Effects of Mycobacterium tuberculosis on B cell responses.
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Background Mycobacterium tuberculosis (M.tb) causes tuberculosis (TB), the leading cause of death from a single pathogen. The success of this microbe has been linked to its subversive effects on host immunity. Indeed, severity of active pulmonary disease (APTB) increases in spite of a robust antibody response. Antibody avidity or the over-all binding strength is an important determinant of antibody-mediated protection. This research study hypothesised that the active TB disease state was associated with less avid antibodies in comparison to latent tuberculosis infection (LTBI), a state in which M.tb infection is controlled. Antibody responses were characterised in individuals of varied M.tb infection state from a TB household contact study in Uganda in order to investigate this hypothesis. Serum samples were not available for all the study participants but supernatants left over from QuantiFERON®-TB Gold in-tube (QFT-GIT) testing for M.tb infection were available. It was therefore necessary to conduct a sub-study to determine whether QFT-GIT could be used for antibody analyses and whether antibody levels in these samples were comparable to those in serum. Concentrations of antibodies against a panel of M.tb secreted and latency associated membrane antigens were evaluated so as to identify the most appropriate for avidity analyses. Rather unexpectedly, APTB was associated with higher concentrations of antibodies to tetanus toxoid, a non-Mycobacterium control antigen included in these investigations. It was hypothesised that M.tb and the related vaccine, Mycobacterium bovis BCG, boosts antibody responses to heterologous pathogens and a further sub-study was established to investigate this. The possibility that Mycobacterium driven non-specific effects may be due to polyclonal activation of memory B cells was also explored. Methods Concentrations of M.tb specific antibodies were determined and compared between QFT-GIT unstimulated (nil) supernatant and serum pairs. Subsequently, concentration and avidity of antibodies against M.tb antigens & heterologous pathogen antigens was determined and compared between uninfected controls, individuals with LTBI and APTB cases. Concentrations of antibodies against heterologous pathogen antigens were also determined and compared between recently BCG vaccinated individuals and their age-matched controls. Antibody ELISAs were used to measure antibody concentration whereas surface plasmon resonance (SPR) and chaotrope-based assays were used to measure relative antibody avidity. Flow cytometry was used to evaluate B cell responses following M.tb antigen stimulation of peripheral blood mononuclear cells (PBMCs) from healthy individuals. Results Use of QuantiFERON®-TB gold in-tube culture supernatants for measurement of antibody responses Antibodies against two M.tb specific diagnostic antigens, CFP-10 and ESAT-6 were evaluated in QFT-GIT unstimulated (nil) supernatant and serum pairs from 68 TB household contacts. There were strong and statistically significant correlations in anti-CFP-10 and anti-ESAT-6 antibody concentrations between QFT-GIT nil supernatants and sera (r=0.89; p<0.0001 for both), and no significant differences in the amounts of anti-CFP-10 and anti-ESAT-6 antibodies between these specimens. Profile of Mycobacterium specific antibody responses in M.tb infected individuals Concentrations of antibodies against M.tb secreted proteins such as Ag85A, crude culture filtrate protein (cCFP) and culture filtrate protein-10/early secretory antigenic target-6 (CFP-10/ESAT-6) fusion protein, and the latency associated dormancy survival regulator (DosR) regulon-encoded proteins, Rv1733c, Rv0081, Rv1735c and Rv1737c, were evaluated in QFT-GIT nil supernatants and/or serum from 68 uninfected individuals, 62 individuals with LTBI, and 107 APTB cases to guide the choice of antigen for antibody avidity investigations. As a control, concentration of antibodies to tetanus toxoid were assessed. Individuals with APTB had higher concentrations of antibodies against the secreted antigens, Ag85A [adjusted geometric ratio (GMR): 3.429, 95% confidence interval (CI): 2.339, 5.026, 𝑝 < 0.001], cCFP (adjusted GMR: 2.876, 95% CI: 2.146, 3.853, 𝑝 < 0.001) and CFP-10/ESAT-6 (adjusted GMR: 2.020, 95% CI: 1.279, 3.190, 𝑝 = 0.003) compared to uninfected controls. There was also higher anti-Ag85A responses in individuals with LTBI (adjusted GMR: 1.517, 95% CI: 1.087, 2.116, 𝑝 = 0.014) compared to uninfected controls. There were no differences in antibody responses to Rv0081, Rv1735c, and Rv1737c DosR regulon-encoded proteins