Immunogenic Activity of a Ribosomal Fraction Obtained from Mycobacterium tuberculosis

Abstract

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.

Binding Between Components of the Tubercle Bacillus and Humoral Antibodies

“…Because of the complex nature of this organism, many fractionation schemes have been devised. Intact cells and culture filtrates have been subjected to chemical fractionation (36)(37)(38)(39), and some components have been isolated by a variety of methods including enzyme digestion, electrophoresis, gel filtration, and chromatography (40)(41)(42)(43)(44)(45)(46)(47)(48). As a result, many fractions have been isolated and partially defined with respect to physical structure and biological properties, but none has been applied to development of a primary test to detect and measure humoral antibodies.…”

Isolation and Partial Characterization of an Immunogenic Moiety Obtained fromSalmonella typhimurium

“…Youmans (34,35) with slight modification. The bacterial suspension was homogenized for 30 sec in an ice-jacketed micro-Waring Blendor.…”

Bacterial crude extracts or ribosomes are recognized similarly by peripheral and mucosal B cells

“…Since the initial experiments of Youmans and Youmans [1], who demonstrated the immunogenic reactivity of ribosomal fractions from Mycobacterium tuberculosis, many studies have confirmed the potential role of such preparations as vaccines. In animal models, ribosomal preparations have been used for intramuscular or intravenous injection, later demonstrated to have induced significant raises in serum antibody levels or peripheral blood T cells reactivity [2].…”