International Journal of Medical Microbiology 299 (2009) 233–246 Genetic stability of vaccine strain Salmonella Typhi Ty21a over 25 ye Dennis J. Kopecko a , Heike Sieber b , Jose A. Ures b , Andreas Fu¨ rer b , Jacqueline Schlup b , Ulrich Knof b , Andre Collioud b , DeQi Xu a , Kevin Colburn a , Guido Dietrich b, a Laboratory of Enteric and Sexually Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, 29 Lincoln Drive, NIH Campus, Bldg. 29/420, HFM440, Bethesda, MD 20892, USA b Berna Biotech AG, Rehhagstr. 79, CH-3018 Berne, Switzerland Received 8 September 2008; accepted 26 September 2008 Abstract The attenuated live bacterial vaccine strain Salmonella enterica Serovar Typhi Ty21a is the main constituent of Vivotif, the only licensed oral vaccine against typhoid fever. The strain was developed in the 1970s by chemical mutagenesis. In the course ofthis mutagenesis, a number ofmutations were introduced into the vaccine strain. Characterisation of the vaccine strain during development as well as release of master- and working seed lots ( and WSL) and commercial batchesis based on phenotypicassaysassessing microbiological and biochemical characteristics of Ty21a. In the current study, we have analysed by DNA sequencing the specific mutations orig correlated with the attenuation of strain Ty21a. These data demonstrate the stability of these mutations for MS WSLs of Ty21a produced between 1980 and 2005. Finally, we have confirmed the correlation of these genetic mutations with the expected phenotypic attenuations for the seed lots used in vaccine manufacture over 25 ye r 2008 Elsevier GmbH. All rights reserved. Keywords: Typhoid fever vaccine; Vaccine strain Ty21a; Genetic and phenotypic analyses Introduction The use of attenuated live bacterial vaccines consti- tutes quite a successful strategy in vaccine development: attenuated strains administered orally mimic natural infection and stimulateboth mucosaland systemic immune responses (McGhee et al., 1992; Shata etal., 2000).Oral vaccination isgenerally associated with lower rates of adverse effects and higher acceptance, and can be logistically simpler than parenteral administra- tion (Levine, 2003). Foremost, the safety and efficacy of attenuated live vaccines must be evaluated carefully to demonstrate an acceptable balance between immuno- genicity and reactogenicity. The potential risk of reversion to virulence can largely be overcome by the use of multiple,defined (i.e.,nowadays,preferably deletions), attenuating mutations. Nevertheless, regula- tory authoritiesrequirea careful evaluation ofthe potentialimpactof environmental releaseof a live vaccine. Particularly relevant to evaluate is the duration and level of human intestinalshedding,length of survival in the environmentas well as the risk of horizontal gene transfer leading to reestablishment of virulence (Viret et al., 2004). One successful example oflive attenuated bacterial vaccines is the vaccine strain Salmonella enterica serovar Typhi Ty21a.Ty21a is an attenuated mutant strain of ARTICLE IN PRESS www.elsevier.de/ijmm 1438-4221/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2008.09.003 Corresponding author. Presentaddress: GlaxoSmithKline Biolo- gicals,Parc de la Noire Epine, Rue Fleming 20,B-1300 Wavre, Belgium. Tel.: +32 10 85 3625; fax: +32 10 85 9292. E-mail address: guido.h.dietrich@gskbio.com (G. Dietrich).