BCG Antibody Profiles in Tuberculoid and Lepromatous Leprosy

transcript

INFECTION AND IMMUNITY, May 1974, p. 952-958Copyright i 1974 American Society for Microbiology

Vol. 9. No. 5Printed in U.S.A.

BCG Antibody Profiles in Tuberculoid and LepromatousLeprosy

NILS HOLGER AXELSEN, MORTEN HARBOE, OTTO CLOSS, AND TORE GODAL

Protein Laboratory, University of Copenhagen, Denmark; Institute for Experimental Medical Research,University of Oslo, Norway; and Armauer Hansen Research Institute, Addis Ababa, Ethiopia

Received for publication 29 January 1974

In sera from 12 patients with polar tuberculoid leprosy, 12 with subpolartuberculoid leprosy, and 16 with lepromatous leprosy were demonstrated a totalnumber of 125 anti-BCG precipitins by means of crossed immunoelectrophoresiswith intermediate gel. Up to 14 different precipitins were found in individualsera, and the complexity in antibody response was higher thanpreviously realized. The specificity of 69% of the antibodies was defined, andthese antibodies were titrated in three arbitrary titer units. A highly significantdifference (P < 0.002) was found in antibody response between the tuberculoidand the lepromatous group. Due to simplicity, sensitivity, and high resolution,the method used is a promising tool for providing exact data to be used as

guidelines for purification of important individual mycobacterial antigens. Theneed for reference antisera is emphasized.

A considerable body of evidence suggests thatthe expression of immunity to intracellularbacteria such as Mycobacterium leprae dependson cell-mediated immune reactions. Humoralantibody responses to M. leprae, on the otherhand, appear to be involved in immunologicalcomplications of leprosy such as erythema no-dosum leprosum (8, 18) and may also be in-volved in the specific defect of cell-mediatedimmunity (CMI) in lepromatous leprosy, al-though they do not appear to operate throughthe mechanism of immunological enhancement(5, 6). To throw light on these and otherimmunological phenomena in leprosy, the studyof circulating antibodies to M. leprae and theircorresponding antigens appears to be of con-siderable interest. The main hindrances forexact studies of this kind have been the lack ofappropriate and enough material from M.leprae for antigen production and lack of sensi-tive methods with high resolution for complexantigen-antibody systems. In the past theseproblems have been partly solved by utilizingantigen from mycobacteria related to M. lepraeand diffusion-in-gel ad modum Ouchterlony.Rees et al. (11) observed that lepromatous incontrast to tuberculoid sera contained strongantibodies against M. tuberculosis, and that theamount of antibody decreased upon treatment.These observations suggested that the antibodyresponse is proportional to the antigenic load,i.e., the amount of bacilli in the tissues. Norlinet al. (12) studied the reactivity of leprosy sera

with different cross-reacting mycobacteria andfound that M. smegmatis, M. kansasii and M.phlei gave most precipitin bands and that manylepromatous sera contained antibodies to thebeta and delta antigens which were common toseveral mycobacterial species. Navalkar (10)extended these studies and showed that M.leprae in addition to the beta and delta con-tained three antigens which were specific forthis species. In the first antibody study (B.Myrvang, C. M. Feek, and T. Godal, manu-script in preparation) using the Ridley-Joplingclassification of leprosy (13, 14), it was demon-strated that the proportion of leprosy sera withprecipitins increased gradually from the tuber-culoid towards the lepromatous end of thespectrum. The precipitin formation is thus in-versely related to the CMI response to M.leprae (9; Myrvang et al., manuscript in prep-aration).The recent development of the sensitive and

highly resolving quantitative immunoelectro-phoretic methods (4) makes more precise stud-ies of even very complex antigen-antibody sys-tems possible. In the present report we in-troduce the crossed immunoelectrophoresiswith intermediate gel (1) for investigation ofantibodies in leprosy.

