Vaccine 30 (2012) 853–861 Contents lists available at SciVerse ScienceDirect Vaccine jou rn al h om epa ge: www.elsevier.com/locate/vaccine Production of a conjugate vaccine for Salmonella enterica serovar Typhi from Citrobacter Vi F. Micoli a,∗ , S. Rondini a , I. Pisoni a , C. Giannelli a , V. Di Cioccio a , P. Costantino a,b , A. Saul a , L.B. Martin a a Novartis Vaccines Institute for Global Health, Via Fiorentina 1, 53100 Siena, Italy b Novartis Vaccines and Diagnostics Research Center, Via Fiorentina 1, 53100 Siena, Italy a r t i c l e i n f o Article history: Received 12 August 2011 Received in revised form 2 November 2011 Accepted 29 November 2011 Available online 13 December 2011 Keywords: Vi polysaccharide Vi CRM197 Conjugate vaccine Salmonella Typhi a b s t r a c t A conjugate vaccine for Salmonella enterica serovar Typhi was produced by chemically linking Vi, purified from Citrobacter, to the non-toxic mutant diphtheria toxin CRM 197 via an adipic dihydrazide spacer using N-(3-Dimethylaminopropyl)-N ′ -ethylcarbodiimide coupling chemistry. The polysaccharide purification process was developed based on Vi precipitation from culture supernatant with cetyl trimethylammo- nium bromide (CTAB), solubilization of the CTA-polysaccharide salt with ethanol followed by exchange of the CTA + counter ion with Na + . The purified Vi polysaccharide was fully O-acetylated and with high purity. The conjugation process was optimized to obtain a scalable process that has been used for GMP production at pilot scale of vaccine currently in clinical trials. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Typhoid fever is a systemic infection caused by the enteric pathogen Salmonella enterica serovar Typhi. It remains a common and serious disease, causing illness and death in many areas of the world where sanitation and clean water are not available, particu- larly in the Indian subcontinent. In 2004, WHO estimated the global typhoid fever disease burden at 21 million cases annually, resulting in an estimated 216,000–600,000 deaths per year, predominantly in children of school age or younger [1] with a significant num- ber of severe cases in children <2 years of age in India, Pakistan and Bangladesh [2–4]. Vaccines based on the Vi capsular polysac- charide (PS) of S. Typhi were assessed in the late 1980s in field trials in typhoid-endemic areas in Nepal and South Africa [5,6] and licensed globally. In adults, a single dose of Vi vaccine provides approximately 70% protection falling to 50% after 3 years [7]. How- ever, Vi induces only short-lived antibody responses in children >2 years old and does not elicit protective antibody levels in younger children [8]. To improve immunogenicity, especially in young children, Vi was conjugated to recombinant mutant Pseudomonas aeruginosa exoprotein A (rEPA) [9]. This vaccine was safe, had enhanced immunogenicity in adults, children 5–14 years old and infants, and elicited a booster response in children 2–5 years old [10–12]. Because of the lack of regulatory precedent for rEPA in licensing ∗ Corresponding author. Tel.: +39 0577 539087; fax: +39 0577 243540. E-mail address: francesca.micoli@novartis.com (F. Micoli). vaccines, a similar conjugation approach for obtaining Vi rEPA has been used by the International Vaccine Institute (Seoul, Korea), with diphtheria toxoid (DT) as carrier protein [13] and full scale production batches are planned [14]. Another vaccine, Vi conjugated to tetanus toxoid (Vi TT, Peda Typh TM , BioMed Pty Ltd.) was licensed in 2008 for infants ≥12 weeks, but its use is limited to India [15]. The Novartis Vaccines Institute for Global Health (NVGH) has a program aimed to develop a scal- able process for the production of an effective S. Typhi vaccine, particularly for use in all ages and affordable in developing coun- tries. Recently, we showed that Vi CRM 197 , a conjugate of Vi derived from Citrobacter freundii WR7011 with CRM 197 [16] as the carrier protein, was well tolerated and immunogenic in mice and rab- bits. In the absence of adjuvant, it induced serum antibody levels comparable to Vi rEPA and Vi TT conjugates [17]. Vaccination of mice with this Vi CRM 197 reduced bacterial colonization following experimental challenge with S. Typhimurium genetically modified to express S. Typhi Vi [18]. Vi from Citrobacter is structurally similar and immunologically indistinguishable to Vi from S. Typhi [19]. Citrobacter, a BSL-1 organ- ism, constitutes a safer source for Vi production than the BSL-3 S. Typhi. In particular, Citrobacter NVGH 328 isolate was chosen for large scale production of Vi (Rondini et al., submitted). Citrobacter NVGH 328, in place of WR7011, is a non genetically modified strain, that produces Vi in a stable manner, at higher levels than WR7011 and also grows in simple chemically defined medium. Growth con- ditions were optimized in order to reach high level production from 0.5 to 1 g/L of crude Vi. 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.11.108