ABSTRACT
We compared the abilities of different Salmonella enterica var. Typhimurium (S. typhimurium) strains harboring mutations in the genes aroA, aroAD,purA, ompR, htrA, and cya crp to present the heterologous antigen, C fragment of tetanus toxin, to the mouse immune system. Plasmid pTETtac4, encoding C fragment, was transferred into the various S. typhimuriummutants, and the levels of antigen expression were found to be equivalent. After primary oral immunization of BALB/c mice, all attenuated strains were capable of penetrating the gut epithelium and colonizing the Peyer’s patches and spleens of mice. Of all strains compared, the ΔpurA mutant colonized and persisted in the Peyer’s patches at the lowest level, whereas the ΔhtrAmutant colonized and persisted in the spleen at the lowest level. The level of specific antibody elicited by the different strains against either S. typhimurium lipopolysaccharide or tetanus toxoid was strain dependent and did not directly correlate to the mutants’ ability to colonize the spleen. The level of immunoglobulin G1 (IgG1) and IgG2a antibody specific for tetanus toxoid was determined in mice immunized with four S. typhimurium mutants. The level of antigen-specific IgG1 and IgG2a was significantly lower in animals immunized with S. typhimurium ΔpurA. Antigen-specific T-cell proliferation assays indicated a degree of variability in the capacity of some strains to elicit T cells to the heterologous antigen. Cytokine profiles (gamma interferon and interleukin-5) revealed that the four S. typhimuriummutants tested induced a Th1-type immune response. Mice were challenged with a lethal dose of tetanus toxin 96 days after oral immunization. With the exception of the S. typhimuriumΔpurA mutant, all strains elicited a protective immune response. These data indicate that the level of total Ig specific for the carried antigen, C fragment, does not correlate with the relative invasiveness of the vector, but it is determined by the carrier mutation and the background of the S. typhimurium strain.
Salmonella typhi is the predominant cause of enteric fever (17) and is transmissible via ingestion of contaminated food or water. S. typhi, once ingested, invades from the small bowel into the reticuloendothelial system, wherein the bacteria replicate in a variety of host cells (11). Live attenuated S. typhimurium strains have been studied extensively in the murine model to obtain insight into the optimal construction of live rationally attenuated S. typhivaccines (30). Live attenuated Salmonellavaccines have been shown to confer better protection against virulentSalmonella infections than traditional whole-cell killed vaccines (5, 9). The best-characterized of the live rationally attenuated salmonellae are those harboring mutations in the prechorismate pathway (15). Prechorismate or aromutants do not produce chorismate, an essential intermediate in the de novo synthesis of aromatic compounds including aromatic amino acids. These S. typhimurium aro mutant vaccines, as well as inducing protective immunity against virulent Salmonellainfection, have been shown to elicit immune responses to a large number of heterologous antigens from a range of pathogens (30).
A number of nonaromatic rationally attenuated S. typhimuriummutants have been generated and studied in the murine model, albeit to various degrees. These mutants include deletions and insertions in genes encoding regulators (cya/crp and ompR) (6, 8), purine metabolism enzymes (purA andpurE) (23, 26), and a heat shock protease (htrA) (4). Several of these nonaromatic mutants have been studied for their capacity to act as vaccine vectors. AnS. typhimurium strain that harbors mutations in the genescya and crp has been used extensively as a vaccine vector and for this reason has been included in our study (7, 16, 28, 31). The genes cya and crpencode adenylate cyclase and cyclic AMP receptor protein, respectively, which regulate expression of a number of Salmonella genes. Similarly, the htrA mutant has been investigated for its ability to carry heterologous antigens, although to a lesser extent.S. typhimurium ΔhtrA strains harbor a deletion in a serine protease gene, and their avirulence may be due to their relative incapacity to mount a complete stress response. Chabalgoity et al. (3) successfully used the htrA mutant as a vaccine vector and demonstrated protection against herpes simplex virus following immunization of mice with S. typhimuriumΔhtrA strains expressing a fusion protein comprising the C fragment of tetanus toxin and the glycoprotein D of herpes simplex virus.
The antigen selected for our study has been extensively investigated inaro-attenuated salmonellae. The C fragment of tetanus toxin (TT) is the immunogenic, nontoxic, binding portion of TT (14). Fairweather et al. (10) reported that two oral doses of an ΔaroA S. typhimurium mutant expressing C fragment from the expression plasmid pTETtac4 successfully immunized mice against lethal challenge with TT. The aim of this study was to assess the capacity of a number of isogenic attenuatedSalmonella strains to act as vaccine vectors by correlating their ability to elicit immune responses with virulence, as measured by in vivo invasion and bacterial persistence.
