Andreia Soares

Specific T Cell Frequency and Cytokine Expression Profile Do Not Correlate with Protection against Tuberculosis after Bacillus Calmette-Guérin Vaccination of Newborns

RATIONALE Immunogenicity of new tuberculosis (TB) vaccines is commonly assessed by measuring the frequency and cytokine expression profile of T cells. OBJECTIVES We tested whether this outcome correlates with protection against childhood TB disease after newborn vaccination with bacillus Calmette-Guérin (BCG). METHODS Whole blood from 10-week-old infants, routinely vaccinated with BCG at birth, was incubated with BCG for 12 hours, followed by cryopreservation for intracellular cytokine analysis. Infants were followed for 2 years to identify those who developed culture-positive TB-these infants were regarded as not protected against TB. Infants who did not develop TB disease despite exposure to TB in the household, and another group of randomly selected infants who were never evaluated for TB, were also identified-these groups were regarded as protected against TB. Cells from these groups were thawed, and CD4, CD8, and γδ T cell-specific expression of IFN-γ, TNF-α, IL-2, and IL-17 measured by flow cytometry. MEASUREMENTS AND MAIN RESULTS A total of 5,662 infants were enrolled; 29 unprotected and two groups of 55 protected infants were identified. There was no difference in frequencies of BCG-specific CD4, CD8, and γδ T cells between the three groups of infants. Although BCG induced complex patterns of intracellular cytokine expression, there were no differences between protected and unprotected infants. CONCLUSIONS The frequency and cytokine profile of mycobacteria-specific T cells did not correlate with protection against TB. Critical components of immunity against Mycobacterium tuberculosis, such as CD4 T cell IFN-γ production, may not necessarily translate into immune correlates of protection against TB disease.

Bacillus Calmette-Guérin Vaccination of Human Newborns Induces T Cells with Complex Cytokine and Phenotypic Profiles

The immune response to vaccination with bacillus Calmette-Guérin (BCG), the only tuberculosis vaccine available, has not been fully characterized. We used multiparameter flow cytometry to examine specific T cell cytokine production and phenotypic profiles in blood from 10-wk-old infants routinely vaccinated with BCG at birth. Ex vivo stimulation of whole blood with BCG for 12 h induced expression of predominantly IFN-γ, IL-2, and TNF-α in CD4+ T cells in seven distinct cytokine combinations. IL-4 and IL-10 expression was detected in CD4+ T cells at low frequencies and only in cells that did not coexpress type 1 cytokines. Specific CD8+ T cells were less frequent than CD4+ T cells and produced mainly IFN-γ and/or IL-2 and less TNF-α, IL-4, and IL-10. Importantly, many mycobacteria-specific CD4+ and CD8+ T cells did not produce IFN-γ. The predominant phenotype of BCG-specific type 1 T cells was that of effector cells, i.e., CD45RA−CCR7−CD27+, which may reflect persistence of Mycobacterium bovis BCG in infants until 10 wk of age. Among five phenotypic patterns of CD4+ T cells, central memory cells were more likely to be IL-2+ and effector cells were more likely to be IFN-γ+. We concluded that neonatal vaccination with BCG induces T cells with a complex pattern of cytokine expression and phenotypes. Measuring IFN-γ production alone underestimates the magnitude and complexity of the host cytokine response to BCG vaccination and may not be an optimal readout in studies of BCG and novel tuberculosis vaccination.

Novel application of Ki67 to quantify antigen-specific in vitro lymphoproliferation

Antigen-specific proliferation is a critical function of memory T cells that is often utilised to measure vaccine immunogenicity and T cell function. We proposed that measurement of intracellular expression of the nuclear protein, Ki67, could reliably assess specific T cell proliferation in vitro. Ki67 was expressed in CD4+ and CD8+ T cells that had undergone in vitro proliferation after 6-day culture of human whole blood or PBMC with antigens. T cells cultured with no antigen did not express Ki67. When compared to current flow cytometry based proliferation assays, Ki67 detected proliferating cells with greater sensitivity than BrdU incorporation, whereas its sensitivity was similar to dye dilution of Oregon Green (OG), a CFSE derivative. Overall, the magnitude and cytokine expression profile of proliferating T cells detected by Ki67 expression correlated strongly with T cells detected with BrdU or OG. The intra-assay variability of Ki67 proliferation was 2–3% for CD4+ T cells, and 10–16% for CD8+ T cells. Finally, we demonstrate that the Ki67 assay detects tetanus toxoid-specific CD4+ T cell proliferation after infant vaccination with tetanus toxoid (TT). Overall our data suggest that intracellular Ki67 expression provides a specific, quantitative and reproducible measure of antigen-specific T cell proliferation in vitro.

