This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jin, Z.
Right arrow Articles by Schneerson, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jin, Z.
Right arrow Articles by Schneerson, R.

 Previous Article  |  Next Article 

Infection and Immunity, September 2003, p. 5115-5120, Vol. 71, No. 9
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.9.5115-5120.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Zhigang Jin,* Chiayung Chu, John B. Robbins, and Rachel Schneerson

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


arrow
ABSTRACT
 
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 GAMPCNBrAH and GAMPCDAPAH. These derivatives were bound to BSA by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form GAMPCNBrAH-BSA and GAMPCDAPAH-BSA. Second, ADH was bound to BSA with EDC to form AHBSA. AHBSA was bound to activated GAMP to form GAMPCNBr-AHBSA and GAMPCDAP-AHBSA. The yield of GAMPCDAP-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 GAMPCDAP-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. GAMPCDAP-AHBSA elicited bactericidal activity roughly proportional to the level of IgG anti-GAMP.


arrow
INTRODUCTION
 
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 (1->6)-{alpha}-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.


arrow
MATERIALS AND METHODS
 
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-17CDAP-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-17CDAP-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.


arrow
RESULTS
 
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 GAMPCNBrAH was 1.69 mmol/mg, and that of GAMPCDAPAH 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 Kd 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 this table:
[in this window]
[in a new window]
  		TABLE 1. Compositions of GAMP, GAMP-AH, and conjugates



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.

The AH content of AHBSA was ~4% (4.38, 3.90, and 3.81%), and both the native BSA and AHBSA gave a line of identity with the BSA antiserum (not shown). The Kd of AHBSA was 0.56.

Compositions of the conjugates (Table 1). Two representative gel filtration profiles are shown in Fig. 2. Figure 2A shows GAMPCNBrAH-BSA with two distinct peaks. The peak at V0 contained both GAMP and BSA, while the peak at Kd 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).

Figure 2B shows that GAMPCDAP-AHBSA had two peaks of GAMP and BSA. The first, emerging in the void volume, had a protein/PS ratio of 1.3 with a yield of 40% (based upon recovery of PS in the conjugate). Immunodiffusion of the conjugate (Fig. 3) showed a line of identity with H49 and anti-BSA sera. The second peak gave a similar immunodiffusion pattern but elicited lower levels of GAMP antibodies (not shown).



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.

The average yield of GAMPCDAP-AHBSA (35 to 40%) was higher than those of the other conjugates (5 to 20%).

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).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Serum IgG GAMP antibodies elicited in mice by three subcutaneous injections 2 weeks apart of conjugates in salinea

All GAMP conjugates elicited statistically significant IgG anti-GAMP levels compared to GAMP (P < 0.0001). The highest anti-GAMP immune response among the four conjugates was induced by GAMPCDAP-AHBSA (11.4 EU). Significant differences were observed for GAMPCDAP-AHBSA (11.4 EU) versus GAMPCNBr-AHBSA (4.34 EU) or GAMPCDAPAH-BSA (5.16 EU) (P < 0.01) and GAMPCNBrAH-BSA (8.50 EU) versus GAMPCNBr-AHBSA (4.34) (P < 0.01), but not between GAMPCNBrAH-BSA (8.50) and GAMPCDAP-AHBSA (11.4 EU). There was a dosage effect, as 5.0 µg elicited higher levels than 2.5 µg (11.3 and 6.68 EU, respectively) for GAMPCNBrAH-BSA and GAMPCDAPAH-BSA; however, these differences were not statistically significant (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 GAMPCDAP-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.


