Physical and Chemical Characterization and Immunologic Properties of Salmonella enterica Serovar Typhi Capsular Polysaccharide-Diphtheria Toxoid Conjugates

Vi purification and characterization.Vi was extracted and purified from S. Typhi strain Ty2 (ATCC 19430) as described previously (54). Briefly, the culture supernatant was mixed with 0.2% hexadecyl-trimethyl-ammonium bromide (Cetavlon; Sigma Life Science). The precipitate was washed with 0.1 M NaCl and extracted with 1.0 M NaCl. The supernatant was brought to 25% ethanol and centrifuged to remove nucleic acids and then was brought to 75% ethanol. The precipitate was washed with 98% ethanol, freeze-dried, and extracted four times with buffered phenol at 10°C. The water phase of the extraction was precipitated with 75% ethanol, dialyzed extensively against pyrogen-free deionized water, and freeze-dried.

The antigenicity of Vi was confirmed by immunodiffusion reaction with burro Vi hyperimmune serum (provided by NIH) (30). The molecular size of Vi was estimated by the size exclusion chromatography (SEC) method in a 1.5- by 95-cm Sepharose CL-4B column (GE Health). The O-acetyl content of Vi was determined by Hestrin assay, using acetylcholine chloride as a standard (50). The nucleic acid content was determined with UV spectroscopy at a wavelength of 260 nm. The protein concentration was measured by the Coomassie protein assay (Thermo Scientific), using bovine serum albumin (BSA) as a standard (5). The gel-clotting method for the Limulus amebocyte lysate (LAL) test was used to estimate the contamination of lipopolysaccharide (LPS) (Associates of Cape Code Inc.).

Derivatization of DT.Bulk DT vaccines were provided by the Lanzhou Institute of Biological Products, People's Republic of China, for conjugates C1 and C2 and by Shantha Biotech Inc., India, for conjugates C3, C4, and C5. DT was concentrated by passing it through Vivaspin 6 centrifugal concentrators (10,000-molecular-weight cutoff; Sartorius), and buffer was exchanged for saline in preparation for derivatization.

Ten mg/ml of DT was mixed with ADH (Sigma) at 35 mg/ml. 1-Ethyl-3(3-dimethylaminopropyl) carbodiimide (EDAC) (Sigma-Aldrich Fine Chemicals) was added as powder to a final concentration of 2 mg/ml. The pH was maintained at 5.6 to 5.8 with 0.1 M HCl for 45 min at room temperature (24, 47). The reaction mixture was passed through a Vivaspin 6 (MWCO 10K) 5 times to remove EDAC and unbound ADH. The derivatized DT was designated DT_AH.

The level of derivatization was measured by colorimetric reactions. The acid hydrazide (AH) concentration was determined by reactions with 2,4,6-trinitrobenzenesulfonic acid (TNBS) (Fluka) using ADH as a standard (24, 44, 50), and the protein concentration was measured by Coomassie protein assay. The degree of derivatization was expressed as a percentage of AH by weight and the molar ratio of AH to DT.

Conjugation.Vi (3 mg/ml) was dissolved in 70 mM MES (morpholineethanesulfonic acid) buffer (pH 5.5; Sigma Life Science) overnight. EDAC was added as powder to a final concentration of 10 mM and mixed at room temperature for 5 min. DT_AH solution was added dropwise to a final concentration of 3 mg/ml, and the reaction proceeded at room temperature for 3 hours. The reaction mixture was passed through Sephacryl S-1000 (2.5 by 95 cm; GE Healthcare) in 0.2 M NaCl. Fractions containing both Vi and DT, with a partition coefficient (K_av) of <0.40, were pooled and designated Vi-DT, lots C1 to C5. Thimerosal (Sigma Life Science) solution was added to a final concentration of 0.01%, and the conjugates were stored at 4°C (47, 50).

The content of Vi in conjugates was determined by Hestrin assay, using Vi as a standard (24). The DT content was determined by Coomassie protein assay.

Stability tests.The accelerated method was applied for assay of stability (16, 18). Conjugate C3 was stored at 4°C, 25°C, and 37°C for 5 weeks or subjected to 20 cycles of freeze-thaw at −20°C. High-pressure liquid chromatography (HPLC) with an Ohpak SB-806 M HQ column (Shodex, Japan) was used to estimate the molecular sizes of the conjugates. Dextrans 110, 500, and 2000 (Pharmacia) were used as references.

Immunization.Outbred female mice (CD1; Charles River Laboratories) were used for all experiments unless otherwise specified. Hyperimmune Vi sera were raised in 8-week-old mice by multiple subcutaneous (s.c.) injections of formalin-inactivated S. Typhi (1, 30). DT hyperimmune sera were raised in 8-week-old mice by multiple s.c. injections of DT with incomplete Freund adjuvant (Pierce Biotechnology), as described previously (24).

