Expression of enterotoxigenic Escherichia coli colonization factors in Vibrio cholerae

Vaccine 24 (2006) 4354–4368

Expression of enterotoxigenic Escherichia coli colonization factors in Vibrio cholerae Didier Favre a,∗ , Stefan L¨udi b , Michael Stoffel b , Joachim Frey c , Michael P. Horn a,1 , Guido Dietrich a , Simone Spreng a , Jean-Franc¸ois Viret a a

Berna Biotech Ltd., Department of Live Bacterial Vaccines, Rehhagstrasse 79, 3018 Bern, Switzerland b Veterinary Anatomy, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland c Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland Received 23 December 2005; received in revised form 22 February 2006; accepted 28 February 2006 Available online 20 March 2006

Abstract As a first step towards a vaccine against diarrhoeal disease caused by enterotoxigenic Escherichia coli (ETEC), we have studied the expression of several ETEC antigens in the live attenuated Vibrio cholerae vaccine strain CVD 103-HgR. Colonization factors (CF) CFA/I, CS3, and CS6 were expressed at the surface of V. cholerae CVD 103-HgR. Both CFA/I and CS3 required the co-expression of a positive regulator for expression, while CS6 was expressed without regulation. Up-regulation of CF expression in V. cholerae was very efficient, so that high amounts of CFA/I and CS3 similar to those in wild-type ETEC were synthesized from chromosomally integrated CF and positive regulator loci. Increasing either the operon and/or the positive regulator gene dosage resulted in only a small increase in CFA/I and CS3 expression. In contrast, the level of expression of the non-regulated CS6 fimbriae appeared to be more dependent on gene dosage. While CF expression in wild-type ETEC is known to be tightly thermoregulated and medium dependent, it seems to be less stringent in V. cholerae. Finally, co-expression of two or three CFs in the same strain was efficient even under the control of one single regulator gene. © 2006 Elsevier Ltd. All rights reserved. Keywords: ETEC; V. cholerae carrier; Fimbriae expression

1. Introduction Enterotoxigenic Escherichia coli (ETEC) is one of the major causes of traveler’s diarrhoea, usually hitting travelers in the first week of travel [1]. Current prophylaxis and treatment include attention to diet and the intake of certain antibiotics. However, non-compliance and side-effects reduce the effectiveness of these measures. Evidence that a vaccine strategy is possible comes from the observation that natural infection provides subsequent immunity [2] and from numerous clinical and field studies with a variety of ETEC vaccine candidates [1,3]. ∗

Corresponding author. Tel.: +41 31 980 62 83; fax: +41 31 980 64 86. E-mail address: [email protected] (D. Favre). 1 Present address: Molecular Diagnostics, Inselspital, 3010 Bern, Switzerland. 0264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2006.02.052

The onset of diarrhoea requires colonization of the gut mucosa by ETEC bacteria that adhere to the gut epithelial cells through a variety of colonization factors (CFs), classified as coli surface antigens (CS) or colonization factor antigens (CFA), most of which are fimbrial in nature, while others consist of pili or fibrillae. The bacteria subsequently elaborate a heat-labile enterotoxin (LT) and/or a heat-stable enterotoxin (ST) [4] which are responsible for profuse watery diarrhoea. Epidemiological studies have allowed the detection of at least 22 different ETEC CFs from human strains, some of which occur together in the same cell. Thus, CFA/II expresses CS3 alone or in combination with CS1 or CS2, while CFA/IV expresses CS6 alone or in combination with CS4 or CS5 [5–9]. Genetically, ETEC CFs are characteristically organized in operons comprising one or two structural genes, as well as chaperone and usher genes, involved in folding, transport, and the correct assembly of the fimbrial subunits

D. Favre et al. / Vaccine 24 (2006) 4354–4368

to form complete structures [7]. CFs are usually plasmidborne and their expression is often, but not always, regulated by various positive regulators which share a high percentage of similarity and are largely interchangeable [10,11]. Such positive regulators include Rns, which regulates the expression of CS1 and CS2 [12], CfaR (sometimes also called CfaD), which regulates CFA/I expression [59], CsvR, which regulates the expression of CS4 and CS5 [13], and CsfR, which regulates the expression of CS4 [6]. All these regulators belong to the AraC family of regulatory proteins [14]. Rns interaction with cognate DNA is known in detail from the literature and was found to bind at two asymmetric DNA sites upstream of the CS1 genes [15,16]. Rns also regulates its own synthesis by binding to two DNA sites centered at 44 and 112 bp upstream from the Rns transcription start site [15]. At least two of the ETEC CF positive regulators, Rns and CfaR, have been shown to activate the transcription of CF operons, at least partly, by relieving transcription suppression mediated by the global regulator H-NS [17–19]. Comparison with other sequences upstream of genes controlled by Rns-like proteins has uncovered a significant degree of similarity with the Rns binding sequences [15]. Various strategies have been devised to create ETEC vaccines, all of which are based on LT and/or the CFs, including the oral administration of microencapsulated purified fimbriae [20–22], transcutaneous inoculation of purified fimbriae and LT [23,24], transgenic plants expressing the B-subunit of LT (LT-B) [25,26], DNA-based vaccines [27,28], and killed whole [29–33] and live attenuated ETEC cells [34]. Finally, an intensively investigated avenue is the use of bacterial carriers expressing ETEC antigens. So far, CFA/I has been successfully expressed in attenuated Salmonella serovar Typhimurium and E. coli [35], CFA/I and CS3 in Salmonella serovar Typhi [36], and CFA/I, CS2, CS3, and CS4, together with a mutant LT in Shigella flexneri vaccine strains [37–42]. The live attenuated classical O1 Vibrio cholerae vaccine strain CVD 103-HgR [43–45] has been in use for the vaccination of humans for decades and has proven to be safe and highly effective. CVD 103-HgR has also been used with success as a carrier for the presentation of the O-polysaccharide of S. sonnei [46,47] and V. cholerae O139 [48] as well as of the B-subunit of the Shiga-like toxin of S. dysenteriae 1 [49]. The rationale of a candidate ETEC vaccine based on CVD 103-HgR as a carrier is essentially that cholera and ETEC diarrhoea are closely related diseases. Both are caused by non-invasive organisms, are toxin-mediated, and elicit antitoxic and antibacterial immunity through mucosal IgAs [50]. In addition, CVD 103-HgR expresses the B subunit of cholera toxin (CT-B) which is highly homologous to the B subunit of HLT (LT-B). Therefore, V. cholerae seems to be ideally suited to deliver ETEC antigens in an immunologically active form, thus mimicking the natural infection route of ETEC wild-type.

4355

With the goal of creating an effective V. cholerae-based ETEC vaccine, we have studied the expression and immunogenicity of ETEC CFA/I, CS3, and CS6 colonization factor in V. cholerae CVD103-HgR.

2. Materials and methods 2.1. Bacterial strains, plasmids, and growth media Wild-type and recombinant E. coli and V. cholerae strains, as well as plasmids used in this study, are presented in Table 1. All strains were grown in LB medium [51]. For selection of integrants during the gene integration procedure, LBS-N consisted of LB without NaCl and with 5% sucrose added. CF expression was obtained by growing the recombinant E. coli or V. cholerae strains in CF medium consisting of 10.0 g/L casamino acids, 1.5 g/L yeast extracts, 50 mg/L MgSO4 ·7H2 O, and 5 mg/L MnCl2 ·4H2 O. For solid media, 2% agar was added. Antibiotics were used at the following concentrations: chloramphenicol (Cm) 17 ␮g/mL, kanamycin (Km) 50 ␮g/mL, and ampicillin (Ap) 100 ␮g/mL. 2.2. Molecular techniques Chromosomal isolation was according to the protocol of Pitcher et al. [52]. Plasmid minipreps and maxipreps were prepared using commercial kits according to the manufacturer’s instructions (Promega Corp., Madison, USA, and QIAGEN Ltd., Hombrechtikon, Switzerland). Endonuclease restriction, modification of the resulting DNA by either Klenow enzyme or Mung Bean Nuclease, and isolation of DNA fragments from low-melting point agarose gels were performed according to published methods [51,53] or using commercial kits (QIAGEN Ltd.). Non-radioactive probe labeling with digoxigenin was performed by using a commercial kit (DIG High-prime, Roche Applied Science, Rotkreuz, Switzerland) and detection was with an anti-DIG alkaline phosphatase-conjugated antibody followed by incubation of the filters with an alkaline phosphatase chemoluminescent substrate (CSPD, Roche Applied Science). 2.3. Chromosomal integration of ETEC genes The procedure for integration of ETEC loci in the chromosome of V. cholerae CVD 103-HgR has been described previously [54] and was used with some slight modifications. Briefly, the integration plasmids were transferred in CVD 103-HgR by either transformation or conjugation via E. coli K12 S17.1. The resultant strains were inoculated in 2 mL 0.9% NaCl. Dilutions of the suspensions were plated onto pre-warmed chloramphenicol-containing LB (LB Cm) plates and incubated at 42 ◦ C for 16 h. Individual colonies were then gathered and streaked onto two 42 ◦ C pre-warmed LB Cm plates, which were incubated at 42 ◦ C. Several of the

