IN Ii RNA I IONA! int !RN^(0 I.I 1•10)S1^ Volume 48, Number 4

i'rinted in the

Antigenic Evaluation of Mycobacterium vaccae inRelation to Mycobacterium leprae"

Ram G. Navalkar, Sotiros D. Chaparas, C. K. Lakshiminarayana,and M. V. Kanchana'

The inability of Mycobacterium leprue togrow in vitro has of necessity directed ef-forts toward studying the antigenic com-position of other mycohacteria with a viewto determining the antigens that these or-ganisms may share with M. leprac. Thismay permit the recognition of an antigeni-cally closely associated mycohacterium(ia)that could be used for studies directed to-ward vaccine preparation. In an effort toelucidate the antigenic mosaic of Al. /ep-rae, Abe (') and Abe, et a/. ( 2 ) and Naval-kar (". '") and Navalkar and associates (")were able to demonstrate Al. /mire specif-ic antigen(s) as well as antigens shared by ,

the leprosy bacillus with other mycobacte-rial species ( 9.10. H.12.13.11) in cell extractsprepared from lepromatous nodules. Sub-sequent studies by other investigators( 3. 7' have also demonstrated such sharedmycobacterial antigens. A few years ago,Stanford and associates ( 15 • '") presentedevidence of immunological linkage betweenAl. /eprae and Al. vaccae, a Runyon GroupI rapid growing mycobacterium, on the ba-sis of which they proposed that Al. /epraewas more closely associated antigenically

' Received for publication on 2 June 1980: acceptedfor publication on 7 August 1980.

R. G. Navalkar, Ph.D., Professor, Department ofMicrobiology, Meharry Medical College, Nashville,Tennessee 37208, U.S.A.: S. D. Chaparas, Ph.D..Director, Mycobacterial and Fungal Antigens Branch,Bureau of Biologics, NIII, Bethesda, Maryland 20205,U.S.A.: C. K. Lakshiminarayana, M.D., ResearchAssociate: M. V. Kanchana, M.Sc., Research Assis-tant. Department of Microbiology, Meharry MedicalCollege, Nashville, Tennessee 37208, U.S.A. Dr. Na-valkar's present address is Department of Microbiol-ogy and Immunology, School of Medicine. MorehouseCollege, Atlanta, Georgia 30314, U.S.A. Dr. Lakshi-minarayana's present address is Stanley Medical Col-lege, Madras, India. Mr. Kanchana's present addressis Department of Microbiology, Madras Medical Col-lege. Madras, India.

Part of this work was presented at the XI Inter-natiomil Leprosy Congress. Mexico City, Mexico,November 1978.

with Al. vaccae than with other mycobac-teria. Since then, this organism has beenstudied by several workers ( 5 . 22 ) to estab-lish such a relationship. These studies wereprimarily directed toward the evaluation ofcell-mediated immune response in animalssensitized with Al. vacua(' or Al. /cprae andskin tested with homologous and heterolo-gous antigens and also to determine thepossibility of an antileprosy vaccine.

The studies reported here are concernedwith the antigenic evaluation of A/. •accaeand the determination of those antigens thatare shared by Al. vaccae and other myco-bacteria, including Al. /eprac.

MATERIALS AND METHODS

Mycobacterial strains. The strains usedwere Al. /epraernarium (Hawaii), M. lep-rae (human and armadillo derived), Al.paratuberculo.s . is (A/. johnei), Al. smegma-lis (no. 8159), Al. kansasii (no. 24178), M.inarinutn (no. 691), ICRC bacillus, Al. tu-berculosis (no. FL 7 Rv), Al. raccae (SN920,923, 931, and 956), M. (whim (no. 724), Al.intraccIlulare (no. 146), Al. firrtaitam (no.1529), Al. cheloni (no. 1544), Al. ph/ei (no.1523), Al. ,:,, ordonac (no. 1324), A/. New-f ./dace/n (no. 1316), and Boone-Battey ba-cillus (no. 1403). The first three organismslisted were separated from infected tissue,and the remainder were grown in vitro.

