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Several emulsified and two nonemulsified incomplete adjuvants were examined for their adjuvant activity by use of mycobacterial ribosomal fractions as a substrate. A good adjuvant is defined as one which produces a high immunological response with the ribosomal fraction in mice to infection with virulent tubercle bacilli. Freund's incomplete adjuvant, consisting of Aquaphor and heavy mineral oil, and Arlacel A plus hexadecane were the best adjuvants tested. Aquaphor plus light mineral oil and Arlacel A plus 7-n-hexyloctadecane were not quite as effective. Peanut oil was not satisfactory when emulsified with either Aquaphor or Arlacel A. A moderate degree of immunity was produced in mice vaccinated with ribosomal fraction mixed with aluminum hydroxide gel. Sodium alginate mixed with ribosomal fraction produced a low degree of immunity only with the highest vaccinating dose. It was found that the effectiveness of the emulsified type of adjuvant depended upon the method of preparation. Careful standardization of technique to produce uniform and complete emulsification was essential for maximal adjuvant activity using minimal vaccinating doses. A rapid and practical method of preparing emulsified adjuvants is given. The mode of action of incomplete adjuvants as employed in these experiments is discussed, and it is thought that they acted primarily by protecting the ribosomes from being inactivated by host ribonuclease before they were engulfed by the macrophages.

Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Nature of the labile immunogenic substance in the particulate fraction isolated from Mycobacterium tuberculosis. J. Bacteriol. 88:1030-1037. 1964.-Deoxyribonuclease had no effect on the immunogenic activity of the labile particulate fraction isolated from ruptured viable cells of the H37Ra strain of Mycobacterium tuberculosis, but decreased the ropiness of the ruptured cellular mass. Ribonuclease, in a high concentration, decreased the immunogenic activity slightly. Addition of yeast ribonucleic acid to particulate fraction incubated at 37 C prevented the decrease in immunogenic activity which normally occurs at this temperature, suggesting that endogenous ribonuclease may be involved in the reduction of activity. Differential centrifugation by the use of Brodie's (1962) method showed that the particles which sedimented at 56,550 x g were immunogenically active. Experiments were done to determine whether the integrity of the structure of the particle was necessary for immunogenic activity. It was found that sonic oscillation, freezing and thawing several times, the addition of surface-active agents (sodium lauryl sulfate or deoxycholate), and preparation of the particulate fraction in hypotonic solutions either decreased or destroyed immunogenic activity. This strengthens the evidence that a structural unit is necessary for activity. In addition, both a waxy sediment and the smallest particles which sedimented only at 144,000 x g were highly immunogenic if incorporated into Freund's incomplete adjuvant. In the absence of adjuvant, neither produced any immunity.

Guinea pigs were injected subcutaneously with mycobacterial ribosomal fraction incorporated in Freund's incomplete adjuvant and tested 6 and 12 weeks later by the intradermal injection of 0.5 mug (25 TU) of Purified Protein Derivative. No evidence of delayed-type hypersensitivity could be detected in these animals, although large necrotic reactions were obtained in guinea pigs sensitized with living, attenuated mycobacterial cells. Mice also were vaccinated by the intraperitoneal injection of mycobacterial ribosomal fraction or ribonucleic acid (RNA) and tested for sensitivity to tuberculin at various subsequent times. No evidence of true tuberculin hypersensitivity could be detected at any time, although what appeared to be small Arthus type reactions were seen in mice given the largest vaccinating doses. Attempts to recall tuberculin sensitivity in vaccinated mice by the intravenous injection, 4 weeks after vaccination of living cells, of either the virulent or attenuated mycobacterial strains were unsuccessful. Instead, when the virulent cells were injected, a suppression of footpad reactivity was noted in animals made sensitive to tuberculin by the previous intraperitoneal injection of viable attenuated mycobacterial cells. Both guinea pigs and mice, vaccinated as described above, were also skin tested or footpad tested, respectively, with 2 mug of the ribosomal fraction or RNA used for vaccination. No evidence of true tuberculin hypersensitivity could be obtained; instead, in guinea pig skin very small dermonecrotic areas were noted, and in mice swelling and redness of the footpad occurred to an equal extent in both vaccinated and nonvaccinated mice. The possible role of tuberculin hypersensitivity in acquired immunity to tuberculosis is discussed, and the conclusion is reached that its part, if any, is minor.

The relationship of the dose of vaccine to the immune response was determined in CF-1 mice vaccinated intraperitoneally with viable cells of the attenuated H37Ra strain of Mycobacterium tuberculosis and in mice vaccinated with cells of the same strain killed by autoclaving at 121 C for 15 min. The results showed, in terms of increased resistance to tuberculous infection, that the immune response with both living and killed cells was dependent upon the dose of vaccine, whereas only the living cells were dependent upon the time of challenge after vaccination. The dose response curves show dramatically that viable cells, which do not multiply in vivo, are several hundred times more effective immunizing agents against tuberculous infection than are autoclaved cells. Viable 2-week-old H37Ra cells were far more immunogenic than viable 4-week-old cells. Autoclaved 2-week-old cells, however, were no more immunogenic than autoclaved 4-week-old cells. H37Ra cells killed by boiling (98 C), exposure to 65 C for 30 min, treating with 2% phenol, or by being dried with acetone also lost most of their capacity to immunize mice. The effect of adjuvant on the immune response of mice to tuberculous infection was tested by incorporating both viable and autoclaved cells in Freund's incomplete adjuvant. We found that this vehicle had little or no effect on the immunizing capacity of either viable or heat-killed mycobacterial cells. The relationship of all the findings to the specificity of the immune response to tuberculosis is discussed.

Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Effect of mitochondrial stabilizers on the immunogenicity of the particulate fraction isolated from Mycobacterium tuberculosis. J. Bacteriol. 87:1346-1354. 1964.-A number of substances which have been used to stabilize mammalian mitochondrial preparations were tested to determine whether they would similarly affect the immunogenicity of a particulate fraction prepared from ruptured viable attenuated mycobacterial cells. The use of 0.44 m sucrose and the presence of 3 x 10(-2)m MgCl(2) during the preparatory processes markedly increased the immunogenicity of the particulate fraction. The increase was so great that immunogenic preparations were then consistently obtained which, in adequate dosage, were more immunogenic in CF-1 male mice than were viable attenuated mycobacterial cells. On the other hand, adenosine triphosphate (ATP), citrate, and polyvinylpyrrolidone when present during the preparatory processes reduced the immunogenicity. The addition of MgCl(2), ethylene-diaminetetraacetate, or ATP to the particulate fraction after it had been prepared did not increase its immunogenicity. When the particles were prepared in the 0.44 m sucrose buffer alone, incorporated in Freund's adjuvant, and injected intraperitoneally, immunogenicity was increased. However, this increase was not significantly greater than that obtained when the particles were prepared in the sucrose buffer containing MgCl(2). The immune state engendered in mice by the intraperitoneal injection of the particulate fraction persisted for at least 12 weeks.

Youmans, Anne S. (Northwestern University Medical School, Chicago, Ill.), and Guy P. Youmans. Effect of trypsin and ribonuclease on the immunogenic activity of ribosomes and ribonucleic acid isolated from Myobacterium tuberculosis. J. Bacteriol. 91:2146-2154. 1966.-The ribosomal fraction of the attenuated strain, H37Ra, of Mycobacterium tuberculosis was treated with trypsin alone, ethylenediaminetetraacetic 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 mug (dry weight) will protect approximately 80% of the mice, and as little as 0.5 mug 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.

The migration inhibition technique has been used to study delayed hypersensitivity in vitro by using peritoneal exudate cells and splenic lymphocytes from mice vaccinated with viable cells of the attenuated H37Ra strain of Mycobacterium tuberculosis and from mice vaccinated with ribonucleic acid (myc RNA) preparations obtained from viable mycobacterial cells of the same strain. Inhibition of macrophage migration was noted when purified protein derivative (PPD) or viable H37Ra cells were added to peritoneal exudate cells obtained from mice immunized with viable H37Ra cells and not from mice immunized with myc RNA. Splenic lymphocyte cultures were exposed to the same antigens in vitro. Filtered supernatant fluids from these lymphocyte cultures, when added to peritoneal exudate cells obtained from nonimmunized mice, inhibited migration only when they were obtained from lymphocytes which came from mice immunized with viable H37Ra cells. Injection of PPD intravenously into vaccinated mice resulted in inhibitory supernatant fluids from splenic lymphocyte cultures only when the lymphocytes came from mice immunized with viable H37Ra cells. However, intravenous injection of either viable H37Ra cells or of myc RNA preparations into mice vaccinated with myc RNA occasionally produced inhibitory supernatant fluids when lymphocytes were obtained from these mice. On the other hand, mice vaccinated with myc RNA or viable H37Ra cell preparations were consistently and equally protected against intravenous challenge with the virulent H37Rv strain. Thus, although some evidence was obtained for a delayed type hypersensitivity in mice vaccinated with H37Ra cells or with myc RNA to ribosomal proteins or other proteins associated with the RNA preparation, no evidence of tuberculin hypersensitivity could be detected in any mice vaccinated with the myc RNA. These results argue against a role for tuberculin hypersensitivity in immunity to tuberculous infection.

Acquired cellular immunity to infection with Mycobacterium tuberculosis is believed to reside in the capacity of mononuclear phagocytes of immunized animals to inhibit intracellular multiplication of the parasite. However, in macrophage tissue culture systems, it has been customary to employ streptomycin in the medium for the purpose of restricting extracellular, but not intracellular, growth of M. tuberculosis. In contrast, our data show that small amounts of streptomycin markedly inhibit intracellular as well as extracellular growth of M. tuberculosis in normal mouse peritoneal macrophages, and that the degree of this inhibition is directly proportional to the concentration of streptomycin used. In the absence of streptomycin, virulent tubercle bacilli grew as rapidly in "immune" macrophages as in normal macrophages. "Immune" macrophages, however, were slightly more resistant to destruction by the intracellularly multiplying mycobacteria. In the presence of streptomycin, however, intracellular mycobacterial growth was inhibited more in "immune" macrophages than in normal macrophages, and this effect also was directly proportional to the concentration of streptomycin used. Virulent mycobacteria grew somewhat more slowly within mouse peritoneal macrophages obtained after induction of a peritoneal exudate with glycogen than in noninduced cells. The rate of multiplication, though, was the same within normal and "immune" induced peritoneal cells except in the presence of streptomycin. As with noninduced macrophages, this drug inhibited the intracellular multiplication of virulent tubercle bacilli more effectively within "immune" induced than within normal induced cells. It would appear, therefore, that the greater inhibition of intracellular multiplication of virulent tubercle bacilli in "immune" macrophages in tissue culture noted by a number of investigators in the past may have been an artifact created by the use of streptomycin in the tissue culture medium.

