Virginie Verscheure

Bordetella pertussis Infection in 2-Month-Old Infants Promotes Type 1 T Cell Responses

Neonatal immaturity of the immune system is currently believed to generally limit the induction of immune responses to vaccine Ags and to skew them toward type 2 responses. We demonstrated here that Bordetella pertussis infection in very young infants (median, 2 mo old) as well as the first administration of whole-cell pertussis vaccine induces B. pertussis Ag-specific IFN-γ secretion by the PBMC of these infants. IFN-γ was secreted by both CD4+ and CD8+ T lymphocytes, and the levels of Ag-induced IFN-γ secretion did not correlate with the age of the infants. Appearance of the specific Th-1 cell-mediated immunity was accompanied by a general shift of the cytokine secretion profile of these infants toward a stronger Th1 profile, as evidenced by the response to a polyclonal stimulation. We conclude that the immune system of 2-mo-old infants is developmentally mature enough to develop Th1 responses in vivo upon infection by B. pertussis or vaccination with whole-cell pertussis vaccines.

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.

Risk stratification of latent tuberculosis defined by combined interferon gamma release assays.

Background Most individuals infected with Mycobacterium tuberculosis develop latent tuberculosis infection (LTBI). Some may progress to active disease and would benefit from preventive treatment yet no means currently exists to predict who will reactivate. Here, we provide an approach to stratify LTBI based on IFN-γ responses to two antigens, the recombinant Early-Secreted Antigen Target-6 (rESAT-6) and the latency antigen Heparin-Binding Haemagglutinin (HBHA). Methods We retrospectively analyzed results from in-house IFN-γ-release assays with HBHA (HBHA-IGRA) and rESAT-6 (rESAT-6-IGRA) performed during a 12-year period on serial blood samples (3 to 9) collected from 23 LTBI subjects in a low-TB incidence country. Both the kinetics of the absolute IFN-γ concentrations secreted in response to each antigen and the dynamics of HBHA/rESAT-6-induced IFN-γ concentrations ratios were examined. Results This analysis allowed the identification among the LTBI subjects of three major groups. Group A featured stable HBHA and rESAT-6-IGRA profiles with an HBHA/rESAT-6 ratio persistently higher than 1, and with high HBHA- and usually negative rESAT-6-IGRA responses throughout the study. Group B had changing HBHA/rESAT-6 ratios fluctuating from 0.0001 to 10,000, with both HBHA and rESAT-6 responses varying over time at least once during the follow-up. Group C was characterized by a progressive disappearance of all responses. Conclusions By combining the measures of IFN-γ concentrations secreted in response to an early and a latency antigens, LTBI subjects can be stratified into different risk groups. We propose that disappearing responses indicate cure, that persistent responses to HBHA with HBHA/rESAT-6 ratios ≥1 represent stable LTBI subjects, whereas subjects with ratios varying from >1 to <1 should be closely monitored as they may represent the highest-risk group, as illustrated by a case report, and should therefore be prioritized for preventive treatment.

Optimal kinetics for quantification of antigen-induced cytokines in human peripheral blood mononuclear cells by real-time PCR and by ELISA.

Real-time polymerase chain reaction (PCR) has recently been described as a new tool to measure and accurately quantify mRNA levels. In this study, we have applied this technique to evaluate cytokine mRNA synthesis induced by antigenic stimulation with purified protein derivative (PPD) or heparin-binding haemagglutinin (HBHA) in human peripheral blood mononuclear cells (PBMC) from Mycobacterium tuberculosis-infected individuals. Whereas PPD and HBHA optimally induced IL-2 mRNA after respectively 8 and 16 to 24 h of in vitro stimulation, longer in vitro stimulation times were necessary for optimal induction of interferon-gamma (IFN-gamma) mRNA, respectively 16 to 24 h for PPD and 24 to 96 h for HBHA. IL-13 mRNA was optimally induced by in vitro stimulation after 16-48 h for PPD and after 48 to 96 h for HBHA. Comparison of antigen-induced Th1 and Th2 cytokines appears, therefore, valuable only if both cytokine types are analysed at their optimal time point of production, which, for a given cytokine, may differ for each antigen tested. Results obtained by real-time PCR for IFN-gamma and IL-13 mRNA correlated well with those obtained by measuring the cytokine concentrations in cell culture supernatants, provided they were high enough to be detected. We conclude that real-time PCR can be successfully applied to the quantification of antigen-induced cytokine mRNA and to the evaluation of the Th1/Th2 balance, only if the kinetics of cytokine mRNA appearance are taken into account and evaluated for each cytokine measured and each antigen analysed.

