Scand. J. Immunol. 36, 371-384, 1992
Antibodies against Secreted and Non-Secreted Antigens in Mice after Infection with Live Mycobacterium tuberculosis A. VERBON*t. S, KUIJPER*, H. M. JANSENf, P. SPEELMANj & A. H. J. KOLK* *N, H, Swellcngrebel Laboratory orTropical Hygiene, Royal Tropical Institute, and Divisions of tPulmonary and ^Internal Medicine, Academic Medical Center, Amsterdam. The Netherlands
Verbon A, Kuijper S, Jansen HM.Speelman P. Kolk AH J, Antibodies against Secreted and NonSecreted Antigens in Mice after Itileetion with Live Mvcohacleriiim luhcrcuiosi.s. Scand J [mmunol I992;36:37I 84 Mice from four different inbred strains were infected with live Mycobacierium luhcrcuiosis and the immune response to M. /u/jfr^Wavw was followed for 24 weeks, using Western blotting. Nearly all miec, irrespective of H-2 lype, reacted with the 3S-kDa protein band. Antibodies ;igainst this secreted 3f:. A. Verbon. N. H. Swi'lk'nurebcl Laboratory of Tropical Hygiene. Royal Tropical Jnstiiute. Meibergtlreef39, 1105 AZ Anisicnlani, The ,\'eilicrhiiid\
An association between HLA haplotype and antibody production in humans has been observed in malaria and Rhesus immunization [1, 2], In patients with tuberculosis an association has been found between DR2 and litres of antibodies to the M. tuherculo.sis specific epitope of the 38-kDa protein defined by TB72 [3], In mice, H-2 linked genes are known to influence the antibody repertoire following hyperimmunization with live M. tiibercidosi.s
[4], live M.
bacilli [5]. Culture filtrate antigens, or culture supernatant antigens, are those antigens which are secreted into the medium during growth and antigens from lysed dead bacilli, Differenees in amount and composition of proteins in culture supernatant are almost inevitable given the wide range of culture conditions used: pellicle growth in Proskauer and Beck medium [8]; growth on the surface ofSauton medium [9, 10]; cultivation in
hovis
roller bottles and addition of 0,05'MJ Tween 80 to
BCG (5], M. tuherculo.sis extract [6] or culture filtrate antigens [7], However, the 3S-kDa protein is preferentially recognized by mice of different strains [4].
Sauton liquid medium [11]; cultivation in Eagle's and Sauton medium in tissue culture plates [12] and shaking culture [13], Proteins found in culture fluid can be described as secreted oreytosolic according to their localization index [14].
Mice infected with live M. hovis BCG develop antibodies directed against antigens pre.scnt in culture filtrate. Such antibodies cannot be detected in sera from mice infected with dead
To determine which antigens provide the first stimulus for the early humoral immune response, we infected miee from four different inbred
371
372
A. Verbon et at. TABLE I. Reactivity and shift in molecular weights ofprotein bands in five different mycobaeterial species and E. coli as identified by monoclonal antibodies in Western blot Sonicate of Monoclonal antibody F67-18 F29-29 F67-I9 F!26-2 F67-IJ FI16-5 F29-47 F23-49
M. tub* M. avi M. gord M. kan M. ter E. coli
65t 42 38 34 33 24 21 \h
65 — — 34 33 26 23 —
65 — 36 33 — — —
65 — — 35 33 24 23 —
65 — — 36 33 NT —
65 — — 37 — — —
—
—
Abbreviaiions used: —, no reaction: NT, not tested. * M. tub. M. Iuhercutosi.s: M. avi, M. arium; M. gord, M. gordonae: M. kan. M. tiun.sasii; M. ter. M. terrae. t Apparent molecular weights in kDa are indicated. X Results obtained with MoAbs F67-i, F67-5 and FI26-5, all cross-reaetive with the 36-kDa proline-rich protein of M. teprae [21], are combined.
strains with live M. tuberculosis, and followed the immune response to M. tubercutosis sonicate and culture supernatant for 24 weeks, using Western blotting. The specificity of the antibodies was tested by Western blotting with five different mycobacterial sonicates. It is hoped thai these studies will identify antigens which may be ttseful in serodiagnosis of tuberculosis in man.
MATERIALS A N D M E T H O D S Mice. Five CBA/ea (H-2'^). five BALB/c (H-2''), live C57bl/6 (H-:") and five Swiss (H-2') 8-week-old mice were obtained from the central laboratory for animal facililies in Amsterdam. M. ttibereulosis infection. M. tuberculosis was grown for 2 weeks on Lowenstein-Jensen medium at 37 C and dilutedinphosphate-buflered saline (PBS). pH 7,4. Five mice of each strain were injected intrapcritoneally with approximately 5 ! 0 x 10'' live bacilli in 0.5 ml of PBS. Biood was oblained from the retrobulbar sinus on day 0 (negaliveeontml)andafter2. 4, 6, 8, 10, 12, 15, 18.21 and 24 weeks using heparin capillaries. The blood was separated by centrifugation at 13,000 g for 2 min and the plasma was stored at — 20 C. Mice were killed at 24 weeks. Second injeetion. Eighteen weeks after the infection with M. tubercutosis. two mice of each strain were again injected intraperitoneally with 5 lOx 10' freshly prepared live M. tubercutosis in 0.5 ml of PBS. Preparation ot culture supernatant and sonicate. The M. tubercutosis strain used was isolaled from a palient with tuberculosis. The bacteria were eultured on Lowenstein-Jensen medium at 37 C and subcultured every 2 months. M. tuberculosis was first adapted to Sauton medium by transfer from Lowenstein-Jensen medium
to a 25-ml eapped bottle containing 10 ml of Sauton liquid medium (2 mM MgS04, lOmM citric aud, 2.5 mM K;HPO4, 30 mM asparagine, 0.1 mM ferric ammonium eitrate, S30 mM giycerol, in Milli Q water, final pM 7.2). After ineubalion at 37 C for 3 weeks, the bacterial suspension was transferred lo ii 1-litre flask eontaining 250 ml of Sauton liquid medium and ineubated in a horizontal position at 37 C for 3 weeks (designated prccullure). For preparation ol' the culture supcrnatani, 30 ml of preculture was inoculated into 500 ml of Sauton liquid medium in a 1-litre conical flask, with a cotton plug. This suspension was cultured at 37 C for 39 days with shaking at 120 rpm, using a gyrorotary shaker (New Brunswick Scientific, Edison, NJ, USA). Bacteria were killed by pasteurization of the suspension in a water bath at 75 C for 15 min: the temperature of the suspension rose from 20 C to 71 C in 10 min and remained at 71 C for 5 min. The suspension was centrifuged at 16,000 g for 20 min at 4 C. The supernatant was centrifuged again under the same conditions. Baeteria in the pellet were washed twice with distilled water, sonicated in distilled water for I h at 50% duty cycle with 100 W power, using a Branson sonitier, and then lyophilized. The supernatant was concentrated in a vacuum rotary evaporator. The remaining volume was dialysed for 2 days against distilled water, using a dialysis membrane with a cut-off point of 10 kDa, and subsequently lyophtlized. Antigens detectable in sodium dodeeyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and Western blot analysis were shown to be a selection of antigens found in sonicates of M. tubercutosis [13]. Sonicates of M. tuherculo.Kis., M. kansasii, M. avium serotype 4, M. terrae. M. gordonae and E. coli were prepared as deseribed for M. tubercutosis. Sodium dodecyt .sulphate polyacrylamide gel etectroptwresis. SDS-PAGE was done as described by Laemmli [15] on 13"A> gels.
