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

Laboratory of Developmental and Molecular Immunity, National Institute of Child Health and Development, National Institutes of Health, Bethesda, Maryland 20892

Received 10 March 2003/ Returned for modification 24 April 2003/ Accepted 30 May 2003

	ABSTRACT

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Epidemic and endemic meningitis caused by group A Neisseria meningitidis remains a problem in sub-Saharan 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_CNBrAH and GAMP_CDAPAH. These derivatives were bound to BSA by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form GAMP_CNBrAH-BSA and GAMP_CDAPAH-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 µg 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-µg dose elicited a higher GM IgG anti-GAMP than the 2.5-µg 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.

	INTRODUCTION

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The incidence of endemic meningitis and the frequency of epidemic meningitis caused by group A Neisseria meningitidis (GAM) are increasing in the "meningitis belt" of sub-Saharan Africa (17, 28, 35, 37, 46, 47). In addition, the meningitis belt is widening and spreading to cities within Africa (Nairobi, Kenya) and to other continents (11, 28, 46, 47). For example, in 1996, 250,000 cases of meningococcal disease with 25,000 deaths, caused mainly by GAM, were reported to the World Health Organization from countries of the meningitis belt (46). Between the end of January and the end of June 2001, an epidemic of 20,945 cases of GAM meningitis had killed >2,408 people in the two West African countries of Burkina Faso and Niger alone (47). Globally, 300,000 cases and 30,000 deaths are caused by this pathogen annually (46, 47).

The licensed GAM polysaccharide (GAMP) vaccine is highly effective at all ages when administered as recommended: two injections of monovalent GAMP at 3 and 6 months, followed by meningococcal group A, C, W135, and Y vaccines at 2 and 5 years (14, 16, 26, 31, 35, 37, 46, 47). Three injections of GAMP before the age of 2 years have no advantage (15). This four-dose GAMP schedule differs from the World Health Organization's Extended Program on Immunization for bacterial vaccines and would require additional visits and personnel to administer. As has been shown for Haemophilus influenzae type b, protein conjugates are more immunogenic than the polysaccharide (PS) at all ages, elicit protective antibody levels in infants, and can be incorporated into the Extended Program on Immunization schedule (34, 35, 36, 37).

To improve immunogenicity, conjugates of GAMP have been synthesized (4, 6, 7, 10, 19, 24). One of two GAMP conjugates, composed of a partially hydrolyzed, size-fractionated oligosaccharide, was less immunologic than GAMP (10, 24). Reports of the other GAMP conjugates provide only limited information on their syntheses and compositions (5-7).

The immunogenicity of PS-protein conjugate vaccines is related to the size of the PS (4, 12, 13, 22, 30, 32, 40, 43). The immunogenicity of GAMP, a linear homopolymer of (16)--D-ManpNAc-1-PO₄ O-acetylated at C-3, is also related to its molecular size and to its O-acetyl content (3, 41, 45). In this study, conjugates of GAMP were bound to bovine serum albumin (BSA), using adipic acid dihydrazide (ADH) as a linker, by two different methods. In addition, conjugates of the cross-reacting Escherichia coli K93 and Bacillus pumilus Sh-17 were synthesized, characterized, and evaluated for their immunogenicities in mice.

	MATERIALS AND METHODS

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Bacteria. GAM strains A:21 and F8238 were kindly provided by Carl Frasch, CBER, U.S. Food and Drug Administration; E. coli K93 and B. pumilus Sh-17 have been described previously (2, 18, 42).

PSs. The bacteria were cultivated on a modified Frantz medium, and their PSs were purified as described previously (45). GAMP, obtained from the Lanzhou Institute of Biological Products, Lanzhou, People's Republic of China, (lot 2001101) and from Chiron Biochine, Siena, Italy, were of clinical grade (45).

Reagents. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), ADH, 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP), agarose, BSA, rabbit anti-BSA serum, Brij 35, MES (morpholineethanesulfonic acid) sodium salt, and MES hydrate were from Sigma Chemical Co., St. Louis, Mo.; Sepharose CL-6B and Sephadex G-50 were from Pharmacia AB, Uppsala, Sweden; triethylamine (TEA) was from Pierce, Rockford, Ill.; acetonitrile was from T. J. Baker, Inc., Philipsburg, N.J.; dialysis membranes (molecular weight cutoff, 6,000 to 8,000) were from Spectra-Por, Laguna Hills, Calif.; ultrafiltration membranes (YM10) were from Amicon, Inc.; alkaline phosphatase-labeled goat anti-mouse immunoglobulin G (IgG) was from KPL Inc. Gaithersburg, MD; rabbit serum (complement) was from Pel-Freez Clinical Systems, LLC; Newborn calf serum was from Biofluids; and methylated human serum albumin and GAMP were from Carl Frasch. Pyrogen-free water (PFW) and pyrogen-free saline (PFS) were used in all experiments. Equine hyperimmune GAM serum (H49) was described previously (42).

