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Escherichia coli O157, an emerging pathogen, causes severe hemorrhagic colitis and the extraintestinal complication of hemolytic uremic syndrome (HUS) in 5 to 10% of patients (11, 16, 26). Pathogens that cause hemorrhagic colitis and HUS, including E. coli O157, O111, and O26 and Shigella dysenteriae type 1, produce Shiga toxin 1 or 2 (Stx1 or Stx2, respectively), or both (6, 7, 10, 19, 25, 28). The source of E. coli O157 is mainly contamination of food or of drinking water by bovine feces (5, 26). Treatment of infection with E. coli O157 has been difficult because antibiotics do not change the course of the enteritis of E. coli O157 or S. dysenteriae type 1 and may increase the incidence of HUS caused by these two pathogens (5, 22). This untoward effect has been proposed to be mediated by antibiotic-induced bacteriolysis and release of intracellular Shiga toxins.

O-specific polysaccharide (O-SP) conjugates for E. coli O157 infections are designed to elicit serum immunoglobulin G (IgG) anti-lipopolysaccharide (anti-LPS) that will inactivate the inoculum on the intestinal epithelium (12, 14, 24). A phase 1 clinical trial of E. coli O157 O-SP bound to Pseudomonas aeruginosa recombinant exoprotein A (rEPA) showed these conjugates to be safe and to elicit statistically significant increases in levels of IgG anti-LPS with bactericidal activity in all of 87 adult recipients: 80% responded with 4-fold increases in anti-LPS levels within 1 week (14).

One concern related to the LPS-based vaccine is that induction of IgG anti-LPS with bactericidal activity could increase the incidence of HUS through toxin released upon lysis of the pathogen. Accordingly, in this study we synthesized conjugates of E. coli O157 O-SP bound to the nontoxic B subunit of Stx1 (Stx1B) that could induce both serum IgG anti-LPS with bactericidal activity and neutralizing antibodies to Stx1 (1, 2, 29). We modified the conjugation schemes from our earlier studies for the following reasons. (i) In the new bivalent conjugate, it was just as important to retain and to improve the immunogenicity of Stx1B as it was to retain and improve that of O-SP. (ii) Stx1B is substantially smaller than the carrier protein rEPA used in a previous clinical study and thus contains fewer reaction sites for conjugation than rEPA. Furthermore, based on our past experiences, the molecular weight of the carrier protein influences the immunogenicity and the final yield of the conjugate. We have therefore chosen conjugation schemes that would minimize modifications to the carrier protein and would result in higher yield.

E. coli O157 O-SP was prepared by treatment of LPS with acetic acid (12, 14, 20). Stx1B was synthesized by Vibrio cholerae 0395-N1 (pSBC32 containing the Stx1B gene) and purified by affinity chromatography (1, 3, 17, 21). Sodium dodecyl sulfate-7% polyacrylamide gel electrophoresis of Stx1B showed one major band at 9 kDa and a faint band with a slightly higher molecular mass which does not correspond to the A subunit of Stx1 (data not shown).

For conjugation, O157 O-SP was bound to the Stx1B directly by treatment with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) or by carbodiimide-mediated condensation, with adipic acid dihydrazide (ADH) as the linker (13, 15). For direct conjugation, CDAP (100 mg/ml in acetonitrile) was added to the O-SP in saline (5 mg/ml) at 0.3/1 (wt/wt) at room temperature, pH 5.8 to 6.0. Sixty microliters of 0.2 M triethylamine was added to bring the pH to 7.0, a level which was maintained for 2 min. An equal weight of Stx1B was added to the CDAP-treated O-SP, and the pH was maintained at 8.0 to 8.5 for 2 h. The reaction mixture was passed through a 1.5- by 90-cm Sepharose 6B column in 0.2 M NaCl, and the void volume fractions were collected together and designated OSP-Stx1B.

Conjugate with ADH as the linker was prepared by modifying the scheme for O-SP-rEPA conjugate described previously (12, 14). Briefly, after the addition of triethylamine in the above procedure, an equal volume of 0.8 M ADH in 0.5 M NaHCO₃ was added and the pH was maintained at 8.0 to 8.5 for 2 h. The reaction mixture was dialyzed against saline overnight at 4°C and passed through a 2.5- by 31-cm P10 column in water. The void volume fractions were pooled, freeze-dried, and together designated OSP-AH. The level of derivatization of O-SP with ADH was similar (3.1 [wt/wt]) to that of our previous E. coli O157 preparations (14). OSP-AH (10 mg), dissolved in 2 ml of saline, was added to an equal weight of Stx1B, and the pH was brought to 5.1. The reaction mixture was put on ice, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was added to a final concentration of 0.05 M, and the pH was maintained at 5.1 to 5.5 for 2 h. The reaction mixture was passed through a 1.5- by 90-cm Sepharose 6B column in 0.2 M NaCl, and the void volume fractions were collected and together designated OSP-AH-Stx1B. The saccharide/protein ratios were 0.5 (wt/wt) and 0.51 (wt/wt) for OSP-Stx1B and OSP-AH-Stx1B, respectively. The yields, based on saccharide in the conjugates, were 2.3% for OSP-Stx1B and 3.4% for OSP-AH-Stx1B. Both OSP-AH-Stx1B and OSP-Stx1B formed lines of identity when reacting with rabbit anti-Stx1 and mouse hyperimmune serum against E. coli O157 (data not shown).

