BCG Culture Filtrate Research Articles

Antibody repertoire against culture filtrate antigens in wild house mice infected withMycobacterium bovisBCG

Wild house mice (Mus domesticus) captured in a Flemish pigsty were infected intravenously with 4 x 10(6) variable units of Mycobacterium bovis BCG and examined by Western blot analysis for IgG secretion against BCG culture filtrate (CF) antigens. Wild mice showed a marked individual variation in antibody pattern when tested 4, 6 and 8 weeks after infection. Some animals reacted to a wide range of antigens and others only to a limited number. Most wild mice recognized preferentially antigens with molecular weight of 24 kD, 32 kD, 37-38-40 kD, 65 kD and 82 kD, i.e. the major CF antigens known to be recognized by sera from BCG-infected inbred laboratory strains, BALB/c, DBA/2, CBA/Ca and C57BL/6. The 32-kD fibronectin-binding protein and the 65-kD heat-shock protein appeared as very immunodominant in wild mice. Furthermore, about 20-25% of the mice reacted strongly with a unique antigen of 35 kD estimated molecular weight, to which the tested inbred laboratory mice did not respond. Monitoring the size of the bacterial population in the spleen indicated that the BCG inoculum did not replicate in wild mice, suggesting that the Bcgr allele is expressed in this population.

Allergic Sensitisation in Tuberculosis and Leprosy Patients

Background: A negative association has been observed between infections and allergy in several studies. The aim of the present study was to examine whether tuberculosis and leprosy patients have more or fewer allergies than healthy individuals. Method:Sera from tuberculosis patients, leprosy patients and healthy controls were analysed by ELISA and Pharmacia Unicap^® for serological markers for allergy and mycobacterial infection. The serological markers for allergy were total IgE, specific IgE using Phadiatop and specific IgE to the dust mite allergen Dermatophagoides pteronyssinus 1 (Der p 1). Serological markers for mycobacterial infections included specific IgG to a mixture of bacille Calmette-Guérin culture filtrate antigens, to purified mannose-capped lipoarabinomannan (manLAM) and to purified secreted antigen 85B. Results:Both tuberculosis and leprosy patients had significantly higher levels of total IgE than controls. Furthermore, a significantly higher level of specific IgE (Phadiatop) was also found in the tuberculosis patients compared with controls. A similar result, but not statistically significant, was observed for the leprosy group. Specific IgG to antigen 85B and to manLAM was found to be significantly higher in both tuberculosis and leprosy patients compared with controls. In addition, leprosy patients had significantly more IgG to the BCG culture filtrate antigen than controls. Conclusions:The results indicate that patients with mycobacterial infections have allergic sensitisation more frequently compared with healthy controls. This is seemingly in contrast with the notion that there is a negative association between allergy and infection (‘hygiene hypothesis’). However, since only one in ten of those infected with Mycobacterium tuberculosis will develop the disease, patients with active mycobacterial disease represent a selected group. A similar relationship applies for leprosy. It is conceivable that those predisposed to allergy are less resistant to mycobacterial infections.

Multicomponent antituberculous subunit vaccine based on immunodominant antigens of Mycobacterium tuberculosis

The low molecular mass polypeptides of secretory proteome of Mycobacterium tuberculosis are dominant targets for recognition by lymphocytes of human models of immunity to tuberculosis. In the present study, we evaluated the inherent immunogenicity of 102 individual polypeptides purified from low molecular mass region below 40 kDa in mouse model of immunization. The aim of this study was to identify molecules relevant for development of subunit vaccine against tuberculosis based on high degree of immunogenecity. Here, we demonstrate that experimental multicomponent subunit vaccine (MSV) consisting of five immunodominant polypeptides with high immunogenicity (CFP-25, CFP-20.5, Ag85B, Ag85A and CPF-32) induced both cellular and humoral immune responses characterized by Th1 and Th2 cytokine induction and imparted significant protection when administered with DDA-MPL adjuvants in C57BL/6J mice. The degree of protection imparted by experimental MSV on the basis of decrease in CFU's from target organs (lungs and spleen) was comparable to BCG and total mycobacterial culture filtrate proteins (CFPs) based vaccines. These results, therefore, suggest the potential of multicomponent subunit vaccination against tuberculosis based on strongly immunogenic proteins of M. tuberculosis.