between individuals with LTBI and APTB, and those who were uninfected but LTBI was associated with increased anti-Rv1733c antibody responses in female participants (adjusted GMR: 1.812, 95% CI: 1.105, 2.973, 𝑝 = 0.019) but not in males (p value for interaction = 0.060). Rather unexpectedly, it was found that concentrations of antibodies against tetanus toxoid (TT) were higher among APTB cases in comparison to the uninfected controls (adjusted GMR: 1.579, 95% CI: 1.028, 2.425, 𝑝 = 0.037). Characterising antibody avidity in individuals of varied M.tb infection status The antigen, Ag85A, was selected for use in antibody avidity studies because antibodies against this protein were elevated in both latent and active states of M.tb infection. Samples from 30 uninfected controls, 34 individuals with LTBI and 75 APTB cases were tested. Chaotrope-based assays indicated that APTB was associated with a higher antibody avidity compared to uninfected controls (adjusted GMR: 1.641, 95% CI: 1.153, 2.337, 𝑝 = 0.006) and to individuals with LTBI (adjusted GMR: 1.604, 95% CI: 1.282, 2.006, 𝑝 < 0.001). The SPR assays showed that APTB and LTBI were both associated with slower antibody-antigen dissociation rates, an indication of higher avidity, compared to uninfected controls (adjusted GMR: 0.871, 95% CI: 0.763, 0.994, 𝑝 = 0.041 and adjusted GMR: 0.796, 95% CI: 0.681, 0.932, 𝑝 = 0.004 respectively) and detected no statistically significant differences between the two. Effects of Mycobacterium tuberculosis infection on antibody and B cell responses to heterologous pathogens Antibodies against several heterologous pathogens were measured in 68 uninfected individuals, 62 with LTBI, and 107 APTB cases and 12 recently vaccinated adolescents from the UK and 12 age-matched controls. Active TB was associated with higher concentrations of antibodies to diphtheria toxin (adjusted GMR: 1.581, 95% CI: 1.136, 2.201, 𝑝 = 0.007), respiratory syncytial virus (adjusted GMR: 1.161, 95% CI: 1.056, 1.276, 𝑝 = 0.002), measles virus (adjusted GMR: 1.665, 95% CI: 1.328, 2.088, 𝑝 < 0.001) and Kaposi’s sarcoma herpesvirus ORF73 (adjusted GMR: 7.680, 95% CI: 1.898, 31.068, 𝑝 = 0.004) compared to uninfected controls but there were no associations with BCG vaccination. Avidity of TT and measles virus specific antibodies was also higher in APTB cases in comparison to uninfected controls. Stimulation of PBMCs from 14 healthy individuals with PPD from M.tb led to a significant decrease in the proportions of resting memory B cells, however activated memory B cell and plasmablast proportions did not changed significantly. Conclusions This PhD study showed that QFT-GIT nil supernatants may be used in the place of sera when measuring anti-mycobacterial antibody responses. This finding allowed the use of QFT-GIT nil supernatants for subsequent investigations. The use of these samples could help reduce blood volumes drawn from study participants. The assessment of antibody responses to various M.tb antigens revealed that APTB was strongly associated with higher responses to secreted antigens with responses against the immunodominant secreted antigen, Ag85A in particular being associated with both APTB and LTBI. Avidity analyses revealed that antibodies of increased avidity are generated against Ag85A in both APTB and LTBI. This result provides evidence of affinity maturation in M.tb infection and does not support the hypothesis that M.tb interferes with this process in active TB disease. The observed increase in antibody avidity in TB disease puts into question the importance of avidity of Mycobacterium-specific antibodies in the control of M.tb growth. Further exploration of the relationship between antibody avidity and humoral immunity against TB is recommended. The increase in antibody responses to heterologous pathogens in terms of concentration and avidity in APTB indicate that M.tb may non-specifically boost recall antibody responses in humans and that memory B cells are their likely source. The fact that BCG vaccination did not have a similar effect on levels of antibodies to the pathogens investigated indicates that this property may be mediated by M.tb specific antigens during infection. Unfortunately, exploration of the effects of M.tb antigen stimulation on memory B cell activation did not yield conclusive results. It is not clear how the M.tb pathogen benefits from the rise in antibody responses to unrelated recall antigens, however, if this process can be exploited it may lead to the development of immunotherapeutics that maintain serological memory to past infections and vaccines. Further research is needed to ascertain the mechanisms underlying memory B cell polyclonal activation.