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The humoral immune response after BCG vaccination: an immunoblotting study using two purified antigens
Tuber&
und Lung Ihrase
C1992) 73, 1Y-140
The humoral immune response after BCG vaccination: an immunoblotting study using two purified antigens A. Drowart”, Vooren”
J. Selleslaghs*,
J-C. Yemault”,
C.Valcke*, J. De Bruynt,
K. Huygent,
C-M. Farber*, and J-P. Van
* Universite’ libre de Bruxelles and tlnstitut Pasteur du Brabant, Brussels, Belgium
S UMMA R Y. The specific IgG response induced by BCG vaccination was investigated in 27 adults using two purified BCG antigens in a dot immunoblotting assay. The reflectance values increased significantly after vaccination when P64 but not when P32 was used as the antigen. The values measured after vaccination were compared with those obtained when testing sera from patients with tuberculosis. We observed that the level of anti-P64 IgG reached after BCG immunization was high enough to preclude a serological differential diagnosis with tuberculosis. La reponse IgG specifique provoquee par la vaccination BCG a CtCCtudiee par une analyse de type ‘dot immuno-blotting’ chez 27 adultes, en utilisant 2 antigenes purifies de BCG. Les valeurs mesurees ont augmente d’une facon significative apt-es vaccination en utilisant la P64 comme antigene, mais non pas avec la P32. Les valeurs mesurees apt-es vaccination ont Cte compardes avec celles obtenues en testant le serum de tuberculeux actifs. Le niveau atteint par les IgG anti P64 apt& immunisation par le BCG est assez ClevCpour em&her le diagnostic serologique de tuberculose avec cet antigene.
R&S UMk.
R ES UM E N. En 27 adultos se investig6 la respuesta IgG especifia inducida por la vacunaci6n BCG mediante
un test de dot inmunoblot, utilizando 2 antigenos BCG purificados. Los valores observados aumentaron significativamente despues de la vacunacidn cuando se empleb el antigen0 P64, y no cuando se emple6 el antigen0 P32. Los valores medidos despues de la vacunaci6n fueron comparados con aquellos obtenidos en el suero de paciente con tuberculosis. Observamos que el nivel de IgG anti-P64 alcanzado despues de la inmunizaci6n BCG era suficientemente elevado coma para impedir un diagnktico diferencial serol6gico con la tuberculosis.
Human tuberculosis remains a major disease in developing countries.’ In Europe and in the USA, the incidence, low during the last 10 years, is on the increase following the emergence of AIDS.* The development of the enzyme linked immunosorbent assay3 and more recently of the immunoblotting assay using purified or non-purified antigens allowed the study of the humoral response.4 Many authors have demonstrated that the Immunoglobulin G isotype was the most discriminatory when comparing tuberculous with non-tuberculous patients.3 It is not quite established if a previous BCG vaccination, commonly performed in developing countries, induces a measurable response as most of the studies were performed on a limited number of subjects.’
Correspondence to: J-P. Van Vooren, Chest Department, Erasme, Rte de Lennik, 808, 1070-Brussels, Belgium.
We recently investigated the humoral response induced by BCG vaccination using PPD and the ELISA technique.6 Serum samples were obtained at different times after BCG administration. We demonstrated a significant rise in specific IgG antibody levels: this had direct implications for the serodiagnosis of tuberculosis since many values measured in tuberculous patients were not different from those measured in healthy recently vaccinated subjects. In the present study we measured the IgG response following vaccination in young adults using our wellstandardized dot immunobinding assay and two purified BCG antigens, P32 (antigen 85A in the Closs reference system)7 and the heat shock protein P64 (antigen 82).7 Sera samples were collected after a set time of 6 weeks.
