INPECFION AND IMMUNITY, Nov. 1970, p. 659-668 Vol. 2, No. 5Copyright © 1970 American Society for Microbiology Printed In U.S.A.

Immunogenic Mycobacterial Ribosomal andRibonucleic Acid Preparations: Chemical

and Physical CharacteristicsANNE S. YOUMANS AND GUY P. YOUMANS

Department of Microbiology, Northwestern University Medical School, Chicago, Illinois 60611

Received for publication 2 March 1970

Five to 20% linear sucrose gradients were used to obtain sedimentation patternsof mycobacterial ribosomes, ribosomal subfractions, and ribonucleic acid (RNA)preparations. Classical 705 ribosomes were obtained when 10-' M magnesiumchloride was used. These, when dialyzed against 104 M MgCl2, yielded typical 50S,30S, and smaller ribosomal subunits. The 30S subunits were the most immunogenicunder these conditions. A ribosomal preparation containing subunits which variedfrom 2.5 to 40S was fractionated by collecting five fractions from a sucrose gradient;based upon the amount of nucleic acid present, the fraction containing the 40Sparticles was most immunogenic. Physical and chemical evidence suggested thatmycobacterial RNA preparations extracted with 65% ethyl alcohol from the ribo-somes and diluted in distilled water, were either double-stranded, or mostly double-helical, or had a highly organized secondary structure. This was based on the follow-ing observations. (i) Native RNA was resistant to trace amounts of ribonuclease.(ii) The approximate Tm value in SSC buffer (0.15 M NaCl plus 0.015 M sodiumcitrate) was greater than 85 C and in 0.1 SSC buffer was 55 C; the RNA diluted inSSC buffer produced a hypochromic effect on cooling at room temperature. (iii)Formaldehyde, in the presence of SSC buffer, decreased the Tm of the RNA toapproximately 55 C, and there was no hypochromic effect on cooling. (iv) Formalde-hyde did not increase the wavelength of maximal adsorption of the RNA. (v) Thepurine/pyrimidine ratio was close to one. (vi) The major peak of the RNA sedi-mented in the more dense zones of the sucrose gradients. There was a relationshipbetween the sedimentation pattern obtained with the RNA-protein subunits onsucrose gradients and immunogenicity; several examples are given. RNA-proteincomplexes of approximately 14 to 20S, and occasionally 23S in the major peak,appeared to produce the highest immune response. Smaller RNA-protein complexessuch as 6S, which were obtained when the RNA preparation was diluted in certainbuffers, were much less immunogenic. This was confirmed by collecting five frac-tions from sucrose gradients and finding the third fraction (containing RNA-proteincomplexes approximately 15 to 16S) the most immunogenic. Immunogenic activitywas apparently related to the structure of the RNA since it was maximal when theRNA appeared to be either double stranded, double helical, or had a highly or-ganized structure.

We have reported recently (41) that carefully of these to immunogenic activity. We will alsoprepared mycobacterial ribosomal and ribo- present evidence which indicates that highlynucleic acid (RNA) fractions can be obtained immunogenic mycobacterial RNA preparationswhich are as immunogenic, on a weight basis, contain nucleic acid which exhibits characteris-as the viable cells from which they were prepared. tics of double-stranded RNA, or, highly organizedA high correlation also was found between the double-helical secondary structures.quality of the RNA and immunogenic activity. In addition, we have included data showingIt is the purpose of the present paper to present that 70S particles can readily be isolated frommore of the chemical and physical characteristics mycobacterial particulate fractions, providedof these preparations and to try to relate certain adequate concentrations of magnesium chloride

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YOUMANS AND YOUMANS

are employed. It will also be shown that, whenthe concentration of magnesium chloride islowered, these 70S particles break up intothe classical 50 and 30S as well as smaller sub-units. Finally, we wish to stress that our concernin this paper with the characteristics of prepara-tions containing ribosomal subunits, rather thanwith 70S particles, is solely because the prepara-tions containing subunits have appeared moreimmunogenic (38). Our primary interest is incharacterizing immunogenic material, not inpreparing and characterizing such classical bac-terial structures as ribosomes.

