The role of novel Neisseria meningitidis antigens in the pathogenesis of infection and their potential as vaccine candidates

Abstract

Neisseria meningitidis is a major cause of bacterial meningitis and septicaemia. Although vaccines against meningococcal A, C, Y and W are available, there is no vaccine for serogroup B infection. The vaccine potential of two proteins, Neisseria meningitidis-macrophage infectivity potentiator (MIP, NMB1567) and Neisseria meningitidis-adhesin complex protein (ACP, NMB2095) were investigated. The gene encoding each protein was cloned in the pRSETA system and propagated in E.coli DH5?. Recombinant MIP (rMIP) and recombinant ACP (rACP) proteins were expressed in E. coli BL21(DE3)pLysS and purified to high purity and yield by nickel column affinity chromatography under native and denaturing conditions, respectively. For animal immunisation, the pure proteins were refolded into i) liposomes and ii) Zwittergent 3-14 micelles, both with and without the adjuvant monophosphoryl lipid A (MPLA), iii) adsorbed onto aluminium hydroxide and iv) diluted in saline alone. Antisera of immunised animals were subjected to immunological analyses. High antibody titres against recombinant proteins and against protein in the outer membrane (OM) were detected by ELISA and western blot. Both proteins induced serum bactericidal antibody (SBA) against homologous strain (titres from 256-1024). However, the optimal preparations for eliciting heterologous SBA were proteins in saline or liposomes alone without addition of adjuvants (titres of 256-1024). Both proteins were highly conserved amongst meningococci and showed similar expression levels amongst a collection of 13 strains surveyed. In particular, the role of ACP in pathogenesis was investigated by generating ACP knockout mutant (MC58?ACP) and complementation strains for in vitro infection assays using a variety of human cells. MC58?ACP showed significantly decreased association with epithelial cells, endothelial cells and meningioma cells compared to MC58 in a capsulated background (p<0.05). Using an acapsular ACP mutant strain, the protein mediated internalisation of meningococci by epithelial cells and endothelial cells. In particular, a tropism for epithelial cell interactions mediated by ACP expression was observed. In conclusion, both MIP and ACP are highly conserved, surface-exposed proteins capable of eliciting cross-protective SBA and they deserve to be considered as vaccine antigens, possibly in a multi-component vaccine. Notably, ACP is a new adhesin and invasin that shows specific cell tropism and antibodies against ACP might provide multi-level protection against meningococcal infection