What Is the Optimal Regimen for BCG Intravesical Therapy?

Objective: For more than 20 years, BCG intravesical therapy schedule has included 6 weekly instillations. Very few studies have, however, analyzed the rationale of this regimen. We previously demonstrated that intravesical BCG induced an increased peripheral immune response against mycobacterial antigens as compared to pretreatment values. In the present work, we have studied the weekly evolution of this immune response induced by intravesical BCG instillations.Materials and Methods: The evolution of the lymphoproliferative response of peripheral blood mononuclear cells against BCG culture filtrate (CF), tuberculin (PPD) and BCG extract (EXT) was tested before, every week during the BCG instillations and at 3 and 6 months follow–up in 9 patients with superficial bladder cancer treated with 6 weekly BCG instillations. Lymphoproliferation was measured by means of a tritiated thymidine incorporation test.Results: A significant increase in the lymphoproliferative response against PPD, CF and EXT was observed in 9, 8 and 7 of the 9 patients, respectively, as compared to pre–BCG values. The maximal lymphoproliferation was achieved after 4 instillations in 4/5 patients initially reactive against mycobacterial antigens whereas 2 of 4 initially nonreactive patients required 6 instillations. At 6 months’ follow–up, lymphoproliferation against BCG and the other mycobacterial antigens returned to pre–BCG values in all patients. In 3 patients who received additional instillations because of tumor recurrence within 1 year of follow–up, the maximum immune response was observed already after 2 instillations.Conclusion: In most patients, the maximal peripheral immune response is already observed after 4 weekly instillations. However, patients not previously immunized against mycobacterial antigens may require 6 weekly instillations to achieve a maximum stimulation level. Our data support the need to further evaluate the role of this status before starting BCG instillations. It could be of interest to study whether 6 BCG instillations are really necessary in patients previously immune against mycobacterial antigens.

Ribosomal protein L7 included in tuberculin purified protein derivative (PPD) is a major heat-resistant protein inducing strong delayed-type hypersensitivity.

The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis. It consists of more than 100 denatured proteins in a culture filtrate of a heated culture of Mycobacterium tuberculosis. In two-dimensional electrophoretic analysis of PPDs from M. tuberculosis and M. bovis BCG, most proteins were diffusely separated and could not be seen as spots because of denaturation, whereas a few proteins showed relatively clear spots, indicating heat resistance. Two such proteins corresponded to ribosomal proteins L7 and L12. The mixture of these proteins L7/L2 induced a strong delayed-type hypersensitivity reaction. Another protein showing a clear spot was a GroES analogue, but this did not induce delayed-type hypersensitivity. There were a few other unidentified proteins. It is well known that L7 and L12 are encoded by the same gene and that they differ from each other only by an acetylic post-translational modification that occurs at the N-terminus of L12 converting it to L7 in Escherichia coli. L12, but not L7, was found in two-dimensional electrophoresis of BCG ribosomes, although we found two proteins corresponding to L7 and L12 in PPDs and a native culture filtrate of BCG. We compared the delayed-type hypersensitivity reaction elicited by L7/L12 derived from a culture filtrate of BCG and L12 derived from BCG ribosomes. L7/L12 from the culture filtrate could induce delayed-type hypersensitivity, but L12 from ribosomes could not, indicating that L7 was attributable to the induction of delayed-type hypersensitivity. The activity of L7/L12 was heat resistant. Neither glycosylation nor phosphorylation of L7/L12 from a culture filtrate could be detected. The acetylation at N-terminal of L12 was essential for the delayed-type hypersensitivity activity.

Cellular responses and Mycobacterium bovis BCG growth inhibition by bovine lymphocytes.

