Kamiel Palfliet

Mapping of murine Th1 helper T-cell epitopes of mycolyl transferases Ag85A, Ag85B, and Ag85C from Mycobacterium tuberculosis

ABSTRACT BALB/c (H-2d) and C57BL/6 (H-2b) mice were infected intravenously with Mycobacterium tuberculosis H37Rv or vaccinated intramuscularly with plasmid DNA encoding each of the three mycolyl transferases Ag85A, Ag85B, and Ag85C from M. tuberculosis. Th1-type spleen cell cytokine secretion of interleukin-2 (IL-2) and gamma interferon (IFN-γ) was analyzed in response to purified Ag85 components and synthetic overlapping peptides covering the three mature sequences. Tuberculosis-infected C57BL/6 mice reacted strongly to some peptides from Ag85A and Ag85B but not from Ag85C, whereas tuberculosis-infected BALB/c mice reacted only to peptides from Ag85A. In contrast, spleen cells from both mouse strains produced elevated levels of IL-2 and IFN-γ following vaccination with Ag85A, Ag85B, and Ag85C DNA in response to peptides of the three Ag85 proteins, and the epitope repertoire was broader than in infected mice. Despite pronounced sequence homology, a number of immunodominant regions contained component specific epitopes. Thus, BALB/c mice vaccinated with all three Ag85 genes reacted against the same amino acid region, 101 to 120, that was also immunodominant for Ag85A in M. bovis BCG-vaccinated and tuberculosis-infected H-2d haplotype mice, but responses were completely component specific. In C57BL/6 mice, a cross-reactive T-cell response was detected against two carboxy-terminal peptides spanning amino acids 241 to 260 and 261 to 280 of Ag85A and Ag85B. These regions were not recognized at all in C57BL/6 mice vaccinated with Ag85C DNA. Our results underline the need for comparative analysis of all three Ag85 components in future vaccination studies.

What is the optimal regimen for BCG intravesical therapy? Are six weekly instillations necessary?

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.

Humoral response against heat shock proteins and other mycobacterial antigens after intravesical treatment with bacille Calmette–Guérin (BCG) in patients with superficial bladder cancer

Few studies have analysed the antibody response during intravesical BCG immunotherapy for superficial bladder cancer. We have examined the evolution in serum antibody response against several heat shock proteins (hsp), including the recombinant mycobacterial hsp65 and the native protein P64 from BCG, GroEL from Escherichia coli (hsp60 family), recombinant mycobacterial hsp70 and the E. coli DnaK (hsp70 family), against purified protein derivative of tuberculin (PPD) and the AG85 complex of Mycobacterium bovis BCG, as well as against tetanus toxoid in 42 patients with a superficial bladder tumour, 28 treated with six intravesical BCG instillations and 14 patients used as controls. We also analysed the lymphoproliferative response of peripheral blood mononuclear cells against PPD in this population. Data of antibody responses at 6 weeks post BCG were available in all 28 patients, and at 4 month follow up in 17 patients. All patients who demonstrated a significant increase in IgG antibodies against PPD at 4 months follow up had a significant increase already at 6 weeks of follow up. In contrast, IgG antibodies against hsp increased significantly from 6 weeks to 4 months post‐treatment. A significant increase in IgG antibodies against PPD, hsp65, P64, GroEL, and hsp70 at 4 months follow up was observed in 10/17, 8/17, 10/17, 4/17 and 8/17 patients. Native P64 protein elicited a higher antibody response than recombinant mycobacterial hsp65. No increase in antibody response was observed against Dnak from E. coli, against AG85 or tetanus toxoid after BCG therapy. An increase in IgG antibodies against P64 at 4 months follow up compared with pretreatment values was found to be a significant predictor of tumour recurrence (P < 0.01). Further studies with a larger number of patients are needed to confirm the value of the antibody response against P64 as a clinical independent prognostic factor.

Strain variation in interferon γ production of BCG-sensitized mice challenged with PPD: II. Importance of one major autosomal locus and additional sexual influences

Abstract Interferon-γ (IFN-γ) production in BCG-sensitized mice challenged with PPD was examined in the sera from BALB/c and C57BL/6 mice. C57BL/6 mice produce about ten times more IFN-γ than BALB/c mice. Studies on F 1 , F 2 , and backcross generations indicate that one partially dominant autosomal locus is involved. Furthermore, females consistently produce more IFN-γ than males in all of these crosses, though the X chromosome cannot be held responsible for this.

