Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody

Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5–15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA.The Gram-negative encapsulated bacterium Neisseria meningitidis causes severe sepsis and meningococcal meningitis. Invasive meningococcal disease (IMD) is associated with 5–15% mortality; furthermore, devastating long-term sequelae such as amputations, hearing loss, and neurodevelopmental disabilities are observed in 11–19% of IMD survivors (1). Meningococcal serogroups are distinguished by the composition of their capsular polysaccharides. The five serogroups most commonly associated with invasive disease are A, B, C, W, and Y. (2). Effective mono- or polyvalent-conjugated polysaccharide vaccines against N. meningitidis serogroups A, C, W, and Y have been available since the early 1990s (3). However, serogroup B meningococcus (MenB) is responsible for the majority of endemic and epidemic meningococcal disease in developed countries (4–6). The development of an efficient capsular polysaccharide-based vaccine against MenB has been hampered by potential autoimmunity issues, namely, the structural similarity between the MenB capsular polysaccharide and the neuraminic acid present on the surface of human fetal neural tissues (7).In early 2013 the European Medicines Agency approved 4CMenB, to our knowledge the first broadly protective vaccine against MenB, for the prevention of IMD in all age groups. 4CMenB is a multicomponent vaccine formulation composed of three surface-exposed meningococcal proteins originally identified by the reverse vaccinology approach (8) plus outer membrane vesicles from the New Zealand epidemic clone. The three antigenic proteins are factor H-binding protein (fHbp), neisserial heparin-binding antigen (NHBA), and neisserial adhesin A (NadA) (9, 10).The gene encoding NadA is present in ∼30% of pathogenic meningococcal isolates and is associated mostly with strains that belong to three of the four hypervirulent serogroup B lineages (11–14). NadA expression levels can vary among isolates by more than 100-fold, and its expression is up-regulated in vivo by niche-specific signals (15). NadA induces high levels of bactericidal antibodies in humans (16–18) and is recognized by serum antibodies of children convalescent after IMD (19), suggesting that it is expressed and is immunogenic during IMD. Two main genetically distinct groups of NadA have been identified that share overall amino acid sequence identities of 45–50%. Group I includes the three most common variants (NadA1, NadA2, and NadA3, the latter being the vaccine variant), which share ∼95% sequence identity and are immunologically cross-reactive (11). Group II includes three rarer variants: NadA4, primarily associated with carriage strains (11); NadA5, found mainly in strains of clonal complex 213 (20, 21); and NadA6 (Fig. S1A); these three share ∼90% sequence identity (Fig. S1B) (22).Functionally, NadA3 expressed on the surface of Escherichia coli promotes adhesion to and invasion of Chang epithelial cells (23). This adhesive activity has been mapped, at least partially, to an N-terminal region extending to residue T132 (23, 24). Recently, interactions of NadA3 with β-1 integrin (25) and with the heat shock protein Hsp90 (26) have been reported.Structurally, NadA belongs to the class of trimeric autotransporter adhesins (TAAs) (27, 28), which are known to mediate adhesion through interaction with extracellular matrix proteins and are involved in invasion of target cells (29). TAAs are obligate homotrimers, and accordingly the recombinant NadA3 vaccine antigen, lacking the C-terminal membrane anchor region, forms soluble, stable trimers (23, 30). TAAs generally are made of a conserved C-terminal integral membrane β-barrel, which anchors the proteins to the outer membrane, and an N-terminal “passenger” domain responsible for adhesion (31). The TAA passenger domain typically is made of a central α-helical domain (stalk) that forms coiled-coil structures and a distinct N-terminal domain (head) that is mainly responsible for binding to host cellular receptors.Here we present the X-ray structure of a large ectodomain fragment of NadA5 and a structural analysis by transmission electron microscopy (TEM) of the vaccine variant NadA3. In addition, epitope mapping shows that the head of NadA3 contains immunogenic regions responsible for the generation of a protective bactericidal response.