Immunological studies with sulpholipids of mycqbacteria

63 (1982) 1 0 7 ~11 ( Lor~(~lnanGroup I td

0041 3s

"[uber

IMMUI~IOLOGICAL

STUDIES

WITH

SULPHOLIPIDS

OF M Y C O B A C T E R I A

G. K. Khuller, Urea Malik and [3. Subrahmanyam D~;porrment #f Biochemtstr V, Poslflradu,'He institute {d Medfcal Educatior~ and Re:,,.ar~? Chandllqat/J 160012. /ztdza

Summary Antibodies to sulpholipids of mycobacteria were p~oduced m rabbits when rejected as sulpholipid-MBSA complexes and were detected by kaolin agglutination and double diffusion techniques. Sulpholipid antibodies did not cross react witil any other lipads of n|ycnbacteria except cord-factor. The antigenicity of sulpholipids appears to be due to ~-D-trehalose and sulohate groups. Guinea pigs irrmlunized with sutpholipid-MBSA complexes showed partial protection against tuberculous infection with M tuberculosis H~,Rv, as revealed by mortality rate and score of lesions. R6sum6 Des anticorps vis-a-vis des sulpholipides des mycobact#.r=esant ere prodults chez des tapins en iniectant ces sulpholipides sous forme de compJexes sulpholipides-MBSA Cos anticorps ant 6t~ d6cetes par agglutination our kaolin et par ies techniques de double diffusion Les anticorps vis-a-vis des sHiphohpides n'ont pas donn#~de.r~.actions C(OlS(~esavei: d'autres hpides des mycnbact~ries, saul avec le facteur responsabte de ta formation de serpentine. L'antig6nicit# des sulpholipides semble due aux substances des groupes des selphates et de I' z- D-trehalose

Des cobayes immunis{~s avec los complexes sulpholipides-MBSA one ~t6 proteges partietlement centre I'infection tuberculeuse par M. tuberculosis H37Rv ainsi que l'ont montr~ lea taux de mortalit~ et lee deg~s d'extension des I~.sions. Resumen Se produjeron anticuerpos a los sulpholfpidos de las micobacteria~ en conejos inyectados con complejus sulpholipides-MBSA Estos anticuerpos fueton detectados par aglutinaci6n sabre kaolin y par t#cnica de doble difusi~n. Los anticuerpos a los sulpholipidos no dieron reacciones cruzadas con arras Ill)trios de las micobacterias, salvo con el factor responsable de la formaci6n de cuerdas en serpentin. La antigenicidad de los sulpholipidos parece deberse a ]as substancias de los grupos sulphate y :~-D-trehalosa. Los conejillos de Indias inmunizados con conqplejos sulphotipidos-MBSA fueron protegidos parciahnente contra la infecci6n tebercutosa par M. t u b e r c u l o s i s H37Rv coma fue demonstrado pal las tasas de mortalidad y la extensi6n de las le'~iones. I ntroduction

Sulpho!ipids, phosphatictyfinositomannosides, cordfactor and mycosides are the main glycolipids of mycobacteria [1, 2], Among these lipids phosphatidylinositomannosides are tile 1}rincip'fl li~,~dantigens of mycobacteria that elicit antibody response in tuberculous patients and

10S

KhulltTr and oil t i~

m e:..pt rm~unEci; tmmdJis L~%] C/>lc,!ilc[ot h#iS ~i[SO i)t.)Uli sh()wn lo be il'rlnlurlogenlc and ~iffolds p!olec lieu ,qgai~rst tubel(tilous !rrt~,d;boll .,v:th /~QCO~)rH.'td/I~HIZ tuborculosJs H.,Rv [4].