MATERIALS AND METHODS

Mycobacterial cultures. The attenuated H37Rastrain of Mycobacteriwn tuberculosis was used as thesource of the ribosomal and RNA preparations.The highly virulent H37Rv strain of M. tuberculosiswas used to challenge mice. Both strains were main-tained, and the suspensions made, as described previ-ously (41).

Preparation of ribosomal subunits and RNA.These were done as described in detail previously(41). Briefly, the particulate fraction was obtained bydifferential centrifugation of ruptured viable H37Racells. A given concentration (50 mg/ml, wet weight)was added to an equal volume of 0.5% sodium dode-cyl sulfate (SDS) at room temperature. This wasrecentrifuged at 144,700 X g for 3 hr to sedimentthe ribosomes. To obtain 70S ribosomes withoutsome dissociation into 50 or 30S subunits, it wasnecessary to use 10-1 M MgCl2 in the sucrose bufferin which the cells were ruptured instead of the 10" MMgCl2 used by other investigators (7, 8, 37). The70S ribosomes were dialyzed overnight against asolution containing 10-4 M MgCl2. After dialysis,the material was recentrifuged at 144,000 X g tosediment the 50 and 30S particles. RNA was pre-pared from the ribosomal fraction by ethanol pre-cipitation, a modification of the method of Crest-field et al. (6).

Chemical and physical methods. The preparationswere standardized by measurement of absorbance at260 to 280 nm with a Beckman DU-2 spectrophotome-ter and by the orcinol method of Dische as discussedby Ashwell (1) by using mycobacterial RNA as thestandard. Protein was determined by the method ofLowry et al. (23) with crystalline bovine albumin asa standard. The quality of each preparation of RNAwas determined by the per cent increase in hyper-chromicity of a ribonuclease hydrolyzed portion, asdescribed earlier (41). The tests used to determinewhether the RNA was single or double stranded werebased primarily on those of Lampson et al. (19)and were as follows. (a) A small amount of ribo-nuclease (0.2 ,ug/20 ,ug of RNA) was used uponnative RNA and upon RNA which had been heatdenatured at 110 C for 7 to 8 min and then placedin ice to prevent reannealing of the strands. After theribonuclease was added to each of these two samples,and to single-stranded yeast RNA, absorption read-

ings at 260 nm were taken at 5-min intervals at roomtemperature to observe increase in hyperchromicity.(b) Three per cent formaldehyde was added to anequal volume (40 ,g/ml) of mycobacterial RNAand to yeast RNA and incubated at 35 C for 4 hr;controls were included in which water was addedinstead of formaldehyde. The absorption of eachwas measured at 5-nm intervals from 220 through280 nm. (c) The effect of formaldehyde, in a concen-tration of 2.76%, upon the thermal transition point(Tm) of the RNA preparation was determined. (d)The approximate Tm was also measured in twoionic concentrations of 0.15 M NaCl plus 0.015 Msodium citrate (SSC), pH 7.0, and 0.1 SSC.The base composition of the RNA was determined

by a modification of the paper chromatographymethod of Bendich, by using an isopropanol-hydro-chloride solvent system (3).

Sucrose gradients. Linear 5 to 20% sucrose gra-dients were prepared approximately 18 hr before useand refrigerated.Volumes of 0.4 and 0.5 ml containing 2.0 and 2.5

mg of ribosomal fraction and RNA, respectively,were layered upon the gradients. Alkaline phospha-tase, used as a point of reference, was contained in avolume of 0.1 ml and was layered on top of the ribo-somal and RNA preparations. Before use, the alka-line phosphatase was diluted 1:10 with 1.0 M this(hydroxymethyl)aminomethane (Tris) buffer, pH8.0, and dialyzed against Tris buffer for 24 hr toremove the ammonium sulfate present.