Cellular responses of a group of cattle immunized subcutaneously with a low dose of Mycobacterium bovis bacille Calmette-Guerin vaccine (BCG) were measured in vitro and compared with nonimmunized control animals. PBMC taken from immunized animals proliferated and produced IFN-gamma in the presence of M. bovis BCG culture filtrate proteins. The addition of PBMC from immunized animals to M. bovis BCG-infected autologous macrophages also resulted in secretion of IFN-gamma. In contrast, the responses of PBMC from control animals were comparatively low over the period of study. In experiments to study the interaction of non-adherent lymphocytes with infected macrophages, M. bovis BCG growth was inhibited in cultures containing autologous PBMC from immunized and non-immunized control animals. The degree of inhibition was related to lymphocyte concentration but did not correlate with IFN-gamma production. Treatment of macrophages with recombinant IFN-gamma prior to, or postinfection did not alter the intracellular growth kinetics of mycobacteria. It appears, therefore, that although M. bovis BCG immunization of cattle stimulates the generation of a T cell-mediated immune response to M. bovis BCG, the cattle may already possess a high level of innate resistance to M. bovis BCG that requires the presence of lymphocytes.

Priming to mycobacterial antigen in vivo using antigen-pulsed antigen presenting cells generated in vitro is influenced by the dose and presence of IL-4 in APC cultures.

Antigen presenting cells (APC) similar to immature dendritic cells can be generated in vitro from bone marrow precursors. The authors have compared the yield, the phenotype and the function of murine bone marrow cells cultured for 7 or 11 days in either granulocyte macrophage colony stimulating factor alone (GM BMAPC) or in combination with interleukin-4 (GM/IL-4 BMAPC). The results showed that GM/IL-4 BMAPC expressed the highest levels of MHC Class 2 molecules, CD86/B7-2 and CD80/B7-1 co-stimulatory molecules and the lowest levels of F4/80 macrophage marker. However, when these APC were pulsed with BCG culture filtrate antigen or PPD they were not correspondingly more effective at stimulating activated T lymphocytes in vitro or priming naive T lymphocytes in vivo. Also, in contrast to GM BMAPC, high backgrounds recorded following injections of GM/IL-4 BMAPC without antigen were not consistently reduced by lowering the dose and irradiating the cells prior to administration. The authors conclude that the degree of maturity of BMAPC varies with culture conditions and that this may be an important consideration where BMAPC are to be used in vivo in immunotherapeutic regimens.

Identification of a second Mycobacterium tuberculosis gene cluster encoding proteins of an ABC phosphate transporter

Following the identification of a M. tuberculosis phosphate transporter belonging to the superfamily of ABC transporters, we report on the cloning and sequencing of two additional genes, called pstS-3 and pstC-2, encoding proteins homologous to PstS and PstC of Escherichia coli, respectively. Together with the previously isolated M. tuberculosis gene similar to the E. coli pstA, these are included in a cluster encoding a second putative phosphate transport system. We demonstrate that pstS-3 encodes the previously described Ag 88, a 40 kDa M. bovis BCG culture filtrate antigen (immunodominant in H-2 b haplotype type mice). Finally, a signature motif identifying integral transmembrane proteins of prokaryotic phosphate binding-dependent permeases is proposed.

Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47-kilodalton secreted antigen complex