Sent. Thirty-three sera from leprosy pa-tients and four sera from normal healthyindividuals were used. Of the 33 leprosysera, 16 were from lepromatous cases, eightfrom tuberculoid cases, and nine from bor-derline cases.

Preparation of cell extract (CE). Cell ex-tracts from mechanically disrupted cellswere obtained for all strains execept Al. to-berculosi.s. (strain H„ 7 Rv). The H 97 Rv cul-ture filtrate system was supplied by Tru-deau Institute, Inc., Saranac Lake, NewYork, U.S.A.

388

48. 4^ Nara/kar, et al.: Analysis of M. vaccae^ 389

For the preparation of extracts of my-cobacterial strains, the bacilli were grownin vitro on Sauton ( 2") medium for a periodof 6 to 8 weeks. The cells were separatedfrom the medium by means of Buchnerfunnels and washed three times in 0.15NI sodium chloride. After the third washingthe pellet was weighed, and the moist cellswere resuspended in isotonic saline at aconcentration of 12 mg per ml. The result-ing suspension was homogenized in a Tef-lon grinder and then disrupted in a refrig-erated cell fractionator (Sorvall) at 45,000p.s.i. The material was then stored at —20°Cafter addition of 1:10,000 merthiolate, untilused. For the Al. vaccae strains, thecells were collected from growth on Low-enstein-Jensen medium since none of thestrains showed adequate growth on Sautonmedium, either under static or shake cul-ture conditions.

A similar method was used to prepare CEfrom M. lepraemurium, M. paratuhercu-losis, and Al. leprac' grown in vivo. Themethod for the separation of Al. lepracfrom infected tissues has been describedpreviously ("). M. /effete/um -him and Al.paratuberculosis were obtained from Dr.A. M. Dhople and Dr. R. Merkal, respec-tively. A4. paratuberculosis were separatedfrom bovine intestinal mucosa, and Al. hp-/we/tuff/um were obtained from infected ratlivers.

Preparation of reference antisera. Anti-sera were prepared against the CE of themycobacterial strains grown or processedin our laboratories. After the collection ofa serum sample, male rabbits, weighing 1.5to 2 kg, were inoculated with 1 ml of a mix-ture of equal volumes of CE with Freund'sincomplete adjuvant. The animals were giv-en weekly subcutaneous injections for 6weeks, and in some instances, up to 8weeks. Three rabbits were used for eachpreparation. One week after the last injec-tion, serum was collected to determine thelevel of antibodies in each rabbit by im-munodiffusion analysis. Only rabbits thatshowed satisfactory and identical antibodyresponses to a given immunizing prepara-tion were used to supply serum. The ani-mals were exsanguinated a week after thepreliminary sample had been taken andtheir serum pooled. Scrum was stored at—20°C in desired aliquots after the addition

of merthiolate. Only Al. yucca(' SN920 wasused to prepare a homologous antiserum.No antisera were raised against the otherA/. vaccae strains.

Serological tests. The comparative im-munodiffusion (CID) technique of Ouchter-lony (''), as modified by Hansen ("), wasused initially to analyze the various seraand to determine the antigenic constituents.Subsequently, the immunoanalysis wascarried out using the fused rocket immu-noelectrophoresis (FRIEP) technique, asdescribed by Svendsen C') and modified byChaparas, et al. (I)

RESULTSAs a first step toward recognizing the

various antigens of Al. vaccae (SN920), theCE was tested by immunodiffusion againstthe corresponding antiserum prepared inrabbits. Nine lines of precipitation were ob-served, indicating at least nine antigens.The three other strains of M. vaccae(SN923, 931, and 956) showed identical an-tigenic composition as Al. vac•ae (SN920).The next step was to determine by CIDanalysis the number of antigens shared byA/. caeca(' and other mycobacterial species.