It was necessary to use 10-l M MgCI2 in the 5 to20% sucrose gradients to obtain 70S ribosomeswithout some dissociation into 50 and 30S subunits.After dialysis and recentrifugation at 144,000 X gfor 3 hr, the 50 and 30S subunits were centrifuged in5 to 20% sucrose gradients containing 104 M MgC12.The centrifugations were done for 1.5 hr in a SW-39Spinco rotor at 39,000 rev/min. Two markers wereused, alkaline phosphatase, and an 80S mammalianbrain ribosomal marker (20) (kindly supplied byTerry C. Johnson).

Since we had found that by using higher concen-trations of magnesium ions the particulate fractionswere not as immunogenic (38), smaller ribosomalsubunits were obtained by centrifugation of the ribo-somal preparations in sucrose gradients to which nomagnesium ions had been added.The ribosomal subunit preparations other than the

50 and 30S were centrifuged at 39,000 rev/min in aSpinco SW-39 rotor for 2 hr, and the RNA prepara-tions were spun for 4 hr. At the end of this time, thegradients were collected manually from the bottomby gravity flow by using a very short 20-gauge needleto puncture the bottom of the tube. Each 2-dropfraction was collected in 3 ml of distilled water, andthe adsorption of each was measured at 260 nm.Alkaline phosphatase was measured by using freshlyprepared 0.001 M p-nitrophenyl phosphate in 1.0 MTris buffer, pH 8.0. In the assay, 0.1 ml of sucrosegradient fraction was added to 3.0 ml substratemixture and read at 410 nm, after 10 min of incuba-tion at room temperature.The approximate S values of the ribosomal and

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RNA-protein complexes were determined by usingthe alkaline phosphatase as a standard which hadan S value of 6.3 (11). The approximate molecularweight of the RNA-protein complexes was determinedby use of the method of Martin and Ames (24).

Vaccines. All ribosomal and RNA-protein vaccineswere incorporated into Freund's incomplete adjuvant(Aquaphor mixed with extra heavy mineral oil)just before vaccination, as we have described previ-ously (40). Viable H37Ra cells, from which the ribo-somes, ribosomal subunits, and RNA-protein com-plexes were prepared were also injected into micein each experiment; a fine suspension of these micro-organisms was made by grinding 2-week-old pelliclesmechanically in a mortar with a pestle with 0.01 Mphosphate buffer, pH 7.0 (42). The largest vaccinatingdose of each vaccine, including the H37Ra cells,was made equivalent to 50,g of RNA. This wasequivalent to 1.0 mg, moist weight, of both the H37Racells and the ribosomal fractions.

Animal experiments. Male CF-I mice, weighing 18to 22 g, housed ten to a cage, were vaccinated intra-peritoneally. They were challenged 4 weeks later bythe intravenous route with 1.0 mg of the highlyvirulent strain, H37Rv, of M. tuberculosis. The micewere observed daily, and the time of death of eachmouse was recorded. The number of mice whichsurvived 30 days indicated the effectiveness of thevaccine since all or most of the control, nonvaccinatedmice were dead by this time. We have given elsewhere(42) the reliability and validity of measuring theimmunogenic activity of vaccines by this procedure.Statistical comparisons between groups of mice weredone by using the chi square test.

RESULTSSedimentation patterns of ribosomal prepara-

tions: 5 to 20% linear sucrose gradients. Theclassical 70, 50, and 30S ribosomal particles (31)have been shown by others to be present in the

H37Ra strain (7, 8, 34). We also have isolatedthese structures (Fig. 1). In Fig. 1, when the Svalues were calculated by using either of the twomarkers, the values were similar. The 80S stand-ard was centrifuged on a separate 5 to 20%sucrose gradient at the same time and in the samerotor as the 70, or the 50 and 30S particles.Under these conditions, the 30S subunits werethe most immunogenic. A spectrum of myco-bacterial ribosomal subunits containing approxi-mately 67% RNA and 33%0 protein were formedin the presence of 0.25% SDS in 0.01 M phos-phate and 10-4 M magnesium chloride, pH 7.0.These were fractionated also by centrifugationby using a 5 to 20c% linear sucrose gradient.The patterns observed were similar to that shownin Fig. 2. A major faster moving peak, whichconsisted of particles approximately 20S, and aminor peak, which consisted of subunits approxi-mately 7-9S, were obtained. There was somevariation between the preparations since theparticles in the ranges of 40, 30, 25, 22, and, avery small particle, 2.55 were not always present.Noll and Stutz (27) have obtained similar sub-units from Escherichia coli ribosomes. The lowconcentration of magnesium ions was usedbecause we had found that higher concentrationsof magnesium in the buffer decreased the immuno-genic activity of the particulate fraction (38).Hosokawa et al. (17) found also that by using10-4 M magnesium ions, 40 and 23S subunitswere formed.Many of the chemical characteristics of these

