Crystal Structure of an Anti-meningococcal Subtype P1.4 PorA Antibody Provides Basis for Peptide–Vaccine Design Clasien J. Oomen 1,2 , Peter Hoogerhout 2 , Betsy Kuipers 2 Gestur Vidarsson 2,3 , Loek van Alphen 2 and Piet Gros 1 * 1 Department of Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University Padualaan 8, 3584 CH Utrecht The Netherlands 2 Netherlands Vaccine Institute (NVI), PO Box 457, 3720 AL Bilthoven, The Netherlands 3 Department of Immunology University Medical Center Utrecht, Lundlaan 6, 3584 EA Utrecht, The Netherlands In various western countries, subtype P1.4 of Neisseria meningitidis serogroup B causes the greatest incidence of meningococcal disease. To investigate the molecular recognition of this subtype, we crystallised a peptide ( P1 HVVVNNKVATH P11 ), corresponding to the subtype P1.4 epitope sequence of outer membrane protein PorA, in complex with a Fab fragment of the bactericidal antibody MN20B9.34 directed against this epitope. Structure determination at 1.95 A ˚ resolution revealed a unique complex of one P1.4 antigen peptide bound to two identical Fab fragments. One Fab recognises the putative epitope residues in a 2:2 type I b-turn at residues P5 NNKV P8 , whereas the other Fab binds the C-terminal residues of the peptide that we consider a crystallisation artefact. Interestingly, recognition of the P1.4 epitope peptide is mediated almost exclusively through the complementarity-determining regions of the heavy chain. We exploited the observed turn conformation for designing conformationally restricted cyclic peptides for use as a peptide vaccine. The conformational stability of the two peptide designs was assessed by molecular dynamics simulations. Unlike the linear peptide, both cyclic peptides, conjugated to tetanus toxoid as a carrier protein, elicited antibody responses in mice that recognised meningococci of subtype P1.7-2,4. Serum bactericidal assays showed that some, but not all, of the sera induced with the cyclic peptide conjugates could activate the complement system with titres that were very high compared to the titres induced by complete PorA protein in its native conformation administered in outer membrane vesicles. q 2005 Elsevier Ltd. All rights reserved. Keywords: Fab structure; peptide vaccine; structure-based design; Neisseria meningitidis *Corresponding author Introduction The human pathogen Neisseria meningitidis can cause life-threatening meningitis and sepsis. Despite medical treatment and antibiotics, still 5–10% of the cases are fatal, corresponding to an estimated 170,000 deaths worldwide per year. 1 Vaccines based on the polysaccharide capsule of serogroups A and C have proven to be effective in prevention of meningococcal disease caused by these serogroups. 1 However, no vaccine is available for serogroup B, a serogroup that causes the majority of cases of meningococcal disease in many countries. As the polysaccharide capsule of serogroup B meningococci is similar to that expressed in human tissue, 2 most vaccine research has been focussed on other surface antigens, such as outer membrane proteins. PorA is a highly abundant and immunogenic outer membrane protein of N. meningitidis. Novel vaccines based on this protein administered in outer membrane vesicles (OMV) are currently used in a phase III clinical trial. 3–5 Though no crystal 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Abbreviations used: CDR, complementarity-determin- ing region; C H 1 and C L , constant domains of heavy chain and light chain, respectively; Fab, antigen-recognising fragment of an antibody; K*ZLys-SAMA, N 3 -(S-acetyl- mercaptoacetyl) lysyl; OMV, outer membrane vesicle; V H and V L , variable domains of heavy chain and light chain, respectively; VR, variable region; ELISA, enzyme-linked immunosorbent assay. E-mail address of the corresponding author: p.gros@chem.uu.nl doi:10.1016/j.jmb.2005.06.061 J. Mol. Biol. (2005) 351, 1070–1080