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Bruttini. Contributor Information E. ad hoc protein microarray to study the immune response induced by the three major 4CMenB antigenic components (fHbp, NHBA and NadA) in individual sera from vaccinated infants, adolescents and adults. The resulting 4CMenB protein antigen fingerprinting allowed the identification of specific human antibody repertoire correlating with the bactericidal response elicited in each subject. This work represents an example of epitope mapping of the immune response induced by a multicomponent vaccine in different age groups with the identification of protective signatures. It shows the high flexibility of this microarray based methodology in terms of high-throughput information and minimal volume of biological samples needed. Subject terms: Antibodies, Protein vaccines, Bacterial infection 4CMenB is an approved multi-component vaccine against Serogroup B meningococcus. Here the authors develop a protein microarray for three major 4CMenB antigenic components (fHbp, NHBA and NadA) and describe antibody repertoires in sera from vaccinated infants, adolescents and adults correlating with bactericidal response. Introduction The development of effective vaccines against of serogroup B (MenB) has been challenging due to the poor immunogenicity of its capsular polysaccharide and to the variability of the major outer membrane proteins. The identification of sub-capsular antigens, more conserved in sequence and able to induce bactericidal antibodies, has been possible through the reverse vaccinology approach1,2. This has led to the development and registration of the multicomponent, recombinant, 4CMenB vaccine, containing two fusion proteins, Neisserial heparin-binding antigen-GNA1030 (NHBA-GNA1030) and GNA2091-factor H-binding protein (GNA2091-fHbp), plus the recombinant adhesin A (NadA), in combination with the detergent-extracted outer membrane vesicles (OMV) derived from the epidemic meningococcal NZ98/254 strain3,4. Bactericidal activity, that is the Rabbit polyclonal to RAB14 ability of the antibodies to kill bacteria in presence of complement, is the established correlate of protection for MenB5,6. However, little is known about the nature of the protein epitopes or antigenic domains inducing bactericidal antibodies in different age groups. This Zoledronic acid monohydrate knowledge is important because different epitopes can show different degree of immunogenicity depending on age7. Therefore, improved vaccine efficacy could be achieved with a deeper understanding of the kinetics of antibody immunity from infancy to adulthood8. Various techniques, such as X-ray crystallography9,10, nuclear magnetic resonance (NMR) spectroscopy11,12, and hydrogenCdeuterium exchange mass spectrometry (HDX-MS)13,14 have been applied successfully to map antibody-recognised epitopes. Although these approaches are extremely informative, they are generally time and sample consuming and require highly specific purified monoclonal antibodies, as they are not easily applicable to the epitope mapping of polyclonal sera. Other methods include the phage display technology described firstly by Smith15, that has been used for decades to investigate proteinCprotein interactions including antigenCantibody recognition16C18. An alternative approach is based on peptide libraries, i.e. arrays of overlapping synthetic peptides encompassing the entire primary structure of the target antigen19. These peptides are Zoledronic acid monohydrate generally of only 12C15 aa in length, therefore present mainly linear epitopes whereas they are not representative of more complex epitopes made by residues located on discontinuous regions. Nevertheless, the use of peptide libraries has been successfully applied to the identification of protein domains or epitopes involved in binding to antibodies generated by vaccination20,21 or autoantibodies22C24. Nowadays, a faster and more powerful technology to perform epitope mapping is represented by protein microarrays, which allow analysing simultaneously short and long peptides that are representative of all immunogenic regions of an antigen, including both linear and conformational epitopes, with the further advantage of using only minimal volumes of biological samples. Proteomic microarrays generated by spotting full-length antigens are largely used to profile responses to Zoledronic acid monohydrate bacterial infections25,26 or following vaccination27C29 or as diagnostic tool30. Differently, only few studies have attempted at characterising the antibody repertoires in vaccinees based on antigenic fingerprinting31 or at analysing the pattern of epitopes that are preferentially targeted by antibodies in subjects of different age..