ribosomal preparations have been given previ-ously (38, 39, 41, 43). In addition, an electronmicrograph of equivalent preparations has beenpublished by us (43).

E vuC I

0 1

o025 20 o 10 0NOttOF

BotmFraction Number

FIG. 1. Sedimentation patterns of (A) 70S mycobacterial ribosomes, sedimented in a 5 to 20% sucrose gradientcontaining 10-1 M MgCI2; (B) 70S ribosomes, dialyzed against 10-4 .X MgCI2 overntiglht, anid sedimenited in a 5to 20% sucrose gradient containing 10-4 M MgCl2; and (C) 80S mammalian brain ribosomes sedimented in a 5 to20%,' sucrose gradient containing 10-2 M MgCI2. Alkaline phosphatase marker, O.

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5 10 15 20 25 30 35Top

Fraction NumberFIG. 2. Sedimentation pattern of mycobacterial ribosomal sutbuniits obtainied onz a 5 to 20'S sucrose gradielnt

(solid line), alkaline phosplhatase marker (brokenz line).

The gradient shown in Fig. 2 was separatedinto five fractions, and each of these fractions,in two vaccinating doses, was tested for im-munogenic activity. The pooled data of twosimilar experiments are given in Table 1. Alsogiven in the table for comparison are three ex-

periments which use the unfractionated ribosomalfractions as the immunogen.

It was found that, although some immunogenicactivity was present in all the fractions, fraction I(40S particles) had the highest immunogenicactivity based on the amount of RNA injected.As little as 0.05 ,ug of ribosomal RNA of thisfraction protected approximately 50% of themice. In comparison, by using the non-frac-tionated ribosomal subunits, 100 times as muchnucleic acid was needed to produce a similarimmune response. The larger vaccinating dose offraction I was significantly less effective (P =

<0.05) than the lower dose. This may have beendue to suppression of the immune response dueto excess immunogen or to an adverse effecton the animals. In contrast, as the ribosomalparticles became smaller the higher vaccinatingdose was more immunogenic than the lowerdose. Fraction II (25 to 30S particles) and frac-tion III (20 to 25S particles) needed more RNAto produce an immune response similar to thatobtained with a 0.05-,ug dose of fraction I.Sixteen jig of RNA of fraction III protected only46% of the mice which, using unfractionatedribosomes, would protect approximately 70% ofthe mice. As the particles became smaller infractions IV (9S particles) and V (2.5S particles)

TABLE 1. Immuunogenicity offive fractions collectedfrom sucrose gradients containing mycobacterial

ribosomal preparations

Immunizingprepn

Ribosomalfraction

I

II

III

IV

V

Controls

AnIcNeo. NTo. ofR~NA S-30

mice mice'

50g

50.05.00.50.50.056.00.616.01.67.30.731.30.13

8980886049455950585849585760

72392818241920231721111694

Percent

of S-30mice

81493230b49b4234462936222816c7c

ApproximateS value

40S

30S, 25S

9S

2.5S

a S-30 mice = number of mice which survived>30 days.

b p = <0.05.c P = >0.05; all other values are statistically

significantly different from the controls.

their immunogenic activities decreased evenmore. These results suggested that, although theimmunogen may be spread through the fivefractions, the size of the ribosomal particle maybe important for the production of a high im-mune response; further experiments are beingdone to explore this possibility.