For immunogenicity evaluation, 6-week-old mice (10/group) were injected twice s.c. at 4-week intervals with 100 μl of the immunogen in saline containing 2.5 μg of Vi as a conjugate or as a polysaccharide. Blood samples were collected through eye bleeding 2 weeks after each injection.

Immunogenicity of Vi-DT C1 was compared with that of Vi-rEPA (lot 13106), a clinical lot prepared at NIH. Following the NIH research protocol, 5- to 6-week-old female mice (Swiss Webster; 10/group) were injected s.c. twice 2 weeks apart with conjugates containing 2.5 μg of Vi per injection. The mice were exsanguinated 7 days after each injection. The negative controls were mice injected with 100 μl saline.

Vi as a priming or boosting vaccine.The effect of Vi on the immunogenicity of Vi conjugate was evaluated in five groups of mice (6 weeks old; 10/group) by injecting Vi polysaccharide s.c. at the first or the second dose and was compared with those receiving 2 doses of conjugate C5. Vi and C5 were injected into mice in various sequences (two injections 4 weeks apart): Vi/C5, C5/Vi, and C5/C5. Serum samples were collected via eye bleeding 2 weeks after each injection. The control groups were those injected with C5 once or Vi alone. Serum samples were taken at 2 and 6 weeks after the injection.

Dosage study.Three groups of mice (6 weeks old; 10/group) were injected s.c. with various amounts of C4 containing 2.5 μg, 1.25 μg, and 0.5 μg of Vi, respectively. Mice were boosted with the same dosage 4 weeks later. Blood samples were taken 2 weeks after each injection via eye bleeding.

Serology.Double immunodiffusion was performed in 1% agarose in saline. Serum IgG Vi and DT antibody levels were determined by enzyme-linked immunosorbent assay (ELISA) using Nunc Maxisorp plates as described previously (13, 48). ELISA data were processed with Program ELISA for Windows (Centers for Disease Control and Prevention) (33). IgG anti-Vi and IgG anti-DT levels were expressed in ELISA units (EU) using hyperimmune sera as references. International Vaccine Institute (IVI) anti-Vi hyperimmune serum was calibrated with NIH reference serum (100 EU) and assigned a value of 82 EU (47-50). IVI anti-DT hyperimmune serum was assigned 100 EU.

The results were tabulated as the geometric mean (GM) and 95% confidence interval (CI).

Statistical evaluation.Antibody levels were expressed as the GM) of EU. Sera with antibodies below detectable levels were assigned 0.02 EU (one-half of the lowest detectable level) for the purpose of GM calculation. Statistical significance was calculated using a two-tailed Student's t test with a confidence level of 95% (P = 0.05).

Vi polysaccharide.The purified Vi met WHO requirements (in parentheses) for polysaccharide typhoid vaccine: the O-acetyl content was 2.56 mmol/g of Vi (≥2.0 mmol/g of Vi), and 57.6% of Vi (≥50%) was eluted from a Sepharose CL-4B column before the K_av reached 0.25. The purity of the polysaccharide was as follows: nucleic acid content < 2.0% (<2.0%), protein content < 0.25% (<1.0%), and an endotoxin level < 0.3 EU per μg of Vi (54).

Characterization of DT_AH.The level of derivatization of DT_AH was between 2.9% and 3.5% AH/DT (wt/wt), corresponding to an AH/DT molar ratio of 10 to 12 (Table 1). Based on the 14% SDS-PAGE pattern, the molecular weight of DT_AH was similar to that of the native DT (data not shown). Double immunodiffusion with DT antiserum showed a line of identity between DT and DT_AH (Fig. 1).

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FIG. 1.

Double immunodiffusion in 1% agarose. DT_AH showed a line of identity to DT against DT antiserum.

Chemical compositions of Vi-DT conjugates (Table 1).The Sephacryl S1000 gel filtration profiles of all five lots of the conjugate reaction mixtures were similar and were exemplified by C2 in Fig. 2. One major peak was eluted from the void volume (K_av = 0) and trailed through the rest of the column. Only the fractions eluted before the K_av = 0.4 were pooled as the final conjugate C2. Since C2 was eluted before Vi and DT_AH, this indicated C2 had a higher molecular weight than the individual components. The size distribution was further confirmed by the HPLC analysis (Fig. 3, left). All five conjugates showed similar profiles, with a single major peak at 6.06 min (range, 5.04 to 7.18 min), ahead of both Vi and DT.

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FIG. 2.

Gel filtration profiles of Vi, DT, and Vi-DT conjugate (exemplified by C2) through a 2.5- by 95-cm column of Sephacryl S-1000 in 0.2 M NaCl (pH 7.4). Fractions (15 ml/fraction) in which K_av ≤ 0.40 were collected as conjugates. Detector wavelength, 280 nm (milliabsorbance unit).

