Effect of Trypsin and Ribonuclease on the Immunogenic Activity of Ribosomes and Ribonucleic Acid Isolated from Mycobacterium tuberculosis

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Abstract
The ribosomal fraction of the attenuated strain, H37Ra, of Mycobacterium tuberculosis was treated with trypsin alone, ethylenedia-minetetraacetatic acid (EDTA) alone, EDTA and pancreatic ribonuclease, or with trypsin and ribonuclease. After each of these treatments, the ribosomal fractions were injected intraperitoneally into male CF-1 mice to test their capacity to produce an immune response to infection with virulent tubercle bacilli, strain H37Rv. Removal of protein with trypsin left the immunogenicity unchanged; EDTA alone reduced immunogenicity in the smaller vaccinating doses; EDTA plus ribonuclease reduced the immunogenicity by approximately 50% in the highest (1.0 mg) vaccinating dose; ribonuclease alone, after treatment with trypsin, reduced immunogenicity also approximately 50%. A crude mycobacterial ribonucleic acid (RNA) was prepared by extraction of the ribosomal fraction with alcohol. This RNA preparation was as effective in producing an immune response as the ribosomal fraction from which it was prepared, unless the RNA was partially or completely degraded during the preparation. The effect of ribonuclease on the immunogenicity of the RNA was similar to that obtained with the ribosomal fractions, except that ribonuclease completely destroyed the immunogenicity of a partially degraded RNA. RNA appears to be an essential part of an immunizing substance in attenuated tubercle bacilli, which produces a high degree of immunity in mice; 50 [mu]g (dry weight) will protect approximately 80% of the mice, and as little as 0.5 [mu]g will protect approximately 30% of the mice. Mycobacterial RNA not incorporated in Freund''s incomplete adjuvant was nonimmunogenic. Yeast RNA incorporated in Freund''s incomplete adjuvant was not immunogenic.