Hopital
137
138
Tubercle and Lung Disease
SUBJECTS, MATERIAL AND METHODS Study population Vaccinated subjects 27 skin test-negative students (17 males and 10 females)
attending a medical examination agreed to participate in the study. Their mean age was 23 years (range 22-26). According to local regulations they had to receive a BCG vaccine. The skin test consisted of an intradermal injection of purified protein derivative (PPD 2 i.u., RT23, Copenhagen, Denmark). Those were repeated 3 months after the BCG vaccination. An induration of at least 6 mm (mean diameter) was considered as positive. The vaccination was performed by intradermal injection of lyophilized BCG vaccine (Institut Pasteur du Brabant). One dose of vaccine contained l-4 x lo5 colony-forming units. The sera were obtained before the first skin test and before the second skin test, 6 weeks after vaccination. Tuberculous patients
The sera of 30 untreated patients with active pulmonary tuberculosis were collected. The diagnosis was assessed in all cases by positive auramine staining of a sputum specimen and was confirmed by culture. Mycobacteria were identified as M. tuberculosis var. Hominis in each case.
Antigens P32 and P64 protein antigens were purified as previously described by successive hydrophobic chromatography on phenyl-sepharose, ion-exchange on DEAE-sephacel and molecular sieving on Sephadex from unheated culture filtrates of M. bovis BCG (substrain 1173~2) grown for 14 days on zinc-deprived Sauton medium.8*g
Dot blot assay We performed this assay as we previously described.” 300 ng/well of purified P32 or P64 antigens were adsorbed on a nitrocellulose membrane placed in a Bio Dot apparatus (Bio Rad laboratories). Serum samples were tested in duplicate. In order to reduce the effect of inter-assay variations, the pre- and post-vaccination sera were tested in the same run. The nitrocellulose sheet was further incubated in a peroxidase conjugated rabbit antiserum diluted l/500, and afterwards in an a-chloronaphtol containing solute. Three washes were performed between each step. A reflectance densitometer (Bio Rad laboratories) was used to quantify the peroxidase stain on the immunoblots.
Statistical analysis The Wilcoxon test for paired data was used to compare reflectance values before and after vaccination.
The Mann-Whitney U test was used for comparison between tuberculous patients’ and healthy subjects’ groups.
RESULTS Using P32, the dilution giving the best discrimination between tuberculous and non-tuberculous patients is l/100, the limit of positivity being 0.2 reflectance units.” Using P64, the working dilution was determined in preliminary experiments by testing two-fold serial dilutions of sera. It was also finally set at l/100. The reflectance values obtained in paired sera taken before and 6 weeks after BCG vaccination are shown in the Figure. When comparing paired sera, a significant increase (P < 0.05) was found with P64, not with P32. Women and men responded alike. Three months after vaccination, 14 (52%) individuals were skin test-positive. There was no difference in antibody levels between the two groups (skin test-negative and skin test-positive). Whatever antigen was used, tuberculous patients had higher antibody levels than healthy subjects (P c 0.001). 21 of the 30 tuberculous patients had anti-P32 IgG antibody levels above or equal with the limit of positivity (defined previously), compared to O/27 vaccinated subjects. A cut-off point for positivity was never determined using the P64 as the antigen. Considering the values reported in the Figure, we could assess that tuberculous patients did not differentiate well from recently vaccinated subjects.
DISCUSSION The aim of this present study was to evaluate the specific humoral response induced by BCG vaccination using two purified proteins as antigens. We previously described that an anti-PPD IgG antibody increase was detectable early 2 weeks after BCG administration; this response was sustained for 10 weeks.6 These preliminary data were confirmed using P32 and P64 as antigens (unpublished data). We decided to collect the second serum samples after a set time of 6 weeks. P32 corresponds to the antigen 85A of the 85 complex in the Closs reference system.7 A similar coding sequence for this protein has recently been described in both M. bovis BCG and M. tuberculosis.” This antigen, which seems limited to Mycobacteria,13 is secreted in large amounts in culture filtrates and could be useful in the serodiagnosis of tuberculosis.14 The protein antigen called P64 or 65 kDa belongs to the family of stress proteins9 It is very immunoreactive and has common epitopes with proteins of unrelated bacteria, plants and humans.” High antibody levels to this antigen are frequently measured in rheumatoid arthritis patients sera
The humoral immune response after BCG vaccination:
an immunoblotting
study using two purified antigens
R
139
l
1.0
P64
P32
0.6 .