Mice were immunized intraperitoneally with 5.0 mg of living and 5.0 mg of heat-killed H37Ra cells of the attenuated strain Mycobacterium tuberculosis and challenged intraperitoneally with Listeria monocytogenes and Klebsiella pneumoniae. The period of protection provided by the living and heat-killed H37Ra cells against both heterologous infections was the same. When mice were immunized intraperitoneally with graded doses of living and heat-killed H37Ra and challenged intraperitoneally with listeria or klebsiella, the lowest immunizing dose providing protection against both klebsiella and listeria challenge was the same for living and heat-killed cells. Living and heat-killed cells also immunized equally effectively when the routes of immunization and challenge were different. Mice also were immunized intraperitoneally with mycobacterial ribosomal fraction, mycobacterial cell walls, and several nonspecific agents (Escherichia coli endotoxin, mineral oil emulsion, and Freund's incomplete adjuvant). The mice were challenged intraperitoneally with listeria or klebsiella at varying times after immunization. The mycobacterial components and all the nonspecific agents provided transitory protection lasting no longer than 4 days after immunization. Only the mycobacterial cell walls and the endotoxin provided protection against listeria challenge. It was concluded that the protection provided by the mycobacterial ribosomal fraction is specific for tuberculosis infection, since this fraction provided no protection against listeria infection and only transitory protection against klebsiella. It was also concluded that the mycobacterial component providing protection against heterologous infections is heat stable and probably is found in the cell wall.

The synthetic polyribonucleotides adenylic acid (poly A), uridylic acid (poly U), cytidylic acid (poly C), and inosinic acid (poly I), whether single- or double-stranded (poly A:U, poly I:C), cannot replace mycobacterial ribonucleic acid (RNA) in the production of a high immune response in CF-1 mice against tuberculous disease. These conclusions are based on the results of several types of experiments. (i) Poly A and poly U, used either singly or in combination, did not increase the immunogenicity of mycobacterial RNA preparations whether emulsified in Freund's incomplete adjuvant (FIA) or not emulsified. (ii) Mycobacterial ribosomal protein, extracted with 2-chloroethanol, was not immunogenic; the addition of poly A:U to the protein did not produce an immune response and FIA did not affect these results. (iii) The RNA left after the protein was extracted was partially immunogenic when emulsified in FIA even though it was partially degraded. (iv) Mycobacterial RNA prepared with ethyl alcohol and partially degraded with ribonuclease had a significantly lower immunogenic activity, and the original higher immune response was not restored by the addition of poly A:U. (v) Mycobacterial RNA totally degraded by weak alkali was not immunogenic, the original immunogenic activity was not restored by the addition of poly A:U or poly I:C, and FIA again did not influence the results. These findings suggest that (i) protein, polypeptides, or other antigenic fragments, if present, are not the specific immunogens; and (ii) mycobacterial RNA is responsible for the high immunogenic activity of mycobacterial ribosomal and RNA preparations. In addition, since the double-stranded forms of these synthetic polynucleotides markedly potentiate the formation of circulating antibodies, these results also reemphasize the lack of correlation between conventional antibody formation and immunity against tuberculosis.

Youmans, Guy P. (Northwestern University Medical School, Chicago, Ill.) and Anne S. Youmans. Effect of mycosuppressin on the course of experimental tuberculosis in mice. J. Bacteriol. 84:701-707. 1962.-When mycosuppressin was administered to normal mice prior to infection with virulent tubercle bacilli, no indication of a favorable effect on the course of the infection could be obtained. However, when suspensions of virulent tubercle bacilli were exposed to mycosuppressin for from 1.25 to 3.25 hr prior to infection, survival of the mice was greatly prolonged in comparison with mice receiving untreated tubercle bacilli. Microbial enumeration studies with bovine serum agar medium revealed that the number of viable particles of tubercle bacilli in the mycosuppressin-treated suspensions did not differ significantly from the number in untreated suspensions. When the microbial enumeration studies were conducted using the same medium without serum, the number of viable particles in the mycosuppressin-treated suspensions appeared to be (1/5) to 1/20 of the number in the untreated suspensions. It was concluded that, under the conditions of these experiments, the action of mycosuppressin was primarily bacteriostatic and that this bacteriostasis persisted for a time in vivo following infection of the mice. The possible relation of these findings to the phenomenon of acquired immunity to tuberculosis also is discussed.