Identification of Major Factors Influencing ELISpot-Based Monitoring of Cellular Responses to Antigens from Mycobacterium tuberculosis

A number of different interferon-γ ELISpot protocols are in use in laboratories studying antigen-specific immune responses. It is therefore unclear how results from different assays compare, and what factors most significantly influence assay outcome. One such difference is that some laboratories use a short in vitro stimulation period of cells before they are transferred to the ELISpot plate; this is commonly done in the case of frozen cells, in order to enhance assay sensitivity. Other differences that may be significant include antibody coating of plates, the use of media with or without serum, the serum source and the number of cells added to the wells. The aim of this paper was to identify which components of the different ELISpot protocols influenced assay sensitivity and inter-laboratory variation. Four laboratories provided protocols for quantifying numbers of interferon-γ spot forming cells in human peripheral blood mononuclear cells stimulated with Mycobacterium tuberculosis derived antigens. The differences in the protocols were compared directly. We found that several sources of variation in assay protocols can be eliminated, for example by avoiding serum supplementation and using AIM-V serum free medium. In addition, the number of cells added to ELISpot wells should also be standardised. Importantly, delays in peripheral blood mononuclear cell processing before stimulation had a marked effect on the number of detectable spot forming cells; processing delay thus should be minimised as well as standardised. Finally, a pre-stimulation culture period improved the sensitivity of the assay, however this effect may be both antigen and donor dependent. In conclusion, small differences in ELISpot protocols in routine use can affect the results obtained and care should be given to conditions selected for use in a given study. A pre-stimulation step may improve the sensitivity of the assay, particularly when cells have been previously frozen.

Cellular immune responses of preterm infants after vaccination with whole-cell or acellular pertussis vaccines.

ABSTRACT Based on studies reporting specific antibody titers, it is recommended to vaccinate preterm infants against Bordetella pertussis according to their chronological age. However, as specific T-cell responses also are involved in the protection against B. pertussis, we have determined whether highly preterm infants (<31 weeks) are able to mount these immune responses during vaccination. Forty-eight premature infants were vaccinated at 2, 3, and 4 months of their chronological age with an acellular (Pa; n = 24) or a whole-cell (Pw; n = 24) tetravalent diphtheria-tetanus-pertussis-polio vaccine, and blood samples were collected at 2, 3, and 6 months of age. Most of the Pa- and Pw-vaccinated infants developed at 3 or 6 months of age a gamma interferon (IFN-γ) response to the B. pertussis antigens, accompanied by an interleukin-5 (IL-5) and IL-13 secretion for the Pa-vaccinated infants. No association was found between a very low infant birth weight, the occurrence of severe infections, and corticosteroid treatment or the administration of gammaglobulins with a low level of antigen-induced IFN-γ secretion. We conclude that like full-term infants, most preterm infants are able to mount a specific cellular immune response to the administration of the first doses of an acellular or a whole-cell pertussis vaccine.

Cytokine and antibody profiles in 1-year-old children vaccinated with either acellular or whole-cell pertussis vaccine during infancy

Two different types of pertussis vaccines are currently available to protect children against whooping cough, the first-generation whole-cell (Pw) vaccines and the more recent acellular (Pa) vaccines. Both types provide good protection, yet induce different types of immune responses in 6-month-old infants, with a strong Th1 response induced by Pw vaccines compared to a mixed Th1/Th2 response and a delay in non-specific IFN-gamma secretions after the administration of Pa vaccines. We show here that at 13 months of age, most Pw- or Pa-vaccinated children display Bordetella pertussis-specific T-cell responses, in addition to significant antibody levels, although a higher Th2/Th1 cytokine ratio remained in Pa recipients compared to Pw recipients. In contrast, the proportion of children with tetanus toxin-specific T-cell responses was lower in Pa than in Pw vaccine recipients, although most children had protective anti-tetanus toxin IgG levels. In addition, the global Th2 bias observed in 6-month-old infants vaccinated with a Pa vaccine was normalized at 13 months.