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Base composition of mycobacterial ribosomalRNA. The relative amount of each of the baseswas determined and the data was pooled fromseveral experiments. There was 21.7% adenine(A), 23.8% cytosine (C), 31.8% guanine (G),and 22.6% uracil (U) present. The A to U ratiowas 0.96%; the G to C ratio was 1.3, the purineto pyrimidine ratio was 1.15; the G plus C toA plus U ratio was 1.26; and the G plus U toA plus C ratio was 1.2. These values were similarto those found by others for Mycobacterium(Corynebacterium) vadosum Kras, but varied alittle from the values found for the bovine strain(Bacillus Calmette Guerin) and the saprophyticstrain, M. phlei (2).

General characteristics of mycobacterial RNApreparations. The E = 1 %/cm is approximately222. The RNA has a typical nucleic acid absorp-tion curve and peaks between 258 and 260 nm.The 260:280 ratio is approximately 2; the235:280 ratio is approximately 1.6. The RNAwas resistant to deoxyribonuclease and nodeoxyribonucleic acid could be detected chemi-cally. The RNA was partially resistant to alarge amount of ribonuclease (1 mg/mg RNA).About 30% protein was present. No polysac-charides could be detected chemically. TheRNA was standardized by ultraviolet absorp-tion at 260 nm and by the orcinol reaction. Thevalues obtained were in close agreement. Muchof this has been covered in our recent publications(38, 39, 41, 43).

Effect of trace amounts of ribonuclease on RNA.Ribonuclease (0.2 ,sg/20 jig RNA) was addedto native RNA and to heat-denatured RNA(110 C for 7-8 minutes). As shown in Fig. 3, the

25

ribonuclease had little effect on the native RNA,but hydrolyzed the heat-denatured RNA toabout the same degree as single-stranded yeastRNA. This suggested that the native RNA mightbe double stranded (14, 33).Tm of the RNA. The RNA was diluted in

SSC and in 0.1 SSC buffers. The Tm value ofmycobacterial RNA in SSC buffer was greaterthan 85 C. Generally, there was little change inthe ultraviolet absorption at 260 nm until 50 to60 C where an increase in absorption occurred.The Tm of the RNA diluted in 0.1 SSC wasapproximately 55 C (Fig. 4). Single-strandedyeast RNA was treated in a similar fashion, andthere was little difference between the Tm valuesobtained by using these two buffers; the valuewas approximately 50 to 55 C. These resultsagain provided evidence which suggested thatmycobacterial RNA might be double stranded(14, 15, 16).After reaching 100 C, the RNA had a hyper-

chromicity of approximately 28%. The tubeswere cooled in ice to determine whether theremight be a hypochromic effect from the finalreading at 100 C. There was a slight decreaseof about 4 to 5 %. In contrast, if the RNA werecooled gradually at room temperature, a markedhypochromic effect occurred so that the opticaldensity reading was almost the same as thatoriginally observed in the SSC buffer; however,there was no hypochromic effect if the RNA werediluted with the 0.1 SSC buffer. This was inline with what would be expected if mycobacterialRNA were double stranded, as yeast RNA (13)dropped about 5% in the SSC buffer and nonein the 0.1 SSC buffer.Formaldehyde was added to an equal volume

of the RNA (40 ,ug/ml), diluted in SSC buffer,so that the final concentration of formaldehyde

E 30

C\j

a

0 10 20

Time in Minutes

C 20

0-

' 03

~o

-0c o0

30 0-a-

FIG. 3. Effect of 0.2 ,ug of ribonuclease on 20,ug ofRNA at 25 C. Undenatured mycobacterial RNA,0; heat-denatured mycobacterial RNA, 0; yeastRNA, A.

20 40 60

Temperature, °C

FIG. 4. Thermal transition curves of mycobacterialRNA in SSC buffer, *; in 0.1 SSC buffer, 0; inSSC buffer containing 2.76% formaldehyde, A.

E

CQ0

CD

a-s

c

8if

20

15

10

5

0

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was 2.76%. The formaldehyde should reactwith the free amino groups (9, 16). The Tmwas measured both with and without the form-aldehyde. Several experiments were done and theresults were remarkably consistent. The averageTm of the RNA in the presence of formaldehydewas 55 C (Fig. 4) and there was no hypochromiceffect on cooling, showing that the amino groupsof the RNA had been bound by the formalde-hyde which prevented the reannealing of thetwo strands (16). This further supported thehypothesis that the mycobacterial RNA wasdouble stranded or had a double helical struc-ture.