Analytical methods. Protein, nucleic acid, phosphorus, and O-acetyl were measured as described previously (9, 20, 44, 45). PS was measured by the anthrone reaction using the homologous PS as a standard, and hydrazide was measured by the trinitrobenzene sulfonic acid assay using ADH as a standard (8, 9). Double immunodiffusion was performed in 0.8% agarose-0.15 M NaCl-0.01% sodium azide with H49 and rabbit anti-BSA sera.

Synthesis of PS-protein conjugates. All reactions were performed at least three times to confirm their reproducibility, and the data for a representative lot are shown.

Derivatization of GAMP. GAMP was activated with cyanogen bromide (CNBr) or CDAP and derivatized with ADH as described previously (9, 23, 40). Briefly, GAMP (10 mg/ml of PFW) was treated with CNBr (1 mg/mg of PS) at pH 10.5 for 6 min at room temperature in a pH-stat. The reaction mixture was brought to pH 8.5 by the addition of an equal volume of 0.5 M ADH in 0.5 M NaHCO₃. The mixture was tumbled for 20 h at 4°C and deionized passed through a 2.5- by 90-cm column of Sephadex G-50 with PFW as the eluant. The void volume fractions were pooled, dialyzed against 6 liters of water at 4°C with two changes for 3 days, and freeze-dried.

CDAP was made at 100 mg/ml in anhydrous acetonitrile and stored at -20°C for up to 1 month (21, 25, 39). CDAP (1 mg/mg of PS) was slowly pipetted into a vortexed solution of PS (10 mg/ml), and 30 s later, a volume of 0.2 M TEA equal to that of the CDAP was added. After 2.5 min an equal volume of 0.5 M ADH in 0.5 M NaHCO₃ was added, and the mixture was tumbled for 20 h at 4°C. The reaction mixture was passed through a 2.5- by 90-cm column of Sephadex G-50 in PFW, and the void volume fractions were pooled, dialyzed against PFW (see above), and freeze-dried.

Derivatization of BSA and K93. ADH was added to 10 mg of BSA or K93 PS/ml at room temperature to a final concentration of 0.2 M, followed by addition of EDC to 0.02 M (38). The reaction was carried out for 4 h with the pH maintained at 6.2 with 0.1 M HCl. The reaction mixture was dialyzed for 48 h at 4°C against 0.2 M NaCl and passed through a 2.5- by 90-cm column of Sephadex G-50 in PFS. The void volume fractions, designated AHBSA or K93AH, were concentrated, sterile filtered, and stored at 4°C.

GAMPAH conjugates of BSA. GAMPAH conjugates of BSA were prepared as described previously (23). GAMPAH and BSA were mixed to a final concentration of 10 mg/ml each in PFS, EDC was added to a final concentration of 0.1 M, and the mixture was stirred at room temperature for 4 h with the pH maintained at 5.0. The reaction mixture was dialyzed against 6 liters of 0.2 M NaCl overnight and passed through a 1.0- by 90-cm column of Sepharose CL-6B in 0.2 M NaCl. The void volume fractions were sterile filtered and assayed for phosphorus, protein, and antigenicity by immunodiffusion.

GAMP conjugates of AHBSA. GAMP (10 mg/ml of PFW) was treated with CNBr (1 mg/mg of PS) at pH 10.5 for 6 min at room temperature in a pH-stat. AHBSA (10 mg/ml of 0.5 M NaHCO₃) was added, and the pH was brought to 8.5. After being tumbled for 20 h at 4°C, the mixture was passed through a 1- by 90-cm column of CL-6B Sepharose in 0.2 M NaCl. The void volume fractions were sterilely filtered and assayed for phosphorus, protein, and antigenicity by immunodiffusion.

CDAP (0.5 mg/mg of PS) was pipetted slowly into a vortexed solution of 1 ml of GAMP (26 mg/ml in 0.2 M MES buffer). Thirty seconds later, a volume of 0.2 M TEA equal to that of the CDAP was added. At 2.5 min, 1 ml of AHBSA (26 mg/ml) was added, and the pH was maintained at 8.0 in a pH-stat with 0.1 M NaOH at room temperature for 4 h (22, 23, 25, 39). The reaction mixture was dialyzed against 0.2 M NaCl at 4°C for 72 h and passed through a 1- by 90-cm column of Sepharose CL-6B in 0.2 M NaCl, and the fractions were assayed for protein, phosphorus, and antigenicity by immunodiffusion. Fractions containing GAMP-AHBSA were pooled.