View this table:
[in this window]
[in a new window]
  		TABLE 3. IgG anti-GAMP and bactericidal titers of individual sera from mice injected with GAMPCDAP-AHBSA and GAMP

Absorption of the GAMPCDAP-AHBSA-induced IgG with GAMP removed almost all bactericidal activity.


arrow
DISCUSSION
 
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 (1->6)-{alpha}-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.


arrow
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.


arrow
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. Back

Editor: J. N. Weiser


arrow
REFERENCES
 
    1
  1. Anderson, P. W., M. E. Pichichero, and R. A. Insel. 1985. Immunogens consisting of oligosaccharides from the capsule of Haemophilus influenzae type b coupled to diphtheria toxoid or the toxin protein CRM197. J. Clin. Investig. 76:52-59.
    2. 2
  2. Bax, A., M. F. Summers, N. Guirgis, R. Schneerson, J. B. Robbins, F. Ørskov, I. Ørskov, W. F. Vann, and W. Egan. 1986. Structural studies of the Escherichia coli K93 and K53 capsular polysaccharides. Carbohydr. Res. 173:53-64.
    3. 3
  3. Berry, D. S., F. Lynn, C. H. Lee, C. E. Frasch, and M. C. Bash. 2002. Effect of acetylation of Neisseria meningitidis serogroup A capsular polysaccharide on development of functional immune response. Infect. Immun. 70:3707-3713.[Abstract/Free Full Text]
    4. 4
  4. Beuvery, E. C., A. Van Den Kaaden, V. Kanhai, and A. B. Leussink. 1983. Physicochemical and immunological characterization of meningococcal group A polysaccharide-tetanus toxoid conjugates prepared by two methods. Vaccine 1:31-36.[CrossRef][Medline]
    5. 5
  5. Borrow, R., N. Andrews, D. Goldblatt, and E. Miller. 2001. Serologic basis for use of meningococcal serogroup C conjugate vaccines in the United Kingdom. Infect. Immun. 69:1568-1573.[Abstract/Free Full Text]
    6. 6
  6. Campbell, J. D., R. Edelman, J. C. King, Jr., T. Papa, R. Ryall, and M. B. Rennels. 2002. Safety, reactogenicity, and immunogenicity of a tetravalent meningococcal polysaccharide-diphtheria toxoid conjugate vaccine given to healthy adults. J. Infect. Dis. 186:1848-1851.[CrossRef][Medline]
    7. 7
  7. Campagne, G., A. Garba, P. Fabre, A. Schuchat, R. Ryall, D. Boulanger, M. Bybel, G. Carlone, P. Briantais, B. Ivanoff, B. Xerri, and J. P. Chippaux. 2000. Safety and immunogenicity of three doses of a Neisseria meningitidis A+C diphtheria conjugate vaccine in infants from Niger. Pediatr. Infect. Dis. J. 19:144-150.[CrossRef][Medline]
    8. 8
  8. Carroll, N. V., R. W. Longley, and J. H. Roe. 1956. The determination of glycogen in liver and muscle by use of anthrone reagent. J. Biol. Chem. 220:583-593.[Free Full Text]
    9. 9
  9. Chu, C., R. Schneerson, J. B. Robbins, and S. Rastogi. 1983. Further studies on the immunogenicity of Haemophilus influenzae type b and pneumococcal type 6A polysaccharide protein conjugates. Infect. Immun. 40:245-256.[Abstract/Free Full Text]
    10. 10
  10. Costantino, P., S. Viti, A. Podda, M. A. Velmonte, L. Nencioni, and R. Rappuoli. 1992. Development and phase 1 clinical testing of a conjugate vaccine against meningococcus A and C. Vaccine 10:691-698.[CrossRef][Medline]
    11. 11
  11. Counts, G. W., D. F. Gregory, and J. G. Spearman. 1984. Group A meningococcal disease in the U. S. Pacific Northwest: epidemiology, clinical features, and effect of a vaccination control program. Rev. Infect. Dis. 6:640-648.[Medline]
    12. 12