Effect of formaldehyde on optical density atdifferent wave lengths. Formaldehyde (a finalconcentration of 1.5%) was added to equalportions of mycobacterial and yeast RNA (40,g/ml), diluted in distilled water, and incubatedat 35 C for 4 hr. Suitable blanks were included.The optical density readings were taken at 5-nmintervals between 220 and 280 nm to determinewhether the formaldehyde would combine withthe free amino groups on the RNA chain andthereby shift the maximal absorbancy to longerwave lengths (10, 14, 15, 28). It was found thatthe wavelength of maximal absorption withmycobacterial RNA was 255 nm, whereas themaximal absorption with the single-strandedyeast RNA was 260 nm (Fig. 5). These resultsindicated that mycobacterial RNA had fewerexposed free amino groups than yeast RNA and

125 I

1.0

E 15$C 0.750NJ

0

that a double-stranded or double-helical struc-ture might exist.

Sedimentation patterns of RNA preparationswith 5 to 20% linear sucrose gradients. Thesedimentation pattern of RNA preparationswashed three times with ethyl alcohol-sodiumchloride solution is given in Fig. 6. This wascharacterized by a single major peak which wascollected in the 14th to 19th fractions. The Svalue, depending on the preparation, varied from14 to 19. A minor peak was present in approxi-mately the 25th fraction and had an S value ofapproximately 6. The approximate molecularweight, therefore, of the larger component wouldbe around 300,000 as determined by the method

>~0.6

ca)

_ 0.4

0.2

A

220 240 260 280 220 240

Wavelength, n m260 280

FIG. 5. Effect of 1.5% formaldehyde on the ultra-violet absorption spectra of (A) mycobacterial RNAin water, @; in formaldehyde, 0; (B) yeast RNA inwater, 0; in formaldehyde, 0.

Fraction NumberFIG. 6. Sedimentation pattern and fractions obtained on a 5 to 20% sucrose gradient with recent preparations

of mycobacterial RNA washed three times with 65% ethyl alcohol-0.013 M NaCI and diluted with distilled water(solid line), alkaline phosphatase marker (broken line).

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of Martin and Ames (24). RNA preparationsgiving this type of sedimentation pattern weremore stable and were of good quality with little orno degradation since the increase in hyperchro-micity was 30 to 34%. This was the preparationwhich appeared to contain double-stranded RNAas determined by the chemical and physicaltests mentioned earlier. In addition, it was con-sistently highly immunogenic (41).The RNA preparations were divided into 5

fractions (Fig. 6), and each fraction was assayedfor immunogenicity. The pooled data from threeexperiments are given in Table 2. The immunogenagain appeared to be spread throughout thegradient as each dose protected a significantnumber of mice as compared with the controlnonvaccinated mice. Analysis of the data, how-ever, revealed marked differences. As was notedwith the ribosomal fraction I, the lower vaccinat-ing dose of the RNA fraction III containing the15 to 16S RNA components was significantlymore immunogenic (P = <0.025) than itshigher vaccinating dose. Fraction II, havingRNA-protein complexes varying from 16.5 to20S, had a relatively low degree of immunogenicactivity which was similar to that obtained withthe higher vaccinating dose of fraction III. It maybe that particles in this size range adverselyaffected the animal in some manner. This was alsoindicated by the fact that the lighter fraction IVwas significantly (P = <0.025) more immuno-genic than fraction II. Again, as was found with

TABLE 2. Immunogenicity offive fractions collectedfrom sucrose gradients containing extracted

mycobacterial RNA-Protein complexes

Immunizing Amt No. of No. of Per cent Approx-S-30 of S-30 imate Sprepn injected mice micea mice values

RNA

I

II

III

IV

V

Controls

5g50.05.00.52.00.24.00.415.01.54.00.41.50.15

4946908889878588808878788890

372540322620192132342520197

76544436292322b2440393226228b

155

9S

6.35

the ribosomal fractions, the smallest RNA com-ponents did not appear as efficient in producingan immune response. In none of the fractions,considering the amount of nucleic acid injected,did the immune responses equal that obtainedwith the unfractionated RNA preparations.