MATERIALS AND METHODS

Leprosy patients and sera. Sera were obtainedfrom selected leprosy patients at the All AfricaLeprosy and Rehabilitation Training Centre

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BCG ANTIBODY PROFILES IN LEPROSY

(ALERT). The great majority were untreated. Diag-nosis was in each case based on clinical and histopath-ological examination, and classification was madeaccording to the criteria of Ridley and Jopling (13)and Ridley and Waters (14). Criteria for distinctionbetween polar tuberculoid (TT) and subpolar tuber-culoid (TT/BT) cases have been described elsewhere(9). Sera from 12 TT, 12 TT/BT, and 16 patients withlepromatous leprosy (LL) were examined in the pres-

ent study. One of the LL patients suffered fromerythema nodosum leprosum. Venous blood sampleswere obtained, and the sera were stored at -25 C forperiods of up to 3 years before examination.BCG antigen. BCG bacilli, strain Bergen, were

supplied by the BCG Laboratory, Bergen, Norway, as

lyophilized vaccine containing 20 mg (wet weight) ofbacilli per ampoule. The contents of 10 ampouleswere suspended in 0.15 M NaCl and sonically treatedin a Branson sonifier (model B-12) for 30 min at an

output effect of 100 W. The sonic extract was cen-

trifuged at 10,000 x g for 20 min and the supernatantwas collected. The protein concentration of thesupernatant determined by a modified Folin-Ciocal-teau method (7) was 0.8 mg/ml, using human immuno-globulin G as the standard. The supernatant was

stored at - 20 C.Rabbit anti-BCG sera. Four rabbits were immu-

nized according to the following protocol. Each injec-tion was divided into three doses, two given intramus-cularly in each hind leg and one given subcutaneouslyin the neck. Initially 2 ml of a suspension of BCG,strain Bergen, in incomplete Freund adjuvant, con-

taining 4 mg (wet weight) of bacilli per ml, was

injected into each rabbit. After 3.5 weeks, 1 ml ofsonically treated BCG containing 12 mg (wet weight)of bacilli per ml was given. This injection was

repeated at 8 and 13 weeks after the first injection.Ten days after the fourth injection the animals were

bled from the ear. Sera were stored at -20 C.Crossed immunoelectrophoresis. For testing the

rabbit anti-BCG sera, crossed immunoelectrophoresiswas carried out in a micro-modification using glassplates measuring 5 by 5 cm as described by Weeke(17). The electrophoreses were performed using theDL immunoelectrophoresis equipment (Dansk Labo-ratorieudstyr Ltd., Copenhagen, Denmark) and 1%(wt/vol) agarose gel (batch AGS 091 A, Litex, Glost-rup, Denmark) in barbital buffer (pH 8.6), ionicstrength 0.02. The temperature of the cooling waterwas 12 C, and the gel thickness was 1 mm. Theamount of BCG-antigen applied was 10 gliters, andthe separation (first-dimension electrophoresis) was

carried out with a potential gradient of 10 V cm'-I for25 min. The second-dimension electrophoresis wasperformed with a potential gradient of 2 V cm- '

overnight; the second dimension gel contained 12.5,Lliters of rabbit serum per ml.

After electrophoresis the plates were pressed underfilter paper and soft blotting paper (20 min), driedunder a hair drier (15 min), stained with Coomassiebrilliant blue R (Michrome no. 1137, Edward GurrLtd., London) (5 min), destained (5 min, three times),and finally dried again (10 min).

Crossed immunoelectrophoresis with intermedi-ate gel. In this modification of crossed immunoelec-

trophoresis a gel with patient serum is interposedbetween the first-dimension gel and the gel containingthe rabbit antibodies (reference gel) against theantigens. This allows a direct comparison of rabbitand patient antibodies. The theoretical and technicaldetails of this method have been recently reviewed(1).

In the present study the immunoelectrophoreseswere carried out in a micro-modification using glassplates (5 by 5 cm) as described above. The dimensionsof the gels appear in Fig. 2A. In the test plates theintermediate gels contained 16.7 gliters of patientserum per cm 2; in the control plates the same amountof control serum obtained from a healthy Danishperson who had never suffered from leprosy or tuber-culosis was used (she was BCG vaccinated as a childand had a positive Mantoux reaction). The referencegel contained 16.7 gliters of rabbit anti-BCG serumper cm2. In our set-up (1), eight first-dimension elec-trophoreses were run simultaneously in the sameapparatus: one was used for a control plate, the restwere used for test plates. First- and second-dimen-sion electrophoreses and the pressing, washing, andstaining of the plates were carried out as describedabove.To minimize bias, the sera were randomized and

coded prior to the investigation; the code was brokenafter the evaluation of the plates. Examination of theprecipitate patterns was performed by looking fordifferences between the test plate and the correspond-ing control plate: the differences were interpreted bythe method of Axelsen (1). The identified humanprecipitins were titrated semi-quantitativelv by com-paring each precipitate of the test plates with corre-sponding precipitates of plates in which the referenceanti-BCG serum was incorporated in the intermediategel. The human titers were expressed in arbitrarvunits referring to two concentrations of the referencerabbit anti-BCG in the intermediate gel: titer 3 _ 8.3,uliters/cm2 > titer 2 2 0.83 jiliters/cm2 > titer1 > titer 0 = 0 zliters/cm2 (control plate).

