Immunogenic Capacity of Ribosomes of Salmonella typhi

INFECTION AND IMMUNITY, Apr. 1978, p. 161-166

Vol. 20, No. 1

0019-9567/78/0020-0161$02.00/0 Copyright © 1978 American Society for Microbiology

Printed in U.S.A.

Immunogenic Capacity of Ribosomes of Salmonella typhi Interfered with by a Flagellin-Like Material Contaminant GRACIELA COFRE, INgS CALDERON, AND GUIDO C. MORA* Laboratorio de Micro biologia, Instituto de Ciencias Biol6gicas, Universidad Cat6lica de Chile, Santiago, Chile Received for publication 28 November 1977

The double-immunodiffusion technique and sodium dodecyl sulfate-polyacrylamide electrophoresis were used to demonstrate the presence of flagellin-like material strongly attached to ribosomes of Salmonella typhi Ty 2. This flagellinlike material contaminating the ribosome preparation interferes with the induction of antiribosome serum, promoting the formation of antisera reacting either only with flagellin or in some cases with flagellin and ribosomes, but giving a very weak reaction with the latter. The interference is also observed when purified ribosomes from a nonflagellated mutant of S. typhi (S. typhi 0-901) mixed with purified S. typhi Ty 2 flagellin are utilized as antigens. The antiribosome sera obtained with ribosomes from S. typhi 0-901 have a considerably higher titer than those that are interfered with. These sera were able to react with ribosomes obtained from several related species and did not react with flagella-derived flagellin of S. typhi Ty 2.

properties of S. typhi Ty 2 as a potential agent to obtain protection against typhoid fever. Therefore, it has been necessary to establish some properties of our ribosome preparations. In this paper, we show that strongly bound flagellin-like material is found as a contaminant in S. typhi Ty 2 purified ribosomes. This contaminant interferes with the immunogenic capacity of ribosomes inoculated into rabbits. The antisera thus induced have only antiflagellin activity. However, in some cases they have, in addition, a very weak antiribosome activity. This is in contrast to the high titer of the antiribosome sera elicited by flagellin-free ribosomes from S. typhi 0-901. The presence of flagellin is further confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis.

Previous reports have shown that bacterial ribosomes are excellent antigens and can induce protection against bacterial infections. Youmans and Youmans (32, 34) were the first to study the immunogenic activity of the ribosomal fraction, and they have shown that when ribosomes obtained from Mycobacterium tuberculosis are injected into mice, they protect them against postinfection with live tubercle bacillus. This finding was followed by other studies on the protective capacity of ribosomes of bacterial species such as: Staphylococcus aureus (31), Pseudomonas aeruginosa (24, 30), Diplococcus pneumoniae (26), Neisseria meningitidis (25),

Salmonella typhimurium (7, 10, 11, 15, 16, 19, 23, 27), Escherichia coli (20), Brucella abortus (1), Streptococcus pyogenes (22), Haemophilus influenzae (14), and Salmonella typhi Ty 2 (17, 18). Studies to find out which are the immunogenic determinants of the ribosome were also carried out. Youmans and Youmans (34-36) have found that ribonucleic acid (RNA) is responsible for the immunogenicity of the ribosomes, but Johnson (10, 11) has found a protein involved in the immunity process. Finally, Smith and Bigley (23) have suggested that an RNA-protein complex is the effective immunogen. More recently, Hoops et al. (6) have reported an extrinsic factor contaminating the ribosomes of S. typhimurium, which could be the immunogenic determinant in these preparations. We have been interested in the immunogenic

MATERIALS AND METHODS Bacterial strains. S. typhi Ty 2 and S. typhi 0901 (nonflagellated mutant) were a kind gift from the

Instituto Bacteriol6gico de Chile. S. typhimurium, S. gallinarum, and E. coli B were from our strain collection.