  12. Daum, R. S., D. Hogerman, M. B. Rennels, K. Bewley, F. Malinoski, E. Rothstein, K. Reisinger, S. Block, H. Keyserling, and M. Steinhoff. 1997. Infant immunization with pneumococcal CRM vaccines: effect of saccharide size on immunogenicity and interactions with simultaneously administered vaccines. J. Infect. Dis. 176:445-455.[Medline]
    13. 13
  13. Fattom, A., C. Lue, S. C. Szu, J. Mestecky, G. Schiffman, D. Bryla, W. F. Vann, D. Watson, L. M. Lorne, L. M. Kimzey, J. B. Robbins, and R. Schneerson. 1990. Serum antibody response in adult volunteers elicited by injection of Streptococcus pneumoniae type 12F polysaccharide alone or conjugated to diphtheria toxoid. Infect. Immun. 58:2309-2312.[Abstract/Free Full Text]
    14. 14
  14. Gold, R., M. L. Lepow, I. Goldschneider, T. F. Draper, and E. C. Gotschlich. 1979. Kinetics of antibody production to group A and group C meningococcal polysaccharide vaccines administered during the first six years of life: prospects for routine immunization of infants and children. J. Infect. Dis. 140:690-697.[Medline]
    15. 15
  15. Gold, R., M. L. Lepow, I. Goldschneider, T. F. Draper, and E. C. Gotschlich. 1978. Antibody responses of human infants to three doses of group A Neisseria meningitidis polysaccharide vaccine administered at two, four, and six months of age. J. Infect. Dis. 138:731-735.[Medline]
    16. 16
  16. Goldschneider, I., E. C. Gotschlich, and M. S. Artenstein. 1969. Human immunity to the meningococcus. I. The role of humoral antibodies. J. Exp. Med. 129:1307-1326.[Abstract]
    17. 17
  17. Guibourdenche, M., E. A. Høiby, J.-Y. Riou, F. Varaine, C. Jogue, and D. A. Vaugant. 1996. Epidemics of serogroup A Neisseria meningitidis of subgroup III in Africa, 1989-1994. Epidemiol. Infect. 116:115-120.[Medline]
    18. 18
  18. Guirgis, N., R. Schneerson, A. Bax, W. Egan, J. B. Robbins, I. Ørskov, and F. Ørskov. 1985. Escherichia coli K51 and K93 capsular polysaccharides cross-reactive with group A meningococcal polysaccharide. Immunochemical, structural and epidemiological studies. J. Exp. Med. 162:1837-1862.[Abstract/Free Full Text]
    19. 19
  19. Jennings, H. J., and C. Lugowski. 1981. Immunochemistry of groups A, B, and C meningococcal polysaccharide-tetanus toxoid conjugates. J. Immunol. 127:1011-1018.[Abstract]
    20. 20
  20. Kabat, E., and M. Mayer. 1971. Experimental immunochemistry, p. 493-495. Charles C. Thomas, Springfield, Ill.
    21. 21
  21. Kohn, J., and M. Wilchek. 1983. 1-Cyano-4-dimethylamino pyridinium tetrafluoroborate as a cyanylating agent for the covalent attachment of ligand to polysaccharide resins. FEBS Lett. 154:209-210.[CrossRef]
    22. 22
  22. Konadu, E., J. Shiloach, D. A. Bryla, J. B. Robbins, and S. C. Szu. 1996. Synthesis, characterization, and immunological properties in mice of conjugates composed of detoxified lipopolysaccharide of Salmonella paratyphi A bound to tetanus toxoid, with emphasis on the role of O acetyls. Infect. Immun. 64:2709-2715.[Abstract]
    23. 23
  23. Kossaczka, Z., J. Shiloach, Y. Johnson, D. N. Taylor, R. A. Finkelstein, J. B. Robbins, and S. C. Szu. 2000. Vibrio cholerae O139 conjugate vaccines: synthesis and immunogenicity of V. cholerae O139 capsular polysaccharide conjugates with recombinant diphtheria toxin mutant in mice. Infect. Immun. 68:5037-5043.[Abstract/Free Full Text]
    24. 24
  24. Leach, A., P. A. Twumasi, S. Kumah, W. S. Banya, S. Jaffar, B. D. Forrest, D. M. Granoff, D. E. LiButti, G. M. Carlone, L. B. Pais, C. V. Broome, and B. M. Greenwood. 1997. Induction of immunologic memory in Gambian children by vaccination in infancy with a group A plus group C meningococcal polysaccharide-protein conjugate vaccine. J. Infect. Dis. 175:200-204.[Medline]
