The highly immunogenic particulate fraction obtained from mechanically ruptured cells of the H37Ra strain of Mycobacterium tuberculosis was suspended and centrifuged at 20,360 X g. The supernatant liquid from this centrifugation was centrifuged at 56,550 X g to remove the larger particles, and the supernatant liquid from this was centrifuged at 144,000 X g to obtain a ribosomal fraction. The sediments from the first two centrifugations were highly immunogenic, but the ribosomal fraction showed only slight capacity to immunize mice. However, when the ribosomal fraction was mixed with Freund's incomplete adjuvant, the immunogenic activity was equivalent to the particulate fraction from which it was prepared. To test the hypothesis that some membranous substance in the particulate fraction was acting as an adjuvant for the smaller particles in the ribosomal fraction, portions of the particulate fraction were treated separately with each of the membrane-disrupting agents, sodium deoxycholate, sodium lauryl sulfate, and 1 M sodium chloride. The treated materials were then centrifuged at 144,000 X g, and the sediments were tested for immunogenicity both with and without the addition of Freund's incomplete adjuvant. Without the adjuvant, the immunizing activities were very weak or absent; with the adjuvant, they were equivalent to that of the particulate fraction from which they were prepared. Other factors which have been found to damage or destroy membranes, such as freezing and thawing, and heat, also significantly decreased the immunogenic activity of the particulate fraction unless it was incorporated into Freund's incomplete adjuvant. The larger particles which sedimented at 56,550 X g were also treated with sodium laurvl sulfate and sodium chloride. Again, immunogenicity was greatly reduced but was fully restored by use of Freund's incomplete adjuvant. The data suggest, then, that the immunizing component of the particulate fraction is a substance (ribosomal?) which sediments at 144,000 X g, but for maximal immunizing activity a labile, possibly membranous, moiety of the mycobacterial cell, which has the properties of an adjuvant, is required.

No abstract

AND GuY P. YOUMANS. Effect of trypsin and ribonuclease on the immunogenic activity of ribosomes and ribonucleic acid isolated from Myobacterium tuberculosis.

By following careful procedures, mycobacterial ribosomal fractions and ribonucleic acid (RNA) prepared by ethyl alcohol precipitation were obtained which have immunogenic activities similar to the viable attenuated H37Ra cells of Mycobacterium tuberculosis from which they were obtained. This comparison was based on the amount of ribonucleic acid (RNA) present. These preparations consisted of approximately 63% RNA and 37% protein; no deoxyribonucleic acid or polysaccharide was detected by chemical tests. A high correlation was found between the immunogenic activity of a preparation and the per cent increase in hyperchromicity at 260 nm of a ribonuclease-hydrolyzed portion. Final concentrations of sodium dodecyl sulfate higher than 0.25% when used for the preparation of the ribosomal fractions and RNA resulted in significantly lower immune responses and greater variation between experiments. This was not related to the amount of protein present. The stability of the ribosomal and RNA preparations was tested under a variety of conditions. The need for a good protective adjuvant again was shown since mouse serum readily hydrolyzed the RNA. Equal immunity was obtained after immunization by the intraperitoneal and subcutaneous routes; however, no immune response was obtained when the intravenous route was used. Preliminary results with RNA prepared with phenol showed that it was more easily degraded during preparation. This resulted in a lower immune response than was obtained with the RNA prepared with ethyl alcohol.

Ribosomal fractions of Mycobacterium tuberculosis, strain H37Ra, were prepared by treatment of the intracellular particulate fraction with 0.25 or 0.5% sodium dodecylsulfate (SDS) followed by centrifugation at 144,700 X g for 3 hr. This procedure has greatly simplified the preparation of ribosomal fractions and has given fractions composed of approximately 50% ribonucleic acid (RNA) and 15 to 20% protein. When incorporated into Freund's incomplete adjuvant and injected intraperitoneally into CF-1 mice, the SDS ribosomal fractions were more immunogenic than the particulate fractions from which they were prepared. They were as much as 100 times more immunogenic than ribosomal fractions prepared by differential centrifugation, 1 ,ug (dry weight) per mouse being sufficient for the induction of some immunity. However, none of these ribosomal preparations, in comparable doses, was as immunogenic as the living cells from which they were prepared. It was also shown that the addition of 10-4 M MgCI2 to the final diluent increased immunogenic activity, whereas larger concentrations (10-3 M) reduced immunogenic activity. Preparation of the ribosomal fraction from ruptured cells in one continuous process during the course of 1 day increased the activity. Twoweek-old H37Ra cells contained more RNA and were more immunogenic than the older cultures which have been used in the past.