In an attempt to find a diluent that wouldstabilize the RNA-protein complexes more thandistilled water, three other buffers were used.The sedimentation patterns of the RNA-proteincomplexes diluted in each of the three bufferson sucrose gradients were obtained, and theimmunogenic activity of each preparation wasdetermined.

If the RNA-protein preparations were dilutedin 0.01 M phosphate buffer, pH 7.0, containing104 M magnesium chloride, the sedimentationpattern changed (so that under these circum-stances the major peak generally appeared inabout the 10th fraction with an approximate Svalue of 20. There was a lower peak which wasconsistent in position and contained particles ofapproximately 6S. The presence of the phos-phate and magnesium ions, therefore, apparentlycaused some aggregation of the larger particles(18), but this did not affect the immunogenicactivity.

If the RNA-protein complexes were dilutedwith SSC buffer, a different sedimentation pat-tern was noted (Fig. 7). There were a majorpeak, which was collected in the 26th fractionand had an S value of approximately 5, and somevery minor peaks, which were collected ap-proximately between the 5th and 18th fractionsand had S values which varied from 12.6 to22.8. The increase in the hyperchromicity wasabout 22%, denoting some degradation of the

E

D

0

Fraction Number

FIG. 7. Sedimentation pattern of mycobacterialRNA preparations diluted in SSC buffer, @; alkalinephosphatase, 0.

a S-30 mice = number of mice which survived>30 days.

b p = <0.05; all other values were statisticallysignificantly different from the controls.

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RNA. The immunogenic activity of this RNApreparation also was lower (41); in contrast,the same preparation of RNA diluted withdistilled water was highly immunogenic.

In two experiments, the RNA preparationswere diluted in 0.01 M NaCi-0.001 M disodiumethylenediaminetetraacetate (EDTA). The sedi-mentation pattern was similar to that obtainedwhen the RNA preparations were diluted inthe SSC buffer; there were, however, no minorpeaks and only one major peak containing com-ponents of approximately 6S. The increasein hyperchromicity was 29%0,, but only the 50-,gvaccinating dose was moderately immunogenic.In this case, this low degree of activity did notcorrelate with the hyperchromic value (41);therefore, the salt-EDTA apparently affectedthe RNA in some manner, other than by pro-ducing degradation, such as changing the RNAstructure by removing magnesium ions.As noted above, RNA-protein components of

approximately 6S in size were not effective inproducing a high immune response.

DISCUSSIONWith the finding that undegraded mycobac-

terial RNA was essential for high immuno-genic activity against tuberculosis by using micro-gram vaccinating doses of the RNA preparation(41), it was important to determine some of itschemical and physical characteristics, andwhether it contained single- or double-strandedRNA. This information, in turn, might be usefulin determining the mechanism by which myco-bacterial RNA-protein complexes immunizedagainst tuberculosis. We had found previously(39) that immunogenic RNA-protein complexeswere partially resistant to a relatively largeamount of ribonuclease, since the immunogenicactivity of the treated RNA-protein preparationswas reduced by only approximately 50%0. Thisfinding suggested that if mycobacterial RNAwas the immunogenic moiety in these prepara-tions, it might be active only in the double-stranded form.

In more recent experiments, we have usedmany of the chemical and physical tests used byothers (4, 14, 19, 26, 34), to determine whethermycobacterial RNA was single or doublestranded. The RNA preparations used for thesetests were diluted in distilled water and had Svalues which varied from approximately 15 to20 and occasionally 23S, depending on thepreparation. The results from these studiesindicated that mycobacterial RNA was eitherdouble-stranded, or mainly double-helical, andresistant to conformational change. However,there was a possibility that the RNA actually

was single stranded and, by overlapping, hadformed double-helical secondary or tertiarystructures which were highly organized andquite stable (31). The type of RNA structuresfound in different diluents provided some evi-dence against this latter possibility. Severalinvestigators (15, 22, 29-31) have discussed thestructures of single-stranded RNA under differ-ent conditions. Single-stranded RNA diluted indistilled water should form a loose randomlycoiled structure or an extended chain. Gavrilovaet al. (12) found that E. coli ribosomal RNAhad S values of 3 to 6 when diluted in deionizedwater. As ions were added to RNA diluted indistilled water, secondary structures formedwhich had helical regions held together by hy-drogen bonds; this type of RNA became moreresistant to heat (16, 29, 30).

