Simple, Direct Conjugation of Bacterial O-SP–Core Antigens to Proteins: Development of Cholera Conjugate Vaccines

Abstract

Bacterial O-SP–core antigens can be conjugated to proteins in the same, simple way as synthetic, linker-equipped carbohydrates by applying squaric acid chemistry. Introduction of spacers (linkers) to either O-SP–core antigens or protein carriers, which is involved in commonly applied protocols, is not required. The newly developed method here described consists of preparation of a squaric acid monoester derivative of O-SP–core antigen, utilizing the amino group inherent in the core, and reaction of the monoester with the carrier protein. The intermediate monoester can be easily purified, its conjugation can be monitored by SELDI-TOF mass spectrometry and, thus, readily controlled, since the conjugation can be terminated when the desired carbohydrate–protein ratio is reached. Here we describe production of conjugates containing the O-SP-core antigen of Vibrio cholerae O1, the major cause of cholera, a severe dehydrating diarrheal disease of humans. The resultant products are recognized by convalescent phase sera from patients recovering from cholera in Bangladesh, and anti-O-SP-core-protein responses correlate with plasma anti-lipopolysaccharide and vibriocidal responses, which are the primary markers of protection from cholera. The results suggest that such conjugates have potential as vaccines for cholera and other bacterial diseases.

Comparison of Immune Responses to the O-Specific Polysaccharide and Lipopolysaccharide of Vibrio cholerae O1 in Bangladeshi Adult Patients with Cholera

“…We prepared V. cholerae LPS, OSPc, and OSPc:BSA conjugates as previously described (52). Briefly, we obtained LPS from V. cholerae O1, Ogawa (strain X-25049) and Inaba (strain T19479) by protein denaturation by hot phenol-water extraction followed by enzymatic treatment (proteinase K, DNase I, and RNase A), and ultracentrifugation (100,000 ϫ g for 3 h) as previously described (52). The samples were dialyzed against distilled water, freeze dried, and stored for subsequent use.…”

“…cholerae LPS consists of the O-specific polysaccharide (OSP) connected through a core oligosaccharide to lipid A (endotoxin) that is integrated into the bacterial outer membrane (52). Lipid A and the core are similar across all V. cholerae serogroups (5,6,48), with the lipid A component of El Tor organisms undergoing glycine modification (13).…”

“…To assess these immune responses, we utilized O1 Ogawa and Inaba OSP-core-bovine serum albumin (OSPc:BSA) conjugates. The OSPc:BSA conjugates were prepared using squaric acid chemistry as previously described (52). Immune responses targeting OSP were then evaluated in adult patients with cholera caused by infections with the V. cholerae O1 El Tor serotypes Inaba and Ogawa in Dhaka, Bangladesh.…”

Immune Responses to the O-Specific Polysaccharide Antigen in Children Who Received a Killed Oral Cholera Vaccine Compared to Responses following Natural Cholera Infection in Bangladesh

“…Plasma LPS-and OSP-specific IgA, IgG, and IgM antibody responses in stored plasma were measured using a standardized enzyme-linked immunosorbent assay (ELISA) technique as described previously (22). Briefly, we prepared V. cholerae O1 Ogawa LPS and OSP:BSA conjugates as described previously (17). We coated 96-well polystyrene plates (Nunc F) with either V. cholerae O1 Ogawa LPS (2.5 g/ml) dissolved in phosphatebuffered saline (PBS) or Ogawa OSP:BSA (1 g of the conjugated product/ml) dissolved in carbonate buffer (pH 9.6).…”

“…Thus, we purified the O-specific polysaccharide-core oligosaccharide fragment (OSPc) of the outer membrane of V. cholerae O1 LPS and conjugated it to bovine serum albumin (BSA) to facilitate immunological analyses of the sugar component of LPS (17). We have previously shown that immune responses target the OSP moiety in this construct (16), and henceforth we refer to this antigen as OSP.…”