Multilayer regulation of Neisseria meningitidis NHBA at physiologically relevant temperatures

This research was sponsored by GlaxoSmithKline Biologicals SA. A.F.H. was funded by Marie Sklodowska-Curie Intra-European Fellowships (PIEF-GA-2012-328377).Neisseria meningitidis colonizes the nasopharynx of humans, and pathogenic strains can disseminate into the bloodstream, causing septicemia and meningitis. NHBA is a surface-exposed lipoprotein expressed by all N. meningitidis strains in different isoforms. Diverse roles have been reported for NHBA in heparin-mediated serum resistance, biofilm formation, and adherence to host tissues. We determined that temperature controls the expression of NHBA in all strains tested, with increased levels at 30–32 °C compared to 37 °C. Higher NHBA expression at lower temperatures was measurable both at mRNA and protein levels, resulting in higher surface exposure. Detailed molecular analysis indicated that multiple molecular mechanisms are responsible for the thermoregulated NHBA expression. The comparison of mRNA steady-state levels and half-lives at 30 °C and 37 °C demonstrated an increased mRNA stability/translatability at lower temperatures. Protein stability was also impacted, resulting in higher NHBA stability at lower temperatures. Ultimately, increased NHBA expression resulted in higher susceptibility to complement-mediated killing. We propose that NHBA regulation in response to temperature downshift might be physiologically relevant during transmission and the initial step(s) of interaction within the host nasopharynx. Together these data describe the importance of NHBA both as a virulence factor and as a vaccine antigen during neisserial colonization and invasion.Publisher PDFPeer reviewe

Characterization of the immune response and cross protection activity elicited by the Neisserial Heparin Binding Antigen (NHBA), a component of the 4CMenB vaccine

Invasive disease caused by capsular group B Neisseria meningitidis (MenB) is life threating disease causing hundred thousands of deaths every year, still remaining an unmet medical need in many countries. Although disease can be observed at all age groups, infants and adolescents are the most at risk populations showing the highest incidence in case numbers. Since the MenB capsule was not-immunogenic the development of a MenB vaccine which makes the use of other antigens becomes necessary. 4CMenB is a multicomponent vaccine against serogroup B N. meningitidis composed by three major protein antigens, factor H-binding protein (fHbp), Neisserial Heparin-Binding Antigen (NHBA) and Neisserial adhesin A (NadA), combined with outer membrane vesicles (OMVs) from the New-Zealand epidemic strain (NZ98/254). Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein expressed by all N. meningitidis strains analyzed so far and is composed of two major domains, a highly variable amino-terminal (N-term) domain which anchors the protein on the bacterial outer membrane through the lipobox motif, and a highly conserved carboxyl-terminal (C-term) domain. These domains are separated by a short and quite conserved Arginine-rich (Arg-rich) motif which has been reported to be involved in different mechanisms that mediate meningococci adhesion, infection and survival within the host’s blood stream. NHBA is susceptible to cleavage by NalP, a bacterial protease which has its cleavage site upstream of the arginine region. Moreover human proteases such as human lactoferrin (hLf) and kallikrein are able to process NHBA downstream the the Arg-rich region. Both bacterial and human proteases-mediated cleavage releases the C-term of NHBA in the supernatant, while the N-term of the protein remains anchored on the bacterial surface. NalP cleavage did not impact SBA titers elicited by anti-NHBA antibodies but little is known about the impact that host’s proteases have on bactericidal titers. Based on sequence analysis it has been reported that NHBA has two major alleles, the so called “short” and “long” variants, which differentiate by the presence or absence of a 190 bp long fragment. Despite its sequence variability, NHBA is able to induce a robust and broad immune response against meningococcal strains expressing vaccine homologous and heterologous variants. Although anti-NHBA antibodies are able to induce bacterial killing when tested in serum bactericidal activity assay (SBA), the regions involved in eliciting cross protective immune response remain still unknown. Aims of this study were to use monoclonal antibodies (mAbs) raised against the NHBA vaccine variant peptide 2 (NHBAp2) to (i) map the NHBA regions involved in eliciting the functional response, (ii) test their ability to induce cross protection against strains expressing epidemiologically relevant homologous and heterologous NHBA variants, and (iii) investigate the molecular mechanism of NHBA-mediated bactericidal activity. To this end we used a panel of anti-NHBA mAbs selected to recognize different regions of the protein. Our results showed that only anti-N-term mAbs were able to induce killing of bacterial strains expressing the homologous NHBAp2 and closely related heterologous NHBA variants. Synergy between monoclonal antibodies targeting the N-term and the C-term of NHBA resulted in a significant increase of bactericidal titers but cross protection remained restricted to closely phylogenetic NHBA variants. Anti C-term mAbs were not able to induce SBA activity when tested individually, but surprisingly they became bactericidal when tested in combination. Moreover they were able to induce full cross protection against a panel of strains expressing phylogenetically distant heterologous NHBA variants. Our results suggest that the partial release of the NHBA C-terminal portion upon NalP and serum proteases could explain why anti-C-term mAbs are not able to induce complement mediated bactericidal killing when tested individually. However, the simultaneous binding of C-term mapping mAbs on the same NHBA molecule can induce the formation of a very stable ternary complex that probably allows a more efficient C1q engagement and C3 deposition, thus leading to the observed co-operative bactericidal activity. These results suggest that synergy between anti-NHBA antibodies is at the basis of the mechanism of NHBA-induced bactericidal activity, which could explain the robust and cross-protective immune response elicited by anti-NHBA polyclonal antibodies following immunization. Collectively, the body of experimental data suggests that both domains of NHBA are required to elicit complement mediated bactericidal activity against strains expressing the vaccine homologous and heterologous NHBA variants