i ---_
y
_____:___ .___A& ---_mmu!e_Ny& --- -&_ N:22 N;l N:12 ,__ N:6 PRE
_^- -.POST
. _i
i _-A--
N:l
POST
Figure-IgG antibodies to P32 (left panel) and P64 (right panel) levels expressed in reflectance units (R). pre-post: 27 healthy subjects before and after BCG immunization. T: 30 patients with active tuberculosis. Limit of specific IgG detection : 0.05 reflectance units. -: sensitivity limit for P32 antibodies.
so that its role in the appearance of auto-immune diseases has been suspected.16 When comparing paired sera, a significant increase in the anti-P64 but not in the anti-P32 IgG antibody levels was induced by BCG as assayed on the sera drawn 6 weeks after BCG vaccination. This rise was not related to another disease as the subjects reporting an intercurrent event were excluded from the study. In accordance with our previous work6 and to the study of Neveu et al,” no relation was noted between the humoral and delayed type hypersensitivity responses to BCG vaccination. The results obtained using unpurified antigens showing a significant increase in antibody levels induced by BCG administration could possibly be related to the presence in such preparations of widely cross-reactive proteins such as P64. The significance of the induction in some patients of an IgG antibody response directed against this antigen following BCG vaccination, and the possible consequence for the development of autoimmune diseases, need further investigation. Finally, as we previously suggested, a humoral response directed against the P32 antigen observed in 70% of our tuberculous patients seems to be specific of an active mycobacterial infection. We do not yet know if anti-P32 antibodies are detectable in severe mycobacterial infections not due to A4. tuberculosis.
References 1. Glassrotb J, Robins A G, De Snider A. Tuberculosis in the 1980’s. N Engl J Med 1980; 302: 1441. 2. Chaisson R E, Sutkin G. Tuberculosis and human immunodeficiency virus infection. J Infect Dis 1989; 159: 96. 3. Daniel TM, Debanne S M, The serodiagnosis of tuberculosis and other mycobacterial diseases by enzyme linked immunosorbent assay. Am Rev Respir Dis 1987; 135: 1137. 4. Van Vooren J P, Turneer M, Yemault J C, et al. A multidot immunobinding assay for the serodiagnosis of tuberculosis: comparison with an enzyme linked immunosorbent assay. J Immunol Methods 1988; 113: 45. 5. Mauch H, Brehmer W. Mycobacterial antibodies after tuberculin testing, BCG vaccination, BCG immunotherapy and against cross-reacting antigens in a solid phase radioimmunoassay. Ztl Bakt Hyg I Abt Grig 1982; A251: 380. 6. Turneer M, Van Vooren J P, Nyabenda 1 et al. The humoral immune response after BCG vaccination in humans: consequences for the serodiagnosis of tuberculosis. Eur Respir J 1988; 1: 589. 7. Closs 0, Harboe M, Axelsen N H, Bunch Kristensen K, Magnusson M. The antigens of Mycobacterium bovis strain BCG, studied by crossed immunoelectrophoresis: a reference system. Stand J Immunol 1980; 12: 249. 8. De Bmyn J, Huygen K, Bosmans R et al. Purification, characterisation and identification of a 32 kDa protein antigen of Mycobacfetium bovis BCG. Microb Pathog 1987; 2: 351. 9. De Bruyn J, Bosmans R, Turneer M et al. Purification, partial characterisation and identification of a skin reactive protein antigen of Mycobacrerium bovis BCG. Infect Immun 1987; 55: 24. 10. Van Vooren J P, Farber C M, Noel E, et al. Local anti-P32 humoral response in tuberculous meningitis. Tubercle 1989; 70: 123. 11. Drowart A, Huygen K, De Bruyn J, Yemault J C, Farber C M, Van Vooren J P. Antibody levels to whole culture filtrate and to
140
Tubercle and Lung Disease
purified P32 during treatment of smear positive tuberculosis. Chest 1991; 100: 685. 12. De Wit L, de la Cuvellerie A, Ooms J, Content J. Nucleotide sequence of the 32 kDa protein gene (antigen 85A) of Mycobacterium bovis BCG. Nucleic Acids Res 1990; 18: 3995. 13. De Bruyn J, Bosmans R, Nyabenda J, Van Vooren J P. Effect of Zinc deficiency on the appearance of two immunodominant protein antigens (32 kDa and 65 kDa) in culture filtrates of mycobacteria. J Gen Microbial 1989; 135: 79. 14. Turneer M, Van Vooren J P, De Bruyn J, Serruys E, Dir&x P, Yernault J C. Humoral immune response in human tuberculosis: Immunoglobulin G, A and M directed against the purified P32 protein antigen of Mycobactert’um bovis bacillus Calmette et Guerin. J Clin Microbial 1988; 26: 1714.