Statistical analysis. The significance of the differ-ence, in number of precipitins per serum, between thethree groups of patients was established by using theMann-Whitney U test. P, the probabilitv of differencebeing due to chance, was obtained from tables for U(16).

RESULTSCrossed immunoelectrophoresis of the BCG

antigen against sera from each of four rabbitsimmunized with this antigen showed that one ofthe rabbits had a far stronger precipitin re-sponse than the other animals, i.e., more pre-cipitins and higher titers. It was therefore de-cided to use this antiserum as reference for thestudy of human antibodies.

Figure 1A shows the crossed immunoelectro-phoresis with control serum in the intermediategel and rabbit anti-BCG in the reference gel. Itappears (Fig. 2A) that 19 precipitates could beseen in the control plate although some werevery faint. Precipitates numbers 11, 16, and 17

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were "fuzzy" and antigen number 16 was com-posed of at least two antigenically relatedcomponents since a spur was observed at theanodic "leg" of the precipitate.

FIG. 2. (A) Drawing of Fig. IA with enumeration ofthe reference precipitates. (B) Drawing of Fig. 1B.After identification the human precipitins of theintermediate gel were given arbitrary titers by com-parison to standard plates with fixed amounts ofreference anti-BCG in the intermediate gel: anti-5(titer 1), anti-6 (titer 2), anti-12 (titer 2), anti-17(titer3), anti-18 (titer 1), anti-19 (titer 3). The precipitatesmarked with dots are due to human precipitins whichcould not be identified in this system.

Serum from a patient with lepromatous lep-rosy was included in the intermediate gel of thetest plate of Fig. 1B. Two strong precipitinswere identified as anti-1/ and anti-19 (Fig. 2).Weak antibodies with specificities for antigennumbers 5, 6, 12, and 18 were demonstrated asdeflections (1) of the reference precipitates. Thethree distinct precipitates marked with dotsrepresent human antibodies with no counter-part in the reference antiserum and thereforeare "unidentified" in terms of the rabbit refer-ence antibody specificities. The unidentifiedprecipitins were not analyzed in detail but somegave very characteristic precipitates, e.g., themost anodic one of Fig. 1B which was present inseveral sera as judged from form and position.A total number of 125 antibodies were found

in the 40 sera from the patients: 6 in the 12 TTsera, 17 in the 12 TT/BT sera, and 102 in the 16LL sera. Figure 3A shows the distribution ofpatients according to the total number of anti-bodies found in each serum (criterium A); the"total" number of antibodies includes regularprecipitins seen in the intermediate gel as wellas low-titer antibodies (titer 1) which were onlydemonstrated by deflections of the referenceprecipitates.The specificity of 86 of the 125 human anti-

bodies (69%) could be defined in terms of the19 rabbit precipitins: 5 in the TT sera, 13 inthe TT/BT sera, and 68 in the LL sera. Figure3B shows the distribution of patients by thenumber of these identified antibodies perserum.The remaining 39 human antibodies could

not be identified in terms of rabbit antibodyspecificities, but according to position and mor-phology of the precipitates it was estimated thatthey belonged to about six different specifici-ties. Figure 3C shows the distribution of pa-tients by the number of unidentified antibodiesin each serum.The highly significant differences (P < 0.002)

between the LL and the TT/BT groups and theLL and the TT groups are shown in Table 1. Nostatistically significant differences were foundbetween the TT/BT and the TT groups. The LLgroup had a relatively small overlapping to theother groups (25%T), using the criteria A and B(Table 1) (95% confidence limits: 7.3%7e to52.3%).The specificities of the 86 identified anti-

bodies were confined to 11 of the 19 antigensenumerated in Fig. 2. The distribution of the

FIG. 1. Crossed immunoelectrophoresis with intermediate gel using BCG as antigen and rabbit anti-BCG asreference antiserum. First-dimension electrophoresis with anode at the right and second-dimension electropho-resis with anode at the top. (A) Control plate with serum from a control person in the intermediate gel. (B) Testplate with serum from a patient with lepromatous leprosy. For details see the text and Fig. 2.

