Linda Petrone

Correlates of tuberculosis risk: predictive biomarkers for progression to active tuberculosis

New approaches to control the spread of tuberculosis (TB) are needed, including tools to predict development of active TB from latent TB infection (LTBI). Recent studies have described potential correlates of risk, in order to inform the development of prognostic tests for TB disease progression. These efforts have included unbiased approaches employing “omics” technologies, as well as more directed, hypothesis-driven approaches assessing a small set or even individual selected markers as candidate correlates of TB risk. Unbiased high-throughput screening of blood RNAseq profiles identified signatures of active TB risk in individuals with LTBI, ≥1 year before diagnosis. A recent infant vaccination study identified enhanced expression of T-cell activation markers as a correlate of risk prior to developing TB; conversely, high levels of Ag85A antibodies and high frequencies of interferon (IFN)-γ specific T-cells were associated with reduced risk of disease. Others have described CD27−IFN-γ+CD4+ T-cells as possibly predictive markers of TB disease. T-cell responses to TB latency antigens, including heparin-binding haemagglutinin and DosR-regulon-encoded antigens have also been correlated with protection. Further studies are needed to determine whether correlates of risk can be used to prevent active TB through targeted prophylactic treatment, or to allow targeted enrolment into efficacy trials of new TB vaccines and therapeutic drugs. Promising biomarkers may allow accurate prediction of progression from infection to active TB disease http://ow.ly/OzCL304ezfk

Higher Frequency of T-Cell Response to M. tuberculosis Latency Antigen Rv2628 at the Site of Active Tuberculosis Disease than in Peripheral Blood

Rationale Due to the invasive nature of the procedures involved, most studies of Mycobacterium tuberculosis (Mtb)-specific immunity in humans have focused on the periphery rather than the site of active infection, the lung. Recently, antigens associated with Mtb-latency and -dormancy have been described using peripheral blood (PB) cells; however their response in the lung is unknown. The objective of this report was to evaluate, in patients prospectively enrolled with suspected active tuberculosis (TB), whether the latency antigen Rv2628 induces local-specific immune response in bronchoalveolar lavage (BAL) cells compared to PB cells. Material/Methods Among the 41 subjects enrolled, 20 resulted with active TB. Among the 21 without active disease, 9 were defined as subjects with latent TB-infection (LTBI) [Quantiferon TB Gold In-tube positive]. Cytokine responses to Rv2628 were evaluated by enzyme linked immunospot (ELISPOT) assay and flow cytometric (FACS) analysis. RD1-secreted antigen stimulation was used as control. Results There was a significantly higher frequency of Rv2628- and RD1-specific CD4+ T-cells in the BAL of active TB patients than in PB. However the trend of the response to Rv2628 in subjects with LTBI was higher than in active TB in both PB and BAL, although this difference was not significant. In active TB, Rv2628 and RD1 induced a cytokine-response profile mainly consisting of interferon (IFN)-γ-single-positive over double-IFN-γ/interleukin (IL)-2 T-cells in both PB and BAL. Finally, BAL-specific CD4+ T-cells were mostly effector memory (EM), while peripheral T-cell phenotypes were distributed among naïve, central memory and terminally differentiated effector memory T-cells. Conclusions In this observational study, we show that there is a high frequency of specific T-cells for Mtb-latency and RD1-secreted antigens (mostly IFN-γ-single-positive specific T-cells with an EM phenotype) in the BAL of active TB patients. These data may be important for better understanding the pathogenesis of TB in the lung.

Anti-tumor CD8+ T cell immunity elicited by HIV-1-based virus-like particles incorporating HPV-16 E7 protein.

Here we report a novel strategy for the induction of CD8(+) T cell adaptive immune response against viral and tumor antigens. This approach relies on high levels of incorporation in HIV-1 VLPs of a mutant of HIV-1 Nef (Nef(mut)) which can act as anchoring element for foreign proteins. By in vitro assay, we found that VLP-associated Nef(mut) is efficiently cross-presented by antigen presenting cells. Inoculation in mice of VLPs incorporating the HPV-16 E7 protein fused to Nef(mut) led to an anti-E7 CD8(+) T cell response much stronger than that elicited by E7 recombinant protein inoculated with incomplete Freunds adjuvant and correlating with well-detectable anti-E7 CTL activity. Most relevantly, mice immunized with Nef(mut)-E7 VLPs developed a protective immune response against tumors induced by E7 expressing tumor cells. These results make Nef(mut) VLPs a promising candidate for new vaccine strategies focused on the induction of CD8(+) T cell immunity.

