salmonella vaccine vectors displaying delayed antigen synthesis in vivo to enhance immunogenicity

INFECTION AND IMMUNITY, Sept. 2010, p. 3969–3980 Vol. 78, No. 90019-9567/10/$12.00 doi:10.1128/IAI.00444-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Salmonella Vaccine Vectors Displaying Delayed Antigen SynthesisIn Vivo To Enhance Immunogenicity�†

Shifeng Wang,1,2 Yuhua Li,1 Giorgio Scarpellini,1,2 Wei Kong,1,2 HuoYing Shi,1 Chang-Ho Baek,1,2

Bronwyn Gunn,1,2 Soo-Young Wanda,1,2 Kenneth L. Roland,1 Xin Zhang,2Patti Senechal-Willis,1 and Roy Curtiss III1,2*

Center for Infectious Diseases and Vaccinology, The Biodesign Institute and School of Life Sciences, Arizona State University,Tempe, Arizona 85287,1 and Department of Biology, Washington University, St. Louis, Missouri 631302

Received 29 April 2010/Returned for modification 7 June 2010/Accepted 24 June 2010

We have developed a regulated delayed antigen synthesis (RDAS) system for use in recombinant attenuatedSalmonella vaccine (RASV) strains to enhance immune responses by reducing the adverse effects of high-levelantigen synthesis. This system includes a chromosomal repressor gene, lacI, expressed from the arabinose-regulated araC PBAD promoter. LacI serves to regulate expression from a plasmid promoter, Ptrc, that directsantigen synthesis. In the presence of arabinose LacI is produced, which binds to Ptrc, blocking antigensynthesis. In vivo, an arabinose-poor environment, the concentration of LacI decreases with each cell division,allowing increased antigen synthesis. To optimize the system and for comparison, we altered the lacI ribosome-binding site, start codon, and/or codon content to construct RDAS strains �9095, �9959, and �9241, synthe-sizing from low to high levels of LacI, respectively, and non-RDAS strain �9555 as a control. We evaluated thissystem with two test antigens, the green fluorescent protein for initial in vitro assessment and the Streptococcuspneumoniae PspA protein for validation of our system in mice. All RASV strains expressing PspA generatedhigh antilipopolysaccharide antibody titers, indicating that expression of lacI did not interfere with the capacityto induce an immune response. Strain �9241 induced significantly higher anti-PspA IgG and IgA antibodytiters than strain �9555, which expressed PspA constitutively. Anti-PspA antibody titers were inversely cor-related to the level of LacI synthesis. Strain �9241 also induced significantly greater protective efficacy againstchallenge with virulent S. pneumoniae. These results suggest that regulated delayed antigen synthesis is usefulfor improving immunogenicity of RASV strains.

Antigens delivered by recombinant attenuated Salmonellavaccine (RASV) strains induce strong systemic and mucosalimmune responses that are dependent on several factors, in-cluding route of immunization (21, 32, 66), expression level(2), cellular location (34), presentation (52), strain background(18, 41), the inherent structural and immunogenic propertiesof antigen, and the host genetic background. Generally, achiev-ing maximal immune responses to the vectored antigen is di-rectly correlated with the amount of the antigen produced (2,74), and thus it is important that the immunizing vector strainproduce adequate levels of antigen. However, for RASVstrains, this need must be weighed against the fact that high-level antigen production can be a drain on the nutrient andenergy resources of the cell, leading to reduced growth ratesand a compromised ability to colonize effector lymphoid tis-sues to induce an immune response (23). In addition, someantigens are inherently toxic to vaccine strains for other rea-sons, leading to a severe inhibition of growth rate, host-colo-nizing potential and, in some cases, death of the RASV strains.Overexpression of foreign proteins can also result in mutations

in the antigen gene promoter or coding sequence, thus reduc-ing or compromising the desired immune response. Severalapproaches have been used to address these problems, includ-ing truncating the antigen, reducing plasmid copy number,chromosomal expression, reducing promoter activity (33),codon optimization of the antigen sequence (72), or exportingthe antigen to an extracellular compartment (25). Anotherpopular approach is the use of in vivo inducible promoters,including pagC (31), nirB (10), and spv and dps promoters (44).In principle, the advantage of using in vivo inducible promotersis that only low levels of antigen are produced during in vitrogrowth and the initial stages of infection, allowing the Salmo-nella vaccine strain to direct its resources toward establishingan infection, an important step for eliciting a robust immuneresponse in the host. These promoters then upregulate antigenexpression once the Salmonella reaches immunocompetent siteswithin the host, thus inducing the desired antigen-specific im-mune response. However, in vivo inducible promoters are ofteneither too weak in vivo or too strong in vitro and can be limited bythe mode of attenuation (9, 10). Therefore, it would be useful todevelop a single system with a promoter that is weakly active invitro, is capable of strong expression in vivo, and whose function isnot influenced by the mode of attenuation.

In previous work, we utilized RASV strains that includedregulated delayed antigen synthesis as one of their features(43, 67, 71). However, until now, we have not fully describedthe system nor presented a clear demonstration of its advan-tages. In this work, we describe the construction of the system,

* Corresponding author. Mailing address: The Biodesign Institute,Center for Infectious Diseases and Vaccinology. Arizona State Uni-versity, P.O. Box 875401, 1001 S. McAllister Avenue, Tempe, AZ85287-5401. Phone: (480) 727-0445. Fax: (480) 727-0466. E-mail:[email protected].

† Supplemental material for this article may be found at http://iai.asm.org/.

� Published ahead of print on 6 July 2010.

3969

utilizing the strong LacI-repressible Ptrc promoter for antigengene expression and attenuated Salmonella enterica serovar Ty-phimurium strains synthesizing different levels of LacI under tran-scriptional control of an arabinose-regulated promoter. Two testantigens were used for evaluation. The green fluorescent protein(GFP) was used for in vitro evaluation of the system, and the�-helical fragment of the Streptococcus pneumoniae PspA protein(7, 36, 51) was used as the test antigen in immunogenicity studies.

Salmonella strains were constructed and evaluated for level ofLacI synthesis, antigen synthesis, and the ability to induce a pro-tective immune response in mice.

