Identification of pertussis-specific effector memory T cells in preschool children

Whooping cough remains a problem despite vaccination, and worldwide resurgence of pertussis is evident. Since cellular immunity plays a role in long-term protection against pertussis, we studied pertussis-specific T-cell responses. Around the time of the preschool acellular pertussis (aP) booster dose at 4 years of age, T-cell memory responses were compared in children who were primed during infancy with either a whole-cell pertussis (wP) or an aP vaccine. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with pertussis vaccine antigens for 5 days. T cells were characterized by flow-based analysis of carboxyfluorescein succinimidyl ester (CFSE) dilution and CD4, CD3, CD45RA, CCR7, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) expression. Before the aP preschool booster vaccination, both the proliferated pertussis toxin (PT)-specific CD4⁺ and CD8⁺ T-cell fractions (CFSE^dim) were higher in aP-than in wP-primed children. Post-booster vaccination, more pertussis-specific CD4⁺ effector memory cells (CD45RA^- CCR7^-) were induced in aP-primed children than in those primed with wP. The booster vaccination did not appear to significantly affect the T-cell memory subsets and functionality in aP-primed or wP-primed children. Although the percentages of Th1 cytokine-producing cells were alike in aP- and wP-primed children pre-booster vaccination, aP-primed children produced more Th1 cytokines due to higher numbers of proliferated pertussis-specific effector memory cells. At present, infant vaccinations with four aP vaccines in the first year of life result in pertussis-specific CD4⁺ and CD8⁺ effector memory T-cell responses that persist in children until 4 years of age and are higher than those in wP-primed children. The booster at 4 years of age is therefore questionable; this may be postponed to 6 years of age.

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-gamma secretion by the PBMC of these infants. IFN-gamma was secreted by both CD4(+) and CD8(+) T lymphocytes, and the levels of Ag-induced IFN-gamma 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, from an extrapulmonary dissemination factor to a powerful diagnostic and protective antigen against tuberculosis.

Interactions of Mycobacterium tuberculosis with macrophages have long been recognized to be crucial to the pathogenesis of tuberculosis. The role of non-phagocytic cells is less well known. We have discovered a M. tuberculosis surface protein that interacts specifically with non-phagocytic cells, expresses hemagglutination activity and binds to sulfated glycoconjugates. It is therefore called heparin-binding hemagglutinin (HBHA). HBHA-deficient M. tuberculosis mutant strains are significantly impaired in their ability to disseminate from the lungs to other tissues, suggesting that the interaction with non-phagocytic cells, such as pulmonary epithelial cells, may play an important role in the extrapulmonary dissemination of the tubercle bacillus, one of the key steps that may lead to latency. Latently infected human individuals mount a strong T cell response to HBHA, whereas patients with active disease do not, suggesting that HBHA is a good marker for the immunodiagnosis of latent tuberculosis, and that HBHA-specific Th1 responses may contribute to protective immunity against active tuberculosis. Strong HBHA-mediated immuno-protection was shown in mouse challenge models. HBHA is a methylated protein and its antigenicity in latently infected subjects, as well as its protective immunogenicity strongly depends on the methylation pattern of HBHA. In both mice and man, the HBHA-specific IFN-gamma was produced by both the CD4(+) and the CD8(+) T cells. Furthermore, the HBHA-specific CD8(+) T cells expressed bactericidal and cytotoxic activities to mycobacteria-infected macrophages. This latter activity is most likely perforin mediated. Together, these observations strongly support the potential of methylated HBHA as an important component in future, acellular vaccines against tuberculosis.

Modulation of the infant immune responses by the first pertussis vaccine administrations.