4356

D. Favre et al. / Vaccine 24 (2006) 4354–4368

Table 1 Strains and plasmids Strains

Relevant genotype and characteristics

Source or reference

araD139, ∆(ara-leu7697), galU, galK, mcrA, ∆(mrr-hsdRMS-mcrBC), rpsL, deoR, Φ80dlacZ∆M15, endA1, nupG, recA1 thi-1 pro hsdR Tpr Smr RP4-2 [Tc::Mu (Km::Tn7)]

[75]

ETEC H10407 CD79a DS7-3 DS198-1 B7A

cfaABCE (CFA/I) cfaABCE (CFA/I) cstABC (CS3) cstABC (CS3) cssABCD (CS6)

[76], W.Gaastra [77] M.K. Wolf M.K. Wolf M.K. Wolf

V. cholerae CVD 103-HgR CHcfaI-1 CHCS3-2 CHCS6-1 CHcfaI-R1 CHCS3-R2 BB06

∆ctxA hlyA::mer (HgR) CVD 103-HgR hlyB::cfaABCE CVD 103-HgR hlyB::cstABC CVD 103-HgR hlyB::cssABCD CHcfaI-1 hlyB::rns-1/cfaABCE CHCS3-2 hlyB::rns/cstABC CHCS3-R2 hlyA::cfaABCE

[44] This work This work This work This work This work

High copy number cloning vector, KmR Low-copy number cloning vector, SpcR Low copy number suicide integration vector, CmR Low copy number suicide integration vector, CmR Low copy number cloning vector, KmR High copy number vector pUC19 carrying cssABCD (CS6) locus, ApR Vector pBR322 carrying cstABC (CS3) locus, ApR Vector pACYC184 carrying the csfR regulator gene, CmR Source of the rns regulator gene, CmR Source of hlyA::mer and hlyB loci, ApR Gene bank clone based on vector pCL1920 and expressing the cfa operon pCL1920 with the 1.3 kb HindIII–EcoRI rns fragment from pEU2040 Plasmid pK18 carrying the cfa locus Plasmid pSSVI215 carrying the cfa locus Plasmid pSSVI215 carrying the CS3 locus Plasmid pSSVI215 carrying the CS6 locus hlyB integration vector based on suicide vector pMAKSACA Integration clone based on pMAKSACAhly carrying the cfa locus Integration clone based on pMAKSACAhly carrying the CS3 locus pMAKSACAhly carrying the CS6 locus Integration clone based on pMAKSACAhly carrying the CS6 locus Integration vector for the positive regulator rns gene Integration clone based on pMAKSACBhlyAR carrying the rns gene Integration clone based on pMAKSACBhlyAR carrying the rns gene. The rns gene in opposite orientation with respect to that in pMAKrns-1 Cosmid clone containing the hly::mer region of CVD 103-HgR hlyA integration vector for the cfa locus in strain CHCS3-R2 Integration clone based on pMAKSACBhlyAL carrying the cfa locus

[78] [79] [54] [54] [55] M.K. Wolf D.A. Scott M.K. Wolf [73], J.R. Scott [44] This work

E. coli K12 DH10B S17.1

Plasmids pK18 pCL1920 pMAKSACA pMAKSACB pSSVI215 pM295 pGB1 pM392 pEU2040 pJMK10 pCL1920/CFASmaI pCLRNS pK18cfaI-a pSSVI215-cfaI pSSVI215-CS3 pSSVI215-CS6 pMAKSACAhly pMAKcfaI-1 pMAKCS3-2 pMAKCS6-Km1 pMAKCS6-1 pMAKSACBhlyAR pMAKrns-1 pMAKrns-2 pSSVI207-2 pMAKSACBhlyAL pMAKhlyAcfaIS

resulting colonies were inoculated in 2 mL of liquid LBS-N and incubated at 30 ◦ C for 3–4 h, during which time extensive lysis occurred. Dilutions of the resulting cultures were plated out onto LBS-N plates, which were incubated at 30 ◦ C for 16–24 h. Cm-sensitive colonies were then identified by replica-plating. The above described integration procedure allowed production of 95–100% of Cm-sensitive colonies. A number of Cm-sensitive colonies were subsequently blotted

This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work

onto nylon filter and the presence of the integrated locus was tested by colony hybridization using specific DIG-labeled probes. 2.4. Construction of integration vectors In order to chromosomally co-integrate one CF locus and a positive regulator gene, two different integration vectors were

D. Favre et al. / Vaccine 24 (2006) 4354–4368

constructed. A further vector was constructed to integrate a second CF operon in the same strain. The vector for integration of the primary CF loci, pMAKSACAhly, was constructed by cloning the 3559 bp PinAI–PstI fragment from plasmid pJMK10 into plasmid pMAKSACA restricted by XmaI and PstI. The latter fragment overlaps the hlyA–hlyB–lipA region of V. cholerae. The plasmid contains a single NruI cloning site within hlyB homology regions of almost identical sizes (1778 and 1786 bp). For the integration of a second CF operon, the integration vector pMAKSACBhlyAL was constructed by cloning a 2.23 kb ScaI–BglII fragment from plasmid pSSVI2072 into vector pMAKSACB cut with BamHI and SmaI. The ScaI–BglII fragment encompasses 0.93 kb of sequence upstream of the hlyA gene, the 5 part of the hlyA gene, the merR gene, and the 5 part of the mer operon in CVD 103HgR. In this context integration of a foreign gene can be effected at the single NruI site located within hlyA, 203 bp downstream from the start codon. The integration vector for the positive regulator gene was based on the suicide integration plasmid pMAKSACB which was restricted with BamHI and NsiI. The homology region corresponded to a 1653 bp BamHI–NsiI fragment derived from pJMK10 encompassing the 3 -end of the hlyA gene and the 5 -end of the hlyB gene. The latter fragment was cloned into pMAKSACB to create pMAKSACBhlyAR. The latter vector contains a unique EcoRV cloning site within the homology region, providing 885 and 772 bp of homology arms, and located 245 bp downstream of the ATG start codon of the hlyB gene. 2.5. Integration of the CF loci 2.5.1. CFA/I For cloning of the cfa locus a gene bank of SmaI-restricted chromosomal fragments of wild-type ETEC strain CD79a was established in the low-copy number plasmid pCL1920 cut by SmaI. Colonies bearing the cfa locus were detected by colony hybridization using a DIG-labeled internal fragment of the cfa operon as the probe. A clone hybridizing to the probe was found and the corresponding plasmid called pCL1920/CFASmaI. The cfa locus was then excised as a 7.5 kb AclI fragment from pCL1920/CFASmaI and cloned into SmaI-cut pK18 resulting in plasmid pK18cfaI-a. The cfa locus was subcloned as a 7.3 kb SacI fragment in the SacI-cut low-copy number vector pSSVI215. The resulting plasmid was called pSSVI215-cfaI. In the latter plasmid, the vectorinsert junction is located 268 bp upstream from the start of cfaA, the first gene of the cfa locus. The cfa locus was further subcloned from pSSVI215-cfaI in the middle of the hlyB gene in the integration plasmid pMAKSACAhly cut by NruI. The kanamycin-resistant gene was removed from one plasmid with the cfa genes in the same orientation than hlyB by SceI restriction and self-ligation as described in a previous article [55]. The resultant plasmid was called pMAKcfaI-1. Using the latter plasmid, the cfa operon was then chromoso-

4357

mally integrated within the hlyB gene of CVD 103-HgR. One integrant was called CHcfaI-1 and used for further work. 2.5.2. CS3 The CS3 operon was excised from plasmid pGB1 as a 4.7 kb HindIII fragment. This fragment was first subcloned into the low-copy number vector pSSVI215 partially restricted by HindIII. The resulting plasmid was called pSSVI215-CS3 and contains the CS3 operon in tandem with a kanamycin-resistance gene (KmR ) which serves as a positive marker for the subsequent subcloning. In the latter plasmid, the vector-insert junction is located 372 bp upstream from the start of the first gene of the CS3 locus. The ca. 6 kb CS3 operon-KmR gene fragment was further isolated by restricting pSSVI215-CS3 with SwaI and subcloned in pMAKSACAhly restricted by NruI. The resulting plasmid was excised by SceI and the large fragment was self-ligated. Integration of this plasmid in V. cholerae CVD 103-HgR produced strain CHCS3-2 in which the CS3 operon is transcribed in opposite direction with respect to hlyB. 2.5.3. CS6 The CS6 operon was excised from plasmid pM295 as a 4.5 kb SacI fragment. This fragment was first cloned in pSSVI215 restricted by SacI. The resulting plasmid was called pSSVI215-CS6. The 6 kb CS6 operon and the adjacent KmR gene were further isolated from pSSVI215-CS6 by restriction with SwaI and subcloned in pMAKSACAhly restricted by NruI. One plasmid called pMAKCS6-Km1 had the cssA, B, C, and D genes of the CS6 operon in the same orientation as the hlyB gene. Restriction by SceI followed by self-ligation allowed removal of the KmR gene resulting in the final 14.5 kb integration plasmid called pMAKCS61. The latter plasmid was introduced into V. cholerae CVD 103-HgR and the integration procedure was carried out as described. One integrant was called CHCS6-1 and used for further work. 2.6. Integration of the rns positive regulator gene The rns regulator gene was cloned as a 1.2 kb klenowed EcoRI–HindIII fragment from plasmid pEU2040 into pMAKSACBhlyAR restricted by EcoRV. Two orientations of the gene within the vector were found called pMAKrns-1 and pMAKrns-2. In plasmid pMAKrns-1, rns is transcribed in the reverse orientation with respect to hlyB gene transcription, whereas in pMAKrns-2, rns is transcribed in the same orientation as hlyB. The two plasmids were introduced in either CHcfaI-1 or CHCS3-2 and the positive regulators were integrated by the same procedure as for the pili operons. Although we attempted to isolate strains possessing the positive regulator in both orientations, only one orientation produced strains which were able to synthesize CFA/I or CS3 CFs. Thus, CHcfaI-1 integrants from pMAKrns-1 but not from pMAKrns-2 were able to synthesize the CFA/I pili, while CHCS3-2 integrants from pMAKrns-2 but not