Ribosomal preparation. Bacteria were grown for 18 h in Roux bottles containing 400 ml of nutrient agar composed of: yeast extract, 5.0 g; peptone, 10.0 g; glucose, 10.0 g; sodium citrate, 8.5 g; sodium thiosulfate, 8.5 g; agar, 17.5 g; and water to 1,000 ml. The cells were collected and washed four times in POM buffer (0.02 M phosphate buffer-0.01 M MgCI2 [pH 7.0]) (11), resuspended in 15 ml of POM buffer, and disrupted at 8,000 lb/in2 in a French press. The

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COFRE, CALDERON, AND MORA

ribosomes were obtained according to the procedure of Kurland (12), using POM buffer instead of TSM buffer [0.01 M tris(hydroxymethyl)aminomethane-0.003 M succinic acid-0.01 M MgCl2 (pH 8.0)]. Chemical assays. Protein was determined by the method of Lowry et al. (4). RNA was determined by the Orcinol reaction (2). RNA from yeast and bovine serum albumin from BDH biochemicals (British Drug Houses, Ltd.) were used as standards. Flagellin preparation. Flagellin was obtained from S. typhi Ty 2 according to the procedure of Nossal and Ada (21), who used a mild agitation of the bacteria and acidification of the flagellar suspension with 0.1 N HCl to yield flagellin. Animals. New Zealand female rabbits of approximately 2 kg in weight were used to produce antisera. Animals with natural titer against the bacterial strains used were discarded. Immunizations. Groups of three rabbits each were immunized by five to six injections of ribosomes containing the equivalent of 3 to 4 mg of proteins each. The injections were given every 10 days, and the rabbits were bled by cardiac puncture 30 days after the last injection. The two first injections consisted of 0.5 ml of ribosomes and 0.5 ml of complete Freund adjuvant (Calbiochem; Perrin's modification) injected in the following way: 0.2 ml through the left footpad, 0.2 through the right footpad, and 0.3 ml intramuscularly in each leg. The remaining injections (three or four) consisted of 0.5 ml of ribosomes, which were given intradermally through four abdominal areas. Trial bleedings (5 ml each) were performed by cardiac puncture every 15 days. The clot formed at room temperature was then separated by centrifugation at 3,000 rpm for 15 min in a Sorvall SS-34 rotor. Sodium azide (0.02%, wt/vol) was added to the sera and stored at 4°C. Agglutination and the immunodiffusion test were used to determine serum activity. The antiflagellin sera were obtained by the same schedule explained above, injecting 0.6 mg of flagellin each time. Sera were also obtained against mixtures of purified S. typhi 0-901 ribosomes and S. typhi Ty 2 flagellin. Three groups, A, B, and C, of two rabbits each were immunized with mixtures containing ribosomes (equivalent to 4.2 mg of proteins) with 0.08, 0.4, and 0.8 mg of purified flagellin, respectively. The immunization schedule described above was followed. Agar double-diffusion test. Antisera activity was tested by the micromethod of Hirschfeld (5). The agar was prepared with 2% (wt/vol) agarose in distilled water, heated to 100°C for 10 min, and cooled to 550C. Then 1 volume of POM buffer (twofold concentrated) at 550C was added, 3 ml of this solution was layered on a glass microscopic slide (75 by 25 mm), and the agar was allowed to spread uniformly over the slides. After the agar hardened, a central well with six peripheral wells of 30-,lI capacity, whose edges are 3 mm apart, were punched with a template. All the slides were incubated in a humid chamber for 24 h at 40C. Antiserum titration. Sera were titrated essentially as described by Williams (29), using the agar double-diffusion assay. The central well contained 30 ,ul (1 mg/ml) of antigen, either ribosome or flagellin,

and the six peripheral wells contained 30 ,d of increasing twofold serial dilutions of the sera, ranging from 1 to 1:32,768. Absorption experiments. Anti-S. typhi Ty 2 ribosome sera were successively absorbed with 0.6% Formalin-treated S. typhi Ty 2 bacteria. Two milliliters of serum mixed with 2 ml of a heavy suspension of bacteria was incubated at 370C for 6 h and left at 4VC overnight. After centrifugation of the suspension obtained, the pellet was discarded and the supernatant was tested by agglutination assays with S. typhi Ty 2 bacteria. Four to five absorptions were done before the serum failed to agglutinate the bacteria. The diluted sera, concentrated by lyophilization and dissolved in 2 ml of saline solution (0.8% NaCl), was assayed by using the agar double-diffusion test against flagella-derived flagellin and S. typhi Ty 2 ribosomes. SDS-polyacrylamide slab gel electrophoresis. The gels were run according to the method of Laemmli (13). The ribosome samples were mixed and denatured at 90°C for 2 to 3 min with a solution containing: tris(hydroxymethyl)aminomethane hydrochloride, 0.0625 M (pH 6.8); glycerol, 15%; 2-mercaptoethanol, 5%; SDS, 5%. Then, 1 lg per protein band was used to load the gels. RESULTS