15. Aguas A P, Esaguy N, Sunkel C E, Silva M T. Cross-reactivity and sequence homology between the 65kDalton mycobacterial heat-shock protein and human lactoferrin, transferrin and DRbeta subsets of major histocompatibility complex class II molecules. Infect Immun 1990; 58: 1461. 16. Babr G M, Rook G A W, Al-Saffar M, Van Embden J, Stanford J L, Behbehani K. Antibody levels to Mycobacteria in relation to HLA type: evidence for non-HLA linked high levels of antibody to the 65 kDa heat shock protein of M. bovis in rheumatoid arthritis. Clin Immunol 1988; 74: 2 Il. 17. Neveu P J, Buscot N, Soulillou J P. Dissociation between humoral and cellular responses to PPD after BCG vaccination. Int Arch Allergy Appl Immunol 1980; 62: 409.
und Lung Ihrase
C1992) 73, 1Y-140
The humoral immune response after BCG vaccination: an immunoblotting study using two purified antigens A. Drowart”, Vooren”
J. Selleslaghs*,
J-C. Yemault”,
C.Valcke*, J. De Bruynt,
K. Huygent,
C-M. Farber*, and J-P. Van
* Universite’ libre de Bruxelles and tlnstitut Pasteur du Brabant, Brussels, Belgium
S UMMA R Y. The specific IgG response induced by BCG vaccination was investigated in 27 adults using two purified BCG antigens in a dot immunoblotting assay. The reflectance values increased significantly after vaccination when P64 but not when P32 was used as the antigen. The values measured after vaccination were compared with those obtained when testing sera from patients with tuberculosis. We observed that the level of anti-P64 IgG reached after BCG immunization was high enough to preclude a serological differential diagnosis with tuberculosis. La reponse IgG specifique provoquee par la vaccination BCG a CtCCtudiee par une analyse de type ‘dot immuno-blotting’ chez 27 adultes, en utilisant 2 antigenes purifies de BCG. Les valeurs mesurees ont augmente d’une facon significative apt-es vaccination en utilisant la P64 comme antigene, mais non pas avec la P32. Les valeurs mesurees apt-es vaccination ont Cte compardes avec celles obtenues en testant le serum de tuberculeux actifs. Le niveau atteint par les IgG anti P64 apt& immunisation par le BCG est assez ClevCpour em&her le diagnostic serologique de tuberculose avec cet antigene.
R&S UMk.
R ES UM E N. En 27 adultos se investig6 la respuesta IgG especifia inducida por la vacunaci6n BCG mediante
un test de dot inmunoblot, utilizando 2 antigenos BCG purificados. Los valores observados aumentaron significativamente despues de la vacunacidn cuando se empleb el antigen0 P64, y no cuando se emple6 el antigen0 P32. Los valores medidos despues de la vacunaci6n fueron comparados con aquellos obtenidos en el suero de paciente con tuberculosis. Observamos que el nivel de IgG anti-P64 alcanzado despues de la inmunizaci6n BCG era suficientemente elevado coma para impedir un diagnktico diferencial serol6gico con la tuberculosis.