MATERIALS AND METHODS

Bacterial strains, plasmids, media, and growth conditions. Bacterial strainsand plasmids used are listed in Table 1. Bacteria were grown statically overnightat 37°C in LB broth (5), 3XD broth, a buffered Casamino Acids medium that

TABLE 1. Strains and plasmids used in this research

Strain or plasmid Relevant characteristicsa or genotype Source orreference

E. coli strainsBL21(DE3) F� ompT hsdSB(rB

� mB�) gal dcm (DE3) Novagen

Top10 F� mcrA �(mrr-hsdRMS-mcrBC) �80lacZ�M15 �lacX74 recA1 araD139 �(ara-leu)7697galU galK rpsL (Strr) endA1 nupG

Invitrogen

XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac �F� proAB lacIq �lacZ(M15) Tn10 (Tetr)� Stratagene�7213 thi-1 thr-1 leuB6 fhuA21 lacY1 glnV44 �asdA4 recA1 RP4 2-Tc::Mu �pir�; Kmr 61

S. enterica serovar Typhimuriumstrains

�3761 Wild-type UK-1 14�8767 �3761 �araBAD23 13�8914 �3761 �pabA1516 �pabB232 �asdA16 73�8989 �3761 �endA19::araC PBAD lacI TT, GTG-lacI �3761�8990 �3761 �relA196::araC PBAD lacI TT, GTG-lacI �3761�9080 �3761 �relA197::araC PBAD lacI TT, ATG-lacI �3761�9226 �3761 �relA198::araC PBAD lacI TT, codon-optimized lacI 3�9097 �pabA1516 �pabB232 �asdA16 �araBAD23 70�9555 �9097 �relA1123 �9097�9095 �9097 �relA196::araC PBAD lacI TT 70�9101 �9097 �relA197::araC PBAD lacI TT, same genotype as �9959 but with spontaneous

mutation allowing faster growth rate70

�9959 �9097 �relA197::araC PBAD lacI TT �9097�9241 �9097 �relA198::araC PBAD lacI TT 70�9506 �relA1123 �araBAD23 �8767�9507 �relA196::araC PBAD lacI TT �araBAD23 �8990�9508 �relA197::araC PBAD lacI TT �araBAD23 �9080�9509 �relA198::araC PBAD lacI TT �araBAD23 �9226

S. pneumoniae WU2 Wild type, virulent, encapsulated type 3 7

PlasmidspBAD-HisA Expression vector, PBAD promoter; Apr InvitrogenpET30a Expression vector, T7 promoter; Kmr NovagenpYA3341 Asd, pUC ori 36pYA3342 Asd, pBR ori 36pYA3493 Asd vector with �-lactamase N-terminal signal sequence, Ptrc, pBR ori 36pYA3494 pYA3493 with pspA gene (aa 3 to 257), pBR ori 36pYA3552 Asd vector expressing gfp3, p15A ori Lab stockpYA3599 Suicide vector to generate �araBAD23 mutation, sacRB mobRP4 oriR6K; Cmr Apr 11, 13pYA3635 Asd vector expressing codon-optimized pspA gene (aa 3 to 257), pBR ori 12pYA3679 Suicide vector to generate �relA1123 mutation, sacB mobRP4 oriR6K; Cmr 39pYA3784 Suicide vector to generate GTG-lacI �relA196::araC PBAD lacI TT mutation, sacB mobRP4

oriR6K; Cmr70

pYA3856 pBAD-HisA with GTG start codon lacI, His tagged; Apr This studypYA3879 Suicide vector to generate ATG-lacI �relA197::araC PBAD lacI TT mutation, sacB mobRP4

oriR6K; Cmr70

pYA4064 Suicide vector to generate codon-optimized lacI �relA198::araC PBAD lacI TT mutation,sacB mobRP4 oriR6K; Cmr

70

pYA4085 pET30a with codon-optimized pspA gene (aa 3 to 285), His tagged, Kmr This studypYA4088 Asd vector with �-lactamase N-terminal secretion signal specifying PspA (aa 3 to 285) in

pYA3493, Ptrc, pBR oriThis study

pYA4090 Asd vector for gfp3 expression in pYA3342, Ptrc, pBR ori This studypYA4349 Asd with weak SD sequence AAGGC and GTG start codon for asdA gene in pYA3341 This studypYA4365 Asd vector with �-lactamase N-terminal secretion signal specifying PspA (aa 3 to 285) in

pYA4349, Ptrc, pUC oriThis study

a Apr, ampicillin resistance; Cmr, chloramphenicol resistance; Kmr, kanamycin resistance.

3970 WANG ET AL. INFECT. IMMUN.

included glycerol as the carbon source (22), or nutrient broth (NB; Difco) asindicated. The next day, the cultures were diluted 1:100 into prewarmed mediumwith aeration at 37°C unless specified. When required, antibiotics and supple-ments were added at the following concentrations: chloramphenicol, 30 �g/ml;diaminopimelic acid (DAP) 50 �g/ml (50); p-aminobenzoic acid (PABA), 1�g/ml. LB agar without NaCl and containing 5% sucrose was used for sacBgene-based counterselection in allelic exchange experiments (24). S. pneumoniaeWU2 was cultured on brain heart infusion agar containing 5% sheep blood or inTodd-Hewitt broth plus 0.5% yeast extract (7).

General DNA procedures. DNA manipulations were carried out as describedby Sambrook et al. (63). Transformation of bacterial strains was routinely doneby electroporation (53) using a Gene Pulser Xcell system (Bio-Rad, Hercules,CA). Transformants containing Asd plasmids were selected on LB agar plateswithout DAP. Only clones containing the recombinant plasmids were able togrow under these conditions. Suicide vectors and P22-mediated transductionwere used to generate defined deletion/deletion-insertion mutations (19, 35, 64).Transfer of recombinant suicide plasmids to Salmonella was accomplished byconjugation using Escherichia coli �7213 (Asd�) as the plasmid donor (61).Bacteriophage P22HTint-mediated general transduction was performed by standardmethods (69). PCR amplification was employed to obtain DNA fragments forcloning and for verification of chromosomal deletion mutations. Nucleotide se-quencing reactions were performed by the DNA lab at Arizona State University.

Suicide plasmid for LacI mutations. Details of the construction of suicideplasmids pYA3784 (GTG-lacI), pYA3879 (ATG-lacI), and pYA4064 (codon-optimized lacI) have been described previously (71). Allele �relA196::araC PBAD

lacI TT (GTG-lacI) has the native AGGG Shine-Dalgarno (SD) sequence andGTG start codon, �relA197::araC PBAD lacI TT (ATG-lacI) has the AGGA SDsequence and ATG start codon, and �relA198::araC PBAD lacI TT (codon-optimized lacI) has an AGGA SD sequence, ATG start codon, and 15 optimizedcodons for increasing expression in Salmonella.