Characterization of the ribosomes. Ribosomes obtained according to the method of Kurland (12) were characterized by determination of their RNA and protein content. The values found were about 60% RNA and 40% proteins, similar to values reported previously (12). These ribosomes were routinely centrifuged in sucrose gradients and analyzed in an ISCO apparatus at 254 n. An absorbance profile corresponding to that of a monosome was found when a 10 to 30% sucrose gradient in POM buffer was used. Agglutination assays. Antiribosome serum (AsR Ty 2) was prepared from rabbits whose sensitivity was checked before the first injection with ribosomes. Then it was assayed against the O and H antigens (Difco) of S. typhi Ty 2. The reactions were negative and positive, respectively. These results gave us the first indication of contamination of our ribosomal preparations with flagellar antigen (Table 1). Double-immunodiffusion assays. (i) Antiribosome serum, S. typhi Ty 2 (AsR Ty 2). AsR Ty 2 was assayed against its own antigen (R Ty 2) and also against ribosome preparations obtained from the following strains: S. typhi 0TABLE 1. Antiribosome serum versus S. typhi Ty 2 0 and H antigens Antiribosome serum

0 antigen

H antigen

AsO

+

-

AsH AsR Ty 2

-

+ +

-

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901 (R Ty 0-901), nonflagellated mutant; S. gallinarum (R-G); S. typhimurium (R-Tym); and E. coli B (R-C). A positive reaction was obtained with ribosomes of S. typhi Ty 2 and with flagellin derived from S. typhi Ty 2 flagella. The precipitation bands were identical (Fig. 1). To eliminate the contaminating flagellin-like material, ribosomes from S. typhi Ty 2 cells, which had previously lost most of the flagella by shaking and washing, were isolated. These efforts were useless, since a positive reaction was always found between these ribosomes and AsR Ty 2. The precipitation line that appeared was identical to lines formed by AsR Ty 2 versus R Ty 2 and AsR Ty 2 versus flagellin (Fig. 2). These results suggest that there is a common antigen in the preparations of flagella-derived flagellin and ribosomes prepared from S. typhi Ty 2. In contrast, this antigen is absent in ribosomes prepared from S. typhi 0-901 and the other related species tested. FIG. 2. Gel diffusion pattern showing the persist(ii) Antiribosome serum, S. typhi 0-901 (AsR 0-901). It was necessary to prepare an ent contamination of ribosomes ofS. typhi Ty 2 deflamechanically. Ribosomal S. typhi Ty 2 anantiserum against ribosomes of the nonflagel- gellated is in the central well. The outside tiserum (AsR lated mutant of S. typhi, 0-901. This AsR 0-901 wells contain Ty-2) flagellin (F) and ribosomes of S. typhi antiserum, assayed by immunodiffusion, reacted Ty 2 (Ty-2), S. typhi 0-901 (0-901), S. typhimurium positively with the following ribosome prepara- (Tym), E. coli (C), and S. typhi Ty 2 deflagellated tions: R 0-901, R-G, R-Tym, R-C, and R Ty 2 mechanically (D). (Fig. 3). As expected, AsR 0-901 reacted negatively with flagellin (Fig. 3). The precipitation

FIG. 1. Gel diffusion pattern showing the contamination of S. typhi Ty 2 ribosomes by flagellin of the same origin. Ribosomal S. typhi Ty 2 antiserum (AsR Ty-2) is in the central well. The outside wells contain flagellin (F) and ribosomes of: S. typhi Ty 2 (Ty-2), S. typhi 0-901 (0-901), S. gallinarum (G), S. typhimurium (Tym), and E. coli (C).

FIG. 3. Gel diffusion test of ribosomal S. typhi 0901 antiserum (AsR 0-901, central well). The outside wells contain flagellin (F) and ribosomes of: S. typhi Ty 2 (Ty-2), S. typhi 0-901 (0-901), S. gallinarum (G), S. typhimurium (Tym), and E. coli (C).

