significant IgA response against OprI in the Peyer's patch cells was only detected in the animals that had received only oral immunization. Oral immunization followed by i.p. booster injections with OprI led predominantly to an IgG antibody response in the spleen. No P. aeruginosa specific immune response could be detected in Peyer's patch cells and spleen cells of the control animals that had received the nonrecombinant S. dublin. In the spleens of immunosuppressed animals, a rapid decline of the number of IgG spots could be observed for the combined orally/i.p, immunized group. Where l l2IgG spot-forming cells per 106 mononuclear cells could be measured on day 5, the number of spot-forming cells declined to 81 at day 6, 45 at day 11, 46 at day 12, 12 at day 24 and 7 at day 25 (spot-forming cells were determined in the spleens of single mice at the indicated days following immunosuppression). No significant number of IgA spots could be detected after immunosuppression during the 26-day observation period in both groups and no IgG spots in the group that had received oral immunization only. From the Peyer's patches no cells could be isolated between days 5 and 12 following immunosuppression. Spot-forming cells remained below the detection limit for the entire observation period (26 days). Protection

To determine the effectiveness of the different immune responses in protection against translocation of P. aeruginosa from the gut, mice were immunosuppressed with cyclophosphamide and challenged orally with four different doses (4.7 x 104, 4.7 x 105, 4.7 x 106, 4.7 x l0 T) of P. aeruginosa serogroup 1. This dose range had been evaluated in pilot experiments. The survival curves of the animals are shown in Figure 3. Calculated LDs0 values (group A; 38.27x 10('; group B; 3.82x 106; group C; 3.82 x 106) revealed that after eight oral immunizations with recombinant OprI expressing S. dublin mice were protected highly significantly (p ~<0.001) against a 58-fold LD50 challenge with P. aeruginosa. In contrast, micc immunized only twice orally and having received six booster injections i.p. with purified Owl were protected only against a sixfold LDso challenge (p>~0.05).

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The relevance of the outer membrane protein l for protection against P. aeruginosa has already been shown in previous reports by our group 17'26. Attenuated Salmonella mutants have been used for immunization with heterologous antigens of various micro-organisms, such as Eseherichia coli 31, Streptococcus pyogenes 32, Franc&ella tularensis 33, Bordetella pertussis 34 and Slreploeoecus sobrinus 3s and reviewed by Curtiss in 199013 and Sch6del in 199214. The present results show that recombinant OprI expressing S. dublin bacteria can be used as a vehicle to induce a systemic and local response against P. aeruginosa.

Oral immunization against Pseudomonas aeruginosa: A. Toth et al. Table 1 ELISPOT data for anti-P, aeruginosa IgG and IgA after oral (group A) or combined oral and i.p. (group C) immunization. Animals received five immunizations at 2-week intervals as described in Materials and methods and were killed for the ELISPOT test 1 week later. No spot-forming cells could be detected in group B immunized with the Oprl negative S. dublin carrier strain Group A

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HOURS POST INFECTION Figure 3 Survival rate of Balb/c mice after oral immunization with recombinant Oprl expressing S. dublin (group A, X ), normal S. dublin (group B,/k) or oral immunization with recombinant Oprl and i.p. immunization with recombinant Oprl (group C, *), followed by oral challenge with four different doses of P. aeruginosa serogroup 1

In a recent paper, published by Forrest et al. in 199236, it was shown that a systemic IgA response induced by oral immunization can be enhanced by a subcutaneous booster injection. This opens the possibility to compare the protective ability of IgA antibodies against P. aeruginosa present in the serum with those induced in the Peyer's patches by oral immunization. We therefore compared two different immunization protocols: first, oral immunization only with recombinant OprI expressing S. dublin, and in the second protocol two oral immunizations with recombinant S. dublin followed by six i.p. immunizations with purified OprI. As shown in Figure 2, serum IgA antibodies titres against P. aeruginosa in the group with oral immunization only were about 1/10 of the titres observed in the combined group. Similar results could be measured for the IgG antibody titres. IgA and IgG antibody titres against P. aeruginosa measured in the control group immunized with the Salmonella carrier strain were not significantly different from those measured in the preimmunization pool serum. These findings could be confirmed by Western blotting.