Many efforts are currently made to prepare combined vaccines against most infectious pathogens, that may be administered early in life to protect infants against infectious diseases as early as possible. However, little is known about the general immune modulation induced by early vaccination. Here, we have analyzed the cytokine secretion profiles of two groups of 6-month-old infants having received as primary immunization either a whole-cell (Pw) or an acellular (Pa) pertussis vaccine in a tetravalent formulation of pertussis-tetanus-diphtheria-poliomyelitis vaccines. Both groups of infants secreted IFN-gamma in response to the Bordetella pertussis antigens filamentous haemagglutinin and pertussis toxin, and this response was correlated with antigen-specific IL-12p70 secretion, indicating that both pertussis vaccines induced Th1 cytokines. However, Pa recipients also developed a strong Th2-type cytokine response to the B. pertussis antigens, as noted previously. In addition, they induced Th2-type cytokines to the co-administrated antigen tetanus toxoïd, as well as to the food antigen beta-lactoglobulin. Furthermore, the general cytokine profile of the Pa recipients was strongly Th2-skewed at 6 months, as indicated by the cytokines induced by the mitogen phytohaemagglutinin. These data demonstrate that the cytokine profile of 6-month-old infants is influenced by the type of formulation of the pertussis vaccine they received at 2, 3 and 4 months of life. Large prospective studies would be warranted to evaluate the possible long-term consequences of this early modulation of the cytokine responses in infants.

HBHA-specific IFN-{gamma} 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. OBJECTIVE: The mycobacterial antigen heparin-binding-hemagglutinin (HBHA) induces strong interferon-gamma (IFN-gamma) responses by circulating lymphocytes from Mycobacterium tuberculosis latently infected subjects, and low responses associated with CD4(+) regulatory T (Treg) cells in TB patients. Here, we investigated HBHA-specific IFN-gamma 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 and/or pleural TB. HBHA-specific IFN-gamma 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-gamma(+) 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-gamma. This local secretion was not suppressed by Treg lymphocytes, contrasting with previously reported data on circulating lymphocytes. CONCLUSION: TB patients 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 to 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.

Different T cell memory in preadolescents after whole-cell or acellular pertussis vaccination.

To better understand vaccine-induced protection and its potential failure in light of recent whooping cough resurgence, we evaluated quantity as well as quality of memory T cell responses in B. pertussis-vaccinated preadolescent children. Using a technique based on flow cytometry to detect proliferation, cytokine production and phenotype of antigen-specific cells, we evaluated residual T cell memory in a cohort of preadolescents who received a whole-cell pertussis (wP; n=11) or an acellular pertussis vaccine (aP; n=13) during infancy, and with a median of 4 years elapsed from the last pertussis booster vaccine, which was aP for all children. We demonstrated that B. pertussis-specific memory T cells are detectable in the majority of preadolescent children several years after vaccination. CD4(+) and CD8(+) T cell proliferation in response to pertussis toxin and/or filamentous hemagglutinin was detected in 79% and 60% of the children respectively, and interferon-γ or tumor necrosis factor-α producing CD4(+) T cells were detected in 65% and 53% of the children respectively. Phenotyping of the responding cells showed that the majority of antigen-specific cells, whether defined by proliferation or cytokine production, were CD45RA(-)CCR7(-) effector memory T cells. Although the time since the last booster vaccine was significantly longer for wP-compared to aP-vaccinated children, their proliferation capacity in response to antigenic stimulation was comparable, and more children had a detectable cytokine response after wP- compared to aP-vaccination. This study supports at the immunological level recent epidemiological studies indicating that infant vaccination with wP induces longer lasting immunity than vaccination with aP-vaccines.

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.

Bordetella pertussis from functional genomics to intranasal vaccination.

