Annie Drowart

Immunogenicity and protective efficacy of a tuberculosis DNA vaccine

Tuberculosis is the most widespread and lethal infectious disease affecting humans. Immunization of mice with plasmid DNA constructs encoding one of the secreted components of Mycobacterium tuberculosis, antigen 85 (Ag85), induced substantial humoral and cell–mediated immune responses and conferred significant protection against challenge with live M. tuberculosis and M. bovis bacille Calmette–Guérin (BCG). These results indicate that immunization with DNA encoding a mycobacterial antigen provides an efficient and simple method for generating protective immunity and that this technique may be useful for defining the protective antigens of M. tuberculosis, leading to the development of a more effective vaccine.

Correlation and prognostic significance of p53, p21WAF1/CIP1 and Ki-67 expression in patients with superficial bladder tumors treated with bacillus Calmette-Guerin intravesical therapy.

PURPOSE We determine if, before intravesical bacillus-Calmette Guerin (BCG) therapy, p53, p21WAF-1-CIP1 (a critical downstream effector of p53 pathway of cell growth control, inhibiting cyclin dependent kinases) and the cell proliferation marker Ki-67 (MIB-1) could be used as prognostic markers of response to BCG in patients with superficial bladder tumors. MATERIALS AND METHODS The study included 47 patients with superficial bladder tumors at high risk for recurrence or progression treated with 6 weekly intravesical BCG instillations. We analyzed p53, p21 and Ki-67 on paraffin embedded samples by immunohistochemistry and the percentage of positive cells was determined in a blinded fashion. Quantitative immunostaining was analyzed in relation to time to recurrence and progression using univariate or multivariate analysis and the Kaplan-Meier method. RESULTS During a mean followup of 24.6 months 23 of the 47 patients (48.9%) presented with tumor recurrence and 10 (21.2%) had later progression to invasive disease. A p21 over expression (greater than 10%) was observed in 23 tumors (48.9%) and positively correlated with p53 (p = 0.0097) but not with Ki-67 (p = 0.327). Of the tumors 18 (38.2%) were p53 and p21 negative. Among p21 positive tumors 15 (65.2%) were p53 and p21 positive, suggesting that p21 may also be regulated by p53 independent pathways. However, p53 did not act as a predictor of recurrence or progression. In contrast, using Kaplan-Meier curves p21 over expression (greater than 10%) and Ki-67 at a 25% cutoff were associated with shorter recurrence-free survival (both p = 0.02 log rank test) but they did not predict additional information about risk of progression. However, multivariate analysis failed to demonstrate any independent prognostic value for p21 or Ki-67 in contrast to tumor stage. CONCLUSIONS Our results indicate that p21WAF-1-CIP1 seems to be regulated by p53 independent pathways in superficial bladder cancer. The present study did not indicate an independent prognostic significance in patients treated with BCG for p53, p21WAF-1-CIP1 or Ki-67 markers. Larger prospective studies are needed to evaluate further the independent value of these biological markers in superficial bladder cancer management.

Methylation-dependent T cell immunity to Mycobacterium tuberculosis heparin-binding hemagglutinin

Although post-translational modifications of protein antigens may be important componenets of some B cell epitopes, the determinants of T cell immunity are generally nonmodified peptides. Here we show that methylation of the Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA) by the bacterium is essential for effective T cell immunity to this antigen in infected healthy humans and in mice. Methylated HBHA provides high levels of protection against M. tuberculosis challenge in mice, whereas nonmethylated HBHA does not. Protective immunity induced by methylated HBHA is comparable to that afforded by vaccination with bacille Calmette et Guérin, the only available anti-tuberculosis vaccine. Thus, post-translational modifications of proteins may be crucial for their ability to induce protective T cell-mediated immunity against infectious diseases such as tuberculosis.

Differential T and B Cell Responses against Mycobacterium tuberculosis Heparin-Binding Hemagglutinin Adhesin in Infected Healthy Individuals and Patients with Tuberculosis

Because only 10% of individuals infected with Mycobacterium tuberculosis will eventually develop disease, antigens that are recognized differently by the immune systems of infected healthy and diseased subjects may constitute potential vaccine candidates. Here, the heparin-binding hemagglutinin adhesin (HBHA) is identified as such an antigen. Lymphocytes from 60% of healthy infected individuals (n=25) produced interferon (IFN)-gamma after stimulation with HBHA, compared with only 4% of patients with active tuberculosis (n=24). In the responders, both CD4(+) and CD8(+) cells secreted HBHA-specific IFN-gamma, and the antigen was presented by both major histocompatibility complex class I and II molecules. In contrast to the reduced ability of patients with tuberculosis to produce HBHA-specific IFN-gamma, most of them (82%) produced anti-HBHA antibodies, compared with 36% of the infected healthy subjects. These observations indicate that HBHA is recognized differently by the immune systems of patients with tuberculosis and infected healthy individuals and might provide a marker for protection against tuberculosis.

