Auto-transporter A protein of Neisseria meningitidis: a potent CD4+ T-cell and B-cell stimulating antigen detected by expression cloning

Abstract

A meningococcal genomic expression library was screened for potent CD4+ T-cell antigens, using patients' peripheral blood lymphocytes (PBLs). One of the most promising positive clones was fully characterized. The recombinant meningococcal DNA contained a single, incomplete, open reading frame (ORF), which was fully reconstructed with reference to available genomic sequence data. The gene was designated autA (auto-transporter A) as its peptide sequence shares molecular characteristics of the auto-transporter family of proteins. Only a single copy of this gene was detected in the meningococcal, and none in the gonococcal, genomic sequence databases. The complete autA gene, when cloned into an expression vector, expressed a protein of approximately 68 kDa. Purified rAutA recalled strong secondary T-cell responses in PBLs of patients and some healthy donors, and induced strong primary T-cell responses in healthy donors. The human B-cell immunogenicity and cross-reactivity of AutA, purified under native conditions, was confirmed in dot immunoblot experiments. Immunoblots with rabbit polyclonal antibodies to rAutA demonstrated the conserved nature, antigenicity and cross-reactivity of AutA amongst meningococci of different serogroups and strains representing different hypervirulent lineages. AutA showed homology with another meningococcal and gonococcal ORF (designated AutB). AutB was cloned and expressed and used to raise an autB-specific antiserum. Immunoblot experiments indicated that AutB is not expressed in meningococci and does not cross-react with AutA. Thus, AutA, being a potent CD4+ T-cell and B-cell-stimulating antigen, which is highly conserved, deserves further investigation as a potential vaccine candidate.