INTRODUCTION

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Development of Vibrio cholerae as a vector organism capable of expressing heterologous antigens at mucosal surfaces is attractive. V. cholerae is a noninvasive organism that effectively colonizes the intestinal mucosa of humans, and infection with V. cholerae results in immune responses that are long-lasting (10, 15). Live, attenuated vaccine strains of V. cholerae can be administered orally, and such strains have been well characterized and shown to be both safe and immunogenic in humans (11, 12, 14, 22, 24). V. cholerae vaccines strains can secrete immunoadjuvants in vivo, such as the nontoxic immunoadjuvantive mutant of Escherichia coli heat-labile enterotoxin LT_(R192G) (19), and V. cholerae vaccines strains can express large quantities of heterologous antigens in a balanced lethal plasmid expression system (20). V. cholerae vaccine strains can also efficiently express and secrete both large and small heterologous antigens (2, 3, 17, 18), and a mouse model of V. cholerae infection that permits rapid preliminary evaluation of V. cholerae vaccine and vector strains in vivo has been developed (4, 6). The optimal promoter for in vivo expression of heterologous antigens by vaccine and vector strains of V. cholerae is, however, unclear.

Constitutive promoters can drive high-level expression of certain antigens; however, such expression can be toxic to bacterial cells (5). In contrast, in vivo-induced promoters may have no or low-level activity when evaluated in vitro, but such promoters may be extremely active in vivo (5, 16). Strains expressing heterologous antigens from in vivo-induced promoters may be less compromised than those expressing antigens from constitutive promoters, and in vivo expression of heterologous antigens by in vivo-induced promoters may exceed that of constitutive promoters (5, 16).

To examine optimal promoter activity in V. cholerae vaccine and vector strains, we compared in vitro and in vivo activities of a number of promoters. We used derivatives of V. cholerae vaccine strain Peru2 (V. cholerae O1 El Tor C6709 attRS1) expressing a nontoxic B subunit (CtxB) of cholera toxin from a plasmid; strains used in this study were identical except for the identity of the promoter driving CtxB expression. We evaluated the tac promoter, which is constitutively active in V. cholerae since V. cholerae lacks the lacI^q repressor (1). We also examined two in vivo-induced V. cholerae promoters: the heat shock htpG promoter (induced under conditions of environmental stress) (13) and the V. cholerae iron-regulated irgA promoter (induced under low-iron conditions) (7, 9). We confirmed appropriate in vitro regulation of these promoters, and we analyzed systemic and mucosal immune responses to CtxB in mice inoculated with the various vaccine strains of V. cholerae.

	MATERIALS AND METHODS

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Bacterial strains and media. The bacterial strains and plasmids used in this study are described in Table 1. 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).

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 unique NheI 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 of ctxB, 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 the EcoRI and PstI sites of pKK223-3 immediately downstream of the tac promoter.

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. cholerae Peru2(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).

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

	RESULTS AND DISCUSSION

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In vitro analysis of CtxB expression. Cultures of V. cholerae Peru2 containing plasmid pETR1, pETR13, pETR12, or control plasmid pMCSETR1B were grown under a number of in vitro conditions, and supernatants were analyzed for CtxB (Table 2). Peru2(pETR1), with the tac promoter controlling CtxB expression, secreted CtxB to the supernatant at a range of temperatures (25, 30, and 37°C). Peru2(pETR1) expressed approximately 2 to 5 µg/ml/OD₆₀₀ at 30 and 37°C. Lower expression (0.5 to 1 µg/ml/OD₆₀₀) occurred at 25°C. CtxB was not detected in culture supernatants of Peru2(pETR1) grown at 42°C. Strain Peru2(pETR13), with the V. cholerae heat shock htpG promoter controlling CtxB expression, did not secrete detectable amounts of CtxB when grown at 25 or 30°C but did express CtxB (approximately 60 ng/ml/OD₆₀₀) when grown at 37°C. Peru2(pETR12), with the irgA promoter controlling CtxB expression, secreted approximately 40- to 50-fold more CtxB into supernatants when grown under low-iron conditions (approximately 4 to 5 µg/ml/OD₆₀₀) compared to expression during normal iron conditions (approximately 100 ng/ml/OD₆₀₀). Peru2(pMCSETR1B), containing a promoterless ctxB gene, produced no CtxB under any in vitro condition.

Measurement of vibriocidal antibody responses. Vibriocidal antibodies were measured in serum samples collected on day 84 (Fig. 1). Vibriocidal antibodies are directed against V. cholerae organisms themselves and reflect the ability of V. cholerae strains to both survive in vivo and colonize the intestinal surface (20). Vibriocidal antibody levels were equivalent in all groups of animals, suggesting that in vivo survival patterns of the various V. cholerae vaccine strains, including Peru2(pETR1), in which the tac promoter drives high-level constitutive expression of CtxB, were equivalent, as well.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Geometric mean titers (GMT) of vibriocidal antibody responses on day 84 samples. Groups of mice were inoculated with Peru2(pMCSETR1B) (CONTROL), Peru2(pETR1) (tacp  ctxB), Peru2(pETR13) (htpGp  ctxB), or Peru2(pETR12) (irgAp  ctxB). See text for details. Error bars represent standard errors of the mean for each group. Vibriocidal responses were comparable in all groups of mice.

Measurement of systemic and mucosal anti-CtxB antibodies. Systemic (serum) and mucosal (stool and bile) anti-CtxB antibodies were measured in samples collected on day 84 (Fig. 2). Compared to responses in mice that received control strain Peru2(pMCSETR1B), mice that received vaccine strains expressing CtxB under the control of the tac or irgA promoter had significant serum anti-CtxB IgG responses (P  0.05), confirming previous results (3). Compared to responses in mice that received Peru2(pETR13) expressing CtxB under the control of the heat shock htpG promoter, and compared to responses in mice that received Peru2(pETR13) expressing CtxB under the control of the iron-regulated irgA promoter, anti-CtxB responses were highest in mice that received Peru2(pETR1), the strain expressing CtxB under the control of the tac promoter. Mice that received V. cholerae Peru2(pETR1) had the highest level of anti-CtxB responses in all samples: serum IgG (P  0.05), serum IgA (P  0.05), stool IgA (P  0.05), and bile IgA (P  0.05).

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Anti-CtxB enzyme-linked immunosorbent assay results on day 84 samples. Mice were inoculated orally with Peru2(pMCSETR1B) (CONTROL), Peru2(pETR1) (tacp  ctxB), Peru2(pETR13) (htpGp  ctxB), or Peru2(pETR12) (irgAp  ctxB). The geometric mean and standard error of the mean are reported for each group. *, P  0.05 compared to mice that received Peru2(pETR13) and compared to mice that received Peru2(pETR12).