Sera from animals immunized with salmonellas expressing OprI recognized the 8 kDa band in immunoblots while control animals immunized with the Salmonella carrier strain alone did not display OprI reactive serum antibodies (data not shown). Oral immunization induced a superior local IgA response in the gut mucosa as demonstrated by the ELISPOT assay. Only within an orally immunized group could a significant number of IgA-specific spots be detected (Table 1). As already mentioned, translocation of P. aeruginosa from the gut is an increasing problem after immunosuppression in bone marrow transplant patients 2, liver transplant patients 37 and in patients after major intestinal surgery 38. The aim of the study was therefore to investigate whether translocation during immunosuppression could be prevented by a local secretory IgA response against OprI in the Peyer's patches. After oral or i.p. immunization with OprI the mice received three doses of cyclophosphamide (150 mg kg- 1) every second day followed by oral challenge with P. aeruginosa. P. aeruginosa challenge dose-dependent mortality is demonstrated in Figure 3. In spite of the finding that the

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O r a l i m m u n i z a t i o n a g a i n s t P s e u d o m o n a s a e r u g i n o s a : A. Toth et al.

animals that had received the combined immunization regimen had developed tenfold higher levels of circulating IgA and IgG antibody titres, this group was only protected against sixfold LDso of P. aeruginosa compared with 57-fold LDso observed for the group immunized orally only. This observation demonstrates the importance of a locally induced immunity for protection against translocation of P. aeruginosa. Using appropriate Salmonella vectors, oral immunization against outer membrane components of P. aeruginosa may be beneficial for some of the abovementioned risk patients. Since the number of cells which could be isolated from the Peyer's patches during immunosuppression was too low for the performance of the ELISPOT assay, we have no data concerning the half-life of IgA-secreting B cells in the Peyer's patches during cyclophosphamide treatment. The observation that in spite of peripheral leucocyte counts below 800 mm- 3 until day 7, protection was observed and in the spleen the number of IgG spot-forming B cells had dropped to 50% only, may indicate that during the critical period synthesis of secretory IgA in the Peyer's patches was still effective to prevent adhesion of P. aeruginosa at the mucosal surface. Ampicillin-resistant S. dublin synthesizing OprI could only be recovered from stool specimens for 24 h after oral immunization. The expression plasmid appeared to be more stable in spleens and Peyer's patches after oral immunization, however. The relatively low in vivo stability of the plasmid may have contributed to the low initial antibody titres observed. In the present set of experiments animals were immunized biweekly. Work is now in progress to analyse alternative carrier strain plasmid combinations and to perform a more thorough analysis of carrier strain persistence, in vivo plasmid stability and the kinetics of secretory immune responses at other mucosal sites after single or multiple oral immunizations.

ACKNOWLEDGEMENTS This work was supported by grant Sp 170/5-1 from the Deutsche Forschungsgemeinschaft to B.-U. von Specht and by grant 01KI8910/4 from the Bundesministerium ftir Forschung und Technologie to H. Domdey and B.-U. von Specht and by grant E/B41G/L0407/L5921 from the Bundesministerium fiir Verteidigung to B.-U. von Specht.

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REFERENCES 1 Andremont, A., Marang, B., Tancrede, C., Baume, D. and Hill, C. Antibiotic treatment and intestinal colonization by Pseudomonas aeruginosa in cancer patients. Antimicrob. Agents Chemother. 1989, 33, 1400-1402 2 Remington, J.S., Schimpff, S.C., Hughes, W.T., Armstrong, D., Klastersky, J. and Bodey, G.P. Life-threatening infections in the compromised host. In: Current Chemotherapy. Proc. lOth International Congress of Chemotherapy Vol. 1 (Eds Siegenthaler, W. and L0thy, R.) American Society for Microbiology, Washington, DC, 1978, pp. 37-42 3 Schimpff, S.C., Young, V.M., Greene, W.H., Vermeulen, G.D., Moody, M.R. and Wiernik, P.H. Origin of infection in acute nonlymphocytic leukemia: significance of hospital acquisition of potential pathogens. Ann. Intern. Med. 1972, 77, 707-714 4 Schimpff, S.C., Alsner, J. and Wiernik, P.H. Infection in

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