Murine Antibodies to Live M. tuberculosis Western bloi analysis. Antigens separated by SDS PAGE were transferred to nitrocellulose and used for Western blotting as described previously [16]. Mouse sera were diluted 1 ; 100, Horseradish peroxidaselabelled anti-mouse immunoglobulin G heavy and light chams (lgG H + L) was obtained from the Pasteur Institute. Pans. France. The following proteins were identified with monoclonal aniibodies (MoAbs): 65 kDa with F67-18 [17]; 42 kDa with F29-29. 19 kDa with F29-47. 16 kDa with F23-49 [18]: 30 .11 kDa wilh MoAb 3A8 [19] and with anti-p32 rabbit antiserum [20]: 33 kDa with F67-I [21]: 38 kDa with F67-19. 34 kDa with FI26-2, 24 kDa in mycobacterial sonicates and 24 kDa and 30 kDa in culture supernatant with FII6-5 [13]: 10 kDa with MoAb SA-12 |22]. Cross-reactivity of these MoAbs and the possible shift in molecular weight of the recognized proteins in different species is shown ill Table I, MoAb SA-12 was kindly supplied by P. Minden. La Jolla. California, USA and P. Kelleher. The Woodlands, Texas. USA and MoAb 3A8 by P. Das. Amsterdam, The Netherlands. Anti-p32 rabbit antiscrum was a generous gift from J. de Bruyn, Brussels, Belgium. The 65-kDa protein has also been designated 2T: the 38-kDaprotein.3T: the 33-kDii antigen, 7T, the 30 31kDa protein, 9T: and the l9-kDa protein, 12T [23]. ELISA wilh recombinuni DNA aniigens. Recombinant .18 kDa protein (24] and recombinant 65 kDa protein kindly supplied from the WHO IMMTtJB. IMMLEP Bank by Dr J, van Embden. Bilthoven. The Netherlands. MPT59 [25] was a generous gift from S. Nagai, Tokyo. Japan. The coating concentrations of the recombinant antigens, diluted in 0.05 M sodium bicarbonate buffer. pH 9.6. were 1 /ig, ml for 38 kDa. 3 /ig, ml for 65 kDa and 4/ig, mi for MPT59, Plasmas of the mice were diluted 1:100. The ELISA technique used has been described in detail elsewhere (13]. Colony forming units (CFli). Spleen, liver and lymph nodes were harvested from the mice 24 weeks after ihe infection with M. luhercuhisi.s and homogenized wilh a glass tube and leilon pestle in respectively I ml. 2 ml and 300 /il of sterile PBS. Tenfold serial dilutions of the homogenate were plated on Middlebrook 7HII agar (Difco. Detroit. Ml) enriched with: oleic acid, albumin, dextrose, catalase and Triton (OADC and WRL1.19). The plates were incubaled for 5 weeks at 37 C and colonies were counted.
RESULTS Antibody repertoire of different mouse strains AtO, 2,4, 6. 8. 10. 12, 15. 18.21 and 24 weeks after infection with live M, tuberculosis the antibody response in fourdifferent strains of mice was tested by Western blotting. None of the mice reacted with M. tuberculosis sonicate before infection with live M. tuberculosis. Mice from different strains showed a pronounced dilTerence in antibody repertoire at 8 weeks after infection (Fig. I). Apparent molecular weights of the
373
protein bands recognized were established by the use of MoAbs against known mycobacterial proteins (Fig, 1. lane 23). The bands seen in Western blot were all from protein antigens, since pronase digestion of the M, tuberculosis sonicate prior to SDS-PAGE abolished all reactions (results not shown). Among the individual mice within the same strain minor differences were seen in the BALB c. Swiss and CBA/ca mice. BALB/c and Swiss mice reacted with a variety of protein bands (Fig, I, lanes 7 11 and 12-16. respectively). Four of the five CBA/ca mice (Fig. 1. lanes 19-22) reacted only with the 38-kDa protein band, one mouse (lane 18} reacted with the 38-kDa and 33kDa protein bands. Three C57BI/6 mice (Fig. I, lanes 1. 2, 4) recognized protein bands of 38 kDa and 19 kDa. One mouse recognized the 19-kDa protein band and some antigens of higher molecular weights (lane 3). The last C57BI/6 mouse (lane 5) did not recognize M. tuberculosis antigens in Western blots, although mycobactcria could be cultured from the spleen and lytnph nodes of this mouse. All mice recognized fewer protein bands in culturesupcrnatant (Fig. 2) thaninsonicateof A/. tuberculosis (Fig. 1). Interestingly, none of the mice showed reaction with a protein band of 3031 kDa. This antigen was present in both sonicate and culture supernatant as shown by the reactions of MoAb 3A8 [19] and the rabbit antiserum against the p32 protein (20]. The reaction of these antibodies with the 30 31-kDa protein band in culture supernatant is shown in Fig. 3. Virtually all mice recognized the 38-kDa protein band (Table II). The 19-kDa protein band was recognized by 12 mice from three strains, but not by any of the CBA/ca mice, A reaction with the 33-kDa protein band was found in 10 mice, eight of whieh were from the Swiss and BALB/c strain. Reaction of the mice with other proteins was strain related (Fig. I), For example, only Swiss mice reacted strongly with the 65-kDa protein band (Table II), Reaction of different mou.se strains during, the course of infection Following intraperitoneal infection, the lgG (H -I- L) antibody response was not detectable at 2 weeks, was weak at 4 weeks and thereafter increased both in intensity and in the number of protein bands. A representative example is shown in Fig. 4. There was no subsequent decrease in
374
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19 21 25 zn :^z
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65
43
42 F29-29 38 F67-19 34 F126-2 33 F67-1
30
20
F67-18
24
F116-5
19
F29-47
•16
F23-49
•10
SA-12
FIG. 1. Antibody response 10 M. tuberculosis sonicate 8 weeks after infection with live M. tuherculo.\is. lesled in Western blot. Lanes I .•>. C57BL;6 mice: lanes 7 1 L BALB/c mice; lanes 12 16. Swiss mice; lanes 18 22.CBA,ai miee; lane 23. reactiviiy of MoAbs. Lanes 6 and 17 arc filled only wilh diluent and conjugate and show the weak reaction of the conjugate at 65 kDa. .14 kDa. 16 kDa and 10 kDa. The positions of ihe molecular mass markers are shown on the left. Protein bands with which the MoAbs reacted are indicated on the right.
