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.

The treatment of latent tuberculosis infection (LTBI) in target populations is one of the current WHO strategies for preventing active tuberculosis (TB) infection and reducing theT he screening and treatment for LTBI in target populations in order to prevent TB and reduce the Mycobacterium tuberculosis reservoir are some of the main strategies of the WHO's Global Plan to Stop TB (http://www.who.int/tb/publications/global _report/en). However, a major obstacle to the instauration and effectiveness of these preventive measures resides in the lack of a gold standard LTBI screening tool.For several decades, the tuberculin skin test (TST) has been the main screening test for LTBI despite its lack of both sensitivity and specificity (1). Subsequently, T-cell-based gamma interferon release assays (IGRAs) in response to antigens encoded in the M. tuberculosis genomic region of difference 1 (RD-1) and RD-11 were developed and commercialized (QuantiFERON-TB Gold In-Tube [QFT-GIT] and T-SPOT.TB tests), with the objective of offering a more powerful diagnostic tool for LTBI. These tests offer a higher specificity than TST particularly in countries with high Mycobacterium bovis BCG vaccination coverage (2). However, recent studies suggest that these short-incubation RD-1-based IGRAs may have suboptimal sensitivities (3, 4).An alternative IGRA in response to the native mycobacterial antigen heparin-binding hemagglutinin (nHBHA-IGRA) that uses a longer incubation time than the commercialized IGRA has been validated in immunocompetent adults in the screening for LTBI (5). This assay not only demonstrates a high sensitivity and specificity for LTBI diagnosis but also a capacity to detect remote M. tuberculosis infections, a substantial advantage over the commercialized IGRAs (5-7).Remote M. tuberculosis infections are generally believed to be identified through central memory T-cell (Tcm) responses detected with long-incubation IGRAs (3, 4). Here, however, we demonstrate that both recent and remote M. tuberculosis infections can be identified through effector memory T-cell (Tem) responses using a short-incubation nHBHA-IGRA, the upgraded assay presented in this study. Our results suggest that the detection of IFN-␥-producing CD4 ϩ Tem in response to nHBHA reflects the persistence of M. tuberculosis antigens and therefore a true state of latency.(These results were presented in part at the European Congress of Immunology, Glasgow, Scotland, 5 to 8 September 2012, and at the MycoClub,

Intracellular cytokine staining combined with flow cytometry is one of a number of assays designed to assess T-cell immune responses. It has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation and functional parameters pertaining to responding T-cells, most notably, the expression of multiple effector cytokines. These attributes make the technique particularly suitable for the assessment of T-cell immune responses induced by novel tuberculosis vaccines in clinical trials. However, depending upon the particular nature of a given vaccine and trial setting, there are approaches that may be taken at different stages of the assay that are more suitable than other alternatives. In this paper, the Tuberculosis Vaccine Initiative (TBVI) TB Biomarker Working group reports on efforts to assess the conditions that will determine when particular assay approaches should be employed. We have found that choices relating to the use of fresh whole blood or peripheral blood mononuclear cells (PBMC) and frozen PBMC; use of serum-containing or serum-free medium; length of stimulation period and use of co-stimulatory antibodies can all affect the sensitivity of intracellular cytokine assays. In the case of sample material, frozen PBMC, despite some loss of sensitivity, may be more advantageous for batch analysis. We also recommend that for multi-site studies, common antibody panels, gating strategies and analysis approaches should be employed for better comparability.

Measurement of antigen-specific T cell responses is an adjunctive parameter to evaluate protection induced by a previous Bordetella pertussis infection or vaccination. The assessment of T cell responses is technically complex and usually performed on fresh peripheral blood mononuclear cells (PBMC). The objective of this study was to identify simplified methods to assess pertussis specific T cell responses and verify if these assays could be performed using frozen/thawed (frozen) PBMC. Three read-outs to measure proliferation were compared: the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) dilution test, the number of blast cells defined by physical parameters, and the incorporation of (3)H-thymidine. The results of pertussis-specific assays performed on fresh PBMC were compared to the results on frozen PBMC from the same donor. High concordance was obtained when the results of CFSE and blast read-outs were compared, an encouraging result since blast analysis allows the identification of proliferating cells and does not require any use of radioactive tracer as well as any staining. The results obtained using fresh and frozen PBMC from the same donor in the different T cell assays, including IFNγ and TNFα cytokine production, did not show significant differences, suggesting that a careful cryopreservation process of PBMC would not significantly influence T cell response evaluation. Adopting blast analysis and frozen PBMC, the possibility to test T cell responses is simplified and might be applied in population studies, providing for new instruments to better define correlates of protection still elusive in pertussis.

CD4+ T cells are prominent effector cells in controlling Mycobacterium tuberculosis (Mtb) infection but may also contribute to immunopathology. Studies probing the CD4+ T cell response from individuals latently infected with Mtb or patients with active tuberculosis using either small or proteome-wide antigen screens so far revealed a multi-antigenic, yet mostly invariable repertoire of immunogenic Mtb proteins. Recent developments in mass spectrometry-based proteomics have highlighted the occurrence of numerous types of post-translational modifications (PTMs) in proteomes of prokaryotes, including Mtb. The well-known PTMs in Mtb are glycosylation, lipidation, or phosphorylation, known regulators of protein function or compartmentalization. Other PTMs include methylation, acetylation, and pupylation, involved in protein stability. While all PTMs add variability to the Mtb proteome, relatively little is understood about their role in the anti-Mtb immune responses. Here, we review Mtb protein PTMs and methods to assess their role in protective immunity against Mtb.