Effective protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by previous immunization with living attenuated mycobacteria, and it has been hypothesized that secreted proteins serve as major targets in the specific immune response. To identify and purify molecules present in culture medium filtrate which are dominant antigens during effective vaccination, a two-step selection procedure was used to select antigens able to interact with T lymphocytes and/or antibodies induced by immunization with living bacteria and to counterselect antigens interacting with the immune effectors induced by immunization with dead bacteria. A Mycobacterium bovis BCG 45/47-kDa antigen complex, present in BCG culture filtrate, has been previously identified and isolated (F. Romain, A. Laqueyrerie, P. Militzer, P. Pescher, P. Chavarot, M. Lagranderie, G. Auregan, M. Gheorghiu, and G. Marchal, Infect. Immun. 61:742-750, 1993). Since the cognate antibodies recognize the very same antigens present in M. tuberculosis culture medium filtrates, a project was undertaken to clone, express, and sequence the corresponding gene of M. tuberculosis. An M. tuberculosis shuttle cosmid library was transferred in Mycobacterium smegmatis and screened with a competitive enzyme-linked immunosorbent assay to detect the clones expressing the proteins. A clone containing a 40-kb DNA insert was selected, and by means of subcloning in Escherichia coli, a 2-kb fragment that coded for the molecules was identified. An open reading frame in the 2,061-nucleotide sequence codes for a secreted protein with a consensus signal peptide of 39 amino acids and a predicted molecular mass of 28,779 Da. The gene was referred to as apa because of the high percentages of proline (21.7%) and alanine (19%) in the purified protein. Southern hybridization analysis of digested total genomic DNA from M. tuberculosis (reference strains H37Rv and H37Ra) indicated that the apa gene was present as a single copy on the genome. The N-terminal identity or homology of the M. tuberculosis and M. bovis BCG purified molecules and their similar global and deduced amino acid compositions demonstrated the perfect correspondence between the molecular and chemical analyses. The presence of a high percentage of proline (21.7%) was confirmed and explained the apparent higher molecular mass (45/47 kDa) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis resulting from the increased rigidity of molecules due to proline residues.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the Gene Encoding the MPB51, One of the Major Secreted Protein Antigens of Mycobacterium bovis BCG, and Identification of the Secreted Protein Closely Related to the Fibronectin Binding 85 Complex

The secreted protein MPB51 is one of the major proteins in the culture filtrate of Mycobacterium bovis BCG (BCG) and is a protein immunologically cross-reacting with the fibronectin binding 85 complex secreted by this bacterium. The gene encoding MPB51 (mpb51) was cloned, sequenced, and expressed in Escherichia coli. The mpb51 gene was mapped downstream of the gene for 85A component with 179 bp spaces. The mpb51 gene encoded 299 amino acids, including 33 amino acids for the signal peptide, followed by 266 amino acids for the mature protein with a molecular mass of 27807.37 Da. This is the first complete sequence of MPB51. MPB51 showed 37-43% homology to the components of 85 complex. Two-dimensional electrophoresis of culture fluids of BCG and Western blotting indicated the existence of the other novel protein(s) which strongly cross-reacted with the alpha antigen (85B) and MPB51.

IgG humoral response against the antigen 85 complex homologues in leprosy.

Antigen 85 complex is the major protein component present in M. bovis BCG culture filtrate (CF). It consists of a family of three proteins: 85A, 85B and 85C. Combining isoelectric focusing and Western blot analysis, we have previously identified different antigenically related proteins present in the CF of other mycobacteria (M. tuberculosis, M. kansasii, M. avium, M. gordonae, M. fortuitum and M. phlei) using monoclonal antibodies (MoAbs) directed against the antigen 85 complex of M. bovis BCG. Humoral immune response directed against these cross-reactive homologues was analysed in sera from 20 patients with multibacillary leprosy (BL/LL), from 20 patients with paucibacillary leprosy (BT/TT) and from 15 healthy leprosy contacts. All the antigen 85 homologues identified in the seven CFs by MoAbs were also recognized by IgG present in sera from multibacillary leprosy patients, but not or very faintly in sera from paucibacillary leprosy patients or from healthy subjects. These results suggest that some of the M. leprae epitopes inducing a significant humoral response in multibacillary leprosy are common to the various 85 antigenically related proteins present in all mycobacterial species.

Antigenic characterization of Mycobacterium bovis BCG culture filtrate

Mycobacterium bovis BCG culture filtrate, on gelfiltration chromatography revealed four prominent regions. Of these 4, region II possessed the highest antigenic reactivity by enzyme-linked immunosorbent assay (ELISA) and delayed type hypersensitivity (DTH) skin testing in guinea pigs. On anion-exchange chromatography, region II was resolved into 5 prominent fractions of which fraction II was found to have the highest antigenic reactivity. On sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), M. bovis BCG culture filtrate revealed 11 structural polypeptides. Fraction II yielded a homogenous polypeptide of 15.3 kDa molecular weight. All fractions cross-reacted with various mycobacteria used in this study except a 15.3 kDa polypeptide which was specific to M. bovis BCG.