MATERIALS AND METHODS
Bacterial strains and plasmids.The bacterial strains used in this study are described in Table 1. Five of the six mutant strains studied are isogenic mutants of S. typhimurium SL1344, whereas one, harboring the cya crpmutation (χ4064), is in the S. typhimurium SR-11 background (Table 1.). S. typhimurium BRD175, BRD509, BRD578, BRD726, and LB5010 and pTETtac4 (10) were the generous gift of G. Dougan, Imperial College, London, England, and S. Chatfield and M. Roberts, Medeva Vaccine Research Unit, Imperial College. S. typhimurium SL3261 was kindly supplied by B. A. D. Stocker (Stanford University, Palo Alto, Calif.), and χ4064 was supplied by R. Curtiss III (Washington University, St. Louis, Mo.).
Attenuated S. typhimurium strains
Expression of C fragment by attenuated S. typhimuriumstrains.Plasmid pTETtac4 was electroporated into the r− m+ strain S. typhimurium LB5010 (2) and then transduced by using bacteriophage P22 (Int−) as previously described (37) into BRD175, BRD509, BRD578, BRD726, and SL3261. S. typhimuriumχ4064 was directly electrotransformed with pTETtac4 (1).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli (21) was performed with a 12.5% gel. Western immunoblot analyses were carried out by the method of Towbin et al. (35). Rabbit anti-TT antiserum (Calbiochem, San Diego, Calif.) was diluted at 1/1,000 in phosphate-buffered saline (PBS). Bound antibody was detected with sheep anti-rabbit–horseradish peroxidase (HRP) conjugate diluted 1/1,000 (Silenus Laboratories, Hawthorn, Victoria, Australia), and C fragment was visualized by the chromogen 4-chloro-1-naphthol (Bio-Rad, Hercules, Calif.), with H2O2 as the substrate.
Immunization of BALB/c mice with attenuatedSalmonella strains.Female 6- to 8-week-old BALB/c mice were obtained from The University of Melbourne, Department of Microbiology animal facility. For oral immunizations, bacteria were grown for 24 h without shaking before being resuspended in aliquots of PBS (200 μl/mouse). Mice were orally immunized via a gastric lavage needle. The dose given to each mouse was determined by retrospective viable count. Thirty minutes prior to oral inoculation, mice were administered 100 μl of 10% sodium bicarbonate to neutralize stomach acidity. For antibody and challenge studies, mice were immunized on day 0 and were boosted again on day 28. In colonization studies and in T-cell proliferation and cytokine assays, mice were immunized at day 0 only.
Isolation of salmonellae from spleens and Peyer’s patches of mice.On days 7, 14, and 21 after oral administration of attenuated Salmonella strains (1 × 1010 to 3 × 1010 bacteria per mouse), mice were killed and their spleens and Peyer’s patches were removed. Spleens and Peyer’s patches were homogenized in 5 ml of sterile PBS, using a Stomacher 80 (Seward Medical, London, England) homogenizer. The number of salmonellae present in the organs was determined by viable counting of serial dilutions on Luria-Bertani (LB) agar plates containing antibiotic selection for the attenuated Salmonella strain with and without ampicillin (75 μg/ml).
Measurement of antibody responses by ELISA.Following oral immunization on day 0 (7 × 109 to 2 × 1010 bacteria per mouse), mice were bled weekly from the retro-orbital sinus from days 14 to 56. The titers of antibody present in mouse sera were determined by using a standard enzyme-linked immunosorbent assay (ELISA) or a kinetic ELISA (20, 36). Ninety-six-well Maxisorp immunoplates (Nunc A/S, Kamstrup, Denmark) were coated overnight at 4°C with either S. typhimuriumlipopolysaccharide (LPS; Sigma, St. Louis, Mo.) at a concentration of 10 μg/ml in PBS or tetanus toxoid (Commonwealth Serum Laboratories Ltd. [CSL], Melbourne, Victoria, Australia) at a concentration of 2 flocculating units/ml in carbonate coating buffer) (pH 9.6). In kinetic ELISAs, bound antibody was detected with either a sheep anti-mouse immunoglobulin (Ig)–HRP conjugate (Silenus Laboratories) or an affinity-purified rabbit anti-mouse IgA–HRP conjugate (ICN Biomedicals, Inc., Costa Mesa, Calif.). To determine IgG1 and IgG2a subclass titers by endpoint ELISA, rabbit anti-mouse IgG1 and IgG2a antibodies (Dako, Carpinteria, Calif.) were used, and bound antibody was detected with an anti-rabbit Ig–HRP conjugate (Silenus Laboratories). Serum subclass antibody titer was designated the reciprocal of the dilution of serum that gave an optical density at 492 nm (OD492) value threefold above the value obtained for preimmune serum. ELISAs were developed by using Immunopure OPD (Pierce, Rockford, Ill.), with H2O2 as the substrate.