antibody response to any of the antigens up to 24 weeks wheti the mice were killed. In all four strains of mice antibodies against the 38-kDa protein band were the first antibodies to appear. Later in the infection the 33-kDa and 19-kDa protein bands were recognized. To study the effeet of a second challenge, two mice (presented iti Fig. I in lanes I and 2, 7 and 8, 12 and 13, 20 and 22) from eaeh strain received a second injection with live M. tuherculo.'iis at week 18. Plasma samples from these mice were taken at week 21 and mice were killed at week 24. The intensity of the antibody response was increased compared with the reaction before the second injection (data not shown). More protein bands were recognized by all mice after the second
injection. Irrespective of the strain, all eight mice reacted with protein bands of 38 kDa. 33 kDa and 19 kDa, although inter-strain differences were still present (Table III). There was no difference in the reactions of plasma samples taken at week 21 and at week 24. Reaction with recombinant antigens in ELISA None of the miee reacted witb the 38-kDa recombinant protein in ELISA before the infection with M. tuberculosis (Fig. 5). Plasma from miee that reacted with the 38-kDa protein band in Western blot also reacted with the 38-kDa recombinant protein in FLISA (Fig. 5). C57B1/6 mice 3 and 5 did not react with the 38-kDa protein band
Murine Antibodies to Live M. tiibereulosis 1 Oi US n? 09 II (]2 ii'4 116 on 1(1
IJ 12
n u
17 ir>
in
375
19 21 ^u
9467
- 65
F67-18
38 34 30
F67-19 F126-2 F116-S
24
F116-5
19
F29-47
10
SA-i:3
43
30
20
14-
FIG. 2. Antibody response to M. /W/KTCH/O.^K culture supernatant 8 weeks after infcelion with live M- tuberculosis, tested In Western biol. Lanes 1 5. C57BL/6 miee; lanes 7 11. BALB/c miee; lanes 12 16. Swiss mice; lanes !8-22.CBA,ca mice; lane 2."^. reactivity of MoAbs. Liines 6 and Ware lilledonly wit h diluent and conjugate and show the weak reaction of the conjugate at 65 kDa. 34 kDa and 10 kDa. The positions of the molecular mass markers are shown on the left. Protein bands with which the MoAbs reacted are indicated on the right.
in Western blot or with the 38-kDa reeombinant protein in ELISA. None of the mice reaeted with a 30-31-kDa protein band in Western blot and none showed a reaction with the purified MPT59 in ELISA (Fig. 6). MoAbs 3A8 and FI16-.^. rcaetive with a 30-kDa band in culture supernatant in Western blot, both reacted with the purified MPT59 in ELISA. The reeombinant 65kDa protein was recognized only by Swiss mice 12 weeks after infection (Fig. 7). None of the other mice showed any reaction with the 65-kDa antigen in ELISA or Western blot. M. tuberculosis colony counts in organs of the injected mice Macroscopic examination showed no granulo-
mata or other signs of infection in liver, spleen or lymph nodes of the infected mice. However, following culture on solid medium a considerable number of viable bacilli could be demonstrated 24 weeks after inoculation (Fig. 8). At least 10' live M. tuberculosis were found in all the spleens. Infection was greatest in the organs of the CBA/ ca mice. The number of M. tuberculosis in the spleens of BALB/c and C57B1,6 miee was cotnparable to that in the other two strains, but tbe number in liver and lymph nodes was somewhat lower. In the organs of mice injected for a second time with live M. tuberculosis, the number of viable bacilli was the same as in mice injected only once. Ziehl-Neelsen staining of the organs correlated with the colony counts, indicating that few dead bacteria were present. These results indicate
376
A. Verbon el at.
1
94 67
-
43
-
30
-
that all miee from the four strains were slill exposed to live M. lubercutosis 24 weeks after in fee tion.
2
Species-specificity oJ antibodies
20
-
14
FIG. 2. Reactivity of monoclonal antibody 3A8 and anti-p32 rabbit antiserum (both directed against the antigen 85 complex) with M. tuberculosis culture supernatant in Western blot, l.anc 1. MoAb 3A8; lane 2, rabbit antiserum directed against p32. The positions of the molecular mass markers are shown on the left.
TABLE 11. Recognition m Western blot of some M. tuberculosis protein bands, found in sonicate and culture supernatant, by differeni mouse sirains 8 weeks after infection wilh live M. tuberculosis Protein band* Mouse strain
65
38
33
19
C57BL/6(M = 5) BALB/c (n = 5) Swiss (n — 5) CBA/ca (M = 5) Total (n = 20)
Of
3 5 5
1 3 5 1 10
4 3 5 0 12
0 5 0 5
5 IS
• Apparent molecular weights in kDa are indicated, t Number of mice recognizing the protein band.
The antibody repertoire of the same two mice from each strain (presented in Fig. I in lanes 1 and 3, 7 and 9. 12 and 14, 21 and 22) was followed in Western blot using sonicates of M. tuberculosis. M. atium. M. gordotiae. M. kansasii, M. terrae and E. coli. MoAbs were used to identify corresponding protein bands in the sonicates, when possible (Table I). Plasma samples were taken before and at 6, 15 and 24 weeks after infection with M. tubcrcttto.'iis to study the specificity ofthe antibodies. A representative example of plasma from one mouse from each strain taken 24 weeks after infection is shown in Fig. 9. There were minor differences in the reactions ofthc two mice from each strain. None ofthe mice tested showed any reaction with M. tuberculosis or E. coli sonicate prior to infection with M. tuberculosis. Sera from CBA/ca mice reacted only with the 38kDa protein band of M. tuberculosis, but not at any time with the other mycobacterial strains or with E. coti (Fig. 9 and Table IV). This implies that the antibodies in CBA/ca mice against the 3S-kDa protein band are specific for M. tubercuto.sis. BALB/c and C57Bi/6 mice showed some reaction with a 19-kDa protein band in M. kansasii. 6 and 24 weeks after infection, indicating a limited specificity of antibodies against this antigen (Table IV). No cross-reaction of antibodies directed against the 33-kDa protein band of M. tuberculosis was seen in any of the mice, although a similar 33-kDa protein was present in M. kansasii as shown with the cross-reactive MoAb F67-I (Table I). Swiss mice reacted with many protein bands in M. tuberculosis sonicate 6 weeks after infection and also reacted with protein bands in M. avium and M. katuasii. Twentyfour weeks after infection cross-reaction was found with proteins from all other mycobacterial strains and even with E. coli. These findings suggest that the murine antibodies directed against the 38-kDa and 33-kDa protein bands are specific for M. tubercutosis. Antibodies against the 19-kDa protein band showed limited specificity.
Murine Antibodies to Lire M. tuberculosis 01
nj 02
riii
()•> 07 09 1 , 13 13 17 19 21 i ri6 08 10 12 U \f, Ifl ?0 ?2
94 67 43
- 65
F67-18
- 42
F29-29
- 38 F67-19 i- 34 F126-2 i- 33 F67-1
3O
20
14
FIG. 4. Antibody response over time in a Swiss mouse (presented in Fig. 1, lane 15) to M. tuhercutosis sonicate (lanes t 11) and culture supernatant (lanes 12 22) after a single injection with live M. tuberculosis, tested in Western blot. Lanes: I and 12, at U weeks; 2 and 13, at 2 weeks after infection; 3 and 14,4 weeks: 4 and 15, 6 weeks: 5 and 16, 8 weeks; 6 and 17, iO weeks; 7 and IS, 12 weeks; 8 and 19, 15 weeks; 9 and 20, 18 weeks; 10 and 21, 21 weeks; 11 and 22, 24 weeks; lane 23, reactivilv of MoAbs with sonicate as described in Fig. I. The posilions ofthe molecular mass markers are shown on the left. Protein bands wilh whieh the MoAbs reacted are indicated on the right.
TABLI; III. Recognition in Western blot of immunodominant M. tubercuto.sis protein bands by diflerent mouse strains after a second injection with M. tuberculosis at week 24 Protein bands* Mouse strain
65
50 42
40
38
33 28
26
19
16
C57BL/6(« = 2) BALB/c (« = 2) Swiss (rt = 2) CBA/ca (« = 2) Total (n = 8)
Of 2 2 0 4
I 2 2 0 5
1 0 0 0 1
2 2
2
2
0
r-l
Ol
Ol
2 2 8
2
1 2 2
?
1
6
1
2 2 8
1 I 4
Ol
2 6
2 8
0 r-l
0
* Apparent moleeular weights in kDa are indicated. t Number of mice recognizing the protein band.
