Identification and Characterization of TspA, a Major CD4⁺T-Cell- and B-Cell-StimulatingNeisseria-Specific Antigen

Abstract

In search for novel T-cell immunogens involved in protection against invasive meningococcal disease, we screened fractionated proteins of Neisseria meningitidis (strain SD, B:15:P1.16) by using peripheral blood mononuclear cells (PBMCs) and specific T-cell lines obtained from normal individuals and patients convalescing fromN. meningitidis infection. Proteins of iron-depleted meningococci produced higher PBMC proliferation indices than proteins of iron-replete organisms, indicating that iron-regulated proteins are T-cell immunogens. Insoluble proteins of the iron-depleted cells, which produced better T-cell stimulation than soluble ones, were fractionated by using sodium dodecyl sulfate-polyacrylamide gels and recovered as five fractions (F1 to F5) corresponding to decreasing molecular weight ranges. The proteins were purified (by elution and precipitation) or electroblotted onto nitrocellulose membranes (dissolved and precipitated) before use in further T-cell proliferation assays. One of the fractions (F1), containing high-molecular-mass proteins (>130 kDa), consistently showed the strongest T-cell proliferation responses in all of the T-cell lines examined. F1 proteins were subdivided into four smaller fractions (F1A to F1D) which were reexamined in T-cell proliferation assays, and F1C induced the strongest responses in patients’ T-cell lines. Rabbit polyclonal antibodies to F1C components were used to screen a genomic expression library of N. meningitidis. Two major clones (C1 and C24) of recombinant meningococcal DNA were identified and fully sequenced. Sequence analysis showed that C24 (1,874 bp) consisted of a single open reading frame (ORF), which was included in clone C1 (2,778 bp). The strong CD4+ T-cell-stimulating effect of the polypeptide product of this ORF (named TspA) was confirmed, using a patient T-cell line. Immunogenicity for B cells was confirmed by showing that convalescent patients’ serum antibodies recognized TspA on Western blots. Additional genetic sequence downstream of C24 was obtained from the meningococcal genomic sequence database (Sanger Centre), enabling the whole gene of 2,761 bp to be reconstructed. The DNA and deduced amino acid sequence data for tspA failed to show significant homology to any known gene, except for a corresponding (uncharacterized) gene in Neisseria gonorrhoeae genome sequences, suggesting that tspA is unique to the genusNeisseria. The DNA and deduced amino acid sequence of the second ORF of clone C1 showed significant homology to gloA, encoding glyoxalase I enzyme, of Salmonella typhimurium andEscherichia coli. Thus, we have identified a novel neisserial protein (TspA) which proved to be a strong CD4+T-cell- and B-cell-stimulating immunogen with potential as a possible vaccine candidate.

Identification and characterization of App: an immunogenic autotransporter protein of Neisseria meningitidis

“…Other OMPs that have been investigated include the transferrin-binding proteins, the class 5 proteins (Opc and Opa) and a Neisseria-specific surface protein, NspA. In addition, we have recently described TspA and AutA, two highly conserved Neisseria-specific proteins that are potent T-cell-stimulating antigens (Kizil et al, 1999;Ait-Tahar et al, 2000). It is likely that a successful vaccine will need to contain multiple, but well-characterized antigens.…”

T-cell stimulating protein A (TspA) of Neisseria meningitidis is required for optimal adhesion to human cells

“…There is currently still a need for a protective vaccine against serogroup B N. meningitidis, which is likely to require T-cell and B-cell memory-inducing components. To search for these, we previously screened the proteome of N. meningitidis and identified TspA (Kizil et al, 1999). The tspA gene was cloned, the encoded protein expressed in E. coli and a specific polyclonal antiserum produced (Robinson et al, 2005).…”

“…Genes corresponding to tspA in the genome sequences of meningococcal strains MC58 (GenBank Accession No. NC_003112; Tettelin et al, 2000), Z2491 (GenBank NC_003116; Parkhill et al, 2000) and the unpublished FAM18 sequence were compared with that of strain SD (GenBank AJ010113; Kizil et al, 1999). Over 98% identity was observed between the protein sequences of these strains, the only variation being mainly located in the C-terminal region (Fig.…”

“…T-cell stimulating protein A (TspA) is an 875 amino acid (aa) T-and B-cell stimulating protein that was identified by screening the N. meningitidis strain SD proteome for molecules that stimulated the proliferation of peripheral blood mononuclear cells taken from patients who had recovered from invasive disease and from healthy donors (Kizil et al, 1999). TspA has been cloned, expressed in Escherichia coli and a specific polyclonal antiserum produced.…”

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

“…These were fully characterized with multilocus enzyme electrophoresis (MLEE) and multilocus sequence typing (MLST) (Maiden et al, 1998). Total protein extracts and OMPs were prepared as described previously (Kizil et al, 1999).…”