Production of recombinant C fragment.Recombinant C fragment was produced in Escherichia coli JM101 and purified by using a polyhistidine affinity tag located at the carboxy terminus of the protein. Briefly, a DNA sequence encoding a His6 tag was genetically fused to the 3′ end of the DNA encoding C fragment in the expression construct pTETtac115 (22) by PCR amplification. Mid-log 500-ml cultures of JM101 harboring the expression construct were grown in LB at 37°C with agitation, and C fragment expression was induced by the addition of isopropylthiogalactopyranoside. After a 4-h induction, cells were harvested and resuspended in 20 ml of lysis buffer (2 M urea, 0.1 M sodium phosphate, 0.01 M Tris-HCl [pH 8.0]). Cell lysis was completed by two passages through a French press at 1,000 lb/in2. Insoluble proteins were removed by centrifugation, and the supernatant containing soluble proteins was collected. A suspension of 50% Ni-nitrilotriacetic acid resin (2 ml; Qiagen, Chatsworth, Calif.) was added to the soluble protein fraction. Recombinant C fragment was then bound, washed under denaturing conditions, and eluted by using a pH gradient essentially as described by the manufacturer (Qiagen). The purified denatured protein was refolded by gradual dialysis against decreasing concentrations of urea and then freeze-dried prior to use. Purity was greater than 95% as determined by Coomassie blue staining of SDS-polyacrylamide gels.
Cytokine detection and antigen-induced T-cell proliferation. (i) Splenocytes.Twenty-one days after oral immunization (1 × 1010 to 3 × 1010 bacteria per mouse), groups of three mice were killed and the spleens were removed. Single-cell suspensions of splenocytes were prepared by sieving spleens through wire mesh and washing cells three times in Hanks buffered saline containing 10% fetal calf serum. Cells were seeded in 48-well trays at a concentration of 5 × 106/ml in a volume of 0.5 ml of tissue culture medium (RPMI 1640; CSL), 10% fetal calf serum (Gibco BRL, Gaithersburg, Md.), l-glutamine (2 mM), pyruvate (1 mM), 2-mercaptoethanol (5 × 10−5 M) penicilin (100 U/ml), and streptomycin (100 μg/ml). C fragment was added to wells at concentrations of 10 and 1 μg/ml; media and concanavalin A (ConA; 5 μg/ml) were added to control wells. After incubation of cells for 42 h (37°C in 5% CO2 in air), supernatants were collected and used in cytokine ELISAs. Gamma interferon (IFN-γ) and interleukin-5 (IL-5) enzyme immunoassays were supplied by CSL and were carried out according to the manufacturer’s specifications. The detection limit of the IFN-γ ELISA was >0.014 ng/ml, whereas the detection limit of the IL-5 ELISA was >0.005 ng/ml.
(ii) Enriched T cells.Twenty-eight days after oral immunization (2.5 × 1010 to 3.5 × 1010 bacteria per mouse), groups of five mice were killed and their spleens were removed and pooled. Single-cell suspensions of splenocytes were prepared by sieving spleens through wire mesh. Enriched T-cell suspensions were collected after passage of splenocytes through nylon wool columns (18). Enriched T cells were seeded into flat-bottom 96-well tissue culture trays (Nunc A/S) at a concentration of 3 × 105 in a volume of 100 μl in tissue culture medium. Gamma-irradiated (300 rads) syngeneic normal spleen cells were added at a concentration of 3 × 105(in 100 μl of tissue culture medium) as a source of antigen-presenting cells. Recombinant C fragment (0.5 μg) was added to the first well and was twofold serially diluted across the plate. Control wells contained either 5 μg of ConA (Sigma) per ml or medium alone. Cells were incubated with antigen for 72 h at 37°C in 5% CO2 in air. Then 1 μCi of tritiated [3H]thymidine was added to all wells, and cells were incubated for a further 18 h before being harvested onto glass fiber filters (Packard, Meriden, Conn.). Incorporation of radioactive label was determined by direct beta counting in a Packard Matrix 9600.