4358

D. Favre et al. / Vaccine 24 (2006) 4354–4368

from pMAKrns-1 were able to synthesize the CS3 fibrillae. The new strains were named CHcfaI-R1 and CHCS3-R2, respectively.

of the integration sites, and identifies for each vector which ETEC loci were subcloned and integrated. 2.8. Evaluation of plasmid copy number

2.7. Integration of a second CF operon in CHCS3-R2 For integration in CHCS3-R2, the cfa locus was cloned from plasmid pK18cfaI-a as a 5563 bp EcoRV–SacI fragment into the integration vector pMAKSACBhlyAL via plasmid pSSVI215. The integration clone was named pMAKhlyAcfaIS. The cfa genes are in the same orientation as hlyA. The plasmid was electroporated into CHCS3-R2 and chromosomally integrated. Colonies of CHCS3-R2 having an integrated copy of the cfa operon were identified and called BB06. Fig. 1 summarizes the construction of the integration vectors, including the homology fragments used and the location

Cultures of the relevant E. coli K12 and V. cholerae recombinant strains were grown in CF medium at 37 ◦ C for 18 h. The cell densities were then adjusted to an OD600 of 3.0 and plasmid DNA minipreps were made from 1 mL cultures. The plasmid DNA was then diluted in two-fold steps and 10 ␮L of each dilution analyzed by agarose gel electrophoresis. The amount of plasmid DNA was evaluated by comparing the band intensities of the plasmid dilutions to the band intensities of a defined amount of a phage ␭ DNA HindIII digest. For the evaluation of copy number, we used plasmid pSSVI215 isolated from E. coli K12 DH10B as the reference. Plasmid pSSVI215 possesses the ori101 origin of replication which

Fig. 1. Construction scheme of integration vectors and precise genetic location of integrated loci. The names of the integrated loci are identified by thick-lined boxes. The integration vectors are identified by thin-lined boxes. Filled boxes: homology regions. Open arrows depict the genes present in the hlyA::mer region of the CVD 103-HgR chromosome, as well as selected integration vector genes.

D. Favre et al. / Vaccine 24 (2006) 4354–4368

drives the production of about 6 copies/cell of plasmids in E. coli K12 [56]. 2.9. Production of polyclonal antibodies For the production of CF-specific polyclonal antisera, wild-type ETEC strains H10407 (CFA/I), DS7-3 (CS3), and B7A (CS6) were grown on CF plates at 37 ◦ C. Cell suspensions of each strain were made in 0.9% NaCl to an OD600 of about 0.17 and used for vaccination of rabbits. 0.2, 0.5, and 1.0 mL of the suspensions were subcutaneously administered on days 1, 3, and 6. On days 8, 10, 17, 23, and 29, 1.0 mL of a 1:10 dilution of fresh cell suspensions was administered. The whole blood was collected 3–7 days after the last immunization. Polyclonal antibodies were adsorbed three times against E. coli K12 DH10B, V. cholerae CVD 103-HgR, and a heterologous ETEC wildtype strain in order to remove irrelevant cross-reacting antibodies. 2.10. Production of monoclonal antibodies Monoclonal antibodies (MAbs) against CFA/I, CS3, and CS6 CFs were generated in Balb/c mice according to standard methods [57]. Briefly, Balb/c mice were immunized three times subcutaneously with 10 ␮g and a fourth time intraperitoneally with 100 ␮g of purified CFA/I, CS3, or CS6 CFs. For fusion, spleen cells were mixed with Ag8 fusion partner cells in a ratio of 2:1 in a PEG/DMSO solution. Following fusion, the cells were cultivated in the presence of feeder cells (mouse peritoneal macrophages) under selection pressure of azaserine and hypoxanthine (both from Sigma, Buchs, Switzerland). Following a primary screening, positive cells were expanded and finally cloned twice by limiting dilution. MAbs anti-CFA/I 2E5 and anti-CS6 4C9 are of the IgG1 isotype, while MAb anti-CS3 1G3 is of the IgG2b isotype. MAb 2E5 was able to efficiently agglutinate a CFA/Iexpressing ETEC wild-type strain, while the CS3 and CS6 MAbs were unable to agglutinate corresponding wild-type strains. 2.11. Protein crude extracts and Western blotting For protein crude extracts, the strains were grown at 37 ◦ C in either liquid or solid CF medium. Liquid cultures were centrifuged and resuspended in 0.9% NaCl to an OD600 of 3.0. Cells grown on solid CF were collected with a cotton swab and resuspended in 0.9% NaCl to an OD600 of 3.0. Cell suspensions were mixed with one volume of 2× SDSPAGE sample buffer. The samples were boiled 5 min and 10–15 ␮L was immediately loaded on a 4–15% SDS-PAGE gel (Bio-Rad, Glattbrugg, Switzerland). Western blotting was performed as previously described with some modifications [47]. The blots were blocked in 10% powdered dried milk, labeled with CF-specific monoclonal and polyclonal anti-

4359

bodies, and the specific bands were finally detected using horseradish peroxidase-conjugated goat anti-mouse or antirabbit IgG (Bio-Rad), and a commercial kit for chemoluminescent detection (ChemiGlowTM , AlphaInnotech Corp., San Leandro, USA). 2.12. Electron microscopy and immunogold-labeling of CFs Bacteria grown onto CF plates under conditions permissive for CF expression were rinsed off the plate with saline and fixed immediately by adding an equal volume of 0.2% paraformaldehyde in 0.01 M PBS, pH 7.4. Bacteria were then adsorbed onto parlodion-coated copper or nickel grids. The grids were washed for 5 min with H2 O. For negative staining, the grids were floated on a drop of 0.25% phosphotungstic acid, 0.01% BSA for 30–40 s. After blotting off the excess liquid, grids were air dried. For immunolabeling, grids were floated for 60 min on a 1:50–1:100 dilution in PBS/0.2% BSA/0.05% Tween 20 solution (PBS-BT) of the relevant primary antibody, washed in PBS-BT, and further incubated for 60 min on a drop of either goat 15 nm gold-conjugated anti-mouse IgG (Sigma) diluted 1:100 in PBS or on a drop of goat 15 nm gold-conjugated anti-rabbit IgG (British Biocell International, Brunschwig, Germany), depending on the origin of the primary antibody. For double labeling a mixture of the relevant primary antibodies was used, followed by incubation with a mixture of appropriate secondary antibodies conjugated to 10 and 15 nm gold particles, respectively. The grids were then washed for 5 min in H2 O. Excess liquid was then removed and the grids air-dried. Samples were finally assessed using a Zeiss 109 transmission electron microscope. 2.13. Whole cell pili ELISA Bacterial strains were either grown in liquid CF at 37 ◦ C and pelleted by centrifugation or bacterial lawns were scraped off a CF plate. The cells were resuspended to an OD600 of 0.1 or 0.2 in 0.9% NaCl/0.5% formaldehyde. ELISA plates (Maxisorp, Nunc, Rochester, USA) were coated in triplicate with 100 ␮L of cell suspension and the plate was incubated for 2 h at 37 ◦ C. Wells were washed thrice with 150 ␮L 0.9% NaCl and blocked 30 min with 200 ␮L 1% human serum albumin in 0.9% NaCl (HSA/NaCl). One hundred microliter of a 1:1000 dilution of the appropriate MAb in HSA/NaCl was added to the wells for 30 min incubation at 37 ◦ C. After washing, 100 ␮L of a 1:1000 dilution of a horseradish peroxidase-conjugated anti-mouse IgG antibody was added and the plate incubated at 37 ◦ C for another 30 min. After washing, 50 ␮L of 3,3 ,5,5 -tetramethylbenzidine (Promega Corp.) horseradish peroxidase substrate was added. After 2.5 min the reaction was stopped by 50 ␮L 1 M sulfuric acid and the color measured at 450 nm in a Spectramax plate reader (Spectramax Plus, Bucher Biotech AG, Basel, Switzerland).

