Synthesis and immunological properties of Vi and di-O-acetyl pectin protein conjugates with adipic acid dihydrazide as the linker

Abstract

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.

Safety and Immunogenicity of Vi Conjugate Vaccines for Typhoid Fever in Adults, Teenagers, and 2- to 4-Year-Old Children in Vietnam

“…To improve its immunogenicity, Vi was conjugated to proteins with N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (35,36). Recently, we used another method, in which carrier proteins were treated with adipic acid dihydrazide (ADH) and bound to Vi in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (19,32). Vi conjugates synthesized with ADH proved to be more immunogenic in mice and guinea pigs than those prepared with SPDP (19).…”

“…Recently, we used another method, in which carrier proteins were treated with adipic acid dihydrazide (ADH) and bound to Vi in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (19,32). Vi conjugates synthesized with ADH proved to be more immunogenic in mice and guinea pigs than those prepared with SPDP (19). In this study, the safety and immunogenicity of Vi conjugates prepared by these Blood samples were taken from all volunteers before and 6 and 26 weeks after the first injection.…”

Evaluation of alum-based adjuvant on the immunogenicity ofsalmonella entericaserovar typhi conjugates vaccines

“…Vaccines TCV was prepared by conjugation of Vi into DT or TT following a standard protocol. 32 To prepare TCV formulated in AlPO 4 , 50 mg of each conjugate was mixed with 3 mg AlPO 4 . These vaccines were designated Vi-DT (AlPO 4 ) and Vi-TT (AlPO 4 ).…”

“…PBMCs were isolated from whole blood via density gradient centrifugation, 400g for 30 minutes at 18-20 C using Ficoll-Paque Premium (GE Healthcare Life Science), washed with PBS and then resuspended in RPMI-1640 media (Gibco) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco). 32…”

“…Cytokine productions in mice receiving immunization with TCV (formulated in adjuvant or PBS) were characterized by intracellular cytokine staining as described previously. 32 Briefly, 10 6 PBMCs from immunized mice were cultured in microtubes and stimulated with the vaccine that was used for immunization or medium alone. The co-stimulatory molecules, anti-CD28 (BioLegend, Cat.…”