Bacillus Calmette Guerin Vaccination of Human Newborns Induces a Specific, Functional CD8+ T Cell Response

Mounting evidence points to CD8+ T cells playing an important role in protective immunity against Mycobacterium tuberculosis. The only available vaccine against tuberculosis, bacillus Calmette Guérin (BCG), has traditionally been viewed not to induce these cells optimally. In this study, we show that vaccination of human newborns with BCG does indeed induce a specific CD8+ T cell response. These cells degranulated or secreted IFN-γ, but not both, when infant blood was incubated with BCG. This stimulation also resulted in proliferation and up-regulation of cytotoxic molecules. Overall, the specific CD8+ T cell response was quantitatively smaller than the BCG-induced CD4+ T cell response. Incubation of whole blood with M. tuberculosis also caused CD8+ T cell IFN-γ expression. We conclude that BCG induces a robust CD8+ T cell response, which may contribute to vaccination-induced protection against tuberculosis.

Longitudinal Changes in CD4+ T-Cell Memory Responses Induced by BCG Vaccination of Newborns

BACKGROUND Improved vaccination strategies against tuberculosis are needed, such as approaches to boost immunity induced by the current vaccine, BCG. Design of these strategies has been hampered by a lack of knowledge of the kinetics of the human host response induced by neonatal BCG vaccination. Furthermore, the functional and phenotypic attributes of BCG-induced long-lived memory T-cell responses remain unclear. METHODS We assessed the longitudinal CD4 T-cell response following BCG vaccination of human newborns. The kinetics, function, and phenotype of these cells were measured using flow cytometric whole-blood assays. RESULTS We showed that the BCG-specific CD4 T-cell response peaked 6-10 weeks after vaccination and gradually waned over the first year of life. Highly activated T-helper 1 cells, predominantly expressing interferon γ, tumor necrosis factor α, and/or interleukin 2, were present at the peak response. Following contraction, BCG-specific CD4 T cells expressed high levels of Bcl-2 and displayed a predominant CD45RACCR7 central memory phenotype. However, cytokine and cytotoxic marker expression by these cells was more characteristic of effector memory cells. CONCLUSIONS Our findings suggest that boosting of BCG-primed CD4 T cells with heterologous tuberculosis vaccines may be best after 14 weeks of age, once an established memory response has developed.

Impact of human immunodeficiency virus 1 infection and inflammation on the composition and yield of cervical mononuclear cells in the female genital tract.

Cervical cytobrush sampling is a relatively non‐invasive method for obtaining mucosal cells from the female genital tract. To define mucosal immune cells sampled by cervical cytobrushing and to validate this approach for local immunity studies, we investigated the impact of human immunodeficiency virus (HIV) status and inflammation on the yield and composition of cervical cytobrush specimens. Cervical cytobrush samples were obtained from 89 chronically HIV‐infected and 46 HIV‐negative women. The HIV‐infected women had significantly higher yields of CD3+, CD45+, CD19+, CD14+, Langerin+ and CD24+ cells than the uninfected women. While cytobrush‐derived T cells from uninfected women were predominantly CD4+ (4·2 CD4 : 1 CD8), CD8+ T cells were predominant in HIV‐infected women (0·6 CD4 : 1 CD8). The majority of CD4+ and CD8+ T cells from HIV‐infected and uninfected women were of the effector memory (CD45RA− CCR7− CD27−) phenotype. HIV‐infected women had significantly elevated levels of interleukin (IL)‐1β, IL‐6 and IL‐8 in cervical supernatants compared with uninfected women. We observed a significant positive correlation between T‐cell counts and IL‐1β, tumour necrosis factor (TNF)‐α and IL‐12 concentrations. Neutrophil counts correlated significantly with cervical concentrations of IL‐1β, TNF‐α, IL‐8, IL‐6 and IL‐10. Antigen‐presenting cell numbers correlated significantly with TNF‐α and IL‐12 concentrations. HIV‐infected women on antiretroviral therapy had similar levels of cervical lymphocyte infiltration and inflammation to women naïve to therapy. In conclusion, we suggest that inflammation at the cervix and HIV infection are likely to be key determinants in the absolute number of mucosal immune cells recovered by cervical cytobrushing.

Qualification of a whole blood intracellular cytokine staining assay to measure mycobacteria-specific CD4 and CD8 T cell immunity by flow cytometry.

BACKGROUND Qualified or validated assays are essential in clinical trials. Short-term stimulation of whole blood and intracellular cytokine staining assay is commonly used to measure immunogenicity in tuberculosis vaccine clinical trials. Previously, the short-term stimulation process of whole blood with BCG was optimized. We aimed to qualify the intracellular cytokine staining process and assess the effects of long-term cryopreservation. Our hypotheses were that the assay is robust in the measurement of the mycobacteria-specific T cells, and long-term cryopreservation of fixed cells from stimulated whole blood would not compromise reliable measurement of mycobacteria induced CD4 T cell immunity. METHODS Whole blood from healthy adults was collected in sodium heparinized tubes. The blood was left unstimulated or stimulated with mycobacterial antigens or mitogens for 12h. Cells were harvested, fixed and multiple aliquots from each participant cryopreserved. Later, mycobacteria-specific CD4 and CD8 T cells expressing IFN-γ, TNF-α, IL-2 and IL-17 were quantitated by flow cytometry. Assay performance characteristics evaluated included limit of quantification and detection, reproducibility, precision, robustness, specificity and sensitivity. To assess the effects of long-term cryopreservation, fixed cells from the stimulated bloods were analysed one week post-cryopreservation and at 3-month intervals over a 3-year period. RESULTS The limit of quantification for the different cytokines was variable: 0.04% for frequencies of IFN-γ- and IL-2-expressing T cells and less than 0.01% for TNF-α- and IL-17-expressing T cells. When measurement of the mycobacteria-specific T cells was assessed at levels above the detection limit, the whole blood intracellular cytokine assay showed high precision that was operator-independent. The assay was also robust: variation in staining conditions including temperature (4 °C or 20-23 °C) and time (45, 60 or 90 min) did not markedly affect quantification of specific T cells. Finally, prolonged periods of cryopreservation also did not significantly influence quantification of mycobacteria-specific CD4 T cells. CONCLUSIONS The whole blood intracellular cytokine assay is robust and reliable in quantification of the mycobacteria-specific T cells and is not significantly affected by cryopreservation of fixed cells.