Evolution and Clinical Significance of the T Cell Proliferative and Cytokine Response Directed Against the Fibronectin Binding Antigen 85 Complex of Bacillus Calmette-Guerin During Intravesical Treatment of Superficial Bladder Cancer

PURPOSE The antitumorigenic effect of intravesical bacillus Calmette-Guerin (BCG) in superficial bladder cancer was reported to be initiated by the attachment of BCG to the bladder wall via fibronectin. The antigen 85 complex secreted in BCG culture filtrate binds specifically to fibronectin and is a powerful T cell stimulus. Therefore, we investigated the evolution and clinical significance of the cellular proliferative response and cytokine production during intravesical BCG therapy against this purified antigen 85 complex. MATERIALS AND METHODS Evolution of the lymphoproliferation, interleukin-2 and interferon-gamma production of peripheral blood lymphocytes against tuberculin (purified protein derivative), purified antigen 85, BCG culture filtrate, whole BCG bacilli and pokeweed mitogen was tested before and after 6 weekly intravesical BCG instillations in 29 patients with superficial bladder cancer at intermediate or high risk for recurrence. RESULTS A major increase in the lymphoproliferative response against purified protein derivative, antigen 85, BCG culture filtrate, whole BCG and pokeweed mitogen was observed in 69.0, 65.5, 79.3, 48.3 and 65.3% of the patients, respectively, analyzed after BCG therapy. Reactivity returned to baseline values at 6 months of followup. Of the patients who received a second BCG course because of tumor recurrence 66% had a novel increase in lymphoproliferation against antigen 85. An increase in the production of interleukin-2 and interferon-gamma by peripheral lymphocytes against antigen 85 was noted in 42.1 and 50% of the treated patients, respectively, after a single BCG course. During a mean followup of 23.11 months 48.5% of the patients remained tumor-free. No correlation could be found between the immunological response against any of the BCG antigens and the clinical evolution of the response. CONCLUSIONS Intravesical BCG instillations induce a transient (less than 6 months) peripheral immune activation against several purified BCG antigens and among them the fibronectin binding antigen 85 complex. Reactivation is observed in most cases after additional BCG courses. The absence of long lasting immune activation after a single 6-week course of BCG could be related to the increased clinical efficacy observed with BCG maintenance instillations.

CROSS-REACTIVE IMMUNE RESPONSES AGAINST MYCOBACTERIUM BOVIS BCG IN MICE INFECTED WITH NON-TUBERCULOUS MYCOBACTERIA BELONGING TO THE MAIS-GROUP

Two bacillus Calmette–Guérin (BCG)‐susceptible mouse strains, BALB/c and C57BL/6, were infected intravenously with Mycobacterium intracellulare, M. avium or M. scrofulaceum and monitored during 3 months for mycobacterial replication and antibody and Th1‐type cytokine production in response to cytoplasmic and secreted antigens from M. bovis BCG. Whereas initial colony‐forming unit (CFU) counts of M. intracellulare and M. avium were higher in lungs than in spleen, the opposite was observed for M. scrofulaceum. Mycobacterium intracellulare was the most virulent species and its replication could not be controlled in either mouse strain. It also induced the strongest antibody response. Mycobacterium avium was eliminated in both mouse strains and M. scrofulaceum finally was eliminated in C57BL/6 but multiplied in spleen from BALB/c mice. Significant sustained interleukin‐2 and interferon‐γ production towards BCG antigens was only found in M. scrofulaceum infection. As in BCG‐vaccination, M. scrofulaceum‐infected C57BL/6 mice demonstrated a higher response towards whole BCG culture filtrate, BCG extract and purified antigen 85 complex (Ag85) from BCG than did BALB/c mice. The data suggest that the presence of M. scrofulaceum in the environment may possibly interfere in genetically predisposed subjects with BCG vaccine and its protective efficacy against M. tuberculosis.

Strain variation in interferon gamma production of BCG-sensitized mice challenged with PPD. I. CBA/Ca mice are low producers in vivo, but high producers in vitro.