377
378
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•e 2 . 0 -
1.0
•A* 0 12 C57BI/6
0 12 BALB/c
0 12 Swiss
0 12 weeks CBA/ca
FIG. 5. Antibodies to the reeombinant 38-kDa protein determined in mice from four different stratns before and 12 weeks after infection with M. tuberculosis as determined by ELISA. Plasmas were diluted 1:100; each point represents one motise.
E 3.0 c in o CD
U
c
e 2.0 H
o n n
1,0-
TT 0 12 C57BI/6
0 12 BALB/c
0 12 Swiss
0 12 weeks CBA/oa
FIG. 6. Antibodies to the MPT59 protein (aniigen 85B) in mice from four different strains before and 12 weeks after infection wilh M. tuhcnulosis as determined by ELISA. Plasmas were diluted 1:100; each point represents one mouse.
Murine Antibodies to Live M, tuberculosis
379
3.0i
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BALB/c
0
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Swiss
0
12 weeks
CBA/ca
FtG. 7. Antibodies to the recombinant 6.S-kDa protein determined in mice from four difVercnl strains before atid 12 weeks after infection with A7./H/JCOH/O.V/A as determined hy ELISA. Plasmas were diluted 1:100: each point represents one mouse.
DISCUSSION We sttidicd the early antibody response to M. tuberculosis iintigcns by infecting inbred mice of diflcrent H-2 types with live M. luhcriulosis. Using Western blot analysis and MoAbs against known antigen.s. we identified the protein bands recognized by the mice. Our rcsttlts in EI.ISA with Ihe recombinant 65-kDa and 38-kDa proteins and the MPT59 protein suggest that the infected mice and our reference MoAbs react with the same proteins as shown in Western blot (Figs 1 and 2), Our results infer, but do not prove, that the other proteins recognized by the infected mice are the same a.s those recognized by the reference MoAbs. The antibody repertoire found in trtice of four dilferent H-2 types showed pronounced dilVerences, but in all four strains antibodies against a 38-kDa protein band were the first to appear. The 38-kDa protein i,s known to be secreted in vitro [13, 14, 26], Our results suggest that this protein is also secreted in vivo by M. tuberculosis and is therefore available for recognition by B cells at an early stage ofinfection. The 38-kDa protein was also recognized by mice after infection with live
M. hovis BCG [5], Although the immune response to A/, tuberculosis probably dificrs between species, the 38-kDa antigen seems lo be irnmunodominant at the B-cell level in man [27], iti guinea pigs [2H] and in mice (this study) [4]. The 19-kDa protein has previously been described as the secreted 21-kDa aniigen 113] and was shown to have a signal sequence [29], The protein band corresponding to ihis antigen was recognized by nearly all BALB/c. Swis.s and C57BL/6 tnice after infection with M. tuberculosis. The aniibodies against the 19-kDa prolein band appear relatively early, although later than anhbodies against the 38-kDa protein. In humans the antibody response against the 19-kDa antigen also suggests that this secreted antigen evokes an early immune response after infectioti with M. tuberculosis [30]. Later in Ihe infection a protein band of 33 kDa is recognized mainly by BALB/c and Swiss mice. We found that the 33-kDa antigen is present in sonicate, but not in culture supernatant of .M. tuberculosis and. thus, is not secreted [13], The 33kDa antigen of M. tuberculosis corresponds lo the proline-rich 36-kDa antigen of M. leprae [21 ]. The apparent molecular weight of this antigen deter-
380
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CFU spleen
CFU Iver
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FIG. 8, Colony forming units in liver, spleen and lymph nodes of (A) CBA/ca, (B) BALB/c. (C) Swiss and (D)C57BI/'6 mice. The organs were harvested 24 weeks after infection with 5 10 x 10^ live M. luberculo.sis. Each point represents the total number of colony forming units in the organ of one mouse: • , mice injected only once with live M. tuberculosis; | . mice injected twice with live M. mbercuiosis.
CBA 1 2 3 4 5 6
BALB/c 1 2 3 4 5 6
Swiss 1 2 3 4 5 6
C57B1/6 1 2 3 4 5 6
94-
67-!
43-i 30-1
14-1
FIG. 9, Western blot analysis of different mycobacterial sonicates and E. coli sonicate with sera from CBA/ ca and BALB/c mice 24 weeks after the lirst injection wilh live M. tuberculosis and from Swiss and C57B1/6 miee 6 weeks after the second injection. Lane I, M. luberculo.sis: lane 2. M. aviunn lane 3. M. gordouac; lane 4. M. kan.saxii: lane 5, M. lerrae: lane 6. E. coli. The positions ofthe molecular mass markers are shown on the left of each two lanes.
Murine Antibodies to Live M. tuberculosis
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mined by SDS-polyacrylamide gel electrophoresis immunoperoxidasc assay using MoAb F47-9 was 36 kDa [31], but in Western blot the molecular weight was 33 kDa [21], A similar protein was identified in other mycobacterial species using cross-reactive MoAbs F67-I, F67-5 and F126-5 (Table I) [21]. This antigen of M. leprae is immunodominant in patients with lepromatous leprosy [31]. The value of the 33-kDa antigen in a serodiagnostic test for tuberculosis has not yet been established. Not all mice reacted with the 65-kDa heat shock protein. This immunodominant prolein [32] indticed strong antibody responses in Swiss and Balb/c mice after the second injection hut not in the other two strains, implying thai the antibody response against the 65-kDa antigen is strain dependent. This strain dependency was also found by Hiiygen ct al. [5]. after infection with live M. hori.s BCG. but in all outbred mice tested antibodies against Ihc (i5-kDa protein band were present [33],
viable M. tubcrculosi.s. Twenty-four weeks after the initial infection we found comparable amounts of colony forming units in three ofthe four mice strains. CBA/ca mice had higher counts of bacilli in all ihree organs; this was also found after infection with live M. hovis BCG [35]. Few dead mycobacteria were present. The marked difference in antibody repertoire is thus due lo strain differences and not to nurnbers of infecting bacilli. Specics-speciticily of the antibody response after infection with M. tuberculosis was tested in Western biol with four other mycobacterial sonicates and E. coli sonicate. In CBA/ca mice the response to the antigen of 38 kDa was specific for M. tubercidosis. Inothermouse strains antibodies against the 3S-kDa antigen also seemed to be specific for M. tuberculosis. So far almost all the polyclonal rabbit sera and murine MoAbs against the 38-kDa antigen are unique for the M. tuberculosi.s complex [7. 36, 37]. although a similar protein is present in other mycobaclcria [38], The antibody response against the 33-kDa protein band was M. tuberculosis specific. This may be explained by the fact that we infected mice with live M. tuberculosis. We have prepared crossreactive MoAbs against this prolein [21], but our MoAbs were produced after immunization with dead bacilli in Freund's adjuvant,
In our study mice did not react with the 30-31kDa secreted protein family in Western blot. although we proved that this antigen was present in both sonicate and culture supernatant (Fig, 3), This secreted protein family is also known as the antigen 85 complex [25] and consists of three components encoded by three separate genes [34], In ELISA none of the mice reacted with MPT59 Murine antibodies against the 38-kDa prolein (antigen H5B). In contrast, antibodies against the were present early in Ihc infection and seemed to antigen 85 complex have been detected after be specific for ihc M. tuberculosis complex. immunization with live M. bovis BCG [5], The Antibodies against the 33-kDa protein were humoral immune response against two mycobacpresent in all mice after a second injection with terial species, both belonging to the M. tubcrculolive M. tuberculosis. These antibodies did not ,si.