E. coli K93 and B. pumilus Sh-17 AHBSA conjugates. K93AH-BSA and Sh-17_CDAP-AHBSA were prepared as for GAMP (see above), except that the final concentration was 5 mg/ml for K93AH-BSA and 20 mg/ml for Sh-17_CDAP-AHBSA.

Immunization. Groups of 10 5- to 6-week-old female NIH mice were injected subcutaneously with 0.1 ml of saline solution of 2.5 or 5 µg of PS alone or a conjugate on days 0, 14, and 28. The mice were exsanguinated 7 days after the third injection (9).

ELISA. Serum IgG GAMP antibodies were measured by enzyme-linked immunosorbent assay (ELISA) (27). The A₄₀₅ was measured using an MRX Dynatech reader. Hyperimmune GAM murine serum (14-1-A,2D7-B5B5), used as a reference, was obtained from Wendell Zollinger, Walter Reed Army Institute of Research, Washington, D.C., and assigned a value of 100 ELISA units (EU).

Results were computed with an ELISA data-processing program provided by B. D. Plikaytis, Biostatistics and Information Management Branch, Centers for Disease Control and Prevention, Atlanta, Ga. (33). Anti-GAMP IgG levels are expressed as geometric means (GM).

Complement-mediated bactericidal assay. Some modifications were used for the published protocol (27). Each well of a 24-well flat-bottom tissue culture plate was filled in sequence with 100 µl of sera diluted twofold with Dulbecco phosphate-buffered saline containing 0.1% glucose, 100 µl of strain F8238 containing 50 to 100 CFU/100 µl, and 50 µl of rabbit serum (complement). The reaction mixture was incubated at 37°C for 60 min, and modified Frantz medium-agarose was added. Colonies were counted after incubation at 37°C overnight. The titer was expressed as the reciprocal of the serum dilution yielding >50% killing compared to the numbers of bacteria in the controls. Adsorption of sera was performed by adding GAMP (100 µg/ml) in the diluting buffer.

Statistics. ELISA values are expressed as the GM and 25th to 75th centiles. Student's t test with Bonferroni correction was used to compare values between groups of mice. Statistical significance was defined as a P value of <0.05.

	RESULTS

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Adipic acid hydrazide (AH) derivatives. CNBr-activated GAMP-AH had an average content of 1.62% AH, and CDAP-activated GAMP-AH had 2.99% (Table 1). Both GAMPAH derivatives yielded a line of identity with native GAMP and H49 antiserum (Fig. 1). The O-acetyl content of GAMP was 1.90 mmol/mg, that of GAMP_CNBrAH was 1.69 mmol/mg, and that of GAMP_CDAPAH was 1.83 mmol/mg. The O-acetyl contents of GAMP and GAMP-AH before conjugation are shown in Table 1. The molecular sizes, as assessed from the K_d through CL-6B Sepharose, were 0.23 for GAMP, 0.43 for CDAP-activated GAMP, 0.64 for CNBr-activated GAMP, and 0.51 for BSA.

View larger version (69K): [in this window] [in a new window]   FIG. 1. Double immunodiffusion of GAMP and GAMPAH (0.1 mg/ml) following activation by CDAP. 1, GAMP; 2, GAMPAH; C, equine H49 meningococcal group A antiserum.

Compositions of the conjugates (Table 1). Two representative gel filtration profiles are shown in Fig. 2. Figure 2A shows GAMP_CNBrAH-BSA with two distinct peaks. The peak at V₀ contained both GAMP and BSA, while the peak at K_d 0.41 contained mostly GAMP: almost all the BSA was in the void volume. Immunodiffusion (not shown) revealed a line of identity of the conjugate with the H49 and anti-BSA sera. There was a slight line over the anti-GAMP line of the conjugate, indicating some free GAMP.

View larger version (12K): [in this window] [in a new window]   FIG. 2. Gel filtration of GAMPCNBrAH-BSA and GAMPCDAP-AHBSA conjugates. (A) GAMPCNBrAH-BSA; (B) GAMPCDAP-AHBSA. Through a 1.0- by 90-cm column of Sepharose CL-6B in 0.2 M NaCl, 1.0-ml samples were applied and 2.3-ml fractions were collected. Protein (A280) and PS were measured as described previously (9, 43).

View larger version (78K): [in this window] [in a new window]   FIG. 3. Double immunodiffusion of GAMPCDAP-AHBSA. C, GAMPCDAP-AHBSA (0.2 mg of PS/ml); 1, equine H49 group A meningococcal antiserum; 2, rabbit BSA antiserum.

