Preparation and Characterization of Group A Meningococcal Capsular Polysaccharide Conjugates and Evaluation of Their Immunogenicity in Mice

Abstract

Epidemic and endemic meningitis caused by group A Neisseria meningitidis remains a problem in subSaharan Africa. Although group A meningococcal capsular polysaccharide (GAMP) vaccine confers immunity at all ages, the improved immunogenicity of a conjugate and its compatibility with the World Health Organization's Extended Program on Immunization offers advantages over GAMP alone. Conjugates of GAMP bound to bovine serum albumin (BSA) were synthesized, characterized, and evaluated for their immunogenicities in mice. Two methods, involving adipic acid dihydrazide (ADH) as a linker, were used. First, ADH was bound to GAMP activated with cyanogen bromide (CNBr) or with 1-cyano-4(dimethylamino)-pyridinium tetrafluoroborate (CDAP) to form GAMP CNBr AH and GAMP CDAP AH. These derivatives were bound to BSA by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form GAMP CNBr AH-BSA and GAMP CDAP AH-BSA. Second, ADH was bound to BSA with EDC to form AHBSA. AHBSA was bound to activated GAMP to form GAMP CNBr -AHBSA and GAMP CDAP -AHBSA. The yield of GAMP CDAP -AHBSA (35 to 40%) was higher than those of the other conjugates (5 to 20%). GAMP conjugates elicited immunoglobulin G (IgG) anti-GAMP in all mice after three injections of 2.5 or 5.0 g of GAMP: the geometric mean (GM) was highest in recipients of GAMP CDAP -AHBSA (11.40 enzyme-linked immunosorbent assay units). Although the difference was not statistically significant, the 5.0-g dose elicited a higher GM IgG anti-GAMP than the 2.5-g dose. Low levels of anti-GAMP were elicited by GAMP alone. GAMP CDAP -AHBSA elicited bactericidal activity roughly proportional to the level of IgG anti-GAMP.

Conjugates of Group A and W135 Capsular Polysaccharides ofNeisseria meningitidisBound to RecombinantStaphylococcus aureusEnterotoxin C1: Preparation, Physicochemical Characterization, and Immunological Properties in Mice

“…We previously reported on several GAM CP (GAMP) conjugates using bovine serum albumin (BSA) as a carrier and evaluated their immunogenicity in mice (18). The synthesis used CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate)-activated polysaccharide bound to adipic acid dihydrazide (AH)-derivatized protein.…”

“…Protein, nucleic acid, phosphorus, CP concentration, AH derivatization of protein, and the partitian coefficient of column chromatography (K d ) were measured as described previously (18). O-acetyl (OAc) content was analyzed by the Hestrin reaction (17), and the 7-OAc and 9-OAc contents of W135P were measured by NMR.…”

“…rSEC was derivatized with ADH as described previously (18). Six lots were prepared, and their derivatizations ranged from 1.23 to 5.54% (see Table 3).…”

“…The GAMP conjugates were prepared at pH 7 as described previously (18), and W135-rSEC conjugates were prepared at pH 7 or pH 8. Three W135P preparations were coupled to the rSEC: native (W135P-rSEC), hydrazinetreated [W135P(H)-rSEC], and sonicated [W135P(S)-rSEC].…”

“…Serum IgG anti-CP and anti-rSEC levels were expressed as geometric means (GM). The complement-mediated bactericidal assay was done as described previously (18).…”