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lines formed with the ribosomal antigens tested were identical. (iii) Antiflagellin serum, S. typhi Ty-2 (AsF). This serum, prepared with flagella-derived flagellin of S. typhi Ty 2, gave a positive reaction with flagellin, the R Ty 2, and S. typhi Ty 2 ribosomes isolated from cells that had lost their flagella by shaking the cultures. All three bands obtained were identical. No reaction was seen between AsF and the ribosome preparations R 0-901, R-G, R-Tym, and R-C. It is evident from our results that ribosomes contaminated with flagellin have induced antisera that are mainly antiflagellin. Somehow, flagellin interferes with the immune response of the ribosome itself. However, it must be stated that in some of the sera, a weak antiribosome activity was found in the undiluted preparations. The immunogenic capacity of the ribosome is only evident when the antiserum is induced with ribosomes isolated from the nonflagellated mutant S. typhi 0-901. (iv) Antiserum against mixed S. typhi 0901 ribosomes and S. typhi Ty 2 flagellin (AsRF). To prove that flagellin interferes with the immunogenic response elicited by ribosomes, purified ribosomes were mixed with flagellin, and the mixture was used as antigen; Mixtures containing constant amounts of ribosomes (equivalent to 4.2 mg of proteins) were added with increasing amounts of flagellin (group A, 0.08 mg; group B, 0.4 mg; and group C, 0.8 mg) and injected into groups of two rabbits each. It has been assumed from SDS-polyacrylamide slab gels that contaminating flagellin is present in S. typhi Ty 2 ribosomes in an amount similar to that in other structural ribosomal proteins. Therefore, the amount of flagellin added to flagellin-free ribosomes corresponds to 1/50 of 4.2 mg, which is the amount of ribosomal proteins present in the mixtures, 50 being the approximate number of proteins present in the ribosome. Titration of the AsRF sera, as well as the control sera, is shown in Table 2. It can be seen that flagellin added to purified ribosomes produces interference and matches very closely the values obtained when purified ribosomes from the flagellated strain are used to produce the sera. The higher antiribosome titer is found in AsR 0-901 serum, which is produced by the purified flagellin-free ribosomes. On the other hand, the higher antiflagellin titer is found in AsF serum, which is produced by flagella-derived flagellin. The six sera elicited by the ribosome-flagellin mixtures gave the same titers. SDS-Polyacrylamide slab gels. Electro-

INFFCT. IMMUN.

phoresis of the ribosomal proteins confirmed the presence of flagellin in our ribosomal preparations. A band was obtained (Fig. 4) which migrates in the same position as the flagella-derived flagellin included as a control in the same

slab.

TABLE 2. Antiserum titers tested by twofold dilutions against flagellin-free ribosomes and

flagellina

.ntiribosom

serum

Antiribosome ac-

tivity

Antiflagellin

activity

AsRF (A) 1 1:8 AsRF (B) 1 1:8 AsRF (C) 1 1:8 0-1 AsRTy 2 1:8 AsR 0-901 1:16 AsF 1:16 a Antisera titration by the agar double-diffusion assay. AsRF corresponds to the sera induced by a mixture of ribosome and flagellin. Three types of mixtures (A, B, and C) were used. AsR Ty 2 and AsR 0-901 are the sera induced by flagellin-contaminated ribosomes and flagellin-free ribosomes, respectively. AsF is the serum induced by flagellin.

123 FIG. 4. SDS-polyacrylamide gel electrophoresis patterns of S. typhi Ty 2 flagellin (gels 1 and 3) and ribosomal proteins (gel 2).

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IMMUNOGENIC CAPACITY OF RIBOSOMES

The electrophoretic pattern of S. typhi 0-901 ribosomal proteins does not show the homologous flagellin band. The ribosomal protein patterns of S. typhi Ty 2 and S. typhi 0-901 differ not only in the flagellin band, but also in a few others that we think are related to the flagellin-like material present. The protein pattern of strain 0-901 shows no difference from one obtained with E. coli B ribosomal proteins. Absorption experiments. Successive absorptions of AsR Ty 2 with Formalin-treated S. typhi Ty 2 bacteria were performed until no reaction of the serum against these bacteria was evident. Then, the absorbed sera were lyophilized and dissolved in 2 ml of saline solution. Titration of the absorbed AsR Ty 2 against S. typhi 0-901 ribosomes gave either a weak positive reaction with the undiluted serum or no reaction at all. Titration against flagellin failed to give any reaction.