    25. 25
  25. Lees, A., B. L. Nelson, and J. J. Mond. 1996. Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridium tetrafluoroborate for use in protein-polysaccharide conjugate vaccines and immunological reagents. Vaccine 14:190-198.[CrossRef][Medline]
    26. 26
  26. Lennon, D., B. Gellin, D. Hood, L. Voss, H. Heffernan, and S. Thakur. 1992. Successful intervention in a group A meningococcal outbreak in Auckland, New Zealand. Pediatr. Infect. Dis. J. 11:617-623.[Medline]
    27. 27
  27. Maslanka, S. E., L. L. Gheesling, D. E. Libutti, K. B. J. Donaldson, H. S. Harakeh, J. K. Dykes, F. F. Arhin, S. J. N. Devi, C. E. Frasch, J. C. Huang, P. Kriz-Kuzemenska, R. D. Lemmon, M. Lorange, C. C. A. M. Peeters, S. Quataert, J. Tai, G. M. Carlone, and the Multilaboratory Study Group. 1997. Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. Clin. Diagn. Lab. Immunol. 4:156-167.[Abstract]
    28. 28
  28. Molesworth, A. M., M. C. Thomson, S. J. Connor, M. P. Cresswell, A. P. Morse, P. Shears, C. A. Hart, and L. E. Cuevas. 2002. Where is the meningitis belt? Defining an area at risk of epidemic meningitis in Africa. Trans. R. Soc. Trop. Med. Hyg. 96:242-249.[CrossRef][Medline]
    29. 29
  29. Pavliakova, D., J. S. Moncrief, D. M. Lyerly, G. Schiffman, D. A. Bryla, J. B. Robbins, and R. Schneerson. 2000. Clostridium difficile recombinant toxin A repeating units as a carrier protein for conjugate vaccines: studies of pneumococcal type 14, Escherichia coli K1, and Shigella flexneri type 2a polysaccharides in mice. Infect. Immun. 68:2161-2166.[Abstract/Free Full Text]
    30. 30
  30. Peeters, C. C., A. M. Tenbergen-Meekers, D. E. Evenberg, J. Poolman, B. J. M. Zegers, and G. T. Rijkers. 1991. A comparative study of the immunogenicity of pneumococcal type 4 polysaccharide and oligosaccharide tetanus toxoid conjugates in adult mice. J. Immunol. 146:4308-4314.[Abstract]
    31. 31
  31. Peltola, H., P. H. Mäkelä, H. Käyhty, H. Jousimies, E. Herva, K. Hällström, A. Sivonen, O.-V. Renkonen, O. Pettay, V. Karanko, P. Ahvonen, and S. Sarna. 1997. Clinical efficacy of meningococcus group A capsular polysaccharide vaccine in children three months to five years of age. N. Engl. J. Med. 297:686-691.
    32. 32
  32. Pichichero, M. E., S. Porcelli, J. Treanor, and P. Anderson. 1988. Serum antibody responses of weanling mice and two-year-old children to pneumococcal-type-6A-protein conjugate vaccines of differing saccharide chain lengths. Vaccine 16:83-91.
    33. 33
  33. Plikaytis, B. D., P. F. Holder, and G. M. Carlone. 1996. Program ELISA for Windows: user's manual 12, version 1.00. Centers for Disease Control and Prevention, Atlanta, Ga.
    34. 34
  34. Robbins, J. B., R. Schneerson, P. Anderson, and D. H. Smith. 1996. Prevention of systemic infections, especially meningitis, caused by Haemophilus influenzae type b: impact on public health and implications for other polysaccharide-based vaccines. JAMA 276:1181-1185.[Abstract/Free Full Text]
    35. 35
  35. Robbins, J. B., R. Schneerson, and E. C. Gotschlich. 2000. A rebuttal: epidemic and endemic meningococcal meningitis in sub-Saharan Africa can be prevented now by routine immunization with group A meningococcal capsular polysaccharide vaccine. Pediatr. Infect. Dis. J. 19:945-953.[Medline]
    36. 36