Construction of pYA3856, pYA4088, and pYA4090. The lacI gene was clonedinto pBAD-HisA to generate plasmid pYA3856 to synthesize His-tagged LacI(GTG-lacI) for protein purification (70). Plasmid pYA3493 is a pYA3342 deriv-ative that encodes the first 23 amino acids (aa) of the �-lactamase gene (36).Plasmid pYA4088, derived from pYA3493, carries a cloned fragment of the S.pneumoniae pspA gene, encompassing aa 3 to 285, that has been codon-opti-mized for expression in Salmonella and fused to aa 1 to 23 of the �-lactamasegene. The pspA gene was originally cloned from S. pneumoniae strain Rx1.Plasmid pYA3494 carries aa 3 to 257 of native pspA fused to the first 23 aa of blato direct secretion of the resulting protein into the periplasm and supernatant(36). Eleven codons were optimized according to Salmonella codon usage with-out changing the amino acid sequence to create plasmid pYA3635 using thesame PCR methods described above for lacI (71). Using pYA3635 as the startingmaterial, we then extended the optimized pspA sequence by an additional 28codons to include a recently identified B-cell epitope (16). The extended codon-optimized pspA gene was constructed in three steps. First, the optimized pspAsequence was amplified using the primer set PspA Rx1 forward (5�-TCTCCGGTAGCCAGTCAGTCTAAAGCTGAG-3�) and PspA Rx1-a1 (5�-CTAATTCAGCTTTTTTAGCAGCAATAGTTTTCTCTAAACCTTCTTTAAAGTAGTCTTCTACATTATTGTTTTCTTC-3�). The resulting 820-bp PCR fragment wasused as template in a second PCR using primer set PspA forward (5�-TCTCCGGTAGCCAGTCAGTCTAAAGCTGAG-3�) and PspA Rx1-a2 (5�-TGCTTTCTTAAGGTCAGCTTCAGTTTTTTCTAATTCAGCTTTTTTAGCAGCAATAGTTTTCTC-3�). The resulting 849-bp PCR product was used as template fora third amplification with the primer set PspA Rx1-EcoRI-s (5�-GGAATTCTCTCCGGTAGCCAGTCAGTCT-3�) and PspA Rx1-HindIII-a (5�-TTCAAGCTTATTATGCTTTCTTAAGGTCAGCTTC-3�). The 869-bp PCR product fromthat reaction was cloned into plasmid pYA3493 using EcoRI-HindIII restrictionsites to generate pYA4088 and also into plasmid pET30a to generate pYA4085for expression of His6-PspA Rx1. Plasmid pYA3342 is an Asd expression vectorwith promoter Ptrc (36). Plasmid pYA4090 is a pYA3342 derivative that codes forgfp3 expression from the Ptrc promoter. A plasmid clone of gfp3, pYA3552, wasprovided by Ho-Young Kang. Plasmid pYA4090 was constructed by PCR am-plification of the 740-bp gfp3 gene by using plasmid pYA3552 as template withprimer set GFP-EcoRI-s (5�-GGGAATTCCGATGAGTAAAGGAGAAGAACTTTTC-3�) and GFP-HindIII-a (5�-CGGTGCAAGCTTATTATTTGTATAGTTCATCCATG-3�) and then cloned into EcoRI-HindIII-digested pYA3342.The plasmid structure was verified by DNA sequencing and restriction enzymedigestion.

Construction of plasmids pYA4349 and pYA4365. The asd gene was amplifiedwith the primer set (5�-ACAGTCTAGACTAGGCCAACTGGCGCAGCATTC-3� and 5�-CTCTTCCGGAAGATCCGCACATCTCTTTGAAGGCAAAAAACGCTGTGAAA-3�) with pYA3341 as template and used to replace asd in

pYA3341 to generate plasmid pYA4349. The asd gene in pYA4349 has a weakSD sequence and a weak start codon, GTG, to reduce the level of Asd synthesis.The bla-SS-pspA gene fusion in pYA4088 was then cloned into pYA4349 togenerate pYA4365.

Construction and phenotypic characterization of S. Typhimurium vaccinestrains. Strain �8767 was constructed by conjugating �3761 with �7213(pYA3599)(35). The �araBAD23 mutation was introduced into strain �8914 to generatestrain �9097 (70). The mutation was verified by PCR and the inability to fermentarabinose, as deduced from its white colony phenotype on MacConkey agarsupplemented with 1% arabinose. To verify the phenotype of �pabA �pabBmutants, strains were streaked onto minimal agar with or without p-aminoben-zoate. The presence of the �asdA mutation in Salmonella was confirmed by itsdependence on DAP for growth (50). The presence of the 3.3-kb deletion-insertion of �relA deletion/insertion mutations was confirmed by PCR withprimer set RelA N-HindIIISacI-5� (5�-CCCAAGCTTGAGCTCGAGGGCGTTCCGGCGCTGGTAGAA-3�) and RelA C-KpnI-3� (5�-CGGGTACCCCAGATATTTTCCAGATCTTCAC-3�), and cell lysates were subjected to Western blotanalysis. Blots were probed with anti-LacI serum. Lipopolysaccharide (LPS)profiles of Salmonella strains were examined as described previously (30). The�relA1123 mutation was introduced into �9097 to generate �9555 by conjugatingwith �7213(pYA3679). The �relA196::araC PBAD lacI TT, �relA197::araC PBAD

lacI TT, and �relA198::araC PBAD lacI TT mutations were introduced into S.Typhimurium strain �3761 by allelic exchange to yield �8990, �9080, and �9226and into RASV strain �9097 to generate �9095, �9959, and �9241, respectively.

Generation of polyclonal antibodies against LacI and PspA. RecombinantHis6-LacI was expressed from pYA3856 in Top10 cells according to the productmanual (Invitrogen, Carlsbad, CA). Recombinant His6-PspA was synthesized inBL21(DE3) cells harboring plasmid pYA4085. Recombinant proteins were pu-rified using the CelLytic B-cell lysis reagent (Sigma, St. Louis, MO) and aHIS-Select nickel affinity gel (Sigma) according to the product manual. Therecombinant proteins were used to raise antisera in rabbits.

Western blot analysis. Protein samples were prepared from equal numbers ofcells, separated on a 12% SDS-PAGE gel, and transferred to a nitrocellulosemembrane using a Trans-Blot SD semidry transfer cell (Bio-Rad, Hercules, CA).Detection of LacI and PspA bands were based on rabbit polyclonal anti-LacI andanti-PspA primary antiserum at a 1:10,000 dilution and a secondary anti-rabbitalkaline phosphatase-conjugated antibody (Sigma) at a 1:10,000 dilution. Bandswere visualized using nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphos-phate (Sigma). The bands were scanned, and densitometry was measured usingQuantity One software (Bio-Rad).

LacI stability analysis. S. Typhimurium strains �8990, �9080, and �9226 weregrown in 3XD medium containing 0.2% arabinose, and E. coli strain XL1-Bluewas grown in LB broth without arabinose. Standing overnight cultures of eachstrain were grown at 37°C, diluted 1:100 into fresh medium, and grown withaeration to an optical density at 600 nm (OD600) of 0.6. Cells were washed twotimes with fresh medium. Chloramphenicol was added to 50 �g/ml. Samplestaken before adding chloramphenicol (pre-0), just after adding chloramphenicol(0), and at 1, 2, 4, 6, 8, and 24 h were analyzed by Western blotting. The sampleswere normalized by cell number before loading onto the gels.