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INFECT. IMMUNrrY

patients by the specificity and titer of theantibodies is shown in Fig. 4. It appears that theLL patients, with one exception, accounted forall antibodies with titers 2 and 3. Although theLL group accounted for all antibodies withspecificity for antigens numbers 4, 8, 11, 12, 13,18, and 19, there were 4 LL patients who had noantibodies against any of these 7 antigens. Thedominating antigens, as regards antibody re-sponse in this material, were numbers 5, 6, 17,18, and 19.

12j:IC B IDENTIFIED

10. C:UNIDENTIFIED

4.~~~~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~~~..

NUMBER OF ANTIBODIESFIG. 3. Distribution of 40 leprosy patients by three

criteria. (A) Total number of antibodies in each serum(identified + unidentified); (B) number of identifiedantibodies per serum; (C) number of unidentifiedantibodies per serum. Symbols: 0, 12 TT patients; ,12 TT/BT patients; X, 16 LL patients.

TABLE 1. Statistical analysis of the differencesbetween the three groups of patients according to

number of antibodies per serum

Criterium Comparison Resulta

A LL-TT/BT P < 0.002LL-TT P < 0.002TT/BT-TT NSb

B LL-TT/BT P < 0.002LL-TT P < 0.002TT/BT-TT NS

C LL-TT/BT P < 0.002LL-TT P < 0.002TT/BT-TT NS

a Mann-Whitney U test.b NS, Not significant.

Although "free" antigens in serum, or im-muno-complexes in antigen excess, might havebeen demonstrated with this method (3), nonewas found.

DISCUSSIONCrossed immunoelectrophoresis with inter-

mediate gel (1) is especially suited for studies ofhuman antibodies in infectious diseases since itis possible by this method to obtain informationon the number, specificities, and titers of pre-cipitins in terms of a reference system. Also, freeantigens in serum can be demonstrated (3), andthe sensitivity and resolving power of themethod are higher than obtained with theconventional diffusion-in-gel methods.As BCG is antigenically related to M. Ieprae

and commonly available, we decided to use thisantigen in the present study. Our demonstra-tion of 19 antigens in BCG contrasts to thefinding of Roberts et al. (15) of 48 antigens inthis species. The reason for this discrepancy isnot obvious since it may be attributed both todifferences in antigen preparation, immuniza-tion procedure, and antibody response of indi-vidual rabbits. Reference reagents, above allreference antisera, are therefore highly neededin such complex systems (2, 15) to make studiesin different laboratories comparable. Such ref-erence antisera should define certain antigens,but need not necessarily contain antibodiesagainst all known antigenic components.

Since BCG antibodies in this study weredemonstrated in all lepromatous sera, themethod seems promising as a tool for furtherstudies in this field. In previous studies themaximal number of precipitins to BCG was 5 ina single serum (Myrvang et al., in press),whereas we found that 75% of the sera in thisstudy ('316) had more than 5, and that up to 14different antibody specificities were demonstra-ble in individual sera. The previously demon-strated difference in antibody response in tuber-culoid and lepromatous leprosy (11, 12; Myr-vang et al., in press) was strongly supported byour findings (Table 1).The specificity of the antibodies varied con-

siderably in individual sera, and the complexityin antibody response was higher than expected.On the whole, however, antibodies against anti-gen numbers 5, 6, 17, 18, and 19 were mostfrequently found.The finding of human BCG antibodies which

could not be identified in the reference systemare of interest since they may represent ahuman-specific or perhaps a leprosy-specificantibody response; the majority of unidentifiedantibodies occurred in lepromatous sera (Fig.3C).

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PATIENTS WITH ANTIBODIES AGAINST ANTIGEN NO.:TITER 4 5TTR4 5 6 8 II 12 13 14 17 18 19

le le is '0 0

2 0 0 e 0 771

2 * * x j f

.§~2~Io~I x 3x~ ~ ~ ~ :

FIG. 4. Distribution of 40 leprosy patients according to specificity and titer of 86 serum antibodies. Noantibodies with specificity for antigen numbers 1, 2, 3, 7, 9, 10, 15, and 16 were demonstrated. Each markrepresents one patient. Symbols TT x TT/BT; 0, LL.