Su:pbOilpitJS, WlliC:,/ ,'~efe e~)llicl thougi~t lo Lie plesent only hi 71.8.tube#cu/oszs H~Rv. have recto;i,, beerl kierlbfied in ~iN the ,.4rains of nlycobacteria [5]. AItllough the antigens of m,~9 !ravc iJeef/ e~:[en5 rely studieci, the inlnlul}cllogical properties of suipholJpids are -,[i] ;e b~. ciu uid~ited. Recently we hl]ve denloi/strated the glesence of antibodies to sulphotipids r~ :cJbul,~uh)us patiems [6] 7i~e p:esent I~aper dedls with the antiganicityofsulpboNpids in iI/i;![s 1 ,iu~s { #,;" ,'u~,rl:ulosls', H Rv M. smumatis ATCC 607, M. ph/ei, and BCG), ',!,e m~ d~r;ds used m ;heir cultiv~itioq and exeac.tioH of ~[UpholJpids were as described elsewhere [7.1 ind,viquai lJ[ited ucc;ordillg to the procedure of Inoue and "h.,!m~a [ o ] Rabbits {1 5 to 2 0 k{4 body wt) were mmlunized with the sulpholipid-MBSA eom;}iezes oh'speeded ir~ Fleane :~ uicouli?lete adiuvant as described in all earlier commuoi~:auon [3] Ea::h rabbit~eceived 5 7 mgofsulphotipids. Blood wastaken weekly by cardiac !Jcu;cture fr.nm ea(:h rabbit stamilg a week atteI the ;ast ejection,

Tie ,nqtiselem WaS analysed Py kaoiu:-agglutinatlon and double+difftision techniques of [akr.lilashJ c,t at [9] and Ouchlerionv [10] ~ r ~qgiubnation-mhJOition studies absorpbon of the antiserum with different antigens was ~:arrled out by mixing equal volumes of the antigen suspensio~ with the antisertim and inct.Ihabnrf ~t 3'7 C for 30 nqinutas The absorbed antiserum was stored at 4 C overnight and :errtrifuged at 3000 g for 16 mhlutes and the !;u[leroatant wa~ used for immunochemicat studies PIr ~.'xperl#reD! Guinea plfrs (200-250 gm body wt) were divided into two groups of 10 to 20 animals each. Or, e group was immunized with 2 mg of total sulpbohpids- M BSA complexes as outlined above. Or, P. week ~fter the last injection both ibe qroups were challenged by injecting into the groin w~th LD-50 dose of the M. tubercu/osL~ H.,Rv. Mortality race of the animals was noted. .At pr)st-mortem examination, a score of 0 to 4 was given to different organs like lungs, liver and spleen according to Gupta and Cha~
Results and discussion ]mmunochesmc,,;1 studies tmmunizatim7 of rabbit~ with total sidpbolipids resulted in a peak antibody title of 1 : 256 to 1 : 512 by agglutination technique. The antiserum also gave a positive precipitin reaction in agar gels against total sulphobpids Autiserum to total sulpholipids showed reactivity with individual sulpholipids viz. SI_ I arid SL-III as shown by precipitation and agglutination reactJorls