4360

D. Favre et al. / Vaccine 24 (2006) 4354–4368

2.14. Immunogenicity studies Groups of five mice were immunized subcutaneously with 100 ␮L containing 109 live recombinant V. cholerae strains resuspended in 100 ␮L 0.9% NaCl. CVD103-HgR and appropriate wild-type ETEC strains served as negative and positive controls, respectively. Booster immunizations were administered at days 14 and 28 and mice were sacrificed at day 35. For serum analysis all steps were at room temperature. 2.5 ␮g/mL of purified CFs (a gift of F. Cassels) was coated onto an ELISA plate (Nunc Maxisorp, Milian, Switzerland). After washing and blocking with 1% human serum albumin in PBS, 100 ␮L of serial dilutions of mice sera was added and the plate incubated for 2 h. The plate was washed again, 100 ␮L of a peroxidase-labeled goat anti-mouse IgG antibody was added at a 1:2000 dilution, and the plate was further incubated for 1 h. Finally 100 ␮L of a peroxidase substrate solution was added and the plate incubated for 10–15 min. The results were then recorded in a plate reader (Spectramax Plus) set at 405 nm. ELISA titers are defined as the reciprocals of the geometric mean of the serum dilutions producing an OD405 of 0.4.

3. Results 3.1. Construction of CF expressing V. cholerae CVD 103-HgR We have chosen CFA/I, CS3, and CS6 for expression in the live attenuated V. cholerae strain CVD103-HgR since they are the most frequently encountered CFs of ETEC in patients with ETEC diarrhoea. Corresponding CF operons and the positive ETEC pili regulator have been subcloned into low- and high-copy number vectors, as well as integrated in the chromosome of the V. cholerae vaccine strain CVD 103-HgR within the hlyA and/or the hlyB gene. The latter genes were selected for integration due to the fact that the carrier strain already harbors a mercury resistance genetic determinant inserted in the hlyA locus. The hlyA locus is naturally inactive in classical strains of V. cholerae [58] and further mutations in hlyA did not modify the immune response to potential V. cholerae vaccine strains in human volunteers [59]. Fig. 1 shows the strategy for construction of integration vectors pMAKSACAhly, pMAKSACBhlyAL, and pMAKSACBhlyAR and for subsequent integration of the CF operons and the positive regulator in the chromosome of CVD 103-HgR. The detailed cloning and construction of strains and plasmids are described in Section 2. 3.2. Expression of CFs in V. cholerae CVD 103-HgR CF expression in CVD103-HgR was detected by Western blotting of bacterial lysates probed with CFA/I, CS3, and CS6-specific antibodies and was compared to CF expression in corresponding wild-type strains (Fig. 2). While CS6

Fig. 2. Western blot analysis of CF expression in CVD103-HgR recombinant strains grown on CF plates at 37 ◦ C. CF proteins were identified by use of specific polyclonal rabbit antisera raised against purified CFs. Panel CFA/I—lanes: (1) wild-type ETEC H10407; (2) CHcfaI-1; (3) CHcfaI-R1; (4) CVD103-HgR. Panel CS3—lanes: (1) ETEC DS7-3; (2) CHCS3-2; (3) CHCS3-R2; (4) CVD 103-HgR. Panel CS6—lanes: (1) ETEC B7A; (2) CVD103-HgR (pSSVI215-CS6); (3) CVD103-HgR (pSSVI215CS6/pM392); (4) CVD103-HgR (pSSVI215).

was well expressed in CVD103-HgR (CS6 panel, lane 2) in amounts comparable to wild-type ETEC B7A (CS6 panel, lane 1), CFA/I and CS3 were only poorly or not expressed in CHcfaI-1 (CFA/I panel, lane 2) and CHCS3-2 (CS3 panel, lane 2), respectively, contrasting with the powerful CFA/I and CS3 expression in ETEC wild-type strains (panels CFA/I and CS3, lane 1). These results were irrespective of chromosomal or plasmid localization of the CF operons (data not shown). 3.3. Regulation of CF expression in V. cholerae CVD 103-HgR To test whether CFA/I and CS3 pili expression is regulated in V. cholerae, the genes encoding two ETEC regulators, CsfR and Rns, have been evaluated. Rns and CsfR regulators were interchangeable without any significant effect on CF expression (data not shown). At a later stage, the Rns regulator was selected for chromosomal integration in CFA/I and CS3-expressing strains. The individual expression of CFA/I, CS3, and CS6 pilins in V. cholerae CVD 103-HgR under the control of a positive

D. Favre et al. / Vaccine 24 (2006) 4354–4368

4361

Fig. 3. Expression of CFs in CVD 103-HgR and ETEC H10407 as revealed by negative staining (panel A) or immunolabeling (panels B–D). (A) Wild-type ETEC strain H10407; (B) CVD 103-HgR (pSSVI215-cfaI/pM392); (C) CVD 103-HgR (pSSVI215-CS3/pM392); (D) CVD 103-HgR (pM295). Arrows depict the corresponding CF structures. Scale bars—panel A: 1 ␮m and panels B–D: 0.5 ␮m.

regulator was studied by Western blotting (Fig. 2, lane 3). For CFA/I and CS3 expression, large amounts of the pilins, similar in magnitude to those made by the wild-type strains (lane 1), are synthesized from strains CHcfaI-R1 and CHCS3R2, respectively, containing the Rns positive regulator gene integrated in their chromosome. The situation of CS6 expression appears to be different. As described above, CHCS6-1 (panel CS6, lane 2), which does not contain an ETEC CF positive regulator, expresses CS6 pilin at levels similar to those of the wild-type strain B7A (lane 1). However, there is no visible increase in CS6 expression when a positive regulator gene is provided to the recombinant V. cholerae, even on a medium copy number plasmid (lane 3). The latter result corroborates earlier observations that CS6 in ETEC is not regulated [60,61]. The apparent sizes of the pilins expressed by CVD 103-HgR are similar to those of the pilins expressed by the corresponding wild-type ETEC strains.

face structures, electron microscopy was performed (Fig. 3). CFA/I, CS3, and CS6 pili could be detected at the surface of recombinant V. cholerae CVD103-HgR, albeit exhibiting different phenotypes. Negatively stained wild-type ETEC strain H10407 is presented as a control of CFA/I expression, showing typical rod-like pili (panel A). Panel B reveals abundant CFA/I pili synthesized by CVD 103-HgR (pSSVI215-cfaI/pM392), which appear as long, rather flexible filaments untypical of the rod-like appearance in wildtype ETEC. CS3 fibrillae synthesized by CVD 103-HgR (pSSVI215-CS3/pM392) display locally formed patches of dense material (panel C). In contrast, CS6 fibrillae synthesized by CVD 103-HgR (pM295) manifested as sparse depositions on the cell surface of gold particles from the labeled anti-CS6 antibodies with no clear fimbrial structure visible (panel D). Immunofluorescence detection further revealed that most, if not all, cells produce the CFs (data not shown).

3.4. Identification of individually expressed CFs by electron microscopy

3.5. Effect of CF operon and regulator gene dosages on CF expression

To correlate the presence of pilin in the recombinant CF expressing V. cholerae strains to the synthesis of sur-

To find out whether CF operon dosage had an influence on the amount of surface CF synthesized, we compared CF

4362

D. Favre et al. / Vaccine 24 (2006) 4354–4368

Fig. 4. Western blot analysis of CF pilin expression from different CF operon dosages: panel CFA/I—lanes: (1) CVD 103-HgR (pSSVI215cfaI/pM392) and (2) CHcfaI-1 (pM392). Panel CS3—lanes: (1) CVD 103-HgR (pSSVI215-CS3/pM392) and (2) CHCS3-2 (pM392). Panel CS6—lanes: (1) CVD 103-HgR (pSSVI215-CS6) and (2) CHCS6-1.