Strong CMI against M. leprae in patientswith tuberculoid leprosy and the continuousdecrease in CMI towards the lepromatous end ofthe spectrum has been demonstrated in vitroand in vivo using whole M. leprae bacilli asantigen (9). The studies of the overall antibodyresponse showing the inverse picture of increas-ing response towards the lepromatous end of thespectrum is based on studies on extracts ofmycobacteria containing a multitude of solubleantigens (12; Myrvang et al., in press). Furtherinformation on the aberration of the immuneresponse in leprosy requires study of CMI andantibody response against the same individualmycobacterial antigens. The method intro-duced in the present study and other refinedimmunoelectrophoretic methods (4) can pro-vide exact data to be used as guidelines forpurification of those mycobacterial antigensthat are prone to induce formation of precipitat-ing antibodies in leprosy patients. After purifi-cation, these antigens should then be studiedwith regard to CMI in different patients to see ifthe inverse relationship between CMI and anti-body production is a feature which also is trueon the level of individual mycobacterial antigenmolecules.

ACKNOWLEDGMENT

Birgitte Jacobsen carefully performed the immunoelectro-phoreses.

LITERATURE CITED

1. Axelsen, N. H. 1973. Intermediate gel in crossed and infused rocket immunoelectrophoresis. Scand. J. Im-munol. 2(suppl. 1):71-77.

2. Axelsen, N. H. 1973. Quantitative immunoelectropho-retic methods as tools for a polyvalent approach tostandardization in the immunochemistry of Candidaalbicans. Infect. Immunity 7:949-960.

3. Axelsen, N. H., and C. H. Kirkpatrick. 1973. Simultane-ous characterization of free Candida antigens andCandida antibodies in a patient's serum by means ofcrossed immunoelectrophoresis with intermediate gel.J. Immunol. Methods 2:245-249.

4. Axelsen, N. H., J. Kr0ll, and B. Weeke (ed.). 1973. Amanual of quantitative immunoelectrophoresis,methods and applications. Universitets for laget,Oslo (alias Scand. J. Immunol. 2 (suppl. 1):1-169).

5. Godal, T., B. Myklestad, D. R. Samuel, and B. Myrvang.1971. Characterization of the cellular immune defect inlepromatous leprosy: a specific lack of circulatingMycobacterium leprae-reactive lymphocytes. Clin.Exp. Immunol. 9:821-832.

6. Godal, T., B. Myrvang, S. S. Fr0land, J. Shao, and G.Melaku. 1972. Evidence that the mechanism of immu-nological tolerance ("central failure") is operative inthe lack of host resistance in lepromatous leprosy.Scand. J. Immunol. 1:311-321.

7. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

8. Moran, C. J., G. Ryder, J. L. Turk, and M. F. R. Waters.1972. Evidence for circulating immune complexes inlepromatous leprosy. Lancet ii:572-573.

9. Myrvang, B., T. Godal, D. S. Ridley, S. S. Froland, andY. K. Song. 1973. Immune responsiveness to Myco-bacterium leprae and other mycobacterial antigensthroughout the clinical and histopathological spectrumof leprosy. Clin. Exp. Immunol. 14:541-553.

10. Navalkar, R. G. 1971. Immunologic analysis of Mycobac-terium leprae antigens by means of diffusion-in-gelmethods. Int. J. Leprosy 39:105-112.

11. Norlin, M., R. G. Navalkar, 0. Ouchterlony, and A. Lind.1966. Characterization of leprosy sera with variousmycobacterial antigens using double diffusion-in-gelanalysis. III. Acta Pathol. Microbiol. Scand.67:555-562.

12. Rees, R. J. W., K. R. Chatterjee, J. Pepys, and R. R. Tee.1965. Some immunologic aspects of leprosy. Amer.Rev. Resp. Dis. 92(suppl.):139-149.

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13. Ridlev, D. S. and W. H. Jopling. 1966. Classification ofleprosy according to immunity. A five group svstem.Int. J. Leprosy 34:255-273.

14. Ridley, D. S.. and M. F. R. Waters. 1969. Significance ofvariations within the lepromatous group. Leprosy Rev.40:143-152.

15. Roberts, D. B., G. L. Wright, Jr., L. F. Affronti, and M.Reich. 1972. Characterization and comparison of myco-bacterial antigens by two-dimensional immunoelectro-

INFECT. IMMUNITY

phoresis. Infect. Immunity 6:564-57,3.16. Siegel, S. 1956. Nonparametric statistics for the behav-

ioral sciences. McGraw Hill Book Co., Inc., New York.17. Weeke, B. 1973. Crossed immunoelectrophoresis.

Scand. J. Immunol. 2:(suppl. 1):47-56.18. Wemambu, S. N. C., J. L. Turk, M. F. R. Waters, and R.

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