SA LIVA RY ANTIBODY RESPONSES IN RHESUS MONKEYS IMMUNIZED WITH STREPTOCOCCUS MUTANS BY THE ORAL, SUBMUCOSAL OR SUBCUTANEOUS ROUTES S. J. CHALLACOMBE and T. LEHNER Department of Oral Immunology and Microbiology, Guy’s Hospital Medical and Dental Schools, London SE1 9RT, England Summary-Antibodies in whole saliva were investigated in 43 rhesus monkeys which had been immunized subcutaneously (SC), submucosally (SM) or orally with Streptococcus mutans. Ail monkeys before immunization had detectable natural agglutinating activity against zyxwvutsrqponmlkjihg Strep. mutans which resided in the macromolecular fractions of saliva. Submucosal immunization with whole cells in Freund’s incomplete adjuvant induced a significant increase in salivary aggluti- nating activity to whole organisms and haemagglutination of antigen-coated red cells, whereas SC immunization did not. SC immunization with pronased cell-wall preparations of Strep. mutuns led to greater increases in agglutinating activity in saliva than immunization with whole cells. Immunization by the oral route, with bacteria in gelatin capsules, resulted in increases in salivary agglutinating activity lasting for 25-30 days. Secondary immunization resulted in a more rapid response, but again of about 25 days. Administration of Strep. mutans cells in the drinking water led to a salivary antibody response in both whole and parotid saliva within 28 days. Antibody titres fell to pre-immune levels within 42 days of cessation of antigen adminis- tration. Oral immunization appears to lead to a salivary antibody response of short duration and is therefore unlikely to be useful in the protection against dental caries. Chromatographic analysis revealed that the increased agglutinating activity in saliva following immunization was mainly due to antibodies of the IgA class. INTRODUCTION Immunity to dental caries in rhesus monkeys has been achieved by immunization with Streptococcus mutans (Lehner, Challacombe and Caldwell, 1975a, 1976). Subcutaneous immunization in Freund’s incomplete adjuvant (FIA) induced both serum anti- bodies and cellular immunity (Lehner er a[., 1976). Protection against dental caries appears to be associ- ated primarily with serum IgG antibodies (Lehner et al., 1976) which may enter the gingival crevice in an intact form from plasma (Challacombe, Russell and Hawkes, 1978). Passive transfer of serum IgG anti- bodies and cellular immunity (Lehner et al., 1976). Protection against dental caries appears to be associ- transfer of serum IgA or IgM antibodies (Lehner et al., 1978). In the irus monkey, systemic immunization has also led to significant protection against caries which is presumably mediated through serum anti- bodies (Bowen, et al., 1975, Schick et a!., 1978). In rodents, protection against caries following immunization with Strep. mutans has been associated with the induction of salivary antibodies. Local immunization of hamsters and rats with whole cells or glucosyltransferase fractions from Strep. mutans results in salivary IgA antibody in addition to serum antibody and a reduction in smooth surface caries (Taubman and Smith. 1974, 1977; McGhee et al.. 1975). Ingestion of whole cells of Strep. mutans by gnotobiotic rats may induce antibodies in saliva in the absence of detectable serum antibody, and these secretory antibodies may lead to protection against caries (Michalek er al., 1976). In addition. passive transfer of IgG and IgA antibodies from rat dams to their offspring in colostrum and milk resulted in pro- tection against caries (Michalek, McGhee and Babb, 1977). From these experiments, it appears that protection against caries in non-human primates can be achieved by the induction of serum antibodies predominantly of the IgG class, and protection in rats can be achieved by the induction of locally-produced anti- bodies of both the IgA and IgG classes. However, in the rhesus monkey, the role of salivary antibodies in animals immunized and protected against caries has not been fully examined, and the objective of this investigation was to compare the effects of different routes of immunization and different antigenic prep- arations on the induction of antibodies in saliva. MATERIALS AND METHODS Rhesus monkeys Forty-three young rhesus monkeys (Mucaca mulatta) initially weighing between 1.6 and 2.2 Kg were caged and maintained on a cariogenic human type of diet containing approximately 15 per cent su- crose as previously described (Lehner, Challacombe and Caldwell, 1975b). All monkeys had a fully erupted deciduous dentition and in some the first permanent molars were erupting or erupted. The experiment was divided into three sections (Table 1). (I) Comparison of subcutaneous (SC) and submu- cosal (SM) immunization on the induction of salivary antibodies. 917