expression in strains expressing CF operons either as single copy/cell (chromosomal integration) or expressed at several copies/cell from plasmids. Evaluation of plasmid copy number in V. cholerae allowed to determine that ColE1- and P15A-based replicons normally replicating as medium to high copy numbers in E. coli K12 were replicating at rather low copy numbers (5–7 copies/cell) in V. cholerae (data not shown). Given a fixed regulator gene dosage, more pilin was not produced from CVD 103-HgR expressing plasmid-borne CFA/I and CS3 operons (Fig. 4, panels CFA/I and CS3, lane 1) than from corresponding strains harboring only one operon copy/cell (panels CFA/I and CS3, lane 2). In contrast, there was more CS6 pilin produced when the operon was plasmidborne (CS6 panel, lane 1) than as single chromosomal copy (CS6 panel, lane 2). We subsequently compared surface expression of CS3 and CS6 CFs in corresponding recombinant V. cholerae CVD 103-HgR when CF operons were present either at 1 copy/cell as chromosomal integrants or expressed from

plasmids with low copy numbers (5–7 copies/cell). In addition, to assess the influence of various regulator dosages, these studies were performed for the regulated CS3 operon in the context of the positive regulator expressed either from a chromosome location or plasmid-borne (pM392). Detection of expressed CFs was made using the whole cell pili ELISA assay (see Section 2), which enabled us to compare relative amounts of the tested antigen expressed on the bacterial cell surface (Table 2). A single copy of the CS3 operon under the control of a single regulator copy in strain CHCS3-R2 led to very good surface expression as judged from the high ELISA titer compared to strain CHCS3-2 which does not harbor a regulator and to CVD 103-HgR which was used as the negative control. A less than two-fold increase was seen when the CS3 operon dosage was increased from 1 to 6–8 copies/cell as present in CHCS3-R2 containing plasmid pSSVI215-CS3. Therefore, increasing the CF operon dosage did not translate in a corresponding increase in CF expression. Increasing the regulator gene dosage in the presence of a single CS3 operon also led to some level of increased CF expression. However, a concomitant increase in the CS3 operon dosage did not translate into a corresponding increase in CS3 expression. In contrast to the above results, the pili ELISA titer of unregulated CS6 CF was largely increased as the CS6 operon dosage was increased from 1 to 3–5 copies/cell. Under the conditions of the assay, there was no detectable surface expression when the CS6 operon was present at 1 copy/ cell. Taken together, these results indicate that positive regulation of ETEC CF expression in V. cholerae nearly abolishes the dependence of expression on CF gene dosage, while unregulated CF expression remains dependent on CF gene dosage.

Table 2 Effect of CS3 and CS6 operon dosage on fibrillae expression in V. cholerae strains carrying the positive regulator gene on a plasmid or chromosomally integrated Straina

CF expressionb

Copy number CS3 locus

Regulator

CVD 103-HgR CHCS3-2 CHCS3-R2 CHCS3-R2 (pSSVI215-CS3)

0 1 1 6–8

0 0 1 1

0.00 0.01 1.12 1.75

CHCS3-2 (pM392) CVD 103-HgR (pSSVI215-CS3/pM392)

1 5–7

5–7 5–7

1.87 ± 0.04 1.91 ± 0.09

CVD 103-HgR CHCS6-1 CVD 103-HgR (pSSVI215-CS6)

0 1 5–7

NAc NA NA

0.02 ± 0.01 0.01 ± 0.01 0.14 ± 0.03

± ± ± ±

0.00 0.00 0.09 0.08

CS6 locus

Growth of all strains was at 37 ◦ C in liquid CF medium with appropriate antibiotics. The bacterial suspensions used to coat ELISA plates were at an OD600 of 0.1. CVD 103-HgR served as the negative control. b The CS3 and CS6 antigens were probed by use of CS3-specific MAb 1G3 and 4C9, respectively. Each strain was tested in triplicate. CF expression is expressed as the average ELISA titer at OD450 . c NA: not applicable. a

D. Favre et al. / Vaccine 24 (2006) 4354–4368

4363

Table 3 Effect of temperature on CF expression in wild-type ETEC and recombinant V. cholerae strains Strain

CD79a CHcfaI-R1 DS198-1 CHCS3-R2 B7A CVD103-HgR (pM295)

CF

CFA/I CFA/I CS3 CS3 CS6 CS6

Percent expressiona 37 ◦ C

30 ◦ C

24 ◦ C

100 100 100 100 100 100

46 28 5 41 30 27

13 16 1 51 1 24

a The strains were grown at the indicated temperature on solid CF medium. The cells were then scraped from the agar and directly resuspended in 0.9% NaCl containing 0.5% formaldehyde. The cell suspensions were adjusted to an OD600 of 0.2 and used for analysis by whole cell pili ELISA. The results are expressed as the percentage of CF expression at 30 and 24 ◦ C relative to expression at 37 ◦ C.

Fig. 5. Western blot analysis of CF pilin expression at various growth phases in recombinant strains grown in liquid cultures at 37 ◦ C. The pilins were identified by use of polyclonal antisera. Panel CFA/I—lanes: (1) ETEC H10407 grown on solid CF; (2) CHcfaI-1 (pM392) grown on solid CF; (3) CHcfaI-1 (pM392) in liquid CF collected at OD600 = 0.5; (4) CHcfaI-1 (pM392) in liquid CF collected at OD600 = 1.5; (5) CHcfaI-1 (pM392) in liquid CF collected after 16 h incubation; (6) CVD103-HgR grown on solid CF. Panel CS3—lanes: (1) ETEC DS7-3 grown on solid CF; (2) CHCS3-2 (pM392) grown on solid CF; (3) CHCS3-2 (pM392) collected at OD600 = 0.5; (4) CHCS3-2 (pM392) CF collected at OD600 = 1.5; (5) CHCS3-2 (pM392) in liquid CF collected after 16 h incubation; (6) CVD103-HgR grown on solid CF. Panel CS6—lanes: (1) ETEC B7A grown on solid CF; (2) CHCS6-1 grown on solid CF; (3) CHCS6-1 in liquid CF collected at OD600 = 0.5; (4) CHCS6-1 grown in liquid CF collected at OD600 = 1.5; (5) CHCS6-1 in liquid CF collected after 16 h incubation; (6) CVD103-HgR grown on solid CF.

3.6. Conditions for ETEC CF expression in V. cholerae CVD103-HgR It is known that CF expression in ETEC is relatively tightly controlled [6]. As a consequence, temperature and medium composition are, in addition to the presence of the positive regulator, critical factors for a balanced expression of CF. To study the conditions leading to optimal expression of ETEC CFs in V. cholerae, we first investigated the effect of medium. In ETEC, pili such as CFA/I are known to be expressed better from solid CF agar compared with liquid CF [36,62]. This did not prove to be the case in CVD 103HgR for any of the three fimbriae tested (Fig. 5). CF pilin expression at 37 ◦ C from recombinant V. cholerae CHcfaI-1 (pM392), CHCS3-2 (pM392), or CHCS6-1, which express

CFA/I, CS3, and CS6, respectively, was similar from cells scraped from solid agar (lane 2) or from liquid medium (lanes 3–5). In addition, similar pilin synthesis for all three CFs was observed from samples collected at OD600 of 0.5, 1.5, and after overnight incubation (corresponding to exponential, early, and late stationary phases, respectively), indicating that CF pilin synthesis in these recombinant V. cholerae strains starts during exponential phase and persists well into stationary phase (lanes 3–5). Cultivation of CHcfaI-1 (pM392) in LB medium did only result in minimal pili expression (data not shown), showing the specificity of the regulation by CF medium. In ETEC, a temperature of 20 ◦ C is inhibiting for CFA/I synthesis compared with 37 ◦ C [18]. CF expression was analyzed by whole cell pili ELISA after growth of wild-type ETEC and recombinant V. cholerae strains expressing CS3 or CS6 on solid CF medium at 24, 30, and 37 ◦ C (Table 3). We found that similar temperature regulation applies to wild-type CFA/I-, CS3-, and CS6-expressing ETEC strains (CD79a, DS198-1, and B7A) which display decreased expression from 37 to 30 ◦ C and a further decrease from 30 to 24 ◦ C. In CF-expressing V. cholerae strains, a similar expression decrease was noted for all three CF types between 37 and 30 ◦ C. However, while a further expression decrease was observed for CFA/I in CHcfaI-R1 between 30 and 24 ◦ C, expression remained similar at 30 and 24 ◦ C for CS3 and CS6 in strains CHCS3-R2 and CVD 103-HgR (pM295), respectively. 3.7. Co-expression of multiple CFs in the same carrier strain Towards the goal of rationalizing the construction of a multivalent ETEC vaccine strain, we wanted to know whether it is also possible to achieve optimal expression of two pilins even when only one regulator copy is present. Using CHCS3R2 as the primary host, we constructed strain BB06, in which the CFA/I operon is chromosomally inserted within the hlyA gene (see Fig. 1). Fig. 6A shows abundant expression of both

4364

D. Favre et al. / Vaccine 24 (2006) 4354–4368 Table 4 Immunogenicity of the trivalent strain BB06 (pM295) in mice Antigen tested

Immunizing strains

Antibody titera (range)

CFA/I

CD79a BB06 (pM295) CVD103-HgR

82924 (71579–96872) 34923 (16735–61179) 0

CS3

DS198-1 BB06 (pM295) CVD103-HgR

72007 (64314–82996) 47279 (37378–60386) 0

CS6

B7A BB06 (pM295) CVD103-HgR

20352 (7978–30437) 3494 (1227–8481) 0

a Reciprocal value of the geometric mean of the serum dilution corresponding to an OD405 of 0.4.