Three different mouse strains--BALB/c, C57BL/6J, and CBA/Ca--were compared for their production of viral interferon upon Newcastle disease virus (NDV) inoculation and of interferon gamma upon antigenic stimulation with PPD following BCG sensitization. BALB/c mice were found to be low producers for NDV-induced IFN-alpha, beta as well as for in vivo (serum) and in vitro (spleen cell culture) induced IFN-gamma. C57BL/6 mice were high producers under the three conditions tested and CBA/Ca mice, finally, were high IFN-alpha, beta and in vitro IFN-gamma producers, but low in vivo IFN-gamma producers. Development of splenomegaly following BCG inoculation was not directly related to IFN-gamma responsiveness.

Antibody repertoire against culture filtrate antigens in wild house mice infected with Mycobacterium bovis BCG

Wild house mice (Mus domesticus) captured in a Flemish pigsty were infected intravenously with 4 × l06 viable 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‐infectcd 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 allcle is expressed in this population.

SUPERFICIAL BLADDER TUMORS AND INCREASED REACTIVITY AGAINST MYCOBACTERIAL ANTIGENS BEFORE BACILLUS CALMETTE-GUERIN THERAPY

PURPOSE The precise mechanism of action of bacillus Calmette-Guerin (BCG) in bladder cancer treatment remains poorly understood. Whether bladder tumor cells are destroyed by nonspecific mechanisms or targeted by specifically activated lymphocytes recognizing cognate antigens is unclear. To investigate a possible cross-reactivity between BCG and bladder cell tumors, we tested before BCG treatment the lymphoproliferation of peripheral blood lymphocytes against several mycobacterial antigens, including the secreted fibronectin binding antigen 85 complex from BCG (AG 85) in patients with superficial bladder tumors compared to control matched patients. MATERIALS AND METHODS Using a whole blood assay, T cell response against purified protein derivative, BCG extract, whole BCG, purified AG 85, and the nonspecific mitogens pokeweed and phytohemagglutinin was investigated in 79 patients with superficial bladder tumors before BCG and in 39 control subjects without malignancy matched for age and sex. Neither group had a history of tuberculosis. Lymphoproliferation was measured with a tritiated thymidine uptake assay on day 7 of culture. RESULTS Of the 79 patients with superficial transitional cell carcinoma, a significant lymphoproliferative response before BCG against PPD, BCG extract, whole BCG and AG 85 was observed in 65 (82.2%), 67 (84.81%), 30 (37.97%) and 49 (62.02%) patients, respectively. Of the 39 controls only 26 (64.1%), 23 (58.9%), 3 (7.7%) and 3 (7.7%) patients, respectively, had a significant lymphoproliferation against PPD, BCG extract, BCG and AG 85 (p >0.05, p = 0.004, p = 0.00001 and p = 0.00001, respectively). In terms of lymphoproliferative levels, patients with superficial transitional cell carcinoma also showed a significantly higher response against PPD (p = 0.000012), BCG extract (p = 0.000001), AG 85 (p = 0.000001), whole BCG (p = 0.00001) and pokeweed (p = 0.01) than controls but not against phytohemagglutinin. CONCLUSIONS Patients with superficial transitional cell carcinoma demonstrate an increased lymphoproliferation against mycobacterial antigens before BCG compared to control subjects. Although a nonspecific activation of the immune system cannot be excluded at this stage, our data may suggest the possible existence of bladder cancer antigens cross-reactive with mycobacterial antigens responsible for boosting precursor cells witnessing previous contacts with mycobacteria. The implication of these findings in the antitumoral mechanism of action of BCG are under investigation.

Influence of in vivo interferon treatment on mitogen responsiveness of T- and B-cells is dependent on the mouse genotype

Intravenous inoculation of moderate doses of viral interferon (up to 10(4) I.U./mouse) induced subsequent modification of the mitogen responsiveness of T-and B-lymphocytes from the spleen. The modulation, however, was dependent on the mouse genotype. Whereas BALB/c mice demonstrated enhanced T-cell responses (especially to PHA), C57B1/6 mice were generally suppressed in their T-cell response by the same treatment. B-cell proliferation to LPS showed a moderate enhancement in both strains, probably due to activation of accessory cells.