In Vitro and In Vivo Analyses of Constitutive and In Vivo-Induced Promoters in Attenuated Vaccine and Vector Strains ofVibrio cholerae

Bacterial strains and media.The bacterial strains and plasmids used in this study are described in Table1. All strains were maintained at −70°C in Luria-Bertani (LB) broth (21) containing 15% glycerol. All cultures contained either streptomycin (100 μg/ml) or ampicillin (100 μg/ml). Cultures were grown at indicated temperatures with aeration.

Recombinant DNA methods.Isolation of plasmid DNA, restriction digestions, and agarose gel electrophoresis were performed using standard procedures (21). DNA sequencing was performed at the DNA Sequencing Core Facility, Department of Molecular Biology, Massachusetts General Hospital, using ABI Prism DiTerminator cycle sequencing with AmpliTaq DNA polymerase FS with an ABI 377 DNA sequencer (Perkin-Elmer Applied Biosystems Division, Foster City, Calif.) (17).

Plasmids were transformed into E. coli JM105 by using standard techniques or were electroporated into V. choleraewith a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.) as instructed by the manufacturer and modified for electroporation intoV. cholerae as previously described (8). Electroporation conditions were 2,500 V at 25-mF capacitance, producing time constants of 4.8 to 4.9 ms.

DNA restriction endonucleases, T4 DNA ligase, calf intestinal alkaline phosphatase, and Klenow fragment of DNA polymerase I were used as specified by manufacturers. Pfu DNA polymerase (Stratagene, La Jolla, Calif.) was used for thermocyclic DNA amplification, using reaction mixtures and protocols as previously described (18). Restriction enzyme-digested plasmid DNA fragments were fractionated on 1% agarose gels; required DNA fragments were removed under UV illumination and recovered using GenElute agarose spin columns (Supelco Inc., Bellefonte, Pa.).

Plasmid constructions.Plasmid pETR1 was constructed by recombinant mutagenic PCR using two oligonucleotide primer pairs (primers 1 plus 3 and primers 2 plus 4) to amplify ctxB from the genome of V. cholerae C6709 and to introduce a uniqueNheI site 6 bp 3′ to the coding sequence of the CtxB leader peptide. Primer 1 (5′-ACTGACAGCTGGGAATTCTAAGGATGAATTATGATTAAATTAAAA-3′) and primer 3 (5′-AGTAATATTTTGGCTAGCAGGTGTTCCATGTGCATATGC-3′), including EcoRI and NheI restriction enzyme sites, respectively (underlined), were initially used to amplify a 110-bp PCR product extending from the Shine-Dalgarno sequence to that of the leader peptide of CtxB, inclusively. Primer 2 (5′-TCAGACTTCAGACTGCAGGGGCAAAACGGTTGCTTCTCATCATCG-3′) and primer 4 (5′-CATGGAACACCTGCTAGCCAAAATAATACTGATTTGTGT-3′), including PstI and NheI restriction enzyme sites, respectively (underlined), were used to amplify a 406-bp PCR product that extended from the leader sequence to the stop codon ofctxB, inclusively. Overlap extension PCR, using primers 1 and 2, was performed with equimolar concentrations of gel-purified 110- and 406-bp PCR products for 10 cycles (94°C, 1 min; 50°C, 1 min; 72°C, 30 s). The final 393-bp fragment was cloned into theEcoRI and PstI sites of pKK223-3 immediately downstream of the tac promoter.

To construct pMCSETR1B, a BamHI-EcoRI DNA fragment containing the tac promoter of plasmid pETR1 was replaced with a polylinker that includedBamHI-NsiI-XbaI-EcoRV-BglII-SpeI-EcoRI-Pst-I restriction enzyme sites (18). To prohibit translational read-through into ctxB, the 3′ end of the multiple cloning site included stop codons in all three reading frames. Plasmid pMCSETR1B, therefore, contained a multiple cloning site immediately upstream of a 393-bp promoterless ctxB gene from V. cholerae C6709.

Plasmid pVC100 contains the toxR-htpG intergenic region ofV. cholerae El Tor E7946 as anEcoRI-DraI DNA fragment inserted into theHincII restriction site of the polylinker of pUC19 (13). Plasmid pETR13 was constructed by cloning an approximately 600-bp BamHI-XbaI DNA fragment containing the toxR-htpG intergenic region from pVC100 into pMCSETR1B, such that the modified ctxB gene was placed under the transcriptional control of the htpG promoter.

Plasmid pMBG126 contains a 510-bpHindIII-SmaI DNA fragment that includes the iron-regulated irgBA genes from V. cholerae O395; this fragment had previously been blunt-end ligated into theHindIII-SphI sites of the polylinker of pUC18 (2). To construct plasmid pETR12, a 438-bpHindIII-EcoRV DNA fragment from pMBG126 containing the irgA promoter was blunt-end ligated into theEcoRV site of pMCSETR1B. The 438-bp insert was then truncated with NcoI and EcoRV, treated with the Klenow fragment of DNA polymerase I, and religated; this truncation removed a 183-bp nonpromoter fragment of DNA and placed ctxBunder the transcriptional control of a 255-bp insert containing theirgA promoter.

Plasmids pMCSETR1B, pETR1, pETR13, and pETR12 were confirmed by restriction enzyme digestion and sequence analysis; plasmids were electroporated into V. cholerae vaccine strain Peru2.