3.8. Immunogenicity of the trivalent strain BB06 (pM295) in mice

Fig. 6. (Panel A) Multivalent pilin expression in CVD 103-HgR. The specific primary antibody used is indicated. The same cell extracts were loaded in each panel. Lanes: (1) CHcfaI-R1; (2) CHCS3-R2; (3) CVD103-HgR (pSSVI215-CS6); (4) CVD 103-HgR; (5) CD79a; (6) DS198-1; (7) B7A; (8) BB06. (Panel B) Electron micrograph of immunogold-labeled CFA/I and CS3 CFs expressed by strain BB06. The CFA/I pili were probed by use of the 2E5 MAb and detected using a secondary antibody conjugated with 10 nm gold particles. The CS3 fibrillae were probed by use of a rabbit CS3-specific polyclonal antibody and detected using a secondary antibody conjugated with 15 nm gold particles. The arrows depict the CFs as indicated. Bar = 0.2 ␮m.

CFA/I and CS3 pilins in BB06 (Fig. 6A, lane 8) which was comparable in magnitude to the synthesis of pili in corresponding monovalent strains CHcfaI-R1 (CFA/I panel, lane 1) and CHCS3-R2 (panel CS3, lane 2). In addition, both CFA/I and CS3 are synthesized in even higher amounts than the wild-type CFA/I-expressing strain CD79a (CFA/I panel, lane 5) and CS3-expressing strain DS198-1 (CS3 panel, lane 6). Using electron microscopy (Fig. 6B), we could further show that pilin expression of CFA/I and CS3 fimbriae in BB06 correlates with the expression of the corresponding surface structures exhibiting a similar phenotype as seen in monovalent strains CHcfaI-R1 and CHCS3-R2, respectively (see Fig. 3). We could also achieve CS6 expression in BB06 following electroporation of plasmid pM295 in the latter strain. Using Western blot and pili ELISA analyses, we could show that CS6 pilin, as well as the corresponding antigen, was expressed. However, the presence of CS6 could not be detected by electron microscopy (results not shown).

We tested the immunogenicity of the CFs in BB06 (pM295) following subcutaneous immunization of mice. CFs purified from the corresponding wild-type strains were used as coating antigens in the ELISA assay. Results shown in Table 4 indicate that CFA/I and CS3 expressed from BB06 (pM295) were nearly as immunogenic as from the corresponding wild-type ETEC strains. CS6 immunogenicity from BB06 (pM295), while significant, was somewhat lower compared to that from the wild-type CS6 ETEC strain B7A. Taken together these results indicate that at least three CFs can be expressed in immunogenic form in V. cholerae.

4. Discussion The present contribution relates to the expression of ETEC CFs in the live attenuated V. cholerae vaccine strain CVD 103-HgR with the aim of creating a carrier-based ETEC vaccine. The choice of CVD 103-HgR as the carrier strain was guided by the facts that: (i) the molecular mechanisms of pathogenicity of ETEC and V. cholerae are very similar, (ii) the high level of safety and immunogenicity of CVD 103-HgR as a cholera vaccine [63] has been extensively documented making it a promising carrier for the development of new vaccines, and (iii) previous studies from us and others have shown that V. cholerae can efficiently express foreign antigenic determinants [46–48,64–69]. In line with the observation that a positive regulator is required for CFA/I expression in ETEC [11,70,71], CFA/I pilin and corresponding surface structures were very efficiently synthesized in V. cholerae CVD 103-HgR, providing that positive regulation was available. Likewise, we found that optimal CS3 expression was also dependent on positive regulation. This was surprising since the CS3 operon, like the CS6 operon, is thought to function without positive regulation in ETEC [12,60]. Our experiments supported that the presence of a positive regulator did not enhance CS6 expression in recombinant V. cholerae. The need for regulation of CS3 in

D. Favre et al. / Vaccine 24 (2006) 4354–4368

V. cholerae, on the other hand, raises the possibility that, contrary to the current view, CS3 might be also regulated in wildtype ETEC. However, this remains to be demonstrated. The level of pilin expression from integrated CF operons in CVD 103-HgR was similar to that in the ETEC wild-type strains indicating that the V. cholerae transcription–translation machinery is quite efficient in this context, provided that the suitable heterologous regulatory elements were present. The pilin level translated into explicit expression of CFA/I, CS3, and CS6 on the surface of V. cholerae as observed by various methods including electron microscopy. Interestingly, electron microscopy examination revealed that the pattern of surface exposure and the structure of CFA/I pili made by V. cholerae CVD 103-HgR were different than CFA/I pili made by wild-type ETEC. It is likely that a homologous V. cholerae pili assembly machinery, possibly that of the toxin co-regulated pili (TCP), co-operates in the assembly of CFA/I pili. This may also be the case for CS3, which displayed a patchy surface occurrence in V. cholerae while in ETEC CS3 fibrillae are more uniformly spread over the entire bacterial surface [72]. However, in spite of these morphological differences, CFA/I and CS3 synthesized in V. cholerae CVD103-HgR were able to bind antibodies produced against the corresponding wild-type fimbriae, indicating that they maintained the same epitopes. Immunolabeling of CS6 produced more sparse labeling, probably due to a lower level of expression than CFA/I and CS3 or to differences in surface exposure. To optimize CF expression in V. cholerae we sought to better understand the contribution of positive regulation. Various combinations of CF and regulator gene dosages only led to relatively modest increases in CF expression compared to the situation where both the CF operon and the positive regulator are present at 1 copy/cell. This, together with the equally efficient concomitant expression of two different CF operons from one single, chromosomally integrated regulator, reflects the very efficient CF expression obtained by the single loci combination. Apparently, a single copy of the positive regulator gene allows the synthesis of sufficient regulator molecules to efficiently activate the transcription of several copies of the CF operons. This is compatible with the results of Froehlich et al. [73] who determined that the rns promoter in ETEC is strong. Since cells containing plasmid-borne copies of both the CF operon and the positive regulator did not show a corresponding increase in CF production, we expect that there is an upper limit to the amount of surface CF that can be produced. It is likely that the putative limiting factor may be at the level of transcription and/or translation of the CF operon rather than at the level of the CF export pathway since the amount of CF pilin as visualized in Western blot was not increased when CF operon dosage was increased in the presence of a plasmid-borne regulator gene. The up-regulation of ETEC CFs in V. cholerae appears to be a highly efficient process even if the amount of surface expressed CF is limited by some event occurring possibly at the level of transcription and/or translation. Nevertheless,

4365

in contrast to the unregulated CS6 expression, regulated CF expression appears to be largely independent of CF operon dosage. The conditions for optimal CF expression in ETEC are known and seem, at least in vitro, to be rather specific. Thus, CFs are not well expressed at 22 ◦ C, in media other than CF, or in liquid medium, even CF [36,62]. In V. cholerae, CF expression was also specific in regard to medium composition and growth temperature. However, importantly, the expression of all three tested CFs was similar in liquid or solid CF medium. The high expression in liquid medium may have broad potential advantages for vaccine production. In the case of live vaccines, this may enable the generation of large quantities of vaccine cells. In the case of vaccines based on purified fimbriae, the large-scale manufacture of fimbriaeted cells will also have a favorable economic impact. Together with the fact that parenteral administration of a trivalent recombinant strain in mice was highly immunogenic for all three expressed CFs, our results indicate that V. cholerae is a favorable carrier system for the expression of ETEC CFs. In other organisms such as S. typhi with CFA/I and CS3 [36], and S. flexneri with CFA/I and CS3 [41] and CFA/I and CS6 [74] the expression of two CFs has been shown and we confirmed that this is possible in V. cholerae as well. Our observations that V. cholerae is able to co-express multiple CFs paves the way for a multivalent ETEC vaccine, which would be a great economic advantage in view of the largescale manufacture of the vaccine. In conclusion, we have shown that the live attenuated V. cholerae vaccine strain CVD 103-HgR is able to efficiently synthesize various types of ETEC CFs and that more than one CF can be produced per strain. These results open the way for the testing of a prototype live oral carrier-based ETEC vaccine in humans.

Acknowledgements We gratefully acknowledge the excellent technical assistance of F. Rubi, M. Wyss, C. Furer, and S. K¨onig. We thank J. Scott for the gift of pEU2040, M. Wolf for ETEC wild-type strains DS7-3, DS198-1, and B7A, as well as for plasmids pM392 and pM295, D. Scott for pGB1, W. Gaastra and J. Blanco for the wild-type ETEC strains H10407 and CD79a, respectively, and F. Cassels for the gift of purified CFA/I, CS3, and CS6 fimbriae. This study was supported in part by Grant Nr. KTI-7304.1 LSPP-LS of the Swiss Federal Office for Industrial Education and Technology to J. Frey and M. Stoffel.

References [1] Steffen R, Castelli F, Dieter NH, Rombo L, Jane ZN. Vaccination against enterotoxigenic Escherichia coli, a cause of travelers’ diarrhea. J Travel Med 2005;12(2):102–7.