In marked contrast, mycobacterial RNA ap-peared to have a very stable structure when thepreparations were diluted with distilled water andhad S values which varied between 10 to 16 andoccasionally 23 S, depending on the preparation.This type of RNA preparation was highly im-munogenic. Mycobacterial RNA diluted inbuffers containing ions, such as SSC or 0.01 MNaCl-0.001 M EDTA, changed in configurationso that the major RNA-containing fractionsedimented in the lighter zones of the sucrosegradients, had an S value of approximately 5, andwas 100 times less immunogenic. Boedtker (5)reported that salt-EDTA prevented aggregationof the RNA as RNA aggregated easily in con-centrations above 1 mg/ml. The EDTA may haveprevented aggregation of the mycobacterialRNA-protein complexes and decreased the sedi-mentation value. Another explanation, however,is that the EDTA and the SSC buffer removed themagnesium ions, thereby decreasing the sedi-mentation value by unfolding the RNA. Mialland Walker (25) found that the presence of 5 mmof EDTA decreased the S value of ribosomalRNA prepared from E. coli from 16.3 to 3.7S.Our results are similar to theirs. In addition,Miall and Walker found no loss of secondarystructure, although the RNA was more flexible.If comparable, this type of unfolding markedlyreduced the immunogenic activity of mycobac-terial RNA preparations.

In addition, there was an apparent relationshipbetween ribosomal particle size, size of the RNA-protein complexes, and immunogenic activity.By the use of salt buffers and fractionation onsucrose gradients of each preparation, it wasfound that the larger molecular complexes weremore efficient in producing a high immune re-sponse than the smaller complexes. The 25 to 40Sribosomal subunits and the 15 to 16S RNA-

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MYCOBACTERIAL RIBOSOMAL AND RNA VACCINES

protein complexes were the best inducers of animmune response. Thus, these two groups ofsubunits are apparently related. In this connec-tion, Kurland (18) reported that the 30S ribo-somal subunits from E. coli yielded a mixture of16- and 23S-RNA components in various propor-tions. Lessie (21) has shown, however, that ribo-somal RNA extracted from Rhodopseudomonasspheroides did not contain the 23S componentbut only the 16S component, except when higherconcentrations of magnesium ions were used.In accordance with their results, we have notobserved the 23S RNA component except whenthe RNA protein preparations were diluted in aphosphate-MgCl2 buffer or in SSC buffer.Venneman and Bigley (32) and Venneman

et al. (33) have recently published results usingribosomal fractions and RNA preparations fromSalmonella typhimurium to vaccinate mice. Theyfound that each preparation produced an im-munity similar to that obtained with living cellsand that the degree of immunity was not affectedby proteolytic enzymes. However, they found thatribonuclease did not reduce the immunogenicactivity; they did not find a correlation betweenparticle size and immunogenic activity, for theimmunogen appeared evenly spread throughoutthe sucrose gradient. In addition, Freund's in-complete adjuvant was unnecessary for immuno-genicity. It is possible that the ribosomal im-munogens obtained from the two microorganismsmay produce immune responses which aremediated by different mechanisms. Winston andBerry also have prepared immunogenic ribosomalvaccines from both Staphylococcus aureus andPseudomonas aeruginosa (35, 36).

ACKNOWLEDGMENTS

This investigation was supported by Public Health Serviceresearch grant AI-01636 from the National Institute of Allergyand Infectious Diseases.We would also like to acknowledge the help of Ruth Deddish,

Verlen Peterson, and Stewart Spies who helped do many of thechemical and physical tests.

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