Growth curves. Standing overnight 37°C cultures of RASV strains with andwithout plasmid were grown in LB or LB plus DAP, respectively, or in NBcontaining different arabinose concentrations as indicated. The culture was ad-justed to an OD600 of 0.6 in the same prewarmed medium and then diluted 1:100into prewarmed LB, LB plus DAP broth, or NB with the designated arabinoseconcentration. The OD600 was measured every 40 min. At the final time point,samples of each strain were taken and used for Western blot analysis withanti-LacI and/or anti-PspA antiserum. In some cases, growth curves were calcu-lated using the automated growth curve device Bioscreen C (Growth CurvesUSA, Piscataway, NJ), at 10-min intervals.

Flow cytometry analysis. Standing overnight cultures of �9555(pYA4090),�9095(pYA4090), �9959(pYA4090), and �9241(pYA4090) were grown at 37°Cin nutrient broth without arabinose. Then, 3 105 CFU of each strain wereadded to 3 ml of fresh medium containing 2%, 0.2%, 0.02%, 0.002%, or 0%arabinose and grown to an OD600 of 0.4. The cultures were diluted 1:10 inphosphate-buffered saline (PBS) and subjected to flow cytometry analysis usinga Cytomics FC500 apparatus (Beckman Coulter, Inc., Fullerton, CA). The datawere analyzed by using CXP analysis software (Beckman Coulter, Inc.).

Kinetics of LacI disappearance and antigen synthesis in preinduced culturesgrown without arabinose. Overnight cultures of strains �9555, �9095, �9959, and�9241 carrying either plasmid pYA4088 or plasmid pYA4090 were grown innutrient broth with or without 0.2% arabinose. Each culture was adjusted to anOD600 of 0.6 and diluted 1:10 into the same prewarmed medium at 37°C. Whencultures reached an OD600 of 0.6, the cultures were washed once with nutrient

VOL. 78, 2010 RDAS SYSTEM FOR DEVELOPMENT OF SALMONELLA VACCINES 3971

broth without arabinose, diluted 1:10 into prewarmed nutrient broth without arabi-nose, and grown to an OD600 of 0.6. The cultures were diluted into fresh medium,and the process was repeated four times (for pYA4090 cultures) or three times more(for pYA4088 cultures). Samples were taken at the end of each growth cycle.Samples of strains carrying pYA4088 were normalized according to cell number andanalyzed by Western blotting. Bands were scanned, and densitometry was measuredusing Quantity One software (Bio-Rad, Hercules, CA). Samples of strains carryingpYA4090 were analyzed by flow cytometry as described above.

Animals. Seven-week-old female BALB/c mice were obtained from CharlesRiver Laboratories (Wilmington, MA). All animal protocols were approved bythe ASU IACUC and complied with rules and regulations of the AmericanAssociation for Accreditation of Laboratory Animal Care. The mice were accli-mated for 7 days after arrival before the experiments were started.

Virulence test. For determination of the 50% lethal dose (LD50), bacteria weregrown statically overnight at 37°C in LB broth with or without 0.2% arabinose,diluted 1:50 in fresh medium with or without 0.2% arabinose, and grown to anOD600 of 0.8 to 0.9 at 37°C. Bacterial cells were harvested at room temperatureand resuspended in buffered saline with gelatin (BSG). Groups of five mice eachwere infected orally with 20 �l containing various doses of S. Typhimurium�3761 or its derivatives, ranging from 103 CFU to 105 CFU. The animals wereobserved for 4 weeks postinfection, and deaths were recorded daily.

Tests for immunogenicity and protection in mice. Bacteria were prepared, andexact numbers for each dose were determined as previously described (43).Groups of female BALB/c mice were orally immunized with approximately 1 109 CFU of the test vaccine and boosted at 6 weeks. Mice were bled at 0, 2, 4,6, and 8 weeks. Ten weeks after immunization, mice were challenged by intra-peritoneal injection with 100 LD50 of virulent S. pneumoniae WU2 and observeddaily for 21 days.

ELISA. Rcombinant PspA (rPspA) protein was purified as described previ-ously (36). S. Typhimurium LPS was obtained from Sigma. The procedure for theenzyme-linked immunosorbent assay (ELISA) has been described before (36).Titers were recorded as the last dilution that resulted in an OD405 of 0.1 greaterthan background.

Statistics. Statistical analyses were performed by using GraphPad’s (San Di-ego, CA) Prism 5 software package. Antibody titers were expressed as means �standard errors. The means were evaluated with two-way analysis of variance andBonferroni tests for multiple comparisons among groups. Differences were con-sidered significant at a P level of �0.05.

RESULTS

Rationale for the RDAS system. The Ptrc promoter is com-monly used to express genes of interest in Salmonella vaccines(50, 65). Ptrc and the similar Ptac are strong promoters in vivo(8), constitutive under most environmental conditions, andmore transcriptionally active both anaerobically and aerobi-cally than the nirB promoter (10). Although the Ptrc promoterhas been widely used (1), constitutive antigen expression fromthe similar Ptac promoter can affect the colonization capabilityof RASV strains (9), an important step in eliciting a robustimmune response. Thus, we hypothesized that downregulatinggene expression from Ptrc would be beneficial during the earlystages of infection. The Ptrc promoter can be repressed byLacI. In Escherichia coli, the LacI repressor is typically synthe-sized at approximately 5 to 10 copies per cell (9, 27, 48) andregulates expression of the lactose metabolic genes by bindingto the lacO operator sequence, repressing lac expression in theabsence of lactose (42). Typically, induction of expression fromLacI-repressed promoters is accomplished by the addition ofchemical agents that bind to LacI, causing an allosteric changein the protein that leads to its release from lacO. However, thisis not a practical method for induction in vivo. Our approachwas to modulate lacI expression by placing it under the controlof the arabinose-inducible araC PBAD promoter (Fig. 1A) (29).When RASV is grown in culture with arabinose, LacI is syn-thesized, repressing transcription from Ptrc. Once inside thehost, transcription from araC PBAD ceases, because free arabi-

nose is not available in animal tissues (37, 39) and no addi-tional LacI is produced. The concentration of LacI will de-crease by dilution as the RASV cells divide and derepressantigen gene expression.