Mycobacterial sulpholipids

109

Antiserunr 1o sulpholipids of M. [uberr:ulo~,t,~ H ; R v 9ave a selologically posilive les~ with :,ulpt)ohpJds from nonpathogerJic stratus (M. 5/rregfriatl's ATCC 6 0 7 , / V / p h i a l ) indicatilbg the uresence of common antigens. These antibodies did not close-react with other myeobacteti~]l phospholipids Le. mannosides, cordiolipin and i'Jhosphatidylethanolamine except cord factor However, antiserum did irot lose its icactlvlty against sulpholqrids even aflel being absorbed with pure cord factor. The cross-reactivlt betwuen Slllpholii)ld5 and corcJ factor was thou(.fi;t to be due to the common tlehalose cole. 7his was confirmed by partial mh,bition of the seloiog~u response to the antisera with trehalose, whereas no mhibitlorl was noted with glucose and maltose tetrapalmdate (Fig. 1 ). In a wide variety of 91ycollpid arlligens antigenlcdy has been strewn to be (.Jue 10 t!le CalbC)hydrate lesidues. Studies with glycolipid alltlgerls such as nlannophosphoi~/ositidef. [13]. galactocerebroside and cytotipin [14] lead to evidence of assoclatlofr of anticlenici!y w~th lh~: ealbohydrate residues. However, fatty acids seem to be essential for tilt reactivity of suIphohpid antigens, silica their water soluble products obtained I.~y mild alkaline hvdroiysts did not react with tile antiserum. Likewise for cardiotipin antigeH, Inoue and Noiima [15] and Do Bruijn [t 6 ] concluded that although antigenic specdicity seemed to reside m the polar-head borti[m, tl~ fatty acid~ were essential for reactwlly. The large amount of inh;bition observed wHh p-nitrocatecholsulphate and trehalose sulphate emphasizes tile role of sulphate proL~p ii1 2'-posJtio~r.The importance of sulphate in the antigeoicJtv of brain-sutphatide ha~ already bne~ suggested byZalcetal [17lend Hakomori [18] Furthermore. the role of sulphoiipid antibodies in alfording protection in animals agaim4 tuberculous infectiol~ was examined. Table I shows the survival time of immunized eRI nonimmtmized guinea pigs against M. tuberculosis infection I1 is clear that about 90 to 100 per cent of tile animals die, d in the control group, while 35 1o 40 per cent ef the immunized animals survived. This could be due to an enhanced resistance in immunized guhTe,a pi.qs

2' I \ , " ~

................

[

L5

.

-'----~o

k

06i

L ....................... o

50

~oo

150

200

CONCENTRATIONCF INHIBITORSADDEOIN mM ( 4tul~ I. h~hihillOllof anti sulpho]if)id ~ntibodiss by diffe~en{seuctutaI ana]ogue.~. 9 9 GI co~,a P,atos(,'x ...... x.Te aloseL ........ " l)-nit oca~echolsu{phate:. -........ .:;T~ehalosesulphme

110

Khuller and others

"]'able h Protection of guinea pigs as revealed by mortality against experimental infection with M. tuberculosis by immunization with sulpholipid-MBSA complex Days after i/fraction

Protective effect (No. of survivors/number teared) Non-vaccinated control

Vaccinated with SL ,MBSA complex

Expefimem--i

7 14 21 30

13/14 6/14 1/14 0/14

14/14 10/14 8/14 5/14'

Experiment--II

7 14 21 30

20/20 14/20 7/20 0/20

20/2O 18/20 12/20 8/20'

The significance Of the results is denoted by * g value -:.O.al.

Table II. P.otective effect* of sulpholipid-M BSA immunization against experimental infection with M . t u b e r c u l o s i s Experiment number

Non-vaccinated control

Veccizxated animals

I~ II ~

0.81 • 0.07 0.73• 0.07

0.67• 0.09** 0.56• 0.02**

* Protectiveeffect ts expressedas 'roar index evaluatedaccording to Gupta and Chakravarti [11 ] and Mitchison et al [12 ]. a) The results are mean • S,D. of root indices of14 guinea pigs in each group. b) The results are mean • S.D. of root indices of 20 guinea pigs in each group. The significance of tile results is denoted by ** p value <0.001.

compared with the control animals. The significance of these observations was tested by 7-=-test. Further, the average root-index for the control and immunized animals is compared in Table II. It clearly shows that the rate of progression of the disease is greater iv] nonimmunized than immunized animals. Several lipid fractions like various waxes [19] and cord factor [4] have been shown to be protective against tuberculous infection. In this study, the sulfolipid-M BSA complex proved to be immunogenic against experimental tuberculosis in animals. What part this antigen plays in the immune response to tuberculosis in man remains to be determined. Acknowledgements

This study was financed in part by a grant from the Department of Science and Technology, India. One author ( U M ) thanks the Indian Council of Medical Research for the award of a Senior Research Fellowship. ~ferencem 1 Batksdale, L., ~ KJm, K. S. (1977). ~ycoPacterium. Bacteriologica/Reviewx, 41. 217.

