Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori ; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen–antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis . The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of ∼1,000 Å ² on fHbp, including >20 fHbp residues distributed on both N- and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen–antibody interfaces are required to understand and design effective immunogens.

Listeria monocytogenes is a food-borne pathogen that is able to form biofilms in food processing facilities. Biofilms are generally more resistant to antimicrobial agents, making it difficult to eradicate them during cleanup procedures. So far, little is known about the function of stress resistance mechanisms in biofilm formation and their resistance to disinfectants. In this study, we investigated the role of sigB , which encodes a major transcriptional regulator of stress response genes, in L. monocytogenes static and continuous-flow biofilm formation and its function in the resistance of biofilm cells to the disinfectants benzalkonium chloride and peracetic acid. Quantitative real-time PCR and promoter reporter studies showed that sigB is activated in static and continuous-flow biofilms. Biofilm formation studies using an in-frame sigB deletion mutant and complementation mutant showed that the presence of SigB is required to obtain wild-type levels of both static and continuous-flow biofilms. Finally, disinfection treatments of planktonically grown cells and cells dispersed from static and continuous-flow biofilms showed that SigB is involved in the resistance of both planktonic cells and biofilms to the disinfectants benzalkonium chloride and peracetic acid.

The food-borne pathogen Listeria monocytogenes has the ability to survive extreme environmental conditions due to an extensive interacting network of stress responses. It is able to grow and survive at relatively high temperatures in comparison with other non-sporulating food-borne pathogens. To investigate the heat-shock response of L. monocytogenes, whole-genome expression profiles of cells that were grown at 37 °C and exposed to 48 °C were examined using DNA microarrays. Transcription levels were measured over a 40 min period after exposure of the culture to 48 °C and compared with those of unexposed cultures at 37 °C. After 3 min, 25 % of all genes were differentially expressed, while after 40 min only 2 % of all genes showed differential expression, indicative of the transient nature of the heat-shock response. The global transcriptional response was validated by analysing the expression of a set of 13 genes by quantitative PCR. Genes previously identified as part of the class I and class III heat-shock response and the class II stress response showed induction at one or more of the time points investigated. This is believed to be the first study to report that several heat-shock-induced genes are part of the SOS response in L. monocytogenes. Furthermore, numerous differentially expressed genes that have roles in the cell division machinery or cell wall synthesis were down-regulated. This expression pattern is in line with the observation that heat shock results in cell elongation and prevention of cell division.