Challenge of immunized mice with TT.Mice were challenged subcutaneously with TT (batch SS620; CSL) which had been semipurified by ammonium sulfate precipitation and reconstituted in PBS at a concentration of approximately 500 50% lethal doses (LD50)/ml. Mice were given 200 μl of TT which contained 100 times the murine LD50. Mice were monitored regularly and at the first sign of paralysis were killed and designated not protected. Mice which showed no sign of paralysis for over 4 weeks were designated protected against lethal TT challenge.
Statistical analysis.Unrelated groups of data were compared by using the unpaired Student t test. When the standard deviations of data groups were significantly different, the unrelated groups were compared by using the nonparametric Mann-Whitney test. A probability (P) value of less than 0.05 indicated that the groups were significantly different. To perform statistics on data groups which contained values below the point of detection for the particular assay, arbitrary values below the detection point were used. For example, when statistics were performed on the IgG2a subclass titers in Fig. 6, the points below the level of detection (100) were arbitrarily assigned values of 50.
RESULTS
Expression of C fragment in S. typhimuriummutants.Plasmid pTETtac4 (10) was electrotransformed into S. typhimurium LB5010 (2) and transduced from this mutant into five different S. typhimuriumattenuated strains by using Int− P22 bacteriophage-mediated transduction. One attenuated strain (χ4064) was electrotransformed with pTETtac4. C-fragment expression in the sixS. typhimurium mutant strains, BRD509, SL3261, BRD726, BRD578, BRD175, and χ4064 harboring pTETtac4, was determined by immunoblotting. Whole-cell protein preparations of BRD509(pTETtac4), SL3261(pTETtac4), BRD726(pTETtac4), BRD578(pTETtac4), BRD175(pTETtac4), and χ4064(pTETtac4) were electrophoresed in an SDS–12.5% polyacrylamide gel, and the proteins were transferred to nitrocellulose. A protein of approximately 50 kDa of equivalent intensity was detected in all mutant strains containing pTETtac4 (Fig.1), using rabbit polyclonal anti-TT antiserum, and was absent from the mutant strains alone (data not shown). The size of C fragment expressed by S. typhimuriumcorresponded to the size of purified commercial C fragment fromClostridium tetani on a Western immunoblot (data not shown).
Western blot showing the expression of C fragment by various S. typhimurium mutants. Cell lysates of S. typhimurium mutants containing pTETtac4 were subjected to SDS-PAGE, and the proteins were transferred to nitrocellulose which was subsequently probed with polyclonal anti-TT antiserum. Lanes: 1, molecular mass markers (positions are indicated in kilodaltons); 2, BRD509; 3, SL3261(pTETtac4); 4, BRD509(pTETtac4); 5, BRD726(pTETtac4); 6, BRD578(pTETtac4); 7, BRD175(pTETtac4); 8, χ4064(pTETtac4) (a nonisogenic mutant).
Persistence of the Salmonella mutants in the spleen and Peyer’s patches.BALB/c mice were orally immunized with attenuated S. typhimurium expressing C fragment, and the kinetics of colonization and persistence of the bacteria in vivo was investigated. On days 7, 14, and 21, three to four mice from each group were killed and the numbers of bacteria present in the spleen (Fig.2A) and Peyer’s patches (Fig. 2B) were determined by viable count. pTETtac4 appeared to be stably maintained by salmonellae in vivo since the numbers of bacteria isolated from the spleen and Peyer’s patches were equivalent on media with and without antibiotic selection for the plasmid.
Time course of colonization and persistence of spleen and Peyer’s patches. Groups of mice were immunized orally with variousS. typhimurium mutants expressing C fragment:aroA [SL3261(pTETtac4)], aroAD[BRD509(pTETtac4)], ompR [BRD578(pTETtac4)],purA [BRD175(pTETtac4)], htrA[BRD726(pTETtac4)], and cya crp[χ4064(pTETtac4)] strains. Seven, 14, and 21 days later, the mice were killed and the numbers of bacteria present in the spleen (A) and Peyer’s patches (B) of each animal were determined by isolating bacteria on LB agar (open shapes) or LB agar containing ampicillin (closed shapes). Each point denotes the number of bacteria isolated from either spleen or Peyer’s patches for one mouse. ∗, the number of bacteria isolated from the organ is significantly lower than the number of bacteria isolated from all other immunized mice. The dashed line in each graph represents the level of detection, which is <5 organisms. #, nonisogenic mutant.
