Multidimensional analysis of immune responses identified biomarkers of recent Mycobacterium tuberculosis infection

The risk of tuberculosis (TB) disease is higher in individuals with recent Mycobacterium tuberculosis (M.tb) infection compared to individuals with more remote, established infection. We aimed to define blood-based biomarkers to distinguish between recent and remote infection, which would allow targeting of recently infected individuals for preventive TB treatment. We hypothesized that integration of multiple immune measurements would outperform the diagnostic performance of a single biomarker. Analysis was performed on different components of the immune system, including adaptive and innate responses to mycobacteria, measured on recently and remotely M.tb infected adolescents. The datasets were standardized using variance stabilizing scaling and missing values were imputed using a multiple factor analysis-based approach. For data integration, we compared the performance of a Multiple Tuning Parameter Elastic Net (MTP-EN) to a standard EN model, which was built to the individual adaptive and innate datasets. Biomarkers with non-zero coefficients from the optimal single data EN models were then isolated to build logistic regression models. A decision tree and random forest model were used for statistical confirmation. We found no difference in the predictive performances of the optimal MTP-EN model and the EN model [average area under the receiver operating curve (AUROC) = 0.93]. EN models built to the integrated dataset and the adaptive dataset yielded identically high AUROC values (average AUROC = 0.91), while the innate data EN model performed poorly (average AUROC = 0.62). Results also indicated that integration of adaptive and innate biomarkers did not outperform the adaptive biomarkers alone (Likelihood Ratio Test χ² = 6.09, p = 0.808). From a total of 193 variables, the level of HLA-DR on ESAT6/CFP10-specific Th1 cytokine-expressing CD4 cells was the strongest biomarker for recent M.tb infection. The discriminatory ability of this variable was confirmed in both tree-based models.A single biomarker measuring M.tb-specific T cell activation yielded excellent diagnostic potential to distinguish between recent and remote M.tb infection.     

Mycobacterium abscessus ESX-3 plays an important role in host inflammatory and pathological responses during infection

Mycobacterial ESX systems are often related to pathogenesis during infection. However, little is known about the function of ESX systems of Mycobacterium abscessus (Mab). This study focuses on the Mab ESX-3 cluster, which contains major genes such as esxH (Rv0288, low molecular weight protein antigen 7; CFP-7) and esxG (Rv0287, ESAT-6 like protein). An esx-3 (MAB 2224c-2234c)-deletional mutant of Mab (Δesx) was constructed and used to infect murine and human macrophages. We then investigated whether Mab Δesx modulated innate host immune responses in macrophages. Mab Δesx infection resulted in less pathological and inflammatory responses. Additionally, Δesx resulted in significantly decreased activation of inflammatory signaling and cytokine production in macrophages compared to WT. Moreover, recombinant EsxG·EsxH (rEsxGH) proteins encoded by the ESX-3 region showed synergistic enhancement of inflammatory cytokine generation in macrophages infected with Δesx. Taken together, our data suggest that Mab ESX-3 plays an important role in inflammatory and pathological responses during Mab infection.     

Onchocerca volvulus: comparative analysis of antibody responses to recombinant antigens in two animal models of onchocerciasis

Experimental infections of chimpanzees with Onchocerca volvulus and cattle with Onchocerca ochengi provide model systems for research in human onchocerciasis. These infections share many similarities from the standpoint of parasite biology, but little is known about the comparability of immune responses in the two systems. To make a direct comparison between the models in terms of immune responsiveness to defined parasite products, three recombinant antigens of O. volvulus (Ov7, Ov103, and B20) were used to analyze the kinetics of antibody production following experimental infection. Each of the antigens was derived from adult cDNA libraries following immunoscreening with sera from chimpanzees (Ov7, Ov103) or cattle (B20). All chimpanzees (n = 12) and cattle (n = 8) displayed responses to Ov7 and Ov103, and all cattle, but only 33% of chimpanzees, showed responses to B20. The dynamics of the response to individual antigens showed further similarities between the chimpanzees and the cattle, with responses to Ov7 and Ov103 peaking after, and B20 before, the onset of patent infections. We conclude that there is good preliminary evidence of concordance in the kinetics of serological responses in the two models. However, individual antigens many be more or less immunogenic in the two systems, making it inadvisable to extrapolate between models concerning the relative immunodominance of specific parasite products.     

Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection

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Evaluation of immune responses raised against Tc13 antigens of Trypanosoma cruzi in the outcome of murine experimental infection

We have previously reported that genetic immunization with Tc13Tul antigen of Trypanosoma cruzi, the aetiological agent of Chagas' disease, triggers harmful effects and non-protective immune responses. In order to confirm the role of Tc13 antigens during T. cruzi infection, herein we studied the humoral and cellular immune responses to the Tc13Tul molecule and its EPKSA C-terminal portion in BALB/c T. cruzi-infected mice or mice immunized with recombinant Tc13Tul. Analysis of the antibody response showed that B-cell epitopes that stimulate a sustained IgM production along the infection and high levels of IgG in the acute phase are mainly located at the Tc13 N- and C-terminal domains, respectively. DTH assays showed that T-cell epitopes are mainly at the Tc13 N-terminal segment and that they do not elicit an efficient memory response. Recombinant Tc13Tul did not induce IFN-gamma secretion in either infected or immunized mice. However, a putative CD8+Tc13Tul-derived peptide was found to elicit IFN-gamma production in chronically infected animals. Immunization with recombinant Tc13Tul did not induce pathology in tissues and neither did it protect against the infection. Our results show that in the outcome of T. cruzi infection the Tc13 family protein mainly triggers non-protective immune responses.     

Genome analysis reveals three genomospecies in Mycobacterium abscessus

Background

Mycobacterium abscessus complex, the third most frequent mycobacterial complex responsible for community- and health care-associated infections in developed countries, comprises of M. abscessus subsp. abscessus and M. abscessus subsp. bolletii reviously referred as Mycobacterium bolletii and Mycobacterium massiliense. The diversity of this group of opportunistic pathogens is poorly described.

Results

In-depth analysis of 14 published M. abscessus complex genomes found a pan-genome of 6,153 proteins and core-genome of 3,947 (64.1%) proteins, indicating a non-conservative genome. Analysing the average percentage of amino-acid sequence identity (from 94.19% to 98.58%) discriminates three main clusters C1, C2 and C3: C1 comprises strains belonging to M. abscessus, C2 comprises strains belonging to M. massiliense and C3 comprises strains belonging to M. bolletii; and two sub-clusters in clusters C2 and C3. The phylogenomic network confirms these three clusters. The genome length (from 4.8 to 5.51-Mb) varies from 5.07-Mb in C1, 4.89-Mb in C2A, 5.01-Mb in C2B and 5.28-Mb in C3. The mean number of prophage regions (from 0 to 7) is 2 in C1; 1.33 in C2A; 3.5 in C2B and five in C3. A total of 36 genes are uniquely present in C1, 15 in C2 and 15 in C3. These genes could be used for the detection and identification of organisms in each cluster. Further, the mean number of host-interaction factors (including PE, PPE, LpqH, MCE, Yrbe and type VII secretion system ESX3 and ESX4) varies from 70 in cluster C1, 80 in cluster C2A, 74 in cluster C2B and 93 in clusters C3A and C3B. No significant differences in antibiotic resistance genes were observed between clusters, in contrast to previously reported in-vitro patterns of drug resistance. They encode both penicillin-binding proteins targeted by β-lactam antibiotics and an Ambler class A β-lactamase for which inhibitors exist.

Conclusions

Our comparative analysis indicates that M. abscessus complex comprises three genomospecies, corresponding to M. abscessus, M. bolletii, and M. massiliense. The genomics data here reported indicate differences in virulence of medical interest; and suggest targets for the refined detection and identification of M. abscessus.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-359) contains supplementary material, which is available to authorized users.     

Mycobacterium abscessus infection of a Norplant contraceptive implant site.