Influence of genes from the major histocompatibility complex on the antibody repertoire against culture filtrate antigens in mice infected with live Mycobacterium bovis BCG.

C57BL/10 and C57BL/6 mice (H-2b); B10 congenic mice with f, k, p, q, r, and s H-2 haplotypes; B10 mice with recombinant g2, o2, a, h2, h4, i5, and bq1 H-2 haplotypes; and B6 mice with major histocompatibility complex (MHC) mutant bm1 and bm13 (class I) and bm12 (class II) haplotypes were infected intravenously with 4 x 10(6) CFU of live Mycobacterium bovis BCG and examined by Western immunoblot analysis for serum antibodies against BCG culture filtrate antigens, following a boost injection with live BCG or with BCG culture filtrate. Parental B10 and B6 mice reacted very intensely with three culture filtrate protein bands with estimated molecular masses of 37, 38, and 40 kDa. Response against the 40-kDa protein was stronger following a boost injection with live BCG than following a boost with culture filtrate. Sera from mice with f, p, i5, bm1, and bm13 haplotypes reacted strongly, with both the 37-38- and 40-kDa antigens, and sera from mice with q and bq1 haplotypes showed a somewhat weaker reaction. Sera from mice with r, s, and bm12 haplotypes reacted against the 37-38-kDa antigen but not against the 40-kDa antigen, and sera from mice with the h2 haplotype reacted only with the 40-kDa antigen but not with the 37-38-kDa antigen. Sera from mice with the k, g2, o2, a, and h4 haplotypes showed, at most, a very weak reaction with the 37-38- and 40-kDa antigens. These results demonstrate that MHC genes profoundly affect the antibody repertoire used against culture filtrate antigens in mice infected with live M. bovis BCG. In particular, as shown in mice with the recombinant H-2 haplotype and in class II mutant bm12 mice, the I-A heterodimer controls the recognition of the immunodominant 40-kDa antigen. By using crossed immunoelectrophoresis, this 40-kDa antigen was identified as antigen 88 according to the reference system of Closs et al. for BCG antigens.

Purification of MPB70 and production of specific monoclonal antibodies.

MPB70 is a protein secreted into the culture filtrate of Mycobacterium bovis BCG (substrain Tokyo 172), which is able to induce a delayed-type hypersensitivity (DTH) skin reaction in guinea pigs immunized with BCG-Tokyo. By high-pressure chromatofocusing and size-exclusion high performance liquid chromatography, a further purified MPB70 protein was obtained, which was visualized as a single band with a molecular mass of 22 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis. A series of hybridoma cell lines that produced monoclonal antibodies (MAbs) against the purified MPB70 protein was prepared, and three MAbs, Bov-1, Bov-2, and Bov-3, with strong antigen-binding capacities were established. Bov-1 was the most potent MAb among them and binds to only a 22 kDa protein band in culture filtrates of M. bovis, but not to bands in those of M. tuberculosis by Western immunoblotting analysis, suggesting that Bov-1 recognize different epitope of MPB70 from MAbs that have been shown previously to recognize several species of molecules in culture filtrates of M. bovis. The purified MPB70 protein elicited a strong DTH skin reaction in guinea pigs sensitized with BCG-Tokyo vaccine. Bov-1 had no inhibitory effect on generation of the DTH skin reaction, showing that MAb bound to an epitope distinct from that inducing the skin reaction. All of the three MAbs were specific to MPB70 and each recognized a different epitope on MPB70. MPB70 was not detected in the culture filtrate of M. tuberculosis H37Rv. Thus, these MAbs may be useful for detecting MPB70 in studies on discriminating infection with M. bovis in domestic animals or in distinguishing vaccination with BCG-Tokyo from other mycobacterial infections in humans.

Spleen cell cytokine secretion in Mycobacterium bovis BCG-infected mice.