Antibodies against Secreted and Non-Secreted Antigens in Mice after Infection with Live Mycobacterium tuberculosis A. VERBON*t. S, KUIJPER*, H. M. JANSENf, P. SPEELMANj & A. H. J. KOLK* *N, H, Swellcngrebel Laboratory orTropical Hygiene, Royal Tropical Institute, and Divisions of tPulmonary and ^Internal Medicine, Academic Medical Center, Amsterdam. The Netherlands
Verbon A, Kuijper S, Jansen HM.Speelman P. Kolk AH J, Antibodies against Secreted and NonSecreted Antigens in Mice after Itileetion with Live Mvcohacleriiim luhcrcuiosi.s. Scand J [mmunol I992;36:37I 84 Mice from four different inbred strains were infected with live Mycobacierium luhcrcuiosis and the immune response to M. /u/jfr^Wavw was followed for 24 weeks, using Western blotting. Nearly all miec, irrespective of H-2 lype, reacted with the 3S-kDa protein band. Antibodies ;igainst this secreted 3f:. A. Verbon. N. H. Swi'lk'nurebcl Laboratory of Tropical Hygiene. Royal Tropical Jnstiiute. Meibergtlreef39, 1105 AZ Anisicnlani, The ,\'eilicrhiiid\
An association between HLA haplotype and antibody production in humans has been observed in malaria and Rhesus immunization [1, 2], In patients with tuberculosis an association has been found between DR2 and litres of antibodies to the M. tuherculo.sis specific epitope of the 38-kDa protein defined by TB72 [3], In mice, H-2 linked genes are known to influence the antibody repertoire following hyperimmunization with live M. tiibercidosi.s
[4], live M.
bacilli [5]. Culture filtrate antigens, or culture supernatant antigens, are those antigens which are secreted into the medium during growth and antigens from lysed dead bacilli, Differenees in amount and composition of proteins in culture supernatant are almost inevitable given the wide range of culture conditions used: pellicle growth in Proskauer and Beck medium [8]; growth on the surface ofSauton medium [9, 10]; cultivation in
hovis
roller bottles and addition of 0,05'MJ Tween 80 to
BCG (5], M. tuherculo.sis extract [6] or culture filtrate antigens [7], However, the 3S-kDa protein is preferentially recognized by mice of different strains [4].
Sauton liquid medium [11]; cultivation in Eagle's and Sauton medium in tissue culture plates [12] and shaking culture [13], Proteins found in culture fluid can be described as secreted oreytosolic according to their localization index [14].
Mice infected with live M. hovis BCG develop antibodies directed against antigens pre.scnt in culture filtrate. Such antibodies cannot be detected in sera from mice infected with dead
To determine which antigens provide the first stimulus for the early humoral immune response, we infected miee from four different inbred
371
372
A. Verbon et at. TABLE I. Reactivity and shift in molecular weights ofprotein bands in five different mycobaeterial species and E. coli as identified by monoclonal antibodies in Western blot Sonicate of Monoclonal antibody F67-18 F29-29 F67-I9 F!26-2 F67-IJ FI16-5 F29-47 F23-49
M. tub* M. avi M. gord M. kan M. ter E. coli
65t 42 38 34 33 24 21 \h
65 — — 34 33 26 23 —
65 — 36 33 — — —
65 — — 35 33 24 23 —
65 — — 36 33 NT —
65 — — 37 — — —
—
—
Abbreviaiions used: —, no reaction: NT, not tested. * M. tub. M. Iuhercutosi.s: M. avi, M. arium; M. gord, M. gordonae: M. kan. M. tiun.sasii; M. ter. M. terrae. t Apparent molecular weights in kDa are indicated. X Results obtained with MoAbs F67-i, F67-5 and FI26-5, all cross-reaetive with the 36-kDa proline-rich protein of M. teprae [21], are combined.
strains with live M. tuberculosis, and followed the immune response to M. tubercutosis sonicate and culture supernatant for 24 weeks, using Western blotting. The specificity of the antibodies was tested by Western blotting with five different mycobacterial sonicates. It is hoped thai these studies will identify antigens which may be ttseful in serodiagnosis of tuberculosis in man.
MATERIALS A N D M E T H O D S Mice. Five CBA/ea (H-2'^). five BALB/c (H-2''), live C57bl/6 (H-:") and five Swiss (H-2') 8-week-old mice were obtained from the central laboratory for animal facililies in Amsterdam. M. ttibereulosis infection. M. tuberculosis was grown for 2 weeks on Lowenstein-Jensen medium at 37 C and dilutedinphosphate-buflered saline (PBS). pH 7,4. Five mice of each strain were injected intrapcritoneally with approximately 5 ! 0 x 10'' live bacilli in 0.5 ml of PBS. Biood was oblained from the retrobulbar sinus on day 0 (negaliveeontml)andafter2. 4, 6, 8, 10, 12, 15, 18.21 and 24 weeks using heparin capillaries. The blood was separated by centrifugation at 13,000 g for 2 min and the plasma was stored at — 20 C. Mice were killed at 24 weeks. Second injeetion. Eighteen weeks after the infection with M. tubercutosis. two mice of each strain were again injected intraperitoneally with 5 lOx 10' freshly prepared live M. tubercutosis in 0.5 ml of PBS. Preparation ot culture supernatant and sonicate. The M. tubercutosis strain used was isolaled from a palient with tuberculosis. The bacteria were eultured on Lowenstein-Jensen medium at 37 C and subcultured every 2 months. M. tuberculosis was first adapted to Sauton medium by transfer from Lowenstein-Jensen medium
to a 25-ml eapped bottle containing 10 ml of Sauton liquid medium (2 mM MgS04, lOmM citric aud, 2.5 mM K;HPO4, 30 mM asparagine, 0.1 mM ferric ammonium eitrate, S30 mM giycerol, in Milli Q water, final pM 7.2). After ineubalion at 37 C for 3 weeks, the bacterial suspension was transferred lo ii 1-litre flask eontaining 250 ml of Sauton liquid medium and ineubated in a horizontal position at 37 C for 3 weeks (designated prccullure). For preparation ol' the culture supcrnatani, 30 ml of preculture was inoculated into 500 ml of Sauton liquid medium in a 1-litre conical flask, with a cotton plug. This suspension was cultured at 37 C for 39 days with shaking at 120 rpm, using a gyrorotary shaker (New Brunswick Scientific, Edison, NJ, USA). Bacteria were killed by pasteurization of the suspension in a water bath at 75 C for 15 min: the temperature of the suspension rose from 20 C to 71 C in 10 min and remained at 71 C for 5 min. The suspension was centrifuged at 16,000 g for 20 min at 4 C. The supernatant was centrifuged again under the same conditions. Baeteria in the pellet were washed twice with distilled water, sonicated in distilled water for I h at 50% duty cycle with 100 W power, using a Branson sonitier, and then lyophilized. The supernatant was concentrated in a vacuum rotary evaporator. The remaining volume was dialysed for 2 days against distilled water, using a dialysis membrane with a cut-off point of 10 kDa, and subsequently lyophtlized. Antigens detectable in sodium dodeeyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and Western blot analysis were shown to be a selection of antigens found in sonicates of M. tubercutosis [13]. Sonicates of M. tuherculo.Kis., M. kansasii, M. avium serotype 4, M. terrae. M. gordonae and E. coli were prepared as deseribed for M. tubercutosis. Sodium dodecyt .sulphate polyacrylamide gel etectroptwresis. SDS-PAGE was done as described by Laemmli [15] on 13"A> gels.