Heparin-binding, Hemagglutinin-specific IFN-γ Synthesis at the Site of Infection during Active Tuberculosis in Humans

RATIONALE Tuberculosis (TB) remains a major cause of mortality. A better understanding of the immune responses to mycobacterial antigens may be helpful to develop improved vaccines and diagnostics. OBJECTIVES The mycobacterial antigen heparin-binding hemagglutinin (HBHA) induces strong IFN-γ responses by circulating lymphocytes from subjects latently infected with Mycobacterium tuberculosis, and low responses associated with CD4(+) regulatory T (Treg) cells in patients with TB. Here, we investigated HBHA-specific IFN-γ responses at the site of the TB disease. METHODS Bronchoalveolar lavages, pleural fluids, and blood were prospectively collected from 61 patients with a possible diagnosis of pulmonary or pleural TB. HBHA-specific IFN-γ production was analyzed by flow cytometry and ELISA. The suppressive effect of pleural Treg cells was investigated by depletion experiments. MEASUREMENTS AND MAIN RESULTS The percentages of HBHA-induced IFN-γ(+) alveolar and pleural lymphocytes were higher for pulmonary (P < 0.0001) and for pleural (P < 0.01) TB than for non-TB controls. Local CD4(+) and CD8(+) T cells produced the HBHA-specific IFN-γ. This local secretion was not suppressed by Treg lymphocytes, contrasting with previously reported data on circulating lymphocytes. CONCLUSIONS Patients with TB display differential effector and regulatory T-cell responses to HBHA in local and circulating lymphocytes with a predominant effector CD4(+) and CD8(+) response locally, compared with a predominant Treg response among circulating lymphocytes. These findings may be helpful for the design of new vaccines against TB, and the detection of HBHA-specific T cells at the site of the infection may be a promising tool for the rapid diagnosis of active TB.

Monocyte-Derived Interleukin-10 Depresses the Bordetella pertussis- Specific Gamma Interferon Response in Vaccinated Infants

ABSTRACT Antigen-specific gamma interferon (IFN-γ) has been demonstrated to participate in protection against Bordetella pertussis infection. Circulating mononuclear cells from B. pertussis-infected and from pertussis-vaccinated infants secrete high amounts of IFN-γ after in vitro stimulation by B. pertussis antigens, but with a large variation in the secreted IFN-γ levels between individuals. We show here that the inhibition of the specific IFN-γ response can be at least partially attributed to IL-10 secretion by monocytes. This IL-10 secretion was not associated with polymorphisms at positions −1082, −819, and −592 of the IL-10 gene promoter, suggesting that other genetic or environmental factors affect IL-10 expression and secretion.

Both CD4+ and CD8+ Lymphocytes Participate in the IFN-γ Response to Filamentous Hemagglutinin from Bordetella pertussis in Infants, Children, and Adults

Infant CD4+ T-cell responses to bacterial infections or vaccines have been extensively studied, whereas studies on CD8+ T-cell responses focused mainly on viral and intracellular parasite infections. Here we investigated CD8+ T-cell responses upon Bordetella pertussis infection in infants, children, and adults and pertussis vaccination in infants. Filamentous hemagglutinin-specific IFN-γ secretion by circulating lymphocytes was blocked by anti-MHC-I or -MHC-II antibodies, suggesting that CD4+ and CD8+ T lymphocytes are involved in IFN-γ production. Flow cytometry analyses confirmed that both cell types synthesized antigen-specific IFN-γ, although CD4+ lymphocytes were the major source of this cytokine. IFN-γ synthesis by CD8+ cells was CD4+ T cell dependent, as evidenced by selective depletion experiments. Furthermore, IFN-γ synthesis by CD4+ cells was sometimes inhibited by CD8+ lymphocytes, suggesting the presence of CD8+ regulatory T cells. The role of this dual IFN-γ secretion by CD4+ and CD8+ T lymphocytes in pertussis remains to be investigated.