The authors report a case of Mycobacterium abscessus infection of a subdermal levonogestrel implant (Norplant) site. The infection lasted 12 weeks and was indolent, skin manifestations were low grade and difficult to detect. Culture of exudate samples showed that M. abscessus was the only causative agent. After the implant was removed the patient''s arm healed uneventfully without antimycobacterial therapy. The authors recommend that if Gram staining of apparently infected material from an implant site does not reveal a causative organism, then cultures should be done for mycobacteria and fungi. Kinyoun staining for acid-fast bacteria and calcoflour-white staining for fungi should also be performed. The implant should be removed and the patient given antimicrobial therapy as indicated. The authors emphasize the need to be aware of the potential for M. abscessus infection of implant sites and stress that appropriate microbiologic culture procedures are essential for accurate diagnosis.     

Expression of small RNAs of Mycobacterium tuberculosis in murine models of tuberculosis infection

The determination of the mechanisms contributing to the survival of pathogenic bacteria in the infected organism and the possible ways of their blocking is a promising approach to the development of new methods of affecting these bacteria. Among these mechanisms, the regulation of bacterial metabolism by small RNAs attracts particular interest since it has been found recently to play an important role in the bacterial pathogenesis. We have studied the expression of three most highly expressed small RNAs of Mycobacterium tuberculosis: MTS0997, MTS1338, and MTS2823 during tuberculosis progression in the strains of mice having different genetic resistance to the disease. It has been shown that the maximum expression of these small RNAs occurs at earlier stages of infection.     

IFNG-mediated immune responses enhance autophagy against Mycobacterium tuberculosis antigens in patients with active tuberculosis

Protective immunity against Mycobacterium tuberculosis (Mtb) requires IFNG. Besides, IFNG-mediated induction of autophagy suppresses survival of virulent Mtb in macrophage cell lines. We investigated the contribution of autophagy to the defense against Mtb antigen (Mtb-Ag) in cells from tuberculosis patients and healthy donors (HD). Patients were classified as high responders (HR) if their T cells produced significant IFNG against Mtb-Ag; and low responders (LR) when patients showed weak or no T cell responses to Mtb-Ag. The highest autophagy levels were detected in HD cells whereas the lowest quantities were observed in LR patients. Interestingly, upon Mtb-Ag stimulation, we detected a positive correlation between IFNG and MAP1LC3B-II/LC3-II levels. Actually, blockage of Mtb-Ag-induced IFNG markedly reduced autophagy in HR patients whereas addition of limited amounts of IFNG significantly increased autophagy in LR patients. Therefore, autophagy collaborates with human immune responses against Mtb in close association with specific IFNG secreted against the pathogen.     

IFNG-mediated immune responses enhance autophagy against Mycobacterium tuberculosis antigens in patients with active tuberculosis

Protective immunity against Mycobacterium tuberculosis (Mtb) requires IFNG. Besides, IFNG-mediated induction of autophagy suppresses survival of virulent Mtb in macrophage cell lines. We investigated the contribution of autophagy to the defense against Mtb antigen (Mtb-Ag) in cells from tuberculosis patients and healthy donors (HD). Patients were classified as high responders (HR) if their T cells produced significant IFNG against Mtb-Ag; and low responders (LR) when patients showed weak or no T cell responses to Mtb-Ag. The highest autophagy levels were detected in HD cells whereas the lowest quantities were observed in LR patients. Interestingly, upon Mtb-Ag stimulation, we detected a positive correlation between IFNG and MAP1LC3B-II/LC3-II levels. Actually, blockage of Mtb-Ag-induced IFNG markedly reduced autophagy in HR patients whereas addition of limited amounts of IFNG significantly increased autophagy in LR patients. Therefore, autophagy collaborates with human immune responses against Mtb in close association with specific IFNG secreted against the pathogen.     

Comparative functional analysis of helper T lymphocyte responses to soluble and particulate antigens

An adaptable and sensitive assay to analyze the roles of helper T lymphocytes (TH) which recognize soluble or cell-surface bound antigens in the induction of cytotoxic T lymphocyte precursors (CTLp) is described. Long-term T cell lines that recognize purified protein derivative, keyhole limpet hemocyanin, or Corynebacterium parvum were used in these studies. The ability of T cells from these lines to induce cytotoxic T lymphocyte or antibody responses were compared with their ability to proliferate or release interleukin 2 (IL 2). The results demonstrate that these T cell lines are able to react to soluble antigen by proliferation and IL 2 release. Moreover, the same cell lines are able to interact with CTLp or with the precursors of antibody-secreting B cells to induce a response. In the induction of CTLp we observed an inverse correlation between the number of TH cells required and the concentration of antigen used to pulse the antigen presenting cells. However the correlation between the ability of TH lines to proliferate specifically in response to antigen and to act as helpers for CTLp and B cells was not absolute as cells with compromised proliferative capacity were able to efficiently deliver inductive signals.     

