Identification of B- and T-cell epitopes of BB, a carrier protein derived from the G protein of Streptococcus strain G148

Abstract

Most conventional vaccines consist of killed organisms or purified antigenic proteins. Such molecules are generally poorly immunogenic and need to be coupled to carrier proteins. We have identified a new carrier molecule, BB, derived from the G protein of Streptococcus strain G148. We show that BB is able to induce strong antibody responses when conjugated to peptides or polysaccharides. In order to localize T and B cell epitopes in BB and match them with the albumin-binding region of the molecule, we immunized mice with BB, performed B and T pepscan analyses, and compared the results with pepscan done with sera and cells from humans. Our results indicate that BB has two distinct T helper epitopes, seven linear B-cell epitopes, and one conformational B-cell epitope in BALB/c mice. Four linear B-cell epitopes were identified from human sera, three of which overlapped mouse B-cell epitopes. Finally, three human T-cell epitopes were detected on the BB protein. One of these T-cell epitopes is common to BALB/c mice and humans and was localized in the region that contains the albumin-binding site. These data are of interest for the optimization of new carrier molecules derived from BB.

FIG. 1.

Anti-peptide (A), anti-polysaccharide (B), or anti-carrier (C) serum antibody responses. Mice were immunized three times with 10 μg of G5 or 0.5 μg of Hib alone or conjugated to BB or TT in presence of aluminum hydroxide. After each immunization, sera were collected and anti-G5, anti-Hib, or anti-carrier IgG content was measured by ELISA. (D) Specific epitopic suppression studies. Naive mice or mice primed, respectively, with TT or BB were then immunized three times with 0.1 μg of G5 conjugated, respectively, to TT or BB. Anti-G5 IgG titers were measured by ELISA. The results are expressed as the geometric means ± the standard deviation (n = 5). ∗, P < 0.05.

FIG. 2.

Structure of the BB protein. The green region correspond to the HSA-binding region. The large letters in this region are residues critical for HSA binding. The red lines indicate the BALB/c T-cell epitopes, and the broken lines indicate the human epitopes.

FIG. 3.

BB reactivities of polyclonal sera from BALB/c mice immunized three times s.c. with 3 μg of BB adjuvanted in complete Freund adjuvant for the first immunization and in incomplete Freund adjuvant for the second and third injections (A) or supernatants from the 5G7, an anti-BB monoclonal MAb generated by mouse immunization with 10 μg of BB adjuvanted as previously described (B) or polyclonal sera from a normal volunteer (C) against overlapping 12-mer peptides spanning the entire BB protein, with the exception of peptides from the region from positions 128 to 175. The mouse serum was a pool of sera from five mice and was diluted 1/5,000. Human serum was diluted 1/5,000. The supernatant from the 5G7 MAb was undiluted. The first two residues of every third peptide are indicated in the x axis. Reactivity peaks are numbered to facilitate direct comparison of the various sera; the numbers correspond to the first amino acid residues of the implicated peptide. The horizontal line indicates the background as defined in Materials and Methods.

FIG. 4.

Proliferative response of T cells derived from BB-immunized BALB/c mice to stimulation with BB or a panel of overlapping BB peptides. The first residues of every nine peptides are indicated in the x axis. Reactivity peaks are numbered to facilitate correlations with the sequence shown in Fig. 1; the numbers correspond to the first amino acid residues of the implicated peptide. The results are reported as the average of the proliferative index of triplicate sample experiments. This experiment is representative of three separate experiments.

FIG. 5.

Proliferative response of T cells derived from PBS-, BB-, peptide 14-, or peptide 41-immunized mice to stimulation with BB, peptide 14, or peptide 41. The results are reported as the average of triplicate proliferative index values.

FIG. 6.

Proliferative response of human PBMC to peptides of the BB protein. PBMC from four of twelve donors with reactivity to the BB protein were cultured in the presence of peptides 33 (▨), 39 (▩), 14 (░⃞), or 41 (▪) at 50 μg/ml; BB (□) at 20 μg/ml; or medium alone. The data are presented as the mean values obtained from duplicate experiments.