Fig. 1 summarizes the results of the CIDstudies. M. yucca(' shared one of its anti-gens with Al. ma•inwn, which was grownat 37°C, and with M. ,c, ordortae; two of itsantigens with Al. ma•inwn grown at 30°C,the ICRC bacillus, and Al. /epraemteriremthree each with A/. paratuberculo.sis andAl. kansasii; four each with Al. leprac de-rived from both human and armadillo tis-sues, M. phlei, A/. smegmatis, and Al. tu-berculosis (strain H,Rv); five each with A/.scrolUlaccum and Al. cheloni; and six eachwith Al. avian and A/. intracellulare. Theresults given are those obtained with thecell extracts of all strains, except Al. tu-berculosis (strain H„Rv) where concen-trated culture filtrate was used as the anti-gen. Fig. 2 is a photograph of a CIDanalysis between A/. vaccae and Al. infra-cellular(' systems.

The Table summarizes the results of theantigenic evaluation of Al. vaccae and com-parative antigen analysis by fused rocketimmunoelectrophoresis (FRIEP). With thistechnique, using only the antisera againstother myeobacteria, it was possible to de-tect anywhere between 13 and 16 antigens of

390^ Inierna !Unita Journal of Lepro.s\'^

1980

1UNYONGROUPS

MYCOBACTERIALREFERENCESSYSTEMS^ SHARED ANTIGENS_ VAC CAL

SN920TE T^TRAIN

I

M KANSASII

M MARINUM(30. C)

M MARINUMM. E)

II

III

M GORDON AE

■M SCROFULACEUM

M. CHELONEI

PA 11VI UM

M INTRACELLULARE

I \M PHL El

M SMEGMATIB

M LEPRAE (H) .

M LEPRAE (A) .

M LEPRAE MURIA.

M JOHNE I

M TUBERCULOSIS(STAIN H371/0

ICRC BACILLUS

NON-SHARED ANTIGENS

TOTAL NUMBER OFDEMONSTRABLEANTIGENSI I\

k ^I\

\\ ININ‘111\

\i\ ii\\\\\\\\\ ‘

rici. I. Antigenic relationships between Mvcobacleruem rumor and of ter mycobacteria, as shown by com-parative inummodausion (CID) analysis. Shared antigens arc indicated )y dark squares unit non-shared byhatched ones. All strains except .1/. rubercrt/oAiN (no. 1-1,,Itv) examined as CE Preparations. WY' = humanderived; IA)' =armadill o deri ved.

M. raccac, depending on the strain used.This was more than those detected by im-munodiffusion. A comparative analysis ofthe antigens with other mycobacterial anti-sera also showed increased numbers ofshared antigens. For example, the maxi-mum number shared was with the I3atteybacillus and M. ph/el (12 each), .1/. .cme,e-mails ( I I), and M. s•rofulaccum, M.cheloni, M. (Muni ,tr -id M. leprac ( I0 each).

intracellulare (9), .11. JOrlialum (8). andM. kw/xi/Ali (7) fall into an intermediategroup as far as sharine, is concerned whereasM. , ,,, ordonae (2) and M. marimem (2) exhibitthe lowest number of shared antigens. Fig.3 is a photograph of FRI EP analysis of A/.raccay against anti-M. intracellulare, A/.avian!, ,,11. Ncrojithweam, A/. /eprae, A/.vaccae, and ,11. Nmegmati.c.

When the ti. vaccae system was usedagainst sera from leprosy patients, only thebacillary positive cases showed lines of pre-cipitins. Not all sera, however, from the

bacillary positive cases gave positive re-sult s. Of the 16 sera from lepromatous pa-tients, eight showed strong lines of at leasttwo precipitins whereas two showed veryfaint, almost undetectable reactions. Of thenine sera from borderline cases, positiveprecipitin reactions were seen with onlythree cases. In our earlier studies ( 0 '. "), allof the bacillary positive cases selected haddemonstrated the presence of either one ortwo immunoprecipitates, and some of thebacillary' negative cases had exhibited oneimmunoprecipitate when various mycohac-terial reference systems were used for CIDassay.