Immunization. For simplicity, IgG anti-GAMP levels are shown only after the third injection. GAMP elicited low antibody levels close to the limit of detection (Table 2).

The E. coli K93 and B. pumilus conjugates did not elicit statistically significant levels of IgG anti-GAMP or provide a significant increment when combined with GAMP conjugates (not shown).

Bactericidal levels. Mice injected three times with GAMP_CDAP-AHBSA had bactericidal levels that correlated roughly with their IgG antibody levels (Table 3). GAMP alone elicited low levels of bactericidal activity that seemed to exceed the IgG levels as determined by ELISA.

	DISCUSSION

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Reliable and efficient methods for preparing GAMP conjugates with ADH as a linker are described. The conjugates were prepared by two methods. One used AH derivatives of GAMP (GAMP-AH), prepared by activation with either CNBr or CDAP and then bound to BSA by condensation with EDC. The other derivatized BSA with ADH by EDC and then bound the resultant derivative (AHBSA) directly to CNBr or CDAP-activated GAMP (23, 38). All conjugates elicited IgG GAMP antibodies when injected in a saline solution by the subcutaneous route at a fraction of the human dose. The GAMP conjugates elicited booster responses and antibody levels significantly higher than those elicited by GAMP alone. Accordingly, it can be expected that these conjugates will be more effective vaccines than GAMP in humans (34). Immunogenicity data from adults may not distinguish differences between the immunogenicities of similar conjugates. It follows that GAMP conjugates must be characterized in infants and young children (1). Clinical evaluation of these conjugates, using new medically useful carriers, such as Clostridium difficile exoprotein A, is planned (29).

The immunogenicity of GAMP, a linear homopolymer of (16)--D-ManpNAc-1-PO₄ O-acetylated at C-3, is related to its molecular size and its O-acetyl content (3, 41, 45). GAMP is comparatively unstable due to the ability of its phosphodiester bond (41, 45). Hydrolysis of this bond is favored by alkaline and acidic conditions and elevated temperature. For this reason, GAMP vaccine is stored at 4°C in the freeze-dried state with a saccharide that competes for the residual moisture in order to prevent hydrolysis and preserve its immunogenicity (41). CDAP-mediated activation at pH 8.0 is preferred over CNBr-mediated activation, which requires pH levels of 10.5, as shown by the lesser effect of CDAP upon the molecular size of GAMP. CDAP activation at close to neutrality makes it useful for pH-sensitive PSs (39). Berry et al. showed the essential role of O-acetyls in eliciting maximal levels of GAMP antibodies (3). Activation of GAMP with either CNBr or CDAP retained most of it O-acetyl content. Probably because of the ability of CDAP to activate GAMP at a pH closer to neutrality, the O-acetyl level was higher after CDAP activation than after CNBr activation.

The finding of cross-reacting PSs has been proposed to account for the wide prevalence of GAMP antibodies in young adults, despite the absence of GAM in the United States for the past 50 years (2, 18, 34, 42). GAMP antibodies were elicited by these cross-reacting bacteria (E. coli K93 and K51, B. pumilus, and Streptococcus faecium), freshly prepared, formalin inactivated, and injected intravenously multiple times into rabbits and horses (2). We were disappointed at the failure of the cross-reacting E. coli K93 and B. pumilus Sh-17 PS conjugates to elicit GAMP IgG antibodies or to significantly increase the immunogenicities of the GAMP conjugates. These conjugates induced homologous K93 or Sh-17 antibodies (not shown). These cross-reacting PSs, on bacteria, probably serve as a stimulant for GAMP antibodies only when present in the intestine for prolonged periods.

In summary, GAMP conjugates with immmunogenicities improved over that of GAMP have been prepared and standardized. CDAP seemed to be a more useful activating reagent, because the treated GAMP had a higher molecular weight and content of O-acetyl than that activated with CNBr. The most immunogenic conjugate, and that with the highest yield, was the CDAP-activated GAMP bound to the ADH-derivatized protein.

	ACKNOWLEDGMENTS

 
We thank Dolores A. Bryla for assistance with the statistical evaluation of the data, Chunyan Guo for ELISA, and Andrew Lees for his review of the manuscript.

	FOOTNOTES

 
* Corresponding author. Mailing address: National Institute of Child Health and Development, National Institutes of Health, Building 6, Room 424, Bethesda, MD 20892-2720. Phone: (301) 496-6141. Fax: (301) 402-9108. E-mail: jinz{at}mail.nih.gov.

Editor: J. N. Weiser

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