DISCUSSION Contamination of S. typhi Ty 2 ribosomes by flagellin-like material, strongly suggested by the double-immunodiffusion technique, was confirmed by SDS-polyacrylamide slab gel electrophoresis of the ribosomal proteins. Ribosomes of the nonflagellated mutant of S. typhi were not contaminated. Since flagellin is a basic protein with a molecular weight of approximately 40,000 (8), it is not very surprising to find it associated with ribosomes, but it is interesting to observe that this interaction is stable throughout the drastic washing process of ribosomes which we used to isolate them. We found that when these flagellin-contaminated ribosomes were injected into rabbits, an antiserum that was mainly antiflagellin was obtained and a weak antiribosomal reaction, if any, was detected. Therefore, under our experimental conditions we observed an interference of the immunogenic capacity of the ribosomes, which is further confirmed by the use of in vitromade mixtures of purified ribosomes and flagellin to produce the antisera. These mixture-induced sera have a considerable lower antiribosome titer than the sera induced by essentially equal amounts of flagellin-free ribosomes. Contamination of ribosomal preparations by extrinsic factors such as 0 antigen (3), lipids, carbohydrates, and proteins (9), and other nonchemically defined components (6) has been reported. From our results it seems that the 0 antigen is not present serologically in our preparations. Care must be taken when ribosomes are used as vaccines, since one or more contaminants

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might be real immunogens masking the immunogenic capacity of the ribosome itself. However, it may be possible that the ribosome-extrinsic factor(s) association constitutes an efficient immunogenic unit capable of eliciting better protection against bacterial infection. ACKNOWLEDGMENTS S. typhi Ty 2, S. typhi 0-901, 0 antiserum, and H antiserum were a kind gift of D. Castillo, A. Contreras, and D. Pinto, Instituto Bacteriol6gico de Chile. We specially thank D. Castillo for his advice and interest in this project. We also thank A. De Ioannes and A. Yudelevich for the critical reading of this manuscript. This research was supported by grant 200-74 from the Direcci6n de Investigaciones, Universidad Cat6lica de Chile. LITERATURE CITED 1. Corbel, M. J. 1976. The immunogenic activity of ribosomal fractions derived from Brucella abortus. J. Hyg. 76:65-74. 2. Dische, Z. 1955. Color reactions of nucleic acid components, p. 285-305. In E. Chargaff and J. N. Davidson (ed.), Nucleic acids, vol. 1. Academic Press Inc., New York. 3. Eisenstein, T. K. 1975. Evidence for 0 antigens as the antigenic determinants in "ribosomal" vaccines prepared from Salmonella. Infect. Immun. 12:364-377. 4. Hartree, E. F. 1972. Determination of protein: a modification of the Lowry Method that gives a linear photometric response. Anal. Biochem. 48:422-427. 5. Hirschfeld, J. 1963. Double diffusion in plates. Micromethods. Methods Immunol. Immunochem. 3:152-154. 6. Hoops, P., N. E. Prather, L. J. Berry, and J. M. Ravel. 1976. Evidence for an extrinsic immunogen in effective ribosomal vaccines from Salmonella typhimurium. Infect. Immun. 13:1184-1192. 7. Houchens, D. P., and G. L. Wright, Jr. 1973. Immunity to Salmonella typhimurium infection: characterization of antigens in active protection by polyacrylamide gel electrophoresis. Infect. Immun. 7:507-511. 8. Iino, T. 1969. Genetics and chemistry of bacterial flagella. Bacteriol. Rev. 33:454-475. 9. Jensen, R. B., G. J. Naylor, and P. Actor. 1972. Isolation of protective somatic antigens from Vibrio cholerae (Ogawa) ribosomal preparations. Infect. Immun. 6:156-161. 10. Johson, W. 1972. Ribosomal vaccines. I. Immunogenicity of ribosomal fractions isolated from Salmonella typhimurium and Yersinia pestis. Infect. Immun. 3:947-952. 11. Johnson, W. 1973. Ribosomal vaccines. II. Specificity of the immune response to ribonucleic acid and protein isolated from Salmonella typhimurium. Infect. Immun. 8:395-400. 12. Kurland, C. G. 1966. The requirements for specific sRNA binding by ribosomes. J. Mol. Biol. 18:90-108. 13. Laeummli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 14. Lynn, M., R. P. Tewari, and M. Solotorovsky. 1977. Immunoprotective activity of ribosomes from Haemophilus influenzae. Infect. Immun. 15:453-460. 15. Margolis, J. M., and N. J. Bigley. 1972. Cytophilic macroglobulin reactive with bacterial protein in mice immunized with ribonucleic acid-protein fractions of virulent Salmonella typhimurium. Infect. Immun. 6:390-397. 16. Medina, S., S. I. Vas, and H. G. Robson. 1975. Effect of nonspecific stimulation on the defense mechanism of inbred mice. J. Immunol. 114:1720-1725.

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