DISCUSSION

Employing two different techniques ofimmunoanalysis, M. vaecae were shown topossess a minimum of nine (ID) and a max-imum of 13 (FRI EP) demonstrable anti-gens. Thus the FRI EP technique of antigenanalysis was more sensitive in eliciting the

A. M. INTRACELLULARE A. M. VACCAE A. M. INTRACELLULARE

M. INTRACELLULARE M VACCAE

48, 4^ Nartilkar. et al.: tliudy.sis a/ ^vaccae^ 391

A.M.Intr. A.M.Vac. A.M.Intr.

M.Intr.^M.Vac.

Fit,. 2. Comparative precipitation analysis of M. vacua(• and .1f. int/ace/hi/are systems with schematicrepresentation, using the CID technique.

antigenic structure of a given organism.This was also evident in the comparativeantigenic studies using other mycobacterialsystems. However, with the FRIEP tech-nique it was difficult to establish a reactionof identity between antigens because of theclose proximity of the immunoprecipitinbands. These difficulties were not encoun-tered with the five-well comparative ID(CID) method. This facilitated the identifi-

cation of the two most commonly sharedantigens between M. vaccae and other my-cobacterial species, i.e., the beta and deltaantigens, which we have described previ-ously. Antibodies to these antigens havebeen shown to he present in the majority ofsera from leprosy patients ( 1 ". ). Confir-mation of the presence of beta and deltaantigens in M. raccae was obtained usingleprosy sera. 'Hie precipitin positive sera

392^ International Journal of Leprosy^ 1980

ANTISEURUM TO

M SCROF-^M AVIUMUL ACEUM

M. INTRA-^M. SMEG -^M. VACCAECELLUARE^MATIS

VACCAE VACCAE C

M. VACCAE ANTIGEN

FIG. 3. Precipitation analysis with schematic representation of M. vacua(' antigen and antisera preparedagainst various mycohacterial species, using the FRIED technique.

showed antibodies to these antigens whenscreened simultaneously against the M.vaccae system and M. stne,f,, matis, ourstandard reference system ( '").

vaccae appeared to share some of itsdemonstrable antigens with other mycobac-teria. With the CID technique the maxi-mum number of antigens that were shownto be shared were with the Runyon GroupIII mycobacteria, particularly with M. (w-him and A/. intracellalare. ,11. ,t,, ordonae.which showed a minimal number of shared

antigens, either with the CID or the FRI EPtechnique, was exceptional. In this regard,our data appear to confirm those of Stan-ford and Rook (' 8). The absence of mycolicacids of the mycobacterial type and thenonreactivity of the skin test agent in lep-rosy patients described by these authors ('")may be the reasons for such limited sharingof antigens with M. gordonae.

When one compares the sharing of anti-gens between /11. vaccae and other myco-bacteria, as demonstrated either by the CID

48, 4^Naraikar, et al.: Analysis of M. vaccae^ 393

THE TABLE. Antigenic sharing of M. vaccae (SN920) as demonstrated by the fusedrocket immunoele•trophoretic (FRIEP) method, using antisera against various myco-bacteria.

Runyon Group Mycobacterlantisera

No. of sharedantigens

Total no. ofdemonstrablehomologous

antigens

I M. kativisii 7 25Al. marintim 30°C 3 29M. null -intim 37°C 20

11 AI. I:or -do/toe 16M. serojithweitm 10 23M. cheloili 10 31

111 A/.^(mile'', It) 30;U. introcellulare 9Battey boone bacillus 16

IV t/. piaci 12Amegmatis 11 31

t!. lorluitiumi 8

M. litherettlois^H„ ; Rv) 6 24A/. /(prue (human tissue derived) 10 18A/. /eprae (armadillo tissue derived) 11 18

Test strain: A/. vaccae (SN920) 16

or the FRIEP techniques, one recognizesthat such a sharing on a large scale is ratherbroad based and is not specific to any par-ticular group. The taxonomical significanceof this is rather unclear at this time sinceM. vaccae tend to be photochromogenicbut exhibit the same general biochemicalpatterns as M. piaci (W. D. Jones, personalcommunication).