Three susceptible mouse strains, i.e., BALB/c (H-2d), C57BL/6 (H-2b), and major histocompatibility complex-congenic BALB.B10 (H-2b), were infected intravenously with 4 x 10(6) CFU of live Mycobacterium bovis BCG and analyzed 4 weeks later for in vitro spleen cell cytokine secretion in response to purified protein derivative (PPD), BCG culture filtrate (CF), BCG cellular extract, total BCG, the purified extracellular 30-32-kDa antigen (the fibronectin-binding antigen 85), or the intracellular 65-kDa heat shock protein. C57BL/6 and BALB.B10 mice produced 5- to 10-fold more gamma interferon and interleukin-2 (IL-2) when stimulated with CF, PPD, and antigen 85 than BALB/c mice did. When stimulated with BCG extract and whole BCG, gamma interferon and IL-2 levels were generally lower and comparable in the three strains. IL-4 was detected in spleen cell culture supernatants from infected BALB/c mice but not from C57BL/6 or BALB.B10 mice. IL-5 could not be detected. C57BL/6 and BALB.B10 spleen cells also produced more tumor necrosis factor alpha and IL-6 after stimulation with PPD and CF than BALB/c cells did. Finally, BCG vaccination generated efficient protective immunity in C57BL/6 and BALB.B10 mice but not in BALB/c mice. These data suggest that secreted mycobacterial CF antigens selectively induce a strong TH1 response in BCG-infected C57BL/6 and BALB.B10 mice, whereas in BALB/c mice this response is partly counterbalanced by TH2 cells.

Antibody Levels to Whole Culture Filtrate Antigens and to Purified P32 during Treatment of Smear-Positive Tuberculosis

Antimycobacterial IgG levels were measured repeatedly during treatment in 12 patients with moderate or severe pulmonary tuberculous disease using a dot immunobinding assay. We used reflectance densitometry equipment to quantify the immunoperoxidase staining and a Mycobacterium bovis BCG culture filtrate and the purified P32 protein as antigens. Antibody response against whole culture filtrate and P32 antigen increased after a three-month period of treatment. After this antibiotherapy was completed, the estimated amount of antibodies directed against the P32 decreased while those against the whole culture filtrate remained at the same level. A serologic test using P32 as the antigen seems to allow a better discrimination between active and healed tuberculosis.

Repertoires of antibodies to culture filtrate antigens in different mouse strains infected with Mycobacterium bovis BCG

Two susceptible (Bcgs) mouse strains, BALB/c and C57BL/6, were compared by Western blot (immunoblot) analysis for their immunoglobulin G response to 14-day-old BCG culture filtrate (CF) following intravenous infection with live Mycobacterium bovis BCG. The two strains demonstrated a completely different antibody repertoire. BALB/c antibodies were directed against a wide range of CF antigens between 20 and about 100 kilodaltons (kDa), with a preferential recognition of the 65-kDa heat shock protein and the 32-kDa fibronectin-binding protein. C57BL/6 sera, on the other hand, showed a much more restricted antibody pattern, almost exclusively directed against three antigens with estimated molecular sizes of 37, 38, and 40 kDa. Whereas the 37- and 38-kDa antigens were also recognized by BALB/c mice, the 40-kDa antigen was very intensely stained by C57BL/6 sera only. F1 mice had the restricted antibody pattern of C57BL/6 after one injection of BCG and had a hybrid BALB/c-C57BL/6 phenotype following a boost injection of BCG 2 months after the initial infection. Analysis of seven recombinant inbred strains derived from the BALB/c x C57BL/6 cross and of congenic mice differing in major histocompatibility complex-coding chromosome 17 fragments suggests that a gene in the K-IA region of the H-2 locus is associated with the preferential recognition of certain CF antigens. Inoculation with the same dose of killed BCG failed to elicit an antibody response to these filtrate antigens.