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FIG. 3.

HPLC profiles of Vi-DTs using refractive index detectors (milliabsorbance units). The column was a Shodex OHpak SB-806 HQ (8 by 300 mm), the eluent was 0.2 N saline (pH 7.2), and the elution speed was 1 ml/minute. (Left) All five conjugates showed similar profiles, with a single major peak at 6.06 min (spanning from 5.04 to 7.18 min, ahead of both Vi and DT). (Right) Conjugate C3 underwent repeated freeze-thaw cycles 20 times and showed a profile identical to that of C3 kept at 4°C. In contrast, C3 stored at 25°C or 37°C for 5 weeks showed obvious size reduction. No dissociation of C3 was observed when it was stored at −20°C for the same period.

The conjugates failed to enter the 14% SDS-polyacrylamide gel, and no free protein was detected (data not shown).

The Vi/DT ratios of the conjugates ranged from 0.80 to 1.09 by weight, equivalent to 198 to 270 Vi repeating units per DT molecule. Based on the recovery of Vi, the yield was between 42% and 52% (Table 1).

The conjugates formed a line of identity with Vi when reacting with anti-Vi serum in immunodiffusion (data not shown). In contrast, none of the conjugates formed visible precipitation when reacting with anti-DT in immunodiffusion.

Stability test.Conjugate C3 underwent repeated freeze-thaw cycles 20 times and showed no visible change in solubility and immunogenicity (data not shown). The HPLC profile of the treated C3 was identical to that of C3 kept at 4°C. In contrast, C3 stored at 25°C or 37°C for 5 weeks showed obvious size reduction, evident from the appearance of trailing in the HPLC profile. No dissociation of C3 was observed when it was stored at −20°C for the same length of time (Fig. 3, right).

Vi elicited 0.39 EU (GM) of serum IgG Vi antibodies. All Vi-DT conjugates elicited levels of IgG anti-Vi antibodies in mice significantly higher than those elicited by Vi alone: anti-Vi levels were about 16 and 42 times higher than those in the Vi groups after the first and second injections, respectively (P < 0.001). The IgG Vi antibody levels increased from 6.5 to 11.1 EU after the first injection and to 16.9 to 18.7 EU after the second injection (1st versus 2nd injections, P < 0.05 for all conjugates except C4, P = 0.19). The GM levels of IgG Vi antibodies after the 1st and 2nd injections were similar among the 5 conjugates (P > 0.083 and P > 0.69, respectively).

One injection of the conjugates in mice did not elicit detectable IgG anti-DT antibodies. After the second injection, anti-DT antibodies were detected in all groups, with C3 having the highest level. Since dosages of DT varied in different groups, no statistical analysis was performed.

As observed in humans injected with Vi-rEPA, the serum IgG Vi antibody response was related to the dosage of Vi-DT: the higher the dosage, the higher the level of antibodies after each of the two injections (7). The group of mice that received 2.5 μg elicited levels of antibodies significantly higher than in those that received 0.5 μg after each of the two injections (P < 0.001). There was a difference between the groups that received 2.5 μg and those that received 1.25 μg after the first injection (P < 0.05), but there was no difference following the second injection (P > 0.3). IgG anti-Vi levels were significantly higher in the 1.25-μg group than in the 0.5-μg group after either of the two injections (P < 0.002 and P < 0.003, respectively).

After one injection, as observed before, groups that received C5 had significantly higher anti-Vi levels than those that received Vi (11.32, 12.95, and 13.20 versus 0.35 and 0.20; P < 0.001) (47-50). After 2 injections, the C5/C5 group had significantly higher levels of anti-Vi than those injected with either Vi/C5 or C5/Vi (16.88 versus 6. 31 and 5.81; P < 0.001). However, IgG anti-Vi levels were comparable in the C5 and Vi/C5 groups 2 weeks after C5 injection (11.32 versus 5.81; P = 0.12), indicating that previous immunization with Vi did not impair or enhance the immune response to Vi-DT.

There was a slight increase in the IgG anti-Vi level in the C5/Vi group compared with those that received C5 only, but the difference was not statistically significant (6.31 versus 2.66; P > 0.05).

The effects of different carriers on immunogenicity were examined by comparing conjugates C1 and Vi-rEPA in mice. The polysaccharide/protein molar ratios were similar in C1 and Vi-rEPA. One week after each of the two injections, the IgG anti-Vi level induced by Vi-rEPA was higher than that elicited by C1, but the difference was not statistically significant (43.99 versus 35.01; P = 0.58). Both conjugates induced levels of IgG anti-Vi statistically higher than those elicited by Vi (P < 0.001) (Table 5). The slightly higher response in C1 (35.01 EU) than those observed in immunogenicity evaluation (18.74 EU) (Table 2) could be due to the different strains of mice used in the studies.