Apparently M. yucca(' shares a rather lim-ited number of its antigens with either hu-man- or armadillo-derived M. leprae or forthat matter with any of the typical strainsexamined regardless of the technique used.Further support for this observation comesfrom the studies using leprosy sera in whichonly antibodies against the most commonlyshared antigens were detectable. The seraselected for the assays, especially thosefrom the bacillary positive cases, had allshown positive precipitin reactions againstthe various mycobacterial systems used inour previous studies. Yet in the presentstudy, only a limited number of these seragave positive results whether they werescreened with the M. vaccae system alone,with the M. smeimititis reference system,or with other mycobacterial reference sys-tems. The sera tested have been from a col-

lection that has been with us over the past12 years and have been stored at —20°Cthroughout, except when needed, with min-imum frequency of thawing and refreezing.It is likely that such prolonged storagecould have led to the discrepancies in ourpresent results and those observed previ-ously.

Although M. vaccae is a rapid growingphotochromogenic organism, it appears toshare very few antigenic determinants withany of the two photochromogens used inthe screening. This is not unexpected sinceNorlin ('") has shown that not all specieswithin a given group will demonstrate themaximum number of shared antigens.

The present study has indicated that M.vaccae may be considered as a rapid grow-er, which is antigenically more similar tothe Mycobacterium avium-intracellularecomplex than to the typical group IV my-cobacteria. It does not, however, indicateany particularly close relationship with M./eprae by the methods we have employedalthough Stanford and associates ( 18 . I")have established such an immunologicallinkage through their studies. Studies byCollins, et al. ( 5) and Watson, et al. ( 22 )have also shown such an absence of rela-

394^ International Journal of Lepro.sy^ 1980

tionship. The closeness of skin reactions inleprosy patients and in presensitized ani-mals with skin test antigens prepared fromthese two mycobactcria observed by Stan-ford and coworkers ( 18 . 1 ") could have beendue to a M. leprae antigen that may beshared specifically by Al. vaccae. This hy-pothesis is supported by our earlier studieson the antigens of M. leprae (") and on thehumoral immune response in Al. leprae in-fected mice ('').

In these studies ("• one of the mate-rials we used was designated as Fraction C,which contained a single antigen of Al. lep-rae when analyzed by immunodiffusion.This fraction was strongly immunoactive,as evidenced by the delayed-type hypersen-sitivity in Al. leprae sensitized guinea pigs,the number of plaque-forming cells elicited,and by the ability to transfer cutaneous ana-phylaxis. This fraction also elicited positivereactions in I3CG sensitized animals but notin animals sensitized to a variety of othermycohacteria. It is likely that M. varcaepossess this antigen which is shared by Al.leprae and 13CG: we have not, however,analyzed Fraction C using the anti-Al. retr-eat antiserum. On the other hand, it is like-ly that Al. vacrae may he sharing a differ-ent antigen with M. leprae that perhaps isnot common to other mycobacteria. Mostinvestigators of mycobacterial antigenshave as yet failed to demonstrate any anti-gen found in Al. leprae only: although somedo suggest that they may he dealing withsuch an antigen (").

From the data presented, we concludethat M. vaccae do possess the two mostcommonly shared antigens between variousmycobacterial species, the beta and thedelta-antigens previously described byus (". 111 . 12). We have, however, not beenable to establish any particularly close im-munological relationship between Al. vac-ua(' and M. leprae that would suggest theuse of Al. varrae as a possible antileprosyvaccine source.

SUMMARYImmunodiffusion analysis of Mycobac-

terium •accae indicated the presence of atleast nine antigens. Using the technique offused rocket immunoelectrophoresis, atleast 13 such antigens were detected. Com-parative analysis of the Al. vaerae antigen-

antibody system with similar systems es-tablished for other mycobacterial speciesshowed that A/. raccar shared a significantnumber of antigens with both typical andatypical mycobacteria. These included thealready described beta and delta antigensthat are common to the majority of myco-bacterial species. Confirmation of the pres-ence of these two antigens was obtainedthrough comparative analysis of sera fromleprosy patients, using A/. raccae and Al.smegmatis conjointly.