Some variation was observed in the abilities of the different mutants to colonize and persist in the spleen (Fig. 2A). BRD726(pTETtac4), thehtrA mutant, was the only strain which was unable to colonize the spleen of all inoculated mice. On day 14, thehtrA mutant colonized the spleen at a significantly lower level than the aroA, aroAD, ompR, andcya crp mutants (P < 0.05), and by day 21 all BRD726(pTETtac4) cells had been cleared from the spleen. Even though BRD726(pTETtac4) was not detectable in the spleen on day 21, it was still possible to isolate the htrA mutant from the Peyer’s patches at this time point (Fig. 2B). All mutants were capable of colonizing the Peyer’s patches; however, the purA mutant [BRD175(pTETtac4)] was unable to persist in the mice for 21 days (Fig. 2B). BRD175(pTETtac4) colonized the Peyer’s patches on day 7 at a significantly lower level (10- to 100-fold) than all otherSalmonella mutants tested (P < 0.05). By day 21, the level of bacteria detected in the Peyer’s patches ofpurA-immunized mice was comparatively low (P< 0.05), with all mice having cleared plasmid-bearing bacteria.
Anti-Salmonella and anti-tetanus toxoid antibody responses generated in BALB/c mice immunized with S. typhimurium mutants expressing C fragment.Groups of five BALB/c mice were immunized orally with attenuated S. typhimurium expressing C fragment and were similarly boosted on day 28. All mice were bled weekly from days 14 to 56, and the serum antibody response was examined by using a kinetic ELISA againstSalmonella LPS and TT (Fig.3).
LPS-specific and tetanus toxoid-specific serum antibody responses. Groups of mice were immunized orally with various S. typhimurium mutants expressing C fragment (for identities, see the legend to Fig. 2). Serum samples were collected at two weekly intervals prior to secondary immunization on day 28; after the boost, sera were collected weekly until day 56. The amounts of S. typhimuriumLPS-specific (A) and tetanus toxoid-specific (B) total Ig present in serum were determined by using a kinetic ELISA with sera diluted 1/1,000. Each point represents the mean and standard deviation of sera from five mice. #, nonisogenic mutant; ∗, the anti-tetanus toxoid antibody titer detected in the sera of purA[BRD175(pTETtac4)]-immunized mice is significantly different (P < 0.05) than the antibody titers detected in all other mice.
LPS-specific serum antibody (total Ig) was detected in all mice immunized with the six different attenuated S. typhimuriumstrains expressing C fragment (Fig. 3A). In all mice, the anti-LPS response took 14 days after immunization to increase, and then the antibody titer peaked either on day 35 or on day 42. On day 35, theompR mutant induced the highest (P < 0.05) anti-LPS antibody titer compared with all other S. typhimurium mutants. The purA mutant induced significantly lower antibody titers than the ompR mutant on days 28 to 56 (P < 0.05). The htrA mutant which colonized and persisted poorly in the spleen was still capable of inducing an anti-LPS response equivalent to that induced by the four other isogenic mutants, ΔaroA, ΔaroAD,ΔompR, and ΔpurA, all of which demonstrated greater splenic persistence. These results suggest that the level of splenic colonization and persistence does not correlate with the homologous antibody response induced by the Salmonellacarrier.
The levels of antibody induced against the heterologous antigen C fragment expressed in the six different mutants are shown in Fig. 3B. Four isogenic mutants, harboring mutations in aroA,aroAD, ompR, and htrA[SL3261(pTETtac4), BRD509(pTETtac4), BRD578(pTETtac4), and BRD726(pTETtac4), respectively], induced a higher level of anti-tetanus toxoid total Ig than the purA mutant [BRD175(pTETtac4)] (P < 0.05). This result suggests that the type of attenuating mutation does affect the strains’ ability to present a heterologous antigen to the host immune system. ThehtrA mutant, BRD726(pTETtac4), which colonized and persisted poorly in the spleen, induced a higher level of antibody against tetanus toxoid than the purA mutant [BRD175(pTETtac4)] (P < 0.05), which remained detectable in the spleen on day 21. This observation suggests that splenic persistence is not essential for the induction of a high antibody titer against the carried antigen. In addition to inducing the lowest level of antibody against tetanus toxoid (P < 0.05), the purA mutant [BRD175(pTETtac4)] colonized and persisted poorly in the Peyer’s patches compared with the other isogenic mutants. This result indicates that Peyer’s patch colonization and persistence of the Salmonella carrier may be necessary to induce a high antibody response against the carried antigen.