  36. Robbins, J. B., R. Schneerson, S. C. Szu, D. A. Bryla, F.-Y. Lin, and E. C. Gotschlich. 1998. Standardization may suffice for licensure of conjugate vaccines. Dev. Biol. Stand. 95:161-167.[Medline]
    37. 37
  37. Robbins, J. B., D. W. Towne, E. C. Gotschlich, and R. Schneerson. 1997. Love's labours lost: failure to implement mass vaccination against group A meningococcal meningitis in sub-Saharan Africa. Lancet 350:880-882.[CrossRef][Medline]
    38. 38
  38. Schneerson, R., O. Barrera, A. Sutton, and J. B. Robbins. 1980. Preparation, characterization, and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates. J. Exp. Med. 152:361-369.[Abstract/Free Full Text]
    39. 39
  39. Shafer, D. E., B. Toll, R. F. Schuman, B. L. Nelson, J. J. Mond, and A. Lees. 2001. Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) for use in protein-polysaccharide conjugate vaccines and immunological reagents. II. Selective crosslinking of proteins to CDAP-activated polysaccharides. Vaccine 19:1547-1558.[CrossRef][Medline]
    40. 40
  40. Szu, S. C., X. Li, R. Schneerson, J. Vickers, and J. B. Robbins. 1989. Comparative immunogenicities of Vi polysaccharide-protein conjugates composed of cholera toxin or its B-subunit as a carrier bound to high or lower molecular weight Vi. Infect. Immun. 57:3823-3827.[Abstract/Free Full Text]
    41. 41
  41. Tiesjema, R. H., E. C. Beuvery, and B. J. Te Pas. 1977. Enhanced stability of meningococcal polysaccharide vaccines by using lactose as a menstruum for lyophilization. Bull. W. H. O. 55:43-48.[Medline]
    42. 42
  42. Vann, W. F., T.-Y. Liu, and J. B. Robbins. 1976. Bacillus pumilus polysaccharide cross-reactive with meningococcal group A polysaccharide. Infect. Immun. 13:1654-1662.[Abstract/Free Full Text]
    43. 43
  43. Wessels, M. R., L. C. Paoletti, H.-K. Guttormsen, F. Michon, A. J. D'Ambra, and D. L. Kasper. 1998. Structural properties of group B streptococcal type III polysaccharide conjugate vaccines that influence immunogenicity and efficacy. Infect. Immun. 66:2186-2192.[Abstract/Free Full Text]
    44. 44
  44. Wong, K. H., O. Barrera, A. Sutton, J. May, D. H. Hochstein, J. B. Robbins, P. D. Parkman, and E. B. J. Seligmann. 1977. Standardization and control of meningococcal vaccines, group A and group C polysaccharides. J. Biol. Stand. 5:197-215.[CrossRef][Medline]
    45. 45
  45. World Health Organization. 1976. Requirements for meningococcal polysaccharide vaccine. World Health Organ. Tech. Rep. Ser. 594:50-75.
    46. 46
  46. World Health Organization. 1999. Group A and C meningococcal vaccines. Wkly. Epidemiol. Rec. 74:297-304.[Medline]
    47. 47
  47. World Health Organization. 2001. Epidemics of meningococcal disease, African meningitis belt, 2001. Wkly. Epidemiol. Rec. 76:282-288.[Medline]

Infection and Immunity, September 2003, p. 5115-5120, Vol. 71, No. 9
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.9.5115-5120.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Jin, Z., Bohach, G. A., Shiloach, J., Norris, S. E., Freedberg, D. I., Deobald, C., Coxon, B., Robbins, J. B., Schneerson, R. (2005). Conjugates of Group A and W135 Capsular Polysaccharides of Neisseria meningitidis Bound to Recombinant Staphylococcus aureus Enterotoxin C1: Preparation, Physicochemical Characterization, and Immunological Properties in Mice. Infect. Immun. 73: 7887-7893 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jin, Z.
Right arrow Articles by Schneerson, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jin, Z.
Right arrow Articles by Schneerson, R.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»