It has been previously reported that the distribution of various fibronectin-binding proteins (e.g., SfbI [protein F1], SfbII [SOF], protein F2, and PFBP) correlates with M serotypes of S. pyogenes
). The most common serotypes of strains isolated worldwide from pharyngitis and TSLS patients are M1 and M1/M3, respectively. However, these strains seemed not to possess fibronectin-binding proteins and/or their genes (12
). In this study, we showed that all of the test strains from TSLS and non-TSLS patients possess the fbp54
gene with different serotypes of M protein (Fig. A and B). It was also found that FBP54 is expressed in different quantities on the cell surface of S. pyogenes
, as revealed by ELISA using rabbit anti-FBP54 antibody (Fig. C). Since common epitopes or proteins are better targets for the production of a universal vaccine, the FBP54 protein could be a better candidate than the M protein. Therefore, we chose to evaluate the efficacy of FBP54 as a vaccine in this study.
Sera from human volunteers were found to contain high levels of antibodies specific for FBP54 and low levels of SPE-B-specific antibodies. Regarding this finding, two hypotheses could be proposed. One is that FBP54 is a better immunogen than SPE-B in human beings, and the other is that some microorganisms induce antibodies cross-reactive to FBP54, as they seem to possess an epitope similar to the FBP54 protein. A homology search of the GenBank database revealed that S. pneumoniae PavA and S. gordonii FlpA possess amino acid sequence similarities of 62 and 63%, respectively.
Nasal, s.c., and oral immunizations with FBP54 resulted in induction of FBP54-specific IgG antibodies in sera (Fig. ). The immune responses elicited by s.c. immunization with FBP54 clearly protect the mice against intraperitoneal challenges with a lethal dose of S. pyogenes (P < 0.001; Fig. ). These results indicate that serum IgG antibody is the most important for protection against S. pyogenes infection. Induction of salivary IgA following nasal and oral immunizations may also be beneficial in protection against S. pyogenes infection, in addition to the serum IgG-dominant systemic immunity, since S. pyogenes initially adheres to and subsequently propagates on the mucosal surface of the oropharynx.
Nasal and oral immunizations with FBP54 alone resulted in an induction of FBP54-specific IgG and IgA antibodies in saliva on day 49 following the first immunization (results not shown). This phenomenon suggests that FBP54 has mucosal adjuvanticity. Medina et al. (29
) have reported that nasal immunization of mice with SfbI (fibronectin-binding protein)-ovalbumin conjugant elicited ovalbumin-specific IgA antibody in lung wash. Bacterial fibronectin-binding proteins may exhibit mucosal adjuvant activity, and fibronectin-binding domains may be responsible for this particular activity. Investigations are ongoing in our laboratory to determine the functional domain.
Although rabbit anti-M protein serum promoted almost complete killing of S. pyogenes
during the 3-h incubation with whole blood (2
), significant but not complete inhibition of bacterial growth was seen with rabbit anti-FBP54 serum (Fig. ). An explanation for this could be that, of the M3-type strains used in this study, strains SSI-35, M2938, SSI-7, and SSI-8 possess a high level of cell-associated protein which reacted with anti-FBP54 serum, compared with strain SSI-1, used in Fig. (Fig. ). If a high titer of anti-FBP54 serum or strains with high FBP54 expression had been employed for this bactericidal assay, S. pyogenes
growth might have been inhibited more strongly than in our assay or even completely.
M protein-specific antibodies have been reported to be involved in serotype-specific opsonization and to protect against group A streptococcal infection; however, these antibodies cross-react with some host tissue components, such as myosin, tropomyocin, and vimentin (36
). Dale et al. (7
) constructed a recombinant multivalent M protein vaccine containing amino-terminal subunits with protective epitopes alone, but it was found to be tissue cross-reactive. Isolates from patients with TSLS mainly show the M1 and M3 types worldwide. Furthermore, passive immunization with antibodies to FBP54 results in rescue of mice infected with a lethal dose of S. pyogenes
. Therefore, from a therapeutical point of view, a vaccine that contains M1- and M3-specific and FBP54-specific antibodies, along with the administration of antibiotics may rescue TSLS patients from death.
The fibronectin-binding domain of FBP54 has been suggested to be the N-terminal 1 to 89 residues of the molecule (5
). Furthermore, the antibody to the N-terminal residue 1 to 89 peptide segment of FBP54 binds to S. pyogenes
), suggesting that this repeat region is expressed on the surface of the cell. Thus, this functional domain within the N-terminal region may be a useful, short segment for a peptide vaccine against S. pyogenes
in the future. In conclusion, FBP54 possesses considerable antigenicity in humans and immunization of mice with this protein induces protective immune responses irrespective of different M types.