Immunoadjuvant activities of cell walls, their water-soluble fractions and peptidoglycan subunits, prepared from various gram-positive bacteria, and of synthetic n-acetylmuramyl peptides.

1. The cell walls from some 20 species of gram-positive bacteria, with only few exceptions, were found to be definitely adjuvant-active in both stimulation of increased serum antibody levels and induction of delayed-type hypersensitivity to ovalbumin when administered to guinea pigs in the form of a water-in-oil emulsion. 2. By the use of various cell wall lytic enzymes, the immunoadjuvant principles were solubilized with the full retention of adjuvant activities observed with the cell walls of S. aureus, Str. pyogenes, Str. mutans, L. plantarum, C. diphtheriae, Myc. smegmatis and A. viscosus. N-acetylmuramyl peptide monomers (either L-Lys or meso-Dap type) were shown to be the unit chemical structure responsible for the manifestation of adjuvant activities to stimulate both antibody-mediated and cell-mediated immune responses. 3. Several N-Acetylmuramyl peptides were prepared by condensation of benzyl N-acetyl-4,6-O-benzylidene-alpha-muramide with each peptide benzyl ester by means of dicyclohexylcarbodiimide-N-hydroxysuccinimide method and removal of the protecting groups by hydrogenolysis. N-acetylmuramyl-L-alanyl-D-isoglutamine was identified as the minimum structural entity essential for the immunoadjuvant activities characteristic of bacterial cell walls. Neither synthetic N-acetylmuramyl-L-alanine nor L-alanyl-D-isoglutaminyl-L-lysyl-D-alanin was found to be active.