Construction of strains synthesizing LacI. Based on thisconcept, a tightly regulated araC PBAD lacI TT cassette wasintegrated into the S. Typhimurium UK-1 chromosome in therelA gene (Fig. 1B), generating the relA196 allele in strain�8990. We chose relA as the integration site because a relAmutation is not attenuating for virulence, nor does it have aneffect on colonization (57). The native lacI gene has a GTGstart codon and an AGGG Shine-Dalgarno sequence, leadingto synthesis of only 5 to 10 molecules each generation (27).Because the functional form of LacI repressor is a tetramer(47) and plasmids with a ColE1 (pBR) replicon are present at20 to 30 copies per cell, we estimate that at least 80 to 120copies of LacI are needed to repress the operator sequences inour pBR-based expression plasmid to achieve adequate repres-sion. Therefore, in addition to strain �8990, which encodes thenative lacI sequence (GTG-lacI), we modified the start codonfrom GTG to ATG and the SD sequence from AGGG to thecanonical ribosome-binding site sequence, AGGA, resulting inthe relA197 allele in strain �9080. This modification increasedLacI expression about 2-fold (Fig. 1C). To enhance expressionfurther, we optimized the codons of lacI according to thecodon usage for highly expressed genes in Salmonella to yieldthe relA198 allele in strain �9226. Depending on the arabinoseconcentration, this modification increased LacI expression ap-proximately 2-fold (2% and 0.2% arabinose) to 40-fold(0.002% arabinose) over a strain with the relA196 construction(Fig. 1C). The expression levels of LacI for relA196 (GTG-lacI,low lacI expression RDAS strain), relA197 (ATG-lacI, mediumlacI expression RDAS strain), and relA198 (codon optimizedATG-lacI, high lacI expression RDAS strain) were propor-tional to the arabinose concentration (Fig. 1C). We anticipatethat different antigens may require different levels of repres-sion, so these three relA alleles provide the flexibility needed toproduce different amounts of LacI repressor to meet variedrequirements (Table 2). We also generated the �relA1123 mu-tation, in which the entire relA gene is deleted, as a control(Fig. 1B).

Determination of LacI stability in RASV strains. LacI is notnormally synthesized in S. Typhimurium, and in E. coli it isexpressed only at low levels. Because of the central role LacIplays in our system, we wanted to investigate its stability in ourstrains, as that could have an impact on the timing of antigensynthesis in vivo. Chloramphenicol was added to mid-exponen-tial-phase cultures of Salmonella strains �8990 (�relA196),�9080 (�relA197), and �9226 (�relA198) and E. coli strainXL1-Blue. The stability of LacI was similar for all strains (Fig.2). The amount of LacI declined by 50% over the first 2 to 4 hin all strains, after which the amount declined very slowly,reaching approximately 20% of the starting levels by 24 h forthe S. Typhimurium strains and 40% for the E. coli strain.These results indicate that LacI is relatively stable overshort intervals, in agreement with a previous report showingthat LacI is stable for 2 h (6, 58) and that its stability issimilar in both S. Typhimurium and E. coli. However, therewas substantial degradation by 24 h. Therefore, during col-onization of host tissues over the course of several days, we


expect that the concentration of LacI in our strains willdecrease due to a combination of dilution, as a result of celldivision, and protein degradation.

High levels of LacI synthesis do not affect growth and pro-vide a growth advantage when strains harbor plasmids encod-ing heterologous antigens. The Salmonella chromosome doesnot encode the lac operon. Consequently, the affect of lacIexpression or overexpression on growth was investigated, be-cause this might translate into a reduction in immunogenicityfor vaccine strains. The relA alleles were moved into the atten-uated RASV vector strain �9097 (Table 1) to generate �9095(relA196, GTG-lacI, low LacI), �9959 (relA197, ATG-lacI, me-dium LacI), and �9241 (relA198, codon-optimized ATG-lacI,high LacI) for further evaluation. The growth of strains �9095,�9959, and �9241 was evaluated in LB broth with DAP and

FIG. 1. (A) Principle of RDAS. This system includes a chromosomal repressor gene, lacI, expressed from the arabinose-regulated araC PBADpromoter. LacI regulates antigen synthesis from the plasmid promoter Ptrc. When grown in the presence of arabinose, LacI is produced, whichbinds to Ptrc, blocking antigen gene expression. In animal tissues, an arabinose-poor environment, the concentration of LacI decreases with eachcell division, allowing increased antigen synthesis. (B) Chromosomal deletion map for lacI strains. Different levels of LacI synthesis due to alteredSD sequence, start codon, or codon usage in the lacI gene were inserted into the relA gene in the Salmonella genome. The deletion-insertionmutation deleted 2,247 bp in relA (relA �12 to relA 2235) and inserted 2,429 bp containing the araC PBAD lacI TT cassette. relA, wild-type relAgene; �relA1123, defined deletion of the relA gene; �relA196, RDAS deletion-insertion mutation with the native GTG start codon and native SDsequence AGGG; �relA197, RDAS deletion-insertion mutation with an ATG start codon and improved SD sequence AGGA; �relA198, RDASdeletion-insertion mutation with an ATG start codon, improved SD sequence AGGA, and optimized lacI codons. The start codon change, SDsequence change, and codon optimization of lacI were used to achieve a higher level of expression in Salmonella. (C) LacI synthesis in strainscarrying various �relA::araC PBAD lacI mutations, detected by Western blotting. Strains �8990 (�relA196, GTG-lacI), �9080 (�relA197, ATG-lacI),and �9226 (�relA198, codon-optimized ATG-lacI) were grown in 3XD medium containing the indicated concentration of arabinose. Samples werenormalized based on cell number. Western blots were probed with anti-LacI antibody. Densitometry readings were quantified using Quantity Onesoftware. The number indicates the densitometry of each band. The data represent one of three independent experiments.

TABLE 2. Relative amount of LacI produced by each relA allele

Allele Relative amt ofLacI produced

�relA1123 ..................................................................................None�relA196 ....................................................................................Low�relA197 ....................................................................................Medium�relA198 ....................................................................................High

FIG. 2. In vivo stability of LacI. XL1-Blue (lacIq E. coli strain) wasgrown in LB medium. Strains �8990 (GTG-lacI), �9080 (ATG-lacI),and �9226 (codon-optimized lacI) were grown in 3XD medium with0.2% arabinose to an OD600 of 0.6 and washed two times with 3XDmedium without arabinose. Chloramphenicol was added to 50 �g/ml.Samples were taken before washing (pre 0), just after adding chlor-amphenicol (0), and at 1, 2, 4, 6, 8, and 24 h and subjected to Westernblot analysis. The samples were normalized based on cell number.Western blots were probed with anti-LacI sera. Densitometry readingswere quantified using Quantity One software. The data represent av-erage results of three independent experiments. Error bars representstandard deviations.


with or without 0.2% arabinose and compared to strain �9555(�relA1123), which does not produce LacI. Without plasmids, allfour strains had similar growth rates with or without 0.2% arabi-nose (Fig. 3A and B). Growth at lower concentrations of arabi-nose (0.02%, 0.05%, or 0.1%) gave similar results (data notshown). However, the addition of 2% arabinose led to reducedgrowth of all the LacI-producing strains (data not shown), prob-ably due to acid formation from arabinose catabolism, the over-production of LacI, or a combined effect of both factors.