It has been concluded that Al. yucca('possess the two most commonly shared an-tigens among various mycobacterial species,in addition to many others. It has not beenpossible with the methods employed to es-tablish any particularly close immunologi-cal relationship between Al. yucca(' and M.leprae that would indicate the use of M.tweeter as a possible antileprosy vaccinesource although this possibility has beensuggested by other investigators.

RESUNIENEl analisis por innitmodifusitin del Mycobacterium

tartar indict') la presencia de cuando menus 9 corn-pommies antigenicos. Usando una tecnica n uts sensi-

ble ("fused rocket inummoclectrophoresis - 1 se logra-run demostrar cuando menus 13 antigenos. El estudio

comparative del sistema antigeno-anticuerpo corres-

pondiente al vaccae con sisternits similares esta-blecidos pant otras especies micobacterianas demos-

trLi quc el yucca(' comparte un niimero signilicantede antigenos con otras micobacterias tipicas y ittipicas.

Estos incluyen los antigenos beta y delta previamente

descritos, que son connines a la mayoria Lie las espe-cies micobacterianas. I.a presencia dc estos dos anti-

genus se confirms') por el analisis comparative de sue-r°, de pacientes con lepra usando sinniltaneamente

los sistemas de M. vaccae y M. .smegmativ.

Se concluyo que el yucca(' posee los dos anti-

genus mas comunmente compartidos por varias es-pecies micobaeterianas, adernas de machos otros. Sin

embargo, con los metodos empleados, no file posible

establecer alguna relaciOn innumokigicit particular-mente importante entre tt. vaccue y linear que

pudiera indicar el use del yucca(' como Puente deuna posible vactinit contra la lepra aim cuando esta

posibilidad ha sido sugerida por otros investigadores.

RESUMELes etudes d'iminimodiffusion de Mycobacterium

vaccae ont revels la presence d'au moins neuf anti-

genes. En utilisant une technique plus sensibled'immunoelectrophorese croisee ("fused rocker . ). ona pa mettre en evidence an moins treize antigenes.

48, 4^Nara/kar. et al.: Analysis of M. vaccae^395

12.

line analyse comparative du systeme antigenes-anti-

corps de M. raccae, avec d'autres systemes sembla-

hies etablis pour &mitres especes mycobacteriennes,

out montre que M. mica' partageait un nombre sig-

nificant' d'antigenes, tam avec les mycobacteries ty-piques qu'avec les mycobacteries atypiques. Ces an-

tigenes comprenaient les antigenes et delta qui

out deja etc decrits et qui sons communs a la majoritedes especes mycobacteriennes. La confirmation de la

presence de ces deux antigenes a ete obtenue par une

analyse comparative d'echantillons de serum obtenus

chez des mat:ides de la lepre, avec des systemes an-

tigenes-anticorps pour M. raccae et M. .one.t,onatis

utilises conjointement.

On en a conch] que M. vaccae possede les deux

antigenes qui sons partages le plus souvent par tomesles especes mycobacteriennes, outre de nonthreux

mitres antigenes. Avec les methodes utilisees, it n'a

pas etc possible d'etablir (uicune relation inummolo-

gigue particulierement etroite entre M. mica( et M.

/eprae, pouvant suggerer que vacua' pourrait ser-

vir de source pour la mise au point eventuelle d'un

vaccin anti-lepreux. Ceci est en contradiction avec lessuggestions faites par &mitres chercheurs qui (ivaient

mentionne cette possihilite.

AcknoN■ledgements. This investigation was support-ed by the U.S. Leprosy Panel of the U.S.-Japan Co-

operative Medical Science Program, administered bythe Geographic Medicine Branch, National Institute

of Allergy and Infectious Diseases (Grant R22 Al-

08647).Grateful appreciation is extended to Dr. J. Gaily for

his helpful comments and suggestions during prepa-

ration of this manuscript. The authors extend theirsincere thanks to Ms. Martha L. Hendricks for doing

an excellent job of typing this manuscript.

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