Human tuberculosis remains a major disease in developing countries.’ In Europe and in the USA, the incidence, low during the last 10 years, is on the increase following the emergence of AIDS.* The development of the enzyme linked immunosorbent assay3 and more recently of the immunoblotting assay using purified or non-purified antigens allowed the study of the humoral response.4 Many authors have demonstrated that the Immunoglobulin G isotype was the most discriminatory when comparing tuberculous with non-tuberculous patients.3 It is not quite established if a previous BCG vaccination, commonly performed in developing countries, induces a measurable response as most of the studies were performed on a limited number of subjects.’
Correspondence to: J-P. Van Vooren, Chest Department, Erasme, Rte de Lennik, 808, 1070-Brussels, Belgium.
We recently investigated the humoral response induced by BCG vaccination using PPD and the ELISA technique.6 Serum samples were obtained at different times after BCG administration. We demonstrated a significant rise in specific IgG antibody levels: this had direct implications for the serodiagnosis of tuberculosis since many values measured in tuberculous patients were not different from those measured in healthy recently vaccinated subjects. In the present study we measured the IgG response following vaccination in young adults using our wellstandardized dot immunobinding assay and two purified BCG antigens, P32 (antigen 85A in the Closs reference system)7 and the heat shock protein P64 (antigen 82).7 Sera samples were collected after a set time of 6 weeks.
Hopital
137
138
Tubercle and Lung Disease
SUBJECTS, MATERIAL AND METHODS Study population Vaccinated subjects 27 skin test-negative students (17 males and 10 females)
attending a medical examination agreed to participate in the study. Their mean age was 23 years (range 22-26). According to local regulations they had to receive a BCG vaccine. The skin test consisted of an intradermal injection of purified protein derivative (PPD 2 i.u., RT23, Copenhagen, Denmark). Those were repeated 3 months after the BCG vaccination. An induration of at least 6 mm (mean diameter) was considered as positive. The vaccination was performed by intradermal injection of lyophilized BCG vaccine (Institut Pasteur du Brabant). One dose of vaccine contained l-4 x lo5 colony-forming units. The sera were obtained before the first skin test and before the second skin test, 6 weeks after vaccination. Tuberculous patients
The sera of 30 untreated patients with active pulmonary tuberculosis were collected. The diagnosis was assessed in all cases by positive auramine staining of a sputum specimen and was confirmed by culture. Mycobacteria were identified as M. tuberculosis var. Hominis in each case.
Antigens P32 and P64 protein antigens were purified as previously described by successive hydrophobic chromatography on phenyl-sepharose, ion-exchange on DEAE-sephacel and molecular sieving on Sephadex from unheated culture filtrates of M. bovis BCG (substrain 1173~2) grown for 14 days on zinc-deprived Sauton medium.8*g
Dot blot assay We performed this assay as we previously described.” 300 ng/well of purified P32 or P64 antigens were adsorbed on a nitrocellulose membrane placed in a Bio Dot apparatus (Bio Rad laboratories). Serum samples were tested in duplicate. In order to reduce the effect of inter-assay variations, the pre- and post-vaccination sera were tested in the same run. The nitrocellulose sheet was further incubated in a peroxidase conjugated rabbit antiserum diluted l/500, and afterwards in an a-chloronaphtol containing solute. Three washes were performed between each step. A reflectance densitometer (Bio Rad laboratories) was used to quantify the peroxidase stain on the immunoblots.
Statistical analysis The Wilcoxon test for paired data was used to compare reflectance values before and after vaccination.
The Mann-Whitney U test was used for comparison between tuberculous patients’ and healthy subjects’ groups.