4366

D. Favre et al. / Vaccine 24 (2006) 4354–4368

[2] Tacket CO, Reid RH, Boedeker EC, Losonsky G, Nataro JP, Bhagat H, et al. Enteral immunization and challenge of volunteers given enterotoxigenic E. coli CFA/II encapsulated in biodegradable microspheres. Vaccine 1994;12(14):1270–4. [3] Qadri F, Svennerholm A-M, Faruque ASG, Sack RB. Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev 2005;18(3):465–83. [4] Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev 1998;11(1):142–201. [5] Boedeker EC. Vaccines for enterotoxigenic Escherichia coli: current status. Curr Opin Gastroenterol 2005;21(1):15–9. [6] Cassels FJ, Wolf MK. Colonization factors of diarrheagenic E. coli and their intestinal receptors. J Ind Microbiol 1995;15(3):214–26. [7] Gaastra W, Svennerholm AM. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol 1996;4(11):444–52. [8] Sizemore DR, Roland KL, Ryan US. Enterotoxigenic Escherichia coli virulence factors and vaccine approaches. Expert Rev Vaccines 2004;3(5):585–95. [9] Torres AG, Zhou X, Kaper JB. Adherence of diarrheagenic Escherichia coli strains to epithelial cells. Infect Immun 2005;73(1): 18–29. [10] Munson GP, Holcomb LG, Scott JR. Novel group of virulence activators within the AraC family that are not restricted to upstream binding sites. Infect Immun 2001;69(1):186–93. [11] Savelkoul PH, Willshaw GA, McConnell MM, Smith HR, Hamers AM, van der Zeijst BA, et al. Expression of CFA/I fimbriae is positively regulated. Microb Pathog 1990;8(2):91–9. [12] Caron J, Coffield LM, Scott JR. A plasmid-encoded regulatory gene, rns, required for expression of the CS1 and CS2 adhesins of enterotoxigenic Escherichia coli. Proc Natl Acad Sci USA 1989;86(3):963–7. [13] de Haan LA, Willshaw GA, van der Zeijst BA, Gaastra W. The nucleotide sequence of a regulatory gene present on a plasmid in an enterotoxigenic Escherichia coli strain of serotype O167:H5. FEMS Microbiol Lett 1991;67(3):341–6. [14] Gallegos MT, Schleif R, Bairoch A, Hofmann K, Ramos JL. Arac/XylS family of transcriptional regulators. Microbiol Mol Biol Rev 1997;61(4):393–410. [15] Munson GP, Scott JR. Binding site recognition by Rns, a virulence regulator in the AraC family. J Bacteriol 1999;181(7):2110–7. [16] Munson GP, Scott JR. Rns, a virulence regulator within the AraC family, requires binding sites upstream and downstream of its own promoter to function as an activator. Mol Microbiol 2000;36(6):1391–402. [17] Murphree D, Froehlich B, Scott JR. Transcriptional control of genes encoding CS1 pili: negative regulation by a silencer and positive regulation by Rns. J Bacteriol 1997;179(18):5736–43. [18] Jordi BJ, Dagberg B, de Haan LA, Hamers AM, van der Zeijst BA, Gaastra W, et al. The positive regulator CfaD overcomes the repression mediated by histone-like protein H-NS (H1) in the CFA/I fimbrial operon of Escherichia coli. EMBO J 1992;11(7):2627–32. [19] Jordi BJ, van der Zeijst BA, Gaastra W. Regions of the CFA/I promoter involved in the activation by the transcriptional activator CfaD and repression by the histone-like protein H-NS. Biochimie 1994;76(10–11):1052–4. [20] de Lorimier AJ, Byrd W, Hall ER, Vaughan WM, Tang D, Roberts ZJ, et al. Murine antibody response to intranasally administered enterotoxigenic Escherichia coli colonization factor CS6. Vaccine 2003;21(19–20):2548–55. [21] Katz DE, DeLorimier AJ, Wolf MK, Hall ER, Cassels FJ, van Hamont JE, et al. Oral immunization of adult volunteers with microencapsulated enterotoxigenic Escherichia coli (ETEC) CS6 antigen. Vaccine 2003;21(5–6):341–6. [22] Tacket CO, Levine MM. Vaccines against enterotoxigenic Escherichia coli infections, part ii, live oral vaccines and subunits

[23]

[24]

[25]

[26] [27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

(purified fimbriae and toxin subunits) vaccines. In: Levine MM, Woodrow GC, Kaper JB, Cobon GS, editors. New generation vaccines. New York: Marcel Dekker Inc.; 1987. p. 875–83. Guerena-Burgueno F, Hall ER, Taylor DN, Cassels FJ, Scott DA, Wolf MK, et al. Safety and immunogenicity of a prototype enterotoxigenic Escherichia coli vaccine administered transcutaneously. Infect Immun 2002;70(4):1874–80. Yu J, Cassels F, Scharton-Kersten T, Hammond SA, Hartman A, Angov E, et al. Transcutaneous immunization using colonization factor and heat-labile enterotoxin induces correlates of protective immunity for enterotoxigenic Escherichia coli. Infect Immun 2002;70(3):1056–68. Lee JY, Yu J, Henderson D, Langridge WH. Plant-synthesized E. coli CFA/I fimbrial protein protects Caco-2 cells from bacterial attachment. Vaccine 2004;23(2):222–31. Tacket CO. Plant-derived vaccines against diarrhoeal diseases. Expert Opin Biol Ther 2004;4(5):719–28. Lasaro MO, Luiz WB, Sbrogio-Almeida ME, Nishimura LS, Guth BE, Ferreira LC. Combined vaccine regimen based on parenteral priming with a DNA vaccine and administration of an oral booster consisting of a recombinant Salmonella enterica serovar Typhimurium vaccine strain for immunization against infection with human-derived enterotoxigenic Escherichia coli strains. Infect Immun 2004;72(11):6480–91. Lasaro MO, Luiz WB, Sbrogio-Almeida ME, Ferreira LC. Primeboost vaccine regimen confers protective immunity to human-derived enterotoxigenic Escherichia coli. Vaccine 2005;23(19):2430–8. Cohen D, Orr N, Haim M, Ashkenazi S, Robin G, Green MS, et al. Safety and immunogenicity of two different lots of the oral, killed enterotoxigenic Escherichia coli-cholera toxin B subunit vaccine in Israeli young adults. Infect Immun 2000;68(8):4492–7. Jertborn M, Ahren C, Holmgren J, Svennerholm AM. Safety and immunogenicity of an oral inactivated enterotoxigenic Escherichia coli vaccine. Vaccine 1998;16(2–3):255–60. Qadri F, Wenneras C, Ahmed F, Asaduzzaman M, Saha D, Albert MJ, et al. Safety and immunogenicity of an oral, inactivated enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine in Bangladeshi adults and children. Vaccine 2000;18(24):2704–12. Savarino SJ, Hall ER, Bassily S, Wierzba TF, Youssef FG, Peruski Jr LF, et al. Introductory evaluation of an oral, killed whole cell enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine in Egyptian infants. Pediatr Infect Dis J 2002;21(4):322–30. Svennerholm AM, Ahren C, Jertborn M. Vaccines against enterotoxigenic Escherichia coli infections, part i, oral inactivated vaccines against enterotoxigenic Escherichia coli. In: Levine MM, Woodrow GC, Kaper JB, Cobon GS, editors. New generation vaccines. New York: Marcel Dekker Inc.; 1997. p. 875–83. Turner AK, Terry TD, Sack DA, Londono-Arcila P, Darsley MJ. Construction and characterization of genetically defined aro omp mutants of enterotoxigenic Escherichia coli and preliminary studies of safety and immunogenicity in humans. Infect Immun 2001;69(8):4969–79. Wu S, Pascual DW, Vancott JL, McGhee JR, Maneval Jr DR, Levine MM, Hone DM. Immune responses to novel Escherichia coli and Salmonella typhimurium vectors that express colonization factor antigen I (CFA/I) of enterotoxigenic E. coli in the absence of the CFA/I positive regulator cfaR. Infect Immun 1995;63(12):4933–8. Giron JA, Xu JG, Gonzalez CR, Hone D, Kaper JB, Levine MM. Simultaneous expression of CFA/I and CS3 colonization factor antigens of enterotoxigenic Escherichia coli by delta aroC, delta aroD Salmonella typhi vaccine strain CVD 908. Vaccine 1995;13(10):939–46. Altboum Z, Barry EM, Losonsky G, Galen JE, Levine MM. Attenuated Shigella flexneri 2a Delta guaBA strain CVD 1204 expressing enterotoxigenic Escherichia coli (ETEC) CS2 and CS3 fimbriae as a live mucosal vaccine against Shigella and ETEC infection. Infect Immun 2001;69(5):3150–8.

