Vaccine 30 (2012) 5812–5823 Contents lists available at SciVerse ScienceDirect Vaccine j ourna l ho me pag e: www.elsevier.com/locate/vaccine Characterization of size, structure and purity of serogroup X Neisseria meningitidis polysaccharide, and development of an assay for quantification of human antibodies Ouli Xie a , Barbara Bolgiano b , Fang Gao b , Kay Lockyer b , Carolyn Swann c , Christopher Jones c , Isabelle Delrieu d , Berthe-Marie Njanpop-Lafourcade d , Tsidi Agbeko Tamekloe e , Andrew J. Pollard a , Gunnstein Norheim a,∗ a Oxford Vaccine Group, Department of Paediatrics, University of Oxford, and the NIHR Oxford Biomedical Research Centre, Oxford OX3 7TU, UK b Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts. EN6 3QG, UK c Laboratory for Molecular Structure, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts. EN6 3QG, UK d Agence de Médicine Préventive, Paris, France e Ministry of Health, Lome, Togo a r t i c l e i n f o Article history: Received 24 January 2012 Received in revised form 16 June 2012 Accepted 13 July 2012 Available online 24 July 2012 Keywords: Neisseria meningitidis Meningococcal disease Polysaccharide NMR MALLS HPAEC–PAD a b s t r a c t Serogroup X Neisseria meningitidis (MenX) has recently emerged as a cause of localized disease outbreaks in sub-Saharan Africa. In order to prepare for vaccine development, MenX polysaccharide (MenX PS) was purified by standard methods and analyzed for identity and structure by NMR spectroscopy. This study presents the first full assignment of the structure of the MenX PS using 13 C, 1 H and 31 P NMR spectroscopy and total correlation spectroscopy (TOCSY) and 1 H– 13 C heteronuclear single quantum coherence (HSQC). Molecular size distribution analysis using HPLC–SEC with multi-angle laser light scattering (MALLS) found the single peak of MenX PS to have a weight-average molar mass of 247,000 g/mol, slightly higher than a reference preparation of purified serogroup C meningococcal polysaccharide. MenX PS tended to be more thermostable than serogroup A PS. A method for the quantification of MenX PS was developed by use of high performance anion exchange chromatography with pulsed amperometric detection (HPAEC–PAD). A novel and specific ELISA assay for quantification of human anti-MenX PS IgG based on covalent linkage of the MenX PS to functionally modified microtitre plates was developed and found valid for the assessment of the specific antibody concentrations produced in response to MenX vaccination or natural infection. The current work thus provides the necessary background for the development of a MenX PS-based vaccine to prevent meningococcal infection caused by bacteria bearing this capsule. © 2012 Elsevier Ltd. All rights reserved. Abbreviations: dOMVs, deoxycholate extracted outer membrane vesicles; EDAC, N-(3-dimethylaminopropyl)-N ′ -ethylcarbodiimide hydrochloride; GMCs, geomet- ric mean concentrations; ICH, International Conference on the Harmonisation of the Requirements for Technical Requirements for Registration of Pharmaceuticals for Human Use; LPS, lipopolysaccharide; MALLS, multi-angle laser light scatter- ing; MenX, serogroup X Neisseria meningitidis; MLST, multi-locus sequence typing; NFDM, non-fat dried skim milk; NMR, nuclear magnetic resonance; PS, polysac- charide; RI, refraction index; SBA, serum bactericidal activity; SEC, size exclusion chromatography; TBS, Tris-buffered saline; TSDS-PAGE, tricine-sodium dodecyl sul- phate polyacrylamide gel electrophoresis. ∗ Corresponding author at: Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, 0403 Oslo, Norway. Tel.: +47 21 07 65 35; fax: +47 21 07 65 18. E-mail address: gunnstein.norheim@fhi.no (G. Norheim). 1. Introduction Serogroup X Neisseria meningitidis (MenX), previously a rare cause of sporadic cases of meningitis, has recently been associ- ated with increased incidence of meningococcal disease and has emerged as a cause of large outbreaks in the “Meningitis Belt” of Africa. Outbreaks have been documented in Niger [1], Burkina Faso [2], Togo and Ghana [3] and have varied in size. In the meningi- tis season of 2010 over 6500 meningitis cases were reported in Burkina Faso, and it is reasonable to assume that at least 1000 of these cases were due to MenX with a locally reported inci- dence of 120 cases per 100,000 [2,4]. In the first 30 weeks of the 2011 season, 3155 cases of meningitis were reported in Burkina Faso after introduction of the monovalent serogroup A conjugate vaccine [5] and 60% of 236 patients verified as having meningo- coccal meningitis were found to be infected with MenX bacteria (15% of all verified meningitis cases). Earlier, several patients were 0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2012.07.032