RESULTS Using P32, the dilution giving the best discrimination between tuberculous and non-tuberculous patients is l/100, the limit of positivity being 0.2 reflectance units.” Using P64, the working dilution was determined in preliminary experiments by testing two-fold serial dilutions of sera. It was also finally set at l/100. The reflectance values obtained in paired sera taken before and 6 weeks after BCG vaccination are shown in the Figure. When comparing paired sera, a significant increase (P < 0.05) was found with P64, not with P32. Women and men responded alike. Three months after vaccination, 14 (52%) individuals were skin test-positive. There was no difference in antibody levels between the two groups (skin test-negative and skin test-positive). Whatever antigen was used, tuberculous patients had higher antibody levels than healthy subjects (P c 0.001). 21 of the 30 tuberculous patients had anti-P32 IgG antibody levels above or equal with the limit of positivity (defined previously), compared to O/27 vaccinated subjects. A cut-off point for positivity was never determined using the P64 as the antigen. Considering the values reported in the Figure, we could assess that tuberculous patients did not differentiate well from recently vaccinated subjects.
DISCUSSION The aim of this present study was to evaluate the specific humoral response induced by BCG vaccination using two purified proteins as antigens. We previously described that an anti-PPD IgG antibody increase was detectable early 2 weeks after BCG administration; this response was sustained for 10 weeks.6 These preliminary data were confirmed using P32 and P64 as antigens (unpublished data). We decided to collect the second serum samples after a set time of 6 weeks. P32 corresponds to the antigen 85A of the 85 complex in the Closs reference system.7 A similar coding sequence for this protein has recently been described in both M. bovis BCG and M. tuberculosis.” This antigen, which seems limited to Mycobacteria,13 is secreted in large amounts in culture filtrates and could be useful in the serodiagnosis of tuberculosis.14 The protein antigen called P64 or 65 kDa belongs to the family of stress proteins9 It is very immunoreactive and has common epitopes with proteins of unrelated bacteria, plants and humans.” High antibody levels to this antigen are frequently measured in rheumatoid arthritis patients sera
The humoral immune response after BCG vaccination:
an immunoblotting
study using two purified antigens
R
139
l
1.0
P64
P32
0.6 .
i ---_
y
_____:___ .___A& ---_mmu!e_Ny& --- -&_ N:22 N;l N:12 ,__ N:6 PRE
_^- -.POST
. _i
i _-A--
N:l
POST
Figure-IgG antibodies to P32 (left panel) and P64 (right panel) levels expressed in reflectance units (R). pre-post: 27 healthy subjects before and after BCG immunization. T: 30 patients with active tuberculosis. Limit of specific IgG detection : 0.05 reflectance units. -: sensitivity limit for P32 antibodies.
so that its role in the appearance of auto-immune diseases has been suspected.16 When comparing paired sera, a significant increase in the anti-P64 but not in the anti-P32 IgG antibody levels was induced by BCG as assayed on the sera drawn 6 weeks after BCG vaccination. This rise was not related to another disease as the subjects reporting an intercurrent event were excluded from the study. In accordance with our previous work6 and to the study of Neveu et al,” no relation was noted between the humoral and delayed type hypersensitivity responses to BCG vaccination. The results obtained using unpurified antigens showing a significant increase in antibody levels induced by BCG administration could possibly be related to the presence in such preparations of widely cross-reactive proteins such as P64. The significance of the induction in some patients of an IgG antibody response directed against this antigen following BCG vaccination, and the possible consequence for the development of autoimmune diseases, need further investigation. Finally, as we previously suggested, a humoral response directed against the P32 antigen observed in 70% of our tuberculous patients seems to be specific of an active mycobacterial infection. We do not yet know if anti-P32 antibodies are detectable in severe mycobacterial infections not due to A4. tuberculosis.
