Downloaded from www.microbiologyresearch.org by IP: 54.80.173.208 On: Sun, 02 Oct 2016 19:44:55 Microbio/ogy (1 996), 142,3269-3274 Printed in Great Britain Sequence variability of FrpB, a major iron-regulatedouter-membrane protein in the pathogenic neisseriae Peter van der Ley, Jenny van der Biezen, Roger Sutmuller, Peter Hoogerhout and Jan T. Poolman Author for correspondence: Peter van der Ley. Tel: +31 30 2742533. Fax: +31 30 2744429. e-mail : peterl@rivm.nl Laboratory of Vaccine Development and Immune Mechanisms, National Institute of Public Health and the Environment, Antonie van Leeuwenhoeklaan 9, PO Box 1,3720 BA Bilthoven, The Netherlands The FrpB protein from pathogenic neisseriae is a 77 kDa iron-regulatedouter- membrane protein that belongs to the family of TonB-dependent receptors and may have potential as a vaccine component. Comparison between the fipB gene from three different meningococcal strains and a publishedgonococcal one revealed that the region from residues 350 to 390 displays pronounced sequence variability. In a model for the topology of FrpB in the outer membrane, this region corresponds to loop 7, the longest of the predicted 13 surface-exposed loops. Binding of four out of a total of eight bactericidal monoclonal antibodies to synthetic peptides corresponding to loop 7 showed that their epitopes are located here. The fipB genes from five additional meningococcal strains were cloned and sequenced in this region. Pairwise comparisons showed different degrees of similarity. Keywords : Neiisiseria meningitidiis, iron limitation, antigenic variation, outer-membrane vaccine INTRODUCTION Disease caused by Neisseria rneningitidis remains a significant public health problem worldwide. At present, there is no vaccine available against group B organisms, which are the predominant cause of meningococcal disease. The use of the group B capsular polysaccharide as a vaccine has been hindered by the presence of identical structures on neural cell adhesion molecules in the human host, raising the possibility of inducing autoimmune pathology with such a vaccine. As an alternative, vaccines based on meningococcal outer membranes are currently under investigation (Diaz Romero & Outschoorn, 1994 ; Poolman, 1995; Frasch, 1995). Efficacy trials in humans with such vaccines have demonstrated partial protection against meningococcal disease. They consist of outer- membrane vesicles prepared from one particular strain ; since many of the major outer-membrane proteins (OMPs) are variable among different strains, a large proportion of the protective antibodies induced will be Abbreviation: OMP, outer-membrane protein. The GenBank accession numbersfor the sequences determined in this work are: US5377 (29961, U55378 (892257), U67312 (8702271, U67310 (8704461, U67314 (M982), U67311 (M990) and U67313 (53446). strain-specific. In addition, not all outer-membrane com- ponents contribute to the induction of bactericidal antibodies. For these reasons, we are attempting to improve outer-membrane vesicle vaccines, by manipu- lating the OMP composition of a particular strain (van der Ley & Poolman, 1992; van der Ley et al., 1995). Since the class 1 protein appears to be an important target for bactericidal antibodies, derivatives of strain H44/76 carrying three copies of the porA gene have been constructed, with each copy encoding an antigenically different form of the class 1 OMP. Expression of the class 3 and 4 OMPs was abolished because these proteins do not contribute much to the induction of bactericidal antibodies and, in the case of class 4 OMP, might even be detrimental due to the induction of blocking antibodies. Other candidates for inclusion are the several OMPs induced by growth under iron limitation, including receptors for such iron sources as lactoferrin, transferrin and haemoglobin (Legrain et al., 1993; Pettersson et al., 1994; Stojiljkovic et al., 1995). In particular, the FrpB protein appears to be a good target for bactericidal antibodies (Pettersson et al., 1990). The sequence of this 77 kDa protein shows the characteristics of a TonB- dependent outer-membrane receptor ; however, no role in iron uptake has yet been demonstrated (Pettersson et al., 1995). Since bactericidal monoclonal antibodies (mAbs) 0002-0873 0 1996 SGM 3269