H-2-linked control of in vitro gamma interferon production in response to a 32-kilodalton antigen (P32) of Mycobacterium bovis bacillus Calmette-Guérin

A 32-kilodalton protein antigen (P32) was previously purified to homogeneity from culture filtrate of Mycobacterium bovis BCG (J. De Bruyn, K. Huygen, R. Bosmans, M. Fauville, R. Lippens, J. P. Van Vooren, P. Falmagne, H. G. Wiker, M. Harboe, and M. Turneer, Microb. Pathog. 2:351-366, 1987). Spleen cells from BCG-sensitized mice produce significant amounts of gamma interferon (IFN-gamma) in response to this P32 protein. The amount of secreted IFN-gamma is influenced by mouse genotype, with C57BL/6 (H-2b), C57BL/10 (H-2b), and 129/Sv (H-2b) mice producing about four times more than BALB/c (H-2d), CBF1 (H-2d/b), and DBA/2 (H-2d) mice do. Analysis of seven recombinant inbred strains derived from the BALB/c x C57BL/6 cross and of congenic mice differing in major histocompatibility complex-coding chromosome 17 fragments indicates a probable H-2-linked control of this IFN-gamma induction, with H-2b cells producing high titers and H-2d cells producing low titers in response to the P32 antigen.

Purification, characterization and identification of a 32 kDa protein antigen of Mycobacterium bovis BCG

An immunogenic protein called P32 has been purified from Sauton zinc deficient culture filtrate of Mycobacterium bovis BCG using successively hydrophobic chromatography on Phenyl-Sepharose, ion exchange on DEAE-Sephacel and molecular sieving on Sephadex G-100. The final preparation was found to be homogeneous as based on several analyses. This P32 protein was a constituent of BCG cells grown in normal conditions. It represented about 3% of the soluble fraction of a cellular extract, and appeared as the major protein released in normal Sauton culture filtrate. This protein was found to have a molecular weight of 32000 by SDS-polyacrylamide gel electrophoresis and in molecular sieving. Its amino acid composition showed an abundance of acidic amino acids (or their amides). The NH2-terminal amino acid sequence (6 amino acids) was determined. Purified P32 was tested by various crossed immunoelectrophoresis techniques, and was shown to belong to the antigen 85 complex in the reference system for BCG antigens. It was more precisely identified as antigen 85A. The protein antigen elicited a weak delayed hypersensitivity reaction in guinea pigs sensitized with heat-killed or living BCG. No delayed hypersensitivity reaction was observed in living BCG sensitized mice, however, it induced significant amounts of gamma Interferon in cultured spleen cells from BCG-sensitized mice. Moreover, P32 either pure or as part of BCG soluble extract promoted substantial antibody levels when injected in rabbits.

Purification, partial characterization, and identification of a skin-reactive protein antigen of Mycobacterium bovis BCG

An immunogenic and skin-reactive protein called P64 was purified from Sauton zinc-deficient culture filtrate of Mycobacterium bovis BCG by using successively hydrophobic chromatography on phenyl-Sepharose, ion exchange on DEAE-Sephacel, and molecular sieving on Sephadex G-200. The final P64 preparation was found to be homogeneous based on several analyses. Protein P64 was a constituent of BCG cells since it was present in soluble cellular extract from normally grown BCG cells. It represented 8 to 9% of the soluble proteins of the extract and appeared as the major soluble protein antigen of BCG. This protein was found to have a molecular weight of 64,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in molecular sieving it eluted at a volume corresponding to a molecular weight of 246,000. An abnormal UV spectrum was observed for this protein. Its amino acid composition showed an abundance of acidic amino acids (or their amides). Aromatic amino acids represented only 3% of the total amino acid residues. The NH2-terminal amino acid sequence of this protein (10 amino acids) was determined. Its sugar content measured with the phenol-sulfuric acid test was lower than 0.3% (wt/wt.) Isolated P64 was tested by various crossed-immunoelectrophoresis techniques and was shown to correspond to antigen 82 in the reference system for BCG antigens. The protein antigen P64 elicited a delayed cutaneous reaction in guinea pigs sensitized with either living or heat-killed BCG. Its potency in skin reaction was, respectively, two- and threefold that of the BCG purified protein derivative. The two types of sensitization used for skin test reactions promoted significant immunoglobulin G antibody production against the protein antigen P64 in guinea pigs 7 weeks after sensitization.