Indian J Med Res 119 (Suppl) May 2004, pp 126-130 Antibody classes & subclasses induced by mucosal immunization of mice with Streptococcus pyogenes M6 protein & oligodeoxynucleotides containing CpG motifs R. Teloni, C. von Hunolstein, S. Mariotti, S. Donati, G. Orefici & R. Nisini Istituto Superiore di Sanità, Rome, Italy Received August 7, 2003 Background & objectives: Type-specific antibodies against M protein are critical for human protection as they enhance phagocytosis and are protective. An ideal vaccine for the protection against Streptococcus pyogenes would warrant mucosal immunity, but mucosally administered M-protein has been shown to be poorly immunogenic in animals. We used a recombinant M type 6 protein to immunize mice in the presence of synthetic oligodeoxynucleotides containing CpG motifs (immunostimulatory sequences: ISS) or cholera toxin (CT) to explore its possible usage in a mucosal vaccine. Methods: Mice were immunized by intranasal (in) or intradermal (id) administration with four doses at weekly intervals of M6-protein (10 μg/mouse) with or without adjuvant (ISS, 10 μg/mouse or CT, 0,5 μg/mouse). M6 specific antibodies were measured by enzyme linked immunosorbent assay using class and subclass specific monoclonal antibodies. Results: The use of ISS induced an impressive anti M-protein serum IgG response but when id administered was not detectable in the absence of adjuvant. When used in, M-protein in the presence of both ISS and CT induced anti M-protein IgA in the bronchoalveolar lavage, as well as specific IgG in the serum. IgG were able to react with serotype M6 strains of S. pyogenes. The level of antibodies obtained by immunizing mice in with M-protein and CT was higher in comparison to M-protein and ISS. The analysis of anti-M protein specific IgG subclasses showed high levels of IgG1, IgG2a and IgG2b, and low levels of IgG3 when ISS were used as adjuvant. Thus, in the presence of ISS, the ratio IgG2a/IgG1 and (IgG2a+IgG3)/IgG1 >1 indicated a type 1-like response obtained both in mucosally or systemically vaccinated mice. Interpretation & conclusion: Our study offers a reproducible model of anti-M protein vaccination that could be applied to test new antigenic formulations to induce an anti-group A Streptococcus (GAS) vaccination suitable for protection against the different diseases caused by this bacterium. Key words Anti GAS vaccination - M6 protein - mucosal immunity - Streptococcus pyogenes M protein is considered the main determinant factor present on the surface of group A Streptococcus (GAS) 1 . In humans and animals only M protein type- specific antibodies have the capacity to opsonize streptococci and to override the anti-phagocytic property of M protein. The development of an efficacious and safe vaccine against GAS infections was prevented by two main factors: the high number of different, non cross- reactive serotypes and the fact that some portions of the protein (B repetitive domain) contain autoantibodies- inducing epitopes against cardiac and skeletal tissues 2 . An ideal vaccine for the protection against GAS would warrant muscosal immunity, but mucosally administered M-protein has been shown to be poorly immunogenic in 126