Murine Antibodies to Live M. tuberculosis Western bloi analysis. Antigens separated by SDS PAGE were transferred to nitrocellulose and used for Western blotting as described previously [16]. Mouse sera were diluted 1 ; 100, Horseradish peroxidaselabelled anti-mouse immunoglobulin G heavy and light chams (lgG H + L) was obtained from the Pasteur Institute. Pans. France. The following proteins were identified with monoclonal aniibodies (MoAbs): 65 kDa with F67-18 [17]; 42 kDa with F29-29. 19 kDa with F29-47. 16 kDa with F23-49 [18]: 30 .11 kDa wilh MoAb 3A8 [19] and with anti-p32 rabbit antiserum [20]: 33 kDa with F67-I [21]: 38 kDa with F67-19. 34 kDa with FI26-2, 24 kDa in mycobacterial sonicates and 24 kDa and 30 kDa in culture supernatant with FII6-5 [13]: 10 kDa with MoAb SA-12 |22]. Cross-reactivity of these MoAbs and the possible shift in molecular weight of the recognized proteins in different species is shown ill Table I, MoAb SA-12 was kindly supplied by P. Minden. La Jolla. California, USA and P. Kelleher. The Woodlands, Texas. USA and MoAb 3A8 by P. Das. Amsterdam, The Netherlands. Anti-p32 rabbit antiscrum was a generous gift from J. de Bruyn, Brussels, Belgium. The 65-kDa protein has also been designated 2T: the 38-kDaprotein.3T: the 33-kDii antigen, 7T, the 30 31kDa protein, 9T: and the l9-kDa protein, 12T [23]. ELISA wilh recombinuni DNA aniigens. Recombinant .18 kDa protein (24] and recombinant 65 kDa protein kindly supplied from the WHO IMMTtJB. IMMLEP Bank by Dr J, van Embden. Bilthoven. The Netherlands. MPT59 [25] was a generous gift from S. Nagai, Tokyo. Japan. The coating concentrations of the recombinant antigens, diluted in 0.05 M sodium bicarbonate buffer. pH 9.6. were 1 /ig, ml for 38 kDa. 3 /ig, ml for 65 kDa and 4/ig, mi for MPT59, Plasmas of the mice were diluted 1:100. The ELISA technique used has been described in detail elsewhere (13]. Colony forming units (CFli). Spleen, liver and lymph nodes were harvested from the mice 24 weeks after ihe infection with M. luhercuhisi.s and homogenized wilh a glass tube and leilon pestle in respectively I ml. 2 ml and 300 /il of sterile PBS. Tenfold serial dilutions of the homogenate were plated on Middlebrook 7HII agar (Difco. Detroit. Ml) enriched with: oleic acid, albumin, dextrose, catalase and Triton (OADC and WRL1.19). The plates were incubaled for 5 weeks at 37 C and colonies were counted.
RESULTS Antibody repertoire of different mouse strains AtO, 2,4, 6. 8. 10. 12, 15. 18.21 and 24 weeks after infection with live M, tuberculosis the antibody response in fourdifferent strains of mice was tested by Western blotting. None of the mice reacted with M. tuberculosis sonicate before infection with live M. tuberculosis. Mice from different strains showed a pronounced dilTerence in antibody repertoire at 8 weeks after infection (Fig. I). Apparent molecular weights of the
373
protein bands recognized were established by the use of MoAbs against known mycobacterial proteins (Fig, 1. lane 23). The bands seen in Western blot were all from protein antigens, since pronase digestion of the M, tuberculosis sonicate prior to SDS-PAGE abolished all reactions (results not shown). Among the individual mice within the same strain minor differences were seen in the BALB c. Swiss and CBA/ca mice. BALB/c and Swiss mice reacted with a variety of protein bands (Fig, I, lanes 7 11 and 12-16. respectively). Four of the five CBA/ca mice (Fig. 1. lanes 19-22) reacted only with the 38-kDa protein band, one mouse (lane 18} reacted with the 38-kDa and 33kDa protein bands. Three C57BI/6 mice (Fig. I, lanes 1. 2, 4) recognized protein bands of 38 kDa and 19 kDa. One mouse recognized the 19-kDa protein band and some antigens of higher molecular weights (lane 3). The last C57BI/6 mouse (lane 5) did not recognize M. tuberculosis antigens in Western blots, although mycobactcria could be cultured from the spleen and lytnph nodes of this mouse. All mice recognized fewer protein bands in culturesupcrnatant (Fig. 2) thaninsonicateof A/. tuberculosis (Fig. 1). Interestingly, none of the mice showed reaction with a protein band of 3031 kDa. This antigen was present in both sonicate and culture supernatant as shown by the reactions of MoAb 3A8 [19] and the rabbit antiserum against the p32 protein (20]. The reaction of these antibodies with the 30 31-kDa protein band in culture supernatant is shown in Fig. 3. Virtually all mice recognized the 38-kDa protein band (Table II). The 19-kDa protein band was recognized by 12 mice from three strains, but not by any of the CBA/ca mice, A reaction with the 33-kDa protein band was found in 10 mice, eight of whieh were from the Swiss and BALB/c strain. Reaction of the mice with other proteins was strain related (Fig. I), For example, only Swiss mice reacted strongly with the 65-kDa protein band (Table II), Reaction of different mou.se strains during, the course of infection Following intraperitoneal infection, the lgG (H -I- L) antibody response was not detectable at 2 weeks, was weak at 4 weeks and thereafter increased both in intensity and in the number of protein bands. A representative example is shown in Fig. 4. There was no subsequent decrease in
374
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65
43
42 F29-29 38 F67-19 34 F126-2 33 F67-1
30
20
F67-18
24
F116-5
19
F29-47
•16
F23-49
•10
SA-12
FIG. 1. Antibody response 10 M. tuberculosis sonicate 8 weeks after infection with live M. tuherculo.\is. lesled in Western blot. Lanes I .•>. C57BL;6 mice: lanes 7 1 L BALB/c mice; lanes 12 16. Swiss mice; lanes 18 22.CBA,ai miee; lane 23. reactiviiy of MoAbs. Lanes 6 and 17 arc filled only wilh diluent and conjugate and show the weak reaction of the conjugate at 65 kDa. .14 kDa. 16 kDa and 10 kDa. The positions of ihe molecular mass markers are shown on the left. Protein bands with which the MoAbs reacted are indicated on the right.