Similar levels of LPS-specific IgA antibody were detected in the sera of mice immunized with all of the isogenic S. typhimuriummutants expressing C fragment [SL3261(pTETtac4), BRD509(pTETtac4), BRD578(pTETtac4), BRD726(pTETtac4), and BRD175(pTETtac4)], whereas the level of tetanus toxoid-specific serum IgA antibody detected in mice immunized with the purA mutant expressing C fragment was considerably less than that detected in the serum of all other immunized mice (data not shown).
χ4064(pTETtac4) is a nonisogenic mutant harboring deletions in the genes cya and crp of S. typhimuriumSR-11. This strain was capable of inducing a low-level antibody response to S. typhimurium LPS which was equivalent to the level induced by the purA mutant (P > 0.05); in contrast, χ4064(pTETtac4) induced a significantly higher anti-tetanus toxoid antibody titer than the purA mutant expressing C fragment (P < 0.05).
Challenge of mice immunized with S. typhimurium mutants expressing C fragment with 100 LD50 of tetanus toxin.On day 96, mice that had previously been immunized with S. typhimurium mutants expressing C fragment on days 0 and 28 were challenged with 100 LD50 of TT. Five mice from each group were challenged subcutaneously with TT and then observed for signs of paralysis. Four of the five mice immunized with BRD175(pTETtac4), harboring a mutation in the purA gene, showed signs of paralysis. Two mice showed paralysis on day 1 after challenge, one showed paralysis on day 2, and one showed paralysis on day 5. The mice that displayed paralysis upon challenge corresponded to those animals with the lowest levels of anti-tetanus toxoid antibody detected in serum at day 56 (Fig. 4).
Challenge of immunized mice with TT. The amount of tetanus toxoid-specific total Ig in sera from individual mice on day 56 after immunization with S. typhimurium aroA[SL3261(pTETtac4)] (□), aroAD[BRD509 (pTETtac4)] (○), ompR[BRD578(pTETtac4)] (▵), htrA [BRD726(pTETtac4)] (✙),purA [BRD175(pTETtac4)] (▹), and cya crp[χ4064(pTETtac4)] (◂) strains expressing C fragment, as determined by kinetic ELISA when serum is diluted 1/1,000. The symbols that lie beneath the dashed line denote individual mice that showed signs of paralysis when challenged on day 96 with 100 LD50of tetanus toxin; the symbols that lie above the dashed line denote mice that were protected against lethal TT challenge. #, nonisogenic mutant.
Anti-tetanus toxoid IgG subclass responses induced in the serum of BALB/c mice immunized with S. typhimurium mutants expressing C fragment.To further define humoral responses induced after S. typhimurium immunization, the titers of serum IgG subclass antibodies, IgG1 and IgG2a specific for tetanus toxoid, were determined in mice 42 days after immunization with the six different S. typhimurium mutants harboring pTETtac4 (Fig.5). IgG1 antibody specific for tetanus toxoid was detected in the sera of all mice; however, the titer of IgG1 detected in mice immunized with BRD175(pTETtac4) was significantly lower than in all other immunized mice tested (P < 0.05). Mice immunized with SL3261(pTETtac4), BRD509(pTETtac4), BRD578(pTETtac4), BRD726(pTETtac4), and χ4064(pTETtac4) had detectable amounts of tetanus toxoid-specific IgG2a in serum on day 42. IgG2a specific for tetanus toxoid was detected in only one of five mice immunized with BRD175(pTETtac4). The subclass analysis revealed that thepurA mutant induced a lower IgG1 and IgG2a antibody response (P < 0.05) than the five other mutants expressing C fragment, with a predominance of IgG1 specific for tetanus toxoid.
Anti-tetanus toxoid-specific IgG1 and IgG2a antibody. The amount of tetanus toxoid-specific IgG1 and IgG2a present in sera from individual mice on day 42 after immunization with aroA[SL3261(pTETtac4)] (▵), aroAD [BRD509(pTETtac4)] (•), htrA [BRD726(pTETtac4)] (◊),