2 Goren, M. a., E~arennan, P. J. (1979). Mycobacterial )ipids. Chemislry and Biological activities, In Tuberculosis ed G, P. Yournans,W. B. Saunders, Philadelphia,136,

Mycobacterial sulpholipids

111

3 Kh~tler, G. K., ~" Subtahmar~yam, D. (1971). Antig~,icity of Phosphalidylinositom~r~flosides of Mycobactetmm tuberculosis, Immunochemistty, 8, 251. 4 Mate, M. (1973). Effect of anti-cord factor antibody on experimental tuberculosqs in mice Infection and immunity, 7, 14. 5 Malik, U., Prabhudesai, A. V., Khuller, G. K., ~t Subrahmanyarn, D, (1981). Sullolipids of Mycobacteria. (Submitted for publication). 6 Khuller, G. K,, Malik. U., Et Nalini, P. (1951). Antibodies to mycobaoterial sulfolipids ill human tuberculous sara IRCS Medical Sciences, 9, 492. 7 P~ebbudesai,A. V., Malik, U., $ubrahmar~y~m,D., ~" Kh~lleh G K. (1981). ~sotati~n and pu~ification of sulfofiplds of Mycobactetium tuberculosis, H3~Rv, fndian Joumal ef Bioch~mi~try and Biophysirs, 18. 71 8 Inoue, K, ~- Nojima, S. (1967), tmmunochemical studies of phospholipids. III, Production of antibody to cardiolipin Biochemica 8iophysica, Acta, 144. 409 9 Takahashi, Y, (1962). Specific serum agglutination of Kaolin particles sensitized with tubercle phosphatide and its clinical evaluation as a serodiagnostic test of tuberculosis. Am~Hcan Review of Respiratory Diseases, 85, 708 10 Ouchter]ony, O, (1958). Diffu.~ion in gel. Methods for Immunological Analysis, In Progress in Allergy, 5 , 1 11 Guptg, $. K., ~' Chaktavarti, R, N. (1855). The therapeutic activity of some sulphones, sulphoxides in experJmenlal tuberculosis in guinea pigs, B:,:t/sh Jou:~a! of P#ermecologF, ~0, ~13. 12 Mitchiso :~ D.A., Bhatia, ,~, L,, Radhakrishnan, S., 8alkon, J. B., Subbaiah, T, V., 6' Wallace, J C (1961 ). The vgulence in juinea pigs of tubercle bacilll isolated before treatment from south. Indian patients with pulmonary tuberculosis. ~, HomogeneiW of the investigation and a cbtique of the virulence test, Bulletin of World Health Otganisation, 25, 285. 13 8aneqee, B., ~ Subrahmanyam, D. (1978), Antigsncity of the phosphatidylinositomannosides of rnycobacteria Immunochemistry, 15, 359. 14 Rapport, M. M., ~ G=af, L. (1989). Imrnunochemica~reactions of lip=ds,progress in Allergy, 13, 273. 15 Inc,ue, K., ~1-NojJm~, 8. (1969). (mmunochemical studies of pho~pholipids ~V. The reacPvities of antisera ~gamsl natural cardiolipin and synthetic cardiolipin analogues containing antigens, Chemistry and Physics of Llpids, 3, 70. 16 De [~ruijn, J. H. (1966). Chemical structure and serological activity of natural and synthetic cardiolipin and related compounds. British Journal of Venereal Disease ~, 4Z, 125 17 Zalc. B,, Jaque, C,. Radin, N. S., f:t Dupouey (1977). hnmunogencity of sulfatide. Immunochemistry. 14, 775 18 Hakomori, S, I. (1974). Preparation and ploparties of anti-sulfatide serum. Journal of Immunology, 112, 424. 19 Cro.vle, A. J. (1952). Tubercle bacillary extracts immunogenio for mice 4. Lipids. Proceedings of Society of Experi, mental Biology, 109, 969.