Restoration of CD4+ Responses to Copathogens in HIV-Infected Individuals on Antiretroviral Therapy Is Dependent on T Cell Memory Phenotype

Antiretroviral therapy (ART) induces rapid suppression of viral replication and a progressive replenishment of CD4+ T cells in HIV-infected individuals. However, the effect of ART on restoring pre-existing memory CD4+ T cells specific for common copathogens is still unclear. To better understand the dynamics of Ag-specific CD4+ T cells during ART, we assessed the frequency, functional capacity, and memory profile of CD4+ T cells specific for Mycobacterium tuberculosis and CMV in 15 HIV-infected individuals before and 1 y after ART initiation. After ART initiation, the frequency of M. tuberculosis–specific CD4+ T cells showed little change, whereas CMV-specific CD4+ T cells were significantly lower (p = 0.003). There was no difference in the polyfunctional or memory profile of Ag-specific CD4+ T cells before and after ART. The replenishment of Ag-specific CD4+ T cells correlated with the memory differentiation profile of these cells prior to ART. Pathogen-specific CD4+ T cells exhibiting a late differentiated profile (CD45RO+CD27−) had a lower capacity to replenish (p = 0.019; r = −0.5) compared with cells with an early differentiated profile (CD45RO+CD27+; p = 0.04; r = 0.45). In conclusion, restoration of copathogen-specific memory CD4+ T cells during treated HIV infection is related to their memory phenotype, in which early differentiated cells (such as most M. tuberculosis–specific cells) have a higher replenishment capacity compared with late differentiated cells (such as most CMV-specific cells). These data identify an important, hitherto unrecognized, factor that may limit restoration of copathogen immunity in HIV-infected individuals on ART.

HIV Skews the Lineage-Defining Transcriptional Profile of Mycobacterium tuberculosis-Specific CD4(+) T Cells

HIV-infected persons are at greater risk of developing tuberculosis (TB) even before profound CD4 loss occurs, suggesting that HIV alters CD4+ T cell functions capable of containing bacterial replication. An effective immune response to Mycobacterium tuberculosis most likely relies on the development of a balanced CD4 response, in which distinct CD4+ Th subsets act in synergy to control the infection. To define the diversity of M. tuberculosis–specific CD4+ Th subsets and determine whether HIV infection impacts such responses, the expression of lineage-defining transcription factors T-bet, Gata3, RORγt, and Foxp3 was measured in M. tuberculosis–specific CD4+ T cells in HIV-uninfected (n = 20) and HIV-infected individuals (n = 20) with latent TB infection. Our results show that, upon 5-d restimulation in vitro, M. tuberculosis–specific CD4+ T cells from healthy individuals have the ability to exhibit a broad spectrum of Th subsets, defined by specific patterns of transcription factor coexpression. These transcription factor profiles were skewed in HIV-infected individuals where the proportion of T-bethighFoxp3+ M. tuberculosis–specific CD4+ T cells was significantly decreased (p = 0.002) compared with HIV-uninfected individuals, a change that correlated inversely with HIV viral load (p = 0.0007) and plasma TNF-α (p = 0.027). Our data demonstrate an important balance in Th subset diversity defined by lineage-defining transcription factor coexpression profiles that is disrupted by HIV infection and suggest a role for HIV in impairing TB immunity by altering the equilibrium of M. tuberculosis–specific CD4+ Th subsets.

The novel capripoxvirus vector lumpy skin disease virus efficiently boosts modified vaccinia Ankara human immunodeficiency virus responses in rhesus macaques.

Poxvirus vectors represent promising human immunodeficiency virus (HIV) vaccine candidates and were a component of the only successful HIV vaccine efficacy trial to date. We tested the immunogenicity of a novel recombinant capripoxvirus vector, lumpy skin disease virus (LSDV), in combination with modified vaccinia Ankara (MVA), both expressing genes from HIV-1. Here, we demonstrated that the combination regimen was immunogenic in rhesus macaques, inducing high-magnitude, broad and balanced CD4(+) and CD8(+) T-cell responses, and transient activation of the immune response. These studies support further development of LSDV as a vaccine vector.