Plasmid pYA4088, a pBR ori plasmid encoding the pneumo-coccal pspA gene, was introduced into attenuated vaccine strains�9555 (�relA1123), �9095 (�relA196), �9959 (�relA197), and�9241 (�relA198), and their growth rates in LB broth with andwithout added arabinose were compared (Fig. 3C and D). Whengrown in the presence of 0.2% arabinose, all of the LacI-produc-ing strains had a doubling time of 22 to 24 min, while strain�9555(pYA4088) had a doubling time of 35 min, indicating thatrepressing antigen gene expression results in faster growth(Fig. 3D). Strain �9241(pYA4088) also grew faster than�9555(pYA4088) in the absence of added arabinose (Fig. 3C).

This may be due to trace amounts of arabinose present in theyeast extract used to prepare the LB broth medium, approxi-mately 0.0034% (71). Although the amount of LacI producedunder these conditions was undetectable by Western blotting(Fig. 1C), there may have been sufficient LacI present to ac-count for the observed growth advantage. This supposition wasconfirmed when we grew the same strains in NB, a mediumthat does not contain arabinose. All four strains grew with thesame doubling time in NB (Fig. 3E). When arabinose wasadded to the growth medium, the three strains expressing lacIgrew faster than �9555(pYA4088), which does not express lacI(Fig. 3F).

In a separate experiment, we evaluated the ability of this systemto regulate expression from high-copy-number plasmids with apUC replicon. Plasmid pYA4365 is similar to plasmid pYA4088,except that it has a pUC replicon instead of a pBR replicon andtherefore has a higher copy number. Cells carrying this plasmidproduce more PspA and maintain a higher copy number of plas-mid than cells carrying pYA4088 (data not shown), thereby ex-erting a greater metabolic burden on the cell. Plasmid pYA4365

FIG. 3. Effects of LacI synthesis on growth. Strains were grown in LB medium with 0% (dashed line; A and C) or 0.2% arabinose (solid line;B and D) or NB medium without (E) or with (F) 0.2% arabinose. The OD600 was measured at 40-min intervals. �, �9555 (�relA1123, no LacI);Œ, �9095 (�relA196, low LacI), ●, �9959 (�relA197, medium LacI), f, �9241 (�relA198, high LacI). (A and B) Results for strains without plasmid.DAP was included in the growth medium for these strains. (C to F) Results for strains carrying the antigen synthesis plasmid pYA4088.


was transferred into �9555 (�relA1123), �9095 (�relA196), �9101(�relA197) (same genotype as �9959 but with a spontaneousmutation allowing a faster growth rate), and �9241 (�relA198).The growth of these vaccine strains was evaluated using the Bio-screen C system. Strain �9555(pYA4365) could not grow at anyconcentration of arabinose (see Fig. S1A, B, C, and D in thesupplemental material). Overall, the more LacI produced bythe strain, the faster the rate of growth [�9241(pYA4365) (highLacI) � �9101(pYA4365) (medium LacI) � �9095(pYA4365)(low LacI) � �9555(pYA4365) (no LacI)] in the presence of0.2% arabinose (see Fig. S1B). The trend was the same withdifferent arabinose concentrations, 0.05% or 0.02% (see Fig.S1C and D) and even without arabinose (see Fig. S1A). Wealso attempted to repeat this experiment in NB but found thatnone of the strains would grow in the absence of arabinose,similar to the results for �9555(pYA4088) shown in Fig. S1A inthe supplemental material (data not shown). Taken together,these results showed that synthesis of LacI provides a growthbenefit for RASV strains expressing antigen genes from a Ptrc

promoter, especially when the antigen gene is present at highcopy number.

Virulence of strains with RDAS in mice. The RDAS systemgives vaccine strains a growth advantage when they harbor anantigen-encoding plasmid in vitro. To evaluate the effect of theRDAS on virulence in BALB/c mice, we determined the LD50

of �3761 (wild-type) derivatives carrying araC PBAD lacI inser-tions (Table 3). The parent strain �3761 was highly virulent,with an LD50 of 1 104 CFU. The LD50 of strain �8990(�relA196::araC PBAD lacI TT) was similar (Table 3). A similarLD50 was observed for strain �8989 (�endA19::araC PBAD lacITT), in which lacI in �endA19 is GTG-lacI, the same as the lacIconstruct in �relA196. We then tested the virulence of theLacI-producing strains grown in the presence of 0.2% arabi-nose. Growth of strains in the presence of arabinose resultsin acid production that can causes cessation of growth. Toprevent the breakdown of arabinose, we introduced the �ar-aBAD23 mutation (4, 59). The LD50 of strain �8767 (�ar-aBAD23) was similar to the parent strain �3761, indicatingthat the �araBAD23 mutation does not affect the virulenceof these strains. We then determined the LD50 for strainsexpressing lacI and carrying the �araBAD23 mutation grownin LB with 0.2% arabinose. The LD50 of strain �9506(�relA1123 �araBAD23) was similar to that of wild-type�3761 and �8767(�araBAD23) (Table 3). The strains ex-

pressing lacI, �9507 (�relA196), �9508 (�relA197), and�9509 (�relA198), were attenuated for virulence, with LD50sof �2 105 CFU. These results indicate that LacI synthesisis attenuating, consistent with a previous report showingthat LacI is an antivirulence factor that inhibits expressionof genes in Salmonella pathogenicity island 2 (20).

Antigen synthesis is inversely correlated with lacI expres-sion. To evaluate the relationship between antigen synthesisand arabinose concentration, we introduced plasmidpYA4090, which encodes the gfp3 gene under transcriptionalcontrol of Ptrc, into S. Typhimurium strains �9555 (�relA1123,no LacI), �9095 (�relA196, low LacI), �9959 (�relA197, me-dium LacI), and �9241 (�relA198, high LacI). Transformantswere grown in LB with various arabinose concentrations andsubjected to fluorescence-activated cell sorting (FACS) analy-sis. The araC PBAD promoter is subject to autocatalytic regu-lation, and therefore we were able to evaluate the fraction ofcells expressing GFP as a measure of induction (68). As ex-pected, there was no effect of arabinose on gfp3 gene expres-sion in strain �9555(pYA4090), which does not encode lacI(Fig. 4A). When strains �9095(pYA4090), �9959(pYA4090),and �9241(pYA4090) were grown without arabinose, nearly allthe cells expressed GFP. No decrease in the number of GFP-positive cells was observed when 0.002% arabinose was in-cluded in the growth medium, but repression was evident with0.02% arabinose for all strains. As the arabinose concentrationincreased, the number of GFP-positive cells dropped substan-tially for all strains with arabinose-regulated expression of lacI.The greatest level of repression was seen in strain �9241(�relA198), with only 7.6% GFP-positive cells in the presenceof 2% arabinose. These results are consistent with our expec-tation that antigen synthesis should be inversely proportionalto arabinose concentration and lacI expression (Fig. 4A). How-ever, it was also noted that although the lacI constructs in�9095 (�relA196) and �9959 (�relA197) produced differentamounts of LacI (Fig. 1C), there was no significant differencein gfp gene expression between the two strains.