In vitro analysis of various promoters controlling CtxB expression.In vitro expression of CtxB by the various V. cholerae vaccine strains was analyzed by measuring CtxB concentrations in culture supernatants as previously described (6, 18, 19). Overnight cultures of V. choleraePeru2(pMCSETR1B), Peru2(pETR1), and Peru2(pETR13) were grown at 25, 30, 37, and 42°C. Overnight cultures of Peru2(pMCSETR1B) and Peru2(pETR12) were grown at 37°C in LB containing ampicillin (normal iron conditions) and in LB containing ampicillin and 0.150 mM 2,2′-dipyridyl, an iron chelator producing low-iron conditions (7). Cell-free supernatants of overnight cultures were serially diluted in phosphate-buffered saline (PBS)–0.05% Tween 20 (Sigma Chemical Co., St. Louis, Mo.) (PBS-T; undiluted to 1:128), applied to 96-well microtiter plates previously coated with 100 ng of type III ganglioside (Sigma) per well in 50 mM carbonate buffer (pH 9.6), and subsequently blocked with PBS–1% bovine serum albumin (Sigma). Plates were incubated at 37°C for 1 h and washed with PBS-T, and a 1:2,000 dilution of goat anti-CtxB (List Biological Laboratories, Inc., Campbell, Calif.) in PBS-T was applied to each well. Following incubation at 37°C for 1 h, plates were washed with PBS-T, and a 1:2,000 dilution of anti-goat immunoglobulin G (IgG)-horseradish peroxidase conjugate (Southern Biotechnology Associates Inc., Birmingham, Ala.) was added to each well. Plates were then incubated at 37°C for 1 h, washed with PBS-T, and developed with a 1-mg/ml solution of 2,2′-azinobis(ethylbenzthiazolinesulfonic acid) (ABTS; Sigma) with 0.1% H₂O₂ (Sigma). The optical density at 405 nm (OD₄₀₅) was read in a Vmax microplate reader (Molecular Devices Corp., Sunnyvale, Calif.) and compared with that of a standard curve generated using dilutions of purified CtxB (List) in PBS-T.

Inoculation of germfree mice.Immediately upon removal from their shipping container, four groups of 5 to 19 germfree female Swiss mice, 3 to 4 weeks old (Taconic Farms, Inc., Germantown, N.Y.), were orally inoculated by gastric intubation with 250 μl of inoculum containing approximately 10⁹ CFU of Peru2(pMCSETR1B), Peru2(pETR1), Peru2(PETR13), or Peru2(pETR12) resuspended in 0.5 M NaHCO₃ (pH 8.0) (6). Mice were subsequently housed in non-germfree conditions. All groups of mice received a primary oral vaccination series administered on days 0, 2, 4, and 6; oral booster inoculations of 10⁹ CFU in 125 μl were administered on days 28, 42, 56, and 70.

Immunological sampling.Mice were sacrificed on day 84, at which time blood, bile, and stool samples were collected, processed, aliquoted, and stored as previously described (19).

Detection of immune responses.Serum vibriocidal antibody titers were measured in a microassay as previously described (18). Anti-CtxB antibody responses were detected using microtiter plates previously coated with ganglioside and CtxB (18, 19). To detect anti-CtxB IgG and IgA antibodies in sera, duplicate samples of 1:200 dilutions of sera in PBS-T were added to wells previously coated with ganglioside-CtxB. Following overnight incubation, goat anti-mouse IgG antibody conjugated to biotin or goat anti-mouse IgA antibody conjugated to biotin (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) was applied to each well. After the addition of a 1:4,000 dilution of streptavidin-horseradish peroxidase conjugate (Zymed Laboratories, Inc., South San Francisco, Calif.) in PBS-T, plates were developed for peroxidase activity as described above. The OD₄₀₅ was determined kinetically with the Vmax microplate reader. Plates were read for 5 min at 19-s intervals, and the maximum slope for an OD change of 0.2 U was reported as milli-OD units per minute (6, 17).

To detect specific IgA antibody responses in stool and bile, concentrations of total IgA in stool and bile were first measured. Duplicate samples of serial twofold dilutions of processed stool (1:100 to 1:800) or bile (1:800 to 1:6,400) in PBS-T were added to wells previously coated with 100 ng of rat monoclonal anti-mouse IgA antibody R5-140 (Pharmingen, San Diego, Calif.) (19). After the addition of a 1:2,000 dilution of goat anti-mouse IgA antibody conjugated to biotin and streptavidin-horseradish peroxidase conjugate, plates were developed for peroxidase activity as described above. Comparison was made to a mouse IgA standard (Kappa TEPC 15; Sigma). To detect specific anti-CtxB antibodies in stool and bile, 200 ng of total biliary IgA (single) or 200 ng of total stool IgA (duplicate) in PBS-T was added to wells previously coated with ganglioside-CtxB (19, 20). Plates were incubated overnight at room temperature and washed with PBS-T, following which goat anti-mouse IgA antibody conjugated to biotin and streptavidin-horseradish peroxidase conjugate were added. Plates were developed for peroxidase activity, and the OD₄₀₅ was determined kinetically.

Statistics and graphs.Statistical analysis for the comparison of geometric means was performed with the Mann-WhitneyU test for nonparametric data using SPSS for Windows 8.0. Data were plotted using GraphPad PRISM, version 3 (GraphPad Software Inc., San Diego, Calif.).