Patients with Tuberculosis Have a Dysfunctional Circulating B-Cell Compartment, Which Normalizes following Successful Treatment

B-cells not only produce immunoglobulins and present antigens to T-cells, but also additional key roles in the immune system. Current knowledge on the role of B-cells in infections caused by intracellular bacteria is fragmentary and contradictory. We therefore analysed the phenotypical and functional properties of B-cells during infection and disease caused by Mycobacterium tuberculosis (Mtb), the bacillus causing tuberculosis (TB), and included individuals with latent TB infection (LTBI), active TB, individuals treated successfully for TB, and healthy controls. Patients with active or treated TB disease had an increased proportion of antibodies reactive with mycobacteria. Patients with active TB had reduced circulating B-cell frequencies, whereas only minor increases in B-cells were detected in the lungs of individuals deceased from TB. Both active TB patients and individuals with LTBI had increased relative fractions of B-cells with an atypical phenotype. Importantly, these B-cells displayed impaired proliferation, immunoglobulin- and cytokine- production. These defects disappeared upon successful treatment. Moreover, T-cell activity was strongest in individuals successfully treated for TB, compared to active TB patients and LTBI subjects, and was dependent on the presence of functionally competent B-cells as shown by cellular depletion experiments. Thus, our results reveal that general B-cell function is impaired during active TB and LTBI, and that this B-cell dysfunction compromises cellular host immunity during Mtb infection. These new insights may provide novel strategies for correcting Mtb infection-induced immune dysfunction towards restored protective immunity.

Blood or urine IP-10 cannot discriminate between active tuberculosis and respiratory diseases different from tuberculosis in children

Objectives. Interferon-γ inducible protein 10 (IP-10), either in blood or in urine, has been proposed as a tuberculosis (TB) biomarker for adults. This study aims to evaluate the potential of IP-10 diagnostics in children from Uganda, a high TB-endemic country. Methods. IP-10 was measured in the blood and urine concomitantly taken from children who were prospectively enrolled with suspected active TB, with or without HIV infection. Clinical/microbiological parameters and commercially available TB-immune assays (tuberculin skin test (TST) and QuantiFERON TB-Gold In-Tube (QFT-IT)) were concomitantly evaluated. Results. One hundred twenty-eight children were prospectively enrolled. The analysis was performed on 111 children: 80 (72%) of them were HIV-uninfected and 31 (27.9%) were HIV-infected. Thirty-three healthy adult donors (HAD) were included as controls. The data showed that IP-10 is detectable in the urine and blood of children with active TB, independent of HIV status and age. However, although IP-10 levels were higher in active TB children compared to HAD, the accuracy of identifying “active TB” was low and similar to the TST and QFT-IT. Conclusion. IP-10 levels are higher in children with respiratory illness compared to controls, independent of “TB status” suggesting that the evaluation of this parameter can be used as an inflammatory marker more than a TB test.

Free hemoglobin: a dangerous signal for the immune system in patients with carotid atherosclerosis?

Abstract:  Atherosclerosis is a chronic inflammatory multifactorial disease in which immune responses are key pathogenetic factors. T cell–mediated immunity contributes to the initiation and progression of atherosclerotic disease, but the nature of antigens responsible for immune cell activation is still not completely elucidated. Convincing evidence supports a determinant role of autoimmune responses to self‐structures in shaping the progression of the disease. Autoimmune responses may be directed against altered self‐structures, such as oxidized low‐density lipoproteins (LDL). Oxidative stress, increasingly reported in patients with atherosclerosis, is the major event causing protein structural modification, thus inducing the appearance of neo/cryptic epitopes on the molecule. Intraplaque hemorrhage, a common event in advanced lesions, causes the deposition of large amounts of hemoglobin (Hb). The pro‐oxidative intraplaque microenvironment may induce structural changes in extra‐erythrocytic free Hb, thus generating novel/cryptic autoantigenic epitopes. We demonstrated that an oxidized Hb preparation enriched in hemichromes expands IFN‐γ‐secreting T lymphocytes in patients with advanced carotid atherosclerosis and enhances the phenotypical and functional maturation of human monocyte‐derived dendritic cells induced by lipopolysaccharide (LPS). Overall, our findings suggest that oxidized forms of Hb could act as a dangerous signal for the immune system, thus contributing to the inflammatory process that takes place within the atherosclerotic plaque.

Assessment of CD27 expression as a tool for active and latent tuberculosis diagnosis

UNLABELLED There are still no reliable tests to distinguish active tuberculosis (TB) from latent TB infection (LTBI). Assessment of CD27 modulation on CD4⁺ T-cells has been suggested as a tool to diagnose different TB stages. OBJECTIVES To use several cytometric approaches to evaluate CD27 expression on Mycobacterium tuberculosis (Mtb)-specific CD4⁺ T-cells to differentiate TB stages. METHODS 55 HIV-uninfected subjects were enrolled: 13 active TB; 12 cured TB; 30 LTBI. Whole blood was stimulated with RD1-proteins or Cytomegalovirus-lysate (CMV). Interferon (IFN)-γ response was evaluated by cytometry. The proportion of CD27(±) within the IFN-γ⁺ CD4⁺ T-cells or RATIO of the CD27-median fluorescence intensity (MFI) of CD4⁺ T-cells over the CD27 MFI of IFN-γ⁺ CD4⁺ T-cells was evaluated. RESULTS The greatest diagnostic accuracy in discriminating active TB vs. LTBI or cured TB was reached by evaluating the CD27(+) CD45RA(-) cells within the IFN-γ⁺ CD4⁺ T-cell subset (76.92 sensitivity for both, and 90% and 91.67% specificity, respectively), although the use of the CD27 MFI RATIO allows for stricter data analysis, independent of the operator. CONCLUSIONS the study of CD27 expression using different approaches, whether it involves evaluation of CD45RA expression or not, is a robust biomarker for discriminating TB stages.