Time course for the induction of GFP synthesis. To evaluatethe kinetics of the induction of antigen synthesis after growthin arabinose, the GFP-producing strains were grown in nutri-ent broth with 0.2% arabinose. Nutrient broth was chosen asthe growth medium because it is derived from animal tissueand should better mimic the low arabinose conditions found inhost tissues than in LB broth. The arabinose-grown cells werediluted 1:10 into fresh nutrient broth without arabinose andgrown to an OD600 of 0.6. The cells were diluted and passagedfour more times in the same way. Each round of growth rep-resented approximately 3.32 cell divisions, for a total of 16.6generations of growth in the absence of arabinose. Sampleswere taken at the end of each growth cycle and analyzed byFACS (Fig. 4B). The results showed that although expressionof gfp was not fully repressed in any of the strains, maximumrepression was achieved in the strain carrying �relA198(�9241), the allele that provides for the most LacI synthesis(Fig. 1C). The derivatives of strains �9095 (�relA196) and�9959 (�relA197) had similar kinetics, achieving full inductionby 6.64 generations, while the �9241 derivative was fully in-duced by 9.96 generations.

Time course for the induction of PspA synthesis. As shownabove, our system worked according to our expectations when

TABLE 3. Virulence of S. Typhimurium strains expressing lacI

Strain Genotype LD50 (CFU)Presence of 0.2%

arabinose inmedium

�3761 Wild type �1.5 104 No�8767 �araBAD23 �2.0 104 Yes�8989 �endA19::araC PBAD lacI TT �1.0 104 No�8990 �relA196::araC PBAD lacI TT �1.0 104 No�9506 �relA1123 �araBAD23 �2.4 104 Yes�9507 �relA196::araC PBAD lacI TT

�araBAD23�2.7 105 Yes

�9508 �relA197::araC PBAD lacI TT�araBAD23

�2.4 105 Yes

�9509 �relA198::araC PBAD lacI TT�araBAD23

�2.5 105 Yes


using gfp3 as a model. We next evaluated our system in thecontext of a clinically relevant antigen, the S. pneumoniae PspAprotein, which has been shown to be a potent and protectiveimmunogen (36). LacI and PspA synthesis were directly eval-uated in cells grown in 0.2% arabinose. This concentration waschosen because there was only a small difference in repressionlevels between 2% and 0.2% arabinose (Fig. 4A) and, as men-tioned above, the addition of 2% arabinose resulted in a re-duction in growth rate. The amount of PspA synthesis wasinversely correlated with LacI synthesis, as expected (Fig. 4C).PspA synthesis in strain �9241(pYA4088), which producedthe most LacI, was approximately 30-fold less than the�9555(pYA4088) control (Fig. 4C, time zero). We evaluatedthe kinetics of induction and found that all of the strainsproduced the same amount of PspA as �9555(pYA4088) after9.9 generations of growth, indicating full derepression of Ptrc

(Fig. 4C). Strains �9095 and �9959 had similar derepressionkinetics, while strain �9241 was slightly slower. These resultsare consistent with the GFP results (Fig. 4B).

RDAS enhances immunogenicity and protection comparedto constitutive antigen expression. We evaluated strains�9555(pYA4088) (�relA1123), �9095(pYA4088) (�relA196),�9959(pYA4088) (�relA197), �9241(pYA4088) (�relA198),and control strains carrying pYA3493 (parent plasmid ofpYA4088) for immunogenicity in mice. All the RASV strainscarrying pYA4088 induced a strong anti-PspA serum IgGresponse (Fig. 5A) in immunized mice, with strain�9241(pYA4088) inducing slightly higher titers than theother strains by week 8. The most interesting differenceswere seen in the mucosal IgA titers, where antibody re-sponses correlated well with the amount of LacI synthesizedby each strain. The two strains producing the most LacI,�9959(pYA4088) and �9241(pYA4088), elicited the highestanti-PspA titers at weeks 6 and 8 (Fig. 5B), while the re-sponses in mice immunized with strains �9555(pYA4088)(no lacI) or �9095 (pYA4088) (�relA196) produced lowerIgA titers. No anti-PspA antibodies were detected in serumor vaginal secretions of mice immunized with control strains

FIG. 4. Effects of arabinose on antigen synthesis in strains containing the RDAS system. (A) Modulation of GFP synthesis by arabinoseconcentration. Plasmid pYA4090 encodes gfp3 expressed from the Ptrc promoter. Overnight nutrient broth cultures of strains carrying pYA4090grown without arabinose were diluted 1:10 into prewarmed nutrient broth containing the indicated concentrations of arabinose and grown to anOD600 of 0.4. Samples were diluted 1:10 into PBS and subjected to FACS analysis. The data represent averages of three independent experiments.(B) Kinetics of GFP expression. Overnight cultures grown in nutrient broth with 0.2% arabinose were diluted 1:10 into prewarmed nutrient brothwith arabinose and grown to an OD600 of 0.6. This culture was designated generation 0. These cells were diluted 1:10 into prewarmed nutrient brothwithout arabinose and grown to an OD600 of 0.6. The process was repeated two more times, and when cultures reached an OD600 of 0.6 sampleswere diluted 1:200 into PBS and subjected to FACS analysis. The data represent averages of three independent experiments. (C) Kinetics of LacIdisappearance and PspA synthesis in cells grown in the absence of arabinose. The indicated strains were grown as described for panel B. For eachdilution, followed by regrowth to an OD600 of 0.6, equal samples were taken for Western blot analysis using anti-LacI and anti-PspA antisera.Densitometry readings were quantified using Quantity One software. The data represent averages of three independent experiments. Error barsrepresent standard deviations.


that did not express pspA. All immunized mice developedhigh titers against S. Typhimurium LPS (Fig. 5C). The anti-LPS response developed more quickly (week 4) in miceimmunized with the RDAS strains than in mice immunizedwith �9555(pYA4088).

The serum immune responses to rPspA were further exam-ined by measuring the levels of IgG isotype subclasses IgG1and IgG2a. The Th1 cells direct cellular immunity and pro-mote class switching to IgG2a, and Th2 cells provide potenthelp for humoral antibody production and promote classswitching to IgG1 (17, 28). Th1-type dominant immune re-sponses are frequently observed after immunization with at-tenuated Salmonella strains (55, 56, 60). At different weekspostimmunization, the IgG2a titers were always higher thanIgG1 titers for all strains, regardless of whether or not theysynthesized LacI (see Fig. S2 in the supplemental material).These results indicated that a Th1 response was induced in allthe strains. The RDAS system therefore does not affect the Thelper cell pathways.