antibody response to any of the antigens up to 24 weeks wheti the mice were killed. In all four strains of mice antibodies against the 38-kDa protein band were the first antibodies to appear. Later in the infection the 33-kDa and 19-kDa protein bands were recognized. To study the effeet of a second challenge, two mice (presented iti Fig. I in lanes I and 2, 7 and 8, 12 and 13, 20 and 22) from eaeh strain received a second injection with live M. tuherculo.'iis at week 18. Plasma samples from these mice were taken at week 21 and mice were killed at week 24. The intensity of the antibody response was increased compared with the reaction before the second injection (data not shown). More protein bands were recognized by all mice after the second
injection. Irrespective of the strain, all eight mice reacted with protein bands of 38 kDa. 33 kDa and 19 kDa, although inter-strain differences were still present (Table III). There was no difference in the reactions of plasma samples taken at week 21 and at week 24. Reaction with recombinant antigens in ELISA None of the miee reacted witb the 38-kDa recombinant protein in ELISA before the infection with M. tuberculosis (Fig. 5). Plasma from miee that reacted with the 38-kDa protein band in Western blot also reacted with the 38-kDa recombinant protein in FLISA (Fig. 5). C57B1/6 mice 3 and 5 did not react with the 38-kDa protein band
Murine Antibodies to Live M. tiibereulosis 1 Oi US n? 09 II (]2 ii'4 116 on 1(1
IJ 12
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19 21 ^u
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- 65
F67-18
38 34 30
F67-19 F126-2 F116-S
24
F116-5
19
F29-47
10
SA-i:3
43
30
20
14-
FIG. 2. Antibody response to M. /W/KTCH/O.^K culture supernatant 8 weeks after infcelion with live M- tuberculosis, tested In Western biol. Lanes 1 5. C57BL/6 miee; lanes 7 11. BALB/c miee; lanes 12 16. Swiss mice; lanes !8-22.CBA,ca mice; lane 2."^. reactivity of MoAbs. Liines 6 and Ware lilledonly wit h diluent and conjugate and show the weak reaction of the conjugate at 65 kDa. 34 kDa and 10 kDa. The positions of the molecular mass markers are shown on the left. Protein bands with which the MoAbs reacted are indicated on the right.
in Western blot or with the 38-kDa reeombinant protein in ELISA. None of the mice reaeted with a 30-31-kDa protein band in Western blot and none showed a reaction with the purified MPT59 in ELISA (Fig. 6). MoAbs 3A8 and FI16-.^. rcaetive with a 30-kDa band in culture supernatant in Western blot, both reacted with the purified MPT59 in ELISA. The reeombinant 65kDa protein was recognized only by Swiss mice 12 weeks after infection (Fig. 7). None of the other mice showed any reaction with the 65-kDa antigen in ELISA or Western blot. M. tuberculosis colony counts in organs of the injected mice Macroscopic examination showed no granulo-
mata or other signs of infection in liver, spleen or lymph nodes of the infected mice. However, following culture on solid medium a considerable number of viable bacilli could be demonstrated 24 weeks after inoculation (Fig. 8). At least 10' live M. tuberculosis were found in all the spleens. Infection was greatest in the organs of the CBA/ ca mice. The number of M. tuberculosis in the spleens of BALB/c and C57B1,6 miee was cotnparable to that in the other two strains, but tbe number in liver and lymph nodes was somewhat lower. In the organs of mice injected for a second time with live M. tuberculosis, the number of viable bacilli was the same as in mice injected only once. Ziehl-Neelsen staining of the organs correlated with the colony counts, indicating that few dead bacteria were present. These results indicate
376
A. Verbon el at.
1
94 67
-
43
-
30
-
that all miee from the four strains were slill exposed to live M. lubercutosis 24 weeks after in fee tion.
2
Species-specificity oJ antibodies
20
-
14
FIG. 2. Reactivity of monoclonal antibody 3A8 and anti-p32 rabbit antiserum (both directed against the antigen 85 complex) with M. tuberculosis culture supernatant in Western blot, l.anc 1. MoAb 3A8; lane 2, rabbit antiserum directed against p32. The positions of the molecular mass markers are shown on the left.
TABLE 11. Recognition m Western blot of some M. tuberculosis protein bands, found in sonicate and culture supernatant, by differeni mouse sirains 8 weeks after infection wilh live M. tuberculosis Protein band* Mouse strain
65
38
33
19
C57BL/6(M = 5) BALB/c (n = 5) Swiss (n — 5) CBA/ca (M = 5) Total (n = 20)
Of
3 5 5
1 3 5 1 10
4 3 5 0 12
0 5 0 5
5 IS
• Apparent molecular weights in kDa are indicated, t Number of mice recognizing the protein band.
The antibody repertoire of the same two mice from each strain (presented in Fig. I in lanes 1 and 3, 7 and 9. 12 and 14, 21 and 22) was followed in Western blot using sonicates of M. tuberculosis. M. atium. M. gordotiae. M. kansasii, M. terrae and E. coli. MoAbs were used to identify corresponding protein bands in the sonicates, when possible (Table I). Plasma samples were taken before and at 6, 15 and 24 weeks after infection with M. tubcrcttto.'iis to study the specificity ofthe antibodies. A representative example of plasma from one mouse from each strain taken 24 weeks after infection is shown in Fig. 9. There were minor differences in the reactions ofthc two mice from each strain. None ofthe mice tested showed any reaction with M. tuberculosis or E. coli sonicate prior to infection with M. tuberculosis. Sera from CBA/ca mice reacted only with the 38kDa protein band of M. tuberculosis, but not at any time with the other mycobacterial strains or with E. coti (Fig. 9 and Table IV). This implies that the antibodies in CBA/ca mice against the 3S-kDa protein band are specific for M. tubercuto.sis. BALB/c and C57Bi/6 mice showed some reaction with a 19-kDa protein band in M. kansasii. 6 and 24 weeks after infection, indicating a limited specificity of antibodies against this antigen (Table IV). No cross-reaction of antibodies directed against the 33-kDa protein band of M. tuberculosis was seen in any of the mice, although a similar 33-kDa protein was present in M. kansasii as shown with the cross-reactive MoAb F67-I (Table I). Swiss mice reacted with many protein bands in M. tuberculosis sonicate 6 weeks after infection and also reacted with protein bands in M. avium and M. katuasii. Twentyfour weeks after infection cross-reaction was found with proteins from all other mycobacterial strains and even with E. coli. These findings suggest that the murine antibodies directed against the 38-kDa and 33-kDa protein bands are specific for M. tubercutosis. Antibodies against the 19-kDa protein band showed limited specificity.
Murine Antibodies to Lire M. tuberculosis 01
nj 02
riii
()•> 07 09 1 , 13 13 17 19 21 i ri6 08 10 12 U \f, Ifl ?0 ?2
94 67 43
- 65
F67-18
- 42
F29-29
- 38 F67-19 i- 34 F126-2 i- 33 F67-1
3O
20
14
FIG. 4. Antibody response over time in a Swiss mouse (presented in Fig. 1, lane 15) to M. tuhercutosis sonicate (lanes t 11) and culture supernatant (lanes 12 22) after a single injection with live M. tuberculosis, tested in Western blot. Lanes: I and 12, at U weeks; 2 and 13, at 2 weeks after infection; 3 and 14,4 weeks: 4 and 15, 6 weeks: 5 and 16, 8 weeks; 6 and 17, iO weeks; 7 and IS, 12 weeks; 8 and 19, 15 weeks; 9 and 20, 18 weeks; 10 and 21, 21 weeks; 11 and 22, 24 weeks; lane 23, reactivilv of MoAbs with sonicate as described in Fig. I. The posilions ofthe molecular mass markers are shown on the left. Protein bands wilh whieh the MoAbs reacted are indicated on the right.
TABLI; III. Recognition in Western blot of immunodominant M. tubercuto.sis protein bands by diflerent mouse strains after a second injection with M. tuberculosis at week 24 Protein bands* Mouse strain
65
50 42
40
38
33 28
26
19
16
C57BL/6(« = 2) BALB/c (« = 2) Swiss (rt = 2) CBA/ca (« = 2) Total (n = 8)
Of 2 2 0 4
I 2 2 0 5
1 0 0 0 1
2 2
2
2
0
r-l
Ol
Ol
2 2 8
2
1 2 2
?
1
6
1
2 2 8
1 I 4
Ol
2 6
2 8
0 r-l
0
* Apparent moleeular weights in kDa are indicated. t Number of mice recognizing the protein band.
