The Vi-rEPA conjugate typhoid vaccine is safe and immunogenic and has more than 90 percent efficacy in children two to five years old. The antibody responses and the efficacy suggest that this vaccine should be at least as protective in persons who are more than five years old.

The capsular polysaccharide of Salmonella typhi, Vi, is an essential virulence factor and a protective vaccine for people older than 5 years. The safety and immunogenicity of two investigational Vi conjugate vaccines were evaluated in adults, 5- to 14-year-old children, and 2- to 4-year-old children in Vietnam. The conjugates were prepared with Pseudomonas aeruginosa recombinant exoprotein A (rEPA) as the carrier, using eitherN-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP; Vi-rEPA1) or adipic acid dihydrazide (ADH; Vi-rEPA2) as linkers. None of the recipients experienced a temperature of >38.5°C or significant local reactions. One injection of Vi-rEPA2 into adults elicited a geometric mean (GM) increase in anti-Vi immunoglobulin G (IgG) from 9.62 enzyme-linked immunosorbent assay units/ml (EU) to 465 EU at 6 weeks; this level fell to 119 EU after 26 weeks. In the 5- to 14-year-old children, anti-Vi IgG levels at 6 weeks elicited by Vi-rEPA2, Vi-rEPA1, and Vi were 169, 22.8, and 18.9 EU, respectively (P = 0.0001 for Vi-rEPA1 and Vi with respect to Vi-rEPA2). At 26 weeks, the anti-Vi IgG levels for recipients of Vi-rEPA2, Vi-rEPA1, and Vi were 30.0, 10.8, and 13.4 EU, respectively (P < 0.001 for Vi-rEPA1 and Vi with respect to Vi-rEPA2); all were higher than the preinjection levels (P = 0.0001). Vi-rEPA2 also elicited the highest anti-Vi IgM and IgA levels of the three vaccines. In the 2- to 4-year-old children at 6 weeks following the first injection, Vi-rEPA2 elicited an anti-Vi IgG level of 69.9 EU compared to 28.9 EU for Vi-rEPA1(P = 0.0001). Reinjection increased Vi antibody levels from 69.9 to 95.4 EU for Vi-rEPA2 and from 28.9 to 83.0 EU for Vi-rEPA1. At 26 weeks, anti-Vi IgG levels remained higher than those at preinjection (30.6 versus 0.18 for Vi-rEPA2 and 12.8 versus 0.33 for Vi-rEPA1; P = 0.0001 for both). Vi vaccine is recommended for individuals of 5 years of age or older. In the present study, the GM level of anti-Vi IgG elicited by two injections of Vi-rEPA2 in the 2- to 4-year-old children was higher than that elicited by Vi in the 5- to 14-year-old children (30.6 versus 13.4; P = 0.0001). The safety and immunogenicity of the Vi-rEPA2conjugate warrant further investigation.

Epidemiologic and experimental data provide evidence that a critical level of serum immunoglobulin G (IgG) antibodies to the surface polysaccharide of Vibrio cholerae O1 (lipopolysaccharide) and of Vibrio cholerae O139 (capsular polysaccharide [CPS]) is associated with immunity to the homologous pathogen. The immunogenicity of polysaccharides, especially in infants, may be enhanced by their covalent attachment to proteins (conjugates). Two synthetic schemes, involving 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents, were adapted to prepare four conjugates of V. cholerae O139 CPS with the recombinant diphtheria toxin mutant, CRMH21G. Adipic acid dihydrazide was used as a linker. When injected subcutaneously into young outbred mice by a clinically relevant dose and schedule, these conjugates elicited serum CPS antibodies of the IgG and IgM classes with vibriocidal activity to strains of capsulated V. cholerae O139. Treatment of these sera with 2-mercaptoethanol (2-ME) reduced, but did not eliminate, their vibriocidal activity. These results indicate that the conjugates elicited IgG with vibriocidal activity. Conjugates also elicited high levels of serum diphtheria toxin IgG. Convalescent sera from 20 cholera patients infected with V. cholerae O139 had vibriocidal titers ranging from 100 to 3,200: absorption with the CPS reduced the vibriocidal titer of all sera to <50. Treatment with 2-ME reduced the titers of 17 of 20 patients to <50. These data show that, like infection with V. cholerae O1, infection with V. cholerae O139 induces vibriocidal antibodies specific to the surface polysaccharide of this bacterium (CPS) that are mostly of IgM class. Based on these data, clinical trials with the V. cholerae O139 CPS conjugates with recombinant diphtheria toxin are planned.It has been proposed that a critical level of serum immunoglobulin G (IgG) to the surface polysaccharides of Vibrio cholerae O1 and V. cholerae O139 confers serotype-specific immunity to cholera (3, 7, 17, 24, 25, 28, 29-32, 38, 39, 43, 44). The surface polysaccharide of V. cholerae O1 is a lipopolysaccharide (LPS). V. cholerae O139, in contrast, has a capsular polysaccharide (CPS) composed of a hexasaccharide repeating unit containing a trisaccharide backbone and two branches (3,4,8,10,12,16,17,19,26,28,30,31,35,38,42,43,45). The repeating unit contains two negatively charged groups: a carboxyl of galactouronic acid and a phosphate cyclic diester. In our preliminary studies we found that CPS did not elicit serum antibodies after three injections in mice (Z. Kossaczka and S. C. Szu, submitted for publication). To improve its immunogenicity, CPS was covalently bound by different synthetic schemes to chicken serum albumin, as a model protein. The resultant conjugates induced serum anti-CPS IgG in mice with vibriocidal activity (Kossaczka and Szu, submitted).We chose the two most successful schemes to prepare V. cholerae O139 CPS conjugates with the recombinant diphth...

The Vi capsular polysaccharide of Salmonella typhi, a licensed vaccine for typhoid fever in individuals >5 years old, induces low and short-lived antibodies in children, and reinjection does not elicit booster responses at any age. Its immunogenicity was improved by binding Vi to proteins by using N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) as a linker. Similar findings were observed with the structurally related, di-O-acetyl derivative of pectin [poly-␣(134)-D-GalpA] designated OAcP. Protein conjugates of Vi and OAcP were synthesized by carbodiimide-mediated synthesis with adipic acid dihydrazide (ADH) as the linker. Hydrazide groups were introduced into proteins (bovine serum albumin or recombinant Pseudomonas aeruginosa exoprotein A) by treatment with ADH and 1-ethyl-3(3-dimethylaminopropyl carbodiimide (EDC). The resultant adipic acid hydrazide derivatives (AH-proteins), containing 2.3 to 3.4% AH, had antigenic and physicochemical properties similar to those of the native proteins. The AH-proteins were bound to Vi and OAcP by treatment with EDC. The immunogenicity of Vi or OAcP, alone or as protein conjugates, was evaluated in young outbred mice and guinea pigs by subcutaneous injection of 2.5 and 5.0 g, respectively, of polysaccharide, and antibodies were measured by enzyme-linked immunosorbent assay. All conjugates were significantly more immunogenic than Vi or OAcP alone and induced booster responses with 5-to 25-fold increases of antibodies. Vi conjugates were significantly more immunogenic than their OAcP analogs. A carboxymethyl derivative of yeast ␤-glucan enhanced the anti-Vi response elicited by an OAcP conjugate but had no effect on the immunogenicity of Vi or of OAcP alone. Vi and OAcP conjugates synthesized by this scheme will be evaluated clinically.