When vaccinated mice were challenged with virulent S.pneumoniae WU2, all groups that received PspA-producingstrains were significantly protected compared with the same

strain carrying the empty vector, pYA3493 (P � 0.01) (Fig. 6).Among these protected groups, protection in mice vaccinatedwith strains �9959(pYA4088) (�relA197, medium LacI) or�9241(pYA4088) (�relA198, high LacI) was significantlygreater than in mice vaccinated with the other strains (P �0.05). Immunization with non-RDAS strain �9555(pYA4088)(�relA1123, no LacI) and RDAS strain �9095(pYA4088)(�relA196, low LacI), which produces the least amount of LacI,provided similar levels of protection. These results showed thatthe level of protection roughly correlated with the amount ofLacI produced by each strain.

DISCUSSION

We have developed a regulated delayed antigen synthesissystem to minimize the negative effects of antigen expressionon the host strain and to enhance immunity. An araC PBAD

lacI TT cassette was engineered to synthesize different levels ofLacI when strains with these constructions were grown in thepresence of arabinose (Fig. 1C). The amount of LacI producedwas proportional to the amount of arabinose present in themedium up to 2%, the maximum concentration tested (Fig.

FIG. 5. Reciprocal mucosal and serum antibody titers against rPspA and Salmonella LPS. Serum IgG (A) and mucosal IgA (B) titers againstrPspA and serum IgG responses against S. Typhimurium LPS (C) were determined by ELISA. Female BALB/c mice were inoculated with theindicated strains grown in LB broth containing 0.05% arabinose. The data represent antibody levels in pooled sera from mice orally immunizedwith RASV strains carrying either a control vector or psaA expression plasmids. ***, P � 0.001.


1C). These results differ from what has been observed in E.coli, in which protein synthesis from PBAD reaches a maximumwith 0.2% arabinose (29). There are several possible reasonsfor this difference. First, our source of the araC PBAD promoter/activator cassette was E. coli K-12, while in the previous studiesthe promoter was derived from E. coli B/r. We have observedthat regulation is tighter with a K-12 cassette than a B/r cas-sette (39). The previous studies were performed using plasmidcopies of PBAD. Apparent differences in regulation have beenobserved for some promoters when they are present in multi-ple copies (K. Roland, unpublished results). Finally, differ-ences in the arabinose transport system between E. coli andSalmonella may have also played a role. Salmonella has onlyone L-arabinose transport system, encoded by araE (40, 45),which has a low affinity for arabinose, while E. coli has botharaE and the high-affinity transport system encoded byaraFGH (38, 40). Therefore, in Salmonella, higher concentra-tions of arabinose are likely to be required for full PBAD pro-moter induction than in E. coli (40).

Although 2% arabinose induced the maximum amount ofLacI synthesis, repression of antigen synthesis was not com-plete (Fig. 4). Only in strain �9241, which produced 3- to 4-foldmore LacI than the other strains, was antigen synthesis nearlyshut off completely (Fig. 4A). We hypothesize that there wasnot enough LacI produced in our strains to completely repressPtrc, since the Ptrc promoter is present on a multicopy plasmid,while LacI is specified by a single gene copy on the chromo-some. An additional consideration is that in the native E. colilac operon, there are three adjacent operator sequences, facil-itating cooperative binding interactions between three LacItetramers (26), while there is only one operator in our plasmid(46, 49, 54). We also note that the lacO sequence in Ptrc is10-fold less than a perfectly symmetrical, “ideal” lac operator(62). Thus, it may be possible to improve the efficiency ofantigen gene repression by optimizing lacO or by introducingmultiple copies of lacO to attain tighter repressor binding.However, changing the lacO sequence may also affect thestrength of the promoter (15). LacI has a nearly optimal bind-ing affinity to its cognate operator (15); therefore, we may haveto modify both LacI and lacO to achieve higher repression and

higher expression. In addition, as we already discussed above,one can also modify the system by adjusting the amount ofarabinose in the growth medium, thereby changing the amountof LacI in the cell. This strategy should be balanced against thenegative impact of increased LacI concentration on growth,which could lead to overattenuation and loss of immunogenic-ity (20). The optimal repression/expression should be achievedby balancing promoter strength, operator binding affinity, andarabinose concentration.

We developed three strains, each synthesizing differentamounts of LacI, with the idea that different antigens mayrequire more or less LacI to achieve an appropriate balancebetween the health of the RASV strain and the optimal anti-gen expression required for induction of protective immuneresponses. The presence of LacI in all strains reduced pspAexpression in vitro, resulting in a faster growth rate (Fig. 3).While all the RDAS RASV strains grew faster than the controlstrain that constitutively expressed pspA, the results from eachstrain were different with respect to the serum immune re-sponse and protective immunity (Fig. 5 and 6). This may be areflection of differences in the amount of antigen produced byeach strain and the timing of the induction of antigen synthesisor that other factors such as cellular immunity might be moreimportant for conferring protective immunity (Fig. 4C).

In conclusion, we have developed a system that reduces thenegative effects of antigen expression during in vitro growth,thereby improving the overall health of the vaccine strain,while allowing for maximum antigen expression in host tissues.This technology should be particularly useful for inducing im-mune responses to antigens that are toxic to the vaccine strainsynthesizing them. We plan to continue evaluating ways tofurther optimize this system and include comparisons withother in vivo inducible promoters (S. Wang, Y. Li, H. Shi, K. L.Roland, and R. Curtiss III, submitted for publication).

ACKNOWLEDGMENTS

The work was supported by NIH R01 AI24533, AI057885, andAI056289 and the Bill and Melinda Gates Foundation grant 37863.

We thank David E. Briles and Susan K. Hollingshead (University ofAlabama at Birmingham) for providing S. pneumoniae strains, Qing

FIG. 6. Survival curve after challenge with virulent S. pneumoniae WU2 strain. Female BALB/c mice were immunized with the indicated strainsgrown in LB containing 0.05% arabinose. Ten weeks after immunization, mice were challenged with 100 LD50 of virulent S. pneumoniae WU2.All mice immunized with PspA-expressing strains were significantly protected (P � 0.05). Mice vaccinated with strain �9241(pYA4088) showedsignificantly greater protection than mice vaccinated with non-RDAS strain �9555 (P � 0.01). Blank, mice immunized with BSG. Numbers showthe number of surviving per total treated mice.*, P � 0.05 compared with �9555(pYA4088); #, P � 0.05 compared with �9095(pYA4088); ##,P � 0.01 compared with �9095(pYA4088).


Liu for providing purifying PspA, and Erika Arch (Arizona StateUniversity) for her assistance with the manuscript.

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