Factors Affecting Immunogenic Activity of Mycobacterial Ribosomal and Ribonucleic Acid Preparations

Abstract

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.

Delayed Hypersensitivity Reactions Provoked by Ribosomes from Acid-Fast Bacilli I. Ribosomal Isolation, Characterization, Delayed Hypersensitivity, and Specificity

“…No membranes could be detected in preparations of ribosomes studied by electron microscopy. The RNA and protein contents of the ribosomes and ribosomal subunits isolated by the present procedures, as well as the yield obtained, are in agreement with those obtained by Youmans and Youmans (40,42) and Worcel et al (38). It is difficult to explain the differences obtained in the studies of Youmans and Youmans (41) and those herein reported.…”

Purification of Immunogenically Active Ribonucleic Acid Preparations of Salmonella typhimurium : Molecular-Sieve and Anion-Exchange Chromatography

“…These results suggest that the immunogenic moiety of the crude nucleic acid fraction is either RNA or an as yet undefined polysaccharide of greater than 300,000 molecular weight.Within the past 5 years, an increasing body of evidence has accumulated to indicate that bacterial subcellular preparations are effective in inducing resistance to several bacterial parasites. Youmans and Youmans have elegantly demonstrated that ribosomal and crude ethanol-precipitated ribonucleic acid (RNA) preparations of Mycobacterium tuberculosis induce an effective immune response in mice to challenge infection with the homologous organism (42)(43)(44)(45). In more recent work (46), they have further suggested that the immunogenic activity of the fractions may be associated with one particular species of nucleic acid.…”

Effect of Polybasic Amines on the Immunogenicity of Mycobacterial Ribonucleic Acid

“…Although a complex was formed between each of the compounds and mycRNA as denoted by the heavy precipitate which was formed, only slight adjuvant activity was shown. It may be that (i) the complexes are fairly labile in vivo; (ii) the polybasic amines may not have the capacity to protect mycRNA against the nucleases of the host, since it has been found that animal serum contains more potent enzymes which can degrade double-stranded RNA (2,3,28,30,37); or (iii) to be a good adjuvant, the material must be somewhat nonbiodegradable.…”

“…We have reported that mycobacterial ribonucleic acid (mycRNA) preparations can be as effective for immunization of mice against infection with Mycobacterium tuberculosis as the living attenuated cells of the H37Ra strain of M. tuberculosis from which they were prepared (33,34,37,38,41). This comparison was based on the amount of ribonucleic acid (RNA) present in each vaccine.…”

“…week-old cells were used because cells of this age are in the logarithmic growth phase and contain the maximum amount of RNA (36). The method of preparation and the chemical and physical characteristics of mycRNA have been given in detail elsewhere (37,38). The mycRNA was standardized by absorption at 260 nm with a Beckman DU-2 spectrophotometer and by the orcinol reaction of Dische as described by Ashwell (4), using mycRNA as a standard.…”