377
378
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•e 2 . 0 -
1.0
•A* 0 12 C57BI/6
0 12 BALB/c
0 12 Swiss
0 12 weeks CBA/ca
FIG. 5. Antibodies to the reeombinant 38-kDa protein determined in mice from four different stratns before and 12 weeks after infection with M. tuberculosis as determined by ELISA. Plasmas were diluted 1:100; each point represents one motise.
E 3.0 c in o CD
U
c
e 2.0 H
o n n
1,0-
TT 0 12 C57BI/6
0 12 BALB/c
0 12 Swiss
0 12 weeks CBA/oa
FIG. 6. Antibodies to the MPT59 protein (aniigen 85B) in mice from four different strains before and 12 weeks after infection wilh M. tuhcnulosis as determined by ELISA. Plasmas were diluted 1:100; each point represents one mouse.
Murine Antibodies to Live M, tuberculosis
379
3.0i
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BALB/c
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0
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FtG. 7. Antibodies to the recombinant 6.S-kDa protein determined in mice from four difVercnl strains before atid 12 weeks after infection with A7./H/JCOH/O.V/A as determined hy ELISA. Plasmas were diluted 1:100: each point represents one mouse.
DISCUSSION We sttidicd the early antibody response to M. tuberculosis iintigcns by infecting inbred mice of diflcrent H-2 types with live M. luhcriulosis. Using Western blot analysis and MoAbs against known antigen.s. we identified the protein bands recognized by the mice. Our rcsttlts in EI.ISA with Ihe recombinant 65-kDa and 38-kDa proteins and the MPT59 protein suggest that the infected mice and our reference MoAbs react with the same proteins as shown in Western blot (Figs 1 and 2), Our results infer, but do not prove, that the other proteins recognized by the infected mice are the same a.s those recognized by the reference MoAbs. The antibody repertoire found in trtice of four dilferent H-2 types showed pronounced dilVerences, but in all four strains antibodies against a 38-kDa protein band were the first to appear. The 38-kDa protein i,s known to be secreted in vitro [13, 14, 26], Our results suggest that this protein is also secreted in vivo by M. tuberculosis and is therefore available for recognition by B cells at an early stage ofinfection. The 38-kDa protein was also recognized by mice after infection with live
M. hovis BCG [5], Although the immune response to A/, tuberculosis probably dificrs between species, the 38-kDa antigen seems lo be irnmunodominant at the B-cell level in man [27], iti guinea pigs [2H] and in mice (this study) [4]. The 19-kDa protein has previously been described as the secreted 21-kDa aniigen 113] and was shown to have a signal sequence [29], The protein band corresponding to ihis antigen was recognized by nearly all BALB/c. Swis.s and C57BL/6 tnice after infection with M. tuberculosis. The aniibodies against the 19-kDa prolein band appear relatively early, although later than anhbodies against the 38-kDa protein. In humans the antibody response against the 19-kDa antigen also suggests that this secreted antigen evokes an early immune response after infectioti with M. tuberculosis [30]. Later in Ihe infection a protein band of 33 kDa is recognized mainly by BALB/c and Swiss mice. We found that the 33-kDa antigen is present in sonicate, but not in culture supernatant of .M. tuberculosis and. thus, is not secreted [13], The 33kDa antigen of M. tuberculosis corresponds lo the proline-rich 36-kDa antigen of M. leprae [21 ]. The apparent molecular weight of this antigen deter-
380
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FIG. 8, Colony forming units in liver, spleen and lymph nodes of (A) CBA/ca, (B) BALB/c. (C) Swiss and (D)C57BI/'6 mice. The organs were harvested 24 weeks after infection with 5 10 x 10^ live M. luberculo.sis. Each point represents the total number of colony forming units in the organ of one mouse: • , mice injected only once with live M. tuberculosis; | . mice injected twice with live M. mbercuiosis.
CBA 1 2 3 4 5 6
BALB/c 1 2 3 4 5 6
Swiss 1 2 3 4 5 6
C57B1/6 1 2 3 4 5 6
94-
67-!
43-i 30-1
14-1
FIG. 9, Western blot analysis of different mycobacterial sonicates and E. coli sonicate with sera from CBA/ ca and BALB/c mice 24 weeks after the lirst injection wilh live M. tuberculosis and from Swiss and C57B1/6 miee 6 weeks after the second injection. Lane I, M. luberculo.sis: lane 2. M. aviunn lane 3. M. gordouac; lane 4. M. kan.saxii: lane 5, M. lerrae: lane 6. E. coli. The positions ofthe molecular mass markers are shown on the left of each two lanes.
Murine Antibodies to Live M. tuberculosis
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A. Verbon et al.
mined by SDS-polyacrylamide gel electrophoresis immunoperoxidasc assay using MoAb F47-9 was 36 kDa [31], but in Western blot the molecular weight was 33 kDa [21], A similar protein was identified in other mycobacterial species using cross-reactive MoAbs F67-I, F67-5 and F126-5 (Table I) [21]. This antigen of M. leprae is immunodominant in patients with lepromatous leprosy [31]. The value of the 33-kDa antigen in a serodiagnostic test for tuberculosis has not yet been established. Not all mice reacted with the 65-kDa heat shock protein. This immunodominant prolein [32] indticed strong antibody responses in Swiss and Balb/c mice after the second injection hut not in the other two strains, implying thai the antibody response against the 65-kDa antigen is strain dependent. This strain dependency was also found by Hiiygen ct al. [5]. after infection with live M. hori.s BCG. but in all outbred mice tested antibodies against Ihc (i5-kDa protein band were present [33],
viable M. tubcrculosi.s. Twenty-four weeks after the initial infection we found comparable amounts of colony forming units in three ofthe four mice strains. CBA/ca mice had higher counts of bacilli in all ihree organs; this was also found after infection with live M. hovis BCG [35]. Few dead mycobacteria were present. The marked difference in antibody repertoire is thus due lo strain differences and not to nurnbers of infecting bacilli. Specics-speciticily of the antibody response after infection with M. tuberculosis was tested in Western biol with four other mycobacterial sonicates and E. coli sonicate. In CBA/ca mice the response to the antigen of 38 kDa was specific for M. tubercidosis. Inothermouse strains antibodies against the 3S-kDa antigen also seemed to be specific for M. tuberculosis. So far almost all the polyclonal rabbit sera and murine MoAbs against the 38-kDa antigen are unique for the M. tuberculosi.s complex [7. 36, 37]. although a similar protein is present in other mycobaclcria [38], The antibody response against the 33-kDa protein band was M. tuberculosis specific. This may be explained by the fact that we infected mice with live M. tuberculosis. We have prepared crossreactive MoAbs against this prolein [21], but our MoAbs were produced after immunization with dead bacilli in Freund's adjuvant,
In our study mice did not react with the 30-31kDa secreted protein family in Western blot. although we proved that this antigen was present in both sonicate and culture supernatant (Fig, 3), This secreted protein family is also known as the antigen 85 complex [25] and consists of three components encoded by three separate genes [34], In ELISA none of the mice reacted with MPT59 Murine antibodies against the 38-kDa prolein (antigen H5B). In contrast, antibodies against the were present early in Ihc infection and seemed to antigen 85 complex have been detected after be specific for ihc M. tuberculosis complex. immunization with live M. bovis BCG [5], The Antibodies against the 33-kDa protein were humoral immune response against two mycobacpresent in all mice after a second injection with terial species, both belonging to the M. tubcrculolive M. tuberculosis. These antibodies did not ,si.