Recombinant HPV16 E7 assembled into particles induces an immune response and specific tumour protection administered without adjuvant in an animal model.

BackgroundThe HPV16 E7 protein is both a tumour-specific and a tumour-rejection antigen, the ideal target for developing therapeutic vaccines for the treatment of HPV16-associated cancer and its precursor lesions. E7, which plays a key role in virus-associated carcinogenesis, contains 98 amino acids and has two finger-type structures which bind a Zn++ ion. The ability of an Escherichia coli-produced E7-preparation, assembled into particles, to induce protective immunity against a HPV16-related tumour in the TC-1-C57BL/6 mouse tumour model, was evaluated.MethodsE7 was expressed in E. coli, purified via a one-step denaturing protocol and prepared as a soluble suspension state after dialysis in native buffer. The presence in the E7 preparation of particulate forms was analysed by non-reducing SDS-PAGE and negative staining electron microscopy (EM). The Zn++ ion content was analysed by mass-spectrometry. Ten μg of protein per mouse was administered to groups of animals, once, twice or three times without adjuvant. The E7-specific humoral response was monitored in mice sera using an E7-based ELISA while the cell-mediated immune response was analysed in mice splenocytes with lymphoproliferation and IFN-γ ELISPOT assays. The E7 immunized mice were challenged with TC-1 tumour cells and the tumour growth monitored for two months.ResultsIn western blot analysis E7 appears in multimers and high molecular mass oligomers. The EM micrographs show the protein dispersed as aggregates of different shape and size. The protein appears clustered in micro-, nano-aggregates, and structured particles. Mice immunised with this protein preparation show a significant E7-specific humoral and cell-mediated immune response of mixed Th1/Th2 type. The mice are fully protected from the tumour growth after vaccination with three E7-doses of 10 μg without any added adjuvant.ConclusionsThis report shows that a particulate form of HPV16 E7 is able to induce, without adjuvant, an E7-specific tumour protection in C57BL/6 mice. The protective immunity is sustained by both humoral and cell-mediated immune responses. The E. coli-derived HPV16 E7 assembled in vitro into micro- and nanoparticles represents not only a good substrate for antigen-presenting cell uptake and processing, but also a cost-effective means for the production of a new generation of HPV subunit vaccines.

Redox imbalance of red blood cells impacts T lymphocyte homeostasis: implication in carotid atherosclerosis

Oxidative stress and immune/inflammatory responses are key pathogenetic factors of atherosclerotic disease. In this contest, mechanisms that regulate survival and death of immune cells may be relevant. Previous studies have demonstrated that red blood cells (RBCs) are physiologically able to inhibit apoptosis and to promote proliferation of activated T lymphocytes from healthy subjects. The aim of the present study was to evaluate whether RBCs from patients with carotid atherosclerosis maintain their property to modulate T cell homeostasis. Peripheral blood lymphocytes (PBLs) obtained from healthy subjects were activated in vitro by phytohemagglutinin in the presence/absence of RBCs from patients with carotid atherosclerosis or of in vitro oxidised RBCs from healthy subjects. Levels of reactive oxygen species (ROS) and aging markers of RBCs as well as susceptibility to apoptosis of PBLs were evaluated by flow cytometry. PBL proliferation was evaluated by 3H-methyl-thymidine incorporation assay whereas secretion of cytokines, analysed in view of their key role in T cell function, was assessed by ELISA. Levels of ROS and phosphatidyl-serine externalisation, a sign of RBC aging, resulted significantly higher in RBCs from patients than in those from healthy subjects, whereas surface glycophorin A expression and reduced glutathione content did the opposite. Unlike RBCs obtained from healthy subjects, RBCs from patients and in vitro oxidised RBCs did not protect activated T lymphocytes from apoptosis. Hence, RBCs from patients with carotid atherosclerosis, probably due to their oxidative imbalance, impact T cell integrity and function. Our results suggest a new regulatory role for RBCs in atherosclerosis.

Use of several immunological markers to model the probability of active tuberculosis

Blood-based biomarkers tests are attractive alternative for diagnosing tuberculosis to assays depending on mycobacteria detection. Given several immunological markers we used logistic regression to model the probability of active tuberculosis in a cohort of patients with active or latent tuberculosis, showing an increased accuracy in distinguishing active from latent tuberculosis.