D. Favre et al. / Vaccine 24 (2006) 4354–4368 [38] Altboum Z, Levine MM, Galen JE, Barry EM. Genetic characterization and immunogenicity of coli surface antigen 4 from enterotoxigenic Escherichia coli when it is expressed in a Shigella live-vector strain. Infect Immun 2003;71(3):1352–60. [39] Barry EM, Altboum Z, Losonsky G, Levine MM. Immune responses elicited against multiple enterotoxigenic Escherichia coli fimbriae and mutant LT expressed in attenuated Shigella vaccine strains. Vaccine 2003;21(5–6):333–40. [40] Koprowski H, Levine MM, Anderson RJ, Losonsky G, Pizza M, Barry EM. Attenuated Shigella flexneri 2a vaccine strain CVD 1204 expressing colonization factor antigen I and mutant heatlabile enterotoxin of enterotoxigenic Escherichia coli. Infect Immun 2000;68(9):4884–92. [41] Noriega FR, Losonsky G, Wang JY, Formal SB, Levine MM. Further characterization of delta aroA delta virG Shigella flexneri 2a strain CVD 1203 as a mucosal Shigella vaccine and as a live-vector vaccine for delivering antigens of enterotoxigenic Escherichia coli. Infect Immun 1996;64(1):23–7. [42] Ranallo RT, Fonseka CP, Cassels F, Srinivasan J, Venkatesan MM. Construction and characterization of bivalent Shigella flexneri 2a vaccine strains SC608(pCFAI) and SC608(pCFAI/LTB) that express antigens from enterotoxigenic Escherichia coli. Infect Immun 2005;73(1):258–67. [43] Favre D, Struck MM, Cryz Jr SJ, Viret JF. Further molecular characterization and stability of the live oral attenuated cholera vaccine strain CVD103-HgR. Vaccine 1996;14(6):526–31. [44] Ketley JM, Michalski J, Galen J, Levine MM, Kaper JB. Construction of genetically marked Vibrio cholerae O1 vaccine strains. FEMS Microbiol Lett 1993;111(1):15–21. [45] Levine MM, Kaper JB. Live oral vaccines against cholera: an update. Vaccine 1993;11(2):207–12. [46] Favre D, Cryz Jr SJ, Viret JF. Development of Shigella sonnei live oral vaccines based on defined rfb Inaba deletion mutants of Vibrio cholerae expressing the Shigella serotype D O polysaccharide. Infect Immun 1996;64(2):576–84. [47] Viret JF, Cryz Jr SJ, Favre D. Expression of Shigella sonnei lipopolysaccharide in Vibrio cholerae. Mol Microbiol 1996;19(5):949–63. [48] Favre D, Cryz Jr SJ, Viret JF. Construction and characterization of a potential live oral carrier-based vaccine against Vibrio cholerae O139. Infect Immun 1996;64(9):3565–70. [49] Acheson DW, Levine MM, Kaper JB, Keusch GT. Protective immunity to Shiga-like toxin I following oral immunization with Shiga-like toxin I B-subunit-producing Vibrio cholerae CVD 103-HgR. Infect Immun 1996;64(1):355–7. [50] Tribble DR, Hale TL, Taylor DN. ETEC and enteric vaccines. In: Jong EC, Zuckerman J, editors. Traveler’s vaccines. Hamilton, Canada: BC Dekker Inc.; 2004. p. 275–97. [51] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989. [52] Pitcher DG, Saunders NA, Owen RJ. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 1989;8:151–6. [53] Favre D. Improved phenol-based method for the isolation of DNA fragments from low melting temperature agarose gels. Biotechniques 1992;13(1):22, 25-2, 26. [54] Favre D, Viret JF. Gene replacement in Gram-negative bacteria: the pMAKSAC vectors. Biotechniques 2000;28(2):198–200, 202, 204. [55] Favre D, Viret JF. Versatile cosmid vectors for facilitated analysis and subcloning of the insert. Gene 1996;178(1–2):43–9. [56] Stoker NG, Fairweather NF, Spratt BG. Versatile low-copynumber plasmid vectors for cloning in Escherichia coli. Gene 1982;18(3):335–41. [57] Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256(5517):495–7. [58] Alm RA, Stroeher UH, Manning PA. Extracellular proteins of Vibrio cholerae: nucleotide sequence of the structural gene (hlyA) for the

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66] [67] [68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

4367

haemolysin of the haemolytic El Tor strain 017 and characterization of the hlyA mutation in the non-haemolytic classical strain 569B. Mol Microbiol 1988;2(4):481–8. Kaper JB, Mobley HL, Michalski J, Herrington DA, Levine MM. Recent advances in developing a safe and effective live oral attenuated Vibrio cholerae vaccine. In: Othomo N, Sack RB, editors. Advances in research on cholera and related diseases. Tokyo, Japan: KTK Scientific Publishers; 1988. p. 161–7. Caron J, Scott JR. A rns-like regulatory gene for colonization factor antigen I (CFA/I) that controls expression of CFA/I pilin. Infect Immun 1990;58(4):874–8. Willshaw GA, McConnell MM, Smith HR, Rowe B. Structural and regulatory genes for coli surface associated antigen 4 (CS4) are encoded by separate plasmids in enterotoxigenic Escherichia coli strains of serotype 0.25.H42. FEMS Microbiol Lett 1990;56(3):255–60. Evans DG, Evans Jr DJ, Tjoa W. Hemagglutination of human group A erythrocytes by enterotoxigenic Escherichia coli isolated from adults with diarrhea: correlation with colonization factor. Infect Immun 1977;18(2):330–7. WHO. New frontiers in the development of vaccines against enterotoxigenic (ETEC) and enterohaemorrhagic (EHEC) E. coli infections. Weekly Ep Rec 1999;74:97–104. Butterton JR, Ryan ET, Acheson DW, Calderwood SB. Coexpression of the B subunit of Shiga toxin 1 and EaeA from enterohemorrhagic Escherichia coli in Vibrio cholerae vaccine strains. Infect Immun 1997;65(6):2127–35. Killeen K, Spriggs D, Mekalanos J. Bacterial mucosal vaccines: Vibrio cholerae as a live attenuated vaccine/vector paradigm. Curr Top Microbiol Immunol 1999;236:237–54. Kochi SK, Killeen KP, Ryan US. Advances in the development of bacterial vector technology. Expert Rev Vaccines 2003;2(1):31–43. Kotton CN, Hohmann EL. Enteric pathogens as vaccine vectors for foreign antigen delivery. Infect Immun 2004;72(10):5535–47. Ryan ET, Butterton JR, Zhang T, Baker MA, Stanley Jr SL, Calderwood SB. Oral immunization with attenuated vaccine strains of Vibrio cholerae expressing a dodecapeptide repeat of the serine-rich Entamoeba histolytica protein fused to the cholera toxin B subunit induces systemic and mucosal antiamebic and anti-V. cholerae antibody responses in mice. Infect Immun 1997;65(8):3118–25. Silva AJ, Mohan A, Benitez JA. Cholera vaccine candidate 638: intranasal immunogenicity and expression of a foreign antigen from the pulmonary pathogen Coccidioides immitis. Vaccine 2003;21(32):4715–21. Grewal HM, Gaastra W, Svennerholm AM, Roli J, Sommerfelt H. Induction of colonization factor antigen I (CFA/I) and coli surface antigen 4 (CS4) of enterotoxigenic Escherichia coli: relevance for vaccine production. Vaccine 1993;11(2):221–6. Hibberd ML, McConnell MM, Willshaw GA, Smith HR, Rowe B. Positive regulation of colonization factor antigen I (CFA/I) production by enterotoxigenic Escherichia coli producing the colonization factors CS5, CS6, CS7, CS17, PCFO9, PCFO159:H4 and PCFO166. J Gen Microbiol 1991;137(8):1963–70. Levine MM, Ristaino P, Marley G, Smyth C, Knutton S, Boedeker E, et al. Coli surface antigens 1 and 3 of colonization factor antigen IIpositive enterotoxigenic Escherichia coli: morphology, purification, and immune responses in humans. Infect Immun 1984;44(2):409–20. Froehlich B, Husmann L, Caron J, Scott JR. Regulation of rns, a positive regulatory factor for pili of enterotoxigenic Escherichia coli. J Bacteriol 1994;176(17):5385–92. Zheng JP, Wang LC, Wang P, Luo G, Li SQ, Duan HQ, et al. Coexpression of CFA/I and CS6 of enterotoxigenic Escherichia coli (ETEC) in Shigella flexneri 2a T32 derivative strain FWL01. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao 2003;35(11):1005–10. Simon R, Priefer U, P¨uhler A. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Biotechnology 1983;1(1):784–91.

4368

D. Favre et al. / Vaccine 24 (2006) 4354–4368

[76] Evans DG, Silver RP, Evans Jr DJ, Chase DG, Gorbach SL. Plasmid-controlled colonization factor associated with virulence in Escherichia coli enterotoxigenic for humans. Infect Immun 1975;12(3):656–67. [77] Blanco J, Gonzalez EA, Blanco M, Garabal JI, Alonso MP, Fernandez S, et al. Enterotoxigenic Escherichia coli associated with infant diarrhoea in Galicia, north-western Spain. J Med Microbiol 1991;35(3):162–7.

[78] Pridmore RD. New and versatile cloning vectors with kanamycinresistance marker. Gene 1987;56(2–3):309–12. [79] Lerner CG, Inouye M. Low copy number plasmids for regulated lowlevel expression of cloned genes in Escherichia coli with blue/white insert screening capability. Nucleic Acids Res 1990;18(15): 4631.