Heparin-Binding-Hemagglutinin-Induced IFN-γ Release as a Diagnostic Tool for Latent Tuberculosis

Background The detection of latent tuberculosis infection (LTBI) is a major component of tuberculosis (TB) control strategies. In addition to the tuberculosis skin test (TST), novel blood tests, based on in vitro release of IFN-γ in response to Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP-10 (IGRAs), are used for TB diagnosis. However, neither IGRAs nor the TST can separate acute TB from LTBI, and there is concern that responses in IGRAs may decline with time after infection. We have therefore evaluated the potential of the novel antigen heparin-binding hemagglutinin (HBHA) for in vitro detection of LTBI. Methodology and Principal Findings HBHA was compared to purified protein derivative (PPD) and ESAT-6 in IGRAs on lymphocytes drawn from 205 individuals living in Belgium, a country with low TB prevalence, where BCG vaccination is not routinely used. Among these subjects, 89 had active TB, 65 had LTBI, based on well-standardized TST reactions and 51 were negative controls. HBHA was significantly more sensitive than ESAT-6 and more specific than PPD for the detection of LTBI. PPD-based tests yielded 90.00% sensitivity and 70.00% specificity for the detection of LTBI, whereas the sensitivity and specificity for the ESAT-6-based tests were 40.74% and 90.91%, and those for the HBHA-based tests were 92.06% and 93.88%, respectively. The QuantiFERON-TB Gold In-Tube (QFT-IT) test applied on 20 LTBI subjects yielded 50% sensitivity. The HBHA IGRA was not influenced by prior BCG vaccination, and, in contrast to the QFT-IT test, remote (>2 years) infections were detected as well as recent (<2 years) infections by the HBHA-specific test. Conclusions The use of ESAT-6- and CFP-10-based IGRAs may underestimate the incidence of LTBI, whereas the use of HBHA may combine the operational advantages of IGRAs with high sensitivity and specificity for latent infection.

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.

What are the immunologically active components of Bacille Calmette-Guérin in therapy of superficial bladder cancer?

The subcomponents of bacille Calmette‐Guérin (BCG) involved in the mechanism of action of intravesical BCG immunotherapy used for prophylaxis of superficial bladder cancer recurrences have been poorly investigated. We purified various BCG subcomponents and analyzed in vitro their ability to enhance a Th1 polarized immune response as well as to increase lymphocyte‐mediated cytotoxicity against bladder tumors. Human peripheral blood mononuclear cells (PBMCs) from healthy purified protein derivative–positive subjects were incubated for 7 days with whole BCG and various fractions (BCG cell wall, plasma membrane, cytosol, purified polysaccharides as glucan or arabinomannan, purified native proteins from BCG culture filtrate, recombinant 22 kDa protein, phosphate transporter PstS‐2 and ‐3 proteins). IFN‐γ, IL‐12, IL‐2, and IL‐6 production by stimulated PBMCs was compared to unstimulated controls and the phenotype of expanded cells analyzed by flow cytometry (FACS analysis). A 51Cr‐release assay monitored the cytotoxicity of amplified effector cells against T24 bladder tumor cells. Live BCG and most of its subcomponents (with the exception of cytosol, PstS‐2 and ‐3) significantly enhanced IFN‐γ and IL‐12 secretion, expanded CD3–CD56+ cells and the non‐MHC‐restricted cytotoxicity against bladder tumor cells compared to unstimulated controls (all P < 0.001, t‐test). IL‐2 receptor blockage resulted in a clear reduction in the cytotoxic activity of stimulated PBMCs. Numerous BCG subcomponents thus provide positive stimuli for Th1 cell differentiation and enhance in vitro, non‐MHC‐restricted cytotoxicity against bladder tumor cells. Our findings provide the basis for the therapeutic use of several of these subfractions in experimental animal models bearing bladder tumors. Int. J. Cancer 87:844–852, 2000.

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.

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.