Whooping cough still represents a major health problem, despite the use of effective vaccines for several decades. Being classically a typical childhood disease, whooping cough in young adults is now more common than it used to be, suggesting that protection after vaccination wanes during adolescence. As an alternative to the current vaccines, we wish to develop live attenuated vaccines to be delivered by the nasal route, such as to mimic the natural route of infection and to induce long lasting immunity. Bordetella pertussis, the etiological agent of whooping cough, produces a number of virulence factors, including toxins. Its recently determined genome sequence makes it now possible to apply functional genomics, such as transcriptomics and systematic knock-out mutagenesis. The expression of most known B. pertussis virulence genes is controlled by the two-component system BvgA/S. DNA microarray analyses have led to the identification of novel genes in the BvgA/S regulon, some of which are activated by BvgA/S and others are repressed by BvgA/S. In addition, some genes appear to be differentially modulated by nicotinic acid and MgSO4, both known to modulate the expression of BvgA/S-regulated genes. Among others, the functional genomics approach has uncovered two strongly BvgA/S-activated genes, named hotA and hotB (for 'homolog of toxin'), the products of which show high sequence similarities to pertussis toxin subunits. The identification of the full array of virulence factors, as well as an integrated understanding of the bacterial physiology should allow us to design attenuated B. pertussis strains useful for intranasal vaccination. A first generation of attenuated strains has already shown full protection in mice after a single intranasal administration. Such strains may also serve as vaccine carriers for heterologous antigens, in order to vaccinate against several different pathogens simultaneously.

Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin.

In addition to antibodies, Th1-type T cell responses are also important for long-lasting protection against pertussis. However, upon immunization with the current acellular vaccines, many children fail to induce Th1-type responses, potentially due to immunomodulatory effects of some vaccine antigens, such as filamentous haemagglutinin (FHA). We therefore analysed the ability of FHA to modulate immune functions of human monocyte-derived dendritic cells (MDDC). FHA was purified from pertussis toxin (PTX)-deficient or from PTX- and adenylate cyclase-deficient Bordetella pertussis strains, and residual endotoxin was neutralized with polymyxin B. FHA from both strains induced phenotypic maturation of human MDDC and cytokine secretion (IL-10, IL-12p40, IL-12p70, IL-23 and IL-6). To identify the FHA domains responsible for MDDC immunomodulation, MDDC were stimulated with FHA containing a Gly→Ala substitution at its RGD site (FHA-RAD) or with an 80-kDa N-terminal moiety of FHA (Fha44), containing its heparin-binding site. Whereas FHA-RAD induced maturation and cytokine production comparable to those of FHA, Fha44 did not induce IL-10 production, but maturated MDDC at least partially. Nevertheless, Fha44 induced the secretion of IL-12p40, IL-12p70, IL-23 and IL-6 by MDDC, albeit at lower levels than FHA. Thus, FHA can modulate MDDC responses in multiple ways, and IL-10 induction can be dissociated from the induction of other cytokines.

The protective effect of heparin-binding haemagglutinin

info:eu-repo/semantics/nonPublished

The heparin-binding haemagglutinin, a latency antigen useful for vaccine development

info:eu-repo/semantics/nonPublished

Early cellular immune response to a new candidate mycobacterial vaccine antigen in childhood tuberculosis

The search for novel vaccines against tuberculosis (TB) would benefit from in-depths knowledge of the human immune responses to Mycobacterium tuberculosis (Mtb) infection. Here, we characterised in a low TB incidence country, the immune responses to a new candidate vaccine antigen against TB, the heparin-binding haemagglutinin (HBHA), in young children in contact with an active TB case (aTB). Children with no history of BCG vaccination were compared to those vaccinated at birth to compare the initial immune responses to HBHA with secondary immune responses. Fifty-eight children with aTB and 76 with latent TB infection (LTBI) were included and they were compared to 90 non-infected children. Whereas Mtb-infected children globally secreted more interferon-gamma (IFN-γ) in response to HBHA compared to the non-infected children, these IFN-γ concentrations were higher in previously BCG-vaccinated compared to non-vaccinated children. The IFN-γ concentrations were similar in LTBI and aTB children, but appeared to differ qualitatively. Whereas the IFN-γ secretion induced by native methylated and recombinant non-methylated HBHA were well correlated for aTB, this was not the case for LTBI children. Thus, Mtb-infected young children develop IFN-γ responses to HBHA that are enhanced by prior BCG vaccination, indicating BCG-induced priming, thereby supporting a prime-boost strategy for HBHA-based vaccines. The qualitative differences between aTB and LTBI in their HBHA-induced IFN-γ responses may perhaps be exploited for diagnostic purposes.

Both CD4+ and CD8+ lymphocytes participate in the IFN-y 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. © 2012 Violette Dirix et al.