NMR dynamics and antibody recognition of the meningococcal lipidated outer membrane protein LP2086 in micellar solution.

Neisseria meningitidis is a major cause of meningitis. Although protective vaccination is available against some pathogenic serogroups, serogroup B meningococci have been a challenge for vaccinologists. A family of outer membrane lipoproteins, LP2086 (or factor H binding proteins, fHbp), has been shown to elicit bactericidal antibodies and is currently part of a cocktail vaccine candidate. The NMR structure of the variant LP2086-B01 in micellar solution provided insights on the topology of this family of proteins on the biological membrane. Based on flow cytometry experiments on whole meningococcal cells, binding experiments with monoclonal antibodies, and the NMR structure in micellar solution, we previously proposed that LP2086-B01 anchors the outer bacterial membrane through its lipidated N-terminal cysteine, while a flexible 20 residue linker positions the protein above the layer of lipo-oligosaccharides that surrounds the bacteria. This topology was suggested to increase the antigen exposure to the immune system. In the present work, using micellar solution as a membrane mimicking system, we characterized the backbone dynamics of the variant LP2086-B01 in both its lipidated and unlipidated forms. In addition, binding experiments with a Fab fragment derived from the monoclonal MN86-1042-2 were also performed. Our data suggests that due to the length and flexibility of the N-terminal linker, the antigen is not in contact with the micelle, thus making both N- and C-domains highly available to the host immune system. This dynamic model, combined with the binding data obtained with MN86-1042-2, supports our previously proposed arrangement that LP2086-B01 exposes one face to the extracellular space. Binding of MN86-1042-2 antibody shows that the N-domain is the primary target of this monoclonal, providing further indication that this domain is immunologically important for this family of proteins.

Challenges for the evaluation of Staphylococcus aureus protein based vaccines: monitoring antigenic diversity.

Clumping factors A (ClfA) and B (ClfB) are Staphylococcus aureus virulence proteins that are displayed on the cell surface of the organism and have potential as vaccine antigens for the prevention of S. aureus disease. Here we evaluate the phylogeny of S. aureus in the context of antigenic variation of these two surface proteins. ClfA and ClfB gene sequences, along with epidemiological markers (MLST, spa and capsule genotype) were obtained for 224 S. aureus isolates including both historical strains and a collection representative of current MRSA isolates from the United States. Variation within ClfA and ClfB was consistent with the established population biology of S. aureus, namely, that S. aureus strains belong to a relatively small number of clonal lineages, with evolution proceeding mainly by mutation and with little to no recombination between clades. Thus most variation in ClfA and ClfB occurs between but not within lineages, and particular groups of ClfA and ClfB variants are closely linked. This has important implications for vaccine development and assessment as it suggests that a relatively small survey of strains will be representative of the total population variation, whereas for species that evolve mainly by recombination, such as Neisseria meningitidis, analysis of a much larger number of strains is needed to accomplish the same purpose. Our study also revealed evidence for the de-evolution of ClfB and therefore its reduced suitability as a target for vaccine development compared to ClfA.

Capsular polysaccharides are an important immune evasion mechanism for Staphylococcus aureus.

Staphylococcus aureus can cause severe life threatening invasive diseases. The principal immune effector mechanism by which humans are protected from Gram positive bacteria such as S. aureus is antigen specific antibody- and complement-dependent opsonophagocytosis. This process can be measured in vitro using the opsonophagocytic antibody assay (OPA), which is a complex assay composed of live S. aureus bacteria, a complement source, phagocytic effector cells such as differentiated HL-60 cells, and test serum. In this report, we investigated the impact on the OPA of S. aureus surface antigens capsular polysaccharides (CP) and protein A (SpA). We demonstrated that higher CP expression renders bacteria more resistant to non-specific opsonophagocytic killing than increased SpA expression, suggesting that the expression of capsular polysaccharides may be the more important immune evasion strategy for S. aureus. Bacteria that were not fully encapsulated were highly susceptible to non-specific killing in the assay in the absence of immune serum. This non-specific killing was prevented by growing the bacteria under conditions that increased capsular polysaccharide levels on the surface of the bacteria. In contrast, the level of SpA expression had no detectable effect on non-specific killing in OPA. Using anti-CP antibodies we demonstrated type-specific killing in OPA of both MRSA and MSSA clinical isolates. SpA expression on the cell surface did not interfere with OPA activity, providing evidence that despite the role of SpA in sequestering antibodies by their Fc region, killing is easily accomplished in the presence of high titered anti-capsular polysaccharide antibodies. This highlights the role of CP as an important immune evasion mechanism and supports the inclusion of capsular polysaccharide antigens in the formulation of multi-component prophylactic vaccines against S. aureus.

Sequence diversity of the factor H binding protein vaccine candidate in epidemiologically relevant strains of serogroup B Neisseria meningitidis.

BACKGROUND: Recombinant forms of Neisseria meningitidis human factor H binding protein (fHBP) are undergoing clinical trials in candidate vaccines against invasive meningococcal serogroup B disease. We report an extensive survey and phylogenetic analysis of the diversity of fhbp genes and predicted protein sequences in invasive clinical isolates obtained in the period 2000-2006. METHODS: Nucleotide sequences of fhbp genes were obtained from 1837 invasive N. meningitidis serogroup B (MnB) strains from the United States, Europe, New Zealand, and South Africa. Multilocus sequence typing (MLST) analysis was performed on a subset of the strains. RESULTS: Every strain contained the fhbp gene. All sequences fell into 1 of 2 subfamilies (A or B), with 60%-75% amino acid identity between subfamilies and at least 83% identity within each subfamily. One fHBP sequence may have arisen via inter-subfamily recombination. Subfamily B sequences were found in 70% of the isolates, and subfamily A sequences were found in 30%. Multiple fHBP variants were detected in each of the common MLST clonal complexes. All major MLST complexes include strains in both subfamily A and subfamily B. CONCLUSIONS: The diversity of strains observed underscores the importance of studying the distribution of the vaccine antigen itself rather than relying on common epidemiological surrogates such as MLST.