Phase 1 and phase 2 studies of Salmonella enterica serovar paratyphi A O-specific polysaccharide-tetanus toxoid conjugates in adults, teenagers, and 2- to 4-year-old children in Vietnam.

Salmonella enterica serovar Paratyphi A O-specific polysaccharide (O-SP) was activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) and bound to tetanus toxoid (TT) with adipic acid dihydrazide as a linker (SPA-TT(1)) or directly (SPA-TT(2)). In mice, these two conjugates elicited high levels of immunoglobulin G (IgG) anti-lipopolysaccharide (LPS) in serum with bactericidal activity (E. Konadu, J. Shiloach, D. A. Bryla, J. B. Robbins, and S. C. Szu, Infect. Immun. 64:2709-2715, 1996). The safety and immunogenicity of the two conjugates were then evaluated sequentially in Vietnamese adults, teenagers, and 2- to 4-year-old children. None of the vaccinees experienced significant side effects, and all had preexisting LPS antibodies. At 4 weeks after injection, there were significant increases of the geometric mean IgG and IgM anti-LPS levels in the adults and teenagers: both conjugates elicited a greater than fourfold rise in the IgG anti-LPS level in serum in >/=80% of the volunteers. SPA-TT(2) elicited slightly higher, though not statistically significantly, levels of IgG anti-LPS than did SPA-TT(1) in these age groups. Accordingly, only SPA-TT(2) was evaluated in the 2- to 4-year-old children. On a random basis, one or two injections were administered 6 weeks apart to the children. No significant side effects were observed, and the levels of preexisting anti-LPS in serum were similar in children of all ages. A significant rise in the IgG anti-LPS titer was elicited by the first injection (P = 0.0001); a second injection did not elicit a booster response. Representative sera from all groups had bactericidal activity that could be adsorbed by S. enterica serovar Paratyphi A LPS.

Syntheses and immunologic properties of Escherichia coli O157 O-specific polysaccharide and Shiga toxin 1 B subunit conjugates in mice.

Escherichia coli O157 is the major cause of diarrhea-associated hemolytic uremic syndrome (HUS). Strains causing HUS contain either Shiga toxin 1 (Stx1) or Stx2, or both. In adult volunteers, conjugate vaccines of detoxified lipopolysaccharide (LPS) elicited bactericidal antibodies to E. coli O157. Here, the detoxified LPS was conjugated with improved schemes to the nontoxic B subunit of Stx1. Mice injected with these bivalent conjugates elicited both bactericidal antibodies to E. coli O157 and neutralization antibodies to Stx1.

Investigational vaccine for Escherichia coli O157: phase 1 study of O157 O-specific polysaccharide-Pseudomonas aeruginosa recombinant exoprotein A conjugates in adults.

Escherichia coli O157 causes severe enteritis and the extraintestinal complication hemolytic-uremic syndrome. Serum IgG against the surface polysaccharide antigen, the O-specific polysaccharide of lipopolysaccharide (LPS), may confer protective immunity by lysing the inocula. In a phase 1 clinical study, three investigational vaccines were studied in 87 healthy adults. The vaccines were prepared by covalently binding E. coli O157 O-specific polysaccharide with Pseudomonas aeruginosa recombinant exoprotein A. No significant reactions were reported. Most volunteers (81%) responded with a > 4-fold increase in IgG LPS antibodies 1 week after vaccination; all volunteers responded with a > 4-fold rise at 4 weeks and this level was sustained for 26 weeks after injection. All three vaccines elicited high titers of serum bactericidal activity that roughly correlated with the serum IgG and IgM LPS antibody levels. A phase 2 study in young children is planned.

Synthesis, characterization, and immunological properties in mice of conjugates composed of detoxified lipopolysaccharide of Salmonella paratyphi A bound to tetanus toxoid with emphasis on the role of O acetyls.

Salmonella paratyphi A, the second most common cause of enteric fever in Southeast Asia, is a habitant of and a pathogen for humans only. Lipopolysaccharides (LPS) are both essential virulence factors and protective antigens for systemic infections caused by groups A, B, C, and D nontyphoidal salmonellae. The O-specific polysaccharide of S. paratyphi A is composed of a trisaccharide, -->2-alpha-D)-Manp-(1-->4)-alpha-L-Rhap-(1-->3)-alpha-D-Galp -(1-->, with a branch of D-paratose from the C-3 of alpha-D-mannose, and the C-3 of beta-L-rhamnose is partially O acetylated (C. G. Hellerqvist, B. Lindberg, K. Samuelsson, and A. A. Lindberg, Acta Chem. Scand. 25:955-961, 1971). On the basis of data from our investigational vaccines for enteric bacterial pathogens, including group B salmonellae (D. C. Watson, J. B. Robbins, and S. C. Szu, Infect. Immun. 60:4679-4686, 1992), conjugates composed of the detoxified LPS of S. paratyphi A bound to tetanus toxoid (TT) were prepared by several schemes. LPS was detoxified with acetic acid or with hydrazine; the latter removed O acetyls from the O-specific polysaccharide. The detoxified polysaccharides were activated with cyanogen bromide (CNBr) or with 1-cyano-4-dimethylaminopyridinium tetratfluoroborate (CDAP) and bound to TT with or without a spacer. Solutions of 2.5 microgram of saccharide, alone or as a conjugate, were injected subcutaneously into young mice, and LPS and TT antibodies were measured by enzyme-linked immunosorbent assaying. A conjugate synthesized with higher-molecular-weight O-SP elicited the highest anti-LPS levels. Only conjugates with O acetyls elicited serum immunoglobulin G anti-LPS with bactericidal activity. There were no statistically significant differences between LPS antibody levels elicited by conjugates synthesized with or without a spacer. The conjugate with O-specific polysaccharide activated by CDAP and bound to TT without a spacer elicited the highest level of TT antibodies. Clinical evaluation (if S. paratyphi A conjugates is planned.

Preparation, characterization, and immunological properties in mice of Escherichia coli O157 O-specific polysaccharide-protein conjugate vaccines.

Escherichia coli O157 causes severe enteritis and the extraintestinal complication of hemolytic-uremic syndrome, with their highest incidence occurring in children. We postulated that serum immunoglobulin G (IgG) antibodies to the O-specific polysaccharide of lipopolysaccharide (LPS) may confer protective immunity to enteric pathogens by inducing bactericidal reactions against the ingested organisms in the jejunum (J. B. Robbins, C. Chu, and R. Schneerson, Clin. Infect. Dis. 15:346-361, 1992; S. C. Szu, R. Gupta, and J. B. Robbins, p. 381-394, in I. K. Wachsmuth, P. A. Blake, and O. Olsvik, ed., Vibrio cholerae, 1994). Because polysaccharide-protein conjugates induce serum IgG antibodies in infants, we bound the O-specific polysaccharide of E. coli O157 to proteins. E. coli O157 LPS, treated with acetic acid or hydrazine, was derivatized with adipic acid dihydrazide and bound to proteins by carbodiimide-mediated condensation. Conjugates of these adipic hydrazide derivative were prepared with bovine serum albumin, formalin-treated exotoxin C of Clostridium welchii (Pig Bel toxoid), or Pseudomonas aeruginosa recombinant exoprotein A. The conjugates had low levels of endotoxin and elicited serum antibodies with bactericidal activity to the O157 LPS. The largest increase in LPS antibodies was of the IgG class. Clinical evaluation of E. coli O157-toxoid conjugates is planned.