References 1. Glassrotb J, Robins A G, De Snider A. Tuberculosis in the 1980’s. N Engl J Med 1980; 302: 1441. 2. Chaisson R E, Sutkin G. Tuberculosis and human immunodeficiency virus infection. J Infect Dis 1989; 159: 96. 3. Daniel TM, Debanne S M, The serodiagnosis of tuberculosis and other mycobacterial diseases by enzyme linked immunosorbent assay. Am Rev Respir Dis 1987; 135: 1137. 4. Van Vooren J P, Turneer M, Yemault J C, et al. A multidot immunobinding assay for the serodiagnosis of tuberculosis: comparison with an enzyme linked immunosorbent assay. J Immunol Methods 1988; 113: 45. 5. Mauch H, Brehmer W. Mycobacterial antibodies after tuberculin testing, BCG vaccination, BCG immunotherapy and against cross-reacting antigens in a solid phase radioimmunoassay. Ztl Bakt Hyg I Abt Grig 1982; A251: 380. 6. Turneer M, Van Vooren J P, Nyabenda 1 et al. The humoral immune response after BCG vaccination in humans: consequences for the serodiagnosis of tuberculosis. Eur Respir J 1988; 1: 589. 7. Closs 0, Harboe M, Axelsen N H, Bunch Kristensen K, Magnusson M. The antigens of Mycobacterium bovis strain BCG, studied by crossed immunoelectrophoresis: a reference system. Stand J Immunol 1980; 12: 249. 8. De Bmyn J, Huygen K, Bosmans R et al. Purification, characterisation and identification of a 32 kDa protein antigen of Mycobacfetium bovis BCG. Microb Pathog 1987; 2: 351. 9. De Bruyn J, Bosmans R, Turneer M et al. Purification, partial characterisation and identification of a skin reactive protein antigen of Mycobacrerium bovis BCG. Infect Immun 1987; 55: 24. 10. Van Vooren J P, Farber C M, Noel E, et al. Local anti-P32 humoral response in tuberculous meningitis. Tubercle 1989; 70: 123. 11. Drowart A, Huygen K, De Bruyn J, Yemault J C, Farber C M, Van Vooren J P. Antibody levels to whole culture filtrate and to
140
Tubercle and Lung Disease
purified P32 during treatment of smear positive tuberculosis. Chest 1991; 100: 685. 12. De Wit L, de la Cuvellerie A, Ooms J, Content J. Nucleotide sequence of the 32 kDa protein gene (antigen 85A) of Mycobacterium bovis BCG. Nucleic Acids Res 1990; 18: 3995. 13. De Bruyn J, Bosmans R, Nyabenda J, Van Vooren J P. Effect of Zinc deficiency on the appearance of two immunodominant protein antigens (32 kDa and 65 kDa) in culture filtrates of mycobacteria. J Gen Microbial 1989; 135: 79. 14. Turneer M, Van Vooren J P, De Bruyn J, Serruys E, Dir&x P, Yernault J C. Humoral immune response in human tuberculosis: Immunoglobulin G, A and M directed against the purified P32 protein antigen of Mycobactert’um bovis bacillus Calmette et Guerin. J Clin Microbial 1988; 26: 1714.
15. Aguas A P, Esaguy N, Sunkel C E, Silva M T. Cross-reactivity and sequence homology between the 65kDalton mycobacterial heat-shock protein and human lactoferrin, transferrin and DRbeta subsets of major histocompatibility complex class II molecules. Infect Immun 1990; 58: 1461. 16. Babr G M, Rook G A W, Al-Saffar M, Van Embden J, Stanford J L, Behbehani K. Antibody levels to Mycobacteria in relation to HLA type: evidence for non-HLA linked high levels of antibody to the 65 kDa heat shock protein of M. bovis in rheumatoid arthritis. Clin Immunol 1988; 74: 2 Il. 17. Neveu P J, Buscot N, Soulillou J P. Dissociation between humoral and cellular responses to PPD after BCG vaccination. Int Arch Allergy Appl Immunol 1980; 62: 409.