Female general purpose mice (n = 10/group) were injected subcutaneously with saline or one of the conjugates containing 2.5 µg of saccharide on days 0, 14, and 28. The mice were exsanguinated 7 days after each injection. Pooled sera from hyperimmunized mice were used as a reference, and 1,000 enzyme-linked immunosorbent assay units (EU) were assigned to each IgG and IgM. Neutralization of Stx1 and Stx2 was measured with HeLa (CCL-2) cell monolayers in 96-well, flat-bottom microtiter plates (9). Each well was seeded with 1 × 10⁴ to 6 × 10⁴ cells in a 0.1-ml volume. Monolayers were established by overnight incubation in 5% CO₂. Toxin neutralization was determined by incubating dilutions of mouse serum with Stx1 or Stx2, each at a final concentration of 100 pg/ml. The serum-and-toxin mixture was incubated at room temperature for 30 min and a 0.1-ml volume was added to each well. Following incubation overnight, the surviving cells were determined spectrophotometrically by crystal violet staining (9). Toxin neutralization was determined from the dose-response curve of either Stx1 or Stx2 on each 96-well plate, and titer was expressed as the highest serum dilution to yield 50% neutralization. Representative serum samples (two from each group) were assayed for their complement-assisted bactericidal activity as described previously (12, 14).

After three injections, both conjugates elicited statistically significant increases in levels of IgG and IgM anti-LPS (Table 1). The geometric mean (GM) anti-LPS levels elicited by OSP-Stx1B were 63 EU for IgG and 14 EU for IgM, and those elicited by OSP-AH-Stx1B were 166 EU for IgG and 25 EU for IgM: the differences between two conjugates were not statistically significant. There was no detectable complement-dependent bactericidal activity in sera from mice injected with saline. Sera from mice injected with OSP-Stx1B or OSP-AH-Stx1B elicited bactericidal antibodies to E. coli O157 (Table 2). This activity was removed by absorption with E. coli O157 LPS.

Sera from mice injected with saline showed no neutralization of Stx1 or Stx2. All sera from mice injected with conjugates showed neutralization of Stx1 (Table 3). At a 1/1,000 dilution, the GM level of neutralization was 90% for OSP-AH-Stx1B and 98% for OSP-Stx1B (not shown). Mice injected with OSP-Stx1B had a significantly higher GM neutralization antibody titer than those injected with OSP-AH-Stx1B (14,000 versus 8,040; P = 0.03). None of the serum samples from mice injected with either conjugate showed neutralization of Stx2.

In summary, both conjugates composed of OSP and Stx1B elicited anti-LPS with bactericidal activity to E. coli O157 and neutralizing antibodies to Stx1. The immunogenicity of each component of the conjugate was related to configuration and the chemical linkage between polysaccharide and protein. OSP-Stx1B, prepared by direct conjugation of the saccharide to the protein, elicited higher levels of neutralizing antibodies to Stx1 than OSP-AH-Stx1B that used ADH as a linker. One possible explanation is that in OSP-AH-Stx1B synthesis, the ADH-derivatized polysaccharide was bound to the carboxyl amino acids on Stx1B through EDC condensation. Carboxyl amino acids may be important for the immunogenicity of Stx1B. In addition, treatment with EDC condensation during synthesis of OSP-AH-Stx1B could have caused protein aggregation and altered its immunologic properties. OSP-Stx1B, on the other hand, was synthesized without utilizing EDC, and synthesis was mostly through the lysine groups when Stx1B was conjugated with the polysaccharide. Similar results have been found in conjugates of Salmonella paratyphi A O-SP and Staphylococcus aureus capsular polysaccharides (13). Direct conjugation of O-SP to the protein seems to be the best compromise for synthesizing these bifunctional conjugates.

As predicted, the B subunit elicited neutralizing antibodies to the homologous Stx1 but not neutralizing antibodies to Stx2 (25, 29). Our experience with conjugates of the capsular polysaccharide from Salmonella typhi, Vi, with cholera toxin and its B subunit is that the holotoxin is a more effective carrier than its B subunit for both saccharide and neutralizing antibodies (27). Accordingly, we plan to use nontoxic mutants of Shiga holotoxins 1 and 2 as carrier proteins for E. coli O157 O-SP conjugates (17, 18). Concurrent vaccination with these proteins plus our E. coli O157 O-SP-Shiga mutant toxin conjugates could enhance the immune responses of both the anti-LPS and the antitoxins (4, 27). These anti-toxins could also be used to neutralize other non-O157 Shiga toxin-producing organisms (6, 7, 10, 23, 28). Shigella sonnei-rEPA conjugates were effective in significantly reducing the number of shigellosis cases in adults within 7 to 14 days of vaccination (8). Similarly, in the clinical trial of E. coli O157, the conjugate vaccines elicited significant antibody level increases within 1 week (12). We propose that E. coli O157 conjugate vaccines may be useful in controlling outbreaks and epidemics. Such conjugate formulations could also be considered for making hyperimmune therapeutic sera.