Genetic control of murine T cell proliferative responses to Mycobacterium leprae. V. Evidence for cross-reactivity between host antigens and Mycobacterium leprae

T cell proliferative responses to Mycobacterium leprae were measured by immunization of mice at the base of the tail with Ag and challenging lymphocytes from draining lymph nodes in culture with M. leprae. C57BL/10J and B10.BR mice were identified as low responder mice and the congenic strains B10.M, B10.Q, and B10.AKM as high responders whereas F1 (high x low) hybrid mice were found to be low responders. The cellular basis of low responsiveness did not appear to result from a defect in Ag-presenting cells or the activation of suppressor T cells by M. leprae. The influence of the environment in which T cells developed on responsiveness to M. leprae was analyzed in chimeric mice prepared by irradiating F1(C57BL/10J x B10.M) mice and reconstituting with bone marrow from C57BL/10J, B10.M, or F1 donors. Six weeks later, chimeric mice were immunized with M. leprae, lymph node cells were subsequently prepared, and H-2 phenotyped and challenged in culture with M. leprae Ag. T cell proliferative responses were found to be low in all cases, similar to those observed using lymph node cells from F1 hybrid mice. These results suggested that high responder B10.M lymphocytes developing in the irradiated F1 mice became tolerized to antigenic determinants found on M. leprae. This implied cross-reactive epitopes existed between some mouse strains and M. leprae. Low responsiveness to M. leprae in low responder and F1 hybrid mice may result from tolerance to H-2-encoded Ag that show cross-reactivity with M. leprae.     

Long-term control of Mycobacterium tuberculosis infection is mediated by dynamic immune responses

The primary goal of this study was to determine how chronic exposure to Ag influences the functionality of Mycobacterium tuberculosis-specific T cell responses. The frequency of IFN-gamma-producing effector CD4(+) and CD8(+) T cells dynamically changed during persistent M. tuberculosis infection. CD8(+) T cells used differential effector functions during acute and chronic phases of the immune response, where CD8(+) T cells produced negligible amounts of IFN-gamma early in infection, but switched to cytokine production during the chronic stage of infection. Using limiting dilution analysis, CD8(+) T cells isolated during the initial phase of infection demonstrated lytic potential, but this waned in the chronic stage. The apparent loss of cytotoxic activity was not associated with the lack of perforin. Ag dose could potentially govern the functional program of CD8(+) T cells. Collectively, these results depict a host immune response mounted against M. tuberculosis of a significantly more dynamic nature than previously recognized.     

Murine immune responses to recombinant Toxoplasma gondii antigens

Recombinant proteins of the RH strain of Toxoplasma gondii were produced by expression in Escherichia coli as glutathione S-transferase (GST) fusion proteins. Enzyme-linked immunosorbent assays were established using 2 of these fusion proteins termed H4/GST and H11/GST. The assays were able to detect antibodies in the sera of mice orally infected with either the cyst or oocyst stage of a pork isolate of T. gondii. In addition, the sera from mice infected with 1 of 4 different T. gondii isolates were investigated for their binding to these fusion proteins. Antibodies in the sera of all mice bound to H11/GST, but not all sera recognized H4/GST. Delayed-type hypersensitivity (DTH) responses to the fusion proteins were found when the mice were sensitized intradermally with H4/GST and H11/GST and challenged with the homologous fusion protein. However, no DTH response was recorded when mice were challenged with homologous fusion proteins after infection with T. gondii, or after immunization with a sonicate of the RH strain of the parasite. In addition, cellular responses were not stimulated against either of the fusion proteins in in vitro assays. These 2 fusion proteins were recognized by anti-T. gondii antibodies in experimental murine infections, and they are therefore potential candidates as antigens in assays for the diagnosis of human toxoplasmosis.